low-Km Phosphodiesterase Activity Research Articles

Failure of insulin to antagonize cAMP-mediated glycogenolysis in rat ventricular cardiomyocytes

Isolated rat ventricular cardiomyocytes were used to study the effects of insulin on glycogen metabolism in cells treated with various agents that activate adenosine 3',5'-cyclic monophosphate (cAMP)-dependent protein kinase. Incubation of myocytes with isoproterenol produced a rapid concentration-dependent increase in cAMP concentration, cAMP-dependent protein kinase activity, and phosphorylase activity and a simultaneous decrease in the glycogen synthase activity ratio. Various cAMP analogues also produced a concentration-dependent increase in phosphorylase activity and a decline in the glycogen synthase activity ratio. Incubation of cells with insulin produced no change in basal phosphorylase activity but produced a rapid 40% increase in the glycogen synthase activity ratio. Inclusion of insulin in cell incubations containing increasing concentrations of isoproterenol did not modify the increases in cAMP concentration, protein kinase activity, or phosphorylase activity. Insulin also did not antagonize the ability of any of the cAMP analogues tested to activate phosphorylase, irrespective of the suitability of the particular cAMP analogue as a substrate for cAMP phosphodiesterases. The failure of insulin to antagonize the glycogenolytic effects of isoproterenol or cAMP analogues was paralleled by its failure to activate low-Km phosphodiesterase activity, but the cAMP analogue, 8-parachlorophenylthio-cAMP produced a small reproducible activation of the low-Km enzyme. In contrast to hepatocytes and adipocytes, where some effects of insulin appear to be due to activation of the phosphodiesterase and hydrolysis of cAMP, the effects in cardiomyocytes appear to be independent of an insulin-sensitive phosphodiesterase or of the effects on other components of the cAMP cascade.

Activation of the particulate low Km phosphodiesterase of adipocytes by addition of cAMP-dependent protein kinase.

The purified catalytic subunit (C) of cAMP-dependent protein kinase produced a 2-fold activation of the low Km phosphodiesterase in crude microsomes (P-2 pellet) of rat adipocytes. This activation was C subunit concentration-dependent, ATP-dependent, blocked by a specific peptide inhibitor, and lost if the C subunit was first heat denatured. The concentration of ATP necessary for half-maximal activation of the low Km phosphodiesterase was 4.50 +/- 1.1 microM, which was nearly the same as the known Km of C subunit for ATP (3.1 microM) using other substrates. The concentration of C subunit producing half-maximal activation of phosphodiesterase was 0.22 +/- 0.04 microM, slightly less than the measured concentration of total C subunit in adipocytes (0.45 microM). The activation of the low Km phosphodiesterase by C subunit was specific, since on an equimolar basis, myosin light chain kinase, cGMP-dependent protein kinase, or Ca2+/calmodulin-dependent protein kinase II did not activate the enzyme. The percent stimulation of phosphodiesterase by C subunit was about the same as that produced by incubation of adipocytes with a cAMP analog, and the enzyme first activated in vivo with the analog was not activated to the same extent (on a percentage basis) by in vitro treatment with C subunit. Treatment of the crude microsomes with trypsin resulted in transfer of phosphodiesterase catalytic activity from the particulate to the supernatant fraction, but the enzyme in the supernatant was minimally activated by C subunit, suggesting either loss or dislocation of the regulatory component. The C subunit-mediated activation of phosphodiesterase was preserved after either transfer of phosphodiesterase activity to the supernatant fraction by nonionic detergents or partial purification of the transferred enzyme. The present findings are consistent with the suggestion that protein kinase regulates the concentration of cAMP through phosphodiesterase activation and provide direct evidence that the mechanism of activation involves phosphorylation.

Short-term feedback regulation of cAMP by accelerated degradation in rat tissues.

