Dual role of BAD in insulin secretion and beta cell survival

 描述（由申请方提供）：葡萄糖代谢对β细胞功能和存活的调节至关重要。我们已经发现BCL-2家族蛋白BAD在其螺旋BH 3结构域内的磷酸化赋予β细胞细胞自主保护作用。模拟磷酸-BAD BH 3螺旋的遗传和药理学策略的益处包括对由炎症和氧化应激诱导的死亡的抗性，以及增加的葡萄糖处理和胰岛素分泌能力。从长远来看，这些益处表现在供体胰岛在移植的糖尿病小鼠中的上级植入和功能性β细胞质量的伴随增加。磷酸化BAD的β细胞保护作用依赖于葡萄糖代谢酶葡萄糖激酶（GK），并通过磷酸化BAD BH 3螺旋直接结合和激活该酶介导。我们提出的研究解决了源于这些发现的一个关键问题：磷酸化BAD如何通过GK依赖性葡萄糖代谢介导β细胞保留效应？我们将在两个高度整合的分子层上解决这个问题。首先，使用代谢通量分析，我们将在丙酮酸脱氢酶（PDH）和丙酮酸羧化酶（PC）途径通量的水平上鉴定葡萄糖衍生的丙酮酸的线粒体代谢中的磷酸-BAD依赖性变化，因为β细胞经受限定的应激刺激，并且确定它们对磷酸-BAD保护β细胞免于应激诱导的功能障碍和死亡的能力的贡献（目的1）。第二，我们将询问确定数量的MODY衍生的GK突变体的体外性质以及它们在磷酸化BAD存在下对β细胞存活的影响（目的2）。这些突变体因其接近GK上BAD BH 3螺旋的结合界面而被选择，将揭示磷酸化BAD模拟对GK调节的其他分子方面，并提供一个补充的实验系统来检查GK依赖性葡萄糖代谢对BAD磷酸化的β细胞保护作用的影响。这些研究将提供连接磷酸化BAD依赖性GK激活和β细胞抵抗应激诱导的功能障碍和死亡的途径的综合图片。随着时间的推移，了解这一途径将产生有价值的翻译见解，以最有效的策略来捕获和模拟葡萄糖信号传导的保护方面，以保存/恢复功能性β细胞群。

 DESCRIPTION (provided by applicant): Glucose metabolism is central to regulation of β-cell function and survival. We have found that phosphorylation of the BCL-2 family protein BAD within its helical BH3 domain imparts cell autonomous protective effects to β-cells. The benefits of genetic and pharmacologic strategies that mimic the phospho-BAD BH3 helix include resistance to death induced by inflammatory and oxidative stress, as well as increased glucose handling and insulin secretory capacities. In the long-term, these benefits manifest in superior engraftment of donor islets in transplanted diabetic mice and an attendant increase in functional β-cell mass. The β-cell protective effect of phospho-BAD is dependent on the glucose-metabolizing enzyme glucokinase (GK), and is mediated by direct binding and activation of this enzyme by the phospho-BAD BH3 helix. Our proposed studies address a key question that stems from these findings: How is the β-cell sparing effect of phospho-BAD mediated by GK-dependent glucose metabolism? We will address this question at two highly integrative molecular layers. First, using metabolic flux analyses we will identify phospho-BAD dependent changes in mitochondrial metabolism of glucose-derived pyruvate at the level of pyruvate dehydrogenase (PDH) and pyruvate carboxylase (PC) pathway fluxes as β-cells are subjected to defined stress stimuli, and define their contribution to phospho-BAD's capacity to shield β-cells from stress-induced dysfunction and death (aim 1). Second, we will interrogate a defined number of MODY-derived GK mutants for their in vitro properties as well as their effect on β-cell survival in the presence of phospho-BAD (aim 2). These mutants, which are selected for their proximity near the binding interface of the BAD BH3 helix on GK, will reveal additional molecular aspects of GK regulation by phospho-BAD mimicry and provide a complementary experimental system to examine the effect of GK-dependent glucose metabolism on the β-cell protective effect of BAD phosphorylation. These studies will provide an integrated picture of the pathway connecting phospho-BAD-dependent GK activation and β-cell resistance to stress-induced dysfunction and death. In the fullness of time, understanding this pathway will yield valuable translational insights into the most effective strategies to capture and mimic the protective aspects of glucose signaling for preservation/restoration of functional β-cell mass.