% Sulfonylurea-induced effect left in the presence of diazoxide/nitrendipine |
|---|
Effect | Glibenclamide Diazoxide | Glibenclamide Nitrendipine | Amaryl Diazoxide | Amaryl Nitrendipine |
|---|
Lipogenesis | 27 ± 8 | 61 ± 11 | 80 ± 6 | 95 ± 6 |
Glycogenesis | 38 ± 11 | 58 ± 7 | 79 ± 8 | 90 ± 6 |
Glucose Transport | 52 ± 14 | 65 ± 10 | 89 ± 8 | 98 ± 8 |
GS Dephosphorylation | 44 ± 9 | 68 ± 9 | 81 ± 5 | 94 ± 7 |
IRS-1 Phosphorylation | 57 ± 10 | 69 ± 10 | 85 ± 7 | 92 ± 10 |
Caveolin Phosphorylation | 35 ± 6 | 56 ± 9 | 92 ± 8 | 96 ± 8 |
pp59Lyn Activation | 42 ± 7 | 55 ± 6 | 89 ± 6 | 90 ± 5 |
Cytosolic Ca2+ | 6 ± 1 | 47 ± 7 | 10 ± 2 | 41 ± 7 |
- Isolated rat adipocytes prepared as described previously (51) were treated with 10 µM Amaryl or glibenclamide alone or in combination with 10 µM diazoxide or 30 µM nitrendipine for 30 min at 30°C prior to assaying lipogenesis for 90 min (136), glycogenesis for 60 min (42), 2-deoxyglucose transport for 20 min (51), GS dephosphorylation for 15 min (125), IRS-1 and caveolin tyrosine phosphorylation for 10 min (40,137), and cytosolic Ca2+ levels for 5 min. Prior to fluorometric Ca2+ measurement, the adipocytes were washed three times (by flotation) with Hepes-based salt solution (HBSS); 140 mM NaCl, 0.8 mM MgSO4, 1.8 mM CaCl2, 0.9 mM NaH2PO4, 4 mM NaHCO3, 5 mM glucose, 2 mM sodium pyruvate, 2 mM glutamine, 20 mM N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid (HEPES), 1% bovine serum albumin]. The cells were then loaded with Fura-2-acetoxymethyl ester (10 µM) in the same buffer for 45 min at 37°C in the dark with continuous shaking. For removal of extracellular dye, the cells were washed three times with HBSS and resuspended in HBSS at 3.5×105 cells/ml. Cytosolic Ca2+ was determined using dual excitation (340 and 380 nm) and single emission (510 nm) fluorometry. After the establishment of stable baseline, the response to Amaryl and glibenclamide was determined. Digitonin (25 µM) and Tris/ethylene glycoe tetraammonium acetate (EGTA) (100 mM) were used to measure maximal and minimal fluorescence to calibrate the signals. Cytosolic Ca2+ was calculated by the equation of Grynkiewicz et al. (138). The sulfonylurea-induced effect (difference of presence and absence of drug) in the absence of diazoxide and nitrendipine was set at 100% for each parameter for each sulfonylurea. The values represent means ± standard deviation (SD) from (at least) three different adipocyte preparations, each, with determinations in quadruplicate. GS, glycogen synthase; IRS-1, insulin receptor substrate -1.
