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Type 2 diabetes is characterized by insulin resistance, hyperglycemia, and progressive β cell dysfunction. Excess glucose and lipid impair β cell function in islet cell lines, cultured rodent and human islets, and in vivo rodent models. Here, we examined the mechanistic consequences of glucotoxic and lipotoxic conditions on human islets in vivo and developed and/or used 3 complementary models that allowed comparison of the effects of hyperglycemic and/or insulin-resistant metabolic stress conditions on human and mouse islets, which responded quite differently to these challenges. Hyperglycemia and/or insulin resistance impaired insulin secretion only from human islets in vivo. In human grafts, chronic insulin resistance decreased antioxidant enzyme expression and increased superoxide and amyloid formation. In human islet grafts, expression of transcription factors NKX6.1 and MAFB was decreased by chronic insulin resistance, but only MAFB decreased under chronic hyperglycemia. Knockdown of NKX6.1 or MAFB expression in a human β cell line recapitulated the insulin secretion defect seen in vivo. Contrary to rodent islet studies, neither insulin resistance nor hyperglycemia led to human β cell proliferation or apoptosis. These results demonstrate profound differences in how excess glucose or lipid influence mouse and human insulin secretion and β cell activity and show that reduced expression of key islet-enriched transcription factors is an important mediator of glucotoxicity and lipotoxicity.

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.

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.

Intraislet Endothelial Cells Contribute to Revascularization of Transplanted Pancreatic Islets Marcela Brissova 1 , Michael Fowler 1 , Peter Wiebe 2 , Alena Shostak 1 , Masakazu Shiota 2 , Aramandla Radhika 1 , P. Charles Lin 3 , Maureen Gannon 1 2 and Alvin C. Powers 1 2 4 1 Department of Medicine, Division of Diabetes, Endocrinology, and Metabolism, Vanderbilt University Medical Center, Nashville, Tennessee 2 Department of Molecular Physiology and Biophysics, Vanderbilt University Medical Center, Nashville, Tennessee 3 Department of Radiation Oncology, Vanderbilt University Medical Center, Nashville, Tennessee 4 VA Tennessee Valley Healthcare System, Nashville, Tennessee Address correspondencereprint requests to Alvin C. Powers, Division of Diabetes, Endocrinology,Metabolism, 715 PRB, Vanderbilt University, Nashville, TN 37232. E-mail: al.powers{at}vanderbilt.edu Abstract Pancreatic islet transplantation is an emerging therapy for type 1 diabetes. To survive and function, transplanted islets must revascularize because islet isolation severs arterial and venous connections; the current paradigm is that islet revascularization originates from the transplant recipient. Because isolated islets retain intraislet endothelial cells, we determined whether these endothelial cells contribute to the revascularization using a murine model with tagged endothelial cells (lacZ knock-in to Flk-1/VEGFR2 gene) and using transplanted human islets. At 3–5 weeks after transplantation beneath the renal capsule, we found that islets were revascularized and that the transplant recipient vasculature indeed contributed to the revascularization process. Using the lacZ-tagged endothelial cell model, we found that intraislet endothelial cells not only survived after transplantation but became a functional part of revascularized islet graft. A similar contribution of intraislet endothelial cells was also seen with human islets transplanted into an immunodeficient mouse model. In the murine model, individual blood vessels within the islet graft consisted of donor or recipient endothelial cells or were a chimera of donor and recipient endothelial cells, indicating that both sources of endothelial cells contribute to the new vasculature. These observations suggest that interventions to activate, amplify, or sustain intraislet endothelial cells before and after transplantation may facilitate islet revascularization, enhance islet survival, and improve islet transplantation. PECAM, platelet endothelial cell adhesion molecule Footnotes Additional information for this article can be found in an online appendix at http://diabetes.diabetesjournals.org . Accepted December 23, 2003. Received October 17, 2003. DIABETES

Dipeptidase-1 (DPEP1) is highly upregulated in colorectal cancer (CRC), with its enzymatic function linked to invasion and metastasis. More recently, DPEP1 was found to serve as a receptor for neutrophils when expressed by activated endothelial cells. It is unknown whether neutrophils bind to DPEP1-expressing CRC cells and whether this impacts features of CRC. Neutrophils have been shown to be tumor promoting in cancers including CRC, where they act to exclude CD8+ T cells. Herein, we show that neutrophils bind DPEP1-expressing CRC cells. In addition, DPEP1 is preferentially expressed in microsatellite-stable (MSS) CRCs, in which there are a paucity of CD8+ T cells, whereas DPEP1 is negatively correlated with microsatellite-unstable (MSI-H) CRCs, which are T cell rich and are more responsive to immunotherapy. Remarkably, carcinogen-treated Dpep1-null mice develop multiple, large, plaque-like, locally invasive adenocarcinomas and squamous cell cancers in the distal colon. These adenocarcinomas exhibit a marked reduction in neutrophils and an influx CD8+ T cells, along with reduced expression of mismatch repair proteins, consistent with features of MSI-H CRC. These results establish DPEP1's importance in maintaining MSS CRC and its ability to shape the tumor microenvironment.

