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In liver, the glucose-responsive transcription factor ChREBP plays a critical role in converting excess carbohydrates into triglycerides through de novo lipogenesis. Although the importance of ChREBP in glucose sensing and hepatic energy utilization is strongly supported, the mechanism driving its activation in response to glucose in the liver is not fully understood. Indeed, the current model of ChREBP activation, which depends on Serine 196 and Threonine 666 dephosphorylation, phosphatase 2A (PP2A) activity, and xylulose 5-phosphate (X5P) as a signaling metabolite, has been challenged. We inhibited PP2A activity in HepG2 cells through the overexpression of SV40 small t antigen and addressed the importance of ChREBP dephosphorylation on Ser-196 using a phospho-specific antibody. To identify the exact nature of the metabolite signal required for ChREBP activity in liver, we focused on the importance of G6P synthesis in liver cells, through the modulation of glucose 6-phosphate dehydrogenase (G6PDH) activity, the rate-limiting enzyme of the pentose phosphate pathway in hepatocytes, and in HepG2 cells using both adenoviral and siRNA approaches. In contrast to the current proposed model, our study reports that PP2A activity is dispensable for ChREBP activation in response to glucose and that dephosphorylation on Ser-196 is not sufficient to promote ChREBP nuclear translocation in the absence of a rise in glucose metabolism. By deciphering the respective roles of G6P and X5P as signaling metabolites, our study reveals that G6P produced by GK, but not X5P, is essential for both ChREBP nuclear translocation and transcriptional activity in response to glucose in liver cells. Altogether, our study, by reporting that G6P is the glucose-signaling metabolite, challenges the PP2A/X5P-dependent model currently described for ChREBP activation in response to glucose in liver. Show less

Pentoxifylline is in clinical trials for non-alcoholic fatty liver disease and diabetic nephropathy. Metabolic and hepatic effects of pentoxifylline were assessed in a murine model of obesity and type 2 diabetes. Pentoxifylline (100 mg·kg(-1) ·day(-1)) was administered for 4 days or 3 weeks in lean and obese/diabetic ob/ob mice. Plasma lipids, glucose, other metabolites and relevant enzymes were measured by standard assays. Hepatic lipids in vivo were assessed with magnetic resonance spectroscopy and by histology. Hepatic extracts were also analysed with RT-PCR and Western blotting. Four days of pentoxifylline treatment slightly increased liver lipids in ob/ob mice. After 3 weeks, pentoxifylline exacerbated fatty liver and plasma transaminases in ob/ob mice but did not induce liver steatosis in lean mice. Plasma glucose was highest in fed, but not fasted, ob/ob mice treated with pentoxifylline. During the first 10 min of an oral glucose tolerance test, blood glucose increased more rapidly in pentoxifylline-treated mice. Jejunal expression of glucose transporter 2 isoform was increased in pentoxifylline-treated obese mice. Hepatic activity of carbohydrate response element binding protein (ChREBP) increased after pentoxifylline in ob/ob, but not lean, mice. Hepatic expression of lipogenic enzymes was highest in pentoxifylline-treated ob/ob mice. However, pentoxifylline reduced markers of oxidative stress and inflammation in ob/ob liver. Pentoxifylline exacerbated fatty liver in ob/ob mice through enhanced intestinal glucose absorption, increased postprandial glycaemia and activation of hepatic lipogenesis. Long-term treatment with pentoxifylline could worsen fatty liver in some patients with pre-existing hyperglycaemia. Show less

Carbohydrate-responsive element-binding protein (ChREBP) is a regulator of pancreatic β-cell gene expression and an important mediator of glucotoxicity. Glucose increases the activity and nuclear localization of ChREBP by still ill-defined mechanisms. Here we reveal, using both MIN6 and primary mouse β-cells, a unique mechanism behind ChREBP nuclear translocation. At low glucose concentrations, ChREBP interacts with sorcin, a penta EF hand Ca(2+) binding protein, and is sequestered in the cytosol. Sorcin overexpression inhibits ChREBP nuclear accumulation at high glucose and reduced the activity of L-type pyruvate kinase (L-PK) and TxNIP promoters, two well-characterized ChREBP target genes. Sorcin inactivation by RNA interference increases ChREBP nuclear localization and in vivo binding to the L-PK promoter at low glucose concentrations. Ca(2+) influx was essential for this process since Ca(2+) chelation with EGTA, or pharmacological inhibition with diazoxide and nifedipine, blocked the effects of glucose. Conversely, mobilization of intracellular Ca(2+) with ATP caused the nuclear accumulation of ChREBP. Finally, sorcin silencing inhibited ATP-induced increases in intracellular Ca(2+) and glucose-stimulated insulin secretion. We therefore conclude that sorcin retains ChREBP in the cytosol at low glucose concentrations and may act as a Ca(2+) sensor for glucose-induced nuclear translocation and the activation of ChREBP-dependent genes. Show less

