Physiology and pharmacology of the enteroendocrine hormone glucagon-like peptide-2.

Glucagon-like peptide-2 (GLP-2) is a 33-amino-acid proglucagon-derived peptide secreted from enteroendocrine L cells. GLP-2 circulates at low basal levels in the fasting period, and plasma levels rise rapidly after food ingestion. Renal clearance and enzymatic inactivation control the elimination of bioactive GLP-2. GLP-2 increases mesenteric blood flow and activates proabsorptive pathways in the gut, facilitating nutrient absorption. GLP-2 also enhances gut barrier function and induces proliferative and cytoprotective pathways in the small bowel. The actions of GLP-2 are transduced via a single G protein-coupled receptor (GLP-2R), expressed predominantly within the gastrointestinal tract. Disruption of GLP-2R signaling increases susceptibility to gut injury and impairs the adaptive mucosal response to refeeding. Sustained augmentation of GLP-2R signaling reduces the requirement for parenteral nutrition in human subjects with short-bowel syndrome. Hence GLP-2 integrates nutrient-derived signals to optimize mucosal integrity and energy absorption.

Functional consequences of glucagon-like peptide-1 receptor cross-talk and trafficking.

The signaling capacity of seven-transmembrane/G-protein-coupled receptors (7TM/GPCRs) can be regulated through ligand-mediated receptor trafficking. Classically, the recycling of internalized receptors is associated with resensitization, whereas receptor degradation terminates signaling. We have shown previously that the incretin glucagon-like peptide-1 receptor (GLP-1R) internalizes fast and is primarily resensitized through recycling back to the cell surface. GLP-1R is expressed in pancreatic islets together with the closely related glucose-dependent insulinotropic polypeptide (GIPR) and glucagon (GCGR) receptors. The interaction and cross-talk between coexpressed receptors is a wide phenomenon of the 7TM/GPCR superfamily. Numerous reports show functional consequences for signaling and trafficking of the involved receptors. On the basis of the high structural similarity and tissue coexpression, we here investigated the potential cross-talk between GLP-1R and GIPR or GCGR in both trafficking and signaling pathways. Using a real-time time-resolved FRET-based internalization assay, we show that GLP-1R, GIPR, and GCGR internalize with differential properties. Remarkably, upon coexpression of the internalizing GLP-1R and the non-internalizing GIPR, GLP-1-mediated GLP-1R internalization was impaired in a GIPR concentration-dependent manner. As a functional consequence of such impaired internalization capability, GLP-1-mediated GLP-1R signaling was abrogated. A similar compromised signaling was found when GLP-1R internalization was abrogated by a dominant-negative version of dynamin (dynamin-1 K44E), which provides a mechanistic link between GLP-1R trafficking and signaling. This study highlights the importance of receptor internalization for full functionality of GLP-1R. Moreover, cross-talk between the two incretin receptors GLP-1R and GIPR is shown to alter receptor trafficking with functional consequences for GLP-1R signaling.

Glucagon-like-peptide-1 receptor expression in normal and diseased human thyroid and pancreas.

Glucagon-like-peptide-1 (GLP1) analogs may induce thyroid or pancreatic diseases in animals, raising questions about their use in diabetic patients. There is, however, controversy regarding expression of GLP1 receptors (GLP1R) in human normal and diseased thyroid and pancreas. Here, 221 human thyroid and pancreas samples were analyzed for GLP1R immunohistochemistry and compared with quantitative in vitro GLP1R autoradiography. Neither normal nor hyperplastic human thyroids containing parafollicular C cells express GLP1R with either method. Papillary thyroid cancer do not, and medullary thyroid carcinomas rarely express GLP1R. Insulin- and somatostatin-producing cells in the normal pancreas express a high density of GLP1R, whereas acinar cells express them in low amounts. Ductal epithelial cells do not express GLP1R. All benign insulinomas express high densities of GLP1R, whereas malignant insulinomas rarely express them. All ductal pancreatic carcinomas are GLP1R negative, whereas 6/20 PanIN 1/2 and 0/12 PanIN 3 express GLP1R. Therefore, normal thyroid, including normal and hyperplastic C cells, or papillary thyroid cancer are not targets for GLP1 analogs in humans. Conversely, all pancreatic insulin- and somatostatin-producing cells are physiological GLP1 targets, as well as most acini. As normal ductal epithelial cells or PanIN 3 or ductal pancreatic carcinomas do not express GLP1R, it seems unlikely that GLP1R is related to neoplastic transformation in pancreas. GLP1R-positive medullary thyroid carcinomas and all benign insulinomas are candidates for in vivo GLP1R targeting.

