Aldo Grefhorst

The Farnesoid X Receptor Modulates Adiposity and Peripheral Insulin Sensitivity in Mice

The farnesoid X receptor (FXR) is a bile acid (BA)-activated nuclear receptor that plays a major role in the regulation of BA and lipid metabolism. Recently, several studies have suggested a potential role of FXR in the control of hepatic carbohydrate metabolism, but its contribution to the maintenance of peripheral glucose homeostasis remains to be established. FXR-deficient mice display decreased adipose tissue mass, lower serum leptin concentrations, and elevated plasma free fatty acid levels. Glucose and insulin tolerance tests revealed that FXR deficiency is associated with impaired glucose tolerance and insulin resistance. Moreover, whole-body glucose disposal during a hyperinsulinemic euglycemic clamp is decreased in FXR-deficient mice. In parallel, FXR deficiency alters distal insulin signaling, as reflected by decreased insulin-dependent Akt phosphorylation in both white adipose tissue and skeletal muscle. Whereas FXR is not expressed in skeletal muscle, it was detected at a low level in white adipose tissue in vivo and induced during adipocyte differentiation in vitro. Moreover, mouse embryonic fibroblasts derived from FXR-deficient mice displayed impaired adipocyte differentiation, identifying a direct role for FXR in adipocyte function. Treatment of differentiated 3T3-L1 adipocytes with the FXR-specific synthetic agonist GW4064 enhanced insulin signaling and insulin-stimulated glucose uptake. Finally, treatment with GW4064 improved insulin resistance in genetically obese ob/ob mice in vivo. Although the underlying molecular mechanisms remain to be unraveled, these results clearly identify a novel role of FXR in the regulation of peripheral insulin sensitivity and adipocyte function. This unexpected function of FXR opens new perspectives for the treatment of type 2 diabetes.

Pharmacological Inhibition of Glucosylceramide Synthase Enhances Insulin Sensitivity

A growing body of evidence implicates ceramide and/or its glycosphingolipid metabolites in the pathogenesis of insulin resistance. We have developed a highly specific small molecule inhibitor of glucosylceramide synthase, an enzyme that catalyzes a necessary step in the conversion of ceramide to glycosphingolipids. In cultured 3T3-L1 adipocytes, the iminosugar derivative N-(5′-adamantane-1′-yl-methoxy)-pentyl-1-deoxynojirimycin (AMP-DNM) counteracted tumor necrosis factor-α–induced abnormalities in glycosphingolipid concentrations and concomitantly reversed abnormalities in insulin signal transduction. When administered to mice and rats, AMP-DNM significantly reduced glycosphingolipid but not ceramide concentrations in various tissues. Treatment of ob/ob mice with AMP-DNM normalized their elevated tissue glucosylceramide levels, markedly lowered circulating glucose levels, improved oral glucose tolerance, reduced A1C, and improved insulin sensitivity in muscle and liver. Similarly beneficial metabolic effects were seen in high fat–fed mice and ZDF rats. These findings provide further evidence that glycosphingolipid metabolites of ceramide may be involved in mediating the link between obesity and insulin resistance and that interference with glycosphingolipid biosynthesis might present a novel approach to the therapy of states of impaired insulin action such as type 2 diabetes.

Hepatic PCSK9 Expression Is Regulated by Nutritional Status via Insulin and Sterol Regulatory Element-binding Protein 1c

Familial autosomal dominant hypercholesterolemia is associated with high risk for cardiovascular accidents and is related to mutations in the low density lipoprotein receptor or its ligand apolipoprotein B (apoB). Mutations in a third gene, proprotein convertase subtilisin kexin 9 (PCSK9), were recently associated to this disease. PCSK9 acts as a natural inhibitor of the low density lipoprotein receptor pathway, and both genes are regulated by depletion of cholesterol cell content and statins, via sterol regulatory element-binding protein (SREBP). Here we investigated the regulation of PCSK9 gene expression during nutritional changes. We showed that PCSK9 mRNA quantity is decreased by 73% in mice after 24 h of fasting, leading to a 2-fold decrease in protein level. In contrast PCSK9 expression was restored upon high carbohydrate refeeding. PCSK9 mRNA increased by 4–5-fold in presence of insulin in rodent primary hepatocytes, whereas glucose had no effect. Moreover, insulin up-regulated hepatic PCSK9 expression in vivo during a hyperinsulinemic-euglycemic clamp in mice. Adenoviral mediated overexpression of a dominant or negative form of SREBP-1c confirmed the implication of this transcription factor in insulin-mediated stimulation of PCSK9 expression. Liver X receptor agonist T0901317 also regulated PCSK9 expression via this same pathway (a 2-fold increase in PCSK9 mRNA of primary hepatocytes cultured for 24 h in presence of 1 μm T0901317). As our last investigation, we isolated PCSK9 proximal promoter and verified the functionality of a SREBP-1c responsive element located from 335 bp to 355 bp upstream of the ATG. Together, these results show that PCSK9 expression is regulated by nutritional status and insulinemia.

