Inhibition of MD2‐dependent inflammation attenuates the progression of non‐alcoholic fatty liver disease

Non‐alcoholic fatty liver disease (NAFLD) can progress to the more serious non‐alcoholic steatohepatitis (NASH), characterized by inflammatory injury and fibrosis. The pathogenic basis of NAFLD progressing to NASH is currently unknown, but growing evidence suggests MD2 (myeloid differentiation factor 2), an accessory protein of TLR4, is an important signalling component contributing to this disease. We evaluated the effectiveness of the specific MD2 inhibitor, L6H21, in reducing inflammatory liver injury in a relevant high‐fat diet (HFD) mouse model of NASH and in the palmitic acid (PA)‐stimulated human liver cell line (HepG2). For study, genetic knockout (MD2^?/?) mice were fed a HFD or control diet for 24 weeks, or wild‐type mice placed on a similar diet regimen and treated with L6H21 for the last 8 or 16 weeks. Results indicated that MD2 inhibition with L6H21 was as effective as MD2 knockout in preventing the HFD‐induced hepatic lipid accumulation, pro‐fibrotic changes and expression of pro‐inflammatory molecules. Direct challenge of HepG2 with PA (200 μM) increased MD2‐TLR4 complex formation and expression of pro‐inflammatory and pro‐fibrotic genes and L6H21 pre‐treatment prevented these PA‐induced responses. Interestingly, MD2 knockout or L6H21 increased expression of the anti‐inflammatory molecule, PPARγ, in liver tissue and the liver cell line. Our results provide further evidence for the critical role of MD2 in the development of NASH and conclude that MD2 could be a potential therapeutic target for NAFLD/NASH treatment. Moreover, the small molecule MD2 inhibitor, L6H21, was an effective and selective investigative agent for future mechanistic studies of MD2.     

L-tryptophan-mediated enhancement of susceptibility to nonalcoholic fatty liver disease is dependent on the mammalian target of rapamycin

Nonalcoholic fatty liver disease is one of the most common liver diseases. L-tryptophan and its metabolite serotonin are involved in hepatic lipid metabolism and inflammation. However, it is unclear whether L-tryptophan promotes hepatic steatosis. To explore this issue, we examined the role of L-tryptophan in mouse hepatic steatosis by using a high fat and high fructose diet (HFHFD) model. L-tryptophan treatment in combination with an HFHFD exacerbated hepatic steatosis, expression of HNE-modified proteins, hydroxyproline content, and serum alanine aminotransaminase levels, whereas L-tryptophan alone did not result in these effects. We also found that L-tryptophan treatment increases serum serotonin levels. The introduction of adenoviral aromatic amino acid decarboxylase, which stimulates the serotonin synthesis from L-tryptophan, aggravated hepatic steatosis induced by the HFHFD. The fatty acid-induced accumulation of lipid was further increased by serotonin treatment in cultured hepatocytes. These results suggest that L-tryptophan increases the sensitivity to hepatic steatosis through serotonin production. Furthermore, L-tryptophan treatment, adenoviral AADC introduction, and serotonin treatment induced phosphorylation of the mammalian target of rapamycin (mTOR), and a potent mTOR inhibitor rapamycin attenuated hepatocyte lipid accumulation induced by fatty acid with serotonin. These results suggest the importance of mTOR activation for the exacerbation of hepatic steatosis. In conclusion, L-tryptophan exacerbates hepatic steatosis induced by HFHFD through serotonin-mediated activation of mTOR.     

Hepatic recruitment of macrophages promotes nonalcoholic steatohepatitis through CCR2

Inflammatory cell infiltration in the liver is a hallmark of nonalcoholic steatohepatitis (NASH). The chemokine-chemokine receptor interaction induces inflammatory cell recruitment. CC-chemokine receptor (CCR)2 is expressed on hepatic macrophages and hepatic stellate cells. This study aims to investigate the therapeutic potential of CCR2 to NASH. Twenty-two weeks on a choline-deficient amino acid-defined (CDAA) diet induced steatosis, inflammatory cell infiltration, and liver fibrosis with increased CCR2 and monocyte chemoattractant protein (MCP)-1 expression in the wild-type livers. The infiltrated macrophages expressed CD68, CCR2, and a marker of bone marrow-derived monocytes, Ly6C. CCR2(-/-) mice had less steatosis, inflammatory cell infiltration, and fibrosis, and hepatic macrophages expressing CD68 and Ly6C were decreased. Toll-like receptor (TLR)4(-/-), TLR9(-/-), and MyD88(-/-) mice had reduced hepatic macrophage infiltration with decreased MCP-1 and CCR2 expression because TLR signaling is a potent inducer of MCP-1. To assess the role of Kupffer cells at the onset of NASH, Kupffer cells were depleted by liposomal clodronate. The Kupffer cell depletion ameliorated steatohepatitis with a decrease in the MCP-1 expression and recruitment of Ly6C-expressing macrophages at the onset of NASH. Finally, to test the therapeutic potential of targeting CCR2, a CCR2 inhibitor was administered to mice on a CDAA diet. The pharmaceutical inhibition of CCR2 prevented infiltration of the Ly6C-positive macrophages, resulting in an inhibition of liver inflammation and fibrosis. We concluded that CCR2 and Kupffer cells contribute to the progression of NASH by recruiting bone marrow-derived monocytes.     