We combined multimodal imaging (bioluminescence, X-ray computed tomography, and PET), tomographic reconstruction of bioluminescent sources, and two unique, complementary models to evaluate three previously synthesized PET radiotracers thought to target pancreatic beta cells. The three radiotracers {[18F]fluoropropyl-(+)-dihydrotetrabenazine ([18F]FP-DTBZ), [18F](+)-2-oxiranyl-3-isobutyl-9-(3-fluoropropoxy)-10-methoxy-2,3,4,6,7,11b-hexahydro-1H-pyrido[2,1-a]isoquinoline (18F-AV-266), and (2S,3R,11bR)-9-(3-fluoropropoxy)-2-(hydroxymethyl)-3-isobutyl-10-methoxy-2,3,4,6,7,11b-hexahydro-1H-pyrido[2,1-a]isoquinolin-2-ol (18F-AV-300)} bind vesicular monoamine transporter 2. Tomographic reconstruction of the bioluminescent signal in mice expressing luciferase only in pancreatic beta cells was used to delineate the pancreas and was coregistered with PET and X-ray computed tomography images. This strategy enabled unambiguous identification of the pancreas on PET images, permitting accurate quantification of the pancreatic PET signal. We show here that, after conditional, specific, and rapid mouse beta-cell ablation, beta-cell loss was detected by bioluminescence imaging but not by PET imaging, given that the pancreatic signal provided by three PET radiotracers was not altered. To determine whether these ligands bound human beta cells in vivo, we imaged mice transplanted with luciferase-expressing human islets. The human islets were imaged by bioluminescence but not with the PET ligands, indicating that these vesicular monoamine transporter 2-directed ligands did not specifically bind beta cells. These data demonstrate the utility of coregistered multimodal imaging as a platform for evaluation and validation of candidate ligands for imaging islets.

Type 2 diabetes mellitus (T2D), a major cause of worldwide morbidity and mortality, is characterized by dysfunction of insulin-producing pancreatic islet β cells 1 , 2 . T2D genome-wide association studies (GWAS) have identified hundreds of signals in non-coding and β cell regulatory genomic regions, but deciphering their biological mechanisms remains challenging 3 – 5 . Here, to identify early disease-driving events, we performed traditional and multiplexed pancreatic tissue imaging, sorted-islet cell transcriptomics and islet functional analysis of early-stage T2D and control donors. By integrating diverse modalities, we show that early-stage T2D is characterized by β cell-intrinsic defects that can be proportioned into gene regulatory modules with enrichment in signals of genetic risk. After identifying the β cell hub gene and transcription factor RFX6 within one such module, we demonstrated multiple layers of genetic risk that converge on an RFX6 -mediated network to reduce insulin secretion by β cells. RFX6 perturbation in primary human islet cells alters β cell chromatin architecture at regions enriched for T2D GWAS signals, and population-scale genetic analyses causally link genetically predicted reduced RFX6 expression with increased T2D risk. Understanding the molecular mechanisms of complex, systemic diseases necessitates integration of signals from multiple molecules, cells, organs and individuals, and thus we anticipate that this approach will be a useful template to identify and validate key regulatory networks and master hub genes for other diseases or traits using GWAS data. Integration of multiomics data with functional analysis of pancreatic tissues from individuals with early-stage type 2 diabetes indicates that the genetic risk converges on RFX6 , which regulates chromatin architecture at multiple risk loci.

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.

Posttransplantation diabetes mellitus (PTDM) is a common and significant complication related to immunosuppressive agents required to prevent organ or cell transplant rejection. To elucidate the effects of 2 commonly used agents, the calcineurin inhibitor tacrolimus (TAC) and the mTOR inhibitor sirolimus (SIR), on islet function and test whether these effects could be reversed or prevented, we investigated human islets transplanted into immunodeficient mice treated with TAC or SIR at clinically relevant levels. Both TAC and SIR impaired insulin secretion in fasted and/or stimulated conditions. Treatment with TAC or SIR increased amyloid deposition and islet macrophages, disrupted insulin granule formation, and induced broad transcriptional dysregulation related to peptide processing, ion/calcium flux, and the extracellular matrix; however, it did not affect regulation of β cell mass. Interestingly, these β cell abnormalities reversed after withdrawal of drug treatment. Furthermore, cotreatment with a GLP-1 receptor agonist completely prevented TAC-induced β cell dysfunction and partially prevented SIR-induced β cell dysfunction. These results highlight the importance of both calcineurin and mTOR signaling in normal human β cell function in vivo and suggest that modulation of these pathways may prevent or ameliorate PTDM.