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.     

Astaxanthin reduces hepatic lipid accumulations in high-fat-fed C57BL/6J mice via activation of peroxisome proliferator-activated receptor (PPAR) alpha and inhibition of PPAR gamma and Akt

We have previously reported that astaxanthin (AX), a dietary carotenoid, directly interacts with peroxisome proliferator-activated receptors PPARα and PPARγ, activating PPARα while inhibiting PPARγ, and thus reduces lipid accumulation in hepatocytes in vitro. To investigate the effects of AX in vivo, high-fat diet (HFD)-fed C57BL/6J mice were orally administered AX (6 or 30 mg/kg body weight) or vehicle for 8 weeks. AX significantly reduced the levels of triglyceride both in plasma and in liver compared with the control HFD mice. AX significantly improved liver histology and thus reduced both steatosis and inflammation scores of livers with hematoxylin and eosin staining. The number of inflammatory macrophages and Kupffer cells were reduced in livers by AX administration assessed with F4/80 staining. Hepatic PPARα-responsive genes involved in fatty acid uptake and β-oxidation were upregulated, whereas inflammatory genes were downregulated by AX administration. In vitro radiolabeled assays revealed that hepatic fatty acid oxidation was induced by AX administration, whereas fatty acid synthesis was not changed in hepatocytes. In mechanism studies, AX inhibited Akt activity and thus decreased SREBP1 phosphorylation and induced Insig-2a expression, both of which delayed nuclear translocation of SREBP1 and subsequent hepatic lipogenesis. Additionally, inhibition of the Akt-mTORC1 signaling axis by AX stimulated hepatic autophagy that could promote degradation of lipid droplets. These suggest that AX lowers hepatic lipid accumulation in HFD-fed mice via multiple mechanisms. In addition to the previously reported differential regulation of PPARα and PPARγ, inhibition of Akt activity and activation of hepatic autophagy reduced hepatic steatosis in mouse livers.     

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.     

Promising role of ferulic acid,atorvastatin and their combination in ameliorating high fat diet-induced stress in mice

AimsThe present study evaluated a comparative and combined hepatoprotective effect of atorvastatin (AS) and ferulic acid (F) against high fat diet (HFD) induced oxidative stress in terms of hyperlipidemia, anti-oxidative status, lipid peroxidation and inflammation.Main methodsMale Swiss albino mice were given a diet containing high fat (H) (23.9% wt/wt), supplemented with AS (10 mg/kg) or F (100 mg/kg) and both (10 and 100 mg/kg) for 8 weeks. The control mice (C) were fed with normal diet.Key findingsThe H mice exhibited increased body weight; hyperlipidemia; serum level of tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6); hepatic lipid profile; lipid accumulation; reactive oxygen species (ROS) of hepatocytes, lipid peroxidation and liver antioxidant capacity was decreased. Immunofluorescent and Western blot assay revealed activation of nuclear factor kappa B (NF-κB) signaling pathway. The addition of F or AS and both in the diet significantly counteracted HFD induced body weight gain; hyperlipidemia; TNF-α, IL-6; hepatic lipid profile; fatty infiltration; NF-κB signaling pathway; ROS; lipid peroxidation and moreover elevated levels of hepatic antioxidant enzymes activity were observed.SignificanceSimultaneous treatment with AS, F and their combination protected against HFD induced weight gain and oxidative stress. The protection may be attributed to the hypolipidemic and free radical scavenging activity of AS or F and their combination. This study illustrates that AS and F have relatively similar hypolipidemic, antioxidative, anti-inflammatory actions and the AS + F combination along with HFD has shown outstanding effects as compared to other treated groups.     

A high-fat diet temporarily renders Sod1-deficient mice resistant to an oxidative insult

Patients with nonalcoholic fatty liver disease may subsequently develop nonalcoholic steatohepatitis after suffering from a second insult, such as oxidative stress. Aim of this study was to investigate the pathogenesis of the liver injury caused when lipids accumulate under conditions of intrinsic oxidative stress using mice that are deficient in superoxide dismutase 1 (SOD1) and the leptin receptor (Lepr). We established Sod1^−/−::Lepr^db/db mice and carried out analyses of four groups of genetically modified mice, namely, wild type, Sod1^−/−, Lepr^db/db and Sod1^−/−::Lepr^db/db mice. Mice with defects in the SOD1 or Lepr gene are vulnerable to developing fatty livers, even when fed a normal diet. Feeding a high-fat diet (HFD) caused an increase in the number of lipid droplets in the liver to different extents in each genotypic mouse. an HFD caused the accelerated death of db/db mice, but contradictory to our expectations, the death rates for the Sod1-deficient mice were decreased by feeding HFD. Consistent with the improved probability of survival, liver damage was significantly ameliorated by feeding an HFD compared to a normal diet in the mice with an Sod1-deficient background. Oxidative stress markers, hyperoxidized peroxiredoxin and lipid peroxidation products, were decreased somewhat in Sod1^−/− mice by feeding HFD. We conclude that lipids reacted with reactive oxygen species and eliminated them in the livers of the young mice, which resulted in the alleviation of oxidative stress, but in advanced age oxidized products accumulated, leading to the aggravation of the liver injury and an increase in fatality rate.     

