characterisation and biological activity of glu3 amino acid substituted gip receptor antagonists

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ABBArchives of Biochemistry and Biophysics 461 (2007) 263–274

Characterisation and biological activity of Glu3 amino acidsubstituted GIP receptor antagonists

Victor A. Gault a, Kerry Hunter a, Nigel Irwin a,*, Brian D. Green a, Brett Greer b,Patrick Harriott b, Finbarr P.M. O’Harte a, Peter R. Flatt a

a School of Biomedical Sciences, University of Ulster, Cromore Road, Coleraine BT52 1SA, Northern Ireland, UKb School of Biological Sciences, Queen’s University of Belfast, Lisburn Road, Belfast BT9 7BL, Northern Ireland, UK

Received 13 February 2007, and in revised form 7 March 2007Available online 28 March 2007

Abstract

Glucose-dependent insulinotropic polypeptide (GIP) is an important gastrointestinal hormone, which regulates insulin release andglucose homeostasis, but is rapidly inactivated by enzymatic N-terminal truncation. Here we report the enzyme resistance and biologicalactivity of several Glu3-substituted analogues of GIP namely; (Ala3)GIP, (Lys3)GIP, (Phe3)GIP, (Trp3)GIP and (Tyr3)GIP. Only (Lys3)-GIP demonstrated moderately enhanced resistance to DPP-IV (p < 0.05 to p < 0.01) compared to native GIP. All analogues demon-strated a decreased potency in cAMP production (EC50 1.47 to 11.02 nM; p < 0.01 to p < 0.001) with (Lys3)GIP and (Phe3)GIPsignificantly inhibiting GIP-stimulated cAMP production (p < 0.05). In BRIN-BD11 cells, (Lys3)GIP, (Phe3)GIP, (Trp3)GIP and (Tyr3)-GIP did not stimulate insulin secretion with both (Lys3)GIP and (Phe3)GIP significantly inhibiting GIP-stimulated insulin secretion(p < 0.05). Injection of each GIP analogue together with glucose in ob/ob mice significantly increased the glycaemic excursion comparedto control (p < 0.05 to p < 0.001). This was associated with lack of significant insulin responses. (Ala3)GIP, (Phe3)GIP and (Tyr3)GIP,when administered together with GIP, significantly reduced plasma insulin (p < 0.05 to p < 0.01) and impaired the glucose-lowering abil-ity (p < 0.05 to p < 0.01) of the native peptide. The DPP-IV resistance and GIP antagonism observed were similar but less pronouncedthan (Pro3)GIP. These data demonstrate that position 3 amino acid substitution of GIP with (Ala3), (Phe3), (Tyr3) or (Pro3) provides anew class of functional GIP receptor antagonists.� 2007 Elsevier Inc. All rights reserved.

Keywords: DPP-IV; GIP; Gastrointestinal; Insulin secretion; Diabetes

Over the last number of years, glucose-dependent insuli-notropic polypeptide (GIP)1 has come forward as a prom-ising candidate for diabetes therapy [1,2]. GIP is a 42amino acid hormone secreted from enteroendocrine K-cellsin response to food and nutrient absorption [1]. Uponrelease, the primary action of GIP is the stimulation of glu-cose-dependent insulin secretion through interaction withspecific heterotrimeric G-protein-coupled GIP receptorson pancreatic b-cells [3]. This glucose-dependent action

0003-9861/$ - see front matter � 2007 Elsevier Inc. All rights reserved.

doi:10.1016/j.abb.2007.03.001

* Corresponding author. Fax: +44 0 28 70324965.E-mail address: [email protected] (N. Irwin).

1 Abbreviations used: GIP, glucose-dependent insulinotropic polypep-tide; CHL, Chinese Hamster Lung; RIA, radioimmunoassay; t1/2, half-life.

on insulin secretion means there is a significantly reducedlikelihood of hypoglycaemic episodes often associated withother insulin-releasing agents used in the treatment ofT2DM [4]. Additionally, the pleiotropic nature of GIP act-ing synergistically as both a growth and anti-apoptotic fac-tor for pancreatic b-cells [5–8], further support its possibletherapeutic application in T2DM.

One of the crucial difficulties in attempting to developGIP as a therapeutic agent is its short biological half-life,which is primarily due to degradation by the ubiquitousenzyme dipeptidyl peptidase-IV (DPP-IV) [9]. DPP-IV(CD26; E.C.3.4.14.5) is a homodimeric class II proteinbelonging to the prolyl oligopeptidase family [10],ubiquitously expressed in mammalian tissues and organs

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264 V.A. Gault et al. / Archives of Biochemistry and Biophysics 461 (2007) 263–274

[11]. It is important to note that DPP-IV action is not spe-cific to GIP alone as natural substrates in vitro also includecytokines, chemokines, endomorphins and almost all mem-bers of the PACAP/glucagon peptide superfamily, includ-ing the sister incretin hormone GLP-1 [12]. DPP-IV,therefore, appears to play an important role in the regula-tion of a number of key physiological processes, whichinclude effects on pain regulation, metabolism, cell adhe-sion, immune response and the cardiovascular system.

