Proglucagon signalling in the rat Dorsomedial Hypothalamus - Physiology and high-fat diet-mediated alterations.

A relatively new pharmacological target in obesity treatment has been the preproglucagon (PPG) signalling, predominantly with glucagon-like peptide (GLP) 1 receptor agonists. As far as the PPG role within the digestive system is well recognised, its actions in the brain remain understudied. Here, we investigated PPG signalling in the Dorsomedial Hypothalamus (DMH), a structure involved in feeding regulation and metabolism, using in situ hybridisation, electrophysiology, and immunohistochemistry. Our experiments were performed on animals fed both control, and high-fat diet (HFD), uncovering HFD-mediated alterations. First, sensitivity to exendin-4 (Exn4, a GLP1R agonist) was shown to increase under HFD, with a higher number of responsive neurons. The amplitude of the response to both Exn4 and oxyntomodulin (Oxm) was also altered, diminishing its relationship with the cells' spontaneous firing rate. Not only neuronal sensitivity, but also GLP1 presence, and therefore possibly release, was influenced by HFD. Immunofluorescent labelling of the GLP1 showed changes in its density depending on the metabolic state (fasted/fed), but this effect was eliminated by HFD feeding. Interestingly, these dietary differences were absent after a period of restricted feeding, allowing for an anticipation of the alternating metabolic states, which suggests possible prevention of such outcome.

GIP and GLP-2 together improve bone turnover in humans supporting GIPR-GLP-2R co-agonists as future osteoporosis treatment.

The intestinal hormones glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-2 (GLP-2) are key regulators of postprandial bone turnover in humans. We hypothesized that GIP and GLP-2 co-administration would provide stronger effect on bone turnover than administration of the hormones separately, and tested this using subcutaneous injections of GIP and GLP-2 alone or in combination in humans. Guided by these findings, we designed series of GIPR-GLP-2R co-agonists as template for new osteoporosis treatment. The clinical experiment was a randomized cross-over design including 10 healthy men administered subcutaneous injections of GIP and GLP-2 alone or in combination. The GIPR-GLP-2R co-agonists were characterized in terms of binding and activation profiles on human and rodent GIP and GLP-2 receptors, and their pharmacokinetic (PK) profiles were improved by dipeptidyl peptidase-4 protection and site-directed lipidation. Co-administration of GIP and GLP-2 in humans resulted in an additive reduction in bone resorption superior to each hormone individually. The GIPR-GLP-2R co-agonists, designed by combining regions of importance for cognate receptor activation, obtained similar efficacies as the two native hormones and nanomolar potencies on both human receptors. The PK-improved co-agonists maintained receptor activity along with their prolonged half-lives. Finally, we found that the GIPR-GLP-2R co-agonists optimized toward the human receptors for bone remodeling are not feasible for use in rodent models. The successful development of potent and efficacious GIPR-GLP-2R co-agonists, combined with the improved effect on bone metabolism in humans by co-administration, support these co-agonists as a future osteoporosis treatment.

[Gly²]-GLP-2, But Not Glucagon or [D-Ala²]-GLP-1, Controls Collagen Crosslinking in Murine Osteoblast Cultures.

Bone tissue is organized at the molecular level to resist fracture with the minimum of bone material. This implies that several modifications of the extracellular matrix, including enzymatic collagen crosslinking, take place. We previously highlighted the role of several gut hormones in enhancing collagen maturity and bone strength. The present study investigated the effect of proglucagon-derived peptides on osteoblast-mediated collagen post-processing. Briefly, MC3T3-E1 murine osteoblasts were cultured in the presence of glucagon (GCG), [D-Ala²]-glucagon-like peptide-1 ([D-Ala²]-GLP-1), and [Gly²]-glucagon-like peptide-2 ([Gly²]-GLP-2). Gut hormone receptor expression at the mRNA and protein levels were investigated by qPCR and Western blot. Extent of collagen postprocessing was examined by Fourier transform infrared microspectroscopy. GCG and GLP-1 receptors were not evidenced in osteoblast cells at the mRNA and protein levels. However, it is not clear whether the known GLP-2 receptor is expressed. Nevertheless, administration of [Gly²]-GLP-2, but not GCG or [D-Ala²]-GLP-1, led to a dose-dependent increase in collagen maturity and an acceleration of collagen post-processing. This mechanism was dependent on adenylyl cyclase activation. In conclusion, the present study highlighted a direct effect of [Gly²]-GLP-2 to enhance collagen post-processing and crosslinking maturation in murine osteoblast cultures. Whether this effect is translatable to human osteoblasts remains to be elucidated.

