Insulin Antagonism of Catecholamine Stimulation of Fatty Acid Transport in the Adipocyte STUDIES ON ITS MECHANISM OF ACTION*

Insulin at physiological concentrations can suppress catecholamine activation of the membrane transport of long chain fatty acids in the adipocyte. We have previously shown that the stimulatory effect of cate- cholamines was mediated by a &receptor interaction and cAMP (Abumrad, N. A., Park, C. R., and Whitesell, R. R. (1986) J. Biol. Chem. 261, 13082-13086). In this study we have investigated the mechanism of insulin action to antagonize transport activation. Fatty acid transport was stimulated using different cAMP derivatives with varying susceptibilities to hydrolysis by the CAMP-degrading enzyme phosphodiesterase. Insulin was effective in antagonizing the effect of cAMP analogs which were good substrates for the phospho- diesterase and failed to suppress the effect of those which were poorly hydrolyzed by the enzyme. Addition of increasing concentrations (1-100 PM) of the phos- phodiesterase inhibitor methylisobutylxanthine (MIX) to norepinephrine (0.1 wg/ml) abolished insulin’s antagonism. Insulin was completely ineffective in inhibiting stimulation by norepinephrine and 20 I.IM methylisobutylxanthine. Also consistent with involve- ment of cAMP lowering in insulin action was the finding that adenosine removal greatly diminished insulin’s responsiveness. (1

Insulin at physiological concentrations can suppress catecholamine activation of the membrane transport of long chain fatty acids in the adipocyte. We have previously shown that the stimulatory effect of catecholamines was mediated by a &receptor interaction and cAMP (Abumrad, N. A., Park, C. R., and Whitesell, R. R. (1986) J. Biol. Chem. 261,[13082][13083][13084][13085][13086]. In this study we have investigated the mechanism of insulin action to antagonize transport activation. Fatty acid transport was stimulated using different cAMP derivatives with varying susceptibilities to hydrolysis by the CAMP-degrading enzyme phosphodiesterase. Insulin was effective in antagonizing the effect of cAMP analogs which were good substrates for the phosphodiesterase and failed to suppress the effect of those which were poorly hydrolyzed by the enzyme. Addition of increasing concentrations (1-100 PM) of the phosphodiesterase inhibitor methylisobutylxanthine (MIX) to norepinephrine (0.1 wg/ml) gradually abolished insulin's antagonism. Insulin was completely ineffective in inhibiting stimulation by norepinephrine and 20 I.IM methylisobutylxanthine. Also consistent with involvement of cAMP lowering in insulin action was the finding that adenosine removal greatly diminished insulin's responsiveness. Treatment of cells with adenosine deaminase (1 unit/ml) enhanced the effect of norepinephrine by about 30%. A 10-fold higher range of insulin concentrations was then required to produce inhibition of fatty acid transport. The effect of adenosine removal was reversed by addition of phenylisopropyladenosine (500 nM), which is resistant to hydrolysis by the deaminase. Finally, exposure of insulintreated cells (1 nM for 5 min) to dinitrophenol (1 mM for 5 min) reversed insulin action, consistent with reports of reversal of insulin's activation of the phosphodiesterase. In conclusion, our studies support the involvement of cAMP lowering in insulin's antagonism of fatty acid transport stimulation in the adipocyte.
Insulin can effectively antagonize the stimulatory effect of catecholamines on the membrane transport of long chain fatty acids in the adipocyte (1). As in the case of lipolysis, the effect * This work was supported by National Institutes of Health Grant DK33301 and by Grants 186651 and 187653 from the Juvenile Diabetes Association. The costs of publication of this article were defrayed in part by the payment of page charges. This article must. therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. of catecholamines on fatty acid transport was shown to be mediated by cAMP and its protein kinase (2). The antagonistic influence of insulin on the transport system was very sensitive and was observed at very low insulin concentrations (10-50 PM) which were similar to those shown to be antilipolytic (3). In this study we have investigated the mechanism of insulin action to influence fatty acid transport. First, we have tested whether insulin's effect was mediated by an acceleration of cAMP hydrolysis via activation of phosphodiesterase. The effectivenss of insulin in antagonizing stimulation of fatty acid transport by a number of cAMP analogs with ranging susceptibilities to hydrolysis by the enzyme phosphodiesterase (4) was examined. Second, we have examined the ability of adenosine and methylisobutylxanthine to modulate the insulin effect.

