Berberine prevents progression from hepatic steatosis to steatohepatitis and fibrosis by reducing endoplasmic reticulum stress.
ABSTRACT: The histological spectrum of nonalcoholic fatty liver diseases (NAFLD) ranges from hepatic steatosis to steatohepatitis and fibrosis. Berberine (BBR) is known for its therapeutic effect on obesity, hyperglycaemia and dyslipidaemia; however, its effect on NAFLD has yet to be thoroughly explored. Db/db mice and methionine-choline-deficient diet-fed mice were administered BBR via gavage. We found that BBR-treated mice were more resistant to steatosis in the liver than vehicle-treated mice and that BBR significantly reduced hepatic inflammation, fibrosis and lipid peroxides. The beneficial effect of BBR was associated with suppressing endoplasmic reticulum (ER) stress. Additionally, BBR decreased the free fatty acid-induced lipid accumulation and tunicamycin-induced ER stress in primary hepatocytes and hepatocyte cell lines. We demonstrated that BBR exhibited chaperone activity, reduced protein aggregation in vitro and alleviated tunicamycin-induced triglyceride and collagen deposition in vivo. Finally, we showed that BBR could reverse ER stress-activated lipogenesis through the ATF6/SREBP-1c pathway in vitro. These results indicated that BBR may be a new therapeutic strategy against hepatic steatosis and non-alcoholic steatohepatitis.
Project description:Mitochondrial glycerol 3-phosphate dehydrogenase (mGPDH) is an integral component of the respiratory chain, and recent studies have suggested that it plays an important role in hepatic glucose homeostasis. However, its function in hepatic lipid metabolism is unclear. Here, we identified a role for mGPDH in nonalcoholic fatty liver disease (NAFLD). Specifically, mGPDH expression and activity were lower in fatty livers from patients and mice with NAFLD (ob/ob, high-fat diet [HFD] and db/db). Liver-specific depletion of mGPDH in mice or mGPDH knockdown in cultured hepatocytes exacerbated diet-induced triglyceride accumulation and steatosis through enhanced lipogenesis. RNA-sequencing revealed that mGPDH regulated endoplasmic reticulum (ER)-related proteins and processes. mGPDH deletion exacerbated tunicamycin (ER stress inducer)-induced hepatic steatosis, whereas tauroursodeoxycholic acid (ER stress inhibitor) rescued mGPDH depletion-induced steatosis on an HFD. Moreover, ER stress induced by mGPDH depletion could be abrogated by the intracellular Ca2+ chelator 1,2-bis (2-aminophenoxy) ethane N,N,N´,N´-tetraacetic acid acetoxymethyl ester, mitochondrial permeability transition pore (mPTP) inhibitor cyclosporine A, or cyclophilin-D (Cyp-D) knockdown. mGPDH promoting Cyp-D ubiquitination was also observed. Finally, liver-specific mGPDH overexpression attenuated hepatic steatosis in ob/ob and HFD mice. Conclusion: mGPDH is a pivotal regulator of hepatic lipid metabolism. Its deficiency induces ER stress by suppressing Cyp-D ubiquitination, a key regulator of the mitochondrial Ca2+ conductance channel mPTP, and results in hepatic steatosis. mGPDH may be a potential therapeutic target for the treatment of NAFLD.
Project description:UNLABELLED: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-4?) 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-4?/CES2 pathway may be useful for treatment of NAFLD. (Hepatology 2016;63:1860-1874).
Project description:Oxidative stress is thought to be critical for the pathogenesis of hepatic steatosis and its progress to non-alcoholic steatohepatitis. Berberine (BBR) can improve hepatic steatosis. In this study, we investigated the role of BBR in ameliorating oxidative stress. Lipid accumulation was measured in the livers of C57BL/6 mice fed a high fat diet (HFD) or a normal diet for 8 weeks, then either received BBR or vehicle for the study duration. Nrf2 distribution was detected in male Sprague-Dawley rats' livers in vivo and in Huh7 cells in vitro. ROS generation and mitochondrial complex expression was measured in Huh7 cells. HepG2 cells were employed for the measurement of oxygen consumption rates. Our results showed that BBR reduced triglyceride accumulation in the liver of HFD-fed mice. The activation and nuclear distribution of Nrf2 was decreased in the hepatocytes of rats that received BBR treatment, while on a HFD. BBR also markedly reduced Nox2-dependent cytoplasmic ROS production and mitochondrial ROS production, which was mediated by the down-regulation of Complex I and III expression. In conclusion, BBR has a great potential to reduce the effects of oxidative stress, which likely contributes to its protective effect in inhibiting the progression of hepatic steatosis to steatohepatitis.
