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.

Paracrine regulation of somatostatin secretion by insulin and glucagon in mouse pancreatic islets.

AIMS/HYPOTHESIS: The endocrine pancreas comprises the islets of Langerhans, primarily consisting of beta cells, alpha cells and delta cells responsible for secretion of insulin, glucagon and somatostatin, respectively. A certain level of intra-islet communication is thought to exist, where the individual hormones may reach the other islet cells and regulate their secretion. Glucagon has been demonstrated to importantly regulate insulin secretion, while somatostatin powerfully inhibits both insulin and glucagon secretion. In this study we investigated how secretion of somatostatin is regulated by paracrine signalling from glucagon and insulin. METHODS: Somatostatin secretion was measured from perfused mouse pancreases isolated from wild-type as well as diphtheria toxin-induced alpha cell knockdown, and global glucagon receptor knockout (Gcgr-/-) mice. We studied the effects of varying glucose concentrations together with infusions of arginine, glucagon, insulin and somatostatin, as well as infusions of antagonists of insulin, somatostatin and glucagon-like peptide 1 (GLP-1) receptors. RESULTS: A tonic inhibitory role of somatostatin was demonstrated with infusion of somatostatin receptor antagonists, which significantly increased glucagon secretion at low and high glucose, whereas insulin secretion was only increased at high glucose levels. Infusion of glucagon dose-dependently increased somatostatin secretion approximately twofold in control mice. Exogenous glucagon had no effect on somatostatin secretion in Gcgr-/- mice, and a reduced effect when combined with the GLP-1 receptor antagonist exendin 9-39. Diphtheria toxin-induced knockdown of glucagon producing cells led to reduced somatostatin secretion in response to 12 mmol/l glucose and arginine infusions. In Gcgr-/- mice (where glucagon levels are dramatically increased) overall somatostatin secretion was increased. However, infusion of exendin 9-39 in Gcgr-/- mice completely abolished somatostatin secretion in response to glucose and arginine. Neither insulin nor an insulin receptor antagonist (S961) had any effect on somatostatin secretion. CONCLUSIONS/INTERPRETATION: Our findings demonstrate that somatostatin and glucagon secretion are linked in a reciprocal feedback cycle with somatostatin inhibiting glucagon secretion at low and high glucose levels, and glucagon stimulating somatostatin secretion via the glucagon and GLP-1 receptors. Graphical abstract.

Tracing the cellular basis of islet specification in mouse pancreas.

Pancreatic islets play an essential role in regulating blood glucose level. Although the molecular pathways underlying islet cell differentiation are beginning to be resolved, the cellular basis of islet morphogenesis and fate allocation remain unclear. By combining unbiased and targeted lineage tracing, we address the events leading to islet formation in the mouse. From the statistical analysis of clones induced at multiple embryonic timepoints, here we show that, during the secondary transition, islet formation involves the aggregation of multiple equipotent endocrine progenitors that transition from a phase of stochastic amplification by cell division into a phase of sublineage restriction and limited islet fission. Together, these results explain quantitatively the heterogeneous size distribution and degree of polyclonality of maturing islets, as well as dispersion of progenitors within and between islets. Further, our results show that, during the secondary transition, α- and ß-cells are generated in a contemporary manner. Together, these findings provide insight into the cellular basis of islet development.

Spontaneous restoration of functional ß-cell mass in obese SM/J mice.

Maintenance of functional ß-cell mass is critical to preventing diabetes, but the physiological mechanisms that cause ß-cell populations to thrive or fail in the context of obesity are unknown. High fat-fed SM/J mice spontaneously transition from hyperglycemic-obese to normoglycemic-obese with age, providing a unique opportunity to study ß-cell adaptation. Here, we characterize insulin homeostasis, islet morphology, and ß-cell function during SM/J's diabetic remission. As they resolve hyperglycemia, obese SM/J mice dramatically increase circulating and pancreatic insulin levels while improving insulin sensitivity. Immunostaining of pancreatic sections reveals that obese SM/J mice selectively increase ß-cell mass but not α-cell mass. Obese SM/J mice do not show elevated ß-cell mitotic index, but rather elevated α-cell mitotic index. Functional assessment of isolated islets reveals that obese SM/J mice increase glucose-stimulated insulin secretion, decrease basal insulin secretion, and increase islet insulin content. These results establish that ß-cell mass expansion and improved ß-cell function underlie the resolution of hyperglycemia, indicating that obese SM/J mice are a valuable tool for exploring how functional ß-cell mass can be recovered in the context of obesity.

