Antioxidative effects of Alisma orientale extract in palmitate-induced cellular injury

Context: Alisma orientale (Sam.) Juzepczuk (Alismataceae) is an indigenous medicinal herb that has been traditionally used for diuretic, hypolipidemic, anti-inflammatory, and antidiabetic proposes in northern and eastern Asia. Objective: This study examined the mechanisms underlying the cytoprotective effect of an aqueous extract of A. orientale (AEAO) against long-chain saturated fatty acid-induced cellular injury. Materials and methods: HepG2 cells were treated with 0.5 mM palmitate to generate a cellular model of nonalcoholic fatty liver disease (NAFLD). Using this cellular model, the cytoprotective effect of AEAO (100 μg/mL) against long-chain saturated fatty acid-induced cellular injury was evaluated by measuring the steatosis, ROS accumulation, and apoptosis. Results: AEAO significantly attenuated palmitate-induced intracellular steatosis and cellular damage up to 54 and 33%, respectively. Palmitate-induced intracellular levels of reactive oxygen species (ROS) and reactive aldehydes were significantly reduced in the presence of AEAO to 40 and 75%, respectively, suggesting that oxidative stress plays a role in the palmitate-induced damage. AEAO inhibited the palmitate-mediated activation of c-Jun NH(2)-terminal kinase (JNK), a kinase that is correlated with NAFLD. Inhibition of JNK by SP600125 or addition of AEAO significantly reduced palmitate-induced steatosis, ROS accumulation, and apoptosis, indicating that the protective effects of AEAO against palmitate-induced cellular damage result from blocking ROS-activated JNK signaling. Discussion and conclusion: The combined properties of AEAO in cellular steatosis and ROS production are beneficial for treating NAFLD, which includes complex metabolic changes, such that modulation of a single target is often not sufficient to achieve the desired therapeutic effect.     

Palmitate increases the susceptibility of cells to drug-induced toxicity: an in vitro method to identify drugs with potential contraindications in patients with metabolic disease

Fatty acids are an important source of energy. Excessive energy intake results in elevated levels of free fatty acids that are thought to be the pathogenic factors causing metabolic disorders such as dyslipidemia, obesity, insulin resistance, diabetes, and fatty liver. Underlying metabolic disorders have been suggested to be a predisposing factor for drug-induced liver injury. The steadily expanding population with metabolic disease may pose a higher risk for drug-induced toxicity. In order to understand the interaction of free fatty acids and drug-induced toxicity at the cellular level, we explored whether the saturated free fatty acid palmitate could modulate drug-induced cytotoxicity in HepG2 cells. A number of drugs known to induce hepatotoxicity in humans were selected to test this hypothesis. Drugs without reported hepatotoxicity were also tested to evaluate the specificity of the palmitate-induced effects. We demonstrate that palmitate, at sublethal concentrations, was able to potentiate the cytotoxicity and/or apoptosis induced by some but not all drugs tested. The palmitate and drug coincubation potentiated toxicity, which when combined with the plasma maximum concentration (C (max)), allowed us to identify idiosyncratic toxic drugs that were not flagged in previously deployed cytotoxicity assays. Our data suggest that treatment of cells with palmitate improves the sensitivity to detect compounds with risk of inducing idiosyncratic liver toxicity. Furthermore, this assay may be used to identify compounds that have higher safety risks in a population with metabolic syndrome.     

Effects of palmitate and astaxanthin on cell viability and proinflammatory characteristics of mesenchymal stem cells

Mesenchymal stem cells (MSCs) have broad immunomodulatory activities. These cells are a stable source of cytokine production such as interleukin-6 (IL6), monocyte chemoattractant protein-1 (MCP-1/CCL2) and vascular endothelial growth factor (VEGF). Fatty acid elevation in chronic metabolic diseases alters the microenvironment of MSCs and thereby, might affect their survival and cytokine production. In the present study, we investigated the effects of palmitate, the most abundant saturated free fatty acid (FFA) in plasma, and astaxanthin, a potent antioxidant, on cell viability and apoptosis in human bone marrow-driven mesenchymal stem cells. We also elucidated how palmitate and astaxanthin influence the inflammation in MSCs. Human mesenchymal stem cells were collected from an aspirate of the femurs and tibias marrow compartment. The effect of palmitate on cell viability, caspase activity and pro-inflammatory cytokines expression and secretion were evaluated. In addition, activation of the MAP kinases and NF-kB signaling pathways were investigated. The results showed that astaxanthin protected MSCs from palmitate-induced cell death. We found that palmitate significantly enhanced IL-6, VEGF and MCP-1 expression, and secretion in MSC cells. Increased cytokine expression was parallel to the enhanced phosphorylation of P38, ERK and IKKα-IKKβ. In addition, pretreatment with JNK, ERK, P38, and NF-kB inhibitors could correspondingly attenuate palmitate-induced expression of VEGF, IL-6, and MCP-1. Our results demonstrated that fatty acid exposure causes inflammatory responses in MSCs that can be alleviated favorably by astaxanthin treatment.     

