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mRNA is thought to predominantly reside in the cytoplasm, where it is translated and eventually degraded. Although nuclear retention of mRNA has a regulatory potential, it is considered extremely rare in mammals. Here, to explore the extent of mRNA retention in metabolic tissues, we combine deep sequencing of nuclear and cytoplasmic RNA fractions with single-molecule transcript imaging in mouse beta cells, liver, and gut. We identify a wide range of protein-coding genes for which the levels of spliced polyadenylated mRNA are higher in the nucleus than in the cytoplasm. These include genes such as the transcription factor ChREBP, Nlrp6, Glucokinase, and Glucagon receptor. We demonstrate that nuclear retention of mRNA can efficiently buffer cytoplasmic transcript levels from noise that emanates from transcriptional bursts. Our study challenges the view that transcripts predominantly reside in the cytoplasm and reveals a role of the nucleus in dampening gene expression noise. Show less

Mammals are composed of hundreds of different cell types with specialized functions. Each of these cellular phenotypes are controlled by different combinations of transcription factors. Using a human non islet cell insulinoma cell line (TC-YIK) which expresses insulin and the majority of known pancreatic beta cell specific genes as an example, we describe a general approach to identify key cell-type-specific transcription factors (TFs) and their direct and indirect targets. By ranking all human TFs by their level of enriched expression in TC-YIK relative to a broad collection of samples (FANTOM5), we confirmed known key regulators of pancreatic function and development. Systematic siRNA mediated perturbation of these TFs followed by qRT-PCR revealed their interconnections with NEUROD1 at the top of the regulation hierarchy and its depletion drastically reducing insulin levels. For 15 of the TF knock-downs (KD), we then used Cap Analysis of Gene Expression (CAGE) to identify thousands of their targets genome-wide (KD-CAGE). The data confirm NEUROD1 as a key positive regulator in the transcriptional regulatory network (TRN), and ISL1, and PROX1 as antagonists. As a complimentary approach we used ChIP-seq on four of these factors to identify NEUROD1, LMX1A, PAX6, and RFX6 binding sites in the human genome. Examining the overlap between genes perturbed in the KD-CAGE experiments and genes with a ChIP-seq peak within 50 kb of their promoter, we identified direct transcriptional targets of these TFs. Integration of KD-CAGE and ChIP-seq data shows that both NEUROD1 and LMX1A work as the main transcriptional activators. In the core TRN (i.e., TF-TF only), NEUROD1 directly transcriptionally activates the pancreatic TFs HSF4, INSM1, MLXIPL, MYT1, NKX6-3, ONECUT2, PAX4, PROX1, RFX6, ST18, DACH1, and SHOX2, while LMX1A directly transcriptionally activates DACH1, SHOX2, PAX6, and PDX1. Analysis of these complementary datasets suggests the need for caution in interpreting ChIP-seq datasets. (1) A large fraction of binding sites are at distal enhancer sites and cannot be directly associated to their targets, without chromatin conformation data. (2) Many peaks may be non-functional: even when there is a peak at a promoter, the expression of the gene may not be affected in the matching perturbation experiment. Show less

Metabolic stress and changes in nutrient levels modulate many aspects of skeletal muscle function during aging and disease. Growth factors and cytokines secreted by skeletal muscle, known as myokines, are important signaling factors, but it is largely unknown whether they modulate muscle growth and differentiation in response to nutrients. Here, we found that changes in glucose levels increase the activity of the glucose-responsive transcription factor MLX (Max-like protein X), which promotes and is necessary for myoblast fusion. MLX promotes myogenesis not via an adjustment of glucose metabolism but rather by inducing the expression of several myokines, including insulin-like growth factor 2 (IGF2), whereas RNAi and dominant-negative MLX reduce IGF2 expression and block myogenesis. This phenotype is rescued by conditioned medium from control muscle cells and by recombinant IGF2, which activates the myogenic kinase Akt. Importantly, MLX-null mice display decreased IGF2 induction and diminished muscle regeneration in response to injury, indicating that the myogenic function of MLX is manifested in vivo. Thus, glucose is a signaling molecule that regulates myogenesis and muscle regeneration via MLX/IGF2/Akt signaling. Show less

Since glucose is the principal energy source for most cells, many organisms have evolved numerous and sophisticated mechanisms to sense glucose and respond to it appropriately. In this context, cloning of the carbohydrate responsive element binding protein has unraveled a critical molecular link between glucose metabolism and transcriptional reprogramming induced by glucose. In this review, we detail major findings that have advanced our knowledge of glucose sensing. Show less

