Oxidative Stress-mediated, Post-translational Loss of MafA Protein as a Contributing Mechanism to Loss of Insulin Gene Expression in Glucotoxic Beta Cells

Jamie S Harmon,Roland Stein,R Paul Robertson

doi:10.1074/jbc.m410345200

Abstract

Glucose toxicity in pancreatic islet beta cells causes loss of insulin gene expression, content, and secretion due to loss of binding of transcription factors, most notably PDX-1 and RIPE-3b1 activator, to the promoter region of the insulin gene. Recently, RIPE-3b1 activator was cloned and identified as the mammalian homologue of avian MafA/Maf-L (MafA). This enabled us to carry out more extensive studies of the role of MafA in glucotoxicity than were hitherto possible. Northern analysis of glucotoxic HIT-T15 cells revealed normal amounts of MafA mRNA, but Western analysis demonstrated a 97 +/- 1% reduction in MafA protein (p < 0.0001). The proteasome is a likely site for MafA degradation as lactacystin, an irreversible proteasome inhibitor, caused an accumulation of MafA protein. Antioxidants have previously been shown to prevent the adverse effects of glucose toxicity on beta cell function both in vivo and in vitro. In the current study, chronic culturing of HIT-T15 cells with the antioxidant N-acetylcysteine (NAC) prevented loss of MafA protein (late passage = 18.9 +/- 10.4% of early passage, p < 0.001; late passage with NAC = 68.7 +/- 19.7% of early passage, p = not significant) and loss of DNA binding (late passage = 63.7 +/- 9% of early passage, p < 0.02; late passage with NAC = 116 +/- 10% of early passage, p = not significant). Additionally, transient transfection of PDX-1 or MafA cDNA into glucotoxic cells increased PDX-1 and MafA protein levels and individually increased insulin promoter activity (untreated = 34%, PDX-1 = 70%, MafA = 78%; percentage of activity of early passage cells), whereas the combined transfection of MafA and PDX-1 completely restored insulin promoter activity. This recovery of promoter activity following transient transfection had no effect on endogenous insulin mRNA. However, adenoviral infection of MafA and PDX-1 significantly increased endogenous insulin mRNA levels by 93% (121 +/- 9 versus 233 +/- 18 density light units; n = 5, p < 0.001). We conclude that the absence of MafA protein from beta cells via chronic oxidative stress contributes importantly to the loss of endogenous insulin gene expression as glucose toxicity develops.

Highlights

Glucose toxicity in pancreatic islet beta cells causes loss of insulin gene expression, content, and secretion due to loss of binding of transcription factors, most notably PDX-1 and RIPE-3b1 activator, to the promoter region of the insulin gene
Western and Northern Analyses for MafA—MafA protein levels were measured in nuclear extracts from HIT-T15 cells (Fig. 1) using early passage (p71–75; lane 1) and late passage (p123– 128; lane 2) cells chronically cultured in glucotoxic conditions (RPMI media containing 11.1 mM glucose)
The accumulation of MafA protein was augmented by the addition of the proteasome inhibitor in cells cultured both in 0.8 mM glucose (3147.3 Ϯ 380.3 versus 4812 Ϯ 617.2 DLU; no lactacystin versus lactacystin; p Ͻ 0.03, n ϭ 4) and in 11.1 mM glucose (4468.3 Ϯ 659.7 versus 6712.8 Ϯ 1281.4 DLU; p Ͻ 0.04, n ϭ 4; Fig. 3A), suggesting that degradation occurs via the proteasome pathway

Summary

Introduction

Glucose toxicity in pancreatic islet beta cells causes loss of insulin gene expression, content, and secretion due to loss of binding of transcription factors, most notably PDX-1 and RIPE-3b1 activator, to the promoter region of the insulin gene. With the recent cloning and identification of RIPE-3b1 activator as MafA (10 –12), we have been able to perform new studies to examine 1) whether levels of MafA mRNA and protein are decreased in glucotoxic beta cells; 2) the mechanism of MafA protein degradation; 3) whether the antioxidant, N-acetylcysteine, can prevent glucotoxicity-induced loss of MafA protein and binding to the insulin promoter; and 4) whether overexpression of MafA and PDX-1 together can restore insulin promoter activity and mRNA levels more fully than PDX-1 alone. Glucose toxicity in the pancreatic islet beta cell secondarily leads to further

Methods

Results

Conclusion