Urocortin 3 contributes to paracrine inhibition of islet alpha cells in mice.

Pancreatic alpha cell activity and glucagon secretion lower as glucose levels increase. While part of the decrease is regulated by glucose itself, paracrine signaling by their neighboring beta and delta cells also plays an important role. Somatostatin from delta cells is an important local inhibitor of alpha cells at high glucose. Additionally, urocortin 3 (UCN3) is a hormone that is co-released from beta cells with insulin and acts locally to potentiate somatostatin secretion from delta cells. UCN3 thus inhibits insulin secretion via a negative feedback loop with delta cells, but its role with respect to alpha cells and glucagon secretion is not understood. We hypothesize that the somatostatin-driven glucagon inhibition at high glucose is regulated in part by UCN3 from beta cells. Here, we use a combination of live functional Ca2+ and cAMP imaging as well as direct glucagon secretion measurement, all from alpha cells in intact mouse islets, to determine the contributions of UCN3 to alpha cell behavior. Exogenous UCN3 treatment decreased alpha cell Ca2+ and cAMP levels and inhibited glucagon release. Blocking endogenous UCN3 signaling increased alpha cell Ca2+ by 26.8 ± 7.6%, but this did not result in increased glucagon release at high glucose. Furthermore, constitutive deletion of Ucn3 did not increase Ca2+ activity or glucagon secretion relative to controls. UCN3 is thus capable of inhibiting mouse alpha cells, but, given the subtle effects of endogenous UCN3 signaling on alpha cells, we propose that UCN3-driven somatostatin may serve to regulate local paracrine glucagon levels in the islet instead of inhibiting gross systemic glucagon release.

Paracrine signalling by pancreatic δ cells determines the glycaemic set point in mice.

While pancreatic ß and α cells are considered the main drivers of blood glucose homeostasis through insulin and glucagon secretion, the contribution of δ cells and somatostatin (SST) secretion to glucose homeostasis remains unresolved. Here we provide a quantitative assessment of the physiological contribution of δ cells to the glycaemic set point in mice. Employing three orthogonal mouse models to remove SST signalling within the pancreas or transplanted islets, we demonstrate that ablating δ cells or SST leads to a sustained decrease in the glycaemic set point. This reduction coincides with a decreased glucose threshold for insulin response from ß cells, leading to increased insulin secretion to the same glucose challenge. Our data demonstrate that ß cells are sufficient to maintain stable glycaemia and reveal that the physiological role of δ cells is to provide tonic feedback inhibition that reduces the ß cell glucose threshold and consequently lowers the glycaemic set point in vivo.

Coordination between ECM and cell-cell adhesion regulates the development of islet aggregation, architecture, and functional maturation.

Pancreatic islets are three-dimensional cell aggregates consisting of unique cellular composition, cell-to-cell contacts, and interactions with blood vessels. Cell aggregation is essential for islet endocrine function; however, it remains unclear how developing islets establish aggregation. By combining genetic animal models, imaging tools, and gene expression profiling, we demonstrate that islet aggregation is regulated by extracellular matrix signaling and cell-cell adhesion. Islet endocrine cell-specific inactivation of extracellular matrix receptor integrin ß1 disrupted blood vessel interactions but promoted cell-cell adhesion and the formation of larger islets. In contrast, ablation of cell-cell adhesion molecule α-catenin promoted blood vessel interactions yet compromised islet clustering. Simultaneous removal of integrin ß1 and α-catenin disrupts islet aggregation and the endocrine cell maturation process, demonstrating that establishment of islet aggregates is essential for functional maturation. Our study provides new insights into understanding the fundamental self-organizing mechanism for islet aggregation, architecture, and functional maturation.

Species-specific roles for the MAFA and MAFB transcription factors in regulating islet ß cell identity.

Type 2 diabetes (T2D) is associated with compromised identity of insulin-producing pancreatic islet ß cells, characterized by inappropriate production of other islet cell-enriched hormones. Here, we examined how hormone misexpression was influenced by the MAFA and MAFB transcription factors, closely related proteins that maintain islet cell function. Mice specifically lacking MafA in ß cells demonstrated broad, population-wide changes in hormone gene expression with an overall gene signature closely resembling islet gastrin+ (Gast+) cells generated under conditions of chronic hyperglycemia and obesity. A human ß cell line deficient in MAFB, but not one lacking MAFA, also produced a GAST+ gene expression pattern. In addition, GAST was detected in human T2D ß cells with low levels of MAFB. Moreover, evidence is provided that human MAFB can directly repress GAST gene transcription. These results support a potentially novel, species-specific role for MafA and MAFB in maintaining adult mouse and human ß cell identity, respectively. Here, we discuss the possibility that induction of Gast/GAST and other non-ß cell hormones, by reduction in the levels of these transcription factors, represents a dysfunctional ß cell signature.

