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Autocrine selection of GLP-1 binding site

Posted in Cell Biology, Signaling & Cell Circuits, Chemical Biology and its relations to Metabolic Disease, Clinical & Translational, Cognition, CT, Diabetes Mellitus, tagged autocrine selection, endocrine control, GLP-1, hyperglycemia, peptides, T2D on December 9, 2015| Leave a Comment »

Autocrine selection of GLP-1 binding site

Larry H. Bernstein, MD, FCAP, Curator

LPBI

Update 12/15/2015

TSRI Team Finds Unique Anti-Diabetes Compound

Scientists from The Scripps Research Institute (TSRI) have deployed a powerful new drug discovery technique to identify an anti-diabetes compound with a novel mechanism of action

http://www.technologynetworks.com/HTS/news.aspx?ID=186055

The finding may lead to a new type of diabetes treatment. Just as importantly, it demonstrates the potential of the new technique, which enables researchers to quickly find drug candidates that activate cellular receptors in desired ways.

“In principle, we can apply this technique to hundreds of other receptors like the one we targeted in this study to find disease treatments that are more potent and have fewer side effects than existing therapies. It has been a very productive cross-campus collaboration, so we’re hoping to build on its success as we continue to collaborate on interrogating potential therapeutic targets,” said Patricia H. McDonald, an assistant professor at TSRI’s Jupiter, Florida campus and a senior investigator of the study.

McDonald’s laboratory collaborated on the study with the laboratory of Richard A. Lerner, the Lita Annenberg Hazen Professor of Immunochemistry at TSRI’s La Jolla campus, and with other TSRI groups. Lerner has pioneered techniques for generating and screening large libraries of antibodies or proteins to find new therapies.

In Search of a Better Activator

Three years ago, Lerner and colleagues devised a technique called autocrine selection, which enables scientists to screen very large libraries of molecules to find those that not only bind a given cellular receptor but also activate it to bring about a desired therapeutic effect. Since then, the Lerner laboratory and collaborating scientists have used the technique to find new molecules that block cold virus infection, boost red blood cell production and kill cancer cells, among other effects.

For the new study, Lerner and his laboratory used the technique to target a receptor linked to type 2 diabetes, a life-shortening disease estimated to affect 30 million people in the US alone.

The GLP-1 receptor, as it is known, is expressed by insulin-producing “beta cells” in the pancreas. Several drugs that activate this receptor—drugs called GLP-1 receptor agonists—are already approved for treating type 2 diabetes. In this case, the TSRI team’s aim was to find a molecule that activates the GLP-1 receptor in a unique way.

The GLP-1 receptor belongs to a large class of receptors known as G protein-coupled receptors (GPCRs). Scientists recently have come to understand that when a molecule activates a GPCR, it doesn’t necessarily trigger a single chain of biochemical signals within the cell. In fact, most GPCR agonists trigger signals via multiple distinct pathways—one being via a so-called G protein and another via a protein known as beta-arrestin. In some cases, a “biased agonist” that principally activates just one of these pathways would work better than one that activates both.

In this case, Lerner and his laboratory teamed up with McDonald, an expert on GPCRs and metabolic disease, to find a molecule that would preferentially activate the GLP-1 receptor’s G protein pathway.

To start, researchers in Lerner’s laboratory, including Hongkai Zhang, a senior staff scientist and co-first author of the study, generated a library of candidate molecules—based on a known GLP-1 receptor agonist, Exendin-4, a small protein (peptide) originally found in the venom of Gila monster lizards; a synthetic version of this protein is now used as a type 2 diabetes medication. Zhang created about one million new peptides by randomly varying one end of Exendin-4—the end that normally activates the G protein and beta arrestin pathways.

“The idea was that at least one of these many variants would induce a change in the shape of the GLP-1 receptor that would activate the G-protein pathway without activating the beta arrestin pathway,” Zhang said.

Using the autocrine selection system, Zhang and colleagues rapidly screened these variant peptides and eventually isolated one, P5, that potently and selectively activated the GLP-1 receptor’s G-protein pathway. An initial test in healthy mice showed that P5 worked well at boosting glucose tolerance—at about one-hundredth the dose of Exendin-4 needed for the same effect.

Protein expert Philip E. Dawson, an associate professor at TSRI’s La Jolla campus, synthesized sufficient quantities of P5, and McDonald and her laboratory performed more advanced tests in cultured cells and in mice.

A Different Mechanism

Exendin-4 and and other GLP-1 receptor agonists work in part by strongly stimulating pancreatic beta cells to produce more insulin—which signals muscle and fat cells to draw glucose from the blood, thus lowering blood glucose levels.

McDonald and her team found that although P5 equals or outperforms Exendin-4 in standard mouse models of diabetes, it stimulates insulin production only weakly.

“We didn’t expect that, but in fact, it was a nice finding because less reliance on stimulating insulin could mean less stress on the beta cells,” said Emmanuel Sturchler, staff scientist in the McDonald laboratory and co-first author of the study.

Investigating further, the team found that while the peptide doesn’t make mice fatter or heavier, it triggers the growth of new fat cells. In typical obesity-related diabetes, fat cells grow larger, not more numerous, and as they grow larger, they lose their ability to respond to insulin (insulin resistance). The proliferation of fat cells with P5 was accompanied by signs of increased insulin sensitivity in those cells, suggesting that the peptide works in part by alleviating insulin resistance.

Exendin-4 induces a feeling of satiety, causing mice (and people) to modestly lower food intake and thus lose weight. But the researchers found that P5 lacks this mechanism and appears to have no effect on appetite or weight.

“P5’s mechanisms of action turned out to be quite different from Exendin-4’s, and we think that this finding could lead to new therapeutics,” Sturchler said.

The team will now look for opportunities to develop P5 into a new diabetes drug. The researchers also see this as the first of many discoveries of GPCR-targeting compounds with unique and potentially valuable properties—as well as discoveries in basic GPCR biology.

New screening tech at Scripps spotlights diabetes drug candidates

Wednesday, December 9, 2015 | By John Carrol

The Scripps Research Institute has used a new drug screening platform to identify a drug which researchers believe has strong potential for treating diabetes.

Working with a technique dubbed autocrine selection, investigators are able to screen molecules in search of targets that can bind to and activate cellular receptors in order to achieve a sought-after drug effect.

In this latest study, published in Nature Communications, the Scripps team went after the GLP-1 receptor, which is already the target of a number of GLP-1 agonists. Scripps, though, wanted to activate the GLP-1 receptor’s G protein pathway.

Hongkai Zhang focused on the GLP-1 agonist Extendin-4, whipping up a million peptides that could alter the end of the protein that activates the G protein and beta arrestin pathways.

They then identified the one in a million that improved glucose tolerance at a radically reduced dose of Extendin-4, testing it on mice.

“P5’s mechanisms of action turned out to be quite different from Exendin-4’s, and we think that this finding could lead to new therapeutics,” said Emmanuel Sturchler, a staff scientist in the McDonald laboratory and co-first author of the study.

https://www.scripps.edu/news/press/2015/20151207lerner-mcdonald.html

Scientists from The Scripps Research Institute (TSRI) have deployed a powerful new drug discovery technique to identify an anti-diabetes compound with a novel mechanism of action.

The finding, which appeared online ahead of print in Nature Communications, may lead to a new type of diabetes treatment. Just as importantly, it demonstrates the potential of the new technique, which enables researchers to quickly find drug candidates that activate cellular receptors in desired ways.

‘Fingerprints’ for Major Drug Development Targets

For the first time, scientists from the Florida campus of The Scripps Research Institute (TSRI) have created detailed “fingerprints” of a class of surface receptors that have proven highly useful for drug development.

http://www.technologynetworks.com/HTS/news.aspx?ID=185860

These detailed “fingerprints” show the surprising complexity of how these receptors activate their binding partners to produce a wide range of signaling actions.

The study focuses on interactions of G protein-coupled receptors (GPCRs) with their signaling mediators known as G proteins. GPCRs—currently accounting for about 40 percent of all prescription pharmaceuticals on the market—play key roles in many physiological functions because they transmit signals from outside the cell to the interior. When an outside substance binds to a GPCR, it activates a G protein inside the cell to release components and create a specific cellular response.

“Until now, it was generally believed that GPCRs are very selective, activating only a few G proteins they were designed to work with,” said TSRI Associate Professor Kirill Martemyanov, who led the study. “It turns out the reality is much more complex.”

Ikuo Masuho, a senior research associate in the Martemyanov lab, added, “Our imaging technology opens a unique avenue of developing drugs that would precisely control complex GPCR-G protein coupling, maximizing therapeutic potency by activating G proteins that contribute to therapeutic efficacy while inhibiting other G proteins that cause adverse side effects.”

The study found that individual GPCRs engage multiple G proteins with varying efficacy and rates, much like a dance where the most desirable partner, the GPCR, is surrounded by 14 suitors all vying for attention. The results, as in any dance, depend on which G proteins bind to the receptor—and for how long. The same receptor changes G protein partners—and the signaling outcome—depending on the action of the signal received from outside of the cell.

This finding was made possible by novel imaging technology used by the Martemyanov lab to monitor G protein activation in live cells. Using a pair of light-emitting proteins, one attached to the G protein, the other attached to what’s known as a reporter molecule, Martemyanov and his colleagues were able to measure simultaneously both the signal and activation rates of most G proteins present in the body.

“Our approach looks at 14 different types of G proteins at once—and we only have 16 in our bodies,” he said. “This is as close as it can get to what is actually happening in real time.”

In the accompanying commentary in Science Signaling, Alan Smrcka, a professor at University of Rochester Medical School and a prominent GPCR researcher, wrote, “[The findings] suggest the power of the GPCR fingerprinting approach, in that it could predict the G protein coupling specificity of a GPCR in a native system, which was previously undetected by conventional analysis. This could be very helpful for identifying previously unappreciated signaling pathways downstream of individual GPCRs that could be useful therapeutically or identified as potential side effects of GPCRs.”

Long-Acting Glucagon-Like Peptide 1 Receptor Agonists

A review of their efficacy and tolerability

Alan J. Garber, MD, PHD⇓

Diabetes Care May 2011; 34(Supplement 2): S279-S284 http://dx.doi.org/10.2337/dc11-s231

Targeting the incretin system has become an important therapeutic approach for treating type 2 diabetes. Two drug classes have been developed: glucagon-like peptide (GLP)-1 receptor agonists and dipeptidyl peptidase 4 (DPP-4) inhibitors. Clinical data have revealed that these therapies improve glycemic control while reducing body weight (GLP-1 receptor agonists, specifically) and systolic blood pressure (SBP) in patients with type 2 diabetes. Furthermore, incidence of hypoglycemia is relatively low with these treatments (except when used in combination with a sulfonylurea) because of their glucose-dependent mechanism of action. There are currently two GLP-1 receptor agonists available (exenatide and liraglutide), with several more currently being developed. This review considers the efficacy and safety of both the short- and long-acting GLP-1 receptor agonists. Head-to-head clinical trial data suggest that long-acting GLP-1 receptor agonists produce superior glycemic control when compared with their short-acting counterparts. Furthermore, these long-acting GLP-1 receptor agonists were generally well tolerated, with transient nausea being the most frequently reported adverse effect.

Careful consideration should be given to the selection of therapies for managing type 2 diabetes. In particular, antidiabetic agents that offer improved glycemic control without increasing cardiovascular risk factors or rates of hypoglycemia are warranted. At present, many available treatments for type 2 diabetes fail to maintain glycemic control in the longer term because of gradual disease progression as β-cell function declines. Where sulfonylureas or thiazolidinediones (common oral antidiabetic drugs) are used, the risk of hypoglycemia and weight gain can increase (1,2). The development of new therapies for the treatment of type 2 diabetes that, in addition to maintaining glycemic control, could reduce body weight and hypoglycemia risk (3,4), may help with patient management. Indeed, guidelines have been developed that support the consensus that blood pressure, weight reduction, and avoidance of hypoglycemic events should be targeted in type 2 diabetes management alongside glycemic targets. For example, the American Diabetes Association (ADA) defines multiple goals of therapy that include A1C <7.0% and SBP <130 mmHg and no weight gain (or, in the case of obese subjects, weight loss) (5). In particular, incretin-based therapies (GLP-1 receptor agonists, specifically) can help meet these new targets by offering weight reduction, blood pressure reduction, and reduced hypoglycemia in addition to glycemic control.

WHAT IS GLP-1?

The incretin effect, responsible for 50–70% of total insulin secretion after oral glucose administration, is defined as the difference in insulin secretory response from an oral glucose load compared with intravenous glucose administration (6) (Supplementary Fig. 1).

There are two naturally occurring incretin hormones that play a role in the maintenance of glycemic control: glucose-dependent insulinotropic polypeptide and GLP-1, both of which have a short half-life because of their rapid inactivation by DPP-4 (7). In patients with type 2 diabetes, the incretin effect is reduced or, in some cases, absent (8). In particular, the insulinoptropic action of glucose-dependent insulinotropic polypeptide is lost in patients with type 2 diabetes. However, it has been shown that, after administration of pharmacological levels of GLP-1, the insulin secretory function can be restored in this population (9), and thus GLP-1 has become an important target for research into new therapies for type 2 diabetes.

GLP-1 has multiple physiological effects that make it an attractive candidate for type 2 diabetes therapy. It increases insulin secretion while inhibiting glucagon release, but only when glucose levels are elevated (6,10), thus offering the potential to lower plasma glucose while reducing the likelihood of hypoglycemia. Furthermore, gastric emptying is delayed (10) and food intake is decreased after GLP-1 administration. Indeed, in a 6-week study investigating continuous GLP-1 infusion, patients with type 2 diabetes achieved a significant weight loss of 1.9 kg and a reduction in appetite from baseline compared with patients receiving placebo, where there was no significant change in weight or appetite (11). Preclinical studies reveal other potential benefits of GLP-1 receptor agonist treatment in individuals with type 2 diabetes, which include the promotion of β-cell proliferation (12) and reduced β-cell apoptosis (13). These preclinical results indicate that GLP-1 could be beneficial in treating patients with type 2 diabetes. However, because native GLP-1 is rapidly inactivated and degraded by the enzyme DPP-4 and has a very short half-life of 1.5 min (14), to achieve the clinical potential for native GLP-1, patients would require 24-h administration of native GLP-1 (15). Because this is impractical as a therapeutic option for type 2 diabetes, it was necessary to develop longer-acting derivatives of GLP-1.

Previous Section Next Section

DEVELOPMENT OF DPP-4–RESISTANT GLP-1 RECEPTOR AGONISTS

Two classes of incretin-based therapy have been developed to overcome the clinical limitations of native GLP-1: GLP-1 receptor agonists (e.g., liraglutide and exenatide), which exhibit increased resistance to DPP-4 degradation and thus provide pharmacological levels of GLP-1, and DPP-4 inhibitors (e.g., sitagliptin, vildagliptin, saxagliptin), which reduce endogenous GLP-1 degradation, thereby providing physiological levels of GLP-1. In this review, we focus on the GLP-1 receptor agonist class of incretin-based therapies. The efficacy and tolerability of the DPP-4 inhibitors have been reviewed elsewhere (16). Two GLP-1 receptor agonists are licensed at present in Europe, the U.S., and Japan: exenatide (Byetta, Eli Lilly) (17) and liraglutide (Victoza, Novo Nordisk) (18). For the purposes of this review, we refer to “short-acting” GLP-1 receptor agonists as those agents having duration of action of <24 h and “long-acting” as those agents with duration of action >24 h (Table 1).

….. more http://care.diabetesjournals.org/content/34/Supplement_2/S279.full.pdf+html

Autocrine selection of a GLP-1R G-protein biased agonist with potent antidiabetic effects

Hongkai Zhang, Emmanuel Sturchler, Jiang Zhu, Ainhoa Nieto, Philip A. Cistrone,…., Patricia H. McDonald & Richard A. Lerner
Nature Communications Dec 2015; 6(8918) http://dx.doi.org:/10.1038/ncomms9918

Glucagon-like peptide-1 (GLP-1) receptor (GLP-1R) agonists have emerged as treatment options for type 2 diabetes mellitus (T2DM). GLP-1R signals through G-protein-dependent, and G-protein-independent pathways by engaging the scaffold protein β-arrestin; preferential signalling of ligands through one or the other of these branches is known as ‘ligand bias’. Here we report the discovery of the potent and selective GLP-1R G-protein-biased agonist, P5. We identified P5 in a high-throughput autocrine-based screening of large combinatorial peptide libraries, and show that P5 promotes G-protein signalling comparable to GLP-1 and Exendin-4, but exhibited a significantly reduced β-arrestin response. Preclinical studies using different mouse models of T2DM demonstrate that P5 is a weak insulin secretagogue. Nevertheless, chronic treatment of diabetic mice with P5 increased adipogenesis, reduced adipose tissue inflammation as well as hepatic steatosis and was more effective at correcting hyperglycemia and lowering hemoglobin A_1clevels than Exendin-4, suggesting that GLP-1R G-protein-biased agonists may provide a novel therapeutic approach to T2DM.

Figure 1: Autocrine-based system for selection of agonists from large combinatorial peptide libraries

Autocrine-based system for selection of agonists from large combinatorial peptide libraries.

(a) Schematic representation of the peptide libraries. (b) Schematic representation of the membrane-tethered Exendin-4 (top) and FACS analysis of mCherry and GFP expression 2 days after transduction of HEK293-GLP-1R-GFP cells with the membrane-tethered Exendin-4 displaying different linker size (bottom). (c) Schematic representation of the autocrine-based selection of combinatorial peptide library. The lentivirus peptide libraries are preparred from lentiviral plasmids (step 1). The CRE-responsive GLP-1R reporter cell line is transduced with lentiviral library (step 2). GFP expressing cells are sorted (step 3) and peptide-encoding genes are amplified from genomic DNA of sorted cells to make the library for the next selection round (step 4). After iterative rounds of selection, enriched peptide sequences are analysed by deep sequencing (step 5). (d) Enrichment of GFP positive cells during three rounds of FACS selection. (e) N termini sequences of top 13 peptides (frequency>1.0% representation).

Type 2 diabetes mellitus (T2DM) is a complex metabolic disorder characterized by hyperglycaemia arising from a combination of insufficient insulin secretion together with the development of insulin resistance. The incretin, glucagon-like peptide-1 (GLP-1) is an endogenous peptide hormone secreted from intestinal endocrine cells in response to food intake¹. GLP-1 lowers postprandial glucose excursion by potentiating glucose-stimulated insulin secretion from pancreatic β-cells and has also recently been shown to promote β-cell survival in rodents². In addition, GLP-1 exerts extra-pancreatic actions such as promoting gastric emptying, weight loss and increasing insulin sensitivity in peripheral tissues³. Hence, incretin-based therapies represent a strategy for the treatment of T2DM.

GLP-1 exerts its action through the GLP-1 receptor (GLP-1R)⁴ expressed in the pancreas, other peripheral tissues, and the central nervous system. Activation of GLP-1R triggers Gαs-protein coupling leading to an elevation of cyclic AMP (cAMP), modulates intracellular calcium concentration⁵ and induces β-arrestin recruitment^{6, 7}. Historically, β-arrestins were believed to serve an exclusive role in G-protein coupled receptor (GPCR) desensitization⁸. However, it has since been shown that β-arrestins can also function to activate signalling cascades^{9, 10}. In this regard, in the pancreatic β-cell, elevation of both cAMP and cytosolic Ca²⁺ and β-arrestin signalling downstream of GLP-1R activation are critical events in promoting glucose-dependent insulin secretion.

Recently, the concept of ‘functional selectivity’ or ‘ligand bias’ has emerged whereby ligand binding promotes engagement of only a particular subset of the full GPCR signalling repertoire to the exclusion of others¹¹. A better understanding of GLP-1R pleiotropic signalling and the underlying physiological consequences might provide new avenues for the development of drugs with novel modes of action that have the potential to provide greater therapeutic value while possibly avoiding unwanted side effects^{12, 13}. Therefore we developed an autocrine-based system, to screen large and diverse, combinatorial peptide libraries containing up to 100 million different members with the aim of identifying potent, selective, G-protein-biased GLP-1R agonists. We identified one such ligand, designated P5 and have characterized its in vitro pharmacological phenotype, and explored its therapeutic potential.

P5 is a selective and potent G-protein-biased GLP-1R agonist

To assess potential signalling bias, the active peptides were further characterized in vitro using distinct assays that monitor receptor proximal signals. Cell-based assays for Gαs-protein (cAMP production), Gαq-protein (intracellular Ca²⁺ mobilization) and β-arrestin (1 and 2) signalling were used to determine the potency (EC₅₀; effector concentration for half-maximum response) and maximal efficacy (E_max (%)) of peptides relative to the reference ligand Ex4 (Table 1). Peptides P1, P2, P5 and P10 all stimulated cAMP production. However, only P5 functioned as a full agonist (E_max=100%) displaying sub-nanomolar potency at both the human (hGLP-1R) and mouse receptor (mGLP-1R) (Fig. 2a,b; Table 1). The P5 EC₅₀ was similar to the endogenous ligand GLP-1 but was slightly right shifted when compared with the reference peptide Ex4 (Fig. 2a,b; Table 1). Importantly, P5-induced cAMP production was inhibited by the selective GLP-1R antagonist Ex 9–39 in a concentration-dependent manner (Supplementary Fig. 1a,b). In addition, P5-induced cAMP production was negligible in HEK293 cells expressing the human glucagon receptor (Supplementary Fig. 1c). These data suggest that P5 selectively interacts with the GLP-1R.

In line with previous reports^{43, 44, 45} our data support the notion that non β-cell actions of GLP-1 agonists can improve glycaemic control. Importantly, GLP-1R is expressed in adipose tissue, in both the stromal vascular and the adipocyte fraction and its expression level has been found to correlate with the degree of insulin resistance⁴⁶. In addition, the GLP-1 peptide has been reported to regulate adipogenesis in vitro^{47, 48}. Given that P5, a G-protein-biased agonist with a severely blunted β-arrestin response has less propensity to induce GLP-1R desensitization, sustained activation of the receptor in adipose tissue may lead to the changes we observed in eWAT. Consistent with this notion, increased expression of adipogenic genes and a decrease in resistin expression was reported in β-arrestin 1 knockout mice⁴⁹. Nevertheless, considering the multitude of metabolic pathways regulated by β-arrestin, further studies are warranted to determine the role of β-arrestin signalling downstream of GLP-1R activation in adipogenesis. Additionally, we found that chronic treatment with P5 increased circulating level of GIP to a greater extent than Ex4. Several studies demonstrated that GIP acts as an insulin sensitizer in adipocytes and disruption of the GIP/GIP-R axis has been reported in insulin-resistant states such as obesity^{50, 51}. Interestingly, PPARγ activation was shown to increase GIP-R levels during adipocyte differentiation⁵². Thus, by increasing GIP and PPARγ levels, P5 chronic treatment may restore GIP/GIP-R signalling in adipocytes. Furthermore, previous studies have demonstrated that the simultaneous activation of the GLP-1R and the GIP-R results in enhanced glycaemic control, and lower HbA1c levels in human and rat, when compared with GLP-1R alone⁵³, suggesting a GIP and GLP-1 synergism. Thus, the superior glycaemic control observed with the G-protein-biased agonist may result from P5-induced increases in GIP level and concomitant receptor activation. In addition, the GLP-1R can form homodimers as well as ligand-induced heterodimers with the GIP-R⁵⁴. It is conceivable, that P5 may promote the formation of new and pharmacologically distinct homo/heterodimers displaying different signalling capacity. However, further studies are required to delineate more precisely the molecular and cellular mechanisms and the consequences of P5-induced increase in GIP levels.

In summary, high-throughput autocrine-based functional screening of combinatorial peptide libraries enabled the discovery of a high potency G-protein-biased GLP-1R agonist demonstrating new pharmacological virtues. In a series of translational preclinical studies we demonstrate that P5 is a weak insulin secretagogue yet displays superior antidiabetic effect (Fig. 7). Thus, GLP-1R G-protein-biased ligands may offer new and unappreciated advantages in the context of chronic treatment such as promoting adipocyte hyperplasia, restoring insulin responsiveness and long-term glycaemic control while preserving pancreatic β-cell function by minimizing the insulin secretory burden.

Figure 7: Schematic depicting the identification and characterization of a novel GLP-1R-biased agonist.

Schematic depicting the identification and characterization of a novel GLP-1R-biased agonist.

Using an autocrine-based system coupled to FACS, we screened large, diverse, combinatorial peptide libraries and identified P5, a potent and selective G-protein-biased GLP-1R agonist. P5 displayed a decreased insulinotropic effect, yet significantly improved glucose tolerance and insulin responsiveness by promoting white adipocyte tissue hyperplasia.

Exendin-4 Is a High Potency Agonist and Truncated Exendin-(9-39)- amide an Antagonist at the Glucagon-like Peptide 1-(7-36)-amide Receptor of Insulin-secreting ,&Cells*

Riidiger Goke, Hans-Christoph Fehmann, Thomas LinnS, Harald Schmidt, Michael Krause9, John EngT, and Burkhard GokeII
J Biol Chem Sept 1993;268(26):19650-19655 http://www.jbc.org/content/268/26/19650.full.pdf

Exendin-4 purified from Heloderma suspecturn venom shows structural relationship to the important incretin hormone glucagon-like peptide 1-(7-36)- amide (GLP-1). We demonstrate that exendin-4 and truncated exendin-(9-39)-amide specifically interact with the GLP-1 receptor on insulinoma-derived cells and on lung membranes. Exendin-4 displaced “‘IGLP- 1, and unlabeled GLP- 1 displaced lZ6I-exendin-4 from the binding site at rat insulinoma-derived RINmSF cells. Exendin-4 had, like GLP-1, a pronounced effect on intracellular CAMP generation, which was reduced by exendin-(9-39)-amide. When combined, GLP-1 and exendin-4 showed additive action on CAMP. They each competed with the radiolabeled version of the other peptide in cross-linking experiments. The apparent molecular mass of the respective ligand-binding protein complex was 63,000 Da. Exendin-(9-39)-amide abolished the cross-linking of both peptides. Exendin-4, like GLP-1, stimulated dose dependently the glucose-induced insulin wcretion in isolated rat islets, and, in mouse insulinoma TC-1 cells, both peptides stimulated the proinsulin gene expression at the level of transcription. Exendin- (9-39)-amide reduced these effects. In conclusion, exendin-4 is an agonist and exendin-(9-39)-amide is a specific GLP- 1 receptor antagonist.

Glucagon-like peptide-1 receptor agonists for the treatment of type 2 diabetes mellitus

Kathleen Dungan, MD, Anthony DeSantis, MD
http://www.uptodate.com/contents/glucagon-like-peptide-1-receptor-agonists-for-the-treatment-of-type-2-diabetes-mellitus

Despite advances in options for the treatment of diabetes, optimal glycemic control is often not achieved. Hypoglycemia and weight gain associated with many antidiabetic medications may interfere with the implementation and long-term application of “intensive” therapies [1]. Current treatments have centered on increasing insulin availability (either through direct insulin administration or through agents that promote insulin secretion), improving sensitivity to insulin, delaying the delivery and absorption of carbohydrate from the gastrointestinal tract, or increasing urinary glucose excretion.

Glucagon-like peptide-1 (GLP-1)-based therapies (eg, GLP-1 receptor agonists, dipeptidyl peptidase 4 [DPP-4] inhibitors) affect glucose control through several mechanisms, including enhancement of glucose-dependent insulin secretion, slowed gastric emptying, and reduction of postprandial glucagon and of food intake (table 1). These agents do not usually cause hypoglycemia in the absence of therapies that otherwise cause hypoglycemia.

This topic will review the mechanism of action and therapeutic utility of GLP-1 receptor agonists for the treatment of type 2 diabetes mellitus. DPP-4 inhibitors are discussed separately. A general discussion of the initial management of blood glucose and the management of persistent hyperglycemia in adults with type 2 diabetes is also presented separately. (See “Dipeptidyl peptidase 4 (DPP-4) inhibitors for the treatment of type 2 diabetes mellitus”.)

GLUCAGON-LIKE PEPTIDE-1

Glucose homeostasis is dependent upon a complex interplay of multiple hormones: insulin and amylin, produced by pancreatic beta cells; glucagon, produced by pancreatic alpha cells; and gastrointestinal peptides, including glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP; gastric inhibitory polypeptide) (figure 1). Abnormal regulation of these substances may contribute to the clinical presentation of diabetes. The role of GLP-1 in glucose homeostasis is illustrative of the incretin effect, in which oral glucose has a greater stimulatory effect on insulin secretion than intravenous glucose [2]. This effect is mediated by several gastrointestinal peptides, particularly GLP-1, that are released in the setting of a meal and stimulate insulin synthesis and insulin secretion, which does not occur when carbohydrate is administered intravenously.

GLP-1 is produced from the proglucagon gene in L-cells of the small intestine and is secreted in response to nutrients (figure 1) [3]. GLP-1 binds to a specific GLP-1 receptor, which is expressed in various tissues including pancreatic beta cells, pancreatic ducts, gastric mucosa, kidney, lung, heart, skin, immune cells, and the hypothalamus [2,4]. GLP-1 exerts its main effect by stimulating glucose-dependent insulin release from the pancreatic islets [2]. It has also been shown to slow gastric emptying [5], inhibit inappropriate post-meal glucagon release [3,6], and reduce food intake (table 1) [3]. Owing in part to the effects of GLP-1 on slowed gastric emptying and appetite centers in the brain, therapy with GLP-1 and its receptor agonists is associated with weight loss, even among patients without significant nausea and vomiting.

Exendin-4, a glucagon-like peptide-1 receptor agonist, reduces Alzheimer disease-associated tau hyperphosphorylation in the hippocampus of rats with type 2 diabetes.

Xu W¹, Yang Y, Yuan G, Zhu W, Ma D, Hu S. Author information

J Investig Med. 2015 Feb;63(2):267-72. http://dx.doi.org:/10.1097/JIM.0000000000000129.

Impaired insulin signaling pathway in the brain in type 2 diabetes (T2D) is a risk factor for Alzheimer disease (AD). Glucagon-like peptide-1 (GLP-1) and its receptor agonist are widely used for treatment of T2D. Here we studied whether the effects of exendin-4 (EX-4), a long-lasting GLP-1 receptor agonist, could reduce the risk of AD in T2D. RESULTS: The levels of phosphorylated tau protein at site Ser199/202 and Thr217 level in the hippocampus of T2D rats were found to be raised notably and evidently decreased after EX-4 intervention. In addition, brain insulin signaling pathway was ameliorated after EX-4 treatment, and this result was reflected by a decreased activity of PI3K/AKT and an increased activity of GSK-3β in the hippocampus of T2D rats as well as a rise in PI3K/AKT activity and a decline in GSK-3β activity after 4 weeks intervention of EX-4. CONCLUSIONS: These results demonstrate that multiple days with EX-4 appears to prevent the hyperphosphorylation of AD-associated tau protein due to increased insulin signaling pathway in the brain. These findings support the potential use of GLP-1 for the prevention and treatment of AD in individuals with T2D.

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Peptides and anti-Cancer activity

Posted in CANCER BIOLOGY & Innovations in Cancer Therapy, Cell Biology, Signaling & Cell Circuits, Chemical Biology and its relations to Metabolic Disease, Curation, Cytoskeleton, Innovations, Lipid metabolism, Lipids, Metabolism, Metabolomics, Proteins, Proteomics, Signaling & Cell Circuits, tagged anticancer activity, antimicrobial activity, glycerophospholipids, lipid bilayer, membrane permeability, membrane poration, MP1 peptide, peptides, phosphoethanolamine, phosphoserine, PIM2-dependent phosphorylation of PKM2 on October 31, 2015| Leave a Comment »

Peptides and anti-Cancer activity

Larry H. Bernstein, MD, FCAP, Curator

LPBI

PE and PS Lipids Synergistically Enhance Membrane Poration by a Peptide with Anticancer Properties

Natália Bueno Leite, Anders Aufderhorst-Roberts, Mario Sergio Palma, Simon D. Connell, João Ruggiero Neto, Paul A. Beales

Biophys J 1 Sept 2015; 109(5):936–947. DOI: http://dx.doi.org/10.1016/j.bpj.2015.07.033

Polybia-MP1 (MP1) is a bioactive host-defense peptide with known anticancer properties. Its activity is attributed to excess serine (phosphatidylserine (PS)) on the outer leaflet of cancer cells. Recently, higher quantities of phosphatidylethanolamine (PE) were also found at these cells’ surface. We investigate the interaction of MP1 with model membranes in the presence and absence of POPS (PS) and DOPE (PE) to understand the role of lipid composition in MP1’s anticancer characteristics. Indeed we find that PS lipids significantly enhance the bound concentration of peptide on the membrane by a factor of 7–8. However, through a combination of membrane permeability assays and imaging techniques we find that PE significantly increases the susceptibility of the membrane to disruption by these peptides and causes an order-of-magnitude increase in membrane permeability by facilitating the formation of larger transmembrane pores. Significantly, atomic-force microscopy imaging reveals differences in the pore formation mechanism with and without the presence of PE. Therefore, PS and PE lipids synergistically combine to enhance membrane poration by MP1, implying that the combined enrichment of both these lipids in the outer leaflet of cancer cells is highly significant for MP1’s anticancer action. These mechanistic insights could aid development of novel chemotherapeutics that target pathological changes in the lipid composition of cancerous cells.

The antimicrobial peptide Polybia-MP1 (IDWKKLLDAAKQIL-NH2), or simply MP1, has unexpectedly been shown to exhibit selective inhibition against several types of cancerous cells and therefore could prove advantageous in the development of novel chemotherapies. Extracted from the Brazilian waspPolybia paulista, MP1 has a broad spectrum of bactericidal activities against Gram-negative and Gram-positive bacteria without being hemolytic and cytotoxic (1). Surprisingly, MP1 also selectively inhibits proliferating bladder and prostate cancer cells (2), and multidrug-resistant leukemic cells (3). Recently, it has been observed that this peptide is cytotoxic against leukemic T lymphocytes and very selective in recognizing these cells compared to healthy lymphocytes (4).

Cancer cell membranes are now known to lose the asymmetric transmembrane distribution of phospholipids that is observed in healthy cells (5, 6). In healthy mammalian cells, the anionic aminophospholipid PS (phosphatidylserine) is predominant in the inner membrane leaflet and zwitterionic phospholipids are predominant in outer membrane leaflet. In such cells, the phospholipid asymmetry is maintained by a family of aminophospholipid translocases that catalyze the transport of PS from the outer to the inner membrane leaflets (7). However, in apoptotic and cancer cells, PS is found to also be located in the outer monolayer of the plasma membrane in significant proportions (5, 6).

The molecular-scale mechanistic basis for MP1’s anticancer properties is yet to be established. Changes in the distribution and/or composition of lipids (e.g., PS) within the plasma membrane of malignant cells could be the origin of MP1’s cancer selectivity. This is a reasonable hypothesis, based upon the well-established selectivity of antimicrobial peptides for bacterial membranes over eukaryotic membranes due to their higher anionic lipid content (8, 9, 10, 11). Recently, the effect of PS on the pore-forming activity of MP1 was investigated by multiple techniques, namely, conductance measurements in planar bilayer lipid membranes, binding assays, and lytic activity on large unilamellar vesicles (4). Although an increase in affinity and lytic activity of MP1 for lipid vesicles containing PS was observed, MP1’s pore-formation activity in BLM showed no difference between PC (phosphatidylcholine) and mixed PC/PS bilayers. Significantly, it was recently reported that PE (phosphatidylethanolamine) lipids, naturally found on the inner plasma membrane of normal cells, are also externalized to the outer monolayer of the plasma membrane of apoptotic and tumor endothelial cells due to both PS and PE lipids being coregulated by the same transporters (7). These authors observed that the exposure to the outer monolayer of one of these phospholipids leads to the exposure of the other. Therefore, it is important for future work to establish the role of increased concentrations of both PE and PS lipids in the interaction of MP1 with membranes.

In this work, we address this challenge by establishing the roles of PE and PS lipids in the effects of MP1 on the structure and permeability of model membranes. Primarily, we study the permeability of giant unilamellar vesicles (GUVs) at the single vesicle level. Fluorescence confocal microscopy was used to determine the size-dependent macromolecular permeability of lipid membranes in GUV model systems by analyzing the influx of three fluorescent dyes with molecular masses of 0.37, 3.0, and 10.0 kDa into these vesicles (Fig. 1). We deconvolve the effects of PS and PE lipids by exploring their effects within DOPC (PC) membranes both separately and in combination: DOPC/POPS 80:20 (PC/PS), DOPC/DOPE 90:10 (PC/PE), and DOPC/DOPE/POPS 70:10:20 (PC/PE/PS). These experiments are corroborated by circular dichroism (CD) spectroscopy to quantify peptide binding to the membrane, fluorescence spectroscopy experiments to establish the leakage mechanism in an ensemble system of nanoscale large unilamellar vesicles (LUVs), and atomic-force microscopy (AFM) imaging of supported lipid bilayers to reveal the nanoscale perturbations of membrane structure induced by the peptide. By combining these approaches, we show that, while PS lipids significantly enhance MP1’s binding onto the membrane, PE lipids impart the most significant contribution to the rate and extent of membrane permeabilization by MP1, facilitating the opening of larger membrane defects than in bilayers lacking in PE.

Thumbnail image of Figure 1. Opens large image

http://www.cell.com/cms/attachment/2035808117/2051293431/gr1.jpgFigure 1

Schematic representation of membrane disruption by peptides and the experimental system. The helical peptide Polybia-MP1 is shown according to the helical wheel projections. Amino acids: (blue) polar with positive net charge; (purple) polar with negative net charge; (red) polar noncharged; and (green) nonpolar. Confocal microscopy was performed to investigate the influx of three dyes with distinct sizes in GUVs in the presence and absence of PE lipids: 0.37 kDa CF (green), 3k-CB (blue), 10k-AF647 (magenta), and the scale bars correspond to 10 μm. Lipid membranes are labeled with Rh-DOPE (red). The peptide interacts with the GUVs, disturbs their structure, and then enables the passage of fluorescent dyes by formation of pore-like structures. To see this figure in color, go online.

Jump to Section
Introduction
Materials and Methods
  Materials
  Peptide synthesis and purification
  Mass spectrometry analysis
  GUV formation
  LUV preparation
  CD spectroscopy for binding isotherms
  Confocal microscopy
  Analysis of confocal images and movies
  ANTS/DPX requenching measurements
  AFM
  Phase contrast microscopy
Results
  PS lipids significantly enhance peptide binding to the membrane
  MP1 dose-response studies reveal that PE and PS lipids enhance membrane permeability at lower peptide concentrations
  Confirmation of the pore-formation hypothesis in lipid vesicles
  Synergistic enhancement of GUV leakage kinetics by PE and PS lipids
  PE lipids significantly enhance pore size and membrane permeability
  Direct imaging of peptide-induced pores by AFM
Discussion
  Biophysical implications for MP1-lipid membrane interactions
  Implications for the chemotherapeutic potential of MP1 peptides
Author Contributions
Supporting Material
References

Results

PS lipids significantly enhance peptide binding to the membrane

The overall efficacy of a peptide at disrupting a target membrane can be broken down into the combination of two sequential steps: 1) binding of the peptide to the membrane surface, and 2) the efficiency of membrane disruption by the bound peptide resulting in membrane poration or leakage. First, we investigate the membrane binding isotherms of the MP1 peptide to our four lipid compositions of interest by CD spectroscopy by titrating a 10 μM MP1 solution with increasing lipid (LUV) concentrations (Fig. 2). Fitting these binding isotherms revealed that the partition coefficient (K_p) of the peptide was 7–8 times higher for membrane compositions containing PS (K_p values were PC 4600 M⁻¹, PC/PE 4000 M⁻¹, PC/PS 33,000 M⁻¹, and PC/PE/PS 30,000 M⁻¹). It is also interesting to note from this data that PE lipids slightly suppress peptide binding by a factor of ∼10%. Due to the cationic nature of MP1 (net charge of +2e), it is highly likely that the enhanced peptide binding to anionic-PS-containing membranes is primarily driven by electrostatic interactions.

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Figure 2

Binding isoterms show that MP1 has a higher affinity for PS-containing membranes. The binding isoterms and the partition coefficients (K_p) obtained using CD by lipid titration at 10 μM MP1 solution. LUVs are composed of (a) PC, (b) PC/PE, (c) PC/PS, and (d) PC/PE/PS.

MP1 dose-response studies reveal that PE and PS lipids enhance membrane permeability at lower peptide concentrations

To investigate the efficiency of membrane disruption, we measured the leakage of macromolecules across GUV model membranes by confocal fluorescence microscopy. Fluorescent passive leakage markers of different sizes were simultaneously employed: 0.37 kDa carboxyfluorescein (CF), 3 kDa dextran labeled with Cascade Blue (3k-CB), and 10 kDa dextran labeled with Alexa Fluor 647 (10k-AF647). GUVs were composed of PC, PC/PS, PC/PE, or PC/PE/PS. The dose-response of the membranes to the addition of MP1 was characterized for each membrane composition and passive leakage marker by evaluating the normalized fluorescence intensities of the probes in the intravesicular lumen of the GUVs after 30 min incubation time (Fig. 3 and Fig. S1 in the Supporting Material). Each data point in Figs. 3 and S1 shows the mean leakage of 50 individual GUVs from a minimum of two independent experiments. For determining the percentage of leaked vesicles (Fig. 3), a threshold of 20% leakage (normalized to the background probe concentration) was used to define a filled vesicle. Alternatively, this data can be analyzed in terms of the average leakage into GUVs as a percentage of the probe concentration in the external medium (Fig. S1).

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Figure 3

Dose-response curves show increased leakage of PE containing GUVs at lower peptide concentrations. (a and c) Percentage of GUVs filled by CF (0.37 kDa) after 30 min incubation time with MP1. (b and d) Percentage of GUVs filled by 10k-AF647 after 30 min incubation time with MP1. All vesicles presenting >20% of dye entry were accounted as filled. The data is plotted as a function of (a and b) total peptide concentration and (c and d) the concentration of peptide bound to the membranes. Fifty GUVs were used to construct each data point. Vesicles are composed of PC, PC/PS, PC/PE, and PC/PE/PS. To see this figure in color, go online.

The integrity of membranes containing both PE and PS lipids is perturbed by lower concentrations of MP1 peptide than the other membrane compositions we investigated. PC/PE/PS GUVs show significant (40–65%) leakage to the CF probe at 0.4 and 1.2 μM MP1 concentrations, whereas other membrane compositions studied leaked <30% within this concentration range (Fig. S1a).

Larger pore defects, evidenced by leakage of the larger 10k-AF647 probe, are shown to be significantly enhanced in membranes containing 10% PE. Almost all GUVs (98%) containing PE lipids are observed to leak the 10k-AF647 probe when in the presence of 4.0 μM MP1, compared to <60% of GUVs for other membrane compositions at the same peptide concentration (Fig. 3b). This is the most significant enhancement in selective perturbation for specific lipid membrane compositions observed within the dose-response data in Figs. 3 andS1. At this MP1 concentration, membranes under native conditions would be susceptible to the leakage of biological macromolecules such as small proteins and RNAs.

Interestingly, we also plot the GUV leakage data as a function of the concentration of bound peptide on the membrane using the specific partition coefficients of the peptide for different lipid compositions that were calculated inFig. 2 (see also Figs. 3, c and d, and S1, c and d). This representation of the data clearly shows that PE lipids increase the susceptibility of PC membranes to disruption by the MP1 peptide, with PC/PE lipids leaking at significantly lower bound peptide concentrations. Due to the higher bound concentration of peptide to membranes containing PS lipids, this lipid decreases the apparent susceptibility of the membrane to leakage as observed by the onset of leakage shifting to higher bound peptide concentrations. For PC/PE/PS GUVs, the apparent competing effects of PE and PS lipids on the membrane’s leakage susceptibility roughly cancel each other out, leading to intermediate membrane disruption susceptibility for a given bound peptide concentration. However, the effect of increased bound peptide concentrations due to PS far outweighs its apparent inhibition of membrane leakage, making PC/PE/PS GUVs the most susceptible to leakage for a given total peptide concentration. Therefore, the combined roles of PS in increasing membrane binding and PE in increasing the susceptibility of the membrane are both important in increasing the membrane disruptive efficacy of MP1.

Confirmation of the pore-formation hypothesis in lipid vesicles

Fluorescence spectroscopy experiments using LUVs give ensemble-averaged measurements with high statistics on a large population of vesicles, complementing single-vesicle GUV imaging experiments that inherently have lower statistics but yield information on the distribution of behaviors and rare events within a sample. The fluorescence requenching method (18) enables us to distinguish the type of leakage mechanism induced by MP1 for the lipid compositions under investigation. One possibility is the all-or-none mechanism where some vesicles release all of their internal contents while the others remain intact. This is attributed to pore-formation mechanisms of membrane perturbation, or complete vesicle lysis. Another possibility is the gradual leakage mechanism where vesicles only release a fraction of their encapsulated contents during a leakage event. This is associated with transient perturbations of the membrane. A fluorophore (ANTS) and a quencher (DPX) are encapsulated within lipid vesicles at high concentrations such that the fluorescence is initially quenched; vesicle leakage results in the release of both ANTS and DPX, but quenching is decreased due to dilution of these probes. The externalized ANTS fluorescence can be suppressed by additional titration of DPX such that the remaining fluorescence signal is only due to the ANTS inside intact vesicles. The data can be represented by a plot of the degree of quenching (Q_in) against the released ANTS fraction (f_out). In the case of an all-or-none leakage mechanism, the plot of Q_in versus f_out will show no dependence of Q_in on f_out. In contrast, the gradual leakage mechanism causes release of only a fraction of the encapsulated contents within individual vesicles and so Q_in increases with increasing f_out (18).

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Figure 4

Fluorescence requenching assays for MP1 reveal all-or-none leakage in the four lipid compositions studied. Q_in is constant as a function of f_out for MP1, which is in agreement with the all-or-none mechanism of dye release.) (Lines) Theoretical curves for ideal graded and all-or-none dye release (18). To see this figure in color, go online.

Fig. 4 shows that the values of Q_in remain constant with the increase of f_out and the consequent increase of peptide/lipid molar ratios. This clearly shows that MP1 exhibits the all-or-none leakage mechanism for all lipid compositions studied, which is in contrast to what has been observed for antimicrobial peptides mastoparan X and mastoparan MP (19). We propose that this all-or-none leakage is related to peptide-induced pore formation (20, 21, 22, 23), where the vesicles are able to release all their internal contents through pore-like structures that are sufficiently long lived (23, 24, 25, 26, 27, 28). We do not solely attribute the all-or-none leakage to lysis of the vesicles because nonlysed, leaky vesicles are observed in our GUV experiments (Figs. 3 and S1). However, we do not discount the possibility that lysis might play a role in the LUV leakage at the highest peptide concentrations used in this assay. Furthermore, pore-like activity of MP1 has previously been identified from electrophysiology measurements in planar lipid bilayers composed of phytanoyl-PC and phytanoyl-PC/PS (70:30) (4).

Our fluorescence requenching results show that stable pores form with a lifetime that persists long enough for the dye efflux to reach equilibrium in LUV systems. However, this does not discount the possibility that pores might be transient over longer timescales, for example during the leakage of much larger vesicles such as GUVs where the encapsulated volume of dye that needs to be released during a leakage event is ∼10⁶ times greater than for the LUV model system. Indeed, we will see some evidence for transient pore events and dynamic changes in membrane permeability in the single GUV leakage kinetics data that follows. Nevertheless, all-or-none leakage is clearly evident in GUVs after 30 min incubation with 1.2 and 4.0 μM MP1. Leakage histograms of the individual GUVs (an alternative representation of data shown in Figs. 3 and S1) predominantly show either Math Eq

20% (unleaked) or Math Eq

80% (fully leaked) leakage (Fig. S2).

Synergistic enhancement of GUV leakage kinetics by PE and PS lipids

Analysis of the time delay from the start of our GUV experiments (addition of the peptide) to observations of the onset of GUV leakage reveals a synergistic reduction in this lag time for PC/PE/PS membranes (Table 1 and Fig. 5). In these GUV experiments, we add 4.0 μM MP1 to our samples and monitor the time taken for initial leakage events of GUVs to the 0.37 kDa CF probe to occur (t₀ − t_CF). This MP1 concentration is chosen as it causes significant leakage of GUVs within 30 min of peptide addition across all four lipid compositions of interest. The onset of leakage occurs approximately twice as quickly for PC/PE/PS GUVs than for other membrane compositions, with only a very slight reduction of the lag time for PC/PS membranes compared with PC/PE and PC GUVs. Therefore, this is not a purely electrostatic effect from the increased rate and extent of peptide binding to anionic PS-containing membranes; it also requires the presence of PE to significantly increase the susceptibility of the membrane to permeabilization.

Table 1Lag times between the onset of leakage of each dye and the time interval before the initial leakage takes place after the addition of peptide
Time Delays (s)	PC/PE	PC/PE/PS	PC/PS	PC
t₀ − t_CF	1600 ± 110	760 ± 120	1400 ± 60	1600
t_CF – t_3k-CB	1.8 ± 0.6	1.5 ± 0.3	41 ± 5^a	160 ± 110
t_CF – t_10k-AF647	4.2 ± 1.5	2.0 ± 0.4	52 ± 6	220 ± 66
t_3k-CB – t_10k-AF647	2.7 ± 0.9	1.4 ± 0.4	9.6 ± 1.2	60 ± 42

The errors represent the standard deviation of the observed GUV data set.

aFor PC/PS GUVs, the t_CF – t_3k-CB data only includes GUVs that leaked to all three dyes; these sample conditions contained two distinct populations of GUV leakage behaviors where a second population of GUVs only leaked to the CF and 3k-CB dyes with a time delay of t_CF – t_3k-CB = 4.8 ± 0.6 s.

