ABSTRACT
Basic research on types 1 and 2 diabetes mellitus require early stage studies using beta cells or cell lines, ideally of human origin and with preserved insulin secretion in response to glucose. The 1.1E7 cells are a hybrid cell line resulting from the electrofusion of dispersed human islets and PANC-1 cells, capable of secreting insulin in response to glucose, but their survival and function under toxic conditions remains untested. This characterization is the purpose of the present study. We treated these cells with a cytokine mix, high glucose, palmitate, and the latter two combined. Under these conditions, we measured cell viability and apoptosis (MTT, Caspase Glo and TUNEL assays, as well as caspase-8 and -9 levels by Western blotting), endoplasmic reticulum stress markers (EIF2AK3, HSPA4, EIF2a, and HSPA5) by real-time PCR, and insulin secretion with a glucose challenge. All of these stimuli (i) induce apoptosis and ER stress markers expression, (ii) reduce mRNA amounts of 2-5 components of genes involved in the insulin secretory pathway, and (iii) abrogate the insulin release capability of 1.1E7 cells in response to glucose. The most pronounced effects were observed with cytokines and with palmitate and high glucose combined. This characterization may well serve as the starting point for those choosing this cell line for future basic research on certain aspects of diabetes.
Subject(s)
Cytokines/toxicity , Glucose/toxicity , Insulin Secretion/drug effects , Insulin-Secreting Cells/drug effects , Insulin-Secreting Cells/metabolism , Palmitates/toxicity , Apoptosis/drug effects , Cell Line , Cell Survival/drug effects , Cytokines/administration & dosage , Cytokines/metabolism , Diabetes Mellitus/etiology , Diabetes Mellitus/metabolism , Diabetes Mellitus/pathology , Endoplasmic Reticulum Chaperone BiP , Endoplasmic Reticulum Stress/drug effects , Endoplasmic Reticulum Stress/genetics , Gene Expression/drug effects , Glucose/administration & dosage , Glucose/metabolism , Humans , Insulin Secretion/genetics , Insulin-Secreting Cells/cytology , Palmitates/administration & dosage , Palmitates/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolismABSTRACT
Ghrelin is a peptidic hormone, which stimulates cell proliferation and inhibits apoptosis in several tissues, including pancreas. In preclinical stage of type 1 diabetes, proinflammatory cytokines generate a destructive environment for ß-cells known as insulitis, which results in loss of ß-cell mass and impaired insulin secretion, leading to diabetes. Our aim was to demonstrate that ghrelin could preserve ß-cell viability, turnover rate, and insulin secretion acting as a counter balance of cytokines. In the present work we reproduced proinflammatory milieu found in insulitis stage by treating murine cell line INS-1E and rat islets with a cytokine cocktail including IL-1ß, IFNγ, and TNFα and/or ghrelin. Several proteins involved in survival pathways (ERK 1/2 and Akt/PKB) and apoptosis (caspases and Bcl-2 protein family and endoplasmic reticulum stress markers) as well as insulin secretion were analyzed. Our results show that ghrelin alone has no remarkable effects on ß-cells in basal conditions, but interestingly it activates cell survival pathways, downregulates apoptotic mediators and endoplasmic reticulum stress, and restores insulin secretion in response to glucose when beta-cells are cytokine-exposed. These data suggest a potential role of ghrelin in preventing or slowing down the transition from a preclinical to clinically established diabetes by ameliorating the effects of insulitis on ß-cells.
