Alterations in response of rat white adipocytes to insulin, noradrenaline, corticotropin and glucagon after adrenalectomy. Correction of these changes by adenosine deaminase.

1. Adipocytes isolated from rats 6--9 days after adrenalectomy had significantly increased sensitivity to insulin action against noradrenaline-stimulated lipolysis. In the presence of adenosine deaminase there was no significant difference in insulin sensitivity between cells from adrenalectomized and sham-operated rats. 2. Adipocytes from adrenalectomized rats had decreased lipolytic responses to all concentrations of noradrenaline and glucagon tested and a decreased lipolytic response to low but not high concentrations of corticotropin. There was no difference in lipolytic response to theophylline after adrenalectomy. Adenosine deaminase corrected the differences in response to noradrenaline and glucagon resulting from adrenalectomy. 3. In the presence of adenosine deaminase rates of lipolysis, after stimulation by high concentrations of noradrenaline, glucagon, corticotropin or theophylline, were the same in cells from adrenalectomized or sham-operated rats. 4. These findings and previously reported effects of adenosine and adrenalectomy on adipocyte function are discussed. It is proposed that changes in adipocyte hormone responsiveness after adrenalectomy may result from changes in adenosine metabolism or release.     

Adrenalectomy-induced alterations in glucagon binding and lipolysis in isolated rat adipocytes.

Adipocytes from adrenalectomized rats nearly lost their lipolytic response to glucagon concomitant with a 90% decrease in the number of glucagon receptors per cell. Quantitative analysis of the relation between amount of cell-bound glucagon and hormone-stimulated lipolysis revealed that the ability of the remaining 10% of glucagon receptors to induce lipolysis was not impaired. Binding of the beta-adrenergic antagonist [3H]dihydroalprenolol and maximal lipolysis induced by (-)-isoproterenol, (Bu)2cAMP, 3-isobutyl-1-methylxanthine, and adenosine deaminase were reduced only 10 to 20% after adrenalectomy. Furthermore, glucagon-stimulated cAMP production was greatly decreased in adrenalectomized animals, but isoproterenol-stimulated cAMP production was not. Hydrocortisone replacement in adrenalectomized rats only partially prevented the loss of glucagon receptors and glucagon effects on both cAMP production and lipolysis. These findings suggest that lipolytic cascade distal to hormone receptors was not greatly impaired in adipocytes after adrenalectomy and that the unresponsiveness of these cells to glucagon was mostly due to a marked reduction in the number of glucagon receptors.     

Studies on adrenaline-induced lipolysis in adrenalectomized rats.

In a short-term study, adrenaline-induced lipolysis was less in adrenalectomized rats than in controls, though the cyclic AMP accumulation was not different. In adrenalectomized rats treated with corticosterone, lipase activity was as low as in untreated adrenalectomized rats, although adrenaline-induced lipolysis was not reduced. In a long-term study, no reduction in adrenaline-induced lipolysis or cyclic AMP accumulation was observed in adrenalectomized rats. The mechanism of the effect of adrenalectomy on adrenaline-induced lipolysis is discussed on the basis of these results.     

Opposite effects of adrenalectomy on eicosanoid release in rat peritoneal macrophages and spleen

The effect of adrenalectomy on the formation of cyclo-oxygenase and lipoxygenase products by activated peritoneal rat macrophages was determined and compared with that of the spleen. After isolation, the cells and tissues were incubated with [1-14C] arachidonic acid and the Ca-ionophore A23187 and the metabolites isolated by HPLC chromatography. The main components formed in the macrophages of the controls are 6-keto-PGF1 alpha, TxB2 and 12-HETE. One peak represents 5, 12 di HETE. Smaller amounts of PGF2 alpha, PGE2, PGD2, LTB4 and 15-HETE are also present. After adrenalectomy, a considerable increase occurs in the amounts of LTB4, 15-HETE and 12-HETE. The increase in the PG is smaller. The compounds formed from endogenous arachidonic acid are also determined. In the cells of the controls, the formation of LTB4 is considerably increased after adrenalectomy. In the spleen, PGD2 and 12-HETE are decreased after adrenalectomy. The effect of the macrophages is most probably related to a diminished amount or inactivation of lipocortin, a glucocorticosteroid induced peptide with PlA2 inhibitory activity in adrenalectomized animals. In the decrease in formation in the spleen, the absence of the permissive effect of glucocorticosteroids on the hormone-induced lipolysis may play a role.     

