Pancreatic α- and ß-cellular clocks have distinct molecular properties and impact on islet hormone secretion and gene expression.

A critical role of circadian oscillators in orchestrating insulin secretion and islet gene transcription has been demonstrated recently. However, these studies focused on whole islets and did not explore the interplay between α-cell and ß-cell clocks. We performed a parallel analysis of the molecular properties of α-cell and ß-cell oscillators using a mouse model expressing three reporter genes: one labeling α cells, one specific for ß cells, and a third monitoring circadian gene expression. Thus, phase entrainment properties, gene expression, and functional outputs of the α-cell and ß-cell clockworks could be assessed in vivo and in vitro at the population and single-cell level. These experiments showed that α-cellular and ß-cellular clocks are oscillating with distinct phases in vivo and in vitro. Diurnal transcriptome analysis in separated α and ß cells revealed that a high number of genes with key roles in islet physiology, including regulators of glucose sensing and hormone secretion, are differentially expressed in these cell types. Moreover, temporal insulin and glucagon secretion exhibited distinct oscillatory profiles both in vivo and in vitro. Altogether, our data indicate that differential entrainment characteristics of circadian α-cell and ß-cell clocks are an important feature in the temporal coordination of endocrine function and gene expression.

An extended minimal model of OGTT: estimation of α- and ß-cell dysfunction, insulin resistance, and the incretin effect.

Loss of insulin sensitivity, α- and ß-cell dysfunction, and impairment in incretin effect have all been implicated in the pathophysiology of type 2 diabetes (T2D). Parsimonious mathematical models are useful in quantifying parameters related to the pathophysiology of T2D. Here, we extend the minimum model developed to describe the glucose-insulin-glucagon dynamics in the isoglycemic intravenous glucose infusion (IIGI) experiment to the oral glucose tolerance test (OGTT). The extended model describes glucose and hormone dynamics in OGTT including the contribution of the incretin hormones, glucose-dependent insulinotropic polypeptide (GIP), and glucagon-like peptide-1 (GLP-1), to insulin secretion. A new function describing glucose arrival from the gut is introduced. The model is fitted to OGTT data from eight individuals with T2D and eight weight-matched controls (CS) without diabetes to obtain parameters related to insulin sensitivity, ß- and α-cell function. The parameters, i.e., measures of insulin sensitivity, a1, suppression of glucagon secretion, k1, magnitude of glucagon secretion, γ2, and incretin-dependent insulin secretion, γ3, were found to be different between CS and T2D with P values < 0.002, <0.017, <0.009, <0.004, respectively. A new rubric for estimating the incretin effect directly from modeling the OGTT is presented. The average incretin effect correlated well with the experimentally determined incretin effect with a Spearman rank test correlation coefficient of 0.67 (P < 0.012). The average incretin effect was found to be different between CS and T2D (P < 0.032). The developed model is shown to be effective in quantifying the factors relevant to T2D pathophysiology.NEW & NOTEWORTHY A new extended model of oral glucose tolerance test (OGTT) has been developed that includes glucagon dynamics and incretin contribution to insulin secretion. The model allows the estimation of parameters related to α- and ß-cell dysfunction, insulin sensitivity, and incretin action. A new function describing the influx of glucose from the gut has been introduced. A new rubric for estimating the incretin effect directly from the OGTT experiment has been developed. The effect of glucose dose was also investigated.

Regulatory Role of Fatty Acid Metabolism on Glucose-Induced Changes in Insulin and Glucagon Secretion by Pancreatic Islet Cells.

