Publications by authors named "Marcela Brissova"

61 Publications

Integrated Analysis of the Pancreas and Islets Reveals Unexpected Findings in Human Male With Type 1 Diabetes.

J Endocr Soc 2021 Dec 29;5(12):bvab162. Epub 2021 Oct 29.

Department of Molecular Physiology and Biophysics, Vanderbilt University, Nashville, TN, USA.

Clinical and pathologic heterogeneity in type 1 diabetes is increasingly being recognized. Findings in the islets and pancreas of a 22-year-old male with 8 years of type 1 diabetes were discordant with expected results and clinical history (islet autoantibodies negative, hemoglobin A1c 11.9%) and led to comprehensive investigation to define the functional, molecular, genetic, and architectural features of the islets and pancreas to understand the cause of the donor's diabetes. Examination of the donor's pancreatic tissue found substantial but reduced β-cell mass with some islets devoid of β cells (29.3% of 311 islets) while other islets had many β cells. Surprisingly, isolated islets from the donor pancreas had substantial insulin secretion, which is uncommon for type 1 diabetes of this duration. Targeted and whole-genome sequencing and analysis did not uncover monogenic causes of diabetes but did identify high-risk human leukocyte antigen haplotypes and a genetic risk score suggestive of type 1 diabetes. Further review of pancreatic tissue found islet inflammation and some previously described α-cell molecular features seen in type 1 diabetes. By integrating analysis of isolated islets, histological evaluation of the pancreas, and genetic information, we concluded that the donor's clinical insulin deficiency was most likely the result autoimmune-mediated β-cell loss but that the constellation of findings was not typical for type 1 diabetes. This report highlights the pathologic and functional heterogeneity that can be present in type 1 diabetes.
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http://dx.doi.org/10.1210/jendso/bvab162DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8633619PMC
December 2021

Microvessels enhance vascularization and function of transplanted insulin-producing cells.

Cell Metab 2021 Nov;33(11):2103-2105

Division of Diabetes, Endocrinology and Metabolism, Department of Medicine, Vanderbilt University Medical Center, Nashville, TN 37232, USA. Electronic address:

Transplantation of insulin-producing cells is an emerging treatment for type 1 diabetes. A recent report in Cell Stem Cell (Aghazadeh et al., 2021) outlines a new approach that accelerates the engraftment and improves the survival and function of such cell transplants by mixing adipose tissue-derived ready-made microvessels with human pancreatic progenitor cells or cadaveric islets prior to transplantation.
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http://dx.doi.org/10.1016/j.cmet.2021.10.013DOI Listing
November 2021

Combinatorial transcription factor profiles predict mature and functional human islet α and β cells.

JCI Insight 2021 09 22;6(18). Epub 2021 Sep 22.

Division of Diabetes, Endocrinology and Metabolism, Department of Medicine, Vanderbilt University Medical Center, Nashville, Tennessee, USA.

Islet-enriched transcription factors (TFs) exert broad control over cellular processes in pancreatic α and β cells, and changes in their expression are associated with developmental state and diabetes. However, the implications of heterogeneity in TF expression across islet cell populations are not well understood. To define this TF heterogeneity and its consequences for cellular function, we profiled more than 40,000 cells from normal human islets by single-cell RNA-Seq and stratified α and β cells based on combinatorial TF expression. Subpopulations of islet cells coexpressing ARX/MAFB (α cells) and MAFA/MAFB (β cells) exhibited greater expression of key genes related to glucose sensing and hormone secretion relative to subpopulations expressing only one or neither TF. Moreover, all subpopulations were identified in native pancreatic tissue from multiple donors. By Patch-Seq, MAFA/MAFB-coexpressing β cells showed enhanced electrophysiological activity. Thus, these results indicate that combinatorial TF expression in islet α and β cells predicts highly functional, mature subpopulations.
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http://dx.doi.org/10.1172/jci.insight.151621DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8492318PMC
September 2021

The Human Islet: Mini-Organ With Mega-Impact.

Endocr Rev 2021 Sep;42(5):605-657

Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, Tennessee, USA.

This review focuses on the human pancreatic islet-including its structure, cell composition, development, function, and dysfunction. After providing a historical timeline of key discoveries about human islets over the past century, we describe new research approaches and technologies that are being used to study human islets and how these are providing insight into human islet physiology and pathophysiology. We also describe changes or adaptations in human islets in response to physiologic challenges such as pregnancy, aging, and insulin resistance and discuss islet changes in human diabetes of many forms. We outline current and future interventions being developed to protect, restore, or replace human islets. The review also highlights unresolved questions about human islets and proposes areas where additional research on human islets is needed.
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http://dx.doi.org/10.1210/endrev/bnab010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8476939PMC
September 2021

Coordinated interactions between endothelial cells and macrophages in the islet microenvironment promote β cell regeneration.

NPJ Regen Med 2021 Apr 6;6(1):22. Epub 2021 Apr 6.

