Regulation Of Glucagon Secretion Research Articles

12248 Leptin Signaling Deficiency At The Central Nervous System Reduces Hepatic Glucagon Sensibility Disturbing Liver Amino Acid Metabolism Leading To Hyperaminoacidemia And Hyperglucagonemia In Male Adult Rats

Abstract Disclosure: C. Pintado Losa: None. L. Mazuecos Fernández-Pacheco: None. Ó. Gómez Torres: None. S. Artigas Jerónimo: None. E. Burgos Ramos: None. I. Ramos: None. J. Poveda Colado: None. C. Arribas Mocoroa: None. A. Andres: None. N. Gallardo: None. Leptin regulates glucagon secretion, but the contribution of central leptin resistance to circulating glucagon levels and hepatic glucagon signaling is poorly studied. To analyze the effects of deficient hypothalamic leptin signaling on glucagon levels and hepatic glucagon signaling, we infused intracerebroventricularly a rat-specific leptin receptor antagonist SLA (0,2 µg/day) for 21 days to male 3-month-old Wistar rats (n=12) or PBS (n=12). At day 21 of treatment and 30 min before sacrifice, a glucagon challenge (100 µg/kg) was performed in 16h fasted rats. After sacrifice, blood and liver samples were collected for analysis. Serum hormones, urea, and total and branched-chain amino acids (BCAA) levels were determined using commercial kits and by RP-HPLC. Hepatic neutral lipid was extracted and quantified using an enzymatic kit and the hepatic content of mRNA and proteins involved in lipid and amino acid metabolism were measured by RT-PCR and Western-Blot. Additionally, the HOMA-IR and glucagon-alanine index were determined in all rats. Blocking central leptin signaling led to impaired fat oxidation and triglyceride export capacity in the liver, resulting in a 50% increase in hepatic triglyceride content in SLA-treated rats. Glucagonemia increased in SLA-treated rats, indicating impaired hepatic glucagon sensitivity. In fact, in response to exogenous glucagon, the hepatic phosphorylation of CREB was reduced by 55% in SLA-infused compared to control rats. Importantly, SLA treatment significantly decreases the expression and the activity of BCKDH (branched-chain alpha-keto acid dehydrogenase) in the liver, leading to a reduction in the BCAA degradation in SLA-treated rats, hyperaminoacidemia, and decreased serum urea levels. In conclusion, deficient hypothalamic leptin signaling alters glucose, lipid, and amino acid homeostasis, highlighting the relevance of central leptin action in the control of peripheral metabolism and glucagon sensitivity. Funding: Work supported by Ministerio de Ciencia, Innovación y Universidades under project grants RTI2018-098643-B-I00, PID2021-128243OB-I00, and from the University of Castilla-La Mancha and the European Regional Development Fund (FEDER) under project grant 2023- GRIN-34280. Presentation: 6/1/2024

Intra-islet α-cell Gs signaling promotes glucagon release

Glucagon, a hormone released from pancreatic α-cells, is critical for maintaining euglycemia and plays a key role in the pathophysiology of diabetes. To stimulate the development of new classes of therapeutic agents targeting glucagon release, key α-cell signaling pathways that regulate glucagon secretion need to be identified. Here, we focused on the potential importance of α-cell Gs signaling on modulating α-cell function. Studies with α-cell-specific mouse models showed that activation of α-cell Gs signaling causes a marked increase in glucagon secretion. We also found that intra-islet adenosine plays an unexpected autocrine/paracrine role in promoting glucagon release via activation of α−cell Gs-coupled A2A adenosine receptors. Studies with α-cell-specific Gαs knockout mice showed that α-cell Gs also plays an essential role in stimulating the activity of the Gcg gene, thus ensuring proper islet glucagon content. Our data suggest that α-cell enriched Gs-coupled receptors represent potential targets for modulating α-cell function for therapeutic purposes.

Metabolic regulation of glucagon secretion.

Since the discovery of glucagon 100 years ago, the hormone and the pancreatic islet alpha cells that produce it have remained enigmatic relative to insulin-producing beta cells. Canonically, alpha cells have been described in the context of glucagon's role in glucose metabolism in liver, with glucose as the primary nutrient signal regulating alpha cell function. However, current data reveal a more holistic model of metabolic signalling, involving glucagon-regulated metabolism of multiple nutrients by the liver and other tissues, including amino acids and lipids, providing reciprocal feedback to regulate glucagon secretion and even alpha cell mass. Here we describe how various nutrients are sensed, transported and metabolised in alpha cells, providing an integrative model for the metabolic regulation of glucagon secretion and action. Importantly, we discuss where these nutrient-sensing pathways intersect to regulate alpha cell function and highlight key areas for future research.