A detailed study of palmitate metabolism in pancreatic islets subject to different experimental conditions, like varying concentrations of glucose, as well as fed or starved conditions, has allowed us to explore the interaction between the two main plasma nutrients and its consequences on hormone secretion. Palmitate potentiates glucose-induced insulin secretion in a concentration-dependent manner, in a physiological range of both palmitate (0-2 mM) and glucose (6-20 mM) concentrations; at glucose concentrations lower than 6 mM, no metabolic interaction with palmitate was apparent. Starvation (48 h) increased islet palmitate oxidation two-fold, and the effect was resistant to its inhibition by glucose (6-20 mM). Consequently, labelled palmitate and glucose incorporation into complex lipids were strongly suppressed, as well as glucose-induced insulin secretion and its potentiation by palmitate. 2-bromostearate, a palmitate oxidation inhibitor, fully recovered the synthesis of complex lipids and insulin secretion. We concluded that palmitate potentiation of the insulin response to glucose is not attributable to its catabolic mitochondrial oxidation but to its anabolism to complex lipids: islet lipid biosynthesis is dependent on the uptake of plasma fatty acids and the supply of α-glycerol phosphate from glycolysis. Islet secretion of glucagon and somatostatin showed a similar dependence on palmitate anabolism as insulin. The possible mechanisms implicated in the metabolic coupling between glucose and palmitate were commented on. Moreover, possible mechanisms responsible for islet gluco- or lipotoxicity after a long-term stimulation of insulin secretion were also discussed. Our own data on the simultaneous stimulation of insulin, glucagon, and somatostatin by glucose, as well as their modification by 2-bromostearate in perifused rat islets, give support to the conclusion that increased FFA anabolism, rather than its mitochondrial oxidation, results in a potentiation of their stimulated release. Starvation, besides suppressing glucose stimulation of insulin secretion, also blocks the inhibitory effect of glucose on glucagon secretion: this suggests that glucagon inhibition might be an indirect or direct effect of insulin, but not of glucose. In summary, there seems to exist three mechanisms of glucagon secretion stimulation: 1. glucagon stimulation through the same secretion coupling mechanism as insulin, but in a different range of glucose concentrations (0 to 5 mM). 2. Direct or indirect inhibition by secreted insulin in response to glucose (5-20 mM). 3. Stimulation by increased FFA anabolism in glucose intolerance or diabetes in the context of hyperlipidemia, hyperglycemia, and hypo-insulinemia. These conclusions were discussed and compared with previous published data in the literature. Specially, we discussed the mechanism for inhibition of glucagon release by glucose, which was apparently contradictory with the secretion coupling mechanism of its stimulation.

Incretin hormones and type 2 diabetes.

Incretin hormones (glucose-dependent insulinotropic polypeptide [GIP] and glucagon-like peptide-1 [GLP-1]) play a role in the pathophysiology of type 2 diabetes. Along with their derivatives they have shown therapeutic success in type 2 diabetes, with the potential for further improvements in glycaemic, cardiorenal and body weight-related outcomes. In type 2 diabetes, the incretin effect (greater insulin secretory response after oral glucose than with 'isoglycaemic' i.v. glucose, i.e. with an identical glycaemic stimulus) is markedly reduced or absent. This appears to be because of a reduced ability of GIP to stimulate insulin secretion, related either to an overall impairment of beta cell function or to specific defects in the GIP signalling pathway. It is likely that a reduced incretin effect impacts on postprandial glycaemic excursions and, thus, may play a role in the deterioration of glycaemic control. In contrast, the insulinotropic potency of GLP-1 appears to be much less impaired, such that exogenous GLP-1 can stimulate insulin secretion, suppress glucagon secretion and reduce plasma glucose concentrations in the fasting and postprandial states. This has led to the development of incretin-based glucose-lowering medications (selective GLP-1 receptor agonists or, more recently, co-agonists, e.g. that stimulate GIP and GLP-1 receptors). Tirzepatide (a GIP/GLP-1 receptor co-agonist), for example, reduces HbA1c and body weight in individuals with type 2 diabetes more effectively than selective GLP-1 receptor agonists (e.g. semaglutide). The mechanisms by which GIP receptor agonism may contribute to better glycaemic control and weight loss after long-term exposure to tirzepatide are a matter of active research and may change the pessimistic view that developed after the disappointing lack of insulinotropic activity in people with type 2 diabetes when exposed to GIP in short-term experiments. Future medications that stimulate incretin hormone and other receptors simultaneously may have the potential to further increase the ability to control plasma glucose concentrations and induce weight loss.

