【作者】 孙伟霞；

【导师】 傅耀文； 蔡露；

【作者基本信息】 吉林大学 ， 内科学， 2014， 博士

【摘要】 糖尿病（Diabetes mellitus，DM）是由遗传和环境因素共同作用而引起的一组以糖代谢紊乱为主要表现的临床综合征。胰岛素分泌缺陷、生物作用受损或两者同时存在引起碳水化合物、脂肪、蛋白质、水和电解质代谢紊乱；慢性高血糖为其共同特征。长期糖尿病可引起多个器官的慢性并发症，导致功能障碍和衰竭，成为致残或致死的主要原因。世界卫生组织预计到2030年，全世界范围内糖尿病患者的人数将超过550亿，将给社会带来沉重的经济负担。因此，如何有效治疗糖尿病及其并发症成为基础及临床研究中亟待解决的问题。糖尿病肾病（Diabetic nephropathy，DN）是糖尿病最严重的并发症之一。它也是导致终末期肾脏疾病最常见的原因，与糖尿病患者死亡率的增加密切相关。高血糖及相关的代谢紊乱是DN发生发展的关键因素。目前认为，高糖主要通过激活蛋白激酶C、形成糖基化终末产物、活化多元醇通路及己糖胺通路四条途径引起肾脏组织ROS产生增多、炎症反应及纤维化，最终导致肾小球基底膜弥漫增厚、系膜基质增生及肾小球硬化，从而推动DN的发生、发展。增加胰岛素信号的敏感性，改善糖代谢，从而纠正高血糖及相关代谢紊乱所致的肾脏组织损伤，成为近年DN临床研究的热点。锌（Zinc，Zn）是一种微量元素，在维持机体多种酶和转录因子的正常功能中起重要作用。临床上Zn是治疗其他疾病的常规用药，潜在的毒副作用非常小；且糖尿病患者由于尿Zn分泌增加，肠道Zn的吸收减少以及严格的饮食控制，常伴有Zn的缺乏。因此，补Zn可能有利于糖尿病患者的治疗。最近的Meta分析及临床综述显示，补Zn对糖尿病患者有益。在人群实验中，Zn能降低微量蛋白尿的2型糖尿病患者尿蛋白的分泌。然而Zn是否对DN有治疗作用及其可能的机制，至今尚不明确，为此我们进行了一系列研究。首先，我们以3个月龄OVE261型糖尿病小鼠作为自发DN模型及建立补Zn模型，通过检测肾功能、肾脏病理学改变及Akt介导的代谢相关信号、金属硫蛋白、Akt负性调节因子的表达，明确Zn对DN的治疗作用及Zn治疗DN的可能机制，即Zn对Akt介导的代谢相关信号的激活作用。因为Akt2在胰岛素介导的葡萄糖代谢中起关键性作用，随后我们采用2个月龄Akt2基因敲除小鼠建立补Zn模型，明确Zn对DN的治疗作用是否依赖于Akt2。最后，我们用MT基因敲除建立补Zn模型，进一步明确Zn对Akt及其下游相关代谢信号的激活作用与MT表达之间的关系。结果如下：1，在OVE261型糖尿病小鼠及Akt2-KO2型糖尿病小鼠中，通过检测补Zn后尿蛋白分泌水平，发现糖尿病小鼠尿蛋白水平明显增加，补Zn后无上述改变，说明Zn能改善DN导致的肾功能异常。通过观察各组小鼠补Zn后肾脏组织纤维化、炎症反应及氧化应激相关指标的变化，结果显示补Zn减轻了DN小鼠肾脏组织炎症反应、氧化应激及纤维化水平，对DN具有保护作用。2，通过检测各组小鼠肾脏组织Akt及其介导的代谢相关分子的表达，结果显示糖尿病导致的肾脏组织病理学变化及肾脏Akt磷酸化水平下降，通过补Zn可以得到明显改善，且在OVE26及Akt2-KO糖尿病小鼠中，Zn几乎完全保留了Akt磷酸化水平。Zn提高OVE26及Akt2-KO糖尿病小鼠肾脏组织Akt磷酸化水平的同时，也保存了正常的GSK-3β磷酸化水平及HKII表达。3，糖尿病导致Akt负性调节因子PTEN、PTP1B及TRB3上调，补Zn后能够改善糖尿病导致的Akt负性调节因子的变化，但生理状态下补Zn对Akt负性调节因子无影响。4，通过检测Akt2-KO小鼠补Zn后肾脏功能、肾脏病理学改变及Akt介导的相关代谢信号，发现Akt2缺失后并不影响Zn对糖尿病导致的肾脏功能异常及肾脏病理学改变的保护作用，也不影响Zn对Akt介导的相关代谢信号的激活作用，即Zn对DN的治疗作用是不依赖于Akt2的。5，通过检测MT-KO小鼠补Zn后磷酸化Akt、GSK-3β及HKII表达，结果显示生理条件下MT基因敲除后阻断Zn对磷酸化Akt、GSK-3β及HKII的诱导作用，说明Zn对肾脏的保护作用是MT依赖。综上所述，我们得出结论：Zn能够改善OVE26及Akt2-KO糖尿病小鼠肾脏功能，可能的机制是Zn激活了Akt介导的代谢相关分子的表达。Zn对Akt介导的代谢信号的刺激作用不是Akt2依赖的，而是MT依赖的。糖尿病状态下Zn对Akt介导的代谢信号的激活作用可能与其抑制Akt负性调节因子磷酸化PTEN、PTP1B及TRB3有关。糖尿病患者由于尿Zn排泄增加及肠道对Zn的吸收减少，常伴有Zn缺乏。我们的研究说明，常规监测糖尿病患者Zn的水平及给予Zn水平低的患者补充足够的Zn将对减缓DN的进展至关重要。创新性：（1）明确Zn能够改善糖尿病导致的肾功能异常及肾脏病理学改变，从而保护DN。（2）阐明Zn保护DN的可能机制，即Zn激活了DN小鼠肾脏组织Akt介导的代谢相关信号及MT表达；Zn对Akt信号的激活作用与其对Akt负性调节因子PTEN、PTP1B及TRB3的抑制作用有关。（3）证明Akt2缺失后，并不影响Zn对糖尿病导致的肾脏功能异常及肾脏病理学改变的保护作用，也不影响Zn对Akt介导的相关代谢信号的激活作用，即Zn对DN的治疗作用是不依赖于Akt2的。（4）证明在生理状态下Zn对Akt及其下游相关代谢信号的激活作用是MT依赖的。更多还原

【Abstract】 Diabetes mellitus (DM) is a clinical syndrome that is hypofuction of islet induced bypathogenic factors included genetic factors and environmental factors. Insulin deficiencyresulting from a defect in insulin secretion, insulin action, or both, in turn leads to chronichyperglycaemia with disturbances of carbohydrate, fat and protein metabolism. As the diseaseprogresses tissue or vascular damage ensues leading to severe diabetic complications, whichwill finally lead to the dysfuction and failure of organs and become the main cause ofmorbidity and mortality. The World Health Organization states that worldwide the number ofindividuals with diabetes is expected to surpass550million by2030. This will bring a heavyfinancial burden to society and family. How to effectively treat diabetes and its complicationswill be an urgent clinical problem need to conquer.Diabetic nephropathy (DN) is