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Am J Physiol Endocrinol Metab 292: E1674-E1682, 2007. First published February 13, 2007; doi:10.1152/ajpendo.00391.2006
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Antagonistic effects of two novel GIP analogs, (Hyp3)GIP and (Hyp3)GIPLys16PAL, on the biological actions of GIP and longer-term effects in diabetic ob/ob mice

Finbarr P. M. O'Harte,1 Kerry Hunter,1 Victor A. Gault,1 Nigel Irwin,1 Brian D. Green,2 Brett Greer,2 Patrick Harriott,2 Clifford J. Bailey,3 and Peter R. Flatt1

1School of Biomedical Sciences, University of Ulster, Coleraine; 2School of Biology and Biochemistry, The Queen's University of Belfast, Belfast, Northern Ireland; and 3School of Life and Health Sciences, Aston University, Birmingham, United Kingdom

Submitted 3 August 2006 ; accepted in final form 6 February 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
This study examines the actions of the novel enzyme-resistant, NH2-terminally modified GIP analog (Hyp3)GIP and its fatty acid-derivatized analog (Hyp3)GIPLys16PAL. Acute effects are compared with the established GIP receptor antagonist (Pro3)GIP. All three peptides exhibited DPP IV resistance, and significantly inhibited GIP stimulated cAMP formation and insulin secretion in GIP receptor-transfected fibroblasts and in clonal pancreatic BRIN-BD11 cells, respectively. Likewise, in obese diabetic ob/ob mice, intraperitoneal administration of GIP analogs significantly inhibited the acute antihyperglycemic and insulin-releasing effects of native GIP. Administration of once daily injections of (Hyp3)GIP or (Hyp3)GIPLys16PAL for 14 days resulted in significantly lower plasma glucose levels (P < 0.05) after (Hyp3)GIP on days 12 and 14 and enhanced glucose tolerance (P < 0.05) and insulin sensitivity (P < 0.05 to P < 0.001) in both groups by day 14. Both (Hyp3)GIP and (Hyp3)GIPLys16PAL treatment also reduced pancreatic insulin (P < 0.05 to P < 0.01) without affecting islet number. These data indicate that (Hyp3)GIP and (Hyp3)GIPLys16PAL function as GIP receptor antagonists with potential for ameliorating obesity-related diabetes. Acylation of (Hyp3)GIP to extend bioactivity does not appear to be of any additional benefit.

glucose-dependent insulinotropic polypeptide receptor antagonists; insulin secretion; antihyperglycemic activity; dipeptidyl peptidase IV; fatty acid conjugation

GLUCOSE-DEPENDENT INSULINOTROPIC POLYPEPTIDE (GIP), along with its counterpart glucagon-like peptide-1 (GLP-1), is one of the major mediators of postprandial glucose-induced insulin secretion (4). As such, GIP is implicated as one of the factors responsible for hyperinsulinemia that occurs in obesity and type 2 diabetes, possibly as a result of an overactive enteroinsular axis (5). Elevated plasma GIP concentrations are reported in both obese diabetic ob/ob mice (14) and in subjects with type 2 diabetes (5, 35).

GIP is released postprandially following nutrient ingestion, especially fat, which makes it a potentially important anabolic agent in obesity etiology either directly by promoting hyperinsulinemia or indirectly through effects on lipoprotein lipase (LPL) activity. LPL plays a key role in the hydrolysis of circulating triacylglycerol, liberating nonesterified fatty acids for uptake and storage within the adipocyte (40). GIP stimulates the synthesis and secretion of LPL in rat adipose tissue (10, 23). Therefore, high circulating concentrations of GIP accompanied by high-fat energy feeding facilitate the uptake of triacylglycerol, all of which may contribute to increased adiposity (9, 10, 23).

A likely link exists between GIP and obesity, which is further supported by studies that demonstrate that GIP enhanced insulin-stimulated glucose transport through increased adipocyte sensitivity (36), increased fatty acid synthesis in adipose tissue explants (1, 30), and reduced glucagon-stimulated lipolysis (8).

Recent studies using GIP receptor (GIP-R) knockout mice demonstrated a phenotype associated with intolerance and diminished insulin secretion (29). Furthermore, knockout of the GIP-R conferred resistance to diet-induced obesity and protection from insulin resistance (28). Cross-breeding with obese diabetic ob/ob mice resulted in decreased body weight gain and significant amelioration of adiposity and insulin resistance despite hyperphagia comparable with control ob/ob mice (28).

