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.     

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.     

Rat fat-cells have three types of adenosine receptors (Ra, Ri and P). Differential effects of pertussis toxin.

Activation of rat adipocyte R1 adenosine receptors by phenylisopropyladenosine (PIA) decreased cyclic AMP and lipolysis; this effect was blocked in cells from pertussis-toxin-treated rats. In contrast, the ability of 2',5'-dideoxyadenosine to decrease cyclic AMP was not affected by pertussis-toxin treatment. Addition of adenosine deaminase to the medium in which adipocytes from control animals were incubated resulted in activation of lipolysis. Interestingly, adipocytes from toxin-treated rats (which had an already increased basal lipolysis) responded in an opposite fashion to the addition of adenosine deaminase, i.e. the enzyme decreased lipolysis, which suggested that adenosine might be increasing lipolysis in these cells. Studies with the selective agonists N-ethylcarboxamidoadenosine (NECA) and PIA indicated that adenosine increases lipolysis and cyclic AMP accumulation in these cells and that these actions are mediated through Ra adenosine receptors. Adenosine-mediated accumulation of cyclic AMP was also observed in cells preincubated with pertussis toxin (2 micrograms/ml) for 3 h. In these studies NECA was also more effective than PIA. Our results indicate that there are three types of adenosine receptors in fat-cells, whose actions are affected differently by pertussis toxin, i.e. Ri-mediated actions are abolished, Ra-mediated actions are revealed and P-mediated actions are not affected.     

Role of growth hormone in the adaptations of lipolysis in rat adipocytes during recovery from lactation.

Removal of the litter from lactating rats results in a fall in the rate of noradrenaline-stimulated lipolysis of adipocytes. This adaptation can be prevented by administration of growth hormone (somatotropin) to such rats and mimicked by injecting lactating rats with an antiserum to growth hormone, whereas lowering serum prolactin by injecting bromocriptine had no effect. The anti-lipolytic effect of adenosine is increased during lactation and is still increased by 2 days after litter removal. Injection of growth hormone into lactating rats decreased slightly the response to adenosine, whereas injection of growth hormone into rats after removal of their litters resulted in a much greater decrease in the response to adenosine, to that found in virgin and pregnant rats.     

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.     

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.     

Impaired insulin-mediated inhibition of lipolysis and glucose transport with aging

Regulation of hormone action with aging has been extensively studied; adipocytes provide an interesting model for some of these questions. We have compared the ability of insulin to stimulate glucose uptake and suppress lipolysis in adipocytes isolated from two month and twelve month-old rats. The ability of insulin to stimulate maximal glucose transport was decreased in adipocytes from the older rats (P less than 0.001); as well, insulin's EC50 was also higher (P less than 0.01) in these cells. Furthermore, these defects were present when insulin-stimulated glucose transport was measured in the presence or absence of adenosine deaminase which metabolizes endogenously released adenosine. Endogenously released adenosine is a stimulator of glucose transport and an inhibitor of lipolysis. Maximal suppression of isoproterenol-induced lipolysis by insulin was similar when adipocytes isolated from the two age groups were incubated in the absence of adenosine deaminase. However, maximal insulin-mediated suppression of lipolysis was found to be significantly decreased (P less than 0.001) in adipocytes isolated from older rats when the experiments were done in the presence of adenosine deaminase; also, insulin's EC50 was increased in these cells under these conditions (P less than 0.001). These results emphasize the importance of the adenosine receptor in modulating the response of isolated adipocytes to insulin, particularly for lipolysis, and document the presence of age-associated defects in insulin regulation of both glucose transport and lipolysis.     