Chromatin accessibility differences between alpha, beta, and delta cells identifies common and cell type-specific enhancers.

BACKGROUND: High throughput sequencing has enabled the interrogation of the transcriptomic landscape of glucagon-secreting alpha cells, insulin-secreting beta cells, and somatostatin-secreting delta cells. These approaches have furthered our understanding of expression patterns that define healthy or diseased islet cell types and helped explicate some of the intricacies between major islet cell crosstalk and glucose regulation. All three endocrine cell types derive from a common pancreatic progenitor, yet alpha and beta cells have partially opposing functions, and delta cells modulate and control insulin and glucagon release. While gene expression signatures that define and maintain cellular identity have been widely explored, the underlying epigenetic components are incompletely characterized and understood. However, chromatin accessibility and remodeling is a dynamic attribute that plays a critical role to determine and maintain cellular identity. RESULTS: Here, we compare and contrast the chromatin landscape between mouse alpha, beta, and delta cells using ATAC-Seq to evaluate the significant differences in chromatin accessibility. The similarities and differences in chromatin accessibility between these related islet endocrine cells help define their fate in support of their distinct functional roles. We identify patterns that suggest that both alpha and delta cells are poised, but repressed, from becoming beta-like. We also identify patterns in differentially enriched chromatin that have transcription factor motifs preferentially associated with different regions of the genome. Finally, we not only confirm and visualize previously discovered common endocrine- and cell specific- enhancer regions across differentially enriched chromatin, but identify novel regions as well. We compiled our chromatin accessibility data in a freely accessible database of common endocrine- and cell specific-enhancer regions that can be navigated with minimal bioinformatics expertise. CONCLUSIONS: Both alpha and delta cells appear poised, but repressed, from becoming beta cells in murine pancreatic islets. These data broadly support earlier findings on the plasticity in identity of non-beta cells under certain circumstances. Furthermore, differential chromatin accessibility shows preferentially enriched distal-intergenic regions in beta cells, when compared to either alpha or delta cells.

A beta cell subset with enhanced insulin secretion and glucose metabolism is reduced in type 2 diabetes.

The pancreatic islets are composed of discrete hormone-producing cells that orchestrate systemic glucose homeostasis. Here we identify subsets of beta cells using a single-cell transcriptomic approach. One subset of beta cells marked by high CD63 expression is enriched for the expression of mitochondrial metabolism genes and exhibits higher mitochondrial respiration compared with CD63lo beta cells. Human and murine pseudo-islets derived from CD63hi beta cells demonstrate enhanced glucose-stimulated insulin secretion compared with pseudo-islets from CD63lo beta cells. We show that CD63hi beta cells are diminished in mouse models of and in humans with type 2 diabetes. Finally, transplantation of pseudo-islets generated from CD63hi but not CD63lo beta cells into diabetic mice restores glucose homeostasis. These findings suggest that loss of a specific subset of beta cells may lead to diabetes. Strategies to reconstitute or maintain CD63hi beta cells may represent a potential anti-diabetic therapy.

Nutrient regulation of the islet epigenome controls adaptive insulin secretion.

Adaptation of the islet ß cell insulin-secretory response to changing insulin demand is critical for blood glucose homeostasis, yet the mechanisms underlying this adaptation are unknown. Here, we have shown that nutrient-stimulated histone acetylation plays a key role in adapting insulin secretion through regulation of genes involved in ß cell nutrient sensing and metabolism. Nutrient regulation of the epigenome occurred at sites occupied by the chromatin-modifying enzyme lysine-specific demethylase 1 (Lsd1) in islets. ß Cell-specific deletion of Lsd1 led to insulin hypersecretion, aberrant expression of nutrient-response genes, and histone hyperacetylation. Islets from mice adapted to chronically increased insulin demand exhibited shared epigenetic and transcriptional changes. Moreover, we found that genetic variants associated with type 2 diabetes were enriched at LSD1-bound sites in human islets, suggesting that interpretation of nutrient signals is genetically determined and clinically relevant. Overall, these studies revealed that adaptive insulin secretion involves Lsd1-mediated coupling of nutrient state to regulation of the islet epigenome.