Due to the rapid inactivation of GLP-1 and GIP, DPP-IV inhibitors are currently being developed as a strategy toprolong the biological activity of incretin hormones.Encouraging effects have been reported with several inhib-itors, including improvements in glucose tolerance, hyper-insulinaemia, b-cell glucose responsiveness and peripheralinsulin sensitivity [13–16]. One important advantage ofDPP-IV inhibitors is the prospect that they can be admin-istered orally. However, one major disadvantage is thatthey also pose a possible threat from long-term administra-tion and undesirable side effects, due to prolonged inhibi-tion of other DPP-IV mediated processes [10,17,18]. Thusalthough DPP IV inhibitors in clinical development showgood specificity, non-selective inhibition of the DPP-IVrelated enzymes, DPP-8 and DPP-9, has been shown toresult in toxicity in preclinical species [19]. Hence, giventhat information concerning the substrate specificity ofDPP-IV is well documented, an attractive alternativeapproach involves the generation of specific GLP-1 andGIP analogues modified in the region around the enzymecleavage site to impart improved DPP-IV resistance[1,2,20,21]. The potential of this approach is demonstratedby the stable GLP-1 mimetic exendin 1–39 (Byetta), whichhas been approved for the clinical treatment of T2DM [22].

Until now, various reports have demonstrated thetherapeutic potential of enzyme-resistant forms of GIP inexperimental diabetic animal models [1,2,23]. In essence,structural modifications performed at the Tyr1 residue inGIP generate analogues that are completely resistant to theactions of DPP-IV and have markedly enhanced insulino-tropic and antihyperglycaemic activity [24,25]. Substitutionof Ala2 produced analogues with varying degrees of DPP-IV resistance and biological activity, however, their efficacywas not as impressive as that of the Tyr1-modified analogues[26–30]. Such ‘super GIP’ analogues may be useful for thetreatment of T2DM, by acting mostly through the stimula-tion of insulin secretion, analogous to the actions of GLP-1but without effects on gastric emptying [31,32].

An exciting finding has been the development of a GIPreceptor antagonist through the substitution of Glu3 with aPro residue [33]. (Pro3)GIP is completely resistant to theactions of DPP-IV and even more notably, effectively andspecifically countered the insulin-releasing actions of thenative hormone in vivo [33,34]. Recent evidence has demon-strated that genetic knockout of GIP can prevent insulinresistance in high-fat fed mice [35]. Furthermore, a recentpaper published from our laboratory has shown thatchemical ablation of GIP receptor action with daily (Pro3)-

GIP administration ameliorated insulin resistance and sig-nificantly improved glucose tolerance, pancreatic b-cellfunction and disturbances of islet morphology in ob/ob

mice [36]. Comparable to the therapeutic actions ofmetformin and other insulin sensitisers, specific GIPreceptor antagonists are currently being considered as anunexpected new class of drugs for alleviation of insulinresistance and treatment of T2DM [1].

In the present study, five Glu3-substituted GIPanalogues were designed, synthesised and tested for theirability to serve as effective GIP receptor antagonists. Theenzyme stability of each analogue together with theirabilities to affect cAMP production and insulin secretionin vitro was investigated. Furthermore, the insulin-releasingand antihyperglycaemic activities of all GIP analogueswere examined in obese diabetic ob/ob mice.

Materials and methods

Peptide synthesis

Human GIP and related GIP analogues were synthesised on anApplied BioSystems automated peptide synthesiser (Model 432A Synergy;Warrington, Cheshire, UK) with a preloaded Fmoc-Gln(Trt)-Wang resin(Calbiochem Novabiochem, Beeston, Nottingham, UK) using standardsolid-phase Fmoc chemistry as described previously [33]. Crude peptideswere purified by semi-preparative followed by analytical HPLC on aWaters Millennium 2010 Chromatography System [33]. The hydrophilicnegatively charged Glu3 residue of GIP was substituted with; either a non-polar hydrophobic residue (Ala), a positively charged hydrophilic residue(Lys), an aromatic hydrophobic non-polar residue (Phe, Trp), or ahydrophilic neutral aromatic residue (Tyr). (Pro3)GIP was also synthes-ised and included as a positive control.

Structural confirmation of GIP peptides by ESI–MS

Peptide samples were dissolved in 100 ll of H2O (�400 pM) andapplied to the LCQ/MS (Finnigan MAT, Hemel Hempstead, UK).Spectra were obtained from the quadrupole ion trap mass analyser andcollected using full ion scan mode over the mass-to-charge (m/z) range150–2000. The molecular masses of the GIP peptides were determinedfrom the prominent multiple charged ions and the following equationapplied: Mr = iMi � iMh (where Mr, molecular mass; Mi, m/z ratio; i,

number of charges; Mh, mass of a proton).

Degradation of GIP and GIP analogues by DPP-IV

GIP and related peptides (15 lg) were incubated (n = 3) at 37 �C withDPP-IV (5 mU; Sigma, Poole, Dorset, UK) for 0, 2, 4 and 8 h in 50 mMtriethanolamine–HCl (500 ll), pH 7.8 (final peptide concentration 2 lM).Enzymatic reactions were terminated by the addition of 10 ll of 10% (v/v)TFA/H2O and stored at �20 �C prior to HPLC analysis as describedpreviously [33]. The absorbance was monitored at 206 nm on a SpectraSystem UV2000 detector (Thermoquest Limited, Manchester, UK). Thepercentage of intact peptide remaining at each time point was calculatedfrom HPLC peak areas of GIP(3–42) and either native GIP or GIPanalogues.