Neuroprotective and anti-apoptotic effects of liraglutide on SH-SY5Y cells exposed to methylglyoxal stress.

Glucagon-like peptide 1 (GLP-1) is a growth factor that has demonstrated neuroprotective properties in a range of studies. In an APPswe/PS1ΔE9 mouse model of Alzheimer's disease (AD), we previously found protective effects on memory formation, synaptic plasticity, synapse survival and a reduction of amyloid synthesis and plaque load in the brain. Here, we analyse the neuroprotective properties of the GLP-1 analogue liraglutide in human neuroblastoma cell line SH-SY5Y during methyl glyoxal stress. We show for the first time that cell viability was enhanced by liraglutide (XTT assay) in a dose-dependent way, while cytotoxicity (LDH assay) and apoptosis were reduced. Expression of the pro-survival Mcl1 signaling protein was increased, as was activation of cell survival kinases Akt, MEK1/2 and the transcription factor p90RSK. Liraglutide also decreased pro-apoptotic Bax and Bik expression. In addition, the membrane potential and the influx of calcium into the cell were enhanced by liraglutide. GLP-1 receptor expression was also increased by the drug. The results demonstrate a range of growth factor-related cytoprotective processes induced by liraglutide, which is currently on the market as a treatment for type 2 diabetes (Victoza®). It is also tested in clinical trials in patients with Alzheimer disease.

Stimulation of intestinal growth and function with DPP4 inhibition in a mouse short bowel syndrome model.

Glucagon-like peptide-2 (GLP-2) has been shown to be effective in patients with short bowel syndrome (SBS), but it is rapidly inactivated by dipeptidyl peptidase IV (DPP4). We used an orally active DPP4 inhibitor (DPP4-I), MK-0626, to determine the efficacy of this approach to promote adaptation after SBS, determined optimal dosing, and identified further functional actions in a mouse model of SBS. Ten-week-old mice underwent a 50% proximal small bowel resection. Dose optimization was determined over a 3-day post-small bowel resection period. The established optimal dose was given for 7, 30, and 90 days and for 7 days followed by a 23-day washout period. Adaptive response was assessed by morphology, intestinal epithelial cell (IEC) proliferation (proliferating cell nuclear antigen), epithelial barrier function (transepithelial resistance), RT-PCR for intestinal transport proteins and GLP-2 receptor, IGF type 1 receptor, and GLP-2 plasma levels. Glucose-stimulated sodium transport was assessed for intestinal absorptive function. Seven days of DPP4-I treatment facilitated an increase in GLP-2 receptor levels, intestinal growth, and IEC proliferation. Treatment led to differential effects over time, with greater absorptive function at early time points and enhanced proliferation at later time points. Interestingly, adaptation continued in the group treated for 7 days followed by a 23-day washout. DPP4-I enhanced IEC proliferative action up to 90 days postresection, but this action seemed to peak by 30 days, as did GLP-2 plasma levels. Thus DPP4-I treatment may prove to be a viable option for accelerating intestinal adaptation with SBS.

Fluorescent exendin-4 derivatives for pancreatic ß-cell analysis.

The ability to reliably identify pancreatic ß-cells would have far reaching implications for a greater understanding of ß-cell biology, measurement of ß-cell mass in diabetes, islet transplantation, and drug development. The glucagon-like peptide-1 receptor (GLP1R) is highly expressed on the surface of insulin producing pancreatic ß-cells. Using systematic modifications of the GLP1R ligand, exendin-4, we screened over 25 compounds and identified a palette of fluorescent exendin-4 with high GLP1R binding affinity. We show considerable differences in affinity, as well as utility of the top candidates for flow cytometry and microscopy of ß-cells. Some of the developed compounds should be particularly useful for basic and translational ß-cell research.

