| Background and ObjectiveIn recent decades,regardless inthe developed country or the developing nation,obesity has become a global threaten of human health and attr-acted more and more attention.Specific pathogenic entities contributing to obesity risk,such as hyperphagia,poor diet,drinking too much,inactivity,obesity-prone gene,Heating and lifestyle change have been well characterized.It has been widely accepted that chronic low-grade inflammation and the increased mass of adipose tissue are two major characteristics in obesity object.There is mounting evidence that from both epidemiological and clinical studies show that chronic low-grade inflammation is related to central obesity,insulin resistance,dyslipidemia,hypertension and other clinical syndrome syndrome(Metabolic,syndrome,MS).In recent years,experimental studies indicate that a large number of adipokines and inflammation involved in the pathogenesis of obesity,.As an adipokine,ASP attracts plenty of attention in last few years.Direct combination of ASP and C5L2,activate PLC?,promote the expression of PKC ?and PKC ? PKC alpha phosphorylation protein,increase the activity of DGAT and promote GLUT4 translocation,through PLC and PI3K-AKT,involved in the synthesis of acylation stimulating protein mediated triglyceride.A variety of inflammatory factors and hormone promoting ASP production,down regulation the expression of C5L2 mRNA,interference the phosphorylation of C5L2 and ASP-C5L2 ligand receptor binding,result in ASP resistance.Reduce TG synthesis and increase fat decompose,a large number of FFA overflow,chronic high blood free fatty acid can lead to pancreatic beta cells apoptosis,nuclear transcription factor(NFkappaB)inflammatory pathway activate,promote the inflammatory factor release,forming a vicious spiral.The formation of obesity causes the inflammatory response,leading to changes in the endocrine environment,such as insulin resistance,leptin resistance,ASP resistance,and chronic low-grade inflammation.while,chronic low-grade inflammation may be the root cause of obesity.A large number of clinical and experimental studies revealed that the cholinergic system is involved in the inflammatory reflex,alpha 7 nicotinic cholinergic receptor(alpha 7 nAChR)is one of the most important cholinergic receptors can respond to vagus nerve branch and spleen specific subsets of T cells secrete acetylcholine.The cholinergic anti-inflammatory pathway,to prevent uncontrolled inflammation.The down regulation of alpha 7 nAChR cholinergic pathway in obese is the base of chronic low-grade inflammation,so explorate the mechanism of cholinergic system reduce the synthesis of triglycerides may provide a new direction for the prevention and treatment of obesity.In this paper,we study the mechanism of regulation of triglyceride synthesis by alpha 7 nAChR.In vivo experiments,mice received nicotine,methyllycaconitine,Hexamethonium chloride injection for 3 weeks,detecte nicotine effect on body weight,blood glucose level,blood triglycerides,blood ASP and the expression of C5L2 mRNA;detect the level of alpha 7nAChR expression in HFD and LFD mice adipose tissue;in vitro,pretreatment adipose tissue with PNU282987 for 24 hours and(or)wortmannin for 30 minutes,detect the level of AKT/PKB phosphorylation of Ser 473 induced by ASP.To study the effect of alpha 7nAChR cholinergic anti-inflammatory pathway on the synthesis of triglyceride in the downstream pathway of ASP-C5L2 signaling,to provide a theoretical basis for search a new target with alpha 7nAChR to treatment of obesity.Methods1.Male C57BL/6J mice at 5-6weeks of age were randomly divided into two groups :HFD group and LFD group with either High Fat Diet(HFD)or Low fat diet(LFD)for 16-18 weeks.The daily diet,stool,activity,fur,and mental state of mice were robserved every day.Body weight were recorded every week.2.After feeding for 12 weeks,HFD mice and LFD mice were randomly divided into four groups respectively.NS group: normal saline was given via intraperitoneally at10ml/kg.NIC group: Nicotine was given via intraperitoneally at 1 mg/kg/day,Bid;NIC + MLA group: Methyllycaconitine citrate salt hydrate was given via intraperitoneally 30 prior to nicotine at 3 mg/kg/day,Bid;NIC + HEX group:Hexamethonium chloride was given via intraperitoneally 30 prior to nicotine at 5mg/kg/day,Bid.The effect of nicotine on body weight gain were measured weekly during the drug treatment for 3 weeks.Administration time: 9: 30 and 22:30.The body weight was measured every day.3.Different groups of mice were given intraperitoneal injection of glucose tolerance test(IPGTT)and intraperitoneal insulin tolerance test(IPITT).4.At the end of protocol,Blood was collected via orbital venous at fed state for measure the level of serum ASP and TG.visceral adipose,liver,kidney and skeletal muscle were quickly collected and stored at-80 ? for measure the level of C5L2 mRNA by RT-qPCR.The level of ?7nAChR from HFD and LFD mice were measured by Western Blot.5.In vitro,adipose tissue from HFD mice were divided into four groups: PBS group ?ASP group ?PNU282987+ASP group ?PNU282987+ Wortmannin +ASP group,each group has 2 holes.ASP group: add ASP 100nM/L into medium,incubation for 10 minutes;ASP+ PNU group: pretreatment adipose tissue with PNU282987 10nM/L for 24 h,changing the freash culture medium,add ASP100nM/L into medium,incubation for 10 minutes;PNU+WOR+ASP group:pretreatment adipose tissue with wortmannin 200nM/L into medium for 30 minutes,changing the freash culture medium,added PNU282987 10nM/L for 24 h,and then changing the culture medium,add ASP 100nM/L into medium,incubation for 10 minutes.The level of AKT/PKB Ser 473 phosphorylation was measured by WesternBlot.6.Statistical analysis of mice weight increase,IPGTT,IPITT