Circadian Rhythm Disruption Influenced Hepatic Lipid Metabolism, Gut Microbiota and Promoted Cholesterol Gallstone Formation in Mice

BackgroundHepatic lipid metabolism regulates biliary composition and influences the formation of cholesterol gallstones. The genes Hmgcr and Cyp7a1, which encode key liver enzymes, are regulated by circadian rhythm-related transcription factors. We aimed to investigate the effect of circadian rhythm disruption on hepatic cholesterol and bile acid metabolism and the incidence of cholesterol stone formation.MethodsAdult male C57BL/6J mice were fed either a lithogenic diet (LD) only during the sleep phase (time-restricted lithogenic diet feeding, TRF) or an LD ad libitum (non-time-restricted lithogenic diet feeding, nTRF) for 4 weeks. Food consumption, body mass gain, and the incidence of gallstones were assessed. Circulating metabolic parameters, lipid accumulation in the liver, the circadian expression of hepatic clock and metabolic genes, and the gut microbiota were analyzed.ResultsTRF caused a dysregulation of the circadian rhythm in the mice, characterized by significant differences in the circadian expression patterns of clock-related genes. In TRF mice, the circadian rhythms in the expression of genes involved in bile acid and cholesterol metabolism were disrupted, as was the circadian rhythm of the gut microbiota. These changes were associated with high biliary cholesterol content, which promoted gallstone formation in the TRF mice.ConclusionDisordered circadian rhythm is associated with abnormal hepatic bile acid and cholesterol metabolism in mice, which promotes gallstone formation.

Changes and Correlations of the Intestinal Flora and Liver Metabolite Profiles in Mice With Gallstones

There is increasing appreciation for the roles of the gut-liver axis in liver and gall diseases. Specific gut microbes are associated with susceptibility to gallstone diseases, while the relationship between intestinal flora and liver metabolism in the formation of gallstones remains unclear. In this study, an experimental group of model mice was given a lithogenic diet, and a control group was given a normal diet. Both groups were fed for 8 weeks. Integrating 16S rRNA gene sequencing and non-targeted metabolomics to explore the impact of the lithogenic diet on intestinal flora and liver metabolism, Spearman correlation analysis reveals the network of relationships between the intestine and liver. Our findings showed that the gut microbiome and liver metabolome compositions of the test group were significantly changed compared with those of the normal group. Through our research, biomarkers of gallstones were identified at the phylum (5), class (5), order (5), family (7), and genus levels. We predicted the function of the differential flora. We analyzed the liver metabolism of mice with gallstones paired with their flora, and the results showed that there were 138 different metabolites between the two groups. The metabolic pathways enriched by these differential metabolites are highly consistent with the functions of the disordered flora. We focused on an analysis of the relationship between deoxycholic acid, asymmetric dimethylarginine, glucosamine, tauroursodeoxycholic acid, and the disordered flora. This provides a basis for the establishment of the intestine-liver axis in gallstone disease. This research provides a theoretical basis for the research and development of probiotics and prebiotics.

Thyroid Hormone Deficiency Modifies Hepatic Lipid Droplet Morphology and Molecular Properties in Lithogenic-Diet Supplemented Mice

Objective Thyroid hormones have been associated with a hepatic lipid lowering effect and thyroid function has been shown to play a substantial role in development of non-alcoholic fatty liver disease. Hepatic lipid droplets differ in the number, size and molecular properties depending on metabolic state or pathological condition. However, in how far thyroid hormone deficiency affects hepatic lipid droplet morphology and molecular properties is still poorly understood. Therefore, we performed a study in mice using a lithogenic diet model of steatohepatitis and modulated the thyroid hormone status. Methods Male and female three months old C57BL/6 mice were divided into a euthyroid (control), a lithogenic (litho) and a lithogenic+thyroid hormone deficient (litho+hypo) group and treated for six weeks. Hepatic transmission electron microscopy and gene expression analysis of lipid-droplet associated proteins were performed. Results Increased mean diameters of hepatic lipid droplets and a shift towards raised electron-density in lipid droplets was observed under thyroid hormone deficiency. Furthermore thyroid hormone deficiency altered hepatic expression of genes involved in lipophagy and triacylglycerol mobilization. Interestingly, while the impact of thyroid hormone deficiency on lipid droplet morphology seems to be sex-independent, hepatic lipid droplet-associated gene expression differed significantly between both sexes. Conclusion This study demonstrates that thyroid hormone deficiency alters hepatic lipid droplet morphology and hepatic gene expression of lipid droplet-associated proteins in a lithogenic diet mouse model of steatohepatitis.

