Genetic risk converges on regulatory networks mediating early type 2 diabetes.

Type 2 diabetes mellitus (T2D), a major cause of worldwide morbidity and mortality, is characterized by dysfunction of insulin-producing pancreatic islet ß cells1,2. T2D genome-wide association studies (GWAS) have identified hundreds of signals in non-coding and ß cell regulatory genomic regions, but deciphering their biological mechanisms remains challenging3-5. 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 ß cell-intrinsic defects that can be proportioned into gene regulatory modules with enrichment in signals of genetic risk. After identifying the ß 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 ß cells. RFX6 perturbation in primary human islet cells alters ß 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.

Human Pseudoislet System for Synchronous Assessment of Fluorescent Biosensor Dynamics and Hormone Secretory Profiles.

The pancreatic islets of Langerhans, which are small 3D collections of specialized endocrine and supporting cells interspersed throughout the pancreas, have a central role in the control of glucose homeostasis through the secretion of insulin by beta cells, which lowers blood glucose, and glucagon by alpha cells, which raises blood glucose. Intracellular signaling pathways, including those mediated by cAMP, are key for regulated alpha and beta cell hormone secretion. The 3D islet structure, while essential for coordinated islet function, presents experimental challenges for mechanistic studies of the intracellular signaling pathways in primary human islet cells. To overcome these challenges and limitations, this protocol describes an integrated live-cell imaging and microfluidic platform using primary human pseudoislets generated from donors without diabetes that resemble native islets in their morphology, composition, and function. These pseudoislets are size-controlled through the dispersion and reaggregation process of primary human islet cells. In the dispersed state, islet cell gene expression can be manipulated; for example, biosensors such as the genetically encoded cAMP biosensor, cADDis, can be introduced. Once formed, pseudoislets expressing a genetically encoded biosensor, in combination with confocal microscopy and a microperifusion platform, allow for the synchronous assessment of fluorescent biosensor dynamics and alpha and beta cell hormone secretory profiles to provide more insight into cellular processes and function.

Exocrine pancreas in type 1 and type 2 diabetes: different patterns of fibrosis, metaplasia, angiopathy, and adiposity.

The endocrine and exocrine compartments of the pancreas are spatially related but functionally distinct. Multiple diseases affect both compartments, including type 1 diabetes (T1D), pancreatitis, cystic fibrosis, and pancreatic cancer. To better understand how the exocrine pancreas changes with age, obesity, and diabetes, we performed systematic analysis of wellpreserved tissue sections from the pancreatic head, body, and tail of organ donors with T1D (n = 20), type 2 diabetes (T2D, n = 25), and donors with no diabetes (ND, n = 74). Among ND donors, we found that acinar-to-ductal metaplasia (ADM), angiopathy, and pancreatic adiposity increased with age, while ADM and adiposity also increased with BMI. Compared to age- and sex-matched ND organs, T1D pancreata had greater acinar atrophy and angiopathy with fewer intralobular adipocytes. T2D pancreata had greater ADM, angiopathy, and total T lymphocytes, but no difference in adipocyte number, compared to ND organs. While total pancreatic fibrosis was increased in both T1D and T2D, the pattern was different with T1D pancreata having greater periductal and perivascular fibrosis, whereas T2D pancreata had greater lobular and parenchymal fibrosis. Thus, the exocrine pancreas undergoes distinct changes as individuals age or develop T1D or T2D.

Human pancreatic capillaries and nerve fibers persist in type 1 diabetes despite beta cell loss.

