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Glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide 1 (GLP-1) are gut-derived incretins that potentiate glucose clearance following nutrient ingestion. Elimination of incretin receptor action in GIPR(-/-) or GLP-1R(-/-) mice produces only modest impairment in glucose homeostasis, perhaps due to compensatory upregulation of the remaining incretin. We have now studied glucose homeostasis in double incretin receptor knockout (DIRKO) mice. DIRKO mice exhibit normal body weight and fail to exhibit an improved glycemic response after exogenous administration of GIP or the GLP-1R agonist exendin-4. Plasma glucagon and the hypoglycemic response to exogenous insulin were normal in DIRKO mice. Glycemic excursion was abnormally increased and levels of glucose-stimulated insulin secretion were decreased following oral but not intraperitoneal glucose challenge in DIRKO compared with GIPR(-/-) or GLP-1R(-/-) mice. Similarly, glucose-stimulated insulin secretion and the response to forskolin were well preserved in perifused DIRKO islets. Although the dipeptidyl peptidase-IV (DPP-IV) inhibitors valine pyrrolidide (Val-Pyr) and SYR106124 lowered glucose and increased plasma insulin in wild-type and single incretin receptor knockout mice, the glucose-lowering actions of DPP-IV inhibitors were eliminated in DIRKO mice. These findings demonstrate that glucose-stimulated insulin secretion is maintained despite complete absence of both incretin receptors, and they delineate a critical role for incretin receptors as essential downstream targets for the acute glucoregulatory actions of DPP-IV inhibitors. Show less

Glucose-dependent insulinotropic polypeptide (GIP) is secreted postprandially and acts in concert with glucose to stimulate insulin secretion from the pancreas. Here, we describe a novel pathway for the regulation of GIP receptor (GIPR) expression within clonal beta-cell lines, pancreatic islets, and in vivo. High (25 mM) glucose was able to significantly reduce GIPR mRNA levels in INS(832/13) cells after only 6 h. In contrast, palmitic acid (2 mM) and WY 14643 (100 microM) stimulated approximate doublings of GIPR expression in INS(832/13) cells under low (5.5 mM), but not high (25 mM), glucose conditions, suggesting that fat can regulate GIPR expression via PPARalpha in a glucose-dependent manner. Both MK-886, an antagonist of PPARalpha, and a dominant negative form of PPARalpha transfected into INS(832/13) cells caused a significant reduction in GIPR expression in low, but not high, glucose conditions. Finally, in hyperglycemic clamped rats, there was a 70% reduction in GIPR expression in the islets and a 71% reduction in GIP-stimulated insulin secretion from the perfused pancreas. Thus, evidence is presented that the GIPR is controlled at normoglycemia by the fatty acid load on the islet; however, when exposed to hyperglycemic conditions, the GIPR is down-regulated, which may contribute to the decreased responsiveness to GIP that is observed in type 2 diabetes. Show less

We have previously characterized the receptor for glucose-dependent insulinotropic polypeptide (GIPR) in vascular endothelial cells (EC). Different EC types were found to contain distinct GIPR splice variants. To determine whether activation of the GIPR splice variants resulted in different cellular responses, we examined GIP effects on human umbilical vein endothelial cells (HUVEC), which contain two GIPR splice variants, and compared them with a spontaneously transformed human umbilical vein EC line, ECV 304, which contains four GIPR splice variants. GIP dose-dependently stimulated HUVEC and ECV 304 proliferation as measured by [3H]thymidine incorporation. GIP increased endothelin-1 (ET-1) secretion from HUVEC but not from ECV 304. Use of the endothelin B receptor blocker BQ-788 resulted in an inhibition of [3H]thymidine incorporation in HUVEC but not in ECV 304. These findings suggest that, although GIP increases [3H]thymidine incorporation in both HUVEC and ECV 304, this proliferative response is mediated by ET-1 only in HUVEC. These differences in cellular response to GIP may be related to differences in activation of GIPR splice variants. Show less

