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.     

β-Cryptoxanthin Alleviates Diet-Induced Nonalcoholic Steatohepatitis by Suppressing Inflammatory Gene Expression in Mice

Recent nutritional epidemiological surveys showed that serum β-cryptoxanthin inversely associates with the risks for insulin resistance and liver dysfunction. Consumption of β-cryptoxanthin possibly prevents nonalcoholic steatohepatitis (NASH), which is suggested to be caused by insulin resistance and oxidative stress from nonalcoholic fatty liver disease. To evaluate the effect of β-cryptoxanthin on diet-induced NASH, we fed a high-cholesterol and high-fat diet (CL diet) with or without 0.003% β-cryptoxanthin to C56BL/6J mice for 12 weeks. After feeding, β-cryptoxanthin attenuated fat accumulation, increases in Kupffer and activated stellate cells, and fibrosis in CL diet-induced NASH in the mice. Comprehensive gene expression analysis showed that although β-cryptoxanthin histochemically reduced steatosis, it was more effective in inhibiting inflammatory gene expression change in NASH. β-Cryptoxanthin reduced the alteration of expression of genes associated with cell death, inflammatory responses, infiltration and activation of macrophages and other leukocytes, quantity of T cells, and free radical scavenging. However, it showed little effect on the expression of genes related to cholesterol and other lipid metabolism. The expression of markers of M1 and M2 macrophages, T helper cells, and cytotoxic T cells was significantly induced in NASH and reduced by β-cryptoxanthin. β-Cryptoxanthin suppressed the expression of lipopolysaccharide (LPS)-inducible and/or TNFα-inducible genes in NASH. Increased levels of the oxidative stress marker thiobarbituric acid reactive substances (TBARS) were reduced by β-cryptoxanthin in NASH. Thus, β-cryptoxanthin suppresses inflammation and the resulting fibrosis probably by primarily suppressing the increase and activation of macrophages and other immune cells. Reducing oxidative stress is likely to be a major mechanism of inflammation and injury suppression in the livers of mice with NASH.     

Direct monitoring of in vivo ER stress during the development of insulin resistance with ER stress-activated indicator transgenic mice

Type 2 diabetes is one of the most prevalent and serious metabolic diseases in the world, and insulin resistance and pancreatic β-cell dysfunction are the hallmarks of the disease. It has been suggested that endoplasmic reticulum (ER) stress is provoked under diabetic conditions and is possibly involved in the development of insulin resistance. In this study, using ER stress-activated indicator (ERAI) transgenic mice which express green fluorescent protein (GFP) under ER stress conditions, we directly monitored in vivo ER stress in various insulin target tissues such as liver, fat, and muscle in diabetic mice with insulin resistance induced by high fat and high sucrose (HF/HS) diet treatment. In the liver of the ERAI transgenic mice, ERAI fluorescence activity was clearly observed as early as after 4 weeks of HF/HS diet treatment, whereas it was not detected at all in the fat and muscle even after 12 weeks of HF/HS diet treatment. These results suggest that induction of ER stress is associated with the development of insulin resistance and that ER stress in the liver may facilitate the development of insulin resistance in the whole body. This is the first report to directly monitor in vivo ER stress in various insulin target tissues during the development of insulin resistance. In addition, our present results suggest that ERAI transgenic mice are very useful for evaluating in vivo ER stress, especially in the liver, during the development of insulin resistance.     

Network pharmacology-based identification of significant pathway for protection of Yinhuang granule in a mice model of metabolic-associated fatty liver disease

BackgroundMetabolic-associated fatty liver disease (MAFLD) is a spectrum of liver disorders. Nonalcoholic steatohepatitis (NASH) is defined as a more serious process of MAFLD with liver inflammation.PurposeThis study aims to observe the alleviation of Yinhuang granule (YHG), a Chinese patent medicine, on methionine and choline-deficient diet (MCD)-induced MAFLD in mice.MethodsNetwork pharmacology was used to analyze the improving effect of YHG on MAFLD and possible targets. MAFLD was induced in mice by MCD diet feeding for 6 weeks. In the last 2 weeks, the mice were orally given with YHG (400, 800 mg/kg) every day. Biochemical parameters of serum and liver, as well as hepatic gene expression were detected.ResultsNetwork pharmacology showed that YHG could improve MAFLD, inflammation, liver fibrosis, and oxidative stress. In animal experiments, YHG reduced hepatocellular damage and hepatic lipids accumulation which induced by MCD. In terms of liver inflammation, YHG attenuated MCD-induced liver inflammation in mice. YHG also inhibited the activation of hepatic stellate cells (HSCs) and alleviated liver fibrosis in MCD-fed mice. Through nuclear factor erythroid 2-related factor 2 (Nrf2) signaling pathway, YHG alleviated liver oxidative stress injury in mice which induced by MCD.ConclusionYHG ameliorated MCD-induced MAFLD in mice by reducing hepatic lipids accumulation, alleviating liver oxidative, inflammatory injury and attenuating hepatic fibrosis.     

