The antilipolytic effect of insulin does not require adenylate cyclase or phosphodiesterase action

Insulin antagonized the lipolytic actions of epinephrine in rat epididymal adipocytes when the phosphodiesterase inhibitor, Ro 20-1724, was present. Adipocytes were depleted of functional cAMP by inhibiting adenylate cyclase with N6-phenylisopropyladenosine in the presence of adenosine deaminase such that Ro 20-1724 no longer stimulated lipolysis. The cAMP analogs 8-thioisopropyl-cAMP or 8-thiomethyl-cAMP, which are resistant to phosphodiesterase hydrolysis, were subsequently added to bypass adenylate cyclase and phosphodiesterase action. Under these conditions, insulin antagonized the lipolytic effects of these analogs, even in the presence of Ro 20-1724.     

Mitochondrial,but not peroxisomal, β-oxidation of fatty acids is conserved in coenzyme A-Deficient rat liver

Growth hormone (GH) exerts acute insulin-like effects, such as increased lipogenesis and inhibition of catecholamine-induced lipolysis, in rat adipocytes that have not been exposed to GH during the preceding three hours. We found that OPC3911, a highly specific inhibitor of the cGMP-inhibited cAMP phosphodiesterase, completely blocked the antilipolytic but not the lipogenic effect of GH. This indicates that the antilipolytic effect of GH is mediated through activation of this phosphodiesterase leading to reduction of cAMP levels in the same manner as has been shown for insulin.     

Effects of cyclic nucleotides on lipid biosynthesis in mouse mammary gland explants

The effects of dibutyryl cAMP, 1-methyl-3-isobutyl xanthine (MIX), cGMP, dibutyryl cGMP, and 8-bromo cGMP on the rate of lipid synthesis in mouse mammary gland explants were studied. Dibutyryl cAMP at 10(-4) M selectively inhibited the effect of prolactin on the rate of [14C]acetate incorporation into lipids. At 10(-3) M, dB-cAMP inhibited the effects of insulin, insulin plus cortisol, and prolactin. The phosphodiesterase inhibitor, MIX, inhibited both basal and prolactin-stimulated incorporation rates in a concentration-dependent fashion. These data suggest an inhibitory role for cAMP in the regulation of lipogenesis in the mammary gland. Cyclic GMP, db-cGMP, and 8-bromo cGMP were all without effect on either basal or prolactin-stimulated incorporation rates. Therefore, it appears that cGMP, by itself, is not involved in the regulation of lipogenesis in the mammary gland.     

Inhibition of basal and hormone-stimulated adenylate cyclase in adipocyte ghosts by the prostaglandin endoperoxide prostaglandin H2.

The prostaglandin endoperoxide PGH2 (15-hydroxy-9alpha, 11alpha-peroxidoprosta-5,13-dienoic acid), at a concentration of 2.8 x 10(-5) M inhibited basal adenylate cyclase activity 11% and epinephrine-stimulated activity 30 to 35%. PGH2 inhibited epinephrine-stimulated enzyme activity in the presence of 10 mM theophylline, 2.5 mM adenosine 3':5'-monophosphate (cAMP), or in the absence of inhibitors or substrates of the cAMP phosphodiesterase. When the cAMP phosphodiesterase was assayed directly using 62 nM and 1.1 muM cAMP, PGH2 did not affect the 100,000 x g particulate cAMP phosphodiesterase from fat cells. The inhibition of adenylate cyclase by PGH2 was readily reversible. A 6-min preincubation of ghost membranes with PGH2, followed by washing, did not alter subsequent epinephrine-stimulated adenylate cyclase activity. During epinephrine stimulation, the PGH2 inhibition was apparent on initial rates of cAMP synthesis, and the addition of PGH2 to the enzyme system at any point during an assay markedly reduced the rate of cAMP synthesis. Between 2.8 x 10(-7) M and 2.8 x 10(-5) M, PGH2 inhibited epinephrine-stimulated enzyme activity in a concentration-dependent manner. The stimulation of adenylate cyclase by thyroid-stimulating hormone, glucagon, and adrenocorticotropic hormone as well as by epinephrine was antagonized by PGH2, suggesting that PGH2 may be an endogenous feedback regulator of hormone-stimulated lipolysis in adipose tissue.     