Fenofibrate Simultaneously Induces Hepatic Fatty Acid Oxidation, Synthesis, and Elongation in Mice

A growing body of evidence indicates that peroxisome proliferator-activated receptor α (PPARα) 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 PPARα 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 β-oxidation and lipogenesis are induced simultaneously upon fenofibrate treatment. These observations may reflect a physiological mechanism by which PPARα and SREBP-1c collectively ensure proper handling of fatty acids to protect the liver against cytotoxic damage.

Transient impairment of the adaptive response to fasting in FXR‐deficient mice

The farnesoid X receptor (FXR) has been suggested to play a role in gluconeogenesis. To determine whether FXR modulates the response to fasting in vivo, FXR‐deficient (FXR−/−) and wild‐type mice were submitted to fasting for 48 h. Our results demonstrate that FXR modulates the kinetics of alterations of glucose homeostasis during fasting, with FXR−/− mice displaying an early, accelerated hypoglycaemia response. Basal hepatic glucose production rate was lower in FXR−/− mice, together with a decrease in hepatic glycogen content. Moreover, hepatic PEPCK gene expression was transiently lower in FXR−/−mice after 6 h of fasting and was decreased in FXR−/−hepatocytes. FXR therefore plays an unexpected role in the control of fuel availability upon fasting.

The liver X receptor : Control of cellular lipid homeostasis and beyond Implications for drug design

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.

An Increased Flux through the Glucose 6-Phosphate Pool in Enterocytes Delays Glucose Absorption in Fxr(-/-) Mice

The farnesoid X receptor (FXR) is involved in regulation of bile acid and lipid metabolism. Recently, a role for FXR in control of glucose metabolism became evident. Because FXR is expressed along the length of the small intestine, we evaluated the potential role of FXR in glucose absorption and processing. During intravenous infusion of a trace amount of d-[6,6-2H2]glucose, a d-[U-13C]glucose-enriched oral glucose bolus was given, and glucose kinetics were determined in wild-type and Fxr–/– mice. Compared with wild-type mice, Fxr–/– mice showed a delayed plasma appearance of orally administered glucose. Multicompartmental kinetic modeling revealed that this delay was caused by an increased flux through the glucose 6-phosphate pool in enterocytes. Thus, our results show involvement of FXR in intestinal glucose absorption, representing a novel physiological function for this nuclear receptor.

Lxrα Deficiency Hampers the Hepatic Adaptive Response to Fasting in Mice

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. Lxrα-/- 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. Lxrα-/- 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 Lxrα-/- mice as compared with controls. Although hepatic gluconeogenic gene expression was increased in 9-h fasted Lxrα-/- mice compared with wild-type controls, the actual gluconeogenic flux was not affected by Lxrα deficiency. Hepatic and peripheral insulin sensitivity were similar in Lxrα-/- and wild-type mice. Compared with wild-type controls, the induction of hepatic lipogenic gene expression was blunted in carbohydrate-refed Lxrα-/- mice, which was associated with lower plasma triglyceride concentrations. Yet, expression of “classic” LXR target genes Abca1, Abcg5, and Abcg8 was not affected by Lxrα deficiency in carbohydrate-refed mice. In summary, these studies identify LXRα 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.

