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Glucagon is released from pancreatic α-cells to activate pathways that raise blood glucose. Its secretion is regulated by α-cell-intrinsic glucose sensing and paracrine control through insulin and somatostatin. To understand the inadequately high glucagon levels that contribute to hyperglycemia in type-2 diabetes (T2D), we analyzed granule behavior, exocytosis and membrane excitability in α-cells of 68 non-diabetic and 21 T2D human donors. We report that exocytosis is moderately reduced in α-cells of T2D donors, without changes in voltage-dependent ion currents or granule trafficking. Dispersed α-cells have a non-physiological V-shaped dose response to glucose, with maximal exocytosis at hyperglycemia. Within intact islets, hyperglycemia instead inhibits α-cell exocytosis, but not in T2D or when paracrine inhibition by insulin or somatostatin is blocked. Surface expression of somatostatin-receptor-2 is reduced in T2D, suggesting a mechanism for the observed somatostatin resistance. Thus, elevated glucagon in human T2D may reflect α-cell insensitivity to paracrine inhibition at hyperglycemia. Glucagon is elevated Type-2 diabetes, which contributes to poor glucose control in patients with the disease. Here the authors report that secretion of the hormone is controlled by paracrine inhibition, and that resistance of α-cells to somatostatin can explain hyperglucagonemia in type-2 diabetes.

Insulin is produced by pancreatic β-cells, and once released to the blood, the hormone stimulates glucose uptake and suppresses glucose production. Defects in both the availability and action of insulin lead to elevated plasma glucose levels and are major hallmarks of type-2 diabetes. Insulin is stored in secretory granules that form at the trans-Golgi network. The granules undergo extensive modifications en route to their release sites at the plasma membrane, including changes in both protein and lipid composition of the granule membrane and lumen. In parallel, the insulin molecules also undergo extensive modifications that render the hormone biologically active. In this review, we summarize current understanding of insulin secretory granule biogenesis, maturation, transport, docking, priming and eventual fusion with the plasma membrane. We discuss how different pools of granules form and how these pools contribute to insulin secretion under different conditions. We also highlight the role of the β-cell in the development of type-2 diabetes and discuss how dysregulation of one or several steps in the insulin granule life cycle may contribute to disease development or progression.

Regulated exocytosis establishes a narrow fusion pore as initial aqueous connection to the extracellular space, through which small transmitter molecules such as ATP can exit. Co-release of polypeptides and hormones like insulin requires further expansion of the pore. There is evidence that pore expansion is regulated and can fail in diabetes and neurodegenerative disease. Here, we report that the cAMP-sensor Epac2 (Rap-GEF4) controls fusion pore behavior by acutely recruiting two pore-restricting proteins, amisyn and dynamin-1, to the exocytosis site in insulin-secreting beta-cells. cAMP elevation restricts and slows fusion pore expansion and peptide release, but not when Epac2 is inactivated pharmacologically or in Epac2 -/- ( Rapgef4 -/- ) mice. Consistently, overexpression of Epac2 impedes pore expansion. Widely used antidiabetic drugs (GLP-1 receptor agonists and sulfonylureas) activate this pathway and thereby paradoxically restrict hormone release. We conclude that Epac2/cAMP controls fusion pore expansion and thus the balance of hormone and transmitter release during insulin granule exocytosis. Insulin is the hormone that signals to the body to take up sugar from the blood. Specialized cells in the pancreas – known as β-cells – release insulin after a meal. Before that, insulin molecules are stored in tiny granules inside the β-cells; these granules must fuse with the cells’ surface membranes to release their contents. The first step in this process creates a narrow pore that allows small molecules, but not the larger insulin molecules, to seep out. The pore then widens to release the insulin. Since the small molecules are known to act locally in the pancreas, it is possible that this “molecular sieve” is biologically important. Yet it is not clear how the pore widens. One of the problems for people with type 2 diabetes is that they release less insulin into the bloodstream. Two kinds of drugs used to treat these patients work by stimulating β-cells to release their insulin. One way to achieve this is by raising the levels of a small molecule called cAMP, which is well known to help prepare insulin granules for release. The cAMP molecule also seems to slow the widening of the pore, and Gucek et al. have now investigated how this happens at a molecular level. By observing individual granules of human β-cells using a special microscope, Gucek et al. could watch how different drugs affect pore widening and content release. They also saw that cAMP activated a protein called Epac2, which then recruited two other proteins – amisyn and dynamin – to the small pores. These two proteins together then closed the pore, rather than expanding it to let insulin out. Type 2 diabetes patients sometimes have high levels of amisyn in their β-cells, which could explain why they do not release enough insulin. The microscopy experiments also revealed that two common anti-diabetic drugs activate Epac2 and prevent the pores from widening, thereby counteracting their positive effect on insulin release. The combined effect is likely a shift in the balance between insulin and the locally acting small molecules. These findings suggest that two common anti-diabetic drugs activate a common mechanism that may lead to unexpected outcomes, possibly even reducing how much insulin the β-cells can release. Future studies in mice and humans will have to investigate these effects in whole organisms.