The autonomic nervous system regulates pancreatic function. Islet capillaries are essential for the extension of axonal projections into islets, and both of these structures are important for appropriate islet hormone secretion. Because beta cells provide important paracrine cues for islet glucagon secretion and neurovascular development, we postulated that beta cell loss in type 1 diabetes (T1D) would lead to a decline in intraislet capillaries and reduction of islet innervation, possibly contributing to abnormal glucagon secretion. To define morphological characteristics of capillaries and nerve fibers in islets and acinar tissue compartments, we analyzed neurovascular assembly across the largest cohort of T1D and normal individuals studied thus far. Because innervation has been studied extensively in rodent models of T1D, we also compared the neurovascular architecture between mouse and human pancreas and assembled transcriptomic profiles of molecules guiding islet angiogenesis and neuronal development. We found striking interspecies differences in islet neurovascular assembly but relatively modest differences at transcriptome level, suggesting that posttranscriptional regulation may be involved in this process. To determine whether islet neurovascular arrangement is altered after beta cell loss in T1D, we compared pancreatic tissues from non-diabetic, recent-onset T1D (<10-yr duration), and longstanding T1D (>10-yr duration) donors. Recent-onset T1D showed greater islet and acinar capillary density compared to non-diabetic and longstanding T1D donors. Both recent-onset and longstanding T1D had greater islet nerve fiber density compared to non-diabetic donors. We did not detect changes in sympathetic axons in either T1D cohort. Additionally, nerve fibers overlapped with extracellular matrix (ECM), supporting its role in the formation and function of axonal processes. These results indicate that pancreatic capillaries and nerve fibers persist in T1D despite beta cell loss, suggesting that alpha cell secretory changes may be decoupled from neurovascular components.NEW & NOTEWORTHY Defining the neurovascular architecture in the pancreas of individuals with type 1 diabetes (T1D) is crucial to understanding the mechanisms of dysregulated glucagon secretion. In the largest T1D cohort of biobanked tissues analyzed to date, we found that pancreatic capillaries and nerve fibers persist in human T1D despite beta cell loss, suggesting that alpha cell secretory changes may be decoupled from neurovascular components. Because innervation has been studied extensively in rodent T1D models, our studies also provide the first rigorous direct comparisons of neurovascular assembly in mouse and human, indicating dramatic interspecies differences.

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

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 ß-cell mass with some islets devoid of ß cells (29.3% of 311 islets) while other islets had many ß 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 ß-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.

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

Islet-enriched transcription factors (TFs) exert broad control over cellular processes in pancreatic α and ß 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 ß cells based on combinatorial TF expression. Subpopulations of islet cells coexpressing ARX/MAFB (α cells) and MAFA/MAFB (ß 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 ß cells showed enhanced electrophysiological activity. Thus, these results indicate that combinatorial TF expression in islet α and ß cells predicts highly functional, mature subpopulations.

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

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

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

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 ß cells. This would require binding and entry of SARS-CoV-2 into ß 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 ß cells. In pancreatic sections, ACE2 and TMPRSS2 protein was not detected in ß 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 ß cells in vivo through ACE2 and TMPRSS2.

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

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 ß cells. This would require binding and entry of SARS-CoV-2 into host ß 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. ACE2 and TMPRSS2 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 ß 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 ß cells through these cell entry proteins.

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

Pancreatic islets secrete insulin from ß 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 ß 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 ß and α cells.

Tacrolimus- and sirolimus-induced human ß cell dysfunction is reversible and preventable.

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 ß cell mass. Interestingly, these ß cell abnormalities reversed after withdrawal of drug treatment. Furthermore, cotreatment with a GLP-1 receptor agonist completely prevented TAC-induced ß cell dysfunction and partially prevented SIR-induced ß cell dysfunction. These results highlight the importance of both calcineurin and mTOR signaling in normal human ß cell function in vivo and suggest that modulation of these pathways may prevent or ameliorate PTDM.

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

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 ß 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.

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

Identification of cell-surface markers specific to human pancreatic ß 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 ß 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 ß cells. Given its specificity and membrane localization, we utilized an NTPDase3 antibody for purification of live human ß cells as confirmed by transcriptional profiling, and, in addition, for in vivo imaging of transplanted human ß cells. Thus, NTPDase3 is a cell-surface biomarker of adult human ß cells, and the antibody directed to this protein should be a useful new reagent for ß cell sorting, in vivo imaging, and targeting.

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

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 ß cell CFTR deletion and normal and CF human pancreas and islets. Specific deletion of CFTR from murine ß cells did not affect ß 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 ß 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 ß cell loss and intraislet inflammation in the setting of a complex pleiotropic disease and not by intrinsic islet dysfunction from CFTR mutation.

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

Many patients with type 1 diabetes (T1D) have residual ß 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 ß 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 ß 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 ß 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-ß cell conversion. These results suggest an explanation for the disordered T1D counterregulatory glucagon response to hypoglycemia.

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

Numerous compounds stimulate rodent ß-cell proliferation; however, translating these findings to human ß-cells remains a challenge. To examine human ß-cell proliferation in response to such compounds, we developed a medium-throughput in vitro method of quantifying adult human ß-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 ß-cells with high specificity using both nuclear and cytoplasmic markers. ß-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-1ß 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+ ß-cells, whereas treatment with other compounds had limited to no effect on human ß-cell proliferation. This new scalable approach reduces the time and effort required for sensitive and specific evaluation of human ß-cell proliferation, thus allowing for increased testing of candidate human ß-cell mitogens.

