Stimulation by epinephrine of the membrane transport of long chain fatty acid in the adipocyte.

In isolated rat adipocytes, epinephrine rapidly stimulates the transport of long chain fatty acid across the plasma membrane. At a concentration of unbound oleate of 0.1 microM ([fatty acid]/[albumin] = 1) and 5 min exposure to the hormone, the minimal effective concentration of epinephrine is 0.03 and the optimal concentration 0.3 microM (0.01 and 0.1 microgram/ml). The stimulated rates are 5-10-fold the basal rate of influx or efflux. The hormone effect is on the transport process specifically as shown by isolation of the product of transport in either direction as unesterified fatty acid and inhibition by the transport inhibitors phloretin and 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid. This effect of epinephrine on transport coordinates physiologically with lipase activation to bring about fatty acid release from adipose tissue.

Long chain fatty acids are the major energy providing substrate for most tissues. Their release from adipose tissue and uptake by liver, heart, and skeletal muscle are increased in exercise, fasting, and diabetes. We have recently identified (1) and characterized (2) a transport system for long chain fatty acid in the plasma membrane of isolated adipocytes. The process is freely reversible, does not require metabolic energy, and has other characteristics of facilitated diffusion. It is strongly inhibited by phloretin and DIDS.' The possibility that fatty acid transport is regulated by hormones or metabolic factors, as are glucose and amino acid transport, appeared likely and is the subject of this study. Such a regulation in adipocytes could be important for release of fatty acid and could have relevance to clinical problems in which lipid metabolism is disturbed. We report here a strong stimulation by epinephrine of fatty acid influx and efflux in isolated rat adipocytes. Evidence is provided that the hormonal effect is exerted on the membrane transport process.
* This work was supported by a grant from the Kroc Foundation and by National Institutes of Health Grant AM 33301. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduertisernent" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Methods
Isolation of Fat Cells-Adipocytes were prepared from the epididymal fat of a fed 170-200 g Sprague-Dawley rat (Harlan Industries). The tissue was digested for 45 min at 37 "C with collagenase (1 mg/ 1.2 ml, Worthington) in modified Krebs Ringer medium (3) buffered with 25 mM bicarbonate, pH 7.4. The digestion medium also contained 20 mg/ml bovine serum albumin (BSA, Fraction V, Sigma) and 2 mM glucose. The washed cells were resuspended (30%, v/v) in Krebs Ringer solution buffered with 10 mM Hepes, pH 7.4, and containing 30 p~ BSA (Fraction V, Sigma).
Assay of Fatty Acid Uptake-Influx and efflux of [3H]oleate were measured as described in detail previously (1, 2) using ice-cold modified Krebs-Ringer medium buffered with Hepes buffer containing phloretin (200 p~) as stop solution. The cells were incubated with the isotope for 6-60 s for measurement of basal transport and for 1-30 s for epinephrine-stimulated transport. A metronome (2 beats/s) was used to estimate these time intervals. The cells were recovered and separated from medium on glass fiber filters (Gelman Type A/E 61630, Gelman) using less than 10 mm Hg filtration pressure. The filters were washed twice with I ml of stop solution and transferred to scintillation vials containing 6 ml of scintillation fluid (ACS, Amersham) for counting. Zero-time radioactivity was substracted (1, 2) except as noted.
The incubation medium was prepared by first adding with stirring a few microliters of ethanol containing labeled fatty acid of appropriate concentration and specific activity to water (1-2 ml). Bovine serum albumin was then added from a 10% stock solution in water buffered with Hepes (10 mM). The medium was made isotonic by addition of an equal volume of 2 X concentrated buffer.
Separation of Cellular Lipids-To determine whether [3H]oleate was incorporated into triglyceride during the assay period, the filters with cells were rapidly transferred to 4 ml of heptane and washed with another 4 ml. The combined extracts were then evaporated to dryness and resuspended in 200 p1 of heptane. An aliquot (10 pl) was spotted as a narrow band on Silica Gel G plates (Brinkman) which were then developed with chloroform/methanol/acetic acid (80: 200.2). Oleic acid was used as a reference. It was mixed with a sample of extract, spotted on a parallel channel, and visualized with rhodamine G. Radioactivity was checked throughout the plate divided in horizontal bands. In an alternative separation procedure, an aliquot of the heptane extract was evaporated and the residue taken up in ethanolic NaOH followed by petroleum ether according to the procedure of Borgstrom (4) to separate fatty acid from neutral lipid. Aliquots of the aqueous (fatty acid) and organic (triglyceride) phases were then assayed for radioactivity.

Materials
All radioactive materials were obtained from New England Nuclear. DIDS and epinephrine (Na bitartrate salt) were purchased from Sigma. All reagents for buffer preparations were obtained from Fisher. RHC 80267 was a generous gift from Dr. Charles Sutherland of the Revlon Care Group, Tuckahoe, NY.

