Role of pH-mediated metabolic reprogramming in β cell failure in Type 2 Diabetes Mellitus

DEI增刊R01-DK128844：增刊摘要 2型糖尿病(T2 DM)表现为空腹和餐后高血糖 其病因可归结为胰腺β细胞未能维持适当的葡萄糖刺激 胰岛素分泌(即β细胞功能)以补偿胰岛素作用的下降(即胰岛素抵抗)。 我们正在进行的部分研究表明，SLC4A4及其蛋白产物(电生钠偶联 β细胞中的碳酸氢协转运蛋白(NBCE1)参与了T2 DM时β细胞功能的下降。作为这个NBCe1蛋白 在T2D中被不适当地激活，并应引起细胞内缓冲和细胞内pH(Phi)的变化 法规，这一补充培训车寻求直接测量在NBCE1被敲打的β细胞中的PHI 过度表达的或过度表达的这项工作不仅应该揭示β细胞的新陈代谢状态，还应该允许我们 更直接地确定细胞内pH调节、细胞内缓冲或其他方面是否发生了变化 NBCE1生理学将β细胞表型转变为T2D状态。 为了解决这一假设，我们将专注于原始特定目标2的部分(细胞pH调节与 遗传功能增益工具)和3(利用新的遗传功能丧失动物模型)来测试治疗 抑制β细胞中SLC4A4/NBCE1表达/活性作为减轻β细胞衰竭和 在糖尿病发生的条件下改善整体的葡萄糖代谢。从特定的AIM 2，INS1细胞带有 基因编码的pH传感器将在有和没有NBCe1遗传功能增益的情况下进行评估以记录 PH-改变并确定PHI改变本身是否通过损害β细胞功能而导致细胞功能衰竭 线粒体的代谢和功能。同样，对于特定目标3，新的遗传功能丧失动物 模型(β细胞中的NBCE1丢失)将与一种新型的pH传感器小鼠杂交，以确定pH和缓冲 完整胰岛内的β细胞。目前的项目将发现新的分子/生理学 诱导β细胞功能障碍的潜在机制并测试潜在的新治疗策略 减轻T2 DM患者β细胞衰竭。 父项目摘要/摘要 2型糖尿病(T2 DM)表现为空腹和餐后高血糖 其病因可归结为胰腺β细胞未能维持适当的葡萄糖刺激 胰岛素分泌(即β细胞功能)以补偿胰岛素作用的下降(即胰岛素抵抗)。 因此，β细胞功能的保存被认为是成功发展的关键障碍 预防和治疗策略，以抗击T2 DM患病率的上升。研究表明，β细胞 T2 DM的功能障碍与代谢重新编程/向非氧化葡萄糖升高的转变有关 新陈代谢和线粒体功能降低类似于在癌细胞中观察到的适应性特征。 因此，癌细胞通过以下方式促进糖酵解通量的增加和随后代谢酸产量的增加 上调离子泵/转运蛋白的表达，增强细胞的缓冲能力，促进 细胞碱化(phi增加)，如SLC4家族的碳酸氢盐转运体。虽然，以前的 研究已经证实了PHI对于正常的β细胞功能的重要性，目前尚不清楚细胞内是否 碱化或pH缓冲增加是T2 DM患者β细胞功能下降的原因之一。因此，关键是 目前这项提议的目的是检验一种新的T2 DM基因的异常诱导假设 β细胞中SLC4A4及其蛋白产物(钠离子偶联转运蛋白NBCE1)的作用 提示T2 DM患者β细胞功能下降。为了解决这一假设，具体目标1)将执行详细的 利用我们独特的尸检胰腺标本检测SLC4A4/NBCe1的表达 来自肥胖、糖尿病前期和T2 DM患者和2)阐明了介导异常的分子机制 糖尿病应激源诱导SLC4A4/NBCe1细胞β《特定目标2》将利用小说 遗传功能增益工具检验β细胞增加所介导的细胞内碱化的假说 SLC4A4/NBCE1的表达通过线粒体代谢障碍导致β细胞功能衰竭 和功能。最后，特殊目标3将利用新的遗传功能丧失动物模型和T2 DM人类 胰岛抑制β细胞中SLC4A4/NBCE1表达/活性的治疗潜力 在糖尿病发生的条件下，减轻β细胞衰竭，改善整体葡萄糖代谢。海流 该项目将揭示诱导β细胞功能障碍的新分子/生理机制 测试一种潜在的新的治疗策略来减轻T2 DM患者的β细胞衰竭。

DEI Supplement R01-DK128844: Supplement abstract Type 2 diabetes mellitus (T2DM) manifests through the development of fasting and postprandial hyperglycemia the etiology of which can be distilled to failure of pancreatic β cells to maintain appropriate glucose-stimulated insulin secretion (i.e., β cell function) to compensate for the decline in insulin action (i.e. insulin resistance). Part of our ongoing studies indicate that SLC4A4 and its protein product (electrogenic Na+ -coupled HCO3- cotransporter, NBCe1) in β cells contributes to β cell functional decline in T2DM. As this NBCe1 protein is inappropriately activated in T2D and should elicit changes of intracellular buffering and intracellular pH (pHi) regulation, this Supplement training vehicle seeks to directly measure pHi in β cells that have NBCe1 knocked out or overexpressed. This work should not only reveal the metabolic state of the β cells but also allow us to more directly determine if altered intracellular pH-regulation, intracellular buffering or some other aspect of NBCe1 physiology changes the β cell phenotype to a T2D-state. To address this hypothesis, we will focus on parts of the original Specific Aims 2 (cellular pH regulation with genetic gain-of-function tools) and 3 (utilize novel genetic loss-of-function animal models) to test therapeutic potential of inhibiting SLC4A4/NBCe1 expression/activity in β cells as means to attenuate β cell failure and improve overall glucose metabolism under diabetogenic conditions. From Specific Aim 2, INS1 cells with a genetically encoded pH-sensor will be evaluated with and without NBCe1 genetic gain-of-function to document pH-changes