Activation of hepatic Nogo-B receptor expression—A new anti-liver steatosis mechanism of statins

Deficiency of hepatic Nogo-B receptor (NgBR) expression activates liver X receptor α (LXRα) in an adenosine monophosphate-activated protein kinase α (AMPKα)-dependent manner, thereby inducing severe hepatic lipid accumulation and hypertriglyceridemia. Statins have been demonstrated non-cholesterol lowering effects including anti-nonalcoholic fatty liver disease (NAFLD). Herein, we investigated if the anti-NAFLD function of statins depends on activation of NgBR expression. In vivo, atorvastatin protected apoE deficient or NgBR floxed, but not hepatic NgBR deficient mice, against Western diet (WD)-increased triglyceride levels in liver and serum. In vitro, statins reduced lipid accumulation in nonsilencing small hairpin RNA-transfected (shNSi), but not in NgBR small hairpin RNA-transfected (shNgBRi) HepG2 cells. Inhibition of cellular lipid accumulation by atorvastatin is related to activation of AMPKα, and inactivation of LXRα and lipogenic genes. Statin also inhibited expression of oxysterol producing enzymes. Associated with changes of hepatic lipid levels by WD or atorvastatin, NgBR expression was inversely regulated. At cellular levels, statins increased NgBR mRNA and protein expression, and NgBR protein stability. In contrast to reduced cellular cholesterol levels by statin or β-cyclodextrin, increased cellular cholesterol levels decreased NgBR expression suggesting cholesterol or its synthesis intermediates inhibit NgBR expression. Indeed, mevalonate, geranylgeraniol or geranylgeranyl pyrophosphate, but not farnesyl pyrophosphate or farnesol, blocked atorvastatin-induced NgBR expression. Furthermore, we determined that induction of hepatic NgBR expression by atorvastatin mainly depended on inactivation of extracellular signal-regulated kinases 1/2 (ERK1/2) and protein kinase B (Akt). Taken together, our study demonstrates that statins inhibit NAFLD mainly through activation of NgBR expression.     

Activation of AMPK by triptolide alleviates nonalcoholic fatty liver disease by improving hepatic lipid metabolism,inflammation and fibrosis

BackgroundTriptolide is naturally isolated from Tripterygium wilfordii Hook F., possessing multiple biological activities. Hepatotoxicity is one of the main side effects of triptolide. However, the effect of triptolide on nonalcoholic fatty liver disease remains unknown (NAFLD).PurposeThis study aimed to observe the amelioration of triptolide against NAFLD and investigate the engaged mechanism.MethodsTwo typical animal models of NAFLD, obese db/db mice and methionine/choline-deficient (MCD) diet-fed mice, were used. Hepatic steatosis, inflammation, and fibrosis were evaluated by H&E and Masson staining. Oil red O staining and lipid extraction analysis were used to detect fat content in mice livers. Expression of lipid metabolism, inflammatory and fibrogenic genes was also detected by Real-time PCR and Western blotting, respectively. Phosphoproteomics, molecular docking, and TR-FRET assay were performed to provide further insight into how triptolide improved NAFLD.ResultsIntraperitoneal injection of triptolide at a daily dose of 50 μg/kg significantly alleviated MCD diet-induced nonalcoholic steatohepatitis (NASH), but 100 μg/kg triptolide caused severe hepatotoxicity. Pathological staining confirmed low-dose triptolide treatment reducing hepatic lipid deposition, inflammation, and fibrosis in NASH. Serum biochemical analysis revealed a reduction in the level of liver enzymes and bilirubin. MCD also induced rising expression of typical genes and proteins related to fibrosis (fibronectin, α-SMA, collagens, TGF-β) and inflammation (ILs, TNF-α, MCP-1), which was suppressed by low-dose triptolide. Data from the proteomics/phosphoproteomics and TR-FRET assay indicated triptolide was a potential allosteric AMPK agonist to increase the phosphorylation on Thr172 residue, with the EC₅₀ of 277.78 μM and 231.02 μM for AMPKα1 and AMPKα2, respectively. Moreover, triptolide exhibited an ability to activate AMPK and further led to increasing ACC1 phosphorylation in the liver. The positive results that triptolide ameliorated hepatic lipogenesis, fatty acid oxidation, and fibrosis of NAFLD via activating AMPK were further confirmed in db/db mice with 10-week intervention (50 μg/kg, i.v., twice a week).ConclusionThis study demonstrates that dose-related triptolide as an allosteric AMPK agonist has the potential to alleviate NAFLD without hepatotoxicity.     

