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Posts Tagged ‘coronary heart disease’


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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Stem Cell Therapy for Coronary Artery Disease (CAD)

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

and

Curator: Aviva Lev-Ari, PhD, RN

 

There is great interest and future promise for stem cell therapy in ischemic heart disease.  This is another report for the active work in cardiology with stem cell therapy by MA Gaballa and associates at University of Arizona.

Stem Cell Therapy for Coronary Heart Disease

Julia N. E. Sunkomat and Mohamed A. Gaballa

The University ofArizona Sarver Heart Center, Section of Cardiology, Tucson, Ar
Cardiovascular Drug Reviews 2003: 21(4): 327–342

Keywords: Angiogenesis — Cardiac therapy — Coronary heart disease — Heart failure — Myoblasts — Myocardial ischemia — Myocardial regenera­tion — Stem cells

ABSTRACT

Coronary artery disease (CAD) remains the leading cause of death in the Western world. The high impact of its main sequelae, acute myocardial infarction and congestive heart failure (CHF), on the quality of life of patients and the cost of health care drives the search for new therapies. The recent finding that

stem cells contribute to neovascularization and possibly improve cardiac function after myocardial infarction makes stem cell therapy the most highly active research area in cardiology. Although the concept of stem cell therapy may revolutionize heart failure treatment, several obstacles need to be ad­dressed. To name a few:

  1.  Which patient population should be considered for stem cell therapy?
  2.  What type of stem cell should be used?
  3.  What is the best route for cell de­livery?
  4.  What is the optimum number of cells that should be used to achieve functional effects?
  5.  Is stem cell therapy safer and more effective than conventional therapies?

The published studies vary significantly in design, making it difficult to draw conclusions on the efficacy of this treatment. For example, different models of

  1. ischemia,
  2. species of donors and recipients,
  3. techniques of cell delivery,
  4. cell types,
  5. cell numbers and
  6. timing of the experiments

have been used. However, these studies highlight the landmark concept that stem cell therapy may play a major role in treating cardiovascular diseases in the near future. It should be noted that stem cell therapy is not limited to the treatment of ischemic cardiac disease.

  • Non-ischemic cardiomyopathy,
  • peripheral vascular disease, and
  • aging may be treated by stem cells.

Stem cells could be used as vehicle for gene therapy and eliminate the use of viral vectors. Finally, stem cell therapy may be combined with phar­macological, surgical, and interventional therapy to improve outcome. Here we attempt a systematic overview of the science of stem cells and their effects when transplanted into ischemic myocardium.

INTRODUCTION

Background

Congestive heart failure (CHF) is the leading discharge diagnosis in patients over the age of 65 with estimates of $24 billion spent on health care in the US (1,11). The number one cause of CHF is coronary artery diseases (CAD). Coronary care units, reperfusion therapy (lytic and percutaneous coronary intervention) and medical therapy with anti-pla­telet agents, statins, ACE-inhibitors and â-adrenoceptor antagonists all significantly reduce morbidity and mortality of CAD and CHF (9), but it is very difficult to regenerate new viable myocardium and new blood vessels.

Identification of circulating endothelial progenitor cells in peripheral blood that incor­porated into foci of neovascularization in hindlimb ischemia (4) and the successful engraftment of embryonic stem cells into myocardium of adult dystrophic mice (31) intro­duced a new therapeutic strategy to the field of cardiovascular diseases: tissue regeneration. This approach is supported by the discovery of primitive cells of extracardiac origin in cardiac tissues after sex-mismatched transplants suggesting that an endogenous repair mechanism may exist in the heart (35,45,54). The number of recruited cells varied significantly from 0 (19) to 18% (54), but the natural course of ischemic cardiomyopathy implies that cell recruitment for tissue repair in most cases is insufficient to prevent heart failure. Therefore, investigational efforts are geared towards

  • augmenting the number of multipotent stem cells and endothelial and myocardial progenitor cells at the site of ischemia to induce clinically significant angiogenesis and potentially myogenesis.

Stem and Progenitor Cells

Stem cells are defined by their ability to give rise to identical stem cells and progenitor cells that continue to differentiate into a specific tissue cell phenotype (23,33). The po­tential of mammalian stem cells varies with stage of development and age (Table 1).

