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.     

Evolution of the vertebrate glucose-dependent insulinotropic polypeptide (GIP) gene

The glucose-dependent insulinotropic polypeptide (GIP) gene is believed to have originated from a gene duplication event very early in vertebrate evolution that also produced the proglucagon gene, yet so far GIP has only been described within mammals. Here we report the identification of GIP genes in chicken, frogs, and zebrafish. The chicken and frog genes are organized in a similar fashion to mammalian GIP genes and contain 6 exons and 5 introns in homologous locations. These genes can also potentially be proteolytically processed in identical patterns as observed in the mammalian sequences that would yield a GIP hormone that is only one amino shorter than the mammalian sequences due to the removal of an extra basic residue by carboxypeptidase E. The zebrafish GIP gene and precursor protein is shorter than other vertebrate GIP genes and is missing exon 5. The predicted zebrafish GIP hormone is also shorter, being only 31 amino acids in length. The zebrafish GIP hormone is similar in length to the proglucagon-derived peptide hormones, peptides encoded from the gene most closely related to GIP. We suggest that the structure of zebrafish GIP is more similar to the ancestral gene, and that tetrapod GIP has been extended. The mammalian GIP hormone has also undergone a period of rapid sequence evolution early in mammalian evolution. The discovery of a conserved GIP in diverse vertebrate suggests that it has an essential role in physiology in diverse vertebrates, although it may have only recently evolved a role as an incretin hormone.     

Glucose-dependent insulinotropic peptide signaling pathways in endothelial cells

Glucose-dependent insulinotropic peptide (GIP) potentiates glucose-induced insulin secretion. In addition, GIP has vasoconstrictive or vasodilatory properties depending on the vascular bed affected. In order to assess whether this effect could be related to differences in GIP receptor expression, several different endothelial cell types were examined for GIP receptor expression. GIP receptor splice variants were detected and varied depending on the endothelial cell type. Furthermore, stimulation of these cells with GIP led to cell type dependent differences in activation of the calcium and cAMP signaling pathways. To our knowledge this is the first physiological characterization of receptors for GIP in endothelial cells.     

GIP, a Petunia hybrida GA-induced cysteine-rich protein: a possible role in shoot elongation and transition to flowering

The Petunia hybrida GA-induced proteins (GIPs) belong to a large group of proteins identified in numerous plant species. These proteins share a similar C-terminal region containing 12 cysteine residues in conserved positions. To date, the function of these proteins remains unclear. We previously found that GIP1 expression coincides with cell elongation in stems and flowers and is induced by gibberellic acid (GA3). Transient expression of a GIP1:green fluorescent protein (GFP) fusion in tobacco bright yellow 2 (BY2) cells and immunoblot analyses suggest microsomal compartmentalization with possible endoplasmic reticulum (ER) localization. However, the polyclonal anti-GIP1 antibodies also reacted with proteins extracted from the cell wall. Three novel GIP homologs, GIP2, GIP4, and GIP5, were isolated. While GIP4, similar to GIP1, is putatively localized to the ER membrane, the cleavable hydrophobic N-terminal sequences of GIP2 and GIP5 suggest cell wall localization. GIP1 and GIP2 are expressed during cell elongation, whereas GIP4 and GIP5 are expressed during cell division; nevertheless, they all were induced by GA3. We generated transgenic petunia in which we repressed the putative cell wall protein GIP2. The transgenic plants exhibited late flowering and reduced stem elongation. These phenotypic alterations were found under low, but not moderate-high temperatures, suggesting functional redundancy under normal growth conditions. The expression pattern and cellular localization of GIP2, its regulation by GA, and the phenotype of the transgenic plants suggest a role in GA-mediated cell elongation and transition to flowering.     

GIP as a potential therapeutic agent?