Antioxidative effects of Alisma orientale extract in palmitate-induced cellular injury

Context: Alisma orientale (Sam.) Juzepczuk (Alismataceae) is an indigenous medicinal herb that has been traditionally used for diuretic, hypolipidemic, anti-inflammatory, and antidiabetic proposes in northern and eastern Asia.

Objective: This study examined the mechanisms underlying the cytoprotective effect of an aqueous extract of A. orientale (AEAO) against long-chain saturated fatty acid–induced cellular injury.

Materials and methods: HepG2 cells were treated with 0.5 mM palmitate to generate a cellular model of nonalcoholic fatty liver disease (NAFLD). Using this cellular model, the cytoprotective effect of AEAO (100 µg/mL) against long-chain saturated fatty acid–induced cellular injury was evaluated by measuring the steatosis, ROS accumulation, and apoptosis.

Results: AEAO significantly attenuated palmitate-induced intracellular steatosis and cellular damage up to 54 and 33%, respectively. Palmitate-induced intracellular levels of reactive oxygen species (ROS) and reactive aldehydes were significantly reduced in the presence of AEAO to 40 and 75%, respectively, suggesting that oxidative stress plays a role in the palmitate-induced damage. AEAO inhibited the palmitate-mediated activation of c-Jun NH₂-terminal kinase (JNK), a kinase that is correlated with NAFLD. Inhibition of JNK by SP600125 or addition of AEAO significantly reduced palmitate-induced steatosis, ROS accumulation, and apoptosis, indicating that the protective effects of AEAO against palmitate-induced cellular damage result from blocking ROS-activated JNK signaling.

Discussion and conclusion: The combined properties of AEAO in cellular steatosis and ROS production are beneficial for treating NAFLD, which includes complex metabolic changes, such that modulation of a single target is often not sufficient to achieve the desired therapeutic effect.     

NO-1886 ameliorates glycogen metabolism in insulin-resistant HepG2 cells by GSK-3β signalling

Objectives The aim of the study was to elucidate the possible role and mechanism of NO‐1886 (ibrolipim, a lipoprotein lipase activator) in ameliorating insulin resistance induced by high palmitate. Methods HepG2 cells were cultured in RPMI 1640 medium and were treated with palmitate to induce insulin resistance. Free fatty acids (FFAs), glucose, glycogen, cell viability and mRNA and protein levels were analysed separately. Key findings We found that HepG2 cells treated with 0.5 mm palmitate for 48 h led to a significant decrease of insulin‐induced glucose consumption (from 2.89 ± 0.85 mm in the control to 0.57 ± 0.44 mm in palmitate). Insulin resistance (IR) of HepG2 cells was induced by 0.5 mm palmitate for 48 h. NO‐1886 stimulated glucose consumption, glycogen synthesis and FFA absorption in insulin‐resistant HepG2 cells. Maximum stimulation effects were observed with 10 µm NO‐1886 for 24 h. Compared with the dimethyl sulfoxide‐treated group, 2.5 µm NO‐1886 or higher could induce the mRNA expression of lipoprotein lipase. Meanwhile, NO‐1886 increased the protein content of P‐GSK‐3βser⁹ and decreased the protein level of GSK‐3β in insulin‐resistant HepG2 cells, but NO‐1886 didn't change the protein levels of PI3‐Kp85 and Akt2. Conclusion Lipoprotein lipase activator NO‐1886 could increase glycogen synthesis in HepG2 cells and could ameliorate the insulin resistance, which was associated with GSK‐3 signalling.     

