Human Proglucagon Research Articles

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.

Yapsin 1 immunoreactivity in {alpha}-cells of human pancreatic islets: implications for the processing of human proglucagon by mammalian aspartic proteases.

Yapsin 1 is an aspartic protease from Saccharomyces cerevisiae and belongs to a class of aspartic proteases that demonstrate specificity for basic amino acids. It is capable of processing prohormone substrates at specific basic residue cleavage sites, similar to that of the prohormone convertases, to generate bioactive peptide hormones. An antibody raised against yapsin 1 was previously shown to immunostain endocrine cells of rat pituitary and brain as well as lysates from bovine pituitary secretory granules demonstrating the existence of yapsin 1-like aspartic proteases in mammalian endocrine tissues, potentially involved in peptide hormone production. Here, we show the specific staining of yapsin 1 immunoreactivity in the α-cells of human pancreatic islets. No staining was observed in the β- or δ-cells, indicating a specificity of the staining for glucagon-producing and not insulin- or somatostatin-producing cells. Purified yapsin 1 was also shown to process proglucagon into glucagon in vitro, demonstrating that the prototypical enzyme of this subclass of enzymes can correctly process proglucagon to glucagon. These findings suggest the existence of a yapsin 1-like enzyme exclusively in the α-cells of the islets of Langerhans in humans, which may play a role in the production of glucagon in that tissue.

Cloning of porcine proglucagon and effect of commensal bacteria on relative gene expression in the intestine of gnotobiotic pigs

Porcine proglucagon mRNA sequence was determined by designing primers based on homologous regions in bovine and human genes. The porcine sequence shared 90% nucleotide identity with human proglucagon; however, predicted Ser159Arg and Leu160Lys substitutions were observed. This newly identified sequence suggests that the intestinal trophic peptide, glucagon-like peptide 2 (GLP-2), is actually 35 amino acids in the pig rather than 33 amino acids, as previously reported. To determine the effect of different bacteria on intestinal proglucagon gene expression, 16 piglets were reared in gnotobiotic isolators maintained germ-free (GF), monoassociated with Lactobacillus fermentum (LF) or Escherichia coli (EC), or conventionalized (CV) in two separate experiments for 13 d. Cultured cecal contents confirmed microbial status with the exception of GF pigs in exp. 2, which were contaminated with Staphylococcus epidermidis (SE). Mean fold differences in ileal proglucagon expression (CV set to 1) were 1.0ab, 1.6a, 0.7b, 1.0ab for GF, LF, EC and CV in exp. 1 and 4.0a, 2.0b, 0.6c, 1.0c for SE, LF, EC and CV groups in exp. 2, respectively. This study, for the first time, accurately describes the full nucleotide sequence for porcine proglucagon, and shows that proglucagon expression is differentially regulated by bacteria colonizing intestine of neonatal piglets. Key words: Proglucagon, gnotobiotic, pig, gene expression, intestine

Biology of Incretins: GLP-1 and GIP

This review focuses on the mechanisms regulating the synthesis, secretion, biological actions, and therapeutic relevance of the incretin peptides glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1). The published literature was reviewed, with emphasis on recent advances in our understanding of the biology of GIP and GLP-1. GIP and GLP-1 are both secreted within minutes of nutrient ingestion and facilitate the rapid disposal of ingested nutrients. Both peptides share common actions on islet beta-cells acting through structurally distinct yet related receptors. Incretin-receptor activation leads to glucose-dependent insulin secretion, induction of beta-cell proliferation, and enhanced resistance to apoptosis. GIP also promotes energy storage via direct actions on adipose tissue, and enhances bone formation via stimulation of osteoblast proliferation and inhibition of apoptosis. In contrast, GLP-1 exerts glucoregulatory actions via slowing of gastric emptying and glucose-dependent inhibition of glucagon secretion. GLP-1 also promotes satiety and sustained GLP-1-receptor activation is associated with weight loss in both preclinical and clinical studies. The rapid degradation of both GIP and GLP-1 by the enzyme dipeptidyl peptidase-4 has led to the development of degradation-resistant GLP-1-receptor agonists and dipeptidyl peptidase-4 inhibitors for the treatment of type 2 diabetes. These agents decrease hemoglobin A1c (HbA1c) safely without weight gain in subjects with type 2 diabetes. GLP-1 and GIP integrate nutrient-derived signals to control food intake, energy absorption, and assimilation. Recently approved therapeutic agents based on potentiation of incretin action provide new physiologically based approaches for the treatment of type 2 diabetes.