A recent study showed that cAMP analogs lowered cAMP levels in rat hepatocytes (Corbin, J.D., Beebe, S.J., and Blackmore, P.F. (1985) J. Biol. Chem. 260, 8731-8735). The present work demonstrates that cAMP analogs also lowered cAMP in a rapid, concentration-dependent manner in heart and fat cells. In order to determine if the cAMP-dependent protein kinase mediated this effect, techniques were developed to assay the protein kinase activity ratio in hepatocytes treated with cAMP analogs. The activation of protein kinase and phosphorylase in hepatocytes by 8-pCl phi S-cAMP (where 8-pCl phi S- indicates 8-parachlorothiophenyl-) was concentration-dependent and occurred in parallel to proportionate decreases in cAMP. More than 20% of the cAMP binding sites on the protein kinase were unoccupied at concentrations of 8-pCl phi S-cAMP that produced maximal cAMP lowering. Thus, the possibility that 8-pCl phi S-cAMP lowered cAMP by displacing it from protein kinase binding sites, making it available for hydrolysis, seemed unlikely. In adipocytes, the lowering of cAMP by 8-pCl phi S-cAMP occurred in parallel with increases in lipolysis and activation of low Km phosphodiesterase, suggesting that the phosphodiesterase was responsible for the cAMP lowering. Further evidence for this assertion was the finding that in hepatocytes preloaded with low concentrations of 8-pCl phi S-cAMP, glucagon lowered 8-pCl phi S-cAMP by about 50%, an amount similar to the cAMP lowering observed with 8-pCl phi S-cAMP treatment. The results were consistent with a cAMP-dependent protein kinase-catalyzed activation of a phosphodiesterase and suggested that 8-pCl phi S-cAMP-mediated hydrolysis of cAMP mimicked a physiologically significant response. The observation of this phenomenon in several tissues further suggested that it may be a general mechanism for dampening and terminating the hormonal signal through accelerated degradation of cAMP.

Acute in vivo stimulation of low-Km cyclic AMP phosphodiesterase activity by insulin in rat-liver Golgi fractions.

A low-Km phosphodiesterase activity, which is acutely stimulated by insulin in vivo, has been identified in plasma membranes and Golgi fractions prepared from rat liver homogenates in isotonic sucrose. Within seconds after insulin injection (25 micrograms/100 g body weight) cAMP phosphodiesterase activity increases by 30-60% in Golgi fractions and by 25% in plasma membranes; activity in crude particulate and microsomal fractions is unaffected. The increase in activity is short-lived in the light and intermediate Golgi fractions, but persists for at least 10 min in the heavy Golgi fraction. It precedes the translocation of insulin and insulin receptors to these fractions, which is maximal at 5 min. The doses of insulin required for half-maximal and maximal activation are, respectively, 7.5 micrograms/100 g and 25 micrograms/100 g body weight. Golgi-associated cAMP phosphodiesterase activity shows non-linear kinetics; a high-affinity component (Vmax, 13 pmol min-1 mg protein-1; Km, 0.35 microM) is detectable. Insulin treatment increases the Vmax 60-70%, but does not affect the Km. Unlike the low-Km cAMP phosphodiesterase associated with crude particulate fractions, the Golgi-associated activity is not easily extractable by solutions of low or high ionic strength. On analytical sucrose density gradients, low-Km cAMP phosphodiesterase associated with the total particulate fraction equilibrates at lower densities than endoplasmic reticulum and lysosomal markers, but at a higher densities than plasma membrane, Golgi markers and insulin receptors. Insulin treatment increases the specific activity of the enzyme by 20-60% at densities below 1.12 g cm-3, and by 20-40% in the density interval 1.23-1.25 g cm-3. Such treatment also causes a slight, but significant shift in the distribution of phosphodiesterase towards lower densities. It is suggested that Golgi elements or physically similar subcellular structures are a major site of localization of insulin-sensitive cAMP phosphodiesterase in rat liver. However, internalization of the insulin-receptor complex is probably not required for enzyme activation.

