The effects of gastric inhibitory polypeptide (GIP) on the release of anterior pituitary hormones

Several members of the secretin family of hormones have been demonstrated to alter anterior pituitary hormone secretion. Here we report the action of gastric inhibitory polypeptide (GIP) on gonadotropin and somatotropin release. Intraventricular injection of 1 microgram (0.2 nmole) GIP (2.5 microliters) produced a significant decrease in plasma FSH at 30 (p less than 0.02) and 60 min after its injection (p less than 0.01). The FSH-lowering effect of a higher dose of 5 micrograms (1 nmole) of GIP was already developed at 15 min (p less than 0.01) and was prolonged until the end of the experiment (60 min, p less than 0.05). No change in plasma LH was detected at any time during the experimental period. If 5 micrograms of estradiol-benzoate were given SC 48 hr prior to experiment, the initial values of FSH and LH were markedly decreased. In these animals GIP failed to influence plasma FSH and LH. When dispersed anterior pituitary cells from OVX rats were cultured overnight and incubated in vitro with GIP, the peptide was found to induce both FSH and LH release. Highly significant release occurred with the lowest dose tested of 10(-7) M and there was a dose-response effect for both hormones. The slope of the dose-response curve was similar for both FSH and LH release. GIP was less potent than LHRH which produced a greater stimulation of both FSH and LH release at a dose of 10(-9) M than did 10(-7) M GIP. The two peptides had an additive effect on the release of both FSH and LH.(ABSTRACT TRUNCATED AT 250 WORDS)     

Understanding interactions of gastric inhibitory polypeptide (GIP) with its G-protein coupled receptor through NMR and molecular modeling.

Gastric inhibitory polypeptide (GIP, or glucose-dependent insulinotropic polypeptide) is a 42-amino acid incretin hormone moderating glucose-induced insulin secretion. Antidiabetic therapy based on GIP holds great promise because of the fact that its insulinotropic action is highly dependent on the level of glucose, overcoming the sideeffects of hypoglycemia associated with the current therapy of Type 2 diabetes. The truncated peptide, GIP(1-30)NH2, has the same activity as the full length native peptide. We have studied the structure of GIP(1-30)NH2 and built a model of its G-protein coupled receptor (GPCR). The structure of GIP(1-30)NH2 in DMSO-d6 and H2O has been studied using 2D NMR (total correlation spectroscopy (TOCSY), nuclear overhauser effect spectroscopy (NOESY), double quantum filtered-COSY (DQF-COSY), 13C-heteronuclear single quantum correlation (HSQC) experiments, and its conformation built by MD simulations with the NMR data as constraints. The peptide in DMSO-d6 exhibits an alpha-helix between residues Ile12 and Lys30 with a discontinuity at residues Gln19 and Gln20. In H2O, the alpha-helix starts at Ile7, breaks off at Gln19, and then continues right through to Lys30. GIP(1-30)NH2 has all the structural features of peptides belonging to family B1 GPCRs, which are characterized by a coil at the N-terminal and a long C-terminal alpha-helix with or without a break. A model of the seven transmembrane (TM) helices of the GIP receptor (GIPR) has been built on the principles of comparative protein modeling, using the crystal structure of bovine rhodopsin as a template. The N-terminal domain of GIPR has been constructed from the NMR structure of the N-terminal of corticoptropin releasing factor receptor (CRFR), a family B1 GCPR. The intra and extra cellular loops and the C-terminal have been modeled from fragments retrieved from the PDB. On the basis of the experimental data available for some members of family B1 GPCRs, four pairs of constraints between GIP(1-30)NH2 and its receptor were used in the FTDOCK program, to build the complete model of the GIP(1-30)NH2:GIPR complex. The model can rationalize the various experimental observations including the potency of the truncated GIP peptide. This work is the first complete model at the atomic level of GIP(1-30)NH2 and of the complex with its GPCR.     