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Figure 5

GUV permeabilization kinetics are synergistically enhanced by PE and PS lipids. (a) Comparison between dye influx kinetics of three distinct dyes (CF-0.37kDa, 3k-CB, and 10k-AF647) for PC/PE/PS and PC/PS GUVs. The time axis represents the time after peptide addition. These individual GUV leakage profiles were chosen as they represent the average behavior of the GUVs observed under these conditions. (b) Schematic representation of the dye influx kinetics for PC/PS and PC/PE/PS GUVs in the presence of CF and 10k-AF647 passive leakage markers. This shows the average lag times for GUV leakage after the addition of 4.0 μM MP1 and the typical average leakage extent of the GUVs that resulted in these experiments. To see this figure in color, go online.

We also quantify the average delay times between leakage of the different-sized fluorescent probes between CF and 3k-CB (t_CF – t_3k-CB), CF and 10k-AF647 (t_CF– t_10k-AF647), and 3k-CB and 10k-AF647 (t_3k-CB – t_10k-AF647). Once the initial leakage event occurs, PE-containing GUVs rapidly become leaky to fluorescent probes of larger sizes (3 and 10 kDa). For PC/PE/PS and PC/PE membranes, GUVs become leaky to larger 3k-CB, then 10k-AF647 passive leakage markers within seconds of permeabilization to the smallest CF (0.37 kDa) probe (Table 1). The consecutive delay times between CF and 3k-CB probes and 3k-CB and 10k-AF647 probes were approximately an order-of-magnitude longer for PC/PS membranes, and almost two orders-of-magnitude longer for purely PC membranes. This strongly implies that the presence of PE significantly enhances the favorability and rate of formation of larger membrane defects or pores.

PE lipids significantly enhance pore size and membrane permeability

We use time-series confocal microscopy imaging to quantify the membrane permeability of GUVs during initial leakage. Quantification of the fluorescence intensity of the leakage markers in the intravesicular and extravesicular medium allows us to calculate the fractional leakage of individual GUVs as a function of time. The leakage kinetics of individual GUVs are monitored for up to 30 min after the addition of 4.0 μM MP1. This concentration is chosen as all lipid compositions show significant leakage within 30 min; a higher MP1 concentration of 10 μM is observed to induce significant lysis of GUV samples (Fig. S3). These experiments are conducted on GUVs of all four membrane compositions under investigation, using the CF, 3k-CB, and 10k-AF647 leakage markers simultaneously. This allows the time evolution of membrane permeability to different molecular sizes to be simultaneously measured for individual GUVs (Figs. 5a and S4). To the best of our knowledge, this is the first example of simultaneous size-dependent permeability measurements in GUVs for three different-sized leakage markers.

Typical leakage kinetic profiles for different membrane compositions and probe sizes are shown in Figs. S4 and 5a. It can be qualitatively seen from these example profiles that membrane compositions containing 10% PE exhibit full and rapid membrane leakage for all three sizes of leakage marker, consistent with the leakage kinetics data in Table 1, which is also outlined in Fig. 5b. For membrane compositions lacking PE, the leakage rates can sometimes be seen to increase and decrease intermittently, sometime plateauing before full leakage is achieved; this is particularly evident in the leakage profile of a single PC/PS GUV shown in Fig. S5. We attribute these observations to membrane self-healing events, where the pores/defects reseal and the membrane regains its permeability barrier, followed by later phases of increased leakage. This is particularly observed for the larger 3k-CB and 10k-AF647 leakage probes. Therefore, the membrane permeability for PC and PC/PS GUVs, in particular, can change dynamically during the observed leakage events; this is a result of the competition between the lipid bilayer and peptides in maintaining their barrier properties and inducing membrane pores, respectively (Fig. S5).

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Figure 6

PE lipids facilitate much greater membrane permeability in GUV membranes. (a) Typical log-linear plot of time-dependent dye influx: −R/3ln(1−c) (10⁻5) versus time, for the three dyes in a single GUV of PC/PS. (b) Distributions of the obtained permeabilities in single GUVs composed of PC/PS. (c) Typical log-linear plot of time-dependent dye influx: −R/3ln(1−c) (10⁻⁵) versus time, for the three dyes in a single GUV of PC/PE/PS. (d) Distributions of the obtained permeabilities in single GUVs composed of PC/PE/PS. The permeabilities are obtained from the slopes of the log-linear plots of the time-dependent influx of dyes into single GUVs. To see this figure in color, go online.

Our leakage kinetic profiles were used to calculate the membrane permeability to the different-sized probes using a diffusional model for membrane translocation (29); the membrane permeability is the gradient of the log-linear plot as seen in the example data in Fig. 6, a and c. Average permeability values for each membrane composition to each probe size during the initial leakage events are shown in Table 2. It can be seen that, for all membrane compositions tested, average permeability decreases with increasing probe size. However, the most significant finding from this data is the large, one-order-of-magnitude increase, in membrane permeability for membrane compositions containing 10% PE. This can be observed for all three leakage markers studied. It can also be seen that the presence of PS in the membrane imparts a modest, but significant, increase in permeability on the membranes upon perturbation by MP1. This effect can be seen further in Fig. 6, b and d, which show the distributions of permeability measurements for PC/PS and PC/PE/PS GUVs to the CF and 10k-AF647 leakage markers, respectively. For both probe sizes, the majority of permeability measurements for PC/PS membranes were in the 0–25 nm/s range, whereas when PE was included in the membrane formulations, a large proportion of permeability measurements were >500 nm/s.

Table 2Average permeability values (〈P_m〉) and the average fractional permeated area per vesicle (〈A_p〉/〈A_v〉) obtained from the average permeability values for each probe size and membrane composition
〈P_m〉_CF(nm/s)	〈A_p〉/〈A_v〉_CF(10⁻⁶)	〈P_m〉_3k-CB(nm/s)	〈A_p〉/〈A_v〉_3k-CB(10⁻⁶)	〈P_m〉_10k-AF647(nm/s)	〈A_p〉/〈A_v〉_10k-AF647 (10⁻⁶)
PC	46 ± 14	0.45 ± 0.14	17 ± 6	0.50 ± 0.16	8 ± 2	0.44 ± 0.08
PC/PS	59 ± 12	0.58 ± 0.12	29 ± 6	0.90 ± 0.20	23 ± 4	1.30 ± 0.20
PC/PE	466 ± 143	4.60 ± 1.40	207 ± 102	6.50 ± 3.40	158 ± 53	8.80 ± 2.90
PC/PE/PS	589 ± 142	5.80 ± 1.40	333 ± 73	10.40 ± 2.80	169 ± 52	9.40 ± 2.90

The errors represent the standard deviation of the observed GUV data set.

It should be noted that the observed permeability distributions (Fig. 6, b and d) are broad due to the fact that peptide-induced pores do not have well-defined structures, pore formation events are stochastic, and the membrane interfaces are fluid, giving rise to this wide distribution of individual permeability events when measured at the single vesicle level. Indeed, it has previously been reported that the initial pores that form during peptide-induced pore formation might be far from equilibrium and can, for example, relax to a smaller size over longer timescales as has been observed for the peptides Bax-α5 (23) and magainin 2 (30).

The permeability data was used to calculate the effective fractional permeable area of the membrane for each probe size using the expression (29)

where A_p is the permeable area of membrane on a GUV, A_v is the total area of the vesicle, P_m is the permeability, and δ is the thickness of the membrane. The Stokes-Einstein diffusion constant of the leakage marker is D₀ = kT/6πηR₀, where kT is the thermal energy, η is the solvent viscosity, and R₀ is the hydrodynamic radius of the fluorescent dye that was estimated with the relationR₀ = 0.0332(M_w)^0.463 in nanometers (31); M_w is the molecular weight of the dye. A brief derivation of this equation is presented in the Supporting Material. It should be noted that this equation is most accurate for the formation of large membrane pores as it assumes that the diffusion constant of the dye within the pore is the same as its diffusion constant in bulk solution. However, we believe this to be a reasonable assumption because these passive leakage markers will have a very short residence time within the pore itself due to the bilayer only being ∼5-nm thick; these solutes are not expected to interact strongly with the membrane itself.

Values of the fractional permeable areas are shown in Table 2. The fractional permeable areas were also found to be an order-of-magnitude greater for membrane compositions containing PE than for those that did not. Note that slightly larger permeable areas were measured for the larger leakage markers; these represent a later time point in the membrane disruption of GUVs by MP1 as the smaller leakage markers translocate the membrane at earlier times (Table 1). This extended delay time therefore allows for a greater area of membrane disruption to occur before the initiation of leakage to the larger M_w dyes.

Besides the order-of-magnitude increase in membrane permeabilization in the presence of PE lipids, we found an interesting correlation between PE content and membrane morphological response to MP1. Without PE, PC and PC/PS GUVs exhibited bright spots of fluorescent lipids at specific locations on the membrane surface in the presence of 4.0 μM MP1 (Fig. 7). We attribute these observations to local aggregation of peptides and lipids at the GUV surface. These peptide-induced lipid aggregates may be in competition with the pore-/defect-forming activity of the peptides. Such dense lipid structures were not seen on GUVs containing PE (PC/PE and PC/PE/PS) upon introduction of the peptide. Therefore, we speculate that the PE suppresses the intramembrane lipid aggregation by more easily facilitating the poration of the membrane.

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Figure 7

Lipid aggregation is observed within the membranes of GUVs lacking in PE. Images of local lipid aggregation at the GUV surface (bright localized spots of fluorescence) seen after peptide addition (C_p = 4.0 μM). This effect is frequently observed in PC and PC/PS GUVs, but not for the lipid mixtures containing 10 mol % PE. To see this figure in color, go online.

While localized lipid aggregation was not observed on the surface of the PE-containing GUVs, these GUVs were observed to decrease in diameter by ∼10–15% over a period of ∼1 h after peptide addition (Fig. S6). Contrary to this, PC and PC/PS GUVs remained at a constant size for up to 2 h after addition of MP1. Therefore, MP1 results in the significant loss of lipid from only those GUVs that contain PE lipids.

Direct imaging of peptide-induced pores by AFM

AFM imaging of supported lipid bilayers confirms the role of PE in potentiating the formation of larger transmembrane pores. MP1 was added at 10 μM concentration to induce significant pore formation on the relatively small patches of membrane imaged by the AFM within a reasonable experimental timescale (<2 h); resultant pores/defects were observed to be much larger in PC/PE/PS membranes (250 ± 110 nm in diameter) compared to PC/PS (54 ± 30 nm in diameter) (Fig. 8). Similar-sized transmembrane pores were observed in PC/PE/PS and PC/PE membranes (290 ± 200 nm in diameter), but significantly fewer defects formed in PC/PE membranes. Note that the large standard deviations in these average pore diameters represent a significant size polydispersity in the defects formed. No pores were evident in PC membranes 2 h after peptide addition (Fig. S7); however, pores would need to be several nanometers in diameter to be observable by AFM, considerably larger than those that can be detected by passive dye influx into the GUVs we used to investigate the early stages of GUV poration.

Our AFM studies also clearly show a difference in pore formation and growth mechanisms dependent on the presence of PE. The large transmembrane pores in PC/PE/PS and PC/PE membranes are seen to grow by the stepwise loss of lipid aggregates from the edge of the pore, implying that vesicle micellization is important for pore growth in the these membranes (Fig. S8). This is consistent with the small decrease (within experimental error) in GUV size observed for PE-containing GUVs by phase contrast microscopy (Fig. S6). Conversely, in PC/PS membranes, raised areas of lipid are first seen to form on the membrane (Fig. S9), which may correlate to the dense lipid structures observed in Fig. 7. These raised areas of membrane later evolve into comparatively small pores; many of the defects seen in Fig. 8 (bottom left) only span half the bilayer, with only the center of a few of these defects showing full bilayer pores (Fig. S10). This indicates that pores in these membranes may form via a half-membrane intermediate state. Finally the timescale for observation of membrane defects by AFM was much faster for PC/PE/PS membranes than for other lipid mixtures, with defects observed almost immediately after peptide addition (Fig S7), compared with a few tens of minutes for other mixtures.

Discussion

Biophysical implications for MP1-lipid membrane interactions

We have shown a synergistic enhancement of the rate and extent of membrane permeabilization by MP1 peptides when PE and PS lipids are present in the lipid membrane. This picture is confirmed and corroborated by complementary experiments using three different model membrane systems: LUVs, GUVs, and planar-supported bilayers. We consider the perturbation of the membrane by MP1 peptides in two steps: 1) binding of the peptides to the membrane, and 2) perturbation of the bilayer structure by bound peptides to induce leakage.

Binding isotherms (Fig. 2) reveal that PS lipids cause a 7–8-fold increase in peptide bound to the membrane. This strongly outweighs the small ∼10% reduction in bound peptide concentration caused by the PE lipids. Therefore, we find that the dominant role of PS lipids’ contribution to the membrane disruption by MP1 is a large increase in peptide binding to the membrane.

The role of PE lipids in MP1-induced membrane disruption is twofold: 1) PE increases the susceptibility of the membrane to permeabilization by bound peptides, and 2) PE facilitates the formation of larger transmembrane pores. First, when the extent of GUV leakage is normalized to bound peptide concentration in the dose-response curves in Fig. 3, c and d, it can be seen that 4–5 times lower bound peptide concentration is required to induce a similar leakage response compared to comparable GUVs without PE lipids. Second, GUV and AFM experiments corroborate the effect of larger pores forming in the presence of PE. Quantitative analysis of GUV leakage profiles in Fig. 6 andTable 2 reveal that the presence of PE increases the permeability of membranes by an order of magnitude compared to membranes lacking in PE. Furthermore, once pores formed in GUVs, they quickly (within seconds) grew large enough in size to allow larger macromolecules (3 and 10 kDa) to permeate the membrane (Table 1); this compared to several tens of seconds for larger pores to form in GUVs lacking PE. Crucially, the formation of larger pores for PE-containing membranes is directly visualized by AFM (Fig. 8), where the observed pore diameters are ∼5 times larger in the presence of PE (and hence ∼20–30 times larger in average pore area, consistent with the order-of-magnitude increase in permeability reported for the GUVs).

The formation of transmembrane pores was confirmed by complementary experimental systems and techniques. Rapid translocation of membrane-impermeable leakage markers across GUV membranes, an all-or-none LUV fluorescence leakage assay, and direct visualization of transmembrane defects by AFM imaging of planar bilayers, all confirm this to be true. While these pores are fairly long lived, the membranes were sometimes observed to temporarily reseal, regaining their barrier properties. This can clearly be seen in the leakage profiles of individual GUVs in Figs. 5a, S4, and S5. GUV and planar bilayer imaging experiments also strongly suggest differences in the mechanism of pore formation depending on whether PE lipids are present. Images of GUVs that did not contain PE lipids often exhibited bright spots of increased local lipid concentrations on the membrane, which we interpret to be local aggregation of peptides and lipid (Fig. 7). Similarly, AFM images showed locally raised regions of lipid scattered across the membrane for these lipid compositions (Fig. S9) before the formation of pores (Fig. S10). This contrasted to the pore-formation mechanism observed in the presence of PE, where local aggregates were not directly observed on the GUV surface and time-resolved AFM imaging showed pore growth to occur by the stepwise micellization and loss of lipid from the edge of the pores (Fig. S8).

Besides the increased binding due to PS and the increased membrane susceptibility and pore size due to PE, the synergistic enhancement of membrane disruption facilitated by these lipids can be observed in the kinetics of initial permeabilization events. GUV experiments showed that PC/PE/PS GUVs leaked a factor-of-two quicker than other membrane compositions (Table 1). This is again corroborated by the AFM studies where defects were observed in PC/PE/PS membranes almost immediately after peptide addition, whereas perturbations of other membrane compositions took a few 10 s of minutes to evolve. The complementary pore-promoting effects of PS on bound peptide concentrations and PE on membrane susceptibility far outweigh their slight inhibitory effects on each other’s roles (PE causes a slight reduction in binding affinity (Fig. 2) and PS causes a decrease in the membrane susceptibility to bound peptide (Fig. 3, c and d)). This is apparent from the effects of MP1 on GUVs, where PC/PE/PS membranes experience the greatest membrane perturbation for any given total peptide concentration (Fig. 3, a and b) and the larger number of pores observed on the membrane surface by AFM (Fig. 8). Therefore, our combined results provide a detailed mechanistic picture of the importance of increased PS and PE lipid concentrations in synergistically enhancing the membrane’s propensity for significant disruption of its barrier properties by MP1 peptides.

Variations in lipid composition are responsible for differences in membrane properties such as charge, fluidity, lateral pressure profiles, and mechanical moduli. Changes in these fundamental membrane properties directly affect their interactions with extraneous compounds, such as antimicrobial peptides. The cationic nature of the MP1 peptide is likely an important component in the initial step of peptide action, in which the peptide recognizes the target membrane due to electrostatic interactions and binds to it in a structured form, most of the time as a helix. Therefore, the inclusion of anionic PS lipids in these membranes increases these electrostatic interactions with the MP1 peptide (net charge = +2e). However, MP1-membrane interactions cannot be solely driven by electrostatics as these peptides were also found to disrupt zwitterionic PC and PC/PE membranes, likely through secondary hydrophobic initial binding interactions that lead to a significantly lower bound concentration of peptide compared to the anionic membranes.

Next, insertion of the peptide into the bilayer likely takes place due to the hydrophobic effect, where nonpolar MP1 residues insert into the bilayer core, and defects may then be opened within the membrane structure, leading to its disruption. Furthermore, taking account of the fact that MP1 is a short peptide (14 amino acids) and hence not long enough to form a bilayer-spanning barrel stave pore (9, 32), we anticipate that these pores will be disorganized toroidal structures formed by lipids and peptides, as described by many molecular-dynamics studies (33, 34). PE is known to significantly modulate the lateral pressure profile through membranes and thereby induce negative curvature stress in the bilayer. Negative curvature stress has been shown to enhance the formation of toroidal lipid pores within a membrane by stabilizing the curvature of these structures (35). Therefore, PE would be expected to favor the formation of pore-like defects in the membrane, consistent with the increase susceptibility of these membranes to MP1-induced poration and the order-of-magnitude increase in membrane permeability that we find for PE-containing membranes upon interaction with MP1 peptides.

Implications for the chemotherapeutic potential of MP1 peptides

The MP1 peptide has been shown to have selective inhibition against numerous cancer lines compared to healthy cells (2, 3). Such malignant cells are also known to have increased expression of PS and PE lipids on their outer plasma membrane (5, 6, 7). This study strongly correlates the enhanced tumor inhibitory effects of these peptides with this pathological change in plasma membrane lipid composition, where the upregulation of PS and PE lipids can synergistically enhance the membrane-permeabilizing activity of MP1. This membrane permeabilization is likely to be the primary mechanism of cancer cell death induced by these peptides.

This suggests that MP1 might be a candidate therapeutic for development of novel cancer therapies, or at least guide the development of novel lead compounds for treatment of these diseases. One challenge for the application of antimicrobial peptides in medicine is that they often do not show high enough selectivity to their target cells to result in a favorable therapeutic index for these compounds (36). However, MP1 does not exhibit hemolytic activity to rat erythrocytes but presents chemotaxis for polymorphonucleated leukocytes and potent antimicrobial action against Gram-positive and Gram-negative bacteria (12), suggesting it could have favorable selectivity. It may also be of interest to test MP1 in a combination therapy with other chemotherapeutics that have intracellular targets. The selectivity of the MP1 peptide to disrupt the membranes of cancer cells may act synergistically with these other drugs to significantly enhance the therapeutic potency. Therefore, the therapeutic potential of this and other membrane-active peptides within the field of oncology is worthy of further investigation.

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Vaccines, Small Peptides, aptamers and Immunotherapy [9]

Posted in Biomarkers & Medical Diagnostics, Cancer and Current Therapeutics, CANCER BIOLOGY & Innovations in Cancer Therapy, Cancer Informatics, Cancer Prevention: Research & Programs, Cancer Screening, Chemical Biology and its relations to Metabolic Disease, Chemical Genetics, Clinical & Translational, Clinical Diagnostics, Clinical Genomics, Curation, Cytoskeleton, Diagnostic Immunology, Diagnostics and Lab Tests, Disease Biology, Drug Toxicity, Genomic Expression, Monoclonal Immunotherapy, tagged Cancer - General, Cancer immunotherapy, Monoclonal antibodies, oligonucleotides, peptides, quantum dots, T- and B- cells, vaccines on May 12, 2015| Leave a Comment »

Vaccines, Small Peptides, aptamers and Immunotherapy [9]

Writer and Curator: Larry H. Bernstein, MD, FCAP

This contribution has the following structure:

9.1.1 Viruses in carcinogenesis

9.1.2 Simultaneous Humoral and Cellular Immune Response against Cancer–Testis Antigen NY-ESO-1: Definition of Human Histocompatibility Leukocyte Antigen (HLA)-A2–binding Peptide Epitopes

9.1.3 Monoclonal Antibodies in Cancer Therapy

9.1.4 Aptamers

9.1.5 Tumor Suppressors

9.1 Vaccines

9.1.1 Viruses in carcinogenesis

HPV-associated cervical cancer
HPV-associated head and neck cancer: a virus-related cancer epidemic

The contributions of hepatitis B virus and hepatitis C virus infections to cirrhosis and primary liver cancer worldwide

Risk of pancreatic cancer among individuals with hepatitis C or hepatitis B virus infection: a nationwide study in Sweden.

HIV Infection and Cancer Risk

HIV and cancer of the cervix

Anal cancer: an HIV-associated cancer

The therapeutic potential of CXCR4 antagonists in the treatment of HIV infection, cancer metastasis and rheumatoid arthritis

Types of Cancer: AIDS/HIV related malignancies

Cytokines in cancer pathogenesis and cancer therapy

Dendritic Cells as Therapeutic Vaccines against Cancer

9.1.2 Simultaneous Humoral and Cellular Immune Response against Cancer–Testis Antigen NY-ESO-1: Definition of Human Histocompatibility Leukocyte Antigen (HLA)-A2–binding Peptide Epitopes

9.1.3 Monoclonal antibodies

Monoclonal antibodies in cancer therapy

Monoclonal Antibodies in Cancer Therapy: 25 Years of Progress

9.1.4 Aptamers

Nanocarriers as an emerging platform for cancer therapy

Quantum Dot−Aptamer Conjugates for Synchronous Cancer Imaging, Therapy, and Sensing of Drug Delivery Based on Bi-Fluorescence Resonance Energy Transfer

Oligonucleotide Aptamers: New Tools for Targeted Cancer Therapy

9.1 Vaccines

9.1.1 Viruses in carcinogenesis

HPV-associated cervical cancer

http://www.cancer.net/navigating-cancer-care/prevention-and-healthy-living/hpv-and-cancer

Human papillomavirus (HPV) is a virus that is usually passed on during direct skin-to-skin contact, most commonly sex. In fact, HPV is the most common sexually transmitted disease in the United States. Most men and women are not aware they have an HPV infection because they do not develop any symptoms or health problems. Certain HPV types can cause precancerous lesions (areas of abnormal tissue) or cancer.

More than 40 of the viruses are called “genital type” HPVs. These viruses are spread from person to person when their genitals come into contact, usually during vaginal or anal sex. They can also be passed on through oral sex.

Genital HPV types can infect the genital area of women, including the vulva (outer portion of the vagina), the vagina, and the cervix (the lower, narrow part of a woman’s uterus), as well as the genital area of men, including the penis. In both men and women, genital HPV can infect the anus and some areas of the head and neck.

Nearly all cervical cancers are caused by HPV infection. Strong scientific evidence shows that a lasting HPV infection is required for cervical cancer to begin developing. Whether a woman who is infected with HPV will develop cervical cancer depends on a number of factors, including the type of HPV infection she has. Of the cervical cancers related to HPV, about 70% are caused by two strains, HPV-16 or HPV-18. In women who have HPV, smoking may increase the risk of cervical cancer.

Warts and precancerous lesions can be removed through cryotherapy (freezing); loop electrosurgical excision procedure (LEEP), which uses electric current to remove abnormal tissue; or surgery.

Receiving an HPV vaccine reduces your risk of infection. The U.S. Food and Drug Administration (FDA) approved two vaccines that help prevent HPV infection: Gardasil and Cervarix. It is important to note that the vaccines cannot cure an existing HPV infection.

Purpose of the vaccines. The goal of HPV vaccination is to prevent a lasting HPV infection after a person is exposed to the virus. Gardasil, introduced in 2006, helps prevent infection from the two HPVs known to cause most cervical cancers and precancerous lesions in the cervix. The vaccine also prevents against the two low-risk HPVs known to cause 90% of genital warts. Gardasil is approved for the prevention of cervical, vaginal, and vulvar cancers in girls and women ages nine to 26. It is also approved to prevent anal cancer in women and men and genital warts in men and boys in the same age range. Cervarix, introduced in 2009, is approved for the prevention of cervical cancer in girls and women ages 10 to 25.

Effectiveness and safety of the vaccines. Data show the HPV vaccinations are safe and highly effective in preventing a lasting infection of the HPV types they target. Because it takes many years before a precancerous lesion develops into an invasive cancer, it will likely take several more years before there is evidence that the number of cancer cases in vaccinated individuals has been reduced.

HPV-associated head and neck cancer: a virus-related cancer epidemic
Shanthi Marur, Gypsyamber D’Souza, William H Westra, Arlene A Forastiere
Lancet Oncol 2010; 11: 781–89
http://dx.doi.org:/10.1016/S1470-2045(10)70017-6

A rise in incidence of oropharyngeal squamous cell cancer—specifically of the lingual and palatine tonsils—in white men younger than age 50 years who have no history of alcohol or tobacco use has been recorded over the past decade. This malignant disease is associated with human papillomavirus (HPV) 16 infection. The biology of HPV-positive oropharyngeal cancer is distinct with P53 degradation, retinoblastoma RB pathway inactivation, and P16 upregulation. By contrast, tobacco-related oropharyngeal cancer is characterized by TP53 mutation and downregulation of CDKN2A (encoding P16). The best method to detect virus in tumor is controversial, and both in-situ hybridization and PCR are commonly used; P16 immunohistochemistry could serve as a potential surrogate marker. HPV-positive oropharyngeal cancer seems to be more responsive to chemotherapy and radiation than HPV-negative disease. HPV 16 is a prognostic marker for enhanced overall and disease-free survival, but its use as a predictive marker has not yet been proven. Many questions about the natural history of oral HPV infection remain under investigation. For example, why does the increase in HPV-related oropharyngeal cancer dominate in men? What is the potential of HPV vaccines for primary prevention? Could an accurate method to detect HPV in tumor be developed? Which treatment strategies reduce toxic effects without compromising survival? Our aim with this review is to highlight current understanding of the epidemiology, biology, detection, and management of HPV-related oropharyngeal head and neck squamous cell carcinoma, and to describe unresolved issues.

Cancers of the head and neck arise from mucosa lining the oral cavity, oropharynx, hypopharynx, larynx, sinonasal tract, and nasophaynx. By far the most common histological type is squamous cell carcinoma, and grade can vary from well-differentiated keratinizing to undifferentiated non-keratinizing. An increase in incidence of oropharyngeal squamous cell carcinoma—specifically in the tonsil and tongue base—has been seen in the USA, most notably in individuals aged 40–55 years. Patients with oropharyngeal cancer are mainly white men. Unlike most tobacco-related head and neck tumors, patients with oropharyngeal carcinoma usually do not have a history of tobacco or alcohol use. Instead, their tumors are positive for oncogenic forms of the human papillomavirus (HPV), particularly 16 type. About 60% of oropharyngeal squamous cell cancers in the USA are positive for HPV 16. HPV-associated head and neck squamous cell carcinoma seems to be a distinct clinical entity, and this malignant disease has a better prognosis than HPV-negative tumors, due in part to increased sensitivity of cancers to chemotherapy and radiation therapy. Although HPV is now recognized as a causative agent for a subset of oropharyngeal squamous cell carcinomas, the biology and natural history of oropharyngeal HPV infection and the best clinical management of patients with HPV-related head and neck squamous cell tumors is not well understood.

Head and neck cancer is the sixth most common cancer worldwide, with an estimated annual burden of 563 826 incident cases (including 274 850 oral cavity cancers, 159 363 laryngeal cancers, and 52 100 oropharyngeal cancers) and 301 408 deaths.¹ Although HPV has been long known to be an important cause of anogenital cancer, only in recent times has it been recognized as a cause of a subset of head and neck squamous cell carcinomas.² More than 100 different types of HPV exist,3 and at least 15 types are thought to have oncogenic potential.4 However, most (>90%) HPV-associated head and neck squamous cell cancers are caused by one virus type, HPV 16, the same type that leads to HPV-associated anogenital cancers. The proportion of head and neck squamous cell carcinomas caused by HPV varies widely (figure 1),^5–16 largely because of the burden of tobacco-associated disease in this population of tumors. Tobacco, alcohol, poor oral hygiene, and genetics remain important risk factors for head and neck tumors overall, but HPV is now recognized as one of the primary causes of oropharyngeal squamous cell cancers. In the USA, about 40–80% of oropharyngeal cancers are caused by HPV, whereas in Europe the proportion varies from around 90% in Sweden to less than 20% in communities with the highest rates of tobacco use (figure 1).

The incidence of head and neck cancers overall in the USA has fallen in recent years, consistent with the decrease in tobacco use in this region. By contrast, incidence of HPV-associated oropharyngeal cancer seems to be rising, highlighting the increasing importance of this causal association.^17–19 In a US study in which data of the Surveillance, Epidemiology, and End Results (SEER) program were used, incidence of oropharyngeal tumors (which are most likely to be HPV-associated) rose by 1·3% for base of tongue cancers and by 0·6% for tonsillar cancers every year between 1973 and 2004. By contrast, incidence of oral cavity cancers (not associated with HPV) declined by 1·9% every year during the same period.¹⁷The age-adjusted incidence of tonsillar cancer increased 3·5-fold in women and 2·6-fold in men between 1970 and 2002.²⁴ Augmented incidence of HPV-associated oropharyngeal cancers represents an emerging viral epidemic of cancer.

Why is increased incidence of HPV-associated oropharyngeal cancer most pronounced in young individuals? This effect could be attributable to changes in sexual norms (i.e., more oral sex partners or oral sex at an earlier age in recent than past generations) combined with fewer tobacco-associated cancers in young cohorts, making the outcomes of HPV-positive cancers more visible. Can the higher rates of HPV-associated oropharyngeal cancers in men compared with women be accounted for solely by differences in sexual behavior, or are biological differences in viral clearance present that could contribute to the higher burden of these cancers in men? HPV prevalence in cervical rather than penile tissue might boost the chances of HPV infection when performing oral sex on a woman, contributing to the higher rate of HPV-associated oropharyngeal cancer in men.

Tobacco use has fallen in past decades, and the corresponding rise in proportion of head and neck cancers that are oropharyngeal in origin has been striking, both in the USA and internationally. SEER data suggest that about 18% of all head and neck carcinomas in the USA were located in the oropharynx in 1973, compared with 31% of such squamous cell tumors in 2004.¹⁹ Similarly, in Sweden, the proportion of oropharyngeal cancers caused by HPV has steadily increased, from 23% in the 1970s to 57% in the 1990s, and as high as 93% in 2007.13,25 These data indicate that HPV is now the primary cause of tonsillar malignant disease in North America and Europe.

Findings of initial studies suggest that oral HPV frequency increases with age. Prevalent oral HPV infection is detected in 3–5% of adolescents^26–28and 5–10% of adults.^14,29 We do not yet know whether the natural history of oral HPV or risk factors for persistent HPV infection in the oropharynx differ from those known for anogenital HPV infection (table 1). Data suggest oral HPV prevalence is amplified with number of sexual partners and is more typical in men, in HIV-infected individuals, and in current tobacco users.^{26–28,30,31}

In view of the importance of tobacco use in head and neck squamous cell carcinoma, most cases of this malignant disease seen in non-smokers are unsurprisingly HPV-related. However, oral HPV infection is common in smokers and non-smokers and is an important cause of oropharyngeal cancer in both groups. For example, in case series, only 13–16% of individuals with HPV-positive head and neck squamous cell cancer did not smoke or drink alcohol.^32,33 Although a higher proportion of individuals with HPV-positive compared with HPV-negative tumors are non-smokers or neither smoke nor drink alcohol, many with HPV-positive disease have a history of alcohol and tobacco use. In fact, 10–30% of HPV-positive head and neck squamous cell carcinomas were recorded in heavy tobacco and alcohol users.^32,33 This finding underscores that HPV-associated malignant disease not only arises in people who do not smoke or drink alcohol but also occurs in people with the traditional risk factors of tobacco and alcohol use.

HPV detection may ultimately serve a more comprehensive role than mere prognostication. Detection of HPV is emerging as a valid biomarker for discerning the presence and progress of disease encompassing all aspects of patients’ care, from early cancer detection,41 to more accurate tumor staging (e.g., localization of tumor origin),^42,43 to selection of patients most likely to benefit from specific treatments,⁴⁴ to post-treatment tumor surveillance.^45,46 Consequently, there is a pressing need for a method of HPV detection that is highly accurate, reproducible from one diagnostic laboratory to the next, and practical for universal application in the clinical arena. Despite growing calls for routine HPV testing of all oropharyngeal carcinomas, the best method for HPV detection is not established. Various techniques are currently in use, ranging from consensus and type-specific PCR methods, real-time PCR assays to quantify viral load, type-specific DNA in-situ hybridization, detection of serum antibodies directed against HPV epitopes, and immunohistochemical detection of surrogate biomarkers (e.g., P16 protein). Although PCR-based detection of HPV E6 oncogene expression in frozen tissue samples is generally regarded as the gold standard for establishing the presence of HPV, selection of assays for clinical use will ultimately be influenced by concerns relating to sensitivity, specificity, reproducibility, cost, and feasibility. Development of non-fluorescent chromogens has enabled visualization of DNA hybridization by conventional light microscope; furthermore, adaptation of in-situ hybridization to formalin-fixed and paraffin-embedded tissues has made this technique compatible with standard tissue-processing procedures and amenable to retrospective analysis of archival tissue blocks. Most PCR-based methods, on the other hand, need a high level of technical skill and are best used with fresh-frozen samples.

Limitations of any one detection assay can be offset by algorithms that combine the strengths of complementary assays.⁵⁰ A highly feasible strategy incorporates P16 immunohistochemistry and HPV in-situ hybridization. In view of sensitivity that approaches 100%, P16 immunostaining is a good first-line assay for elimination of HPV-negative cases from any additional analysis. Since specificity is almost 100%, a finding positive for HPV 16 on in-situ hybridization reduces the number of false-positive cases by P16 staining alone. A P16-positive, HPV 16-negative result singles out a subset of tumors that qualifies for rigorous analysis for other (i.e., non-HPV 16) oncogenic virus types.

HPV in-situ hybridisation and P16 immunostaining as a practical diagnostic approach to discernment of HPV status can be applied readily to cytological preparations, including fi ne-needle aspirates from patients with cervical lymph-node metastases.^41,52 Further expansion of HPV testing to blood and other body fl uids would advance the role of HPV as a clinically relevant biomarker, but these specimens would need other detection platforms. PCR-based detection of HPV DNA in blood (53) and saliva (54) of patients after treatment of their HPV-positive cancers suggests a future role in tumour surveillance. Detection of serum antibodies to HPV-related epitopes can predict the HPV status of head and neck cancers, and this method has been advocated as a way to project clinical outcomes and guide treatment without the constraints of tissue acquisition.^53,55

The increasing prevalence of oropharyngeal cancer in young populations and substantially amplified survival rates with current treatment approaches stands in contrast to survival achieved in older individuals with comorbid disorders associated with tobacco and alcohol history. Several characteristics of patients with head and neck cancer have been linked with favorable prognosis, including non-smoker, minimum exposure to alcohol, good performance status, and no comorbid disorders, all of which are related to HPV-positive tumor status. Findings of retrospective analyses suggest that individuals with HPV-positive oropharyngeal cancer have higher response rates to chemotherapy and radiation and increased survival^62–65compared with those with HPV-negative tumors. Augmented sensitivity to chemotherapy and radiotherapy has been attributed to absence of exposure to tobacco and presence of functional unmutated TP53.^63,64,66 Increased survival of patients with HPV-positive cancer is also possibly attributable in part to absence of field cancerization related to tobacco and alcohol exposure.⁶⁷

Survival outcomes for individuals with HPV 16-positive and P16-positive oropharyngeal tumors were similar. Failure data indicated significantly diminished rates of locoregional failure and second primary tumour in patients with HPV-positive oropharyngeal cancer compared with those with HPV-negative tumors; distant metastases did not differ between the two groups. When survival was assessed after adjustment for tobacco exposure, in individuals who smoked, those with HPV-positive oropharyngeal tumors and fewer than 20 pack-years had 2-year overall survival of 95%, compared with 80% in those with HPV-positive cancers and 20 pack-years or more, and 63% in HPV-negative cancers and 20 pack-years or more. By comparison with people with HPV-positive oropharyngeal tumors who smoked and had fewer than 20 pack-years, the hazard of death was raised for those with HPV-negative tumors and 20 pack-years or more (hazard ratio 4·33) and those with HPV-positive cancers and 20 pack-years or more (1·79). These data indicate clearly that tobacco exposure alters the biology of HPV-positive oropharyngeal tumors and is an important prognostic factor.

An association between HPV-positive, P16-positive oropharyngeal tumors and survival outcomes was reported in another retrospective analysis of a large phase 3 trial of chemoradiation, which included more than 800 patients enrolled from international sites.72 This substudy analysis looked at 195 available tumor samples in patients with an oropharyngeal primary cancer, of which 28% were HPV-positive and 58% were P16-positive. Individuals with HPV-positive cancers had 2-year overall survival of 94% and 2-year failure-free survival of 86% compared with 77% (p=0·007) and 75% (p=0·035), respectively, in those with HPV-negative tumors. When co-expression of HPV and P16 was correlated with survival outcomes, individuals with HPV-positive, P16-positive tumors had 2-year overall survival of 95% compared with 88% in those with HPV-negative, P16-positive cancers and 71% (p=0·003) in those with HPV-negative, P16-negative tumors. Similar results were noted for 2-year failure-free survival (89%, 86%, and 69%, respectively; p=0·002) and time to locoregional failure (93%, 95%, and 84%, respectively; p=0·051). By multivariable analysis, HPV 16 and P16 were identified as independent prognostic factors.

ECOG proposes induction chemotherapy with a triple drug regimen to reduce tumor burden to subclinical disease (clinical complete response at primary site) followed by lower dose radiation (total dose 54 Gy) and concurrent cetuximab. Overall survival and progression-free survival outcomes will be assessed and compared with results of the 2008 ECOG study.⁷⁰The main aim of this planned study is to assess potential for a lower dose of radiation to control disease and to investigate toxic effects and quality-of-life variables.

In summary, tumor HPV status is a prognostic factor for overall survival and progression-free survival and might also be a predictive marker of response to treatment. The method of in-situ hybridization provides a feasible approach for implementation in most diagnostic pathology laboratories, and immunohistochemical staining for P16 could be useful as a surrogate marker for HPV status. Seemingly, locoregional recurrence—but not the rate of distant disease—is diminished in patients with HPV-positive tumors. Smoking and tobacco exposure might modify survival and recurrence of HPV-positive tumors and should be considered in future trials for risk stratification of patients with HPV-positive malignant disease.

HCV and cancer

The contributions of hepatitis B virus and hepatitis C virus infections to cirrhosis and primary liver cancer worldwide
Joseph F. Perz, Armstrong GL, Farrington LA, Hutin YJF, Bell BP
J Hepatol 2006; 45:529-538
http://dx.doi.org:/10.1016/j.jhep.2006.05.013

End-stage liver disease accounts for one in forty deaths worldwide. Chronic infections with hepatitis B virus (HBV) and hepatitis C virus (HCV) are well-recognized risk factors for cirrhosis and liver cancer, but estimates of their contributions to worldwide disease burden have been lacking. Methods: The prevalence of serologic markers of HBV and HCV infections among patients diagnosed with cirrhosis or hepatocellular carcinoma (HCC) was obtained from representative samples of published reports. Attributable fractions of cirrhosis and HCC due to these infections were estimated for 11 WHO-based regions. Results: Globally, 57% of cirrhosis was attributable to either HBV (30%) or HCV (27%) and 78% of HCC was attributable to HBV (53%) or HCV (25%). Regionally, these infections usually accounted for >50% of HCC and cirrhosis. Applied to 2002 worldwide mortality estimates, these fractions represent 929,000 deaths due to chronic HBV and HCV infections, including 446,000 cirrhosis deaths (HBV: n = 235,000; HCV: n = 211,000) and 483,000 liver cancer deaths (HBV: n = 328,000; HCV: n = 155,000). Conclusions: HBV and HCV infections account for the majority of cirrhosis and primary liver cancer throughout most of the world, highlighting the need for programs to prevent new infections and provide medical management and treatment for those already infected.

Among primary liver cancers occurring worldwide, hepatocellular carcinoma (HCC) represents the major histologic type and likely accounts for 70% to 85% of cases [2]. Cirrhosis precedes most cases of HCC, and may exert a promotional effect via hepatocyte regeneration [3,4]. Compared with other causes of cirrhosis, chronic infection with hepatitis B virus (HBV) or hepatitis C virus (HCV) is associated with a higher risk of developing HCC [3,5]. Alcohol abuse represents a leading cause of cirrhosis and is also a major contributor. dietary aflatoxin exposure in parts of Africa and Asia has been associated with primary liver cancer, especially in hosts with chronic HBV infection [8].

An understanding of the relative contribution of various etiologies to disease burden is important for setting public health priorities and guiding prevention programs [10,11]. The World Health Organization’s Global Burden of Disease (GBD) 2000 project aims to quantify the burden of premature morbidity and mortality from over 130 major causes [1,12]. Liver cancer and cirrhosis are included in the analysis, but with the exception of alcohol, the etiologies underlying these diseases have not been well accounted for [1,11,13]. In particular, HBV and HCV infections have been poorly characterized in previous WHO estimates since these were based primarily on the acute effects of infection and omitted the associated burdens of chronic liver disease [10,11].

The attributable fraction represents the proportion of disease occurrence that potentially would be prevented by eliminating a given risk factor. For cirrhosis, a systematic analysis of attributable fractions has been lacking altogether. For HCC, previous estimates of the attributable fractions due to HBV and HCV are available but are not comprehensive and do not correspond to the regional designations and related conventions of the current GBD project [14].

The prevalence of HBV and HCV infection among cirrhosis and HCC patients varied considerably within and between regions (Tables 2 and 3). These variations tended to reflect known patterns of HBV and HCV infection endemicity [99,100]. For example, in countries where HCV infection has long been endemic, such as Japan and Egypt, there were high prevalences of HCV infection among cirrhosis and HCC patients. The same held true for China and most of the African nations in our sample regarding HBV infection. Areas such as these, where HBV infection predominated, appeared to have a younger population of HCC cases, which is thought to reflect the preponderance of infections acquired early in life (e.g., perinatal HBV transmission) [8]. Patterns of HBV and HCV co-infection were also notable.

When we applied the HBV- and HCV-attributable fractions we derived to 2002 worldwide mortality estimates [1], we found that approximately 929,000 deaths from cirrhosis (n = 446,000) and primary liver cancer (n = 483,000) were likely due to chronic viral hepatitis infections. HBV infection accounted for 563,000 deaths (235,000 from cirrhosis and 328,000 from liver cancer) and HCV infection accounted for 366,000 deaths (211,000 from cirrhosis and 155,000 from liver cancer).

We showed that chronic viral hepatitis infections likely account for the majority of both cirrhosis and HCC globally and in nearly all regions of the world. One of the strengths of our analysis was that it employed simple and transparent methods. Our estimates of attributable fractions were derived from reviews of published studies reporting the prevalence of HBV and HCV infections in patients with cirrhosis or HCC in all regions of the world. Alternate approaches rely on estimates of the prevalence of risk factors and corresponding relative risks in the source populations. However, errors associated with extrapolating exposure or hazard from one population to another are a major source of uncertainty in efforts to characterize international health risks [12]. Given the lack of representative data regarding HBV and HCV infection prevalences worldwide along with uncertainties in deriving region specific risk estimates, we believe ours is the preferred approach.

Our findings help illustrate the great need for programs aimed at preventing HBV or HCV transmission. In 1992, WHO recommended that all countries include hepatitis B vaccine in their routine infant immunization programs. As of 2003, WHO/UNICEF estimated 42% hepatitis B vaccination coverage among the global birth cohort [106]. Therefore, implementation of this strategy, which represents the most effective way of preventing chronic HBV infection and related end stage liver disease, is far from complete [107,108]. Other key primary prevention strategies include screening blood donors and maintaining infection control practices to prevent the transmission of healthcare-related HBV and HCV infections [105,109,110]. In countries where these activities have not been fully implemented, they should be given a high priority. In most developed countries, injection drug use and high-risk sexual behaviors represent the major risk factors for HCV infection and HBV infection, respectively, indicating the importance of related prevention efforts (e.g., reducing the numbers of new initiates to injection drug use).

The role of programs to identify, counsel, and provide medical management for the many persons already infected with HBV or HCV requires careful consideration [105,110]. Counseling that includes advice regarding avoidance of alcohol and education regarding modes of transmission can help reduce the risks for developing chronic disease or spreading infection to susceptible persons. The widespread application of therapeutic interventions also has the potential to accelerate the declines in end-stage liver disease that will eventually follow from hepatitis B vaccination and other primary prevention efforts [104,107]. Recent advances have occurred in the therapeutic management of chronic hepatitis B and chronic hepatitis C, but treatments are long and involve substantial costs and side effects [111–113]. Countries will need to consider the potential benefits of treatment while insuring that scarce healthcare resources are allocated in a manner that does not undermine primary prevention efforts [114].

Risk of pancreatic cancer among individuals with hepatitis C or hepatitis B virus infection: a nationwide study in Sweden.

Huang J¹, Magnusson M, Törner A, Ye W, Duberg AS.
Br J Cancer. 2013 Nov 26; 109(11):2917-23.
http://dx.doi.org:/10.1038/bjc.2013.689

A few studies indicated that hepatitis C and hepatitis B virus (HCV/HBV) might be associated with pancreatic cancer risk. The aim of this nationwide cohort study was to examine this possible association. Methods: Hepatitis C virus-
and hepatitis B virus-infected individuals were identified from the national surveillance database from 1990 to 2006, and followed to the end of 2008. The pancreatic cancer risk in the study population was compared with the general population by calculation of Standardized Incidence Ratios (SIRs), and with a matched reference population using a Cox proportional hazards regression model to calculate hazard ratios (HRs). Results: In total 340 819 person-years in the HCV cohort and 102 295 in the HBV cohort were accumulated, with 34 and 5 pancreatic cancers identified, respectively. The SIRHCV was 2.1 (95% confidence interval, CI: 1.4, 2.9) and the SIRHBV was 1.4 (0.5, 3.3). In the Cox model analysis, the HR for HCV infection was 1.9 (95% CI: 1.3, 2.7), diminishing to 1.6 (1.04, 2.4) after adjustment for potential confounders.
Conclusion: Our results indicated that HCV infection might be associated with an increased risk of pancreatic cancer but further studies are needed to verify such association. The results in the HBV cohort indicated an excess risk, however, without statistical significance due to lack of power.

Pancreatic cancer is one of the most rapidly fatal malignancies with a 5-year survival rate below 5%. The long-term survival is poor also for early diagnosed patients treated with resection surgery (Jemal et al, 2010). In Europe, it was estimated in a prediction model that in the year 2012 there would be 75 000–80 000 deaths from pancreatic cancer, which is the fourth most common cause of cancer-associated death for both men and women (Malvezzi et al, 2012). The incidence of pancreatic cancer is higher in the Nordic countries and Central Europe than in other parts of the world (Bosetti et al, 2012).

Tobacco smoking is a well-established risk factor for pancreatic cancer (Iodice et al, 2008), and a similar magnitude of excess risk as smoking was found among the users of Scandinavian snus (moist snuff) (Boffettaet al, 2005; Luo et al, 2007). Besides, accumulating evidence consistently shows that old age, male sex, diabetes mellitus, hereditary pancreatitis, chronic pancreatitis and family history are positively associated with this carcinoma (Pandol et al, 2012). Albeit the biological mechanism is unclear, recent epidemiological studies indicated that some infections, such as exposure to Helicobacter pylori (Trikudanathan et al, 2011), poor oral health (Michaud et al, 2007), hepatitis C virus (HCV) (Hassan et al, 2008; El-Serag et al, 2009) or hepatitis B virus (HBV) (Hassan et al, 2008; Iloeje et al, 2010; Wang et al, 2012a, 2012b) might be associated with pancreatic cancer risk.

Globally, ∼170 million people are chronically infected with HCV (World Health Organization, 1997) and an estimated 350 million with HBV (Custer et al, 2004). The prevalence rates of HCV and HBV infection vary widely in the world, and Sweden is a low endemic country with an estimated 0.5% of the population infected with HCV (Duberg et al, 2008a) and even lower rate for HBV infection. Both chronic HCV and HBV infections are main causes of hepatocellular carcinoma (HCC). Previous findings demonstrated that HBV may replicate within the pancreas (Shimoda et al, 1981; Yoshimura et al, 1981) and that HCV could be associated with pancreatitis (Alvares-Da-Silva et al, 2000; Torbenson et al, 2007). Some studies support that HCV and HBV may have a role in the development of pancreatic cancer, but the evidence is far from conclusive (Hassan et al, 2008; El-Serag et al, 2009; Iloeje et al, 2010; Wang et al, 2012a, 2012b), and more studies are needed. Towards this end, we utilised Swedish population-based nationwide registers, with documentation of all diagnosed HCV- and HBV-infected individuals in Sweden, to explore the association of HCV or HBV infection and the risk of pancreatic cancer.

