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GIP is an important hormonal link between nutrition and bone formation. We show for the first time that BMSCs express functional GIP receptors, that expression decreases with aging, and that elevations in GIP can prevent age-associated bone loss. We previously showed that C57BL/6 mice lose bone mass as they age, particularly between 18 and 24 mo of age. The mechanisms involved in this age-dependent induced bone loss are probably multifactorial, but adequate nutrition and nutritional signals seem to be important. Glucose-dependent insulinotropic peptide (GIP) is an enteric hormone whose receptors are present in osteoblasts, and GIP is known to stimulate osteoblastic activity in vitro. In vivo, GIP-overexpressing C57BL/6 transgenic (GIP Tg(+)) mice have increased bone mass compared with controls. Bone histomorphometric data suggest that GIP increases osteoblast number, possibly by preventing osteoblastic apoptosis. However, potential GIP effects on osteoblastic precursors, bone marrow stromal cells (BMSCs), had not previously been examined. In addition, effects of GIP on age-induced bone loss were not known. Changes in BMD, biomechanics, biomarkers of bone turnover, and bone histology were assessed in C57BL/6 GIP Tg(+) versus Tg(-) (littermate) mice between the ages of 1 and 24 mo of age. In addition, age-related changes in GIP receptor (GIPR) expression and GIP effects on differentiation of BMSCs were also assessed as potential causal factors in aging-induced bone loss. We report that bone mass and bone strength in GIP Tg(+) mice did not drop in a similar age-dependent fashion as in controls. In addition, biomarker measurements showed that GIP Tg(+) mice had increased osteoblastic activity compared with wildtype control mice. Finally, we report for the first time that BMSCs express GIPR, that the expression decreases in an age-dependent manner, and that stimulation of BMSCs with GIP led to increased osteoblastic differentiation. Our data show that elevated GIP levels prevent age-related loss of bone mass and bone strength and suggest that age-related decreases in GIP receptor expression in BMSCs may play a pathophysiological role in this bone loss. We conclude that elevations in GIP may be an effective countermeasure to age-induced bone loss. Show less

Transgenic mice overexpressing a dominant negative glucose-dependent insulinotropic polypeptide receptor (GIPR(dn)) have recently been shown to develop diabetes mellitus due to disturbed postnatal development of the endocrine pancreas. In this study, the effects of feeding a high fibre/low calorie diet on the diabetic phenotype of GIPR(dn) transgenic mice were examined. Transgenic and control animals received either a conventional breeding diet (BD) or a high fibre diet (HF). Both fasting and postprandial blood glucose levels and HbA1C levels were largely elevated in transgenic mice vs. controls (p<0.05), irrespective of the diet fed. Food and water intake and the daily urine volume of GIPR(dn) transgenic mice were higher than that of control mice (p<0.05). Transgenic animals receiving the HF diet showed significantly lower blood glucose and HbA1C levels as well as less food and water intake than transgenic mice fed BD. The 365-day survival of transgenic mice was significantly lower than that of control mice. Transgenic animals fed the HF diet lived significantly longer than their counterparts receiving BD. GIPR(dn) transgenic mice develop a severe diabetic phenotype which can be ameliorated by a HF diet, thereby resembling some aspects of the pathophysiology of human type 2 diabetes mellitus. Show less

Gastric inhibitory polypeptide (GIP) is an insulinotropic duodenal hormone released in response to meals. Recent studies in rodents suggested that GIP directly links overnutrition to obesity. Despite evidence for GIP effects on fat metabolism in humans, the GIP receptor (GIPR) has not been identified in fat tissues. We identified the GIPR gene in human subcutaneous and visceral fat tissues and tested the hypothesis that that the expression of this gene is influenced by central obesity and weight loss. GIPR gene mRNA expression in subcutaneous fat tissue biopsies (n=70) and in paired subcutaneous and visceral fat tissue samples (n=25) of non-diabetic postmenopausal women was studied by real-time reverse transcription polymerase chain reaction. The effect of weight reduction on GIPR gene expression in subcutaneous fat tissue was studied in a subset of 14 women. GIPR adipose tissue gene expression was significantly lower in insulin resistant obese non-diabetic women (p=0.004). The GIPR mRNA expression was higher in the visceral fat tissue compared with subcutaneous fat (p<0.001). Despite adjustment for obesity-associated variables, waist circumference was the most significant predictor of GIPR gene expression in subcutaneous fat depot (F=4.066; beta=-0.997; p=0.0001) and, together with fasting insulin levels, in visceral fat (F=3.553; beta=-0.507 and beta=0.495; p=0.0001). Moderate weight reduction did not change gene expression levels of the GIPR gene (p=0.085). Decreased expression of the GIPR gene in subcutaneous fat tissue is associated with signs of insulin resistance in non-diabetic women with central obesity and demonstrates that fasting hyperinsulinemia is a possible negative regulator of GIPR gene expression in subcutaneous fat. Higher GIPR gene expression levels in visceral fat vs. subcutaneous fat reflect regional differences in adipose tissue biology. Moderate weight reduction did not change gene expression levels of GIPR in subcutaneous fat. Show less

