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.     

Altered lipolytic response to glucagon and adenosine deaminase in adipocytes from starved rats.

1. Adipocytes isolated from epididymal adipose tissue of fed or 24 h-starved rats were incubated with a range of glucagon concentrations in the presence and absence of adenosine deaminase (4 munits/ml). 2. With adenosine deaminase present, the lipolytic response to low concentrations of glucagon (1-6 ng/ml) was considerably enhanced in cells from starved rats. 3. The effect of adenosine deaminase on basal lipolysis was altered after starvation. 4. D-3-Hydroxybutyrate (5 mM) decreased the sensitivity of lipolysis to glucagon. 5. The possible involvement of glucagon-stimulated lipolysis in the regulation of ketogenesis is briefly discussed.     

Streptozotocin diabetes results in increased responsiveness of adipocyte lipolysis to glucagon

Adipocytes from streptozotocin-diabetic rats are approximately 50-times more sensitive to the lipolytic action of glucagon. This change is only perceived in the presence of a small quantity of adenosine deaminase which itself has little effect on basal lipolysis. Insulin treatment restores glucagon sensitivity to normal.     

Sensitivity of adipocyte lipolysis to stimulatory and inhibitory agonists in hypothyroidism and starvation.

The responsiveness of lipolysis to the stimulatory agonists noradrenaline, corticotropin and glucagon and to the inhibitory agonists N6-phenylisopropyladenosine, prostaglandin E1 and nicotinic acid was investigated with rat white adipocytes incubated with a high concentration of adenosine deaminase (1 unit/ml). The cells were obtained from fed or 48 h-starved euthyroid animals or from fed or starved animals rendered hypothyroid by 4 weeks of treatment with low-iodine diet and propylthiouracil. Hypothyroidism increased sensitivity to and efficacy of all three inhibitory agonists in their opposition of noradrenaline-stimulated lipolysis. Starvation decreased sensitivity to all three inhibitory agonists when opposing basal lipolysis. Hypothyroidism decreased sensitivity to noradrenaline, glucagon and corticotropin by 37-, 4- and 4-fold respectively and decreased the maximum response to these agonists by approx. 50%, 50% and 75% respectively. Starvation reversed decreases in maximum response to these agonists in hypothyroidism. Starvation in the euthyroid state increased sensitivity to glucagon and noradrenaline, but did not alter sensitivity to corticotropin. Cells from hypothyroid rats were relatively insensitive to Bordetella pertussis toxin, which substantially increased basal lipolysis in the euthyroid state.     

Changes in the sensitivity to glucagon of lipolysis in adipocytes from pregnant and lactating rats.

1. Rates of lipolysis were measured at different concentrations of glucagon in adipocytes prepared from parametrial adipose tissue of fed or starved rats in different reproductive states. All experiments were performed in the presence of a high concentration of adenosine deaminase (1 unit/ml). 2. Maximal rates of lipolysis (elicited by 25 nM-glucagon in each instance) were higher in adipocytes from peak-lactating rats than those from pregnant animals in both the fed and starved states. 3. Of adipocytes from fed animals, those from peak-lactating rats were the most sensitive to glucagon, whereas those from late-pregnant and early-lactating rats were 1-2 orders of magnitude less sensitive. 4. Adipocytes from 24 h-starved rats showed a much smaller stimulation of lipolysis by glucagon, making the assessment of sensitivity difficult. Therefore, rates of lipolysis were also measured in the presence of a maximally anti-lipolytic dose of insulin. The presence of insulin did not alter the relative sensitivities to glucagon of adipocytes from fed animals in different reproductive states, although all dose-response curves were shifted to the right. When lipolysis in adipocytes from starved animals was measured in the presence of insulin, it became evident that starvation for 24 h markedly increased the sensitivity of adipocytes from late-pregnant rats to glucagon, but did not affect that of cells from animals in the other reproductive states. 5. It is concluded that the large changes in sensitivity to glucagon that occurred during the reproductive cycle may enable the modulation of adipose-tissue lipolysis in vivo to satisfy the different metabolic requirements of the animal in the transition from pregnancy to peak lactation.     

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 adenosine deaminase, N6-phenylisopropyladenosine and hypothyroidism on the responsiveness of rat brown adipocytes to noradrenaline.