Anti-inflammatory effect of exendin-4 postconditioning during myocardial ischemia and reperfusion.

High mobility group box 1 protein (HMGB1) plays an important role in myocardial ischemia and reperfusion (I/R) injury. Preconditioning of exendin-4 (Ex), a glucagon-like peptide-1 receptor agonist, has been reported to attenuate myocardial I/R injury. The current study investigated whether Ex postconditioning also attenuated myocardial I/R injury and the potential mechanisms. Anesthetized male rats were subjected to ischemia for 30 min and treated with Ex (5 µg/kg, i.v.) 5 min before reperfusion, in the absence and/or presence of exendin (9-39) (an antagonist of glucagon-like peptide-1 receptor, 5 µg/kg, i.v.), followed by reperfusion for 4 h. Lactate dehydrogenase (LDH), creatine kinase (CK), tumor necrosis factor-α, interleukin-6, and infarct size were measured. HMGB1 expression was assessed by immunoblotting. Postconditioning with Ex significantly decreased infarct size and levels of LDH and CK after 4 h reperfusion (all p < 0.05). Ex also significantly inhibited the increase in malondialdehyde level and decreased the level of superoxide dismutase (both p < 0.05). In addition, the increase in HMGB1 expression induced by I/R was significantly attenuated by Ex postconditioning. Administration of exendin (9-39) abolished the protective effect of Ex postconditioning (all p < 0.05). The present study suggests that Ex postconditioning may attenuate myocardial I/R injury, which may in turn be associated with inhibiting inflammation.

DPP-4 inhibitors repress NLRP3 inflammasome and interleukin-1beta via GLP-1 receptor in macrophages through protein kinase C pathway.

BACKGROUND: Anti-atherosclerotic effects of dipeptidyl peptidase-4 (DPP-4) inhibitors have been shown in many studies. Since inflammation and immune response play a key role in atherogenesis, we examined the effect of DPP-4 inhibitors on the expression of nod-like receptor family, pyrin domain containing 3 (NLRP3) Inflammasome and Interleukin-1beta (IL-1ß) in human macrophages. METHODS AND RESULTS: THP-1 macrophages were incubated with oxidized low density lipoprotein (ox-LDL) with or without DPP-4 inhibitors (sitagliptin and NVPDPP728). The effects of DPP-4 inhibitors on the expression of NLRP3, toll-like receptor 4 (TLR4) and pro-inflammatory cytokine IL-1ß were studied. Both DPP-4 inhibitors induced a significant reduction in NLRP3, TLR4 and IL-1ß expression; concurrently, there was an increase in glucagon like peptide 1 receptor (GLP-1R) expression. Simultaneously, DPP-4 inhibitors reduced phosphorylated-PKC, but not PKA, levels. To determine the role of PKC activation in the effects of DPP-4 inhibitors, cells were treated with PMA- which blocked the effect of DPP-4 inhibitors on NLRP3 and IL-1ß as well as TLR4 and GLP-1R. Over-expression of GLP-1R in macrophages with its agonist liraglutide also blocked the effects of PMA. CONCLUSION: DPP-4 inhibitors suppress NLRP3, TLR4 and IL-1ß in human macrophages through inhibition of PKC activity. This study provides novel insights into the mechanism of inhibition of inflammatory state and immune response in atherosclerosis by DPP-4 inhibitors.

Ectopic expression of glucagon receptor in skeletal muscles improves glucose homeostasis in a mouse model of diabetes.

