ChREBP Research Articles

Carbohydrate Sensing Through the Transcription Factor ChREBP.

Carbohydrate response element binding protein (ChREBP) is a carbohydrate-signaling transcription factor that in the past years has emerged as a central metabolic regulator. ChREBP expression is mostly abundant in active sites of de novo lipogenesis including liver and white and brown adipose tissues. ChREBP is also expressed in pancreatic islets, small intestine and to a lesser extent in the kidney and the brain. In response to glucose, ChREBP undergoes several post-translational modifications (PTMs) (phosphorylation, acetylation and/or O-GlcNAcylation) that will either modulate its cellular location, stability and/or its transcriptional activity. ChREBPβ is a shorter isoform of ChREBP that was first described in adipose tissue and later found to be expressed in other sites including liver and pancreatic β cells. ChREBPβ lacks an important regulatory inhibitory domain, known as LID (low glucose inhibitory domain), in its N-terminal domain and is therefore reported as a highly active isoform. In this review, we recapitulate a recent progress concerning the mechanisms governing the activity of the ChREBP isoforms, including PTMs, partners/cofactors as well as novel metabolic pathways regulated by ChREBP in key metabolic tissues, by discussing phenotypes associated with tissue-specific deletion of ChREBP in knockout mice.

2191-P: PPM1K Regulates ß-Cell Proliferation, Insulin Content, and GSIS

A single nucleotide polymorphism in the gene encoding the protein phosphatase m1K (PPm1K) is associated with increased risk for the development of type 2 diabetes but not insulin resistance in humans. We recently discovered that expression of the PPM1K gene in liver is suppressed by the carbohydrate response element binding protein (ChREBP) which controls transcriptional responses to changes in carbohydrate availability. Taken together these findings suggest that PPM1K might play an important role in the glucose responsive pancreatic β cell; however, little is known about the role of PPm1K in this cell type. Here we studied how changes in glucose concentration affect PPm1K expression in β cells and explored the effect of modulating PPM1K expression on β cell proliferation, insulin content and glucose stimulated insulin secretion. Studies were carried out in INS1-832/13 β cells in culture and key findings were validated in isolated rat islets. In line with our previous observations in liver, we found that increasing glucose concentrations in INS1 cells results in elevated ChREBP activity, measured by expression of the ChREBP β isoform, and lower PPM1K mRNA expression (P<0.01). Treatment of INS-1 cells with adenovirus expressing recombinant human PPM1K for 96h increased β cell proliferation (P<0.05) but robustly lowered GSIS (P<0.001). This effect was due to substantially lower insulin content in the Ad-PPm1K treated cells (P<0.01). Importantly, there was no effect of the control GFP adenovirus compared to non-transfected cells for any of these measures. Moreover, siRNA mediated knock down of PPM1K resulted in increased expression of INS1 and INS2 mRNA suggesting that altered transcription of the insulin gene likely accounts for the effect of PPm1K on β cell insulin content and release. In conclusion, our data clearly demonstrate that PPM1K plays an important role in the regulation of β cell insulin production and proliferation. Disclosure Y. Deleye: None. J. Herring: None. K.H. Hess: None. B.R. Leininger: None. S.A. Hannou: None. I. Astapova: None. M.A. Herman: None. J.S. Tessem: None. P.J. White: None. Funding American Diabetes Association/Pathway to Stop Diabetes (1-16-INI-17 to P.J.W.)

2196-P: HB-EGF Signaling Is Required for Glucose-Induced Pancreatic ß-Cell Proliferation in Rats

