Abstract
ObjectiveTo investigate the role of matrix metalloproteinase 2 (MMP2) in pancreatic beta cell injury induced by oxidative stress.MethodsRat pancreatic beta cell line INS-1 cells were treated with advanced glycation end-products (AGE) to induce intracellular oxidative stress. Intracellular MMP2 expression and activity were determined by quantitative reverse transcription polymerase chain reaction (RT-PCR), Western blotting, and zymography, respectively. MMP2 expression and activity were manipulated by over-expression with recombinant MMP2 plasmids or knockdown with either MMP2 specific siRNA or inhibitors, and effects on apoptosis and insulin-secretion were measured by flow cytometry and ELISA.ResultsAGE treatment induced intracellular oxidative stress in INS-1 cells, as indicated by elevated ROS levels, apoptotic cell death, and suppressed insulin secretion. This was accompanied by increased MMP2 expression and activity. However, Antioxidant N-acetylcysteine (NAC) treatment inhibited MMP2 expression and activity, and partially reversed cell apoptosis and insulin secretion dysfunction induced by AGE. Forced expression of MMP2 mimicked the effects of AGE treatment while inhibition of MMP2 either by a specific MMP2 inhibitor or MMP2 siRNA protected oxidative stress induced by AGE.ConclusionMMP2 expression and intracellular activity are increased by oxidative stress, contributing to cellular dysfunction and apoptosis in INS-1 cells after AGE challenge.
Highlights
Type 2 diabetes mellitus (T2DM) is generally considered to be caused by the dysfunction of pancreatic beta cells and insulin resistance
matrix metalloproteinase 2 (MMP2) expression and activity, INS-1 cells were challenged by an intracellular oxidative stress with Glycated bovine serum albumin (GA), which acts as an Advanced glycation end-products (AGE)
The protein levels of MMP2 were elevated in Annexin V positive cells representing apoptotic cells were mainly located in the right upper quadrant and right lower quadrant. (d) Average apoptotic rate in the three groups. (e) Insulin secretion of INS-1 cells stimulated with 3.3 mmol/L glucose or 16.7 mmol/L glucose in the different groups. *P,0.05 vs the corresponding control group; #P,0.05 vs corresponding GA-treated group (f) Insulin release index (IRI) of each group
Summary
Type 2 diabetes mellitus (T2DM) is generally considered to be caused by the dysfunction of pancreatic beta cells and insulin resistance. The mechanisms underlying pancreatic beta cell dysfunction and loss are still unclear. Accumulating evidence has indicated that pancreatic beta cell loss in T2DM partially results from oxidative stress [3,4,5]. The factors responsible for pancreatic beta cell damage that lead to cell dysfunction via oxidative stress remain unknown. Reactive oxygen species (ROS) can oxidize or nitrify proteins, lipids and DNA by direct chemical modification, leading to dysfunction of crucial proteins including signal transduction factors, ribosomal subunits, DNA repair enzymes, and proteases associated with energy metabolism in pancreatic beta cells [8]. Whether factors other than direct chemical modification might contribute to pancreatic beta cell dysfunction in oxidative stress is largely unknown
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