ChREBP Research Articles

Beneficial effects of cherry consumption as a dietary intervention for metabolic, hepatic and vascular complications in type 2 diabetic rats

BackgroundOxidative stress (OS) plays an important role in type 2 diabetes (T2D) pathogenesis and its complications. New therapies target natural antioxidants as an alternative and/or supplemental strategy to prevent and control them. Our previous chemical and biological studies highlighted the important antioxidant activities of cherries, among other fruits and vegetables, thus we aimed to determine in vivo effects of 2-month long cherry consumption using a high-fat/high-fructose (HFHF) model of diabetic-rats (Lozano et al. in Nutr Metab 13:15, 2016).MethodsAfter 2 months of HFHF, male Wistar rats were divided into: HFHF and HFHF enriched in cherry (nutritional approach) or standard diet ND (lifestyle measures) and ND plus cherry during 2 months. Metabolic, lipidic, oxidative parameters were quantified. Tissues (liver, pancreas and vessels) OS were assessed and hepatic (steatosis, fibrosis, inflammation) and vascular (endothelial dysfunction) complications were characterized.ResultsT2D was induced after 2 months of HFHF diet, characterized by systemic hyperglycaemia, hyperinsulinemia, glucose intolerance, dyslipidaemia, hyperleptinemia, and oxidative stress associated with endothelial dysfunction and hepatic complications. Cherry consumption for 2 months, in addition to lifestyle measures, in T2D-rats decreased and normalized the systemic disturbances, including oxidative stress complications. Moreover, in the vessel, cherry consumption decreased oxidative stress and increased endothelial nitric oxide (NO) synthase levels, thus increasing NO bioavailability, ensuring vascular homeostasis. In the liver, cherry consumption decreased oxidative stress by inhibiting NADPH oxidase subunit p22phox expression, nuclear factor erythroid-2 related factor 2 (Nrf2) degradation and the formation of reactive oxygen species. It inhibited the activation of sterol regulatory element-binding proteins (1c and 2) and carbohydrate-responsive element-binding protein, and thus decreased steatosis as observed in T2D rats. This led to the improvement of metabolic profiles, together with endothelial and hepatic function improvements.ConclusionCherry consumption normalized vascular function and controlled hepatic complications, thus reduced the risk of diabetic metabolic disorders. These results demonstrate that a nutritional intervention with a focus on OS could prevent and/or delay the onset of vascular and hepatic complications related to T2D.

Carbohydrate Response Element Binding Protein (ChREBP) Promotes Inflammation and Apoptosis of Mesangial Cells

Objective: 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. Materials and Methods: 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. Results: The ChREBP and several cytokines (TNFα, IL-1β, and IL-6) were up-regulated in T2DM patients (P<0.05). The mRNA and protein levels of ChREBP were 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. Conclusions: 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. Disclosure Y. Chen: None. S. Zhao: None. Y. Wang: None.

Localization of ChREBPa and ChREBPß in Pancreatic ß Cells

Glucose is a key physiological driver of adaptive β-cell mass expansion, promoting β-cell proliferation both in vitro and in vivo. Carbohydrate response element binding protein (ChREBP) is required for glucose-stimulated β-cell proliferation in both rodent and human β-cells. ChREBP has two splice isoforms - α and β. Translation of the ChREBPβ mRNA generates a truncated protein, resulting in a potent, constitutively active isoform. To study the role of the 2 splice isoforms in β-cell biology, we performed numerous experiments including immunostaining and chromatin immunoprecipitation with antibodies that recognize only ChREBPα or both isoforms. We found that ChREBPα is only transiently expressed in the nucleus. By contrast, ChREBPβ, once expressed at high enough levels to detect, is constitutively nuclear. Our data indicate that mice fed on high fat diet and diabetic db/db mice have abundant ChREBPβ localized in the nucleus while control mice have mostly cytoplasmic ChREBPα. We generated Ins-1 cell lines where we can distinguish between the splice isoforms using CRISPR/Cas9: 1. The C-terminus of ChREBPα and ChREBPβ was labeled with eGFP; 2. The N-terminus of ChREBPα was labeled by mCherry; and 3. ChREBPα was labeled with mCherry and eGFP on the N- and C- terminus, respectively and ChREBPβ is labeled with eGFP on the C-terminus. We validated the three cell lines by sequencing, ChIP, qRT-PCR and also showed that the glucose response is not altered in these cells. We found that in low glucose, ChREBPβ is present at very low levels in the nucleus. By contrast, in high glucose, ChREBPβ is present at high levels in the nucleus. Our study suggests that in response to glucose, ChREBPα, which is mostly cytoplasmic, transiently enters the nucleus, binds to a carbohydrate response element (ChoRE), and induces ChREBPβ expression, which is mostly nuclear. We propose that while the induction of ChREBPβ drives adaptive beta cell expansion, ChREBPβ contributes to glucose toxicity after prolonged exposure to high glucose and in diabetes. Disclosure L.S. Katz: None. A.K. Laxman: None. P. Zhang: None. D. Scott: None.