A novel element regulates expression of the proximal human proglucagon promoter in islet cells

The human and rat proglucagon gene proximal promoter regions have differing transcriptional activities in pancreatic islet cell lines, with 300 bases of rat proglucagon 5′ flanking sequence being sufficient to support expression in rodent islet cell lines, while the homologous human sequences are transcriptionally silent. To better understand the changes in promoter activity between human and rat we have used a comparative approach and cloned promoters from diverse mammalian species and tested their transcriptional activities. Proglucagon gene proximal promoter regions from species representing three orders of mammals (rodents, artiodactyls, and carnivores) support transcription in rodent islet cell lines, while promoters from primates (human and rhesus monkey), despite significant sequence conservation, failed to drive reporter gene expression. These results suggest that nucleotide changes have occurred to the sequence of the proximal promoter region of the proglucagon gene during the evolution of primates that prevent them from supporting expression in rodent islet cell lines. Using hybrid human-rat proglucagon promoters and site-directed mutagenesis we identified a novel regulatory element in the human proglucagon proximal promoter, located between the G2 and G3 enhancer elements that is responsible for most of the difference in transcriptional activity between the human and rat proximal proglucagon promoters.

Intron 1 sequences are required for pancreatic expression of the human proglucagon gene

The mammalian proglucagon gene is expressed in pancreatic islet A-cells, intestinal L-cells, and select neurons of the brain, where posttranslational processing results in the liberation of a unique profile of peptides. Despite the importance of proglucagon-derived peptides in human biology, little is known about the regulation of the human gene, as the rat gene has been the preferred model for understanding the regulation of proglucagon gene expression. Previously, we have shown that although the immediate promoter region of the rat proglucagon gene is sufficient for expression in pancreatic islet cells, the homologous human proglucagon promoter sequences are not sufficient. We have now used a comparative genomic approach to identify noncoding sequences near the human proglucagon gene that are conserved among mammals, and thus potentially are regulatory sequences. Our alignments identified three evolutionarily conserved noncoding regions (ECR), one is the immediate promoter region (ECR1), the second is about 5 kb 5' to the mRNA start site (ECR2), and the third is near the 3' end of the first intron (ECR3). Our in vitro transient transfection assays with reporter gene constructs that include the human ECR3 support expression in rodent islet cell lines. Complementary studies with transgenic mice possessing a reporter gene regulated by a human proglucagon gene promoter-intron 1 (including ECR3) sequences express the reporter gene in the pancreas, as well as the intestine and selected neurons. These studies suggest that conserved sequences within intron 1 of the human proglucagon gene are important for expression in the pancreas.

Structure and expression of the chicken proglucagon gene

The rat and human proglucagon gene immediate promoter regions have differing transcriptional activities in islet cell lines. It is unclear upon which lineage the expression changes have occurred, or which base changes and transcription factors explain the changes in promoter activity. We have studied the expression of the chicken proglucagon gene to better understand changes in mammalian proglucagon gene promoter activity. The chicken proglucagon gene is composed of seven exons, and contains an additional exon that contains much of intervening peptide 1 that has not been seen in any other proglucagon gene. Despite very limited sequence identity between chicken and rat proglucagon promoters, the chicken proglucagon promoter supports reporter gene expression in rodent glucagon-expressing cell lines. These results suggest that the immediate proglucagon promoter region likely has retained ability to be expressed in islet cells, but a few changes to this sequence on the human lineage have extinguished expression of the immediate promoter region. Our work also demonstrate that in contrast to the conserved human proglucagon promoter region which has lost promoter activity that significant changes in proglucagon promoter sequence can occur yet retain expression potential.