Background: Glucose is a major β-cell mitogen. Despite recent progress, the underlying mechanisms remain unclear. In a rat model of nutrient excess we previously showed that nutrient-induced β-cell proliferation is blocked when either EGF receptor (EGFR) or mTOR signaling is inhibited. Parallel transcriptomic analyses identified the EGFR ligand, HB-EGF as a potential mediator of nutrient-induced β-cell proliferation. Objective: To determine the role of HB-EGF in glucose-induced β-cell proliferation. Methods: HB-EGF mRNA levels were assessed by real-time PCR in isolated rat islets following a 24-h exposure to 2.8 or 16.7 mM glucose. The Carbohydrate-Responsive Element-Binding Protein (ChREBP) transcription factor was down-regulated by siRNA in dispersed rat islets. For β-cell proliferation studies islets were exposed to 16.7 mM glucose or HB-EGF (100 ng/ml) in the presence of 2.8 mM glucose for 72 h. Islets were co-cultured with the EGFR inhibitor AG1478 (300 nM) or the HB-EGF inhibitor CRM197 (10 ug/ml). shRNA was used to knockdown HB-EGF in isolated islets and either cultured ex vivo or transplanted under the kidney capsule of glucose-infused rats. β-cell proliferation was assessed by immunohistochemistry for Ki67 and insulin. Results: Glucose increased HB-EGF mRNA levels and this was prevented by ChREBP knockdown. HB-EGF potently stimulated β-cell proliferation. Inhibition of the EGFR or HB-EGF completely blocked not only the proliferative response to HB-EGF but also the response to 16.7 mM glucose. Knockdown of HB-EGF blocked the β-cell proliferative response to glucose in isolated rat islets as well as in transplanted islets. Conclusion: HB-EGF is a potent β-cell mitogen in rat islets. Glucose increases HB-EGF gene expression via ChREBP. The proliferative response to glucose requires an intact HB-EGF - EGFR pathway. Our findings identify a novel player in the complex mechanisms controlling β-cell proliferation in response to glucose. Disclosure H. Maachi: None. D. Scott: None. J. Ghislain: None. V. Poitout: None. Funding National Institutes of Health; Fonds de recherche du Québec-Santé

363-OR: ChREBP-Beta Controls Both Proliferation and Cell Death of Pancreatic Beta Cells

Glucose is a key physiological driver of adaptive beta cell mass expansion, promoting beta cell proliferation both in vitro and in vivo. Carbohydrate response element binding protein β (ChREBPβ) is upregulated in response to glucose and is required for glucose-stimulated beta cell proliferation. To study the role of ChREBPβ in beta cells, we knocked out (KO) or overexpressed (OE) ChREBPβ in mouse pancreatic beta cells using Cre LoxP approaches with either MIP-CreERT (induced by tamoxifen at 3 months of age), or the RIP-Cre model which leads to recombination specifically in β cells, early during embryogenesis (E9). We performed intraperitoneal glucose tolerance tests (IPGTTs) and assessed pancreata for proliferation (Ki67), cell death (TUNEL) and beta cell mass in the 4 mouse models. ChREBPβ MIP-CreERT KO mice displayed impaired glucose tolerance, with an increase of 25.3 ± 7.9% (N=3-4, P<0.05) in the area under the curve (AUC) of IPGTT. Heterozygous or complete Chrebpβ KOs in the RIP-Cre model resulted in an increase in the AUC of IPGTT compared with control littermates (20.1 ± 13.7% and 56 ± 7.1%, respectively; n=2-4). Beta cells from isolated ChREBPβ KO islets had 60.4 ± 17.4% less proliferation compared to control islets in 6 mM glucose, and 22.9 ± 2.4% (P<0.05, N=5) less in 20 mM glucose. Inducible OE of ChREBPβ resulted in 97.7 ± 6% increase in AUC of IPGTT (N=3, p<0.01) and fasting blood glucose levels were increased by 89 ± 13.2% (P<0.01). Similarly, when ChREBPβ was OE early in the embryogenesis of β cells, we observed AUC for GTT was increased by 2.4 ± 0.5 fold compared to non-Cre expressing litter mates (N=5, p<0.05). In both mouse models, the OE of ChREBPβ resulted in β cell death as assessed by TUNEL along with increased in Ki67 staining. Remarkably, the RIP-Cre mice had almost no remaining β cells at 4 months of age and were overtly diabetic. Taken these data together, we propose a molecular regulatory mechanism where ChREBPβ controls both beta cell proliferation and cell death depending on its abundance. Disclosure L.S. Katz: None. G. Brill: None. M.A. Herman: None. D. Scott: None. Funding National Institutes of Health

A Diet-Induced Pediatric Model of Non Alcoholic Fatty Liver Disease Using Neonatal Iberian Pigs (OR26-02-19)

