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Am J Physiol Endocrinol Metab 291: E468-E475, 2006. First published April 11, 2006; doi:10.1152/ajpendo.00577.2005
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GIP-(3–42) does not antagonize insulinotropic effects of GIP at physiological concentrations

Carolyn F. Deacon,1 Astrid Plamboeck,1 Mette M. Rosenkilde,2 Jocelyn de Heer,1 and Jens J. Holst1

1Department of Medical Physiology and 2Laboratory for Molecular Pharmacology, Department of Pharmacology, The Panum Institute, University of Copenhagen, Copenhagen, Denmark

Submitted 23 November 2005 ; accepted in final form 6 April 2006


   ABSTRACT
TOP
 ABSTRACT
RESEARCH DESIGN AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Glucose-dependent insulinotropic polypeptide [GIP-(1–42)] is degraded by dipeptidyl peptidase IV (DPP IV), forming GIP-(3–42). In mice, high concentrations of synthetic GIP-(3–42) may function as a GIP receptor antagonist, but it is unclear whether this occurs at physiological concentrations. In COS-7 cells transiently transfected with the human GIP receptor, GIP-(1–42) and -(3–42) bind with affinities (IC50) of 5.2 and 22 nM, respectively. GIP-(1–42) was a potent agonist, stimulating cAMP accumulation (EC50, 13.5 pM); GIP-(3–42) alone had no effect. When incubated together with native GIP, GIP-(3–42) behaved as a weak antagonist (IC50, 92 and 731 nM for inhibition of cAMP accumulation elicited by 10 pM and 1 nM native GIP, respectively). In the isolated perfused rat pancreas, GIP-(3–42) alone had no effect on insulin output and only reduced the response to GIP (1 nM) when coinfused in >50-fold molar excess (IC50, 138 nM). The ability of GIP-(3–42) to affect the antihyperglycemic or insulinotropic actions of GIP-(1–42) was examined in chloralose-anesthetized pigs given intravenous glucose. Endogenous DPP IV activity was inhibited to reduce degradation of the infused GIP-(1–42), which was infused alone and together with GIP-(3–42), at rates sufficient to mimic postprandial concentrations of each peptide. Glucose, insulin, and glucagon responses were identical irrespective of whether GIP-(1–42) was infused alone or together with GIP-(3–42). We conclude that, although GIP-(3–42) can weakly antagonize cAMP accumulation and insulin output in vitro, it does not behave as a physiological antagonist in vivo.

glucose homeostasis; dipeptidyl peptidase IV; inhibitor; valine-pyrrolidide

GLUCOSE-DEPENDENT INSULINOTROPIC POLYPEPTIDE (GIP), like glucagon-like peptide-1 (GLP-1), is degraded by dipeptidyl peptidase IV (DPP IV) in vivo to form an NH2-terminally truncated metabolite (6, 7, 15). This metabolite, GIP-(3–42), has been reported both to lack insulinotropic activity (3, 33) and to act as a GIP receptor antagonist in mice (10).

In previous studies (4, 5), our group showed that DPP IV inhibition reduces the NH2-terminal degradation of both incretin hormones in vivo, resulting in a potentiation of their insulinotropic effects. Furthermore, DPP IV inhibition increases the levels of endogenous intact GLP-1 and GIP in the circulation (9, 22), and this is associated with improved glucose tolerance in rodents and man (1, 2, 29, 30, 34). These effects could be due to the enhanced levels of intact incretins found after DPP IV inhibition, the reduced levels of potentially antagonistic metabolites, or a combination of both factors. It was previously not possible to assess the true efficacy of the incretin hormones independently of their metabolites in vivo, because both endogenous and exogenous GLP-1 and GIP are rapidly degraded by endogenous DPP IV. Any observed effect, therefore, is the result of the combination of effects of the intact forms of each peptide together with any effects of their primary metabolites. However, the present availability of DPP IV inhibitors means that it is now possible to prevent this degradation, thereby making it possible to assess the independent effects of the intact peptides in vivo, and by additional infusion of the metabolites, any modifying effects can then be assessed.