Glucose-dependent insulinotropic polypeptide (GIP) is released from K-cells in the gut after meal ingestion, and acts in concert with glucagon-like peptide 1 (GLP-1) to augment glucose-stimulated insulin secretion. While derivatives of GLP-1 are under active investigation for the treatment of type 2 diabetes, the case is different for GIP. Indeed, the insulinotropic effect of GIP is almost absent in patients with type 2 diabetes. In addition, the unfavourable pharmacokinetic profile of native GIP obviates its clinical application. Different analogues of GIP exhibiting prolonged stability and enhanced biological potency have been generated in order improve the anti-diabetic properties of GIP. However, glucose-normalisation, as is typically observed during the intravenous administration of GLP-1 in patients with type 2 diabetes, has not yet been achieved with GIP or its derivatives. Since GIP appears to play a role in lipid physiology and elevated levels of GIP have been associated with obesity, antagonising GIP action has been proposed as a therapeutic strategy for obesity. This concept has recently been reinforced by the observation that GIP receptor knock-out mice are protected from high-fat diet-induced obesity. However, eliminating the effect of endogenous GIP may at the same time impair postprandial insulin secretion, thereby severely disturbing glucose homeostasis. Therefore, therapeutic strategies based on either augmenting or antagonising GIP action are far from being established alternatives for the future therapy of type 2 diabetes or obesity.     

Glucose-dependent insulinotropic polypeptide (GIP) inhibits signaling pathways of advanced glycation end products (AGEs) in endothelial cells via its antioxidative properties

Glucose-dependent insulinotropic polypeptide (GIP) is one of the incretins, a gut hormone secreted from K cells in the intestine in response to food intake. It could be a potential therapeutic target for the treatment of patients with type 2 diabetes. However, effects of GIP on vascular injury remain unknown. Since interaction of advanced glycation end products (AGEs) with their receptor RAGE has been shown to play a crucial role in vascular damage in diabetes, this study investigated whether and how GIP blocked the deleterious effects of AGEs on human umbilical vein endothelial cells (HUVECs). GIP receptor was expressed in HUVECs. GIP, an analogue of cyclic AMP or inhibitors of NADPH oxidase inhibited the AGE-induced reactive oxygen species (ROS) generation in HUVECs. Furthermore, GIP reduced both RAGE mRNA and protein levels in HUVECs. GLP-1 also blocked the AGE-induced increase in mRNA levels of vascular cell adhesion molecule-1 (VCAM-1) and plasminogen activator inhibitor-1 in HUVECs. In addition, an antioxidant N-acetylcysteine mimicked the effects of GIP on RAGE and VCAM-1 gene expression in HUVECs. Our present study suggests that GIP could block the signal pathways of AGEs in HUVECs by reducing ROS generation and subsequent RAGE expression probably via GIP receptor-cyclic AMP axis.     

Glucose-dependent insulinotropic peptide: differential effects on hepatic artery vs. portal vein endothelial cells

Glucose-dependent insulinotropic peptide (GIP) has been reported to have opposing effects on splanchnic blood flow. GIP infusion in dogs results in an increase in portal vein circulation but a drop in hepatic artery blood flow. In an effort to evaluate whether these different responses were related to intrinsic differences in GIP effects, we isolated canine hepatic artery (HAEC) and portal vein endothelial cells (PVEC). We report that there are differences in GIP activation of the signal transduction pathways in these two cell types. GIP stimulates secretion of endothelin-1 (ET-1), a potent vasoconstrictor, from HAEC (EC50 0.28 nM) but not from PVEC. This effect could be abolished by preventing a rise in intracellular calcium, demonstrating the calcium dependence of GIP-induced ET-1 secretion from HAEC. The GIP effect was specific, as a GIP receptor antagonist blocked it. In contrast, GIP stimulated nitric oxide production from PVEC (EC50 0.09 nM) but not from HAEC. Taken together, our data demonstrate distinct differences in GIP effects on HAEC from those on PVEC. We conclude that differences in GIP stimulation of ET-1 vs. nitric oxide production in different vascular beds may account for some of the observed differences in its physiological effects.     

