Theo H. van Dijk

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.

Activation of the liver X receptor stimulates trans-intestinal excretion of plasma cholesterol.

Recent studies have indicated that direct intestinal secretion of plasma cholesterol significantly contributes to fecal neutral sterol loss in mice. The physiological relevance of this novel route, which represents a part of the reverse cholesterol transport pathway, has not been directly established in vivo as yet. We have developed a method to quantify the fractional and absolute contributions of several cholesterol fluxes to total fecal neutral sterol loss in vivo in mice, by assessing the kinetics of orally and intravenously administered stable isotopically labeled cholesterol combined with an isotopic approach to assess the fate of de novo synthesized cholesterol. Our results show that trans-intestinal cholesterol excretion significantly contributes to removal of blood-derived free cholesterol in C57Bl6/J mice (33% of 231 μmol/kg/day) and that pharmacological activation of LXR with T0901317 strongly stimulates this pathway (63% of 706 μmol/kg/day). Trans-intestinal cholesterol excretion is impaired in mice lacking Abcg5 (−4%), suggesting that the cholesterol transporting Abcg5/Abcg8 heterodimer is involved in this pathway. Our data demonstrate that intestinal excretion represents a quantitatively important route for fecal removal of neutral sterols independent of biliary secretion in mice. This pathway is sensitive to pharmacological activation of the LXR system. These data support the concept that the intestine substantially contributes to reverse cholesterol transport.

Short-Chain Fatty Acids protect against High-Fat Diet-Induced Obesity via a PPARγ-dependent switch from lipogenesis to fat oxidation

Short-chain fatty acids (SCFAs) are the main products of dietary fiber fermentation and are believed to drive the fiber-related prevention of the metabolic syndrome. Here we show that dietary SCFAs induce a peroxisome proliferator–activated receptor-γ (PPARγ)–dependent switch from lipid synthesis to utilization. Dietary SCFA supplementation prevented and reversed high-fat diet–induced metabolic abnormalities in mice by decreasing PPARγ expression and activity. This increased the expression of mitochondrial uncoupling protein 2 and raised the AMP-to-ATP ratio, thereby stimulating oxidative metabolism in liver and adipose tissue via AMPK. The SCFA-induced reduction in body weight and stimulation of insulin sensitivity were absent in mice with adipose-specific disruption of PPARγ. Similarly, SCFA-induced reduction of hepatic steatosis was absent in mice lacking hepatic PPARγ. These results demonstrate that adipose and hepatic PPARγ are critical mediators of the beneficial effects of SCFAs on the metabolic syndrome, with clearly distinct and complementary roles. Our findings indicate that SCFAs may be used therapeutically as cheap and selective PPARγ modulators.

Farnesoid X Receptor Deficiency Improves Glucose Homeostasis in Mouse Models of Obesity

OBJECTIVE Bile acids (BA) participate in the maintenance of metabolic homeostasis acting through different signaling pathways. The nuclear BA receptor farnesoid X receptor (FXR) regulates pathways in BA, lipid, glucose, and energy metabolism, which become dysregulated in obesity. However, the role of FXR in obesity and associated complications, such as dyslipidemia and insulin resistance, has not been directly assessed. RESEARCH DESIGN AND METHODS Here, we evaluate the consequences of FXR deficiency on body weight development, lipid metabolism, and insulin resistance in murine models of genetic and diet-induced obesity. RESULTS FXR deficiency attenuated body weight gain and reduced adipose tissue mass in both models. Surprisingly, glucose homeostasis improved as a result of an enhanced glucose clearance and adipose tissue insulin sensitivity. In contrast, hepatic insulin sensitivity did not change, and liver steatosis aggravated as a result of the repression of β-oxidation genes. In agreement, liver-specific FXR deficiency did not protect from diet-induced obesity and insulin resistance, indicating a role for nonhepatic FXR in the control of glucose homeostasis in obesity. Decreasing elevated plasma BA concentrations in obese FXR-deficient mice by administration of the BA sequestrant colesevelam improved glucose homeostasis in a FXR-dependent manner, indicating that the observed improvements by FXR deficiency are not a result of indirect effects of altered BA metabolism. CONCLUSIONS Overall, FXR deficiency in obesity beneficially affects body weight development and glucose homeostasis.

