Objective
This research proposal aims to investigate the significance and unravel the molecular mechanisms involved in enhancing insulin signaling in beta-cells as a potential therapeutic approach to increase beta-cell numbers to combat type 1 diabetes (T1D). The proposed methodology consists of several key steps. Firstly, I will assess the phenotype of a genetic T1D mouse model that selectively overexpresses insulin receptors solely in beta-cells. This analysis will encompass evaluating metabolic characteristics, islet morphology, and immune cell profiles in the mouse model. Secondly, I will delve into the mechanism(s) underlying beta-cell replication and survival in this specific mouse model, utilizing advanced techniques such as RNA-sequencing and phosphoproteomics. These investigations will focus on examining purified beta-cells and immune cells. Thirdly, to ascertain the relevance of these molecular mechanisms in humans, I will employ lentiviral gene delivery and CRISPR technologies to generate genetically engineered human beta-cell lines and human islets. These modified cells will be utilized to enhance insulin signaling and perform functional assays to evaluate their effects. Finally, I will transplant these modified cells into the kidney capsule of humanized mouse models with diabetes and monitor whether augmented insulin signaling can facilitate human beta-cell proliferation and survival.
Background Rationale
Type 1 diabetes (T1D), also known as juvenile diabetes, is characterized by a complete deficiency of insulin caused by the targeted destruction of beta-cells by self-reactive T-cells, leading to high blood sugar levels. The release of beta-cell autoantigens, such as glutamate decarboxylase (GAD) or insulin, triggered by genetic variations and/or environmental stress, initiates the death of beta-cells through apoptosis, contributing to the development of insulitis. Insulitis, a characteristic pathological feature of T1D, involves the infiltration of various types of immune cells into the islets of Langerhans, ultimately resulting in the selective destruction of insulin-producing beta-cells. As the loss of beta-cells reaches a critical threshold, the production of insulin becomes insufficient to regulate blood glucose levels, leading to hyperglycemia and the clinical manifestation of T1D.
Emerging evidence suggests that even individuals with long-standing type 1 diabetes (T1D) may retain residual beta-cells, as observed in the Joslin Medalist Study. This discovery indicates that beta-cell mass can undergo dynamic changes in response to physiological or pathological demands. Consequently, researchers have been exploring strategies to stimulate beta-cell proliferation as a means of compensating for beta-cell loss and addressing the absolute insulin deficiency characteristic of T1D. Recent studies have shed light on the pivotal role of beta-cells themselves in the development of T1D, revealing that the metabolic status of beta-cells influences the immunological self-tolerance of pancreatic islets. Notably, our group and others have recently demonstrated that inducing beta-cell proliferation or dedifferentiation prior to immune cell infiltration can modulate beta-cell autoantigen expression, decrease effector T-cell activity, and prevent the onset of diabetes in T1D mouse models.
These investigations present exciting possibilities for therapeutic interventions in type 1 diabetes (T1D), wherein the modulation of signaling pathways within beta-cells prior to the onset of insulitis may stimulate the regeneration of endogenous beta-cells. Building upon these prior investigations focused on T1D and insulin signaling in beta-cells, my objective is to explore the relevance of enhancing insulin signaling in promoting beta-cell proliferation and survival, particularly in the context of autoimmune-induced beta-cell death, and to elucidate its potential implications for T1D treatment.
Description of Project
Diabetes is a chronic and serious illness that hampers an individual's ability to effectively perceive, utilize, and store food. It is characterized by the body's inability to maintain normal blood glucose levels, either due to organs not responding adequately to insulin (as in type 2 diabetes) or insufficient insulin production by the body (as in type 1 diabetes). Both types of diabetes ultimately lead to the failure of beta-cells, impacting millions of people worldwide, with incidences significantly increasing since the onset of COVID-19.
Type 1 diabetes (T1D), also known as juvenile diabetes, is distinguished by early beta-cell failure, resulting in limited or no insulin production due to the targeted destruction of insulin-secreting beta-cells by autoreactive immune cells. Current therapies for T1D focus on administering exogenous insulin and/or performing islet transplantation. However, these treatments have limitations such as unpredictable hypoglycemia and graft malfunction or rejection by the immune system. Therefore, it is crucial to understand the biology of beta-cells and develop strategies that promote their survival for the treatment of T1D. These strategies should aim to increase the number of beta-cells, enhance their secretory function, evade immune rejection, counteract the loss of existing beta-cells (beta-cell regeneration), and explore the transplantation of insulin-producing beta-like cells derived from human embryonic or induced pluripotent stem cells (beta-cell replacement).
