L-Glutamine → (2R,3S,4R,5R)-2,3,4,5,6-pentahydroxyhexanal

Differentiated adipocytes were incubated for a period of 3 days in DMEM (containing 2 mM L-glutamine, 100 iu/ml penicillin, 100 μg/ml streptomycin, 1 nM insulin and 10% FCS) supplemented with or without CLA and/or compound As. The cells were then washed three times with DMEM (containing 1 mM deoxyglucose). Insulin stimulated glucose uptake was carried out according to Iwata et al (Diabetes 2001 50: 1083-1092). Briefly, cells were stimulated with 1 nM insulin in Krebs Ringer Phosphate (KRP) and HEPES buffer [10 mM HEPES, 131.2 mM NaCl, 4.7 mM KCl, 2.5 mM CaCl2, 1.2 mM MgSO4, 2.5 mM sodium phosphate buffer, pH 7.4] with 1% BSA at 37° C. After 1-30 minutes the assay was initiated by addition of 10 μl KRP containing 2.5 μCi [3H]2deoxyglucose and 1 mM deoxyglucose. After 1 hr the reaction was terminated by washing the cells 3× with ice-cold DMEM. Cells were then solubilised with 500 μl/well of 1% Triton X-100, 0.2% SDS and 0.2N NaOH for 20 minutes at 37° C. Radioactive incorporation was measured by scintillation counting of 100 μl of each cell lysate.

1. washThe cells were then washed three times
2. additionwith DMEM (containing 1 mM deoxyglucose)
3. customwere stimulated with 1 nM insulin in Krebs Ringer Phosphate (KRP) and HEPES buffer [10 mM HEPES, 131.2 mM NaCl, 4.7 mM KCl, 2.5 mM CaCl2, 1.2 mM MgSO4, 2.5 mM sodium phosphate buffer, pH 7.4] with 1% BSA at 37° C
4. additionAfter 1-30 minutes the assay was initiated by addition of 10 μl KRP
5. additioncontaining 2.5 μCi [3H]2deoxyglucose and 1 mM deoxyglucose
6. customAfter 1 hr the reaction was terminated
7. washby washing the cells 3× with ice-cold DMEM
8. customfor 20 minutes
9. customat 37° C

ORD record

ord-5e68a6e5540248b59d5deaf822976fbf

Source dataset

uspto-grants-2005_05

DOI

10.6084/m9.figshare.5104873.v1

Patent number

US06888016B2