Carbohydrate Response Element-binding Protein Activity Research Articles

Sweet dreams could be made of this: carbohydrate-responsive element-binding protein (ChREBP) as a target for hepatocellular carcinoma therapy.

Rewiring of cellular metabolism is now fully recognized as a hallmark of cancer. Tumor cells reprogram metabolic pathways to meet the energetic and macromolecular demands to support unrestricted growth and survival under unfavorable conditions. It is becoming apparent that these adaptations underpin most of the traits that define a cancer cell's identity, including the ability to avoid immune surveillance, endure nutrient and oxygen restrictions, detach and migrate from their natural histological niche, and avert human-made aggressions (i.e., therapy). In a recent study, Benichou and collaborators identify carbohydrate-responsive element-binding protein (ChREBP), a master regulator of physiological glucose metabolism, as an oncogene in hepatocellular carcinoma (HCC) development. Upregulation of ChREBP expression results in a self-stimulatory loop interconnecting PI3K/AKT signaling and glucose metabolism to feed fatty acid and nucleotide synthesis supporting tumorigenesis. Importantly, pharmacological inhibition of ChREBP activity quells in vivo HCC tumor growth without causing systemic toxicity. This study identifies novel oncometabolic pathways and open up new avenues to improve the treatment of a deadly tumor.

Activation of skeletal carbohydrate-response element binding protein (ChREBP)-mediated de novo lipogenesis increases intramuscular fat content in chickens

The intracellular lipids in muscle cells of farm animals play a crucial role in determining the overall intramuscular fat (IMF) content, which has a positive impact on meat quality. However, the mechanisms underlying the deposition of lipids in muscle cells of farm animals are not yet fully understood. The purpose of this study was to determine the roles of carbohydrate-response element binding protein (ChREBP) and fructose in IMF deposition of chickens. For virus-mediated ChREBP overexpression in tibialis anterior (TA) muscle of chickens, seven 5-d-old male yellow-feather chickens were used. At 10 d after virus injection, the chickens were slaughtered to obtain TA muscles for analysis. For fructose administration trial, sixty 9-wk-old male yellow-feather chickens were randomly divided into 2 groups, with 6 replicates per group and 5 chickens per replicate. The chickens were fed either a basal diet or a basal diet supplemented with 10% fructose (purity ≥ 99%). At 4 wk later, the chickens were slaughtered, and breast and thigh muscles were collected for analysis. The results showed that the skeletal ChREBP mRNA levels were positively associated with IMF content in multiple species, including the chickens, pigs, and mice (P < 0.05). ChREBP overexpression increased lipid accumulation in both muscle cells in vitro and the TA muscles of mice and chickens in vivo (P < 0.05), by activation of the de novo lipogenesis (DNL) pathway. Moreover, activation of ChREBP by dietary fructose administration also resulted in increased IMF content in mice and notably chickens (P < 0.05). Furthermore, the lipidomics analysis revealed that ChREBP activation altered the lipid composition of chicken IMF and tented to improve the flavor profile of the meat. In conclusion, this study found that ChREBP plays a pivotal role in mediating the deposition of fat in chicken muscles in response to fructose-rich diets, which provides a novel strategy for improving meat quality in the livestock industry.

The transcription factor ChREBP Orchestrates liver carcinogenesis by coordinating the PI3K/AKT signaling and cancer metabolism

Cancer cells integrate multiple biosynthetic demands to drive unrestricted proliferation. How these cellular processes crosstalk to fuel cancer cell growth is still not fully understood. Here, we uncover the mechanisms by which the transcription factor Carbohydrate responsive element binding protein (ChREBP) functions as an oncogene during hepatocellular carcinoma (HCC) development. Mechanistically, ChREBP triggers the expression of the PI3K regulatory subunit p85α, to sustain the activity of the pro-oncogenic PI3K/AKT signaling pathway in HCC. In parallel, increased ChREBP activity reroutes glucose and glutamine metabolic fluxes into fatty acid and nucleic acid synthesis to support PI3K/AKT-mediated HCC growth. Thus, HCC cells have a ChREBP-driven circuitry that ensures balanced coordination between PI3K/AKT signaling and appropriate cell anabolism to support HCC development. Finally, pharmacological inhibition of ChREBP by SBI-993 significantly suppresses in vivo HCC tumor growth. Overall, we show that targeting ChREBP with specific inhibitors provides an attractive therapeutic window for HCC treatment.