Tissue culture

Chinese Hamster Lung (CHL) fibroblast cells stably transfected withthe human GIP-R [37] were cultured in DMEM tissue culture mediumcontaining 10% (v/v) fetal bovine serum, 1% (v/v) antibiotics (100 U/ml

V.A. Gault et al. / Archives of Biochemistry and Biophysics 461 (2007) 263–274 265

penicillin, 0.1 mg/ml streptomycin) (all from Gibco, Paisley, Strathclyde,Scotland). BRIN-BD11 cells were cultured in RPMI-1640 tissue culturemedium containing 10% (v/v) fetal bovine serum, 1% (v/v) antibiotics(100 U/ml penicillin, 0.1 mg/ml streptomycin) and 11.1 mM glucose. Theorigin and secretory characteristics of these cells have been described indetail previously [38]. The cells were maintained in sterile tissue cultureflasks (Corning Glass Works, Sunderland, UK) at 37 �C in an atmosphereof 5% CO2 and 95% air using a LEEC incubator (Laboratory TechnicalEngineering, Nottingham, UK).

cAMP stimulation

Receptor activation by GIP and GIP analogues in CHL cells trans-fected with the human GIP-R was performed according to publishedmethodologies [33]. Briefly, GIP-R transfected CHL cells seeded into 24-well plates (Nunc, Roskilde, Denmark) at a density of 3.0 · 105 cells perwell were loaded with tritiated adenine (2 lCi; TRK311; Amersham,Buckinghamshire, UK) and allowed to grow for 18 h at 37 �C. Theculture medium was removed and cells subsequently washed twice with2 ml ice-cold HBS buffer (130 mM NaCl, 20 mM Hepes, 0.9 mM NaH-PO4, 0.8 mM MgSO4, 5.4 mM KCl, 1.8 mM CaCl2, 5.6 mM glucose and25 lM phenol red) (pH 7.4). The cells were then exposed for 20 min at37 �C to forskolin (10 lM; Sigma, Poole, Dorset, UK) or GIP/GIPanalogues (10�13 to 10�7 M) in the presence or absence of native GIP(10�7 M) in HBS buffer containing 1 mM IBMX (Sigma, Poole, Dorset,UK). The medium was subsequently removed and the cells lysed with1 ml of lysing solution (5% TCA, 3% SDS, 92% H2O, also containing0.1 mM unlabelled cAMP and 0.1 mM unlabelled ATP) (Sigma, Poole,Dorset, UK). The plates were then left on a shaker at room temperaturefor 30 min and tritiated cAMP formation determined by column chro-matography using Dowex and alumina ion exchange columns (BioRadLife Science Research, Alpha Analytical, Larne, UK) as previouslydescribed [33].

Insulin secretion

Insulin release from BRIN-BD11 cells was determined by use of cellmonolayers as described previously [33]. In brief, BRIN-BD11 cells wereseeded into 24-well plates (Nunc, Roskilde, Denmark) at a density of1.5 · 105 cells per well and allowed to attach overnight in RPMI-1640culture medium at 37 �C. Acute tests for insulin secretion were precededby 40 min pre-incubation at 37 �C in 1.0 ml Krebs Ringer BicarbonateBuffer (KRBB, 115 mM NaCl, 4.7 mM KCl, 1.28 mM CaCl2, 1.2 mMMgSO4, 1.2 mM KH2PO4, 25 mM Hepes and 10 mM NaHCO3; pH 7.4with NaOH) supplemented with 0.1% (w/v) BSA and 1.1 mM glucose.Test incubations were performed (n = 8) in the presence of 5.6 mMglucose over a range of concentrations (10�13 to 10�7 M) of GIP/GIPanalogues in the presence or absence of native GIP (10�7 M). After 20 minincubation, the buffer was removed and used for measurement of insulinby radioimmunoassay (RIA) [39].

In vivo biological activity

Plasma glucose and insulin responses were evaluated using14–18 week old obese diabetic ob/ob mice [40] following intraperitonealinjection of native GIP or GIP analogues (25 nmol/kg bw) immediatelyfollowing the combined injection of GIP (25 nmoles/kg bw) togetherwith glucose (18 mmol/kg bw). Previous studies using GIP analogueshave shown that they exert in vivo effects within this dose range [1,2].All test solutions were administered in a final volume of 5 ml/kg bodyweight. Blood samples were collected from the cut tip of the tail vein ofconscious mice into chilled fluoride/heparin coated glucose microcen-trifuge tubes (Sarstedt, Numbrecht, Germany) immediately prior toinjection and at 15, 30 and 60 min post injection. Blood samples wereimmediately centrifuged using a Beckman microcentrifuge (BeckmanInstruments, High Wycombe, Buckinghamshire, UK) for 30 s at13,000g. Plasma glucose was assayed using a Beckman Glucose Ana-

lyser II (Beckman Instruments, High Wycombe, Buckinghamshire, UK)[41] and plasma insulin was determined by RIA [39]. All animal studieswere carried out in accordance with the UK Animals (ScientificProcedures) Act 1986.

