GLP-1 analogs reduce hepatocyte steatosis and improve survival by enhancing the unfolded protein response and promoting macroautophagy

Background

Nonalcoholic fatty liver disease (NAFLD) is a known outcome of hepatosteatosis. Free fatty acids (FFA) induce the unfolded protein response (UPR) or endoplasmic reticulum (ER) stress that may induce apoptosis. Recent data indicate ER stress to be a major player in the progression of fatty liver to more aggressive lesions. Autophagy on the other hand has been demonstrated to be protective against ER stress- induced cell death. We hypothesized that exendin-4 (GLP-1 analog) treatment of fat loaded hepatocytes can reduce steatosis by autophagy which leads to reduced ER stress-related hepatocyte apoptosis.

Methodology/Principal Findings

Primary human hepatocytes were loaded with saturated, cis- and trans-unsaturated fatty acids (palmitic, oleic and elaidic acid respectively). Steatosis, induced with all three fatty acids, was significantly resolved after exendin-4 treatment. Exendin-4 sustained levels of GRP78 expression in fat-loaded cells when compared to untreated fat-loaded cells alone. In contrast, CHOP (C/EBP homologous protein); the penultimate protein that leads to ER stress-related cell death was significantly decreased by exendin-4 in hepatocytes loaded with fatty acids. Finally, exendin-4 in fat loaded hepatocytes clearly promoted gene products associated with macroautophagy as measured by enhanced production of both Beclin-1 and LC3B-II, markers for autophagy; and visualized by transmission electron microscopy (TEM). Similar observations were made in mouse liver lysates after mice were fed with high fat high fructose diet and treated with a long acting GLP-1 receptor agonist, liraglutide.

Conclusions/Significance

GLP-1 proteins appear to protect hepatocytes from fatty acid-related death by prohibition of a dysfunctional ER stress response; and reduce fatty acid accumulation, by activation of both macro-and chaperone-mediated autophagy. These findings provide a novel role for GLP-1 proteins in halting the progression of more aggressive lesions from underlying steatosis in humans afflicted with NAFLD.     

Exendin-4 improves steatohepatitis by increasing Sirt1 expression in high-fat diet-induced obese C57BL/6J mice

The effects of exendin-4 on Sirt1 expression as a mechanism of reducing fatty liver have not been previously reported. Therefore, we investigated whether the beneficial effects of exendin-4 treatment on fatty liver are mediated via Sirt1 in high-fat (HF) diet-induced obese C57BL/6J mice and related cell culture models. Exendin-4 treatment decreased body weight, serum free fatty acid (FA), and triglyceride levels in HF-induced obese C57BL/6J mice. Histological analysis showed that exendin-4 reversed HF-induced hepatic accumulation of lipids and inflammation. Exendin-4 treatment increased mRNA and protein expression of Sirt1 and its downstream factor, AMPK, in vivo and also induced genes associated with FA oxidation and glucose metabolism. In addition, a significant increase in the hepatic expression of Lkb1 and Nampt mRNA was observed in exendin-4-treated groups. We also observed increased expression of phospho-Foxo1 and GLUT2, which are involved in hepatic glucose metabolism. In HepG2 and Huh7 cells, mRNA and protein expressions of GLP-1R were increased by exendin-4 treatment in a dose-dependent manner. Exendin-4 enhanced protein expression of Sirt1 and phospho-AMPKα in HepG2 cells treated with 0.4 mM palmitic acid. We also found that Sirt1 was an upstream regulator of AMPK in hepatocytes. A novel finding of this study was the observation that expression of GLP-1R is proportional to exendin-4 concentration and exendin-4 could attenuate fatty liver through activation of Sirt1.     

Tumor necrosis factor-α accelerates apoptosis of steatotic hepatocytes from a murine model of non-alcoholic fatty liver disease

Non-alcoholic steatohepatitis (NASH) develops in a subset of patients with non-alcoholic fatty liver disease (NAFLD), but the exact mechanisms involved in the progression of NAFLD to NASH remain poorly understood. We investigated the role of tumor necrosis factor-α (TNF-α) in the apoptosis of hepatocytes that is related to the severity of NASH. We separated primary hepatocytes from the NAFLD liver caused by a high-fat diet. The production of intracellular reactive oxygen species was increased in steatotic hepatocytes, which were also sensitive to TNF-α. This factor induced significant apoptosis through the signal-regulating kinase 1 (ASK1) and c-Jun N-terminal kinase (JNK) pathway. We describe here a novel culture model of steatotic hepatocytes separated from the NAFLD liver, and demonstrate that TNF-α induces their apoptosis in vitro.     

