Intra-islet glucagon signalling regulates beta-cell connectivity, first-phase insulin secretion and glucose homoeostasis.

OBJECTIVE: Type 2 diabetes (T2D) is characterised by the loss of first-phase insulin secretion. We studied mice with ß-cell selective loss of the glucagon receptor (Gcgrfl/fl X Ins-1Cre), to investigate the role of intra-islet glucagon receptor (GCGR) signalling on pan-islet [Ca2+]I activity and insulin secretion. METHODS: Metabolic profiling was conducted on Gcgrß-cell-/- and littermate controls. Crossing with GCaMP6f (STOP flox) animals further allowed for ß-cell specific expression of a fluorescent calcium indicator. These islets were functionally imaged in vitro and in vivo. Wild-type mice were transplanted with islets expressing GCaMP6f in ß-cells into the anterior eye chamber and placed on a high fat diet. Part of the cohort received a glucagon analogue (GCG-analogue) for 40 days and the control group were fed to achieve weight matching. Calcium imaging was performed regularly during the development of hyperglycaemia and in response to GCG-analogue treatment. RESULTS: Gcgrß-cell-/- mice exhibited higher glucose levels following intraperitoneal glucose challenge (control 12.7 mmol/L ± 0.6 vs. Gcgrß-cell-/- 15.4 mmol/L ± 0.0 at 15 min, p = 0.002); fasting glycaemia was not different to controls. In vitro, Gcgrß-cell-/- islets showed profound loss of pan-islet [Ca2+]I waves in response to glucose which was only partially rescued in vivo. Diet induced obesity and hyperglycaemia also resulted in a loss of co-ordinated [Ca2+]I waves in transplanted islets. This was reversed with GCG-analogue treatment, independently of weight-loss (n = 8). CONCLUSION: These data provide novel evidence for the role of intra-islet GCGR signalling in sustaining synchronised [Ca2+]I waves and support a possible therapeutic role for glucagonergic agents to restore the insulin secretory capacity lost in T2D.

Glucagon-like peptide 1 (GLP-1).

BACKGROUND: The glucagon-like peptide-1 (GLP-1) is a multifaceted hormone with broad pharmacological potential. Among the numerous metabolic effects of GLP-1 are the glucose-dependent stimulation of insulin secretion, decrease of gastric emptying, inhibition of food intake, increase of natriuresis and diuresis, and modulation of rodent ß-cell proliferation. GLP-1 also has cardio- and neuroprotective effects, decreases inflammation and apoptosis, and has implications for learning and memory, reward behavior, and palatability. Biochemically modified for enhanced potency and sustained action, GLP-1 receptor agonists are successfully in clinical use for the treatment of type-2 diabetes, and several GLP-1-based pharmacotherapies are in clinical evaluation for the treatment of obesity. SCOPE OF REVIEW: In this review, we provide a detailed overview on the multifaceted nature of GLP-1 and its pharmacology and discuss its therapeutic implications on various diseases. MAJOR CONCLUSIONS: Since its discovery, GLP-1 has emerged as a pleiotropic hormone with a myriad of metabolic functions that go well beyond its classical identification as an incretin hormone. The numerous beneficial effects of GLP-1 render this hormone an interesting candidate for the development of pharmacotherapies to treat obesity, diabetes, and neurodegenerative disorders.

Regulation of glucose kinetics during exercise by the glucagon-like peptide-1 receptor.