A high throughput screen for pharmacological inhibitors of the carbohydrate response element

A central regulator of metabolism, transcription factor carbohydrate response element binding protein (ChREBP) senses and responds to dietary glucose levels by stimulating the transcription of glycolytic and lipogenic enzymes. Genetic depletion of ChREBP rescues β-cell dysfunction arising from high glucose levels, suggesting that inhibiting ChREBP might represent an attractive therapeutic approach to manage diabetes and other metabolic diseases. However, the molecular mechanisms governing ChREBP activation are poorly understood and chemical tools to probe the cellular activity of ChREBP are lacking. Here, we report a high-throughput pharmacological screen in INS-1E β-cells that identified novel inhibitors of ChREBP-driven transcription at carbohydrate response element sites, including three putative covalent inhibitors and two likely non-covalent chemical scaffolds. This work affords a pharmacological toolkit to help uncover the signaling logic controlling ChREBP activation and may ultimately reveal potential therapeutic approaches for treating metabolic disease.

F6P/G6P-mediated ChREBP activation promotes the insulin resistance-driven hepatic lipid deposition in zebrafish

Insulin-sensitive lipogenesis dominates the body lipid deposition; however, nonalcoholic fatty liver disease (NAFLD) develops in the insulin-resistant state. The regulation mechanism of insulin resistance-driven NAFLD remains elusive. Using zebrafish model of insulin resistance (ZIR, insrb−/−) and mouse hepatocytes (NCTC 1469), we explored the regulation mechanism of insulin resistance-driven hepatic lipid deposition under the stimulation of carbohydrate diet (CHD). In ZIR model, insulin resistance induced hyperlipidemia and elevated hepatic lipid deposition via elevating the gene/protein expressions of lipogenic enzymes, that was activated by carbohydrate response element binding protein (ChREBP), rather than sterol regulatory element binding proteins 1c (SREBP-1c). The metabolomic analysis in zebrafish and silencing of chrebp in mouse hepatocytes revealed that the increased hepatic frucotose-6-phosphate (F6P) and glucose-6-phosphate (G6P) promoted the ChREBP-mediated lipid deposition. We further identified that F6P alone was sufficient to activate ChREBP-mediated lipid deposition by a SREBP-1c-independent manner. Moreover, we clarified the suppressed hepatic phosphofructokinase/glucose-6-phosphatase functions and the normal glucokinase function preserved by glucose transporter 2 (GLUT2) manipulated the increased F6P/G6P content in ZIR. In conclusion, the present study revealed that insulin resistance promoted hepatic lipid deposition via the F6P/G6P-mediated ChREBP activation. Our findings deciphered the main regulation pathway for the liver lipid deposition in the insulin-resistant state and identified F6P as a new potential regulator for ChREBP.

Normalization of hepatic ChREBP activity does not protect against liver disease progression in a mouse model for Glycogen Storage Disease type Ia