Amino acid transporters revisited: New views in health and disease.

Amino acid transporters (AATs) are membrane-bound transport proteins that mediate transfer of amino acids into and out of cells or cellular organelles. AATs have diverse functional roles ranging from neurotransmission to acid-base balance, intracellular energy metabolism, and anabolic and catabolic reactions. In cancer cells and diabetes, dysregulation of AATs leads to metabolic reprogramming, which changes intracellular amino acid levels, contributing to the pathogenesis of cancer, obesity and diabetes. Indeed, the neutral amino acid transporters (NATs) SLC7A5/LAT1 and SLC1A5/ASCT2 are likely involved in several human malignancies. However, a clinical therapy that directly targets AATs has not yet been developed. The purpose of this review is to highlight the structural and functional diversity of AATs, their diverse physiological roles in different tissues and organs, their wide-ranging implications in human diseases and the emerging strategies and tools that will be necessary to target AATs therapeutically.

Screening of Metabolism-Disrupting Chemicals on Pancreatic α-Cells Using In Vitro Methods.

Metabolism-disrupting chemicals (MDCs) are endocrine disruptors with obesogenic and/or diabetogenic action. There is mounting evidence linking exposure to MDCs to increased susceptibility to diabetes. Despite the important role of glucagon in glucose homeostasis, there is little information on the effects of MDCs on α-cells. Furthermore, there are no methods to identify and test MDCs with the potential to alter α-cell viability and function. Here, we used the mouse α-cell line αTC1-9 to evaluate the effects of MDCs on cell viability and glucagon secretion. We tested six chemicals at concentrations within human exposure (from 0.1 pM to 1 µM): bisphenol-A (BPA), tributyltin (TBT), perfluorooctanoic acid (PFOA), triphenylphosphate (TPP), triclosan (TCS), and dichlorodiphenyldichloroethylene (DDE). Using two different approaches, MTT assay and DNA-binding dyes, we observed that BPA and TBT decreased α-cell viability via a mechanism that depends on the activation of estrogen receptors and PPARγ, respectively. These two chemicals induced ROS production, but barely altered the expression of endoplasmic reticulum (ER) stress markers. Although PFOA, TPP, TCS, and DDE did not alter cell viability nor induced ROS generation or ER stress, all four compounds negatively affected glucagon secretion. Our findings suggest that αTC1-9 cells seem to be an appropriate model to test chemicals with metabolism-disrupting activity and that the improvement of the test methods proposed herein could be incorporated into protocols for the screening of diabetogenic MDCs.

Do sodium-glucose co-transporter-2 inhibitors increase plasma glucagon by direct actions on the alpha cell? And does the increase matter for the associated increase in endogenous glucose production?

Sodium-glucose co-transporter-2 inhibitors (SGLT2is) lower blood glucose and are used for treatment of type 2 diabetes. However, SGLT2is have been associated with increases in endogenous glucose production (EGP) by mechanisms that have been proposed to result from SGLT2i-mediated increases in circulating glucagon concentrations, but the relative importance of this effect is debated, and mechanisms possibly coupling SGLT2is to increased plasma glucagon are unclear. A direct effect on alpha-cell activity has been proposed, but data on alpha-cell SGLT2 expression are inconsistent, and studies investigating the direct effects of SGLT2 inhibition on glucagon secretion are conflicting. By contrast, alpha-cell sodium-glucose co-transporter-1 (SGLT1) expression has been found more consistently and appears to be more prominent, pointing to an underappreciated role for this transporter. Nevertheless, the selectivity of most SGLT2is does not support interference with SGLT1 during therapy. Paracrine effects mediated by secretion of glucagonotropic/static molecules from beta and/or delta cells have also been suggested to be involved in SGLT2i-induced increase in plasma glucagon, but studies are few and arrive at different conclusions. It is also possible that the effect on glucagon is secondary to drug-induced increases in urinary glucose excretion and lowering of blood glucose, as shown in experiments with glucose clamping where SGLT2i-associated increases in plasma glucagon are prevented. However, regardless of the mechanisms involved, the current balance of evidence does not support that SGLT2 plays a crucial role for alpha-cell physiology or that SGLT2i-induced glucagon secretion is important for the associated increased EGP, particularly because the increase in EGP occurs before any rise in plasma glucagon.

