cAMP-dependent protein kinase and lipolysis in rat adipocytes. III. Multiple modes of insulin regulation of lipolysis and regulation of insulin responses by adenylate cyclase regulators

The relationship between cAMP-dependent protein kinase (A-kinase) activity ratios and lipolysis in the presence of insulin was compared to the standard relationship between these two parameters established with a variety of adenylate cyclase modulators (Honnor, R. C., Dhillon, G., and Londos, C. (1985) J. Biol. Chem. 260, 15130-15138). Three phases of insulin action were observed. First, when tested in control cells exhibiting A-kinase activity ratios up to approximately 0.25, insulin inhibition of lipolysis could be accounted for by the decrease in A-kinase activity. Second, in cells exhibiting A-kinase activity ratios greater than 0.3, the decrease in kinase activity by insulin did not account for the decrease in lipolysis. Finally, as the A-kinase activity ratio approached 0.6 the insulin effect on lipolysis was lost. The data suggest that protein phosphatase activation accounts for the cAMP-independent insulin action. Moreover, the insulin effect not accounted for by a decrease in A-kinase activity appears to be elicited only upon elevation of A-kinase activity. The method by which cells were stimulated determined the IC50 for insulin inhibition of: 1) A-kinase activity ratios, 2) lipolysis explained by the decrease in A-kinase activity ratios, and 3) lipolysis not explained by a decrease in A-kinase activity ratios. For all three parameters, cells stimulated by lipolytic hormones were approximately 5 times more sensitive to insulin than cells stimulated by incubation in a ligand-free environment achieved with adenosine deaminase; insulin IC50 values were approximately 120 and 600 pM, respectively. Such data establish a link between insulin actions in modifying cAMP concentrations and in modifying events apparently independent of changes in cAMP. It is proposed that the receptors and regulatory components associated with adipocyte adenylate cyclase are associated also with components of the insulin response system separate from cyclase.     

Regulation of insulin-stimulated glucose transport in the isolated rat adipocyte. cAMP-independent effects of lipolytic and antilipolytic agents

This paper examines the modulation of insulin-stimulated glucose transport activity in rat adipose cells by ligands for receptors (R) that mediate stimulation (Rs; lipolytic) or inhibition (Ri; antilipolytic) of adenylate cyclase. The changes in glucose transport activity and cAMP, as assessed by 3-O-methylglucose uptake and (-/+) cAMP-dependent protein kinase (A-kinase) activity ratios, respectively, were monitored under conditions that maintain steady-state A-kinase activity ratios (Honnor, R. C., Dhillon, G. S., and Londos, C. (1985) J. Biol. Chem. 260, 15122-15129). Removal of endogenous adenosine with adenosine deaminase decreased insulin-stimulated glucose transport activity by approximately 30%, which was prevented or restored with Ri agonists such as phenylisopropyladenosine, nicotinic acid, and prostaglandin E1. These changes in transport activity were not accompanied by changes in A-kinase activity ratios, indicating that Ri-mediated effects on transport are independent of cAMP changes. Addition of an Rs ligand, isoproterenol, in the presence of adenosine increased kinase activity but did not change glucose transport activity. Conversely, upon removal of adenosine, addition of Rs ligands such as isoproterenol, adrenocorticotropic hormone, or glucagon strongly inhibited transport (approximately 50%) and stimulated kinase activity. However, subsequent addition of phenylisopropyladenosine nearly restored transport activity without alteration of A-kinase activity. These data and additional kinetic experiments suggest that Rs-mediated glucose transport modulations are also independent of cAMP. The interchangeability of ligands for both Rs and Ri receptors in modulating transport activity suggests that these cAMP-independent effects are mediated by the stimulatory (Ns) and inhibitory (Ni) guanyl nucleotide-binding regulatory proteins of adenylate cyclase. All Rs-and Ri-induced changes in transport activity occurred without a change in glucose transporter distribution, as assessed by D-glucose-inhibitable cytochalasin B binding, suggesting that Rs and Ri ligands modulate the intrinsic activity of the glucose transporter present in the plasma membrane.     