Alleviation of Liver Dysfunction,Oxidative Stress,and Inflammation Underlines the Protective Effects of Polysaccharides from Cordyceps cicadae on High Sugar/High Fat Diet-Induced Metabolic Syndrome in Rats

This study investigated the protective effects of two polysaccharides (CPA-1 and CPB-2) from Cordyceps cicadae against high fructose/high fat diet (HF/HFD) induced obesity and metabolic disorders in rats. Rats were either fed with normal diet or HF/HFD and treated with CPA-1 and CPB-2 (100 and 300 mg/kg) for 11 weeks. Administration of CPA-1 and CPB-2 significantly and dose dependently reduced body and liver weight, insulin and glucose tolerance, serum insulin and glucose levels. Furthermore, serum and hepatic lipid profiles, liver function enzymes and proinflammatory cytokines (TNF-α, IL-1β and IL-6) were markedly reduced. Additionally, CPA-1 and CPB-2 treatment alleviated hepatic oxidative stress by reducing lipid peroxidation level (MDA) and upregulating glutathione peroxidase (GSH-Px), superoxide dismutase (SOD) and catalase (CAT) activities as well as ameliorated histological alterations through the reduction of hepatic lipid accumulation. These results suggested that the polysaccharides from C. cicadae showed protective effects against HF/HFD induced metabolic disturbances and may be considered as a dietary supplement for treating obesity.     

高脂及MCD饮食诱导非酒精性脂肪性肝炎动物模型的比较

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

The Selective SGLT2 Inhibitor Ipragliflozin Has a Therapeutic Effect on Nonalcoholic Steatohepatitis in Mice

Background & Aims

In recent years, nonalcoholic steatohepatitis (NASH) has become a considerable healthcare burden worldwide. Pathogenesis of NASH is associated with type 2 diabetes mellitus (T2DM) and insulin resistance. However, a specific drug to treat NASH is lacking. We investigated the effect of the selective sodium glucose cotransporter 2 inhibitor (SGLT2I) ipragliflozin on NASH in mice.

Methods

We used the Amylin liver NASH model (AMLN), which is a diet-induced model of NASH that results in obesity and T2DM. AMLN mice were fed an AMLN diet for 20 weeks. SGLT2I mice were fed an AMLN diet for 12 weeks and an AMLN diet with 40 mg ipragliflozin/kg for 8 weeks.

Results

AMLN mice showed steatosis, inflammation, and fibrosis in the liver as well as obesity and insulin resistance, features that are recognized in human NASH. Ipragliflozin improved insulin resistance and liver injury. Ipragliflozin decreased serum levels of free fatty acids, hepatic lipid content, the number of apoptotic cells, and areas of fibrosis; it also increased lipid outflow from the liver.

Conclusions

Ipragliflozin improved the pathogenesis of NASH by reducing insulin resistance and lipotoxicity in NASH-model mice. Our results suggest that ipragliflozin has a therapeutic effect on NASH with T2DM.     

Time-restricted feeding mice a high-fat diet induces a unique lipidomic profile

Time-restricted feeding (TRF) can reduce adiposity and lessen the co-morbidities of obesity. Mice consuming obesogenic high-fat (HF) diets develop insulin resistance and hepatic steatosis, but have elevated indices of long-chain polyunsaturated fatty acids (LCPUFA) that may be beneficial. While TRF impacts lipid metabolism, scant data exist regarding the impact of TRF upon lipidomic composition of tissues. We (1) tested the hypothesis that TRF of a HF diet elevates LCPUFA indices while preventing insulin resistance and hepatic steatosis and (2) determined the impact of TRF upon the lipidome in plasma, liver, and adipose tissue. For 12 weeks, male, adult mice were fed a control diet ad libitum, a HF diet ad libitum (HF-AL), or a HF diet with TRF, 12 hours during the dark phase (HF-TRF). HF-TRF prevented insulin resistance and hepatic steatosis resulting from by HF-AL treatment. TRF-blocked plasma increases in LCPUFA induced by HF-AL treatment but elevated concentrations of triacylglycerols and non-esterified saturated fatty acids. Analysis of the hepatic lipidome demonstrated that TRF did not elevate LCPUFA while reducing steatosis. However, TRF created (1) a separate hepatic lipid signature for triacylglycerols, phosphatidylcholine, and phosphatidylethanolamine species and (2) modified gene and protein expression consistent with reduced fatty acid synthesis and restoration of diurnal gene signaling. TRF increased the saturated fatty acid content in visceral adipose tissue. In summary, TRF of a HF diet alters the lipidomic profile of plasma, liver, and adipose tissue, creating a third distinct lipid metabolic state indicative of positive metabolic adaptations following HF intake.     

