Urocortin 3 contributes to paracrine inhibition of islet alpha cells in mice.

Pancreatic alpha cell activity and glucagon secretion lower as glucose levels increase. While part of the decrease is regulated by glucose itself, paracrine signaling by their neighboring beta and delta cells also plays an important role. Somatostatin from delta cells is an important local inhibitor of alpha cells at high glucose. Additionally, urocortin 3 (UCN3) is a hormone that is co-released from beta cells with insulin and acts locally to potentiate somatostatin secretion from delta cells. UCN3 thus inhibits insulin secretion via a negative feedback loop with delta cells, but its role with respect to alpha cells and glucagon secretion is not understood. We hypothesize that the somatostatin-driven glucagon inhibition at high glucose is regulated in part by UCN3 from beta cells. Here, we use a combination of live functional Ca2+ and cAMP imaging as well as direct glucagon secretion measurement, all from alpha cells in intact mouse islets, to determine the contributions of UCN3 to alpha cell behavior. Exogenous UCN3 treatment decreased alpha cell Ca2+ and cAMP levels and inhibited glucagon release. Blocking endogenous UCN3 signaling increased alpha cell Ca2+ by 26.8 ± 7.6%, but this did not result in increased glucagon release at high glucose. Furthermore, constitutive deletion of Ucn3 did not increase Ca2+ activity or glucagon secretion relative to controls. UCN3 is thus capable of inhibiting mouse alpha cells, but, given the subtle effects of endogenous UCN3 signaling on alpha cells, we propose that UCN3-driven somatostatin may serve to regulate local paracrine glucagon levels in the islet instead of inhibiting gross systemic glucagon release.

Prenatal exposure to a mixture of PAHs causes the dysfunction of islet cells in adult male mice: Association with type 1 diabetes mellitus.

Polycyclic aromatic hydrocarbons (PAHs) have been detected throughout the human body. Whether exposure to PAHs is associated with the incidence of type 1 diabetes mellitus should be investigated. To this end, pregnant mice were exposed to mixed PAHs (5, 50, or 500 µg/kg) once every other day during gestation. The adult male offspring displayed impaired glucose tolerance and reduced serum levels of glucagon and insulin. Immunohistochemical staining revealed increased numbers of apoptotic ß-cells and a reduced ß-cell mass in these males. The downregulated expression of pancreatic estrogen receptor α, androgen receptor, and transcription factor PDX1 was responsible for impacting ß-cell development. The relatively reduced α-cell area was associated with downregulated ARX expression. The transcription of Isn2 and Gcg in pancreatic tissue was downregulated, which indicated that the function of ß-cells and α-cells was impaired. Methylation levels in the Isn2 promotor were significantly elevated in mice prenatally exposed to 500 µg/kg PAHs, which was consistent with the change in its mRNA levels. The number of macrophages infiltrating islets was significantly increased, indicating that prenatal PAH exposure might reduce islet cell numbers in an autoimmune manner. This study shows that prenatal exposure to PAHs may promote the pathogenesis of type 1 diabetes mellitus.

Live-cell imaging of glucose-induced metabolic coupling of ß and α cell metabolism in health and type 2 diabetes.

Type 2 diabetes is characterized by ß and α cell dysfunction. We used phasor-FLIM (Fluorescence Lifetime Imaging Microscopy) to monitor oxidative phosphorylation and glycolysis in living islet cells before and after glucose stimulation. In healthy cells, glucose enhanced oxidative phosphorylation in ß cells and suppressed oxidative phosphorylation in α cells. In Type 2 diabetes, glucose increased glycolysis in ß cells, and only partially suppressed oxidative phosphorylation in α cells. FLIM uncovers key perturbations in glucose induced metabolism in living islet cells and provides a sensitive tool for drug discovery in diabetes.

HDL (High-Density Lipoprotein) and ApoA-1 (Apolipoprotein A-1) Potentially Modulate Pancreatic α-Cell Glucagon Secretion.

