Pirfenidone attenuates nonalcoholic fatty liver disease through activation of the nuclear factor erythroid 2-related factor 2 (Nrf2) signaling pathway

Nonalcoholic fatty liver disease (NAFLD) originates from the hepatopathy of fatty liver. Pirfenidone is a novel broad-spectrum anti-fibrosis agent used for treating various kinds of tissue fibrosis. The present study will evaluate the effects of Pirfenidone on liver injury in high-fat diet (HFD)-fed mice to evaluate the value of Pirfenidone in treating NAFLD. The pathology of NAFLD was simulated by feeding mice with an HFD in the present study, followed by treating the HFD mice with 150 and 300 mg/kg/day Pirfenidone once a day. The pathological state of HFD mice was identified by the elevated liver weight, promoted serum triglyceride (TG), total cholesterol (TC), and low-density lipoprotein cholesterol (LDL-C) levels, declined serum high-density lipoprotein cholesterol (HDL-C) levels, increased alanine aminotransferase and aspartate aminotransferase activity, and histopathological changes to the liver tissues, all of which were dramatically ameliorated by 150 and 300 mg/kg Pirfenidone administration. Furthermore, the excessive production of interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), and IL-6, as well as upregulated phosphorylated nuclear factor kappa-B (p- NF-κB p65), were observed in HFD-fed mice, but significantly reversed by Pirfenidone. Finally, activated oxidative stress, identified by promoted malondialdehyde (MDA) levels and declined catalase (CAT) activity, was observed in HFD-fed mice, accompanied by the downregulation of nuclear factor erythroid 2-related factor 2 (Nrf2) and sterol-regulatory element-binding proteins-1c (SREBP-1c). After the treatment with Pirfenidone, oxidative stress was greatly mitigated. Our results imply that Pirfenidone ameliorated the progression of NAFLD by mediating inflammation and oxidative stress.     

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.     

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.     

Mechanisms underlying different responses of plasma triglyceride to high-fat diets in hamsters and mice: Roles of hepatic MTP and triglyceride secretion

Hypertriglyceridemia, closely associated with insulin resistance, is induced on high-fat diets (HFD) in humans but not in mouse models. Mechanisms underlying this species difference are still unclear. Hamsters resemble humans in lipoprotein metabolism. Here by comparing the responses to HFD in hamsters and mice, we found that hepatic TG secretion, MTP expression and plasma free fatty acid (FFA) level were increased in hamsters on HFD feeding but decreased in mice. Although hepatic steatosis and de novo lipogenesis were induced by HFD feeding in both models, cholesterol biosynthesis was inhibited in mice but not in hamsters. Moreover, in insulin deficient state, HFD increased plasma TG level, hepatic TG secretion, MTP expression and plasma FFA level in both models. In summary, distinct changes of MTP expression, in correlation with hepatic TG secretion, underlie the opposite responses of plasma TG levels to high-fat diets in hamsters and mice. Furthermore, hepatic TG secretion and MTP expression seems to be associated with plasma FFA level and cholesterol biosynthesis but not hepatic steatosis or de novo lipogenesis.     

Targeting the gut microbiota with resveratrol: a demonstration of novel evidence for the management of hepatic steatosis

Resveratrol is a natural polyphenol that has been reported to reduce the risk of obesity and nonalcoholic fatty liver disease (NAFLD). Recent evidence has demonstrated that the gut microbiota plays an important role in the protection against NAFLD and other metabolic diseases. The present study aimed to investigate the relationship between the gut microbiota and the beneficial effects of resveratrol on the amelioration of NAFLD in mice. We observed marked decreases in body weight and liver steatosis and improved insulin resistance in high-fat diet (HFD)-fed mice treated with resveratrol. Furthermore, we found that resveratrol treatment alleviated NAFLD in HFD-fed mice by improving the intestinal microenvironment, including gut barrier function and gut microbiota composition. On the one hand, resveratrol improved gut intestinal barrier integrity through the repair of intestinal mucosal morphology and increased the expression of physical barrier- and physiochemical barrier-related factors in HFD-fed mice. On the other hand, in HFD-fed mice, resveratrol supplementation modulated the gut bacterial composition. The resveratrol-induced gut microbiota was characterized by a decreased abundance of harmful bacteria, including Desulfovibrio, Lachnospiraceae_NK4A316_group and Alistipes, as well as an increased abundance of short-chain fatty acid (SCFA)-producing bacteria, such as Allobaculum, Bacteroides and Blautia. Moreover, transplantation of the HFDR-microbiota into HFD-fed mice sufficiently decreased body weight, liver steatosis and low-grade inflammation and improved hepatic lipid metabolism. Collectively, resveratrol would provide a potentially dietary intervention strategy against NAFLD through modulating the intestinal microenvironment.     