The incretin hormones glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) control glucose homeostasis through well-defined actions on the islet beta cell via stimulation of insulin secretion and preservation and expansion of beta cell mass. We examined the importance of endogenous incretin receptors for control of glucose homeostasis through analysis of Glp1r(-/-), Gipr(-/-), and double incretin receptor knockout (DIRKO) mice fed a high-fat (HF) diet. DIRKO mice failed to upregulate levels of plasma insulin, pancreatic insulin mRNA transcripts, and insulin content following several months of HF feeding. Both single incretin receptor knockout and DIRKO mice exhibited resistance to diet-induced obesity, preservation of insulin sensitivity, and increased energy expenditure associated with increased locomotor activity. Moreover, plasma levels of plasminogen activator inhibitor-1 and resistin failed to increase significantly in DIRKO mice after HF feeding, and the GIP receptor agonist [D-Ala(2)]GIP, but not the GLP-1 receptor agonist exendin-4, increased the levels of plasma resistin in studies of both acute and chronic administration. These findings extend our understanding of how endogenous incretin circuits regulate glucose homeostasis independent of the beta cell via control of adipokine secretion and energy expenditure. Show less

In addition to its role as the primary mediator of the enteroinsular axis, glucose-dependent insulinotropic polypeptide (GIP) may play a critical role in the development of obesity. The purpose of these studies was to characterize the effects of GIP and its receptor (GIPR) in adipocyte development and signaling. Effects of GIP and GIPR on differentiated 3T3-L1 cells were analyzed using Western blot analysis, Oil-Red-O staining, cyclic adenosine monophosphate radioimmunoassay, immunofluorescence microscopy, and glucose uptake measurements. To determine whether GIP and GIPR are important components in adipocyte development, the expression profile of GIPR during differentiation was examined. GIPR protein expression was enhanced during the differentiation process, and coincubation with its ligand GIP augmented the expression of aP2, a fat cell marker. Conversely, the suppression of GIPR expression by a specific short hairpin RNA attenuated Oil-Red-O staining and aP2 expression, suggesting that the GIPR may play a critical role in adipocyte development. To investigate specific signaling components that may mediate the effects of GIP, we analyzed Akt, glucose transporter-4, and glucose uptake, all of which are modulated by insulin in fat cells. Like insulin, GIP induced the activation of Akt in a concentration-dependent manner, promoted membrane glucose transporter-4 accumulation, and enhanced [(3)H]-2-deoxyglucose uptake. These studies provide further evidence for an important physiologic role for GIP in lipid homeostasis and possibly in the pathogenesis of obesity. Furthermore, our data indicate that the GIPR might represent a suitable target for the treatment of obesity. Show less

The aim of this study was to evaluate an association of carrier status of common inherited thrombophilic genetic mutations and implantation failure after assisted reproduction (ART): IVF and ICSI. Sixty seven women with failure of embryo implantation and ninety six controls--women without obstetric complication were investigated for carriage of factor V Leiden (FVL), G20210A prothrombin gene mutation, genetic variant C677T in methylentetrahydrofolate reductase gene (MTHFR) and polymorphism A2 in platelet glycoprotein IIb/IIIa (GIPr IIb/IIIa). A significantly higher prevalence of polymorphism A2 in GIPr IIb/IIIa was found in women with implantation failure in ART compared to controls (respectively 26.1% and 12.5%; OR: 2.571, 95% CI: 1.066-6.258, p = 0.033). A higher but not significant prevalence of G20210A prothrombin gene mutation carriage was found inpatients compared to controls (respectively 5.8% and 3.13%, OR: 1.968, 95% CI 0.356-11.539). The carriage of FVL was a little but not significantly higher in controls. The carriage of genetic variant C677T in MTHFR was the same in both groups. These data suggest that polymorphism A2 in GIPr IIb/IIIa and G20210A prothrombin gene mutation could be play a role in the etiology of IVF failures and the carriers of GIPr IIb/IIIa A1/A2 and G20210A prothrombin gene mutation are at higher risk of implantation failure and not successful ART outcome. The carriage of these two genetic defects should be investigated in women undergoing IVF and the antithrombotic or anticoagulant prophylaxis should be indicated for carriers of these two factors. Show less

Aging is associated with increased fat mass and decreased lean mass, which is strongly associated with the development of insulin resistance. Gastric inhibitory polypeptide (GIP) is known to promote efficient storage of ingested nutrients into adipose tissue; we examined aging-associated changes in body composition using 10-week-old and 50-week-old wild-type (WT) and GIP receptor knockout (Gipr-/-) mice on a normal diet, which show no difference in body weight. We found that Gipr-/- mice showed significantly reduced fat mass without reduction of lean mass or food intake, while WT mice showed increased fat mass and decreased lean mass associated with aging. Moreover, aged Gipr-/- mice showed improved insulin sensitivity, which is associated with amelioration in glucose tolerance, higher plasma adiponectin levels, and increased spontaneous physical activity. We therefore conclude that genetic inactivation of GIP signaling can prevent the development of aging-associated insulin resistance through body composition changes. Show less

