Carbohydrate Response Element Binding Protein mRNA Research Articles

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.

Carbohydrate response element binding protein (ChREBP) correlates with colon cancer progression and contributes to cell proliferation

Cancers are characterized by reprogrammed glucose metabolisms to fuel cell growth and proliferation. Carbohydrate response element binding protein (ChREBP) is a glucose-mediated transcription factor that strongly regulates glycolytic and lipogenic pathways. It has been shown to associate with metabolic diseases, such as obesity, diabetes and non-alcoholic fatty liver diseases. However, how it associates with cancers has not been well understood. In this study, ChREBP expression was assessed by immunohistochemistry in colon tissue arrays containing normal colon tissue and cancer tissue at different clinical stages. Tissue mRNA levels of ChREBP were also measured in a cohort of colon cancer patients. We found that ChREBP mRNA and protein expression were significantly increased in colon cancer tissue compared to healthy colon (p < 0.001), and their expression was positively correlated to colon malignancy (for mRNA, p = 0.002; for protein p < 0.001). Expression of lipogenic genes (ELOVL6 and SCD1) in colon cancer was also positively associated with colon malignancy (for both genes, p < 0.001). In vitro, ChREBP knockdown with siRNA transfection inhibited cell proliferation and induced cell cycle arrest without changes in apoptosis in colon cancer cell lines (HT29, DLD1 and SW480). Glycolytic and lipogenic pathways were inhibited but the p53 pathway was activated after ChREBP knockdown. Taken together, ChREBP expression is associated with colon malignancy and it might contribute to cell proliferation via promoting anabolic pathways and inhibiting p53. In addition, ChREBP might represent a novel clinical useful biomarker to evaluate the malignancy of colon cancer.

Role of carbohydrate response element-binding protein (ChREBP) in generating an aerobic metabolic phenotype and in breast cancer progression.

Background:The lipogenic transcription factor carbohydrate response element-binding protein (ChREBP) may play a key role in malignant progression of breast cancer by allowing metabolic adaptations to take place in response to changes in oxygenation.Methods:Immunohistochemical analysis of ChREBP was carried out in human breast tumour tissue microarrays representative of malignant progression from normal breast through to metastatic cancer. The ChREBP protein and mRNA expressions were then analysed in a series of breast cancers for correlative analysis with common and breast-specific hypoxia signatures, and survival.Results:In invasive ductal carcinoma, ChREBP correlated significantly with mean ‘downregulated' hypoxia scores (r=0.3, P<0.015, n=67) and in two distinct breast progression arrays, ChREBP protein also increased with malignant progression (P<0.001). However, bioinformatic analysis of a large data set (2136 cases) revealed an apparent reversal in the relationship between ChREBP mRNA level and clinical outcome – not only being significantly correlated with increased survival (log rank P<0.001), but also downregulated in malignant tissue compared with adjacent normal tissue.Conclusion:The ChREBP expression may be reflective of an aerobic metabolic phenotype that may conflict with hypoxia-induced signalling but provide a mechanism for growth at the oxygenated edge of the tumours.

Molecular Mechanisms of Estrogen Receptors' Suppression of Lipogenesis in Pancreatic β-Cells

The gonadal steroid, 17β-estradiol (E2), suppresses pancreatic islet fatty acid and glycerolipid synthesis and prevents β-cell failure in rodent models of type 2 diabetes. β-Cell estrogen receptors (ER) mediate these actions by suppressing the expression and enzymatic activity of fatty acid synthase (FAS). Here, we explored the mechanism of FAS suppression. We show that E2, and pharmacological agonists for ERα, ERβ, and the G protein-coupled ER, suppress mRNA and protein expression of the transcriptional regulators of FAS, namely, sterol regulatory element-binding protein 1c (SREBP1c) and carbohydrate response element binding protein (ChREBP) in insulin-secreting INS-1 cells. ER suppress SREBP1c and ChREBP mRNA and protein expression via an extranuclear localization. Using two mouse lines with pancreas-specific null deletion of either ERα or the signal transducer and activator of transcription 3 (STAT3), we show that ERα activation in vivo reduces SREBP1c and ChREBP mRNA expression via a direct islet action involving STAT3 activation. The master regulators of lipogenesis, liver X receptor (LXR) α and β, transcriptionally up-regulate SREBP1c and ChREBP. We find that activation of ERα, ERβ, and G protein-coupled ER suppresses LXR's mRNA expression in INS-1 cells. We also observe that activation of ERα in mouse islets in vivo suppresses LXR mRNA in a STAT3-dependent manner. Finally, we show that E2 also activates and uses AMP-activated protein kinase in INS-1 cells to suppress SREBP1c protein expression. This study identifies extranuclear ER pathways involving STAT3 and AMP-activated protein kinase in the genetic control of lipogenesis with therapeutic implications to protect β-cells in type 2 diabetes.