OBJECTIVE: Subjects with low levels of HDL (high-density lipoprotein) and ApoA-1 (apolipoprotein A-1) have increased risk to develop type 2 diabetes. HDL levels are an independent predictor of ß-cell function and positively modulate it. Type 2 diabetes is characterized by defects in both ß and α-cell function, but the effect of HDL and ApoA1 on α-cell function is unknown. Approach and Results: We observed a significant negative correlation (r=-0.422, P<0.0001) between HDL levels and fasting glucagon in a cohort of 132 Italian subjects. In a multivariable regression analysis including potential confounders such as age, sex, BMI, triglycerides, total cholesterol, fasting and 2-hour postload glucose, and fasting insulin, the association between HDL and fasting glucagon remained statistically significant (ß=-0.318, P=0.006). CD1 mice treated with HDL or ApoA-1 for 3 consecutive days showed a 32% (P<0.001) and 23% (P<0.05) reduction, respectively, in glucagon levels following insulin-induced hypoglycemia, compared with controls. Treatment of pancreatic αTC1 clone 6 cells with HDL or ApoA-1 for 24 hours resulted in a significant reduction of glucagon expression (P<0.04) and secretion (P<0.01) after an hypoglycemic stimulus and increased Akt (RAC-alpha serine/threonine-protein kinase) and FoxO1 (forkhead/winged helix box gene, group O-1) phosphorylation. Pretreatment with Akt inhibitor VIII, PI3K (phosphatidylinositol 3-kinase) inhibitor LY294002, and HDL receptor SCARB-1 (scavenger receptor class B type 1) inhibitor BLT-1 (block lipid transport-1) restored αTC1 cell response to low glucose levels. CONCLUSIONS: These results support the notion that HDL and ApoA-1 modulate glucagon expression and secretion by binding their cognate receptor SCARB-1, and activating the PI3K/Akt/FoxO1 signaling cascade in an in vitro α-cell model. Overall, these results raise the hypothesis that HDL and ApoA-1 may have a role in modulating glucagon secretion.

Co-impairment of autonomic and glucagon responses to insulin-induced hypoglycemia in dogs with naturally occurring insulin-dependent diabetes mellitus.

This study aimed to investigate the contributions of two factors potentially impairing glucagon response to insulin-induced hypoglycemia (IIH) in insulin-deficient diabetes: 1) loss of paracrine disinhibition by intra-islet insulin and 2) defects in the activation of the autonomic inputs to the islet. Plasma glucagon responses during hyperinsulinemic-hypoglycemic clamps ([Formula: see text]40 mg/dL) were assessed in dogs with spontaneous diabetes (n = 13) and in healthy nondiabetic dogs (n = 6). Plasma C-peptide responses to intravenous glucagon were measured to assess endogenous insulin secretion. Plasma pancreatic polypeptide, epinephrine, and norepinephrine were measured as indices of parasympathetic and sympathoadrenal autonomic responses to IIH. In 8 of the 13 diabetic dogs, glucagon did not increase during IIH (diabetic nonresponder [DMN]; ∆ = -6 ± 12 pg/mL). In five other diabetic dogs (diabetic responder [DMR]), glucagon responses (∆ = +26 ± 12) were within the range of nondiabetic control dogs (∆ = +27 ± 16 pg/mL). C-peptide responses to intravenous glucagon were absent in diabetic dogs. Activation of all three autonomic responses were impaired in DMN dogs but remained intact in DMR dogs. Each of the three autonomic responses to IIH was positively correlated with glucagon responses across the three groups. The study conclusions are as follows: 1) Impairment of glucagon responses in DMN dogs is not due to generalized impairment of α-cell function. 2) Loss of tonic inhibition of glucagon secretion by insulin is not sufficient to produce loss of the glucagon response; impairment of autonomic activation is also required. 3) In dogs with major ß-cell function loss, activation of the autonomic inputs is sufficient to mediate an intact glucagon response to IIH.NEW & NOTEWORTHY In dogs with naturally occurring, insulin-dependent (C-peptide negative) diabetes mellitus, impairment of glucagon responses is not due to generalized impairment of α-cell function. Loss of tonic inhibition of glucagon secretion by insulin is not sufficient, by itself, to produce loss of the glucagon response. Rather, impaired activation of the parasympathetic and sympathoadrenal autonomic inputs to the pancreas is also required. Activation of the autonomic inputs to the pancreas is sufficient to mediate an intact glucagon response to insulin-induced hypoglycemia in dogs with naturally occurring diabetes mellitus. These results have important implications that include leading to a greater understanding and insight into the pathophysiology, prevention, and treatment of hypoglycemia during insulin treatment of diabetes in companion dogs and in human patients.