Variation in the Evolution and Sequences of Proglucagon and the Receptors for Proglucagon-Derived Peptides in Mammals.

The mammalian proglucagon gene (Gcg) encodes three glucagon like sequences, glucagon, glucagon-like peptide-1 (GLP-1), and glucagon-like peptide-2 that are of similar length and share sequence similarity, with these hormones having cell surface receptors, glucagon receptor (Gcgr), GLP-1 receptor (Glp1r), and GLP-2 receptor (Glp2r), respectively. Gcgr, Glp1r, and Glp2r are all class B1 G protein-coupled receptors (GPCRs). Despite their sequence and structural similarity, analyses of sequences from rodents have found differences in patterns of sequence conservation and evolution. To determine whether these were rodent-specific traits or general features of these genes in mammals I analyzed coding and protein sequences for proglucagon and the receptors for proglucagon-derived peptides from the genomes of 168 mammalian species. Single copy genes for each gene were found in almost all genomes. In addition to glucagon sequences within Hystricognath rodents (e.g., guinea pig), glucagon sequences from a few other groups (e.g., pangolins and some bats) as well as changes in the proteolytic processing of GLP-1 in some bats are suggested to have functional effects. GLP-2 sequences display increased variability but accepted few substitutions that are predicted to have functional consequences. In parallel, Glp2r sequences display the most rapid protein sequence evolution, and show greater variability in amino acids at sites involved in ligand interaction, however most were not predicted to have a functional consequence. These observations suggest that a greater diversity in biological functions for proglucagon-derived peptides might exist in mammals.

GLP-2 Is Locally Produced From Human Islets and Balances Inflammation Through an Inter-Islet-Immune Cell Crosstalk.

Glucagon-like peptide-1 (GLP-1) shows robust protective effects on ß-cell survival and function and GLP-1 based therapies are successfully applied for type-2 diabetes (T2D) and obesity. Another cleavage product of pro-glucagon, Glucagon-like peptide-2 (GLP-2; both GLP-1 and GLP-2 are inactivated by DPP-4) has received little attention in its action inside pancreatic islets. In this study, we investigated GLP-2 production, GLP-2 receptor (GLP-2R) expression and the effect of GLP-2R activation in human islets. Isolated human islets from non-diabetic donors were exposed to diabetogenic conditions: high glucose, palmitate, cytokine mix (IL-1ß/IFN-γ) or Lipopolysaccharide (LPS) in the presence or absence of the DPP4-inhibitor linagliptin, the TLR4 inhibitor TAK-242, the GLP-2R agonist teduglutide and/or its antagonist GLP-2(3-33). Human islets under control conditions secreted active GLP-2 (full-length, non-cleaved by DPP4) into the culture media, which was increased by combined high glucose/palmitate, the cytokine mix and LPS and highly potentiated by linagliptin. Low but reproducible GLP-2R mRNA expression was found in all analyzed human islet isolations from 10 donors, which was reduced by pro-inflammatory stimuli: the cytokine mix and LPS. GLP-2R activation by teduglutide neither affected acute or glucose stimulated insulin secretion nor insulin content. Also, teduglutide had no effect on high glucose/palmitate- or LPS-induced dysfunction in cultured human islets but dampened LPS-induced macrophage-dependent IL1B and IL10 expression, while its antagonist GLP-2(3-33) abolished such reduction. In contrast, the expression of islet macrophage-independent cytokines IL6, IL8 and TNF was not affected by teduglutide. Medium conditioned by teduglutide-exposed human islets attenuated M1-like polarization of human monocyte-derived macrophages, evidenced by a lower mRNA expression of pro-inflammatory cytokines, compared to vehicle treated islets, and a reduced production of itaconate and succinate, marker metabolites of pro-inflammatory macrophages. Our results reveal intra-islet production of GLP-2 and GLP-2R expression in human islets. Despite no impact on ß-cell function, local GLP-2R activation reduced islet inflammation which might be mediated by a crosstalk between endocrine cells and macrophages.

