Stimulation and inhibition of lipolysis in isolated rat adipocytes: evidence for age-related changes in responses to forskolin and PGE1

The ability of a number of hormones to activate cellular responses in a variety of cells declines with age. The mechanisms responsible for these alterations are complex and incompletely understood. Rat adipocytes have served as an important model to study blunted responses to stimulatory hormones which function by activating cAMP accumulation. We have previously found that the blunted lipolytic response of adipocytes from older rats to the beta adrenergic receptor agonist isoproterenol appeared to be due to a lessened ability of isoproterenol to activate cAMP accumulation. Further, the blunted response to isoproterenol was apparently caused by an accentuated inhibition of lipolysis, mediated by adenosine receptors activated by endogenously released adenosine. The present studies were designed to test and extend those conclusions. We have utilized forskolin to augment the cAMP accumulation that occurs in the presence of isoproterenol. Isoproterenol-activated lipolysis was greater in adipocytes from 2 month old rats compared with those from 12 month old rats (603 +/- 32 vs 450 +/- 29 nmol/10(5) cells/hr, P less than 0.01). However, in the presence of forskolin (10(-6) M), there was no significant difference in the response to isoproterenol between the two groups (646 +/- 23 vs 615 +/- 29 nmoles/10(5) cells/hr). As we had seen previously, the adenosine receptor agonist phenylisopropyladenosine more effectively inhibited lipolysis in the adipocytes from older rats. We now also find that PGE1 more efficaciously inhibits lipolysis in the cells from older rats. These data confirm that diminished cAMP accumulation in adipocytes from older rats appears to be a rate-limiting alteration in the regulation of lipolysis.(ABSTRACT TRUNCATED AT 250 WORDS)     

The effects of lipolytic hormones on calcium uptake in endoplasmic reticulum of adipocytes

To explore interrelationship between the roles of cAMP and calcium ion in hormone-stimulated lipolysis, cAMP accumulation in rat adipocytes and calcium binding in the endoplasmic reticulum were investigated with special reference to the effects of lipolytic hormones under various conditions. ACTH, isoproterenol, DBcAMP and aminophylline significantly increased ATP-dependent calcium uptake in adipocyte endoplasmic reticulum, but only after they were incubated with intact cells and not when they were added after homogenization. In vivo dexamethasone treatment and A-23187 accelerated, while 2.4-dinitrophenol blunted ACTH-stimulated lipolysis, cAMP accumulation and microsomal calcium uptake in parallel. Adrenalectomy, Mn2+ and adenosine enhanced ACTH-stimulated cAMP accumulation in adipocytes but lowered the calcium uptake and lipolysis. Thus, there was consistent parallelism between hormone-stimulated lipolysis and microsomal calcium uptake throughout the study. These data suggest that changes in the microsomal calcium uptake plays a crucial role in the regulation of hormone-induced lipolysis, irrespective of whether or not the intracellular cAMP concentration is involved in the lipolytic mechanism.     

Calciotropic hormones and lipolysis of human adipose tissue: role of extracellular calcium as conditioning but not regulating factor

The influences of different calcium concentrations (0, 0.924 and 2.772 mMol/l) on lipolysis of in vitro incubated human adipose tissue slices or adipocytes were studied under the conditions of stimulation with isoproterenol and parathyroid hormone preparations or inhibition by insulin. Extractive bovine PTH (as well as synthetic PTH 1--34) stimulated glycerol release in a biphasic pattern similarly to isoproterenol; PTH was about half as potent as isoproterenol. The optimal conditions for lipolysis were observed using a calcium concentration of 0.924 mMol/l, whereas lipolysis was distinctly impaired at concentrations of 0 or 2.772 mMol/l; this was true for basal as well as isoproterenol- and PTH stimulated lipolysis or the inhibitory effect of insulin. In contrast to partially purified extractive calcitonin, pure synthetic calcitonin did not inhibit lipolysis. Isoproterenol- and PTH-administrations led to cAMP accumulation in the adipose tissue, this process was also diminished at the non-optimal calcium concentrations. The results suggest a conditioning, but not a regulating significance of extracellular calcium for lipolysis, whereas the importance of the lipolytic potency of PTH remains to be elucidated.     