Liver fat is increased in carriers of the minor G allele in rs738409 (I148M amino acid substitution) in patatin-like phospholipase domain-containing 3 (PNPLA3)/adiponutrin. We studied transcriptional regulation of PNPLA3 in immortalized human hepatocytes (IHH) and human hepatoma cells (HuH7) and the impact of PNPLA3 I148M mutant on hepatocyte triglyceride metabolism. Studies in IHH showed that silencing of the carbohydrate response element-binding protein (ChREBP) abolished induction of PNPLA3 mRNA by glucose. Glucose-dependent binding of ChREBP to a newly identified carbohydrate response element in the PNPLA3 promoter was demonstrated by chromatin immunoprecipitation. Adenoviral overexpression of mouse ChREBP in IHH failed to induce PNPLA3 mRNA. [(3)H]acetate or [(3)H]oleate incorporation with 1-h pulse labeling or 18-h [(3)H]oleate labeling in HuH7 cells showed no effect of PNPLA3 I148M on triglyceride (TG) synthesis in the absence of free fatty acid (FFA) loading. Increased [(3)H]oleate accumulation into triglycerides in I148M-expressing cells was observed after 18 h of labeling in the presence of 200 μM FFA-albumin complexes. This was accompanied by increased PNPLA3 protein levels. The rate of hydrolysis of [(3)H]TG during lipid depletion was decreased significantly by PNPLA3 I148M. Our results suggest that PNPLA3 is regulated in human hepatocytes by glucose via ChREBP. PNPLA3 I148M enhances cellular accumulation of [(3)H]TG in the presence of excess FFA, which is known to stabilize PNPLA3 protein. These data do not exclude an effect of PNPLA3 I148M on hepatocyte lipogenesis but show that the mutant increases the stability of triglycerides. Show less

Following acute hepatic injury, the metabolic capacity of the liver is altered during the process of compensatory hepatocyte proliferation by undefined mechanisms. In this study, we examined the regulation of de novo lipogenesis by cyclin D1, a key mediator of hepatocyte cell cycle progression. In primary hepatocytes, cyclin D1 significantly impaired lipogenesis in response to glucose stimulation. Cyclin D1 inhibited the glucose-mediated induction of key lipogenic genes, and similar effects were seen using a mutant (D1-KE) that does not activate cdk4 or induce cell cycle progression. Cyclin D1 (but not D1-KE) inhibited the activity of the carbohydrate response element-binding protein (ChREBP) by regulating the glucose-sensing motif of this transcription factor. Because changes in ChREBP activity could not fully explain the effect of cyclin D1, we examined hepatocyte nuclear factor 4α (HNF4α), which regulates numerous differentiated functions in the liver including lipid metabolism. We found that both cyclins D1 and D1-KE bound to HNF4α and significantly inhibited its recruitment to the promoter region of lipogenic genes in hepatocytes. Conversely, knockdown of cyclin D1 in the AML12 hepatocyte cell line promoted HNF4α activity and lipogenesis. In mouse liver, HNF4α bound to a central domain of cyclin D1 involved in transcriptional repression. Cyclin D1 inhibited lipogenic gene expression in the liver following carbohydrate feeding. Similar findings were observed in the setting of physiologic cyclin D1 expression in the regenerating liver. In conclusion, these studies demonstrate that cyclin D1 represses ChREBP and HNF4α function in hepatocytes via Cdk4-dependent and -independent mechanisms. These findings provide a direct link between the cell cycle machinery and the transcriptional control of metabolic function of the liver. Show less

While several type 2 diabetes mellitus (T2DM) susceptibility loci identified through genome-wide association studies (GWAS) have been replicated in many populations, their association in Arabs has not been reported. For this reason, the present study looked at the contribution of ENNP1 (rs1044498), IGF2BP2 (rs1470579), KCNJ11 (rs5219), MLXIPL (rs7800944), PPARγ (rs1801282), SLC30A8 (rs13266634) and TCF7L2 (rs7903146) SNPs to the risk of T2DM in Lebanese and Tunisian Arabs. Study subjects (case/controls) were Lebanese (751/918) and Tunisians (1470/838). Genotyping was carried out by the allelic discrimination method. In Lebanese and Tunisians, neither ENNP1 nor MLXIPL was associated with T2DM, whereas TCF7L2 was significantly associated with an increased risk of T2DM in both the Lebanese [P < 0.001; OR (95% CI): 1.38 (1.20-1.59)] and Tunisians [P < 0.001; OR (95% CI): 1.36 (1.18-1.56)]. Differential associations of IGF2BP2, KCNJ11, PPARγ and SLC30A8 with T2DM were noted in the two populations. IGF2BP2 [P = 1.3 × 10(-5); OR (95% CI): 1.66 (1.42-1.94)] and PPARγ [P = 0.005; OR (95% CI): 1.41 (1.10-1.80)] were associated with T2DM in the Lebanese, but not Tunisians, while KCNJ11 [P = 8.0 × 10(-4); OR (95% CI): 1.27 (1.09-1.47)] and SLC30A8 [P = 1.6 × 10(-5); OR (95% CI): 1.37 (1.15-1.62)] were associated with T2DM in the Tunisians, but not Lebanese, after adjusting for gender and body mass index. T2DM susceptibility loci SNPs identified through GWAS showed differential associations with T2DM in two Arab populations, thus further confirming the ethnic contributions of these variants to T2DM susceptibility. Show less

