Effect of PCO2 on epinephrine-induced lipolysis in isolated fat cells.

The effect of different pHs obtained by changing the PCO2 and the effect of PCO2 at constant pH on the lipolysis induced by epinephrine in isolated fat cells have been investigated. An inhibition of activated lipolysis was found in acidosis while in alkalosis no significant change was detected. When the experiments were performed at different PCO2s but at constant pH, the results showed an inhibition of lipolysis by high PCO2 whereas low PCO2 did not affect it. It is concluded that either acidosis or high PCO2 lead to an inhibition of the lipolysis induced by epinephrine in isolated fat cells. As regards alkalosis and low PCO2 it seems likely that the intracellular pH is not affected to the same extent as in alkalosis by high [HCO(-3)] or under the conditions of the present experiments the [H+] needed to alterate lipolysis was not reached.     

Effect of aging and cellularity on lipolysis in isolated mouse fat cells

The effects of age and cellularity on lipolysis have been investigated in isolated epididymal fat cells from both Swiss albino mice and Sprague-Dawley rats. No significant lipolytic response to glucagon could be demonstrated with adipocytes from either young or old mice, while glycerol output was increased by this hormone with fat cells from young rats. Larger adipocytes from older mice showed significantly greater isoproterenol-stimulated lipolysis than those from younger animals if the glycerol output was expressed on a per cell basis. However, the lipolytic response per cell appeared to be equivalent in young and old rat adipocytes with either isoproterenol or ACTH-(1-24). In a complete aging study, relationships between body weight, epididymal fat pad weight and cellularity were examined covering the life span of the mouse. ACTH-(1-24)- and dibutyryl cyclic AMP-stimulated lipolysis increased with age and cell size but fell at senescence when adipocyte size diminished. Although an effect of aging per se cannot be ruled out with the experimental techniques used in the present study, a dominant influence of adipocyte size on the lipolytic process was demonstrated.     

An assay for beta-adrenergic receptors in isolated human fat cells

The beta-adrenergic receptors have been characterized in isolated human adipocytes using a potent beta-adrenergic antagonist (-)-[3H]dihydroalprenolol. Binding of (-)-[3H]dihydroalprenolol to isolated fat cells was stereospecific and saturable, the maximum number of binding sites calculated being 7.8 +/- 2.2 pmol of bound ligand/10(7) cells, corresponding to 450,000 binding sites/cell. The dissociation constant was estimated to be 2.7 +/- 1.1 nM. The results with competition-inhibition experiments using beta-adrenergic agonists and antagonists indicated that the binding sites in isolated adipocytes were predominantly of the beta1-subtype; about 80% of the receptors were of this type. With the present method, specific beta-adrenergic receptor number and affinity in isolated human adipocytes could be determined in about 1 g of human adipose tissue.     

A multichannel perifusion system for the continuous monitoring of glycogenolysis and gluconeogenesis in isolated rat hepatocytes

A multichannel perifusion system for isolated rat hepatocytes entrapped in a Sephadex matrix is described and criteria for the choice of matrices are discussed. This system overcomes the usual problem of clogged filters and impaired flow rates encountered in suspension perifusion systems, and is assembled from standard widely available components. Gluconeogenic capability and mitochondrial respiratory control ratios were unaltered. Decreases in trypan blue viability index and respiration rate were small when compared with flask-incubated hepatocytes. The endogenous rate of glycogenolysis was slightly higher in perifused hepatocytes but hormone response, as judged by glucagon stimulation of glycogenolysis, was unimpaired. The potential of this system is indicated by experiments monitoring glycogenolysis and gluconeogenesis in recycling and non-recycling modes.     

Column perifusion system for steroidogenesis in isolated rat adrenal cells

A perifusion system using a plastic column into which isolated rat adrenal cells had been installed was attempted. After ACTH or cAMP was administered to the column, the corticosterone concentration in the eluate was determined. ACTH in 10(-13) and 10(-12) M did not promote corticosterone production, whereas 10(-11) and 10(-10) M showed a dose dependent production of corticosterone. By iterative infusion of 10(-11) or 10(-9) M of ACTH, very clear responses to restimulation of ACTH were noted. Following the administrations of 10(-3) or 10(-2) M of dibutyryl adenosine 3',5'-cyclic monophosphate (dbcAMP), the production of corticosterone increased dose-dependently. These results suggest that this perifusion system is effective for examining the effects of ACTH or cAMP on steroidogenesis of cells.     