The NH2-terminally truncated metabolite of GLP-1, GLP-1-(9–36) amide, binds to the GLP-1 receptor, but with an affinity of only 1% compared with that of the intact peptide, and is able to weakly antagonize the parent peptide's stimulatory effect on adenylyl cyclase activity (16). However, it has been shown that GLP-1-(9–36) amide does not antagonize the insulinotropic activity of GLP-1-(7–36) amide in vivo in pigs (8) or humans (35). Moreover, in pigs (8), but not in humans (35) or mice (31), the metabolite possesses weak antihyperglycemic activity that appears to be independent of insulin secretion (8). The situation regarding the metabolite of GIP, GIP-(3–42), is less clear. In vitro studies using the cloned GIP receptor have reported that GIP-(3–42) can behave as a good (10) or a poor antagonist (13), whereas antagonism was not apparent in isolated rat islets (33). In vivo studies have produced conflicting results, showing both the presence (10) and absence (13) of antagonistic effects. Therefore, this study aimed to examine whether GIP-(3–42) can antagonize GIP's action in vitro (using the cloned human GIP receptor and the perfused rat pancreas) and in vivo (in the anesthetized pig) and, furthermore, to assess whether it, too, possesses any inherent activity. The antihyperglycemic and insulinotropic effects of GIP-(1–42), given alone and coinfused with GIP-(3–42), were examined in anesthetized pigs in which endogenous DPP IV activity was inhibited using valine-pyrrolidide.


   RESEARCH DESIGN AND METHODS
TOP
 ABSTRACT
 RESEARCH DESIGN AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Reagents

The valine-pyrrolidide used in these studies was kindly provided by Dr. Lise Christiansen (Novo Nordisk, Bagsværd, Denmark). It is a competitive inhibitor of DPP IV with an inhibition contant (Ki) of ~0.4 µM (25).

Synthetic porcine GIP was purchased from Bachem (Bubendorf, Switzerland). Synthetic porcine GIP-(3–42) was custom synthesized by PolyPeptide Laboratories (Wolfenbüttel, Germany). Its authenticity was confirmed by amino acid analysis, analytical reversed-phase high performance liquid chromatography (HPLC), and plasma desorption mass spectrometry, and its purity was shown to be >99% by HPLC with detection at 214 nm.

GIP Receptor Signaling

Receptor signaling experiments were carried out using COS-7 cells transfected with the human GIP receptor. The COS-7 cells were grown at 10% CO2 and 37°C in Dulbecco's modified Eagle's medium with glutamax (catalog no. 21885-025; GIBCO) adjusted with 10% fetal bovine serum, 180 U/ml penicillin, and 45 µg/ml streptomycin. Transfection of the COS-7 cells was performed using the calcium phosphate precipitation method (32). For the cAMP accumulation assay, the transiently transfected cells (2.5 x 105 cells/well) were incubated for 24 h with 2 µCi/ml of [3H]adenine in 0.5 ml of growth medium per well. Cells were washed twice in HBS buffer [25 mM HEPES, pH 7.2, supplemented with 0.75 mM NaH2PO4, 140 mM NaCl, and 0.05% (wt/vol) bovine serum albumin], and 0.5 ml of HBS buffer supplemented with 1 mM of the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (Sigma Chemical, St. Louis, MO) were added together with increasing concentrations of the different ligands. After 15 min of incubation at 37°C, the cells were placed on ice, the medium was removed, and the cells were lysed in 1 ml of 5% (wt/vol) trichloroacetic acid supplemented with 0.1 mM cAMP and 0.1 mM ATP for 30 min. The lysate mixtures were loaded onto Dowex columns (Bio-Rad, Hercules, CA), which were washed with 2 ml of water and placed onto the top of alumina columns (Sigma) and washed again with 10 ml of water. The alumina columns were eluted with 6 ml of 0.1 M imidazole into 15 ml of scintillation fluid (Highsafe III). Columns were reused up to 15 times. Dowex columns were regenerated by adding 10 ml of 2 N HCl followed by 10 ml of water; the alumina columns were regenerated by adding 2 ml of 1 M imidazole, 10 ml of 0.1 M imidazole, and, finally, 5 ml of water. Determinations were made in duplicate.

Competition Binding

Competition binding experiments were carried out in COS-7 cells transiently transfected with the human GIP receptor. The cells were grown and transfected as described in GIP Receptor Signaling. 125I-labeled GIP (Amersham Pharmacia Biotech, Little Chalfont, UK) was used as radioligand, and the competition binding analysis was performed on intact cells, seeded at a concentration of 50,000 cells/well, on the second day after transfection. Briefly, the cells were incubated for 16 h at 4°C in 0.25 ml of buffer consisting of 25 mM Tris·HCl, pH 7.4, and 5 mM MgCl2, using 15 pM 125I-GIP as radioligand. Increasing concentrations of unlabeled full-length GIP-(1–42) and truncated GIP-(3–42), ranging from 10–11 to 10–6 M, were used as competitors. The competition binding was terminated by washing the cells once with 1 ml of binding buffer, and subsequently, the cells were lysed by the addition of 1 ml of lysis buffer (8 M carbamide, 3 M acetic acid, 2% Nonidet-P 40). The binding data were analyzed and IC50 values determined using nonlinear regression analysis with GraphPrism (GraphPad Software, San Diego, CA). Kd and Ki values were calculated from competition binding experiments using the equations Kd = IC50 – L (where L is the concentration of free radioligand) and Ki = IC50/[1 + (L/Kd)].