No direct effect of SGLT2 activity on glucagon secretion.

AIMS/HYPOTHESIS: Sodium-glucose cotransporter (SGLT) 2 inhibitors constitute a new class of glucose-lowering drugs, but they increase glucagon secretion, which may counteract their glucose-lowering effect. Previous studies using static incubation of isolated human islets or the glucagon-secreting cell line α-TC1 suggested that this results from direct inhibition of alpha cell SGLT1/2-activity. The aim of this study was to test whether the effects of SGLT2 on glucagon secretion demonstrated in vitro could be reproduced in a more physiological setting. METHODS: We explored the effect of SGLT2 activity on glucagon secretion using isolated perfused rat pancreas, a physiological model for glucagon secretion. Furthermore, we investigated Slc5a2 (the gene encoding SGLT2) expression in rat islets as well as in mouse and human islets and in mouse and human alpha, beta and delta cells to test for potential inter-species variations. SGLT2 protein content was also investigated in mouse, rat and human islets. RESULTS: Glucagon output decreased three- to fivefold within minutes of shifting from low (3.5 mmol/l) to high (10 mmol/l) glucose (4.0 ± 0.5 pmol/15 min vs 1.3 ± 0.3 pmol/15 min, p < 0.05). The output was unaffected by inhibition of SGLT1/2 with dapagliflozin or phloridzin or by addition of the SGLT1/2 substrate α-methylglucopyranoside, whether at low or high glucose concentrations (p = 0.29-0.99). Insulin and somatostatin secretion (potential paracrine regulators) was also unaffected. Slc5a2 expression and SGLT2 protein were marginal or below detection limit in rat, mouse and human islets and in mouse and human alpha, beta and delta cells. CONCLUSIONS/INTERPRETATION: Our combined data show that increased plasma glucagon during SGLT2 inhibitor treatment is unlikely to result from direct inhibition of SGLT2 in alpha cells, but instead may occur downstream of their blood glucose-lowering effects.

Glucose intolerance in monosodium glutamate obesity is linked to hyperglucagonemia and insulin resistance in α cells.

Obesity predisposes to glucose intolerance and type 2 diabetes (T2D). This disease is often characterized by insulin resistance, changes in insulin clearance, and ß-cell dysfunction. However, studies indicate that, for T2D development, disruptions in glucagon physiology also occur. Herein, we investigated the involvement of glucagon in impaired glycemia control in monosodium glutamate (MSG)-obese mice. Male Swiss mice were subcutaneously injected daily, during the first 5 days after birth, with MSG (4 mg/g body weight [BW]) or saline (1.25 mg/g BW). At 90 days of age, MSG-obese mice were hyperglycemic, hyperinsulinemic, and hyperglucagonemic and had lost the capacity to increase their insulin/glucagon ratio when transitioning from the fasting to fed state, exacerbating hepatic glucose output. Furthermore, hepatic protein expressions of phosphorylated (p)-protein kinase A (PKA) and cAMP response element-binding protein (pCREB), and of phosphoenolpyruvate carboxykinase (PEPCK) enzyme were higher in fed MSG, before and after glucagon stimulation. Increased pPKA and phosphorylated hormone-sensitive lipase content were also observed in white fat of MSG. MSG islets hypersecreted glucagon in response to 11.1 and 0.5 mmol/L glucose, a phenomenon that persisted in the presence of insulin. Additionally, MSG α cells were hypertrophic displaying increased α-cell mass and immunoreactivity to phosphorylated mammalian target of rapamycin (pmTOR) protein. Therefore, severe glucose intolerance in MSG-obese mice was associated with increased hepatic glucose output, in association with hyperglucagonemia, caused by the refractory actions of glucose and insulin in α cells and via an effect that may be due to enhanced mTOR activation.

