👤 M H Oosterveer

Stable isotope studies have shown that hepatic de novo lipogenesis (DNL) plays an important role in the pathogenesis of intrahepatic lipid (IHL) deposition. Furthermore, previous research has demonstrated that fructose 1-phosphate (F1P) not only serves as a substrate for DNL, but also acts as a signalling metabolite that stimulates DNL from glucose. The aim of this study was to elucidate the mediators of F1P-stimulated DNL, with special focus on two key regulators of intrahepatic glucose metabolism, i.e., glucokinase regulatory protein (GKRP) and carbohydrate response element binding protein (ChREBP). Aldolase B deficient mice (Aldob Aldob Both GKRP and ChREBP mediate F1P-stimulated DNL in aldolase B deficient mice. Further studies are needed to unravel the role of GKRP and hepatic ChREBP in regulating IHL accumulation in aldolase B deficiency. Show less

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

Glycogen 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. Liver-specific G6pc-knockout (L-G6pc Attenuation 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. Show less

It is well established that, besides facilitating lipid absorption, bile acids act as signaling molecules that modulate glucose and lipid metabolism. Bile acid metabolism, in turn, is controlled by several nutrient-sensitive transcription factors. Altered intrahepatic glucose signaling in type 2 diabetes associates with perturbed bile acid synthesis. We aimed to characterize the regulatory role of the primary intracellular metabolite of glucose, glucose-6-phosphate (G6P), on bile acid metabolism. Hepatic gene expression patterns and bile acid composition were analyzed in mice that accumulate G6P in the liver, that is, liver-specific glucose-6-phosphatase knockout (L-G6pc Show less

Fructose has become a major constituent of our modern diet and is implicated as an underlying cause in the development of metabolic diseases. The fructose transporter GLUT5 (SLC2A5) is required for intestinal fructose absorption. GLUT5 expression is induced in the intestine and skeletal muscle of type 2 diabetes (T2D) patients and in certain cancers that are dependent on fructose metabolism, indicating that modulation of GLUT5 levels could have potential in the treatment of these diseases. Using an unbiased screen for transcriptional control of the human GLUT5 promoter we identified a strong and specific regulation by liver X receptor α (LXRα, NR1H3). Using promoter truncations and site-directed mutagenesis we identified a functional LXR response element (LXRE) in the human GLUT5 promoter, located at -385 bp relative to the transcriptional start site (TSS). Finally, mice treated with LXR agonist T0901317 showed an increase in Glut5 mRNA and protein levels in duodenum and adipose tissue, underscoring the in vivo relevance of its regulation by LXR. Together, our findings show that LXRα regulates GLUT5 in mice and humans. As a ligand-activated transcription factor, LXRα might provide novel pharmacologic strategies for the selective modulation of GLUT5 activity in the treatment of metabolic disease as well as cancer. Show less

The hepatic glucose-sensing system is a functional network of enzymes and transcription factors that is critical for the maintenance of energy homeostasis and systemic glycemia. Here we review the recent literature on its components and metabolic actions. Glucokinase (GCK) is generally considered as the initial postprandial glucose-sensing component, which acts as the gatekeeper for hepatic glucose metabolism and provides metabolites that activate the transcription factor carbohydrate response element binding protein (ChREBP). Recently, liver receptor homolog 1 (LRH-1) has emerged as an upstream regulator of the central GCK-ChREBP axis, with a critical role in the integration of hepatic intermediary metabolism in response to glucose. Evidence is also accumulating that O-linked β-N-acetylglucosaminylation (O-GlcNAcylation) and acetylation can act as glucose-sensitive modifications that may contribute to hepatic glucose sensing by targeting regulatory proteins and the epigenome. Further elucidation of the components and functional roles of the hepatic glucose-sensing system may contribute to the future treatment of liver diseases associated with deregulated glucose sensors. Show less

Since high expression of farnesoid X receptor (FXR) has been detected in glucocorticoid-producing adrenocortical cells, we evaluated the potential role of FXR in adrenal glucocorticoid production. FXR agonist GW4064 increased fasting plasma corticosterone levels (+45%; P<0.01) in C57BL/6 mice, indicative of enhanced adrenal steroidogenesis. GW4064 treatment did not affect plasma ACTH levels, adrenal weight, or adrenal expression of steroidogenic genes. Scavenger receptor BI (SR-BI) mRNA and protein expression, respectively, increased 1.9-fold (P<0.01) and 1.5-fold, which suggests a stimulated lipoprotein-associated cholesterol uptake into the adrenals upon GW4064 treatment. In line with an enhanced flux of cellular cholesterol into the steroidogenic pathway, adrenal unesterified and esterified cholesterol stores were 21-41% decreased (P<0.01) upon GW4064 treatment. In conclusion, we have shown that the FXR agonist GW4064 stimulates plasma corticosterone levels in C57BL/6 mice. Our findings suggest a novel role for FXR in the modulation of adrenal cholesterol metabolism and glucocorticoid synthesis in mice. Show less