Central energy metabolism remains robust in acute steatotic hepatocytes challenged by a high free fatty acid load

Overnutrition is one of the major causes of non-alcoholic fatty liver disease (NAFLD). NAFLD is characterized by an accumulation of lipids (triglycerides) in hepatocytes and is often accompanied by high plasma levels of free fatty acids (FFA). In this study, we compared the energy metabolism in acute steatotic and non-steatotic primary mouse hepatocytes. Acute steatosis was induced by pre-incubation with high concentrations of oleate and palmitate. Labeling experiments were conducted using [U-(13)C(5),U-(15)N(2)] glutamine. Metabolite concentrations and mass isotopomer distributions of intracellular metabolites were measured and applied for metabolic flux estimation using transient 13C metabolic flux analysis. FFAs were efficiently taken up and almost completely incorporated into triglycerides (TAGs). In spite of high FFA uptake rates and the high synthesis rate of TAGs, central energy metabolism was not significantly changed in acute steatotic cells. Fatty acid β-oxidation does not significantly contribute to the detoxification of FFAs under the applied conditions.     

S-Allyl cysteine attenuates free fatty acid-induced lipogenesis in human HepG2 cells through activation of the AMP-activated protein kinase-dependent pathway

S-Allyl cysteine (SAC), a nontoxic garlic compound, has a variety of pharmacological properties, including antioxidant and hepatoprotective properties. In this report, we provide evidence that SAC prevented free fatty acid (FFA)-induced lipid accumulation and lipotoxicity in hepatocytes. SAC significantly reduced FFA-induced generation of reactive oxygen species, caspase activation and subsequent cell death. Also, SAC mitigated total cellular lipid and triglyceride accumulation in steatotic HepG2 cells. SAC significantly increased the phosphorylation of AMP-activated protein kinase (AMPK) and acetyl-CoA carboxylase (ACC) in HepG2 cells. Additionally, SAC down-regulated the levels of sterol regulatory element binding protein-1 (SREBP-1) and its target genes, including ACC and fatty acid synthase. Use of a specific inhibitor showed that SAC activated AMPK via calcium/calmodulin-dependent kinase kinase (CaMKK) and silent information regulator T1. Our results demonstrate that SAC activates AMPK through CaMKK and inhibits SREBP-1-mediated hepatic lipogenesis. Therefore, SAC has therapeutic potential for preventing nonalcoholic fatty liver disease.     

PPARalpha activation increases triglyceride mass and adipose differentiation-related protein in hepatocytes

Adipose differentiation-related protein (ADRP) is a lipid droplet-associated protein that is expressed in various tissues. In mice treated with the peroxisome proliferator-activated receptor alpha (PPARalpha) agonist Wy14,643 (Wy), hepatic mRNA and protein levels of ADRP as well as hepatic triglyceride content increased. Also in primary mouse hepatocytes, Wy increased ADRP expression and intracellular triglyceride mass. The triglyceride mass increased in spite of unchanged triglyceride biosynthesis and increased palmitic acid oxidation. However, Wy incubation decreased the secretion of newly synthesized triglycerides, whereas apolipoprotein B secretion increased. Thus, decreased availability of triglycerides for VLDL assembly could help to explain the cellular accumulation of triglycerides after Wy treatment. We hypothesized that this effect could be mediated by increased ADRP expression. Similar to PPARalpha activation, adenovirus-mediated ADRP overexpression in mouse hepatocytes enhanced cellular triglyceride mass and decreased the secretion of newly synthesized triglycerides. In ADRP-overexpressing cells, Wy incubation resulted in a further decrease in triglyceride secretion. This effect of Wy was not attributable to decreased cellular triglycerides after increased fatty acid oxidation because the triglyceride mass in Wy-treated ADRP-overexpressing cells was unchanged. In summary, PPARalpha activation prevents the availability of triglycerides for VLDL assembly and increases hepatic triglyceride content in part by increasing the expression of ADRP.     