BackgroundGlycogen storage disease type 1a (GSD Ia) is an inborn error of metabolism caused by a defect in glucose-6-phosphatase (G6PC1) activity, which induces severe hepatomegaly and increases the risk for liver cancer. Hepatic GSD Ia is characterized by constitutive activation of Carbohydrate Response Element Binding Protein (ChREBP), a glucose-sensitive transcription factor. Previously, we showed that ChREBP activation limits non-alcoholic fatty liver disease (NAFLD) in hepatic GSD Ia. As ChREBP has been proposed as a pro-oncogenic molecular switch that supports tumour progression, we hypothesized that ChREBP normalization protects against liver disease progression in hepatic GSD Ia.MethodsHepatocyte-specific G6pc knockout (L-G6pc−/−) mice were treated with AAV-shChREBP to normalize hepatic ChREBP activity.ResultsHepatic ChREBP normalization in GSD Ia mice induced dysplastic liver growth, massively increased hepatocyte size, and was associated with increased hepatic inflammation. Furthermore, nuclear levels of the oncoprotein Yes Associated Protein (YAP) were increased and its transcriptional targets were induced in ChREBP-normalized GSD Ia mice. Hepatic ChREBP normalization furthermore induced DNA damage and mitotic activity in GSD Ia mice, while gene signatures of chromosomal instability, the cytosolic DNA-sensing cGAS-STING pathway, senescence, and hepatocyte dedifferentiation emerged.ConclusionsIn conclusion, our findings indicate that ChREBP activity limits hepatomegaly while decelerating liver disease progression and protecting against chromosomal instability in hepatic GSD Ia. These results disqualify ChREBP as a therapeutic target for treatment of liver disease in GSD Ia. In addition, they underline the importance of establishing the context-specific roles of hepatic ChREBP to define its therapeutic potential to prevent or treat advanced liver disease.

The role of ChREBP in carbohydrate sensing and NAFLD development.

Excessive sugar consumption and defective glucose sensing by hepatocytes contribute to the development of metabolic diseases including type 2 diabetes mellitus (T2DM) and nonalcoholic fatty liver disease (NAFLD). Hepatic metabolism of carbohydrates into lipids is largely dependent on the carbohydrate-responsive element binding protein (ChREBP), a transcription factor that senses intracellular carbohydrates and activates many different target genes, through the activation of de novo lipogenesis (DNL). This process is crucial for the storage of energy as triglycerides in hepatocytes. Furthermore, ChREBP and its downstream targets represent promising targets for the development of therapies for the treatment of NAFLD and T2DM. Although lipogenic inhibitors (for example, inhibitors of fatty acid synthase, acetyl-CoA carboxylase or ATP citrate lyase) are currently under investigation, targeting lipogenesis remains a topic of discussion for NAFLD treatment. In this Review, we discuss mechanisms that regulate ChREBP activity in a tissue-specific manner and their respective roles in controlling DNL and beyond. We also provide in-depth discussion of the roles of ChREBP in the onset and progression of NAFLD and consider emerging targets for NAFLD therapeutics.

PS-BPB06-3: EXAMINATION OF THE EFFECTIVENESS OF DIABETIC NEPHROPATHY TREATMENT TARGETING CHREBP ACTIVITY

Introduction: Diabetic nephropathy (DN) is the main cause of chronic kidney disease (CKD), and is characterized by elevated urinary albumin to creatinine ratio (UACR) and serum creatinine, and decreased glomerular filtration rate. Anti hypertensive agents are used for DN treatment, however, the effects of these drugs are insufficient, and the establishment of more effective treatment methods is desired. Carbohydrate responsive element binding protein (ChREBP) is one of the factors reported to be involved in the onset and progression of DN. Furthermore, whole body ablation of ChREBP suggested that the progression of DN was suppressed in the rodent model. On the other hand, the effectiveness of pharmaceutical ChREBP suppression is unclear, thus, we performed administration experiment to DN model mice using ChREBP activity inhibitor and examined the effects of pharmaceutical ChREBP activity suppression on renal function of diabetic mice. Results: First, we established high-throughput screening system capable of detecting ChREBP activity and performed screening of Tohoku university compound library. The result compound, D-532, was ChREBP activity specific inhibitor without cell toxicity. D-532 directly binds to DNA-binding domain of ChREBP, and inhibits the transcriptional activity. We next performed administration experiment using type 1 DN model mice. D-532 treatment group showed an improvement tendency of the low body weight compared with that of vehicle treatment group, while it had no effect on blood glucose level. About renal functions, both of UACR and serum creatinine were significantly lower in D-532 treatment group. Importantly, renal tissues such as glomeruli and renal tubules were preserved in D-532 treatment group compared with in vehicle treatment group. In vivo as well, D-532 suppressed ChREBP target genes mRNA expression, and RNA-sequence analysis with mice renal cortex RNA at the end of D-532 administration experiment indicated that D-532 suppressed excessive oxidative stress through the ChREBP activation under diabetes. Conclusion: These results indicate that ChREBP suppression is effective on DN rodent model, and ChREBP activity inhibitor D-532 may have potential as a new treatment for DN in the future.

