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Posts Tagged ‘pathophysiology’


Diet and Diabetes

Writer and Curator: Larry H Bernstein, MD, FCAP 

 

Bile acid signaling in lipid metabolism: Metabolomic and lipidomic analysis of lipid and bile acid markers linked to anti-obesity and anti-diabetes in mice

Yunpeng Qi, Changtao Jiang, Jie Cheng, Kristopher W. Krausz, et al.

Biochimica et Biophysica Acta 1851 (2015) 19–29

http://dx.doi.org/10.1016/j.bbalip.2014.04.008

Bile acid synthesis is the major pathway for catabolism of cholesterol. Cholesterol 7α-hydroxylase (CYP7A1) is the rate-limiting enzyme in the bile acid biosynthetic pathway in the liver and plays an important role in regulating lipid, glucose and energy metabolism. Transgenic mice overexpressing CYP7A1 (CYP7A1-tg mice) were resistant to high fat diet (HFD)-induced obesity, fatty liver, and diabetes. However the mechanism of resistance to HFD-induced obesity of CYP7A1-tg mice has not been determined. In this study, metabolomic and lipidomic profiles of CYP7A1-tg mice were analyzed to explore the metabolic alterations in CYP7A1-tg mice that govern the protection against obesity and insulin resistance by using ultra-performance liquid chromatography-coupled with electrospray ionization quadrupole time-of-flight mass spectrometry combined with multivariate analyses. Lipidomics analysis identified seven lipid markers including lysophosphatidylcholines, phosphatidylcholines, sphingomyelins and ceramides that were significantly decreased in serum of HFD-fed CYP7A1-tgmice.Metabolomics analysis identified 13metabolites in bile acid synthesis including taurochenodeoxy-cholic acid, taurodeoxycholic acid, tauroursodeoxycholic acid, taurocholic acid, and tauro-β-muricholic acid (T-β-MCA) that differed between CYP7A1-tg and wild-type mice. Notably, T-β-MCA, an antagonist of the farnesoid X receptor (FXR) was significantly increased in intestine of CYP7A1-tg mice. This study suggests that reducing 12α-hydroxylated bile acids and increasing intestinal T-β-MCA may reduce high fat diet-induced increase of phospholipids, sphingomyelins and ceramides, and ameliorate diabetes and obesity. This article is part of a Special Issue entitled Linking transcription to physiology in lipidomics.

Bile acid synthesis is the major pathway for catabolism of cholesterol to bile acids. In the liver, cholesterol 7α-hydroxylase (CYP7A1) is the first and rate-limiting enzyme of the bile acid biosynthetic pathway producing two primary bile acids, cholic acid (CA, 3α, 7α, 12α-OH) and chenodeoxycholic acid (CDCA, 3α, 7α-OH) in humans. Sterol-12α hydroxylase (CYP8B1) catalyzes the synthesis of CA. In mice, CDCA is converted to α-muricholic acid (α-MCA: 3α, 6β, 7α-OH) and β-muricholic acid (β-MCA: 3α, 6β, 7β-OH). Bile acids are conjugated to taurine or glycine, secreted into the bile and stored in the gallbladder. After a meal, bile acids are released into the gastrointestinal tract. In the intestine, conjugated bile acids are first de-conjugated and then 7α-dehydroxylase activity in the gut flora converts CA to deoxycholic acid (DCA: 3α, 12α), and CDCA to lithocholic acid (LCA: 3α), two major secondary bile acids in humans.

In humans, most bile acids are glycine or taurine-conjugated and CA, CDCA and DCA are the most abundant bile acids. In mice, most bile acids are taurine-conjugated and CA and α- and β-MCAs are the most abundant bile acids. Bile acids facilitate absorption of dietary fats, steroids, and lipid soluble vitamins into enterocytes and are transported via portal circulation to the liver for metabolism and distribution to other tissues and organs. About 95% of bile acids are reabsorbed in the ileum and transported to the liver to inhibit CYP7A1 and bile acid synthesis. Enterohepatic circulation of bile acids provides a negative feedback mechanism to maintain bile acid homeostasis. Alteration of bile acid synthesis, secretion and transport causes cholestatic liver diseases, gallstone diseases, fatty liver disease, diabetes and obesity.

 Bile acid synthesis

 

Bile acid synthesis. In the classic bile acid synthesis pathway, cholesterol is converted to cholic acid (CA, 3α, 7α, 12α) and chenodeoxycholic acid (CDCA, 3α, 7α). CYP7A1 is the rate-limiting enzyme and CYP8B1 catalyzes the synthesis of CA. In mouse liver, CDCA is converted to α-muricholic acid (α-MCA, 3α, 6β, 7α) and β-MCA (3α, 6β, 7β). Most bile acids in mice are taurine (T)-conjugated and secreted into bile. In the intestine, gut bacteria de-conjugate bile acids and then remove the 7α-hydroxyl group from CA and CDCA to form secondary bile acids deoxycholic acid (DCA, 3α, 12α) and lithocholic acid (LCA, 3α), respectively. T-α-MCA and T-β-MCA are converted to T-hyodeoxycholic acid (THDCA, 3α, 6α), T-ursodeoxycholic acid (TUDCA, 3α, 7β), T-hyocholic acid (THCA, 3α, 6α, 7α) and T-murideoxycholic acid (TMDCA, 3α, 6β). These secondary bile acids are reabsorbed and circulated to liver to contribute to the bile acid pool. Secondary bile acids ω-MCA (3α, 6α, 7β) and LCA are excreted into feces.

Two FXR-dependent mechanisms are known to inhibit bile acid synthesis.  In the liver bile acid-activated FXR induces a negative receptor small heterodimer partner (SHP) to inhibit trans-activation activity of hepatic nuclear factor 4α(HNF4α) and liver receptor homologue-1 (LRH-1) that bind to the bile acid response element in the CYP7A1 and CYP8B1 gene promoters (Fig. 2, Pathway 1). In the intestine, bile acids activate FXR to induce fibroblast growth factor (mouse FGF15, or human FGF19), which activates hepatic FGF receptor 4 (FGFR4) and cJun N-terminal kinase 1/2 (JNK1/2) and extracellular-regulated kinase (ERK1/2) signaling of mitogen-activated protein kinase (MAPK) pathways to inhibit trans-activation of CYP7A1/CYP8B1 gene by HNF4α (Pathway 2). Several FXR-independent cell-signaling pathways have been reported and are shown as Pathway 3 (Fig. 2). Conjugated bile acids are known to activate several protein kinase Cs (PKC) and growth factor receptors, epidermal growth factor receptor (EGFR), and insulin receptor (IR) signaling to inhibit CYP7A1/CYP8B1 and bile acid synthesis via activating the ERK1/2, p38 and JNK1/2 pathways.

 

Bile acid signaling pathways. Bile acids activate FXR, TGR5 and cell signaling pathways to inhibit CYP7A1 and CYP8B1 gene transcription.

1) Hepatic FXR/SHP pathway: bile acid activated-FXR induces SHP, which inhibits HNF4α and LRH-1 trans-activation of CYP7A1 and CYP8B1 gene transcription in hepatocytes. Bile acid response element binds HNF4α and LRH-1.

2) Intestinal FXR/FGF19/FGFR4 pathway: in the intestine, FXR induces FGF15 (mouse)/FGF19 (human), which is secreted into portal circulation to activate FGF receptor 4 (FGFR4) in hepatocytes. FGFR4 signaling stimulates JNK1/2 and ERK1/2 pathways of MAPK signaling to inhibit CYP7A1 gene transcription by phosphorylation and inhibition of HNF4α binding activity.

3) FXR-independent signaling pathways: Conjugated bile acids activate PKCs,which activate the MAPK pathways to inhibit CYP7A1. Bile acids also activate insulin receptor (IR) signaling IRS/PI3K/PDK1/AKT, possibly via activation of epidermal growth factor receptor (EGFR) signaling, MAPKs (MEK, MEKK), to inhibit CYP7A1 gene transcription. The secondary bile acid TLCA activates TGR5 signaling in Kupffer cells. TGR5 signaling may regulate CYP7A1 by an unknown mechanism. TCA activates sphingosine-1-phosphate (S1P) receptor 2 (S1PR2), which may activate AKT and ERK1/2 to inhibit CYP7A1. S1P kinase 1 (Sphk1) phosphorylates sphingosine (Sph) to S-1-P, which activates S1PR2. On the other hand, nuclear SphK2 interacts with and inhibits histone deacetylase (HDAC1/2) and may induce CYP7A1. The role of S1P, SphK2, and S1PR2 signaling in regulation of bile acid synthesis is not known.

 

When challenged with an HFD, CYP7A1-tg mice had lower body fat mass and higher lean mass compared to wild-type mice. As a platform for comprehensive and quantitative description of the set of lipid species, lipidomics was used to investigate the mechanism of this phenotype. By use of an unsupervised PCA model with the cumulative R2X 0.677 for serum and 0.593 for liver, CYP7A1-tg and wild-type mice were clearly separated based on the scores plot (Supplementary Fig. S2), indicating that these two groups have distinct lipidomic profiles. Supervised PLS-DA models were then established to maximize the difference of metabolic profiles between CYP7A1-tg and wild-type groups as well as to facilitate the screening of lipid marker metabolites (Fig. 3).

PLS-DA analysis of CYP7A1-tg and wild-type (WT)mice challenged with HFD. Based on the score plots, distinct lipidomic profiles of male CYP7A1-tg and wild-type groups were shown for serum (A) and liver samples (B). Based on the loading plots (C for serum and D for liver) the most significant ions that led to the separation between CYP7A1-tg and wild-type groups were obtained and identified as follows: 1. LPC16:0; 2. LPC18:0; 3. LPC18:1; 4. LPC 18:2; 5. PC16:0-20:4; 6. PC16:0-22:6; 7. SM16:0. (not shown)

Fig. 5. OPLS-DA highlighted thirteen markers in bile acid pathway that contribute significantly to the clustering of CYP7A1-tg and wild-type (WT) mice. Ileum bile acids are shown. (not shown)

(A) In the score plot, female CYP7A1-tg andWTmicewere well separated;

(B) using a statistically significant thresholds of variable confidence approximately 0.75 in the S-plot, a number of ions were screened out as potential markers, which were later identified as 13 bile acid metabolites, including α-MCA, TCA, CDCA, and TCDCA etc.

Our recent study of CYP7A1-tg mice revealed that increased CYP7A1 expression and enlarged bile acid pool resulted in significant improvement of lipid homeostasis and resistance to high-fed diet-induced hepatic steatosis, insulin resistance, and obesity in CYP7A1-tg mice. In this study, metabolomics and lipidomics were employed to characterize the metabolic profiles of CYP7A1-tg mice and to provide new insights into the critical role of bile acids in regulation of lipid metabolism and metabolic diseases. Lipidomics analysis of serum lipid profiles of high fat diet-fed CYP7A1-tg identified 7 lipidomic markers that were reduced in CYP7A1-tg mice compared to wild type mice. Metabolomics analysis identified 13 bile acid metabolites that were altered in CYP7A1-tg mice. In CYP7A1-tg mice, TCA and TDCA were reduced, whereas T-β-MCA was increased in the intestine compared to that of wild type mice. The decrease of serum LPC, PC, SM and CER, and 12α-hydroxylated bile acids, and increase of T-β-MCA may contribute to the resistance to diet-induced obesity and diabetes in CYP7A1-tg mice (Fig. 8).

The present metabolomics and lipidomics analysis revealed that even upon challenging with HFD, CYP7A1-tg mice had reduced lipid levels including LPC, PC, SM and CER. Metabolomics studies of human steatotic liver tissues and HFD-fed mice showed that serum and liver LPC and PC and other lipids levels were increased compared with non-steatotic livers, suggesting altered lipid metabolism contributes to non-alcoholic fatty liver disease (NAFLD). In HFD-fed CYP7A1-tg mice, reduced serum PC, LPC, SM and CER levels suggest a role for bile acids in maintaining phospholipid homeostasis to prevent NAFLD. SMs are important membrane phospholipids that interact with cholesterol in membrane rafts and regulate cholesterol distribution and homeostasis. A role for SM and CER in the pathogenesis of insulin resistance, diabetes and obesity and development of atherosclerosis has been reported. CER has a wide range of biological functions in cellular signaling such as activating protein kinase C and c-Jun N-terminal kinase (JNK), induction of β-cell apoptosis and insulin resistance. CER increases reactive oxidizing species and activates the NF-κB pathway, which induces proinflammatory cytokines, diabetes and insulin resistance. CER is synthesized from serine and palmitoyl-CoA or hydrolysis of SM by acid sphingomyelinase (ASM). HFD is known to increase CER and SM in liver. The present observation of decreased SM and CER levels in HFD-fed CYP7A1-tg mice indicated that bile acids might reduce HFD-induced increase of SM and CER. DCA activates an ASM to convert SM to CER, and Asm−/− hepatocytes are resistant to DCA induction of CER and activation of the JNK pathway [65]. In CYP7A1-tg mice, enlarged bile acid pool inhibits CYP8B1 and reduces CA and DCA levels. Thus, decreasing DCA may reduce ASM activity and SM and CER levels, and contribute to reducing inflammation and improving insulin sensitivity in CYP7A1-tg mice. It has been reported recently that in diabetic patients, serum 12α-hydroxylated bile acids are increased and correlated to insulin resistance [66].

Fig. 8. Mechanisms of anti-diabetic and anti-obesity function of bile acids in CYP7A1-tg mice. In CYP7A1-tg mice, overexpressing CYP7A1 increases bile acid pool size and reduces cholic acid by inhibiting CYP8B1. Lipidomics analysis revealed decreased serum LPC, PC, SM and CER. These lipidomic markers are increased in hepatic steatosis and NAFLD. Bile acids may reduce LPC, PC, SM and CER levels and protect against high fat diet-induced insulin resistance and obesity in CYP7A1-tgmice. Metabolomics analysis showed decreased intestinal TCA and TDCA and increased intestinal T-β-MCA in CYP7A1-tgmice.High fat diets are known to increase CA synthesis and intestinal inflammation. It is proposed that decreasing CA and  DCA synthesis may increase intestinal T-β-MCA,which antagonizes FXR signaling to increase bile acid synthesis and prevent high fat diet-induced insulin resistance and obesity. (not shown)

In conclusion,metabolomics and lipidomicswere employed to characterize the metabolic profiles of CYP7A1-tg mice, aiming to provide new insights into the mechanism of bile acid signaling in regulation of lipid metabolism and maintain lipid homeostasis. A number of lipid and bile acid markers were unveiled in this study. Decreasing of lipid markers, especially SM and CER may explain the improved insulin sensitivity and obesity in CYP7A1-tg mice. Furthermore, this study uncovered that enlarged bile acid pool size and altered bile acid composition may reduce de-conjugation by gut microbiota and increase tauroconjugated muricholic acids, which partially inhibit intestinal FXR signaling without affecting hepatic FXR signaling. This study is significant in applying metabolomics for diagnosis of lipid biomarkers for fatty liver diseases, obesity and diabetes. Increasing CYP7A1 activity and bile acid synthesis coupled to decreasing CYP8B1 and 12α-hydroxylated bile acids may be a therapeutic strategy for treating diabetes and obesity.