Baseline characteristics of the HCV and HBV cohorts are presented in Table 1. In the HCV and chronic HBV cohorts the mean follow-up time were 9.1 and 9.4 years, with a total of 360 154 and 107 986 person-years at risk, respectively. There was a clear male dominance in the HCV cohort, and median age at entry into the HCV or HBV cohorts (notification date) was 38 and 31 years, respectively. A marked difference between cohorts was observed regarding the aspect of country of origin; HCV-infected individuals were more likely from Nordic countries, but persons with chronic HBV infection were often immigrants from non-Nordic countries.

Hepatitis C virus cohort

In the HCV cohort, there were 34 pancreatic cancer cases observed during 340 819 person-years of follow-up (first 6 months of follow-up excluded), whereas 16.5 were expected, yielding a statistically significant increased risk of pancreatic cancer (SIR: 2.1; 95% CI: 1.4, 2.9). The SIR did not alter substantially across sex or estimated duration of HCV infection (Table 2). The majority of cases were among the patients who were born before 1960.

From the Cox regression model, an ∼90% excessive risk for pancreatic cancer (HR 1.9; 95% CI: 1.3, 2.7) was observed after adjustment for age, sex and county of residence, which is similar to the result from the SIR analysis. This excess risk diminished somewhat but remained statistically significant after further adjustment for potential confounders (HR 1.6; 95% CI: 1.04, 2.4). The results did not vary markedly when stratified by sex (Table 3). In the additional analyses, excluding all individuals ever hospitalized with acute and/or chronic pancreatitis, the results did not alter notably (data not shown).

In the HCV cohort, the Standardized Incidence Ratio (SIR) for lung cancer was 2.3 (95% CI: 1.9, 2.7) and the Hazard Ratio (HR) for lung cancer was 2.2 (95% CI: 1.8, 2.7), decreasing to 1.6 (95% CI: 1.3, 2.1) after adjustment for the potential confounders used in the pancreatic cancer analyses.

Chronic HBV cohort

A total of five pancreatic cancer cases were found during 102 295 person-years of follow-up (first 6 months excluded), whereas 3.5 were expected. Compared with the age- and sex-matched Swedish general population, a 40% excess risk of pancreatic cancer was found in the chronic HBV cohort (SIR: 1.4; 95% CI: 0.5, 3.3), but without statistical significance. Because of the small number of pancreatic cancer cases, there was not enough power for additional stratified analyses (Table 4).

The Cox regression model revealed similar results as the SIR analysis. The point estimates were somewhat higher (HR=2.0 from the model adjusted for only matching factors and HR=1.8 from the fully adjusted model), but still statistically non-significant (Table 5). The SIR for lung cancer in the chronic HBV infection cohort was 1.7 (95% CI: 1.1, 2.5).

This population-based large cohort study revealed a doubled risk of pancreatic cancer among HCV-infected patients compared with the Swedish general population. The excess risk was persistent across strata by sex or duration of infection. Although further adjustment for potential confounders, i.e., chronic obstructive pulmonary disease (related to smoking), diabetes mellitus, chronic pancreatitis and alcohol-related disease, resulted in an attenuated relative risk, this finding still supports the hypothesis that HCV infection might be associated with an increased risk of pancreatic cancer. Besides, the result indicated a moderate excessive risk of pancreatic cancer among HBV-infected patients according to different statistical approaches, but the size of the study cohort and the observed number of cancers were too small to draw a sound conclusion. Pancreatic cancer is more common in older age groups, and the small number of pancreatic cancers among the HBV cohort was probably an effect of the relatively young cohort, concordant with the epidemiology of chronic hepatitis B in Sweden.

The strengths of this register-based study include population-based cohort design, relatively large sample size, independently collected data on documentation of HCV/HBV notifications and pancreatic cancer occurrence and high completeness of follow-up.

The parallel (laboratory and clinician) notification system of HCV/HBV infections in Sweden has a high coverage of those with a diagnosed infection; it is estimated that about 75–80% of HCV infections are diagnosed, but there still remain unknown infections, not yet diagnosed or documented. In addition, a small portion of the reported patients could have a resolved infection, spontaneously or by treatment, this could (probably insignificant) lower the risk in the HCV and HBV cohort.

The number of unidentified HCV/HBV-coinfected individuals is probably low in the studied cohorts. However, in the HCV cohort there could be some patients who were never diagnosed with hepatitis B but have serologic markers of a past HBV infection. In these patients we cannot exclude the possibility of occult hepatitis B.

The biological mechanism of the association between HCV and pancreatic cancer is unclear. However, virtually, the pancreas and liver share the common blood vessels and ducts, and prior evidence demonstrated that the pancreas is a remote location for hepatitis virus inhabitation and replication (Hassan et al, 2008). HCV infection is associated with type 2 diabetes, which is both a risk factor and might be a consequence of pancreatic cancer (Mehta et al, 2000; Sangiorgio et al, 2000). Besides, previous studies reported that subclinical/acute pancreatitis (Katakura et al, 2005) and hyperlipasemia (Yoffe et al, 2003) may be extrahepatic manifestations of HCV infection. In addition, pancreatic involvement was observed among patients who suffered from chronic hepatitis infection, resulting in mild pancreatic damage accompanied with increased serum levels of pancreatic enzyme (Taranto et al, 1989; Katakura et al, 2005). Immune response may lead to chronic inflammation in the targeted organs after long time persistent infection with HCV. Therefore, hepatitis C virus conceivably serves as a biological agent that may indirectly have a role in inflammation-associated pancreatic carcinogenesis. Although still unclear to what extent chronic inflammation contributes to pancreatic cancer development, it is postulated that HCV can induce inflammatory microenvironment with high concentration of growth factors and cytokines. This may exert effects by accumulating alterations in driver genes and promoting cancer cell growth and proliferation.

HIV AIDS and Cancer

http://www.cancer.gov/cancertopics/causes-prevention/risk/infectious-agents/hiv-fact-sheet

Key Points

People infected with human immunodeficiency virus (HIV) have a higher risk of some types of cancer than uninfected people.
A weakened immune system caused by infection with HIV, infection with other viruses, and traditional risk factors such as smoking all contribute to this higher cancer risk.
Highly active antiretroviral therapy and lifestyle changes may reduce the risk of some types of cancer in people infected with HIV.
The National Cancer Institute (NCI) conducts and supports a number of research programs aimed at understanding, preventing, and treating HIV infection, acquired immunodeficiency syndrome-related cancers, and cancer-associated viral diseases.

Do people infected with human immunodeficiency virus (HIV) have an increased risk of cancer?

Yes. People infected with HIV have a substantially higher risk of some types of cancer compared with uninfected people of the same age (1). Three of these cancers are known as “acquired immunodeficiency syndrome (AIDS)-defining cancers” or “AIDS-defining malignancies”: Kaposi sarcoma, non-Hodgkin lymphoma, and cervical cancer. A diagnosis of any one of these cancers marks the point at which HIV infection has progressed to AIDS.

People infected with HIV are several thousand times more likely than uninfected people to be diagnosed with Kaposi sarcoma, at least 70 times more likely to be diagnosed with non-Hodgkin lymphoma, and, among women, at least 5 times more likely to be diagnosed with cervical cancer (1).

In addition, people infected with HIV are at higher risk of several other types of cancer (1). These other malignancies include anal, liver, and lung cancer, and Hodgkin lymphoma.

People infected with HIV are at least 25 times more likely to be diagnosed with anal cancer than uninfected people, 5 times as likely to be diagnosed with liver cancer, 3 times as likely to be diagnosed with lung cancer, and at least 10 times more likely to be diagnosed with Hodgkin lymphoma (1).

People infected with HIV do not have increased risks of breast, colorectal, prostate, or many other common types of cancer (1). Screening for these cancers in HIV-infected people should follow current guidelines for the general population

HIV and cancer of the cervix

Z.M. Chirenje
bestpracticeobgyn April 2005; 19(2):269–276
http://dx.doi.org/10.1016/j.bpobgyn.2004.10.002

Cancer of the cervix is the second most common cause of cancer-related death in women worldwide, and in some low resource countries accounts for the highest cancer mortality in women. The highest burden of the HIV/AIDS epidemic is currently in sub-Saharan Africa, where more than half of the people infected are women who have no access to cervical cancer screening. The association between HIV and invasive cervical cancer is complex, with several studies now clearly demonstrating an increased risk of pre-invasive cervical lesions among HIV-infected women. However, there have not been significantly higher incidence rates of invasive cervical cancer associated with the HIV epidemic. The highest numbers of HIV-infected women are in poorly-resourced countries, where the natural progression of HIV disease in the absence of highly active antiretroviral treatment sometimes results in deaths from opportunistic infections before the onset of invasive cervical cancer. This chapter will discuss the association of HIV and cervical intraepithelial neoplasia, the treatment of pre-invasive lesions, and invasive cervical cancer in HIV-infected women. The role of screening and the impact of antiretroviral treatment on the progression of pre-invasive and invasive cancer will also be discussed.

Anal cancer: an HIV-associated cancer

Klencke BJ, Palefsky JM
Hematology/oncology Clinics of North America [2003, 17(3):859-872]
http://dx.doi.org:/1016/S0889-8588(03)00039-X

Although not yet included in the Centers for Disease Control definition of AIDS, anal cancer clearly occurs more commonly in HIV-infected patients. An effective screening program for those groups who are at highest risk might be expected to impact rates of anal cancer just as significantly as did cervical Pap screening programs for the incidence of cervical cancer. Despite a relatively low rate of progression from AIN to invasive cancer, the scope of the problem is enormous based on the prevalence of anal HPV infection and the size of the HIV-infected, at-risk population. Thus, the potential benefits of screening, detection, and the development of more effective therapy also are enormous. Currently, therapeutic HPV vaccines for AIN represent an exciting avenue of research in HPV-related anogenital disease. Invasive anal cancer and HSIL (which is believed to be the precursor lesion) are expected to become increasingly important health problems for both HIV-infected men and women as their life expectancy lengthens. Although HAART may have improved the ability of many to tolerate CMT, it appears that toxicity of this therapy continues to be a problem for a proportion of HIV-infected subjects. The acute side effects present specific challenges to the clinician and patient, have an immediate impact on the patient’s plan of care and dose intensity of the treatment, and ultimately may impact the outcome of the planned treatment. Late toxicity may influence the long-term quality of life. Small patient numbers, variable radiation therapy doses, limited information about viral load, and a potential confounding effect of higher CD4+ levels make it difficult to draw any conclusions about the effect of HAART on anal cancer outcome. Large, prospective studies will be required before solid conclusions about the impact of various factors on anal cancer prognosis and outcome can be drawn.

The therapeutic potential of CXCR4 antagonists in the treatment of HIV infection, cancer metastasis and rheumatoid arthritis

Hirokazu Tamamura, and Nobutaka Fujii
Exp Opin on Ther Targets Dec 2005; 9(6): 1267-1282 http://dx.doi.org:/10.1517/14728222.9.6.1267

CXCR4 is the receptor of the chemokine CXCL12, which is involved in progression and metastasis of several types of cancer cells, HIV infection and rheumatoid arthritis. The authors developed selective CXCR4 antagonists, T22 and T140, initially as anti-HIV agents, which inhibit T cell line-tropic (X4-) HIV-1 infection through their specific binding to CXCR4. Recently, T140 analogues have also been shown to inhibit CXCL12-induced migration of breast cancer cells, leukaemia T cells, pancreatic cancer cells, small cell lung cancer cells, chronic lymphocytic leukaemia B cells, pre-B acute lymphoblastic leukaemia cells and so on in vitro. Biostable T140 analogues significantly suppressed pulmonary metastasis of breast cancer cells and melanoma cells in mice. Furthermore, these compounds significantly suppressed the delayed-type hypersensitivity response induced by sheep red blood cells and collagen-induced arthritis, which represent in vivo mouse models of arthritis. Thus, T140 analogues proved to be attractive lead compounds for chemotherapy of these problematic diseases. This article reviews recent research on T140 analogues, referring to several other CXCR4 antagonists.

Types of Cancer: AIDS/HIV related malignancies

http://cancer.northwestern.edu/cancertypes/cancer_type.cfm?category=1

People with HIV/AIDS are at high risk for developing certain cancers, such as Kaposi’s sarcoma, non-Hodgkin lymphoma, and cervical cancer. For people with HIV, these three cancers are often called “AIDS-defining conditions,” meaning that if a person with HIV has one of these cancers it can signify the development of AIDS. The connection between HIV/AIDS and certain cancers is not completely understood, but the link likely depends on a weakened immune system. Most types of cancer begin when normal cells begin to change and grow uncontrollably, forming a mass called a tumor. A tumor can be benign (noncancerous) or malignant (cancerous, meaning it can spread to other parts of the body). The types of cancer most common for people with HIV/AIDS are described in more detail below.

Kaposi’s sarcoma

Kaposi’s sarcoma is a type of skin cancer, which has traditionally occurred in older men of Jewish or Mediterranean descent, young men in Africa, or people who have received organ transplantation. Today, Kaposi’s sarcoma is found most often in homosexual men with HIV/AIDS and related to an infection with the human herpesvirus 8 (HHV-8). Kaposi’s sarcoma in people with HIV is often called epidemic Kaposi’s sarcoma. HIV/AIDS-related Kaposi’s sarcoma causes lesions to arise in multiple sites in the body, including the skin, lymph nodes, and organs such as the liver, spleen, lungs, and digestive tract.

Non-Hodgkin lymphoma

HIV/AIDS-related NHL is the second most common cancer associated with HIV/AIDS, after Kaposi’s sarcoma. There are many different subtypes of NHL. The most common subtypes of NHL in people with HIV/AIDS are primary central nervous system lymphoma (affecting the brain and spinal fluid), found in 20% of all NHL cases in people with HIV/AIDS, primary effusion lymphoma (causing fluid to accumulate around the lungs or in the abdomen), or intermediate and high-grade lymphoma. More than 80% of lymphomas in people with HIV/AIDS are high-grade B-cell lymphoma, while 10% to 15% of lymphomas among people with cancer who do not have HIV/AIDS are of this type. It is estimated that between 4% and 10% of people with HIV/AIDS develop NHL.

Other types of cancer

Other, less common types of cancer that may develop in people with HIV/AIDS are Hodgkin’s lymphoma, angiosarcoma (a type of cancer that begins in the lining of the blood vessels), anal cancer, liver cancer, mouth cancer, throat cancer, lung cancer, testicular cancer, colorectal cancer, and multiple types of skin cancer including basal cell carcinoma, squamous cell carcinoma, and melanoma.

Treatment options for the most common treatments for HIV/AIDS-related cancers are listed by the specific type of cancer. Treatment options and recommendations depend on several factors, including the type and stage of cancer, possible side effects, and the patient’s preferences and overall health.

Palliative care can help a person at any stage of illness. People often receive treatment for the cancer and treatment to ease side effects at the same time. In fact, patients who receive both often have less severe symptoms, better quality of life, and report they are more satisfied with treatment.

Palliative treatments vary widely and often include medication, nutritional changes, relaxation techniques, and other therapies. You may also receive palliative treatments similar to those meant to eliminate the cancer, such as chemotherapy, surgery, and radiation therapy.

It is extremely important that all patients with HIV/AIDS and an associated cancer receive treatment with highly active antiretroviral treatment (HAART) both during the cancer treatments and afterwards. HAART can effectively control the virus in most patients. Better control of the HIV infection decreases the side effects of many of the treatments, may decrease the chance of a recurrence, and can improve a patient’s chance of recovery from the cancer.

The treatment of HIV/AIDS-related Kaposi sarcoma usually cannot cure the cancer, but it can help relieve pain or other symptoms. This can be followed by palliative care for Kaposi sarcoma. Antiviral treatment for HIV/AIDS helps reduce a person’s chance of getting Kaposi sarcoma and can reduce the severity of Kaposi sarcoma. HAART helps treat the tumor and reduce the symptoms associated with Kaposi sarcoma for people with HIV/AIDS. It is usually used before other treatments, such as chemotherapy.

Curettage and electrodesiccation. In this procedure, the cancer is removed with a curette, a sharp, spoon-shaped instrument. The area can then be treated with electrodesiccation, which uses an electric current to control bleeding and kill any remaining cancer cells. Many patients have a flat, pale scar from this procedure.

Cryosurgery. Cryosurgery, also called cryotherapy or cryoablation, uses liquid nitrogen to freeze and kill cells. The skin will later blister and shed off. This procedure will sometimes leave a pale scar. More than one freezing may be needed.

In photodynamic therapy, a light-sensitive substance is injected into the lesion that stays longer in cancer cells than in normal cells. A laser is directed at the lesion to destroy the cancer cells.

Radiation therapy is the use of high-energy x-rays or other particles to destroy cancer cells. A doctor who specializes in giving radiation therapy to treat cancer is called a radiation oncologist. The most common type of radiation treatment is called external-beam radiation therapy, which is radiation given from a machine outside the body. When radiation therapy is given using implants, it is called internal radiation therapy or brachytherapy. External-beam radiation therapy may be given as a palliative treatment. A radiation therapy regimen (schedule) usually consists of a specific number of treatments given over a set period of time.

Side effects from radiation therapy may include fatigue, mild skin reactions, upset stomach, and loose bowel movements. Most side effects go away soon after treatment is finished. Learn more about radiation therapy.

Chemotherapy may help control advanced disease, although curing HIV/AIDS-related Kaposi sarcoma with chemotherapy is extremely rare. Usually, for HIV/AIDS-related Kaposi sarcoma, chemotherapy is used to help relieve symptoms and to lengthen a patient’s life. Common drugs for Kaposi sarcoma include: liposomal doxorubicin (Doxil), paclitaxel (Taxol, LEP-ETU, Abraxane), and vinorelbine (Navelbine, Alocrest).

The side effects of chemotherapy depend on the individual and the dose used, but they can include fatigue, risk of infection, nausea and vomiting, hair loss, loss of appetite, and diarrhea. These side effects usually go away once treatment is finished.

HIV/AIDS-related Kaposi sarcoma may receive alpha-interferon (Roferon-A, Intron A, Alferon), which appears to work by changing the surface proteins of cancer cells and by slowing their growth. Immunotherapy is generally used for people who are in the good-risk category in the immune system (I) factor of the TIS staging system (see Stages). The most common side effects of alpha-interferon are low levels of white blood cells and flu-like symptoms.

The main treatments for HIV/AIDS-related non-Hodgkin lymphoma are chemotherapy, targeted therapy, and radiation therapy.

Treatments for women with the precancerous condition called CIN (see Overview) are generally not as effective for women with HIV/AIDS because of a weakened immune system. Often, the standard treatment for HIV/AIDS can lower the symptoms of CIN.

Women with invasive cervical cancer and HIV/AIDS that is well-controlled with medication, generally receive the same treatments as women who do not have HIV/AIDS. Common treatment options include surgery, radiation therapy, and chemotherapy.

Cytokines in cancer pathogenesis and cancer therapy

Glenn Dranoff
Nature Reviews Cancer Jan 2004; 4(11-22) http://dx.doi.org:/10.1038/nrc1252

The mixture of cytokines that is produced in the tumor microenvironment has an important role in cancer pathogenesis. Cytokines that are released in response to infection, inflammation and immunity can function to inhibit tumor development and progression. Alternatively, cancer cells can respond to host-derived cytokines that promote growth, attenuate apoptosis and facilitate invasion and metastasis. A more detailed understanding of cytokine–tumor-cell interactions provides new opportunities for improving cancer immunotherapy.

Dendritic Cells as Therapeutic Vaccines against Cancer
Jacques Banchereau and A. Karolina Palucka
Nature Reviews Immunology APR 2005; 5:296-306
http://cnc.cj.uc.pt/BEB/private/pdfs/2007-2008/Immunology/E/Rev_paper_E4.pdf

Mouse studies have shown that the immune system can reject tumours, and the identification of tumor antigens that can be recognized by human T cells has facilitated the development of immunotherapy protocols. Vaccines against cancer aim to induce tumor-specific effector T cells that can reduce the tumor mass, as well as tumor-specific memory T cells that can control tumor relapse. Owing to their capacity to regulate T-cell immunity, dendritic cells are increasingly used as adjuvants for vaccination, and the immunogenicity of antigens delivered by dendritic cells has now been shown in patients with cancer. A better understanding of how dendritic cells regulate immune responses will allow us to better exploit these cells to induce effective anti-tumor immunity.

Vaccines against infectious agents are one success of immunology and have spared countless individuals from diseases such as polio, measles, hepatitis B and tetanus⁸ . However, progress in the development of vaccines against infectious agents has been largely empirical and not always successful, as many infectious diseases still evade the immune system, particularly chronic infections such as tuberculosis, malaria and HIV infection. Further progress will be made through rational design based on our increased understanding of how the immune system works and how the induction of protective immunity is regulated. The same principle applies to vaccines against cancer, particularly as cancer is a chronic disease, and when it becomes clinically visible, tumor cells and their products have already been interacting with and affecting host cells for a considerable time to ensure the survival of the tumor. Ex vivo-generated, antigen-loaded DCs have now been used as vaccines to improve immunity⁹. Numerous studies in mice have shown that DCs loaded with tumor antigens can induce therapeutic and protective antitumor immunity¹⁰. The immunogenicity of antigens delivered by DCs has been shown in patients with cancer9 or chronic HIV infection¹¹, thereby providing proof of principle that using DCs as vaccines can work. Despite this, the efficacy of therapeutic vaccination against cancer has recently been questioned¹²because of the undeniably limited rate of objective tumor regressions that has been observed in clinical studies so far. However, the question is not whether DC vaccines work but how to orient further studies to refine the immunological and clinical parameters of vaccination with DCs to improve its efficacy.

Vaccines against cancer Early studies in mice showed that the immune system can recognize and reject tumours¹³and that immunodeficient mice (lacking interferon-γ (IFN-γ) and recombination-activating gene 2) have an increased incidence of cancer¹⁴ (BOX 1). In humans, the incidence of some cancers is increased in immunodeficient patients¹⁵ and is increased with age, owing to Immunosenescence¹⁶. These observations support the scientific rationale for immunotherapy for cancer. The term immunotherapy refers to any approach that seeks to mobilize or manipulate the immune system of a patient for therapeutic benefit¹⁷. In this regard, there are numerous strategies for improving the resistance of a patient to cancer. These include non-specific activation of the immune system with microbial components or cytokines, antigen-specific adoptive immunotherapy with antibodies and/or T cells, and antigen-specific active immunotherapy (that is, vaccination). The main limitation of using antibodies is that target proteins need to be expressed at the cell surface. By contrast, targets for T cells are usually peptides derived from intracellular proteins, which are presented at the cell surface in complexes with MHC molecules¹⁸. The identification of defined tumor antigens in humans^19,20 prompted the development of adoptive T-cell therapy. Yet, the most attractive strategy is vaccination, which is expected to induce both therapeutic T-cell immunity (in the form of tumor-specific effector T cells) and protective T-cell immunity (in the form of tumor-specific memory T cells that can control tumor relapse)^21–23. Numerous approaches for the therapeutic vaccination of individuals who have cancer have been developed, including the use of the following: autologous and allogeneic tumor cells (which are often modified to express various cytokines), peptides, proteins and DNA vaccines^9,23–26. The observed results are variable; however, in many cases, a tumour-specific immune response has been induced, and tumor regressions, albeit limited, have occurred. These approaches rely on random encounter of the vaccine with host DCs. A lack of encounter of the vaccine antigen with DCs might result in the absence of an immune response. Alternatively, an inappropriate encounter — for example, with unactivated DCs or with the ‘wrong’ subset of DCs — might lead to silencing of the immune response²⁷. Both of these situations could explain some of the shortcomings of current cancer vaccines. Furthermore, we do not know how tumor antigens need to be delivered to DCs in vivo to elicit an appropriate immune response.

Immature and mature dendritic cells have different functions. A | Immature dendritic cells (DCs) induce tolerance. Tissue DCs constantly sample their environment, capture antigens and migrate in small numbers to draining lymph nodes. In the absence of inflammation, the DCs remain in an immature state, and antigens are presented to T cells in the lymph node without costimulation, leading to either the deletion of T cells or the generation of inducible regulatory T cells. B | Mature DCs induce immunity. Tissue inflammation induces the maturation of DCs and the migration of large numbers of mature DCs to draining lymph nodes. The mature DCs express peptide–MHC complexes at the cell surface, as well as appropriate co-stimulatory molecules. This allows the priming of CD4+ T helper cells and CD8+ cytotoxic T lymphocytes (CTLs), the activation of B cells and the initiation of an adaptive immune response. To control the immune response, CD4+CD25+ regulatory T (TReg)-cell populations are also expanded. [ADCC, antibody-dependent cell-mediated cytotoxicity; NK, natural killer; TCR, T-cell receptor].

Box 1 |

Mice

The immune system can reject tumors
Immune-mediated rejection of chemically induced tumours¹³
Increased cancer incidence in immunodeficient mice¹⁴

Humans

Increased cancer incidence in immunodeficient patients¹⁵
Increased cancer incidence with age (immunosenescence)¹⁶
Cancer regression in patients with paraneoplastic neurological disorders that are mediated by onconeuronal antibodies and specific CD8+ T cells¹³⁶

Dendritic cells DC subsets. There are thought to be two main pathways of differentiation into DCs^2,31 (FIG. 2). The myeloid pathway generates two subsets: Langerhans cells, which are found in stratified epithelia such as the skin; and interstitial DCs, which are found in all other tissues³². The lymphoid pathway generates plasmacytoid DCs (pDCs), which secrete large amounts of type I IFNs (IFN-α and IFN-β) after viral infection^33,34. DCs and their precursors show remarkable functional plasticity. For example, pDCs form one of the first barriers to the expansion of intruding viruses, thereby functioning, through the release of type I IFNs, as part of the innate immune response. Subsequently, these cells differentiate into DCs that can present antigens to T cells, thereby functioning as members of the adaptive immune system^35,36. Monocytes can differentiate into either macrophages, which function as scavengers, or DCs that induce specific immune responses^37,38. Different cytokines skew the in vitro differentiation of monocytes into DCs with different phenotypes and functions (FIG. 3). So, after activation (for example, by granulocyte/ macrophage colony-stimulating factor, GM-CSF), monocytes that encounter interleukin-4 (IL-4) become DCs known as IL-4-DCs^29,30,39. By contrast, after encounter with IFN-α, tumour-necrosis factor (TNF) or IL-15, activated monocytes differentiate into IFN-α-DCs^40–43, TNF-DCs44 or IL-15-DCs45, respectively. Whether, in vivo, all interstitial DCs are derived from monocytes remains to be established, but myeloid DCs that are isolated from human peripheral blood also give rise to different DC types after exposure to different cytokines. Each of these DC subsets has both common and unique biological functions, which are determined by a unique combination of cell-surface molecule expression and cytokine secretion. For example, whereas IL-4-DCs are a homologous population of immature cells that is devoid of Langerhans cells, TNFDCs are heterogeneous and include both CD1a+ Langerhans cells and CD14+ interstitial DCs⁴⁴.In vitro experiments showed that Langerhans cells and interstitial DCs that were generated from cultures of CD34+ hematopoietic progenitors differ in their capacity to activate lymphocytes: interstitial DCs induce the differentiation of naive B cells into immunoglobulin-secreting plasma cells^4,32, whereas Langerhans cells seem to be particularly efficient activators of cytotoxic CD8+ T cells. They also differ in their cytokine-secretion pattern (only interstitial DCs produce IL-10) and their enzymatic activity^4,32, which might be fundamental for the selection of peptides that are presented to T cells. Indeed, different enzymes are likely to degrade a given antigen into different sets of peptides, as has recently been shown for the HIV protein Nef 46. This then leads to different sets of peptide–MHC complexes being presented and thereby to distinct repertoires of antigen-specific T cells. So, these unique DCs are likely to yield unique immune effectors, thereby allowing the broad immune response that is required to combat permanently evolving microorganisms and tumors.

Distinct DC subsets induce distinct types of immune response. DCs have a crucial role in determining the type of response that is induced. There is evidence that either polarized DCs or distinct DC subsets might provide T cells with different signals that determine the class of immune response³¹. So, in mice, splenic CD8α+ DCs prime naive CD4+ T cells to produce TH1 cytokines in a process that involves IL-12, whereas splenic CD8α– DCs prime naive CD4+ T cells to produce TH2 cytokines^47,48. Furthermore, this polarization into different T-cell subsets also depends on the signal received by a DC, as shown by the induction of IL-12 production and polarization towards TH1 cells when DCs are activated with Escherichia coli lipopolysaccharide (LPS), but the absence of IL-12 production and polarization towards TH2 cells when the same type of DC is exposed to LPS from Porphyromonas gingivalis ⁴⁹. In humans, CD40 ligand (CD40L)-activated monocyte-derived DCs prime TH1-cell responses through an IL-12-dependent mechanism, whereas pDCs activated with IL-3 and CD40L have been shown to secrete negligible amounts of IL-12 and to prime TH2-cell responses⁵⁰. So, both the type of DC subset and the activation signals to which DCs are exposed are important for polarization of T cells.

Mouse proof-of-principle in vivo studies

Ex vivo-generated, antigen-loaded dendritic cells (DCs) induce antigen-specific T-cell immunity¹³⁷
Ex vivo gene-loaded DCs can induce humoral immunity¹³⁸
Ex vivo-generated, antigen-loaded DCs induce tumor-specific immunity^139,140
Ex vivo-generated DCs are superior to other types of vaccine¹⁴¹
Ex vivo-generated immature DCs induce tolerance¹⁴²
Combination therapy with ex vivo-generated DCs improves vaccine efficacy^112,113

This is an important parameter in vaccination against cancer, as type 1 immunity (including IFN-γ secretion) is desirable, whereas type 2 immunity (including IL-4 or IL-10 secretion) is considered deleterious. DCs and immune tolerance. DCs can induce and maintain immune tolerance27, both central and peripheral.

Central Tolerance depends on mature thymic DCs, which are essential for the deletion of newly generated T cells that have a receptor that recognizes self-components51. However, central tolerance might not be effective for all antigens. Furthermore, many self-antigens might not have access to the thymus, and others are only expressed later in life. So, there is a requirement for Peripheral Tolerance, which occurs in lymphoid organs and is mediated by immature DCs (FIG. 1a). Immature DCs present tissue antigens to T cells in the absence of appropriate co-stimulation, leading to T-cell Anergy or deletion²⁷ or to the development of IL-10-secreting Inducible Regulatory T Cells^52,53. The research groups of Nussenzweig and Steinman⁵⁴ have elegantly shown that fusion proteins targeted to immature DCs lead to the induction of antigen-specific tolerance. By contrast, concomitant activation of these DCs with CD40- specific antibody results in a potent immune response, because the DCs are induced to express a large number of co-stimulatory molecules55. However, mature DCs might also contribute to peripheral tolerance by promoting the clonal expansion of naturally occurring CD4+CD25+ REGULATORY T (TReg) CELLS56, as discussed later. Therefore, the biology of DCs offers several targets for the control of cellular immunity. The parameters that need to be considered include DC-related factors, host-related factors and combining DC vaccines with other therapies.

Subsets of human dendritic cells. (Fig not shown). The population of dendritic cells (DCs) in the peripheral blood, which can be mobilized by treatment with FLT3L (fms-related tyrosine kinase 3 ligand), contains both CD11c+ myeloid DCs and CD11c– plasmacytoid DCs. So far, most studies of DCs have been carried out with DCs generated by culturing monocytes with granulocyte/macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4); this simple procedure yields a homogenous population of DCs that resemble interstitial DCs, and the population is devoid of Langerhans cells. These DCs are immature and require exogenous factors for maturation. DCs can also be generated by culturing CD34+ haematopoietic progenitor cells (HPCs) or peripheral-blood monocytes with GM-CSF and tumour-necrosis factor (TNF). In this way, two DC subsets can be obtained: Langerhans cells, which might have improved efficacy for eliciting cytotoxic T lymphocytes; and interstitial DCs, which resemble monocyte-derived DCs. Adding IL-4 to CD34+ HPC cultures in the presence of GM-CSF and TNF inhibits the differentiation of Langerhans cells. [Green boxes indicate cell types that can be induced by culture with GM-CSF and TNF. Yellow boxes indicate cell types that can be induced by culture or mobilization with FLT3L].

Plasticity of monocyte-derived dendritic cells. (Fig not shown). Activated monocytes can differentiate into different types of dendritic cell (DC) after encounter with different cytokines. These distinct DCs will influence the differentiation of lymphocytes into immune effectors with different functions, leading to varied immune responses. For example, interleukin-15-DCs (IL-15- DCs) are remarkably more efficient at priming and maturation of rare antigen-specific cytotoxic T lymphocytes (CTLs) than are IL-4-DCs. Thymic stromal lymphopoietin-DCs (TSLP-DCs) induce CD4+ T cells to differentiate into pro-inflammatory T helper 2 (TH2) cells, which secrete large amounts of IL-13 and tumor-necrosis factor (TNF)¹⁴³, whereas interferon-α-DCs (IFN-α-DCs) induce CD4+ T cells to differentiate into TH1 cells, which secrete IFN-γ and IL-10. The properties and function of TNF-DCs remain to be determined. [FLT3L, fms-related tyrosine kinase 3 ligand; GM-CSF, granulocyte/macrophage colony-stimulating factor].

Antigen loading. Loading MHC class I and class II molecules at the cell surface of DCs with peptides derived from defined antigens is the most commonly used strategy for DC-based vaccination^22,87. Although this technique is important for proof-of-principle studies, the use of peptides has limitations: the restriction of a peptide to a given HLA type; the limited number of well-characterized Tumor-Associated Antigens; the relatively rapid turnover of exogenous peptide– MHC complexes, resulting in comparatively low antigen presentation by the time that the DC arrives in the draining lymph node after injection; and the induction of a restricted repertoire of T-cell clones, thereby limiting the ability of the immune system to control tumor-antigen variation. Yet another level of complexity is brought about by the use of MODIFIED HETEROCLITIC PEPTIDES. Some synthetic peptides, even those derived from immune-dominant antigens, do not bind MHC class I molecules with high affinity, possibly explaining their limited immunogenicity in vivo88. Therefore, the generation of peptide analogues with increased affinity for MHC class I molecules (known as heteroclitic peptides) could be used to improve peptide immunogenicity^89,90. However, recent elegant studies in patients with malignant melanoma show that T cells elicited in vivo by vaccination with heteroclitic MART1 (melanoma antigen recognized by autologous T cells) or glycoprotein 100 (gp100) peptide show poor recognition of the endogenous melanoma-derived peptide and less efficient tumor-cell lysis compared with T cells specific for the native peptide⁹¹.

Immunoregulatory mechanisms

Naturally occurring CD4+CD25+ regulatory T cells

Cell-mediated suppression independent of interleukin-10 (IL-10) and/or transforming growth factor-β (TGF-β);
clonal expansion is regulated by mature dendritic cells (DCs)

Inducible regulatory T cells

Cytokine-mediated suppression through IL-10 and/or TGF-β; induction and clonal expansion is regulated by immature DCs

Natural killer T cells

Cytokine-mediated suppression through IL-13

Vaccine-induced B cells?

Cytokine-mediated regulation through IL-4, IL-6 and IL-10; competition with DCs for antigen uptake

Tumor-specific interferon-γ-secreting T cells?

Immunoediting and selection of escape variants (not discussed in main text)

Immune correlates of efficacy of dendritic-cell-based vaccines

Induction of broad tumour-specific T-cell immunity: T cells specific for several tumour antigens
Induction of effector T cells: T cells with immediate capacity to recognize tumour antigens and secrete cytokines such as tumour-necrosis factor and interferon-γ
Induction of memory T cells: T cells that secrete interleukin-2 and proliferate on re-exposure to tumour antigen
Induction of T cells that kill tumour cells
Decreased number of T cells with regulatory function

DCs are an attractive target for therapeutic manipulation of the immune system to increase otherwise insufficient immune responses to tumour antigens. However, the complexity of the DC system requires rational manipulation of DCs to achieve protective or therapeutic immunity. So, further research is needed to analyse the immune responses induced in patients by distinct ex vivo-generated DC subsets that are activated through different pathways. The ultimate ex vivo-generated DC vaccine will be heterogeneous and composed of several subsets, each of which will target a specific immune effector. These ex vivo strategies should help to identify the parameters for in vivo targeting of DCs, which is the next step in the development of DC-based vaccination. Indeed, distinct DC subsets express unique cell-surface molecules, such as different lectins¹³¹: Langerhans cells express langerin, which is crucial for the formation of Birbeck granules^132,133; interstitial DCs express DCSIGN (dendritic-cell-specific intercellular-adhesionmolecule-3-grabbing non-integrin), which is involved in interactions with T cells and in DC migration but is also used by pathogens (such as HIV) to hijack the immune system; and pDCs express yet another lectin, BDCA2 (blood DC antigen 2)^134,135. Such differential expression of cell-surface molecules might allow specific in vivo targeting of DC subsets for induction of the desired type of immune response.

9.1.2 Simultaneous Humoral and Cellular Immune Response against Cancer–Testis Antigen NY-ESO-1: Definition of Human Histocompatibility Leukocyte Antigen (HLA)-A2–binding Peptide Epitopes

Elke Jäger, Yao-Tseng Chen, Jan W. Drijfhout, Julia Karbach, et al.
J Exp Med. 1998 Jan 19; 187(2): 265–270.
A growing number of human tumor antigens have been described that can be recognized by cytotoxic T lymphocytes (CTLs) in a major histocompatibility complex (MHC) class I–restricted fashion. Serological screening of cDNA expression libraries, SEREX, has recently been shown to provide another route for defining immunogenic human tumor antigens. The detection of antibody responses against known CTL-defined tumor antigens, e.g., MAGE-1 and tyrosinase, raised the question whether antibody and CTL responses against a defined tumor antigen can occur simultaneously in a single patient. In this paper, we report on a melanoma patient with a high-titer antibody response against the “cancer–testis” antigen NY-ESO-1. Concurrently, a strong MHC class I–restricted CTL reactivity against the autologous NY-ESO-1–positive tumor cell line was found. A stable CTL line (NW38-IVS-1) was established from this patient that reacted with autologous melanoma cells and with allogeneic human histocompatibility leukocyte antigen (HLA)-A2⁻, NY-ESO-1–positive, but not NY-ESO-1–negative, melanoma cells. Screening of NY-ESO-1 transfectants with NW38-IVS-1 revealed NY-ESO-1 as the relevant CTL target presented by HLA-A2. Computer calculation identified 26 peptides with HLA-A2–binding motifs encoded by NY-ESO-1. Of these, three peptides were efficiently recognized by NW38-IVS-1. Thus, we show that antigen-specific humoral and cellular immune responses against human tumor antigens may occur simultaneously. In addition, our analysis provides a general strategy for identifying the CTL-recognizing peptides of tumor antigens initially defined by autologous antibody.

There is growing evidence for humoral and cellular immune recognition of cancer by the autologous human host (1–6). Based on CTL-dependent lysis of cultured melanoma cell lines, several categories of autoimmunogenic tumor antigens have been characterized, including differentiation antigens of specific cell lineages (7–9), individual antigens caused by point mutations (10, 11), and tumor antigens, such as MAGE, which are expressed in a variable proportion of different tumor types, but are silent in most normal tissues except the testis (12). CTL responses against melanoma antigens induced by peptide vaccines in vivo have been associated with a favorable development of advanced melanoma in some patients (6, 13). As immunoselection of antigen-negative tumor cell variants has been observed during peptide vaccination (14), the molecular characterization of additional CTL-defined tumor antigens is needed to develop polyvalent vaccines with broader immunotherapeutic effects.

Sahin et al. have recently introduced a powerful new methodology for identifying human tumor antigens eliciting humoral immune response (5). The method has been called SEREX, for serological expression cloning of recombinant cDNA libraries of human tumors. Novel and previously defined tumor antigens have been identified by the SEREX method, including MAGE-1 and tyrosinase, both originally identified by cloning the epitopes recognized by CTLs. Thus, antibody screening of cDNA libraries prepared from human tumors can be used to identify antigens eliciting a cellular immune response, including CTLs, circumventing the need for established cultured autologous cell lines and stable CTL lines.

We have recently identified a novel human tumor antigen by SEREX analysis of a human esophageal cancer (15). The antigen, NY-ESO-1, belongs to a growing number of human tumor antigens we have called “cancer–testis” antigens that include MAGE, GAGE, BAGE (1), and SSX2 (HOM-MEL-40) (5, 16). These antigens have the following characteristics: (a) they are expressed in a variable portion of a wide range of cancers, (b) their normal tissue expression is generally restricted to the testis, and (c) they are generally coded for by genes on the X chromosome. In a recent survey of sera from normal individuals and cancer patients, antibodies against NY-ESO-1 were found in ∼10% of patients with melanoma, ovarian cancer, and other cancers, but not in normal individuals (Stockert, E., manuscript in preparation). One patient with a high NY-ESO-1 antibody response was found to have specific CTL reactivity against cultured autologous melanoma cells. In the present study, we report that NY-ESO-1 encodes the CTL target in this patient and identify the NY-ESO-1 peptides that are recognized.

High-titer Antibody Reactivity against NY-ESO-1.

Melanoma patient NW38 presented with extensive metastases to inguinal lymph nodes having large areas of necrosis. Reverse transcriptase PCR of tumor RNA showed that this tumor expressed NY-ESO-1. Based on the hypothesis that exposure of the immune system to large amounts of intracellular tumor proteins released from the necrotic tumor might elicit a strong humoral immune response, the serum of patient NW38 was tested for specific reactivity against recombinant NY-ESO-1 protein. Fig. Fig.11 shows the reactivity of NW38 serum with the recombinant NY-ESO-1 protein, with a lysate of NY-ESO-1–transfected COS-7 cells, and with a lysate of the autologous NY-ESO-1 messenger RNA–positive tumor cell line NW-MEL-38. A 22-kD protein species was identified in both cell lysates, and comigrated with the purified recombinant NY-ESO-1 protein. The identity of this protein species as NY-ESO-1 was further confirmed by using an anti–NY-ESO-1 mouse monoclonal antibody. Reactivity against recombinant NY-ESO-1 protein was still detectable at a serum dilution of 1:100,000. No reactivity was detected against a lysate of untransfected COS-7 cells.
The correlation between NY-ESO-1 expression and NW38-IVS-1 reactivity suggested NY-ESO-1 as the antigenic target. To prove this, COS-7 cells were transfected with NY-ESO-1 cDNA and different MHC class I molecules and used as targets for NW38-IVS-1. Reactivity was measured in a standard TNF-α release assay. TNF release was found after stimulation of NW38-IVS-1 with COS-7 cells cotransfected with HLA-A2⁻ and NY-ESO-1 cDNA. No reactivity was detected after stimulation with cotransfectants of pcDNA3.1(−)-NY-ESO-1⁻ and pcDNA1Amp-HLA-A1 cDNA, COS-7 cells transfected with pcDNA3.1(−), or untransfected COS-7 cells (Fig. (Fig.3).3).

Peptide-specific CTLs.

26 different peptides encoded by NY-ESO-1 with theoretical binding motifs to the HLA-A2.1 molecule were tested for specific recognition by NW38-IVS-1. The target cells were peptide-pulsed T2 cells. Of these 26 peptides, three were recognized by NW38-IVS-1 as determined by a standard⁵¹Cr–release assay (Table (Table1).1). The peptide sequences SLLMWITQCFL, SLLMWITQC, and QLSLLMWIT are located between positions 155 and 167 of the NY-ESO-1 protein (15), and show overlapping sequences. The 11-mer SLLMWITQCFL (2 in Table Table1)1) and the 9-mer SLLMWITQC (12 in Table Table1)1) consist of identical amino acids at positions 1–9.

To provide additional confirmation of the peptide specificity, the 26 synthetic peptides were individually incubated with HLA-A2–transfected COS-7 cells and tested in the TNF release assay. Consistent with the results of ⁵¹Cr–release assay, specific TNF-α release was detected in tests with peptides SLLMWITQCFL, SLLMWITQC, and QLSLLMWIT. NY-ESO-1/HLA-A2 transfectants were used as a positive control in these assays (Fig. (Fig.4).4).

The search for tumor antigens that induce specific immune responses in cancer patients is the ongoing challenge in tumor immunology. Evidence for a specific humoral response to human cancer came from serological analysis of cell surface reactivity of sera from cancer patients for autologous cancer cells, an approach called autologous typing (4). However, with only a few exceptions, this approach did not allow for the structural definition of the antigenic target. An autologous typing system also provided the first evidence for the development of CTLs with specificity for human melanoma cells (3, 17, 21–24). Using specific antitumor CTLs as probes, a number of CTL targets have been cloned on the basis of MHC class I–restricted recognition (1, 6). However, this approach involves cultured cancer cell lines and stable CTL lines from the same patient, two requirements that cannot easily be met with many tumor types. With the demonstration that genes coding for CTL-recognized tumor antigens elicit humoral immunity and can be cloned by SEREX methodology, a technically less demanding approach defining immunogenic tumor antigens is now available, one that extends the range of analysis to tumor types that are not easily adaptable to in vitro growth and are not sensitive targets for CTLs. A number of novel tumor antigens have been defined by SEREX, including two new members of the cancer–testis antigenic family, SSX2 (HOM-MEL-40) (5, 16), and NY-ESO-1 (15).

In this study, we identified a melanoma patient, NW38, with high-titered antibody against NY-ESO-1. This patient had a large and highly necrotic tumor, and the sustained release of intracellular antigens that are usually inaccessible to the immune system may account for the high NY-ESO-1 titer. The establishment of an autologous cell line that typed NY-ESO-1 positive provided target cells for assessing CTL reactivity in this patient. A CTL line was established from this patient that lysed the autologous melanoma cell line in an HLA-A2–restricted fashion. Using target cells transfected with NY-ESO-1 and HLA-A2, the specificity of CTL reactivity was found to be coded by NY-ESO-1. Computer analysis of the NY-ESO-1 sequence identified 26 peptides with HLA-A2–binding motifs. Screening of these peptides presented by T2 cells identified three sequences that were confirmed to be specifically recognized by NW38-IVS-1. This is the first conclusive demonstration of simultaneous antibody and CTL responses against a cancer–testis antigen in a single patient.

The strategy used in this study to generate and analyze CTL reactivity to a SEREX-defined antigen can be used as a model for investigating cellular immune responses to the growing list of other SEREX antigens. Identification of clones in SEREX requires high-titered IgG antibody, and the development of such antibodies requires the help of CD4⁺ T cells. In this sense, SEREX can be thought of as a method to define the CD4⁺ T cell repertoire to human tumor antigens. Also, the presence of both NY-ESO-1 antibody and CTLs in patient NW38 suggests that screening for an antibody response may be a simple and effective way to identify patients with concomitant CTL reactivity, and this possibility is now being tested in other patients with NY-ESO-1 antibody. In the absence of autologous tumor cell lines, CD8⁺ T cells can be stimulated with autologous antigen-presenting cells that have been transfected with the coding gene or fed purified protein antigens. A similar strategy can be used to identify peptide targets for CD4⁺ T cells.

A major objective in defining immunogenic human tumor targets is to explore their use in the development of cancer vaccines, and a number of clinical trials with various vaccine constructs are currently underway. Although tumor regression is the desired goal of a therapeutic vaccine, this end point cannot be expected to be an effective way to develop maximally immunogenic tumor vaccines. For this purpose, reliable immunological assays are needed to monitor the specificity and strength of specific immune reactions generated by the vaccine. With the exception of vaccines aimed at inducing a humoral immune response such as GM2 ganglioside vaccines, most vaccine trials are designed to stimulate cellular immunity, particularly the development of CTLs and CD4⁺ T cells. These have been difficult to detect in vaccine trials with MAGE peptides (25), and difficult to interpret in trials with vaccines containing melanocyte differentiation antigens, since CTLs against these antigens can be generated in vitro from nonvaccinated melanoma patients as well as normal individuals (26, 27). However, de novo induction and increase of preexisting CTL reactivity have been detected after vaccination with melanocyte differentiation antigens and observed to be associated with cancer regressions in a limited number of patients (13). The demonstration of a simultaneous antibody and CTL response to NY-ESO-1 in the same patient suggests that serological methods may be useful in monitoring vaccine trials with NY-ESO-1 and other tumor antigens eliciting a humoral immune response.

9.1.3 Monoclonal Antibodies in Cancer Therapy

R K Oldham
JCO September 1983; 1(9): 582-590
http://jco.ascopubs.org/content/1/9/582.short

The need for improved specificity in cancer therapy is apparent. With the advent of monoclonal antibodies, the possibility of specifically targeted therapy is being considered. Early trials of monoclonal antibody in experimental animals and humans have indicated its ability to traffic to specific tumor sites and to localize on or around the tumor cells displaying antigens to which the antibody is directed. This evidence of specific targeting, along with preliminary evidence of therapeutic efficacy for monoclonal antibodies and immunoconjugates with drugs, toxins, and isotopes is encouraging. The current status of clinical trials with monoclonal antibodies is reviewed and an example of the experimental approach for the development of immunoconjugates in animal models is presented.

Monoclonal Antibodies in Cancer Therapy: 25 Years of Progress

Robert K. Oldham, Robert O. Dillman
JCO Apr 10, 2008: 26(11): 1774-1777
http://dx.doi.org:/10.1200/JCO.2007.15.7438

In 1983, it was apparent that a major problem with current modalities of cancer treatment was the lack of specificity for the cancer cell.¹ It was predicted that a major advancement in treatment of cancer would be the development of a class of agents that would have a greater degree of specificity for the tumor cell. Based on many animal studies and the treatment of fewer than 100 patients, it was evident in 1983 that monoclonal antibodies would be that major advance.