Adrenalectomy Reduces Adiposity by Decreasing Feed Efficiency,Not Direct Effects on White Adipose Tissue

EDENS, N. K., A. MOSHIRFAR, G. M. POTTER, S. K. FRIED, AND T. W. CASTONGUAY. Adrenalectomy reduces adiposity by decreasing feed efficiency, not direct effects on white adipose tissue. Obes Res. Objective: This study was conducted to establish the effects of adrenalectomy (ADX) on adipose tissue metabolism in male Sprague—Dawley rats fed a standard chow diet. Research Methods and Procedures: The effects of adrenalectomy on adipose cell size, lipoprotein lipase activity, and basal and insulin-stimulated glucose conversion to lipid and lipolysis were measured. Results: ADX decreased body weight gain during the postoperative period in the absence of changes in food intake; feed efficiency was decreased significantly. ADX decreased adipocyte size by 30%. ADX increased adipocyte response to the effect of submaximal concentrations of insulin on lipid synthesis and lipolysis. ADX decreased maximally insulin-stimulated lipid synthesis, but this effect was accounted for by decreased adipocyte size. In contrast, ADX had no effect on maximally insulin-inhibited lipolysis. ADX did not affect heparin-releasable LPL. The small effect of ADX on residual extractable adipose tissue LPL activity was accounted for by decreased fat cell size. Discussion: ADX decreased adiposity in the absence of changes in food intake, lipoprotein lipase activity, and adipocyte lipid metabolism. The effect is best attributed to decreased feed efficiency.     

Correction by dexamethasone treatment of the altered lipolytic cascade induced by adrenalectomy in rat adipocytes

The ability of glucocorticoid-treatment to reverse the metabolic alterations caused by adrenalectomy in rat adipocytes was studied. Correction of the enhanced adenosine antilipolytic effect and of the defect in lipolysis, cyclic AMP and adenylate cyclase responsiveness to guanine nucleotides were all achieved after a 24 h dexamethasone treatment, whereas correction of the defect in beta-adrenoceptor-density required a 48 h treatment. The latter treatment, however, failed to reverse the defect in both the adenylate cyclase catalytic activity and protein content per fat cell. These different kinetics of restoration indicate that correction by dexamethasone of the defective cyclic AMP and lipolytic responses on one hand and of the guanine nucleotide control of adenylate cyclase on the other one are two related phenomenoms.     

Opposite effects of adrenalectomy on eicosanoid release in rat peritoneal macrophages and spleen

The effect of adrenalectomy on the formation of cyclo-oxygenase and lipoxygenase products by activated peritoneal rat macrophages was determined and compared with that of the spleen. After isolation, the cells and tissues were incubated with [1-¹⁴C] arachidonic acid and the Ca-ionophore A23187 and the metabolites isolated by HPLC chromatography. The main components formed in the macrophages of the controls are 6-keto-PGF_1α, TxB₂ and 12-HETE. One peak represents 5, 12 di HETE. Smaller amounts of PGF_2α, PGE₂, PGD₂, LTB₄ and 15-HETE are also present. After adrenalectomy, a considerable increase occurs in the amounts of LTB₄, 15-HETE and 12-HETE. The increase in the PG is smaller. The compounds formed from endogenous arachidonic acid are also determined. In the cells of the controls, the formation of LTB₄ is considerably increased after adrenalectomy. In the spleen, PGD₂ and 12-HETE are decreased after adrenalectomy.The effect of the macrophages is most probably related to a diminished amount or inactivation of lipocortin, a glucocorticosteroid induced peptide with PlA₂ inhibitory activity in adrenalectomized animals. In the decrease in formation in the spleen, the absence of the permissive effect of glucocorticosteroids on the hormone-induced lipolysis may play a role.     