Green tea extract attenuates hepatic steatosis by decreasing adipose lipogenesis and enhancing hepatic antioxidant defenses in ob/ob mice

Excess hepatic lipid accumulation and oxidative stress contribute to nonalcoholic fatty liver disease (NAFLD). Thus, we hypothesized that the hypolipidemic and antioxidant activities of green tea extract (GTE) would attenuate events leading to NAFLD. Obese mice (ob/ob; 5 weeks old, n=38) and their lean littermates (n=12) were fed 0%, 0.5% or 1% GTE for 6 weeks. Then, hepatic steatosis, oxidative stress and inflammatory markers were measured. Obese mice, compared to lean controls, had greater hepatic lipids and serum alanine aminotransferase (ALT). GTE at 1% lowered (P<.05) hepatic lipids and ALT in obese mice. The GTE-mediated attenuation in hepatic steatosis was accompanied by decreased mRNA expression of adipose sterol regulatory element-binding protein-1c, fatty acid synthase, stearoyl CoA desaturase-1, and hormone-sensitive lipase and decreased serum nonesterified fatty acid concentrations. Immunohistochemical data indicated that steatotic livers from obese mice had extensive accumulation of tumor necrosis factor-α (TNF-α), whereas GTE at 1% decreased hepatic TNF-α protein and inhibited adipose TNF-α mRNA expression. Hepatic total glutathione, malondialdehyde and Mn- and Cu/Zn-superoxide dismutase activities in obese mice fed GTE were normalized to the levels of lean littermates. Also, GTE increased hepatic catalase and glutathione peroxidase activities, and these activities were inversely correlated with ALT and liver lipids. Collectively, GTE mitigated NAFLD and hepatic injury in ob/ob mice by decreasing the release of fatty acids from adipose and inhibiting hepatic lipid peroxidation as well as restoring antioxidant defenses and decreasing inflammatory responses. These findings suggest that GTE may be used as an effective dietary strategy to mitigate obesity-triggered NAFLD.     

Hepatoprotective effects of berberine on liver fibrosis via activation of AMP-activated protein kinase

Aims

We investigated the protective effect of berberine (BBR) on chronic liver fibrosis in mice and the potential mechanism underlying the activation of AMP-activated protein kinase (AMPK) pathway.

Main methods

CCl4-induced chronic liver fibrosis model in mice was established and activated rat hepatic stellate cell was treated with BBR. Cell viability was evaluated by SRB assay and protein expressions were detected by Western blot.

Key findings

Our results showed that BBR ameliorated the liver fibrosis in mice with CCl4-induced liver injury and inhibited the proliferation of hepatic stellate cell in dose- and time-dependent manner. BBR decreased the enzyme release of ALT, AST, and ALP in serum, elevated SOD and reduced MDA content of liver tissue in CCl4-induced liver fibrosis model. BBR delayed the formation of regenerative nodules and reduced necrotic areas compared to CCl4 group. Moreover, BBR treatment activated AMPK, decreased the protein expression of Nox4, TGF-β1 and the phosphorylated Akt. The expression of smooth muscle actin (α-SMA), the marker of activated hepatic stellate cell, was also reduced by BBR treatment.

Significance

Our studies firstly demonstrated that BBR exerted hepatoprotective effects possibly via activation of AMPK, blocking Nox4 and Akt expression. Our findings may benefit the development of new strategies in the prevention of chronic liver disease.     

