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Protein intake potently increases body temperature and energy expenditure, but the underlying mechanism thereof remains incompletely understood. Simultaneously, protein intake potently stimulates glucagon-like peptide-1 (GLP-1) secretion. Here, we examined the involvement of GLP-1 in the thermic effects of dietary proteins in rodents by measuring rectal temperature and energy expenditure and modulating GLP-1 signaling. Rectal temperature of rats or mice fasted for 4 or 5 hours were measured using a thermocouple thermometer before and after an oral administration of nutrients. Oxygen consumption after oral protein administration was also measured in rats. Rectal temperature measurements in rats confirmed an increase in core body temperature after refeeding, and the thermic effect of the oral administration of protein was greater than that of a representative carbohydrate or lipid. Among the five dietary proteins examined (casein, whey, rice, egg, and soy), soy protein had the highest thermic effect. The thermic effect of soy protein was also demonstrated by increased oxygen consumption. Studies using a nonselective β-adrenergic receptor antagonist and thermal camera suggested that brown adipose tissue did not contribute to soy protein-induced increase in rectal temperature. Furthermore, the thermic effect of soy protein was completely abolished by antagonism and knockout of the GLP-1 receptor, yet potentiated via augmentation of intact GLP-1 levels through inhibition of dipeptidyl peptidase-4 activity. These results indicate that GLP-1 signaling is essential for the thermic effects of dietary proteins in rats and mice, and extend the metabolic actions of GLP-1 ensuing from nutrient ingestion to encompass the thermic response to ingested protein., (© The Author(s) 2023. Published by Oxford University Press on behalf of the Endocrine Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Glucagon-like peptide-1 (GLP-1) controls islet hormone secretion, gut motility, and body weight, supporting development of GLP-1 receptor agonists (GLP-1RA) for the treatment of type 2 diabetes (T2D) and obesity. GLP-1RA exhibit a favorable safety profile and reduce the incidence of major adverse cardiovascular events in people with T2D. Considerable preclinical data, supported by the results of clinical trials, link therapy with GLP-RA to reduction of hepatic inflammation, steatosis, and fibrosis. Mechanistically, the actions of GLP-1 on the liver are primarily indirect, as hepatocytes, Kupffer cells, and stellate cells do not express the canonical GLP-1R. GLP-1RA reduce appetite and body weight, decrease postprandial lipoprotein secretion, and attenuate systemic and tissue inflammation, actions that may contribute to attenuation of metabolic-associated fatty liver disease (MAFLD). Here we discuss evolving concepts of GLP-1 action that improve liver health and highlight evidence that links sustained GLP-1R activation in distinct cell types to control of hepatic glucose and lipid metabolism, and reduction of experimental and clinical nonalcoholic steatohepatitis (NASH). The therapeutic potential of GLP-1RA alone, or in combination with peptide agonists, or new small molecule therapeutics is discussed in the context of potential efficacy and safety. Ongoing trials in people with obesity will further clarify the safety of GLP-1RA, and pivotal studies underway in people with NASH will define whether GLP-1-based medicines represent effective and safe therapies for people with MAFLD., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Endocrine Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Glucagon-like peptide-1 (GLP-1) is produced in gut endocrine cells and in the brain, and acts through hormonal and neural pathways to regulate islet function, satiety, and gut motility, supporting development of GLP-1 receptor (GLP-1R) agonists for the treatment of diabetes and obesity. Classic notions of GLP-1 acting as a meal-stimulated hormone from the distal gut are challenged by data supporting production of GLP-1 in the endocrine pancreas, and by the importance of brain-derived GLP-1 in the control of neural activity. Moreover, attribution of direct vs indirect actions of GLP-1 is difficult, as many tissue and cellular targets of GLP-1 action do not exhibit robust or detectable GLP-1R expression. Furthermore, reliable detection of the GLP-1R is technically challenging, highly method dependent, and subject to misinterpretation. Here we revisit the actions of GLP-1, scrutinizing key concepts supporting gut vs extra-intestinal GLP-1 synthesis and secretion. We discuss new insights refining cellular localization of GLP-1R expression and integrate recent data to refine our understanding of how and where GLP-1 acts to control inflammation, cardiovascular function, islet hormone secretion, gastric emptying, appetite, and body weight. These findings update our knowledge of cell types and mechanisms linking endogenous vs pharmacological GLP-1 action to activation of the canonical GLP-1R, and the control of metabolic activity in multiple organs., (© The Author(s) 2020. Published by Oxford University Press on behalf of the Endocrine Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

A number of disease states, including type 2 diabetes (T2D), are associated with an increased risk of pulmonary infection. Glucagon-like peptide-1 (GLP-1) receptor agonists are used to treat T2D and exert anti-inflammatory actions through a single, well-defined GLP-1 receptor (GLP-1R). Although highly expressed in the lung, little is known about the role of the GLP-1R in the context of pulmonary inflammation. Here we examined the consequences of gain or loss of GLP-1R activity in infectious and noninfectious lung inflammation. We studied wild-type mice treated with a GLP-1R agonist, and Glp1r-/- mice, in the setting of bleomycin-induced noninfectious lung injury and influenza virus infection. Loss of the GLP-1R attenuated the severity of bleomycin-induced lung injury, whereas activation of GLP-1R signaling increased pulmonary inflammation via the sympathetic nervous system. In contrast, GLP-1R agonism reduced the pathogen load in mice with experimental influenza virus infection in association with increased expression of intracellular interferon-inducible GTPases. Notably, the GLP-1 receptor agonist liraglutide improved the survival rate after influenza virus infection. Our results reveal context-dependent roles for the GLP-1 system in the response to lung injury. Notably, the therapeutic response of GLP-1R agonism in the setting of experimental influenza virus infection may have relevance for ongoing studies of GLP-1R agonism in people with T2D susceptible to viral lung injury., (© The Author(s) 2020. Published by Oxford University Press on behalf of the Endocrine Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Individuals with diabetes are at increased risk for bacterial, mycotic, parasitic, and viral infections. The severe acute respiratory syndrome (SARS)-CoV-2 (also referred to as COVID-19) coronavirus pandemic highlights the importance of understanding shared disease pathophysiology potentially informing therapeutic choices in individuals with type 2 diabetes (T2D). Two coronavirus receptor proteins, angiotensin-converting enzyme 2 (ACE2) and dipeptidyl peptidase-4 (DPP4) are also established transducers of metabolic signals and pathways regulating inflammation, renal and cardiovascular physiology, and glucose homeostasis. Moreover, glucose-lowering agents such as the DPP4 inhibitors, widely used in subjects with T2D, are known to modify the biological activities of multiple immunomodulatory substrates. Here, we review the basic and clinical science spanning the intersections of diabetes, coronavirus infections, ACE2, and DPP4 biology, highlighting clinical relevance and evolving areas of uncertainty underlying the pathophysiology and treatment of T2D in the context of coronavirus infection., (© Endocrine Society 2020. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Proglucagon-derived peptides (PGDPs) and related gut hormones exemplified by glucose-dependent insulinotropic polypeptide (GIP) regulate energy disposal and storage through actions on metabolically sensitive organs, including adipose tissue. The actions of glucagon, glucagon-like peptide (GLP)-1, GLP-2, GIP, and their rate-limiting enzyme dipeptidyl peptidase-4, include direct and indirect regulation of islet hormone secretion, food intake, body weight, all contributing to control of white and brown adipose tissue activity. Moreover, agents mimicking actions of these peptides are in use for the therapy of metabolic disorders with disordered energy homeostasis such as diabetes, obesity, and intestinal failure. Here we highlight current concepts and mechanisms for direct and indirect actions of these peptides on adipose tissue depots. The available data highlight the importance of indirect peptide actions for control of adipose tissue biology, consistent with the very low level of endogenous peptide receptor expression within white and brown adipose tissue depots. Finally, we discuss limitations and challenges for the interpretation of available experimental observations, coupled to identification of enduring concepts supported by more robust evidence., (© Endocrine Society 2019. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Glucagon-like peptide-2 (GLP-2), secreted from enteroendocrine cells, attenuates gut motility, enhances barrier function, and augments nutrient absorption, actions mediated by a single GLP-2 receptor (GLP-2R). Despite extensive analyses, the precise distribution and cellular localization of GLP-2R expression remains controversial, confounded by the lack of suitable GLP-2R antisera. Here, we reassessed murine Glp2r expression using regular and real-time quantitative PCR (qPCR), in situ hybridization (ISH), and a Glp2rLacZ reporter mouse. Glp2r mRNA expression was detected from the stomach to the rectum and most abundant in the jejunum. Glp2r transcripts were also detected in cerebral cortex, mesenteric lymph nodes, gallbladder, urinary bladder, and mesenteric fat. Surprisingly, Glp2r mRNA was found in testis by qPCR at levels similar to jejunum. However, the testis Glp2r transcripts, detected by different primer pairs and qPCR, lacked 5' mRNA coding sequences, and only a minute proportion of them corresponded to full-length Glp2r mRNA. Within the gut, Glp2r-driven LacZ expression was localized to enteric neurons and lamina propria stromal cells, findings confirmed by ISH analysis of the endogenous Glp2r mRNA. Unexpectedly, vascular Glp2rLacZ expression was localized to mesenteric veins and not arteries. Moreover, mesenteric fat Glp2rLacZ expression was detected within blood vessels and not adipocytes. Reporter LacZ expression was not detected in all tissues expressing an endogenous Glp2r transcript, such as gallbladder, urinary bladder, and mesenteric lymph nodes. Collectively, these findings extend our understanding of the cellular domains of Glp2r expression and highlight limitations inherent in application of commonly used technologies to infer analysis of gene expression., (Copyright © 2019 Endocrine Society.)

