Proteomic screening of glucose-responsive and glucose non-responsive MIN-6 beta cells reveals differential expression of proteins involved in protein folding, secretion and oxidative stress

Abstract

The glucose-sensitive insulin-secretion (GSIS) phenotype is relatively unstable in long-term culture of beta cells. The purpose of this study was to investigate relative changes in the proteome between glucose-responsive (low passage) and glucose non-responsive (high passage) murine MIN-6 pancreatic beta cells. The 2D-DIGE and subsequent DeCyder analysis detected 3351 protein spots in the pH range of 4-7. Comparing MIN-6(H) to MIN-6(L) and using a threshold of 1.2-fold, the number of proteins with a decrease in expression level was 152 (4.5%), similar was 3140 (93.7%) and increased 59 (1.8%). From the differentially expressed proteins identified in this study, groups of proteins associated with the endoplasmic reticulum (ER) and proteins involved in oxidative stress were found to be significantly decreased in the high-passage (H passage) cells. These proteins included endoplasmic reticulum protein 29 (ERp29); 78-kDa glucoserelated protein, (GRP78); 94-kDa glucose-related protein (GRP94); protein disulphide isomerase; carbonyl reductase 3; peroxidoxin 4 and superoxide dismutase 1. These results suggest that non-GSIS MIN-6 cells do not have the same ability/capacity of glucose-responsive MIN-6 cells to successfully fold, modify or secrete proteins and counteract the problems associated with oxidative stress.

GRP78, but Not Protein-disulfide Isomerase, Partially Reverses Hyperglycemia-induced Inhibition of Insulin Synthesis and Secretion in Pancreatic β-Cells

“…To our knowledge, this is the first report that shows that PDI affects proinsulin disulfide bond formation in living cells. Interestingly, the levels of PDI are reduced in cultured MIN6 ␤-cells that lose glucose-responsive insulin secretion after long term passage (41), suggesting that PDI is important for maintaining normal ␤-cell function. PDI is a member of a large family of oxidoreductases, many of which are expressed in multiple cell types (26).…”

Proteomic analysis of conditioned media from glucose responsive and glucose non‐responsive phenotypes reveals a panel of secreted proteins associated with beta cell dysfunction

“…Excision of protein spots, trypsin digestion, and protein identification by mass spectrometric analysis using an Ettan MALDI-TOF Pro instrument from Amersham Biosciences was performed according to an established methodology [5]. Preparative gels containing 300 mg of protein were fixed in 7.5% v/v acetic acid, 10% v/v methanol overnight.…”

“…Previous work by us and others have shown that low-passage MIN-6 cells respond to changes in glucose concentrations, producing an approximately 5.5-fold glucose-stimulated insulin secretion (GSIS) in response to 26.7 mmol/L, compared with 3.3 mmol/L, glucose. After continuous culture, this GSIS was no longer present and thus represents an excellent model system for investigating beta cell dysfunction [5,6].…”

Antioxidant Levels Represent a Major Determinant in the Regenerative Capacity of Muscle Stem Cells

“…MDSCs can be maintained in culture through 200 population doublings with no evidence of neoplastic transformation, chromosome abnormalities, or decrease in regenerative capacity (Deasy et al, 2005). There is no evidence that MDSCs obtain increased antioxidant capacity with increasing passage number; indeed, just the opposite has been reported in multiple groups that have demonstrated that low passage cell populations have an increased resistance to stress, antioxidant capacity, and ability to withstand adverse environmental conditions compared with higher passaged cell populations (Luce and Cristofalo, 1992;Yuan et al, 1996;Kaneko et al, 2001;Gurjala et al, 2005;Dowling et al, 2006).Inflammation clearly is one of the most important hurdles to overcome in order to increase the efficacy of cellular therapies. A major source of the destructive power of inflammation is the direct and indirect generation of ROS and free radicals after the inflammatory cytokine response (Dhalla et al, 1999).…”