Metabolomics of the interaction between PPAR‐α and age in the PPAR‐α‐null mouse

Regulation between the fed and fasted states in mammals is partially controlled by peroxisome proliferator‐activated receptor‐α (PPAR‐α). Expression of the receptor is high in the liver, heart and skeletal muscle, but decreases with age. A combined ¹H nuclear magnetic resonance (NMR) spectroscopy and gas chromatography‐mass spectrometry metabolomic approach has been used to examine metabolism in the liver, heart, skeletal muscle and adipose tissue in PPAR‐α‐null mice and wild‐type controls during ageing between 3 and 13 months. For the PPAR‐α‐null mouse, multivariate statistics highlighted hepatic steatosis, reductions in the concentrations of glucose and glycogen in both the liver and muscle tissue, and profound changes in lipid metabolism in each tissue, reflecting known expression targets of the PPAR‐α receptor. Hepatic glycogen and glucose also decreased with age for both genotypes. These findings indicate the development of age‐related hepatic steatosis in the PPAR‐α‐null mouse, with the normal metabolic changes associated with ageing exacerbating changes associated with genotype. Furthermore, the combined metabolomic and multivariate statistics approach provides a robust method for examining the interaction between age and genotype.     

Resveratrol alleviates alcoholic fatty liver in mice

Alcoholic fatty liver is associated with inhibition of sirtuin 1 (SIRT1) and AMP-activated kinase (AMPK), two critical signaling molecules regulating the pathways of hepatic lipid metabolism in animals. Resveratrol, a dietary polyphenol, has been identified as a potent activator for both SIRT1 and AMPK. In the present study, we have carried out in vivo animal experiments that test the ability of resveratrol to reverse the inhibitory effects of chronic ethanol feeding on hepatic SIRT1-AMPK signaling system and to prevent the development of alcoholic liver steatosis. Resveratrol treatment increased SIRT1 expression levels and stimulated AMPK activity in livers of ethanol-fed mice. The resveratrol-mediated increase in activities of SIRT1 and AMPK was associated with suppression of sterol regulatory element binding protein 1 (SREBP-1) and activation of peroxisome proliferator-activated receptor gamma coactivator alpha (PGC-1alpha). In parallel, in ethanol-fed mice, resveratrol administration markedly increased circulating adiponectin levels and enhanced mRNA expression of hepatic adiponectin receptors (AdipoR1/R2). In conclusion, resveratrol treatment led to reduced lipid synthesis and increased rates of fatty acid oxidation and prevented alcoholic liver steatosis. The protective action of resveratrol is in whole or in part mediated through the upregulation of a SIRT1-AMPK signaling system in the livers of ethanol-fed mice. Our study suggests that resveratrol may serve as a promising agent for preventing or treating human alcoholic fatty liver disease.     

Pyruvate dehydrogenase kinase 4 mediates lipogenesis and contributes to the pathogenesis of nonalcoholic steatohepatitis

Nonalcoholic steatohepatitis (NASH) is a progressive disease and poses a high risk of severe liver damage. However, the pathogenesis of NASH is still unclear. Accumulation of lipid droplets and insulin resistance is the hallmark of NASH. Pyruvate dehydrogenase kinase isoenzyme 4 (PDK4) plays key role in glucose metabolism via regulating the activity of pyruvate dehydrogenase complex (PDC). Here, we demonstrated a novel of PDK4 in NASH by regulating hepatic steatosis and insulin signaling pathway in methionine and choline deficient (MCD) diet induced NASH model. Hepatic PDK4 levels were highly induced in human patients with NASH and MCD diet fed mice, as well as in hepatocytes treated with oleic acid. The glucose and lipid metabolism were impaired in Pdk4^?/? mice. Pdk4 deficiency ameliorated the hepatic steatosis significantly in NASH mice. Pdk4^?/?-MCD mice had reduced liver weights and triglyceride (TG) levels. And Pdk4 deficiency dramatically reduced the expression of genes related to fatty acid uptake, synthesis and gluconeogenesis. In addition, elevated phosphorylated AMPK (p-AMPK), p-SAPK/JNK and diminished p-ERK, p-P38, p-Akt and p-mTOR/p-4EBP1 proteins were observed. In conclusion, our data indicated that PDK4 potentially contributes to the hepatic steatosis in NASH via regulating several signaling pathway and PDK4 may be a new therapeutic strategy against NAFLD.     