ObjectivesThe prevalence of non-alcoholic fatty liver disease (NAFLD) in children has increased over the past decades, creating a need for animal models that recapitulate the features of the pediatric disease. Iberian pigs have a leptin-resistant phenotype characterized by hyperleptinemia, hyperphagia, and extreme adipogenesis. We hypothesized that neonatal Iberian pigs fed a high-fat high-fructose (HFF) diet will develop a pattern of liver injury resembling pediatric NAFLD. In addition, we sought to determine if a mixture of probiotics would prevent the disease. MethodsAnimals were fed 1 of 4 diets containing (g/kg body weight × d) 0 g fructose, 11 g fat and 199 kcal (CON-N; n = 8), 22 g fructose, 16 g fat and 300 kcal (HFF-N; n = 6), CON + probiotic (CON-P; n = 6), or HFF + probiotic (HFF-P; n = 6) every 6 h for 70 d. The probiotic mixture (6.2 × 104cfu/mL) contained Pediococcus, Lactobacillus and Bacillus. Body weight was recorded every 3 d. Serum markers of liver injury and dyslipidemia were measured on d 65 at 2 h post feeding. Fasting leptin, insulin, glucose and homeostatic model assessment (HOMA) values were assessed on d 70. Liver tissue was collected on d 70 for histology, triacylglyceride (TG) quantification, and gene expression of tumor necrosis factor (TNF) α, tumor growth factor (TGF) β, interleukin (IL) 1α, peroxisome proliferator-activated receptor (PPAR) γ, carbohydrate-responsive element-binding protein (ChREBP) and sterol regulatory element-binding protein (SREBP) 1c. ResultsBody weight was higher in CON-P, and insulin and HOMA values in HFF-P and CON-P (P ≤ 0.05). Leptin, alanine and aspartate aminotransferases, alkaline phosphatase, lactate dehydrogenase and total bilirubin were increased (P ≤ 0.001), and high and low density lipoproteins decreased (P ≤ 0.05) in HFF-N and HFF-P. Livers in HFF-P and HFF-N had higher relative weight and TG (P ≤ 0.001), micro and macrovesicular steatosis, ballooning degeneration, Mallory-denk bodies, inflammation and necrosis, increased gene expression of TNFα, TGFβ, IL1α and PPARγ (P ≤ 0.001), and decreased ChREBP (P ≤ 0.001). ConclusionsIberian pigs fed a HFF diet recapitulate many pediatric NAFLD-associated features, in the absence of obesity and independently of probiotic supplementation, suggesting a potentially suitable model for pediatric NAFLD research. Funding SourcesARI, AcornSeekers.

Adipocyte ACLY Facilitates Dietary Carbohydrate Handling to Maintain Metabolic Homeostasis in Females

SUMMARYSugars and refined carbohydrates are major components of the modern diet. ATP-citrate lyase (ACLY) is upregulated in adipocytes in response to carbohydrate consumption and generates acetyl-coenzyme A (CoA) for both lipid synthesis and acetylation reactions. Here, we investigate the role of ACLY in the metabolic and transcriptional responses to carbohydrates in adipocytes and unexpectedly uncover a sexually dimorphic function in maintaining systemic metabolic homeostasis. When fed a high-sucrose diet, AclyFAT−/− females exhibit a lipodystrophy-like phenotype, with minimal fat accumulation, insulin resistance, and hepatic lipid accumulation, whereas AclyFAT−/− males have only mild metabolic phenotypes. We find that ACLY is crucial for nutrient-dependent carbohydrate response element-binding protein (ChREBP) activation in adipocytes and plays a key role, particularly in females, in the storage of newly synthesized fatty acids in adipose tissue. The data indicate that adipocyte ACLY is important in females for the systemic handling of dietary carbohydrates and for the preservation of metabolic homeostasis.

Nutritional Regulation of Gene Expression: Carbohydrate-, Fat- and Amino Acid-Dependent Modulation of Transcriptional Activity.

The ability to detect changes in nutrient levels and generate an adequate response to these changes is essential for the proper functioning of living organisms. Adaptation to the high degree of variability in nutrient intake requires precise control of metabolic pathways. Mammals have developed different mechanisms to detect the abundance of nutrients such as sugars, lipids and amino acids and provide an integrated response. These mechanisms include the control of gene expression (from transcription to translation). This review reports the main molecular mechanisms that connect nutrients’ levels, gene expression and metabolism in health. The manuscript is focused on sugars’ signaling through the carbohydrate-responsive element binding protein (ChREBP), the role of peroxisome proliferator-activated receptors (PPARs) in the response to fat and GCN2/activating transcription factor 4 (ATF4) and mTORC1 pathways that sense amino acid concentrations. Frequently, alterations in these pathways underlie the onset of several metabolic pathologies such as obesity, insulin resistance, type 2 diabetes, cardiovascular diseases or cancer. In this context, the complete understanding of these mechanisms may improve our knowledge of metabolic diseases and may offer new therapeutic approaches based on nutritional interventions and individual genetic makeup.