 

Bile acids are nutrient signaling hormones

Huiping Zhou, Phillip B. Hylemon
Steroids 86 (2014) 62–68
http://dx.doi.org/10.1016/j.steroids.2014.04.016

Bile salts play crucial roles in allowing the gastrointestinal system to digest, transport and metabolize nutrients. They function as nutrient signaling hormones by activating specific nuclear receptors (FXR, PXR, Vitamin D) and G-protein coupled receptors [TGR5, sphingosine-1 phosphate receptor 2 (S1PR2), muscarinic receptors]. Bile acids and insulin appear to collaborate in regulating the metabolism of nutrients in the liver. They both activate the AKT and ERK1/2 signaling pathways. Bile acid induction of the FXR-a target gene, small heterodimer partner (SHP), is highly dependent on the activation PKCf, a branch of the insulin signaling pathway. SHP is an important regulator of glucose and lipid metabolism in the liver. One might hypothesize that chronic low grade inflammation which is associated with insulin resistance, may inhibit bile acid signaling and disrupt lipid metabolism. The disruption of these signaling pathways may increase the risk of fatty liver and non-alcoholic fatty liver disease (NAFLD). Finally, conjugated bile acids appear to promote cholangiocarcinoma growth via the activation of S1PR2.

 

In the past, bile salts were considered to be just detergent molecules that were required for the solubilization of cholesterol in the gall bladder, promoting the digestion of dietary lipids and stimulating the absorption of lipids, cholesterol and fat-soluble vitamins in the intestines. Bile salts were also known to stimulate bile flow, promote cholesterol secretion from the liver, and have antibacterial properties. However, in 1999, three independent laboratories reported that bile acids were natural ligands for the farnesoid X receptor (FXR-α) . The recognition that bile acids activated specific nuclear receptors started a renaissance in the field of bile acid research. Since 1999, bile acids have been reported to activate other nuclear receptors (pregnane X receptor, vitamin D receptor), G protein coupled receptors [TGR5, sphingosine-1-phosphate receptor 2 (S1PR2), muscarinic receptor 2 (M2)] and cell signaling pathways (JNK1/2, AKT, and ERK1/2). Deoxycholic acid (DCA), a secondary bile acid, has also been reported to activate the epidermal growth factor receptor (EGFR). It is now clear that bile acids function as hormones or nutrient signaling molecules that help to regulate glucose, lipid, lipoprotein, and energy metabolism as well as inflammatory responses.

Bile acids are synthesized from cholesterol in liver hepatocytes, conjugated to either glycine or taurine and actively secreted via ABC transporters on the canalicular membrane into biliary bile. Conjugated bile acids are often referred to as bile salts. Bile acid synthesis represents a major output pathway of cholesterol from the body. Bile acids are actively secreted from hepatocytes via the bile salt export protein (BSEP, ABCB11) along with phospholipids by ABCB4 and cholesterol by ABCG5/ABCG8 in a fairly constant ratio under normal conditions. Bile acids are detergent molecules and form mixed micelles with cholesterol and phospholipids, which help to keep cholesterol in solution in the gall bladder. Eating stimulates the gall bladder to contract, emptying its contents into the small intestines. Bile salts are crucial for the solubilization and absorption of cholesterol and lipids as well as lipid soluble vitamins (A, D, E, and K). They activate pancreatic enzymes and form mixed micelles with lipids in the small intestines, promoting their absorption. Bile acids are efficiently recovered from the intestines, primarily the ileum, by the apical sodium dependent transporter (ASBT). Bile acids are secreted from ileocytes, on the basolateral side, by the organic solute OSTα/OSTβ transporter. Secondary bile acids, formed by 7α-dehydroxylation of primary bile acids by anaerobic gut bacteria, can be passively absorbed from the large bowel or secreted in the feces. Absorbed bile acids return to the liver via the portal blood where they are actively transported into hepatocytes primarily via the sodium taurocholate cotransporting polypeptide (NTCP, SLC10A1). Bile acids are again actively secreted from the hepatocytes into the bile, stimulating bile flow and the secretion of cholesterol and phospholipids. Bile acids undergo enterohepatic circulation several times each day (Fig. 1). During their enterohepatic circulation approximately 500–600 mg/day are lost via fecal excretion and must be replaced by new bile acid synthesis in the liver. The bile acid pool size is tightly regulated as excess bile acids can be highly toxic to mammalian cells.

Enterohepatic circulation of bile acids

 

Enterohepatic circulation of bile acids. Bile acids are synthesized and conjugated mainly to glycine or taurine in hepatocytes. Bile acids travel to the gall bladder for storage during the fasting state. During digestion, bile acids travel to the duodenum via the common bile duct. 95% of the bile acids delivered to the duodenum are absorbed back into blood within the ileum and circulate back to the liver through the portal vein. 5% of bile acids are lost in feces.

There are two pathways of bile acid synthesis in the liver, the neutral pathway and the acidic pathway (Fig. 2). The neutral pathway is believed to be the major pathway of bile acid synthesis in humans under normal physiological conditions. The neutral pathway is initiated by cholesterol 7α-hydroxylase (CYP7A1), which is the rate-limiting step in this biochemical pathway. CYP7A1 is a cytochrome P450 monooxygenase, and the gene encoding this enzyme is highly regulated by a feed-back repressive mechanism involving the FXR-dependent induction of fibroblast growth factor 15/19 (FGF15/19) by bile acids in the intestines. FGF15/19 binds to the fibroblast growth factor receptor 4 (FGFR4)/β-Klotho complex in hepatocytes activating both the JNK1/2 and ERK1/2 signaling cascades. Activation of the JNK1/2 pathway has been reported to down-regulate CYP7A1 mRNA in hepatocytes. FGFR4 and β-Klotho mice have increased levels of CYP7A1 and upregulated bile acid synthesis. Moreover, treatment of FXR mice with a specific FXR agonist failed to repress CYP7A1 in the liver. These results support an important role of FGF15, synthesized in the intestines by activation of FXR, in the regulation of CYP7A1 and bile acid synthesis in the liver. CYP7A1 has also been reported to be down-regulated by glucagon and pro-inflammatory cytokines and up-regulated by glucose and insulin during the postprandial period.

Fig. 2. (not shown) Biosynthetic pathways of bile acids. Two major pathways are involved in bile acid synthesis. The neutral (or classic) pathway is controlled by cholesterol 7α-hydroxylase (CYP7A1) in the endoplasmic reticulum. The acidic (or alternative) pathway is controlled by sterol 27-hydroxylase (CYP27A1) in mitochondria. The sterol 12α-hydroxylase (CYP8B1) is required to synthesis of cholic acid (CA). The oxysterol 7α-hydroxylase (CYP7B1) is involved in the formation of chenodeoxycholic acid (CDCA) in acidic pathway. The neutral pathway is also able to form CDCA by CYP27A1.

The neutral pathway of bile acid synthesis produces both cholic acid (CA) and chenodeoxycholic acid (CDCA) (Fig. 2). The ratio of CA and CDCA is primarily determined by the activity of sterol 12α-hydroxylase (CYP8B1). The gene encoding CYP8B1 is also highly regulated by bile acids. Bile acids induce the gene encoding small heterodimer partner (SHP) in the liver via activation of the farnesoid X receptor (FXR-α). SHP is an orphan nuclear receptor without a DNA binding domain. It interacts with several transcription factors, including hepatocyte nuclear factor 4 (HNF4α) and liver-related homolog-1 (LRH-1), and acts as a dominant negative protein to inhibit transcription. In this regard, a liver specific knockout of LRH-1 completely abolished the expression of CYP8B1, but had little effect on CYP7A1. These results suggest that the interaction of SHP with LRH-1, caused by bile acids, may be the key regulator of hepatic CYP8B1 and the ratio of CA/CDCA. The acidic or alternative pathway of bile acid synthesis is initiated in the inner membrane of mitochondria by sterol 27-hydroxylase (CYP27A1). This enzyme also has low sterol 25-hydroxylase activity. CYP27A1 is capable of further oxidizing the 27-hydroxy group to a carboxylic acid. Unlike, CYP7A1, CYP27A1 is widely expressed in various tissues in the body where it may produce regulatory oxysterols. Even though CYP27A1 is the initial enzyme in the acidic pathway of bile acid synthesis, it may not be the rate limiting step. The inner mitochondrial membrane is very low in cholesterol content. Hence, cholesterol transport into the mitochondria appears to be the rate limiting step.

The acidic pathway of bile acid synthesis is now being viewed as an important pathway for generating regulatory oxysterols. For example, 25-hydroxy-cholesterol and 27-hydroxycholesterol are natural ligands for the liver X receptor (LXR), which is involved in regulating cholesterol and lipid metabolism. Moreover, recent studies report that 25-hydroxycholesterol, formed by CYP27A1, can be converted into 5-cholesten-3β-25-diol-3-sulfate in the liver. The sulfated 25-hydroxycholesterol is a regulator of inflammatory responses, lipid metabolism and cell proliferation, and is located in the liver. Recent evidence suggests that sulfated 25-hydroxycholesterol is a ligand for peroxisome proliferator-activated receptor gamma (PPARc), which is a major regulator of inflammation and lipid metabolism. The 7α-hydroxylation of oxysterols is catalyzed by oxysterol 7α-hydroxylase (CYP7B1). This biotransformation allows some of these oxysterols to be converted to bile acids. Finally, oxysterols generated in extrahepatic tissues can be transported to the liver and metabolized into bile acids.

Bile acids can activate several different nuclear receptors (FXR, PXR and Vitamin D) and GPCRs (TGR5, S1PR2, and [M2] Muscarinic receptor). The ability of different bile acids to activate FXR-α occurs in the following order CDCA > LCA = DCA > CA; for the pregnane X receptor (PXR) LCA > DCA > CA and the vitamin D receptor, 3-oxo-LCA > LCA > DCA > CA. LCA is the best activator of PXR and the vitamin D receptor which correlates with the hydrophobicity and toxicity of this bile acid toward mammalian cells. Activation of PXR and the vitamin D receptor induces genes encoding enzymes which metabolize LCA into a more hydrophilic and less toxic metabolite. These nuclear receptors appear to function in the protection of cells from hydrophobic bile acids. In contrast, FXR-α appears to play a much more extensive role in the body by regulating bile acid synthesis, transport, and enterohepatic circulation. Moreover, FXR-α also participates in the regulation of glucose, lipoprotein and lipid metabolism in the liver as well as a suppressor of inflammation in the liver and intestines.

TGR5, also referred to as membrane-type bile acid receptor (MBAR), was the first GPCR to be reported to be activated by bile acids in the order LCA > DCA > CDCA > CA. TGR5 is a Gas type receptor which activates adenyl cyclase activity increasing the rate of the synthesis of c-AMP. TGR5 is widely expressed in human tissues, including: intestinal neuroendocrine cells, gall bladder, spleen, brown adipose tissue, macrophages and cholangiocytes, but not hepatocytes. TGR5 may play a role in various physiological processes in the body. TGR5 appears to be important in regulating energy metabolism. It has been postulated that bile acids may activate TGR5 in brown adipose tissue, activating type 2-iodothyroxine deiodinase and leading to increased levels of thyroid hormone and stimulation of energy metabolism. Moreover, TGR5 has been reported to promote the release of glucagon-like peptide-1 release from neuroendocrine cells, which increases insulin release in the pancreas. These results suggest that TGR5 may play a role in glucose homeostasis in the body. TGR5 is a potential target for drug development for treating type 2 diabetes and other metabolic disorders.

Interrelationship between sphingosine 1-phosphate receptor 2 and the insulin signaling pathway

 

Interrelationship between sphingosine 1-phosphate receptor 2 and the insulin signaling pathway in regulating hepatic nutrient metabolism. S1PR2, sphingosine 1-phosphate receptor 2; Src, Src Kinase; EGFR, epidermal growth factor receptor; PPARa, peroxisome proliferator-activated receptor alpha; NTCP, Na+/taurocholate cotransporting polypeptide; BSEP, bile salt export pump; PC, phosphotidylcholine; PECK, phosphoenolpyruvate carboxykinase; G6Pase, glucose-6-phosphatase; PDK1, phosphoinositide-dependent protein kinase 1; AKT, protein kinase B; SREBP, sterol regulatory element-binding protein; PKCf, protein kinase C zeta; FXR, farnesoid X receptor; SHP, small heterodimeric partner; MDR3, phospholipid transporter (ABCB4); GSK3b, glycogen synthase kinase 3 beta.

 

Both unconjugated and conjugated bile acids activate the insulin signaling (AKT) and ERK1/2 pathways in hepatocytes. Interesting, insulin and bile acids both activated glycogen synthase activity to a similar extent in primary rat hepatocytes. Moreover, the addition of both insulin and bile acids to the culture medium resulted in an additive effect on activation of glycogen synthase activity in primary hepatocytes. Infusion of taurocholate (TCA) into the chronic bile fistula rat rapidly activated the AKT and ERK1/2 signaling pathway and glycogen synthase activity. In addition, there was a rapid down-regulation of the gluconeogenic genes, PEP carboxykinase (PEPCK) and glucose-6-phophatase (G-6-Pase) and a marked up-regulation of SHP mRNA in these sample livers. These results suggest that TCA functions much like insulin to regulate hepatic glucose metabolism both in vitro and in vivo.

It has been reported that PKCζ phosphorylates FXR-α and may allow for its activation of target gene expression. In contrast, phosphorylation of FXR-α by AMPK inhibits the ability of FXR to induce target genes. PKCζ has been reported to be important for the translocation of the bile acid transporters NTCP (SLC10A1) and BSEP (ABC B11) to the basolateral and canalicular membranes, respectively. Finally, it has been recently reported that PKCζ phosphorylates SHP allowing both to translocate to the nucleus and down-regulate genes via epigenetic mechanisms. In total, these results all suggest that the insulin signaling pathway is an important regulator of FXR-α activation and bile acid signaling in the liver.