Project description:Endoplasmic reticulum (ER) stress is associated with the pathogenesis of hepatic steatosis. Alisma orientale Juzepzuk is a traditional medicinal herb for diuretics, diabetes, hepatitis, and inflammation. In this study, we investigated the protective effects of methanol extract of the tuber of Alisma orientale (MEAO) against ER stress-induced hepatic steatosis in vitro and in vivo. MEAO inhibited the tunicamycin-induced increase in luciferase activity of ER stress-reporter constructs containing ER stress response element and ATF6 response element. MEAO significantly inhibited tunicamycin-induced ER stress marker expression including GRP78, CHOP, and XBP-1 in tunicamycin-treated Human hepatocellular carcinoma (HepG2) cells and the livers of tunicamycin-injected mice. It also inhibited tunicamycin-induced accumulation of cellular triglyceride. Similar observations were made under physiological ER stress conditions such as in palmitate (PA)-treated HepG2 cells and the livers of high-fat diet (HFD)-induced obese mice. MEAO repressed hepatic lipogenic gene expression in PA-treated HepG2 cells and the livers of HFD obese mice. Furthermore, MEAO repressed very low-density lipoprotein receptor (VLDLR) expression and improved ApoB secretion in the livers of tunicamycin-injected mice or HFD obese mice as well as in tunicamycin or PA-treated HepG2 cells. Alismol, a guaiane-type sesquiterpenes in Alisma orientale, inhibited GRP78 expression in tunicamycin-treated HepG2 cells. In conclusion, MEAO attenuates ER stress and prevents hepatic steatosis pathogenesis via inhibition of expression of the hepatic lipogenic genes and VLDLR, and enhancement of ApoB secretion.
Project description:Hepatic steatosis is the initial stage of nonalcoholic fatty liver disease (NAFLD) and may predispose to more severe hepatic disease, including hepatocellular carcinoma. Endoplasmic reticulum (ER) stress has been recently implicated as a novel mechanism that may lead to NAFLD, although the genetic factors invoking ER stress are largely unknown. During a screen for liver defects from a zebrafish insertional mutant library, we isolated the mutant cdipthi559Tg/+ (hi559). CDIPT is known to play an indispensable role in phosphatidylinositol (PtdIns) synthesis. Here we show that cdipt is expressed in the developing liver, and its disruption in hi559 mutants abrogates de novo PtdIns synthesis, resulting in hepatomegaly at 5 days postfertilization. The hi559 hepatocytes display features of NAFLD, including macrovesicular steatosis, ballooning, and necroapoptosis. Gene set enrichment of microarray profiling revealed significant enrichment of endoplasmic reticulum stress response (ERSR) genes in hi559 mutants. ER stress markers, including atf6, hspa5, calr, and xbp1, are selectively up-regulated in the mutant liver. The hi559 expression profile showed significant overlap with that of mammalian hepatic ER stress and NAFLD. Ultrastructurally, the hi559 hepatocytes display marked disruption of ER architecture with hallmarks of chronic unresolved ER stress. Induction of ER stress by tunicamycin in wild-type larvae results in a fatty liver similar to hi559, suggesting that ER stress could be a fundamental mechanism contributing to hepatic steatosis.cdipt-deficient zebrafish exhibit hepatic ER stress and NAFLD pathologies, implicating a novel link between PtdIns, ER stress, and steatosis. The tractability of hi559 mutant provides a valuable tool to dissect ERSR components, their contribution to molecular pathogenesis, and evaluation of novel therapeutics of NAFLD.
