Awardees 2015

PI: Cristina Aguayo- Mazzucato, MD PhD
Abstract: β-cell aging markers and functional decline in type 2 Diabetes
This proposal focuses on understanding the beta cell aging process and how this contributes to the development of type 2 diabetes (T2D). T2D is related to aging and is partly characterized by dysfunctional insulin secretion. The direct interaction between age and altered beta cell function remains poorly understood but its importance is illustrated by a continued beta cell functional decline as diabetes progresses coupled with an inability of the beta cell population to regenerate. Such a lack of response to mitogenic stimuli is considered cellular senescence and has been linked to the expression of p16Ink4a. Therefore, a powerful new tool to study the role of aged cells is the INK-ATTAC (p16Ink4a mediated Apoptosis Through Targeted Activation of Caspase) mouse, which can be used to mark p16Ink4a -positive senescent cells with enhanced green fluorescence protein (EGFP) allowing their detection and collection. In addition, this cell population can be selectively cleared in the presence of the synthetic drug AP20187, which induces apoptosis in p16Ink4a-positive cells, both in vitro and in vivo. Our hypothesis is that with age, the senescent population of beta cells accumulates and contributes to the pathophysiology of diabetes. The overall goal of this project is to use INK-ATTAC mouse as a model to study beta cell aging by identifying markers of young and old beta cells, understanding their functional changes and determining how this process influences the progression of T2D.

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PI: Sean Burns, M.D
Abstract: Identifying diabetes genes regulating beta cell function using a pooled insulin secretion assay
Type 2 diabetes (T2D) is characterized by impaired insulin action and the failure of compensatory insulin secretion.  Determining the genetic pathways that regulate insulin secretion would bring us closer to a cure.  Recently, >85 common DNA variants have been identified through genome-wide association studies (GWAS) that increase risk for T2D, many of which appear to affect beta cell function.  While genes near these variants are likely to play a role in insulin secretion, for most implicated regions, the variants are noncoding, and the causal genes remain undefined.

Understanding which candidate T2D genes affect insulin secretion requires a systematic and comprehensive approach, in which the expression of each gene is perturbed in a beta cell model,and the effect on glucose response quantified.  As the number of potential causal genes near disease-associated variants is large (i.e., >1100 using conservative thresholds), attempts to assay the role of each gene in an arrayed format are impractical.

 

To overcome this bottleneck, we recently developed a fluorescent reporter of insulin secretion that enables pooled assays of beta-cell function.  Fluorescence of each beta cell expressing the reporter tracks closely with the amount of insulin secreted per cell, providing a potential means to identify and isolate those cells with altered secretion resulting from genetic manipulation.

 

To explore the feasibility of using this assay to perform genetic investigations of beta-cell function, I propose to:

  1. Validate the assay for use with a few genetic perturbations, focusing on positive control genes of known importance in regulating insulin secretion;
  2. Apply the assay to test the effects of silencing a small subset of candidate T2D genes harboring variants linked to insulin secretion in humans;
  3. Perform a pooled CRISPR/Cas9 nuclease screen of all 1169 candidate T2D genes within a 500kb radius of disease-associated variants.

 

Should the assay prove useful for testing the effect of genetic perturbations on insulin secretion, I intend to apply for an ADA Junior Faculty Award to comprehensively screen all candidate diabetes genes in human induced pluripotent stem (iPS) cell-derived beta cells.  As I have no current NIH funding, support from the BADERC to complete this pilot study will be will be vital to establishing sufficient preliminary data to be competitive for the ADA grant.

 

Relevance to public health

Identification of the genes that control insulin secretion in type 2 diabetes will help us to develop new targeted preventative and therapeutic measures for the disease.