Roles of alpha and beta adrenergic receptors in control of glucose oxidation in hamster epididymal adipocytes.

Oxidation of [14C] glucose in isolated epididymal adipocytes from Golden hamsters was stimulated by isoproterenol, epinephrine and norepinephrine, which all interact with beta-adrenergic receptors and by adrenocorticotrophic hormone. In contrast alpha-receptor agonists, such as phenylephrine, methoxamine or clonidine did not increase basal glucose oxidation. The beta-adrenergic blocking drug propranolol inhibited both lipolysis and glucose oxidation when these had been stimulated by isoproterenol, epinephrine or norepinephrine. Conversely, the alpha-adrenergic blocking drugs phentolamine and phenoxybenzamine did not influence lipolysis or glucose oxidation when isoproterenol provided the stimulus and increased both lipolysis and glucose metabolism in the present of either epinephrine or norepinephrine. All alpha-adrenergic agonists tested (phenylephrine, methoxamine and clonidine) lowered lipolysis and glucose oxidation isolated adipocytes exposed to isoproterenol. However, when adrenocorticotropin provided the stimulus for glucose oxidation and lipolysis, only clonidine produced a significant reduction in lipolysis and glucose oxidation. None of the alpha-agonists influenced glucose metabolism which had been increased by insulin. These data confirm the presence of both alpha and beta adrenergic receptors on hamster epididymal adipocytes and suggest that they exert antagonistic influences on lipolysis and glucose oxidation. These data are also consistent with the view that adrenergic stimulation of glucose oxidation and lipolysis in adipocytes are both mediated through beta receptors.     

Effect of insulin and adrenergic agonists on glucose transport of porcine adipocytes

1. Insulin increased basal 2-deoxyglucose uptake in isolated swine adipocytes by 75%. In the absence of insulin, isoproterenol did not inhibit basal 2-deoxyglucose transport. 2. Adenosine deaminase plus isoproterenol or theophylline alone reduced insulin effect by 10 and 40%, respectively. Isoproterenol alone or with 2-chloroadenosine did not inhibit insulin effect on glucose transport activity. 3. Insulin effect was inhibited by isoproterenol in the presence of theophylline but not in the presence of adenosine deaminase. 4. These results suggest that catecholamines do not counter-regulate basal and insulin-stimulated glucose transport in swine adipocytes.     

Roles of alpha and beta adrenergic receptors in control of glucose oxidation in hamster epididymal adipocytes

Oxidation of [¹⁴C]glucose in isolated epididymal adipocytes from Golden hamsters was stimulated by isoproterenol and norepinephrine, which all interact with β-adrenergic receptors and by adrenorticotrophic hormone. In contrast α-receptor agonists, such as phenylephrine, methoxamine or clonidine did not increase basal glucose oxidation. The β-adrenergic blocking drug propranolol inhibited both lipolysis and glucose oxidation when these had been stimulated by isoproterenol, ephinephrine and phenoxybenzamine did not the α-adrenergic blocking drugs phentolamine and phenoxybenzamine did not influence lipolysis or glucose oxidation when isoproterenol provided the stimulus and increased both liposlysis and glucose metabolism in the presence of either epinephrine or norepinephrine. All α-adrenergic agonists tested (phenylephrine, methoxamine and clonidine) lowered liposlysis and glucose oxidation in isolated adipocytes exposed to isoproterenol. However, when adrenorcortropin provided the stimulus for glucose oxidation and lipolysis, only clonidine produced a significant reduction in lipolysis and glucose oxidation. None of the α-agonists influenced glucose metabolism which had been increased by insulin. These data confirm the presence of both α and β adrenergic receptors on hamster epididymal adipocytes and suggests that they exert antagonistic influences on lipolysis and glucose oxidation. These data are also consistent with the view that adrenergic stimulation of glucose oxidation and lipolysis in adipocytes are both mediated through β receptors.     

