Expression of the GCG gene and secretion of active glucagon-like peptide-1 varies along the length of intestinal tract in horses.

BACKGROUND: Active glucagon-like peptide-1 (aGLP-1) has been implicated in the pathogenesis of equine insulin dysregulation (ID), but its role is unclear. Cleavage of proglucagon (coded by the GCG gene) produces aGLP-1 in enteral L cells. OBJECTIVES: The aim in vivo was to examine the sequence of the exons of GCG in horses with and without ID, where aGLP-1 was higher in the group with ID. The aims in vitro were to identify and quantify the expression of GCG in the equine intestine (as a marker of L cells) and determine intestinal secretion of aGLP-1. STUDY DESIGN: Genomic studies were case-control studies. Expression and secretion studies in vitro were cross-sectional. METHODS: The GCG gene sequence of the exons was determined using a hybridisation capture protocol. Expression and quantification of GCG in samples of stomach duodenum, jejunum, ileum, caecum and ascending and descending colon was achieved with droplet digital PCR. For secretory studies tissue explants were incubated with 12 mM glucose and aGLP-1 secretion was measured with an ELISA. RESULTS: Although the median [IQR] post-prandial aGLP-1 concentrations were higher (p = 0.03) in animals with ID (10.2 [8.79-15.5]), compared with healthy animals (8.47 [6.12-11.7]), there was 100% pairwise identity of the exons of the GCG sequence for the cohort. The mRNA concentrations of GCG and secretion of aGLP-1 differed (p < 0.001) throughout the intestine. MAIN LIMITATIONS: Only the exons of the GCG gene were sequenced and breeds were not compared. The horses used for the study in vitro were not assessed for ID and different horses were used for the small, and large, intestinal studies. CONCLUSIONS: Differences in post-prandial aGLP-1 concentration were not due to a variant in the exons of the GCG gene sequence in this cohort. Both the large and small intestine are sites of GLP-1 secretion.

Targeted Analysis of Three Hormonal Systems Identifies Molecules Associated with the Presence and Severity of NAFLD.

AIMS: To investigate circulating levels and liver gene expression of 3 hormonal pathways associated with obesity, insulin resistance, and inflammation to identify leads towards potential diagnostic markers and therapeutic targets in patients with nonalcoholic fatty liver disease (NAFLD). METHODS: We compared circulating levels of (1) proglucagon-derived hormones (glucagon-like peptide [GLP]-1, GLP-2, glicentin, oxyntomodulin, glucagon, major proglucagon fragment [MPGF]), (2) follistatins-activins (follistatin-like [FSTL]3, activin B), (3) IGF axis (insulin-like growth factor [IGF]-1, total and intact IGF binding protein [IGFBP]-3 and IGFBP-4, and pregnancy-associated plasma protein [PAPP]-A) in 2 studies: (1) 18 individuals with early stage NAFLD versus 14 controls (study 1; early NAFLD study) and in (2) 31 individuals with biopsy proven NAFLD (15 with simple steatosis [SS] and 16 with nonalcoholic steatohepatitis [NASH]), vs 50 controls (24 lean and 26 obese) (study 2). Liver gene expression was assessed in 22 subjects (12 controls, 5 NASH, 5 NASH-related cirrhosis). RESULTS: Patients in early stages of NAFLD demonstrate higher fasting MPGF and lower incremental increase of glicentin during oral glucose tolerance test than controls. In more advanced stages, FSTL3 levels are higher in NASH than simple steatosis and, within NAFLD patients, in those with more severe lobular and portal inflammation. The IGF-1/intact IGFBP-3 ratio is lower in patients with liver fibrosis. Genes encoding follistatin, activin A, activin B, and the IGF-1 receptor are higher in NASH. CONCLUSION: MPGF and glicentin may be involved in early stages of NAFLD, whereas FSTL3 and IGF-1/intact IGFBP3 in the progression to NASH and liver fibrosis respectively, suggesting potential as diagnostic markers or therapeutic targets.

Postabsorptive hyperglucagonemia in patients with type 2 diabetes mellitus analyzed with a novel enzyme-linked immunosorbent assay.

AIMS/INTRODUCTION: The aims of the present study were to investigate the performance of a novel sandwich enzyme-linked immunosorbent assay (ELISA) for measuring glucagon (1-29) with monoclonal antibodies against both the C- and N-terminal regions of glucagon (1-29), and to analyze the differences in plasma levels and responses of glucagon (1-29) to oral glucose loading in normal glucose tolerance (NGT) subjects and patients with type 2 diabetes mellitus. MATERIALS AND METHODS: The cross-reactivity against proglucagon fragments using the ELISA kit and two types of conventional radioimmunoassay (RIA) kits was evaluated. A 75-g oral glucose tolerance test was carried out with NGT subjects and patients with type 2 diabetes mellitus, and the glucagon (1-29) concentration was measured using three types of kit. RESULTS: The ELISA kit clearly had the lowest cross-reactivity against miniglucagon (19-29) and glicentin (1-61). The oral glucose tolerance test was carried out with 30 NGT and 17 patients with type 2 diabetes mellitus. The glucagon (1-29) levels measured by the ELISA kit after glucose loading were significantly higher at all time-points in the type 2 diabetes mellitus group than in the NGT group. However, the glucagon (1-29) levels measured by one RIA kit were significantly higher in the NGT group, and those measured with the other RIA kit were approximately the same among the groups. CONCLUSIONS: The novel sandwich ELISA accurately determines plasma glucagon (1-29) concentrations with much less cross-reactivity against other proglucagon fragments than conventional RIA kits.

