Pilot Feasibility & Study - Current Recipients

Ingolf Bach, Ph.D.
Associate Professor
Program in Gene Function and Expression
FUNDING PERIOD: April 1, 2008 thru present

TITLE OF STUDY
Roles of nuclear LIM cofactors for pancreas development

PILOT RESEARCH SUMMARY:
The successful production of β cells in vitro requires a thorough understanding of the molecular networks that direct normal β cell development. Isl1 is a LIM homeodomain (LIM-HD) transcription factor that plays crucial roles for the development of β -cells. However, little is known on how Isl1 is regulated during pancreas development and the identity of its target genes. LIM-HD cofactors CLIM and SSDP1 form complexes with and regulate the biological activity of Isl1 in the nervous system. Our preliminary analysis of expression and function of CLIM and SSDP1 indicates a differential recruitment of these cofactors by Isl1 in the developing zebrafish pancreas when compared with neuronal tissues, suggesting that Isl1 is regulated by distinct mechanisms in different cell types. The proposed research project uses overexpression and morpholino experiments early during zebrafish development in combination with the identification of target genes via microarray analysis to elucidate protein networks and molecular mechanisms that direct normal β cell development.

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Eric Huseby, Ph.D.
Assistant Professor
Department of Pathology
FUNDING PERIOD: April 1, 2008 thru present

TITLE OF STUDY
Do auto-reactive CD8 T cell responses to non beta cell proteins within the pancreatic induce Type 1 diabetes?

PILOT RESEARCH SUMMARY:
The overall goal of this grant is to identify and characterize CD8 T cells specific for neuronal proteins expressed in the pancreas and to determine if the in vivo activation of these neuronal CD8 T cells induce Type 1 diabetes. Our hypothesis is that CD8 T cells specific for proteins expressed by neuronal cells of the pancreas can induce -islet cell death and initiate T1D.  To test our hypothesis, we are identifying CD8 T cells that target Schwann cells and peri-islet Schwann cells of the pancreas, and determine the ability of these CD8 T cells in inducing T1D.   We have currently isolated and characterized novel CD8 T cells specific for MAG, MOG, GAD65 and GFAP.  If an additional year of funding from the UMass DERC Pilot and Feasibility program is obtained, our focus for the following next year will be to determine whether these CD8 T cells are capable of inducing T1D.  Because the targeting of neuronal cells by T cells occurs in T1D patients and because T1D patients have an increased risk to develop Multiple Sclerosis, auto-reactive T cell responses specific for nervous system and endocrine system proteins appear to be linked.  Identifying whether neuronal protein specific CD8 T cells are capable of inducing T1D will determine whether the targeting of neuronal cells in T1D patients can have a causative impact on T1D disease progression or whether the expansion of these T cells in T1D patients is a characteristic of a general breakdown in immune tolerance mechanisms.

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Olga Hardy, M.D.
Assistant Professor
Department of Pediatrics/Endocrinology Division
FUNDING PERIOD: April 1, 2009 thru present

TITLE OF STUDY
The role of adipose inflammation in the metabolic sequelae associated with childhood obesity

