Liya Yin

Identification of Novel Pathways That Control Farnesoid X Receptor-mediated Hypocholesterolemia

Farnesoid X receptor (FXR) plays important regulatory roles in bile acid, lipoprotein, and glucose homeostasis. Here, we have utilized Fxr−/− mice and mice deficient in scavenger receptor class B type I (SR-BI), together with an FXR-specific agonist and adenovirus expressing hepatocyte nuclear factor 4α or constitutively active FXR, to identify the mechanisms by which activation of FXR results in hypocholesterolemia. We identify a novel pathway linking FXR to changes in hepatic p-JNK, hepatocyte nuclear factor 4α, and finally SR-BI. Importantly, we demonstrate that the FXR-dependent increase in SR-BI results in both hypocholesterolemia and an increase in reverse cholesterol transport, a process involving the transport of cholesterol from peripheral macrophages to the liver for excretion into the feces. In addition, we demonstrate that FXR activation also induces an SR-BI-independent increase in reverse cholesterol transport and reduces intestinal cholesterol absorption. Together, these data indicate that FXR is a promising therapeutic target for treatment of hypercholesterolemia and coronary heart disease.

Hepatic Hepatocyte Nuclear Factor 4α Is Essential for Maintaining Triglyceride and Cholesterol Homeostasis

Objective—Loss-of-function mutations in human hepatocyte nuclear factor 4&agr; (HNF4&agr;) are associated with maturity-onset diabetes of the young and lipid disorders. However, the mechanisms underlying the lipid disorders are poorly understood. In this study, we determined the effect of acute loss or augmentation of hepatic HNF4&agr; function on lipid homeostasis. Methods and Results—We generated an adenovirus expressing LacZ (Ad-shLacZ) or short hairpin RNA of Hnf4&agr; (Ad-shHnf4&agr;). Tail vain injection of C57BL/6J mice with Ad-shHnf4&agr; reduced hepatic Hnf4&agr; expression and resulted in striking phenotypes, including the development of fatty liver and a >80% decrease in plasma levels of triglycerides, total cholesterol, and high-density lipoprotein cholesterol. These latter changes were associated with reduced hepatic lipogenesis and impaired very-low-density lipoprotein secretion. Deficiency in hepatic Hnf4&agr; did not affect intestinal cholesterol absorption despite decreased expression of genes involved in bile acid synthesis. Consistent with the loss-of-function data, overexpression of Hnf4&agr; induced numerous genes involved in lipid metabolism in isolated primary hepatocytes. Interestingly, many of these HNF4&agr;-regulated genes were not induced in wild-type mice that overexpressed hepatic Hnf4&agr;. Because of selective gene regulation, mice overexpressing hepatic Hnf4&agr; had unchanged plasma triglyceride levels and decreased plasma cholesterol levels. Conclusion—Loss of hepatic HNF4&agr; results in severe lipid disorder as a result of dysregulation of multiple genes involved in lipid metabolism. In contrast, augmentation of hepatic HNF4&agr; activity lowers plasma cholesterol levels but has no effect on plasma triglyceride levels because of selective gene regulation. Our data indicate that hepatic HNF4&agr; is essential for controlling the basal expression of numerous genes involved in lipid metabolism and is indispensable for maintaining normal lipid homeostasis.

A metabolic stress-inducible miR-34a-HNF4α pathway regulates lipid and lipoprotein metabolism

Summary Non-alcoholic fatty liver disease (NAFLD) is one of the most common liver diseases, but its underlying mechanism is poorly understood. Here we show that hepatocyte nuclear factor 4α (HNF4α), a liver-enriched nuclear hormone receptor, is markedly inhibited whereas miR-34a is highly induced in patients with non-alcoholic steatohepatitis, diabetic mice and mice fed a high fat diet. miR-34a is essential for HNF4α expression and regulates triglyceride accumulation in human and murine hepatocytes. miR-34a inhibits very low-density lipoprotein secretion and promotes liver steatosis and hypolipidemia in an HNF4α-dependent manner. As a result, increased miR-34a or reduced HNF4α expression in the liver attenuates the development of atherosclerosis in Apoe−/− or Ldlr−/− mice. These data indicate that the miR-34a-HNF4α pathway is activated under common conditions of metabolic stress and may have a role in the pathogenesis of NAFLD and in regulating plasma lipoprotein metabolism. Targeting this pathway may represent a novel approach for the treatment of NAFLD.