Diets high in sucrose/fructose or fat can result in hepatic steatosis (fatty liver). Mice fed a high-fat diet, especially that of saturated-fat-rich oil, develop fatty liver with an increase in peroxisome proliferator-activated receptor (PPAR) γ2 protein in liver. The fatty liver induced by a high-fat diet is improved by knockdown of liver PPARγ2. In this study, we investigated whether β-conglycinin (a major protein of soy protein) could reduce PPARγ2 protein and prevent high-fat-diet-induced fatty liver in ddY mice. Mice were fed a high-starch diet (70 energy% [en%] starch) plus 20% (wt/wt) sucrose in their drinking water or a high-safflower-oil diet (60 en%) or a high-butter diet (60 en%) for 11 weeks, by which fatty liver is developed. As a control, mice were fed a high-starch diet with drinking water. Either β-conglycinin or casein (control) was given as dietary protein. β-Conglycinin supplementation completely prevented fatty liver induced by each type of diet, along with a reduction in adipose tissue weight. β-Conglycinin decreased sterol regulatory element-binding protein (SREBP)-1c and carbohydrate response element-binding protein (ChREBP) messenger RNAs (mRNAs) in sucrose-supplemented mice, whereas it decreased PPARγ2 mRNA (and its target genes CD36 and FSP27), but did not decrease SREBP-1c and ChREBP mRNAs, in mice fed a high-fat diet. β-Conglycinin decreased PPARγ2 protein and liver triglyceride (TG) concentration in a dose-dependent manner in mice fed a high-butter diet; a significant decrease in liver TG concentration was observed at a concentration of 15 en%. In conclusion, β-conglycinin effectively prevents fatty liver induced by a high-fat diet through a decrease in liver PPARγ2 protein. Show less

The prevalence of obesity and type 2 diabetes is increasing worldwide and threatens to shorten lifespan. Impaired insulin action in peripheral tissues is a major pathogenic factor. Insulin stimulates glucose uptake in adipose tissue through the GLUT4 (also known as SLC2A4) glucose transporter, and alterations in adipose tissue GLUT4 expression or function regulate systemic insulin sensitivity. Downregulation of human and mouse adipose tissue GLUT4 occurs early in diabetes development. Here we report that adipose tissue GLUT4 regulates the expression of carbohydrate-responsive-element-binding protein (ChREBP; also known as MLXIPL), a transcriptional regulator of lipogenic and glycolytic genes. Furthermore, adipose ChREBP is a major determinant of adipose tissue fatty acid synthesis and systemic insulin sensitivity. We find a new mechanism for glucose regulation of ChREBP: glucose-mediated activation of the canonical ChREBP isoform (ChREBP-α) induces expression of a novel, potent isoform (ChREBP-β) that is transcribed from an alternative promoter. ChREBP-β expression in human adipose tissue predicts insulin sensitivity, indicating that it may be an effective target for treating diabetes. Show less

Glucose metabolism in the liver activates the transcription of various genes encoding enzymes of glycolysis and lipogenesis and also G6pc (glucose-6-phosphatase). Allosteric mechanisms involving glucose 6-phosphate or xylulose 5-phosphate and covalent modification of ChREBP (carbohydrate-response element-binding protein) have been implicated in this mechanism. However, evidence supporting an essential role for a specific metabolite or pathway in hepatocytes remains equivocal. By using diverse substrates and inhibitors and a kinase-deficient bisphosphatase-active variant of the bifunctional enzyme PFK2/FBP2 (6-phosphofructo-2-kinase-fructose-2,6-bisphosphatase), we demonstrate an essential role for fructose 2,6-bisphosphate in the induction of G6pc and other ChREBP target genes by glucose. Selective depletion of fructose 2,6-bisphosphate inhibits glucose-induced recruitment of ChREBP to the G6pc promoter and also induction of G6pc by xylitol and gluconeogenic precursors. The requirement for fructose 2,6-bisphosphate for ChREBP recruitment to the promoter does not exclude the involvement of additional metabolites acting either co-ordinately or at downstream sites. Glucose raises fructose 2,6-bisphosphate levels in hepatocytes by reversing the phosphorylation of PFK2/FBP2 at Ser32, but also independently of Ser32 dephosphorylation. This supports a role for the bifunctional enzyme as the phosphometabolite sensor and for its product, fructose 2,6-bisphosphate, as the metabolic signal for substrate-regulated ChREBP-mediated expression of G6pc and other ChREBP target genes. Show less

