Somatostatin, produced by pancreatic islet δ cells, is a key intra-islet paracrine factor that regulates the secretion of the glucoregulatory hormones insulin and glucagon from β cells and α cells, respectively. Here, we show that glutamate and glucagon released by α cells cooperatively activate neighbouring δ cells through AMPA and glucagon receptors, thereby enabling spatiotemporal feedback control of glucagon secretion. Crucially, prior hypoglycaemia enhances this mechanism by sensitizing δ cells to α cell-derived factors and inducing long-lasting structural and functional changes that facilitate δ cell and α cell paracrine interaction. This culminates in somatostatin hypersecretion that impairs counter-regulatory glucagon release. These hypoglycaemia-driven effects were emulated by chemogenetic activation of α cells or high concentrations of exogenous glucagon but prevented by inhibitors of glucagon receptors or the transcription factor CREB. This plasticity represents a key component of the islet’s ‘metabolic memory’, which, through impaired counter-regulatory glucagon secretion, increases the occurrence of recurrent hypoglycaemia that complicates the management of insulin-dependent diabetes. Prior hypoglycemia alters the paracrine interaction between islet α and δ cells, leading to impaired counter-regulatory glucagon secretion through somatostatin hypersecretion, increasing the risk of recurrent hypoglycemia.

Liraglutide, a glucagon-like peptide-1 receptor (GLP-1R) agonist for type 2 diabetes and obesity management, shows variable patient responses. We investigated the metabolic state-dependent mechanisms underlying this heterogeneity and how liraglutide’s mode of action shifts across stages of metabolic dysfunction. We employed human pancreatic islets from donors across metabolic states (normoglycaemic [HbA1c <42 mmol/l (<6.0

In the article cited above, affiliation School of Biomedical Sciences, Ulster University, Coleraine, U.K., was inadvertently omitted at submission for author Andrei I. Tarosov. The authors apologize for the error. The online version of the article (https://doi.org/10.2337/db25-0302) has been updated with the correct affiliation information.

At birth, blood glucose levels drop sharply before rising to normoglycemic adult levels. This is accompanied by functional maturation of pancreatic β-cells and the acquisition of glucose-responsive insulin secretion. Although transcriptional programs have been well characterized, the metabolic adjustments that enhance insulin secretion in neonatal β-cells remain poorly understood. Neonatal islets exhibit a high rate of basal insulin secretion, and elevating glucose to high concentrations produces little additional stimulation (unlike adult islets). This study aimed to elucidate how neonatal pancreatic β-cells become glucose-responsive, using an in vitro protocol involving culture of neonatal islets at low glucose to emulate systemic hypoglycemia. Low-glucose exposure markedly improved glucose responsiveness of neonatal islets by selectively lowering insulin secretion at low glucose concentrations. Unlike adult β-cells, neonatal β-cells were depolarized and generated spontaneous action potentials at low glucose, resulting in elevated [Ca2+]i and a blunted response to high glucose. These changes were reversed by brief (1-2 hours) low-glucose culture, which was accompanied by increased KATP channel surface expression and activity. The effects of low-glucose incubation were mimicked by the AMPK agonist AICAR. These insights may inform strategies to restore glucose-regulated insulin secretion in type 2 diabetes.

