Effects Of Different Concentrations Of Glucose,Insulin And Oxidative Stress On Visfatin Expression In 3T3-L1 Adipocytes

Objective: Visfatin is a novel adipocytokine that correlates with the development of insulin resistance and type 2 diabetes mellitus. It is important to illustrate the regulation mechanism of visfatin to reveal the pathogenesis of insulin resistance and diabetes coused by obesity and how to control the disease. In this paper,we investigated the effects of different concentrations of glucose,insulin and prolonged oxidative stress on the expressions of visfatin mRNA and protein in 3T3-L1 adipocytes to know the relationships of visfatin with insulin resistance and diabetes mellitus.Methods: 3T3-L1 preadipocyte cells (purchased from Shanghai Cell Bank)were taken as the investigative object. 3T3-L1 cells were induced to differentiate. The cells were stained with oil red 0 to identify wheather preadipocyte cells were fully differentiated. The mature adipocytes were serum starved by high glucose DMEM with 0.5% BSA for 6 hours.Then different concentrations of glucose(5mmol/L,25mmol/L,45mmol/L,60mmol/L) and insulin(0.01μmol/L,0.1μmol/L, 1μmol/L) were added for 48 hours. Different concentrations of hydrogen peroxide(0.2mmol/L,0.5mmol/L,lmmol/L) were added for 10 minutes. After that,this group was cultured in high glucose DMEM with 10% fetal bovine serum for another 6 hours. Cell viability was determined by MTT assay. Total cellular RNA and protein were extracted. Then the expressions of visfatin mRNA and protein were measured by semi-quantitative RT-PCR and western blot withβ-actin as internal control. All the experimental processes were repeated for 4 times.Results: 1.Cell viability determined by MTT There was no statistical difference between intervention groups and control group (P>0.05).2.The effects of different concentrations of glucose, hydrogen peroxide and insulin on the expression of visfatin in 3T3-L1 adipocytesa. The effects of different concentrations of glucose on the expression of visfatin in 3T3-L1 adipocytesThere were decreases of 16.45% (P<0.05),23.58% (P<0.05),56.27% (P <0.01 ) and 58.61% (P<0.01 ) in visfatin mRNA expressions in 5mmol/L,25mmol/L,45mmol/L and 60mmol/L glucose groups compeared with control group. The expressions of visfatin protein were suppressed by 33.97%,38.30%,54.32% and 56.94% (P<0.01) in 5 mmol/L,25 mmol/L,45 mmol/L and 60 mmol/L glucose groups compeared with control group. Moreover, the negative effect of glucose was time dependent. When 5 mmol/L glucose group compared with 25 mmol/L glucose group, 45 mmol/L glucose group compared with 60 mmol/L glucose group, there was also no statistical difference. But when 45 mmol/L glucose group compared with 5 mmol/L glucose group and 25 mmol/L glucose group,the decreased expressions of visfatin mRNA were 47.67% and 42.78% (P<0.01) .Visfatin protein were decreased by 30.82% and 25.96% (P<0.01) . When 60 mmol/L glucose group compared with 5 mmol/L glucose group and 25 mmol/L glucose group,the expressions of visfatin mRNA were decreased by 50.46% and 45.83% (P <0.01) ,and protein were 34.79% and 30.22% (P<0.05) .b. The effects of different concentrations of hydrogen peroxide on the expression ofvisfatinin 3T3-L1 adipocytesSignificant 70.17%,67.35% and 57.32% reductions of visfatin mRNA were seen in 0.2mmol/L,0.5mmol/L and 1mmol/L hydrogen peroxide groups compared with control group. Hydrogen peroxide significantly suppressed protein experessions of visfatin by 22.79%,35.38% and 30.26% in 0.2mmol/L,0.5mmol/L and 1mmol/L hydrogen peroxide groups. The expressions of visfatin mRNA and protein rose with the increased concentrations of hydrogen peroxide,but there was no statistical difference (P>0.05).c. The effects of different concentrations of insulin on the expression of visfatin in 3T3-L1 adipocytesThe mRNA and protein experessions of visfatin were decreased by insulin, but there was no significance (P>0.05).And there was also no statistical difference between different intervention groups (P>0.05).Conclusion: The expressions of visfatin mRNA and protein in 3T3-L1 adipocytes in vitro can be inhibited by high level glucose. The decreased expression of visfatin may be one of the mechanisms of insulin resistance caused by high level glucose. Prolonged oxidative stress can also down-regulate visfatin expression,which illustrates that visfatin takes part in the process of insulin resistance aggravated by oxidative stress. The change of insulin concentrations does not affect visfatin expression. Maybe insulin is not a main factor to impact the expression of visfatin in vitro.