Novel dipeptidyl peptidase IV resistant analogues of glucagon-like peptide-1(7-36)amide have preserved biological activities in vitro conferring improved glucose-lowering 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 Ala(8)-substituted analogues of GLP-1, (Abu(8))GLP-1 and (Val(8) GLP-1 which were completely resistant to inactivation by DPP IV or human plasma. (Abu(8))GLP-1 and (Val(8))GLP-1 exhibited moderate affinities (IC50: 4.76 and 81.1 nM, respectively) for the human GLP-1 receptor compared with native GLP-1 (IC50: 0.37 nM). (Abu(8))GLP-1 and (Val(8))GLP-1 dose-dependently stimulated cAMP in insulin-secreting BRIN BD11 cells with reduced potency compared with native GLP-1 (1.5- and 3.5-fold, respectively). Consistent with other mechanisms of action, the analogues showed similar, or in the case of (Val(8))GLP-1 slightly impaired insulin releasing activity in BRIN BD11 cells. Using adult obese (ob/ob) mice, (Abu(8))GLP-1 had similar glucose-lowering potency to native GLP-1 whereas the action of (Val(8))GLP-1 was enhanced by 37%. The in vivo insulin-releasing activities were similar. These data indicate that substitution of Ala(8) in GLP-1 with Abu or Val confers resistance to DPP IV inactivation and that (Val(8))GLP-1 is a particularly potent N-terminally modified GLP-1 analogue of possible use in type 2 diabetes.

Nitrate reductase activity in macroalgae and its vertical distribution in macroalgal epiphytes of seagrasses.

Macroalgal epiphytes within seagrass meadows make a significant contribution to total primary production by assimilating water column N and transferring organic N to sediments. Assimilation of NO3 – requires nitrate reductase (NR, EC 1.6.6.1); NR activity represents the capacity for NO3 – assimilation. An optimised in vitro assay for determining NR activity in algal extracts was applied to a wide range of macroalgae and detected NR activity in all 22 species tested with activity 2 to 290 nmolNO3 – min–1 g–1 frozen thallus. With liquid-N2 freezing immediately after sample collection, this method was practical for estimating NR activity in field samples. Vertical distribution of NR activity in macroalgal epiphytes was compared in contrasting Posidonia sinuosa and Amphibolis antarctica seagrass meadows. Epiphytes on P. sinuosa had higher mass-specific NR activity than those on A. antarctica. In P. sinuosa canopies, NR activity increased with distance from the sediment surface and was negatively correlated with [NH4 +] in the water but uncorrelated with [NO3 –]. This supported the hypothesis that NH4 + released from the sediment suppresses NR in epiphytic algae. In contrast, the vertical variation in NR activity in macroalgae on A. antarctica was not statistically significant although there was a weak correlation with [NO3 –], which increased with distance from the sediment. Estimated capacities for NO3 – assimilation in macroalgae epiphytic on seagrasses during summer (24 and 46 mmolN m–2 d–1 for P. sinuosa and A. antarctica, respectively) were more than twice the estimated N assimilation rates in similar seagrasses. When the estimates were based on annual average epiphyte loads for seagrass meadows in other locations, they were comparable to those of seagrasses. We conclude that epiphytic algae represent a potentially important sink for water-column nitrate within seagrass meadows.

Triclabendazole-resistant Fasciola hepatica: β-tubulin and response to in vitro treatment with triclabendazole

Resistance in Fasciola hepatica to triclabendazole (Fasinex) has emerged in several countries. Benzimidazole resistance in parasitic nematodes has been linked to a single amino acid substitution (phenylalanine to tyrosine) at position 200 on the [beta]-tubulin molecule. Sequencing of [beta]-tubulin cDNAs from triclabendazole-susceptible and triclabendazole-resistant flukes revealed no amino acid differences between their respective primary amino acid sequences. In order to investigate the mechanism of triclabendazole resistance, triclabendazole-susceptible and triclabendazole-resistant flukes were incubated in vitro with triclabendazole sulphoxide (50 [mu]g/ml). Scanning and transmission electron microscopy revealed extensive damage to the tegument of triclabendazole-susceptible F. hepatica, whereas triclabendazole-resistant flukes showed only localized and relatively minor disruption of the tegument covering the spines. Immunocytochemical studies, using an anti-tubulin antibody, showed that tubulin organization was disrupted in the tegument of triclabendazole-susceptible flukes. No such disruption was evident in triclabendazole-resistant F. hepatica. The significance of these findings is discussed with regard to the mechanism of triclabendazole resistance in F. hepatica.

Interaction of glucose and long chain fatty acids (C18) on antioxidant defences and free radical damage in porcine vascular smooth muscle cells in vitro.

Aims/hypothesis: Abnormalities of glucose and fatty acid metabolism in diabetes are believed to contribute to the development of oxidative stress and the long term vascular complications of the disease therefore the interactions of glucose and long chain fatty acids on free radical damage and endogenous antioxidant defences were investigated in vascular smooth muscle cells. Methods: Porcine vascular smooth muscle cells were cultured in 5 mmol/l or 25 mmol/l glucose for ten days. Fatty acids, stearic acid (18:0), oleic acid (18:1), linoleic acid (18:2) and gamma-linolenic acid (18:3) were added with defatted bovine serum albumin as a carrier for the final three days. Results. Glucose (25 mmol/l) alone caused oxidative stress in the cells as evidenced by free radical-mediated damage to DNA, lipids, and proteins. The addition of fatty acids (0.2 mmol/l) altered the profile of free radical damage; the response was J-shaped with respect to the degree of unsaturation of each acid, and oleic acid was associated with least damage. The more physiological concentration (0.01 mmol/l) of gamma-linolenic acids was markedly different in that, when added to 25 mmol/l glucose it resulted in a decrease in free radical damage to DNA, lipids and proteins. This was due to a marked increase in levels of the antioxidant, glutathione, and increased gene expression of the rate-limiting enzyme in glutathione synthesis, gamma-glutamylcysteine synthetase. Conclusion/Interpretation: The results clearly show that glucose and fatty acids interact in the production of oxidative stress in vascular smooth muscle cells.