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PI: Hans Dooms, Ph.D.
Abstract: Dysregulated cellular metabolism predisposes to Type 1 Diabetes
Type 1 Diabetes (T1D) is an autoimmune disease caused by T-cell mediated destruction of the b-cells in the pancreas, but the initial events leading to activation of islet-specific T cells in susceptible individuals are poorly understood. Environmental factors likely play an important role in the breakdown of immune tolerance that causes T1D but a convincing model linking the initiation of autoreactive T cell responses with an environmental insult is still missing. Our preliminary data reveal aberrant responses to fatty acids and inflammatory cytokine stimulation in fibroblasts and immune cells from human T1D patients and pre-diabetic NOD mice, suggesting an underlying metabolic predisposition to autoimmunity. Therefore, we will analyze metabolism in PBMCs from T1D subjects and immune cells from NOD mice. Metabolic defects may lead to inappropriate responses to elevations in cytokines and free fatty acids, for example during viral infection, ultimately triggering autoimmunity. The objectives of this proposal are (1) to determine whether immune cell types in T1D patients and NOD mice are broadly affected by the metabolic phenotype or if this re-wiring is limited to specific immune cell subsets; (2) to test if increased lipid accumulation, lipid peroxidation and Ca2+ signal transduction can be reversed in affected immune cells using bezafibrate, medium-chain triglycerides and catalase or glutathione peroxidase overexpression, and determine the functional significance of such interventions on autoimmune responses. Importantly, our novel concept has direct translational potential to develop new strategies for early diagnosis and prevention of T1D in at-risk individuals by measuring and correcting cellular metabolic parameters.

 

Relevance to public health

Type 1 Diabetes (T1D) is an autoimmune disease caused by the complex interplay of genetic, immunologic and environmental factors. We discovered that immune cells of diabetic patients and mice show dysregulated metabolic pathways. In this project we aim to explore how this altered metabolism promotes autoimmunity and to test whether interventions that correct cellular metabolism can be used as a novel therapy for T1D, a disease that is globally on the rise.

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PI: Lydia Lynch, Ph.D.
Abstract: Novel insight into the req uirement of adipose iNKT cells in the actions of GLP-1 analog therapy
My reason for choosing science as a career was to make a difference to human health. The major focus of my research is to understand the interface of the immune system and metabolism, especially in the setting of inflammation, and this has opened many new pathways for exploration in obesity, insulin resistance, type 2 diabetes. My specific long-term objectives are 1) to understand   the effects of obesity on the immune system which leads to poorer immune surveillance and increased risk of cancers and infections in certain obese individuals, and 2) to investigate the effects of the local adipose immune system on metabolic and weight control, 3) Develop therapeutic strategies to safely manipulate the immune system in obesity, type 2 diabetes and obesity-induced inflammatory disease. These goals are very timely as obesity and metabolic disorders continue to be a major human health burden which threatens to shorten human lifespan by 5-20 years.

Research
design and methods:
I have >8 years experience in studying the dysregulation of the immune system in obesity and inflammatory diseases in humans and mice, and in the effects of weight loss through diet intervention or bariatric surgery on the immune system (Lynch et al, Obesity, 2008, Eur. J. Immunology, 2009, Immunity, 2012, Nature Immunology, 2014). To achieve my research goals, I have begun to  develop state-of-the-art immunometabolic facility in Harvard Medical School to specifically study immune and metabolic systems   in vivo in obesity and insulin resistance models. These include Complete Lab Animal Monitoring System (CLAMS), MRI and calorimetry for animal studies which allows us to measure the effects of manipulating the immune system and inflammation in the adipose tissue and other organs, and on whole body metabolism. I have also developed novel ways to study leukocyte traffic in adipose tissue including parabiosis and 2-photon intravital microscopy in live adipose tissue, which allows us to visualize immune and adipocyte interactions in real time in vivo. I have established excellent collaborative relationships with clinicians and surgeons in Brigham and Women’s Hospital including bariatric surgeon Dr. Ali Tavakkoli and vascular surgeon Dr. Keith Ozaki, as well with  my mentor Prof. Michael Brenner, a leader in the field of iNKT cells, and Prof. Ulrich von Andrian, a world leader in leukocyte trafficking and intravital microscopy. Important for this application, I have established a collaboration with Prof. Daniel Drucker at University of Toronto, who is a leading expert in GLP-1 research. My next goal is to obtaining further funding from NIH, as well as the ADA and private organizations to lead and develop a research group in cutting edge science in immunometabolism.

Relevance to public health:
Over
1.4 billion adults and 40 million children under age 5 are overweight or obese worldwide, and obesity is a major risk factor for many serious diseases such as cardiovascular disease, diabetes, and cancer. Inflammation is an underlying cause or contributor to many of these diseases, and thus, preventing obesity-induced inflammation should be a key priority in tacking the obesity burden. This project will try to determine what starts this inflammation and if it can be lessened or prevented, by using or manipulating the bodies own immune system.

