Shannon Boehme

Glucose and Insulin Induction of Bile Acid Synthesis MECHANISMS AND IMPLICATION IN DIABETES AND OBESITY

Background: Bile acid synthesis plays an important role in nutrient absorption and maintaining metabolic homeostasis under normal physiology. Results: Glucose induces cholesterol 7α-hydroxylase activity and postprandial bile acid synthesis via insulin signaling and epigenetic mechanisms. Conclusion: Glucose and insulin are major postprandial factors that stimulate bile acid synthesis to maintain hepatic metabolic homeostasis. Significance: Nutrient regulation of bile acid synthesis is impaired in diabetes and obesity. Bile acids facilitate postprandial absorption of nutrients. Bile acids also activate the farnesoid X receptor (FXR) and the G protein-coupled receptor TGR5 and play a major role in regulating lipid, glucose, and energy metabolism. Transgenic expression of cholesterol 7α-hydroxylase (CYP7A1) prevented high fat diet-induced diabetes and obesity in mice. In this study, we investigated the nutrient effects on bile acid synthesis. Refeeding of a chow diet to fasted mice increased CYP7A1 expression, bile acid pool size, and serum bile acids in wild type and humanized CYP7A1-transgenic mice. Chromatin immunoprecipitation assays showed that glucose increased histone acetylation and decreased histone methylation on the CYP7A1 gene promoter. Refeeding also induced CYP7A1 in fxr-deficient mice, indicating that FXR signaling did not play a role in postprandial regulation of bile acid synthesis. In streptozocin-induced type I diabetic mice and genetically obese type II diabetic ob/ob mice, hyperglycemia increased histone acetylation status on the CYP7A1 gene promoter, leading to elevated basal Cyp7a1 expression and an enlarged bile acid pool with altered bile acid composition. However, refeeding did not further increase CYP7A1 expression in diabetic mice. In summary, this study demonstrates that glucose and insulin are major postprandial factors that induce CYP7A1 gene expression and bile acid synthesis. Glucose induces CYP7A1 gene expression mainly by epigenetic mechanisms. In diabetic mice, CYP7A1 chromatin is hyperacetylated, and fasting to refeeding response is impaired and may exacerbate metabolic disorders in diabetes.

Overexpression of Cholesterol 7α-hydroxylase promotes hepatic bile acid synthesis and secretion and maintains cholesterol homeostasis

We reported previously that mice overexpressing cytochrome P450 7a1 (Cyp7a1; Cyp7a1‐tg mice) are protected against high fat diet–induced hypercholesterolemia, obesity, and insulin resistance. Here, we investigated the underlying mechanism of bile acid signaling in maintaining cholesterol homeostasis in Cyp7a1‐tg mice. Cyp7a1‐tg mice had two‐fold higher Cyp7a1 activity and bile acid pool than did wild‐type mice. Gallbladder bile acid composition changed from predominantly cholic acid (57%) in wild‐type to chenodeoxycholic acid (54%) in Cyp7a1‐tg mice. Cyp7a1‐tg mice had higher biliary and fecal cholesterol and bile acid secretion rates than did wild‐type mice. Surprisingly, hepatic de novo cholesterol synthesis was markedly induced in Cyp7a1‐tg mice but intestine fractional cholesterol absorption in Cyp7a1‐tg mice remained the same as wild‐type mice despite the presence of increased intestine bile acids. Interestingly, hepatic but not intestinal expression of several cholesterol (adenosine triphosphate–binding cassette G5/G8 [ABCG5/G8], scavenger receptor class B, member 1) and bile acid (ABCB11) transporters were significantly induced in Cyp7a1‐tg mice. Treatment of mouse or human hepatocytes with a farnesoid X receptor (FXR) agonist GW4064 or bile acids induced hepatic Abcg5/g8 expression. A functional FXR binding site was identified in the Abcg5 gene promoter. Study of tissue‐specific Fxr knockout mice demonstrated that loss of the Fxr gene in the liver attenuated bile acid induction of hepatic Abcg5/g8 and gallbladder cholesterol content, suggesting a role of FXR in the regulation of cholesterol transport. Conclusion: This study revealed a new mechanism by which increased Cyp7a1 activity expands the hydrophobic bile acid pool, stimulating hepatic cholesterol synthesis and biliary cholesterol secretion without increasing intestinal cholesterol absorption. This study demonstrated that Cyp7a1 plays a critical role in maintaining cholesterol homeostasis and underscores the importance of bile acid signaling in regulating overall cholesterol homeostasis. (HEPATOLOGY 2011)

Regulation of cholesterol and bile acid homeostasis by the cholesterol 7α-hydroxylase/steroid response element-binding protein 2/microRNA-33a axis in mice.