Expression, purification and molecular dynamics simulation of extracellular domain of glucagon-like peptide-2 receptor linked to teduglutide.

Teduglutide is the only drug approved for long-term treatment of short bowel syndrome (SBS). This drug exerts its pharmacological effects via binding to the GLP-2 receptors (ECD-GLP2R) located in intestinal tissue. The three dimensional (3D) structure of ECD-GLP2R hasn't been determined yet and hence its mode of interaction with agonists/antagonists is not clear. Therefore, it would be of great importance to develop a structural scaffold for investigation of ECD-GLP2R interactions with its binders. For this, the current study aimed to produce fusion protein of ECD-GLP2R-teduglutide. The ECD-GLP2R-teduglutide protein was expressed in bacterial expression system and purified using affinity and size exclusion chromatography techniques. Using circular dichroism the secondary structure content of purified protein was determined which was comparable to that of theoretical calculations. The low structural stability of purified protein (ΔG = 3.64 kJ.mol-1) was elucidated by monitoring its fluorescence emission at the presence of various concentrations of GdnHCl as a denaturant. Finally, a 3D model for ECD-GLP2R-teduglutide protein was generated and validated using molecular dynamics simulation whose information alongside the experimental studies can be useful for providing new insight into the mode of interaction of ECD-GLP2R with its specific ligands in order to design potent and specific GLP2R agonists.

Dietary Fiber Is Essential to Maintain Intestinal Size, L-Cell Secretion, and Intestinal Integrity in Mice.

Dietary fiber has been linked to improved gut health, yet the mechanisms behind this association remain poorly understood. One proposed mechanism is through its influence on the secretion of gut hormones, including glucagon-like peptide-1 (GLP-1) and glucagon-like peptide-2 (GLP-2). We aimed to: 1) investigate the impact of a fiber deficient diet on the intestinal morphological homeostasis; 2) evaluate L-cell secretion; and 3) to ascertain the role of GLP-1, GLP-2 and Takeda G protein-receptor-5 (TGR5) signaling in the response using GLP-1 receptor, GLP-2 receptor and TGR5 knockout mice. Female C57BL/6JRj mice (n = 8) either received a standard chow diet or were switched to a crude fiber-deficient diet for a short (21 days) and long (112 days) study period. Subsequent identical experiments were performed in GLP-1 receptor, GLP-2 receptor and TGR5 knockout mice. The removal of fiber from the diet for 21 days resulted in a decrease in small intestinal weight (p < 0.01) and a corresponding decrease in intestinal crypt depth in the duodenum, jejunum and ileum (p < 0.001, p < 0.05, and p < 0.01, respectively). Additionally, colon weight was decreased (p < 0.01). These changes were associated with a decrease in extractable GLP-1, GLP-2 and PYY in the colon (p < 0.05, p < 0.01, and p < 0.01). However, we could not show that the fiber-dependent size decrease was dependent on GLP-1 receptor, GLP-2 receptor or TGR5 signaling. Intestinal permeability was increased following the removal of fiber for 112 days. In conclusion, our study highlights the importance of dietary fiber to maintain intestinal weight, colonic L-cell secretion and intestinal integrity.

Epicardial, pericardial fat and glucagon-like peptide-1 and -2 receptors expression in stable patients with multivessel coronary artery disease: an association with the renin-angiotensin-aldosterone system.