Adenosine deaminase (1 unit/ml) potentiated the lipolytic action of noradrenaline in adipocytes isolated from brown adipose tissue of 1- and 6-week-old rats by decreasing the EC50 (concn. giving 50% of maximal effect) for noradrenaline by 3-4-fold. With cells from neonatal rabbit tissue, adenosine deaminase only had a small, non-significant, effect on the EC50 for noradrenaline. Lipolysis in rat brown adipocytes was inhibited by low concentrations of N6-phenylisopropyladenosine (PIA). Rabbit cells were far less sensitive to PIA. PIA, prostaglandin E1 and nicotinate all inhibited noradrenaline-stimulated respiration in rat brown adipocytes. Hypothyroidism diminished the maximum response of respiration and lipolysis to noradrenaline in rat cells and increased the EC50 for noradrenaline. Responsiveness of lipolysis to noradrenaline was particularly decreased in hypothyroidism and was partially restored by addition of adenosine deaminase. Lipolysis in cells from hypothyroid rats was more sensitive to the anti-lipolytic action of PIA. Bordetella pertussis toxin increased lipolysis in the presence of PIA, suggesting an involvement of the Ni guanine-nucleotide-binding protein in the control of brown-adipocyte metabolism.     

Impaired hormonal regulation of lipolysis and the interference with adenosine in adipose tissue from hyperglycemic sand rats in vitro

Sand rats (Psammomys obesus) developed in response to different food intake various states of hyperglycemia and hyperinsulinism. 12 normo- and 10 hyperglycemic animals were selected by means of a weekly control of plasma glucose and plasma insulin over a period of 12 weeks after separation from the mother. During this time also the development of body weight gain was checked. In both groups of rats the hormonal regulation of glycerol release by incubated adipose tissue was investigated. In any case, the fat tissue from hyperglycemic sand rats showed a lower lipolytic responsiveness to noradrenaline stimulation than that of their normoglycemic controls. This correlates well with previous results in hyperglycemic sand rats in which the catecholamine-stimulated cAMP production was disturbed (Knospe and K?hler 1981). Degradation of released adenosine by addition of adenosine deaminase significantly enhanced the noradrenaline action on glycerol release in both groups of sand rats. Even though the noradrenaline-stimulated lipolytic activity of adipose tissue from normo- and hyperglycemic animals was enhanced in the presence of adenosine deaminase, the hormone resistance of adipose tissue from hyperglycemic sand rats was nevertheless not abolished. The theophylline-mediated adenosine receptor blockade gave further evidence that particularly endogenous adenosine released during incubation of adipose tissue from sand rats inhibited the noradrenaline action on lipolysis. The antilipolytic action of insulin on glycerol release is negligibly low in normoglycemic as well as hyperglycemic sand rats. The degradation of adenosine by adenosine deaminase failed to improve the insulin action. Adenosine addition completely blocked the stimulating effects of noradrenaline on glycerol release.(ABSTRACT TRUNCATED AT 250 WORDS)     

Lipolysis in rat adipocytes during recovery from lactation. Response to noradrenaline and adenosine.

The replenishment of lipid reserves of adipocytes following the removal of litters from lactating rats is associated with a 3-fold decrease in both the response of lipolysis to noradrenaline and the maximum rate of lipolysis (measured in the presence of noradrenaline plus adenosine deaminase); these adaptations do not appear to result from the changes in serum prolactin and insulin concentrations that occur on litter removal.     

Changes in the anti-lipolytic action and binding to plasma membranes of N6-L-phenylisopropyladenosine in adipocytes from starved and hypothyroid rats.

The anti-lipolytic effect of the adenosine analogue N6-L-phenylisopropyladenosine was studied with rat adipocytes incubated with a high concentration of adenosine deaminase (0.5 unit/ml, approx. 2.5 micrograms/ml) and concentrations of noradrenaline that were equieffective in different physiological states. These studies were performed to compare the fed and starved (24h) states and to compare a hypothyroid state (induced by feeding propylthiouracil + a low-iodine diet) with the euthyroid state. Starvation increased sensitivity of the cells to the lipolytic action of noradrenaline, while decreasing sensitivity to the antilipolytic action of phenylisopropyladenosine. Hypothyroidism resulted in decreased sensitivity to noradrenaline and increased sensitivity to phenylisopropyladenosine. Studies of the binding of [3H]phenylisopropyladenosine to adipocyte plasma membranes indicated heterogeneity of binding sites or negative co-operativity in the binding. Starvation did not change [3H]phenylisopropyladenosine binding to membranes, whereas hypothyroidism caused an unexpected decrease in both the number and affinity of the binding sites. These observations are discussed in terms of the dual regulation of adipose-tissue lipolysis by lipolytic and anti-lipolytic agents.     