Dynamic Ins2 Gene Activity Defines ß-Cell Maturity States.

Transcriptional and functional cellular specialization has been described for insulin-secreting ß-cells of the endocrine pancreas. However, it is not clear whether ß-cell heterogeneity is stable or reflects dynamic cellular states. We investigated the temporal kinetics of endogenous insulin gene activity using live cell imaging, with complementary experiments using FACS and single-cell RNA sequencing, in ß-cells from Ins2GFP knockin mice. In vivo staining and FACS analysis of islets from Ins2GFP mice confirmed that at a given moment, ∼25% of ß-cells exhibited significantly higher activity at the evolutionarily conserved insulin gene, Ins2. Live cell imaging over days captured Ins2 gene activity dynamics in single ß-cells. Autocorrelation analysis revealed a subset of oscillating cells, with mean oscillation periods of 17 h. Increased glucose concentrations stimulated more cells to oscillate and resulted in higher average Ins2 gene activity per cell. Single-cell RNA sequencing showed that Ins2(GFP)HIGH ß-cells were enriched for markers of ß-cell maturity. Ins2(GFP)HIGH ß-cells were also significantly less viable at all glucose concentrations and in the context of endoplasmic reticulum stress. Collectively, our results demonstrate that the heterogeneity of insulin production, observed in mouse and human ß-cells, can be accounted for by dynamic states of insulin gene activity.

ß-Cell Succinate Dehydrogenase Deficiency Triggers Metabolic Dysfunction and Insulinopenic Diabetes.

Mitochondrial dysfunction plays a central role in type 2 diabetes (T2D); however, the pathogenic mechanisms in pancreatic ß-cells are incompletely elucidated. Succinate dehydrogenase (SDH) is a key mitochondrial enzyme with dual functions in the tricarboxylic acid cycle and electron transport chain. Using samples from human with diabetes and a mouse model of ß-cell-specific SDH ablation (SDHBßKO), we define SDH deficiency as a driver of mitochondrial dysfunction in ß-cell failure and insulinopenic diabetes. ß-Cell SDH deficiency impairs glucose-induced respiratory oxidative phosphorylation and mitochondrial membrane potential collapse, thereby compromising glucose-stimulated ATP production, insulin secretion, and ß-cell growth. Mechanistically, metabolomic and transcriptomic studies reveal that the loss of SDH causes excess succinate accumulation, which inappropriately activates mammalian target of rapamycin (mTOR) complex 1-regulated metabolic anabolism, including increased SREBP-regulated lipid synthesis. These alterations, which mirror diabetes-associated human ß-cell dysfunction, are partially reversed by acute mTOR inhibition with rapamycin. We propose SDH deficiency as a contributing mechanism to the progressive ß-cell failure of diabetes and identify mTOR complex 1 inhibition as a potential mitigation strategy.

Urocortin3: Local inducer of somatostatin release and bellwether of beta cell maturity.

Urocortin 3 (UCN3) is a peptide hormone expressed in pancreatic islets of Langerhans of both human alpha and human beta cells and solely in murine beta cells. UCN3 signaling acts locally within the islet to activate its cognate receptor, corticotropin releasing hormone receptor 2 (CRHR2), which is expressed by delta cells, to potentiate somatostatin (SST) negative feedback to reduce islet cell hormone output. The functional importance of UCN3 signaling in the islet is to modulate the amount of SST tone allowing for finely tuned regulation of insulin and glucagon secretion. UCN3 signaling is a hallmark of functional beta cell maturation, increasing the beta cell glucose threshold for insulin secretion. In doing so, UCN3 plays a relevant functional role in accurately maintaining blood glucose homeostasis. Additionally, UCN3 acts as an indicator of beta cell maturation and health, as UCN3 is not expressed in immature beta cells and is downregulated in dedifferentiated and dysfunctional beta cell states. Here, we review the mechanistic underpinnings of UCN3 signaling, its net effect on islet cell hormone output, as well as its value as a marker for beta cell maturation and functional status.

IAPP-induced beta cell stress recapitulates the islet transcriptome in type 2 diabetes.

