Experimental evidence for therapeutic potential of taurine in the treatment of nonalcoholic fatty liver disease

The incidence of obesity is now at epidemic proportions and has resulted in the emergence of nonalcoholic fatty liver disease (NAFLD) as a common metabolic disorder that can lead to liver injury and cirrhosis. Excess sucrose and long-chain saturated fatty acids in the diet may play a role in the development and progression of NAFLD. One factor linking sucrose and saturated fatty acids to liver damage is dysfunction of the endoplasmic reticulum (ER). Although there is currently no proven, effective therapy for NAFLD, the amino sulfonic acid taurine is protective against various metabolic disturbances, including alcohol-induced liver damage. The present study was undertaken to evaluate the therapeutic potential of taurine to serve as a preventative treatment for diet-induced NAFLD. We report that taurine significantly mitigated palmitate-mediated caspase-3 activity, cell death, ER stress, and oxidative stress in H4IIE liver cells and primary hepatocytes. In rats fed a high-sucrose diet, dietary taurine supplementation significantly reduced hepatic lipid accumulation, liver injury, inflammation, plasma triglycerides, and insulin levels. The high-sucrose diet resulted in an induction of multiple components of the unfolded protein response in the liver consistent with ER stress, which was ameliorated by taurine supplementation. Treatment of mice with the ER stress-inducing agent tunicamycin resulted in liver injury, unfolded protein response induction, and hepatic lipid accumulation that was significantly ameliorated by dietary supplementation with taurine. Our results indicate that dietary supplementation with taurine offers significant potential as a preventative treatment for NAFLD.     

N-3 PUFAs inhibited hepatic ER stress induced by feeding of a high-saturated fat diet accompanied by the expression LOX-1

Excessive consumption of saturated fat leads to non-alcoholic fatty liver disease (NAFLD), which is attenuated by supplementation of n-3 polyunsaturated fatty acids (PUFAs). Endoplasmic reticulum (ER) stress is crucial in the development of NAFLD, but how high-saturated fat diet (HFD) causes ER stress and NAFLD remains unclear. Lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) is involved in hepatic ER stress. We aimed to explore the roles of LOX-1 in HFD-induced ER stress. Male Sprague–Dawley rats were fed an HFD without or with supplementation of fish oil for 16 weeks. The effects of n-3 PUFAs on hepatic ER stress degrees and the expression levels of LOX-1 were examined. Then human L02 hepatoma cells were treated with palmitate or palmitate and DHA to determine the ER stress and LOX-1 expression levels in vitro. After that the expression of LOX-1 in L02 cells was either knocked-down or overexpressed to analyze the roles of LOX-1 in palmitate-induced ER stress. The feeding of HFD induced NAFLD development and ER stress in the liver, and LOX-1 expressing level, which were all reversed by fish oil supplementation. In vitro, DHA treatment reduced the expression of LOX-1, and palmitate-induced ER stress. SiRNA-mediated knock-down of LOX-1 inhibited palmitate-induced ER stress, whereas overexpression of LOX-1 dramatically induced ER stress in L02 cells.LOX-1 is critical for HFD-induced ER stress, and inhibition of its expression under the treatment of n-3 PUFAs could ameliorate HFD-induced NAFLD.     

Endoplasmic reticulum stress and Oxidative stress in the pathogenesis of Non-alcoholic fatty liver disease

Non-alcoholic fatty liver disease (NAFLD) is the hepatic manifestation of metabolic syndrome. The underlying causes of the disease progression in NAFLD are unclear. Recent evidences suggest endoplasmic reticulum stress in the development of lipid droplets (steatosis) and subsequent generation of reactive oxygen species (ROS) in the progression to non-alcoholic steatohepatitis (NASH). The signalling pathway activated by disruption of endoplasmic reticulum (ER) homoeostasis, called as unfolded protein response, is linked with membrane biosynthesis, insulin action, inflammation and apoptosis. ROS are important mediators of inflammation. Protein folding in ER is linked to ROS. Therefore understanding the basic mechanisms that lead to ER stress and ROS in NAFLD have become the topics of immense interest. The present review focuses on the role of ER stress and ROS in the pathogenesis of NAFLD. We also highlight the cross talk between ER stress and oxidative stress which suggest and encourage the development of therapeutics for NAFLD. Further we have reviewed various strategies used for the management of NAFLD/NASH and limitations of such strategies. Our review therefore highlights the need for newer strategies with regards to ER stress and oxidative stress.     