Altering ß-Cell Proliferation In Vivo by Pharmacological Manipulation of the Nrf2 Pathway

Glucose is a natural mitogen that drives adaptive β-cell mass expansion by promoting proliferation. We previously demonstrated that induction of Nrf2 is required for glucose-stimulated and carbohydrate response element binding protein (ChREBPa)-augmented β-cell proliferation. Here we found that adenoviral delivery of Nrf2 increased human β-cell proliferation by 8.9 ± 0.2 fold (n=6), which exceeded the effect of 20 mM glucose (4.3 ± 0.4 fold). To test if activation of the Nrf2 pathway is necessary for adaptive β-cell expansion in vivo, we used pharmacological inhibitors and activators of Nrf2. Mice were placed on a high fat diet (HFD) or standard chow diet (SD) for 7 days and treated with vehicle or brusatol, which depletes Nrf2 by a post-translational mechanism. Treatment with brusatol effectively diminished Nrf2 abundance in β-cells as determined by immunofluorescence. Further, brusatol treatment ameliorated the increased β-cell proliferation normally seen with a one-week HFD (2.7±03% ki67 positive β-cells in control HFD mice, vs. 1.6±0.3% in HFD mice treated with brusatol, p < 0.05, n=5). Brusatol-treated mice also displayed significantly diminished glucose tolerance on a HFD by IPGTT, without altering the food intake or weight of the animals. Using an activator of Nrf2, bardoxolone methyl (CDDO-Me), for 7 days in the presence or absence of HFD. We found, consistent with previous reports, improved glycemic levels and decreased body weight in mice treated with CDDO-Me on a HFD. Remarkably, we also found increased β-cell proliferation in mice fed a SD and treated with CDDO-Me (1.7 ± 0.1% controls compared to 2.6±0.1% ki67 positive, insulin positive β-cells in mice treated with CDDO-Me). Crucially, in human β-cells, brusatol inhibited glucose-stimulated proliferation (1.9 fold vs. 12.7 fold, n=2) and CDDO-Me increased proliferation in 5 mM glucose (37.5 fold, n=2). Thus, we conclude that the Nrf2 pathway is an exciting novel target for manipulation of β-cell proliferation in vivo. Disclosure L.S. Katz: None. A. Garcia-Ocana: None. D. Scott: None.

T3 and Glucose Increase Expression of ChREBPß to Induce Expression of Phosphoenolpyruvate Carboxykinase (Pck1) Leading to Increased ß-Cell Proliferation

Thyroid hormone (T3) is required for islet development and function. Both T3 and high glucose concentrations are critical components of differentiation protocols of stem cells to β-cells. We previously demonstrated that in brown adipose tissue, T3 and glucose synergistically regulate carbohydrate response element binding protein (ChREBP), which in turn upregulates Ucp1, Glut4 and Fasn, resulting in increased thermogenesis, decreased body weight and improved glycemic levels. In the present study, we asked whether T3 and glucose signaling pathways can coordinately regulate transcription of genes important for β-cell function and mass. RNA-seq analysis was performed on cadaveric human islets from five different donors in response to low and high glucose concentrations (6 and 20 mM, respectively) and in the presence or absence of T3 (10 nM). We found that T3 and glucose coordinately regulate the expression of ChREBPβ and Pck1 (phosphoenolpyruvate carboxykinase-1). Typically, it is thought that Pck1 is not expressed in pancreatic β-cells, but it is a known target of T3 in hepatocytes. However, in FACS sorted adult human β-cells, we found that high concentrations of glucose (20 mM) and T3 (10 nM) induced the expression of Pck1 by 17.2±1.3 fold for mRNA levels. In addition, we found that overexpression of Pck1 together with its substrate dimethyl malate (DMM) increased β- cell proliferation in human islets (0.49±0.03% for human islets overexpressing Pck1, treated with DMM compared with 0.01±0.01% for control islets, n=4). Finally, using a sh-RNA knockdown approach, we demonstrated that in Ins-1 cells, ChREBPβ is necessary for Pck1 dependent β-cell proliferation. We conclude that T3 and glucose act together to regulate ChREBPβ which in turn upregulates expression of Pck1. In turn, Pck1 activity leads to increased β-cell mass. Disclosure L.S. Katz: None. D. Scott: None.

