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Carbohydrate Response Element Binding Protein (ChREBP) is a glucose 6-phosphate (G6P)-sensitive transcription factor that acts as a metabolic switch to maintain intracellular glucose and phosphate homeostasis. Hepatic ChREBP is well-known for its regulatory role in glycolysis, the pentose phosphate pathway, and de novo lipogenesis. The physiological role of ChREBP in hepatic glycogen metabolism and blood glucose regulation has not been assessed in detail, and ChREBP's contribution to carbohydrate flux adaptations in hepatic Glycogen Storage Disease type 1 (GSD I) requires further investigation. The current study aimed to investigate the role of ChREBP as a regulator of glycogen metabolism in response to hepatic G6P accumulation, using a model for acute hepatic GSD type Ib. The immediate biochemical and regulatory responses to hepatic G6P accumulation were evaluated upon G6P transporter inhibition by the chlorogenic acid S4048 in mice that were either treated with a short hairpin RNA (shRNA) directed against ChREBP (shChREBP) or a scrambled shRNA (shSCR). Complementary stable isotope experiments were performed to quantify hepatic carbohydrate fluxes in vivo. ShChREBP treatment normalized the S4048-mediated induction of hepatic ChREBP target genes to levels observed in vehicle- and shSCR-treated controls. In parallel, hepatic shChREBP treatment in S4048-infused mice resulted in a more pronounced accumulation of hepatic glycogen and further reduction of blood glucose levels compared to shSCR treatment. Hepatic ChREBP knockdown modestly increased glucokinase (GCK) flux in S4048-treated mice while it enhanced UDP-glucose turnover as well as glycogen synthase and phosphorylase fluxes. Hepatic GCK mRNA and protein levels were induced by shChREBP treatment in both vehicle- and S4048-treated mice, while glycogen synthase 2 (GYS2) and glycogen phosphorylase (PYGL) mRNA and protein levels were reduced. Finally, knockdown of hepatic ChREBP expression reduced starch domain binding protein 1 (STBD1) mRNA and protein levels while it inhibited acid alpha-glucosidase (GAA) activity, suggesting reduced capacity for lysosomal glycogen breakdown. Our data show that ChREBP activation controls hepatic glycogen and blood glucose levels in acute hepatic GSD Ib through concomitant regulation of glucose phosphorylation, glycogenesis, and glycogenolysis. ChREBP-mediated control of GCK enzyme levels aligns with corresponding adaptations in GCK flux. In contrast, ChREBP activation in response to acute hepatic GSD Ib exerts opposite effects on GYS2/PYGL enzyme levels and their corresponding fluxes, indicating that GYS2/PYGL expression levels are not limiting to their respective fluxes under these conditions. Show less

Brown fat activation accelerates the uptake of cholesterol-enriched remnants by the liver and thereby lowers plasma cholesterol, consequently protecting against atherosclerosis development. Hepatic cholesterol is then converted into bile acids (BAs) that are secreted into the intestine and largely maintained within the enterohepatic circulation. We now aimed to evaluate the effects of prolonged brown fat activation combined with inhibition of intestinal BA reabsorption on plasma cholesterol metabolism and atherosclerosis development. APOE*3-Leiden.CETP mice with humanized lipoprotein metabolism were treated for 9 weeks with the selective β3-adrenergic receptor (AR) agonist CL316,243 to substantially activate brown fat. Prolonged β3-AR agonism reduced faecal BA excretion (-31%), while markedly increasing plasma levels of total BAs (+258%), cholic acid-derived BAs (+295%), and chenodeoxycholic acid-derived BAs (+217%), and decreasing the expression of hepatic genes involved in BA production. In subsequent experiments, mice were additionally treated with the BA sequestrant Colesevelam to inhibit BA reabsorption. Concomitant intestinal BA sequestration increased faecal BA excretion, normalized plasma BA levels, and reduced hepatic cholesterol. Moreover, concomitant BA sequestration further reduced plasma total cholesterol (-49%) and non-high-density lipoprotein cholesterol (-56%), tended to further attenuate atherosclerotic lesion area (-54%). Concomitant BA sequestration further increased the proportion of lesion-free valves (+34%) and decreased the relative macrophage area within the lesion (-26%), thereby further increasing the plaque stability index (+44%). BA sequestration prevents the marked accumulation of plasma BAs as induced by prolonged brown fat activation, thereby further improving cholesterol metabolism and reducing atherosclerosis development. These data suggest that combining brown fat activation with BA sequestration is a promising new therapeutic strategy to reduce hyperlipidaemia and cardiovascular diseases. Show less

