100 years of glucagon anniversary.

The year 2023 marks 100 years since publication of the first report of a hyperglycemic factor in pancreatic extracts which C P Kimball and John R Murlin named glucagon (from GLUCose AGONist). Glucagon has a range of profound effects on metabolism including, but not limited to, stimulation of hepatic glucose production. Dysregulation of glucagon secretion is a key feature of both major forms of diabetes, leading to the concept that diabetes is a bihormonal disorder. Still, the work to fully understand the production and biological effects of glucagon has proceeded at a slower pace compared to that of insulin. A recent resurgence of interest in the islet alpha (α) cell, the predominant site of glucagon production, has been facilitated in part by technological innovations. This work has led to significant developments in the field, from defining how alpha cells develop and how glucagon secretion from pancreatic alpha cells is regulated to determining the role of glucagon in metabolic homeostasis and the progression of both major forms of diabetes. In addition, glucagon is considered to be a promising target for diabetes therapy, with many new potential applications arising from research in this field. This collection of reviews, led by Guest Editors James Cantley, Vincent Poitout and Rebecca Hull-Meichle, is intended to capture the field's current understanding of glucagon and alpha cell biology, as well stimulate additional interest and research on this important hormone.

Vitamin K-dependent carboxylation regulates Ca2+ flux and adaptation to metabolic stress in ß cells.

Vitamin K is a micronutrient necessary for γ-carboxylation of glutamic acids. This post-translational modification occurs in the endoplasmic reticulum (ER) and affects secreted proteins. Recent clinical studies implicate vitamin K in the pathophysiology of diabetes, but the underlying molecular mechanism remains unknown. Here, we show that mouse ß cells lacking γ-carboxylation fail to adapt their insulin secretion in the context of age-related insulin resistance or diet-induced ß cell stress. In human islets, γ-carboxylase expression positively correlates with improved insulin secretion in response to glucose. We identify endoplasmic reticulum Gla protein (ERGP) as a γ-carboxylated ER-resident Ca2+-binding protein expressed in ß cells. Mechanistically, γ-carboxylation of ERGP protects cells against Ca2+ overfilling by diminishing STIM1 and Orai1 interaction and restraining store-operated Ca2+ entry. These results reveal a critical role of vitamin K-dependent carboxylation in regulation of Ca2+ flux in ß cells and in their capacity to adapt to metabolic stress.

Single-Cell RNA Sequencing Reveals a Role for Reactive Oxygen Species and Peroxiredoxins in Fatty Acid-Induced Rat ß-Cell Proliferation.

The functional mass of insulin-secreting pancreatic ß-cells expands to maintain glucose homeostasis in the face of nutrient excess, in part via replication of existing ß-cells. Type 2 diabetes appears when these compensatory mechanisms fail. Nutrients including glucose and fatty acids are important contributors to the ß-cell compensatory response, but their underlying mechanisms of action remain poorly understood. We investigated the transcriptional mechanisms of ß-cell proliferation in response to fatty acids. Isolated rat islets were exposed to 16.7 mmol/L glucose with or without 0.5 mmol/L oleate (C18:1) or palmitate (C16:0) for 48 h. The islet transcriptome was assessed by single-cell RNA sequencing. ß-Cell proliferation was measured by flow cytometry. Unsupervised clustering of pooled ß-cells identified different subclusters, including proliferating ß-cells. ß-Cell proliferation increased in response to oleate but not palmitate. Both fatty acids enhanced the expression of genes involved in energy metabolism and mitochondrial activity. Comparison of proliferating versus nonproliferating ß-cells and pseudotime ordering suggested the involvement of reactive oxygen species (ROS) and peroxiredoxin signaling. Accordingly, N-acetyl cysteine and the peroxiredoxin inhibitor conoidin A both blocked oleate-induced ß-cell proliferation. Our study reveals a key role for ROS signaling through peroxiredoxin activation in oleate-induced ß-cell proliferation.

ß Cell mass expansion during puberty involves serotonin signaling and determines glucose homeostasis in adulthood.

