Characterisation and biological activity of Glu(3) amino acid substituted GIP receptor antagonists

Glucose-dependent insulinotropic polypeptide (GIP) is an important gastrointestinal hormone, which regulates insulin release and glucose homeostasis, but is rapidly inactivated by enzymatic N-terminal truncation. Here we report the enzyme resistance and biological activity of several Glu(3) -substituted analogues of GIP namely; (Ala(3))GIP, (Lys(3))GIP, (Phe(3))GIP, (Trp(3))GIP and (Tyr(3))GIP. Only (Lys(3))- GIP demonstrated moderately enhanced resistance to DPP-IV (p <0.05 to p <0.01) compared to native GIP. All analogues demonstrated a decreased potency in cAMP production (EC50 1.47 to 11.02 nM; p <0.01 to p <0.001) with (Lys(3))GIP and (Phe(3))GIP significantly inhibiting GIP-stimulated cAMP production (p <0.05). In BRIN-BD11 cells, (Lys(3))GIP, (Phe(3))GIP, (Trp(3))GIP and (Tyr(3))- GIP did not stimulate insulin secretion with both (Lys(3))GIP and (Phe(3))GIP significantly inhibiting GIP-stimulated insulin secretion (p <0.05). Injection of each GIP analogue together with glucose in oblob mice significantly increased the glycaemic excursion compared to control (p <0.05 to p <0.001). This was associated with lack of significant insulin responses. (Ala(3))GIP, (Phe(3))GIP and (Tyr(3))GIP, when administered together with GIP, significantly reduced plasma insulin (p <0.05 top <0.01) and impaired the glucose-lowering ability (p <0.05 to p <0.01) of the native peptide. The DPP-IV resistance and GIP antagonism observed were similar but less pronounced than (Pro(3))GIP. These data demonstrate that position 3 amino acid substitution of GIP with (Ala(3)), (Phe(3)), (Tyr(3)) or (Pro(3)) provides a new class of functional GIP receptor antagonists. (C) 2007 Elsevier Inc. All rights reserved.

Comparison of the anti-diabetic effects of GIP- and GLP-1-receptor activation in obese diabetic (ob/ob) mice: studies with DPP IV resistant N-AcGIP and exendin(1-39)amide

Background The two major incretin hormones, glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1) are being actively explored as anti-diabetic agents because they lower blood glucose through multiple mechanisms. The rapid inactivation of GIP and GLP-1 by the ubiquitous enzyme, dipeptidyl peptidase IV (DPP IV) makes their biological actions short-lived, but stable agonists such as N-acetylated GIP (N-AcGIP) and exendin(1-39)amide have been advocated as stable and specific GIP and GLP-1 analogues.

Characterisation and glucoregulatory actions of a novel acylated form of the (Pro(3))GIP receptor antagonist in type 2 diabetes

In this study, we tested the biological activity of a novel acylated form of (Pro(3))glucose-dependent insulinotropic polypetide [(Pro3)GIP] prepared by conjugating palmitic acid to Lys(16) to enhance its efficacy in vivo by promoting binding to albumin and extending its biological actions. Like the parent molecule (Pro(3))GIP, (Pro(3))GIPLys(16)PAL was completely stable to the actions of DPP-IV and significantly (p <0.01 to p <0.001) inhibited GIP-stimulated cAMP production and cellular insulin secretion. Furthermore, acute administration of (Pro(3))GIPLys(16)PAL also significantly (p <0.05 to p <0.001) countered the glucose-lowering and insulin-releasing actions of GIP in ob/ob mice. Daily injection of (Pro(3))GIPLys(16)PAL (25 nmol/kg bw) in 14-18-week-old ob/ob mice over 14 days had no effect on body weight, food intake or non-fasting plasma glucose and insulin concentrations. (Pro(3))GIPLys(16)PAL treatment also failed to significantly alter the glycaemic response to an i.p. glucose load or test meal, but insulin concentrations were significantly reduced (1.5-fold; p <0.05) after the glucose load. Insulin sensitivity was enhanced (1.3-fold; p <0.05) and pancreatic insulin was significantly reduced (p <0.05) in the (Pro(3))GIPLys(16)PAL-treated mice. These data demonstrate that acylation of Lys(16) with palmitic acid in (Pro(3))GIP does not improve its biological effectiveness as a GIP receptor antagonist.

Early administration of the glucose-dependent insulinotropic polypeptide receptor antagonist (Pro(3))GIP prevents the development of diabetes and related metabolic abnormalities associated with genetically inherited obesity in ob/ob mice

Aims/hypothesis Ablation of gastric inhibitory polypeptide ( GIP) receptor action is reported to protect against obesity and associated metabolic abnormalities. The aim of this study was to use prediabetic ob/ob mice to examine whether 60 days of chemical GIP receptor ablation with (Pro(3)) GIP is able to counter the development of genetic obesity-related diabetes.