Fatty acid and fat synthesis in the liver is a highly regulated metabolic pathway that is important for very low-density lipoprotein (VLDL) production and thus energy distribution to other tissues. Having common features at their promoter regions, lipogenic genes are coordinately regulated at the transcriptional level. Transcription factors, such as upstream stimulatory factors (USFs), sterol regulatory element-binding protein 1C (SREBP1C), liver X receptors (LXRs) and carbohydrate-responsive element-binding protein (ChREBP) have crucial roles in this process. Recently, insights have been gained into the signalling pathways that regulate these transcription factors. After feeding, high blood glucose and insulin levels activate lipogenic genes through several pathways, including the DNA-dependent protein kinase (DNA-PK), atypical protein kinase C (aPKC) and AKT-mTOR pathways. These pathways control the post-translational modifications of transcription factors and co-regulators, such as phosphorylation, acetylation or ubiquitylation, that affect their function, stability and/or localization. Dysregulation of lipogenesis can contribute to hepatosteatosis, which is associated with obesity and insulin resistance. Show less

Enhanced glucose utilization can be visualized in atherosclerotic lesions and may reflect a high glycolytic rate in lesional macrophages, but its causative role in plaque progression remains unclear. We observe that the activity of the carbohydrate-responsive element binding protein ChREBP is rapidly downregulated upon TLR4 activation in macrophages. ChREBP inactivation refocuses cellular metabolism to a high redox state favoring enhanced inflammatory responses after TLR4 activation and increased cell death after TLR4 activation or oxidized LDL loading. Targeted deletion of ChREBP in bone marrow cells resulted in accelerated atherosclerosis progression in Ldlr(-/-) mice with increased monocytosis, lesional macrophage accumulation, and plaque necrosis. Thus, ChREBP-dependent macrophage metabolic reprogramming hinders plaque progression and establishes a causative role for leukocyte glucose metabolism in atherosclerosis. Show less

Carbohydrate-responsive element-binding protein (ChREBP) is a glucose-sensing transcription factor required for glucose-stimulated proliferation of pancreatic β-cells in rodents and humans. The full-length isoform (ChREBPα) has a low glucose inhibitory domain (LID) that restrains the transactivation domain when glucose catabolism is minimal. A novel isoform of ChREBP (ChREBPβ) was recently described that lacks the LID domain and is therefore constitutively and more potently active. ChREBPβ has not been described in β-cells nor has its role in glucose-stimulated proliferation been determined. We found that ChREBPβ is highly expressed in response to glucose, particularly with prolonged culture in hyperglycemic conditions. In addition, small interfering RNAs that knocked down ChREBPβ transcripts without affecting ChREBPα expression or activity decreased glucose-stimulated expression of carbohydrate response element-containing genes and glucose-stimulated proliferation in INS-1 cells and in isolated rat islets. Quantitative chromatin immunoprecipitation, electrophoretic mobility shift assays, and luciferase reporter assays were used to demonstrate that ChREBP binds to a newly identified powerful carbohydrate response element in β-cells and hepatocytes, distinct from that in differentiated 3T3-L1 adipocytes. We conclude that ChREBPβ contributes to glucose-stimulated gene expression and proliferation in β-cells, with recruitment of ChREBPα to tissue-specific elements of the ChREBPβ isoform promoter. Show less

To assess potential effects of variants in six lipid modulating genes (SORT1, HMGCR, MLXIPL, FADS2, APOE and MAFB) on early development of dyslipidemia independent of the degree of obesity in children, we investigated their association with total (TC), low density lipoprotein (LDL-C), high density lipoprotein (HDL-C) cholesterol and triglyceride (TG) levels in 594 children. Furthermore, we evaluated the expression profile of the candidate genes during human adipocyte differentiation. Expression of selected genes increased 10(1) to >10(4) fold during human adipocyte differentiation, suggesting a potential link with adipogenesis. In genetic association studies adjusted for age, BMI SDS and sex, we identified significant associations for rs599839 near SORT1 with TC and LDL-C and for rs4420638 near APOE with TC and LDL-C. We performed Bayesian modelling of the combined lipid phenotype of HDL-C, LDL-C and TG to identify potentially causal polygenic effects on this multi-dimensional phenotype and considering obesity, age and sex as a-priori modulating factors. This analysis confirmed that rs599839 and rs4420638 affect LDL-C. We show that lipid modulating genes are dynamically regulated during adipogenesis and that variants near SORT1 and APOE influence lipid levels independent of obesity in children. Bayesian modelling suggests causal effects of these variants. Show less

PANcreatic-DERived factor (PANDER, FAM3B) has been shown to regulate glycemic levels via interactions with both pancreatic islets and the liver. Although PANDER is predominantly expressed from the endocrine pancreas, recent work has provided sufficient evidence that the liver may also be an additional tissue source of PANDER production. At physiological levels, PANDER is capable of disrupting insulin signaling and promoting increased hepatic glucose production. As shown in some animal models, strong expression of PANDER, induced by viral delivery within the liver, induces hepatic steatosis. However, no studies to date have explicitly characterized the transcriptional regulation of PANDER from the liver. Therefore, our investigation elucidated the nutrient and hormonal regulation of the hepatic PANDER promoter. Initial RNA-ligated rapid amplification of cDNA ends identified a novel transcription start site (TSS) approximately 26 bp upstream of the PANDER translational start codon not previously revealed in pancreatic β-cell lines. Western evaluation of various murine tissues demonstrated robust expression in the liver and brain. Promoter analysis identified strong tissue-specific activity of the PANDER promoter in both human and murine liver-derived cell lines. The minimal element responsible for maximal promoter activity within hepatic cell lines was located between -293 and -3 of the identified TSS. PANDER promoter activity was inhibited by both insulin and palmitate, whereas glucose strongly increased expression. The minimal element was responsible for maximal glucose-responsive and basal activity. Co-transfection reporter assays, chromatin-immunoprecipitation (ChIP) and site-directed mutagenesis revealed that the carbohydrate-responsive element binding protein (ChREBP) increased PANDER promoter activity and interacted with the PANDER promoter. E-box 3 was shown to be critical for basal and glucose responsive expression. In summary, in-vitro and in-vivo glucose is a potent stimulator of the PANDER promoter within the liver and this response may be facilitated by ChREBP. Show less