Adenosine receptor down-regulation and insulin resistance following prolonged incubation of adipocytes with an A1 adenosine receptor agonist

Adenosine, via interaction with A1 adenosine receptors, increases insulin sensitivity and inhibits lipolysis in adipocytes. To investigate regulation of this system, adipocytes were incubated for up to 72 h with the nonmetabolizable adenosine receptor agonist, N6-phenylisopropyl adenosine (PIA). Adenosine receptors were measured by the binding of 125I-hydroxyphenylisopropyl adenosine to membranes. PIA down-regulated adenosine receptors, decreasing the number of binding sites with no change in affinity. Adipocytes were incubated for 48 h without or with 100 nM PIA to down-regulate the A1 receptors by approximately 60%. The cells were washed, and lipolysis and glucose transport were assessed. The ability of PIA to inhibit lipolysis was markedly attenuated in the down-regulated cells. Furthermore, the EC50 of insulin was increased approximately 3-fold in the PIA-treated cells. 125I-Insulin binding to the PIA-treated cells was unchanged, demonstrating that the decreased insulin sensitivity is not due to decreased insulin receptor binding. Pertussis toxin catalyzed ADP-ribosylation of a 41-kDa protein thought to be the alpha-subunit of Gi. This 41-kDa protein was decreased in membranes from cells treated with PIA, with a maximal 50% loss. This suggests that Gi is down-regulated and that loss of both the A1 adenosine receptor and Gi are involved in the metabolic changes observed after PIA treatment.     

Prolonged treatment with prostaglandin E1 increases the rate of lipolysis in rat adipocytes

Prolonged treatment of adipocytes with certain inhibitors of lipolysis, including N(6)-phenylisopropyl adenosine (PIA) and prostaglandin E(1) (PGE(1)) leads to down-regulation of G(i). Prolonged treatment with PIA increases the rate of lipolysis, and we have reported that tumor necrosis factor-alpha (TNF alpha) stimulates lipolysis by down-regulating G(i). To determine the relationship between G(i) concentration and lipolysis, we have investigated the effect of two other acute inhibitors of lipolysis; PGE(1), which down-regulates G(i), and nicotinic acid (NA), which does not down-regulate G(i). Rat adipocytes were incubated with PIA (300 nM), PGE(1) (3 microM) or nicotinic acid (1 mM) for 24 h. The rate of lipolysis (glycerol release) was increased approximately 2 to 3-fold in PIA-treated cells, and in PGE(1)-treated cells. Conversely, the rate of lipolysis was not altered by the prolonged nicotinic acid treatment. These findings support the hypothesis that the rate of lipolysis in adipocytes is determined, at least partly, by the cellular concentration of G(i) proteins.     

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.     

Antilipolytic effects of N6-phenylisopropyladenosine and prostaglandin E2 in fat-cells of obese volunteers before and during energy restriction.

The antilipolytic effects of N6-phenylisopropyladenosine and of prostaglandin E2 were studied with adipocytes of obese volunteers before and after 4 weeks of severe energy restriction [1250 kJ (300 cal)/day] in the presence and absence of adenosine deaminase (1.6 micrograms/ml, corresponding to 320 m-units/ml). The studies were undertaken to define more clearly the role that local modulators might play in adaptation of lipid mobilization to starvation in humans. Starvation was associated with an approx. 3-fold increase in non-stimulated lipolysis. Removal of endogenous adenosine resulted in a similar increase in basal glycerol release under both conditions, averaging 2 and 2.2 mumol/180 min per 10(6) cells respectively. The sensitivity of the cells to N6-phenylisopropyladenosine and to prostaglandin E2 was not changed by starvation in the presence of adenosine deaminase. These results are discussed in terms of the possible role that local regulators might play during dietary adaption in human fat-cells in vitro.     

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.     

Does glucocorticoid deprivation promote the expression of adenosine receptor-sites stimulating adenylate cyclase in rat adipocyte membranes?