In response to oral glucose, glucagon-like peptide-1 receptor (Glp1r) knockout (Glp1r−/−) mice become hyperglycaemic due to impaired insulin secretion. Exercise also induces hyperglycaemia in Glp1r−/− mice. In contrast to oral glucose, exercise decreases insulin secretion. This implies that exercise-induced hyperglycaemia in Glp1r−/− mice results from the loss of a non-insulinotropic effect mediated by the Glp1r. Muscle glucose uptake (MGU) is normal in exercising Glp1r−/− mice. Thus, we hypothesize that exercise-induced hyperglycaemia in Glp1r−/− mice is due to excessive hepatic glucose production (HGP). Wild-type (Glp1r+/+) and Glp1r−/− mice implanted with venous and arterial catheters underwent treadmill exercise or remained sedentary for 30 min. [3-3H]glucose was used to estimate rates of glucose appearance (Ra), an index of HGP, and disappearance (Rd). 2[14C]deoxyglucose was used to assess MGU. Glp1r−/− mice displayed exercise-induced hyperglycaemia due to an excessive increase in Ra but normal Rd and MGU. Exercise-induced glucagon levels were ∼2-fold higher in Glp1r−/− mice, resulting in a ∼2-fold higher glucagon:insulin ratio. Since inhibition of the central Glp1r stimulates HGP, we tested whether intracerebroventricular (ICV) infusion of the Glp1r antagonist exendin(9­39) (Ex9) in Glp1r+/+ mice would result in exercise-induced hyperglycaemia. ICV Ex9 did not enhance glucose levels or HGP during exercise, suggesting that glucoregulatory effects of Glp1 during exercise are mediated via the pancreatic Glp1r. In conclusion, functional disruption of the Glp1r results in exercise-induced hyperglycaemia associated with an excessive increase in glucagon secretion and HGP. These results suggest an essential role for basal Glp1r signalling in the suppression of alpha cell secretion during exercise.

Glucagon-like peptide-1 (GLP-1) receptor agonists, obesity and psoriasis: diabetes meets dermatology.

Type 2 diabetes mellitus is characterised by beta cell failure, which frequently develops in the setting of insulin resistance. Inflammation contributes to the pathophysiology of type 2 diabetes by impairing insulin action in peripheral tissues and via reduction of beta cell function. Inflammation may also play an important role in the development of complications that arise in patients with type 2 diabetes. Hence, the anti-inflammatory actions of commonly used glucose-lowering drugs may contribute, indirectly, to their mechanisms of action and therapeutic benefit. Herein we highlight the anti-inflammatory actions of glucagon-like peptide-1 (GLP-1), which exerts direct and indirect actions on immune function. The observations that GLP-1 receptor agonists exert anti-inflammatory actions in preclinical studies, taken together with case reports linking improvements in psoriasis with GLP-1 receptor agonist therapy, illustrates the emerging clinical implications of non-classical anti-inflammatory actions of incretin-based therapeutics.

Metformin regulates the incretin receptor axis via a pathway dependent on peroxisome proliferator-activated receptor-α in mice.

AIMS/HYPOTHESIS: Metformin is widely used for the treatment of type 2 diabetes. Although it reduces hepatic glucose production, clinical studies show that metformin may reduce plasma dipeptidyl peptidase-4 activity and increase circulating levels of glucagon-like peptide 1 (GLP-1). We examined whether metformin exerts glucoregulatory actions via modulation of the incretin axis. METHODS: Metformin action was assessed in Glp1r(-/-), Gipr(-/-), Glp1r:Gipr(-/-), Pparα (also known as Ppara)(-/-) and hyperglycaemic obese wild-type mice with or without the GLP-1 receptor (GLP1R) antagonist exendin(9-39). Experimental endpoints included glucose tolerance, plasma insulin levels, gastric emptying and food intake. Incretin receptor expression was assessed in isolated islets from metformin-treated wild-type and Pparα(-/-) mice, and in INS-1 832/3 beta cells with or without peroxisome proliferator-activated receptor (PPAR)-α or AMP-activated protein kinase (AMPK) antagonists. RESULTS: In wild-type mice, metformin acutely increased plasma levels of GLP-1, but not those of gastric inhibitory polypeptide or peptide YY; it also improved oral glucose tolerance and reduced gastric emptying. Metformin significantly improved oral glucose tolerance despite loss of incretin action in Glp1r(-/-), Gipr(-/-) and Glp1r(-/-) :Gipr(-/-) mice, and in wild-type mice fed a high-fat diet and treated with exendin(9-39). Levels of mRNA transcripts for Glp1r, Gipr and Pparα were significantly increased in islets from metformin-treated mice. Metformin directly increased Glp1r expression in INS-1 beta cells via a PPAR-α-dependent, AMPK-independent mechanism. Metformin failed to induce incretin receptor gene expression in islets from Pparα(-/-) mice. CONCLUSIONS/INTERPRETATION: As metformin modulates multiple components of the incretin axis, and enhances expression of the Glp1r and related insulinotropic islet receptors through a mechanism requiring PPAR-α, metformin may be mechanistically well suited for combination with incretin-based therapies.