A link between fructose drinking and nonalcoholic fatty liver disease (NAFLD) has been demonstrated in human and rodent animals. The aim of the present study was to investigate whether endoplasmic reticulum (ER) stress is mediated in the development of fructose-induced NAFLD. Female CD-1 mice were fed with 30% fructose solution for eight weeks. Hepatic lipid accumulation was assessed. Hepatic nuclear sterol regulatory element-binding protein (SREBP)-1c was measured. Results showed that hepatic SREBP-1c was activated in mice fed with fructose solution. Fatty acid synthase (fas) and acetyl-CoA carboxylase (acc), two target genes of SREBP-1c, were up-regulated. Fructose-evoked hepatic SREBP-1c activation seemed to be associated with insulin-induced gene (Insig)-1 depletion. An ER stress and unfolded protein response (UPR), as determined by an increased glucose-regulated protein (GRP78) expression and an increased eIF2α and PERK phosphorylation, were observed in liver of mice fed with fructose solution. Phenylbutyric acid (PBA), an ER chemical chaperone, not only significantly attenuated ER stress, but also alleviated fructose-induced hepatic Insig-1 depletion. PBA inhibited fructose-evoked hepatic SREBP-1c activation and the expression of SREBP-1c target genes, and protected against hepatic lipid accumulation. In conclusion, ER stress contributes, at least in part, to hepatic SREBP-1c activation and lipid accumulation in fructose-evoked NAFLD. Show less

The aim of this study was to determine the effects of high-glucose, high-fructose and high-sucrose diets on weight gain, liver lipid metabolism and gene expression of proteins involved with hepatic fat metabolism. Rats were fed a diet containing either 60% glucose, 60% fructose, 60% sucrose, or a standard chow for 28 days. Results indicated that high-fructose and high-sucrose diets were associated with higher mRNA levels of gene transcripts involved with fat synthesis; ACC, FAS and ChREBP, with no change in SREBP-1C mRNA. The protein level of ChREBP and SREBP1c was similar in liver homogenates from all groups, but were higher in nuclear fractions from the liver of high-fructose and high-sucrose fed rats. The mRNA level of gene transcripts involved with fat oxidation was the same in all three diets, whilst a high-fructose diet was associated with greater amount of mRNA of the fat transporter CD36. Despite the changes in mRNA of lipogenic proteins, the body weight of animals from each group was the same and the livers from rats fed high-fructose and high-sucrose diets did not contain more fat than control diet livers. In conclusion, changing the composition of the principal monosaccharide in the diet to a fructose containing sugar elicits changes in the level of hepatic mRNA of lipogenic and fat transport proteins and protein levels of their transcriptional regulators; however this is not associated with any changes in body weight or liver fat content. Show less

Glucose stimulates rodent and human β-cell replication, but the intracellular signaling mechanisms are poorly understood. Carbohydrate response element-binding protein (ChREBP) is a lipogenic glucose-sensing transcription factor with unknown functions in pancreatic β-cells. We tested the hypothesis that ChREBP is required for glucose-stimulated β-cell proliferation. The relative expression of ChREBP was determined in liver and β-cells using quantitative RT-PCR (qRT-PCR), immunoblotting, and immunohistochemistry. Loss- and gain-of-function studies were performed using small interfering RNA and genetic deletion of ChREBP and adenoviral overexpression of ChREBP in rodent and human β-cells. Proliferation was measured by 5-bromo-2'-deoxyuridine incorporation, [(3)H]thymidine incorporation, and fluorescence-activated cell sorter analysis. In addition, the expression of cell cycle regulatory genes was measured by qRT-PCR and immunoblotting. ChREBP expression was comparable with liver in mouse pancreata and in rat and human islets. Depletion of ChREBP decreased glucose-stimulated proliferation in β-cells isolated from ChREBP(-/-) mice, in INS-1-derived 832/13 cells, and in primary rat and human β-cells. Furthermore, depletion of ChREBP decreased the glucose-stimulated expression of cell cycle accelerators. Overexpression of ChREBP amplified glucose-stimulated proliferation in rat and human β-cells, with concomitant increases in cyclin gene expression. In conclusion, ChREBP mediates glucose-stimulated proliferation in pancreatic β-cells. Show less

Excessive dietary fructose intake may have an important role in the current epidemics of fatty liver, obesity and diabetes as its intake parallels the development of these syndromes and because it can induce features of metabolic syndrome. The effects of fructose to induce fatty liver, hypertriglyceridemia and insulin resistance, however, vary dramatically among individuals. The first step in fructose metabolism is mediated by fructokinase (KHK), which phosphorylates fructose to fructose-1-phosphate; intracellular uric acid is also generated as a consequence of the transient ATP depletion that occurs during this reaction. Here we show in human hepatocytes that uric acid up-regulates KHK expression thus leading to the amplification of the lipogenic effects of fructose. Inhibition of uric acid production markedly blocked fructose-induced triglyceride accumulation in hepatocytes in vitro and in vivo. The mechanism whereby uric acid stimulates KHK expression involves the activation of the transcription factor ChREBP, which, in turn, results in the transcriptional activation of KHK by binding to a specific sequence within its promoter. Since subjects sensitive to fructose often develop phenotypes associated with hyperuricemia, uric acid may be an underlying factor in sensitizing hepatocytes to fructose metabolism during the development of fatty liver. Show less