Bile acid synthesis not only produces physiological detergents required for intestinal nutrient absorption, but also plays a critical role in regulating hepatic and whole‐body metabolic homeostasis. We recently reported that overexpression of cholesterol 7α‐hydroxylase (CYP7A1) in the liver resulted in improved metabolic homeostasis in Cyp7a1 transgenic (Cyp7a1‐tg) mice. This study further investigated the molecular links between bile acid metabolism and lipid homeostasis. Microarray gene profiling revealed that CYP7A1 overexpression led to marked activation of the steroid response element‐binding protein 2 (SREBP2)‐regulated cholesterol metabolic network and absence of bile acid repression of lipogenic gene expression in livers of Cyp7a1‐tg mice. Interestingly, Cyp7a1‐tg mice showed significantly elevated hepatic cholesterol synthesis rates, but reduced hepatic fatty acid synthesis rates, which was accompanied by increased 14C‐glucose‐derived acetyl‐coenzyme A incorporation into sterols for fecal excretion. Induction of SREBP2 also coinduces intronic microRNA‐33a (miR‐33a) in the SREBP2 gene in Cyp7a1‐tg mice. Overexpression of miR‐33a in the liver resulted in decreased bile acid pool, increased hepatic cholesterol content, and lowered serum cholesterol in mice. Conclusion: This study suggests that a CYP7A1/SREBP2/miR‐33a axis plays a critical role in regulation of hepatic cholesterol, bile acid, and fatty acid synthesis. Antagonism of miR‐33a may be a potential strategy to increase bile acid synthesis to maintain lipid homeostasis and prevent nonalcoholic fatty liver disease, diabetes, and obesity. (Hepatology 2013;53:1111–1121)

Farnesoid X receptor induces Takeda G-protein receptor 5 Crosstalk to regulate Bile Acid Synthesis and Hepatic Metabolism

The bile acid-activated receptors, nuclear farnesoid X receptor (FXR) and the membrane Takeda G-protein receptor 5 (TGR5), are known to improve glucose and insulin sensitivity in obese and diabetic mice. However, the metabolic roles of these two receptors and the underlying mechanisms are incompletely understood. Here, we studied the effects of the dual FXR and TGR5 agonist INT-767 on hepatic bile acid synthesis and intestinal secretion of glucagon-like peptide-1 (GLP-1) in wild-type, Fxr−/−, and Tgr5−/− mice. INT-767 efficaciously stimulated intracellular Ca2+ levels, cAMP activity, and GLP-1 secretion and improved glucose and lipid metabolism more than did the FXR-selective obeticholic acid and TGR5-selective INT-777 agonists. Interestingly, INT-767 reduced expression of the genes in the classic bile acid synthesis pathway but induced those in the alternative pathway, which is consistent with decreased taurocholic acid and increased tauromuricholic acids in bile. Furthermore, FXR activation induced expression of FXR target genes, including fibroblast growth factor 15, and unexpectedly Tgr5 and prohormone convertase 1/3 gene expression in the ileum. We identified an FXR-responsive element on the Tgr5 gene promoter. Fxr−/− and Tgr5−/− mice exhibited reduced GLP-1 secretion, which was stimulated by INT-767 in the Tgr5−/− mice but not in the Fxr−/− mice. Our findings uncovered a novel mechanism in which INT-767 activation of FXR induces Tgr5 gene expression and increases Ca2+ levels and cAMP activity to stimulate GLP-1 secretion and improve hepatic glucose and lipid metabolism in high-fat diet-induced obese mice. Activation of both FXR and TGR5 may therefore represent an effective therapy for managing hepatic steatosis, obesity, and diabetes.

G‐protein‐coupled bile acid receptor plays a key role in bile acid metabolism and fasting‐induced hepatic steatosis in mice

