Novel Molecular Determinants of Insulin Clearance

胰岛素清除率的新分子决定因素

• 批准号: 10609503
• 负责人: Sonia M. Najjar
• 金额: $ 47.77万
• 依托单位: WESTERN UNIVERSITY OF HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-04-25 至 2026-01-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10609503
• 关键词: Ablation; Adipose tissue; Affect; Animals; Antidiabetic Drugs; Area; Attention; Automobile Driving; Beta Cell; Binding; Blindness; C-Peptide; CEACAM1; Cell Line; Cells; Circulation; Collection; Compensatory Hyperinsulinemia; Defect; Dependovirus; Development; Diabetes Mellitus; Diabetes prevention; Down-Regulation; Endosomes; Epidemic; Etiology; Excision; Foundations; Functional disorder; Genes; Genetic; Genetic Determinism; Hepatic; Hepatocyte; Heritability; Homeostasis; Human; Hybrids; Hyperinsulinism; Impairment; Inbred Strains Mice; Individual; Insulin; Insulin Receptor; Insulin Resistance; Investigation; Kidney Failure; Knowledge; Liver; Measures; Mediating; Mediator; Metabolic; Metabolism; Molecular; Mus; Muscle; Non-Insulin-Dependent Diabetes Mellitus; Obese Mice; Obesity; Pathway interactions; Phosphorylation; Physiological; Population; Prediabetes syndrome; Prevalence; Prevention; Process; Regulation; Research; Risk; Role; Signal Transduction; Testing; Text; Therapeutic; Variant; Work; blood glucose regulation; cardiovascular disorder risk; cell growth regulation; db/db mouse; diabetes risk; diet-induced obesity; follow-up; gain of function; gene function; genetic approach; genome wide association study; hepatoma cell; improved; insight; insulin regulation; insulin secretion; limb amputation; longitudinal analysis; loss of function; novel; novel therapeutic intervention; overexpression; pancreatic juice; pharmacologic; receptor; trafficking; transcriptome; translational potential

Enter the text here that is the new abstract information for your application. This section must be no longer than 30 lines of text. Type 2 diabetes (T2D) is associated with elevated risk of cardiovascular disease and a major cause of blindness, limb amputation and kidney failure. It has reached epidemic proportions worldwide with a prevalence exceeding 10% in the US. The pathophysiology of T2D involves defects in insulin homeostasis including insufficient insulin secretion from the pancreas, impaired insulin action in muscle, liver and adipose tissues (insulin resistance), and reduced insulin removal from the circulation (insulin clearance) predominantly by the liver. Understanding the cellular and molecular mechanisms of insulin secretion and insulin resistance has been a major focus of investigations for decades, which has resulted in successful antidiabetic therapeutic strategies. In contrast, insulin clearance has been a relatively understudied area in diabetes research. Its molecular determinants are incompletely understood and its role in the etiology of T2D remains unclear. Reduced insulin clearance may represent a beneficial metabolic adaptation to insulin resistance that promotes compensatory hyperinsulinemia to limit the burden on -cells and likely protect against T2D. Conversely, it has been proposed that impaired insulin clearance may exacerbate insulin resistance by driving hyperinsulinemia- mediated downregulation of insulin receptors. It has also been hypothesized that genetically reduced hepatic insulin clearance constitute a primary causal factor in the development of T2D. Clearly, the mechanisms and physiological correlates of insulin clearance deserve further attention. A limitation of experimental investigations of insulin clearance is the relative dearth of known molecular determinants that regulate this process. In previous studies, we identified CEACAM1 as a critical factor in hepatic insulin clearance. While the role of CEACAM1 in receptor-mediated