[3H]norepinephrine binding and lipolysis by isolated fat cells

[³H]norepinephrine was shown to bind to specific sites on isolated fat cells. A Scatchard plot of norepinephrine binding showed two apparent K_a of 1.9 · 10⁶ and 1.2 · 10⁵ LM^?. 1.4 · 10^?4 M Norepinephrine covalently-linked to agarose beads reduced [³H]norepinephrine binding by over 50%. Several structurally related drugs were compared as inhibitors of [³H]norepinephrine binding and as stimulators of lipolysis in preparations of similarly prepared cells. Dose-response curves for norepinephrine, epinephrine and isoproterenol showed the affinities for binding inhibition and for stimulation of lipolysis to be in the same range of 6 · 10^?7-2 · 10^?6 M. Dopamine and dopa were potent inhibitors of [³H]norepinephrine binding at 8.5 · 10^?7 M and 2.0 · 10^?6 M respectively, but did not stimulate lipolysis even at 10^?4 M. Propranolol, a β-adrenergic antagonist, had no effect on [³H]norepinephrine binding at 10^?4 M but completely inhibited catecholamine-stimulated lipolysis at 10^?5 M. Phentolamine, an α-adrenergic antagonist, did not inhibit binding or catecholamine-stimulated lipolysis at 10^?4 M. Ephedrine, metaraminol, phenylephrine and normetanephrine were also ineffective both as [³H]norepinephrine binding inhibitors and as stimulators of lipolysis. The results suggested the catechol ring of catecholamines is more important than the ethanolamine side chain as a requirement for binding, while both an intact catechol moiety and ethanolamine function appear necessary for physiological effect.     

Regulation of lipolysis and cyclic AMP synthesis through energy supply in isolated human fat cells.

The effects of glucose and of various inhibitors of glycolysis or of oxidative phosphorylation on stimulated lipolysis and on intracellular cyclic AMP and ATP levels were investigated in isolated human fat cells. The glycolysis inhibitors, NaF and monoiodoacetate, inhibited epinephrine or theophylline-stimulated lipolysis and parallely reduced the intracellular cyclic AMP and ATP levels; however, neither NaF nor monoidoacetate significantly affected dibutyryl cyclic AMP-induced lipolysis. Removal of glucose from the medium also reduced the rate of epinephrine-stimulated lipolysis and the intracellular cyclic AMP and ATP levels but failed to modify the lipolytic activity of dibutyryl cyclic AMP. The oxidative phosphorylation inhibitors, antimycin A and, under fixed conditions, 2,4-dinitrophenol also strongly decreased the adipocyte cyclic AMP and ATP levels but inhibited as well the rate of epinephrine- and of dibutyryl cyclic AMP-induced lipolysis. N-Ethylmaleimide, a mixed glycolysis and oxidative phosphorylation inhibitor, not only reduced the intracellular cyclic AMP and ATP levels and epinephrine- or theophylline-induced lipolysis, but also that stimulated by dibutyryl cyclic AMP. When glycolysis was almost fully inhibited, human fat cells were insensitive to epinephrine but remained fully responsive to dibutyryl cyclic AMP. These results, showing a relationship between ATP availability, cyclic AMP synthesis and lipolysis, suggest a different ATP requirement for cyclic AMP synthesis and triacylglycerol lipase activation, a difference which could explain why ATP issued from glucose breakdown appears to be a determinant factor for cyclic AMP synthesis, but not for triacylglycerol lipase activation in human fat cells.     

Membrane phospholipids and hormone-sensitive lipolysis in fat cells

Isolated fat cells from rats which have been made hypothyroid do not give a lipolytic response to catecholamines. A recent report has suggested that catecholamine-sensitive lipolysis may be correlated with an “unmasking” of receptors by linoleic acid rich phospholipids in the fat cell membrane. No apparent differences in phospholipid fatty acid composition could be found in membrane “ghosts” prepared from normal and hypothyroid rats.     

Effects of insulin on adrenoceptor binding and the rate of catecholamine-induced lipolysis in isolated human fat cells

