Palmitate-induced Lipoapoptosis Research Articles

Monounsaturated fatty acids protect against palmitate-induced lipoapoptosis in human umbilical vein endothelial cells

Diets high in saturated fatty acids are linked to increased cardiovascular disease risk, whereas monounsaturated fatty acids have been associated with improved cardiovascular outcomes. Accordingly, cell culture studies have demonstrated that saturated fatty acids, particularly long chain saturated fatty acids such as palmitate, induce dysfunction and cell death in a variety of cell types, and monounsaturated fatty acids may confer protection against palmitate-mediated damage. The aim of the present study was to examine whether monounsaturated fatty acids could protect against palmitate-mediated cell death in endothelial cells, to determine if AMPK inactivation and activation (via compound C and AICAR, respectively) underlies both palmitate-induced damage and monounsaturated fatty acid-mediated protection, and to explore the role of ER stress in this context. Human umbilical vein endothelial cells were examined for cell viability and apoptosis following treatment for 24 hours with palmitate (0.25 and 0.5mM) alone or in combination with the monounsaturated fatty acids oleate or palmitoleate (0.25 and 0.5mM), AICAR, compound C, 4μ8C, or TUDCA. Compared to control cells, palmitate significantly decreased cell viability and increased apoptosis in a dose-dependent manner. The monounsaturated fatty acids oleate and palmitoleate completely prevented the cytotoxic effects of palmitate. Although palmitate induced markers of ER stress, chemical inhibition of ER stress did not prevent palmitate-induced lipoapoptosis. Conversely, the AMPK activator AICAR (0.1 and 0.5mM) conferred protection from palmitate mediated-alterations in viability, apoptosis and ER stress, whereas the AMPK inhibitor compound C (20 and 40μM) significantly exacerbated palmitate-mediated damage. Lastly, co-incubation with palmitate, monounsaturated fatty acids, and compound C significantly mitigated the protective effects of both oleate and palmitoleate. In conclusion, monounsaturated fatty acids confer protection against the cytotoxic effects of palmitate in vascular endothelial cells; and palmitate-mediated damage, as well as monounsaturated-mediated protection, are due in part to inactivation and activation, respectively, of the metabolic regulator AMPK. These results may have implications for understanding the deleterious effects of high saturated fat diets on cardiovascular dysfunction and disease risk.

The functional role of Bax/Bak in palmitate-induced lipoapoptosis

Induction of programmed cell death, mainly apoptosis (lipoapoptosis) is a major cellular consequence of the lipotoxicity, a harmful effect resulting from the overload of lipids. Both Endoplasmic reticulum (ER) stress and autophagy have been suggested to play important role in the regulation of lipoapoptosis. However, the exact mechanisms underlying lipoapoptosis remain unclear. In the present study, we aimed to investigate the functional role of Bax/Bak in lipoapoptosis using mouse embryonic fibroblasts (MEFs) cell culture model. Results showed that palmitate induced caspase-dependent apoptosis in wild-type Bax/Bak MEF cells, whereas a caspase-independent cell death was induced by palmitate in Bax/Bak knockout MEF cells, suggesting requirement of Bax/Bak in palmitate-induced caspase activation. More importantly, we found that the status of Bax/Bak is a determinant that governs the decision between the pro-survival or pro-death function of autophagy in response to palmitate exposure, and Bax/Bak is required for palmitate-induced activation of endoplasmic reticulum (ER) stress and subsequently ER stress-mediated apoptosis. The findings of the present study provided novel insights into understanding the mechanisms involved in the regulation of palmitate-induced lipoapoptosis.

Glycycoumarin inhibits hepatocyte lipoapoptosis through activation of autophagy and inhibition of ER stress/GSK-3-mediated mitochondrial pathway.