Role of AMP-activated protein kinase in cyclic AMP-dependent lipolysis In 3T3-L1 adipocytes

AMP-activated protein kinase (AMPK) is a phylogenetically conserved intracellular energy sensor that has been implicated as a major regulator of glucose and lipid metabolism in mammals. However, its possible role in mediating or influencing the adrenergic control of lipolysis in adipocytes remains uncertain. In this study, we utilized the murine cultured preadipocyte line 3T3-L1 to examine this question. Treatment of adipocytes with isoproterenol or forskolin promoted the phosphorylation of AMPK at a critical activating Thr-172 residue in a dose- and time-dependent manner. This correlated well with a stimulation of the activity of AMPK, as measured in the immune complex. Analogs of cAMP mimicked the effect of isoproterenol and forskolin on AMPK phosphorylation. Treatment of adipocytes with insulin reduced both basal and forskolin-induced AMPK phosphorylation via a pathway dependent on phosphatidylinositol 3'-kinase. Overexpression of a dominant-inhibitory mutant of AMPK blocked isoproterenol-induced lipolysis by approximately 50%. These data indicate that there exists a novel pathway by which cAMP can lead to the activation of AMPK, and in adipocytes, this is required for maximal activation of lipolysis.     

Calyculin and okadaic acid promote perilipin phosphorylation and increase lipolysis in primary rat adipocytes

Lipolysis is primarily regulated by protein kinase A (PKA), which phosphorylates perilipin and hormone-sensitive lipase (HSL), and causes translocation of HSL from cytosol to lipid droplets in adipocytes. Perilipin coats lipid droplet surface and assumes to prevent lipase access to triacylglycerols, thus inhibiting basal lipolysis; phosphorylated perilipin facilitates lipolysis on PKA activation. Here, we induced lipolysis in primary rat adipocytes by inhibiting protein serine/threonine phosphatase with specific inhibitors, okadaic acid and calyculin. The incubation with calyculin promotes incorporation of 32Pi into perilipins, thus, confirming that perilipin is hyperphosphorylated. The lipolysis response to calyculin is gradually accompanied by increased accumulation of phosphorylated perilipin A in a concentration- and time-responsive manner. When perilipin phosphorylation is abrogated by the addition of N-ethylmaleimide, lipolysis ceases. Different from a considerable translocation of HSL upon PKA activation with isoproterenol, calyculin does not alter HSL redistribution in primary or differentiated adipocytes, as confirmed by both immunostaining and immunoblotting. Thus, we suggest that inhibition of the phosphatase by calyculin activates lipolysis via promoting perilipin phosphorylation rather than eliciting HSL translocation in adipocytes. Further, we show that when the endogenous phosphatase is inhibited by calyculin, simultaneous PKA activation with isoproterenol converts most of the perilipin to the hyperphosphorylated species, and induces enhanced lipolysis. Apparently, as PKA phosphorylates perilipin and stimulates lipolysis, the phosphatase acts to dephosphorylate perilipin and attenuate lipolysis. This suggests a two-step strategy governed by a kinase and a phosphatase to modulate the steady state of perilipin phosphorylation and hence the lipolysis response to hormonal stimulation.     

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.     

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.     

