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.     

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.     

Conformational, receptor interaction and alanine scan studies of glucose-dependent insulinotropic polypeptide

Glucose-dependent insulinotropic polypeptide (GIP) is an insulinotropic incretin hormone that stimulates insulin secretion during a meal. GIP has glucose lowering abilities and hence is considered as a potential target molecule for type 2 diabetes therapy. In this article, we present the solution structure of GIP in membrane-mimicking environments by proton NMR spectroscopy and molecular modelling. GIP adopts an α-helical conformation between residues Phe(6)-Gly(31) and Ala(13)-Gln(29) for micellar and bicellar media, respectively. Previously we examined the effect of N-terminal Ala substitution in GIP, but here eight GIP analogues were synthesised by replacing individual residues within the central 8-18 region with alanine. These studies showed relatively minor changes in biological activity as assessed by insulin releasing potency. However, at higher concentration, GIP(Ala(16)), and GIP(Ala(18)) showed insulin secreting activity higher than the native GIP (P<0.01 to P<0.001) in cultured pancreatic BRIN-BD11 cells. Receptor interaction studies of the native GIP with the extracellular domain of its receptor were performed by using two different docking algorithms. At the optimised docking conformation, the complex was stabilised by the presence of hydrophobic interactions and intermolecular hydrogen bonding. Further, we have identified some potentially important additional C-terminal interactions of GIP with its N-terminal extracellular receptor domain.     

Functional analysis of glucose-dependent insulinotropic polypeptide fusion proteins

To generate functional fluorescently tagged glucose-dependent insulinotropic polypeptide (GIP), a series of GIP expression constructs were devised. These included G1 (complete preprohormone), G2 (lacking the C-terminal extension), G3 (lacking both N- and C-terminal extensions), G4 (G2 fused to green fluorescent protein, GFP), and G5 (G3 fused to GFP). Expression of G5 in bacteria generated immunopositive GIP together with GFP fluorescence, while G4 generated only fluorescence without immunoreactivity. Transfection of NIH3T3 cells with cDNAs of G1, G3, G5, but not G2, G4, and EGFP, resulted in immunologically detectable GIP formation, although fluorescence could be detected in the latter two. GIP as well as GIP-GFP secreted by NIH3T3 cells significantly stimulated intracellular cAMP accumulation and Ca(2+) mobilization in SaOS2 cells. The GIP receptor antagonist GIP(7-30) abolished these responses. These results suggest that a GIP-GFP fusion protein seven times larger than the native peptide retains function and may be used as an in vivo probe to detect GIP receptor distribution and to explore GIP's biological roles.     

Structure-activity relationships of glucose-dependent insulinotropic polypeptide (GIP)

Six GIP(1-NH2) analogs were synthesized with modifications (de-protonation, N-methylation, reversed chirality, and substitution) at positions 1, 3, and 4 of the N-terminus, and additionally, a cyclized GIP derivative was synthesized. The relationship between altered structure to biological activity was assessed by measuring receptor binding affinity and ability to stimulate adenylyl cyclase in CHO-K1 cells transfected with the wild-type GIP receptor (wtGIPR). These structure-activity relationship studies demonstrate the importance of the GIP N-terminus and highlight structural constraints that can be introduced in GIP analogs. These analogs may be useful starting points for design of peptides with enhanced in vivo bioactivity.     

Alterations of glucose-dependent insulinotropic polypeptide (GIP) during cold acclimation