Animal Studies

All animal studies were conducted in accordance with international guidelines (National Institutes of Health Publication no. 85-23, revised 1985) and Danish legislation governing animal experimentation (1987) and were carried out after permission had been granted by the Animal Experiments Inspectorate, Ministry of Justice, Denmark.

Isolated perfused pancreas. Male Wistar rats (390–440 g, 12 wk old, n = 4; Charles River, Sulzfeld, Germany) were anesthetized with an intraperitoneal injection of pentobarbital sodium (50 mg/kg; Royal Veterinary and Agriculture University, Frederiksberg, Denmark), and the pancreata were dissected and perfused in situ, as previously described (20). The perfusion medium consisted of a modified Krebs-Ringer bicarbonate buffer, containing in addition 5% human serum albumin (HSA), 10 mM glucose, and 5 mM each of pyruvate, fumarate, and glutamate. After a 30-min equilibration period, the venous effluent was collected for 1-min intervals and stored at –20°C until analysis. GIP-(1–42) or GIP-(3–42) was dissolved in 0.9% NaCl containing 1% HSA and infused into the arterial line with the use of a syringe pump to give final perfusate concentrations of 1 nM GIP-(1–42) or 5 and 50 nM GIP-(3–42). After a basal period, peptides were infused alone or together [1 nM GIP-(1–42), 5 nM GIP-(3–42), 1 nM GIP-(1–42) + 5 nM GIP-(3–42), 1 nM GIP-(1–42) + 50 nM GIP-(3–42), 1 nM GIP-(1–42), 50 nM GIP-(3–42)] for 10-min periods separated by 15-min rest periods, during which time endocrine secretion returned to basal levels. In subsequent experiments (n = 3), the effects of higher concentrations of GIP-(3–42) were tested, and GIP-(1–42) (1 nM) was infused alone and together with 100 nM and 1 µM GIP-(3–42).

Anesthetized pigs. Nonfasted Danish LYY strain pigs (28–30 kg, n = 6; Lars Jonssen, Lynge, Denmark) were used, following the protocol previously described (5, 8). Briefly, animals were anesthetized with intravenous {alpha}-chloralose (66 mg/kg; Merck, Darmstadt, Germany) and ventilated with intermittent positive pressure using N2O2/O2. Catheters were placed in the right carotid artery for sampling of arterial blood, in a left ear vein for peptide infusion, and in a right ear vein for glucose and valine-pyrrolidide administration. After surgical preparation, a saline drip was set up in an ear vein (5 ml/min), and the animals were heparinized and left undisturbed for 30 min. Anesthesia was maintained with additional chloralose as necessary.

Six animals were given valine-pyrrolidide [300 µmol/kg, dissolved in 0.9% NaCl; a dose previously shown to inhibit plasma DPP IV activity by >95% for at least 220 min (5)] as a bolus intravenous injection over 2 min, commencing at minute –20. The animals then received two separate intravenous infusions of GIP-(1–42), one with and one without coinfusion of GIP-(3–42), in a crossover design with 90 min between each infusion. In this manner, three animals received GIP-(1–42) alone as the first infusion, and three received it as the second infusion. Peptides were dissolved in 0.9% NaCl containing 1% HSA (Behringwerke, Marburg, Germany) and infused over 57 min with a syringe pump. GIP-(1–42) was given as a bolus dose of 0.22 nmol followed by an infusion at a rate of 0.58 pmol·kg–1·min–1. For infusion of both peptides, animals concomitantly received GIP-(3–42), given as a bolus of 0.53 nmol followed by infusion at a rate of 1.42 pmol·kg–1·min–1. Bolus doses and infusion rates were calculated to achieve approximately the same plasma concentrations of GIP-(1–42) and GIP-(3–42) as in our group's original study infusing GIP alone in the absence of valine-pyrrolidide (4). An intravenous glucose infusion (0.2 g/kg; 50% solution) was administered between minutes 38 and 47. Arterial blood samples (4 ml) were taken at –30, –20, –10, 0, 10, 20, 30, 35, 42, 47, 49, 52, 54, and 57 min from the start of the infusion. After 57 min, the GIP infusion was stopped, and further blood samples were taken at 5, 10, 15, 20, 30, 45, 60, and 90 min. Ninety minutes after cessation of the first infusion, the second infusion was started, and the protocol was repeated for blood sampling and the glucose infusion. The volume of blood taken did not exceed 5% of the total blood volume, and this fluid loss was replaced by flushing the catheters with 5 ml of 0.9% NaCl after each sample was taken. This protocol has previously been shown not to affect heart rate or blood pressure (4, 5).

