Publications by authors named "Klaus H Kaestner"

321 Publications

Insulin-degrading enzyme ablation in mouse pancreatic alpha cells triggers cell proliferation, hyperplasia and glucagon secretion dysregulation.

Diabetologia 2022 Jun 2. Epub 2022 Jun 2.

Unidad de Excelencia Instituto de Biología y Genética Molecular (University of Valladolid-CSIC), Valladolid, Spain.

Aims/hypothesis: Type 2 diabetes is characterised by hyperglucagonaemia and perturbed function of pancreatic glucagon-secreting alpha cells but the molecular mechanisms contributing to these phenotypes are poorly understood. Insulin-degrading enzyme (IDE) is present within all islet cells, mostly in alpha cells, in both mice and humans. Furthermore, IDE can degrade glucagon as well as insulin, suggesting that IDE may play an important role in alpha cell function in vivo.

Methods: We have generated and characterised a novel mouse model with alpha cell-specific deletion of Ide, the A-IDE-KO mouse line. Glucose metabolism and glucagon secretion in vivo was characterised; isolated islets were tested for glucagon and insulin secretion; alpha cell mass, alpha cell proliferation and α-synuclein levels were determined in pancreas sections by immunostaining.

Results: Targeted deletion of Ide exclusively in alpha cells triggers hyperglucagonaemia and alpha cell hyperplasia, resulting in elevated constitutive glucagon secretion. The hyperglucagonaemia is attributable in part to dysregulation of glucagon secretion, specifically an impaired ability of IDE-deficient alpha cells to suppress glucagon release in the presence of high glucose or insulin. IDE deficiency also leads to α-synuclein aggregation in alpha cells, which may contribute to impaired glucagon secretion via cytoskeletal dysfunction. We showed further that IDE deficiency triggers impairments in cilia formation, inducing alpha cell hyperplasia and possibly also contributing to dysregulated glucagon secretion and hyperglucagonaemia.

Conclusions/interpretation: We propose that loss of IDE function in alpha cells contributes to hyperglucagonaemia in type 2 diabetes.
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http://dx.doi.org/10.1007/s00125-022-05729-yDOI Listing
June 2022

α Cell dysfunction in islets from nondiabetic, glutamic acid decarboxylase autoantibody-positive individuals.

J Clin Invest 2022 Jun;132(11)

Institute for Diabetes, Obesity, and Metabolism.

BACKGROUNDMultiple islet autoantibodies (AAbs) predict the development of type 1 diabetes (T1D) and hyperglycemia within 10 years. By contrast, T1D develops in only approximately 15% of individuals who are positive for single AAbs (generally against glutamic acid decarboxylase [GADA]); hence, the single GADA+ state may represent an early stage of T1D.METHODSHere, we functionally, histologically, and molecularly phenotyped human islets from nondiabetic GADA+ and T1D donors.RESULTSSimilar to the few remaining β cells in the T1D islets, GADA+ donor islets demonstrated a preserved insulin secretory response. By contrast, α cell glucagon secretion was dysregulated in both GADA+ and T1D islets, with impaired glucose suppression of glucagon secretion. Single-cell RNA-Seq of GADA+ α cells revealed distinct abnormalities in glycolysis and oxidative phosphorylation pathways and a marked downregulation of cAMP-dependent protein kinase inhibitor β (PKIB), providing a molecular basis for the loss of glucose suppression and the increased effect of 3-isobutyl-1-methylxanthine (IBMX) observed in GADA+ donor islets.CONCLUSIONWe found that α cell dysfunction was present during the early stages of islet autoimmunity at a time when β cell mass was still normal, raising important questions about the role of early α cell dysfunction in the progression of T1D.FUNDINGThis work was supported by grants from the NIH (3UC4DK112217-01S1, U01DK123594-02, UC4DK112217, UC4DK112232, U01DK123716, and P30 DK019525) and the Vanderbilt Diabetes Research and Training Center (DK20593).
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http://dx.doi.org/10.1172/JCI156243DOI Listing
June 2022

Variant-to-gene-mapping analyses reveal a role for pancreatic islet cells in conferring genetic susceptibility to sleep-related traits.

Sleep 2022 May 10. Epub 2022 May 10.

Center for Spatial and Functional Genomics, Children's Hospital of Philadelphia, Philadelphia, PA, USA.

We investigated the potential role of sleep-trait associated genetic loci in conferring a degree of their effect via pancreatic α- and β- cells, given that both sleep disturbances and metabolic disorders, including type 2 diabetes and obesity, involve polygenic contributions and complex interactions. We determined genetic commonalities between sleep and metabolic disorders, conducting linkage disequilibrium genetic correlation analyses with publicly available GWAS summary statistics. Then we investigated possible enrichment of sleep-trait associated SNPs in promoter-interacting open chromatin regions within α- and β- cells, intersecting public GWAS reports with our own ATAC-seq and high-resolution promoter-focused Capture C data generated from both sorted human α-cells and an established human beta-cell line (EndoC-βH1). Finally, we identified putative effector genes physically interacting with sleep-trait associated variants in α- and EndoC-βH1cells running variant-to-gene mapping and establish pathways in which these genes are significantly involved. We observed that insomnia, short and long sleep - but not morningness - were significantly correlated with type 2 diabetes, obesity and other metabolic traits. Both the EndoC-βH1 and α-cells were enriched for insomnia loci (P=0.01; P=0.0076), short sleep loci (P=0.017; P=0.022) and morningness loci (P=2.2x10 -7; P=0.0016), while the α-cells were also enriched for long sleep loci (P=0.034). Utilizing our promoter contact data, we identified 63 putative effector genes in EndoC-βH1 and 76 putative effector genes in α-cells, with these genes showing significant enrichment for organonitrogen and organophosphate biosynthesis, phosphatidylinositol and phosphorylation, intracellular transport and signaling, stress responses and cell differentiation. Our data suggest that a subset of sleep-related loci confer their effects via cells in pancreatic islets.
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http://dx.doi.org/10.1093/sleep/zsac109DOI Listing
May 2022

β-Hydroxybutyrate suppresses colorectal cancer.