Glucagon-like peptide-1 receptor signalling selectively regulates murine lymphocyte proliferation and maintenance of peripheral regulatory T cells.

AIMS/HYPOTHESIS: Glucagon-like peptide-1 receptor (GLP-1R) agonists improve glucose control in animals and humans with type 1 diabetes. However, there is little information on the role of the GLP-1R in the immune system. We studied the role of the GLP-1R in immune function in wild-type (WT) and nonobese diabetic (NOD) and Glp1r-/- mice. METHODS: Glp1r mRNA expression was examined in sorted immune subpopulations by RT-PCR. The effects of GLP-1R activation were assessed on cAMP production and proliferation, migration and survival of primary immune cells from WT and NOD mice. The ability of primary cells from Glp1r-/- mice to proliferate, migrate or survive apoptosis was determined. Immunophenotyping studies were performed to assess the frequency of immune subpopulations in Glp1r-/- mice. RESULTS: Ex vivo activation of the GLP-1R resulted in a modest but significant elevation of cAMP in primary thymocytes and splenocytes from both WT and NOD mice. GLP-1R activation did not increase proliferation of primary thymocytes, splenocytes or peripheral lymph node cells. In contrast, Glp1r-/- thymocytes exhibited a hypoproliferative response, whilst peripheral Glp1r-/- lymphocytes were hyperproliferative in response to mitogenic stimulation. Activation or loss of GLP-1R signalling did not modify apoptosis or chemotaxis in primary lymphocytes. Male Glp1r-/- mice exhibited a significantly lower percentage of peripheral regulatory T cells, although no differences were observed in the numbers of CD4+ and CD8+ T cells and B cells in the spleen and lymph nodes of Glp1r-/- mice. CONCLUSIONS/INTERPRETATION: These studies establish that GLP-1R signalling may regulate lymphocyte proliferation and maintenance of peripheral regulatory T cells.

The glucagon-like peptide 1 receptor is essential for postprandial lipoprotein synthesis and secretion in hamsters and mice.

AIMS/HYPOTHESIS: Glucagon-like peptide 1 (GLP-1) receptor (GLP-1R) agonists and dipeptidyl peptidase-4 (DPP-4) inhibitors attenuate postprandial lipaemia through mechanisms that remain unclear. As dyslipidaemia is a contributing risk factor for cardiovascular disease in type 2 diabetes, we examined the mechanisms linking pharmacological and physiological regulation of GLP-1 action to control of postprandial lipid metabolism. METHODS: Postprandial lipid synthesis and secretion were assessed in normal and fructose-fed hamsters and in wild-type mice that were treated with or without sitagliptin. Apolipoprotein B-48 (ApoB-48) synthesis and secretion were also examined in primary enterocyte cultures. The importance of exogenous vs endogenous GLP-1R signalling for regulation of intestinal lipoprotein synthesis and secretion was assessed in mice and hamsters treated with the GLP-1R agonist exendin-4, the GLP-1R antagonist exendin(9-39) and in Glp1r (+/+) vs Glp1r (-/-) mice. RESULTS: Sitagliptin decreased fasting plasma triacylglycerol, predominantly in the VLDL fraction, as well as postprandial triacylglycerol-rich lipoprotein (TRL)-triacylglycerol, TRL-cholesterol and TRL-ApoB-48 in hamsters and mice. GLP-1R activation with exendin-4 alone also decreased plasma and TRL-ApoB-48 in hamsters and mice, and reduced secretion of ApoB-48 in hamster enterocyte cultures. Conversely, blockade of endogenous GLP-1R signalling by the antagonist exendin(9-39) or genetic elimination of GLP-1R signalling in Glp1r (-/-) mice enhanced TRL-ApoB-48 secretion in vivo. Co-administration of exendin(9-39) also abolished the hypolipidaemic effect of sitagliptin. CONCLUSIONS/INTERPRETATION: Potentiation of endogenous incretin action via DPP-4 inhibition or pharmacological augmentation of GLP-1R signalling reduces intestinal secretion of triacylglycerol, cholesterol and ApoB-48. Moreover, endogenous GLP-1R signalling is essential for the control of intestinal lipoprotein biosynthesis and secretion.