Stress-impaired transcription factor expression and insulin secretion in transplanted human islets.

Type 2 diabetes is characterized by insulin resistance, hyperglycemia, and progressive ß cell dysfunction. Excess glucose and lipid impair ß 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 ß cell line recapitulated the insulin secretion defect seen in vivo. Contrary to rodent islet studies, neither insulin resistance nor hyperglycemia led to human ß cell proliferation or apoptosis. These results demonstrate profound differences in how excess glucose or lipid influence mouse and human insulin secretion and ß cell activity and show that reduced expression of key islet-enriched transcription factors is an important mediator of glucotoxicity and lipotoxicity.

Human islet preparations distributed for research exhibit a variety of insulin-secretory profiles.

Human islet research is providing new insights into human islet biology and diabetes, using islets isolated at multiple US centers from donors with varying characteristics. This creates challenges for understanding, interpreting, and integrating research findings from the many laboratories that use these islets. In what is, to our knowledge, the first standardized assessment of human islet preparations from multiple isolation centers, we measured insulin secretion from 202 preparations isolated at 15 centers over 11 years and noted five distinct patterns of insulin secretion. Approximately three quarters were appropriately responsive to stimuli, but one quarter were dysfunctional, with unstable basal insulin secretion and/or an impairment in stimulated insulin secretion. Importantly, the patterns of insulin secretion by responsive human islet preparations (stable Baseline and Fold stimulation of insulin secretion) isolated at different centers were similar and improved slightly over the years studied. When all preparations studied were considered, basal and stimulated insulin secretion did not correlate with isolation center, biological differences of the islet donor, or differences in isolation, such as Cold Ischemia Time. Dysfunctional islet preparations could not be predicted from the information provided by the isolation center and had altered expression of genes encoding components of the glucose-sensing pathway, but not of insulin production or cell death. These results indicate that insulin secretion by most preparations from multiple centers is similar but that in vitro responsiveness of human islets cannot be predicted, necessitating preexperimental human islet assessment. These results should be considered when one is designing, interpreting, and integrating experiments using human islets.

Vascular endothelial growth factor coordinates islet innervation via vascular scaffolding.

Neurovascular alignment is a common anatomical feature of organs, but the mechanisms leading to this arrangement are incompletely understood. Here, we show that vascular endothelial growth factor (VEGF) signaling profoundly affects both vascularization and innervation of the pancreatic islet. In mature islets, nerves are closely associated with capillaries, but the islet vascularization process during embryonic organogenesis significantly precedes islet innervation. Although a simple neuronal meshwork interconnects the developing islet clusters as they begin to form at E14.5, the substantial ingrowth of nerve fibers into islets occurs postnatally, when islet vascularization is already complete. Using genetic mouse models, we demonstrate that VEGF regulates islet innervation indirectly through its effects on intra-islet endothelial cells. Our data indicate that formation of a VEGF-directed, intra-islet vascular plexus is required for development of islet innervation, and that VEGF-induced islet hypervascularization leads to increased nerve fiber ingrowth. Transcriptome analysis of hypervascularized islets revealed an increased expression of extracellular matrix components and axon guidance molecules, with these transcripts being enriched in the islet-derived endothelial cell population. We propose a mechanism for coordinated neurovascular development within pancreatic islets, in which endocrine cell-derived VEGF directs the patterning of intra-islet capillaries during embryogenesis, forming a scaffold for the postnatal ingrowth of essential autonomic nerve fibers.

Islet microenvironment, modulated by vascular endothelial growth factor-A signaling, promotes ß cell regeneration.

Pancreatic islet endocrine cell and endothelial cell (EC) interactions mediated by vascular endothelial growth factor-A (VEGF-A) signaling are important for islet differentiation and the formation of highly vascularized islets. To dissect how VEGF-A signaling modulates intra-islet vasculature, islet microenvironment, and ß cell mass, we transiently increased VEGF-A production by ß cells. VEGF-A induction dramatically increased the number of intra-islet ECs but led to ß cell loss. After withdrawal of the VEGF-A stimulus, ß cell mass, function, and islet structure normalized as a result of a robust, but transient, burst in proliferation of pre-existing ß cells. Bone marrow-derived macrophages (MΦs) recruited to the site of ß cell injury were crucial for the ß cell proliferation, which was independent of pancreatic location and circulating factors such as glucose. Identification of the signals responsible for the proliferation of adult, terminally differentiated ß cells will improve strategies aimed at ß cell regeneration and expansion.