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Biomedical subjects

R A Mooney

Publications and source records attributed to R A Mooney.

At least 55 records · Page 3Linked to original sources
PubMed1986

Coupling of adenylate cyclase to lipolysis in permeabilized adipocytes: direct evidence that an antilipolytic effect of insulin is independent of adenylate cyclase.

In digitonin-permeabilized adipocyte suspensions, adenylate cyclase activation was analyzed by an in situ assay and compared to lipolytic activity which was determined under essentially identical assay conditions. This approach permitted direct comparison of the regulatory responses of these two processes. Adenylate cyclase was activated by isoproterenol in permeabilized adipocytes, but digitonin caused a decrease in sensitivity to the beta-agonist. On the other hand, activation of adenylate cyclase by forskolin and NaF was unaffected by digitonin. Inhibition of isoproterenol-activated adenylate cyclase by propranolol was also unaffected by digitonin. These observations are consistent with a partial uncoupling by digitonin of the interaction between occupied beta-adrenergic receptors and the stimulatory GTP-binding component (Ns). Despite permeabilization, adenylate cyclase activity retained the capability to activate the lipolytic pathway. Activation of adenylate cyclase and lipolysis by isoproterenol was first detectable at 10(-8) M. At 10(-7) M isoproterenol, when the cyclase activity increase was 11 pmol cAMP formed/min X 10(6) cells (21% increase over basal), lipolysis was increased 153%. Forskolin was a more effective activator of adenylate cyclase than isoproterenol, increasing activity maximally by greater than 7-fold in contrast to a maximum of 4.5-fold activation by the beta-agonist. Forskolin-dependent adenylate cyclase activity increased by 63 pmol cAMP/min X 10(6) cells, however, before an increase in lipolysis was first detected (33% increase). Thus, cAMP generated by the two lipolytic agents was not identical in its lipolytic potential. Maximum rates of lipolysis were equal for the two agents. Insulin (10(-9) M) exerted an antilipolytic effect on lipolysis in permeabilized adipocytes whether the lipolytic activator was isoproterenol or forskolin. At 10(-6) and 10(-4) M isoproterenol, the magnitudes of the antilipolytic effect were 35% and 21%, respectively, whereas forskolin-activated lipolysis (at 10(-6) M) was inhibited 22%. Insulin had no effect on adenylate cyclase activity stimulated by either agent and assayed under essentially identical conditions. In summary, direct comparison of adenylate cyclase activity and lipolysis in digitonin-permeabilized adipocytes demonstrated a more efficient coupling of adenylate cyclase to lipolysis when the enzyme was activated by isoproterenol rather than forskolin. Additionally, the antilipolytic effect of insulin was preserved in the permeabilized adipocytes, but this effect did not involve inhibition of adenylate cyclase.

Adenylyl Cyclases↗
PubMed1986

Clinical utility of measuring free thyroxin and free triiodothyronine in serum of critically ill patients by ultrafiltration.

Free thyroxin (FT4) measurements by ultrafiltration were compared with results obtained by equilibrium dialysis and commercial RIA methods. In a group of critically ill patients, the mean +/- 2 SD FT4 value by ultrafiltration (26.5 +/- 21.4 ng/L) was higher than in the healthy reference population (19.3 +/- 9.8 ng/L, p less than 0.0001), and correlated well with equilibrium dialysis (23.8 +/- 20 ng/L, r = 0.87). By a two-step RIA method (Clinical Assays), the FT4 value was greater in the critically ill (14.7 +/- 9.6 ng/L) than in the reference population (12.8 +/- 5 ng/L, p less than 0.001) and was also correlated with ultrafiltration results (r = 0.71). FT4 values for the ill patients were lower than for the reference population in two one-step RIA methods (Clinical Assays: 8.6 +/- 7.1 ng/L; Corning Medical: 12.7 +/- 9.4 ng/L), neither of which gave results correlated with ultrafiltration (r = 0.16 and 0.22, respectively). The concentration of free triiodothyronine (FT3), also measured by ultrafiltration, was 4.5 +/- 2.2 ng/L in healthy subjects, 12.5 +/- 14.2 ng/L in hyperthyroidism, 1.7 +/- 2.0 ng/L in hypothyroidism, 2.2 +/- 1.9 ng/L in the critically ill subjects, and 3.8 +/- 0.9 ng/L in pregnancy. Thus, FT4 and FT3 measured by ultrafiltration accurately affects assessment of the thyroid status of the patient except in critical illnesses in which FT3 values are indistinguishable from those in hypothyroidism.