CREB and ChREBP oppositely regulate SIRT1 expression in response to energy availability.

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

Thyroid Hormone Receptor β (TRβ) and Liver X Receptor (LXR) Regulate Carbohydrate-response Element-binding Protein (ChREBP) Expression in a Tissue-selective Manner

Thyroid hormone (TR) and liver X (LXR) receptors are transcription factors involved in lipogenesis. Both receptors recognize the same consensus DNA-response element in vitro. It was previously shown that their signaling pathways interact in the control of cholesterol elimination in the liver. In the present study, carbohydrate-response element-binding protein (ChREBP), a major transcription factor controlling the activation of glucose-induced lipogenesis in liver, is characterized as a direct target of thyroid hormones (TH) in liver and white adipose tissue (WAT), the two main lipogenic tissues in mice. Using genetic and molecular approaches, ChREBP is shown to be specifically regulated by TRbeta but not by TRalpha in vivo, even in WAT where both TR isoforms are expressed. However, this isotype specificity is not found in vitro. This TRbeta specific regulation correlates with the loss of TH-induced lipogenesis in TRbeta(-/-) mice. Fasting/refeeding experiments show that TRbeta is not required for the activation of ChREBP expression particularly marked in WAT following refeeding. However, TH can stimulate ChREBP expression in WAT even under fasting conditions, suggesting completely independent pathways. Because ChREBP has been described as an LXR target, the interaction of LXR and TRbeta in ChREBP regulation was assayed both in vitro and in vivo. Each receptor recognizes a different response element on the ChREBP promoter, located only 8 bp apart. There is a cross-talk between LXR and TRbeta signaling on the ChREBP promoter in liver but not in WAT where LXR does not regulate ChREBP expression. The molecular basis for this cross-talk has been determined in in vitro systems.

Hepatic FoxO1 Ablation Exacerbates Lipid Abnormalities during Hyperglycemia

Patients with diabetes suffer disproportionately from impaired lipid metabolism and cardiovascular disease, but the relevant roles of insulin resistance and hyperglycemia in these processes are unclear. Transcription factor FoxO1 is regulated dually by insulin and nutrients. In this study, we addressed the hypothesis that, in addition to its established role to regulate hepatic glucose production, FoxO1 controls aspects of lipid metabolism in the diabetic liver. Mice with a liver-specific deletion of FoxO1 (L-FoxO1) and their control littermates were rendered hyperglycemic by streptozotocin administration. Subsequently, we monitored serum lipids, liver VLDL secretion, and hepatic expression of genes related to lipid metabolism. Hepatic FoxO1 ablation resulted in increased VLDL secretion, increased cholesterol, and increased plasma free fatty acids, three hallmarks of the diabetic state. l-FoxO1 mice expressed increased levels of SREBP-2 and FGF21 without affecting lipogenic genes. We propose that FoxO1 fine tunes lipolysis through its actions on FGF21 and that hepatic FoxO1 ablation increases availability of substrates for hepatic triglyceride and cholesterol synthesis and VLDL secretion. The implications of these findings are that FoxO1 protects against excessive hepatic lipid production during hyperglycemia and that its inhibition by intensive insulin treatment may exacerbate paradoxically the lipid abnormalities of diabetes.