Mesenchymal stromal cells protect hepatocytes from lipotoxicity through alleviation of endoplasmic reticulum stress by restoring SERCA activity

The aim of this study was to investigate how mesenchymal stromal cells (MSCs) modulate metabolic balance and attenuate hepatic lipotoxicity in the context of non-alcoholic fatty liver disease (NAFLD). In vivo, male SD rats were fed with high-fat diet (HFD) to develop NAFLD; then, they were treated twice by intravenous injections of rat bone marrow MSCs. In vitro, HepG2 cells were cocultured with MSCs by transwell and exposed to palmitic acid (PA) for 24 hours. The endoplasmic reticulum (ER) stressor thapsigargin and sarco/ER Ca²⁺-ATPase (SERCA2)–specific siRNA were used to explore the regulation of ER stress by MSCs. We found that MSC administration improved hepatic steatosis, restored systemic hepatic lipid and glucose homeostasis, and inhibited hepatic ER stress in HFD-fed rats. In hepatocytes, MSCs effectively alleviated the cellular lipotoxicity. Particularly, MSCs remarkably ameliorated the ER stress and intracellular calcium homeostasis induced by either PA or thapsigargin in HepG2 cells. Additionally, long-term HFD or PA stimulation would activate pyroptosis in hepatocytes, which may contribute to the cell death and liver dysfunction during the process of NAFLD, and MSC treatment effectively ameliorates these deleterious effects. SERCA2 silencing obviously abolished the ability of MSCs against the PA-induced lipotoxicity. Conclusively, our study demonstrated that MSCs were able to ameliorate liver lipotoxicity and metabolic disturbance in the context of NAFLD, in which the regulation of ER stress and the calcium homeostasis via SERCA has played a key role.     

Inhibition of MAP4K4 signaling initiates metabolic reprogramming to protect hepatocytes from lipotoxic damage

The primary hepatic consequence of obesity is non-alcoholic fatty liver disease (NAFLD), affecting about 25% of the global adult population. Non-alcoholic steatohepatitis (NASH) is a severe form of NAFLD characterized by liver lipid accumulation, inflammation, and hepatocyte ballooning, with a different degree of hepatic fibrosis. In the light of rapidly increasing prevalence of NAFLD and NASH, there is an urgent need for improved understanding of the molecular pathogenesis of these diseases. The aim of this study was to decipher the possible role of STE20-type kinase MAP4K4 in the regulation of hepatocellular lipotoxicity and susceptibility to NAFLD. We found that MAP4K4 mRNA expression in human liver biopsies was positively correlated with key hallmarks of NAFLD (i.e., liver steatosis, lobular inflammation, hepatocellular ballooning, and fibrosis). We also found that the silencing of MAP4K4 suppressed lipid deposition in human hepatocytes by stimulating β-oxidation and triacylglycerol secretion, while attenuating fatty acid influx and lipid synthesis. Furthermore, downregulation of MAP4K4 markedly reduced the glycolysis rate and lowered incidences of oxidative/endoplasmic reticulum stress. In parallel, we observed suppressed JNK and ERK and increased AKT phosphorylation in MAP4K4-deficient hepatocytes. Together, these results provide the first experimental evidence supporting the potential involvement of STE20-type kinase MAP4K4 as a component of the hepatocellular lipotoxic milieu promoting NAFLD susceptibility.     

