| Part 1Serum Glypican4,Proneurotensin and Adiponectin are associated with adipose tissue insulin resistanceObjectivesObesity has become one of the global public health problems,the situation is very serious,a serious threat to human health and life expectancy.Adipose tissue insulin resistance is a common feature of obesity-related metabolic diseases,which may precede liver and skeletal muscle insulin resistance,and is one of the important pathophysiological components of type 2 diabetes mellitus(T2DM).As an important endocrine organ,adipose tissue secretes a variety of adipokines,which are closely related to glucolipid metabolism.However,whether these adipokines are related to adipose tissue insulin resistance has not been reported.Glypican 4(GPC4)is a novel adipokine strongly associated with obesity and insulin resistance.Glycosylphosphatidylinositol-specific phospholipase D(GPLD1)has been suggested to cleave GPC4.Proneurotensin(PNT)is an adipokine that may be associated with diabetes and coronary heart disease.Adiponectin(ADP),as one of the most widely studied adipokine,is closely related to insulin resistance and glucose and lipid metabolism.The purpose of this study was to investigate the changes of serum GPC4,GPLD1,PNT,and ADP levels in obese subjects with different glucose metabolism status,and to further analyze the relationship between them and adipose tissue insulin resistance.MethodsA total of 221 obese subjects with different glucose metabolism status from the Endocrine clinic in the Peking Union Medical College Hospital and 37 normal controls(NC)from the Medical Center were recruited.According to the levels of blood glucose and insulin,the obese subjects were divided into four groups:normal insulin(NI)group,obese subjects with normal glucose tolerance(NGT)and normal insulin levels(n=66);hyperinsulinemia(HI)group,obese subjects with NGT and hyperinsulinemia(n=50);impaired glucose tolerance(IGT)group,obese subjects with IGT(n=63);and diabetes mellitus(DM)group,obese subjects with T2DM(n=42).Serum GPC4,GPLD1,PNT,and ADP were determined by commercially available enzyme-linked immunosorbent assay(ELISA)kits.The adipose tissue insulin resistance index(Adipo-IR)was calculated by multiplying the fasting insulin(FINS)concentration by the fasting free fatty acids(FFA)concentration.One-way ANOVA analysis and Bonferroni post hoc analysis were used for comparison between multiple groups.Pearson correlation analysis was used to evaluate the bivariate relationships between GPC4,GPLD1,PNT,ADP and clinical parameters.Multiple linear regression analysis was applied for screening of GPC4,GPLD1,PNT,and ADP independent risk factors.Logistic regression analysis was performed to evaluate the relationships between serum GPC4,GPLD1,ADP levels and Adipo-IR.Results1.Adipo-IR was significantly higher in insulin resistance subjects(HI,IGT,and DM groups)than that in insulin sensitivity subjects(NC and NI groups)(P<0.05).Adipo-IR increased 4.2-fold in HI group and 3.1-fold in IGT group and 5.1-fold in DM group versus NC group(9.27[6.22,13.71],6.77[3.91,10.64],11.09[7.52,18.02]vs 2.19[1.26,3.28],all P<0.05).2.Serum GPC4 levels in obese patients(NI,HI,IGT,and DM groups)were significantly higher than those in NC group(1.93±0.34 ng/mL,2.27±0.58 ng/mL,2.21 ± 0.60 ng/mL,2.49±0.67 ng/mL vs.1.70 ± 0.33 ng/mL,P<0.05),increased by 13.53%,33.53%,30.00%and 46.47%,respectively.In addition,serum GPC4 levels in obese patients with insulin resistance(HI,IGT,and DM groups)were higher than those in the NI