Short-term high-fat diet intake leads to exacerbation of concanavalin A-induced liver injury through the induction of procoagulation state

Obesity and high-fat diet (HFD) are known to cause proinflammatory and procoagulation states and suggested to become a risk of developing thromboembolic diseases. Non-alcoholic fatty liver disease (NAFLD) is usually associated with obesity and HFD, and a part of NAFLD is known to progress to nonalcoholic steatohepatitis (NASH), the pathogenesis of which has not been fully elucidated. In the current study, we examined the influence of short-term HFD on hepatic expression of the molecules related to inflammation, coagulation, metabolism, and cellular stresses from the perspective that HFD itself can be a risk for the development to NASH. In the analysis in short-term (4 days to 14 days) HFD-fed mice, we found out that HFD increased hepatic expression of IFN-γ, TNF-α, IL-10, monocyte chemotactic protein-1 (MCP-1), tissue factor (TF), plasminogen activator inhibitor-1 (PAI-1) mRNAs, and fibrin/fibrinogen deposition in the liver tissues. And it was suggested that metabolic alterations and endoplasmic reticulum (ER) stresses induced by the HFD intake were associated with this proinflammatory and procoagulation states. When we administered concanavalin A (Con A) to these HFD-fed mice, the extent of liver injury was dramatically exacerbated in HFD-fed mice. Heparin treatment to Con A-administered, HFD-fed mice (for 4 days) profoundly ameliorated the extent of liver injury. These suggest that even short-term of HFD intake induces proinflammatory and procoagulation states in the liver and thereby increases the susceptibility of the liver to circulating inflammatory stimuli. We think that it may explain a part of NASH pathogenesis.     

GCN2 deficiency protects against high fat diet induced hepatic steatosis and insulin resistance in mice

Nonalcoholic fatty liver disease (NAFLD) is characterized by hepatic lipid deposition and oxidative stress. It has been demonstrated that general control nonderepressible 2 (GCN2) is required to maintain hepatic fatty acid homeostasis under conditions of amino acid deprivation. However, the impact of GCN2 on the development of NAFLD has not been investigated. In this study, we used Gcn2^?/? mice to investigate the effect of GCN2 on high fat diet (HFD)-induced hepatic steatosis. After HFD feeding for 12?weeks, Gcn2^?/? mice were less obese than wild-type (WT) mice, and Gcn2^?/? significantly attenuated HFD-induced liver dysfunction, hepatic steatosis and insulin resistance. In the livers of the HFD-fed mice, GCN2 deficiency resulted in higher levels of lipolysis genes, lower expression of genes related to FA synthesis, transport and lipogenesis, and less induction of oxidative stress. Furthermore, we found that knockdown of GCN2 attenuated, whereas overexpression of GCN2 exacerbated, palmitic acid-induced steatosis, oxidative & ER stress, and changes of peroxisome proliferator-activated receptor gamma (PPARγ), fatty acid synthase (FAS) and metallothionein (MT) expression in HepG2 cells. Collectively, our data provide evidences that GCN2 deficiency protects against HFD-induced hepatic steatosis by inhibiting lipogenesis and reducing oxidative stress. Our findings suggest that strategies to inhibit GCN2 activity in the liver may provide a novel approach to attenuate NAFLD development.     