Glucagonlike peptide-1 receptor (GLP-1R) agonists, which are used to treat type 2 diabetes and obesity, reduce the rates of myocardial infarction and cardiovascular death. GLP-1R has been localized to the human sinoatrial node; however, its expression in ventricular tissue remains uncertain. Here we studied GLP-1R expression in the human heart using GLP-1R-directed antisera, quantitative polymerase chain reaction (PCR), reverse transcription PCR to detect full-length messenger RNA (mRNA) transcripts, and in situ hybridization (ISH). GLP1R mRNA transcripts, encompassing the entire open reading frame, were detected in all four cardiac chambers from 15 hearts at levels approximating those detected in human pancreas. In contrast, cardiac GLP2R expression was relatively lower, and cardiac GCGR expression was sporadic and not detected in the left ventricle. GLP1R mRNA transcripts were not detected in RNA from human cardiac fibroblasts, coronary artery endothelial, or vascular smooth muscle cells. Human Brunner glands and pancreatic islets exhibited GLP-1R immunopositivity and abundant expression of GLP1R mRNA transcripts by ISH. GLP1R transcripts were also detected by ISH in human cardiac sinoatrial node tissue. However, definitive cellular localization of GLP1R mRNA transcripts or immunoreactive GLP-1R protein within human cardiomyocytes or cardiac blood vessels remained elusive. Moreover, validated GLP-1R antisera lacked sufficient sensitivity to detect expression of the endogenous islet or cardiac GLP-1R by Western blotting. Hence, although human cardiac ventricles express the GLP1R, the identity of one or more ventricular cell type(s) that express a translated GLP1R protein requires further clarification with highly sensitive methods of detection.

Regulatory peptides produced in islet and gut endocrine cells, including glucagon, glucagon-like peptide-1 (GLP-1), GLP-2, and glucose-dependent insulinotropic polypeptide, exert actions with considerable metabolic importance and translational relevance. Although the clinical development of GLP-1 receptor agonists and dipeptidyl peptidase-4 inhibitors has fostered research into how these hormones act on the normal and diseased heart, less is known about the actions of these peptides on blood vessels. Here we review the effects of these peptide hormones on normal blood vessels and highlight their vascular actions in the setting of experimental and clinical vascular injury. The cellular localization and signal transduction properties of the receptors for glucagon, GLP-1, GLP-2, and glucose-dependent insulinotropic polypeptide are discussed, with emphasis on endothelial cells and vascular smooth muscle cells. The actions of these peptides on the control of blood flow, blood pressure, angiogenesis, atherosclerosis, and vascular inflammation are reviewed with a focus on elucidating direct and indirect mechanisms of action. How these peptides traverse the blood-brain barrier is highlighted, with relevance to the use of GLP-1 receptor agonists to treat obesity and neurodegenerative disorders. Wherever possible, we compare actions identified in cell lines and primary cell culture with data from preclinical studies and, when available, results of human investigation, including studies in subjects with diabetes, obesity, and cardiovascular disease. Throughout the review, we discuss pitfalls, limitations, and challenges of the existing literature and highlight areas of controversy and uncertainty. The increasing use of peptide-based therapies for the treatment of diabetes and obesity underscores the importance of understanding the vascular biology of peptide hormone action.

Context: Classical enteroenteroendocrine cell (EEC) biology evolved historically from identification of scattered hormone-producing endocrine cells within the epithelial mucosa of the stomach, small and large intestine. Purification of functional EEC hormones from intestinal extracts, coupled with molecular cloning of cDNAs and genes expressed within EECs has greatly expanded the complexity of EEC endocrinology, with implications for understanding the contribution of EECs to disease pathophysiology., Evidence Acquisition: Pubmed searches identified manuscripts highlighting new concepts illuminating the molecular biology, classification and functional role(s) of EECs and their hormonal products., Evidence Synthesis: Molecular interrogation of EECs has been transformed over the past decade, raising multiple new questions that challenge historical concepts of EEC biology. Evidence for evolution of the EEC from a unihormonal cell type with classical endocrine actions, to a complex plurihormonal dynamic cell with pleiotropic interactive functional networks within the gastrointestinal mucosa is critically assessed. We discuss gaps in understanding how EECs sense and respond to nutrients, cytokines, toxins, pathogens, the microbiota, and the microbial metabolome, and highlight the expanding translational relevance of EECs in the pathophysiology and therapy of metabolic and inflammatory disorders., Conclusions: The EEC system represents the largest specialized endocrine network in human physiology, integrating environmental and nutrient cues, enabling neural and hormonal control of metabolic homeostasis. Updating EEC classification systems will enable more accurate comparative analyses of EEC subpopulations and endocrine networks in multiple regions of the gastrointestinal tract.

Dipeptidyl peptidase-4 (DPP4) is a widely expressed enzyme transducing actions through an anchored transmembrane molecule and a soluble circulating protein. Both membrane-associated and soluble DPP4 exert catalytic activity, cleaving proteins containing a position 2 alanine or proline. DPP4-mediated enzymatic cleavage alternatively inactivates peptides or generates new bioactive moieties that may exert competing or novel activities. The widespread use of selective DPP4 inhibitors for the treatment of type 2 diabetes has heightened interest in the molecular mechanisms through which DPP4 inhibitors exert their pleiotropic actions. Here we review the biology of DPP4 with a focus on: 1) identification of pharmacological vs physiological DPP4 substrates; and 2) elucidation of mechanisms of actions of DPP4 in studies employing genetic elimination or chemical reduction of DPP4 activity. We review data identifying the roles of key DPP4 substrates in transducing the glucoregulatory, anti-inflammatory, and cardiometabolic actions of DPP4 inhibitors in both preclinical and clinical studies. Finally, we highlight experimental pitfalls and technical challenges encountered in studies designed to understand the mechanisms of action and downstream targets activated by inhibition of DPP4.

Glucagon-like peptide-1 receptor (GLP-1R) agonists reduce lipid accumulation in peripheral tissues, attenuating atherosclerosis and hepatic steatosis in preclinical studies. We examined whether GLP-1R activation decreases atherosclerosis progression in high-fat diet-fed male ApoE(-/-) mice after administration of streptozotocin and treatment with the long-acting GLP-1R agonist taspoglutide administered once monthly vs. metformin in the drinking water for 12 wk. Taspoglutide did not reduce plaque area or lipid content in the aortic arch or abdominal aorta, and no significant change in aortic macrophage accumulation was detected after taspoglutide or metformin. In contrast, hepatic triglyceride levels were significantly reduced in livers from taspoglutide-treated mice. Both peripheral and intracerebroventricular administration of exendin-4 rapidly decreased plasma triglyceride levels in fasted mice, and taspoglutide therapy in ApoE(-/-) mice modulated the expression of hepatic genes controlling fatty acid uptake and oxidation. We were unable to detect expression of the entire Glp1r coding sequence in macrophages isolated from ApoE(-/-), C57BL/6, and IL10(-/-) mice. Similarly, Glp1r mRNA transcripts were not detected in RNA from isolated murine hepatocytes. Using Western blotting and tissue extracts from Glp1r(+/+) and Glp1r(-/-) mice, and cells transfected with a tagged murine GLP-1R cDNA, we could not validate the sensitivity and specificity of three different GLP-1R antisera commonly used for the detection of GLP-1R protein. Taken together, these findings illustrate divergent actions of GLP-1R agonists on atherosclerosis progression and accumulation of ectopic lipid in ApoE(-/-) mice and highlight the importance of indirect GLP-1R actions for the control of hepatic lipid accumulation.