Hepatic HuR protects against the pathogenesis of non-alcoholic fatty liver disease by targeting PTEN

The liver plays an important role in lipid and glucose metabolism. Here, we show the role of human antigen R (HuR), an RNA regulator protein, in hepatocyte steatosis and glucose metabolism. We investigated the level of HuR in the liver of mice fed a normal chow diet (NCD) and a high-fat diet (HFD). HuR was downregulated in the livers of HFD-fed mice. Liver-specific HuR knockout (HuR^LKO) mice showed exacerbated HFD-induced hepatic steatosis along with enhanced glucose tolerance as compared with control mice. Mechanistically, HuR could bind to the adenylate uridylate-rich elements of phosphatase and tensin homolog deleted on the chromosome 10 (PTEN) mRNA 3′ untranslated region, resulting in the increased stability of Pten mRNA; genetic knockdown of HuR decreased the expression of PTEN. Finally, lentiviral overexpression of PTEN alleviated the development of hepatic steatosis in HuR^LKO mice in vivo. Overall, HuR regulates lipid and glucose metabolism by targeting PTEN.Subject terms: Type 2 diabetes, Dyslipidaemias     

Treatment of growth hormone attenuates hepatic steatosis in hyperlipidemic mice via downregulation of hepatic CD36 expression

ABSTRACT

The recombinant human growth hormone (GH) has been used for the treatment of growth hormone deficiency (GHD) and diverse short stature state, and its physiological and therapeutic effects are well documented. However, since the effect of GH treatment on metabolic disorders has not been well characterized, we injected GH to Western diet-fed low-density lipoprotein receptor-deficient (Ldlr ^?/?) mice to understand the exact effect of GH on metabolic diseases including atherosclerosis, hepatic steatosis, and obesity. Exogenous GH treatment increased plasma IGF-1 concentration and decreased body weight without affecting serum lipid profiles. GH treatment changed neither atherosclerotic lesion size nor collagen and smooth muscle cells accumulation in the lesion. GH treatment reduced macrophage accumulation in adipose tissue. Importantly, GH treatment attenuated hepatic steatosis and inflammation. The hepatic expression IL-1β mRNA were decreased by GH treatment. The mRNA and protein levels of CD36 were markedly decreased in GH treated mice without significant changes in other molecules related to lipid metabolism. Therefore, the treatment of GH treatment could attenuate hepatic steatosis and inflammation with downregulation of CD36 expression in hyperlipidemic condition.     

Celastrol exerts anti‐inflammatory effect in liver fibrosis via activation of AMPK‐SIRT3 signalling

Celastrol, a pentacyclic tritepene extracted from Tripterygium Wilfordi plant, showing potent liver protection effects on several liver‐related diseases. However, the anti‐inflammatory potential of celastrol in liver fibrosis and the detailed mechanisms remain uncovered. This study was to investigate the anti‐inflammatory effect of celastrol in liver fibrosis and to further reveal mechanisms of celastrol‐induced anti‐inflammatory effects with a focus on AMPK‐SIRT3 signalling. Celastrol showed potent ameliorative effects on liver fibrosis both in activated hepatic stellate cells (HSCs) and in fibrotic liver. Celastrol remarkably suppressed inflammation in vivo and inhibited the secretion of inflammatory factors in vitro. Interestingly, celastrol increased SIRT3 promoter activity and SIRT3 expression both in fibrotic liver and in activated HSCs. Furthermore, SIRT3 silencing evidently ameliorated the anti‐inflammatory potential of celastrol. Besides, we found that celastrol could increase the AMPK phosphorylation. Further investigation showed that SIRT3 siRNA decreased SIRT3 expression but had no obvious effect on phosphorylation of AMPK. In addition, inhibition of AMPK by employing compound C (an AMPK inhibitor) or AMPK1α siRNA significantly suppressed SIRT3 expression, suggesting that AMPK was an up‐stream protein of SIRT3 in liver fibrosis. We further found that depletion of AMPK significantly attenuated the inhibitory effect of celastrol on inflammation. Collectively, celastrol attenuated liver fibrosis mainly through inhibition of inflammation by activating AMPK‐SIRT3 signalling, which makes celastrol be a potential candidate compound in treating or protecting against liver fibrosis.     