The influence of N⁶-phenylisopropyladenosine (PIA) on adenylate cyclase was compared in adipocyte membranes from adrenalectomized and sham operated rats. In the presence of 100 mM sodium, 10 μM GTP and adenosine deaminase, PIA inhibited basal adenylate cyclase activity in sham rats, but elicited biphasic effects in adrenalectomized rats: at concentrations up to 10 nM, PIA first stimulated the enzyme, after which higher concentrations produced inhibition. In the presence of theophylline, these biphasic effects could not be observed. When isoproterenol maximally-stimulated adenylate cyclase was studied, the same biphasic effects of PIA were also observed in adrenalectomized rats, provided that no sodium was added in the assay, since with 100 mM sodium, only inhibition was seen. Finally, the stimulatory but not the inhibitory effect of PIA was prevented by glucocorticoid administration, a phenomenon which suggests that glucocorticoid deprivation may promote the expression of adenosine receptorsites which activate adenylate cyclase and which are normally absent, cryptic or unfunctional in normal adipocytes.     

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.     

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.     

Phosphatidic acid and phosphatidylinositol labelling in adipose tissue. The role of endogenously formed adenosine.

Incorporation of [32P]Pi into phosphatidic acid and phosphatidylinositol of hamster epididymal adipocytes was partially inhibited by 3-isobutyl-1-methylxanthine. This effect of 3-isobutyl-1-methylxanthine was antagonized by isopropyl-N6-phenyladenosine but not by 2',5'-dideoxyadenosine, prostaglandin E1 or clonidine. N6-Phenylisopropyladenosine did not affect incorporation of [32P]Pi into phosphatidic acid or phosphatidylinositol when 3-isobutyl-1-methylxanthine was not present. In contrast with 3-isobutyl-1-methylxanthine inhibition of [32P]Pi incorporation into phospholipids, which was blocked only by N6-phenylisopropyladenosine, accelerated lipolysis was blocked by prostaglandin E1, clonidine and 2',5'-dideoxyadenosine as well as by N6-phenylisopropyladenosine. Phospholipid labelling was also decreased in the presence of adenosine deaminase, but not in the presence of isoprenaline (isoproterenol). The stimulatory effect of N6-phenylisopropyladenosine on [32P]Pi incorporation into phospholipids in cells exposed to 3-isobutyl-1-methylxanthine was evident as soon as 3 min after addition of the adenosine analogue and maximum 10 min after its addition. As observed by others, [32P]Pi incorporation into phospholipids was increased by the alpha 1-selective agonist methoxamine. The stimulatory effect of methoxamine occurred with a time course similar to that of N6-phenylisopropyladenosine and was present at nearly equal magnitude in the absence or presence of 3-isobutyl-1-methylxanthine. The inhibitory effects of 3-isobutyl-1-methylxanthine and adenosine deaminase on phospholipid labelling are attributed to blockade of the action, or to the enzymic removal, of adenosine formed in and released from the fat-cells during their incubation. Supporting this view is the selective reversal of the actions of 3-isobutyl-1-methylxanthine and of adenosine deaminase by N6-phenylisopropyladenosine. These findings suggest an important role for endogenous adenosine in regulation of phospholipid turnover in adipocytes.     

Leptin-induced lipolysis opposes the tonic inhibition of endogenous adenosine in white adipocytes.

The aim of the present study was to gain insight into the signaling pathway used by leptin to stimulate lipolysis. The lipolytic rate of white adipocytes from sex- and age-matched lean (+/+) and fa/fa rats was determined in the absence or presence of leptin together with a number of agents acting at different levels of the signaling cascade. Leptin did not modify FSK-, dbcAMP-, and IBMX-stimulated lipolysis. Lipolysis can also be maximally stimulated by lowering media adenosine levels with adenosine deaminase (ADA), i.e., in the ligand-free state. Although ADA produced near maximal lipolysis in adipocytes of lean animals, only half of the maximal lipolytic rate (50.9+/-3.2%) was achieved in fat cells from fa/fa rats (P=0.0034). In adipocytes from lean animals preincubated with ADA, leptin caused a concentration-related stimulation of lipolysis (P=0.0001). However, leptin had no effect on the lipolytic activity of adipocytes in the ligand-free state from fa/fa rats. The adenosine A1 receptor agonist CPA effectively inhibited basal lipolysis in both lean and obese adipocytes (P=0.0001 and P=0.0090, respectively). Leptin had no effect on the lipolytic rate of adipocytes isolated from fa/fa rats and preincubated with CPA. When adipocytes were incubated with the A1 receptor antagonist DPCPX, a significant increase in glycerol release was observed in fa/fa fat cells (P=0.009), whereas cells isolated from lean rats showed no differences to ADA-stimulated lipolysis. After pretreatment with PTX, which inactivates receptor-mediated Gi function, adipocytes of obese rats became as responsive to the stimulatory actions of ISO as cells from lean rats (P=0.0090 vs. ISO in fa/fa rats; P=0.2416 vs. lean rats, respectively). PTX treatment of lean cells, however, did not alter their response to this lipolytic agent. It can be concluded that the lipolytic effect of leptin is located at the adenylate cyclase/Gi proteins level and that leptin-induced lipolysis opposes the tonic inhibition of endogenous adenosine in white adipocytes.     