Hepatic carboxylesterase 1 is essential for both normal and farnesoid X receptor‐controlled lipid homeostasis

Nonalcoholic fatty liver disease (NAFLD) is one of the major health concerns worldwide. Farnesoid X receptor (FXR) is considered a therapeutic target for treatment of NAFLD. However, the mechanism by which activation of FXR lowers hepatic triglyceride (TG) levels remains unknown. Here we investigated the role of hepatic carboxylesterase 1 (CES1) in regulating both normal and FXR‐controlled lipid homeostasis. Overexpression of hepatic CES1 lowered hepatic TG and plasma glucose levels in both wild‐type and diabetic mice. In contrast, knockdown of hepatic CES1 increased hepatic TG and plasma cholesterol levels. These effects likely resulted from the TG hydrolase activity of CES1, with subsequent changes in fatty acid oxidation and/or de novo lipogenesis. Activation of FXR induced hepatic CES1, and reduced the levels of hepatic and plasma TG as well as plasma cholesterol in a CES1‐dependent manner. Conclusion: Hepatic CES1 plays a critical role in regulating both lipid and carbohydrate metabolism and FXR‐controlled lipid homeostasis. (Hepatology 2014;59:1761–1771)

Aldo-keto reductase 1B7 is a target gene of FXR and regulates lipid and glucose homeostasis

Aldo-keto reductase 1B7 (AKR1B7) is proposed to play a role in detoxification of by-products of lipid peroxidation. In this article, we show that activation of the nuclear receptor farnesoid X receptor (FXR) induces AKR1B7 expression in the liver and intestine, and reduces the levels of malondialdehyde (MDA), the end product of lipid peroxidation, in the intestine but not in the liver. To determine whether AKR1B7 regulates MDA levels in vivo, we overexpressed AKR1B7 in the liver. Overexpression of AKR1B7 in the liver had no effect on hepatic or plasma MDA levels. Interestingly, hepatic expression of AKR1B7 significantly lowered plasma glucose levels in both wild-type and diabetic db/db mice, which was associated with reduced hepatic gluconeogenesis. Hepatic expression of AKR1B7 also significantly lowered hepatic triglyceride and cholesterol levels in db/db mice. These data reveal a novel function for AKR1B7 in lipid and glucose metabolism and suggest that AKR1B7 may not play a role in detoxification of lipid peroxides in the liver. AKR1B7 may be a therapeutic target for treatment of fatty liver disease associated with diabetes mellitus.

Farnesoid X receptor activation increases reverse cholesterol transport by modulating bile acid composition and cholesterol absorption in mice

Activation of farnesoid X receptor (FXR) markedly attenuates development of atherosclerosis in animal models. However, the underlying mechanism is not well elucidated. Here, we show that the FXR agonist, obeticholic acid (OCA), increases fecal cholesterol excretion and macrophage reverse cholesterol transport (RCT) dependent on activation of hepatic FXR. OCA does not increase biliary cholesterol secretion, but inhibits intestinal cholesterol absorption. OCA markedly inhibits hepatic cholesterol 7α‐hydroxylase (Cyp7a1) and sterol 12α‐hydroxylase (Cyp8b1) partly through inducing small heterodimer partner, leading to reduced bile acid pool size and altered bile acid composition, with the α/β‐muricholic acid proportion in bile increased by 2.6‐fold and taurocholic acid (TCA) level reduced by 71%. Overexpression of Cyp8b1 or concurrent overexpression of Cyp7a1 and Cyp8b1 normalizes TCA level, bile acid composition, and intestinal cholesterol absorption. Conclusion: Activation of FXR inhibits intestinal cholesterol absorption by modulation of bile acid pool size and composition, thus leading to increased RCT. Targeting hepatic FXR and/or bile acids may be useful for boosting RCT and preventing the development of atherosclerosis. (Hepatology 2016;64:1072‐1085)

FXR Activation Increases Reverse Cholesterol Transport by Modulating Bile Acid Composition and Cholesterol Absorption

Activation of farnesoid X receptor (FXR) markedly attenuates development of atherosclerosis in animal models. However, the underlying mechanism is not well elucidated. Here, we show that the FXR agonist, obeticholic acid (OCA), increases fecal cholesterol excretion and macrophage reverse cholesterol transport (RCT) dependent on activation of hepatic FXR. OCA does not increase biliary cholesterol secretion, but inhibits intestinal cholesterol absorption. OCA markedly inhibits hepatic cholesterol 7α‐hydroxylase (Cyp7a1) and sterol 12α‐hydroxylase (Cyp8b1) partly through inducing small heterodimer partner, leading to reduced bile acid pool size and altered bile acid composition, with the α/β‐muricholic acid proportion in bile increased by 2.6‐fold and taurocholic acid (TCA) level reduced by 71%. Overexpression of Cyp8b1 or concurrent overexpression of Cyp7a1 and Cyp8b1 normalizes TCA level, bile acid composition, and intestinal cholesterol absorption. Conclusion: Activation of FXR inhibits intestinal cholesterol absorption by modulation of bile acid pool size and composition, thus leading to increased RCT. Targeting hepatic FXR and/or bile acids may be useful for boosting RCT and preventing the development of atherosclerosis. (Hepatology 2016;64:1072‐1085)

