Involvement of G protein-coupled receptor kinase 2 (GRK2) in the development of non-alcoholic steatosis and steatohepatitis in mice and humans

Insulin resistance (IR) and obesity are important risk factors for non-alcoholic fatty liver disease (NAFLD). G protein-coupled receptor kinase 2 (GRK2) is involved in the development of IR and obesity in vivo. However, its possible contribution to NAFLD and/or non-alcoholic steatohepatitis (NASH) independently of its role on IR or fat mass accretion has not been explored. Here, we used wild-type (WT) or GRK2 hemizygous (GRK2±) mice fed a high-fat diet (HFD) or a methionine and choline-deficient diet (MCD) as a model of NASH independent of adiposity and IR. GRK2± mice were protected from HFD-induced NAFLD. Moreover, MCD feeding caused an increased in triglyceride content and liver-to-body weight ratio in WT mice, features that were attenuated in GRK2± mice. According to their NAFLD activity score, MCD-fed GRK2± mice were diagnosed with simple steatosis and not overt NASH. They also showed reduced expression of lipogenic and lipid-uptake markers and less signs of inflammation in the liver. GRK2± mice preserved hepatic protective mechanisms as enhanced autophagy and mitochondrial fusion and biogenesis, together with reduced endoplasmic reticulum stress. GRK2 protein was increased in MCD-fed WT but not in GRK2± mice, and enhanced GRK2 expression potentiated palmitic acid-triggered lipid accumulation in human hepatocytes directly relating GRK2 levels to steatosis. GRK2 protein and mRNA levels were increased in human liver biopsies from simple steatosis or NASH patients in two different human cohorts. Our results describe a functional relationship between GRK2 levels and hepatic lipid accumulation and implicate GRK2 in the establishment and/or development of NASH.     

Lipotoxicity and steatohepatitis in an overfed mouse model for non-alcoholic fatty liver disease

The major risk factors for non-alcoholic fatty liver disease (NAFLD) are obesity, insulin resistance and dyslipidemia. The cause for progression from the steatosis stage to the inflammatory condition (non-alcoholic steatohepatitis (NASH)) remains elusive at present. Aim of this study was to test whether the different stages of NAFLD as well as the associated metabolic abnormalities can be recreated in time in an overfed mouse model and study the mechanisms underlying the transition from steatosis to NASH.Male C57Bl/6J mice were subjected to continuous intragastric overfeeding with a high-fat liquid diet (HFLD) for different time periods. Mice fed a solid high-fat diet (HFD) ad libitum served as controls. Liver histology and metabolic characteristics of liver, white adipose tisue (WAT) and plasma were studied.Both HFD-fed and HFLD-overfed mice initially developed liver steatosis, but only the latter progressed in time to NASH. NASH coincided with obesity, hyperinsulinemia, loss of liver glycogen and hepatic endoplasmatic reticulum stress. Peroxisome proliferator-activated receptor γ (Pparγ), fibroblast growth factor 21 (Fgf21), fatty acid binding protein (Fabp) and fatty acid translocase (CD36) were induced exclusively in the livers of the HFLD-overfed mice. Inflammation, reduced adiponectin expression and altered expression of genes that influence adipogenic capacity were only observed in WAT of HFLD-overfed mice.In conclusion: this dietary mouse model displays the different stages and the metabolic settings often found in human NAFLD. Lipotoxicity due to compromised adipose tissue function is likely associated with the progression to NASH, but whether this is cause or consequence remains to be established.     

The Annexin A2-Notch regulatory loop in hepatocytes promotes liver fibrosis in NAFLD by increasing osteopontin expression

BackgroundThe mechanisms underlying the progression of liver disease from simple hepatic steatosis to advanced nonalcoholic steatohepatitis (NASH) and liver fibrosis warrant further investigation. Increased mRNA levels of Annexin A2 protein (Anxa2) have been observed in patients with NASH. However, the role of Anxa2 in NASH remains unclear.MethodsThe protein levels of Anxa2 were analyzed in the livers of mice and patients with NASH. Anxa2-knockout and -knockdown mice were generated, and NASH was induced through a high fructose, palmitate, and cholesterol (FPC) diet or methionine- and choline-deficient (MCD) diet.FindingsWe found elevated expression of Anxa2 in the livers of patients and mice with NASH. Anxa2 knockdown but not knockout ameliorated liver fibrosis in both FPC and MCD diet–fed mice. Liver-specific Anxa2 overexpression increased collagen deposition in mice fed a normal diet. Mechanistically, Anxa2 overexpression in hepatocytes promoted hepatic stellate cell activation in a paracrine manner by increasing osteopontin expression. Notch inhibition suppressed the exogenous overexpression of Anxa2-induced osteopontin and endogenous Anxa2 expression. Additionally, Anxa2 overexpression accelerated the progression of nonalcoholic fatty liver disease (NAFLD) in mice fed a high-fat diet. Moreover, Anxa2 levels were higher in NAFLD patients with advanced liver fibrosis than in those with mild liver fibrosis, as determined using the Gene Expression Omnibus database.InterpretationIn conclusion, we found increased Anxa2 expression in hepatocytes promoted liver fibrosis in NASH mice by increasing osteopontin expression. The Anxa2-Notch positive regulatory loop contributes to this process and represents a novel target for the treatment of NASH-related liver fibrosis.     

