Mutational Landscape of the Proglucagon-Derived Peptides.

Strong efforts have been placed on understanding the physiological roles and therapeutic potential of the proglucagon peptide hormones including glucagon, GLP-1 and GLP-2. However, little is known about the extent and magnitude of variability in the amino acid composition of the proglucagon precursor and its mature peptides. Here, we identified 184 unique missense variants in the human proglucagon gene GCG obtained from exome and whole-genome sequencing of more than 450,000 individuals across diverse sub-populations. This provides an unprecedented source of population-wide genetic variation data on missense mutations and insights into the evolutionary constraint spectrum of proglucagon-derived peptides. We show that the stereotypical peptides glucagon, GLP-1 and GLP-2 display fewer evolutionary alterations and are more likely to be functionally affected by genetic variation compared to the rest of the gene products. Elucidating the spectrum of genetic variations and estimating the impact of how a peptide variant may influence human physiology and pathophysiology through changes in ligand binding and/or receptor signalling, are vital and serve as the first important step in understanding variability in glucose homeostasis, amino acid metabolism, intestinal epithelial growth, bone strength, appetite regulation, and other key physiological parameters controlled by these hormones.

Deficiency of glucagon gene-derived peptides induces peripheral polyneuropathy in mice.

Although diabetic polyneuropathy (DPN) is the commonest diabetic complication, its pathology remains to be clarified. As previous papers have suggested the neuroprotective effects of glucagon-like peptide-1 in DPN, the current study investigated the physiological indispensability of glucagon gene-derived peptides (GCGDPs) including glucagon-like peptide-1 in the peripheral nervous system (PNS). Neurological functions and neuropathological changes of GCGDP deficient (gcg-/-) mice were examined. The gcg-/- mice showed tactile allodynia and thermal hyperalgesia at 12-18 weeks old, followed by tactile and thermal hypoalgesia at 36 weeks old. Nerve conduction studies revealed a decrease in sensory nerve conduction velocity at 36 weeks old. Pathological findings showed a decrease in intraepidermal nerve fiber densities. Electron microscopy revealed a decrease in circularity and an increase in g-ratio of myelinated fibers and a decrease of unmyelinated fibers in the sural nerves of the gcg-/- mice. Effects of glucagon on neurite outgrowth were examined using an ex vivo culture of dorsal root ganglia. A supraphysiological concentration of glucagon promoted neurite outgrowth. In conclusion, the mice with deficiency of GCGDPs developed peripheral neuropathy with age. Furthermore, glucagon might have neuroprotective effects on the PNS of mice. GCGDPs might be involved in the pathology of DPN.

Role of glucagon-like peptides in inflammatory bowel diseases-current knowledge and future perspectives.

Inflammatory bowel diseases (IBD), including Crohn's disease (CD) and ulcerative colitis (UC), are chronic, relapsing, intestinal inflammatory disorders with complex and yet unrevealed pathogenesis in which genetic, immunological, and environmental factors play a role. Nowadays, a higher proportion of elderly IBD patients with coexisting conditions, such as cardiovascular disease and/or diabetes is recorded, who require more complex treatment and became a great challenge for gastroenterologists. Furthermore, some patients do not respond to anti-IBD therapy. These facts, together with increasing comorbidities in patients with IBD, imply that urgent, more complex, novel therapeutic strategies in the treatment are needed. Glucagon-like peptides (GLPs) possess numerous functions in the human body such as lowering blood glucose level, controlling body weight, inhibiting gastric emptying, reducing food ingestion, increasing crypt cell proliferation, and improving intestinal growth and nutrient absorption. Thus, GLPs and dipeptidyl peptidase IV (DPP-IV) inhibitors have recently gained attention in IBD research. Several animal models showed that treatment with GLPs may lead to improvement of colitis. This review presents data on the multitude effects of GLPs in the inflammatory intestinal diseases and summarizes the current knowledge on GLPs, which have the potential to become a novel therapeutic option in IBD therapy.

Roles of Gut-Derived Secretory Factors in the Pathogenesis of Non-Alcoholic Fatty Liver Disease and Their Possible Clinical Applications.

