Insulin and isoproterenol induce phosphorylation of the particulate cyclic GMP-inhibited, low Km cyclic AMP phosphodiesterase (cGI PDE) in 3T3-L1 adipocytes.

The cGI PDE in particulate fractions of differentiated adipocytes (but not control 3T3-L1 fibroblasts) was cross-reactive with a polyclonal antibody raised against the bovine adipose cGI PDE. The 3T3-L1 adipocyte cGI PDE is a 135 kDa protein which is phosphorylated in 32P-labeled cells in response to beta-agonist or insulin. These results indicate that the 3T3-L1 cGI PDE is similar in structure and hormonal regulation to the analogous enzyme in the rat adipocyte.     

Hormonal activation of the cGMP-inhibited low-Km cyclic AMP phosphodiesterase of rat adipocytes from different sites: influence of ovariectomy.

Hormonal activation of the cGMP-inhibited low Km cyclic AMP phosphodiesterase isoenzyme (cGI.PDE) by effectors, acting either through the cAMP-independent (insulin) or through cAMP-dependent (isoproterenol, forskolin ACTH and 8Br-cAMP) mechanisms, were compared in parametrial (PM) and femoral subcutaneous (SC) adipocytes from sham-operated (SHAM) and ovariectomized (OVX) rats. In SHAM rats, the basal cGI.PDE activity was 50% higher in PM than in SC adipocytes. In OVX rats, the cGi.PDE activatory responses to all the effectors tested remained unchanged in SC, but were completely suppressed in PM adipocytes. The mechanism underlying these defective cGI.PDE activatory responses to cAMP-dependent effectors observed in PM adipocytes after OVX seems to involve protein kinase A, since a decreased activation of cGI.PDE by protein kinase A was also found in these cells. Treatment of OVX rats with both estradiol and progesterone reversed the defective cAMP-dependent activation of cGI.PDE, but not the refractoriness of this isoenzyme to insulin activation. Taken together with previous observations from this laboratory on the fat cell adenylate cyclase system (Lacasa et al. (1991) Endocrinology 128, 747-753), these results: (a) demonstrate that the influence of the ovarian status on the key enzymes controlling cAMP metabolism in fat cells depends on the anatomical origin of these cells, and; (b) provide a biochemical explanation to the insensitivity of the SC adipocyte lipolytic system to ovarian hormones.     

The insulin- and glucagon-stimulated ''dense-vesicle'' high-affinity cyclic AMP phosphodiesterase from rat liver. Purification, characterization and inhibitor sensitivity.

The hormone-stimulated 'dense-vesicle' cyclic AMP phosphodiesterase was solubilized as a proteolytically 'clipped' species, and purified to apparent homogeneity from rat liver with a 2000-3000-fold purification and a 13-18% yield. It appeared to be a dimer (Mr 112,000), of two Mr-57,000 subunits. Solubilization of either a liver or a hepatocyte membrane fraction, with sodium cholate in the presence of the protein inhibitor benzamidine, identified three protein bands which could be immunoprecipitated by a polyclonal antibody raised against the pure enzyme. The major band at Mr 62,000 is suggested to be the native 'dense-vesicle' enzyme, having a Mr-5000 extension which serves to anchor this enzyme to the membrane and which is cleaved off during proteolytic solubilization; the Mr-200,000 band is an aggregate of the Mr-62,000 species, and the Mr-63,000 species is possibly a precursor. The purified 'clipped' enzyme hydrolysed cyclic AMP with kinetics indicative of apparent negative co-operativity, with a Hill coefficient (h) of 0.43 and limiting kinetic constants of Km1 = 0.3 +/- 0.05 microM, Km2 = 29 +/- 6 microM, Vmax.1 = 0.114 +/- 0.015 unit/mg of protein and Vmax.2 = 0.633 +/- 0.054 unit/mg of protein. It hydrolysed cyclic GMP with Michaelis kinetics, Km = 10 +/- 1 microM and Vmax. = 4.1 +/- 0.2 units/mg of protein. Cyclic GMP was a potent inhibitor of cyclic AMP hydrolysis, with an IC50 (concn. giving 50% inhibition) of 0.20 +/- 0.01 microM-cyclic GMP when assayed at 0.1 microM-cyclic AMP. This enzyme was inhibited potently by several drugs known to exert positive inotropic effects on the heart, was extremely thermolabile, with a half-life of 4.5 +/- 0.5 min at 40 degrees C, and was shown to be distinct from the rat liver insulin-stimulated peripheral-plasma-membrane cyclic AMP phosphodiesterase [Marchmont, Ayad & Houslay (1981) Biochem. J. 195, 645-652].     