Carboxylesterase 2 prevents liver steatosis by modulating lipolysis, endoplasmic reticulum stress, and lipogenesis and is regulated by hepatocyte nuclear factor 4 alpha in mice

Nonalcoholic fatty liver disease (NAFLD) is a common liver disease that ranges from simple steatosis to nonalcoholic steatohepatitis (NASH). So far, the underlying mechanism remains poorly understood. Here, we show that hepatic carboxylesterase 2 (CES2) is markedly reduced in NASH patients, diabetic db/db mice, and high‐fat diet (HFD)‐fed mice. Restoration of hepatic CES2 expression in db/db or HFD‐fed mice markedly ameliorates liver steatosis and insulin resistance. In contrast, knockdown of hepatic CES2 causes liver steatosis and damage in chow‐ or Western diet‐fed C57BL/6 mice. Mechanistically, we demonstrate that CES2 has triglyceride hydrolase activity. As a result, gain of hepatic CES2 function increases fatty acid oxidation and inhibits lipogenesis, whereas loss of hepatic CES2 stimulates lipogenesis by inducing endoplasmic reticulum stress. We further show that loss of hepatic CES2 stimulates lipogenesis in a sterol regulatory element‐binding protein 1 (SREBP‐1)‐dependent manner. Finally, we show that hepatocyte nuclear factor 4 alpha (HNF‐4α) plays a key role in controlling hepatic CES2 expression in diabetes, obesity, or NASH. Conclusion: CES2 plays a protective role in development of NAFLD. Targeting the HNF‐4α/CES2 pathway may be useful for treatment of NAFLD. (Hepatology 2016;63:1860‐1874)

Carboxylesterase 1 Is Regulated by Hepatocyte Nuclear Factor 4α and Protects Against Alcohol- and MCD diet-induced Liver Injury

The liver is a major organ that controls hepatic and systemic homeostasis. Dysregulation of liver metabolism may cause liver injury. Previous studies have demonstrated that carboxylesterase 1 (CES1) regulates hepatic triglyceride metabolism and protects against liver steatosis. In the present study, we investigated whether CES1 played a role in the development of alcoholic liver disease (ALD) and methionine and choline-deficient (MCD) diet-induced liver injury. Both hepatocyte nuclear factor 4α (HNF4α) and CES1 were markedly reduced in patients with alcoholic steatohepatitis. Alcohol repressed both HNF4α and CES1 expression in primary hepatocytes. HNF4α regulated CES1 expression by directly binding to the proximal promoter of CES1. Global inactivation of CES1 aggravated alcohol- or MCD diet-induced liver inflammation and liver injury, likely as a result of increased production of acetaldehyde and reactive oxygen species and mitochondrial dysfunctions. Knockdown of hepatic CES1 exacerbated ethanol-induced steatohepatitis. These data indicate that CES1 plays a crucial role in protection against alcohol- or MCD diet-induced liver injury.

Reversal of metabolic disorders by pharmacological activation of bile acid receptors TGR5 and FXR

Objectives Activation of the bile acid (BA) receptors farnesoid X receptor (FXR) or G protein-coupled bile acid receptor (GPBAR1; TGR5) improves metabolic homeostasis. In this study, we aim to determine the impact of pharmacological activation of bile acid receptors by INT-767 on reversal of diet-induced metabolic disorders, and the relative contribution of FXR vs. TGR5 to INT-767s effects on metabolic parameters. Methods Wild-type (WT), Tgr5−/−, Fxr−/−, Apoe−/− and Shp−/− mice were used to investigate whether and how BA receptor activation by INT-767, a semisynthetic agonist for both FXR and TGR5, could reverse diet-induced metabolic disorders. Results INT-767 reversed HFD-induced obesity dependent on activation of both TGR5 and FXR and also reversed the development of atherosclerosis and non-alcoholic fatty liver disease (NAFLD). Mechanistically, INT-767 improved hypercholesterolemia by activation of FXR and induced thermogenic genes via activation of TGR5 and/or FXR. Furthermore, INT-767 inhibited several lipogenic genes and de novo lipogenesis in the liver via activation of FXR. We identified peroxisome proliferation-activated receptor γ (PPARγ) and CCAAT/enhancer-binding protein α (CEBPα) as novel FXR-regulated genes. FXR inhibited PPARγ expression by inducing small heterodimer partner (SHP) whereas the inhibition of CEBPα by FXR was SHP-independent. Conclusions BA receptor activation can reverse obesity, NAFLD, and atherosclerosis by specific activation of FXR or TGR5. Our data suggest that, compared to activation of FXR or TGR5 only, dual activation of both FXR and TGR5 is a more attractive strategy for treatment of common metabolic disorders.