Glucose Controls Glucagon Secretion by Regulating Fatty Acid Oxidation in Pancreatic α-Cells.

It has become clear that dysregulation of glucagon secretion and α-cell function plays an important role in the development of diabetes, but we do not know how glucagon secretion is regulated. Here we asked whether glucose inhibits fatty acid oxidation in α-cells to regulate glucagon secretion. We found that fatty acid oxidation is required for the inhibitory effects of glucose on glucagon secretion through reductions in ATP. These findings provide a new framework for the regulation of glucagon secretion by glucose.

200-OR: Role of Insulin-Degrading Enzyme (IDE) in the Tubulin–Primary Cilium Axis to Regulate Glucagon Secretion

Hyperglucagonemia is a hallmark of T2D, although molecular mechanisms underlying glucagon secretion dysregulation are poorly understood. We have recently reported that genetic ablation of IDE in α-cells (A-IDE-KO mouse) leads to constitutive glucagon secretion, hypertrophy, hyperplasia and increased proliferation. αTC1.9-KD-IDE cells (~30% IDE-knock-down) displayed reduced tubulin and acetylated-α-tubulin protein levels, impaired primary cilium and increased proliferation. Thus, we aimed to elucidate the role of IDE in α-cells tubulin network and ciliogenesis, and how this axis may be part of the glucagon secretion machinery. We have studied by western-blot IDE, tubulin, acetylated-α-tubulin and Arl13b (primary cilium marker) levels in αTC1.9 cells under low- and high-glucose conditions. We have found that high-glucose lowers IDE protein levels by 50% compared to low-glucose conditions, leading to a similar decrease in tubulin, acetylated-α-tubulin and Arl13b protein levels. These findings agree with our previous results using αTC1.9-KD-IDE cells, suggesting a relevant role of IDE in microtubule dynamics to control glucagon granules exocytosis. On the other hand, αTC1.9-KD-Arl13b cells (~70% Arl13b-knock-down) were used as a model of impaired ciliogenesis, showing 50% decrease in tubulin, and dysregulated glucagon secretion, mimicking αTC1.9-KD-IDE cells phenotype. In parallel, insulin-mediated inhibition of glucagon secretion is lost and insulin receptor protein levels were decreased by 40% in both, αTC1.9-KD-IDE and αTC1.9-KD-Arl13b cells, showing the relevance of IDE and primary cilium in the paracrine regulation of α-cells. Furthermore, these results have been reproduced in A-IDE-KO mice isolated islets. In conclusion, our results show that IDE-tubulin-primary cilium axis is regulated during glucose-stimulated glucagon secretion, this axis is also required for the paracrine action of insulin on α-cells to inhibit glucagon secretion. Disclosure E.Casanueva-alvarez: None. A.Sanz-gonzález: None. B.Merino: None. G.Perdomo: None. I.Cozar-castellano: None. Funding Ministerio de Ciencia e Innovación-Spain (PID2019-110496RB-C21)

α-cell electrophysiology and the regulation of glucagon secretion.

Glucagon is the principal glucose-elevating hormone that forms the first-line defence against hypoglycaemia. Along with insulin, glucagon also plays a key role in maintaining systemic glucose homeostasis. The cells that secrete glucagon, pancreatic α-cells, are electrically excitable cells and use electrical activity to couple its hormone secretion to changes in ambient glucose levels. Exactly how glucose regulates α-cells has been a topic of debate for decades but it is clear that electrical signals generated by the cells play an important role in glucagon secretory response. Decades of studies have already revealed the key players involved in the generation of these electrical signals and possible mechanisms controlling them to tune glucagon release. This has offered the opportunity to fully understand the enigmatic α-cell physiology. In this review, we describe the current knowledge on cellular electrophysiology and factors regulating excitability, glucose sensing, and glucagon secretion. We also discuss α-cell pathophysiology and the perspective of addressing glucagon secretory defects in diabetes for developing better diabetes treatment, which bears the hope of eliminating hypoglycaemia as a clinical problem in diabetes care.

The expanding incretin universe: from basic biology to clinical translation.