Ectopic expression of GIP in pancreatic ß-cells maintains enhanced insulin secretion in mice with complete absence of proglucagon-derived peptides.

Glucagon and glucagon-like peptide-1 (GLP-1) are produced in pancreatic α-cells and enteroendocrine L-cells, respectively, in a tissue-specific manner from the same precursor, proglucagon, that is encoded by glucagon gene (Gcg), and play critical roles in glucose homeostasis. Here, we studied glucose homeostasis and ß-cell function of Gcg-deficient mice that are homozygous for a Gcg-GFP knock-in allele (Gcg(gfp/gfp)). The Gcg(gfp/gfp) mice displayed improved glucose tolerance and enhanced insulin secretion, as assessed by both oral glucose tolerance test (OGTT) and intraperitoneal glucose tolerance test (IPGTT). Responses of glucose-dependent insulinotropic polypeptide (GIP) to both oral and intraperitoneal glucose loads were unexpectedly enhanced in Gcg(gfp/gfp) mice, and immunohistochemistry localized GIP to pancreatic ß-cells of Gcg(gfp/gfp) mice. Furthermore, secretion of GIP in response to glucose was detected in isolated islets of Gcg(gfp/gfp) mice. Blockade of GIP action in vitro and in vivo by cAMP antagonism and genetic deletion of the GIP receptor, respectively, almost completely abrogated enhanced insulin secretion in Gcg(gfp/gfp) mice. These results indicate that ectopic GIP expression in ß-cells maintains insulin secretion in the absence of proglucagon-derived peptides (PGDPs), revealing a novel compensatory mechanism for sustaining incretin hormone action in islets.

Short communication: expression of peptide YY, proglucagon, neuropeptide Y receptor Y2, and glucagon-like peptide-1 receptor in bovine peripheral tissues.

The role of distal gut signals in control of feed intake and metabolism in cattle has received scant attention. Peptide YY (PYY) and glucagon-like peptide-1, which are secreted from enteroendocrine cells of the distal gut in monogastrics have several functions, including regulation of energy balance. However, little is known of the tissue expression of these peptides and their receptors in cattle. The aim of the current study was to characterize the tissue distribution of PYY, neuropeptide Y receptor Y2 (Y2), proglucagon (GCG), and glucagon-like peptide-1 receptor (GLP1R) in various peripheral tissues of cattle. Four male 7-wk-old dairy calves were euthanized and 16 peripheral tissues were collected. Conventional PCR and quantitative real-time PCR were performed to confirm tissue expression and quantify the transcript abundance in various tissues. The results of conventional PCR revealed that mRNA for both PYY and Y2 was detectable in the rumen, abomasum, duodenum, jejunum, ileum, and colon but not in other tissues. Quantitative real-time PCR data demonstrated that PYY mRNA was 2- to 3-fold greater in the pancreas, kidney, and heart relative to the liver. By conventional PCR, GCG mRNA was detected in the abomasum, duodenum, jejunum, ileum, and colon and GLP1R mRNA was expressed in all gut segments, pancreas, spleen, and kidney. Quantitative real-time PCR data demonstrated that, relative to transcript abundance in the liver, GCG mRNA was 4- to 40-fold higher from abomasum to colon, and GLP1R mRNA was 50- to 300-fold higher from the rumen to colon, 14-fold greater in the pancreas, 18-fold higher in the spleen, and 166-fold greater in the kidney. The tissue distribution of PYY, GCG, and their receptors observed in the current study is, in general, consistent with expression patterns in monogastrics. The predominant expression of PYY, Y2, and GCG in the gut, and the presence of GLP1R in multiple peripheral tissues suggest a role for PYY in controlling gut functions and for GLP-1 in regulating multiple physiological functions in cattle.

Effects of 2-hydroxy-4-methylthiobutyrate on portal plasma flow and net portal appearance of amino acids in piglets.

To determine whether portal plasma flow (PPF) and net portal appearance of amino acids (AA) could be affected by 2-hydroxy-4-methylthiobutyrate (HMB), six barrows (35-day-old, 8.6+/-1.4 kg), implanted with arterial, portal and mesenteric catheters, were fed a DL-methionine (as the control) or HMB-supplemented diet once hourly and infused intramesenterically with 1% p-amino hippurate. PPF was numerically 9% higher (P=0.09) in HMB-fed pigs than in controls over a 4-6 h period. Compared with controls, pigs fed the HMB diet had increased (P<0.05) net portal balance and/or appearance of leucine, isoleucine, histidine, arginine and alanine, but had decreased (P<0.05) portal appearance of glutamate over a 6-h period. The concentration of acetate in the lumen of the distal small intestine was higher (P=0.01) in HMB-fed pigs than in controls (25.14 vs. 7.64 mmol/kg). mRNA levels for proglucagon and endothelial nitric-oxide synthase (eNOS) in stomach and proximal small intestine, and mRNA levels for GLP-2 receptor (GLP-2R) in stomach were higher (P<0.05) in HMB-fed pigs compared with those in controls. Collectively, HMB supplementation increased concentrations of short-chain fatty acids in intestinal lumen, expression of proglucagon, GLP-2R, and eNOS genes, and net portal absorption of AA. These novel findings from the study with pigs may also have important implications for intestinal nutrition and health in humans.