Acute Disease↗
PubMed1984

Insulin-mediated antilipolysis in permeabilized rat adipocytes.

Elucidating the mechanism by which insulin inhibits lipolysis has been hampered by the unavailability of a broken cell preparation in which the intact cell responses to the hormone could be duplicated. Here we report, using digitonin-permeabilized rat adipocytes, that physiological concentrations of insulin inhibit cyclic AMP-activated lipolysis despite the absence of cytosolic and plasma membrane integrity. Cyclic AMP (1.0 mM) maximally activates lipolysis in permeabilized adipocytes greater than 10-fold. Insulin inhibits this activation in a biphasic manner with maximum inhibition of 59 +/- 8% (N = 7) at 10(-9) M. At the submaximal concentrations of cyclic AMP (1.0 to 10 microM), insulin (10(-9) M) inhibits lipolysis 80 to 90%. Additionally, the antilipolytic effect of insulin is rapid (3 min) and it is specific, with the relatively inactive desoctapeptide analogue of insulin being three orders of magnitude less inhibitory than native insulin. In contrast to permeabilized cells, intact cells demonstrate only a small lipolytic response to cyclic AMP which is insensitive to insulin. These findings suggest the following about insulin's antilipolytic effects: 1) an intact cell is not required; 2) the intracellular mechanism of action does not require physiological concentrations of the freely diffusible cytosolic components; and 3) a site of insulin action independent of adenylate cyclase may play a major role.

Adipose Tissue↗
PubMed1984

Effect of phenylephrine on lipolysis in rat adipocytes: no evidence for an alpha-adrenergic mechanism.

Phenylephrine, a strong alpha 1-adrenergic agonist, exerted a concentration dependent antilipolytic effect against isoproterenol-activated lipolysis in rat adipocytes with the effect decreasing as the isoproterenol concentration increased. The alpha-adrenergic antagonists phentolamine and phenoxybenzamine did not reverse phenylephrine's antilipolytic effect. Phenylephrine alone activated lipolysis at concentrations above 10(-5) M and at 5 X 10(-4) M the rate of lipolysis was increased 3.4-fold. Propranolol abolished this effect. In the presence of sub-maximum concentrations of dibutyryl cyclic-AMP (less than 10(-4) M), 10(-4) M phenylephrine increased the rate of lipolysis above that activated by dibutyryl cyclic-AMP alone. At maximum dibutyryl cyclic-AMP concentrations, or in the presence of propranolol, phenylephrine had no effect on dibutyryl cyclic-AMP-dependent lipolysis. There is no evidence to support an alpha 1-adrenergic mechanism for regulation of lipolysis in the rat adipocyte. All effects of the alpha-adrenergic agonist phenylephrine appear to be due to its weak beta-adrenergic activity.

Adipose Tissue↗
PubMed1983

Novel approach to the study of the regulation of hormone-sensitive lipase in rat adipocytes. Permeabilization of cells with digitonin.

