Henry M. Colecraft, PhD, Professor of Physiology and Cell Biology
Ca2+ influx through voltage-gated calcium channels (VGCCs) is necessary for glucose-stimulated insulin secretion in pancreatic β-cells (PβCs). VGCCs are thus a potential locus for both PβC-dependent pathophysiology and therapy. CaVβ subunits (β1-β4) are powerful regulators of VGCCs by controlling α1 subunit trafficking and tuning channel gating. A central unresolved question is: how does Ca2+ influx via VGCCs give rise to divergent functions in PβCs such as insulin secretion and excitation-transcription coupling? The PI hypothesized that in PβCs distinct CaVβs are instrumental in organizing VGCCs into discrete macromolecular complexes with specialized functions. A significant barrier to rigorously assess the functional roles of CaVβ molecular diversity in excitable cells, including PβCs, is the inability to inhibit VGCCs based on the identity of their resident CaVβ. The PI will develop novel genetically-encoded CaV channel blockers that enable inhibition of CaVβ-specific VGCC complexes, and apply them to decipher signaling functions of CaVβs in PβCs. The approach exploits a bioengineering method to generate genetically-encoded VGCC inhibitors by pairing the specificity of single-domain antibodies (or nanobodies) with the consequential catalytic activity of an E3 ubiquitin ligase. Using Cavβ isoform-selective nanobodies with molecular biology, electrophysiology, flow cytometry, fluorescence resonance energy transfer (FRET), ion channel engineering, and biochemistry, the PI will develop and engineer nanobodies to selectively inhibit VGCCs on the basis of their resident CaVβ, and elucidate the functions of CaVβ specific VGCC complexes in pancreatic β-cells.
Joshua Cook, MD, PhD, Instructor in Medicine
Bile acids (BA) have emerged as promising and manipulatable mediators of the dual threats posed by insulin resistance (IR) underlying type 2 diabetes: chronic hyperglycemia and cardiovascular disease. Levels of 12ahydroxylated bile acids (12-HBA) strongly correlate with measures of IR in humans and depletion of 12-HBA in animals improves glucose tolerance. However, hepatic IR also appears to cause increased 12-HBA in animals. Better understanding the mechanisms behind this apparent pathologic cycle in humans is essential
for developing novel cardioprotective diabetes treatments. We propose short-circuiting this cycle with two complementary patient-oriented research studies of growth hormone (GH) excess, a secondary cause of insulin resistance featuring both clear pathogenesis and reversibility, and its consequences for BA metabolism. First, we will prospectively study patients with acromegaly (“GH-oma”), measuring IR indices and BA profiles before and after surgical and/or medical treatment. Second, we will conduct a randomized, placebo-controlled prospective clinical trial in which we administer recombinant human GH (rhGH) to healthy volunteers in order to evaluate the impact of temporarily induced IR on BA metabolism – especially 12-HBA content. We will also assess the ability of the BA sequestrant colesevelam to mitigate rhGH-induced IR by offsetting adverse BA reprogramming. In each case we will perform our BA analysis using advanced mass spectrometric techniques capable of identifying >150 individual BA species, most as yet unstudied. These patient-oriented studies will yield new mechanistic insights into the direction(s) of causality in the relationship between IR and BA, particularly 12-HBA, with major implications for drug design.
Andrea Califano, PhD, Professor of Systems Biology
The aim of this proposal is to identify driver networks of pancreatic β-cell failure in humans, and perform an unbiased drug screen to test whether it is reversible by pharmacological treatment. In preliminary data, using a combinatorial approach of single-cell RNA sequencing (scRNA-Seq) with human T2D islets and computational regulatory network analysis, the PI identified potential master regulatory networks associated with diabetes-unique b-cell sub-populations. Using this information, the PI proposes to functionally test the effects of candidate master regulators using
a candidate gene perturbation screen in primary islets. This information will be used to carry out an unbiased screen of a limited subset of FDA-approved drugs in primary islets, using an integrated high-throughput screening (HTS)/RNA-seq platform (PlateSeq) for the high-multiplex, low-cost RNA-seq profiling of compound perturbations in cellular systems. The findings will increase our understanding of cellular heterogeneity in human T2D islets, determine driver networks that control cell fate transition, and begin to define actionable therapeutic pathways to intervene on the disease process.