Mir-128 Targets Thioredoxin-Interacting Protein 3’UTR and Downregulates Its Expression in Pancreatic Beta Cells

Thioredoxin-interacting protein (TXNIP) expression is induced by glucose, promotes beta cell apoptosis and is essential for glucotoxicity-induced beta cell death, whereas TXNIP deficiency protects against diabetes. Carbohydrate-response element-binding protein (ChREBP/MondoA) is the major transcription factor conferring glucose-induced TXNIP expression. However, we recently discovered that glucose was still able to induce TXNIP in ChREBP knockout mouse islets suggesting the existence of other factors involved in glucose-induced TXNIP regulation. Analyses of the TXNIP 3’UTR revealed several potential miRNA binding sites including miR-93, miR-106b and miR-128. However, using INS-1 beta cells, primary mouse islets and T2D human islets, we found that only miR-128 was downregulated by glucose or diabetes. This suggested that glucose might decrease miR-128 and thereby promote TXNIP expression. In fact, we found overexpression of miR-128 significantly reduced TXNIP mRNA and protein expression whereas inhibition of miR-128 increased beta cell TXNIP expression. Furthermore, luciferase assays using reporter constructs with the wild type TXNIP 3’UTR or a mutated TXNIP 3’UTR confirmed that TXNIP is indeed a direct target of miR-128. Taken together, the results of these studies have identified a novel microRNA directly targeting TXNIP and revealed an alternative, post-transcriptional pathway by which glucose induces TXNIP expression. Disclosure G. Jing: None. J. Chen: None. G. Xu: None. A. Shalev: None.

Oncogene-dependent addiction to carbohydrate-responsive element binding protein in hepatocellular carcinoma

ABSTRACTMetabolic reprogramming is a hallmark of many cancer types, including hepatocellular carcinoma (HCC). Identifying the critical players in this process might be crucial for the generation of novel and effective anti-neoplastic therapies. In the present investigation, we determined the importance of carbohydrate responsive element binding protein (ChREBP), a central player in the regulation of lipid and glucose metabolism in the liver, on the development of HCC in in vitro and in vivo models. We found that genetic deletion of ChREBP (that will be referred to as ChREBPKO mice) strongly delays or impairs hepatocarcinogenesis driven by AKT or AKT/c-Met overexpression in mice, respectively. In contrast, HCC development was found to be completely unaffected by ChREBP depletion in mice co-expressing AKT and N-Ras protooncogenes. In mouse and human HCC cell lines, suppression of ChREBP via specific small interfering RNAs (siRNAs) resulted in decreased proliferation and induction of apoptosis. Of note, these cellular events were strongly augmented by concomitant inhibition of the mitogen-activated protein kinase (MAPK) pathway. The present data indicate that ChREBP activity might be required or dispensable for HCC growth, depending on the oncogenes involved. In particular, the activation of Ras/MAPK signaling might represent a possible mechanism of resistance to ChREBP depletion in this tumor type. Additional studies are needed to unravel the molecular mechanisms rendering HCC cells insensitive to ChREBP suppression.

The effect of maternal intake of sucrose or high-fructose corn syrup (HFCS)-55 during gestation and lactation on lipogenic gene expression in rat offspring at 3 and 12 weeks of age.

Perinatal exposure to sucrose or high-fructose corn syrup-55 (HFCS-55) in rats has previously been associated with altered hepatic fat content and composition post-weaning, although the effects on hepatic metabolism are unknown. The current study aimed to determine the sex-specific effects of maternal consumption of sucrose or HFCS-55 on the expression of hepatic lipogenic genes in the offspring. Liver samples were collected from offspring of albino Wistar rats provided with ad libitum access to either water (control), 10% sucrose or 10% HFCS-55 solution during pregnancy and lactation at 3 weeks (control n=16, sucrose n=22, HFCS-55 n=16) and 12 weeks (control n=16, sucrose n=10, HFCS-55 n=16) of age. Hepatic expression of the transcription factors such as carbohydrate response element-binding protein, sterol regulatory element-binding protein-1c and downstream genes was determined by quantitative real-time PCR. Sucrose-exposed offspring had higher hepatic SREBP-1c messenger RNA expression compared with control and HFCS-55 groups at both 3 weeks (P=0.01) and 12 weeks (P=0.03) of age. There were no differences in the expression of other hepatic lipogenic genes between groups at either 3 or 12 weeks. Thus, perinatal exposure to sucrose may be more detrimental to offspring hepatic metabolism compared with HFCS-55, independent of sex, and it will be important to evaluate the longer-term effects of perinatal sucrose exposure in future studies.

Mori Cortex extract ameliorates nonalcoholic fatty liver disease (NAFLD) and insulin resistance in high-fat-diet/streptozotocin-induced type 2 diabetes in rats

Nonalcoholic fatty liver disease (NAFLD) and type 2 Diabetes Mellitus (T2DM) are highly prevalent diseases and are closely associated, with NAFLD being present in the majority of T2DM patients. In Asian traditional medicine, Mori Cortex is widely used for the treatment of diabetes and hyperlipidemia. However, whether it has a therapeutic effect on T2DM associated with NAFLD is still unknown. The present study showed that the oral treatment with Mori Cortex extract (MCE; 10 g·kg−1·d−1) lowered the blood lipid levels and reversed insulin resistance (IR) in high fat-diet/streptozotocin-induced type 2 diabetes in rats. The expression levels of sterol receptor element-binding protein-1c (SREBP-1c) and carbohydrate-responsive element binding protein (ChREBP), which are involved in steatosis in NAFLD rats, were measured in the liver samples. MCE decreased the protein and mRNA expression levels of SREBP-1c and ChREBP. In conclusion, down-regulation of SREBP-1c and ChREBP might contribute to the protective effect of MCE on hepatic injury and IR in the rats with T2DM associated with NAFLD.