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.     

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.     

GLP-1 analogs reduce hepatocyte steatosis and improve survival by enhancing the unfolded protein response and promoting macroautophagy

Background

Nonalcoholic fatty liver disease (NAFLD) is a known outcome of hepatosteatosis. Free fatty acids (FFA) induce the unfolded protein response (UPR) or endoplasmic reticulum (ER) stress that may induce apoptosis. Recent data indicate ER stress to be a major player in the progression of fatty liver to more aggressive lesions. Autophagy on the other hand has been demonstrated to be protective against ER stress- induced cell death. We hypothesized that exendin-4 (GLP-1 analog) treatment of fat loaded hepatocytes can reduce steatosis by autophagy which leads to reduced ER stress-related hepatocyte apoptosis.

Methodology/Principal Findings

Primary human hepatocytes were loaded with saturated, cis- and trans-unsaturated fatty acids (palmitic, oleic and elaidic acid respectively). Steatosis, induced with all three fatty acids, was significantly resolved after exendin-4 treatment. Exendin-4 sustained levels of GRP78 expression in fat-loaded cells when compared to untreated fat-loaded cells alone. In contrast, CHOP (C/EBP homologous protein); the penultimate protein that leads to ER stress-related cell death was significantly decreased by exendin-4 in hepatocytes loaded with fatty acids. Finally, exendin-4 in fat loaded hepatocytes clearly promoted gene products associated with macroautophagy as measured by enhanced production of both Beclin-1 and LC3B-II, markers for autophagy; and visualized by transmission electron microscopy (TEM). Similar observations were made in mouse liver lysates after mice were fed with high fat high fructose diet and treated with a long acting GLP-1 receptor agonist, liraglutide.

Conclusions/Significance

GLP-1 proteins appear to protect hepatocytes from fatty acid-related death by prohibition of a dysfunctional ER stress response; and reduce fatty acid accumulation, by activation of both macro-and chaperone-mediated autophagy. These findings provide a novel role for GLP-1 proteins in halting the progression of more aggressive lesions from underlying steatosis in humans afflicted with NAFLD.     

Impaired autophagy contributes to hepatocellular damage during ischemia/reperfusion: Heme oxygenase-1 as a possible regulator

Liver ischemia and reperfusion (I/R) injury is characterized by oxidative stress that is accompanied by alterations of the endogenous defensive system. Emerging evidence suggests a protective role for autophagy induced by multiple stressors including reactive oxygen species. Meanwhile, heme oxygenase-1 (HO-1) has long been implicated in cytoprotection against oxidative stress in vitro and in vivo. Therefore, we investigated the impact of autophagy in the pathogenesis of liver I/R and its molecular mechanisms, particularly its linkage to HO-1. By using transmission electron microscopic analysis and biochemical autophagic flux assays with microtubule-associated protein 1 light chain 3-II, and beclin-1, representative autophagy markers, and p62, a selective substrate for autophagy, we found that reperfusion reduced autophagy both in the rat liver and in primary cultured hepatocytes. When autophagy was further inhibited with chloroquine or wortmannin, I/R-induced hepatocellular injury was aggravated. While livers that underwent I/R showed increased levels of mammalian target of rapamaycin and calpain 1 and 2, inhibition of calpain 1 and 2 induced an autophagic response in hepatocytes subjected to hypoxia/reoxygenation. HO-1 increased autophagy, and HO-1 reduced I/R-induced calcium overload in hepatocytes and prevented calpain 2 activation both in vivo and in vitro. Taken together, these findings suggest that the impaired autophagy during liver I/R, which is mediated by calcium overload and calpain activation, contributes to hepatocellular damage and the HO-1 system protects the liver from I/R injury through enhancing autophagy.     