A new pathway for glucose-dependent insulinotropic polypeptide (GIP) receptor signaling: evidence for the involvement of phospholipase A2 in GIP-stimulated insulin secretion

The hormone glucose-dependent insulinotropic polypeptide (GIP) is an important regulator of insulin secretion. GIP has been shown to increase adenylyl cyclase activity, elevate intracellular Ca(2+) levels, and stimulate a mitogen-activated protein kinase pathway in the pancreatic beta-cell. In the current study we demonstrate a role for arachidonic acid in GIP-mediated signal transduction. Static incubations revealed that both GIP (100 nm) and ATP (5 microm) significantly increased [(3)H]arachidonic acid ([(3)H]AA) efflux from transfected Chinese hamster ovary K1 cells expressing the GIP receptor (basal, 128 +/- 11 cpm/well; GIP, 212 +/- 32 cpm/well; ATP, 263 +/- 35 cpm/well; n = 4; p < 0.05). In addition, GIP receptors were shown for the first time to be capable of functionally coupling to AA production through Gbetagamma dimers in Chinese hamster ovary K1 cells. In a beta-cell model (betaTC-3), GIP was found to elicit [(3)H]AA release, independent of glucose, in a concentration-dependent manner (EC(50) value of 1.4 +/- 0.62 nm; n = 3). Although GIP did not potentiate insulin release under extracellular Ca(2+)-free conditions, it was still capable of elevating intracellular cAMP and stimulating [(3)H]AA release. Our data suggest that cAMP is the proximal signaling intermediate responsible for GIP-stimulated AA release. Finally, stimulation of GIP-mediated AA production was shown to be mediated via a Ca(2+)-independent phospholipase A(2). Arachidonic acid is therefore a new component of GIP-mediated signal transduction in the beta-cell.     

Regulation of GIP and GLP1 receptor cell surface expression by N-glycosylation and receptor heteromerization

In response to a meal, Glucose-dependent Insulinotropic Polypeptide (GIP) and Glucagon-like Peptide-1 (GLP-1) are released from gut endocrine cells into the circulation and interact with their cognate G-protein coupled receptors (GPCRs). Receptor activation results in tissue-selective pleiotropic responses that include augmentation of glucose-induced insulin secretion from pancreatic beta cells. N-glycosylation and receptor oligomerization are co-translational processes that are thought to regulate the exit of functional GPCRs from the ER and their maintenance at the plasma membrane. Despite the importance of these regulatory processes, their impact on functional expression of GIP and GLP-1 receptors has not been well studied. Like many family B GPCRs, both the GIP and GLP-1 receptors possess a large extracellular N-terminus with multiple consensus sites for Asn-linked (N)-glycosylation. Here, we show that each of these Asn residues is glycosylated when either human receptor is expressed in Chinese hamster ovary cells. N-glycosylation enhances cell surface expression and function in parallel but exerts stronger control over the GIP receptor than the GLP-1 receptor. N-glycosylation mainly lengthens receptor half-life by reducing degradation in the endoplasmic reticulum. N-glycosylation is also required for expression of the GIP receptor at the plasma membrane and efficient GIP potentiation of glucose-induced insulin secretion from the INS-1 pancreatic beta cell line. Functional expression of a GIP receptor mutant lacking N-glycosylation is rescued by co-expressed wild type GLP1 receptor, which, together with data obtained using Bioluminescence Resonance Energy Transfer, suggests formation of a GIP-GLP1 receptor heteromer.     

Overexpression, Purification, and Characterization of Glutaminase-Interacting Protein, a PDZ-Domain Protein from Human Brain

A human brain cDNA clone coding for a novel PDZ-domain protein of 124 amino acids has been previously isolated in our laboratory. The protein was termed GIP (glutaminase-interacting protein) because it interacts with the C-terminal region of the human brain glutaminase L. Here we report the heterologous expression of GIP as a histidine-tagged fusion protein in Escherichia coli cells. The induction conditions (temperature and isopropyl beta-d-thiogalactopyranoside concentrations) were optimized in such a way that GIP accounted for about 20% of the total E. coli protein. A simple and rapid procedure for purification was developed, which yielded 17 mg of purified GIP per liter of bacterial cell culture. The apparent molecular mass of the protein by SDS-PAGE was 16 kDa, whereas in native form it was determined to be 28 kDa, which suggests dimer formation. The nature and integrity of the recombinant protein were verified by mass spectrometry analysis. The functionality of the GIP protein was tested with an in vitro activity assay: after being pulled down with glutathione S-transferase-glutaminase, GIP was revealed by Western blot using anti-GIP antibodies. Furthermore, the glutaminase activity in crude rat liver extracts was inhibited by the presence of recombinant purified GIP protein.     

