LEPTIN REGULATION OF BETA CELL PROLIFERATION

加强1R01DK128098-01A1的未来努力 我明年的目标是确定瘦素对胰岛三角洲细胞功能的调控的分子和转录机制。通过抑制α细胞和β细胞的生长抑素(SST)的旁分泌作用，Delta细胞已成为胰岛功能的重要调节因子。然而，我们对三角洲细胞中执行基因表达和代谢结果的内分泌信号知之甚少。瘦素抑制胰岛素的分泌；然而，瘦素受体(Lepr)仅在人胰岛的三角洲细胞上表达，这表明瘦素可能间接作用于β细胞。我们的初步数据首次表明，瘦素刺激人类胰岛的SST分泌，进而减少β细胞的胰岛素分泌。因此，我们提出了一个模型，在这个模型中，瘦素通过刺激三角洲细胞释放SST来作为β细胞胰岛素分泌的负反馈调节因子。我们将通过实现以下目标来构建我们的模型： 证实了我们的中心假设，即瘦素刺激三角洲细胞SST分泌以抑制β细胞功能。我们的初步数据表明，瘦素刺激人和小鼠胰岛的SST分泌，而在静态条件下抑制胰岛素的分泌。我们将利用胰岛灌流研究来测试激素分泌和旁分泌信号的动态变化，最终建立更好的活体胰岛反应模型。 -我将访问杜克大学，向乔纳森·坎贝尔博士和大卫·达莱西奥博士学习胰岛灌输。这些努力将使我能够定义接触瘦素后的动态胰岛反应和旁分泌信号。 -我将利用胰岛灌流方面的新专业知识来确定瘦素(功能丧失：SST-CRE；Lepr-LP/LP小鼠(先前产生)或siRNA介导的人类胰岛Lepr缺失)对三角洲细胞的特定影响，以及SST对胰岛素和高血糖素分泌的旁分泌影响。 这些数据将为我的新R01应用奠定坚实的前提，即瘦素通过刺激三角洲细胞SST分泌来抑制β细胞功能。 确定瘦素在三角洲细胞中作用的分子机制。胰岛激素释放涉及膜去极化和细胞内钙升高，以调节激素胞吐。虽然瘦素通过Lepr的信号涉及酪氨酸激酶活性和JAK-STAT信号，但有证据表明神经元中的瘦素增加细胞内钙(PMID：30304668)。在这里，我们将测试瘦素是否会增加三角洲细胞中的钙以调节SST的分泌。 -我与马克·惠辛博士建立了一个合作关系，以确定瘦素刺激的SST分泌是否涉及经典的胞吐信号，最显著的是细胞内钙的增加。在三角洲细胞中表达钙指示剂GCaMP6的胰岛(Sst-Cre；rosa26-LSL-GCaMP；PMID：28380380，PMID：27408771)将用瘦素处理，GCaMP6的激活将通过荧光成像读出。 揭示瘦素对三角洲细胞钙水平的影响将为未来的研究定位，以确定导致SST分泌的Lepr下游信号。 确定三角洲细胞中瘦素作用的转录机制。我们的初步数据表明，瘦素诱导的SST分泌需要德尔塔细胞表达STAT3。与对照组相比，SST-CRE；STAT3-fl/fl(DStat3KO)小鼠的胰岛在用瘦素处理时SST的分泌明显减弱。 -我们将在人(STAT3抑制剂，TTI-101；Tvardi Treeutics)或小鼠(DStat3 KO)的胰岛中，在瘦素存在的情况下，在胰岛灌流期间阻断STAT3，以评估对激素分泌的影响。 -用Leptin+/-STAT3抑制剂(TTI-101)治疗的人胰岛将接受scRNA-seq分析(以前作为PREP单独手稿的一部分建立的专业知识)，以确定定义Delta细胞活动的Leptin-STAT3信号的三角洲细胞特定基因目标。我们还将发现其他转录因子，并填补定义三角洲细胞调控的关键知识空白。 意义重大。我们拟议的研究将揭示瘦素刺激三角洲细胞中的JAK-STAT信号准备SST分泌，从而抑制β细胞胰岛素的分泌。瘦素是能量平衡和葡萄糖稳态的关键调节因子，因此，这些发现将为了解瘦素在生理性(餐后)和病理性(肥胖、糖尿病)状态下的作用提供关键的见解。我们还将确定抑制STAT3的潜在翻译益处。我们修订的前提侧重于胰岛功能，结合使用创新工具的新初步数据，将显著加强我们在2022年末或2023年初的新R01应用。我们计划在明年进行的研究将揭示有关三角洲细胞的新生物学，并确定瘦素调节胰岛功能的分子和转录机制。

Future Efforts to Strengthen 1R01DK128098-01A1 My goal for the next year is to define molecular and transcriptional mechanisms that govern leptin regulation of islet delta cell functions. The delta cell has emerged as an important regulator of islet function through paracrine actions of somatostatin (SST) that inhibit alpha and beta cells. However, we know little about endocrine signals that perform gene expression and metabolic outcomes in delta cells. Leptin suppresses insulin secretion; however, the leptin receptor (LepR) is exclusively expressed on delta cells of human islets, suggesting leptin may act indirectly on beta cells. Our preliminary data demonstrate for the first time that leptin stimulates SST secretion from human islets which, in turn, decreases insulin secretion from the beta cell. Thus, we propose a model whereby leptin serves as a negative feedback regulator of beta cell insulin secretion by stimulating SST release from delta cells. We will build out our model by achieving the following goals: Cement our central hypothesis that leptin stimulates delta cell SST secretion to inhibit beta cell function. Our preliminary data demonstrate that leptin stimulates SST secretion from human and mouse islets, which suppresses insulin secretion under static conditions. We will employ islet perifusion studies to test dynamic changes in hormone secretion and paracrine signaling that, ultimately, better model in vivo islet responses. - I will visit Duke University to learn islet perifusion from Drs. Jonathan Campbell and David D’Alessio. These efforts will empower me to define dynamic islet responses