Treatment with myo-inositol attenuates binding of the carbohydrate-responsive element-binding protein to the ChREBP-β and FASN genes in rat nonalcoholic fatty liver induced by high-fructose diet

Dietary supplementation with the major lipotrope myo-inositol (MI) potently reduces triglyceride (TG) content and expression levels of the fatty acid synthesis genes, for example, fatty acid synthase (FASN), in rat nonalcoholic fatty liver induced by high-fructose diet. Fatty acid synthesis genes are regulated by the carbohydrate-responsive element-binding protein (ChREBP) that exists in 2 isoforms: ChREBP-α and ChREBP-β. The gene encoding the latter isoform is more responsive to fructose. Because MI repressed the induction of fatty acid synthesis gene expression by high-fructose diet, we hypothesized that MI may reduce binding of ChREBP to the carbohydrate response elements (ChoREs) in the ChREBP-β gene as well as in fatty acid synthesis genes in the liver. Rats were fed high-glucose, high-fructose, or high-fructose diets supplemented with MI (0.05% and 0.25%) for 2 weeks. Hepatic TG content and expression levels of the glucose-6-phosphate dehydrogenase, malic enzyme 1, FASN, acetyl-CoA carboxylase alpha, S14, and ChREBP-β were remarkably elevated in rats fed with high fructose compared with the corresponding levels in high-glucose group. Notably, elevated values of these parameters in high-fructose group were reduced by MI. Similarly, high-fructose–induced ChREBP binding to the ChoREs of the ChREBP-β and FASN genes was nominally decreased by MI. This study showed that treatment with MI reduced elevated TG content and expression of genes related to fatty acid synthesis, such as FASN and ChREBP-β, in rat nonalcoholic fatty liver induced by high-fructose diet. Furthermore, MI treatment nominally decreased increased binding of ChREBP to the ChoREs of ChREBP-β and FASN genes.

3: Pregnancy high fructose diet: pathways involved in fetal programming of metabolic syndrome in adult offspring

We, as well as others, have shown that maternal intake of high fructose has been shown to lead to sex- specific fetal programming of adult obesity, hypertension and metabolic dysfunction in mice. Our objective was to test the hypothesis that these outcomes are related to altered fructose metabolism, lipid accumulation and inflammation in the liver of adult offspring. Pregnant C57BL/6J dams were randomly allocated to fructose solution (10% W/V; n=10) as only drinking liquid or water (n=10), starting at ED 1 and until birth. After weaning, pups were then started on regular chow. Offspring were sacrificed when they reached adult stage (1 year of life) and their liver harvested. Protein and mRNA levels of ketohexokinase (KHK; a crucial enzyme in fructose metabolism that promotes insulin resistance and hepatic inflammation through toll like receptor-4: TLR-4), of transcription factors that lead to hepatic lipid accumulation (CHREBP: carbohydrate responsive element-binding protein; SREBP-1c: sterol regulatory element-binding protein-1c; PPAR-g: peroxisome proliferator-activated receptors-gamma), and of an enzyme responsible for tissue fat entrapment (CD36: fatty acid translocase) were assayed using Western blotting and PCR techniques. Normality was tested using Kurtosis. T-test or Mann-Whitney rank sum were used for statistical analysis. P< 0.05 was considered significant.Data are reported as mean ± SEM or median [IQR]. Female adult mice born to dams in the fructose group had significantly higher liver CHREBP, SREBP-1c & KHK protein levels compared to controls (Figure 1A). No significant differences were noted in the male counterparts (Figure 1B). Despite lack of significant changes in liver protein levels of CD36 and PPAR-g, exposure to fructose was associated with upregulation of CD36, KHK & TLR-4 mRNA in females (Figure 2 A, B & C), and PPAR-g mRNA in males (Figure 2 D). Exposure to high fructose diet in utero leads to gender-specific activation of molecular pathways responsible for insulin resistance, liver inflammation and liver steatosis in adults. High fructose enriched diets in pregnancy can have significant adverse long-term impact on health. Understanding the pathways involved can guide development of preventive and therapeutic approaches.View Large Image Figure ViewerDownload Hi-res image Download (PPT)

Carbohydrate response element binding protein (ChREBP) modulates the inflammatory response of mesangial cells in response to glucose.

Diabetic nephropathy (DN) is one of the most devastating complications of diabetes mellitus. Carbohydrate response element binding protein (ChREBP) is a basic helix–loop–helix leucine zipper transcription factor that primarily mediates glucose homeostasis in the body. The present study investigated the role of ChREBP in the pathogenesis of DN. The expression of ChREBP was detected in patients with type 2 diabetes mellitus (T2DM), diabetic mice, and mesangial cells. ELISA was used to measure cytokine production in mesangial cells. Flow cytometry analysis was performed to detect the apoptosis of mesangial cells in the presence of high glucose. The expression levels of ChREBP and several cytokines (TNF-α, IL-1β, and IL-6) were up-regulated in T2DM patients. The mRNA and protein levels of ChREBP were also significantly elevated in the kidneys of diabetic mice. Moreover, glucose treatment promoted mRNA levels of TNF-α, IL-1β, and IL-6 in mesangial cells. Glucose stimulation induced significant apoptosis of SV40 MES 13 cells. In addition, transfection with ChREBP siRNA significantly inhibited ChREBP expression. Consequently, the inflammatory responses and apoptosis were inhibited in SV40 MES 13 cells. These results demonstrated that ChREBP could mediate the inflammatory response and apoptosis of mesangial cells, suggesting that ChREBP may be involved in the pathogenesis of DN.