Endoplasmic reticulum stress in adipose tissue augments lipolysis

The endoplasmic reticulum (ER) is an organelle important for protein synthesis and folding, lipid synthesis and Ca²⁺ homoeostasis. Consequently, ER stress or dysfunction affects numerous cellular processes and has been implicated as a contributing factor in several pathophysiological conditions. Tunicamycin induces ER stress in various cell types in vitro as well as in vivo. In mice, a hallmark of tunicamycin administration is the development of fatty livers within 24–48 hrs accompanied by hepatic ER stress. We hypothesized that tunicamycin would induce ER stress in adipose tissue that would lead to increased lipolysis and subsequently to fatty infiltration of the liver and hepatomegaly. Our results show that intraperitoneal administration of tunicamycin rapidly induced an ER stress response in adipose tissue that correlated with increased circulating free fatty acids (FFAs) and glycerol along with decreased adipose tissue mass and lipid droplet size. Furthermore, we found that in addition to fatty infiltration of the liver as well as hepatomegaly, lipid accumulation was also present in the heart, skeletal muscle and kidney. To corroborate our findings to a clinical setting, we examined adipose tissue from burned patients where increases in lipolysis and the development of fatty livers have been well documented. We found that burned patients displayed significant ER stress within adipose tissue and that ER stress augments lipolysis in cultured human adipocytes. Our results indicate a possible role for ER stress induced lipolysis in adipose tissue as an underlying mechanism contributing to increases in circulating FFAs and fatty infiltration into other organs.     

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.     

Alpha-lipoic acid attenuates methionine choline deficient diet-induced steatohepatitis in C57BL/6 mice

AimsNon-alcoholic steatohepatitis (NASH) is a liver disease that causes fat accumulation, inflammation and fibrosis. Increased oxidative stress contributes to hepatic inflammation and fibrosis by upregulation of Cytochrome P450 2E1 (CYP2E1), endoplasmic reticulum (ER) stress and mitogen-activated protein kinase (MAPK) activity. This study examined whether alpha-lipoic acid (ALA), a naturally occurring thiol antioxidant, prevents steatohepatitis through the inhibition of several pathways involved in hepatic inflammation and fibrosis.Main MethodsC57BL/6 mice were fed an MCD diet with or without ALA for 4 weeks. Liver sections from mice on control or MCD diets with or without ALA were stained with hematoxylin-eosin, oil red O, and anti-4-HNE antibody. The effects of ALA on methionine-choline deficient MCD-diet induced plasma AST and ALT as well as tissue TBARS were measured. The effects of ALA on CYP2E1 expression, ER stress, MAPK levels, and NF-κB activity in MCD diet-fed mice liver were measured by northern and western blot analysis.Key findingsDietary supplementation with ALA reduced MCD diet-induced hepatic lipid accumulation, hepatic inflammation, TBARS, 4-HNE, and plasma ALT and AST levels. These effects were associated with a reduced expression of CYP2E1 and reduced ER stress and MAPK and NF-κB activity.SignificanceTaken together, the results of the present study indicate that ALA attenuates steatohepatitis through inhibition of several pathways, and provide the possibility that ALA can be used to prevent the development and progression of non-alcoholic fatty liver disease in patients who have strong risk factors for NASH.     

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.     

Mesenteric adipose tissue B lymphocytes promote local and hepatic inflammation in non‐alcoholic fatty liver disease mice

