Mechanism Underlying Regulation Of Triglyceride Metabolism By Apolipoprotein A5 And Clinical Implication

Background:Elevated plasma triglyceride(TG) is an independent risk factor of coronary heart disease.The common causes for hypertriglyceridemia (HTG) include high fructose diet,acute phase response(APR) and metabolic syndrome(MS).Plasma TG reduction will help to prevent coronary heart disease.Therefore,it will help to reduce the morbidity and mortality of coronary heart disease by the treatment of high fructose diet-induced HTG.However,the underlying mechanism for high fructose diet-induced HTG remains to be clarified.Fibrate,statin and Xuezhikang are three clinically widely-used hypotriglyceridemic agents.The recently identified apolipoprotein A5(apoA5) is implicated in TG-lowering by the considerable body of data.Peroxisome proliferator-activated receptor-α(PPARα) and liver X receptor-α(LXRα) are two key nuclear receptors in regulation of apoA5 expression.PPARαcan upregulate apoA5 expression but otherwise for LXRα.However,it is unclear for the role of apoA5 in HTG induced by high fructose diet,APR and MS.Furthermore,it remains unknown the association of apoA5 with the hypotriglyceridemic effects of fibrate, statin and Xuezhikang. Objective:The aim of this study was to define the role of apoA5 in HTG induced by high fructose diet,APR and MS;to investigate the association of apoA5 with the hypotriglyceridemic effects of fibrate,statin and Xuezhikang;to determine the involvement of PPARαand LXRα.Methods:Ⅰ.Animal and cell experiments for fructose-induced HTGForty healthy male Sprague-Dawley rats were randomized into five groups(n=8 each group):(1) control group,with no special treatment;(2) fructose group,with 10%fructose water for two weeks to establish animal HTG model,and with 10%fructose water for next four weeks;(3) fibrate group,with 10%fructose water for two weeks and supplemented with fenofibrate 100 mg/kg/d for next four weeks;(4) statin group,with 10%fructose water for two weeks and supplemented with atorvastatin 10 mg/kg/d for next four weeks;and(5) combination group,with 10% fructose water for two weeks and supplemented with fenofibrate 100 mg/kg/d and atorvastatin 10 mg/kg/d for next four weeks.The measurements were performed at 0,2 and 6 week for weight and fasting plasma lipids,blood sugar and insulin in animals.Liver samples were obtained at 6 week.The expressions of hepatic apoA5 gene and protein were measured using immunohistochemistry,RT-PCR and Western Blot analysis respectively.Besides,hepatic expressions of PPARαand LXRα were measured by RT-PCR and Western Blot analysis.In the experiments in vitro,we tested the effects on TG and the expressions of apoA5,PPARαand LXRαin HepG2 cells by fructose, fenofibrate and atorvastatin,respectively.Furthermore,we investigated the mechanisms of the three chemicals in regulation of apoA5 expression in cells by treatment with antagonists or inhibitors of PPARαand LXRα.Ⅱ.Human,animal and cell experiments for APR-induced HTG1.ACS experiment:ACS patients(n=228) and healthy volunteers(n=232) were included. After plasma samples obtained,plasma apoAV level was measured by an ELISA method.Besides,plasma lipids and C-reactive protein(CRP) were measured.2.Acute inflammation experiments:Thirty two healthy male C57BL/6 mice were randomized into four groups(n=8 each group):(1) control group,with normal diet for four weeks;(2) inflammation group,with normal diet for four weeks and then intraperitoneal injection with 100μg LPS to establish acute inflammatory animal model;(3) statin group,with atorvastatin 10 mg/kg/d for four weeks and then intraperitoneal injection with 100μg LPS;(4) fibrate group,with fenofibrate 100 mg/kg/d for four weeks and then intraperitoneal injection with 100μg LPS.The measurements of fasting plasma lipids and tumor necrosis factor-α(TNFα) were performed at 0 week,4 week before LPS injection,and after LPS injection.The expressions of hepatic gene and protein of apoA5 and PPARαwere measured using RT-PCR and Western Blot analysis respectively.In the experiments in vitro,we tested the effects on TG and the expressions of apoA5,PPARαand LXRαin HepG2 cells by human recombinant TNFα.Furthermore,we investigated the effects and mechanisms of atorvastatin and fenofibrate on apoA5 expression-induced by TNFα.