Nonalcoholic Hepatic Steatosis in Zucker Diabetic Rats: Spontaneous Evolution and Effects of Metformin and Fenofibrate

No specific treatment for nonalcoholic hepatic fatty liver disease has been defined. We followed the spontaneous evolution of liver steatosis and tested the therapeutic usefulness of metformin and fenofibrate in a model of steatosis, the Zucker diabetic fatty (ZDF) rat. ZDF and control rats were studied at 7, 14, and 21 weeks. After initial study at 7 weeks, ZDF rats received no treatment, metformin or fenofibrate until studies at 14 or 21 weeks. ZDF rats were obese, hypertriglyceridemic, insulin resistant at 7 weeks, type 2 diabetic at 14, diabetic with insulin deficiency at 21. They had steatosis at 7 weeks with increased hepatic expression and activity of lipogenesis. Steatosis was unchanged at 14 and 21 weeks despite lower expression and activity of lipogenesis. Metformin and fenofibrate did not modify energy intake or expenditure or the evolution of diabetes. Both compounds decreased plasma triacylglycerol (TAG) concentrations. Hepatic TAG content was reduced by fenofibrate at 14 and 21 weeks but only at 21 weeks by metformin. Metformin had no significant effects on the expression in liver of genes of fatty acids metabolism. The beneficial effect of fenofibrate occurred despite increased expression of genes involved in the uptake and activation of fatty acids. Acyl‐CoA oxidase (ACO) and carnitine palmitoyltransferase I (CPTI) mRNA levels were increased by fenofibrate showing evidence of increased lipid oxidation. To conclude, metformin had only moderate effects on liver steatosis. The effects of fenofibrate was more marked but remained mild.     

Tremella fuciformis Crude Polysaccharides Attenuates Steatosis and Suppresses Inflammation in Diet-Induced NAFLD Mice

Nonalcoholic fatty liver disease (NAFLD) is a chronic liver disorder characterized by an enhanced accumulation of lipids, which affects around 40% of the world’s population. The T. fuciformis fungus possesses immunomodulatory activity and other beneficial properties that may alleviate steatosis through a different mechanism. The present study was designed to evaluate the effect T. fuciformis crude polysaccharides (TFCP) on inflammatory and lipid metabolism gene expression, oxidative stress, and lipid profile. Mice were divided into groups receiving (a) a normal chow diet (NCD), (b) a methionine–choline-deficient (MCD) diet, and (c) a MCD diet with TFCP. Liver histopathology was performed, and the hepatic gene expression levels were estimated using qRT-PCR. The lipid profiles, ALT, AST, and efficient oxidative enzymes were analyzed using ELISA. The TFCP administration in the MCD-fed mice suppressed hepatic lipid accumulation, lipid metabolism-associated genes (HMGCR, FABP, SREBP, ACC, and FAS), and inflammation-associated genes (IL-1β, TLR4, TNF-α, and IL-6) whilst enhancing the expression of HNF4α genes. TFCP mitigated against oxidative stress and normalized healthy lipid profiles. These results highlighted that TFCP prevents NAFLD through the inhibition of oxidative stress and inflammation, suggesting TFCP would potentially be an effective therapeutic agent against NAFLD progression.     

Lipocalin-2 (LCN2) regulates PLIN5 expression and intracellular lipid droplet formation in the liver

Lipocalin-2 (LCN2) belongs to the superfamily of lipocalins and plays critical roles in the control of cellular homeostasis during inflammation and in responses to cellular stress or injury. In the liver, LCN2 triggers protective effects following acute or chronic injury, and its expression is a reliable indicator of liver damage. However, little is known about LCN2's functions in the homeostasis and metabolism of hepatic lipids or in the development of steatosis. In this study, we fed wild type (WT) and LCN2-deficient (Lcn2^−/−) mice a methionine- and choline-deficient (MCD) diet as a nutritional model of non-alcoholic steatohepatitis, and compared intrahepatic lipid accumulation, lipid droplet formation, mitochondrial content, and expression of the Perilipin proteins that regulate cellular lipid metabolism. We found that Lcn2^−/− mice fed an MCD diet accumulated more lipids in the liver than WT controls, and that the basal expression of the lipid droplet coat protein Perilipin 5 (PLIN5, also known as OXPAT) was significantly reduced in these animals. Similarly, the overexpression of LCN2 and PLIN5 were also found in animals that were fed with a high fat diet. Furthermore, the loss of LCN2 and/or PLIN5 in hepatocytes prevented normal intracellular lipid droplet formation both in vitro and in vivo. Restoration of LCN2 in Lcn2^−/− primary hepatocytes by either transfection or adenoviral vector infection induced PLIN5 expression and restored proper lipid droplet formation. Our data indicate that LCN2 is a key modulator of hepatic lipid homeostasis that controls the formation of intracellular lipid droplets by regulating PLIN5 expression. LCN2 may therefore represent a novel therapeutic drug target for the treatment of liver diseases associated with elevated fat accumulation and steatosis.     

