Under normal physiological conditions, glucagon is released from pancreatic alpha cells to elevate circulating glucose levels in response to hypoglycemia. In patients with type 2 diabetes, glucagon secretion is dysregulated, but the underlying mechanisms remain unclear. Several hypotheses have been suggested to explain the coupling of blood glucose sensing to electrical activity and glucagon secretion from alpha cells. Here, we show that glucose rapidly regulates mitochondrial motility and localization in alpha cells. Under conditions of low glucose, mitochondria are arrested in positions further from the nucleus, correlating with increased ATP/ADP in the sub-plasma membrane space. We also find that knockdown (KD) of Mitochondrial Rho GTPase 2 (Miro2), but not Miro1, reduces mitochondrial motility in alpha cells and impairs glucose-induced inhibition of glucagon secretion without effects on insulin secretion or mitochondrial motility in non-alpha islet cells. These findings highlight the significance of mitochondrial motility for alpha cell function and reveal fundamental differences between alpha and beta cells.

Using 13C6 glucose labeling coupled to gas chromatography-mass spectrometry and 2D 1H-13C heteronuclear single quantum coherence NMR spectroscopy, we have obtained a comparative high-resolution map of glucose fate underpinning β cell function. In both mouse and human islets, the contribution of glucose to the tricarboxylic acid (TCA) cycle is similar. Pyruvate fueling of the TCA cycle is primarily mediated by the activity of pyruvate dehydrogenase, with lower flux through pyruvate carboxylase. While the conversion of pyruvate to lactate by lactate dehydrogenase (LDH) can be detected in islets of both species, lactate accumulation is 6-fold higher in human islets. Human islets express LDH, with low-moderate LDHA expression and β cell-specific LDHB expression. LDHB inhibition amplifies LDHA-dependent lactate generation in mouse and human β cells and increases basal insulin release. Lastly, cis-instrument Mendelian randomization shows that low LDHB expression levels correlate with elevated fasting insulin in humans. Thus, LDHB limits lactate generation in β cells to maintain appropriate insulin release.

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.

Abstract Pancreas and islet cell transplantation are used in the treatment of patients with diabetes complications. During static cold storage (SCS) ATP is depleted, and by-products of anaerobic respiration accumulate. Ischaemia-reperfusion injury (IRI) affects quantity and viability of islets and it is characterised by acinar necrosis, oedema, and endothelial disruption (graft pancreatitis). The introduction of HMPO2 in liver & kidney preservation has demonstrated a significant reduction in the consequences of IRI. Pancreas HMPO2 was shown to be feasible in pre-clinical studies. We aimed to compare a ‘continuous’ to an ‘end-ischaemic’ approach of HMPO2 in pancreases using a porcine circulatory death model. Porcine pancreases were either started on HMPO2 for the totality of the cold storage time (‘Continuous’ group, n=6) or for the last two hours of the cold storage time (‘End’ group, n=6). All pancreases then underwent normothermic reperfusion (NR) to mimic transplantation. Glucose stimulated insulin secretion (GSIS) was measured using Mercodia human insulin ELISA (n=3 in each group). Both groups had no significant change of wet-to-dry ratio during the experiment, despite an increase in gross weight. Flows were higher during NR in the Continuous group. Amylase, Lipase and LDH increased throughout the study for all pancreases and showed no statistically significant difference between both groups. The Continuous group had a significantly greater insulin secretion in response to glucose stimulation and followed a biphasic pattern. Continuous HMPO2 preserved islet function and was a superior mode of preservation, showing no statistically significant difference in oedema or markers of damage, with improved perfusion parameters.