Department of Medicine, Division of Diabetes, Endocrinology, and Metabolism, Vanderbilt University Medical Center, Nashville, TN, USA.

Endogenous β cell regeneration could alleviate diabetes, but proliferative stimuli within the islet microenvironment are incompletely understood. We previously found that β cell recovery following hypervascularization-induced β cell loss involves interactions with endothelial cells (ECs) and macrophages (MΦs). Here we show that proliferative ECs modulate MΦ infiltration and phenotype during β cell loss, and recruited MΦs are essential for β cell recovery. Furthermore, VEGFR2 inactivation in quiescent ECs accelerates islet vascular regression during β cell recovery and leads to increased β cell proliferation without changes in MΦ phenotype or number. Transcriptome analysis of β cells, ECs, and MΦs reveals that β cell proliferation coincides with elevated expression of extracellular matrix remodeling molecules and growth factors likely driving activation of proliferative signaling pathways in β cells. Collectively, these findings suggest a new β cell regeneration paradigm whereby coordinated interactions between intra-islet MΦs, ECs, and extracellular matrix mediate β cell self-renewal.
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http://dx.doi.org/10.1038/s41536-021-00129-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8024255PMC
April 2021

Dopamine regulates pancreatic glucagon and insulin secretion via adrenergic and dopaminergic receptors.

Transl Psychiatry 2021 02 16;11(1):59. Epub 2021 Feb 16.

Translational Neuroscience Program, Department of Psychiatry, University of Pittsburgh, Pittsburgh, PA, USA.

Dopamine (DA) and norepinephrine (NE) are catecholamines primarily studied in the central nervous system that also act in the pancreas as peripheral regulators of metabolism. Pancreatic catecholamine signaling has also been increasingly implicated as a mechanism responsible for the metabolic disturbances produced by antipsychotic drugs (APDs). Critically, however, the mechanisms by which catecholamines modulate pancreatic hormone release are not completely understood. We show that human and mouse pancreatic α- and β-cells express the catecholamine biosynthetic and signaling machinery, and that α-cells synthesize DA de novo. This locally-produced pancreatic DA signals via both α- and β-cell adrenergic and dopaminergic receptors with different affinities to regulate glucagon and insulin release. Significantly, we show DA functions as a biased agonist at α-adrenergic receptors, preferentially signaling via the canonical G protein-mediated pathway. Our findings highlight the interplay between DA and NE signaling as a novel form of regulation to modulate pancreatic hormone release. Lastly, pharmacological blockade of DA D-like receptors in human islets with APDs significantly raises insulin and glucagon release. This offers a new mechanism where APDs act directly on islet α- and β-cell targets to produce metabolic disturbances.
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http://dx.doi.org/10.1038/s41398-020-01171-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7884786PMC
February 2021

Pancreatlas: Applying an Adaptable Framework to Map the Human Pancreas in Health and Disease.

Patterns (N Y) 2020 Nov 5;1(8):100120. Epub 2020 Oct 5.

Division of Diabetes, Endocrinology, and Metabolism, Department of Medicine, Vanderbilt University Medical Center, Nashville, TN, USA.

Human tissue phenotyping generates complex spatial information from numerous imaging modalities, yet images typically become static figures for publication, and original data and metadata are rarely available. While comprehensive image maps exist for some organs, most resources have limited support for multiplexed imaging or have non-intuitive user interfaces. Therefore, we built a Pancreatlas resource that integrates several technologies into a unique interface, allowing users to access richly annotated web pages, drill down to individual images, and deeply explore data online. The current version of Pancreatlas contains over 800 unique images acquired by whole-slide scanning, confocal microscopy, and imaging mass cytometry, and is available at https://www.pancreatlas.org. To create this human pancreas-specific biological imaging resource, we developed a React-based web application and Python-based application programming interface, collectively called Flexible Framework for Integrating and Navigating Data (FFIND), which can be adapted beyond Pancreatlas to meet countless imaging or other structured data-management needs.
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http://dx.doi.org/10.1016/j.patter.2020.100120DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7691395PMC
November 2020

SARS-CoV-2 Cell Entry Factors ACE2 and TMPRSS2 Are Expressed in the Microvasculature and Ducts of Human Pancreas but Are Not Enriched in β Cells.

Cell Metab 2020 12 13;32(6):1028-1040.e4. Epub 2020 Nov 13.

