Therapeutic Target For Non-alcoholic Fatty Liver Disease Research Articles

Protective effect of metformin against palmitate-induced hepatic cell death

Lipotoxicity causes hepatic cell death and therefore plays an important role in the pathogenesis of non-alcoholic fatty liver disease (NAFLD). Metformin, a first-line anti-diabetic drug, has shown a potential protective effect against NAFLD. However, the underlying mechanism is still not clear. In this study, we aim to understand the molecular mechanism of the protective effect of metformin in NAFLD, focusing on lipotoxicity. Cell death was studied in HepG2 cells and primary rat hepatocytes exposed to palmitate and metformin. Metformin ameliorated palmitate-induced necrosis and apoptosis (decreased caspase-3/7 activity by 52% and 57% respectively) in HepG2 cells. Metformin also reduced palmitate-induced necrosis in primary rat hepatocytes (P < 0.05). The protective effect of metformin is not due to reducing intracellular lipid content or activation of AMPK signaling pathways. Metformin and a low concentration (0.1 μmol/L) of rotenone showed moderate inhibition on mitochondrial respiration indicated by reduced basal and maximal mitochondrial respiration and proton leak in HepG2 cells. Moreover, metformin and rotenone (0.1 μmol/L) preserved mitochondrial membrane potential in both HepG2 cells and primary rat hepatocytes. In addition, metformin and rotenone (0.1 μmol/L) also reduces reactive oxygen species (ROS) production and increase superoxide dismutase 2 (SOD2) expression. Our results establish that metformin AMPK-independently protects against palmitate-induced hepatic cell death by moderate inhibition of the mitochondrial respiratory chain, recovering mitochondrial function, decreasing cellular ROS production, and inducing SOD2 expression, indicating that metformin may have beneficial actions beyond its glucose-lowering effect and also suggests that mitochondrial complex І may be a therapeutic target in NAFLD.

Inhibition of receptor-interacting protein kinase 1 improves experimental non-alcoholic fatty liver disease

In non-alcoholic fatty liver disease (NAFLD), hepatocytes can undergo necroptosis: a regulated form of necrotic cell death mediated by the receptor-interacting protein kinase (RIPK) 1. Herein, we assessed the potential for RIPK1 and its downstream effector mixed lineage kinase domain-like protein (MLKL) to act as therapeutic targets and markers of activity in NAFLD. C57/BL6J-mice were fed a normal chow diet or a high-fat diet (HFD). The effect of RIPA-56, a highly specific inhibitor of RIPK1, was evaluated in HFD-fed mice and in primary human steatotic hepatocytes. RIPK1 and MLKL concentrations were measured in the serum of patients with NAFLD. When used as either a prophylactic or curative treatment for HFD-fed mice, RIPA-56 caused a downregulation of MLKL and a reduction of liver injury, inflammation and fibrosis, characteristic of non-alcoholic steatohepatitis (NASH), as well as of steatosis. This latter effect was reproduced by treating primary human steatotic hepatocytes with RIPA-56 or necrosulfonamide, a specific inhibitor of human MLKL, and by knockout (KO) of Mlklin fat-loaded AML-12 mouse hepatocytes. Mlkl-KO led to activation of mitochondrial respiration and an increase in β-oxidation in steatotic hepatocytes. Along with decreased MLKL activation, Ripk3-KO mice exhibited increased activities of the liver mitochondrial respiratory chain complexes in experimental NASH. In patients with NAFLD, serum concentrations of RIPK1 and MLKL increased in correlation with activity. The inhibition of RIPK1 improves NASH features in HFD-fed mice and reverses steatosis via an MLKL-dependent mechanism that, at least partly, involves an increase in mitochondrial respiration. RIPK1 and MLKL are potential serum markers of activity and promising therapeutic targets in NAFLD. There are currently no pharmacological treatment options for non-alcoholic fatty liver disease (NAFLD), which is now the most frequent liver disease. Necroptosis is a regulated process of cell death that can occur in hepatocytes during NAFLD. Herein, we show that RIPK1, a gatekeeper of the necroptosis pathway that is activated in NAFLD, can be inhibited by RIPA-56 to reduce not only liver injury, inflammation and fibrosis, but also steatosis in experimental models. These results highlight the potential of RIPK1 as a therapeutic target in NAFLD.