乙酰辅酶A羧化酶抑制剂联合非诺贝特对小鼠非酒精性脂肪肝的治疗效果

摘要 目的：探讨乙酰辅酶A羧化酶抑制剂（MK-4074）联合非诺贝特对小鼠非酒精性脂肪肝（NAFLD）的脂质含量以及肝功能的改善效果。方法：20只C57BL/6小鼠给予60%高脂饲料连续喂养8周构建NAFLD小鼠模型后，随机分为安慰剂组、MK-4074组、非诺贝特组以及MK-4074联合非诺贝特治疗组，每组各5只，继续高脂喂养并分别给予安慰剂（Placebo）、MK-4074（10 mg/kg/天）、非诺贝特（30 mg/kg/天）、以及MK-4074（10 mg/kg/天）+ 非诺贝特（30 mg/kg/天）治疗持续8周。治疗结束后对小鼠体重、肝指数、肝脏脂质含量、肝功能以及肝脏病理和肝脏中性粒细胞和巨噬细胞浸润情况进行分析。结果：与安慰剂组相比，单用MK-4074治疗可显著降低肝指数、肝脏甘油三酯（TG）、胆固醇（TC）、非酯化脂肪酸（NEFA）的含量以及血清ALT和AST水平，而对小鼠体重和血清TC没有显著影响；单用非诺贝特可显著降低小鼠体重，肝脏TG、TC、NEFA以及血清TG、 ALT和AST水平，对小鼠的肝指数、血清TC没有显著影响；而MK-4074与非诺贝特联合治疗可显著降低小鼠体重、肝脏TG、TC、NEFA，以及血清TG、ALT和AST水平，降低肝脏脂质积累以及中性粒细胞与巨噬细胞浸润，效果优于MK-4074或非诺贝特单药治疗。结论：MK-4074联合非诺贝特可显著减少NAFLD小鼠肝脏的脂质含量，改善肝功能。     

S100A16, a novel lipogenesis promoting factor in livers of mice and hepatocytes in vitro

To investigate the role of S100 calcium-binding protein A16 (S100A16) in hepatic lipid metabolism, S100a16 transgenic, S100a16 knockdown, and wildtype C57BL/6 mice were fed either a high-fat diet (HFD) or normal-fat diet (NFD) for 16 weeks. The results showed that for HFD-fed mice, S100a16 transgenic mice showed significantly more severe fatty liver than other HFD-fed mice, with a significant increase in serum triglyceride (TG) concentration, with more and larger lipid droplets in the liver, whereas S100a16 knockdown mice were completely opposite, with liver fat lesions and TG serological changes being the mildest; for NFD-fed mice, liver fat accumulation and serum TG concentrations were significantly lower than those fed HFD, and no significant lipid droplets were found in the liver. Further, we found that calmodulin (CaM) interacts with S100A16, a member of the AMP-activated protein kinase (AMPK) pathway. Our research found that S100A16 regulates the AMPK pathway-associated protein by interacting with CaM to regulate liver lipid synthesis. S100A16 regulates liver lipid metabolism through the CaM/CAMKK2/AMPK pathway. Overexpression of S100A16 promotes the deterioration of fatty liver induced by HFD, and low expression of S100A16 can attenuate fatty liver.     

Macrophage-specific MyD88 deletion and pharmacological inhibition prevents liver damage in non-alcoholic fatty liver disease via reducing inflammatory response

Activation of the innate immune system through toll-like receptors (TLRs) has been repeatedly demonstrated in non-alcoholic fatty liver disease (NAFLD) and several TLRs have been shown to contribute. Myeloid differentiation primary response 88 (MyD88) is as an adapter protein for the activation of TLRs and bridges TLRs to NF-κB-mediated inflammation in macrophages. However, whether myeloid cell MyD88 contributes to NAFLD are largely unknown. To test this approach, we generated macrophage-specific MyD88 knockout mice and show that these mice are protected against high-fat diet (HFD)-induced hepatic injury, lipid accumulation, and fibrosis. These protective effects were associated with reduced macrophage numbers in liver tissues and surpassed inflammatory responses. In cultured macrophages, saturated fatty acid palmitate utilizes MyD88 to activate NF-κB and induce inflammatory and fibrogenic factors. In hepatocytes, these factors may cause lipid accumulation and a further elaboration of inflammatory cytokines. In hepatic stellate cells, macrophage-derived factors, especially TGF-β, cause activation and hepatic fibrosis. We further show that pharmacological inhibition of MyD88 is also able to reduce NAFLD injury in HFD-fed mice. Therefore, our study has provided empirical evidence that macrophage MyD88 participates in HFD-induced NAFLD and could be targeted to prevent the development and progression of NAFLD/NASH.     