Reduction of TIP47 improves hepatic steatosis and glucose homeostasis in mice

Lipid droplets in the liver are coated with the perilipin family of proteins, notably adipocyte differentiation-related protein (ADRP) and tail-interacting protein of 47 kDa (TIP47). ADRP is increased in hepatic steatosis and is associated with hyperlipidemia, insulin resistance, and glucose intolerance. We have shown that reducing ADRP in the liver via antisense oligonucleotide (ASO) treatment attenuates steatosis and improves insulin sensitivity and glucose tolerance. We hypothesized that TIP47 has similar effects on hepatic lipid and glucose metabolism. We found that TIP47 mRNA and protein levels were increased in response to a high-fat diet (HFD) in C57BL/6J mice. TIP47 ASO treatment decreased liver TIP47 mRNA and protein levels without altering ADRP levels. Low-dose TIP47 ASO (15 mg/kg) and high-dose TIP47 ASO (50 mg/kg) decreased triglyceride content in the liver by 35% and 52%, respectively. Liver histology showed a drastic reduction in hepatic steatosis following TIP47 ASO treatment. The high dose of TIP47 ASO significantly blunted hepatic triglyceride secretion, improved glucose tolerance, and increased insulin sensitivity in liver, adipose tissue, and muscle. These findings show that TIP47 affects hepatic lipid and glucose metabolism and may be a target for the treatment of nonalcoholic fatty liver and related metabolic disorders.     

Silymarin alleviates hepatic oxidative stress and protects against metabolic disorders in high-fat diet-fed mice

Silymarin is a potent antioxidant medicine and has been widely used for the treatment of liver diseases over 30 years. Recent studies suggest that silymarin may benefit patients with glucose intolerance. However, the mechanism underlying the action of silymarin is not clarified. The aim of this work was to assess the impact of silymarin on glucose intolerance in high-fat diet (HFD)-fed mice, and explore the potential therapeutic mechanisms. C57BL/6 mice were fed with HFD for 12 weeks, randomized, and treated orally with vehicle saline or silymarin (30?mg/kg) daily for 30 days. We found that silymarin significantly improved HFD-induced body weight gain, glucose intolerance, and insulin resistance in mice. Silymarin treatment reduced HFD-increased oxidative stress indicators (reactive oxygen species, lipid peroxidation, protein oxidation) and restored HFD-down-regulated activities of antioxidant enzymes (superoxide dismutase, catalase, glutathione peroxidase) in the plasma and/or liver of the HFD-fed mice. Furthermore, silymarin decreased HFD-up-regulated hepatic NADPH oxidase expression and NF-κB activation in mice. Additionally, silymarin treatment mitigated HFD-increased plasma IL-1β, TNF-α levels, and HFD-enhanced hepatic NO, TLR4, and iNOS expression in mice. These novel data indicate that silymarin has potent anti-diabetic actions through alleviating oxidative stress and inflammatory response, partially by inhibiting hepatic NADPH oxidase expression and the NF-κB signaling.     

Identification of isotschimgine as a novel farnesoid X receptor agonist with potency for the treatment of obesity in mice

Obesity and its associated non-alcoholic fatty liver disease (NAFLD) have become epidemic medical problems worldwide; however, the current available therapeutic options are limited. Farnesoid X receptor (FXR) has recently emerged as an attractive target for obesity treatment. Here we demonstrate that isotschimgine (ITG), a constituent in genus Ferula, as a novel FXR agonist with anti-obesity and anti-hepatic steatosis effects. The results showed that ITG activated the FXR transactivity and bound with the ligand binding dormain (LBD) of FXR with gene reporter assays and AlphaScreen assays. In high-fat diet-induced obese (DIO) mice, ITG lowered body weight and fat mass, improved insulin resistance and hepatic steatosis. Mechanistic studies showed that ITG altered the expression levels of FXR downstream genes, lipid synthesis and energy metabolism genes in the liver of mice. Our findings suggest that ITG is a novel FXR agonist and may be a potential therapeutic choice for obesity associated with NAFLD.