AIMS/HYPOTHESIS: Excessive secretion of glucagon partially contributes to the development of diabetic hyperglycaemia. However, complete blocking of glucagon action will lead to adverse effects, since glucagon exerts certain beneficial effects via its receptor in many organs. We aimed to study the effects of a 'decoy receptor' for circulating glucagon on modulating beta cell function and glucose homeostasis in mice by over-producing the glucagon receptor (GCGR) in skeletal muscles. METHODS: We generated transgenic mice in which the expression of Gcgr is driven by the muscle specific creatine kinase (Mck) promoter, and assessed the effects of glucagon on the modulation of glucose homeostasis under conditions of extremes of glucose influx or efflux. RESULTS: Mck/Gcgr mice showed increased circulating levels of glucagon and insulin, resulting in an unchanged ratio of glucagon-to-insulin. The levels of hepatic glucose-6-phosphatase (G6PC) and fructose-1,6-bisphosphatase (F1,6P2ase) were significantly decreased, whereas the phosphorylation level of pancreatic cAMP-response-element-binding-protein (CREB) was significantly increased in these transgenic mice. Under basal conditions, the mice displayed normal blood glucose levels and unchanged glucose tolerance and insulin sensitivity when compared with their age-matched wild-type (WT) littermates. However, following multiple low-dose streptozotocin injections, Mck/Gcgr mice exhibited a delay in the onset of hyperglycaemia compared with the WT controls. This was associated with preserved beta cell mass and beta cell secretory capacity in response to glucose challenge. CONCLUSIONS/INTERPRETATION: We suggest that mild and chronic hyperglucagonaemia, through a strategy involving neutralising peripheral glucagon action, provides beneficial effects on beta cell function and glucose homeostasis. Mck/Gcgr mice thus represent a novel mouse model for studying the physiological effects of glucagon.

GLP-2 receptor in POMC neurons suppresses feeding behavior and gastric motility.

Glucagon-like peptides (GLP-1/2) are cosecreted from endocrine L cells in the gut and preproglucagonergic neurons in the brain. Peripheral GLP-2 action is essential for maintaining intestinal homeostasis, improving absorption efficiency and blood flow, promoting immune defense, and producing efficacy in treatment of gastrointestinal diseases. However, it is unknown if CNS GLP-2 plays a physiological role in the control of energy homeostasis. Since GLP-1/2 are cotranslated from preproglucagongene and coproduced by prohormone convertase-1, it is challenging to knockout GLP-2 only. Instead, our laboratory has generated a Glp2r-floxed mouse line to dissect cell-specific GLP-2 receptor GLP-2R) action in the regulation of energy balance. Our objective was to determine if GLP-2R in the hypothalamus modulates feeding behavior and gastric emptying. We show that Glp2r mRNA and protein are highly expressed in the arcuate nucleus and dorsomedial nucleus of the mouse hypothalamus. Using the Cre-LoxP system, we generated mice that lack Glp2r expression in POMC neurons (KO; mainly in the hypothalamus). The KO mice showed hyperphagic behavior (such as increases in food intake and meal frequency), accelerated gastric emptying (assessed by [(13)C]octanoic acid breath test), and late-onset obesity, yet there was no decrease in basal metabolic rate. Infusion of GLP-2 (2.5 nmol into the 4th ventricle) suppressed food intake and gastric emptying, while GLP-2-mediated effects were abolished in the melanocortin receptor-4 (MC4R) KO mice. We conclude that Glp2r deletion in POMC neurons enhances feeding behavior and gastric motility, whereas icv GLP-2R activation suppresses food intake and gastric emptying through the MC4R signaling pathway. This study indicates that CNS GLP-2R plays a physiological role in the control of feeding behavior and gastric emptying and that this is mediated probably through the melanocortin system.

Two small molecule agonists of glucagon-like peptide-1 receptor modulate the receptor activation response differently.