and paracrine signals following exposure to leptin. - I will leverage new expertise in islet perifusion to define the delta cell specific effects of leptin (loss of function: SST-Cre;LepR-lp/lp mice (previously generated) or siRNA-mediated LepR deletion in human islets) and consequent paracrine effects of SST on insulin and glucagon secretion. These data will firmly establish the premise for my new R01 application that leptin suppresses beta cell function through stimulation of delta cell SST secretion. Define molecular mechanisms of leptin action in delta cells. Islet hormone release involves membrane depolarization and an elevation in intracellular calcium to mediate hormone exocytosis. Although leptin signaling through LepR involves tyrosine kinase activity and JAK-STAT signaling, there is evidence in neurons that leptin increases intracellular calcium (PMID: 30304668). Here, we will test whether leptin increases calcium in delta cells to mediate SST secretion. - I set up a collaboration with Dr. Mark Huising to determine if leptin stimulated SST secretion involves classical exocytotic signals, most notably increased intracellular calcium. Islets expressing the calcium indicator GCaMP6 in delta cells (SST-Cre;Rosa26-lsl-GCaMP; PMID: 28380380, PMID: 27408771) ) will be treated with leptin and GCaMP6 activation will be read out by fluorescence imaging. Revealing leptin effects on calcium levels in delta cells will position future studies to define the signals downstream of LepR that lead to SST secretion. Define transcriptional mechanisms of leptin action in delta cells. Our preliminary data suggest delta cell expression of Stat3 is required for leptin-induced SST secretion. Islets from SST-Cre;Stat3-fl/fl (dStat3 KO) mice exhibit a significantly blunted secretion of SST when treated with leptin compared to control islets. - We will block STAT3 during islet perifusion in the presence of leptin in human (STAT3 inhibitor, TTI-101; Tvardi Therapeutics) or mouse (dStat3 KO) islets to assess the impacts on hormone secretion. - Human islets treated with leptin +/- STAT3 inhibitor (TTI-101) will be subjected to scRNA-seq analysis (expertise previously established as part of a separate manuscript in prep) to determine the delta cell specific gene targets of leptin-STAT3 signaling that define delta cell activities. We will also uncover other transcription factors and close key knowledge gaps that define the regulation of delta cells. Significance. Our proposed studies will reveal leptin stimulates JAK-STAT signaling in delta cells to ready SST secretion, which suppresses beta cell insulin secretion. Leptin is a critical regulator of energy balance and glucose homeostasis and therefore, these findings will provide key insights into leptin action during physiologic (post-prandial) and pathologic (obesity, diabetes) states. We will also determine the potential translational benefits of STAT3 inhibition. Our revised premise focused on islet function combined with new preliminary data using innovative tools will significantly strengthen our new R01 application for late 2022 or early 2023. Our proposed studies for the next year will reveal new biology about delta cells and define molecular and transcriptional mechanisms through which leptin regulates islet function.