Glucose-mediated inhibition of calcium-activated potassium channels limits α-cell calcium influx and glucagon secretion.

Pancreatic α-cells exhibit oscillations in cytosolic Ca2+ (Ca2+c), which control pulsatile glucagon (GCG) secretion. However, the mechanisms that modulate α-cell Ca2+c oscillations have not been elucidated. As ß-cell Ca2+c oscillations are regulated in part by Ca2+-activated K+ (Kslow) currents, this work investigated the role of Kslow in α-cell Ca2+ handling and GCG secretion. α-Cells displayed Kslow currents that were dependent on Ca2+ influx through L- and P/Q-type voltage-dependent Ca2+ channels (VDCCs) as well as Ca2+ released from endoplasmic reticulum stores. α-Cell Kslow was decreased by small-conductance Ca2+-activated K+ (SK) channel inhibitors apamin and UCL 1684, large-conductance Ca2+-activated K+ (BK) channel inhibitor iberiotoxin (IbTx), and intermediate-conductance Ca2+-activated K+ (IK) channel inhibitor TRAM 34. Moreover, partial inhibition of α-cell Kslow with apamin depolarized membrane potential ( Vm) (3.8 ± 0.7 mV) and reduced action potential (AP) amplitude (10.4 ± 1.9 mV). Although apamin transiently increased Ca2+ influx into α-cells at low glucose (42.9 ± 10.6%), sustained SK (38.5 ± 10.4%) or BK channel inhibition (31.0 ± 11.7%) decreased α-cell Ca2+ influx. Total α-cell Ca2+c was similarly reduced (28.3 ± 11.1%) following prolonged treatment with high glucose, but it was not decreased further by SK or BK channel inhibition. Consistent with reduced α-cell Ca2+c following prolonged Kslow inhibition, apamin decreased GCG secretion from mouse (20.4 ± 4.2%) and human (27.7 ± 13.1%) islets at low glucose. These data demonstrate that Kslow activation provides a hyperpolarizing influence on α-cell Vm that sustains Ca2+ entry during hypoglycemic conditions, presumably by preventing voltage-dependent inactivation of P/Q-type VDCCs. Thus, when α-cell Ca2+c is elevated during secretagogue stimulation, Kslow activation helps to preserve GCG secretion.

Glucagon and insulin secretion, insulin clearance, and fasting glucose in GIP receptor and GLP-1 receptor knockout mice.

It is not known whether GIP receptor and GLP-1 receptor knockout (KO) mice have perturbations in glucagon secretion or insulin clearance, and studies on impact on fasting glycemia have previously been inconsistent in these mice. We therefore studied glucagon secretion after oral whey protein (60 mg) and intravenous arginine (6.25 mg), insulin clearance after intravenous glucose (0.35 g/kg) and fasting glucose, insulin, and glucagon levels after standardized 5-h fasting in female GIP receptor and GLP-1 receptor KO mice and their wild-type (WT) littermates. Compared with WT controls, GIP receptor KO mice had normal glucagon responses to oral protein and intravenous arginine, except for an enhanced 1-min response to arginine, whereas glucagon levels after oral protein and intravenous arginine were enhanced in GLP-1 receptor KO mice. Furthermore, the intravenous glucose test revealed normal insulin clearance in both GIP receptor and GLP-1 receptor KO mice, whereas ß-cell glucose sensitivity was enhanced in GIP receptor KO mice and reduced in GLP-1 receptor KO mice. Finally, GIP receptor KO mice had reduced fasting glucose (6.7 ± 0.1, n = 56, vs. 7.4 ± 0.1 mmol/l, n = 59, P = 0.001), whereas GLP-1 receptor KO mice had increased fasting glucose (9.1 ± 0.2, n = 44, vs. 7.7 ± 0.1 mmol/l, n = 41, P < 0.001). We therefore suggest that GIP has a limited role for glucagon secretion in mice, whereas GLP-1 is of importance for glucagon regulation, that GIP and GLP-1 are of importance for the regulation of ß-cell function beyond their role as incretin hormones, and that they are both of importance for fasting glucose.

