Study On Effects And Mechanisms Of Nicotine On Vascular Function And Insulin Sensitivity

【Objective】Smoking is a leading cause of atherosclerosis, hypertension, diabetes and other cardiovascular and endocrine diseases. Nicotine is one of the major components of cigarette with complex effects on cardiovascular systems, and many mechanisms still left unkown. Up to now, most studies showed that nicotine impaired vascular fuction and insulin action, for it reduced endothelium-independent vascular relaxation and led to insulin resistance. However, in chonic nicotine treatment rat model established in our department, aortic reactivity showed that endothelium-dependent relaxation was not altered, but endothelium-independent relaxation induced by sodium nitroprusside (a donor of nitrc oxide) was significantly enhanced. Besides, the insulin resistance index of this model was reduced. These indicated that nicotine not only enhanced the sensitivity of nitric oxide pathway in vascular smooth muscle but also insulin sensitivity.Nicotine produces most of its effects through nicotine acetylcholine receptors (nAChRs). Nicotine acetylcholine receptors are ligand-gated ion channels, they form five aggregates composed of five subunits. There are seventeen subunits (α1-10,β1-4,γ/ε,δ), and ligands binding site are onαsubunit. Traditionally, Receptors of the nicotinic subclass can be distinguished as"muscle"or"neuronal". The former mediates nerve-muscle connector transmission, and the latter mediates or modulates nerve system synapsis transmission. Recent studies pointed out that nAChRs are also expressed in many non-neuro cells. Moreover, the fact that nicotine receptors and their function were discovered in non-neuro cells has already attracted scientists in different fields. For example, the clarification ofα7 nicotinic receptor in immune cells triggered the studies on therapeutics significance of nicotinic receptor agonist treating infectious and imflammatory diseases. In smooth muscle cells, chronic nicotine expoture enhances insulin-induced mitogenic signaling via up-regulation ofα7 Nicotinic receptors. Currently, study nAChRs and their function in non-neuro cells will reveal new fuction of nAChRs. Meanwhile, determining specific nicotinic receptor is of most importance for therapeutic use of nicotine. Above all, this study was to investigate the effects and mechanisms of nicotine on vascular fuction and insulin sensitivity. This will not only contribute to the understanding of the complex effect of nicotine on cardiovascular and endocrine systems, but also has significance in reveal the new fuction of nicotinic receptors and new targets in nitric oxide signaling pathway.【Methods】PartⅠ: Effect of nicotine on vascular fuction and its mechanism. First, in nicotine treated rat, anticontraction effect of adventitia and endothelium were tested; endothelium-dependent relaxation effect was tested; as well as smooth muscle contraction and endothelium-independent relaxation were tested. Then, we further investigated the mechanism of nicotine enhancing nitroxide induced relaxation. On the one hand, time course effect of nicotine treatment on aortic reactivity was first studied in SD rats and C57 mice to find out whether this effect of nicotine was generally and chronic. We also observe the relaxation effect of aorta smooth muscle induced by insulin in nicotine treated and untreated groups. Next, we studied its mechanism. In organ bath experiment, we used sGC inhibitor, PKG inhibitor and cGMP analog. Meanwhile, we detected PKG expression and activity to identify whether this effect was through cGMP dependent PKG pathway. Thirdly, we tried to find out the possible target nicotine altered in the down stream of nitric oxide signaling pathway by gene chip analysis accompanied with gradually excluding from old pathway. After selecting the possible targets, such as cyclooxygenase-2, sarcoplamic reticulium Ca2+-ATPase, amp-activated protein kinase, Rho, etc., we carried out western-blot and organ bath experiment to verify and confirm the targets. On the other hand, we made rat model treated at the same time with nicotine and hexamethonium that blocked nicotinic receptors exceptα7 receptor. We also treated alpha 7 knockout mice with nicotine. These two experiment were to find out the possible nicotinic receptor mediated this effect. At last, we investigated the blood pressure indicators in nicotine treated and untreated groups by conscious blood pressure detection technique. PartⅡ: Effect of nicotine on insulin sensitivity and its mechanism. First, we confirm the insulin sensitivity enhancement effect of nicotine on normal rats through HOMA-index, insulin tolerance test and glucose tolerance test. Then, we studied its mechanism. On the one hand, we weighed the fat tissue from various parts of rats. On the other hand, we analysis the gene chip from PartⅠto find out insulin sentivity related gene alteration. Meanwhile, we detected key protein expression (PPAR-gamma, COX-2, AMPK) in four insulin sensitive tissue (skeletal muscle, liver, visceral fat and subcutaneous fat) in nicotine treated rat model. Moreover, we made hexamethonium plus nicotine treated rat model to study the central or periphery receptor mechanism of nicotine on insulin sensitiviy.