Effect Of CXCR2 On Glucose Metabolism In Hepatocytes Induced By Adipose Tissue In Different Regions

Objectives:The purpose of this study is to explore whether CXCR2 plays a part in glucose metabolism in hepatocytes induced by adipose tissue(AT)in different regions.Methods:First of all,CXCR2-specific small interfering RNA(siRNA)and CXCR2 overexpressed plasmid were used to transfect HepG2 cells to construct the HepG2 cells of silenced,overexpressed CXCR2;secondly,visceral adipose tissue(VAT)and subcutaneous adipose tissue(SAT),which release adipokines stably,were co-cultured with HepG2 cells after interfering with CXCR2 expression through transwell chamber;thirdly,HepG2 cells in each group were collected after co-culture,and quantitative real-time PCR(qRT-PCR)was used to detect CXCR2 expression of HepG2 cells after co-culture,so as to verify the intervention effect of CXCR2 expression of HepG2 cells induced by AT in different regions.At the same time,the supernatants of each group after co-culture were collected for lactate dehydrogenase(LDH)cytotoxicity assay to detect the activity of LDH in the supernatant after co-culture.The growth of HepG2 was observed under an inverted microscope to learn about the apoptosis of HepG2 cells after co-culture,and further experiments were conducted using hepatocytes;and finally,HepG2 cells in each group were collected after co-culture under basic state and insulin stimulation state,and stained by fluorescent glucose analog 2-NBDG,the proportion of fluorescently labeled HepG2 cells was observed by flow cytometry(FCM),and the glucose uptake rate of HepG2 cells was evaluated.At the same time,the glycogen distribution in HepG2 cells was observed by period-acid-shiff stain(PAS)staining,and the glycogen content in HepG2 cells was determined by anthrone assay.Results:(a)Verification of intervention effects of CXCR2 expression of HepG2 cells induced by AT in different regions:Compared with HepG2 cells treated with AT,the expression level of CXCR2 was significantly decreased in HepG2 cells transfected with CXCR2-siRNA induced by AT(p<0.001),while the expression level of CXCR2 was markedly elevated in HepG2 cells transfected with CXCR2-overexpressed plasmid induced by AT(p<0.001).(b)Determination results of LDH activity and cell growth of supernatant of HepG2 cells induced by AT in different regions:1)Compared with HepG2 Group,the LDH levels of supernatant of HepG2 cells induced by AT in different regions all went up(P<0.05);2)Under an inverted microscope,HepG2 cells had good morphology and refraction,and the results of cell count indicated that HepG2 cells in each group still maintained certain activity.(c)Effect of CXCR2 on the glucose uptake rate of HepG2 cells induced by AT in different regions:1)Under basic state and insulin stimulation state,compared with HepG2 cells induced by AT,the glucose uptake rate of HepG2 cells transfected with CXCR2-siRNA induced by AT went up(P<0.001);2)Under basic state and insulin stimulation state,compared with HepG2 cells induced by AT,the glucose uptake rate of HepG2 cells transfected with CXCR2-overexpressed plasmid induced by AT decreased(P<0.001);3)Taking HepG2 group as the baseline,the glucose uptake rate of the HepG2 cells transfected with CXCR2-overexpressed plasmid induced by AT showed the greatest reduction,that of HepG2 cells induced by AT came second,the glucose uptake of HepG2 cells transfected with CXCR2-siRNA induced by AT decreased the least(P<0.001),and the reduction of VAT group was more pronounced than that of SAT group(P<0.001);4)After insulin stimulation,compared with that under basic state,the glucose uptake rates of HepG2 cells induced by AT in different locations all went up(P<0.001).(d)Effect of CXCR2 on the glycogen content of HepG2 cells induced by AT in different regions and glycogen distribution:1)Under basic state and insulin stimulation state,compared with HepG2 cells induced by AT,the glycogen content of HepG2 cells transfected with CXCR2-siRNA induced by AT went up(P<0.001);2)Under basic state and insulin stimulation state,compared with HepG2 cells induced by AT,the glycogen content of HepG2 cells transfected with CXCR2-overexpressed plasmid induced by AT decreased(P<0.001);3)Taking HepG2 group as the baseline,the glycogen content of the HepG2 cells transfected with CXCR2-overexpressed plasmid induced by AT showed the greatest reduction,that of HepG2 cells induced by AT came second,the glycogen content of HepG2 cells transfected with CXCR2-siRNA induced by AT decreased the least(P<0.001),and the reduction of VAT group was more pronounced than that of SAT group(P<0.001);4)After insulin stimulation,compared with that under basic state,the glycogen content of HepG2 cells induced by AT in different locations all went up4(P<0.001);5)Under an inverted microscope,the glycogen distribution of HepG2 was observed to be consistent with the determination result of glycogen content.Conclusion:1)The glucose uptake and glycogen synthesis of hepatocytes were affected by AT in different regions,and VAT was more effective than SAT in inhibiting the glucose uptake and glycogen synthesis of hepatocytes.2)CXCR2 negatively regulates the glucose uptake and glycogen synthesis of hepatocytes induced by AT,suggesting that CXCR2 is an essential participant in AT mediating the dysfunction of glucose uptake and glycogen synthesis in hepatocytes.The enhanced expression of CXCR2 of hepatocytes induced by AT may interfere with the normal glucose uptake and glycogen synthesis of hepatocytes;3)Insulin can be helpful to improve AT-induced glucose uptake and glycogen synthesis;4)IL-8-CXCR2 may be a vital pathway in the development of obesity-related T2DM.