Salsalate (salicylsalicylic acid) is an anti-inflammatory drug that was recently found to exert beneficial metabolic effects on glucose and lipid metabolism. Although its utility in the prevention and management of a wide range of vascular disorders, including type 2 diabetes and metabolic syndrome has been suggested before, the potential of salsalate to protect against non-alcoholic steatohepatitis (NASH) remains unclear. The aim of the present study was therefore to ascertain the effects of salsalate on the development of NASH. Transgenic APOE*3Leiden.CETP mice were fed a high-fat and high-cholesterol diet with or without salsalate for 12 and 20 weeks. The effects on body weight, plasma biochemical variables, liver histology and hepatic gene expression were assessed. Salsalate prevented weight gain, improved dyslipidemia and insulin resistance and ameliorated diet-induced NASH, as shown by decreased hepatic microvesicular and macrovesicular steatosis, reduced hepatic inflammation and reduced development of fibrosis. Salsalate affected lipid metabolism by increasing β-oxidation and decreasing lipogenesis, as shown by the activation of PPAR-α, PPAR-γ co-activator 1β, RXR-α and inhibition of genes controlled by the transcription factor MLXIPL/ChREBP. Inflammation was reduced by down-regulation of the NF-κB pathway, and fibrosis development was prevented by down-regulation of TGF-β signalling. Salsalate exerted a preventive effect on the development of NASH and progression to fibrosis. These data suggest a clinical application of salsalate in preventing NASH. Show less

It is suggested that C771G (His241Gln) polymorphism of MLXIPL gene might be a genetic risk factor for coronary artery disease (CAD); therefore, the aim of the present study was to investigate the association between C771G polymorphism of MLXIPL gene and the pathogenesis of CAD in Iranian patients with coronary artery stenosis and control subjects. Two hundred and five patients with coronary artery stenosis and 195 healthy control subjects were included in this study. MLXIPL genotypes were determined by polymerase chain reaction and restriction fragment length polymorphism (RFLP). There was an association between the MLXIPL polymorphism and quantitative lipid traits in patient group. Distribution of the CC genotype of MLXIPL was more frequent in patients, (χ2=5.13; p<0.005) and after adjustment for classical CAD risk factors, the MLXIPL CC genotype was independently associated with CAD (OR=1.98, 95% CI, 1.12-4.11; p=0.02). Distribution of MLXIPL genotypes were significantly different as compared with the severity of stenosis (χ2=6.34; p<0.05). These results suggest that C771G polymorphism of MLXIPL gene is associated with stenosis and its severity. Show less

Carbohydrate response element binding protein (ChREBP) regulates cellular glucose and lipid homeostasis. Although ChREBP is highly expressed in many key metabolic tissues, the role of ChREBP in most of those tissues and the consequent effects on whole-body glucose and lipid metabolism are not well understood. Therefore, we generated a transgenic mouse that overexpresses a constitutively active ChREBP isoform under the control of the fatty acid binding protein 4-Cre-driven promoter (FaChOX). Weight gain was blunted in male, but not female, FaChOX mice when placed on either a normal chow diet or an obesogenic Western diet. Respiratory exchange ratios were increased in Western diet-fed FaChOX mice, indicating a shift in whole-body substrate use favoring carbohydrate metabolism. Western diet-fed FaChOX mice showed improved insulin sensitivity and glucose tolerance in comparison with controls. Hepatic triglyceride content was reduced in Western diet-fed FaChOX mice in comparison with controls, suggesting protection from fatty liver. Epididymal adipose tissue exhibited differential expression of genes involved in differentiation, browning, metabolism, lipid homeostasis, and inflammation between Western diet-fed FaChOX mice and controls. Our findings support a role for ChREBP in modulating adipocyte differentiation and adipose tissue metabolism and inflammation as well as consequent risks for obesity and insulin resistance. Show less

