Saturated fatty acids promote endoplasmic reticulum stress and liver injury in rats with hepatic steatosis

Nonalcoholic fatty liver disease is a relatively new hepatic sequela of obesity and type 2 diabetes. The pathogenesis of liver injury and disease progression in nonalcoholic fatty liver disease, however, is poorly understood. The present study examined the hypothesis that the composition of fatty acids in the steatotic liver promotes liver injury. Using dietary models of hepatic steatosis characterized by similar accumulation of total triglyceride but different composition of fatty acids, we show that hepatic steatosis characterized by increased saturated fatty acids is associated with increased liver injury and markers of endoplasmic reticulum stress (e.g. X-box binding protein-1 mRNA splicing and glucose-regulated protein 78 expression). These changes preceded and/or occurred independently of obesity and differences in leptin, TNFalpha, insulin action, and mitochondrial function. In addition, hepatic steatosis characterized by increased saturated fatty acids reduced proliferative capacity in response to partial hepatectomy and increased liver injury in response to lipopolysaccharide. These data suggest that the composition of fatty acids in the steatotic liver is an important determinant of susceptibility to liver injury.     

Neue Aspekte zur Pathogenese der NASH

Non-alcoholic fatty liver disease (NAFLD) und non-alcoholic steatohepatitis (NASH) represent etiologically not completely understood disease entities for which an established therapy is not yet available. This is a challenge in medicine because a progressive course of the disorder from simple steatosis to steatohepatitis with development of fibrosis and finally cirrhosis with possible development of liver cancer has been described. Overload of fatty acids, e.g. by high caloric diet or release from adipose tissue, leads to triglyceride accumulation in lipid droplets of hepatocytes. Saturated fatty acids within lipid droplets cause endoplasmic reticulum (ER) stress which induces insulin resistance, inflammation and apoptosis by intracellular signal cascades (clinical features of NASH). The responsible intracellular switch is activated JNK1. The subsequent deactivation of FXR perpetuates the lipid droplet accumulation via insulin resistance, maintains inflammation and leads to hyperglycemia. Moreover, apoptotic cell death is triggered by JNK1 directly or indirectly by release of TNFα (amplified by activation of liver macrophages). Besides the involvement of defined signal cascades, the activation of mitochondrial phospholipase A2 (iPLA 2) resulting in release of lysophosphatidylcholine (LPC) is the key player for apoptosis induction. Thus, future therapeutic concepts should focus on prevention of ER stress, maintenance of FXR activation and inhibition of iPLA2 activity.     

Palmitate induces insulin resistance without significant intracellular triglyceride accumulation in HepG2 cells

Previous studies showed that increased release of free fatty acids from adipocytes leads to insulin resistance and triglyceride (TG) accumulation in the liver, which may progress into hepatic steatohepatitis. We and other investigators have previously reported that palmitate induces endoplasmic reticulum stress-mediated toxicity in several tissues. This work investigated whether palmitate could induce insulin resistance and steatosis in HepG2 cells. We treated cells with either saturated fatty acid (palmitate) or unsaturated fatty acid (oleate), and observed that palmitate significantly activated c-jun N-terminal kinase and inactivated protein kinase B. Both 4-phenylbutyric acid and glycerol significantly activated protein kinase B, confirming the involvement of endoplasmic reticulum stress in palmitate-mediated insulin resistance. Oleate, but not palmitate, significantly induced intracellular TG deposition and activated sterol regulatory element binding protein-1. Instead, diacylglycerol level and protein kinase C ? activity were significantly increased by palmitate, suggesting the possible role of diacylglycerol in palmitate-mediated lipotoxicity. Therefore, the present study clearly showed that palmitate impairs insulin resistance, but does not induce significant TG accumulation in HepG2 cells.     

Ursodeoxycholic acid improves insulin sensitivity and hepatic steatosis by inducing the excretion of hepatic lipids in high-fat diet-fed KK-Ay mice