Diabetes mellitus involves both insufficient insulin secretion and dysregulation of glucagon secretion1. In healthy people, a fall in plasma glucose stimulates glucagon release and thereby increases counter-regulatory hepatic glucose production. This response is absent in many patients with type-1 diabetes (T1D)2, which predisposes to severe hypoglycaemia that may be fatal and accounts for up to 10% of the mortality in patients with T1D3. In rats with chemically induced or autoimmune diabetes, counter-regulatory glucagon secretion can be restored by SSTR antagonists4-7 but both the underlying cellular mechanism and whether it can be extended to humans remain unestablished. Here, we show that glucagon secretion is not stimulated by low glucose in isolated human islets from donors with T1D, a defect recapitulated in non-obese diabetic mice with T1D. This occurs because of hypersecretion of somatostatin, leading to aberrant paracrine inhibition of glucagon secretion. Normally, KATP channel-dependent hyperpolarization of β-cells at low glucose extends into the δ-cells through gap junctions, culminating in suppression of action potential firing and inhibition of somatostatin secretion. This 'electric brake' is lost following autoimmune destruction of the β-cells, resulting in impaired counter-regulation. This scenario accounts for the clinical observation that residual β-cell function correlates with reduced hypoglycaemia risk8.

Aims/hypothesis Diabetes mellitus is associated with impaired insulin secretion, often aggravated by oversecretion of glucagon. Therapeutic interventions should ideally correct both defects. Glucagon-like peptide 1 (GLP-1) has this capability but exactly how it exerts its glucagonostatic effect remains obscure. Following its release GLP-1 is rapidly degraded from GLP-1(7–36) to GLP-1(9–36). We hypothesised that the metabolite GLP-1(9–36) (previously believed to be biologically inactive) exerts a direct inhibitory effect on glucagon secretion and that this mechanism becomes impaired in diabetes. Methods We used a combination of glucagon secretion measurements in mouse and human islets (including islets from donors with type 2 diabetes), total internal reflection fluorescence microscopy imaging of secretory granule dynamics, recordings of cytoplasmic Ca 2+ and measurements of protein kinase A activity, immunocytochemistry, in vivo physiology and GTP-binding protein dissociation studies to explore how GLP-1 exerts its inhibitory effect on glucagon secretion and the role of the metabolite GLP-1(9–36). Results GLP-1(7–36) inhibited glucagon secretion in isolated islets with an IC 50 of 2.5 pmol/l. The effect was particularly strong at low glucose concentrations. The degradation product GLP-1(9–36) shared this capacity. GLP-1(9–36) retained its glucagonostatic effects after genetic/pharmacological inactivation of the GLP-1 receptor. GLP-1(9–36) also potently inhibited glucagon secretion evoked by β-adrenergic stimulation, amino acids and membrane depolarisation. In islet alpha cells, GLP-1(9–36) led to inhibition of Ca 2+ entry via voltage-gated Ca 2+ channels sensitive to ω-agatoxin, with consequential pertussis-toxin-sensitive depletion of the docked pool of secretory granules, effects that were prevented by the glucagon receptor antagonists REMD2.59 and L-168049. The capacity of GLP-1(9–36) to inhibit glucagon secretion and reduce the number of docked granules was lost in alpha cells from human donors with type 2 diabetes. In vivo, high exogenous concentrations of GLP-1(9–36) (>100 pmol/l) resulted in a small (30%) lowering of circulating glucagon during insulin-induced hypoglycaemia. This effect was abolished by REMD2.59, which promptly increased circulating glucagon by >225% (adjusted for the change in plasma glucose) without affecting pancreatic glucagon content. Conclusions/interpretation We conclude that the GLP-1 metabolite GLP-1(9–36) is a systemic inhibitor of glucagon secretion. We propose that the increase in circulating glucagon observed following genetic/pharmacological inactivation of glucagon signalling in mice and in people with type 2 diabetes reflects the removal of GLP-1(9–36)’s glucagonostatic action. Graphical Abstract