These studies suggest that disruption of the GIP signaling pathway by use of GIP-R antagonists could theoretically prevent obesity and insulin resistance, making it a target worth investigating for treatment of type 2 diabetes. NH2-terminally truncated GIP fragment peptides such as GIP-(3–42) have been shown to act as antagonists in vivo (17), although this has not been found to be the case in vivo in a recent report (7). However, of most utility thus far is (Pro3)GIP, which is a specific GIP-R antagonist capable of abolishing GIP's actions both in vitro and in vivo (18, 22). Acute administration of (Pro3)GIP with an intraperitoneal injection of glucose exacerbates glycemic and insulinotropic excursions thought to be due to both inhibition of the insulinotropic actions of high levels of endogenous GIP (14, 15) and blocking of other extrapancreatic glucose-lowering actions (31).

Long-term administration of (Pro3)GIP to ob/ob mice resulted in reductions of basal glucose and insulin concentrations and improvement of glucose and meal tolerance together with a change toward normalization of islet morphology and beta-cell function (20). These actions are underlined by significant improvement by (Pro3)GIP of insulin resistance that is otherwise particularly severe in this animal model of type 2 diabetes (2).

The present study has investigated the acute and long-term effects of another novel Glu3-substituted GIP-related peptide analog, (Hyp3)GIP. Furthermore, a daughter analog modified to contain a [C16]palmitate fatty acid at the position Lys16 residue was produced with the aim of providing a longer-acting molecule due to promotion of albumin binding in vivo. Evaluation of the in vitro properties of these novel GIP analogs was carried out alongside parallel studies using the established antagonist (Pro3)GIP (18). Finally, the effects of 14 days of treatment with daily (Hyp3)GIP and (Hyp3)GIPLys16PAL were examined in obese diabetic ob/ob mice to establish their antidiabetic potential.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Reagents. HPLC grade acetonitrile was obtained from Rathburn (Walkersburn, Scotland). Sequencing grade trifluoroacetic acid (TFA), dipeptidyl peptidase IV (DPP IV), 3-isobutyl-1-methylxanthine (IBMX), adenosine 3,5,-cyclic monophosphate (cAMP), adenosine 5'-triphosphate (ATP), and {alpha}-cyano-4-hydroxycinnamic acid were all purchased from Sigma (Poole, Dorset, UK). {alpha}-Fluorenylmethyloxycarbonyl (Fmoc)-protected amino acids and peptide synthesis reagents were obtained from Calbiochem Novabiochem (Beeston, Nottingham, UK). RPMI 1640 and DMEM tissue culture medium, fetal bovine serum, penicillin, and streptomycin were all purchased from GIBCO (Paisley, Strathclyde, Scotland). The chromatography columns used for cAMP assay, Dowex AG 50WX and neutral alumina AG7, were obtained from Bio-Rad (Life Science Research, Alpha Analytical, Larne, Northern Ireland). Tritiated adenine (TRK311) was obtained from Amersham Pharmacia Biotech (Amersham, Bucks, UK). All water used in these experiments was purified using a Milli-Q water purification system (Millipore, Milford, MA). All other chemicals used were of the highest available purity.

Synthesis, purification, and characterization of GIP peptides. Native GIP was sequentially synthesized, as described previously (32), on an Applied Biosystems automated peptide synthesizer (Model 432A; Synergy, Foster City, CA) with a preloaded Fmoc-Gln(Trt)-Wang resin using standard solid-phase Fmoc peptide chemistry (12). (Pro3)GIP, (Hyp3)GIP, and (Hyp3)GIPLys16PAL were sequentially synthesized in the same way but with the exception that the glutamic acid residue at position 3 was substituted with either proline or hydroxyproline (Hyp), and in addition, the lysine residue at position 16 was conjugated to an Fmoc-protected [C16]palmitate fatty acid in the synthesis of (Hyp3)LysPAL16GIP. Purity was determined by reversed-phase HPLC on a Waters Millennium 2010 chromatography system (Software version 2.1.5) and subsequently characterized using electrospray ionisation mass spectrometry as described previously (18). The molecular masses of GIP, (Pro3)GIP, (Hyp3)GIP, and (Hyp3)GIPLys16PAL were very close to the theoretical masses, indicating successful peptide synthesis (Table 1).