group(P<0.05).Serum GPLD1 levels in obese patients(NI,HI,IGT,and DM groups)were significantly higher than those in NC group(12.48±4.67 ug/mL,15.14±4.47 ug/mL,13.63±5.40 ug/mL,15.47±4.92 ug/mL vs.8.87±2.37 ug/mL,P<0.05),increased by 40.70%,70.69%,53.66%and 74.41%,respectively.Serum GPLD1 levels were higher in the HI and DM groups than those in the NI group(P<0.05).The IGT group had higher serum PNT levels than the NC group(45.02 ± 10.38 pg/mL vs.38.15±7.31 pg/mL,P<0.05).Conversely,serum ADP levels in NI,HI,and DM groups were significantly lower than those in the NC group(17.77±6.68 ug/mL,15.53±6.41 ug/mL,14.31±6.09 ug/mL vs.20.49±6.74 ug/mL,P<0.05).Serum ADP levels in the DM group were the lowest,which decreased by 30.16%,19.47%,and 23.93%compared with NC,NI,and IGT groups respectively(P<0.05).3.Bivariate correlation analysis in all subjects showed that serum GPC4 levels were positively correlated with body mass index(BMI)(r=0.266),waist circumference(WC)(r=0.306),diastolic blood pressure(DBP)(r=0.151),alanine transaminase(ALT)(r=0.352),aspartate aminotransferase(AST)(r=0.307),uric acid(UA)(r=0.152),triglyceride(TG)(r=0.234),FFA(r=0.201),FINS(r=0.254),homeostasis model assessment of insulin(HOMA-IR)(r=0.256),Adipo-IR(r=0.359),homeostasis model assessment of adiponectin(HOMA-AD)(r=0.247)and GPLD1(r=0.464)(P<0.05),and were negatively correlated with Revised quantitative insulin sensitivity check index(Revised-QUICKI)(r=-0.279)(P<0.05).Serum GPLDI was positively correlated with age(r=0.146),BMI(r=0.303),WC(r=0.278),systolic blood pressure(SBP)(r=0.201),DBP(r=0.205),ALT(r=0.232),AST(r=0.237),total cholesterol(TC)(r=0.268),TG(r=0.300),FFA(r=0.239),FINS(r=0.245),HOMA-IR r=0.241),HOMA-AD(r=0.260),and Adipo-IR(r=0.336),and were negatively correlated with high-density lipoprotein cholesterol(HDL-C)and Revised-QUICKI(r=-0.264)(r=-0.158)(P<0.05).Serum PNT were positively correlated with age(r=0.128),TC(r=0.123),and TG(r=0.128)(P<0.05).By contrast,serum ADP levels were positively correlated with age(r=0.210),gender(r=0.303),HDL-C(r=0.336),and Revised-QUICKI(r=0.390),and were negatively correlated with BMI(r=-0.174),WC(r=-0.263),SBP(r=-0.146),DBP(r=-0.208),ALT(r=-0.223),AST(r=-0.143),UA(r=-0.291),TG(r=-0.386),FFA(r=-0.264),FINS(r=-0.304),fasting blood glucose(FBG)(r=-0.205),2h-postprandial blood glucose(2hPBG)(r=-0.192),HOMA-IR(r=-0.330),Adipo-IR(r=-0.373),HOMA-AD(r=-0.739),GPC4(r=-0.187)and GPLD1(r=-0.180)(P<0.05).4.In multiple linear regression analysis,after adjusting age,gender,BMI,SBP,AST,creatinine(Cr),TG,HDL-C,low-density lipoprotein cholesterol(LDL-C),FBG,and ADP,we found that GPLD1(?=0.559),Adipo-IR(?=0.190),and UA(?=0.128)were independent influencing factors to serum GPC4 levels in all subjects(P<0.05).GPLD1 was the most important influencing factor for serum GPC4 levels.TG(P=0.467),GPLD1(?=0.208),Adipo-IR(?=0.197),and age(?=0.107)were independent influencing factors to serum GPLD1 levels after adjusting for gender,BMI,SBP,AST,Cr,UA,HDL-C,LDL-C,FBG,and ADP in all subjects(P<0.05),and TG was the most important independent influence factor for serum GPLD1 levels.Serum PNT levels were independently correlated with TG(?=0.140)and age((3=0.122)after adjusting for gender,BMI,SBP,AST,Cr,UA,HDL-C,LDL-C,FBG,Adipo-IR,and ADP in all subjects(P<0.05),and TG was the most important factor for serum PNT levels.Serum ADP levels were independently correlated with Adipo-IR(?=-0.183),HDL-C(?=0.173),gender(?=0.145),TG(?