Is increased basal lipolysis in adipose tissue of fasted-refed rats related to cyclic-AMP-dependent mechanisms?

The possible contribution of the cyclic AMP/protein kinase system to the enhanced basal lipolytic activity of rat adipose tissue in vitro after fasting and refeeding a carbohydrate-rich diet was investigated. 1 After 120 h of fasting/48 h of refeeding, the basal lipolytic rate was fourfold above normal, and the protein kinase activity ratio was significantly elevated. However, basal adenylate cyclase activity was not significantly different from normal, and basal cyclic AMP levels were unchanged despite an increased (+40%) low-Kmphosphodiesterase activity. 2 Insulin (500 μ/ml) stimulated (50%) low-Km, but not high-Kmphosphodiesterase activity in normal adipose tissue, but had no further stimulatory effect on the elevated low-Kmphosphodiesterase activity in the fasted-refed tissue. At the same concentration, insulin completely blocked enhanced basal lipolysis in the refed state and caused a small transient decrease of the cyclic AMP level (-16%) and of the protein kinase activity ratio (-10%), whereas it did not significantly influence any of the three latter indices in the normal tissue. Although adenosine (0.1 mM), like insulin, completely blocked enhanced lipolysis in the refed tissue, it did not lower tissue cyclic AMP or the protein kinase activity ratio. 3 After 72 h of fasting/72h of refeeding, the basal lipolytic activity of the adipose tissue was enhanced threefold above normal. However, the tissue cyclic AMP level and the protein kinase activity ratio were not different from those of normal tissue. 4 Lipolytic activity in homogenates from fasted-refed tissue previously exposed to epinephrine was twofold higher than in homogenates from the untreated tissue. It corresponded to the epinephrine-stimulated lipolytic rate of the ‘intact’ tissue and was accompanied by a drastic rise of the tissue cyclic AMP level and of the protein kinase activity ratio. In contrast, the pronounced inhibitory effect of insulin on enhanced basal lipolysis of refed tissue did not persist in homogenates of the same tissue preincubated with this hormone. These results reveal a number of discrepancies that are difficult to reconcile with an obvious and simple cyclic-AMP-dependent activation of basal lipolysis in the refed state. Furthermore, they suggest that the antilipolytic effect of insulin on fasted-refed-adipose tissue is unlikely to solely mediated by the cyclic AMP/protein kinase system.

Thiazide diuretics do not potentiate cAMP response to parathyroid hormone

We evaluated the hypothesis that thiazide-induced hypercalcemia reflects potentiation of the cAMP response to parathyroid hormone (PTH) consequent to inhibition of phosphodiesterase in bone and kidney. A panel of thiazide diuretics did inhibit low-Km phosphodiesterase activity from bone homogenates. However, furosemide, a nonthiazide diuretic that does not promote calcium retention, was more potent a phosphodiesterase inhibitor than either chloro- or hydrochlorothiazide (CTZ, HCTZ). Thiazides did not influence basal or PTH-stimulated cAMP levels in incubated calvaria or renal cortical slices. Administration of CTZ or HCTZ to rats for 4 days did not affect basal cAMP, nor did such treatment potentiate the cAMP response in Calvaria to infusion of parathyroid extract in vivo. CTZ, HCTZ, and furosemide increased basal adenylate cyclase from renal cortex but did not affect PTH-stimulated activity. Adenylate cyclase from bone was not affected by thiazides but was inhibited by furosemide. Thiazide treatment potentiated the calcemic response to parathyroid extract in vivo but did not affect the calcemic response to dibutyryl cAMP. We conclude that potentiation of the cAMP response to PTH does not underlie the unique effects of thiazides on calcium metabolism.