Division of Diabetes, Endocrinology and Metabolism, Department of Medicine, Vanderbilt University Medical Center, Nashville, TN 37232, USA; VA Tennessee Valley Healthcare System, Nashville, TN 37212, USA; Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, TN 37232, USA. Electronic address:

Isolated reports of new-onset diabetes in individuals with COVID-19 have led to the hypothesis that SARS-CoV-2 is directly cytotoxic to pancreatic islet β cells. This would require binding and entry of SARS-CoV-2 into β cells via co-expression of its canonical cell entry factors, angiotensin-converting enzyme 2 (ACE2) and transmembrane serine protease 2 (TMPRSS2); however, their expression in human pancreas has not been clearly defined. We analyzed six transcriptional datasets of primary human islet cells and found that ACE2 and TMPRSS2 were not co-expressed in single β cells. In pancreatic sections, ACE2 and TMPRSS2 protein was not detected in β cells from donors with and without diabetes. Instead, ACE2 protein was expressed in islet and exocrine tissue microvasculature and in a subset of pancreatic ducts, whereas TMPRSS2 protein was restricted to ductal cells. These findings reduce the likelihood that SARS-CoV-2 directly infects β cells in vivo through ACE2 and TMPRSS2.
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http://dx.doi.org/10.1016/j.cmet.2020.11.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7664344PMC
December 2020

SARS-CoV-2 Cell Entry Factors ACE2 and TMPRSS2 are Expressed in the Pancreas but are Not Enriched in Islet Endocrine Cells.

bioRxiv 2020 Oct 20. Epub 2020 Oct 20.

Reports of new-onset diabetes and diabetic ketoacidosis in individuals with COVID-19 have led to the hypothesis that SARS-CoV-2, the virus that causes COVID-19, is directly cytotoxic to pancreatic islet β cells. This would require binding and entry of SARS-CoV-2 into host β cells via cell surface co-expression of ACE2 and TMPRSS2, the putative receptor and effector protease, respectively. To define ACE2 and TMPRSS2 expression in the human pancreas, we examined six transcriptional datasets from primary human islet cells and assessed protein expression by immunofluorescence in pancreata from donors with and without diabetes. and transcripts were low or undetectable in pancreatic islet endocrine cells as determined by bulk or single cell RNA sequencing, and neither protein was detected in α or β cells from these donors. Instead, ACE2 protein was expressed in the islet and exocrine tissue microvasculature and also found in a subset of pancreatic ducts, whereas TMPRSS2 protein was restricted to ductal cells. The absence of significant ACE2 and TMPRSS2 co-expression in islet endocrine cells reduces the likelihood that SARS-CoV-2 directly infects pancreatic islet β cells through these cell entry proteins.
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http://dx.doi.org/10.1101/2020.08.31.275719DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7587777PMC
October 2020

Decreased pancreatic acinar cell number in type 1 diabetes.

Diabetologia 2020 07 9;63(7):1418-1423. Epub 2020 May 9.

Division of Diabetes, Endocrinology, and Metabolism, Department of Medicine, Vanderbilt University Medical Center, 7465 Medical Research Bldg IV, 2215 Garland Avenue, Nashville, TN, 37232-0475, USA.

Aims/hypothesis: Individuals with longstanding and recent-onset type 1 diabetes have a smaller pancreas. Since beta cells represent a very small portion of the pancreas, the loss of pancreas volume in diabetes is primarily due to the loss of pancreatic exocrine mass. However, the structural changes in the exocrine pancreas in diabetes are not well understood.

Methods: To characterise the pancreatic endocrine and exocrine compartments in diabetes, we studied pancreases from adult donors with type 1 diabetes compared with similarly aged donors without diabetes. Islet cell mass, islet morphometry, exocrine mass, acinar cell size and number and pancreas fibrosis were assessed by immunohistochemical staining. To better understand possible mechanisms of altered pancreas size, we measured pancreas size in three mouse models of insulin deficiency.

Results: Pancreases from donors with type 1 diabetes were approximately 45% smaller than those from donors without diabetes (47.4 ± 2.6 vs 85.7 ± 3.7 g), independent of diabetes duration or age of onset. Diabetic donor pancreases had decreased beta cell mass (0.061 ± 0.025 vs 0.94 ± 0.21 g) and reduced total exocrine mass (42.0 ± 4.9 vs 96.1 ± 6.5 g). Diabetic acinar cells were similar in size but fewer in number compared with those in pancreases from non-diabetic donors (63.7 ± 8.1 × 10 vs 121.6 ± 12.2 × 10 cells/pancreas), likely accounting for the difference in pancreas size. Within the type 1 diabetes exocrine tissue, there was a greater degree of fibrosis. The pancreases in three mouse models of insulin deficiency were similar in size to those in control mice.

Conclusions/interpretation: Pancreases from donors with type 1 diabetes are smaller than normal donor pancreases because exocrine cells are fewer in number rather than smaller in size; these changes occur early in the disease process. Our mouse data suggest that decreased pancreas size in type 1 diabetes is not directly caused by insulin deficiency, but the precise mechanism responsible remains unclear.
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http://dx.doi.org/10.1007/s00125-020-05155-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8403487PMC
July 2020

Myt Transcription Factors Prevent Stress-Response Gene Overactivation to Enable Postnatal Pancreatic β Cell Proliferation, Function, and Survival.

Dev Cell 2020 05 30;53(4):390-405.e10. Epub 2020 Apr 30.