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  		Table 1. Molecular identity and susceptibility of GIP and related analogues to DPP IV degradation

 
Degradation of GIP peptides by DPP IV. GIP and related peptides (15 µg) were incubated (n = 3) at 37°C with DPP IV (5 mU) in 50 mM triethanolamine-HCl (500 µl), pH 7.8 (final peptide concentration 2 µM). Enzymatic reactions were terminated by the addition of 10 µl of 10% (vol/vol) TFA/H2O and stored at –20°C prior to HPLC analysis. The reaction products were applied to a Vydac C-18 [(Pro3)GIP and (Hyp3)GIP] or Vydac C-4 column [(Hyp3)LysPAL16GIP, 4.6 x 250 mm] equilibrated with 0.12% TFA/H2O at a flow rate of 1.0 ml/min. Using 0.1% TFA in 70% acetonitrile-water, the concentration of acetonitrile in the eluting solvent was raised from 0 to 28% over 10 min and from 28 to 38.5% over 20 min for the GIP, (Pro3)GIP, and (Hyp3)GIP incubations and from 0 to 28% over 10 min and 28 to 56% over 20 min for the (Hyp3)GIPLys16PAL incubations. The absorbance was monitored at 214 nm on a SpectraSystem UV2000 detector (Thermoquest Limited, Manchester, UK).

Cell culture. Chinese hamster lung fibroblasts (CHL cells, ATCC CCL39) stably transfected with the human GIP-R (25) were cultured in DMEM tissue culture medium containing, 10% (vol/vol) fetal bovine serum (FBS), 1% (vol/vol) antibiotic (100 U/ml penicillin, 0.1 mg/ml streptomycin), and 1.1 mM glucose. BRIN-BD11 cells were cultured in RPMI 1640 tissue culture medium containing 10% (vol/vol) FBS, 1% (vol/vol) antibiotic (100 U/ml penicillin, 0.1 mg/ml streptomycin), and 11.1 mM glucose as described previously (27). Cells were cultured in sterile vented tissue culture flasks (Corning Glass Works, Corning, NY) at 37°C in an atmosphere of 5% CO2 and 95% air using a LEEC incubator (Laboratory Technical Engineering, Nottingham, UK).

cAMP production. CHL cells were harvested and seeded into 24-well plates (Nunc, Roskilde, Denmark) at a density of 3.0 x 105 cells per well loaded with TRK311 (2 µCi, TRK311; Amersham, Buckinghamshire, UK) and allowed to grow for 18 h. The culture medium was removed and cells subsequently washed twice with 2 ml of ice-cold Hanks’ buffered saline (HBS) buffer (130 mM NaCl, 20 mM HEPES, 0.9 mM NaHPO4, 0.8 mM MgSO4, 5.4 mM KCl, 1.8 mM CaCl2, 5.6 mM glucose, and 25 µmol/l phenol red, pH 7.4). The cells were then exposed to varying concentrations of GIP peptides(10–13 to 10–7 M) in HBS buffer supplemented with 1 mM IBMX for 20 min at 37°C. Similarly for the antagonist studies, varying concentrations (10–13 to 10–7 M) of GIP peptides were added in the presence of GIP (10–7 M). The medium was subsequently removed and the cells lysed with 1 ml of lysing solution (5% TCA, 3% SDS, and 92% H2O, also containing 0.1 mM unlabeled cAMP and 0.1 mM unlabeled ATP). The intracellular tritiated cAMP was then separated on Dowex and alumina exchange resins, as described previously (39).

Insulin secretion. BRIN-BD11 cells were harvested and seeded into 24-well plates (Nunc) at a density of 1.5 x 105 cells/well and allowed to attach overnight in culture medium at 37°C. Culture medium was then removed, and 1.0 ml Krebs-Ringer bicarbonate buffer (KRB, 115 mM NaCl, 4.7 mM KCl, 1.28 mM CaCl2, 1.2 mM MgSO4, 1.2 mM KH2PO4, 25 mM HEPES, and 10 mM NaHCO3, pH 7.4 with NaOH) supplemented with 0.1% (wt/vol) BSA and 1.1 mM glucose was added to each well. The cells were then incubated for 40 min at 37°C, after which the buffer was removed and replaced with fresh KRB (1.0 ml) supplemented with 5.6 mM glucose and a range of concentrations (10–13 to 10–7 M) of GIP or GIP analogs. Likewise, varying concentrations of GIP peptides (10–13 to 10–7 M) were added with GIP (10–7 M) for the antagonist studies. After a 20-min incubation at 37°C, the buffer was removed from each well (900 µl) and (200 µl) aliquots used for measurement of insulin by RIA (13).