The glucagon-like peptide-1 receptor (GLP-1R) is a target for type 2 diabetes treatment. Due to the inconvenience of peptide therapeutics, small-molecule GLP-1R agonists have been studied. Compound 2 (6,7-dichloro-2-methylsulfonyl-2-N-tert-butylaminoquinoxaline) and compound B (4-(3-(benzyloxy)phenyl)-2-(ethylsulfinyl)-6-(trifluoromethyl)pyrimidine) have been described as small molecule, ago-allosteric modulators of GLP-1R. However, their modes of action at the GLP-1R have not been elucidated. Thus, in this study, we compared the mechanisms of action between these two compounds. When compound 2 was treated with endogenous or exogenous peptide agonists (GLP-1 and exenatide) or fragments of peptide agonists (GLP-1(9-36), Ex3, Ex4, and Ex5), the response curve of these peptide agonists shifted left without a change in maximum efficacy. In contrast, compound B potentiated the response and increased maximum efficacy. However, N-terminal truncated orthosteric antagonists including Ex7, Ex9, and Ex10, augmented the response of compound 2 at the GLP-1R but did not alter compound B activity. Intriguingly, when we co-treated compound 2 with compound B in CHO cells expressing full-length hGLP-1R or N-terminal extracellular domain-truncated GLP-1R, the activation of both types of receptors increased additively, implying that the N-terminus of the receptor is not involved in the modulation by compound agonists. We confirmed that these two compounds increased calcium influx by different patterns in CHO cells expressing GLP-1R. Taken together, our findings suggest that compounds 2 and B have different modes of action to activate GLP-1R. Further study to identify the putative binding sites will help in the discovery of orally available GLP-1R agonists.

Oleic acid and glucose regulate glucagon-like peptide 1 receptor expression in a rat pancreatic ductal cell line.

The glucagon-like peptide 1 receptor (GLP1R) plays a critical role in glucose metabolism and has become an important target for a growing class of drugs designed to treat type 2 diabetes. In vitro studies were designed to investigate the effect of the GLP1R agonist, exenatide (Ex4), in "on-target" RIN-5mF (islet) cells as well as in "off-target" AR42J (acinar) and DSL-6A/C1 (ductal) cells in a diabetic environment. Ex4 increased islet cell proliferation but did not affect acinar cells or ductal cells at relevant concentrations. A high caloric, high fat diet is a risk factor for impaired glucose tolerance and type-2 diabetes. An in vitro Oleic acid (OA) model was used to investigate the effect of Ex4 in a high calorie, high fat environment. At 0.1 and 0.4mM, OA mildly decreased the proliferation of all pancreatic cell types. Ex4 did not potentiate the inhibitory effect of OA on cell proliferation. Akt phosphorylation in response to Ex4 was diminished in OA-treated ductal cells. GLP1R protein detected by western blot was time and concentration dependently decreased after glucose stimulation in OA-treated ductal cells. In ductal cells, OA treatment altered the intracellular localization of GLP1R and its co-localization with early endosome and recycling endosomes. Chloroquine (lysosomal inhibitor), N-acetyl-l-cysteine (reactive oxygen species scavenger) and wortmannin (a phosphatidylinositol-3-kinase inhibitor), fully or partially, rescued GLP1R protein in OA-pretreated, glucose-stimulated ductal cells. The impact of altered regulation on phenotype/function is presently unknown. However, these data suggest that GLP1R regulation in ductal cells can be altered by a high fat, high calorie environment.

Glucose exposure pattern determines glucagon-like peptide 1 receptor expression and signaling through endoplasmic reticulum stress in rat insulinoma cells.

Repeated fluctuation in plasma glucose levels, as well as chronic hyperglycemia, is an important phenomenon frequently observed in diabetic patients. Recently, several studies have reported that glucose fluctuation, compared to chronic hyperglycemia, mediates more adverse effects due to induced oxidative and/or endoplasmic reticulum (ER) stress. In type 2 diabetes, stimulation of insulin secretion by glucagon-like peptide-1 (GLP-1) has been found to be reduced, and the results of recent studies have shown that the expression of the GLP-1 receptor (GLP-1R) is reduced by chronic hyperglycemia. However, GLP-1R signaling in glucose fluctuation has not been elucidated clearly. In this study, we hypothesized that intermittent high glucose (IHG) conditions also reduced GLP-1-mediated cellular signaling via reduction in GLP-1R expression. To evaluate this hypothesis, rat insulinoma cells (INS-1) were exposed for 72 h to either sustained high glucose (SHG) conditions (30 mM glucose) or IHG conditions (11 and 30 mM glucose, alternating every 12h). In comparison to both the SHG and control groups, IHG conditions induced a more significant impairment of insulin release and calcium influx in response to 1nM GLP-1 treatment. In addition, the activity of caspase 3/7 as well as the gene expression of binding protein (Bip) and C/EBP homologous protein (CHOP), molecular markers of ER stress, was significantly higher in IHG-treated cells than in SHG-treated cells. Interestingly, the expression level of GLP-1R was significantly lower under IHG conditions than under SHG conditions. Collectively, these findings indicated that glucose fluctuation reduces GLP-1R expression through ER stress more profoundly than sustained hyperglycemia, which may contribute to the diminished response of GLP-1.