BACKGROUND:Hypoglycaemia, a common complication of insulin-treated diabetes, especially type 1 diabetes (T1D), is caused by impaired counterregulatory increases in plasma glucagon, leading to decreased hepatic glucose production. Alpha-cells in the pancreatic islets, which have sulfonylurea-sensitive ATP-regulated potassium (KATP) channels, secrete glucagon at low plasma glucose levels in response to increased electrical activity. Activation of KATP channels inhibits alpha-cell electrical activity and consequently reduces glucagon secretion. Excessive secretion of somatostatin from neighbouring delta-cells (which are sensitive to the SGLT2 inhibitor dapagliflozin) also causes inappropriate paracrine inhibition of glucagon secretion at low glucose levels. We hypothesise that increased KATP channel activity and/or intra-islet somatostatin may contribute to the glucagon secretion defects seen in T1D and that these issues can be addressed with sulfonylureas or SGLT2 inhibitors. METHODS:We first tested this hypothesis in vitro by comparing glucagon secretion in response to increasing concentrations of the sulfonylurea tolbutamide-used to titrate KATP channel activity-in isolated human islets from donors with T1D and healthy donors without a history of diabetes. Next, we conducted a single-centre, randomised, triple crossover, phase 2a superiority trial in people with T1D to evaluate the effectiveness of oral glibenclamide (0.3, 0.6, and 3 mg/day, over 14-18 days per dose) or an acute single dose of dapagliflozin (10 mg) in restoring glucagon counter-regulation. This was assessed using a hyperinsulinaemic-hypoglycaemic clamp in 12 male participants with T1D, with responses from 6 healthy controls (4 males and 2 females) serving as a reference. The primary endpoint was the change in plasma glucagon levels during hypoglycaemia from baseline (no medication) to post-treatment (glibenclamide or dapagliflozin). All participants were included in the analyses. The study is registered with ISRCTN (58098350). FINDINGS:In islets from 9 healthy male donors, lowering glucose from 6 to 1 mM (to emulate hypoglycaemia) stimulated glucagon secretion by 120%. This effect was not observed in islets from 7 donors with T1D (4 males and 3 females). In these islets, a low concentration (10 μM) but not a high (100 μM) concentration of the sulfonylurea tolbutamide stimulated glucagon release by 70%. During the trial, insulin-induced hypoglycaemia (plasma glucose: <3 mM), in the absence of glibenclamide or dapagliflozin, produced a >700% increase in circulating glucagon in the control group, but only a 54% increase in participants with T1D. The latter value fell to <10% in the 8 participants with low circulating C-peptide levels (≤6 pM). In these participants, glibenclamide (0.3-0.6 mg/day for 14-18 days) raised circulating glucagon during hypoglycaemia by 125-150%; this correlated with reduced glucose infusion requirements, reflecting stimulation of endogenous glucose production. No effect on either parameter was seen with 3 mg/day glibenclamide. These increases in circulating glucagon were associated with better glucose control, as measured by continuous glucose monitoring during the last 7 days of each treatment period. Similar effects on plasma glucagon secretion and glucose infusion rates were observed with the SGLT2 inhibitor dapagliflozin. INTERPRETATION:Enhanced KATP channel activity in alpha-cells and/or increased paracrine inhibition by somatostatin impairs the counterregulatory glucagon response in T1D. These issues can be partially corrected by glibenclamide or dapagliflozin. We suggest that these agents be further tested as adjuvants to insulin therapy in T1D to help reduce the occurrence of iatrogenic hypoglycaemia and the risks it poses. FUNDING:Supported by The Leona M. and Harry B. Helmsley Charitable Trust. The funding body did not influence the study's design, data analysis, or interpretation.

Type 2 diabetes (T2D) is a devastating chronic disease marked by pancreatic β cell dysfunction and insulin resistance, whose pathophysiology remains poorly understood. HNF1A, which encodes transcription factor hepatocyte nuclear factor-1 alpha, is the most commonly mutated gene in Mendelian diabetes. HNF1A also carries loss- or gain-of-function coding variants that respectively predispose to or protect against polygenic T2D. The mechanisms underlying HNF1A-deficient diabetes, however, are still unclear. We now demonstrate that diabetes arises from β cell-autonomous defects and identify direct β cell genomic targets of HNF1A. This uncovered a regulatory axis where HNF1A controls transcription of A1CF, which orchestrates an RNA splicing program encompassing genes that regulate β cell function. This HNF1A-A1CF transcription-splicing axis is suppressed in β cells from T2D individuals, while genetic variants reducing pancreatic islet A1CF are associated with increased glycemia and T2D susceptibility. Our findings, therefore, identify a linear hierarchy that coordinates β cell-specific transcription and splicing programs and link this pathway to T2D pathogenesis.

Gliflozins, such as dapagliflozin, belong to a class of drugs that inhibit the sodium-glucose cotransporter 2. Gliflozins have been found to raise glucagon levels, a hormone secreted from pancreatic islet α-cells, which can trigger ketosis. However, the precise mechanisms through which gliflozins increase glucagon secretion remain poorly understood. In addition, gliflozins induce osmotic diuresis, resulting in increased urine volume and plasma osmolality. In this study, we investigated the hypothesis that a compensatory increase in arginine-vasopressin (AVP) mediates dapagliflozin-induced increases in glucagon in vivo. We show that dapagliflozin does not increase glucagon secretion in the perfused mouse pancreas, neither at clinical nor at supra-clinical doses. In contrast, AVP potently increases glucagon secretion. In vivo, dapagliflozin increased plasma glucagon, osmolality, and AVP. An oral load with hypertonic saline amplified dapagliflozin-induced glucagon secretion. Notably, a similar increase in glucagon could also be elicited by dehydration, evoked by 24-h water restriction. Conversely, blockade of vasopressin 1b receptor signaling, with either pharmacological antagonism or knockout of the receptor, resulted in reduced dapagliflozin-induced glucagon secretion in response to both dapagliflozin and dehydration. Finally, blocking vasopressin 1b receptor signaling in a mouse model of type 1 diabetes diminished the glucagon-promoting and ketogenic effects of dapagliflozin. Collectively, our data suggest that AVP is an important regulator of glucagon release during both drug-induced and physiological dehydration.NEW & NOTEWORTHY Gliflozin-induced ketogenic effects partly result from increased glucagon levels. This study shows that dapagliflozin-triggered glucagon secretion is not directly mediated by the pancreas but rather linked to arginine-vasopressin (AVP). Dehydration, common in diabetic ketoacidosis, elevates AVP, potentially explaining the increased ketoacidosis risk in gliflozin-treated patients. Thus, our results highlight AVP as a potential therapeutic target to mitigate the risk of ketoacidosis associated with gliflozin treatments in patients with diabetes.