The rising prevalence of non-alcoholic fatty liver disease (NAFLD) parallels the global increase in the number of people diagnosed with obesity and metabolic syndrome. The gut-liver axis (GLA) plays an important role in the pathogenesis of NAFLD/non-alcoholic steatohepatitis (NASH). In this review, we discuss the clinical significance and underlying mechanisms of action of gut-derived secretory factors in NAFLD/NASH, focusing on recent human studies. Several studies have identified potential causal associations between gut-derived secretory factors and NAFLD/NASH, as well as the underlying mechanisms. The effects of gut-derived hormone-associated drugs, such as glucagon-like peptide-1 analog and recombinant variant of fibroblast growth factor 19, and other new treatment strategies for NAFLD/NASH have also been reported. A growing body of evidence highlights the role of GLA in the pathogenesis of NAFLD/NASH. Larger and longitudinal studies as well as translational research are expected to provide additional insights into the role of gut-derived secretory factors in the pathogenesis of NAFLD/NASH, possibly providing novel markers and therapeutic targets in patients with NAFLD/NASH.

Discovery of a novel glucagon-like peptide (GCGL) and its receptor (GCGLR) in chickens: evidence for the existence of GCGL and GCGLR genes in nonmammalian vertebrates.

Glucagon (GCG), glucagon-related peptides, and their receptors have been reported to play important roles including the regulation of glucose homeostasis, gastrointestinal activity, and food intake in vertebrates. In this study, we identified genes encoding a novel glucagon-like peptide (named GCGL) and its receptor (GCGLR) from adult chicken brain using RACE and/or RT-PCR. GCGL was predicted to encode a peptide of 29 amino acids (cGCGL(1-29)), which shares high amino acid sequence identity with mammalian and chicken GCG (62-66%). GCGLR is a receptor of 430 amino acids and shares relatively high amino acid sequence identity (53-55%) with the vertebrate GCG receptor (GCGR). Using a pGL3-CRE-luciferase reporter system, we demonstrated that synthetic cGCGL(1-29), but not its structurally related peptides, i.e. exendin-4 and GCG, could potently activate GCGLR (EC(50): 0.10 nm) expressed in Chinese hamster ovary cells, indicating that GCGLR can function as a GCGL-specific receptor. RT-PCR assay revealed that GCGL expression is mainly restricted to several tissues including various brain regions, spinal cord, and testes, whereas GCGLR mRNA is widely expressed in adult chicken tissues with abundant expression noted in the pituitary, spinal cord, and various brain regions. Using synteny analysis, GCGL and GCGLR genes were also identified in the genomes of fugu, tetraodon, tilapia, medaka, coelacanth, and Xenopus tropicalis. As a whole, the discovery of GCGL and GCGLR genes in chickens and other nonmammalian vertebrates clearly indicates a previously unidentified role of GCGL-GCGLR in nonmammalian vertebrates and provides important clues to the evolutionary history of GCG and GCGL genes in vertebrates.

Structural and functional development of small intestine in intrauterine growth retarded porcine offspring born to gilts fed diets with differing protein ratios throughout pregnancy.

Protein level in the maternal diet plays a crucial role in fetal programming during pregnancy. Low or high protein level increases the risk of intrauterine growth retardation (IUGR). The aim of this study was to investigate the structural and functional development of the small intestine in piglets from sows fed a control (C, 12.1% protein), a high protein (HP, 30% protein), or a low protein (LP, 6.5% protein) diet during pregnancy. Newborns were classified as IUGR (birth weight ≤1.18 kg) and non-IUGR (birth weight >1.18 kg). The piglets were euthanized on postnatal day (PD)1, PD28 and PD188. The LP diet in non-IUGR neonates resulted in decreased body weight on PD1. The LP and HP diets resulted in both decreased body weight and delayed catch-up growth in the IUGR piglets. The HP and LP-diets increased the length of villi on PD1 in non-IUGRs but not in IUGRs. At birth, the expressions of Ki67 and active caspase 3 in mid-jejunum epithelium of HP and LP non-IUGR neonates were significantly lower as compared to C non-IUGRs whilst in IUGRs the respective expressions were as high as in C non-IUGRs. The postnatal dynamics of brush border enzyme activities and vacuolated enterocytes disappearance showed significant drop in enterocyte maturation in IUGR as compared to non-IUGR neonates. In conclusion, both HP and LP diets led to retarded development of non-IUGR piglets. In IUGR piglets both HP and LP diets resulted in delayed catch-up growth, without adaptive changes in brush border digestive enzymes.