PILOT RESEARCH ABSTRACT:
Obese children of all ages have evidence of a low-grade chronic inflammatory state.  The inflammatory response in obesity is predominantly a function of macrophages which are recruited into adipose tissue by chemokines and are responsible for a significant proportion of circulating cytokines.  Studies in adult patients provide evidence that systemic circulation of these cytokines may play a role in obesity-related comorbidities. Little is known about this association in children.  The study of inflammation coupled with excessive adipose tissue in childhood obesity is vital to understanding obesity and modifying its impact.
Children with similar degrees of obesity may manifest markedly different metabolic consequences.  We hypothesize that a subset of obese children with low-grade inflammation who are at high risk for obesity-related co-morbidities will be detected by biologic and genetic factors. 
This longitudinal pilot study will evaluate 15 healthy obese patients and 15 obese patients with at least 2 features of metabolic syndrome (MS).  Fasting serum (adipokines, cytokines and chemokines) and adipose biopsy samples obtained via needle aspiration will be obtained at baseline, 6 months and 12 months later.  Adipose tissue will be processed for histology, mophometry and gene expression analysis.  We anticipate that a high-risk subset of children will have (a) increased adipocyte cell size, (b) elevated chemokine expression in adipose tissue, (c) elevated levels of circulatory chemokines resulting in macrophage and T cell infiltration into adipose tissue and (d) obesity-related comorbidities including insulin resistance, dyslipidemia, and hypertension. Gene expression analysis and other outcome measures will be stratified by clinical status: (a) worsening metabolic syndrome, (b) improvement in metabolic syndrome, (c) no change in metabolic syndrome.
Identifying early surrogate markers of macrophage infiltration in adipose tissue in children opens new perspectives in the research of the pathophysiological mechanisms involved in the development of obesity comorbidities.  This study offers the unique opportunity to study both the mechanism of obesity-related inflammation and as pilot data to develop studies targeting therapy to a subset of youth at risk for serious co-morbidities. 

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Joonsoo Kang, Ph.D.
Assistant Professor
Department of Pathology
FUNDING PERIOD: April 1, 2009 thru present

TITLE OF STUDY
Determination of the function of diabetes-susceptibility gene Ctla4

PILOT RESEARCH ABSTRACT:
Type 1 Diabetes (T1D) is a T cell-mediated organ-specific autoimmune disease that targets the destruction of insulin producing -islet cells in the pancreas. The genetic basis of T1D has been intensely investigated and in rodents more than 20 T1D-susceptibility genes have been mapped, with MHC polymorphisms providing the best predictive measure of risk in humans and mice. Ctla4 is the only other T1D susceptibility gene common to humans and mice. It encodes for a protein belonging to the T cell costimulatory family whose expression is induced upon T cell activation and functions to rein in unfettered T cell proliferation and effector activities. CTLA-4 is also expressed constitutively on regulatory CD4+ T (Treg) cell subset that expresses the transcription factor FOXP3 and has been suggested to be critical for its function in vivo. Mice lacking CTLA-4 or FOXP3 succumb to multi-organ failure caused by unchecked self-reactive T cell expansion and infiltration into tissues and die before 4 weeks of age. So far, humans lacking CTLA-4 have not been identified, whereas Foxp3 null mutations are observed in patients with Immunodysregulation, Polyendocrinopathy, Enteropathy, X-linked (IPEX) syndrome.
Given our current knowledge of CTLA-4 function and where it is expressed, how malfunctions in CTLA-4 can lead to T1D was conceptually easy to grasp. However, exactly when and to what extent aberrations in CTLA-4 function can specifically lead to T cell mediated destruction of the -islet cells are unresolved. Much of this uncertainty exists from the current difficulty in dissecting where and when CTLA-4 functions in normal individuals to maintain T cell homeostasis. It has been particularly difficult to study CTLA-4 functions in conventional effector T cells independent of Treg cells in vivo since manipulations of the latter subset result in profound dysregulation of T cell homeostasis and early mortality. To overcome these hurdles we have generated several mouse models that target CTLA-4 expression to distinct T cell subsets. Using these new animal models, we have learned that CTLA-4 is absolutely required on Treg cells to maintain naive T cell homeostasis (Friedline, R., Chambers, C. and Kang, J., manuscript in press, J. Exp. Med). Further, although C57BL/6 mice expressing functional CTLA-4 in effector T cells, but not in Treg cells, are replete with activated self-reactive T cells, no T cell infiltration or expansion in non-lymphoid organs are observed, indicating that CTLA-4 is required in aberrantly activated T cells to prevent organ destruction. As a result, mice that contain CTLA-4-deficient Treg cells live at least 8-12 months instead of 3-4 weeks for Ctla4-/- mice, permitting more detailed studies of CTLA-4 function in vivo (Jain, N., Chambers, C. and Kang, J., revised manuscript submitted, J. Exp. Med). Critically, we have identified two tissues refractory to CTLA-4-mediated protection: the gut and the pancreas, resulting in enteritis and insulitis, but not full-fledged diabetes. Based on these results we hypothesize that CTLA-4 is required on Treg cells to regulate naïve T cell activation and CTLA-4 functions distinctly in effector T cells to prevent the accumulation of aberrantly activated self-reactive T cells in non-lymphoid organs. Our data suggest that the pancreas is especially dependent on CTLA-4 to prevent self-reactive T cell activation and infiltration into tissues. Using the newly developed series of mice in which CTLA-4 function can be altered in a cell type-specific and temporally controlled fashion we propose to model diabetes in a CTLA-4-dependent manner in diabetes resistant B6 mice by introducing the MHC allele of diabetes-susceptible NOD, H-2g7, in B6 mice. The proposed studies will for the first time determine the contribution of CTLA-4 in distinct T cell subsets and at different stages of T1D progression to maintain T cell homeostasis so that therapies involving modulations of costimulatory signals or Treg cells to treat T1D can be more rationally formulated.