By stimulating hepatic glucose production, glucagon (released by islet α-cells) restores normal blood glucose levels when they fall below the normal range. We used optogenetics in conjunction with electrophysiology, [Ca2+]i imaging and hormone release measurements to explore the intrinsic and paracrine regulation of glucagon secretion. Many α-cells were spontaneously active at 1mM glucose. However, up to ~50% of the α-cells were electrically silent. KATP channel blockade, amino acids and somatostatin receptor (SSTR) antagonism restored electrical activity in such α-cells. Termination of optoactivation resulted in KATP channel-dependent (tolbutamide-sensitive) membrane repolarization in active α-cells but long-lasting membrane depolarization and action potential firing in silent α-cells. The latter effect was associated with an increased cytoplasmic ATP:ADP-ratio. Optoactivation or -inhibition of somatostatin-releasing δ-cells inhibits and stimulates electrical activity in adjacent (but not distal) α-cells. There is an inverse relationship between basal glucagon secretion (a measure of the fraction active α-cells) and the relative stimulatory effects of amino acids. We conclude that islet α-cells are functionally heterogenous and that their electrical excitability and glucagon release are determined by K+ channel activity due to variable mosaic of KATP and somatostatin-sensitive K+ channels reflecting metabolic state and proximity to δ-cells, respectively.

Smoking is widely regarded as a risk factor for type 2 diabetes because nicotine contributes to insulin resistance by desensitizing the insulin receptors in muscle, liver, or fat. Little is known, however, about the immediate regulation of islet hormonal output by nicotine, an agonist of ionotropic cholinergic receptors. We investigated this by imaging cytosolic Ca2+ dynamics in mouse and human islets using confocal microscopy and measuring glucagon secretion in response to the alkaloid from isolated mouse islets. Nicotine acutely stimulated cytosolic Ca2+ in glucagon-secreting α-cells but not in insulin-secreting β-cells. The 2.8- ± 0.5-fold (P < 0.05) increase in Ca2+, observed in >70% of α-cells, correlated well with a 2.5- ± 0.3-fold stimulation of glucagon secretion. Nicotine-induced elevation of cytosolic Ca2+ relied on influx from the extracellular compartment rather than release of the cation from intracellular depots. Metabotropic cholinergic signaling, monitored at the level of intracellular diacylglycerol, was limited to 69% of α-cells versus 94% of β-cells. We conclude that parasympathetic regulation of pancreatic islet hormone release uses different signaling pathways in β-cells (metabotropic) and α-cells (metabotropic and ionotropic), resulting in the fine-tuning of acetylcholine-induced glucagon exocytosis. Sustained nicotinic stimulation is, therefore, likely to attenuate insulin sensitivity by increasing glucagon release. ARTICLE HIGHLIGHTS:

Insulin secretion from vesicles within pancreatic beta cells occurs through the rapid (≤10 ms) process of exocytosis. A crucial final step in this process is the formation of a fusion pore, which connects the insulin vesicle interior to the extracellular space and insulin secretion rate. We employed real-time single-cell amperometry (SCA) with microsensors to quantitatively and dynamically monitor serotonin secretion (used as a proxy for insulin) and alterations in the dynamics of fusion pore formation, including opening, duration, and closing times, during single exocytosis events. Additionally, total internal reflection fluorescence (TIRF) microscopy was used to track docked vesicles and optically measure insulin release. In beta cells from human organ donors with type 2 diabetes (T2D), the fusion pore opening was prematurely aborted, leading to diminished cargo release. Furthermore, the number of docked vesicles per beta cell differed between healthy donors and individuals with T2D. In conclusion, our combined super-sensitive optical and electrochemical analyses from a new perspective highlight a link between defective fusion pore dynamics and reduced insulin secretion (a hallmark of T2D) that has not been previously reported. These findings reveal the role of impaired fusion pore dynamics in T2D, independent of its underlying etiology, with potential therapeutic implications.