Phytotherapy using blueberry leaf polyphenols to alleviate non-alcoholic fatty liver disease through improving mitochondrial function and oxidative defense

BackgroundSince non-alcoholic fatty liver disease (NAFLD) pathogenesis is multi-factorial, pharmacotherapy with a specific target commonly exhibits limited efficacy. Phytotherapy, whose therapeutic efficacy is based on the combined action of several active compounds, offers new treatment opportunity for NAFLD. As a representative, many natural polyphenols could be utilized in phytotherapy for NAFLD.PurposeIn present work, we aimed to investigate the therapeutic effects and underlying mechanism of polyphenols in blueberry leaves (PBL) on NAFLD from a mitochondria-centric perspective since mitochondrial dysfunction could play a dominant role in NAFLD.MethodsIdentification and quantification of PBL were performed using liquid chromatography coupled with tandem mass spectrometry. The beneficial effects, especially improving mitochondrial function, and potential mechanism of PBL on NAFLD were studied by in vitro and in vivo study.ResultsPolyphenols were abundant in blueberry leaves making it advantaged in NAFLD phytotherapy. PBL effectively alleviated hepatic steatosis, oxidative stress and inflammation as indicated by both in vitro and in vivo study. Furthermore, PBL mediated improvement of mitochondrial dysfunction and antioxidant capability through activation of AMPK/PGC-1α/SIRT3 signaling axis.ConclusionConsidering that mitochondrial dysfunction takes precedence over hepatic steatosis and induces NAFLD development, we conclude that PBL improve mitochondrial dysfunction and oxidative defense, subsequently alleviate hepatic steatosis, oxidative stress and inflammation, and eventually alleviate NAFLD.     

Food restriction improves glucose and lipid metabolism through Sirt1 expression: a study using a new rat model with obesity and severe hypertension

AimsTo determine the effects of food restriction (FR) on the expression of Sirt1 and its down-stream factors related to lipid and glucose metabolism in obese and hypertensive rats (SHRSP/IDmcr-fa), as a model of human metabolic syndrome.Main methodsMale, 10-week-old SHRSP/IDmcr-fa rats were treated with 85% FR for 2 weeks. Metabolic parameters, serum adipocytokines and distribution of serum adiponectin multimers were investigated. Sirt1 expression was determined in epididymal adipose tissue, liver and skeletal muscle. We also determined the expression of PPARα, γ and other adipocyte-related genes in epididymal adipose tissue, and glucose transporters (GLUT2 and GLUT4) in the liver and skeletal muscle.Key findingsFR improved the general conditions as well as blood chemistry of SHRSP/IDmcr-fa rats. In the epididymal adipose tissue of the FR rats, Sirt1 expression was enhanced, as was adiponectin, whereas leptin was downregulation, findings that were paralleled by the serum protein levels. Furthermore, the serum ratio of high to total adiponectin was increased in the FR group. The mRNA expression of Sirt1 was upregulated in the adipose tissue in the FR group. Sirt1 mRNA expression was downregulated, while PPARα and GLUT2 expression was enhanced in the liver. No differences were found in terms of Sirt1, PPAR or GLUT4 expression in skeletal muscle.SignificanceThese results indicate that FR corrects adipokine dysfunction by activating PPARγ via Sirt1 in adipose tissue. Furthermore, glucose and lipid metabolism are activated by upregulation of GLUT2 via the activation of PPARα in the liver.     

Efficacy of Berberine in Patients with Non-Alcoholic Fatty Liver Disease

Objectives

A randomized, parallel controlled, open-label clinical trial was conducted to evaluate the effect of a botanic compound berberine (BBR) on NAFLD.