INTRODUCTION: The expression of glucagon­like peptide receptors (GLP­Rs) in epicardial fat (EF) and pericardial fat (PF) depots might be involved in the pathogenesis of cardiovascular diseases. OBJECTIVES: We sought to evaluate the messenger RNA (mRNA) expressions of GLP­1R and GLP­2R in EF and PF and their associations with the renin­angiotensin­aldosterone system (RAAS) in patients with multivessel coronary artery disease (CAD). PATIENTS AND METHODS: Consecutive stable patients with multivessel CAD requiring elective coronary artery bypass grafting were enrolled. Clinical data, anthropometric parameters, and the quantity of fat depots (assessed by cardiovascular magnetic resonance and abdominal ultrasound) were obtained. Fat samples (EF, PF, subcutaneous fat) were taken from patients during cardiac surgery. Relative mRNA expression of GLP­1R, GLP­2R, and RAAS components (angiotensin II receptor type 1, angiotensinogen, angiotensin I-converting enzyme 1, and angiotensinI-converting enzyme 2) were assessed in those fat depots. RESULTS: Fifty­three patients (64.7 [7.4] years) were included in the final analysis. We found that only the relative expression of GLP­2R was lower in PF compared with subcutaneous fat (reference). Ultrasound abdominal fat depots were associated with both GLP­1R and GLP­2R in PF. GLP­1R and GLP­2R showed significant correlations with RAAS components in both EF and PF. CONCLUSIONS: In stable patients with MVD, the relative mRNA expression for both GLP receptors revealed significant associations with majority of analysed RAAS components.

A cross-platform approach identifies genetic regulators of human metabolism and health.

In cross-platform analyses of 174 metabolites, we identify 499 associations (P < 4.9 × 10-10) characterized by pleiotropy, allelic heterogeneity, large and nonlinear effects and enrichment for nonsynonymous variation. We identify a signal at GLP2R (p.Asp470Asn) shared among higher citrulline levels, body mass index, fasting glucose-dependent insulinotropic peptide and type 2 diabetes, with ß-arrestin signaling as the underlying mechanism. Genetically higher serine levels are shown to reduce the likelihood (by 95%) and predict development of macular telangiectasia type 2, a rare degenerative retinal disease. Integration of genomic and small molecule data across platforms enables the discovery of regulators of human metabolism and translation into clinical insights.

A Prognostic Model Based on the Immune-related Genes in Colon Adenocarcinoma.

Background: Immune-related genes (IRGs) are critically involved in the tumor microenvironment (TME) of colon adenocarcinoma (COAD). Here, the study was mainly designed to establish a prognostic model of IRGs to predict the survival of COAD patients. Methods: The Cancer Genome Atlas (TCGA), Immunology Database and Analysis Portal (ImmPort) database, and Cistrome database were utilized for extracting data regarding the expression of immune gene- and tumor-related transcription factors (TFs), aimed at the identification of differentially expressed genes (DEGs), differentially expressed IRGs (DEIRGs), and differentially expressed TFs (DETFs). Univariate Cox regression analysis was subsequently performed for the acquisition of prognosis-related IRGs, followed by establishment of TF regulatory network for uncovering the possible molecular regulatory association in COAD. Subsequently, multivariate Cox regression analysis was conducted to further determine the role of prognosis-related IRGs for prognostic prediction in COAD. Finally, the feasibility of a prognostic model with immunocytes was explored by immunocyte infiltration analysis. Results: A total of 2450 DEGs, 8 DETFs, and 79 DEIRGs were extracted from the corresponding databases. Univariate Cox regression analysis revealed 11 prognosis-related IRGs, followed by establishment of a regulatory network on prognosis-related IRGs at transcriptional levels. Functionally, IRG GLP2R was negatively modulated by TF MYH11, whereas IRG TDGF1 was positively modulated by TF TFAP2A. Multivariate Cox regression analysis was subsequently performed to establish a prognostic model on the basis of seven prognosis-related IRGs (GLP2R, ESM1, TDGF1, SLC10A2, INHBA, STC2, and CXCL1). Moreover, correlation analysis of immunocyte infiltration also revealed that the seven-IRG prognostic model was positively associated with five types of immunocytes (dendritic cell, macrophage, CD4 T cell, CD8 T cell, and neutrophil), which may directly reflect tumor immune state in COAD. Conclusions: Our present findings indicate that the prognostic model based on prognosis-related IRGs plays a crucial role in the clinical supervision and prognostic prediction of COAD patients at both molecular and cellular levels.

Pharmacological activation of TGR5 promotes intestinal growth via a GLP-2-dependent pathway in mice.