Acute in vivo biological activity. Following an 18-h fast, plasma glucose and insulin responses were evaluated using 14- to 18-wk-old obese diabetic ob/ob mice (2). Responses were measured immediately prior to and following administration by intraperitoneal injection (n = 8) of glucose alone (18 mmol/kg body wt) as control or glucose together with GIP or GIP peptides (25 nmol/kg body wt). These tests were performed to evaluate whether either of the two Hyp-substituted analogs had any GIP agonist activity. In addition, to assess the antagonistic properties of these peptides, a double dose of GIP (50 nmol/kg body wt) or GIP (25 nmol/kg body wt) in combination with each GIP analog peptide (25 nmol/kg body wt) was given by intraperitoneal injection. Fasted mice were used to maximize glycemic and insulin responses to enable evaluation of the effects of GIP peptides. All animal studies were carried out in accordance with the UK Animals (Scientific Procedures) Act 1986. Protocol approval was granted under appropriate project licences by HPSS in Northern Ireland.

Longer-term in vivo studies. Obese diabetic ob/ob mice aged 14- to 18-wk (22) were randomly divided into groups that received once daily intraperitoneal injections (1700) of saline vehicle, (Hyp3)GIP, or (Hyp3)GIPLys16PAL (25 nmol/kg body wt) over 14 days. Food intake and body weights were recorded daily. Blood samples were taken at 0900 at 3- to 4-day intervals for plasma glucose and insulin analysis. A number of metabolic tests were carried out after 14 days treatment and again 14 days after cessation of treatment. These included evaluation of the effects in nonfasted mice of intraperitoneal glucose (18 mmol/kg body wt), glucose together with GIP (25 nmol/kg body wt), or insulin (50 U/kg body wt). Mice fasted for 18 h were used to evaluate responses to 15-min refeeding. In one experimental series, the pancreases of mice treated for 14 days were resected for determination of insulin content and islet morphometry, as described previously (20).

In both acute and longer-term studies, test solutions were administered in a final volume of 5 ml/kg body wt. Blood samples were collected from the cut tip of the tail vein of conscious mice into chilled fluoride/heparin coated glucose microcentrifuge tubes (Sarstedt, Nümbrecht, Germany) at the times indicated in the figures. Blood samples were immediately centrifuged using a Beckman microcentrifuge (Beckman Instruments, High Wycombe, Buckinghamshire, UK) for 30 s at 13,000 g. The resulting plasma was then aliquoted into fresh Eppendorf tubes and stored at –20°C prior to glucose determination using a Beckman Glucose Analyzer II (37) and insulin determination by RIA (13).

Statistical analysis. Results are expressed as means ± SE. Data were compared using Student's t-test or ANOVA, followed by the Student-Newman-Keuls post hoc test, as appropriate. Groups of data were considered to be significantly different if P < 0.05.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Degradation by DPP IV. As shown in Table 1, native GIP was rapidly degraded by DPP IV to the truncated metabolite GIP-(3–42), with an estimated in vitro half-life of 1.3 h. In contrast, (Pro3)GIP, (Hyp3)GIP, and (Hyp3)GIPLys16PAL remained fully intact following 8-h incubation with DPP IV (Table 1).

cAMP production in vitro. GIP analogs tested at 10–7 M concentrations were considerably less potent than native GIP in stimulating cAMP production in GIP-R-transfected CHL fibroblasts (Table 2). The cAMP production at 10–12 M GIP was 758 ± 83 dpm, which was only 5.05 ± 0.55% of the maximal GIP response at 10–7 M GIP. EC50 values for native GIP, (Pro3)GIP, (Hyp3)GIP, and (Hyp3)GIPLys16PAL were 0.47 nM, 2.07 nM, 13.4 nM, and 1.45 nM, respectively. Indeed, in the presence of 10–7 M GIP, (Pro3)GIP, (Hyp3)GIP, and (Hyp3)GIPLys16PAL significantly inhibited cAMP formation 34–52% (P < 0.01 to P < 0.001).