Sensitization of vascular smooth muscle cell to TNF-alpha-mediated death in the presence of palmitate

Saturated free fatty acids (FFAs), including palmitate, can activate the intrinsic death pathway in cells. However, the relationship between FFAs and receptor-mediated death pathway is still unknown. In this study, we have investigated whether FFAs are able to trigger receptor-mediated death. In addition, to clarify the mechanisms responsible for the activation, we examined the biochemical changes in dying vascular smooth muscle cell (VSMC) and the effects of various molecules to the receptor-mediated VSMC death. Tumor necrosis factor (TNF)-alpha-mediated VSMC death occurred in the presence of sub-cytotoxic concentration of palmitate as determined by assessing viability and DNA degradation, while the cytokine did not influence VSMC viability in the presence of oleate. The VSMC death was inhibited by the gene transfer of a dominant-negative Fas-associated death domain-containing protein and the baculovirus p35, but not by the bcl-xL or the c-Jun N-terminal kinase (JNK) binding domain of JNK-interacting protein-1, in tests utilizing recombinant adenoviruses. The VSMC death was also inhibited by a neutralizing anti-TNF receptor 1 antibody, the caspase inhibitor z-VAD, and the cathepsin B inhibitor CA074, a finding indicative of the role of both caspases and cathepsin B in this process. Consistent with this finding, caspase-3 activation and an increase in cytosolic cathepsin B activity were detected in the dying VSMC. Palmitate inhibited an increase of TNF-alpha-mediated nuclear factor kappa B (NF-kappaB) activity, the survival pathway activated by the cytokine, by hindering the translocation of the NF-kappaB subunit of p65 from the cytosol into the nucleus. The gene transfer of inhibitor of NF-kappaB predisposed VSMC to palmitate-induced cell death. To the best of our knowledge, this study is the first report to demonstrate the activation of TNF-alpha-mediated cell death in the presence of palmitate. The current study proposes that FFAs would take part in deleterious vascular consequences of such patients with elevated levels of FFAs as diabetics and obese individuals via the triggering of receptor-mediated death pathways of VSMC.     

JNK在棕榈酸造成骨骼肌细胞胰岛素抵抗中的作用

目的:探讨棕榈酸造成L6GLUT4myc骨骼肌细胞胰岛素抵抗的机制中,c-Jun N-末端激酶(JNK)的作用。方法:第一部分将细胞分两组,分别用0.3 mmol／L的棕榈酸盐或用溶剂牛血清白蛋白（BSA）孵育16 h;第二部分在如上处理的后30 min加入JNK的抑制剂,用或不用胰岛素刺激后,检测细胞膜上GLUT4myc的含量以及蛋白激酶B(Akt)、JNK和胰岛素受体底物1（IRS1）的磷酸化。结果：与溶剂组相比,棕榈酸组中胰岛素刺激的GLUT4myc转位和Akt的磷酸化降低(P<0.05);JNK和IRS1Ser307的磷酸化水平则没有变化。结论：棕榈酸导致L6骨骼肌细胞胰岛素抵抗的机制可能不涉及JNK或JNK的作用很小。     

软脂酸诱导的HepG2细胞糖脂代谢紊乱及机制研究

目的探讨软脂酸诱导HepG2细胞糖脂代谢紊乱特征及二甲双胍和辛伐他丁对糖脂代谢的影响。方法以软脂酸诱导HepG2细胞产生糖脂代谢紊乱,用二甲双胍和辛伐他丁进行治疗,检测各组糖原、总胆固醇、甘油三酯累积量,用荧光定量PCR检测糖脂代谢相关基因AMP活化蛋白激酶(AMPK)α-2、葡萄糖转运体(GLUT)2、肝脂肪酶(HL)、磷酸烯醇丙酮酸羧激酶(PEPCK)、固醇调节元件结合蛋白(SREBP)1、乙酰辅酶A羧化酶(ACC)2等的表达。结果软脂酸诱导HepG2细胞总胆固醇、甘油三酯升高(P<0.05),糖脂代谢相关基因表达异常。二甲双胍和辛伐他丁治疗后,细胞总胆固醇、甘油三酯累积明显下降,糖原累积上升;AMPKα-2、GLUT2、HL mRNA表达上升,G6pase、PEPCK、SREBP1、ACC2 mRNA表达下降。结论软脂酸诱导HepG2细胞出现糖脂代谢异常,经二甲双胍和辛伐他丁治疗后异常情况改善。     