LBODP050 Examination Of The Effectiveness Of Diabetic Nephropathy Treatment Targeting Chrebp Activity

Abstract Diabetic nephropathy (DN) is the main cause of chronic kidney disease (CKD) and is characterized by elevated urinary albumin to creatinine ratio (UACR) and serum creatinine, and decreased glomerular filtration rate. Anti hypertensive agents are used for DN treatment, however, the effects of these drugs are insufficient, and the establishment of more effective treatment methods is desired. Carbohydrate responsive element binding protein (ChREBP) is one of the factors reported to be involved in the onset and progression of DN (1). Furthermore, whole body ablation of ChREBP suggested that the progression of DN was suppressed in the rodent model (2). On the other hand, the effectiveness of pharmaceutical ChREBP suppression is unclear, thus, we performed administration experiment to DN model mice using ChREBP activity inhibitor and examined the effects of pharmaceutical ChREBP activity suppression on renal function of diabetic mice. First, we established high-throughput screening system capable of detecting ChREBP activity and performed screening of Tohoku university compound library. The result compound, D-532, was ChREBP activity specific inhibitor without cell toxicity. D-532 directly binds to DNA-binding domain of ChREBP, and inhibits the transcriptional activity. We next performed administration experiment using type I DN model mice. D-532 treatment group showed an improvement tendency of the low body weight compared with that of vehicle treatment group, while it had no effect on blood glucose level. About renal functions, both of UACR and serum creatinine were significantly lower in D-532 treatment group. Importantly, renal tissues such as glomeruli and renal tubules were preserved in D-532 treatment group compared with in vehicle treatment group. In vivo as well, D-532 suppressed ChREBP target genes mRNA expression, and RNA-seq analysis with mice renal cortex RNA at the end of D-532 administration experiment indicated that D-532 suppressed excessive oxidative stress through the ChREBP activation under diabetes. These results indicate that ChREBP suppression is effective on DN rodent model, and ChREBP activity inhibitor D-532 may have potential as a new treatment for DN in the future. Reference: (1) Suzuki et al., Endocr J. 2019 Mar 28;67(3): 335-345. (2) Zhang et al., Eur Rev Med Pharmacol Sci. 2017 Jan;21(1): 42-47. Presentation: No date and time listed

CHREBP suppresses gastric cancer progression via the cyclin D1-Rb-E2F1 pathway

Accumulating evidence has demonstrated that carbohydrate response element binding protein (CHREBP) has a crucial function in tumor pathology. In this study, we found CHREBP downregulation in gastric cancer (GC) tissues, and CHREBP was determined to be an independent diagnostic marker of GC. The downregulation of CHREBP promoted cell proliferation and inhibited apoptosis. Moreover, the level of cyclin D1 was significantly correlated with CHREBP expression in GC and paracancerous normal samples. In addition, CHREBP transcriptionally inhibited cyclin D1 expression in GC cells. Tumor suppressor activity of CHREBP could be affected by the upregulation of cyclin D1. In summary, CHREBP was found to be an independent diagnostic marker of GC and to influence GC growth and apoptosis via targeting the cyclin D1-Rb-E2F1 pathway.

The Roles of Carbohydrate Response Element Binding Protein in the Relationship between Carbohydrate Intake and Diseases.