Endotoxin, zymosan, and cytokines decrease the expression of the transcription factor, carbohydrate response element binding protein, and its target genes

Carbohydrate response element binding protein (ChREBP) is a recently discovered transcription factor whose levels and activity are increased by glucose leading to the activation of target genes, which include acetyl-CoA carboxylase, fatty acid synthase, and liver-type pyruvate kinase. Here, we demonstrate that lipopolysaccharide (LPS) treatment causes a marked decrease in ChREBP mRNA and protein levels in the liver of mice fed a normal chow diet or in mice fasted for 24 h and then re-fed a high carbohydrate diet. This decrease occurs rapidly and is a sensitive response (half-maximal dose 0.1 μg/mouse). The decrease in ChREBP is accompanied by a decrease in the expression of ChREBP target genes. Zymosan and turpentine treatment also decrease hepatic ChREBP levels and the expression of its target genes. Additionally, tumor necrosis factor alpha (TNF-α) and interleukin-1 beta (IL-1β) decrease liver ChREBP expression both in vivo and in Hep3B cells in culture. Finally, LPS decreased ChREBP expression in muscle and adipose tissue. These studies demonstrate that ChREBP is down-regulated during the acute phase response resulting in alterations in the expression of ChREBP regulated target genes. Thus, ChREBP joins a growing list of transcription factors that are regulated during the acute phase response.

Insulin Stimulates the Expression of Carbohydrate Response Element Binding Protein (ChREBP) by Attenuating the Repressive Effect of Pit-1, Oct-1/Oct-2, and Unc-86 Homeodomain Protein Octamer Transcription Factor-1

The carbohydrate response element binding protein (ChREBP) has been recognized as a key controller of hepatic lipogenesis. Whereas the function of ChREBP has been extensively investigated, mechanisms underlying its transcription remain largely unknown, although ChREBP production is elevated in a hyperinsulinemic mouse model. We located a conserved Pit-1, Oct-1/Oct-2, and Unc-86 (POU) protein binding site (ATGCTAAT) within the proximal promoter region of human ChREBP. This site interacts with the POU homeodomain protein octamer transcription factor-1 (Oct-1), as detected by gel shift and chromatin immunoprecipitation assays. Oct-1 cotransfection in the human HepG2 cell line repressed ChREBP promoter activity approximately 50-75% (P < 0.01 to P < 0.001), and this repression was dependent on the existence of the POU binding site. Furthermore, overexpression of Oct-1 repressed endogenous ChREBP mRNA and protein expression, whereas knockdown of Oct-1 expression, using a lentivirus-based small hairpin RNA approach, led to increased ChREBP mRNA and protein expression. In contrast, HepG2 cells treated with 10 or 100 nM insulin for 4 or 8 h resulted in an approximately 2-fold increase of ChREBP promoter activity (P < 0.05 to P < 0.01). Insulin (10 nM) also stimulated endogenous ChREBP expression in HepG2 and primary hamster hepatocytes. More importantly, we found that the stimulatory effect of insulin on ChREBP promoter activity was dependent on the presence of the POU binding site, and insulin treatment reduced Oct-1 expression levels. Our observations therefore identify Oct-1 as a transcriptional repressor of ChREBP and suggest that insulin stimulates ChREBP expression via attenuating the repressive effect of Oct-1.

Carbohydrate Response Element Binding Protein Gene Expression Is Positively Regulated by Thyroid Hormone

The molecular mechanism of thyroid hormone (TH) effects to fatty acid metabolism in liver is yet to be clear. The carbohydrate response element-binding protein (ChREBP) as well as sterol response element-binding protein (SREBP)-1c plays a pivotal role in hepatic lipogenesis. Both SREBP-1c and ChREBP are target genes of liver X receptors (LXRs). Because LXRs and TH receptors (TRs) cross talk mutually in many aspects of transcription, we examined whether TRs regulate the mouse ChREBP gene expression. In the current study, we demonstrated that TH up-regulated mouse ChREBP mRNA and protein expression in liver. Run-on and luciferase assays showed that TH and TR-beta1 positively regulated the ChREBP gene transcription. The mouse ChREBP gene promoter contains two direct repeat-4 sites (LXRE1 and LXRE2) and EMSAs demonstrated that LXR-alpha and TR-beta1 prefer to bind LXRE1 and LXRE2, respectively. The direct repeat-4 deletion and LXRE2 mutants of the promoter deteriorate the positive regulation by TR-beta1, indicating that LXRE2 is functionally important for the regulation. We also showed that human ChREBP gene expression and promoter activities were up-regulated by TH. These data suggest that ChREBP mRNA expression is positively regulated by TR-beta1 and TH at the transcriptional level in mammals. This novel observation indicates that TH fine-tunes hepatic lipogenesis via regulating SREBP-1c and ChREBP gene expression reciprocally.