Bimodal effect of insulin on hormone-stimulated lipolysis: relation to intracellular 3',5'-cyclic adenylic acid and free fatty acid levels

The present study was undertaken to determine the relationship between the antilipolytic and lipolytic effects of insulin on hormone-stimulated lipolysis and the mechanisms of these reactions. The dose-response curve of norepinephrine-stimulated lipolysis in rat adipocytes was not sigmoidal but biphasic in nature. Intracellular free fatty acid levels were linearly related to lipolytic rate and also described a biphasic profile in response to increments in norepinephrine concentration. Intracellular 3',5'-cyclic AMP levels measured 10 min after addition of increasing concentrations of norepinephrine showed a rise and a plateau followed by a secondary rise. Insulin was antilipolytic at low concentrations of norepinephrine and distinctly lipolytic at high concentrations. The combined antilipolytic and lipolytic effect of insulin is termed the "bimodal" effect of insulin on hormone-stimulated lipolysis. The bimodal effect of insulin correlated positively with changes in peak intracellular 3',5'-cyclic AMP levels. In the presence of glucose, insulin invariably enhanced lipolysis. It is suggested that the antilipolytic effect of insulin is achieved by both inhibition of adenyl cyclase activity and activation of low-K(m) 3',5'-cyclic AMP phosphodiesterase, the net effect being a low accumulation of 3',5'-cyclic AMP. On the other hand, the lipolytic effect of insulin probably reflects enhancement of adenyl cyclase activity to an extent that overrides any activation of low-K(m) 3',5'-cyclic AMP phosphodiesterase activity, resulting in an increase in peak adipocyte 3',5'-cyclic AMP levels.     

Evidence that metabolically active synaptosomes lack functional cyclic AMP-dependent protein kinase.

Cyclic adenosine monophosphate (cAMP)-mediated signal transduction was evaluated in synaptosomes prepared from rat brain cortex. Adenylate cyclase was responsive to known adenylate cyclase stimulators including peptides (CRH and VIP), catecholamines (norepinephrine and isoproterenol) and ligands that directly stimulate adenylate cyclase (forskolin). Cyclic AMP accumulation also increased approximately 2 to 3-fold, but none of the agonists was able significantly to activate cyclic AMP-dependent protein kinase (A-kinase) in cortical synaptosomes. However, in parallel studies with slices prepared from rat brain cortex, adenylate cyclase activity, cAMP accumulation and A-kinase activity were all stimulated by CRH, VIP, norepinephrine, isoproterenol and forskolin. These data suggest that, in intact synaptosomes, either the cellular machinery which facilitates binding of cAMP to the regulatory subunit of A-kinase is missing or the cAMP produced by adenylate cyclase is not accessible to A-kinase.     

Angiopoietin-like 4 (Angptl4) protein is a physiological mediator of intracellular lipolysis in murine adipocytes

Intracellular triacylglycerol (TG) hydrolysis and fatty acid release by the white adipose tissue (WAT) during a fast is stimulated by counter-regulatory factors acting in concert, although how adipocytes integrate these lipolytic inputs is unknown. We tested the role of angiopoietin-like 4 (Angptl4), a secreted protein induced by fasting or glucocorticoid treatment, in modulating intracellular adipocyte lipolysis. Glucocorticoid receptor blockade prevented fasting-induced tissue Angptl4 expression and WAT TG hydrolysis in mice, and TG hydrolysis induced by fasts of 6 or 24 h was greatly reduced in mice lacking Angptl4 (Angptl4(-/-)). Glucocorticoid treatment mimicked the lipolytic effects of fasting, although with slower kinetics, and this too required Angptl4. Thus, fasting-induced WAT TG hydrolysis requires glucocorticoid action and Angptl4. Both fasting and glucocorticoid treatment also increased WAT cAMP levels and downstream phosphorylation of lipolytic enzymes. Angptl4 deficiency markedly reduced these effects, suggesting that Angptl4 may stimulate lipolysis by modulating cAMP-dependent signaling. In support of this, cAMP levels and TG hydrolysis were reduced in primary Angptl4(-/-) murine adipocytes treated with catecholamines, which stimulate cAMP-dependent signaling to promote lipolysis, and was restored by treatment with purified human ANGPTL4. Remarkably, human ANGPTL4 treatment alone increased cAMP levels and induced lipolysis in these cells. Pharmacologic agents revealed that Angptl4 modulation of cAMP-dependent signaling occurs upstream of adenylate cyclase and downstream of receptor activation. We show that Angptl4 is a glucocorticoid-responsive mediator of fasting-induced intracellular lipolysis and stimulates cAMP signaling in adipocytes. Such a role is relevant to diseases of aberrant lipolysis, such as insulin resistance.     