Characterization of antilipolytic action of polyamines in isolated rat adipocytes.

The interactions of polyamines with the lipolytic system were studied in isolated rat adipocytes. Spermine, spermidine and putrescine significantly inhibited adenosine deaminase-stimulated lipolysis. An antilipolytic effect of spermine was detectable at a concentration of 0.25 mM (P less than 0.05). At a concentration of 10 mM all three polyamines inhibited the stimulated lipolysis by 50-60% (P less than 0.001). In addition, spermine enhanced the antilipolytic sensitivity of insulin. Spermine (1 mM) decreased the half-maximal inhibitory concentration of insulin from 320 +/- 70 pM to 56 +/- 20 pM (P less than 0.01). The antilipolytic effects and the cyclic-AMP-lowering effects of the polyamines were almost completely prevented in the presence of different phosphodiesterase (PDE) inhibitors (3-isobutyl-1-methylxanthine and RO 20-1724) and, in addition, polyamines had no effect on lipolysis stimulated by dibutyryl cyclic AMP, indicating that polyamines may inhibit lipolysis by activating the PDE enzyme. This latter suggestion was confirmed by demonstrating that spermine (5 mM) significantly enhanced the low-Km PDE enzyme activity (P less than 0.01). Finally, the amounts of polyamines present in isolated adipocytes were measured, and the estimated cytoplasmic concentrations were 0.02 mM (putrescine), 0.86 mM (spermidine), and 1.0 mM (spermine). It is concluded that polyamines may possibly be involved in the physiological regulation of triacylglycerol mobilization in adipocytes.     

Evidence that protein kinase C may not be involved in the insulin action on cAMP phosphodiesterase: studies with electroporated rat adipocytes that were highly responsive to insulin.

Partially permeabilized rat adipocytes with a high responsiveness to insulin were prepared by electroporation and used to study the effect of 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7) on insulin actions in adipocytes. H-7 is a well-documented inhibitor of several protein kinases, including protein kinase C; however, it does not rapidly enter adipocytes protected with the intact plasma membrane. The cells were suspended in Buffer X [4.74 mM NaCl, 118.0 mM KCl, 0.38 mM CaCl2, 1.00 mM EGTA, 1.19 mM Mg2SO4, 1.19 mM KH2PO4, 25.0 mM Hepes/K, 20 mg/ml bovine serum albumin, and 3 mM pyruvate/Na, pH 7.4] and electroporated six times with a Gene-Pulser (from Bio-Rad) set at 25 microF and 2 kV/cm. In cells electroporated as above, insulin stimulated (a) membrane-bound, cAMP phosphodiesterase approximately 2.6-fold when the hormone concentration was 10 nM and (b) glucose transport activity approximately 4.5-fold when the hormone concentration was raised to 100 nM. H-7 strongly inhibited the actions of insulin on both glucose transport (apparent Ki = 0.3 mM) and cAMP phosphodiesterase (apparent Ki = 1.2 mM) in electroporated adipocytes. H-7 also inhibited lipolysis in adipocytes; the apparent Ki value for the reaction in intact cells was 0.45 mM, and that in electroporated cells was 0.075 mM. It is suggested that a certain protein kinase or kinases that are significantly sensitive to H-7 may be involved in the insulin-dependent stimulation of glucose transport and that of phosphodiesterase. However, protein kinase C (or Ca2+/phospholipid-dependent protein kinase) may not be involved, at least, in the hormonal action on phosphodiesterase since the apparent Ki value of H-7 for the reaction is too high.     