Molecular cloning and expression of chicken carbohydrate response element binding protein and Max-like protein X gene homologues

Carbohydrate response element binding protein (ChREBP) and sterol regulatory element binding protein-1c (SREBP-1c) are transcription factors that are known to be key regulators of glucose metabolism and lipid synthesis in mammals. Since ChREBP and its co-activator Max-like protein X (Mlx) have not been identified in birds, the objectives of this work were to clone, sequence, and characterize the genomic organization of ChREBP and Mlx genes and to determine the expression of ChREBP, Mlx, and several related genes including liver X receptor (LXR), SREBP-1 and thyroid hormone responsive Spot 14 (Spot 14) in chickens. Alternative splicing resulted in two ChREBP mRNA transcript variants that code for predicted proteins of 895 and 869 amino acids. The chicken Mlx gene produced a single mRNA transcript that codes for a predicted protein of 245 amino acids. Chicken ChREBP and Mlx predicted proteins shared high amino acid homology with select portions of corresponding mammalian proteins. In chickens, Mlx, SREBP-1, and LXR were expressed at comparable levels in all tissues examined. However, ChREBP demonstrated significant tissue-specific expression with the highest mRNA levels found in liver and duodenum and Spot 14 was expressed predominantly in liver and abdominal fat. Using Western blotting, the presence of ChREBP protein was detected in chicken liver tissue. Our findings add new insight into a potential role for specific transcription factors such as ChREBP and Mlx in the glucose-dependent regulation of lipogenesis in birds.

The Liver X Receptor (LXR) and Hepatic Lipogenesis: THE CARBOHYDRATE-RESPONSE ELEMENT-BINDING PROTEIN IS A TARGET GENE OF LXR

The liver X receptors, LXRalpha (NR1H3) and LXRbeta (NR1H2), are ligand-activated transcription factors that belong to the nuclear hormone receptor superfamily. LXRs play a critical role in cholesterol homeostasis and bile acid metabolism. In addition, oral administration of LXR agonists to mice results in elevated hepatic fatty acid synthesis and steatosis and increased secretion of triglyceride-rich very low density lipoprotein resulting in hypertriglyceridemia. This increased hepatic lipogenesis has been largely attributed to the LXR-dependent up-regulation of sterol regulatory element-binding protein 1c (SREBP-1c) expression. However, it has been reported that treating Srebp-1c null mice with the synthetic LXR agonist T0901317 still results in enhanced expression of many lipogenic genes, suggesting additional mechanisms by which LXR can enhance hepatic lipogenesis. In this report, we identify the carbohydrate response element-binding protein (ChREBP) as an LXR target that independently enhances the up-regulation of select lipogenic genes. The ChREBP promoter contains functional LXR-binding sites that confer receptor-dependent binding and transactivation. We show that T0901317 treatment of mice is associated with up-regulation of the ChREBP target gene, liver-type pyruvate kinase. Therefore, activation of LXR not only increases ChREBP mRNA via enhanced transcription but also modulates ChREBP activity. This establishes LXR as a master lipogenic transcription factor, as it directly regulates both SREBP-1c and ChREBP to enhance hepatic fatty acid synthesis.

Effect of insulin on transcription of lipogenic and lipolytic-related genes and lipid metabolism in primary porcine adipocytes

AbstractPrimary adipocytes from subcutaneous adipose tissue samples obtained from 7-day-old Yorkshire×Landrace crossbreed piglets were exposed to 0–400 nmol/l of insulin for 48 h. The accumulated triglyceride was measured through Oil Red O staining and the cumulative glycerol released was determined to assess lipolytic activity in adipocytes. Transcription levels of sterol regulatory element binding protein (SREBP)-1c, carbohydrate response element binding protein (ChREBP), acetyl-CoA carboxylase 1 (ACC1), fatty acid synthase (FAS), and hormone-sensitive lipase (HSL) were assessed using reverse transcriptase-polymerase chain reaction (RT-PCR). The results showed that ChREBP and ACC1 mRNA levels were not influenced by insulin alone under low glucose (5 nmol/l). FAS mRNA level was markedly stimulated by all doses of insulin except 200 nmol/l, and SREBP-1c mRNA level increased with 100–300 nmol/l insulin. High insulin doses (300 and 400 nmol/l) increased the HSL mRNA level as well as lipolytic activity.