Hormone-sensitive particulate cAMP phosphodiesterase activity in 3T3-L1 adipocytes. Regulation of responsiveness by dexamethasone

Confluent 3T3-L1 fibroblasts incubated for 72 h with methylisobutylxanthine, dexamethasone, and insulin differentiate and acquire phenotypic characteristics of mature adipocytes, including hormone-sensitive cAMP phosphodiesterase activity located in a particulate fraction of homogenates. About 10 days after initiating differentiation, a maximally effective concentration of insulin (100 pM) increased particulate cAMP phosphodiesterase activity 40 to 60% in 8 min; activation persisted for at least 30 min in the presence of insulin. Incubation of adipocytes for 6-8 min with agents that increased cAMP, e.g. 1 microM epinephrine, 0.1 microM isoproterenol, corticotropin (2 mu units/ml), or thyroid-stimulating hormone (15 ng/ml), also increased particulate phosphodiesterase activity 40-60%. Changes in phosphodiesterase activity produced by epinephrine tended to lag behind changes in cAMP. Insulin, epinephrine, and corticotropin increased Vmax, not Km (0.5 microM), for cAMP. Particulate phosphodiesterase activity, solubilized with detergent, eluted in a single peak from DEAE-Bio-Gel. Insulin and epinephrine increased the activity eluted in this peak. Neither insulin nor lipolytic hormones increased activity in soluble fractions from differentiated cells or particulate or soluble fractions from undifferentiated cells. Incubation of adipocytes for 48 h with 1 microM dexamethasone prevented insulin-induced activation of the particulate phosphodiesterase and did not alter basal activity. After incubation for 72 h with 0.1 microM dexamethasone, insulin and epinephrine activation were abolished. These effects of dexamethasone on hormonal regulation of particulate phosphodiesterase activity could account for some of the so-called permissive effects of glucocorticoids on cAMP-mediated processes as well as the "anti-insulin" effects of glucocorticoids.     

Activation and phosphorylation of the ''dense-vesicle'' high-affinity cyclic AMP phosphodiesterase by cyclic AMP-dependent protein kinase.

Incubation of a hepatocyte particulate fraction with ATP and the isolated catalytic unit of cyclic AMP-dependent protein kinase (A-kinase) selectively activated the high-affinity 'dense-vesicle' cycle AMP phosphodiesterase. Such activation only occurred if the membranes had been pre-treated with Mg2+. Mg2+ pre-treatment appeared to function by stimulating endogenous phosphatases and did not affect phosphodiesterase activity. Using the antiserum DV4, which specifically immunoprecipitated the 51 and 57 kDa components of the 'dense-vesicle' phosphodiesterase from a detergent-solubilized membrane extract, we isolated a 32P-labelled phosphoprotein from 32P-labelled hepatocytes. MgCl2 treatment of such labelled membranes removed 32P from the immunoprecipitated protein. Incubation of the Mg2+-pre-treated membranes with [32P]ATP and A-kinase led to the time-dependent incorporation of label into the 'dense-vesicle' phosphodiesterase, as detected by specific immunoprecipitation with the antiserum DV4. The time-dependences of phosphodiesterase activation and incorporation of label were similar. It is suggested (i) that phosphorylation of the 'dense-vesicle' phosphodiesterase by A-kinase leads to its activation, and that such a process accounts for the ability of glucagon and other hormones, which increase intracellular cyclic AMP concentrations, to activate this enzyme, and (ii) that an as yet unidentified kinase can phosphorylate this enzyme without causing any significant change in enzyme activity but which prevents activation and phosphorylation of the phosphodiesterase by A-kinase.     