Glucose-dependent insulinotropic polypeptide (GIP) and peptide YY (PYY) are secreted from the intestinal K- and L-cells, respectively, following a meal. Both peptides are believed to play a key role in glucose homeostasis and energy expenditure. This study investigated the effects of daily administration of the stable and specific GIP-R antagonist, (Pro(3))GIP (25 nmol/kg) and the endogenous truncated form of PYY, PYY(3-36) (50 nmol/kg), in mice fed with a high fat diet. Daily i.p. injection of (Pro(3))GIP, PYY(3-36) or combined peptide administration over 24 days significantly (P<0.05-0.01) decreased body weight compared with saline-treated controls without change in food intake. Plasma glucose levels and glucose tolerance were significantly (P<0.05) lowered by (Pro(3))GIP treatment alone, and in combination with PYY(3-36). These changes were accompanied by a slight improvement of insulin sensitivity in all of the treatment groups. (Pro(3))GIP treatment significantly reduced plasma corticosterone (P<0.05), while combined administration with PYY(3-36) significantly lowered serum glucagon (P<0.05). No appreciable changes were observed in either circulating or glucose-stimulated insulin secretion in all treatment groups. (Pro(3))GIP-treated mice had significantly (P<0.01) lowered fasting glucose levels and an improved (P<0.05) glycemic response to feeding. These comparative data indicate that chemical ablation of GIP receptor action using (Pro(3))GIP provides an especially effective means of countering obesity and related abnormalities induced by consumption of high fat energy rich diet. Show less

Glucose-dependent insulinotropic polypeptide (GIP) stimulates insulin release via interaction with its pancreatic receptor (GIP receptor (GIPR)). GIP also acts as vasoactive protein. To investigate whether variations in GIP and GIPR genes are associated with risk factors of the metabolic syndrome we sequenced gene regions and identified two coding SNPs (GIP Ser103Gly, GIPR Glu354Gln) and one splice site SNP (GIP rs2291726) in 47 subjects. Interestingly, in silico analyses revealed that splice site SNP rs2291726 results in a truncated protein and classified GIPR variant Glu354Gln as a functional amino acid change. Association analyses were performed in a case-cohort study of incident cardiovascular disease (CVD) nested in the EPIC-Potsdam cohort. No significant associations between incident CVD and GIP Ser103Gly and rs2291726 were found. For GIPR Glu354Gln, we obtained a nominal association of heterozygous minor allele carrier with CVD in a codominant model adjusted for BMI, sex, and age (OR: 0.67, CI: 0.50-0.91, p = 0.01) or additional covariates of CVD (OR: 0.72, CI: 0.52-0.97, p = 0.03). In conclusion, we identified a common splice site mutation (rs2291726) of the GIP gene which results in a truncated protein and provide preliminary evidence for an association of the heterozygous GIPR Glu354Gln genotype with CVD. Show less

Glucose-dependent insulinotropic polypeptide (GIP) is a gastrointestinal hormone that has a potent stimulatory effect on insulin release under conditions of normal glucose tolerance. However, its insulinotropic effect is reduced or even absent entirely in type 2 diabetic patients. In this study, we addressed the role of glucose concentration in the diabetic range of >or=11 mM, i.e., hyperglycemia per se, as a cause of the lack of response to GIP. Culturing rat and human pancreatic islets in >or=11 mM glucose for up to 24 h resulted in prevention of GIP-mediated intracellular cAMP increase compared with culturing in 5 mM glucose. Western blot analysis revealed a selective 67 +/- 2% (rat) and 60 +/- 8% (human) decrease of GIP-R expression in islets exposed to >or=11 mM glucose compared with 5 mM glucose (P < 0.001). We further immunoprecipitated GIP-R from islets and found that GIP-R was targeted for ubiquitination in a glucose- and time-dependent manner. Downregulation of GIP-R was rescued by treating isolated islets with proteasomal inhibitors lactacystin and MG-132, and the islets were once again capable of increasing intracellular cAMP levels in response to GIP. These results suggest that the GIP-R is ubiquitated, resulting in downregulation of the actions of GIP. Show less