The histone demethylase Phf2 acts as a molecular checkpoint to prevent NAFLD progression during obesity

Aberrant histone methylation profile is reported to correlate with the development and progression of NAFLD during obesity. However, the identification of specific epigenetic modifiers involved in this process remains poorly understood. Here, we identify the histone demethylase Plant Homeodomain Finger 2 (Phf2) as a new transcriptional co-activator of the transcription factor Carbohydrate Responsive Element Binding Protein (ChREBP). By specifically erasing H3K9me2 methyl-marks on the promoter of ChREBP-regulated genes, Phf2 facilitates incorporation of metabolic precursors into mono-unsaturated fatty acids, leading to hepatosteatosis development in the absence of inflammation and insulin resistance. Moreover, the Phf2-mediated activation of the transcription factor NF-E2-related factor 2 (Nrf2) further reroutes glucose fluxes toward the pentose phosphate pathway and glutathione biosynthesis, protecting the liver from oxidative stress and fibrogenesis in response to diet-induced obesity. Overall, our findings establish a downstream epigenetic checkpoint, whereby Phf2, through facilitating H3K9me2 demethylation at specific gene promoters, protects liver from the pathogenesis progression of NAFLD.

Histone modifications in fatty acid synthase modulated by carbohydrate responsive element binding protein are associated with non‑alcoholic fatty liver disease.

Non-alcoholic fatty liver disease (NAFLD) is a manifestation of metabolic syndrome in the liver and is closely associated with diabetes; however, its pathogenesis remains to be elucidated. Carbohydrate responsive element binding protein (ChREBP), the hub of glucolipid metabolism, regulates the induction of fatty acid synthase (FASN), the key enzyme of de novo lipogenesis, by directly binding to carbohydrate response element (ChoRE) in its promoter. Investigations of histone modifications on NAFLD remain in their infancy. In the present study, by using ChIP, the association between histone modifications and FASN transcription was investigated and histone modifications in FASN modulated by ChREBP were measured. It was demonstrated that ChREBP induced FASN ChREBP-ChoRE binding to accelerate the expression of FASN, leading to hepatocellular steatosis by facilitating H3 and H4 acetylation, H3K4 trimethylation and the phosphorylation of H3S10, but inhibiting the trimethylation of H3K9 and H4K20 in FASN promoter regions of HepG2 and L02 cells. It was also found that ChREBP-ChoRE binding of FASN relied on histone acetylation and that the transcriptional activity of ChREBP on FASN is required, based on the premise that histone acetylation causes conformational changes in FASN chromatin. This indicated histone acetylation as a crucial mechanism involved in the transcription of FASN modulated by ChREBP. Consequently, the present study provides further insight into the pathophysiology and a novel therapeutic potential of NAFLD based on epigenetic mechanisms.

Activation of Nrf2 Is Required for Normal and ChREBPα-Augmented Glucose-Stimulated β-Cell Proliferation.

Patients with both major forms of diabetes would benefit from therapies that increase β-cell mass. Glucose, a natural mitogen, drives adaptive expansion of β-cell mass by promoting β-cell proliferation. We previously demonstrated that a carbohydrate response element–binding protein (ChREBPα) is required for glucose-stimulated β-cell proliferation and that overexpression of ChREBPα amplifies the proliferative effect of glucose. Here we found that ChREBPα reprogrammed anabolic metabolism to promote proliferation. ChREBPα increased mitochondrial biogenesis, oxygen consumption rates, and ATP production. Proliferation augmentation by ChREBPα required the presence of ChREBPβ. ChREBPα increased the expression and activity of Nrf2, initiating antioxidant and mitochondrial biogenic programs. The induction of Nrf2 was required for ChREBPα-mediated mitochondrial biogenesis and for glucose-stimulated and ChREBPα-augmented β-cell proliferation. Overexpression of Nrf2 was sufficient to drive human β-cell proliferation in vitro; this confirms the importance of this pathway. Our results reveal a novel pathway necessary for β-cell proliferation that may be exploited for therapeutic β-cell regeneration.

Reduction in Secretion of Very Low Density Lipoprotein-Triacylglycerol by a Matrix Metalloproteinase Inhibitor in a Rat Model of Diet-Induced Hypertriglyceridemia.