Cyclic AMP phosphodiesterase and cyclic GMP phosphodiesterase activities of rat mammary tissue.

Cyclic nucleotide phosphodiesterase activity in mammary tissue from rats in midlactation was resolved by DEAE-cellulose chromatography into three functionally distinct fractions: a Ca2+/calmodulin-stimulated cyclic GMP phosphodiesterase, a cyclic GMP-stimulated low-affinity cyclic nucleotide phosphodiesterase, and a high-affinity cyclic AMP-specific phosphodiesterase. The absolute activities and relative proportions of high- and low-affinity enzymes resemble those found, for example, in liver, as distinct from those in excitable tissues. Three functional characteristics are described which are peculiar to mammary-tissue phosphodiesterases. Firstly, the concentration of free Ca2+ required to achieve half-maximal activation of the Ca2+/calmodulin-stimulated phosphodiesterase is somewhat higher than for the analogous enzyme in other tissues; secondly, the activity of this enzyme towards cyclic AMP relative to that towards cyclic GMP is unusually low, and thirdly, the low-affinity cyclic nucleotide phosphodiesterase is inhibited by low concentrations of free Ca2+.     

Proteolytic activation and solubilization of endoplasmic-reticulum cyclic AMP phosphodiesterase activity.

Dithiothreitol led to the activation and solubilization of the cyclic nucleotide phosphodiesterase activities associated with the smooth and various rough subfractions of rat liver endoplasmic reticulum. The activity in each of the subfractions exhibited somewhat different time courses, and sensitivities to dithiothreitol concentration, in respect of their solubilization and activation. Both activation and solubilization by dithiothreitol could be blocked by either thiol proteinase inhibitors or excess bovine serum albumin. Freeze-thaw solubilization was not blocked by the thiol proteinase inhibitor antipain and did not lead to the activation of the enzyme. After dithiothreitol-induced solubilization, all of the enzymes exhibited non-linear Lineweaver-Burk plots indicative of apparent negative co-operativity. In contrast, after freeze-thaw solubilization the enzyme in the smooth-endoplasmic-reticulum-plus-Golgi fraction still obeys Michaelis kinetics, as does the membrane-bound enzyme. It is possible to mimic the action of dithiothreitol in solubilizing and activating the enzyme by limited proteolysis with trypsin. Triton X-100 is highly efficient at solubilizing these enzymes, yet has little effect on their activities. Charged detergents exhibit highly selective effects on the enzymes as regards their solubilization and activity expressed.     

Kinetic and electrophoretic abnormality of cyclic AMP phosphodiesterase in genetically obese mouse adipocytes

In the soluble fraction of adipocytes from Bar Harbor ob/ob mice, phosphodiesterase exhibits abnormal kinetics for cyclic AMP as compared to adipocyte extracts from normal littermates: a 10 fold increase of the high Km enzyme and a 2 fold increase of the corresponding Vmax. Upon starch gel electrophoresis an abnormal pattern was seen. These abnormalities were found regardless the nutritional status of the animals. There was no kinetic abnormality with cyclic GMP.     

Properties and distribution of cyclic AMP phosphodiesterase from rat liver.