Inhibition of adipocyte lipolysis by papaverine: papaverine can inhibit the redistribution of hormone-sensitive lipase

Papaverine, despite being a potent phosphodiesterase inhibitor, actually blocks adipocyte lipolysis. The present study was designed to clarify the mechanism of the inhibitory effect of papaverine on lipolysis. Lipolysis, stimulated by either 10 microM isoproterenol or 5 mM dibutyryl cAMP, was significantly inhibited by papaverine (100 microM and above). Papaverine, however, did not affect the isoproterenol-induced increase in the protein kinase A (A-kinase) activity ratio. In cell-free extract from non-stimulated adipocytes, cAMP-stimulated A-kinase activities were almost completely blocked by H-89, a potent inhibitor of A-kinase, but not by papaverine. Thus, the inhibitory effect of papaverine on lipolysis could be responsible for a deficit in step(s) distal to A-kinase activity. Hormone-sensitive lipase activities in the infranatant fraction of centrifuged homogenates of cells, which were maximally stimulated with isoproterenol were significantly reduced. This result indicates that hormone-sensitive lipase redistributes from cytosol to its substrate in lipolytically stimulated cells. Papaverine completely blocked the isoproterenol-induced decrease in lipase activity in the infranatant fraction. These results suggest that papaverine blocks lipolysis through its inhibitory effect on the redistribution of hormone-sensitive lipase.     

Characterization of the yeast low Km cAMP-phosphodiesterase with cAMP analogues. Applications in mammalian cells that express the yeast PDE2 gene

The essential interactions between cAMP and the yeast low Km cAMP-phosphodiesterase have been analyzed using cAMP analogues and phosphodiesterase inhibitors. cAMP specificity is conferred by hydrogen bonding at the N-6 and N-7 positions. In contrast to the other yeast phosphodiesterase, (Rp)-adenosine 3',5'-monophosphorothioate is not hydrolyzed. Eleven standard phosphodiesterase inhibitors were not highly effective. In Chinese hamster ovary (CHO) cells that express the yeast cAMP-phosphodiesterase (PDE2) gene, cAMP levels cannot be raised by cholera toxin. cAMP analogues that are efficiently hydrolyzed by the yeast cAMP-phosphodiesterase had no effect on the growth of CHO cells that express the PDE2 gene, even though they block the growth and alter the morphology of control cells. cAMP analogues that are not hydrolyzed by the yeast enzyme inhibited the growth and changed the morphology of both control and PDE2 expressing CHO cells. We have developed a method for creating cell lines in which cAMP levels can be reduced by expression of an exogenous cAMP-phosphodiesterase gene. By employing cAMP analogues that are not hydrolyzed by this phosphodiesterase, the inhibitory effects of the enzyme can be bypassed.     

Wortmannin, a PI3-kinase inhibitor: promoting effect on insulin secretion from pancreatic beta cells through a cAMP-dependent pathway

To determine the role of phosphatidylinositol 3-kinase (PI3-kinase) in the regulation of insulin secretion, we examined the effect of wortmannin, a PI3-kinase inhibitor, on insulin secretion using the isolated perfused rat pancreas and freshly isolated islets. In the perfused pancreas, 10(-8) M wortmannin significantly enhanced the insulin secretion induced by the combination of 8.3 mM glucose and 10(-5) M forskolin. In isolated islets, cyclic AMP (cAMP) content was significantly increased by wortmannin in the presence of 3.3 mM, 8.3 mM, and 16.7 mM glucose with or without forskolin. In the presence of 16.7 mM glucose with or without forskolin, wortmannin promoted insulin secretion significantly. On the other hand, in the presence of 8.3 mM glucose with forskolin, wortmannin augmented insulin secretion significantly; although wortmannin tended to promote insulin secretion in the presence of glucose alone, it was not significant. To determine if wortmannin increases cAMP content by promoting cAMP production or by inhibiting cAMP reduction, we examined the effects of wortmannin on 10(-4) M 3-isobutyl-1-methylxantine (IBMX)-induced insulin secretion and cAMP content. In contrast to the effect on forskolin-induced secretion, wortmannin had no effect on IBMX-induced insulin secretion or cAMP content. Moreover, wortmannin had no effect on nonhydrolyzable cAMP analog-induced insulin secretion in the perfusion study. These data indicate that wortmannin induces insulin secretion by inhibiting phosphodiesterase to increase cAMP content, and suggest that PI3-kinase inhibits insulin secretion by activating phosphodiesterase to reduce cAMP content.     

