The Role Of Adiponectin Receptor In Nicotine-induced Adipokine Dysfunction And The Involved Mechanism

Objective:The morbidity and mortality of cardiovascular diseases always occupy the first place in all kinds of diseases.Smoking is an important independent risk factor for cardiovascular disease.In recent years,the increasing of sudden death rate caused by electronic cigarette(e-cigarettes)which became popular created a great threat to human health.Nicotine is an important component of cigarettes and e-cigarettes.Therefore,the specific mechanisms of nicotine damage to the cardiovascular system needs further research.Studies have shown that the abnormal adipose function caused by smoking leads to weight loss,but the influence on cardiovascular morbidity and the course of diabetes mellitus are not reduced.Therefore,the relationship between smoking and the adipose endocrine function and whether it is related to vascular injury is unknown.Therefore,this subject is dedicated to clarifying the effect of nicotine,the main component of tobacco and e-cigarettes,on adipose endocrine function and further revealing its relationship with cardiovascular diseases,and on this basis,to explore the specific mechanism of nicotine causing vascular damage in diabetic models.It will provide experimental evidence or potential diagnosis and treatment ideas for vascular injury in clinical smokers or e-cigarette users and diabetic smokers.Methods:1.In clinical epidemiology:we collected more than 30 healthy male smokers and non-smokers,recorded smoking status,age,height,weight,blood pressure,blood glucose,blood lipid,liver function and other related indicators of the two groups,preserved 5ml blood samples for adiponectin(APN)and C1qTNF-related protein superfamily(CTRPs).Analysis of the above indicators between the two groups to study whether smoking has an impact on human lipid metabolism and adipose endocrine function.2.Nicotine animal and cell model studies:(1)We used animal models to study nicotine’s effect on adipose secretion.Adult male C57BL/6 mice were divided into two groups:vehicle and nicotine groups.Small animal micropumps were implanted subcutaneously in both groups.0.9%saline was inserted into the pump in the vehicle group,and nicotine solution with a dose of 1.5mg/(kg·d)was inserted into the pump in the nicotine group.We analyzed the changes of blood glucose,blood lipids,adiponectin and insulin levels in the two groups at different time periods and used the vasoconstriction and relaxation experiment to measure the vascular response to exogenous globular adiponectin(gAPN)in the two groups.(2)We used animal and cell models to study the mechanism of nicotine-induced vascular adiponectin resistance.In nicotine animal model,the changes of adiponectin receptor 1(AdipoR1)in aortic vascular tissues of mice of two groups were measured to determine the changes of AdipoR1 when blood vessels developed adiponectin resistance.Human Umbilical Vein Endothelial Cells(HUVECs)were divided into control and nicotine groups.We measured the changes of AdipoR1 in two cells groups,used RT~2 Profiler PCR Array kit to detect the changes of related genes m RNA in the two groups,determined the change of SOCS3 levels in the two cells/vascular tissues groups and used immunofluorescence to determine the level and location of AdipoR1 and SOCS3 in the two groups;after using MG132 interfere the cells,Western Blot was used to determine the change of AdipoR1 in the two groups,immunofluorescence method was used to determine whether AdipoR1 and SOCS3 occur co-localization and Co-immunoprecipitation experiments was used to determine whether AdipoR1 combine SOCS3 and ubiquitinated.3.Diabetic animal and cell model:we used high-fat diet or high-sugar/high-fat to create diabetes model,and further investigated whether nicotine can aggravate adiponectin resistance in diabetes model and the mechanism of that.After using siRNA-SOCS3,the effects of SOCS3 knockout on the ubiquitination of AdipoR1 and the response to gAPN were tested.Results:1.Smoking and its main component,nicotine,led to the abnormal metabolic index,adipokine endocrine dysfunction,and vascular adiponectin resistance.(1)Clinical epidemiological studies showed that there were no significant differences in age,blood pressure and liver function between the smokers and non-smokers groups;smoking caused increasing blood glucose,abnormal blood lipids and abnormal lipoendocrine function:serum CTRP1 and CTRP5 were increased,CTRP15 was decreased,and APN level was significantly decreased in the smoking group.(2)Studies on animal models of nicotine have shown that nicotine-induced adiponectin signal dysfunction,in mice,nicotine caused the APN level increased at 6 weeks,reached a peak at 8 weeks,and decreased significantly at 12 weeks.(3)Nicotine decreased the vasodilation reaction to gAPN which illustrated nicotine induced vascular adiponectin resistance.2.Nicotine reduced the expression of AdipoR1 and increased the expression of SOCS3in blood vessels.(1)Nicotine significantly reduced the expression of AdipoR1 and the reactivity to gAPN(phosphorylation of AMPK and ERK1/2);(2)RT~2 Profiler PCR Array showed that nicotine significantly increased the m RNA of SOCS3;(3)Nicotine increased the protein level of SOCS3 and the cell immunofluorescence method further verified nicotine increased the expression level of SOCS3 in the cytoplasm.3.Nicotine increased the ubiquitin-mediated degradation of AdipoR1 via SOCS3 in endothelial cells.(1)MG132(proteasome inhibitor)inhibited the down-regulation of AdipoR1 induced by nicotine;(2)Co-immunoprecipitation and immunofluorescence staining assay revealed that the interaction between SOCS3 and AdipoR1 were significantly increased in the present of nicotine.4.In the diabetes model,nicotine significantly aggravated metabolic disorders and vascular adiponectin resistance.(1)In the diabetes model,nicotine further aggravated the decline of AdipoR1 level;(2)In the diabetic model,the increasing of SOCS3 level in vascular endothelial cells induced by nicotine and the ubiquitination of AdipoR1 were further aggravated;(3)Knockout of SOCS3 reduced the ubiquitination degradation of AdipoR1 and the decrease of the reactivity to adiponectin(phosphorylation of ERK1/2)caused by nicotine.Conclusion:This study proved that(1)nicotine significantly induced adipose endocrine dysfunction and vascular adiponectin resistance which significantly aggravated in diabetes;(2)ubiquitin degradation of adiponectin receptors is an important factor in cardiovascular damage caused by smoking and its further aggravation in diabetic body;(3)SOCS3-mediated AdipoR1ubiquitination played an important role in adiponectin resistance.