In mammals, the fertilized oocyte and blastomere cells of embryos of the two to eight cell stage can generate a complete organism when implanted into the uterus; they are called totipotent stem cells. After the blastocyst stage, embryonic stem cells retain the ability to differentiate into all cell types, but

  • cannot generate a complete organism and thus are denoted pluripotent stem cells.

Other examples of pluripotent stem cells are embryo­nic germ cells that are derived from the gonadal ridge of aborted embryos and embryonic carcinoma cells that are found in gonadal tumors (teratocarcinomas) (23,33). Both these cell types can also differentiate into cells of all three germ layers, but are not as well inves­tigated as embryonic stem cells.

It is well established that embryonic stem cells can differentiate into cardiomyocytes (7,10,13,14,31,37,76), endothelial cells (55), and smooth muscle cells (5,22,78) in vitro, but it is unclear whether

  • pure populations of embryonic stem cell-derived cardiomyocytes can integrate and function appropriately in the heart after transplantation.
  • one study reported arrhythmogenic potential of embryonic stem cell-derived cardiomyocytes in vitro (80).

Adult somatic stem cells are cells that have already committed to one of the three germ layers: endoderm, ectoderm, or mesoderm (76). While embryonic stem cells are defined by their origin (the inner cell mass of the blastocyst), the origin of adult stem cells in mature tissues is still unknown. The primary role of adult stem cells in a living organism is thought to be maintaining and repairing the tissue in which they reside. They are the source of more identical stem cells and cells with a progressively more distinct phenotype of specialized tissue cells (progenitor and precursor cells) (Fig. 1). Until recently adult stem cells were thought to be lineage-specific, meaning that they can only differentiate into the cell-type of their original tissue. This concept has now been challenged with the discovery of multipotent stem and progenitor cells (26, 50, 51).

The presence of multipotent stem and progenitor cells in adult mammals has vast im­plications on the availability of stem cells to research and clinical medicine. Recent publi­cations, however, have questioned whether the adaptation of a phenotype in those dogma-challenging studies is really a result of trans-differentiation or rather a result of cell and nuclear fusion (60,68,75,79). Spontaneous fusion between mammalian cells was first re­ported in 1961 (8), but how frequently fusion occurs and whether it occurs in vivo is not clear.

The bone marrow is a known source of stem cells. Hematopoietic stem cells are fre­quently used in the field of hematology. Surface receptors are used to differentiate hematopoietic stem and progenitor cells from mature cells. For example, virtually all

  • hematopoietic stem and progenitor cells express the CD34+ glycoprotein antigen on their cell membrane (73),

though a small proportion of primitive cells have been shown to be CD34 negative (58).

The function of the CD34+ receptor is not yet fully understood. It has been suggested that it may act as a regulator of hematopoietic cell adhesion in the bone marrow microenvironment. It also appears to be involved in the maintenance of the hematopoietic stem/progenitor cell phenotype and function (16,21). The frequency of immature CD34+ cells in peripheral circulation diminishes with age.

  • It is the highest (up to 11%) in utero (69) and decreases to 1% of nucleated cells in term cord blood (63).
  • This equals the per­centage of CD34+ cells in adult bone marrow.
  • The number of circulating stem cells in adult peripheral blood is even lower at 0.1% of nucleated cells.

Since hematopoietic stem cells have been identified as endothelial progenitor cells (29,30,32) their low density in adult bone marrow and blood could explain the inadequacy of endogenous recruitment of cells to injured organs such as an ischemic heart. The bone marrow is also home to another stem cell population the so-called mesenchymal stem cells. These may constitute a subset of the bone marrow stromal cells (2,43). Bone marrow stromal cells are a mixed cell popu­lation that generates

  1. bone,
  2. cartilage,
  3. fat,
  4. connective tissue, and
  5. reticular network that sup­ports cell formation (23).

Mesenchymal stem cells have been described as multipotent (51,52) and as a source of myocardial progenitor cells (41,59). They are, however, much less defined than the hematopoietic stem cells and a characteristic antigen constellation has not yet been identified (44).