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  		Table 2. cAMP production and insulin secretory effects of GIP and related analogues

 
Insulin secretion in vitro. Table 2 shows that GIP analogs exhibited weak insulin-releasing activity compared with native GIP. (Pro3)GIP, (Hyp3)GIP, and (Hyp3)GIPLys16PAL inhibited maximal GIP-induced insulin secretion by 29–38% (P < 0.05 to P < 0.001). There were no significant differences between any of the in vitro properties of the three GIP analogs (Table 2). The basal insulin secretion as measured in an acute test was (means ± SE, n = 8) 1.82 ± 0.14 ng·106 cell–1·20 min–1 at 5.6 mM glucose alone (control). This was 66 ± 2% of the maximum insulin response observed with 10–7 M native GIP.

Acute metabolic effects in ob/ob mice. Figure 1A shows the plasma glucose responses to intraperitoneal glucose alone or in combination with GIP or its analogs. Injection of GIP reduced the glycemic excursion at 30 and 60 min and significantly lowered (P < 0.001) area under the curve (AUC) values (Fig. 1B). In contrast, glucose concentrations and AUC measurements were significantly greater following (Pro3)GIP, (Hyp3)GIP, or (Hyp3)GIPLys16PAL (P < 0.01 to P < 0.001). The effects were significantly greater for (Pro3)GIP (P < 0.01 to P < 0.001) than other GIP analogs, indicating that (Pro3)GIP was the most potent GIP-R antagonist under the conditions tested. The corresponding insulin release data are shown in Fig. 1C. The overall insulin responses to (Pro3)GIP, (Hyp3)GIP, or (Hyp3)GIPLys16PAL were significantly less (P < 0.05 to P < 0.01) than to GIP (Fig. 1D). Native GIP caused significantly greater insulin release than glucose alone (P < 0.05).


Figure 1
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  		Fig. 1. Effects of glucose-dependent insulinotropic polypeptide (GIP) peptides on glucose and insulin in 18-h-fasted ob/ob mice. Plasma glucose (A) and insulin (C) were measured prior to and after ip administration of glucose alone (18 mmol/kg body wt) or in combination with GIP, (Pro3)GIP, (Hyp3)GIP, and (Hyp3)GIPLys16PAL (25 nmol/kg). The time of injection is indicated by the arrow (0 min). Plasma glucose (B) or insulin (D) area under the curve (AUC) values for 0–60 min postinjection are shown. Values represent means ± SE for 8 mice. *P < 0.05; **P < 0.01; ***P < 0.001 compared with glucose alone; {Delta}P < 0.05; {Delta}{Delta}P < 0.01; {Delta}{Delta}{Delta}P < 0.001 compared with native GIP; ++P < 0.01; +++P < 0.001 compared with (Pro3)GIP.

 
The effects of (Pro3)GIP, (Hyp3)GIP, and (Hyp3)GIPLys16PAL on the glucose-lowering and insulinotropic actions of GIP administered together with glucose are shown in Fig. 2, A–D. Glucose levels were significantly higher (P < 0.05 to P < 0.001) at 15, 30, and 60 min after injection compared with native GIP alone. AUC values (Fig. 2B) showed that the overall glucose responses were 1.8-fold greater (P < 0.001) than for native GIP. Insulinotropic actions of GIP were diminished 1.2- to 1.8-fold (P < 0.05 to P < 0.001) by coadministration of each of the three GIP analogs over the 0- to 60-min study period (Fig. 2D). The effects of (Hyp3)GIP, (Hyp3)GIPLys16PAL, and (Pro3)GIP on the antihyperglycemic and insulin-releasing effects of native GIP were not significantly different.


Figure 2
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  		Fig. 2. Antagonistic effects of GIP peptides on the glucose-lowering and insulinotropic effects of GIP in 18-h-fasted ob/ob mice. Plasma glucose (A) and insulin (C) were measured prior to and after ip administration of glucose (18 mmol/kg body wt) and GIP (25 nmol/kg), alone or in combination with GIP, (Pro3)GIP, (Hyp3)GIP, and (Hyp3)GIPLys16PAL (25 nmol/kg). The time of injection is indicated by the arrow (0 min). Plasma glucose (B) and insulin (D) AUC values for 0–60 min postinjection are shown. Values represent means ± SE for 8 mice. *P < 0.05; ***P < 0.001 compared with glucose alone; {Delta}P < 0.05; {Delta}{Delta}P < 0.01; {Delta}{Delta}{Delta}P < 0.001 compared with native GIP.