Abstract Background Frailty is common in patients with chronic heart failure (CHF). Frail patients are at high risk of poor clinical outcomes which might be attributable to the presence of multiple comorbidities. The impact of comorbidities on mortality in frail patients with CHF is not well described. Aim To compare the burden and patterns of comorbidities in frail vs non-frail patients with CHF and their impact on mortality. Methods We studied consecutive patients attending a routine follow-up visit to a HF clinic. Frailty was assessed using the Clinical Frailty Scale (CFS); those with CFS>4 were classified as frail. Patients were classified into 6 comorbidity groups including: metabolic (obesity, diabetes); respiratory; renal; cancer; neuropsychiatric (depression, dementia); and degenerative (falls, arthritis, fragility fractures). We investigated the relation between frailty, comorbidity groups and all-cause mortality in patients with CHF. Results Amongst 467 patients with CHF [67% male, median (IQR) age 76 (69–82) years, NTproBNP 1156 (469–2463) ng/L], 291 patients had HF with reduced ejection fraction (HFrEF, LVEF <40%), and 176 had HF with preserved ejection fraction (HFpEF, LVEF ≥40%). Frailty was more common in HFpEF vs HFrEF (51 vs 40%). 64% of patients had >4 comorbidities (36% 5–6, 21% 7–9 and 7% >9 comorbidities). Frail patients were more likely to have multiple comorbidities than non-frail patients (85% vs 48% with >4 comorbidities, p<0.001). The number of comorbidities increased with worsening frailty severity (Figure 1). Those with HFrEF were more like to suffer from cancer, whereas those with HFpEF were more likely to have neuropsychiatric, metabolic and degenerative comorbidities. During a median follow up of 554 days, 82 (18%) patients died. Increasing number of comorbidities was associated with increasing mortality. (Figure 2) Patients who were frail with >4 comorbidities had a 6-fold increased risk of mortality compared to those who were neither frail nor had multiple comorbidities [HR (95% CI) 6.6 (3.2–13.9), p<0.001]. In a model adjusted for age, sex, logNTproBNP and NYHA class, amongst comorbidity groups, the presence of renal and neuropsychiatric comorbidities were independent predictors of higher mortality. Conclusion Frail patients with CHF have a high comorbidity burden. The co-existence of frailty and multiple comorbidities predisposes to higher risk of mortality. Future studies should investigate whether treatment focusing on comorbidities improve outcomes. Funding Acknowledgement Type of funding sources: None.

Background: Tetraspanin-7 (Tspan7) is an islet autoantigen involved in autoimmune type 1 diabetes and known to regulate beta-cell L-type Ca-2(+) channel activity. However, the role of Tspan7 in pancreatic beta-cell function is not yet fully understood. Methods: Histological analyses were conducted using immunostaining. Whole-body metabolism was tested using glucose tolerance test. Islet hormone secretion was quantified using static batch incubation or dynamic perifusion. beta-cell transmembrane currents, electrical activity and exocytosis were measured using whole-cell patch-clamping and capacitance measurements. Gene expression was studied using m RNA-sequencing and quantitative PCR. Results: Tspan7 is expressed in insulin-containing granules of pancreatic beta-cells and glucagon-producing alpha-cells. Tspan7 knockout mice (Tspan(gamma/-) mouse) exhibit reduced body weight and ad libitum plasma glucose but normal glucose tolerance. Tspan(gamma/- )islets have normal insulin content and glucose- or tolbutamide-stimulated insulin secretion. Depolarisation-triggered Ca2+ current was enhanced in Tspan(gamma/-) beta-cells, but beta-cell electrical activity and depolarisation-evoked exocytosis were unchanged suggesting that exocytosis was less sensitive to Ca2+. TSPAN7 knockdown (KD) in human pseudo-islets led to a significant reduction in insulin secretion stimulated by 20 mM Transcriptomic analyses show that TSPAN7 KD in human pseudo-islets correlated with changes in genes involved in hormone secretion, apoptosis and ER stress. Consistent with rodent beta-cells, exocytotic Ca2+ sensitivity was reduced in a human beta-cell line (EndoC-beta H1) following Tspan7 KD. Conclusion: Tspan7 is involved in the regulation of Ca2+-dependent exocytosis in beta-cells. Its function is more significant in human beta-cells than their rodent counterparts.