Blood glucose was measured immediately (One Touch II; Lifescan, Lyngby, Denmark). Blood samples for hormonal analysis were collected into chilled tubes containing EDTA (7.4 mM final concentration) and valine-pyrrolidide (0.01 mM final concentration). All samples were kept on ice until centrifugation at 4°C, after which the plasma was separated and stored at –20°C until analysis.

Hormonal Analysis

Insulin concentrations in venous effluent from the perfused pancreas were assayed using antiserum 2006 and rat insulin standards (Novo Nordisk).

Total GIP was measured using the COOH-terminally directed antiserum R65 (17, 18), which reacts fully with intact GIP and the NH2-terminally truncated metabolite GIP-(3–42) but not with the so-called 8-kDa GIP, whose chemical nature and relation to GIP secretion is uncertain. The assay has a detection limit of 2 pM. Intact, biologically active GIP was measured using antiserum 98171 (7), which is specific for the intact NH2 terminus of GIP and cross-reacts <0.1% with GIP-(3–42). It has a detection limit of 5 pM. For both assays, human GIP (Peninsula Laboratories Europe, St. Helens, UK) was used as standard, and radiolabeled GIP was obtained from Amersham Pharmacia Biotech (Little Chalfont, UK). Separation of bound from free peptide was achieved using plasma-coated charcoal (27). Plasma samples were extracted with ethanol (70% vol/vol final concentration), giving recoveries of 85% and intra-assay variations of <6%. Insulin immunoreactivity was measured in unextracted plasma using antiserum 2004 (28), and glucagon immunoreactivity was determined after ethanol extraction using the COOH-terminally directed antiserum 4305, which measures glucagon of pancreatic origin (28).

Calculations and Statistical Analysis

The area under the insulin curve was calculated using the trapezoidal method for the first phase and second phase of insulin secretion, defined as the periods when insulin concentrations in the venous effluent increased by >5% above the prestimulus value.

The plasma half-life (t1/2) of GIP was calculated by loge-linear regression analysis of peptide concentrations (after subtraction of endogenous values) in samples collected after the end of the infusion. The incremental areas under the curve (AUC) for GIP, glucose, insulin, and glucagon were calculated using the trapezoidal method either after subtraction of the basal concentrations measured in samples before the start of each GIP infusion (GIP and glucose) or immediately before the glucose infusion (insulin and glucagon). The fractional clearance (k) for glucose was calculated using the formula k = 0.693/t1/2, where the t1/2 was calculated from loge-linear regression analysis of glucose concentrations during the period between minutes 47 and 57 of each infusion.

Data are expressed as means ± SE. Data from the perfused pancreas were analyzed by repeated-measures ANOVA followed by Tukey-Kramer multiple comparison. In vivo data were analyzed by ANOVA followed by Dunnet's multiple comparison or two-tailed t-tests for paired data, as appropriate, using GraphPad Instat software (version 3.05; San Diego, CA). Values of P < 0.05 were considered to be significant.


   RESULTS
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 ABSTRACT
 RESEARCH DESIGN AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
GIP Receptor Activation

As expected, GIP-(1–42) was a potent agonist and stimulated cAMP accumulation with very high potency (EC50 = 13.5 pM), whereas GIP-(3–42) did not stimulate cAMP accumulation, even at concentrations as high as 1 µM (Fig. 1A). Rather, GIP-(3–42) acted as a weak antagonist, shifting the concentration response curve to GIP-(1–42) to the right in a dose-dependent manner (Fig. 1B). The potency by which GIP-(3–42) inhibited GIP-(1–42) activity was measured for several concentrations of GIP-(1–42). Thus an IC50 value of 92, 134, and 731 nM for GIP-(3–42) was found for the inhibition of cAMP accumulation elicited by 10 pM, 100 pM, and 1 nM GIP-(1–42), respectively [i.e., at doses of GIP-(1–42) giving rise to ~50, 75, and 90% of maximal stimulation; Fig. 1C].


Figure 1
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  		Fig. 1. Whole cell cAMP accumulation was measured in transiently transfected COS-7 cells expressing the human glucose-dependent insulinotropic polypeptide (GIP) receptor. A: dose-response curves for GIP-(1–42) (bullet) and GIP-(3–42) ({circ}). B: dose-dependent shift of the dose-response curve for GIP-(1–42) (bullet) by the presence of increasing concentrations of GIP-(3–42): 10 nM ({square}), 100 nM ({triangleup}), and 1 µM ({lozenge}). C: dose-response curves for GIP-(3–42) in the presence of 10 pM ({blacksquare}), 100 pM (bullet), and 1 nM ({blacktriangleup}) GIP-(1–42). Data are shown as means ± SE; n = 2–4.

