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.
“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.
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.
“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.
‘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.
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 A1clevels 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
(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 recruitment6, 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 cascades9, 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 effects12, 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 (Emax (%)) 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 (Emax=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 reports43, 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 vitro47, 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 obesity50, 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.
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.
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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