Type 2 diabetes mellitus is frequently accompanied by fatty liver/nonalcoholic fatty liver disease. Hence, accumulation of lipids in the liver is considered to be one of the risk factors for insulin resistance and metabolic syndrome. Ursodeoxycholic acid (UDCA) is widely used for the treatment of liver dysfunction. We investigated the therapeutic effects of UDCA on type 2 diabetes mellitus exacerbating hepatic steatosis and the underlying mechanisms of its action using KK-A(y) mice fed a high-fat diet. KK-A(y) mice were prefed a high-fat diet; and 50, 150, and 450 mg/kg of UDCA was orally administered for 2 or 3 weeks. Administration of UDCA decreased fasting hyperglycemia and hyperinsulinemia. Hyperinsulinemic-euglycemic clamp analyses showed that UDCA improved hepatic (but not peripheral) insulin resistance. Hepatic triglyceride and cholesterol contents were significantly reduced by treatment with UDCA, although the genes involved in the synthesis of fatty acids and cholesterol, including fatty acid synthase and 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase, were upregulated. Fecal levels of bile acids, neutral sterols, fatty acids, and phospholipids were significantly increased by UDCA treatment. The gene expression levels and protein phosphorylation levels of endoplasmic reticulum stress markers were not changed by UDCA treatment. These results indicate that UDCA ameliorates hyperglycemia and hyperinsulinemia by improving hepatic insulin resistance and steatosis in high-fat diet-fed KK-A(y) mice. Reduction of hepatic lipids might be due to their excretion in feces, followed by enhanced utilization of glucose for the synthesis of fatty acids and cholesterol. Ursodeoxycholic acid should be effective for the treatment of type 2 diabetes mellitus accompanying hepatic steatosis.     

From chronic overfeeding to hepatic injury: role of endoplasmic reticulum stress and inflammation

We analyse how chronic overfeeding, by increasing circulating fatty acids, might lead to inflammation, insulin resistance (IR) and injury in the liver. Chronic overfeeding causes an increase in adipose tissue depots and is characterised by an increased presence of hypertrophic adipocytes when adipose tissue expandability is inadequate. Adipocyte hypertrophy is a possible stress condition for the endoplasmic reticulum (ER), which will activate inflammatory and apoptotic pathways and cause IR in adipose tissue. Insulin-resistant adipocytes, being more lipolytic and less liposynthetic, induce an increase in circulating free fatty acids. Moreover, the strongly compromised secretion/function of the adipocyte hormones, adiponectin and leptin, decreases lipid oxidation, particularly in the liver, causing lipid accumulation, ER stress and IR in hepatocytes. ER stress may lead to reduced very-low-density lipoprotein (VLDL) secretion and increased lipogenic gene expression despite the presence of IR. These events and reduced lipid oxidation may lead to further hepatic lipid accumulation. When the triglyceride storage capacity of hepatocytes is exceeded, hepatic injury may occur. ER-stressed steatotic hepatocytes activate apoptotic and inflammatory pathways, which trigger IR and the release of chemokines and cytokines, and these, in turn, elicit an increased influx of Kupffer cells (KCs) and hepatic stellate cells (HSCs) around dying hepatocytes. Soluble mediators, secreted mainly by ER-stressed steatotic hepatocytes and activated KCs, induce the transdifferentiation of HSCs to myofibroblasts, which secrete fibrogenic cytokines and matrix components that trigger fibrosis. In conclusion, chronic lipid overloading due to inadequate fat-storing capacity of adipose tissue can induce hepatic injury when triglyceride storage capacity of hepatocytes is exceeded.     

Non-alcoholic fatty liver disease and insulin resistance: From bench to bedside

Non-alcoholic fatty liver disease (NAFLD) is now the most frequent chronic liver disease in the developed countries. There is also growing evidence from basic and clinical research that NAFLD has a strong relationship to insulin resistance, which is a key factor in the development of type 2 diabetes. The aim of this review is to summarize the recent important findings linking NAFLD and insulin resistance. Lipid accumulation, particularly of diacylglycerol, appears to be of major importance in this process. Mitochondrial dysfunction, through decreased mitochondrial biogenesis, increases oxidative stress, and ageing also plays an important role. Finally, endoplasmic reticulum stress and inflammation also probably contribute to the development of insulin resistance via mechanisms that are still not well understood. Clinical aspects of NAFLD, such as its diagnosis and management, are also investigated in this review.     

Role of endoplasmic reticulum stress in the pathogenesis of nonalcoholic fatty liver disease

Nonalcoholic fatty liver disease(NAFLD)has emerged as a common public health problem in recent decades.However,the underlying mechanisms leading to the development of NAFLD are not fully understood.The endoplasmic reticulum(ER)stress response has recently been proposed to play a crucial role in both the development of steatosis and progression to nonalcoholic steatohepatitis.ER stress is activated to regulate protein synthesis and restore homeostatic equilibrium when the cell is stressed due to the accumulation of unfolded or misfolded proteins.However,delayed or insufficient responses to ER stress may turn physiological mechanisms into pathological consequences,including fat accumulation,insulin resistance,inflammation,and apoptosis,all of which play important roles in the pathogenesis of NAFLD.Therefore,understanding the role of ER stress in the pathogenesis of NAFLD has become a topic of intense investigation.This review highlights the recent findings linking ER stress signaling pathways to the pathogenesis of NAFLD.     