Incretin hormones, principally glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1(GLP-1), potentiate meal-stimulated insulin secretion through direct (GIP + GLP-1) and indirect (GLP-1) actions on islet β-cells. GIP and GLP-1 also regulate glucagon secretion, through direct and indirect pathways. The incretin hormone receptors (GIPR and GLP-1R) are widely distributed beyond the pancreas, principally in the brain, cardiovascular and immune systems, gut and kidney, consistent with a broad array of extrapancreatic incretin actions. Notably, the glucoregulatory and anorectic activities of GIP and GLP-1 have supported development of incretin-based therapies for the treatment of type 2 diabetes and obesity. Here we review evolving concepts of incretin action, focusing predominantly on GLP-1, from discovery, to clinical proof of concept, to therapeutic outcomes. We identify established vs uncertain mechanisms of action, highlighting biology conserved across species, while illuminating areas of active investigation and uncertainty that require additional clarification.

Roles of mixed nutrient intakes on metabolic syndrome among korean adults 19-80 years old: molecular mechanisms involved.

We aim to identify the association between nutrient intake mixtures (22 micro-macro nutrients) and metabolic syndrome (MetS) or its components, including molecular mechanisms involved, among 16,807 Korean adults aged 19-80. The associations of mixed nutrient intakes on MetS or its components were identified using linear regression models, WQS regression, qgcomp, and BKMR regression. Genes, transcription factors, miRNA, biological processes, and pathways were assessed using GeneMania, CHEA3, MIENTURNET, and ToppFun functions. We found that the overall effect of mixed nutrient intakes was also related to MetS and its components. In silico analysis, we found that a mixture of nutrients interacted with the IL6 gene and was linked with MetS. Physical interactions were the key interactions (77%) involved in the mutual genes and MetS targeted by a mixture of nutrients. IL6 related pathways, "positive regulation of type B pancreatic cell apoptotic process", "regulation of glucagon secretion", "LDL pathway during atherogenesis", and "IL-10 anti-inflammatory signaling pathway" were identified as key molecular mechanisms that may be targeted by mixed nutrients implicated in MetS. The key miRNAs and transcription factors involved in the process of MetS targeted by a mixture of nutrients were also described. The cutoff levels for nutrient intake levels associated with MetS and its components were also described. Our findings will pave the way for further research to evaluate the interactions between a mixture of nutrients and non-communicable diseases. The online version contains supplementary material available at 10.1007/s40200-022-01158-1.

RhoA as a Signaling Hub Controlling Glucagon Secretion From Pancreatic α-Cells.

Glucagon hypersecretion from pancreatic islet α-cells exacerbates hyperglycemia in type 1 diabetes (T1D) and type 2 diabetes. Still, the underlying mechanistic pathways that regulate glucagon secretion remain controversial. Among the three complementary main mechanisms (intrinsic, paracrine, and juxtacrine) proposed to regulate glucagon release from α-cells, juxtacrine interactions are the least studied. It is known that tonic stimulation of α-cell EphA receptors by ephrin-A ligands (EphA forward signaling) inhibits glucagon secretion in mouse and human islets and restores glucose inhibition of glucagon secretion in sorted mouse α-cells, and these effects correlate with increased F-actin density. Here, we elucidate the downstream target of EphA signaling in α-cells. We demonstrate that RhoA, a Rho family GTPase, plays a key role in this pathway. Pharmacological inhibition of RhoA disrupts glucose inhibition of glucagon secretion in islets and decreases cortical F-actin density in dispersed α-cells and α-cells in intact islets. Quantitative FRET biosensor imaging shows that increased RhoA activity follows directly from EphA stimulation. We show that in addition to modulating F-actin density, EphA forward signaling and RhoA activity affect α-cell Ca2+ activity in a novel mechanistic pathway. Finally, we show that stimulating EphA forward signaling restores glucose inhibition of glucagon secretion from human T1D donor islets.

229-LB: G6PC2, a GWAS Locus for Fasting Glucose and HbA1c, Regulates Glucose-Suppression of Glucagon Secretion in a-Cells