Puberty is associated with transient insulin resistance that normally recedes at the end of puberty; however, in overweight children, insulin resistance persists, leading to an increased risk of type 2 diabetes. The mechanisms whereby pancreatic ß cells adapt to pubertal insulin resistance, and how they are affected by the metabolic status, have not been investigated. Here, we show that puberty is associated with a transient increase in ß cell proliferation in rats and humans of both sexes. In rats, ß cell proliferation correlated with a rise in growth hormone (GH) levels. Serum from pubertal rats and humans promoted ß cell proliferation, suggesting the implication of a circulating factor. In pubertal rat islets, expression of genes of the GH/serotonin (5-hydroxytryptamine [5-HT]) pathway underwent changes consistent with a proliferative effect. Inhibition of the pro-proliferative 5-HT receptor isoform HTR2B blocked the increase in ß cell proliferation in pubertal islets ex vivo and in vivo. Peripubertal metabolic stress blunted ß cell proliferation during puberty and led to altered glucose homeostasis later in life. This study identifies a role of GH/GH receptor/5-HT/HTR2B signaling in the control of ß cell mass expansion during puberty and identifies a mechanistic link between pubertal obesity and the risk of developing type 2 diabetes.

Very-Long-Chain Unsaturated Sphingolipids Mediate Oleate-Induced Rat ß-Cell Proliferation.

Fatty acid (FA) signaling contributes to ß-cell mass expansion in response to nutrient excess, but the underlying mechanisms are poorly understood. In the presence of elevated glucose, FA metabolism is shifted toward synthesis of complex lipids, including sphingolipids. Here, we tested the hypothesis that sphingolipids are involved in the ß-cell proliferative response to FA. Isolated rat islets were exposed to FA and 16.7 mmol/L glucose for 48-72 h, and the contribution of the de novo sphingolipid synthesis pathway was tested using the serine palmitoyltransferase inhibitor myriocin, the sphingosine kinase (SphK) inhibitor SKI II, or knockdown of SphK, fatty acid elongase 1 (ELOVL1) and acyl-CoA-binding protein (ACBP). Rats were infused with glucose and the lipid emulsion ClinOleic and received SKI II by gavage. ß-Cell proliferation was assessed by immunochemistry or flow cytometry. Sphingolipids were analyzed by liquid chromatography-tandem mass spectrometry. Among the FAs tested, only oleate increased ß-cell proliferation. Myriocin, SKI II, and SphK knockdown all decreased oleate-induced ß-cell proliferation. Oleate exposure did not increase the total amount of sphingolipids but led to a specific rise in 24:1 species. Knockdown of ACBP or ELOVL1 inhibited oleate-induced ß-cell proliferation. We conclude that unsaturated very-long-chain sphingolipids produced from the available C24:1 acyl-CoA pool mediate oleate-induced ß-cell proliferation in rats.

The Tetracycline-Controlled Transactivator (Tet-On/Off) System in ß-Cells Reduces Insulin Expression and Secretion in Mice.

Controllable genetic manipulation is an indispensable tool in research, greatly advancing our understanding of cell biology and physiology. However in ß-cells, transgene silencing, low inducibility, ectopic expression, and off-targets effects are persistent challenges. In this study, we investigated whether an inducible Tetracycline (Tet)-Off system with ß-cell-specific mouse insulin promoter (MIP)-itTA-driven expression of tetracycline operon (TetO)-CreJaw/J could circumvent previous issues of specificity and efficacy. Following assessment of tissue-specific gene recombination, ß-cell architecture, in vitro and in vivo glucose-stimulated insulin secretion, and whole-body glucose homeostasis, we discovered that expression of any tetracycline-controlled transactivator (e.g., improved itTA, reverse rtTA, or tTA) in ß-cells significantly reduced Insulin gene expression and decreased insulin content. This translated into lower pancreatic insulin levels and reduced insulin secretion in mice carrying any tTA transgene, independent of Cre recombinase expression or doxycycline exposure. Our study echoes ongoing challenges faced by fundamental researchers working with ß-cells and highlights the need for consistent and comprehensive controls when using the tetracycline-controlled transactivator systems (Tet-On or Tet-Off) for genome editing.