Naringenin Promotes Thermogenic Gene Expression in Human White Adipose Tissue.

ObjectiveNaringenin, a citrus flavonoid, prevents diet-induced weight gain and improves glucose and lipid metabolism in rodents. There is evidence that naringenin activates brown fat and increases energy expenditure in mice, but little is known about its effects in humans. Our goal was to examine the effects of naringenin on energy expenditure in adipose tissue.MethodsHuman white adipocyte cultures (hADSC), and subcutaneous abdominal adipose tissue (pWAT) were treated with naringenin for 7–14 days. Expression (qRT-PCR, immunoblotting) of candidate genes involved in thermogenesis and glucose metabolism was measured. Oxygen consumption rate (OCR) was measured in hADSC using a Seahorse Flux analyzer.ResultsIn hADSC, naringenin increased expression of the genes associated with thermogenesis and fat oxidation including uncoupling protein 1, adipose triglyceride lipase, and key factors associated with insulin sensitivity including glucose transporter 4, adiponectin, and carbohydrate response element binding protein (p<0.01). Similar responses were observed in pWAT. Basal, ATP-linked, maximal, and reserve OCR increased in the naringenin-treated hADSC (p<0.01).ConclusionsNaringenin increases energy expenditure in hADSC and stimulates expression of key enzymes involved in thermogenesis and insulin sensitivity in hADSC and pWAT. Naringenin may promote conversion of human white adipose tissue to a brown/beige phenotype.

Methionine deficiency decreases hepatic lipid exportation and induces liver lipid accumulation in broilers

The objective of the study was to investigate the effects of deficiency or high level addition of methionine (Met) in diet on homocysteine (Hcy) metabolism and hepatic lipid metabolism in broiler. A completely random design was used with 3 dietary treatments with the addition of Met from DL-methionine (DLM) at 0%, 0.22%, and 0.32% in a basal diet. The analyzed dietary Met was 0.271%, 0.485%, and 0.584% respectively for the Met-deficient, sufficient, and excessive diet. A total of 360 one-day-old male Ross 308 broiler chicks were randomly assigned to the treatments. The trial was last for 21 d. Compared to the Met-sufficient diet, the Met-deficient diet significantly resulted the decrease of 1 to 21 d growth performance and the increase of liver ether extract (EE). Compared to the Met-sufficient group, the expression of lipid transport gene apolipoprotein B (APOB) in both Met-deficient and Met-excessive group was decreased (P < 0.05). However, compared to the Met-sufficient group, a decreased of serum low-density lipoprotein cholesterol (LDLC) was only shown in birds fed a Met-deficient diet (P < 0.05). Although the plasma Hcy content was decreased, the expression of Cystathionine β-synthase (CβS) and hepatic inflammatory cytokines included interleukin-1 (IL-1) and interleukin-8 (IL-8) was significantly decreased in birds fed the Met-deficient diet when compared to those in Met-sufficient group (P < 0.05). When compared to the Met-sufficient group, the broilers in high dietary Met group had increased levels of catabolic enzyme genes expression especially acyl-coenzyme A oxidase 1 (ACOX1) and carbohydrate response element-binding protein (CHREBP) (P < 0.05), and a decreased content of liver EE. In conclusion, dietary Met deficiency decreased the hepatic lipid export and subsequently increased the hepatic lipid accumulation. High dietary Met addition increased the hepatic lipid catabolism and subsequently decreased the liver lipid accumulation.

Glucagon attenuates lipid accumulation in cow hepatocytes through AMPK signaling pathway activation.