Lina Sui, PhD, Assistant Professor of Pediatrics
The human genome contains a large number of genetic variants associated with disease risk, including for diabetes. However, for most variants, the functional consequence has not been demonstrated in human cells, and functional assays to evaluate the consequences in animal models are limited to conserved regions. The PI has collected a set of novel variants predicted to alter the function of proteins implicated in familial diabetes. She proposes to study variants in two genes TRIB3 and SEL1L for detailed characterization, because of their likely role in endoplasmic reticulum (ER) function. The ER is critical for proinsulin folding and insulin secretion from beta cells. There is increasing evidence that the presence of unfolded proteins is of central importance to the failure of beta cells in all forms of diabetes, including type 1 and type 2 diabetes. The PI will introduce mutations identified in patients into human pluripotent stem cells, and test their developmental and functional properties of iPS-derived beta cells in vitro as well as after grafting into mice in vivo. She has established an efficient experimental system for beta cell differentiation and grafting without the formation of teratomas that allows the functional study of human genotypes in a physiologically relevant environment. The proposed studies have the potential to identify two novel diabetes genes, and enable the study of mechanisms by which protein folding stress causes beta cells to fail. The proposed work is therefore relevant to all forms of diabetes.
Gordana Vunjak-Novakovic, PhD Professor of Biomedical Engineering
Goal of this project is to develop small 3D microphysiological systems devices that surpass currently available systems in physiological relevance using compartmentalized beta cells and circulating immune cells separated by a layer of endothelial cells. This system will be used to define the conditions that determine the recruitment of immune cells that mediate the sequential phases of autoimmune assault on beta cells. The PI’s hypothesis is that the earliest signals leading to the recruitment of immune cells originate form genome instability resulting from replication-transcription conflicts at highly transcribed genes, such as insulin, in highly proliferating cells. The PI proposes that activation of the cGAS-STING and TLR9 pathways by damaged DNA results in the production of chemokynes. Streamlined production of cellular components into miniature and personalized microphysiological devices will provide a powerful and affordable precision medicine platform to evaluate drugs and perform diagnostic testing.
Long non-coding RNAs in beta cell function
Luis Arnes, PhD, Assistant Professor of Pathology and Cell Biology
Considerable effort has been directed towards deciphering the molecular mechanisms that regulate β-cell formation, maturation and function. Past studies of pancreas development and β-cell function have largely focused on coding genes. In recent years, integrated analyses of islet-specific transcription factor binding sites, epigenetic modifications and gene expression profiles have uncovered the existence of chromatin domains and long non-coding RNAs (lncRNAs) with largely unknown functions. Notably, many of them are located in the genome nearby islet-specific genes, conserved across species and dynamically regulated by glucose concentration and throughout pancreas development. Although these features suggest a regulatory role in β-cell specification and function, few of them have been examined. The PI hypothesized that lncRNAs affect gene regulation in pancreas development and β-cell function. To test this hypothesis, he proposed to define the transcriptome profile of pancreatic lineages at single cell resolution using in vitro differentiation of human embryonic stem cells. Moreover, he will study the regulation and function of positionally-conserved lncRNAs in the specification of insulin-producing β-cells. The results from these studies will help generate alternative sources for β-cell replacement therapies.