Statistical analyses

Data are expressed as means ± SEM and the values compared usingthe Student’s unpaired t-test. Where appropriate, data were comparedusing repeated measures ANOVA or one-way ANOVA, followed by theStudent–Newman–Keuls post hoc test. Groups of data were considered tobe significantly different if p < 0.05. Integrated glucose and insulinresponses were calculated by the trapezoidal method, using the algorithmincluded in the software package Prism (version 3.02; GraphPad, SanDiego, CA), with basal levels as base line.

Results

Structural characterisation of GIP and GIP analogues by

ESI–MS

Following solid-phase peptide synthesis and extensiveHPLC purification, the monoisotopic molecular mass ofeach peptide was determined using ESI–MS (Table 1). Pep-tides were purified to >95% purity by repeated HPLC andmolecular masses measured correlated well with predictedtheoretical molecular weights, thereby confirming molecu-lar identity (Table 1).

Peptide stability

As shown in Table 2, native GIP was rapidly hydrolysedby DPP-IV with only 20.9 ± 1.6% remaining intact after2 h incubation. After 8 h, native GIP was completelydegraded with an estimated half-life (t1/2) of 1.3 h. (Ala3)-GIP was significantly less resistant to DPP-IV, with almostcomplete degradation occurring after just 2 h (5.9 ± 1.7%intact; p < 0.05; t1/2 1.1 h). (Phe3)GIP and (Trp3)GIP dis-played similar degradation profiles to the native peptidewith 15.0 ± 5.2 and 22.5 ± 1.7% of peptide remainingintact, respectively, after 2 h incubation with both com-pletely degraded after 8 h (t1/2 1.2 and 1.3 h, respectively).(Tyr3)GIP displayed improved resistance to DPP-IV(34.1 ± 4.1% after 2 h), although total degradation alsooccurred after 8 h (t1/2 1.5 h). (Lys3)GIP was significantlymore resistant to DPP-IV degradation compared to nativeGIP (41.4 ± 0.3% after 2 h; p < 0.01), although was com-pletely degraded after 8 h (t1/2 1.7 h). In contrast, (Pro3)-GIP remained fully intact (100%; p < 0.001) for up to 8 has no degradation fragment was evident (t1/2 > 8 h).

cAMP studies

cAMP stimulation by native GIP and GIP analogues inreceptor-transfected CHL cells is shown in Fig. 1, andreceptor activation statistics shown in Table 3. FromFig. 1, GIP concentration-dependently (10�13 to 10�7 M)stimulated cAMP production with an EC50 value of0.5 nM. In contrast, all of the Glu3-substituted analogues

Table 1Summary of the theoretical and measured molecular masses of GIP and related analogues

Peptide ESI–MS multiple charged species Molecular mass (Da)

(M + 3H)3+ (M + 4H)4+ Measured Theoretical

GIP 1661.6 1246.8 4982.5 4980.5(Ala3)GIP 1642.6 1232.4 4925.2 4924.0(Lys3)GIP 1661.3 1246.5 4981.5 4981.1(Phe3)GIP 1667.6 1251.3 5000.5 5000.0(Trp3)GIP 1680.6 1260.8 5039 5039.0(Tyr3)GIP 1673.0 1255.0 5016.0 5016.0(Pro3)GIP 1238.6 1650.8 4949.9 4948.5

Table 2Percentage intact peptide remaining and estimated half-life of GIP and related analogues after incubation with DPP-IV

Peptide Intact peptide remaining (%) Estimated half-life (h)

0 h 2 h 4 h 8 h

GIP 100 20.9 ± 1.6 4.9 ± 1.2 0 1.3(Ala3)GIP 100 5.9 ± 1.7* 2.7 ± 0.1 0 1.1(Lys3)GIP 100 41.4 ± 0.3** 14.9 ± 1.1* 0 1.7(Phe3)GIP 100 15.0 ± 5.2 2.5 ± 0.1 0 1.2(Trp3)GIP 100 22.5 ± 1.7 4.7 ± 2.4 0 1.3(Tyr3)GIP 100 34.1 ± 4.1 6.3 ± 3.1 0 1.5(Pro3)GIP 100 100*** 100*** 100*** >8

Data represent the percentage of intact peptide remaining (following HPLC separation) relative to the major degradation fragment GIP(3–42) afterincubation with DPP-IV. The reactions were performed in triplicate and the results expressed as means ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001compared to native GIP.

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Fig. 1. Effects of GIP analogues on basal cAMP production in stablytransfected GIP-receptor CHL cells. GIP-receptor CHL cells were exposedfor 20 min to various concentrations (10�13 to 10�7 M) of GIP or GIPanalogues. Each experiment was performed in triplicate and the dataexpressed as means ± SEM. Refer to Table 3 for numerical EC50

parameters and statistical analysis. s, GIP; �, (Ala3)GIP; j, (Lys3)GIP;m, (Phe3)GIP; �, (Trp3)GIP; ., (Tyr3)GIP; $, (Pro3)GIP.


tested displayed weaker cAMP activation responses withincreased EC50 values (1.0–294.5 nM; p < 0.001; Table 3).When incubated in the presence of a stimulatory concen-tration of GIP (10�7 M), (Ala3)GIP, (Trp3)GIP and (Tyr3)-GIP did not significantly inhibit cAMP production (Fig. 2;Table 3). However, (Lys3)GIP, (Phe3)GIP and (Pro3)GIPsignificantly inhibited (p < 0.05 to p < 0.01) GIP-stimulated

cAMP production with maximal inhibitions of 36.5 ± 4,38 ± 3 and 50 ± 4%, respectively (Fig. 2; Table 3).

