| BackgroundHigh-density lipoprotein cholesterol (HDL-C) level is negatively correlate with the incidence of coronary heart disease.Increased HDL-C levels can delay or prevent the occurrence of atherosclerosis.HDL-C against atherosclerosis is primarily related to its role in reverse cholesterol transport (RCT). In recent years, the worldwide epidemic of obesity has become a public health problem of global concern.Obesity is accompnied with lower HDL-C.Statins and fibrates are widely used in clinical lipid-lowering drugs which can elevate HDL-C levels and improve the RCT.Apolipoprotein M (apoM) is a newly discovered apolipoprotein mainly associated with HDL-C,which play an important role in the metabolism of HDL-C.It affects the RCT process mainly through the regulation of pre-β-HDL. Hepatocyte nuclear factor-1α(HNF-1α), forkhead box A2(FOXA2) and liver X receptor-α(LXRα) are three key nuclear receptors regulating apoM expression.The first two increase the expression of apoM, the latter reduce apoM expression.It is unclear whether the effect of statins and fibrates on HDL-C and RCT related to apoM or not;It is not clear whether apoM changes in the state of obesity.Obesity is a chronic inflammatory disease, In addition, obesity often accompanied with hypoadiponectinemia.However, there is no reports about the relationship between inflammatory factors, adipone-ctin and apolipoprotein M.ObjectiveTo observe the changes of apolipoprotein M in obese mice and to explore the relationship with adiponectin and its mechanism;To observe the level of apoM in obesity and explore the relationship between apoM and inflammatory factor, vascular endohelial function.To examine the effects and mechanism of simvastatin, fenofibrate and combination of the two drugs on the expression of apolipoprotein M.Methods1.changes of apolipoprotein M in obese mice and the relationship with adiponectin1) Animal experimentsThree-week-old male C57BL/6N mice (n=32) were randomly divided into 4 groups :control group:normal diet; obese group:high-fat feed, fed for 12 weeks; obesity-intervention group:high-fat feed, fed for 12 weeks and then received an intraperitoneal injection of adiponectin for 7 days.General intervention group:normal diet,received an intraperitoneal injection of adiponectin for 7 days. Body weight, adiponectin, glucose, insulin levels were measured in the 0 week, the 12 week and after the intervention of adiponectin, respectively. Apolipoprotein M, forkhead box A2(FoxA2)mRNA and protein expression were measured by real-time PCR(real-time reverse transcript PCR)and Western blot at end of the experiment.2) cell experimentsHigh concentrations of insulin-induced insulin resistance in HepG2 cells to buid cell model, insulin-resistance HepG2 cells and HepG2 cells were interfered with adiponectin for 24hrs, Apolipoprotein M, FoxA2-mRNA and protein expression were measured in all cell groups.2.Apolipoprotein M levels and its relationship with inflammatory factors in obesityObese patients(n=40)and healthy volunteers(n=58) were included. blood pressure, weight, height were measured.After plasma samples obtained,plasma apoM level was measured by an ELISA method. Besides, fasting plasma lipids, glucose, insulin, Interleukin-6 (IL-6), creactive protein (CRP), tumor necrosis factor-a (TNF-a), adiponectin were measured. Body mass index and insulin resistance index were calculated.Furthermore, brachial artery endothelial function was detected.3.Effects and mechanism of simvastatin and fenofibrate on the expression of apolipoprotein M1) Animal experimentsHealthy male C57BL/6N mice (n=32) were randomly divided into four groups (n= 8 each group):(1)control group, with no special treat- ment; (2)statin group:with simvastatin (10mg/kg/day) for 4 weeks; (3)brate group:with fenofibrate (1 OOmg/kg/day) for 4 weeks; (4) combin-ation group:with simvastatin (10mg/kg/day) and fenofibrate (100mg/kg /day) for 4 weeks;fasting plasma lipid were measured 0 and 4 week. Liver samples were obtained at 4 week.The expression of hepatic apoM, HNF-la and LXRa gene and protein were measured using real time RT-PCR and Western Blot analysis respectively.2) cell experimentsThe HepG2 cells were incubated with different concentration of simvastatin (0,1,5,10,25μmol/L),fenofibrate (0,50,100mmol /L),simvastatin (5.0μmol/L)+fenofibrate(50mmol/L), simvastatin (5.0μmol/L)+HNF-1αinhibitor simvastatin (5.0μmol/L)+LXRa antagonist, fenofibrate(50mmol/L)+HNF-1αinhibitor, fenofibrate (50mmol/L)+LXRa inhibitor for 24h, respectively. Total RNA and protein of HepG2 cells were extracted. The levels of apoM,HNF-la and LXRa gene and protein were measured by real time RT-PCR and Western blot.Results1.Compared with the control group, body weight of obese group and obese intervention group were significantly higher.The weight of visceral fat, fasting glucose, fasting insulin, HOMA-IR in obesity group were significantly higher than control group.Compared with control group, plasma adiponectin levels significantly decreased in obese group.Blood glucose, insulin, insulin resistance index in obesity intervention group was significantly lower than the obese group. Plasma adiponectin levels in obesity intervention group were significantly higher than the obese group.2.compared with control group,apolipoprotein M mRNA and protein expression were significantly decreased in obese mice (all P<0.05);3.compared with control group,FOXA2mRNA and protein expression were significantly decreased in obese mice(all P＜0.05);4.apolipoprotein MmRNA and protein expression were signifi-cantly increased after the intervention of adiponectin in mice(all P＜0.05);5.FOXA2 mRNA and protein expression were significantly increased after the intervention of adiponectin in mice(all P<0.05),Plasma adiponectin levels was significantly increased. Fasting glucose, fasting insulin, HOMA-IR were significantly decreased.