Vanderbilt Program in Developmental Biology, Department of Cell and Developmental Biology, and Center for Stem Cell Biology, Vanderbilt University School of Medicine, Nashville, TN 37232, USA. Electronic address:

Although cellular stress response is important for maintaining function and survival, overactivation of late-stage stress effectors cause dysfunction and death. We show that the myelin transcription factors (TFs) Myt1 (Nzf2), Myt2 (Myt1l, Nztf1, and Png-1), and Myt3 (St18 and Nzf3) prevent such overactivation in islet β cells. Thus, we found that co-inactivating the Myt TFs in mouse pancreatic progenitors compromised postnatal β cell function, proliferation, and survival, preceded by upregulation of late-stage stress-response genes activating transcription factors (e.g., Atf4) and heat-shock proteins (Hsps). Myt1 binds putative enhancers of Atf4 and Hsps, whose overexpression largely recapitulated the Myt-mutant phenotypes. Moreover, Myt(MYT)-TF levels were upregulated in mouse and human β cells during metabolic stress-induced compensation but downregulated in dysfunctional type 2 diabetic (T2D) human β cells. Lastly, MYT knockdown caused stress-gene overactivation and death in human EndoC-βH1 cells. These findings suggest that Myt TFs are essential restrictors of stress-response overactivity.
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http://dx.doi.org/10.1016/j.devcel.2020.04.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7278035PMC
May 2020

Integrated human pseudoislet system and microfluidic platform demonstrate differences in GPCR signaling in islet cells.

JCI Insight 2020 05 21;5(10). Epub 2020 May 21.

Division of Diabetes, Endocrinology and Metabolism, Department of Medicine, Vanderbilt University Medical Center, Nashville, Tennessee, USA.

Pancreatic islets secrete insulin from β cells and glucagon from α cells, and dysregulated secretion of these hormones is a central component of diabetes. Thus, an improved understanding of the pathways governing coordinated β and α cell hormone secretion will provide insight into islet dysfunction in diabetes. However, the 3D multicellular islet architecture, essential for coordinated islet function, presents experimental challenges for mechanistic studies of intracellular signaling pathways in primary islet cells. Here, we developed an integrated approach to study the function of primary human islet cells using genetically modified pseudoislets that resemble native islets across multiple parameters. Further, we developed a microperifusion system that allowed synchronous acquisition of GCaMP6f biosensor signal and hormone secretory profiles. We demonstrate the utility of this experimental approach by studying the effects of Gi and Gq GPCR pathways on insulin and glucagon secretion by expressing the designer receptors exclusively activated by designer drugs (DREADDs) hM4Di or hM3Dq. Activation of Gi signaling reduced insulin and glucagon secretion, while activation of Gq signaling stimulated glucagon secretion but had both stimulatory and inhibitory effects on insulin secretion, which occur through changes in intracellular Ca2+. The experimental approach of combining pseudoislets with a microfluidic system allowed the coregistration of intracellular signaling dynamics and hormone secretion and demonstrated differences in GPCR signaling pathways between human β and α cells.
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http://dx.doi.org/10.1172/jci.insight.137017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7259531PMC
May 2020

Modeling Monogenic Diabetes using Human ESCs Reveals Developmental and Metabolic Deficiencies Caused by Mutations in HNF1A.

Cell Stem Cell 2019 08;25(2):273-289.e5

Center for Cellular and Molecular Therapeutics, The Children's Hospital of Philadelphia, Philadelphia, PA, USA; Department of Pathology and Laboratory Medicine, The Children's Hospital of Philadelphia, Philadelphia, PA, USA. Electronic address:

Human monogenic diabetes, caused by mutations in genes involved in beta cell development and function, has been a challenge to study because multiple mouse models have not fully recapitulated the human disease. Here, we use genome edited human embryonic stem cells to understand the most common form of monogenic diabetes, MODY3, caused by mutations in the transcription factor HNF1A. We found that HNF1A is necessary to repress an alpha cell gene expression signature, maintain endocrine cell function, and regulate cellular metabolism. In addition, we identified the human-specific long non-coding RNA, LINKA, as an HNF1A target necessary for normal mitochondrial respiration. These findings provide a possible explanation for the species difference in disease phenotypes observed with HNF1A mutations and offer mechanistic insights into how the HNF1A gene may also influence type 2 diabetes.
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http://dx.doi.org/10.1016/j.stem.2019.07.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6785828PMC
August 2019

Imaging mass spectrometry enables molecular profiling of mouse and human pancreatic tissue.

Diabetologia 2019 06 6;62(6):1036-1047. Epub 2019 Apr 6.

9160 MRB III, Department of Biochemistry, Vanderbilt University, Nashville, TN, 37232, USA.

Aims/hypothesis: The molecular response and function of pancreatic islet cells during metabolic stress is a complex process. The anatomical location and small size of pancreatic islets coupled with current methodological limitations have prevented the achievement of a complete, coherent picture of the role that lipids and proteins play in cellular processes under normal conditions and in diseased states. Herein, we describe the development of untargeted tissue imaging mass spectrometry (IMS) technologies for the study of in situ protein and, more specifically, lipid distributions in murine and human pancreases.