Nature 2022 05 27;605(7908):160-165. Epub 2022 Apr 27.

Department of Biostatistics Epidemiology and Informatics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA.

Colorectal cancer (CRC) is among the most frequent forms of cancer, and new strategies for its prevention and therapy are urgently needed. Here we identify a metabolite signalling pathway that provides actionable insights towards this goal. We perform a dietary screen in autochthonous animal models of CRC and find that ketogenic diets exhibit a strong tumour-inhibitory effect. These properties of ketogenic diets are recapitulated by the ketone body β-hydroxybutyrate (BHB), which reduces the proliferation of colonic crypt cells and potently suppresses intestinal tumour growth. We find that BHB acts through the surface receptor Hcar2 and induces the transcriptional regulator Hopx, thereby altering gene expression and inhibiting cell proliferation. Cancer organoid assays and single-cell RNA sequencing of biopsies from patients with CRC provide evidence that elevated BHB levels and active HOPX are associated with reduced intestinal epithelial proliferation in humans. This study thus identifies a BHB-triggered pathway regulating intestinal tumorigenesis and indicates that oral or systemic interventions with a single metabolite may complement current prevention and treatment strategies for CRC.
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http://dx.doi.org/10.1038/s41586-022-04649-6DOI Listing
May 2022

Development of a Beta Cell-Specific Expression Control Element for Recombinant Adeno-Associated Virus.

Hum Gene Ther 2022 May 10. Epub 2022 May 10.

Papé Family Pediatric Research Institute, Oregon Stem Cell Center, Oregon Health & Science University, Portland, Oregon, USA.

Diabetes mellitus, caused by loss or dysfunction of the insulin-producing beta cells of the pancreas, is a promising target for recombinant adeno-associated virus (rAAV)-mediated gene therapy. To target potential therapeutic payloads specifically to beta cells, a cell type-specific expression control element is needed. In this study, we tested a series of rAAV vectors designed to express transgenes specifically in human beta cells using the islet-tropic rAAV-KP1 capsid. A small promoter, consisting of only 84 bp of the insulin core promoter was not beta cell-specific in AAV, but highly active in multiple cell types, including tissues outside the pancreas. A larger 363 bp fragment of the insulin promoter (INS) also lacked beta cell specificity. However, beta cell-specific expression was achieved by combining two regulatory elements, a promoter consisting of two copies of INS (INS × 2) and microRNA (miRNA) recognition elements (MREs). The INS × 2 promoter alone showed some beta cell preference, but not tight specificity. To reduce unspecific transgene expression in alpha cells, negative regulation by miRNAs was applied. MREs that are recognized by miRNAs abundant in alpha cells effectively downregulated the transgene expression in these cells. The INS2 × -MRE expression vector was highly specific to human beta cells and stem cell-derived beta cells.
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http://dx.doi.org/10.1089/hum.2021.219DOI Listing
May 2022

Single-cell multi-omics analysis of human pancreatic islets reveals novel cellular states in type 1 diabetes.

Nat Metab 2022 02 28;4(2):284-299. Epub 2022 Feb 28.

Department of Genetics, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA, USA.

Type 1 diabetes (T1D) is an autoimmune disease in which immune cells destroy insulin-producing beta cells. The aetiology of this complex disease is dependent on the interplay of multiple heterogeneous cell types in the pancreatic environment. Here, we provide a single-cell atlas of pancreatic islets of 24 T1D, autoantibody-positive and nondiabetic organ donors across multiple quantitative modalities including ~80,000 cells using single-cell transcriptomics, ~7,000,000 cells using cytometry by time of flight and ~1,000,000 cells using in situ imaging mass cytometry. We develop an advanced integrative analytical strategy to assess pancreatic islets and identify canonical cell types. We show that a subset of exocrine ductal cells acquires a signature of tolerogenic dendritic cells in an apparent attempt at immune suppression in T1D donors. Our multimodal analyses delineate cell types and processes that may contribute to T1D immunopathogenesis and provide an integrative procedure for exploration and discovery of human pancreatic function.
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http://dx.doi.org/10.1038/s42255-022-00531-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8938904PMC
February 2022

Heterogenous impairment of α cell function in type 2 diabetes is linked to cell maturation state.

