LEPTIN REGULATION OF BETA CELL PROLIFERATION

瘦素对 β 细胞增殖的调节

• 批准号: 10625939
• 负责人: RICHARD A COX
• 金额: $ 20万
• 依托单位: BAYLOR COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-07-19 至 2023-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10625939
• 关键词: Affect; Alpha Cell; Beta Cell; Biology; Blood Glucose; Calcium; Cell Proliferation; Cell physiology; Chronic Disease; Collaborations; D Cells; Data; Diabetes Mellitus; Endocrine; Equilibrium; Exhibits; Exocytosis; Exposure to; Feedback; Future; Gene Expression; Gene Targeting; Genetic Transcription; Glucagon; Goals; Hormone secretion; Hormones; Human; Insulin; Islet Cell; Knockout Mice; Knowledge; Lead; Learning; Leptin; Manuscripts; Mediating; Membrane; Metabolic; Modeling; Molecular; Mus; Neurons; Obesity; Outcome; Pancreatic delta Cell; Paracrine Communication; Pathologic; Patients; Persons; Physiological; Play; Positioning Attribute; Protein Tyrosine Kinase; Regulation; Role; STAT3 gene; Signal Transduction; Small Interfering RNA; Somatostatin; Testing; Therapeutic; Time; Universities; Visit; blood glucose regulation; calcium indicator; energy balance; fluorescence imaging; in vivo Model; inhibitor; innovation; insight; insulin secretion; islet; leptin receptor; loss of function; paracrine; response; single-cell RNA sequencing; tool; transcription factor; translational potential

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.

今后加强1 R 01 DK 128098 - 01 A1的努力 我明年的目标是确定控制瘦素调节胰岛δ细胞功能的分子和转录机制。通过抑制α和β细胞的生长抑素（SST）的旁分泌作用，δ细胞已成为胰岛功能的重要调节剂。然而，我们对在δ细胞中执行基因表达和代谢结果的内分泌信号知之甚少。瘦素抑制胰岛素分泌;然而，瘦素受体（LepR）仅在人类胰岛的δ细胞上表达，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SST分泌从人类胰岛，这反过来又减少胰岛素分泌从β细胞。因此，我们提出了一个模型，其中瘦素作为β细胞胰岛素分泌的负反馈调节刺激SST从δ细胞释放。我们将通过实现以下目标来构建我们的模型： 巩固我们的中心假设，即瘦素刺激δ细胞SST分泌，抑制β细胞功能。我们的初步数据表明，瘦素刺激SST分泌从人类和小鼠胰岛，抑制胰岛素分泌在静态条件下。我们将采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，最终更好地模拟体内胰岛反应。 - 我将访问杜克大学，向乔纳森坎贝尔和大卫达莱西奥博士学习胰岛灌注。这些努力将使我能够定义动态胰岛反应和旁分泌信号暴露于瘦素。 - 我将利用胰岛灌流的新专业知识来定义瘦素的δ细胞特异性作用（功能丧失：SST-Cre;LepR-lp/lp小鼠（先前生成）或siRNA介导的人类胰岛LepR缺失）以及SST对胰岛素和胰高血糖素分泌的后续旁分泌作用。 这些数据将为我的新R 01应用建立坚实的前提，即瘦素通过刺激δ细胞SST分泌来抑制β细胞功能。 定义瘦素在δ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离子升高以介导激素胞吐作用。尽管通过LepR的瘦素信号传导涉及酪氨酸激酶活性和JAK-STAT信号传导，但在神经元中有证据表明瘦素增加细胞内钙（PMID：30304668）。在这里，我们将测试瘦素是否增加δ细胞中的钙来介导SST分泌。 - 我与Mark Huising博士建立了合作关系，以确定瘦素刺激SST分泌是否涉及经典的胞吐信号，最明显的是增加细胞内钙。将用瘦素处理在δ细胞中表达钙指示剂GCaMP 6的胰岛（SST-Cre; Rosa 26-lsl-GCaMP; PMID：28380380，PMID：27408771），并通过荧光成像读出GCaMP 6活化。 揭示瘦素对δ细胞中钙水平的影响将使未来的研究定位于确定导致SST分泌的LepR下游信号。 定义δ细胞中瘦素作用的转录机制。我们的初步数据表明，δ细胞表达的Stat 3所需的瘦素诱导的SST分泌。与对照胰岛相比，当用瘦素处理时，来自SST-Cre; Stat 3-fl/fl（dStat 3 KO）小鼠的胰岛表现出显著钝化的SST分泌。 - 我们将在人（STAT 3抑制剂，TTI-101; Tvardi Therapeutics）或小鼠（dStat 3 KO）胰岛中存在瘦素的情况下在胰岛灌流期间阻断STAT 3以评估对激素分泌的影响。 - 将对用瘦素+/-STAT 3抑制剂（TTI-101）处理的人胰岛进行scRNA-seq分析（先前作为制备中的单独手稿的一部分建立的专业知识），以确定定义δ细胞活性的瘦素-STAT 3信号传导的δ细胞特异性基因靶标。我们还将发现其他转录因子，并缩小定义δ细胞调控的关键知识差距。 意义我们提出的研究将揭示瘦素刺激δ细胞中的JAK-STAT信号以准备SST分泌，其抑制β细胞胰岛素分泌。瘦素是能量平衡和葡萄糖稳态的关键调节剂，因此，这些发现将提供生理（餐后）和病理（肥胖症，糖尿病）状态下瘦素作用的关键见解。我们还将确定STAT 3抑制的潜在翻译益处。我们修改后的前提侧重于胰岛功能，结合使用创新工具的新初步数据，将大大加强我们在2022年底或2023年初的新R 01应用。我们明年的研究将揭示δ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。