(all P＜0.05);6.apolipoprotein M, FOXA2mRNA and protein expression were significantly decreased in insulin-resistance HepG2 cells(all P＜0.05);7.apolipoprotein M, FOXA2mRNA and protein expression increased significantly after the intervention of adiponectin in insulin-resistance HepG2 cells(all P<0.05);apolipoprotein M, FOXA2mRNA and protein expression had no significant change after the intervention of adiponectin in HepG2 cells(all P>0.05)8.Compared with controls, obesity had lower plasma apoM, lower HDL-C, lower plasma adiponectin level(P<0.05). Conversely, Fasting insulin, IL-6, TNF-α, CRP markedly increased in obesity than controls(P<0.05).9.In obesity, apoM was positively related to HDL-C level and negat-ively related to BMI, insulin, HOMA-IR, IL-6, TNF-α, CRP. Plasma apoM had no significant correlation with plasma adiponectin, LDL-C, TC, TG, blood pressure, blood sugar and FMD. In control group,apoM was positively related to HDL-C level and negatively related to BMI, Plasma apoM had no significant correlation with insulin, HOMA-IR, IL-6, TNF-a, CRP,plasma adiponectin, LDL-C, TC, TG, blood pressure, blood sugar, FMD.10.Compared with the control group, plasma HDL-C was significantly elevated in three drug treatment group.Combination group was more effective than statin group and brate group (P< 0.05). The expression of apoM was significantly elevated in three drug treament group. Combination group was more effective than statin group and brate group(P<0.05). both of simvastatin and fenofibrate can dose-dependently increase the expression of apolipoprotein M in HepG2 cells. Combination group obtained more effects than either agent (P< 0.05). The expression of apolipoprotein M in simvastatin+HNF-1αinhibitor, simvastatin+ LXRa antagonist group were significantly lower than the same concent-ration simvastatin group (P< 0.05). The expression of apolipoprotein M in fenofibrate+ HNF-la inhibitor was significantly lower than the same concentration fenofibrate group (P< 0.05).The expression of apolipo-protein M in fenofibrate+LXRa inhibitor was significantly higher than the same concentration fenofibrate group (P< 0.05).11.Compared with the control group,the expression of HNF-1αwas significantly elevated in three drug treatment group.Combination group was more effective than statin group and brate group (P< 0.05). both of simvastatin and fenofibrate can dose-dependently increase the expression of HNF-la in HepG2 cells.Combination group obtained more effects than either agent (P<0.05).12.Compared with the control group,the expression of LXRa gene and protein was significantly decreased in statin group, the expression of LXRa was significantly elevated in brate group.However, no significant difference in LXRa expression was seen between combination and control (P<0.05).simvastatin can dose-dependently decrease the expression of LXRa in HepG2 cells. Fenofibrate can dose-dependently inc-rease the expression of LXRa in HepG2 cells (P＜0.05).No significant difference in LXRa expression was seen between combination and control in HepG2 cells.Conclusions 1.Apolipoprotein M, FOXA2 gene and protein expression signi-ficantly decreased in obese mice. Decrease apolipoprotein M expression may be related to decreased FOXA2.2.Plasma adiponectin level was significantly decreased in obese mice,visceral fat weight, fasting glucose, fasting insulin, HOMA-IR were significantly increased in obese mice.3.Adiponectin in obese mice can upregulate the expression of apolipoprotein M and its mechanism may be related to its effect on insulin resistance.4.Adiponectin increases apolipoprotein M expression in insulin resistance HepG2 cells, but has no effect on apolipoprotein M expression in HepG2 cells.This suggests that adiponectin regulates apolipoprotein M by indirectly affecting insulin resistance.5.Obese patients had lower apoM plasma. Lower apoM levels may be one of the underlying mechanisms account for lower HDL-C in obese patients.6.ApoM levels was closely related to CRP, TNF-a and IL-6 levels in obese patients. apoM may be regulated by these inflammatory factors, its mechanism may be related to inflammatory factors in the promotion of insulin resistance.7.ApoM levels of obese patients had no significant correlation with FMD, suggesting that apoM can not serve as an early predictor of atherosclerosis.8.Upregulation of apoM expression by simvastatin and fenofibrate may contribute to their effect on HDL-C.Combination is more effective.9.There is a mechanic compensation in regulation of apoM expression, i.e. Simvastatin increases HNF-1αand inhibits LXRa, but fenofibrate simultaneously induces the expression of HNF-1αand LXRa.It indicates the combination of statin and fibrate will obtain more HDL and apoM elevation effects than either agent.
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