Novel Molecular Determinants of Insulin Clearance

在此输入文本，它是您的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(T2D)与心血管疾病风险增加有关，也是失明、截肢和肾衰竭的主要原因。它已经在世界范围内达到了流行的程度，在美国的流行率超过了10%。T2D的病理生理学涉及胰岛素稳态的缺陷，包括胰腺胰岛素分泌不足，肌肉、肝脏和脂肪组织的胰岛素作用受损(胰岛素抵抗)，以及主要由肝脏从循环中清除胰岛素的减少。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重点，这导致了成功的抗糖尿病治疗策略。相比之下，在糖尿病研究中，胰岛素清除一直是一个相对较少的研究领域。其分子决定因素尚不完全清楚，其在T2D病因学中的作用尚不清楚。胰岛素清除减少可能代表了对胰岛素抵抗的有益代谢适应，促进了代偿性高胰岛素血症，从而限制了细胞的负担，并可能保护其免受T2D的影响。相反，已有研究提出，胰岛素清除障碍可能通过驱动高胰岛素血症介导的胰岛素受体下调而加重胰岛素抵抗。也有人假设，遗传性降低的肝脏胰岛素清除率是T2D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。 胰岛素清除的实验研究的一个局限性是相对缺乏调节这一过程的已知分子决定因素。在以前的研究中，我们发现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、细胞胰岛素清除的第一步和代谢稳态中的作用现在已经很清楚，但细胞胰岛素运输后续步骤的分子机制和介体还不是很清楚。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性胰岛素/葡萄糖稳态中的作用来扩大我们对这一过程的理解。 受观察到的人类胰岛素清除的高遗传性的激励，我们开始了一种无假说的遗传方法。利用杂交小鼠多样性小组收集的约100个近交系小鼠，我们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)摩尔比相关的基因和分子途径，C/I摩尔比是衡量胰岛素清除的替代指标。我们的初步结果提供了几个新的见解。首先，他们发现AMPK通路参与调节细胞内胰岛素的清除，并将这一过程确定为先前未知的AMPK信号代谢靶点。此外，我们发现Tme175是小鼠体内胰岛素清除的一种新的遗传决定因素。虽然以前没有在胰岛素代谢的背景下描述Tmem175的特征，但它与内体pH调节有关，这是胰岛素和胰岛素受体(INSR)降解和细胞内转运的关键过程。此外，TMEM175染色体区域与人类T2D相关，这支持了TMEM175活性变异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。我们将跟进初步结果，如下所述： 具体目的1：探讨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。将使用药理学和遗传学方法来调节AMPK的活性，并将评估其对胰岛素结合、内化和降解以及INSR的内吞运输的影响。为了确定AMPK信号在调节胰岛素清除中的下游效应，我们将使用获得和功能丧失的方法测试直接AMPK磷酸化靶标在细胞胰岛素清除中的作用。AMPK信号对全身胰岛素清除的影响将通过药理学激活C57BL/6J小鼠和肝脏特异性基因消融AMPK的小鼠来评估。 具体目的2：研究Tem175在胰岛素清除和代谢稳态中的作用。为了从机制上深入了解Tmem175在细胞胰岛素代谢中的作用，我们将使用获得和失去功能的方法来评估胰岛素在原代肝细胞和肝癌细胞系中的结合、内化、降解和INSR运输。将在全身缺乏Tem175的小鼠中评估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的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)小鼠和已建立T2D的遗传性肥胖db/db小鼠，通过对糖尿病前期背景下的胰岛素和葡萄糖稳态的纵向分析，评估Tmem175缺乏对代谢的影响。 我们预计这些研究将为胰岛素清除的调节及其在T2D的病理生理学中的作用提供新的机械性见解。此外，我们的工作将为基于胰岛素清除操作的T2D的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.