Bile acids are signaling molecules that play a critical role in regulation of hepatic metabolic homeostasis by activating nuclear farnesoid X receptor (Fxr) and membrane G‐protein‐coupled receptor (Takeda G‐protein‐coupled receptor 5; Tgr5). The role of FXR in regulation of bile acid synthesis and hepatic metabolism has been studied extensively. However, the role of TGR5 in hepatic metabolism has not been explored. The liver plays a central role in lipid metabolism, and impaired response to fasting and feeding contributes to steatosis and nonalcoholic fatty liver and obesity. We have performed a detailed analysis of gallbladder bile acid and lipid metabolism in Tgr5−/− mice in both free‐fed and fasted conditions. Lipid profiles of serum, liver and adipose tissues, bile acid composition, energy metabolism, and messenger RNA and protein expression of the genes involved in lipid metabolism were analyzed. Results showed that deficiency of the Tgr5 gene in mice alleviated fasting‐induced hepatic lipid accumulation. Expression of liver oxysterol 7α‐hydroxylase in the alternative bile acid synthesis pathway was reduced. Analysis of gallbladder bile acid composition showed marked increase of taurocholic acid and decrease of tauro‐α and β‐muricholic acid in Tgr5−/− mice. Tgr5−/− mice had increased hepatic fatty acid oxidation rate and decreased hepatic fatty acid uptake. Interestingly, fasting induction of fibroblast growth factor 21 in liver was attenuated. In addition, fasted Tgr5−/− mice had increased activation of hepatic growth hormone‐signal transducer and activator of transcription 5 (GH‐Stat5) signaling compared to wild‐type mice. Conclusion: TGR5 may play a role in determining bile acid composition and in fasting‐induced hepatic steatosis through a novel mechanism involving activation of the GH‐Stat5 signaling pathway. (Hepatology 2017;65:813‐827)

Cholesterol 7α-hydroxylase protects the liver from inflammation and fibrosis by maintaining cholesterol homeostasis

Cholesterol 7α-hydroxylase (CYP7A1) plays a critical role in control of bile acid and cholesterol homeostasis. Bile acids activate farnesoid X receptor (FXR) and Takeda G protein-coupled receptor 5 (TGR5) to regulate lipid, glucose, and energy metabolism. However, the role of bile acids in hepatic inflammation and fibrosis remains unclear. In this study, we showed that adenovirus-mediated overexpression of Cyp7a1 ameliorated lipopolysaccharide (LPS)-induced inflammatory cell infiltration and pro-inflammatory cytokine production in WT and TGR5-deficient (Tgr5−/−) mice, but not in FXR-deficient (Fxr−/−) mice, suggesting that bile acid signaling through FXR protects against hepatic inflammation. Nuclear factor κ light-chain enhancer of activated B cells (NF-κB)-luciferase reporter assay showed that FXR agonists significantly inhibited TNF-α-induced NF-κB activity. Furthermore, chromatin immunoprecipitation and mammalian two-hybrid assays showed that ligand-activated FXR interacted with NF-κB and blocked recruitment of steroid receptor coactivator-1 to cytokine promoter and resulted in inhibition of NF-κB activity. Methionine/choline-deficient (MCD) diet increased hepatic inflammation, free cholesterol, oxidative stress, apoptosis, and fibrosis in CYP7A1-deficient (Cyp7a1−/−) mice compared with WT mice. Remarkably, adenovirus-mediated overexpression of Cyp7a1 effectively reduced hepatic free cholesterol and oxidative stress and reversed hepatic inflammation and fibrosis in MCD diet-fed Cyp7a1−/− mice. Current studies suggest that increased Cyp7a1 expression and bile acid synthesis ameliorate hepatic inflammation through activation of FXR, whereas reduced bile acid synthesis aggravates MCD diet-induced hepatic inflammation and fibrosis. Maintaining bile acid and cholesterol homeostasis is important for protecting against liver injury and nonalcoholic fatty liver disease.

Cholesterol 7α-hydroxylase-deficient mice are protected from high-fat/high-cholesterol diet-induced metabolic disorders

Cholesterol 7α-hydroxylase (CYP7A1) is the first and rate-limiting enzyme in the conversion of cholesterol to bile acids in the liver. In addition to absorption and digestion of nutrients, bile acids play a critical role in the regulation of lipid, glucose, and energy homeostasis. We have backcrossed Cyp7a1−/− mice in a mixed B6/129Sv genetic background to C57BL/6J mice to generate Cyp7a1−/− mice in a near-pure C57BL/6J background. These mice survive well and have normal growth and a bile acid pool size ∼60% of WT mice. The expression of the genes in the alternative bile acid synthesis pathway are upregulated, resulting in a more hydrophilic bile acid composition with reduced cholic acid (CA). Surprisingly, Cyp7a1−/− mice have improved glucose sensitivity with reduced liver triglycerides and fecal bile acid excretion, but increased fecal fatty acid excretion and respiratory exchange ratio (RER) when fed a high-fat/high-cholesterol diet. Supplementing chow and Western diets with CA restored bile acid composition, reversed the glucose tolerant phenotype, and reduced the RER. Our current study points to a critical role of bile acid composition, rather than bile acid pool size, in regulation of glucose, lipid, and energy metabolism to improve glucose and insulin tolerance, maintain metabolic homeostasis, and prevent high-fat diet-induced metabolic disorders.