Pronounced proliferation of non-beta cells in response to beta-cell mitogens in isolated human islets of Langerhans.

The potential to treat diabetes by increasing beta-cell mass is driving a major effort to identify beta-cell mitogens. Demonstration of mitogen activity in human beta cells is frequently performed in ex vivo assays. However, reported disparities in the efficacy of beta-cell mitogens led us to investigate the sources of this variability. We studied 35 male (23) and female (12) human islet batches covering a range of donor ages and BMI. Islets were kept intact or dispersed into single cells and cultured in the presence of harmine, glucose, or heparin-binding epidermal growth factor-like growth factor (HB-EGF), and subsequently analyzed by immunohistochemistry or flow cytometry. Proliferating cells were identified by double labeling with EdU and Ki67 and glucagon, c-peptide or Nkx6.1, and cytokeratin-19 to respectively label alpha, beta, and ductal cells. Harmine and HB-EGF stimulated human beta-cell proliferation, but the effect of glucose was dependent on the assay and the donor. Harmine potently stimulated alpha-cell proliferation and both harmine and HB-EGF increased proliferation of insulin- and glucagon-negative cells, including cytokeratin 19-positive cells. Given the abundance of non-beta cells in human islet preparations, our results suggest that assessment of beta-cell mitogens requires complementary approaches and rigorous identification of cell identity using multiple markers.

Combined Deletion of Free Fatty-Acid Receptors 1 and 4 Minimally Impacts Glucose Homeostasis in Mice.

The free fatty-acid receptors FFAR1 (GPR40) and FFAR4 (GPR120) are implicated in the regulation of insulin secretion and insulin sensitivity, respectively. Although GPR120 and GPR40 share similar ligands, few studies have addressed possible interactions between these 2 receptors in the control of glucose homeostasis. Here we generated mice deficient in gpr120 (Gpr120KO) or gpr40 (Gpr40KO), alone or in combination (Gpr120/40KO), and metabolically phenotyped male and female mice fed a normal chow or high-fat diet. We assessed insulin secretion in isolated mouse islets exposed to selective GPR120 and GPR40 agonists singly or in combination. Following normal chow feeding, body weight and energy intake were unaffected by deletion of either receptor, although fat mass increased in Gpr120KO females. Fasting blood glucose levels were mildly increased in Gpr120/40KO mice and in a sex-dependent manner in Gpr120KO and Gpr40KO animals. Oral glucose tolerance was slightly reduced in male Gpr120/40KO mice and in Gpr120KO females, whereas insulin secretion and insulin sensitivity were unaffected. In hyperglycemic clamps, the glucose infusion rate was lower in male Gpr120/40KO mice, but insulin and c-peptide levels were unaffected. No changes in glucose tolerance were observed in either single or double knock-out animals under high-fat feeding. In isolated islets from wild-type mice, the combination of selective GPR120 and GPR40 agonists additively increased insulin secretion. We conclude that while simultaneous activation of GPR120 and GPR40 enhances insulin secretion ex vivo, combined deletion of these 2 receptors only minimally affects glucose homeostasis in vivo in mice.

Free fatty acid receptor 4 inhibitory signaling in delta cells regulates islet hormone secretion in mice.