Repositioning the Alpha Cell in Postprandial Metabolism.

Glucose homeostasis is maintained in large part due to the actions of the pancreatic islet hormones insulin and glucagon, secreted from ß- and α-cells, respectively. The historical narrative positions these hormones in opposition, with insulin primarily responsible for glucose-lowering and glucagon-driving elevations in glucose. Recent progress in this area has revealed a more complex relationship between insulin and glucagon, highlighted by data demonstrating that α-cell input is essential for ß-cell function and glucose homeostasis. Moreover, the common perception that glucagon levels decrease following a nutrient challenge is largely shaped by the inhibitory effects of glucose administration alone on the α-cell. Largely overlooked is that a mixed nutrient challenge, which is more representative of typical human feeding, actually stimulates glucagon secretion. Thus, postprandial metabolism is associated with elevations, not decreases, in α-cell activity. This review discusses the recent advances in our understanding of how α-cells regulate metabolism, with a particular focus on the postprandial state. We highlight α- to ß-cell communication, a term that describes how α-cell input into ß-cells is a critical axis that regulates insulin secretion and glucose homeostasis. Finally, we discuss the open questions that have the potential to advance this field and continue to evolve our understanding of the role that α-cells play in postprandial metabolism.

Purification of pancreatic endocrine subsets reveals increased iron metabolism in beta cells.

OBJECTIVES: The main endocrine cell types in pancreatic islets are alpha, beta, and delta cells. Although these cell types have distinct roles in the regulation of glucose homeostasis, inadequate purification methods preclude the study of cell type-specific effects. We developed a reliable approach that enables simultaneous sorting of live alpha, beta, and delta cells from mouse islets for downstream analyses. METHODS: We developed an antibody panel against cell surface antigens to enable isolation of highly purified endocrine subsets from mouse islets based on the specific differential expression of CD71 on beta cells and CD24 on delta cells. We rigorously demonstrated the reliability and validity of our approach using bulk and single cell qPCR, immunocytochemistry, reporter mice, and transcriptomics. RESULTS: Pancreatic alpha, beta, and delta cells can be separated based on beta cell-specific CD71 surface expression and high expression of CD24 on delta cells. We applied our new sorting strategy to demonstrate that CD71, which is the transferrin receptor mediating the uptake of transferrin-bound iron, is upregulated in beta cells during early postnatal weeks. We found that beta cells express higher levels of several other genes implicated in iron metabolism and iron deprivation significantly impaired beta cell function. In human beta cells, CD71 is similarly required for iron uptake and CD71 surface expression is regulated in a glucose-dependent manner. CONCLUSIONS: This study provides a novel and efficient purification method for murine alpha, beta, and delta cells, identifies for the first time CD71 as a postnatal beta cell-specific marker, and demonstrates a central role of iron metabolism in beta cell function.

Single-cell transcriptomics of human islet ontogeny defines the molecular basis of ß-cell dedifferentiation in T2D.

OBJECTIVE: Dedifferentiation of pancreatic ß-cells may reduce islet function in type 2 diabetes (T2D). However, the prevalence, plasticity and functional consequences of this cellular state remain unknown. METHODS: We employed single-cell RNAseq to detail the maturation program of α- and ß-cells during human ontogeny. We also compared islets from non-diabetic and T2D individuals. RESULTS: Both α- and ß-cells mature in part by repressing non-endocrine genes; however, α-cells retain hallmarks of an immature state, while ß-cells attain a full ß-cell specific gene expression program. In islets from T2D donors, both α- and ß-cells have a less mature expression profile, de-repressing the juvenile genetic program and exocrine genes and increasing expression of exocytosis, inflammation and stress response signalling pathways. These changes are consistent with the increased proportion of ß-cells displaying suboptimal function observed in T2D islets. CONCLUSIONS: These findings provide new insights into the molecular program underlying islet cell maturation during human ontogeny and the loss of transcriptomic maturity that occurs in islets of type 2 diabetics.

