Purification and chemical characterization of a glicentin-related pancreatic peptide (proglucagon fragment) from porcine pancreas.
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Amino Acid Sequence↗
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Ontogeny of glucagon messenger RNA in the rat pancreas.
The synthesis of proglucagon mRNA was studied in rat pancreas from day 11 of fetal gestation (E11) to maturity. Proglucagon mRNA was first detected on E11, the time that the pancreatic bud forms in developing rats. The synthesis of proglucagon mRNA and its translation product at this early time point in pancreatic development suggests an early differentiation of A cell function. Between E17 prenatally and day 10-14 postnatally, pancreatic proglucagon mRNA abundance was higher than in adult pancreas. Regulation of the abundance of pancreatic proglucagon mRNA therefore appears to underlie the previously documented increases in serum and pancreatic glucagon immunoreactivity in the late fetal and perinatal periods. By day 20 postnatally, pancreatic proglucagon mRNA declined to levels found in adult pancreas. Prenatally between E17 and E21, changes in proglucagon mRNA abundance did not parallel previously reported developmental changes in relative mass of proglucagon-producing pancreatic A cells. This suggests that changes in proglucagon mRNA abundance during these times may be attributed to changes in proglucagon gene transcription or proglucagon mRNA stability per cell. In contrast between E21 and maturity, changes in proglucagon mRNA abundance paralleled previously reported changes in relative A cell mass, suggesting no major changes in proglucagon gene transcription or mRNA stability per cell during these times.
Animals↗
Cellular localization of proglucagon/glucagon-like peptide I messenger RNAs in rat brain.
Techniques of in situ hybridization histochemistry, Northern blot hybridization, and immunocytochemistry were used to investigate the biosynthesis of glucagon-like immunoreactants (GLIs) in rat brain. Cells in the nucleus tractus solitarius of the medulla oblongata of adult rat brain hybridized to a synthetic oligonucleotide probe (GLP-I oligomer) corresponding to nucleotide sequences in pancreatic proglucagon mRNA encoding glucagon-like peptide I (GLP-I), and stained with antisera specific for two antigenic determinants of pancreatic proglucagon, glucagon, and GLP-I. These data suggest that there is de novo synthesis of proglucagon in cells of the nucleus tractus solitarius via expression of a proglucagon mRNA similar to that produced in pancreas. Previous studies have shown that cells in hypothalamus stain with GLP-I antisera, but not with glucagon antisera. However, cells in the hypothalamus did not hybridize with the GLP-I oligomer and may therefore produce a GLP-I immunoreactant that is encoded by a mRNA different from the pancreatic proglucagon-mRNA-encoding glucagon and GLP-I. Northern blot hybridizations with a cDNA probe encoding the entire pancreatic proglucagon sequence did not detect proglucagon/GLP-I mRNAs in polyadenylated RNAs (Poly A RNA) from adult rat brainstem and hypothalamus, probably because of their low abundance. Poly A RNAs from fetal rat brain, however, contained two mRNAs that hybridized to the proglucagon cDNA probe. One mRNA of 1,300 bases is the same size as pancreatic proglucagon mRNA. The second mRNA of 1,500 bases may encode the GLP-I immunoreactant detected in the hypothalamus of adult rat brain. The presence of neurons with glucagon and glucagon-like peptides in the nucleus tractus solitarius suggests a role of these peptides in gustatory and/or cardiopulmonary control.
Animals↗
Molecular pathophysiology of glucagon-SV40 T antigen transgenic mice.
The gene encoding proglucagon is expressed in the pancreas, intestine, and brain. The molecular determinants of proglucagon gene expression and the biological activities of the proglucagon-derived peptides (PGDPs) have been examined using transgenic mice harboring a glucagon-SV40 large T antigen (GLUTag) transgene. These experiments have delineated DNA sequences important for intestinal-specific proglucagon gene transcription. GLUTag mice develop neuroendocrine tumors of the pancreas and large bowel, leading to elevated plasma levels of the PGDPs and suppression of endogenous proglucagon gene expression. Transplantation of the large bowel tumors subcutaneously into nude mice provides additional evidence for inhibition of endogenous pancreatic proglucagon gene expression by one or more of the tumor-derived PGDPs. The large bowel GLUTag tumors exhibit abnormalities in the posttranslational processing of proglucagon. GLUTag tumors may be passaged in vivo and in vitro and have been used to generate stable cell lines that express the proglucagon gene at high levels. Taken together, these studies highlight the utility of transgenic systems for the physiological analysis of hormone action and the molecular determinants of peptide hormone gene expression.