The enteroendocrine and enteric nervous systems convey signals through an overlapping network of regulatory peptides that act either as circulating hormones or as localized neurotransmitters within the gastrointestinal tract. Because recent studies invoke an important role for vasoactive intestinal peptide (VIP) as a downstream mediator of glucagon-like peptide-2 (GLP-2) action in the gut, we examined the importance of the VIP-GLP-2 interaction through analysis of Vip(-/-) mice. Unexpectedly, we detected abnormal villous architecture, expansion of the crypt compartment, increased crypt cell proliferation, enhanced Igf1 and Kgf gene expression, and reduced expression of Paneth cell products in the Vip(-/-) small bowel. These abnormalities were not reproduced by antagonizing VIP action in wild-type mice, and VIP administration did not reverse the intestinal phenotype of Vip(-/-) mice. Exogenous administration of GLP-2 induced the expression of ErbB ligands and immediate-early genes to similar levels in Vip(+/+) vs. Vip(-/-) mice. Moreover, GLP-2 significantly increased crypt cell proliferation and small bowel growth to comparable levels in Vip(+/+) vs. Vip(-/-) mice. Unexpectedly, exogenous GLP-2 administration had no therapeutic effect in mice with dextran sulfate-induced colitis; the severity of colonic injury and weight loss was modestly reduced in female but not male Vip(-/-) mice. Taken together, these findings extend our understanding of the complex intestinal phenotype arising from loss of the Vip gene. Furthermore, although VIP action may be important for the antiinflammatory actions of GLP-2, the Vip gene is not required for induction of a gene expression program linked to small bowel growth after enhancement of GLP-2 receptor signaling.

Glucagon-like peptide-1 (GLP-1) is an incretin hormone that enhances glucose-stimulated insulin secretion and exerts direct and indirect actions on the cardiovascular system. GLP-1 and its related incretin hormone, glucose-dependent insulinotropic polypeptide, are rapidly inactivated by the enzyme dipeptidyl peptidase 4 (DPP-4), a key determinant of incretin bioactivity. Two classes of medications that enhance incretin action, GLP-1 receptor (GLP-1R) agonists and DPP-4 inhibitors, are used for the treatment of type 2 diabetes mellitus. We review herein the cardiovascular biology of GLP-1R agonists and DPP-4 inhibitors, including direct and indirect effects on cardiomyocytes, blood vessels, adipocytes, the control of blood pressure, and postprandial lipoprotein secretion. Both GLP-1R activation and DPP-4 inhibition exert multiple cardioprotective actions in preclinical models of cardiovascular dysfunction, and short-term studies in human subjects appear to demonstrate modest yet beneficial actions on cardiac function in subjects with ischemic heart disease. Incretin-based agents control body weight, improve glycemic control with a low risk of hypoglycemia, decrease blood pressure, inhibit the secretion of intestinal chylomicrons, and reduce inflammation in preclinical studies. Nevertheless, there is limited information on the cardiovascular actions of these agents in patients with diabetes and established cardiovascular disease. Hence, a more complete understanding of the cardiovascular risk to benefit ratio of incretin-based therapies will require completion of long-term cardiovascular outcome studies currently underway in patients with type 2 diabetes mellitus.

Exogenous glucagon-like peptide-2 receptor (GLP-2R) activation elicits proliferative and cytoprotective responses in the gastrointestinal mucosa and ameliorates experimental small and large bowel gut injury. Nevertheless, the essential physiological role(s) of the endogenous GLP-2R remain poorly understood. We studied the importance of the GLP-2R for gut growth, epithelial cell lineage allocation, the response to mucosal injury, and host-bacterial interactions in Glp2r(-/-) and littermate control Glp2r(+/+) mice. Glp2r(-/-) mice exhibit normal somatic growth and preserved small and large bowel responses to IGF-I and keratinocyte growth factor. However, Glp2r(-/-) mice failed to up-regulate intestinal epithelial c-fos expression in response to acute GLP-2 administration and do not exhibit changes in small bowel conductance or small or large bowel growth after administration of GLP-2R agonists. The crypt and villus compartment and the numbers and localization of Paneth, enteroendocrine, and goblet cells were comparable in Glp2r(+/+) vs. Glp2r(-/-) mice. Although the severity and extent of colonic mucosal injury in response to 3% oral dextran sulfate was similar across Glp2r genotypes, Glp2r(-/-) mice exhibited significantly increased morbidity and mortality and increased bacterial translocation after induction of enteritis with indomethacin and enhanced mucosal injury in response to irinotecan. Moreover, bacterial colonization of the small bowel was significantly increased, expression of Paneth cell antimicrobial gene products was reduced, and mucosal bactericidal activity was impaired in Glp2r(-/-) mice. Although the Glp2r is dispensable for gut development and the response to colonic injury, Glp2r(-/-) mice exhibit enhanced sensitivity to small bowel injury, and abnormal host-bacterial interactions in the small bowel.

Obesity, accompanying or independent of type 2 diabetes mellitus (T2DM), is associated with higher rates of malignancy. Hence, there is considerable interest in understanding whether therapies used to treat obese patients with T2DM impact cancer cell growth. Glucagon-like peptide-1 (GLP-1) is produced in enteroendocrine cells and secreted after meal ingestion. GLP-1 regulates blood glucose through multiple mechanisms, principally inhibition of glucagon and stimulation of insulin secretion. GLP-1 also exerts independent effects promoting cell growth and survival, and sustained activation of GLP-1 receptor (GLP-1R) signaling in rodent thyroid glands leads to C-cell hyperplasia and medullary thyroid cancer. Hence, whether therapies based on GLP-1R activation modify growth or survival of cancer cells is of ongoing interest. We studied the biological actions of GLP-1 in mouse CT26 colon cancer cells that express a functional GLP-1R. The GLP-1R agonist exendin (Ex)-4 (exenatide) increased intracellular cAMP levels and inhibited the activity of signaling kinases glycogen synthase kinase 3 and ERK1/2 in CT26 cells. The Ex-4-induced inactivation of glycogen synthase kinase 3, but not ERK1/2, was dependent on protein kinase A and blocked by the GLP-1R antagonist Ex(9-39). Furthermore, Ex-4 altered cell morphology, induced apoptosis, and inhibited proliferation of CT26 cells in vitro. Moreover Ex-4 decreased CT26 colony formation in soft agar and augmented apoptosis induced by irinotecan. Twice-daily treatment of CT26 tumor-bearing BALB/c mice with Ex-4 for 2 wk increased tumor apoptosis. Hence, GLP-1R activation reduces growth and survival in CT26 colon cancer cells that express the endogenous classical GLP-1R.

The stimulatory G protein α-subunit (G(s)α) couples hormone and other receptors to the generation of intracellular cAMP. We previously showed that mice with liver-specific G(s)α deficiency [liver-specific G(s)α knockout (LGsKO) mice] had reduced adiposity and improved glucose tolerance associated with increased glucose-stimulated insulin secretion, pancreatic islet hyperplasia, and very high serum glucagon and glucagon-like peptide 1 (GLP-1) levels. Because GLP-1 is known to stimulate insulin secretion and to have effects on energy balance, we mated LGsKO mice with germline GLP-1 receptor (GLP-1R) knockout mice (Glp1r(-/-)) and compared LGsKO to double-knockout (LGs/Glp1r(-/-)) mice to determine the contribution of excess GLP-1R signaling to the LGsKO phenotype. Loss of the GLP-1R failed to reverse most of the metabolic features of LGsKO mice, including reduced fat mass, increased glucose tolerance, and second-phase glucose-stimulated insulin secretion, islet cell hyperplasia, and very high glucagon and GLP-1 levels. However, loss of GLP-1R impaired first-phase insulin secretion in mice with or without liver-specific G(s)α deficiency. Thus, excess GLP-1 action (or at least through GLP-1R) does not contribute to the LGsKO metabolic phenotype, and other unknown factors involved in the cross talk between the liver G(s)α/cAMP pathway and pancreatic islet function need to be further elucidated.

