Hexose Uptake in Adipocytes To determine glucose transport activity, hAT was grossly minced and digested at 37C under shaking in 20?mL of Krebs-Ringer medium containing 0.015?mg/mL liberase (type TM, Roche Diagnostics), 15?mM sodium bicarbonate, 10?mM HEPES, and 3.5% bovine serum albumin. This indicates that various phenolic compounds block downstream effects of H2O2 produced by biogenic or exogenous amine oxidation without directly inhibiting AO. Phenolic compounds remain of interest regarding their capacity to limit oxidative stress rather than inhibiting AO. 1. Introduction Resveratrol is a well-known nonenzymatic antioxidant molecule and it has been reported to exert neuroprotective actions for more than a decade . Recently,transcistranstrans= 16) and 15800 1770?dpm (= 12), respectively. These absolute values, which varied substantially from one individual to another, were set as 100% reference in each subject for the calculation of OAC2 percentages of inhibition. 2.4. Hexose Uptake in Adipocytes To determine glucose transport activity, hAT was grossly minced and digested at 37C under shaking in 20?mL of Krebs-Ringer medium containing 0.015?mg/mL liberase (type TM, Roche Diagnostics), 15?mM sodium bicarbonate, 10?mM HEPES, and 3.5% bovine serum albumin. Buoyant adipocytes were separated by filtration through nylon screen and carefully washed in the same medium at pH 7. 4 without liberase to obtain adipocyte suspensions as already described . Freshly isolated adipocytes were incubated for 45?min with the tested agents just before [3H]-2-deoxy-glucose uptake assays (PerkinElmer) performed in 10?min at 37C in plasticware as already described . 2.5. Chemicals Tyramine hydrochloride, benzylamine hydrochloride, amine oxidase inhibitors, quercetin,transtrans 0.05. IC50 values were calculated by nonlinear regression using GraphPad Prism (CA, USA). 3. Results 3.1. ROS Release by Human Subcutaneous Adipose Depots in Response to Amines Spontaneous and amine-stimulated hydrogen peroxide production by hAT preparations was measured on 30?min incubation (Figure 1). When prolonging incubation conditions it was observed that such ROS release was linear with time for at least one hour (not shown). Benzylamine, and tyramine to a lesser extent, significantly increased the amount of detected hydrogen peroxide. Since the chromogenic mixture was already present in the incubation medium at time 0 when the amines were added and since the net fluorescent intensity was calculated as the difference betweentt 0.05 and 0.001. The amine-induced hydrogen peroxide production was then used to test a putative interaction of phenolic OAC2 compounds with human MAO and SSAO. 3.2. Interactions between Phenolic Compounds and MAO- or SSAO-Induced Hydrogen Peroxide Production It was tested whether the response to tyramine was sensitive to reference inhibitors: pargyline (MAO-selective) and semicarbazide (SSAO-selective). The former inhibited dose-dependently tyramine action, while the latter was totally inefficient (Figure 2(a)). Of note, the combination of both inhibitors did not inhibit more than pargyline alone, leaving unaltered approximately 30% of the production found in the presence of tyramine. This confirmed that, in human fat stores, tyramine was mainly oxidized by MAO. When phenolic compounds were studied in identical conditions, all the four tested molecules (see Figure 7 for chemical structures) reached at 1?mM the same maximal inhibition of tyramine-induced H2O2 release (Figure 2(b)). At the 1?ttttcistransinteractions are marked with light blue dotted lines, while OAC2 H-bond interactions are marked with yellow dotted lines. In the case of MAO-A, all the tested agents could dock in a close vicinity to the FAD. Possible aromatic interactions were observed with Phe208, Phe352, Tyr407, and Tyr444. Some H-bonds were also formed between the hydroxyl groups of the ligands and polar amino acids Asn181, Tyr197, and Tyr444. Docking scores calculated by glide varied between ?8.300 and ?5.682 with the standard precision method (SP) and between ?11.568 and ?5.563 with the extra precision level (XP). The predicted activity order was as follows: quercetin cistransinteractions was indicated with the aromatic rings of Tyr326, Tyr398, and Tyr435; H-bonds were found with Cys 172, Tyr188, and Tyr435. The backbone oxygen atom of Pro102 seemed to be an H-bond acceptor for several ligands. Scores were slightly higher than those with MAO-A, since SP scores ranged from ?9.312 to ?6.849 and XP scores ranged from ?11.020 to ?6.997. Regarding SSAO, bothcistransstacking Rabbit polyclonal to IL18R1 interactions were formed with OAC2 the following residues: Phe173, Tyr176, Phe389, and Tyr394; H-bonds were formed with Asp180, Thr210, and Tyr394. Backbone oxygen atom of Thr467 and backbone nitrogen atom of Leu469 could also form additional H-bonds. Docking scores were weaker than those with MAO-A and MAO-B: SP scores varied between ?6.857 and ?5.156, and XP scores varied between ?9.261 and ?4.100. These weaker scores of the tested polyphenols were.
Hexose Uptake in Adipocytes To determine glucose transport activity, hAT was grossly minced and digested at 37C under shaking in 20?mL of Krebs-Ringer medium containing 0