Fructose induces nonalcoholic fatty liver disease (NAFLD). Citrulline (Cit) may exert a beneficial effect on steatosis. We compared the effects of Cit and an isonitrogenous mixture of nonessential amino acids (NEAAs) on fructose-induced NAFLD. Twenty-two male Sprague Dawley rats were randomly assigned into 4 groups (n = 4-6) to receive for 8 wk a 60% fructose diet, either alone or supplemented with Cit (1 g · kg(-1) · d(-1)), or an isonitrogenous amount of NEAAs, or the same NEAA-supplemented diet with starch and maltodextrin instead of fructose (controls). Nutritional and metabolic status, liver function, and expression of genes of hepatic lipid metabolism were determined. Compared with controls, fructose led to NAFLD with significantly higher visceral fat mass (128%), lower lean body mass (-7%), insulin resistance (135%), increased plasma triglycerides (TGs; 67%), and altered plasma amino acid concentrations with decreased Arg bioavailability (-27%). This was corrected by both NEAA and Cit supplementation. Fructose caused a 2-fold increase in the gene expression of fatty acid synthase (Fas) and 70% and 90% decreases in that of carnitine palmitoyl-transferase 1a and microsomal TG transfer protein via a nearly 10-fold higher gene expression of sterol regulatory element-binding protein-1c (Srebp1c) and carbohydrate-responsive element-binding protein (Chrebp), and a 90% lower gene expression of peroxisome proliferator-activated receptor α (Ppara). NEAA or Cit supplementation led to a Ppara gene expression similar to controls and decreased those of Srebp1c and Chrebp in the liver by 50-60%. Only Cit led to Fas gene expression and Arg bioavailability similar to controls. In our rat model, Cit and NEAAs effectively prevented fructose-induced NAFLD. On the basis of literature data and our findings, we propose that NEAAs may exert their effects specifically on the liver, whereas Cit presumably acts at both the hepatic and whole-body level, in part via improved peripheral Arg metabolism. Show less

Reduced de novo lipogenesis in adipose tissue, often observed in obese individuals, is thought to contribute to insulin resistance. Besides trapping excess glucose and providing for triglycerides and energy storage, endogenously synthesized lipids can function as potent signaling molecules. Indeed, several specific lipids and their molecular targets that mediate insulin sensitivity have been recently identified. Here, we report that carbohydrate-response element-binding protein (ChREBP), a transcriptional inducer of glucose use and de novo lipogenesis, controls the activity of the adipogenic master regulator peroxisome proliferator-activated receptor (PPAR)γ. Expression of constitutive-active ChREBP in precursor cells activated endogenous PPARγ and promoted adipocyte differentiation. Intriguingly, ChREBP-constitutive-active ChREBP expression induced PPARγ activity in a fatty acid synthase-dependent manner and by trans-activating the PPARγ ligand-binding domain. Reducing endogenous ChREBP activity by either small interfering RNA-mediated depletion, exposure to low-glucose concentrations, or expressing a dominant-negative ChREBP impaired differentiation. In adipocytes, ChREBP regulated the expression of PPARγ target genes, in particular those involved in thermogenesis, similar to synthetic PPARγ ligands. In summary, our data suggest that ChREBP controls the generation of endogenous fatty acid species that activate PPARγ. Thus, increasing ChREBP activity in adipose tissue by therapeutic interventions may promote insulin sensitivity through PPARγ. Show less

Metformin has been considered a potential adjunctive therapy in treating poorly controlled type 1 diabetes with obesity and insulin resistance, owing to its potent effects on improving insulin sensitivity. However, the underlying mechanism of metformin's vascular protective effects remains obscure. Thioredoxin-interacting protein (TXNIP), a key regulator of cellular redox state induced by high-glucose concentration, decreases thioredoxin reductase activity and mediates apoptosis induced by oxidative stress. Here we report that high glucose-induced endothelial dysfunction is associated with induction of TXNIP expression in primary human aortic endothelial cells exposed to high-glucose conditions, whereas the metformin treatment suppresses high-glucose-induced TXNIP expression at mRNA and protein levels. We further show that metformin decreases the high-glucose-stimulated nuclear entry rate of two transcription factors, carbohydrate response element-binding protein (ChREBP) and forkhead box O1 (FOXO1), as well as their recruitment on the TXNIP promoter. An AMP-activated protein kinase inhibitor partially compromised these metformin effects. Our data suggest that endothelial dysfunction resulting from high-glucose concentrations is associated with TXNIP expression. Metformin down-regulates high-glucose-induced TXNIP transcription by inactivating ChREBP and FOXO1 in endothelial cells, partially through AMP-activated protein kinase activation. Show less