Role of TTC39B in Mediating NAFLD in Insulin-Resistant States
Joanne Hsieh, PhD, Assistant Professor of Medicine
Non-alcoholic fatty liver disease (NAFLD) is very common in diabetes and insulin resistance and one feature is increased de novo lipogenesis (DNL). However, the mechanism for upregulated hepatic DNL is not well-understood. Deficiency in the scaffolding protein TTC39B results in protection from NAFLD in mice, associated with decreased hepatic lipogenic gene expression. TTC39B regulates hepatic lipogenic gene expression by interacting with and promoting the degradation of RB1. TTC39B also interacts with 14-3-3γ, which may sequester from the nucleus. Based on these observations, the PI hypothesizes that insulin resistance activates a phosphatase that releases TTC39B from 14-3-3γ binding, allowing TTC39B to enter the nucleus to degrade RB1, resulting in lipogenic gene expression. Aim 1 will use hepatic RB1-deficient mice to determine its contribution to hepatic lipogenic gene expression and NAFLD pathogenesis in vivo. RNA-Sequencing and ATAC-Sequencing analysis will be used to identify promoter and enhancer motifs that are affected in the TTC39B/RB1-dependent pathway. Aim 2 will use unbiased approaches to identify how TTC39B’s interaction with kinases and phosphatases change as insulin resistance and NAFLD develops. This study has the potential to identify a novel pathway that explains the upregulated DNL in insulin resistant individuals, and inform the development of new therapies to treat
Transcriptional regulation of pancreatic beta cell failure by single cell RNA-Seq
Submitter: James C. Lo, MD, PhD, Assistant Professor of Medicine, Weill-Cornell Medical College
This project examines the gradual decline of beta cell function in diabetes. There is evidence that transcriptional dysregulation of beta cells leads to beta cell failure. Pancreatic islets are composed of a heterogeneous group of specialized endocrine cells. Hence, traditional strategies of RNA profiling on bulk islet cells have likely not captured the complexity of individual pancreatic islet cells in normal physiology and especially the stresses of T2DM. The PI will assess single cell transcriptomes of ~10,000 pancreatic islet cells from healthy control and diabetic mice. He will use these data to map transcriptional networks perturbed in T2DM with an eye on identifying pathways disrupted in beta cell failure.
The Role of ER-Mitochondria Contacts in the AKT Signaling Pathway
Submitter: Estela Area-Gomez, PhD, Assistant Professor of Neurology
This project examines the link between insulin resistance and AKT-dependent mitochondrial signaling. AKT can translocate to the outer mitochondrial membrane (OMM), where it activates hexokinase-II (HK-II), the rate-limiting step of glycolysis, promotes its binding to the mitochondrial voltage-dependent anion channel (VDAC). Thus, localization of AKT to OMM regions bridges ER and mitochondria to regulate glycolysis and calcium transfer between ER and mitochondria. Areas of the ER in close apposition to mitochondria are referred to as mitochondria-associated ER membranes, or MAMs. MAMs are functional domains that regulate lipid metabolism and have been shown to modulate hepatic insulin action and AKT activation. This project will examine how targeting of AKT and its kinase mTORC2 to MAMs regulates hepatic insulin sensitivity, HK-II function, and calcium fluxes.
Hepatic Dach1 regulates lipid metabolism
Submitter: Lale Ozcan, MD, Assistant Professor of Medical Sciences, Department of Medicine
Individuals with type 2 diabetes have a 2–4-fold increase in their lifetime risk of cardiovascular diseases, arising from lipid abnormalities. The PI has identified a pathway activated by calcium/calmodulin-dependent protein kinase II (CaMKII) that inhibits insulin signaling. She went on to characterize the corepressor Dachshund homolog 1 (Dach1) as a key effector of the calmodulin-kinase pathway in liver. Hepatic Dach1 levels increase in obese mice and humans, and Dach1 inhibition protects against hyperinsulinemia and hyperglycemia. In addition, hepatocyte-specific Dach1 deletion leads to a decrease of plasma cholesterol levels. In this PF grant, the PI is investigating whether Dach1-mediated co-repression links insulin resistance with abnormal lipid and cholesterol metabolism.