The first patient treated in the United States with monoclonal antibody therapy was a patient with non-Hodgkin’s lymphoma.² Nadler et al² described the treatment using a murine monoclonal antibody designated AB 89. Although treatment was not successful in inducing a significant clinical response, it did represent the first proof of principle in humans that a monoclonal antibody could induce transient decreases in the number of circulating tumor cells, induce circulating dead cells, and form complexes with circulating antigen, all with minimal toxicity to the patient. Antibody could be detected on the surface of circulating lymphoma cells, and free antigen in the serum decreased with each infusion of antibody. After two courses of milligram doses of AB 89, a final and third course with 1.5 g of antibody was administered during a 6-hour period. A marked reduction in circulating antigen was noted, but these studies suggested to the authors that the quantity of circulating antigen was too great to effectively deliver AB 89 to the patient’s tumor cells in a therapeutically effective manner.²

In the Journal of Clinical Oncology review article cited earlier,¹ evidence was reviewed from animal tumor models that clearly demonstrated both specificity and therapeutic efficacy with little serious toxicity. Whereas passive serotherapy of human cancer had shown little success,³ it was apparent in the earlier review that monoclonal antibodies could be used in the treatment of leukemia and lymphoma.^4,5 In 1983, a review of the literature revealed approximately 10 published studies and one in-press article of therapeutic trials of monoclonal antibody therapy in humans. All of these studies used murine monoclonal antibodies and were phase I/II studies. Most were in leukemia or lymphoma, but the earliest solid tumor studies were also underway in melanoma⁶ and GI cancer.¹

By 1983, the pioneers in monoclonal antibody research believed that a new era of cancer therapy had begun, and for the first time, true specific and targeted therapy was underway using hybridoma technology to produce monoclonal antibodies with exquisite specificity. It was also apparent, based on animal model studies, that monoclonal antibodies could be a vehicle to bring immunoconjugate therapy to the clinic by conjugating monoclonal antibodies to drugs, toxins, and radioisotopes using the specificity of the monoclonal antibody to carry enhanced killing capacity directly to the tumor cells. Thus, the era of monoclonal antibody therapy, as well as immunoconjugate therapy, had begun.

Although there was much excitement (and skepticism) about this new treatment modality (the use of a form of biologic therapy with great specificity in patients with advanced cancer) there were also problems and limitations. As presented in Table 1, there were clinical toxicities with murine monoclonal antibodies, most of which were secondary to the interaction with the target antigen.⁷ However, the major limitation was their immunogenicity. Murine proteins are highly immunogenic, and it was soon found that only a few infusions of these foreign proteins could be given to patients with cancer because of the development of human antimouse antibody.⁸ Another problem quickly became apparent, in that some of the antigens on cancer cell surfaces modulated off the surface and into the circulation when antibody attached. Modulation could also cause internalization of the complex. It was recognized that this could represent a therapeutic advantage by using the antibody as carrier to internalize the toxic component of an immunoconjugate, potentially making it more therapeutically active.

In 1983, few specific antigens found only in cancer cells had been identified, and there was much debate about the specificity of these antigens. Many of the antigens to which monoclonal antibodies were made were embryonic antigens or shared antigens found on cancer cells and some normal cells. Therefore, although the specificity of the antibody was exquisite for the antigen, the specificity for the antibody or immunoconjugate for cancer was not absolute. One fairly clear exception occurred early in the 1980s when Levy et al⁹ developed monoclonal antibodies to the idiotype of B-lymphoma cells. The first patient given this anti-idiotypic antibody had a complete response to therapy, and his lymphoma went into a sustained remission that lasted for years. As a direct result of these early studies with anti-idiotypic antibodies, there is now a series of idiotype vaccines that are in phase III trials in patients with low-grade follicular lymphomas.¹⁰ These anti-idiotype vaccines will likely be the first truly custom-tailored, personalized anticancer vaccines to be approved for therapeutic use.

The major limitation of murine monoclonal antibody therapy was the immunogenicity of the mouse protein; a variety of investigators postulated that for monoclonal antibody therapy to be truly successful, human or humanized antibodies would be necessary. It was also known 25 years ago that the half-life of murine antibodies in the circulation was brief, and because of human antimouse antibody, became briefer with each infusion of murine monoclonal antibody. Previous studies of human immunoglobulin in clinical trials had demonstrated a much longer half-life for human immunoglobulin, which predicted that once human or humanized antibodies were available, the therapeutic efficacy of monoclonal antibodies and their immunoconjugates might be considerably enhanced.¹
How has the field of monoclonal antibody and immunoconjugate therapy fared since the predictions of the early 1980s? Twenty-five years later, considerable progress has been made in this field.^11,12 The US Food and Drug Administration has approved 21 monoclonal antibody products, with six of these biologic drugs approved specifically for cancer (Table 2). It was a landmark date in November 1997 when rituximab became the first monoclonal antibody approved specifically for cancer therapy.¹³ In addition to these six unconjugated monoclonal antibody therapies, one drug immunoconjugate, gemtuzumab ozogamicin (Mylotarg; Wyeth-Ayerst, Madison, NJ), has been approved. This humanized monoclonal antibody to CD33 is approved for use in acute myelogenous leukemia and uses the antibody conjugated to calicheamicin, a potent enediyene antibiotic originally isolated from aMicromonospora echoinospora.¹⁴ Two radioisotope-antibody conjugates, ytrrium-90 ibritumomab tiuxetan (Zevalin; Cell Therapeutics Inc, Seattle, WA) and iodine-131 tositumomab (Bexxar; GlaxoSmithKline, Middlesex, United Kingdom) have been approved.¹⁵ The murine form of these antibodies was retained in order to expedite clearance from the circulation. Both radiolabeled antibodies target the CD20 antigen on lymphoma cells.

Unlike the immunoconjugates, which are currently infrequently used, each of the six unconjugated antibodies approved for cancer therapy is currently frequently used in the treatment of humans with cancer. The use of techniques to humanize or chimarize monoclonal antibodies to decrease their murine components has been an important advance in the field. These molecules have a long half-life in the blood stream, and can interact with human complement or effector cells of the patient’s immune system. They behave in a manner similar to naturally occurring immunoglobulin and work along the lines of our normal antibody-based immune response as effective agents in treating patients with cancer.16

Rituximab has become the largest-selling biologic drug in clinical oncology, and is active in a variety of human lymphomas and chronic lymphocytic leukemia.17,18 This is a chimeric monoclonal antibody targeting the CD20 antigen found on both normal B cells and on most low-grade and some higher grade B-cell lymphomas. It is effective as a single agent in induction and maintenance therapy. It is primarily used, however, in combination with standard chemotherapies in the treatment of patients with non-Hodgkin’s B-cell lymphomas and chronic lymphocytic leukemia.19-22

A second monoclonal antibody that has proven highly effective in the clinic is trastuzumab, a humanized antibody that reacts with the second part of the human epidermal growth factor receptor 2.23 Like rituximab, it is effective as a single agent in induction and maintenance therapy, but is used primarily in conjunction with chemotherapy for patients with human epidermal growth factor receptor 2/neu–positive breast cancer.24,25

Alemtuzumab is a humanized monoclonal antibody targeting the CD52 antigen found on B lymphocytes and is used primarily for chronic lymphocytic leukemia.26 Like the two previously cited monoclonal antibody therapies, alemtuzumab is effective as induction and maintenance therapy. Alemtuzumab is also reactive with T lymphocytes, and unlike the other two antibodies, it is typically not combined with chemotherapy because of the increased risk of infection.(26)

Another humanized monoclonal antibody, bevacizumab, has been applied more broadly in human solid tumors because it targets vascular endothelial growth factor, which is the ligand for a receptor found on blood vessels.(27) Because this receptor is on endothelial cells, bevacizumab seems to be effective by reducing the blood supply to tumor nodules, thereby slowing or interrupting growth. Initially approved for advanced colorectal cancer,(28) it is now used in a variety of human solid tumors including cancers of the lung, kidney, and breast.(29-31)

The last two antibodies approved for clinical use were cetuximab (a chimeric antibody), and panitumumab (a completely human antibody). Both target the epidermal growth factor receptors found on a variety of human tumors.(32,33) Cetuximab was originally approved for use in combination with chemotherapy in metastatic colorectal cancer.(34) It also enhances chemotherapy and radiation therapy of squamous cell cancers of the head and neck.(35) Panitumumab was approved based on its single-agent activity in refractory colorectal cancer and is being combined with chemotherapy as well.

At the end of 2007, 25 years of clinical studies have resulted in the approval of six unconjugated, humanized, or chimeric monoclonal antibodies for cancer therapy along with one drug immunoconjugate and two radioisotope immunoconjugates. Although few in number, these monoclonal antibodies are changing the face of cancer therapy, bringing us closer to more specific and more effective biologic therapy of cancer as opposed to nonspecific cytotoxic chemicals.

Modern recombinant techniques have made it possible to rapidly produce both chimeric antibodies and humanized antibodies, and totally human antibodies are also being produced. Identification of surface receptors that are integral to proliferation and apoptosis has provided more targets for monoclonal antibodies beyond those originally identified by the murine immune system. In 2008, there are more than 100 monoclonal antibody–based biologic drugs in hundreds of clinical trials. Many of these are in phase II and phase III and will be coming before the US Food and Drug Administration for approval in the next few months and years. At long last, immunoconjugates are proving efficacious with acceptable toxicity and will extend our diagnostic (36) and therapeutic armamentarium (37) from mainly unconjugated monoclonal antibodies to a broad array of highly active and specific immunoconjugates.

On this silver anniversary for our 1983 review, “Monoclonal Antibodies in Cancer Therapy, ” we can confidently predict that progress toward more specific and less toxic therapy for human cancer is in our near future. The developments during the past 25 years in both biologic drugs and targeted small molecules place us on the verge of more cures with less toxicity for our patients with cancer.

9.1.4 Aptamers

Nanocarriers as an emerging platform for cancer therapy

Dan Peer^1,7, Jeffrey M. Karp^2,3,7, Seungpyo Hong, et al.
Nature Nanotechnology 2, 751 – 760 (2007)
http://dx.doi.org:/10.1038/nnano.2007.387

Nanotechnology has the potential to revolutionize cancer diagnosis and therapy. Advances in protein engineering and materials science have contributed to novel nanoscale targeting approaches that may bring new hope to cancer patients. Several therapeutic nanocarriers have been approved for clinical use. However, to date, there are only a few clinically approved nanocarriers that incorporate molecules to selectively bind and target cancer cells. This review examines some of the approved formulations and discusses the challenges in translating basic research to the clinic. We detail the arsenal of nanocarriers and molecules available for selective tumor targeting, and emphasize the challenges in cancer treatment.

Quantum Dot−Aptamer Conjugates for Synchronous Cancer Imaging, Therapy, and Sensing of Drug Delivery Based on Bi-Fluorescence Resonance Energy Transfer
Vaishali Bagalkot, L Zhang, E Levy-Nissenbaum, S Jon, PW Kantoff, et al.
Nano Letters 2007; 7(10):3065-3070
http://dx.doi.org:/10.1021/nl071546n

We report a novel quantum dot (QD)−aptamer(Apt)−doxorubicin (Dox) conjugate [QD−Apt(Dox)] as a targeted cancer imaging, therapy, and

sensing system. By functionalizing the surface of fluorescent QD with the A10 RNA aptamer, which recognizes the extracellular domain of the prostate specific membrane antigen (PSMA), we developed a targeted QD imaging system (QD−Apt) that is capable of differential uptake and imaging of prostate cancer cells that express the PSMA protein. The intercalation of Dox, a widely used antineoplastic anthracycline drug with fluorescent properties, in the double-stranded stem of the A10 aptamer results in a targeted QD−Apt(Dox) conjugate with reversible self-quenching properties based on a Bi-FRET mechanism. A donor−acceptor model fluorescence resonance energy transfer (FRET) between QD and Dox and a donor−quencher model FRET between Dox and aptamer result when Dox intercalated within the A10 aptamer. This simple multifunctional nanoparticle system can deliver Dox to the targeted prostate cancer cells and sense the delivery of Dox by activating the fluorescence of QD, which concurrently images the cancer cells. We demonstrate the specificity and sensitivity of this nanoparticle conjugate as a cancer imaging, therapy and sensing system in vitro.

Semiconductor nanocrystals known as quantum dots (QDs)

have been increasingly utilized as biological imaging and labeling probes because of their unique optical properties, including broad absorption with narrow photoluminescence spectra, high quantum yield, low photobleaching, and resistance to chemical degradation. In some cases, these unique properties have conferred advantages over traditional fluorophores such as organic dyes.^1-4 The surface modification of QDs with antibodies, aptamers, peptides, or small

molecules that bind to antigens present on the target cells or tissues has resulted in the development of sensitive and specific targeted imaging and diagnostic modalities for in vitro and in vivo applications.^5-7 More recently, QDs have been engineered to carry distinct classes of therapeutic agents for simultaneous imaging and therapeutic applications.^8,9While these combined imaging therapy nanoparticles represent an exciting advance in the field of nanomedicine, it would be ideal to engineer “smart” multifunctional nanoparticles that are capable of performing these tasks while sensing the delivery of drugs in a simple and easily detectable manner. One way to achieve this goal is to develop multifunctional nanoparticles capable of sensing the release of the therapeutic modality by a change in the fluorescence of the imaging modality.

Figure 1. (a) Schematic illustration of QD-Apt(Dox) Bi-FRET system. In the first step, the CdSe/ZnS core-shell QD are surface functionalized with the A10 PSMA aptamer. The intercalation of Dox within the A10 PSMA aptamer on the surface of QDs results in the formation of the QD-Apt(Dox) and quenching of both QD and Dox fluorescence through a Bi-FRET mechanism: the fluorescence of the QD is quenched by Dox while simultaneously the fluorescence of Dox is quenched by intercalation within the A10 PSMA aptamer resulting in the “OFF” state. (b)

Schematic illustration of specific uptake of QD-Apt(Dox) conjugates into target cancer cell through PSMA mediate endocytosis. The release of Dox from the QD-Apt(Dox) conjugates induces the recovery of fluorescence from both QD and Dox (“ON” state), thereby sensing the intracellular delivery of Dox and enabling the synchronous fluorescent localization and killing of cancer cells.

Figure 3. Fluorescence spectra. (a) QD-Apt conjugate (1 µM) with increasing molar ratio of Dox (from top to bottom: 0, 0.1, 0.3, 0.6, 1, 1.5, 2.1, 2.8, 3.5, 4.5, 5.5, 7, and 8) at an excitation of 350 nm. (b) Dox (10 µM) with increasing molar ratio of QD-Apt conjugate (from top to bottom: 0.02, 0.04, 0.07, 0.09, 0.12, 0.14, and 0.16) at an excitation of 480 nm.

In conclusion, herein we report to our knowledge the first example of a multifunctional nanoparticle that can detect cancer cells at a single cell level while intracellularly releasing a cytotoxic dose of a therapeutic agent in a reportable manner. We demonstrate the specificity and sensitivity of this cancer imaging, therapy and sensing nanoparticle conjugate system in vitro by using PCa cell lines. By functionalizing the surface of fluorescent QD with the A10 PSMA aptamer, and intercalating Dox into the double-stranded CG sequence of the A10 PSMA aptamer, we developed a targeted QD-Apt(Dox) conjugate with reversible Bi-FRET properties. The incorporation of multiple CG sequences within the stem of the aptamers may further increase the loading efficiency of Dox on these conjugates. The presence of additional Dox may enhance the selfquenching effect of QD-Apt(Dox) conjugates thereby improving their imaging sensitivity, while the higher dose of Dox may enhance the therapeutic efficacy of the conjugates. Furthermore, through the use of other disease-specific aptamers or other targeting molecules, similar multifunctional nanoparticles may potentially be developed for additional important medical applications

Oligonucleotide Aptamers: New Tools for Targeted Cancer Therapy

Hongguang Sun¹, Xun Zhu², Patrick Y Lu³, Roberto R Rosato, et al.
Molecular Therapy Nucleic Acids(2014) 3, e182;
http://dx.doi.org:/10.1038/mtna.2014.32

Aptamers are a class of small nucleic acid ligands that are composed of RNA or single-stranded DNA oligonucleotides and have high specificity and affinity for their targets. Similar to antibodies, aptamers interact with their targets by recognizing a specific three-dimensional structure and are thus termed “chemical antibodies.” In contrast to protein antibodies, aptamers offer unique chemical and biological characteristics based on their oligonucleotide properties. Hence, they are more suitable for the development of novel clinical applications. Aptamer technology has been widely investigated in various biomedical fields for biomarker discovery, in vitro diagnosis, in vivo imaging, and targeted therapy. This review will discuss the potential applications of aptamer technology as a new tool for targeted cancer therapy with emphasis on the development of aptamers that are able to specifically target cell surface biomarkers. Additionally, we will describe several approaches for the use of aptamers in targeted therapeutics, including aptamer-drug conjugation, aptamer-nanoparticle conjugation, aptamer-mediated targeted gene therapy, aptamer-mediated immunotherapy, and aptamer-mediated biotherapy.

The terms “aptamer” and “SELEX” were introduced by two independent groups in 1990.^1,2 The term “aptamer” refers to small nucleic acid ligands that exhibit specific therapeutic functions and an unambiguous binding affinity for their targets. Conversely, Systematic Evolution of Ligands by EXponential enrichment (SELEX) technology is the method used for aptamer development. Although using small molecule nucleic acids as therapeutics has been explored for decades, development of SELEX and aptamer technology revolutionized this field.

The most important property of an aptamer, from the Latin aptus (to fit), is its high target selectivity. These short, chemically synthesized, single-stranded (ss) RNA or DNA oligonucleotides fold into specific three-dimensional (3D) structures with dissociation constants usually in the pico- to nano-molar range.³ Moreover, in contrast to other nucleic acid molecular probes, aptamers interact with and bind to their targets through structural recognition (Figure 1), a process similar to that of an antigen-antibody reaction. Thus, aptamers are also referred to as “chemical antibodies.”

Figure 1.

Schematic diagram of aptamer binding to its target.

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Due to their small size and oligonucleotide properties, aptamers offer several advantages over protein antibodies in both their extensive clinical applicability and a less challenging industrial synthesis process. Specifically, (i) aptamers can penetrate tissues faster and more efficiently due to their significantly lower molecular weight (8–25 kDa aptamers versus ~150 kDa of antibodies). Therefore, aptamers penetrate tissues barriers and reach their target sites in vivo more efficiently than the larger-sized protein antibodies. (ii) Aptamers are virtually nonimmunogenic in vivo. In principal, as aptamers are oligonucleotides they should not be recognized by the immune system. In practice, a recent clinical study showed that aptamers did not stimulate an immune response in vivo,^4,5 as compared to protein antibodies that are highly immunogenic, especially following repeat injections. (iii) Aptamers are thermally stable. Based on the intrinsic property of oligonucleotides, even after a 95 °C denaturation, aptamers can refold into their correct 3D conformations once cooled to room temperature. In comparison, protein-based antibodies permanently lose their activity at high temperatures. More importantly, a well-established synthesis protocol and chemical modification technology lead to (iv) rapid, large-scale aptamer synthesis and modification capacity that includes a variety of functional moieties; (v) low structural variation during chemical synthesis; and (vi) have lower production costs. Moreover, aptamers specifically recognize a wide range of targets, such as ions, drugs, toxins, peptides, proteins, viruses, bacteria, cells, and even tissues.^{6,7,8,9,10,11,12} In the clinic, aptamer-based therapeutics are gaining momentum. For example, Macugen, a modified RNA aptamer, specifically targets vascular endothelial growth factor. It has been approved by the US Food and Drug Administration (FDA)¹³ for the treatment of wet age-related macular degeneration and is under evaluation for other conditions.¹⁴ In the cancer setting, AS1411 targets nucleolin, a protein over-expressed in a variety of tumors. It is currently being evaluated as a potential treatment option in solid tumors and acute myeloid leukemia.¹⁵ An updated list of therapeutic aptamers undergoing clinical trials is included in ref. 16 and Table 1. Taken together, these clinical studies highlight many possible uses that aptamers may have in a variety of biomedical fields, including therapeutics.¹⁷
Table 1 – A list of therapeutic aptamers undergoing clinical trials.

Since aptamer technology was first introduced, the RNA-based sequence library has been widely used for SELEX. Based on the existing evidence, it is believed that the presence of a 2′-OH group and non-Watson-Crick base pairing allows RNA aptamer oligonucleotides to fold into more diverse 3D structures than ssDNA molecules. Consequently, using the more flexible RNA sequences simplifies the development of high-affinity and -specificity aptamers. Despite their advantages, RNA sequences are very sensitive to nucleases present in biological environments and can be rapidly degraded.¹⁸ To increase nuclease resistance of RNA-based aptamers, several chemical modifications have been investigated. Evidence shows that 2′-OH group and phosphodiester linkages of RNA sequences are the sites of nuclease hydrolysis. Subsequently, substitutions of the 2′-OH functional group by 2′-fluoro, 2′-amino, or 2′-O-methoxy motifs, and/or changes to the phosphodiester backbone with boranophosphate or phosphorothioate are the most common modifications aimed at increasing nuclease resistance.¹⁹ More recently, Wu et al. developed a novel chemical modification method to increase siRNA stability, in which phosphorodithioate and 2′-O-Methyl were simultaneously substituted in the same nucleotide.²⁰
This modification method significantly enhanced siRNA stability and represents a potential new direction for utilization of RNA-based therapies in complex biological systems. Other effective modifications recently reported utilize the locked nucleic acid technology^16,21 or generate “mirror” RNA sequence structures, termed spiegelmers.²² These modifications result in structural changes to the RNA sequences, which cannot be digested by nucleases.

In addition to RNA aptamers, ssDNA-based aptamers have also been developed. Due to their lack of 2′-OH groups, DNA molecules are naturally resistant to 2′-endonucleases and are stable in biological environments. Recently, our group developed a biostable DNA-based aptamer specific for CD30, a protein biomarker that is over-expressed in Hodgkin and anaplastic large cell lymphomas. Functional analysis demonstrated that this ssDNA-based aptamer exhibited high CD30 binding affinity as low as 2 nmol/l and was stable in human serum for up to 8 hours. Conversely, an RNA-based CD30 aptamer was digested within 10 minutes under similar conditions.²³
In summary, unique chemical features and biological functions have made aptamers a very attractive tool in biomedical research over the past two decades. Currently, there are over 4,000 published articles referenced in the PubMed database that include the term “aptamer.” Research areas that include aptamer technology cover bioassays, drug development, cell detection, tissue staining, in vitro and in vivo imaging, nanotechnology, and targeted therapy. As chemical antibodies, aptamers represent an excellent alternative to replace or supplement protein antibodies, which have been extensively used in the clinic.

Aptamers Specifically Targeting Cell Surface Biomarkers

Using SELEX technology to develop aptamers for cell surface biomarkers

SELEX, the methodology used to develop aptamers specific for a target of interest, is based on a repetitive amplification and enrichment process. The SELEX process follows several steps: first, a random ssDNA oligonucleotide library is chemically synthesized to contain between 10¹⁴–10¹⁵ unique random sequences flanked by conserved primer binding sites. This step utilizes the following universal scheme: 5′-sense primer sequence-(random sequence)-antisense primer sequence-3′, where the primer sequence ranges from 18 to 22 bases and the random sequence contains 20–40 nucleic acids. The general procedure consists of labeling the 5′-sense primer with a fluorochrome reporter for monitoring aptamer selection, while the 3′-antisense primer is labeled with an affinity molecule, such as biotin, that is used to separate single-stranded oligonucleotides generated in each amplification round. This random ssDNA library can be used directly to select an initial pool of DNA aptamers. Conversely, generation of RNA aptamers requires two extra steps. Specifically, a pool of random ssDNA oligonucleotides is generated, T7 RNA polymerase promoter sequence is added to the 5′-sense primer, and the DNA is then used as a template for T7 RNA polymerase-based transcription in the 5′ to 3′ direction. During the second SELEX step, the oligonucleotide library is heated and rapidly cooled to promote the formation of 3D structures. The library is then mixed with the target of interest for specific binding enrichment. In the third step, the unbound sequences are discarded through the use of membranes, columns, magnetic beads, and capillary electrophoresis.^6,24,25 In the fourth step, the enriched sequences are amplified in vitro by either PCR (DNA aptamers) or RT-PCR (RNA aptamers) to generate a new sequence library for the next round of SELEX. The amplified sequence library may go through further negative-target selection, which eliminates the nonspecific sequences generated by binding of nontarget moieties. Lastly, aptamer selection goes through 4–20 rounds of amplification and enrichment. The exact number of required amplification and selection steps depends on the aptamer target being a purified protein or a living cell, and on the evolution of the aptamer sequence library, as that established by gel electrophoresis, flow cytometry (for target binding), classical cloning or sequencing methods, or by high throughput Next-Generation Sequencing (NGS). In recent years, the traditional SELEX method had also been modified to include the capillary electrophoresis (CE) SELEX, toggle selection, photo-SELEX, bead-based selection, X-Aptamers, and Slow Off-rate Modified Aptamers (SOMAmers) in order to maximize affinity and specificity, to improve the speed of selection and success rate, and to provide additional properties to the selected aptamers.^{26,27,28,29,30,31}

Similar to protein antibody development, purified recombinant proteins or peptides expressed in prokaryotic or eukaryotic systems can be used as targets for aptamers selected by the SELEX method. However, because of the posttranslational modifications, especially in the case of highly glycosylated proteins, purified proteins or peptides often cannot fold into the correct 3D structure that is formed under physiologic conditions.³² Consequently, the newly synthesized aptamers may not be able to selectively recognize and interact with their corresponding targets, which would result in failure of the biomedical application. As this is a common problem, it is very important to choose biomarkers in their native conformation for aptamers selection. Taking this issue into an account, a modified SELEX technology that uses whole living cells, Cell-based SELEX (or Cell-SELEX), was recently established.³³ To develop cell-specific aptamers, the Cell-SELEX method uses whole living cells that express surface biomarkers of interest. However, the presence of many different cell surface molecules in addition to the target biomarker(s) results in the synthesis of many unrelated/unwanted aptamers. Therefore, in addition to all the SELEX steps described above, Cell-SELEX technology also utilizes control cells that do not express the target biomarker(s) during the counter-selection step.³³
Well-characterized biomarkers that are endogenously expressed at high levels, such as the ErbB superfamily, MUC1, EpCAM, and CD30, offer the best potential for cell-based aptamer development. Subsequently, cell lines that have high endogenous expression of cell-specific or cancer type-specific biomarker(s) are commonly used for Cell-SELEX. However, if such cell lines are unavailable, a biomarker of interest could be over-expressed in a particular cell line via gene transfection and the parental cells used for counter-selection. Using this approach, aptamers targeting the cancer stem cell (CSC) biomarker CD133 have been recently developed.³⁴ In this study, CD133 cDNA was transfected into HEK293T cells that were then used for aptamer enrichment, with the parental HEK293T cells serving as a negative control. Similarly, an aptamer specific for the human receptor tyrosine kinase was recently developed.³⁵

Figure 2.

Schematic diagram of our hybrid-SELEX method for selection of CD30-specific ssDNA aptamer. In our experiment, the hybrid-SELEX process is divided into (a) the cell-based SELEX selection and (b) CD30 protein-based SELEX enrichment. First, CD30-expressing lymphoma cells are used for positive selection and CD30-negative Jurkat cells are used in negative counter-selection. After 20 rounds of selection, the enriched aptamer pool is incubated with CD30 protein immobilized on magnetic beads for five additional rounds of enrichment. SELEX, Systematic Evolution of Ligands by EXponential enrichment.

Full figure and legend (183K)

Aptamers specific for cell surface biomarkers

Cell surface biomarkers are functionally important molecules involved in many biological processes, such as signal transduction, cell adhesion and migration, cell–cell interactions, and communication between the intra- and extra-cellular environments. An abnormal expression of cell surface biomarkers is often related to tumorigenesis.⁵⁰ Clinically, it is estimated that about 60% of cancer-targeting drugs, including therapeutic antibodies and small molecule inhibitors, target cell surface biomarkers,⁵¹ making them attractive for disease treatment. In the last decade, many aptamers targeting cell surface biomarkers have been developed through the advancement of both the protein- and/or cell-based SELEX technologies (see Table 2 for detailed list). These aptamers have been extensively studied for diagnosis and/or treatment of hematological malignancies,^7,23,49 lung,^52,53,54 liver,⁵⁵ breast,^56,57 ovarian,⁵⁸ brain,^59,60colorectal,⁶¹ and pancreatic cancers,⁴⁶ as well as for identification and characterization of CSCs.^34,62

Aptamer-Mediated Targeted Therapies

Traditional cancer treatment approaches, such as chemotherapy, radiotherapy, photodynamic therapy, and photothermal therapy can cause serious side effects in patients due to their associated nonspecific toxicity. To minimize these side effects, a concept of personalized, targeted therapy has been gaining momentum. One of the main clinical approaches for targeted cancer therapy employs antibody-based drugs. Although antibody-mediated therapy is highly specific and results in fewer side effects, potential immunogenicity and high cost of production may limit its clinical applications. To overcome these obstacles, oligonucleotide aptamer-based targeted therapeutics and specific drug delivery systems have recently been explored. These studies revealed numerous advantages offered by the aptamer technology over protein-based antibody therapies, with some of these described in the section below.
Aptamer-drug conjugates

Aptamer-drug conjugation (ApDC) is a very simple yet effective model of noncovalently or covalently conjugating aptamer sequences directly with therapeutic agents (Figure 3). For example, aptamer-conjugated Doxorubicin (Dox), a chemotherapeutic agent extensively used in the treatment of various cancers, has recently been shown to have enhanced therapeutic efficacy over Dox alone. Mechanistically, Dox cytotoxicity is caused by its intercalation into the nucleic acid structure at the preferred paired CG or GC sites with subsequent inhibition of cancer cell proliferation. Taking advantage of its propensity for intercalation, Dox can be noncovalently conjugated to oligonucleotide aptamers containing CG/GC sequences through a simple incubation step. A recent report by Subramanian et al. describes the effectiveness of aptamer-Dox conjugates in the treatment of retinoblastoma.⁶³ In their study, a 2′-fluoro modified RNA aptamer EpDT3 (specific for EpCAM, a CSC marker), was noncovalently conjugated with Dox. After binding to EpCAM molecules expressed at the cancer cell surface, the EpDT3-Dox conjugates were preferentially internalized by the cancer and not by the healthy cells, greatly enhancing therapeutic efficacy and reducing treatment-associated side effects. Several other studies also utilized aptamer-Dox conjugates for cancer therapy, such as HER2 aptamer-Dox conjugates targeting breast cancer,⁶⁴ MUC1 aptamer-Dox conjugates targeting lung cancer,⁶⁵ and PSMA aptamer-Dox conjugates targeting prostate cancer.⁶⁶ Despite their obvious advantages, several concerns related to the use of aptamer-Dox conjugate have been raised. These include (i) instability of the aptamer-drug conjugate due to the reversible nature of noncovalent conjugation process; (ii) short circulating half-life of aptamer-drug conjugates in vivo due to their low molecular weight; and (iii) poor drug payload capacity due to a very simple structure of aptamers. These three disadvantages and technological approaches to improve them are described in greater detail below.

Figure 3.

Schematic diagram of noncovalent or covalent aptamer-drug conjugation.

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To enhance the stability of drug loading, Dox can be covalently conjugated to aptamer sequences via a functional linker moiety. For example, the DNA aptamer sgc8 possesses a strong affinity for PTK7 kinase that is abundantly expressed on the surface of CCRF-CEM T-cell acute lymphoblastic leukemia cells. To enhance its stability, this aptamer was covalently conjugated with Dox through an acid-labile linker.⁶⁷ Once the sgc8 aptamer-Dox conjugate was preferentially bound and internalized by the target cells, the acid-labile linker was easily cleaved in the acidic lysosomal environment, releasing Dox and effectively killing target cells.⁶⁷ On the other side of the spectrum, covalent conjugation is the most commonly used method of aptamer-drug conjugation, especially for agents that cannot intercalate into the nucleic acid structure or whose intercalation would disrupt aptamer structure.⁶⁸ Evidence suggests that these covalently conjugated aptamer-drug compounds are significantly more stable than the corresponding noncovalently conjugated intercalations.⁶⁹

Conjugation of aptamers with high molecular weight polymers, such as polyethylene glycol (PEG), has been examined in order to increase aptamer molecular weight. Specifically, PEG has been widely used in drug modifications, including synthesis of Macugen aptamers. This modification, resulting in PEGylated aptamers, not only increased the aptamer molecular weight and prolonged its circulating half-life, but also enhanced its stability and decreased its toxic accumulation in nontarget tissues.^70,71

Finally, in order to increase aptamer-drug payload capacity, an innovative model named aptamer-tethered DNA nanotrains (aptNTrs) was recently introduced by Zhu et al. to deliver Dox to cancer cells.⁷² In this study, structure of the sgc8 aptamer that targets PTK7 was modified by adding a DNA trigger probe on the 5′-end. Consequently, the modified aptamer acted as a locomotive for targeting, while two hairpin monomers containing Dox intercalation sites acted as boxcars to deliver the drug. After self-assembly, the newly synthesized sgc8 aptamer-NTrs displayed high drug payload capacity, with the drug/sgc8 aptamer-NTr molar ratio of 50:1. Importantly, sgc8 aptamer-NTrs-Dox conjugates were preferentially internalized by the target cells, thereby inhibiting tumor cell growth in vitro and in vivo.⁷²

Another strategy for increasing the aptamer payload capacity involves the construction of polyvalent aptamers. Polyvalent aptamers exhibit an increased target affinity and are more rapidly internalized by their target cells. To demonstrate this, Boyacioglu et al. developed a new DNA aptamer they termed SZTI₀₁ against PSMA.⁶⁹ First, a dimeric aptamer complex (DAC) was created for specific delivery of Dox to PSMA-expressing cancer cells. Then, the SZTI₀₁aptamer was modified on the 3′-terminus with either a dA16 or dT16 single-stranded tail that contained CpG sites for loading Dox, and the two monomers were annealed in a 1:1 ratio to form the DAC structure. The results of the study showed that DACs have a high Dox payload capacity with the Dox/DAC molar ratio of about 4:1, and the DACs-Dox conjugates were stable under physiological conditions for up to 8 hours.⁶⁹ In another study, a DNA aptamer targeting MUC1 was truncated and an aptamer containing three repeats of the active targeting region, termed L3, was synthesized. Although the Dox payload capacity was not specifically modified in the L3 aptamer, the L3-Dox conjugates showed a stronger affinity to target cells and lower cytotoxicity to off-target cells than the parental MUC1 aptamer.⁷³ Finally, polyvalent aptamers can also be constructed through the rolling circle amplification (RCA) technology. Using the RCA method and the sgc8 aptamer sequence as a circular template, a polyvalent sgc8 aptamer, termed Poly-Aptamer-Drug, was synthesized.⁷⁴ It was determined that the Dox payload capacity of the polyvalent sgc8 aptamer increased tenfold, as compared to the monovalent sgc8 aptamer. Moreover, because of their 40-fold greater binding affinity, the Poly-Aptamer-Drug conjugates were more effective than their monovalent counterparts in targeting and killing leukemia cells.⁷⁴

Although Dox presents itself as a very attractive chemotherapeutic agent for use in aptamer conjugation, other drugs, such as Gemcitabine (Gem) and photosensitizers, can also be targeted to cancer cells through the aptamer technology. Gem is an FDA-approved deoxycytidine analog (dFdC) used for anticancer therapy. To deliver Gem specifically to pancreatic cancer cells, Ray et al. developed a novel aptamer-Gem polymer model. In this model, a single-stranded RNA polymer contained Gem that was enzymatically synthesized through a mutant T7 RNA polymerase-mediated transcription reaction and fused with a nuclease-resistant 2′-fluoro-modified RNA aptamer (E07) that selectively binds to EGFR on pancreatic cancer cells. The E07 aptamer structure was modified by introducing a 24-nucleotide sequence at the 3′ end and using it as an adaptor for Gem polymer binding. Following an annealing step, the Gem polymer complementary bound with the E07 aptamer and preferentially targeted the EGFR-expressing pancreatic cancer cells, inhibiting cell proliferation.⁷⁵

Compared with the traditional chemotherapeutic agents, controlled conditional prodrug photosensitizers have also been extensively used for aptamer-mediated drug delivery. In this therapeutic approach, termed photodynamic therapy, or photodynamic therapy, photosensitizers are activated by light irradiation and induce production of intracellular reactive oxygen species, resulting in cytotoxicity. A study by Ferreira et al. describes the development of a DNA aptamer specific for MUC1 and covalently conjugated at the 5′ end with the photosensitizer chlorin e6.⁷⁶ Upon light irradiation, MUC1-expressing epithelial cancer cells were preferentially killed with cytotoxicity about 500-fold higher than that of the control cells. Similar studies have reported using a necleolin aptamer (AS1411)-TMPyP4 for targeting breast cancer⁷⁷ and the EGFR aptamer (R13)-TF70 for treatment of lung cancer.⁷⁸

Finally, approaches to extend the scope of aptamer application have also been developed. Similar to bi-specific antibodies, bi-specific or even tri-specific aptamers can be constructed. A bi-specific aptamer for targeting different cells was recently described by Zhu et al. In their study, specific DNA aptamers sgc8 and sgd5a were conjugated through a dsDNA linker. Compared to each mono-aptamer, this bi-specific aptamer (named SD) could recognize its target cell simultaneously with equal specificity and affinity, while Dox intercalation into the dsDNA induced target cell cytotoxicity.⁷⁹ In the same study, a Y-shape dsDNA linker was used to construct a tri-specific aptamer that also recognized its target cells with high specificity and affinity.⁷⁹ Clinically, Min et al. proposed using a bi-specific aptamer for prostate cancer therapy. It is well established that prostate tumors may contain both PSMA-positive and -negative cell types. Thus, this study utilized two aptamers, a 2′-fluoro modified RNA aptamer targeting PSMA-expressing cells and a DUP-1 peptide aptamer specific to PSMA-negative cells, conjugated through streptavidin. Moreover, intercalating Dox into the PSMA aptamer of this bi-specific aptamer model could serve as a tool to target all prostate cancer cell types.⁸⁰

Aptamer-nanoparticle therapeutics

Nanoparticles (NPs) are attractive vehicles to increase both the half-life and the drug payload capacity of aptamer-mediated drug delivery. In addition to their common features, such as biocompatibility for clinical applications, large surface for enhanced aptamer and drug loading, and uniform size and shape for excellent biodistribution, NPs have other individual physical and chemical properties defined by their materials. For example, copolymers and liposomes are biodegradable, while metal materials offer exceptional photothermal and magnetic performance.

Conclusion

Antibody-based targeted therapeutics provide high target specificity and affinity. However, their potential for immunogenicity is of a great concern, as is their high production cost, both of which have limited their clinical applicability. As discussed in this review, when compared to protein antibodies, oligonucleotide aptamers offer many advantages, including simple chemical synthesis, virtual nonimmunogenicity, smaller size, faster tissue penetration, ease of modification with different functional moieties, low cost of production, and high biological stability. Therefore, aptamers have become a promising new class of molecular ligands that could replace or supplement protein antibodies. In summary, aptamer technology has a strong market value and may be applied in various biomedical fields, including in vitro cancer cell detection, in vivo tumor imaging, and targeted cancer therapy (Figure 7).

Figure 7.

Summary of various aptamer applications.

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Although aptamer technology has a great potential in the biomedical field, several technical challenges remain and must be addressed. These include: (i) how can aptamers be rapidly adapted for specific targets by decreasing false-positive/-negative selection? Primarily dependent on the natural properties of targets of interest, such as proteins versus cells or tissues, the process of aptamer selection is usually time-consuming, and the success rate is sometimes low. To improve the speed and success rate, novel methods for aptamer selection have been recently described. They include bead-based selection, that can select aptamers as rapidly as a single round of selection,^27,28 and the SOMAmer, which improves the aptamer production success rate from less than 30% to over 50%.^29,30 More recently, a study by Cho et al. devised a Quantitative Parallel Aptamer Selection System (QPASS) method, which integrates microfluidic selection, NGS, and in situ-synthesized aptamer arrays. This approach allows for the simultaneous measurement of affinity and specificity for thousands of candidate aptamers in parallel.¹¹⁶ In addition to QPASS, evolving modifications to the Cell-SELEX approach are beginning to address difficulties with successful removal of the influence stemming from the presence of dead cells, slow enrichment aptamers recognizing targets of interest, and contamination with unwanted aptamer sequences. As described above, utilization of the above-mentioned FACS-mediated SELEX^44,45 and hybrid-SELEX²³ offers novel approaches that address these technical challenges.

(ii) How can we select cancer-relevant targets for aptamer development and clinical applications? Tumorigenesis is a dynamic process that includes multiple constantly changing factors. Therefore, a one-size-fits-all cancer-specific biomarker is unlikely to ever be identified. Yet, it has been established that certain biomarkers present in healthy tissues are highly expressed in cancer cells. Moreover, certain biomarkers are associated with particular cancer cell types making them to be considered as useful targets for development of targeted cancer therapy. However, while use of cancer cells to identify biomarkers and to develop therapeutic agents is a reasonable approach, cultured cells, especially immortalized cell lines, greatly differ from tumor tissues in vivo. To overcome these limitations and to select more reliable cancer-relevant biomarkers for aptamer development, several innovative SELEX methods have been recently described. Of particular interest are the tissue-based SELEX¹¹⁷ and the in vivo-SELEX,¹¹⁸ which offer target selection under more relevant pathologic conditions. This cell/tissue-specific biomarker selection can also be utilized for development of noncancer related therapies, as shown for aptamers targeting the adipose tissue in obesity¹¹⁹ and for aptamers designed to penetrate the blood-brain barrier in order to combat brain diseases.¹²⁰ Hence, we believe that the careful selection of cancer-associated biomarkers and cell/tissue type-specific biomarkers will expand the scopes of aptamer applicability and improve the feasibility of clinical applications.

(iii) What methods could improve aptamer biostability in vivo? Unmodified RNA-based aptamers are very susceptible to the nuclease-mediated degradation in vivo. Although many chemical modifications aimed at increasing biostability of the RNA aptamers have been developed, including 2′-modifications, 3′-modifications, phosphodiester backbone modifications,^19,20 and utilizations of novel nucleic acids (locked nucleic acid and Spiegelmers),^16,21,22 their effectiveness is still limited. When it was first described, PEGylation was a very attractive strategy for prolonging aptamer circulation half-life and enhancing their biostability. However, a recent report showed that the in vivo use of PEGylated aptamers induced production of anti-PEG antibodies,¹²¹ emphasizing the need for the development of alternative approaches.

(iv) How can aptamer technology be modified to achieve a more effective drug delivery? Many drug delivery systems described in this review are tested in vitroor in animal models. Yet, as with any compound that is translated from the bench to the bedside, aptamer-drug conjugates may behave differently in a human patient than they do in laboratory animals. Therefore, aptamer-drug conjugation remains an important challenge that must be considered. Specifically, various coupling approaches lead to different pharmacokinetics, biodistribution, and tolerability in vivo, which in turn greatly affect treatment effectiveness. In the same vein, we must consider the effectiveness of aptamer-mediated target gene therapy. Gene therapy, including siRNA and miRNA aimed at silencing specific genes, is considered the next generation therapeutic approach. However, silencing a single pathogenic gene may not be a viable therapeutic option because tumorigenesis is a process regulated by multiple genes and signaling pathways. Therefore, combining targeted therapeutics with gene therapy may represent the most effective strategy. Such combinational therapy approaches can greatly improve the therapeutic efficacy while reducing the required dosages of both drugs and small molecule RNAs,¹²² and, more importantly, may offer new alternatives to combat chemotherapy-resistant cancers.¹¹⁰

(v) The last important point to consider is whether aptamer-mediated biotherapies can become effective, FDA-approved medications. Following Macugen approval by the FDA, many aptamer-mediated biotherapies have been evaluated in clinical trials. Of particular interest is AS1411, an antitumor aptamer that has completed several Phase I clinical trials.¹⁵ Trial results are promising and offer useful insights into further modifications that could be applied to therapeutic aptamer development.

Taken together, although some technical challenges remain to be addressed, oligonucleotide aptamers have become an attractive and promising tool for targeted cancer therapy. As more clinical data are accumulated, we and others will be better equipped to optimize aptamer formulations, leading to the expansion of aptamer use in the clinic.

9.1.5 Tumor Suppressors

Intrinsic Disorder in PTEN and its Interactome Confers Structural Plasticity and Functional Versatility
Prerna Malaney, Ravi R Pathak, Bin Xue, VN Uversky & Vrushank Davé
Scientific Reports 20 June 2013; 3(2035)
http://dx.doi.org:/10.1038/srep02035

IDPs, while structurally poor, are functionally rich by virtue of their flexibility and modularity. However, how mutations in IDPs elicit diseases, remain elusive. Herein, we have identified tumor suppressor PTEN as an intrinsically disordered protein (IDP) and elucidated the molecular principles by which its intrinsically disordered region (IDR) at the carboxyl-terminus (C-tail) executes its functions. Post-translational modifications, conserved eukaryotic linear motifs and molecular recognition features present in the C-tail IDR enhance PTEN’s protein-protein interactions that are required for its myriad cellular functions. PTEN primary and secondary interactomes are also enriched in IDPs, most being cancer related, revealing that PTEN functions emanate from and are nucleated by the C-tail IDR, which form pliable network-hubs. Together, PTEN higher order functional networks operate via multiple IDP-IDP interactions facilitated by its C-tail IDR. Targeting PTEN IDR and its interaction hubs emerges as a new paradigm for treatment of PTEN related pathologies.

The concept of “Intrinsic Disorder” in proteins has rapidly gained attention as the preponderance and functional roles of IDPs are increasingly being identified in eukaryotic proteomes^1, 2. Structured proteins adopt energetically stable three-dimensional conformations with minimum free energy. In contrast, IDPs, due to their unique amino acid sequence arrangements, cannot adopt energetically favorable conformations and, thus, lack stable tertiary structure in vitro³. This structural plasticity allows IDPs to operate within numerous functional pathways, conferring multiple regulatory functions^4, 5, 6. Indeed, mutations in and dysregulation of IDPs are associated with many diseases including cancer^1, 6, 7, signifying that IDPs play vital roles in functional pathways. Evidence suggests that ~80% of proteins participating in processes driving cancer contain IDRs⁶. For example, tumor suppressor p53 as an IDP, functions via its C-terminal IDR, which simultaneously exists in different conformations, each of which function differently¹. Since PTEN is the second most frequently mutated tumor suppressor with versatile functions⁸, we hypothesized that PTEN may contain IDR(s) that can be exploited for therapeutic targeting in cancers and diseases associated with pathogenic PI3K/Akt/mTOR (Phosphoinositide 3-Kinase/Akt/ mammalian Target of Rapamycin) signaling^9, 10, 11.

PTEN (phosphatase and tensin homolog), a 403 amino acid dual protein/lipid phosphatase converts phosphatidylinositol(3,4,5)-triphosphate (PIP3) to phosphatidylinositol(4,5)-bisphosphate (PIP2), thereby regulating the PI3K/Akt/mTOR pathway involved in oncogenic signaling, cell proliferation, survival and apoptosis¹². PTEN, as a protein phosphatase, autodephosphorylates itself¹³. Deficiency or dysregulation of PTEN drives endometrial, prostate, brain and lung cancers, and causes neurological defects^14, 15. PTEN is activated after membrane association¹⁶, providing conformational accessibility to the catalytic phosphatase domain (PD) that converts PIP3 to PIP2¹⁶(Figure 1a). Because PTEN reduces PIP3 levels and inhibits pathogenic PI3K signaling, therapeutically targeting PTEN to the membrane to enhance its activity is of significance in treating several pathologies including cancer.

Figure 1: PTEN: A newly identified IDP.

PTEN – A newly identified IDP. srep02035-f1

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(a) Diagrammatic representation of PTEN structure. PTEN, a 403 amino acid protein, comprises of PBM: PIP2 Binding Module (AA 1–13; in green), a phosphatase Domain (AA 14–185; in pink), C2 Domain (AA 190–350; in blue), C-terminal region or Tail (AA 351–400; in orange) and a PDZ binding domain (AA 401–403; in dark blue). The PDZ-binding motif is considered as a part of the C-terminal region. *Figure not to scale. (b) Crystal structure of PTEN. Only the phosphatase (in pink) and C2 domain (in blue) are amenable to crystallization. The first seven residues and the last 50 residues represent unstructured/loosely-folded regions that are yet to be crystallized. These regions represent the N- and C-termini of PTEN, respectively. (Source: RCSB Protein Data Bank). (c) Disorder analysis of PTEN. PONDR-VLXT and PONDR-FIT prediction tools were used to determine the disorder score of PTEN. Any value above 0.5 indicates intrinsic disorder. There are several disordered stretches within the PTEN protein, however, the most prominent of these disordered regions is a 50 amino-acid stretch located at the C-terminus of the PTEN protein. (d) IDPs are enriched in polar (R, Q, S, T, E, K, D, H) and structure breaking (G, P) amino acids and are depleted in hydrophobic (I, L, V, M, A), aromatic (Y, W, F) and cysteine (C) and asparagine (N) residues. The amino acid sequence of PTEN highlights these classes of residues with their relative distribution. (e) Composition profiling for full-length PTEN (in green), its ordered domain (in yellow) and its IDR (in red). The tool used is Composition Profiler (Vacic et al, 2007). As shown in the graph, the disordered region in PTEN is enriched in polar residues (specifically H, T, D, S and E), structure breaking residues (specifically P) and is depleted in all hydrophobic residues, cysteine and all aromatic residues. (f) Histogram representing the percentage of hydrophobic, polar, aromatic, structure breaking, cysteine and asparagines residues in ordered vs. disordered regions. The disordered region has an amino acid composition in line with the definition of IDPs.

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PTEN crystal structure revealed that the PD and membrane-binding C2 domains are ordered (Figure 1b); however, the structures of the N-terminus, the CBR3 loop and the 50 amino-acid C-tail remain undetermined¹⁷. The C-tail is of particular significance due to its ability to regulate PTEN membrane association, activity, function, stability^{18, 19, 20, 21}. Herein, we identify PTEN as an IDP with its C-tail being intrinsically disordered. The PTEN C-tail IDR is heavily phosphorylated by a number of kinases and regulates the majority of PTEN functions, including a large number of PPIs that forms the PTEN primary and secondary interactomes, comprising critical functional protein hubs, most of which are related to cancer. Our analysis provides a mechanistic insight into the functioning of the PTEN C-tail IDR at the systems level, including inter- and intra-molecular interactions that will aid in designing drugs to enhance the lipid phosphatase activity of PTEN for the pharmacotherapy of cancers and pathological conditions driven by hyperactive PI3K-signaling.