Combination of metformin and genistein alleviates non-alcoholic fatty liver disease in high-fat diet-fed mice

Metformin (MET) and genistein (GEN) have a beneficial role in alleviating non-alcoholic fatty liver disease (NAFLD), but their combined effect on this disease has not yet been studied. The present study aimed to investigate the potential protective effects of combined MET and GEN on NAFLD in high-fat diet (HFD) fed mice. C57BL/6 male mice were fed on an HFD for 10 weeks. Animals were then divided into different groups and treated with MET (0.23%), GEN (0.2%) and MET+GEN (0.23% + 0.2%) for 3 months. Treatment with MET and GEN, alone or in combination significantly lowered body and liver weights and fasting blood glucose (FBG) in HFD mice. Combination therapy reduced liver triglyceride (TG) level and this effect was correlated with increased expression of carnitine palmitoyl transferase 1 (CPT1) gene, and reduced expression of fatty-acid synthase (FAS)and sterol regulatory element-binding protein-1c (SREBP-1c) genes. Combination therapy also affects gluconeogenesis pathway through decreasing expression of Glucose 6-phosphatase (G6Pase) and increasing phosphorylation of Glycogen synthase kinase 3β (GSK-3β). Furthermore, combination of MET and GEN ameliorates liver inflammation by switching macrophage into M2 phenotype, decreasing macrophage infiltration, reducing expression of pro-inflammatory cytokines and decreasing nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) activity. In addition, combination therapy enhances phosphorylation of 5′ adenosine monophosphate-activated protein kinase (AMPK). Taken together, these findings suggest that the combination of MET and GEN have beneficial effects against NAFLD in HFD-fed model.     

Capsaicin suppresses liver fat accumulation in high-fat diet-induced NAFLD mice

ABSTRACT

Dietary capsaicin exhibits anti-steatosis activity in obese mice. High-fat diet (HFD)-induced mice is a highly studied approach to develop non-alcoholic fatty liver disease (NAFLD). In this study, we determined whether the topical application of capsaicin can improve lesions of NAFLD. The HFD-induced mice were treated with daily topical application of capsaicin for 8 weeks. Topical application of capsaicin reduced liver fat in HFD-fed mice. Capsaicin stimulated carnitine palmitoyl transferase (CPT)-1 and CD36 expression, which are associated with β-oxidation and fatty acids influx of liver while it decreased the expression of key enzymes involved in the synthesis of fatty acids, such as acetyl Co-A carboxylase (ACC) and fatty acid synthase (FAS). Immunohistochemical analysis revealed the elevated level of adiponectin in liver tissue of the capsaicin-treated mice. These results suggest that the topical application of capsaicin suppresses liver fat accumulation through the upregulation of β-oxidation and de novo lipogenesis in HFD-induced NAFLD mice.     

Nobiletin alleviates high-fat diet-induced nonalcoholic fatty liver disease by modulating AdipoR1 and gp91phox expression in rats

Nobiletin, one of the polymethoxylated flavonoids isolated from citrus peels, is reported to possess various biological activities. The current study investigates the effect and possible mechanisms of nobiletin on nonalcoholic fatty liver disease (NAFLD) in high-fat diet (HFD)-fed rats. Male Sprague-Dawley rats were administrated with HFD and fructose (15%) in drinking water for 16 weeks to induce NAFLD. HFD-fed rats were treated with nobiletin (20 or 40 mg/kg/day) or vehicle for the last 4 weeks. Treatment of HFD-fed rats with nobiletin significantly reduced systolic blood pressure, adiposity, hyperlipidemia, insulin resistance, hepatic lipids content, NAFLD activity score and liver fibrosis. Nobiletin significantly increased plasma adiponectin levels, together with up-regulation of liver adiponectin receptor 1 (AdipoR1) expression. Additionally, decreased malondialdehyde levels and increased superoxide dismutase activity in plasma and hepatic tissue, consistent with down-regulation of liver NADPH oxidase subunit gp91^phox expression, were also observed after nobiletin treatment. Furthermore, high dose of nobiletin exhibited higher therapeutic effect as a compared to low dose. These findings suggest that nobiletin alleviates HFD-induced NAFLD and metabolic dysfunction in rats. There might be an association between the observed inhibitory effect of nobiletin on NAFLD and modulation of AdipoR1 and gp91^phox.     

A moderate increase in carnitine palmitoyltransferase 1a activity is sufficient to substantially reduce hepatic triglyceride levels

