POTENTIAL MECHANISM OF INSULIN RESISTANCE IN AGING - IMPAIRED INSULIN-STIMULATED GLUCOSE-TRANSPORT DUE TO A DEPLETION OF THE INTRACELLULAR POOL OF GLUCOSE TRANSPORTERS IN FISCHER RAT ADIPOCYTES

To examine the cellular mechanism responsible for impaired insulin action in ageing, we determined various in-vitro parameters involved in the pathogenesis of insulin resistance, i.e. basal and insulin-stimulated [14C]3-O-methylglucose transport (3OMG), 125I-labelled insulin binding, activation of insulin receptor kinase (IRKA) in intact cells, and number and subcellular distribution of glucose transporters in subcellular membrane fractions of adipocytes from 6- (FR-6) and 24- (FR-24) month-old Fischer rats. Ageing had no effect on basal 3OMG (12 ± 4 vs 13 ± 3 fmol/5 x 104 cells, means ±S.E.M.); in contrast, in FR-24 rats insulin-stimulated 3OMG was markedly decreased by 43% when compared with that in FR-6 rats (158 ± 14 vs 90 ± 8 fmol/5 x 104 cells; P<0.01). Insulin binding to adipocytes from FR-6 rats was 2.40 ± 0.38% compared with 2.28 ± 0.47% in FR-24 (P not significant). Moreover, ageing had no significant effect on IRKA, as determined by insulin-stimulated (0, 1, 4 and 500 ng insulin/ml) 32P-incorporation into histone 2B. In subcellular membrane fractions, low density microsomes and plasma membranes, glucose transporter numbers were determined using [3H]cytochalasin B binding and immunodetection using an antiserum against the C-terminal peptide of the hepatoma-G2-glucose transporter. Cytochalasin B binding revealed that in the basal state the intracellular pool of glucose transporters was depleted in FR-24 by about 39% compared with low density microsomes from FR-6: (48.6 ± 7.2 vs 29.8 ± 5.5 pmol/mg membrane protein; P<0.01). In consequence, in FR-24 there were fewer glucose transporters available for insulin-induced translocation to the plasma membrane (insulin-treated plasma membrane: 23.9 ± 4.2 (FR-6) vs 14.4 ± 3.1 (FR-24) pmol/mg membrane protein; P<0.01). These results were confirmed by immunoblotting. In conclusion, (1) maximal insulin-stimulated 3OMG was decreased by 43% in cells from FR-24 rats compared with those from FR-6 rats, while basal 3OMG was similar in both groups, (2) neither insulin binding nor IRKA were significantly altered in cells from FR-24 rats, and (3) impaired insulin-stimulated 3OMG was associated with reduced numbers of glucose transporters in the plasma membrane as a consequence of a depletion of the intracellular pool of glucose transporters in cells from FR-24 rats.