Project description:Acid sphingomyelinase (ASMase) is activated in non-alcoholic steatohepatitis (NASH). However, the contribution of ASMase to NASH is poorly understood and limited to hepatic steatosis and glucose metabolism. Here we examined the role of ASMase in high fat diet (HFD)-induced NASH.Autophagy, endoplasmic reticulum (ER) stress and lysosomal membrane permeabilization (LMP) were determined in ASMase(-/-) mice fed a HFD. The impact of pharmacological ASMase inhibition on NASH was analyzed in wild type mice fed a HFD.ASMase deficiency determined resistance to hepatic steatosis mediated by a HFD or methionine-choline deficient diet. ASMase(-/-) mice were resistant to HFD-induced hepatic ER stress, but sensitive to tunicamycin-mediated ER stress, indicating selectivity in the resistance of ASMase(-/-) mice to ER stress and steatosis. Autophagic flux, determined in the presence of rapamycin and/or chloroquine, was lower in primary mouse hepatocytes (PMH) from ASMase(-/-) mice and accompanied by increased p62 levels, suggesting autophagic impairment. Moreover, autophagy suppression by chloroquine and brefeldin A caused ER stress in PMH from ASMase(+/+) mice but not in ASMase(-/-) mice. ASMase(-/-) PMH exhibited increased lysosomal cholesterol loading, decreased LMP and apoptosis resistance induced by O-methyl-serine dodecylamide hydrochloride or palmitic acid, effects that were reversed by decreasing cholesterol levels by oxysterol 25-hydroxycholesterol. In vivo pharmacological ASMase inhibition by amitriptyline, a widely used tricyclic antidepressant, protected wild type mice against HFD-induced hepatic steatosis, fibrosis, and liver damage, effects indicative of early-stage NASH.These findings underscore a critical role for ASMase in diet-induced NASH and suggest the potential of amitriptyline as a treatment for patients with NASH.
Project description:Endoplasmic reticulum (ER) stress is highly associated with liver steatosis. B-cell receptor-associated protein 31 (BAP31) has been reported to be involved in ER homeostasis, and plays key roles in hepatic lipid metabolism in high-fat diet-induced obese mice. However, whether BAP31 modulates hepatic lipid metabolism via regulating ER stress is still uncertain. In this study, wild-type and liver-specific BAP31-depleted mice were administrated with ER stress activator of Tunicamycin, the markers of ER stress, liver steatosis, and the underlying molecular mechanisms were determined. BAP31 deficiency increased Tunicamycin-induced hepatic lipid accumulation, aggravated liver dysfunction, and increased the mRNA levels of ER stress markers, including glucose-regulated protein 78 (GRP78), X-box binding protein 1 (XBP1), inositol-requiring protein-1? (IRE1?) and C/EBP homologous protein (CHOP), thus promoting ER stress in vivo and in vitro. Hepatic lipid export via very low-density lipoprotein (VLDL) secretion was impaired in BAP31-depleted mice, accompanied by reduced Apolipoprotein B (APOB) and microsomal triglyceride transfer protein (MTTP) expression. Exogenous lipid clearance was also inhibited, along with impaired gene expression related to fatty acid transportation and fatty acid ?-oxidation. Finally, BAP31 deficiency increased Tunicamycin-induced hepatic inflammatory response. These results demonstrate that BAP31 deficiency increased Tunicamycin-induced ER stress, impaired VLDL secretion and exogenous lipid clearance, and reduced fatty acid ?-oxidation, which eventually resulted in liver steatosis.