insulin internalization, an initial step in cellular insulin clearance, and metabolic homeostasis is now well established, the molecular mechanisms and mediators of subsequent steps in cellular insulin trafficking are less well understood. Thus, the overall objective of the present proposal is to extend our understanding of this process through the discovery of novel molecular determinants and characterization of their role in systemic insulin/glucose homeostasis. Motivated by the high heritability of insulin clearance observed in humans, we embarked on a hypothesis-free genetic approach. Using a collection of ~100 inbred mouse strains in the Hybrid Mouse Diversity Panel, we performed transcriptome and genome-wide association studies (GWAS) to identify genes and molecular pathways associated with steady-state C-peptide/insulin (C/I) molar ratio, a surrogate measure of insulin clearance. Our preliminary results provide several novel insights. First, they implicate the AMPK pathway in the regulation of cellular insulin clearance and identify this process as a previously unrecognized metabolic target of AMPK signaling. Moreover, we identified Tmem175 as a novel genetic determinant of insulin clearance in the mouse. While Tmem175 has not previously been characterized in the context of insulin metabolism, it has been implicated in endosomal pH regulation, a key process in the degradation and intracellular trafficking of insulin and the insulin receptor (INSR). Furthermore, the TMEM175 chromosomal region is associated with T2D in human populations, which lends support to the hypothesis that variation in TMEM175 activity may affect diabetes risk and highlights the potential translational relevance of this project. We will follow up on our preliminary results as described below: Specific Aim 1: To investigate the role of AMPK pathway in insulin clearance. We will use primary hepatocytes to investigate the cellular mechanisms responsible for the regulation of insulin clearance by AMPK. Pharmacological and genetic approaches will be employed to modulate AMPK activity and the impact on insulin binding, internalization and degradation, as well as the endocytic trafficking of INSR will be assessed. To identify downstream effectors of AMPK signaling in the regulation of insulin clearance, we will test the role of direct AMPK phosphorylation targets in cellular insulin clearance using gain- and loss-of- function approaches. The effect of AMPK signaling on systemic insulin clearance will be evaluated through pharmacological activation of AMPK in C57BL/6J mice and in mice with liver-specific genetic ablation of AMPK. Specific Aim 2: To investigate the role of Tmem175 in insulin clearance and metabolic homeostasis. To gain mechanistic insights into the role of Tmem175 in cellular insulin metabolism, we will assess insulin binding, internalization, degradation and INSR trafficking in primary hepatocytes and hepatoma cell lines using gain- and loss-of-function approaches. The role of Tmem175 in systemic insulin clearance will be assessed in whole-body Tmem175-deficient mice and its impact on hepatic insulin extraction will be investigated in mice with adeno-associated virus (AAV)-mediated liver-specific suppression. Furthermore, we will evaluate the metabolic impact of Tmem175 deficiency through longitudinal analyses of insulin and glucose homeostasis in the context of pre-diabetes using diet-induced obese (DIO) mice and the genetically obese db/db mice with established T2D. We anticipate that these studies will provide novel mechanistic insights into the regulation of insulin clearance and its role in the pathophysiology of T2D. Furthermore, our work will lay the foundation for novel therapeutic approaches in T2D based on the manipulation of insulin clearance.