Matrix metalloproteinase inhibitors (MMPIs) reduced serum triacylglycerol (TAG) levels in streptozotocin-induced diabetic rats and Zucker fa/fa rats in our previous study. However, the mechanisms underlying TAG reduction by MMPIs remain unclear. The present study aimed to elucidate the mechanism by which F81-1144b, an MMPI, lowers serum TAG levels in an animal model of high-sucrose diet (HSD)-induced hypertriglyceridemia. F81-1144b was repeatedly administered to rats fed HSD, and its effects were evaluated on TAG levels in serum and the liver, very low density lipoprotein (VLDL) secretion, de novo fatty acid (FA) synthesis in the liver, and the expression of genes regulating the metabolism of FA, TAG, and VLDL in the liver and serum. F81-1144b lowered TAG levels in serum and the liver, VLDL-TAG secretion, de novo FA synthesis in the liver, and serum levels of insulin and glucose. F81-1144b suppressed the expression of genes related to the de novo synthesis of FA and TAG, key proteins (lipin 1 and apolipoprotein CIII) responsible for VLDL metabolism, and sterol regulatory element-binding protein-1c and carbohydrate response element-binding protein. F81-1144b little affected the expression of genes related directly to the degradation of TAG or FA, but it upregulated that of gene for uncoupling protein 2 in the liver. These results suggest that MMPIs are a novel type of therapeutic agent for the treatment of hypertriglyceridemia, because the metabolic effects of F81-1144b expected from changes in the expression of genes regulating lipid metabolism would alter metabolism differently from those induced by fibrates, niacin, or n-3 FAs.

Involvement of glucocorticoid prereceptor metabolism and signaling in rat visceral adipose tissue lipid metabolism after chronic stress combined with high-fructose diet.

Both fructose overconsumption and increased glucocorticoids secondary to chronic stress may contribute to overall dyslipidemia. In this study we specifically assessed the effects and interactions of dietary fructose and chronic stress on lipid metabolism in the visceral adipose tissue (VAT) of male Wistar rats. We analyzed the effects of 9-week 20% high fructose diet and 4-week chronic unpredictable stress, separately and in combination, on VAT histology, glucocorticoid prereceptor metabolism, glucocorticoid receptor subcellular redistribution and expression of major metabolic genes. Blood triglycerides and fatty acid composition were also measured to assess hepatic Δ9 desaturase activity. The results showed that fructose diet increased blood triglycerides and Δ9 desaturase activity. On the other hand, stress led to corticosterone elevation, glucocorticoid receptor activation and decrease in adipocyte size, while phosphoenolpyruvate carboxykinase, adipose tissue triglyceride lipase, FAT/CD36 and sterol regulatory element binding protein-1c (SREBP-1c) were increased, pointing to VAT lipolysis and glyceroneogenesis. The combination of stress and fructose diet was associated with marked stimulation of fatty acid synthase and acetyl-CoA carboxylase mRNA level and with increased 11β-hydroxysteroid dehydrogenase type 1 and hexose-6-phosphate dehydrogenase protein levels, suggesting a coordinated increase in hexose monophosphate shunt and de novo lipogenesis. It however did not influence the level of peroxisome proliferator-activated receptor-gamma, SREBP-1c and carbohydrate responsive element-binding protein. In conclusion, our results showed that only combination of dietary fructose and stress increase glucocorticoid prereceptor metabolism and stimulates lipogenic enzyme expression suggesting that interaction between stress and fructose may be instrumental in promoting VAT expansion and dysfunction.

Insulin suppresses the AMPK signaling pathway to regulate lipid metabolism in primary cultured hepatocytes of dairy cows.

Dairy cows with type II ketosis display hepatic fat accumulation and hyperinsulinemia, but the underlying mechanism is not completely clear. This study aimed to clarify the regulation of lipid metabolism by insulin in cow hepatocytes. In vitro, cow hepatocytes were treated with 0, 1, 10, or 100 nm insulin in the presence or absence of AICAR (an AMP-activated protein kinase alpha (AMPKα) activator). The results showed that insulin decreased AMPKα phosphorylation. This inactivation of AMPKα increased the gene and protein expression levels of carbohydrate responsive element-binding protein (ChREBP) and sterol regulatory element-binding protein-1c (SREBP-1c), which downregulated the expression of lipogenic genes, thereby decreasing lipid biosynthesis. Furthermore, AMPKα inactivation decreased the gene and protein expression levels of peroxisome proliferator-activated receptor-α (PPARα), which upregulated the expression of lipid oxidation genes, thereby increasing lipid oxidation. In addition, insulin decreased the very low density lipoprotein (VLDL) assembly. Consequently, triglyceride content was significantly increased in insulin treated hepatocytes. Activation of AMPKα induced by AICAR could reverse the effect of insulin on PPARα, SREBP-1c, and ChREBP, thereby decreasing triglyceride content. These results indicate that insulin inhibits the AMPKα signaling pathway to increase lipid synthesis and decrease lipid oxidation and VLDL assembly in cow hepatocytes, thereby inducing TG accumulation. This mechanism could partly explain the causal relationship between hepatic fat accumulation and hyperinsulinemia in dairy cows with type II ketosis.