Phosphorylation of eIF2α signaling pathway attenuates obesity-induced non-alcoholic fatty liver disease in an ER stress and autophagy-dependent manner

Non-alcoholic fatty liver disease (NAFLD) is the most common liver disorder and frequently exacerbates in postmenopausal women. In NAFLD, the endoplasmic reticulum (ER) plays an important role in lipid metabolism, in which salubrinal is a selective inhibitor of eIF2α de-phosphorylation in response to ER stress. To determine the potential mechanism of obesity-induced NAFLD, we employed salubrinal and evaluated the effect of ER stress and autophagy on lipid metabolism. Ninety-five female C57BL/6 mice were randomly divided into five groups: standard chow diet, high-fat (HF) diet, HF with salubrinal, HF with ovariectomy, and HF with ovariectomy and salubrinal. All mice except for SC were given HF diet. After the 8-week obesity induction, salubrinal was subcutaneously injected for the next 8 weeks. The expression of ER stress and autophagy markers was evaluated in vivo and in vitro. Compared to the normal mice, the serum lipid level and adipose tissue were increased in obese mice, while salubrinal attenuated obesity by blocking lipid disorder. Also, the histological severity of hepatic steatosis and fibrosis in the liver and lipidosis was suppressed in response to salubrinal. Furthermore, salubrinal inhibited ER stress by increasing the expression of p-eIF2α and ATF4 with a decrease in the level of CHOP. It promoted autophagy by increasing LC3II/I and inhibiting p62. Correlation analysis indicated that lipogenesis in the development of NAFLD was associated with ER stress. Collectively, we demonstrated that eIF2α played a key role in obesity-induced NAFLD, and salubrinal alleviated hepatic steatosis and lipid metabolism by altering ER stress and autophagy through eIF2α signaling.Subject terms: Obesity, Metabolic syndrome, Outcomes research     

ER Stress and Autophagy Dysfunction Contribute to Fatty Liver in Diabetic Mice

Diabetes mellitus and nonalcoholic fatty liver disease (NAFLD) are often identified in patients simultaneously. Recent evidence suggests that endoplasmic reticulum (ER) stress and autophagy dysfunction play an important role in hepatocytes injury and hepatic lipid metabolism, however the mechanistic interaction between diabetes and NAFLD is largely unknown. In this study, we used a diabetic mouse model to study the interplay between ER stress and autophagy during the pathogenic transformation of NAFLD. The coexist of inflammatory hepatic injury and hepatic accumulation of triglycerides (TGs) stored in lipid droplets indicated development of steatohepatitis in the diabetic mice. The alterations of components for ER stress signaling including ATF6, GRP78, CHOP and caspase12 indicated increased ER stress in liver tissues in early stage but blunted in the later stage during the development of diabetes. Likewise, autophagy functioned well in the early stage but suppressed in the later stage. The inactivation of unfolded protein response and suppression of autophagy were positively related to the development of steatohepatitis, which linked to metabolic abnormalities in the compromised hepatic tissues in diabetic condition. We conclude that the adaption of ER stress and impairment of autophagy play an important role to exacerbate lipid metabolic disorder contributing to steatohepatitis in diabetes.     

Chromium attenuates high-fat diet-induced nonalcoholic fatty liver disease in KK/HlJ mice

Nonalcoholic fatty liver disease (NAFLD) is a chronic liver disease associated with insulin resistance, oxidative stress, and inflammation. Evidence indicates that chromium has a role in the regulation of glucose and lipid metabolism and may improve insulin sensitivity. In this study, we report that chromium supplementation has a beneficial effect against NAFLD. We found that KK/HlJ mice developed obesity and progressed to NAFLD after feeding with high-fat diet for 8 weeks. High-fat-fed KK/HlJ mice showed hepatocyte injury and hepatic triglyceride accumulation, which was accompanied by insulin resistance, oxidative stress, and inflammation. Chromium supplementation prevented progression of NAFLD and the beneficial effects were accompanied by reduction of hepatic triglyceride accumulation, elevation of hepatic lipid catabolic enzyme, improvement of glucose and lipid metabolism, suppression of inflammation as well as resolution of oxidative stress, probably through enhancement of insulin signaling. Our findings suggest that chromium could serve as a hepatoprotective agent against NAFLD.     