 
GIP Receptor Binding

The binding affinities for the interaction of GIP-(1–42) and GIP-(3–42) with the human GIP receptor were determined in the same cellular background as the signaling analyses described above. Thus the competition binding experiments were carried out in transiently transfected COS-7 cells using 125I-GIP as radioligand. For the homologous competition binding, an IC50 value of 5.2 nM (log IC50 ± SE = –8.3 ± 0.11, n = 3) was observed, whereas the affinity (IC50) for GIP-(3–42) was lower (22 nM; log IC50 ± SE = –7.7 ± 0.13, n = 3, Fig. 2A). The corresponding Kd and Ki values were similar to the IC50 values because of the low radioligand concentration (15 pM) compared with the observed affinities (IC50). The above values were determined for the competition binding at 4°C, and similar values were obtained for the binding at 37°C (Fig. 2B). Thus IC50 values of 3.5 nM (log IC50 ± SE = –8.5 ± 0.09, n = 3) and 14 nM (log IC50 ± SE = –7.8 ± 0.07, n = 3) for GIP-(1–42) and GIP-(3–42), respectively, were observed.


Figure 2
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  		Fig. 2. Competition binding was carried out in transiently transfected COS-7 cells expressing the human GIP receptor, using 125I-GIP and increasing concentrations of GIP-(1–42) ({blacksquare}) and GIP-(3–42) (bullet) at 4°C (A) and 37°C (B). Data are shown as means ± SE; n = 3.

 
Isolated Perfused Pancreas

Perfusion of the pancreas with 1 nM GIP-(1–42) increased insulin secretion significantly (P < 0.001) relative to basal secretion during perfusion with 10 mM glucose alone (Fig. 3). There was no significant difference (AUC, paired t-test) in insulin output in response to the first compared with the second GIP stimulation, so the mean response to GIP alone for each experiment was used in subsequent statistical analysis. Infusion of GIP-(3–42) (5 and 50 nM) alone had no effect on insulin output. The response to GIP-(1–42) (AUC: 127.6 ± 36.4 nM/min) was not changed significantly during coinfusion with GIP-(3–42) in 5- or 50-fold molar excesses (AUC: 103.0 ± 34.9 and 98.6 ± 52.2 nM/min, respectively; Fig. 2). However, at higher concentrations of GIP-(3–42), insulin output was reduced significantly, giving rise to an IC50 value of 138 nM.


Figure 3
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  		Fig. 3. Insulin output from the perfused rat pancreas during perfusion with 10 mM glucose and GIP-(1–42) (1 nM) alone, GIP-(3–42) (5 and 50 nM) alone, and GIP-(1–42) (1 nM) together with GIP-(3–42) (5 and 50 nM). Data are means ± SE; n = 4. Top: dose-response effect of GIP-(3–42) to inhibit the insulin secretion elicited by 1 nM GIP-(1–42) (n = 3–4).

 
GIP Pharmacokinetics in Anesthetized Pigs

Plasma concentration curves for GIP, measured with NH2-terminal (intact peptide) and COOH-terminal (intact + NH2-terminally degraded peptide) RIAs are shown in Fig. 4. Under basal conditions (before inhibitor administration), endogenous GIP concentrations measured with the NH2-terminal assay (1.7 ± 0.4 pM) were lower than those determined with the COOH-terminal assay (12.8 ± 4.1 pM, P = 0.0457). COOH-terminal concentrations were unaffected by valine-pyrrolidide administration (14.0 ± 1.8 pM), whereas NH2-terminal concentrations increased significantly (to 8.2 ± 1.6 pM, P = 0.0143). However, despite this increase, endogenous NH2-terminal GIP concentrations were still significantly (P = 0.0037) lower than COOH-terminal concentrations during DPP IV inhibition.


Figure 4
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  		Fig. 4. Plasma GIP immunoreactivity in blood samples from the carotid artery measured with COOH-terminally ({blacktriangleup}) and NH2-terminally (bullet) directed RIAs. Animals were given valine-pyrrolidide (val-pyd; 300 µmol/kg) 20 min before commencement of crossover intravenous infusions of GIP-(1–42) alone (bolus dose of 0.22 nmol followed by 0.58 pmol·kg–1·min–1; A) or in combination with GIP-(3–42) (bolus dose of 0.53 nmol followed by 1.52 pmol·kg–1·min–1; B). The horizontal arrows indicate the periods of the infusions. Data are means ± SE; n = 6.