Divergent effects of lycopene on pancreatic alpha and beta cells.

Lycopene is an antioxidant which has potential anti-diabetic activity, but the cellular mechanisms have not been clarified. In this study, different concentrations of lycopene were used to treat pancreatic alpha and beta cell lines, and the changes of cell growth, cell apoptosis, cell cycle, reactive oxygen species (ROS), ATP levels and expression of related cytokines were determined. The results exhibited that lycopene did not affect cell growth, cell apoptosis, cell cycle, ROS and ATP levels of alpha cells, while it promoted the growth of beta cells, increased the ratio of S phase, reduced the ROS levels and increased the ATP levels of beta cells. At the same time, lycopene treatment elevated the mRNA expression levels of tnfα, tgfß and hif1α in beta cells. These findings suggest that lycopene plays cell-specific role and activates pancreatic beta cells, supporting its application in diabetes therapy.

Alanine, arginine, cysteine, and proline, but not glutamine, are substrates for, and acute mediators of, the liver-α-cell axis in female mice.

The aim of this study was to identify the amino acids that stimulate glucagon secretion in mice and whose metabolism depends on glucagon receptor signaling. Pancreata of female C57BL/6JRj mice were perfused with 19 individual amino acids and pyruvate (at 10 mM), and secretion of glucagon was assessed using a specific glucagon radioimmunoassay. Separately, a glucagon receptor antagonist (GRA; 25-2648, 100 mg/kg) or vehicle was administered to female C57BL/6JRj mice 3 h before an intraperitoneal injection of four different isomolar amino acid mixtures (in total 7 µmol/g body wt) as follows: mixture 1 contained alanine, arginine, cysteine, and proline; mixture 2 contained aspartate, glutamate, histidine, and lysine; mixture 3 contained citrulline, methionine, serine, and threonine; and mixture 4 contained glutamine, leucine, isoleucine, and valine. Blood glucose, plasma glucagon, amino acid, and insulin concentrations were measured using well-characterized methodologies. Alanine (P = 0.03), arginine (P < 0.0001), cysteine (P = 0.01), glycine (P = 0.02), lysine (P = 0.02), and proline (P = 0.03), but not glutamine (P = 0.9), stimulated glucagon secretion from the perfused mouse pancreas. However, when the four isomolar amino acid mixtures were administered in vivo, the four mixtures elicited similar glucagon responses (P > 0.5). Plasma concentrations of total amino acids in vivo were higher after administration of GRA when mixture 1 (P = 0.004) or mixture 3 (P = 0.04) were injected. Our data suggest that alanine, arginine, cysteine, and proline, but not glutamine, are involved in the acute regulation of the liver-α-cell axis in female mice, as they all increased glucagon secretion and their disappearance rate was altered by GRA.

Stathmin-2 Mediates Glucagon Secretion From Pancreatic α-Cells.

Inhibition of glucagon hypersecretion from pancreatic α-cells is an appealing strategy for the treatment of diabetes. Our hypothesis is that proteins that associate with glucagon within alpha cell secretory granules will regulate glucagon secretion, and may provide druggable targets for controlling abnormal glucagon secretion in diabetes. Recently, we identified a dynamic glucagon interactome within the secretory granules of the α cell line, αTC1-6, and showed that select proteins within the interactome could modulate glucagon secretion. In the present study, we show that one of these interactome proteins, the neuronal protein stathmin-2, is expressed in αTC1-6 cells and in mouse pancreatic alpha cells, and is a novel regulator of glucagon secretion. The secretion of both glucagon and Stmn2 was significantly enhanced in response to 55 mM K+, and immunofluorescence confocal microscopy showed co-localization of stathmin-2 with glucagon and the secretory granule markers chromogranin A and VAMP-2 in αTC1-6 cells. In mouse pancreatic islets, Stathmin-2 co-localized with glucagon, but not with insulin, and co-localized with secretory pathway markers. To show a function for stathmin-2 in regulating glucagon secretion, we showed that siRNA-mediated depletion of stathmin-2 in αTC1-6 cells caused glucagon secretion to become constitutive without any effect on proglucagon mRNA levels, while overexpression of stathmin-2 completely abolished both basal and K+-stimulated glucagon secretion. Overexpression of stathmin-2 increased the localization of glucagon into the endosomal-lysosomal compartment, while depletion of stathmin-2 reduced the endosomal localization of glucagon. Therefore, we describe stathmin-2 as having a novel role as an alpha cell secretory granule protein that modulates glucagon secretion via trafficking through the endosomal-lysosomal system. These findings describe a potential new pathway for the regulation of glucagon secretion, and may have implications for controlling glucagon hypersecretion in diabetes.

