It is generally accepted that cyclic AMP (cAMP) plays a major role in mediating the action of lipolytic hormones on fat cells. It is also accepted that insulin antagonizes the action of those hormones. There is a good deal of controversy over the question as to whether or not the insulin effect is exerted primarily through changes in cAMP accumulation. We believe that the complexity of the system is such that there is inevitable ambiguity in the collected data if insufficient attention is given to the efficiency with which cAMP activates protein kinase and to the time dependent variation of cAMP accumulation. We have developed techniques for the analysis of cAMP accumulation time courses and cAMP turnover in cultured fibroblasts. Preliminary data indicate that these techniques can be used for studies on rat fat cell systems. The purpose of this proposed research is to determine the extent and the manner in which insulin action impinges on cAMP accumulation and to determine quantitatively the compatibility of that action with concurrent changes in lipolysis. This will be achieved by the following studies. The basic kinetic parameters that describe cAMP accumulation in fat cells (the rate constants for synthesis, degradation and desensitization) will be determined. The changes in those rate constants brought about by insulin action will be determined, and the relationships between cAMP accumulation, protein kinase activation and lipolysis will be determined. The effect of insulin on those relationships will be measured. These studies will allow the elucidation of the extent to which insulin acts directly via the cAMP-mediated lipolysis system. Similar approaches will be used with other anti-lipolytic agents which also lower cAMP levels.