 
During infusion of GIP-(1–42) alone (0.58 pmol·kg–1·min–1) with concomitant DPP IV inhibition, NH2-terminal GIP immunoreactivity (44 ± 4 pM) remained lower than COOH-terminal immunoreactivity (63 ± 6 pM), although this difference was not significant (P = 0.0871). However, even though plasma DPP IV activity was inhibited, the NH2-terminal plasma t1/2 was shorter than that determined by the COOH-terminal assay (6.8 ± 0.5 vs. 10.2 ± 0.9 min, P = 0.0068, NH2- and COOH-terminal immunoreactivity, respectively).

Exogenous intact GIP concentrations were unaltered by coinfusion of GIP-(3–42) [44 ± 2 pM, not significant, vs. infusion of GIP-(1–42) alone], whereas concentrations determined with the COOH-terminal assay increased (282 ± 13 pM) and were higher (P < 0.0001) than those determined using the NH2-terminal assay. Neither the AUC nor the plasma t1/2 for intact GIP-(1–42) (determined using the NH2-terminal assay) was affected by coinfusion of the metabolite [AUC0–147 min: 3,324 ± 302 vs. 3,632 ± 147 pM/min; t1/2: 6.8 ± 0.5 vs. 7.2 ± 0.7 min; GIP-(1–42) alone and together with GIP-(3–42), respectively].

GIP Pharmacodynamics in Anesthetized Pigs

Glucose concentrations are shown in Fig. 5A. There were no significant differences between the glucose profiles, and neither the glucose excursion [glucose {Delta}AUC35–77 min: 60 ± 13 and 77 ± 8 mM/min, GIP-(1–42) alone and with GIP-(3–42), respectively] nor the glucose elimination rate [k: 12.5 ± 1.6 and 10.1 ± 0.5%/min, GIP-(1–42) alone and with GIP-(3–42)] were significantly affected by coinfusion of GIP-(3–42).


Figure 5
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  		Fig. 5. Blood glucose (A), plasma insulin (B), and glucagon concentrations (C) measured during and after crossover intravenous infusions of either of GIP-(1–42) alone (bolus dose of 0.22 nmol followed by 0.58 pmol·kg–1·min–1; {square}) or in combination with GIP-(3–42) (bolus dose of 0.53 nmol followed by 1.52 pmol·kg–1·min–1; {blacksquare}). Animals received valine-pyrrolidide (val-pyd; 300 µmol/kg) 20 min before commencement of infusions, and an intravenous glucose load (0.2 g/kg) was administered during minutes 38–47 of each infusion. The horizontal arrows indicate the periods of the infusions. Data are means ± SE; n = 6.

 
Insulin concentrations are shown in Fig. 5B. There were no significant differences at any time point during infusion of GIP-(1–42) alone or in combination with GIP-(3–42), and the amounts of insulin secreted in response to the glucose load (AUC35–77 min) were similar during both infusions [2,895 ± 450 vs. 2,745 ± 448 pM/min, GIP-(1–42) alone and together with GIP-(3–42)].

The plasma glucagon concentrations (Fig. 5C) were similar during infusion of GIP-(1–42) alone and together with GIP-(3–42) [AUC35–77 min: –50 ± 27 vs. –106 ± 51 pM/min, GIP-(1–42) and together with GIP-(3–42)].


   DISCUSSION
TOP
 ABSTRACT
 RESEARCH DESIGN AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
GIP is, like its fellow incretin hormone GLP-1, rapidly degraded in vivo by the widespread serine protease DPP IV, and it has been speculated that the truncated metabolites may act as antagonists. In the case of GLP-1, the metabolite has been shown to bind to the GLP-1 receptor, albeit with reduced affinity (1% of that of the intact peptide) and to be able to antagonize the parent peptide's stimulatory effect on adenylyl cyclase activity in vitro (16). However, this ability to prevent GLP-1-stimulated cAMP production in isolated cells transfected with the pancreatic GLP-1 receptor does not translate to antagonism of GLP-1's insulinotropic effects in vivo (8, 35).

In the present study, native GIP stimulated cAMP production in COS-7 cells transfected with the human GIP receptor with an EC50 value in the picomolar range (13.5 pM), in agreement with studies reported by Wheeler et al. (36), Hinke et al. (13, 14), and Gelling et al. (11) using Chinese hamster ovary (CHO-K1) cells transfected with the rat pancreatic GIP receptor (69, 373, 245, and 310 pM, respectively), by Moens et al. (23) using isolated rat beta-cells (200 pM), by Maletti et al. (21) using human insulinoma plasma membranes (<100 pM), and by Gremlich et al. (12) using Chinese hamster lung (CHL) fibroblast cells transfected with the human pancreatic GIP receptor (600–800 pM). In contrast, Gault et al. (10) reported EC50 values in the nanomolar range (18.2 nM) in CHL cells transfected with the human GIP receptor.