Effects of a plant product consisting of green tea and curcuma extract on milk production and the expression of hepatic genes involved in endoplasmic stress response and inflammation in dairy cows

During the periparturient phase, cows are typically in an inflammation-like condition, and it has been proposed that inflammation associated with the induction of stress of the endoplasmic reticulum (ER) in the liver contributes to the development of fatty liver syndrome and ketosis. In the present study, the hypothesis that supplementation of dairy cows with a plant product consisting of green tea (95%) and curcuma extract (5%) rich in polyphenols attenuates inflammation and ER stress in the liver during early lactation was investigated. Twenty-seven cows were assigned to two groups, either a control group (n = 14) or a treatment group (n = 13). Both groups of cows received a total mixed ration, and the ration of the treatment group was supplemented with 0.175 g of the plant product per kg dry matter from week 3 prepartum to week 9 postpartum. Dry matter intake and energy balance during week 2 to week 9 postpartum were not different between the two groups. However, cows supplemented with the plant product had a greater amount of energy-corrected milk during week 2 to week 9 postpartum and lower concentrations of triacylglycerols and cholesterol in the liver in week 1 and week 3 postpartum than cows of the control group (p < 0.05). Cows supplemented with the plant product showed a trend towards a reduced mRNA concentration of haptoglobin (p < 0.10), while relative mRNA concentrations of eight genes of the unfolded protein response considered in the liver were not different between the two groups of cows. Relative hepatic mRNA concentration of fibroblast growth factor, a stress hormone induced by various stress conditions, was reduced at week 1 and week 3 postpartum in cows supplemented with the plant product (p < 0.05). Overall, the data of this study suggest that – although there were only minor effects on the occurrence of ER stress and inflammation – a supplementation of polyphenols might be useful to improve milk yield and prevent fatty liver syndrome in dairy cows.     

Cranberry extract attenuates hepatic inflammation in high-fat-fed obese mice

Cranberry (Vaccinium macrocarpon) consumption has been associated with health beneficial effects. Nonalcoholic fatty liver disease (NAFLD) is a comorbidity of obesity. In the present study, we investigated the effect of a polyphenol-rich cranberry extract (CBE) on hepatic inflammation in high fat (HF)-fed obese C57BL/6J mice. Following dietary treatment with 0.8% CBE for 10 weeks, we observed no change in body weight or visceral fat mass in CBE-supplemented mice compared to HF-fed control mice. We did observe a significant decrease in plasma alanine aminotransferase (31%) and histological severity of NAFLD (33% decrease in area of involvement, 29% decrease in lipid droplet size) compared to HF-fed controls. Hepatic protein levels of tumor necrosis factor α and C-C chemokine ligand 2 were reduced by 28% and 19%, respectively, following CBE supplementation. CBE significantly decreased hepatic mRNA levels of toll-like receptor 4 (TLR4, 63%) and nuclear factor κB (NFκB, 24%), as well as a number of genes related to the nucleotide-binding domain, leucine-rich-containing family, pyrin domain-containing 3 inflammasome. In conclusion, CBE reduced NAFLD and hepatic inflammation in HF-fed obese C57BL/6J mice. These effects appear to be related to mitigation of TLR4-NFκB related signaling; however, further studies into the underlying mechanisms of these hepatoprotective effects are needed.     