The effect of glucocorticoids on adipocyte corticotropin receptors and adipocyte responses

A specific and sensitive assay for determining the binding of adrenocorticotropin (ACTH) to isolated rat adipocytes has been developed and utilized to study the effect of glucocorticoids on ACTH receptor. Measurement of the binding of tritiated ACTH (spec. act. 90 Ci/mmol) to adipocytes isolated from normal, adrenalectomized, and adrenalectomized dexamethasone-treated rats indicated that there are no differences among these three populations in either the magnitude or the affinity of the binding reaction. The binding interaction was found to be of high affinity (K_d = 5.23 + 1.92 · 10^?9 M) and paralleled closely the stimulation of lipolysis (K_m = 2.09 ± 0.35 · 10^?9 M). About 16 300 receptors were calculated to be present per adipocyte. Hormone-induced cyclic 3′,5′-adenosine monophosphate production remained intact after adrenalectomy, thereby confirming that receptors are not lost during steroid deprivation. The lipolytic response did, however, become less sensitive to both ACTH and epinephrine following adrenalectomy. Pre-treatment of adrenalectomized rats with dexamethasone resulted in an increase in basal and hormone-stimulated levels of cyclic AMP and glycerol production to super-normal values. In adipocyte ghost preparations, ACTH and epinephrine sensitive adenylate cyclase activity was not decreased by adrenalectomy and dexamethasone administration did not result in a selective enhancement of ACTH sensitive adenylate cyclase activity. Our results indicate that glucocorticoids do not cause their permissive effects by specific regulation of the ACTH receptor on the adipocyte.     

Corticotropin-releasing factor (CRF) and vasopressin concentration in major brain regions after adrenalectomy and their involvement in adrenalectomy-induced ACTH elevation

CRF and vasopressin concentrations in major brain regions after bilateral adrenalectomy and their involvement in adrenalectomy-induced ACTH secretion were investigated. At 5, 14 and 28 days after bilateral adrenalectomy, the plasma ACTH level was greatly elevated, whereas hypothalamic CRF content was reduced at 5 days and was not changed at 14 and 28 days after adrenalectomy. The CRF concentration in the medulla oblongata was reduced at 2-4 weeks after adrenalectomy. On the other hand, the arginine vasopressin (AVP) concentration was significantly elevated 2-4 weeks after adrenalectomy. An intrajugular administration of anti-ovine or anti-rat CRF serum significantly suppressed the elevated plasma ACTH level in adrenalectomized, freely moving rats, whereas anti-AVP serum or antipressor AVP antagonist, dpTyr(Me)AVP did not suppress the ACTH level. These results indicate that CRF played an important role in the adrenalectomy-induced ACTH elevation but that vasopressin was not involved.     

Comparative studies of the role of hormone-sensitive lipase and adipose triglyceride lipase in human fat cell lipolysis

Hormone-sensitive lipase (HSL) and adipose triglyceride lipase (ATGL) regulate adipocyte lipolysis in rodents. The purpose of this study was to compare the roles of these lipases for lipolysis in human adipocytes. Subcutaneous adipose tissue was investigated. HSL and ATGL protein expression were related to lipolysis in isolated mature fat cells. ATGL or HSL were knocked down by RNA interference (RNAi) or selectively inhibited, and effects on lipolysis were studied in differentiated preadipocytes or adipocytes derived from human mesenchymal stem cells (hMSC). Subjects were all women. There were 12 lean controls, 8 lean with polycystic ovary syndrome (PCOS), and 27 otherwise healthy obese subjects. We found that norepinephrine-induced lipolysis was positively correlated with HSL protein levels (P < 0.0001) but not with ATGL protein. Women with PCOS or obesity had significantly decreased norepinephrine-induced lipolysis and HSL protein expression but no change in ATGL protein expression. HSL knock down by RNAi reduced basal and catecholamine-induced lipolysis. Knock down of ATGL decreased basal lipolysis but did not change catecholamine-stimulated lipolysis. Treatment of hMSC with a selective HSL inhibitor during and/or after differentiation in adipocytes reduced basal lipolysis by 50%, but stimulated lipolysis was inhibited completely. In contrast to findings in rodents, ATGL is of less importance than HSL in regulating catecholamine-induced lipolysis and cannot replace HSL when this enzyme is continuously inhibited. However, both lipases regulate basal lipolysis in human adipocytes. ATGL expression, unlike HSL, is not influenced by obesity or PCOS.     