Carbohydrates are macronutrients that serve as energy sources. Many studies have shown that carbohydrate intake is nonlinearly associated with mortality. Moreover, high-fructose corn syrup (HFCS) consumption is positively associated with obesity, cardiovascular disease, and type 2 diabetes mellitus (T2DM). Accordingly, products with equal amounts of glucose and fructose have the worst effects on caloric intake, body weight gain, and glucose intolerance, suggesting that carbohydrate amount, kind, and form determine mortality. Understanding the role of carbohydrate response element binding protein (ChREBP) in glucose and lipid metabolism will be beneficial for elucidating the harmful effects of high-fructose corn syrup (HFCS), as this glucose-activated transcription factor regulates glycolytic and lipogenic gene expression. Glucose and fructose coordinately supply the metabolites necessary for ChREBP activation and de novo lipogenesis. Chrebp overexpression causes fatty liver and lower plasma glucose levels, and ChREBP deletion prevents obesity and fatty liver. Intestinal ChREBP regulates fructose absorption and catabolism, and adipose-specific Chrebp-knockout mice show insulin resistance. ChREBP also regulates the appetite for sweets by controlling fibroblast growth factor 21, which promotes energy expenditure. Thus, ChREBP partly mimics the effects of carbohydrate, especially HFCS. The relationship between carbohydrate intake and diseases partly resembles those between ChREBP activity and diseases.

NF-κB p65 regulates hepatic lipogenesis by promoting nuclear entry of ChREBP in response to a high carbohydrate diet

Overconsumption of sucrose and other sugars has been associated with nonalcoholic fatty liver disease (NAFLD). Reports suggest hepatic de novo lipogenesis (DNL) as an important contributor to and regulator of carbohydrate-induced hepatic lipid accumulation in NAFLD. The mechanisms responsible for the increase in hepatic DNL due to overconsumption of carbohydrate diet are less than clear; however, literatures suggest high carbohydrate diet to activate the lipogenic transcription factor carbohydrate response element-binding protein (ChREBP), which further transcribes genes involved in DNL. Here, we provide an evidence of an unknown link between nuclear factor kappa-light chain enhancer of activated B cells (NF-κB) activation and increased DNL. Our data indicates high carbohydrate diet to enforce nuclear shuttling of hepatic NF-κB p65 and repress transcript levels of sorcin, a cytosolic interacting partner of ChREBP. Reduced sorcin levels, further prompted ChREBP nuclear translocation, leading to enhanced DNL and intrahepatic lipid accumulation both in vivo and in vitro. We further report that pharmacological inhibition of NF-κB abrogated high carbohydrate diet–mediated sorcin repression and thereby prevented ChREBP nuclear translocation and this, in turn, attenuated hepatic lipid accumulation both in in vitro and in vivo. Additionally, sorcin knockdown blunted the lipid-lowering ability of the NF-κB inhibitor in vitro. Together, these data suggest a heretofore unknown role for NF-κB in regulating ChREBP nuclear localization and activation, in response to high carbohydrate diet, for further explorations in lines of NAFLD therapeutics.

The glucose-sensing transcription factor ChREBP is targeted by proline hydroxylation

Cellular energy demands are met by uptake and metabolism of nutrients like glucose. The principal transcriptional regulator for adapting glycolytic flux and downstream pathways like de novo lipogenesis to glucose availability in many cell types is carbohydrate response element-binding protein (ChREBP). ChREBP is activated by glucose metabolites and post-translational modifications, inducing nuclear accumulation and regulation of target genes. Here we report that ChREBP is modified by proline hydroxylation at several residues. Proline hydroxylation targets both ectopically expressed ChREBP in cells and endogenous ChREBP in mouse liver. Functionally, we found that specific hydroxylated prolines were dispensable for protein stability but required for the adequate activation of ChREBP upon exposure to high glucose. Accordingly, ChREBP target gene expression was rescued by re-expressing WT but not ChREBP that lacks hydroxylated prolines in ChREBP-deleted hepatocytes. Thus, proline hydroxylation of ChREBP is a novel post-translational modification that may allow for therapeutic interference in metabolic diseases.