Perilipin 5 Protects against Cellular Oxidative Stress by Enhancing Mitochondrial Function in HepG2 Cells.

Non-alcoholic fatty liver disease (NAFLD) is one of the most common liver diseases worldwide. Reactive oxygen species (ROS), as potent oxidants in cells, have been shown to promote the development of NAFLD. Previous studies reported that for ROS-induced cellular oxidative stress, promoting lipid droplet (LD) accumulation is associated with the cellular antioxidation process. However, the regulatory role of LDs in relieving cellular oxidative stress is poorly understood. Here, we showed that Perilipin 5 (PLIN5), a key LD protein related to mitochondria–LD contact, reduced ROS levels and improved mitochondrial function in HepG2 cells. Both mRNA and protein levels of PLIN5 were significantly increased in cells with hydrogen peroxide or lipopolysaccharide (LPS) treatment (p < 0.05). Additionally, the overexpression of PLIN5 promoted LD formation and mitochondria–LD contact, reduced cellular ROS levels and up-regulated mitochondrial function-related genes such as COX and CS. Knockdown PLIN5, meanwhile, showed opposite effects. Furthermore, we identified that cellular oxidative stress up-regulated PLIN5 expression via the JNK-p38-ATF pathway. This study shows that the up-regulation of PLIN5 is a kind of survival strategy for cells in response to stress. PLIN5 can be a potential therapeutic target in NAFLD.

Insulin-like growth factor-1 and non-alcoholic fatty liver disease: a systemic review and meta-analysis.

The prevalence of non-alcoholic fatty liver disease (NAFLD) is rapidly increasing worldwide. A number of researchers have studied the relationship between Insulin-like growth factor-1(IGF-1) and NAFLD. However, the results are controversial. This meta-analysis, aimed to systemically evaluate the correlation between IGF-1 and NAFLD. We searched for four online databases: PubMed, Web of Science, Embase and CNKI up to Feb 2018. We then applied a random-effects model to evaluate the overall effect sizes by calculating Standard mean difference (SMD) and its 95% confidence intervals (CIs). Twelve articles were included in this meta-analysis. The pooled analysis showed that the level of IGF-1 in the control group was significantly higher than that in the NAFLD group. (SMD: 1.00, 95% CI: 0.54-1.46, P < 0.00001). However, significant heterogeneity was discovered among the included studies (P < 0.00001, I2 = 96%). Then a series of subgroup analyses were performed. Compared to the nonalcoholic steatohepatitis (NASH) group, the level of IGF-1 was significantly higher in the Non- or probable-NASH group (SMD: 1.42, 95% CI: 0.25-2.58, P = 0.02). The level of IGF-1 in patients with increased insulin resistance (SMD: 0.49; 95% CI: 0.36-0.63; P < 0.00001) and high Body Mass Index (SMD: 0.50; 95% CI: 0.22-0.79; P < 0.05) were significantly lower than healthy control. In addition, the same conclusion were found in studies carried out in Asia and Europe (Asia: SMD: 0.69, 95% CI: -0.29-1.66, P = 0.17; Europe: SMD: 0.89, 95% CI: 0.41-1.38, P < 0.05). The level of IGF-1 is down-regulated in NAFLD patients compared to healthy controls, suggesting that IGF-1 might be used as a potential biomarker and therapeutic target for NAFLD.

FBXW7 suppresses HMGB1-mediated innate immune signaling to attenuate hepatic inflammation and insulin resistance in a mouse model of nonalcoholic fatty liver disease