The relationship between the concentration of adenosine 3′:5′-cyclic monophosphate and the anti-lipolytic action of insulin in isolated rat fat-cells

The relationship between cyclic AMP content and lipolysis, as measured by glycerol formation, was studied in isolated rat fat-cells. Inhibition of lipolysis by insulin in the presence of a low concentration of adrenaline was accompanied by little or no lowering of cyclic AMP content, measured after 15min incubation. The time-course of cyclic AMP content after addition of adrenaline showed that the effect of insulin in lowering cyclic AMP content measured after 2-5min was gradually lost over the next hour, mainly because of the fall in cyclic AMP content after an early peak in the presence of adrenaline alone. There was a 44% loss of immunoreactive insulin, from an initial concentration of 0.3nm, during a 1h incubation with fat-cells. Insulin did not affect partitioning of cyclic AMP between cells and incubation medium. When the correlation between cyclic AMP content and rate of lipolysis was investigated for a wide range of adrenaline concentrations, it was found that the lowering of cyclic AMP content by insulin was much less than that required to account for the amount of inhibition of lipolysis. It is concluded that inhibition of adrenaline-stimulated lipolysis by insulin involves factors in addition to a decrease in intracellular cyclic AMP concentration.     

Rat insulin-like growth factor-I and -II mRNAs are unchanged during compensatory adrenal growth but decrease during ACTH-induced adrenal growth

The insulin-like growth factors (IGFs) may be important autocrine and paracrine mediators of organ growth. We used solution-hybridization/ribonuclease protection assays to examine IGF-I and IGF-II mRNA abundance during hypertrophy or the rat adrenal gland induced by unilateral adrenalectomy or by adrenocorticotropic hormone (ACTH) infusion. Adrenal IGF-I mRNA did not change during the period of rapid organ growth at 18 or 66 h after unilateral adrenalectomy. ACTH infusion induced a time- and dose-dependent decrease in adrenal IGF-I mRNA despite significant increases in gland size. IGF-II mRNA also remained unchanged after unilateral adrenalectomy and decreased after ACTH infusion, to a greater extent than IGF-I mRNA. Liver IGF-I mRNA did not change with ACTH exposure, indicating an effect specific to the adrenal. We also measured adrenal P450scc mRNA as a marker of steroidogenic capacity. P450scc mRNA was unchanged after unilateral adrenalectomy and increased with ACTH infusion. Thus IGF-I and IGF-II mRNAs respond in parallel, but in different fashions with different stimuli for adrenal growth. The decrease in IGF mRNA after exposure to ACTH may be a factor in the ACTH-induced inhibition of compensatory hypertrophy after unilateral adrenalectomy.     

Changes of lipase-catalyzed lipolytic rates in a batch reactor

A dramatic change of the reaction rate was observed for the lipase-catalyzed hyrolysis of tributyrin in a batch reactor. Immediately after the addition of the enzyme, the lipolysis rate increased continuously until a maximal reaction rate was reached. The duration of the induction was mainly controlled by the bulk enzyme concentration and the reactor stirring speed. The reaction rate dropped sharply after reaching its maximal value. The lipolysis decayed at a rate of about 0.012 min(-1), and was not affected by changes of the stirring speed. This decay was attributed to the fast deactivation of the surface-adsorbed lipase, and possibly to the extremely slow desorption of the inactivated species. For reaction time longer than 120 minutes, the lipolysis decreased at a much slower rate. Several mechanisms for the decay of the lipolysis rate were discussed.     