Inhibition of adipocyte lipolysis by papaverine: papaverine can inhibit the redistribution of hormone-sensitive lipase

Papaverine, despite being a potent phosphodiesterase inhibitor, actually blocks adipocyte lipolysis. The present study was designed to clarify the mechanism of the inhibitory effect of papaverine on lipolysis. Lipolysis, stimulated by either 10 microM isoproterenol or 5 mM dibutyryl cAMP, was significantly inhibited by papaverine (100 microM and above). Papaverine, however, did not affect the isoproterenol-induced increase in the protein kinase A (A-kinase) activity ratio. In cell-free extract from non-stimulated adipocytes, cAMP-stimulated A-kinase activities were almost completely blocked by H-89, a potent inhibitor of A-kinase, but not by papaverine. Thus, the inhibitory effect of papaverine on lipolysis could be responsible for a deficit in step(s) distal to A-kinase activity. Hormone-sensitive lipase activities in the infranatant fraction of centrifuged homogenates of cells, which were maximally stimulated with isoproterenol were significantly reduced. This result indicates that hormone-sensitive lipase redistributes from cytosol to its substrate in lipolytically stimulated cells. Papaverine completely blocked the isoproterenol-induced decrease in lipase activity in the infranatant fraction. These results suggest that papaverine blocks lipolysis through its inhibitory effect on the redistribution of hormone-sensitive lipase.     

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.     

Enhanced coupling of adenylate cyclase to lipolysis in permeabilized adipocytes from trained rats.

Digitonin-permeabilized adipocytes were used to study the coupling of adenylate cyclase (AC) to lipolysis in exercise-trained rats. Isoproterenol-(IPR) stimulated lipolysis in permeabilized cells was significantly greater in trained than in control rats. Under essentially identical conditions, the dose-response curve for IPR stimulation of AC activity in the absence of 3-isobutyl-1-methylxanthine was similar in trained and control rats. However, the potency of stimulation by IPR as a percentage of the basal level was greater in trained rats. AC activity and lipolysis in the presence of 3-isobutyl-1-methylxanthine were also significantly greater in trained than in control rats. Least-squares analysis by plotting the log AC vs. lipolysis values showed that the regression coefficient was about three-fold greater in trained than in control rats. The concentration of endogenous adenosine 3',5'-cyclic monophosphate (cAMP) needed to produce a half-maximal lipolytic response was 18.58 and 10.81 pmol.min-1.10(6) cells-1 in control and trained rats, respectively. Thus a positive relationship existed between lipolysis and AC activity, with a tighter coupling in trained rats. Lipolysis in response to exogenous cAMP tended to be greater in trained than in control rats, and the difference was statistically significant for 50 microM and 10 mM cAMP. Our finding support the concept that the major mechanism of enhanced lipolysis in trained rats was an increase in the activity of enzymatic step(s) distal to cAMP.     

Response of white adipocyte of mouse and rabbit to catecholamines and ACTH

The isolated intact white adipocyte of the Swiss mouse responds to both ACTH and catecholamines by an elevation of cAMP levels and an increase in lipolysis. However, in the isolated plasma membrane of the mouse adipocyte, adenylate cyclase loses its responsiveness to ACTH but retains its ability to respond to catecholamines. This lack of responsiveness to ACTH by adenylate cyclase of mouse adipocyte plasma membrane can be overcome, at least partially, by addition of GPP (NH)p, an analog of GTP, to the assay medium. The data on mouse adipocyte membrane suggests that the coupling of ACTH receptor to adenylate cyclase is dependent on GTP and that catecholamine-activation of adenylate cyclase is less dependent on this nucleotide. The isolated intact white adipocyte of adult New Zealand rabbit responds to ACTH, but does not (or only weakly) respond to catecholamines. In contrast to the mouse plasma membrane preparation, adenylate cyclase of adipocyte membrane of the rabbit responds to ACTH. And the addition of GPP(NH)P is not required to demonstrate the CTH: sensitive adenylate cyclase activity. The difference between mouse and rabbit adipocyte membrane in the requirement for GPP(NH)P in ACTH action is not readily explained. The lack of catecholamine sensitivity of rabbit membrane enzyme cannot be reversed by addition of GPP(NH)P or adenosine deaminase. These two adenylate cyclase model systems using mouse and rabbit adipocyte plasma membrane may be useful tools for the study of the specificity and mechanism of action of lipolytic hormones such as ACTH and catecholamines.     

Inhibition of rat and human adipocyte adenylate cyclase in the antilipolytic action of insulin, clofibrate, and nicotinic acid.

