Objective
While our previous findings suggest the potential for a therapeutic application of APOA4 in type 1 diabetes, the mechanisms underlying its anti-hyperglycemic effects remain largely unknown. The objectives of this application are to determine APOA4’s anti-hyperglycemic action in pancreatic β cells and to elucidate the underlying mechanism(s) in autoimmune diabetic mice.
Background Rationale
Type 1 diabetes (T1D) results from insulin insufficiency due to islet death and dysfunction following T cell-mediated autoimmune attack. For nearly a century, exogenous insulin has been the mainstay of therapy in T1D. While it is effective, insulin therapy has its shortcomings. For example, it requires complex injectable regimens that can result in hypoglycemia. In fact, less than 33% of Americans with T1D consistently achieve target blood-glucose control levels. Additionally, weight gain is a common side effect for people who take insulin. Therefore, there is a persistent need to find additional therapeutic options for patients with T1D that may enhance effectiveness, improve tolerability, and/or decrease the frequency of chronic complications.
Our laboratory has shown that apolipoprotein A4 knockout (apoA4 KO) mice are glucose intolerant and secrete less insulin in response to a glucose challenge, both of which could be restored by exogenous APOA4. In contrast, an increased level of APOA4 in apoA4 transgenic (apoA4-Tg) mice, after streptozotocin (STZ) treatment to induce a type 1 diabetic mouse model, resulted in significantly attenuated hyperglycemia, compared to wild type mice. More importantly, chronic administration of APOA4 to nonobese diabetic (NOD) mice, the gold standard model of human T1D, significantly delayed the onset of T1D, decreased the incidence of T1D, and reduced number of CD3+ T cells infiltrating into pancreatic islets. Unlike insulin, APOA4 administration does not induce hypoglycemia or weight gain in mice at its effective doses. While these findings suggest the potential for a therapeutic application of APOA4 in T1D, the mechanisms underlying these actions of APOA4 in T1D mice remain unclear. Thus, the objectives of this application are to determine APOA4’s anti-hyperglycemic action in pancreatic β cells and to elucidate the underlying mechanism(s) in T1D animal models
The proposed research is the first time to systemically study the mechanistic roles of APOA4 in pancreatic α and β cells of T1D mice. Supported by our previous studies and preliminary data, prolonged treatment with APOA4 is safe and not associated with liver or renal toxicity, as determined by plasma alanine amino transferase and creatinine levels (data not shown). Unlike insulin, APOA4 administration does not induce hypoglycemia or weight gain in mice at its effective doses (2). The rationale for the proposed research is that once the mechanisms of apoA4’s anti-hyperglycemic actions are understood, the key components of apoA4 signaling could be manipulated pharmacologically, leading to novel targets for the treatment of type 1 diabetes.
Description of Project
Type 1 diabetes is an autoimmune disorder and a major health concern in the U.S. and worldwide. According to the Juvenile Diabetes Research Foundation (JDRF), type 1 diabetes mellitus (T1D) afflicts 1.6 million people in the U.S. alone. Unfortunately, the incidence of T1D has been increasing at an alarming rate, and 5 million Americans are expected to have T1D by 2050. This corresponds to T1D-associated healthcare expenditures of $16 billion in the U.S. Once diagnosed, patients require lifelong insulin treatment and can experience insulin-associated complications. For example, hypoglycemia, a condition in which a patient’s blood glucose level is lower than the standard range, is a significant concern because the average patient with type 1 diabetes experiences two episodes of symptomatic hypoglycemia each week. Additionally, weight gain after insulin treatment is common, nearly half of type 1 diabetes patients treated with insulin become obese. These highlight the critical need for new therapies or modifications to existing insulin therapies.
Apolipoprotein A4 (APOA4), a protein produced in the intestine, plays important roles in lipid and glucose metabolisms. In our previous studies, we have found that mice lacking apolipoprotein A4 (apoA4 KO mice) are glucose intolerant and secrete less insulin in response to a glucose challenge compared with control wildtype mice. Administering exogenous APOA4 restores glucose tolerance and increases insulin secretion in apoA4 KO mice. In contrast, an increased level of APOA4 in apoA4 transgenic (apoA4-Tg) mice, after the treatment with streptozotocin (STZ), a cytotoxic glucose analogue that destroys pancreatic β cells, to induce a type 1 diabetic mouse model, resulted in significantly attenuated hyperglycemia, compared to wild type mice. Chronic administration of APOA4 to nonobese diabetic (NOD) mice, a polygenic model for autoimmune type 1 diabetes, significantly delayed the onset of diabetes and reduced the incidence of diabetes. However, the mechanisms underlying these actions of APOA4 in the type 1 diabetic mice remain unclear.
The objectives of this application are to determine APOA4’s anti-hyperglycemic action(s) in pancreatic β cells and to elucidate the underlying mechanism(s) in NOD mice. Because our recently published studies have demonstrated that APOA4 binds to Low-Density Lipoprotein Receptor Related Protein 1 (LRP1), a cell surface receptor, our central hypothesis is that APOA4 ameliorates hyperglycemia in NOD mice by suppressing autoimmune responses and promoting β cell survival through LRP1. This hypothesis will be tested in two specific aims. Aim 1 will determine the therapeutic effects of APOA4 administration alone or combined with low-dose insulin in NOD mice with hyperglycemia. Aim 2 will elucidate the underlying mechanisms by determining if APOA4 attenuates autoimmune responses in pancreatic β cells and promotes β cell survival through LRP1 in the NOD mice. The proposed research is highly innovative because it is the first time to focus on APOA4’s anti-hyperglycemic action in pancreatic β cells of a type 1 diabetic mouse model. The proposed work is significant because the findings from the proposed research may provide new preventative and therapeutic targets for the treatment of type 1 diabetes patients, without common insulin side effects.