Cold acclimation is initially associated with shivering thermogenesis in skeletal muscle followed by adaptive non-shivering thermogenesis, particularly in brown adipose tissue (BAT). In response, hyperphagia occurs to meet increased metabolic demand and thermoregulation. The present study investigates the effects of cold (4 ± 1 °C) acclimation and hyperphagia on circulating and intestinal levels of gastric inhibitory polypeptide (GIP) in rats. Pair fed animals were used as additional controls in some experiments. Cold acclimation for 42 days significantly (p<0.01) increased daily food intake. There was no corresponding change in body weight. However, body weights of pair fed cold exposed rats were significantly (p<0.01) reduced compared to controls and ad libitum fed cold exposed rats. By day 42, non-fasting plasma glucose was increased (p<0.05) by chronic cold exposure regardless of food intake. Corresponding plasma insulin concentrations were significantly (p<0.01) lower in pair fed cold exposed rats. Circulating GIP levels were elevated (p<0.05) in ad libitum fed cold acclimated rats on days 18 and 24, but returned to normal levels by the end of the study. The glycaemic response to oral glucose was improved (p<0.01) in all cold exposed rats, with significantly (p<0.05) elevated GIP responses in ad libitum fed rats and significantly (p<0.05) reduced insulin responses in pair fed rats. In keeping with this, insulin sensitivity was enhanced (p<0.05) in cold exposed rats compared to controls. By the end of the study, cold acclimated rats had significantly (p<0.01) increased BAT mass and intestinal concentrations of GIP and GLP-1 compared to controls, independent of food intake. These data indicate that changes in the secretion and actions of GIP may be involved in the metabolic adaptations to cold acclimation in rats.     

NMR and alanine scan studies of glucose-dependent insulinotropic polypeptide in water

Glucose-dependent insulinotropic polypeptide (GIP) is an incretin hormone that stimulates the secretion of insulin after ingestion of food. GIP also promotes the synthesis of fatty acids in adipose tissue. Therefore, it is not surprising that numerous literature reports have shown that GIP is linked to diabetes and obesity-related diseases. In this study, we present the solution structure of GIP in water determined by NMR spectroscopy. The calculated structure is characterized by the presence of an alpha-helical motif between residues Ser(11) and Gln(29). The helical conformation of GIP is further supported by CD spectroscopic studies. Six GIP-(1-42)Ala(1-7) analogues were synthesized by replacing individual N-terminal residues with alanine. Alanine scan studies of these N-terminal residues showed that the GIP-(1-42)Ala(6) was the only analogue to show insulin-secreting activity similar to that of the native GIP. However, when compared with glucose, its insulinotropic ability was reduced. For the first time, these NMR and modeling results contribute to the understanding of the structural requirements for the biological activity of GIP.     

Transgenic rescue of adipocyte glucose-dependent insulinotropic polypeptide receptor expression restores high fat diet-induced body weight gain

The glucose-dependent insulinotropic polypeptide receptor (GIPr) has been implicated in high fat diet-induced obesity and is proposed as an anti-obesity target despite an uncertainty regarding the mechanism of action. To independently investigate the contribution of the insulinotropic effects and the direct effects on adipose tissue, we generated transgenic mice with targeted expression of the human GIPr to white adipose tissue or beta-cells, respectively. These mice were then cross-bred with the GIPr knock-out strain. The central findings of the study are that mice with GIPr expression targeted to adipose tissue have a similar high fat diet -induced body weight gain as control mice, significantly greater than the weight gain in mice with a general ablation of the receptor. Surprisingly, this difference was due to an increase in total lean body mass rather than a gain in total fat mass that was similar between the groups. In contrast, glucose-dependent insulinotropic polypeptide-mediated insulin secretion does not seem to be important for regulation of body weight after high fat feeding. The study supports a role of the adipocyte GIPr in nutrient-dependent regulation of body weight and lean mass, but it does not support a direct and independent role for the adipocyte or beta-cell GIPr in promoting adipogenesis.     

Characterization and biological actions of N-terminal truncated forms of glucose-dependent insulinotropic polypeptide