BackgroundInnate immune dysfunction contributes to the development and progression of nonalcoholic fatty liver disease (NAFLD), however, its pathogenesis is still incompletely understood. Identifying the key innate immune component responsible for the pathogenesis of NAFLD and clarifying the underlying mechanisms may provide therapeutic targets for NAFLD. Recently, F-box- and WD repeat domain-containing 7 (FBXW7) exhibits a regulatory role in hepatic glucose and lipid metabolism. This study aims to investigate whether FBXW7 controls high-mobility group box 1 protein (HMGB1)-mediated innate immune signaling to improve NAFLD and the mechanism underlying this action.MethodsMice were fed a high-fat diet (HFD) for 12 or 20 weeks to establish NAFLD model. Hepatic overexpression or knockdown of FBXW7 was induced by tail-vein injection of recombinant adenovirus. Some Ad-FBXW7-injected mice fed a HFD were injected intraperitoneally with recombinant mouse HMGB1 to confirm the protective role of FBXW7 in NAFLD via inhibition of HMGB1.ResultsFBXW7 improves NAFLD and related metabolic parameters without remarkable influence of body weight and food intake. Moreover, FBXW7 markedly ameliorated hepatic inflammation and insulin resistance in the HFD-fed mice. Furthermore, FBXW7 dramatically attenuated the expression and release of HMGB1 in the livers of HFD-fed mice, which is associated with inhibition of protein kinase R (PKR) signaling. Thereby, FBXW7 restrains Toll-like receptor 4 (TLR4) and receptor for advanced glycation end products (RAGE) signaling in HFD-fed mouse livers. In addition, exogenous HMGB1 treatment abolished FBXW7-mediated inhibition of hepatic inflammation and insulin resistance in HFD-fed mouse livers.ConclusionsOur results demonstrate a protective role of FBXW7 in NAFLD by abating HMGB1-mediated innate immune signaling to suppress inflammation and consequent insulin resistance, suggesting that FBXW7 is a potential target for therapeutic intervention in NAFLD development.

Bone Morphogenetic Protein-8B Expression is Induced in Steatotic Hepatocytes and Promotes Hepatic Steatosis and Inflammation In Vitro

Non-alcoholic fatty liver disease (NAFLD) is considered to be the hepatic manifestation of the metabolic syndrome. The bone morphogenetic protein-8B (BMP8B) has been shown to be expressed in brown adipose tissues and the hypothalamus and to affect thermogenesis and susceptibility to diet-induced obesity. Here, we aimed to analyze BMP8B expression in NAFLD and to gain insight into BMP8B effects on pathophysiological steps of NAFLD progression. BMP8B mRNA and protein expression were dose-dependently induced in primary human hepatocytes in vitro upon incubation with fatty acids. Furthermore, hepatic BMP8B expression was significantly increased in a murine NAFLD model and in NAFLD patients compared with controls. Incubation with recombinant BMP8B further enhanced the fatty acid-induced cellular lipid accumulation as well as NFκB activation and pro-inflammatory gene expression in hepatocytes, while siRNA-mediated BMP8B depletion ameliorated these fatty acid-induced effects. Analysis of the expression of key factors of hepatocellular lipid transport and metabolisms indicated that BMP8B effects on fatty acid uptake as well as de novo lipogenesis contributed to hepatocellular accumulation of fatty acids leading to increased storage in the form of triglycerides and enhanced combustion by beta oxidation. In conclusion, our data indicate that BMP8B enhances different pathophysiological steps of NAFLD progression and suggest BMP8B as a promising prognostic marker and therapeutic target for NAFLD and, potentially, also for other chronic liver diseases.

Dual-Specificity Phosphatase 26 Protects Against Nonalcoholic Fatty Liver Disease in Mice Through Transforming Growth Factor Beta-Activated Kinase 1 Suppression.

Nonalcoholic fatty liver disease (NAFLD), which has a wide global distribution, includes different stages ranging from simple steatosis to nonalcoholic steatohepatitis, advanced fibrosis, and liver cirrhosis according to the degree of severity. Chronic low‐grade inflammation, insulin resistance, and lipid accumulation are the leading causes of NAFLD. To date, no effective medicine for NAFLD has been approved by governmental agencies. Our study demonstrated that the expression of dual‐specificity phosphatase 26 (Dusp26), a member of the Dusp protein family, was decreased in the liver tissue of mice with hepatic steatosis and genetically obese (ob/ob) mice. In our study, hepatic steatosis, inflammatory responses, and insulin resistance were exacerbated in liver‐specific Dusp26‐knockout (KO) mice but ameliorated in liver‐specific Dusp26‐transgenic mice induced by a high‐fat diet. In addition, the degree of liver fibrosis was aggravated in high‐fat high‐cholesterol diet–induced Dusp26‐KO mice. We further found that the binding of Dusp26 to transforming growth factor beta–activated kinase 1 (TAK1) to block the phosphorylation of TAK1 regulated the TAK1–p38/c‐Jun NH2‐terminal kinase signaling axis to alleviate hepatic steatosis and metabolic disturbance. Conclusion: These findings suggest that Dusp26 is a good TAK1‐dependent therapeutic target for NAFLD.