The isolation and sequencing of human gastric inhibitory peptide (GIP)

Human GIP 1-42 and fragments of human GIP corresponding to GIP 10-42, GIP 11-42, and GIP 17-42 were isolated from acid-ethanol extracts of human small intestines with the aid of an anti-GIP serum specific for the extreme C-terminal portion of the GIP molecule. The full sequence of human GIP has been established by Edman degradation of these peptides and fragments thereof by automatic gas-phase sequencing. Human GIP differs from porcine GIP at residues 18 and 34. The sequence of human GIP is thus: (Formula: see text) Amino acid residues 18 and 34 are Arg and Ser, respectively, in porcine GIP.     

Regulation of glucose-dependent insulinotropic polypeptide release by protein in the rat

Glucose-dependent insulinotropic polypeptide (GIP) release has been demonstrated predominantly after ingestion of carbohydrate and fat. These studies were conducted to determine the effects of protein on GIP expression in the rat. Whereas no significant changes in duodenal mucosal GIP mRNA levels were detected in response to peptone, the duodenal GIP concentration increased from 8.4+/-1.5 to 19.8+/-3.2 ng GIP/mg protein at 120 min (P<0.01). Plasma GIP levels also increased from 95+/-5.2 pg/ml to a peak of 289+/-56.1 pg/ml at 120 min (P<0.01). To determine whether the effects of protein on GIP were due to stimulation of acid secretion, rats were pretreated with 10 mg/kg omeprazole, after which mucosal and plasma GIP concentrations were partially attenuated. To further examine the effects of luminal acid, rats were administered intraduodenal 0.1 M HCl for 120 min, which significantly enhanced GIP expression. These studies indicate that nutrient protein provides a potent stimulus for GIP expression in the rat, an effect that occurs at the posttranslational level and may be mediated in part through the acid-stimulatory properties of protein. The effects of acid on GIP are consistent with a role for GIP as an enterogastrone in the rat.     

Effects of glicentine on insulin secretion

The glucagon-like immunoreactivity of the gastrointestinal tract is heterogeneous, probably including several different peptides. One of these peptides, glicentine, has recently been extracted and highly purified. Furthermore, by immunocytochemistry a glicentine-like peptide has been reported to occur in the glucagon cell of the pancreatic islets. In the present study we investigated the effects of pure glicentine on insulin release in vivo in mice. The effects were compared with effects of two other peptides, glucagon and GIP. It was found that glicentine had no influence on basal insulin secretion. This was in contrast to equimolar doses of glucagon and GIP, which both stimulated the secretion of insulin. Glucose-induced insulin release was partially inhibited by glicentine. D-glucose, in a dose selected to give a response of 25% of its maximal, raised the plasma insulin concentrations by 44.0 +/- 5.9 microU/ml. The corresponding rise for glicentine plus D-glucose was 22.3 +/- 3.7 microU/ml, i.e. glicentine inhibited glucose-induced insulin released by about 50% (p < 0.01). GIP, on the other hand, enhanced glucose-induced insulin release. This enhancement was diminished by glicentine, a reflection of the inhibition by glicentine of the glucose-induced insulin release. Neither glicentine nor GIP in the doses tested had any effect on insulin secretion induced by cholinergic stimulation. In conclusion, glicentine seems to have no effect on basal insulin release in the mouse, but it partially inhibits glucose-induced insulin secretion. Thus, if the recently demonstrated glicentine-like peptide in the glucagon cell is authentic glicentine, the glucagon cell of the pancreatic islets may contain peptides with stimulatory (glucagon) as well as inhibitory (glicentine) effects on insulin secretion induced by glucose.     