Cell Metab 2022 02;34(2):256-268.e5

Department of Pharmacology, University of Alberta, Edmonton, AB T6G2R3, Canada; Alberta Diabetes Institute, University of Alberta, Edmonton, AB T6G2R3, Canada. Electronic address:

In diabetes, glucagon secretion from pancreatic α cells is dysregulated. The underlying mechanisms, and whether dysfunction occurs uniformly among cells, remain unclear. We examined α cells from human donors and mice using electrophysiological, transcriptomic, and computational approaches. Rising glucose suppresses α cell exocytosis by reducing P/Q-type Ca channel activity, and this is disrupted in type 2 diabetes (T2D). Upon high-fat feeding of mice, α cells shift toward a "β cell-like" electrophysiological profile in concert with indications of impaired identity. In human α cells we identified links between cell membrane properties and cell surface signaling receptors, mitochondrial respiratory chain complex assembly, and cell maturation. Cell-type classification using machine learning of electrophysiology data demonstrated a heterogenous loss of "electrophysiologic identity" in α cells from donors with type 2 diabetes. Indeed, a subset of α cells with impaired exocytosis is defined by an enrichment in progenitor and lineage markers and upregulation of an immature transcriptomic phenotype, suggesting important links between α cell maturation state and dysfunction.
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http://dx.doi.org/10.1016/j.cmet.2021.12.021DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8852281PMC
February 2022

Paternal Exercise Improves the Metabolic Health of Offspring via Epigenetic Modulation of the Germline.

Int J Mol Sci 2021 Dec 21;23(1). Epub 2021 Dec 21.

Department of Genetics and Institute for Diabetes, Obesity and Metabolism, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA.

Background/aims: Epigenetic regulation is considered the main molecular mechanism underlying the developmental origin of health and disease's (DOHAD) hypothesis. Previous studies that have investigated the role of paternal exercise on the metabolic health of the offspring did not control for the amount and intensity of the training or possible effects of adaptation to exercise and produced conflicting results regarding the benefits of parental exercise to the next generation. We employed a precisely regulated exercise regimen to study the transgenerational inheritance of improved metabolic health.

Methods: We subjected male mice to a well-controlled exercise -training program to investigate the effects of paternal exercise on glucose tolerance and insulin sensitivity in their adult progeny. To investigate the molecular mechanisms of epigenetic inheritance, we determined chromatin markers in the skeletal muscle of the offspring and the paternal sperm.

Results: Offspring of trained male mice exhibited improved glucose homeostasis and insulin sensitivity. Paternal exercise modulated the DNA methylation profile of PI3Kca and the imprinted H19/Igf2 locus at specific differentially methylated regions (DMRs) in the skeletal muscle of the offspring, which affected their gene expression. Remarkably, a similar DNA methylation profile at the PI3Kca, H19, and Igf2 genes was present in the progenitor sperm indicating that exercise-induced epigenetic changes that occurred during germ cell development contributed to transgenerational transmission.

Conclusion: Paternal exercise might be considered as a strategy that could promote metabolic health in the offspring as the benefits can be inherited transgenerationally.
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http://dx.doi.org/10.3390/ijms23010001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8744992PMC
December 2021

Offspring from trained male mice inherit improved muscle mitochondrial function through PPAR co-repressor modulation.

Life Sci 2022 Feb 21;291:120239. Epub 2021 Dec 21.

Obesity and Comorbidities Research Center (OCRC), Department of Functional and Structural Biology, Institute of Biology, University of Campinas (UNICAMP), Campinas, SP, Brazil. Electronic address:

Aim Investigate whether inheritance of improved skeletal muscle mitochondrial function and its association with glycemic control are multigenerational benefits of exercise.

Main Methods: Male Swiss mice were subjected to 8 weeks of endurance training and mated with untrained females.

Key Findings: Trained fathers displayed typical endurance training-induced adaptations. Remarkably, offspring from trained fathers also exhibited higher endurance performance, mitochondrial oxygen consumption, glucose tolerance and insulin sensitivity. However, PGC-1α expression was not increased in the offspring. In the offspring, the expression of the co-repressor NCoR1 was reduced, increasing activation of PGC-1α target genes. These effects correlated with higher DNA methylation at the NCoR1 promoter in both, the sperm of trained fathers and in the skeletal muscle of their offspring.

Significance: Higher skeletal muscle mitochondrial function is inherited by epigenetic de-activation of a key PGC-1α co-repressor.
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http://dx.doi.org/10.1016/j.lfs.2021.120239DOI Listing
February 2022

Cancer stem cells: advances in biology and clinical translation-a Keystone Symposia report.

Ann N Y Acad Sci 2021 12 30;1506(1):142-163. Epub 2021 Nov 30.

Institute for Stem Cell Biology and Regenerative Medicine, Ludwig Center for Cancer Stem Cell Research, Stanford University, Stanford, California.

The test for the cancer stem cell (CSC) hypothesis is to find a target expressed on all, and only CSCs in a patient tumor, then eliminate all cells with that target that eliminates the cancer. That test has not yet been achieved, but CSC diagnostics and targets found on CSCs and some other cells have resulted in a few clinically relevant therapies. However, it has become apparent that eliminating the subset of tumor cells characterized by self-renewal properties is essential for long-term tumor control. CSCs are able to regenerate and initiate tumor growth, recapitulating the heterogeneity present in the tumor before treatment. As great progress has been made in identifying and elucidating the biology of CSCs as well as their interactions with the tumor microenvironment, the time seems ripe for novel therapeutic strategies that target CSCs to find clinical applicability. On May 19-21, 2021, researchers in cancer stem cells met virtually for the Keystone eSymposium "Cancer Stem Cells: Advances in Biology and Clinical Translation" to discuss recent advances in the understanding of CSCs as well as clinical efforts to target these populations.
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http://dx.doi.org/10.1111/nyas.14719DOI Listing
December 2021

CMGH: The year in review.