The activation of the insulin signaling pathway and FXR-α appear to collaborate in the coordinate regulation of glucose, bile acid and lipid metabolism in the liver. SHP, an FXR target gene, is an important pleotropic regulator of multiple metabolic pathways in the liver (Fig. 3). The S1PR2 appears to be an important regulator of hepatic lipid metabolism as S1PR2 mice rapidly (2 weeks) develop overt fatty livers on a high fat diet as compared to wild type mice (unpublished data). It is well established that inflammation and the synthesis of inflammatory cytokines i.e. TNFα inhibit insulin signaling by activation of the JNK1/2 signaling pathway, which phosphorylates insulin receptor substrate 1. Inflammation is believed to be an important factor in the development of type 2 diabetes and fatty liver disease. A Western diet is correlated with low grade chronic inflammation and insulin resistance. Inhibition of the insulin signaling pathway may decrease the ability of bile acids to activate FXR-α, induce SHP and other FXR target genes, leading to an increased risk of fatty liver and non-alcoholic fatty liver disease (NAFLD).

There appears to be extensive interplay between bile salts and insulin signaling in the regulation of nutrient metabolism in both the intestines and liver. Bile salts play a key role in the solubilization and absorption of nutrients from the intestines. The absorption of nutrients stimulates the secretion of insulin from the pancreas. Moreover, bile acids may also stimulate the secretion of insulin by activating TGR5 in intestinal neuroendocrine cells resulting in the secretion of glucagon-like peptide-1. In the liver, bile salts and insulin both activate the AKT and ERK1/2 signaling pathways which yields a stronger signal than either alone. The activation of PKCζ, a branch of the insulin signaling pathway, is required for the optimal induction of FXR target genes and the regulation of the cellular location of bile acid transporters

 

Fruit and vegetable consumption and risk of type 2 diabetes mellitus: A dose-response meta-analysis of prospective cohort studies

  1. Wu, D. Zhang, X. Jiang, W. Jiang
    Nutrition, Metabolism & Cardiovascular Diseases (2015) 25, 140-147
    http://dx.doi.org/10.1016/j.numecd.2014.10.004

Background and aims: We conducted a dose-response meta-analysis to summarize the evidence from prospective cohort studies regarding the association of fruit and vegetable consumption with risk of type 2 diabetes mellitus (T2DM). Methods and results: Pertinent studies were identified by searching Embase and PubMed through June 2014. Study-specific results were pooled using a random-effect model. The dose-response relationship was assessed by the restricted cubic spline model and the multivariate random-effect meta-regression. We standardized all data using a standard portion size of 106 g. The Relative Risk (95% confidence interval) [RR (95% CI)] of T2DM was 0.99 (0.98-1.00) for every 1 serving/day increment in fruit and vegetable (FV) (P < 0.18), 0.98 (0.95-1.01) for vegetable (P < 0.12), and 0.99 (0.97-1.00) for fruit (P < 0.05). The RR (95%CI) of T2DM was 0.99 (0.97-1.01), 0.98 (0.96-1.01), 0.97 (0.93-1.01), 0.96 (0.92-1.01), 0.96 (0.91-1.01) and 0.96 (0.91-1.01) for 1, 2, 3, 4, 5 and 6 servings/day of FV (P for non-linearity < 0.44). The T2DM risk was 0.96 (0.95-0.99), 0.94 (0.90-0.98), 0.94 (0.89-0.98), 0.96 (0.91-1.01), 0.98 (0.92-1.05) and 1.00 (0.93-1.08) for 1, 2, 3, 4, 5 and 6 servings/day of vegetable (P for non-linearity < 0.01). The T2DM risk was 0.95 (0.93-0.97), 0.91 (0.89-0.94), 0.88 (0.85-0.92), 0.92 (0.88-0.96) and 0.96 (0.92-1.01) for 0.5, 1, 2, 3 and 4 servings/day of fruit (P for non-linearity < 0.01). Conclusions: Two-three servings/day of vegetable and 2 servings/day of fruit conferred a lower risk of T2DM than other levels of vegetable and fruit consumption, respectively.

dose-response analysis between total fruit and vegetable consumption and risk of type 2 diabetes mellitus

 

The dose-response analysis between total fruit and vegetable consumption and risk of type 2 diabetes mellitus. The solid line and the long dash line represent the estimated relative risk and its 95% confidence interval.

 

Healthy behaviours and 10-year incidence of diabetes: A population cohort study

G.H. Long , I. Johansson , O. Rolandsson , …, E. Fhärm, L.Weinehall, et al.
Preventive Medicine 71 (2015) 121–127
http://dx.doi.org/10.1016/j.ypmed.2014.12.013

Objective. To examine the association between meeting behavioral goals and diabetes incidence over 10 years in a large, representative Swedish population. Methods. Population-based prospective cohort study of 32,120 individuals aged 35 to 55 years participating in a health promotion intervention in Västerbotten County, Sweden (1990 to 2013). Participants underwent an oral glucose tolerance test, clinical measures, and completed diet and activity questionnaires. Poisson regression quantified the association between achieving six behavioral goals at baseline – body mass index (BMI) < 25 kg/m2, moderate physical activity, non-smoker, fat intake  < 30% of energy, fibre intake ≥15 g/4184 kJ and alcohol intake ≤ 20 g/day – and diabetes incidence over 10 years. Results. Median interquartile range (IQR) follow-up time was 9.9 (0.3) years; 2211 individuals (7%) developed diabetes. Only 4.4% of participants met all 6 goals (n = 1245) and compared to these individuals, participants meeting 0/1 goals had a 3.74 times higher diabetes incidence (95% confidence interval (CI) = 2.50 to 5.59), adjusting for sex, age, calendar period, education, family history of diabetes, history of myocardial infarction and long-term illness. If everyone achieved at least four behavioral goals, 14.1% (95% CI: 11.7 to 16.5%) of incident diabetes cases might be avoided. Conclusion. Interventions promoting the achievement of behavioral goals in the general population could significantly reduce diabetes incidence.

 

Long term nutritional intake and the risk for non-alcoholic fatty liver disease (NAFLD): A population based study

Shira Zelber-Sagi, Dorit Nitzan-Kaluski, Rebecca Goldsmith, et al.
Journal of Hepatology 47 (2007) 711–717
http://dx.doi.org:/10.1016/j.jhep.2007.06.020

Background/Aims: Weight loss is considered therapeutic for patients with NAFLD. However, there is no epidemiological evidence that dietary habits are associated with NAFLD. Dietary patterns associated with primary NAFLD were investigated. Methods: A cross-sectional study of a sub-sample (n = 375) of the Israeli National Health and Nutrition Survey. Exclusion criteria were any known etiology for secondary NAFLD. Participants underwent an abdominal ultrasound, biochemical tests, dietary and anthropometric evaluations. A semi-quantitative food-frequency questionnaire was administered. Results: After exclusion, 349 volunteers (52.7% male, mean age 50.7 ± 10.4, 30.9% primary NAFLD) were included. The NAFLD group consumed almost twice the amount of soft drinks (P = 0.03) and 27% more meat (P < 0.001). In contrast, the NAFLD group consumed somewhat less fish rich in omega-3 (P = 0.056). Adjusting for age, gender, BMI and total calories, intake of soft drinks and meat was significantly associated with an increased risk for NAFLD (OR = 1.45, 1.13–1.85 95% CI and OR = 1.37, 1.04–1.83 95% CI, respectively). Conclusions: NAFLD patients have a higher intake of soft drinks and meat and a tendency towards a lower intake of fish rich in omega-3. Moreover, a higher intake of soft drinks and meat is associated with an increased risk of NAFLD, independently of age, gender, BMI and total calories.

 

The association between types of eating behavior and dispositional mindfulness in adults with diabetes. Results from Diabetes MILES. The Netherlands

Sanne R. Tak, Christel Hendrieckx, Giesje Nefs, Ivan Nyklícek, et al.
Appetite 87 (2015) 288–295
http://dx.doi.org/10.1016/j.appet.2015.01.006

Although healthy food choices are important in the management of diabetes, making dietary adaptations is often challenging. Previous research has shown that people with type 2 diabetes are less likely to benefit from dietary advice if they tend to eat in response to emotions or external cues. Since high levels of dispositional mindfulness have been associated with greater awareness of healthy dietary practices in students and in the general population, it is relevant to study the association between dispositional mindfulness and eating behavior in people with type 1 or 2 diabetes. We analyzed data from Diabetes MILES – The Netherlands, a national observational survey in which 634 adults with type 1 or 2 diabetes completed the Dutch Eating Behavior Questionnaire (to assess restrained, external and emotional eating behavior) and the Five Facet Mindfulness Questionnaire-Short Form (to assess dispositional mindfulness), in addition to other psychosocial measures. After controlling for potential confounders, including  demographics, clinical variables and emotional distress, hierarchical linear regression analyses showed that higher levels of dispositional mindfulness were associated with eating behaviors that were more restrained (β = 0.10) and less external (β = −0.11) and emotional (β = −0.20). The mindfulness subscale ‘acting with awareness’ was the strongest predictor of both external and emotional eating behavior, whereas for emotional eating, ‘describing’ and ‘being non-judgmental’ were also predictive. These findings suggest that there is an association between dispositional mindfulness and eating behavior in adults with type 1 or 2 diabetes. Since mindfulness interventions increase levels of dispositional mindfulness, future studies could examine if these interventions are also effective in helping people with diabetes to reduce emotional or external eating behavior, and to improve the quality of their diet.

 

Soft drink consumption is associated with fatty liver disease independent of metabolic syndrome

Ali Abid, Ola Taha, William Nseir, Raymond Farah, Maria Grosovski, Nimer Assy
Journal of Hepatology 51 (2009) 918–924
http://dx.doi.org:/10.1016/j.jhep.2009.05.033

Background/Aims: The independent role of soft drink consumption in non-alcoholic fatty liver disease (NAFLD) patients remains unclear. We aimed to assess the association between consumption of soft drinks and fatty liver in patients with or without metabolic syndrome. Methods: We recruited 31 patients (age: 43 ± 12 years) with NAFLD and risk factors for metabolic syndrome, 29 patients with NAFLD and without risk factors for metabolic syndrome, and 30 gender- and age-matched individuals without NAFLD. The degree of fatty infiltration was measured by ultrasound. Data on physical activity and intake of food and soft drinks were collected during two 7-day periods over 6 months using a food questionnaire. Insulin resistance, inflammation, and oxidant–antioxidant markers were measured.
Results: We found that 80% of patients with NAFLD had excessive intake of  soft drink beverages (>500 cm3/day) compared to 17% of healthy controls (p < 0.001). The NAFLD group consumed five times more carbohydrates from soft drinks compared to healthy controls (40% vs. 8%, p < 0.001). Seven percent of patients consumed one soft drink per day, 55% consumed two or three soft drinks per day, and 38% consumed more than four soft drinks per day for most days and for the 6-month period. The most common soft drinks were Coca-Cola (regular: 32%; diet: 21%) followed by fruit juices (47%). Patients with NAFLD with metabolic syndrome had similar malonyldialdehyde, paraoxonase, and C-reactive protein (CRP) levels but higher homeostasis model assessment (HOMA) and higher ferritin than NAFLD patients without metabolic syndrome (HOMA: 8.3 ± 8 vs. 3.7 ± 3.7 mg/dL, p < 0.001; ferritin: 186 ± 192 vs. 87 ± 84 mg/dL, p < 0.01). Logistic regression analysis showed that soft drink consumption is a strong predictor of fatty liver (odds ratio: 2.0; p < 0.04) independent of metabolic syndrome and CRP level. Conclusions: NAFLD patients display higher soft drink consumption independent of metabolic syndrome diagnosis. These findings might optimize NAFLD risk stratification.

 

Dietary predictors of arterial stiffness in a cohort with type 1 and type 2 diabetes

K.S. Petersen, J.B. Keogh, P.J. Meikle, M.L. Garg, P.M. Clifton
Atherosclerosis 238 (2015) 175-181
http://dx.doi.org/10.1016/j.atherosclerosis.2014.12.012

Objective: To determine the dietary predictors of central blood pressure, augmentation index and pulse wave velocity (PWV) in subjects with type 1 and type 2 diabetes. Methods: Participants were diagnosed with type 1 or type 2 diabetes and had PWV and/or pulse wave analysis performed. Dietary intake was measured using the Dietary Questionnaire for Epidemiological Studies Version 2 Food Frequency Questionnaire. Serum lipid species and carotenoids were measured, using liquid chromatography electrospray ionization- tandem mass spectrometry and high performance liquid chromatography, as biomarkers  of dairy and vegetable intake, respectively. Associations were determined using linear regression adjusted for potential confounders. Results: PWV (n = 95) was inversely associated with reduced fat dairy intake (β = -0.01; 95% CI -0.02, -0.01; p = 0 < 0.05) in particular yoghurt consumption (β = 0.04; 95% CI -0.09, -0.01; p = 0 < 0.05) after multivariate adjustment. Total vegetable consumption was negatively associated with PWV in the whole cohort after full adjustment (β =0.04; 95% CI -0.07, -0.01; p < 0.05). Individual lipid species, particularly those containing 14:0, 15:0, 16:0, 17:0 and 17:1 fatty acids, known to be of ruminant origin, in lysophosphatidylcholine, cholesterol ester, diacylglycerol, phosphatidylcholine, sphingomyelin and triacylglycerol classes were positively associated with intake of full fat dairy, after adjustment for multiple comparisons. However, there was no association between serum lipid species and PWV. There were no dietary predictors of central blood pressure or augmentation index after multivariate adjustment. Conclusion: In this cohort of subjects with diabetes reduced fat dairy intake and vegetable consumption were inversely associated with PWV. The lack of a relationship between serum lipid species and PWV suggests that the fatty acid composition of dairy may not explain the beneficial effect.

In this cohort with type 1 and type 2 diabetes there was an inverse association between reduced fat dairy intake, in particular yoghurt consumption, and PWV, which persisted after multivariate adjustment. Serum lipid species, known to be of ruminant origin, were positively associated with full fat dairy consumption; however there was no association between these lipid species and PWV. In addition, higher vegetable intake was also associated with lower PWV. There were no dietary predictors of central blood pressure or augmentation index identified in this cohort.

In this study there was no relationship between augmentation index and PWV, which has been previously reported. Augmentation index is not a direct measure of arterial stiffness and is influenced by the timing and magnitude of the wave reflection. In contrast, PWV is a robust measure of arterial stiffness as it is determined by measuring the velocity of the waveform between the carotid and femoral arteries. Previously, it has been shown that in a population with diabetes PWV was elevated compared with healthy controls, however augmentation index was not different. Lacy et al.  concluded that augmentation index is not a reliable measure of arterial stiffness in people with diabetes. This may explain why we did not see an association between augmentation index and dietary intake, despite seeing correlations with PWV.