Another example of an adult tissue containing stem cells is the skeletal muscle. The cells responsible for renewal and growth of the skeletal muscle are called satellite cells or myoblasts and are located between the sarcolemma and the basal lamina of the muscle fiber (5). Since skeletal muscle and cardiac muscle share similar characteristics such as they both are striated muscle cells, satellite cells are considered good candidates for the repair of damaged myocardium and have been extensively studied (20,25,38–40,48,56, 64–67). Myoblasts are particularly attractive, because they can be autotransplanted, so that issues of donor availability, ethics, tumorigenesis and immunological compatibility can be avoided. They also have been shown to have a high growth potential in vitro and a strong resistance to ischemia in vivo (20). On the down side

  • they may have more arrhythmogenic potential when transplanted into myocardium than bone marrow or peripheral blood de­rived stem cells and progenitor cells (40).

Isolation of Cells Prior to Transplantation

Hematopoietic stem and progenitor cells are commonly identified by the expression of a profile of surface receptors (cell antigens). For example, human hematopoietic stem cells are defined as CD34+/CD59+/Thy-1+/CD38low//c-kit/low/lin, while mouse hema-topoietic stem cells are defined as CD34low//Sca-1+/Thy-1+/low/CD38+/c-kit+/lin (23). Additional cell surface receptors have been identified as markers for subgroups of hema-topoietic stem cells with the ability to differentiate into non-hematopoetic tissues, such as endothelial cells (57,78). These can be specifically targeted by isolation methods that use the receptors for cell selection (positive selection with antibody coated magnetic beads or fluorescence-activated cell sorting, FACS). Other stem cell populations are identified by their behavior in cell culture (mesenchymal stem cells) or dye exclusion (SP cells). Finally, embryonic stem cells are isolated from the inner cell mass of the blastocyst and skeletal myoblasts are mechanically and enzymatically dissociated from an easily acces­sible skeletal muscle and expanded in cell culture.

FIG. 1. Maturation process of adult stem cells: with acquisition of a certain phenotype the cell gradually loses its self-renewal capability.  (unable to transfer)

METHODICAL APPROACHES 

j.1527-3466.2003.tb00125.x  fig stem cell

FIG. 2. Intramyocardial injection:

the cells are injected directly into the myocardium through the epicardium. Usually a thoracotomy or sternotomy is required. Transendocardial injection: access can be gained from the ar­terial vasculature. Cells are injected through the endocardium into the myocardium, ideally after identifying the ischemic myocardium by perfusion studies and/or electromechanical mapping. Intracoronary injection: the coronary artery is accessed from the arterial vasculature. Stem cells are injected into the lumen of the coronary artery. Proximal washout is prevented by inflation of a balloon. Cells are then distributed through the capillary system. They eventually cross the endothelium and migrate towards ischemic areas.

The intracoronary delivery of stem cells (Fig. 2) and distribution through the coronary system has also been explored (6,62,74). This approach was pioneered by Robinson et al. (56), who demonstrated successful engraftment within the coronary distribution after intracoronary delivery of genetically labeled skeletal myoblasts. The risk of intracoronary injection is comparable to that of a coronary angiogram and percutaneous transluminal coronary angioplasty (PTCA) (62), which are safe and clinically well established.

RESULTS IN ANIMAL STUDIES AND HUMAN TRIALS

Dif­ferentiation into cardiomyocytes was observed after transplantation of embryonic stem cells, mesenchymal stem cells, lin/c-kit+ and SP cells. The induction of angiogenesis was observed after transplantation of embryonic stem cells, mesenchymal stem cells, bone marrow-derived mononuclear cells, circulating endothelial progenitor cells, SP cells and lin/c-kit+ cells.

The use of embryonic stem cells in ischemia was examined in two studies (42,43). These studies demonstrated that mice embryonic stem cells transplanted into rat myo­cardium exhibited cardiomyocyte phenotype at 6 weeks after transplantation. In addition, generation of myocardium and angiogenesis were observed at 32 weeks after allogenic transplantation in rats. In these two studies no arrhythmias or cardiac tumors were reported.