Transplantation of pancreatic islet cells derived from human pluripotent stem cells is a promising treatment for diabetes. Despite progress in the generation of stem-cell-derived islets (SC-islets), no detailed characterization of their functional properties has been conducted. Here, we generated functionally mature SC-islets using an optimized protocol and benchmarked them comprehensively against primary adult islets. Biphasic glucose-stimulated insulin secretion developed during in vitro maturation, associated with cytoarchitectural reorganization and the increasing presence of alpha cells. Electrophysiology, signaling and exocytosis of SC-islets were similar to those of adult islets. Glucose-responsive insulin secretion was achieved despite differences in glycolytic and mitochondrial glucose metabolism. Single-cell transcriptomics of SC-islets in vitro and throughout 6 months of engraftment in mice revealed a continuous maturation trajectory culminating in a transcriptional landscape closely resembling that of primary islets. Our thorough evaluation of SC-islet maturation highlights their advanced degree of functionality and supports their use in further efforts to understand and combat diabetes. Pancreatic islets derived from stem cells are benchmarked against primary cells.

White adipocytes function as major energy reservoirs in humans by storing substantial amounts of triglycerides, and their dysfunction is associated with metabolic disorders; however, the mechanisms underlying cellular specialization during adipogenesis remain unknown. Here, we generate a spatiotemporal proteomic atlas of human adipogenesis, which elucidates cellular remodelling as well as the spatial reorganization of metabolic pathways to optimize cells for lipid accumulation and highlights the coordinated regulation of protein localization and abundance during adipocyte formation. We identify compartment-specific regulation of protein levels and localization changes of metabolic enzymes to reprogramme branched-chain amino acids and one-carbon metabolism to provide building blocks and reduction equivalents. Additionally, we identify C19orf12 as a differentiation-induced adipocyte lipid droplet protein that interacts with the translocase of the outer membrane complex of lipid droplet-associated mitochondria and regulates adipocyte lipid storage by determining the capacity of mitochondria to metabolize fatty acids. Overall, our study provides a comprehensive resource for understanding human adipogenesis and for future discoveries in the field. Klingelhuber, Frendo-Cumbo et al. develop a proteomic atlas elucidating the intracellular spatiotemporal changes in protein levels and localizations during human adipogenesis.

Human pancreatic islets highly express CD59, which is a glycosylphosphatidylinositol (GPI)-anchored cell-surface protein and is required for insulin secretion. How cell-surface CD59 could interact with intracellular exocytotic machinery has so far not been described. We now demonstrate the existence of CD59 splice variants in human pancreatic islets, which have unique C-terminal domains replacing the GPI-anchoring signal sequence. These isoforms are found in the cytosol of β-cells, interact with SNARE proteins VAMP2 and SNAP25, colocalize with insulin granules, and rescue insulin secretion in CD59-knockout (KO) cells. We therefore named these isoforms IRIS-1 and IRIS-2 (Isoforms Rescuing Insulin Secretion 1 and 2). Antibodies raised against each isoform revealed that expression of both IRIS-1 and IRIS-2 is significantly lower in islets isolated from human type 2 diabetes (T2D) patients, as compared to healthy controls. Further, glucotoxicity induced in primary, healthy human islets led to a significant decrease of IRIS-1 expression, suggesting that hyperglycemia (raised glucose levels) and subsequent decreased IRIS-1 expression may contribute to relative insulin deficiency in T2D patients. Similar isoforms were also identified in the mouse CD59B gene, and targeted CRISPR/Cas9-mediated knockout showed that these intracellular isoforms, but not canonical CD59B, are involved in insulin secretion from mouse β-cells. Mouse IRIS-2 is also down-regulated in diabetic db/db mouse islets. These findings establish the endogenous existence of previously undescribed non–GPI-anchored intracellular isoforms of human CD59 and mouse CD59B, which are required for normal insulin secretion.