Metabolic stability, receptor binding, cAMP generation, insulin secretion and antihyperglycaemic activity of novel N-terminal Glu(9)-substituted analogues of glucagon-like peptide-1

Glucagonlike peptide-1(7 36)amide (GLP-1) is an incretin hormone with therapeutic potential for type 2 diabetes. Rapid removal of the Nterminal dipeptide, His7-Ala8, by the ubiquitous enzyme dipeptidyl peptidase IV (DPP IV) curtails the biological activity of GLP-1. Chemical modifications or substitutions of GLP-1 at His7 or Ala8 improve resistance to DPPIV action, but this often reduces potency. Little attention has focused on the metabolic stability and functional activity of GLP-1 analogues with amino acid substitution at Glu9, adjacent to the DPP IV cleavage site. We generated three novel Glu9-substituted GLP-1 analogues, (Pro9)GLP-1, (Phe9)GLP-1 and (Tyr9)GLP-1 and show for the first time that Glu9 of GLP-1 is important in DPP IV degradation, since replacing this amino acid, particularly with proline, substantially reduced susceptibility to degradation. All three novel GLP-1 analogues showed similar or slightly enhanced insulinotropic activity compared with native GLP-1 despite a moderate 4 10-fold reduction in receptor binding and cAMP generation. In addition, (Pro9)GLP 1 showed significant ability to moderate the plasma glucose excursion and increase circulating insulin concentrations in severely insulin resistant obese diabetic (ob/ob) mice. These observations indicate the importance of Glu9 for the biological activity of GLP-1 and susceptibility to DPP IVmediated degradation.

Degradation, receptor binding, insulin secreting and antihyperglycaemic actions of palmitate-derivatised native and Ala(8)-substituted GLP-1 analogues

The hormone glucagonlike peptide-1(736)amide (GLP-1) is released in response to ingested nutrients and acts to promote glucosedependent insulin secretion ensuring efficient postprandial glucose homeostasis. Unfortunately, the beneficial actions of GLP-1 which give this hormone many of the desirable properties of an antidiabetic drug are short lived due to degradation by dipeptidylpeptidase IV (DPP IV) and rapid clearance by renal filtration. In this study we have attempted to extend GLP-1 action through the attachment of palmitoyl moieties to the epsilon-amino group in the side chain of the Lys(26) residue and to combine this modification with substitutions of the Ala(8) residue, namely Val or aminobutyric acid (Abu). In contrast to native GLP-1, which was rapidly degraded, [Lys(pal)(26)]GLP-1, [Abu(8),Lys(pal)(26)]GLP-1 and [Val(8),Lys(pal)(26)]GLP-1 all exhibited profound stability during 12 h incubations with DPP IV and human plasma. Receptor binding affinity and the ability to increase cyclic AMP in the clonal beta-cell line BRIN-BD11 were decreased by 86- to 167-fold and 15- to 62-fold, respectively compared with native GLP-1. However, insulin secretory potency tested using BRIN-BD11 cells was similar, or in the case of [Val(8),Lys(pal)(26)]GLP-1 enhanced. Furthermore, when administered in vivo together with glucose to diabetic (ob/ob) mice, [Lys(pal)(26)]GLP-1, [Abu(8),Lys(pal)(26)]GLP-1 and [Val8,Lys(pal)26]GLP-1 did not demonstrate acute glucoselowering or insulinotropic activity as observed with native GLP-1. These studies support the potential usefulness of fatty acid linked analogues of GLP-1 but indicate the importance of chain length for peptide kinetics and bioavailability.

Evidence that the major degradation product of glucose-dependent insulinotropic polypeptide, GIP(3-42), is a GIP receptor antagonist in vivo

The incretin hormone glucose-dependent insulinotropic polypeptide (GIP) is rapidly degraded in the circulation by dipeptidyl peptidase IV forming the N-terminally truncated peptide GIP(3-42). The present study examined the biological activity of this abundant circulating fragment peptide to establish its possible role in GIP action. Human GIP and GIP(3-42) were synthesised by Fmoc solid-phase peptide synthesis, purified by HPLC and characterised by electrospray ionisation-mass spectrometry. In GIP receptor-transfected Chinese hamster lung fibroblasts, GIP(3-42) dose dependently inhibited GIP-stimulated (10(-7) M) cAMP production (up to 75.4 +/-5.4%; P

Improved stability, insulin-releasing activity and antidiabetic potential of two novel N-terminal analogues of gastric inhibitory polypeptide: N-acetyl-GIP and pGlu-GIP

Aims/hypothesis: This study examined the plasma stability, biological activity and antidiabetic potential of two novel N-terminally modified analogues of gastric inhibitory polypeptide (GIP).

Methods: Degradation studies were carried out on GIP, N-acetyl-GIP (Ac-GIP) and N-pyroglutamyl-GIP (pGlu-GIP) in vitro following incubation with either dipeptidylpeptidase IV or human plasma. Cyclic adenosine 3'5' monophosphate (cAMP) production was assessed in Chinese hamster lung fibroblast cells transfected with the human GIP receptor. Insulin-releasing ability was assessed in vitro in BRIN-BD11 cells and in obese diabetic (ob/ob) mice.