Effects of long-acting GIP, xenin and oxyntomodulin peptide analogues on alpha-cell transdifferentiation in insulin-deficient diabetic GluCreERT2;ROSA26-eYFP mice.

Enzyme-resistant long-acting forms of the gut-derived peptide hormones, glucose-dependent insulinotropic polypeptide (GIP), xenin and oxyntomodulin (Oxm) have been generated, and exert beneficial effects on diabetes control and pancreatic islet architecture. The current study has employed alpha-cell lineage tracing in GluCreERT2;ROSA26-eYFP transgenic mice to investigate the extent to which these positive pancreatic effects are associated with alpha- to beta-cell transdifferentiation. Twice-daily administration of (D-Ala2)GIP, xenin-25[Lys13PAL] or (D-Ser2)-Oxm[Lys38PAL] for 10 days to streptozotocin (STZ)-induced diabetic mice did not affect body weight, food intake or blood glucose levels, but (D-Ser2)-Oxm[Lys38PAL] reduced (P < 0.05 to P < 0.001) fluid intake and circulating glucagon. (D-Ala2)GIP and (D-Ser2)-Oxm[Lys38PAL] also augmented (P < 0.05 and P < 0.01, respectively) pancreatic insulin content. Detrimental changes of pancreatic morphology induced by STZ in GluCreERT2;ROSA26-eYFP mice were partially reversed by all treatment interventions. This was associated with reduced (P < 0.05) apoptosis and increased (P < 0.05 to P < 0.01) proliferation of beta-cells, alongside opposing effects on alpha-cells, with (D-Ala2)GIP and (D-Ser2)-Oxm[Lys38PAL] being particularly effective in this regard. Alpha-cell lineage tracing revealed that induction of diabetes was accompanied by increased (P < 0.01) transdifferentiation of glucagon positive alpha-cells to insulin positive beta-cells. This islet cell transitioning process was augmented (P < 0.01 and P < 0.001, respectively) by (D-Ala2)GIP and (D-Ser2)-Oxm[Lys38PAL]. (D-Ser2)-Oxm[Lys38PAL] also significantly (P < 0.05) promoted loss of alpha-cell identity in favour of other endocrine islet cells. These data highlight intra-islet benefits of (D-Ala2)GIP, xenin-25[Lys13PAL] and (D-Ser2)-Oxm[Lys38PAL] in diabetes with beta-cell loss induced by STZ. The effects appear to be independent of glycaemic change, and associated with alpha- to beta-cell transdifferentiation for the GIP and Oxm analogues.

The role of GIP in α-cells and glucagon secretion.

Glucose-dependent insulinotropic polypeptide (GIP) is an intestinally derived peptide that is secreted in response to feeding. The GIP receptor (GIPR) is expressed in many cell types involved in the regulation of metabolism, including α- and ß-cells. Glucagon and insulin exert tremendous control over glucose metabolism. Thus, GIP action in islets strongly dictates metabolic control in the postprandial state. Loss of GIPR activity in ß-cells is a characteristic of type 2 diabetes (T2D) which associates with reduced postprandial insulin secretion and hyperglycemia. Less is known about GIPR activity in α-cells or the control of glucagon secretion. GIP stimulates glucagon secretion in a glucose-dependent manner in healthy people, with enhanced activity at lower glycemia. However, GIP stimulates glucagon secretion even at hyperglycemia in people with T2D, suggesting that inappropriate GIPR activity in α-cells contributes to the pathogenesis of T2D. Here, we review the literature describing GIP action and GIPR activity in the α-cell, detailing the basic science that has shaped the view of how GIP regulates glucagon secretion. We also contrast the effects of GIP on glucagon secretion in healthy and T2D people. Finally, we contextualize these observations in light of recent work that redefines the role of glucagon in glucose homeostasis, suggesting that hyperglucagonemia per se does not drive hyperglycemia. As new medications for T2D that incorporate GIPR activity are being developed, it is clear that a better understanding of GIPR activity beyond the ß-cell is necessary. This work highlights the importance of focusing on the GIPR in α-cells.