Insulin antagonism of catecholamine stimulation of fatty acid transport in the adipocyte. Studies on its mechanism of action

Insulin at physiological concentrations can suppress catecholamine activation of the membrane transport of long chain fatty acids in the adipocyte. We have previously shown that the stimulatory effect of catecholamines was mediated by a beta-receptor interaction and cAMP (Abumrad, N.A., Park, C.R., and Whitesell, R. R. (1986) J. Biol. Chem. 261, 13082-13086). In this study we have investigated the mechanism of insulin action to antagonize transport activation. Fatty acid transport was stimulated using different cAMP derivatives with varying susceptibilities to hydrolysis by the cAMP-degrading enzyme phosphodiesterase. Insulin was effective in antagonizing the effect of cAMP analogs which were good substrates for the phosphodiesterase and failed to suppress the effect of those which were poorly hydrolyzed by the enzyme. Addition of increasing concentrations (1-100 microM) of the phosphodiesterase inhibitor methylisobutylxanthine (MIX) to norepinephrine (0.1 microgram/ml) gradually abolished insulin's antagonism. Insulin was completely ineffective in inhibiting stimulation by norepinephrine and 20 microM methylisobutylxanthine. Also consistent with involvement of cAMP lowering in insulin action was the finding that adenosine removal greatly diminished insulin's responsiveness. Treatment of cells with adenosine deaminase (1 unit/ml) enhanced the effect of norepinephrine by about 30%. A 10-fold higher range of insulin concentrations was then required to produce inhibition of fatty acid transport. The effect of adenosine removal was reversed by addition of phenylisopropyladenosine (500 nM), which is resistant to hydrolysis by the deaminase. Finally, exposure of insulin-treated cells (1 nM for 5 min) to dinitrophenol (1 mM for 5 min) reversed insulin action, consistent with reports of reversal of insulin's activation of the phosphodiesterase. In conclusion, our studies support the involvement of cAMP lowering in insulin's antagonism of fatty acid transport stimulation in the adipocyte.     

Characterization of lipolytic responses of isolated white adipocytes from hamsters.

1. Lipolysis by isolated white adipocytes from hamsters, as measured by glycerol production, was stimulated by corticotropin, isopropylnorepinephrine (INE), norepinephrine, or epinephrine (EPI), in a dose-dependent fashion. 2. Lipolysis was stimulated by five inhibitors of cyclic 3',5'-adenosine monophosphate phosphodiesterase: caffeine, theophylline, 1-methyl-3-isobutyl xanthine, 1-ethyl-4-(isopropylidenehydrazine)-1H-pyrazolo-(3,4,-b)-pyridine-5-carboxylic acid ethyl ester (SQ 20009), and 4-(3,4-dimethoxybenzyl)-2-imidazolidinone (Ro 7-2956). Caffeine-stimulated lipolysis consistently attained higher rates than did hormone-stimulated lipolysis. However, when cells were stimulated by both caffeine and a hormone, lipolytic rates were consistently lower than those attained under the influence of caffeine alone. 3. Isolated white adipocytes from hamsters were sensitive to both alpha- and beta-adrenergic antagonists. The beta-adrenergic antagonist propranolol could completely inhibit norepinephrine-stimulated glycerol production. The alpha-adrenergic antagonist phentolamine, on the other hand, had a biphasic effect on the cells. At 5-10(-7) M or 5-10(-6) M, phentolamine enhanced norepinephrine-stimulated lipolysis, while concentrations higher than 5-10(-5) M caused inhibition. 4. The effects of two different concentrations of six antilipolytic agents, prostaglandin E1, nicotinic acid, phenylisopropyladenosine, 5-methylpyrazole-3-carboxylic acid, adenosine and insulin, were measured. With the exception of insulin, all of these agents showed much more potent inhibition of caffeine-stimulated lipolysis than of hormone-stimulated lipolysis. Insulin, in contrast, showed only modest inhibition of hormone-stimulated lipolysis and virtually no inhibition of caffeine-stimulated lipolysis.     