Glucagon and glucagon-like peptide receptors as drug targets.

Glucagon and the glucagon-like peptides are derived from a common proglucagon precursor, and regulate energy homeostasis through interaction with a family of distinct G protein coupled receptors. Three proglucagon-derived peptides, glucagon, GLP-1, and GLP-2, play important roles in energy intake, absorption, and disposal, as elucidated through studies utilizing peptide antagonists and receptor knockout mice. The essential role of glucagon in the control of hepatic glucose production, taken together with data from studies employing glucagon antagonists, glucagon receptor antisense oligonucleotides, and glucagon receptor knockout mice, suggest that reducing glucagon action may be a useful strategy for the treatment of type 2 diabetes. GLP-1 secreted from gut endocrine cells controls glucose homeostasis through glucose-dependent enhancement of beta-cell function and reduction of glucagon secretion and gastric emptying. GLP-1 administration is also associated with reduction of food intake, prevention of weight gain, and expansion of beta-cell mass through stimulation of beta-cell proliferation, and prevention of apoptosis. GLP-1R agonists, as well as enzyme inhibitors that prevent GLP-1 degradation, are in late stage clinical trials for the treatment of type 2 diabetes. Exenatide (Exendin-4) has been approved for the treatment of type 2 diabetes in the United States in April 2005. GLP-2 promotes energy absorption, inhibits gastric acid secretion and gut motility, and preserves mucosal epithelial integrity through enhancement of crypt cell proliferation and reduction of epithelial apoptosis. A GLP-2R agonist is being evaluated in clinical trials for the treatment of inflammatory bowel disease and short bowel syndrome. Taken together, the separate receptors for glucagon, GLP-1, and GLP-2 represent important targets for developing novel therapeutic agents for the treatment of disorders of energy homeostasis.

The HeLa cell glucagon-like peptide-2 receptor is coupled to regulation of apoptosis and ERK1/2 activation through divergent signaling pathways.

Glucagon-like peptide-2 (GLP-2) regulates proliferative and cytoprotective pathways in the intestine; however GLP-2 receptor (GLP-2R) signal transduction remains poorly understood, and cell lines that express the endogenous GLP-2R have not yet been isolated. We have now identified several expressed sequence tags from human cervical carcinoma cDNA libraries that correspond to GLP-2R nucleotide sequences. GLP-2R mRNA transcripts were detected by RT-PCR in two human cervical carcinoma cell lines, including HeLa cells. GLP-2 increased cAMP accumulation and activated ERK1/2 in HeLa cells transiently expressing the cloned human HeLa cell GLP-2R cDNA. However, the GLP-2R-induced activation of ERK1/2 was not mediated through Galphas, adenylyl cyclase, or transactivation of the epidermal growth factor receptor, but was pertussis toxin sensitive, inhibited by dominant negative Ras, and dependent on betagamma-subunits. GLP-2 also induced a significant increase in bromodeoxyuridine incorporation that was blocked by dominant negative Ras. Furthermore, GLP-2 inhibited HeLa cell apoptosis induced by LY294002 in a protein kinase A-dependent, but ERK-independent, manner. These findings demonstrate that the HeLa cell GLP-2R differentially signals through both Galphas/cAMP- and Gi/Go-dependent pathways, illustrating for the first time that the GLP-2R is capable of coupling to multiple heterotrimeric G proteins defining distinct GLP-2R-dependent biological actions.

