The Effects Of Rosiglitazone On Glucosamine-induced Alterations Of Mitochondrial Function In HIT-T15 Cells

Objective 1.This experiment aims at researching whether glucosamine (GLcN) have effects on apoptosis, insulin release ,morphologic changes of HIT-T15 cells ultrastructure, NRFs and PGC-1 mRNA expression in HIT-T15 cells. 2.To investigate the effects of rosiglitazone(RSG )on GLcN-induced alterations of mitochondrial function in HIT-T15 cells and the possible mechanisms of GLcN and RSG involved.Methods The HIT-T15 cells were selected to be our objects and the following aspects were studied:1. ultrastructure of HIT-T15 cells was observed with transmission electron microscope. 2.Apoptosis peaks were assessed by flow cytometry and apoptosis cells were determined by terminal deoxynucleotidyl transferasse-mediated dUTP nick end labeling (TUNEL). 3.To investigate the ATP/ADP by hyper performance liquid chromatography(HPLC). 4.The levels of insulin in supernatant were measured with radioimmunity method. 5.NRF and PGC-1 mRNA were detected by means of TaqMan real time quantitative reverse transcription (RT-PCR).The HIT-T15 cells were cultured and divided into 7 groups according to the drugs in culture media, each group had been cultured for 48h: (1).control group ,(2).High glucose group: 11mM glucose,(3).high glucose +RSG10^-6M group ,(4).high glucose+RSG10^-8M group, (5).8mMGLcN+llmM glucose group,(6).8rnMGLcN+llmMglucose+RSG10"6M group,(7).8mMGLcN+llmMglucose +RSG10⁸M group. Result l.The HIT-T15 cells in GLcN group showed apoptosis,more necrosis cells,endoplasmic reticulums vaculation through transmission electro-telescope assay,and Majority of mitochondrion was illegible even swelling with exception,and intermittent space of nuclear was wide and endoplasmic reticulum was degenerated with vacuolated pathologic change. The HIT-T15 cells In RSG10"8M group, the pathology of ultrastructure was distinctively improved whereas the RSG10"6M group hadn't. 2. 8mM GLcN could induce HIT-T15 cells apoptosis(measured by flowcytometry:8.83±0.81% vs 8.37± 1.88% ;TUNEL: 7.73+0.49% vs 5.18 + 0.94% ,respectively.),RSG could prevent the procedure moderately ,There was a marked reduction in RSG10"8M group ( measured by flow cytometry :5.10+0.42% vs 8.83 + 0.81%, P<0.05;TUNEL: 5.74+0.65% vs 7.73 + 0.49% ,P<0.05. respectively) . 3. HIT-T15 cells in 8mM GLcN produced a reduction of ATP leavels and ATP/ADP ratio compared wih contronl groups (1.20+1.03 vs 4.49 + 0.53, P<0.05) .There was not statistically different between 8mM GLcN group and RSG10"6M or RSG10" M group. But there was also a marked tendency of ascensus. 4. 8mM GLcN could inhibit the secretion of insulin from HIT-T15 cells after cocultured for 48 hours and subsequently stimulation by hyperglycemia (22mM) for 1 hour in either normal or llmM of glucose concentration, but there exsisted no significant difference, when pre-dispose with RSG ,the release of insulin by 8mMGLcN would partly be affected. Insulin concentration of 48 hours were increased in RSG10"6M +8mMGLcN and RSG10"8M+8mMGLcN, reaching 1.09 + 0.08ng/ml and 1.13 +0.09ng/ml,respectively. after cocultured with high glucose (22mM) for 1 hours, the results were increased ,reaching 0.09 + 0.12ng/ml and 0.09 + 0.06ng/ml respectively,showed no stastically significance compared with 8mM GLcN group (0.01 ± 0.02ng/ml P>0.05).But there was a marked tendency of ascensus. 5. PGC-lmRNA and NRF-lmRNA were both expressed in HIT-T15 cells which markedly increase especially in 8mM GLcN group in contrast with the normal control ones (1.33 + 0.58 vs 4.85 + 1.20 and 0.42 + 0.50 vs 1.27 + 0.24) . while RSG10"6M and RSG10"8M could proportionally contradict the upregulation of 8mM GLcN on the expression ofPGC-1 mRNA and NRF-lmRNA (2.33 + 1.53 and 2.14+1.63 vs 4.85 + 1.20, P<0.05;0.31+0.27 and 0.10 + 0.05 vs 1.27 + 0.24, P<0.05) . Conclusion GLcN could induce HIT-T15 cells to apoptosis and damage the hyperglycemia-stimulated insulin secretion and decrease ATP/ADP. GLcN destroy the ultra microstructure of HIT-T15 cells and which could be protected by appropriate concentration of RSG efficiently.Both NRF-lmRNA and PGC-lmRNA were measured in HIT-T15 cells and RSG could down-regulate the expression of NRF-lmRNA and PGC-lmRNA that induced by GLcN. We can conclude that the toxicity on HIT-T15 cells induced by GLcN was probably through by damaging the mitochondrial. When add rosiglitazone in advance with appropriate concentration ,the aforementioned damage will be partly been abolished.