The incretins glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1) are gut hormones that act via the enteroinsular axis to potentiate insulin secretion from the pancreas in a glucose-dependent manner. Both GLP-1 receptor and GIP receptor knockout mice (GLP-1R(-/-) and GIPR(-/-), respectively) have been generated to investigate the physiological importance of this axis. Although reduced GIP action is a component of type 2 diabetes, GIPR-deficient mice exhibit only moderately impaired glucose tolerance. The present study was directed at investigating possible compensatory mechanisms that take place within the enteroinsular axis in the absence of GIP action. Although serum total GLP-1 levels in GIPR knockout mice were unaltered, insulin responses to GLP-1 from pancreas perfusions and static islet incubations were significantly greater (40-60%) in GIPR(-/-) than in wild-type (GIPR(+/+)) mice. Furthermore, GLP-1-induced cAMP production was also elevated twofold in the islets of the knockout animals. Pancreatic insulin content and gene expression were reduced in GIPR(-/-) mice compared with GIPR(+/+) mice. Paradoxically, immunocytochemical studies showed a significant increase in beta-cell area in the GIPR-null mice but with less intense staining for insulin. In conclusion, GIPR(-/-) mice exhibit altered islet structure and topography and increased islet sensitivity to GLP-1 despite a decrease in pancreatic insulin content and gene expression. Show less

Secretion of gastric inhibitory polypeptide (GIP), a duodenal hormone, is primarily induced by absorption of ingested fat. Here we describe a novel pathway of obesity promotion via GIP. Wild-type mice fed a high-fat diet exhibited both hypersecretion of GIP and extreme visceral and subcutaneous fat deposition with insulin resistance. In contrast, mice lacking the GIP receptor (Gipr(-/-)) fed a high-fat diet were clearly protected from both the obesity and the insulin resistance. Moreover, double-homozygous mice (Gipr(-/-), Lep(ob)/Lep(ob)) generated by crossbreeding Gipr(-/-) and obese ob/ob (Lep(ob)/Lep(ob)) mice gained less weight and had lower adiposity than Lep(ob)/Lep(ob) mice. The Gipr(-/-) mice had a lower respiratory quotient and used fat as the preferred energy substrate, and were thus resistant to obesity. Therefore, GIP directly links overnutrition to obesity and it is a potential target for anti-obesity drugs. Show less

Control of cortisol secretion by the abnormal expression of the gastric inhibitory polypeptide receptor (GIP-R) have been observed in some rare cases of ACTH-independent, food-dependent Cushing's syndrome (FD-ACS) due to adrenal adenoma (AA) or bilateral macronodular hyperplasia (AIMAH). This study was performed to determine the prevalence of GIP-R ectopic expression in ACS and its correlation with fasting cortisol levels. GIP-R expression was studied by RT-PCR in 30 unilateral adrenal tumors [16 AA and 14 adrenocortical cancer (AC)] and 8 AIMAH tissues. Fasting and postprandial cortisol levels were assayed, respectively, at 0800 and 1200 h in AA, AC, and AIMAH, and 1 h after a morning standard meal in 6 AIMAH patients. Similar expression of 2 GIP-R isoforms was observed in 1 of 16 AA, 0 of 14 AC, and 4 of 8 AIMAH as well as in the 4 insulinomas used as positive controls. In vitro study of the GIP-R-expressing AA showed stimulation of cortisol secretion and cAMP production by GIP. The fasting 0800-h plasma cortisol level was above 276 nmol/liter in all patients except 1 AA case and 1 AIMAH case, both of whom expressed GIP-R. In the 3 additional AIMAH cases that expressed the GIP-R, fasting plasma cortisol levels were above 276 nmol/liter. This study demonstrates that ectopic expression of GIP-R is rare in AA and is usually associated with the low fasting plasma cortisol levels that characterize FD-ACS. In contrast, GIP-R expression is frequent in AIMAH and might not always be associated with a low fasting plasma cortisol level. This suggests that maintenance of hypercortisolemia in GIP-R- expressing AIMAH does not always depend solely on GIP-R, and that simultaneous abnormal expression of other membrane receptors might be present. The expression of GIP-R could not be observed during malignant transformation of the adrenal cortex. This study highlighted the major role of cAMP alterations secondary to GIP-R ectopic expression in the pathophysiology of AIMAH and in some rare cases of well differentiated benign adrenocortical tumors. Show less

