The regulation of adipocyte lipolysis by insulin

描述(申请人提供)：在禁食期间，脂肪细胞中以甘油三酯形式储存的能量被释放出来，供肌肉、肝脏和其他组织使用。这一过程被称为脂肪分解，受到荷尔蒙、神经和营养输入的高度调控。有证据表明，抵抗胰岛素的抗脂解作用是2型糖尿病和代谢综合征发生的早期和重要的一步。细胞内最常用于传递脂解增加信号的第二信使是环磷酸腺苷，它在对β-肾上腺素能药物的反应中积聚，并激活蛋白激酶A。关于蛋白激酶A下游的信号转导已知很多，包括存在于甘油三酯脂滴上的主要底物Perilipin。胰岛素拮抗脂肪分解的过程，有一个被普遍接受的模型来解释脂肪细胞脂肪分解的急性抑制，尽管还没有详细的研究。胰岛素信号传导的经典模型描述了肌醇磷脂3‘-依赖的蛋白激酶B激活Akt，进而磷酸化并刺激磷酸二酯酶3B(PDE3B)，导致细胞内环磷酸腺苷的减少。然而，首席研究员的实验室之前的工作揭示了胰岛素作用的另一种途径，该途径在脂肪分解的次极大刺激下发挥作用。初步数据表明，这种信号级联作用独立于Akt，关键的信号事件发生在表面，即脂滴。这项拨款提案中描述的研究将正式测试抗脂信号替代途径的假说。实验包括使用FRET生物传感器来确定培养脂肪细胞中PKA激活的准确细胞内位置，以及识别针对脂滴调节分子的支架蛋白。通过遗传功能丧失实验首次测试Akt和PDE3B在抑制脂解过程中对磷酸化的要求。最后，这一新途径对抑制脂肪细胞脂肪分解的贡献将通过转基因小鼠的体内实验来评估其生理意义。

DESCRIPTION (provided by applicant): During times of fasting, energy stored in the adipocyte as triglyceride is released for use by muscle, liver and other tissues. This process, termed lipolysis, is highly regulated by hormonal, neural and nutritional inputs. There is subtantial evdence that resistance to the actions of insulin to antagonize lipolysis is an early and important step in the development of type 2 diabetes mellitus and the metabolic syndrome. The intracellular second messenger most frequently used to convey the signal for increased lipolysis is cyclic AMP, which accumulates in reponse to beta-adrenergic agents and activates protein kinase A. Much is known about signaling downstream of protein kinase A, including a major substrate, perilipin, present on the triglyceride lipid droplet. Insulin antagonizes the process of lipolysis, and there is a generally accepted model to explain the acute suppression of adipocyte lipolysis, though it has not been studied in detail. The canonical model of insulin signaling describes phosphoinositide 3'-kinase-dependent activation of Akt (protein kinase B), which then phosphorylates and stimulates phosphodiesterase 3B (PDE3B), leading to a decrease in intracellular cyclic AMP. Previous work from the laboratory of the principal investigator has revealed, however, an alternative pathway of insulin action that comes into play upon sub-maximal stimulation of lipolysis. Preliminary data indicate that this signaling cascade functions independently of Akt and that the critical signaling events occur on the surface the lipid droplet. The studies described in this grant proposal will formally test the hypothesis of an alternative pathway for anti-lipolytic signaling. Experiments include the use of FRET biosensors to determine the precise intracellular sites of PKA activation in cultured adipocytes and the identification of scaffolding proteins that target regulatory molecules ot the lipid droplet. The requirement for Akt and PDE3B phosphorylation in the suppression of lipolysis will be tested for the first time by genetic loss of function experiments. Lastly, the contribution of this novel pathway for the suppression of fat cell lipolysis will be assessed for physiological relevance through in vivo experiments in genetically modified mice.