RESULTS
Effect of Epinephrine on Permeation of fH]Oleate-Incubation of isolated adipocytes with epinephrine-stimulated oleate influx 5-lo-fold (Fig. 1). Reduction of assay temperature from 23 to 4 "C decreased both basal and stimulated rates about 90% but the fold stimulation by epinephrine was not significantly altered.
Since epinephrine is a potent stimulator of triglyceride lipolysis, the increase in fatty acid influx could have resulted from a trans effect of accumulated intracellular free fatty acids. The following experiment, however, showed this not to be the case. Adipocytes were pretreated for 15 min with the  lipolytic inhibitor, RHC 80267, before exposure to the hormone. This compound is a powerful inhibitor of tri-and diglyceride lipases (5). In separate experiments, using the same incubation and assay conditions, it was determined that a 15-min preincubation with RHC 80267 (10 p~) inhibited the stimulatory effect of epinephrine (1 pglml) on glycerol production by 85% (data not shown). No effects of the compound itself on fatty acid or glucose transport were observed. As shown in Fig. 1 (triangular symbols) inhibition of lipolysis by RHC 80267 did not alter the epinephrine effect. This suggested that accumulation of intracellular fatty acid after epinephrine treatment could not explain the transport activation.
Of particular importance physiologically was the observa- Krebs-Ringer medium buffered with Hepes buffer containing 2 mM glucose and 0.2% BSA were incubated without or with DIDS (300 p~) for 45 min at 37 "C in the dark. RHC 80267 was then added (10 p~) and the cells were left at 37 "C for 15 min more. Aliquots from control and DIDS-treated cells were then exposed to epinephrine (1 pg/ml) for 5 min at 37 "C. In some experiments the cells were washed in BSA containing medium following DIDS treatment to remove the unbound compound and were then treated with RHC 80267 and epinephrine as described above. Since this washing did not alter the results, it was subsequently omitted. A and A, basal cells; 0 and 0, epinephrine treated; A and 0, DIDS treated. The data is a composite of three experiments.
tion that epinephrine also stimulated fatty acid efflux 5-10fold from adipocytes preloaded with [3H]oleate, as shown in Fig. 2. In this situation, trans stimulation could not play a role. Any accumulation of intracellular fatty acid would only delay the efflux process by dilution of the isotope. Dose Response of Epinephrine Effect on Fatty Acid Transport-Epinephrine was optimally effective at a concentration of about 0.1 pg/ml and marginally effective at 20 ng/ml (Fig.  3). These concentrations are higher than blood levels of epinephrine (0.1-10 ng/ml (6)) but blood levels are probably not as important physiologically as release of catecholamine in the tissue at nerve endings.
Evidence that Epinephrine Stimulates Transport Specifically-The action of epinephrine on both influx and efflux of fatty acid argues strongly for an effect on transport since this is a freely reversible process (1,2). The completeness of isotope efflux in the epinephrine case (Fig. 2) indicated that the fatty acid taken up during the preincubation period of 12 s was still present in the unesterified form (since esterification is slow at 23 "C). It could therefore be released back into the Epinephrine Effect on Fatty Acid Transport 9971 medium whereas esterified fatty acid would be retained. Further evidence that the epinephrine effect was not due to a stimulation of intracellular disposal of fatty acid was obtained by thin layer chromatography of lipid extracts from basaland epinephrine-treated cells. In these experiments, the isotope taken up was quantitatively recovered on the silica plate in the free fatty acid band. This result was confirmed by extracting cells in ethanolic NaOH and petroleum ether (see "Methods"). All radioactivity partitioned in the aqueous phase. Effect of DIDS-The permeation of long chain fatty acid in basal adipocytes involves to a limited extent a component with kinetics of simple diffusion as well as the carrier mediated system (1). To determine whether epinephrine might be stimulating the diffusion component the effect of DIDS' on permeation in epinephrine-treated cells was investigated.
As shown in Fig. 4, DIDS inhibited fatty acid permeation by more than 80%. This effect of DIDS is similar in magnitude to its effect in basal cells (2) and indicates that epinephrine stimulates the carrier mediated component of FA permeation. Phloretin also strongly inhibits epinephrine-activated transport but the inhibition is not complete (data not shown).

DISCUSSION
The failure of epinephrine treatment to overcome the inhibitory effect of DIDS (Fig. 4) argues against the stimulation of transport by translocation of carriers to the plasma membrane from an intracellular reservoir as hypothesized for insulin activation of glucose transport (7). Since DIDS does not penetrate into the cell, intracellular carriers, if present, would be protected during exposure to DIDS which, in our studies, preceded epinephrine treatment.
The mechanism of epinephrine action on fatty acid transport could involve the same factors known to mediate its *This is the well-known inhibitor of band 3 anion transport in human erythrocytes. Its action in intact cell systems, like that of phloretin, is largely or exclusively on the plasma membrane.
lipolytic effect (8); namely binding of the catecholamine to the receptors leading to increased activation of cAMP production. Our studies, to be reported later, indicate this to be the case.
It is probable, therefore, that transport will be stimulated by other hormones which raise CAMP. We have recent evidence which indicates that insulin (1 nM) can counteract very effectively the epinephrine stimulatory e f f e~t .~ I t is wellknown that insulin can reduce cAMP levels in epinephrineactivated adipose cells. Thus it appears that changes in the activity of fatty acid transport are physiologically coordinated with changes in lipase activity to bring about the lipolytic and anti-lipolytic effects of epinephrine and insulin in intact cells. Hormonal regulation of fatty acid transport, therefore, could have great significance physiologically for substrate supply and utilization in fasting, feeding, exercise, stress, and diabetes.