An optical method to evaluate both mass and functional competence of pancreatic α- and ß-cells.

Imbalanced glucagon and insulin release leads to the onset of type 2 diabetes. To pinpoint the underlying primary driving force, here we have developed a fast, non-biased optical method to measure ratios of pancreatic α- and ß-cell mass and function simultaneously. We firstly label both primary α- and ß-cells with the red fluorescent probe ZinRhodaLactam-1 (ZRL1), and then highlight α-cells by selectively quenching the ZRL1 signal from ß-cells. Based on the signals before and after quenching, we calculate the ratio of the α-cell to ß-cell mass within live islets, which we found matched the results from immunohistochemistry. From the same islets, glucagon and insulin release capability can be concomitantly measured. Thus, we were able to measure the ratio of α-cell to ß-cell mass and their function in wild-type and diabetic Lepr(db)/Lepr(db) (denoted db/db) mice at different ages. We find that the initial glucose intolerance that appears in 10-week-old db/db mice is associated with further expansion of α-cell mass prior to deterioration in functional ß-cell mass. Our method is extendable to studies of islet mass and function in other type 2 diabetes animal models, which shall benefit mechanistic studies of imbalanced hormone secretion during type 2 diabetes progression.

Antioxidant effects of epigallocatechin-3-gallate on the aTC1-6 pancreatic alpha cell line.

Hypoglycemia is a major barrier to achieving stable metabolic control in patients with diabetes which is a serious clinical concern. With progression of diabetes, the ability of pancreatic α-cells which respond to hypoglycemia becomes impaired; However, it is not clear whether the dysfunctional responses of α-cells during hypoglycemia are related with oxidative stress. In the present study, we investigated whether epigallocatechin-3-gallate (EGCG) has antioxidant potential on pancreatic alpha TC1-6 (αTC1-6) cell lines and protect the normal function of α-cells from H2O2 induced oxidative stress. ROS production, cell viability, glucagon secretion, and cell apoptosis were assessed. EGCG reduced ROS production and cell apoptosis, while restored cell viability and glucagon secretion within a particular concentration range. Moreover, EGCG activated Akt signaling and inhibited P38 as well as JNK mitogen-activated protein kinase (MAPK) pathway. Taken together, EGCG prevented αTC1-6 cells from H2O2 induced oxidative stress, restored dysfunction of glucagon secretion and inhibited cell apoptosis via the activation of Akt signaling and suppression of P38 and JNK pathway. These results provide rationale for combining the conventional anti-hyperglycemia therapy and antioxidant therapy in order to avert hypoglycemia in clinical treatment of diabetes.

Decreased intracellular granule movement and glucagon secretion in pancreatic α cells attached to superior cervical ganglion neurites.

Autonomic neurons innervate pancreatic islets of Langerhans and participate in the maintenance of blood glucose concentrations by controlling hormone levels through attachment with islet cells. We previously found that stimulated superior cervical ganglia (SCG) could induce Ca2+ oscillation in α cells via neuropeptide substance P using an in vitro co-culture model. In this study, we studied the effect of SCG neurite adhesion on intracellular secretory granule movement and glucagon secretion in α cells stimulated by low glucose concentration. Spinning disk microscopic analysis revealed that the mean velocity of intracellular granules was significantly lower in α cells attached to SCG neurites than that in those without neurites under low (2 mM), middle (10 mM), and high (20 mM) glucose concentrations. Stimulation by a low (2 mM) glucose concentration significantly increased glucagon secretion in α cells lacking neurites but not in those bound to neurites. These results suggest that adhesion to SCG neurites decreases low glucose-induced glucagon secretion in pancreatic α cells by attenuating intracellular granule movement activity.

The role of somatostatin in GLP-1-induced inhibition of glucagon secretion in mice.