Methods
Preparation of Fat Cells-Adipocytes were prepared from the epididymal fat of one to two 170-200-g Sprague-Dawley rats (Harlan Industries, Inc). Cell isolation followed the methodology detailed previously (2). The washed cells were suspended (30%, v/v) in Krebs-Ringer Hepes buffer (KRH) containing 0.2% fatty acid-free albumin (Sigma, fraction V, fatty acid free) and glucose (2 mM) except where indicated. The cell suspension was allowed to cool to room temperature (3-5 min) before the fatty acid transport assay was performed.
Transport Assay-Membrane transport of [3H]oleate (DuPont-New England Nuclear) was measured as described in detail previously (5,6). Medium (20 pl) containing labeled and unlabeled fatty acid (42 p~, about 3000 cpm/pl) complexed to 10 p~ bovine serum albumin (BSA)' was prepipetted onto the bottom of polystyrene tubes. The cell suspension was mixed before sampling with the use of an automatic pipette with the plastic tip enlarged to avoid cell breakage. The assay was started by rapidly ejecting 30 p1 of cell suspension onto the 20-pl medium and this was followed by gentle swirling. At the desired time, stop solution (5 ml of ice-cold KRH buffer containing 200 pM phloretin) was added. The cells were separated from medium and washed by filtration as described in detail previously (6). Controls for fatty acid absorption to cells and filters were routinely subtracted. Controls were samples where isotope and stop solution were premixed before cell addition.
Stimulation of Fatty Acid Transport-Cells (30% (v/v) in KRH with 0.2% BSA and 2 mM glucose) were incubated at 37 "c for 10 min in the case of epinephrine and norepinephrine and for 20 min in the case of cAMP analogs. The various hormones or agents were added as a few microliters from a concentrated stock. At the end of the incubation period the cells were allowed to cool to room temperature before assay of fatty acid transport. Insulin Treatment-Insulin was added at the doses indicated, usually at the same time as the stimulatory hormone or agent, unless indicated otherwise. The insulin used was lot Fz3695 (Schwarz/ Mann). The various insulin stocks or dilutions were made only a few minutes before actual use in KRH containing 0.2% BSA.
Treatment with Lipolytic Inhibitor RHC80267"When inhibition of triglyceride mobilization was desired, the isolated cells were suspended in KRH buffer at 30% (v/v) and were divided into two batches. RHC80267 (15 p~) was added to one batch, and both were incubated at 37 "C for 15 min. Under these conditions RHC80267 inhibits NEgenerated glycerol release by 90% (1). Following this incubation, each batch was subdivided into aliquots for the various hormonal treatments and processed as indicated for the particular condition.