A non-mitogenic FGF4 analog alleviates non-alcoholic steatohepatitis through an AMPK-dependent pathway

Background & aimsNon-alcoholic fatty liver disease (NAFLD) has emerged as a major liver disease increasingly in association with non-alcoholic steatohepatitis (NASH), cirrhosis and hepatocellular carcinoma (HCC). However, there are currently no approved therapies for treating NAFLD and NASH. Fibroblast growth factor 4 (FGF4) has recently been shown as a promising drug candidate for several metabolic diseases.MethodsMice fed a high-fat diet with high fructose/glucose drinking water (HF/HFG, Western-like diet) for 21 weeks were intraperitoneally injected with non-mitogenic recombinant FGF4^△NT (rFGF4^△NT, 1.0 mg/kg body weight) every other day for 8 weeks. Primary mouse hepatocytes cultured in medium containing high glucose/palmitic acid (HG/PA) or TNFα/cyclohexane (TNFα/CHX) were treated with 1.0 μg/ml rFGF4^△NT. Changes in parameters for histopathology, lipid metabolism, inflammation, hepatocellular apoptosis and fibrosis were determined. The Caspase6 activity and AMPK pathway were assessed.ResultsAdministration of rFGF4^△NT significantly attenuated the Western-like diet-induced hepatic steatosis, inflammation, liver injury and fibrosis in mice. rFGF4^△NT treatment reduced fatty acid-induced lipid accumulation and lipotoxicity-induced hepatocyte apoptosis, which were associated with inhibition of Caspase6 cleavage and activation. Inhibition of AMP-activated protein kinase (AMPK) by Compound C or deficiency of Ampk abrogated rFGF4^△NT–induced hepatoprotection in primary hepatocytes and in mice with NASH.ConclusionrFGF4^△NT exerts significant protective effects on NASH via an AMPK-dependent signaling pathway. Our study indicates that FGF4 analogs may have therapeutic potential for the Western-like diet induced NASH.     

S100A9: A Potential Biomarker for the Progression of Non-Alcoholic Fatty Liver Disease and the Diagnosis of Non-Alcoholic Steatohepatitis

Non-alcoholic fatty liver (NAFL) has the potential to progress to non-alcoholic steatohepatitis (NASH) or to promote type 2 diabetes mellitus (T2DM). However, NASH and T2DM do not always develop coordinately. Additionally, there are no definite noninvasive methods for NASH diagnosis currently. We established rat models of NAFL, NASH, and NAFL + T2DM to recapitulate different phenotypes associated with non-alcoholic fatty liver disease (NAFLD) and its progression. Histologic features of rat livers were scored according to criteria established by the Nonalcoholic Steatohepatitis Clinical Research Network. Microarray was performed to assess gene expression changes in rat livers. We find that gene expression of s100a9 was higher in NAFL group compared with control, and was increased in NASH groups and decreased in NAFL + T2DM group compared with NAFL. In contrast, srebf1, tbx21, and gimap4 only showed limited discriminating abilities in different groups. There is a significant positive correlation between serum levels of S100A9 and NAFLD Activity Score (NAS), the severity of hepatic steatosis, and lobular inflammation (r = 0.80, 0.64 and 0.86, P < 0.001). These findings suggest that S100A9 may be extremely useful in the diagnosis of NASH (AUROC: 0.947, CI: 0.845-1.049). Additionally, serum S100A9 levels displayed a strong correlation with ALT, AST and TBil (r = 0.81, 0.89 and 0.91, P < 0.001) but a weak correlation with FBG, HOMA-IR, TG, and TC (r = -0.41, -0.40, 0.47 and 0.49, P < 0.05). Conclusions: The results we provide here suggest that S100A9 may be useful as a biomarker for the hepatic and metabolic progression of NAFLD and the non-invasive diagnosis of NASH.     

高脂及MCD饮食诱导非酒精性脂肪性肝炎动物模型的比较

目的:对高脂饮食诱发的大鼠NASH模型与蛋氨酸胆碱缺乏饮食诱发的小鼠NASH模型进行血清学及病理学比较,并初步探讨两种模型的发病过程及机制。方法:高脂饮食喂养SD大鼠8周,蛋氨酸胆碱缺乏饮食喂养C57BL/6小鼠2周,以制备NASH模型。取材后,血清用比色法对TG、CHO、FPG的含量进行检测,用放免法对FINS的含量进行检测,并对HOMA-IR指数进行计算;肝组织制成石蜡切片及冰冻切片进行HE及油红O染色,并根据"NAFLD活动度积分"对各组肝组织进行NASH分级评估。结果:高脂饮食大鼠血清中TG、CHO、FPG、FINS的含量显著升高,经计算HOMA-IR指数显著升高;MCD小鼠血清中TG、CHO的含量显著下降,FPG、FINS的含量未发生显著性改变,经计算HOMA-IR指数未发生显著性改变。HE染色、油红O染色及NAFLD活动度积分结果显示,高脂饮食大鼠及MCD小鼠的肝组织均已发展到NASH阶段。结论:两种造模方法均可稳定的模拟人类NASH疾病的血清学及病理学变化,其中高脂饮食诱发的大鼠NASH模型可模拟人类的发病过程及机制,能够复制胰岛素抵抗、氧化应激等人类全身代谢紊乱表现,在NASH研究领域更占优势。     