The G6PC2 locus, encoding the catalytic subunit of the islet-specific glucose-6-phosphatase enzyme, is an established risk locus for HbA1c and fasting glucose levels. A critical role of glycolytic flux in regulating glucagon secretion was established through manipulation of the activity and expression of the glycolytic enzyme glucokinase (GCK) which controls the setpoint for glucose-suppression of glucagon secretion (GSGS) . G6PC2 opposes the action of GCK by hydrolyzing glucose-6-phosphate back to glucose and creating a futile substrate cycle. Previous studies from germline and β-cell-specific G6PC2 null mice have demonstrated that G6PC2 ablation reduces glucose-6-phosphatase activity, glucose cycling, and glycolytic flux, resulting in a leftward shift of dose-response curve for glucose-stimulated insulin secretion. Through cell type specific chromatin accessibility maps of sorted human islet cells, we established that G6PC2 risk SNPs are in open chromatin regions in human α-cells, suggesting that they might not only affect β-cell but also α-cell expression of this critical gene. To test whether α-cell G6PC2 impacts glycemic control, we derived a new mouse line that induces ablation of the first three exons of G6PC2 after Cre activation – resulting in a null allele (G6pc2 loxP/loxP; Gcg-CreERT2; referred to as “α-G6PC2KO”) . Assessment of glucagonemia during glucose tolerance tests revealed a decrease in glucagon levels in α-G6PC2KO mice relative to controls. Intriguingly, fasting blood glucose levels were significantly lower in α-G6PC2KO mice. α-G6PC2KO mice recovered more slowly from hypoglycemia during an insulin tolerance test, and presented lower glucagon levels relative to control mice following the insulin bolus. α-G6PC2KO islets secreted less glucagon relative to control islets when incubated in low-glucose media, independent of alterations in insulin secretion or islet hormone content. Collectively, our data demonstrate that the G6PC2 locus impacts glycemic control via its action in α-cells. Disclosure V. Bahl: None. C. L. May: None. Human pancreas analysis program: n/a. K. H. Kaestner: None.

Protein Kinase C (Pkc)-δ Mediates Arginine-Induced Glucagon Secretion in Pancreatic α-Cells.

The pathophysiology of type 2 diabetes involves insulin and glucagon. Protein kinase C (Pkc)-δ, a serine–threonine kinase, is ubiquitously expressed and involved in regulating cell death and proliferation. However, the role of Pkcδ in regulating glucagon secretion in pancreatic α-cells remains unclear. Therefore, this study aimed to elucidate the physiological role of Pkcδ in glucagon secretion from pancreatic α-cells. Glucagon secretions were investigated in Pkcδ-knockdown InR1G9 cells and pancreatic α-cell-specific Pkcδ-knockout (αPkcδKO) mice. Knockdown of Pkcδ in the glucagon-secreting cell line InR1G9 cells reduced glucagon secretion. The basic amino acid arginine enhances glucagon secretion via voltage-dependent calcium channels (VDCC). Furthermore, we showed that arginine increased Pkcδ phosphorylation at Thr505, which is critical for Pkcδ activation. Interestingly, the knockdown of Pkcδ in InR1G9 cells reduced arginine-induced glucagon secretion. Moreover, arginine-induced glucagon secretions were decreased in αPkcδKO mice and islets from αPkcδKO mice. Pkcδ is essential for arginine-induced glucagon secretion in pancreatic α-cells. Therefore, this study may contribute to the elucidation of the molecular mechanism of amino acid-induced glucagon secretion and the development of novel antidiabetic drugs targeting Pkcδ and glucagon.

Acetyl-CoA-carboxylase 1 (ACC1) plays a critical role in glucagon secretion

Dysregulated glucagon secretion from pancreatic alpha-cells is a key feature of type-1 and type-2 diabetes (T1D and T2D), yet our mechanistic understanding of alpha-cell function is underdeveloped relative to insulin-secreting beta-cells. Here we show that the enzyme acetyl-CoA-carboxylase 1 (ACC1), which couples glucose metabolism to lipogenesis, plays a key role in the regulation of glucagon secretion. Pharmacological inhibition of ACC1 in mouse islets or αTC9 cells impaired glucagon secretion at low glucose (1 mmol/l). Likewise, deletion of ACC1 in alpha-cells in mice reduced glucagon secretion at low glucose in isolated islets, and in response to fasting or insulin-induced hypoglycaemia in vivo. Electrophysiological recordings identified impaired KATP channel activity and P/Q- and L-type calcium currents in alpha-cells lacking ACC1, explaining the loss of glucose-sensing. ACC-dependent alterations in S-acylation of the KATP channel subunit, Kir6.2, were identified by acyl-biotin exchange assays. Histological analysis identified that loss of ACC1 caused a reduction in alpha-cell area of the pancreas, glucagon content and individual alpha-cell size, further impairing secretory capacity. Loss of ACC1 also reduced the release of glucagon-like peptide 1 (GLP-1) in primary gastrointestinal crypts. Together, these data reveal a role for the ACC1-coupled pathway in proglucagon-expressing nutrient-responsive endocrine cell function and systemic glucose homeostasis.

Sodium, Glucose and Dysregulated Glucagon Secretion: The Potential of Sodium Glucose Transporters.