Methods

A randomized, parallel controlled, open-label clinical trial was conducted in three medical centers (NIH Registration number: NCT00633282). A total of 184 eligible patients with NAFLD were enrolled and randomly received (i) lifestyle intervention (LSI), (ii) LSI plus pioglitazone (PGZ) 15mg qd, and (iii) LSI plus BBR 0.5g tid, respectively, for 16 weeks. Hepatic fat content (HFC), serum glucose and lipid profiles, liver enzymes and serum and urine BBR concentrations were assessed before and after treatment. We also analyzed hepatic BBR content and expression of genes related to glucose and lipid metabolism in an animal model of NAFLD treated with BBR.

Results

As compared with LSI, BBR treatment plus LSI resulted in a significant reduction of HFC (52.7% vs 36.4%, p = 0.008), paralleled with better improvement in body weight, HOMA-IR, and serum lipid profiles (all p<0.05). BBR was more effective than PGZ 15mg qd in reducing body weight and improving lipid profile. BBR-related adverse events were mild and mainly occurred in digestive system. Serum and urine BBR concentrations were 6.99ng/ml and 79.2ng/ml, respectively, in the BBR-treated subjects. Animal experiments showed that BBR located favorably in the liver and altered hepatic metabolism-related gene expression.

Conclusion

BBR ameliorates NAFLD and related metabolic disorders. The therapeutic effect of BBR on NAFLD may involve a direct regulation of hepatic lipid metabolism.

Trial Registration

ClinicalTrials.gov NCT00633282     

红景天苷调节AMPK/mTOR/ULK1信号通路对结肠癌SW480细胞裸鼠的肝脏损伤的影响

摘要 目的：探讨红景天苷（Sal）调节单磷酸腺苷活化蛋白激酶（AMPK）/哺乳动物雷帕霉素靶蛋白（mTOR）/Unc51样激酶1（ULK1）信号通路对结肠癌SW480细胞裸鼠肝脏损伤的影响。方法：通过皮下注射SW480细胞悬浮液建立肝转移裸鼠模型,将造模后的裸鼠随机分为模型组、Sal低剂量（Sal-L,50 mg/kg Sal）组、Sal中剂量（Sal-M,100 mg/kg Sal）组、Sal高剂量（Sal-H,200 mg/kg Sal）组,Sal-H+AMPK抑制剂（Compound C,200 mg/kg Sal+10 mg/kg Compound C）组,以未接种SW480细胞悬液的裸鼠作为对照组。腹部主动脉取血,检测裸鼠血清中丙氨酸氨基转移酶（AST）、天冬氨酸氨基转移酶（ALT）水平;处死裸鼠,检测肝转移瘤数目及肝脏重量;HE染色观察肝脏组织病理变化;qRT-PCR检测肝脏组织中AMPK、mTOR、ULK1 mRNA表达水平;Western blot检测肝脏组织中自噬（Beclin1、p62）蛋白及通路相关蛋白表达。结果：与对照组相比,模型组裸鼠组织中出现肝转移瘤,肝脏重量、AST、ALT水平、mTORmRNA、ULK1 mRNA、p62表达显著增加（P<0.05）;Beclin1、AMPK mRNA及蛋白表达显著降低（P<0.05）;与模型组相比,Sal-L、Sal-M、Sal-H组肝转移瘤数目、肝脏重量、AST、ALT水平、mTORmRNA、ULK1 mRNA、p62表达显著降低（P<0.05）;Beclin1、AMPK mRNA及蛋白表达显著增加（P<0.05）;与Sal-H组相比,Sal-H+Compound C组肝转移瘤数目、肝脏重量、AST、ALT水平、mTORmRNA、ULK1 mRNA、p62表达显著增加（P<0.05）;Beclin1、AMPK mRNA及蛋白表达显著降低（P<0.05）。结论：Sal可通过减少裸鼠肝转移瘤形成,保护裸鼠肝脏,其机制可能与激活AMPK/mTOR/ULK1信号通路,促进肝脏自噬有关。     

Preventing non-alcoholic fatty liver disease through Lactobacillus johnsonii BS15 by attenuating inflammation and mitochondrial injury and improving gut environment in obese mice