Exposure to Aroclor 1254 differentially affects the survival of pancreatic ß-cells and α-cells in the male mice and the potential reason.

Previous works showed that chronic exposure to Aroclor 1254 disrupted glucose homeostasis and induced insulin resistance in male mice. To further observe the different effects of Aroclor 1254 exposure on the pancreatic α-cells and ß-cells, male mice were exposed to Aroclor 1254 (0, 0.5, 5, 50, 500 µg/kg) for 60 days, the pancreas was performed a histological examination. The results showed that the percentage of apoptosis cell (indicated by TUNEL assay) was increased in both α-cells and ß-cells, as the Aroclor 1254 dose was increased; the proliferation (indicated by PCNA expression) rate of ß-cells was elevated while that of α-cells was not affected, resulting in an increased ß-cell mass and a decreased α-cell mass in a dose-depend manner. The number of Pdx-1 positive ß-cells was significantly increased whereas that of Arx positive α-cells was markedly decreased, indicating an enhanced ß-cell neogenesis and a weakened α-cell neogenesis. The drastically reduction of serum testosterone levels in all the treatments suggested an anti-androgenic potency of Aroclor 1254. The up-regulation of estrogen receptors (ERα and ERß) and androgen receptor in ß-cells might be responsible for the increased ß-cell mass and neogenesis.

Modulatory effect of empagliflozin on cellular parameters of endocrine pancreas in experimental pre-diabetes.

The effect of empagliflozin (EMPA), a sodium-glucose cotransporter 2 inhibitor (SGLT2i), on the structure of endocrine pancreas in pre-diabetes (Pre-DM) is not yet elucidated. In the current study the relatively enlarged islets of Langerhans seen in the Pre-DM group was restored to control size by administration of EMPA. In addition the disbalance in the percentage of ß-cells and α-cells in islets of the Pre-DM was corrected in the Pre-DM + EMPA group with reversal of the significantly increased islet mass, ß-cell mass and neogenesis. Administrating EMPA in Pre-DM decreased level of caspase-3, increased that of Bcl-2 to control level and reduced the significantly increased inflammatory cytokines to levels approximated to those of the control group. In Pre-DM + EMPA group, EMPA had efficiently restored the significantly impaired glucose hemostasis to levels nearly similar to those of the control animals. This may indicate that the modulatory effect of EMPA on cells of the islets in Pre-DM is associated with a local pleotropic effect on inflammatory cytokines.

A New Way for Beta Cell Neogenesis: Transdifferentiation from Alpha Cells Induced by Glucagon-Like Peptide 1.

Recent studies showed that alpha cells, especially immature cells and proalpha cells, might be the precursors of beta cells. Exposure to glucagon-like peptide 1 (GLP1) can ameliorate hyperglycemia in diabetic mice and restore the beta cell mass. In the present study, we adopted single high-dose (60 mg/kg, i.p.) streptozotocin (STZ) to model diabetes mellitus (DM) and randomly assigned short-tail (SD) rats to a normal group, a diabetic group, GLP1 groups (50 µg/kg, 100 µg/kg, and 200 µg/kg), a GLP1 (200 µg/kg) with exendin (9-39) group, and a GLP1 with LY294002 group. We found that the pancreatic insulin-glucagon-positive cell populations increased according to the increase in GLP1 exposure. By contrast, no insulin-amylase-positive cell populations or insulin/pan-cytokeratin cells were observed in the pancreatic sections. The GLP1 receptor antagonist exendin (9-39) and the phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) family inhibitor LY294002 not only suppressed protein kinase B (Akt), pancreatic and duodenal homeobox 1 (Pdx1), forkhead box O 1 (FoxO1), and mast cell function-associated antigen A (MafA) mRNA expression but also increased MAFB expression. We concluded that treatment with GLP1 might result in beta cell neogenesis by promoting the transdifferentiation of alpha cells but not by pancreatic acinar cells, ductal cells, or the self-replication of beta cells. The regulation on the GLP1 receptor and its downstream transcription factor PI3K/AKT/FOXO1 pathway, which causes increased pancreatic and duodenal homeobox 1 (Pdx1) and MafA mRNA expression but causes decreased MAFB expression, may be the mechanism involved in this process.