Carbohydrate response element-binding protein (ChREBP) is an insulin-independent, glucose-responsive transcription factor that is expressed at high levels in liver hepatocytes where it plays a critical role in converting excess carbohydrates to fat for storage. In response to fluctuating glucose levels, hepatic ChREBP activity is regulated in large part by nucleocytoplasmic shuttling of ChREBP protein via interactions with 14-3-3 proteins. The N-terminal ChREBP regulatory region is necessary and sufficient for glucose-responsive ChREBP nuclear import and export. Here, we report the crystal structure of a complex of 14-3-3β bound to the N-terminal regulatory region of ChREBP at 2.4 Å resolution. The crystal structure revealed that the α2 helix of ChREBP (residues 117-137) adopts a well defined α-helical conformation and binds 14-3-3 in a phosphorylation-independent manner that is different from all previously characterized 14-3-3 and target protein-binding modes. ChREBP α2 interacts with 14-3-3 through both electrostatic and van der Waals interactions, and the binding is partially mediated by a free sulfate or phosphate. Structure-based mutagenesis and binding assays indicated that disrupting the observed 14-3-3 and ChREBP α2 interface resulted in a loss of complex formation, thus validating the novel protein interaction mode in the 14-3-3β·ChREBP α2 complex. Show less

In liver, glucose utilization and lipid synthesis are inextricably intertwined. When glucose availability exceeds its utilization, lipogenesis increases, leading to increased intrahepatic lipid content and lipoprotein secretion. Although the fate of three-carbon metabolites is largely determined by flux rate through the relevant enzymes, insulin plays a permissive role in this process. But the mechanism integrating insulin receptor signaling to glucose utilization with lipogenesis is unknown. Forkhead box O1 (FoxO1), a downstream effector of insulin signaling, plays a central role in hepatic glucose metabolism through the regulation of hepatic glucose production. In this study, we investigated the mechanism by which FoxO1 integrates hepatic glucose utilization with lipid synthesis. We show that FoxO1 overexpression in hepatocytes reduces activity of carbohydrate response element binding protein (Chrebp), a key regulator of lipogenesis, by suppressing O-linked glycosylation and reducing the protein stability. FoxO1 inhibits high glucose- or O-GlcNAc transferase (OGT)-induced liver-pyruvate kinase (L-PK) promoter activity by decreasing Chrebp recruitment to the L-PK promoter. Conversely, FoxO1 ablation in liver leads to the enhanced O-glycosylation and increased protein level of Chrebp owing to decreased its ubiquitination. We propose that FoxO1 regulation of Chrebp O-glycosylation is a mechanism linking hepatic glucose utilization with lipid synthesis. Show less

Nonalcoholic fatty liver disease (NAFLD) is associated with all features of the metabolic syndrome. Although deposition of excess triglycerides within liver cells, a hallmark of NAFLD, is associated with a loss of insulin sensitivity, it is not clear which cellular abnormality arises first. We have explored this in mice overexpressing carbohydrate responsive element-binding protein (ChREBP). On a standard diet, mice overexpressing ChREBP remained insulin sensitive, despite increased expression of genes involved in lipogenesis/fatty acid esterification and resultant hepatic steatosis (simple fatty liver). Lipidomic analysis revealed that the steatosis was associated with increased accumulation of monounsaturated fatty acids (MUFAs). In primary cultures of mouse hepatocytes, ChREBP overexpression induced expression of stearoyl-CoA desaturase 1 (Scd1), the enzyme responsible for the conversion of saturated fatty acids (SFAs) into MUFAs. SFA impairment of insulin-responsive Akt phosphorylation was therefore rescued by the elevation of Scd1 levels upon ChREBP overexpression, whereas pharmacological or shRNA-mediated reduction of Scd1 activity decreased the beneficial effect of ChREBP on Akt phosphorylation. Importantly, ChREBP-overexpressing mice fed a high-fat diet showed normal insulin levels and improved insulin signaling and glucose tolerance compared with controls, despite having greater hepatic steatosis. Finally, ChREBP expression in liver biopsies from patients with nonalcoholic steatohepatitis was increased when steatosis was greater than 50% and decreased in the presence of severe insulin resistance. Together, these results demonstrate that increased ChREBP can dissociate hepatic steatosis from insulin resistance, with beneficial effects on both glucose and lipid metabolism. Show less