Results: GIP was rapidly degraded by dipeptidylpeptidase IV and plasma (t1/2 2.3 and 6.2 h, respectively) whereas Ac-GIP and pGlu-GIP remained intact even after 24 h. Both Ac-GIP and pGlu-GIP were extremely potent (p<0.001) at stimulating cAMP production (EC50 values 1.9 and 2.7 nmol/l, respectively), almost a tenfold increase compared to native GIP (18.2 nmol/l). Both Ac-GIP and pGlu-GIP (10–13–10–8 mmol/l) were more potent at stimulating insulin release compared to the native GIP (p<0.001), with 1.3-fold and 1.2-fold increases observed at 10–8 mol/l, respectively. Administration of GIP analogues (25 nmol/kg body weight, i.p.) together with glucose (18 mmol/kg) in (ob/ob) mice lowered (p<0.001) individual glucose values at 60 min together with the areas under the curve for glucose compared to native GIP. This antihyperglycaemic effect was coupled to a raised (p<0.001) and more prolonged insulin response after administration of Ac-GIP and pGlu-GIP (AUC, 644±54 and 576±51 ng·ml–1·min, respectively) compared with native GIP (AUC, 257±29 ng·ml–1·min).

Conclusion/interpretation: Ac-GIP and pGlu-GIP, show resistance to plasma dipeptidylpeptidase IV degradation, resulting in enhanced biological activity and improved antidiabetic potential in vivo, raising the possibility of their use in therapy of Type II (non-insulin-dependent) diabetes mellitus.

Kinetic analysis of the cleavage of human protease-activated receptor-1/2/3 and 4 using quenched-fluorescent peptide substrates

Protease-activated receptors [PARs] are a family of G-protein-coupled seven-transmembrane domain receptors that are activated by proteolytic cleavage of their amino-terminal exodomain. To characterize the cleavage rate of human PAR-1 / 2 / 3 and 4 by trypsin and thrombin, four synthetic quenched-fluorescent peptide substrates have been synthesized. Each substrate consisted of a ten-residue peptide spanning the receptor activation cleavage site and using progress-curve kinetics, k(cat)/K-m values were determined.

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

A novel N-terminally substituted Pro(3) analogue of glucose-dependent insulinotropic polypeptide (GIP) was synthesized and tested for plasma stability and biological activity both in vitro and in vivo. Native GIP was rapidly degraded by human plasma with only 39 +/- 6% remaining intact after 8 h, whereas (Pro(3))GIP was completely stable even after 24 h. In CHL cells expressing the human GIP receptor, (Pro(3))GIP antagonized the cyclic adenosine monophosphate (cAMP) stimulatory ability of 10(-7)M native GIP, with an IC50 value of 2.6 muM. In the clonal pancreatic beta cell line BRIN-BD11, (Pro(3))GIP over the concentration range 10(-13) to 10(-8) M dose dependently inhibited GIP-stimulated (10(-7) M) insulin release (1.2- to 1.7-fold; P <0.05 to P <0.001). In obese diabetic (ob/ob) mice, intraperitoneal administration of (Pro(3))GIP (25 nmol/kg body wt) countered the ability of native GIP to stimulate plasma insulin (2.4-fold decrease; P <0.001) and lower the glycemic excursion (1.5-fold decrease; P <0.001) induced by a glucose load (18 mmol/kg body wt). Collectively these data demonstrate that (Pro(3))GIP is a novel and potent enzyme-resistant GIP receptor antagonist capable of blocking the ability of native GIP to increase cAMP, stimulate insulin secretion, and improve glucose homeostasis in a commonly employed animal model of type 2 diabetes. (C) 2002 Elsevier Science (USA).

Interleukin-8 signaling promotes androgen-independent proliferation of prostate cancer cells via induction of androgen receptor expression and activation

The aim of our study was to assess the importance of the CXC chemokine and interleukin (IL)-8 in promoting the transition of prostate cancer (CaP) to the androgen-independent state. Stimulation of the androgen-dependent cell lines, LNCaP and 22Rv1, with exogenous recombinant human interleukin-8 (rh-IL-8) increased androgen receptor (AR) gene expression at the messenger RNA (mRNA) and protein level, assessed by quantitative polymerase chain reaction and immunoblotting, respectively. Using an androgen response element-luciferase construct, we demonstrated that rh-IL-8 treatment also resulted in increased AR transcriptional activity in both these cell lines, and a subsequent upregulation of prostate-specific antigen and cyclin-dependent kinase 2 mRNA transcript levels in LNCaP cells. Blockade of CXC chemokine receptor-2 signaling using a small molecule antagonist (AZ10397767) attenuated the IL-8-induced increases in AR expression and transcriptional activity. Furthermore, in 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays, coadministration of AZ10397767 reduced the viability of LNCaP and 22Rv1 cells exposed to bicalutamide. Our data show that IL-8 signaling increases AR expression and promotes ligand-independent activation of this receptor in two androgen-dependent cell lines, describing two mechanisms by which this chemokine may assist in promoting the transition of CaP to the androgen-independent state. In addition, our data show that IL-8-promoted regulation of the AR attenuates the effectiveness of the AR antagonist bicalutamide in reducing CaP cell viability.