Three new potential cAMP affinity labels. Inactivation of human platelet low Km cAMP phosphodiesterase by 8-[(4-bromo-2,3-dioxobutyl)thio]adenosine 3',5'-cyclic monophosphate

Three new analogues of cAMP have been synthesized and characterized: 2-[(4-bromo-2,3-dioxobutyl)thio]adenosine 3',5'-cyclic monophosphate (2-BDB-TcAMP), 2-[(3-bromo-2-oxopropyl)thio]-adenosine 3',5'-cyclic monophosphate (2-BOP-tcAMP), and 8-[(4-bromo-2,3-dioxobutyl)thio]adenosine 3',5'-cyclic monophosphate (8-BDB-TcAMP). The bromoketo moiety has the ability to react with the nucleophilic side chains of several amino acids, while the dioxobutyl group can interact with arginine. These cAMP analogues were tested for their ability to inactivate the low Km (high affinity) cAMP phosphodiesterase from human platelets. The 2-BDB-TcAMP and 2-BOP-TcAMP were competitive inhibitors of cAMP hydrolysis by the phosphodiesterase with Ki values of 0.96 +/- 0.12 and 0.70 +/- 0.12 microM, respectively. However, 2-BDB-TcAMP and 2-BOP-TcAMP did not irreversibly inactivate the phosphodiesterase at pH values from 6.0 to 7.5 and at concentrations up to 10 mM. These results indicate that although the 2-substituted TcAMP analogues bind to the enzyme, there are no reactive amino acids in the vicinity of the 2-position of the cAMP binding site. In contrast, incubation of the platelet low Km cAMP phosphodiesterase with 8-BDB-TcAMP resulted in a time-dependent, irreversible inactivation of the enzyme with a second-order rate constant of 0.031 +/- 0.009 min-1 mM1. Addition of the substrates, cAMP and cGMP, and the product, AMP, to the reaction mixture resulted in marked decreases in the inactivation rate, suggesting that the inactivation was due to reaction at the active site of the phosphodiesterase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Methylisobutylxanthine blocks insulin antagonism of cAMP-stimulated glycogenolysis at a site distinct from phosphodiesterase. Evidence favoring an insulin-insensitive calcium release mechanism