Insulin-induced dephosphorylation of hormone-sensitive lipase. Correlation with lipolysis and cAMP-dependent protein kinase activity

The effect of insulin on the state of phosphorylation of hormone-sensitive lipase, cellular cAMP-dependent protein kinase activity and lipolysis was investigated in isolated adipocytes. Increased phosphorylation of hormone-sensitive lipase in response to isoproterenol stimulation was closely paralleled by increased lipolysis. Maximal phosphorylation and lipolysis was obtained when the cAMP-dependent protein kinase activity ratio was greater than or equal to 0.1, and this corresponded to a 50% increase in the state of phosphorylation of hormone-sensitive lipase. Insulin (1 nM) reduced cAMP-dependent protein kinase activity and also reduced lipolysis with both cAMP-dependent and cAMP-independent antilipolytic effects up to an activity ratio of approximately 0.4, above which the antilipolytic effect was lost. Insulin caused a decrease in the state of phosphorylation of hormone-sensitive lipase at all levels of cAMP-dependent protein kinase activity. Under basal conditions, with cAMP-dependent protein kinase activity at a minimum, this reflected a dephosphorylation of the basal phosphorylation site of hormone-sensitive lipase in a manner not mediated by cAMP. When the cAMP-dependent protein kinase was stimulated to phosphorylate the regulatory phosphorylation site of hormone-sensitive lipase, the insulin-induced dephosphorylation occurred both at the basal and regulatory sites. At low levels of cAMP-dependent protein kinase activity ratios (0.05-0.1), dephosphorylation of the regulatory site correlated with reduced cAMP-dependent protein kinase activity, but not at higher activity ratios (greater than 0.1). Stimulation of cells with isoproterenol produced a transient (1-5 min) peak of cAMP-dependent protein kinase activity and of phosphorylation of hormone-sensitive lipase. The state of phosphorylation also showed a transient peak when the protein kinase was maximally and constantly activated. In the presence of raised levels of cellular cAMP, insulin (1 nM) caused a rapid (t1/2 approximately 1 min) dephosphorylation of hormone-sensitive lipase. In unstimulated cells the reduction in phosphorylation caused by insulin was distinctly slower (t1/2 approximately 5 min). These findings are interpreted to suggest that insulin affects the state of phosphorylation of hormone-sensitive lipase and lipolysis through a cAMP-dependent pathway, involving reduction of cAMP, and through a cAMP-independent pathway, involving activation of a protein phosphatase activity that dephosphorylates both the regulatory and basal phosphorylation sites of hormone-sensitive lipase.     

Cyclic nucleotides and lipolysis in rat fat cells. Interaction between calcium ionophore A23187 and FCCP,uncoupler of oxidative phosphorylation

The interaction between calcium ionophore A23187 and carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) has been studied at the level of cyclic-AMP (cAMP), cyclic-GMP (cGMP) and lipolysis in isolated rat fat cells. Ionophore A23187 (1–10 μM) stimulated cGMP accumulation and glycerol release without affecting cAMP level. FCCP (1–100 μM) inhibited the effect of A23187 on cGMP level and glycerol release, but did not affect or increase cAMP. Thus a correlation exists between the changes of cGMP levels and lipolysis and a dissociation of lipolysis from cAMP.     

Both inflammatory and classical lipolytic pathways are involved in lipopolysaccharide-induced lipolysis in human adipocytes

High fat diet-induced endotoxaemia triggers low-grade inflammation and lipid release from adipose tissue. This study aims to unravel the cellular mechanisms leading to the lipopolysaccharide (LPS) effects in human adipocytes. Subcutaneous pre-adipocytes surgically isolated from patients were differentiated into mature adipocytes in vitro. Lipolysis was assessed by measurement of glycerol release and mRNA expression of pro-inflammatory cytokines were evaluated by real-time PCR. Treatment with LPS for 24 h induced a dose-dependent increase in interleukin (IL)-6 and IL-8 mRNA expression. At 1 μg/ml LPS, IL-6 and IL-8 were induced to 19.5 ± 1.8-fold and 662.7 ± 91.5-fold (P < 0.01 vs basal), respectively. From 100 ng/ml to 1 μg/ml, LPS-induced lipolysis increased to a plateau of 3.1-fold above basal level (P < 0.001 vs basal). Co-treatment with inhibitors of inhibitory kappa B kinase kinase beta (IKKβ) or NF-κB inhibited LPS-induced glycerol release. Co-treatment with the protein kinase A (PKA) inhibitor H-89, the lipase inhibitor orlistat or the hormone-sensitive lipase (HSL) inhibitor CAY10499 abolished the lipolytic effects of LPS. Co-treatment with the MAPK inhibitor, U0126 also reduced LPS-induced glycerol release. Inhibition of lipolysis by orlistat or CAY10499 reduced LPS-induced IL-6 and IL-8 mRNA expression. Induction of lipolysis by the synthetic catecholamine isoproterenol or the phosphodiesterase type III inhibitor milrinone did not alter basal IL-6 and IL-8 mRNA expression after 24 treatments whereas these compounds enhanced LPS-induced IL-6 and IL-8 mRNA expression. Both the inflammatory IKKβ/NF-κB pathway and the lipolytic PKA/HSL pathways mediate LPS-induced lipolysis. In turn, LPS-induced lipolysis reinforces the expression of pro-inflammatory cytokines and, thereby, triggers its own lipolytic activity.     