Effects of glucose-dependent insulinotropic peptide on behavior

Glucose-dependent insulinotropic peptide (GIP) is an incretin hormone that rises rapidly in response to nutrient ingestion. The GIP receptor is widely expressed in the brain including the brain stem, telencephalon, diencephalon, olfactory bulb, pituitary, and cerebellum. Until recently it was not clear what the endogenous ligand for this receptor was because no GIP expression had been demonstrated in the brain. GIP synthesis has now been documented in the dentate gyrus of the hippocampus. To define GIP effects on behavior we utilized a mouse model a GIP-overexpressing transgenic mouse (GIP Tg). Specifically, anxiety-related behavior, exploration, memory, and nociception were examined. Compared to age-matched adult male C57BI/6 controls GIP Tg mice displayed enhanced exploratory behavior in the open-field locomotor activity test. GIP Tg mice also demonstrated increased performance in some of the motor function tests. These data suggest that the GIP receptor plays a role in the regulation of locomotor activity and exploration. To our knowledge, this is the first report of effects of GIP on behavior.     

Physiology of GIP--a lesson from GIP receptor knockout mice.

A much greater insulin response is observed after oral glucose load than after intravenous injection of glucose. The hormonal factor(s) implicated as transmitters of signals from the gut to pancreatic beta-cells was referred to incretin; gastric inhibitory polypeptide or glucose-dependent insulinotropic polypeptide (GIP) is identified as one of the incretins. GIP exerts its effects by binding to its specific receptor, the GIP receptor, which is expressed in various tissues including pancreatic islets, adipose tissue, and brain. However, the physiological role of GIP has been generally thought to stimulate insulin secretion from pancreatic beta-cells, and the other actions of GIP have received little attention. We have bred and characterized mice with a targeted mutation of the GIP receptor gene. From these studies, we now know that GIP not only mediates early insulin secretion by acting on pancreatic beta-cells, but also links overnutrition to obesity by acting on adipocytes.     

Review and proposed action of alpha-fetoprotein growth inhibitory peptides as estrogen and cytoskeleton-associated factors

The (H) human growth-promoting factor, alpha-fetoprotein (AFP), has been reported to possess a growth inhibitory motif as an occult epitope in the compactly folded circulating form of the protein. Intermediate unfolded forms of the human HAFP molecule induced by stress, shock, and high ligand concentrations have revealed the presence of an encrypted growth-suppressive segment on the third domain of HAFP. A purified linear synthetic 34-mer segment termed the "growth inhibitory peptide" (GIP) exhibits various oligomeric forms with complex aggregation behaviors, in which dominant trimeric forms were found to be suppressive in assays of estrogen-induced growth. While several amino acid analogs of the cysteines of the GIP retained inhibitory activity, heavy metal binding and pre-incubation of the peptides with a variety of cations and hormone ligands were found to influence the outcomes of growth bioassays. Smaller segments of the original 34-mer were each found to display growth activities of their own, with the middle segment (P149b) also showing hydrophobic dye-binding properties. Studies of amino acid sequence identity further revealed that the GIP sequences displayed identity/similarity matches to both cytoplasmic and nucleus-cytoskeleton-associated proteins, and experimental evidence served to support these findings. That is, the peptide was capable of modulating tubulin polymerization, cell shape, and cell-surface aggregation phenomena reminiscent of a microtubule-associated protein. Immunofluorescence studies further pinpointed the localization of the GIP to cytoplasmic regions of high cytoskeletal density in the cell. Because of the involvement of the GIP in experimental models of the estrogen receptor/cytoskeleton, a mechanism of action is forwarded in which the linear GIP is proposed to be a G-coupled receptor binding ligand that is translocated across the plasma membrane via receptor-mediated endocytosis. Thus, it was predicted that the linear GIP and possibly its peptidic segments serve as decoy ligands to cell-surface receptors in order to gain access to the cytoplasmic compartment of the cell.     

Ultrastructural localization of cholecystokinin in endocrine cells of the dog duodenum by the immunogold technique

Summary Cholecystokinin (CCK) has been localized by the immunogold technique in a type of endocrine cell of the dog duodenum characterized by small (166±38 nm) secretory granules with fairly dense, homogeneous core separated from its enveloping membrane by a thin clear space. The CCK cell is immunocytochemically distinct and cytologically different from other types of endocrine cells, as the secretin, GIP and motilin cells, already identified in the dog duodenum.