Aberrant regulation of human intestinal proglucagon gene expression in the NCI-H716 cell line.

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.

Expression, purification, and PC1-mediated processing of human proglucagon, glicentin, and major proglucagon fragment

To examine the cleavage specificity of different members of the furin/propeptide convertase (PC) family of enzymes, we have selected proglucagon (PG) as a model substrate. PG was selected because it is subject to differential processing in vivo. PG is thought to be cleaved initially at an interdomain site to produce glicentin and the major proglucagon fragment (MPGF). These intermediates are subsequently cleaved, most likely by the convertases PC2 and PC1, respectively. To determine the exact sites within PG that are cleaved by PC1 and PC2, we attempted to produce milligram quantities of human PG, glicentin, and MPGF for use in an in vitro conversion assay. A methionine residue was added to the N-terminus of each protein to initiate translation. Purification was achieved using cation exchange and reversed-phase chromatography, and the integrity of the methionylated proteins was confirmed by both electrospray ionization-mass spectrometry and amino acid analysis. The combined expression and purification scheme is fast, efficient, and results in milligram quantities of ⩾95% pure proglucagon, ⩾95% pure MPGF, and ⩾93% pure glicentin. These prohormones are cleaved by PC1 to produce product peptides consistent with the processing of PG observed in vivo, and should therefore be suitable for further analysis of the post-translational processing of PG.

Transfection of pancreatic-derived beta-cells with a minigene encoding for human glucagon-like peptide-1 regulates glucose-dependent insulin synthesis and secretion.

Glucagon-like peptide-1 (GLP-1) is an incretin hormone derived from the proglucagon gene, capable of regulating the transcription of the three major genes that determine the pancreatic beta-cell-specific phenotype: insulin, GLUT-2, and glucokinase. The aim of this study was to investigate the potential role of GLP-1 for the gene therapy of glucose-insensitive pancreatic beta-cells. We transfected mouse insulinoma cells with a DNA fragment of the human proglucagon gene containing the nucleotide sequence encoding for human GLP-1 but lacking the coding region for glucagon. Two constructs were generated: In one, the expression of GLP-1 was under the control of the cytomegalovirus (CMV) promoter (CMV/GLP-1), and the second was regulated by the rat insulin II promoter (RIP)/GLP-1). Northern blot, HPLC, and RIA analyses confirmed that the minigene was transcribed and the protein appropriately translated, processed, and secreted in the extracellular environment. Gene expression studies revealed that although CMV/GLP-1 cells did not gain a greater glucose sensitivity as a result of the transfection with GLP-1, compared with cells transfected with the plasmid alone, RIP/GLP-1 was capable of regulating the gene expression of insulin and GLP-1 based on the concentration of glucose in the culture medium. Detection of the counterpart proteins (insulin and GLP-1) in the culture medium paralleled the observation derived from the Northern blot analysis. GLP-1 action was mediated by an IDX-1 (islet/duodenum homeobox-1) dependent transactivation of the endogenous insulin promoter, as demonstrated by gel shift analysis. This was further suggested by a significant increase of the glucose-dependent binding of IDX-1 to the insulin promoter in RIP/GLP-1 cells but not in CMV/GLP-1 cells or control cells. Finally, we observed that although the GLP-1-dependent secretion of insulin was mediated by an increase in cAMP levels, the transcription of the insulin gene, in response to GLP-1, was in large part cAMP independent. The present study lays the research foundation to investigate the potential use of GLP-1 for the gene or cell therapy of diabetes.

Human glucagon gene promoter sequences regulating tissue-specific versus nutrient-regulated gene expression.