The increasing prevalence of obesity worldwide is associated with a parallel increase in non-alcoholic fatty liver disease (NAFLD). To investigate the effect of Lactobacillus johnsonii BS15 on NAFLD, 120 male ICR mice were randomly divided into four groups and administrated with BS15 (2?×?10⁷ cfu/0.2 mL or 2?×?10⁸ cfu/0.2 mL) or phosphate buffered saline (PBS) throughout a 17-week experimental period. The mice were fed with normal chow diet (NCD) 5 weeks before the experimental period. Afterward, with the exception of the PBS group, NCD was changed into high-fat diet (HFD) for the remaining experimental period. Results showed that BS15-treated HFD mice were protected from hepatic steatosis and hepatocyte apoptosis. BS15 exhibited a positive effect on liver lipid peroxidation through an anti-oxidative stress activity by enhancing the liver antioxidant defense system. In addition, BS15 inhibited the insulin resistance; decreased the mRNA levels of acetyl–CoA carboxylase 1, fatty acid synthase, and peroxisome proliferator-activated receptor γ; and increased the expression of the fasting-induced adipose factor in livers. Meanwhile, BS15 attenuated mitochondria abnormalities when the content of uncoupling protein-2 decreased and cytochrome c increased in NAFLD mice. BS15 also reduced the level of serum lipopolysaccharide in NAFLD mice by lowering the intestinal permeability and adjusting gut flora, followed by the downregulation of the TNFα mRNA level in liver and the serum level of C-reactive protein. These findings suggest that BS15 may be effective in preventing NAFLD induced by HFD.     

n-3 Fatty acids combined with flavan-3-ols prevent steatosis and liver injury in a murine model of NAFLD

Non-alcoholic fatty liver disease (NAFLD) affects 25% of adults and at present no licensed medication has been approved. Despite its complex patho-physiology, dietary strategies aiming at delaying or preventing NAFLD have taken a reductionist approach, examining the impact of single components. Accumulating evidence suggests that n-3 LC-PUFAs are efficacious in regulating lipogenesis and fatty acid oxidation. In addition, plant derived flavonoids are also emerging as a dietary strategy for NAFLD prevention, with efficacy attributed to their insulin sensitising and indirect antioxidant effects. Based on knowledge of their complementary molecular targets, we aimed to demonstrate that the combination of n-3 LC-PUFA (n-3) and flavan-3-ols (FLAV) prevents NAFLD. In a high-fat high-fructose (HF/HFr) fed C57Bl/6 J mouse model, the independent and interactive impact of n-3 and FLAV on histologically defined NAFLD, insulin sensitivity, weight gain, intestinal and hepatic gene expression, intestinal bile acids were examined. Only the combination of FLAV and n-3 (FLAVn-3) prevented steatosis as evidenced by a strong reduction in hepatocyte ballooning. While FLAV reduced body (? 28–30%), adipose tissue (? 45–50%) weights and serum insulin (? 22–25%) as observed following an intra-peritoneal glucose tolerance test, n-3 downregulated the expression of Srebf1 and the lipogenic genes (Acaca, Fasn). Significant impacts of interventions on intestinal bile acid metabolism, farnesoid X receptor (Fxr) signalling in the intestine and liver, and hepatic expression of fatty acid transporters (Fabp4, Vldlr, Cd36) were also evident. FLAVn-3 may be a novel intervention for NAFLD. Future research should aim to demonstrate its efficacy in the prevention and treatment of human NAFLD.     

n-3 PUFAs protect against adiposity and fatty liver by promoting browning in postnatally overfed male rats: a role for NRG4

