An Effect of Insulin on Cyclic Adenosine 3′:5′-Monophosphate Phosphodiesterase Activity in Fat Cells

Abstract Homogenates of rat fat cells were separated into three fractions by centrifugation: P1 (sedimented at 10,000 x g for 7 min), P2 (sedimented from the 10,000 x g supernatant after 20 min at 100,000 x g), and S (the 100,000 x g supernatant). All fractions contained adenosine 3':5'-monophosphate (cAMP) phosphodiesterase activity. The S fraction was enriched in higher Km (∼15 µm) phosphodiesterase and the P2 fraction in low Km (∼0.2 µm) activity, although both exhibited two apparent Michaelis constants for cAMP. When assayed with 62 nm cAMP, the specific activity of P2 was 2 to 3 times that of the unfractionated homogenate, and this fraction contained more than one-half the total phosphodiesterase activity. Incubation of fat cells with 1.0 milliunit per ml of insulin invariably increased the phosphodiesterase activity assayed in homogenates with l10 µm cAMP. The increment in activity was for the most part confined to the P2 fraction, the specific activity of which was increased 71 ± 5.5% (mean ± S.E.) in 24 experiments. The specific activity of 5'-AMP nucleotidase in P2 (which was 3 to 4 times that in the whole homogenate) was not altered by insulin. The effect on phosphodiesterase activity was essentially maximal after exposure of cells to insulin, 1 milliunit per ml, for 6 to 8 min. It was not diminished by washing the cells four times without insulin but was completely reversed by incubation for 30 min without insulin after washing. Neither prostaglandin E1, 2.8 µm, nor nicotinic acid, 10 µm, mimicked the effect of insulin on fat cell phosphodiesterase activity. The effect of insulin was prevented by anti-insulin serum and insulin treated with dithiothreitol was inactive. When present in the phosphodiesterase assay, dithiothreitol, 0.0042 to 4.2 mm, increased the activity of both P2 and S fractions isolated from control cells, whereas heparin, 8.4 to 420 ng per ml, and ethylene glycol bis(β-aminoethyl ether-N,N'-tetraacetic acid (EGTA), 0.05 to 2.0 mm, increased P2 activity with little or no effect on the activity of the supernatant fraction. Dithiothreitol, heparin, and EGTA increased, and sodium deoxycholate, 25 to 850 µg per ml, inhibited the activity of P2 fractions isolated from control and insulin-treated cells. Triton X-100, 0.0025 to 0.25%, was without effect. Incubation at 45° for 5 min reduced the phosphodiesterase activity of P2 fractions from control and insulin-treated cells to the same level which was about 50% of the initial control value. The activities then were stable for at least 25 min at 45°. Treatment of rats with dexamethasone for 20 hours decreased the phosphodiesterase activity of the isolated fat cells. The decrement in activity of whole homogenates was apparently accounted for by a loss of activity in the P2 fraction. These observations are consistent with the view that the effects of insulin and perhaps corticosteroids on cAMP-mediated processes in fat cells may be the result of alterations in the activity of a membrane-associated phosphodiesterase which has a relatively high affinity for cAMP.