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. Show less

Stimulation of insulin secretion by the incretin hormones glucagon-like peptide 1 (GLP-1) and glucose-dependent insulinotropic peptide (GIP) has been found to be diminished in type 2 diabetes. We hypothesized that this impairment is due to a defect at the receptor level induced by the diabetic state, particularly hyperglycemia. Gene expression of incretin receptors, GLP-1R and GIPR, were significantly decreased in islets of 90% pancreatectomized (Px) hyperglycemic rats, with recovery when glucose levels were normalized by phlorizin. Perifused islets isolated from hyperglycemic Px rats showed reduced insulin responses to GLP-1 and GIP. To examine the acute effect of hyperglycemia on incretin receptor expression, a hyperglycemic clamp study was performed for 96 h with reduction of GLP-1 receptor expression but increase in GIP receptor expression. Similar findings were found when islets were cultured at high glucose concentrations for 48 h. The reduction of GLP-1 receptor expression by high glucose was prevented by dominant-negative protein kinase C (PKC)alpha overexpression, whereas GLP-1 receptor expression was reduced with wild-type PKCalpha overexpression. Taken together, GLP-1 and GIP receptor expression is decreased with chronic hyperglycemia, and this decrease likely contributes to the impaired incretin effects found in diabetes. Show less

Calcium plays a fundamental role as second messenger in intracellular signaling and bone serves as the body's calcium reserve to tightly maintain blood calcium levels. Calcium in ingested meal is the main supply and inadequate calcium intake causes osteoporosis and bone fracture. Here, we describe a novel mechanism of how ingested calcium is deposited on bone. Meal ingestion elicits secretion of the gut hormone gastric inhibitory polypeptide (GIP) from endocrine K cells in the duodenum. Bone histomorphometrical analyses revealed that bone formation parameters in the mice lacking GIP receptor (GIPR(-/-)) were significantly lower than those of wild-type (GIPR(+/+)) mice, and that the number of osteoclasts, especially multinuclear osteoclasts, was significantly increased in GIPR(-/-) mice, indicating that GIPR(-/-) mice have high-turnover osteoporosis. In vitro examination showed the percentage of osteoblastic cells undergoing apoptosis to be significantly decreased in the presence of GIP. Because GIPR(-/-) mice exhibited an increased plasma calcium concentration after meal ingestion, GIP directly links calcium contained in meal to calcium deposition on bone. Show less

The molecular mechanisms responsible for glucose-dependent insulinotrophic peptide receptor or gastric inhibitory polypeptide receptor (GIPR) ectopic expression and function in GIP-dependent Cushing's syndrome (CS) are still unknown. GIPR presumably acts, like the ACTH receptor (ACTHR), through the Gs protein/cyclic AMP/protein kinase A (PKA) pathway to stimulate steroidogenesis. We studied the expression of several genes involved in this pathway in the adrenal tissues of patients with GIP-dependent CS. RNA was extracted from adrenal tissues from nine patients with GIP-dependent CS [seven ACTH-independent bilateral macronodular adrenal hyperplasia (AIMAH), two adenomas], two control whole adult adrenals, two fasciculata cell-enriched preparations from normal adrenals, seven patients with Cushing's disease (CD) and two normal pancreas. Multiplex reverse transcriptase polymerase chain reaction (RT-PCR) evaluated the expression of GIPR, ACTHR, SF-1, Nur77, DAX-1, CYP11A, 3beta-HSD, CYP21, CREB and CREM genes. GIPR mRNA was overexpressed in all GIP-dependent cases. In normal adrenals and in the adrenal tissues from patients with CD, minimal amounts of GIPR mRNA were detected. ACTHR mRNA expression was observed in all GIP-dependent adrenal tissues. The expression of steroidogenic enzymes and some specific and ubiquitous transcription factors (TFs) involved in the ACTHR cascade was significantly reduced. Our results indicate that the expression of ACTHR and other genes located downstream in the ACTHR cascade, including steroidogenic enzymes genes and some transcription factors, are relatively suppressed in GIP-dependent CS. Although the expression of aberrant receptors plays an important role in steroidogenesis and initiation of cell proliferation, additional genetic events might occur, altering the activity of the ACTHR pathway. Show less

Secreted proteins from cancer cells may be potential serologic biomarkers of cancer. It's important to globally identify secreted proteins of cancer cells. This study was to identify secreted proteins of lung cancer cells. Proteins in the conditioned medium of non-small cell lung cancer (NSCLC) cell line A549 was collected and the proteome analysis was subsequently performed. Specific protein spots in A549 cells were identified by peptide mass fingerprints using mass spectrometry and through searching database. The expression of identified secreted proteins was detected by reverse transcription-polymerase chain reaction (RT-PCR) in 15 specimens of NSCLC tissue and paired distant lung tissue. Manganese superoxide dismutase (Mn-SOD) activity in serum and conditioned medium was detected by spectrophotometry. Fourteen secreted proteins were identified, which included peptidyl-prolyl cis-trans isomerase A (PPIA), Mn-SOD, peroxiredoxin 1 (PDX1), phosphatidylethanolamine binding protein (PEBP), glutathione S-transferase P (GSTP1-1), glucose-dependent insulinotropic protein receptor (GIPR), ubiquitin carboxyl-terminal hydrolase isozyme L1 (PGP9.5), alpha enolase (ENO1), dihydrodiol dehydrogenase (DDH), phosphoglycerate mutase 1 (PGAM1), galectin-1 (GAL1). PPIA, DDH, PGAM1, PDX1, PGP9.5, ENO1, and PEBP were overexpressed in cancer tissues. Higher level of Mn-SOD activity was detected in conditioned medium than in control. Serum Mn-SOD activity was significantly higher in NSCLC patients than in healthy controls (P<0.01). Multiple secreted proteins of A549 cells were identified in this study and the overexpression of ENO1 and PEBP in NSCLC was revealed for the first time. Mn-SOD is secreted serologic marker of NSCLC. The results presented here would provide clues to identify new serologic biomarkers of NSCLC. Show less