[The physiology of incretins]. / Az inkretinek elettana.

The discovery of incretins-glucagon-like peptide (GLP)-1 and glucose-dependent insulinotrop peptide (GIP)-, clarification of their physiological properties as well as therapeutic application of incretin-based blood glucose lowering drugs opened new perspectives in the medical management of type 2 diabetes. New results of basic research investigations led to revaluation of the role of GIP in metabolic processes and a more established use of GLP-1 action. The article overviews the most relevant data of production and effects of incretins, as well as future possibilities of their therapeutic use.

Incretin hormones and the expanding families of glucagon-like sequences and their receptors.

Peptide hormones encoded by the proglucagon (Gcg) and glucose-dependent insulinotropic polypeptide (Gip) genes are evolutionarily related glucagon-like sequences and act through a subfamily of G-protein-coupled receptors. A better understanding of the evolutionary history of these hormones and receptors should yield insight into their biological functions. The availability of a large number of near-complete vertebrate genome sequences is a powerful resource to address questions concerning the evolution of sequences; here, we utilize these resources to examine the evolution of glucagon-like sequences and their receptors. These studies led to the discovery of novel genes for a glucagon receptor-like receptor (Grlr) and a glucagon-like sequence (exendin) in vertebrates. Both exendin and GRLR have ancient origins, early in vertebrate evolution, but have been lost on the ancestral lineage leading to extant mammals. We also show that exendin and GRLR are both expressed in the brain of the chicken and Xenopus tropicals, results that suggest that the products of these genes function in this tissue. The lack of exendin or Grlr genes in mammals suggests that other genes may have acquired the functions of exendin and Grlr during mammalian evolution.

The glucagon-like peptides: pleiotropic regulators of nutrient homeostasis.

The glucagon-like peptides, GLP-1 and GLP-2, are cosecreted by intestinal L cells in response to nutrient ingestion. These peptides exert multiple effects on the gastrointestinal tract and pancreas to regulate the digestion, absorption, and assimilation of ingested nutrients, as well as providing feedback signals to the brain to modulate food intake. Tropic effects of GLP-1 and GLP-2 on their major peripheral target tissues, the beta cell and the intestinal epithelium, respectively, further enhance capacity for nutrient handling. When taken together, these findings demonstrate the diverse actions of the intestinal glucagon-like peptides to regulate nutrient homeostasis.

Conserved and non-conserved loci of the glucagon gene in old world ruminating ungulates.

The homology and diversification of genomic sequence encoding glucagon gene among native Egyptian buffalos, camel and sheep were tested using cattle as model. Oligodeoxynucleotide primers designed from the available GenBank data were used for PCR probing of the glucagon gene encoding sequence at different loci. The DNA oligomer probes were constructed to flank either the whole gene encoding sequence or different intra-gene encoding sequences. The PCR products were visualized using agarose gel electrophoresis. All species showed a same size band of prepro-glucagon when PCR was used to amplify the whole gene encoding sequence. In contrary, amplifications of different intra-gene loci failed to give the same results. The results indicated variable degrees of diversity among old world ruminating ungulates in the glucagon gene encoding sequence. Compared with other ruminants, the variation appears predominantly in camel. Surprisingly, the similarity in size between both amplification products of whole gene encoding sequence and the proposed size of glucagon cDNA definitely excludes the possibility of large intervening introns spanning the genomic sequence of the glucagon gene in these species. This indicates that, in contrast to other tested mammals, the glucagon gene includes an essentially full-length copy of glucagon mRNA. The study revealed a possible new aspect of glucagon gene evolution in order to correlate its corresponding protein function among different ruminant species.