PTEN is an IDP

Utilizing two disorder prediction software programs, PONDR-VLXT and PONDR-FIT^22, 23, we have identified PTEN as a bona fide IDP. PTEN has a highly disordered, functionally versatile, C-tail encompassing amino acids 351–403 (Figure 1a and 1c). A PDZ-binding motif (amino acids 401–403) is part of the disordered region. Thus, the PTEN C-tail IDR facilitates interactions with a vast repertoire of PDZ domain-containing proteins (Figs. 1a and 2d). The unique amino acid composition of IDRs dictates their structural plasticity^3, 23, 24. IDRs are enriched in polar and structure-breaking amino acid residues, depleted in hydrophobic and aromatic residues and, rarely, contain Cys and Asn residues^1, 23, 24. The ordered region of PTEN (AA 1–350) has 25% hydrophobic, 43% polar, 9% structure breaking, 13% aromatic and 9% Cys and Asn residues. In contrast, the PTEN C-tail (AA 351–403) is enriched in polar (66%) and structure breaking (11%) residues and is depleted in hydrophobic (11%), aromatic (6%) and Cys and Asn residues (6%), indicating an ideal profile for the IDR (Figs. 1d and 1f ). Further, compositional analysis of PTEN using the Composition Profiler²⁴ reveals that the disordered region in PTEN is enriched in polar residues (specifically H, T, D, S and E) and structure breaking residues (specifically P) but is depleted in all aromatic and hydrophobic residues in addition to cysteine. (Figure 1e), again exhibiting universal characteristics of IDPs. Taken together, we establish the PTEN C-tail as a functional IDR and classify PTEN as a new IDP.

Figure 2: The functional relevance of the PTEN IDR.

The functional relevance of the PTEN IDR. srep02035-f2

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(a) The number of mutations observed in PTEN over its 403 amino-acid stretch is plotted. Fewer mutations are observed in the tail region (in red) possibly indicating the deleterious nature of mutations in the functionally critical C-terminal region. [Source: Sanger Institute Catalogue of Somatic Mutations in Cancer (COSMIC), Human Gene Mutation Database (HGMD)]. (b) Number of mutations in every successive 50 amino-acid stretch of the PTEN protein. The last 50 amino-acid stretch, representing the tail region has at least one-eighth the number of mutations seen in any other 50 amino-acid stretch along PTEN, pointing to its critical function in cell homeostasis. (c) Correlation of mutations with the amino acid composition of PTEN. The ratio of mutations in specific residues in the disordered vs. ordered region are represented in this graph. The residues considered here are those used to define IDRs: hydrophobic, polar, aromatic, structure-breaking, cysteine and asparagine residues. Compared to the other classes of residues, mutations in aromatic residues are much higher in the disordered region when compared to the ordered region. (d) The PTEN primary interactome. Forty proteins interact with known regions of PTEN. There are approximately 340 more proteins that interact with PTEN at sites that are yet to be determined (see Supplementary Table S2). Proteins shown in pink interact with the phosphatase domain, those in blue interact with the C2 domain and those in orange interact with the disordered tail. (Visualization tool: Cytoscape). (e) The PTEN C-tail has a higher propensity for PPIs. Of the 40 mapped proteins, 60% interact with the disordered indicating a strong correlation between degree of disorder and the number of protein interactions. (f) Most proteins within the PTEN interactome are highly disordered. Approximately 80% of PTEN-interacting proteins within the primary interactome are disordered, as indicated in red. The proteins within the interactome that are ordered are indicated in blue.

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Low mutability of PTEN IDR suggests critical biological functions

Mutations in PTEN are associated with several types of cancers¹⁴. To correlate PTEN mutations to its structure, we analyzed all human PTEN mutations deposited in the COSMIC Database (http://www.sanger.ac.uk/genetics/CGP/cosmic/). The disordered PTEN C-tail IDR shows unusually low mutability (~8-fold less) compared to any other 50 amino-acid stretch of PTEN (Figure 2a and 2b). To confirm our finding of the low mutability of the C-tail region, we also analyzed all human PTEN mutations deposited in the Human Gene Mutation Database (HGMD,http://www.hgmd.cf.ac.uk/ac/index.php)²⁵ (Figure 2a), cBioPortal for Cancer Genomics^26, 27(Supplementary Figure S1) and the Roche Cancer Genome Database²⁸ (Supplementary Figure S1) which was consistent with the COSMIC database mutational data. It is likely that evolutionary pressure maintains a survival advantage and ipso facto abrogates progeny with mutations in highly functional protein sequences^29, 30, 31. Thus, the functionally versatile PTEN C-tail IDR cannot afford mutations, hence showing least number of mutations. It is equally likely that mutations in individual residues within the IDR are well tolerated, as the evolutionary pressure may have shifted to maintaining global biophysical properties and structural malleability of the IDR to safeguard the critical protein function²⁹. In either case, on a global scale, the versatile structural pliability of the PTEN IDR dictates functional diversity and biological activities²⁹. Thus, the slightest functional perturbation in the PTEN IDR due to mutations, either within the IDR or in domains interacting with it, could disrupt cellular homeostasis as seen in cancers and neurodegenerative disorders associated with PTEN mutations. This is supported by our data indicating that PTEN, as an IDP when mutated, causes several cancers¹⁴.

Moreover, the PTEN C-tail IDR exhibits preferential mutations in aromatic residues compared to the ordered region (Figure 2c). The ratio of mutations in aromatic residues in the disordered to ordered region is much higher than any other class of residues (structure breaking, hydrophobic, polar, Cys and Asn), likely attributed to the structure-imparting property of aromatic residue³². Specifically, aromatic residues within IDRs engage in stacking interactions, enhancing nucleation between distinct residues at functional protein-protein interaction interfaces³². Thus loss of this critical structural and functional property imparted by aromatic residues is associated with a disease phenotype. In summary, the disordered PTEN C-tail IDR has functionally evolved to contain a combination of peptides that cannot tolerate mutations.

Disorderliness in PTEN primary interactome drives functional networks

Protein-Protein Interactions (PPIs) typically occur between conserved, structurally rigid regions of two or more proteins, particularly ordered proteins that display energetically favorable, highly-folded conformations. Intriguingly, IDPs lack tertiary structure, yet engage in PPIs, albeit with lower affinities but high specificity¹. The lack of structure within IDPs enhances their biophysical landscape, conferring them with the ability to attain structural complementarities required for PPIs. Since IDPs do not conform to a stable structure, they are less compact, providing a larger physical interface and energetic adaptability to interact with multiple proteins^1, 7. Thus, conditional folding within IDPs is effectively utilized for interaction with a multitude of binding partners, enabling them to shuttle between several signaling cascades as efficient “cogs”, mediating and regulating PPIs^{4,7, 33, 34, 35, 36}. Indeed, we discovered that PTEN, being an IDP, interacted with more than 400 proteins (Supplementary Table S1) when a combination of online software, literature search and database mining tools were used. Proteins with known PTEN interaction domains were classified as “mapped” (Figure 2d and Supplementary Table S1), whereas those with uncharacterized/predicted interactions were designated as “unmapped” proteins (Supplementary Table S1). Derivation of PTEN primary interactome from the mapped proteins using Cytoscape (http://www.cytoscape.org/) indicated that PTEN disorderliness is efficiently used for interaction with 40 proteins, most existing in distinct functional pathways (Figure 2d, 2e and Supplementary Table S2).

Interestingly, within the PTEN primary interactome, 60% of interactions occurred within the disordered C-tail region. Furthermore, disorder analysis on the primary interactome revealed that 33 proteins (>82%) were IDPs, of which two-thirds interacted with the C-tail IDR (Figure 2e, 2f andSupplementary Table S3), indicating a high propensity for disorder-disorder (D-D)-type interactions.

In order to study evolutionary conservation of the PTEN C-tail and its interactions across species, several sequence alignments were performed (Figure 3a). Sequence alignment of the entire PTEN protein from different animal species shows a good conservation of the catalytic phosphatase domain between vertebrates and invertebrates with 100% sequence conservation for the dual specificity phosphatase catalytic motif HCKAGKGR⁸ (Supplementary Figure S2). The C-tail shows good conservation in the vertebrate species, likely indicating the recent emergence of the function of PTEN C-tail region in regulating PTEN activity and enriching its PPI potential, translating to its versatile functions. In order to examine the conservation across species for the PTEN C-tail interacting proteins, a literature search was conducted to identify experimentally verified domains/motifs involved in interaction with the C-tail. The domains involved in these interactions with the C-tail for 13 proteins with relevant literature sources for these interactions are part of Supplementary Figure S3. Subsequent sequence alignments for these thirteen proteins (Supplementary Figure S3) shows good sequence homology for the domains/motifs involved in interaction with the PTEN C-tail. These findings support the concept that the PTEN C-tail has evolved in vertebrates to incorporate features that allow it to interact with these proteins.

Figure 3: Sequence conservation in PTEN and its interacting partners reflects functionality.

Sequence conservation in PTEN and its interacting partners reflects functionality. srep02035-f3

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(a) Sequence alignment of the PTEN protein for vertebrate and invertebrate animals. Green color indicates sequence similarity while red indicates sequence dissimilar amino acid residues. All comparisons are made with respect to the human PTEN protein. (b) Network analysis for PTEN was performed to assess its potential as a network hub. The network shows multiple secondary interactions within the 40 mapped proteins, indicating their role in multiple signaling cascades mediated via PTEN. The proteins SMAD2/3, AR, PCAF, ANAPC7, B-arrestin 1 and p53 appear to be critical within these signaling cascades and also happen to be intrinsically disordered (Supplementary Table S3), reinforcing the concept of preferential interactions between disordered proteins. (Analysis Tool: Metacore by GeneGo).

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Further, to assess whether PTEN acts as a functional hub protein and regulates pathways through its protein-binding partners, we performed functional network analysis using the Analyze Network option from MetaCore (GeneGo Inc, Thomson Reuters, 2011) (Figure 3b). The PTEN primary interactome was used as input with PTEN as the central node. We identified multiple interactions not only between PTEN (node) and SMAD2/3, AR, PCAF, ANAPC3, ANAPC4, Caveolin, β-arrestin 1 and p53 (edges), but also amongst the edge proteins themselves (Figure 3b). Interestingly, all the edge proteins are themselves highly disordered (Supplementary Table S3). Further supporting this finding, our functional enrichment revealed that 13 proteins (one-third) of the PTEN primary interactome were cancer-related and highly disordered (Figure 4a, Supplementary Table S3 and S4).

Figure 4: Derivation and disorder analysis of the PTEN cancer interactome.

Derivation and disorder analysis of the PTEN cancer interactome. srep02035-f4

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Derivation of the PTEN Cancer Interactome. Functional enrichment of the PTEN primary interactome identified 13 cancer-related proteins which are also intrinsically disordered. Subsequently, the PTEN secondary interactome was derived from the primary PTEN interacting proteins. A subset of the secondary interactome was designated as the PTEN Cancer Interactome and it represents the proteins that interact with the 13 cancer-related proteins of the primary interactome. (b) PTEN Cancer Interactome. PTEN is the primary node that interacts with the 13 cancer-related proteins representing the partial primary interactome. Proteins that interact with each of the 13 cancer-related proteins comprise the secondary interactome. Disordered proteins are represented in red while ordered proteins are shown in blue. Cancer-related proteins in the PTEN primary interactome were identified using IPA (Ingenuity® Systems, ingenuity.com). (c) We identified 40 proteins that are part of the PTEN primary interactome of which 13 are highly disordered (IDP) and identified as potential cancer network hubs based on functional network analysis. We further identify 299 IDPS from the secondary PTEN interactome. A filter for cancer-related proteins revealed that approximately two-thirds of the IDPs that form the secondary interactome (193 out of 299) are involved in oncogenesis, suggesting a high degree of functional enrichment. (Functional network analysis was performed using IPA (Ingenuity® Systems,www.ingenuity.com).Full size image (805 KB)

Pliant PTEN secondary interactome relays function of the primary network

The disorderliness of the PTEN primary interactome prompted us to investigate the possibility that PTEN radiates its function via a malleable network of IDPs that extends beyond the primary interactome. Therefore, we derived the PTEN secondary interactome (Supplementary Table S5) and ascertained the interaction of 13 cancer-related proteins identified in the primary interactome (Figure 4a). The entire PTEN secondary interactome consisted of 299 IDPs, of which 193 IDPs (two-thirds) were associated with the 13 cancer-related proteins, generating a “PTEN-Cancer Interactome” (Figure 4, Supplementary Table S5 and S6). Thus, two-third of the IDPs within the PTEN secondary interactome associates with one-third of the cancer related IDPs within the PTEN primary interactome, indicating that cancer-related functions are driven by IDPs in the PTEN interactome and that the flexibility of IDP-IDP interactions modulates diverse functions; dysregulation of which causes cancers.

Functional network analysis of the 193 cancer-related IDPs identified 31 proteins that shared multiple nodes (Figure 5a and Supplementary Table S6). We overlaid this network with the cancer-related IDPs of the primary interactome to predict functionally critical protein hubs (indicated in yellow circles in Figure 5a and b). Our analysis revealed 16 proteins as highly populated hubs, most enriched in disordered regions, again demonstrating that a high degree of structural and functional association between the hubs required IDP-IDP interactions (Figure 5b). The involvement of these hubs in multiple, critical oncogenic signaling pathways make them attractive drug targets in the field of clinical oncology. Our bioinformatic analysis resonates well with observed biological phenomena as seen in the case of MDM2 protein, which is a major PPI hub regulating p53. Interaction of the human androgen receptor (AR) protein and MDM2 influences prostate cell growth and apoptosis³⁷. Mdm2-Daxx interaction activates p53 following DNA damage³⁸, and Daxx binds and inhibits AR function³⁹. Conversely, the breast cancer susceptibility gene 1 (BRCA1) interacts directly with AR and enhances AR target genes, such as p21(WAF1/CIP1), that may result in the increase of androgen-induced cell death in prostate cancer cells⁴⁰. Further, BRCA1 complexes with Smad3 and is inactivated, leading to early-onset familial breast and ovarian cancer⁴¹. Within the same network, MDM2 inhibits the transcriptional activity of SMAD proteins including SMAD3⁴², thereby, emerging as a major player in prostrate, breast and ovarian cancer. Loss of PTEN, on the other hand, results in resistance to apoptosis by activating the MDM2-mediated antiapoptotic mechanism. We also identified proteins like NCL, DAXX and SUMO that play critical roles in mediating cancers as being a part of the PTEN centric cancer interactome (Figure 5b). Interestingly, all of the 16 predicted hubs can be traced back to PTEN (either directly or through other signaling adaptors) reinforcing our analysis (Figure 5c). These findings support the prevailing concept of preferential interaction between disordered regions of two distinct proteins; with PTEN being the common disordered interacting hub, giving functional centrality to PTEN in many critical cellular pathways.

Figure 5: Predicting functionally relevant network hubs in the PTEN cancer interactome.

Predicting functionally relevant network hubs in the PTEN cancer interactome. srep02035-f5

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(a) Methodology to identify functional hubs within the PTEN Cancer Interactome. The PTEN Cancer Interactome contains 193 IDPs that are potential hubs. Over-represented IDPs (or IDPs with multiple occurrences) in the PTEN Cancer Interactome would have a greater propensity to function as hubs. Upon sorting for over-represented IDPs the list of 193 proteins is brought down to 31 proteins. In order to assess the possibility of these 31 proteins as functional hubs a network analysis is warranted. (b) We identified 31 potential hubs based on multiple associations from within the 193 cancer-associated IDPs of the PTEN secondary interactome. Regulatory networks derived from these 31 proteins were overlaid with a similar network from the 13 cancer-related proteins. Based on the number of associations within the network, we identify 16 potential functional hubs in the PTEN cancer interactome (indicated in yellow). Regulatory interactions were generated using the Transcriptome Browser tool (Lopez et al, 2008). (c) Functional network analysis of the 16 predicted hubs. In order to assess the functional association of the 16 predicted hubs with PTEN – a network analysis with PTEN as a central node was done. The analysis identifies MDM2 protein, a major regulator of p53, as one of the major PPI hubs in the PTEN cancer interactome. A number of other critical cancer-related proteins, such as AR, SMAD2/3 and PDGFRB that are part of the PTEN primary interactome, feature prominently in the PTEN cancer interactome. We also identified proteins like NCL, DAXX and SUMO that play critical roles in mediating cancers as being a part of the PTEN centric cancer interactome. Interestingly, all of the 16 predicted hubs can be traced back to PTEN (either directly or through other signaling adaptors) reinforcing our analysis. (Functional network analysis was performed using IPA (Ingenuity® Systems, www.ingenuity.com).

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To further validate our methodology in using intrinsic disorder and cancer as filters to identify key signaling hubs, we compared our data sets with a previously published cancer signaling data set. We derived 7 common hubs (Supplementary Table S7), which were extended using the expansive human signaling network described previously^{43, 44, 45, 46} to obtain the PTEN associated cancer interactome (Figure 6a). An extensive disease associated network analysis using IPA validated our predictions as all the seven predicted hubs had an extensive cross-talk across multiple cancer disease types (Figure 6b).

Figure 7: Biochemical features modulating PTEN PPIs.

Biochemical features modulating PTEN PPIs. srep02035-f7

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(a) A PTEN linked cancer network was derived using seven of the 16 predicted cancer hubs that were common with the human cancer associated gene set. The associated partners of the seven hubs were extracted from the human signaling network (Cui et al, 2007, Awan et al, 2007, Li et al, 2012 and Newman et al, 2013). Red color denotes the potential cancer hubs and blue color are their associated partners. Topological analysis identifies p53 as the most significant network hub in the PTEN linked cancer network (Supplementary Table S7). (b) Disease associated network of PTEN cancer hubs. A functional network was constructed with the seven topologically relevant hubs identified previously using the Core Analysis function from the IPA suite to derive the primary network (denoted as MP). A disease network was constructed using the Path Designer option and disease associated biological functions were overlaid on the primary network. Fx denotes the different functions associated with the members of the networks.

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Modulation of PTEN PPIs by linear binding motifs

Recent evidence has shown that IDPs mediate PPIs via short linear amino acid sequences (~20 residues) called Molecular Recognition Elements (MoREs) or Molecular Recognition Features (MoRFs)^35, 47. MoRFs undergo disorder-to-order transitions upon binding and adopt thermodynamically stable well-defined structures⁴⁷, increasing the propensity of IDPs to interact with a vast repertoire of proteins. MoRFs also display molecular recognition elements that capture the binding partner proteins with high specificity. These partner-dependent conformational differences are critical to imparting versatile binding properties to IDRs³⁵.

Since the PTEN IDR engages in multiple PPIs, we tested the possibility for the existence of MoRFs. The MORFP red algorithm⁴⁸ revealed that PTEN contains major MoRF sites at amino acids 273–279 (part of the disordered CBR3 loop of the C2 domain), amino acids 339–347 (in close vicinity of the disordered C-tail) and amino acids 395–403 (part of the disordered C-tail) (Figure 7a and Supplementary Figure S4). The primary restriction of MoRFs to the PTEN C-tail IDR or adjacent regions indicates that these MoRFs directly participate in modulating PPI functions (Figure 7a). However, mutational analysis within MoRFs is required to establish their active role in functional PPIs.

Figure 7: Biochemical features modulating PTEN PPIs.

Biochemical features modulating PTEN PPIs. srep02035-f7

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(a) MoRFs in the PTEN C-tail IDR. MoRFpred (Disfani et al, 2012), a computational tool, was used to identify MoRF regions within the PTEN protein (Supplementary Figure S4). The MoRFs in the vicinity of the C-tail IDR are highlighted in red. Interestingly, all of the major MoRFs (with a length greater than 5 residues) are observed in the vicinity of disordered regions (either part of the disordered CBR3 loop of the C2 domain or the C-tail IDR) indicating a positive correlation between intrinsic disorder and PPIs. (b) ELMs in PTEN C-tail IDR. Eukaryotic Linear Motifs (or ELMs) are 3–11 amino acid long sequences that mediate PPIs. IDRs are particularly enriched in ELMs (Dinkel et al, 2012). The linear motifs occurring in the disordered segment of PTEN (tail + PDZ domain) have been highlighted. The motifs with a high conservation score (>0.75) are indicated in red. Interestingly, all of the motifs with a high conservation score are restricted to the C-tail IDR. (c) Phosphorylation sites in the C-tail IDR. Phosphorylation of PTEN, particularly on serine and threonine residues in the disordered region, regulates the function and stability of PTEN. Phosphorylation occurs at Ser 362, Thr 366, Ser 370, Ser 380, Thr 382, Thr 383, Ser 385 by various enzymes such as Casein Kinase II, Glycogen synthase kinase 3-B and Polo-like kinase 3. Each of these phosphorylation events helps regulate the availability and stability of the PTEN molecule within the cell.

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Protein-protein interactions are also facilitated by very short motifs (3–10 amino acids) called Short Linear Motifs (SLiMs) or Eukaryotic Linear Motifs (ELMs)^49, 50. Because of their short sequences, ELMs arise/disappear by simple point mutations, providing the evolutionary plasticity that the ordered protein domains lack. Thus, ELMs easily adapt to novel interactions in signaling pathways, where rapid assembly/disassembly of multi-protein complexes is a prerequisite. The frequent occurrence of ELMs in a typical proteome indicates their critical cellular functions. Consistent with this notion, a higher density of ELMs are observed in hub proteins and IDPs⁵⁰. Since ELMs have short sequences, they interact with low-affinity, however, they engage in highly cooperative binding in protein complexes, triggering productive signaling⁵⁰. Therefore, at increased intracellular local concentrations they competitively bind to mutually overlapping physiological targets of each other as seen with PDZ, SH2 and PTB interaction domains found in cancer-associated proteins and in IDRs^49, 50. As PTEN contains a PDZ-binding motif within the IDR (Figure 1a and c), we probed for the existence and features of ELMs in PTEN using The Eukaryotic Linear Motif Resource (http://elm.eu.org). We identified 34 different classes of ELMs in PTEN that mediate PPIs (Supplementary Figure S5). Interestingly, the four ELMs that are most conserved (conservation score>0.75) occurred within the PTEN C-tail IDR, indicating its high level of functional/biological significance (Figure 7b). ELM functions are further modulated by post-translational modifications, mainly by phosphorylation⁵⁰. Indeed, the PTEN IDR possesses nine phosphorylation sites^51, 52(Figure 7c).

PTEN phosphorylation modulates intramolecular association and PPI function

Post-translational Modifications (PTMs) in IDPs facilitate PPIs⁵. Modifying enzymes readily dock on structurally flexible IDRs, making them a hot spot for PTMs^{4, 7, 53, 54}. Consistent with this notion, regulatory cancer-associated proteins have twice as much disorder and undergo more frequent phosphorylation/dephosphorylation than other cellular proteins as predicted by DISPHOS (a DISorder-enhanced PHOSphorylation prediction software)⁵⁴, implicating a tight interconnection between protein phosphorylation and disorder. Consistent with the function of PTM in IDRs, clustering of Ser and Thr phosphorylation sites (Figure 7c) in the C-tail IDR regulates PTEN stability, membrane association and activity^19, 20. Phosphorylation in the PEST [proline (P), glutamic acid (E), serine (S) and threonine (T)] domain within the C-tail IDR (amino acids 352 to 399) inhibits degradation of PTEN⁵¹. Casein kinase II (CK II), Glycogen synthase kinase 3-beta (GSK3-β) and PLK3 (Polo-like kinase 3) phosphorylate Ser and Thr residues within the IDR, each providing a distinct function⁵¹ (Figure 7c). The microtubule-associated serine/threonine (MAST), serine/threonine kinase 11(STK11) or LKB1 and casein kinase I (CKI) kinases have also been implicated in PTEN phosphorylation. STK11/LKB1 modifies T383, while CKI modifies T366, S370 and S385⁵². Indeed, our DISPHOS prediction for C-tail IDRs supports these experimental observations (Supplementary Figure S6).

Substrate-kinase interactions are typically of the disordered-ordered (D-O) type and are stabilized by hydrogen bonding (Figure 7c), a hallmark of IDRs⁵⁴. Indeed, computational analysis revealed that large ordered regions comprising the catalytic domains of CKII, GSK3B, PLK3, Rak, and Src kinases interact with the C-tail IDR (Supplementary Table S8), indicating that PTEN engages in D-O type intermolecular interactions with the modifying kinases.

At the intramolecular level, phosphorylation at C-tail residues triggers a conformational change in PTEN, inhibiting its membrane association and, therefore, its lipid phosphatase activity^{18, 19, 21, 55}. The phosphorylated C-tail IDR folds onto the PD and C2 domains giving rise to the “closed-closed” conformation of PTEN (Figure 8a) that is incapable of interaction with the membrane^18, 20. The “closed- closed” form of PTEN is enzymatically inactive and cannot convert PIP3 to PIP2. The identification of the exact resides involved in this intramolecular interaction remains an active area of research^18, 20, 56.

Figure 8: Targeting PTEN C-tail IDR.
http://www.nature.com/srep/2013/130620/srep02035/images_article/srep02035-f8.jpg

Most PTEN functions emanate from the C-tail IDR, including aberrant PPIs that hyper-activate oncogenic pathways. (a) Phosphorylation mediates an intramolecular interaction in the PTEN molecule. Phosphorylation causes a conformational change in PTEN converting it to the enzymatically inactive “closed closed ” form wherein the flexible tail folds onto residues in the C2 and phosphatase domain, thereby making it incapable of interacting with the membrane. Dephosphorylation (by an unknown phosphatase or via auto-dephosphorylation) converts PTEN to the “open-closed” form. Electrostatic interactions, mediated by the PBM, further convert PTEN to the “open-open” form wherein it binds to the membrane and acts as a lipid phosphatase converting PIP3 to PIP2, thereby, abrogating signaling via the PI3K/Akt/mTOR pathways. Subsequent to membrane binding, several E3 ubiquitin ligases polyubiquitinate PTEN marking it for proteasomal degradation. Phosphorylation, by inducing the intramolecular interaction, masks the ubiquitination sites thereby increasing the half-life of the PTEN protein within the cell. Therefore, phosphorylation negatively regulates PTEN function but positively regulates its stability. (b) PTEN IDR engages in PPIs of the disorder:order type (D-O type). As revealed in the present study, this occurs via the use of a MoRF or SLiM region. Therefore, designing a peptidomimetic drug molecule that competes with the PTEN MoRF/SLiM binding to the ordered protein will abrogate PTEN binding, therefore PTEN function. PTEN IDR is highly accessible to multiple kinases that phosphorylate and modulate PTEN function, mainly its inhibition via intra-molecular interactions. PTEN inhibition hyper-activates the PI3K/AKT/mTOR pathway, which increase the oncogenic potential of the cell and drives cancer growth. Therefore, targeting the PTEN C-tail IDR with small molecules that bind and sterically hinder PTEN phosphorylation and/or intra-molecular interactions will be an ideally adjunctive therapy to multiple inhibitor therapy targeting of the PI3/AKT/mTOR pathway.

Full size image (553 KB)

It was recently shown that the phosphorylation events of PTEN occur in two independent cascades of ordered events, with the S380–S385 cluster being modified prior to the S361–S70 cluster⁵². Even within the two clusters, the phosphorylation events follow a specific pattern with a distributive kinetic mechanism. Not surprisingly, distributive kinetics is energetically favorable on protein domains that are highly disordered with multiple ensembles of flexible structures⁵². Thus the dynamic nature of these phosphorylation events is contingent to the inherent flexibility in the PTEN structure driven by intrinsically disordered C-tail crucial for PTEN stability and localization within the cell (Figure 8a).

Targeting intrinsic disorder in PTEN and its interactome

Drug targeting to critical protein regions can mitigate aberrant cellular processes driving oncogenesis⁵⁷. However, despite numerous clinical trials with molecularly targeted therapies, failure rates for cancer treatments remain high. Conventional therapies targeting pathway-specific kinases suffer from “off-target effects” and often fail due to the emergence of compensatory and alternative pathways⁵⁸. As a novel approach, facile drug targeting to IDRs within critical signaling hub proteins is highly plausible^59, 60, 61. Moreover, as IDRs undergo extensive PTMs⁵³ and engage in PPIs^4, 34, 36, the multitude of resulting protein interactions (normal and aberrant) can be targeted concomitantly with a cocktail of distinct inhibitors, which dampens oncogenic signaling⁶⁰.

Indeed, targeting PPIs is a more selective treatment strategy over conventional enzyme inhibitors⁶⁰. However, disruption of multiple ordered interfaces within PPIs by small molecule inhibitors remains challenging⁶². The advantage of targeting IDPs engaged in PPIs is that, unlike ordered proteins, they engage in PPIs via MoRFs or ELMs, which are small peptide regions that bind with low affinity and thus are susceptible to disruption by small molecule inhibitors⁵⁹. Consistent with this notion, small molecules disrupted highly disordered complexes of p53-Mdm2 and c-Myc-Max interactions by inducing order upon binding^60, 63. Likewise, targeting the PTEN C-tail IDR may reduce its intra- and inter-molecular interactions and limit accessibility to enzymes mediating PTMs (Figure 8b), providing a means to increase PTEN activity. Our analysis shows that since the C-tail IDR is rich in conserved MoRFs/SLiMS, targeting these regions will prove to be a rational therapeutic modality for a large number of cancers that show compromised PTEN activity or hyperactivation of the oncogenic PI3K/AKT/mTOR pathway^9, 10, 11. Since reductions in the levels and activity of PTEN are sufficient to drive oncogenesis^11, 14, 15, increasing PTEN activity is an ideal therapy for cancers associated with hyperactive PI3K-signaling.

Discussion

Recent studies on genome- and proteome-wide molecular alterations in diseases indicate that pathological conditions are caused by perturbations in complex, highly interconnected biological networks⁶⁴. Thus, current reductionist approach of studying structure-function relationship in diseases has limited our abilities to discover effective targeted therapeutics. In an attempt to overcome these limitations, in the current study, we have undertaken a novel approach to drug discovery that exploits systems and network biology at the structural, topological and functional level. Using PTEN, a tumor suppressor, we have applied computational and systems biology approaches and integrated extensive data-mining and biochemical properties of IDP interactions to reach a finer understanding of PTEN function. These results have identified PTEN C-tail IDR and several hub proteins in PTEN-driven molecular network implicated in human diseases as therapeutic targets, enhancing the repertoire of clinically relevant biological targets for pharmacotherapy.

Our derivation and analysis of PTEN primary and secondary interactome indicates that altered levels or interactions of IDPs perturb myriad cellular signaling pathways, leading to pathological conditions including cancer. IDPs have the propensity to aggregate and cause cellular toxicity⁶⁵. Therefore, PTEN as an IDP has evolved a mechanism, wherein, the level of active PTEN, its cellular localization and PTEN-PPIs are regulated via phosphorylation of the C-tail IDR. Furthermore, evolutionarily conserved ELMs and MoRFs that we have identified within the C-tail IDR may play a critical role in orchestrating the formation and function of the PTEN interactome.

Increase in complexity of PPIs is either directed by the number and type of proteins or by increasing the number of interactions required to execute cellular functions⁶⁶. To delineate how PTEN executes myriad functions, we first derived the PTEN primary interactome. We found 40 proteins to directly interact on the PTEN molecule, out of which 25 were associated with the C-tail IDR, consistent with the concept that disorderliness within PTEN executes its myriad functions. To enhance our understanding of PTEN functions in the context of multiple distinct pathways at the systems-level, we delineated functional networks operating within the primary interactome. Our findings showed a high degree of cross-talk between edges, implying that shared regulatory modules, comprised of multiple signaling cascades, operate via PTEN-mediated interaction networks. When these networks are altered, diseases ensue with extreme functional penalties. We also found that the edge proteins were themselves highly disordered indicating that disorderliness within the PTEN primary interactome confers functional versatility. Supporting this notion, 13 proteins that were functionally classified as cancer-related were also highly disordered forming a pliable “PTEN-Cancer Interactome”. Thus, PTEN lesions influence the flexibility of IDP-IDP interactions modulating diverse functions, likely causing cancer.

Owing to the inherent ability of PPIs to be flexible while being complex, specific cellular functions are readily fine-tuned as per the biological demands. Emerging evidence suggests that certain features on the IDRs are recognized as a way of conferring plasticity to protein interaction networks. Consistent with this concept, our data suggest that PTEN, a hub protein containing an IDR, likely utilizes MoRFs and ELMs, gets differentially modified via PTMs, acquiring complementary structures to engage and modulate PPI activity by facilitating adaptive binding to multiple protein partners in many cellular pathways. Thus, our present work provide a novel entrée in targeting intrinsic disorder in PTEN and its interactome to dampen the aberrant PI3K-signaling that drives many cancers. First, imparting order to the PTEN structure may help dampen multiple oncogenic signaling pathways mediated via the 16 hub proteins identified in the present study, by limiting their affinity for PPIs. Second, targeting intrinsic disorder in PTEN and its interactome can become an adjunctive or alternative approach to the use of various kinase inhibitors, which are toxic and have many off-target effects when used to mitigate the aberrant hyperactivation of PI3K/AKT/mTOR oncogenic signaling pathway. Taken together, the present findings provide a novel entrée to design strategies for drug discovery and may become a logical intervention in the pharmacotherapy of cancer and other PTEN-associated disease treatment modalities.

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Pancreatic Islets

Posted in Curation, Diabetes Mellitus, Diagnostics and Lab Tests, Disease Biology, Endocrine Diseases, Epigenetics and Cardiovascular Risks, Fatty acids, Frontiers in Cardiology and Cardiovascular Disorders, Gene Regulation, Genomic Endocrinology, Lipid metabolism, Lipids, Metabolism, Nephrology, Neurodegenerative Diseases, Nitric Oxide in Health and Disease, Nutrition, Nutritional Supplements: Atherogenesis, Origins of Cardiovascular Disease, tagged alpha cell, Apoptosis, Beta cell, beta cell amyloid, endocrinology, insulin and glucagon, oligomerization, Pancreas, peptides on February 8, 2015| Leave a Comment »

Pancreatic Islets

Writer and Curator: Larry H. Bernstein, MD, FCAP

Part I. Endocrine Pancreas

The eclipse and rehabilitation of JJR Macleod, Scotland’s insulin laureate

Bliss, M
Journal of the Royal College of Physicians of Edinburgh 2013; 43(4): 1-8

John JR Macleod (1876-1935,) an Aberdonian Scot who had emigrated to North America, shared the 1923 Nobel Prize with Frederick Banting for their discovery of insulin at the University of Toronto in 1921-22. Macleod finished his career as Regius Professor of Physiology at the University of Aberdeen from 1928 to 1935.Macleod’s posthumous reputation was deeply tarnished by the campaigns against him carried out by his fellow laureate, Banting, and by Banting’s student assistant during the insulin research, Charles Best. Banting’s denigration of Macleod was based on their almost total personality conflict; Best’s was based on a hunger for personal recognition. New research indicates how scarred both men were in their obsessions.The rehabilitation of Macleod’s reputation, begun in 1982 with my book, The Discovery of Insulin, has continued in both scholarly and popular circles. By 2012, the ninetieth anniversary of the discovery of insulin, it had become complete both at the University of Toronto and in Canada.

Almost famous: E. Clark Noble, the common thread in the discovery of insulin and vinblastine

Wright Jr., J.R.
CMAJ 2002; 167 (12), pp. 1391-1396

CLARK NOBLE WAS ONE OF THE FIRST members of the University of Toronto insulin team and came within a coin toss of replacing Charles Best as Frederick Banting’s assistant during the summer of 1921. Noble performed important early studies helping to characterize insulin’s action, and he co-authored many of the original papers describing insulin. Because mass production of insulin from livestock pancreata had proved elusive throughout 1922, J.J.R. Macleod hired Noble during the summer of 1923 to help him test and develop a new method for producing commercial quantities of insulin that Macleod believed would revolutionize insulin production. However, commercial production of insulin from fish proved impractical and was dropped by 1924, as methods to produce large quantities of mammalian insulin had improved very rapidly. Noble later played a small but critical role in the most important Canadian contribution to cancer chemotherapy research: the discovery of vinca alkaloids by his brother Robert Laing Noble. Although one might expect that a physician involved in 2 of Canada’s most important medical discoveries during the 20th century must be famous, such was not Clark Noble’s fate. He died without so much as an obituary in CMAJ.

The Pathophysiology of Diabetes and Cardiovascular Disease

Larry H. Bernstein, MD, FCAP, Reviewer and Curator
and Aviva Lev-Ari, PhD, RN, Curator

http://pharmaceuticalintelligence.com/2014/01/15/pathophysiological-effects-of-diabetes-on-ischemic-cardiovascular-disease-and-on-chronic-obstructive-pulmonary-disease-copd/

This is a multipart article that develops the pathological effects of type-2 diabetes in the progression of a systemic inflammatory disease with a development of neuropathy, and fully developing into cardiovascular disease. It also identifies a systemic relationship to the development of chronic obstructive pulmonary disease. In medical school we were taught that syphilis is the great masquerader. The more we learn about diabetes, we learn about its generalized systemic effects.

Part 1. Role of Autonomic Cardiovascular Neuropathy in Pathogenesis

This article is an abstract only of a related publication of the pathogenesis of autonomic neuropathy in diabetics leading to ischemic heart disease.

The role of autonomic cardiovascular neuropathy in pathogenesis of ischemic heart disease in patients with diabetes mellitus

Subjects: Medicine (General), Medicine, Medicine (General), Health Sciences
Authors: Popović-Pejičić Snježana, Todorović-Đilas Ljiljana, Pantelinac Pavle
Publisher: Društvo lekara Vojvodine Srpskog lekarskog društva
Publication: Medicinski Pregled 2006; 59(3-4): Pp 118-123 (2006)
http://dx.doi.org/10.2298/MPNS0604118P

http://www.doiserbia.nb.rs/img/doi/0025-8105/2006/0025-81050604118P.pdf

Keywords: diabetes mellitus, autonomic nervous system diseases, heart diseases, myocardial ischemia, comorbidity

Introduction. Diabetes is strongly associated with macrovascular complications, among which ischemic heart disease is the major cause of mortality. Autonomic neuropathy increases the risk of complications, which calls for an early diagnosis. The aim of this study was to determine both presence and extent of cardiac autonomic neuropathy, in regard to the type of diabetes mellitus, as well as its correlation with coronary disease and major cardiovascular risk factors. Material and methods. We have examined 90 subjects, classified into three groups, with 30 patients each: those with type 1 diabetes, type 2 diabetes and control group of healthy subjects. All patients underwent cardiovascular tests (Valsalva maneuver, deep breathing test, response to standing, blood pressure response to standing sustained, handgrip test), electrocardiogram, treadmill exercise test and filled out a questionnaire referring to major cardiovascular risk factors: smoking, obesity, hypertension, and dyslipidemia. Results. Our results showed that cardiovascular autonomic neuropathy was more frequent in type 2 diabetes, manifesting as autonomic neuropathy. In patients with autonomic neuropathy, regardless of the type of diabetes, the treadmill test was positive, i.e. strongly correlating with coronary disease. In regard to coronary disease risk factors, the most frequent correlation was found for obesity and hypertension. Discussion. Cardiovascular autonomic neuropathy is considered to be the principal cause of arteriosclerosis and coronary disease. Our results showed that the occurrence of cardiovascular autonomic neuropathy increases the risk of coronary disease due to dysfunction of autonomic nervous system. Conclusions. Cardiovascular autonomic neuropathy is a common complication of diabetes that significantly correlates with coronary disease. Early diagnosis of cardiovascular autonomic neuropathy points to increased cardiovascular risk, providing a basis for preventive and therapeutic measures.

Part 2. a longitudinal cohort study of the cardiovascular experience of individuals at high risk for diabetes

Protocol for ADDITION-PRO: a longitudinal cohort study of the cardiovascular experience of individuals at high risk for diabetes recruited from Danish primary care

Subjects: Public aspects of medicine, Medicine, Public Health, Health Sciences
Authors: Johansen Nanna B, Hansen Anne-Louise S, Jensen Troels M, Philipsen Annelotte, Rasmussen Signe S, Jørgensen Marit E, Simmons Rebecca K, Lauritzen Torsten, Sandbæk Annelli, Witte Daniel R
Publisher: BioMed Central Date of publication: 2012 December
Published in: BMC Public Health 2012; 12(1): 1078
ISSN(s): 1471-2458 Added to DOAJ: 2013-03-12 http://dx.doi.org/10.1186/1471-2458-12-1078 http://www.biomedcentral.com/1471-2458/12/1078

Keywords: Diabetes, Cardiovascular disease, Primary care, Complications, Microvascular, Impaired fasting glucose, Impaired glucose intolerance, Aortic stiffness, Physical activity, Body composition.

Background: Screening programmes for type 2 diabetes inevitably find more individuals at high risk for diabetes than people with undiagnosed prevalent disease. While well established guidelines for the treatment of diabetes exist, less is known about treatment or prevention strategies for individuals found at high risk following screening. In order to make better use of the opportunities for primary prevention of diabetes and its complications among this high risk group, it is important to quantify diabetes progression rates and to examine the development of early markers of cardiovascular disease and microvascular diabetic complications. We also require a better understanding of the mechanisms that underlie and drive early changes in cardiometabolic physiology. The ADDITION-PRO study was designed to address these issues among individuals at different levels of diabetes risk recruited from Danish primary care. Methods/Design: ADDITION-PRO is a population-based, longitudinal cohort study of individuals at high risk for diabetes. 16,136 eligible individuals were identified at high risk following participation in a stepwise screening programme in Danish general practice between 2001 and 2006. All individuals with impaired glucose regulation at screening, those who developed diabetes following screening, and a random sub-sample of those at lower levels of diabetes risk were invited to attend a follow-up health assessment in 2009–2011 (n = 4,188), of whom 2,082 (50%) attended. The health assessment included detailed measurement of anthropometry, body composition, biochemistry, physical activity and cardiovascular risk factors including aortic stiffness and central blood pressure. All ADDITION-PRO participants are being followed for incident cardiovascular disease and death. Discussion: The ADDITION-PRO study is designed to increase understanding of cardiovascular risk and its underlying mechanisms among individuals at high risk of diabetes. Key features of this study include (i) a carefully characterised cohort at different levels of diabetes risk; (ii) detailed measurement of cardiovascular and metabolic risk factors; (iii) objective measurement of physical activity behaviour; and (iv) long-term follow-up of hard clinical outcomes including mortality and cardiovascular disease. Results will inform policy recommendations concerning cardiovascular risk reduction and treatment among individuals at high risk for diabetes. The detailed phenotyping of this cohort will also allow a number of research questions concerning early changes in cardiometabolic physiology to be addressed.

Part 3. Clinical significance of cardiovascular dysmetabolic syndrome

This third part is a description of a longitudinal cohort study of individuals at high-risk for diabetes. Unlike the SSA study, the study is not focused on protein-energy malnutrition. This study also addresses the issue of diabetes insulin resistance leading to cardiovascular dysmetabolic syndrome.

Subjects: Diseases of the circulatory (Cardiovascular) system, Specialties of internal medicine, Internal medicine, Medicine, Cardiovascular, Medicine (General), Health Sciences
Authors: Deedwania Prakash C
Publisher: BioMed Central Date of publication: 2002 January
Published in: Trials 2002; 3: 1(2)
ISSN(s): 1468-6708
Added to DOAJ: 2004-06-03
http://dx.doi.org/10.1186/1468-6708-3-2
Full text: http://cvm.controlled-trials.com/content/3/1/2

Keywords: cardiovascular dysmetabolic syndrome, coronary heart disease, diabetes mellitus, hyperinsulinemia, insulin resistance

Although diabetes mellitus is predominantly a metabolic disorder, recent data suggest that it is as much a vascular disorder. Cardiovascular complications are the leading cause of death and disability in patients with diabetes mellitus. A number of recent reports have emphasized that many patients already have atherosclerosis in progression by the time they are diagnosed with clinical evidence of diabetes mellitus. The increased risk of atherosclerosis and cardiovascular complications in diabetic patients is related to the frequently associated dyslipidemia, hypertension, hyperglycemia, hyperinsulinemia, and endothelial dysfunction.

The evolving knowledge regarding the variety of metabolic, hormonal, and hemodynamic abnormalities in patients with diabetes mellitus has led to efforts designed for early identification of individuals at risk of subsequent disease. It has been suggested that insulin resistance, the key abnormality in type II diabetes, often precedes clinical features of diabetes by 5–6 years.

Careful attention to the criteria described for the cardiovascular dysmetabolic syndrome should help identify those at risk at an early stage. The application of nonpharmacologic as well as newer emerging pharmacologic therapies can have beneficial effects in individuals with cardiovascular dysmetabolic syndrome and/or diabetes mellitus by improving insulin sensitivity and related abnormalities. Early identification and implementation of appropriate therapeutic strategies would be necessary to contain the emerging new epidemic of cardiovascular disease related to diabetes.

Part 4. Waist circumference a good indicator of future risk for type 2 diabetes and cardiovascular disease

Subjects: Public aspects of medicine, Medicine, Public Health, Health Sciences Authors: Siren Reijo, Eriksson Johan G, Vanhanen Hannu
Publisher: BioMed Central Date of publication: 2012 August
Published in: BMC Public Health 2012; 12: 1(631)
ISSN(s): 1471-2458
Added to DOAJ: 2013-03-12
http://dx.doi.org/10.1186/1471-2458-12-631
http://www.biomedcentral.com/1471-2458/12/631

Keywords: Waist circumference, Type 2 diabetes, Cardiovascular disease, Middle-aged men.

Background: Abdominal obesity is a more important risk factor than overall obesity in predicting the development of type 2 diabetes and cardiovascular disease. From a preventive and public health point of view it is crucial that risk factors are identified at an early stage, in order to change and modify behaviour and lifestyle in high risk individuals. Methods: Data from a community based study was used to assess the risk for type 2 diabetes, cardiovascular disease and prevalence of metabolic syndrome in middle-aged men. In order to identify those with increased risk for type 2 diabetes and/or cardiovascular disease sensitivity and specificity analysis were performed, including calculation of positive and negative predictive values, and corresponding 95% CI for eleven different cut-off points, with 1 cm intervals (92 to 102 cm), for waist circumference. Results: A waist circumference ≥94 cm in middle-aged men, identified those with increased risk for type 2 diabetes and/or for cardiovascular disease with a sensitivity of 84.4% (95% CI 76.4% to 90.0%), and a specificity of 78.2% (95% CI 68.4% to 85.5%). The positive predictive value was 82.9% (95% CI 74.8% to 88.8%), and negative predictive value 80.0%, respectively (95% CI 70.3% to 87.1%). Conclusions: Measurement of waist circumference in middle-aged men is a reliable test to identify individuals at increased risk for type 2 diabetes and cardiovascular disease. This measurement should be used more frequently in daily practice in primary care in order to identify individuals at risk and when planning health counselling and interventions.

Part 5. Chronic obstructive pulmonary disease and glucose metabolism: a bitter sweet symphony

Subjects: Diseases of the circulatory (Cardiovascular) system, Specialties of internal medicine, Internal medicine, Medicine, Cardiovascular, Medicine (General), Health Sciences
Authors: Mirrakhimov Aibek E
Publisher: BioMed Central
Date of publication: Oct 2012
ISSN(s): 1475-2840
ADDED to DOAJ: 2013-03-12
Published in: Cardiovascular Diabetology 2012; 11(1):132
Journal Language(s): English Country of publication: United Kingdom
http://dx.doi.org:/10.1186/1475-2840-11-132
Full text: http://www.cardiab.com/content/11/1/132

Keywords: COPD, Dysglycemia, Insulin resistance, Obesity, Metabolic syndrome, Diabetes mellitus endothelial dysfunction, Vasculopathy

Chronic obstructive pulmonary disease, metabolic syndrome and diabetes mellitus are common and underdiagnosed medical conditions. It was predicted that chronic obstructive pulmonary disease will be the third leading cause of death worldwide by 2020. The healthcare burden of this disease is even greater if we consider the significant impact of chronic obstructive pulmonary disease on the cardiovascular morbidity and mortality.

Chronic obstructive pulmonary disease may be considered as a novel risk factor for new onset type 2 diabetes mellitus via multiple pathophysiological alterations such as: inflammation and oxidative stress, insulin resistance, weight gain and alterations in metabolism of adipokines.

On the other hand, diabetes may act as an independent factor, negatively affecting pulmonary structure and function. Diabetes is associated with an increased risk of pulmonary infections, disease exacerbations and worsened COPD outcomes. On the top of that, coexistent OSA may increase the risk for type 2 DM in some individuals.

The current scientific data necessitate a greater outlook on chronic obstructive pulmonary disease and chronic obstructive pulmonary disease may be viewed as a risk factor for the new onset type 2 diabetes mellitus. Conversely, both types of diabetes mellitus should be viewed as strong contributing factors for the development of obstructive lung disease. Such approach can potentially improve the outcomes and medical control for both conditions, and, thus, decrease the healthcare burden of these major medical problems.

The Economic Costs of Diabetes: Is It Time for a New Treatment Paradigm?

Commentary: William H. Herman
Diabetes Care Apr 2013; 36: 775-776

In a series of rigorous and exhaustive descriptive cost analyses conducted over the past decade, the American Diabetes Association (ADA) has documented an inexorable increase in the cost of diabetes in the U.S. and its detrimental impact on productivity. For the 2012 study, the ADA estimated that there were 22.3 million Americans diagnosed with diabetes. These patients incurred $306 billion in direct medical costs, more than 1 of 5 dollars spent on medical care in the U.S. The direct medical costs attributed to diabetes, that is, the costs of medical care for people with diabetes in excess of those that would be expected in the absence of diabetes, were $176 billion or approximately 1 of 8 dollars spent on medical care in the U.S. Americans with diagnosed diabetes have annual medical expenditures that are $7,900 or approximately 2.3 times higher than they would be in the absence of diabetes ($13,700 vs. $5,800). Americans with diabetes also incur $69 billion in costs related to absenteeism, reduced productivity while at work or at home, diabetes-related disability, and premature mortality. The increasing economic burden of diabetes is due in large part to the increase in the number of people with diagnosed diabetes.