Alleviation of Cholesterol Gallstones by Lactobacillus Targeting the Gut Microbiota Depends on Global Farnesoid X Receptor-Mediated Bile Acid Metabolism

Background Cholesterol gallstone (CGS) disease is characterized by an imbalance in bile acid (BA) metabolism and is closely associated with gut microbiota disorders. However, the role and mechanism by which probiotics targeting the gut microbiota attenuate CGS are still unknown. In this study, Lactobacillus reuteri CGMCC 17942 (LR) and L. plantarum CGMCC 14407 (LP) were individually administered to lithogenic diet (LD)-fed mice at a dosage of 109 CFU/day for 8 weeks. Results Both Lactobacillus strains significantly reduced LD-induced gallstones, hepatic steatosis, and hyperlipidemia. These strains modulated serum BA profiles, with significantly decreased conjugated primary BA taurine-β-muricholic acid (T-β-MCA), an FXR antagonist. At the molecular level, LR and LP increased Farnesoid X Receptor (FXR) expression in the liver but not in the ileum, increased the levels of ileum and liver fibroblast growth factor 15 (FGF15) and liver FGFR4, small heterodimer partner (SHP), and subsequently reduced cholesterol 7α-hydroxylase (CYP7A1) and cytochrome P450 family 7 subfamily B polypeptide 1 (CYP7B1) to inhibit BA synthesis in the liver. At the same time, the two strains enhanced BA transport by increasing the levels of multidrug-resistance-associated protein homologs (MRP) 3/4, multidrug-resistance-associated protein homologs (MRP) 3/4, hepatic multidrug resistance protein (MDR2) and bile salt export pump (BSEP) mRNA in the liver. In addition, both LR and LP reduced LD-associated gut microbiota dysbiosis. LR increased the relative abundance of Muribaculaceae, while LP increased that of Akkermansia. The changed gut microbiota was significantly negatively correlated with the grade and incidence of gallstones, hyperlipidemia, the level of T-β-MCA in serum, or the gene expression levels of Fxr in liver. Furthermore, the protective effects of the two strains were abolished by a global but not intestinal-specific FXR antagonist. Conclusions Taken together, our results suggested that Lactobacillus might relieve gallstones through FXR-dependent regulation of BA synthesis and transport.

Role of Baicalin and Liver X Receptor Alpha in the Formation of Cholesterol Gallstones in Mice

This study was aimed at investigating the effect of baicalin on experimental cholesterol gallstones in mice. The mouse gallstone model was induced by feeding with a lithogenic diet, and cholesterol stones were found in the gallbladder. The lithogenic diet caused elevation of triglycerides, cholesterol, and low-density lipoprotein concentrations and descent of high-density lipoprotein concentration in serum. Hyperplasia and inflammatory infiltration were observed in the gallbladder wall of lithogenic diet-fed mice. We also found the increase of cholesterol content and the decrease of bile acid in bile. Real-time PCR and western blot results demonstrated that the expression levels of two enzymes (cholesterol 7α-hydroxylase (CYP7a1) and sterol 12α-hydroxylase (CYP8b1)) to catalyze the synthesis of bile acid from cholesterol were decreased and that two cholesterol transporters (ATP-binding cassette transporter G5/G8 (ABCG5/8)) were increased in the liver of lithogenic diet-fed mice. The lithogenic diet also led to enhanced activity of alanine aminotransferase and aspartate aminotransferase in serum; increased concentrations of tumor necrosis factor-α, interleukin- (IL-) 1β, IL-6, and malondialdehyde; and decreased superoxide dismutase activity in the liver, suggesting inflammatory and oxidative stress. In addition, liver X receptor alpha (LXRα) was increased in the liver. After gavage of baicalin, the lithogenic diet-induced gallstones, hyperlipidemia, gallbladder hyperplasia, inflammation, and oxidative stress in liver and cholesterol metabolism disorders were all alleviated to some degree. The expression of LXRα in the liver was inhibited by baicalin. In addition, the LXRα agonist T0901317 aggravated lithogenic diet-induced harmful symptoms in mice, including the increase of gallstone formation, hyperlipidemia, hepatic injury, inflammation, and oxidative stress. In conclusion, we demonstrated that baicalin played a protective role in a lithogenic diet-induced gallstone mouse model, which may be mediated by inhibition of LXRα activity. These findings may provide novel insights for prevention and therapy of gallstones in the clinic.

A novel GPER antagonist protects against the formation of estrogen-induced cholesterol gallstones in female mice

Many clinical studies and epidemiological investigations have clearly demonstrated that women are twice as likely to develop cholesterol gallstones as men, and oral contraceptives and other estrogen therapies dramatically increase that risk. Further, animal studies have revealed that estrogen promotes cholesterol gallstone formation through the estrogen receptor (ER) α, but not ERβ, pathway. More importantly, some genetic and pathophysiological studies have found that G protein-coupled estrogen receptor (GPER) 1 is a new gallstone gene, Lith18, on chromosome 5 in mice and produces additional lithogenic actions, working independently of ERα, to markedly increase cholelithogenesis in female mice. Based on computational modeling of GPER, a novel series of GPER-selective antagonists were designed, synthesized, and subsequently assessed for their therapeutic effects via calcium mobilization, cAMP, and ERα and ERβ fluorescence polarization binding assays. From this series of compounds, one new compound, 2-cyclohexyl-4-isopropyl-N-(4-methoxybenzyl)aniline (CIMBA), exhibits superior antagonism and selectivity exclusively for GPER. Furthermore, CIMBA reduces the formation of 17β-estradiol-induced gallstones in a dose-dependent manner in ovariectomized mice fed a lithogenic diet for 8 weeks. At 32 μg/day/kg CIMBA, no gallstones are found, even in ovariectomized ERα (−/−) mice treated with 6 μg/day 17β-estradiol and fed the lithogenic diet for 8 weeks. In conclusion, CIMBA treatment protects against the formation of estrogen-induced cholesterol gallstones by inhibiting the GPER signaling pathway in female mice. CIMBA may thus be a new agent for effectively treating cholesterol gallstone disease in women.­