Receptors and effects of gut hormones in three osteoblastic cell lines.

BACKGROUND: In recent years the interest on the relationship of gut hormones to bone processes has increased and represents one of the most interesting aspects in skeletal research. The proportion of bone mass to soft tissue is a relationship that seems to be controlled by delicate and subtle regulations that imply "cross-talks" between the nutrient intake and tissues like fat. Thus, recognition of the mechanisms that integrate a gastrointestinal-fat-bone axis and its application to several aspects of human health is vital for improving treatments related to bone diseases. This work analysed the effects of gut hormones in cell cultures of three osteoblastic cell lines which represent different stages in osteoblastic development. Also, this is the first time that there is a report on the direct effects of glucagon-like peptide 2, and obestatin on osteoblast-like cells. METHODS: mRNA expression levels of five gut hormone receptors (glucose-dependent insulinotropic peptide [GIP], glucagon-like peptide 1 [GLP-1], glucagon-like peptide 2 [GLP-2], ghrelin [GHR] and obestatin [OB]) were analysed in three osteoblastic cell lines (Saos-2, TE-85 and MG-63) showing different stages of osteoblast development using reverse transcription and real time polymerase chain reaction. The responses to the gut peptides were studied using assays for cell viability, and biochemical bone markers: alkaline phosphatase (ALP), procollagen type 1 amino-terminal propeptides (P1NP), and osteocalcin production. RESULTS: The gut hormone receptor mRNA displayed the highest levels for GIP in Saos-2 and the lowest levels in MG-63, whereas GHR and GPR39 (the putative obestatin receptor) expression was higher in TE-85 and MG-63 and lower in Saos-2. GLP-1 and GLP-2 were expressed only in MG-63 and TE-85. Treatment of gut hormones to cell lines showed differential responses: higher levels in cell viability in Saos-2 after GIP, in TE-85 and MG-63 after GLP-1, GLP-2, ghrelin and obestatin. ALP showed higher levels in Saos-2 after GIP, GHR and OB and in TE-85 after GHR. P1NP showed higher levels after GIP and OB in Saos-2. Decreased levels of P1NP were observed in TE-85 and MG-63 after GLP-1, GLP-2 and OB. MG-63 showed opposite responses in osteocalcin levels after GLP-2. CONCLUSIONS: These results suggest that osteoblast activity modulation varies according to different development stage under different nutrition related-peptides.

Pharmacological characterization of human incretin receptor missense variants.

Glucagon-like peptide 1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) are gut-derived incretin hormones that regulate blood glucose levels. In addition to their widely accepted insulinotropic role, there is evidence that GLP-1 modulates feeding behavior and GIP regulates lipid metabolism, thereby promoting postprandial fat deposition. In this study, we investigated whether naturally occurring polymorphisms in the GLP-1 receptor (GLP-1R) and the GIP receptor (GIP-R) affect the pharmacological properties of these proteins. After transient expression of the receptors in human embryonic kidney 293 cells, basal and ligand-induced cAMP production were assessed by use of luciferase reporter gene assays. Our data reveal that the wild-type GIP-R displays a considerable degree of ligand-independent activity. In comparison, the GIP-R variants C46S, G198C, R316L, and E354Q show a marked decrease in basal signaling that may, at least in part, be explained by reduced cell surface expression. When stimulated with GIP, the C46S and R316L mutants display significantly reduced potency (>1000 and 25- fold, respectively) compared with wild type. Complementary competition binding assays further demonstrate that the C46S variant fails to bind radio-iodinated GIP, whereas all other GIP-R mutants maintain normal ligand affinity. In contrast to the GIP-R, the wild-type GLP-1R lacks constitutive activity. Furthermore, none of the 10 GLP-1R missense mutations showed an alteration in pharmacological properties versus wild type. The extent to which abnormalities in GIP-R function may lead to physiological changes or affect drug sensitivity in selected populations (e.g., obese, diabetic individuals) remains to be further investigated.