Embryonic stem (ES) cells can be differentiated into insulin-producing cells by conditioning the culture media. However, the number of insulin-expressing cells and amount of insulin released is very low. Glucose-dependent insulinotropic polypeptide (GIP) enhances the growth and differentiation of pancreatic beta-cells. This study examined the potential of the stable analogue GIP(LysPAL16) to enhance the differentiation of mouse ES cells into insulin-producing cells using a five-stage culturing strategy. Semi-quantitative PCR indicated mRNA expression of islet development markers (nestin, Pdx1, Nkx6.1, Oct4), mature pancreatic beta-cell markers (insulin, glucagon, Glut2, Sur1, Kir6.1) and the GIP receptor gene GIP-R in undifferentiated (stage 1) cells, with increasing levels in differentiated stages 4 and 5. IAPP and somatostatin genes were only expressed in differentiated stages. Immunohistochemical studies confirmed the presence of insulin, glucagon, somatostatin and IAPP in differentiated ES cells. After supplementation with GIP(LysPAL16), ES cells at stage 4 released insulin in response to secretagogues and glucose in a concentration-dependent manner, with 35-100% increases in insulin release. Cellular C-peptide content also increased by 45% at stages 4 and 5. We conclude that the stable GIP analogue enhanced differentiation of mouse ES cells towards a phenotype expressing specific beta-cell genes and releasing insulin. Show less

The mechanisms responsible for the ectopic adrenal expression of glucose-dependent insulinotropic peptide (GIP) receptor (GIPR) in GIP-dependent Cushing's syndrome (CS) are unknown. Chronic adrenal stimulation by ACTH in Cushing's disease or GIP in GIP-dependent ACTH-independent macronodular adrenal hyperplasia both lead to the induction of genes implicated in adrenal proliferation and steroidogenesis. The objective of the study was to identify genes differentially expressed specifically in GIP-dependent CS that could be implicated in the ectopic expression of GIPR. We used the Affymetrix U133 plus 2.0 microarray oligochips to compare the whole genome expression profile of adrenal tissues from five cases of GIP-dependent bilateral ACTH-independent macronodular adrenal hyperplasia with CS, one case of GIP-dependent unilateral adenoma with CS, five cases of ACTH-dependent hyperplasias, and a pool of adrenals from 62 normal individuals. After data normalization and statistical filtering, 723 genes with differential expression were identified, including 461 genes or sequences with a known functional implication, classified in eight dominant functional classes. Specific findings include repression of perilipin, the overexpression of 13 G protein-coupled receptors, and the potential involvement of Rho-GTPases. We also isolated 94 probe sets potentially linked to the formation of GIP-dependent nodules adjacent to the diffuse hyperplasia. These included probe sets related to the linker histone H1 and repression of RXRa and CCND2. The expression profiles for eight genes were confirmed by real-time RT-PCR. This study identified an extensive series of potentially novel target candidate genes that could be implicated in the molecular mechanisms of ectopic expression of the GIPR as well as in the multistep progression of GIP-dependent CS. Show less

The physiological effects of glucose-dependent insulinotropic polypeptide (GIP) are mediated through specific receptors expressed on target cells. Because aberrant GIP receptor (GIPR) expression has been implicated in abnormal GIP responses associated with type 2 diabetes mellitus and food-induced Cushing's syndrome, we sought to identify factors that regulate the GIPR. We previously demonstrated that sequences between -1 and -100 of the GIPR gene were sufficient to direct transcription in a rat insulinoma cell line (RIN38). In the present study, we compared the 5'-flanking regions of the rat and human GIPR gene and demonstrated 88% identity within the first 92 bp. Subsequent serial deletion analyses showed that the region between -85 and -40 is essential for maximal promoter activity. Within this region, we identified three putative Sp1 binding motifs, located at positions -77, -60, and -50, that can specifically bind both Sp1 and Sp3. Whereas mutation of the Sp1 sites at -50 and -60 led to 36 and 40% reduction in promoter activity, respectively, mutation of the Sp1 motif at -70 did not affect promoter activity. Cotransfection of S2 Schneider cells with GIPR-luciferase chimeric constructs and either Sp1 or Sp3 expression vectors indicated that both Sp1 and the long form of Sp3 activate transcription through binding to the Sp1 sites located between -100 and -40. Lastly, chromatin immunoprecipitation analyses revealed that both Sp1 and Sp3 bind to the GIPR promoter region in RIN38 cells. These results indicate that cell-specific expression of GIPR is associated with the binding of the transcription factors Sp1 and Sp3 to the GIPR promoter. Show less