Mesenteric adipose tissue (MAT) inflammation is associated with non‐alcoholic fatty liver disease (NAFLD), and immune cells play pivotal roles in the inflammation of adipose tissue. Here, we investigated the roles of MAT B lymphocytes in NAFLD. Mice fed with high‐fat diet (HFD) and normal diet (ND) were killed in time gradients (4, 8 and 12 weeks). Compared with ND‐fed mice, intra‐hepatic CD45⁺CD19⁺ B lymphocytes increased after 4 weeks (P < 0.01) of HFD feeding, and lasted until the 12th week, infiltrated earlier than CD45⁺CD3⁺ T lymphocytes and CD45⁺F4/80⁺ macrophages. The mRNA expression of tumour necrosis factor (TNF)‐α, interleukin (IL)‐6 and monocyte chemotactic protein (MCP)‐1 decreased in MAT of B^null HFD‐fed mice compared to that in wild‐type HFD‐fed mice, along with lesser macrophages. Mesenteric adipose tissue B cells from HFD‐fed mice promoted macrophage differentiation to type‐Ι macrophages and expression of pro‐inflammatory cytokines in vitro. Macrophages pre‐treated with MAT B cells from HFD‐fed mice showed elevated mRNA expression of IL‐6 and TNF‐α and declined IL‐10 levels in adipocytes compared to ND MAT B cell pre‐treated macrophages. Besides, internal near‐infrared scanning and external transwell assay showed that HFD MAT B cells migrated to the liver more than ND MAT B cells. High‐fat diet MAT B cells induced higher MCP‐1 and lower IL‐10 expression in primary hepatocytes compared to ND MAT B cells in co‐culture experiment. These data indicate that B lymphocytes infiltrate early in MAT during the development of NAFLD, which may not only promote MAT inflammation by regulating macrophages but also migrate to the liver and induce hepatocytes inflammation.     

Chitosan reduces inflammation and protects against oxidative stress in a hyperlipidemic rat model: relevance to nonalcoholic fatty liver disease

Background

An altered lipid profile may lead to the development of inflammation and NAFLD (Non-alcoholic fatty liver disease). Although statins have a positive effect on blood lipid levels their long-term use is known to cause adverse effects, in this backdrop there is an interest in natural compounds which may affect lipid metabolism and prevent NAFLD. We have examined the effect of Chitosan on rats subjected to a high-fat diet.

Methods and results

Male Wistar middle aged rats (12–16 months) were treated with high-fat diet orally for two months for creating a NAFLD model. Rats were also supplemented with Chitosan (2% chitosan daily) for 2 months. We assessed the activity of antioxidant enzymes, the histopathological profile of the liver. Inflammatory cytokines and adiponectin levels were also measured in serum. HFD induced significant changes in liver tissue and inflammatory markers (Il-6, TNF- alpha, NF-KB). Chitosan treatment protected rats from HFD induced alterations.

Conclusions

The findings suggest that Chitosan can effectively improve liver lipid metabolism by normalizing cholesterol, triglyceride, lowering NF-KB expression, and protecting the liver from oxidative stress by improving hepatic function. Chitosan also regulates genes related to lipidemic stress i,e leptin and adiponectin.

     

Tissue-dependent preventive effect of metallothionein against DNA damage in dyslipidemic mice under repeated stresses of fasting or restraint

AimsTo investigate the effect of repeated stress on DNA damage in seven organs of dyslipidemic mice, and the preventive role of metallothionein (MT).Main methodsFemale adult 129/Sv wild-type and MT-null mice fed high-fat diet (HFD) were repeatedly subjected to mild stress of fasting or restraint in weeks 2 to 4 of 4-week study period. Serum cholesterol level, DNA damage in the liver, pancreas, spleen, bone marrow, kidney, lung and gastric mucosa, and other parameters were determined.Key findingsBody weights were increased in both types of mice fed HFD compared to those fed standard diet (STD), and further increased by 12 h-fasting, while they were markedly decreased by 1–3 h-restraint. Fasting accelerated accumulation of fat in the liver, and increase in serum cholesterol of both types of mice fed HFD. Feeding of HFD increased DNA damage in the pancreas, spleen and bone marrow of both types of mice, compared with those fed STD. In the wild-type mice fed HFD, 24 h-fasting increased DNA damage in the liver and spleen, while restraint increased the damage in the liver, pancreas, spleen and bone marrow. DNA damage in the cells of organs was markedly increased in the MT-null mice. Specifically, damage in the liver, pancreas, spleen and bone marrow was greatly increased with the intensity of stress increased, and the damage was much greater in the restraint mice than in the fasting mice.SignificanceMT plays a tissue-dependent preventive role against DNA damage in various murine organs induced by repeated stress.     

Mitochondrial DNA Damage and Dysfunction,and Oxidative Stress Are Associated with Endoplasmic Reticulum Stress,Protein Degradation and Apoptosis in High Fat Diet-Induced Insulin Resistance Mice

Background

Recent studies showed a link between a high fat diet (HFD)-induced obesity and lipid accumulation in non-adipose tissues, such as skeletal muscle and liver, and insulin resistance (IR). Although the mechanisms responsible for IR in those tissues are different, oxidative stress and mitochondrial dysfunction have been implicated in the disease process. We tested the hypothesis that HFD induced mitochondrial DNA (mtDNA) damage and that this damage is associated with mitochondrial dysfunction, oxidative stress, and induction of markers of endoplasmic reticulum (ER) stress, protein degradation and apoptosis in skeletal muscle and liver in a mouse model of obesity-induced IR.