Clofibrate (Atromid-S), nicotinic acid, and insulin are known to be potent hypolipidemic and antilipolytic agents. The present study was undertaken to define the mechanism of action of this latter effect on isolated rat and human fat cells. Sodium clofibrate (0.42 mM), nicotinic acid (0.42 mM), and insulin (100 microU/mL) were shown to inhibit norepinephrine-stimulated lipolysis in rat and human adipose cells and this inhibition was associated with a reduction in intracellular 3',5'-cyclic AMP levels. A similar cyclic AMP lowering effect was demonstrated with insulin in the presence of procaine-HCL, which uncouples the adenylate cyclase system from lipolysis. This insulin effect was attributed to inhibition of adenylate cyclase. A direct and significant inhibition of adenylate cyclase in membrane fractions obtained from isolated human adipocytes was demonstrated for all three antilipolytic agents. The common membrane site of action of these agents whereby adenylate cyclase activity is depressed, thus decreasing cyclic AMP production and free fatty acid (FFA) mobilization from adipose stores, implies a central role for the adenylate cyclase system. These findings are consistent with the view that the hypotriglyceridemic effects of clofibrate, nicotinic acid, and insulin may be partly explained by deprivation of FFA substrate for hepatic very low density lipoprotein synthesis.     

Free fatty acids as feedback regulators of adenylate cyclase and cyclic 3':5'-AMP accumulation in rat fat cells.

Rat fat cells incubated with lipolytic agents released substances to the medium which acted as feedback regulators of cyclic adenosine 3':5'-monophosphate (cyclic AMP) accumulation. The feedback regulators were not removed by adenosine deaminase. Dialyzed medium that had previously been incubated with fat cells in the presence of norepinephrine markedly inhibited cyclic AMP accumulation by fresh cells, whereas dialyzed medium from control cells did not inhibit cyclic AMP accumulation. The effects of lipolytic agents could be mimicked by adding dialyzed medium previously incubated with fat cells in the presence of oleic acid. This suggested that free fatty acids were the nondialyzable and adenosine deaminase-insensitive inhibitors of cyclic AMP accumulation released to the medium by fat cells incubated with lipolytic agents. The regulatory function of free fatty acids was related to the molar ratio of fatty acid to albumin. Profound inhibition of both lipolysis and cyclic AMP accumulation was seen as the free fatty acid/albumin ratio exceeded 3. The inhibition of cyclic AMP accumulation by oleate was seen as soon as there was a detectable increase in cyclic AMP due to lipolytic agents. Protein kinase activity (in the presence of cyclic AMP) of the infranatant obtained after centrifugation of fat cell homogenates at 48,000 x g was inhibited by medium from cells incubated with lipolytic agents or added oleate. Adenylate cyclase activity of rat fat cell ghosts was also inhibited by dialyzed or nondialyzed medium that previously had been incubated with lipolytic agents or added fatty acids. The direct addition of oleate markedly inhibited adenylate cyclase activity as the free fatty acid/albumin ratio exceeded 2. These data suggest that the prolonged drop in cyclic AMP accumulation seen during the incubation of rat fat cells with lipolytic agents is due to the inhibition of adenylate cyclase. This occurs when the free fatty acid/albumin ratio exceeds 3.     

cAMP-dependent protein kinase and lipolysis in rat adipocytes. I. Cell preparation, manipulation, and predictability in behavior

With the use of -cAMP/+cAMP activity ratios of cAMP-dependent protein kinase (A-kinase) in fat cell extracts as an index of cellular cAMP concentrations, it is apparent from both the current literature and from data presented in this paper that classical cell isolation procedures yield cells whose behavior is unpredictable from day to day. Herein, procedures are described for isolating adipocytes, preparing cytosolic extracts, and assaying A-kinase that result in kinase activity ratios in isolated cells equal to those in the fat pad from which cells are derived, approximately 0.05. An important modification in the procedure is the inclusion of 200 nM exogenous Ado in all cell manipulation media, and the data indicate that variable removal of contaminating endogenous Ado accounts for unpredictable results with standard cell isolation techniques. A further benefit of Ado inclusion is greatly reduced cell lysis. Acute removal of Ado with adenosine deaminase results in rapid elevation of A-kinase activity ratios and lipolysis which, in fasted animals, equals that achieved with lipolytic hormones. Cells from fed animals exhibit poor predictability in behavior. Moreover, A-kinase activity ratios exhibit seasonal tendencies in response to Ado removal, with cells isolated in spring being more activated than cells isolated later in the year. The information and procedures in this paper form the basis for succeeding papers on the regulation of adipocyte metabolism by hormones.     