the most common microvascular complication of diabetesmellitus. It is a leading cause of end-stage renal disease and a contributor to significantmorbidity and mortality in patients with diabetes. Several mechanisms are thought to beinvolved in the pathogenesis of diabetic nephropathy and its complications, all of themoriginating from hyperglycemia. It induces activation of protein kinase C, increasedproduction of advanced glycosylation end products, and diacylglycerol synthesis.Inflammation, oxidative stress and fibrosis may be activated by hyperglycemia in kidneytissues which eventually leads to basement membranes thickening, extracellular matrixaccumulation and glomerular sclerosis. All these changes plays an important role in thedevelopment and progression of DN. Increasing the sensitivity of insulin signal, andimproving glucose metabolism become a clinical hotspot recently in dealing with diabetickidney disease.Zinc (Zn) is a trace element that plays a pivotal role in the proper functioning of manyenzymes and transcription factors. Zn has been used clinically in the treatment of severaldiseases. Its low toxicity profile makes it possible to use in pediatric patients. Diabeticpatients are often Zn deficient owing to elevated urinary Zn excretion,decreased intestinal Znabsorption, and are stricted diet. So Zn supplementation may be beneficial for the treatment ofDN. Recently, meta-analysis and systematic review of clinical data showed the beneficial effects of Zn supplementation for diabetic patients. Human study proves that Znsupplementation reduces albumin excretion in micro albuminuric type2diabetic patients.However, the mechanism by which zinc protects the kidney from diabetes remains unknown,so we conducted a series of research.Firstly, we examined whether Zn can provide a therapeutic effect against DN and thepossible mechanism of it using the OVE26type1diabetic mouse model. We have examinedthe renal pathological and functional changes, Akt-mediated molecules related to metabolism,MT and Akt negative regulators in the kidney of OVE26type1diabetic mice. Because of theimportant role of Akt2in insulin-mediated glucose metabolism, We then examined whetherthe therapeutic effect of Zn on the diabetic kidney is dependent on Akt2using an Akt2-KOmouse model. Finally, we examined if MT is required for Zn stimulation of Akt and itsdownstream pathways by using mice with an MT gene deletion (MT-KO). The results ispresented:(1) We found ACR continually increase in both the DM group and the DM/Zn group, butit was slightly lower in DM/Zn mice than in DM mice at6months. The renal fibroticresponse, inflammation and oxidative damage were significantly increased in DM mice, butwas significantly prevented by Zn treatment. The above results have shown Znsupplementation can significantly improve diabetes-induced renal functional and pathologicalchanges in OVE26and Akt2-KO diabetic models;(2) We demonstrated that decreased renal Akt phosphorylation along with diabetesinduced pathological changes in the kidney which were significantly attenuated by Zntreatment, such that Akt phosphorylation in both OVE26and Akt2-KO diabetic mouse modelswas almost completely preserved. Preservation of renal Akt phosphorylation by Zn treatmentwas associated with normal GSK-3βphosphorylation and HKII expression in both OVE26andAkt2-KO diabetic mice.