Calcium antagonists and lipolysis in isolated rat epididymal adipocytes: effects of tetracaine, manganese, cobaltous and lanthanum ions and D600.

The present study reports the effects on lipolysis occurring in isolated rat epididymal adipocytes of several agents which have each been found to interfere with membrane calcium transport in a variety of tissues. As reported by other workers, the local tetracaine was a strong inhibitor of hormone accelerated but not of basal lipolysis. The bivalent cations Mn2+ and Co2+ were similarly found to inhibit lipolysis stimulated with either epinephrine, ACTH, theophylline or dibutyryl cyclic AMP, whereas basal lipolysis was not markedly altered. This effect of Mn2+ and Co2+ was not mimicked by either Sr2+, Ba2+, Mg2+ or Ca2+. Cyclic AMP levels in adipocytes stimulated with epinephrine or ACTH tended to be higher in the presence of Mn2+ and Co2+. It is concluded, therefore, that Mn2+ and Co2+ inhibit lipolysis by uncoupling cyclic AMP accumulation from activation of triglyceride lipase. In contrast to Mn2+ and Co2+, the calcium antagonists La3+ and D600 were without effect on lipolysis. The antilipolytic effect of tetracaine, Mn2+ and Co2+ was found to persist in the absence of extracellular calcium, suggesting therefore that the antilipolytic effect of these drugs is unrelated to inhibition of calcium influx into adipocytes. The possibility is discussed that lipolytic agents cause an intracellular redistribution of calcium ion and that local anesthetics, Mn2+ and Co2+ interfere with lipolysis by preventing this intracellular redistribution of calcium.     

Berberine attenuates cAMP-induced lipolysis via reducing the inhibition of phosphodiesterase in 3T3-L1 adipocytes

Berberine, a hypoglycemic agent, has been shown to decrease plasma free fatty acids (FFAs) level in insulin-resistant rats. In the present study, we explored the mechanism responsible for the antilipolytic effect of berberine in 3T3-L1 adipocytes. It was shown that berberine attenuated lipolysis induced by catecholamines, cAMP-raising agents, and a hydrolyzable cAMP analog, but not by tumor necrosis factor α and a nonhydrolyzable cAMP analog. Unlike insulin, the inhibitory effect of berberine on lipolysis in response to isoproterenol was not abrogated by wortmannin, an inhibitor of phosphatidylinositol 3-kinase, but additive to that of PD98059, an extracellular signal-regulated kinase kinase inhibitor. Prior exposure of adipocytes to berberine decreased the intracellular cAMP production induced by isoproterenol, forskolin, and 3-isobutyl-1-methylxanthine (IBMX), along with hormone-sensitive lipase (HSL) Ser-563 and Ser-660 dephosphorylation, but had no effect on perilipin phosphorylation. Berberine stimulated HSL Ser-565 as well as adenosine monophosphate-activated protein kinase (AMPK) phosphorylation. However, compound C, an AMPK inhibitor, did not reverse the regulatory effect of berberine on HSL Ser-563, Ser-660, and Ser-565 phosphorylation, nor the antilipolytic effect of berberine. Knockdown of AMPK using RNA interference also failed to restore berberine-suppressed lipolysis. cAMP-raising agents increased AMPK activity, which was not additive to that of berberine. Stimulation of adipocytes with berberine increased phosphodiesterase (PDE) 3B and PDE4 activity measured by hydrolysis of ³[H]cAMP. These results suggest that berberine exerts an antilipolytic effect mainly by reducing the inhibition of PDE, leading to a decrease in cAMP and HSL phosphorylation independent of AMPK pathway.     