High Fat Feeding Induces Hepatic Fatty Acid Elongation in Mice

Background High-fat diets promote hepatic lipid accumulation. Paradoxically, these diets also induce lipogenic gene expression in rodent liver. Whether high expression of these genes actually results in an increased flux through the de novo lipogenic pathway in vivo has not been demonstrated. Methodology/Principal Findings To interrogate this apparent paradox, we have quantified de novo lipogenesis in C57Bl/6J mice fed either chow, a high-fat or a n-3 polyunsaturated fatty acid (PUFA)-enriched high-fat diet. A novel approach based on mass isotopomer distribution analysis (MIDA) following 1-13C acetate infusion was applied to simultaneously determine de novo lipogenesis, fatty acid elongation as well as cholesterol synthesis. Furthermore, we measured very low density lipoprotein-triglyceride (VLDL-TG) production rates. High-fat feeding promoted hepatic lipid accumulation and induced the expression of lipogenic and cholesterogenic genes compared to chow-fed mice: induction of gene expression was found to translate into increased oleate synthesis. Interestingly, this higher lipogenic flux (+74 µg/g/h for oleic acid) in mice fed the high-fat diet was mainly due to an increased hepatic elongation of unlabeled palmitate (+66 µg/g/h) rather than to elongation of de novo synthesized palmitate. In addition, fractional cholesterol synthesis was increased, i.e. 5.8±0.4% vs. 8.1±0.6% for control and high fat-fed animals, respectively. Hepatic VLDL-TG production was not affected by high-fat feeding. Partial replacement of saturated fat by fish oil completely reversed the lipogenic effects of high-fat feeding: hepatic lipogenic and cholesterogenic gene expression levels as well as fatty acid and cholesterol synthesis rates were normalized. Conclusions/Significance High-fat feeding induces hepatic fatty acid synthesis in mice, by chain elongation and subsequent desaturation rather than de novo synthesis, while VLDL-TG output remains unaffected. Suppression of lipogenic fluxes by fish oil prevents from high fat diet-induced hepatic steatosis in mice.

Gut-derived short-chain fatty acids are vividly assimilated into host carbohydrates and lipids.

Acetate, propionate, and butyrate are the main short-chain fatty acids (SCFAs) that arise from the fermentation of fibers by the colonic microbiota. While many studies focus on the regulatory role of SCFAs, their quantitative role as a catabolic or anabolic substrate for the host has received relatively little attention. To investigate this aspect, we infused conscious mice with physiological quantities of stable isotopes [1-(13)C]acetate, [2-(13)C]propionate, or [2,4-(13)C2]butyrate directly in the cecum, which is the natural production site in mice, and analyzed their interconversion by the microbiota as well as their metabolism by the host. Cecal interconversion, pointing to microbial cross-feeding, was high between acetate and butyrate, low between butyrate and propionate, and almost absent between acetate and propionate. As much as 62% of infused propionate was used in whole body glucose production, in line with its role as gluconeogenic substrate. Conversely, glucose synthesis from propionate accounted for 69% of total glucose production. The synthesis of palmitate and cholesterol in the liver was high from cecal acetate (2.8 and 0.7%, respectively) and butyrate (2.7 and 0.9%, respectively) as substrates, but low or absent from propionate (0.6 and 0.0%, respectively). Label incorporation due to chain elongation of stearate was approximately eightfold higher than de novo synthesis of stearate. Microarray data suggested that SCFAs exert a mild regulatory effect on the expression of genes involved in hepatic metabolic pathways during the 6-h infusion period. Altogether, gut-derived acetate, propionate, and butyrate play important roles as substrates for glucose, cholesterol, and lipid metabolism.

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.

LRH-1–dependent glucose sensing determines intermediary metabolism in liver

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.

Bile salt sequestration induces hepatic de novo lipogenesis through farnesoid X receptor– and liver X receptorα–controlled metabolic pathways in mice

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 α (LXRα). 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 Lxrα−/− mice compared with wild‐type littermates, in line with reports indicating a regulatory role of FXR and LXRα in bile salt–mediated regulation of hepatic lipid metabolism. Conclusion: Bile salt sequestration by colesevelam induces the lipogenic pathway in an FXR‐ and LXRα‐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. (HEPATOLOGY 2010.)