The role of AMPKα2 in the HFD-induced nonalcoholic steatohepatitis

Nonalcoholic fatty liver disease (NAFLD) is associated with hepatic steatosis, inflammation and liver fibrosis and has become one of the leading causes of hepatocellular carcinoma and liver failure. However, the underlying molecular mechanism of hepatic steatosis and the progression to nonalcoholic steatohepatitis (NASH) are not fully understood. Herein, we discovered that AMPKα2 catalytic subunit showed reduced expression in the liver following high fat diet (HFD) feeding to mice. Importantly, knockout of AMPKα2 in mice aggravated NAFLD, hepatic steatosis, inflammation and fibrosis. On the other hand, hepatocyte-targeted overexpression of AMPKα2 prevented or reversed NAFLD indications. In vivo mechanistic studies revealed that increased phosphorylation of IKKα/β and NF-κB in HFD-fed AMPKα2^−/− mice compared to WT mice, and treatment of these mouse cohorts with an inhibitor of NF-κB signaling for 4 weeks, effectively attenuated the progression of steatohepatitis and metabolic disorder features. In summary, AMPKα2 provides a protective role in the process of hepatic steatosis to NASH progression through suppression of liver NF-κB signaling.     

Alpha-lipoic acid attenuates methionine choline deficient diet-induced steatohepatitis in C57BL/6 mice

AimsNon-alcoholic steatohepatitis (NASH) is a liver disease that causes fat accumulation, inflammation and fibrosis. Increased oxidative stress contributes to hepatic inflammation and fibrosis by upregulation of Cytochrome P450 2E1 (CYP2E1), endoplasmic reticulum (ER) stress and mitogen-activated protein kinase (MAPK) activity. This study examined whether alpha-lipoic acid (ALA), a naturally occurring thiol antioxidant, prevents steatohepatitis through the inhibition of several pathways involved in hepatic inflammation and fibrosis.Main MethodsC57BL/6 mice were fed an MCD diet with or without ALA for 4 weeks. Liver sections from mice on control or MCD diets with or without ALA were stained with hematoxylin-eosin, oil red O, and anti-4-HNE antibody. The effects of ALA on methionine-choline deficient MCD-diet induced plasma AST and ALT as well as tissue TBARS were measured. The effects of ALA on CYP2E1 expression, ER stress, MAPK levels, and NF-κB activity in MCD diet-fed mice liver were measured by northern and western blot analysis.Key findingsDietary supplementation with ALA reduced MCD diet-induced hepatic lipid accumulation, hepatic inflammation, TBARS, 4-HNE, and plasma ALT and AST levels. These effects were associated with a reduced expression of CYP2E1 and reduced ER stress and MAPK and NF-κB activity.SignificanceTaken together, the results of the present study indicate that ALA attenuates steatohepatitis through inhibition of several pathways, and provide the possibility that ALA can be used to prevent the development and progression of non-alcoholic fatty liver disease in patients who have strong risk factors for NASH.     

The water extract of Sophorae tonkinensis Radix et Rhizoma alleviates non-alcoholic fatty liver disease and its mechanism

BackgroundSophorae tonkinensis Radix et Rhizoma is traditionally used for clearing away heat and toxic materials in China.PurposeThis study aims to observe the amelioration of Sophorae tonkinensis water extract (STR) against non-alcoholic fatty liver disease (NAFLD) and the engaged mechanism.MethodsNAFLD was induced in mice fed by methionine and choline deficient (MCD) diet. Liver histological observation, Oil Red O, Masson's trichrome and F4/80 immunohistochemical staining were performed. Serum and liver biochemical parameters, hepatic gene and protein expression were detected. Cellular lipids accumulation in human normal liver l-02 and hepatoma HepRG cells were induced by 0.5 mM nonestesterified fatty acid (NEFA). The contents of matrine (MT) and oxymatrine (OMT) in STR were detected by using high-performance liquid chromatography (HPLC). Carnitine palmitoyltransferase 1A (CPT1A) expression and enzymatic activity were detected both in vivo and in vitro.ResultsSerum alanine/aspartate aminotransferase (ALT/AST) activity, hepatic malondialdehyde (MDA) content and liver histological observation showed that STR alleviated hepatocellular damage in mice fed with MCD diet. Hepatic triglyceride (TG), total cholesterol (TC) and NEFA amounts, and Oil Red O staining showed that STR reduced hepatic lipids accumulation in mice fed with MCD diet. STR and its main compounds including MT and OMT decreased NEFA-induced cellular lipids accumulation in hepatocytes. STR enhanced CPT1A activity both in vivo and in vitro. MT and OMT also enhanced cellular CPT1A activity in l-02 hepatocytes treated with NEFA. Moreover, the CTP1A inhibitor etomoxir (ETO) reduced the lipid-lowering activity provided by STR, MT or OMT in vitro. Liver myeloperoxidase (MPO) activity and hydroxyproline content, Masson's trichrome and F4/80 immunohistochemical staining, and hepatic mRNA expression of some molecules involved in regulating inflammation or fibrosis demonstrated that STR alleviated hepatic inflammation and liver fibrosis in mice fed with MCD diet.ConclusionSTR alleviated NAFLD by inhibiting hepatic inflammation and liver fibrosis, and reducing hepatic lipids accumulation through promoting fatty acids β-oxidation by enhancing liver CPT1A activity. MT and OMT may be the main active compounds contributing to the lipid-lowering activity provided by STR.     