Inhibition of acetyl-CoA carboxylase activity in isolated rat adipocytes incubated with glucagon. Interactions with the effects of insulin, adrenaline and adenosine deaminase

1. Adipocytes isolated from epididymal fat-pads of fed rats were incubated with different concentrations of glucagon, insulin, adrenaline and adenosine deaminase, and the effects of these agents on the ;initial' activity of acetyl-CoA carboxylase in the cells were studied. 2. Glucagon (at concentrations between 0.1 and 10nm) inhibited acetyl-CoA carboxylase activity. Maximal inhibition was approx. 70% of the ;control' activity in the absence of added hormone, and the concentration of hormone required for half-maximal inhibition was 0.3-0.5nm-glucagon. 3. Incubation of cells with adenosine deaminase resulted in a similar inhibition of acetyl-CoA carboxylase activity. Preincubation of adipocytes with adenosine deaminase did not alter either the sensitivity of carboxylase activity to increasing concentrations of glucagon or the maximal extent of inhibition. 4. Adrenaline inhibited acetyl-CoA carboxylase to the same extent as glucagon. Preincubation of the cells with glucagon did not alter the sensitivity of enzyme activity to adrenaline or the degree of maximal inhibition. 5. Insulin activated the enzyme by 70-80% of ;control' activity. Preincubation of the cells with glucagon did not alter the concentration of insulin required to produce half the maximal stimulatory effect (about 12muunits of insulin/ml). The effects of insulin and glucagon appeared to be mediated completely independently, and were approximately quantitatively similar but opposite. These characteristics resulted in the mutual cancellation of the effects of the two hormones when they were both present at equally effective concentrations. 6. The implications of these findings with regard to current concepts about the mechanism of regulation of acetyl-CoA carboxylase and to the regulation of the enzyme in vivo are discussed.     

Short-term fasting and lipolytic activity in rat adipocytes.

The aim of this experiment was to study the influence of 18-hour food deprivation on basal and stimulated lipolysis in adipocytes obtained from young male Wistar rats. Fat cells from fed and fasted rats were isolated from the epididymal adipose tissue by collagenase digestion. Adipocytes were incubated in Krebs-Ringer buffer (pH 7.4, 37 degrees C) without agents affecting lipolysis and with different lipolytic stimulators (epinephrine, forskolin, dibutyryl-cAMP, theophylline, DPCPX, amrinone) or inhibitors (PIA, H-89, insulin). After 60 min of incubation, glycerol and, in some cases, also fatty acids released from adipocytes to the incubation medium were determined. Basal lipolysis was substantially potentiated in cells of fasted rats in comparison to adipocytes isolated from fed animals. The inhibition of protein kinase A activity by H-89 partially suppressed lipolysis in both groups of adipocytes, but did not eliminate this difference. The agonist of adenosine A (1) receptor also did not suppress fasting-enhanced basal lipolysis. The epinephrine-induced triglyceride breakdown was also enhanced by fasting. Similarly, the direct activation of adenylyl cyclase by forskolin or protein kinase A by dibutyryl-cAMP resulted in a higher lipolytic response in cells derived from fasted animals. These results indicate that the fasting-induced rise in lipolysis results predominantly from changes in the lipolytic cascade downstream from protein kinase A. The antagonism of the adenosine A (1) receptor and the inhibition of cAMP phosphodiesterase also induced lipolysis, which was potentiated by food deprivation. Moreover, the rise in basal and epinephrine-stimulated lipolysis in adipocytes of fasted rats was shown to be associated with a diminished non-esterified fatty acids/glycerol molar ratio. This effect was presumably due to increased re-esterification of triglyceride-derived fatty acids in cells of fasted rats. Comparing fed and fasted rats for the antilipolytic effect of insulin in adipocytes revealed that short-term food deprivation resulted in a substantial deterioration of the ability of insulin to suppress epinephrine-induced lipolysis.     