Several studies have shown retardation of LV remodeling and improvement of cardiac function after administration of bone marrow-derived mononuclear cells. For example, decreases in infarct size, and increase in ejection fraction (EF), and left ventricular (LV) time rate change of pressure (dP/dtmax) were observed after direct injection of bone marrow-derived mononuclear cells 60 min after ischemia in swine (28). In humans, intra-coronary delivery and transendocardial injection of mononuclear cells leads to a decrease in LV dimensions and improvement of cardiac function and perfusion (49,62). A decrease in end systolic volume (ESV) and an increase in EF as well as regional wall motion were observed following intracoronary administration of CD34+/CD45+ human circulating en­dothelial cells (6). Injection of circulating human CD34+/CD117+ cells into infarcted rat myocardium induced neoangiogenesis and improved cardiac function (32). This study suggests that the improvement in LV remodeling after infarction appears to be in part me­diated by a decrease in apoptosis within the noninfarcted myocardium. Two other studies reported increased fractional shortening, improved regional wall motion and decreased left ventricular dimensions after transplantation of human CD34+ cells (29,30). Improved global left ventricular function and infarct perfusion was demonstrated after intramyo-cardial injection of autologous endothelial progenitor cells in humans (61).

DISCUSSION AND OUTLOOK

The idea of replacing damaged myocardium by healthy cardiac tissue is exciting and has received much attention in the medical field and the media. Therefore, it is important for the scientist to know what is established and what is based on premature conclusions. Currently, there are data from animal studies and human trials (Table 2). However, some of these data are not very concrete. For example,

  • many animal studies do not report the level of achieved neoangiogenesis and/or regeneration of myocardium.
  • In studies where the numbers of neovessels and new cardiomyocytes are specified, these numbers are often very low.

While these experiments confirm the concept that bone marrow and peripheral blood-derived stem and progenitor cells can differentiate into cardiomyocytes and endo­thelial cells when transplanted into ischemic myocardium, they also raise the question how effective this treatment is.

The results of the clinical trials that have been conducted are encouraging, but they need to be interpreted with caution. The common endpoints of these studies include left ventricular dimensions, perfusion, wall motion and hemodynamic function. While all studies report improvement after mononuclear cell, myoblast or endothelial progenitor cell transplantation, it is difficult to separate the effects of stem cell transplantation from the effects of the state-of-the art medical care that the patients typically received.

CONCLUSION

While the majority of studies demonstrate neoangiogenesis and some studies also show regeneration of myocardium after stem/progenitor cell transplantation, it remains unclear whether the currently achieved level of tissue regeneration is sufficient to affect clinical outcome. Long-term follow-up of patients that received stem/progenitor cells in clinical trials will provide important information on the potential risks of neoplasm and arrhythmias and, therefore, safety of this treatment. Ultimately, postmortem histological confirmation of scar tissue repair by transplanted cells and randomized placebo control trials with long-term follow-up are required to prove efficacy of this treatment.

REFERENCES (10)

1. American Heart Association Disease and Stroke Statistics-2003 Update, Dallas TX, American Heart Associ­ation; 2002 http://http://www.americanheart.org/downloadable/heart/10461207852142003HDSStatsBook.pdf

2. Arai A, Sheikh F, Agyeman K, et al. Lack of benefit from cytokine mobilized stem cell therapy for acute myocardial infarction in nonhuman primates. J Am Coll Cardiol 2003;41(Suppl 6A):371.

3. Asahara T, Masuda H, Takahashi T, et al. Bone marrow origin of endothelial progenitor cells responsible for postnatal vasculogenesis in physiological and pathological neovascularization. Circ Res 1999;85:221–228.

4. Asahara T, Murohara T, Sullivan A, et al. Isolation of putative progenitor endothelial cells for angiogenesis. Science 1997;275:964–967.

5. Asakura A, Seale P, Girgis-Gabardo A, Rudnicki M. Myogenic specification of side population cells in skeletal muscle. J Cell Biol 2002;159(1):123–134.

6. Assmus B, Schaechinger V, Teupe C, et al. Transplantation of progenitor cells and regeneration en­hancement in acute myocardial infarction (TOPCARE-AMI). Circulation 2002;106:r53–r61.

7. Bader A, Al-Dubai H, Weitzer G. Leukemia inhibitory factor modulates cardiogenesis in embryoid bodies in opposite fashions. Circ Res 2000;86(7):787–794.

8. Barski G, Sorieul S, Cornefert F. “Hybrid” type cells in combined cultures of two different mammalian cell strains. J Natl Cancer Inst 1961;26:1269–1291.