Herbal medicine as an alternative approach in the treatment of disease has drawn growing attention. Identification of the active ingredient is needed for effective utilization of the herbal medicine. Licorice is a popular herbal plant that is widely used to treat various diseases including liver diseases. Glycycoumarin (GCM) is a representative of courmarin compounds isolated from licorice. In the present study, the protective effect of GCM on hepatocyte lipoapoptosis has been evaluated using both cell culture model of palmitate-induced lipoapoptosis and animal model of non-alcoholic steatohepatitis (NASH). The results demonstrated for the first time that GCM was highly effective in suppressing hepatocyte lipoapoptosis in both in vitro and in vivo. Mechanistically, GCM was able to re-activate the impaired autophagy by lipid metabolic disorders. In line with the activation of autophagy, ER stress-mediated JNK and mitochondrial apoptotic pathway activation was inhibited by GCM both in vitro and in vivo. In addition, inactivation of GSK-3 might also contribute to the protective effect of GCM on hepatocyte lipoapoptosis. Our findings supported GCM as a novel active component of licorice against non-alcoholic fatty liver disease (NAFLD).

Metformin attenuates palmitate-induced endoplasmic reticulum stress, serine phosphorylation of IRS-1 and apoptosis in rat insulinoma cells.

Lipotoxicity refers to cellular dysfunctions caused by elevated free fatty acid levels playing a central role in the development and progression of obesity related diseases. Saturated fatty acids cause insulin resistance and reduce insulin production in the pancreatic islets, thereby generating a vicious cycle, which potentially culminates in type 2 diabetes. The underlying endoplasmic reticulum (ER) stress response can lead to even β-cell death (lipoapoptosis). Since improvement of β-cell viability is a promising anti-diabetic strategy, the protective effect of metformin, a known insulin sensitizer was studied in rat insulinoma cells. Assessment of palmitate-induced lipoapoptosis by fluorescent microscopy and by detection of caspase-3 showed a significant decrease in metformin treated cells. Attenuation of β-cell lipotoxicity was also revealed by lower induction/activation of various ER stress markers, e.g. phosphorylation of eukaryotic initiation factor 2α (eIF2α), c-Jun N-terminal kinase (JNK), insulin receptor substrate-1 (IRS-1) and induction of CCAAT/enhancer binding protein homologous protein (CHOP). Our results indicate that the β-cell protective activity of metformin in lipotoxicity can be at least partly attributed to suppression of ER stress.

ER calcium release promotes mitochondrial dysfunction and hepatic cell lipotoxicity in response to palmitate overload.

Palmitate overload induces hepatic cell dysfunction characterized by enhanced apoptosis and altered citric acid cycle (CAC) metabolism; however, the mechanism of how this occurs is incompletely understood. We hypothesize that elevated doses of palmitate disrupt intracellular calcium homeostasis resulting in a net flux of calcium from the ER to mitochondria, activating aberrant oxidative metabolism. We treated primary hepatocytes and H4IIEC3 cells with palmitate and calcium chelators to identify the roles of intracellular calcium flux in lipotoxicity. We then applied 13C metabolic flux analysis (MFA) to determine the impact of calcium in promoting palmitate-stimulated mitochondrial alterations. Co-treatment with the calcium-specific chelator BAPTA resulted in a suppression of markers for apoptosis and oxygen consumption. Additionally, 13C MFA revealed that BAPTA co-treated cells had reduced CAC fluxes compared to cells treated with palmitate alone. Our results demonstrate that palmitate-induced lipoapoptosis is dependent on calcium-stimulated mitochondrial activation, which induces oxidative stress.

Saturated Free Fatty Acid Sodium Palmitate-Induced Lipoapoptosis by Targeting Glycogen Synthase Kinase-3β Activation in Human Liver Cells