Glucagon-like peptide (GLP)-1 and -2-secreting L cells have been shown to express the bile acid receptor Takeda G protein-receptor-5 (TGR5) and increase secretion upon receptor activation. Previous studies have explored GLP-1 secretion following acute TGR5 activation, but chronic activation and GLP-2 responses have not been characterized. In this study, we aimed to investigate the consequences of pharmacological TGR5 receptor activation on L cell hormone production in vivo using the specific TGR5 agonist RO5527239 and the GLP-2 receptor knockout mouse. Here, we show that 1) TGR5 receptor activation led to increased GLP-1 and GLP-2 content in the colon, which 2) was associated with an increased small intestinal weight that 3) was GLP-2 dependent. Additionally, we report that TGR5-mediated gallbladder filling occurred independently of GLP-2 signaling. In conclusion, we demonstrate that pharmacological TGR5 receptor activation stimulates L cells, triggering GLP-2-dependent intestinal adaption in mice.NEW & NOTEWORTHY Using the specific Takeda G protein-receptor-5 (TGR5) agonist RO5527239 and GLP-2 receptor knockout mice, we show that activation of TGR5 led to the increase in colonic GLP-1 and GLP-2 concomitant with a GLP-2 dependent growth response in the proximal portion of the small intestine.

Potentiation of Glucagon-Like Peptide-2 Dynamics by Methotrexate Administration in Rat Small Intestine.

The aim of this study was to clarify the relationship between chemotherapy-induced mucositis and endogenous glucagon-like peptide-2 (GLP-2) dynamics in the small intestine following treatment with either methotrexate or 5-fluorouracil. Rats were injected intraperitoneally with a single dose of either 50 mg/kg methotrexate or 100 mg/kg 5-fluorouracil. At 24 and 72 h after drug administration, ileal tissues and plasma were used to investigate GLP-2 dynamics. Administration of methotrexate caused moderate but not significant intestinal injury within 72 h, while administration of 5-fluorouracil caused severe injury in a time-dependent manner. Methotrexate significantly increased proglucagon mRNA expression and the number of anti-GLP-2 antibody-positive cells in the ileal tissue at 24 h after administration. Methotrexate also significantly induced GLP-2 receptor, insulin-like growth factor-1 (IGF-1), and transforming growth factor-ß2 (TGF-ß2) mRNA expression in ileal tissue. In contrast, 5-fluorouracil significantly inhibited proglucagon, GLP-2 receptor, IGF-1, and TGF-ß2 mRNA expression as well as the number of anti-GLP-2 antibody-positive cells. Methotrexate slightly increased dipeptidyl peptidase IV (DPP-4) mRNA expression, whereas 5-fluorouracil significantly increased DPP-4 mRNA expression. These results suggest that potentiation of endogenous GLP-2 dynamics by methotrexate is associated with a mechanism that preserves gastrointestinal mucosal integrity at a moderate level.

Aspartame supplementation in starter accelerates small intestinal epithelial cell cycle and stimulates secretion of glucagon-like peptide-2 in pre-weaned lambs.

The objective of this study was to test the hypothesis that aspartame supplementation in starter diet accelerates small intestinal cell cycle by stimulating secretion and expression of glucagon-like peptide -2 (GLP-2) in pre-weaned lambs using animal and cell culture experiments. In vivo, twelve 14-day-old lambs were selected and allocated randomly to two groups; one was treated with plain starter diet (Con, n = 6) and the other was treated with starter supplemented with 200 mg of aspartame/kg starter (APM, n = 6). Results showed that the lambs received APM treatment for 35 d had higher (p < .05) GLP-2 concentration in the plasma and greater jejunum weight/live body weight (BW) and jejunal crypt depth. Furthermore, APM treatment significantly upregulated (p < .05) the mRNA expression of cyclin D1 in duodenum; and cyclin A2, cyclin D1, cyclin-dependent kinases 6 (CDK6) in jejunum; and cyclin A2, cyclin D1, CDK4 in ileum. Moreover, APM treatment increased (p < .05) the mRNA expression of glucagon (GCG), insulin-like growth factor 1 (IGF-1) in the jejunum and ileum and mRNA expression of GLP-2 receptor (GLP-2R) in the jejunum. In vitro, when jejunal cells were treated with GLP-2 for 2 hr, the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) OD, IGF-1 concentration, and the mRNA expression of IGF-1, cyclin D1 and CDK6 were increased (p < .05). Furthermore, IGF-1 receptor (IGF-1R) inhibitor decreased (p < .05) the mRNA expression of IGF-1, cyclin A2, cyclin D1 and CDK6 in GLP-2 treatment jejunal cells. These results suggest that aspartame supplementation in starter accelerates small intestinal cell cycle that may, in part, be related to stimulate secretion and expression of GLP-2 in pre-weaning lambs. Furthermore, GLP-2 can indirectly promote the proliferation of jejunal cells mainly through the IGF-1 pathway. These findings provide new insights into nutritional interventions that promote the development of small intestines in young ruminants.

