转化生长因子β对胰高血糖素信号通路的协同效应及其在2型糖尿病中的作用

Glucagon, a peptide hormone secreted from pancreatic α cells in response to hypoglycemia, promotes hepatic glucose production by stimulating gluconeogenesis/glycogenolysis in the liver. Accumulating evidence indicate that elevated glucagon level could be a key factor of fasting hyperglycemia in type 2 diabetes, suggesting that suppression of hepatic glucagon signaling and function may be an effective strategy to reduce glucagon-mediated diabetic hyperglycemia. However, inhibition or blocking of conventional glucagon signaling in hepatocytes is associated with pancreatic cell hyperplasia and abnormal lipid metabolism, suggesting that directly targeting hepatic glucagon signing pathway may not be practical for developing effective therapeutic treatment for type 2 diabetes. Thus, it is the time to fully understand hepatic glucagon signaling by elucidating alternative pathway(s) that may amplify conventional glucagon signaling or work independently to control hepatic glucose output. .Several findings confirmed that high levels of circulating transforming growth factor β (TGFβ) are associated with obesity and diabetes in humans and that inhibition of hepatic TGFβ signaling reduces diet-induced fasting hyperglycemia in mice. Our previous studies further showed that, in mouse primary hepatocytes, a) TGFβ promotes glucagon-induced dissociation of PKA regulatory subunit (PKA-R) from the catalytic subunit (PKA-C) and glucagon-induced hepatic glucose production; b) the effects of TGFβ on glucagon signaling are dependent on TGFβ receptor activity; and c) overexpression of Smad3 promotes glucagon-induced PKA signaling and gluconeogenesis. So we hypothesize that TGFβ signaling may promote hyperglycemia by sensitizing hepatic glucagon-stimulated PKA activity and function in vivo. In this project, firstly, we will use liver-specific TGFβ type II receptor (TGFβRII) knockout mice to investigate whether liver-specific disrupting TGFβRII expression reduces fasting hyperglycemia in high fat diet-fed mice. Secondly, we will determine if overexpression of constitutively activated Smad3 promotes PKA activity and enhances hepatic glucose production in vivo. Thirdly, we will elucidate if disrupting Smad3/PKA-R interaction impairs the cross talk between TGFβ and glucagon signaling and action in mouse liver. Finally, we will further explore the clinical relevance of synergistic effects of TGFβ and glucagon signaling in onset and progression of type 2 diabetes in Chinese. These findings will shed new light on the pathogenesis of hyperglycemia and type 2 diabetes, and will open a new avenue for the treatment of hyperglycemia and type 2 diabetes.

胰高血糖素诱导的肝糖生成增加是导致高血糖的重要原因。我们前期发现小鼠原代肝细胞中TGFβ可增加胰高血糖素刺激的肝糖生成，促进胰高血糖素诱导的PKA调节亚基（PKA-R）和催化亚基解离；TGFβ对胰高血糖素信号通路的作用依赖于TGFβ受体活性，且TGFβ信号通路中Smad3可与PKA-R结合。杂合型肝脏特异性敲除TGFβ II型受体（TGFβRII）小鼠空腹血糖较对照小鼠降低。因此，我们提出肝脏TGFβ信号通路可协同胰高血糖素信号通路，增加肝糖生成，导致高血糖。本项目将研究TGFβRII、Smad3以及Smad3/PKA相互作用在TGFβ对胰高血糖素信号通路的协同效应中的作用及分子机制，同时探讨中国人群TGFβ与胰高血糖素信号通路的相关性及与2型糖尿病的临床关联。研究结果将阐明TGFβ信号通路与胰高血糖素信号通路的对话在2型糖尿病发生中的作用和机制，为寻求2型糖尿病的药物新靶标提供科学依据。

肝脏是内源性葡萄糖产生的主要部位，尤其是在长期禁食的状态下。但是，糖异生增强也是2型糖尿病（T2D）的特征。因此，阐明调节肝糖异生的信号传导途径，将更好地了解内源性葡萄糖产生的过程，以及T2D中糖异生增强的病因。转化生长因子β（TGF-β）已显示可促进肝糖异生，但具体机制尚不清楚。本研究旨在探讨TGF-β1在肝糖异生中的具体机制和作用。我们选择健康对照和T2D观察其糖代谢状况的变化，并通过ELISA法检测其血清TGF-β1水平。收集非肥胖T2D和非糖尿病对照患者的肝组织，通过Western blot检测Smad3磷酸化，并通过ELISA和Western blot检测cAMP / PKA / CREB的活化水平。在小鼠原代肝细胞中检测了胰高血糖素和TGF-β1信号通路，通过免疫共沉淀检测Smad3和PKA的结合以及PKA亚基的解离。利用Smad3基因敲除小鼠模型，评估胰高血糖素耐量试验中的葡萄糖水平。结果显示，与健康对照组相比，无肥胖的T2D患者TGF-β1水平明显升高，HbA1c，空腹血糖和餐后饮食与TGF-β1水平呈正相关，线性回归表明TGF-β1水平是独立糖代谢指标的独立影响因素。胰高血糖素信号通路和TGF-β1/ Smad3信号通路均在T2D患者的肝脏中被激活。用TGF-β1处理小鼠原代肝细胞可大大增强胰高血糖素刺激的PKA-CREB磷酸化和肝糖异生。TGF-β促进Smad3与PKA调节亚基（PKA-R）结合，并增强胰高血糖素诱导的PKA-R从PKA催化亚基（PKA-C）的解离，从而导致PKA活化和糖异生。提示TGF-β1通过促进Smad3与肝细胞中PKA-R结合，PKA-R解离，增强胰高血糖素刺激的体内PKA信号通路来增加肝糖异生，在T2D发病中起到致病作用。胰高血糖素是引起高血糖和T2D的关键因素，以传统的胰高血糖素信号通路作为T2D的靶标存在诸多不良反应，本项目从非传统选择性通路的新视角发掘出TGFβ信号通路对胰高血糖素信号通路的协同效应，为更好地理解高血糖和T2D的发病机制和验证TGFβ信号通路是否可作为将来干预这些疾病的药物靶标提供重要实验依据，提出针对TGFβ信号通路关键靶点分子可形成治疗高血糖及T2D的新概念、新策略。

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