Immunohistochemical localization of gastrin C-terminus, gastric inhibitory peptide (GIP) and endorphin in the pancreas of lizards with special reference to the hibernation period

Gastrin C-terminus and GIP immunoreactive cells were observed in the pancreas of the desert lizard (Uromastyx aegyptia) captured during the hibernation period, but not in those collected in the active period. These cell types were encountered among the exocrine parenchyma, especially around ducts and among the ductal epithelial cells. Occasionally a few GIP cells were seen to occupy the islet periphery. No gastrin C-terminus or GIP immunoreactive cells were observed in the pancreas of the grass lizard (Mabuya quinquetaeniata)--which does not hibernate--collected in winter and in summer. In both species of lizards endorphin-like immunoreactivity was localized in the pancreatic PP-cells in specimens collected in winter and summer. It was assumed that the presence of the gastrin C-terminus and GIP cells in the desert lizard pancreas represents a response to the peculiar physiological state through which these lizards pass in hibernation.     

Influence of gastric inhibitory polypeptide (GIP) and glucose on the regulation of glucagon secretion by pancreatic alpha cells

The effects of glucose and GIP on glucagon secretion were studied in perifused microdissected murine pancreatic islets. Glucagon levels were determined in effluent samples collected at 1-min intervals by radioimmunoassay using the glucagon-specific antibody, 30 K. There was no significant difference in the total amount (7740 +/- 212 pg vs 8630 +/- 36 pg, n = 10) of glucagon secreted over a 20 min period when the glucose concentration was alternately shifted between 5.5 mM and 11.1 mM, respectively. However, 22.2 mM glucose profoundly suppressed glucagon secretion. The suppressive effect of high glucose on glucagon release was partially, yet significantly, reversed by the presence of GIP, as glucagon secretion increased from a non-detectable level at 22.2 mM glucose alone to 10,175 +/- 145 pg, n = 10 (P less than 0.01). The glucagonotropic effect of GIP was dose-dependent in the range of 2 x 10(-9) - 2 x 10(-7) M, at 11.1 mM glucose. Our data show that GIP is able to substantially reverse the suppressive effect of a high glucose load on glucagon secretion.     

Synthesis and intracellular transport of proteins in the exocrine pancreas of the frog (Rana esculenta)

Summary Frog pancreatic tissue was pulse-labelled in vitro with ³H-leucine and protein transport was studied in exocrine cells by electron microscope autoradiography. The proteins appeared to be synthesized in the RER and transported to the secretory granules along a similar route and with the same velocity as previously described under in vitro conditions.Evidence was obtained for the involvement of the vesicular and tubular elements at the periphery of the Golgi system in transferring protein from the RER to the Golgi cisternae.Kinetics of the release of newly synthesized proteins from the RER and their appearance in the condensing vacuoles are discussed and related to results reported from other tissues.The transport velocity in this poikilothermic system was studied in relation to the incubation temperature and compared with results reported from its mammalian counterpart. At temperatures between 20 and 30° C intracellular protein transport occurs faster in the frog than in the Guinea pig pancreas. At higher temperature the transport process was severely disturbed in the frog.     

Response of gastric inhibitory polypeptide (GIP) to oral administration of nutrients in normal and pancreatectomized dogs

In order to clarify the response of plasma gastric inhibitory polypeptide (GIP) to various nutrients and to investigate the relationship between the pancreas and GIP secretion, an experimental study was performed using normal and pancreatectomized dogs. Oral administration of glucose (2 g/kg) or butter (2 g/kg) resulted in an increase of plasma GIP in five normal dogs. In contrast, oral administration of arginine (1 g/kg) did not produce any discernible changes in plasma GIP in normal dogs. In a group of nine pancreatectomized dogs, the fasting level of plasma GIP did not differ from that of the control group. Furthermore, glucose ingestion in the pancreatectomized group resulted in the same pattern and the same degree of change in plasma GIP as it did in the normal controls. In contrast, plasma GIP did not change at all following fat loading in the pancreatectomized group. However, butter with pancreatic enzymes elicited a significant rise of plasma GIP in the pancreatectomized dogs. The present study indicates that plasma GIP increases following oral administration of glucose or fat but not arginine. Furthermore, it is demonstrated that GIP secretion following fat ingestion occurs only after fat digestion by pancreatic enzymes. In addition, the findings observed in the present study do not support the existence of feedback effect of insulin on GIP secretion.     