The ketotic cows displayed hepatic lipid metabolic disorder and high blood concentration of glucagon. Importantly, adenosine monophosphate-activated protein kinase (AMPK) signaling pathway plays an important role in the hepatic lipid homeostasis. Therefore, the aim of this study was to investigate the effect of glucagon on AMPK pathway and its underlying mechanism on lipid metabolism in cow hepatocytes. Cow hepatocytes were cultured and treated with glucagon and AMPK inhibitor (BML-275). The results showed that glucagon significantly promoted the expression of glucagon receptor and increased the phosphorylation level and activity of AMPKα. Activated AMPKα increased the expression level and transcriptional activity of peroxisome proliferator-activated receptor α, which further increased the expression of fatty acid oxidation genes and lipid oxidation. Furthermore, activated AMPKα inhibited the expression level and transcriptional activity of sterol regulatory element binding protein-1c and carbohydrate response element binding protein, which decreased the expression of lipogenic genes, thereby decreasing lipid synthesis. In addition, glucagon also increased the expression of very-low-density lipoprotein (VLDL) assembly to export intracellular triglycerides (TG). Consequently, the content of intracellular TG was significantly decreased in cow hepatocytes. These results indicate that glucagon activates the AMPK signaling pathway to increase lipid oxidation and VLDL assembly and decrease lipid synthesis in cow hepatocytes, thereby reducing liver fat accumulation.

ChREBP Reciprocally Regulates Liver and Plasma Triacylglycerol Levels in Different Manners.

Carbohydrate response element-binding protein (ChREBP) has an important role in the carbohydrate-mediated regulation of hepatic de novo lipogenesis, but the mechanism for how it regulates plasma triacylglycerol (TAG) levels has not been established. This study aimed to clarify the role of ChREBP in regulation of plasma TAG levels. We analyzed the metabolic changes in mice infected with an adenovirus expressing ChREBP Δ196 (Ad-ChREBP). Compared with adenovirus harboring green fluorescent protein infected mice, Ad-ChREBP-infected mice had higher plasma free fatty acid levels and paradoxically lower plasma 3-hydroxybutyrate levels through decreased fatty acid oxidation, rather than ketogenesis. Consistent with their hepatomegaly and increased lipogenic gene expression, the liver TAG contents were much higher. Regarding lipid composition, C16:0 was much lower and C18:1n-9 was much higher, compatible with increased stearoyl CoA desaturase-1 and ELOVL fatty acid elongase 6 expression. Furthermore, Ad-ChREBP-infected mice had decreased plasma TAG and very low density lipoprotein (VLDL)-TAG levels, consistent with decreased Angiopoietin-like protein 3 (Angptl3) and increased fibroblast growth factor (Fgf21) mRNA and protein levels. Finally, Ad-ChREBP infection increased white adipose tissue Ucp1 mRNA levels with increased plasma Fgf21 levels. Because Fgf21 and Angptl3 are known to activate and suppress lipolysis in adipose tissues and oxidative tissues, ChREBP appears to regulate plasma TAG levels by modulating Fgf21 and Angptl3 levels. Thus, ChREBP overexpression led to dissociation of hepatic steatosis from hyperlipidemia.

SIRT1 overexpression attenuates offspring metabolic and liver disorders as a result of maternal high-fat feeding.

Maternal high-fat diet (MHF) consumption led to metabolic and liver disorders in male offspring, which are associated with reduced sirtuin (SIRT)1 expression and activity in the offspring liver SIRT1 overexpression in MHF offspring reduced their body weight and adiposity and normalized lipid metabolic markers in epididymal and retroperitoneal adipose tissues SIRT1 overexpression in MHF offspring improved glucose tolerance, as well as systemic and hepatic insulin sensitivity SIRT1 overexpression ameliorated MHF-induced lipogenesis, oxidative stress and fibrogenesis in the liver of offspring. Maternal obesity can increase the risk of metabolic disorders in the offspring. However, the underlying mechanism responsible for this is not clearly understood. Previous evidence implied that sirtuin (SIRT)1, a potent regulator of energy metabolism and stress responses, may play an important role. In the present study, we have shown, in C57BL/6 mice, that maternal high-fat diet (HFD) consumption can induce a pre-diabetic and non-alcoholic fatty liver disease phenotype in the offspring, associated with reduced SIRT1 expression in the hypothalamus, white adipose tissues (WAT) and liver. Importantly, the overexpression of SIRT1 in these offspring significantly attenuated the excessive accumulation of epididymal (Epi) white adipose tissue (WAT) and retroperitoneal (Rp)WAT (P<0.001), glucose intolerance and insulin resistance (both P<0.05) at weaning age. These changes were associated with the suppression of peroxisome proliferator-activated receptor gamma (PPAR)γ (P<0.01), PPARγ-coactivator 1-alpha (P<0.05) and sterol regulatory element-binding protein-1c in EpiWAT (P<0.01), whereas there was increased expression of PPARγ in RpWAT (P<0.05). In the liver, PPARγ mRNA expression, as well as Akt protein expression and activity, were increased (P<0.05), whereas fatty acid synthase and carbohydrate response element binding protein were downregulated (P<0.05), supporting increased insulin sensitivity and reduced lipogenesis in the liver. In addition, hepatic expression of endogenous anti-oxidants, including glutathione peroxidase 1 and catalase, was increased (P<0.01 and P<0.05 respectively), whereas collagen and fibronectin deposition was suppressed (P<0.01). Collectively, the present study provides direct evidence of the mechanistic significance of SIRT1 in maternal HFD-induced metabolic dysfunction in offspring and suggests that SIRT1 is a promising target for fetal reprogramming.