AIMS/HYPOTHESIS: Glucagon-like peptide-1 (GLP-1) receptor agonists are currently used for the treatment of type 2 diabetes. Their main mechanism of action is enhancement of glucose-induced insulin secretion (from increased beta cell glucose sensitivity) and inhibition of glucagon secretion. The latter has been demonstrated to account for about half of their blood glucose-lowering activity. Whereas the effect of GLP-1 on insulin secretion is clearly dependent on ambient glucose concentrations and has been described in detail, the mechanism responsible for the inhibitory effect of GLP-1 on glucagon secretion is heavily debated. Glucagon inhibition is also said to be glucose-dependent, although it is unclear what is meant by this. We hypothesise here that GLP-1 does not inhibit glucagon secretion during hypoglycaemia because the inhibition depends on somatostatin secretion, which in turn is dependent on glucose levels. METHODS: We used the perfused mouse pancreas model to investigate this hypothesis. RESULTS: We found that, in this model, GLP-1 was able to significantly inhibit glucagon secretion from pancreatic alpha cells at all glucose levels tested: 6.0, 1.5 and 0.5 mmol/l (-27.0%, -37.1%, and -23.6%, respectively), and the decrease in glucagon secretion was invariably accompanied by an increase in somatostatin secretion (+286.8%, +158.7%, and +118.8%, respectively). Specific blockade of somatostatin receptor 2 increased glucagon secretion (+118.8% at 1.5 mmol/l glucose and +162.9% at 6.0 mmol/l glucose) and completely eliminated the inhibitory effect of GLP-1. CONCLUSIONS/INTERPRETATION: We have shown here that the glucagon-lowering effect of GLP-1 is entirely mediated through the paracrine actions of somatostatin in the perfused mouse pancreas. However, in this model, the inhibitory effect of GLP-1 was preserved at hypoglycaemic levels, leaving unanswered the question of how this is avoided in vivo in individuals treated with GLP-1 receptor agonists.

Current Therapies That Modify Glucagon Secretion: What Is the Therapeutic Effect of Such Modifications?

PURPOSE OF REVIEW: Hyperglucagonemia contributes significantly to hyperglycemia in type 2 diabetes and suppressed glucagon levels may increase the risk of hypoglycemia. Here, we give a brief overview of glucagon physiology and the role of glucagon in the pathophysiology of type 2 diabetes and provide insights into how antidiabetic drugs influence glucagon secretion as well as a perspective on the future of glucagon-targeting drugs. RECENT FINDINGS: Several older as well as recent investigations have evaluated the effect of antidiabetic agents on glucagon secretion to understand how glucagon may be involved in the drugs' efficacy and safety profiles. Based on these findings, modulation of glucagon secretion seems to play a hitherto underestimated role in the efficacy and safety of several glucose-lowering drugs. Numerous drugs currently available to diabetologists are capable of altering glucagon secretion: metformin, sulfonylurea compounds, insulin, glucagon-like peptide-1 receptor agonists, dipeptidyl peptidase-4 inhibitors, sodium-glucose cotransporter 2 inhibitors and amylin mimetics. Their diverse effects on glucagon secretion are of importance for their individual efficacy and safety profiles. Understanding how these drugs interact with glucagon secretion may help to optimize treatment.

Addition of a dipeptidyl peptidase-4 inhibitor, sitagliptin, to ongoing therapy with the glucagon-like peptide-1 receptor agonist liraglutide: A randomized controlled trial in patients with type 2 diabetes.