The N-terminal domain of glucose-dependent insulinotropic polypeptide (GIP) plays an important role in regulating biological activity. This study examined biological properties of several N-terminal truncated forms of GIP and two novel forms with substitutions at Phe position-6 with Arg or Val. GIP(6-42), GIP(R6-42), GIP(V6-42), GIP(7-42) and GIP(9-42) stimulated cAMP production in BRIN-BD11 cells similar to native GIP, whereas responses to GIP(3-42), GIP(4-42), GIP(5-42) and GIP(8-42) were reduced (P < 0.01 to P < 0.001). GIP-induced cyclic AMP production was significantly inhibited by GIP(3-42), GIP(4-42), GIP(5-42), GIP(6-42), GIP(R6-42), GIP(7-42) and GIP(8-42) (P < 0.001). Compared with native GIP, in vitro insulinotropic activity of GIP(3-42), GIP(4-42), GIP(5-42), GIP(7-42) and GIP(8-42) was reduced (P < 0.05 to P < 0.001), with GIP(4-42), GIP(5-42), GIP(7-42) and GIP(8-42) also potently inhibiting GIP-stimulated insulin secretion (P < 0.001). In ob/ob mice, GIP(4-42) and GIP(8-42) increased (P < 0.05 to P < 0.01) plasma glucose concentrations compared to the glucose-lowering action of native GIP. When GIP(8-42) was co-administered with native GIP it countered the ability of the native peptide to lower plasma glucose and increase circulating insulin concentrations. These data confirm the importance of the N-terminal region of GIP in regulating bioactivity and reveal that sequential truncation of the peptide yields novel GIP receptor antagonists which may have functional significance.     

The bioactive conformation of glucose-dependent insulinotropic polypeptide by NMR and CD spectroscopy

Glucose-dependent insulinotropic polypeptide (GIP) is a gastrointestinal incretin hormone, which modulates physiological insulin secretion. Because of its glucose-sensitive insulinotropic activity, there has been a considerable interest in utilizing the hormone as a potential treatment for type 2 diabetes. Structural parameters obtained from NMR spectroscopy combined with molecular modeling techniques play a vital role in the design of new therapeutic drugs. Therefore, to understand the structural requirements for the biological activity of GIP, the solution structure of GIP was investigated by circular dichroism (CD) followed by proton nuclear magnetic resonance (NMR) spectroscopy. CD studies showed an increase in the helical character of the peptide with increasing concentration of trifluoroethanol (TFE) up to 50%. Therefore, the solution structure of GIP in 50% TFE was determined. It was found that there was an alpha-helix between residues 6 and 29, which tends to extend further up to residue 36. The implications of the C-terminal extended helical segment in the inhibitory properties of GIP on gastric acid secretion are discussed. It is shown that the adoption by GIP of an alpha-helical secondary structure is a requirement for its biological activity. Knowledge of the solution structure of GIP will help in the understanding of how the peptide interacts with its receptor and aids in the design of new therapeutic agents useful for the treatment of diabetes.     

Diets influence the diabetic phenotype of transgenic mice expressing a dominant negative glucose-dependent insulinotropic polypeptide receptor (GIPRdn)

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.     

Contribution of site-specific PEGylation to the dipeptidyl peptidase IV stability of glucose-dependent insulinotropic polypeptide

The effects of PEGylation of glucose-dependent insulinotropic polypeptide (GIP) on potency and dipeptidyl peptidase IV (DPPIV) stability are reported. N-terminal modification of GIP(1-30) with 40 kDa polyethylene glycol (PEG) abrogates functional activity. In contrast, C-terminal PEGylation of GIP(1-30) maintains full agonism and reasonable potency at the GIP receptor and confers a high level of DPPIV resistance. Moreover, the dual modification of N-terminal palmitoylation and C-terminal PEGylation results in a full agonist of comparable potency to native GIP that is stable to DPPIV cleavage. The results provide the basis for the development of long acting, PEGylated GIP, GIP variants, or GIP-based hybrid peptide therapeutics.     

Characterization of the carboxyl-terminal domain of the rat glucose-dependent insulinotropic polypeptide (GIP) receptor. A role for serines 426 and 427 in regulating the rate of internalization.

