Objective
One strategy for the treatment of T1D involves the stimulation of β-cell growth. To date, inhibiting the activity of elastase has been recognized as a successful method to stimulate mouse β-cell proliferation in cultured pancreatic islets. Despite these studies, the fine molecular mechanisms that links the inhibition of elastase activity and proliferation of β-cells still need to be clarified also in human samples, in order to consider antidiabetic therapies for treating T1D patients. The principal objectives of our proposal are to examine the effects of blocking pancreatic elastase (PE) activity in rat islets and to explore the optimum mode of administration in a rat model of type 1 diabetes to regulate β-cell proliferation and functionality, and define the safety profile of the compound for safe use in vivo. To address the aims we will treat isolated rat islets with the compound. Subsequently we will administer the compound by different routes to define the optimum dose to promote survival of beta cells. Finally we will administer very high dose of the compound in a rat model of type 1 diabetes to examine the safety profile determine off target effects. These data will inform planning future studies to assess whether blocking PE activity could prevent the development of T1D in humans.
Background Rationale
Patients with T1D have defective immune system cells, which attack and kill pancreatic endocrine cells. These individuals develop childhood-onset endocrine pancreas insufficiency, resulting in deficiency of insulin, the only known hormone, normally secreted after a meal that activates cells to take up glucose from the blood circulation, in order to maintain normoglycemia. The effects of lack of insulin lead to a persistent hyperglycemic state that requires T1D patients to be injected with exogenous insulin after every meal. Restoring the population of insulin-secreting cells (β-cells) is one of the major research aims to plan improved therapies for T1D. Several groups of scientists have identified multiple molecular mechanisms that can be modulated to stimulate β-cell proliferation. Among these, a role for SerpinB1 in regulating β-cell growth has been demonstrated. SerpinB1 is a protein that blocks the activity of enzymes that digest proteins, called proteases, such as pancreatic elastase (PE). SerpinB1 is overexpressed by the liver in conditions of overt insulin resistance, a typical state of patients with diabetes, and secreted in the systemic circulation. The delivery of SerpinB1 within the pancreatic islets causes a decrease in PE activity, consequently promoting β-cell proliferation. The link between elastase activity and diabetes was proved also by other studies, which reported that patients affected by genetic forms of diabetes characterized by pancreas dysfunctions, have altered PE levels in the blood. Since treating pancreatic islets with an established PE inhibitor, called sivelestat, led to an increase of proliferation of β-cells comparable to the SerpinB1 effect, we hypothesized that inhibiting the activity of PE within the islets represents an optimal strategy to induce regeneration of human insulin-producing cells and treat diabetes in patients. For this purpose, we identified telaprevir, an antiviral drug, as a potent PE inhibitor, after a screening within libraries of chemical compounds. We lately found that telaprevir was able to promote β-cell proliferation in human islets. However, the effect of telaprevir in restoring normoglycemia in animal models transplanted with human samples needs to be clarified and further investigations are needed to identify the molecular mechanism(s) that link PE activity with β-cell proliferation. This proposal warrants the assessment of the effects of blocking PE on human β-cell proliferation, survival and secretory function in animal models of T1D, using the compound identified in a screening assay as a PE inhibitor.
Description of Project
Diabetes is a severe chronic disease affecting millions of people worldwide. This disease exhibits when the body is unable to produce functional insulin to maintain blood sugar in the normal range. The most common forms of diabetes include type 1 and type 2 diabetes. While the latter is mostly caused by a defective response to insulin by different organs of the body, causing hyperglycemia and affecting mainly the adult population, type 1 diabetes (T1D) occurs in young individuals because of early β-cell failure and consequently poor or null production of insulin. Indeed, the pancreatic cells that are deputies to produce insulin, called β-cells, are attacked and disrupted by defective immune cells that recognize β-cells as the target. Regeneration of β-cells and the enhancement of insulin secretion in patients affected by T1D, are some of the most challenging strategies towards fighting diabetes. So far, these strategies have been carried out in animal models studies, which discovered different genes and proteins whose expression or activity could be regulated to promote β-cell growth. Among these, SerpinB1, an inhibitor of pancreatic elastase (PE), has been identified as an enhancer of human β-cell proliferation. Despite these findings, the exact mechanism by which blockade of PE, through inhibitors as SerpinB1, regulates human β-cell regeneration is still unclear. Furthermore, additional investigations are needed to evaluate the potential of inhibitors of PE in restoring normal blood glucose levels in patients with diabetes. For these purposes, we will use a compound as an inhibitor of human PE activity, identified through a screening of collections of chemical compounds aimed to discover PE inhibitors. This project aims to 1) evaluate the effects of a PE inhibitor on viability, proliferation and function of rat islet, 2) identify the appropriate dose of the PE inhibitor that can effectively modulate beta cell viability and proliferation of rat islets, and 3) define the toxic and side effects due to off targets of the PE inhibitor when administered at a high dose in the rats. These observations will be critical for developing treatment strategies to prevent and/or cure T1D.
Anticipated Outcome
The availability of a specific PE inhibitor will give us the opportunity to gain more insight about the effects of PE inhibition. In particular, the application of this drug on rat islets will provide more data about the regulatory mechanisms on β-cell proliferation. The subsequent experiments proposed in this study have the potential to define the optimum route of administration and list the off target effects to insure the safety profile of the compound. This study also has the potential to make a significant contribution to the field by demonstrating the role of PE inhibition in promoting β-cell secretory function and regeneration in rescuing affected rats from T1D.
Relevance to T1D
The studies in this project are directly related to therapies aimed at preventing and/or curing type 1 diabetes (T1D). One of the principal strategies to compensate for the gradual β-cell death typical of T1D is regulating cellular processes to stimulate β-cell growth. A continuing challenge in the treatment of T1D is to prevent β-cell death while also promoting β-cell proliferation with effective results. Among the pathways and molecular mechanisms that regulate β-cell survival, the role of the PE is not fully investigated. The availability of a human PE inhibitor provides us with an exclusive occasion to gain insights into the relevance of modulating PE activity in rat model that will allow future studies aimed at the regulation of human β-cell survival and proliferation in the context of restoring functional β-cell mass and insulin secretion in T1D.