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Norman Kennedy, Ph.D.
Assistant Professor
Program in Molecular Medicine
FUNDING PERIOD: April 1, 2009 thru present

TITLE OF STUDY
Mechanisms of Insulin Resistance

PILOT RESEARCH ABSTRACT:
Insulin resistance is characterized by dysregulation of glucose homeostasis and often portends the development of type 2 diabetes.  While substantial research efforts have defined insulin signaling pathways, the molecular mechanisms that promote insulin resistance remain poorly understood.
Accumulating evidence suggests that the c-Jun N-terminal kinase (JNK) pathway may contribute to the development of insulin resistance.  For example, JNK activity is elevated in tissues under conditions of insulin resistance.  Additionally, JNK inhibits signaling through the insulin receptor.
However, our understanding of the involvement of JNK proteins in the induction of insulin resistance is incomplete.  In particular, previous studies have focused the analysis of Jnk1 knockout mice.  Since JNK1 is deleted globally during embryogenesis, results are complicated by the potential of abnormal development in the absence of JNK1 or by the possibility that insulin responsiveness in one tissue is affected by Jnk1 deletion elsewhere.
This proposal aims to examine the role of JNK in the development of insulin resistance through the analysis of mice that conditionally lack expression of JNK proteins in fat. To accomplish this aim, we have generated RosaCreJnk1f/fJnk2-/- mice.  Administration of 4-hydroxytamoxifen causes deletion of Jnk1 in vitro or in vivo and allows for the analysis of insulin resistance in tissues that acutely lack expression of JNK proteins.
A further goal of this project is to uncover molecular mechanisms used by JNK to promote insulin resistance in adipocytes.   I propose to accomplish this goal through the use of a chemical genetics approach to identify substrates of JNK in adipocytes.  This strategy uses a “shokatized” JNK and N6-modified thio-ATP to tag JNK substrates for identification using mass spectrometry.  The identification of JNK substrates in adipocytes may reveal novel aspects of JNK regulation of insulin signaling that may lead to the development of new therapeutic strategies to treat diabetes.

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Joel Richter, Ph.D.
Professor
Program in Molecular Medicine
FUNDING PERIOD: April 1, 2009 thru present