Cortisol secretion is usually under the control of ACTH. However, cortisol secretion occurs in response to gastric inhibitory polypeptide (GIP) in rare cases of food-dependent Cushing's syndrome (CS). We have investigated whether chronic ACTH stimulation or activation of the ACTH signaling pathway might be associated with GIP receptor (GIPR) expression. RT-PCR analysis and primary culture of hyperplastic adrenals. All patients presented with CS: 20 unilateral adrenal adenomas, five Cushing's disease, one food-dependent CS. RT-PCR revealed GIPR expression in all hyperplastic adrenals studied. No RT-PCR product could be detected in two normal adrenals or 20 hyperfunctioning adrenal adenomas. Primary culture revealed a significant cAMP response to ACTH in all adrenals available for study (EC50, 8.1 x 10(-10) M in normals, 4.7 x 10(-10) M in Cushing's disease, and 4.4 x 10(-10) M in food-dependent disease). However, cultures taken from all four ACTH-dependent and the one food-dependent hyperplastic adrenals studied were also responsive to GIP (EC50 for cAMP, 1.3 x 10(-9) M in Cushing's disease and 4.1 x 10(-10) M in food-dependent disease). Fasting cortisol levels were low in the case of food-dependant Cushing's, rising postprandially as predicted. However, there was no trend toward low fasting or high postprandial cortisol in the other cases, suggesting that the presence of detectable GIPR alone, albeit with definite function in vitro, is not sufficient to cause clinically food-dependent CS. These data are consistent with the hypothesis that chronic ACTH stimulation or constitutive activation of the ACTH signaling pathway may be associated with aberrant GIPR expression, and suggest one mechanism for the pathogenesis of this phenomenon. Show less

Gut hormone gastric inhibitory polypeptide (GIP) stimulates insulin secretion from pancreatic beta-cells upon ingestion of nutrients. Inhibition of GIP signaling prevents the onset of obesity and consequent insulin resistance induced by high-fat diet. In this study, we investigated the role of GIP in accumulation of triglycerides into adipocytes and in fat oxidation peripherally using insulin receptor substrate (IRS)-1-deficient mice and revealed that IRS-1(-/-)GIPR(-/-) mice exhibited both reduced adiposity and ameliorated insulin resistance. Furthermore, increased gene expression of CD36 and UCP2 in liver, and increased expression and enzyme activity of 3-hydroxyacyl-CoA dehydrogenase in skeletal muscle of IRS-1(-/-)GIPR(-/-) mice might contribute to the lower respiratory quotient and the higher fat oxidation in light phase. These results suggest that GIP plays a crucial role in switching from fat oxidation to fat accumulation under the diminished insulin action as a potential target for secondary prevention of insulin resistance. Show less

Glucose-dependent insulinotropic polypeptide (GIP) is an incretin hormone, which is secreted from endocrine cells in the small intestine after meal ingestion. GIP has been shown to affect osteoblastic function in vitro; however, the in vivo effects of GIP on bone remodeling remain unclear. In the present study, we investigated the role of GIP in modulating bone turnover, by evaluating serum markers of bone turnover, bone density, bone morphology, and changes in biomechanical bone strength over time (one to five months) in GIP receptor knockout mice (GIPR-/- mice). The GIPR-/- mice showed a decreased bone size, lower bone mass, altered bone microarchitecture and biomechanical properties, and altered parameters for bone turnover, especially in bone formation. Moreover, the effects of GIP on bone mass were site-specific and compensatory mechanism developed over time and ameliorated the impact of the loss of GIP signaling on bone mass. Further, GIPR-/- mice had earlier age-related changes than wild-type mice in body composition, including bone mass, lean body mass, and fat percentage. In summary, our results indicate that GIP has an anabolic effect on bone mass and bone quality and suggests that GIP may be a hormonal link between nutrient ingestion and utilization. Show less

The best characterized effect of glucose-dependent insulinotropic polypeptide (GIP) is its stimulatory effect on insulin secretion by pancreatic beta-cells. Recently, it was demonstrated that some cases of primary adrenal Cushing's syndrome were secondary to the ectopic expression of non-mutated GIP receptor (GIP-R) in bilateral adrenal hyperplasias or unilateral adrenal adenomas, resulting in food-dependent steroidogenesis. Using a human multiple-expression tissue array, GIP-R was found to be expressed in a large number of human adult and fetal tissues, but not in the adrenal gland. The analysis of the promoter region of human (h) GIP-R gene revealed six consensus sequences important in regulating the reporter gene activity and capable of binding to Sp1 and Sp3 transcription factors. Data obtained by gene array and semi-quantitative RT-PCR showed an increase in the expression of Sp3 and CRSP9 (co-regulator of Sp1 transcription factor, subunit 9) in the adrenal adenomas or bilateral macronodular hyperplasias of patients with GIP-dependent Cushing's syndrome; they were, however, also increased in some patients with non-GIP-dependent cortisol-secreting adenomas or with ACTH-dependent Cushing's disease. This study represents the first step in our understanding of the mechanisms involved in the regulation of the expression of the hGIP-R gene. Show less