Aims/hypothesis Regulatory factor X 6 (RFX6) is crucial for pancreatic endocrine development and differentiation. The RFX6 variant p.His293LeufsTer7 is significantly enriched in the Finnish population, with almost 1:250 individuals as a carrier. Importantly, the FinnGen study indicates a high predisposition for heterozygous carriers to develop type 2 and gestational diabetes. However, the precise mechanism of this predisposition remains unknown. Methods To understand the role of this variant in beta cell development and function, we used CRISPR technology to generate allelic series of pluripotent stem cells. We created two isogenic stem cell models: a human embryonic stem cell model; and a patient-derived stem cell model. Both were differentiated into pancreatic islet lineages (stem-cell-derived islets, SC-islets), followed by implantation in immunocompromised NOD-SCID-Gamma mice. Results Stem cell models of the homozygous variant RFX6 −/− predictably failed to generate insulin-secreting pancreatic beta cells, mirroring the phenotype observed in Mitchell–Riley syndrome. Notably, at the pancreatic endocrine stage, there was an upregulation of precursor markers NEUROG3 and SOX9 , accompanied by increased apoptosis. Intriguingly, heterozygous RFX6 +/− SC-islets exhibited RFX6 haploinsufficiency (54.2% reduction in protein expression), associated with reduced beta cell maturation markers, altered calcium signalling and impaired insulin secretion (62% and 54% reduction in basal and high glucose conditions, respectively). However, RFX6 haploinsufficiency did not have an impact on beta cell number or insulin content. The reduced insulin secretion persisted after in vivo implantation in mice, aligning with the increased risk of variant carriers to develop diabetes. Conclusions/interpretation Our allelic series isogenic SC-islet models represent a powerful tool to elucidate specific aetiologies of diabetes in humans, enabling the sensitive detection of aberrations in both beta cell development and function. We highlight the critical role of RFX6 in augmenting and maintaining the pancreatic progenitor pool, with an endocrine roadblock and increased cell death upon its loss. We demonstrate that RFX6 haploinsufficiency does not affect beta cell number or insulin content but does impair function, predisposing heterozygous carriers of loss-of-function variants to diabetes. Data availability Ultra-deep bulk RNA-seq data for pancreatic differentiation stages 3, 5 and 7 of H1 RFX6 genotypes are deposited in the Gene Expression Omnibus database with accession code GSE234289. Original western blot images are deposited at Mendeley ( https://data.mendeley.com/datasets/g75drr3mgw/2 ). Graphical Abstract

Glutamine and glutamate are interconverted by several enzymes and alterations in this metabolic cycle are linked to cardiometabolic traits. Herein, we show that obesity-associated insulin resistance is characterized by decreased plasma and white adipose tissue glutamine-to-glutamate ratios. We couple these stoichiometric changes to perturbed fat cell glutaminase and glutamine synthase messenger RNA and protein abundance, which together promote glutaminolysis. In human white adipocytes, reductions in glutaminase activity promote aerobic glycolysis and mitochondrial oxidative capacity via increases in hypoxia-inducible factor 1α abundance, lactate levels and p38 mitogen-activated protein kinase signalling. Systemic glutaminase inhibition in male and female mice, or genetically in adipocytes of male mice, triggers the activation of thermogenic gene programs in inguinal adipocytes. Consequently, the knockout mice display higher energy expenditure and improved glucose tolerance compared to control littermates, even under high-fat diet conditions. Altogether, our findings highlight white adipocyte glutamine turnover as an important determinant of energy expenditure and metabolic health. Lecoutre, Maqdasy and Rizo-Roca show that whole-body pharmacological inhibition or adipocyte-specific deletion of glutaminase in mice activates thermogenesis in inguinal adipocytes and promotes metabolic health. They also link decreased plasma and adipose tissue glutamine-to-glutamate ratios to insulin resistance in humans with obesity.