Diabetes relief in mice by glucose-sensing insulin-secreting human α-cells.

Cell-identity switches, in which terminally differentiated cells are converted into different cell types when stressed, represent a widespread regenerative strategy in animals, yet they are poorly documented in mammals. In mice, some glucagon-producing pancreatic α-cells and somatostatin-producing δ-cells become insulin-expressing cells after the ablation of insulin-secreting ß-cells, thus promoting diabetes recovery. Whether human islets also display this plasticity, especially in diabetic conditions, remains unknown. Here we show that islet non-ß-cells, namely α-cells and pancreatic polypeptide (PPY)-producing γ-cells, obtained from deceased non-diabetic or diabetic human donors, can be lineage-traced and reprogrammed by the transcription factors PDX1 and MAFA to produce and secrete insulin in response to glucose. When transplanted into diabetic mice, converted human α-cells reverse diabetes and continue to produce insulin even after six months. Notably, insulin-producing α-cells maintain expression of α-cell markers, as seen by deep transcriptomic and proteomic characterization. These observations provide conceptual evidence and a molecular framework for a mechanistic understanding of in situ cell plasticity as a treatment for diabetes and other degenerative diseases.

A Novel Dipeptidyl Peptidase IV Inhibitory Tea Peptide Improves Pancreatic ß-Cell Function and Reduces α-Cell Proliferation in Streptozotocin-Induced Diabetic Mice.

Dipeptidyl peptidase IV (DPP-IV) inhibitors occupy a growing place in the drugs used for the management of type 2 diabetes. Recently, food components, including food-derived bioactive peptides, have been suggested as sources of DPP-IV inhibitors without side effects. Chinese black tea is a traditional health beverage, and it was used for finding DPP-IV inhibitory peptides in this study. The ultra-filtrated fractions isolated from the aqueous extracts of black tea revealed DPP-IV inhibitory activity in vitro. Four peptides under 1 kDa were identified by SDS-PAGE and LC-MS/MS (Liquid Chromatography-Mass Spectrometry-Mass Spectrometry) from the ultra-filtrate. The peptide II (sequence: AGFAGDDAPR), with a molecular mass of 976 Da, showed the greatest DPP-IV inhibitory activity (in vitro) among the four peptides. After administration of peptide II (400 mg/day) for 57 days to streptozotocin (STZ)-induced hyperglycemic mice, the concentration of glucagon-like peptide-1 (GLP-1) in the blood increased from 9.85 ± 1.96 pmol/L to 19.22 ± 6.79 pmol/L, and the insulin level was increased 4.3-fold compared to that in STZ control mice. Immunohistochemistry revealed the improved function of pancreatic beta-cells and suppressed proliferation of pancreatic alpha-cells. This study provides new insight into the use of black tea as a potential resource of food-derived DPP-IV inhibitory peptides for the management of type 2 diabetes.

GLP-1 suppresses glucagon secretion in human pancreatic alpha-cells by inhibition of P/Q-type Ca2+ channels.