Hepatic Carbohydrate Response Element Binding Protein Activation Limits Nonalcoholic Fatty Liver Disease Development in a Mouse Model for Glycogen Storage Disease Type 1a.

Background and AimsGlycogen storage disease (GSD) type 1a is an inborn error of metabolism caused by defective glucose‐6‐phosphatase catalytic subunit (G6PC) activity. Patients with GSD 1a exhibit severe hepatomegaly due to glycogen and triglyceride (TG) accumulation in the liver. We have shown that the activity of carbohydrate response element binding protein (ChREBP), a key regulator of glycolysis and de novo lipogenesis, is increased in GSD 1a. In the current study, we assessed the contribution of ChREBP to nonalcoholic fatty liver disease (NAFLD) development in a mouse model for hepatic GSD 1a.Approach and ResultsLiver‐specific G6pc–knockout (L‐G6pc −/−) mice were treated with adeno‐associated viruses (AAVs) 2 or 8 directed against short hairpin ChREBP to normalize hepatic ChREBP activity to levels observed in wild‐type mice receiving AAV8–scrambled short hairpin RNA (shSCR). Hepatic ChREBP knockdown markedly increased liver weight and hepatocyte size in L‐G6pc −/− mice. This was associated with hepatic accumulation of G6P, glycogen, and lipids, whereas the expression of glycolytic and lipogenic genes was reduced. Enzyme activities, flux measurements, hepatic metabolite analysis and very low density lipoprotein (VLDL)‐TG secretion assays revealed that hepatic ChREBP knockdown reduced downstream glycolysis and de novo lipogenesis but also strongly suppressed hepatic VLDL lipidation, hence promoting the storage of “old fat.” Interestingly, enhanced VLDL‐TG secretion in shSCR‐treated L‐G6pc −/− mice associated with a ChREBP‐dependent induction of the VLDL lipidation proteins microsomal TG transfer protein and transmembrane 6 superfamily member 2 (TM6SF2), the latter being confirmed by ChIP‐qPCR.ConclusionsAttenuation of hepatic ChREBP induction in GSD 1a liver aggravates hepatomegaly because of further accumulation of glycogen and lipids as a result of reduced glycolysis and suppressed VLDL‐TG secretion. TM6SF2, critical for VLDL formation, was identified as a ChREBP target in mouse liver. Altogether, our data show that enhanced ChREBP activity limits NAFLD development in GSD 1a by balancing hepatic TG production and secretion.

The structure of importin α and the nuclear localization peptide of ChREBP, and small compound inhibitors of ChREBP–importin α interactions

The carbohydrate response element binding protein (ChREBP) is a glucose-responsive transcription factor that plays a critical role in glucose-mediated induction of genes involved in hepatic glycolysis and lipogenesis. In response to fluctuating blood glucose levels ChREBP activity is regulated mainly by nucleocytoplasmic shuttling of ChREBP. Under high glucose ChREBP binds to importin α and importin β and translocates into the nucleus to initiate transcription. We have previously shown that the nuclear localization signal site (NLS) for ChREBP is bipartite with the NLS extending from Arg158 to Lys190. Here, we report the 2.5 Å crystal structure of the ChREBP-NLS peptide bound to importin α. The structure revealed that the NLS binding is monopartite, with the amino acid residues K171RRI174 from the ChREBP-NLS interacting with ARM2–ARM5 on importin α. We discovered that importin α also binds to the primary binding site of the 14-3-3 proteins with high affinity, which suggests that both importin α and 14-3-3 are each competing with the other for this broad-binding region (residues 117–196) on ChREBP. We screened a small compound library and identified two novel compounds that inhibit the ChREBP-NLS/importin α interaction, nuclear localization, and transcription activities of ChREBP. These candidate molecules support developing inhibitors of ChREBP that may be useful in treatment of obesity and the associated diseases.