Comparative effects of the endogenous agonist glucagon-like peptide-1 (GLP-1)-(7-36) amide and the small-molecule ago-allosteric agent "compound 2" at the GLP-1 receptor.

Glucagon-like peptide-1 (GLP-1) mediates antidiabetogenic effects through the GLP-1 receptor (GLP-1R), which is targeted for the treatment of type 2 diabetes. Small-molecule GLP-1R agonists have been sought due to difficulties with peptide therapeutics. Recently, 6,7-dichloro-2-methylsulfonyl-3-N-tert-butylaminoquinoxaline (compound 2) has been described as a GLP-1R allosteric modulator and agonist. Using human embryonic kidney-293 cells expressing human GLP-1Rs, we extended this work to consider the impact of compound 2 on G protein activation, Ca(2+) signaling and receptor internalization and particularly to compare compound 2 and GLP-1 across a range of functional assays in intact cells. GLP-1 and compound 2 activated Galpha(s) in cell membranes and increased cellular cAMP in intact cells, with compound 2 being a partial and almost full agonist, respectively. GLP-1 increased intracellular [Ca(2+)] by release from intracellular stores, which was mimicked by compound 2, with slower kinetics. In either intact cells or membranes, the orthosteric antagonist exendin-(9-39), inhibited GLP-1 cAMP generation but increased the efficacy of compound 2. GLP-1 internalized enhanced green fluorescent protein-tagged GLP-1Rs, but the speed and magnitude evoked by compound 2 were less. Exendin-(9-39) inhibited internalization by GLP-1 and also surprisingly that by compound 2. Compound 2 displays GLP-1R agonism consistent with action at an allosteric site, although an orthosteric antagonist increased its efficacy on cAMP and blocked compound 2-mediated receptor internalization. Full assessment of the properties of compound 2 was potentially hampered by damaging effects that were particularly manifest in either longer term assays with intact cells or in acute assays with membranes.

Glucagon receptor signaling is essential for control of murine hepatocyte survival.

BACKGROUND & AIMS: Glucagon action in the liver is essential for control of glucose homeostasis and the counterregulatory response to hypoglycemia. Because receptors for the related peptides glucagon-like peptide-1 and glucagon-like peptide-2 regulate beta-cell and enterocyte apoptosis, respectively, we examined whether glucagon receptor (Gcgr) signaling modulates hepatocyte survival. METHODS: The importance of the Gcgr for hepatocyte cell survival was examined using Gcgr+/+ and Gcgr-/- mice in vivo, and murine hepatocyte cultures in vitro. RESULTS: Gcgr-/- mice showed enhanced susceptibility to experimental liver injury induced by either Fas Ligord activation or a methionine- and choline-deficient diet. Restoration of hepatic Gcgr expression in Gcgr-/- mice attenuated the development of hepatocellular injury. Furthermore, exogenous glucagon administration reduced Jo2-induced apoptosis in wild-type mice and decreased caspase activation in fibroblasts expressing a heterologous Gcgr and in primary murine hepatocyte cultures. The anti-apoptotic actions of glucagon were independent of protein kinase A, phosphatidylinositol-3K, and mitogen-activated protein kinase, and were mimicked by the exchange protein directly activated by the cyclic AMP agonist 8-(4-chloro-phenylthio)-2'-O-methyladenosine-3', 5'-cyclic monophosphate-cAMP. CONCLUSIONS: These findings extend the essential actions of the Gcgr beyond the metabolic control of glucose homeostasis to encompass the regulation of hepatocyte survival.

An albumin-exendin-4 conjugate engages central and peripheral circuits regulating murine energy and glucose homeostasis.