=-0.190),and age(p=0.151)alter adjusting for BMI,SBP,AST,Cr,UA,LDL-C,and FBG in all subjects.Adipo-IR was the important influencing factor for serum ADP levels.Adipo-IR was independently related to serum GPC4,GPLD1,and ADP levels,respectively.5.Logistic regression analysis was used to explore the relationships between serum GPC4,GPLD1,and ADP levels and Adipo-IR.All subjects were stratifed into trisections according to serum GPC4 levels tertiles(lowest GPC4 group:?1.81 ng/mL,medium GPC4 group:1.82-2.18 ng/mL.highest GPC4 group:?2.19 ng/mL).Subjects with the highest GPC4 levels were 1.974-fold more likely to have adipose tissue insulin resistance than those with the lowest GPC4 levels(OR=2.974(1.262-7.011),P=0.013)after adjusting for age,gender,BMI,SBP,AST,FBG,TC,TG,and HDL-C.The results indicated that the increase in serum GPC4 level is a risk factor for adipose tissue insulin resistance.Then,all subjects were stratifed into trisections according to serum GPLD1 levels tertiles(lowest GPLD1 group:? 10.71 p,g/mL;medium GPLD1 group:10.72-15.05 ?g/mL;highest GPLD1 group:?15.06 ?g/mL).Compared with the lowest GPLD1 group(reference=1.00),the risk of adipose tissue insulin resistance in the highest GPLD1 group was 3.568-fold(95%CI 1.545-8.239,P=0.003)higher than that in the lowest GPLD1 group after adjusting for age,gender,BMI,SBP.AST,and FBG.However,the increased risk of adipose tissue insulin resistance in the highest GPLD1 group disappeared after further adjusting for TC,TG,and HDL-C,indicating that high serum GPLD1 levels are more likely to have adipose tissue insulin resistance,which may be based on dyslipidemia.Eventually,all subjects were stratifed into trisections according to serum ADP levels tertiles(lowest ADP group:?12.99?g/mL;medium ADP group:13.00-19.84 ?g/mL;highest ADP group:?19.85 ?g/mL).Compared with the highest ADP group(reference=1.00),the risk of adipose tissue insulin resistance in the lowest ADP group was 2.266-fold(95%CI 1.080-4.755.P=0.040)higher than that in the highest ADP group after adjusting for age,gender,and BMI.However,the increased risk of adipose tissue insulin resistance in the lowest ADP group disappeared after further adjusting for SBP,AST,FBG,TC,TG,and HDL-C,indicating that low serum ADP levels are more likely to have adipose tissue insulin resistance,which is based on other abnormalities of glucose and lipid metabolism.Conclusions1.Adipo-IR progressively increased in the transition from NGT to IGT and T2DM.suggesting that Adipo-IR plays an important role in the pathogenesis of T2DM.2.Serum GPC4 levels were significantly increased in obese subjects,especially in obese subjects with insulin resistance.The changing trend of serum GPLD1 was similar to that of serum GPC4.GPLD1 was the most important independent influencing factor for serum GPC4,which supports the hypothesis that GPLD1 can cleave GPC4.Both GPC4 and GPLD1 were independently positively correlated with Adipo-IR.Subjects with high GPC4 levels were more likely to have adipose tissue insulin resistance after adjusting for confounders.These results indicate that serum GPC4 is a biomarker of adipose tissue insulin resistance in Chinese north populations.3.Serum PNT levels were closely related to lipid metabolism but not with adipose tissue insulin resistance.4.Serum ADP levels were closely related to glucose and lipid metabolism-related variables and negatively correlated with Adipo-IR adipose tissue insulin resistance.Part 2The mechanism of insulin resistance of adipocytes driven by the interaction between adipokine and inflammatory microenvironmentObjectivesObesity