Reduced Nogo expression inhibits diet-induced metabolic disorders by regulating ChREBP and insulin activity

Chronic overconsumption of a high-carbohydrate diet leads to steatosis and its associated metabolic disorder and, eventually, to non-alcoholic fatty liver disease. Carbohydrate-responsive element binding protein (ChREBP) and insulin regulate de novo lipogenesis from glucose. Herein, we studied the effect of reticulon-4 (Nogo) expression on diet-induced metabolic disorders in mice. Nogo-deficient (Nogo-/-) and littermate control [wild-type (WT)] mice were fed a high-glucose or high-fructose diet (HGD/HFrD) to induce metabolic disorders. The effects of Nogo small interfering (si) RNA (siRNA) on HFrD-induced metabolic disorders were investigated in C57BL/6J mice. HGD/HFrD induced steatosis and its associated metabolic disorders in WT mice by activating ChREBP and impairing insulin sensitivity. They also activated Nogo-B expression, which in turn inhibited insulin activity. In response to HGD/HFrD feeding, Nogo deficiency enhanced insulin sensitivity and energy metabolism to reduce the expression of ChREBP and lipogenic molecules, activated AMP-activated catalytic subunit α, peroxisome proliferator activated receptor α and fibroblast growth factor 21, and reduced endoplasmic reticulum (ER) stress and inflammation, thereby blocking HGD/HFrD-induced hepatic lipid accumulation, insulin resistance and other metabolic disorders. Injection of Nogo siRNA protected C57BL/6J mice against HFrD-induced metabolic disorders by ameliorating insulin sensitivity, ChREBP activity, ER stress and inflammation. Our study identified Nogo as an important mediator of insulin sensitivity and ChREBP activity. Reduction of Nogo expression is a potential strategy for the treatment of high-carbohydrate diet-induced metabolic complications. Nogo deficiency blocks high-carbohydrate diet-induced glucose intolerance and insulin resistance, while increasing glucose/lipid utilisation and energy expenditure. Thus, reduction of Nogo expression protects against high-carbohydrate diet-induced body-weight gain, hepatic lipid accumulation and the associated metabolic disorders, indicating that approaches inhibiting Nogo expression can be applied for the treatment of diseases associated with metabolic disorders.

Carb-Loading: Freeze-Induced Activation of the Glucose-Responsive ChREBP Transcriptional Network in Wood Frogs.

The freeze-tolerant wood frog, Rana sylvatica, is one of few vertebrate organisms that can tolerate freezing, with up to 70% of its total body water being converted into extracellular ice. Physiologically, wood frogs show no signs of muscle movement, breathing, heartbeat, or brain activity for several weeks or months at a time but emerge unharmed upon thawing. Given that wood frogs rely mainly on carbohydrate metabolism during freezing, the involvement of the carbohydrate-responsive element-binding protein (ChREBP) in response to freezing is of interest. In liver tissue, protein and transcript levels of ChREBP increased by and , respectively, and nuclear distribution and DNA-binding activity rose by and , respectively. This enhanced transcriptional activity of ChREBP was corroborated by increased transcript expression of select downstream genes of fatty acid synthase, pyruvate kinase, and stearoyl-CoA desaturase in liver tissue. While liver tissue displayed ChREBP activation, in muscle tissue, ChREBP protein levels, DNA-binding activity, and downstream gene targets were generally found to decrease or to remain unchanged during freezing. Overall, our results demonstrate that ChREBP regulates metabolism in a tissue-dependent manner during freezing, when its activity is required for liver tissue but not for skeletal muscle tissue in wood frogs.

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&amp;lt;0.01). Treatment of INS-1 cells with adenovirus expressing recombinant human PPM1K for 96h increased β cell proliferation (P&amp;lt;0.05) but robustly lowered GSIS (P&amp;lt;0.001). This effect was due to substantially lower insulin content in the Ad-PPm1K treated cells (P&amp;lt;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.)

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.

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.