Glucose-dependent insulinotropic polypeptide (GIP) is a gastrointestinal hormone involved in the regulation of insulin secretion. In non-insulin-dependent diabetes mellitus insulin responses to GIP are blunted, possibly due to altered signal transduction or reduced receptor number. Site-directed mutagenesis was used to construct truncated GIP receptors to study the importance of the carboxyl-terminal tail (CT) in binding, signaling, and receptor internalization. Receptors truncated at amino acids 425, 418, and 405, expressed in COS-7 or CHO-K1 cells, exhibited similar binding to wild type receptors. GIP-dependent cAMP production with the 405 mutant was decreased in COS-7 cells. Maximal cAMP production in CHO-K1 cells was reduced with all truncated forms. Binding was undetectable with a receptor truncated at amino acid 400; increasing tail length by adding 5 alanines restored binding and signaling. Mutants produced by alanine scanning of residues 394-401, adjacent to transmembrane domain 7, were all functional. CT truncation by 30 or more amino acids, mutation of serines 426/427, singly or combined, or complete CT serine knockout all reduced receptor internalization rate. The majority of the GIP receptor CT is therefore not required for signaling, a minimum chain length of approximately 405 amino acids is needed for receptor expression, and serines 426 and 427 are important for regulating rate of receptor internalization.     

Prohormone convertase 1/3 is essential for processing of the glucose-dependent insulinotropic polypeptide precursor

The physiology of the incretin hormones, glucagon-like peptide 1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP), and their role in type 2 diabetes currently attract great interest. Recently we reported an essential role for prohormone convertase (PC) 1/3 in the cleavage of intestinal proglucagon, resulting in formation of GLP-1, as demonstrated in PC1/3-deficient mice. However, little is known about the endoproteolytic processing of the GIP precursor. This study investigates the processing of proGIP in PC1/3 and PC2 null mice and in cell lines using adenovirus-mediated overexpression. Supporting a role for PC1/3 in proGIP processing, we found co-localization of GIP and PC1/3 but not PC2 in intestinal sections by immunohistochemistry, and analysis of intestinal extracts from PC1/3-deficient animals demonstrated severely impaired processing to GIP, whereas processing to GIP was unaltered in PC2-deficient mice. Accordingly, overexpression of preproGIP in the neuroendocrine AtT-20 cell line that expresses high levels of endogenous PC1/3 and negligible levels of PC2 resulted in production of GIP. Similar results were obtained after co-expression of preproGIP and PC1/3 in GH4 cells that express no PC2 and only low levels of PC1/3. In addition, studies in GH4 cells and the alpha-TC1.9 cell line, expressing PC2 but not PC1/3, indicate that PC2 can mediate processing to GIP but also to other fragments not found in intestinal extracts. Taken together, our data indicate that PC1/3 is essential and sufficient for the production of the intestinal incretin hormone GIP, whereas PC2, although capable of cleaving proGIP, does not participate in intestinal proGIP processing and is not found in intestinal GIP-expressing cells.     

Molecular cloning of rat glucose-dependent insulinotropic peptide (GIP).

A cDNA clone encoding glucose-dependent insulinotropic peptide (GIP) was identified that consisted of 34 bp of 5' untranslated sequence, an open reading frame of 432 bp and 115 bp in the 3' untranslated region. The deduced amino acid sequence revealed a 144 amino acid preprohormone consisting of a 43 amino acid N-terminal extension including a signal peptide, a 42 amino acid hormone, and a 59 amino acid C-terminal extension. Rat GIP differs from the human hormone by two amino acid substitutions: arginine for histidine at position 18 and leucine for isoleucine at position 40. A single mRNA from small intestine of approximately 800 bases was identified on Northern blot analysis in equivalent amounts in proximal and distal small intestine.     