Gut bacteria contributes to NAFLD pathogenesis by promoting secondary bile acids biosynthesis

ObjectiveThe gut microbiota affect liver metabolism and health through its metabolites. We previously observed that the microbiome may have a negative effect on non‐alcoholic fatty liver disease (NAFLD) through elevated secondary bile acids (BAs) production, and consequently suppressed BA‐mediated signaling. In this study, we examined the whole genome sequencing (WGS) data from NAFLD microbiome aiming to identify the species that contribute to the altered BA metabolism in NAFLD.MethodsThe metagenome data of the gut microbiota from 79 NAFLD patients were compared to those from 18 healthy controls. Metaphlan2 and HUMAnN were used to examine the gene‐based taxonomic and functional profiles of gut microbiota. Genome‐based analysis was further performed to obtain insights on the microbial genomes and to identify BA metabolizing microbes. Statistical tests, feature selection and microbial interaction analysis were integrated to identify disease markers and structural alterations in NAFLD gut microbiome.ResultsThe gut microbiota of NAFLD were less diverse than those of healthy subjects. In addition, interactions among the members of the gut community in NAFLD were less than those in healthy subjects. We identified 37 differential species markers, including Ruminococcus torques, Clostridium leptum, and Escherichia coli; and 32 differential pathway markers including secondary BA biosynthesis and arachidonic acid metabolism, that distinguish NAFLD from healthy controls. Subsequently, 115 high‐quality microbial genomes (HQMGs) were re‐assembled, and taxonomy determined at strain level. Notably, metabolic potentials analysis identified 15 HQMGs, from Clostridium saccharolyticum and Ruminococcus bromii, that are involved in secondary BA biosynthesis, and were dominant in NAFLD and interplayed as a synergetic ecological guild.ConclusionsIn‐depth analysis of the WGS data from NAFLD patients and healthy controls revealed the structural and functional alterations of the gut microbiota. We identified the bacterial strains responsible for elevated secondary BA production in NAFLD, supporting our hypothesis that elevated secondary BA biosynthesis contributes to the development of NAFLD. The 15 BA‐metabolizing strains dominant in NAFLD could be novel therapeutic targets for NAFLD. In addition, our WGS study revealed strain‐specific functional differences within certain gut microbial species.Support or Funding InformationThis work was supported by National Natural Science Foundation of China 81770571 (to LZ), 81774152(to RZ), 41530105 (to RZ).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Covering the Cover

Covering the Cover

Hepatic fat as clinical outcome and therapeutic target for nonalcoholic fatty liver disease

Nonalcoholic fatty liver disease (NAFLD) is rapidly becoming the leading cause of advanced liver disease in Western countries. NAFLD is defined in the presence of increased hepatic fat content, which is mainly stored under the form of neutral lipids within intracellular droplets and is not explained by at risk alcohol intake. In order to understand the pathogenesis, monitor the progression and find novel treatments for this condition, previous research efforts mainly addressed the role of inflammation. However, very recent data seem to suggest that hepatic lipid accumulation may be involved in NAFLD pathogenesis by driving secondary inflammation and fibrosis progression. Here, we will briefly review the novel results derived from natural history, genetics, imaging studies and therapeutic trials that support the notion that hepatic fat accumulation may represent a major clinical outcome and therapeutic target for NAFLD. Indeed, prospective and genetic data are consistent with hepatic fat being a driver of NAFLD progression. Furthermore, new technologies will render possible to monitor hepatic fat content without the need of invasive assessment, thereby allowing to identify patients at higher risk, and to monitor the response to drugs that act by decreasing hepatic lipid accumulation.