Randomized controlled clinical trials have demonstrated that intensive glycemic management can delay the onset of microvascular, neuropathic, and cardiovascular complications in people with both type 1 and type 2 diabetes, and that the benefits of early intensive treatment persist over time. Randomized controlled clinical trials have also demonstrated that blood pressure management (target blood pressure 135/80 mmHg) and lipid management using statin medications can delay or prevent the development of adverse cardiovascular outcomes.

The growing economic and societal burden of diabetes as documented by the ADA in this issue of Diabetes Care highlights the urgent need to implement interventions to delay the development of type 2 diabetes. Both intensive lifestyle and pharmacologic interventions are proven effective and cost-effective. Health policy should support their implementation.

Complimentary societal interventions to delay the onset of type 2 diabetes include school-based health promotion programs and interventions that address advertising, food availability and price, the built and workplace environment, and even tax policy. In addition, early aggressive management of glycemia and cardiovascular risk factors must be implemented for persons diagnosed with diabetes. Increasing access to care, including self management education and nutritional counseling, and ensuring access to necessary treatments and supplies are critical, especially in light of the proven value of early intensive treatment in preventing chronic complications. The cost estimates provided by the ADA from 2002, 2007, and 2012 show that the economic and societal burden of diabetes is growing in the U.S. This trend underscores the importance of prevention and interventions to mitigate the complications of diabetes.

Insulin regulates carboxypeptidase E by modulating translation initiation scaffolding protein eIF4G1 in pancreatic β cells

Liew, C.W., Assmann, A., Templin, A.T., (…), Urano, F., Kulkarni, R.N
2014 Proc National Academy of Sciences USA 111 (22), pp. E2319-E2328

Insulin resistance, hyperinsulinemia, and hyperproinsulinemia occur early in the pathogenesis of type 2 diabetes (T2D). Elevated levels of proinsulin and proinsulin intermediates are markers of β-cell dysfunction and are strongly associated with development of T2D in humans. However, the mechanism(s) underlying β-cell dysfunction leading to hyperproinsulinemia is poorly understood. Here, we show that disruption of insulin receptor (IR) expression in β cells has a direct impact on the expression of the convertase enzyme carboxypeptidase E (CPE) by inhibition of the eukaryotic translation initiation factor 4 gamma 1 translation initiation complex scaffolding protein that is mediated by the key transcription factors pancreatic and duodenal homeobox 1 and sterol regulatory element-binding protein 1, together leading to poor proinsulin processing. Reexpression of IR or restoring CPE expression each independently reverses the phenotype. Our results reveal the identity of key players that establish a previously unknown link between insulin signaling, translation initiation, and proinsulin processing, and provide previously unidentified mechanistic insight into the development of hyperproinsulinemia in insulin-resistant states.

Disruption of growth factor receptor-binding protein 10 in the pancreas enhances β-cell proliferation and protects mice from streptozotocin-induced β-cell apoptosis

Zhang, J., Zhang, N., Liu, M., (…), Lu, X.-Y., Liu, F.
2014 Environmental Science and Technology 48 (9), pp. 5179-5186

It has been reported that organotin compounds such as triphenyltin or tributyltin (TBT) induce diabetes and insulin resistance. However, histopathological effects of organotin compounds on the Islets of Langerhans and exocrine pancreas are still unclear. In the present study, male KM mice were orally administered with TBT (0.5, 5, and 50 µg/kg) once every 3 days. The fasting plasma glucose levels significantly elevated, and the levels of serum insulin or glucagon decreased in the animals treated with TBT for 60 days. In animals treated for 45 days, the number of apoptotic cells in the islets and exocrine pancreas was elevated in a dose-dependent manner. The percentage of proliferating (PCNA-positive) cells was decreased in the islets, while it was increased in exocrine acinar cells. Immunohistochemistry analysis showed that estrogen receptor (ER) and androgen receptor (AR) were present in vascular endothelium, ductal cells, and islet cells, but absent from pancreatic exocrine cells. TBT exposure decreased the production of estradiol and triiodothyronine and elevated the concentration of testosterone, and resulted in a decrease of ERβ expression and an elevation of AR in the pancreas measured by Western blotting. The results suggested that TBT inhibited the proliferation and induced the apoptosis of islet cells via multipathways, causing a decrease of relative islet area in the animals treated for 60 days, which could result in a disruption of glucose homeostasis. The different presence of ERs and AR between the islets and exocrine pancreas might be one of reasons causing different effects on cell proliferation

Pancreatic alpha-cell dysfunction contributes to the disruption of glucose homeostasis and compensatory insulin hypersecretion in glucocorticoid-treated rats

Rafacho, A., Gonçalves-Neto, L.M., Santos-Silva, J.C., (…), Nadal, A., Quesada, I.
2014 Journal of Biological Chemistry 289 (9), pp. 6028-604

In α-cells, syntaxin (Syn)-1A interacts with SUR1 to inhibit ATP-sensitive potassium channels (KATP channels). PIP2 binds the Kir6.2 subunit to open KATP channels. PIP2 also modifies Syn-1A clustering in plasma membrane (PM) that may alter Syn-1A actions on PM proteins like SUR1. Here, we assessed whether the actions of PIP 2 on activating KATP channels is contributed by sequestering Syn-1A from binding SUR1. In vitro binding showed that PIP 2 dose-dependently disrupted Syn-1A·SUR1 complexes, corroborated by an in vivo Forster resonance energy transfer assay showing disruption of SUR1-(-EGFP)/Syn-1A(-mCherry) interaction along with increased Syn-1A cluster formation. Electrophysiological studies of rat α-cells, INS-1, and SUR1/Kir6.2-expressing HEK293 cells showed that PIP2 dose-dependent activation of KATP currents was uniformly reduced by Syn-1A. To unequivocally distinguish between PIP2 actions on Syn-1A and Kir6.2, we employed several strategies. First, we showed that PIP 2-insensitive Syn-1A-5RK/A mutant complex with SUR1 could not be disrupted by PIP2, consequently reducing PIP2 activation of KATP channels. Next, Syn-1A·SUR1 complex modulation of KATP channels could be observed at a physiologically low PIP 2 concentration that did not disrupt the Syn-1A·SUR1 complex, compared with higher PIP2 concentrations acting directly on Kir6.2. These effects were specific to PIP2 and not observed with physiologic concentrations of other phospholipids. Finally, depleting endogenous PIP 2 with polyphosphoinositide phosphatase synaptojanin-1, known to disperse Syn-1A clusters, freed Syn-1A from Syn-1A clusters to bind SUR1, causing inhibition of KATP channels that could no longer be further inhibited by exogenous Syn-1A. These results taken together indicate that PIP2 affects islet β-cell KATP channels not only by its actions on Kir6.2 but also by sequestering Syn-1A to modulate Syn-1A availability and its interactions with SUR1 on PM.

Aging and sleep deprivation induce the unfolded protein response in the pancreas: Implications for metabolism

Naidoo, N., Davis, J.G., Zhu, J., (…), Agarwal, B., Baur, J.A.
2014 Aging Cell 13 (1), pp. 131-141

Sleep disruption has detrimental effects on glucose metabolism through pathways that remain poorly defined. Although numerous studies have examined the consequences of sleep deprivation (SD) in the brain, few have directly tested its effects on peripheral organs. We examined several tissues in mice for induction of the unfolded protein response (UPR) following acute SD. In young animals, we found a robust induction of BiP in the pancreas, indicating an active UPR. At baseline, pancreata from aged animals exhibited a marked increase in a pro-apoptotic transcription factor, CHOP, that was amplified by SD, whereas BiP induction was not observed, suggesting a maladaptive response to cellular stress with age. Acute SD increased plasma glucose levels in both young and old animals. However, this change was not overtly related to stress in the pancreatic beta cells, as plasma insulin levels were not lower following acute SD. Accordingly, animals subjected to acute SD remained tolerant to a glucose challenge. In a chronic SD experiment, young mice were found to be sensitized to insulin and have improved glycemic control, whereas aged animals became hyperglycemic and failed to maintain appropriate plasma insulin concentrations. Our results show that both age and SD cooperate to induce the UPR in pancreatic tissue. While changes in insulin secretion are unlikely to play a major role in the acute effects of SD, CHOP induction in pancreatic tissues suggests that chronic SD may contribute to the loss or dysfunction of endocrine cells and that these effects may be exacerbated by normal aging

Bayesian total internal reflection fluorescence correlation spectroscopy reveals hIAPP-induced plasma membrane domain organization in live cells

Guo, S.-M., Bag, N., Mishra, A., Wohland, T., Bathe, M.
2014 Biophysical Journal 106 (1), pp. 190-200

Amyloid fibril deposition of human islet amyloid polypeptide (hIAPP) in pancreatic islet cells is implicated in the pathogenesis of type II diabetes. A growing number of studies suggest that small peptide aggregates are cytotoxic via their interaction with the plasma membrane, which leads to membrane permeabilization or disruption. A recent study using imaging total internal reflection-fluorescence correlation spectroscopy (ITIR-FCS) showed that monomeric hIAPP induced the formation of cellular plasma membrane microdomains containing dense lipids, in addition to the modulation of membrane fluidity. However, the spatial organization of microdomains and their temporal evolution were only partially characterized due to limitations in the conventional analysis and interpretation of imaging FCS datasets. Here, we apply a previously developed Bayesian analysis procedure to ITIR-FCS data to resolve hIAPP-induced microdomain spatial organization and temporal dynamics. Our analysis enables the visualization of the temporal evolution of multiple diffusing species in the spatially heterogeneous cell membrane, lending support to the carpet model for the association mode of hIAPP aggregates with the plasma membrane. The presented Bayesian analysis procedure provides an automated and general approach to unbiased model-based interpretation of imaging FCS data, with broad applicability to resolving the heterogeneous spatial-temporal organization of biological membrane systems.

SMAD2 disruption in mouse pancreatic beta cells leads to islet hyperplasia and impaired insulin secretion due to the attenuation of ATP-sensitive K + channel activity

Nomura, M., Zhu, H.-L., Wang, L., (…), Takayanagi, R., Teramoto, N.
2014 Diabetologia 57 (1), pp. 157-166

Aims/hypothesis: The TGF-β superfamily of ligands provides important signals for the development of pancreas islets. However, it is not yet known whether the TGF-β family signalling pathway is required for essential islet functions in the adult pancreas. Methods: To identify distinct roles for the downstream components of the canonical TGF-β signalling pathway, a Cre-loxP system was used to disrupt SMAD2, an intracellular transducer of TGF-β signals, in pancreatic beta cells (i.e. Smad2-β- knockout [KO] mice). The activity of ATP-sensitive K+ channels (KATP channels) was recorded in mutant beta cells using patch-clamp techniques. Results: The Smad2-β-KO mice exhibited defective insulin secretion in response to glucose and overt diabetes. Interestingly, disruption of SMAD2 in β-cells was associated with a striking islet hyperplasia and increased pancreatic insulin content, together with defective glucose-responsive insulin secretion. The activity of KATP channels was decreased in mutant β-cells. Conclusions/interpretation: These results suggest that in the adult pancreas, TGF-β signalling through SMAD2 is crucial for not only the determination of beta cell mass but also the maintenance of defining features of mature pancreatic beta cells, and that this involves modulation of KATP channel activity.

Disruption of protein-tyrosine phosphatase 1B expression in the pancreas affects β-cell function

Liu, S., Xi, Y., Bettaieb, A., (…), Kulkarni, R.N., Haj, F.G.
2014 Endocrinology 155 (9), pp. 3329-3338

Protein-tyrosine phosphatase 1B (PTP1B) is a physiological regulator of glucose homeostasis and energy balance. However, the role of PTP1B in pancreatic endocrine function remains largely unknown. To investigate the metabolic role of pancreatic PTP1B, we generated mice with pancreas PTP1B deletion (panc-PTP1B KO). Mice were fed regular chow or a high-fat diet, and metabolic parameters, insulin secretion and glucose tolerance were determined. On regular chow, panc-PTP1B KO and control mice exhibited comparable glucose tolerance whereas aged panc-PTP1B KO exhibited mild glucose intolerance. Furthermore, high-fat feeding promoted earlier impairment of glucose tolerance and attenuated glucose-stimulated insulin secretion in panc-PTP1B KO mice. The secretory defect in glucose-stimulated insulin secretion was recapitulated in primary islets ex vivo, suggesting that the effects were likely cell-autonomous. At the molecular level, PTP1B deficiency in vivo enhanced basal and glucose-stimulated tyrosyl phosphorylation of EphA5 in islets. Consistently, PTP1B overexpression in the glucose-responsive MIN6 β-cell line attenuated EphA5 tyrosyl phosphorylation, and substrate trapping identified EphA5 as a PTP1B substrate. In summary, these studies identify a novel role forPTP1Bin pancreatic endocrine function.

Fluorescence recovery after photobleaching reveals regulation and distribution of connexin36 gap junction coupling within mouse islets of Langerhans

Farnsworth, N.L., Hemmati, A., Pozzoli, M., Benninger, R.K.P.
2014 Journal of Physiology 592 (20), pp. 4431-4446

Key points: Gap junctions provide electrical coupling that is critical to the function of pancreatic islets. Disruptions to connexin36 (Cx36) have been suggested to occur in diabetes. No accurate and non-invasive technique has yet been established to quantify changes in Cx36 gap junction coupling in the intact islet. This study developed fluorescence recovery after photobleaching (FRAP) as a non-invasive technique for quantifying Cx36 gap junction coupling in living islets. The study identified treatments that modulate gap junction coupling, confirmed that the cellular distribution of coupling throughout the islet is highly heterogeneous and confirmed that β-cells and β-cells do not form functional Cx36 gap junctions. This technique will enable future studies to examine the regulation of Cx36 gap junction coupling and its disruption in diabetes, and to uncover potential novel therapeutic targets associated with gap junction coupling. The pancreatic islets are central to the maintenance of glucose homeostasis through insulin secretion. Glucose-stimulated insulin secretion is tightly linked to electrical activity in β-cells within the islet. Gap junctions, composed of connexin36 (Cx36), form intercellular channels between β-cells, synchronizing electrical activity and insulin secretion. Loss of gap junction coupling leads to altered insulin secretion dynamics and disrupted glucose homeostasis. Gap junction coupling is known to be disrupted in mouse models of pre-diabetes. Although approaches to measure gap junction coupling have been devised, they either lack cell specificity, suitable quantification of coupling or spatial resolution, or are invasive. The purpose of this study was to develop fluorescence recovery after photobleaching (FRAP) as a technique to accurately and robustly measure gap junction coupling in the islet. The cationic dye Rhodamine 123 was used with FRAP to quantify dye diffusion between islet β-cells as a measure of Cx36 gap junction coupling. Measurements in islets with reduced Cx36 verified the accuracy of this technique in distinguishing between distinct levels of gap junction coupling. Analysis of individual cells revealed that the distribution of coupling across the islet is highly heterogeneous. Analysis of several modulators of gap junction coupling revealed glucose- and cAMP-dependent modulation of gap junction coupling in islets. Finally, FRAP was used to determine cell population specific coupling, where no functional gap junction coupling was observed between β-cells and β-cells in the islet. The results of this study show FRAP to be a robust technique which provides the cellular resolution to quantify the distribution and regulation of Cx36 gap junction coupling in specific cell populations within the islet. Future studies utilizing this technique may elucidate the role of gap junction coupling in the progression of diabetes and identify mechanisms of gap junction regulation for potential therapies.

Glucocorticoid treatment and endocrine pancreas function: Implications for glucose homeostasis, insulin resistance and diabetes

Rafacho, A., Ortsäter, H., Nadal, A., Quesada, I.
2014 Journal of Endocrinology 223 (3), pp. R49-R62

Glucocorticoids (GCs) are broadly prescribed for numerous pathological conditions because of their anti-inflammatory, antiallergic and immunosuppressive effects, among other actions. Nevertheless, GCs can produce undesired diabetogenic side effects through interactions with the regulation of glucose homeostasis. Under conditions of excess and/or long-term treatment, GCs can induce peripheral insulin resistance (IR) by impairing insulin signalling, which results in reduced glucose disposal and augmented endogenous glucose production. In addition, GCs can promote abdominal obesity, elevate plasma fatty acids and triglycerides, and suppress osteocalcin synthesis in bone tissue. In response to GC-induced peripheral IR and in an attempt to maintain normoglycaemia, pancreatic β-cells undergo several morphofunctional adaptations that result in hyperinsulinaemia. Failure of β-cells to compensate for this situation favours glucose homeostasis disruption, which can result in hyperglycaemia, particularly in susceptible individuals. GC treatment does not only alter pancreatic β-cell function but also affect them by their actions that can lead to hyperglucagonaemia, further contributing to glucose homeostasis imbalance and hyperglycaemia. In addition, the release of other islet hormones, such as somatostatin, amylin and ghrelin, is also affected by GC administration. These undesired GC actions merit further consideration for the design of improved GC therapies without diabetogenic effects. In summary, in this review, we consider the implication of GC treatment on peripheral IR, islet function and glucose homeostasis.

β-Cell failure in type 2 diabetes: Postulated mechanisms and prospects for prevention and treatment

Halban, P.A., Polonsky, K.S., Bowden, D.W., (…), Sussel, L., Weir, G.C.
2014 Journal of Clinical Endocrinology and Metabolism 99 (6), pp. 1983-1992

OBJECTIVE: This article examines the foundation of β-cell failure in type 2 diabetes (T2D) and suggests areas for future research on the underlying mechanisms that may lead to improved prevention and treatment. RESEARCH DESIGN AND METHODS: A group of experts participated in a conference on 14-16 October 2013 cosponsored by the Endocrine Society and the American Diabetes Association. A writing group prepared this summary and recommendations. RESULTS: The writing group based this article on conference presentations, discussion, and debate. Topics covered include genetic predisposition, foundations of β-cell failure, natural history of β-cell failure, and impact of therapeutic interventions. CONCLUSIONS: β-Cell failure is central to the development and progression of T2D. It antedates and predicts diabetes onset and progression, is in part genetically determined, and often can be identified with accuracy even though current tests are cumbersome and not well standardized. Multiple pathways underlie decreased β-cell function and mass, some of which may be shared and may also be a consequence of processes that initially caused dysfunction. Goals for future research include to 1) impact the natural history of β-cell failure; 2) identify and characterize genetic loci for T2D; 3) target β-cell signaling, metabolic, and genetic pathways to improve function/mass; 4) develop alternative sources of β-cells for cell-based therapy; 5) focus on metabolic environment to provide indirect benefit to β-cells; 6) improve understanding of the physiology of responses to bypass surgery; and 7) identify circulating factors and neuronal circuits underlying the axis of communication between the brain and β-cells.

Metabolic effects of sleep disruption, links to obesity and diabetes

Nedeltcheva, A.V., Scheer, F.A.J.L
2014 Current Opinion in Endocrinology, Diabetes and Obesity 21 (4), pp. 293-298

Purpose of Review: To highlight the adverse metabolic effects of sleep disruption and to open ground for research aimed at preventive measures. This area of research is especially relevant given the increasing prevalence of voluntary sleep curtailment, sleep disorders, diabetes, and obesity. Recent Findings: Epidemiological studies have established an association between decreased self-reported sleep duration and an increased incidence of type 2 diabetes (T2D), obesity, and cardiovascular disease. Experimental laboratory studies have demonstrated that decreasing either the amount or quality of sleep decreases insulin sensitivity and decreases glucose tolerance. Experimental sleep restriction also causes physiological and behavioral changes that promote a positive energy balance. Although sleep restriction increases energy expenditure because of increased wakefulness, it can lead to a disproportionate increase in food intake, decrease in physical activity, and weight gain. SUMMARY: Sleep disruption has detrimental effects on metabolic health. These insights may help in the development of new preventive and therapeutic approaches against obesity and T2D based on increasing the quality and/or quantity of sleep. Video abstract http://links.lww.com/COE/A6.

Impaired proteostasis: Role in the pathogenesis of diabetes mellitus

Jaisson, S., Gillery, P.
2014 Diabetologia 57 (8), pp. 1517-1527

In living organisms, proteins are regularly exposed to ‘molecular ageing’, which corresponds to a set of non-enzymatic modifications that progressively cause irreversible damage to proteins. This phenomenon is greatly amplified under pathological conditions, such as diabetes mellitus. For their survival and optimal functioning, cells have to maintain protein homeostasis, also called ‘proteostasis’. This process acts to maintain a high proportion of functional and undamaged proteins. Different mechanisms are involved in proteostasis, among them degradation systems (the main intracellular proteolytic systems being proteasome and lysosomes), folding systems (including molecular chaperones), and enzymatic mechanisms of protein repair. There is growing evidence that the disruption of proteostasis may constitute a determining event in pathophysiology. The aim of this review is to demonstrate how such a dysregulation may be involved in the pathogenesis of diabetes mellitus and in the onset of its long-term complications.

Influence of miRNA in insulin signaling pathway and insulin resistance: Micro-molecules with a major role in type-2 diabetes

Chakraborty, C., Doss, C.G.P., Bandyopadhyay, S., Agoramoorthy, G.
2014 Wiley Interdisciplinary Reviews: RNA 5 (5), pp. 697-712

The prevalence of type-2 diabetes (T2D) is increasing significantly throughout the globe since the last decade. This heterogeneous and multifactorial disease, also known as insulin resistance, is caused by the disruption of the insulin signaling pathway. In this review, we discuss the existence of various miRNAs involved in regulating the main protein cascades in the insulin signaling pathway that affect insulin resistance. The influence of miRNAs (miR-7, miR-124α, miR-9, miR-96, miR-15α/β, miR-34α, miR-195, miR-376, miR-103, miR-107, and miR-146) in insulin secretion and beta (β) cell development has been well discussed. Here, we highlight the role of miRNAs in different significant protein cascades within the insulin signaling pathway such as miR-320, miR-383, miR-181β with IGF-1, and its receptor (IGF1R); miR-128α, miR-96, miR-126 with insulin receptor substrate (IRS) proteins; miR-29, miR-384-5p, miR-1 with phosphatidylinositol 3-kinase (PI3K); miR-143, miR-145, miR-29, miR-383, miR-33α/β miR-21 with AKT/protein kinase B (PKB) and miR-133α/β, miR-223, miR-143 with glucose transporter 4 (GLUT4). Insulin resistance, obesity, and hyperlipidemia (high lipid levels in the blood) have a strong connection with T2D and several miRNAs influence these clinical outcomes such as miR-143, miR-103, and miR-107, miR-29α, and miR-27β. We also corroborate from previous evidence how these interactions are related to insulin resistance and T2D. The insights highlighted in this review will provide a better understanding on the impact of miRNA in the insulin signaling pathway and insulin resistance-associated diagnostics and therapeutics for T2D

Genetic disruption of sod1 gene causes glucose intolerance and impairs β-cell function

Muscogiuri, G., Salmon, A.B., Aguayo-Mazzucato, C., (…), Van Remmen, H., Musi, N.
2013 Diabetes 62 (12), pp. 4201-4207

Oxidative stress has been associated with insulin resistance and type 2 diabetes. However, it is not clear whether oxidative damage is a cause or a consequence of the metabolic abnormalities present in diabetic subjects. The goal of this study was to determine whether inducing oxidative damage through genetic ablation of superoxide dismutase 1 (SOD1) leads to abnormalities in glucose homeostasis. We studied SOD1-null mice and wild-type (WT) littermates. Glucose tolerance was evaluated with intraperitoneal glucose tolerance tests. Peripheral and hepatic insulin sensitivity was quantitated with the euglycemic-hyperinsulinemic clamp. β-Cell function was determined with the hyperglycemic clamp and morphometric analysis of pancreatic islets. Genetic ablation of SOD1 caused glucose intolerance, which was associated with reduced in vivo β-cell insulin secretion and decreased b-cell volume. Peripheral and hepatic insulin sensitivity were not significantly altered in SOD1-null mice. High-fat diet caused glucose intolerance in WT mice but did not further worsen the glucose intolerance observed in standard chow-fed SOD1-null mice. Our findings suggest that oxidative stress per se does not play a major role in the pathogenesis of insulin resistance and demonstrate that oxidative stress caused by SOD1 ablation leads to glucose intolerance secondary to β-cell dysfunction.

VHL-mediated disruption of Sox9 activity compromises β-cell identity and results in diabetes mellitus

Puri, S., Akiyama, H., Hebrok, M.
2013 Genes and Development 27 (23), pp. 2563-2575

Precise functioning of the pancreatic β cell is paramount to whole-body glucose homeostasis, and β-cell dysfunction contributes significantly to diabetes mellitus. Using transgenic mouse models, we demonstrate that deletion of the von Hippel-Lindau (Vhlh) gene (encoding an E3 ubiquitin ligase implicated in, among other functions, oxygen sensing in pancreatic β cells) is deleterious to canonical β-cell gene expression. This triggers erroneous expression of factors normally active in progenitor cells, including effectors of the Notch, Wnt, and Hedgehog signaling cascades. Significantly, an up-regulation of the transcription factor Sox9, normally excluded from functional β cells, occurs upon deletion of Vhlh. Sox9 plays important roles during pancreas development but does not have a described role in the adult β cell. β-Cell-specific ectopic expression of Sox9 results in diabetes mellitus from similar perturbations in β-cell identity. These findings reveal that assaults on the β cell that impact the differentiation state of the cell have clear implications toward our understanding of diabetes mellitus

Second generation antipsychotic-induced type 2 diabetes: A role for the muscarinic M3 receptor

Weston-Green, K., Huang, X.-F., Deng, C.
2013 CNS Drugs 27 (12), pp. 1069-1080

Second generation antipsychotics (SGAs) are widely prescribed to treat various disorders, most notably schizophrenia and bipolar disorder; however, SGAs can cause abnormal glucose metabolism that can lead to insulin-resistance and type 2 diabetes mellitus side-effects by largely unknown mechanisms. This review explores the potential candidature of the acetylcholine (ACh) muscarinic M3 receptor (M3R) as a prime mechanistic and possible therapeutic target of interest in SGA-induced insulin dysregulation. Studies have identified that SGA binding affinity to the M3R is a predictor of diabetes risk; indeed, olanzapine and clozapine, SGAs with the highest clinical incidence of diabetes side-effects, are potent M3R antagonists. Pancreatic M3Rs regulate the glucose-stimulated cholinergic pathway of insulin secretion; their activation on β-cells stimulates insulin secretion, while M3R blockade decreases insulin secretion. Genetic modification of M3Rs causes robust alterations in insulin levels and glucose tolerance in mice. Olanzapine alters M3R density in discrete nuclei of the hypothalamus and caudal brainstem, regions that regulate glucose homeostasis and insulin secretion through vagal innervation of the pancreas. Furthermore, studies have demonstrated a dynamic sensitivity of hypothalamic and brainstem M3Rs to altered glucometabolic status of the body. Therefore, the M3R is in a prime position to influence glucose homeostasis through direct effects on pancreatic β-cells and by potentially altering signaling in the hypothalamus and brainstem. SGA-induced insulin dysregulation may be partly due to blockade of central and peripheral M3Rs, causing an initial disruption to insulin secretion and glucose homeostasis that can progressively lead to insulin resistance and diabetes during chronic treatment.

Islet amyloid polypeptide toxicity and membrane interactions

Cao, P., Abedini, A., Wang, H., (…), Schmidt, A.M., Raleigh, D.P.
2013 Proc National Academy of Sciences USA 110 (48), pp. 19279-19284

Islet amyloid polypeptide (IAPP) is responsible for amyloid formation in type 2 diabetes and contributes to the failure of islet cell transplants, however the mechanisms of IAPP-induced cytotoxicity are not known. Interactions with model anionic membranes are known to catalyze IAPP amyloid formation in vitro. Human IAPP damages anionic membranes, promoting vesicle leakage, but the features that control IAPP-membrane interactions and the connection with cellular toxicity are not clear. Kinetic studies with wild type IAPP and IAPP mutants demonstrate that membrane leakage is induced by prefibrillar IAPP species and continues over the course of amyloid formation, correlating additional membrane disruption with fibril growth. Analyses of a set of designed mutants reveal that membrane leakage does not require the formation of α-sheet or α-helical structures. A His-18 to Arg substitution enhances leakage, whereas replacement of all of the aromatic residues via a triple leucine mutant has no effect. Biophysical measurements in conjunction with cytotoxicity studies show that nonamyloidogenic rat IAPP is as effective as human IAPP at disrupting standard anionic model membranes under conditions where rat IAPP does not induce cellular toxicity. Similar results are obtained with more complex model membranes, including ternary systems that contain cholesterol and are capable of forming lipid rafts. A designed point mutant, I26P-IAPP; a designed double mutant, G24P, I26P-IAPP; a double N-methylated variant; and pramlintide, a US Food and Drug Administration-approved IAPP variant all induce membrane leakage, but are not cytotoxic, showing that there is no one-to-one relationship between disruption of model membranes and induction of cellular toxicity.

Diabetes and beta cell function: From mechanisms to evaluation and clinical implications

Cernea, S., Dobreanu, M.
2013 Biochemia Medica 23 (3), pp. 266-280

Diabetes is a complex, heterogeneous condition that has beta cell dysfunction at its core. Many factors (e.g. hyperglycemia/glucotoxicity, lipotoxicity, autoimmunity, inflammation, adipokines, islet amyloid, incretins and insulin resistance) influence the function of pancreatic beta cells. Chronic hyperglycemia may result in detrimental effects on insulin synthesis/secretion, cell survival and insulin sensitivity through multiple mechanisms: gradual loss of insulin gene expression and other beta-cell specific genes; chronic endoplasmic reticulum stress and oxidative stress; changes in mitochondrial number, morphology and function; disruption in calcium homeostasis. In the presence of hyperglycemia, prolonged exposure to increased free fatty acids result in accumulation of toxic metabolites in the cells (“lipotoxicity”), finally causing decreased insulin gene expression and impairment of insulin secretion. The rest of the factors/mechanisms which impact on the course of the disease are also discusses in detail. The correct assessment of beta cell function requires a concomitant quantification of insulin secretion and insulin sensitivity, because the two variables are closely interrelated. In order to better understand the fundamental pathogenetic mechanisms that contribute to disease development in a certain individual with diabetes, additional markers could be used, apart from those that evaluate beta cell function. The aim of the paper was to overview the relevant mechanisms/factors that influence beta cell function and to discuss the available methods of its assessment. In addition, clinical considerations are made regarding the therapeutical options that have potential protective effects on beta cell function/mass by targeting various underlying factors and mechanisms with a role in disease progression.

The PACAP-regulated gene selenoprotein T is abundantly expressed in mouse and human β-cells and its targeted inactivation impairs glucose tolerance

Prevost, G., Arabo, A., Jian, L., (…), Pattou, F., Anouar, Y
2013 Endocrinology 154 (10), pp. 3796-3806

Selenoproteins are involved in the regulation of redox status, which affects several cellular processes, including cell survival and homeostasis. Considerable interest has arisen recently concerning the role of selenoproteins in the regulation of glucose metabolism. Here, we found that selenoprotein T (SelT), a new thioredoxin-like protein of the endoplasmic reticulum, is present at high levels in human and mouse pancreas as revealed by immunofluorescence and quantitative PCR. Confocal immunohistochemistry studies revealed that SelT is mostly confined to insulin- and somatostatin-producing cells in mouse and human islets. To elucidate the role of SelT in β-cells, we generated, using a Cre-Lox strategy, a conditional pancreatic β-cell SelT-knockout C57BL/6J mice (SelT-insKO) in which SelT gene disruption is under the control of the rat insulin promoter Cre gene. Glucose administration revealed that male SelT-insKO mice display impaired glucose tolerance. Although insulin sensitivity was not modified in the mutant mice, the ratio of glucose to insulin was significantly higher in the SelT-insKO mice compared with wild-type littermates, pointing to a deficit in insulin production/secretion in mutant mice. In addition, morphometric analysis showed that islets from SelT-insKO mice were smaller and that their number was significantly increased compared with islets from their wild-type littermates. Finally, we found that SelT is up-regulated by pituitary adenylate cyclase-activating polypeptide (PACAP) in β-pancreatic cells and that SelT could act by facilitating a feed-forward mechanism to potentiate insulin secretion induced by the neuropeptide. Our findings are the first to show that the PACAP-regulated SelT is localized in pancreatic α- and β-cells and is involved in the control of glucose homeostasis

SIRT1 deacetylates FOXA2 and is critical for Pdx1 transcription and β-cell formation

Wang, R.-H., Xu, X., Kim, H.-S., Xiao, Z., Deng, C.-X.
2013 International Journal of Biological Sciences 9 (9), pp. 934-946

Pancreas duodenum homeobox 1 (PDX1) is essential for pancreas development and β-cell formation; however more studies are needed to clearly illustrate the precise mechanism regarding spatiotemporal regulation of Pdx1 expression during β-cell formation and development. Here, we demonstrate that SIRT1, FOXA2 and a number of proteins form a protein complex on the promoter of the Pdx1 gene. SIRT1 and PDX1 are expressed in the same set of cells during β-cell differentiation and maturation. Pancreas-specific disruption of SIRT1 diminished PDX1 expression and impaired islet development. Consequently, SIRT1 mutant mice develop progressive hyperglycemia, glucose intolerance, and insulin insufficiency, which directly correlate with the extent of SIRT1 deletion. We further show that SIRT1 interacts with and deacetylates FOXA2 on the promoter of the Pdx1gene, and positively regulates its transcription. These results uncover an essential role of SIRT1 in β-cell formation by maintaining expression of PDX1 and its downstream genes, and identify pancreas-specific SIRT1 mutant mice as a relevant model for studying insulin insufficiency.

NOX, NOX who is there? The contribution of NADPH oxidase one to beta cell dysfunction

Taylor-Fishwick, D.A.
2013 Frontiers in Endocrinology 4 (APR), Article 40

Predictions of diabetes prevalence over the next decades warrant the aggressive discovery of new approaches to stop or reverse loss of functional beta cell mass. Beta cells are recognized to have a relatively high sensitivity to reactive oxygen species (ROS) and become dysfunctional under oxidative stress conditions. New discoveries have identified NADPH oxidases in beta cells as contributors to elevated cellular ROS. Reviewed are recent reports that evidence a role for NADPH oxidase-1 (NOX-1) in β-cell dysfunction. NOX-1 is stimulated by inflammatory cytokines that are elevated in diabetes. First, regulation of cytokine-stimulated NOX-1 expression has been linked to inflammatory lipid mediators derived from 12-lipoxygenase activity. For the first time in beta cells these data integrate distinct pathways associated with beta cell dysfunction. Second, regulation of NOX-1 in
β-cells involves feed-forward control linked to elevated ROS and Src-kinase activation. This potentially results in unbridled ROS generation and identifies candidate targets for pharmacologic intervention. Third, consideration is provided of new, first-in-class, selective inhibitors of NOX-1. These compounds could have an important role in assessing a disruption of NOX-1/ROS signaling as a new approach to preserve and protect beta cell mass in diabetes.

Retinoblastoma tumor suppressor protein in pancreatic progenitors controls α- and β-cell fate

Cai, E.P., Wu, X., Schroer, S.A., (…), Zacksenhaus, E., Woo, M.
2013 Proc National Academy of Sciences USA 110 (36), pp. 14723-14728

Pancreatic endocrine cells expand rapidly during embryogenesis by neogenesis and proliferation, but during adulthood, islet cells have a very slow turnover. Disruption of murine retinoblastoma tumor suppressor protein (Rb) in mature pancreatic β-cells has a limited effect on cell proliferation. Here we show that deletion of Rb during embryogenesis in islet progenitors leads to an increase in the neurogenin 3-expressing precursor cell population, which persists in the postnatal period and is associated with increased β-cell mass in adults. In contrast, Rb-deficient islet precursors, through repression of the cell fate factor aristaless related homeobox, result in decreased β-cell mass. The opposing effect on survival of Rb-deficient β- and β-cells was a result of opposing effects on p53 in these cell types. As a consequence, loss of Rb in islet precursors led to a reduced α- to β-cell ratio, leading to improved glucose homeostasis and protection against diabetes.

Statin therapy and new-onset diabetes: Molecular mechanisms and clinical relevance

Banach, M., Malodobra-Mazur, M., Gluba, A., (…), Rysz, J., Dobrzyn, A.
2013 Current Pharmaceutical Design 19 (27), pp. 4904-4912

Despite positive effects on the plasma lipid profile and vascular events, statin use is associated with various side effects. Among these, statins might cause a disruption of a number of regulatory pathways including insulin signaling. This may affect insulin sensitivity, pancreatic beta-cell function and adipokine secretion. The statin-associated risk of new-onset diabetes (NOD) appears to be a dose-dependent class effect. It still remains unclear whether statin treatment is associated with increased risk of NOD in the general population or if there are groups of individuals at particular risk. However, according to the available data it seems that cardiovascular (CV) benefits in high-risk individuals strongly favor statin therapy since it outweighs other risks. Whether statins should be used for primary prevention among patients with a relatively low baseline CV risk is still questionable, however the results of primary prevention trials have shown reductions in mortality in this population. Thus, there is a need for randomized, placebo-controlled statin studies with carefully selected groups of patients and NOD as a key end point in order to resolve queries concerning this issue.

Basement membrane extract preserves islet viability and activity in vitro by up-regulating α3 integrin and its signal

Miao, G., Zhao, Y., Li, Y., (…), Li, J., Wei, J
2013 Pancreas 42 (6), pp. 971-976

OBJECTIVE: Survival of transplanted islets is limited partly because of the disruption of the islet basement membrane (BM) occurring during isolation. We hypothesized that the embedment of BM extract (BME) could induce a viable cell mass and prolong islet functionality before transplantation. METHODS: A special reconstituted BME that solidifies into a gel at 37 C was used to embed isolated islets in this study. The strategy was used to re-establish the interaction between the islets and peri-islet BM. RESULTS: Islets embedded in BME showed lower caspase-3 levels and higher Akt activity than those in suspension. Moreover, we found for the first time that the expression of β3 integrin and focal adhesion kinase (FAK) and FAK activity was up-regulated in islets after BME embedment. The reverse effect was observed on islet apoptosis when islets rescued from a 24-hour suspension culture were embedded in BME for the next 24 hours. In addition, expression of pancreatic duodenal homeobox factor-1 and phospho-extracellular signal-regulated kinase 1/2 was partially preserved, suggesting the positive effect of BME on islet development. CONCLUSIONS: These results indicate that BME embedment of islets can up-regulate the expression of β3 integrin and its signal transduction, which may improve islet viability.

Involvement of the Clock Gene Rev-erb alpha in the Regulation of Glucagon Secretion in Pancreatic Alpha-Cells

Vieira, E., Marroquí, L., Figueroa, A.C., (…), Gomis, R., Quesada, I.
2013 PLoS ONE 8 (7), e6993

Disruption of pancreatic clock genes impairs pancreatic β-cell function, leading to the onset of diabetes. Despite the importance of pancreatic α-cells in the regulation of glucose homeostasis and in diabetes pathophysiology, nothing is known about the role of clock genes in these cells. Here, we identify the clock gene Rev-erbα as a new intracellular regulator of glucagon secretion. Rev-erbα down-regulation by siRNA (60-70% inhibition) in alphaTC1-9 cells inhibited low-glucose induced glucagon secretion (p<0.05) and led to a decrease in key genes of the exocytotic machinery. The Rev-erbα agonist GSK4112 increased glucagon secretion (1.6 fold) and intracellular calcium signals in αTC1-9 cells and mouse primary alpha-cells, whereas the Rev-erbα antagonist SR8278 produced the opposite effect. At 0.5 mM glucose, alphaTC1-9 cells exhibited intrinsic circadian Rev-erbα expression oscillations that were inhibited by 11 mM glucose. In mouse primary alpha-cells, glucose induced similar effects (p<0.001). High glucose inhibited key genes controlled by AMPK such as Nampt, Sirt1 and PGC-1 alpha in alphaTC1-9 cells (p<0.05). AMPK activation by metformin completely reversed the inhibitory effect of glucose on Nampt-Sirt1-PGC-1 alpha and Rev-erb alpha. Nampt inhibition decreased Sirt1, PGC-1 alpha and Rev-erb alpha mRNA expression (p<0.01) and glucagon release (p<0.05). These findings identify Rev-erb alpha as a new intracellular regulator of glucagon secretion via AMPK/Nampt/Sirt1 pathway.

Bmal1 and β-cell clock are required for adaptation to circadian disruption, and their loss of function leads to oxidative stress- induced β-cell failure in mice

Lee, J., Moulik, M., Fang, Z., (…), Moore, D.D., Yechoor, V.K.
2013 Molecular and Cellular Biology 33 (11), pp. 2327-2338

Circadian disruption has deleterious effects on metabolism. Global deletion of Bmal1, a core clock gene, results in β-cell dysfunction and diabetes. But it is unknown if this is due to loss of cell-autonomous function of Bmal1 in β cells. To address this, we generated mice with β-cell clock disruption by deleting Bmal1 in β cells (β-Bmal1-/-). β-Bmal1-/- mice develop diabetes due to loss of glucose-stimulated insulin secretion (GSIS). This loss of GSIS is due to the accumulation of reactive oxygen species (ROS) and consequent mitochondrial uncoupling, as it is fully rescued by scavenging of the ROS or by inhibition of uncoupling protein 2. The expression of the master antioxidant regulatory factor Nrf2 (nuclear factor erythroid 2-related factor 2) and its targets, Sesn2, Prdx3, Gclc, and Gclm, was decreased in β-Bmal1-/- islets, which may contribute to the observed increase in ROS accumulation. In addition, by chromatin immunoprecipitation experiments, we show that Nrf2 is a direct transcriptional target of Bmal1. Interestingly, simulation of shift work-induced circadian misalignment in mice recapitulates many of the defects seen in Bmal1-deficient islets.

Thus, the cell-autonomous function of Bmal1 is required for normal β-cell function by mitigating oxidative stress and serves to preserve β-cell function in the face of circadian misalignment.

A common landscape for membraneactive peptides

Last, N.B., Schlamadinger, D.E., Miranker, A.D.
2013 Protein Science 22 (7), pp. 870-882

Three families of membrane-active peptides are commonly found in nature and are classified according to their initial apparent activity. Antimicrobial peptides are ancient components of the innate immune system and typically act by disruption of microbial membranes leading to cell death. Amyloid peptides contribute to the pathology of diverse diseases from Alzheimer’s to type II diabetes. Preamyloid states of these peptides can act as toxins by binding to and permeabilizing cellular membranes. Cell-penetrating peptides are natural or engineered short sequences that can spontaneously translocate across a membrane. Despite these differences in classification, many similarities in sequence, structure, and activity suggest that peptides from all three classes act through a small, common set of physical principles. Namely, these peptides alter the Brownian properties of phospholipid bilayers, enhancing the sampling of intrinsic fluctuations that include membrane defects. A complete energy landscape for such systems can be described by the innate membrane properties, differential partition, and the associated kinetics of peptides dividing between surface and defect regions of the bilayer. The goal of this review is to argue that the activities of these membrane-active families of peptides simply represent different facets of what is a shared energy landscape.

Membrane disordering is not sufficient for membrane permeabilization by islet amyloid polypeptide: Studies of IAPP(20-29) fragments

Brender, J.R., Heyl, D.L., Samisetti, S., (…), Pesaru, R.R., Ramamoorthy, A.
2013 Physical Chemistry Chemical Physics 15 (23), pp. 8908-8915

A key factor in the development of type II diabetes is the loss of insulin-producing beta-cells. Human islet amyloid polypeptide protein (human-IAPP) is believed to play a crucial role in this process by forming small aggregates that exhibit toxicity by disrupting the cell membrane. The actual mechanism of membrane disruption is complex and appears to involve an early component before fiber formation and a later component associated with fiber formation on the membrane. By comparing the peptide-lipid interactions derived from solid-state NMR experiments of two IAPP fragments that cause membrane disordering to IAPP derived peptides known to cause significant early membrane permeabilization, we show here that membrane disordering is not likely to be sufficient by itself to cause the early membrane permeabilization observed by IAPP, and may play a lesser role in IAPP membrane disruption than expected.
Downregulation of Fas activity rescues early onset of diabetes in c-KitWv/+ mice

Feng, Z.-C., Riopel, M., Li, J., Donnelly, L., Wang, R.
2013 American Journal of Physiology – Endocrinology and Metabolism 304 (6), pp. E557-E565

c-Kit and its ligand stem cell factor (SCF) are important for β-cell survival and maturation; meanwhile, interactions between the Fas receptor (Fas) and Fas ligand are capable of triggering β-cell apoptosis. Disruption of c-Kit signaling leads to severe loss of β-cell mass and function with upregulation of Fas expression in c-KitWv/++ mouse islets, suggesting that there is a critical balance between c-Kit and Fas activation in β-cells. In the present study, we investigated the interrelationship between c-Kit and Fas activation that mediates β-cell survival and function. We generated double mutant, c-KitWv/++;Faslpr/lpr (Wv-/-), mice to study the physiological and functional role of Fas with respect to β-cell function in c-KitWv/++ mice. Isolated islets from these mice and the INS-1 cell line were used. We observed that islets in c-KitWv/++ mice showed a significant increase in β-cell apoptosis along with upregulated p53 and Fas expression. These results were verified in vitro in INS-1 cells treated with SCF or c-Kit siRNA combined with a p53 inhibitor and Fas siRNA. In vivo, Wv-/- mice displayed improved β-cell function, with significantly enhanced insulin secretion and increased β-cell mass and proliferation compared with Wv+/+ mice. This improvement was associated with downregulation of the Fas-mediated caspase-dependent apoptotic pathway and upregulation of the cFlip/NF-?B pathway. These findings demonstrate that a balance between the c-Kit and Fas signaling pathways is critical in the regulation of β-cell survival and function.
Study Suggests Genetic Susceptibility to T2D May Have Shifted with Human Migration

May 24, 2013 By a GenomeWeb staff reporter

NEW YORK (GenomeWeb News) – The apparent genetic risk for type 2 diabetes seems to vary between human populations from different parts of the world, new research suggests, with populations in Africa and East Asia showing particularly pronounced differences in T2D susceptibility.

A pair of papers appearing online — both led by investigators at Stanford University — outline the approaches and analyses used to reach that conclusion.

For the first study, published in PLOS Genetics, researchers trolled through data on more than 1,000 individuals from around the world who’d been genotyped for the Human Genome Diversity Panel project. Patterns in that data revealed geography or population-related differentiation in the genetic risk associated with certain diseases.

“We demonstrated that differences in genetic risk for multiple diseases go well beyond what is expected by genetic drift,” the study authors noted. “In addition, using a human population phylogenetic tree allowed us to elucidate a substructure of worldwide relationships.”

In the East Asian population, for instance, the team saw diminished genetic risk for both T2D and pancreatic cancer. On the other hand, individuals of African ancestry appeared to be more apt to carry T2D risk alleles, results of the analysis suggest, pointing to possible migration-related shifts in genetic susceptibility to T2D.

For their PLOS Genetics analysis, the researchers used data for 1,043 individuals genotyped for the HGDP to delve into the genetic risk associated with more than 100 diseases, including T2D.

Because the individuals hailed from 51 different populations around the world, the group was able to get a glimpse at relationships between these genetic risk contributors and human migration and population patterns.

From that data, investigators saw at least 11 conditions for which risk variant profiles differed across human populations, researchers reported, including ulcerative colitis, bladder cancer, lupus, and inflammatory bowel disease.

For T2D, that genetic differentiation appeared to correspond with population patterns stemming from human migrations out of Africa and into other parts of the world. For instance, the analysis indicated that genetic risk for T2D dips in East Asian populations but tends to be elevated in populations from Africa — particularly the Mandinka population, which appeared to be at highest genetic risk of T2D.

“East Asians definitely get diabetes,” Stanford University’s Atul Butte, senior author on the study, said in a statement.

Nevertheless, he added, it’s possible that there are population-specific differences in the risk alleles and genetic pathways involved, potentially producing somewhat distinct forms of the disease.

Those involved in the study noted that additional, follow-up research is needed, including whole-genome sequencing analysis, which can offer a look at larger structural variants contributing to disease risk in different populations, for instance.

But if findings from the current analysis hold in future studies, that may ultimately prompt a shift in researchers’ understanding of T2D and the factors contributing to it.

“Other fields of medicine have undergone a radical rethinking in disease taxonomy,” Butte said in a statement, “but this has not happened yet for diabetes, one of the world’s public health menaces.”

“If these are separate diseases at a molecular level, we need to try to understand that,” he added.

A related study in the journal Diabetes Care, also by Stanford’s Butte and his colleagues, touched on the consequences of such genetic differences. That work highlighted apparent clinical differences in T2D-related traits — particularly in insulin resistance and insulin response — in African, East Asian, and Caucasian populations.

More generally, Butte and his colleagues put together a so-called “Genetic Risk World Map” to tie together the information generated from their study of disease risk genetics in the context of human migration. The resource is available online through a Stanford website.
Use of pioglitazone in the treatment of diabetes: effect on cardiovascular risk

Authors: Zou C, Hu H
Published Date: 25 July 2013; 9: 429 – 433
DOI: http://dx.doi.org/10.2147/VHRM.S34421

Pioglitazone and other thiazolidinediones (TZDs) initially showed great promise as unique receptor-mediated oral therapy for type 2 diabetes, but a host of serious side effects, primarily cardiovascular, have limited their utility. It is crucial at this point to perform a risk–benefit analysis to determine what role pioglitazone should play in our current treatment of type 2 diabetes and where the future of this class of drugs is headed. This review provides a comprehensive overview of the present literature. Clinical data currently available indicate that pioglitazone is an effective and generally well-tolerated treatment option for use in patients with type 2 diabetes. Pioglitazone can still reduce adverse cardiovascular risk.