OBJECTIVE: Maintenance of glucose homeostasis requires the precise regulation of hormone secretion from the endocrine pancreas. Free fatty acid receptor 4 (FFAR4/GPR120) is a G protein-coupled receptor whose activation in islets of Langerhans promotes insulin and glucagon secretion and inhibits somatostatin secretion. However, the contribution of individual islet cell types (α, ß, and δ cells) to the insulinotropic and glucagonotropic effects of GPR120 remains unclear. As gpr120 mRNA is enriched in somatostatin-secreting δ cells, we hypothesized that GPR120 activation stimulates insulin and glucagon secretion via inhibition of somatostatin release. METHODS: Glucose tolerance tests were performed in mice after administration of selective GPR120 agonist Compound A. Insulin, glucagon, and somatostatin secretion were measured in static incubations of isolated mouse islets in response to endogenous (ω-3 polyunsaturated fatty acids) and/or pharmacological (Compound A and AZ-13581837) GPR120 agonists. The effect of Compound A on hormone secretion was tested further in islets isolated from mice with global or somatostatin cell-specific knock-out of gpr120. Gpr120 expression was assessed in pancreatic sections by RNA in situ hybridization. Cyclic AMP (cAMP) and calcium dynamics in response to pharmacological GPR120 agonists were measured specifically in α, ß, and δ cells in intact islets using cAMPER and GCaMP6 reporter mice, respectively. RESULTS: Acute exposure to Compound A increased glucose tolerance, circulating insulin, and glucagon levels in vivo. Endogenous and/or pharmacological GPR120 agonists reduced somatostatin secretion in isolated islets and concomitantly demonstrated dose-dependent potentiation of glucose-stimulated insulin secretion and arginine-stimulated glucagon secretion. Gpr120 was enriched in δ cells. Pharmacological GPR120 agonists reduced cAMP and calcium levels in δ cells but increased these signals in α and ß cells. Compound A-mediated inhibition of somatostatin secretion was insensitive to pertussis toxin. The effect of Compound A on hormone secretion was completely absent in islets from mice with either global or somatostatin cell-specific deletion of gpr120 and partially reduced upon blockade of somatostatin receptor signaling by cyclosomatostatin. CONCLUSIONS: Inhibitory GPR120 signaling in δ cells contributes to both insulin and glucagon secretion in part by mitigating somatostatin release.

Targeting lipid GPCRs to treat type 2 diabetes mellitus - progress and challenges.

Therapeutic approaches to the treatment of type 2 diabetes mellitus that are designed to increase insulin secretion either directly target ß-cells or indirectly target gastrointestinal enteroendocrine cells (EECs), which release hormones that modulate insulin secretion (for example, incretins). Given that ß-cells and EECs both express a large array of G protein-coupled receptors (GPCRs) that modulate insulin secretion, considerable research and development efforts have been undertaken to design therapeutic drugs targeting these GPCRs. Among them are GPCRs specific for free fatty acid ligands (lipid GPCRs), including free fatty acid receptor 1 (FFA1, otherwise known as GPR40), FFA2 (GPR43), FFA3 (GPR41) and FFA4 (GPR120), as well as the lipid metabolite binding glucose-dependent insulinotropic receptor (GPR119). These lipid GPCRs have demonstrated important roles in the control of islet and gut hormone secretion. Advances in lipid GPCR pharmacology have led to the identification of a number of synthetic agonists that exert beneficial effects on glucose homeostasis in preclinical studies. Yet, translation of these promising results to the clinic has so far been disappointing. In this Review, we present the physiological roles, pharmacology and clinical studies of these lipid receptors and discuss the challenges associated with their clinical development for the treatment of type 2 diabetes mellitus.

HB-EGF Signaling Is Required for Glucose-Induced Pancreatic ß-Cell Proliferation in Rats.

The molecular mechanisms of ß-cell compensation to metabolic stress are poorly understood. We previously observed that nutrient-induced ß-cell proliferation in rats is dependent on epidermal growth factor receptor (EGFR) signaling. The aim of this study was to determine the role of the EGFR ligand heparin-binding EGF-like growth factor (HB-EGF) in the ß-cell proliferative response to glucose, a ß-cell mitogen and key regulator of ß-cell mass in response to increased insulin demand. We show that exposure of isolated rat and human islets to HB-EGF stimulates ß-cell proliferation. In rat islets, inhibition of EGFR or HB-EGF blocks the proliferative response not only to HB-EGF but also to glucose. Furthermore, knockdown of HB-EGF in rat islets blocks ß-cell proliferation in response to glucose ex vivo and in vivo in transplanted glucose-infused rats. Mechanistically, we demonstrate that HB-EGF mRNA levels are increased in ß-cells in response to glucose in a carbohydrate-response element-binding protein (ChREBP)-dependent manner. In addition, chromatin immunoprecipitation studies identified ChREBP binding sites in proximity to the HB-EGF gene. Finally, inhibition of Src family kinases, known to be involved in HB-EGF processing, abrogated glucose-induced ß-cell proliferation. Our findings identify a novel glucose/HB-EGF/EGFR axis implicated in ß-cell compensation to increased metabolic demand.