Cell Mol Gastroenterol Hepatol 2021 ;12(5):1873-1874

Editor-in-Chief, CMGH. Electronic address:

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http://dx.doi.org/10.1016/j.jcmgh.2021.09.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8591182PMC
March 2022

Gq signaling in α cells is critical for maintaining euglycemia.

JCI Insight 2021 12 22;6(24). Epub 2021 Dec 22.

Molecular Signaling Section, Laboratory of Bioorganic Chemistry, National Institute of Diabetes and Digestive and Kidney Diseases, NIH, Bethesda, Maryland, USA.

Glucagon, a hormone released from pancreatic α cells, plays a key role in maintaining euglycemia. New insights into the signaling pathways that control glucagon secretion may stimulate the development of novel therapeutic agents. In this study, we investigated the potential regulation of α cell function by G proteins of the Gq family. The use of a chemogenetic strategy allowed us to selectively activate Gq signaling in mouse α cells in vitro and in vivo. Acute stimulation of α cell Gq signaling led to elevated plasma glucagon levels, accompanied by increased insulin release and improved glucose tolerance. Moreover, chronic activation of this pathway greatly improved glucose tolerance in obese mice. We also identified an endogenous Gq-coupled receptor (vasopressin 1b receptor; V1bR) that was enriched in mouse and human α cells. Agonist-induced activation of the V1bR strongly stimulated glucagon release in a Gq-dependent fashion. In vivo studies indicated that V1bR-mediated glucagon release played a key role in the counterregulatory hyperglucagonemia under hypoglycemic and glucopenic conditions. These data indicate that α cell Gq signaling represents an important regulator of glucagon secretion, resulting in multiple beneficial metabolic effects. Thus, drugs that target α cell-enriched Gq-coupled receptors may prove useful to restore euglycemia in various pathophysiological conditions.
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http://dx.doi.org/10.1172/jci.insight.152852DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8783673PMC
December 2021

Single-cell analysis of the human pancreas in type 2 diabetes using multi-spectral imaging mass cytometry.

Cell Rep 2021 11;37(5):109919

Department of Genetics, University of Pennsylvania, Philadelphia, PA 19104, USA; Institute for Diabetes, Obesity, and Metabolism, University of Pennsylvania, Philadelphia, PA 19104, USA. Electronic address:

Type 2 diabetes mellitus (T2D) is a chronic age-related disorder characterized by hyperglycemia due to the failure of pancreatic beta cells to compensate for increased insulin demand. Despite decades of research, the pathogenic mechanisms underlying T2D remain poorly defined. Here, we use imaging mass cytometry (IMC) with a panel of 34 antibodies to simultaneously quantify markers of pancreatic exocrine, islet, and immune cells and stromal components. We analyze over 2 million cells from 16 pancreata obtained from donors with T2D and 13 pancreata from age-similar non-diabetic controls. In the T2D pancreata, we observe significant alterations in islet architecture, endocrine cell composition, and immune cell constituents. Thus, both HLA-DR-positive CD8 T cells and macrophages are enriched intra-islet in the T2D pancreas. These efforts demonstrate the utility of IMC for investigating complex events at the cellular level in order to provide insights into the pathophysiology of T2D.
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http://dx.doi.org/10.1016/j.celrep.2021.109919DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8609965PMC
November 2021

Highly Multiplexed Image Analysis of Intestinal Tissue Sections in Patients With Inflammatory Bowel Disease.

Gastroenterology 2021 12 14;161(6):1940-1952. Epub 2021 Sep 14.

Department of Genetics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania. Electronic address:

Background & Aims: Significant progress has been made since the first report of inflammatory bowel disease (IBD) in 1859, after decades of research that have contributed to the understanding of the genetic and environmental factors involved in IBD pathogenesis. Today, a range of treatments is available for directed therapy, mostly targeting the overactive immune response. However, the mechanisms by which the immune system contributes to disease pathogenesis and progression are not fully understood. One challenge hindering IBD research is the heterogeneous nature of the disease and the lack of understanding of how immune cells interact with one another in the gut mucosa. Introduction of a technology that enables expansive characterization of the inflammatory environment of human IBD tissues may address this gap in knowledge.

Methods: We used the imaging mass cytometry platform to perform highly multiplex image analysis of IBD and healthy deidentified intestine sections (6 Crohn's disease compared to 6 control ileum; 6 ulcerative colitis compared to 6 control colon). The acquired images were graded for inflammation severity by analysis of adjacent H&E tissue sections. We assigned more than 300,000 cells to unique cell types and performed analyses of tissue integrity, epithelial activity, and immune cell composition.

Results: The intestinal epithelia of patients with IBD exhibited increased proliferation rates and expression of HLA-DR compared to control tissues, and both features were positively correlated with the severity of inflammation. The neighborhood analysis determined enrichment of regulatory T cell interactions with CD68 macrophages, CD4 T cells, and plasma cells in both forms of IBD, whereas activated lysozyme C macrophages were preferred regulatory T cell neighbors in Crohn's disease but not ulcerative colitis.

Conclusions: Altogether, our study shows the power of imaging mass cytometry and its ability to both quantify immune cell types and characterize their spatial interactions within the inflammatory environment by a single analysis platform.
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http://dx.doi.org/10.1053/j.gastro.2021.08.055DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8606000PMC
December 2021

What is a β cell? - Chapter I in the Human Islet Research Network (HIRN) review series.