Project description:Melatonin, an indole produced by pineal and extrapineal tissues, but also taken with a vegetarian diet, has strong anti-oxidant, anti-inflammatory and anti-obesogenic potentials. Non-alcoholic fatty liver disease (NAFLD) is the hepatic side of the metabolic syndrome. NAFLD is a still reversible phase but may evolve into steatohepatitis (NASH), cirrhosis and carcinoma. Currently, an effective therapy for blocking NAFLD staging is lacking. Silent information regulator 1 (SIRT1), a NAD+ dependent histone deacetylase, modulates the energetic metabolism in the liver. Micro-RNA-34a-5p, a direct inhibitor of SIRT1, is an emerging indicator of NAFLD grading. Thus, here we analyzed the effects of oral melatonin against NAFLD and underlying molecular mechanisms, focusing on steatosis, ER stress, mitochondrial shape and autophagy. Male C57BL/6J (WT) and SIRT1 heterozygous (HET) mice were placed either on a high-fat diet (58.4% energy from lard) (HFD) or on a standard maintenance diet (8.4% energy from lipids) for 16 weeks, drinking melatonin (10 mg/kg) or not. Indirect calorimetry, glucose tolerance, steatosis, inflammation, ER stress, mitochondrial changes, autophagy and microRNA-34a-5p expression were estimated. Melatonin improved hepatic metabolism and steatosis, influenced ER stress and mitochondrial shape, and promoted autophagy in WT HFD mice. Conversely, melatonin was ineffective in HET HFD mice, maintaining NASH changes. Indeed, autophagy was inconsistent in HET HFD or starved mice, as indicated by LC3II/LC3I ratio, p62/SQSTM1 and autophagosomes estimation. The beneficial role of melatonin in dietary induced NAFLD/NASH in mice was related to reduced expression of microRNA-34a-5p and sterol regulatory element-binding protein (SREBP1) but only in the presence of full SIRT1 availability.
Project description:Hepatic steatosis is the accumulation of excess fat in the liver. Recently, hepatic steatosis has become more important because it occurs in the patients with obesity, type 2 diabetes, and hyperlipidemia and is associated with endoplasmic reticulum (ER) stress and insulin resistance. C-C chemokine receptor 2 (CCR2) inhibitor has been reported to improve inflammation and glucose intolerance in diabetes, but its mechanisms remained unknown in hepatic steatosis. We examined whether CCR2 inhibitor improves ER stress-induced hepatic steatosis in type 2 diabetic mice. In this study, db/db and db/m (n = 9) mice were fed CCR2 inhibitor (2 mg/kg/day) for 9 weeks. In diabetic mice, CCR2 inhibitor decreased plasma and hepatic triglycerides levels and improved insulin sensitivity. Moreover, CCR2 inhibitor treatment decreased ER stress markers (e.g., BiP, ATF4, CHOP, and XBP-1) and inflammatory cytokines (e.g., TNF?, IL-6, and MCP-1) while increasing markers of mitochondrial biogenesis (e.g., PGC-1?, Tfam, and COX1) in the liver. We suggest that CCR2 inhibitor may ameliorate hepatic steatosis by reducing ER stress and inflammation in type 2 diabetes mellitus.
Project description:Increasing evidence demonstrates that berberine (BBR) is beneficial for obesity-associated non-alcoholic fatty liver disease (NAFLD). However, it remains to be elucidated how BBR improves aspects of NAFLD. Here we revealed an AMP-activated protein kinase (AMPK)-independent mechanism for BBR to suppress obesity-associated inflammation and improve hepatic steatosis. In C57BL/6J mice fed a high-fat diet (HFD), treatment with BBR decreased inflammation in both the liver and adipose tissue as indicated by reduction of the phosphorylation state of JNK1 and the mRNA levels of proinflammatory cytokines. BBR treatment also decreased hepatic steatosis, as well as the expression of acetyl-CoA carboxylase and fatty acid synthase. Interestingly, treatment with BBR did not significantly alter the phosphorylation state of AMPK in both the liver and adipose tissue of HFD-fed mice. Consistently, BBR treatment significantly decreased the phosphorylation state of JNK1 in both hepatoma H4IIE cells and mouse primary hepatocytes in both dose-dependent and time-dependent manners, which was independent of AMPK phosphorylation. BBR treatment also caused a decrease in palmitate-induced fat deposition in primary mouse hepatocytes. Taken together, these results suggest that BBR actions on improving aspects of NAFLD are largely attributable to BBR suppression of inflammation, which is independent of AMPK.