Diabetes is defined by hyperglycaemia due to progressive insulin resistance and compromised insulin release. In parallel, alpha cells develop dysregulation of glucagon secretion. Diabetic patients have insufficient glucagon secretion during hypoglycaemia and a lack of inhibition of glucagon secretion at higher blood glucose levels resulting in postprandial hyperglucagonaemia, which contributes to the development of hyperglycaemia. Sodium-glucose co-transporter 2 (SGLT2) inhibitors are an efficient pharmacologic approach for the treatment of hyperglycaemia in type 2 diabetes. While SGLT2 inhibitors aim at increasing glycosuria to decrease blood glucose levels, these inhibitors also increase circulating glucagon concentrations. Here, we review recent advances in our understanding of how SGLTs are involved in the regulation of glucagon secretion. Sodium plays an important role for alpha cell function, and a tight regulation of intracellular sodium levels is important for maintaining plasma membrane potential and intracellular pH. This involves the sodium-potassium pump, sodium-proton exchangers and SGLTs. While the expression of SGLT2 in alpha cells remains controversial, SGLT1 seems to play a central role for alpha cell function. Under hyperglycaemic conditions, SGLT1 mediated accumulation of sodium results in alpha cell dysregulation due to altered cellular acidification and ATP production. Taken together, this suggests that SGLT1 could be a promising, yet highly underappreciated drug target to restore alpha cell function and improve treatment of both type 1 and 2 diabetes.

The Glucagon-Like Adipokinetic Hormone in Drosophila melanogaster - Biosynthesis and Secretion.

Metabolic homeostasis requires the precise regulation of circulating sugar titers. In mammals, homeostatic control of circulating sugar titers requires the coordinated secretion and systemic activities of glucagon and insulin. Metabolic homeostasis is similarly regulated in Drosophila melanogaster through the glucagon-like adipokinetic hormone (AKH) and the Drosophila insulin-like peptides (DILPs). In flies and mammals, glucagon and AKH are biosynthesized in and secreted from specialized endocrine cells. KATP channels borne on these cells respond to fluctuations in circulating glucose titers and thereby regulate glucagon secretion. The influence of glucagon in the pathogenesis of type 2 diabetes mellitus is now recognized, and a crucial mechanism that regulates glucagon secretion was reported nearly a decade ago. Ongoing efforts to develop D. melanogaster models for metabolic syndrome must build upon this seminal work. These efforts make a critical review of AKH physiology timely. This review focuses on AKH biosynthesis and the regulation of glucose-responsive AKH secretion through changes in CC cell electrical activity. Future directions for AKH research in flies are discussed, including the development of models for hyperglucagonemia and epigenetic inheritance of acquired metabolic traits. Many avenues of AKH physiology remain to be explored and thus present great potential for improving the utility of D. melanogaster in metabolic research.

Misrouting of glucagon and stathmin-2 towards lysosomal system of α-cells in glucagon hypersecretion of diabetes

ABSTRACT Glucagon hypersecretion from the pancreatic α-cell is a characteristic sign of diabetes, which exacerbates fasting hyperglycemia. Thus, targeting glucagon secretion from α-cells may be a promising approach for combating hyperglucagonemia. We have recently identified stathmin-2 as an α-cell protein that regulates glucagon secretion by directing glucagon toward the endolysosomal system in αTC1-6 cells. We hypothesized that disruption of Stmn2-mediated trafficking of glucagon to the endolysosomes in diabetes contributes to hyperglucagonemia. In isolated islets from male mice treated with streptozotocin (STZ), glucagon secretion and cellular content were augmented, but cellular Stmn2 levels were reduced (p < .01), as measured by both ELISA and immunofluorescence intensity. Using confocal immunofluorescence microscopy, the colocalization of glucagon and Stmn2 in Lamp2A+ lysosomes was dramatically reduced (p < .001) in islets from diabetic mice, and the colocalization of Stmn2, but not glucagon, with the late endosome marker, Rab7, significantly (p < .01) increased. Further studies were conducted in αTC1-6 cells cultured in media containing high glucose (16.7 mM) for 2 weeks to mimic glucagon hypersecretion of diabetes. Surprisingly, treatment of αTC1-6 cells with the lysosomal inhibitor bafilomycin A1 reduced K+-induced glucagon secretion, suggesting that high glucose may induce glucagon secretion from another lysosomal compartment. Both glucagon and Stmn2 co-localized with Lamp1, which marks secretory lysosomes, in cells cultured in high glucose. We propose that, in addition to enhanced trafficking and secretion through the regulated secretory pathway, the hyperglucagonemia of diabetes may also be due to re-routing of glucagon from the degradative Lamp2A+ lysosome toward the secretory Lamp1+ lysosome.