Lipolysis Measurement-Lipolysis was estimated by assaying the glycerol released into the medium and also by measuring intracellular fatty acids. For these measurements, cells (30% (v/v) in KRH with 0.2% BSA and 2 mM glucose) were suspended and incubated under the same conditions described previously for hormonal treatments. At the desired time, the cell suspensions were mixed and an aliquot (100 pl) of cell suspension was ejected into 5 ml of ice-cold stop solution. This was followed by filtration and washing as described for the assay of fatty acid transport. Filters were immediately transferred to extraction tubes containing 2.4 ml of isopropanol. The tubes were vortexed for 30 s, and then heptane and sulfuric acid (0.05 N) were added in the volumes (0.6 and 0.06 ml, respectively) necessary to bring their concentrations to those present in 3 ml of Dole's extraction mixture (7). The vials were vortexed again for 30 s; then 2 ml of heptane and 3.5 ml of water were added. The heptane layer (2 ml) was transferred to clean tubes and washed once with an equal volume of 0.05 N sulfuric acid. An aliquot (0.6-1 ml) was then evaporated at 35 "C under nitrogen and resuspended in 125 pl of heptane, 100 pl of which was used for measurement of free fatty acids according to Ho and Meng (8). For measurement of medium fatty acid or glycerol, an aliquot of cell suspension (350-500 pl) was centrifuged for 15 s in microfuge tubes. The infranatant below the cells was transferred to tubes containing 3 ml of Dole's mixture (for fatty acid extraction and titration) or to chilled tubes for later assay of glycerol. For glycerol estimation, 200 p1 of medium were deproteinized with 40 pl of 5 N perchloric acid, and the extract was neutralized on ice with 10 N KOH. Glycerol was determined enzymatically (9). Sensitivity of the assay was rt0.005 pmol/ml. Measurement of Protein Kinase Actiuity Ratios-Cells were maintained under conditions identical to those used for the assay of fatty acid transport (see legend to Table 11). Following incubation at 37 "C with the various agents tested, the cells were added to homogenizing buffer at 23 'C (10 mM potassium phosphate, 0.5 mM MIX, 10 mM EDTA, 0.5 mM DTT, 0.05% BSA). The cells were homogenized (10 strokes) in a glass homogenizer (Kontes) with a Teflon pestle (clearance B). The homogenate was centrifuged at 4 "C (10,000 rpm for 10 min). Protein kinase activity in the presence or absence of cAMP was assayed in the infranatant below the fat layer using Kemptide (Peninsula Laboratories) as substrate following the methodology of Beebe et al. (10). The protein kinase activity ratio is defined as the ratio of activity measured in the absence and presence of CAMP.
Cellular ATP was measured according to standard procedures (11).

Materials
All [3H]oleate (9 Ci/mmol), [32P]ATP (protein kinase assay), and 63Ni (fatty acid measurements) were obtained from Du Pont-New England Nuclear. Analogs of cAMP were generously donated by Dr. Jackie Corbin at Vanderbilt University or were purchased from Sigma. RHC80267 was a generous gift from Dr. Charles Sutherland of the Revlon Care Group, Hershey, PA.

RESULTS
Insulin Antagonism of Fatty Acid Transport Stimulation by CAMP Anubgs-As reported previously (2) permeable cAMP analogs stimulate fatty acid transport with a varying degree of potency. We tested the ability of insulin to antagonize the effect of a number of CAMP analogs with differing sensitivities to hydrolysis by the cAMP degrading enzyme phosphodiesterase (4). A correlation between insulin's effect and susceptibility of the analogs to hydrolysis would suggest that insulin's antagonistic effect on fatty acid transport is mediated by a lowering of cAMP via stimulation of its degradation. As shown in Fig. 1 insulin at the optimally effective dose of 1 nM (1) was unable to antagonize the stimulatory effect of N6benzoyl cAMP and 8-aminohexylamino cAMP (AHA CAMP) (Fig. 1, A and B, respectively). This was apparent over a wide range of analog concentrations. Higher insulin concentrations of 10 and 100 nM were still ineffective (not shown). Also, pretreating cells with the lipolytic inhibitor RHC80267 did not modify the results, indicating that insulin's unresponsiveness was not related to the potency of the analog in increasing intracellular fatty acid (data not shown). As determined by Beebe et al. (4), N6-benzoyl and 8-AHA cAMP are poor substrates for the low K,,, phosphodiesterase (4). On the other hand insulin inhibited fatty acid transport stimulation by 8thioethyl cAMP (not shown), 8-bromo CAMP, and 8-CPT cAMP (Fig 1, C and D, respectively). These analogs are good substrates for the phosphodiesterase. In all three cases insulin was able to suppress fatty acid transport, when stimulated by the analogs by 8to 10-fold, down to the basal rate. However, when the concentration of each analog was increased above its optimal dose, there was a decrease in insulin's ability t o antagonize transport stimulation which was similar in the absence or presence of RHC80267. The data are consistent with insulin exerting its effect through activation of the low K,,, phosphodiesterase and consequently decreasing cAMP levels. When the levels of the nucleotide become too high, the antagonistic effect of insulin is partially lost. It is completely lost when the levels of cAMP analog cannot be lowered, as in the case of 8-aminohexylamino-and N6-benzoyl-CAMP which are resistant to the action of the phosphodiesterase. Insulin's effectiveness as an antagonist was not related to whether the cAMP derivatives were specific for site 1 (8-modified derivatives) or site 2 (6-modified derivatives) on the CAMP-dependent protein kinase.