Differences in the interindividual response to dietary intervention could be modified by genetic variation in nutrient-sensitive genes. This study examined single nucleotide polymorphisms (SNPs) in presumed nutrient-sensitive candidate genes for obesity and obesity-related diseases for main and dietary interaction effects on weight, waist circumference, and fat mass regain over 6 mo. In total, 742 participants who had lost ≥ 8% of their initial body weight were randomly assigned to follow 1 of 5 different ad libitum diets with different glycemic indexes and contents of dietary protein. The SNP main and SNP-diet interaction effects were analyzed by using linear regression models, corrected for multiple testing by using Bonferroni correction and evaluated by using quantile-quantile (Q-Q) plots. After correction for multiple testing, none of the SNPs were significantly associated with weight, waist circumference, or fat mass regain. Q-Q plots showed that ALOX5AP rs4769873 showed a higher observed than predicted P value for the association with less waist circumference regain over 6 mo (-3.1 cm/allele; 95% CI: -4.6, -1.6; P/Bonferroni-corrected P = 0.000039/0.076), independently of diet. Additional associations were identified by using Q-Q plots for SNPs in ALOX5AP, TNF, and KCNJ11 for main effects; in LPL and TUB for glycemic index interaction effects on waist circumference regain; in GHRL, CCK, MLXIPL, and LEPR on weight; in PPARC1A, PCK2, ALOX5AP, PYY, and ADRB3 on waist circumference; and in PPARD, FABP1, PLAUR, and LPIN1 on fat mass regain for dietary protein interaction. The observed effects of SNP-diet interactions on weight, waist, and fat mass regain suggest that genetic variation in nutrient-sensitive genes can modify the response to diet. This trial was registered at clinicaltrials.gov as NCT00390637. Show less

The GTPase ADP-ribosylation factor-related protein 1 (ARFRP1) is located at the trans-Golgi compartment and regulates the recruitment of Arf-like 1 (ARL1) and its effector golgin-245 to this compartment. Here, we show that liver-specific knockout of Arfrp1 in the mouse (Arfrp1(liv-/-)) resulted in early growth retardation, which was associated with reduced hepatic insulin-like growth factor 1 (IGF1) secretion. Accordingly, suppression of Arfrp1 in primary hepatocytes resulted in a significant reduction of IGF1 release. However, the hepatic secretion of IGF-binding protein 2 (IGFBP2) was not affected in the absence of ARFRP1. In addition, Arfrp1(liv-/-) mice exhibited decreased glucose transport into the liver, leading to a 50% reduction of glycogen stores as well as a marked retardation of glycogen storage after fasting and refeeding. These abnormalities in glucose metabolism were attributable to reduced protein levels and intracellular retention of the glucose transporter GLUT2 in Arfrp1(liv-/-) livers. As a consequence of impaired glucose uptake into the liver, the expression levels of carbohydrate response element binding protein (ChREBP), a transcription factor regulated by glucose concentration, and its target genes (glucokinase and pyruvate kinase) were markedly reduced. Our data indicate that ARFRP1 in the liver is involved in the regulation of IGF1 secretion and GLUT2 sorting and is thereby essential for normal growth and glycogen storage. Show less

Adipogenesis is a continuous process even in adult adipose tissue for the presence of preadipocytes that, when subjected to appropriate stimuli can proliferate and differentiate. ChREBP, the essential transcription factor for lipogenesis, is expressed in all tissues, but mainly in lipogenic organs. In this study, we focused on ChREBP expression during preadipocytes differentiation. Since it was found that cyanidin-3 reduces body weight in mice even in the presence of a high-fat diet, by decreasing levels of blood glucose and by improving insulin sensitivity, we studied the effect of this substance on adipogenic differentiation. For this purpose we used preadipocytes obtained from subcutaneous and visceral human adipose explant tissue, characterized and stimulated to differentiate in selective media. On cytofluorimetric analysis these cells showed mesenchymal markers (CD29, CD90, CD44), whereas they were negative for hematopoietic markers (CD45, CD10, CD117,CD31). ChREBP expression levels were quantified by immunoelectron-microscopy and western blotting analysis. In this report we show that ChREBP is expressed in preadipocytes (both nuclear and cytoplasmic compartments); the cytoplasmic level of ChREBP increased by 50 percent on day seven of differentiation into mature adipocytes. Cyanidin reduced differentiation by 20 percent (as evaluated by red oil O staining) and the expression of ChREBP. In addition, cyanidin-treated cells showed abnormal morphology, a square shape with irregular size, probably due to the fact that cyanidin may interfere with the extracellular matrix. These findings suggest that dietary cyanidin, may have inhibitory effects on adipogenesis. Show less

Liver receptor homolog 1 (LRH-1), an established regulator of cholesterol and bile acid homeostasis, has recently emerged as a potential drug target for liver disease. Although LRH-1 activation may protect the liver against diet-induced steatosis and insulin resistance, little is known about how LRH-1 controls hepatic glucose and fatty acid metabolism under physiological conditions. We therefore assessed the role of LRH-1 in hepatic intermediary metabolism. In mice with conditional deletion of Lrh1 in liver, analysis of hepatic glucose fluxes revealed reduced glucokinase (GCK) and glycogen synthase fluxes as compared with those of wild-type littermates. These changes were attributed to direct transcriptional regulation of Gck by LRH-1. Impaired glucokinase-mediated glucose phosphorylation in LRH-1-deficient livers was also associated with reduced glycogen synthesis, glycolysis, and de novo lipogenesis in response to acute and prolonged glucose exposure. Accordingly, hepatic carbohydrate response element-binding protein activity was reduced in these animals. Cumulatively, these data identify LRH-1 as a key regulatory component of the hepatic glucose-sensing system required for proper integration of postprandial glucose and lipid metabolism. Show less