Characterization and functional role of voltage gated cation conductances in the glucagon-like peptide-1 secreting GLUTag cell line.

Glucagon-like peptide-1 (GLP-1) is released from intestinal L-cells in response to nutrient ingestion. It is currently under therapeutic evaluation because it enhances insulin secretion in type 2 diabetes. Previous studies using the GLP-1 secreting cell line GLUTag have shown that the cells are electrically active, and that the action potential frequency is regulated by nutrients. In this study we characterize voltage gated currents underlying this electrical activity and correlate the electrophysiological findings with gene expression determined by microarrays. Whole cell voltage clamp experiments designed to separate different ionic components revealed rapidly inactivating sodium currents sensitive to tetrodotoxin, calcium currents sensitive to nifedipine and omega-conotoxin GVIA, and sustained as well as rapidly inactivating potassium currents, which were sensitive to TEA and 4-AP, respectively. In perforated patch experiments we also observed hyperpolarization-activated currents which were inhibited by ZD7288. The amplitude of the sodium current was approximately 10 times that of the other depolarizing currents and tetrodotoxin abolished action potential firing. In secretion experiments, however, nifedipine, but not tetrodotoxin, omega-conotoxin GVIA or ZD7288, inhibited glucose-induced GLP-1 release. Consistent with this finding, the intracellular Ca2+ response to glucose was impaired by nifedipine but not by tetrodotoxin. Thus, in GLUTag cells, GLP-1 release is not dependent on the firing of Na+-carrying action potentials but requires membrane depolarization and Ca2+ entry through L-type Ca2+ channels. Understanding the characteristics of the currents and the molecular identification of the underlying channels in GLP-1 secreting cells might facilitate the development of agents to enhance GLP-1 secretion in vivo.

Double incretin receptor knockout (DIRKO) mice reveal an essential role for the enteroinsular axis in transducing the glucoregulatory actions of DPP-IV inhibitors.

Glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide 1 (GLP-1) are gut-derived incretins that potentiate glucose clearance following nutrient ingestion. Elimination of incretin receptor action in GIPR(-/-) or GLP-1R(-/-) mice produces only modest impairment in glucose homeostasis, perhaps due to compensatory upregulation of the remaining incretin. We have now studied glucose homeostasis in double incretin receptor knockout (DIRKO) mice. DIRKO mice exhibit normal body weight and fail to exhibit an improved glycemic response after exogenous administration of GIP or the GLP-1R agonist exendin-4. Plasma glucagon and the hypoglycemic response to exogenous insulin were normal in DIRKO mice. Glycemic excursion was abnormally increased and levels of glucose-stimulated insulin secretion were decreased following oral but not intraperitoneal glucose challenge in DIRKO compared with GIPR(-/-) or GLP-1R(-/-) mice. Similarly, glucose-stimulated insulin secretion and the response to forskolin were well preserved in perifused DIRKO islets. Although the dipeptidyl peptidase-IV (DPP-IV) inhibitors valine pyrrolidide (Val-Pyr) and SYR106124 lowered glucose and increased plasma insulin in wild-type and single incretin receptor knockout mice, the glucose-lowering actions of DPP-IV inhibitors were eliminated in DIRKO mice. These findings demonstrate that glucose-stimulated insulin secretion is maintained despite complete absence of both incretin receptors, and they delineate a critical role for incretin receptors as essential downstream targets for the acute glucoregulatory actions of DPP-IV inhibitors.

Extrahypothalamic expression of the glucagon-like peptide-2 receptor is coupled to reduction of glutamate-induced cell death in cultured hippocampal cells.