Effect of Adenosine Deaminase and Adenosine-Londos and co-workers (12) provided evidence recently to support the view that the effective concentrations of insulin as an antilipolytic hormone were greatly dependent on the level of adenosine in the cell suspension. Adenosine is an inhibitor of adenylate cyclase and, as such, it would be expected to alter effects which are mediated by changes in cAMP levels. We examined the effects that adenosine removal by treatment with adenosine deaminase (ADA), and subsequent adenosine addition, in the form of the nonhydrolyzable phenylisopropyladenosine (PIA), had on the regulation of fatty acid transport, by norepinephrine plus insulin. Enhancement of insulin's effectiveness by adenosine would further support involvement of cAMP lowering in insulin action. Addition of adenosine deaminase (1 unit/ml) did not significantly stimulate basal fatty acid transport (not shown), but it produced a 30% enhancement of the stimulatory effect of norepinephrine (Fig.  2). Sensitivity to insulin was decreased. For example 100 PM insulin was only 25% inhibitory and 1-5 nM insulin reduced the effect of NE by 65%. Pretreatment of cells with the lipolytic inhibitor RHC80267 (15 p~) before exposure to adenosine deaminase did not improve insulin's effectiveness (data not shown) although RHC80267 decreased intracellular fatty acid by 65% (Table I). In control experiments it was determined that RHC did not alter the protein kinase activity ratios (Table 11) nor did it affect the activity of brain adenylate cyclase measured in vitro (data not shown). The effect of adenosine deaminase was due to adenosine removal since it was reversed by addition of PIA, which is resistant to hydrolysis. PIA addition to adenosine deaminase-treated cells, returned insulin sensitivity (Fig. 2) almost fully to the levels we had reported earlier (1). More specifically, insulin at the concentration of 50 pM was 70% inhibitory in the presence of   had not been treated with adenosine deaminase did not increase insulin sensitivity tested over a range of insulin concentrations (data not shown). This is consistent with the finding that our methods of cell handling result in minimal activation of adenylate cyclase as shown by the measurement of low protein kinase activity ratios (Table 11).

Effect of Methylisobutylxanthine (MZX) on Insulin's Antag-
onism-MIX is an inhibitor of the CAMP-degrading enzyme phosphodiesterase. It has been shown previously to potentiate stimulation of fatty acid transport by suboptimal concentrations of catecholamines (2). We tested the effect of cell treatment with MIX on insulin's antagonism of fatty acid  Cells,suspended 30% (v/v) in KRH containing 0.2% BSA and 2 mM glucose, were incubated at 37 "C with RHC80267 for 10 min or with NE and NE plus MIX for 5 min. An equal volume of homogenizing buffer was then added at 23 "C and the cells were homogenized manually (10 strokes). Protein kinase activity ratios in the absence or presence of cAMP was measured in the infranatant obtained from centrifuging the homogenate according to the methodology of Beebe et al. (10). The data shown are a composite from three to four experiments done in tridicates.

RHC80267
NE antagonizing epinephrine and norepinephrine (Fig. 4) at extremely high doses (10-1000 pglml). This could indicate that in our assay system of a concentrated cell suspension and low BSA in the medium there is early negative feedback of accumulated fatty acid (Table 11) or of adenosine on adenylate cyclase which prevents further production of cAMP as the catecholamine concentration is increased. Pretreatment of cells with RHC80267 did not modify the effectivness of insulin in spite of a 70% decrease in intracellular fatty acid in both Insulin was about 50% inhibitory at 1000 pg/ml norepinephrine (data points were not included). Pretreating cells for 15 min at 37 "C with the lipolytic inhibitor RHC80267 did not modify the results. Fatty acid transport was assayed at 23 "C. The data presented are a composite from three experiments. basal and NE-treated cells. The increase in intracellular free fatty acid generated by NE was antagonized by insulin when it inhibited transport irrespective of the initial concentration of intracellular fatty acid. For example, when cells were incubated in 4% BSA instead of the usual 0.2%, intracellular fatty acid dropped significantly (Table I). Under both conditions, however, insulin was equally effective in inhibiting stimulation of fatty acid transport and lipolysis.