Combined metabolic activators therapy ameliorates liver fat in nonalcoholic fatty liver disease patients

Nonalcoholic fatty liver disease (NAFLD) refers to excess fat accumulation in the liver. In animal experiments and human kinetic study, we found that administration of combined metabolic activators (CMAs) promotes the oxidation of fat, attenuates the resulting oxidative stress, activates mitochondria, and eventually removes excess fat from the liver. Here, we tested the safety and efficacy of CMA in NAFLD patients in a placebo‐controlled 10‐week study. We found that CMA significantly decreased hepatic steatosis and levels of aspartate aminotransferase, alanine aminotransferase, uric acid, and creatinine, whereas found no differences on these variables in the placebo group after adjustment for weight loss. By integrating clinical data with plasma metabolomics and inflammatory proteomics as well as oral and gut metagenomic data, we revealed the underlying molecular mechanisms associated with the reduced hepatic fat and inflammation in NAFLD patients and identified the key players involved in the host–microbiome interactions. In conclusion, we showed that CMA can be used to develop a pharmacological treatment strategy in NAFLD patients.     

Interleukin-38 alleviates hepatic steatosis through AMPK/autophagy-mediated suppression of endoplasmic reticulum stress in obesity models

Interleukin-38 (IL-38), recently recognized as a cytokine with anti-inflammatory properties that mitigate type 2 diabetes, has been associated with indicators of insulin resistance and nonalcoholic fatty liver disease (NAFLD). This study investigated the impact of IL-38 on hepatic lipid metabolism and endoplasmic reticulum (ER) stress. We assessed protein expression levels using Western blot analysis, while monodansylcadaverine staining was employed to detect autophagosomes in hepatocytes. Oil red O staining was utilized to examine lipid deposition. The study revealed elevated serum IL-38 levels in high-fat diet (HFD)-fed mice and IL-38 secretion from mouse keratinocytes. IL-38 treatment attenuated lipogenic lipid accumulation and ER stress markers in hepatocytes exposed to palmitate. Furthermore, IL-38 treatment increased AMP-activated protein kinase (AMPK) phosphorylation and autophagy. The effects of IL-38 on lipogenic lipid deposition and ER stress were nullified in cultured hepatocytes by suppressing AMPK through small interfering (si) RNA or 3-methyladenine (3MA). In animal studies, IL-38 administration mitigated hepatic steatosis by suppressing the expression of lipogenic proteins and ER stress markers while reversing AMPK phosphorylation and autophagy markers in the livers of HFD-fed mice. Additionally, AMPK siRNA, but not 3MA, mitigated IL-38-enhanced fatty acid oxidation in hepatocytes. In summary, IL-38 alleviates hepatic steatosis through AMPK/autophagy signaling-dependent attenuation of ER stress and enhancement of fatty acid oxidation via the AMPK pathway, suggesting a therapeutic strategy for treating NAFLD.