Inhibition of dipeptidyl peptidase-4 (DPP-4) activity improves glucose homeostasis through a mode of action related to the stabilization of the active forms of DPP-4-sensitive hormones such as the incretins that enhance glucose-induced insulin secretion. However, the DPP-4 enzyme is highly expressed on the surface of intestinal epithelial cells; hence, the role of intestinal vs. systemic DPP-4 remains unclear. To analyze mechanisms through which the DPP-4 inhibitor sitagliptin regulates glycemia in mice, we administered low oral doses of the DPP-4 inhibitor sitagliptin that selectively reduced DPP-4 activity in the intestine. Glp1r(-/-) and Gipr(-/-) mice were studied and glucagon-like peptide (GLP)-1 receptor (GLP-1R) signaling was blocked by an i.v. infusion of the corresponding receptor antagonist exendin (9-39). The role of the dipeptides His-Ala and Tyr-Ala as DPP-4-generated GLP-1 and glucose-dependent insulinotropic peptide (GIP) degradation products was studied in vivo and in vitro on isolated islets. We demonstrate that very low doses of oral sitagliptin improve glucose tolerance and plasma insulin levels with selective reduction of intestinal but not systemic DPP-4 activity. The glucoregulatory action of sitagliptin was associated with increased vagus nerve activity and was diminished in wild-type mice treated with the GLP-1R antagonist exendin (9-39) and in Glp1r(-/-) and Gipr(-/-) mice. Furthermore, the dipeptides liberated from GLP-1 (His-Ala) and GIP (Tyr-Ala) deteriorated glucose tolerance, reduced insulin, and increased portal glucagon levels. The predominant mechanism through which DPP-4 inhibitors regulate glycemia involves local inhibition of intestinal DPP-4 activity, activation of incretin receptors, reduced liberation of bioactive dipeptides, and activation of the gut-to-pancreas neural axis.

Context: Antidiabetic therapies based on potentiation of incretin action are now widely used; however, understanding of their long-term safety remains incomplete., Evidence Acquisition: We searched articles in PubMed for data assessing the safety of incretin-based therapies., Evidence Synthesis: Three major areas of interest are reviewed: incretin action in the cardiovascular system, pancreatitis, and cancer. Incretin therapies reduce weight gain, minimize hypoglycemia, decrease inflammation, and are cardioprotective in preclinical studies. However, data permitting conclusions about whether incretin therapies modify the development of cardiovascular events in humans are not available. Case reports link incretin therapies to pancreatitis, but retrospective case control studies do not associate pancreatitis with glucagon-like peptide-1 receptor (GLP-1R) agonists or dipeptidyl peptidase-4 inhibitors. Preclinical studies of pancreatitis have yielded conflicting results, and mechanisms linking incretin receptor activation to pancreatic inflammation have not yet been forthcoming. GLP-1R activation promotes C-cell hyperplasia and medullary thyroid cancer in rodents; however, long-term clinical studies of sufficient size and duration to permit conclusions regarding cancer and incretin therapeutics have not yet been completed., Conclusions: The available data on incretin action and incidence of cardiovascular events, pancreatitis, or cancer are not yet sufficient or robust enough to permit firm conclusions regarding associations with incretin-based therapies in humans with diabetes. The forthcoming results of long-term cardiovascular safety studies should provide more conclusive information about the safety of GLP-1R agonists and dipeptidyl peptidase-4 inhibitors in diabetic patients.

Patients with type 2 diabetes mellitus have an increased risk of fracture that can be further exacerbated by thiazolidinediones. A new class of antidiabetic agents control glucose through reduction of dipeptidyl peptidase-4 (DPP-4) activity; however the importance of DPP-4 for the control of bone quality has not been extensively characterized. We compared the effects of the thiazolidinedione pioglitazone and the DPP-4 inhibitor sitagliptin on bone quality in high-fat diet (HFD)-fed wild-type mice. In complementary studies, we examined bone quality in Dpp4(+/+) vs. Dpp4(-/-) mice. Pioglitazone produced yellow bones with greater bone marrow adiposity and significantly reduced vertebral bone mechanics in male, female, and ovariectomized (OVX) HFD fed female mice. Pioglitazone negatively affected vertebral volumetric bone mineral density, trabecular architecture, and mineral apposition rate in male mice. Sitagliptin treatment of HFD-fed wild-type mice significantly improved vertebral volumetric bone mineral density and trabecular architecture in female mice, but these improvements were lost in females after OVX. Genetic inactivation of Dpp4 did not produce a major bone phenotype in male and female Dpp4(-/-) mice; however, OVX Dpp4(-/-) mice exhibited significantly reduced femoral size and mechanics. These findings delineate the skeletal consequences of pharmacological and genetic reduction of DPP-4 activity and reveal significant differences in the effects of pioglitazone vs. sitagliptin vs. genetic Dpp4 inactivation on bone mechanics in mice.

G protein-coupled receptor 119 (GPR119) was originally identified as a β-cell receptor. However, GPR119 activation also promotes incretin secretion and enhances peptide YY action. We examined whether GPR119-dependent control of glucose homeostasis requires preservation of peptidergic pathways in vivo. Insulin secretion was assessed directly in islets, and glucoregulation was examined in wild-type (WT), single incretin receptor (IR) and dual IR knockout (DIRKO) mice. Experimental endpoints included plasma glucose, insulin, glucagon, glucagon-like peptide-1 (GLP-1), glucose-dependent insulinotropic peptide (GIP), and peptide YY. Gastric emptying was assessed in WT, Glp1r-/-, DIRKO, Glp2r-/-, and GPR119-/- mice treated with the GPR119 agonist AR231453. AR231453 stimulated insulin secretion from WT and DIRKO islets in a glucose-dependent manner, improved glucose homeostasis, and augmented plasma levels of GLP-1, GIP, and insulin in WT and Gipr-/- mice. In contrast, although AR231453 increased levels of GLP-1, GIP, and insulin, it failed to lower glucose in Glp1r-/- and DIRKO mice. Furthermore, AR231453 did not improve ip glucose tolerance and had no effect on insulin action in WT and DIRKO mice. Acute GPR119 activation with AR231453 inhibited gastric emptying in Glp1r-/-, DIRKO, Glp2r-/-, and in WT mice independent of the Y2 receptor (Y2R); however, AR231453 did not control gastric emptying in GPR119-/- mice. Our findings demonstrate that GPR119 activation directly stimulates insulin secretion from islets in vitro, yet requires intact IR signaling and enteral glucose exposure for optimal control of glucose tolerance in vivo. In contrast, AR231453 inhibits gastric emptying independent of incretin, Y2R, or Glp2 receptors through GPR119-dependent pathways. Hence, GPR119 engages multiple complementary pathways for control of glucose homeostasis.

Glucagon-like peptide-1 augments nutrient-stimulated insulin secretion. Chow-fed mice lacking the glucagon-like peptide-1 receptor (Glp1r) exhibit enhanced insulin-stimulated muscle glucose uptake but impaired suppression of endogenous glucose appearance (endoRa). This proposes a novel role for the Glp1r to regulate the balance of glucose disposal in muscle and liver by modulating insulin action. Whether this is maintained in an insulin-resistant state is unknown. The present studies tested the hypothesis that disruption of Glp1r expression overcomes high-fat (HF) diet-induced muscle insulin resistance and exacerbates HF diet-induced hepatic insulin resistance. Mice with a functional disruption of the Glp1r (Glp1r-/-) were compared with wild-type littermates (Glp1r+/+) after 12 wk on a regular chow diet or a HF diet. Arterial and venous catheters were implanted for sampling and infusions. Hyperinsulinemic-euglycemic clamps were performed on weight-matched male mice. [3-(3)H]glucose was used to determine glucose turnover, and 2[14C]deoxyglucose was used to measure the glucose metabolic index, an indicator of glucose uptake. Glp1r-/- mice exhibited increased glucose disappearance and muscle glucose metabolic index on either diet. This was associated with enhanced activation of muscle Akt and AMP-activated protein kinase and reduced muscle triglycerides in HF-fed Glp1r-/- mice. Chow-fed Glp1r-/- mice exhibited impaired suppression of endoRa and hepatic insulin signaling. In contrast, HF-fed Glp1r-/- mice exhibited improved suppression of endoRa and hepatic Akt activation. This was associated with decreased hepatic triglycerides and impaired activation of sterol regulatory element-binding protein-1. These results show that mice lacking the Glp1r are protected from HF diet-induced muscle and hepatic insulin resistance independent of effects on total fat mass.