Animals↗
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.
Aging↗
Decrease in plasma GLP-1 immunoreactivity in starved rats.
To determine whether starvation affects the metabolism of glucagon-like peptide-1 (GLP-1), we measured the plasma levels of proglucagon-derived peptides and the biosynthesis and posttranslational processing of proglucagon in groups of six rats starved for 1, 3 and 5 days. The plasma levels of GLP-1 immunoreactivity (GLP-1 IR) and glucagon-like immunoreactivity (GLI) decreased during starvation reaching 79 and 56% of the respective control values by day 5 (P less than 0.05 and less than 0.01 vs control). The same is true of the plasma IRI level. The ileal contents of GLP-1 IR and GLI were 50.8 +/- 3.8 pmol/g wet weight and 161.8 +/- 13.2 pmol/g wet weight, respectively, on day 5 of starvation, which were significantly lower (P less than 0.01) than the respective values of 94.8 +/- 16.6 pmol/g wet weight and 262.7 +/- 28.1 pmol/g wet weight in control rats. However, the pancreatic contents of proglucagon-derived peptides tended to increase during starvation, although their increases were not statistically significant. No significant change in the posttranslational processing of proglucagon was detected during starvation. The decrease in the ileal proglucagon-derived peptides content was not associated with a decrease in intestinal proglucagon mRNA transcripts. These results suggested that decreased synthesis of proglucagon-derived peptides by the intestine was largely responsible for the reductions in their circulating levels in starved rats.
Analysis of Variance↗
Inhibition of pancreatic glucagon gene expression in mice bearing a subcutaneous glucagon-producing GLUTag transplantable tumor.
Transgenic mice that express a glucagon gene-simian virus-40 large T-antigen (GLUTag) fusion gene develop neuroendocrine carcinoma of the large bowel. This glucagon-producing tumor was implanted sc and reproducibly formed tumors in nude mice. The transplanted GLUTag tumor expressed large amounts of proglucagon mRNA transcripts, and the levels of proglucagon mRNA transcripts remained constant during 2-8 weeks of tumor growth. The posttranslational processing of proglucagon in the transplantable tumor resembled that detected in the original transgenic tumor, with the liberation of glicentin, oxyntomodulin, glucagon, glucagon-like peptide (1-37) [GLP-1-(1-37)] and GLP-1-(7-37). Tumor-bearing mice demonstrated progressive elevations in the plasma levels of proglucagon-derived peptides. Elevated plasma levels of glucagon-like immunoreactive peptides and immunoreactive glucagon were associated with a marked reduction in the levels of pancreatic glucagon mRNA transcripts by 4 weeks, and after 8 weeks of tumor growth, the levels of glucagon mRNA transcripts in the pancreas were not detectable by Northern blot analysis. Synthesis of the proglucagon-derived peptides was also significantly suppressed at 4-8 weeks in the pancreas of tumor-bearing animals. Histological examination of the endocrine pancreas in mice carrying the GLUTag tumor for 6-8 weeks demonstrated a marked reduction in the number and size of the islets of Langerhans and a disproportionately greater decrease in the number of cells exhibiting glucagon immunoreactivity. By electron microscopy, the residual A-cells were small, compressed at the periphery of the islets, and had poorly developed cytoplasmic organelles. In contrast, no changes in mouse glucagon gene expression or islet morphology were detected in control animals without tumors or mice carrying a sc v-jun-induced fibrosarcoma. The suppression of pancreatic A-cell function and islet size in mice with elevated plasma levels of the proglucagon-derived peptides raises the possibility that a proglucagon-derived peptide may participate in a negative feedback loop, inhibiting expression of the glucagon gene in the A-cells of the endocrine pancreas.
Animals↗
Fermentable dietary fiber increases GLP-1 secretion and improves glucose homeostasis despite increased intestinal glucose transport capacity in healthy dogs.