Effects of bile duct ligation and cholic acid treatment on fatty liver in two rat models of non-alcoholic fatty liver disease

Background

Non-alcoholic fatty liver disease, one of the most prevalent liver disorders in Western countries, is characterized by hepatic accumulation of triglycerides. Bile acids have long been known to affect triglyceride homeostasis through a not completely understood mechanism.

Aim

To analyse the effects of two different manipulations of bile acid circulation on non-alcoholic fatty liver disease.

Methods

Two animal models of non-alcoholic fatty liver disease were developed by either feeding rats with a choline deficient or with a high fat diet. After 4 weeks, rats were randomized to undergo either bile duct ligation, sham operation or cholic acid administration.

Results

During cholestasis there was an increased CYP7A1 expression, the rate limiting enzyme in bile acid synthesis, and a reduction of hepatic concentration of oxysterols, ligands of the liver X receptors. Target genes of the liver X receptors, involved in fatty acid and triglyceride synthesis, were down-regulated in association with decreased hepatic triglyceride content and improvement of fatty liver. Administration of cholic acid, ligand of farnesoid X receptor, also had a beneficial effect on fatty liver in rats on choline deficient diet.

Conclusion

These results indicate that pharmacological approaches increasing the expression of CYP7A1 or stimulating farnesoid X receptor pathway could represent a promising treatment for non-alcoholic fatty liver disease.     

内质网应激参与蛋氨酸-胆碱缺乏饮食诱导大鼠脂肪性肝纤维化的发生与恢复

目的 探讨内质网应激在非酒精性脂肪性肝纤维化形成中的作用及饮食控制对脂肪性肝纤维化恢复的影响.方法 蛋氨酸-胆碱缺乏饮食 (MCDD) 喂养10周诱导非酒精性脂肪性肝纤维化大鼠模型,恢复组于第9周开始将MCDD转变为蛋氨酸-胆碱对照饮食 (MCCD) 喂养2周.纤维化和炎症程度采用组织病理染色方法检测,纤维化和炎症相关因子采用Western blot和实时定量聚合酶链反应 (RT-PCR) 方法检测,内质网应激标记物半胱氨酸天冬氨酸蛋白酶 (caspase)12、7和葡萄糖调节蛋白78 (GRP78) 采用免疫组织化学、Western blot和RT-PCR方法检测.计量资料采用统计分析软件SPSS13.0中的ANOVA程序进行单因素方差分析或q检验,并用LSD进行两两比较.结果 饮食由MCDD转变为MCCD后,组织病理学结果显示肝细胞脂肪沉积及炎症反应明显减轻,伴随着活化的肝星状细胞和枯否细胞减少,肝纤维化程度明显减轻.模型组大鼠肝细胞凋亡数为每5个高倍视野 (68.2±15.9) 个,明显高于正常组 (40.3±8.3) 个,P<0.05;肝细胞增殖/凋亡比值 (0.10±0.03) 明显低于正常组(0.19±0.03),P<0.01.恢复组大鼠肝细胞凋亡数为每5个高倍视野 (48.0±6.5)个,明显低于模型组 (68.2±15.9) 个,P<0.05;增殖数为每5个高倍视野 (17.2±4.4)个,明显高于模型组 (7.5±3.0) 个,P<0.01;肝细胞增殖/凋亡比值 (0.41±0.09) 也明显高于模型组 (0.10±0.03),P<0.01.模型组大鼠肝组织GRP78、caspase12、caspase7和cleaved caspase7蛋白质和mRNA表达均明显高于正常组(P<0.05或P<0.01),恢复组均明显低于模型组 (P<0.05或P<0.01).结论 大鼠饮食由MCDD转变为MCCD后,肝脏的炎症和纤维化程度迅速逆转,提示饮食摄取对于控制肝脏脂肪沉积和病理改变至关重要,且内质网应激参与了这一过程,阻断内质网应激尤其是Caspase12通路也许有助于非酒精性脂肪性肝纤维化的临床治疗.     