Metabolomic linkage reveals functional interaction between glucose-dependent insulinotropic polypeptide and ghrelin in humans

The gastric peptide ghrelin promotes energy storage, appetite, and food intake. Nutrient intake strongly suppresses circulating ghrelin via molecular mechanisms possibly involving insulin and gastrointestinal hormones. On the basis of the growing evidence that glucose-dependent insulinotropic polypeptide (GIP) is involved in the control of fuel metabolism, we hypothesized that GIP and/or insulin, directly or via changes in plasma metabolites, might affect circulating ghrelin. Fourteen obese subjects were infused with GIP (2.0 pmol·kg(-1)·min(-1)) or placebo in the fasting state during either euglycemic hyperinsulinemic (EC) or hyperglycemic hyperinsulinemic clamps (HC). Apart from analysis of plasma ghrelin and insulin levels, GC-TOF/MS analysis was applied to create a hormone-metabolite network for each experiment. The GIP and insulin effects on circulating ghrelin were analyzed within the framework of those networks. In the HC, ghrelin levels decreased in the absence (19.2% vs. baseline, P = 0.028) as well as in the presence of GIP (33.8%, P = 0.018). Ghrelin levels were significantly lower during HC with GIP than with placebo, despite insulin levels not differing significantly. In the GIP network combining data on GIP-infusion, EC+GIP and HC+GIP experiments, ghrelin was integrated into hormone-metabolite networks through a connection to a group of long-chain fatty acids. In contrast, ghrelin was excluded from the network of experiments without GIP. GIP decreased circulating ghrelin and might have affected the ghrelin system via modification of long-chain fatty acid pools. These observations were independent of insulin and offer potential mechanistic underpinnings for the involvement of GIP in systemic control of energy metabolism.     

Dietary regulation of glucose-dependent insulinotropic peptide (GIP) gene expression in rat small intestine

The hormone, glucose-dependent insulinotropic peptide (GIP), is an important incretin regulator of the gastrointestinal tract. To investigate whether diet is important for the control of GIP gene expression in the small intestine, GIP messenger RNA (mRNA) levels were measured in rats during fasting and after glucose or fat administration. Ribonuclease protection analyses revealed that glucose and fat administration increased GIP mRNA levels by 4-fold and 2.5-fold, respectively, compared with the control, and that prolonged fasting decreased GIP mRNA levels to 44% of those of control animals. Glucose infusion increased plasma GIP levels and tended to stimulate an increase in the GIP hormone concentration in the mucosa of the small intestine. Administration of fat also stimulated an increase of plasma GIP levels but did not modify tissue GIP concentrations. Prolonged fasting tended to decrease plasma GIP levels, although GIP tissue concentrations did not change. These data suggest that dietary glucose or fat stimulates GIP synthesis and secretion, and that food deprivation causes a decrease in GIP synthesis and secretion. This regulation involves changes at the pretranslational level and is reflected by modifications of GIP mRNA expression.     

Characterization of the cellular and metabolic effects of a novel enzyme-resistant antagonist of glucose-dependent insulinotropic polypeptide

A novel N-terminally substituted Pro(3) analogue of glucose-dependent insulinotropic polypeptide (GIP) was synthesized and tested for plasma stability and biological activity both in vitro and in vivo. Native GIP was rapidly degraded by human plasma with only 39 +/- 6% remaining intact after 8 h, whereas (Pro(3))GIP was completely stable even after 24 h. In CHL cells expressing the human GIP receptor, (Pro(3))GIP antagonized the cyclic adenosine monophosphate (cAMP) stimulatory ability of 10(-7) M native GIP, with an IC(50) value of 2.6 microM. In the clonal pancreatic beta cell line BRIN-BD11, (Pro(3))GIP over the concentration range 10(-13) to 10(-8) M dose dependently inhibited GIP-stimulated (10(-7) M) insulin release (1.2- to 1.7-fold; P < 0.05 to P < 0.001). In obese diabetic (ob/ob) mice, intraperitoneal administration of (Pro(3))GIP (25 nmol/kg body wt) countered the ability of native GIP to stimulate plasma insulin (2.4-fold decrease; P < 0.001) and lower the glycemic excursion (1.5-fold decrease; P < 0.001) induced by a glucose load (18 mmol/kg body wt). Collectively these data demonstrate that (Pro(3))GIP is a novel and potent enzyme-resistant GIP receptor antagonist capable of blocking the ability of native GIP to increase cAMP, stimulate insulin secretion, and improve glucose homeostasis in a commonly employed animal model of type 2 diabetes.