Effect of Dinitrophenol on Fatty Acid Transport in Celk
Pretreated with Insulin-We have previously shown that pretreatment of cells with dinitrophenol (DNP) abolishes the effects on fatty acid transport of both catecholamines and insulin (3). In this study we show that addition of D N P following the hormonal treatments had a different effect. D N P did not affect transport stimulation by catecholamines. Furthermore, it prevented the return of transport rates to basal levels upon withdrawal of the hormone. In contrast, DNP treatment completely eliminated insulin's antagonistic effect. This was observed when D N P was added 5 min following exposure t o insulin. As shown in Fig. 5, fatty acid transport rates in cells exposed to norepinephrine plus insulin for 5 min and then to dinitrophenol for 5 min were comparable to those Fatty Acid Transport in the Adipocyte I

FIG. 5 .
Effect of DNP addition on fatty acid (FA) transport in adipocytes pretreated with NE plus insulin. Adipocytes were incubated with NE (0.1 pg/ml) plus or minus insulin (1 nM) for 5 or 10 min at 37 "C. DNP (1 mM) was then added and the cells were kept at 37 "C for 5 more min. Fatty acid transport was assayed at 23 "C. For more details regarding the transport assay, see legend to Fig. 1 and "Methods." measured in cells treated with norepinephrine alone. DNP was less effective, however, when added at 10 min following insulin addition. This pattern was similar to that described for DNP's effect on insulin activation of the phosphodiesterase (13). As discussed by Kono (13), it is one of the distinguishing factors between the mechanisms of action of insulin on the phosphodiesterase versus its effect on glucose transport. Glucose transport, once stimulated by insulin, is not altered by DNP treatment. In addition, DNP blocks reversal of the glucose transport stimulation which would otherwise occur upon washout of insulin.

DlSCUSSION
We have previously shown that a variety of cAMP analogs can stimulate long chain fatty acid transport in the rat adipocyte (2). Beebe et al. (4) recently determined the susceptibility of the various analogs to degradation by the low K,,, phosphodiesterase. Furthermore, they showed in the case of a large number of cAMP analogs that insulin's ability to antagonize their antilipolytic effect generally correlated with phosphodiesterase susceptibility. Our findings show a similar correlation with respect to insulin's antagonism of their stimulation of fatty acid transport. These findings, together with the known effect of insulin to stimulate the low K , phosphodiesterase (14-16), implicate cAMP lowering as a necessary component to insulin action. The ability of adenosine to modulate insulin's effectiveness as an antagonist of fatty acid transport stimulation would implicate CAMP, through effects on cyclase. Insulin effectiveness was dramatically decreased by adenosine deaminase and the magnitude of the decrease was similar whether or not the cells were pretreated with the lipolytic inhibitor RHC80267. This indicated that the decreased response of fatty acid transport to insulin observed with adenosine removal is not linked to production of intracellular fatty acid. It also was not a consequence of a depletion of cellular ATP which measured 2.2 and 2.1 mM, respectively, in control and adenosine deaminase plus NE-treated cells.
The decreased insulin response could have been related to the increase in intracellular cAMP levels and protein kinase activity. It is possible that kinase-dependent phosphorylations promote resistance to insulin. This might explain why insulin is most effective against NE when the two hormones are added together. When insulin was added 10 min following NE, a diminished insulin effect was observed two of six times. Furthermore, when insulin was added 20 min after NE, insulin's antagonism was abolished 70% of the time. ' Although insulin effects on fatty acid transport and lipolysis correlated, there did not appear to be any general correlation between absolute levels of intracellular fatty acid and the magnitude of insulin's effectivenss on fatty acid transport. For example, insulin was equally effective when the cells were suspended in 0.2 or 4% albumin in spite of different levels of intracellular fatty acid before and 5 min after incubation with NE (Table I). A lowering of intracellular fatty acid by insulin never occurred in the absence of an effect on fatty acid transport, suggesting that the two effects were a result of the same preceeding event. In contrast, insulin stimulated glucose transport independently of the effects on fatty acid transport (data not shown).