The action of local anaesthetics on lipolysis and on adenosine 3′:5′-cyclic monophosphate content in isolated rat fat-cells

1. Local anaesthetics inhibited hormone-stimulated lipolysis in isolated rat fat-cells. The most potent anaesthetic was dibucaine, which inhibited adrenaline-stimulated lipolysis by 50% at a concentration of 0.16mm. 2. The amount of inhibition produced by a given concentration of anaesthetic was very similar with adrenaline, theophylline and dibutyryl cyclic AMP, at submaximal and maximal concentrations. 3. The inhibitory effect of dibucaine on lipolysis was apparent within 5 min and was constant over 1h. 4. Dibucaine inhibited basal, adrenaline-stimulated and insulin-stimulated glucose uptake at concentrations 6-10-fold higher than those inhibiting lipolysis. 5. The effects of dibucaine on lipolysis and glucose uptake were reversed after removal of anaesthetic and washing of cells. 6. Dibucaine further elevated the concentration of cyclic AMP in the presence of adrenaline or adrenaline plus theophylline. 7. Dibucaine had no effect on ATP content at concentrations causing 80% inhibition of lipolysis, but lowered ATP content at higher concentrations. 8. The relative potency of different local anaesthetics as inhibitors of hormone-stimulated lipolysis paralleled their potency as inhibitors of ion movements in other systems. 9. The possibility is discussed that Ca(2+) ions are involved in the regulation of lipolysis, and that local anaesthetics inhibit lipolysis by interfering with Ca(2+) translocation.     

Evidence for a defect in the number of β-adrenergic receptors and in the adenylate cyclase responsiveness to guanine nucleotides in fat cells after adrenalectomy

Receptor binding studies (?)-[³H]dihydroalprenolol as the ligand revealed, in adrenalectomized rat fat cells, a 50% decrease in the number of β-adrenergic receptors. er cell with no change in the receptor affinity for this ligand. Adrenalectomy caused no change in the binding affinity for isoproterenol of both high affinity and low affinity populations of the β-adrenergic receptors. Guanine nucleotide sensitivity of the agonist binding to β-receptors was also unaltered by adrenalectomy. Adrenalectomy caused a 30–40% decrease in the maximal response of adenylate cyclase to (?)-isoproterenol only when guanine nucleotides were present in the assay, without altering the (?)-isoproterenol concentration giving half-maximal adenylate cyclase stimulation (K_act values). The maximal response of adenylate cyclase to Gpp(NH)p also was lower in adrenalectomized membranes, indicating a defect at the guanine nucleotide regulatory site. Removal of adenosine by addition of adenosine deaminase failed to reverse the decreased adenylate cyclase response to isoproterenol in adrenalectomized rats. However, in intact fat cells, in which cyclic AMP accumulation in response to isoproterenol was decreased by adrenalectomy, removal of adenosine almost completely corrected this defect. These results indicate that the observed changes in the number of β-adrenergic receptors and in the ability of guanine nucleotides to stimulate adenylate cyclase, though explaining the decreased adenylate cyclase responsiveness to catecholamines, do probably not contribute significantly to the mechanism by which adrenalectomy decreases the lipolytic responsiveness of adipocyte to catecholamines. In addition, this study also suggests that the increased sensitivity to adenosine of lipolysis reported in adipocytes from adrenalectomized rats may result from an action of adenosine at a post-adenylate cyclase step, possibly on the cyclic AMP phosphodiesterase.     

Effect of epinephrine and other lipolytic agents on intracellular lipolysis and lipoprotein lipase activity in 3T3-L1 adipocytes

3T3-L1 adipocytes were used to test the hypothesis that hormone-sensitive lipolysis and lipoprotein lipase activity might be regulated in a reciprocal manner. Intracellular lipolysis was stimulated by catecholamine, dibutyryl cAMP, and ACTH, but not by glucagon. The effects of epinephrine on lipolysis were blocked by the beta-antagonist propanolol but not by the alpha-antagonist phentolamine. Hormone-stimulated lipolysis was not changed by acute (45 min) or chronic (2 days) treatment of the cells with insulin whereas the latter treatment augmented lipoprotein lipase activity about fivefold. Epinephrine did not affect the lipoprotein lipase activity of insulin-stimulated cells. Withdrawal of glucose from the medium decreased lipoprotein lipase activity and the effect of epinephrine on lipolysis. Effects of lipolytic agents on activity of lipoprotein lipase were variable and concentration-dependent. Lipoprotein lipase activity was decreased only by concentrations of epinephrine greater than those inducing maximal intracellular lipolysis, and the decrease in activity occurred about 30 min after the increase in glycerol release. There seems to be no relationship between the level of activity of lipoprotein lipase and the maximal rate of hormone-stimulated lipolysis in 3T3-L1 cells. Unlike in adipose tissue and adipocytes of rats, hormone-stimulated lipolysis and lipoprotein lipase activity in murine 3T3-L1 adipocytes appear to be regulated independently.     