The cyclic AMP (cAMP) pathway plays a major role in the development of endocrine tissues and various molecular defects of key components of this pathway (G protein, receptors, PKA, ...) have been observed in endocrine tumors. Hypersecretion of adrenocorticotropin hormone (ACTH), the key activator of the cAMP pathway in adrenal cortex, is associated with adrenocortical hyperplasia and cortisol oversecretion (Cushing's syndrome). The best example of "illegitimate" membrane receptors expression reported is the abnormal expression of the adenylyl cyclase activating gastric inhibitory peptide receptor (GIP-R) in ACTH-independent Cushing's syndrome (ACS). We have observed that ectopic expression of the GIP-R is frequent in ACTH-Independent Macronodular Adrenal Hyperplasia (AIMAH), rare in benign adrenal adenoma (AA), but seems absent in Adrenal Cancer (AC). In vivo systematic screening of AIMAH shows at least one abnormal response of cortisol (suggesting "illegitimate" membrane receptor expression) in almost all patients. Somatic and germ line inactivating mutations of PRKAR1 (regulatory subunit R1A of PKA) can be observed in patient with isolated primary pigmented nodular adrenocortical disease (PPNAD) and AA responsible for ACS. At the nuclear level, the cAMP pathway regulates transcription mainly by PKA-dependent phosphorylation of the cyclic AMP response element binding (CREB) family of transcription factors (CREB, CREM, and ATF-1). Cyclic AMP response element binding protein (CREB) is expressed in normal adrenal cortex. Alterations of CRE binding proteins with loss of CREB expression and compensatory overexpression of CREMtau is observed in the human adrenocortical cancer cell line H295R. Similar alterations are found at the protein level in human malignant adrenocortical tumors. In conclusion, various alterations leading to activation or inactivation of key components of the cAMP signaling pathway can be observed in adrenocortical tumorigenesis. Show less

The pathology of diabetic retinopathy includes dilatation and beading of retinal vessels, and vascular sheathing. To gain a better understanding of the molecular events leading to diabetic retinopathy, we investigated disease-specific gene responses by screening differential expression using cDNA microarray. Male Sprague-Dawley rats were intraperitoneally injected with streptozotocin (STZ, 50 mg/kg) or the control buffer and were maintained for 6 weeks. Total RNA extracted from the retinas of both groups was used for cDNA microarray analysis. Signals from all the spots representing hybridized DNA were quantified and compared between the normal and diabetic rat retinas. Among 1176 genes analyzed, the retinal expression of glucose-dependent insulinotropic polypeptide (GIP) was found to increase in STZ-induced diabetic rats compared to controls. GIP is a secreted protein, known to be released from the small intestine, which potentiates glucose-induced insulin secretion from the pancreas. However, the expression of GIP and its receptor (GIPR) has not been previously noted in the rat retina. To further validate the expression of GIP in the rat retina and to determine its possible role in the development of early diabetic retinopathy, we investigated its expression by RT-PCR, Northern blotting, and immunohistochemistry in normal and diabetic rat retinas. GIP mRNA and protein are not only expressed in the rat retina, but their levels are greater in the diabetic rat as compared to controls. And GIPR expression was also upregulated in the retinas of STZ-induced diabetic rats. We here demonstrate for the first time the expression of GIP and GIPR in the rat retina. And we also revealed some genetic events in the early stage of diabetic retinopathy including the de novo increment of GIP and GIPR expression in the retina. Show less

Glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic peptide (GIP) potentiate glucose-stimulated insulin secretion after enteral nutrient ingestion. We compared the relative incretin and nonincretin actions of GLP-1 and GIP in +/+ and GLP-1R-/- mice using exendin(9-39) and immunopurified anti-GIP receptor antisera (GIPR Ab) to antagonize GLP-1 and GIP action, respectively. Both antagonists produced a significant increase in glycemic excursion after oral glucose loading of +/+ mice (P < 0.05 for antagonists us. controls). Exendin(9-39) also increased blood glucose and decreased glucose-stimulated insulin in +/+ mice after ip glucose loading [0.58 +/- 0.02 vs. 0.47 +/- 0.02 ng/ml in saline- vs. exendin(9-39)-treated mice, respectively, P < 0.05]. In contrast, GIPR Ab had no effect on glucose excursion or insulin secretion, after ip glucose challenge, in +/+ or GLP-1R-/- mice. Repeated administration of exendin(9-39) significantly increased blood glucose and reduced circulating insulin levels but had no effect on levels of pancreatic insulin or insulin messenger RNA transcripts. In contrast, no changes in plasma glucose, circulating insulin, pancreatic insulin content, or insulin messenger RNA were observed in mice, 18 h after administration of GIPR Ab. These findings demonstrate that GLP-1, but not GIP, plays an essential role in regulating glycemia, independent of enteral nutrient ingestion in mice in vivo. Show less

A novel GIP receptor antagonist was developed to evaluate the acute role of glucose-dependent insulinotropic polypeptide (GIP) in the insulin response to oral glucose in rats. Antisera to an extracellular epitope of the GIP receptor (GIPR) detected immunoreactive GIPR on rat pancreatic beta-cells. Purified GIPR antibody (GIPR Ab) specifically displaced GIP binding to the receptor and blocked GIP-mediated increases in intracellular cAMP. When delivered to rats by ip injection, GIPR Ab had a half-life of approximately 4 days. Treatment with GIPR Ab (1 microg/g BW) blocked the potentiation of glucose-stimulated insulin secretion by GIP (60 pmol) but not glucagon-like peptide-1 (GLP-1, 60 pmol) in anesthetized rats. The insulin response to oral glucose was delayed in conscious unrestrained rats that were pretreated with GIPR Ab. Plasma insulin levels were approximately 35% lower at 10 min in GIPR Ab treated animals compared with controls. As a result, the glucose excursion was greater in the GIPR Ab treated group. Fasting plasma glucose levels were not altered by GIPR Ab. We conclude that release of GIP following oral glucose may act as an anticipatory signal to pancreatic beta-cells to promote rapid release of insulin for glucose disposal. Show less

The glucose-dependent insulinotropic polypeptide receptor (GIPR) is a member of class II G protein-coupled receptors. Recent studies have suggested that desensitization of the GIPR might contribute to impaired insulin secretion in type II diabetic patients, but the molecular mechanisms of GIPR signal termination are unknown. Using HEK L293 cells stably transfected with GIPR complementary DNA (L293-GIPR), the mechanisms of GIPR desensitization were investigated. GIP dose dependently increased intracellular cAMP levels in L293-GIPR cells, but this response was abolished (65%) by cotransfection with G protein-coupled receptor kinase 2 (GRK2), but not with GRK5 or GRK6. Beta-arrestin-1 transfection also induced a significantly decrease in GIP-stimulated cAMP production, and this effect was greater with cotransfection of both GRK2 and beta-arrestin-1 than with either alone. In betaTC3 cells, expression of GRK2 or beta-arrestin-1 attenuated GIP-induced insulin release and cAMP production, whereas glucose-stimulated insulin secretion was not affected. GRK2 and beta-arrestin-1 messenger RNAs were identified by Northern blot analysis to be expressed endogenously in betaTC3 and L293 cells. Overexpression of GRK2 enhanced agonist-induced GIPR phosphorylation, but receptor endocytosis was not affected by cotransfection with GRKs or beta-arrestin-1. These results suggest a potential role for GRK2/beta-arrestin-1 system in modulating GIP-mediated insulin secretion in pancreatic islet cells. Furthermore, GRK-mediated receptor phosphorylation is not required for endocytosis of the GIPR. Show less