Glucophage, Glucophage XR

In a US double-blind clinical study of GLUCOPHAGE in patients with type 2 diabetes, a total of 141 patients received GLUCOPHAGE therapy (up to 2550 mg per day) and 145 patients received placebo. Adverse reactions reported in greater than 5% of the GLUCOPHAGE patients, and that were more common in GLUCOPHAGE- than placebo-treated patients are reported.

The following adverse reactions were reported in ≥ 1.0% to ≤ 5.0% of GLUCOPHAGE patients and were more commonly reported with GLUCOPHAGE than placebo:

abnormal stools, – myalgia, – lightheaded, – dyspnea,

the following adverse reactions were reported in ≥ 1.0% to ≤ 5.0% of GLUCOPHAGE XR patients and were more commonly reported with GLUCOPHAGE XR than placebo

dizziness, – More common

Metabolic side effects have included lactic acidosis, which is a potentially fatal metabolic complication. The incidence of lactic acidosis has been about 1.5 cases per 10,000 patient years. The risk of lactic acidosis has been particularly high in patients with underlying renal insufficiency. Cases of lactic acidosis occurring in patients with normal renal function have been rarely reported.

Signs and symptoms of severe acidosis may include bradycardia (lactic acidosis)
lactic acid concentration, serum electrolytes, blood pH

High-Fructose Corn Syrup Linked to Diabetes

By Brenda Goodman, MA WebMD Health News
Reviewed By Louise Chang, MD

In a study published in the journal Global Health, researchers compared the average availability of high-fructose corn syrup to rates of diabetes in 43 countries.

About half the countries in the study had little or no high-fructose corn syrup in their food supply. In the other 20 countries, high-fructose corn syrup in foods ranged from about a pound a year per person in Germany to about 55 pounds each year per person in the United States.

The researchers found that countries using high-fructose corn syrup had rates of diabetes that were about 20% higher than countries that didn’t mix the sweetener into foods. Those differences remained even after researchers took into account data for differences in body size, population, and wealth.

But couldn’t that mean that people in countries that used more high-fructose corn syrup were just eating more sugar or more total calories?

The researchers say no: There were no overall differences in total sugars or total calories between countries that did and didn’t use high-fructose corn syrup, suggesting that there’s an independent relationship between high-fructose corn syrup and diabetes.

“It raises a lot of questions about fructose,” says researcher Michael I. Goran, PhD, co-director of the Diabetes and Obesity Research Institute at the Keck School of Medicine at the University of Southern California, in Los Angeles. Although the study found an association, it doesn’t establish a cause/effect relationship.
Genetic association of ADIPOQ gene variants with type 2 diabetes, obesity and serum adiponectin levels in south Indian population.

Ramya K; Ayyappa KA; Ghosh S; Mohan V; Radha V
Gene 2013 Dec 15;532(2):253-62 (ISSN: 1879-0038)

OBJECTIVE: To investigate the genetic association of eight variants of the adiponectin gene with type 2 diabetes mellitus (T2DM), obesity and serum adiponectin level in the south Indian population. METHODS: The study comprised of 1100 normal glucose tolerant (NGT) and 1100 type 2 diabetic, unrelated subjects randomly selected from the Chennai Urban Rural Epidemiology Study (CURES), in southern India. Fasting serum adiponectin
levels were measured by radioimmunoassay. The variants were screened by polymerase chain reaction-restriction fragment length polymorphism. Linkage disequilibrium was estimated from the estimates of haplotype frequencies. RESULTS: Of the 8 variants, four SNPs namely, +276 G/T (rs1501299), -4522 C/T (rs822393), -11365 C/G (rs266729), and +712 G/A (rs3774261) were significantly associated with T2DM in our study population. The -3971 A/G (rs822396) and -11391 G/A (rs17300539) SNPs’ association with T2DM diabetes was mediated through obesity (where the association with type 2 diabetes was lost after adjusting for BMI). There was an independent
association of +276 G/T (rs1501299) and -3971 A/G (rs822396) SNPs with generalized obesity and +349 A/G (rs2241767) with central obesity. Four SNPs, -3971 A/G (rs822396), +276 G/T (rs1501299), -4522 C/T (rs822393) and Y111H T/C (rs17366743) were significantly associated with hypoadiponectinemia. The haplotypes GCCATGAAT and AGCGTGGGT conferred lower risk of T2DM in this south Indian population. CONCLUSION: The adiponectin gene variants and haplotype contribute to the genetic risk towards the development of type 2 diabetes, obesity and hypoadiponectinemia in the south Indianpopulation. [ 2013.].

Association of family history of type 2 diabetes mellitus with markers of endothelial dysfunction in South Indian population.

Dhananjayan R; Malati T; Brindha G; Kutala VK
Indian J Biochem Biophys 2013 Apr;50(2):93-8 (ISSN: 0301-1208)

Studies indicate that risk for type 2 diabetes mellitus (T2D) or cardiovascular disease is detectable in childhood, though these disorders may not emerge until adulthood. This study was aimed to assess the markers of endothelial dysfunction in patients with the family history of T2D from South Indian population. A total of 450 subjects were included in the study comprising Group I (n = 200) of T2D, Group II (n = 200) of age- and sex-matched healthy controls, Group III (n = 25) of children of T2D patients and Group IV (n = 25) of children of healthy controls. Results showed that intimal medial thickening (IMT) was significantly higher in T2D patients, compared with control subjects with no family history of diabetes. The fasting plasma glucose, glycated hemoglobin, serum total cholesterol, triglyceride, LDL-cholesterol, apolipoprotein B (ApoB) and high-sensitive C-reactive protein (hsCRP) levels were significantly increased, whereas HDL-cholesterol and serum nitrite levels were significantly decreased in T2D patients. However, children of T2D patients who were not diabetic did not show significant increase in the IMT, as compared to those of healthy controls. In conclusion, the present study demonstrate that IMT was significantly higher in the T2D patients and increased with age and family history. The increased levels of lipids, hsCRP, IMT and decreased nitrite levels might contribute to the risk of endothelial dysfunction in patients with T2D. However, further studies are warranted with other biomarkers of endothelial dysfunction in T2D patients with increased sample size.

Hemoglobin A1c variability as an independent correlate of cardiovascular disease in patients with type 2 diabetes: a cross-sectional analysis of the renal insufficiency and cardiovascular events (RIACE) Italian multicenter study.

Penno G; Solini A; Zoppini G; Orsi E; Fondelli C; Zerbini G; Morano S; and
Renal Insufficiency and Cardiovascular Events (RIACE) Study Group.
Cardiovasc Diabetol 2013;12:98 (ISSN: 1475-2840)

BACKGROUND: Previous reports have clearly indicated a significant relationship between hemoglobin (Hb) A1c change from one visit to the next and microvascular complications, especially nephropathy (albuminuria and albuminuric chronic kidney disease, CKD). In contrast, data on macrovascular disease are less clear. This study was aimed at examining the association of HbA1c variability with cardiovascular disease (CVD) in the large cohort of subjects with type 2 diabetes from the Renal Insufficiency and Cardiovascular Events (RIACE) Italian Multicenter Study. METHODS: Serial (3-5) HbA1c values obtained during the 2-year period preceding recruitment, including that obtained at the enrolment, were available from 8,290 subjects from 9 centers (out of 15,773 patients from 19 centers). Average HbA1c and HbA1c variability were calculated as the intra-individual mean (HbA1c-MEAN) and standard deviation (HbA1c-SD), respectively, of 4.52 0.76 values. Prevalent CVD, total and by vascular bed, was assessed from medical history by recording previous documented major acute events. Diabetic retinopathy (DR) was assessed by dilated fundoscopy. CKD was defined based on albuminuria, as measured by immunonephelometry or immunoturbidimetry, and estimated glomerular filtration rate, as calculated from serum creatinine. RESULTS: HbA1c-MEAN, but not HbA1c-SD, was significantly higher (P <0.0001) in subjects with history of any CVD (n. 2,133, 25.7%) than in those without CVD (n. 6,157, 74.3%). Median and interquartile range were 7.78 (7.04-8.56) and 7.49 (6.81-8.31), respectively, for HbA1c-MEAN, and 0.47 (0.29-0.75) and 0.46 (0.28-0.73), respectively, for HbA1c-SD. Logistic regression analyses showed that HbA1c-MEAN, but not HbA1c-SD (and independent of it), was a significant correlate of any CVD. Similar findings were observed in subjects with versus those without any coronary or cerebrovascular event or myocardial infarction. Conversely, none of these measures were associated with stroke, whereas both correlated with any lower limb vascular event and HbA1c-SD alone with ulceration/gangrene. All these associations were independent of known CVD risk factors and microvascular complications (DR and CKD). CONCLUSIONS: In patients with type 2 diabetes, HbA1c variability has not a major impact on macrovascular complications, at variance with average HbA1c, an opposite finding as compared with microvascular disease, and particularly nephropathy. TRIAL REGISTRATION: ClinicalTrials.Gov NCT00715481.

Genetic association of adiponectin gene polymorphisms (+45T/G and +10211T/G) with type 2 diabetes in North Indians.

Saxena M; Srivastava N; Banerjee M
Diabetes Metab Syndr 2012 Apr-Jun;6(2):65-9 (ISSN: 1878-0334)

Adiponectin (ADIPOQ) is an abundant protein hormone which belongs to a family of so-called adipokines. It is expressed mostly by adipocytes and is an important regulator of lipid and glucose metabolism. It was shown that decreased serum adiponectin concentration indicated insulin resistance and type 2 diabetes (T2DM) with the risk of cardiovascular complications. The fact that adiponectin is an insulin-sensitizing hormone with anti-diabetic, anti-inflammatory and anti-atherogenic properties, we proposed to study the association of ADIPOQ gene polymorphisms in subjects with T2DM. DNA was isolated from venous blood samples, quantified and subjected to Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) using suitable primers and restriction endonucleases. Adiponectin levels were measured in serum using ELISA. The genotypic, allelic and carriage rate frequencies distribution in patients and controls were analyzed by PSAW software (ver. 17.0). Odd ratios (OR) with 95% confidence interval (CI) were determined to describe the strength of association by logistic regression model. Out of the two polymorphisms studied, +10211T/G showed significant association (P=0.042), the ‘G’ allele association being highly significant (P=0.022). Further analysis showed that individuals with ‘GG’ haplotype were at increased risk of T2DM up to 15.5 times [P=0.015, OR (95% CI); 15.558 (1.690-143.174)]. The present study showed that the ‘G’ allele of ADIPOQ gene (+10211T/G) plays a prominent role with respect to T2DM susceptibility in North-Indian population. [Copyright 2012 Diabetes India. Published by Elsevier Ltd. All rights reserved.].

Association of RAGE gene polymorphism with vascular complications in Indian type 2 diabetes mellitus patients [In Process Citation]

Tripathi AK; Chawla D; Bansal S; Banerjee BD; Madhu SV; Kalra OP
Diabetes Res Clin Pract 2014 Mar;103(3):474-81 (ISSN: 1872-8227)

AIMS: The study was designed to evaluate the association of -374T/A and -429T/C polymorphism in the promoter region and Gly82Ser polymorphism in exon 3 region of RAGE gene with diabetic vascular complications in Indian population. METHODS: We screened 603 subjects which includes 176 healthy controls, 140 type 2 diabetes mellitus (T2DM) subjects without any vascular complications (DM), 152 T2DM subjects with microvascular complications (DM-micro) and 135 T2DM subjects with macrovascular complications (DM-macro) for -374T/A, -429T/C and Gly82Ser polymorphisms of RAGE gene. DNA isolated from the enrolled subjects were genotyped by PCR-RFLP. Logistic regression analysis was used to evaluate the association of single nucleotide polymorphisms (SNPs). RESULTS: The -429 T/C and Gly82Ser RAGE polymorphisms were found to be significantly associated with the development of macrovascular and microvascular complications, respectively, in T2DM subjects while -374A allele showed reduced risk towards the development of macrovascular complications. Further, -429T/C, -374T/A and Gly82Ser haplotype analysis revealed association of CTG haplotype with development of macrovascular complications while haplotype TAG was observed to be significantly protective towards development of macrovascular complications in T2DM subjects (OR=0.617, p=0.0202). CONCLUSIONS: Our data indicates significant association of RAGE SNPs and haplotypes with vascular complications in North Indian T2DM subjects.
Clinical profile and complications of childhood- and adolescent-onset type 2 diabetes seen at a diabetes center in south India.

Amutha A; Datta M; Unnikrishnan R; Anjana RM; Mohan V
Diabetes Technol Ther 2012 Jun;14(6):497-504 (ISSN: 1557-8593)

OBJECTIVE: This study describes the clinical characteristics of childhood- and adolescent-onset type 2 diabetes mellitus (CAT2DM) seen at a diabetes center in southern India. RESEARCH DESIGN AND METHODS: Between January 1992 and December 2009, 368 CAT2DM patients were registered. Anthropometric measurements were done using standardized techniques. Biochemical investigations included C-peptide measurements and glutamic acid decarboxylase antibody assay wherever feasible. Retinopathy was diagnosed by retinal photography; microalbuminuria, if urinary albumin excretion was between 30 and 299vmg/1/4g of creatinine; nephropathy, if urinary albumin excretion was (yen)300vmg/1/4g; and neuropathy, if vibration perception threshold on biothesiometry was (yen)20vV. RESULTS: The proportion of CAT2DM patients, expressed as percentage of total patients registered at our center, rose from 0.01% in 1992 to 0.35% in 2009 (P <0.001). Among the 368 cases of CAT2DM, 96 (26%) were diagnosed before the age of 15 years. The mean age at first visit and age at diagnosis of the CAT2DM subjects were 22.29.7 and 16.12.5 years, respectively. Using World Health Organization growth reference charts, 56% of boys and 50.4% of girls were > 85(th) percentile of body mass index for age. Prevalence rates of retinopathy, microalbuminuria, nephropathy, and neuropathy were 26.7%, 14.7%, 8.4%, and 14.2%, respectively. Regression analysis revealed female gender, body mass index > 85(th) percentile, parental history of diabetes, serum cholesterol, and blood pressure to be associated with earlier age at onset of CAT2DM. CONCLUSIONS: CAT2DM appears to be increasing in urban India, and the prevalence of microvascular complications is high. Female predominance is seen at younger ages.

Variants of the adiponectin gene and diabetic microvascular complications in patients with type 2 diabetes.

Choe EY; Wang HJ; Kwon O; Kim KJ; Kim BS; Lee BW; Ahn CW; et al.
Metabolism 2013 May;62(5):677-85 (ISSN: 1532-8600)

OBJECTIVE: The aim of this study was to examine the association between common polymorphisms of the adiponectin gene (ADIPOQ) and microvascular complications in patients with type 2 diabetes mellitus (T2DM). RESEARCH DESIGN AND METHODS: Rs2241766 and rs1501299 of ADIPOQ were genotyped in 708 patients with T2DM. Fundus photography, nerve conducting velocity, and urine analysis were performed to check for the presence of microvascular complications including diabetic nephropathy, retinopathy and neuropathy. RESULTS: The prevalence of diabetic nephropathy tended to be different according to rs2241766 genotype (p=0.057) and the GG genotype of rs2241766 was associated with diabetic nephropathy [urine albumin/creatinine ratio (UACR) greater than 30 mg/g] after adjusting for age, sex, body mass index, duration of diabetes, HDL-cholesterol, smoking status, and blood pressure (odds ratio=1.96; 95% confidence interval=1.01-3.82, p=0.049). Also, the G allele of rs2241766 demonstrated a trend to be associated with an increase in UACR (p=0.087). Rs2241766 genotype was not associated with diabetic retinopathy (p=0.955) and neuropathy (p=0.104) or any diabetic microvascular complications (p=0.104). There was no significant association between the rs1501299 genotype of ADIPOQ and the prevalence of diabetic retinopathy and neuropathy or any diabetic microvascular complications even after adjustment. CONCLUSION: These data suggest that the GG genotype at rs2241766 is implicated in the pathogenesis of risk for diabetic nephropathy defined as UACR greater than 30 mg/day in patients with T2DM. [Copyright 2013 Elsevier Inc. All rights reserved.].

The prevalence of presarcopenia in Asian Indian individuals with and without type 2 diabetes.

Anbalagan VP; Venkataraman V; Pradeepa R; Deepa M; Anjana RM; Mohan V
Diabetes Technol Ther 2013 Sep;15(9):768-75 (ISSN: 1557-8593)

OBJECTIVE: This study compared the skeletal muscle mass and prevalence of presarcopenia between Asian Indian individuals with and without type 2 diabetes. SUBJECTS AND METHODS: Participants with type 2 diabetes (n=76) and age- and sex-matched controls without diabetes (n=76) were drawn from the Chennai Urban Rural Epidemiological Study (CURES), which was carried out on a representative sample of Chennai City in South India. Skeletal muscle mass was estimated by dual-energy X-ray absorptiometry, and skeletal muscle mass index (SMI) was calculated by dividing the appendicular skeletal muscle mass by the square of the individual’s height in meters and expressed as kg/m. Presarcopenia was defined as an SMI of 7.26 kg/m² for males and 5.5 kg/m² for females. Biochemical and anthropometric measurements were done using standardized procedures. RESULTS: The 152 participants included 68 women (44.7%). Mean age was 449 years (range, 28-67 years), and the mean body mass index (BMI) was 25.73.8 kg/m². The prevalence rates of presarcopenia among individuals with and without diabetes were 39.5% and 15.8%, respectively (P=0.001). The mean SMI values were significantly lower in those with diabetes (6.841.02 kg/m² compared with participants without diabetes (7.281.01 kg/m²) (P=0.009). SMI showed a positive correlation with BMI and waist circumference but a negative correlation with age, fasting plasma glucose, glycated hemoglobin, and low-density lipoprotien cholesterol in the total study population. Logistic regression analysis showed that diabetes was independently associated with presarcopenia (P=0.001). CONCLUSIONS: Prevalence of presarcopenia is higher among Asian Indian subjects with type 2 diabetes compared with age- and sex-matched participants without diabetes.

Increased risk of type 2 diabetes with ascending social class in urban South Indians is explained by obesity: The Chennai urban rural epidemiology study (CURES-116).

Skar M; Villumsen AB; Christensen DL; Petersen JH; Deepa M; Anjana RM; et al.
Indian J Endocrinol Metab 2013 Nov;17(6):1084-9 (ISSN: 2230-8210)

AIM: The aim of this study is to determine the factors responsible for differences in the prevalence of diabetes mellitus (DM) in subjects of different social class in an urban South Indian population. MATERIALS AND METHODS: Analyses were based on the cross-sectional data from the Chennai Urban Rural Epidemiology Study of 1989 individuals, aged (yen)20 years. Entered in the analyses were information obtained by self-report on (1) household income; (2) family history of diabetes; (3) physical activity; (4) smoking status; (5) alcohol consumption. Biochemical, clinical and anthropometrical measurements were performed and included in the analyses. Social class was classified based on income as low (Rs. <2000) intermediate (Rs. 2000-5000`) and high (Rs. 5000-20000). RESULTS: The prevalence rates of DM were 12.0%, 18.4% and 21.7% in low, intermediate and high social class, respectively (P < 0.001). A significant increase in the risk of diabetes was found with ascending social class (Intermediate class: Odds ratio [OR], 1.7 [confidence interval [CI], 1.2-2.3]; High class: OR, 2.0 [CI-1.4-2.9]). The multivariable adjusted logistic regression analysis revealed that the effect of social class on the risk of diabetes remained significant (P = 0.016) when age, family history of diabetesand blood pressure were included. However, with the inclusion of abdominal obesity in the model, the significant effect of social class disappeared (P = 0.087). CONCLUSION: An increased prevalence of DM was found in the higher social class in this urban South Indian population, which is explained by obesity.

Prevalence of inflammatory markers (high-sensitivity C-reactive protein, nuclear factor-(ordM)B, and adiponectin) in Indian patients with type 2 diabetes mellitus with and without macrovascular complications.

Misra DP; Das S; Sahu PK
Metab Syndr Relat Disord 2012 Jun;10(3):209-13 (ISSN: 1557-8518)

BACKGROUND: Atherosclerosis is more prevalent in subjects with diabetes mellitus. Recent evidence suggests that diabetic atherosclerosis is not simply a disease of hyperlipidemia, but is also an inflammatory disorder. Our aim was to study the prevalence of inflammatory markers such as high-sensitivity C-reactive protein (hsCRP), adiponectin, and nuclear factor-(ordM)B (NF-(ordM)B) expression, in peripheral blood mononuclear cells in Indian patients with type 2 diabetes mellitus (T2DM) with and without macrovascular disease (MVD). METHODS: A total of 29 consecutive cases of T2DM with proven MVD (group A), 28 matched cases without MVD (group B), and 14 healthy controls (group C) were evaluated for the clinical parameters fasting blood glucose (FBG), 2-h postprandial blood glucose (PPBG), glycosylated hemoglobin (HbA1c), lipid profile, and the above-mentioned inflammatory markers. RESULTS: Diabetic subjects with T2DM had higher hsCRP and NF-(ordM)B expression and lower values of adiponectin compared to healthy controls. Group A had significantly higher serum hsCRP than group B (P=0.0001) despite comparable values of BMI, FBG, 2-h PPBG, HbA1c, and lipid parameters. Group A had significantly higher serum hsCRP and NF-(ordM)B expression and significantly lower levels of adiponectin than group C (P=0.0001, 0.007, and 0.02, respectively). In Group A, serum adiponectin negatively correlated with NF-(ordM)B expression. In Group B, adiponectin values correlated negatively with both FBG and 2-h PPBG. CONCLUSIONS: Indian subjects with T2DM with or without MVD had higher hsCRP and lower adiponectin values as compared to healthy controls, whereas hsCRP was significantly higher in those with MVD, suggesting that our patients with T2DM were in a proinflammatory state.

Adiponectin G276T gene polymorphism is associated with cardiovascular disease in Japanese patients with type 2 diabetes.

Katakami N; Kaneto H; Matsuoka TA; Takahara M; Maeda N; Shimizu I; et al.
Atherosclerosis 2012 Feb;220(2):437-42 (ISSN: 1879-1484)

OBJECTIVE: Adiponectin has anti-atherogenic properties and reduced serum adiponectin levels are associated with cardiovascular disease (CVD). In this study, we examined the relationship between CVD and adiponectin (ADIPOQ) gene G276T polymorphism that is associated with serum adiponectin level in a large cohort of type 2 diabetic patients. RESEARCH DESIGN AND METHODS: We enrolled 2637 Japanese type 2 diabetic subjects (males, 61.1%; age, 54.97.9 years old), determined their genotypes regarding ADIPOQ G276T polymorphisms, and evaluated the association between this polymorphism and the prevalence of CVD (myocardial infarction and/or cerebral infarction). RESULTS: The prevalence of CVD tended to be higher as the number of G alleles increased [GG (9.5%), GT (6.8%), TT (5.6%), p value for trend=0.0059] and was significantly higher in the subjects with GG genotype compared to those with GT or TT genotype (9.5% vs. 6.6%, p=0.0060). Multiple logistic regression analyses revealed that the number of G alleles (Odds ratio (OR)=1.49 with 95%CI 1.09-2.05, p=0.0125) and GG genotype (OR=1.66 with 95%CI 1.13-2.43, p=0.0098) were significantly associated with CVD even after adjustment for conventional risk factors. Interestingly, the presence of obesity further and significantly increased the risk of CVD in the subjects with GG genotype (OR=1.67 with 95%CI 1.14-2.44, p=0.0090) but not in the subjects with TT or GT genotype (OR=1.17 with 95%CI 0.73-1.89, NS). CONCLUSIONS: It is likely that the G allele of the ADIPOQ G276T polymorphism is a susceptibility allele for CVD in Japanese type 2 diabetic patients, especially when they accompany obesity. [Copyright 2011 Elsevier Ireland Ltd. All rights reserved.].

A comprehensive investigation of variants in genes encoding adiponectin (ADIPOQ) and its receptors (ADIPOR1/R2), and their association with serum adiponectin, type 2 diabetes, insulin resistance and the metabolic syndrome.

Peters KE; Beilby J; Cadby G; Warrington NM; Bruce DG; Davis WA; et al.
BMC Med Genet 2013;14:15 (ISSN: 1471-2350)

BACKGROUND: Low levels of serum adiponectin have been linked to central obesity, insulin resistance, metabolic syndrome, and type 2 diabetes. Variants in ADIPOQ, the gene encoding adiponectin, have been shown to influence serum adiponectin concentration, and along with variants in theadiponectin receptors (ADIPOR1 and ADIPOR2) have been implicated in metabolic syndrome and type 2 diabetes. This study aimed to comprehensively investigate the association of common variants in ADIPOQ, ADIPOR1 and ADIPOR2 with serum adiponectin and insulin resistance syndromes in a large cohort of European-Australian individuals. METHODS: Sixty-four tagging single nucleotide polymorphisms in ADIPOQ, ADIPOR1 and ADIPOR2 were genotyped in two general population cohorts consisting of 2,355 subjects, and one cohort of 967 subjects with type 2 diabetes. The association of tagSNPs with outcomes were evaluated using linear or logistic modelling. Meta-analysis of the three cohorts was performed by random-effects modelling. RESULTS: Meta-analysis revealed nine genotyped tagSNPs in ADIPOQ significantly associated with serum adiponectinacross all cohorts after adjustment for age, gender and BMI, including rs10937273, rs12637534, rs1648707, rs16861209, rs822395, rs17366568, rs3774261, rs6444175 and rs17373414. The results of haplotype-based analyses were also consistent. Overall, the variants in the ADIPOQ gene explained <5% of the variance in serum adiponectin concentration. None of the ADIPOR1/R2 tagSNPs were associated with serum adiponectin. There was no association between any of the genetic variants and insulin resistance or metabolic syndrome. A multi-SNP genotypic risk score for ADIPOQ alleles revealed an association with 3 independent SNPs, rs12637534, rs16861209, rs17366568 and type 2 diabetes after adjusting foradiponectin levels (OR=0.86, 95% CI=(0.75, 0.99), P=0.0134). CONCLUSIONS: Genetic variation in ADIPOQ, but not its receptors, was associated with altered serum adiponectin. However, genetic variation in ADIPOQ and its receptors does not appear to contribute to the risk of insulin resistance or metabolic syndrome but did for type 2
diabetes in a European-Australian population.
Autophagy: Protection Against T2D?

By Salynn Boyles, Contributing Writer,
MedPage Today Published: Jul 27, 2014 | Updated: Jul 28, 2014

The cellular regulatory system known as autophagy appeared to play a key role in preventing type 2 diabetes by protecting insulin-secreting beta cells from the accumulation of toxic amylin oligomers, researchers reported.

Findings from three independent research teams, published online in the Journal of Clinical Investigation, suggested autophagy boosting therapies could prove to be a novel approach for type 2 diabetes prevention.

Autophagy — derived from the Greek words for “self” (auto) and “to eat” (phagein) — describes the controlled disposal of damaged organelles within the cell. This cell-cleaning process is increasingly being recognized as a potential protective mechanism against many diseases, including Parkinson’s disease, amyotrophic lateral sclerosis, and Alzheimer’s disease.

Earlier studies found autophagy to be important for normal beta-cell functionand autophage activity to be increased in beta cells from patients with type 2 diabetes.

The studies provide new insight into how beta cells are normally protected against amylin (IAPP) toxic oligomers, wrote Dhananjay Gupta, PhD, and Jack L. Leahy, MD, of the University of Vermont in Burlington in an accompanying editorial.

Action Points:

Autophagy appeared to play a key role in preventing type 2 diabetes by protecting insulin-secreting beta cells from the accumulation of toxic amylin oligomers.
Note that the studies suggest that autophagy — controlled disposal of damaged organelles within the cell — boosting therapies could prove to be a novel approach for type 2 diabetes prevention.

Autophagy – continued

IAPP: Co-Expressed With Insulin

Type 2 diabetes is characterized by loss of beta-cell, beta-cell dysfunction, and increased beta-cell apoptosis. Islet pathology in type 2 diabetes is also characterized by accumulation of extracellular islet amyloid derived from islet amyloid polypeptide (IAPP).

“IAPP is a 37-amino acid protein co-expressed and secreted by pancreatic [beta cells] along with insulin,” wrote Peter Butler, MD, from the University of California Los Angeles, and colleagues. “While the extracellular islet amyloid is relatively inert, intracellular membrane-permeant toxic oligomers of IAPP that form within [beta cells in type 2 diabetes] are thought to induce [beta-cell dysfunction and apoptosis].”

In contrast to the human form of IAPP (h-IAPP), which forms toxic membrane-permeant oligomers, the rodent form of IAPP (r-IAPP) is nonamyloidogenic and nontoxic due to proline substitutions. Transgenic expression of h-IAPP in [beta cells] of rodents may lead to development of diabetes as a consequence of [beta-cell] apoptosis and formation of intracellular IAPP oligomers comparable to those found in humans with type 2 diabetes.

In earlier in vitro studies, the authors reported that enhancement of autophagy was protective while attenuated lysosomal degradation rendered beta cells more vulnerable to h-IAPP-induced apoptosis.

In the current study, the researchers determined that beta-cell IAPP content is regulated by autophagy through p62-dependent lysosomal degradation.

“Induction of high levels of human IAPP in mouse [beta cells] resulted in accumulation of this amyloidogenic protein as relatively inert fibrils with cytosolic p62-positive inclusions, which temporarily averts formation of toxic oligomers,” they wrote.

Mice hemizygous for transgenic expression of human IAPP did not develop diabetes. But the loss of beta cell-specific autophagy in the mice induced diabetes as a result of the accumulation of toxic human IAPP oligomers and loss of beta-cell mass, the researchers noted.

“In human IAPP-expressing mice that lack [beta-cell] autophagy, increased oxidative damage and loss of an antioxidant-protective pathway appeared to contribute to increased [beta- cell] apoptosis,” they wrote. “These findings indicate that autophagy/lysosomal degradation defends [beta cells] against proteotoxicity induced by oligomerization-prone human IAPP.”

‘Enhance the Toxic Potential of h-IAPP’

In a separate study, Yoshio Fujitani, PhD, of Juntendo University, Tokyo, and colleagues, examined the pathogenic role of human-IAPP and its relation to autophagy in h-IAPP-knock-in mice.

In animals fed a standard diet, h-IAPP had no toxic effects on beta-cell function. However, h-IAPP-knock-in mice did not exhibit a high-fat diet-induced compensatory increase in beta-cell mass, which was due to limited beta-cell proliferation and enhanced beta-cell apoptosis, the researchers wrote.

Expression of h-IAPP in mice with a beta-cell-specific autophagy defect resulted in substantial deterioration of glucose tolerance and dispersed cytoplasmic expression of p62-associated toxic oligomers, which were otherwise sequestrated within p62-positive inclusions.

“Together, our results indicate that increased insulin resistance in combination with reduced autophagy may enhance the toxic potential of h-IAPP and enhance [beta-cell] dysfunction and progression of type 2 diabetes,” the researchers noted.

Autophagy Enhancers

In the third paper, Myung-Shik Lee, MD, PhD, of the Sungkyunkwan University School of Medicine in Seoul, and colleagues, studied transgenic mice with beta cell-specific expression of h-IAPP to evaluate the contribution of autophagy in type 2 diabetes-associated accumulation of h-IAPP.

In mice with beta-cell-specific expression of h-IAPP, a deficiency in autophagy resulted in development of overt diabetes, which was not observed in mice expressing h-IAPP alone or lacking autophagy alone. Lack of autophagy in h-IAPP-expressing animals also resulted in h-IAPP oligomer and amyloid accumulation in pancreatic islets, leading to increased death and decreased mass of beta cells.

“Expression of h-IAPP in purified monkey islet cells or a murine [beta cell] line resulted in pro-h-IAPP dimer formation, while dimer formation was absent or reduced dramatically in cells expressing either nonamyloidogenic mouse-IAPP or nonfibrillar mutant h-IAPP,” the researchers wrote. “In autophagy-deficient cells, accumulation of pro-h-IAPP dimers increased markedly, and pro-h-IAPP trimers were detected in the detergent-insoluble fraction.”

Enhancement of autophagy also improved the metabolic profile of h-IAPP-expressing mice fed a high-fat diet.

“These results suggest that autophagy promotes clearance of amyloidogenic h-IAPP, autophagy deficiency exacerbates pathogenesis of human [type 2 diabetes], and autophagy enhancers have therapeutic potential for islet amyloid accumulation-associated human [type 2 diabetes],” the researchers concluded.

Building on Previous Work

Gupta and Leahy noted that all three research teams generated human IAPP-expressing mice with a beta-cell-specific deficiency of the autophagy indicator ATG7, and all three found that autophagy-dependent packaging of monomeric or unprocessed IAPP dimers or trimers into p62-associated vacuoles allowed autophagosomes to dispose of these molecules, keeping them nontoxic.

Each team showed the activity of this detoxification system to be increased when a high-fat diet was fed to the mice with hyperexpression of h-IAPP.

The studies build on previous work and the findings that don’t discern – “how and when during the course of type 2 diabetes development this autophagy-dependent detoxification system might be overcome, allowing toxic IAPP oligomers to form.”

“There are many additional mechanisms that have been proposed for [beta-cell] dysfunction and death in type 2 diabetes, including ER stress, oxidative stress, and autoimmune damage, all of which have been linked to IAPP toxicity,” they wrote. “While it is tempting to try and connect the dots through a single, unified mechanism, all of these proposed pathways of [beta-cell] dysfunction have been recapitulated and extensively studied in rodent models of diabetogenic systems, such as high-fat feeding and partial pancreatectomy, or through genetic modification.”

Given the absence of rodent IAPP oligomerization, these mechanisms of reduced beta-cell function clearly do not require IAPP activation, they noted.

These papers have implications for the study of target therapies for type 2 diabetes based on the common link to T2D and IAPP oligomerization.

“Patients with type 2 diabetes have an increased risk of Alzheimer’s disease, suggesting a common pathogenesis,” they wrote. Disordered neuronal autophagy, described in Alzheimer’s, with alteration in the clearance of amyloidogenic proteins may be a tie between these two diseases

They concluded that acceptance of the hypothesis that IAPP oligomer formation and subsequent plaque development are a major cause of type 2 diabetes will require a better understanding of

when this mechanism is activated and
what modulates its destructive potential.

“These current studies may shift the focus away from

the biology of how IAPP oligomers cause [beta cell] destruction
to probing for defects within the protective system against the formation of toxic IAPP oligomers,” they wrote.

Part 2. Pancreatic Islet Cell Dysfunction
N-terminal fragment of probrain natriuretic peptide is associated with diabetes microvascular complications in type 2 diabetes

Kumiko Hamano, Ikue Nakadaira, Jun Suzuki, Megumi Gonai
Vascular Health and Risk Management 2014:10 585–589
http://dx.doi.org/10.2147/VHRM.S67753

Aim/introduction: Circulating levels of N-terminal fragment of probrain natriuretic peptide (NT-proBNP) are established as a risk factor for cardiovascular disease and mortality in patients with diabetes, as well as in the general population. We sought to examine the possibility of NT-proBNP as a biomarker of microvascular complications in patients with type 2 diabetes. Materials and methods: In total, 277 outpatients with type 2 diabetes were consecutively enrolled as a hospital cohort. Two hundred and seventeen of these patients (132 males; mean age, 63.4 years) were designated as cases with any of the diabetic complications (retinopathy, neuropathy, nephropathy, ischemic heart disease, strokes, peripheral artery disease), and 60 (42 males; mean age, 54.1 years) were set as controls without clinical evidence of diabetic complications. Diabetic complications were evaluated by medical record and routine laboratory examinations. NT-proBNP was measured and investigated with regard to the associations with diabetic complications. Results: Mean NT-proBNP levels were significantly higher in patients with any of the diabetic complications (59 versus 33 pg/mL; P,0.0001). In logistic regression analysis, NT-proBNP levels .79 pg/mL, which was the highest tertile, were independently associated with a 5.04 fold increased risk of all complications (P,0.0051) compared to the lowest tertile (NT-proBNP levels ,31 pg/mL). Odd ratios of cardiovascular disease and nephropathy, neuropathy, and retinopathy were 9.33, 6.23, 6.6 and 13.78 respectively, in patients with NT-proBNP values in the highest tertile (.79 pg/mL), independently of age, sex, duration of diabetes or other risk factors, such as body mass index or hemoglobin A1c. In addition, NT-proBNP levels were associated with surrogate markers of atherosclerosis, such as brachial-ankle pulse wave velocity (r=0.449, P,0.0001) and left ventricular hypertrophy (r=0.212, P,0.001). Conclusion: In this hospital-based cohort of type 2 diabetes, the NT-proBNP levels were associated with systemic atherosclerosis and comorbid diabetic microvascular as well as macrovascular complications. It is useful to stratify high-risk diabetic patients by measuring NT-proBNP and to start comprehensive care for preventing the progression of diabetic complications. It is necessary to elucidate the underlying mechanism for the progression of diabetic complications represented by an elevation of NT-proBNP and to demonstrate the ability of NT-proBNP as a predictive global biomarker for diabetic complications in Japanese type 2 diabetic patients.
How are patients with type 2 diabetes and renal disease monitored and managed? Insights from the observational OREDIA study

Alfred Penfornis, J F Blicklé, B Fiquet, S Quéré, S Dejager
Vascular Health and Risk Management 2014:10 341–352
http://dx.doi.org/10.2147/VHRM.S60312

Background and aim: Chronic kidney disease (CKD) is frequent in type 2 diabetes mellitus (T2DM), and therapeutic management of diabetes is more challenging in patients with renal impairment (RI). The place of metformin is of particular interest since most scientific societies now recommend using half the dosage in moderate RI and abstaining from use in severe RI, while the classic contraindication with RI has not been removed from the label. This study aimed to assess the therapeutic management, in particular the use of metformin, of T2DM patients with CKD in real life. Methods: This was a French cross-sectional observational study: 3,704 patients with T2DM diagnosed for over 1 year and pharmacologically treated were recruited in two cohorts (two-thirds were considered to have renal disease [CKD patients] and one-third were not [non-CKD patients]) by 968 physicians (81% general practitioners) in 2012. Results: CKD versus non-CKD patients were significantly older with longer diabetes history, more diabetic complications, and less strict glycemic control (mean glycated hemoglobin [HbA1c] 7.5% versus 7.1%; 25% of CKD patients had HbA1c $8% versus 15% of non-CKD patients). Fifteen percent of CKD patients had severe RI, and 66% moderate RI. Therapeutic management of T2DM was clearly distinct in CKD, with less use of metformin (62% versus 86%) but at similar mean daily doses (∼2 g/d). Of patients with severe RI, 33% were still treated with metformin, at similar doses. For other oral anti-diabetics, a distinct pattern of use was seen across renal function (RF): use of sulfonylureas (32%, 31%, and 20% in normal RF, moderate RI, and severe RI, respectively) and DPP4-i (dipeptidyl peptidase-4 inhibitors) (41%, 36%, and 25%, respectively) decreased with RF, while that of glinides increased (8%, 14%, and 18%, respectively). CKD patients were more frequently treated with insulin (40% versus 16% of non-CKD patients), and use of insulin increased with deterioration of RF (19%, 39%, and 61% of patients with normal RF, moderate RI, and severe RI, respectively). Treatment was modified at the end of the study-visit in 34% of CKD patients, primarily to stop or reduce metformin. However, metformin was stopped in only 40% of the severe RI patients. Conclusion: Despite a fairly good detection of CKD in patients with T2DM, RI was insufficiently taken into account for adjusting anti-diabetic treatment.

Efficacy and safety of insulin glargine added to a fixed-dose combination of metformin and a dipeptidyl peptidase-4 inhibitor: results of the GOLD observational study

Jochen Seufert, Katrin Pegelow, Peter Bramlage
Vascular Health and Risk Management 2013:9 711–717
http://dx.doi.org/10.2147/VHRM.S54362

Background: For patients with type 2 diabetes who are uncontrolled on a combination of two oral antidiabetic agents, addition of the long-acting basal insulin glargine is a well established treatment option. However, data on the efficacy and safety of a combination of metformin, a dipeptidyl peptidase-4 (DPP-4) inhibitor, and insulin glargine are limited in real-world settings. Therefore, the aim of this study was to analyze blood glucose control, rates of hypoglycemia and body weight in a large cohort of patients with type 2 diabetes treated with this combination therapy in real practice. Methods: This noninterventional, multicenter, prospective, observational trial with a follow-up of 20 weeks enrolled insulin-naïve patients who had been on a stable fixed dose of metformin and a DPP-4 inhibitor for at least 3 months, and had a glycosylated hemoglobin (HbA1c) between 7.5% and 10%. Patients were selected at the investigators’ discretion for initiation of insulin glargine at baseline. A total of 1,483 patients were included, of whom 1,262 were considered to be the efficacy set. Primary efficacy parameters were HbA1c and fasting plasma glucose. Secondary outcome measures included achievement of glycemic targets, body weight, rates of hypoglycemia, and other safety parameters, as well as resource consumption. Results: Upon initiation of insulin glargine, mean HbA1c decreased from 8.51% to 7.36% (−1.15%±0.91%; 95% confidence interval [CI] −1.20 to −1.10). An HbA1c level ,6.5% was achieved in 8.2% of patients and a level ,7.0% in 31.5%. Mean fasting plasma glucose decreased from 174±47 mg/dL to 127±31 mg/dL (−47.3±44.1 mg/dL; 95% CI −49.8 to −44.8). In 11.9% of patients, a fasting plasma glucose level ,100 mg/dL was achieved. Bodyweight decreased on average by 0.98±3.90 kg (95% CI 1.19–0.76). Hypoglycemia (blood glucose #70 mg/dL) was observed in 29 patients (2.30%), of whom six (0.48%) had nocturnal hypoglycemia and four (0.32%) had documented severe events (blood glucose ,56 mg/dL). Conclusion: The results of this observational study show that insulin glargine, when added to a fixed-dose combination of metformin and a DPP-4 inhibitor, resulted in a significant and clinically relevant improvement of glycemic control. Importantly, this intervention did not interfere with the action of the DPP-4 inhibitors, resulting in neutral effects on weight and low rates of hypoglycemia. We conclude that this treatment intensification approach may be useful, efficient, and safe in daily clinical practice for patients with type 2 diabetes.

Long-term insulin glargine therapy in type 2 diabetes mellitus: a focus on cardiovascular outcomes

Joshua J Joseph, Thomas W Donner
Vascular Health and Risk Management 2015:11 107–116
http://dx.doi.org/10.2147/VHRM.S50286

Cardiovascular disease is the leading cause of mortality in type 2 diabetes mellitus. Hyperinsulinemia is associated with increased cardiovascular risk, but the effects of exogenous insulin on cardiovascular disease progression have been less well studied. Insulin has been shown to have both cardioprotective and atherosclerosis-promoting effects in laboratory animal studies. Long-term clinical trials using insulin to attain improved diabetes control in younger type 1 and type 2 diabetes patients have shown improved cardiovascular outcomes. Shorter trials of intensive diabetes control with high insulin use in higher risk patients with type 2 diabetes have shown either no cardiovascular benefit or increased all cause and cardiovascular mortality. Glargine insulin is a basal insulin analog widely used to treat patients with type 1 and type 2 diabetes. This review focuses on the effects of glargine on cardiovascular outcomes. Glargine lowers triglycerides, leads to a modest weight gain, causes less hypoglycemia when compared with intermediate-acting insulin, and has a neutral effect on blood pressure. The Outcome Reduction With Initial Glargine Intervention (ORIGIN trial), a 6.2 year dedicated cardiovascular outcomes trial of glargine demonstrated no increased cardiovascular risk.

Visceral obesity is not an independent risk factor of mortality in subjects over 65 years

Frédérique Thomas, Bruno Pannier, Athanase Benetos, Ulrich M Vischer
Vascular Health and Risk Management 2013:9 739–745
http://dx.doi.org/10.2147/VHRM.S49922

The aim of the study was to determine the role of obesity evaluated by body mass index (BMI), waist circumference (WC), and their combined effect on all-cause mortality according to age and related risk factors. This study included 119,090 subjects (79,325 men and 39,765 women), aged from 17 years to 85 years, who had a general health checkup at the Centre d’Investigations Préventives et Cliniques, Paris, France. The mean follow-up was 5.6±2.4 years. The prevalence of obesity, defined by WC and BMI categories, was determined according to age groups (< 55, 55–65, > 65 years). All-cause mortality according to obesity and age was determined using Cox regression analysis, adjusted for related risk factors and previous cardiovascular events.
For the entire population, WC adjusted for BMI, an index of central obesity, was strongly associated with mortality, even after adjustment for hypertension, dyslipidemia, and diabetes. The prevalence of obesity increased with age, notably when defined by WC. Nonetheless, the association between WC adjusted for BMI and mortality was not observed in subjects .65 years old (hazard ratio [HR] =1.010, P=NS) but was found in subjects < 55 (HR =1.030,
P < 0.0001) and 55–65 years old (HR =1.023, P,0.05). By contrast, hypertension
(HR =1.31, P < 0.05), previous cardiovascular events (HR =1.98, P < 0.05), and smoking (HR =1.33, P < 0.05) remained associated with mortality even after
age 65.
In conclusion, WC adjusted for BMI is strongly and independently associated with all-cause mortality before 65 years of age, after taking into account the associated risk factors. This relationship disappears in subjects
> 65 years of age, suggesting a differential impact of visceral fat deposition according to age.

Insulin degludec/insulin aspart combination for the treatment of type 1 and type 2 diabetes

Angela Dardano, Cristina Bianchi, Stefano Del Prato, Roberto Miccoli
Vascular Health and Risk Management 2014:10 465–475
http://dx.doi.org/10.2147/VHRM.S40097

Glycemic control remains the major therapeutic objective to prevent or delay the onset and progression of complications related to diabetes mellitus. Insulin therapy represents a cornerstone in the treatment of diabetes and has been used widely for achieving glycemic goals. Nevertheless, a large portion of the population with diabetes does not meet the internationally agreed glycemic targets. Moreover, insulin treatment, especially if intensive, may be associated with emergency room visits and hospitalization due to hypoglycemic events. Therefore, fear of hypoglycemia or hypoglycemic events represents the main barriers to the attainment of glycemic targets. The burden associated with multiple daily injections also remains a significant obstacle to initiating and maintaining insulin therapy. The most attractive insulin treatment approach should meet the patients’ preference, rather than demanding patients to change or adapt their lifestyle. Insulin degludec/insulin aspart (IDegAsp) is a new combination, formulated with ultra-long-acting insulin degludec and rapid-acting insulin aspart, with peculiar pharmacological features, clinical efficacy, safety, and tolerability. IDegAsp provides similar, noninferior glycemic control to a standard basal–bolus regimen in patients with type 1 diabetes mellitus, with additional benefits of significantly lower episodes of hypoglycemia (particularly nocturnal) and fewer daily insulin injections. Moreover, although treatment strategy and patients’ viewpoint are different in type 1 and type 2 diabetes, trial results suggest that IDegAsp may be an appropriate and reasonable option for initiating insulin therapy in patients with type 2 diabetes inadequately controlled on maximal doses of conventional oral agents. This paper will discuss the role of IDegAsp combination as a novel treatment option in diabetic patients.

UCP2 Regulates the Glucagon Response to Fasting and Starvation

Emma M. Allister, Christine A. Robson-Doucette, Kacey J. Prentice, et al.
Diabetes Feb 22, 2013; p 1-11. http://dx.doi.org:/10.2337/db12-0981
http://diabetes.diabetesjournals.org/lookup/suppl/doi:10.2337/db12-0981/-/DC1

Glucagon is important for maintaining euglycemia during fasting/starvation, and abnormal glucagon secretion is associated with type 1 and type 2 diabetes; however, the mechanisms of hypoglycemia-induced glucagon secretion are poorly understood. We previously demonstrated that global deletion of mitochondrial uncoupling protein 2 (UCP22/2) in mice impaired glucagon secretion from isolated islets. Therefore, UCP2 may contribute to the regulation of hypoglycemia-induced glucagon secretion, which is supported by our current finding that UCP2 expression is increased in nutrient-deprived murine and human islets. Further to this, we created a-cell–specific UCP2 knockout (UCP2AKO) mice, which we used to demonstrate that blood glucose recovery in response to hypoglycemia is impaired owing to attenuated glucagon secretion. UCP2-deleted a-cells have higher levels of intracellular reactive oxygen species (ROS), due to enhanced mitochondrial coupling, which translated into defective stimulus/secretion coupling. The effects of UCP2 deletion were mimicked by the UCP2 inhibitor genipin on both murine and human islets and also by application of exogenous ROS, confirming that changes in oxidative status and electrical activity directly reduce glucagon secretion. Therefore, a-cell UCP2 deletion perturbs the fasting/hypoglycemic glucagon response and shows that UCP2 is necessary for normal a-cell glucose sensing and the maintenance of euglycemia.

Main points:

UCP2 is efficiently deleted specifically from islet a-cells of UCP2AKO mice.
α-Cell UCP2 deletion reduces glucagon secretion in vivo
UCP2AKO mice display normal glucose tolerance and GLP-1 secretion
α-Cell UCP2 deletion reduces the gluconeogenic response of the liver and switches fatty acid usage during a prolonged fast
UCP2 expression is increased after nutrient depletion and glucagon secretion from UCP2AKO islets was impaired.
UCP2AKO α-cells display enhanced hyperpolarization of ΔψC_m and increased superoxide levels
UCP2AKO α-cells have more depolarized plasma membranes and reduced intracellular calcium
UCP2 is required for normal glucagon secretion in response to hypoglycemia

Management of Diabetes Mellitus: Could Simultaneous Targeting of Hyperglycemia and Oxidative Stress Be a Better Panacea?