Expression of STING Is Increased in Liver Tissues From Patients With NAFLD and Promotes Macrophage-Mediated Hepatic Inflammation and Fibrosis in Mice

Transmembrane protein 173 (TMEM173 or STING) signaling by macrophage activates the type I interferon-mediated innate immune response. The innate immune response contributes to hepatic steatosis and non-alcoholic fatty liver disease (NAFLD). We investigated whether STING regulates diet-induced in hepatic steatosis, inflammation, and liver fibrosis in mice. Mice with disruption of Tmem173 (STINGgt) on a C57BL/6J background, mice without disruption of this gene (controls), and mice with disruption of Tmem173 only in myeloid cells were fed a standard chow diet, a high-fat diet (HFD; 60% fat calories), or a methionine- and choline-deficient diet (MCD). Liver tissues were collected and analyzed by histology and immunohistochemistry. Bone marrow cells were isolated from mice, differentiated into macrophages, and incubated with 5,6-dimethylxanthenone-4-acetic acid (DMXAA; an activator of STING) or cyclic guanosine monophosphate-adenosine monophosphate (cGAMP). Macrophages or their media were applied to mouse hepatocytes or human hepatic stellate cells (LX2) cells, which were analyzed for cytokine expression, protein phosphorylation, and fat deposition (by oil red O staining after incubation with palmitate). We obtained liver tissues from patients with and without NAFLD and analyzed these by immunohistochemistry. Non-parenchymal cells of liver tissues from patients with NAFLD had higher levels of STING than cells of liver tissues from patients without NAFLD. STINGgt mice and mice with disruption only in myeloid cells developed less severe hepatic steatosis, inflammation, and/or fibrosis after the HFD or MCD than control mice. Levels of phosphorylated c-Jun N-terminal kinase and p65 and mRNAs encoding tumor necrosis factor and interleukins 1B and 6 (markers of inflammation) were significantly lower in liver tissues from STINGgt mice vs control mice after the HFD or MCD. Transplantation of bone marrow cells from control mice to STINGgt mice restored the severity of steatosis and inflammation after the HFD. Macrophages from control, but not STINGgt, mice increased markers of inflammation in response to lipopolysaccharide and cGAMP. Hepatocytes and stellate cells cocultured with STINGgt macrophages in the presence of DMXAA or incubated with the medium collected from these macrophages had decreased fat deposition and markers of inflammation compared with hepatocytes or stellate cells incubated with control macrophages. Levels of STING were increased in liver tissues from patients with NAFLD and mice with HFD-induced steatosis. In mice, loss of STING from macrophages decreased the severity of liver fibrosis and the inflammatory response. STING might be a therapeutic target for NAFLD.

Mechanistic and therapeutic advances in non-alcoholic fatty liver disease by targeting the gut microbiota.

Non-alcoholic fatty liver disease (NAFLD) is one of the most common metabolic diseases currently in the context of obesity worldwide, which contains a spectrum of chronic liver diseases, including hepatic steatosis, non-alcoholic steatohepatitis and hepatic carcinoma. In addition to the classical "Two-hit" theory, NAFLD has been recognized as a typical gut microbiota-related disease because of the intricate role of gut microbiota in maintaining human health and disease formation. Moreover, gut microbiota is even regarded as a "metabolic organ" that play complementary roles to that of liver in many aspects. The mechanisms underlying gut microbiota-mediated development of NAFLD include modulation of host energy metabolism, insulin sensitivity, and bile acid and choline metabolism. As a result, gut microbiota have been emerging as a novel therapeutic target for NAFLD by manipulating it in various ways, including probiotics, prebiotics, synbiotics, antibiotics, fecal microbiota transplantation, and herbal components. In this review, we summarized the most recent advances in gut microbiota-mediated mechanisms, as well as gut microbiota-targeted therapies on NAFLD.

Down-regulation of microRNA-375 regulates adipokines and inhibits inflammatory cytokines by targeting AdipoR2 in non-alcoholic fatty liver disease.