Anticipated Outcome
Two specific aims are proposed to accomplish the objectives of the application.
Specific Aim 1 will determine the therapeutic effects of APOA4 administration alone or combined with low-dose insulin in NOD mice with hyperglycemia. Supported by our previous reports and preliminary data, we anticipate that 1) like insulin, chronic APOA4 treatment alone or combined with low-dose insulin will 1) ameliorate hyperglycemia, 2) improve glucose tolerance, 3) reduce plasma HbA1c and ketonuria, and 4) have less islets with severe insulitis and lower percentage of infiltrated islets in the NOD mice with hyperglycemia, compared to vehicle treatment. Additionally, unlike insulin monotherapy, APOA4 treatment is anticipated to reduce animal fat mass, as well as the levels of triacylglycerol, cholesterol, and free fatty acids in plasma and tissues (liver and adipose tissue). This is because APOA4 is a satiation factor, which reduces energy intake, and inhibits lipogenic transcription factors and enzymes, whereas insulin enhances lipogenesis. These studies are important because the outcomes could provide a promising potential option for APOA4 therapy alone or as an adjunctive therapy that could bestow comparable glycemic stability without the increase in body fat and up-regulation of lipogenesis observed with insulin monotherapy.
Specific Aim 2 will elucidate the underlying mechanisms by determining if APOA4 attenuates autoimmune responses and promotes β cell survival through LRP1 in the NOD mice. T1D is an autoimmune disease in which insulin-producing pancreatic β-cells are destroyed. We propose to use a unique animal model, the NOD mice with overexpressed APOA4 (apoA4-Tg+/+-NOD mice) to determine the actions of endogenous APOA4 in T1D development and to elucidate the underlying mechanisms. Supported by our preliminary data, we anticipate that apoA4-Tg+/+-NOD mice will have attenuated autoimmune responses, compared to control apoA4-Tg-/--NOD mice, specifically, the apoA4-Tg+/+-NOD mice will have 1) reduced percentages of CD4+ and/or CD8+ T cells in the pancreata, pancreatic lymph nodes, and/or spleen; and 2) increased percentage of regulatory T cells (Tregs) in the pancreata because the Tregs are essential suppressors of unwanted autoimmune responses. We also anticipate that APOA4 will protect β cells against the cytokine-induced cell death and at the same time promote β cell proliferation, specifically, 1) we expect that apoA4-Tg+/+-NOD mice will have an increased β cell area and increased insulin intensity per islet area, implying more β-cells per islet, and/or more insulin content per islet, compared to control apoA4-Tg-/--NOD mice; 2) we anticipate that apoA4-Tg+/+-NOD mice will have fewer apoptotic β cells, as indicated by the lower percentage of TUNEL staining in β cells, as well as an increased percentage of cells double-positive for insulin and Ki67 (a proliferation marker), suggesting that APOA4 promotes β cell proliferation in non-diabetic NOD mice, compared with those of control mice. Since LRP1 (Low-Density Lipoprotein Receptor Related Protein 1) is a new receptor and APOA4, and increased endoplasmic reticulum (ER) stress and cell death have been observed in Lrp1-dificient hepatocytes (7), we anticipate that APOA4 is able to protect against cytokine- and/or thapsigargin (a inducer of ER stress)-induced ER stress and β cell death, and these effects could be mediated by the LRP1.
Relevance to T1D
In this proposal, we propose to use a nonobese diabetic (NOD) mouse strain, which is a polygenic model for autoimmune type 1 diabetes (T1D) and the gold standard model of human T1D. Diabetes in this strain of mouse is characterized by hyperglycemia and insulitis, a leukocytic infiltration of the pancreatic islets. Marked decreases in pancreatic insulin content occur in females at about 12 weeks of age and several weeks later in males. Using this autoimmune mouse model, we will determine the therapeutic effects of APOA4 administration alone or combined with low-dose insulin in the control of hyperglycemia, and to elucidate the underlying mechanisms by attenuating autoimmune responses and promoting β cell survival through LRP1.
Additionally, the proposed research will generate several novel genetically modified mouse models, e.g., NOD mice with overexpressed APOA4 (apoA4-Tg+/+-NOD mice) and apoA4-Tg-/--NOD (for control) mice, as well as tissue-specific Lrp1 gene knockout (KO) mice, e.g., Lrp1 KO in pancreatic β cell (β-Lrp1-KO) mice. These unique animal models plus the sophisticated state-of-the-art experimental methods proposed in this application provide us powerful approaches to study the mechanistic role(s) and impact of APOA4 on cellular physiology of pancreatic β cells in T1D mice.
The objective of this proposal is to characterize apoA-IV’s beneficial action(s) in the control of hyperglycemia and to identify the underlying mechanism(s) in the pancreatic β cells of NOD mice. The proposed research is directly relevant to the JDRF mission to improve lives today and tomorrow by accelerating life-changing breakthroughs to cure, prevent and treat T1D and its complications.