 

Curcumin ameliorates diabetic nephropathy by inhibiting the activation of the SphK1-S1P signaling pathway

Juan Huang, Kaipeng Huang, Tian Lan, Xi Xie, .., Peiqing Liu, Heqing Huang
Molecular and Cellular Endocrinology 365 (2013) 231–240
http://dx.doi.org/10.1016/j.mce.2012.10.024

Curcumin, a major polyphenol from the golden spice Curcuma longa commonly known as turmeric, has been recently discovered to have renoprotective effects on diabetic nephropathy (DN). However, the mechanisms underlying these effects remain unclear. We previously demonstrated that the sphingosine kinase 1-sphingosine 1-phosphate (SphK1-S1P) signaling pathway plays a pivotal role in the pathogenesis of DN. This study aims to investigate whether the renoprotective effects of curcumin on DN are associated with its inhibitory effects on the SphK1-S1P signaling pathway. Our results demonstrated that the expression and activity of SphK1 and the production of S1P were significantly down-regulated by curcumin in diabetic rat kidneys and glomerular mesangial cells (GMCs) exposed to high glucose (HG). Simultaneously, SphK1-S1P-mediated fibronectin (FN) and transforming growth factor-beta 1 (TGF-b1) overproduction were inhibited. In addition, curcumin dose dependently reduced SphK1 expression and activity in GMCs transfected with SphKWT and significantly suppressed the increase in SphK1-mediated FN levels. Furthermore, curcumin inhibited the DNA-binding activity of activator protein 1 (AP-1), and c-Jun small interference RNA (c-Jun-siRNA) reversed the HG-induced up-regulation of SphK1. These findings suggested that down-regulation of the SphK1-S1P pathway is probably a novel mechanism by which curcumin improves the progression of DN. Inhibiting AP-1 activation is one of the therapeutic targets of curcumin to modulate the SphK1-S1P signaling pathway, thereby preventing diabetic renal fibrosis.

The creation of the STZ-induced DN model relies on the level and continuous cycle of high blood glucose in vivo. Long-term hyperglycemia induces significant structural changes in the kidney, including glomerular hypertrophy, GBM thickening, and later glomerulosclerosis and tubulointerstitial fibrosis, leading to microalbuminuria and elevated Cr levels. These effects usually occur at around 8–12 weeks after diabetes formation. In the current study, the experimental diabetic model was induced by a single intraperitoneal injection of STZ (60 mg/kg). When the experiment was terminated at 12 weeks, FBG, KW/BW, BUN, Cr, and UP 24 h were significantly increased and body weight was remarkably decreased in the STZ-induced diabetic rats compared with those in the normal control group. Furthermore, PAS staining of the kidneys revealed the induction of glomerular hypertrophy, mesangial matrix expansion, and increased regional adhesion of the glomerular tuft to the Bowman’s capsule in the diabetic rats. This finding indicated the emergence of the diabetic renal injury model characterized by renal hypertrophy, glomerulus damage, and renal dysfunction. As the limited water solubility of curcumin, various methods such as heat treatment, mild alkali and sodium carboxymethyl cellulose are used to increase the solubility of curcumin before administration. Based on our previous study, we employed 1% sodium carboxymethyl cellulose as the vehicle to solubilize curcumin. Compared with the diabetic group, curcumin treatment slightly reduced FBG level and significantly decreased KW/BW, BUN, Cr, and UP 24 h. Moreover, curcumin remarkably improved glomerular pathological changes in the diabetic kidneys. Consistent with previous studies, the current results demonstrated that curcumin prominently ameliorated renal function and renal parenchymal alterations in the diabetic renal injury model. Previous studies revealed that the amelioration of renal dysfunction in diabetes by curcumin was partly related to its function in inhibiting inflammatory injury. Based on these findings, the current experiment further explored whether the renoprotective effects of curcumin are associated with the regulation of the SphK1-S1P signaling pathway.

S1P is a polar sphingolipid metabolite acting as an extracellular mediator and an intracellular second messenger. Ample evidence proves that S1P participates in cell growth, proliferation, migration, adhesion, molecule expression, and angiogenesis. The formation of S1P is catalyzed by SphK1. Recently, the SphK1-S1P signaling pathway has gained considerable attention because of its potential involvement in the progression of DN. Hyperglycemia, AGE, and oxidative stress can activate SphK1 and can increase the intracellular level of S1P. Geoffroy et al. (2004) reported that the treatment of cells with low AGE concentration increases SphK activity and S1P production, thereby and S1P content were significantly increased simultaneously with the up-regulated expression of FN and TGF-β1 (mRNA and protein) in the diabetic rat kidneys. These findings indicated the activation of the SphK1-S1P signaling pathway and the appearance of pathological alterations, including ECM accumulation. After curcumin treatment for 12 weeks, elevations of the said indexes were significantly inhibited. HG remarkably activated the SphK1-S1P signaling pathway and increased FN and TGF-β1 expressions in GMCs. Curcumin dramatically suppressed the SphK1-S1P pathway as well as FN and TGF-β1 levels in a dose-dependent manner. Overall, these results indicated that curcumin ameliorated the pathogenic progression of DN by inhibiting the activation of the SphK1-S1P signaling pathway, resulting in the down-regulation of TGF-β1 and the subsequent reduction of ECM accumulation.

SphK1 expression is mediated by a novel AP-1 element located within the first intron of the human SphK1 gene. AP-1 sites are also found in rat SphK1 promoter from NCBI. Numerous studies indicated that curcumin can inhibit the activity of AP-1 and is widely used as an AP-1 inhibitor. Therefore, further elucidating the link between the inhibition of the SphK1-S1P signaling pathway by curcumin and the suppression of AP-1 activity is important. The data showed that treatment with c-Jun-siRNA significantly down-regulated the basal levels of SphK1 expression. Thus, inhibiting AP-1 activity is one of the therapeutic targets of curcumin in modulating the SphK1-S1P signaling pathway, thereby inhibiting diabetic renal fibrosis.

In summary, curcumin inhibited SphK1 expression and activity, reduced S1P content, and effectively inhibited increased FN and TGF-β1 expressions mediated by the SphK1-S1P signaling pathway. Moreover, the inhibitory effect of curcumin on SphK1-S1P was independent of its hypoglycemic and anti-oxidant roles and might be closely related to the inhibition of AP-1 activity. Our findings suggested that the SphK1-S1P pathway might be a novel mechanism by which curcumin attenuates renal fibrosis and ameliorates DN. In addition, the present study provides further experimental evidence for the clinical application and new drug exploration of curcumin.

 

Antidiabetic Activity of Hydroalcoholic Extracts of Nardostachys jatamansi in Alloxan-induced Diabetic Rats

  1. A. Aleem, B. Syed Asad, Tasneem Mohammed, et al.
    British Journal of Medicine & Medical Research 4(28): 4665-4673, 2014

A review of literature indicates that diabetes mellitus was fairly well known and well conceived as an entity in India with complications like angiopathy, retinopathy, nephropathy, and causing neurological disorders. The antidiabetic study was carried out to estimate the anti-hyperglycemic potential of Nardostachys Jatamansi rhizome’s hydroalcoholic extracts in alloxan induced diabetic rats over a period of two weeks. The hydroalcoholic extract HAE1 at a dose (500mg/kg) exhibited significant antihyperglycemic activity than extract HAE2 at a dose (500mg/kg) in diabetic rats. The hydroalcoholic extracts showed improvement in different parameters associated with diabetes, like body weight, lipid profile and biochemical parameters. Extracts also showed improvement in regeneration of β-cells of pancreas in diabetic rats. Histopath-ological studies strengthen the healing of pancreas by hydroalcoholic extracts (HAE1& HAE2) of Nardostachys Jatamansi, as a probable mechanism of their antidiabetic activity.
Metabolic syndrome and serum carotenoids : findings of a cross-sectional study in Queensland, Australia

Coyne, T, Ibiebele, T,… McClintock, C and Shaw, J
Brit J Nutrition: Int J Nutr Sci 2009; 102(11). pp. 1668-1677
Several components of the metabolic syndrome, particularly diabetes and cardiovascular disease, are known to be oxidative stress-related conditions and there is research to suggest that antioxidant nutrients may play a protective role in these conditions. Carotenoids are compounds derived primarily from plants and several have been shown to be potent antioxidant nutrients. The aim of this study was to examine the associations between metabolic syndrome status and major serum carotenoids in adult Australians. Data on the presence of the metabolic syndrome, based on International Diabetes Federation criteria, were collected from 1523 adults aged 25 years and over in six randomly selected urban centers in Queensland, Australia, using a cross sectional study design. Weight, height, BMI, waist circumference, blood  pressure, fasting and 2-hour blood glucose and  lipids were determined, as well as five serum carotenoids. Mean serum alpha-carotene, beta-carotene and the sum of the five carotenoid concentrations were significantly lower (p<0.05) in persons with the metabolic syndrome (after adjusting for age, sex, education, BMI status, alcohol intake, smoking, physical activity status and vitamin/mineral use) than persons without the syndrome. Alpha, beta and total carotenoids also decreased significantly (p<0.05) with increased number of components of the metabolic syndrome, after adjusting for these confounders. These differences were significant among former smokers and non-smokers, but not in current smokers. Low concentrations of serum alpha-carotene, beta carotene and the sum of five carotenoids appear to be associated with metabolic syndrome status. Additional research, particularly longitudinal studies, may help to determine if these associations are causally related to the metabolic syndrome, or are a result of the pathologies of the syndrome.

Although there is no universal definition of the metabolic syndrome, it is generally described as a constellation of pathologies or anthropometric conditions, which include central obesity, glucose intolerance, lipid abnormalities, and hypertension. It is, however, universally accepted that the presence of the metabolic syndrome is associated with increased risk of type 2 diabetes and cardiovascular disease. The prevalence of the metabolic syndrome in developed countries varies widely depending upon definitions used and age ranges included, but is estimated to be 24% among adults 20 years and over in the US. Given the impending worldwide epidemic of obesity, diabetes and cardiovascular disease, strategies aimed at greater understanding of the pathology of the syndrome, as well as strategies aimed at preventing or treating persons with the syndrome are urgently required.

Few studies have investigated associations of antioxidant nutrients and the metabolic syndrome. Ford and colleagues reported lower levels of several carotenoids and vitamins C and E among those with metabolic syndrome present compared with those without the syndrome in the Third National Health and Nutrition Examination Survey. Sugiura et al.  suggested that several carotenoids may exert a protective effect against the development of the metabolic syndrome, especially among current smokers. Confirming these findings in another population may add strength to these associations.

Our study showed significantly lower concentrations of β-carotene, α-carotene and the sum of the five carotenoids among those with the metabolic syndrome present compared to those without. We also found decreasing concentrations of all the carotenoids tested as the number of the metabolic syndrome components increased. These findings are consistent with data reported by Ford et al. from the third 262 National Health and Nutrition Examination Survey (NHANES III). In the NHANES III study, significantly lower concentrations of all the carotenoids, except lycopene, were found among persons with the metabolic syndrome compared with those without, after adjusting for  confounding factors similar to those in our study.

 

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

UPDATED 6/01/2019 

Genomics

Unraveling the clonal hierarchy of somatic genomic aberrations

D Prandi, SC Baca, A Romanel, CE Barbieri, Juan-Miguel Mosquera, et al.
Genome Biology 2014, 15:439
http://genomebiology.com/2014/15/8/439

Defining the chronology of molecular alterations may identify milestones in carcinogenesis. To unravel the temporal evolution of aberrations from clinical tumors, we developed CLONET, which upon estimation of tumor admixture and ploidy infers the clonal hierarchy of genomic aberrations. Comparative analysis across 100 sequenced genomes from prostate, melanoma, and lung cancers established diverse evolutionary hierarchies, demonstrating the early disruption of tumor-specific pathways. The analyses highlight the diversity of clonal evolution within and across tumor types that might be informative for risk stratification and patient selection for targeted therapies. CLONET addresses heterogeneous clinical samples seen in the setting of precision medicine.

The Transcription Factor Titration Effect Dictates Level of Gene Expression

RC Brewster, FM Weinert, HG Garcia, D Song, M Rydenfelt, and R Phillips
Cell,  Mar 13, 2014;156: 1312–1323
http://dx.doi.org/10.1016/j.cell.2014.02.022

Models of transcription are often built around a picture of RNA polymerase and transcription factors (TFs) acting on a single copy of a promoter. However, most TFs are shared between multiple genes with varying binding affinities. Beyond that, genes often exist at high copy number—in multiple identical copies on the chromosome or on plasmids or viral vectors with copy numbers in the hundreds. Using a thermodynamic model, we characterize the interplay between TF copy number and the demand for that TF. We demonstrate the parameter-free predictive power of this model as a function of the copy number of the TF and the number and affinities of the available specific binding sites; such predictive control is important for the understanding of transcription and the desire to quantitatively design the output of genetic circuits. Finally, we use these experiments to dynamically measure plasmid copy number through the cell cycle.

Telomere dynamics in human mesenchymal stem cells after exposure to acute oxidative stress

M Harbo, S Koelvraa, N Serakinci, L Bendixa
DNA Repair 2012.  http://dx.doi.org/10.1016/j.dnarep.2012.06.003

A gradual shortening of telomeres due to replication can be measured using the standard telomere restriction fragments (TRF) assay and other methods by measuring the mean length of all the telomeres in a cell. In contrast, stress-induced telomere shortening, which is believed to be just as important for causing cellular senescence, cannot be measured properly using these methods. Stress-induced telomere shortening caused by, e.g. oxidative damage happens in a stochastic manner leaving just a few single telomeres critically short. It is now possible to visualize these few ultra-short telomeres due to the advantages of the newly developed Universal single telomere length assay (STELA), and we therefore believe that this method should be considered the method of choice when measuring the length of telomeres after exposure to oxidative stress. In order to test our hypothesis, cultured human mesenchymal stem cells, either primary or hTERT immortalized, were exposed to sub-lethal doses of hydrogen peroxide, and the short term effect on telomere dynamics was monitored by Universal STELA and TRF measurements. Both telomere measures were then correlated with the percentage of senescent cells estimated by senescence-associated β-galactosidase staining. The exposure to acute oxidative stress resulted in an increased number of ultra-short telomeres, which correlated strongly with the percentage of senescent cells, whereas a correlation between mean telomere length and the percentage of senescent cells was absent. Based on the findings in the present study, it seems reasonable to conclude that Universal STELA is superior to TRF in detecting telomere damage caused by exposure to oxidative stress. The choice of method should therefore be considered carefully in studies examining stress-related telomere shortening as well as in the emerging field of lifestyle studies involving telomere length measurements.

tDNA insulators and the emerging role of TFIIIC in genome organization

Kevin Van Bortle and Victor G. Corces
Transcription Dec 12, 2012; 3(6): 1-8. www.landesbioscience.com

Recent findings provide evidence that tDNAs function as chromatin insulators from yeast to humans. TFIIIC, a transcription factor that interacts with the B-box in tDNAs as well as thousands of ETC sites in the genome, is responsible for insulator function. Though tDNAs are capable of enhancer-blocking and barrier activities for which insulators are defined, new insights into the relationship between insulators and chromatin structure suggest that TFIIIC serves a complex role in genome organization. We review the role of tRNA genes and TFIIIC as chromatin insulators, and highlight recent findings that have broadened our understanding of insulators in genome biology.