9. Boersma E, Mercado N, Poldermans D, Gardien M, Vos J, Simoons M. Acute myocardial infarction. Lancet 2003;361:847–58.

  1. 10.          Boheler K, Czyz J, Tweedie D, Yang H, Anisimov S, Wobus A. Differentiation of pluripotent embryonic stem cells into cardiomyocytes. Circ Res 2002;91:189–201.

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Reporter: Sudipta Saha, Ph.D.

Assessment of the propensity for vascular events has been based on measurement of risk factors predisposing one to vascular injury. These assessments are based on the strong associations between risk factors such as hypertension, cholesterol levels, smoking, and diabetes which were first described almost a half century ago. The more recent discovery of the relationship between ongoing inflammation and clinical outcomes has led to a variety of blood-based assays which may impart additional knowledge about an individual’s propensity for future cardiovascular events. Vascular health is now better represented as a balance between ongoing injury and resultant vascular repair, mediated at least in part by circulating endothelial progenitor cells (http://www.ncbi.nlm.nih.gov/pubmed/19124422). Accurate enumeration of circulating endothelial progenitor cells is essential for their potential application as biomarkers of angiogenesis. Different stem cell markers (CD34, CD133) and endothelial cell antigens (KDR/VEGFR-2, CD31) in different flow cytometric protocols are assessed for the purpose of circulating progenitor endothelial cell quantification (http://www.ncbi.nlm.nih.gov/pubmed/20381496). Enumeration of circulating progenitor endothelial cells are used in the assessment of various diseases and physiological states, such as: type 2 diabetes patients with peripheral vascular disease, certain phases during congestive heart failure, acute myocardial infarction, atherosclerosis, cardiovascular disease, physical training, cessation of smoking. Two modern instruments used now-a-days to measure the circulating progenitor endothelial cells are discussed below:

MACSQuant® Analyzer:

Circulating progenitor endothelial cells are defined by co-expression of the markers CD34, CD309 (VEGFR-2/KDR), and CD133, though CD133 expression is lost during maturation to endothelial cells.8-10 Since circulating progenitor endothelial cells are rare in peripheral blood, EPC enumeration protocols are rather extensive and laborious. To obtain reliable enumeration results for these rare cells, the sensitivity of flow cytometric analysis needs to be increased. This has been achieved by magnetic enrichment of circulating progenitor endothelial cells prior to flow cytometric analysis, which reduces the number of events that have to be analyzed. The circulating progenitor endothelial cell Enrichment and Enumeration Kit have been designed for enumeration of circulating progenitor endothelial cells from peripheral blood, cord blood, bone marrow, or leukapheresis products. In combination with magnetic pre-enrichment and flow cytometric analysis on the MACSQuant® Analyzer, this kit overcomes some of the limitations of circulating progenitor endothelial cell analysis and offers a simple and time effective solution for EPC enumeration. The circulating progenitor endothelial cell Enrichment and Enumeration Kit in combination with pre-enrichment and flow cytometric analysis on the MACSQuant Analyzer is an effective method to enumerate circulating progenitor endothelial cells in 10 mL of whole blood. Based on the calculated starting number of cells, the circulating progenitor endothelial cell Express Mode analysis template automatically calculates the absolute number and concentration of circulating progenitor endothelial cells in 10 mL of starting material, i.e., whole blood, bone marrow, cord blood, or leukapheresis products. The MACSQuant Analyzer has the ability to enrich cells using MACS technology. This capability makes the enumeration of circulating progenitor endothelial cells fast and easy. The entire process takes less than 2 hours to perform from blood draw to analyzed data and drastically reduces the time and difficulty of such a protocol by combining magnetic enrichment and flow cytometric analysis in one streamlined experiment (http://www.miltenyibiotec.com/downloads/6760/6764/18602/31184/MQ_ApplicationFlyer_EPC.pdf).