葛根素联合小檗碱干预非酒精性脂肪性肝炎两种动物模型的病理学研究

目的:从病理学的角度探讨葛根素及小檗碱对非酒精性脂肪性肝炎(NASH)的干预作用。方法:制备高脂饮食诱导的SD大鼠NASH模型及蛋氨酸-胆碱缺乏饮食诱导的C57BL/6小鼠NASH模型,各给药组造模并同时灌胃给药,大鼠持续8周后取材,小鼠持续2周后取材。肝组织制成石蜡切片进行HE染色,制成冰冻切片进行油红O染色,经光镜观察摄片后,采用NAFLD活动度积分对各组进行NASH评估。结果:HE及油红O检测提示葛根素联合小檗碱可减轻两种NASH动物模型肝脏的脂肪变性、抑制肝细胞空泡化的发生,减少肝小叶内炎性病灶的数量。给予葛根素联合小檗碱的SD大鼠NAS总分为2.60±0.52(3),给予葛根素联合小檗碱的C57BL/6小鼠NAS总分为2.80±0.79(3),均已排除NASH。结论:葛根素联合小檗碱可显著抑制NASH相关病理变化的发生发展,对NASH具有一定的干预作用。     

Extracellular vesicles derived from fat-laden hepatocytes undergoing chemical hypoxia promote a pro-fibrotic phenotype in hepatic stellate cells

BackgroundThe transition from steatosis to non-alcoholic steatohepatitis (NASH) is a key issue in non-alcoholic fatty liver disease (NAFLD). Observations in patients with obstructive sleep apnea syndrome (OSAS) suggest that hypoxia contributes to progression to NASH and liver fibrosis, and the release of extracellular vesicles (EVs) by injured hepatocytes has been implicated in NAFLD progression.AimTo evaluate the effects of hypoxia on hepatic pro-fibrotic response and EV release in experimental NAFLD and to assess cellular crosstalk between hepatocytes and human hepatic stellate cells (LX-2).MethodsHepG2 cells were treated with fatty acids and subjected to chemically induced hypoxia using the hypoxia-inducible factor 1 alpha (HIF-1α) stabilizer cobalt chloride (CoCl2). Lipid droplets, oxidative stress, apoptosis and pro-inflammatory and pro-fibrotic-associated genes were assessed. EVs were isolated by ultracentrifugation. LX-2 cells were treated with EVs from hepatocytes. The CDAA-fed mouse model was used to assess the effects of intermittent hypoxia (IH) in experimental NASH.ResultsChemical hypoxia increased steatosis, oxidative stress, apoptosis and pro-inflammatory and pro-fibrotic gene expressions in fat-laden HepG2 cells. Chemical hypoxia also increased the release of EVs from HepG2 cells. Treatment of LX2 cells with EVs from fat-laden HepG2 cells undergoing chemical hypoxia increased expression pro-fibrotic markers. CDAA-fed animals exposed to IH exhibited increased portal inflammation and fibrosis that correlated with an increase in circulating EVs.ConclusionChemical hypoxia promotes hepatocellular damage and pro-inflammatory and pro-fibrotic signaling in steatotic hepatocytes both in vitro and in vivo. EVs from fat-laden hepatocytes undergoing chemical hypoxia evoke pro-fibrotic responses in LX-2 cells.     

The DNA damage checkpoint protein ATM promotes hepatocellular apoptosis and fibrosis in a mouse model of non-alcoholic fatty liver disease