Structure and organization of insulators in eukaryotes. (A) From yeast to mammals, in organisms in which it has been studied, the TFIIIC protein interacts with the B-box sequence in tRNA genes or sites in the genome named ETC sites.

Synthetic CpG islands reveal DNA sequence determinants of chromatin structure

E Wachter, T Quante, C Merusi, A Arczewska, F Stewart, S Webb, A Bird
eLife 2014;3:e03397. http://dx.doi.org:/10.7554/eLife.03397.001

The mammalian genome is punctuated by CpG islands (CGIs), which differ sharply from the bulk genome by being rich in G + C and the dinucleotide CpG. CGIs often include transcription initiation sites and display ‘active’ histone marks, notably histone H3 lysine 4 methylation. In embryonic stem cells (ESCs) some CGIs adopt a ‘bivalent’ chromatin state bearing simultaneous ‘active’ and ‘inactive’ chromatin marks. To determine whether CGI chromatin is developmentally programmed at specific genes or is imposed by shared features of CGI DNA, we integrated artificial CGI-like DNA sequences into the ESC genome. We found that bivalency is the default chromatin structure for CpG-rich, G + C-rich DNA. A high CpG density alone is not sufficient for this effect, as A + T-rich sequence settings invariably provoke de novo DNA methylation leading to loss of CGI signature chromatin. We conclude that both CpG-richness and G + C-richness are required for induction of signature chromatin structures at CGIs.

Locus-specific mutation databases: pitfalls and good practice based on the p53 experience

Thierry Soussi, Chikashi Ishioka, Mireille Claustres and Christophe Béroud
NATURE REVIEWS | CANCER JAN 2006; 6: 83-90.

Between 50,000 and 60,000 mutations have been described in various genes that are associated with a wide variety of diseases. Reporting, storing and analysing these data is an important challenge as such data provide invaluable information for both clinical medicine and basic science.

The practical value of mutation analysis All studies performed to date show that mutations are, in general, not randomly distributed. Hot-spot regions have been demonstrated, corresponding to a region of DNA that is susceptible to mutations (such as CpG dinucleotides), a codon encoding a key residue in the biological function of the protein, or both (BOX 1). Identification of these hot-spot regions and natural mutants is essential to define crucial regions in an unknown protein.

Locus-specific databases have been developed to exploit this huge volume of data. The p53 mutation database is a paradigm, as it constitutes the largest collection of somatic mutations (22,000). However, there are several biases in this database that can lead to serious erroneous interpretations. We describe several rules for mutation database management that could benefit the entire scientific community.

Gene set enrichment analysis: A knowledge-based approach for interpreting genome-wide expression profiles

A Subramaniana, P Tamayo, VK  Mootha, S Mukherjee, BL Ebert, et al.
PNAS  Oct 25, 2005; 102(43): 15545–15550
http://pnas.org/cgi/doi/10.1073/pnas.0506580102

Although genomewide RNA expression analysis has become a routine tool in biomedical research, extracting biological insight from such information remains a major challenge. Here, we describe a powerful analytical method called Gene Set Enrichment Analysis (GSEA) for interpreting gene expression data. The method derives its power by focusing on gene sets, that is, groups of genes that share common biological function, chromosomal location, or regulation. We demonstrate how GSEA yields insights into several cancer-related data sets, including leukemia and lung cancer. Notably, where single-gene analysis finds little similarity between two independent studies of patient survival in lung cancer, GSEA reveals many biological pathways in common. The GSEA method is embodied in a freely available software package, together with an initial database of 1,325 biologically defined gene sets.

Mutational landscape and significance across 12 major cancer types

C Kandoth, MD McLellan, F Vandin, Kai Ye, B Niu, C Lu, et al.
NATURE  OCT 2013; 502: 333-337. http://dx.doi.org:/10.1038/nature12634

The Cancer Genome Atlas (TCGA) has used the latest sequencing and analysis methods to identify somatic variants across thousands of tumours. Here we present data and analytical results for point mutations and small insertions/deletions from 3,281 tumours across 12 tumour types as part of the TCGA Pan-Cancer effort. We illustrate the distributions of mutation frequencies, types and contexts across tumour types, and establish their links to tissues of origin, environmental/ carcinogen influences, and DNA repair defects. Using the integrated data sets, we identified 127 significantly mutated genes from well-known(for example, mitogen-activated protein kinase, phosphatidylinositol-3-OH kinase,Wnt/b-catenin and receptor tyrosine kinase signalling pathways, and cell cycle control) and emerging (for example, histone, histone modification, splicing, metabolism and proteolysis) cellular processes in cancer. The average number of mutations in these significantly mutated genes varies across tumour types; most tumours have two to six, indicating that the numberof driver mutations required during oncogenesis is relatively small. Mutations in transcriptional factors/regulators show tissue specificity, whereas histone modifiers are often mutated across several cancer types. Clinical association analysis identifies genes having a significant effect on survival, and investigations of mutations with respect to clonal/subclonal architecture delineate their temporal orders during tumorigenesis. Taken together, these results lay the groundwork for developing new diagnostics and individualizing cancer treatment.

Molecular insights into RNA and DNA helicase evolution from the determinants of  specificity for a DEAD-box RNA helicase

Anna L. Mallam, David J. Sidote and Alan M. Lambowitz
eLife 2014; http://dx.doi.org:/10.7554/eLife.04630

How different helicase families with a conserved catalytic ‘helicase core’ evolved to function on varied RNA and DNA substrates by diverse mechanisms remains unclear. Here, we used Mss116, a yeast DEAD-box protein that utilizes ATP to locally unwind dsRNA, to investigate helicase specificity and mechanism. Our results define the molecular basis for the substrate specificity of a DEAD-box protein. Additionally, they show that Mss116 has ambiguous substrate-binding properties and interacts with all four NTPs and both RNA and DNA. The efficiency of unwinding correlates with the stability of the ‘closed-state’ helicase core, a complex with nucleotide and nucleic acid that forms as duplexes are unwound. Crystal structures reveal that core stability is modulated by family-specific interactions that favor certain substrates. This suggests how present-day  helicases diversified from an ancestral core with broad specificity by retaining core closure as a common catalytic mechanism while optimizing substrate-binding interactions for different cellular functions.

Identification of human TERT elements necessary for telomerase recruitment to telomeres

Jens C Schmidt, Andrew B Dalby, Thomas R Cech
eLife 2014; http://dx.doi.org/10.7554/eLife.03563

Human chromosomes terminate in telomeres, repetitive DNA sequences bound by the shelterin complex. Shelterin protects chromosome ends, prevents recognition by the DNA damage machinery, and recruits telomerase. A patch of  amino acids, termed the TEL-patch, on the OB-fold domain of the shelterin  component TPP1 is essential to recruit telomerase to telomeres. In contrast, the site on telomerase that interacts with the TPP1 OB-fold is not well defined. Here we identify separation-of-function mutations in the TEN-domain of human telomerase reverse transcriptase (hTERT) that disrupt the interaction of telomerase with TPP1 in vivo and in vitro but have very little effect on the catalytic activity of telomerase. Suppression of a TEN-domain mutation with a compensatory charge-swap mutation in the TEL-patch indicates that their association is direct. Our findings define the interaction interface required for telomerase recruitment to telomeres, an important step towards developing modulators of this interaction as therapeutics for human disease.

Metabolomics

Single Cell Profiling of Circulating Tumor Cells: Transcriptional Heterogeneity and Diversity from Breast Cancer Cell Lines

MN Mindrinos, G Bhanot, SH Dairkee, RW Davis, SS Jeffrey
PLoS ONE 7(5): e33788. http://dx.doi.org:/doi:10.1371/journal.pone.0033788

Background: To improve cancer therapy, it is critical to target metastasizing cells. Circulating tumor cells (CTCs) are rare cells found in the blood of patients with solid tumors and may play a key role in cancer dissemination. Uncovering CTC phenotypes offers a potential avenue to inform treatment. However, CTC transcriptional profiling is limited by leukocyte contamination; an approach to surmount this problem is single cell analysis. Here we demonstrate feasibility of performing high dimensional single CTC profiling, providing early insight into CTC heterogeneity and allowing comparisons to breast cancer cell lines widely used for drug discovery.
Methodology/Principal Findings: We purified CTCs using the MagSweeper, an immunomagnetic enrichment device that isolates live tumor cells from unfractionated blood. CTCs that met stringent criteria for further analysis were obtained from 70% (14/20) of primary and 70% (21/30) of metastatic breast cancer patients; none were captured from patients with nonepithelial cancer (n = 20) or healthy subjects (n = 25). Microfluidic-based single cell transcriptional profiling of 87 cancer associated and reference genes showed heterogeneity among individual CTCs, separating them into two major subgroups, based on 31 highly expressed genes. In contrast, single cells from seven breast cancer cell lines were tightly clustered together by sample ID and ER status. CTC profiles were distinct from those of cancer cell lines, questioning the suitability of such lines for drug discovery efforts for late stage cancer therapy.
Conclusions/Significance: For the first time, we directly measured high dimensional gene expression in individual CTCs without the common practice of pooling such cells. Elevated transcript levels of genes associated with metastasis NPTN, S100A4, S100A9, and with epithelial mesenchymal transition: VIM, TGFß1, ZEB2, FOXC1, CXCR4, were striking compared to cell lines. Our findings demonstrate that profiling CTCs on a cell-by-cell basis is possible and may facilitate the application of ‘liquid biopsies’ to better model drug discovery

Simplifying Disease Complexity part 6 – Bringing Metabolomics into Practice
Dr. Kirk Beebe, Director of Application Science, Metabolon, Inc.

n the previous editions of this 6-part series, we’ve explored numerous example of how metabolomics is bringing success to areas such as cancer, metabolic disease, cardiovascular, and rare disease research. Although we did not devote attention to every area of biology or therapeutic area, the intent of this broad series was not only to convey how metabolomics can be used in a specific area of research (e.g. cancer), but actually, how metabolomics is a central science for interrogating any biological question. So, although it may seem like an oversimplification, to understand whether metabolomics could be used in a research setting one need only ask themselves, “Do I have a biological question that would benefit from a hypothesis-free approach?, am I interested in exploring my system for potential new discoveries? Or do I need a biomarker/better biomarker?

As described in our first part, metabolites have been and continue to be a staple for clinical and in vivo decision making (e.g. cholesterol, glucose, bilirubin, creatinine, thyroid hormone, newborn screening for inborn errors of metabolism (IEMs)). In short, this utility is fundamental to the foundations of biology since metabolism is central to all kingdoms of life and contemporary biology is driven to maintain metabolic homeostasis to maintain the phenotype. An unappreciated point that we leave this series with is that this fundamental nature (the connection of metabolism to the phenotype) confers an important advantage of metabolism for deriving biomarkers and understanding the underlying physiology.

Metabolites are a diagnostic data stream.

Whether a phenotype is driven by a single mutation or a combination of genetic differences, environmental influences or the microbiota, metabolism provides a systems-level diagnostic.

That is, no matter the source of the physiological or phenotypic change (i.e. genes, microbiota, environmental), the change will almost invariably register within metabolism. Thus, modern metabolomic approaches offer the opportunity to more deeply interrogate the “metabolome” to discover more sensitive and specific biomarkers and understand the basis of disease and drug response.

As such, metabolomics has the potential to be able to integrate systems on a number of levels. It is useful through its ability to enrich genomics, transcriptomics and proteomics, thus integrating a number of data streams that provide knowledge and contribute to informed decision-making and patient management1. Using metabolomics, individual tissues can be queried but less invasive sample types (e.g., blood, urine, feces, and/or saliva) can also yield biomarkers and mechanistic insight. The integration of the individual tissues at the level of these more accessible samples can offer an overview of the entire system and inform on important biological pathways. Finally, although the focus of this series was on what metabolomics can bring to biomarker and other related research areas, it should be noted that a combination of metabolomics with other scientific approaches will undoubtedly broaden insight and produce verifiable, validatable biomarkers that track with efficacy and therapy.

As we close this series, we hope that we have conveyed 4 critical points – 1) metabolism is central to biology and hence, key in research and biomarker discovery, 2) the reason for this is due to the fundamental nature of metabolism being central to the development of all life and being the focal point of contemporary biology’s drive to maintain homeostasis, 3) metabolomic is the most powerful way to survey metabolism by offering a simultaneous read-out if hundreds of reactions and pathways, and 4) metabolomics as a practical tool has only recently emerged.

And it is on this last point that we leave the reader with some final considerations. We imagine that, after careful review of the information outlined in this series, many readers will be motivated to explore the use of metabolomics in their research. However, as outlined throughout this series, mature technologies have only recently arisen. Nevertheless, there are many laboratories that perform some version of “metabolomics”. Although the experimental goal often dictates the precise approach, there are 5 critical features  that a metabolomic technology must harbor in order for it to achieve a similar purpose as mature omic technologies (e.g. DNA sequencers) in terms of depth of coverage and data quality. These minimally include:

  1. Must be based on an authenticated chemical library
    2. Must have procedures for eliminated noise from the data
    5. Must have a mechanism to identify novel metabolites
    6. Must have robust QC process from sample preparation through statistical analysis
    4. Must provide a mechanism to abstract information/interpret the data

References

  1. Eckhart, A.D., Beebe, K. & Milburn, M. Metabolomics as a key integrator for “omic” advancement of personalized medicine and future therapies. Clin Transl Sci 5, 285-288

(2012).

  1. Evans, A., Mitchell, M., Dai, H. & DeHaven, C.D. Categorizing Ion –Features in Liquid Chromatography/Mass Spectrometry Metobolomics Data. Metabolomics 2 (2012).
  2. DeHaven, C.D., Evans, A., Dai, H. & Lawton, K.A. in Metabolomics. (ed. U. Roessner) (InTech, 2012).
  3. Dehaven, C.D., Evans, A.M., Dai, H. & Lawton, K.A. Organization of GC/MS and LC/MS metabolomics data into chemical libraries. J Cheminform 2, 9 (2010).
  4. Evans, A.M., DeHaven, C.D., Barrett, T., Mitchell, M. & Milgram, E. Integrated, nontargeted ultrahigh performance liquid chromatography/electrospray ionization tandem mass spectrometry platform for the identification and relative quantification of the small-molecule complement of biological systems. Anal Chem 81, 6656-6667 (2009).

Prediction of intracellular metabolic states from extracellular metabolomic data

MK Aurich, G Paglia, Ottar Rolfsson, S Hrafnsdottir, M  Magnusdottir, MM, et al.