Forskolin as a novel lipolytic agent

Forskolin is a novel lipolytic agent which elevates cAMP and FFA release in rat adipocytes in a manner different from existing lipolytic factors. This effect of Forskolin is potentiated by all lipolytic hormones tested, i.e. epinephrine, ACTH, and glucagon and is also reversible. The same batch of adipocytes can be repeatedly stimulated after washing. The effective concentration of Forskolin is in the micromolar range. Its action is due to an activation of cAMP synthesis by adenylate cyclase. There is no effect on cAMP hydrolysis. In contrast to stimulation by lipolytic hormones, Forskolin-activated membrane adenylate cyclase was not further stimulated by GPP(NH)P. These results suggest that Forskolin may be a useful analytical agent in the study of adenylate cyclase mediated function in intact adipocytes.     

Inhibition of adenosine 3':k'-monophosphate accumulation white fat acids, lactate, and beta-hydroxybutyrate.

The large increase in cyclic AMP accumulation by rat white fat cells seen in the presence of lipolytic agents plus methylxanthines and adenosine deaminase was markedly inhibited by lactate. However, lipolysis was unaffected by lactate. Octanoate, hexanoate, heptanoate, and beta-hydroxybutyrate inhibited both cyclic AMP accumulation and lipolysis by rat fat cells. The mechanism by which these acids inhibit lipolysis differs from that for long chain fatty acids such as oleate. Oleate directly inhibited triglyceride lipase activity of homogenized rat adipose tissue. In contrast, octanoate, beta-hydroxybutyrate, and lacatate had no effect on triglyceride lipase activity. Hormone-stimulated adenylate cyclase activity of rat fat cell ghosts was inhibited by oleate and 4mM octanoate but not by 1.6 mM octanoate, heptanoate, hexanoate, beta-hydroxybutyrate or lactate. None of the acids affected the soluble protein kinase activity of rat adipose tissue. There was no stimulation by lactate, butyrate, beta-hydroxybutyrate, or octanoate of the soluble or particulate cyclic AMP antilipolytic action of a short chain acid such as octanoate or hexanoate was not accompanied by any drop in total fat cell ATP. The mechanism by which lactate lowers cyclic AMP but not lipolysis remains to be established.     

Characteristics of the alpha 2/beta-adrenoceptor-coupled adenylate cyclase system and their relationship with adrenergic responsiveness in hamster fat cells from different anatomical sites.

Various studies have shown that the lipolytic response of white adipocytes to catecholamines was dependent on the anatomical origin of these cells. To provide a biological explanation for this phenomenon, we compared hamster white adipocytes, from femoral subcutaneous and epididymal fat, for their lipolytic activities, cAMP responses and adrenoceptor-coupled adenylate cyclase system. Basal and maximal lipolytic responses to the beta-adrenergic (isoproterenol) and the mixed alpha 2/beta-adrenergic (epinephrine) agonists were lower in femoral subcutaneous cells than in epididymal cells, but the alpha 2-adrenergic antilipolytic response to 5-bromo-6-(2-imidazolin-2-ylamino)quinoxaline bi-tartate (UK14304) was slightly greater in femoral subcutaneous fat cells than in epididymal fat cells. Identical results were observed for cAMP responses, except for the alpha 2-adrenergic inhibitory response which was identical in both fat deposits. Adrenoceptors studies revealed higher density of inhibitory alpha 2-adrenoceptors 2-(2-methoxy-1,4-benzodioxan-2-yl)-2-imidazoline ([3H]RX821002-binding sites) in femoral subcutaneous fat cells than in epididymal fat cells, but identical density of stimulatory beta-adrenoceptors (125I-cyanopindolol-binding sites) and similar subdivision into beta-adrenoceptor subtypes in both adipose deposits. Finally, the level of the alpha-subunits of the stimulatory and inhibitors guanine-nucleotide-binding regulatory proteins, as well as the adenylate cyclase catalytic activity were 40-50% lower in femoral subcutaneous fat cell membranes than in epididymal fat cell membranes. These results suggest that the differences in cAMP and lipolytic responses to catecholamines between epididymal and femoral subcutaneous adipocytes result at least in part from site-related differences in the adenylate cyclase system rather than in the adrenoceptor status.     