Recent Insights Into Mechanisms of ß-Cell Lipo- and Glucolipotoxicity in Type 2 Diabetes.

The deleterious effects of chronically elevated free fatty acid (FFA) levels on glucose homeostasis are referred to as lipotoxicity, and the concurrent exposure to high glucose may cause synergistic glucolipotoxicity. Lipo- and glucolipotoxicity have been studied for over 25 years. Here, we review the current evidence supporting the role of pancreatic ß-cell lipo- and glucolipotoxicity in type 2 diabetes (T2D), including lipid-based interventions in humans, prospective epidemiological studies, and human genetic findings. In addition to total FFA quantity, the quality of FFAs (saturation and chain length) is a key determinant of lipotoxicity. We discuss in vitro and in vivo experimental models to investigate lipo- and glucolipotoxicity in ß-cells and describe experimental pitfalls. Lipo- and glucolipotoxicity adversely affect many steps of the insulin production and secretion process. The molecular mechanisms underpinning lipo- and glucolipotoxic ß-cell dysfunction and death comprise endoplasmic reticulum stress, oxidative stress and mitochondrial dysfunction, impaired autophagy, and inflammation. Crosstalk between these stress pathways exists at multiple levels and may aggravate ß-cell lipo- and glucolipotoxicity. Lipo- and glucolipotoxicity are therapeutic targets as several drugs impact the underlying stress responses in ß-cells, potentially contributing to their glucose-lowering effects in T2D.

A role for PKD1 in insulin secretion downstream of P2Y1 receptor activation in mouse and human islets.

Along with insulin, ß-cells co-secrete the neurotransmitter ATP which acts as a positive autocrine signal via P2Y1 receptors to activate phospholipase C and increase the production of diacylglycerol (DAG). However, the downstream signaling that couples P2Y1 activation to insulin secretion remains to be fully elucidated. Since DAG activates protein kinase D1 (PKD1) to potentiate glucose-stimulated insulin release, we hypothesized that autocrine ATP signaling activates downstream PKD1 to regulate insulin secretion. Indeed, we find that the P2Y1 receptor agonists, MRS2365 and ATP induce, PKD1 phosphorylation at serine 916 in mouse islets. Similarly, direct depolarization of islets by KCl caused PKD1 activation, which is reduced upon P2Y1 antagonism. Potentiation of insulin secretion by P2Y1 activation was lost from PKD1-/- mouse islets, and knockdown of PKD1 reduced the ability of P2Y1 activation to facilitate exocytosis in single mouse ß-cells. Finally, qPCR analysis confirmed PKD1 transcript (PRKD1) expression in human islets, and insulin secretion assays showed that inhibition of either P2Y1 or PKD1 signaling impaired glucose-stimulated insulin secretion. Human islets showed donor-to-donor variation in their responses to both P2Y1 and PKD1 inhibition, however, and we find that the P2Y1 -PKD1 pathway contributes a substantially greater proportion of insulin secretion from islets of overweight and obese donors. Thus, PKD1 promotes increased insulin secretion, likely mediating an autocrine ATP effect via P2Y1 receptor activation which may be more important in islets of donors who are overweight or obese.

The autonomic nervous system regulates pancreatic ß-cell proliferation in adult male rats.