and its related metabolic diseases seriously affect human health and quality of life.Adipose tissue insulin resistance is the pathophysiological basis of obesity.Dysregulation of adipokine secretion and chronic inflammation in adipose tissue are the two most important mechanisms of adipose tissue insulin resistance,and they interact with each other and promote each other.Previous studies have shown that Adiponectin(ADP)has significant anti-inflammatory properties,while it is not clear whether the novel adipokines Glypican 4(GPC4)and Neurotensin(NT)also influence the inflammation of adipose tissue.Therefore,in this study,we simulated the real state of obesity in vivo and established a non-contact co-culture system of adipocytes and macrophages and the chronic inflammation of adipose tissue induced by palmitic acid(PA).We investigated the roles of GPC4,ADP,and NT on PA-induced macrophage inflammation.Methods1.Changes of secretion of adipokines and inflammatory factors after co-culture of adipocytes and macrophages3T3-L1 preadipocytes were induced to differentiate into mature adipocytes in 6-well plates and Transwell inserts,respectively.At the same time,RAW264.7 macrophages were cultured in 6-well plates and Transwell inserts.The adipocytes in Transwell insert and macrophages in the 6-well plate were co-cultured for 24 hours,at the same time,the macrophages in Transwell insert and adipocytes in the 6-well plate were co-cultured for 24 hours.Total RNA was extracted from co-cultured adipocytes,and GPC4,ADP,NT,IRS-1,and Glut4 mRNA levels were detected by RT-qPCR.Total RNA was extracted from co-cultured macrophages,and TNF?,MCP-1,IL-6,and IL-10 mRNA were detected by RT-qPCR.Besides,the adipokines GPC4,ADP,and NT levels and inflammatory cytokines TNFa,MCP-1,IL-6,and IL-10 levels in co-cultured supernatants were determined by commercially available ELISA kit.2.Effects of PA induction on cytokine secretion of adipocytes and macrophages(1)Effect of PA on 3T3-L1 adipocytes:3T3-L1 preadipocytes were induced to differentiate into mature adipocytes and treated with different concentrations of PA(100?M,200 ?M,400 ?M)for 6 hours,12 hours and 24 hours,respectively.Total RNA was extracted from cultured cells,and GPC4,ADP,and NT mRNA levels and insulin sensitivity related genes insulin receptor substrate-1(IRS-1)and glucose transporter 4(Glut4)mRNA levels were detected by quantitative real-time PCR(RT-qPCR).Besides,GPC4,ADP,and NT levels in culture supernatants were determined by commercially available ELISA kit.Adipocytes were treated with 0.2%BSA for 6 hours,12 hours,and 24 hours in the control group.(2)Effect of PA on RAW264 macrophages:RAW264.7 macrophages were treated with different concentrations of PA(100 ?M,200 ?M,400 ?M)for 6 hours,12 hours,and 24 hours,respectively.Total RNA was extracted from cultured cells,and TNF ?,MCP-1,IL-6,and IL-10 mRNA levels were detected by RT-qPCR.Besides,TNF a,MCP-1,IL-6,and IL-10 levels in culture supernatants were determined by commercially available ELISA kit.Macrophages were treated with 0.2%BSA for 6 hours,12 hours,and 24 hours in the control group.3.The cross-talk between adipocytes and macrophages.(1)Effect of macrophages induced by PA on adipocytes:After the RAW264.7 macrophages in Transwell insert were treated with 400 ?M PA for 24 hours,they were replaced with DMEM containing 10%FBS and antibiotics.Then the macrophages co-cultured with 3T3-L1 adipocytes cultured in 6-well plates for 24 hours,while 0.2%BSA was used to treat macrophages in Transwell insert for 24 hours before co-culture in the control group.TNF?,MCP-1,IL-6,and IL-10 mRNA levels in macrophages and GPC4,ADP,NT,IRS-1 and Glut 4 mRNA levels in adipocytes were detected by RT-qPCR.At the same time,GPC4,ADP,NT,TNF?,MCP-1,IL-6,and IL-10 levels in co-cultured supernatants were determined by commercially available ELISA kit.