| Objective 1.This experiment aims at researching whether pioglitazone(PIO) has any effect on the , mitochondrial ultrastructrue , apoptosis and insulin secretion of HIT-T15 cell induced by palmitate. 2. To investigate the influence of palmitate and PIO on the production of ATP and expression of PGC-1mRNA, NRFmRNA of HIT-T15 cell. 3. To discuss the possible protective mechanism of PIO on the impairment of beta cell induced by palmitate.Methods The HIT-T15 were selected to be a model of islet beta cell and palmitate presents free fat acids(FFA). There were 7 groups in this experiment: 1: Matched control group without drugs, 2 : 0. 5mmol/lFFA, 3 : 0. 5mmol/lFFA+PI010-7mol/1, 4 : 0.5mmol/lFFA+PI010-5mol/1, 5 : 1.0 mmol/lFFA,6 : 1.0 mmol/lFFA+PI010-7mol/l, 7: 1.0mmol/lFFA+PI010-5mol/l. The following aspects were studied: . 1: Apoptosis peaks wereassessed by flow cytometry and TUNNEL respectively. 2: The change of HIT-T15 cell mitochondrial ultrastructrue induced by palmitate and PIO were observed by electron-microscope .3: To investigate the ATP/ADP by hyper performance liquid chromatography(HPLC) .4: To evaluate change of the PGC-1mRNA, NRFmRNA by RT-PCR.5: To evaluate the insulin level by radiate-immune assay.Results 1: Palmitate could increase the apoptosis. Detected by flow cytometry and tunnel respectively, the apoptosis ratio increasedto in low concentration palmitate group( 9. 73 ± 0.38%, 8.31 ± 0.32%) and high concentration palmitate group(12. 97 ± 0.73%, 10. 80 ± 0. 27%) comparing with control group(7. 40 ± 0. 96%, 5.93 ± 0. 56%). Pioglitazine could alleviate the effect induced by palmitate, the apoptosis ratio in 0. 5mmol/lFFA±10~7mol/lPI0 group is 7.80 ± 0.35%, 7. 01 ±0.41% checked by flow cytometry and tunnel respectively, and 0.5mmol/lFFA±10-5mol/lPI0 is 6.10 ± 0.17% , 6.79 ±0. 45%, 1. 0mmol/lFFA±10-7mol/lPI0 is 10. 20±0. 06%,7. 70±0. 23%,1. 0mmol/lFFA±10-5mol/lPI0 is 7. 35 ± 0. 26%, 6. 59 ± 0. 21%. 2. It was showed by electron-microscope that the cells and mitochondrion were swollen and there were many apoptotic body cultured with palmitate, and pioglitazone could alleviate those changes. 3. Palmitate could decrease ATP/ADP in HIT-T15 cell from normal control group 3.15 ± 1.14 to 0.70 ±0.14 in low concentration palmitate group and 0.41 ± 0.08 in highconcentration palmitate. Pioglitazone could restore partly the level of ATP/ADP induced by palmitate, FFA0.5mmol/l±PI010-7mol/l increased to 1.71 ± 0.12, and FFA0.5mmol/l±PI010-5mol/l group to 1.58 ± 0.07, and FFA1.0mmol/l±PI010-7mol/l to 1.80 ± 0.36, and FFA1.0mmol/l±PI010-5mol/l group to 1. 94 ± 0. 61. 4. palmitate could induce the upregulation of the expression of relative copies of NRFmRNA and PGC-1mRNA from normal control group 0. 83 ±0.28, 0.81 ±0.16 to 1.94 ±0.91, 2.36 ±0.81 in low concentration paltimate group and 2. 94±1.00, 4.16 ± 1.42 in high concentration palmitate group.Pioglitazone could decrease the change induced by palmitate, especial the group FFA1.0mmol/l±PI010-5mol/l which decreased the NRFmRNA and PGC-1mRNA to 0.45±0.28 和 0.89±0.34 (P<0.05). 5. Basal insulin cultured at concentration of 5.5mmol/lglucose for 48h increased when cultured with palmitate, from normal control group 1.01 ±0. 10ng/ml to 1. 16 ± 0. 20ng/ml in FFAO. 5 mmol/l group and 1.22 ± 0.20ng/ml in FFA1.0mmol/l group. Glucose stimulating insulin secretion (GSIS )by 22mmol/l glucose were decreased induced by palmitate, from normal control group 1. 03 ±0.20 ng/ml decresaed to 0.35±0.05 ng/ml in FFAO. 5 mmol/l group and 0.07 ±0.00 ng/ml in FFA1.0mmol/l group. PIO could alleviate the change induced by palmitate, and GSIS in FFA0.5mmol/l±PI010-5mol/ group were 0.81 ± 0.3ng/ml , and FFA1.0mmol/l±PI010-5mol/l group 0. 67±0.20ng/ml.Conculsion 1.palmitate could induce the HIT-T15cells apoptosis and mitochondrial swollen and decrease the level of ATP/ADP and GSIS, which could be associated with the over-expression of PGC-1mRNA,NRFmRNA. 2. Pioglitazone could meliorate partly those changes induced by palmitate: decreased the apoptosis and alleviated the mitochondrial swollen, and restored the level of ATP/ADP and GSIS. The effect of pioglitazone maybe achieved by the regulation of PPAR y on the expression of PGC-1mRNA and NRFmRNA.
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