Proglucagon-derived glucagon-like peptide-2 (GLP-2) is liberated in enteroendocrine cells and neurons. GLP-2 regulates energy absorption and epithelial integrity in the gastrointestinal tract, whereas GLP-2 action in the central nervous system remains poorly defined. We identified proglucagon and GLP-2 receptor (GLP-2R) mRNA transcripts by RT-PCR in multiple regions of the developing and adult rat central nervous system. GLP-2R mRNA transcripts were localized by in situ hybridization to the hippocampus, hypothalamus, nucleus of the solitary tract, parabrachial nucleus, supramammillary nucleus, and substantia nigra. The bioactive form of GLP-2, GLP-2-(1-33) was detected by RIA and HPLC analysis in the fetal and adult brainstem and hypothalamus. GLP-2 stimulated increases in cAMP accumulation in postnatal d 8, but not embryonic d 14, dispersed neonatal rat brainstem tissues. The actions of GLP-2 were independent of the GLP-1R antagonist exendin-(9-39), and GLP-2 stimulated cAMP accumulation in hippocampal cell cultures from both wild-type and GLP-1R(-/-) mice. GLP-2 significantly reduced glutamate-induced excitotoxic injury in hippocampal cells via a protein kinase A-dependent pathway, but had no effect on the rate of cell proliferation. These findings establish the presence of a functional GLP-2-GLP-2R axis in the developing rodent brain and demonstrate that GLP-2 exerts cytoprotective actions in cells derived from the central nervous system.

Minireview: Glucagon-like peptides regulate cell proliferation and apoptosis in the pancreas, gut, and central nervous system.

Gut peptides exert diverse effects regulating satiety, gastrointestinal motility and acid secretion, epithelial integrity, and both nutrient absorption and disposal. These actions are initiated by activation of specific G protein-coupled receptors and may be mediated by direct or indirect effects on target cells. More recent evidence demonstrates that gut peptides, exemplified by glucagon-like peptides-1 and 2 (GLP-1 and GLP-2), directly regulate signaling pathways coupled to cell proliferation and apoptosis. GLP-1 receptor activation enhances beta-cell proliferation and promotes islet neogenesis via activation of pdx-1 expression. The proliferative effects of GLP-1 appear to involve multiple intracellular pathways, including stimulation of Akt, activation of protein kinase Czeta, and transactivation of the epidermal growth factor receptor through the c-src kinase. GLP-1 receptor activation also promotes cell survival in beta-cells and neurons via increased levels of cAMP leading to cAMP response element binding protein activation, enhanced insulin receptor substrate-2 activity and, ultimately, activation of Akt. These actions of GLP-1 are reflected by expansion of beta-cell mass and enhanced resistance to beta-cell injury in experimental models of diabetes in vivo. GLP-2 also promotes intestinal cell proliferation and confers resistance to cellular injury in a variety of cell types. Administration of GLP-2 to animals with experimental intestinal injury promotes regeneration of the gastrointestinal epithelial mucosa and confers resistance to apoptosis in an indirect manner via yet-to-be identified GLP-2 receptor-dependent regulators of mucosal growth and cell survival. These proliferative and antiapoptotic actions of GLP-1 and GLP-2 may contribute to protective and regenerative actions of these peptides in human subjects with diabetes and intestinal disorders, respectively.

Cellular specificity of proexendin-4 processing in mammalian cells in vitro and in vivo.