The widely expressed dipeptidyl peptidase-4 enzyme rapidly cleaves the gut hormone glucagon-like peptide-1 [GLP-1(7-36)amide] at the N terminus to generate GLP-1(9-36)amide. Both intact GLP-1(7-36)amide and GLP-1(9-36)amide exert cardioprotective actions in rodent hearts; however, the mechanisms underlying the actions of GLP-1(9-36)amide remain poorly understood. We used mass spectrometry of coronary effluents to demonstrate that isolated mouse hearts rapidly convert infused GLP-1(7-36)amide to GLP-1(9-36)amide. After ischemia-reperfusion (I/R) injury of isolated mouse hearts, administration of GLP-1(9-36)amide or exendin-4 improved functional recovery and reduced infarct size. The direct actions of these peptides were studied in cultured neonatal mouse cardiomyocytes. Both GLP-1(9-36)amide and exendin-4 increased levels of cAMP and phosphorylation of ERK1/2 and the phosphoinositide 3-kinase target protein kinase B/Akt. In I/R injury models in vitro, both peptides improved mouse cardiomyocyte viability and reduced lactate dehydrogenase release and caspase-3 activation. These effects were attenuated by inhibitors of ERK1/2 and phosphoinositide 3-kinase. Unexpectedly, the cardioprotective actions of GLP-1(9-36)amide were blocked by exendin(9-39) yet preserved in Glp1r(-/-) cardiomyocytes. Furthermore, GLP-1(9-36)amide, but not exendin-4, improved the survival of human aortic endothelial cells undergoing I/R injury, actions sensitive to the nitric oxide synthase inhibitor, N(G)-nitro-l-arginine methyl ester (L-NAME). In summary, our findings demonstrate separate actions for GLP-1(9-36)amide vs. the GLP-1R agonist exendin-4 and reveal the existence of a GLP-1(9-36)amide-responsive, exendin(9-39)-sensitive, cardioprotective signaling pathway distinct from that associated with the classical GLP-1 receptor.

Liraglutide is a glucagon-like peptide-1 (GLP-1) analog developed for type 2 diabetes. Long-term liraglutide exposure in rodents was associated with thyroid C-cell hyperplasia and tumors. Here, we report data supporting a GLP-1 receptor-mediated mechanism for these changes in rodents. The GLP-1 receptor was localized to rodent C-cells. GLP-1 receptor agonists stimulated calcitonin release, up-regulation of calcitonin gene expression, and subsequently C-cell hyperplasia in rats and, to a lesser extent, in mice. In contrast, humans and/or cynomolgus monkeys had low GLP-1 receptor expression in thyroid C-cells, and GLP-1 receptor agonists did not activate adenylate cyclase or generate calcitonin release in primates. Moreover, 20 months of liraglutide treatment (at >60 times human exposure levels) did not lead to C-cell hyperplasia in monkeys. Mean calcitonin levels in patients exposed to liraglutide for 2 yr remained at the lower end of the normal range, and there was no difference in the proportion of patients with calcitonin levels increasing above the clinically relevant cutoff level of 20 pg/ml. Our findings delineate important species-specific differences in GLP-1 receptor expression and action in the thyroid. Nevertheless, the long-term consequences of sustained GLP-1 receptor activation in the human thyroid remain unknown and merit further investigation.

Glucagon-like peptide-1 (GLP-1) diminishes postmeal glucose excursions by enhancing insulin secretion via activation of the beta-cell GLP-1 receptor (Glp1r). GLP-1 may also control glucose levels through mechanisms that are independent of this incretin effect. The hyperinsulinemic-euglycemic clamp (insulin clamp) and exercise were used to examine the incretin-independent glucoregulatory properties of the Glp1r because both perturbations stimulate glucose flux independent of insulin secretion. Chow-fed mice with a functional disruption of the Glp1r (Glp1r(-/-)) were compared with wild-type littermates (Glp1r(+/+)). Studies were performed on 5-h-fasted mice implanted with arterial and venous catheters for sampling and infusions, respectively. During insulin clamps, [3-(3)H]glucose and 2[(14)C]deoxyglucose were used to determine whole-body glucose turnover and glucose metabolic index (R(g)), an indicator of glucose uptake. R(g) in sedentary and treadmill exercised mice was determined using 2[(3)H]deoxyglucose. Glp1r(-/-) mice exhibited increased glucose disappearance, muscle R(g), and muscle glycogen levels during insulin clamps. This was not associated with enhanced muscle insulin signaling. Glp1r(-/-) mice exhibited impaired suppression of endogenous glucose production and hepatic glycogen accumulation during insulin clamps. This was associated with impaired liver insulin signaling. Glp1r(-/-) mice became significantly hyperglycemic during exercise. Muscle R(g) was normal in exercised Glp1r(-/-) mice, suggesting that hyperglycemia resulted from an added drive to stimulate glucose production. Muscle AMP-activated protein kinase phosphorylation was higher in exercised Glp1r(-/-) mice. This was associated with increased relative exercise intensity and decreased exercise endurance. In conclusion, these results show that the endogenous Glp1r regulates hepatic and muscle glucose flux independent of its ability to enhance insulin secretion.

The development of type 1 diabetes (T1D) has been linked to environmental factors and dietary components. Increasing evidence indicates that the integrity of the gut mucosa plays a role in the development of autoimmune diseases, and evidence from both preclinical and clinical studies demonstrates that increased leakiness of the intestinal epithelium precedes the development of type 1 diabetes. However, there is limited information on modulation of gut barrier function and its relationship to diabetes development. Here we show that the nonobese diabetic (NOD) mouse, a model of T1D, exhibits enhanced intestinal transcellular permeability before the development of autoimmune diabetes. Treatment of NOD mice with a glucagon-like peptide 2 (GLP-2) analog, synthetic human [Gly(2)] glucagon-like peptide-2 (h[Gly(2)]GLP-2, increased the length and weight of the small bowel and significantly improved jejunal transepithelial resistance. However, chronic administration of once daily h[Gly(2)]GLP-2 failed to delay or reverse the onset of T1D when treatment was initiated in young, normoglycemic female NOD mice. Furthermore, h[Gly(2)]GLP-2 administration had no significant effect on lymphocyte subpopulations in NOD mice. These findings demonstrate that h[Gly(2)]GLP-2-mediated enhancement of gut barrier function in normoglycemic NOD mice disease is not sufficient to prevent or delay the development of experimental T1D.

Pharmacological approaches that enhance incretin action for the treatment of type 2 diabetes mellitus have recently been developed, i.e. injectable glucagon-like peptide-1 receptor (GLP-1R) agonists with prolonged plasma half-lives and orally available inhibitors of dipeptidyl peptidase (DPP)-4, the main enzyme responsible for the rapid degradation of circulating glucagon-like peptide-1 and glucose-dependent insulinotropic peptide. The mechanism(s) underlying the glucose-lowering effect of these two pharmacotherapies differs and is not yet fully understood. Here we investigated whether acute GLP-1R activation (exendin-4) or DPP-4 inhibition (des-F-sitagliptin) modulates insulin action in mice using a hyperinsulinemic euglycemic clamp. A single iv bolus of des-F-sitagliptin (11 mg/kg) was administered to mice 15 min after the start of the clamp, and its effect was compared with a 50-ng bolus of exendin-4 or the same volume of saline. Despite matched levels of plasma glucose and insulin, within 15 min the glucose infusion rate required to maintain euglycemia was significantly greater after des-F-sitagliptin compared with saline or exendin-4. This difference was entirely due to enhancement of insulin-mediated suppression of endogenous glucose production by des-F-sitagliptin, with no difference in glucose disposal rate. These findings illustrate that DPP-4 inhibition modulates glucose homeostasis through pathways distinct from those used by GLP-1R agonists in mice.