Metabolomics Aug 14, 2014;  http://dx.doi.org:/10.1007/s11306-014-0721-3
http://link.springer.com/article/10.1007/s11306-014-0721-3/fulltext.html#Sec1

intra- extracellular metabolites

intra- extracellular metabolites

http://link.springer.com/static-content/images/404/
art%253A10.1007%252Fs11306-014-0721-3/MediaObjects/11306_2014_721_Fig1_HTML.gif

Metabolic models can provide a mechanistic framework to analyze information-rich omics data sets, and are increasingly being used to investigate metabolic alternations in human diseases. An expression of the altered metabolic pathway utilization is the selection of metabolites consumed and released by cells. However, methods for the inference of intracellular metabolic states from extracellular measurements in the context of metabolic models remain underdeveloped compared to methods for other omics data. Herein, we describe a workflow for such an integrative analysis emphasizing on extracellular metabolomics data. We demonstrate, using the lymphoblastic leukemia cell lines Molt-4 and CCRF-CEM, how our methods can reveal differences in cell metabolism. Our models explain metabolite uptake and secretion by predicting a more glycolytic phenotype for the CCRF-CEM model and a more oxidative phenotype for the Molt-4 model, which was supported by our experimental data. Gene expression analysis revealed altered expression of gene products at key regulatory steps in those central metabolic pathways, and literature query emphasized the role of these genes in cancer metabolism. Moreover, in silico gene knock-outs identified unique control points for each cell line model, e.g., phosphoglycerate dehydrogenase for the Molt-4 model. Thus, our workflow is well suited to the characterization of cellular metabolic traits based on extracellular metabolomic data, and it allows the integration of multiple omics data sets into a cohesive picture based on a defined model context.

Metabolome Informatics Research

Metabolome Informatics Research

Identification of Metabolites in the Normal Ovary and Their Transformation in Primary and Metastatic Ovarian Cancer MOC vs EOC

Identification of Metabolites in the Normal Ovary and Their Transformation in Primary and Metastatic Ovarian Cancer MOC vs EOC

Genomics and Cancer

Identification of Gene Networks Associated with Acute Myeloid Leukemia by Comparative Molecular Methylation and Expression Profiling

M Dellett, KA O’Hagan, HA Alexandra Colyer and KI Mills
Biomarkers in Cancer 2010:2 43–55  http://www.la-press.com.

Around 80% of acute myeloid leukemia (AML) patients achieve a complete remission, however many will relapse and ultimately die of their disease. The association between karyotype and prognosis has been studied extensively and identified patient cohorts as having favourable [e.g. t(8; 21), inv (16)/t(16; 16), t(15; 17)], intermediate [e.g. cytogenetically normal (NK-AML)] or adverse risk [e.g. complex karyotypes]. Previous studies have shown that gene expression profiling signatures can classify the sub-types of AML, although few reports have shown a similar feature by using methylation markers. The global methylation patterns in 19 diagnostic AML samples were investigated using the Methylated CpG Island Amplification Microarray (MCAM) method and CpG island microarrays containing 12,000 CpG sites. The first analysis, comparing favourable and intermediate cytogenetic risk groups, revealed significantly differentially methylated CpG sites (594 CpG islands) between the two subgroups. Mutations in the NPM1 gene occur at a high frequency (40%) within the NK-AML subgroup and are associated with a more favourable prognosis in these patients. A second analysis comparing the NPM1 mutant and wild-type research study subjects again identified distinct methylation profiles between these two subgroups. Network and pathway analysis revealed possible molecular mechanisms associated with the different risk and/or mutation sub-groups. This may result in a better classification of the risk groups, improved monitoring targets, or the identification of novel molecular therapies.

Molecular Imaging of Proteases in Cancer

Yunan Yang, Hao Hong, Yin Zhang and Weibo Cai
Cancer Growth and Metastasis 2009:2 13–27. http://www.la-press.com

Proteases play important roles during tumor angiogenesis, invasion, and metastasis. Various molecular imaging techniques have been employed for protease imaging: optical (both fluorescence and bioluminescence), magnetic resonance imaging (MRI), single-photon emission computed tomography (SPECT), and positron emission tomography (PET). In this review, we will summarize the current status of imaging proteases in cancer with these techniques. Optical imaging of proteases, in particular with fluorescence, is the most intensively validated and many of the imaging probes are already commercially available. It is generally agreed that the use of activatable probes is the most accurate and appropriate means for measuring protease activity. Molecular imaging of proteases with other techniques (i.e. MRI, SPECT, and PET) has not been well-documented in the literature which certainly deserves much future effort. Optical imaging and molecular MRI of protease activity has very limited potential for clinical investigation. PET/SPECT imaging is suitable for clinical investigation; however the optimal probes for PET/SPECT imaging of proteases in cancer have yet to be developed. Successful development of protease imaging probes with optimal in vivo stability, tumor targeting efficacy, and desirable pharmacokinetics for clinical translation will eventually improve cancer patient management. Not limited to cancer, these protease-targeted imaging probes will also have broad applications in other diseases such as arthritis, atherosclerosis, and myocardial infarction.

Evolutionarily conserved genetic interactions with budding and fission yeast MutS identify orthologous relationships in mismatch repair-deficient cancer cells

E Tosti, JA Katakowski, S Schaetzlein, Hyun-Soo Kim, CJ Ryan, M Shales, et al.
Genome Medicine 2014, 6:68. http://genomemedicine.com/content/6/9/68

Background: The evolutionarily conserved DNA mismatch repair (MMR) system corrects base-substitution and insertion-deletion mutations generated during erroneous replication. The mutation or inactivation of many MMR factors strongly predisposes to cancer, where the resulting tumors often display resistance to standard chemotherapeutics. A new direction to develop targeted therapies is the harnessing of synthetic genetic interactions, where the simultaneous loss of two otherwise non-essential factors leads to reduced cell fitness or death. High-throughput screening in human cells to directly identify such interactors for disease-relevant genes is now widespread, but often requires extensive case-by-case optimization. Here we asked if conserved genetic interactors (CGIs) with MMR genes from two evolutionary distant yeast species (Saccharomyces cerevisiae and Schizosaccharomyzes pombe) can predict orthologous genetic relationships in higher eukaryotes.
Methods: High-throughput screening was used to identify genetic interaction profiles for the MutSα and MutSβ heterodimer subunits (msh2Δ, msh3Δ, msh6Δ) of fission yeast. Selected negative interactors with MutSβ (msh2Δ/msh3Δ) were directly analyzed in budding yeast, and the CGI with SUMO-protease Ulp2 further examined after RNA interference/drug treatment in MSH2-deficient and -proficient human cells.
Results: This study identified distinct genetic profiles for MutSα and MutSβ, and supports a role for the latter in recombinatorial DNA repair. Approximately 28% of orthologous genetic interactions with msh2Δ/msh3Δ are conserved in both yeasts, a degree consistent with global trends across these species. Further, the CGI between budding/fission yeast msh2 and SUMO-protease Ulp2 is maintained in human cells (MSH2/SENP6), and enhanced by Olaparib, a PARP inhibitor that induces the accumulation of single-strand DNA breaks. This identifies SENP6 as a promising new target for the treatment of MMR-deficient cancers.
Conclusion: Our findings demonstrate the utility of employing evolutionary distance in tractable lower eukaryotes to predict orthologous genetic relationships in higher eukaryotes. Moreover, we provide novel insights into the genome maintenance functions of a critical DNA repair complex and propose a promising targeted treatment for MMR deficient tumors.

Cancer Genome Landscapes

B Vogelstein, N Papadopoulos, VE Velculescu, S Zhou, LA Diaz Jr., KW Kinzler, et al.
Science 339, 1546 (2013); http://dx.doi.org:/10.1126/science.1235122

Over the past decade, comprehensive sequencing efforts have revealed the genomic landscapes of common forms of human cancer. For most cancer types, this landscape consists of a small number of “mountains” (genes altered in a high percentage of tumors) and a much larger number of “hills” (genes altered infrequently). To date, these studies have revealed ~140 genes that, when altered by intragenic mutations, can promote or “drive” tumorigenesis. A typical tumor contains two to eight of these “driver gene” mutations; the remaining mutations are passengers that confer no selective growth advantage. Driver genes can be classified into 12 signaling pathways that regulate three core cellular processes: cell fate, cell survival, and genome maintenance. A better understanding of these pathways is one of the most pressing needs in basic cancer research. Even now, however, our knowledge of cancer genomes is sufficient to guide the development of more effective approaches for reducing cancer morbidity and mortality.

Approaches for establishing the function of regulatory genetic variants involved in disease

Julian Charles Knight
Genome Medicine 2014, 6:92.  http://genomemedicine.com/content/6/10/92

The diversity of regulatory genetic variants and their mechanisms of action reflect the complexity and context-specificity of gene regulation. Regulatory variants are important in human disease and defining such variants and establishing mechanism is crucial to the interpretation of disease-association studies. This review describes approaches for identifying and functionally characterizing regulatory variants, illustrated using examples from common diseases. Insights from recent advances in resolving the functional epigenomic regulatory landscape in which variants act are highlighted, showing how this has enabled functional annotation of variants and the generation of hypotheses about mechanism of action. The utility of quantitative trait mapping at the transcript, protein and metabolite level to define association of specific genes with particular variants and further inform disease associations are reviewed. Establishing mechanism of action is an essential step in resolving functional regulatory variants, and this review describes how this is being facilitated by new methods for analyzing allele-specific expression, mapping chromatin interactions and advances in genome editing. Finally, integrative approaches are discussed together with examples highlighting how defining the mechanism of action of regulatory variants and identifying specific modulated genes can maximize the translational utility of genome-wide association studies to understand the pathogenesis of diseases and discover new drug targets or opportunities to repurpose existing drugs to treat them.

Biomarkers

TRIM29 as a Novel Biomarker in Pancreatic Adenocarcinoma

Hongli Sun, Xianwei Dai, and Bing Han
Disease Markers 2014, Article ID 317817, 7 pages
http://dx.doi.org/10.1155/2014/317817

Background and Aim. Tripartite motif-containing 29 (TRIM29) is structurally a member of the tripartite motif family of proteins and is involved in diverse human cancers. However, its role in pancreatic cancer remains unclear.
Methods. The expression pattern of TRIM29 in pancreatic ductal adenocarcinoma was assessed by immunocytochemistry. Multivariate logistic regression analysis was used to investigate the association between TRIM29 and clinical characteristics. In vitro analyses by scratch wound healing assay and invasion assays were performed using the pancreatic cancer cell lines.
Results. Immunohistochemical analysis showed TRIM29 expression in pancreatic cancer tissues was significantly higher (𝑛 = 186) than that in matched adjacent nontumor tissues. TRIM29 protein expression was significantly correlated with lymph node metastasis (𝑃 = 0.019). Patients with positive TRIM29 expression showed both shorter overall survival and shorter recurrence-free survival than those with negative TRIM29 expression. Multivariate analysis revealed that TRIM29 was an independent factor for pancreatic cancer over survival (HR = 2.180, 95% CI: 1.324–4.198, 𝑃 = 0.011). In vitro, TRIM29 knockdown resulted in inhibition of pancreatic cancer cell proliferation, migration, and invasion.
Conclusions. Our results indicate that TRIM29 promotes tumor progression and may be a novel prognostic marker for pancreatic ductal adenocarcinoma.

Bioinformatic identification of proteins with tissue-specific expression for biomarker discovery

I Prassas, CC Chrystoja, S Makawita1, and EP Diamandis
BMC Medicine 2012, 10:39. http://www.biomedcentral.com/1741-7015/10/39

Background: There is an important need for the identification of novel serological biomarkers for the early detection of cancer. Current biomarkers suffer from a lack of tissue specificity, rendering them vulnerable to nondisease-specific increases. The present study details a strategy to rapidly identify tissue-specific proteins using bioinformatics.
Methods: Previous studies have focused on either gene or protein expression databases for the identification of candidates. We developed a strategy that mines six publicly available gene and protein databases for tissue-specific proteins, selects proteins likely to enter the circulation, and integrates proteomic datasets enriched for the cancer secretome to prioritize candidates for further verification and validation studies.
Results: Using colon, lung, pancreatic and prostate cancer as case examples, we identified 48 candidate tissuespecific biomarkers, of which 14 have been previously studied as biomarkers of cancer or benign disease. Twenty six candidate biomarkers for these four cancer types are proposed.
Conclusions: We present a novel strategy using bioinformatics to identify tissue-specific proteins that are potential cancer serum biomarkers. Investigation of the 26 candidates in disease states of the organs is warranted

The Serum Glycome to Discriminate between Early-Stage Epithelial Ovarian Cancer and Benign Ovarian Diseases

K Biskup, E Iona Braicu, J Sehouli, R Tauber, and V Blanchard
Disease Markers 2014, Article ID 238197, 10 pages
http://dx.doi.org/10.1155/2014/238197

Epithelial ovarian cancer (EOC) is the sixth most common cause of cancer deaths in women because the diagnosis occurs mostly when the disease is in its late-stage. Current diagnostic methods of EOC show only a moderate sensitivity, especially at an early-stage of the disease; hence, novel biomarkers are needed to improve the diagnosis. We recently reported that serum glycome modifications observed in late-stage EOC patients by MALDI-TOF-MS could be combined as a glycan score named GLYCOV that was calculated from the relative areas of the 11 N-glycan structures that were significantly modulated. Here, we evaluated the ability of GLYCOV to recognize early-stage EOC in a cohort of 73 individuals comprised of 20 early-stage primary serous EOC, 20 benign ovarian diseases (BOD), and 33 age-matched healthy controls. GLYCOV was able to recognize stage I EOC whereas CA125 values were statistically significant only for stage II EOC patients. In addition, GLYCOV was more sensitive and specific compared to CA125 in distinguishing early-stage EOC from BOD patients, which is of high relevance to clinicians as it is difficult for them to diagnose malignancy prior to operation.

The Clinicopathological Significance of miR-133a in Colorectal Cancer

Timothy Ming-Hun Wan, Colin Siu-Chi Lam, Lui Ng, Ariel Ka-Man Chow, et al.
Disease Markers  2014, Article ID 919283, 8 pages http://dx.doi.org/10.1155/2014/919283

This study determined the expression of microRNA-133a (MiR-133a) in colorectal cancer (CRC) and adjacent normal mucosa samples and evaluated its clinicopathological role in CRC. The expression of miR-133a in 125 pairs of tissue samples was analyzed by quantitative real-time polymerase chain reaction (qRT-PCR) and correlated with patient’s clinicopathological data by statistical analysis. Endogenous expression levels of several potential target genes were determined by qRT-PCR and correlated using Pearson’s method. MiR-133a was downregulated in 83.2% of tumors compared to normal mucosal tissue. Higher miR-133a expression in tumor tissues was associated with development of distant metastasis, advanced Dukes and TNM staging, and poor survival. The unfavorable prognosis of higher miR-133a expression was accompanied by dysregulation of potential miR-133a target genes, LIM and SH3 domain protein 1 (LASP1), Caveolin-1 (CAV1), and Fascin-1 (FSCN1). LASP1 was found to possess a negative correlation (𝛾 = −0.23), whereas CAV1 exhibited a significant positive correlation (𝛾 = 0.27), and a stronger correlation was found in patients who developed distant metastases (𝛾 = 0.42). In addition, a negative correlation of FSCN1 was only found in nonmetastatic patients. In conclusion, miR-133a was downregulated in CRC tissues, but its higher expression correlated with adverse clinical characteristics and poor prognosis.

