Aims/hypothesis. This study examined the biological effects of the GIP receptor antagonist, (Pro 3 )GIP and the GLP-1 receptor antagonist, exendin(9-39)amide. Methods Cyclic AMP production was assessed in Chinese hamster lung fibroblasts transfected with human GIP or GLP-1 receptors, respectively. In vitro insulin release studies were assessed in BRIN-BD11 cells while in vivo insulinotropic and glycaemic responses were measured in obese diabetic (ob/ob) mice. Results. In GIP receptor-transfected fibroblasts, (Pro 3 )-GIP or exendin(9-39)amide inhibited GIP-stimulated cyclic AMP production with maximal inhibition of 70.0±3.5% and 73.5±3.2% at 10 −6 mol/l, respectively. In GLP-1 receptor-transfected fibroblasts, exendin-(9-39)amide inhibited GLP-1-stimulated cyclic AMP production with maximal inhibition of 60±0.7% at 10 −6 mol/l, whereas (Pro 3 )GIP had no effect. (Pro 3 )-GIP specifically inhibited GIP-stimulated insulin release (86%; p<0.001) from clonal BRIN-BD11 cells, but had no effect on GLP-1-stimulated insulin release. In contrast, exendin(9-39)amide inhibited both GIP and GLP-1-stimulated insulin release (57% and 44%, respectively; p<0.001). Administration of (Pro 3 )GIP, exendin(9-39)amide or a combination of both peptides (25 nmol/kg body weight, i.p.) to fasted (ob/ob) mice decreased the plasma insulin responses by 42%, 54% and 49%, respectively (p<0.01 to p<0.001). The hyperinsulinaemia of non-fasted (ob/ob) mice was decreased by 19%, 27% and 18% (p<0.05 to p<0.01) by injection of (Pro 3 )GIP, exendin(9-39)amide or combined peptides but accompanying changes of plasma glucose were small. Conclusions/interpretation. These data show that (Pro 3 )GIP is a specific GIP receptor antagonist. Furthermore, feeding studies in one commonly used animal model of obesity and diabetes, (ob/ob) mice, suggest that GIP is the major physiological component of the enteroinsular axis, contributing approximately 80% to incretin-induced insulin release. [Diabetologia (2003) 46:222-230] Keywords Enteroinsular axis, GIP receptor antagonist, glucagon-like peptide-1, glucose-dependent insulinotropic polypeptide, insulin release. The insulin secretory activity of pancreatic beta cells can be modulated in both a positive or negative manner by several peptide hormones and neurotransmitters [1,2,3]. The two intestinal hormones, glucosedependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1-(7-36)amide (GLP-1) have long been recognised as potent stimulators of insulin secretion under physiological conditions, and for that reason they are considered important incretin hormones [4,5,6]. The effects of both GIP and GLP-1 are glucose-dependent, providing a safeguard against potentially hazardous hypoglycaemic episodes, and as a

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 (t 1/2 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 (EC 50 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. [Diabetologia (2002[Diabetologia ( ) 45:1281[Diabetologia ( -1291 Keywords GIP analogues, antihyperglycaemic effects, insulin secretion, DPP IV stability, BRIN-BD11 cells, obese hyperglycaemic (ob/ob) mice. Corresponding author: Dr. F. P. M. O'Harte, School of Biomedical Sciences, University of Ulster, Coleraine, N. Ireland, UK, BT52 1SA, E-mail: fpm.oharte@ulst.ac.uk Abbreviations: cAMP, Cyclic adenosine 3′ 5′ monophosphate; DPP IV, Dipeptidylpeptidase IV; GIP, gastric inhibitory polypeptide; GLP-1, glucagon-like peptide-1(7-36)amide; TFA, trifluoroacetic acid; ESI-MS, electrospray ionisation mass spectrometry; pGlu, N-pyroglutamyl; Ac, N-acetyl; Fmoc, 9-fluorenylmethoxycarbonyl; CHL, Chinese hamster lung fibroblast; DPA, diprotin A; FSK, forskolin; IBMX, isobutylmethylxanthine Diabetologia (2002) 45:1281-1291 DOI 10.1007 Improved stability, insulin-releasing activity and antidiabetic potential of two novel N-terminal analogues of gastric inhibitory polypeptide: N-acetyl-GIP and pGlu-GIP Type II (non-insulin-dependent) diabetes me...

Gastric inhibitory polypeptide (GIP) is an important insulin-releasing hormone of the enteroinsular axis which is rapidly inactivated by the exopeptidase dipeptidyl peptidase (DPP) IV. The present study has examined the ability of Tyr 1 -glucitol GIP to be protected from plasma degradation and to enhance insulin-releasing and antihyperglycaemic activity in 20-to 25-week-old obese diabetic ob/ob mice. Degradation of GIP by incubation at 37 C with obese mouse plasma was clearly evident after 3 h (35% degraded). After 6 h, more than 61% of GIP was converted to GIP(3-42) whereas N-terminally modified Tyr 1 -glucitol GIP was resistant to degradation in plasma (>99% intact after 6 h). The formation of GIP(3-42) was almost completely abolished by inhibition of plasma DPP IV with diprotin A. Effects of GIP and Tyr -glucitol GIP also enhanced the glucose-lowering ability of 50 units/kg insulin (218·4 30·2 vs insulin alone 133·9 16·2 mmol/l.min; P<0·05). These data demonstrate that Tyr 1 -glucitol GIP displays resistance to plasma DPP IV degradation in a commonly used animal model of type 2 diabetes, resulting in enhanced antihyperglycaemic activity and insulin-releasing action in vivo.