Effect of dry tomato peel supplementation on glucose tolerance,insulin resistance,and hepatic markers in mice fed high-saturated-fat/high-cholesterol diets

Many studies have investigated the effect of crude tomato peel in vivo, but no studies have determined the dose-effect of dry tomato peel (DTP) on glucose intolerance, insulin resistance, and atherogenic dyslipidemia induced by a high-saturated-fat (HSF) diet in vivo. The aim of this study was to investigate the effects of different doses of DTP on the levels of oxidative stress in mice fed an HSF and cholesterol-rich diet for 12 weeks. The main outcomes are glucose and insulin tolerance, plasma lipids, and hepatic steatosis and inflammation. BALB/c male mice (n=40) (8 weeks old, weighing 22.2±1.0 g) were divided into four treatment groups (10 mice/group): (a) high-fat control diet (HF Ctrl), which contains sunflower oil as a sole source of fat; (b) HSF/high-cholesterol (HC) diet; (c) HSF/HC diet supplemented with 9% DTP and (d) HSF/HC diet supplemented with 17% DTP. The HSF/HC diet significantly increased body weight gain, adipose tissue weight, fasting plasma glucose, fasting plasma insulin and lipid peroxidation and caused the development of liver steatosis and inflammation. Supplementation with DTP increased plasma lycopene concentration and reduced the development of indicators of metabolic syndrome, with no consistent effect of the DTP dose. Hepatic steatosis and inflammation were not reversed with DTP supplementation. Among mice fed the HSF/HC diet, DTP supplementation appears to have a beneficial effect on insulin resistance, which confirms the antiatherogenic effect of DTP.     

高脂及MCD饮食诱导非酒精性脂肪性肝炎动物模型的比较

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

Metformin Prevents and Reverses Inflammation in a Non-Diabetic Mouse Model of Nonalcoholic Steatohepatitis

Background

Optimal treatment for nonalcoholic steatohepatitis (NASH) has not yet been established, particularly for individuals without diabetes. We examined the effects of metformin, commonly used to treat patients with type 2 diabetes, on liver pathology in a non-diabetic NASH mouse model.

Methodology/Principal Findings

Eight-week-old C57BL/6 mice were fed a methionine- and choline-deficient plus high fat (MCD+HF) diet with or without 0.1% metformin for 8 weeks. Co-administration of metformin significantly decreased fasting plasma glucose levels, but did not affect glucose tolerance or peripheral insulin sensitivity. Metformin ameliorated MCD+HF diet-induced hepatic steatosis, inflammation, and fibrosis. Furthermore, metformin significantly reversed hepatic steatosis and inflammation when administered after the development of experimental NASH.

Conclusions/Significance

These histological changes were accompanied by reduced hepatic triglyceride content, suppressed hepatic stellate cell activation, and the downregulation of genes involved in fatty acid metabolism, inflammation, and fibrogenesis. Metformin prevented and reversed steatosis and inflammation of NASH in an experimental non-diabetic model without affecting peripheral insulin resistance.     

Fast food diet mouse: novel small animal model of NASH with ballooning, progressive fibrosis, and high physiological fidelity to the human condition

Although there are small animal platforms that recapitulate some of the histological features of nonalcoholic fatty liver disease, there are no small animal models of nonalcoholic steatohepatitis (NASH) with consistent hepatocellular ballooning and progressive fibrosis that also exhibit fidelity to the human condition physiologically. We examined the metabolic and histological effects of a diet on the basis of the composition of "fast food" (high saturated fats, cholesterol, and fructose). Mice (n = 8 in each group) were assigned to diets as follows: 1) standard chow (SC), i.e., 13% energy as fat [1% saturated fatty acids (SFA)], 2) high fat (HF), i.e., 60% energy as fat (1% SFA), and 3) fast food (FF), i.e., 40% energy as fat (12% SFA, 2% cholesterol). All three diets were supplemented with high fructose. All diets produced obesity. The HF and FF diets produced insulin resistance. Liver histology was normal in animals fed the SC diet. Steatohepatitis with pronounced ballooning and progressive fibrosis (stage 2) was observed in mice fed the FF diet. Although the HF diet produced obesity, insulin resistance, and some steatosis; inflammation was minimal, and there was no increase in fibrosis. The FF diet produced a gene expression signature of increased fibrosis, inflammation, and endoplasmic reticulum stress and lipoapoptosis. A diet based on high cholesterol, high saturated fat, and high fructose recapitulates features of the metabolic syndrome and NASH with progressive fibrosis. This represents a novel small animal model of fibrosing NASH with high fidelity to the human condition. These results highlight the contribution of dietary composition to the development of nonalcoholic fatty liver disease and NASH.     