Pertussis toxin effects on adenylate cyclase activity, cyclic AMP accumulation and lipolysis in adipocytes from hypothyroid, euthyroid and hyperthyroid rats

Adipocytes from hypothyroid rats have a decreased responsiveness to agents that activate adenylate cyclase, whereas cells from hyperthyroid rats have an increased responsiveness as compared to the controls. This is reflected in cyclic AMP accumulation as well as lipolysis. Administration of pertussis toxin to rats or its in vitro addition to adipocytes increased basal lipolysis and cyclic AMP accumulation as well as the response to norepinephrine or forskolin. The effects of thyroid status was not abolished by toxin treatment. Pertussis toxin-catalyzed ADP ribosylation of Ni was increased in adipocyte membranes from hypothyroid rats as compared to those from euthyroid rats. However, no change in sensitivity to N6-(phenylisopropyl)adenosine was observed. The data suggest that the amount of Ni might not be rate-limiting for the inhibitory action of adenosine. A consistent decrease in maximal lipolysis was observed in freshly isolated adipocytes from hypothyroid animals as compared to those from the controls. Such defective maximal lipolysis was not corrected by adenosine deaminase or in vivo administration of pertussis toxin. The relationship between cyclic AMP levels and lipolysis suggests that in fat cells from hypothyroid rats either the cyclic AMP-dependent protein kinase or the lipase activity itself may limit maximal lipolysis. There appears to be multiple effects of thyroid status on lipolysis involving factors other than those affecting adenylate cyclase activation.     

Glucagon inhibition of insulin-stimulated 2-deoxyglucose uptake by rat adipocytes in the presence of adenosine deaminase.

Effects of adenosine deaminase and glucagon on insulin-stimulated 2-deoxyglucose uptake by rat adipocytes are reported. (1) Adenosine deaminase (10 micrograms/ml) caused a rightward shift in the dose-response curve for the stimulation by insulin of 2-deoxyglucose uptake, but the enzyme did not alter either the basal or the maximally insulin-stimulated uptake rate. (2) In adipocytes obtained from 24 h-starved rats, glucagon inhibited the effect of insulin on 2-deoxyglucose uptake in the presence (but not in the absence) of adenosine deaminase. Basal uptake rates were unaffected. (3) Glucagon inhibited insulin-stimulated 2-deoxyglucose uptake to a greater extent in cells isolated from starved rats than in cells from fed rats. (4) Adipocytes isolated from fed and from starved rats did not differ in their capacity for degradation of 125I-labelled glucagon. The results suggest that adenosine and glucagon are regulators of insulin action in adipose tissue.     

Factors influencing the altered thermogenic response of rat brown adipose tissue in streptozotocin-diabetes.

1. Adipocytes were isolated from the interscapular brown fat of male rats maintained at 21 degrees C. These animals were controls, streptozotocin-diabetics or 2-day insulin-treated diabetics. 2. With adipocytes from diabetic animals, maximum rates of noradrenaline-stimulated O2 uptake were decreased by 58%, and the Bmax. of [3H]GDP binding to mitochondria was decreased by 55%. Insulin administration reversed both of these changes. 3. Streptozotocin-diabetes increased basal lipolysis in adipocytes incubated with adenosine deaminase (1 unit/ml), decreased the EC50 (concn. giving 50% of maximum effect) for noradrenaline, but did not change the maximum rate of noradrenaline-stimulated lipolysis. Except for some small differences at very low concentrations (10-100 pM), diabetes or insulin treatment did not alter the sensitivity of noradrenaline-stimulated lipolysis or O2 uptake to the inhibitory effect of N6-phenylisopropyladenosine. It is therefore concluded that the lesion(s) in thermogenesis in diabetes are not attributable to any changes in lipolysis. 4. Blood flow through interscapular brown fat, measured by accumulation of [14C]DDT [14C-labelled 1,1,1-trichloro-2,2-bis-(p-chlorophenyl)ethane] was increased by 2.3-fold 70 min after a single administration of insulin to diabetic rats. This treatment decreased blood flow through epididymal white fat by 58%. 5. Propranolol treatment of diabetic rats muted the ability of insulin treatment to increase the maximum rate of noradrenaline-stimulated O2 uptake, suggesting that this action of insulin may be a secondary one rather than a direct effect of the hormone on the adipocytes.     

Adenosine and the control of lipolysis in rat adipocytes during pregnancy and lactation.