Pancreatic cell development is a tightly controlled process. Although information is available regarding the mesodermal signals that control pancreatic development, little is known about the role of environmental factors such as nutrients, including glucose, on pancreatic development. We previously showed that glucose and its metabolism through the hexosamine biosynthesis pathway (HBP) promote pancreatic endocrine cell differentiation. Here, we analysed the role of the transcription factor carbohydrate-responsive element-binding protein (ChREBP) in this process. This transcription factor is activated by glucose, and has been recently described as a target of the HBP. We used an in vitro bioassay in which pancreatic endocrine and exocrine cells develop from rat embryonic pancreas in a way that mimics in vivo pancreatic development. Using this model, gain-of-function and loss-of-function experiments were undertaken. ChREBP was produced in the endocrine lineage during pancreatic development, its abundance increasing with differentiation. When rat embryonic pancreases were cultured in the presence of glucose or xylitol, the production of ChREBP targets was induced. Concomitantly, beta cell differentiation was enhanced. On the other hand, when embryonic pancreases were cultured with inhibitors decreasing ChREBP activity or an adenovirus producing a dominant-negative ChREBP, beta cell differentiation was reduced, indicating that ChREBP activity was necessary for proper beta cell differentiation. Interestingly, adenovirus producing a dominant-negative ChREBP also reduced the positive effect of N-acetylglucosamine, a substrate of the HBP acting on beta cell differentiation. Our work supports the idea that glucose, through the transcription factor ChREBP, controls beta cell differentiation from pancreatic progenitors. Show less

Intraductal papillary mucinous neoplasm (IPMN) is a common pancreatic cystic neoplasm that is often invasive and metastatic, resulting in a poor prognosis. Few molecular alterations unique to IPMN are known. We performed whole-exome sequencing for a primary IPMN tissue, which uncovered somatic mutations in KCNF1, DYNC1H1, PGCP, STAB1, PTPRM, PRPF8, RNASE3, SPHKAP, MLXIPL, VPS13C, PRCC, GNAS, KRAS, RBM10, RNF43, DOCK2, and CENPF. We further analyzed GNAS mutations in archival cases of 118 IPMNs and 32 pancreatic ductal adenocarcinomas (PDAs), which revealed that 48 (40.7%) of the 118 IPMNs but none of the 32 PDAs harbored GNAS mutations. G-protein alpha-subunit encoded by GNAS and its downstream targets, phosphorylated substrates of protein kinase A, were evidently expressed in IPMN; the latter was associated with neoplastic grade. These results indicate that GNAS mutations are common and specific for IPMN, and activation of G-protein signaling appears to play a pivotal role in IPMN. Show less

Thioredoxin-interacting protein (TXNIP) regulates critical biological processes including inflammation, stress and apoptosis. TXNIP is upregulated by glucose and is a critical mediator of hyperglycemia-induced beta-cell apoptosis in diabetes. In contrast, the saturated long-chain fatty acid palmitate, although toxic to the beta-cell, inhibits TXNIP expression. The mechanisms involved in the opposing effects of glucose and fatty acids on TXNIP expression are unknown. We found that both palmitate and oleate inhibited TXNIP in a rat beta-cell line and islets. Palmitate inhibition of TXNIP was independent of fatty acid beta-oxidation or esterification. AMP-activated protein kinase (AMPK) has an important role in cellular energy sensing and control of metabolic homeostasis; therefore we investigated its involvement in nutrient regulation of TXNIP. As expected, glucose inhibited whereas palmitate stimulated AMPK. Pharmacologic activators of AMPK mimicked fatty acids by inhibiting TXNIP. AMPK knockdown increased TXNIP expression in presence of high glucose with and without palmitate, indicating that nutrient (glucose and fatty acids) effects on TXNIP are mediated in part via modulation of AMPK activity. TXNIP is transcriptionally regulated by carbohydrate response element-binding protein (ChREBP). Palmitate inhibited glucose-stimulated ChREBP nuclear entry and recruitment to the Txnip promoter, thereby inhibiting Txnip transcription. We conclude that AMPK is an important regulator of Txnip transcription via modulation of ChREBP activity. The divergent effects of glucose and fatty acids on TXNIP expression result in part from their opposing effects on AMPK activity. In light of the important role of TXNIP in beta-cell apoptosis, its inhibition by fatty acids can be regarded as an adaptive/protective response to glucolipotoxicity. The finding that AMPK mediates nutrient regulation of TXNIP may have important implications for the pathophysiology and treatment of diabetes. Show less