Mitochondrial uncoupling protein-2 deficiency protects steatotic mouse hepatocytes from hypoxia/reoxygenation

Steatotic livers are sensitive to ischemic events and associated ATP depletion. Hepatocellular necrosis following these events may result from mitochondrial uncoupling protein-2 (UCP2) expression. To test this hypothesis, we developed a model of in vitro steatosis using primary hepatocytes from wild-type (WT) and UCP2 knockout (KO) mice and subjected them to hypoxia/reoxygenation (H/R). Using cultured hepatocytes treated with emulsified fatty acids for 24 h, generating a steatotic phenotype (i.e., microvesicular and broad-spectrum fatty acid accumulation), we found that the phenotype of the WT and UCP2 KO were the same; however, cellular viability was increased in the steatotic KO hepatocytes following 4 h of hypoxia and 24 h of reoxygenation; Hepatocellular ATP levels decreased during hypoxia and recovered after reoxygenation in the control and UCP2 KO steatotic hepatocytes but not in the WT steatotic hepatocytes; mitochondrial membrane potential in WT and UCP2 KO steatotic groups was less than control groups but higher than UCP2 KO hepatocytes. Following reoxygenation, lipid peroxidation, as measured by thiobarbituric acid reactive substances, increased in all groups but to a greater extent in the steatotic hepatocytes, regardless of UCP2 expression. These results demonstrate that UCP2 sensitizes steatotic hepatocytes to H/R through mitochondrial depolarization and ATP depletion but not lipid peroxidation.     

Mechanism of preventive effects of exendin-4 and des-fluoro-sitagliptin in a murine model of fructose-induced prediabetes

Protective effects of exendin-4 (glucagon-like peptide-1 -GLP-1- receptor agonist) and des-fluoro-sitagliptin (dipeptidyl peptidase-4 inhibitor) on fructose-induced hepatic disturbances were evaluated in prediabetic rats. Complementary, a possible direct effect of exendin-4 in human hepatoblastoma-derived cell line HepG2 incubated with fructose in presence/absence of exendin-9-39 (GLP-1 receptor antagonist) was investigated. In vivo, after 21 days of fructose rich diet, we determined: glycemia, insulinemia, and triglyceridemia; hepatic fructokinase, AMP-deaminase, and G-6-P dehydrogenase (G-6-P DH) activities; carbohydrate-responsive element-binding protein (ChREBP) expression; triglyceride content and lipogenic gene expression (glycerol-3-phosphate acyltransferase -GPAT-, fatty acid synthase -FAS-, sterol regulatory element-binding protein-1c -SREBP-1c); oxidative stress and inflammatory markers expression. In HepG2 cells we measured fructokinase activity and triglyceride content. Hypertriglyceridemia, hyperinsulinemia, enhanced liver fructokinase, AMP-deaminase, and G-6-P DH activities, increased ChREBP and lipogenic genes expression, enhanced triglyceride level, oxidative stress and inflammatory markers recorded in fructose fed animals, were prevented by co-administration of either exendin-4 or des-fluoro-sitagliptin. Exendin-4 prevented fructose-induced increase in fructokinase activity and triglyceride contain in HepG2 cells. These effects were blunted co-incubating with exendin-9-39. The results demonstrated for the first time that exendin-4/des-fluro-sitagliptin prevented fructose-induced endocrine-metabolic oxidative stress and inflammatory changes probably acting on the purine degradation pathway. Exendin 9–39 blunted in vitro protective exendin-4 effects, thereby suggesting a direct effect of this compound on hepatocytes through GLP-1 receptor. Direct effect on fructokinase and AMP-deaminase activities, with a key role in the pathogenesis of liver dysfunction induced by fructose, suggests purine degradation pathway constitute a potential therapeutic objective for GLP-1 receptor agonists.     

Endoplasmic reticulum stress leads to lipid accumulation through upregulation of SREBP-1c in normal hepatic and hepatoma cells