Nonalcoholic fatty liver disease (NAFLD), hypertriglyceridemia, and elevated free fatty acids are present in the majority of patients with metabolic syndrome and type 2 diabetes mellitus and are strongly associated with hepatic insulin resistance. In the current study, we tested the hypothesis that an increased rate of fatty acid oxidation in liver would prevent the potentially harmful effects of fatty acid elevation, including hepatic triglyceride (TG) accumulation and elevated TG secretion. Primary rat hepatocytes were transduced with adenovirus encoding carnitine palmitoyltransferase 1a (Adv-CPT-1a) or control adenoviruses encoding either beta-galactosidase (Adv-beta-gal) or carnitine palmitoyltransferase 2 (Adv-CPT-2). Overexpression of CPT-1a increased the rate of beta-oxidation and ketogenesis by approximately 70%, whereas esterification of exogenous fatty acids and de novo lipogenesis were unchanged. Importantly, CPT-1a overexpression was accompanied by a 35% reduction in TG accumulation and a 60% decrease in TG secretion by hepatocytes. There were no changes in secretion of apolipoprotein B (apoB), suggesting the synthesis of smaller, less atherogenic VLDL particles. To evaluate the effect of increasing hepatic CPT-1a activity in vivo, we injected lean or obese male rats with Adv-CPT-1a, Adv-beta-gal, or Adv-CPT-2. Hepatic CPT-1a activity was increased by approximately 46%, and the rate of fatty acid oxidation was increased by approximately 44% in lean and approximately 36% in obese CPT-1a-overexpressing animals compared with Adv-CPT-2- or Adv-beta-gal-treated rats. Similar to observations in vitro, liver TG content was reduced by approximately 37% (lean) and approximately 69% (obese) by this in vivo intervention. We conclude that a moderate stimulation of fatty acid oxidation achieved by an increase in CPT-1a activity is sufficient to substantially reduce hepatic TG accumulation both in vitro and in vivo. Therefore, interventions that increase CPT-1a activity could have potential benefits in the treatment of NAFLD.     

Short-term high fat feeding increases organ injury and mortality after polymicrobial sepsis

The purpose of this study was to examine the effect of short-term high fat feeding on the inflammatory response in polymicrobial sepsis. Male C57BL/6 mice at 6 weeks of age were randomized to a high-fat diet (HFD) (60% kcal fat) or control diet (CD) (16% kcal fat) for 3 weeks. After 3 weeks of feeding, sepsis was induced by cecal ligation and puncture (CLP) and animals were monitored for survival. In a separate experiment, after 3 weeks of feeding mice underwent CLP and were sacrificed at various time points thereafter. Tissue was collected for biochemical studies. Mice fed a HFD gained more weight and had a greater fat mass compared to CD-fed mice. Mice on a HFD had a lower probability of survival and more severe lung injury compared with CD-fed mice following sepsis. Myeloperoxidase (MPO) activity, an indicator of neutrophil infiltration, was increased in the lung and liver after CLP in HFD-fed mice compared with CD (P < 0.05). The plasma cytokines tumor necrosis factor-α (TNF-α) and interleukin (IL)-6 were increased in both groups after CLP, however, TNF-α and IL-6 levels were lower in HFD mice at 3 h after CLP compared with CD and consistent with lung, but not liver, messenger RNA (mRNA) expression. Leptin levels were higher in HFD-fed mice at 18 h after sepsis compared to baseline levels (P < 0.05). Polymicrobial sepsis increased hepatic nuclear factor-κB (NF-κB) activation in HFD-fed mice after CLP vs. CD-fed mice. Short duration high fat feeding increases mortality and organ injury following polymicrobial sepsis. These effects correspond to changes in NF-κB.     

Intestinal acyl-CoA:diacylglycerol acyltransferase 2 overexpression enhances postprandial triglyceridemic response and exacerbates high fat diet-induced hepatic triacylglycerol storage

Intestinal acyl-CoA:diacylglycerol acyltransferase 2 (DGAT2) is important in the cellular and physiological responses to dietary fat. To determine the effect of increased intestinal DGAT2 on cellular and physiological responses to acute and chronic dietary fat challenges, we generated mice with intestine-specific overexpression of DGAT2 and compared them with intestine-specific overexpression of DGAT1 and wild-type (WT) mice. We found that when intestinal DGAT2 is present in excess, triacylglycerol (TG) secretion from enterocytes is enhanced compared to WT mice; however, TG storage within enterocytes is similar compared to WT mice. We found that when intestinal DGAT2 is present in excess, mRNA levels of genes involved in fatty acid oxidation were reduced. This result suggests that reduced fatty acid oxidation may contribute to increased TG secretion by overexpression of DGAT2 in intestine. Furthermore, this enhanced supply of TG for secretion in Dgat2^Int mice may be a significant contributing factor to the elevated fasting plasma TG and exacerbated hepatic TG storage in response to a chronic HFD. These results highlight that altering fatty acid and TG metabolism within enterocytes has the capacity to alter systemic delivery of dietary fat and may serve as an effective target for preventing and treating metabolic diseases such as hepatic steatosis.