Effects of excess dietary iron and fat on glucose and lipid metabolism

PurposeDiets rich in fat and energy are associated with metabolic syndrome (MS). Increased body iron stores have been recognized as a feature of MS. High-fat diets (HFs), excess iron loading and MS are closely associated, but the mechanism linking them has not been clearly defined. We investigated the interaction between dietary fat and dietary Fe in the context of glucose and lipid metabolism in the body.MethodsC57BL6/J mice were divided into four groups and fed the modified AIN-93G low-fat diet (LF) and HF with adequate or excess Fe for 7 weeks. The Fe contents were increased by adding carbonyl iron (2% of diet weight) (LF+Fe and HF+Fe).ResultsHigh iron levels increased blood glucose levels but decreased high-density lipoprotein cholesterol levels. The HF group showed increases in plasma levels of glucose and insulin and insulin resistance. HF+Fe mice showed greater changes. Representative indices of iron status, such hepatic and plasma Fe levels, were not altered further by the HF. However, both the HF and excess iron loading changed the hepatic expression of hepcidin and ferroportin. The LF+Fe, HF and HF+Fe groups showed greater hepatic fat accumulation compared with the LF group. These changes were paralleled by alterations in the levels of enzymes related to hepatic gluconeogenesis and lipid synthesis, which could be due to increases in mitochondrial dysfunction and oxidative stress.ConclusionsHigh-fat diets and iron overload are associated with insulin resistance, modified hepatic lipid and iron metabolism and increased mitochondrial dysfunction and oxidative stress.     

Dietary cholesterol exacerbates hepatic steatosis and inflammation in obese LDL receptor-deficient mice

Non-alcoholic fatty liver disease (NAFLD), the hepatic manifestation of the metabolic syndrome, can progress to steatohepatitis (NASH) and advanced liver disease. Mechanisms that underlie this progression remain poorly understood, partly due to lack of good animal models that resemble human NASH. We previously showed that several metabolic syndrome features that develop in LDL receptor-deficient (LDLR-/-) mice fed a diabetogenic diet are worsened by dietary cholesterol. To test whether dietary cholesterol can alter the hepatic phenotype in the metabolic syndrome, we fed LDLR-/- mice a high-fat, high-carbohydrate diabetogenic diet (DD) without or with added cholesterol (DDC). Both groups of mice developed obesity and insulin resistance. Hyperinsulinemia, dyslipidemia, hepatic triglyceride, and alanine aminotransferase (ALT) elevations were greater with DDC. Livers of DD-fed mice showed histological changes resembling NAFLD, including steatosis and modest fibrotic changes; however, DDC-fed animals developed micro- and macrovesicular steatosis, inflammatory cell foci, and fibrosis resembling human NASH. Dietary cholesterol also exacerbated hepatic macrophage infiltration, apoptosis, and oxidative stress. Thus, LDLR-/- mice fed diabetogenic diets may be useful models for studying human NASH. Dietary cholesterol appears to confer a second "hit" that results in a distinct hepatic phenotype characterized by increased inflammation and oxidative stress.     

Serine prevented high-fat diet-induced oxidative stress by activating AMPK and epigenetically modulating the expression of glutathione synthesis-related genes

Serine deficiency has been observed in patients with nonalcoholic fatty liver disease (NAFLD). Whether serine supplementation has any beneficial effects on the prevention of NAFLD remains unknown. The present study was conducted to investigate the effects of serine supplementation on hepatic oxidative stress and steatosis and its related mechanisms. Forty male C57BL/6J mice (9 week-old) were randomly assigned into four groups (n = 10) and fed: i) a low-fat diet; ii) a low-fat diet supplemented with 1% (wt:vol) serine; iii) a high-fat (HF) diet; and iv) a HF diet supplemented with 1% serine, respectively. Palmitic acid (PA)-treated primary hepatocytes separated from adult mice were also used to study the effects of serine on oxidative stress. The results showed that serine supplementation increased glucose tolerance and insulin sensitivity, and protected mice from hepatic lipid accumulation, but did not significantly decreased HF diet-induced weight gain. In addition, serine supplementation protected glutathione (GSH) antioxidant system and prevented hypermethylation in the promoters of glutathione synthesis-related genes, while decreasing reactive oxygen species (ROS) in mice fed a HF diet. Moreover, we found that serine supplementation increased phosphorylation and S-glutathionylation of AMP-activated protein kinase α subunit (AMPKα), and decreased ROS, malondialdehyde and triglyceride contents in PA-treated primary hepatocytes. However, while AMPK activity or GSH synthesis was inhibited, the abovementioned effects of serine on PA-treated primary hepatocytes were not observed. Our results suggest that serine supplementation could prevent HF diet-induced oxidative stress and steatosis by epigenetically modulating the expression of glutathione synthesis-related genes and through AMPK activation.     

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

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