Methods: We developed matrix-assisted laser desorption/ionisation (MALDI) IMS protocols to study metabolite, lipid and protein distributions in mouse (wild-type and ob/ob mouse models) and human pancreases. IMS allows for the facile discrimination of chemically similar lipid and metabolite isoforms that cannot be distinguished using standard immunohistochemical techniques. Co-registration of MS images with immunofluorescence images acquired from serial tissue sections allowed accurate cross-registration of cell types. By acquiring immunofluorescence images first, this serial section approach guides targeted high spatial resolution IMS analyses (down to 15 μm) of regions of interest and leads to reduced time requirements for data acquisition.

Results: MALDI IMS enabled the molecular identification of specific phospholipid and glycolipid isoforms in pancreatic islets with intra-islet spatial resolution. This technology shows that subtle differences in the chemical structure of phospholipids can dramatically affect their distribution patterns and, presumably, cellular function within the islet and exocrine compartments of the pancreas (e.g. 18:1 vs 18:2 fatty acyl groups in phosphatidylcholine lipids). We also observed the localisation of specific GM3 ganglioside lipids [GM3(d34:1), GM3(d36:1), GM3(d38:1) and GM3(d40:1)] within murine islet cells that were correlated with a higher level of GM3 synthase as verified by immunostaining. However, in human pancreas, GM3 gangliosides were equally distributed in both the endocrine and exocrine tissue, with only one GM3 isoform showing islet-specific localisation.

Conclusions/interpretation: The development of more complete molecular profiles of pancreatic tissue will provide important insight into the molecular state of the pancreas during islet development, normal function, and diseased states. For example, this study demonstrates that these results can provide novel insight into the potential signalling mechanisms involving phospholipids and glycolipids that would be difficult to detect by targeted methods, and can help raise new hypotheses about the types of physiological control exerted on endocrine hormone-producing cells in islets. Importantly, the in situ measurements afforded by IMS do not require a priori knowledge of molecules of interest and are not susceptible to the limitations of immunohistochemistry, providing the opportunity for novel biomarker discovery. Notably, the presence of multiple GM3 isoforms in mouse islets and the differential localisation of lipids in human tissue underscore the important role these molecules play in regulating insulin modulation and suggest species, organ, and cell specificity. This approach demonstrates the importance of both high spatial resolution and high molecular specificity to accurately survey the molecular composition of complex, multi-functional tissues such as the pancreas.
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http://dx.doi.org/10.1007/s00125-019-4855-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6553460PMC
June 2019

HLA Class II Antigen Processing and Presentation Pathway Components Demonstrated by Transcriptome and Protein Analyses of Islet β-Cells From Donors With Type 1 Diabetes.

Diabetes 2019 05 4;68(5):988-1001. Epub 2019 Mar 4.

Division of Diabetes, Department of Medicine, Diabetes Center of Excellence, University of Massachusetts Medical School, Worcester, MA

Type 1 diabetes studies consistently generate data showing islet β-cell dysfunction and T cell-mediated anti-β-cell-specific autoimmunity. To explore the pathogenesis, we interrogated the β-cell transcriptomes from donors with and without type 1 diabetes using both bulk-sorted and single β-cells. Consistent with immunohistological studies, β-cells from donors with type 1 diabetes displayed increased Class I transcripts and associated mRNA species. These β-cells also expressed mRNA for Class II and Class II antigen presentation pathway components, but lacked the macrophage marker CD68. Immunohistological study of three independent cohorts of donors with recent-onset type 1 diabetes showed Class II protein and its transcriptional regulator Class II MHC -activator protein expressed by a subset of insulinCD68 β-cells, specifically found in islets with lymphocytic infiltrates. β-Cell surface expression of HLA Class II was detected on a portion of CD45insulin β-cells from donors with type 1 diabetes by immunofluorescence and flow cytometry. Our data demonstrate that pancreatic β-cells from donors with type 1 diabetes express Class II molecules on selected cells with other key genes in those pathways and inflammation-associated genes. β-Cell expression of Class II molecules suggests that β-cells may interact directly with islet-infiltrating CD4 T cells and may play an immunopathogenic role.
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http://dx.doi.org/10.2337/db18-0686DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6477908PMC
May 2019

Human islets expressing HNF1A variant have defective β cell transcriptional regulatory networks.

J Clin Invest 2019 01 3;129(1):246-251. Epub 2018 Dec 3.

Department of Molecular Physiology and Biophysics, Vanderbilt University, Nashville, Tennessee, USA.