Ileal proglucagon gene expression and postprandial plasma concentrations of proglucagon-derived peptides are reported to change with the type and quantity of dietary fiber ingested by rats. Within the intestine, proglucagon encodes several proglucagon-derived peptides known to modulate intestinal absorption capacity and pancreatic insulin secretion. To determine whether the chronic ingestion of fermentable dietary fiber regulates the expression and synthesis of proglucagon-derived peptides in the distal intestine to modulate glucose homeostasis, the following study was conducted: 16 adult dogs (23 +/- 2 kg) were fed isoenergetic, isonitrogenous diets containing a mixture of high fermentable dietary fibers (HFF) or low fermentable (LFF) wood cellulose for 14 d in a randomized cross-over design. Food was withheld for 16 h before an oral glucose tolerance test was conducted supplying 2 g of glucose/kg body wt, and peripheral blood was collected via a hind-leg catheter at 0, 15, 30, 45, 60, 90 and 120 min for plasma glucose, insulin and glucagon-like peptide-1(7-36)NH2 (GLP-1) analyses. Intestinal samples were collected after the second dietary treatment. Ileal proglucagon mRNA, intestinal (GLP-1) concentrations and the integrated area under the curves (AUC) for plasma GLP-1 and insulin were greater and plasma glucose AUC was reduced when dogs were fed the HFF diet compared to the LFF diet (P < 0.05). Intestinal villi heights, brush border and basolateral glucose transporter protein abundance and jejunal transport capacities were significantly greater when dogs were fed the HFF diet than when fed the LFF diet. In conclusion, improvements in glucose homeostasis are observed in healthy dogs when they ingest fermentable fibers.
Animals↗
Glucagon and related peptides in fetal rat hypothalamus in vivo and in vitro.
Proglucagon-derived peptides are localized in pancreas, intestine, and the nervous system. We have examined the ontogeny of glucagon and related peptides in developing rat hypothalamus and have developed a fetal rat hypothalamic cell culture model to study the synthesis and secretion of these peptides in cells of neural origin. Fetal rat hypothalamus (19-21 day gestation) was found to contain glucagon-like immunoreactive (GLI) peptides including glucagon. The relative amounts of two of the GLI peptides (glicentin and oxyntomodulin) increased with development such that adult hypothalamus contained a predominance of these peptides over glucagon. The ratio of GLI peptides to glucagon increased from 2.6 +/- 0.5 in fetus to 46 +/- 11 in adult (P less than 0.001). When fetal rat hypothalamic cells (FRHC) were placed into primary culture for 7 days, the presence of neurons, glial cells, and glucagon-containing cells was detected by immunohistochemical staining. Analysis of proglucagon gene expression in FRHC cultures by Northern blotting demonstrated the presence of a single proglucagon messenger RNA (mRNA) transcript identical in size and sequence to that detected in fetal pancreas and intestine. RNase protection analysis of RNA from FRHC cultures, brainstem, and intestine confirmed that the proglucagon mRNA transcripts present in these three tissues were identical. Analysis of FRHC content of GLI peptides and immunoreactive glucagon demonstrated that peptide levels were not significantly different from those of whole fetal rat hypothalamus, and did not vary significantly throughout 2 weeks in culture. FRHC cultures were found to contain substantial amounts of glucagon after 1 week of culture. Release of the GLI peptides on day 7 of culture was increased 3-fold (P less than 0.001) by treatment of FRHC for 1 h with 5 mM (Bu)2cAMP. Rat hypothalamus therefore appears to undergo unique changes in posttranslational processing of proglucagon during development. Primary cultures of FRHC thus provide a promising in vitro model to study the molecular control of proglucagon biosynthesis and GLI peptide secretion in the brain.
Animals↗
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.
Animal Nutritional Physiological Phenomena↗
Prolonged gastrointestinal transit in a patient with a glucagon-like peptide (GLP)-1- and -2-producing neuroendocrine tumor.
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.
Female↗
Complete sequences of glucagon-like peptide-1 from human and pig small intestine.
In the small intestine, proglucagon is processed into the previously characterized peptide "glicentin" (proglucagon (PG) 1-69) and two smaller peptides showing about 50% homology with glucagon: glucagon-like peptide-1 and -2. It was assumed that the sites of post-translational cleavage in the small intestine of the proglucagon precursor were determined by pairs of basic amino acid residues flanking the two peptides. Earlier studies have shown that synthetic glucagon-like peptide-1 (GLP-1) synthesized according to the proposed structure (proglucagon 71-108 or because residue 108 is Gly, 72-107 amide) had no physiological effects, whereas a truncated from of GLP-1, corresponding to proglucagon 78-107 amide, strongly stimulated insulin secretion and depressed glucagon secretion. To determine the amino acid sequence of the naturally occurring peptide we isolated GLP-1 from human small intestine by hydrophobic, gel permeation, and reverse-phase high performance liquid chromatography. By analysis of composition and sequence it was determined that the peptide corresponded to PG 78-107. By mass spectrometry the molecular mass was determined to be 3295, corresponding to PG 78-107 amide. Furthermore, mass spectrometry of the methyl-esterified peptide showed an increase in mass compatible with the presence of alpha-carboxyl amidation. Thus, the gut-derived insulinotrophic hormone GLP-1 is shown to be PG 78-107 amide.