Glucose-dependent insulinotropic polypeptide (gastric inhibitory polypeptide [GIP]) is an important incretin hormone secreted by endocrine K-cells in response to nutrient ingestion. In this study, we investigated the effects of chemical ablation of GIP receptor (GIP-R) action on aspects of obesity-related diabetes using a stable and specific GIP-R antagonist, (Pro3)GIP. Young adult ob/ob mice received once-daily intraperitoneal injections of saline vehicle or (Pro3)GIP over an 11-day period. Nonfasting plasma glucose levels and the overall glycemic excursion (area under the curve) to a glucose load were significantly reduced (1.6-fold; P < 0.05) in (Pro3)GIP-treated mice compared with controls. GIP-R ablation also significantly lowered overall plasma glucose (1.4-fold; P < 0.05) and insulin (1.5-fold; P < 0.05) responses to feeding. These changes were associated with significantly enhanced (1.6-fold; P < 0.05) insulin sensitivity in the (Pro3)GIP-treated group. Daily injection of (Pro3)GIP reduced pancreatic insulin content (1.3-fold; P < 0.05) and partially corrected the obesity-related islet hypertrophy and beta-cell hyperplasia of ob/ob mice. These comprehensive beneficial effects of (Pro3)GIP were reversed 9 days after cessation of treatment and were independent of food intake and body weight, which were unchanged. These studies highlight a role for GIP in obesity-related glucose intolerance and emphasize the potential of specific GIP-R antagonists as a new class of drugs for the alleviation of insulin resistance and treatment of type 2 diabetes. Show less

The hormone glucose-dependent insulinotropic polypeptide (GIP) potently stimulates insulin secretion and promotes beta-cell proliferation and cell survival. In the present study we identified Forkhead (Foxo1)-mediated suppression of the bax gene as a critical component of the effects of GIP on cell survival. Treatment of INS-1(832/13) beta-cells with GIP resulted in concentration-dependent activation of the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (PKB)/Foxo1 signaling module. In parallel studies, GIP decreased bax promoter activity. Serial deletion analysis of the bax promoter demonstrated that the region -682 to -320, containing FHRE-II (5AAAACAAACA), was responsible for GIP-mediated effects. Foxo1 bound to FHRE-II in gel mobility shift assays, and Foxo1-FHRE-II interactions conferred GIP responsiveness to the bax promoter. INS-1 cells incubated under proapoptotic and glucolipotoxic conditions demonstrated increased nuclear localization of Foxo1 and bax promoter activity and decreased cytoplasmic phospho-PKB/Foxo1. GIP partially restored expression PKB/Foxo1 and bax promoter activity. Similar protective effects were found with dispersed islet cells from C57BL/6 mice, but not with those from GIP receptor knock-out (GIPR(-/-)) mice. GIP treatment reduced glucolipotoxicity-induced cell death in C57 BL/6 and Bax(-/-) islets, but not GIPR(-/-) mouse islets. Chronic treatment of Vancouver diabetic fatty Zucker rats with GIP resulted in down-regulation of Bax and up-regulation of Bcl-2 in pancreatic beta-cells. The results show that PI3K/PKB/Foxo1 signaling mediates GIP suppression of bax gene expression and that this module is a key pathway by which GIP regulates beta-cell apoptosis in vivo. Show less

Glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1) are gut-derived incretins secreted in response to nutrient ingestion. Both incretins potentiate glucose-dependent insulin secretion and enhance beta-cell mass through regulation of beta-cell proliferation, neogenesis and apoptosis. In contrast, GLP-1, but not GIP, inhibits gastric emptying, glucagon secretion, and food intake. Furthermore, human subjects with Type 2 diabetes exhibit relative resistance to the actions of GIP, but not GLP-1R agonists. The physiological importance of both incretins has been investigated through generation and analysis of incretin receptor knockout mice. Elimination of incretin receptor action in GIPR-/- or GLP-1R-/- mice produces only modest impairment in glucose homeostasis. Similarly, double incretin receptor knockout (DIRKO) mice exhibit normal body weight and normal levels of plasma glucagon and hypoglycemic responses to exogenous insulin. However, glucose-stimulated insulin secretion is significantly decreased following oral but not intraperitoneal glucose challenge in DIRKO mice and the glucose lowering actions of dipeptidyl peptidase-IV (DPP-IV) inhibitors are extinguished in DIRKO mice. Hence, incretin receptor signaling exerts physiologically relevant actions critical for glucose homeostasis, and represents a pharmacologically attractive target for development of agents for the treatment of Type 2 diabetes. Show less