The diabetic environment perpetuates Staphylococcus aureus infection
Submitter: Dane Parker, PhD, Assistant Professor of Microbial Pathogenesis [in Pediatrics]
The hypothesis of this study is that hyperglycemia supports S. aureus infection. To address this question, the PI established a diabetic mouse model of subcutaneous skin infection and will investigate whether diabetes affects (i) clearance, (ii) pathogenesis, and (iii) immune response to S. aureus skin infection. He has shown that diabetic mice have increased dermal necrosis and delayed resolution of infection in response to S. aureus infection. This work establishes a model to investigate S. aureus skin infection in the context of diabetes. Using this model, the PI will determine the effects of glucose on immune cell function and test the hypothesis that hyperglycemia inhibits the phagocytic and killing function of macrophages and neutrophils as well as their ability to properly invoke an inflammatory response. By using cells exposed to varying levels of glucose as well as cells isolated from control and diabetic mice he will investigate whether phagocytes have diminished bacterial killing capacity and reduced ability to induce inflammatory cytokines.
PPARγ Accumulation and Metabolic Decline in Aging
Submitter: Li Qiang, PhD, Assistant Professor of Pathology and Cell Biology
The association of aging with the metabolic syndrome remains unexplained. The PI proposes to investigate a novel mechanism linking insulin resistance with aging, with the aim to identify novel and safer interventions for age-associated metabolic diseases. PPARγ is a master regulator of adipocyte formation and function, and plays a pivotal role in metabolism and insulin action, but its role in aging is unknown. The PI demonstrated a remarkable increase of PPARγ in tissues from aging individuals and aging experimental animals, along with stark differences in the response to the PPARγ ligand thiazolidinedione (TZD) in young vs. aging animals. He hypothesized that the accumulation of PPARγ contributes to the metabolic decline during aging. He proposed to establish the physiological significance of PPARγ accumulation in aging and elucidate its molecular basis. The ultimate goal of this work is to identify new mechanisms connecting aging and metabolic syndrome and provide novel therapeutic candidates for delaying the onset of metabolic diseases in aging.
Redox regulation of diabetic vascular remodeling by s-glutathiolation
Submitter: Ziad Ali, MD, DPhil, Assistant Professor of Medicine Cardiology
Among the many potential catalysts for the pathophysiological manifestations of diabetes, overproduction of reactive oxygen species (ROS) appears to be a common upstream event. Protein S-glutathiolation, the reversible covalent addition of glutathione to cysteine residues on target proteins, is a candidate mechanism by which changes in intracellular redox state and generation of ROS can modulate protein function. The PI hypothesized that the increased ROS generation in type 2 diabetes promotes protein S-glutathiolation, in turn leading to neointimal hyperplasia, accelerated atherosclerosis, and revascularization failure. He identified two highly glutathiolated proteins, annexin A2 and Beta-actin. These proteins are involved in cellular processes critical to vascular remodeling, such as proliferation and migration. Moreover, recent data suggest that these proteins interact dynamically with one another. Thus, the PI is studying the physiological and cellular effects of S-glutathiolation in type 2 diabetes; identifying the pathways involved in S-glutathiolation-mediated phenotypes; and 3) establishing the role of S-glutathiolation as a therapeutic target to prevent diabetes-induced premature revascularization failure.
Differential Effects of Bile Acid Species in Intestine
Submitter: Rebecca A. Haeusler, PhD, Assistant Professor of Pathology and Cell Biology
Bile acids (BA) are produced in the liver from cholesterol to regulate several biological processes. A key site of BA signaling is the intestine, a tissue that secretes a multitude of peptide hormones that act on the pancreas, brain, and liver. At least two gut hormones are secreted in response to BAs: glucagon-like peptide-1 (Glp1), and fibroblast growth factor-19 (Fgf19). It’s unknown whether different BA species affect secretion of these hormones differently, or whether there are additional intracellular or secreted proteins targeted by BAs in intestine. The PI first demonstrated that different BA species have distinct effects on insulin sensitivity and lipid metabolism. In this work, she hypothesized that alterations in BA levels and composition affect gene expression and secretion of proteins in the intestine. She tested this hypothesis by determining the effects of BA levels and composition on intestinal gene expression using intestinal explants, oral gavage, and intestinal perfusion in mice, followed by tissue collection and transcriptional profiling. Moreover, she investigated the effects of BA levels and composition on the gut secretome using intestinal perfusion in mice, followed by mesenteric blood collection and proteomic analysis.