AIMS/HYPOTHESIS: Type 2 diabetes is characterised by islet amyloid and toxic oligomers of islet amyloid polypeptide (IAPP). We posed the questions, (1) does IAPP toxicity induce an islet response comparable to that in humans with type 2 diabetes, and if so, (2) what are the key transcriptional drivers of this response? METHODS: The islet transcriptome was evaluated in five groups of mice: beta cell specific transgenic for (1) human IAPP, (2) rodent IAPP, (3) human calpastatin, (4) human calpastatin and human IAPP, and (5) wild-type mice. RNA sequencing data was analysed by differential expression analysis and gene co-expression network analysis to establish the islet response to adaptation to an increased beta cell workload of soluble rodent IAPP, the islet response to increased expression of oligomeric human IAPP, and the extent to which the latter was rescued by suppression of calpain hyperactivation by calpastatin. Rank-rank hypergeometric overlap analysis was used to compare the transcriptome of islets from human or rodent IAPP transgenic mice vs humans with prediabetes or type 2 diabetes. RESULTS: The islet transcriptomes in humans with prediabetes and type 2 diabetes are remarkably similar. Beta cell overexpression of soluble rodent or oligomer-prone human IAPP induced changes in islet transcriptome present in prediabetes and type 2 diabetes, including decreased expression of genes that confer beta cell identity. Increased expression of human IAPP, but not rodent IAPP, induced islet inflammation present in prediabetes and type 2 diabetes in humans. Key mediators of the injury responses in islets transgenic for human IAPP or those from individuals with type 2 diabetes include STAT3, NF-κB, ESR1 and CTNNB1 by transcription factor analysis and COL3A1, NID1 and ZNF800 by gene regulatory network analysis. CONCLUSIONS/INTERPRETATION: Beta cell injury mediated by IAPP is a plausible mechanism to contribute to islet inflammation and dedifferentiation in type 2 diabetes. Inhibition of IAPP toxicity is a potential therapeutic target in type 2 diabetes.

Heterogeneity of Diabetes: ß-Cells, Phenotypes, and Precision Medicine: Proceedings of an International Symposium of the Canadian Institutes of Health Research's Institute of Nutrition, Metabolism and Diabetes and the U.S. National Institutes of Health's National Institute of Diabetes and Digestive and Kidney Diseases.

One hundred years have passed since the discovery of insulin-an achievement that transformed diabetes from a fatal illness into a manageable chronic condition. The decades since that momentous achievement have brought ever more rapid innovation and advancement in diabetes research and clinical care. To celebrate the important work of the past century and help to chart a course for its continuation into the next, the Canadian Institutes of Health Research's Institute of Nutrition, Metabolism and Diabetes and the U.S. National Institutes of Health's National Institute of Diabetes and Digestive and Kidney Diseases recently held a joint international symposium, bringing together a cohort of researchers with diverse interests and backgrounds from both countries and beyond to discuss their collective quest to better understand the heterogeneity of diabetes and thus gain insights to inform new directions in diabetes treatment and prevention. This article summarizes the proceedings of that symposium, which spanned cutting-edge research into various aspects of islet biology, the heterogeneity of diabetic phenotypes, and the current state of and future prospects for precision medicine in diabetes.

Restoring normal islet mass and function in type 1 diabetes through regenerative medicine and tissue engineering.

Type 1 diabetes is characterised by autoimmune-mediated destruction of pancreatic ß-cell mass. With the advent of insulin therapy a century ago, type 1 diabetes changed from a progressive, fatal disease to one that requires lifelong complex self-management. Replacing the lost ß-cell mass through transplantation has proven successful, but limited donor supply and need for lifelong immunosuppression restricts widespread use. In this Review, we highlight incremental advances over the past 20 years and remaining challenges in regenerative medicine approaches to restoring ß-cell mass and function in type 1 diabetes. We begin by summarising the role of endocrine islets in glucose homoeostasis and how this is altered in disease. We then discuss the potential regenerative capacity of the remaining islet cells and the utility of stem cell-derived ß-like cells to restore ß-cell function. We conclude with tissue engineering approaches that might improve the engraftment, function, and survival of ß-cell replacement therapies.

Genetic deficiency or pharmacological inhibition of soluble epoxide hydrolase ameliorates high fat diet-induced pancreatic ß-cell dysfunction and loss.