Mol Metab 2021 11 17;53:101323. Epub 2021 Aug 17.

Division of Diabetes, Endocrinology, and Metabolism, Department of Medicine, Vanderbilt University Medical Center, and VA Tennessee Valley Healthcare System, Nashville, TN, USA. Electronic address:

Background: The pancreatic β cell, as the sole source of the vital hormone insulin, has been under intensive study for more than a century. Given the potential of newly created insulin-producing cells as a treatment or even cure of type 1 diabetes (T1D) and possibly in severe cases of type 2 diabetes (T2D), multiple academic and commercial laboratories are working to derive surrogate glucose-responsive, insulin-producing cells.

Scope Of Review: The recent development of advanced phenotyping technologies, including molecular, epigenomic, histological, or functional, have greatly improved our understanding of the critical properties of human β cells. Using this information, here we summarize the salient features of normal, fully functional adult human β cells, and propose minimal criteria for what should rightfully be termed 'β cells' as opposed to insulin-producing but not fully-functional surrogates that we propose should be referred to as 'β-like' cells or insulin-producing cells.

Major Conclusions: Clear criteria can be established to differentiate fully functional, mature β cells from 'β-like' surrogates. In addition, we outline important knowledge gaps that must be addressed to enable a greater understanding of the β cell.
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http://dx.doi.org/10.1016/j.molmet.2021.101323DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8452767PMC
November 2021

FoxL1 mesenchymal cells are a critical source of Wnt5a for midgut elongation during mouse embryonic intestinal development.

Cells Dev 2021 03 28;165:203662. Epub 2021 Jan 28.

Department of Genetics and Institute for Diabetes, Obesity and Metabolism, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA. Electronic address:

Wnt5a is a non-canonical Wnt ligand that is essential for normal embryonic development in mammals. The role of Wnt5a in early intestinal development has been examined in gene ablation models, where Wnt5a mice exhibit strikingly shortened intestines. However, the exact cellular source of Wnt5a has remained elusive, until a recent study found that FoxL1-expressing mesenchymal cells (FoxL1 cells), which are localized directly beneath the intestinal epithelium, express Wnt5a. To determine whether FoxL1 cells are a required source of Wnt5a during intestinal development, we derived FoxL1-Cre; Wnt5a mice, which is the first mouse model to ablate Wnt5a in a cell type-specific manner in the intestine in vivo. Our results show that Wnt5a deletion in FoxL1 cells during fetal life causes a shortened gut phenotype in neonatal mice, and that our model is sufficient to increase rate of apoptosis in the elongating epithelium, thus explaining the shortened gut phenotype. However, in contrast to previous studies using Wnt5a null mice, we did not observe dysregulation of epithelial structure or apical-basal protein localization. Altogether, our findings establish a developmental role for FoxL1 mesenchymal cells in controlling non-canonical Wnt signaling during midgut elongation.
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http://dx.doi.org/10.1016/j.cdev.2021.203662DOI Listing
March 2021

Cochlear supporting cells require GAS2 for cytoskeletal architecture and hearing.

Dev Cell 2021 05 7;56(10):1526-1540.e7. Epub 2021 May 7.

Department of Genetics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA. Electronic address:

In mammals, sound is detected by mechanosensory hair cells that are activated in response to vibrations at frequency-dependent positions along the cochlear duct. We demonstrate that inner ear supporting cells provide a structural framework for transmitting sound energy through the cochlear partition. Humans and mice with mutations in GAS2, encoding a cytoskeletal regulatory protein, exhibit hearing loss due to disorganization and destabilization of microtubule bundles in pillar and Deiters' cells, two types of inner ear supporting cells with unique cytoskeletal specializations. Failure to maintain microtubule bundle integrity reduced supporting cell stiffness, which in turn altered cochlear micromechanics in Gas2 mutants. Vibratory responses to sound were measured in cochleae from live mice, revealing defects in the propagation and amplification of the traveling wave in Gas2 mutants. We propose that the microtubule bundling activity of GAS2 imparts supporting cells with mechanical properties for transmitting sound energy through the cochlea.
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http://dx.doi.org/10.1016/j.devcel.2021.04.017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8137675PMC
May 2021

Single cell regulatory landscape of the mouse kidney highlights cellular differentiation programs and disease targets.

Nat Commun 2021 04 15;12(1):2277. Epub 2021 Apr 15.

Renal, Electrolyte, and Hypertension Division, Department of Medicine, University of Pennsylvania, Perelman School of Medicine, Philadelphia, PA, USA.

Determining the epigenetic program that generates unique cell types in the kidney is critical for understanding cell-type heterogeneity during tissue homeostasis and injury response. Here, we profile open chromatin and gene expression in developing and adult mouse kidneys at single cell resolution. We show critical reliance of gene expression on distal regulatory elements (enhancers). We reveal key cell type-specific transcription factors and major gene-regulatory circuits for kidney cells. Dynamic chromatin and expression changes during nephron progenitor differentiation demonstrates that podocyte commitment occurs early and is associated with sustained Foxl1 expression. Renal tubule cells follow a more complex differentiation, where Hfn4a is associated with proximal and Tfap2b with distal fate. Mapping single nucleotide variants associated with human kidney disease implicates critical cell types, developmental stages, genes, and regulatory mechanisms. The single cell multi-omics atlas reveals key chromatin remodeling events and gene expression dynamics associated with kidney development.
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http://dx.doi.org/10.1038/s41467-021-22266-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8050063PMC
April 2021

FoxL1 mesenchymal cells are a critical source of Wnt5a for midgut elongation during mouse embryonic intestinal development.

