The Research Of Uncoupling Protein 2 And Pancreatic β TC3 Cells Impairment Induced By FFA

Objective: UCP2 is a member of UCPs,which is expressed in many tissues,such as adipose tissue and pancreaticβcells.UCP2 is located in mitochondria inner membrane.Through dissipating proton across the inner membrane UCP2 can participate in many mitochondrial functions such as ATP synthesis ROS production.It has been researched that UCP2 can reduce the secretion of insulin.UCP2 gene may associate with obesity, pancreaticβcells functional impairment.Environmental factor,obesity,hyperlipemia and hyperglycosemia may damage the function of pancreaticβcells though hyperoxide-UCP2 pathway. Inβcells, An increase in the ratio of ATP to ADP inhibits ATP-sensitive potassium (KATP) channels and provokes a decrease in the depolarization of the plasma membrane, which opens voltage-gated Ca2+ channels. The increase in the intra-cellular calcium concentration contributes to the exo-cytosis of insulin-containing granules. Overexpression of UCP2 adenovirus transfection impaire Glucose stimulated insulin secretion (GSIS). In UCP2 knocked out obese mice,fasting plasma Glucose and postprandial plasma Glucose decrease significantly.When intraperitoneal injection Glucose, insulinⅠphase secretion improve obviously,which suggest that UCP2 can down-regulate insulin secretion.Type 2 diabetes have lipide metabolic disorder besides glycometabolism disorder,especially hypertriglyceridemia and high-free fatty acidsmia.Oxyradical is generated though enzymatic system and unenzymatic system in metabolic process, which can attack poly-unsaturation free fatty acids of the biomembrane.The free fatty acids attacked generate lipid peroxidation,such as aldehyde group (Maleic Dialdehyde,MDA),ketone group and new oxyradical. Lipid peroxidation can course cascade amplification, evoke cell damage . The level of MDA can reflect indirectly the Severity of body cell attacted by oxyradical. Some research indicate that certain concentration of oleinic acid was gived to myocardial cell cultured in vitro for 48h, then measured the level of MDA, which was high. That indicate oxidative stress is high.Free fatty acid(FFA)is high in type 2 diabetes. Oleinic acid is one of FFA in the blood .This experiment employ oleinic acid of different concentration to affect pancreticβcells for 72h ,then investigate the level of oxidative stress,apoptosis rate,the expression of UCP2 and insulin secretion, and study the impact of oleinic in pancreticβcells, then investigate the mechanism of pancreatic island function impairment by FFA.Methods: pancreaticβ-TC3 cells were cultured in RPMI 1640 medium containing 10 percent heat-inactivated fetal calf serum. The experiment was quadruplet, N=normal control group, A=0.3mmol/l,B=0.4mmol/l,C=0.5mmol/l oleinic acid group. pancreaticβ-TC3 cells were differently exposed to free-serum RPMI 1640 medium containing 2%BSA and containing 0.3mmol/l,0.4mmol/l,0.5mmol/l oleinic acid for 72h. Basic insulin secretion and glucose stimulant insulin secretion were measured though radioimmunity in cell culture fluid.Cell apoptosis rate and mitochondria membrane were detected with flow cytometer. The expresstion of protein UCP2 in pancreaticβ-TC3 cells was detected by western blot. The content of Maleic Dialdehyde (MDA) was measured by Thibabituric Acid test.Result:1 Compared with control group, basic insulin secretion of oleinic acid group stimulated by 2.8mmol/l glucose was obviously increased dependent on oleinic concentration and GSIS(stimulated by 16.7mmol/l glucose)were decreased depadent on oleinic concentration. The basic insulin secretion in control group was 62±7mU/l, although the basic insulin secretion in oleinic acid group was 90±3mU/l, 127±6mU/l, 207±9mU/l. That was 1.45 time, 2.05 time and 2.30 time(P<0.01). The GSIS in control group was 479±16 mU/l, which in oleinic acid group was 1.45 time, 2.05 time and 2.30 time(P<0.01). Each group is significant difference(P<0.01).2 The ratio of pancreaticβ-TC3 cells apoptosis of oleinic acid group was 4.70%±0.15,which was 8.52%±0.29 , 29.11%±0.67,15.20%±0.50( P<0.01). The apoptosis of 0.4mmol/l oleinic acid group is especially high. Each group is significant difference(P<0.01).3 The mitochondrial membrane mean fluorescence(MIF) of pancreaticβ-TC3 cells was 9.98±1.18 in control group, which was 6.64±0.84,3.52±0.80,1.62±0.36( P<0.01)in oleinic acid group. Each group is significant difference(P<0.01).4 UCP2 protein relative gray scale was 0.46±0.05, which was 0.85±0.02, 1.17±0.03 and 1.38±0.06( P<0.01)in oleinic acid group. It was increased dependent on oleinic concentration. Each group is significant difference(P<0.01).5 The content of MDA was 0.80±0.043,which was 1.36±0.027,1.97±0. 048,2.33±0.078( P<0.01)in oleinic acid group.It was increased dependent on oleinic concentration.Each group is significant difference(P<0.01).Conclusion: In vitro pancreaticβcells, the impairment induced by FFA maybe concerned with the increasing of oxidative stress, apoptosis ratio and the expression of the protein UCP2.The increase of UCP2 in expression and effect cause the reduce of mitochondrial membrane and insulin secretion.