Using an integrated approach to characterize the pancreatic tissue and isolated islets from a 33-year-old with 17 years of type 1 diabetes (T1D), we found that donor islets contained β cells without insulitis and lacked glucose-stimulated insulin secretion despite a normal insulin response to cAMP-evoked stimulation. With these unexpected findings for T1D, we sequenced the donor DNA and found a pathogenic heterozygous variant in the gene encoding hepatocyte nuclear factor-1α (HNF1A). In one of the first studies of human pancreatic islets with a disease-causing HNF1A variant associated with the most common form of monogenic diabetes, we found that HNF1A dysfunction leads to insulin-insufficient diabetes reminiscent of T1D by impacting the regulatory processes critical for glucose-stimulated insulin secretion and suggest a rationale for a therapeutic alternative to current treatment.
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http://dx.doi.org/10.1172/JCI121994DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6307934PMC
January 2019

Ectonucleoside Triphosphate Diphosphohydrolase-3 Antibody Targets Adult Human Pancreatic β Cells for In Vitro and In Vivo Analysis.

Cell Metab 2019 03 15;29(3):745-754.e4. Epub 2018 Nov 15.

Department of Molecular Physiology and Biophysics, Vanderbilt University, Nashville, TN 37240, USA; Department of Medicine, Division of Diabetes, Endocrinology, and Metabolism, Vanderbilt University Medical Center, Nashville, TN 37232, USA; VA Tennessee Valley Healthcare, Nashville, TN 37212, USA. Electronic address:

Identification of cell-surface markers specific to human pancreatic β cells would allow in vivo analysis and imaging. Here we introduce a biomarker, ectonucleoside triphosphate diphosphohydrolase-3 (NTPDase3), that is expressed on the cell surface of essentially all adult human β cells, including those from individuals with type 1 or type 2 diabetes. NTPDase3 is expressed dynamically during postnatal human pancreas development, appearing first in acinar cells at birth, but several months later its expression declines in acinar cells while concurrently emerging in islet β cells. Given its specificity and membrane localization, we utilized an NTPDase3 antibody for purification of live human β cells as confirmed by transcriptional profiling, and, in addition, for in vivo imaging of transplanted human β cells. Thus, NTPDase3 is a cell-surface biomarker of adult human β cells, and the antibody directed to this protein should be a useful new reagent for β cell sorting, in vivo imaging, and targeting.
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http://dx.doi.org/10.1016/j.cmet.2018.10.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6402969PMC
March 2019

Examining How the MAFB Transcription Factor Affects Islet β-Cell Function Postnatally.

Diabetes 2019 02 13;68(2):337-348. Epub 2018 Nov 13.

Department of Molecular Physiology and Biophysics, Vanderbilt University, Nashville, TN

The sustained expression of the MAFB transcription factor in human islet β-cells represents a distinct difference in mice. Moreover, mRNA expression of closely related and islet β-cell-enriched MAFA does not peak in humans until after 9 years of age. We show that the MAFA protein also is weakly produced within the juvenile human islet β-cell population and that expression is postnatally restricted in mouse β-cells by de novo DNA methylation. To gain insight into how MAFB affects human β-cells, we developed a mouse model to ectopically express in adult mouse β-cells using transcriptional control sequences. Coexpression of MafB with MafA had no overt impact on mouse β-cells, suggesting that the human adult β-cell MAFA/MAFB heterodimer is functionally equivalent to the mouse MafA homodimer. However, MafB alone was unable to rescue the islet β-cell defects in a mouse mutant lacking MafA in β-cells. Of note, transgenic production of MafB in β-cells elevated tryptophan hydroxylase 1 mRNA production during pregnancy, which drives the serotonin biosynthesis critical for adaptive maternal β-cell responses. Together, these studies provide novel insight into the role of MAFB in human islet β-cells.
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http://dx.doi.org/10.2337/db18-0903DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6341297PMC
February 2019

β-Cell DNA Damage Response Promotes Islet Inflammation in Type 1 Diabetes.

Diabetes 2018 11 27;67(11):2305-2318. Epub 2018 Aug 27.

Department of Developmental Biology and Cancer Research, The Hebrew University, Jerusalem, Israel

Type 1 diabetes (T1D) is an autoimmune disease where pancreatic β-cells are destroyed by islet-infiltrating T cells. Although a role for β-cell defects has been suspected, β-cell abnormalities are difficult to demonstrate. We show a β-cell DNA damage response (DDR), presented by activation of the 53BP1 protein and accumulation of p53, in biopsy and autopsy material from patients with recently diagnosed T1D as well as a rat model of human T1D. The β-cell DDR is more frequent in islets infiltrated by CD45 immune cells, suggesting a link to islet inflammation. The β-cell toxin streptozotocin (STZ) elicits DDR in islets, both in vivo and ex vivo, and causes elevation of the proinflammatory molecules IL-1β and Cxcl10. β-Cell-specific inactivation of the master DNA repair gene ataxia telangiectasia mutated (ATM) in STZ-treated mice decreases the expression of proinflammatory cytokines in islets and attenuates the development of hyperglycemia. Together, these data suggest that β-cell DDR is an early event in T1D, possibly contributing to autoimmunity.
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http://dx.doi.org/10.2337/db17-1006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6198335PMC
November 2018

Cystic fibrosis-related diabetes is caused by islet loss and inflammation.