The Glucose-dependent insulinotropic polypeptide receptor (GIPR) is a member of the secretin-vasoactive intestinal polypeptide family of G-protein coupled receptors possessing seven transmembrane domains. We report here the cloning and the exon-intron structure of the rat GIPR gene, along with the identification and characterization of its 5'-flanking region. The coding region of the GIPR gene spans approximately 10.2 kilobases and contains 13 exons. Three additional exons, two encoding either 5' or 3' untranslated sequences and one contained in a novel alternatively spliced mRNA, were identified. The 5'-flanking sequences contained a number of transcription factor binding motifs, including a cAMP response element, an octamer binding site, three SP1 sites and an initiator element. However, neither a CAAT motif nor TATA box were found. Transient transfection assays demonstrated that the 5'-flanking region of the GIPR gene can efficiently promote transcription in RIN38 cells and that deletion of 50 base pairs containing a potential SPI binding sites leads to a 2.4-fold loss of transcriptional activity. In addition, transient transfection experiments comparing the relative promoter activities of 5'-flanking sequences of the GIPR gene in RIN38 and rat-2 cells suggests that distal negative regulatory sequences may control cell-specific expression. Show less

Mice with a targeted mutation of the gastric inhibitory polypeptide (GIP) receptor gene (GIPR) were generated to determine the role of GIP as a mediator of signals from the gut to pancreatic beta cells. GIPR-/- mice have higher blood glucose levels with impaired initial insulin response after oral glucose load. Although blood glucose levels after meal ingestion are not increased by high-fat diet in GIPR+/+ mice because of compensatory higher insulin secretion, they are significantly increased in GIPR-/- mice because of the lack of such enhancement. Accordingly, early insulin secretion mediated by GIP determines glucose tolerance after oral glucose load in vivo, and because GIP plays an important role in the compensatory enhancement of insulin secretion produced by a high insulin demand, a defect in this entero-insular axis may contribute to the pathogenesis of diabetes. Show less

Gastric inhibitory polypeptide (GIP)-dependent Cushing's syndrome has been reported to occur either in unilateral adrenal adenoma or in bilateral macronodular adrenal hyperplasia. A 33-yr-old woman with Cushing's syndrome was found to have two 2.5- to 3-cm nodules in the right adrenal on computed tomography scan; the left adrenal appeared normal except for the presence of a small 0.8 x 0.6-cm nodule. Uptake of iodocholesterol was limited to the right adrenal. Plasma morning cortisol was 279 nmol/L fasting and 991 nmol/L postprandially, and ACTH remained suppressed. Plasma cortisol increased after oral glucose (202%) or a lipid-rich meal (183%), but not after a protein-rich meal (95%) or iv glucose (93%); the response to oral glucose was blunted by pretreatment with 100 microg octreotide, sc. Plasma cortisol and GIP levels were positively correlated (r = 0.95; P = 0.0001); cortisol was stimulated by the administration of human GIP iv (225%), but not by GLP-1, insulin, TRH, GnRH, glucagon, arginine vasopressin, upright posture, or cisapride orally. A right adrenalectomy was performed; GIP receptor messenger ribonucleic acid was overexpressed in both adrenal nodules and in the adjacent cortex. Histopathology revealed diffuse macronodular adrenal hyperplasia without internodular atrophy. Three months after surgery, fasting plasma ACTH and cortisol were suppressed, but cortisol increased 3.6-fold after oral glucose, whereas ACTH remained suppressed; this was inhibited by octreotide pretreatment, suggesting that cortisol secretion by the left adrenal is also GIP dependent. We conclude that GIP-dependent nodular hyperplasia can progress in an asynchronous manner and that GIPR overexpression is an early event in this syndrome. Show less