Human duodenal enteroendocrine cells: source of both incretin peptides, GLP-1 and GIP.

Among the products of enteroendocrine cells are the incretins glucagon-like peptide-1 (GLP-1, secreted by L cells) and glucose-dependent insulinotropic peptide (GIP, secreted by K cells). These are key modulators of insulin secretion, glucose homeostasis, and gastric emptying. Because of the rapid early rise of GLP-1 in plasma after oral glucose, we wished to definitively establish the absence or presence of L cells, as well as the relative distribution of the incretin cell types in human duodenum. We confirmed the presence of proglucagon and pro-GIP genes, their products, and glucosensory molecules by tissue immunohistochemistry and RT-PCR of laser-captured, single duodenal cells. We also assayed plasma glucose, incretin, and insulin levels in subjects with normal glucose tolerance and type 2 diabetes for 120 min after they ingested 75 g of glucose. Subjects with normal glucose tolerance (n=14) had as many L cells (15+/-1), expressed per 1,000 gut epithelial cells, as K cells (13+/-1), with some containing both hormones (L/K cells, 5+/-1). In type 2 diabetes, the number of L and L/K cells was increased (26+/-2; P<0.001 and 9+/-1; P < 0.001, respectively). Both L and K cells contained glucokinase and glucose transporter-1, -2, and -3. Newly diagnosed type 2 diabetic subjects had increased plasma GLP-1 levels between 20 and 80 min, concurrently with rising plasma insulin levels. Significant coexpression of the main incretin peptides occurs in human duodenum. L and K cells are present in equal numbers. New onset type 2 diabetes is associated with a shift to the L phenotype.

Evolution of new hormone function: loss and gain of a receptor.

The vertebrate proglucagon gene encodes three glucagon-like sequences (glucagon, glucagon-like peptide-1 [GLP-1], and glucagon-like peptide 2 [GLP-2]) that have distinct functions in regulating metabolism in mammals. In contrast, glucagon and GLP-1 have similar physiological actions in fish, that of mammalian glucagon. We have identified sequences similar to receptors for proglucagon-derived peptides from the genomes of two fish (pufferfish and zebrafish), a frog (Xenopus tropicalis), and a bird (chicken). Phylogenetic analysis of the receptor sequences suggested an explanation for the divergent function of GLP-1 in fish and mammals. The phylogeny of our predicted and characterized receptors for proglucagon-derived peptides demonstrate that receptors for glucagon, GLP-1, and GLP-2 have an origin before the divergence of fish and mammals; however, fish have lost the gene encoding the GLP-1 class of receptors, and likely the incretin action of GLP-1. Receptors that bind GLP-1, but yield glucagon-like action, have been characterized in goldfish and zebrafish, and these sequences are most closely related to glucagon receptors. Both pufferfish and zebrafish have a second glucagon receptor-like gene that is most closely related to the characterized goldfish glucagon receptor. The phylogeny of glucagon receptor-like genes in fish indicates that a duplication of the glucagon receptor gene occurred on the ancestral fish lineage, and could explain the shared action of glucagon and GLP-1. We suggest that the binding specificity of one of the duplicated glucagon receptors has diverged, yielding receptors for GLP-1 and glucagon, but that ancestral downstream signaling has been maintained, resulting in both receptors retaining glucagon-stimulated downstream effects.

Proglucagon processing in islet and intestinal cell lines.

To investigate the factors involved in the post-translational processing of proglucagon, we have examined the proglucagon-derived peptides (PGDPs) expressed in normal mouse pancreas and intestine, as well as in both islet (InR1-G9, RIN 1056A) and intestinal (STC-1) cell lines. N-terminal proglucagon processing was similar to that of normal mouse pancreas in InR1-G9 cells, but differed in RIN 1056A and STC-1 cells, which contained significant amounts of glucagon as well as the intestinal PGDPs, glicentin and oxyntomodulin. The C-terminal end of proglucagon was processed to small amounts of glucagon-like peptide-1 in InR1-G9 and RIN 1056A cells, as in normal pancreas, while processing was more extensive in both STC-1 cells and normal intestine. Northern blot analysis of mRNA transcripts for the prohormone convertases, PC1 and PC2, in the 3 cell lines demonstrated correlations between PC2 and the presence of glucagon, as well as between PC1 and production of the intestinal PGDPs. These findings provide support for the suggestion that PC1 and PC2 play roles in the tissue-specific post-translational processing of proglucagon.