Glucagon is the body's main hyperglycemic hormone, and its secretion is dysregulated in type 2 diabetes mellitus (T2DM). The incretin hormone glucagon-like peptide-1 (GLP-1) is released from the gut and is used in T2DM therapy. Uniquely, it both stimulates insulin and inhibits glucagon secretion and thereby lowers plasma glucose levels. In this study, we have investigated the action of GLP-1 on glucagon release from human pancreatic islets. Immunocytochemistry revealed that only <0.5% of the α-cells possess detectable GLP-1R immunoreactivity. Despite this, GLP-1 inhibited glucagon secretion by 50-70%. This was due to a direct effect on α-cells, rather than paracrine signaling, because the inhibition was not reversed by the insulin receptor antagonist S961 or the somatostatin receptor-2 antagonist CYN154806. The inhibitory effect of GLP-1 on glucagon secretion was prevented by the PKA-inhibitor Rp-cAMPS and mimicked by the adenylate cyclase activator forskolin. Electrophysiological measurements revealed that GLP-1 decreased action potential height and depolarized interspike membrane potential. Mathematical modeling suggests both effects could result from inhibition of P/Q-type Ca2+ channels. In agreement with this, GLP-1 and ω-agatoxin (a blocker of P/Q-type channels) inhibited glucagon secretion in islets depolarized by 70 mmol/L [K+ ]o , and these effects were not additive. Intracellular application of cAMP inhibited depolarization-evoked exocytosis in individual α-cells by a PKA-dependent (Rp-cAMPS-sensitive) mechanism. We propose that inhibition of glucagon secretion by GLP-1 involves activation of the few GLP-1 receptors present in the α-cell membrane. The resulting small elevation of cAMP leads to PKA-dependent inhibition of P/Q-type Ca2+ channels and suppression of glucagon exocytosis.

Glucagon-Like Peptide 1 Increases ß-Cell Regeneration by Promoting α- to ß-Cell Transdifferentiation.

Glucagon-like peptide 1 (GLP-1) can increase pancreatic ß-cells, and α-cells could be a source for new ß-cell generation. We investigated whether GLP-1 increases ß-cells through α-cell transdifferentiation. New ß-cells originating from non-ß-cells were significantly increased in recombinant adenovirus expressing GLP-1 (rAd-GLP-1)-treated RIP-CreER;R26-YFP mice. Proliferating α-cells were increased in islets of rAd-GLP-1-treated mice and αTC1 clone 9 (αTC1-9) cells treated with exendin-4, a GLP-1 receptor agonist. Insulin+glucagon+ cells were significantly increased by rAd-GLP-1 or exendin-4 treatment in vivo and in vitro. Lineage tracing to label the glucagon-producing α-cells showed a higher proportion of regenerated ß-cells from α-cells in rAd-GLP-1-treated Glucagon-rtTA;Tet-O-Cre;R26-YFP mice than rAd producing ß-galactosidase-treated mice. In addition, exendin-4 increased the expression and secretion of fibroblast growth factor 21 (FGF21) in αTC1-9 cells and ß-cell-ablated islets. FGF21 treatment of ß-cell-ablated islets increased the expression of pancreatic and duodenal homeobox-1 and neurogenin-3 and significantly increased insulin+glucagon+ cells. Generation of insulin+glucagon+ cells by exendin-4 was significantly reduced in islets transfected with FGF21 small interfering RNA or islets of FGF21 knockout mice. Generation of insulin+ cells by rAd-GLP-1 treatment was significantly reduced in FGF21 knockout mice compared with wild-type mice. We suggest that GLP-1 has an important role in α-cell transdifferentiation to generate new ß-cells, which might be mediated, in part, by FGF21 induction.

Insulin regulates glucagon-like peptide-1 secretion by pancreatic alpha cells.

PURPOSE: Proglucagon is expressed in both pancreatic alpha cells and intestinal epithelial L cells and is cleaved into glucagon and glucagon-like peptide-1 (GLP-1) by different prohormone convertases (PCs). Recent studies have shown that α-cells can also secrete GLP-1, which may improve islet function. However, little is known about the factors influencing GLP-1 secretion by α cells. In this study, we investigated whether insulin promotes GLP-1 secretion by α cells, as well as the mechanisms underlying this phenomenon. METHODS: We cultured the alpha-cell line In-R1-G9 in low- or high-glucose medium in the presence or absence of insulin to determine the influence of glucose concentrations on the actions of insulin. We also treated In-R1-G9 cells with insulin for different times and at different doses. Then GLP-1 and glucagon protein expression levels were estimated. Moreover, ERK and phosphatidylinositol-3-kinase/AKT (PI3K/AKT) pathway activity levels and prohormone convertase expression levels were evaluated to elucidate the mechanism underlying the effects of insulin on GLP-1 secretion by α-cells. RESULTS: Insulin promoted GLP-1 secretion in a time- and dose-dependent manner under high-glucose conditions. Inhibiting the PI3K/AKT pathway with LY294002 and the Ras/mitogen-activated protein kinase (RAS/MAPK) pathway with PD98059 reduced GLP-1 secretion, respectively, in inhibitor-treated cells compared with insulin-treated cells. Moreover, insulin increased prohormone convertase 1/3 expression levels in the corresponding group of IN-R1-G9 cells compared with the control group of cells. CONCLUSION: Insulin facilitates GLP-1 secretion by pancreatic alpha cells by inducing PC1/3 expression under high-glucose conditions, a phenomenon that may be associated mainly with PI3K/AKT pathway activation.