Use of a digitonin-permeabilized rat adipocyte preparation overcomes inherent problems which occur when currently used broken cell systems are utilized for studying the regulation of hormone-sensitive lipase. The effect of digitonin on plasma membrane permeability was concentration-dependent being nearly maximum at 20 micrograms/ml as assessed by (a) leakage of 85% cellular lactate dehydrogenase after 30 min, (b) the efflux of 72% preloaded cellular (86Rb) rubidium within 10 min and (c) immediate inhibition of glucose oxidation. Hormone-modulated rates of lipolysis were preserved in this preparation. Following maximal activation of lipolysis in adipocytes with catecholamines, the rate of lipolysis in intact cells and digitonin-treated cells was elevated 26-fold and 20-fold respectively, while the rate in homogenates from these cells was elevated only 2.8-fold. Insulin suppressed catecholamine-dependent activation of lipolysis by at least 90% when subsequently measured in intact cells and digitonin-treated cells. Insulin suppression was only 56% when measured in homogenates. The hormone-sensitive lipase in permeabilized cells, as opposed to intact cells and homogenates, was activated by cyclic AMP to a degree that approached activation by catecholamines. In homogenates, cyclic AMP (1.0 mM) plus ATP (0.25 mM) activated the lipase only 36%, while neither alone had any effect. In digitonin-permeabilized cells, however, exogenous cyclic AMP alone activated lipolysis in a concentration-dependent manner with 1 microM, 30 microM and 1.0 mM cyclic AMP activating lipolysis by 41%, 250% and 1300% respectively. In contrast, lipolysis in intact cells was activated by 0%, 25% and 250% by 1 microM, 30 microM and 1.0 mM cyclic AMP. Also in digitonin-treated preparations, ATP alone activated lipolysis 40%, but ATP plus cyclic AMP activated lipolysis to only 74% of the level due to cyclic AMP alone. These studies indicate that the permeabilized adipocyte preparation is an excellent system for investigating the mechanism of regulation of the hormone-sensitive lipase by permitting manipulation of the intracellular environment while preserving the physiological response of the lipase.

Adenosine Triphosphate↗
PubMed1981

Formation and turnover of triglyceride-rich vesicles in the chick liver cell. Effects of cAMP and carnitine on triglyceride mobilization and conversion to ketones.

Refractile cytoplasmic vesicles are formed in less than 10 h when chick liver cell monolayers are incubated with serum-free medium containing 0.9 mM oleate. These vesicles are identical in microscopic appearance to those formed in monolayers by de novo fatty acid synthesis (Tarlow, D. M., Watkins, P. A., Reed, R. E., Miller, R. S., Zwergel, E. E., and Lane, M. D. (1977) J. Cell Biol. 73, 332-353), but require about one-seventh the incubation time to achieve comparable size. After release from the cells by lysis in hypotonic medium, the vesicles can be isolated by flotation at 27,000 X g. Electron microscopy reveals that the isolated vesicles are rimmed by a membrane. Analysis of vesicles isolated from cells labeled with [14C]oleate or [14C]acetate showed that greater than 95% of their 14C content was in the form of triglyceride and that most cellular [14C]triglyceride was contained in the triglyceride-rich vesicles. Exposure of cells to dibytyryl-cAMP after removal of oleate from the medium caused the disappearance of triglyceride-rich vesicles within 36 h. In the absence of cyclic nucleotide, the vesicles persist. Consistent with this morphological change, dibutyryl-cAMP caused a 5.5-fold activation of the apparent rate of mobilization of cellular [14C]triglyceride from cells previously labeled with [14C]oleate. L-(--)-Carnitine alone had no effect; however, when added with dibutyryl-cAMP, cellular triglyceride mobilization was activated 7.4-fold. Although [14C]triglyceride was the principal 14C-labeled product secreted in the absence of cyclic nucleotide and comprised 90% of the total, [14C]acetoacetate and [14C] beta-hydroxybutyrate became major products when cells were treated with dibutyryl-cAMP. Thus, dibytyryl-cAMP activated ketogenesis from cellular [14C]triglyceride by 200-fold and when added with L-(--)-carnitine, by 400-fold. Cells containing triglyceride-rich vesicles labeled with [2-glyceryl-3H]triglyceride were generated by incubation with medium containing [2-3H]glycerol. A comparison of the rates of loss of cellular [1-oleoyl-14C- and [2-glyceryl-3H]triglyceride revealed that substantial re-esterification, i.e. recycling, of 14C-fatty acid released by lipolysis occurred. Under conditions where recycling of 3H label ws minimal, it was determined that 15% of the cellular [2-glyceryl-3H]triglyceride was secreted "en bloc," i.e. without prior lipolysis. En bloc secretion was not affected by dibutyryl-cAMP. The rate of lipolysis of vesicle-associated [2-glyceryl-3H]triglyceride was increased 2.2-fold in the presence of dibutyryl-cAmP. Chloroquine markedly inhibited the dibutyryl-cAMP-dependent lipolysis suggesting the participation of lysosomes in the mobilization of triglyceride-rich vesicles. Mechanisms are presented which could account for the effects of cAMP and carnitine on the turnover of vesicle triglyceride both at the level of lipolysis and the utilization of the released fatty acids by mitochondria...