Physiological adaptations resulting in the development of the metabolic syndrome in man occur over a time span of several decades. This combined with the prohibitive financial cost and ethical concerns to measure key metabolic parameters repeatedly in subjects for the major part of their life span makes that comprehensive longitudinal human data sets are virtually nonexistent. While experimental mice are often used, little is known whether this species is in fact an adequate model to better understand the mechanisms that drive the metabolic syndrome in man. We took up the challenge to study the response of male apoE*3-Leiden.CETP mice (with a humanized lipid profile) to a high-fat high-cholesterol diet for 6 months. Study parameters include body weight, food intake, plasma and liver lipids, hepatic transcriptome, VLDL - triglyceride production and importantly the use of stable isotopes to measure hepatic de novo lipogenesis, gluconeogenesis, and biliary/fecal sterol secretion to assess metabolic fluxes. The key observations include (1) high inter-individual variation; (2) a largely unaffected hepatic transcriptome at 2, 3, and 6 months; (3) a biphasic response curve of the main metabolic features over time; and (4) maximum insulin resistance preceding dyslipidemia. The biphasic response in plasma triglyceride and total cholesterol appears to mimic that of men in cross-sectional studies. Combined, these observations suggest that studies such as these can help to delineate the causes of metabolic derangements in patients suffering from metabolic syndrome. Show less

GSD-1 (glycogen storage disease type 1) is caused by an inherited defect in glucose-6-phosphatase activity, resulting in a massive accumulation of hepatic glycogen content and an induction of de novo lipogenesis. The chlorogenic acid derivative S4048 is a pharmacological inhibitor of the glucose 6-phosphate transporter, which is part of glucose-6-phosphatase, and allows for mechanistic studies concerning metabolic defects in GSD-1. Treatment of mice with S4048 resulted in an ~60% reduction in blood glucose, increased hepatic glycogen and triacylglycerol (triglyceride) content, and a markedly enhanced hepatic lipogenic gene expression. In mammals, hepatic expression of lipogenic genes is regulated by the co-ordinated action of the transcription factors SREBP (sterol-regulatory-element-binding protein)-1c, LXRα (liver X receptor α) and ChREBP (carbohydrate-response-element-binding protein). Treatment of Lxra-/- mice and Chrebp-/- mice with S4048 demonstrated that ChREBP, but not LXRα, mediates the induction of hepatic lipogenic gene expression in this murine model of GSD-1. Thus ChREBP is an attractive target to alleviate derangements in lipid metabolism observed in patients with GSD-1. Show less

A growing body of evidence indicates that peroxisome proliferator-activated receptor alpha (PPARalpha) not merely serves as a transcriptional regulator of fatty acid catabolism but also exerts a much broader role in hepatic lipid metabolism. We determined adaptations in hepatic lipid metabolism and related aspects of carbohydrate metabolism upon treatment of C57Bl/6 mice with the PPARalpha agonist fenofibrate. Stable isotope procedures were applied to assess hepatic fatty acid synthesis, fatty acid elongation, and carbohydrate metabolism. Fenofibrate treatment strongly induced hepatic de novo lipogenesis and chain elongation (+/-300, 150, and 600% for C16:0, C18:0, and C18:1 synthesis, respectively) in parallel with an increased expression of lipogenic genes. The lipogenic induction in fenofibrate-treated mice was found to depend on sterol regulatory element-binding protein 1c (SREBP-1c) but not carbohydrate response element-binding protein (ChREBP). Fenofibrate treatment resulted in a reduced contribution of glycolysis to acetyl-CoA production, whereas the cycling of glucose 6-phosphate through the pentose phosphate pathway presumably was enhanced. Altogether, our data indicate that beta-oxidation and lipogenesis are induced simultaneously upon fenofibrate treatment. These observations may reflect a physiological mechanism by which PPARalpha and SREBP-1c collectively ensure proper handling of fatty acids to protect the liver against cytotoxic damage. Show less

Besides its well established role in control of cellular cholesterol homeostasis, the liver X receptor (LXR) has been implicated in the regulation of hepatic gluconeogenesis. We investigated the role of the major hepatic LXR isoform in hepatic glucose metabolism during the feeding-to-fasting transition in vivo. In addition, we explored hepatic glucose sensing by LXR during carbohydrate refeeding. Lxralpha(-/-) mice and their wild-type littermates were subjected to a fasting-refeeding protocol and hepatic carbohydrate fluxes as well as whole body insulin sensitivity were determined in vivo by stable isotope procedures. Lxralpha(-/-) mice showed an impaired response to fasting in terms of hepatic glycogen depletion and triglyceride accumulation. Hepatic glucose 6-phosphate turnover was reduced in 9-h fasted Lxralpha(-/-) mice as compared with controls. Although hepatic gluconeogenic gene expression was increased in 9-h fasted Lxralpha(-/-) mice compared with wild-type controls, the actual gluconeogenic flux was not affected by Lxralpha deficiency. Hepatic and peripheral insulin sensitivity were similar in Lxralpha(-/-) and wild-type mice. Compared with wild-type controls, the induction of hepatic lipogenic gene expression was blunted in carbohydrate-refed Lxralpha(-/-) mice, which was associated with lower plasma triglyceride concentrations. Yet, expression of "classic" LXR target genes Abca1, Abcg5, and Abcg8 was not affected by Lxralpha deficiency in carbohydrate-refed mice. In summary, these studies identify LXRalpha as a physiologically relevant mediator of the hepatic response to fasting. However, the data do not support a role for LXR in hepatic glucose sensing. Show less