Despite decades of work, low replication rates of human beta-cells and a lack of knowledge regarding the molecular mechanisms by which beta-cells interact with immune cells in T1D, successfully increasing the number of human beta-cells in patients remains a challenge. In this context, our laboratory has been exploring the significance of proteins in the growth factor (insulin and insulin-like growth factor 1 [IGF-1]) signaling pathways for regulating beta-cell proliferation, survival, and function over the past two decades. We and others have also made progress in understanding the interplay between cellular signaling pathways, such as insulin and IGF-1 signaling, and cell death signaling events induced by cytokines or stress, highlighting their importance in beta-cell survival. A recent study that focused on a protein called “inceptor” has provided further evidence to support a role for insulin signaling in regulating mammalian beta-cell function and growth.
In this proposed study, I aim to build upon our previous work and investigate the molecular components involved in enhancing insulin signaling in beta-cells, thereby creating new therapeutic possibilities for increasing beta-cell numbers to combat T1D. Specifically, I will focus on characterizing the effects of overexpressing the insulin receptor in the beta-cells of a genetically modified mouse model of T1D. The objectives of this research are twofold: 1) analyze the metabolic characteristics, islet morphology, and immune cell profiles in the proposed mouse model, and 2) identify novel mechanisms underlying beta-cell replication and survival and their interplay with immune cells through advanced high-throughput techniques such as RNA-sequencing and phospho-proteomics in the mouse model. Additionally, to evaluate the relevance of these mechanisms in humans, I plan to generate genetically engineered human beta-cell lines and human islets, enhancing insulin signaling and conduct functional assays to examine cell proliferation, programmed cell death, and insulin secretion in response to glucose or hormone stimulation. Furthermore, I plan to transplant these cells into the kidney capsule of mouse models with humanized immune systems to observe whether enhanced insulin signaling can promote the proliferation and survival of human beta-cells in these animals.
Anticipated Outcome
Upon completion of this study, I will have obtained significant data regarding the importance of insulin signaling in increasing beta-cell numbers as a strategy to combat type 1 diabetes (T1D). Specifically, I will have successfully characterized a distinct genetic T1D mouse model that exclusively exhibits enhanced insulin signaling within beta-cells. It is anticipated that this mouse model will demonstrate heightened beta-cell proliferation, safeguarding of beta-cells against autoimmune assault, and prevent the development of T1D. Furthermore, through the utilization of this mouse model, I will have identified crucial gene(s) and protein(s) responsible for mediating the favorable effects of enhanced insulin signaling in beta-cell proliferation and survival. These findings will offer valuable mechanistic insights into the essential signaling pathway(s) involved in the interplay between beta-cells and immune cells. Lastly, I will have validated the translational significance of these gene(s) and protein(s) underlying the increased beta-cell numbers using a human beta-cell culture system and a diabetic humanized mouse model. This investigation will assess the feasibility of leveraging insulin signaling to enhance human beta-cell regeneration. The reagents and models generated throughout this study will be made accessible to the scientific community at large, facilitating the development of novel therapeutic targets for T1D prevention in humans.
Relevance to T1D
While the exact cause of type 1 diabetes (T1D) remains uncertain, the primary factor behind its development is the significant loss of pancreatic beta-cell mass due to immune attack by autoreactive T-cells. To address the absolute insulin deficiency in T1D patients, various strategies have been explored, including increasing beta-cell numbers, enhancing beta-cell function, counteracting beta-cell death (beta-cell regeneration), and transplanting insulin-producing beta-like cells derived from human embryonic or induced pluripotent stem cells (beta-cell replacement). Among these approaches, recent discoveries of growth factors and small molecules have highlighted the potential of modulating the cell cycle to stimulate the generation of new beta-cells (beta-cell proliferation). However, increasing beta-cell numbers in T1D patients remains challenging due to factors such as the low replication rates of human beta-cells and limited understanding of the molecular mechanisms underlying the communication between beta-cells and immune cells in T1D. Recent studies conducted by our group and others have demonstrated that inducing beta-cell proliferation prior to immune cell infiltration into the islets can diminish the activity of autoreactive T-cells, boost beta-cell numbers, and prevent diabetes in T1D mouse models. The research outlined in this proposal holds significant relevance for T1D patients as it seeks to identify effective strategies for inducing human beta-cell proliferation, thereby improving glycemic control and preventing the debilitating complications associated with the disease.