Role of Lipid Peroxidation and PPAR-δ in Amplifying Glucose-Stimulated Insulin Secretion

Abstract

OBJECTIVEPrevious studies show that polyunsaturated fatty acids (PUFAs) increase the insulin secretory capacity of pancreatic β-cells. We aimed at identifying PUFA-derived mediators and their cellular targets that are involved in the amplification of insulin release from β-cells preexposed to high glucose levels.RESEARCH DESIGN AND METHODSThe content of fatty acids in phospholipids of INS-1E β-cells was determined by lipidomics analysis. High-performance liquid chromatography was used to identify peroxidation products in β-cell cultures. Static and dynamic glucose-stimulated insulin secretion (GSIS) assays were performed on isolated rat islets and/or INS-1E cells. The function of peroxisome proliferator–activated receptor-δ (PPAR-δ) in regulating insulin secretion was investigated using pharmacological agents and gene expression manipulations.RESULTSHigh glucose activated cPLA2 and, subsequently, the hydrolysis of arachidonic and linoleic acid (AA and LA, respectively) from phospholipids in INS-1E cells. Glucose also increased the level of reactive oxygen species, which promoted the peroxidation of these PUFAs to generate 4-hydroxy-2E-nonenal (4-HNE). The latter mimicked the GSIS-amplifying effect of high glucose preexposure and of the PPAR-δ agonist GW501516 in INS-1E cells and isolated rat islets. These effects were blocked with GSK0660, a selective PPAR-δ antagonist, and the antioxidant N-acetylcysteine or by silencing PPAR-δ expression. High glucose, 4-HNE, and GW501516 also induced luciferase expression in a PPAR-δ–mediated transactivation assay. Cytotoxic effects of 4-HNE were observed only above the physiologically effective concentration range.CONCLUSIONSElevated glucose levels augment the release of AA and LA from phospholipids and their peroxidation to 4-HNE in β-cells. This molecule is an endogenous ligand for PPAR-δ, which amplifies insulin secretion in β-cells.

Beta cell response to nutrient overload involves phospholipid remodelling and lipid peroxidation

“…Islets of Langerhans were isolated from male Wistar rats (150-175 g; Harlan, Jerusalem, Israel) following collagenase digestion of pancreases, as described [13]. INS-1E cells (passages 65-85) were grown and maintained as described [18].…”

“…INS-1E cells (passages 65-85) were grown and maintained as described [18]. GSIS assays in INS-1E cell cultures and rat isolated islets and insulin RIA (Merck Millipore, Billerica, MA, USA) were performed as described [13].…”

“…The lipidomic analysis of INS-1E cells was performed as described [13]. Fatty acids were identified by G. Cohen and quantified by comparison with standard methylated references.…”

“…Oligonucleotide primers, designed using Primer Express program (Applied Biosystems, Foster City, CA, USA), were synthesised by Sigma-Aldrich (Rehovot, Israel). The PCR protocols have been described previously [13,20]. Primer sequences are given in ESM Table 1.…”

“…We have shown in INS-1E cells that high glucose levels induced remodelling of membrane phospholipids and released AA and LA. Reactive oxygen species (ROS) prompted non-enzymatic peroxidation of AA and LA to produce 4-hydroxy-2E-nonenal (4-HNE), which augments glucose-stimulated insulin secretion (GSIS) via activation of the nuclear receptor peroxisome proliferatoractivated receptor-δ (PPARδ) [13].…”