Cells Dev 2021 Mar 8;165. Epub 2021 Feb 8.

Department of Genetics and Institute for Diabetes, Obesity and Metabolism, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA.

Wnt5a is a non-canonical Wnt ligand that is essential for normal embryonic development in mammals. The role of Wnt5a in early intestinal development has been examined in gene ablation models, where mice exhibit strikingly shortened intestines. However, the exact cellular source of Wnt5a has remained elusive, until a recent study found that FoxL1-expressing mesenchymal cells (FoxL1 cells), which are localized directly beneath the intestinal epithelium, express Wnt5a. To determine whether FoxL1 cells are a required source of Wnt5a during intestinal development, we derived mice, which is the first mouse model to ablate Wnt5a in a cell type-specific manner in the intestine . Our results show that Wnt5a deletion in FoxL1 cells during fetal life causes a shortened gut phenotype in neonatal mice, and that our model is sufficient to increase rate of apoptosis in the elongating epithelium, thus explaining the shortened gut phenotype. However, in contrast to previous studies using Wnt5a null mice, we did not observe dysregulation of epithelial structure or apical-basal protein localization. Altogether, our findings establish a developmental role for FoxL1 mesenchymal cells in controlling non-canonical Wnt signaling during midgut elongation.
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http://dx.doi.org/10.1016/j.cdev.2021.203662DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7988427PMC
March 2021

FoxA-dependent demethylation of DNA initiates epigenetic memory of cellular identity.

Dev Cell 2021 03 25;56(5):602-612.e4. Epub 2021 Feb 25.

Department of Genetics and Institute for Diabetes Obesity and Metabolism, Perelman School of Medicine, University of Pennsylvania, 12-126 Smilow Center for Translational Research, 3400 Civic Center Boulevard, Philadelphia, PA 19104-5156, USA. Electronic address:

Tissue-specific DNA methylation patterns are created by transcription factors that recruit methylation and demethylation enzymes to cis-regulatory elements. To date, it is not known whether transcription factors are needed to continuously maintain methylation profiles in development and mature tissues or whether they only establish these marks during organ development. We queried the role of the pioneer factor FoxA in generating hypomethylated DNA at liver enhancers. We discovered a set of FoxA-binding sites that undergo regional, FoxA-dependent demethylation during organ development. Conditional ablation of FoxA genes in the adult liver demonstrated that continued FoxA presence was not required to maintain the hypomethylated state, even when massive cell proliferation was induced. This study provides strong evidence for the stable, epigenetic nature of tissue-specific DNA methylation patterns directed by lineage-determining transcription factors during organ development.
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http://dx.doi.org/10.1016/j.devcel.2021.02.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8129911PMC
March 2021

Highly multiplexed 2-dimensional imaging mass cytometry analysis of HBV-infected liver.

JCI Insight 2021 04 8;6(7). Epub 2021 Apr 8.

Department of Medicine, University of Pennsylvania Perelman School of Medicine, Philadelphia, Pennsylvania, USA.

Studies of human hepatitis B virus (HBV) immune pathogenesis are hampered by limited access to liver tissues and technologies for detailed analyses. Here, utilizing imaging mass cytometry (IMC) to simultaneously detect 30 immune, viral, and structural markers in liver biopsies from patients with hepatitis B e antigen+ (HBeAg+) chronic hepatitis B, we provide potentially novel comprehensive visualization, quantitation, and phenotypic characterizations of hepatic adaptive and innate immune subsets that correlated with hepatocellular injury, histological fibrosis, and age. We further show marked correlations between adaptive and innate immune cell frequencies and phenotype, highlighting complex immune interactions within the hepatic microenvironment with relevance to HBV pathogenesis.
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http://dx.doi.org/10.1172/jci.insight.146883DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8119221PMC
April 2021

Genetic activation of α-cell glucokinase in mice causes enhanced glucose-suppression of glucagon secretion during normal and diabetic states.

Mol Metab 2021 07 19;49:101193. Epub 2021 Feb 19.

Institute of Diabetes, Obesity, and Metabolism, Perelman School of Medicine, The University of Pennsylvania, 3400 Civic Center Boulevard, Philadelphia, PA 19104, USA; Department of Genetics, Perelman School of Medicine, The University of Pennsylvania, 3400 Civic Center Boulevard, Philadelphia, PA 19104, USA. Electronic address:

Objective: While the molecular events controlling insulin secretion from β-cells have been documented in detail, the exact mechanisms governing glucagon release by α-cells are understood only partially. This is a critical knowledge gap, as the normal suppression of glucagon secretion by elevated glucose levels fails in type 2 diabetes (T2D) patients, contributing to hyperglycemia through stimulation of hepatic glucose production. A critical role of glycolytic flux in regulating glucagon secretion was supported by recent studies in which manipulation of the activity and expression of the glycolytic enzyme glucokinase altered the setpoint for glucose-suppression of glucagon secretion (GSGS). Given this precedent, we hypothesized that genetic activation of glucokinase specifically in α-cells would enhance GSGS and mitigate T2D hyperglucagonemia.