(2)Effect of adipocytes induced by PA on macrophages:3T3-L1 adipocytes were cultured in Transwell insert,RAW264.7 macrophages were cultured in 6-well plate,and other steps were the same as above 3(2).4.Effects of GPC4,NT,and ADP on inflammation of macrophages induced by PA.(1)Effect of GPC4 on inflammation of macrophages induced by PA:RAW264.7 macrophages induced by PA(400 ? M)were treated with different concentrations of mouse GPC4 recombinant protein(1,10,100 ng/mL)for 6 hours,12 hours and 24 hours,while macrophages induced by 400 ?M PA were used in the control group.TNF?,MCP-1,IL-6,and IL-10 mRNA levels in macrophages were detected by RT-qPCR.At the same time,TNF?,MCP-1,IL-6,and IL-10 levels in culture supernatants were determined by commercially available ELISA kit.(2)Effect of NT on the inflammation of macrophages induced by PA:RAW264.7 macrophages induced by PA(400?M)were treated with different concentrations of mouse NT recombinant protein(1,10,100?g/mL)for 6 hours,12 hours and 24 hours.The method was the same as 4(1).(3)Effect of ADP on the inflammation of macrophages induced by PA:RAW264.7 macrophages induced by PA(400 ?M)were treated with different concentrations of mouse ADP recombinant protein(0.5,1,2 ?g/mL)for 6 hours,12 hours and 24 hours.The method was the same as 4(1).Results1.Changes of secretion of adipokines and inflammatory factors after co-culture of adipocytes and macrophages3T3-L1 adipocytes and RAW264.7 macrophages were co-cultured for 24 hours.Compared with the adipocytes cultured alone,GPC4 and NT mRNA levels in adipocytes increased by 0.7 times and 2.2 times(P<0.05),respectively,and ADP mRNA levels decreased by 55.0%(P<0.05).Glut4 mRNA levels decreased by 34.0%(P<0.05),and IRS-1 mRNA levels showed a downward trend,but there was no statistical difference.TNF a,IL-6 and IL-10 mRNA levels in macrophages were 1.6 times,2.0 times,2.4 times higher than that of macrophages cultured alone,respectively.MCP-1 mRNA levels had an increasing trend,but the difference was not statistically significant.Replacing the types of cells in the co-culture system,the above adipokines and inflammatory cytokines mRNA levels showed a consistent trend with the first co-culture approach.The changes of adipokines and inflammatory cytokines levels except MCP-1 in the co-culture supernatant were consistent with the changes of their mRNA,indicating that the co-culture system was constructed successfully.2.Effects of PA induction on cytokine secretion of adipocytes and macrophages(1)Effect of PA on 3T3-L1 adipocytes:3T3-L1 adipocytes were treated with different concentrations of PA(100 ?M,200 ?M,400 ?M)for 6 hours,12 hours,and 24 hours.At 6 hours,ADP mRNA levels in different concentrations of PA group were lower than that in the control group,while GPC4 and NT mRNA levels tended to increase,but there was no significant difference.At 12 hours,ADP mRNA levels in the 400 ?M PA group decreased by 40.8%(P<0.05),and NT mRNA levels increased by 62.0%(P<0.05)compared with the control group.At 24 hours,GPC4 and NT mRNA levels in the 400 ?M PA group increased by 1.1 times and 1.5times(P<0.05),and ADP mRNA levels decreased by 30.1%(P<0.05)compared with the control group.IRS-1 mRNA levels in 400 ?M PA group decreased by 44.6%,39.8%,and 36.0%respectively at 6 hours,12 hours,and 24 hours,and Glut4 mRNA levels decreased by 39.8%and 38.2%at 12 hours and 24 hours,respectively.The changes of GPC4,ADP,and NT levels in the culture supernatant were consistent with the changing trend of their mRNA.