JCI Insight 2018 04 19;3(8). Epub 2018 Apr 19.

Department of Medicine, Division of Diabetes, Endocrinology, and Metabolism, Vanderbilt University Medical Center, Nashville, Tennessee, USA.

Cystic fibrosis-related (CF-related) diabetes (CFRD) is an increasingly common and devastating comorbidity of CF, affecting approximately 35% of adults with CF. However, the underlying causes of CFRD are unclear. Here, we examined cystic fibrosis transmembrane conductance regulator (CFTR) islet expression and whether the CFTR participates in islet endocrine cell function using murine models of β cell CFTR deletion and normal and CF human pancreas and islets. Specific deletion of CFTR from murine β cells did not affect β cell function. In human islets, CFTR mRNA was minimally expressed, and CFTR protein and electrical activity were not detected. Isolated CF/CFRD islets demonstrated appropriate insulin and glucagon secretion, with few changes in key islet-regulatory transcripts. Furthermore, approximately 65% of β cell area was lost in CF donors, compounded by pancreatic remodeling and immune infiltration of the islet. These results indicate that CFRD is caused by β cell loss and intraislet inflammation in the setting of a complex pleiotropic disease and not by intrinsic islet dysfunction from CFTR mutation.
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http://dx.doi.org/10.1172/jci.insight.98240DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5931120PMC
April 2018

α Cell Function and Gene Expression Are Compromised in Type 1 Diabetes.

Cell Rep 2018 03;22(10):2667-2676

Department of Medicine, Division of Diabetes, Endocrinology and Metabolism, Vanderbilt University Medical Center, Nashville, TN, USA; Department of Molecular Physiology and Biophysics, Vanderbilt University, Nashville, TN, USA; Department of Veterans Affairs, Tennessee Valley Healthcare System, Nashville, TN, USA. Electronic address:

Many patients with type 1 diabetes (T1D) have residual β cells producing small amounts of C-peptide long after disease onset but develop an inadequate glucagon response to hypoglycemia following T1D diagnosis. The features of these residual β cells and α cells in the islet endocrine compartment are largely unknown, due to the difficulty of comprehensive investigation. By studying the T1D pancreas and isolated islets, we show that remnant β cells appeared to maintain several aspects of regulated insulin secretion. However, the function of T1D α cells was markedly reduced, and these cells had alterations in transcription factors constituting α and β cell identity. In the native pancreas and after placing the T1D islets into a non-autoimmune, normoglycemic in vivo environment, there was no evidence of α-to-β cell conversion. These results suggest an explanation for the disordered T1D counterregulatory glucagon response to hypoglycemia.
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http://dx.doi.org/10.1016/j.celrep.2018.02.032DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6368357PMC
March 2018

Mouse pancreatic islet macrophages use locally released ATP to monitor beta cell activity.

Diabetologia 2018 Jan 7;61(1):182-192. Epub 2017 Sep 7.

Division of Endocrinology, Diabetes and Metabolism, Department of Medicine, University of Miami Miller School of Medicine, 1580 NW 10th Ave, Miami, FL, 33136, USA.

Aims/hypothesis: Tissue-resident macrophages sense the microenvironment and respond by producing signals that act locally to maintain a stable tissue state. It is now known that pancreatic islets contain their own unique resident macrophages, which have been shown to promote proliferation of the insulin-secreting beta cell. However, it is unclear how beta cells communicate with islet-resident macrophages. Here we hypothesised that islet macrophages sense changes in islet activity by detecting signals derived from beta cells.

Methods: To investigate how islet-resident macrophages respond to cues from the microenvironment, we generated mice expressing a genetically encoded Ca indicator in myeloid cells. We produced living pancreatic slices from these mice and used them to monitor macrophage responses to stimulation of acinar, neural and endocrine cells.

Results: Islet-resident macrophages expressed functional purinergic receptors, making them exquisite sensors of interstitial ATP levels. Indeed, islet-resident macrophages responded selectively to ATP released locally from beta cells that were physiologically activated with high levels of glucose. Because ATP is co-released with insulin and is exclusively secreted by beta cells, the activation of purinergic receptors on resident macrophages facilitates their awareness of beta cell secretory activity.

Conclusions/interpretation: Our results indicate that islet macrophages detect ATP as a proxy signal for the activation state of beta cells. Sensing beta cell activity may allow macrophages to adjust the secretion of factors to promote a stable islet composition and size.
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http://dx.doi.org/10.1007/s00125-017-4416-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5868749PMC
January 2018

Glycoprotein 2 is a specific cell surface marker of human pancreatic progenitors.

Nat Commun 2017 08 24;8(1):331. Epub 2017 Aug 24.