Pancreatic ß-cells are crucial regulators of systemic glucose homeostasis, and their dysfunction and loss are central features in type 2 diabetes. Interventions that rectify ß-cell dysfunction and loss are essential to combat this deadly malady. In the current study, we sought to delineate the role of soluble epoxide hydrolase (sEH) in ß-cells under diet-induced metabolic stress. The expression of sEH was upregulated in murine and macaque diabetes models and islets of diabetic human patients. We postulated that hyperglycemia-induced elevation in sEH leads to a reduction in its substrates, epoxyeicosatrienoic acids (EETs), and attenuates the function of ß-cells. Genetic deficiency of sEH potentiated glucose-stimulated insulin secretion in mice, likely in a cell-autonomous manner, contributing to better systemic glucose control. Consistent with this observation, genetic and pharmacological inactivation of sEH and the treatment with EETs exhibited insulinotropic effects in isolated murine islets ex vivo. Additionally, sEH deficiency enhanced glucose sensing and metabolism with elevated ATP and cAMP concentrations. This phenotype was associated with attenuated oxidative stress and diminished ß-cell death in sEH deficient islets. Moreover, pharmacological inhibition of sEH in vivo mitigated, albeit partly, high fat diet-induced ß-cell loss and dedifferentiation. The current observations provide new insights into the role of sEH in ß-cells and information that may be leveraged for the development of a mechanism-based intervention to rectify ß-cell dysfunction and loss.

Virgin ß-Cells at the Neogenic Niche Proliferate Normally and Mature Slowly.

Proliferation of pancreatic ß-cells has long been known to reach its peak in the neonatal stages and decline during adulthood. However, ß-cell proliferation has been studied under the assumption that all ß-cells constitute a single, homogenous population. It is unknown whether a subpopulation of ß-cells retains the capacity to proliferate at a higher rate and thus contributes disproportionately to the maintenance of mature ß-cell mass in adults. We therefore assessed the proliferative capacity and turnover potential of virgin ß-cells, a novel population of immature ß-cells found at the islet periphery. We demonstrate that virgin ß-cells can proliferate but do so at rates similar to those of mature ß-cells from the same islet under normal and challenged conditions. Virgin ß-cell proliferation rates also conform to the age-dependent decline previously reported for ß-cells at large. We further show that virgin ß-cells represent a long-lived, stable subpopulation of ß-cells with low turnover into mature ß-cells under healthy conditions. Our observations indicate that virgin ß-cells at the islet periphery can divide but do not contribute disproportionately to the maintenance of adult ß-cell mass.

Free fatty acid receptor 4 inhibitory signaling in delta cells regulates islet hormone secretion in mice.

OBJECTIVE: Maintenance of glucose homeostasis requires the precise regulation of hormone secretion from the endocrine pancreas. Free fatty acid receptor 4 (FFAR4/GPR120) is a G protein-coupled receptor whose activation in islets of Langerhans promotes insulin and glucagon secretion and inhibits somatostatin secretion. However, the contribution of individual islet cell types (α, ß, and δ cells) to the insulinotropic and glucagonotropic effects of GPR120 remains unclear. As gpr120 mRNA is enriched in somatostatin-secreting δ cells, we hypothesized that GPR120 activation stimulates insulin and glucagon secretion via inhibition of somatostatin release. METHODS: Glucose tolerance tests were performed in mice after administration of selective GPR120 agonist Compound A. Insulin, glucagon, and somatostatin secretion were measured in static incubations of isolated mouse islets in response to endogenous (ω-3 polyunsaturated fatty acids) and/or pharmacological (Compound A and AZ-13581837) GPR120 agonists. The effect of Compound A on hormone secretion was tested further in islets isolated from mice with global or somatostatin cell-specific knock-out of gpr120. Gpr120 expression was assessed in pancreatic sections by RNA in situ hybridization. Cyclic AMP (cAMP) and calcium dynamics in response to pharmacological GPR120 agonists were measured specifically in α, ß, and δ cells in intact islets using cAMPER and GCaMP6 reporter mice, respectively. RESULTS: Acute exposure to Compound A increased glucose tolerance, circulating insulin, and glucagon levels in vivo. Endogenous and/or pharmacological GPR120 agonists reduced somatostatin secretion in isolated islets and concomitantly demonstrated dose-dependent potentiation of glucose-stimulated insulin secretion and arginine-stimulated glucagon secretion. Gpr120 was enriched in δ cells. Pharmacological GPR120 agonists reduced cAMP and calcium levels in δ cells but increased these signals in α and ß cells. Compound A-mediated inhibition of somatostatin secretion was insensitive to pertussis toxin. The effect of Compound A on hormone secretion was completely absent in islets from mice with either global or somatostatin cell-specific deletion of gpr120 and partially reduced upon blockade of somatostatin receptor signaling by cyclosomatostatin. CONCLUSIONS: Inhibitory GPR120 signaling in δ cells contributes to both insulin and glucagon secretion in part by mitigating somatostatin release.