Down-regulation of Gi sub-types by prolonged incubation of adipocytes with an A1 adenosine receptor agonist

We have reported previously that prolonged incubation of adipocytes with (-)-N6-phenylisopropyl adenosine (PIA) (an A1 adenosine receptor agonist) down-regulates A1 adenosine receptors. There was a concomitant decrease in pertussis toxin catalyzed ADP-ribosylation of a 41-kDa peptide thought to be the alpha-subunit of Gi. To determine whether this represents true down-regulation of the G-protein, and if so which of the three known forms of Gi are down-regulated, we have used antipeptide antisera specific for Gi alpha-subunits. Serum SG1 recognizes alpha i1 and -2, I1C recognizes only alpha i1, and I3B recognizes alpha i3. Rat adipocytes were maintained in primary culture for up to 7 days with 0-1000 nM PIA. Crude membrane preparations were analyzed by Western blots. There was almost complete loss of alpha i1 and -3, and about 50% loss of alpha i2 from PIA-treated cells. The loss of each alpha i was detectable after 24 h with 300 nM PIA and maximal by 4 days. After 4 days, down-regulation was detectable with 3 nM and maximal with 100 nM PIA. Antiserum BN2 demonstrated approximately 50% loss of G-protein beta-subunits in cells treated with 300 nM PIA for 4 days. When cells were incubated for 4 days with 300 nM PIA and then washed to remove PIA, alpha i1, -2, and -3 and beta-subunits returned to control levels within 5 days. Antiserum CS1 detected normal amounts of both the 43- and 47-kDa forms of Gs alpha in PIA-treated cells. We conclude that Gi alpha-subunits are down-regulated along with the adenosine receptor in rat adipocytes.     

Tumor necrosis factor alpha stimulates lipolysis in adipocytes by decreasing Gi protein concentrations

Prolonged treatment (12-24 h) of adipocytes with tumor necrosis factor alpha (TNFalpha) stimulates lipolysis. We have investigated the hypothesis that TNFalpha stimulates lipolysis by blocking the action of endogenous adenosine. Adipocytes were incubated for 48 h with TNFalpha, and lipolysis was measured in the absence or presence of adenosine deaminase. Without adenosine deaminase, the rate of glycerol release was 2-3-fold higher in the TNFalpha-treated cells, but with adenosine deaminase lipolysis increased in the controls to approximately that in the TNFalpha-treated cells. This suggests that TNFalpha blocks adenosine release or prevents its antilipolytic effect. Both N6-phenylisopropyl adenosine and nicotinic acid were less potent and efficacious inhibitors of lipolysis in treated cells. A decrease in the concentration of alpha-subunits of all three Gi subtypes was detected by Western blotting without a change in Gs proteins or beta-subunits. Gi2alpha was about 50% of control, whereas Gi1alpha and Gi3alpha were about 20 and 40% of control values, respectively. The time course of Gi down-regulation correlated with the stimulation of lipolysis. Furthermore, down-regulation of Gi by an alternative approach (prolonged incubation with N6-phenylisopropyl adenosine) stimulated lipolysis. These findings indicate that TNFalpha stimulates lipolysis by blunting endogenous inhibition of lipolysis. The mechanism appears to be a Gi protein down-regulation.