Early-life nutrition plays an important role in regulating adult metabolism. This study evaluated the effects of early nutrition during the suckling and postweaning periods on expression of the adipocytokine Neuregulin 4 (Nrg4) and its relationship with nonalcoholic fatty liver disease (NAFLD) in adulthood. In vivo, male rats were adjusted to litter sizes of three (small litter, SL) or ten (normal litter, NL) on postnatal day 3. Pups were fed control chow (NL and SL groups) or a high-fat diet (NL-HF and SL-HF groups), and SL pups specifically were fed a fish oil diet rich in n-3 polyunsaturated fatty acids (n-3 PUFAs) (SL-FO group), from postnatal weeks 3 to 13. The results demonstrated that postnatal overnutrition increased weight, hepatic de novo lipogenesis (DNL) gene expression and NAFLD and decreased body temperature and Nrg4, Ucp1 and Pgc1a mRNA expression in adipose tissues in SL, SL-HF and NL-HF rats compared to NL rats in adulthood. The opposite trends were observed in SL-FO rats. Moreover, in vitro, recombinant NRG4 protein reduced lipid accumulation by inhibiting DNL gene expression in fatty HepG2 cells stimulated with sodium oleate. In HPAs, eicosapentaenoic acid (EPA) treatment elevated NRG4 production and caused adipocyte browning, and these effects were abrogated by PPARG antagonism. In conclusion, a postweaning n-3 PUFA diet enhanced Nrg4 expression in adipose tissues, associated with attenuation of NAFLD induced by SL rearing. Additionally, external NRG4 reduced lipogenesis in steatotic hepatocytes. Thus, white adipose tissue browning induced by n-3 PUFAs may promote NRG4 production through the PPARG pathway.     

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.     

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.     

Impact of caloric restriction on AMPK and endoplasmic reticulum stress in peripheral tissues and circulating peripheral blood mononuclear cells from Zucker rats

The activation of endoplasmic reticulum (ER) stress and a reduction of AMP-dependent protein kinase (AMPK) phosphorylation have been described in obesity. We hypothesize that a moderate caloric restriction (CR) might contribute to reducing ER stress and increasing AMPK phosphorylation in peripheral tissues from genetically obese Zucker fa/fa rats and in peripheral blood mononuclear cells (PBMCs).Zucker Lean and Zucker fa/fa rats were fed with chow diet either ad libitum (AL) (C, as controls) or 80% of AL (CR) for 2 weeks, giving rise to four experimental groups: Lean C, Lean CR, fa/fa C and fa/fa CR.CR significantly increased AMPK phosphorylation in the liver, perirenal adipose tissue (PRAT) and PBMCs from fa/fa rats but not in the subcutaneous AT (SCAT), suggesting a reduced response of SCAT to CR. Liver samples of fa/fa rats exhibited an increased mRNA expression of PERK, EIF-2α, XBP-1(s), Chop and caspase 3, which was significantly reduced by CR. PRAT exhibited an overexpression of Edem and PDIA-4 in fa/fa rats, but only PDIA-4 expression was reduced by CR. eIF-2α phosphorylation was significantly increased in all studied tissues from fa/fa rats and reduced by CR. A negative correlation was detected between p-AMPK and p-eIF-2α in the liver, PRAT and PBMCs from fa/fa rats but not in SCAT.This study shows that a moderate CR reduces ER stress and improves AMPK phosphorylation in several peripheral tissues and in circulating PBMCs, suggesting that alterations observed in PBMCs could reflect metabolic alterations associated with obesity.     

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.     

Atractylenolide III ameliorates Non-Alcoholic Fatty Liver Disease by activating Hepatic Adiponectin Receptor 1-Mediated AMPK Pathway

