Pancreatic Proglucagon Research Articles

Immunoassays for the incretin hormones GIP and GLP-1

The measurement of the incretin hormones, glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP), using immunologically based assays is made difficult by the fact that the processing of the precursor molecules gives rise to a number of different peptides which cross-react with antisera raised against the two hormones. For GLP-1, the picture is further complicated because of the necessity to differentiate between the intestinal and pancreatic proglucagon products. Finally, once secreted, both incretins are rapidly degraded by the enzyme dipeptidyl peptidase-4 (DPP-4) to generate metabolites which have lost their insulinotropic activities. These metabolites are the major circulating forms of the incretins, accounting for 60-80% of total immunoreactive GLP-1 and GIP in the peripheral plasma, while concentrations of the intact forms can be very low and, in some cases, barely detectable. The use of highly specific assays using well-characterised antisera and careful sample handling is therefore required for a reliable determination of incretin hormone concentrations.

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.

CDNA cloning of proglucagon from the stomach and pancreas of the dog.

In human and rat, tissue-specific proteolytic processing of identical proglucagon precursors yield tissue-specific proglucagon-derived peptides. In contrast, in many non-mammalian vertebrates alternative mRNA splicing yields different proglucagon precursors in different tissues. Thus alternative mRNA splicing, in part, limits the choices of proglucagon-derived peptides that can be produced by proteolytic processing. Stomach proglucagon mRNAs from the rainbow trout and Xenopus laevis were found not to encode the proglucagon-derived peptide glucagon-like peptide 2 (GLP-2). To determine if the absence of GLP-2 was a general feature of stomach proglucagons we isolated and characterized proglucagon cDNAs from the stomach and the pancreas of the dog, a mammal that expresses the proglucagon gene in the stomach. A major proglucagon transcript of about 1100 bases and a minor transcript of about 800 bases were identified in both stomach and pancreas. The coding sequences of both the stomach and pancreatic proglucagon transcripts were identical. Therefore, tissue-specific proteolytic processing, and not alternative mRNA splicing, must regulate the production of tissue-specific proglucagon-derived peptides from the stomach of the dog.

Inactivation of the winged helix transcription factor HNF3alpha affects glucose homeostasis and islet glucagon gene expression in vivo.

Mice homozygous for a null mutation in the winged helix transcription factor HNF3alpha showed severe postnatal growth retardation followed by death between P2 and P12. Homozygous mutant mice were hypoglycemic despite unchanged expression of HNF3 target genes involved in hepatic gluconeogenesis. Whereas insulin and corticosteroid levels were altered as expected, plasma glucagon was reduced markedly in the mutant animals despite the hypoglycemia that should be expected to increase glucagon levels. This correlated with a 70% reduction in pancreatic proglucagon gene expression. We also showed that HNF3alpha could bind to and transactivate the proglucagon gene promoter. These observations invoke a central role for HNF3alpha in the regulatory control of islet genes essential for glucose homeostasis in vivo.

Glucagon response to hypoglycemia is improved by insulin-independent restoration of normoglycemia in diabetic rats.

The aim of this study was to determine whether the impaired glucagon response to insulin-induced hypoglycemia in the diabetic rat can be improved by correction of hyperglycemia independent of insulin. Four groups of age-matched male Sprague-Dawley rats (246 +/- 13 g BW) were studied: 1) normal controls (NC; n = 7); 2) diabetic, untreated (DU; n = 6); 3) diabetic, treated for 5-7 days using sustained release (2-3 U/day) insulin implants (DI; n = 6); and 4) diabetic, treated for 3-4 days with phlorizin (0.4 g/kg), given sc twice daily (DP; n = 7). Diabetes was induced by a single injection of streptozotocin (65 mg/kg). Basal plasma glucose was 7.4 +/- 0.3 mM in NC, but rose to 14.5 +/- 2.2 mM in DU. Basal hyperglycemia was corrected with phlorizin and insulin treatments (5.5 +/- 0.5 and 6.7 +/- 0.8 mM, respectively). NC rats responded to insulin-induced hypoglycemia with a rapid and marked increase in glucagon (peak, 2059 +/- 311 pg/ml). The glucagon response was blunted in DU (635 +/- 180 pg/ml) and was partially improved by prolonged normalization of glycemia in DP (1335 +/- 295 pg/ml; P < 0.05). Plasma somatostatin levels in all diabetic groups were 2- to 3-fold higher in the basal state, but were not different during hypoglycemia, than those in NC rats. Compared to levels in NC rats, diabetes resulted in decreased insulin, but elevated glucagon and somatostatin concentrations in the pancreatic tissue. Treatment with both insulin and phlorizin reversed the changes in the pancreatic content of both glucagon and somatostatin. Pancreatic proglucagon messenger RNA did not show significant differences among the four groups in either state. Insulin treatment in the DI group resulted in a delayed and much smaller increase in the glucagon response (740 +/- 138 pg/ml) to hypoglycemia despite normalization of glycemia. We, therefore, conclude that in streptozotocin-diabetic rats, the impaired glucagon responsiveness to hypoglycemia is significantly improved by insulin-independent correction of hyperglycemia, suggesting the importance of normoglycemia per se in maintaining, at least in part, the glucose sensitivity of pancreatic alpha-cells.