Amino Acid Sequence↗
Glucagon-like immunoreactivity in hypothalamic neurons of the rat.
Antisera specific for three different regions of pancreatic proglucagon were used to examine the distribution of such immunoreactivity in rat hypothalamus. Neurons in the supraoptic and paraventricular nuclei were immunoreactive with an antiserum against glucagon, but not with antisera directed towards the aminoterminal region of proglucagon (glicentin) or the glucagon-like peptide I sequence in the carboxyl-terminal region of proglucagon. These findings confirm a previous report of glucagon-like immunoreactivity in the supraoptic and paraventricular nuclei, but indicate that, while this material is immunochemically related to glucagon, it is not derived from a proglucagon-like precursor.
Animals↗
Glucagon and the glucagon-like peptides.
Glucagon and the glucagon-like peptides are encoded within a larger precursor, proglucagon. The proglucagon gene is expressed in the pancreas, intestine, and brain, giving rise to a single proglucagon mRNA transcript that is identical in all tissues. Tissue-specific posttranslational processing of proglucagon accounts for the different molecular forms of the glucagon-related peptides present in each tissue. Glucagon-like peptide I has been demonstrated to have potent insulinotropic properties, but the biological importance of other glucagon-related peptides remains unclear. The recent advances that have increased our understanding of the biology of glucagon and the glucagon-like peptides are reviewed.
Animals↗
PKA independent and cell type specific activation of the expression of caudal homeobox gene Cdx-2 by cyclic AMP.
Cdx-2 is a transactivator for the proglucagon gene in pancreatic and intestinal endocrine cells. Cdx-2 is also expressed in differentiated intestinal epithelia of nonendocrine origin. Cdx-2-/- mice are embryonic lethal, while Cdx-2+/- mutants show multiple malfunctions including the formation of intestinal polyps. Within the polyps, the remaining wild type Cdx-2 allele ceases its expression, while the expression of both Cdx-2 and proglucagon in the endocrine cells remains unaltered, indicating that Cdx-2 could be haplo-insufficient for nonendocrine cells, but not for proglucagon producing endocrine cells. We propose that mechanisms underlying Cdx-2 expression and auto-regulation [Xu F, Li H & Jin T (1999), J Biol Chem274, 34310-34316] differ in these two types of cells. We show here that forskolin and cAMP upregulate Cdx-2 expression in proglucagon producing cells, but not in colon cancer cells and primary intestinal cell cultures. It is unlikely that the activation is mainly mediated by PKA, because the activation was observed in a PKA deficient cell line. Co-transfecting a dominant negative Ras expression plasmid substantially repressed the Cdx-2 promoter, in contrast to a previous finding that Ras is a negative factor for Cdx-2 expression in colon cancer cells. Furthermore, forskolin activated ERK1/2 phosphorylation in the endocrine cells, and attenuation of ERK1/2 phosphorylation by its inhibitor is associated with attenuated Cdx-2 expression. Finally, an Epac pathway specific cAMP analogue stimulated both ERK1/2 phosphorylation and Cdx-2 expression. Taken together, our observations suggest that Cdx-2 expression is regulated by the second messenger cAMP, cell-type specifically, via the Epac pathway.
Animals↗
Functional characterization of somatostatin receptors expressed on hamster glucagonoma cells.