Role of skeletal muscle lipids in the pathogenesis of insulin resistance of obesity and type 2 diabetes

Obesity predisposes individuals to the development of insulin resistance, which is a risk factor for type 2 diabetes, and muscle plays a central role in this phenomenon. Insulin resistance is associated with: (i) a metabolic inflexibility characterized by a reduced impaired switching from free fatty acid (FA) to carbohydrate substrates; and (ii) an ectopic accumulation of triglyceride in skeletal muscle, generating a cellular “lipotoxicity”, but triglyceride per se, does not contribute to insulin resistance (“athlete’s paradox”). A large body of evidence supports the idea that a decreased mitochondrial capacity to oxidize FA leads to an accretion of intracellular triglyceride and an accumulation of acyl-CoAs, which are used to synthesize diacylglycerol and ceramide. These lipid derivatives activate serine kinases, leading to increase of insulin receptor substrate 1 serine phosphorylation, which impairs insulin signaling. A second model proposes that insulin resistance arises from an excessive mitochondrial FA oxidation. Studies have shown that the type of FA, unsaturated or saturated, is critical in the development of insulin resistance. It should be also stressed that FA oversupply activates inflammatory signals, induces endoplasmic reticulum stress, increases mitochondrial oxidative stress and influences the regulation of genes that contributes to impaired glucose metabolism. These cellular insults are thought to engage stress-sensitive serine kinases disrupting insulin signaling. In conclusion, reduced dietary lipid intake in association with physical exercise could be a therapeutic option to improve insulin sensitivity.     

Bile-acid-induced cell injury and protection

Several studies have characterized the cellular and molecular mechanisms of hepatocyte injury caused by the retention of hydrophobic bile acids （BAs） in cholestatic diseases. BAs may disrupt cell membranes through their detergent action on lipid components and can promote the generation of reactive oxygen species that, in turn, oxidatively modify lipids, proteins, and nucleic acids, and eventually cause hepatocyte necrosis and apoptosis. Several pathways are involved in triggering hepatocyte apoptosis. Toxic BAs can activate hepatocyte death receptors directly and induce oxidative damage, thereby causing mitochondrial dysfunction, and induce endoplasmic reticulum stress. When these compounds are taken up and accumulate inside biliary cells, they can also cause apoptosis. Regarding extrahepatic tissues, the accumulation of BAs in the systemic circulation may contribute to endothelial injury in the kidney and lungs. In gastrointestinal cells, BAs may behave as cancer promoters through an indirect mechanism involving oxidative stress and DNA damage, as well as acting as selection agents for apoptosis-resistant cells. The accumulation of BAs may have also deleterious effects on placental and fetal cells. However, other BAs, such as ursodeoxycholic acid, have been shown to modulate BA-induced injury in hepatocytes. The major beneficial effects of treatment with ursodeoxycholic acid are protection against cytotoxicity due to more toxic BAs; the stimulation of hepatobiliary secretion; antioxidant activity, due in part to an enhancement in glutathione levels; and the inhibition of liver cell apoptosis. Other natural BAs or their derivatives, such as cholyI-N- methylglycine or pharmacological properties. cholylsarcosine, interest owing have also aroused to their protective     

Fructose as a key player in the development of fatty liver disease

We aimed to investigate whether increased consumption of fructose is linked to the increased prevalence of fatty liver.The prevalence of nonalcoholic steatohepatitis(NASH) is 3% and 20% in nonobese and obese subjects,respectively.Obesity is a low-grade chronic inflam-m-atory condition and obesity-related cytokines such as interleukin-6,adiponectin,leptin,and tumor necrosis factor-α may play important roles in the developm-ent of nonalcoholic fatty liver disease(NAFLD).Additionally,the prevalence of NASH associated with both cirrhosis and hepatocellular carcinom-a was reported to be high am-ong patients with type 2 diabetes with or without obesity.Our research group previously showed that consumption of fructose is associated with adverse alterations of plasma lipid profiles and metabolic changes in m-ice,the Am-erican Lifestyle-Induced Obesity Syndrom-e m-odel,which included consum-ption of a high-fructose corn syrup in amounts relevant to that consum-ed by som-e Am-ericans.The observation reinforces the concerns about the role of fructose in the obesity epidem-ic.Increased availability of fructose(e.g.,high-fructose corn syrup) increases not only abnorm-al glucose flux but also fructose m-etabolism-in the hepatocyte.Thus,the anatomic position of the liver places it in a strategic buffering position for absorbed carbohydrates and am-ino acids.Fructose was previously accepted as a beneficial dietary com-ponent because it does not stim-ulate insulin secretion.However,since insulin signaling plays an important role in central m-echanism-s of NAFLD,this property of fructose m-ay be undesirable.Fructose has a selective hepatic m-etabolism,and provokes a hepatic stress response involving activation of c-Jun N-term-inal kinases and subsequent reduced hepatic insulin signaling.As high fat diet alone produces obesity,insulin resistance,and som-e degree of fatty liver with m-inim-al inflam-m-ation and no fibrosis,the fast food diet which includes fructose and fats produces a gene expression signature of increased hepatic     