Effect of endogenous gastric inhibitory polypeptide (GIP) on the removal of triacylglycerol in dogs

In order to clarify the effect of endogenous gastric inhibitory polypeptide (GIP) upon lipid metabolism, the removal of intravenously administered triacylglycerol was investigated following an oral glucose or galactose load in dogs. After an overnight fast, the triacylglycerol emulsion was infused at a constant rate of 1 ml/min for 90 min, and glucose, galactose or tap water was orally administered at 30 min. Blood glucose increased after the glucose load but it did not change following the galactose load or water ingestion. Plasma insulin increased after the glucose load but did not change after galactose or tap water ingestion. Plasma glucagon did not show any discernible change in the three experimental groups. Plasma GIP increased following the glucose or galactose load to 4360 or 1653 pg/ml, respectively. Plasma triacylglycerol increased to the same levels at 30 min in the three experimental groups. The peak levels of plasma triacylglycerol and integrated plasma triacylglycerol for 150 min did not differ in the three groups. Moreover, there was no difference in the removal rate of plasma triacylglycerol following the withdrawal of the fat emulsion. It is concluded from the present study that endogenously released GIP does not elicit any effect upon triacylglycerol removal.     

Pituitary adenylate cyclase-activating polypeptide (PACAP): occurrence in rodent pancreas and effects on insulin and glucagon secretion in the mouse

Summary Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide that occurs in several tissues, e.g., in the gut. We have studied PACAP-like immunoreactivity in the pancreas of rat and mouse, and the effects of PACAP-38 on basal and stimulated insulin and glucagon secretion in the mouse. Immunofluorescence staining demonstrated the presence of PACAP-like immunoreactivity in nerve fibers in both the rat and mouse pancreas. The nerve fibers were seen in the exocrine pancreas and surrounding the islets. Occasionally, the nerve fibers occurred within the islets. Most PACAP-positive nerve fibers innervated the intrapancreatic ganglia, although no nerve cell bodies contained PACAP-like immunoreactivity. In-vivo experiments in mice revealed that basal plasma glucagon levels were increased by PACAP-39 injected intravenously at dose levels exceeding 1.8 nmol/kg. Furthermore, PACAP-38 (7 nmol/kg) potentiated the plasma glucagon response to the cholinergic agonist carbachol (0.16 mol/kg). This potentiation was reduced to simple addition by pretreatment with a combined - and -adrenergic blockade by phentolamine (35 mol/kg) and propranolol (8.5 mol/kg). Moreover, PACAP-38 inhibited a carbachol-induced increase in the level of plasma insulin in the absence but not in the presence of adrenergic blockade. PACAP-38 increased basal plasma insulin levels and increased basal plasma glucose levels 6 min and 10 min, respectively, after injection of the peptide. We conclude that PACAP-like immunoreactivity exists in nerve fibers innervating the mouse and rat pancreas, particularly the intrapancreatic ganglia, and that PACAP-38 augments both basal and carbachol-stimulated glucagon secretion in the mouse.     

Ultrastructural localization of gastric inhibitory polypeptide (GIP) in a well characterized endocrine cell of canine duodenal mucosa

The polypeptide hormone GIP has been localized ultrastructurally by using specific, monoclonal GIP antibodies and an immunogold technique on aldehyde-osmium fixed specimens of dog duodenal mucosa. A single type of cell showing round, homogeneous, fairly osmiophilic granules with closely applied membrane and a mean size of 188 nm +/- 34 SD has been identified as the GIP cell.     