and determine if pHi changes per se leads to β cell functional failure through impairment of mitochondrial metabolism and function. Similarly for Specific Aim 3, novel genetic loss-of-function animal models (NBCe1-loss in β cells) will be crosses with a novel pH-sensor mouse to determine pH and buffering of β cells within intact pancreatic islets. The current project will uncover novel molecular/physiological mechanisms underlying induction of β cell dysfunction and test a potentially novel therapeutic strategy to attenuate of β cell failure in T2DM. Parent PROJECT SUMMARY/ABSTRACT Type 2 diabetes mellitus (T2DM) manifests through the development of fasting and postprandial hyperglycemia the etiology of which can be distilled to failure of pancreatic β cells to maintain appropriate glucose-stimulated insulin secretion (i.e. β cell function) to compensate for the decline in insulin action (i.e. insulin resistance). Thus preservation of β cell function has been identified as a critical barrier for the development of successful preventative and treatment strategies to combat the rise in T2DM prevalence. Studies suggest that β cell dysfunction in T2DM is associated with metabolic reprogramming/shift toward increased non-oxidative glucose metabolism and reduced mitochondrial function similar to an adaptive features observed in cancer cells. Accordingly, cancer cells facilitate increased glycolytic flux and subsequent rise in metabolic acid production by upregulating expression of ion pumps/transporters that enhance cellular buffering capacity and promote cellular alkalinization (increased pHi), such as SLC4 family of bicarbonate transporters. Although, previous studies have confirmed importance of pHi for proper β cell functionality, it is unknown whether intracellular alkalinization or increased pHi buffering contributes to β cell functional decline in T2DM. Thus, the key objective of the current proposal is to test the hypothesis that aberrant induction of a novel T2DM gene SLC4A4 and its protein product (electrogenic Na+ -coupled HCO3- cotransporter, NBCe1) in β cells contributes to β cell functional decline in T2DM. To address this hypothesis, Specific Aim 1 will 1) perform detailed examination of SLC4A4/NBCe1 expression using our unique collection of autopsy-derived human pancreas from patients with obesity, pre-diabetes and T2DM and 2) elucidate molecular mechanisms mediating aberrant β cell induction of SLC4A4/NBCe1 in response to diabetogenic stressors. Specific Aim 2 will utilize novel genetic gain-of-function tools to test the hypothesis that intracellular alkalinization mediated by increased β cell expression of SLC4A4/NBCe1 leads to β cell functional failure through impairment of mitochondrial metabolism and function. Finally, Specific aim 3 will utilize novel genetic loss-of-function animal models and T2DM human islets to test therapeutic potential of inhibiting SLC4A4/NBCe1 expression/activity in β cells as means to attenuate β cell failure and improve overall glucose metabolism under diabetogenic conditions. The current project will uncover novel molecular/physiological mechanisms underlying induction of β cell dysfunction and test a potentially novel therapeutic strategy to attenuate of β cell failure in T2DM.