Thursday, November 21, 2024
11/21/2024
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.
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