Toronto General Hospital Research Institute, University Health Network, Toronto, ON, Canada, M5G 1L7.

PDX1/NKX6-1 pancreatic progenitors (PPs) give rise to endocrine cells both in vitro and in vivo. This cell population can be successfully differentiated from human pluripotent stem cells (hPSCs) and hold the potential to generate an unlimited supply of β cells for diabetes treatment. However, the efficiency of PP generation in vitro is highly variable, negatively impacting reproducibility and validation of in vitro and in vivo studies, and consequently, translation to the clinic. Here, we report the use of a proteomics approach to phenotypically characterize hPSC-derived PPs and distinguish these cells from non-PP populations during differentiation. Our analysis identifies the pancreatic secretory granule membrane major glycoprotein 2 (GP2) as a PP-specific cell surface marker. Remarkably, GP2 is co-expressed with NKX6-1 and PTF1A in human developing pancreata, indicating that it marks the multipotent pancreatic progenitors in vivo. Finally, we show that isolated hPSC-derived GP2 cells generate β-like cells (C-PEPTIDE/NKX6-1) more efficiently compared to GP2 and unsorted populations, underlining the potential therapeutic applications of GP2.Pancreatic progenitors (PPs) can be derived from human pluripotent stem cells in vitro but efficiency of differentiation varies, making it hard to sort for insulin-producing cells. Here, the authors use a proteomic approach to identify the secretory granule membrane glycoprotein 2 as a marker for PDX1+/NKX6-1+ PPs.
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http://dx.doi.org/10.1038/s41467-017-00561-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5569081PMC
August 2017

Analysis of self-antigen specificity of islet-infiltrating T cells from human donors with type 1 diabetes.

Nat Med 2016 12 31;22(12):1482-1487. Epub 2016 Oct 31.

Department of Medicine, Division of Diabetes, Diabetes Center of Excellence, University of Massachusetts Medical School, Worcester, Massachusetts, USA.

A major therapeutic goal for type 1 diabetes (T1D) is to induce autoantigen-specific tolerance of T cells. This could suppress autoimmunity in those at risk for the development of T1D, as well as in those with established disease who receive islet replacement or regeneration therapy. Because functional studies of human autoreactive T cell responses have been limited largely to peripheral blood-derived T cells, it is unclear how representative the peripheral T cell repertoire is of T cells infiltrating the islets. Our knowledge of the insulitic T cell repertoire is derived from histological and immunohistochemical analyses of insulitis, the identification of autoreactive CD8 T cells in situ, in islets of human leukocyte antigen (HLA)-A2 donors and isolation and identification of DQ8 and DQ2-DQ8 heterodimer-restricted, proinsulin-reactive CD4 T cells grown from islets of a single donor with T1D. Here we present an analysis of 50 of a total of 236 CD4 and CD8 T cell lines grown from individual handpicked islets or clones directly sorted from handpicked, dispersed islets from nine donors with T1D. Seventeen of these T cell lines and clones reacted to a broad range of studied native islet antigens and to post-translationally modified peptides. These studies demonstrate the existence of a variety of islet-infiltrating, islet-autoantigen reactive T cells in individuals with T1D, and these data have implications for the design of successful immunotherapies.
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http://dx.doi.org/10.1038/nm.4203DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5140746PMC
December 2016

Development of a reliable automated screening system to identify small molecules and biologics that promote human β-cell regeneration.

Am J Physiol Endocrinol Metab 2016 11 13;311(5):E859-E868. Epub 2016 Sep 13.

Department of Molecular Physiology and Biophysics, Vanderbilt University Medical Center, Nashville, Tennessee.

Numerous compounds stimulate rodent β-cell proliferation; however, translating these findings to human β-cells remains a challenge. To examine human β-cell proliferation in response to such compounds, we developed a medium-throughput in vitro method of quantifying adult human β-cell proliferation markers. This method is based on high-content imaging of dispersed islet cells seeded in 384-well plates and automated cell counting that identifies fluorescently labeled β-cells with high specificity using both nuclear and cytoplasmic markers. β-Cells from each donor were assessed for their function and ability to enter the cell cycle by cotransduction with adenoviruses encoding cell cycle regulators cdk6 and cyclin D3. Using this approach, we tested 12 previously identified mitogens, including neurotransmitters, hormones, growth factors, and molecules, involved in adenosine and Tgf-1β signaling. Each compound was tested in a wide concentration range either in the presence of basal (5 mM) or high (11 mM) glucose. Treatment with the control compound harmine, a Dyrk1a inhibitor, led to a significant increase in Ki-67 β-cells, whereas treatment with other compounds had limited to no effect on human β-cell proliferation. This new scalable approach reduces the time and effort required for sensitive and specific evaluation of human β-cell proliferation, thus allowing for increased testing of candidate human β-cell mitogens.
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http://dx.doi.org/10.1152/ajpendo.00515.2015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5130356PMC
November 2016
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