 
Longer-term effects on body weight, food intake, and nonfasting glucose and insulin in ob/ob mice. Body weight and food intake of ob/ob mice were not changed by daily injection of (Hyp3)GIP or (Hyp3)GIPLys16PAL (25 nmol/kg body wt) for 14 days (Fig. 3, A and B). Plasma glucose was significantly reduced (P < 0.05) following (Hyp3)GIP, but not (Hyp3)GIPLys16PAL, on day 12 (Fig. 4A). As shown in Fig. 4B, insulin concentrations were unaffected by treatment with the GIP analogs.


Figure 3
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  		Fig. 3. Body weights (A) and food intakes (B) in ob/ob mice. Mice received daily injections of saline alone, or GIP peptides (25 nmol/kg body wt), for the 14-day period indicated by the gray bar. Values are mean ± SE for 8 mice.

 

Figure 4
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  		Fig. 4. Effects of daily injection of (Hyp3)GIP and (Hyp3)GIPLys16PAL on plasma glucose (A) and insulin (B) in ob/ob mice. Values are means ± SE for 8 mice. *P < 0.05 compared with saline group.

 
Longer-term effects on glucose and insulin responses to intraperitoneal injection of glucose in ob/ob mice. Figure 5 shows the effect of 14-day treatment with (Hyp3)GIP or (Hyp3)GIPLys16PAL on glucose tolerance and insulin responses. Glucose concentrations were significantly lower (P < 0.01) for both peptide-treated groups compared with saline-treated controls at both basal (t = 0) as well as 15-min postinjection. The glucose concentration for the (Hyp3)GIP-treated group remained significantly reduced at 60-min postinjection (P < 0.05). Plasma insulin was significantly reduced (P < 0.01) at 15-min postinjection in the (Hyp3)GIP-treated group (Fig. 5B). However, plasma insulin was unaffected in the (Hyp3)GIPLys16PAL-treated group (Fig. 5B).


Figure 5
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  		Fig. 5. Effects of daily injection of (Hyp3)GIP and (Hyp3)GIPLys16PAL on glucose tolerance and insulin response to glucose in nonfasted ob/ob mice. Mice received daily injections of saline alone or GIP peptides (25 nmol/kg body wt) for 14 days prior to ip administration of glucose (18 mmol/kg body wt) and subsequent measurement of glucose (A) and insulin (B). The time of injection is indicated by the arrow. Values are means ± SE for 8 mice. *P < 0.05; **P < 0.01 compared with saline control.

 
Longer-term effects on glucose and insulin responses to feeding and insulin sensitivity in ob/ob mice. No significant differences were observed in the glycemic or insulin responses to refeeding in ob/ob mice treated for 14 days with either saline, (Hyp3)GIP, or (Hyp3)GIPLys16PAL (Fig. 6, A and B). As shown in Fig. 7, glucose-lowering effects of insulin at 30 and 60 min postinjection were also enhanced in the (Hyp3)GIP- and (Hyp3)GIPLys16PAL-treated groups (P < 0.05 to P < 0.001) compared with saline controls.


Figure 6
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  		Fig. 6. Effects of daily injection of (Hyp3)GIP and (Hyp3)GIPLys16PAL on metabolic response to feeding in ob/ob mice. Mice received daily injections of saline alone or GIP peptides (25 nmol/kg body wt) for 14 days prior to an overnight fast (18 h) and subsequent 15-min refeeding as indicated by the black bars. Plasma glucose (A) and insulin (B) are shown. Values are means ± SE for 8 mice.

 

Figure 7
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  		Fig. 7. Effects of daily injection of (Hyp3)GIP and (Hyp3)GIPLys16PAL on insulin sensitivity in nonfasted ob/ob mice. Mice received daily injections of saline alone or GIP peptides (25 nmol/kg body wt) for 14 days prior to ip injection of insulin (50 U/kg body wt). The time of injection is indicated by the arrow. Values are means ± SE for 8 mice. *P < 0.05; ***P < 0.001 compared with saline control.