(2)Effect of PA on RAW264 macrophages:RAW264.7 macrophages were treated with different concentrations of PA(100 ?M,200 ?M,400 ?M)for 6 hours,12 hours,and 24 hours.After treatment with 400 ?M PA for 6 hours,TNFa,MCP-1,IL-6,and IL-10 mRNA levels increased by 1.0 time,7.5 times,3.6 times,and 3.4 times compared with the control group;at 12 hours,they increased by 3.8 times,11.6 times,4.9 times and 3.3 times;at 24 hours,they increased by 5.5 times,7.4 times,7.7 times and 6.9 times,respectively.PA increased the mRNA levels of TNF?,MCP-1,IL-6,and IL-10 in a dose-dependent and time-dependent manner.Besides,the changes of TNF?,MCP-1,and IL-6 levels in the culture supernatant were consistent with the changing trend of their mRNA,but the 1L-10 level was not significantly changed.3.The cross-talk between adipocytes and macrophages.(1)Effect of macrophages induced by PA on adipocytes:RAW264.7 macrophages weretreated with 400 ?M PA for 24 hours and then co-cultured with 3T3-L1 adipocytes for 24 hours(experimental group).Compared with the control group,TNF?,MCP-1,IL-6 and IL-10 mRNA levels in macrophages increased by 2.3 times,1.5 times,2.5 times,and 1.6 times,respectively.GPC4 and NT mRNA levels in adipocytes increased by 4.0 times and 7.0 times,respectively(P<0.05),and ADP mRNA levels decreased by 21.2%.Besides,Glut4 mRNA levels decreased by 56.1%,and IRS-1 mRNA levels showed a downward trend,but the difference was not statistically significant.GPC4 and NT mRNA levels increased progressively in adipocytes of the three groups(3T3-L1 cultured alone,control group,and experimental group).Compared with the 3T3-L1 cultured alone group,GPC4 mRNA levels increased by 1.6 times and 6.9 times in the control group and the experimental group,with 1.6 times and 3.6 times increase in NT,respectively.ADP mRNA levels in the experimental group were lower than that in the other two groups,decreased by 54.0%and 40.3%(P<0.05).IRS-1 mRNA levels in the control group and the experimental group decreased by 54.0%and 72.0%respectively compared with the 3T3-L1 cultured alone group,and Glut4 mRNA levels in the experimental group decreased by 34.3%compared with the 3T3-L1cultured alone group(P<0.05).GPC4 and NT levels in the cultured supernatant increased gradually in the above three groups,while the levels of ADP decreased gradually(P<0.05).(2)Effect of adipocytes induced by PA on macrophages:3T3-L1 adipocytes were treated with 400 ?M PA for 24 hours and then co-cultured with RAW264.7 macrophages for 24 hours(experimental group).Compared with the control group,GPC4 and NT mRNA levels in adipocytes increased by 2.6 times and 1.6 times,respectively(P<0.05),and ADP mRNA levels decreased by 43.3%.IRS-1 mRNA levels decreased by 22.1%,and Glut4 mRNA showed a decreasing trend,but the difference was not statistically significant.TNF?,MCP-1,IL-6 and IL-10 mRNA levels in macrophages increased by 0.7 times,0.6 times,1.0 time,and 0.8 times,respectively.TNF?,IL-6,and IL-10 mRNA levels increased progressively in macrophages of the three groups(RAW264.7 cultured alone,control group,and experimental group).Compared with the RAW264.7 cultured alone group,TNF?,IL-6,and IL-10 in the control group and the experimental group increased by 1.6 times and 3.7 times,2.0 times and 4.6 times,2.4 times and 5.7 times,respectively.MCP-1 mRNA levels in the experimental group were 0.5 times higher than that in the RAW264.7 cultured alone group and 0.4 times higher than that in the control group(P<0.05).TNF?,MCP-1,IL-6,and IL-10 levels in the cultured supernatant increased progressively in the above three groups(P<0.05).4.Effects of GPC4,NT,and ADP on inflammation of macrophages induced by PA.