在此处输入文本，该文本是您的应用程序的新摘要信息。此部分的文本长度不得超过 30 行。 2 型糖尿病 (T2D) 与心血管疾病风险升高相关，也是导致失明、截肢和肾衰竭的主要原因。它已在全球范围内达到流行程度，在美国患病率超过 10%。 T2D 的病理生理学涉及胰岛素稳态缺陷，包括胰腺胰岛素分泌不足、肌肉、肝脏和脂肪组织中的胰岛素作用受损（胰岛素抵抗）以及主要由肝脏从循环中清除的胰岛素减少（胰岛素清除）。几十年来，了解胰岛素分泌和胰岛素抵抗的细胞和分子机制一直是研究的主要焦点，这导致了成功的抗糖尿病治疗策略。相比之下，胰岛素清除率一直是糖尿病研究中相对较少研究的领域。其分子决定因素尚不完全清楚，其在 T2D 病因学中的作用仍不清楚。胰岛素清除率降低可能代表对胰岛素抵抗的有益代谢适应，促进代偿性高胰岛素血症，以限制 β 细胞的负担，并可能预防 T2D。相反，有人提出，胰岛素清除受损可能会通过驱动高胰岛素血症介导的胰岛素受体下调而加剧胰岛素抵抗。还假设遗传性肝脏胰岛素清除率降低是导致 2 型糖尿病发生的主要原因。显然，胰岛素清除的机制和生理相关性值得进一步关注。 胰岛素清除实验研究的一个局限性是调节这一过程的已知分子决定因素相对缺乏。在之前的研究中，我们确定 CEACAM1 是肝脏胰岛素清除的关键因素。虽然 CEACAM1 在受体介导的胰岛素内化（细胞胰岛素清除的初始步骤和代谢稳态）中的作用现已得到很好的确定，但细胞胰岛素运输后续步骤的分子机制和介质尚不清楚。因此，本提案的总体目标是通过发现新的分子决定因素并表征它们在全身胰岛素/葡萄糖稳态中的作用来扩展我们对这一过程的理解。 受人类中观察到的胰岛素清除率高遗传性的启发，我们开始采用无假设的遗传方法。我们使用混合小鼠多样性面板中约 100 个近交系小鼠品系进行了转录组和全基因组关联研究 (GWAS)，以确定与稳态 C 肽/胰岛素 (C/I) 摩尔比（胰岛素清除率的替代指标）相关的基因和分子通路。我们的初步结果提供了一些新颖的见解。首先，他们将 AMPK 通路与细胞胰岛素清除率的调节联系起来，并将这一过程确定为以前未被识别的 AMPK 信号传导的代谢靶标。此外，我们确定 Tmem175 是小鼠胰岛素清除率的新遗传决定因素。虽然 Tmem175 之前尚未在胰岛素代谢背景下进行表征，但它与内体 pH 调节有关，这是胰岛素和胰岛素受体 (INSR) 降解和细胞内运输的关键过程。此外，TMEM175 染色体区域与人群中的 T2D 相关，这支持了 TMEM175 活性的变化可能影响糖尿病风险的假设，并强调了该项目的潜在转化相关性。我们将跟进我们的初步结果，如下所述： 具体目标1：研究AMPK通路在胰岛素清除中的作用。我们将使用原代肝细胞来研究 AMPK 调节胰岛素清除的细胞机制。将采用药理学和遗传学方法来调节 AMPK 活性，并评估对胰岛素结合、内化和降解以及 INSR 内吞运输的影响。为了确定 AMPK 信号在胰岛素清除率调节中的下游效应器，我们将使用功能获得和丧失的方法测试直接 AMPK 磷酸化靶标在细胞胰岛素清除率中的作用。 AMPK 信号传导对全身胰岛素清除率的影响将通过 C57BL/6J 小鼠和肝脏特异性 AMPK 基因消除的小鼠中 AMPK 的药理学激活来评估。 具体目标 2：研究 Tmem175 在胰岛素清除和代谢稳态中的作用。为了深入了解 Tmem175 在细胞胰岛素代谢中的作用，我们将使用功能获得和丧失的方法评估原代肝细胞和肝癌细胞系中的胰岛素结合、内化、降解和 INSR 运输。将在全身 Tmem175 缺陷小鼠中评估 Tmem175 在全身胰岛素清除中的作用，并将在腺相关病毒 (AAV) 介导的肝脏特异性抑制小鼠中研究其对肝脏胰岛素提取的影响。此外，我们将使用饮食诱导肥胖 (DIO) 小鼠和患有 T2D 的遗传性肥胖 db/db 小鼠，通过对糖尿病前期背景下胰岛素和葡萄糖稳态的纵向分析来评估 Tmem175 缺乏的代谢影响。 我们预计这些研究将为胰岛素清除率的调节及其在 T2D 病理生理学中的作用提供新的机制见解。此外，我们的工作将为基于胰岛素清除率操纵的 T2D 新型治疗方法奠定基础。