Lactate activation of α-cell KATP channels inhibits glucagon secretion by hyperpolarizing the membrane potential and reducing Ca2+ entry.

OBJECTIVE: Elevations in pancreatic α-cell intracellular Ca2+ ([Ca2+]i) lead to glucagon (GCG) secretion. Although glucose inhibits GCG secretion, how lactate and pyruvate control α-cell Ca2+ handling is unknown. Lactate enters cells through monocarboxylate transporters (MCTs) and is also produced during glycolysis by lactate dehydrogenase A (LDHA), an enzyme expressed in α-cells. As lactate activates ATP-sensitive K+ (KATP) channels in cardiomyocytes, lactate may also modulate α-cell KATP. Therefore, this study investigated how lactate signaling controls α-cell Ca2+ handling and GCG secretion. METHODS: Mouse and human islets were used in combination with confocal microscopy, electrophysiology, GCG immunoassays, and fluorescent thallium flux assays to assess α-cell Ca2+ handling, Vm, KATP currents, and GCG secretion. RESULTS: Lactate-inhibited mouse (75 ± 25%) and human (47 ± 9%) α-cell [Ca2+]i fluctuations only under low-glucose conditions (1 mM) but had no effect on ß- or δ-cells [Ca2+]i. Glyburide inhibition of KATP channels restored α-cell [Ca2+]i fluctuations in the presence of lactate. Lactate transport into α-cells via MCTs hyperpolarized mouse (14 ± 1 mV) and human (12 ± 1 mV) α-cell Vm and activated KATP channels. Interestingly, pyruvate showed a similar KATP activation profile and α-cell [Ca2+]i inhibition as lactate. Lactate-induced inhibition of α-cell [Ca2+]i influx resulted in reduced GCG secretion in mouse (62 ± 6%) and human (43 ± 13%) islets. CONCLUSIONS: These data demonstrate for the first time that lactate entry into α-cells through MCTs results in KATP activation, Vm hyperpolarization, reduced [Ca2+]i, and inhibition of GCG secretion. Thus, taken together, these data indicate that lactate either within α-cells and/or elevated in serum could serve as important modulators of α-cell function.

Characterization of Insulin and Glucagon Genes and Their Producing Endocrine Cells From Pygmy Sperm Whale (Kogia breviceps).

Insulin and glucagon are hormones secreted by pancreatic ß and α cells, respectively, which together regulate glucose homeostasis. Dysregulation of insulin or glucagon can result in loss of blood glucose control, characterized by hyperglycemia or hypoglycemia. To better understand the endocrine physiology of cetaceans, we cloned and characterized the insulin and glucagon genes from pygmy sperm whale (Kogia breviceps). We obtained the complete coding sequences of the preproinsulin and preproglucagon genes, which encodes the preproinsulin protein of 110 amino acid (aa) residues and encodes the preproglucagon protein of 179 aa residues, respectively. Sequence comparison and phylogenetic analyses demonstrate that protein structures were similar to other mammalian orthologs. Immunohistochemistry and immunofluorescence staining using insulin, glucagon, and somatostatin antibodies allowed analysis of pygmy sperm whale islet distribution, architecture, and composition. Our results showed the pygmy sperm whale islet was irregularly shaped and randomly distributed throughout the pancreas. The architecture of α, ß, and δ cells of the pygmy sperm whale was similar to that of artiodactyls species. This is the first report about insulin and glucagon genes in cetaceans, which provides new information about the structural conservation of the insulin and glucagon genes. Furthermore, offers novel information on the properties of endocrine cells in cetacean for further studies.

Molecular and genetic regulation of pig pancreatic islet cell development.