Non-alcoholic fatty liver disease (NAFLD) has been considered as a multi-factorial metabolic syndrome. MicroRNA-375 (MiR-375) was significantly up-regulated in serum of NAFLD patients and the role of miR-375 was addressed as a putative biomarker of NAFLD progression. However, the specific function of miR-375 in the progression of NAFLD is still unclear and the molecular mechanisms underlying NAFLD have yet to be elucidated. Our study aimed at investigating the regulatory role of miR-375 in the molecular mechanisms of the pathogenic progression of NAFLD and to find out whether miR-375 regulates the expression level of adipokines and inflammatory cytokines in NAFLD. We found that miR-375 expression was increased in the serum of high fat diet (HFD)-feeding mice comparing to that in healthy controls, whereas the expression of Adiponectin receptor 2 (AdipoR2) was decreased in mice fed with HFD. Moreover, inhibiton of miR-375 up-regulated the expression of Adiponectin, inhibited the lipid accumulation and down-regulated both the level of Leptin and inflammatory cytokines including tumour necrosis factor (TNF)-α and interleukin (IL)-6 in palmiticacid (PA)-induced human hepatocellular carcinoma HepG2 cells. In addition, we also found that AdipoR2 was a target of miR-375 by binding directly to the 3'UTR of it. Of note, the reduced level of TNF-α, IL-6 as well as Leptin and the production of Adiponectin by miR-375 inhibitors was significantly reversed by silencing of AdipoR2 in PA-induced HepG2 cells. Our findings bring new insight into understanding the complex mechanisms underlying the pathogenesis of NAFLD and provide evidence that miR-375 might represent a novel therapeutic target for NAFLD.

Deletion of NLRX1 increases fatty acid metabolism and prevents diet-induced hepatic steatosis and metabolic syndrome

NOD-like receptor (NLR)X1 (NLRX1) is an ubiquitously expressed inflammasome-independent NLR that is uniquely localized in mitochondria with as yet unknown effects on metabolic diseases. Here, we report that NLRX1 is essential in regulating cellular metabolism in non-immune parenchymal hepatocytes by decreasing mitochondrial fatty acid-dependent oxidative phosphorylation (OXPHOS) and promoting glycolysis. NLRX1 loss in mice has a profound impact on the prevention of diet-induced metabolic syndrome parameters, non-alcoholic fatty liver disease (NAFLD) progression, and renal dysfunction. Despite enhanced caloric intake, NLRX1 deletion in mice fed a western diet (WD) results in protection from liver steatosis, hepatic fibrosis, obesity, insulin resistance, glycosuria and kidney dysfunction parameters independent from inflammation. While mitochondrial content was equal, NLRX1 loss in hepatocytes leads to increased fatty acid oxidation and decreased steatosis. In contrast, glycolysis was decreased in NLRX1-deficient cells versus controls. Thus, although first implicated in immune regulation, we show that NLRX1 function extends to the control of hepatocyte energy metabolism via the restriction of mitochondrial fatty acid-dependent OXPHOS and enhancement of glycolysis. As such NLRX1 may be an attractive novel therapeutic target for NAFLD and metabolic syndrome.

MicroRNA-194 inhibition improves dietary-induced non-alcoholic fatty liver disease in mice through targeting on FXR

Non-alcoholic fatty liver disease (NAFLD) affects obesity-associated metabolic syndrome, which exhibits hepatic steatosis, insulin insensitivity and glucose intolerance. Previous studies indicated that hepatic microRNAs (miRs) play critical roles in the development of NAFLD. In this study, we aim to explore the pathophysiological role of miR-194 in obesity-mediated metabolic dysfunction. Our findings show that the high fat diet or palmitic acid treatment significantly increase hepatic miR-194 levels in vivo and in vitro. Silence of miR-194 protects palmitic acid-induced inflammatory response in cultured hepatocytes, and attenuates structural disorders, lipid deposits and inflammatory response in fatty liver. MiR-194 inhibitor also improves glucose and insulin intolerance in obese mice. Through dual luciferase assay, we demonstrate that miR-194 directly binds to FXR/Nr1h4 3′-UTR, and inhibits gene expression of FXR/Nr1h4. Furthermore, overexpression of miR-194 downregulates FXR/Nr1h4 in cultured hepatocytes, but miR-194 inhibitor reversely increases FXR/Nr1h4 expression in obese mouse liver tissues. On the contrast, silence of FXR/Nr1h4 abolishes the hepatic benefits in obese mice treated with miR-194 inhibitor. Present study provides a novel finding that suppression of miR-194 attenuates dietary-induced NAFLD via upregulation of FXR/Nr1h4. The findings suggest miR-194/FXR are potential diagnostic markers and therapeutic targets for NAFLD.