Interaction of gastric inhibitory polypeptide (GIP) with the insulin-secreting pancreatic beta cell line, In lll: characteristics of GIP binding sites

The interaction of GIP with its receptors in the hamster pancreatic insulin-secreting beta cell line, In lll, has been analyzed. 125I-labelled GIP used as tracer showed the same affinity as native GIP for the GIP binding sites. Binding of the tracer was time, temperature and cell concentration dependent. It was saturable, reversible and highly specific. Under equilibrium conditions, i.e. 2 hours at 13 degrees C, 20% and 25% of the tracer and of GIP binding sites were inactivated, respectively. Native GIP inhibited binding of 125I-labelled GIP in a dose-dependent manner, saturation of the GIP binding sites being obtained at 3. 10(-7) M peptide. Two types of GIP binding sites were found by Scatchard analysis, a small population with a high affinity for GIP (KD = 7 nM) and a large population with a low affinity (KD = 800 nM). The biphasic dissociation process confirmed the GIP binding sites heterogeneity. Apart from GIP, no peptide tested influenced the binding of the 125I-labelled GIP. The present data represents the first analysis of functionally relevant GIP binding sites in a insulin-secreting cell.     

Ultrastructural localization of gastric inhibitory polypeptide (GIP) in a well characterized endocrine cell of canine duodenal mucosa

Summary The polypeptide hormone GIP has been localized ultrastructurally by using specific, monoclonal GIP antibodies and an immunogold technique on aldehyde-osmium fixed specimens of dog duodenal mucosa. A single type of cell showing round, homogeneous, fairly osmiophilic granules with closely applied membrane and a mean size of 188 nm±34 SD has been identified as the GIP cell.     

Development of the duct system during exocrine pancreas differentiation in the grass snake Natrix natrix (Lepidosauria,Serpentes)

We analyzed the development of the pancreatic ducts in grass snake Natrix natrix L. embryos with special focus on the three‐dimensional (3D)‐structure of the duct network, ultrastructural differentiation of ducts with attention to cell types and lumen formation. Our results indicated that the system of ducts in the embryonic pancreas of the grass snake can be divided into extralobular, intralobular, and intercalated ducts, similarly as in other vertebrate species. However, the pattern of branching was different from that in other vertebrates, which was related to the specific topography of the snake's internal organs. The process of duct remodeling in Natrix embryos began when the duct walls started to change from multilayered to single‐layered and ended together with tube formation. It began in the dorsal pancreatic bud and proceeded toward the caudal direction. The lumen of pancreatic ducts differentiated by cavitation because a population of centrally located cells was cleared through cell death resembling anoikis. During embryonic development in the pancreatic duct walls of the grass snake four types of cells were present, that is, principal, endocrine, goblet, and basal cells, which is different from other vertebrate species. The principal cells were electron‐dense, contained indented nuclei with abundant heterochromatin, microvilli and cilia, and were connected by interdigitations of lateral membranes and junctional complexes. The endocrine cells were electron‐translucent and some of them included endocrine granules. The goblet cells were filled with large granules with nonhomogeneous, moderately electron‐dense material. The basal cells were small, electron‐dense, and did not reach the duct lumen.     

Modulation of insulin and glucagon secretion from the perfused rat pancreas by the neurohypophysial hormones and by desamino-D-arginine vasopressin (DDAVP)

Marked stimulation of glucagon release and modest stimulation of insulin release were observed during in situ perfusion of the rat pancreas with AVP or OT. Glucagon release in response to AVP or OT (200 pg/ml) gradually increased over a 45 min perfusion period reaching maxima of 500% and 300% of the pre-stimulatory levels, respectively. Insulin release transiently increased by 100%. In each case release rates returned to control values immediately after withdrawal of the peptides. Total glucagon release was concentration dependent and linear from 20 pg to 20 ng AVP or OT/ml (r greater than .97). Pancreatic response to DDAVP perfused at 20 ng/ml was virtually indistinguishable from that induced by AVP at 200 pg/ml. This demonstration of a glucagonotrophic action of the neurohypophysial hormones in the in situ perfused rat pancreas confirms earlier studies using isolated islets and bolus IV injection.     