Background: Nonalcoholic fatty liver disease (NAFLD) is the most frequent cause of chronic liver diseases worldwide. At present, there are no effective pharmacological therapies for NAFLD except lifestyle intervention-mediated weight loss. Atractylenolide III (ATL III), the major bioactive component found in Atractylode smacrocephala Koidz, has been shown to exert anti-oxidant, anti-tumor, anti-allergic response, anti-bacterial effects and cognitive protection. Here we investigate the therapeutic potential and underlying mechanisms of ATL III for the treatment of NAFLD.Methods: Male C57BL/6J mice were fed a high-fat diet (HFD) and treated with ATL III. Lipid accumulation was analyzed by Oil Red O staining in liver tissues and free fatty acids (FFAs)-treated hepatocytes. AMP-activated protein (AMPK) and sirtuin 1(SIRT1) signaling pathways were inhibited by Compound C and EX527 in vitro, respectively. Small-interfering RNA (siRNA) was used to knockdown adiponectin receptor 1 (AdipoR1) expression in HepG2 cells.Results: ATL III treatment ameliorated liver injury and hepatic lipid accumulation in the HFD-induced NAFLD mouse model as demonstrated by that ATL III administration significantly reduced serum levels of alanine aminotransferase, glutamic oxaloacetic transaminase, triglycerides, total cholesterol and low-density lipoprotein. Furthermore, treatment with ATL III alleviated hepatic oxidative stress, inflammation and fibrosis in the HFD feeding model. To study the underlying mechanisms, we performed Computer Aided Design assay and found that open-formed AdipoR1 and adiponectin receptor 2 were the potential receptors targeted by ATL III. Interestingly, HFD feeding or FFAs treatment only reduced hepatic AdipoR1 expression, while such reduction was abolished by ATL III administration. In addition, in vitro treatment with ATL III activated the AdipoR1 downstream AMPK /SIRT1 signaling pathway and reduced lipid deposition in HepG2 cells, which was diminished by silencing AdipoR1. Finally, inhibition of AMPK or SIRT1, the AdipoR1 downstream signaling, abolished the protective effects of ATL III on lipid deposition and oxidative stress in FFAs-treated HepG2 cells.Conclusion: Our findings suggest that ATL III is a therapeutic drug for the treatment of NAFLD and such protective effect is mediated by activating hepatic AdipoR1-mediated AMPK/SIRT1 signaling pathway.     

Circ_0057558 promotes nonalcoholic fatty liver disease by regulating ROCK1/AMPK signaling through targeting miR-206

Nonalcoholic fatty liver disease (NAFLD) is one of the most prevalent chronic liver disorders that is featured by the extensive deposition of fat in the hepatocytes. Current treatments are very limited due to its unclear pathogenesis. Here, we investigated the function of circ_0057558 and miR-206 in NAFLD. High-fat diet (HFD) feeding mouse was used as an in vivo NAFLD model and long-chain-free fatty acid (FFA)-treated liver cells were used as an in vitro NAFLD model. qRT-PCR was used to measure levels of miR-206, ROCK1 mRNA, and circ_0057558, while Western blotting was employed to determine protein levels of ROCK1, p-AMPK, AMPK, and lipogenesis-related proteins. Immunohistochemistry were performed to examine ROCK1 level. Oil-Red O staining was used to assess the lipid deposition in cells. ELISA was performed to examine secreted triglyceride (TG) level. Dual-luciferase assay was used to validate interactions of miR-206/ROCK1 and circ_0057558/miR-206. RNA immunoprecipitation was employed to confirm the binding of circ_0057558 with miR-206. Circ_0057558 was elevated while miR-206 was reduced in both in vivo and in vitro NAFLD models. miR-206 directly bound with ROCK1 3’-UTR and suppressed lipogenesis and TG secretion through targeting ROCK1/AMPK signaling. Circ_0057558 directly interacted with miR-206 to disinhibit ROCK1/AMPK signaling. Knockdown of circ_0057558 or overexpression of miR-206 inhibited lipogenesis, TG secretion and expression of lipogenesis-related proteins. ROCK1 knockdown reversed the effects of circ_0057558 overexpression. Injection of miR-206 mimics significantly ameliorated NAFLD progression in vivo. Circ_0057558 acts as a miR-206 sponge to de-repress the ROCK1/AMPK signaling and facilitates lipogenesis and TG secretion, which greatly contributes to NAFLD development and progression.Subject terms: Diseases, Gastrointestinal diseases