The proglucagon gene gives rise to multiple peptides that play diverse roles in the control of energy intake, gut motility, and nutrient disposal. Glucagon-like peptide-1 (GLP-1), a 30-amino-acid peptide regulates glucose homeostasis via control of insulin and glucagon secretion and by inhibition of gastric emptying and food intake. Oxyntomodulin (OXM) a 37-amino-acid peptide also derived from the proglucagon gene, binds to both the glucagon and GLP-1 receptor (GLP-1R); however, a separate OXM receptor has not yet been identified. Here we show that OXM, like other GLP-1R agonists, stimulates cAMP formation and lowers blood glucose after both oral and ip glucose administration, actions that require a functional GLP-1R. OXM also directly stimulates insulin secretion from murine islets and INS-1 cells in a glucose- and GLP-1R-dependent manner. Moreover, OXM ameliorates hyperglycemia and significantly reduces apoptosis in murine beta-cells after streptozotocin administration and directly reduces apoptosis in thapsigargin-treated INS-1 cells. Unexpectedly, OXM, but not the GLP-1R agonist exendin-4, increased plasma levels of insulin after oral glucose administration. Moreover, OXM administered at doses that potently lower blood glucose had no effect on inhibition of gastric emptying but reduced food intake in WT mice. Taken together, these findings illustrate that although structurally distinct proglucagon-derived peptides such as GLP-1 and OXM engage the GLP-1R, OXM mimics some but not all of the actions of GLP-1R agonists in vivo. These findings may have implications for therapeutic efforts using OXM as a long-acting GLP-1R agonist for the treatment of metabolic disorders.

Activation of the glucagon-like peptide-1 receptor (GLP-1R) is associated with expansion of beta-cell mass due to stimulation of cell proliferation and induction of antiapoptotic pathways coupled to beta-cell survival. Although the GLP-1R agonist Exenatide (exendin-4) is currently being evaluated in subjects with type 1 diabetes, there is little information available about the efficacy of GLP-1R activation for prevention of experimental type 1 diabetes. We examined the consequences of exendin-4 (Ex-4) administration (100 ng once daily and 2 microg twice daily) on diabetes onset in nonobese diabetic mice beginning at either 4 or 9 wk of age prior to the onset of diabetes. Ex-4 treatment for 26 wk (2 microg twice daily) initiated at 4 wk of age delayed the onset of diabetes (P = 0.007). Ex-4-treated mice also exhibited a significant reduction in insulitis scores, enhanced beta-cell mass, and improved glucose tolerance. Although GLP-1R mRNA transcripts were detected in spleen, thymus, and lymph nodes from nonobese diabetic mice, Ex-4 treatment was not associated with significant changes in the numbers of CD4+ or CD8+ T cells or B cells in the spleen. However, Ex-4 treatment resulted in an increase in the number of CD4+ and CD8+ T cells in the lymph nodes and a reduction in the numbers of CD4+CD25+Foxp3+ regulatory T cells in the thymus but not in lymph nodes. These findings demonstrate that sustained GLP-1R activation in the absence of concomitant immune intervention may be associated with modest but significant delay in diabetes onset in a murine model of type 1 diabetes.

Gastrointestinal hormones including gastric inhibitory polypeptide (GIP), glucagon-like peptide (GLP)-1, and GLP-2 are secreted immediately after meal ingestion, and GIP and GLP-2 have been shown to regulate bone turnover. We hypothesize that endogenous GLP-1 may also be important for control of skeletal homeostasis. We investigated the role of GLP-1 in the regulation of bone metabolism using GLP-1 receptor knockout (Glp-1r(-/-)) mice. A combination of bone density and histomorphometry, osteoclast activation studies, biochemical analysis of calcium and PTH, and RNA analysis was used to characterize bone and mineral homeostasis in Glp-1r(-/-) and Glp-1r(+/+) littermate controls. Glp-1r(-/-) mice have cortical osteopenia and bone fragility by bone densitometry as well as increased osteoclastic numbers and bone resorption activity by bone histomorphometry. Although GLP-1 had no direct effect on osteoclasts and osteoblasts, Glp-1r(-/-) mice exhibited higher levels of urinary deoxypyridinoline, a marker of bone resorption, and reduced levels of calcitonin mRNA transcripts in the thyroid. Moreover, calcitonin treatment effectively suppressed urinary levels of deoxypyridinoline in Glp-1r(-/-), mice and the GLP-1 receptor agonist exendin-4 increased calcitonin gene expression in the thyroid of wild-type mice. These findings establish an essential role for endogenous GLP-1 receptor signaling in the control of bone resorption, likely through a calcitonin-dependent pathway.

Glucagon-like peptide-1(7-36NH2) (GLP-1) and peptide YY(3-36NH2) (PYY(3-36NH2)) are cosecreted from the intestine in response to nutrient ingestion. Peripheral administration of GLP-1 or PYY(3-36NH2) decreases food intake (FI) in rodents and humans; however, the exact mechanisms by which these peptides regulate FI remain unclear. Male C57BL/6 mice were injected (ip) with exendin-4(1-39) (Ex4, a GLP-1 receptor agonist) and/or PYY(3-36NH2) (0.03-3 microg), and FI was determined for up to 24 h. Ex4 and PYY(3-36NH2) alone decreased FI by up to 83 and 26%, respectively (P < 0.05-0.001), whereas a combination of the two peptides (0.06 microg Ex4 plus 3 microg PYY(3-36NH2)) further reduced FI for up to 8 h in a synergistic manner (P < 0.05-0.001). Ex4 and/or PYY(3-36NH2) delayed gastric emptying by a maximum of 19% (P < 0.01-0.001); however, there was no significant effect on locomotor activity nor was there induction of taste aversion. Capsaicin pretreatment prevented the inhibitory effect of Ex4 on FI (P < 0.05), but had no effect on the anorexigenic actions of PYY(3-36NH2). Similarly, exendin-4(9-39) (a GLP-1 receptor antagonist) partially abolished Ex4-induced anorexia (P < 0.05), but did not affect the satiation produced by PYY(3-36NH2). Conversely, BIIE0246 (a Y2 receptor antagonist) completely blocked the anorexigenic effects of PYY(3-36NH2) (P < 0.001), but had no effect on Ex4-induced satiety. Thus, Ex4 and PYY(3-36NH2) suppress FI via independent mechanisms involving a GLP-1 receptor-dependent, sensory afferent pathway (Ex4) and a Y2-receptor mediated pathway (PYY(3-36NH2)). These findings suggest that administration of low doses of Ex4 together with PYY(3-36NH2) may increase the suppression of FI without inducing significant side effects.

In rats, central administration of glucagon-like peptide-1 (GLP-1) elicits symptoms of visceral illness like those caused by the toxin lithium chloride (LiCl), including anorexia, conditioned taste aversion (CTA) formation, and neural activation in the hypothalamus and hindbrain including activation of brainstem preproglucagon cells. Most compellingly, pharmacological antagonists of the GLP-1 receptor (GLP-1R) block several effects of LiCl in rat. The major goal of these experiments was to further test the hypothesis that the central nervous system GLP-1 system is critical to the visceral illness actions of LiCl by using mice with a targeted disruption of the only described GLP-1R. First, we observed that, like the rat, LiCl activates preproglucagon neurons in wild-type mice. Second, GLP-1R -/- mice demonstrated normal anorexic and CTA responses to LiCl. To test the possibility that alternate GLP-1Rs mediate aversive effects, we examined the ability of GLP-1 to produce a CTA in GLP1R -/- mice. Although lateral ventricular GLP-1 produced a CTA in wild-type mice, it did not produce a CTA in GLP-1R -/- mice. Furthermore, the same GLP-1R antagonist that can block the aversive effects of LiCl in the rat failed to do so in the mouse. These results support the conclusion that in mouse, unlike in rat, GLP-1R signaling is not required for the visceral illness response to LiCl. Such species differences are an important consideration when comparing results from rat and mouse studies.