Ⅲ.Study for hypertriglyceridemic mechanism for Xuezhikang in CHD patients1.Trial about lipid-lowering by Xuezhikang:Sixty stable CAD patients were included,and randomly assigned into two groups to receive 600 mg of xuezhikang(300 mg of cholestin per capsule;WBL Peking University Biotech Co,China) twice daily (xuezhikang group;n = 30) or placebo(placebo group;n = 30) for 6 weeks.Plasma apoAV level was measured by an ELISA method at baseline and six week,and fasting plasma lipids were measured.2.Animal and cell experiments:Thirty male Sprague-Dawley rats were randomized into five groups (n=8 each group):(1) control group,with no special treatment;(2) hypertriglyceridemia(HTG) group,with 10%fructose water for two weeks to establish animal HTG model,and with 10%fructose water for next four weeks;(3) low-dose Xuezhikang(L-XZK) group,with 10% fructose water for two weeks and supplemented with Xuezhikang 20 mg/kg/d for next four weeks;(4) high-dose Xuezhikang(H-XZK) group, with 10%fructose water for two weeks and supplemented with Xuezhikang 40 mg/kg/d for next four weeks.The measurements of fasting plasma lipids in rats were performed at 0,2 and 6 week.Liver samples were obtained at 6 week.The gene and protein expressions of hepatic apoA5,PPARαand LXRαwere measured using RT-PCR and WB analysis,respectively.In the experiments in vitro,we tested the mechanism underlying the effects on TG and the expressions of apoA5,PPARαand LXRαin HepG2 cells by Xuezhikang.Ⅳ.Human,animal and cell experiments for HTG in MS1.MS experiment:MS patients(n=340) and healthy volunteers(n=342) were included. After plasma samples obtained,plasma apoAV level was measured by an ELISA method.Besides,fasting plasma lipids,glucose and insulin were measured.Furthermore,homeostasis model assessment of insulin resistance(HOMA-IR) was calculated.2.Animal and cell experiments:Tweenty-four male Sprague-Dawley rats were randomized into three groups(n=8 each group):(1) control group,with no special treatment;(2) MS group,with high- fructose diet(containing 60%fructose) for seven weeks to establish animal MS model;(3) fibrate group,with high-fructose diet and fenofibrate 100 mg/kg/d for seven weeks.The measurements of fasting plasma lipids and tail arterial blood pressure in rats were performed at 0 and 7 week.Then,insulin sensitivity in rats was tested by hyperinsulinemic euglycemic glucose clamp.At the end of study,Liver samples were obtained.The expressions of hepatic apoA5 gene and protein were measured using RT-PCR and Western Blot analysis, respectively.In the experiments in vitro,we established an insulin resistant hepatocyte model by adding high-dose insulin into HepG2 cells.Then, we tested the effects on TG and apoA5 expression in cells during insulin resistance(IR).Furthermore,we investigated the effect and mechanism of fenofibrate on apoA5 expression in cells during IR.Results:Ⅰ.Animal and cell experiments for fructose-induced HTG1.At 6 week,plasma TG was higher in rats in HTG group,but lower in three drug groups with ascending order as follows: statin＜fibrate＜combination(P＜0.05);However,the expression of hepatic apoA5 gene and protein displayed otherwise.HTG group had lower apoA5 gene and protein,but apoA5 expression increased in three drug groups with ascending order as follows: statin＜fibrate＜combination(P＜0.05).Likewise,fructose decreased apoA5 expression in HepG2 cells,which had been reversed by statin and/or fibrate with ascending order as follows: statin＜fibrate＜combination(P＜0.05).2.Hepatic PPARαgene decreased in HTG group,and otherwise in three drug groups with ascending order as follows: statin＜fibrate＜combination(P＜0.05),but no signifcant changes were shown in PPARαprotein expression among all groups.Besides, fructose decreased PPARαgene and protein expression in HepG2 cells,which had been reversed by statin and/or fibrate with ascending order as follows:statin＜fibrate＜combination(P＜0.05).3.Hepatic LXRαgene increased in HTG group and fibrate group,but otherwise in statin group and combination group(P＜0.01).However, there was no significant difference in LXRαprotein expression among all groups.Additionally,fructose and fibrate increased LXRαgene and protein expression in HepG2 cells,and otherwise by treatment of statin(P＜0.01).However,no significant difference in LXRαexpression was seen between combination treatment and control.