1. Phosphodiesterase activity in rat liver supernatant and solubilized rat liver particulate fractions was chromatographed on Q Sepharose and several characteristics of each peak determined. 2. Rat liver supernatant contained four peaks of activity. The first two of these corresponded to type I and II phosphodiesterases. The fourth peaks was similar to a type V activity and the third peak could not be definitely classified. 3. Particulate activity solubilized by mild protease treatment also contained four peaks of activity. The first two corresponded to the first two from the supernatant, the fourth was a type IV enzyme which is the insulin activated phosphodiesterase. The third peak could not be definitely characterized. 4. Particulate activity solubilised by Triton X-100 contained three peaks. Two had the properties of a type IV enzyme but only one of these was immunologically identified as the insulin sensitive enzyme. The remaining activity was similar to the chymotrypsin peak 3 activity. 5. Most of the particulate phosphodiesterase of rat liver is found in a microsomal fraction, and most is the insulin sensitive type IV enzyme.     

The action of adenosine in relation to that of insulin on the low-Km cyclic AMP phosphodiesterase in rat adipocytes.

The adenosine-sensitive cyclic AMP phosphodiesterase of rat adipocytes was found to reside in the same subcellular fraction as the enzyme sensitive to insulin. There were several similarities between the action of adenosine and that of insulin on the enzyme. The action of adenosine on the phosphodiesterase is probably like that of insulin, both being receptor-mediated, although different sites or different receptors could be involved. Adenosine analogues with intact ribose but a modified purine moiety elicited a response similar to that of adenosine. Added Ca2+ was also not a requirement for the action of adenosine. The action of adenosine was not synergistic with that of insulin, neither was adenosine essential for insulin action. Insulin stimulated the enzyme even at low cell concentrations and in the presence of adenosine deaminase. Adenosine, however, enhanced the effect of insulin, but only at insulin concentrations that produced submaximal effects. Thus the mechanisms of action could be similar or related. The time-course effect of a suboptimal concentration of insulin was transitory, like that of adenosine, and was influenced by the presence of adenosine, whereas that of a maximally effective concentration of insulin was sustained for at least 20 min and was not affected by the presence of adenosine. Isoprenaline enhanced phosphodiesterase activity stimulated by optimal concentrations of either adenosine or insulin, suggesting that their effects were mediated through different mechanisms of action.     

Insulin stimulates the tyrosyl phosphorylation and activation of the 52 kDa peripheral plasma-membrane cyclic AMP phosphodiesterase in intact hepatocytes.

The 52 kDa subunit of the peripheral-plasma-membrane insulin-stimulated high-affinity cyclic AMP phosphodiesterase can be specifically detected by the antibody PM1 by Western-blotting procedures and also can be immunoprecipitated from a hepatocyte extract. PM1-mediated immunoprecipitation from hepatocyte extracts showed that insulin treatment of intact 32P-labelled hepatocytes caused the rapid phosphorylation of the peripheral-plasma-membrane cyclic AMP phosphodiesterase. Phosphoamino acid analysis and the use of a phosphotyrosine-specific antibody indicated that phosphorylation occurred on tyrosyl residue(s) of this phosphodiesterase. Prior treatment of hepatocytes with glucagon (10 nM) completely blocked the insulin-mediated tyrosyl phosphorylation of this 52 kDa protein, as detected with both the PM1 and the anti-phosphotyrosine antibodies. Treatment of hepatocytes with glucagon alone did not increase the phosphorylation state of the peripheral-plasma-membrane cyclic AMP phosphodiesterase. The specific anti-phosphotyrosine antibody also detected the insulin-stimulated phosphorylation of proteins of 180 kDa, 95 kDa and 39 kDa. Prior treatment of hepatocytes with glucagon decreased the ability of insulin to phosphorylate the 180 kDa and 39 kDa species, but not the 95 kDa species.     

