mTORC1 regulation in the liver by metformin, AMPK and energy status

描述（由申请人提供）：amp依赖性蛋白激酶（AMPK）被能量剥夺激活，是细胞能量平衡的关键调节剂。AMPK的激活允许细胞在能量压力条件下存活，通过打开产生atp的分解代谢途径，抑制消耗atp的合成代谢过程。相反，哺乳动物雷帕霉素靶点复合物1 （mTORC1）被生长因子和营养物质激活，并促进合成代谢过程，导致细胞生长。最近，AMPK的激活与抗糖尿病药物二甲双胍的有益降血糖作用有关，二甲双胍可诱导能量应激。因此，阐明二甲双胍和AMPK负责这些有益作用的下游功能将极大地影响我们对细胞代谢的理解和更好地控制细胞代谢的能力。为此，细胞培养实验表明AMPK可以通过两种独立的机制抑制mTORC1：激活Tsc1-Tsc2复合物（mTORC1的上游抑制剂）和抑制Raptor （mTORC1的关键成分），这表明mTORC1在这一过程中可能发挥作用。因此，本研究的主要目标是阐明二甲双胍和肝脏能量应激调控mTORC1抑制的关键机制，并确定这种调控的代谢后果。小鼠肝脏或培养肝细胞中Tsc1的条件缺失将为研究Tsc1-2对二甲双胍和其他形式的能量和营养应激对mTORC1的抑制的相对贡献提供一个遗传模型。通过Tsc1抑制mTORC1对二甲双胍的细胞和生理作用的要求也将在该小鼠模型中得到解决。mTORC1和AMPK激活的功能读数将用于识别它们在这些条件下的活性。此外，AMPK在抑制

DESCRIPTION (provided by applicant): AMP-dependent protein kinase (AMPK) is activated by energy deprivation and is a critical regulator of cellular energy balance. Activation of AMPK allows cells to survive under conditions of energy stress by turning on ATP-producing catabolic pathways, and inhibiting ATP-consuming anabolic processes. Conversely, the mammalian target of rapamycin complex 1 (mTORC1) is activated by growth factors and nutrients, and promotes anabolic processes leading to cell growth. Recently, activation of AMPK has been implicated in the beneficial, glucose lowering effects of the anti-diabetic drug metformin, which induces energy stress. Thus, elucidation of the downstream functions of metformin and AMPK responsible for these beneficial effects will greatly impact our understanding of, and ability to better control cellular metabolism. To this end, cell culture experiments have indicated that AMPK can inhibit mTORC1 through two independent mechanisms: activation of the Tsc1-Tsc2 complex (an upstream inhibitor of mTORC1), and inhibition of Raptor (a critical component of mTORC1), suggesting a potential role for mTORC1 in this process. Therefore, the main goal of this proposal is to elucidate the key mechanisms that regulate mTORC1 inhibition by metformin and energy stress in the liver, and to determine the metabolic consequences of this regulation. Conditional deletion of Tsc1 in the mouse liver or cultured hepatocytes will provide a genetic model to study the relative contribution of Tsc1-2 to the inhibition of mTORC1 by Metformin, and other forms of energy and nutrient stress. The requirement for mTORC1 inhibition, through Tsc1, for the cellular and physiological effects of metformin will also be addressed using this mouse model. Functional readouts of mTORC1 and AMPK activation will be used to discern their activities under these conditions. Furthermore, the contribution of AMPK to the inhibition of mTORC1 by metformin will be determined using an additional mouse model with conditional deletion of AMPK in hepatocytes. These experiments address important questions that are crucial to our understanding of the cellular and organismal response to energy fluctuations, and are particularly relevant for further elucidation of the mode of action of metformin, the most commonly prescribed anti-diabetic drug. PUBLIC HEALTH RELEVANCE: The results of this study will impact our understanding of the energy-sensing signaling pathways controlling metabolic homeostasis and will provide novel insights into the therapeutic actions of metformin. Such knowledge is critical to our understanding of type-2 diabetes and to the development of new targeted therapeutics.