Palmitate induces a pro-inflammatory response in human pancreatic islets that mimics CCL2 expression by beta cells in type 2 diabetes

Abstract

Aims/hypothesis Beta cell failure is a crucial component in the pathogenesis of type 2 diabetes. One of the proposed mechanisms of beta cell failure is local inflammation, but the presence of pancreatic islet inflammation in type 2 diabetes and the mechanisms involved remain under debate.Methods Chemokine and cytokine expression was studied by microarray analysis of laser-capture microdissected islets from pancreases obtained from ten non-diabetic and ten type 2 diabetic donors, and by real-time PCR of human islets exposed to oleate or palmitate at 6 or 28 mmol/l glucose. The cellular source of the chemokines was analysed by immunofluorescence of pancreatic sections from individuals without diabetes and with type 2 diabetes.

A local glucagon-like peptide 1 (GLP-1) system in human pancreatic islets

“…The corresponding spectra of the GLP-1(7-37) and GLP-1 (7-36) tryptic sequences were matched with the putative digested sequences of the peptides using the sequence editor implemented on BIOTOOLS. GLP-1(7-37) and GLP-1(7-36) peptides were identified by the species at m/z 1105 (aa [21][22][23][24][25][26][27][28] and at m/z 2098 (aa [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20].…”

“…NCI-H716 cells, a human L cell model [21], were obtained from American Type Culture Collection (Manassas, VA, USA) and used as GLP-1-like positive controls. Samples were analysed by laser confocal microscopy (TCS SP5; Leica Microsystems, Wetzlar, Germany), as previously reported [22].…”

“…Islet isolation and culture Islets were prepared by enzymatic digestion and gradient separation [22,23], and suspended in M199 culture medium (Sigma Chemicals, St Louis, MO, USA) supplemented as detailed elsewhere [22,23]. Islets with a diameter ≥100 μm were handpicked and studied within 3 days of isolation.…”

“…Whole-cell lysate (A-431, SC-2201; Santa Cruz Biotechnologies, Santa Cruz, CA, USA) was used as positive control. Islet glucagon and GLP-1 content was measured from four nondiabetic and three type 2 diabetic preparations after overnight acid-ethanol (1.5% HCl, 79% ethanol [95% pure], 19.5% water) extraction [22,23].…”

“…Quantitative RT-PCR studies The expression of genes encoding insulin, glucagon, PC1/3 and PC2 was assessed by quantitative RT-PCR experiments in alpha and beta cell fractions from eight non-diabetic and three type 2 diabetic islet preparations, according to methods detailed elsewhere [22,23,25]. Briefly, after RNA extraction and reverse transcription, mRNA levels of the genes of interest were quantified and normalised to β-actin in a bioanalyser (7700; Applied Biosystems, Foster City, CA, USA).…”