Coffee, a major dietary source of caffeine, is among the most widely consumed beverages in the world and has received considerable attention regarding health risks and benefits. We conducted a genome-wide (GW) meta-analysis of predominately regular-type coffee consumption (cups per day) among up to 91,462 coffee consumers of European ancestry with top single-nucleotide polymorphisms (SNPs) followed-up in ~30 062 and 7964 coffee consumers of European and African-American ancestry, respectively. Studies from both stages were combined in a trans-ethnic meta-analysis. Confirmed loci were examined for putative functional and biological relevance. Eight loci, including six novel loci, met GW significance (log10Bayes factor (BF)>5.64) with per-allele effect sizes of 0.03-0.14 cups per day. Six are located in or near genes potentially involved in pharmacokinetics (ABCG2, AHR, POR and CYP1A2) and pharmacodynamics (BDNF and SLC6A4) of caffeine. Two map to GCKR and MLXIPL genes related to metabolic traits but lacking known roles in coffee consumption. Enhancer and promoter histone marks populate the regions of many confirmed loci and several potential regulatory SNPs are highly correlated with the lead SNP of each. SNP alleles near GCKR, MLXIPL, BDNF and CYP1A2 that were associated with higher coffee consumption have previously been associated with smoking initiation, higher adiposity and fasting insulin and glucose but lower blood pressure and favorable lipid, inflammatory and liver enzyme profiles (P<5 × 10(-8)).Our genetic findings among European and African-American adults reinforce the role of caffeine in mediating habitual coffee consumption and may point to molecular mechanisms underlying inter-individual variability in pharmacological and health effects of coffee. Show less

Bile acids are signalling molecules, which activate the transmembrane receptor TGR5 and the nuclear receptor FXR. BA sequestrants (BAS) complex bile acids in the intestinal lumen and decrease intestinal FXR activity. The BAS-BA complex also induces glucagon-like peptide-1 (GLP-1) production by L cells which potentiates β-cell glucose-induced insulin secretion. Whether FXR is expressed in L cells and controls GLP-1 production is unknown. Here, we show that FXR activation in L cells decreases proglucagon expression by interfering with the glucose-responsive factor Carbohydrate-Responsive Element Binding Protein (ChREBP) and GLP-1 secretion by inhibiting glycolysis. In vivo, FXR deficiency increases GLP-1 gene expression and secretion in response to glucose hence improving glucose metabolism. Moreover, treatment of ob/ob mice with the BAS colesevelam increases intestinal proglucagon gene expression and improves glycaemia in a FXR-dependent manner. These findings identify the FXR/GLP-1 pathway as a new mechanism of BA control of glucose metabolism and a pharmacological target for type 2 diabetes. Show less

Glycogen storage disease type-Ia (GSD-Ia) is caused by a lack of glucose-6-phosphatase-α (G6Pase-α or G6PC) activity. We have shown that gene therapy mediated by a recombinant adeno-associated virus (rAAV) vector expressing human G6Pase-α normalizes blood glucose homeostasis in the global G6pc knockout (G6pc(-/-)) mice for 70-90 weeks. The treated G6pc(-/-) mice expressing 3-63% of normal hepatic G6Pase-α activity (AAV mice) produce endogenous hepatic glucose levels 61-68% of wild-type littermates, have a leaner phenotype and exhibit fasting blood insulin levels more typical of young adult mice. We now show that unlike wild-type mice, the lean AAV mice have increased caloric intake and do not develop age-related obesity or insulin resistance. Pathway analysis shows that signaling by hepatic carbohydrate response element binding protein that improves glucose tolerance and insulin signaling is activated in AAV mice. In addition, several longevity factors in the calorie restriction pathway, including the NADH shuttle systems, NAD(+) concentrations and the AMP-activated protein kinase/sirtuin 1/peroxisome proliferator-activated receptor-γ coactivator 1α pathway are upregulated in the livers of AAV mice. The finding that partial restoration of hepatic G6Pase-α activity in GSD-Ia mice not only attenuates the phenotype of hepatic G6Pase-α deficiency but also prevents the development of age-related obesity and insulin resistance seen in wild-type mice may suggest relevance of the G6Pase-α enzyme to obesity and diabetes. Show less

The PREDIMED (PREvención con DIeta MEDiterránea) multicenter, randomized, primary prevention trial assessed the long-term effects of the Mediterranean diet (MeDiet) on clinical events of cardiovascular disease (CVD). We randomized 7447 men and women at high CVD risk into three diets: MeDiet supplemented with extra-virgin olive oil (EVOO), MeDiet supplemented with nuts, and control diet (advice on a low-fat diet). No energy restriction and no special intervention on physical activity were applied. We observed 288 CVD events (a composite of myocardial infarction, stroke or CVD death) during a median time of 4.8years; hazard ratios were 0.70 (95% CI, 0.53-0.91) for the MeDiet+EVOO and 0.70 (CI, 0.53-0.94) for the MeDiet+nuts compared to the control group. Respective hazard ratios for incident diabetes (273 cases) among 3541 non-diabetic participants were 0.60 (0.43-0.85) and 0.82 (0.61-1.10) for MeDiet+EVOO and MeDiet+nuts, respectively versus control. Significant improvements in classical and emerging CVD risk factors also supported a favorable effect of both MeDiets on blood pressure, insulin sensitivity, lipid profiles, lipoprotein particles, inflammation, oxidative stress, and carotid atherosclerosis. In nutrigenomic studies beneficial effects of the intervention with MedDiets showed interactions with several genetic variants (TCF7L2, APOA2, MLXIPL, LPL, FTO, M4CR, COX-2, GCKR and SERPINE1) with respect to intermediate and final phenotypes. Thus, the PREDIMED trial provided strong evidence that a vegetable-based MeDiet rich in unsaturated fat and polyphenols can be a sustainable and ideal model for CVD prevention. Show less