The pancreatic ß-cell responds to changes in the nutrient environment to maintain glucose homeostasis by adapting its function and mass. Nutrients can act directly on the ß-cell and also indirectly through the brain via autonomic nerves innervating islets. Despite the importance of the brain-islet axis in insulin secretion, relatively little is known regarding its involvement in ß-cell proliferation. We previously demonstrated that prolonged infusions of nutrients in rats provoke a dramatic increase in ß-cell proliferation in part because of the direct action of nutrients. Here, we addressed the contribution of the autonomic nervous system. In isolated islets, muscarinic stimulation increased, whereas adrenergic stimulation decreased, glucose-induced ß-cell proliferation. Blocking α-adrenergic receptors reversed the effect of epinephrine on glucose + nonesterified fatty acids (NEFA)-induced ß-cell proliferation, whereas activation of ß-adrenergic receptors was without effect. Infusion of glucose + NEFA toward the brain stimulated ß-cell proliferation, and this effect was abrogated following celiac vagotomy. The increase in ß-cell proliferation following peripheral infusions of glucose + NEFA was not inhibited by vagotomy or atropine treatment but was blocked by coinfusion of epinephrine. We conclude that ß-cell proliferation is stimulated by parasympathetic and inhibited by sympathetic signals. Whereas glucose + NEFA in the brain stimulates ß-cell proliferation through the vagus nerve, ß-cell proliferation in response to systemic nutrient excess does not involve parasympathetic signals but may be associated with decreased sympathetic tone.

Increases in bioactive lipids accompany early metabolic changes associated with ß-cell expansion in response to short-term high-fat diet.

Pancreatic ß-cell expansion is a highly regulated metabolic adaptation to increased somatic demands, including obesity and pregnancy; adult ß cells otherwise rarely proliferate. We previously showed that high-fat diet (HFD) feeding induces mouse ß-cell proliferation in less than 1 wk in the absence of insulin resistance. Here we metabolically profiled tissues from a short-term HFD ß-cell expansion mouse model to identify pathways and metabolite changes associated with ß-cell proliferation. Mice fed HFD vs. chow diet (CD) showed a 14.3% increase in body weight after 7 days; ß-cell proliferation increased 1.75-fold without insulin resistance. Plasma from 1-wk HFD-fed mice induced ß-cell proliferation ex vivo. The plasma, as well as liver, skeletal muscle, and bone, were assessed by LC and GC mass-spectrometry for global metabolite changes. Of the 1,283 metabolites detected, 159 showed significant changes [false discovery rate (FDR) < 0.1]. The majority of changes were in liver and muscle. Pathway enrichment analysis revealed key metabolic changes in steroid synthesis and lipid metabolism, including free fatty acids and other bioactive lipids. Other important enrichments included changes in the citric acid cycle and 1-carbon metabolism pathways implicated in DNA methylation. Although the minority of changes were observed in bone and plasma (<20), increased p-cresol sulfate was increased >4 fold in plasma (the largest increase in all tissues), and pantothenate (vitamin B5) decreased >2-fold. The results suggest that HFD-mediated ß-cell expansion is associated with complex, global metabolite changes. The finding could be a significant insight into Type 2 diabetes pathogenesis and potential novel drug targets.

Deletion of Protein Kinase D1 in Pancreatic ß-Cells Impairs Insulin Secretion in High-Fat Diet-Fed Mice.

Βß-Cell adaptation to insulin resistance is necessary to maintain glucose homeostasis in obesity. Failure of this mechanism is a hallmark of type 2 diabetes (T2D). Hence, factors controlling functional ß-cell compensation are potentially important targets for the treatment of T2D. Protein kinase D1 (PKD1) integrates diverse signals in the ß-cell and plays a critical role in the control of insulin secretion. However, the role of ß-cell PKD1 in glucose homeostasis in vivo is essentially unknown. Using ß-cell-specific, inducible PKD1 knockout mice (ßPKD1KO), we examined the role of ß-cell PKD1 under basal conditions and during high-fat feeding. ßPKD1KO mice under a chow diet presented no significant difference in glucose tolerance or insulin secretion compared with mice expressing the Cre transgene alone; however, when compared with wild-type mice, both groups developed glucose intolerance. Under a high-fat diet, deletion of PKD1 in ß-cells worsened hyperglycemia, hyperinsulinemia, and glucose intolerance. This was accompanied by impaired glucose-induced insulin secretion both in vivo in hyperglycemic clamps and ex vivo in isolated islets from high-fat diet-fed ßPKD1KO mice without changes in islet mass. This study demonstrates an essential role for PKD1 in the ß-cell adaptive secretory response to high-fat feeding in mice.