Deciphering and Visualizing Epitope Spreading in Autoimmune Diabetes
Submitter: Remi J. Creusot, PhD, Assistant Professor of Medical Sciences (in Medicine)
Type 1 diabetes results from T cell-mediated immune response against multiple Beta-cell antigens. Although few self-antigens appear to be involved initially, the autoimmune response spreads to other epitopes. Epitope spreading is a common but unexplained mechanism of autoimmune diseases. Mechanisms regulating T cell clonal selection and responsivity to tissue damade are unclear. The PI investigated whether successfully activated T cell clones can boost the immunogenic function of dendritic cells to stimulate low-affinity T cells in the context of “antigen linkage”. Using a novel polyclonal adoptive transfer model with traceable diabetogenic T cell clones, he tested whether linked cooperation can support low-affinity T cells activation. He evaluated how these diabetogenic T cells, with varying degrees of antigen responsiveness, are stimulated in vivo when transferred alone or as a mixed population and allowed to influence one another within DC clusters. The functional analysis of the activation of these different T cells will help design therapeutic strategies aimed at reversing this phenomenon using linked suppression by regulatory T cells.
The PI has published a paper using data from this PF grant. He has obtained 3 new grants, and is a co-investigator on another one. Data obtained through the PF grant were instrumental to the successful competition for two R21 grants. When asked about the role of the PF grant in his budding career, he wrote that: “The PF grant was instrumental to develop an in vivo system to co-express multiple beta cell antigens by antigen-presenting cells (APCs) and analyze the response of multiple antigen-specific T cell clones. We generated constructs producing up to 8 epitopes that can be recognized by T cell receptor-transgenic T cells. As mRNA, we used these constructs to study how signals delivered by APCs upon antigen engagement can cooperate to induce regulatory T cells. This led directly to an R21 grant. As DNA vaccines, we have delivered these constructs by various routes and under different formulations to improve in vivo delivery and tolerogenic presentation. This help has been invaluable in my career development.”
The AF10/DOT1 Complex, O-GlcNAc, and Transcription: From Nematodes to Mammals
Submitter: Alla Grishok, PhD, Assistant Professor of Biochemistry and Molecular Biophysics
Excess flux through the hexosamine biosynthesis pathway increases protein O-linked GlcNAcylation and contributes to insulin resistance. This reaction is catalyzed by O-GlcNAc transferase (OGT) and reversed by O-GlcNAcase (OGA). Mutations of OGA have been associated with type 2 diabetes. The PI showed that the C.elegans chromatin-binding protein ZFP-1 (AF10 in mammals) acts to reduce transcription of metabolic genes. O-GlcNAc modification on chromatin at active gene promoters has been implicated in transcriptional activation. In C. elegans, ZFP-1 co-localizes with and inhibits O-GlcNAc. The PI investigated whether O-GlcNAc modification at active promoters mediates glucose-responsive transcription and whether ZFP-1 and its major interacting partner DOT-1.1 provide feedback inhibition of O-GlcNAc. She evaluated levels of O-GlcNAc modification on chromatin in zfp-1 mutant by genome-wide ChIP-seq and the effect of O-GlcNAc on gene expression by RNA-seq analyses of zfp-1; ogt-1 and zfp-1; oga-1 double mutant worms. She also tested chromatin localization of AF10 and O-GlcNAc in 3T3-L1 murine adipocytes by ChIP-seq.
The PI has two papers in preparation describing the findings. She has applied for two NIH grants (1DP1OD023968-01 and 1R01GM124175-01) that did not receive a fundable score on first submission but have been resubmitted.