Intestine farnesoid X receptor agonist and the gut microbiota activate G-protein bile acid receptor-1 signaling to improve metabolism

Bile acids activate farnesoid X receptor (FXR) and G protein–coupled bile acid receptor‐1 (aka Takeda G protein–coupled receptor‐5 [TGR5]) to regulate bile acid metabolism and glucose and insulin sensitivity. FXR and TGR5 are coexpressed in the enteroendocrine L cells, but their roles in integrated regulation of metabolism are not completely understood. We reported recently that activation of FXR induces TGR5 to stimulate glucagon‐like peptide‐1 (GLP‐1) secretion to improve insulin sensitivity and hepatic metabolism. In this study, we used the intestine‐restricted FXR agonist fexaramine (FEX) to study the effect of activation of intestinal FXR on the gut microbiome, bile acid metabolism, and FXR and TGR5 signaling. The current study revealed that FEX markedly increased taurolithocholic acid, increased secretion of fibroblast growth factors 15 and 21 and GLP‐1, improved insulin and glucose tolerance, and promoted white adipose tissue browning in mice. Analysis of 16S ribosomal RNA sequences of the gut microbiome identified the FEX‐induced and lithocholic acid–producing bacteria Acetatifactor and Bacteroides. Antibiotic treatment completely reversed the FEX‐induced metabolic phenotypes and inhibited taurolithocholic acid synthesis, adipose tissue browning, and liver bile acid synthesis gene expression but further increased intestinal FXR target gene expression. FEX treatment effectively improved lipid profiles, increased GLP‐1 secretion, improved glucose and insulin tolerance, and promoted adipose tissue browning, while antibiotic treatment reversed the beneficial metabolic effects of FEX in obese and diabetic mice. Conclusion: This study uncovered a mechanism in which activation of intestinal FXR shaped the gut microbiota to activate TGR5/GLP‐1 signaling to improve hepatic glucose and insulin sensitivity and increase adipose tissue browning; the gut microbiota plays a critical role in bile acid metabolism and signaling to regulate metabolic homeostasis in health and disease. (Hepatology 2018).

Intestinal Farnesoid X Receptor and Takeda G Protein Couple Receptor 5 Signaling in Metabolic Regulation

Bile acids play a critical role in the regulation of glucose, lipid and energy metabolisms by activating the nuclear bile acid receptor farnesoid X receptor (FXR) and membrane G protein-coupled bile acid receptor-1 (aka takeda G protein couple receptor 5, TGR5) signaling. Paradoxical roles of FXR in the regulation of glucose and lipid metabolism and metabolic disorder have been reported recently. The activation or inhibition of intestinal FXR signaling has been shown to improve insulin and glucose sensitivity and energy metabolism to prevent diabetes, obesity and non-alcoholic fatty liver disease (NAFLD). TGR5 has an anti-inflammatory function in the intestine and stimulates glucagon-like peptide-1 (GLP-1) secretion in the intestine to stimulate insulin secretion from the pancreas. The role of TGR5 in metabolism and metabolic regulation is not clear and warrants further study. FXR and TGR5 are co-expressed in the ileum and colon. These 2 bile acid-activated receptors may cooperate to stimulate GLP-1 secretion and improve hepatic metabolism. FXR and TGR5 dual agonists may have therapeutic potential for treating diabetes and NAFLD.

Deficiency of cholesterol 7α‐hydroxylase in bile acid synthesis exacerbates alcohol‐induced liver injury in mice

Alcoholic fatty liver disease (AFLD) is a major risk factor for cirrhosis‐associated liver diseases. Studies demonstrate that alcohol increases serum bile acids in humans and rodents. AFLD has been linked to cholestasis, although the physiologic relevance of increased bile acids in AFLD and the underlying mechanism of increasing the bile acid pool by alcohol feeding are still unclear. In this study, we used mouse models either deficient of or overexpressing cholesterol 7α‐hydroxylase (Cyp7a1), the rate‐limiting and key regulatory enzyme in bile acid synthesis, to study the effect of alcohol drinking in liver metabolism and inflammation. Mice were challenged with chronic ethanol feeding (10 days) plus a binge dose of alcohol by oral gavage (5 g/kg body weight). Alcohol feeding reduced bile acid synthesis gene expression but increased the bile acid pool size, hepatic triglycerides and cholesterol, and inflammation and injury in wild‐type mice and aggravated liver inflammation and injury in Cyp7a1‐deficient mice. Interestingly, alcohol‐induced hepatic inflammation and injury were ameliorated in Cyp7a1 transgenic mice. Conclusion: Alcohol feeding alters hepatic bile acid and cholesterol metabolism to cause liver inflammation and injury, while maintenance of bile acid and cholesterol homeostasis protect against alcohol‐induced hepatic inflammation and injury. Our findings indicate that CYP7A1 plays a key role in protection against alcohol‐induced steatohepatitis. (Hepatology Communications 2018;2:99–112)