Elevated Carbohydrate Response Element-Binding Protein Beta (ChREBPβ) and Thioredoxin Interacting Protein (TXNIP) Levels in Human Adipocytes Differentiated in High Glucose Concentrations.

Obesity and type 2 diabetes often coexist. The effect of hyperglycemia on adipose tissue is, therefore, of interest. Although studies have shown that high glucose (HG) concentrations do not inhibit adipocyte differentiation, the resulting adipocyte phenotype has not been investigated. In particular, the levels of the glucose-responsive transcription factor carbohydrate-responsive response element binding protein (ChREBP) isoforms have not been assessed. Human preadipocytes were differentiated into adipocytes in either normal glucose (NG) or HG conditions. RNA and protein analyses were used to measure the expression of ChREBP isoforms, thioredoxin interacting protein (TXNIP) and lipogenic genes. Insulin-stimulated glucose uptake was measured. HG- vs. NG-differentiated adipocytes expressed more ChREBPβ and more TXNIP at the mRNA and protein levels. There was no change in lipogenic gene expression. HG- vs. NG-differentiated adipocytes displayed an inhibition of insulin-stimulated glucose uptake. HG-differentiated human adipocytes have distinct molecular differences and are insulin resistant. More studies are warranted to investigate potential mechanisms linking changes in ChREBPβ and TXNIP to insulin responsiveness.

Role of X-Box Binding Protein-1 in Fructose-Induced De Novo Lipogenesis in HepG2 Cells.

Background:A high consumption of fructose leads to hepatic steatosis. About 20–30% of triglycerides are synthesized via de novo lipogenesis. Some studies showed that endoplasmic reticulum stress (ERS) is involved in this process, while others showed that a lipotoxic environment directly influences ER homeostasis. Here, our aim was to investigate the causal relationship between ERS and fatty acid synthesis and the effect of X-box binding protein-1 (XBP-1), one marker of ERS, on hepatic lipid accumulation stimulated by high fructose.Methods:HepG2 cells were incubated with different concentrations of fructose. Upstream regulators of de novo lipogenesis (i.e., carbohydrate response element-binding protein [ChREBP] and sterol regulatory element-binding protein 1c [SREBP-1c]) were measured by polymerase chain reaction and key lipogenic enzymes (acetyl-CoA carboxylase [ACC], fatty acid synthase [FAS], and stearoyl-CoA desaturase-1 [SCD-1]) by Western blotting. The same lipogenesis-associated factors were then evaluated after exposure of HepG2 cells to high fructose followed by the ERS inhibitor tauroursodeoxycholic acid (TUDCA) or the ERS inducer thapsigargin. Finally, the same lipogenesis-associated factors were evaluated in HepG2 cells after XBP-1 upregulation or downregulation through cell transfection.Results:Exposure to high fructose increased triglyceride levels in a dose- and time-dependent manner and significantly increased mRNA levels of SREBP-1c and ChREBP and protein levels of FAS, ACC, and SCD-1, concomitant with XBP-1 conversion to an active spliced form. Lipogenesis-associated factors induced by high fructose were inhibited by TUDCA and induced by thapsigargin. Triglyceride level in XBP-1-deficient group decreased significantly compared with high-fructose group (4.41 ± 0.54 μmol/g vs. 6.52 ± 0.38 μmol/g, P < 0.001), as mRNA expressions of SREBP-1c (2.92 ± 0.46 vs. 5.08 ± 0.41, P < 0.01) and protein levels of FAS (0.53 ± 0.06 vs. 0.85 ± 0.05, P = 0.01), SCD-1 (0.65 ± 0.06 vs. 0.90 ± 0.04, P = 0.04), and ACC (0.38 ± 0.03 vs. 0.95 ± 0.06, P < 0.01) decreased. Conversely, levels of triglyceride (4.22 ± 0.54 μmol/g vs. 2.41 ± 0.35 μmol/g, P < 0.001), mRNA expression of SREBP-1c (2.70 ± 0.33 vs. 1.00 ± 0.00, P < 0.01), and protein expression of SCD-1 (0.93 ± 0.06 vs. 0.26 ± 0.05, P < 0.01), ACC (0.98 ± 0.09 vs. 0.43 ± 0.03, P < 0.01), and FAS (0.90 ± 0.33 vs. 0.71 ± 0.02, P = 0.04) in XBP-1s-upregulated group increased compared with the untransfected group.Conclusions:ERS is associated with de novo lipogenesis, and XBP-1 partially mediates high-fructose-induced lipid accumulation in HepG2 cells through augmentation of de novo lipogenesis.