Reliance on rodents for understanding pancreatic genetics, development and islet function could limit progress in developing interventions for human diseases such as diabetes mellitus. Similarities of pancreas morphology and function suggest that porcine and human pancreas developmental biology may have useful homologies. However, little is known about pig pancreas development. To fill this knowledge gap, we investigated fetal and neonatal pig pancreas at multiple, crucial developmental stages using modern experimental approaches. Purification of islet ß-, α- and δ-cells followed by transcriptome analysis (RNA-seq) and immunohistology identified cell- and stage-specific regulation, and revealed that pig and human islet cells share characteristic features that are not observed in mice. Morphometric analysis also revealed endocrine cell allocation and architectural similarities between pig and human islets. Our analysis unveiled scores of signaling pathways linked to native islet ß-cell functional maturation, including evidence of fetal α-cell GLP-1 production and signaling to ß-cells. Thus, the findings and resources detailed here show how pig pancreatic islet studies complement other systems for understanding the developmental programs that generate functional islet cells, and that are relevant to human pancreatic diseases.

DLL1- and DLL4-Mediated Notch Signaling Is Essential for Adult Pancreatic Islet Homeostasis.

Genes of the Notch signaling pathway are expressed in different cell types and organs at different time points during embryonic development and adulthood. The Notch ligand Delta-like 1 (DLL1) controls the decision between endocrine and exocrine fates of multipotent progenitors in the developing pancreas, and loss of Dll1 leads to premature endocrine differentiation. However, the role of Delta-Notch signaling in adult tissue homeostasis is not well understood. Here, we describe the spatial expression pattern of Notch pathway components in adult murine pancreatic islets and show that DLL1 and DLL4 are specifically expressed in ß-cells, whereas JAGGED1 is expressed in α-cells. We show that mice lacking both DLL1 and DLL4 in adult ß-cells display improved glucose tolerance, increased glucose-stimulated insulin secretion, and hyperglucagonemia. In contrast, overexpression of the intracellular domain of DLL1 in adult murine pancreatic ß-cells results in impaired glucose tolerance and reduced insulin secretion, both in vitro and in vivo. These results suggest that Notch ligands play specific roles in the adult pancreas and highlight a novel function of the Delta/Notch pathway in ß-cell insulin secretion.

How do you make new ß-cells in humans? An attempt to create human ß-cells from α-cells of brain-dead donors.

Ex vivo reprogramming of human α-cells toward functional ß-cells by adenoviral transduction of key transcription factors.

Morphological and functional adaptations of pancreatic alpha-cells during late pregnancy in the mouse.

BACKGROUND: Pregnancy represents a major metabolic challenge for the mother, and involves a compensatory response of the pancreatic beta-cell to maintain normoglycemia. However, although pancreatic alpha-cells play a key role in glucose homeostasis and seem to be involved in gestational diabetes, there is no information about their potential adaptations or changes during pregnancy. MATERIAL AND METHODS: Non-pregnant (controls) and pregnant C57BL/6 mice at gestational day 18.5 (G18.5) and their isolated pancreatic islets were used for in vivo and ex vivo studies, respectively. The effect of pregnancy hormones was tested in glucagon-secreting α-TC1.9 cells. Immunohistochemical analysis was performed in pancreatic slices. Glucagon gene expression was monitored by RT-qPCR. Glucagon secretion and plasma hormones were measured by ELISA. RESULTS: Pregnant mice on G18.5 exhibited alpha-cell hypertrophy as well as augmented alpha-cell area and mass. This alpha-cell mass expansion was mainly due to increased proliferation. No changes in alpha-cell apoptosis, ductal neogenesis, or alpha-to-beta transdifferentiation were found compared with controls. Pregnant mice on G18.5 exhibited hypoglucagonemia. Additionally, in vitro glucagon secretion at low glucose levels was decreased in isolated islets from pregnant animals. Glucagon content was also reduced. Experiments in α-TC1.9 cells indicated that, unlike estradiol and progesterone, placental lactogens and prolactin stimulated alpha-cell proliferation. Placental lactogens, prolactin and estradiol also inhibited glucagon release from α-TC1.9 cells at low glucose levels. CONCLUSIONS: The pancreatic alpha-cell in mice undergoes several morphofunctional changes during late pregnancy, which may contribute to proper glucose homeostasis. Gestational hormones are likely involved in these processes.

Increased mast cell number is associated with a decrease in beta-cell mass and regeneration in type 2 diabetic rats.

