The Change Of Foxo1 And Tsc2 Expression And Cell Function Of Nit-1 Cell Lines Treated With Rosiglitazone In High Glucose Conditions

Objective To study the FoxO1 and TSC2 expression and cell proliferat- ion, secretion of NIT-1 cell line treated with rosiglitazone intervention in different concentrations of glucose.Methods The NIT-1 cells were cultured in RPMI-1640 medium, then divided into five groups according to different concentrations glucose culture medium, in which the first group(containing 5.6mmol/L glucose), the second group(11.1mmol/L), the third group (16.7mmol/L), the fourth group (22.5mmol/ L) and the fifth group(27.6mmol/L) differently.Firstly all groups were cultured with 11.1 mmol/L density of glucose which was called normal culture medium for 48 hours and then changed medium to different concentrateions glucose of culture medium for processing culture to 120 hours. The each groups of NIT-1 cells were added different concentrations of rosiglitazone intervention diferently after 72 hours and continue to culture for 48 hours and then cell proliferation detected with MTT assay,cell insulin secretion measured with radioimmunoas- say,cell apoptosis detected with immunofluorescence,p-FoxO1 expression were checked with western blot and immunofluorescence assay and FoxO1,TSC2 mRNA expression measured with RT-PCR assay.Results The proliferation of NIT-1 cells were raised with glucose concen- tration increased. When the glucose concentration is up to 11.1mmol/L, the rate of cell proliferation reach the highest, when the glucose concentrations were over 11.1mmol/L, the cell proliferation were descended accompany with glucose concentrations growth (the results of cell proliferation rate as follow:5.6mmol/L group was lower than 11.1mmol/L group (P<0.05), 16.7mmol/L group, 22.5 mm ol/L group, 27.6mmol/L group compared with 5.6mmol/L group (P<0.05). Insul- in secretion of the cell is the highest in 11.1 mmol/L group. With glucose concentration increase arrived at 16.7mmol/L, 22.5mmol/L and 27.6mmol/L group the cell secreted insulin reduced gradually, there were significant different between every two groups (P<0.05), but insulin secretion in those of groups were much more than 5.6mmol/L group (P<0.05). The cell apoptosis progressi- vely increased in 16.7mmol/L group, 22.5mmol/L group, 27.6mmol/L(P<0.05) compared with 5.6mmol/L group and 11.1mmol/L group differently and rate of cell apoptosis was lowest in 11.1mmol/L group. p-FoxO1 protein expression was the highest in level of glucose concentration at 11.1 mmol/L, but the level of p-FoxO1 protein expression were descended followed by glucose concentra- tion increased progressively (P<0.05) compered with 11.1mmol/L glucose con- centration. FoxO1 and TSC2 results were showed that the two gene expressed were lowest in the glucose concentration at 5.6mmol/L and FoxO1 and TSC2 expression were increased with glucose concentration increased, it had much differece between each group (P<0.05). Cell proliferation,insulin secretion and p-FoxO1 expression increased after the intervention of rosiglitazone in each group, the rate of cell apoptosis and FoxO1,TSC2 mRNA expression were descended in each group. Conclusions It is most suitful condition for cell culture,in that case, pan- creaticβ-cell growth better, cell proliferation and insulin secretion increased at glucose concentration of 11.1mmol/L. With the glucose concentration(>11.1 mm ol/L)enhenced the NIT-1 cells growth were inhibited but the rate of apoptotic cells were increased,meanwhile the level of insulin secretion, were reduced and caused the emergence of functional defects. After rosiglitazone added the cell proliferation increasing growth and insulin secretion levels was also increased but cell apoptosis significantly decreased. High concentrations of glucose (>11.1 mmol/L)can caused p-FoxO1 protein levels decrease,meanwhile, mRNA expres- sion levels of FoxO1 increased. In this results we speculated by the increased of nuclear localization of FoxO1, on the one hand,it can enhance the transcription of correlative apoptotic genes, which can lead to isletβ-cell apoptosis,and on the other hand by inhibiting the transcription activity of peroxisome proliferator- activated receptorγ(PPARγ), to affect cell insulin secretion and increase cells insulin resistance.After treated with PPARγagonist rosiglitazone FoxO1 expres- sion decreased in NIT-1cell line and TSC2 expressionis alsodecreased. The resu- lts clue to us that maybe have some corelation between TSC2 and FoxO1 in cell signaling pathway. It can be also inferred that rosiglitazone may regulate both FoxO1 and TSC2 in different way to improve the isletβ-cell proliferation,cell secretory function,and to release cell insulin resistance.