Recent insights into the role of ChREBP in intestinal fructose absorption and metabolism

Fructose in the form of sucrose and high fructose corn syrup is absorbed by the intestinal transporter and mainly metabolized in the small intestine. However, excess intake of fructose overwhelms the absorptive capacity of the small intestine, leading to fructose malabsorption. Carbohydrate response element-binding protein (ChREBP) is a basic helix-loop-helix leucine zipper transcription factor that plays a key role in glycolytic and lipogenic gene expression in response to carbohydrate consumption. While ChREBP was initially identified as a glucose-responsive factor in the liver, recent evidence suggests that ChREBP is essential for fructoseinduced lipogenesis and gluconeogenesis in the small intestine as well as in the liver. We recently identified that the loss of ChREBP leads to fructose intolerance via insufficient induction of genes involved in fructose transport and metabolism in the intestine. As fructose consumption is increasing and closely associated with metabolic and gastrointestinal diseases, a comprehensive understanding of cellular fructose sensing and metabolism via ChREBP may uncover new therapeutic opportunities. In this mini review, we briefly summarize recent progress in intestinal fructose metabolism, regulation and function of ChREBP by fructose, and delineate the potential mechanisms by which excessive fructose consumption may lead to irritable bowel syndrome.

A Renewed Focus on the Association Between Thyroid Hormones and Lipid Metabolism.

Thyroid dysfunction, manifesting as either overt or subclinical hypothyroidism, negatively affects lipid metabolism: this leads to hypercholesterolemia which progressively increases the risk for cardiovascular disease and, potentially, mortality. Hypercholesterolemia in hypothyroidism is mainly due to a reduction in low-density lipoprotein (LDL) receptor activity, this accompanied by concomitant diminishing control by triiodothyronine (T3) of sterol regulatory element-binding protein 2 (SREBP-2), which modulates cholesterol biosynthesis by regulating rate-limit degrading enzyme 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMG-CoA) activity. Recently, 3,5-diiodothyronine (T2), a natural thyroid hormone derivative, was found to repress the transcription factor carbohydrate-response element-binding protein (ChREBP) and also to be involved in lipid catabolism and lipogenesis, though via a different pathway than that of T3. While thyroid hormone could therapeutically reverse the dyslipidemic profile commonly occurring in hypothyroidism, it should be borne in mind that the potency of the effects may be age-and sex-dependent. Thyroid hormone administration possibly also sustains and enhances the efficacy of hypolipidemic drugs, such as statins, ezetimibe and proprotein convertase subtilisin/kexin type 9 (PCSK9), in patients with dyslipidemia and hypothyroidism.

MiR-1322 regulates ChREBP expression via binding a 3'-UTR variant (rs1051943).

The carbohydrate response element‐binding protein (ChREBP), also referred to as MLXIPL, plays a crucial role in the regulation of glucose and lipid metabolism. Existing studies have shown an association between genetic variations of the ChREBP gene and lipid levels, such as triglycerides and high‐density lipoprotein cholesterol. However, mechanistic studies of this association are limited. In this study, bioinformatic analysis revealed that the polymorphism rs1051943A occurs in the complementary binding sequence of miR‐1322 in the ChREBP 3′‐untranslated region (UTR). Studies of potential mechanisms showed that the A allele could facilitate miR‐1322 binding, and luciferase activity significantly decreased when co‐transfected with a ChREBP 3′‐UTR luciferase reporter vector and miR‐1322 mimics in HepG2 cells. Furthermore, miR‐1322 significantly regulated the expression of ChREBP downstream genes and reduced the synthesis of lipids. The expression of miR‐1322 was up‐regulated by glucose and palmitic acid stimulation. Population studies showed that rs1051943‐A allele was only found in the Han Chinese and Uighur ethnic groups, different from European populations (G allele frequency = 0.07). In summary, we provide evidence that the rs1051943 A allele creates a functional miR‐1322 binding site in ChREBP 3′‐UTR and post‐transcriptionally down‐regulates its expression, possibly associated with levels of plasma lipids and glucose.