The product of DPP IV-mediated truncation of GIP,GIP-(3–42), has also been shown to bind to the GIP receptor, but with an affinity less than that of the parent peptide. Thus Hinke et al. (13) reported a binding affinity (IC50) value of 58 nM for GIP-(3–42) compared with 3.5 nM for the intact peptide, using CHO-K1 cells transfected with the rat pancreatic islet GIP receptor. These findings were corroborated in the present study using COS-7 cells transfected with the human GIP receptor. However, despite displaying affinity for the GIP receptor, GIP-(3–42) in concentrations up to 50 µM was without effect on cAMP production in vitro (Ref. 13 and present study). Weak agonist activity (<5% of that of native GIP) was reported by Gault et al. (10).

GIP-(3–42) has been reported to inhibit GIP-stimulated cAMP accumulation (10), but in the present study we were only able to demonstrate a weak antagonist effect, thereby confirming the findings of Hinke et al. (13). Therefore, although in receptor activation studies GIP-(3–42) can behave as a poor antagonist, it appears unlikely to have any physiological relevance, because physiological concentrations of GIP-(3–42) tend to be maximally only approximately five to six times higher than those of GIP-(1–42) (4, 9).

In the binding experiments, the affinity estimate [Kd (i.e., the concentration of ligand required to occupy 50% of its receptors at equilibrium) of 3.5 nM] for GIP-(1–42) was much higher than its agonist potency [EC50 (i.e., the concentration of ligand required to elicit 50% of its maximum effect) of 13.5 pM] observed in the cAMP stimulation assay. This discrepancy can be explained by a number of factors, such as the presence of a receptor reserve ("spare receptors;" i.e., an excess of receptors compared with intracellular signal transduction mediators), where receptor activation can occur at lower ligand concentrations than those necessary to saturate the specific binding sites, or the presence of a high-efficacy ligand, which needs only occupy relatively few receptors to produce a maximum response. These same arguments also can be used to explain the difference between the affinity of GIP-(3–42) for the GIP receptor and its inhibitory potency [IC50 (i.e., the concentration of inhibitor/antagonist required to inhibit 50% of the agonist effect)] relative to the potency of GIP-(1–42). Thus much higher concentrations of GIP-(3–42) are required to antagonize the effect of GIP-(1–42) compared with their respective relative affinities, because high agonist concentrations are more difficult for an antagonist to overcome than low agonist concentrations and because with a high-efficacy agonist and reserve receptors, it is possible to inactivate a proportion of the receptors without depressing the maximum concentration response. We therefore predict that the observed affinity of GIP-(3–42) of 14 nM corresponds to the potency of this ligand in its ability to inhibit GIP-(1–42)-mediated signaling. This potency is still 1,000 times lower than the agonist potency, and therefore it would be predicted that physiological levels of GIP-(3–42) could not antagonize endogenous GIP-(1–42).

In agreement with the earlier in vitro studies of Brown et al. (3) using the perfused rat pancreas and Schmidt et al. (33) using cultured pancreatic islets, in the present study GIP-(3–42) was found to be devoid of insulin-releasing activity in the perfused rat pancreas. Furthermore, and in accord with the receptor binding studies, GIP-(3–42) (up to 50 nM) had no effect on the insulinotropic effect of the parent peptide, fully supporting the observations by Schmidt et al. (33), who found no antagonistic effect of GIP-(3–42) on insulin release from isolated pancreatic islets when given at a 10-fold higher concentration relative to the intact peptide. However, when GIP-(3–42) concentrations were increased into the range shown to be capable of antagonizing GIP-(1–42)-stimulated cAMP accumulation, antagonism of the parent peptide's insulinotropic activity became apparent, with the IC50 value in the perfused pancreas (138 nM) being close to that observed in the receptor activation experiments (730 nM). Moreover, in the present study, and in agreement with what could have been predicted from the in vitro results, GIP-(3–42) was found to antagonize neither the antihyperglycemic nor the insulinotropic effects of intact GIP in vivo when given at a dose that raises metabolite concentrations to approximately six times those of the intact peptide, which approximates the endogenous intact-to-metabolite ratio. In contrast to these results, studies in mice have suggested that GIP-(3–42) can antagonize the insulinotropic effect of the parent peptide (10). However, in that study, high doses (25 nmol/kg) of peptides were administered intraperitoneally, which would be expected to raise plasma levels well into the nanomolar range, in contrast to the present study, where concentrations of intact GIP (~50 pM) and the metabolite, GIP-(3–42) (200–250 pM), were similar to those seen after ingestion of a mixed meal in pigs (19), healthy humans (4), and dogs (9). Moreover, in that study (10), the in vivo experimental protocol employed makes it very difficult to assess whether the metabolite actually does possess any antagonistic properties, because the animals received different doses of the parent peptide depending on whether it was given alone (2 x 25 nmol/kg) or concomitantly with the metabolite (25 nmol/kg). In rats, GIP-(3–42) injected subcutaneously at a dose of 8 nmol/kg had no effect on glycemia or insulin release in response to an oral glucose tolerance test, suggesting that the metabolite did not alter the effect of endogenously released GIP-(1–42), where peak levels of endogenous immunoreactive GIP reached 1.68 ng/ml (337 pM), leading the authors to conclude that it was extremely unlikely that GIP-(3–42) would play an antagonistic role in vivo (13). However, the possibility cannot be excluded that higher concentrations of GIP-(3–42) may have had an antagonistic effect in vivo. GIP-(1–42) concentrations [and possibly GIP-(3–42), depending on how quickly GIP is degraded after its release] would be higher close to the site of release than they are peripherally. However, because there is no evidence to suggest that the insulinotropic effect of GIP is mediated via its interaction with afferent nerves within the intestine or hepatoportal region (24, 26), where peptide concentrations would be higher, it is the arterial concentrations that reflect the levels of both peptides reaching the pancreas. The finding that no antagonism was observed with the chosen infusion rates of GIP-(1–42) and -(3–42), which achieved arterial plasma concentrations in the physiological range seen after meal ingestion (4, 9, 19), further supports the conclusion that endogenous GIP-(3–42) is unlikely to behave as an antagonist in vivo.