The glucagon-like peptides (GLPs) are synthesized and secreted in a nutrient-dependent manner in rodents; however, the factors regulating human GLP-1 and GLP-2 biosynthesis remain unclear. To understand how nutrients regulate human proglucagon gene expression, we studied the expression of a human proglucagon promoter-growth hormone (GH) transgene in 1.6 human glucagon-GH transgenic mice. Fasting-refeeding significantly decreased and increased the levels of circulating mouse insulin and transgene-derived hGH (P < 0.05 fasting vs. refeeding) and decreased and upregulated, respectively, the levels of endogenous mouse proglucagon RNA in the ileum but not in the jejunum or colon. High-fiber feeding significantly increased the levels of glucose-stimulated circulating hGH and upregulated levels of mouse intestinal proglucagon gene expression in the jejunum, ileum, and colon (P < 0.05, 0 vs. 30% fiber diet). In contrast, neither fasting-refeeding nor a high-fiber diet upregulated the expression of the human proglucagon promoter-hGH transgene. These findings demonstrate that human proglucagon gene regulatory sequences specifying tissue-specific expression in gut endocrine cells are not sufficient for recognition of energy-derived signals regulating murine glucagon gene expression in enteroendocrine cells in vivo.

Structure, measurement, and secretion of human glucagon-like peptide-2

By using radioimmunoassays toward the cDNA-predicted amino acid sequence of human glucagon-like peptide-2, a peptide was isolated from extracts of human ileum. By mass spectrometry and Edman sequencing, this peptide was identified as human proglucagon 126-158. High-performance liquid chromatography analyses indicated that a similar immunoreactive peptide (iGLP-2) was present in human plasma. Human plasma concentrations of iGLP-2 were elevated 3- to 4-fold at 1 to 2 h after ingestion of 800 to 1200 kcal meals.

Divergent regulation of human and rat proglucagon gene promoters in vivo.

A single mammalian proglucagon gene is expressed in the brain, islets, and intestinal enteroendocrine cells, which gives rise to a unique profile of proglucagon-derived peptides (PGDPs) in each tissue. The biological importance of glucagon, glucagon-like peptide (GLP)-1, and GLP-2 has engendered considerable interest in the factors regulating the synthesis and secretion of the PGDPs in vivo. Although rat proglucagon gene transcription has been extensively studied, the factors important for control of human proglucagon gene expression have not been examined. We now report that, despite conservation of proximal promoter G1-G4 enhancer-like elements, human proglucagon reporter plasmids containing these elements are transcriptionally inactive in islet cell lines. Remarkably, larger human proglucagon promoter fragments, such as the 1604 hGLU-Luc, are expressed in GLUTag enteroendocrine cells but not in islet cell lines. A total of 5775 bases of human proglucagon promoter were required for expression in islet cell lines. Analysis of human proglucagon promoter expression in transgenic mice demonstrated that approximately 1.6 kb of human proglucagon gene sequences directs expression of a human growth hormone reporter gene to the brain and intestinal enteroendocrine cells but not islet cells in vivo. These findings provide the first evidence demonstrating divergence in the mechanisms utilized for tissue-specific regulation of the human and rodent proglucagon genes.

Naturally occurring products of proglucagon 111-160 in the porcine and human small intestine.

Recent studies have revealed that the glucagon gene is expressed in the mammalian intestine. Here it codes for "glicentin" (proglucagon 1-69) and a glucagon-like peptide, proglucagon 78-107, recently isolated from porcine intestine. We studied the fate of the remaining COOH-terminal part of proglucagon (proglucagon 111-160) using radioimmunoassays against proglucagon 111-123 and 126-160. Two peptides were isolated from acid ethanol extracts of porcine ileal mucosa and sequenced: one corresponding to proglucagon 126-158 and one probably corresponding to proglucagon 111-158. By comparing human and porcine proglucagon sequences, Ala117 is replaced by Thr, and Ile138, Ala144, Ile152 and Gln153 are replaced by Val, Thr, Leu, and His. By gel filtration and radioimmunoassay of intestinal extracts it was established that a large part of porcine and virtually all of human proglucagon are processed to release proglucagon 111-123 (designated spacer peptide 2), which, like proglucagon 126-158 must be considered a potential hormonal entity. By isocratic high pressure liquid chromatography human spacer peptide 2 was indistinguishable from synthetic proglucagon 111-122 amide, suggesting that this is the structure of the naturally occurring human peptide.