Trout and chicken proglucagon: alternative splicing generates mRNA transcripts encoding glucagon-like peptide 2.

In mammals, the proglucagon gene is transcribed into a single identical mRNA in pancreas, intestine, and brain. The proglucagon mRNA encodes glucagon and two glucagon-like peptides (GLP 1 and GLP 2), whose production is regulated by tissue-specific proteolytic processing. Previously characterized pancreatic proglucagon cDNAs from birds and fish encode glucagon and only one glucagon-like peptide, GLP 1. The isolation of intestinal proglucagon cDNAs from the rainbow trout, Oncorhynchus mykiss, and chicken, Gallus gallus, shows that the proglucagon gene of fish and birds also contains the sequence of a second glucagon-like peptide, GLP 2. In contrast to the proglucagon mRNAs from mammals, fish and bird proglucagon mRNAs from pancreas and intestine have different 3'-ends that are due to alternative mRNA splicing. The intestinal mRNA was found to be spliced to one or more exons, which encode GLP 2, while the pancreatic mRNA terminates within the intron between the exons encoding GLP 1 and GLP 2. These results show that proglucagon gene expression is regulated at the level of mRNA splicing and serve to reemphasize the potential biological importance of GLP 2.

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.

Inhibition of pancreatic proglucagon gene expression in mice bearing subcutaneous endocrine tumors.

The proglucagon-derived peptides (PGDPs) play key roles in the regulation of carbohydrate metabolism and insulin secretion, yet the factors important for the regulation of proglucagon gene expression remain poorly understood. Recent experiments demonstrated that nude mice carrying subcutaneous proglucagon-SV40 T antigen tumors contained markedly reduced levels of the pancreatic PGDPs. To determine if elevated circulating levels of the PGDPs are consistently associated with inhibition of endogenous pancreatic proglucagon gene expression, we have now studied islet hormone gene expression in mice bearing three different endocrine tumors (InR1-G9, RIN1056A, and STC-1) that express the proglucagon gene. All tumors synthesized large amounts of the PGDPs. Plasma levels of the PGDPs were elevated in all tumor-bearing mice (8- to 22-fold greater than controls, P < 0.001), and this was associated with marked inhibition of mouse pancreatic proglucagon gene expression. The levels of pancreatic PGDPs were also significantly lower in tumor-bearing mice compared with controls (71 +/- 4, 38 +/- 6, and 18 +/- 2% of controls for InR1-G9-, RIN1056A-, and STC-1-bearing mice, respectively, P < 0.05-0.001). This inhibition of proglucagon gene expression was highly specific, in that no consistent change in pancreatic insulin or somatostatin gene expression was detected. Examination of proglucagon posttranslational processing in the tumors demonstrated that proglucagon was processed differently in each tumor to yield a unique profile of PGDPs. These observations demonstrate that elevated circulating levels of the PGDPs are associated with reduction in islet size and inhibition of proglucagon gene expression and PGDP synthesis in the pancreatic A cell.

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.

Pancreatic proglucagon processing: isolation and structures of glucagon and glucagon-like peptide from gene I.

The anglerfish endocrine pancreas expresses two different genes for preproglucagon. The regions of the two proglucagons that correspond to glucagon have different sequences, as do the two glucagon-like peptides (GLPs). The products derived from processing the more abundant proglucagon-II have recently been determined. However, it was not known whether proglucagon-I was processed to similar products. The two major biologically active products of preproglucagon-I processing (glucagon-I and GLP-I) have now been purified to homogeneity. Their structures were determined using automated gas phase Edman degradation, tryptic mapping, and fast atom bombardment mass spectrometry. The preproglucagon-I-processing sites were identified. Glucagon-I represents residues 53-81, and GLP-I corresponds to preproglucagon-I-(91-124) (numbering from the initiator Met).