The widely used phosphodiesterase inhibitor MIX (1-methyl 3-isobutyl xanthine) blocked insulin antagonism of cAMP-stimulated glycogenolysis in rat hepatocytes but other phosphodiesterase inhibitors including Ro 20-1724 had no effect. Dose-response curves for MIX potentiation of cAMP-stimulated glycogenolysis and for MIX inhibition of the effects of insulin on cAMP-stimulated glycogenolysis suggested that at higher concentrations (250 microM) MIX may act at a site other than phosphodiesterase inhibition. MIX, at 250 microM, attenuated the insulin antagonism of glucose release stimulated by 8-bromo-cAMP, an extremely poor substrate for phosphodiesterase; other phosphodiesterase inhibitors did not. The possibility that MIX acts as an adenosine antagonist interfering with a postulated role for adenosine in insulin action was examined using N6-phenylisopropyladenosine (PIA), an Ra adenosine receptor agonist which increases hepatic cAMP levels. MIX inhibited insulin antagonism of PIA-stimulated glycogenolysis under conditions where it did not act as an adenosine antagonist (MIX and Ro 20-1724 both increased the response to PIA equally). The effect of concanavalin A on cAMP-stimulated glycogenolysis was antagonized by MIX, suggesting a post-receptor site of action for MIX. MIX paradoxically increased lactate production in the presence of 8-bromo-cAMP, reminiscent of the reported actions of calcium mobilizing hormones on lactate formation in fed hepatocytes. Cytosolic free Ca2+, as measured in Quin 2-loaded cells, was increased by MIX. In cells depleted of calcium, MIX no longer blocked insulin antagonism of 8-bromo-cAMP-stimulated glucose release, suggesting that MIX may function through an insulin-insensitive release of calcium. MIX greatly potentiated the stimulation of glycogenolysis by phenylephrine but did not alter the response to vasopressin. The relationship of this effect of MIX to the mechanism of insulin action and the ability of insulin to antagonize only alpha-adrenergic responses and not those of vasopressin is discussed.     

In vivo regulation of cyclic AMP phosphodiesterase in Xenopus oocytes. Stimulation by insulin and insulin-like growth factor 1

Cyclic AMP phosphodiesterase activity was measured in vivo after microinjection of [3H]cAMP into intact Xenopus oocytes. This activity was inhibited by extracellular application of methylxanthines, and the dose-dependent inhibition of phosphodiesterase activity correlated with the abilities of isobutylmethylxanthine and theophylline to inhibit oocyte maturation induced by progesterone, with IC50 values of approximately 0.3 and 1.5 mM, respectively. Insulin stimulated in vivo phosphodiesterase activity measured after microinjection of 200 microM [3H]cAMP in a time- and dose-dependent fashion without affecting phosphodiesterase activity measured after microinjection of 2 microM [3H]cAMP. Although progesterone alone had no effect on in vivo phosphodiesterase activity, low concentrations of progesterone (0.01 microM) accelerated the time course of insulin stimulation of both phosphodiesterase activity and oocyte maturation. The EC50 for stimulation of in vivo phosphodiesterase activity by insulin correlated with the IC50 for inhibition of oocyte membrane adenylate cyclase activity measured in vitro (2 and 4 nM, respectively). Twenty-fold higher concentrations of insulin were required to stimulate oocyte maturation. In contrast, insulin-like growth factor 1 stimulated in vivo phosphodiesterase, inhibited in vitro adenylate cyclase, and induced oocyte maturation at concentrations of 0.3-1.0 nM. These results demonstrate a dual regulation of oocyte phosphodiesterase and adenylate cyclase by insulin and insulin-like growth factor 1.     

Regulation by insulin of cyclic nucleotide phosphodiesterase (PDE) from rat luteal cells

The effect of insulin on cyclic nucleotide phosphodiesterase (PDE) in rat luteal cells was studied. Cells were obtained from PMSG/hCG primed rats and further incubated or not with insulin. The hormone produced an increase of enzyme activity after a 10 min incubation of intact cells. Maximal stimulation was achieved at 0.2 nM of insulin. Two peaks of cyclic nucleotide phosphodiesterase activity were resolved after chromatography of cell cytosolic extracts on DEAE-cellulose. These peaks (I and II) were active with cAMP as substrate but only peak I was active with cGMP. The enzyme activity of both peaks was increased in cells treated with insulin. Phosphodiesterase activity in the two peaks show two kinetic components for cAMP hydrolysis, one of high affinity (Km 2-4 microM) and the other of low affinity (47-56 microM). Treatment of the cells with insulin produced a 2 to 8 fold increase of the Vmax of these peaks. In addition after stimulation with insulin, the activation of peak I phosphodiesterase by calmodulin was less effective.     