and area under the curve,serum ASP and TG levels;the expression of C5L2 mRNA gene in adipose tissue,liver,kidney and left leaf liver tissue by fluorescence quantitative PCR;the level of alpha 7nAChR protein in adipose tissue from HFD and LFD mice and AKT/PKB Ser 473 phosphorylation in vitro adipose tissue culture group by Western blot.Results1.the weight of HFD mice and LFD mice increased with the feeding time,and there was no significant difference between the HFD group and the LFD group in the weight for begin 4 weeks.At eightweeks,the weight of HFD mice was significantly increased compared with that of LFD group,the difference was statistically significant(P<0.0001).2.Body Weight Gain: The body weight gain in the NS group with HFD is significantly greater(P<0.05)than that of NS with LFD.In HFD mice,the weight of NIC group was significantly lower than that of NS group(P<0.001);NIC+MLA group(P<0.05)and NIC+HEX group(P<0.001)were significantly higher than that in NIC group.In LFD mice,the weight of NIC group was lower than that of NS group(P< 0.05).There was no significant difference in NIC group,NIC+MLA group and NIC+HEX group.3.Serum TG and ASP: The level of serum TG and ASP in the NS group with HFD were significantly higher(P<0.05)than that of NS group with LFD.In HFD mice,the blood TG level in group NS was significantly higher in group NIC(P<0.05);NIC+MLA group and NIC+HEX group were significantly higher than that in NIC group.In LFD mice,there was no significant difference in serum TG level between NS group,NIC group,NIC+MLA group and NIC+HEX group.The level of serum ASP in the NS group with HFD were significantly higher(P<0.05)than that of NS group with LFD.In HFD mice,the blood ASP level in group NS was significantly lower than that in group NIC(P< 0.05).There was no significantdifference in serum ASP level between NIC group,NIC+MLA group and NIC+HEX group.In LFD mice,there was no significant difference in serum ASP level between NS group,NIC group,NIC+MLA group and NIC+HEX group.4.Blood glucose: compared with HFD mice fasting blood glucose,LFD mice fasting blood glucose was lower,the difference was statistically significant(P<0.05).In HFD mice,the fasting blood glucose level of NIC group was lower than that of NS group,the difference was statistically significant(P<0.05).There was no significant difference between the NIC group,NIC+MLA group and NIC+HEX group.In LFD mice,fasting blood glucose level of NIC group was lower than that of NS group,the difference was statistically significant(P<0.05).Compared with NIC group fasting blood glucose level,that in NIC+MLA group(P<0.001)and NIC+HEX group(P<0.01)were higher,the difference was statistically significant.5.The blood glucose levels of NS group in HFD mice is higher than that in LFD mice,The area under the IPGTT curve of NS group in HFD mice is bigger that in LFD mice;compared with NS group,the blood glucose level of NIC group from HFD and LFD mice is lower,the NIC group had significantly improved the IPGTT curve.In HFD mice,the NIC + MLA group had higher blood glucose level than the NIC group,and there was no significant difference in LFD,NIC+,MLA and NIC+HEX groups.IPITT in HFD mice,compared with NS group,after intraperitoneal injection of insulin 30min?60min and 90 min,the blood glucose levels in NIC group was significantly decreased,there is no significant difference about the level of blood glucose between NIC+MLA group and NIC+HEX group.6.Compared with LFD mice,the AUC in HFD mice increased significantly(P<0.01).In HFD mice,the AUC in NIC group was significantly lower than that in NS group(P<0.01).There was no significant difference in AUC among the NIC group,NIC+MLA group and NIC+HEX group.In LFD mice,the AUC in NIC group was less than that in NS group(P<0.05);there was no significant difference in AUC among the NS group,NIC+MLA group,NIC+HEX group.7.The expression of C5L2 mRNA in different tissue :Compared with LFD mice NS group,the expression of C5L2 mRNA in adipose tissue(P<0.001),liver(P<0.01),kidney(P<0.001),skeletal muscle(P<0.05)was significantly lower in HFD mice NSgroup.In HFD mice,compared with the NS group,the NIC group C5L2 mRNA in the adipose tissue(P<0.05)and muscle tissue(P<0.05)is higher expression,and lower expression in liver tissue(P<0.001);compared with NIC group,the NIC+MLA group C5L2 mRNA is increased expression in liver tissue(P<0.01).In LFD mice,compared with NS group,the NIC group C5L2 mRNA in adipose tissue(P<0.001)?skeletal muscle(P<0.05)and kidney(P<0.001)is decreased expression;compared with NIC group,the expression of C5L2 mRNA in NIC+MLA group(P<0.001)and NIC+HEX group(P<0.01)is higher in liver tissue.The expression of C5L2 among NIC group?NIC +MLA group and NIC +HEX group in adipose tissue,kidney tissue,skeletal muscle tissue is no significant difference.8.a7nAChR protein was expressed in adipose tissue,and the expression level of7 nAChR protein in HFD mice was lower than that in LFD mice(P=0.0379).9.Compared with the PBS group,ASP induced phosphorylation of Akt(P=0.0379),the difference was statistically significant.Compared with ASP group,the Akt phosphorylation level was lower in PNU282987+ASP group(P=0.0292).There is no significantly difference between wortmannin + PNU282987+ASP group and ASP group.Conclusion:1.Long-term nicotine administration reduced weight gain,improve ASP sensitivity in HFD induced obesity mice.2.The expression of 7nAChR protein in obese individuals was decreased.3.Nicotine interacted with ?7nAChR and via PI3K-AKT signaling pathway disturb ASP induced the synthesis of triglycerides.These results show a new view for preventing and treating diet-induced obesity and metabolic syndrome in human. |
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