Uncoupling of lipolysis from cyclic AMP by procaine: a tool for studying the mechanism of action of antilipolytic agents.

The initial rate of net glycerol release in norepinephrine-stimulated adipose tissue fragments was inhibited (40-78%) by procaine-HCl (1-5mM), whereas basal (unstimulated) lipolysis was unaffected. A dose-related inhibition of norepinephrine-induced lipolysis by procaine-HCl (0.1-1 mM) also occurred in adipocytes. Procaine-induced antilipolysis was associated with an augmented rather than a reduced hormone-stimulated increment in intracellular cyclic AMP. The dissociation of lipolysis from cyclic AMP accumulation has been termed the uncoupling effect of procaine. This effect of procaine was employed to define the precise mechanism of action of the antilipolytic drug clofibrate (Atromid-S) which inhibits lipolysis by reducing cyclic AMP. A reduction in cyclic AMP by clofibrate was demonstrated in norepinephrine-stimulated cells exposed to procaine (uncoupled system). Thus, the inhibitory effect of clofibrate on cyclic AMP could not be attributed to accumulation of products of lipolysis. Because neither procaine-HCl nor clofibrate had any effect on the low Km 3':5'-cyclic-AMP phosphodiesterase (EC 3.1.4.17) activity in hormone stimulated cells, the clofibrate-induced reduction in cyclic AMP was attributed to its direct action on adipocyte adenylate cyclase.     

The antilipolytic effect of insulin does not require adenylate cyclase or phosphodiesterase action

Insulin antagonized the lipolytic actions of epinephrine in rat epididymal adipocytes when the phosphodiesterase inhibitor, Ro 20-1724, was present. Adipocytes were depleted of functional cAMP by inhibiting adenylate cyclase with N6-phenylisopropyladenosine in the presence of adenosine deaminase such that Ro 20-1724 no longer stimulated lipolysis. The cAMP analogs 8-thioisopropyl-cAMP or 8-thiomethyl-cAMP, which are resistant to phosphodiesterase hydrolysis, were subsequently added to bypass adenylate cyclase and phosphodiesterase action. Under these conditions, insulin antagonized the lipolytic effects of these analogs, even in the presence of Ro 20-1724.     

Metabolic antagonism between insulin action and activators of adenylate cyclase in rat fat cells

1. Short-term effects of lipolytic agents in the absence or in the presence of insulin on fatty acid biosynthesis have been examined, in terms of the control rate of [1-14C]acetate incorporation into labeled fatty acids in the presence of glucose, as stimulator of lipogenesis by generating NADPH for the process. 2. The relationship between lipogenesis and lipolysis in the absence or in the presence of insulin was compared with a variety of adenylate cyclase activators. 3. The data obtained reveal that a reciprocal relationship exists between lipogenesis and lipolysis. 4. The changes in the activity of hexose monophosphate shunt produced by activation or inhibition of lipogenic process has been studied. 5. The regulation of the hexose monophosphate shunt activity mainly by the intracellular fatty acyl-CoA concentration and NADPH/NADP ratio is discussed.     

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.     

Antilipolytic effect of calcium-sensing receptor in human adipocytes

The extracellular calcium-sensing receptor (CaSR), a seven transmembrane G-protein-coupled receptor, was cloned in 1993. Its activation was first associated to the regulation of calcium homeostasis; however, the presence in tissues unrelated with this role has revealed its participation in numerous other cell functions. We previously described CaSR expression in human adipocytes, and here we investigated the effect of its activation on adipocyte lipolytic activity by measuring glycerol release to the incubation medium. Treatment of adipocytes with CaSR agonists elicited an inhibitory effect on basal lipolysis, which was prevented by a CaSR antagonist. To further corroborate the antilipolytic effect of CaSR activation, lipolysis was evaluated under conditions that interfere with main antilipolytic regulatory pathways. Cells were preincubated with pertussis toxin (PT, a Gialpha protein inhibitor), the phosphatidylinositol 3 kinase (PI3K) inhibitors wortmannin and LY-294002 as well as the cAMP analog 8Br-cAMP, all of which influenced the antilipolytic effect of CaSR stimulation. In light of the current view of adipose tissue as an organ involved in whole-body metabolic control, the role of the CaSR modulating basal lipolysis elicits great interest, given its metabolic sensing capabilities due to the variety of ligands that regulate its activity, and its potential cross-talk with insulin and adipose tissue-secreted factors.