Changes in distribution of molecular weight forms of biologically active and immunoreactive adrenocorticotropic hormone after adrenalectomy in rat anterior pituitary

The concentration of ACTH in extracts of rat anterior pituitary was measured by both radioimmunoassay and bioassay at different stages following adrenalectomy. Both types of ACTH activity decreased the day immediately following adrenalectomy but increased gradually afterwards. Immunological ACTH activity increased to 250% of the control value and biological ACTH activity increased to 490% of control value 3 weeks after adrenalectomy. The increase in biological ACTH activity occurred earlier, and the rate of increase was greater, than that of the immunological ACTH activity. The distributions of molecular weight forms of ACTH in extracts of anterior pituitary lobes was determined by gel filtration. Three molecular weight forms of immunoassayable ACTH were detected. Biological ACTH activity appeared in the 2nd and the 3rd peaks. A striking change was observed after adrenalectomy in the distribution of biologically active forms of ACTH. The ratio of biological ACTH activity to immunological ACTH activity in each peak changed at various stages after adrenalectomy. This indicated the heterogenous nature of the ACTH included in each peak. At 2 and again at 3 weeks, biological activity markedly increased until it exceeded the immunological ACTH activity in the 2nd peak. Dexamethasone had little influence on the elution profile of either immunoassayable and biologically active ACTH in gel filtration. Adrenalectomy may possibly have an effect on the intracellular posttranslational processing of ACTH precursors which leads to the development of biological ACTH activity.     

Post receptor activation of lipolysis in starvation, diabetes mellitus and hyperthyroidism

Activation of lipolysis by cyclic AMP in conditions with accelerated lipid mobilization was examined in subcutaneous adipose tissue incubated in vitro. In (a) 16 obese patients before and during therapeutic starvation, (b) 18 diabetics before and after antidiabetic treatment and (c) 11 hyperthyroid patients before and after anti-thyroid treatment, a positive correlation was found between stimulation of basal cyclic AMP accumulation and stimulation of basal glycerol release using either isopropyl noradrenaline or noradrenaline (r = 0.6-0.9). During antidiabetic treatment stimulation of lipolysis increased in relation to that of cyclic AMP accumulation (F = 10.1, p less than 0.01), whereas during antithyroid therapy there was a decrease (F = 95.2, p less than 0.01). Starvation did not alter the relationship between lipolysis and cyclic AMP in hypogastric adipose tissue whereas in femoral tissue stimulation of lipolysis decreased in relation to that of cyclic AMP accumulation (F = 9.6, p less than 0.01). It is concluded that the amount of cyclic AMP needed to promote lipolysis is increased during starvation and in diabetes mellitus but is decreased in hyperthyroidism. From the studies during starvation it appears that regional differences in the post-receptor activation of lipolysis exist in human adipose tissue.     

Effect of adrenalectomy on acceleration of gluconeogenesis by calcium ions, adenosine 3'':5''-cyclic monophosphate and adrenaline in rat kidney tubules.

1. Gluconeogenesis from pyruvate was measured in renal-cortical-tubules fragments prepared from fed male rats 6-8 days after adrenalectomy or sham adrenalectomy. The response of this process to 3':5'-cyclic AMP and adrenaline was compared in these two states at two Ca2+ concentrations. 2. Adrenalectomy decreased the percentage stimulation of gluconeogenesis by 3':5'-cyclic AMP, but increased percentage stimulation by adrenaline. Cortisol treatment of adrenalectomized rats (50 mg/kg, twice daily for 2 days) did not reverse the change in responsiveness to 3':5'-cyclic AMP and adrenaline. 3. Stimulation of gluconeogenesis by 1 micron-adrenaline was unaffected by 10 micron-propranolol (beta-blocker) in either state. Phentolamine (alpha-blocker; 10 micron) totally blocked stimulation of gluconeogenesis by 1 micron-adrenaline in the sham-operated condition, but was only partially effective in this respect after adrenalectomy.