BACKGROUND & AIMS: Glucagon-like peptide-1 (GLP-1) regulates glucose homeostasis through multiple mechanisms including direct actions on the endocrine pancreas and indirect activation of central nervous system circuits regulating gastric emptying, satiety, and body weight. Because native GLP-1 is rapidly degraded, there is considerable interest in development of more potent GLP-1 receptor (GLP-1R) agonists with sustained activity; however, the extent to which much larger GLP-1R agonists will mimic some or all of the actions of smaller peptides remains uncertain. METHODS: We studied the actions of CJC-1134-PC, a recombinant human serum albumin-exendin-4 conjugated protein, at the GLP-1R using heterologous cells expressing the GLP-1R in vitro and both wild-type and Glp1r(-/-) mice in vivo. RESULTS: CJC-1134-PC activated GLP-1R-dependent signaling in baby hamster kidney-GLP-1R cells and acutely lowered blood glucose in wild-type but not in Glp1r(-/-) mice. Moreover, acute administration of CJC-1134-PC rapidly activated c-Fos expression in multiple regions of the central nervous system, acutely inhibited gastric emptying, and produced sustained inhibition of food intake in a GLP-1R-dependent manner. Furthermore, chronic daily treatment of high-fat diet-fed wild-type mice with CJC-1134-PC for 4 weeks led to improved glucose tolerance, increased levels of glucose-stimulated insulin, decreased HbA1c, and weight loss associated with decreased hepatic triglyceride content. CONCLUSIONS: These findings illustrate that a high-molecular-weight exendin-4-albumin conjugate retains the ability to mimic a full spectrum of GLP-1R-dependent actions, including activation of central nervous system circuits regulating gastric emptying, food intake, and body weight.

Exendin-4 has an anti-hypertensive effect in salt-sensitive mice model.

The improvement of salt-sensitive hypertension is a therapeutic target for various vascular diseases. Glucagon-like peptide 1 (GLP-1), an incretin peptide, has been reported to have natriuretic effect as well as blood glucose lowering effect, although its exact mechanism and clinical usefulness remain unclear. Here, we examined anti-hypertensive effect of exendin-4, a GLP-1 analog, in salt-sensitive obese db/db mice and angiotensin II (angII)-infused C57BLK6/J mice. The treatment of exendin-4 for 12 weeks inhibited the development of hypertension in db/db mice. In db/db mice, the urinary sodium excretion was delayed and blood pressure was elevated in response to a high-salt load, whereas these were attenuated by exendin-4. In db/db mice, intra-renal angII concentration was increased. Furthermore, exendin-4 prevented angII-induced hypertension in non-diabetic mice and inhibited angII-induced phosphorylation of ERK1/2 in cultured renal cells. Considered together, our results indicate that exendin-4 has anti-hypertensive effects through the attenuation of angII-induced high-salt sensitivity.

Expression of the GLP-1 receptor in mouse, rat, and human pancreas.

We studied the intra-islet localization of the glucagon-like peptide 1 receptor (GLP-1R) by colocalization studies of the GLP-1R mRNA and protein with islet cell hormones in mice, rats, and humans. In contrast to previous reports, we show that the GLP-1R is selectively located on the beta cells. The localization of GLP-1R in islets and ducts was studied using ISH and double and triple fluorescence microscopy. In normal pancreatic tissue from mice and rats, GLP-1R mRNA was only detectable in the beta cells. Double and triple immunofluorescence using two different GLP-1R antisera and combinations of insulin, glucagon, pancreatic polypeptide, and somatostatin showed that GLP-1R protein is almost exclusively colocalized with insulin. The same pattern was observed in human pancreas, but the GLP-1R expression was more heterogeneous, with populations of insulin immunoreactive cells with high and low expression. This is the first time that the GLP-1R has been localized in human islets. Furthermore, GLP-1R immunoreactivity was found in the pancreatic ducts in mouse, rat, and human pancreas. As an important confirmation of the specificity of our methods, we found no signals for GLP-1R mRNA or protein in pancreatic tissue from gene-targeted GLP-1R-deficient mice. In conclusion, our data suggest that the GLP-1 receptor is restricted to the pancreatic beta cells and the lack of receptor immunoreactivity on delta cells cannot be explained suitably to correspond with published in vivo and in vitro data. This manuscript contains online supplemental material at http://www.jhc.org. Please visit this article online to view these materials.