Salsalate and adiponectin ameliorate hepatic steatosis by inhibition of the hepatokine fetuin-A

Fetuin-A was recently identified as a novel hepatokine which is associated with obesity, insulin resistance and non-alcoholic fatty liver disease. Salsalate, a prodrug of salicylate with an anti-inflammatory effect and lower side effect profile, significantly lowers glucose and triglyceride levels, and increased adiponectin concentrations in randomized clinical trials. In this study, we examined the effects and regulatory mechanisms of salsalate and full length-adiponectin (fAd) on fetuin-A expression, steatosis and lipid metabolism in palmitate-treated HepG2 cells. Incubation of hepatocytes with palmitate significantly increased fetuin-A and SREBP-1c expression which lead to steatosis and knock-down of fetuin-A by siRNA restored these changes. Salsalate significantly down-regulated palmitate-induced fetuin-A mRNA expression and secretion in a dose- and time-dependent manner. Inhibition of palmitate-induced fetuin-A by salsalate was mediated by AMPK-mediated reduction of NFκB activity, which was blocked by AMPK siRNA or an inhibitor of AMPK. Salsalate attenuated the excessive steatosis by palmitate through SREBP-1c regulation in hepatocytes. Furthermore, fAd also showed suppression of palmitate-induced fetuin-A through the AMPK pathway and improvement of steatosis accompanied by restoration of SREBP-1c, PAPR-α and CD36. In preliminary in vivo experiments, salsalate treatment inhibited high fat diet (HFD)-induced steatosis as well as fetuin-A mRNA and protein expression in SD rats. In conclusion, salsalate and fAd improved palmitate-induced steatosis and impairment of lipid metabolism in hepatocytes via fetuin-A inhibition through the AMPK-NFκB pathway.     

The roles of Akt and MAPK family members in silymarin's protection against UV-induced A375-S2 cell apoptosis

Silymarin is a polyphenolic flavonoid derived from milk thistle (Silybum marianum) and has anti-inflammatory, cytoprotective as well as anticarcinogenic effects [Manna, S.K., Mukhopadlhyay, A., Van, N.T., Aggarwal, B., Silymarin suppresses TNF-induced activation of NF-kappaB, c-Jun N-terminal kinase, and apoptosis. J. Immunol. 1999; 163, 6800-6809.]. In this study, we assessed the effect of silymarin on ultraviolet light (UV)-induced cell apoptosis in human malignant melanoma, A375-S2 cells. Silymarin pre-treatment reversed the effect of UV irradiation on the expression of phosphorylated Akt and phosphorylated p53 (regulated by Akt activation), followed by down-regulation of Bax and up-regulated expressions of Bcl-2 and Bcl-xL proteins in UV-irradiated A375-S2 cells. Akt inhibitor decreased the viability of UV-irradiated cells which was treated with silymarin. In addition, the effect of UV irradiation on the phosphorylation of mitogen-activated protein kinase (MAPK) family members [extracellular signal-regulated kinase (ERK), p38 and c-Jun N-terminal kinase (JNK)] was also reversed by silymarin. Moreover, ERK inhibitor (PD98059) and p38 inhibitor (SB203580) augmented UV-induced apoptosis in silymarin treated A375-S2 cells. Consequently, silymarin partially reduced UV-induced apoptosis by activating the Akt pathway, and silymarin's protective effect was also exerted by MAPK family members.     

Protective effect of curcumin, silymarin and N-acetylcysteine on antitubercular drug-induced hepatotoxicity assessed in an in vitro model