(3) Zn treatment prevents diabetic upregulation of the Akt negative mediators PTEN,PTP1B, and TRB3in OVE26and Akt2-KO diabetic models, but had no effect under normalconditions.(4) By detecting renal functional, pathological changes and Akt-mediated moleculesrelated to metabolism in the kidney tissue of Akt2-KO mouse, we found that deletion of theAkt2gene had no effect on Zn’s ability to provide renal protection against diabetes-inducedfunctional and pathologic changes; furthermore, deletion of the Akt2gene had no effect onZn-induced stimulation of Akt-mediated metabolic signaling. we draw the conclusion that zn protection against diabetes-induced renal damage is independent of Akt2.(5) We also checked phosphorylation of renal Akt and GSK-3βas well as the expression ofHKII in MT-KO mice after Zn supplementation1month, and found that Zn-inducedstimulation of Akt-related metabolic signaling is MT dependent, at least under normalphysiological conditions.In summary, this study shows that Zn stimulation of Akt-mediated renal glucosemetabolism improves renal functionin OVE26and Akt2-KO diabetic models.We furthershowed that the preservation of Akt-mediated metabolic signaling by Zn was not dependenton Akt2, but was dependent on MT. The preservation of Akt-mediated metabolic signalingunder diabetic conditions may be associated with Zn suppression of Akt negative regulators,PTEN phosphorylation, and PTP1B, aswellas TRB3, which needs further investigation.Considering that diabetic patients are often Zn deficient owing to elevated urinary Znexcretion, decreased intestinal Zn absorption, and are stricted diet, this study suggests thatregularly monitoring Zn levels in diabetic patients, as well as adequate Zn supplementation inpatients whose Zn levels are low, would be very important in mitigating the development ofdiabetic nephropathy.Innovations:(1) We demonstrated that Zn supplementation can significantly improve diabetes-inducedrenal functional and pathological changes in OVE26and Akt2-KO diabetic models and havethe protective effects on DN.(2) We elucidated the possible mechanisms of the protective effects of Zn supplementationon DN. The therapeutic effect of Zn treatment on the diabetic kidney is associated with thestimulation of MT expression and Akt-mediated metabolic signaling. Preservation ofAkt-mediated metabolic signaling by Zn treatment may be associated with Zn-mediatedsuppression of Akt negative regulators PTEN phosphorylation and PTP1B and TRB3expression, under diabetic conditions.(3) We demonstrated that deletion of the Akt2gene had no effect on Zn’s ability toprovide renal protection against diabetes-induced functional and pathologic changes;furthermore, deletion of the Akt2gene had no effect on Zn-induced stimulation of Akt-mediated metabolic signaling. zn protection against diabetes-induced renal damage isindependent of Akt2.(4) We elucidated that Zn-induced stimulation of Akt-related metabolic signaling is MT dependent, at least under normal physiological conditions.更多还原