The Clinical Significance of PR, ER, NF-𝜅B, and TNF-𝛼 in Breast Cancer

Xian-Long Zhou, Wei Fan, Gui Yang, and Ming-Xia Yu
Disease Markers 2014, Article ID 494581, 7 pages http://dx.doi.org/10.1155/2014/494581

Objectives. To investigate the expression of estrogen (ER), progesterone receptors (PR), nuclear factor-𝜅B (NF-𝜅B), and tumor necrosis factor-𝛼 (TNF-𝛼) in human breast cancer (BC), and the correlation of these four parameters with clinicopathological features of BC.
Methods and Results. We performed an immunohistochemical SABC method for the identification of ER, PR, NF-𝜅B, and TNF-𝛼 expression in 112 patients with primary BC.The total positive expression rate of ER, PR, NF-𝜅B, and TNF-𝛼 was 67%, 76%, 84%, and 94%, respectively. The expressions of ER and PR were correlated with tumor grade, TNM stage, and lymph node metastasis (𝑃 < 0.01, resp.), but not with age, tumor size, histological subtype, age at menarche, menopause status, number of pregnancies, number of deliveries, and family history of cancer. Expressions of ER and PR were both correlated with NF-𝜅B and TNF-𝛼 expression (𝑃 < 0.05, resp.). Moreover, there was significant correlation between ER and PR (𝑃 < 0.0001) as well as between NF-𝜅B and TNF-𝛼 expression (𝑃 < 0.05).
Conclusion. PR and ER are highly expressed, with significant correlation with NF-𝜅B and TNF-𝛼 expression in breast cancer. The important roles of ER and PR in invasion and metastasis of breast cancer are probably associated with NF-𝜅B and TNF-𝛼 expression.

Serum Protein Profile at Remission Can Accurately Assess Therapeutic Outcomes and Survival for Serous Ovarian Cancer

J Wang, A Sharma, SA Ghamande, S Bush, D Ferris, W Zhi, et la.
PLoS ONE 8(11): e78393. http://dx.doi.org:/10.1371/journal.pone.0078393

Background: Biomarkers play critical roles in early detection, diagnosis and monitoring of therapeutic outcome and recurrence of cancer. Previous biomarker research on ovarian cancer (OC) has mostly focused on the discovery and validation of diagnostic biomarkers. The primary purpose of this study is to identify serum biomarkers for prognosis and therapeutic outcomes of ovarian cancer. Experimental Design: Forty serum proteins were analyzed in 70 serum samples from healthy controls (HC) and 101 serum samples from serous OC patients at three different disease phases: post diagnosis (PD), remission (RM) and recurrence (RC). The utility of serum proteins as OC biomarkers was evaluated using a variety of statistical methods including survival analysis.
Results: Ten serum proteins (PDGF-AB/BB, PDGF-AA, CRP, sFas, CA125, SAA, sTNFRII, sIL-6R, IGFBP6 and MDC) have individually good area-under-the-curve (AUC) values (AUC = 0.69–0.86) and more than 10 three-marker combinations have excellent AUC values (0.91–0.93) in distinguishing active cancer samples (PD & RC) from HC. The mean serum protein levels for RM samples are usually intermediate between HC and OC patients with active cancer (PD & RC). Most importantly, five proteins (sICAM1, RANTES, sgp130, sTNFR-II and sVCAM1) measured at remission  can classify, individually and in combination, serous OC patients into two subsets with significantly different overall survival (best HR = 17, p,1023).
Conclusion: We identified five serum proteins which, when measured at remission, can accurately predict the overall survival of serous OC patients, suggesting that they may be useful for monitoring the therapeutic outcomes for ovarian cancer.

Serum Clusterin as a Tumor Marker and Prognostic Factor for Patients with Esophageal Cancer

Wei Guo, Xiao Ma, Christine Xue, Jianfeng Luo, Xiaoli Zhu, et al.
Disease Markers 2014, Article ID 168960, 7 pages http://dx.doi.org/10.1155/2014/168960

Background. Recent studies have revealed that clusterin is implicated in many physiological and pathological processes, including tumorigenesis. However, the relationship between serum clusterin expression and esophageal squamous cell carcinoma (ESCC) is unclear.
Methods. The serum clusterin concentrations of 87 ESCC patients and 136 healthy individuals were examined. An independent-samples Mann-Whitney 𝑈 test was used to compare serum clusterin concentrations of ESCC patients to those of healthy controls. Univariate analysis was conducted using the log-rank test and multivariate analyses were performed using the Cox proportional hazards model. Results. In healthy controls, the mean clusterin concentration was 288.8 ± 75.1 𝜇g/mL, while in the ESCC patients, the mean clusterin concentration was higher at 412.3±159.4 𝜇g/mL (𝑃 < 0.0001). The 1-, 2-, and 4-year survival rates for the 87 ESCC patients were 89.70%, 80.00%, and 54.50%. Serum clusterin had an optimal diagnostic cut-off point (serum clusterin concentration = 335.5 𝜇g/mL) for esophageal squamous cell carcinoma with sensitivity of 71.26% and specificity of 77.94%. And higher serum clusterin concentration (>500 𝜇g/mL) indicated better prognosis (𝑃 = 0.030).
Conclusions. Clusterin may play a key role during tumorigenesis and tumor progression of ESCC and it could be applied in clinical work as a tumor marker and prognostic factor.

Septin 9 methylated DNA is a sensitive and specific blood test for colorectal cancer

JD Warren, Wei Xiong, AM Bunker, CP Vaughn, LV Furtado, et al.
BMC Medicine 2011, 9:133. http://www.biomedcentral.com/1741-7015/9/133

Background: About half of Americans 50 to 75 years old do not follow recommended colorectal cancer (CRC) screening guidelines, leaving 40 million individuals unscreened. A simple blood test would increase screening compliance, promoting early detection and better patient outcomes. The objective of this study is to demonstrate the performance of an improved sensitivity blood-based Septin 9 (SEPT9) methylated DNA test for colorectal cancer. Study variables include clinical stage, tumor location and histologic grade.
Methods: Plasma samples were collected from 50 untreated CRC patients at 3 institutions; 94 control samples were collected at 4 US institutions; samples were collected from 300 colonoscopy patients at 1 US clinic prior to endoscopy. SEPT9 methylated DNA concentration was tested in analytical specimens, plasma of known CRC cases, healthy control subjects, and plasma collected from colonoscopy patients.
Results: The improved SEPT9 methylated DNA test was more sensitive than previously described methods; the test had an overall sensitivity for CRC of 90% (95% CI, 77.4% to 96.3%) and specificity of 88% (95% CI, 79.6% to 93.7%), detecting CRC in patients of all stages. For early stage cancer (I and II) the test was 87% (95% CI, 71.1% to 95.1%) sensitive. The test identified CRC from all regions, including proximal colon (for example, the cecum) and had a 12% false-positive rate. In a small prospective study, the SEPT9 test detected 12% of adenomas with a false-positive rate of 3%.
Conclusions: A sensitive blood-based CRC screening test using the SEPT9 biomarker specifically detects a majority of CRCs of all stages and colorectal locations. The test could be offered to individuals of average risk for CRC who are unwilling or unable to undergo colonoscopy.

Matrix Metalloproteinases in Cancer: Prognostic Markers and Therapeutic Targets

Pia Vihinen And Veli-Matti K¨Ah¨Ari
Int. J. Cancer 2002; 99: 157–166 http://dx.doi.org:/10.1002/ijc.10329

Degradation of extracellular matrix is crucial for malignant tumour growth, invasion, metastasis and angiogenesis. Matrix metalloproteinases (MMPs) are a family of zinc-dependent neutral endopeptidases collectively capable of degrading essentially all matrix components. Elevated levels of distinct MMPs can be detected in tumour tissue or serumof patients with advanced cancer and their role as prognostic indicators in cancer is studied. In addition, therapeutic intervention of tumour growth and invasion based on inhibition of MMP activity is under intensive investigation and several MMP inhibitors are in clinical trials in cancer. In this review, we discuss the current view on the feasibility of MMPs as prognostic markers and as targets for therapeutic intervention in cancer.

Mass Spectrometric Screening of Ovarian Cancer with Serum Glycans

Jae-Han Kim, Chang Won Park, Dalho Um, Ki Hwang Baek, Yohahn Jo, et al.
Disease Markers  2014, Article ID 634289, 9 pages
http://dx.doi.org/10.1155/2014/634289

development of novel biomarkers based on the glycomic analysis. In this study, N-linked glycans from human serum were quantitatively profiled by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) and compared between healthy controls and ovarian cancer patients. A training set consisting of 40 healthy controls and 40 ovarian cancer cases demonstrated an inverse correlation between 𝑃 value of ANOVA and area under the curve (AUC) of each candidate biomarker peak from MALDI-TOF MS, providing standards for the classification. A multi-biomarker panel composed of 15 MALDI-TOF MS peaks resulted in AUC of 0.89, 80∼90% sensitivity, and 70∼83% specificity in the training set. The performance of the biomarker panel was validated in a separate blind test set composed of 23 healthy controls and 37 ovarian cancer patients, leading to 81∼84% sensitivity and 83% specificity with cut-off values determined by the training set. Sensitivity of CA-125, the most widely used ovarian cancer marker, was 74%in the training set and 78% in the test set, respectively. These results indicate that MALDI-TOF MS-mediated serum N-glycan analysis could provide critical information for the screening of ovarian cancer.

Large, Collaborative Lung Cancer Trial Goes for Precision Medicine Goal

News | June 30, 2014 | Lung Cancer Targets

By Anna Azvolinsky, PhD

In a new biomarker-focused clinical trial, five therapies will be tested to develop new, precision medicine approaches to treat squamous cell lung cancer. The Lung Cancer Master Protocol (Lung-MAP)/SWOG S1400 phase 2/3 clinical trial, brings together the National Cancer Institute (NCI), the Foundation for the National Institutes of Health (FNIH), SWOG Cancer Research, five pharmaceutical companies (Amgen, AstraZeneca, Genentech, MedImmune, and Pfizer), Foundation Medicine (a molecular informatics company), and Friends of Cancer Research, a non-profit foundation.

The trial aims to enroll about 10,000 patients total and will cost about $160 million, of which the NCI is contributing $25 million.

Lung-MAP is unique as this is the first public-private partnership in drug development that includes the NCI, the Food and Drug Administration (FDA), U.S. oncology cooperative groups, and a number of patient advocacy groups according to one of the study investigators, David Gandara, MD, chair of the SWOG lung committee, and thoracic oncologist at the UC Davis Cancer Center. “Funds are made available for every aspect of the trial,” said Gandara. “There is nothing in the history of oncology or drug development like it.”

The clinical trial seeks to identify molecular aberrations in patients with advanced squamous cell lung cancer that can be targeted either by existing therapies or through the development of new ones. The innovation of this trial is a master protocol that will rely on the strength of numbers—up to 1000 patients per year at more than 200 sites throughout the U.S. for more than 200 cancer-related genetic alterations. Testing results will then dictate which experimental trial arm is most appropriate for which patient. Unlike a trial that seeks to enroll patients harboring just one mutation, which limits the access for many patients, the Lung-MAP design better ensures that a patient who is screened will be eligible for a targeted therapy trial arm.

This type of umbrella trial design is particularly suitable for squamous cell lung cancer. Thus far, has not been defined by one or several driver mutations. Instead, these tumors are made of a spectrum of genetic aberrations that are each relatively rare within the squamous lung cancer patient population, making enrollment into targeted therapy clinical trials difficult. According to the NCI, Lung-MAP “aims to establish a model of clinical testing that more efficiently meets the needs of both patients and drug developers,” facilitating more efficient matching of a patient to an investigational targeted therapy trial.

Lung-MAP was specifically designed for squamous cell lung cancer because this lung cancer subtype represents the greatest unmet need for new treatment, Gandara told OncoTherapy Network:

“All of the dramatic advances that have been made in the treatment of lung cancer over the last ten years have occurred in adenocarcinoma, a lung cancer subtype with several recently recognized and ‘druggable oncogenes’ such as EGFR mutations or ALK translocations. However, there have been essentially no advances in squamous cell lung cancer.”

But, recent genome-wide studies have identified several gene alterations in squamous cell lung cancer that are also druggable, including PI3K, FGFR, and CDK mutations, said Gandara. The trial is initially testing four targeted therapies: Genentech’s GDC-0032 (a PI3 kinase inhibitor), Pfizer’s palbociclib (an oral cyclin-dependent-kinase 4/6 inhibitor, AZD4547), an oral fibroblast growth factor receptor inhibitor from AstraZeneca, and rilotumumab, Amgen’s antibody against the human hepatocyte growth factor.

The fifth agent is, MEDI4736, an immune checkpoint inhibitor antibody targeting PD-L1. Patients whose tumors do not harbor a mutation suitable for targeting with one of the four targeted therapies will be enrolled in the MED4736 sub-study.

Once a patient is matched to a specific trial sub-study, randomization will determine whether the patient receives the experimental therapy or standard of care chemotherapy. The planned trial endpoints for each sub-study are overall survival and progression-free survival.

“I cannot overemphasize the importance of the FDA’s participation in this project, since each of these sub-studies is designed to result in approval of a paired biomarker and new drug if that sub-study meets the requirements for improved effectiveness,” said Gandara.

– See more at: http://www.oncotherapynetwork.com/lung-cancer-targets/large-collaborative-lung-cancer-trial-goes-precision-medicine-goal

The BATTLE Trial: Personalizing Therapy for Lung Cancer

Kim, RS. Herbst, II. Wistuba, JJ Lee, GR. Blumenschein Jr., A Tsao, DJ. Stewart, et al.

Authors’ Affiliations: 1Departments of Thoracic/Head and Neck Medical Oncology, 2Pathology, 3Biostatistics, and 4Diagnostic Radiology, The University of Texas MD Anderson Cancer Center, Houston, Texas; 5Winship Cancer Center, Emory University, Atlanta, Georgia; 6Dana-Farber Cancer Institute, Boston, Massachusetts; and 7University of Maryland, Baltimore, Maryland.