We characterized somatostatin receptors expressed in hamster glucagonoma INR1G9 cells and the effects of somatostatin on glucagon secretion, proglucagon gene expression, and the adenosine 3',5'-cyclic monophosphate (cAMP)-dependent signal-transduction cascade. 125I-labeled somatostatin was displaced by somatostatin-14 and somatostatin-28 with a dissociation constant of 2 nmol/l. Stable GTP analogues decreased binding of 125I-somatostatin to its receptors, suggesting an interaction of somatostatin receptors with G proteins. Chemical cross-linking of 125I-somatostatin to its receptor revealed a molecular mass of the ligand-receptor complex of 47 kDa. Somatostatin inhibited forskolin-stimulated activation of adenylate cyclase [2.5 microM forskolin (161%) + 1 microM somatostatin (128%); P < 0.05] and protein kinase A [10 microM forskolin (143%) + 1 microM somatostatin (114%); P < 0.05] but did not influence basal activities of these enzymes. Forskolin-induced stimulation of cAMP generation was reduced by somatostatin [2.5 microM forskolin (306%) + 1 microM somatostatin (145%); P < 0.05]. Somatostatin inhibited forskolin, theophylline, and arginine stimulation of glucagon secretion. Basal as well as forskolin-, theophylline-, and isobutyl methylxanthine-induced proglucagon gene expression was significantly reduced by somatostatin. Our data show that, in INR1G9 cells, somatostatin receptors are at least in part coupled to the adenylate cyclase system. Somatostatin is a potent negative regulator of both basal and forskolin-stimulated proglucagon gene expression. The interaction with forskolin occurs at the level of adenylate cyclase. The effect of somatostatin on basal proglucagon gene transcription is most probably mediated by an unrelated second messenger system. Somatostatin may influence several functions of the pancreatic A cell.
Adenylyl Cyclases↗
Molecular biology of gastrointestinal peptides and growth factors: relevance to intestinal adaptation.
New approaches towards understanding regulation of growth and adaptation of the small intestine are made possible by the isolation and characterization of genes and complementary DNAs (cDNAs) encoding gastrointestinal peptides, growth factors and their receptors. Nucleotide sequencing provides prerequisite structural information. Analyses of gene expression by quantitation and localization of mRNAs provide information about correlations between local alterations in peptide or receptor synthesis and intestinal growth. Analyses of intestinal growth in transgenic animals that overexpress or underexpress growth factor or receptor genes provides direct information about peptide effects on growth. Our recent studies with genes and cDNAs encoding proglucagon and the growth hormone dependent insulin-like growth factor 1 (IGF-I) represent examples of these approaches. Sequences of proglucagon and IGF-I cDNAs provide the primary structures of the peptide precursors. Analyses of proglucagon mRNA during adaptive growth after small bowel resection indicate that increases in proglucagon gene transcription or mRNA stability underly previously observed increases in serum enteroglucagons during adaptive growth. Analyses of IGF-I mRNAs in intestine indicate that small intestine expresses only a subset of the IGF-I mRNAs expressed in liver due to utilization of specific promotors and/or exon splicing mechanisms. Oligomers derived from the 3' end of the rat IGF-I gene detect a novel intestinal specific IGF-I related mRNA that shows an aboral decline in abundance from duodenum to colon and is upregulated in a number of situations of adaptive growth. Transgenic mice that overexpress growth hormone or IGF-I are under analysis to establish the effects of growth hormone and IGF-I on intestinal growth.
Adaptation, Physiological↗
Effects of hypoglycemia and prolonged fasting on insulin and glucagon gene expression. Studies with in situ hybridization.
In situ hybridization of proinsulin and proglucagon mRNA was performed in rat pancreas to assess prohormone gene expression during various glucopenic conditions. During a 4-d fast mean blood glucose declined by 48 mg/dl; proinsulin mRNA signal density remained normal while proglucagon mRNA signal density more than doubled. At the end of a continuous 12-d insulin infusion blood glucose averaged 53 +/- 12 mg/dl; proinsulin mRNA signal density declined to 30% of controls while proglucagon mRNA signal density more than doubled. In insulinoma-bearing NEDH rats blood glucose averaged 34 +/- 3.5 mg/dl; the proinsulin mRNA signal was virtually undetectable and proglucagon mRNA signal density was more than twice the controls. There was no detectable change in either beta-cell area or islet number in rats subjected to fasting or insulin infusion, but in insulinoma-bearing rats beta cell area was markedly reduced. Thus compensation during 4 d of starvation involves an increase in glucagon gene expression without change in insulin gene expression or beta cell mass. In moderate insulin-induced hypoglycemia glucagon gene expression is increased and insulin gene expression decreased. In more profound insulinoma-induced hypoglycemia, in addition to the foregoing changes in hormone gene expression, there is a profound reduction in the number of insulin-expressing cells.
Animals↗