We have previously shown that pre-exposure of rat adipocytes to 10 mM glucose for 30 min results ia a 3-fold acceleration (ACC) of deoxyglucose (DG) uptake. In the last year we determined whether glucose simply initiates the ACC or whether glucose is also required to maintain the ACC. A 2 min exposure of adipocytes to 10 mM glucose resulted in a partial DG ACC when the interval between the addition of glucose and measurement of DG uptake was 25 min, but no ACC when the interval was 2 min. Neither actinomycin D (1 mM) nor puromycin (1 mM) inhibited the DG ACC. The data indicate that glucose is the initiator of a cellular process which results in an ACC of DG uptake 10-15 min later. This ACC is maintained for at least 30 min after glucose is removed from the medium and is independent of transcription and translation of mRNA. In the last year we determined whether sugar transport is inhibited by puromycin by a mechanism that is independent of protein synthesis. Maximum inhibitory effect of puromycin on methylglucose transport was observed by 1 sec. Puromycin inhibited methylglucose transport in a dose-dependent manner and a half-maximal inhibition occurred at a puromycin concentration of 0.20 mM. Kinetic experiments demonstrated that puromycin competitively inhibited methylglucose transport (Ki=0.36 mM). Thus, there is an acute effect of puromycin on sugar transport via competitive inhibition that is independent of an inhibition of protein synthesis. Glucose (GL) transport (TR) is generally assumed to be the rate limiting step in GL uptake and metabolism (MET) in muscle. To test this assumption, hindlimbs from 200-230 g rats were perfused with and without 100 nM insulin (INS) and with 3H-3-GL + 0-40 mM GL for 35 min. The disappearance rate of GL from the medium was calculated from samples taken every 5 min over the last 15 min of incubation. GL clearance rate (CR) was constant up to 2 mM in the presence of INS and 7 mM in its absence. At higher [GL] there was a decline in CR which could be due to either competition or to a shift from TR to some step beyond TR being rate limiting. A decline in CR between a [GL] of 2 mM and 5 mM in the presence of INS but not in its absence indicated that this decreased CR is not due to competition since INS does not change the Ks of GL TR. Thus, the data indicate that maximum INS-stimulated GL TR is not the rate limiting step of GL uptake and MET in the perfused rate hindlimb at physiological [GL].