Pdx-1 plays a key role in the development of the pancreas and the control of islet gene transcription and has also been proposed as a dominant regulator of the alpha- vs. beta-cell phenotype via extinction of proglucagon expression. To ascertain the relationship between Pdx-1 and proglucagon gene expression, we examined the effect of enhanced pdx-1 expression on proglucagon gene expression in murine islet alphaTC-1 and GLUTag enteroendocrine cells. Although adenoviral transduction increased the levels of pdx-1 mRNA transcripts and nuclear Pdx-1 protein, overexpression of pdx-1 did not repress endogenous proglucagon gene expression in alphaTC-1 or GLUTag cells or murine islets. Immunohistochemical analysis of cells transduced with Ad-pdx-1 demonstrated multiple individual islet or enteroendocrine cells exhibiting both nuclear Pdx-1 and cytoplasmic glucagon-like peptide-1 immunopositivity. The failure of pdx-1 to inhibit endogenous proglucagon gene expression was not attributable to defects in Pdx-1 nuclear translocation or DNA binding as demonstrated using Western blotting and EMSA analyses. Furthermore, Ad-pdx-1 transduction did not repress proglucagon promoter activity in alphaTC-1 or GLUTag cells. Taken together, these findings demonstrate that pdx-1 alone is not sufficient for specification of the hormonal phenotype or extinction of proglucagon gene expression in islet or enteroendocrine 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.

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.

Glucagon-like peptide-2 (GLP-2) increases small intestinal growth and function in rodents and human subjects. GLP-2 exerts its effects through a seven-transmembrane domain, G protein-coupled receptor (GLP-2R), stimulating cAMP generation and activating protein kinase A signaling in heterologous cell lines transfected with the GLP-2R. As intestinal cell lines expressing the GLP-2R have not been identified, we developed methods for studying GLP-2R signaling in the rat small intestinal mucosa in vitro. Isolated rat intestinal mucosal cells expressed mRNA transcripts for the GLP-2R, as well as for chromogranin A and beta-tubulin III, markers for enteroendocrine and neural cells, respectively. cAMP production in response to [Gly2]GLP-2, a degradation-resistant analog of GLP-2, was maximal at 10-11 m (268 +/- 93% of control, P < 0.001), with reduced cAMP accumulation observed at higher doses. The cAMP response was diminished by pretreatment with 10-9 m GLP-2, and was abolished by pretreatment with 10-6 m GLP-2 (P < 0.05), indicating receptor desensitization. GLP-2 treatment of isolated mucosal cells increased 3H-thymidine incorporation (to 128 +/- 8% of controls, P < 0.05), and this was prevented by inhibition of the protein kinase A pathway with H89. In contrast, GLP-2 did not affect p44/p42 MAPK phosphorylation or the levels of cytosolic calcium in the mucosal cell preparation. These results provide the first evidence that activation of the endogenous rat mucosal GLP-2 receptor is linked to activation of a cAMP/protein kinase A-dependent, growth-promoting pathway in vitro.

Glucagon-like peptide-1 (GLP-1) acts via its G protein-coupled receptor (GLP-1R) to regulate blood glucose. Although the GLP-1R is widely expressed in peripheral tissues, including the heart, and exogenous GLP-1 administration increases heart rate and blood pressure in rodents, the physiological importance of GLP-1R action in the cardiovascular system remains unclear. We now show that 2-month-old mice with genetic deletion of the GLP-1R (GLP-1R(-/-)) exhibit reduced resting heart rate and elevated left ventricular (LV) end diastolic pressure compared with CD-1 wild-type controls. At the age of 5 months, echocardiography and histology demonstrate increased LV thickness in GLP-1R(-/-) mice. Although baseline hemodynamic parameters of GLP-1R(-/-) did not differ significantly from those of wild type, GLP-1R(-/-) mice displayed impaired LV contractility and diastolic function after insulin administration. The defective cardiovascular response to insulin was not attributable to a generalized defect in the stress response, because GLP-1R(-/-) mice responded appropriately to insulin with increased c-fos expression in the hypothalamus and increased circulating levels of glucagon and epinephrine. Furthermore, LV contractility after exogenous epinephrine infusion was also reduced in GLP-1R(-/-) mice. These findings provide new evidence implicating an essential role for GLP-1R in the control of murine cardiac structure and function in vivo.

Despite interest in understanding glucagon-like peptide-1 (GLP-1) production, the factors important for GLP-1 biosynthesis remain poorly understood. We examined control of human proglucagon gene expression in NCI-H716 cells, a cell line that secretes GLP-1 in a regulated manner. Insulin, phorbol myristate acetate, or forskolin, known regulators of rodent proglucagon gene expression, had no effect, whereas sodium butyrate decreased levels of NCI-H716 proglucagon mRNA transcripts. The inhibitory effect of sodium butyrate was mimicked by trichostatin A but was not detected with sodium acetate or isobutyrate. The actions of butyrate were not diminished by the ERK1/2 inhibitor PD98059, p38 inhibitor SB203580, or soluble guanylate cyclase inhibitor LY83583 or following treatment of cells with KT5823, a selective inhibitor of cGMP-dependent protein kinase. NCI-H716 cells expressed multiple proglucagon gene transcription factors including isl-1, pax-6, pax-2, cdx-2/3, pax-4, hepatocyte nuclear factor (HNF)-3 alpha, HNF-3beta, HNF-3 gamma, and Nkx2.2. Nevertheless, the butyrate-dependent inhibition of proglucagon gene expression was not associated with coordinate changes in transcription factor expression and both the human and rat transfected proglucagon promoters were transcriptionally inactive in NCI-H716 cells. Hence, NCI-H716 cells may not be a physiologically optimal model for studies of human enteroendocrine proglucagon gene transcription.

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.

Neuroendocrine tumors overexpressing the proglucagon- derived peptides have been associated with severe constipation. The relationship between two of the intestinal proglucagon-derived peptides, glucagon-like peptide (GLP)-1 and -2, and delayed gastrointestinal transit, was characterized in a patient with a neuroendocrine proglucagon-derived peptide tumor. A 60-yr-old female presented with intractable constipation and intermittent vomiting. Gastric, oral-ileal and colonic transit times, and plasma hormone levels were determined before tumor resection. Expression of the proglucagon-derived peptides by the tumor was determined by immunohistochemistry, Northern blot analysis, HPLC, and RIA. Oral-cecal transit was more than 3 h, and a barium follow-through study showed dilated and thickened folds with most of the barium concentrated in the ileum at 24 h; residual barium was identified in the colon at 14 d post ingestion. Circulating levels of GLP-1 and -2 were 300- to 400-fold elevated compared with levels in normal human subjects. Normal bowel function was restored by tumor resection. Consistent with the elevated plasma hormone levels, the tumor was found to express the proglucagon gene, and immunoreactive GLP-1 and -2 were detected by both immunohistochemistry and RIA. Overexpression of glucagon-like peptide-1 and -2 is associated with markedly prolonged gastrointestinal transit in humans. These findings are consistent with a role for these peptides in the regulation of gastrointestinal motility.

Glucagon-like peptide 2 (GLP-2) is a 33 amino acid peptide-encoded carboxyterminal to the sequence of GLP-1 in the proglucagon gene. Both GLP-1 and GLP-2 are secreted from gut endocrine cells and promote nutrient absorption through distinct mechanisms of action. GLP-2 regulates gastric motility, gastric acid secretion, intestinal hexose transport, and increases the barrier function of the gut epithelium. GLP-2 significantly enhances the surface area of the mucosal epithelium via stimulation of crypt cell proliferation and inhibition of apoptosis in the enterocyte and crypt compartments. The cytoprotective and reparative effects of GLP-2 are evident in rodent models of experimental intestinal injury. GLP-2 reduces mortality and decreases mucosal injury, cytokine expression, and bacterial septicemia in the setting of small and large bowel inflammation. GLP-2 also enhances nutrient absorption and gut adaptation in rodents or humans with short bowel syndrome. The actions of GLP-2 are transduced by the GLP-2 receptor, a G protein-coupled receptor expressed in gut endocrine cells of the stomach, small bowel, and colon. Activation of GLP-2 receptor signaling in heterologous cells promotes resistance to apoptotic injury in vitro. The cytoprotective, reparative, and energy-retentive properties of GLP-2 suggests that GLP-2 may potentially be useful for the treatment of human disorders characterized by injury and/or dysfunction of the intestinal mucosal epithelium.