Endoplasmic reticulum stress (ERS) has been found in non-alcoholic fatty liver disease. The study was to further explore the mechanistic relationship between ERS and lipid accumulation. To induce ERS, the hepatoblastoma cell line HepG2 and the normal human L02 cell line were exposed to Tg for 48 h. RT-PCR and Western blot were performed to evaluate glucose-regulated protein (GRP-78) expression as a marker of ERS. ER ultrastructure was assessed by electron microscopy. Triglyceride content was examined by Oil Red O staining and quantitative intracellular triglyceride assay. The hepatic nuclear sterol regulatory element-binding protein (SREBP-1c), liver X receptor (LXRs), fatty acid synthase (FAS), and acetyl-coA carboxylase (ACC1) expressions were examined by real-time PCR and Western blot. 4-(2-aminoethyl) benzenesulfonyl fluoride (AEBSF) was used to inhibit S1P serine protease inhibitor, and SREBP-1c cleavage was evaluated under ERS. SREBP-1c was knockdown and its effect on lipid metabolism was observed. Tg treatment upregulated GRP-78 expression and severely damaged the ER structure in L02 and HepG2 cells. ERS increased triglyceride deposition and enhanced the expression of SREBP-1c, FAS, and ACC1, but have no influence on LXR. AEBSF pretreatment abolished Tg-induced SREBP-1c cleavage. Moreover, SREBP-1c silencing reduced triglycerides and downregulated FAS expression. Pharmacological ERS induced by Tg leads to lipid accumulation through upregulation of SREBP-1c in L02 and HepG2 cells.     

Oxidative stress impairs HIF1α activation: a novel mechanism for increased vulnerability of steatotic hepatocytes to hypoxic stress

Steatosis increases the sensitivity of hepatocytes to hypoxic injury. Thus, this study was designed to elucidate the role of hypoxia-inducible factor-1α (HIF1α) in steatotic hepatocytes during hypoxia. AML12 hepatocytes and isolated rat hepatocytes were treated with a free fatty acid mixture of oleate and palmitate (2:1, 1 mM) for 18 h, which generated intrahepatocyte fat accumulation. The cells were then exposed to hypoxia (1% oxygen, 6-24 h). After hypoxia, a further increase in cellular fat accumulation was seen. In steatotic hepatocytes, a decreased HIF1α activation by hypoxia was observed. The capacity of these cells to express HIF1α-dependent genes responsible for the utilization of nutrients for energy was also impaired. This resulted in significantly lower intracellular ATP levels and greater cell death in steatotic hepatocytes compared with control hepatocytes. In contrast, overexpression of constitutively active HIF1α significantly increased cell viability as well as ATP and GLUT1 mRNA levels in steatotic hepatocytes under hypoxia. Hypoxia significantly enhanced HIF1α mRNA levels in control but not in steatotic hepatocytes. Concomitantly, an increase in oxidative stress was found in steatotic hepatocytes under hypoxic conditions compared with control cells. This included higher reactive oxygen species generation, lower cellular and nuclear GSH levels, and higher accumulation of 4-hydroxynonenal protein adducts. Hypoxia-mediated oxidative stress was accompanied by inactivation of basal nuclear factor-κB (NF-κB) DNA binding. Treatment with N-acetyl-l-cysteine, a reducing agent, improved NF-κB DNA-binding capacity and restored HIF1α induction. Conversely, overexpression of an NF-κB super-suppressor in control hepatocytes (IκBαΔN-transfected cells) resulted in complete inhibition of HIF1α expression, confirming that indeed NF-κB regulates HIF1α expression in hypoxic hepatocytes. In conclusion, hypoxia in combination with hepatic steatosis was shown to promote augmented oxidative stress, leading to NF-κB inactivation and impaired HIF1α induction and thereby increased susceptibility to hypoxic injury.     

A theoretical study of lipid accumulation in the liver—implications for nonalcoholic fatty liver disease

A hallmark of the nonalcoholic fatty liver disease is the accumulation of lipids. We developed a mathematical model of the hepatic lipid dynamics to simulate the fate of fatty acids in hepatocytes. Our model involves fatty acid uptake, lipid oxidation, and lipid export. It takes into account that storage of triacylglycerol within hepatocytes leads to cell enlargement reducing the sinusoids radius and impairing hepatic microcirculation. Thus oxygen supply is reduced, which impairs lipid oxidation. The analysis of our model revealed a bistable behavior (two stable steady states) of the system, in agreement with histological observations showing distinct areas of lipid accumulation in lobules. The first (healthy) state is characterized by intact lipid oxidation and a low amount of stored lipids. The second state in our model may correspond to the steatotic cell; it is marked by a high amount of stored lipids and a reduced lipid oxidation caused by impaired oxygen supply. Our model stresses the role of insufficient oxygen supply for the development of steatosis. We discuss implications of our results in regard to the experimental design aimed at exploring lipid metabolism reactions under steatotic conditions. Moreover, the model helps to understand the reversibility of lipid accumulation and predicts the reversible switch to show hysteresis. The system can switch from the steatotic state back to the healthy state by reduction of fatty acid uptake below the threshold at which steatosis started. The reversibility corresponds to the observation that caloric restriction can reduce the lipid content in the liver.     