The glucose-dependent insulinotropic peptide receptor (GIP-R) is a member of G-protein-coupled, seven transmembrane-spanning receptors. Recent studies have shown that elevated serum GIP level in diabetic patients may induce chronic desensitization of the GIP-R, and that this mechanism could contribute to impaired insulin secretion. The cellular basis of down-regulation and chronic desensitization of GIP-R is unclear. To explore the role of the carboxyl terminus of the GIP-R in mediating these processes, five truncated GIP-Rs (T395, T399, T420, T431, T455) were created to delete consecutive serines from the carboxyl end. All mutants except T395 exhibit an identical ligand-binding affinity to the WT receptor. The T395 mutant, which had the entire carboxyl tail removed, does not bind to ligand. Down-regulation and desensitization was assessed by measuring the receptor number and the ability of agonist-induced cAMP or [Ca2+] generation after pre-exposure to 10(-7) M GIP for 24 h. The wild-type (WT) and T421, T431, T455 mutant GIP-Rs are maximally down-regulated by GIP preincubation, whereas T399 mutant does not, indicating that the sequence between amino acids 399 and 420 is critical for this process. Mutation analysis of this area by alanine scanning mutagenesis reveals two critical residues: serine 406 and cysteine 411. Replacement of serine 406 with arginine (S406R) or alanine (S406A) partly attenuates agonist-induced down-regulation and desensitization. In contrast, mutation of the cysteine 411 to glycine (C411G) or alanine (C411A) markedly attenuates both processes. Mutant SCRG, in which both serine 406 and cysteine 411 are mutated, behaves similar to C411G or C4111A. The data suggest that chronic desensitization and down-regulation of the GIP-R may be mediated by similar mechanisms, and that the cysteine in the carboxyl terminus plays an essential role in regulating both processes. Show less

The glucose-dependent insulinotropic peptide receptor (GIP-R) is a member of the G protein-coupled receptors. Recent studies have indicated that elevated serum GIP concentrations in type II diabetic patients might induce desensitization of the GIP-R, and this mechanism could contribute to impaired insulin secretion. The cellular and molecular mechanisms governing GIP desensitization are unknown. Here, we report the results of studies on a new family of proteins known as regulators of G protein signaling (RGS) that have been shown to mediate the desensitization process of other receptors. GIP-R and RGS1, -2, -3, and -4 complementary DNAs were cotransfected into human embryonic kidney cells (L293). GIP-stimulated cAMP generation in control cells and in those coexpressing RGS1, -3, and -4 displayed a dose-dependent increase 10 min after GIP treatment. In contrast, RGS2 expression inhibited the GIP-induced cAMP response by 50%, a response similar to that of cells desensitized by preincubation with 10(-7) M GIP. In betaTC3 cells, preincubation of GIP attenuated GIP-induced insulin release by 45% at 15 min and by 55% at 30 min. Expression of RGS2 in the betaTC3 cells significantly decreased GIP-stimulated insulin secretion, whereas glucose-induced insulin release was not affected. RGS2 messenger RNA was identified by Northern blot analysis to be expressed endogenously in betaTC3 and L293 cells, and its level was significantly induced by GIP treatment in betaTC3 cells. Moreover, RGS2 bound Gs alpha protein in an in vitro system, suggesting that RGS2 attenuated the Gs-adenylate cyclase signaling pathway. These results suggest a potential role for RGS2 in modulating GIP-mediated insulin secretion in pancreatic islet cells. Show less

Abnormal responsiveness of adrenocortical cells to gastric inhibitory polypeptide (GIP) in food-dependent Cushing's syndrome suggested that adrenal expression of ectopic, overexpressed, or mutated GIP receptor (GIPR) underlies this syndrome. The expression of GIPR was studied by RT-PCR in human adrenal tissues from two patients with GIP-dependent Cushing's syndrome (adenoma, bilateral hyperplasia), five fetal or adult controls, one patient with Cushing's disease, and four patients with non-food-dependent cortisol-secreting adenomas or bilateral hyperplasias and compared to that in normal pancreas. Hybridization of the RT-PCR-amplified ribonucleic acids with the human GIPR complementary DNA showed an overexpression of GIPR in the adrenals of the two GIP-dependent Cushing's syndrome patients compared to that in normal adrenal tissues (2-3 orders of magnitude) or pancreas (10-fold); no signal could be seen in adrenal adenomas or macronodular hyperplasia from cases of non-food-dependent Cushing's syndrome. No mutation of the GIPR was identified by sequencing the full-length receptor in GIP-dependent adrenal tissue. New alternative spliced isoforms of the GIPR were found, but are identical in GIP-dependent and normal adrenal tissues. Incubation of adrenal cells with GIP stimulates cortisol secretion in GIP-dependent, but not in normal fetal, adult, or non-food-dependent Cushing's syndrome, adrenals. We conclude that the GIPR overexpression and its coupling to steroidogenesis underlie GIP-dependent Cushing's syndrome. Show less