ChREBP deficiency leads to diarrhea-predominant irritable bowel syndrome

ObjectiveFructose malabsorption is a common digestive disorder in which absorption of fructose in the small intestine is impaired. An abnormality of the main intestinal fructose transporter proteins has been proposed as a cause for fructose malabsorption. However the underlying molecular mechanism for this remains unclear. In this study, we investigated whether carbohydrate response element-binding protein (ChREBP) plays a role in intestinal fructose absorption through the regulation of genes involved in fructose transport and metabolism and ion transport. MethodsWild type (WT) and Chrebp knockout (KO) mice (6 or 8 weeks old) were fed a control diet (55% starch, 15% maltodextrin 10) or high-fructose diet (HFrD, 60% fructose, 10% starch) for 3–12 days. Body weight and food intake were measured, signs of fructose malabsorption were monitored, and the expression of genes involved in fructose transport/metabolism and ion transport was evaluated. Furthermore, transient transfection and chromatin immunoprecipitation were performed to show the direct interaction between ChREBP and carbohydrate response elements in the promoter of Slc2A5, which encodes the fructose transporter GLUT5. ResultsChrebp KO mice fed the control diet maintained a constant body weight, whereas those fed a HFrD showed significant weight loss within 3–5 days. In addition, Chrebp KO mice fed the HFrD exhibited a markedly distended cecum and proximal colon containing both fluid and gas, suggesting incomplete fructose absorption. Fructose-induced increases of genes involved in fructose transport (GLUT5), fructose metabolism (fructokinase, aldolase B, triokinase, and lactate dehydrogenase), and gluconeogenesis (glucose-6-phosphatase and fructose-1,6-bisphosphatase) were observed in the intestine of WT but not of Chrebp KO mice. Moreover the Na+/H+ exchanger NHE3, which is involved in Na+ and water absorption in the intestine, was significantly decreased in HFrD-fed Chrebp KO mice. Consistent with this finding, the high-fructose diet-fed Chrebp KO mice developed severe diarrhea. Results of chromatin immunoprecipitation assays showed a direct interaction of ChREBP with the Glut5 promoter, but not the Nhe3 promoter, in the small intestine. Ectopic co-expression of ChREBP and its heterodimer partner Max-like protein X activated the Glut5 promoter in Caco-2BBE cells. ConclusionsChREBP plays a key role in the dietary fructose transport as well as conversion into lactate and glucose through direct transcriptional control of genes involved in fructose transport, fructolysis, and gluconeogenesis. Moreover, ablation of Chrebp results in a severe diarrhea in mice fed a high-fructose diet, which is associated with the insufficient induction of GLUT5 in the intestine.

Apple Pomace Supplementation Favorably Alters Hepatic Lipid Metabolism in Young Female Sprague‐Dawley Rats fed a Western Diet

Apple pomace is a waste byproduct of apple processing that is high in nutrients including polyphenols, pectin, insoluble fibers, and fructose that may contribute to alleviation of some metabolic diseases. Non‐alcoholic fatty liver disease is the most common chronic liver disease in Western countries with the cause attributed to a high fat, high sucrose (Western diet) consumption. The objective of this study was to determine the effects of apple pomace supplementation on hepatic lipid metabolism. Growing (age 22–29 days) female Sprague‐Dawley rats were randomly assigned (n=8 rats/group) to consume a standard purified rodent diet (AIN‐93G), AIN‐93G +10% kcal apple pomace, Western diet (45% fat, 33% sucrose), or Western + 10% kcal apple pomace diets for 8 weeks. Histological evaluation showed rats consuming Western diets had the highest hepatic steatosis although apple pomace supplementation diet led to a lower degree of fat infiltration. Gene expression of hepatic lipogenesis by RT‐qPCR showed no significant differences of transcription factors, carbohydrate response element binding protein (ChREBP) and sterol element binding protein‐1c (SREBP‐1c), or the enzymes, fatty acid synthase (FAS) and stearoyl‐CoA desasturase‐1 (SCD‐1) among diet treatments. Rats fed Western diet had higher (p<0.01) gene expression of diacylglycerol O‐acyltransferase 2 (DGAT2) than Western + 10% apple pomace. The results suggest lower triacylglycerol synthesis reduced hepatic fat infiltration in rats fed Western diet + 10% apple pomace. In a hypercaloric state, apple pomace supplementation, attenuated hepatic steatosis induced by the consumption of a Western diet, despite the high levels of fructose in this supplement.Support or Funding InformationThis project was supported by Dean Daniel J. Robison, West Virginia University Davis College of Agriculture, Natural Resources, and Design and the West Virginia University Office of the Provost Fellowship.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