Prolonged fasting on the lipolytic activity of isolated adipocytes of the rat epididymis

Male Wistar rats, 6-8 week old, were fasted for 72 hours. The in vitro lipolytic activity of epididymal adipocytes was measured in the presence of adrenalin (a alpha and beta adrenergic agonist), isoprenaline (a pure beta agonist), theophylline (a phosphodiesterase inhibitor) or UK 14304 (a alpha 2 adrenoceptor agonist) associated with adenosine deaminase. The basal lipolytic activity, expressed per 100 mg lipids, was higher in fasted adipocytes than in fed ones. Its stimulation by adrenalin or isoproterenol was decreased by fast. The effects of these drugs were more potentiated by theophylline in fasted adipocytes than in fed ones. The UK 14304 inhibition of adenosine deaminase-stimulated lipolysis was about 20% in fasted adipocytes and 50% in fed adipocytes. The in vitro resistance of fasted adipocytes to the lipolytic effect of adrenalin or isoproterenol may be related to the hypothyroid status of fasted rats.     

Evolution of the sensitivity of isolated adipocytes of ewes to the lipolytic effects of different stimuli during pregnancy and lactation

Four successive biopsies of omental adipose tissue were performed at 43,100,140 days and during the 3rd week of lactation on 6 "Pré-Alpes" ewes. Using isolated adipocyte incubation, we studied the evolution of both basal lipolysis and stimulated lipolysis in response to different stimuli during these physiologic periods. The basal lipolysis increased from 53 +/- 10 micrograms glycerol/4 hr incubation/g total lipids (TL) at 43 days of pregnancy and 55 +/- 11 micrograms/4 hr/g TL at 100 days of pregnancy to a maximum value of 204 +/- 10 micrograms/4 h/g TL observed one week before parturition. Basal lipolysis remained at a high level during lactation: 153 +/- 27 micrograms/4 hr/g TL. The sensitivity of the fat cells to the lipolytic effects of isoproterenol, theophylline and adenosine-deaminase evolved with profiles comparable to that observed for basal lipolysis. The threshold concentration of stimuli necessary to observe an effect was decreased and the maximum response was increased. Bovine growth hormone (bGH) did not exhibit a direct lipolytic effect during pregnancy and lactation. Nevertheless, bGH provoked a significant potentiation of 20% of the lipolysis stimulated by isoproterenol and theophylline at 43 and 100 days of pregnancy. Ovine placental lactogene hormone (oPL) did not modulate, directely or undirectely, lipolysis.     

The antilipolytic effect of insulin in human adipocytes requires activation of the phosphodiesterase

Human fat cells were incubated with two different cAMP analogues, 8-bromocAMP and 6N-monobutyrylcAMP. The former analogue is an excellent substrate for the phosphodiesterase while the latter is resistant to hydrolysis. In the presence of adenosine deaminase, isoproterenol (10(-6)M) stimulated lipolysis 8-10 fold which was similar to the effect exerted by the cAMP analogues. Basal lipolysis and lipolysis activated by 6N-monobutyrylcAMP was not inhibited by insulin even at high concentrations, whereas the effect of 8-bromocAMP was virtually completely inhibited. This effect of insulin was completely prevented by the addition of IBMX. Thus, activation of phosphodiesterase by insulin is necessary to elicit the antilipolytic effect in human adipocytes.     