高果糖、高脂饲料喂养小鼠肝脏脂质合成酶类与内质网应激相关因子蛋白表达变化

目的 探讨和比较高果糖、高脂饮食诱导的小鼠肝脏三酰甘油(TG)的发生机制及其与内质网应激的关系.方法 成年雄性C57BL/J6小鼠45只,质量25～30 g,按随机数字表随机均分为对照组、高脂组及高果糖组,每组15只.对照组予以普通饲料,高果糖组予以高糖饮食,高脂组予以高脂饮食,3组每日进食热量基本相等,经喂养8周后对小鼠行葡萄糖耐量(ipGTT)实验,处死小鼠后测定各组肝脏三酰甘油含量,并测定肝脏脂质合成酶类和内质网应激相关因子的蛋白表达.结果 不同饲料喂养8周后,高果糖组和高脂组的附睾脂肪含量均为(2.0±0.1)g/100 g(质量),明显高于对照组[(1.2±0.1)g/100 g(质量)P＜0.01].高果糖组和高脂组ipGTT实验后的血糖曲线下面积明显高于对照组(P＜0.01).与对照组相比,高果糖组和高脂组的肝脏TG水平显著增高(P＜0.01),其中高果糖组肝脏TG升高更明显,高果糖组肝脏TG水平显著高于高脂组(P＜0.01).与对照组相比,高果糖组的乙酰辅酶A羧化酶( ACC)、脂肪酸合成酶(FAS)、硬脂酰辅酶A去饱和酶(SCD-1)表达增加(P＜0.01),而高脂组的FAS、ACC、SCD-1的表达减少(P＜0.05);反应内质网应激的磷酸化胰腺内质网激酶(p-PERK)、山梨醇要求激酶-1(p-IRE-1/t-IRE-1)和葡萄糖调节蛋白78(GRP78)蛋白表达在高果糖组和高脂组均增加(P值均＜0.01).结论 高果糖饮食和高脂饮食均可引起脂肪肝,二者通过不同机制引起脂肪肝,高果糖饮食促进内源性脂质生成,高脂喂养抑制肝内源性脂质生成,两种饮食均可诱发内质网应激,提示内质网应激与饮食因子诱导的脂肪肝的发生发展有关.     

葡萄糖调节蛋白78在非酒精性脂肪性肝病发病中的作用*

葡萄糖调节蛋白78（GRP78）作为一个重要的内质网分子伴侣,在蛋白质的折叠、转运和内质网应激反应过程中发挥重要作用。GRP78还存在于肿瘤细胞、内皮细胞和单核细胞的表面,可作为病毒进入宿主细胞的受体。内质网应激是非酒精性脂肪性肝病(NAFLD)的一个重要的发病机制。对GRP78功能的深入挖掘,将为探索NAFLD的防御机制提供新思路。     

Adverse physicochemical properties of tripalmitin in beta cells lead to morphological changes and lipotoxicity in vitro

Aims/hypothesis Long-term exposure of beta cells to lipids, particularly saturated fatty acids in vitro, results in cellular dysfunction and apoptosis (lipotoxicity); this could contribute to obesity-related diabetes. Our aims were to relate cell death to intracellular triglyceride concentration, composition and localisation following incubation of INS1 cells in saturated and unsaturated NEFA in high and low glucose concentrations.Materials and methods Insulin-producing INS1 cells were cultured (24 h; 3 and 20 mmol/l glucose) with palmitic, oleic or linoleic acids and the resulting intracellular lipids were analysed by gas chromatography and microscopy. Cell death was determined by quantitative microscopy and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, and glucose-stimulated insulin secretion by ELISA.Results All NEFA (0.5 mmol/l, 0.5% albumin) inhibited glucose-stimulated (20 mmol/l) insulin secretion. Cytotoxicity was evident only with palmitic acid (p<0.05), in which case intracellular triglyceride consisted largely of tripalmitin in angular-shaped dilated endoplasmic reticulum. Cytotoxicity and morphological disruption were reduced by addition of unsaturated NEFA. Triglyceride content (control cells; 14.5 ng/g protein) increased up to 10-fold following incubation in NEFA (oleic acid 153.2 ng/g protein; p<0.05) and triglyceride and phospholipid fractions were both enriched with the specific fatty acid added to the medium (p<0.05).Conclusions/interpretation In INS1 cells, palmitic acid is converted in the endoplasmic reticulum to solid tripalmitin (melting point >65°C), which could induce endoplasmic reticulum stress proteins and signal apoptosis; lipid-induced apoptosis would therefore be a consequence of the physicochemical properties of these triglycerides. Since cellular triglycerides composed of single species of fatty acid are not likely to occur in vivo, destruction of beta cells by saturated fatty acids could be predominantly an in vitro scenario.