In vitro insulin secretion

The effects of GIP and GIP analogues on insulin secretionfrom clonal pancreatic BRIN-BD11 cells are shown in Fig. 3.GIP concentration-dependently (10�10 to 10�7 M) stimu-lated insulin secretion (1.2- to 1.6-fold; p < 0.05 top < 0.01) compared with control incubations (5.6 mM glu-cose alone). Of the analogues tested only (Ala3)GIP, at10�7 M, elicited a significantly enhanced (1.7-fold;p < 0.001) insulin response compared with control. (Lys3)-GIP, (Phe3)GIP, (Trp3)GIP, (Tyr3)GIP and (Pro3)GIP dis-played significantly reduced insulinotropic activity(p < 0.05 to p < 0.001) compared with native GIP between10�9 and 10�7 M (Fig. 3; Table 3). When incubated in thepresence of stimulatory GIP (10�7 M), (Ala3)GIP,(Trp3)GIP and (Tyr3)GIP did not significantly inhibit GIP-induced insulin secretion (Fig. 4; Table 3). (Lys3)GIP,(Phe3)GIP and (Pro3)GIP significantly inhibited (p < 0.05to p < 0.01) GIP-stimulated insulin secretion with maximalinhibitions of 22 ± 4, 27 ± 3 and 56 ± 4%, respectively(Fig. 4; Table 3).

In vivo glucose homeostasis

The acute effects of GIP analogues on glucose homeosta-sis in ob/ob mice are shown in Fig. 5. Administration ofglucose alone (18 mmol/kg bw) resulted in a significant rise

Table 3Summary of cAMP and insulin secretion data from data in Figs. 1–4

Peptide cAMP Insulin secretion

EC50 (nm) % Inhibition of GIP-stimulatedcAMP activity

Maximal insulinresponse (% GIP max)

% Inhibition of GIP-stimulatedinsulin secretion

GIP 0.5 100 100 100(Ala3)GIP 2.3 12 ± 3 111 ± 6** �15 ± 3(Lys3)GIP 2.6 36.5 ± 4* 78 ± 3* 22 ± 4*

(Phe3)GIP 1.0 38 ± 3* 75 ± 3** 27 ± 3*

(Trp3)GIP 3.0 22 ± 2 73 ± 2** 0 ± 5(Tyr3)GIP 1.5 24 ± 4 75 ± 4* 7 ± 2(Pro3)GIP 294.0*** 50 ± 4** 60 ± 3*** 56 ± 4***

cAMP production and insulin releasing activity were measured in GIP-R transfected CHL cells and glucose-responsive BRIN-BD11 cells, respectively.Basal cAMP production was 758 ± 83 DPM which was only 5.05 ± 0.55% of the maximal GIP response. Basal insulin secretion was 1.82 ± 0.14 ng/106

cells/20 min which was only 65.14 ± 1.43% of the maximal GIP response. Data represent means ± SEM (n P 3) and *p < 0.05, **p < 0.01, ***p < 0.001compared to native GIP.


in plasma glucose at 15 min (28.2 ± 1.8 mM) which contin-ued to rise during the remaining 45 min, peaking at38.2 ± 1.1 mM. The glucose response to native GIP was sig-nificantly reduced (p < 0.05 to p < 0.01) at 30 and 60 minwith a significantly lower overall glucose excursion(p < 0.01) compared to glucose alone. In contrast, all GIPanalogues displayed significantly higher plasma glucoseconcentrations (p < 0.05 to p < 0.001) when compared withnative GIP. Furthermore, corresponding plasma glucoseAUC values were significantly elevated (1.3- to 1.7-fold;p < 0.05 to p < 0.001) compared to native GIP. AUC valuesfor (Phe3)GIP and (Pro3)GIP were also greater (1.7-fold;p < 0.05) than glucose alone.

Additionally, GIP analogues were administered in com-bination with native GIP to ob/ob mice to test for receptorantagonistic activity (Fig. 6). Administration of glucose anda double dose of GIP (50 nmoles/kg bw) displayed similarglycaemic profiles as depicted in Fig. 5. (Lys3)GIP and(Trp3)GIP did not significantly alter the relative plasma glu-cose profile compared to native GIP (Fig. 6). Conversely,(Ala3)GIP (Phe3)GIP, (Tyr3)GIP and (Pro3)GIP signifi-cantly inhibited the action of GIP and blood glucose levelswere significantly elevated (p < 0.05 to p < 0.001) during thestudy period compared to the GIP only-treated mice. Fur-thermore, plasma glucose AUC values were significantlyraised (1.4-, 1.4-, 1.4- and 1.7-fold, respectively; p < 0.01to p < 0.001) with these analogues compared to the GIPonly-treated group.