 
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PI: Kathleen J. Sweadner, Ph.D.
Abstract: Resistance to diabetes and hypertension in the Fxyd2 knockout mouse: role of AT2R
Diabetes and hypertension have long been known to be interrelated, but much remains to be understood about the metabolic links. Elevated angiotensin II produces hypertension and insulin resistance, and often precedes the development of Type 2 diabetes (T2D). Angiotensin blockers (ACE inhibitors and receptor antagonists) have been found to be beneficial in prevention of T2D in multiple clinical trials. The classic angiotensin type 1 receptor, AT1R, increases hypertension and metabolic phenotype, but the homologous type 2 AT2R receptor, which acts via different G proteins, opposes them. AT2R has been reported to be highly expressed in islets, and to be protective in streptozotocin challenge. Normal, healthy renal adaptation to excess salt includes a decrease in the functional prominence AT1R and an increase in the influence of AT2R, specifically in the proximal tubule. We have obtained surprising evidence that Fxyd2knockout mice are in a highly desirable state: resistant to salt- or angiotensin II-induced hypertension and to beta cell loss in streptozotocin challenge. FXYD2 is a regulator of Na,K-ATPase ion pumping, but Na,K-ATPase also has signaling roles and has been reported to associate with and regulate angiotensin receptors. Our hypothesis is that in the knockout, there is baseline activation the angiotensin type 2 receptor (AT2R), part of the angiotensin counter-regulatory pathway, and it is responsible for the “super mouse” phenotype in both tissues, islets and kidney. Islets and kidney, incidentally, have the highest levels of FXYD2 in the body, and possibly of AT2R. Preliminary qPCR data show a large increase in AT2R mRNA in the knockout islets. The aims will be to investigate the expression and regulatory status of the two angiotensin receptors in beta cell and proximal tubule tissue from WT and Fxyd2 knockout mice. Systems-level studies will entail infusion of WT and Fxyd2 knockout mice with angiotensin receptor-specific agonists and antagonists; the effect on phenotype will test the hypothesis. The physiological and signaling responses of isolated islets and renal slices in vitro will be studied with the same agonists and antagonists of AT1R and AT2R. Finally, immunoprecipitation will be used to investigate whether there is a direct association of Na,K-ATPase with either Ang II receptor in islets and kidney, and the influence of FXYD2. The work is expected to be predictive of mouse physiology, and to test the potential of FXYD2 as a therapeutic target.


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PI: Mark Yore, Ph.D.
Abstract: Identification pathways of enzymes involved in biosynthesis of a novel class of anti-diabetic lipids
We recently identified a novel class of mammalian lipids which have anti-diabetic and anti-inflammatory effects. These lipids are branched Fatty-Acid-esters-of-Hydroxy-Fatty-Acids (FAHFAs).  A member of this class, Palmitic-Acid-Hydroxy-Stearic-Acid (PAHSA) is present in serum and many mouse and human tissues. PAHSA levels in adipose tissue (AT) are regulated by Carbohydrate-response element binding protein (ChREBP). PAHSA levels are reduced in serum and tissues of obese, insulin-resistant versus lean insulin-sensitive mice. In insulin-resistant versus insulin-sensitive people, PAHSA levels in serum and AT are reduced and levels correlate highly with insulin-sensitivity. In insulin-resistant mice, PAHSA administration lowers blood glucose, stimulates GLP-1 and insulin secretion, and improves glucose-tolerance and insulin-sensitivity. In vitro, PAHSAs augment insulin-stimulated glucose transport in adipocytes through the G-protein coupled receptor, GPR120 and also potentiate glucose-stimulated insulin secretion. PAHSAs could potentially be administered as drugs, or the pathways that regulate them in vivo perturbed to increase their levels for therapeutic effects.

This proposal’s objective is to identify the pathways and biosynthetic enzymes that control PAHSA levels/biosynthesis. This will improve our understanding of their biology, the mechanisms by which PAHSA levels are reduced in insulin-resistant states and their therapeutic potential to treat Type 2 diabetes (T2D). These objectives are in alignment with BADERC’s mission to cure of T2D and associated complications. 

  • Aim #1: Investigate ChREBP’s role in controlling PAHSA levels and biosynthetic activity.
  • Aim #2: Identify pathways/enzymes involved in PAHSA biosynthesis. To achieve these aims, we’ll take a systematic genetic approach in combination with a biosynthetic activity assay developed by our lab.


 





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