Inhibition of mitochondrial fatty acid oxidation in vivo only slightly suppresses gluconeogenesis but enhances clearance of glucose in mice

Mitochondrial fatty acid oxidation (mFAO) is considered to be essential for driving gluconeogenesis (GNG) during fasting. However, quantitative in vivo data on de novo synthesis of glucose‐6‐phosphate upon acute inhibition of mFAO are lacking. We assessed hepatic glucose metabolism in vivo after acute inhibition of mFAO by 30 mg kg−1 2‐tetradecylglycidic acid (TDGA) in hypoketotic hypoglycemic male C57BL/6J mice by the infusion of [U‐13C]glucose, [2‐13C]glycerol, [1‐2H]galactose, and paracetamol for 6 hours, which was followed by mass isotopomer distribution analysis in blood glucose and urinary paracetamol‐glucuronide. During TDGA treatment, endogenous glucose production was unaffected (127 ± 10 versus 118 ± 7 μmol kg−1 minute−1, control versus TDGA, not significant), but the metabolic clearance rate of glucose was significantly enhanced (15.9 ± 0.9 versus 26.3 ± 1.1 mL kg−1 minute−1, control versus TDGA,P < 0.05). In comparison with control mice, de novo synthesis of glucose‐6‐phosphate (G6P) was slightly decreased in TDGA‐treated mice (108 ± 19 versus 85 ± 6 μmol kg−1 minute−1, control versus TDGA, P < 0.05). Recycling of glucose was decreased upon TDGA treatment (26 ± 14 versus 12 ± 4 μmol kg−1 minute−1, control versus TDGA, P < 0.05). Hepatic messenger RNA (mRNA) levels of genes encoding enzymes involved in de novo G6P synthesis were unaltered, whereas glucose‐6‐phosphate hydrolase mRNA expressions were increased in TDGA‐treated mice. Glucokinase and pyruvate kinase mRNA levels were significantly decreased, whereas pyruvate dehydrogenase kinase isozyme 4 expression was increased 30‐fold; this suggested decreased glycolytic activity. Conclusion: Acute pharmacological inhibition of mFAO using TDGA had no effect on endogenous glucose production and only a marginal effect on de novo G6P synthesis. Hence, fully active mFAO is not essential for maintenance of hepatic GNG in vivo in fasted mice.(HEPATOLOGY 2008.)

Direct activating effects of adrenocorticotropic hormone (ACTH) on brown adipose tissue are attenuated by corticosterone

Brown adipose tissue (BAT) and brownlike cells in white adipose tissue (WAT) can dissipate energy through thermogenesis, a process mediated by uncoupling protein 1 (UCP1). We investigated whether stress hormones ACTH and corticosterone contribute to BAT activation and browning of WAT. ACTH and corticosterone were studied in male mice exposed to 4 or 23°C for 24 h. Direct effects were studied in T37i mouse brown adipocytes and primary cultured murine BAT and inguinal WAT (iWAT) cells. In vivo effects were studied using 18F‐deoxyglucose positron emission tomography. Cold exposure doubled serum ACTH concentrations (P=0.03) and fecal corticosterone excretion (P=0.008). In T37i cells, ACTH dose‐dependently increased Ucp1 mRNA (EC50=1.8 nM) but also induced Ucp1 protein content 88% (P=0.02), glycerol release 32% (P=0.03) and uncoupled respiration 40% (P=0.003). In cultured BAT and iWAT, ACTH elevated Ucp1 mRNA by 3‐fold (P=0.03) and 3.7‐fold (P=0.01), respectively. In T37i cells, corticosterone prevented induction of Ucp1 mRNA and Ucp1 protein by both ACTH and norepinephrine in a glucocorticoid receptor (GR)‐dependent fashion. ACTH and GR antagonist RU486 independently doubled BAT 18F‐deoxyglucose uptake (P= 0.0003 and P= 0.004, respectively) in vivo. Our results show that ACTH activates BAT and browning of WAT while corticosterone counteracts this.—Van den Beukel, J. C., Grefhorst, A., Quarta, C., Steenbergen, J., Mastroberardino, P. G., Lombès, M., Delhanty, P. J., Mazza, R., Pagotto, U., van der Lely, A. J., Themmen, A. P. N., Direct activating effects of adrenocorticotropic hormone (ACTH) on brown adipose tissue are attenuated by corticosterone. FASEB J. 28, 4857–4867 (2014). www.fasebj.org