ABSTRACT Using 13 C 6 glucose labeling coupled to GC-MS and 2D 1 H- 13 C HSQC NMR spectroscopy, we have obtained a comparative high-resolution map of glucose fate underpinning β cell function. In both mouse and human islets, the contribution of glucose to the TCA cycle is similar. Pyruvate-fueling of the TCA cycle is primarily mediated by the activity of pyruvate dehydrogenase, with lower flux through pyruvate carboxylase. While conversion of pyruvate to lactate by lactate dehydrogenase (LDH) can be detected in islets of both species, lactate accumulation is six-fold higher in human islets. Human islets express LDH, with low-moderate LDHA expression and β cell-specific LDHB expression. LDHB inhibition increases glucose-dependent lactate generation in mouse and human β cells, and decreases Ca 2+ -spiking frequency without affecting ATP/ADP levels. Thus, we show that LDHB limits glucose-stimulated lactate generation in β cells. Further studies are warranted to understand how lactate impacts β cell metabolism and/or function. HIGHLIGHTS Human and rodent islets generate lactate following glucose stimulation. β cells specifically express LDHB, which acts to limit lactate generation. LDHB inhibition influences Ca 2+ spiking frequency without affecting ATP/ADP ratio. eTOC Cuozzo et al show that glucose-stimulated rodent and human islets generate lactate. Transcriptomic and imaging analyses reveal that LDHB is specifically expressed in β cells and unexpectedly restrains lactate production. LDHB expression and thus regulated lactate generation might reflect a key mechanism underlying β cell metabolism, function and survival.

Background. Whole pancreas transplantation (Tx) is a successful treatment for type 1 diabetes resulting in independence from antidiabetic therapies. Transplant-related factors contributing to pancreatic islet failure are largely unknown; both recurring insulitis and pancreatitis have been implicated. The aim was to determine if cellular changes in islets and exocrine tissue are evident early in Tx, which could contribute to eventual graft failure using well-preserved tissue of grafts explanted from largely normoglycemic recipients. Methods. Histological specimens of explants (n = 31), Tx duration 1 day–8 years (median 29 d), cold ischemia time 7.2–17.3 hours (median 11.1 h), donor age 13–54 years (median 38 y) were examined; sections were labeled for inflammation, islet amyloidosis, and tissue fibrosis, and morphometry performed on immunolabeled insulin and glucagon positive islet cells. Data were related to clinical details of donor, recipient, and features of Tx. Results. Islet inflammation consistent with recurrent insulitis was not seen in any sample. Insulin-labeled islet cell proportion decreased with donor age (P < 0.05) and cold ischemia (P < 0.01) in explants from 26 normoglycemic patients; glucagon-labeled area proportion increased with cold ischemia (P < 0.05). Clinical pancreatitis was the explant reason in 12 of 28 normoglycemic cases. Exocrine fibrotic area/pancreas was variable (0.7%–55%) and unrelated to clinical/pathological features. Islet amyloid was present in 3 normoglycemic cases (donor ages 58, 42, and 31 y; Tx duration 8 y, 31 and 33 d, respectively). In 1 patient receiving antidiabetic therapy, the insulin-labeled area was reduced but with no evidence of islet inflammation. Conclusions. Explant histological changes after short-term Tx are similar to those seen in type 2 diabetes and occur in the absence of immunologic rejection without causing hyperglycemia. This suggests that factors associated with Tx affect islet stability; persistent deterioration of islet integrity and exocrine tissue fibrosis could impact on sustainability of islet function.