Although the expression of hepatic lipogenic genes is enhanced in insulin resistance, the underlying mechanism is unclear. To reveal the details, the aim of this study was to investigate whether the expression of hepatic lipogenic genes are mediated by epigenetic regulation and specific transcription factors in an insulin resistance model of rats. Using a rat model of insulin resistance (SHR/NDmc-cp), we investigated the relationship between hepatic expression of the lipogenic gene fatty-acid synthase (Fasn), binding of the transcription factor carbohydrate-responsive element-binding protein (ChREBP) to the Fasn gene, and histone modifications in the region of the Fasn gene by real-time reverse transcriptase polymerase chain reaction, immunoblotting, and chromatin immunoprecipitation assay. Compared with control rats, Fasn mRNA expression and protein levels were higher in the livers of SHR/NDmc-cp rats, as were protein expression levels and Fasn binding of ChREBP and RNA polymerase II. Moreover, compared with the livers of control rats, levels of mono-methylated histone H3 lysine (K) 4 and acetylated histone H4 were higher in the promoter/enhancer region of the Fasn gene in the livers of SHR/NDmc-cp rats. Levels of trimethylated histone H3K4 and acetylated histone H3 were higher in the transcribed region. The results of this study indicate that expression of the Fasn gene in the livers of insulin-resistant rats is associated with increased H3K4 methylation, increased histone H3 acetylation, and increased H4 acetylation, and also, binding levels of ChREBP to promoter/enhancer region of Fasn gene is involved in the Fasn gene expression caused by hyperglycemia. Show less

We investigated the contributions of dietary fat and dietary carbohydrate to the development of fatty liver induced by western diet (WD). Compared with WD-fed wild type (WT) mice, livers of WD-fed ChREBP(-/-) mice showed lipid droplets of varying sizes around the hepatic lobules, while hepatic triglyceride and cholesterol contents were only modestly decreased. Inflammation and fibrosis were suppressed in ChREBP(-/-) mice. In addition, compared with WD-fed WT mice, ChREBP(-/-) mice showed decreased β-oxidation, ketogenesis and FGF21 production, increased intestinal lipid absorption, and decreased VLDL secretion. These findings suggest that dietary fat and carbohydrate contribute differently to the development of fatty liver. Show less

Although many of the factors and molecules closely associated with non-alcoholic steatohepatitis (NASH) have been reported, the role of inducible nitric oxide synthase (iNOS)-derived nitric oxide (NO) on the progression of NASH remains unclear. We therefore investigated the role of iNOS-derived NO in NASH pathogenesis with a long-term follow-up study using systemic iNOS-knockout mice under high-fat diet (HFD) conditions. iNOS-knockout and wild-type mice were fed a basal or HFD for 10 or 48 weeks. Lipid accumulation, fibrosis, and inflammation were evaluated, and various factors and molecules closely associated with NASH were analyzed. Marked fibrosis and inflammation (indicators of NASH) were observed in the livers of iNOS-knockout mice compared to wild-type mice after 48 weeks of a HFD; however, lipid accumulation in iNOS-knockout mice livers was less than in the wild-type. Increased expressions of various cytokines that are transcriptionally controlled by NF-kB in iNOS-deficient mice livers were observed during HFD conditions. iNOS-derived NO may play a protective role against the progression to NASH during an HFD by preventing fibrosis and inflammation, which are mediated by NF-kB activation in Kupffer cells. A lack of iNOS-derived NO accelerates progression to NASH without excessive lipid accumulation. Show less

Carbohydrate responsive element binding protein (ChREBP) is central for de novo fatty acid synthesis under physiological conditions and in the context of nonalcoholic fatty liver disease. We explored its contribution to alcohol-induced steatosis in a mouse model of binge drinking as acute ethanol (EtOH) intoxication has become an alarming health problem. Within 6 hours, ChREBP acetylation and its recruitment onto target gene promoters were increased in liver of EtOH-fed mice. Acetylation of ChREBP was dependent on alcohol metabolism because inhibition of alcohol dehydrogenase (ADH) activity blunted ChREBP EtOH-induced acetylation in mouse hepatocytes. Transfection of an acetylation-defective mutant of ChREBP (ChREBP(K672A) ) in HepG2 cells impaired the stimulatory effect of EtOH on ChREBP activity. Importantly, ChREBP silencing in the liver of EtOH-fed mice prevented alcohol-induced triglyceride accumulation through an inhibition of the lipogenic pathway but also led, unexpectedly, to hypothermia, increased blood acetaldehyde concentrations, and enhanced lethality. This phenotype was associated with impaired hepatic EtOH metabolism as a consequence of reduced ADH activity. While the expression and activity of the NAD(+) dependent deacetylase sirtuin 1, a ChREBP-negative target, were down-regulated in the liver of alcohol-fed mice, they were restored to control levels upon ChREBP silencing. In turn, ADH acetylation was reduced, suggesting that ChREBP regulates EtOH metabolism and ADH activity through its direct control of sirtuin 1 expression. Indeed, when sirtuin 1 activity was rescued by resveratrol pretreatment in EtOH-treated hepatocytes, a significant decrease in ADH protein content and/or acetylation was observed. our study describes a novel role for ChREBP in EtOH metabolism and unravels its protective effect against severe intoxication in response to binge drinking. Show less