TITLE OF STUDY
CPEB Control of Insulin Signaling

PILOT RESEARCH ABSTRACT:
CPEB is a sequence-specific RNA binding protein that regulates cytoplamsic polyadenylation-induced translation.  Through its regulation of RNA 3’ end formation and translation, CPEB mediates germ cell and embryo development, neuronal synaptic plascticity and learning and memory, and cellular senescence.  To further investigate CPEB function in cellular senescence, we made mouse embryo fibroblasts (MEFs) from WT and CPEB knockout (KO) mice, and centrifuged extracts from the cells through polysome sucrose density gradients.  RNA from the polysome (translating) fractions of the gradients was used to screen microarrays.  We noticed immediately that several RNAs encoding proteins involved in the insulin signaling pathway were mis-regulated in the KO; they were IGF1, IGF2, IGF2 receptor, AKT, PTEN, and SOCS3, among others.  Interestingly, all of these RNAs contain binding sites – CPEs – for CPEB in their 3’ UTRs, indicating that they may be directly regulated by CPEB.  At the protein, we have examined phospho-AKT, which is elevated by about two fold in the KO cells.
To assess whether CPEB KO mice might have altered insulin expression and/or signaling, we have results from preliminary experiments performed with Jason Kim:  CPEB KO animals have about two fold high insulin compared to WT, a glucose tolerance test shows there to be ~50% more glucose in serum of KO vs WT, and the KOs have about 22% more body mass than WT.
We hypthesize that CPEB regulates the translation of several mRNAs whose products are intermediates in the insulin signaling pathway, and that CPEB knockout mice, when challenged with the appropriate diet, will become diabetic.  We propose to test two hypotheses:

1. That CPEB regulates the polyadenylation and translation of mRNAs encoding IGF1 and 2, IGF2 receptor, AKT, PTEN, and SOCS3
2. That CPEB knockout mice become diabetic when fed a high fat diet

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Jie Song, Ph.D.
Assistant Professor
Department of Orthopedics
FUNDING PERIOD: April 1, 2009 thru present

TITLE OF STUDY
Synthetic bone grafts promoting the repair of diabetic bony lesion

PILOT RESEARCH ABSTRACT:
Advances in the detection and treatment of diabetes mellitus (DM), combined with a growing, aging and more obese population, mean more people living with diabetes in the future. It is well documented that diabetic patients are at a 90% higher risk for bone fractures, and diabetic bony lesions are significantly harder to heal than normal ones. Although the specific mechanism for the delayed healing in patients with DM has yet to be elucidated, recent evidence suggests that the lack of insulin may have adversely affected the local expression of growth factors mediating the normal fracture healing.
We hypothesize that the repair of diabetic bony lesions can be stimulated by the localized release of insulinlike, angiogenic and osteogenic growth factors from synthetic composite bone grafts that are implanted to the site of defect. The bioactive polymer-hydroxyapatite composite grafts are designed to provide immediate structural support to the site of defect and to release recombinant protein therapeutics BMP-2/7, IGF-1 and VEGF to promote the recruitment, adhesion and osteognic differentiation of osteoprogenitor cells. The performance of the synthetic graft in promoting the healing of a 5-mm femoral segmental defect in diabetic rats (BB Wistar diabetic rats) will be carried out. The extent of the remodeling, vascularization and osteointegration of the bone graft as a function of graft composition will be analyzed by histology, microcomputed tomography, and torsion tests.
We expect to complete the preparation and in vitro characterization of the grafts in the first 3 months. This will be followed by the proposed in vivo study in diabetic rats. The successful development of synthetic bone grafts possessing the structural and biochemical microenvironment that enable immediate stabilization and expedited healing of diabetic bony defects can offer an exciting new strategy in the treatment of this
debilitating commobidity of DM.

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Eric Baehrecke, Ph.D.
Professor
Cancer Biology Department
FUNDING PERIOD: April 1, 2009 thru present