Localization of Glucagon-Like Peptide-2 Receptor Expression in the Mouse.

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.

The effect of body mass index on glucagon-like peptide receptor gene expression in the post mortem brain from individuals with mood and psychotic disorders.

There is an increasing interest in the putative role of glucagon-like peptide 1 receptor (GLP-1R) agonists as novel therapeutic agents for mental disorders. Herein, we investigated the expressions of GLP-1R and GLP-2R genes, and its relationship with body mass index (BMI), in the post-mortem brain tissue of patients with mood (MD) and psychotic disorders. Brain samples were localized to the dorsolateral prefrontal cortex (dlPFC) (n = 459) and hippocampus (n = 378). After adjustment for age, sex, ethnicity, post-mortem interval (PMI) and BMI, we observed significant differences, between healthy controls and MD subjects, in GLP-1R and GLP-2R gene expression in the dlPFC (ß = 1.504, p = 0.004; and ß = 1.305, p = 0.011, respectively); whereas in the hippocampus, only GLP-1R expression was significantly associated with MD (ß = -1.28, p = 0.029). No significant differences were found in relation to schizophrenia. In addition, we observed a moderating effect of MD diagnosis on the associations between BMI, GLP-1R and GLP-2R expression values in the dlPFC (ß = -0.05, p = 0.003; and ß = -0.04, p = 0.004, respectively). There was a similar moderating effect for GLP-1R in the hippocampus (ß = 0.043, 95% CI 0.003; 0.08 p = 0.03), but in an opposite direction than observed in the dlPFC. This is the first evidence of abnormal gene expression of GLP-1R and GLP-2R in postmortem brain of individuals with MD, providing a rationale for further inquiry and proof of principle interventional studies.

GLP-2 Dysregulates Hepatic Lipoprotein Metabolism, Inducing Fatty Liver and VLDL Overproduction in Male Hamsters and Mice.

Fundamental complications of insulin resistance and type 2 diabetes include the development of nonalcoholic fatty liver disease and an atherogenic fasting dyslipidemic profile, primarily due to increases in hepatic very-low-density lipoprotein (VLDL) production. Recently, central glucagon-like peptide-2 receptor (GLP2R) signaling has been implicated in regulating hepatic insulin sensitivity; however, its role in hepatic lipid and lipoprotein metabolism is unknown. We investigated the role of glucagon-like peptide-2 (GLP-2) in regulating hepatic lipid and lipoprotein metabolism in Syrian golden hamsters, C57BL/6J mice, and Glp2r-/- mice consuming either a normal chow or high-fat diet (HFD). In the chow-fed hamsters, IP GLP-2 administration significantly increased fasting dyslipidemia, hepatic VLDL production, and the expression of key genes involved in hepatic de novo lipogenesis. In HFD-fed hamsters and chow-fed mice, GLP-2 administration exacerbated or induced hepatic lipid accumulation. HFD-fed Glp2r-/- mice displayed reduced glucose tolerance, VLDL secretion, and microsomal transfer protein lipid transfer activity, as well as exacerbated fatty liver. Thus, we conclude that GLP-2 plays a lipogenic role in the liver by increasing lipogenic gene expression and inducing hepatic steatosis, fasting dyslipidemia, and VLDL overproduction. In contrast, the lack of Glp2r appears to interfere with VLDL secretion, resulting in enhanced hepatic lipid accumulation. These studies have uncovered a role for GLP-2 in maintaining hepatic lipid and lipoprotein homeostasis.