Early regional expression and secretion of peptide YY and enteroglucagon after massive resection of small bowel.

BACKGROUND: Previous studies suggest that peptide YY (PYY) and enteroglucagon have an important role in intestinal adaptation after massive small bowel resection. This study was done to define the mechanisms, timing, and anatomic distribution of the PYY and enteroglucagon response. STUDY DESIGN: Lewis rats underwent resection of 70 percent of the small bowel (leaving equal segments of jejunum and ileum), transection, or laparotomy alone. Jejunum, ileum, and colon were compared in resected, transected, and control bowel six hours, 24 hours, one week, and two weeks postoperatively. RESULTS: Analysis of DNA, RNA, and protein per cm of bowel demonstrated hyperplastic changes. Radioimmunoassay revealed plasma PYY and enteroglucagon to be significantly elevated 24 hours after resection and they remained so through week two. In contrast, tissue PYY and enteroglucagon content decreased significantly in all tissues (p < 0.05) after resection. Reverse transcriptase polymerase chain reaction and Southern blot analysis demonstrated an immediate and sustained increase in PYY messenger RNA (mRNA) in both the ileum (fourfold) and in the colon (2.5-fold) at six hours (p < 0.05). A gradual increase in PYY mRNA was also demonstrated in the jejunum with significance at two weeks (p < 0.05). Proglucagon mRNA was significantly higher in the jejunum, compared with the ileum and colon, at 24 hours, one week, and two weeks postresection. CONCLUSIONS: Alterations in PYY and enteroglucagon synthesis occur early in the ileum and colon after massive small bowel resection. The residual jejunum, however, is primarily responsible for the adaptive hyperenteroglucagonemia. These findings suggest that although PYY and enteroglucagon are colocalized to the same cell type, there is a gene-specific response for these two peptides after resection.

Expression of ileal glucagon and peptide tyrosine-tyrosine genes. Response to inhibition of polyamine synthesis in the presence of massive small-bowel resection.

Massive small-bowel resection results in a marked adaptive response in the residual terminal ileum. Increased polyamine synthesis is a necessary component of this response. The ileal L-cell-derived peptides enteroglucagon and peptide tyrosine tyrosine (PYY) have been implicated as humoral mediators of this response. We have previously reported a rapid and sustained increase in glucagon mRNA concentrations after massive small-bowel resection. In this study using an inhibitor of the rate-limiting enzyme in polyamine biosynthesis, ornithine decarboxylase, we have demonstrated that the response of the glucagon and PYY genes to massive small-bowel resection is dependent on polyamine biosynthesis. In addition, we have examined the response of both the ornithine decarboxylase and c-jun genes in this model of intestinal adaptation.

Ontogeny of glucagon messenger RNA and encoded precursor in the rat intestine.

The ontogeny of proglucagon mRNA and encoded precursor was studied in rat intestine from day 11 of fetal gestation (E11) to maturity. The earliest time point for detection of proglucagon antigenic determinants by immunocytochemistry, and of proglucagon mRNA by in situ hybridization histochemistry, was day 14 of fetal gestation (E14), suggesting this time as the point of onset of intestinal proglucagon gene expression and mRNA translation. Between day 17 and 18 of gestation (E17 and E18) there was a significant 10 fold increase in intestinal L cell density, indicating that this time in gestation is one of increased L cell differentiation and/or proliferation. Proglucagon mRNA abundance in developing rat intestine showed a major 8 fold increase between E17 and E18. Similar magnitude of increases in L cell density and proglucagon mRNA abundance suggests that the increase in proglucagon mRNA abundance reflects an increase in L cell numbers rather than increases in proglucagon gene transcription or mRNA stability per cell.