Distribution of glucagonlike peptide I (GLP-I), glucagon, and glicentin in the rat brain: an immunocytochemical study.

Although glucagonlike immunoreactants (GLIs) are present in the central nervous system of several mammalian species, their structural relationship with pancreatic proglucagon is not defined, and their precise anatomical distribution has not been studied extensively. To obtain further information about the structure and biological significance of brain GLIs, the anatomical distribution of three different antigenic determinants of pancreatic proglucagon--glucagonlike peptide I (GLP-I), glucagon, and glicentin--was mapped in the brain of colchicine-treated rats by immunocytochemistry using the avidin-biotin-peroxidase method. Neuronal cell bodies immunoreactive with antisera specific for GLP-I, glucagon, and glicentin were found only in the caudal medulla oblongata. Within the caudal medulla immunostained cell bodies were found at levels from approximately 0.55 mm rostral to the obex to 0.45 mm caudal to the obex, and were located within the nucleus of the solitary tract (NTS) and the dorsal (MdD) and ventral (MdV) parts of the medullary reticular nucleus. The NTS contained three times more immunoreactive cell bodies than the MdD and MdV, and these cell bodies were located in the midline, medial, and lateral subnuclei of the caudal third of the NTS. Immunostaining of the same cell bodies in paired adjacent sections incubated with GLP-I and glucagon antisera or glucagon and glicentin antisera provided evidence for coexistence of the three antigens within the same neurons of the NTS. Nerve fibers and terminals immunoreactive with GLP-I, glucagon, and glicentin antisera were widely distributed throughout the rat brain and there was no discernible difference in the distribution of fibers and terminals immunoreactive with each of the three antisera. The highest densities of immunostained fibers and terminals were observed in the hypothalamus, thalamus, and septal regions, and the lowest in the cortex and hindbrain. The localization of neuronal cell bodies containing GLP-I, glucagon, and glicentin within the NTS and the MdD and MdV, and the extensive distribution of immunoreactive fibers and terminals throughout the rat brain suggest a role for these peptides in the integration of autonomic as well as central nervous system functions.

Distribution of glucagon-like peptide I in canine and feline pancreas and gastrointestinal tract.

Recently, a putative hormone, glucagon-like peptide I (GLP I), has been identified in the predicted sequences of the precursors to pancreatic glucagon in human, rat, hamster, and ox. The distribution of GLP I immunoreactivity in canine and feline pancreas and gastrointestinal tract was examined immunohistochemically and was compared with that of two other antigenic determinants of pancreatic pro-glucagon, i.e., glucagon and the NH2 terminus of glicentin. All three determinants occurred in the same population of islet cells in normal pancreas and in pancreas consisting predominantly of islet tissue from dogs with canine pancreatic acinar atrophy. Northern blot analysis of mRNA from the latter tissue, using a rat pre-pro-glucagon complementary DNA probe, revealed a single mRNA species similar in size to the pre-pro-glucagon mRNA detected in fetal rat pancreas. The three antigenic determinants of pancreatic pro-glucagon were co-localized also in intestinal L-cells and in canine gastric A-cells. Canine and feline pancreatic pro-glucagons therefore resemble those identified in other mammals and may also occur in gastrointestinal endocrine cells. Although there is evidence that the GLP I sequence is not liberated from pancreatic pro-glucagon, our results raise the possibility that this putative hormone may be a cleavage product of pro-glucagon in the gastrointestinal tract.

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.

Isolation and chemical characterization of glicentin C-terminal hexapeptide in porcine pancreas

Using a radioimmunoassay specific for porcine glicentin C-terminal hexapeptide, we isolated a peptide from porcine pancreas and characterized it as the C-terminal 64–69 sequence of glicentin: H-Asn-Lys-Asn-Asn-Ile-Ala-OH. The purification steps included gel filtration, ion-exchange chromatography and HPLC. In each step, the recovery of the desired peptide, radioimmunologically estimated from the respective elution profile, was 71.4–91.7%. The final yield of the hexapeptide was 22 μg (4.3%) from 800 g pancreas. The pancreatic content of this peptide was estimated to be approximately equimolar to that of pancreatic glucagon. No hexapeptide-like component was detected in porcine intestinal extracts. The data confirmed that the processing of pancreatic proglucagon liberates the C-terminal hexapeptide of the intramolecular glicentin sequence in a tissue-specific manner during the production of glucagon. Glicentin Glicentin (64–69) Proglucagon Isolation Radioimmunoassay Sequence analysis Proteolytic processing