Tmbim1 is a multivesicular body regulator that protects against non-alcoholic fatty liver disease in mice and monkeys by targeting the lysosomal degradation of Tlr4.

Non-alcoholic steatohepatitis (NASH) is an increasingly prevalent liver pathology that can progress from non-alcoholic fatty liver disease (NAFLD), and it is a leading cause of cirrhosis and hepatocellular carcinoma. There is currently no pharmacological therapy for NASH. Defective lysosome-mediated protein degradation is a key process that underlies steatohepatitis and a well-recognized drug target in a variety of diseases; however, whether it can serve as a therapeutic target for NAFLD and NASH remains unknown. Here we report that transmembrane BAX inhibitor motif-containing 1 (TMBIM1) is an effective suppressor of steatohepatitis and a previously unknown regulator of the multivesicular body (MVB)-lysosomal pathway. Tmbim1 expression in hepatocytes substantially inhibited high-fat diet-induced insulin resistance, hepatic steatosis and inflammation in mice. Mechanistically, Tmbim1 promoted the lysosomal degradation of toll-like receptor 4 by cooperating with the ESCRT endosomal sorting complex to facilitate MVB formation, and the ubiquitination of Tmbim1 by the E3 ubiquitin ligase Nedd4l was required for this process. We also found that overexpression of Tmbim1 in the liver effectively inhibited a severe form of NAFLD in mice and NASH progression in monkeys. Taken together, these findings could lead to the development of promising strategies to treat NASH by targeting MVB regulators to properly orchestrate the lysosome-mediated protein degradation of key mediators of the disease.

Impact of activating transcription factor 4 signaling on lipogenesis in HepG2 cells.

Non-alcoholic fatty liver disease (NAFLD) is a rapidly growing health threat that has previously been associated with lipogenesis. The direct effect of endoplasmic reticulum stress (ERS) inhibition on the induction of lipogenesis has not been investigated in hepatocytes invitro. The impact of activating transcription factor‑4 (ATF4) on the lipogenic pathway and hepatic insulin transduction in liver cells also requires further investigation. In the present study, the triglyceride (TG) content of HepG2 cells stimulated with fructose was investigated using a commercially available enzymatic assay, and the expression levels of lipogenesis‑associated factors were determined by western blotting and reverse transcription‑quantitative polymerase chain reaction. Notably, the TG content of HepG2 cells was increased following incubation with fructose, which was accompanied by ERS. 4‑Phenylbutyric acid, an inhibitor of ERS, lowered the TG content by reducing the mRNA expression levels of sterol regulatory element‑binding protein 1 (SREBP‑1c) and carbohydrate‑responsive element‑binding protein (ChREBP), and the protein expression levels of fatty acid synthase (FAS), acetyl‑CoA carboxylase (ACC) and stearoyl‑CoA desaturase‑1 (SCD‑1). Conversely, tunicamycin, which is an inducer of ERS, increased the TG content and stimulated the expression of the above lipogeneic markers. ATF4 deficiency relieved TG accumulation and decreased the mRNA expression levels of SREBP‑1c and ChREBP, and protein expression levels of FAS, ACC and SCD‑1 in fructose‑treated HepG2 cells. Conversely, ATF4 overexpression increased the TG content by upregulating the mRNA expression levels of SREBP‑1c and ChREBP and protein expression levels of FAS, ACC and SCD‑1. Inhibition of ERS was shown to protect HepG2 cells against fructose‑induced TG accumulation, whereas induction of ERS stimulated hepatic lipogenesis. As a downstream transcription factor of the unfolded protein response, a deficiency in ATF4 attenuates fructose‑induced lipogenesis; while an overexpression of ATF4 can induce TG accumulation through stimulating hepatic lipogenesis. The results of the present study suggested that ATF4 may exert various physiological roles in lipid metabolism depending on the nutrient composition. In addition, these results suggested that ATF4 has a role in regulating lipogenesis and in the development of NAFLD; thus ATF4 may be considered a therapeutic target for NAFLD.

MiR-149 controls non-alcoholic fatty liver by targeting FGF-21.