Methods: We derived an inducible, α-cell-specific glucokinase activating mutant mouse model (Gck; Gcg-CreER; henceforth referred to as "α-mutGCK") in which the wild-type glucokinase gene (GCK) is conditionally replaced with a glucokinase mutant allele containing the ins454A activating mutation (Gck∗), a mutation that increases the affinity of glucokinase for glucose by almost 7-fold. The effects of α-cell GCK activation on glucose homeostasis, hormone secretion, islet morphology, and islet numbers were assessed using both in vivo and ex vivo assays. Additionally, the effect of α-cell GCK activation on GSGS was investigated under diabetogenic conditions of high-fat diet (HFD) feeding that dysregulate glucagon secretion.

Results: Our study shows that α-mutGCK mice have enhanced GSGS in vivo and ex vivo, independent of alterations in insulin levels and secretion, islet hormone content, islet morphology, or islet number. α-mutGCK mice maintained on HFD displayed improvements in glucagonemia compared to controls, which developed the expected obesity, glucose intolerance, elevated fasting blood glucose, hyperinsulinemia, and hyperglucagonemia.

Conclusions: Using our novel α-cell specific activation of GCK mouse model, we have provided additional support to demonstrate that the glycolytic enzyme glucokinase is a key determinant in glucose sensing within α-cells to regulate glucagon secretion. Our results contribute to our fundamental understanding of α-cell biology by providing greater insight into the regulation of glucagon secretion through α-cell intrinsic mechanisms via glucokinase. Furthermore, our HFD results underscore the potential of glucokinase as a druggable target which, given the ongoing development of allosteric glucokinase activators (GKAs) for T2D treatment, could help mitigate hyperglucagonemia and potentially improve blood glucose homeostasis.
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http://dx.doi.org/10.1016/j.molmet.2021.101193DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7973249PMC
July 2021

Biphasic dynamics of beta cell mass in a mouse model of congenital hyperinsulinism: implications for type 2 diabetes.

Diabetologia 2021 05 9;64(5):1133-1143. Epub 2021 Feb 9.

Department of Endocrinology and Metabolism, Hadassah Medical Center, Jerusalem, Israel.

Aims/hypothesis: Acute hyperglycaemia stimulates pancreatic beta cell proliferation in the mouse whereas chronic hyperglycaemia appears to be toxic. We hypothesise that this toxic effect is mediated by increased beta cell workload, unrelated to hyperglycaemia per se.

Methods: To test this hypothesis, we developed a novel mouse model of cell-autonomous increased beta cell glycolytic flux caused by a conditional heterozygous beta cell-specific mutation that activates glucokinase (GCK), mimicking key aspects of the rare human genetic disease GCK-congenital hyperinsulinism.

Results: In the mutant mice, we observed random and fasting hypoglycaemia (random 4.5-5.4 mmol/l and fasting 3.6 mmol/l) that persisted for 15 months. GCK activation led to increased beta cell proliferation as measured by Ki67 expression (2.7% vs 1.5%, mutant and wild-type (WT), respectively, p < 0.01) that resulted in a 62% increase in beta cell mass in young mice. However, by 8 months of age, mutant mice developed impaired glucose tolerance, which was associated with decreased absolute beta cell mass from 2.9 mg at 1.5 months to 1.8 mg at 8 months of age, with preservation of individual beta cell function. Impaired glucose tolerance was further exacerbated by a high-fat/high-sucrose diet (AUC 1796 vs 966 mmol/l × min, mutant and WT, respectively, p < 0.05). Activation of GCK was associated with an increased DNA damage response and an elevated expression of Chop, suggesting metabolic stress as a contributor to beta cell death.

Conclusions/interpretation: We propose that increased workload-driven biphasic beta cell dynamics contribute to decreased beta cell function observed in long-standing congenital hyperinsulinism and type 2 diabetes.
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http://dx.doi.org/10.1007/s00125-021-05390-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8117185PMC
May 2021

TCR/BCR dual-expressing cells and their associated public BCR clonotype are not enriched in type 1 diabetes.

Cell 2021 02;184(3):827-839.e14

Department of Microbiology, University of Pennsylvania, Perelman School of Medicine, Philadelphia, PA 19104, USA; Institute for Immunology, University of Pennsylvania, Perelman School of Medicine, Philadelphia, PA 19104, USA. Electronic address:

Ahmed and colleagues recently described a novel hybrid lymphocyte expressing both a B and T cell receptor, termed double expresser (DE) cells. DE cells in blood of type 1 diabetes (T1D) subjects were present at increased numbers and enriched for a public B cell clonotype. Here, we attempted to reproduce these findings. While we could identify DE cells by flow cytometry, we found no association between DE cell frequency and T1D status. We were unable to identify the reported public B cell clone, or any similar clone, in bulk B cells or sorted DE cells from T1D subjects or controls. We also did not observe increased usage of the public clone VH or DH genes in B cells or in sorted DE cells. Taken together, our findings suggest that DE cells and their alleged public clonotype are not enriched in T1D. This Matters Arising paper is in response to Ahmed et al. (2019), published in Cell. See also the response by Ahmed et al. (2021), published in this issue.
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http://dx.doi.org/10.1016/j.cell.2020.11.035DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8016147PMC
February 2021

Tumor-infiltrating mast cells are associated with resistance to anti-PD-1 therapy.

Nat Commun 2021 01 12;12(1):346. Epub 2021 Jan 12.

The Wistar Institute, Philadelphia, PA, USA.