The glucagon-like peptides GLP-1 and GLP-2 are produced in enteroendocrine L cells of the small and large intestine and secreted in a nutrient-dependent manner. GLP-1 regulates nutrient assimilation via inhibition of gastric emptying and food intake. GLP-1 controls blood glucose following nutrient absorption via stimulation of glucose-dependent insulin secretion, insulin biosynthesis, islet proliferation, and neogenesis and inhibition of glucagon secretion. Experiments using GLP-1 antagonists and GLP-1 receptor-/- mice indicate that the glucoregulatory actions of GLP-1 are essential for glucose homeostasis. In the central nervous system, GLP-1 regulates hypothalamic-pituitary function and GLP-1-activated circuits mediate the CNS response to aversive stimulation. GLP-2 maintains the integrity of the intestinal mucosal epithelium via effects on gastric motility and nutrient absorption, crypt cell proliferation and apoptosis, and intestinal permeability. Both GLP-1 and GLP-2 are rapidly inactivated in the circulation as a consequence of amino-terminal cleavage by the enzyme dipeptidyl peptidase IV (DP IV). The actions of these peptides on nutrient absorption and energy homeostasis and the efficacy of GLP-1 and GLP-2 in animal models of diabetes and intestinal diseases, respectively, suggest that analogs of these peptides may be clinically useful for the treatment of human disease.

Glucagon-like peptide-2 (GLP-2) is secreted by enteroendocrine cells in the small and large intestines and exerts intestinotropic effects in the gastrointestinal mucosal epithelium of the adult rodent. The actions of GLP-2 are mediated by the GLP-2 receptor, a new member of the G protein-coupled receptor superfamily. To ascertain whether the GLP-2/GLP-2 receptor axis is expressed and functional in the developing intestine, we have studied the synthesis of GLP-2 and the expression of the GLP-2 receptor (GLP-2R) in the fetal and neonatal rat gut. GLP-2 immunoreactivity (GLP-2-IR) was detected in the fetal rat intestine, and fetal rat intestinal cell cultures secreted correctly processed GLP-2(1-33) into the medium. High levels of GLP-2(1-33) were also detected in the circulation of 13-day-old neonatal rats (P < 0.001 vs. adult). Analysis of GLP-2 receptor expression by RT-PCR demonstrated GLP-2R messenger RNA transcripts in fetal intestine and in neonatal stomach, jejunum, ileum, and colon. The levels of GLP-2R messenger RNA transcripts were comparatively higher in the fetal and neonatal intestine (P < 0.05-001 vs. adult) and declined to adult levels by postnatal day 21. Subcutaneous administration of a degradation-resistant GLP-2 analog, h[Gly2]-GLP-2 once daily for 10 days increased stomach (0.009 +/- 0.0003 vs. 0.007 +/- 0.002 g/g body mass, h[Gly2]-GLP-2-treated vs. controls; P < 0.05) and small bowel weight (0.043 +/- 0.0037 vs. 0.031 +/- 0.0030 g/g body mass; P < 0.05). h[Gly2]-GLP-2 also increased both small (2.4 +/- 0.05 vs. 1.8 +/- 0.17 cm/g body mass; P < 0.05) and large bowel length (0.32 +/- 0.01 vs. 0.25 +/- 0.02 cm/g body mass, h[Gly2]-GLP-2-treated vs. saline-treated controls, respectively; P < 0.05) in neonatal rats. These findings demonstrate that both components of the GLP-2/GLP-2 receptor axis are expressed in the fetal and neonatal intestine. The ontogenic regulation and functional integrity of this axis raises the possibility that GLP-2 may play a role in the development and/or maturation of the developing rat intestine.

Glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic peptide (GIP) potentiate glucose-stimulated insulin secretion after enteral nutrient ingestion. We compared the relative incretin and nonincretin actions of GLP-1 and GIP in +/+ and GLP-1R-/- mice using exendin(9-39) and immunopurified anti-GIP receptor antisera (GIPR Ab) to antagonize GLP-1 and GIP action, respectively. Both antagonists produced a significant increase in glycemic excursion after oral glucose loading of +/+ mice (P < 0.05 for antagonists us. controls). Exendin(9-39) also increased blood glucose and decreased glucose-stimulated insulin in +/+ mice after ip glucose loading [0.58 +/- 0.02 vs. 0.47 +/- 0.02 ng/ml in saline- vs. exendin(9-39)-treated mice, respectively, P < 0.05]. In contrast, GIPR Ab had no effect on glucose excursion or insulin secretion, after ip glucose challenge, in +/+ or GLP-1R-/- mice. Repeated administration of exendin(9-39) significantly increased blood glucose and reduced circulating insulin levels but had no effect on levels of pancreatic insulin or insulin messenger RNA transcripts. In contrast, no changes in plasma glucose, circulating insulin, pancreatic insulin content, or insulin messenger RNA were observed in mice, 18 h after administration of GIPR Ab. These findings demonstrate that GLP-1, but not GIP, plays an essential role in regulating glycemia, independent of enteral nutrient ingestion in mice in vivo.

Glucagon-like peptide-1 (GLP-1), a potent regulator of glucose homeostasis, is also produced in the central nervous system, where GLP-1 has been implicated in the neuroendocrine control of hypothalamic-pituitary function, food intake, and the response to stress. The finding that intracerebroventricular GLP-1 stimulates LH, TSH, corticosterone, and vasopressin secretion in rats prompted us to assess the neuroendocrine consequences of disrupting GLP-1 signaling in mice in vivo. Male GLP-1 receptor knockout (GLP-1R-/-) mice exhibit reduced gonadal weights, and females exhibit a slight delay in the onset of puberty; however, male and female GLP-1R-/- animals reproduce successfully and respond appropriately to fluid restriction. Although adrenal weights are reduced in GLP-1R-/- mice, hypothalamic CRH gene expression and circulating levels of corticosterone, thyroid hormone, testosterone, estradiol, and progesterone are normal in the absence of GLP-1R-/- signaling. Intriguingly, GLP-1R-/- mice exhibit paradoxically increased corticosterone responses to stress as well as abnormal responses to acoustic startle that are corrected by glucocorticoid treatment. These findings suggest that although GLP-1R signaling is not essential for development and basal function of the murine hypothalamic-pituitary-adrenal axis, abrogation of GLP-1 signaling is associated with impairment of the behavioral and neuroendocrine responses to stress.

Glucagon-like peptide-1 (GLP-1) released from the intestine is a potent stimulator of glucose-dependent insulin secretion. To elucidate the factors regulating GLP-1 secretion, we have studied the enteroendocrine GLUTag cell line. GLP-1 secretion was stimulated in a dose-dependent fashion by activation of protein kinase A or C with forskolin or phorbol 12,13-dibutyrate, respectively (by 2.3 +/- 0.5-fold at 100 microM and 4.3 +/- 0.6-fold at 0.3 microM, respectively; P < 0.01-0.001). Of the regulatory peptides tested, only glucose-dependent insulinotropic peptide stimulated the release of GLP-1 (by 2.3 +/- 0.2-fold at 0.1 microM; P < 0.001); glucagon was without effect, and paradoxically, the inhibitory neuropeptide somatostatin-14 increased secretion slightly (by 1.6 +/- 0.3-fold at 0.01 microM; P < 0.05). In tests of several neurotransmitters, only the cholinergic agonists carbachol and bethanechol stimulated peptide secretion in a dose-dependent fashion (by 2.3 +/- 0.5- and 1.7 +/- 0.3-fold at 1000 microM; P < 0.05-0.001); the beta-adrenergic agonist isoproterenol and the chloride channel inhibitor gamma-aminobutyric acid did not affect release of GLP-1. Long chain monounsaturated fatty acids (18:1), but not saturated fatty acids (16:0), also stimulated the release of GLP-1 (by 1.7 +/- 0.1-fold at 150 microM; P < 0.001). Consistent with the presence of a cAMP response element in the proglucagon gene, activation of the protein kinase A-dependent pathway with forskolin increased proglucagon messenger RNA transcript levels by 2-fold (P < 0.05); glucose-dependent insulinotropic peptide and phorbol 12,13-dibutyrate were without effect. Therefore, by comparison with results obtained using primary L cell cultures or in vivo models, GLUTag cells appear to respond appropriately to the regulatory mechanisms controlling intestinal GLP-1 secretion.