PARASYMPATHETIC DYSFUNCTION IN OBESITY: EFFECT OF CHOLESTEROL ON VAChT

描述（由申请人提供）：肥胖是美国1和全球2的严重公共卫生问题，是高血压、心律失常、心肌缺血、心力衰竭1，3 - 6和糖尿病4，7的主要风险因素。在肥胖症中，心脏的副交感神经系统（PSNS）调节显著减弱8 - 10，这可能导致肥胖症相关的心律失常和心肌缺血8，11 - 13。然而，迄今为止，肥胖患者PSNS功能降低对心脏的作用机制仍知之甚少。 乙酰胆碱（Ach）是PSNS的主要神经递质。囊泡乙酰胆碱转运体（Vesicular Acetylcholine transporter，VAChT）是PSNS胆碱能末梢中Ach包装的主要载体。高血浆胆固醇（高胆固醇血症）影响大脑中的神经元14 - 16，但其对外周胆碱能系统的影响尚不清楚。我们最近的研究发现，单泛素化参与VAChT再循环和Ach摄取活性。在高脂饮食（HFD）诱导的肥胖小鼠中，VAChT泛素化减少，沿着减弱的PSNS对心脏的调节。 本研究旨在验证一个中心假说，即肥胖时PSNS对心脏的作用减弱与高胆固醇抑制泛素化介导的VAChT再循环有关，导致突触囊泡中VAChT水平和功能降低以及PSNS胆碱能终末Ach释放减少。目的1将测试是否减弱PSNS对HFD诱导的肥胖小鼠心脏的影响涉及减少心脏中副交感神经末梢的Ach释放和VAChT表达。喂食常规饮食（RD）或HFD 6周的小鼠将用作肥胖模型。将检查迷走神经刺激（VS）诱导的心动过缓和心房Ach释放，以及PSNS心脏末梢突触囊泡中VAChT蛋白水平和Ach摄取活性。目的2探讨高胆固醇是否在PSNS胆碱能功能障碍中起作用。将使用饲喂RD、RD+他汀类药物、HFD、HFD+他汀类药物、RD +2%胆固醇和RD +2%胆固醇+他汀类药物的小鼠。将检查PSNS心脏终末对VS和VAChT水平以及Ach摄取的反应，并将其与胆固醇水平相关联。胆固醇对VAChT表达和乙酰胆碱摄取的直接影响也将在胆碱能细胞系SN56中进行测试。目的3将阐明高胆固醇是否抑制单泛素化介导的VAChT再循环，从而在PSNS末端的功能。目的2将在来自六组的心房样品中分析血浆胆固醇水平与VAChT的单泛素化和Ach摄取活性的相关性;将使用SN 56细胞测试胆固醇对VAChT单泛素化、再循环和活性的直接抑制作用。 总体而言，本研究的结果将证明，高胆固醇对泛素介导的VAChT再循环的负面影响有助于降低PSNS胆碱能作用对心脏的肥胖。这一新的概念可能会开辟一个新的研究领域，并可能提出新的策略，预防和治疗肥胖相关的自主神经和心血管疾病。

DESCRIPTION (provided by applicant): Obesity is a serious public health issue in the United States 1 and worldwide 2 and is a major risk factor for hypertension, arrhythmia, myocardial ischemia, heart failure1, 3-6, and diabetes4, 7. In obesity, parasympathetic nervous system (PSNS) regulation of the heart is markedly attenuated 8-10, which may contribute to obesity-related arrhythmia and myocardial ischemia8, 11-13. To date, however, the mechanisms of the reduced PSNS function on the heart in obesity are still poorly understood. Acetylcholine (Ach) is a major neurotransmitter of PSNS. Vesicular acetylcholine transporters (VAChT) are responsible for Ach packaging In PSNS cholinergic terminals. High plasma cholesterol (hypercholesterolemia) affects neurons in the brain14-16 but its effect on the peripheral cholinergic system is unknown. Our recent study identified mono-ubiquitination is involved in VAChT recycling and Ach uptake activity. In high fat diet (HFD)-induced obese mice, VAChT ubiquitination is reduced, along with the attenuated PSNS regulation of the heart. This study is to test a central hypothesis that Attenuated PSNS action on the heart in obesity is related to the inhibitory effect of high cholesterol on the ubiquitination-mediated VAChT recycling, leading to the reduced VAChT level and function in synaptic vesicles and decreased Ach release in PSNS cholinergic terminals. Aim 1 will test whether the attenuated PSNS effect on the heart in HFD-induced obese mice involves reduced Ach release and VAChT expression at parasympathetic terminals in the heart. Mice fed a regular diet (RD) or HFD for 6 weeks will be used as an obesity model. Vagal stimulation (VS)-induced bradycardia and Ach release in atria, and VAChT protein levels and Ach uptake activity in synaptic vesicles from PSNS cardiac terminals will be examined. Aim 2 will elucidate whether high cholesterol plays a role in the PSNS cholinergic dysfunction in the heart. Mice fed RD, RD plus Statin, HFD, HFD plus Statin, RD plus 2% cholesterol and RD plus 2% cholesterol plus Statin will be used. Responses to VS and VAChT level and Ach uptake in the PSNS cardiac terminals will be examined and correlated with cholesterol levels. The direct effect of cholesterol on VAChT expression and Ach uptake will also be tested in a cholinergic cell line, SN56. Aim 3 will delineate whether high cholesterol inhibits the mono-ubiquitination-mediated VAChT recycling and thus function in the PSNS terminals. Correlation of plasma cholesterol levels with mono- ubiquitination and Ach uptake activity of VAChT will be analyzed in atria samples from the six groups in Aim 2; The direct inhibitory effect of cholesterol on VAChT mono-ubiquitination, recycling and activity will be tested using SN56 cells. Overall, results of this study will demonstrate that the negative effect of high cholesterol on the ubiquitination-mediated VAChT recycling contributes to the reduced PSNS cholinergic action on the heart in obesity. This novel concept may open a new area of research and may suggest novel strategies for prevention and treatments of obesity-related autonomic and cardiovascular disorders.