(1)Effect of GPC4 on inflammation of macrophages induced by PA:RAW264.7 macrophages induced by PA(400 ?M)were treated with mouse GPC4 recombinant protein(1 ng/mL,10 ng/mL,100 ng/mL)for 6 hours,12 hours and 24 hours.After 100 ng/mL GPC4 treatment for 6 hours,TNF?,MCP-1,IL-6,and IL-10 mRNA levels increased by 2.0 times,0.8 times,0.7 times,and 0.5 times,respectively.At 12 hours,TNF?,MCP-1,and IL-10 mRNA increased by 1.1 times,0.4 times,and 0.4 times respectively compared with the control group.At 24 hours,TNF?,IL-6,and IL-10 mRNA levels increased by 0.3 times,0.6 times,and 0.5 times compared with the control group,respectively(P<0.05).The changes of TNFa,MCP-1,IL-6,and IL-10 levels in the cultured supernatant were consistent with the changes in their mRNA levels.(2)Effect of NT on the inflammation of macrophages induced by PA:RAW264.7 macrophages induced by PA(400 ?M)were treated with mouse NT recombinant protein(1 ?g/mL,10 ?g/mL,100 ?g/mL)for 6 hours,12 hours and 24 hours.After 100 ?g/mL NT treatment for 6 hours,12 hours,and 24 hours,TNF?,MCP-1,IL-6,and IL-10 mRNA levels were significantly increased compared with the control group.TNFa increased by 0.7 times,0.3 times,and 0.4 times,MCP-1 by 1.6 times,0.3 times,and 0.9 times,IL-6 by 0.7 times,1.3 times,and 1.9 times,and IL-10 by 2.0 times,0.7 times and 0.9 times(P<0.05).The changes of TNF?,MCP-1,IL-6,and IL-10 levels in the cultured supernatant were consistent with the changes in their mRNA levels.(3)Effect of ADP on the inflammation of macrophages induced by PA:RAW264.7 macrophages induced by PA(400 ?M)were treated with mouse ADP recombinant protein(0.5?g/mL,1?g/mL,2 ?g/mL)for 6 hours,12 hours and 24 hours.After 2?g/mL ADP treatment for 6 hours,TNFa and IL-10 mRNA levels increased by 1.7 times and 2.1 times,respectively,while MCP-1 and IL-6 mRNA levels decreased by 70.3%and 42.7%,respectively.At 12 hours,TNF?,MCP-1,and IL-6 mRNA levels decreased by 42.5%,78.4%,and 41.3%,respectively,and IL-10 mRNA levels increased by 1.2 times(P<0.05).At 24 hours,TNF?,MCP-1,and IL-6 mRNA levels decreased by 38.7%,86.2%,and 45.8%,respectively.The changes of TNF?,MCP-1,IL-6,and IL-10 levels in the cultured supernatant were consistent with the changes in their mRNA levels.Conclusions1.Adipocytes and macrophages were co-cultured in the Transwell system.Their interaction through paracrine interactions between cytokines exacerbates the inflammatory state of adipose tissue.2.PA induces adipocyte insulin resistance,promotes the expression and secretion of GPC4 and NT,and inhibits the expression and secretion of ADP.3.PA activates the inflammation of macrophages and affects the expression and secretion of inflammatory cytokines.4.PA induction can further enhance the interaction between adipocytes and macrophages,and then aggravate adipose tissue inflammation.5.GPC4 and NT recombinant proteins exacerbated PA-induced inflammation in macrophages,while ADP recombinant protein slowed PA-induced inflammation in macrophages. |
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