The effectiveness of insulin in antagonizing fatty acid transport stimulation by the phosphodiesterase inhibitor MIX is similar to the finding with caffeine (3). A possible explanation is that these agents do not completely inhibit all the phosphodiesterase present in the cell so that an inhibitory effect of insulin can stili be observed. On the other hand, 1 nM insulin was unable to block the combined effects of MIX plus NE, in cells pretreated with RHC80267, to rule out deleterious effects of fatty acid. This would suggest that cAMP levels which are significantly raised under these conditions (Table 11) cannot be lowered by insulin to levels below those producing stimulation of fatty acid transport. Consistent with this interpretation was the observation that transport stimulation was only increased by 18% when 3 p~ MIX is included with the 0.1 pg/ml NE. However, inhibition by insulin was reduced by MIX from 97 to 59%, suggesting a critical range of cAMP levels which insulin can antagonize. Increasing MIX from 10 to 20 PM in the presence of NE reduced insulin inhibition from 59 to 0% but did not result in a proportional increase in the magnitude of fatty acid transport stimulation in the absence of insulin.
The ability of insulin in our studies to antagonize transport stimulated by high concentrations of NE (1-1000 pg/ml) is at variance with what has been reported for its antagonism of NE-stimulated lipolysis (4, 17, 18). In our studies the effectiveness of insulin on NE-stimulated glycerol production was also observed over a wider range of NE concentrations than that reported previously (4, 5, 18). Insulin (1 nM) was 100% effective at shutting off glycerol release after 0.1 and lpg/ml NE. It was 68 and 33% effective at 50 and 500 pg/rnl NE, respectively, and completely ineffective at 1000 pg/rnl NE (data not shown). The discrepancy between our results and previous ones could be explained by the use in our incubations of concentrated cell suspensions and of low BSA (0.2%) in the medium. Both conditions should favor low cellular CAMP. High cell concentration favors adenosine accumulation which inhibits adenylate cyclase, and low BSA accelerates the increase in the molar ratio of fatty acid to albumin which would feed back to inhibit cAMP accumulation and lipolysis (19)(20)(21). This interpretation is consistent with the low range of activity ratios for CAMP-dependent protein kinase that we measure in our cell homogenates (Table 11)  insulin's effectiveness is enhanced by low cellular CAMP. Under such conditions insulin can lower cAMP and protein kinase to the levels necessary for deactivation of catecholamine-sensitive pathways including fatty acid transport.
The ability of dinitrophenol to block insulin's action when added 5 min after insulin treatment is consistent with mediation of the insulin effect by activation of the low K,,, phosphodiesterase. As shown by Kono (13), phosphodiesterase activation by insulin is abolished by subsequent addition of DNP in contrast to insulin-activated glucose transport which is actually preserved by DNP.
In summary, insulin appears to inhibit catecholamine stimulation of fatty acid transport by accelerating hydrolysis of cAMP through activation of the phosphodiesterase. 1) Insulin can antagonize the effect of up to 1000 pg/ml NE or epinephrine when experimental conditions prevent build up of CAMP, suggesting that the lack of an antilipolytic effect of insulin previously described at high concentrations of NE was related to cAMP accumulation and not to that of fatty acid. 2) Insulin becomes ineffective when cAMP levels are raised by the use of nonhydrolyzable cAMP analogs, by treatment with adenosine deaminase, or by MIX in the presence of NE. Insulin activation of the phosphodiesterase could still be occurring under such conditions but the resultant lowering of cAMP levels is not enough to produce deactivation. Finally, although our studies implicate cAMP lowering in insulin's inhibition of fatty acid transport, they do not rule out the participation of other events in the insulin effect.