The lipolytic stimulation of 3T3-L1 adipocytes promotes the translocation of hormone-sensitive lipase to the surfaces of lipid storage droplets

Hormone-sensitive lipase catalyzes the rate-limiting step in the release of fatty acids from triacylglycerol-rich lipid storage droplets of adipocytes, which contain the body's major energy reserves. Hormonal stimulation of cAMP formation and the activation of cAMP-dependent protein kinase leads to the phosphorylation of hormone-sensitive lipase and a large increase in lipolysis in adipocytes. By contrast, phosphorylation of hormone-sensitive lipase by the kinase in vitro results in a comparatively minor increase in catalytic activity. In this study, we investigate the basis for this discrepancy by using immunofluorescence microscopy to locate hormone-sensitive lipase in lipolytically stimulated and unstimulated 3T3-L1 adipocytes. In unstimulated cells, hormone-sensitive lipase is diffusely distributed throughout the cytosol. Upon stimulation of cells with the beta-adrenergic receptor agonist, isoproterenol, hormone-sensitive lipase translocates from the cytosol to the surfaces of intracellular lipid droplets concomitant with the onset of lipolysis, as measured by the release of glycerol to the culture medium. Both hormone-sensitive lipase translocation and lipolysis are reversed by the incubation of cells with the beta-adrenergic receptor antagonist, propranolol. The treatment of cells with cycloheximide fails to inhibit lipase translocation or lipolysis, indicating that the synthesis of nascent proteins is not required. Cytochalasin D and nocodazole used singly and in combination also failed to have a major effect, thus suggesting that the polymerization of microfilaments and microtubules and the formation of intermediate filament networks is unnecessary. Hormone-sensitive lipase translocation and lipolysis were inhibited by N-ethylmaleimide and a combination of deoxyglucose and sodium azide. We propose that the major consequence of the phosphorylation of hormone-sensitive lipase following the lipolytic stimulation of adipocytes is the translocation of the lipase from the cytosol to the surfaces of lipid storage droplets.     

Enhanced lipolysis is not evident in adipocytes from exercise-trained SHR

Adipocytes from spontaneously hypertensive rats (SHR) are not as responsive to isoproterenol or dibutyryl adenosine 3',5'-cyclic monophosphate (cAMP) stimulation compared with Sprague-Dawley or Wistar-Kyoto rats. Lipolytic activity in adipocytes from trained normotensive rats was enhanced in response to 1 microM isoproterenol and 0.5 mM dibutyryl cAMP but not in adipocytes from trained SHR. Decreases in isoproterenol-stimulated (1 microM) cAMP accumulation were evident in adipocytes from trained normotensive rats but not in adipocytes from trained SHR. Basal and agonist-induced lipolysis in fat cells isolated from both normotensive rats and SHR immediately following a 60-min run was increased in both sedentary and trained rats. Adenylate cyclase activity in fat cell membranes was blunted in sedentary and trained SHR both in the absence and presence of 100 microM 5'-guanylyl imidophosphate. No apparent differences existed in antagonist affinity of binding sites for the antagonist dihydroalprenolol in normal rats or SHR. Evidence for a change in affinity of agonist isoproterenol might be indicated based on the enhanced potency of isoproterenol to stimulate lipolysis in trained normal rats. beta-Adrenergic receptor density and antagonist affinity were not different in normotensive rats and SHR in response to training. However, displacement of [3H]dihydroalprenolol in adipocytes from SHR required greater concentrations of isoproterenol compared with adipocytes from normotensive rats, further suggestive of increased agonist affinity of binding sites in normal rats. These data suggest a postreceptor lesion of the lipolytic pathway in adipocytes from spontaneously hypertensive rats, possibly at the guanine nucleotide regulatory protein level.(ABSTRACT TRUNCATED AT 250 WORDS)