Glucose is an essential nutrient that directly regulates the expression of numerous genes in liver and adipose tissue. The carbohydrate response element-binding protein (ChREBP) links glucose as a signaling molecule to multiple glucose-dependent transcriptional regulatory pathways, particularly genes involved in glycolytic and lipogenic processes. In this study, we used chromatin immunoprecipitation followed by next-generation sequencing to identify specific ChREBP binding targets in liver and white adipose tissue. We found a large number of ChREBP binding sites, which are attributable to 5825 genes in the liver, 2418 genes in white adipose tissue, and 5919 genes in both tissues. The majority of these target genes were involved in known metabolic processes. Pathways in insulin signaling, the adherens junction, and cancers were among the top 5 pathways in both tissues. Motif analysis revealed a consensus sequence CAYGYGnnnnnCRCRTG that was commonly shared by ChREBP binding sites. Putative ChREBP binding sequences were enriched on promoters of genes involved in insulin signaling pathway, insulin resistance, and tumorigenesis. Show less

Liver X receptor (LXR)α and LXRβ play key roles in hepatic de novo lipogenesis through their regulation of lipogenic genes, including sterol regulatory element-binding protein (SREBP)-1c and carbohydrate responsive element-binding protein (ChREBP). LXRs activate lipogenic gene transcription in response to feeding, which is believed to be mediated by insulin. We have previously shown that LXRs are targets for glucose-hexosamine-derived O-linked β-N-acetylglucosamine (O-GlcNAc) modification enhancing their ability to regulate SREBP-1c promoter activity in vitro. To elucidate insulin-independent effects of feeding on LXR-mediated lipogenic gene expression in vivo, we subjected control and streptozotocin-treated LXRα/β(+/+) and LXRα/β(-/-) mice to a fasting-refeeding regime. We show that under hyperglycemic and hypoinsulinemic conditions, LXRs maintain their ability to upregulate the expression of glycolytic and lipogenic enzymes, including glucokinase (GK), SREBP-1c, ChREBPα, and the newly identified shorter isoform ChREBPβ. Furthermore, glucose-dependent increases in LXR/retinoid X receptor-regulated luciferase activity driven by the ChREBPα promoter was mediated, at least in part, by O-GlcNAc transferase (OGT) signaling in Huh7 cells. Moreover, we show that LXR and OGT interact and colocalize in the nucleus and that loss of LXRs profoundly reduced nuclear O-GlcNAc signaling and ChREBPα promoter binding activity in vivo. In summary, our study provides evidence that LXRs act as nutrient and glucose metabolic sensors upstream of ChREBP by modulating GK expression, nuclear O-GlcNAc signaling, and ChREBP expression and activity. Show less

Williams-Beuren syndrome (WBS, OMIM-194050) is a neurodevelopmental disorder with multisystemic manifestations caused by a 1.55-1.83 Mb deletion at 7q11.23 including 26-28 genes. Reported endocrine and metabolic abnormalities include transient hypercalcaemia of infancy, subclinical hypothyroidism in ∼ 30% of children and impaired glucose tolerance in ∼ 75% of adult individuals. The purpose of this study was to further study metabolic alterations in patients with WBS, as well as in several mouse models, to establish potential candidate genes. We analysed several metabolic parameters in a cohort of 154 individuals with WBS (data available from 69 to 151 cases per parameter), as well as in several mouse models with complete and partial deletions of the orthologous WBS locus, and searched for causative genes and potential modifiers. Triglyceride plasma levels were significantly decreased in individuals with WBS while cholesterol levels were slightly decreased compared with controls. Hyperbilirubinemia, mostly unconjugated, was found in 18.3% of WBS cases and correlated with subclinical hypothyroidism and hypotriglyceridemia, suggesting common pathogenic mechanisms. Haploinsufficiency at MLXIPL and increased penetrance for hypomorphic alleles at the UGT1A1 gene promoter might underlie the lipid and bilirubin alterations. Other disturbances included increased protein and iron levels, as well as the known subclinical hypothyroidism and glucose intolerance. Our results show that several unreported biochemical alterations, related to haploinsufficiency for specific genes at 7q11.23, are relatively common in WBS. The early diagnosis, follow-up and management of these metabolic disturbances could prevent long-term complications in this disorder. Show less