In vivo insulinotropic activity

The actions of GIP analogues on insulin-release in ob/ob

mice are shown in Fig. 7. Injection of glucose alone evokeda maximal rise in plasma insulin at 15 min (19.7 ± 1.8 ng/ml),which gradually returned toward basal levels over the fol-lowing 45 min. The overall plasma insulin response in theGIP-treated animals was significantly higher (AUC—1.2-fold; p < 0.05) compared to glucose control. (Ala3)GIPand (Tyr3)GIP did not significantly alter insulin responsecompared to GIP over the 60 min study period. (Lys3)GIP,

(Phe3)GIP, (Trp3)GIP and (Pro3)GIP displayed a signifi-cantly reduced insulinotropic response (35, 43, 40 and56%; p < 0.05 to p < 0.001) compared to GIP.

Fig. 8 displays the insulin-releasing activity of GIP ana-logues when administered in combination with GIP to ob/

ob mice. Administration of a double dose (50 nmoles/kgbw) of GIP resulted in a significant increase in insulin-release (AUC—1.4-fold; p < 0.01) compared with glucosecontrol. (Lys3)GIP and (Trp3)GIP did not significantlyalter GIP-induced insulin release. In contrast, plasmainsulin concentrations of ob/ob mice treated with either(Ala3)GIP, (Phe3)GIP, (Tyr3)GIP and (Pro3)GIP were sig-nificantly decreased (p < 0.05 to p < 0.01) at 15–30 minpost-injection compared with the GIP-treated mice. Fur-thermore, the overall plasma insulin responses, estimatedas AUC, were significantly lower (37, 46, 29 and 51%;p < 0.05 to p < 0.001) compared to GIP.

Discussion

Glucose-dependent insulinotropic polypeptide (GIP) ispresently under renewed interest as a potential therapeuticdrug candidate for the treatment of T2DM [1,2]. One of thekey advantages in exploiting GIP as a therapeutic optionstems from its ability to potentiate glucose-induced insulinsecretion, thereby negating the risk of hypoglycaemic epi-sodes due to excessive insulin-release from the pancreaticb-cells. In addition to its actions on the pancreas, GIP alsoexerts myriad extrapancreatic actions in a range of periph-eral tissues which further augment its ability to lower bloodglucose concentrations [1]. However, the major disadvan-tage in developing GIP as a therapeutic molecule is its rel-atively short biological half-life in the circulation due todegradation by the ubiquitous enzyme, DPP-IV, andadditionally, its rapid elimination from the kidneys [25].

Several studies have reported the metabolic stability andbiological activity of a wide range of GIP analogues mod-ified at positions Tyr1 and Ala2 within the GIP molecule[23–28]. In general, analogues of GIP modified at positionsTyr1 and Ala2 displayed increased metabolic stability to

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Fig. 2. Effects of GIP analogues on GIP-stimulated cAMP production in stably transfected GIP-R CHL cells. GIP-receptor CHL cells were exposed for20 min to various concentrations (10�13 to 10�7 M) of GIP analogues in the presence of 10�7 M GIP. Each experiment was performed in triplicate and thedata expressed as means ± SEM. *p < 0.05, **p < 0.01 compared to native GIP (10�7 M).


DPP-IV and enhanced biological activity compared withnative GIP when tested in several biological systems. Theseproperties translate into significant antihyperglycaemicactions in vivo. Substitution of Glu3 in GIP with a prolineresidue rendered the peptide completely stable to theactions of DPP-IV [34]. Unexpectedly though, this pro-line-substituted analogue, (Pro3)GIP, exhibited potentantagonist actions at the GIP receptor when testedin vitro and in vivo [33,34]. Furthermore, chronic, asopposed to acute, administration of (Pro3)GIP significantlyimproved glucose tolerance and ameliorated insulin resis-tance and abnormalities of islet structure in ob/ob mice[36]. These observations suggest that development of GIPreceptor antagonists may provide a new class of drug offer-ing a safe and exciting therapeutic approach to T2DM[1,36].

In the present study, five Glu3-substituted analogues ofGIP, namely (Ala3)GIP, (Lys3)GIP, (Phe3)GIP,(Trp3)GIP and (Tyr3)GIP, were synthesised and testedfor DPP-IV resistance and biological activity bothin vitro and in vivo. The properties of these analogueswere compared with native GIP and (Pro3)GIP. Substitut-ing the native Glu3 with an Ala, Phe, Trp or Tyr residueresulted in analogues with DPP-IV degradation profilesand half-lives similar to the native peptide, thereby imply-ing that these residues were still capable of fulfilling thestrict substrate specificity required for DPP-IV action. Incontrast, substituting Glu3 with a Lys residue resulted inan analogue that displayed weakly improved enzymestability compared to native GIP. This increased enzymestability is most likely due to the effect of the net positivecharge arising from the side chain of Lys which may cause

0

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4 5.6 mM glucose controlNative GIP(Ala3)GIP

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tion

(ng

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ΔΔ Δ

Δ

ΔΔ ΔΔ Δ Δ ΔΔ

ΔΔΔΔΔΔΔΔ

ΔΔΔ

Fig. 3. Effects of GIP analogues on basal insulin release in clonal pancreatic BRIN-BD11 cells. BRIN-BD11 cells were incubated for 20 min with variousconcentrations (10�13 to 10�7 M) of GIP or GIP analogues. Data are expressed as means ± SEM for 8 separate observations. *p < 0.05, **p < 0.01compared with 5.6 mM glucose control. Dp < 0.05, DDp < 0.01 compared with native GIP at the same concentration.


weak electrostatic interference around the DPP-IV cleav-age site. In contrast to each of the analogues tested,(Pro3)GIP remained completely resistant to the actionsof DPP-IV throughout the entire incubation period,which is entirely consistent with previous studies characte-rising (Pro3)GIP activity [33].