17-ß Estradiol regulates proglucagon-derived peptide secretion in mouse and human α- and L cells.

Clinical and experimental data indicate a beneficial effect of estrogens on energy and glucose homeostasis associated with improved insulin sensitivity and positive effects on insulin secretion. The aim of the study was to investigate the impact of estrogens on proglucagon-producing cells, pancreatic α cells, and enteroendocrine L cells. The consequences of sexual hormone deprivation were evaluated in ovariectomized mice (ovx). Ovx mice exhibited impaired glucose tolerance during oral glucose tolerance tests (OGTT), which was associated with decreased GLP-1 intestinal and pancreatic secretion and content, an effect that was reversed by estradiol (E2) treatment. Indeed, E2 increased oral glucose-induced GLP-1 secretion in vivo and GLP-1 secretion from primary culture of mouse and human α cells through the activation of all 3 estrogen receptors (ERs), whereas E2-induced GLP-1 secretion from mouse and human intestinal explants occurred only by ERß activation. Underlying the implication of ERß, its selective agonist WAY20070 was able to restore glucose tolerance in ovx mice at least partly through plasma GLP-1 increase. We conclude that E2 directly controls both α- and L cells to increase GLP-1 secretion, in addition to its effects on insulin and glucagon secretion, highlighting the potential beneficial role of the estrogenic pathway and, more particularly, of ERß agonists to prevent type 2 diabetes.

Effects on the glucagon response to hypoglycaemia during DPP-4 inhibition in elderly subjects with type 2 diabetes: A randomized, placebo-controlled study.

AIMS: Maintainance of glucagon response to hypoglycaemia is important as a safeguard against hypoglycaemia during glucose-lowering therapy in type 2 diabetes. During recent years, DPP-4 (dipeptidyl peptidase-4) inhibition has become more commonly used in elderly patients. However, whether DPP-4 inhibition affects the glucagon response to hypoglycaemia in the elderly is not known and was the aim of this study. METHODS: In a single-centre, double-blind, randomized, placebo-controlled crossover study, 28 subjects with metformin-treated type 2 diabetes (17 male, 11 female; mean age, 74 years [range 65-86]; mean HbA1c, 51.5 mmol/mol [6.9%]) received sitagliptin (100 mg once daily) as add-on therapy or placebo for 4 weeks with a 4-week washout period in between. After each treatment period, the subjects underwent a standard breakfast test, followed by a 2-step hyperinsulinaemic hypoglycaemic clamp (target 3.5 and 3.0 mmol/L), followed by lunch. RESULTS: Glucagon levels after breakfast and lunch, and the glucagon response at 3.5 mmol/L, were lower after sitagliptin than after placebo. However, the glucagon response to hypoglycaemia at 3.1 mmol/L did not differ significantly between the two. Similarly, the noradrenaline, adrenaline and cortisol responses were lower with sitagliptin than with placebo at 3.5 mmol/L, but not at 3.1 mmol/L glucose. Responses in pancreatic polypeptide did not differ between the two. CONCLUSIONS: Elderly subjects with metformin-treated type 2 diabetes have lower glucagon levels at 3.5 mmol/L glucose, but maintain the glucagon response to hypoglycaemia at 3.1 mmol/L during DPP-4 inhibition, which safeguards against hypoglycaemia and may contribute to decreasing the risk of hypoglycaemia by DPP-4 inhibition in this age group.