Effects Of Vaspin On Autophagy Induced By Palmitic Acid In INS-1 Cells

Objective:To investigate the effects of visceral adipose tissue-derived serpin(vaspin)on autophagy in INS-1 cells induced by palmitic acid(PA)and discuss whether vaspin could affect the proliferation,apoptosis and secretion function of INS-1 cells induced by PA via autophagy,aiming at establishing the theoretical foundation for the treatment of type 2 diabetes mellitus with adipocytokine vaspin and discuss its specific mechanisms.Methods:(1)INS-1 cells were cultured and randomly divided into the normal group: cultured in ordinary medium for 24 hours;the PA group: cultured in ordinary medium and 0.5mmol/L PA for 24 hours;the PA+vaspin group: 320 ng/ml vaspin and ordinary medium were added for 2 hours,then cultured with 0.5 mmol/L PA and 320 ng/ml vaspin and ordinary medium for 24 hours;the PA+vaspin+rapamycin group: cells were pre-cultured with 50 nmol/L rapamycin and ordinary medium for 2 hours,then co-cultured with 0.5mmol/L PA and 320 ng/ml vaspin and ordinary medium for 24 hours;PA+vaspin+3-methyladenine(3-MA)group: pre-cultured with 1 mmol/L 3-MA and ordinary medium for 2 hours,then co-cultured with 0.5 mmol/L PA and 320 ng/ml vaspin and ordinary medium for 24 hours.(2)The experiment of glucose-stimulated insulin secretion(GSIS)was performed to detect insulin level and clarify the effect of vaspin on the secretory function of PA-induced INS-1 cells.(3)The protein levels of mammalian target of rapamycin(mTOR),phos-phorylated mTOR(p-mTOR),Beclin-1,LC3-I,LC3-II and P62 were examined by western blot toclarify the effect of vaspin on PA-induced autophagy in INS-1 cells.(4)The level of autophagic flow was detected by mRFP-GFP-LC3 fusion protein to observe the effect of vaspin on PA-induced autophagic flow in INS-1 cells.(5)CCK-8 kit was used to detect the proliferation of INS-1 cells,and to explore the effect of vaspin on the proliferation of INS-1 cells induced by PA.(6)Cell apoptosis was detected by flow cytometry to observe the effect of vaspin on apoptosis of INS-1 cells induced by PA.Results:(1)Compared with the control group,the insulin level stimulated by 3.3 mmol/L and 16.7 mmol/L glucose in INS-1 cell supernatant of PA group were lower(1.02�0.08 vs.0.54�0.06,P=0.000;1.15�0.13 vs.0.71�0.05,P=0.000),vaspin intervention group was higher than that of PA group(0.76�0.03 vs.0.54�0.06,P=0.001;0.91�0.04 vs.0.71�0.05,P=0.011).(2)PA reduced the ratio of p-mTOR to mTOR protein compared with the normal group(2.04�0.14 vs.1.19�0.09,P=0.000),and vaspin increased the ratio of p-mTOR to mTOR protein(1.51�0.05 vs.1.19�0.09,P=0.005).Meanwhile,in the PA group,autophagy-associated proteins such as Beclin-1 and the ratio of LC3-II to LC3-I were significantly elevated and P62 level was decreased(0.65�0.04 vs.0.81�0.07,P=0.005;1.16�0.11 vs.2.35�0.25,P=0.000;1.04�0.19 vs.0.60�0.08,P=0.000),vaspin intervention antagonized these effects(0.58�0.04 vs.0.81�0.07,P=0.001;1.95�0.08 vs.2.35�0.25,P=0.007;0.82�0.03 vs.0.60�0.08,P=0.032).(3)The number of autolysosomes in PA group was higher than that in normal group(8.33�1.53 vs.0.33�0.58,P=0.000),and this decreased after vaspin intervention(3.67�1.53 vs.8.33 �1.53,P=0.001).(4)INS-1 cells intervened by PA for 24 hours could decrease cell proliferation as compared to the control group(0.50�0.16 vs.1.00�0.00,P=0.000),and vaspin intervention could inhibited the decrease of INS-1 cells induced by PA(0.84�0.76 vs.0.50�0.16,P=0.002).(5)INS-1 cells intervened by PA for 24 hours could increase cell apoptosis as compared to the control group(22.15�2.05 vs.7.91�0.50,P=0.000),and vaspin intervention decreased the apoptosis(13.23�1.97 vs.22.15�2.05,P=0.000).Conclusion:(1)PA induced excessive autophagy and apoptosis of INS-1 cells,inhibited cell proliferation and decreased the secretion function of INS-1 cells.(2)Vaspin inhibits excessive autophagy and apoptosis in INS-1 cells induced by PA,and improves the proliferation and secretion function of INS-1 cell.