Elevated serum saturated fatty acid levels and hepatocyte lipoapoptosis are features of nonalcoholic fatty liver disease (NAFLD). The purpose of this study was to investigate saturated fatty acid induction of lipoapoptosis in human liver cells and the underlying mechanisms. Human liver L02 and HepG2 cells were treated with sodium palmitate, a saturated fatty acid, for up to 48h with or without lithium chloride, a glycogen synthase kinase-3β (GSK-3β) inhibitor, or GSK-3β shRNA transfection. Transmission electron microscopy was used to detect morphological changes, flow cytometry was used to detect apoptosis, a colorimetric assay was used to detect caspase-3 activity, and western blot analysis was used to detect protein expression. The data showed that sodium palmitate was able to induce lipoapoptosis in L02 and HepG2 cells. Western blot analysis showed that sodium palmitate activated GSK-3β protein, which was indicated by dephosphorylation of GSK-3β at Ser-9. However, inhibition of GSK-3β activity with lithium chloride treatment or knockdown of GSK-3β expression with shRNA suppressed sodium palmitate-induced lipoapoptosis in L02 and HepG2 cells. On a molecular level, inhibition of GSK-3β expression or activity suppressed sodium palmitate-induced c-Jun-N-terminal kinase (JNK) phosphorylation and Bax upregulation, whereas GSK-3β inhibition did not affect endoplasmic reticulum stress-induced activation of unfolded protein response. The present data demonstrated that saturated fatty acid sodium palmitate-induced lipoapoptosis in human liver L02 and HepG2 cells was regulated by GSK-3β activation, which led to JNK activation and Bax upregulation. This finding indicates that GSK-3β inhibition may be a potential therapeutic target to control NAFLD.

The protective effect of berberine on β-cell lipoapoptosis

To evaluate the protective effect of berberine on β-cell lipoapoptosis induced by palmitate and to explore the possible underlying mechanisms. HIT-T15 pancreatic β-cells were divided into the following treatment groups: untreated controls; 100 μM berberine; 0.5 mM palmitate; 0.5 mM palmitate + 0.1 μM berberine; 0.5 mM palmitate + 1 μM berberine; 0.5 mM palmitate + 10 μM berberine; and 0.5 mM palmitate + 100 μM berberine. After 48 h, cell apoptosis was assessed by flow cytometry and the Hoechst 33258 fluorescent assay. Basal and glucose-stimulated insulin levels in culture medium were measured by radioimmunoassay. Peroxisome proliferator- activated receptor-γ (PPAR-γ) mRNA and protein levels were determined by real-time PCR and immunocytochemistry, respectively. Apoptosis was significantly increased upon treatment with palmitate as compared to the untreated controls (p<0.0001). In addition, glucose-stimulated insulin secretion (GSIS), PPAR-γ mRNA and protein expression were significantly reduced in response to palmitate (p<0.0001); however, palmitate-induced apoptosis and reduction in PPAR-γ expression were reversed in response to berberine in a dose-dependent manner (p<0.05). Furthermore, there was a non-significant increase in GSIS with increasing berberine dose. Palmitate exerted lipotoxic effects on HIT-T15 cells, inducing apoptosis and reducing GSIS. Berberine reduced palmitate-induced lipoapoptosis and tended to increase GSIS in HIT-T15 cells, possibly through increased PPAR-γ expression.

The role of G protein-coupled receptor 40 in lipoapoptosis in mouse -cell line NIT-1

Free fatty acids (FFAs) exert divergent effects on beta-cells. Acute exposure to FFAs stimulates insulin secretion, whereas chronic exposure impairs beta-cell function and induces apoptosis. The G protein-coupled receptor 40 (GPR40) is preferentially expressed in beta-cells and is activated by a wide range of FFAs. In this study, we used small interfering RNA technology and apoptosis assay in mouse beta-cell NIT-1 to address the role of GPR40 in beta-cell lipoapoptosis and function. Results showed that palmitate induced beta-cell apoptosis, which was not mediated through GPR40, whereas oleate protected NIT-1 cells from palmitate-induced lipoapoptosis, which was mediated at least in part through GPR40. Moreover, by detecting the activation of the phosphatidylinositol 3-kinase and MAP kinase (MAPK) pathways, we found that oleate promoted the activation of extracellular signal-regulated protein kinase-MAPK pathway mainly via GPR40, increased the expression of early growth response gene-1, leading to the anti-lipoapoptotic effect on NIT-1 cells. It was suggested that GPR40 might be implicated in the control of beta-cell mass plasticity and GPR40 probably provide a link between obesity and type 2 diabetes.