Activation of rat parotid low Km cyclic AMP phosphodiesterase by isoproterenol

Approximately 94% of rat parotid cyclic AMP phosphodiesterase activity measured at a substrate concentration of 0.1 microM cyclic AMP was found in the 100,000 X g supernatant while the remaining enzyme activity was in the particulate fraction. Incubation of parotid slices with 10 microM isoproterenol resulted in approximately 40% activation of the cyclic AMP phosphodiesterase activity of the 100,000 X g supernatant. The enzyme activity in the particulate fraction was unaffected. The activation resulted from an increase in the value of the Vmax while the apparent Km (0.51 microM) was unaffected. The concentration of isoproterenol required to give half-maximal activation was 0.34 microM. The activation was rapid, became significant after 2 min and reached maximum after 30 min incubation of the parotid slices with isoproterenol. The activation of the enzyme activity by isoproterenol could be blocked by propanolol but was unaffected by cycloheximide. Dibutyryl-cyclic AMP was also effective while phenylephrine and carbamylcholine were ineffective in increasing the activity of the enzyme.     

Regulation of cyclic AMP phosphodiesterase from Mucor rouxii by phosphorylation and proteolysis. Interrelationship of the activatable and insensitive forms of the enzyme.

DEAE-cellulose chromatography of mycelial extracts of Mucor rouxii grown to mid-exponential phase resolves two types of low-Km cyclic AMP phosphodiesterase (EC 3.1.4.17; PDE): PDE I, highly activatable (4-6-fold) by phosphorylation or proteolysis, and PDE II, unresponsive to activation. The enzymic profile of PDE activity obtained from germlings shows only PDE I activity, whereas PDE activity from mycelia grown to stationary phase is eluted from the DEAE-cellulose column at the position of PDE II, and like PDE II is unresponsive to activation. Endogenous proteolysis or controlled trypsin treatment transforms PDE I into PDE II. The insensitive forms of PDE exhibit a slightly smaller sedimentation coefficient than the activatable forms, as judged by sucrose-gradient centrifugation. The basal activity of the highly activatable form of PDE is elevated almost to the value in the presence of trypsin on storage at 4 degrees C in the absence of proteinase inhibitors. Benzamidine, leupeptin, antipain or EGTA prevents the activation produced by storage. PDE I remains strongly activatable by phosphorylation and proteolysis after resolution by polyacrylamide-gel electrophoresis.     

Plasma membrane-bound cyclic AMP phosphodiesterase activity in 3T3-L1 adipocytes

Plasma membranes were isolated from 3T3-L1 adipocytes. Plasma membrane phosphodiesterase (PM-PDE) was measured in the presence of 5 microM cilostamide. Time course and cAMP dose response ranging from 0 to 2 microM were measured. PM-PDE remained linear up to 20 min. Non-linear curve fitting analysis showed that the low Km cAMP dose data fit a two component curve significantly better than a one component curve, indicating that there are two iso-forms of PDE in the plasma membrane of 3T3-L1 adipocytes, similar to swine adipocytes. The Km and Vmax values for this two component curve were Km1=0.12 microM, Vmax1=3.08 pmol min(-1) mg(-1) protein, and Km2=3.67 microM, Vmax2=83.8 pmol min(-1) mg(-1) protein. Inhibitors of PDE1, PDE2 and PDE5 failed to inhibit PM-PDE, as observed in swine adipocyte plasma membranes. However, PDE4 inhibitors were three-fold more effective at inhibiting PDE in 3T3-L1 PM compared to swine adipocyte PM. One mM 1, 3-dipropyl-8-p-sulfophenylxanthine (DPSPX) inhibited PM-PDE by approximately 75% in both preparations. These data demonstrate that PM-PDE is distinct from microsomal membrane PDE and may be responsible for extracellular cAMP metabolism to AMP in 3T3-L1 adipocytes.     

Detergent solubilisation of rat liver particulate cyclic AMP phosphodiesterase

• 1.1. Detergent solubilisation of particulate rat liver low K_m cyclic AMP phosphodiesterase in the presence of protease inhibitors yields a form of the enzyme with a larger molecular weight than the form solubilised by protease treatment.
• 2.2. The detergent solubilised enzyme could be partially purified by anion exchange chromatography.
• 3.3. It displayed a marked tendency to precipitate from solution when detergent was removed.