Glucagon-like peptide-1 (GLP-1) is a potent stimulator of glucose-dependent insulin secretion. Exendin-4(1-39) (Ex-4), isolated from Gila monster venom, is a highly specific GLP-1 receptor agonist that exhibits a prolonged duration of action in vivo. Although the processing mechanisms underlying liberation of GLP-1 from its prohormone have been elucidated, those for Ex-4 remain unknown. To examine the requirements for proEx-4 processing in mammalian cells, BHK fibroblasts, InR1-G9 islet A cells, and AtT-20 corticotropes, which express different prohormone convertases (furin, prohormone convertase 2, and prohormone convertase 1, respectively) were transfected with full-length lizard proEx-4, and the processing of proexendin was examined by HPLC and RIA (n = 3). All of the transfected cell lines exhibited Ex-4-like immunoreactivity in the media, and Ex-4-like immunoreactivity was detected in extracts of InR1-G9 and AtT-20 cells. However, only media and extracts from AtT-20 cells (not InR1-G9 and BHK cells) contained a single peak by HPLC corresponding to synthetic Ex-4. To establish whether proEx-4 can be processed to Ex-4 in nonimmortalized mammalian cells in vivo, the molecular forms of exendin-4 were examined in mice expressing a metallothionein-proEx-4 transgene (n = 3-6 for both males and females). ProEx4 mRNA transcripts were detected by RT-PCR in a broad range of both endocrine and nonendocrine tissues. Ex-4-like immunoreactivity was detected in pituitary, fat, adrenals, and testes; however HPLC analyses demonstrated that processed Ex-4 was found only in adrenals and testes. These results indicate that lizard proEx-4 is processed to mature bioactive Ex-4 in both rodent endocrine and nonendocrine mammalian cell types in vitro and in murine tissues in vivo. These findings may be useful for engineering cells that express a lizard pro-Ex4 transgene for the treatment of type 2 diabetes.

Gut adaptation and the glucagon-like peptides.

The glucagon-like peptides GLP-1 and GLP-2 are synthesised and then released from enteroendocrine cells in the small and large intestine. GLP-1 promotes efficient nutrient assimilation while GLP-2 regulates energy absorption via effects on nutrient intake, gastric acid secretion and gastric emptying, nutrient absorption, and mucosal permeability. Preliminary human studies indicate that GLP-2 may enhance energy absorption and reduce fluid loss in subjects with short bowel syndrome suggesting that GLP-2 functions as a key regulator of mucosal integrity, permeability, and nutrient absorption. Hence GLP-2 may be therapeutically useful in diseases characterised by injury or dysfunction of the gastrointestinal epithelium.

Structure-function of the glucagon receptor family of G protein-coupled receptors: the glucagon, GIP, GLP-1, and GLP-2 receptors.

The glucagon-like peptides include glucagon, GLP-1, and GLP-2, and exert diverse actions on nutrient intake, gastrointestinal motility, islet hormone secretion, cell proliferation and apoptosis, nutrient absorption, and nutrient assimilation. GIP, a related member of the glucagon peptide superfamily, also regulates nutrient disposal via stimulation of insulin secretion. The actions of these peptides are mediated by distinct members of the glucagon receptor superfamily of G protein-coupled receptors. These receptors exhibit unique patterns of tissue-specific expression, exhibit considerable amino acid sequence identity, and share similar structural and functional properties with respect to ligand binding and signal transduction. This article provides an overview of the biology of these receptors with an emphasis on understanding the unique actions of glucagon-related peptides through studies of the biology of their cognate receptors.

Development of glucagon-like peptide-1-based pharmaceuticals as therapeutic agents for the treatment of diabetes.