Pathways of Glucagon Secretion and Trafficking in the Pancreatic Alpha Cell: Novel Pathways, Proteins, and Targets for Hyperglucagonemia.

Patients with diabetes mellitus exhibit hyperglucagonemia, or excess glucagon secretion, which may be the underlying cause of the hyperglycemia of diabetes. Defective alpha cell secretory responses to glucose and paracrine effectors in both Type 1 and Type 2 diabetes may drive the development of hyperglucagonemia. Therefore, uncovering the mechanisms that regulate glucagon secretion from the pancreatic alpha cell is critical for developing improved treatments for diabetes. In this review, we focus on aspects of alpha cell biology for possible mechanisms for alpha cell dysfunction in diabetes: proglucagon processing, intrinsic and paracrine control of glucagon secretion, secretory granule dynamics, and alterations in intracellular trafficking. We explore possible clues gleaned from these studies in how inhibition of glucagon secretion can be targeted as a treatment for diabetes mellitus.

236-OR: Volagidemab, a Human Glucagon Receptor Antagonist, Improves Glycemic Control in Subjects with Type 1 Diabetes (T1D): A 12-Week, Randomized, Double-Blind, Placebo-Controlled Trial

Abnormal regulation of glucagon secretion contributes to impaired glycemic control in patients with T1D. We conducted two 12-week, double-blind, randomized, placebo-controlled trials to evaluate the efficacy of blocking glucagon action with the glucagon receptor antagonist antibody, volagidemab (VOLA), on glycemic control in patients with T1D. In both trials, participants were randomized in a 1:1:1 fashion to weekly subcutaneous injection with placebo (PBO), VOLA 35 mg, or VOLA 70 mg. Participants in Trial 1 (n=75) were unblinded and participants in Trial 2 (n=78) were blinded to the data from the study-provided continuous glucose monitoring (CGM). All participants could monitor glucose values by fingerstick or their own CGM, if these modalities were part of their usual care before the study. In Trial 1, baseline A1C was 7.5% and decreased by 0.39% (PBO), 0.64% (35 mg, p=0.029), and 0.69% (70 mg, p=0.008) at week 12. An A1C target of ≤ 7.0% was achieved in 43% (PBO), 70% (VOLA 35 mg), and 71% (VOLA 70 mg) of participants. Insulin total daily dose (TDD) decreased by 6.3% (PBO), 12.9% (VOLA 35 mg, NS) and 12.2% (VOLA 70 mg, NS) at week 12. In Trial 2, baseline A1C was 7.9% and decreased by 0.03% (PBO), 0.63% (VOLA 35 mg, p=0.001) and 0.66% (VOLA 70 mg, p=0.001). An A1C target of ≤7.0% was achieved in 17% (PBO), 50% (VOLA 35 mg), and 38% (VOLA 70 mg) of participants. Insulin TDD decreased by 3.5% (PBO), 15.1% (VOLA 35 mg, NS), and 10.2% (VOLA 70 mg, NS). A majority of adverse events were Grade 2 or lower in severity. Intermittent Increases in BP and ALT were observed in VOLA vs. placebo participants during the dosing period and returned to BL after treatment discontinuation. These data demonstrate that treatment with VOLA 35 mg or 70 mg once weekly as an adjunct to insulin is safe, well-tolerated, and decreases A1C with a trend toward decreased insulin dosing in people with T1D. Disclosure J. Pettus: Advisory Panel; Self; Novo Nordisk, Sanofi, Consultant; Self; Diasome Pharmaceuticals, Inc., Insulet Corporation, Lilly Diabetes, MannKind Corporation, Tandem Diabetes Care. B. W. Bode: Advisory Panel; Self; Eli Lilly and Company, Consultant; Self; Bigfoot Biomedical, Inc., Companion Medical, Lexicon Pharmaceuticals, Inc., Medtronic, Novo Nordisk Inc., Zealand Pharma A/S, Research Support; Self; Abvance Therapeutics, Dexcom, Inc., Diasome Pharmaceuticals, Inc., Dompe, Eli Lilly and Company, Eyenuk, Inc., Insulet Corporation, Jaeb Center for Health Research, Medtronic, Nova Biomedical, Novo Nordisk, Provention Bio, Inc., REMD