Human studies indicate that there is a decrease in peripheral insulin sensitivity after puberty. We have previously demonstrated that a significant reduction in glycogen synthesis accounts for 80% of the reduction in insulin-mediated glucose uptake after puberty in rats. We tested the hypothesis that accumulation of fat mass after puberty determines the alteration in intracellular glucose metabolism. We studied 3 groups of Sprague Dawley rats (n=16), post-weaning prepubertal (PRE), 4 mo old ad libitum fed young adults (POST), and 4 mo old calorie restricted (CR) rats (allowing for 60% of the food intake of ad libidum control). Body composition (by3 H2O isotope dilution technique) revealed that in CR, fat mass was decreased by 39% and epididymal fat by 52% as compared to POST. Rate of disappearance (Rd) of 3H glucose by 18 mU/kg/min hyperinsulinemic clamp was increased by 26% in CR to 53±2, as compared with 39±2 mg/kg/min in POST (p<0.001; 63±8 mg/kg/min in PRE). While in CR, glycolysis (rate of conversion of 3H-glucose to 3H2O) was mildly increased (by 5 mg/kg/min as compared to POST;p<0.05), glycogen synthesis(Rd-glycolysis) was increased to 33±2 mg/kg/min from 23±2 mg/kg/min in POST (p<0.001). Thus, CR resulted in a 65% increase in glycogen synthesis, achieving prepubertal levels (38±7 mg/kg/min in PRE). Maintenance of prepubertal glycogen synthesis was further supported by enhanced accumulation of 3H-glucose into glycogen (5.9 ± 0.7× 107 dpm/gm in CR and 4.4 ± 0.7 × 107 in POST; p<0.01). No changes in the enzyme kinetics of glycogen synthase or phosphorylase were observed. Summary: A moderate reduction of fat mass prevented the decline in insulin responsiveness and glycogen synthesis occuring after puberty. Our data suggests that there is a cause-effect relationship between the increased deposition of fat and the reduced ability to store glucose in muscle in post pubertal rats.