Ⅱ.Human,animal and cell experiments for APR-induced HTG1.Compared with controls,ACS patients had higher plasma apoA5,TG and CRP(P＜0.05).2.In ACS patients,plasma apoA5 level was positively correlated with TG(r=0.642,P＜0.001) and CRP(r=0.594,P＜0.001),and TG was also positively correlated with CRP(r=0.572,P＜0.001).3.Multiple logistic regression analysis indicated that plasma apoA5 level may be a protective factor for ACS(OR=0.82,95%CI 0.70-0.95,P=0.02).4.After LPS injection,plasma TG was obviously higher in mice,but TC,HDL-C and LDL-C was sharply reduced(P＜0.001).However, the magnitude of above lipid changes induced by LPS had been reduced by statin and fibrate(P＜0.05).5.After LPS injection,the expressions of apoA5 gene and protein obviously increased in mice,but PPARαexpression was significantly impressed(P＜0.05).Likewise,statin and fibrate attenuated the change magnitude of apoA5 and PPARαexpressions induced by LPS (P＜0.05).6.The treatment of TNFαinduced an elevation of TG in HepG2 cells (P＜0.001),which had been reversed by statin and fibrate(P＜0.05).7.TNFαconsiderably increased the expressions of apoA5 gene and protein in HepG2 cells,but otherwise for PPARαexpression. Conversely,statin and fibrate can attenuated apoA5 upregulation and PPARαdownregulation by TNFα(P＜0.05).Ⅲ.Study for hypertriglyceridemic mechanism for Xuezhikang in CHD patients1.After Xuezhikang therapy,CHD patients had lower plasma TG and higher apoA5 than baseline(P＜0.001).Plasma apoA5 level was significantly inversely correlated with TG in all patients(P＜0.001)2.Xuezhikang dose-dependently increased hepatic expressions of apoA5 gene and protein,and decreased plasma TG level in rats. Furthermore,Xuezhikang dose-dependently increased hepatic PPARαgene expression,but impressed LXRαgene expression(all P＜0.05).3.The treatment of Xuezhikang induced an elevation of apoA5 gene and protein in HepG2 cells,resulting in cell TG reduction(P＜0.01). However,the apoA5 upregulation by Xuezhikang had been obviously attenuated by PPARαinhibitor or LXRαagonist(all P＜0.05).Ⅳ.Human,animal and cell experiments for HTG in MS1.Compared with controls,MS patients had lower plasma apoA5 and higher TG levels(P＜0.05).An inverse correlation was observed between apoA5 and TG(P＜0.05).Besides,HOMA-IR markedly elevated in MS patients and was negatively related to plasma apoA5 level(P＜0.05).2.Multiple logistic regression analysis indicated that plasma apoA5 level may be a protective factor for MS(OR=0.77,95%CI0.64-0.92, P=0.04).3.Compared with controls,MS rats had obviously attenuated insulin sensitivity,higher plasma TG,and reduced expressions of hepatic apoA5 gene and protein(all P＜0.05).4.After fenofibrate treatment,MS rats had considerably improved insulin sensitivity,reduced plasma TG,and up-regulated expressions of hepatic apoA5 gene and protein(all P＜0.05).5.By treatment with high-dose insulin,the expressions of apoA5 gene and protein obviously decreased in HepG2 cells,but cellular TG significantly increased.Conversely,the above changes of cell apoA5 and TG by high-dose insulin was effectively reversed by fenofibrate, that was obviously attenuated by a PPARαinhibitor(all P＜0.05).Conclusions:1.Fructose inhibits hepatic apoA5 expression by PPARαinhibition and LXRαinduction,resulting in HTG.Fenofibrate and atorvastatin can upregulate apoA5 expression for the treatment of HTG induced by fructose,and a mechanic compensation exists in regulation of apoA5 expression for the two agents.2.APR increases plasma TG and apoA5.But the elevation of apoA5 may be a compensation for APR,which could not be influenced by PPARαimpression.Fenofibrate and atorvastatin can ameliorate HTG induced by acute inflammation,which is not mainly dependent on apoA5 but is associated with their anti- inflammatory effects.3.Xuezhikang decreases plasma TG by pregulation of apoA5 expression, which results from PPARαupregulation and LXRαinhibition by this agent.4.During MS,hepatic expression and plasma level of apoA5 are lower resulting in higher plasma TG level,which could increase MS risk.5.Fenofibrate decreases plasma TG level in MS rats by upregulation of hepatic apoA5 expression and improvement of IR.