The glucose-dependent insulinotropic peptide receptor (GIP-R) is a member of the secretin and parathyroid hormone (PTH) family of seven transmembrane-spanning receptors. Point mutations of a histidine at the junction between the first intracellular loop and the second membrane-spanning domain and a threonine in the sixth membrane-spanning domain of the human PTH-receptor have been reported to be associated with constitutive activation of the PTH receptor in Jansen-type metaphyseal chondrodysplasia. In this study, we explored whether such mutations in the GIP-R might similarly induce constitutive, ligand-independent activation of the receptor. Single amino acid substitutions in the GIP receptor were made by site-directed mutagenesis and receptor binding and cAMP levels were measured in transfected human embryonal kidney cell line (L293). Mutation of the threonine at position 340 in the sixth transmembrane spanning domain to proline (T340P) led to agonist-independent constitutive activity and exhibited a four-fold increase in basal cAMP level as compared to the wild-type GIP-R. The increase in cAMP level in T340P mutant was proportional to the amount of transfected plasmid and corresponded to the receptor number on the cell surface. Despite its high basal cAMP level, the T340P mutant could be further stimulated by GIP, with maximal cAMP generation comparable to the wild-type receptor. The change of amino acid histidine at position 169 to arginine (H169R), however, behaved like the wild type receptor and did not possess constitutive activity. These results illustrate that a point mutation of threonine to proline at position 340 results in constitutive activation of the GIP receptor, without affecting its sensitivity to agonist stimulation. Show less

Gastric inhibitory polypeptide, originally isolated from porcine intestine, is a gastrointestinal hormone belonging to the vasoactive intestinal peptide (VIP)/glucagon/secretin family. GIP consists of 42 amino acid residues which is derived by proteolytic processing of a GIP precursor. In vivo and in vitro experiments have indicated that GIP auguments glucose-stimulated insulin secretion, suggesting that GIP plays an important role in the regulation of insulin secretion as an incretin. Thus, GIP now is generally referred to as glucose-dependent insulinotropic polypeptide. It is also suggested that GIP may be involved in the pathogenesis of non insulin-dependent diabetes mellitus (NIDDM). GIP exerts its biological actions by binding to its specific receptors, which appear to be coupled to G proteins. We have isolated a cDNA encoding a GIP receptor from a hamster insulinoma(HIT-T15) cDNA library. The hamster GIP receptor is a 462 amino acid protein having seven transmembrane segments. Expression of recombinant of hamster GIP receptors in Chinese hamster ovary (CHO) cells shows that it binds specifically to GIP with high affinity (IC50 = 9.6 nM) and is positively coupled to adenylate cyclase. RNA blot analysis reveals that a 3.8-kb GIP receptor mRNA is expressed at high levels in rat pancreatic islets as well as in HIT-T15 cells. Show less

Gastric inhibitory polypeptide (GIP), which is released from the gastrointestinal tract, stimulates insulin secretion from pancreatic beta cells and plays a crucial role in the regulation of insulin secretion during the postprandial phase. We have isolated the human gene (GIPR) and cDNA encoding the GIP receptor by a combination of the conventional screening and polymerase chain reaction procedures. Human GIP receptor cDNA encodes a protein of 466 amino acids that is 81.5 and 81.2% identical to the previously cloned hamster and rat GIP receptor, respectively. Hydropathic analysis shows the presence of a signal peptide and seven potential transmembrane domains, a feature characteristic of the VIP/glucagon/secretin receptor family of G protein-coupled receptors. The human GIPR gene is about 13.8 kb long, consists of 14 exons, and carries 17 Alu repeats. Show less