Non‐alcoholic fatty liver disease (NAFLD), a lipid metabolism disorder characterized by the accumulation of intrahepatic fat, has emerged as a global public health problem. However, its underlying molecular mechanism remains unclear. We previously have found that miR‐149 was elevated in NAFLD induced by high‐fat diet mice model, whereas decreased by a 16‐week running programme. Here, we reported that miR‐149 was increased in HepG2 cells treated with long‐chain fatty acid (FFA). In addition, miR‐149 was able to promote lipogenesis in HepG2 cells in the absence of FFA treatment. Moreover, inhibition of miR‐149 was capable of inhibiting lipogenesis in HepG2 cells in the presence of FFA treatment. Meanwhile, fibroblast growth factor‐21 (FGF‐21) was identified as a target gene of miR‐149, which was demonstrated by the fact that miR‐149 could negatively regulate the protein expression level of FGF‐21, and FGF‐21 was also responsible for the effect of miR‐149 inhibitor in decreasing lipogenesis in HepG2 cells in the presence of FFA treatment. These data implicate that miR‐149 might be a novel therapeutic target for NAFLD.

Uric acid regulates hepatic steatosis and insulin resistance through the NLRP3 inflammasome-dependent mechanism

Hyperuricemia significantly increases risk of non-alcoholic fatty liver disease (NAFLD) and insulin resistance. However, the mechanisms responsible for this association are as yet unclear. This study aimed to investigate the effects and underlying mechanisms of uric acid on development of NAFLD and insulin resistance. We initially analyzed the impact of uric acid on the development of hepatic steatosis and insulin resistance in mice and in two cell models, HepG2 and L02. Subsequently, we studied the role of the NOD-like receptor family pyrin domain containing 3 (NLRP3) inflammasome in uric acid-induced fat accumulation and insulin signaling impairment. We found that uric acid directly induces hepatocyte fat accumulation, insulin resistance, and insulin signaling impairment both in vivo and in vitro. We also found that uric acid-induced NLRP3 inflammasome activation, whereas lowering uric acid by allopurinol inhibited NLRP3 inflammasome activation in a high fat diet mouse model of NAFLD. Moreover, knocking down NLRP3 expression significantly attenuated uric acid-induced fat accumulation both in HepG2 cells and L02 cells. Knocking down NLRP3 expression also rescued uric acid-induced insulin signaling impairment in both cell types. Uric acid regulates hepatic steatosis and insulin resistance through the NLRP3 inflammasome. Uric acid may be a new therapeutic target for NAFLD and insulin resistance.

Tyrosol Attenuates High Fat Diet-Induced Hepatic Oxidative Stress: Potential Involvement of Cystathionine β-Synthase and Cystathionine γ-Lyase.

The Mediterranean diet is known for its cardioprotective effects. Recently, its protective qualities have also been reported in patients with non-alcoholic fatty liver disease (NAFLD). Oxidative stress is one of the important factors responsible for the development and progression of NAFLD. Hydrogen sulfide (H2S), a multifaceted gasotransmitter, has emerged as a potential therapeutic target in NAFLD. Cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE) are major enzymes responsible for endogenous H2S synthesis. Since oxidative stress contributes to NAFLD pathogenesis, the objective of this study was to investigate the effect of tyrosol, a major compound in olive oil and white wine, on high fat diet-induced hepatic oxidative stress and the mechanisms involved. Mice (C57BL/6) were fed for 5weeks with a control diet (10% kcal fat), a high fat diet (60% kcal fat, HFD) or a HFD supplemented with tyrosol. High fat diet feeding induced hepatic oxidative stress, as indicated by the significant increase in lipid peroxidation and NADPH oxidase activity. Tyrosol supplementation significantly increased hepatic CBS and CSE expression and H2S synthesis in high fat diet-fed mice. Such effects were associated with the attenuation of high fat diet-induced hepatic lipid peroxidation and the restoration of the redox equilibrium of the antioxidant glutathione. Tyrosol also inhibited palmitic acid-induced oxidative stress in hepatocytes (HepG2 cells). These results suggest that the antioxidant properties of tyrosol may be mediated through functional changes in CBS and CSE activity, which might contribute to the hepatoprotective effect of the Mediterranean diet.