 
Longer-term effects on glucose and insulin responses to intraperitoneal injection of GIP in ob/ob mice. Glucose levels were significantly lower (P < 0.05 to P < 0.01) at basal and 15 min postinjection with GIP for both peptide-treated groups compared with saline controls (Fig. 8A). However, at 60 min, glucose was significantly decreased (P < 0.01) in (Hyp3)GIP-treated mice compared with the (Hyp3)GIPLys16PAL-treated group. There were no significant differences in the corresponding insulin responses following a GIP challenge, as shown in Fig. 8B. Thus this indicates that the action of the fatty acid-linked GIP-R antagonist (Hyp3)GIPLys16PAL may be acting through mechanisms other than blocking the direct insulinotropic actions to induce its potent hyperglycemic effects (Fig. 6B).


Figure 8
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  		Fig. 8. Effects of daily injection of (Hyp3)GIP and (Hyp3)GIPLys16PAL on glucose-lowering and insulinotropic actions of GIP in ob/ob mice. Mice received daily injections of saline alone or GIP peptides (25 nmol/kg) for 14 days prior to ip administration of GIP (25 nmol/kg body wt) together with glucose (18 mmol/kg body wt). The time of injection is indicated by the arrow. Plasma glucose (A) and insulin (B) are shown. Values are means ± SE for 8 mice. *P < 0.05; **P < 0.01; ***P < 0.001 compared with saline control; {Delta}{Delta}P < 0.01 compared with (Hyp3)GIP.

 
Effects of longer-term treatment with GIP analogs on pancreatic tissue. (Hyp3)GIP- and (Hyp3)GIPLys16PAL-treated groups exhibited significantly lower pancreatic insulin content (P < 0.05 to P < 0.01) compared with saline-treated control mice (Table 3). There was no significant difference in the average number of islets per section.


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  		Table 3. Pancreatic insulin content and islet number in ob/ob mice after 14 once daily injections of saline, (Pro3)LysPAL16GIP, or (Hyp3)GIPLys16PAL

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
(Pro3)GIP has been developed as a powerful and effective specific GIP-R antagonist capable of completely abolishing the glucose-lowering and insulinotropic actions of GIP (18). Daily administration of (Pro3)GIP for 11 days has been shown to ameliorate features of the ob/ob syndrome in mice, suggesting a therapeutic utility of GIP-R antagonists in obesity and/or diabetes (20). This effect is pathology related, since chemical or molecular knockout of the GIP-R has little effect on glucose homeostasis in normal mice (22, 28).

The present study has investigated the actions of another Glu3-substituted analog of GIP, (Hyp3)GIP, as a potentially new GIP-R antagonist. Hyp was selected because of its structural similarity to proline and the fact that it is unlikely to form a peptide bond that was susceptible to the action of DPP IV (33). In addition to DPP IV-mediated degradation, recent reports (6) highlight the importance of renal filtration in elimination of GIP from the circulation. Structural modification of GIP to delay removal by the kidneys may therefore lead to a longer half-life. Thus we synthesized a daughter analog with palmitate conjugated to the {epsilon}-amino group of Lys16 of (Hyp3)GIP. This was envisaged to facilitate binding to plasma proteins and thereby prevent clearance by the kidney. Already, such an approach has been used successfully with insulin (16, 25) and other peptide hormones, including GLP-1 (16, 24).

As suspected, substitution of the third NH2-terminal amino acid of GIP with Hyp resulted in a stable enzyme-resistant analog that was unaffected by 8-h incubation with DPP IV. (Hyp3)GIPLys16PAL and, consistent with previous findings (18), (Pro3)GIP also exhibited complete DPP IV resistance. All three GIP analogs only very weakly stimulated cAMP production and insulin secretion. However, in the presence of cAMP and a stimulatory concentration of native GIP, (Hyp3)GIP and (Hyp3)GIPLys16PAL retained some antagonistic activity in vitro. This is consistent with their actions as GIP-R antagonists, as also observed and previously described for (Pro3)GIP (18).

Consistent with this view, (Hyp3)GIP and its acylated analog (Hyp3)GIPLys16PAL and (Pro3)GIP increased basal glucose and decreased insulin levels in ob/ob mice when administered acutely. Coadministration of these peptides with native GIP significantly countered both the antihyperglycemic and insulin-releasing effects of GIP. Although the three GIP analogs appeared to have similar potency in the in vitro tests, (Pro3)GIP appeared to have more pronounced effects when administered in vivo to ob/ob mice.