The expression of a dominant negative glucose-dependent insulinotropic polypeptide receptor (GIPRdn) under the control of the rat pro-insulin gene promoter induces severe diabetes mellitus in transgenic mice. This study aims to gain further insight into the effect of the expression of a dominant negative GIPR on glucose homeostasis and postnatal development of the endocrine pancreas. The diabetic phenotype of GIPRdn transgenic animals was first observed between 14 and 21 days of age (urine glucose>1000 mg/dl). After onset of diabetes, serum glucose was significantly higher and insulin values were significantly lower in GIPRdn transgenic mice vs. non-transgenic littermate controls. Morphometric studies of pancreatic islets and their endocrine cell types were carried out at 10, 30 and 90 days of age. The total islet and total beta-cell volume of transgenic mice was severely reduced as compared to control mice, irrespective of the age at sampling (p<0.05). The total volume of isolated insulin positive cells that were not contained within established islets was significantly reduced in transgenic mice, indicating disturbed islet neogenesis. These findings demonstrate in vivo evidence that intact signaling of G-protein coupled receptors is involved in postnatal islet and beta-cell development and neogenesis of the pancreatic islets. Show less

The role of the gluco-incretin hormones GIP and GLP-1 in the control of beta cell function was studied by analyzing mice with inactivation of each of these hormone receptor genes, or both. Our results demonstrate that glucose intolerance was additively increased during oral glucose absorption when both receptors were inactivated. After intraperitoneal injections, glucose intolerance was more severe in double- as compared to single-receptor KO mice, and euglycemic clamps revealed normal insulin sensitivity, suggesting a defect in insulin secretion. When assessed in vivo or in perfused pancreas, insulin secretion showed a lack of first phase in Glp-1R(-/-) but not in Gipr(-/-) mice. In perifusion experiments, however, first-phase insulin secretion was present in both types of islets. In double-KO islets, kinetics of insulin secretion was normal, but its amplitude was reduced by about 50% because of a defect distal to plasma membrane depolarization. Thus, gluco-incretin hormones control insulin secretion (a) by an acute insulinotropic effect on beta cells after oral glucose absorption (b) through the regulation, by GLP-1, of in vivo first-phase insulin secretion, probably by an action on extra-islet glucose sensors, and (c) by preserving the function of the secretory pathway, as evidenced by a beta cell autonomous secretion defect when both receptors are inactivated. Show less

Regulation of cortisol secretion by aberrant hormone receptors may play a role in the pathogenesis of ACTH-independent Cushing's syndrome. In this study, the topic was evaluated by combining in vivo and in vitro approaches. Cortisol responses to various stimuli (standard meal, GnRH + TRH, cisapride, vasopressin, glucagon) were assessed in 6 patients with clinical or subclinical adrenal Cushing's syndrome, and non-functioning adrenal adenoma in two cases. Abnormal responses were observed in three patients with Cushing's syndrome; one patient showed a gastric inhibitory polypeptide (GIP)-dependent cortisol rise after meal, together with responses after GnRH and cisapride; the second patient showed an LH-dependent cortisol response to GnRH, and in the third cortisol rose after cisapride. The pattern of receptor expression performed by RT-PCR showed that while GIP-R was only expressed in tumor from the responsive patient, 5-hydroxytryptamine type 4 receptor and LH-R were also present in normal adrenal tissues and tissues from non-responsive patients. Interestingly, an activating mutation of Gsalpha gene was identified in one of these tumors. Therefore, cortisol responses to agents operating via Gs protein coupled receptors (in one case associated with Gsalpha mutation) were found in Cushing's patients, while these responses were absent in the others. The finding of receptor expression in normal and non-responsive tumors suggests that different mechanisms are probably involved in inducing in vivo cortisol responses. Show less

Gastric inhibitory polypeptide (GIP)-dependent Cushing's syndrome (CS) results from the ectopic expression of non-mutated GIP receptor (hGIPR) in the adrenal cortex. We evaluated whether mutations or polymorphisms in the regulatory region of the GIPR gene could lead to this aberrant expression. We studied 9.0kb upstream and 1.3kb downstream of the GIPR gene putative promoter (pProm) by sequencing leukocyte DNA from controls and from adrenal tissues of GIP- and non-GIP-dependent CS patients. The putative proximal promoter region (800 bp) and the first exon and intron of the hGIPR gene were sequenced on adrenal DNA from nine GIP-dependent CS, as well as on leukocyte DNA of nine normal controls. Three variations found in this region were found in all patients and controls; at position -4/-5, an insertion of a T was seen in four out of nine patients and in five out of nine controls. Transient transfection studies conducted in rat GC and mouse Y1 cells showed that the TT allele confers loss of 40% in the promoter activity. The analysis of the 8-kb distal pProm region revealed eight distal single nucleotide polymorphisms (SNPs) without probable association with the disease, since frequencies in patients and controls were very similar. In conclusion, mutations or SNPs in the regulatory region of the GIPR gene are unlikely to underlie GIP-dependent CS. Show less