Although the incretin hormone glucagon-like peptide-1 (GLP-1) is a potent stimulator of insulin release, its rapid degradation in vivo by the enzyme dipeptidyl peptidase IV (DPP IV) greatly limits its potential for treatment of type 2 diabetes. Here, we report two novel

Glucagon-like peptide-1(7-36)amide (GLP-1) possesses several unique and beneficial effects for the potential treatment of type 2 diabetes. However, the rapid inactivation of GLP-1 by dipeptidyl peptidase IV (DPP IV) results in a short half-life in vivo (less than 2 min) hindering therapeutic development. In the present study, a novel His 7 -modified analogue of GLP-1, N-pyroglutamyl-GLP-1, as well as N-acetyl-GLP-1 were synthesised and tested for DPP IV stability and biological activity. Incubation of GLP-1 with either DPP IV or human plasma resulted in rapid degradation of native GLP-1 to GLP-1(9-36)amide, while N-acetyl-GLP-1 and N-pyroglutamyl-GLP-1 were completely resistant to degradation. N-acetyl-GLP-1 and N-pyroglutamyl-GLP-1 bound to the GLP-1 receptor but had reduced affinities (IC 50 values 32·9 and 6·7 nM, respectively) compared with native GLP-1 (IC 50 0·37 nM). Similarly, both analogues stimulated cAMP production with EC 50 values of 16·3 and 27 nM respectively compared with GLP-1 (EC 50 4·7 nM). However, N-acetyl-GLP-1 and Npyroglutamyl-GLP-1 exhibited potent insulinotropic activity in vitro at 5·6 mM glucose (P,0·05 to P,0·001) similar to native GLP-1. Both analogues (25 nM/kg body weight) lowered plasma glucose and increased plasma insulin levels when administered in conjunction with glucose (18 nM/kg body weight) to adult obese diabetic (ob/ob) mice. N-pyroglutamyl-GLP-1 was substantially better at lowering plasma glucose compared with the native peptide, while N-acetyl-GLP-1 was significantly more potent at stimulating insulin secretion. These studies indicate that N-terminal modification of GLP-1 results in DPP IV-resistant and biologically potent forms of GLP-1. The particularly powerful antihyperglycaemic action of N-pyroglutamyl-GLP-1 shows potential for the treatment of type 2 diabetes.

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Previous studies have shown that glycation of insulin occurs in pancreatic -cells under conditions of hyperglycaemia and that the site of glycation is the N-terminal Phe 1 of the insulin B-chain. To enable evaluation of glycated insulin in diabetes, specific antibodies were raised in rabbits and guinea-pigs by using two synthetic peptides (A: Phe-Val-Asn-Gln-His-Leu-Cys-Tyr, and B: Phe-ValAsn-Gln-His-Leu-Tyr-Lys) modified by N-terminal glycation and corresponding closely to the N-terminal sequence of the glycated human insulin B-chain. For immunization, the glycated peptides were conjugated either to keyhole limpet haemocyanin or ovalbumin using glutaraldehyde, m-maleimidobenzoyl-N-hydroxysuccinimide ester or 1-ethyl-3-(3-dimethylamino propyl) carbodiimide hydrochloride. Antibody titration curves, obtained using I 125 -tyrosylated tracer prepared from glycated peptide A, revealed high-titre antisera in five groups of animals immunized for 8-28 weeks. The highest titres were observed in rabbits and guinea-pigs immunized with peptide B coupled to ovalbumin using glutaraldehyde. Under radioimmunoassay conditions, these antisera exhibited effective dose (median) (ED 50 ) values for glycated insulin of 0·3-15 ng/ml and 0·9-2·5 ng/ml respectively, with negligible cross-reactivity against insulin or other islet peptides. The degree of cross-reaction with glycated proinsulin was approximately 50%. Glycated insulin in plasma of control and hydrocortisone-treated diabetic rats measured using rabbit 3 antiserum (1:10 000 dilution; sensitivity <19 pg/ml) was 0·08 0·01 and 1·5 0·6 ng/ ml (P<0·01), corresponding to 4 and 16% of total circulating insulin concentration respectively. Immunocytochemistry studies of the pancreas of streptozotocin-treated diabetic rats using a 1:1000 dilution of guinea-pig 2 antiserum revealed clusters of fluorescent positively stained cells in islets. These studies document the successful production of polyclonal antisera specific for glycated insulin and their usefulness in radioimmunoassays and immunocytochemistry. The demonstration of glycated insulin in plasma and islets of animal models of diabetes supports the view that glycation of insulin is involved in the pathogenesis of this disease.

The therapeutic potential of glucagon-like peptide-1