Acute incubations with native GIP showed a concentra-tion-dependent increase in cAMP production from GIP-Rtransfected CHL cells, which corroborates previous resultsusing this cell-line [25]. All of the Glu3-substituted ana-logues tested in this experimental system displayed notablydecreased ability to increase cAMP production comparedto native GIP. However, this is in sharp contrast to (Pro3)-GIP, which had an almost negligible effect, correspondingto approximately 5% of maximal GIP activity at supra-

physiological concentrations. To establish whether thisreduced activity at the GIP-R was indicative of antagonistactivity, analogues were incubated with GIP-R transfectedcells in the presence of a stimulatory concentration ofnative GIP. Concentrations of (Ala3)GIP, (Trp3)GIP and(Tyr3)GIP up to 10�7 m had no effect on GIP-mediatedcAMP production. However, (Lys3)GIP and (Phe3)GIPwere clearly capable of antagonising GIP-stimulatedcAMP production, although these inhibitory actions weremuch less potent than (Pro3)GIP.

Native GIP stimulated insulin secretion from BRIN-BD11 cells in a concentration-dependent manner as notedpreviously [25]. In accordance with the results from cAMPstudies, all of the Glu3-substituted analogues displayed noor significantly reduced insulinotropic activity compared

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10-13 10-12 10-11 10-10 10-9 10-8 10-7

***

****

Peptide Concentration (M)

Insu

lin S

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tion

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)

Fig. 4. Effects of GIP analogues on GIP-stimulated insulin secretion in clonal pancreatic BRIN-BD11 cells. BRIN-BD11 cells were incubated for 20 minwith various concentrations (10�13 to 10�7 M) of GIP analogues in the presence of 10�7 M GIP. Data are expressed as means ± SEM for 8 separateobservations. *p < 0.05, **p < 0.01, ***p < 0.001 compared to native GIP (10�7 M).


with native GIP. (Ala3)GIP was the only analogue whichelicited an insulinotropic response, similar to GIP at thehighest concentration. The reason why (Ala3)GIP shouldinduce such a secretory effect is unclear, especially whenweighed against its actions on cAMP activity in GIP-Rtransfected CHL cells. However, one likely explanationcould be the involvement of additional signal transductionpathways in b-cells [42]. (Ala3)GIP, (Trp3)GIP and (Tyr3)-GIP did not significantly influence GIP-mediated insulinsecretion from BRIN-BD11 cells which concurs withcAMP studies. Similarly, (Lys3)GIP and (Phe3)GIP signif-icantly inhibited GIP-stimulated insulin secretion, andthese inhibitory actions were substantially less potent than(Pro3)GIP.

To determine the acute metabolic actions of GIP ana-logues in vivo, we employed ob/ob mice, a well characterisedanimal model of spontaneous obesity and diabetes [43].Typically, ob/ob mice exhibit hyperphagia, marked obesity,moderate hyperglycaemia, hyperinsulinaemia and severeinsulin resistance [43]. As observed previously [25], admin-istration of native GIP together with glucose elicited a sig-nificantly reduced glycaemic excursion and increasedinsulinotropic response in ob/ob mice. In contrast, all ofthe analogues evaluated in acute tests displayed signifi-cantly elevated plasma glucose concentrations comparedwith native GIP. Interestingly, (Phe3)GIP exhibited asimilar glycaemic profile to (Pro3)GIP with both peptidesactually worsening the glycaemic excursion above that

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Fig. 5. Effects of GIP analogues on glucose tolerance in ob/ob mice. (a) Plasma glucose concentrations after ip administration of glucose alone(18 mmol/kg bw) or in combination with GIP or GIP analogues (25 nmol/kg bw). The time of injection is indicated by the arrow (0 min). (b) Plasmaglucose AUC values for 0–60 min post injection. Data are expressed as means ± SEM for 8 mice. *p < 0.05, **p < 0.01 compared with glucose alone.Dp < 0.05, DDp < 0.01, DDDp < 0.001 compared with native GIP (shown in each panel to facilitate comparison). h, glucose alone; s, GIP; d,(Ala3)GIP; j, (Lys3)GIP; m, (Phe3)GIP; �, (Trp3)GIP; ., (Tyr3)GIP; $, (Pro3)GIP.