Corresponding Author:

Waun K. Hong, The University of Texas MD Anderson Cancer Center, 1515 Holcombe Blvd., Houston, TX 77030. Phone: 713-794-1441; Fax: 1-713-792-4654; E-mail:whong@mdanderson.org

The Biomarker-integrated Approaches of Targeted Therapy for Lung Cancer Elimination (BATTLE) trial represents the first completed prospective, biopsy-mandated, biomarker-based, adaptively randomized study in 255 pretreated lung cancer patients. Following an initial equal randomization period, chemorefractory non–small cell lung cancer (NSCLC) patients were adaptively randomized to erlotinib, vandetanib, erlotinib plus bexarotene, or sorafenib, based on relevant molecular biomarkers analyzed in fresh core needle biopsy specimens. Overall results include a 46% 8-week disease control rate (primary end point), confirm prespecified hypotheses, and show an impressive benefit from sorafenib among mutant-KRAS patients. BATTLE establishes the feasibility of a new paradigm for a personalized approach to lung cancer clinical trials.

(ClinicalTrials.gov numbers:NCT00409968, NCT00411671, NCT00411632, NCT00410059, and   NCT00410189.

Significance: The BATTLE study is the first completed prospective, adaptively randomized study in heavily pretreated NSCLC patients that mandated tumor profiling with “real-time” biopsies, taking a substantial step toward realizing personalized lung cancer therapy by integrating real-time molecular laboratory findings in delineating specific patient populations for individualized treatment. Cancer Discovery; 1(1); 44–53. © 2011 AACR.

Read the Commentary on this article by Sequist et al., p. 14
Read the Commentary on this article by Rubin et al., p. 17
This article is highlighted in the In This Issue feature, p. 4

Pharmacometabolomics in Drug Discovery & Development: Applications and Challenges

Yang and F. Marotta
Metabolomics 2012, 2:5 http://dx.doi.org/10.4172/2153-0769.1000e122

Recently, the concept of pharmaco-metabolomics is mentioned more frequently as an emerging discipline to study the effect of drugs on the whole pattern of small endogenous molecules and in applying the profiles of metabolomics for drug development. For the latter part, metabolomics is majorly used to differentiate patients into responder or non-responder groups in an effort to decrease large inter-individual variation in clinical trials. It is a novel approach that combines metabolite profile and chemo-metrics to model and predict drug targets, efficacy, pharmacokinetics and toxicity on both individual and population basis. It attracts many scientists’ attention because of its intrinsic advantages and promising potentials in drug discovery and development. Considering personalized drug treatment is the desired goal for current drug development, pharmaco-metabolomics provide an effective and inexpensive strategy to evaluate drug efficacy and toxicology, which may make personalized medicine realistic both from scientific and financial perspectives. Furthermore, the FDA also realized that metabolomics coupling with other “Omics” approaches could be a valuable tool in evaluating general toxicology and could eventually replace the use of animals after addressing certain challenges.

Networking metabolites and diseases

Pascal Braun, Edward Rietman, and Marc Vidal
PNAS  July 22, 2008; 105(29): 9849–9850

Diseasome and Drug-Target Network

Recently, Goh et al. constructed a ‘‘diseasome’’ network in which two diseases are linked to each other if they share at least one gene, in which mutations are associated with both diseases. In the resulting network, related disease families cluster tightly together, thus phenotypically defining functional modules. Importantly, for the first time this study applied concepts from network biology to human diseases, thus opening the door for discovering causal relationships between  disregulated networks and resulting ailments.

Subsequently Yilderim et al. linked drugs to protein targets in a drug–target network, which could then be overlaid with the diseasome network. One notable finding was the recent trend toward the development of new compounds directly targeted at disease gene products, whereas previous drugs, often found by trial and error, appear to target proteins only indirectly related to the actual disease molecular mechanisms. An important question that remains in this emerging field of network analysis consists of investigating the extent to which directly targeting the product of mutated genes is an efficient approach or whether targeting network properties instead, and thereby accounting for indirect nonlinear effects of system perturbations by drugs, may prove more fruitful. However, to answer such questions it is important to have a good understanding of the various influences that can lead to diseases.

UPDATED 6/01/2019

Combined hereditary and somatic mutations of replication error repair genes result in rapid onset of ultra-hypermutated cancers

from  2015 Mar;47(3):257-62. doi: 10.1038/ng.3202. Epub 2015 Feb 2.

Shlien A1Campbell BB2de Borja R3Alexandrov LB4Merico D5Wedge D4Van Loo P6Tarpey PS4Coupland P7Behjati S4Pollett A8Lipman T9Heidari A9Deshmukh S9Avitzur N9Meier B10Gerstung M4Hong Y10Merino DM3Ramakrishna M4Remke M11Arnold R3Panigrahi GB3Thakkar NP12Hodel KP13Henninger EE13Göksenin AY13Bakry D14Charames GS15Druker H16Lerner-Ellis J17Mistry M2Dvir R18Grant R14Elhasid R18Farah R19Taylor GP20Nathan PC14Alexander S14Ben-Shachar S21Ling SC22Gallinger S23Constantini S24Dirks P25Huang A26Scherer SW27Grundy RG28Durno C29Aronson M30Gartner A10Meyn MS31Taylor MD25Pursell ZF13Pearson CE12Malkin D32Futreal PA4Stratton MR4Bouffet E26Hawkins C33Campbell PJ34Tabori U35Biallelic Mismatch Repair Deficiency Consortium.

Abstract: DNA replication-associated mutations are repaired by two components: polymerase proofreading and mismatch repair. The mutation/consequences of disruption to both repair components in humans are not well studied. We sequenced cancer genomes from children with inherited biallelic mismatch repair deficiency (bMMRD). High-grade bMMRD brain tumors exhibited massive numbers of substitution mutations (>250/Mb), which was greater than all childhood and most cancers (>7,000 analyzed). All ultra-hypermutated bMMRD cancers acquired early somatic driver mutations in DNA polymerase ɛ or δ. The ensuing mutation signatures and numbers are unique and diagnostic of childhood germ-line bMMRD (P < 10(-13)). Sequential tumor biopsy analysis revealed that bMMRD/polymerase-mutant cancers rapidly amass an excess of simultaneous mutations (∼600 mutations/cell division), reaching but not exceeding ∼20,000 exonic mutations in <6 months. This implies a threshold compatible with cancer-cell survival. We suggest a new mechanism of cancer progression in which mutations develop in a rapid burst after ablation of replication repair.

Genetic changes which occur in spontaneous arising somatic cancers include point mutations, copy number alterations and rearrangements and in general result from a defective DNA repair mechanisms during proliferation/replication over many years however as most somatic cancers are heterogeneous it is difficult to pinpoint the exact repair defects which may be ultimately responsible for such genetic aberrations.

However, early-onset cancers (e.g. pediatric cancers) in patients with hereditary DNA repair defects offer a good view of the mutation types and secondary pathways that drive oncogenesis. bMMRD is a childhood cancer syndrome characterized by early-onset cancers in various organs and caused by biallelic mutations.  In this study, genomes from 17 inherited cancers, by exomic sequencing and microarrays, were analyzed and compared to non-neoplastic tissue genomes from matched patients.  Brain cancers from these patients had an extremely high number of point mutations compared to other childhood cancers and adult cancers.

Mismatch repair was defective in all these cancers therefore it appeared that secondary mutations are required to cause the ultrahypermutated state.  The most frequently mutated gene was POLE (polymerase epsilon), affecting the proofreading ability of this DNA polymerase.  The genomes of tumors with mutant POLE had signature mutational spectrum and the signature occurred early but these signatures had been found in endometrial and colorectal cancers.  The authors concluded, based on serial analysis of other brain cancers with bMMRD and the observation that recurrent brain cancers accumulated mutations over a relatively short period, once the proofreading capability of pol epsilon is compromised in MMR deficient cells there is no defense against rapid and catastrophic accumulations of mutations.  This rapid accumulation of mutations apparently do not lead to apoptosis but rather rapid tumor initiation, and generating multiple subclones of tumor cells.

 

 

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Introduction to Lipid Metabolism

Author, Curator: Larry H. Bernstein, MD, FCAP 

Introduction to Lipid Metabolism

This series of articles is concerned with lipid metabolism. These discussions lay
the groundwork to proceed to discussions that will take on a somewhat different
approach, but they are critical to developing a more complete point of view of life
processes.  I have indicated that there are protein-protein interactions or protein-membrane interactions and associated regulatory features, but the focus of the
discussion or points made were different, and will be returned to.  The role of
lipids in circulating plasma proteins as biomarkers for coronary vascular disease
can be traced to the early work of Frederickson and the classification of lipid disorders.  The very critical role of lipids in membrane structure in health and
disease has had much less attention, despite the enormous importance,
especially in the nervous system.

This portion of the discussions of metabolism will have several topics on lipid
metabolism.  The first is concerned with the basic types of lipids -which are defined structurally and have different carbon chain length, and have
two basic types of indispensible fatty acid derivations – along pro-inflammatory
and anti-inflammatory pathways:

  1. Alpha-linolenic acid (ALA) and LA, n-3 polyunsaturated fatty acids LCPUFAs (EPA, DHA, and AA), eicosanoids,
    delta-3-desaturase, prostaglandins, and leukotrienes.
  2. the role of the mitochondrial electron transport chain in hydrogen transfers
    and oxidative phosphorylation with respect to the oxidation of fatty acids
    and fatty acid synthesis.
  3. The membrane structures of the cell, including
  • the cytoskeleton, essential organelles, and the intercellular matrix, which
    is a critical consideration for
  • cell motility, membrane conductivity, flexibility, and  signaling.
  • The membrane structure involves aggregation of lipids with proteins,
  • and is associated with hydrophobicity.
  1. The pathophysiology of systemic circulating lipid disorders.
  2. The fifth is the pathophysiology of cell structures under oxidative
    stress.
  3. Lipid disposal and storage diseases.

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Reporter and Curator: Dr. Sudipta Saha, Ph.D.

  • Multiple important and complex interactions exist between the endocrine and other systems (e.g. immune, nervous).
  • Definition of hormones: circulating molecules with a site of action distant from site of origin with ability to bind to cellular receptors and initiate signal transduction via conformational changes in the receptor.
  • Hormones participate in growth and development, reproduction, energy metabolism and maintenance of the internal environment.
  • In general, hormones are protein-derived molecules that bind to cell surface receptors or steroid hormones that bind to nuclear receptors. An exemption is thyroid hormone, a modified amino acid that binds to nuclear receptors.
  • Integrated feedback loops are very characteristic to the endocrine system and critical in maintaining normal hormonal function. Two major types of control exist: the hypothalamic-pituitary-peripheral organ unit and the free standing endocrine gland.
  • Pathology in endocrinology is due to abnormal hormone activity or neoplasms, leading to endocrine hyperfunction/hyperfunction or structural abnormalities.

Endocrine pathology is derived from defects found at any point in the hormonal synthesissecretiontransportaction, or regulatory control of a hormone. Endocrine pathology often occurs in one of the following broad categories:

  1. Abnormal Hormone Activity which can be subdivided into:
    • Endocrine organ hypofunction
      • Primary endocrine organ failure can be genetic or acquired
        • Endocrine organ agenesis (absence)
        • Genetic defect in hormone biosynthetic pathway (e.g. adrenal insufficiency due to 21-hydroxylase deficiency)
        • Destruction due to
          • Autoimmune disease (e.g. Hashimoto’s hypothyroidism)
          • A tumor, infection or hemorrhage
        • Deficiency of precursor (e.g. iodine deficiency leading to decreased thyroid hormone synthesis)
      • Production of abnormal hormone resulting in hypofunction (e.g. abnormal glycosylation of TSH). Secondary endocrine organ failure (e.g. hypothyroidism due to hypopituitarism)
    • Endocrine organ hyperfunction
      • Primary endocrine organ process due to a benign condition (e.g. autoimmune thyroid gland stimulation in Graves’ disease) or benign neoplasm (e.g. primary hyperparathyroidism causing hypercalcemia). Endocrine cancers are rare but they may also release hormones that cause endocrine hyperfunction (e.g. adrenocortical carcinoma secreting excessive androgens causing virilization).
        • Benign condition (e.g. thyroid gland stimulation in Graves’ disease by autoantibodies against the TSH receptor)
        • Benign neoplasm (e.g. primary hyperparathyroid adenoma secreting excessive PTH causing hypercalcemia).
        • Endocrine cancers (e.g. adrenocortical carcinoma secreting excessive androgens causing virilization).
      • Secondary due to stimulation by a trophic/stimulatory hormone, most often due to a benign neoplasm (e.g. hypersecretion of cortisol from adrenal cortex due to and ACTH-secreting pituitary adenoma).
      • Less commonly, ectopic production of a hormone may lead to endocrine hyperfunction (e.g. ACTH released from small cell lung cancer cause hypersecretion of cortisol by adrenal glands).
    • Abnormality in hormone transport or metabolism (e.g. genetic defects of abnormal thyroid binding globulin)
    • Abnormal hormone receptor binding and/or signal transduction. Most often causing endocrine hypofunction due to resistance to the action of hormone. The receptor itself being unable to bind the hormone (e.g. thyroid hormone resistance) or there may be a defect in post-receptor signal transduction (e.g. type 2 diabetes mellitus). Occasionally, abnormal hormone signaling may lead to endocrine hyperfunction (e.g. Gs protein mutation leading to unregulated secretion of Growth Hormone).
  2. Neoplasms. They can be both benign or malignant. Symptoms develop either due to
    • Overproduction of hormone by the tumor (e.g. ACTH producing pituitary adenoma causing hypersecretion of cortisol)
    • Underproduction of nearby hormones due to mass effect (e.g. pituitary hormone production is often affected by large pituitary tumors)
    • Structural damage (e.g. hypothalamic-pituitary tumors causing headache, visual problems).
  3. Iatrogenic. Most common iatrogenic cause of endocrine abnormality is exogenous administration of glucocorticoids (give to treat non-endocrine conditions, e.g. rheumatoid arthritis)

Source References:

http://www.vivo.colostate.edu/hbooks/pathphys/endocrine/

http://ocw.tufts.edu/Content/14/lecturenotes/265876

http://intranet.tdmu.edu.ua/data/kafedra/internal/magistr/classes_stud/English/First%20year/Clinical%20Pathophysiology%20of%20Diseases/CLINICAL%20PATHOPHYSIOLOGY%20OF%20THE%20ENDOCRINE%20SYSTEM.htm

Greenspan FS and Gardner DG. Basic and Clinical Endocrinology, 6th edition. Lange Medical Books, McGraw-Hill, 2001.

Wilson, JD, Foster, DW, Kronenberg, HM, and Larsen, PR. Principles of Endocrinology. In: Williams Textbook of Endocrinology, 9th edition, W.B. Saunders, Philadelphia, 1998.

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