TXNIP as a Mediator of Palmitic Acid‐Induced Programmed Cell‐Death in Cardiomyocytes

INTRODUCTIONThioredoxinInteracting Protein (TXNIP), an endogenous inhibitor of the thioredoxinantioxidant system, is known to be elevated in diabetes, a condition characterized by abnormalities in both glucose and lipid metabolism. Underhyperglycemic conditions TXNIP is regulated in part by glucose responsive factors MondoA and ChREBP (carbohydrate response element binding protein) but littleis known about its role in mediating cardiac lipotoxicity. Plasma saturatedfatty acid (SFA) levels are elevated in type 2 diabetes and may lead tocardiomyocyte death and subsequently diabetic cardiomyopathy (DCM), a leading cause of morbidity and mortality in diabetic patients. The current aim is toelucidate the role of TXNIP in SFA‐driven programmed cell death (eitherapoptosis or pyroptosis) in vitro inAC16 cardiomyocytes and to identify potential mechanisms involved.METHODSAND RESULTSAC16 cardiomyocytes were treated with 150μMand 300μM palmitic acid(PA) for a period of 16 hours. TXNIP expression was determined by westernblotting of whole cell lysates in comparison to vehicle (control). Cytosolicand nuclear fractions were prepared using a suitable kit and analyzed bywestern blotting to determine activation of transcription factors MondoA andChREBP in response to PA. Apoptosis was measured by flow cytometry usingAnnexin V/PI staining while pyroptosis was determined by assessing inflammatorymarkers using RT‐PCR. To confirm the regulation of TXNIP by MondoA/ChREBP andthe involvement of TXNIP in PA‐driven cell death, TXNIP was inhibited bypre‐treating (24 hours) cells with 10μMSBI‐477, a chemical inhibitor of MondoA/ChREBP.PA showed adose‐dependent increase in TXNIP protein expression (1.5 and 2.3 ‐fold increaseat 150μM and 300μM PA respectively; ANOVA p<0.01 between groups; n=3) in comparison to the control. PA did not affect MondoA andChREBP protein expression but activated these transcription factors as demonstrated by an approximately 2‐fold increase in their nuclear expression. PA induceddose‐dependent apoptotic cell death in AC16 cardiomyocytes (1.3 and 4.4 ‐fold increase at 150μM and 300μM PA respectively; ANOVA p<0.001 between groups;n=4) which was inhibited by pre‐treatment with inhibitor SBI‐477 (81% declinerelative to 300μM PA alone; t‐test p<0.05; n=3). 300μM PA increased IL‐6 and IL‐8 gene expression by 56 and 15 ‐fold respectively but showed nosignificant change in the pyroptosis marker IL‐18.CONCLUSIONOurdata reflects a central role for TXNIP in SFA‐driven apoptosis. TXNIP could represent a key link between lipotoxicity and cardiomyocyte cell death, providing an attractive therapeutictarget to prevent SFA‐mediated cell death and in doing so stall the development of DCM. Further studies will be needed to establish the key players involved in SFA‐driven cardiomyocyte cell death and determine the programmed cell death pathways that predominate.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Effect of Dietary Alpha‐Linolenic Acid and Long‐Chain n‐3 Polyunsaturated Fatty Acids on Fructose‐Induced Hypertriglyceridemia, Hepatic Oxidative Stress and Inflammation