Objectives were to determine adipose tissue mRNA expression of peroxisome proliferator-activated receptor (PPAR)γ co-regulators, target enzymes and transcription regulators, inflammation-related genes, and adipokines in response to dietary long-chain fatty acids (LCFA). From -21 through 10 d relative to parturition cows were fed no supplemental LCFA (control), saturated LCFA (SFAT; mainly 16:0 and 18:0), or fish oil (FO). Lipid was fed at 250 g/d prepartum or approximately 1.5 to 1.9% of the previous day's dry matter intake postpartum. Transcript profiling of 35 genes via quantitative PCR was conducted on biopsies (n=5 cows/diet) collected at -14 and 11 d from parturition. Despite lower dry matter intake with FO, pre- and postpartal blood nonesterified fatty acids, β-hydroxybutyrate, and liver triacylglycerol were unaffected by treatment but increased after calving regardless of diet. Prepartal expression of adipogenic/lipogenic transcription regulators [CEBPA, CEBPB, RXRA, KLF5, and MLXIPL (formerly ChREBP)] and co-regulators (CARM1, EP300, NCOA1, MED1, NCOR2, and NRIP1) was upregulated by FO and SFAT versus control, whereas most enzymes involved in lipogenesis/triacylglycerol synthesis (FASN, SCD, DGAT2, and LPIN1) had greater expression only with FO. Expression of most adipogenic/lipogenic genes decreased after parturition, but feeding SFAT led to sustained upregulation of CEBPA, CEBPB, RXRA, several PPAR-co-activators, and DGAT2 and SCD, suggesting maintenance of a pro-adipogenic/pro-lipogenic state with SFAT. The co-activator CREBBP was greater in cows fed lipid and did not decrease after parturition, suggesting ligand activation of PPARγ. The greater peripartal expression of NFKB1 and TBK1 due to dietary lipid was suggestive of a local inflammatory response. At amounts fed prepartum, both FO and SFAT were effective in upregulating the adipose tissue PPARγ-gene network. In contrast, only SFAT led to sustaining that response. Overall, the observed expression patterns are suggestive of an adipogenic regulatory mechanism particularly responsive to SFAT. Show less

MLXIPL is a transcription factor integral to the regulation of glycolysis and lipogenesis in the liver. Common variants of the MLXIPL gene (MLXIPL) are known to influence plasma triglyceride levels in people of European descent. As MLXIPL has a key role in energy storage, genetic variations of the MLXIPL may be relevant to physiological adaptations to nutritional stresses that have occurred during the evolution of modern humans. In the present study, we assessed the phenotypic consequences of the Q241H variant of MLXIPL in populations of Asian and Oceanian origin and also surveyed the prevalence of Q241H variant in populations worldwide. Multiple linear regression models based on 2373 individuals of Asian origin showed that the H allele was significantly associated with decreased concentrations of plasma triglycerides (P=0.0003). Direct genotyping of 1455 individuals from Africa, Asia and Oceania showed that the triglyceride-lowering H allele was found at quite low frequencies (0.00-0.16) in most of the populations examined. The exceptions were some Central Asian populations, including Mongolians, Tibetans and Uyghurs, which exhibited much higher frequencies of the H allele (0.21-0.26). The high prevalence of the H allele in Central Asia implies that the Q241H variant of MLXIPL might have been significant for utilization of carbohydrates and fats in the common ancestors of these populations, who successfully adapted to the environment of Central Asia by relying on nomadic livestock herding. Show less

Krüppel-like factor (KLF)-10, is a circadian transcriptional regulator, which links the molecular clock to energy metabolism in the liver. Recently, it was reported that Klf-10 expression is induced by glucose stimulation in mouse hepatocytes. We previously reported that carbohydrate response element-binding protein (ChREBP) plays an important role in the regulation of hepatic lipogenic gene expression. Here, we investigate whether ChREBP, a glucose-activated transcription factor, directly regulates Klf-10 mRNA expression in rat primary hepatocytes. We found that both glucose stimulation and adenoviral overexpression of ChREBP induce Klf-10 mRNA expression in rat primary hepatocytes in a dose-dependent manner. Conversely, overexpression of dominant-negative Max-like protein inhibits glucose-induction expression of Klf-10 mRNA. Deletion analysis using rat Klf-10 promoter in the pGL3 vector combined with a chromatin immunoprecipitation assay against the anti-ChREBP antibody demonstrated that the carbohydrate response element is located between -125 bp and -109 bp in the rat Klf-10 promoter. Conversely, adenoviral overexpression of KLF-10 partly inhibits glucose induction of ChREBP target genes in primary hepatocytes. In conclusion, these data suggest that crosstalk between ChREBP and KLF-10 is involved in the regulation of the lipogenic pathway. Show less

The nicotinamide adenine dinucleotide (NAD(+))-dependent deacetylase SIRT1 is a major metabolic regulator activated by energy stresses such as fasting or calorie restriction. SIRT1 activation during fasting not only relies on the increase in the NAD(+)/NADH ratio caused by energy deprivation but also involves an upregulation of SIRT1 mRNA and protein levels in various metabolic tissues. We demonstrate that SIRT1 expression is controlled systemically by the activation of the cyclic AMP response-element-binding protein upon low nutrient availability. Conversely, in the absence of energetic stress, the carbohydrate response-element-binding protein represses the expression of SIRT1. Altogether, these results demonstrate that SIRT1 expression is tightly controlled at the transcriptional level by nutrient availability and further underscore that SIRT1 is a crucial metabolic checkpoint connecting the energetic status with transcriptional programmes. Show less