Absence of exendin-4 effects on postprandial glucose and lipids in the Gila monster, <Emphasis Type="Italic">Heloderma suspectum</Emphasis>

Circulating nutrients serve as energy resources for functioning tissues throughout the body. While the tight regulation of plasma nutrients has been extensively studied in mammals, investigations into specific metabolic regulators in reptiles have been limited and have revealed conflicting results. The peptide exendin-4, which was isolated from the saliva of Gila monsters, Heloderma suspectum, has demonstrated prolonged plasma glucose-lowering properties in mammals. Although exendin-4 has often been labeled a venom protein, circulating plasma levels of exendin-4 have been shown to increase in response to feeding. Because exendin-4 has glucose-regulating effects in mammals, we hypothesized that post-prandial elevation in circulating exendin-4 levels in Gila monsters reduces plasma glucose and triglycerides. To examine the effect of exendin-4 on circulating nutrients, we measured plasma glucose, triglyceride, and cholesterol levels of Gila monsters in response to one of four treatments: fed live mice (a natural post-prandial increase in exendin-4), force-fed dead mice while anesthetized (no post-prandial exendin-4 increase), force-fed dead mice while anesthetized and injected with exendin-4 immediately after feeding (exogenous increase in exendin-4), and force-fed dead mice while anesthetized and injected with exendin-4 24 h after feeding (delayed exogenous increase in exendin-4). After prey ingestion, glucose and triglyceride levels increased significantly over time in all treatment groups, but there was no significant treatment effect. Plasma exendin-4 levels showed significant time and treatment effects, but did not correspond to glucose and triglyceride levels. Our results demonstrate that plasma nutrient levels in Gila monsters respond relatively slowly to feeding and that exendin-4 does not have the same effect on circulating glucose in Gila monsters as it does in mammals. Further studies are necessary to determine whether circulating exendin-4 has an alternate role in regulating other components of energy metabolism such as nutrient uptake rate in the small intestine.     

Reduced endoplasmic reticulum luminal calcium links saturated fatty acid-mediated endoplasmic reticulum stress and cell death in liver cells

Chronic exposure to elevated free fatty acids, in particular long chain saturated fatty acids, provokes endoplasmic reticulum (ER) stress and cell death in a number of cell types. The perturbations to the ER that instigate ER stress and activation of the unfolded protein in response to fatty acids in hepatocytes have not been identified. The present study employed H4IIE liver cells and primary rat hepatocytes to examine the hypothesis that saturated fatty acids induce ER stress via effects on ER luminal calcium stores. Exposure of H4IIE liver cells and primary hepatocytes to palmitate and stearate reduced thapsigargin-sensitive calcium stores and increased biochemical markers of ER stress over similar time courses (6 h). These changes preceded cell death, which was only observed at later time points (16 h). Co-incubation with oleate prevented the reduction in calcium stores, induction of ER stress markers and cell death observed in response to palmitate. Inclusion of calcium chelators, BAPTA-AM or EGTA, reduced palmitate- and stearate-mediated enrichment of cytochrome c in post-mitochondrial supernatant fractions and cell death. These data suggest that redistribution of ER luminal calcium contributes to long chain saturated fatty acid-mediated ER stress and cell death.     

Epiberberine regulates lipid synthesis through SHP (NR0B2) to improve non-alcoholic steatohepatitis

Epiberberine (EPI), extracted from Rhizome Coptidis, has been shown to attenuate hyperlipidemia in vivo. Herein we have studied the mechanism by which EPI is active against non-alcoholic steatohepatitis (NASH) using, mice fed on a methionine- and choline-deficient (MCD) diet and HepG2 cells exposed to free fatty acids (FFA). We show that small heterodimer partner (SHP) protein is key in the regulation of lipid synthesis. In HepG2 cells and in the livers of MCD-fed mice, EPI elevated SHP levels, and this was accompanied by a reduction in sterol regulatory element-binding protein-1c (SREBP-1c) and FASN. Therefore, EPI reduced triglyceride (TG) accumulation in steatotic hepatocytes, even in HepG2 cells treated with siRNA-SHP, and also improved microbiota. Thus, EPI suppresses hepatic TG synthesis and ameliorates liver steatosis by upregulating SHP and inhibiting the SREBP1/FASN pathway, and improves gut microbiome.