Effects Of Metformin On M RNA Expression Of Vesicle-associated Membrane Protein By Human Omental Adipocytes

Objective:To observe the effects of metformin on the expression of vesicle-associated membrane proteins(VAMP2,3,4,7,8) which the subfailmilies of vescical-souble N-ethymaleimide sensitive factor attachment proteins receptor(v-SNAREs)in human omental adipocytes.Method:1.Omental adipose tissue biopsies were obtained from patients undergoing elective open-abdominal surgery, and preadipocytes was separated from it by collagenase typeⅠdigestion.Preadipocytes were seeded in DMEM/F12(1:1) medium at 37℃ and with CO2 existed with the concentration of 5%.Then induce human preadipocytes into mature adipocytes and proofed by oil red O staining.2.Twenty-four hours after giving different concentration metfomin(0m M, 1m M,10 m M) intervention, we using relative realtime fluorescence quantitative PCR technology to detect the m RNA expression of VAMP2,3,4,5,7,8.3.Values were given as means±SE and analysized by SPSS19.0 softwares.Statistical analysis was undertaken using Student T test for comparison of control versus treatments,differences were considered significant at a value of P<0.05.Results:1.Human preadipocytes were primary cultured successfully,and we successfully induced human preadipocytes into mature adipocytes.2.The results from RT-PCR:The levels of VAMP4,5,8 in 1m M metformin group was increased compared with control group(respectively, P<0.05, P<0.05, P<0.05,), while the levels of VAMP3,7 was decreased(respectively,P<0.05,P<0.01),the expression of VAMP2 do not have statistically significant difference. 10 m M metformin could also increase theVAMP4,5,8 expression,compared with those in control group(respectively, P<0.01,P<0.01, P<0.01,), and the levels of VAMP7 was decreased(P<0.05),while the effection on VAMP2、3 do not have statistically significant difference. Comparison of 1m M group and10 m M group, 10 m M group metformin increase the levels of VAMP3,4,5,8(respectively,P<0.05, P<0.05, P<0,05,P<0.01),but the expression of VAMP2,7 do not have statistically significant difference between the two groups.Conclusions:1.Human preadipocytes are separated from adipocyte tissue by using collagenaseⅠand primary cultured successfully.The feasibility and practicability of the method of culturing preadipocytes that our laboratory established have been verified by our experiments.2.Metformin can increases the levels of VAMP4,5,8 and decreases the levels of VAMP3,7 in adipocytes with a certain degree of dose-dependently, but do not regulate the expression of VAMP2, which show that metformin regulated GLUT4 trafficking may through its regulation of v-SNAREs expression.Our results make a better understanding of the mechanism that how metformin increase insulin sensitivity, and providing clues for further study on the mechanism of how metformin lower blood sugar.