Abnormal lipid levels are considered one of the most significant risk factors for atherosclerosis and coronary artery disease, two of the main causes of death worldwide. Apart from monogenic cases of hypercholesterolemia, most of the common dyslipidemias are caused by a number of low-impact polymorphisms. It has recently been reported that frequent polymorphisms at a large number of loci are significantly associated with one or more blood lipid parameters in many populations. Identifying these associations in different populations and estimating the possible interactions between genetic models are necessary to explain the underlying genetic architecture of the associated loci and their ultimate impact on lipid-associated traits. We estimated the association between 144 common single-nucleotide polymorphisms (SNPs) from published genome-wide association studies and the levels of total cholesterol, low- and high-density lipoprotein-cholesterol, and triglycerides in 1273 individuals from the Genome Database of the Latvian Population. We analyzed a panel of 144 common SNPs with Illumina GoldenGate Genotyping Assays on the Illumina BeadXpress System. Ten SNPs at the CETP locus and two at the MLXIPL locus were associated with reduced high-density lipoprotein-cholesterol levels; one SNP at the TOMM40 locus was associated with increased low-density lipoprotein-cholesterol; and four SNPs at the MLXIPL locus were associated with increased log triglyceride levels. There was also a significant correlation between the number of risk alleles and all the lipid parameters, suggesting that the coexistence of many low-impact SNPs has a greater effect on the dyslipidemia phenotype than the individual effects of found SNPs. We conclude that the CETP, MLXIPL, and TOMM40 loci are the strongest genetic factors underlying the variability in lipid traits in our population. Show less

To explore the effects of serum insulin on the expression of ChREBP, ACC and FAS in vivo, KKAy mice which were characterized with high levels of both serum insulin and glucose and DIO mice which were characterized with high serum insulin level alone were utilized, separately. The age-matched C57BL/6J mice fed with standard chow were used as normal control (Con). Expressions of hepatic ChREBP, ACC and FAS were detected by Western blotting. As the results, in KKAy mice, a positive correlation between the levels of serum insulin and glucose (r = 0.902, P < 0.000), as well as between the levels of serum insulin and TG (r = 0.732, P < 0.000), was observed. Meanwhile, the expressions of hepatic ChREBP, ACC and FAS increased significantly and accompanied with its hyperinsulinemia and hyperglycemia, separately. In DIO mice, correlation between the levels of serum insulin and TG (r = 0.722, P < 0.001) also showed positive, and the expressions of hepatic ChREBP, ACC and FAS increased significantly and also accompanied with its hyperinsulinemia. However, their blood glucose values were almost normal. These demonstrated that hyperinsulinemia may cause glycolipid metabolic disorders by up-regulating the expression of ChREBP in vivo. Show less

Allelic variations in gene expression influence many biological responses and cause phenotypic variations in humans. In this study, Illumina Human Exome BeadChips containing more than 240,000 single nucleotide polymorphisms (SNPs) were used to identify changes in allelic gene expression in hepatocellular carcinoma cells following lipopolysaccharide (LPS) stimulation. We found 17 monoallelically expressed genes, 58 allelic imbalanced genes, and 7 genes showing allele substitution. In addition, we also detected 33 differentially expressed genes following LPS treatment in vitro using these human exome SNP chips. However, alterations in allelic gene expression following LPS treatment were detected in only three genes (MLXIPL, TNC, and MX2), which were observed in one cell line sample only, indicating that changes in allelic gene expression following LPS stimulation of liver cells are rare events. Among a total of 75 genes showing allelic expression in hepatocellular carcinoma cells, either monoallelic or imbalanced, 43 genes (57.33%) had expression quantitative trait loci (eQTL) data, indicating that high-density exome SNP chips are useful and reliable for studying allelic gene expression. Furthermore, most genes showing allelic expression were regulated by cis-acting mechanisms and were also significantly associated with several human diseases. Overall, our study provides a better understanding of allele-specific gene expression in hepatocellular carcinoma cells with and without LPS stimulation and potential clues for the cause of human disease due to alterations in allelic gene expression. Show less

To investigate whether alpha-lipoic acid (ALA) could attenuate the insulin resistance and metabolic disorders in high fat diet-fed mice. Male mice were fed a high fat diet (HFD) plus ALA (100 and 200 mg·kg(-1)·d(-1)) or HFD plus a positive control drug metformin (300 mg·kg(-1)·d(-1)) for 24 weeks. During the treatments, the relevant physiological and metabolic parameters of the mice were measured. After the mice were euthanized, blood samples and livers were collected. The expression of proteins and genes related to glucose metabolism in livers were analyzed by immunoblotting and real time-PCR. HFD induced non-alcoholic fatty liver disease (NAFLD) and abnormal physiological and metabolic parameters in the mice, which were dose-dependently attenuated by ALA. ALA also significantly reduced HFD-induced hyperglycemia and insulin resistance in HFD-fed mice. Furthermore, ALA significantly upregulated the glycolytic enzymes GCK, HK-1 and PK, and the glycogen synthesis enzyme GS, and downregulated the gluconeogenic enzymes PEPCK and G6Pase, thus decreased glucose production, and promoted glycogen synthesis and glucose utilization in livers. Moreover, ALA markedly increased PKB/Akt and GSK3β phosphorylation, and nuclear carbohydrate response element binding protein (ChREBP) expression in livers. Metformin produced similar effects as ALA in HFD-fed mice. ALA is able to sustain glucose homeostasis and prevent the development of NAFLD in HFD-fed mice. Show less