Tuberculosis (TB) is highly endemic in India. The first-line anti-TB therapy (ATT) involving isoniazid (INH), rifampicin and pyrazinamide causes hepatotoxicity in approximately 11.5% of Indian patients. Studies have shown that ATT-induced hepatotoxicity is primarily due to oxidative stress caused by the drugs and metabolites. Herbal drugs with antioxidative properties have been tested in animal studies and clinical trials for the management of hepatotoxicity. The objective of this study was to investigate the role of curcumin (CUR), silymarin (SILY) and N-acetylcysteine (N-ACET) on hepatotoxicity by ATT drugs using an in vitro model of human hepatocellular carcinoma cell line (HepG2). HepG2 cells were treated with ATT drugs alone or along with CUR, SILY or N-ACET for a 48-h duration. The cells were monitored for viability, morphology, respiring mitochondria and cell cycle. Our results suggest that the presence of hepatoprotective drugs during treatment of HepG2 cells with ATT drugs lowers the hepatotoxic effect of the latter. This is observed in terms of (a) increased cell viability, (b) healthy-looking cell morphology as revealed by phase contrast microscopy, (c) active respiring cells as observed with confocal microscopy upon staining with a mitochondrial membrane-specific dye, MitoTracker(?) Red, and reduction in the sub-G(1) peak in cell cycle analysis by flow cytometry. Our results suggest that these hepatoprotective drugs need to be further explored as potential adjuvant therapy along with ATT drugs.     

Benzofuran pyran compound rescues rat and human osteoblast from lipotoxic effect of palmitate by inhibiting lipid biosynthesis and promoting stabilization of RUNX2

Obesity and ageing increases bone marrow fat which in turn is associated with lower bone mass. Marrow adipocytes by secreting cytokines, adipokines and free fatty acids change the bone marrow milieu and thus the number of osteoblasts. Palmitate is the common saturated fatty acid, an unavoidable ingredient we consume with food, which kindles cell apoptosis. Compound 4e is osteogenic in nature. We examine the effect of compound 4e in palmitate induced lipotoxicity in rat osteoblasts. Design of benzofuran Pyran hybrid compound (4e) was found to be effective in inhibiting palmitate induced cell apoptosis. In this study an in vitro model of palmitate was contrived. Anti-apoptotic effect of compound 4e was assessed by Annexin/PI and LDH (Lactate dehydrogenase) assay. Compound 4e also increased osteoblast differentiation and mineralization. It also increased expression of osteogenic markers (RUNX2 and BMP2), assessed by Real time PCR and immunofluorescence, which was impeded by palmitate. Acetyl Co-Carboxylase (ACC) and Fatty acid synthase (FAS), two prominent mediators of lipid biosynthesis were increased by palmitate exposure. Compound 4e modulated lipid biosynthesis by inhibiting ACC and FAS as reflected visually and after quantification of less lipid droplet formation suggesting that 4e is osteogenic and simultaneously anti-lipotoxic.     

Glucagon-like peptide 1 receptor plays an essential role in geniposide attenuating lipotoxicity-induced β-cell apoptosis

β-Cell apoptosis is considered to be a major cause of loss of β cells in diabetes. Geniposide could prevent oxidative stress-induced neuron apoptosis, and improved glucose stimulated insulin secretion by activating glucagon-like peptide 1 receptor (GLP-1R) in INS-1 cells. Here we have investigated whether geniposide can exert a direct effect against pancreatic β-cell lipoapoptosis. The results indicated that pretreatment pancreatic INS-1 cells with geniposide for 7 h attenuated palmitate-induced β-cell apoptosis and active caspase-3 expression, but this effect was disappeared at 18 h. Long-term incubation with palmitate decreased GLP-1R expression in INS-1 cells, and exendin (9-39), an antagonist for GLP-1R, inhibited the effect of geniposide on palmitate-induced apoptosis in INS-1 cells. Moreover, geniposide also improved the impairment of GLP-1R signaling through enhancing the phosphorylation of Akt and Foxo1, and increased the expression of PDX-1 in palmitate-treated INS-1 cells. These results suggest that geniposide inhibits early stage of lipotoxicity-induced β-cell apoptosis, and GLP-1R plays a critical role in geniposide counteracting the action of lipotoxicity in INS-1 pancreatic β cells.     

Effects of silymarin and its combination with succinic acid on brain bioenergetics in rats with experimental inhibition of beta-oxidation of fatty acids

In the tests on rats with a model of encephalopathy caused by 4-pentenoic acid (inhibitor of the beta-oxidation of fatty acids), the hepatoprotective agent silymarin increases the respiratory activity in brain mitochondria, improves oxidative phosphorylation coupling and energization, and inhibits lipid peroxidation. Succinic acid (a regulator of bioenergetics) improves the damaged Krebs cycle metabolic pathways and produces an antioxidant effect. The combined use of silymarin and succinic demonstrated more expressed cerebroprotective action as compared to that of each agent administered separately.     