In vivo expression of carbohydrate responsive element binding protein in lean and obese rats

ChREBP (Carbohydrate response element binding protein) is considered to mediate the stimulatory effect of glucose on the expression of lipogenic genes. Its activity is stimulated by glucose. Less is known on the control of its expression. This expression could be controlled by nutritional (glucose, fatty acids) and hormonal (insulin) factors. We examined the in vivo nutritional control of ChREBP expression in liver and adipose tissue of Wistar rats. Compared respectively to the fed state and to a high carbohydrate diet, ChREBP mRNA concentrations were not modified by fasting or a high fat diet in rat liver and adipose tissue. FAS and ACC1 mRNA concentrations were on the contrary decreased as expected by fasting and high fat diets and these variations of FAS and ACC1 mRNA were positively related to those of SREBP-1c mRNA and protein, but not of ChREBP mRNA. Therefore i) ChREBP expression appears poorly responsive to modifications of nutritional condition, ii) modifications of the expression of ChREBP do not seem implicated in the physiological control of lipogenesis. To investigate the possible role of ChREBP in pathological situations we measured its mRNA concentrations in the liver and adipose tissue of obese Zucker rats. ChREBP expression was increased in the liver but not the adipose tissue of obese rats compared to their lean littermates. These results support a role of ChREBP in the development of hepatic steatosis and hypertriglyceridemia but not of obesity in this experimental model.

Modulation of carbohydrate response element-binding protein gene expression in 3T3-L1 adipocytes and rat adipose tissue.

Carbohydrate response element-binding protein (ChREBP) is a rat homolog of human Williams-Beuren syndrome region 14 and a member of the basic helix-loop-helix leucine zipper transcription factor family. Its activation was found to be inducible by carbohydrate in the liver nuclear extracts from rats fed a high-sucrose diet. ChREBP is able to bind to the carbohydrate response element on the promoter of L-type pyruvate kinase and initiate the gene transcription. The detailed expression profile and transcriptional regulation of the ChREBP gene in adipocytes have not been characterized. In the present study, we provide evidence showing that 1) the ChREBP gene is expressed in differentiated 3T3-L1 adipocytes and rat adipose tissue; 2) insulin, glucose, and the antidiabetic agent troglitazone can significantly upregulate the gene expression of ChREBP in 3T3-L1 adipocytes, whereas free fatty acids suppress its expression in this cell type; 3) fasting followed by refeeding with a high-carbohydrate diet resulted in a 10-fold increase of ChREBP mRNA level in rat adipose tissue; and 4) ChREBP expression in adipose tissue is not significantly affected by the diabetic state. Taken together, the results we present are consistent with the idea that ChREBP is an important modulator of adipocyte biology and that its expression in adipose tissue is subject to combined regulation by glucose and insulin in vivo. The induction of ChREBP may serve as a novel pharmacological pathway for troglitazone-mediated hypoglycemic effects in vivo.

Comparison of the expression and activity of the lipogenic pathway in human and rat adipose tissue

Lipogenesis is considered less active in human than in rat adipose tissue. This could be explained by different nutritional conditions, namely high-carbohydrate (HCHO) diet in rats and high-fat (HF) diet in humans. Adipose tissue was sampled (postabsorptive state) in rats and humans receiving HCHO or HF diets, ad libitum fed humans, and obese subjects. We measured 1) mRNA concentrations of fatty acid synthase (FAS), acetyl-CoA carboxylase 1 (ACC1), sterol regulatory element binding protein 1c (SREBP-1c), and carbohydrate response element binding protein (ChREBP), 2) SREBP-1c protein, and 3) FAS activity. FAS, ACC1, ChREBP, and SREBP1-c mRNA concentrations were unaffected by diet in humans or in rats. FAS and ACC1 mRNA levels were lower in humans than in rats (P < 0.05). FAS activity was unaffected by diet and was lower in humans (P < 0.05). SREBP-1c mRNA concentrations were similar in rats and humans, but the precursor and mature forms of SREBP-1c protein were less abundant in humans (P < 0.05). ChREBP mRNA concentrations were lower in humans than in rats. In conclusion, the lipogenic capacity of adipose tissue is lower in humans than in rats. This is not related to differences in diet and is probably explained by lower abundance of SREBP-1c protein. A decreased expression of ChREBP could also play a role.