Anti-PD-1 therapy is used as a front-line treatment for many cancers, but mechanistic insight into this therapy resistance is still lacking. Here we generate a humanized (Hu)-mouse melanoma model by injecting fetal liver-derived CD34 cells and implanting autologous thymus in immune-deficient NOD-scid IL2Rγ (NSG) mice. Reconstituted Hu-mice are challenged with HLA-matched melanomas and treated with anti-PD-1, which results in restricted tumor growth but not complete regression. Tumor RNA-seq, multiplexed imaging and immunohistology staining show high expression of chemokines, as well as recruitment of FOXP3 Treg and mast cells, in selective tumor regions. Reduced HLA-class I expression and CD8/Granz B T cells homeostasis are observed in tumor regions where FOXP3 Treg and mast cells co-localize, with such features associated with resistance to anti-PD-1 treatment. Combining anti-PD-1 with sunitinib or imatinib results in the depletion of mast cells and complete regression of tumors. Our results thus implicate mast cell depletion for improving the efficacy of anti-PD-1 therapy.
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http://dx.doi.org/10.1038/s41467-020-20600-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7804257PMC
January 2021

Exome-wide evaluation of rare coding variants using electronic health records identifies new gene-phenotype associations.

Nat Med 2021 01 11;27(1):66-72. Epub 2021 Jan 11.

Division of Cardiothoracic Surgery, Michael E. DeBakey Department of Surgery, Baylor College of Medicine, Houston, TX, USA.

The clinical impact of rare loss-of-function variants has yet to be determined for most genes. Integration of DNA sequencing data with electronic health records (EHRs) could enhance our understanding of the contribution of rare genetic variation to human disease. By leveraging 10,900 whole-exome sequences linked to EHR data in the Penn Medicine Biobank, we addressed the association of the cumulative effects of rare predicted loss-of-function variants for each individual gene on human disease on an exome-wide scale, as assessed using a set of diverse EHR phenotypes. After discovering 97 genes with exome-by-phenome-wide significant phenotype associations (P < 10), we replicated 26 of these in the Penn Medicine Biobank, as well as in three other medical biobanks and the population-based UK Biobank. Of these 26 genes, five had associations that have been previously reported and represented positive controls, whereas 21 had phenotype associations not previously reported, among which were genes implicated in glaucoma, aortic ectasia, diabetes mellitus, muscular dystrophy and hearing loss. These findings show the value of aggregating rare predicted loss-of-function variants into 'gene burdens' for identifying new gene-disease associations using EHR phenotypes in a medical biobank. We suggest that application of this approach to even larger numbers of individuals will provide the statistical power required to uncover unexplored relationships between rare genetic variation and disease phenotypes.
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http://dx.doi.org/10.1038/s41591-020-1133-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8775355PMC
January 2021

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

Islet transplantation in the subcutaneous space achieves long-term euglycaemia in preclinical models of type 1 diabetes.

Nat Metab 2020 10 7;2(10):1013-1020. Epub 2020 Sep 7.

Division of Transplantation, Department of Surgery, Hospital of the University of Pennsylvania, Philadelphia, PA, USA.

The intrahepatic milieu is inhospitable to intraportal islet allografts, limiting their applicability for the treatment of type 1 diabetes. Although the subcutaneous space represents an alternate, safe and easily accessible site for pancreatic islet transplantation, lack of neovascularization and the resulting hypoxic cell death have largely limited the longevity of graft survival and function and pose a barrier to the widespread adoption of islet transplantation in the clinic. Here we report the successful subcutaneous transplantation of pancreatic islets admixed with a device-free islet viability matrix, resulting in long-term euglycaemia in diverse immune-competent and immuno-incompetent animal models. We validate sustained normoglycaemia afforded by our transplantation methodology using murine, porcine and human pancreatic islets, and also demonstrate its efficacy in a non-human primate model of syngeneic islet transplantation. Transplantation of the islet-islet viability matrix mixture in the subcutaneous space represents a simple, safe and reproducible method, paving the way for a new therapeutic paradigm for type 1 diabetes.
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http://dx.doi.org/10.1038/s42255-020-0269-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7572844PMC
October 2020

Organisation of the human pancreas in health and in diabetes.

Diabetologia 2020 10 7;63(10):1966-1973. Epub 2020 Sep 7.

Department of Genetics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA.

For much of the last century, our knowledge regarding the pancreas in type 1 and type 2 diabetes was largely derived from autopsy studies of individuals with these disorders or investigations utilising rodent models of either disease. While many important insights emanated from these efforts, the mode for investigation has increasingly seen change due to the availability of transplant-quality organ-donor tissues, improvements in pancreatic imaging, advances in metabolic assessments of living patients, genetic analyses, technological advances for laboratory investigation and more. As a result, many long-standing notions regarding the role for and the changes that occur in the pancreas in individuals with these disorders have come under question, while, at the same time, new issues (e.g., beta cell persistence, disease heterogeneity, exocrine contributions) have arisen. In this article, we will consider the vital role of the pancreas in human health and physiology, including discussion of its anatomical features and dual (exocrine and endocrine) functions. Specifically, we convey changes that occur in the pancreas of those with either type 1 or type 2 diabetes, with careful attention to the facets that may contribute to the pathogenesis of either disorder. Finally, we discuss the emerging unknowns with the belief that understanding the role of the pancreas in type 1 and type 2 diabetes will lead to improvements in disease diagnosis, understanding of disease heterogeneity and optimisation of treatments at a personalised level. Graphical abstract.
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http://dx.doi.org/10.1007/s00125-020-05203-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7565096PMC
October 2020
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