Troglitazone does not protect rat pancreatic beta cells against free fatty acid-induced cytotoxicity

Thiazolidinediones are a novel class of antidiabetic drugs that reduce insulin resistance through interaction with nuclear peroxisome proliferator-activated receptor (PPAR)gamma. One of these agents, troglitazone, was also proposed to protect beta cells against FFA-induced toxicity, but this effect has not yet been directly demonstrated. We recently reported in vitro conditions under which free fatty acids (FFA) cause beta cell death by necrosis or apoptosis. The present study investigates whether troglitazone (10 microM) interferes with this FFA-induced toxicity. Addition of this compound did not protect against oleate- or palmitate-induced toxicity. On the contrary, it increased palmitate-induced necrosis during the first two days of culture, and elevated (increase by 10-20%, P<0.05) both oleate- and palmitate-induced apoptosis after 8 days. These results do not support the view that troglitazone exerts a direct protective effect on beta cells that are exposed to cytotoxic FFA concentrations. They instead indicate that the agent may sensitize pancreatic beta cells to FFA-induced damage, raising the possibility that its use facilitates the deleterious effect of increased FFA levels on the pancreatic beta cell mass.     

阿魏酸抑制p38 MAPK保护棕榈酸诱导的肝细胞脂毒性

目的:研究阿魏酸对棕榈酸诱导的HepG2肝细胞脂毒性的保护作用并初步探究其分子机制。方法:采用棕榈酸诱导HepG2肝细胞建立脂毒性模型并给予阿魏酸进行干预,采用LDH法检测细胞损伤,采用MTT法检测细胞存活率。采用Western Blotting技术对阿魏酸保护作用的分子机制进行研究。结果:不同浓度(25、50、100、200μmol/L)阿魏酸暴露对肝细胞无毒性作用(P>0.05)。阿魏酸干预显著抑制棕榈酸诱导的肝细胞损伤并改善棕榈酸诱导的细胞线粒体膜电位降低(P<0.05)。激活p38可显著增强棕榈酸诱导的肝细胞脂毒性(P<0.05),而抑制p38显著改善棕榈酸诱导的细胞损伤(P<0.05)。此外,阿魏酸显著抑制棕榈酸上调的p38磷酸化(P<0.05),采用p38激活剂处理细胞可阻断阿魏酸对脂毒性的保护作用(P<0.05)。结论:阿魏酸可有效改善脂毒性诱导的肝细胞损伤,该保护作用可能与其抑制p38信号通路有关。阿魏酸可能成为防治以脂毒性为主要病理特征性肝病的功效因子。     

Protective potential [correction of potencial] of Euphorbia hirta against cytotoxicity induced in hepatocytes and a HepG2 cell line

Medicinal plants play a key role in human health care. Frustration over the side effects of allopathic drugs has driven the medical world to take asylum in the plant kingdom for the treatment of various ailments. Euphorbia hirta belonging to the family of Euphorbiacae has been reported to possess antibacterial, antiviral, and anticancer activity. The aim of the present study was to investigate the protective effect of E. hirta against antitubercular drug-induced cytotoxicity in freshly isolated hepatocytes. The extent of cytotoxicity of the plant extracts was also analyzed using human liver derived HepG2 cell line by estimating the viability of cells (MTT assay). The alcoholic plant extract normalized the levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), lactate dehydrogenase (LDH), triacylglycerol (TAG), cholesterol, total protein, albumin, total and direct bilirubin, which were altered due to antitubercular drug intoxication. A dose-dependent increase in percent viability was observed when antitubercular drug exposed HepG2 cells were treated with different concentrations of plant extracts (125, 250, 500 and 1000 microg/mL) which were compared with a standard hepatoprotective drug silymarin. The highest percentage viability of HepG2 was observed at a concentration of 1000 microg/mL. The results suggest that E. hirta exerts protection against antitubercular drug-induced cytotoxicity in this vitro model system.     