Somatostatin is a powerful inhibitor of insulin secretion and β-cell electrical activity but the effects are weak in intact islets, possibly because of high intraislet somatostatin levels. We used optogenetics in conjunction with hormone secretion measurements, electrophysiology and [Ca2+]i imaging to interrogate the relative roles of paracrine and electrical control of β-cells by δ-cells. We confirm that optoactivation and -inhibition of δ-cells stimulated and inhibited their electrical activity and somatostatin secretion, respectively. Unexpectedly, neither optoactivation nor -inhibition of δ-cells had any effect on insulin secretion at 1 or 20 mM glucose. Paradoxically, optoactivation of δ-cells at 6 mM glucose increased insulin secretion by 113%, an effect that correlated with β-cell action potential firing. In [Ca2+]i imaging experiments, optoactivation of δ-cells induced islet-wide β-cell [Ca2+]i transients and synchronized the oscillatory pattern induced by 7 mM glucose. Conversely, optoinhibition of δ-cells and somatostatin secretion reduced rather than increased β-cell electrical activity and [Ca2+]i in the <10% of β-cells situated <20 µm from δ-cells. We propose that δ-cells, in addition to subserving an inhibitory paracrine effect, play a role in the rapid propagation of electrical signals across the islet, possibly contributing to the coordination of β-cell activity.

While liraglutide effectively treats type 2 diabetes (T2D) and obesity, its mechanism of action across disease progression remains poorly understood. Liraglutide selectively enhances GSIS in islets from glucose-intolerant donors and in islets exposed to prediabetic conditions, but not in normoglycemic or T2D islets. In healthy mice, liraglutide's insulinotropic effect requires tanycyte-mediated central transport, whereas in glucose intolerance it acts directly on islets. Additionally, liraglutide reduces blood glucose in normoglycemic mice through insulin-independent mechanisms involving decreased gluconeogenesis and enhanced peripheral glucose uptake. These findings demonstrate that the therapeutic window for liraglutide's pancreatic effects may be optimal during prediabetes, while its central and insulin-independent actions predominate in other metabolic states.

iPSC-derived human β-like cells (BLC) hold promise for both therapy and disease modelling, but their generation remains challenging and their functional analyses beyond transcriptomic and morphological assessments remain limited. Here, we validate an approach using multicellular and single cell electrophysiological tools to evaluate function of BLCs from pioneer protocols that can be easily adapted to more differentiated BLCs. The Multi-Electrode Arrays (MEAs) measuring the extracellular electrical activity revealed that BLCs are electrically coupled, produce slow potential (SP) signals like primary β-cells that are closely linked to insulin secretion. We also used high-resolution single-cell patch-clamp measurements to capture the exocytotic properties, and characterise voltage-gated sodium and calcium currents and found that they were comparable to those in primary β and EndoC-βH1 cells. The KATP channel conductance is greater than in human primary β-cells which may account for the limited glucose responsiveness observed with MEA. We used MEAs to study the impact of the type 2 diabetes protective SLC30A8 allele (p.Lys34Serfs*50) and found that BLCs with this allele have stronger electrical coupling activity. Our data suggest that BLCs can be used to evaluate the functional impact of genetic variants on β-cell function and coupling.

Glucagon is secreted by pancreatic a - cells to counteract hypoglycaemia. How glucose regulates glucagon secretion remains unclear. Here, using mouse islets, we studied the role of transmembrane and endoplasmic reticulum (ER) Ca t+ on intrinsic a - cell glucagon secretion. Blocking isradipine-sensitive L -type voltage -gated Ca t+ (Ca v ) channels abolished a - cell electrical activity but had little impact on its cytosolic Ca t+ oscillations or low -glucose -stimulated glucagon secretion. In contrast, depleting ER Ca t+ with cyclopiazonic acid or blocking ER Ca t+ -releasing ryanodine receptors abolished a - cell glucose sensitivity and low -glucose -stimulated glucagon secretion. ER Ca t+ mobilization in a - cells is regulated by intracellular ATP and likely to be coupled to Ca t+ influx through P/Q-type Ca v channels. u- Agatoxin IVA blocked a - cell ER Ca t+ release and cell exocytosis, but had no additive effect on glucagon secretion when combined with ryanodine. We conclude that glucose regulates glucagon secretion through the control of ER Ca t+ mobilization, a mechanism that can be independent of a - cell electrical activity.