The role of mast cells (MCs) in type 2 diabetes (T2D) is not thoroughly studied as much as in T1D. Therefore in the current study we investigated correlation between these cells and various parameters of islets of Langerhans (IOL) in rats which were equally divided (n = 40) into; control and diabetic groups. We detected a significantly increased (p < 0.05) MC count (MCC) in the diabetic IOL compared to the control, together with a noticeable intra-islet seeding of these cells which displayed a tryptase positive immunostaining. A significant positive correlation (p < 0.05) between MCC and the % of glucagon cells per islet was detected in DM, unlike mass of the islets, mass of ß-cells, and % of ß-cells per islet which were negatively correlated with MCC. Similarly, there was a negative correlation between MCC with ß-cell proliferation and neogenesis frequency in DM. This highlights the potential association between the increased MC number and the diminished islet`s mass as well as regeneration which may fasten the progression of T2D.

The Liver-α-Cell Axis and Type 2 Diabetes.

Both type 2 diabetes (T2D) and nonalcoholic fatty liver disease (NAFLD) strongly associate with increasing body mass index, and together these metabolic diseases affect millions of individuals. In patients with T2D, increased secretion of glucagon (hyperglucagonemia) contributes to diabetic hyperglycemia as proven by the significant lowering of fasting plasma glucose levels following glucagon receptor antagonist administration. Emerging data now indicate that the elevated plasma concentrations of glucagon may also be associated with hepatic steatosis and not necessarily with the presence or absence of T2D. Thus, fatty liver disease, most often secondary to overeating, may result in impaired amino acid turnover, leading to increased plasma concentrations of certain glucagonotropic amino acids (e.g., alanine). This, in turn, causes increased glucagon secretion that may help to restore amino acid turnover and ureagenesis, but it may eventually also lead to increased hepatic glucose production, a hallmark of T2D. Early experimental findings support the hypothesis that hepatic steatosis impairs glucagon's actions on amino acid turnover and ureagenesis. Hepatic steatosis also impairs hepatic insulin sensitivity and clearance that, together with hyperglycemia and hyperaminoacidemia, lead to peripheral hyperinsulinemia; systemic hyperinsulinemia may itself contribute to worsen peripheral insulin resistance. Additionally, obesity is accompanied by an impaired incretin effect, causing meal-related glucose intolerance. Lipid-induced impairment of hepatic sensitivity, not only to insulin but potentially also to glucagon, resulting in both hyperinsulinemia and hyperglucagonemia, may therefore contribute to the development of T2D at least in a subset of individuals with NAFLD.

Replica moulded poly(dimethylsiloxane) microwell arrays induce localized endothelial cell immobilization for coculture with pancreatic islets.

PolyJet three-dimensional (3D) printing allows for the rapid manufacturing of 3D moulds for the fabrication of cross-linked poly(dimethylsiloxane) microwell arrays (PMAs). As this 3D printing technique has a resolution on the micrometer scale, the moulds exhibit a distinct surface roughness. In this study, the authors demonstrate by optical profilometry that the topography of the 3D printed moulds can be transferred to the PMAs and that this roughness induced cell adhesive properties to the material. In particular, the topography facilitated immobilization of endothelial cells on the internal walls of the microwells. The authors also demonstrate that upon immobilization of endothelial cells to the microwells, a second population of cells, namely, pancreatic islets could be introduced, thus producing a 3D coculture platform.

Modelling the endocrine pancreas in health and disease.

Diabetes mellitus is a multifactorial disease affecting increasing numbers of patients worldwide. Progression to insulin-dependent diabetes mellitus is characterized by the loss or dysfunction of pancreatic ß-cells, but the pathomechanisms underlying ß-cell failure in type 1 diabetes mellitus and type 2 diabetes mellitus are still poorly defined. Regeneration of ß-cell mass from residual islet cells or replacement by ß-like cells derived from stem cells holds great promise to stop or reverse disease progression. However, the development of new treatment options is hampered by our limited understanding of human pancreas organogenesis due to the restricted access to primary tissues. Therefore, the challenge is to translate results obtained from preclinical model systems to humans, which requires comparative modelling of ß-cell biology in health and disease. Here, we discuss diverse modelling systems across different species that provide spatial and temporal resolution of cellular and molecular mechanisms to understand the evolutionary conserved genotype-phenotype relationship and translate them to humans. In addition, we summarize the latest knowledge on organoids, stem cell differentiation platforms, primary micro-islets and pseudo-islets, bioengineering and microfluidic systems for studying human pancreas development and homeostasis ex vivo. These new modelling systems and platforms have opened novel avenues for exploring the developmental trajectory, physiology, biology and pathology of the human pancreas.

