| Part 1 The protective effect of metformin on rat pancreatic βcell dysfunction induced by high glucose or high free fatty acidsObjective To test the effect of metformin(MF) on improving β cell insulin secretion function impaired by its chronic exposure to high concentrations of free fatty acids or glucose.Methods Pancreatic islets of male Wistar rats were incubated for 48h in a medium containing 5.5-16.7mmol/L glucose and 0.5mmol/L palmitic acid respectively. The cells were re-incubated for another 24h with or without 0-5ug/ml MF.Basal and stimulated (16.7mmol/L glucose) insulin contents in the medium were measured by radioimmuoassay(RIA). Results Metformin(0-5ug/ml) did not affect basal and glucose-induced insulin release in 5.5mmol/L glucose(control).The islets exposed to high glucose or high FFA concentration showed an increased basal(51.24±8.75 Vs 13.09±4.92 , 60.99± 15.42 Vs 13.09±4.92 , P<0.01) and a decreased glucose-indused insulin release (68.49±17.98 Vs 118.84±20.98, 79.63±21.09 Vs 118.84±20.98,P<0.01) as compared with control islets.However,when these islets pretreated with high glucose or high FFA re-cultured for additional 24h in the presence of 2.5-5ug/ml MF,both basal and glucose-induced insulinsecretion were reversed dosely to the control levels(P<0.01). Conclusion Metformin did not affect β cell insulin release in 5.5mmol/L glucose (control) concentration.But it was able to restore substantially the secretory function of rat pancreatic islets whose secretory function has been impaired by chronic exposure to elevated glucose or free fatty acids levels.These data raise the possibility that metformin may have a direct beneficial effect on the β-cell secretory function.Part 2 The effect of metformin on insulin receptor protein tyrosine kinase activities of HIT-T15 cells exposured to highglucose or high free fatty acids concentrationObjective To study the effect of metformin on insulin receptor(IRc) proteintyrosine kinase(PTK) activities of HIT-T15 cells exposured to high glucose orfree fatty acids concentration,and to explore the mechanism of MF improvingP cell insulin resistance.Methods HIT-T15 cells were incubated for 48h in a medium containing5.5-16.7mmol/L glucose and 0.5mmol/L palmitic acid respectively. The cellswere re-incubated for another 24h with or without 2.5ug/ml MF. PTKactivities of IRc were measured by radioactive enzyme assay.Results The enzymatic activities of IRc PTK were significantly decreased inHIT-T15 cells exposure to high glucose or high FFA concentration(52.5±18.6and 54.6±14 vs 119.4±29.1pmol/min/ug, PO.01 respectively) compared tocontrol. The enzymatic activities of IRc PTK in HIT-T15 cells reincubated for an additional 24h in the presence of 2.5ug/ml MF were significantly increased vs those without MF group (113.0±29.8 vs 52.5±18.6pmol/min/ug, 98.6±26.1 vs 54.6±14.0,P<0.01 respectively),and were no significant difference in comparison with control group(P>0.05).Conclusion The enzymatic activities of IRc PTK was significantly decreased in HIT-T15 cells chronic ally exposure to elevated glucose or free fatty acids levels. Metformin restored approximately normal enzymatic activities of PTK of HIT-T15 cells its PTK activities had been impaired by chronic exposure to high glucose or free fatty acids levels.Part 3 The effect of metformin on insulin signal transduction of HIT-T15 cells chronic ally exposure to high glucose or highfree fatty acidsObjective To study the effect of metformin on insulin signal transduction of HIT-T15 cells( P cell line) exposure chronically to high glucose or free fatty acids concentration.Methods HIT-T15 cells were incubated for 48h in a medium containing 5.5-16.7mmol/L glucose and 0.5mmol/L palmitic acid respectively. The cells were re-incubated for another 24h with or without 2.5ug/ml MF.The expression levels of IRS-1,IRS-2,PI3K and Glut2 mRNA were determined by RT-PCR,and the protein levels of IRc,IRS-l and IRS-2 were detected by Western blotting.Results In high glucose groups the expression levels of IRS-1,IRS-2,PI3K and Glut2 mRNA were significantly decreased to 42. 3%, 30. 6%, 46. 6% and 32. 5%(P<0. 01) of control levels, respectively, and the protein levels of IRc,IRS-l and IRS-2 were significantly decreased to 48.7%,30.3% and 28.9%(P<0.01) of control levels,respectively. In high FFA groups the expression levels of IRS-1,IRS-2,PI3K and Glut2 mRNA were also significantly decreased to34.7%,26.3%,40.5% and 37.6%(P<0.01 of control levels respectively),and the protein levels of IRc,IRS-l and IRS-2 were significantly decreased to 38.2%, 22.2% and 26.7%(P<0.01, of control levels respectively). After reincubated for 24h in the presence of 2.5ug/ml MF, the levels of IRS-1,IRS-2 mRNA and protein were significantly increased in the high FFA group vs MF-free group(P<0.05, respectively),but in hight glucos group only the levels of IRS-2mRNA and protein were significantly increased vs those MF-free group(P<0.05, respectively). The levels of PI3K and Glut2 mRNA and IRc protein in MF treated group were no significant difference in comparison with those without MF group (P>0.05).Conclusion The levels of IRS-l,IRS-2,PI3K,Glut2 mRNA and IRc, IRS-1,IRS-2 protein were significantly decreased in the HIT-T15 cells chronic ally exposure to elevated glucose or free fatty acids levels. Metformin is able to significantly increase the expression levels of IRS-1,IRS-2 mRNA and protein in HIT-T15 cells which has been impaired by chronic ally exposure to high free fatty acids level,and to significantly increase the expression levels of IRS-2 mRNA and protein in HIT-T15 cells impaired by high glucose.
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