AIM: To determine whether the addition of sitagliptin to pre-existing therapy with liraglutide changes glycaemic excursions after a mixed meal. METHODS: A total of 16 patients with type 2 diabetes treated with metformin and liraglutide (1.2 mg/d for ≥2 weeks) were randomized (sealed envelopes), within a cross-over design, to be studied on two occasions, after an overnight fast, with (1) sitagliptin (100 mg orally) and (2) placebo (patients and care givers blinded) administered 60 minutes before a mixed meal, or vice versa. Glucose excursions (incremental area under the curve [AUC]; primary endpoint) and insulin, C-peptide, glucagon and incretin concentrations were measured. The study setting was a metabolic study unit at a specialized diabetes hospital. RESULTS: All 16 patients completed the study and were analysed. Glucose (AUCglucose 319 ± 30 [placebo] vs 315 ± 18 mmol.L-1 .min-1 [sitagliptin], Δ 7 [95% confidence interval -50 to 63] mmol.L-1 .min-1 ), insulin, C-peptide and glucagon concentrations were not affected significantly by sitagliptin treatment ( P = .60-1.00). Intact glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) concentrations were augmented by sitagliptin, by 78.4% and 90.2%, respectively (both P < .0001). The influence of sitagliptin treatment on incretin plasma concentrations was similar to previously published results obtained in patients with type 2 diabetes on metformin treatment only. CONCLUSIONS: Sitagliptin, in patients already treated with a GLP-1 receptor agonist (liraglutide), increased intact GLP-1 and GIP concentrations, but with marginal, non-significant effects on glycaemic control. GLP-1 receptors have probably been maximally stimulated by liraglutide. Our findings do not support combination treatment with GLP-1 receptor agonists and DPP-4 inhibitors, but longer-term trials are needed to support clinical recommendations.

FAM3D inhibits glucagon secretion via MKP1-dependent suppression of ERK1/2 signaling.

Dysregulated glucagon secretion is a hallmark of type 2 diabetes (T2D). To date, few effective therapeutic agents target on deranged glucagon secretion. Family with sequence similarity 3 member D (FAM3D) is a novel gut-derived cytokine-like protein, and its secretion timing is contrary to that of glucagon. However, the roles of FAM3D in metabolic disorder and its biological functions are largely unknown. In the present study, we investigated whether FAM3D modulates glucagon production in mouse pancreatic alpha TC1 clone 6 (αTC1-6) cells. Glucagon secretion, prohormone convertase 2 (PC2) activity, and mitogen-activated protein kinase (MAPK) pathway were assessed. Exogenous FAM3D inhibited glucagon secretion, PC2 activity, as well as extracellular-regulated protein kinase 1/2 (ERK1/2) signaling and induced MAPK phosphatase 1 (MKP1) expression. Moreover, knockdown of MKP1 and inhibition of ERK1/2 abolished and potentiated the inhibitory effect of FAM3D on glucagon secretion, respectively. Taken together, FAM3D inhibits glucagon secretion via MKP1-dependent suppression of ERK1/2 signaling. These results provide rationale for developing the therapeutic potential of FAM3D for dysregulated glucagon secretion and T2D.

Pancreatic polypeptide regulates glucagon release through PPYR1 receptors expressed in mouse and human alpha-cells.

BACKGROUND: Plasma levels of pancreatic polypeptide (PP) rise upon food intake. Although other pancreatic islet hormones, such as insulin and glucagon, have been extensively investigated, PP secretion and actions are still poorly understood. METHODS: The release of PP upon glucose stimulation and the effects of PP on glucagon and insulin secretion were analyzed in isolated pancreatic islets. Expression of PP receptor (PPYR1) was investigated by immunoblotting, quantitative RT-PCR on sorted pancreatic islet cells, and immunohistochemistry. RESULTS: In isolated mouse pancreatic islets, glucose stimulation increased PP release, while insulin secretion was up and glucagon release was down. Direct exposure of islets to PP inhibited glucagon release. In mouse islets, PPYR1 protein was observed by immunoblotting and quantitative RT-PCR revealed PPYR1 expression in the FACS-enriched glucagon alpha-cell fraction. Immunohistochemistry on pancreatic sections showed the presence of PPYR1 in alpha-cells of both mouse and human islets, while the receptor was absent in other islet cell types and exocrine pancreas. CONCLUSIONS: Glucose stimulates PP secretion and PP inhibits glucagon release in mouse pancreatic islets. PP receptors are present in alpha-cells of mouse and human pancreatic islets. GENERAL SIGNIFICANCE: These data demonstrate glucose-regulated secretion of PP and its effects on glucagon release through PPYR1 receptors expressed by alpha-cells.