Stem cell-based model for HNF1A deficiency in human pancreatic beta cells
Submitter: Lina Sui, PhD, Associate Research Scientist, Department of Pediatrics
Stem cells can reflect disease-relevant phenotypes after in vitro differentiation and transplantation into mice. Though monogenetic forms of diabetes are rare (1-5% of all diabetics), they provide a model to test the utility of stem cells as an approach to Beta-cell replacement, and to better understand the mechanisms of Beta-cell dysfunction, as the same or related genes are likely to contribute to the risk of developing Beta-cell failure. Insight into the roles of MODY-causing transcription factors in the formation, survival, and function of beta cells can advance strategies for the prevention and mitigation of more prevalent forms of diabetes. Unlike humans, mice haploinsufficient for HNF1A show no diabetic phenotype, indicating that there are species-specific differences in genotype-phenotype relationship, and pointing to the importance of using human cells for these studies. The PI generated pluripotent stem cells from MODY3 patients and converted them into Beta-cells, then assessed HNF1A function using insulin production, storage, and secretion in vitro and in vivo. She also determined rates of proliferation and cell death and analyzed the transcriptome of MODY3 Beta-cells. These studies have contributed to establishing a platform to improve generation and testing of iPS and ESC-derived Beta-cells.
The PI has not yet published the findings of this PF project nor obtained new grants.
Enteroendocrine Specification in Drosophila and Vertebrates
Submitter: Benjamin Ohlstein, MD, PhD, Associate Professor of Genetics & Development and of Medicine
The intestine is the largest endocrine organ in the human body. Scattered throughout the human gut are enteroendocrine cells that secrete hormones involved in the regulation of various physiological processes such as digestion, intestinal motility, and glucose metabolism. The Drosophila adult intestine, like that of vertebrates, contains enteroendocrine cells that can be distinguished by staining with antibodies to the various hormones that they secrete. By analyzing RNA obtained from wild-type intestines, intestines lacking enteroendocrine cells, and intestines producing enteroendocrine cells in excess, the PI generated a set of candidate genes that regulate enteroendocrine differentiation and function. Remarkably, among these candidates are homologs of important determinants of pancreatic endocrine cell fate: Ngn3, Hes, Pax, and Ptf1. In addition to these homologs, the PI identified additional candidate mediators of enteroendocrine cell differentiations. He is currently involved in functional studies of genes so identified.
The PI has published 2 original papers and one review based on data obtained from this PF grant. The PF grant was also instrumental to the successful award of a new NIDDK R01 grant.
Intelligent glucagon delivery vehicle as adjunct for exogenous insulin therapy
Submitter: Milan Stojanovic, PhD, Associate Professor of Medical Sciences and Biomedical Engineering
This project pursued the development of a novel adjunct to exogenous insulin therapy for the management of hyperglycemia accompanying beta cell insufficiency associated with type 1 and type 2 diabetes. The PI developed nanoscale particles that deliver glucagon or insulin in a glucose- responsive manner and have therapeutically useful in vivo half-lives. Ultimate goal of this proposal is the production of a self-operating molecular machine with medical applications. The approach draws inspiration from the PI’s previous work in DNA nanotechnology and molecular computing. The hypothesis is that nano-objects can be used to sequester functions and that a structural change can be used to reveal new functions. As proof-of-principle the PI investigated glucose control in response to insulin. He created nano-eggs (neggs) composed of two half-shells that open and close in response to environmental glucose concentrations. Once opened, neggs release glucagon in response to falling glucose levels to help maintain euglycemia. This delivery vehicle, added to current insulin formulations, has the potential of circumventing the problem of hypoglycemia and can result in tighter glycemic control in diabetic patients.
The PI has published 2 papers based on this PF grant. He has obtained 2 new grants, and is a co-investigator on 2 more with other DRC members. Most importantly, this PF grant has triggered an ongoing, extremely successful collaboration with two additional PF recipients, Dr. Paul Harris and Dr. Qiao Lin. With Harris, Stojanovic is pursuing imaging methods for the functional visualization of pancreatic islets. With Dr. Lin, they are pursuing glucose sensors. These interactions were entirely catalyzed by the DRC, and by the program enrichment activities in which all PF recipients participate.