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

Abstract

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: In sulin-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-dimethlthiazol-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. Triglyceridecontent (control cells; 14.5 ng/μg protein) increased up to 10-fold following incubation in NEFA (oleic acid 153.2 ng/μg 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 >65C), 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.

Endoplasmic reticulum stress contributes to beta cell apoptosis in type 2 diabetes

“…8 The triggering of ER stress by palmitate is probably not due to nitric oxide generation, which is not apparent in pure beta cell populations such as MIN6 cells [19,23]. Neither is deposition of the saturated triacylglycerol, tripalmitin, in ER [41] likely to be relevant, since, at the relatively low palmitate/BSA ratios used here, the increase in triacylglycerol mass observed in MIN6 cells is due primarily to the incorporation of unsaturated fatty acid side-chains, as the increase in triacylglycerol levels was abolished by desaturase inhibitors [19]. Increased formation of ceramide is a possibility since this is implicated in beta cell lipotoxicity [5,6], and in other cell-types ceramide has been shown to perturb ER Ca 2+ homeostasis [42].…”

Diazoxide-induced β-cell rest reduces endoplasmic reticulum stress in lipotoxic β-cells

“…The findings indicate that other mechanisms for palmitate-induced apoptosis, ER stress related or not, are operational in the pancreatic b-cell. Among such mechanisms, tripalmitin formation has been proposed (Moffitt et al 2005). Esterification of the fatty acid may not necessarily be b-cell toxic, however (Diakogiannaki et al 2007).…”

Palmitate modulates intracellular signaling, induces endoplasmic reticulum stress, and causes apoptosis in mouse 3T3-L1 and rat primary preadipocytes

“…Cells were grown in 96-well plates and treated with indicated conditions. Cell viability was measured using the MTT kit according to the manufacturer's instructions (R&D Systems, Minneapolis, MN) (31).…”