【Results】PartⅠ: Study on effects and mechanisms of nicotine on vascular function1. Chronic nicotine treatment did not affect anticontraction effect of aorta adventitia and endothelium; did not affect endothelium-dependent relaxation effect; did not affect aorta smooth muscle contraction effect induced by phenylephrine but had slight inhibition effect on contraction effect induced by low dose KCl; significantly enhanced endothelium independent smooth muscle relaxation.2. Nicotine did not alter phenyllphrine induced aortic contraction but significantly enhanced sodium nitroprusside (donor of nitroxide) induced endothelium-independent relaxation in Sprague-Dawley (SD) rats and C57BL/6J mice. This phenomenon emerged after three weeks nicotine treatment in rats and four weeks in mice and was stable after 6 weeks nicotine treatment in rats and mice. Nicotine treatment also enhanced insulin induced smooth muscle relaxation. These indicated that the effect of nicotine is chronic rather than acute.3. The relaxation induce by sodium nitroprusside was totally blocked by sGC inhibitor ODQ and PKG inhibitor RP-8-bromo-cGMP. Further the relaxation was mimicked by cGMP analog. PKG expression between nicotine and saline group was the same, and so was the PKG activity. This indicated the alteration was in the PKG downstream pathway.4. Analysis on aorta gene chip showed two nitroxide relaxation pathway related gene altered. Sarcoplamic reticulium Ca2+-ATPase mRNA up regulated by 1.56 folds and heat shock protein 27 mRNA downregulated by 0.51 folds. Besides, another candidate gene COX-2 significant down regulated by 0.09 folds.5. COX-2 protein expression was significantly reduced in aorta smooth mucle in nicotine group. But indomethacin, a nonselective inhibitor of cox-2 and meloxicam, a selective inhibitor of COX-2 could not abolish the relaxation difference between nicotine and saline group. Sarcoplamic reticulium Ca2+-ATPase inhibitor thapsigargin could not cancel the relaxation difference between nicotine and saline group. Hsp 27 protein expression was not altered in nicotine treated rats. These results eliminated SERCA 3,Hsp 27 and COX-2.6. Rho kinase inhibitor Y-27632 could not abolish the relaxation difference between two groups. This indicated Rho was not involved in this mechanism.7. AMPK inhibitor compound C could not abolish the relaxation difference between nicotine and saline groups. This eliminated the involvement of AMPK pathway.8. Non-selective Kca and Kv inhibitor, 20 mM Tetraethylammonium (TEA), could totally abolish the relaxation difference between saline and nicotine group, while selective block KATP and Kir could not affect the difference between two groups. Selective block BKCa and 4-AP sensitive potassium channel could not abolish the relaxation difference between two groups either. In aorta preconstricted by 80 mM KCl, the relaxation difference between two groups induced by SNP was not present.9. Hexamethonium abrogated the relaxation difference between saline and nicotine groups, while in alpha 7 knockout mice, the relaxation difference between two goups was still exist.10. Chronic nicotine treatment did not affect basal systolic blood pressure, diastolic blood pressure and mean blood pressure but extended heart period; did not affect acute sodium nitroprusside (3μg/kg, 10μg/kg, 30μg/kg, 100μg/kg) injection induced blood pressure lowering effect.PartⅡ:1. Chronic nicotine treatment did not affect serum glucose level, but significantly reduce insulin and triglycride levels and significantly reduced HOMA-insulin resistant index.2. Insulin tolerance test and glucose tolerance test in 3 weeks and 6 weeks nicotine and saline treated rats all showed that blood glucose lowering percentage was greater in nicotine treated rats.3. Chronic nicotine treatment significantly reduced subcutaneous fat and visceral fat, especially visceral fat.4. Aorta gene chip showed that after chronic nicotine treatment lipid metabolism related genes (lipase, adrenergic receptorβ3 ) significantly upregulated; energy expenditure related gene ( uncoupling protein 1) significantly upregulated; insulin sensitivity related gene (peroxisomal proliferators activated receptorγ, PPARγ) significantly upregulated.5. Nicotine treatment did not affect PPARγexpression in skeletal muscle, liver, visceral fat and subcutaneous fat.Nicotine treatment significantly reduced COX-2 expression in skeletal muscle, but did not affect COX-2 expression in liver, visceral fat and subcutaneous fat.Nicotine treatment significantly increased AMPK expression in visceral fat, but did not affect AMPK expression in skeletal muscle, liver and subcutaneous6. Periphery nicotinic receptor blocker plus nicotine treatment did not abolish the insulin sensitivity enhancing effect of nicotine, but abolished the triglyceride lowering effect of nicotine. These indicated that nicotine enhance insulin sensitivity major through central mechanism.【Conclusion】In this study, two main new effects and mechanisms were discussed: 1. Chronic nicotine treatment can enhance aortic smooth muscle relaxation induced by nitroxide. Nicotine can affect the down stream target of cGMP dependent protein kinase pathway through non-α7 nicotinic receptor. SERCA 3, Hsp 27 and COX-2 are not involved in this mechanism; Rho and AMPK are not involved either. Nicotine acts through enhancing opening of TEA sensitive potassium channel, which leads to hyperpolarization of the membrane, closing calcium channel and relaxation enhancement. This potassium channel is non-classic PKG activated BKCa or KATP channel, and is not 4-AP sensitive Kv potassium channel or Kir channel either, which is still need to be identified.2. Chronic nicotine treatment can enhance insulin sensitivity. This effect is mainly mediated by central nicotinic receptors. The possible downstream mechanisms are that nicotine could upregulate AMPK protein expression in visceral and hypothalamus; nicotine reduces COX-2 expression in muscle, but it has no relationship with regulating PPARγprotein expression.