The secretion of islet hormones from endocrine cells of the pancreas plays vital roles in maintaining glucose homeostasis. Dysfunction of these cells leads to diabetes, a devastating metabolic disorder affecting millions worldwide, but underlying mechanisms remain poorly understood. In hyperglycemic conditions, β-cells secrete insulin, whereas α-cells secrete an increased amount of glucagon in hypoglycemic conditions. Both insulin and glucagon are stored in secretory granules preceding their release by regulated exocytosis. This process involves several steps, including tethering, docking, priming, and finally, a fusion of the granules with the plasma membrane. Soluble N-ethylmaleimide–sensitive factor attachment protein receptor (SNARE) proteins and phosphoinositides (PIs) drive pancreatic hormone exocytosis and secretion, which follows a biphasic time course. Biphasic secretion is thought to reflect the vastly different release probabilities of individual granules, but direct evidence for this is still lacking. Therefore, this thesis investigates exocytosis in the two main pancreatic cell types with a particular focus on preceding steps docking and priming, to identify rate-limiting steps in health and type-2 diabetes (T2D). Our data indicated that granule docking is critical for sustained secretion in α- and β-cells. Glucagon granule exocytosis had a U-shaped sensitivity to glucose in both healthy and T2D α-cells. However, T2D α-cells exhibited a marginal decrease in exocytosis, as well as docking, and they were markedly insensitive to somatostatin and insulin. T2D β-cells reduced exocytosis dramatically, and docking was compromised and no longer responsive to glucose, which correlated with reduced insulin secretion and elevated donor HbA1c. These results were further strengthened by the finding that expression of a group of genes that are involved explicitly in granule docking was reduced (by RNAseq of islets from over 200 human donors), and overexpression of the corresponding proteins increased granule docking in human β-cells.We further aimed to study the basis for the recruitment of these proteins to the docking site. Here we tested the hypothesis that highly charged lipids mainly PIs act as a hotspot to interact with SNARE proteins that initiate docking. We showed the homogenous distribution of all PIs markers in the plasma membrane, with no PIs microdomains at the exocytotic site during granule docking. However, rapid and local PI(4,5)P2 signaling at fusion sites was crucial for stabilizing fusion pore by binding to proteins related to the release site. These results suggested a role of PI(4,5)P2 in priming and fusion regulation rather than docking. Overall, this work gives new insights into the mechanisms underlying pancreatic hormone secretion in both healthy and diabetic conditions.

Phosphatidylinositol(4,5)bisphosphate (PI(4,5)P2) is an important signaling phospholipid that is required for regulated exocytosis and some forms of endocytosis. The two processes share a topologically similar pore structure that connects the vesicle lumen with the outside. Widening of the fusion pore during exocytosis leads to cargo release, while its closure initiates kiss&run or cavicapture endocytosis. We show here, using live cell TIRF microscopy of insulin granule exocytosis, that transient accumulation of PI(4,5)P2 at the release site recruits components of the endocytic fission machinery, and stalls the late fusion pore expansion that is required for peptide release. The absence of clathrin differentiates this mechanism from clathrin-mediated endocytosis. The PI(4,5)P2 transients result from local phosphatidylinositol-phosphate-5-kinase-1c (PIP5K1c) activity, and knockdown of PIP5K1c, or optogenetic ablation of PI(4,5)P2 promotes fusion pore expansion. Thus, local phospholipid signaling controls fusion pore expansion peptide release through an unconventional endocytic mechanism. Competing Interest Statement The authors have declared no competing interest.