Attenuated maximal activations by forskolin, Mn+. NaF or guanosine 5'-[gamma-thio]triphosphate (GTP[S]) were noted for adenylate cyclase activity in adipocytes from obese (fa/fa) Zucker rats compared with their lean (Fa/Fa) littermates. GTP[S] achieved half-maximal activation of adenylate cyclase at some 10-fold lower concentrations in membranes from lean animals compared with those from obese. Levels of the 42 and 45 kDa forms of Gs were some 40-50% lower in membranes from obese animals, and levels of Gi-1 and Gi-3 were some 62-65% lower. No differences in levels of Gi-2 alpha-subunits or G-protein beta-subunits were observed. Gi function, as assessed by inhibiting forskolin-stimulated adenylate cyclase, achieved by prostaglandin E1, nicotinate and phenylisopropyladenosine, was similar in membranes from both lean and obese animals. Levels of beta-adrenoceptors were some 50% lower in membranes from obese animals. It is suggested that the attenuated activation of adenylate cyclase by stimulatory ligands in membranes from obese animals may be caused by decreases in both Gs and receptors, and that this may contribute to the attenuated lipolytic response seen in adipocytes from such animals.

We have previously reported that modest dietary sodium restriction, as advocated in management guidelines for diabetes, may reduce insulin sensitivity. It has since been suggested that this effect may be mediated via cross-talk between insulin and angiotensin II (AII)-stimulated intracellular second messengers. In order to assess the effect of 5 days of modest sodium restriction (to <80 mmol/day target sodium intake) on insulin sensitivity, 15 healthy males underwent a double-blind, placebo-controlled, randomized, cross-over euglycaemic hyperinsulinaemic clamp study. One phase was supplemented with sodium tablets and the other with matched placebo. Insulin sensitivity (M) was reduced during dietary sodium restriction [median M value, 10.2 mg/kg per min (interquartile range 9.50-13.85) versus 12.8 mg/kg per min (interquartile range 9.60-14.30), P <0.05]. To elucidate potential mechanisms that may explain this observation, we investigated the effect of AII on insulin action in isolated adipocytes obtained from healthy females. No effect of AII on insulin-mediated glucose transport or suppression of lipolysis was observed. In conclusion, despite the observation that dietary sodium restriction was associated with a median 15% reduction in insulin sensitivity, we found no evidence of a direct effect of AII on insulin action in human adipocytes.

Fusion proteins between the human A 1 adenosine receptor and the pertussis toxin resistant (Cys351Gly) mutant of the G-protein K K subunit G i1 K K (A1/Gi), and between the human A 1 adenosine receptor, the Aequorea victoria green fluorescent protein (GFP) and Cys351Gly

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range of vascular pathologies including vascular inflammation and atherosclerosis. While TNF-a has shown to regulate Tie receptors, the chronic impact IL-1b has on Ang-1/Tie receptor signalling pathway and vascular function has not been investigated. Aim To examine the impact IL-1b has on Tie receptor expression and Angiopoietin-1 induced PI3Kinase/AKT activity in endothelial cells. Method Primary Human Umbilical Vein Endothelial Cells (HUVEC) were stimulated with 25ng/ml of IL-1b in the presence or absence of 100ng/ml of human recombinant Ang-1. The treatment times ranged from 0 to 48h. Cell lysates from the treated cells were then subjected to Western blotting to analyze Tie receptor levels and phospho-AKT (pAKT), a signaling molecule associated with Ang-1 cellular transduction. The levels of target proteins were compared between reactions by quantifying mean intensity of bands.In addition, cells were treated with Ang-1 in the absence or presence of IL-1b at various time points. The cell viability assay was performed on the treated cells by following the manufactures protocol. The mean percentage of live to dead cells was calculated from three random fields for each treatment.Data for the Tie receptor and AKT analysis is presented as means and SEM of three independent experiments. Statistical significance represented with p<0.05 using Student's t-test. Data for the cell viability assay is presented as means and SEM of two independent experiments. Results A significant reduction in the levels of Tie-1 receptor was observed at 3h (58.6±8.6%) in HUVECs treated with IL-1b. The cytokine was able to maintain significant low levels of Tie1 up until 48h of treatment, whereas the changes in levels of Tie-2 were insignificant. Interestingly, IL-1b significantly reduced Ang1-induced pAKT activity from 3h onwards with maximum reduction of 73.4±12.8% observed at 48h. The cell viability assays showed reduction in the percentage live to dead cells between Ang-1 and Ang1 + IL-1b for chronic time points tested. Conclusion Long term exposure of Interleukin-1b is capable of altering Tie-1 levels and increasing the Tie-2: Tie-1 ratio in endothelial cells. In contracts, IL-1b reduces Ang-1-PI3Kinase/ AKT signalling and endothelium cell viability. This opposing phenomenon observed where IL-1b impairs Ang-1 protective ability suggests and different mechanism of regulation, independent of the Tie-1 receptor.