Impact of dietary n‐6/n‐3 polyunsaturated fatty acids (PUFA) ratio on the fructose‐induced hypertriglyceridemia, hepatic oxidative stress and inflammation was not explored previously. Here, we report the effect of diets containing n‐6 PUFA & α‐linolenic acid (ALA, 18:3 n‐3 PUFA) and n‐6 PUFA & long chain n‐3 PUFA (docosahexanoic acid and eicosapentaenoic acid, DHA and EPA) in the ratio of 2:1 and 5:1 respectively on fructose‐induced hypertriglyceridemia and hepatic oxidative stress and inflammation in the rats.Weanling Wistar rats were divided in to four groups and fed with diets containing starch (n‐6/n‐3 PUFA ratio 215:1), fructose (n‐6/n‐3 PUFA ratio 215:1) and fructose‐with n‐6/n‐3 PUFA ratio 2:1 (18:3 n‐3 PUFA) and 5:1 (long chain n‐3 PUFA) diets for twenty‐four weeks. Circulatory triglycerides were measured along with the hepatic triglyceride content. Gene expression of proteins belong to pro‐ and anti‐inflammatory cytokines, oxidative and endoplasmic stress along with pre‐receptor metabolism of glucocorticoids were quantified by quantitative PCR. Hepatic glutathione peroxidase and catalase activities were measured along with lipid peroxidation.Decreasing dietary n‐6/n‐3 PUFA ratio by supplementing ALA and LC n‐3 PUFA significantly reduced the fructose‐induced hypertriglyceridemia by 42% and 47% respectively (p≤0.05) without altering the hepatic weight and triglyceride content. ALA and long chain (LC) n‐3 PUFA feeding significantly reduced the fructose‐induced increase in lipogenic gene expression including stearoyl‐CoA desaturase, acetyl‐CoA carboxylase 1, carbohydrate‐responsive element‐binding protein, peroxisome proliferator‐activated receptor ‐ gamma, liver X receptor alpha, sterol regulatory element‐binding transcription factor 1, but only LC n‐3 PUFA feeding reduced the peroxisome proliferator‐activated receptor ‐ alpha, transcription factor regulating beta‐oxidation genes. ALA and LC n‐3 PUFA also reduced hepatic expression of 11betab‐hydroxysteroid dehydrogenase 1, an active glucocorticoid generating enzyme along with p47phox and glucose‐regulated protein 78 genes related to oxidative and ER stress respectively. Interestingly only, LC n‐3 PUFA‐feeding decreased the fructose‐induced expression of inflammatory genes including monocyte chemoattractant protein 1, tumor necrosis factor‐alpha and toll‐like receptor 4. Both ALA and LC n‐3 PUFA corrected the fructose‐induced reduction in glutathione peroxidase and catalase activity. ALA and n‐3 PUFA reduced the elevated lipid peroxidation significantly by 14% and 28% (p≤0.05).We conclude that replacement of dietary n‐6 PUFA with ALA and LC n‐3 PUFA ameliorates fructose‐induced hypertriglyceridemia and hepatic lipogenic gene expression. We also conclude that LC n‐3 PUFA is more protective when compared to ALA from fructose‐induced hepatic lipid peroxidation and only dietary LC n‐3 PUFA reduced fructose‐induced hepatic inflammatory genes. Though both ALA and LC n‐3 PUFA reduced fructose‐induced hypertriglyceridemia, considering protective effects of LC n‐3 PUFA on hepatic oxidative stress and inflammation, and as LC n‐3 PUFA exhibited protective nature at less quantity than ALA, we propose that diets with LC n‐3 PUFA gives better protection against fructose‐induced hepatic oxidative stress and inflammation.Support or Funding InformationThe present study was carried out with the financial support of Department of Biotechnology (DBT), Ministry of Science and Technology, Government of India, and Indian Council of Medical Research (ICMR), Government of India. AS was supported by Council of Scientific and Industrial Research (CSIR).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Targeting hepatic pyruvate dehydrogenase kinases restores insulin signaling and mitigates ChREBP-mediated lipogenesis in diet-induced obese mice

ObjectiveMitochondrial pyruvate dehydrogenase kinases 1–4 (PDKs1–4) negatively regulate activity of the pyruvate dehydrogenase complex (PDC) by reversible phosphorylation. PDKs play a pivotal role in maintaining energy homeostasis and contribute to metabolic flexibility by attenuating PDC activity in various mammalian tissues. Cumulative evidence has shown that the up-regulation of PDK4 expression is tightly associated with obesity and diabetes. In this investigation, we test the central hypothesis that PDKs1-4 are a pharmacological target for lowering glucose levels and restoring insulin sensitivity in obesity and type 2 diabetes (T2D). MethodsDiet-induced obese (DIO) mice were treated with a liver-specific pan-PDK inhibitor 2-[(2,4-dihydroxyphenyl) sulfonyl]isoindoline-4,6-diol (PS10) for four weeks, and results compared with PDK2/PDK4 double knockout (DKO) mice on the same high fat diet (HFD). ResultsBoth PS10-treated DIO mice and HFD-fed DKO mice showed significantly improved glucose, insulin and pyruvate tolerance, compared to DIO controls, with lower plasma insulin levels and increased insulin signaling in liver. In response to lower glucose levels, phosphorylated AMPK in PS10-treated DIO and HFD-fed DKO mice is upregulated, accompanied by decreased nuclear carbohydrate-responsive element binding protein (ChREBP). The reduced ChREBP signaling correlates with down-regulation of hepatic lipogenic enzymes (ACC1, FAS, and SCD1), leading to markedly diminished hepatic steatosis in both study groups, with lower circulating cholesterol and triacylglyceride levels as well as reduced fat mass. PS10-treated DIO as well as DKO mice showed predominant fatty acid over glucose oxidation. However, unlike systemic DKO mice, increased hepatic PDC activity alone in PS10-treated DIO mice does not raise the plasma total ketone body level. ConclusionOur findings establish that specific targeting of hepatic PDKs with the PDK inhibitor PS10 is an effective therapeutic approach to maintaining glucose and lipid homeostasis in obesity and T2D, without the harmful ketoacidosis associated with systemic inhibition of PDKs.