Resveratrol protects muscle cells against palmitate-induced cellular senescence and insulin resistance through ameliorating autophagic flux

Skeletal muscle, a highly metabolic tissue, is particularly vulnerable to increased levels of saturated free fatty acids (FFAs). The role of autophagy in saturated FFAs-induced cellular senescence and insulin resistance in skeletal muscle remains unclear. Therefore, the present study was aimed to explore autophagic flux in cellular senescence and insulin resistance induced by palmitate in muscle cells, and whether resveratrol limited these responses. Our results showed that palmitate induced cellular senescence in both myoblasts and myotubes. In addition, palmitate delayed differentiation in myoblasts and inhibited expression of insulin-stimulated p-AKTSer473 in myotubes. The accumulations of autophagosome assessed by tandem fluorescent-tagged LC3 demonstrated that autophagic flux was impaired in both palmitate-treated myoblasts and myotubes. Resveratrol protected muscle cells from palmitate-induced cellular senescence, apoptosis during differentiation, and insulin resistance via ameliorating autophagic flux. The direct influence of autophagic flux on development of cellular senescence and insulin resistance was confirmed by blockage of autophagic flux with chloroquine. In conclusion, impairment of autophagic flux is crucial for palmitate-induced cellular senescence and insulin resistance in muscle cells. Restoring autophagic flux by resveratrol could be a promising approach to prevent cellular senescence and ameliorate insulin resistance in muscle.     

Ceramide formation as a target in beta-cell survival and function

INTRODUCTION: Ceramide may be synthesized de novo or generated by sphingomyelinase-dependent hydrolysis of sphingomyelin. AREAS COVERED: The role of ceramide, ceramide-sensitive signaling and ion channels in β-cell apoptosis, lipotoxicity and amyloid-induced β-cell death. EXPERT OPINION: Ceramide participates in β-cell dysfunction and apoptosis after exposure to TNFα, IL-1β and IFN-γ, excessive amyloid and islet amyloid polypeptide or non-esterified fatty acids (lipotoxicity). Knockout of sphingomyelin synthase 1, which converts ceramide to sphingomyelin, leads to impairment of insulin secretion. Increased ceramidase activity or pharmacological inhibition of ceramide synthetase, inhibits β-cell apoptosis. Ceramide contributes to endoplasmatic reticulum (ER) stress, decreased mitochondrial membrane potential in insulin-secreting cells and mitochondrial release of cytochrome c into the cytosol, which are all triggers of apoptotic cell death. Ceramide-dependent signaling involves activation of extracellularly regulated kinases 1 and 2 (ERK1/2), downregulation of Period (Per)-aryl hydrocarbon receptor nuclear translocator (Arnt)-single-minded (Sim) kinase (PASK), activation of okadaic-acid-sensitive protein phosphatase 2A (PP2A) and stimulation of NADPH-oxidase with generation of superoxides and lipid peroxides. Ceramide reduces the activity of voltage gated potassium (Kv)-channels in insulin-secreting cells. The role of ceramide in β-cell survival and function may be therapeutically relevant, because ceramide formation can be suppressed by pharmacological inhibition of ceramide synthetase and/or sphingomyelinase.     

Cellular glutathione in fatty liver in vitro models

The range of non-alcoholic fatty liver disease (NAFLD) includes simple hepatic steatosis, the inflammatory non-alcoholic steatohepatitis (NASH), fibrosis and cirrhosis. The accumulation of specific lipids in hepatocytes has been associated with oxidative stress and progression of the disease. Elevated serum free fatty acids and hepatocyte lipotoxicity can be studied in an in vitro cellular model. For this purpose, we cultured the human liver cell line, HepG2/C3A, in medium supplemented with increasing amounts of oleic acid (C18:1) and evaluated oxidative stress by measuring the content of the cellular antioxidant, glutathione (GSH). We observed a dose-dependent steatosis, as determined by Nile Red staining, with concurrent increases of GSH; similar findings were also observed in cultured human hepatocytes. Cells cultured with palmitic acid (C16:0) or the combination oleic/palmitic acids (2:1 ratio) also exhibited a dose-dependent increase of GSH; however palmitic-supplemented cultures did not sustain the GSH increase after 24 h. We also detected an increase in the formation of lipid peroxides (LPO) indicating that the increase of GSH was a cellular mechanism that may be related to the high exposure of fatty acids. The results of this in vitro study suggest an antioxidant response against fat overloading and indicate potential differences in response to specific fatty acid-induced hepatic steatosis and associated lipotoxicity.