The aim of the study was to investigate the regulation affecting the recently described atrial natriuretic peptide (ANP)-dependent lipolytic pathway in comparison with the adrenergic lipolytic cascade. We studied in vivo the effect of a euglycemic-hyperinsulinemic clamp on the changes occurring in the extracellular glycerol concentration (EGC) of subcutaneous adipose tissue (SCAT) during ANP or epinephrine perfusion in a microdialysis probe. Homologous desensitization and the incidence of hyperinsulinemia on the ANP- and catecholaminergic-dependent control of lipolysis were also investigated in vitro on fat cells from SCAT. When perfused in SCAT, epinephrine and ANP promoted an increase in EGC; the EGC increase was significantly lower during the clamp. The reduction of epinephrine-induced lipolysis was limited (18%) when phentolamine (an alpha(2)-adrenergic receptor [AR] antagonist) was perfused together with epinephrine. Unlike the effect of epinephrine, the response to ANP observed during the second perfusion was reduced by 32%. The increase in extracellular guanosine 3',5' -cyclic monophosphate concentration, which reflects ANP activity, was also reduced during the second perfusion. Desensitization of the lipolytic effects of ANP was observed in vitro after a 2-hour period of recovery, while the effects of alpha(2)-AR agonist or of epinephrine were unchanged. Insulin was without any effect on ANP-induced lipolysis and alpha(2)-AR-mediated antilipolysis, while it reduced beta-AR-induced lipolysis. The ANP-dependent lipolytic pathway undergoes desensitization in vitro and in situ. Insulin had no inhibitory effect on either ANP- or alpha(2)-AR-dependent pathways, while it counteracted the beta-AR pathway.