Biotherapeutics, Sanofi, Senseonics, Viacyte, Inc., vTv Therapeutics, Xeris Pharmaceuticals, Inc., Speaker’s Bureau; Self; AstraZeneca, Boehringer Ingelheim Pharmaceuticals, Inc., Eli Lilly and Company, MannKind Corporation, Medtronic, Novo Nordisk, Sanofi, Stock/Shareholder; Self; AgaMatrix, Aseko, Inc., Glytec, LLC. E. D. Bautista: Consultant; Self; REMD Biotherapeutics. H. Yan: Board Member; Self; REMD Biotherapeutics, Other Relationship; Spouse/Partner; REMD Biotherapeutics. D. Thai: Employee; Self; REMD Biotherapeutics. S. K. Garg: Advisory Panel; Self; Eli Lilly and Company, Medtronic, Novo Nordisk Inc., Zealand Pharma A/S, Research Support; Self; Dexcom, Inc., Eli Lilly and Company, Medtronic. S. Klein: Advisory Panel; Self; Altimmune, Merck &amp; Co., Inc., Novo Nordisk Inc., ProSciento, Research Support; Self; Janssen Research &amp; Development, LLC. S. C. Boeder: Consultant; Self; Cecelia Health, Research Support; Self; Dexcom, Inc. M. P. Christiansen: Research Support; Self; Abbott Diabetes, Biolinq, Dexcom, Inc., Eli Lilly and Company, Medtronic, Merck &amp; Co., Inc., Novo Nordisk A/S, Pfizer Inc., Viacyte, Inc. D. S. Denham: None. T. S. Bailey: Consultant; Self; Abbott Diabetes, LifeScan, Novo Nordisk, Sanofi, Research Support; Self; Abbott, Abbott Diabetes, Biolinq, Capillary Biomedical, Inc., Dexcom, Inc., Eli Lilly and Company, Kowa Research Institute, Inc., Lexicon Pharmaceuticals, Inc., Livongo, Medtronic, Medtrum Technologies, Novo Nordisk, REMD Biotherapeutics, Sanofi, Sanvita, Viacyte, Inc., vTv Therapeutics, Zealand Pharma A/S, Speaker’s Bureau; Self; BD, Medtronic, Sanofi. H. K. Akturk: Consultant; Self; American Diabetes Association, Research Support; Self; Dexcom, Inc., Eli Lilly and Company, IM Therapeutics, MannKind Corporation, REMD Biotherapeutics, Senseonics, Speaker’s Bureau; Self; American Diabetes Association. L. J. Klaff: Research Support; Self; Abbott Diabetes, Dong-A ST Co. Ltd., Gan &amp; Lee Pharmaceuticals, Lexicon Pharmaceuticals, Inc., Lilly Diabetes, Medtronic, Novo Nordisk, Oramed Pharmaceuticals, Inc., Pfizer Inc., REMD Biotherapeutics. J. Rosenstock: Board Member; Self; Applied Therapeutics, Boehringer Ingelheim Pharmaceuticals, Inc., Eli Lilly and Company, Intarcia Therapeutics, Inc., Novo Nordisk, Oramed Pharmaceuticals, Inc., Sanofi, Consultant; Self; Applied Therapeutics, Boehringer Ingelheim Pharmaceuticals, Inc., Eli Lilly and Company, Intarcia Therapeutics, Inc., Novo No

Islet sympathetic innervation and islet neuropathology in patients with type 1 diabetes

Dysregulation of glucagon secretion in type 1 diabetes (T1D) involves hypersecretion during postprandial states, but insufficient secretion during hypoglycemia. The sympathetic nervous system regulates glucagon secretion. To investigate islet sympathetic innervation in T1D, sympathetic tyrosine hydroxylase (TH) axons were analyzed in control non-diabetic organ donors, non-diabetic islet autoantibody-positive individuals (AAb), and age-matched persons with T1D. Islet TH axon numbers and density were significantly decreased in AAb compared to T1D with no significant differences observed in exocrine TH axon volume or lengths between groups. TH axons were in close approximation to islet α-cells in T1D individuals with long-standing diabetes. Islet RNA-sequencing and qRT-PCR analyses identified significant alterations in noradrenalin degradation, α-adrenergic signaling, cardiac β-adrenergic signaling, catecholamine biosynthesis, and additional neuropathology pathways. The close approximation of TH axons at islet α-cells supports a model for sympathetic efferent neurons directly regulating glucagon secretion. Sympathetic islet innervation and intrinsic adrenergic signaling pathways could be novel targets for improving glucagon secretion in T1D.