The mechanisms by which insulin inhibits catecholamine-induced lipolysis in fat cells are unknown. In this study the possible role of an interaction between insulin and the adrenoceptors on human fat cells was investigated. Insulin inhibited, in a dose-dependent fashion, the specific binding of hydrophobic as well as hydrophilic nonselective beta-receptor radioligands but had no effect on the binding of alpha 2-selective radioligands. The results of saturation experiments and competition-inhibition experiments under both equilibrium conditions and nonequilibrium conditions revealed that insulin reduced the total number of beta-adrenergic binding sites (maximum effect 25%) without changing the beta-adrenoceptor affinity. This insulin effect was rapid and reversible; one-third of the effect occurred within 1 min of incubation and it was completely reversed within 30 min after withdrawal of insulin. It could be mimicked by a polyclonal rabbit insulin receptor antibody but not by insulin mimickers acting distal to the initial interaction between the hormone and its specific insulin-receptor binding site. The beta-adrenoceptor binding to a plasma membrane-enriched fraction decreased at the same time as it increased to a microsomal enriched fraction after insulin treatment, indicating a redistribution of beta-adrenoceptors in the cell. In lipolysis experiments performed under conditions like those in the binding experiments, insulin inhibited the rate of lipolysis with a lag period of 3 min. Furthermore, the hormone caused a dose-dependent maximum 10-fold shift to the right of the dose-response curve for isoprenaline-induced lipolysis without changing the amplitude of the curve. This effect of insulin was specific for the beta-adrenergic receptors system, since insulin markedly decreased the amplitude of the dose-response curve for parathyroid hormone-induced lipolysis. In addition, the effect of insulin on isoprenaline-induced lipolysis could be mimicked by long-lasting fractional inactivation of the beta-adrenoceptors. The dose-response relationships for the inhibitory effects of insulin on beta-adrenoceptor binding and the lipolytic sensitivity to isoprenaline were almost identical. Half-maximum and maximum effects occurred at about 5 and 100 microunits/ml of insulin, respectively. In conclusion, the exposure of human fat cells to physiological insulin doses is followed by a rapid and dose-dependent translocation of beta-adrenoceptors from the exterior to the interior of the cell and a subsequent dose-dependent decrease in the lipolytic sensitivity to beta-adrenergic agonists, without a change in maximum lipolysis.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effect of pH on lipolysis, cAMP and cAMP-dependent protein kinase activity in isolated rat fat cells

The effect of acidosis and alkalosis on lipolysis, cAMP production and cAMP-dependent protein kinase activity in isolated rat fat cells incubated in the presence of norepinephrine and norepinephrine plus theophylline has been investigated. The pH of the incubation medium was adjusted to 6.8, 7.4 and 7.8 respectively. Acidosis inhibited both norepinephrine- and norepinephrine plus theophylline-induced release of glycerol whereas alkalosis led to slight stimulation. Norepinephrine produced an increase in cAMP and cAMP-dependent protein kinase activity. However, comparison of both parameters in acidosis and alkalosis with those at pH 7.4 indicates that they were higher at pH 7.8 and lower at pH 6.8. Addition of theophylline in combination with norepinephrine increases cAMP production within 5 min, under acidosis to values similar to those obtained at pH 7.4 with norepinephrine. The same effect on protein kinase activity was obtained. In spite of this increment in cAMP and protein kinase activity produced by addition of norepinephrine plus theophylline, lipolysis remains inhibited by acidosis. Addition of theophylline at pH 7.4 and 7.8 induced a much higher cAMP production and cAMP-dependent protein kinase activity although at pH 7.8 there was a statistically significant increase in protein kinase activity at 10 min it did not induce a significant increase in lipolysis. This is discussed and possible mechanisms are suggested to explain the effect of acidosis and alkalosis on the lipolysis induced by norepinephrine in rat fat cells.     

Effects of prolonged incubation of isolated fat cells on their response to hormones stimulating lipolysis and glucose metabolism

1. The metabolism of isolated fat cells from parametrial adipose tissue of starved normal rats was studied during 8hr. incubation. 2. There was a three- to eight-fold increase in conversion of glucose into carbon dioxide, fatty acids and glycerol during the fourth to eighth hours of incubation in 4% albumin buffer over that seen during the first 4hr. of incubation. 3. The addition of growth hormone and dexamethasone to fat cells at the start of the incubation period accelerated lipolysis during the first 4hr. of incubation but no further effect was seen during the fourth to eighth hours of incubation. Addition of growth hormone and dexamethasone to fat cells that had been incubated for 4hr. did not accelerate lipolysis during the next 4hr. whether fat cells were incubated with or without glucose. 4. Fat cells incubated for prolonged periods also displayed a reduced sensitivity to the lipolytic action of adrenocorticotrophic hormone. 5. During prolonged incubation there was no damage to the cells as judged by the retention of two soluble cytoplasmic enzymes, lactate dehydrogenase and malate dehydrogenase, within the cells.     

Potentiation of epinephrine-induced lipolysis in fat cells from estrogen-treated rats

We investigated concomitantly the effects of ethinyl-estradiol (EE₂) at low dose (1.2 μg/animal for 10 days) and high dose (120 μg/animal for 3 days) on body weight, weight of fat stores, triglyceridemia and fat cell lipoprotein lipase and hormone-sensitive lipase activities in female rats. At low dose, EE₂ increased triglyceridemia and lipoprotein lipase activity, whilst high dose decreased both parameters. At low and high doses, and regardless of whether triglyceridemia and lipoprotein lipase activity were increased and decreased, EE₂ caused depletion in fat stores. Fat cells isolated from depleted fat tissue elicited marked increases in the response of hormone-sensitivelipase to epinephrine. Taken together, the data suggest that the potentiation of epinephrine-induced lipolysis in fat cells is likely to represent a major cause to estrogen-induced fat depletion.