In the present study, valine-pyrrolidide was used to reduce the in vivo degradation of the infused GIP-(1–42) by DPP IV, but despite this, NH2-terminal GIP concentrations tended to be slightly lower than COOH-terminal concentrations. This may be an assay artifact (due to the interassay variability associated with using 2 different antibodies), but it also remains a possibility that some NH2-terminal degradation occurred due to enzymes other than DPP IV, which was not inhibited by valine-pyrrolidide, as discussed previously (4). Nevertheless, the difference was not significant, suggesting that in the present study, no or only marginal degradation of the infused peptide occurred.

The reason for the difference between the results of Gault et al. (10) with those of Hinke et al. (13), Schmidt et al. (33), and the present study is unclear. It seems to be unrelated to whether the rat or human GIP receptor was used, because the present study using the human receptor agrees with the findings of Hinke et al. (13) and Schmidt et al. (33), who both used the rat system, but finds opposing results to Gault et al. (10), who also used the human receptor. Moreover, the difference cannot be explained by the use of the human vs. porcine sequence of GIP, because Wheeler et al. (37) found both forms to have similar EC50 values. It is of note that the native GIP used by Gault et al. (10) is apparently less potent in stimulating cAMP production in vitro than that used by others. Although potency estimates may exhibit some variation between studies because of differences in experimental conditions (cell type, incubation time, temperature, etc.), the EC50 concentration (10–7 M) reported by Gault et al. (10) is greater than that required to elicit the maximal response (10–8 M) in the present study and in those of Hinke et al. (13, 14), Gelling et al. (11), and Maletti et al. (21), which may have given scope to observe an effect of a poor antagonist in that study (because it is better able to compete with a weak agonist).

In conclusion, the results of the present study demonstrate that, although GIP-(3–42) can behave as a weak antagonist at the cloned GIP receptor in vitro, this does not translate to physiological antagonism of GIP's insulinotropic effect in vivo, in much the same way as the metabolite of GLP-1 fails to antagonize the insulinotropic effect of GLP-1. However, unlike the metabolite of GLP-1, which possesses weak antihyperglycemic activity in the pig, GIP-(3–42) does not appear to possess any antihyperglycemic activity of its own.


   GRANTS
TOP
 ABSTRACT
 RESEARCH DESIGN AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by grants from the European Foundation for the Study of Diabetes, the Danish Medical Research Council, the Danish Biotechnology Programme, the Novo Nordisk Foundation, the Toyota Foundation, the German Research Foundation, and the Velux Foundation.


   ACKNOWLEDGMENTS
 
The technical assistance of Lene Albæk, Lisbet Elbak, Letty Klarskov, Mette Olesen, and Sophie Pilgaard is gratefully acknowledged.


   FOOTNOTES
 

Address for reprint requests and other correspondence: C. F. Deacon, Dept. of Medical Physiology, The Panum Institute, Blegdamsvej 3, DK-2200 Copenhagen N, Denmark (e-mail: deacon{at}mfi.ku.dk)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 RESEARCH DESIGN AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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