Vasoactive intestinal polypeptide (VIP) in the pig pancreas: role of VIPergic nerves in control of fluid and bicarbonate secretion

Vasoactive intestinal polypeptide (VIP) in the pig pancreas is localized to nerves, many of which travel along the pancreatic ducts. VIP stimulates pancreatic fluid and bicarbonate secretion like secretin. Electrical vagal stimulation in the pig causes an atropine-resistant profuse secretion of bicarbonate-rich pancreatic juice. In an isolated perfused preparation of the pig pancreas with intact vagal nerve supply, electrical vagal stimulation caused an atropine-resistant release of VIP, which accurately parallelled the exocrine secretion of juice and bicarbonate. Perfusion of the pancreas with a potent VIP-antiserum inhibited the effect of vagal stimulation on the exocrine secretion. It is concluded, that VIP is responsible for (at least part of) the neurally controlled fluid and bicarbonate secretion from the pig pancreas.     

The response of plasma gastric-inhibitory polypeptide (GIP) to slow and fast glucose ingestion in Billroth II resected patients and normal controls

Eight Billroth II resected patients and 8 normal controls were given two oral glucose loads, one ingested within 2 min, and the other ingested slowly over 80 min. In the Billroth II resected group, the integrated plasma GIP release was significantly higher after the fast than after the slow glucose ingestion. In this group the integrated plasma GIP release was also significantly higher than in the control group, but only after the fast glucose ingestion. These findings indicate that the rate of glucose delivery into the intestine may be of importance in the plasma GIP response to oral glucose.     

DPP IV resistance and insulin releasing activity of a novel di-substituted analogue of glucose-dependent insulinotropic polypeptide, (Ser2-Asp13)GIP

Structure-function studies suggest that preservation of the N-terminus and secondary structure of glucose-dependent insulinotropic polypeptide (GIP) is important for biological activity. Therefore, a novel di-substituted analogue of GIP, (Ser(2)-Asp(13))GIP, containing a negatively charged Asp residue in place of an Ala in position 13, was synthesised and evaluated for in vitro biological activity. Incubation with dipeptidyl peptidase IV (DPP IV) showed the half-lives of GIP and (Ser(2)-Asp(13))GIP to be 2.3 and >4h, respectively. Insulin releasing studies in clonal pancreatic BRIN-BD11 cells demonstrated that (Ser(2)-Asp(13))GIP (10(-12)to 10(-7)mol/l) was significantly less potent (60-90%; P<0.05 to P<0.001) than native GIP. The peptide failed to display antagonistic properties as it did not significantly alter insulin secretion when incubated in the presence of GIP (10(-7)mol/l). These results demonstrate that despite increased resistance to DPP IV, substituting Ala in position 13 with a negatively charged Asp, thus producing the di-substituted analogue (Ser(2)-Asp(13))GIP, significantly reduces biological activity, most likely due to modifications within the secondary structure.     

Immunohistochemical localization of porcine diazepam-binding inhibitor (DBI) to rat endocrine pancreas

Summary The occurrence of diazepam-binding inhibitor (DBI), isolated and characterized from porcine upper intestine, was examined in the pancreas of Sprague-Dawley albino rats using indirect immunofluorescence. The polypeptide was found in the endocrine Langerhans islets and, utilizing double-labelling controls, it was shown to be present within the peripherally located glucagon-containing cells. Regulation of islet hormone production may therefore be under DBI control.     