Genetic activation of α-cell glucokinase in mice causes enhanced glucose-suppression of glucagon secretion during normal and diabetic states

ObjectiveWhile the molecular events controlling insulin secretion from β-cells have been documented in detail, the exact mechanisms governing glucagon release by α-cells are understood only partially. This is a critical knowledge gap, as the normal suppression of glucagon secretion by elevated glucose levels fails in type 2 diabetes (T2D) patients, contributing to hyperglycemia through stimulation of hepatic glucose production. A critical role of glycolytic flux in regulating glucagon secretion was supported by recent studies in which manipulation of the activity and expression of the glycolytic enzyme glucokinase altered the setpoint for glucose-suppression of glucagon secretion (GSGS). Given this precedent, we hypothesized that genetic activation of glucokinase specifically in α-cells would enhance GSGS and mitigate T2D hyperglucagonemia. MethodsWe derived an inducible, α-cell-specific glucokinase activating mutant mouse model (GckLoxPGck∗/LoxPGck∗; Gcg-CreERT2; henceforth referred to as “α-mutGCK”) in which the wild-type glucokinase gene (GCK) is conditionally replaced with a glucokinase mutant allele containing the ins454A activating mutation (Gck∗), a mutation that increases the affinity of glucokinase for glucose by almost 7-fold. The effects of α-cell GCK activation on glucose homeostasis, hormone secretion, islet morphology, and islet numbers were assessed using both in vivo and ex vivo assays. Additionally, the effect of α-cell GCK activation on GSGS was investigated under diabetogenic conditions of high-fat diet (HFD) feeding that dysregulate glucagon secretion. ResultsOur study shows that α-mutGCK mice have enhanced GSGS in vivo and ex vivo, independent of alterations in insulin levels and secretion, islet hormone content, islet morphology, or islet number. α-mutGCK mice maintained on HFD displayed improvements in glucagonemia compared to controls, which developed the expected obesity, glucose intolerance, elevated fasting blood glucose, hyperinsulinemia, and hyperglucagonemia. ConclusionsUsing our novel α-cell specific activation of GCK mouse model, we have provided additional support to demonstrate that the glycolytic enzyme glucokinase is a key determinant in glucose sensing within α-cells to regulate glucagon secretion. Our results contribute to our fundamental understanding of α-cell biology by providing greater insight into the regulation of glucagon secretion through α-cell intrinsic mechanisms via glucokinase. Furthermore, our HFD results underscore the potential of glucokinase as a druggable target which, given the ongoing development of allosteric glucokinase activators (GKAs) for T2D treatment, could help mitigate hyperglucagonemia and potentially improve blood glucose homeostasis.

Quantification of Insulin Vesicle Dynamics, Fusion Events and Calcium Activity in Intact Mouse Islets

Proper regulation of glucose homeostasis requires the controlled secretion of insulin and glucagon by β- and α-cells, respectively, in the pancreatic islets. The secretion profiles of insulin and glucagon track in opposition to each other with respect to glucose stimulation. These two hormones work as counterregulatory partners to maintain blood glucose homeostasis and the misregulation of both is implicated in the onset of diabetes. Therefore, it is crucial to understand the mechanistic pathways that drive the regulation of both insulin and glucagon release in pancreatic islets. For this, we adopt a multi-pronged approach to investigate the dynamics and fusion events of insulin vesicles, and intracellular Ca2+ activity in β- and α-cells of mouse pancreatic islets. The dynamics of insulin vesicle motion and release were monitored by 2D confocal and 3D dual-view inverted selective plane illumination microscopy (diSPIM) time-lapse imaging on c-peptide-EGFP labeled MIN6 cells and intact islets of transgenic ins1 promoter driven c-peptide-superfolder GFP (c-peptide-sfGFP) expressing mice under different glucose conditions. insulin vesicle fusion events were also quantified with pH-sensitive Vamp2-pHluorin labeled vesicles in MIN6 cells using total internal reflection fluorescence (TIRF) microscopy. Quantification of α- and β-cell Ca2+ activities under glucose stimulation and various conditions were conducted in intact islets of transgenic GCG-iCre and INS-Cre-GCaMP6 mice, respectively, utilizing confocal and diSPIM imaging. in addition, we examine the effects of Complexin 2, a protein kinase A (PKA)-dependent SNARE binding protein, on the regulation of glucagon secretion, along with α-cell intracellular Ca2+ activity, in Complexin2 knockout mice islets. by stepwise delineation of the various mechanistic pathways, we aim to decipher the intricate coordination between the intrinsic, paracrine and juxtacrine mechanisms within individual islet cells for proper islet function.