MicroRNAs in islet hormone secretion.

Pancreatic islet hormone secretion is central in the maintenance of blood glucose homeostasis. During development of hyperglycaemia, the ß-cell is under pressure to release more insulin to compensate for increased insulin resistance. Failure of the ß-cells to secrete enough insulin results in type 2 diabetes (T2D). MicroRNAs (miRNAs) are short non-coding RNA molecules suitable for rapid regulation of the changes in target gene expression needed in ß-cell adaptations. Moreover, miRNAs are involved in the maintenance of α-cell and ß-cell phenotypic identities via cell-specific, or cell-enriched expression. Although many of the abundant miRNAs are highly expressed in both cell types, recent research has focused on the role of miRNAs in ß-cells. It has been shown that highly abundant miRNAs, such as miR-375, are involved in several cellular functions indispensable in maintaining ß-cell phenotypic identity, almost acting as "housekeeping genes" in the context of hormone secretion. Despite the abundance and importance of miR-375, it has not been shown to be differentially expressed in T2D islets. On the contrary, the less abundant miRNAs such as miR-212/miR-132, miR-335, miR-130a/b and miR-152 are deregulated in T2D islets, wherein the latter three miRNAs were shown to play key roles in regulating ß-cell metabolism. In this review, we focus on ß-cell function and describe miRNAs involved in insulin biosynthesis and processing, glucose uptake and metabolism, electrical activity and Ca2+ -influx and exocytosis of the insulin granules. We present current status on miRNA regulation in α-cells, and finally we discuss the involvement of miRNAs in ß-cell dysfunction underlying T2D pathogenesis.

GLP-1 suppresses glucagon secretion in human pancreatic alpha-cells by inhibition of P/Q-type Ca2+ channels.

Glucagon is the body's main hyperglycemic hormone, and its secretion is dysregulated in type 2 diabetes mellitus (T2DM). The incretin hormone glucagon-like peptide-1 (GLP-1) is released from the gut and is used in T2DM therapy. Uniquely, it both stimulates insulin and inhibits glucagon secretion and thereby lowers plasma glucose levels. In this study, we have investigated the action of GLP-1 on glucagon release from human pancreatic islets. Immunocytochemistry revealed that only <0.5% of the α-cells possess detectable GLP-1R immunoreactivity. Despite this, GLP-1 inhibited glucagon secretion by 50-70%. This was due to a direct effect on α-cells, rather than paracrine signaling, because the inhibition was not reversed by the insulin receptor antagonist S961 or the somatostatin receptor-2 antagonist CYN154806. The inhibitory effect of GLP-1 on glucagon secretion was prevented by the PKA-inhibitor Rp-cAMPS and mimicked by the adenylate cyclase activator forskolin. Electrophysiological measurements revealed that GLP-1 decreased action potential height and depolarized interspike membrane potential. Mathematical modeling suggests both effects could result from inhibition of P/Q-type Ca2+ channels. In agreement with this, GLP-1 and ω-agatoxin (a blocker of P/Q-type channels) inhibited glucagon secretion in islets depolarized by 70 mmol/L [K+ ]o , and these effects were not additive. Intracellular application of cAMP inhibited depolarization-evoked exocytosis in individual α-cells by a PKA-dependent (Rp-cAMPS-sensitive) mechanism. We propose that inhibition of glucagon secretion by GLP-1 involves activation of the few GLP-1 receptors present in the α-cell membrane. The resulting small elevation of cAMP leads to PKA-dependent inhibition of P/Q-type Ca2+ channels and suppression of glucagon exocytosis.