Glucagon-like peptide-1 (GLP-1) is released from gut endocrine cells following nutrient ingestion and acts to regulate nutrient assimilation via effects on gastrointestinal motility, islet hormone secretion, and islet cell proliferation. Exogenous administration of GLP-1 lowers blood glucose in normal rodents and in multiple experimental models of diabetes mellitus. Similarly, GLP-1 lowers blood glucose in normal subjects and in patients with type 2 diabetes. The therapeutic utility of the native GLP-1 molecule is limited by its rapid enzymatic degradation by the serine protease dipeptidyl peptidase IV. This review highlights recent advances in our understanding of GLP-1 physiology and GLP-1 receptor signaling, and summarizes current pharmaceutical strategies directed at sustained activation of GLP-1 receptor-dependent actions for glucoregulation in vivo. Given the nutrient-dependent control of GLP-1 release, neutraceuticals or modified diets that enhance GLP-1 release from the enteroendocrine cell may exhibit glucose-lowering properties in human subjects. The utility of GLP-1 derivatives engineered for sustained action and/or DP IV-resistance, and the biological activity of naturally occurring GLP-1-related molecules such as exendin-4 is reviewed. Circumventing DP IV-mediated incretin degradation via inhibitors that target the DP IV enzyme represents a complementary strategy for enhancing GLP-1-mediated actions in vivo. Finally, the current status of alternative GLP-1-delivery systems via the buccal and enteral mucosa is briefly summarized. The findings that the potent glucose-lowering properties of GLP-1 are preserved in diabetic subjects, taken together with the potential for GLP-1 therapy to preserve or augment beta cell mass, provides a powerful impetus for development of GLP-1-based human pharmaceuticals.

Glucagon-like peptide 1 receptor signaling influences topography of islet cells in mice.

Glucagon-like peptide 1 (GLP-1) amplifies glucose-induced insulin release in vivo and in vitro. Activation of GLP-1 receptor (GLP-1R) signaling leads to differentiation of exocrine cells towards a beta-cell phenotype in vitro and stimulation of islet cell proliferation in vitro and in vivo, suggesting a potential role for GLP-1 in the modulation of islet growth and differentiation. To determine whether basal levels of GLP-1R signaling are essential for islet development, we examined islet cell composition and topography in GLP-1R-/- mice. Total beta-cell volume and number are not altered, but the topography of beta cells is markedly different in GLP-1R-/- mice compared with GLP-1R+/+ controls. The distribution of beta cells is shifted from large to small and medium-sized islets in the absence of GLP-1R signaling (large islets: 50 +/- 3% in GLP-1R+/+ vs 28 +/- 4% in GLP- 1R-/-, P < 0.01 and medium islets: 32 +/- 2% in GLP- 1R+/+ vs 48 +/- 3% in GLP-1R-/-, P < 0.001). Furthermore, GLP-1R-/- islets exhibit abnormalities in cell topography, with two to threefold more centrally located alpha cells detected in GLP-1R-/- islets. These alterations in alpha- and beta-cell topography indicate that basal levels of GLP-1 signaling in the normal rodent are involved in the normal cellular organization of the endocrine pancreas.

Identification and characterization of a novel sucrose-non-fermenting protein kinase/AMP-activated protein kinase-related protein kinase, SNARK.

Subtraction hybridization after the exposure of keratinocytes to ultraviolet radiation identified a differentially expressed cDNA that encodes a protein of 630 amino acid residues possessing significant similarity to the catalytic domain of the sucrose-non-fermenting protein kinase (SNF1)/AMP-activated protein kinase (AMPK) family of serine/threonine protein kinases. Northern blotting and reverse-transcriptase-mediated PCR demonstrated that mRNA transcripts for the SNF1/AMPK-related kinase (SNARK) were widely expressed in rodent tissues. The SNARK gene was localized to human chromosome 1q32 by fluorescent in situ hybridization. SNARK was translated in vitro to yield a single protein band of approx. 76 kDa; Western analysis of transfected baby hamster kidney (BHK) cells detected two SNARK-immunoreactive bands of approx. 76-80 kDa. SNARK was capable of autophosphorylation in vitro; immunoprecipitated SNARK exhibited phosphotransferase activity with the synthetic peptide substrate HMRSAMSGLHLVKRR (SAMS) as a kinase substrate. SNARK activity was significantly increased by AMP and 5-amino-4-imidazolecarboxamide riboside (AICAriboside) in rat keratinocyte cells, implying that SNARK might be activated by an AMPK kinase-dependent pathway. Furthermore, glucose deprivation increased SNARK activity 3-fold in BHK fibroblasts. These findings identify SNARK as a glucose- and AICAriboside-regulated member of the AMPK-related gene family that represents a new candidate mediator of the cellular response to metabolic stress.