A novel form of gastric inhibitory polypeptide (GIP) isolated from bovine intestine using a radioreceptor assay. Fragmentation with staphylococcal protease results in GIP1-3 and GIP4-42, fragmentation with enterokinase in GIP1-16 and GIP17-42

Sodium cholate and digitonin were used to solubilize alpha2-adrenergic receptors from rat and calf brain. Sodium cholate extracted 40-50% of the membrane protein and 25-30% of the binding capacity. Digitonin extracted only 20-30% of the membrane protein and only 10-15% of the binding capacity of the native membranes. Both detergents were removed by dialysis in the presence of phospholipids, and the solubilized protein was precipitated upon addition of poly(ethyleneglycol) and magnesium. In the solubilization/reconstitution process no purification of the alpha2-adrenergic receptor was obtained, most probably due to its inactivation by the solubilization conditions. The reconstituted protein(s) tested for binding properties, using p-[3H]aminoclonidine and/or [3H]clonidine, maintained the pharmacological profile of the native alpha2-adrenergic receptor. The potency order of various alpha2-agonists and alpha2-antagonists as well as their stereoselectivity were identical to those of the native alpha2-receptor. Specific receptor binding decreases in the presence of the guanyl nucleotides GTP or guanosine 5'-[beta, gamma-imido]-triphosphate but not ATP, thus indicating a co-solubilization of GTP regulatory components (stimulatory protein Ns or inhibitory protein Ni or both). Adenylate cyclase activity of the reconstituted preparation is stimulated threefold by sodium fluoride, suggesting the presence of both Ns-protein and the catalytic unit (C) in the reconstituted protein(s).     

Effects of secretin and gastric inhibitory polypeptide on human pancreatic growth hormone-releasing factor(1-40)-stimulated growth hormone levels in the rat

Synthetic human pancreatic growth hormone-releasing factor containing 40 amino acids ([hpGRF (1-40)]-OH) significantly stimulated plasma growth hormone (GH) levels in both sodium pentobarbital and urethane anesthetized rats. Synthetic secretin, gastric inhibitory polypeptide (GIP), and glucagon significantly decreased plasma GH levels while synthetic vasoactive intestinal peptide (VIP) had no effect. Secretin and GIP also altered the in vivo plasma GH response to [hpGRF(1-40)]-OH. Whether this effect is the result of an interaction at the pituitary level or is due to an extra-pituitary effect of secretin and GIP awaits further study.     

Impairment of the insulinotropic effect of gastric inhibitory polypeptide (GIP) in obese and diabetic rats is related to the down-regulation of its pancreatic receptors

The association of obesity with type 2 diabetes mellitus has been recognized for years. In type 2 diabetes, there is a possibility that an important part of the impaired insulin secretion is due to the gastric inhibitory polypeptide (GIP) hormone. This study investigated changes that occur in the pancreatic GIP receptors' (GIP-Rs) expression and in GIP secretion in obese and type 2 diabetic rats and its relation to plasma glucose and insulin levels during oral glucose tolerance test (OGTT) compared to control rats. During the first 20 min of the OGTT, both the obese and the diabetic rats had a significant increase in the glucose excursion and a significant decrease in early-insulin secretion compared to the control group, with more prominent changes in the diabetic group. The obese rats had a significant increase in fasting GIP level and in the incremental change of GIP from 0 to 20 min (GIP Delta 0-20: 60.1 + or - 6.66 pmol/l) compared to that of the control (33.96 + or - 4.69 pmol/l) and the diabetic (29.34 + or - 2.62 pmol/l) group, which were not significantly different from each other. However, there was a significant decrease in GIP-Rs expression in both the obese (88.07 + or - 10.36 microg/ml) and diabetic (87.51 + or - 4.72 microg/ml) groups compared to the control group (120.35 + or - 8.06 microg/ml). During the second hour of the OGTT, plasma GIP was decreasing in all groups, however, the obese group had a significant hyperinsulinemia compared to the other two groups. Moreover, the diabetic group had a significantly lower plasma insulin level until the 90 min interval and thereafter it showed a non-significant difference compared to the control group. In conclusion, both obese and diabetic rats had an impaired early-phase insulinotropic effect of GIP due to impaired gene expression of GIP-Rs which could be a potential target to prevent transition of obesity to diabetes and to improve insulin secretion in the latter.