Novel Molecular Determinants of Insulin Clearance

在此输入文本，它是应用程序的新摘要信息。此部分不得超过30行文本。 2型糖尿病（T2 D）与心血管疾病的风险升高有关，是失明、截肢和肾衰竭的主要原因。它已达到全球流行的比例，在美国的患病率超过10%。T2 D的病理生理学涉及胰岛素稳态缺陷，包括胰腺分泌胰岛素不足，肌肉、肝脏和脂肪组织中胰岛素作用受损（胰岛素抵抗），以及主要通过肝脏从循环中清除胰岛素（胰岛素清除）减少。了解胰岛素分泌和胰岛素抵抗的细胞和分子机制是几十年来研究的主要焦点，这导致了成功的抗糖尿病治疗策略。相比之下，胰岛素清除在糖尿病研究中一直是一个相对欠研究的领域。其分子决定因素尚未完全了解，其在T2 D病因学中的作用仍不清楚。降低的胰岛素清除率可能代表对胰岛素抵抗的有益代谢适应，其促进代偿性高胰岛素血症以限制β细胞的负担并可能防止T2 D。相反，已经提出受损的胰岛素清除可能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抗性。还假设遗传性降低的肝脏胰岛素清除率构成T2 D发展的主要致病因素。显然，胰岛素清除的机制和生理相关性值得进一步关注。 胰岛素清除率实验研究的一个局限性是相对缺乏已知的调节这一过程的分子决定因素。在以前的研究中，我们确定CEACAM 1是肝脏胰岛素清除的关键因素。虽然CEACAM 1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在已经很好地建立，但细胞胰岛素运输中后续步骤的分子机制和介导剂还不太清楚。因此，本提案的总体目标是通过发现新的分子决定因素及其在全身胰岛素/葡萄糖稳态中的作用的表征来扩展我们对这一过程的理解。 在人类中观察到的胰岛素清除率的高遗传性的激励下，我们开始了一个无假设的遗传方法。使用杂交小鼠多样性小组中约100个近交系小鼠品系的集合，我们进行了转录组和全基因组关联研究（GWAS），以鉴定与稳态C肽/胰岛素（C/I）摩尔比（胰岛素清除率的替代指标）相关的基因和分子途径。我们的初步结果提供了一些新的见解。首先，他们暗示AMPK通路参与细胞胰岛素清除的调节，并将该过程确定为AMPK信号传导的先前未被识别的代谢靶点。此外，我们确定Tmem 175作为一种新的遗传决定因素的胰岛素清除在小鼠中。虽然Tmem 175以前没有在胰岛素代谢的背景下进行表征，但它涉及内体pH调节，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，TMEM 175染色体区域与人群中的T2 D相关，这支持了TMEM 175活性变化可能影响糖尿病风险的假设，并突出了该项目的潜在翻译相关性。我们将对初步结果进行如下跟踪： 具体目的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信号传导在胰岛素清除调节中的下游效应物，我们将使用功能获得和功能丧失方法测试直接AMPK磷酸化靶点在细胞胰岛素清除中的作用。将通过C57 BL/6 J小鼠和AMPK肝脏特异性基因消融小鼠中AMPK的药理学活化来评价AMPK信号传导对全身胰岛素清除的影响。 具体目标2：研究Tmem 175在胰岛素清除和代谢稳态中的作用。为了获得对Tmem 175在细胞胰岛素代谢中作用的机制性见解，我们将使用功能获得和丧失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。Tmem 175在全身胰岛素清除中的作用将在Tmem 175缺陷小鼠中评估，其对肝脏胰岛素提取的影响将在腺相关病毒（AAV）介导的肝脏特异性抑制小鼠中研究。此外，我们将使用饮食诱导的肥胖（DIO）小鼠和遗传性肥胖的db/db小鼠与建立T2 D的背景下，通过纵向分析胰岛素和葡萄糖稳态，评估Tmem 175缺乏的代谢影响。 我们预计，这些研究将提供新的机制的见解，调节胰岛素清除率及其在2型糖尿病的病理生理学的作用。此外，我们的工作将为基于胰岛素清除操纵的T2 D新治疗方法奠定基础。

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.