The rate of noradrenaline-stimulated lipolysis is lower in fat-cells from lactating than from pregnant rats; this difference is eliminated by the addition of adenosine deaminase [Aitchison, Clegg & Vernon (1982) Biochem. J. 202, 243-247]. The activity of 5'-nucleotidase, and hence the capacity of the cells to synthesize adenosine, was the same in fat-cells and also stromal cells of adipose tissue from pregnant, lactating and male rats. The response and sensitivity of fat-cells to the anti-lipolytic effects of adenosine were measured by incubating cells in the presence of noradrenaline, adenosine deaminase (to remove endogenous adenosine) and various concentrations of the adenosine analogue N6-phenylisopropyladenosine (PIA). PIA caused a greater inhibition of the rate of noradrenaline-stimulated lipolysis in adipocytes from lactating than from pregnant rats. The concentration of PIA required to inhibit by 50% the rate of noradrenaline-stimulated lipolysis fell from over 100 nM for fat-cells from pregnant rats to 30 nM for fat-cells from lactating rats. The decreased rate of noradrenaline-stimulated lipolysis during lactation was not due to the smaller mean cell volume of adipocytes during this state.     

Regulation of lipolysis in isolated adipocytes of rainbow trout (Oncorhynchus mykiss): the role of insulin and glucagon

In the present study, we have examined the effects of insulin and glucagon on the lipolysis of rainbow trout (Oncorhynchus mykiss). To this end, adipocytes were isolated from mesenteric fat and incubated in the absence (basal lipolysis) or presence of different concentrations of insulin and glucagon. In addition, to further elucidate the effects of these hormones in vivo on adipocyte lipolysis, both fasting and intraperitoneal glucagon injection experiments were performed. Basal lipolysis, measured as the glycerol released in the adipocyte medium, increased proportionally with cell concentration and incubation time. Cell viability was verified by measuring the release of lactate dehydrogenase (LDH) activity in the medium. Insulin (at doses of 35 and 350 nM) decreased lipolysis in isolated adipocytes of rainbow trout in vitro, while glucagon was clearly lipolytic at concentrations of 10 and 100 nM. Furthermore, hypoinsulinemia induced by fasting, as well as glucagon injection, significantly increased lipolysis in isolated adipocytes approximately 1.5- and 1.4-fold, respectively, when compared with adipocytes from control fish. Our data demonstrate that lipolysis, as measured in isolated adipocytes of rainbow trout, can be regulated by both insulin and glucagon. These results not only indicate that insulin is an important hormone in lipid deposition via its anti-lipolytic effects on rainbow trout adipocytes, but also reveal glucagon as a lipolytic hormone, as shown by both in vitro and in vivo experiments.     

Lipolysis in rat adipocytes during pregnancy and lactation. The response to noradrenaline.

1. Lipolysis has been measured in parametrial adipocytes from virgin, pregnant and lactating rats. 2. The basal rate and the maximal rate of lipolysis, the latter measured in the presence of noradrenaline and theophylline, remained constant between the three experimental categories, with the exception of a significant transient increase in the basal rate at parturition. 3. The noradrenaline-stimulated lipolysis rate rose above the virgin rate during pregnancy and fell below it during lactation; inclusion of adenosine deaminase in incubations abolished these differences in response to noradrenaline. 4. Cyclic AMP phosphodiesterase activity was lower in adipocytes during pregnancy and lactation than in virgin animals.     

Adenosine effects upon insulin action on lipolysis and glucose transport in human adipocytes

The dose response effect of a new adenosine analogue, GR 79236 (N-[1S trans-2-hydroxycyclopentyl] adenosine) upon insulin sensitivity was examined in human adipocytes. The influence of adenosine upon insulin sensitivity for suppression of lipolysis and stimulation of glucose transport was examined. Removal of adenosine by use of adenosine deaminase stimulated lipolysis to the same extent as did 10^–9 M noradrenaline. GR79236 brought about dose dependent inhibition of lipolysis with half-maximal effect at 11.3±7.8×10^–9 M. When lipolysis was stimulated by noradrenaline alone the subsequent inhibition of lipolysis brought about by GR79236 was significantly greater than that of insulin. To examine adenosine effects on the insulin signalling pathway separately from those on lipolysis, the insulin sensitivity of glucose transport was examined. Removal of adenosine brought about a small but significant increase in the concentration of insulin required for half-maximal stimulation of glucose transport. Adenosine agonists offer promise as new agents for the modulation of metabolism in diabetes and other states of insulin resistance.