Effect of Insulin and Lipolytic Hormones on Cyclic AMP Phosphodiesterase Activity in Normal and Diabetic Rat Adipose Tissue

Insulin has been shown to lower cyclic AMP (cAMP) levels in hormonally sensitive tissue. The mechanism by which this lowering occurs has not yet been fully defined. We studied the effects of insulin on rat adipose tissue cyclic nucleotide phosphodiestrase (PDE) in an incubation system. The adipose tissue used was from both normal animals and animals rendered diabetic by intravenous injections of streptozotocin. Rat epididymal fat pads were incubated in a Krebs-Ringer bicarbonate-4% albumin system with O, 100, 1,000 or 10,000 PU/ml insulin (INS); epinephrine (EPI) or glucagon (GLU) at several different concentrations. After 15 min of incubation, each tissue was homogenized, centrifugated, and the supernatant assayed for cAMP PDE activity using the breakdown of (3-H)cAMP. The data was used to characterize cAMP PDE into apparent high and low K-m PDE components. In the normal animals, INS increased Vmax of the low Km PDE components; 100 pU/ml INS, 30%, 1000 p1/ML INS, 40; and 10,000 pU/ml INS, 20%. In contrast, streptoxotocin diabetes lowered this Vmax by 30%. In the diabetic animals, INS also increased Vmax by 30%. In the diabetic animals, INS also increased Vmax of the low Km PDE component; 100 pU/ml INS, 30%; 1000 pU/ml INS, 50% and 10,000 pU/ml INS, 100%. Epinephrine at 1, 10, and 100 pg/ml stimulated low Km cAMP PDE activity by 67%, 73% and 44% respectively. The stimulatory effect of EPI on both the low and high Km cAMP PDE activity was neutralized by propranolol or adenosine. In comparison to EPI, GLU at very low concentrations, 10-9M, stimulated low Km cAMP PDE. These studies suggest that some of the biologic actions of insulin, an antilipolytic substance, are mediated through activation of low Km PDE. Furthermore, this enzymatic activity is lower in experimental diabetes. The stimulation of low Km PDE by lipolytic hormones may reflect a long-range protective action of these agents.

Effect of Insulin and Lipolytic Agents on Rat Adipocyte Low Km Cyclic Adenosine 3′ : 5′-Monophosphate Phosphodiesterase

Abstract Isolated rat fat cells were incubated with insulin or lipolytic agents and measurements made of the activity of low Km cyclic adenosine 3' : 5'-monophosphate (cyclic AMP) phosphodiesterase in subsequently prepared fat-free homogenates. In the assay [3H]adenosine production from cyclic[3H]AMP was measured. Insulin consistently increased Vmax, did not alter Km (0.3 µm), and was ineffective when added to homogenates. As little as 7.5 microunits per ml of insulin increased phosphodiesterase activity by 18% and 50 microunits produced maximum stimulation of 65%. Incubation of fat cells with isoproterenol, epinephrine, or adrenocorticotropic hormone increased phosphodiesterase activity by 20 to 30 %. The effect of lipolytic hormones on phosphodiesterase activity could be reproduced when fat cells were incubated with 2.0 mm N6, O2'-dibutyryl adenosine 3' : 5'-monophosphate. The effect of insulin on phosphodiesterase activity was apparent within 2 min, was maximum at 5 min, and was sustained for at least 60 min. The response to isoproterenol was equally rapid, reaching a peak at 5 to 10 min but was not sustained. The fall in phosphodiesterase activity observed with isoproterenol was apparently related to intracellular ATP depletion. Homogenates prepared from fat cells preincubated with insulin and isoproterenol were separated into 50,000 x g particulate and supernatant fractions. The low Km phosphodiesterase activity associated with the particulate fraction was increased, while that of the supernatant was unaffected by hormonal preincubation. These data suggest that the antilipolytic effect of insulin may be mediated through activation of low Km phosphodiesterase. As well, the activation of phosphodiesterase produced by lipolytic hormones may be responsible for the failure of these agents to produce sustained increases in adipocyte intracellular cyclic AMP.