Two classes of cAMP analogs which are selective for the two different cAMP-binding sites of type II protein kinase demonstrate synergism when added together to intact adipocytes

Twenty-five cyclic nucleotide analogs were tested individually to act as lipolytic agents and to activate adipocyte protein kinase. The lipolytic potency of individual analogs correlated better with their Ka for protein kinase and their lipophilicity rather than with either parameter alone. Some of the most potent lipolytic analogs had I50 values for the particulate low Km cAMP phosphodiesterase suggesting that their effect was not due to raising endogenous cAMP levels through inhibition of phosphodiesterase. The most potent lipolytic analogs contained a thio moiety at the C-8 or C-6 position. These analogs exhibited concave upward dose-response curves. At high concentrations, some analogs were as effective as optimal concentrations of epinephrine in stimulating glycerol release. The regulatory subunit of protein kinase has two different intrachain cAMP-binding sites and cAMP analogs modified at the C-8 position (C-8 analogs) are generally selective for Site 1 and analogs modified at the C-6 position (C-6 analogs) are generally selective for Site 2 (Rannels, S. R., and Corbin, J. D. (1980) J. Biol. Chem. 255, 7085-7088). Thus, C-8 and C-6 analogs were tested in combination to stimulate lipolysis in intact adipocytes and to activate protein kinase in vitro. Each process was stimulated synergistically by a combination of a C-6 and C-8 analog. Two C-8 analogs or two C-6 analogs added together did not cause synergism of either process. For both lipolysis and protein kinase activation, C-8 thio analogs acted more synergistically than C-8 amino analogs when incubated in combination with C-6 analogs, a characteristic of type II protein kinase. It is concluded that the observed synergism of lipolysis is due to binding of cAMP analogs to both intrachain sites and that it is the type II protein kinase isozyme which is responsible for the lipolytic response.     

A role for soluble cAMP phosphodiesterases in differentiation of 3T3-L1 adipocytes

Differentiation of 3T3-L1 adipocytes, monitored by accumulation of neutral lipid and by increase in alpha-glycerophosphate dehydrogenase activity, is accelerated by incubation of confluent 3T3-L1 fibroblasts in media containing insulin, dexamethasone and isobutylmethylxantine (IBMX). IBMX inhibits cyclic nucleotide phosphodiesterases as well as the binding of adenosine to its receptor. Agents with relatively specific effects were utilized to examine the role of IBMX in differentiation. Ro 20-1724, a selective inhibitor of soluble cAMP phosphodiesterase activities, was as effective as IBMX in increasing alpha-glycerophosphate dehydrogenase activity and fat deposition. Neither cilostamide, which inhibits particulate but not soluble cAMP phosphodiesterase activities, 8-phenyltheophylline, an adenosine receptor antagonist with little inhibitory effect on phosphodiesterase activities, nor N6-(R phenyl-isopropyl) adenosine (PIA), a potent adenosine receptor agonist, were effective in promoting differentiation. In addition, we find that maximal increases in alpha-glycerophosphate dehydrogenase activity and lipid accumulation were observed when differentiation was initiated in the presence of 10 nM dexamethasone. These data suggest that inhibition of soluble cAMP phosphodiesterase activity and subsequent alterations in cAMP may play an important role in the mechanism whereby IBMX enhances differentiation of 3T3-L1 cells.     

Phosphodiesterase inhibition by a gastroprotective agent irsogladine: preferential blockade of cAMP hydrolysis

