STUDIES ON ACTION OF INSULIN IN ISOLATED ADIPOSE TISSUE CELLS .2. 3',5'-NUCLEOTIDE PHOSPHODIESTERASE AND ANTILIPOLYSIS

1. 1. In isolated fat cells the inhibition of lypolysis by insulin in the presence of ACTH, glucagon and epinephrine occured in a similar concentration range of insulin and exhibited noncompetitive characteristics. In contrast, the antagonism to caffeine was competitive. 2. 2. Ado-3′,5′-P caused lipolysis in fat cells incubated in a Tris-bicarbonate-albumin medium. Lipolysis induced by either Ado-3′,5′-P or by its dibutyryl derivative could not be blocked by insulin, indicating that the hormone neither interfered with lipase activation nor activated phosphodiesterase. 3. 3. 10 mM of imidazole lowered lipolysis stimulated by epinephrine and lipolysis from Ado-3′,5′-P but had no significant effect on lipolysis stimulated by N6,O2-di-butyryl-Ado-3′,5′-P. 4. 4. The greater part of the phosphodiesterase activity in the fat cell was found in the 700 × g sediment. When this fraction was subjected to ultrasonication or to mechanical homogenization, the enzyme activity was released and remained in the 100 000 × g supernatant. A pH optimum was found in the region of 7.0-7.5 with a Km=0.5 mM, a dependency on Mg2+ and a preference for cyclic nucleotides with a purine base, similar to the rat liver enzyme. 5. 5. While inhibition of phosphodiesterase activity by theophylline and stimulation by cacodylate and imidazole were readily demonstrated, no stimulation by nicotinic acid or by insulin could be obtained when the compounds were added directly to the test mixture. A previous incubation of fat cells with insulin or nicotinic acid did not result in phosphodiesterase activation in homogenates. The data do not support the concept that insulin or nicotinic acid may act through the activation of phosphodiesterase. © 1969.