Protein expression analysis of insulin producing cells exposed to endoplasmic reticulum stress

Abstract

The prevalence of type 2 diabetes mellitus (T2DM) is increasing rapidly as a result of the obesity epidemic. T2DM develops in individuals who fails to compensate for increased demand of insulin. Obese individuals display alterations in the circulating lipid profile and there is evidence that the elevated levels of fatty acids are detrimental to pancreatic β-cells which may contribute to the progression of β-cell failure via both impaired function and a reduction of the β-cell mass seen in T2DM patients, but the mechanism is not completely known. There is evidence that saturated fatty acids can induce apoptosis, as well as endoplasmic reticulum (ER) stress. As a secretory cell, the pancreatic β-cell is equipped with a highly developed ER to assist in the folding process of newly synthesized proteins, but this cell organelle is sensitive to alteration in homeostasis, and an imbalance between protein load and folding capacity can result in ER stress. This thesis aimed to investigate the protein expression in insulin producing cells exposed to ER stress by the use of proteomics and Western blot.

In paper I, cells were exposed to the ER stress inducer thapsigargin. By the use of proteomic methodology we found decreased levels of the ER localized proteins GRP78/BiP, PDIA3 and PDIA6 following thapsigargin treatment. The decreased protein levels of GRP78/BiP was not a consequence of reduced mRNA expression. Instead the decrease involved a combination of reduced protein synthesis and enhanced degradation by both proteasome and autophagy. The data in paper I provides an explanation to why INS-1E cells are vulnerable to conditions of ER stress. In paper II, cells were exposed to thapsigargin and the saturated fatty acid palmitate. By proteomic investigation we identified 7,786 proteins, of which 6,117 were overlapping in two data sets. 1,000 proteins were regulated by thapsigargin and 227 by palmitate. With bioinformatic analysis we found a set of transcription factors predicted to be regulated in the same manner by the thapsigargin and palmitate. This paper shows that the treatments not only share the induction of ER stress but also share an effect on the lipid handling in the cell by affecting several metabolic master transcription factors involved in lipid and cholesterol biosynthesis. In paper III we investigated pathways involved in the cytotoxic action of glucocorticoids (GCs). Excess of GCs is associated with glucose intolerance and diabetes. The data show that GC activates p38 MAPK and JNK in β-cells, which work in opposite to regulate the cytotoxic effects induced by GCs. The data also suggests that protein phosphatase 5 play a protective role, since reduced PP5 levels in MIN6 cells and islets makes the cells sensitized to the toxic effects induced by GCs. Furthermore, our result indicate that GC exposure 2 compromises the cellular response to ER stress in insulin producing cells which may lead to an impaired capacity to restore the homeostasis in the ER. A deeper understanding of the mechanisms and the mediators of the ER and apoptotic pathways will hopefully provide tools to preserve the β-cell mass and delay the progression to T2DM.