The effect of irsogladine [2,4-diamino-6-(2,5-dichlorophenyl)-s-triazine maleate], an antiulcer drug, on contents of cyclic nucleotides including cAMP and cGMP was investigated in rat stomachs. Irsogladine concentration-dependently increased cAMP content in rat glandula stomach. However, irsogladine at higher concentration (10(-5) M) was unable to further increase cAMP level in the presence of non-selective phosphodiesterase (PDE) inhibitor 3-isobutyl-1-methylxanthine, although 3-isobutyl-1-methylxanthine by itself increased cAMP level. On the other hand, irsogladine had no effect on the glandula cGMP content. Subsequently, the effect of irsogladine on the cyclic nucleotide degradation by purified bovine brain and heart PDEs was investigated. The cAMP degradation by purified bovine brain PDE was partially suppressed by PDE1 inhibitor vinpocetin, PDE2 inhibitor erythro-9-(2-hydroxy-3-nonyl)adenine hydrochloride and PDE4 inhibitor rolipram but not by PDE3 inhibitor cilostamide, and completely inhibited by 3-isobutyl-1-methylxanthine, suggesting that is attributed almost exclusively to PDE1, PDE2 and PDE4. Meanwhile, cGMP degradation by purified bovine brain PDE was partially suppressed by erythro-9-(2-hydroxy-3-nonyl)adenine hydrochloride. Irsogladine preferentially inhibited the response to cAMP degradation compared with cGMP degradation by this brain PDE. The cAMP degradation by bovine heart PDE was almost completely inhibited by the combination with vinpocetine and cilostamide, indicating that is mediated almost exclusively by PDE1 and PDE3. Irsogladine suppressed this cAMP degradation measured in the presence of vinpocetine to almost the same extent as that determined in the presence of cilostamide. These results indicate that irsogladine produces the increase of intracellular cAMP content via non-selective inhibition of PDE isozymes, which may be a key mechanism involved in its gastroprotective actions.     

Purification of the putative hormone-sensitive cyclic AMP phosphodiesterase from rat adipose tissue using a derivative of cilostamide as a novel affinity ligand

A "low Km" cAMP phosphodiesterase with properties of a peripheral membrane protein accounts for approximately 90% of total cAMP phosphodiesterase activity in particulate (100,000 X g) fractions from rat fat cells. Incubation of fat cells with insulin for 10 min increased particulate (but not soluble) cAMP phosphodiesterase activity, with a maximum increase (approximately 100%) at 1 nM insulin. Most of the increase in activity was retained after solubilization (with non-ionic detergent and NaBr) and partial purification (approximately 20-fold) on DEAE-Sephacel. The solubilized enzyme from adipose tissue was purified approximately 65,000-fold to apparent homogeneity (yield approximately 20%) by chromatography on DEAE-Sephacel and Sephadex G-200 and affinity chromatography on aminoethyl agarose conjugated with the N-(2-isothiocyanato)ethyl derivative of the phosphodiesterase inhibitor cilostamide (OPC 3689). A 63,800 +/- 200-Da polypeptide (accounting for greater than 90% of the protein eluted from the affinity column) was identified by polyacrylamide gel electrophoresis in sodium dodecyl sulfate (with or without reduction). Enzyme activity was associated with the single protein band after electrophoresis under nondenaturing conditions. On gel permeation, Mr(app) was 100,000-110,000, suggesting that the holoenzyme is a dimer. A pI of 4.9-5.0 was estimated by isoelectric focusing. At 30 degrees C, the purified enzyme hydrolyzed both cAMP and cGMP with normal Michaelis-Menten kinetics; the pH optimum was 7.5. The Km(app) for cAMP was 0.38 microM and Vmax, 8.5 mumol/min/mg; for cGMP, Km(app) was 0.28 microM and Vmax, 2.0 mumol/min/mg. cGMP competitively inhibited cAMP hydrolysis with a Ki of approximately 0.15 microM. The enzyme was also inhibited by several OPC derivatives and "cardiotonic" drugs, but not by RO 20-1724. It was very sensitive to inhibition by agents which covalently modify protein sulfhydryls, but not by diisopropyl fluorophosphate. The activation by insulin and other findings indicate that the purified enzyme, which seems to belong to a subtype of low Km cAMP phosphodiesterases that is specifically and potently inhibited by cGMP, cilostamide, other OPC derivatives, and certain cardiotonic drugs, is likely to account for the hormone-sensitive particulate low Km cAMP phosphodiesterase activity of rat adipocytes.     

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.