To further evaluate the possible utility of (Hyp3)GIP and beneficial effects of acylation, longer-term studies were performed in which ob/ob mice received once daily injections of either saline, (Hyp3)GIP, or (Hyp3)LysPAL16GIP for 14 days. Food intakes or body weights were not significantly changed compared with saline-treated controls. This corresponds with previous observations made in ob/ob mice treated with (Pro3)GIP (20, 22). More importantly, (Hyp3)GIP but not the acylated analog induced a significant reduction in plasma glucose on days 12 and 14 of this study compared with saline-treated controls. Plasma insulin levels were not appreciably affected by either peptide, although there was a tendency toward lower levels. Such results indicate that peptide half-life was significantly enhanced or that the dose was high enough to overcome a short half-life. Presently available GIP antibodies react poorly with these analogs, and necessary pharmacokinetic studies to discriminate between these two possibilities await generation of new assay methods. These results also are reminiscent of the effects of (Pro3)GIP (20) and have parallel effects with Roux-en-Y gastric bypass in morbidly obese type 2 diabetic patients (34). These individuals suffer loss of functional GIP secretory cells and demonstrate significantly decreased fasting plasma glucose within 2 wk. In contrast, insulin approached but never reached significantly reduced levels after 12 wk (3).

More convincing evidence for impact on blood glucose control comes from results of glucose tolerance tests conducted in ob/ob mice after 14 days treatment. This revealed significantly lower glucose concentrations in both (Hyp3)GIP and (Hyp3)GIPLys16PAL-treated groups. Plasma insulin levels in the (Hyp3)GIPLys16PAL group were significantly lower during the test, suggesting an increased effectiveness of insulin. This was indeed confirmed by increased hypoglycemic response to exogenous insulin in both of the groups treated with GIP analogs. These observations, together with previously published effects of (Pro3)GIP (20), indicate that GIP-R antagonists alleviate obesity-related insulin resistance, which is a major driving force behind glucose intolerance, hyperglycemia, and beta-cell dysfunction (19).

Plasma glucose and plasma insulin responses to feeding following 14-day treatment with either (Hyp3)GIP, (Hyp3)GIPLys16PAL, or saline gave similar plasma glucose concentrations. This was accompanied by unchanged insulin responses, arguing against an important inhibitory effect of GIP-R antagonism on intestinal glucose adsorption, as suggested by the results of some but not all relevant studies (11, 38). This observation may also account for less pronounced lowering of nonfasting glucose than the glycemic responses to a large intraperitoneal glucose load. The possible involvement of changes in the expression of GIP-Rs on target cells in the beneficial effects of these analogs also merits further investigation, especially since downregulation has been reported in diabetic fatty Zucker rats (26).

In conclusion, these studies indicate that (Hyp3)GIP and (Hyp3)GIPLys16PAL are effective GIP-R antagonists in vitro but are less effective than (Pro3)GIP in vivo. Nevertheless, the (Hyp3)-substituted analogs improved glucose tolerance of ob/ob mice after 14 days of treatment by enhanced insulin sensitivity and beta-cell sparing. Acylation of (Hyp3)GIP appears to be of relatively little benefit and resulted in marginal impairment of in vivo activity compared with the parent molecule. Finally, since it might be speculated that GIP has an effect on leptin expression in the adipocyte, it is important to recognize that use of ob/ob mice has the limitation of representing a leptin-deficient model. Nevertheless, preliminary unpublished data indicate similar benefits of GIP-R antagonism in normal mice fed high-fat diets.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This study was supported by the R&D Office of the Health and Personal Social Services for Northern Ireland, Diabetes UK, and University of Ulster Research Strategy Funding.


   ACKNOWLEDGMENTS
 
We thank Bernard Thorens (Institute of Pharmacology, University of Lausanne, Switzerland) for kindly providing the Chinese hamster lung fibroblast cell line transfected with the human GIP-R.


   FOOTNOTES
 

Address for reprint requests and other correspondence: F. P. M. O'Harte, School of Biomedical Sciences, Univ. of Ulster, Coleraine, BT52 1SA, Northern Ireland. (e-mail: fpm.oharte{at}ulster.ac.uk)

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
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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