Animals↗
PubMed1976

Prevention of peritoneal adhesions with aprotinin (trasylol).

An objective assessment of intraperitoneal trasylol in the prevention of abdominal adhesions in 20 patients has shown that Trasylol resulted in a marked reduction in adhesions in 16 patients who underwent 'second look' procedures. No side-effects were noted and all wounds healed without complications.

Adolescent↗
PubMed1975

Intracellular localization of the 3-hydroxy-3-methylglutaryl coenzme A cycle enzymes in liver. Separate cytoplasmic and mitochondrial 3-hydroxy-3-methylglutaryl coenzyme A generating systems for cholesterogenesis and ketogenesis.

Acetoacetyl-CoA thiolase and 3-hydroxy-3-methylglutaryl coenzyme synthase which comprise the 3-hydroxy-3-methylglutaryl-CoA-generating system(s) for hepatic cholesterogenesis and ketogenesis exhibit dual mitochondrial and cytoplasmic localization. Twenty to forty per cent of the thiolase and synthase of avian and rat liver are localized in the cytoplasmic compartment, the remainder residing in the mitochondria. In contrast, 3-hydroxy-3 methylglutaryl-CoA lyase, an enzyme unique to the "3-hydroxy-3-methylglutaryl-CoA cycle" of ketogenesis, appears to be localized in the mitochondrion. The small proportion, 4 to 8 percent, of this enzyme found in the cytoplasmic fraction appears to arise via leakage from the mitochondria during cell fractionation in that its properties, pI and stability, are identical to those of the mitochondrial lyase. These results are consistent with the view that ketogenesis which involves all three enzymes, acetoacetyl-CoA thiolase, 3-hydroxy-3-methylglutaryl-CoA synthase and 3-hydroxy-3-methylglutaryl-CoA lyase, occurs exclusively in the mitochondrion, whereas cholesterogenesis, a pathway which involves only the 3-hydroxy-3-methylglutaryl-CoA synthesizing enzymes, is restricted to the cytoplasm. Further fractionation of isolated mitochondria from chicken and rat liver showed that all three of the 3-hydroxy-3-methylglutaryl-CoA cycle enzymes are soluble and are localized within the matrix compartment of the mitochondrion. Likewise, cytoplasmic acetoacetyl-CoA thiolase and 3-hydroxy-3-methylglutaryl-CoA synthase are soluble cytosolic enzymes, no thiolase or synthase activity being detectable in the microsomal fraction. Chicken liver mitochondrial 3-hydroxy-3methylglutaryl-CoA synthase activity consists of a single enzymic species with a pI of 7.2, whereas the cytoplasmic activity is composed of at least two species with pI values of 4.8 and 6.7. Thus it is evident that the mitochondrial and cytoplasmic species are molecularly distinct as has been shown to be the case for the mitochondrial and cytoplasmic acetoacetyl-CoA thiolases from avian liver (Clinkenbeard, K. D., Sugiyama, T., Moss, J., Reed, W. D., and Lane, M. D. (1973) J. Biol. Chem. 248, 2275). Substantial mitochondrial 3-hydroxy-3-methylglutaryl-CoA lyase activity is present in all tissues surveyed, while only liver and kidney possess significant mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase activity. Therefore, it is proposed that tissues other than liver and kidney are unable to generate acetoacetate because they lack the mitochondrial synthase.

Adenosine Monophosphate↗