Methodology/Principal Findings

C57BL/6J male mice were fed either a HFD (60% fat) or normal chow (NC) (10% fat) for 16 weeks. We found that HFD-induced IR correlated with increased mtDNA damage, mitochondrial dysfunction and markers of oxidative stress in skeletal muscle and liver. Also, a HFD causes a change in the expression level of DNA repair enzymes in both nuclei and mitochondria in skeletal muscle and liver. Furthermore, a HFD leads to activation of ER stress, protein degradation and apoptosis in skeletal muscle and liver, and significantly reduced the content of two major proteins involved in insulin signaling, Akt and IRS-1 in skeletal muscle, and Akt in liver. Basal p-Akt level was not significantly influenced by HFD feeding in skeletal muscle and liver.

Conclusions/Significance

This study provides new evidence that HFD-induced mtDNA damage correlates with mitochondrial dysfunction and increased oxidative stress in skeletal muscle and liver, which is associated with the induction of markers of ER stress, protein degradation and apoptosis.     

Hepatic deficiency of selenoprotein S exacerbates hepatic steatosis and insulin resistance

Nonalcoholic fatty liver disease (NAFLD) is closely associated with insulin resistance (IR) and type 2 diabetes mellitus (T2DM), which are all complex metabolic disorders. Selenoprotein S (SelS) is an endoplasmic reticulum (ER) resident selenoprotein involved in regulating ER stress and has been found to participate in the occurrence and development of IR and T2DM. However, the potential role and mechanism of SelS in NAFLD remains unclear. Here, we analyzed SelS expression in the liver of high-fat diet (HFD)-fed mice and obese T2DM model (db/db) mice and generated hepatocyte-specific SelS knockout (SelS^H-KO) mice using the Cre-loxP system. We showed that hepatic SelS expression levels were significantly downregulated in HFD-fed mice and db/db mice. Hepatic SelS deficiency markedly increased ER stress markers in the liver and caused hepatic steatosis via increased fatty acid uptake and reduced fatty acid oxidation. Impaired insulin signaling was detected in the liver of SelS^H-KO mice with decreased phosphorylation levels of insulin receptor substrate 1 (IRS1) and protein kinase B (PKB/Akt), which ultimately led to disturbed glucose homeostasis. Meanwhile, our results showed hepatic protein kinase Cɛ (PKCɛ) activation participated in the negative regulation of insulin signaling in SelS^H-KO mice. Moreover, the inhibitory effect of SelS on hepatic steatosis and IR was confirmed by SelS overexpression in primary hepatocytes in vitro. Thus, we conclude that hepatic SelS plays a key role in regulating hepatic lipid accumulation and insulin action, suggesting that SelS may be a potential intervention target for the prevention and treatment of NAFLD and T2DM.Subject terms: Metabolic syndrome, Obesity     

N-3 PUFAs inhibited hepatic ER stress induced by feeding of a high-saturated fat diet accompanied by the expression LOX-1

Excessive consumption of saturated fat leads to non-alcoholic fatty liver disease (NAFLD), which is attenuated by supplementation of n-3 polyunsaturated fatty acids (PUFAs). Endoplasmic reticulum (ER) stress is crucial in the development of NAFLD, but how high-saturated fat diet (HFD) causes ER stress and NAFLD remains unclear. Lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) is involved in hepatic ER stress. We aimed to explore the roles of LOX-1 in HFD-induced ER stress. Male Sprague–Dawley rats were fed an HFD without or with supplementation of fish oil for 16 weeks. The effects of n-3 PUFAs on hepatic ER stress degrees and the expression levels of LOX-1 were examined. Then human L02 hepatoma cells were treated with palmitate or palmitate and DHA to determine the ER stress and LOX-1 expression levels in vitro. After that the expression of LOX-1 in L02 cells was either knocked-down or overexpressed to analyze the roles of LOX-1 in palmitate-induced ER stress. The feeding of HFD induced NAFLD development and ER stress in the liver, and LOX-1 expressing level, which were all reversed by fish oil supplementation. In vitro, DHA treatment reduced the expression of LOX-1, and palmitate-induced ER stress. SiRNA-mediated knock-down of LOX-1 inhibited palmitate-induced ER stress, whereas overexpression of LOX-1 dramatically induced ER stress in L02 cells.LOX-1 is critical for HFD-induced ER stress, and inhibition of its expression under the treatment of n-3 PUFAs could ameliorate HFD-induced NAFLD.