0 10 20 30 40 50 60

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Fig. 6. Effects of GIP analogues on antihyperglycaemic actions of native GIP in ob/ob mice. (a) Plasma glucose concentrations after ip administration ofglucose alone (18 mmol/kg bw) or in combination with either native GIP (25 nmol/kg bw) or native GIP (25 nmol/kg bw) plus GIP analogues (25 nmol/kgbw). The time of injection is indicated by the arrow (0 min). (b) Plasma glucose AUC values for 0–60 min post injection. Data are expressed asmeans ± SEM for 8 mice. *p < 0.05, **p 0.01, ***p < 0.001 compared with glucose alone. Dp < 0.05, DDp < 0.01, DDDp < 0.001 compared with nativeGIP(shown in each panel to facilitate comparison). h, glucose alone; s, GIP; d, (Ala3)GIP; j, (Lys3)GIP; m, (Phe3)GIP; �, (Trp3)GIP; ., (Tyr3)GIP; $,(Pro3)GIP.


observed with administration of glucose alone. This wors-ening in glycaemic control was consistently reflected in adecrease in insulin-releasing activity compared with native

GIP. While (Ala3)GIP and (Tyr3)GIP failed to increaseinsulin concentrations above that induced by glucose,(Lys3)GIP, (Phe3)GIP and (Trp3)GIP actually reduced

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Fig. 7. Effects of GIP analogues on plasma insulin response to glucose in ob/ob mice. (a) Plasma insulin concentrations after ip administration of glucosealone (18 mmol/kg bw) or in combination with GIP or GIP analogues (25 nmol/kg bw). The time of injection is indicated by the arrow (0 min). (b) Plasmainsulin AUC values for 0–60 min post injection. Data are expressed as means ± SEM for 8 mice. *p < 0.05 compared with glucose alone. Dp < 0.05,DDp < 0.01, DDDp < 0.001 compared with native GIP (shown in each panel to facilitate comparison). h, glucose alone; s, GIP; d, (Ala3)GIP;j, (Lys3)GIP; m, (Phe3)GIP; �, (Trp3)GIP; ., (Tyr3)GIP; $, (Pro3)GIP.

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Fig. 8. Effects of GIP analogues on insulin-releasing action of native GIP in ob/ob mice. (a) Plasma insulin concentrations after ip administration ofglucose alone (18 mmol/kg bw) or in combination with either native GIP (25 nmol/kg bw) or native GIP (25 nmol/kg bw) plus GIP analogues (25 nmol/kgbw). The time of injection is indicated by the arrow (0 min). (b) Plasma insulin AUC values for 0–60 min post injection. Data are expressed asmeans ± SEM for 8 mice. **p < 0.01 compared with glucose alone. Dp < 0.05, DDp < 0.01, DDDp < 0.001 compared with native GIP (shown in each panel tofacilitate comparison). h, glucose alone; s, GIP; d, (Ala3)GIP; j, (Lys3)GIP; m, (Phe3)GIP; �, (Trp3)GIP; ., (Tyr3)GIP; $, (Pro3)GIP.



plasma insulin concentrations significantly in ob/ob micecompared with the native peptide. However, none of theanalogues tested were as potent in reducing plasma insulinconcentrations as (Pro3)GIP.

Given the actions of GIP analogues administered as asingle dose to ob/ob mice, studies were performed to assesstheir ability to act in vivo as antagonists of GIP-inducedinsulinotropic and antihyperglycaemic actions. (Ala3)GIP,(Phe3)GIP, (Tyr3)GIP and (Pro3)GIP all counteracted theglucose-lowering actions of native GIP, being associatedwith correspondingly decreased insulin responses. Theacute inhibitory actions were most pronounced with (Pro3)-GIP, which also inhibited the insulinotropic response toglucose alone. This together with worsening of glucosetolerance by both (Pro3)GIP and (Phe3)GIP indicates apermissive action of raised endogenous GIP in ob/ob miceon insulin secretion and glucose disposal, including effectsat extrapancreatic sites [44–46].

In contrast to other analogues, (Lys3)GIP and(Trp3)GIP did not modify the antihyperglycaemic actionsof native GIP. This effect of (Trp3)GIP is entirely consis-tent with its profile of actions. However, the lack of GIPantagonism by (Lys3)GIP is surprising given its marginallyenhanced resistance to DPP-IV degradation and ability toinhibit GIP-mediated cAMP production and insulin secre-tion in vitro. Conversely, (Ala3)GIP and (Tyr3)GIP failedto inhibit these acute cellular actions of GIP but provedto be highly effective antagonists of GIP action in ob/ob

mice. A better understanding of the full range of biologicaleffects of GIP at pancreatic and extrapancreatic sites mighthelp explain such observations.

In summary, we have reported the characteristics of arange of Glu3-substituted analogues of GIP. The analoguesdesigned all showed poor resistance to DPP-IV degrada-tion with only (Lys3)GIP showing moderately increasedstability. Of the analogues tested, (Ala3)GIP, (Phe3)GIPand (Tyr3)GIP demonstrated significant GIP-R antagonistactivity in ob/ob mice. Their inhibitory actions though weremuch less pronounced than the already well-establishedGIP-R antagonist, (Pro3)GIP, which has the additionalbenefit of being completely resistant to the actions ofDPP-IV. By exploring further strategies to increase enzymestability and duration of action, for example, through fattyacid derivatisation, such analogues may be developed intomore powerful GIP-R antagonists. As illustrated elsewhereGIP-R antagonists may have potential for developmentinto a new class of drugs for the long-term treatment ofT2DM.

Acknowledgments

These studies were supported by University of UlsterStrategic Research Funding. The authors thank ProfessorBernard Thorens (University of Lausanne, Switzerland)for kindly providing CHL cells transfected with the GIPreceptor and Professor Cliff Bailey (Aston University,UK) for ob/ob mice.

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