BACKGROUND:Amyloid deposits are a typical finding in pancreatic islets from patients with type 2 diabetes. Whether this is linked to the pathogenesis of type 2 diabetes is currently unknown. Therefore, we compared the occurrence of islet amyloid in patients with type 2 diabetes, diabetes secondary to pancreatic disorders, and nondiabetic individuals.PATIENTS AND METHODS:Pancreatic tissue from 15 nondiabetic patients, 22 patients with type 2 diabetes, and 11 patients with diabetes due to exocrine pancreatic disorders (chronic pancreatitis, pancreatic carcinoma) were stained for insulin, amyloid, and apoptosis. β-cell area, amyloid deposits, and β-cell apoptosis were quantified by morphometric analysis.RESULTS:The proportion of islets containing amyloid deposits was significantly higher in both type 2 diabetes and diabetes due to exocrine pancreatic disorders than in healthy subjects. Islets with both amyloid and apoptosis were observed more frequently in type 2 diabetes and significantly more so in diabetes due to exocrine pancreatic disorders. In both diabetic groups, apoptotic ß-cells were found significantly more frequently in islets with more prominent amyloid deposits.CONCLUSIONS:The occurrence of amyloid deposits in both type 2 diabetes and diabetes secondary to exocrine pancreatic disorders suggests that islet amyloid formation is a common feature of diabetes mellitus of different etiologies and may be associated with a loss of pancreatic ß-cells.

The incretin hormone glucagon-like peptide 1(7-36) (GLP-1(7-36)) stimulates insulin and inhibits glucagon secretion. The mechanisms by which GLP-1 suppresses glucagon release are unclear as glucagon-secreting α-cells express GLP-1 receptors (GLP-1Rs) at very low levels. Here, we examine the underlying mechanisms. We find that both GLP-1(7-36) and its degradation product GLP-1(9-36) inhibit glucagon secretion at physiological (pM) concentrations. Whereas the effect of GLP-1(7-36) is sensitive to PKA inhibition, GLP-1(9-36) exerts its effect by a PKA-independent mechanism sensitive to pretreatment with pertussis. The glucagonostatic effects of both GLP-1(7-36) and (9-36) are retained in islets from Glp1r knockout mice but only GLP-1(9-36) remains glucagonostatic in the presence of the DPP-4 (the peptidase catalyzing the formation of GLP-1(9-36)) inhibitor sitagliptin. Glucagon receptor (GCGR) antagonism specifically prevents the inhibitory effects of GLP-1(9-36) whilst not affecting that of GLP-1(7-36). We conclude that GLP-1(7-36) and GLP-1(9-36) regulate glucagon secretion via interaction with GLP-1R and GCGR, respectively. Highlights

SummaryMetabolic dysregulation in multiple tissues alters glucose homeostasis and influences risk for type 2 diabetes (T2D). To identify pathways and tissues influencing T2D-relevant glycemic traits (fasting glucose [FG], fasting insulin [FI], two-hour glucose [2hGlu] and glycated hemoglobin [HbA1c]), we investigated associations of exome-array variants in up to 144,060 individuals without diabetes of multiple ancestries. Single-variant analyses identified novel associations at 21 coding variants in 18 novel loci, whilst gene-based tests revealed signals at two genes, TF (HbA1c) and G6PC (FG, FI). Pathway and tissue enrichment analyses of trait-associated transcripts confirmed the importance of liver and kidney for FI and pancreatic islets for FG regulation, implicated adipose tissue in FI and the gut in 2hGlu, and suggested a role for the non-endocrine pancreas in glucose homeostasis. Functional studies demonstrated that a novel FG/FI association at the liver-enriched G6PC transcript was driven by multiple rare loss-of-function variants. The FG/HbA1c-associated, islet-specific G6PC2 transcript also contained multiple rare functional variants, including two alleles within the same codon with divergent effects on glucose levels. Our findings highlight the value of integrating genomic and functional data to maximize biological inference.Highlights23 novel coding variant associations (single-point and gene-based) for glycemic traits51 effector transcripts highlighted different pathway/tissue signatures for each traitThe exocrine pancreas and gut influence fasting and 2h glucose, respectivelyMultiple variants in liver-enriched G6PC and islet-specific G6PC2 influence glycemia