GLP-1 Receptor Expression Within the Human Heart.

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.

Oral Bifidobacterium longum expressing GLP-2 improves nutrient assimilation and nutritional homeostasis in mice.

Bifidobacterium has been developed for the oral delivery of peptides and has the added beneficial effect on our bodies through its probiotic properties. Here, we utilize Bifidobacterium as a delivery system to orally deliver Glucagon like peptide-2 (GLP-2). We constructed vector derived from pET-31b(+) to construct a Bifidobacterium longum expressing GLP-2. We then determined the bioactivity of recombinant Bifidobacterium in Caco-2 cells. Finally, we quantified newly synthesized ApoB48 and chylomicron production in mice infused with exogenous GLP-2 or Bifidobacterium expressing GLP-2. Results based on secretion of the triglyceride (TG)-rich lipoprotein (TRL)-ApoB48 and secretion of chylomicron revealed that recombinant Bifidobacterium was efficient in treating intestinal dysfunction,suggesting an alternative way to use Bifidobacterium as a delivery system to deliver GLP-2 for gastrointestinal nutrition coordination.

Genetic determinants of circulating GIP and GLP-1 concentrations.

The secretion of insulin and glucagon from the pancreas and the incretin hormones glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic peptide (GIP) from the gastrointestinal tract is essential for glucose homeostasis. Several novel treatment strategies for type 2 diabetes (T2D) mimic GLP-1 actions or inhibit incretin degradation (DPP4 inhibitors), but none is thus far aimed at increasing the secretion of endogenous incretins. In order to identify new potential therapeutic targets for treatment of T2D, we performed a meta-analysis of a GWAS and an exome-wide association study of circulating insulin, glucagon, GIP, and GLP-1 concentrations measured during an oral glucose tolerance test in up to 7,828 individuals. We identified 6 genome-wide significant functional loci associated with plasma incretin concentrations in or near the SLC5A1 (encoding SGLT1), GIPR, ABO, GLP2R, F13A1, and HOXD1 genes and studied the effect of these variants on mRNA expression in pancreatic islet and on metabolic phenotypes. Immunohistochemistry showed expression of GIPR, ABO, and HOXD1 in human enteroendocrine cells and expression of ABO in pancreatic islets, supporting a role in hormone secretion. This study thus provides candidate genes and insight into mechanisms by which secretion and breakdown of GIP and GLP-1 are regulated.

Differential expression of glucagon-like peptide-2 (GLP-2) is involved in pancreatic islet cell adaptations to stress and beta-cell survival.

Recent studies have confirmed that locally released proglucagon derived gene products, other than glucagon, have a major influence on pancreatic endocrine function. We assessed the impact of glucagon-like peptide-2 (GLP-2) on beta-cell secretory function, proliferation and apoptosis, as well as glucose tolerance, feeding behaviour and islet adaptions to chemically-induced insulin deficiency and resistance. The GLP-2 receptor was evidenced on cultured rodent and human beta-cells, rodent alpha-cells and isolated mouse islets. GLP-2 had no effect on insulin secretion from beta-cells, or isolated mouse islets. In vivo, GLP-2 administration significantly (P<0.05 to P<0.01) decreased food intake in mice. Conversely, GLP-2 had no discernible effects on glucose disposal or insulin secretion. As expected, streptozotocin treatment decreased and hydrocortisone increased beta-cell mass in mice. GLP-2 was visualised in mouse islets and intestinal L-cells. Islet GLP-2 co-localisation with glucagon was significantly decreased (P<0.01) by both streptozotocin and hydrocortisone. In contrast, both interventions increased (P<0.05) co-localisation of GLP-2 with somatostatin. Interestingly, GLP-2 positive cells were reduced (P<0.05) in the intestines of streptozotocin, but not hydrocortisone, treated mice. Further in vitro investigations revealed that GLP-2 protected rodent and human 1.1B4 beta-cells against streptozotocin induced DNA damage. Furthermore, GLP-2 augmented (P<0.05) BRIN BD11 beta-cell proliferation, but was less efficacious in 1.1B4 cells. These data highlight the involvement of GLP-2 receptor signalling in the adaptations to pancreatic islet cell stress.