bay 11 sale In the present study five Glu substituted analog
In the present study, five Glu3-substituted analogues of GIP, namely (Ala3)GIP, (Lys3)GIP, (Phe3)GIP, (Trp3)GIP and (Tyr3)GIP, were synthesised and tested for DPP-IV resistance and biological activity both in vitro and in vivo. The properties of these analogues were compared with native GIP and (Pro3)GIP. Substituting the native Glu3 with an Ala, Phe, Trp or Tyr residue resulted in analogues with DPP-IV degradation profiles and half-lives similar to the native peptide, thereby implying that these residues were still capable of fulfilling the strict substrate specificity required for DPP-IV action. In contrast, substituting Glu3 with a Lys residue resulted in an analogue that displayed weakly improved enzyme stability compared to native GIP. This increased enzyme stability is most likely due to the effect of the net positive charge arising from the side chain of Lys which may cause weak electrostatic interference around the DPP-IV cleavage site. In contrast to each of the analogues tested, (Pro3)GIP remained completely resistant to the actions of DPP-IV throughout the entire incubation period, which is entirely consistent with previous studies characterising (Pro3)GIP activity . Acute incubations with native GIP showed a concentration-dependent increase in cAMP production from GIP-R transfected CHL cells, which corroborates previous results using this cell-line . All of the Glu3-substituted analogues tested in this experimental system displayed notably decreased ability to increase cAMP production compared to native GIP. However, this is in sharp contrast to (Pro3)GIP, which had an almost negligible effect, corresponding to approximately 5% of maximal GIP activity at supraphysiological concentrations. To establish whether this reduced activity at the GIP-R was indicative of antagonist activity, analogues were incubated with GIP-R transfected bay 11 sale in the presence of a stimulatory concentration of native GIP. Concentrations of (Ala3)GIP, (Trp3)GIP and (Tyr3)GIP up to 10−7m had no effect on GIP-mediated cAMP production. However, (Lys3)GIP and (Phe3)GIP were clearly capable of antagonising GIP-stimulated cAMP production, although these inhibitory actions were much less potent than (Pro3)GIP. Native GIP stimulated insulin secretion from BRIN-BD11 cells in a concentration-dependent manner as noted previously . In accordance with the results from cAMP studies, all of the Glu3-substituted analogues displayed no or significantly reduced insulinotropic activity compared with native GIP. (Ala3)GIP was the only analogue which elicited an insulinotropic response, similar to GIP at the highest concentration. The reason why (Ala3)GIP should induce such a secretory effect is unclear, especially when weighed against its actions on cAMP activity in GIP-R transfected CHL cells. However, one likely explanation could be the involvement of additional signal transduction pathways in β-cells . (Ala3)GIP, (Trp3)GIP and (Tyr3)GIP did not significantly influence GIP-mediated insulin secretion from BRIN-BD11 cells which concurs with cAMP studies. Similarly, (Lys3)GIP and (Phe3)GIP significantly inhibited GIP-stimulated insulin secretion, and these inhibitory actions were substantially less potent than (Pro3)GIP. To determine the acute metabolic actions of GIP analogues in vivo, we employed ob/ob mice, a well characterised animal model of spontaneous obesity and diabetes . Typically, ob/ob mice exhibit hyperphagia, marked obesity, moderate hyperglycaemia, hyperinsulinaemia and severe insulin resistance . As observed previously , administration of native GIP together with glucose elicited a significantly reduced glycaemic excursion and increased insulinotropic response in ob/ob mice. In contrast, all of the analogues evaluated in acute tests displayed significantly elevated plasma glucose concentrations compared with native GIP. Interestingly, (Phe3)GIP exhibited a similar glycaemic profile to (Pro3)GIP with both peptides actually worsening the glycaemic excursion above that observed with administration of glucose alone. This worsening in glycaemic control was consistently reflected in a decrease in insulin-releasing activity compared with native GIP. While (Ala3)GIP and (Tyr3)GIP failed to increase insulin concentrations above that induced by glucose, (Lys3)GIP, (Phe3)GIP and (Trp3)GIP actually reduced plasma insulin concentrations significantly in ob/ob mice compared with the native peptide. However, none of the analogues tested were as potent in reducing plasma insulin concentrations as (Pro3)GIP.