Steatoapoptosis is a hallmark of non-alcoholic fatty liver disease (NAFLD) and is an important factor in liver disease progression. We hypothesized that increased reactive oxygen species resulting from excess dietary fat contribute to liver disease by causing DNA damage and apoptotic cell death, and tested this by investigating the effects of feeding mice high fat or standard diets for 8 weeks. High fat diet feeding resulted in increased hepatic H₂O₂, superoxide production, and expression of oxidative stress response genes, confirming that the high fat diet induced hepatic oxidative stress. High fat diet feeding also increased hepatic steatosis, hepatitis and DNA damage as exemplified by an increase in the percentage of 8-hydroxyguanosine (8-OHG) positive hepatocytes in high fat diet fed mice. Consistent with reports that the DNA damage checkpoint kinase Ataxia Telangiectasia Mutated (ATM) is activated by oxidative stress, ATM phosphorylation was induced in the livers of wild type mice following high fat diet feeding. We therefore examined the effects of high fat diet feeding in Atm-deficient mice. The prevalence of apoptosis and expression of the pro-apoptotic factor PUMA were significantly reduced in Atm-deficient mice fed the high fat diet when compared with wild type controls. Furthermore, high fat diet fed Atm^−/− mice had significantly less hepatic fibrosis than Atm^+/+ or Atm^+/− mice fed the same diet. Together, these data demonstrate a prominent role for the ATM pathway in the response to hepatic fat accumulation and link ATM activation to fatty liver-induced steatoapoptosis and fibrosis, key features of NAFLD progression.     

Female Mice Are More Susceptible to Nonalcoholic Fatty Liver Disease: Sex-Specific Regulation of the Hepatic AMP-Activated Protein Kinase–Plasminogen Activator Inhibitor 1 Cascade,but Not the Hepatic Endotoxin Response

As significant differences between sexes were found in the susceptibility to alcoholic liver disease in human and animal models, it was the aim of the present study to investigate whether female mice also are more susceptible to the development of non-alcoholic fatty liver disease (NAFLD). Male and female C57BL/6J mice were fed either water or 30% fructose solution ad libitum for 16 wks. Liver damage was evaluated by histological scoring. Portal endotoxin levels and markers of Kupffer cell activation and insulin resistance, plasminogen activator inhibitor 1 (PAI-1) and phosphorylated adenosine monophosphate–activated protein kinase (pAMPK ) were measured in the liver. Adiponectin mRNA expression was determined in adipose tissue. Hepatic steatosis was almost similar between male and female mice; however, inflammation was markedly more pronounced in livers of female mice. Portal endotoxin levels, hepatic levels of myeloid differentiation primary response gene (88) (MyD88) protein and of 4-hydroxynonenal protein adducts were elevated in animals with NAFLD regardless of sex. Expression of insulin receptor substrate 1 and 2 was decreased to a similar extent in livers of male and female mice with NAFLD. The less pronounced susceptibility to liver damage in male mice was associated with a superinduction of hepatic pAMPK in these mice whereas, in livers of female mice with NAFLD, PAI-1 was markedly induced. Expression of adiponectin in visceral fat was significantly lower in female mice with NAFLD but unchanged in male mice compared with respective controls. In conclusion, our data suggest that the sex-specific differences in the susceptibility to NAFLD are associated with differences in the regulation of the adiponectin–AMPK–PAI-1 signaling cascade.     

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.     

Aberrant FGFR4 signaling worsens nonalcoholic steatohepatitis in FGF21KO mice

Background: Nonalcoholic steatohepatitis (NASH) is the most severe form of non-alcoholic fatty liver disease (NAFLD) and a potential precursor of hepatocellular carcinoma (HCC). In our previous studies, we found that endocrine fibroblast growth factor 21 (FGF21) played a key role in preventing the development of NASH, however, the FGF15/19 mediated-FGFR4 signaling worsened NASH and even contributed to the NASH-HCC transition. The aim of this study is to determine whether FGF15/FGFR4 signaling could alleviate or aggravate NASH in the FGF21KO mice.Methods: NASH models were established in FGF21KO mice fed with high fat methionine-choline deficient (HFMCD) diet to investigate FGF15/FGFR4 signaling during early stage NASH and advanced stage NASH. Human hepatocytes, HepG2 and Hep3B cells, were cultured with human enterocytes Caco-2 cells to mimic gut-liver circulation to investigate the potential mechanism of NASH development.Results: Significant increase of FGF15 production was found in the liver of the NASH-FGF21KO mice, however the increased FGF15 protein was unable to alleviate hepatic lipid accumulation. In contrast, up-regulated FGF15/19/FGFR4 signaling was found in the FGF21KO mice with increased NASH severity, as evident by hepatocyte injury/repair, fibrosis and potential malignant events. In in vitro studies, blockage of FGFR4 by BLU9931 treatment attenuated the lipid accumulation, up-regulated cyclin D1, and epithelial-mesenchymal transition (EMT) in the hepatocytes.Conclusion: The increased FGF15 in NASH-FGF21KO mice could not substitute for FGF21 to compensate its lipid metabolic benefits thereby to prevent NASH development. Up-regulated FGFR4 signaling in NASH-FGF21KO mice coupled to proliferation and EMT events which were widely accepted to be associated with carcinogenic transformation.     

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.