Omotayo O. Erejuwa

Int. J. Mol. Sci. 2012, 13, 2965-2972; http://dx.doi.org:/10.3390/ijms13032965

Oxidative stress is defined as an “imbalance between oxidants and antioxidants in favor of the oxidants, potentially leading to damage”. It is implicated in the pathogenesis and complications of diabetes mellitus. The role of oxidative stress is more definite in the pathogenesis of type 2 diabetes mellitus than in type 1 diabetes mellitus. In regard to diabetic complications, there is compelling evidence in support of the role of oxidative stress in both types of diabetes mellitus. Evidence suggests that elevated reactive oxygen species (ROS), which causes oxidative stress, accumulate in certain micro milieu or tissues (such as retina and kidney) where they cause damage or toxicity. In diabetes mellitus, oxidative stress is enhanced through various sources such as hyperglycemia, dyslipidemia, hyperinsulinemia, insulin resistance, impaired antioxidant defense network, uncoupling of ROS-generating enzymes, elevated level of leptin and sedentary lifestyle.

A number of mechanisms or pathways by which hyperglycemia, the major contributing factor of increased ROS production, causes tissue damage or diabetic complications have been identified. These include: hyperglycemia-enhanced polyol pathway; hyperglycemia-enhanced formation of advanced glycation endproducts (AGEs); hyperglycemia-activated protein kinase C (PKC) pathway; hyperglycemia-enhanced hexosamine pathway; and hyperglycemia-activated Poly-ADP ribose polymerase (PARP) pathway. These pathways are activated or enhanced by hyperglycemia-driven mitochondrial superoxide overproduction. Even though oxidative stress plays an important role in its pathogenesis and complications, unlike other diseases characterized by oxidative stress, diabetes mellitus is unique. Its cure (restoration of euglycemia, e.g., via pancreas transplants) does not prevent oxidative stress and diabetic complications. This is very important because hyperglycemia exacerbates oxidative stress which is linked to diabetic complications]. Theoretically, restoration of euglycemia should prevent oxidative stress and diabetic complications. However, this is not the case.

The primary aim of the current management of diabetes mellitus is to achieve and/or maintain a glycated hemoglobin level of ≤6.5%. However, recent evidence indicates that intensive treatment of hyperglycemia is characterized by increased weight gain, severe hypoglycemia and higher mortality. Besides, evidence suggests that it is difficult to achieve and/or maintain optimal glycemic control in many diabetic patients; and that the benefits of intensively-treated hyperglycemia are restricted to microvascular complications only. In view of these adverse effects and limitations of intensive treatment of hyperglycemia in preventing diabetic complications, which is linked to oxidative stress, this commentary proposes a hypothesis that “simultaneous targeting of hyperglycemia and oxidative stress” could be more effective than “intensive treatment of hyperglycemia” in the management of diabetes mellitus.

The Relationship between Inflammation, Oxidative Stress, and Metabolic Risk Factors in Type 2 Diabetic Patients

Fatemeh Azizi Soleiman, N Pahlavani, H Rasad, O Sadeghi, MR Gohari
Iranian Journal Of Diabetes And Obesity 2013; 5(4): 151-156

Increased production of free radicals due to the imbalance between free radicals and antioxidants load may reduce antioxidants levels, partial clearing of free radicals, and cause oxidation of lipids, sugars, proteins and nucleic acids which eventually leads to widespread pathological consequences of diabetes. One of the factors that facilitate formation of atherosclerosis in diabetes is oxidative stress.

Objective: Globally, 3-5.2 percent of people suffer from diabetes which is one of the most serious metabolic disorders resulting in an increase in inflammatory biomarkers e.g. interleukin-6, tumor necrosis factor-alpha, and C-reactive protein. The aim of this study was to investigate the relationship between inflammation, oxidative stress and fasting blood glucose, lipid profile and anthropometric parameters in patients with type 2 diabetes. Material and methods: This study was conducted as a cross sectional study in Tehran through 2009-2010 on 45 men and women aged 35-65 years old with type 2 diabetes. Blood glucose, lipid profile, C-reactive protein, and malonedialdehyde were measured. Independent sample T-test and linear regression analysis were used. Results: Fasting blood glucose, malonedialdehyde, total cholesterol and body mass index were higher in women than in men; but there was no difference between two sexes in other factors. Malonedialdehyde, neither directly or after adjustment for sex was related to fasting blood glucose, total cholesterol, triglycerides and anthropometric indices (weight, body mass index, and body fat mass). Conclusion: This study showed that oxidative stress had no relationship with blood glucose, lipid profile, and anthropometric index, but inflammation was related to glycemia, body mass index, and fat mass. Control of inflammation and oxidative stress is necessary for accelerating treatment process and preventing complications due to them.

This study showed that in diabetic patients, oxidative stress which was measured by MDA, was not significantly associated with fasting blood glucose, lipid profile and anthropometric parameters. However, fasting plasma glucose, body mass index and body fat mass were significant predictors of the inflammatory factor, CRP.

Oxidative Stress as an Underlying Contributor in the Development of Chronic Complications in Diabetes Mellitus

Suziy de M. Bandeira, Lucas José S. da Fonseca, Glaucevane da S. Guedes, et al.
Int. J. Mol. Sci. 2013, 14, 3265-3284; http://dx.doi.doi:/10.3390/ijms14023265

The high prevalence of diabetes mellitus and its increasing incidence worldwide, coupled with several complications observed in its carriers, have become a public health issue of great relevance. Chronic hyperglycemia is the main feature of such a disease, being considered the responsible for the establishment of micro and macrovascular complications observed in diabetes. Several efforts have been directed in order to better comprehend the pathophysiological mechanisms involved in the course of this endocrine disease. Recently, numerous authors have suggested that excess generation of highly reactive oxygen and nitrogen species is a key component in the development of complications invoked by hyperglycemia. Overproduction and/or insufficient removal of these reactive species result in vascular dysfunction, damage to cellular proteins, membrane lipids and nucleic acids, leading different research groups to search for biomarkers which would be capable of a proper and accurate measurement of the oxidative stress (OS) in diabetic patients, especially in the presence of chronic complications.
In the face of this scenario, the present review briefly addresses the role of hyperglycemia in OS, considering basic mechanisms and their effects in diabetes mellitus, describes some of the more commonly used biomarkers of oxidative/nitrosative damage and includes selected examples of studies which evaluated OS biomarkers in patients with diabetes, pointing to the relevance of such biological components in general oxidative stress status of diabetes mellitus carriers.
The role of FOXO1 in β‑cell failure and type 2 diabetes mellitus

Tadahiro Kitamura
Nat. Rev. Endocrinol. 2013; 9, 615–623
http://dx.doi.org:/10.1038/nrendo.2013.157

Over the past two decades, insulin resistance has been considered essential to the etiology of type 2 diabetes mellitus (T2DM). However, insulin resistance does not lead to T2DM unless it is accompanied by pancreatic β‑cell dysfunction, because healthy β cells can compensate for insulin resistance by increasing in number and functional output. Furthermore, β‑cell mass is decreased in patients with diabetes mellitus, suggesting a primary role for β‑cell dysfunction in the pathogenesis of T2DM. The dysfunction of β cells can develop through various mechanisms, including oxidative, endoplasmic reticulum or hypoxic stress, as well as via induction of cytokines; these processes lead to apoptosis, uncontrolled autophagy and failure to proliferate. Transdifferentiation between β cells and α cells occurs under certain pathological conditions, and emerging evidence suggests that β‑cell dedifferentiation or transdifferentiation might account for the reduction in β‑cell mass observed in patients with severe T2DM. FOXO1, a key transcription factor in insulin signaling, is implicated in these mechanisms. This Review discusses advances in our understanding of the contribution of FOXO1 signaling to the development of β‑cell failure in T2DM.

Selective peroxisome proliferator-activated receptor g (PPARg) modulation as a strategy for safer therapeutic PPARg activation

Linda Slanec Higgins and Alex M DePaoli
Am J Clin Nutr 2010;91(suppl):267S–72S.
http://dx.doi.org:/10.3945/ajcn.2009.28449E

Peroxisome proliferator-activated receptor c (PPARc) is a clinically validated target for treatment of insulin resistance. PPARc activation by full agonists such as thiazolidinediones has shown potent and durable glucose-lowering activity in patients with type 2 diabetes without the concern for hypoglycemia or gastrointestinal toxicities associated with some other medications used to treat this disease. However, thiazolidinediones are linked to safety and tolerability issues such as weight gain, fluid retention, edema, congestive heart failure, and bone fracture. Distinctive properties of PPARc provide the opportunity for selective modulation of the receptor such that desirable therapeutic effects may be attained without the unwanted effects of full activation. PPARc is a nuclear receptor that forms a complex with coreceptor RXR and a cell type– and cell state– specific array of coregulators to control gene transcription. PPARc affinity for these components, and hence transcriptional response, is determined by the conformational changes induced by ligand binding within a complex pocket with multiple interaction points. This molecular mechanism thereby offers the opportunity for selective modulation. A desirable selective PPARc modulator profile would include high-affinity interaction with the PPARc-binding pocket in a manner that leads to retention of the insulin-sensitizing activity that is characteristic of full agonists as well as mitigation of the effects leading to increased adiposity, fluid retention, congestive heart failure, and bone fracture. Examples of endogenous and synthetic selective PPARc modulator (SPPARM) ligands have been identified. SPPARM drug candidates are being tested clinically and provide support for this strategy.

Predicting response to incretin-based therapy

Sanjay Kalra, Bharti Kalra, Rakesh Sahay, Navneet Agrawal
Research and Reports in Endocrine Disorders 2011:1 11–19
http://dx.doi.org:/10.2147/RRED.S16282

There are two important incretin hormones, glucose-dependent insulin tropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1). The biological activities of GLP-1 include stimulation of glucose-dependent insulin secretion and insulin biosynthesis, inhibition of glucagon secretion and gastric emptying, and inhibition of food intake. GLP-1 appears to have a number of additional effects in the gastrointestinal tract and central nervous system. Incretin based therapy includes GLP-1 receptor agonists like human GLP-1 analogs (liraglutide) and exendin-4 based molecules (exenatide), as well as DPP-4 inhibitors like sitagliptin, vildagliptin and saxagliptin. Most of the published studies showed a significant reduction in HbA1c using these drugs. A critical analysis of reported data shows that the response rate in terms of target achievers of these drugs is average. One of the first actions identified for GLP-1 was the glucose-dependent stimulation of insulin secretion from islet cell lines. Following the detection of GLP-1 receptors on islet beta cells, a large body of evidence has accumulated illustrating that GLP-1 exerts multiple actions on various signaling pathways and gene products in the β cell. GLP-1 controls glucose homeostasis through well-defined actions on the islet β cell via stimulation of insulin secretion and preservation and expansion of β cell mass. In summary, there are several factors determining the response rate to incretin therapy. Currently minimal clinical data is available to make a conclusion. Key factors appear to be duration of diabetes, obesity, presence of autonomic neuropathy, resting energy expenditure, plasma glucagon levels and plasma free fatty acid levels. More clinical evidence is required to identify the factors affecting response rate to incretin therapy.

Regulation of Large Conductance Ca2⁺-activated K⁺ (BK) Channel β1 Subunit Expression by Muscle RING Finger Protein 1 in Diabetic Vessels

Fu Yi, Huan Wang, Qiang Chai, Xiaoli Wang, et al.
J. Biol. Chem. 2014, 289: 10853-10864
http://dx.doi.org:/10.1074/jbc.M113.520940

Background: Impaired BK channel function in diabetic vessels is associated with decreased BK channel[1]1 subunit (BK-β1) expression. Results: Muscle RING finger protein 1 (MuRF1) physically interacts with BK-β1 and accelerates BK-β1 proteolysis. Conclusion: Increased MuRF1 expression is a novel mechanism underlying diabetic BK channelopathy and vasculopathy. Significance: MuRF1 is a potential therapeutic target of BK channel dysfunction and vascular complications in diabetes.

The large conductance Ca2⁺-activated K⁺ (BK) channel, expressed abundantly in vascular smooth muscle cells (SMCs), is a key determinant of vascular tone. BK channel activity is tightly regulated by its accessory β1 subunit (BK-β1). However, BK channel function is impaired in diabetic vessels by increased ubiquitin/proteasome-dependent BK-β1 protein degradation. Muscle RING finger protein 1 (MuRF1), a muscle-specific ubiquitin ligase, is implicated in many cardiac and skeletal muscle diseases. However, the role of MuRF1 in the regulation of vascular BK channel and coronary function has not been examined. In this study, we hypothesized that MuRF1 participated in BK-β1 proteolysis, leading to the down-regulation of BK channel activation and impaired coronary function in diabetes. Combining patch clamp and molecular biological approaches, we found that MuRF1 expression was enhanced, accompanied by reduced BK-β1 expression, in high glucose-cultured human

coronary SMCs and in diabetic vessels. Knockdown of MuRF1 by siRNA in cultured human SMCs attenuated BK-β1 ubiquitination and increased BK-β1 expression, whereas adenoviral expression of MuRF1 in mouse coronary arteries reduced BK-β1 expression and diminished BK channel-mediated vasodilation. Physical interaction between the N terminus of BK-β1 and the coiled-coil domain of MuRF1 was demonstrated by pulldown assay. Moreover, MuRF1 expression was regulated by NF-κB. Most importantly, pharmacological inhibition of proteasome and NF-κB activities preserved BK-β1 expression and BK-channel-mediated coronary vasodilation in diabetic mice. Hence, our results provide the first evidence that the up-regulation of NF-κB-dependent MuRF1 expression is a novel mechanism that leads to BK channelopathy and vasculopathy in diabetes.
The origin of circulating CD36 in type 2 diabetes

MJ Alkhatatbeh, AK Enjeti, S Acharya, RF Thorne, and LF Lincz
Nutrition and Diabetes (2013) 3, e59; http://dx.doi.org:/10.1038/nutd.2013.1

Objective: Elevated plasma levels of the fatty acid transporter, CD36, have been shown to constitute a novel biomarker for type 2 diabetes mellitus (T2DM). We recently reported such circulating CD36 to be entirely associated with cellular microparticles (MPs) and aim here to determine the absolute levels and cellular origin(s) of these CD36 + MPs in persons with T2DM. Design: An ex vivo case-control study was conducted using plasma samples from 33 obese individuals with T2DM (body mass index (BMI) =39.9±6.4 kgm²; age=57±9 years; 18 male:15 female) and age- and gender-matched lean and obese non-T2DM controls (BMI =23.6±1.8 kgm² and 33.5±5.9 kgm², respectively). Flow cytometry was used to analyse surface expression of CD36 together with tissue-specific markers: CD41, CD235α, CD14, CD105 and phosphatidyl serine on plasma MPs. An enzyme-linked immunosorbent assay was used to quantify absolute CD36 protein concentrations. Results: CD36 + MP levels were significantly higher in obese people with T2DM (P<0.00001) and were primarily derived from erythrocytes (CD235α + = 35.8±14.6%); although this did not correlate with hemoglobin A1c. By contrast, the main source of CD36 + MPs in non-T2DM individuals was endothelial cells (CD105 + = 40.9±8.3% and 33.9±8.3% for lean and obese controls, respectively). Across the entire cohort, plasma CD36 protein concentration varied from undetectable to 22.9 µgml^-1 and was positively correlated with CD36 +MPs measured by flow cytometry (P=0.0006) but only weakly associated with the distribution of controls and T2DM (P=0.021). Multivariate analysis confirmed that plasma CD36 + MP levels were a much better biomarker for diabetes than CD36 protein concentration (P=0.009 vs P=0.398, respectively). Conclusions: Both the levels and cellular profile of CD36 + MPs differ in T2DM compared with controls, suggesting that these specific vesicles could represent distinct biological vectors contributing to the pathology of the disease.
A Novel High-Throughput Assay for Islet Respiration Reveals Uncoupling of Rodent and Human Islets

Jakob D. Wikstrom, Samuel B. Sereda, Linsey Stiles, Alvaro Elorza, et al.
PLoS ONE 7(5): e33023. http://dx.doi.org:/10.1371/journal.pone.0033023

Background: The pancreatic beta cell is unique in its response to nutrient by increased fuel oxidation. Recent studies have demonstrated that oxygen consumption rate (OCR) may be a valuable predictor of islet quality and long term nutrient responsiveness. To date, high-throughput and user-friendly assays for islet respiration are lacking. The aim of this study was to develop such an assay and to examine bioenergetic efficiency of rodent and human islets. Methodology/Principal Findings: The XF24 respirometer platform was adapted to islets by the development of a 24-well plate specifically designed to confine islets. The islet plate generated data with low inter-well variability and enabled stable measurement of oxygen consumption for hours. The F1F0 ATP synthase blocker oligomycin was used to assess uncoupling while rotenone together with myxothiazol/antimycin was used to measure the level of non-mitochondrial respiration. The use of oligomycin in islets was validated by reversing its effect in the presence of the uncoupler FCCP. Respiratory leak averaged to 59% and 49% of basal OCR in islets from C57Bl6/J and FVB/N mice, respectively. In comparison, respiratory leak of INS-1 cells and C2C12 myotubes was measured to 38% and 23% respectively. Islets from a cohort of human donors showed a respiratory leak of 38%, significantly lower than mouse islets. Conclusions/Significance: The assay for islet respiration presented here provides a novel tool that can be used to study islet mitochondrial function in a relatively high-throughput manner. The data obtained in this study shows that rodent islets are less bioenergetically efficient than human islets as well as INS1 cells.

Refeeding and metabolic syndromes: two sides of the same coin

OA Obeid, DH Hachem and JJ Ayoub
Nutrition & Diabetes (2014) 4, e120; http://dx.doi.org:/10.1038/nutd.2014.21

Refeeding syndrome describes the metabolic and clinical changes attributed to aggressive rehabilitation of malnourished subjects. The metabolic changes of refeeding are related to hypophosphatemia, hypokalemia, hypomagnesemia, sodium retention and hyperglycemia, and these are believed to be mainly the result of increased insulin secretion following high carbohydrate intake. In the past few decades, increased consumption of processed food (refined cereals, oils, sugar and sweeteners, and so on) lowered the intake of several macrominerals (mainly phosphorus, potassium and magnesium). This seems to have compromised the postprandial status of these macrominerals, in a manner that mimics low grade refeeding syndrome status. At the pathophysiological level, this condition favored the development of the different components of the metabolic syndrome. Thus, it is reasonable to postulate that metabolic syndrome is the result of long term exposure to a mild refeeding syndrome.

HSP72 protects against obesity-induced insulin resistance

Jason Chung, Anh-Khoi Nguyen, Darren C. Henstridge, Anna G. Holmes, et al.
PNAS Feb 5, 2008; 105(5): 1739–1744
http://www.pnas.org/cgi/doi/10.1073/pnas.0705799105

Patients with type 2 diabetes have reduced gene expression of heat shock protein (HSP) 72, which correlates with reduced insulin sensitivity. Heat therapy, which activates HSP72, improves clinical parameters in these patients. Activation of several inflammatory signaling proteins such as c-jun amino terminal kinase (JNK), inhibitor of B kinase, and tumor necrosis factor-β, can induce insulin resistance, but HSP 72 can block the induction of these molecules in vitro. Accordingly, we examined whether activation of HSP72 can protect against the development of insulin resistance. First, we show that obese, insulin resistant humans have reduced HSP72 protein expression and increased JNK phosphorylation in skeletal muscle. We next used heat shock therapy, transgenic overexpression, and pharmacologic means to overexpress HSP72 either specifically in skeletal muscle or globally in mice. Herein, we show that regardless of the means used to achieve an elevation in HSP72 protein, protection against diet- or obesity induced hyperglycemia, hyperinsulinemia, glucose intolerance, and insulin resistance was observed. This protection was tightly associated with the prevention of JNK phosphorylation. These findings identify an essential role for HSP72 in blocking inflammation and preventing insulin resistance in the context of genetic obesity or high-fat feeding.

pH-responsive modulation of insulin aggregation and structural transformation of the aggregates

Ekaterina Smirnova, I Safenkova, V Stein-Margolina, V Shubin, et al.
Biochimie 109 (2015) 49e59
http://dx.doi.org/10.1016/j.biochi.2014.12.006

Over the past two decades, much information has appeared on electrostatically driven molecular mechanisms of protein self-assembly and formation of aggregates of different morphology, varying from soluble amorphous structures to highly-ordered amyloid-like fibrils. Protein aggregation represents a special tool in biomedicine and biotechnology to produce biological materials for a wide range of applications. This has awakened interest in identification of pH-triggered regulators of transformation of aggregation-prone proteins into structures of higher order. The objective of the present study is to elucidate the effects of low-molecular-weight biogenic agents on aggregation and formation of supramolecular structures of human recombinant insulin, as a model therapeutic protein. Using dynamic light scattering, turbidimetry, circular dichroism, fluorescence spectroscopy, atomic force microscopy, transmission electron microscopy, and nuclear magnetic resonance, we have demonstrated that the amino acid L-arginine (Arg) has the striking potential to influence insulin aggregation propensity. It was shown that modification of the net charge of insulin induced by changes in the pH level of the incubation medium results in dramatic changes in the interaction of the protein with Arg. We have revealed the dual effects of Arg, highly dependent on the pH level of the solution e suppression or acceleration of the aggregation of insulin at pH 7.0 or 8.0, respectively. These effects can be regulated by manipulating the pH of the environment. The results of this study may be of interest for development of appropriate drug formulations and for the more general insight into the functioning of insulin in living systems, as the protein is known to release by exocytosis from pancreatic beta cells in a pH-dependent manner.
Human β-cell proliferation by promoting Wnt signaling

Carol Wilson
Original article Aly, H. et al. A novel strategy to increase the proliferative potential of adult human β-cells while maintaining their differentiated phenotype. PLoS ONE 2013; 8, e66131
Nature Reviews Endocrinology 2013; 9, 502
http://dx.doi.org:/10.1038/nrendo.2013.130

Islet transplantation for patients with type 1 diabetes mellitus typically requires 2–4 donors for one recipient, whereas use of one donor would minimize the risk of immune rejection. Proliferation of adult β cells in vitro could hold the key to providing one donor for one recipient.

“In previous studies, we found that activation of the Wnt/GSK-3/β-catenin pathway by pharmacologic inhibition of GSK-3 in combination with nutrient activation of mTOR, modestly enhanced human β-cell proliferation in vitro,” says lead researcher Haytham Aly of the Washington University School of Medicine in St. Louis, MO, USA. “However, expansion of human islets was associated with a loss of insulin content and secretory function.”

In the current study, the researchers aimed to engage canonical and noncanonical Wnt signalling at the receptor level to increase the proliferation of human β cells in vitro, without losing the capacity of the cells to produce and secrete insulin.

The researchers treated cadaver-derived intact human islets with a conditioned medium from L cells that constitutively produce Wnt-3a, R-spondin-3 and Noggin. A similar medium had previously enabled successful proliferation of mouse colonic intestinal epithelial cells. The researchers added inhibitors of ROCK and RhoA to this medium to augment cell survival.

The conditioned medium with the inhibitors lead to ~20-fold proliferation of the human β cells above that with glucose alone. Crucially, treatment with this conditioned medium did not impair glucose-stimulated insulin secretion or decrease insulin content of the cells.

“This novel strategy has clear potential for use in the in vitro expansion of human islets and the subsequent treatment of impaired β-cell functional mass in type 1 diabetes mellitus and type 2 diabetes mellitus,” concludes Aly.

Betatrophin—inducing β-cell expansion to treat diabetes mellitus?

Elisabeth Kugelberg
Original article Yi, P. et al. Betatrophin: a hormone that controls pancreatic β cell proliferation. Cell http://dx.doi.org:/10.1016/j.cell.2013.04.008
Nature Reviews Endocrinology 2013; 9, 379; http://dx.doi.org:/10.1038/nrendo.2013.98

Betatrophin, a newly identified hormone, increases the production and expansion of insulin-secreting β cells in mice, research from Harvard University suggests.

When insulin resistance develops, pancreatic β cells undergo an expansion in mass and proliferation to compensate for increasing insulin needs. To date, the mechanisms regulating β-cell replication are unclear.

Yi et al. developed a mouse model of insulin resistance using the insulin receptor antagonist S961. Subcutaneous injections of the S961 peptide into mice led to dose-dependent, instant β-cell proliferation and hyperglycemia.

Microarray analysis revealed that a highly conserved mammalian gene, betatrophin, was upregulated fourfold in liver and threefold in white adipose tissue cells in response to the acute peripheral insulin resistance induced by S961.

Yi and coworkers found that Betatrophin encodes a secreted protein that can be detected in human plasma. Intravenous injection of betatrophin-expressing constructs into mice resulted in a 17-fold higher β-cell proliferation rate compared with control vectors, and ultimately led to increased islet size and insulin content, with improvements in glucose tolerance, in betatrophin-injected animals.

The mechanisms of action of betatrophin are still unknown, and the next step is to test the effects of recombinant betatrophin protein on β-cell mass. The authors conclude that the identification of betatrophin and its control of β-cell proliferation opens a new door to possible diabetes therapy.

Blocking RANKL signaling might prevent T2DM

Carol Wilson
Original article Kiechl, S. et al. Blockade of receptor activator of nuclear factor-κB (RANKL) signaling improves hepatic insulin resistance and prevents development of diabetes mellitus. Nat. Med.
http://dx.doi.org:/10.1038/nm.3084

Nature Reviews Endocrinology 2013; 9, 188;
http://dx.doi.org:/10.1038/nrendo.2013.43

Blockade of receptor activator of nuclear factor κB ligand (RANKL) signaling in hepatocytes protects against type 2 diabetes mellitus (T2DM), report researchers.

“It is well known that activation of nuclear factor κB (NF-κB) in the liver is a crucial event in the development of hepatic insulin resistance and T2DM,” explains lead author Stefan Kiechl of the Medical University of Innsbruck, Austria. “RANKL, a member of the tumour necrosis factor superfamily, is a potent activator of NF-κB, and its receptor RANK is expressed on liver cells. We, thus, hypothesized that RANKL is involved in hepatic NF-κB activation, leading to T2DM.”

The researchers studied the association between serum levels of soluble RANKL and osteoprotegerin and subsequent risk of developing T2DM in 844 men and women without T2DM aged 40–79 years. Soluble RANKL was assessed because it has been shown to be functionally active.

During follow-up, between 1990 and 2005, 78 individuals of the cohort developed T2DM. Baseline levels of soluble RANKL between individuals who had and had not developed T2DM differed considerably: risk of T2DM was elevated in the group with the top tertile T2DM of concentrations of soluble RANKL compared with the group with the bottom tertile (OR 4.06, 95% CI 2.01–8.20). Adjustment for lifestyle factors and body composition did not significantly affect the risk association. Interestingly, although concentrations of osteoprotegerin were not elevated preceding T2DM onset, as they were for soluble RANKL, increased levels were found in individuals after disease occurrence.

In a series of mouse models in which RANKL signaling was downregulated systemically or in the liver, the investigators showed that hepatic insulin sensitivity and plasma glucose concentrations improved with blockade of RANKL signaling. In one such experiment, mice with a hepatocyte-specific Rank knockout were fed a high-fat diet for 4 weeks. These mice did not develop insulin resistance, whereas control mice did.

The investigators note that medications for T2DM already available, such as metformin, lower RANKL activity in bone and might also lower RANKL activity in the liver. They speculate that RANKL antagonism could be a yet unknown.

SFRP4—a biomarker for islet dysfunction?

Carol Wilson
Original article Mahdi, T. et al. Secreted frizzled-related protein 4 reduces insulin secretion and is overexpressed in type 2 diabetes. Cell Metab. http://doi.org:/10.1016/j.cmet.2012.10.009

Secreted frizzled-related protein 4 (SFRP4) reduces insulin secretion and is a potential biomarker for islet dysfunction in type 2 diabetes mellitus (T2DM), report researchers.

Mahdi et al. discovered these insights into the pathophysiology of T2DM by the analysis of global gene expression in human pancreatic islets. The researchers identified a group of co-expressed genes (also called a gene co-expression module) associated with T2DM, reduced insulin secretion and elevated HbA1c levels after analysing global microarray expression data from human islets of 48 individuals, including 10 with T2DM. This module was enriched for IL-1-related genes.

The investigators identified SFRP4 as a gene highly expressed in islets from patients with T2DM. The protein encoded by SFRP4 is an extracellular regulator of the Wnt pathway, and has roles in tissue development, cancer and phosphate metabolism. Further study revealed that the expression and release of SFRP4 from islets was stimulated by IL-1β. Furthermore, elevated systemic SFRP4 levels led to reduced glucose tolerance as a result of decreased islet expression of voltage-gated Ca2+ channels and supressed insulin exocytosis.

Interestingly, levels of SFRP4 were elevated in serum of patients a few years before they developed T2DM, which indicates that this protein has potential to be used as a biomarker for T2DM. The researchers also point out that their data suggest that SFRP4 could be a therapeutic target for the treatment of islet dysfunction.

Add-on to metformin in T2DM —linagliptin or glimepiride?

Mikkel Christensen and Filip K. Knop
Nat. Rev. Endocrinol. 2012; 8, 576–578 http://dx.doi.org:/10.1038/nrendo.2012.163

Dipeptidyl peptidase 4 (DPP4) inhibitors, also known as gliptins, are a rapidly expanding class of oral antidiabetic drugs for the treatment of type 2 diabetes mellitus (T2DM). Since 2006, five DPP4 inhibitors have reached the market and, because they can be administered orally and have an almost impeccable safety profile, these drugs have gained widespread use in the treatment of T2DM. The DPP4 inhibitor linagliptin was approved in 2011 by the FDA and the European Medicines Agency (EMA) for use in patients with T2DM as second-line therapy to add on to metformin either alone or in combination with another second-line treatment.

The UK Prospective Diabetes Study trial showed that sulphonylurea treatment was more effective than metformin treatment after 1 year in terms of reducing HbA1c levels; however, after 6 years of treatment, the effectiveness of sulphonylurea treatment declined and metformin treatment was more effective. A decline in the effectiveness of the sulphonylurea treatment over time could be due to sulphonylureas inducing stress and possibly causing apoptosis in β cells. However, in the trial by Gallwitz et al. the sustained efficacies of the add-on treatments with linagliptin and glimepiride were similar after 2 years.

The inhibitors of DPP4 enhance glucose-dependent insulin secretion and could even augment the counter-regulatory glucagon response to hypoglycemia. DPP4 inhibition generally has a neutral effect upon body weight.

The study by Gallwitz et al. included patients whose plasma glucose levels were near-normal whilst they were receiving metformin monotherapy (baseline level 6–7 mmol/l), which could result in increased occurrence of hypoglycemia. Treating patients whose blood glucose levels were, by many standards, already adequately controlled with metformin with a drug known to be associated with inducing hypoglycemia would be expected to increase the frequency of hypoglycemia in this group, inflating the differences in the frequency of this event between the group receiving linagliptin and that receiving glimepiride.

The most groundbreaking findings in the study by Gallwitz et al. are related to cardiovascular outcomes. Although the study was not adequately powered to detect subtle differences in cardiovascular event frequency, significantly fewer patients who received linagliptin than glimepiride experienced major cardiovascular events (12 versus 26 individuals, respectively). This difference was driven by fewer patients experiencing nonfatal myocardial infarctions and nonfatal strokes in the linagliptin-treated group than in the glimepiride-treated group (9 versus 21 individuals, respectively).

Clinicians are responsible for selecting a suitable second-line treatment for patients with type 2 diabetes mellitus when metformin monotherapy fails. New evidence could aid clinicians in deciding between one of the most commonly used second-line agents, glimepiride, and the recently approved dipeptidyl peptidase 4 inhibitor linagliptin.

Relation of Mitochondrial Oxygen Consumption in Peripheral Blood Mononuclear Cells to Vascular Function in Type 2 Diabetes Mellitus

Mor-Li Hartman, Orian S. Shirihai, Monika Holbrook, Guoquan Xu, et al.
Vasc Med. 2014 February ; 19(1): 67–74. http://dx.doi.org:/10.1177/1358863X14521315.

Recent studies have shown mitochondrial dysfunction and increased production of reactive

oxygen species in peripheral blood mononuclear cells (PBMC’s) and endothelial cells from patients with diabetes mellitus. Mitochondria oxygen consumption is coupled to ATP production and also occurs in an uncoupled fashion during formation of reactive oxygen species by components of the electron transport chain and other enzymatic sites. We therefore hypothesized that diabetes would be associated with higher total and uncoupled oxygen consumption in PBMC’s that would correlate with endothelial dysfunction. We developed a method to measure oxygen consumption in freshly isolated PBMC’s and applied it to 26 patients with type 2 diabetes mellitus and 28 non-diabetic controls. Basal (192±47 vs. 161±44 pMoles/min, P=0.01), uncoupled (64±16 vs. 53±16 pMoles/min, P=0.007), and maximal (795±87 vs. 715±128 pMoles/min, P=0.01) oxygen consumption rates were higher in diabetic patients compared to controls. There were no significant correlations between oxygen consumption rates and endothelium-dependent flow-mediated dilation measured by vascular ultrasound. Non-endothelium-dependent nitroglycerin-mediated dilation was lower in diabetics (10.1±6.6 vs. 15.8±4.8%, P=0.03) and correlated with maximal oxygen consumption (R= −0.64, P=0.001). In summary, we found that diabetes mellitus is associated with a pattern of mitochondrial oxygen consumption consistent with higher production of reactive oxygen species. The correlation between oxygen consumption and nitroglycerin-mediated dilation may suggest a link between mitochondrial dysfunction and vascular smooth muscle cell dysfunction that merits further study. Finally, the described method may have utility for assessment of mitochondrial function in larger scale observational and interventional studies in humans.

Musashi expression in b-cells coordinates insulin expression, apoptosis and proliferation in response to endoplasmic reticulum stress in diabetes

M Szabat, TB Kalynyak, GE Lim, KY Chu, YH Yang, A Asadi, BK Gage, et al.
Cell Death and Disease (2011) 2, e232
http://dx.doi.org:/10.1038/cddis.2011.119

Diabetes is associated with the death and dysfunction of insulin-producing pancreatic b-cells. In other systems, Musashi genes regulate cell fate via Notch signaling, which we recently showed regulates b-cell survival. Here we show for the first time that human and mouse adult islet cells express mRNA and protein of both Musashi isoforms, as well Numb/Notch/Hes/neurogenin-3 pathway components. Musashi expression was observed in insulin/glucagon double-positive cells during human fetal development and increased during directed differentiation of human embryonic stem cells (hESCs) to the pancreatic lineage. De-differentiation of b-cells with activin A increased Msi1 expression. Endoplasmic reticulum (ER) stress increased Msi2 and Hes1, while it decreased Ins1 and Ins2 expression, revealing a molecular link between ER stress and b-cell dedifferentiation in type 2 diabetes. These effects were independent of changes in Numb protein levels and Notch activation. Overexpression of MSI1 was sufficient to increase Hes1, stimulate proliferation, inhibit apoptosis and reduce insulin expression, whereas Msi1 knockdown had the converse effects on proliferation and insulin expression. Overexpression of MSI2 resulted in a decrease in MSI1 expression. Taken together, these results demonstrate overlapping, but distinct roles for Musashi-1 and Musashi-2 in the control of insulin expression and b-cell proliferation. Our data also suggest that Musashi is a novel link between ER stress and the compensatory b-cell proliferation and the loss of b-cell gene expression seen in specific phases of the progression to type 2 diabetes.

Cooperation between brain and islet in glucose homeostasis and diabetes

Michael W. Schwartz, RJ Seeley, MH Tschöp, SC Woods, et al.
Nature 7 Nov 2013; 503: 59–66 http://dx.doi.org/10.1038/nature12709

Although a prominent role for the brain in glucose homeostasis was proposed by scientists in the nineteenth century, research throughout most of the twentieth century focused on evidence that the function of pancreatic islets is both necessary and sufficient to explain glucose homeostasis, and that diabetes results from defects of insulin secretion, action or both. However, insulin-independent mechanisms, referred to as ‘glucose effectiveness’, account for roughly 50% of overall glucose disposal, and reduced glucose effectiveness also contributes importantly to diabetes pathogenesis. Although mechanisms underlying glucose effectiveness are poorly understood, growing evidence suggests that the brain can dynamically regulate this process in ways that improve or even normalize glycaemia in rodent models of diabetes. Here we present evidence of a brain-centred glucoregulatory system (BCGS) that can lower blood glucose levels via both insulin-dependent and -independent mechanisms, and propose a model in which complex and highly coordinated interactions between the BCGS and pancreatic islets promote normal glucose homeostasis. Because activation of either regulatory system can compensate for failure of the other, defects in both may be required for diabetes to develop. Consequently, therapies that target the BCGS in addition to conventional approaches based on enhancing insulin effects may have the potential to induce diabetes remission, whereas targeting just one typically does not.

The traditional view holds that diabetes arises as a consequence of damage to, and ultimately failure of, beta-cell function. We propose a two-component model in which failure of glucose homeostasis can begin after initial impairment.

Schematic illustrations of brain- and islet-centred glucoregulatory systems

Schematic illustrations of brain- and islet-centred glucoregulatory systems
The BCGS is proposed to regulate tissue glucose metabolism and plasma glucose levels via mechanisms that are both insulin dependent (for example, by regulating tissue insulin sensitivity) and insulin independent

Proposed contributions of defective brain- and islet-centred glucoregulatory systems to T2D pathogenesis

Insulin’s discovery: New insights on its ninetieth birthday

Jesse Roth, Sana Qureshi, Ian Whitford, Mladen Vranic, et al.
Diabetes Metab Res Rev 2012; 28: 293–304
http://dx.doi.org:/10.1002/dmrr.2300

2012 marks the 90th year since the purification of insulin and the miraculous rescue from death of youngsters with type 1 diabetes. In this review, we highlight several previously unappreciated or unknown events surroundingthe discovery.
(i) We remind readers of the essential contributions of each of the four discoverers – Banting, Macleod, Collip, and Best.
(ii) Banting and Best (each with his own inner circle) worked not only to accrue credit for himself but also to minimize credit to the other discoverers.
(iii) Banting at the time of the insulin research was very likely suffering from post-traumatic stress disorder (PTSD) that originated during his heroic service as a surgeon in World War I on the Western Front in 1918, including an infected shrapnel wound that threatened amputation of his arm. His war record along with the newly discovered evidence of a suicide threat goes along with his paranoia, combativeness, alcohol excess, and depression, symptoms we associate with PTSD.
(iv) Banting’s eureka idea, ligation of the pancreatic duct to preserve the islets, while it energized the early research, was unnecessary and was bypassed early.
(v) Post discovery,Macleod uncovered many features of insulin action that he summarized in his 1925 Nobel Lecture.Macleod closed by raising the question – what is the mechanism of insulin action in the body? – a challenge that attracted many talented investigators but remained unanswered until the latter third of the 20th century.

Genetic Variants Associated With Glycine Metabolism and Their Role in Insulin Sensitivity and Type 2 Diabetes

Weijia Xie, Andrew R. Wood, Valeriya Lyssenko, Michael N. Weedon, et al.
Diabetes 2013; 62:2141–2150 http://dx.doi.org:/10.2337/db12-0876

Circulating metabolites associated with insulin sensitivity may represent useful biomarkers, but their causal role in insulin sensitivity and diabetes is less certain. We previously identified novel metabolites correlated with insulin sensitivity measured by the hyperinsulinemic-euglycemic clamp. The top-ranking metabolites were in the glutathione and glycine biosynthesis pathways. We aimed to identify common genetic variants associated with metabolites in these pathways and test their role in insulin sensitivity and type 2 diabetes. With 1,004 nondiabetic individuals from the RISC study, we performed a genome-wide association study (GWAS) of 14 insulin sensitivity–related metabolites and one metabolite ratio. We replicated our results in the Botnia study (n = 342). We assessed the association of these variants with diabetes-related traits in GWAS meta-analyses (GENESIS [including RISC, EUGENE2, and Stanford], MAGIC, and DIAGRAM). We identified four associations with three metabolites—glycine (rs715 at CPS1), serine (rs478093 at PHGDH), and betaine (rs499368 at SLC6A12; rs17823642 at BHMT)—and one association signal with glycine-to-serine ratio (rs1107366 at ALDH1L1). There was no robust evidence for association between these variants and insulin resistance or diabetes. Genetic variants associated with genes in the glycine biosynthesis pathways do not provide consistent evidence for a role of glycine in diabetes related traits.

Fractalkine (CX3CL1), a new factor protecting b-cells against TNFa

Sabine Rutti, Caroline Arous, Domitille Schvartz, Katharina Timper, et al.
MOLMET164_proof ■ 14 Aug 2014 ■ 1/11
http://dx.doi.org/10.1016/j.molmet.2014.07.007

Objective: We have previously shown the existence of a muscleepancreas intercommunication axis in which CX3CL1 (fractalkine), a CX3C chemokine produced by skeletal muscle cells, could be implicated. It has recently been shown that the fractalkine system modulates murine β-cell function. However, the impact of CX3CL1 on human islet cells especially regarding a protective role against cytokine-induced apoptosis remains to be investigated. Methods: Gene expression was determined using RNA sequencing in human islets, sorted β- and non-β-cells. Glucose-stimulated insulin secretion (GSIS) and glucagon secretion from human islets was measured following 24 h exposure to 1e50 ng/ml CX3CL1. GSIS and specific protein phosphorylation were measured in rat sorted β-cells exposed to CX3CL1 for 48 h alone or in the presence of TNFα (20 ng/ml). Rat and human β-cell apoptosis (TUNEL) and rat β-cell proliferation (BrdU incorporation) were assessed after 24 h treatment with increasing concentrations of CX3CL1. Results: Both CX3CL1 and its receptor CX3CR1 are expressed in human islets. However, CX3CL1 is more expressed in non-β-cells than in b-cells while its receptor is more expressed in β-cells. CX3CL1 decreased human (but not rat) β-cell apoptosis. CX3CL1 inhibited human islet glucagon secretion stimulated by low glucose but did not impact human islet and rat sorted β-cell GSIS. However, CX3CL1 completely prevented the adverse effect of TNFa on GSIS and on molecular mechanisms involved in insulin granule trafficking by restoring the phosphorylation (Akt, AS160, paxillin) and expression (IRS2, ICAM-1, Sorcin, PCSK1) of key proteins involved in these processes. Conclusions: We demonstrate for the first time that human islets express and secrete CX3CL1 and CX3CL1 impacts them by decreasing glucagon secretion without affecting insulin secretion. Moreover, CX3CL1 decreases basal apoptosis of human β-cells. We further demonstrate that CX3CL1 protects β-cells from the adverse effects of TNFa on their function by restoring the expression and phosphorylation of key proteins of the insulin secretion pathway.
Heart Failure, Saxagliptin and Diabetes Mellitus: Observations from the SAVOR – TIMI 53 Randomized Trial

Benjamin M. Scirica; Eugene Braunwald; Itamar Raz, and SAVOR-TIMI 53 Steering Committee and Investigators
Circulation. Sept 4, 2014 http://dx.doi.org:/10.1161/CIRCULATIONAHA.114.010389
Background—Diabetes and heart failure frequently coexist. However, few diabetes trials have prospectively evaluated and adjudicated heart failure as an endpoint. Methods and Results—16,492 patients with type 2 diabetes and a history of, or at risk for, cardiovascular events were randomized to saxagliptin or placebo (mean followup-2.1 years). The primary endpoint was the composite of cardiovascular death, myocardial infarction, or ischemic stroke. Hospitalization for heart failure was a predefined component of the secondary endpoint. Baseline NT-proBNP was measured in 12,301 patients. More patients treated with saxagliptin (289, 3.5%) were hospitalized for heart failure compared to placebo (228, 2.8%) (HR 1.27; 95%CI 1.07-1.51; p=0.007). Corresponding rates at 12-months were 1.9% vs.1.3% (HR 1.46, 95%CI 1.15-1.88, p=0.002, with no significant difference thereafter time-varying interaction
p=0.017). Subjects at greatest risk for hospitalization for heart failure had prior heart failure, EGFR < 60 ml/min and/or elevated baseline levels of NT-proBNP. There was no evidence of heterogeneity between NT-proBNP and saxagliptin (p for interaction=0.46), though the absolute risk excess for heart failure with saxagliptin was greatest in the highest NT-proBNP quartile (2.1%). Even in patients at high-risk for hospitalization for heart failure, the risk of the primary and secondary endpoints were similar between treatment groups. Conclusions—In the context of balanced primary and secondary endpoints, saxagliptin treatment was associated with an increased risk for hospitalization for heart failure. This increase in risk was highest among patients with elevated levels of natriuretic peptides, prior heart failure, or chronic kidney disease.
Angiotensin 1–7 improves insulin sensitivity by increasing skeletal muscle glucose uptake in vivo

Omar Echeverría-Rodríguez, Leonardo Del Valle-Mondragón, Enrique Hong
Peptides 51 (2014) 26– 30 http://dx.doi.org/10.1016/j.peptides.2013.10.022

The renin–angiotensin system (RAS) regulates skeletal muscle insulin sensitivity through different mechanisms. The overactivation of the ACE (angiotensin-converting enzyme)/Ang (angiotensin) II/AT1R (Ang IItype 1 receptor) axis has been associated with the development of insulin resistance, whereas the stimulation of the ACE2/Ang 1–7/MasR (Mas receptor) axis improves insulin sensitivity. The in vivo mechanismsby which this axis enhances skeletal muscle insulin sensitivity are scarcely known. In this work, we investigated whether rat soleus muscle expresses the ACE2/Ang 1–7/MasR axis and determined the effect ofAng 1–7 on rat skeletal muscle glucose uptake in vivo. Western blot analysis revealed the expression ofACE2 and MasR, while Ang 1–7 levels were detected in rat soleus muscle by capillary zone electrophoresis. The euglycemic clamp exhibited that Ang 1–7 by itself did not promote glucose transport, but itincreased insulin-stimulated glucose disposal in the rat. In a similar manner, captopril (an ACE inhibitor) enhanced insulin-induced glucose uptake and this effect was blocked by the MasR antagonist A-779. Our results show for the first time that rat soleus muscle expresses the ACE2/Ang 1–7/MasR axis of the RAS,and Ang 1–7 improves insulin sensitivity by enhancing insulin-stimulated glucose uptake in rat skeletal muscle in vivo. Thus, endogenous (systemic and/or local) Ang 1–7 could regulate insulin-mediated glucose transport in vivo.

Evolving concepts in advanced glycation, diabetic nephropathy, and diabetic vascular disease

George Jerums, S Panagiotopoulos, J Forbes, T Osicka, and Mark Cooper
Archives of Biochemistry and Biophysics 419 (2003) 55–62
http://dx.doi.org:/10.1016/j.abb.2003.08.017

Advanced glycation endproducts (AGEs) have been postulated to play a role in the development of both nephropathy and large vessel disease in diabetes. However, it is still not clear which AGE subtypes play a pathogenetic role and which of several AGE receptors mediate AGE effects on cells. This review summarises the renoprotective effect of inhibitors of AGE formation, including aminoguanidine, and of cross-link breakers, including ALT-711, on experimental diabetic nephropathy and on mesenteric vascular hypertrophy. It also demonstrates similar effects of aminoguanidine and ramipril (an angiotensin converting enzyme inhibitor) on fluorescent and immunoassayable AGE levels, renal protein kinase C activity, nitrotyrosine expression, lysosomal function, and protein handling in experimental diabetes. These findings indicate that inhibition of the renin angiotensin system blocks both upstream and downstream pathways leading to tissue injury. We postulate that the chemical pathways leading to advanced glycation endproduct formation and the renin angiotensin systems may interact through the generation of free radicals, induced both by glucose and angiotensin II. There is also evidence to suggest that AGE-dependent pathways may play a role in the development of tubulointerstitial fibrosis in the diabetic kidney. This effect is mediated through RAGE and is TGF-b and CTGF-dependent.

Preconditioning with Associated Blocking of Ca2+ Inflow Alleviates Hypoxia-Induced Damage to Pancreatic β-Cells

Zuheng Ma, Noah Moruzzi, Sergiu-Bogdan Catrina, Ingrid Hals, et al.
PLoS ONE 8(7): e67498. http://dx.doi.org:/10.1371/journal.pone.0067498

Objective: Beta cells of pancreatic islets are susceptible to functional deficits and damage by hypoxia. Here we aimed to characterize such effects and to test for and pharmacological means to alleviate a negative impact of hypoxia. Methods and Design: Rat and human pancreatic islets were subjected to 5.5 h of hypoxia after which functional and viability parameters were measured subsequent to the hypoxic period and/or following a 22 h re-oxygenation period. Preconditioning with diazoxide or other agents was usually done during a 22 h period prior to hypoxia. Results: Insulin contents decreased by 23% after 5.5 h of hypoxia and by 61% after a re-oxygenation period. Preconditioning with diazoxide time-dependently alleviated these hypoxia effects in rat and human islets. Hypoxia reduced proinsulin biosynthesis (3H-leucine incorporation into proinsulin) by 35%. Preconditioning counteracted this decrease by 91%. Preconditioning reduced hypoxia-induced necrosis by 40%, attenuated lowering of proteins of mitochondrial complexes I–IV and enhanced stimulation of HIF-1-alpha and phosphorylated AMPK proteins. Preconditioning by diazoxide was abolished by co-exposure to tolbutamide or elevated potassium (i.e. conditions which increase Ca2+ inflow). Preconditioning with nifedipine, a calcium channel blocker, partly reproduced effects of diazoxide. Both diazoxide and nifedipine moderately reduced basal glucose oxidation whereas glucose-induced oxygen consumption (tested with diazoxide) was unaffected. Preconditioning with diaxoxide enhanced insulin contents in transplants of rat islets to nondiabetic rats and lowered hyperglycemia vs. non-preconditioned islets in streptozotocin-diabetic rats. Preconditioning of human islet transplants lowered hyperglycemia in streptozotocin-diabetic nude mice.
Conclusions:
1) Prior blocking of Ca2+ inflow associates with lesser hypoxia-induced damage,
2) preconditioning affects basal mitochondrial metabolism and accelerates activation of hypoxia-reactive and potentially protective factors,
3) results indicate that preconditioning by K+-ATP-channel openers has therapeutic potential for islet transplantations.