Attune® Acoustic Focusing Cytometer:

In cancer research, circulating progenitor endothelial cells have been suggested as a noninvasive biomarker for angiogenic activity, providing insight into tumor regrowth, resistance to chemotherapy, early recurrence, and metastasis during or after chemotherapy. In healthy individuals, circulating progenitor endothelial cells are reported to be present in very low numbers: 0.01%–0.0001% of all peripheral blood mononuclear cells. Flow cytometry offers the necessary collection and analysis capabilities for detection of circulating progenitor endothelial cells, but is subject to numerous technical challenges. In comparison to traditional hydrodynamic focusing cytometers, the Attune® Acoustic Focusing Cytometer, with its fast acquisition times and increased precision, overcomes the technological hurdles involved in analyzing circulating progenitor endothelial cells. The method includes a number of conventional ways to improve rare-event detection: a blocking step, a viability stain (SYTOX® AADvanced™ Dead Cell Stain), and the use of a dump channel to eliminate unwanted cells and decrease background fluorescence. The challenge of collecting a large enough number of events in a reasonable amount of time is met by using a collection rate of 1,000 μL/min with the Attune® cytometer. This setting enables the collection of more than 4,000,000 live white blood cell (WBC) events in just 35 minutes; the acquisition time using a traditional hydrodynamic focusing cytometer would be 10–12 times longer, close to 6 hours. Furthermore, this method delivers additional time savings by eliminating wash steps to avoid sample loss and employing a simpler sample preparation method. (http://zh.invitrogen.com/etc/medialib/files/Cell-Analysis/PDFs.Par.54318.File.tmp/CO24210-Human-CEC_cancer.pdf)

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Reported by: Dr. Venkat S. Karra, Ph.D.

Coronary heart disease (CHD) is a condition caused by a build-up of fatty deposits on the inner walls of the blood vessels that supply the heart, causing the affected person to experience pain, usually on exertion (angina). A complete occlusion of the vessel by deposits causes a heart attack (myocardial infarction).

Lifestyle factors, such as diet (particularly one high in fat), contribute to causing CHD.

There are different types of fat, some of which are thought to increase risk of CHD, such as saturated fat, typically found in meat and dairy foods. However, others, such as unsaturated fats (polyunsaturated and monounsaturated fats) found in foods such as vegetable oils, fish, and nuts, may actually help prevent this condition.

Fatty Acids

Although there have been many studies investigating the role of different types of dietary fat in coronary heart disease, it is still not clear whether coronary heart disease can be prevented by changing the type of dietary fat consumed from saturated to unsaturated fats or by lowering all types of dietary fat. Furthermore, many of these studies have relied on participants recalling their dietary intake in questionnaires, which is an unreliable method for different fats.

So in this study, the researchers used an established UK cohort to measure the levels of different types of fatty acids in blood to investigate whether a diet high in saturated fatty acids and low in unsaturated fatty acids increases CHD risk.

These findings suggest that plasma concentrations of saturated fatty acids are associated with increased risk of CHD and that concentrations of omega-6 poly-unsaturated fatty acids are associated with decreased risk of CHD.

These findings are consistent with other studies and with current dietary advice for preventing CHD, which encourages substituting foods high in saturated fat with n-6 polyunsaturated fats. The results also suggest that different fatty acids may relate differently to CHD risk and that the overall balance between different fatty acids is important. However, there are limitations to this study, such as that factors other than diet (genetic differences in metabolism, for example) may cause changes to blood fatty acid levels so a major question is to identify what factors influence blood fatty acid concentrations.

Nevertheless, these findings suggest that individual fatty acids play a role in increasing or decreasing risks of CHD.

read more

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Methylation of the gene F2RL3—which has been linked with platelet activation and inflammation—was lowest in smokers and highest in nonsmokers. Methylation is an important source of variation and regulation in the genome in Epigenetic modification of DNA. In a prospective study in patients with stable coronary heart disease, lower F2RL3 methylation in smokers and former smokers indicated a worse prognosis with excess cardiovascular mortality as well as overall mortality.

The authors found a correlation between F2RL3 methylation intensity and established prognostic markers, including natriuretic peptide, C-reactive protein, and interleukin-6. Current standard medical treatment for coronary artery disease did not affect F2RL3 methylation.

This article was published in European Heart Journal

L. P. Breitling et al., Smoking, F2RL3 methylation, and prognosis in stable coronary heart disease. Eur. Heart J. 17 April 2012

http://eurheartj.oxfordjournals.org/content/early/2012/04/16/eurheartj.ehs091.abstract

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