Our previous study demonstrated that microinjection of leptin into the ventromedial hypothalamus (VMH) dramatically increased glucose uptake in the heart, brown adipose tissue (BAT), and skeletal muscles, but not in white adipose tissue (WAT) in conscious unrestrained rats, as assessed in vivo by the 2-[3H]deoxyglucose method. Here we examined the role of the sympathetic nervous system and insulin in enhanced glucose uptake by tissues after hypothalamic leptin injection. Pretreatment with guanethidine significantly suppressed the increased glucose uptake by the tissues in response to leptin injected into the VMH, whereas bilateral adrenal demedullation had no significant effect. Treatment with propranolol but not phenoxybenzamine also decreased significantly enhanced glucose uptake by the tissues. We further examined the interaction of the effects of hypothalamic leptin and insulin administered peripherally by clamping the glucose concentrations at a constant level. When leptin was injected into the VMH and a maximal dose of insulin was administered intravenously, the rates of glucose uptake by the heart, BAT, and skeletal muscles, but not by WAT, markedly increased beyond the values reached by insulin stimulation alone. Surgical sympathetic denervation of BAT abolished the enhancement of glucose uptake in this tissue, decreasing to the level stimulated by insulin alone. These results appear to indicate that leptin in the hypothalamus enhances glucose uptake in certain peripheral tissues through mediation of a beta-adrenergic mechanism for the sympathetic nerves innervating the tissues and that central leptin and peripheral insulin have a synergistic role in augmenting tissue glucose uptake.