Liver receptor homolog 1 (LRH-1), an established regulator of cholesterol and bile acid homeostasis, has recently emerged as a potential drug target for liver disease. Although LRH-1 activation may protect the liver against diet-induced steatosis and insulin resistance, little is known about how LRH-1 controls hepatic glucose and fatty acid metabolism under physiological conditions. We therefore assessed the role of LRH-1 in hepatic intermediary metabolism. In mice with conditional deletion of Lrh1 in liver, analysis of hepatic glucose fluxes revealed reduced glucokinase (GCK) and glycogen synthase fluxes as compared with those of wild-type littermates. These changes were attributed to direct transcriptional regulation of Gck by LRH-1. Impaired glucokinase-mediated glucose phosphorylation in LRH-1-deficient livers was also associated with reduced glycogen synthesis, glycolysis, and de novo lipogenesis in response to acute and prolonged glucose exposure. Accordingly, hepatic carbohydrate response element-binding protein activity was reduced in these animals. Cumulatively, these data identify LRH-1 as a key regulatory component of the hepatic glucose-sensing system required for proper integration of postprandial glucose and lipid metabolism. Show less

Pharmacological LXR activation has anti-atherosclerotic actions in animal models. Part of these beneficial effects may be explained by accelerated reverse cholesterol transport since both plasma high density lipoprotein (HDL) cholesterol and fecal neutral sterol secretion are higher upon LXR activation. Mechanisms underlying these LXR-mediated effects have not been fully elucidated. We investigated the roles of the isoforms LXRα and LXRβ and the HDL cholesterol uptake receptor SR-B1 in modulation of cholesterol metabolism upon treatment of mice with the LXR ligand T0901317. HDL cholesterol was maximally 60% increased in a time-dependent fashion due to appearance of more and larger HDL particles. Fecal neutral sterol secretion was maximally induced after 1 week treatment. T0901317 treatment induced fecal neutral sterol secretion by ~300% in wild-type but not in Lxrα deficient mice. Surprisingly, LXR activation reduced SR-B1 protein amount in hepatic membranes, suggesting that this might contribute to elevated HDL cholesterol. However, T0901317 still elevated plasma HDL cholesterol in Sr-b1 deficient mice, suggesting that SR-B1 is not the only step involved in LXR-mediated induction of plasma HDL cholesterol. In addition, SR-B1 is not essential for LXR-induced cholesterol removal from the body. Induction of fecal neutral sterol secretion by T0901317 critically depends on LXRα but not on LXRβ. LXR activation reduces SR-B1 in hepatic membranes, probably partly contributing to elevated HDL cholesterol. SR-B1 is not required to enhance fecal neutral sterol secretion. 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

Liver X receptor (LXR) α and β are nuclear receptors that control cellular metabolism. LXRs modulate the expression of genes involved in cholesterol and lipid metabolism in response to changes in cellular cholesterol status. Because of their involvement in cholesterol homeostasis, LXRs have emerged as promising drug targets for anti-atherosclerotic therapies. In rodents, synthetic LXR agonists promote cellular cholesterol efflux, transport and excretion. As a result, the progression of atherosclerosis is halted. However, pharmacological LXR activation also induces hepatic steatosis and promotes the secretion of atherogenic triacylglycerol-rich VLDL particles by the liver, complicating the clinical application of LXR agonists. The more recently emerged roles of LXRs in fat tissue, pituitary and brain may have implications for treatment of obesity and Alzheimer disease. In addition to the improvements in atherosclerosis, LXR activation exerts beneficial effects on glucose control in mouse models of type 2 diabetes. Future therapeutic strategies aiming to exert beneficial effects on cholesterol and glucose homeostasis, while circumventing the undesired effects on hepatic lipid metabolism, should target specific LXR-mediated processes. Therefore, tissue and/or isotype-specific effects of LXR action need to be established. The consequences of combinatorial drug approaches and the identification of the co-regulatory networks involved in the LXR-mediated control of particular genes may contribute to development of novel LXR agonists. Finally, pathway analyses of LXR actions provide tools to evaluate and optimize the effectiveness of novel therapeutic strategies to prevent and/or treat metabolic diseases. Show less

Diabetes is characterized by high blood glucose levels and dyslipidemia. Bile salt sequestration has been found to improve both plasma glycemic control and cholesterol profiles in diabetic patients. Yet bile salt sequestration is also known to affect triglyceride (TG) metabolism, possibly through signaling pathways involving farnesoid X receptor (FXR) and liver X receptor alpha (LXRalpha). We quantitatively assessed kinetic parameters of bile salt metabolism in lean C57Bl/6J and in obese, diabetic db/db mice upon bile salt sequestration using colesevelam HCl (2% wt/wt in diet) and related these to quantitative changes in hepatic lipid metabolism. As expected, bile salt sequestration reduced intestinal bile salt reabsorption. Importantly, bile salt pool size and biliary bile salt secretion remained unchanged upon sequestrant treatment due to compensation by de novo bile salt synthesis in both models. Nevertheless, lean and db/db mice showed increased, mainly periportally confined, hepatic TG contents, increased expression of lipogenic genes, and increased fractional contributions of newly synthesized fatty acids. Lipogenic gene expression was not induced in sequestrant-treated Fxr(-/-) and Lxralpha(-/-) mice compared with wild-type littermates, in line with reports indicating a regulatory role of FXR and LXRalpha in bile salt-mediated regulation of hepatic lipid metabolism. Bile salt sequestration by colesevelam induces the lipogenic pathway in an FXR- and LXRalpha-dependent manner without affecting the total pool size of bile salts in mice. We speculate that a shift from intestinal reabsorption to de novo synthesis as source of bile salts upon bile salt sequestration affects zonation of metabolic processes within the liver acinus. 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