Insulin Insensitivity Due To Increased O-GlcNAc Modification Is Associated With Decreased Levels Of HMW Adiponectin

【Objective】 Through the increase of O-GlcNAc levels in adipocytes,the changes of intracellular adiponectin levels,insulin sensitivity and glucose and lipid metabolism were explored.【Methods】3T3-L1 fibroblasts were used as experimental subjects.The traditional cocktail method was used to induce adipogenic differentiation into mature adipocytes.The cells were stained with oil red on the 10 th day of differentiation to identify whether adipocytes differentiated and matured..(1)Differentiated mature adipocytes were randomly divided into two groups,and each group was repeated at least 3 times.The experimental group was further cultured in DMEM serum-free medium containing PUNUMA,an inhibitor of the O-GlcNAcase(OGA),which is a key enzyme of the O-GlcNAc modification,and the control group was cultured in serum-free DMEM medium for 16 hours.After that,the cell supernatant was taken to measure the residual glucose concentration;the protein was extracted by lysis of RIPA cell lysate pre-dosed with PMSF;the total protein concentration in the cell extract was determined by BCA method;and the protein O-was determined by Western Blotting.The level of GlcNAc modification,glycosylation modification correlated with OGA,OGT levels and total adiponectin,high polymer adiponectin(HMW),p-AMPK/AMPK levels,and p-Akt/Akt levels.(2)After completion of PUGNAc intervention culture or blank control culture for 16 hours,the two groups were gently rinsed twice with sterile PBS buffer,and then were administered with DMEM medium containing 10 nmol/L insulin and glucose concentration of 25 mmol/L.After incubation,the cell culture supernatant was extracted after 1 hour.GLU,HDL,LDL,total cholesterol(Chol)in the cell culture supernatant were detected by biochemical analysis.)and triglyceride(TG)levels;Western blotting was used to detect p-Akt/Akt levels in protein extracts.Adipocytes were stained with a lipid-specific fluorescent stain,Bodipy.Fluorescence immunoassays were used to detect p-AMPK levels in the cells and observed under a fluorescence microscope.【Results】: The results showed:(1)Successful induction of differentiation of fat precursor cells into adipocytes.After differentiation,the cells were divided into two groups for drug PUGNAc intervention and blank control.1 Western blotting showed that compared with the control group,the 3T3-L1 adipocytes in the PUGNAc treated group had significantly increased levels of total protein O-GlcNAc(P<0.05),and the levels of OGA also increased(p< 0.05),the difference was statistically significant;however,there was no significant change in protein OGT levels(P>0.05);(2)The remaining glucose concentration in the supernatant of the cells was measured before and after insulin treatment.The results showed that compared with the control group,PUGNAc treatment The residual glucose concentration in the supernatant of the group cell was higher,and the glucose uptake rate was lower than that in the control group(the glucose uptake rate was equal to the glucose concentration in the initial culture minus the residual glucose concentration at the culture endpoint)(P<0.05);The level of LDL-C in the supernatant was higher than that in the control group(P<0.05).There was significant difference between the two groups.The fluorescence dye Bodipy staining of the adipocytes showed an increase in fat droplets outside the adipocytes in the PUGNAc group;(3)PUGNAc The total adiponectin(APN)level in the treated group was higher than that in the control group,but the level of high molecular adiponectin(HMW)was lower than that in the control group,and the ratio of HMW/total APN was decreased.P values were <0.05,and the difference was significant.(4)Increased O-GlaNAc levels,P-The level of AMPK decreased,and the proportion of P-AMPK/AMPK decreased(P<0.05).Without insulin stimulation,the levels of p-Akt/Akt in the two groups did not change significantly(P>0.05).After insulin stimulation,the p-Akt/Akt levels in the PUGNAc treated group were lower than those in the control group(p<0.05).【Conclusion】The level of protein O-GlcNAc modification in adipocytes is increased,the sensitivity of cells to insulin is decreased,and the function of glucose and lipid metabolism is disordered,which may be related to the decrease of high-molecular-weight adiponectin.