TITLE OF STUDY
Function of Autophagy in Nutrient Homeostasis

PILOT RESEARCH ABSTRACT:
Autophagy is a conserved catabolic process that is used by all cells, and is associated with both cell survival and cell death. We are studying steroid-activated autophagy during development of Drosophila, and are using dying larval tissues as a model. Our hypothesis is that autophagy occurs during developmental cell death as a mechanism to obtain metabolic substrates to complete this cell autonomous process, as well as to provide resources to promote organism development and nutritional homeostasis. We have shown that growth arrest that is regulated by class I PI3 Kinase/TOR signaling is required for autophagy and dying salivary gland degradation, and that autophagy is required for cell degradation. In addition, the death of this tissue is activated by a rise in steroid hormone that regulates known autophagy genes. Therefore, both growth factor withdrawal and steroids contribute to the regulation of autophagy, but the role of autophagy is a mystery. Drosophila larvae spend 3.5 days eating and growing, and then spend the following 3.5 days of metamorphosis destroying larval tissues and building a new adult body without any external nutrients. We believe that this life history and the strength of Drosophila genetics presents the best model for this work at this time, including limited genetic redundancy, available mutations in key conserved genes, as well as steroid- and insulin- regulated autophagy that is associated with a natural part of fly life history during development. Here we propose to determine: (1) the influence of autophagy on energy utilization during development, (2) the role of autophagy in dying larval tissues during development, (3) the influence of mutations in autophagy genes on animal reproductive fitness. The conservation of the pathways that regulate autophagy, cell growth, metabolism and cell death indicate that discoveries in flies will be directly relevant to human health, including diabetes.

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Gregory Martens, Ph.D.
Instructor
Department of Medicine, Division of Pulmonary Medicine
FUNDING PERIOD: April 1, 2009 thru present

TITLE OF STUDY
Influence of Hyperglycemia on Host Defense in Pneumonia

PILOT RESEARCH ABSTRACT:
Increased susceptibility to infectious diseases including pneumonia is a major cause of morbidity and mortality for persons with diabetes mellitus. Pneumonia related hospitalizations have been increasing over the past ten years in the United States of American and there is a dramatic global rise in the incidence of diabetes. Further, the relative risk of diabetic patients with pneumonia being hospitalized ranges from 1.2 to 1.7 with patients with the poorest glycemic control (A1c >7%) having the highest risk. Despite the clinical importance of this phenomenon, remarkably little is known about the mechanisms leading to compromised immunity in diabetes. The goal of this study is to characterize the effects of hyperglycemia on pneumonia susceptibility using a mouse animal model. Hyperglycemia will be induced in mice by streptozotocin treatment and the relative resistance of normoglycemic and hyperglycemic to low-dose aerosol (250 CFU) infection with K. pneumoniae compared. Mice with different durations of hyperglycemia (1 or 3 months) will be challenged as advanced glycation end (AGE) products that have been associated with altered neutrophil, macrophage and T cell function accumulate over time. We will analyze innate and Th17 immune responses to K. pneumoniae that are essential for protective immunity. This well-characterized animal model of infection will then be used as a platform to test the hypothesis that heterologous activation of receptors for advance glycation end products on neutrophils, macrophages and T lymphocytes is responsible for impaired protective immunity to pneumonia in diabetes.

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Claire Standen, Ph.D.
Instructor
Program in Molecular Medicine
FUNDING PERIOD: April 1, 2009 thru present

TITLE OF STUDY
Role of JIP1 in Diet-induced Obesity and Insulin Resistance

PILOT RESEARCH ABSTRACT:
In mice administered a high fat diet, JNK1 and its scaffold JIP1 have been demonstrated to be essential for obesity and the subsequent development of insulin resistance (1, 2). The research to be performed within the scope of this Pilot and Feasibility grant will help to further elucidate the mechanism of JIP1 function during dietinduced obesity and insulin resistance. This will provide a strong foundation for future research, possibly leading to the development of new targets for pharmaceutical therapies. Specifically, the research to be undertaken will address the following aims:

I) Establish the mechanism by which JIP1 influences diet-induced obesity and insulin resistance. Is the function of JIP1 simply to act as a scaffold, potentiating the JNK signal by direct interaction with JNK? Does JIP1 take part in transporting the JNK signaling module, or other proteins involved in metabolic signaling, to specific sub-cellular locations, for example, via the interaction of JIP1 with kinesin? I will test these hypotheses using mice with targeted point mutations in the Jip1 gene.
II) Identify the tissue(s) in which JIP1 acts to regulate obesity and insulin resistance. I will examine the role of JIP1 using mice with conditional (floxed) Jip1 and expression of Cre recombinase in specific tissues.

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