Role of triglyceride-fatty acid cycle in controlling fat metabolism in humans during and after exercise

Transcription

1 ,~ ~ Rle f triglyceride-fatty acid cycle in cntrlling fat metablism in humans during and after exercise ROBERT R. WOLFE, SAMUEL KLEIN, FABIO CARRARO, AND JEAN-MICHEL WEBER Metablism Research Unit, Shriners Burns Institute, and Departments [ Surgery, Anesthesilgy, Internal Medicine, and Preventive Medicine, The University [ Texas Medical Branch, Galvestn, Texas WOLFE, ROBERTR., SAMUELKLEIN, FABIOCARRARO,AND JEAN-MICHELWEBER. Rle f triglyceride-fatty acid cycle in cntrlling fat metablism in humans during and after exercise. Am. J. Physil. 258 (Endcrinl. Metab. 21): E382-E389, We have investigated the rle f triglyceride-fatty acid cycling in amplifying cntrl f the net flux f fatty acids in respnse t exercise and in recvery frm exercise. Five nrmal vlunteers were infused with [l-13c]palmitate and D-5-glycerl thrughut rest, 4 h f treadmill exercise at 40% maximum O2 cnsumptin, and 2 h f recvery. Ttal fat xidatin was quantified by indirect calrimetry. Liplysis (rate f appearance f glycerl) increased frm 2.1 :t 0.3 t 6.0 :t 1.2 ).Lml.kg-1. min-1 after 30 min f exercise and prgressively increased thereafter t a value f 10.5 :t 0.8 ).Lml.kg-1.min-1 after 4 h. Liplysis decreased rapidly during the first 20 min f recvery, but it was still significantly elevated after 2 h f recvery. The rate f appearance f free fatty acids fllwed the same pattern f respnse. Seventy percent f released fatty acids were reesterified at rest, and this value decreased t 25% within the first 30 min f exercise. Reesterificatin remained <35% f liplysis until the start f recvery, at which time the value rse t 90%. In exercise, mre than ne-half the increase in fat xidatin culd be attributed t the reductin in the percent reesterificatin. Mst f the change in percent reesterificatin during exercise and recvery was caused by changes in extracellular cycling f fatty acids released int plasma. We cnclude that triglyceride-fatty acid cycling plays an imprtant rle in enabling a rapid respnse f fatty acid metablism t majr changes in energy metablism fies the respnse f substrate flux t a given change in regulatr (e.g., hrmne) (17). The mst dramatic change in the demand fr fat as an xidative substrate ccurs in respnse t exercise and in the recvery perid immediately after exercise. Therefre, amplificatin f cntrl f net substrate flux by TG-FA cycling wuld be imprtant during exercise and recvery frm exercise. The precise relatinship between changes in liplysis, reesterificatin, and the availability f ttal fatty acids fr xidatin has nt been quantified previusly in exercising humans. A small amunt f infrmatin is available regarding the rate f fatty acid reesterificatin in exercising dgs (5, 20), but the results f these studies are cnflicting and nly address the rate f reesterificatin within the adipcyte. Our previus studies have shwn that the rate f this intracellular reesterificatin is generally <20% f the ttal whle bdy reesterificatin f fatty acids (27). The primary gal f this experiment was t assess the imprtance and the energy cst f ttal TG- FA cycling in crdinating the availability f fatty acids with energy requirements during exercise and recvery. An additinal gal was t determine if changes in TG-FA cycling that ccur in exercise and recvery primarily invlve intracellular r extracellular recycling, s that mechanisms invlved in the cntrl f TG- FA cycling culd be better understd. stable istpes; liplysis; reesterificatin METHODS Subjects A SUBSTRATECYCLEexists when ppsing, nnequilib- This study was apprved by the Institutinal Review rium reactins, catalyzed by different enzymes, are active Bard f The University f Texas MedicaI Branch at simultaneusly (17). These cycles require energy and Galvestn. Five male subjects participated after giving prduce heat but d nt result in any increase in the net their infrmed written cnsent. They were physically flux f prduct. The triglyceride- fatty acid (TG- FA) cycle active, in gd health, and had n histryf metablic is ne such cycle that has received cnsiderable attentin recently. In this cycle, fatty acids released during the diseases. Three subjects jgged 5 km abut twice a week. Each vlunteer was admitted t the Clinical Research prcess f liplysis are subsequently reesterified rather than xidized. Reesterificatin can ccur within the adi- Center at 11 A.M.n day 1 fthe study. Maximum xygen cnsumptin C'V02max)was measured in the afternn pcyte ("intracellular" recycling), r the fatty acid can during a stepwise exercise prtcl n a treadmill (Quinbe released and reesterified elsewhere (e.g., liver) ("ex- tn Q55 with Q645 cntrl bard, Seattle, WA). Exercise tracellular" recycling). TG-FA cycling is under bth hr- was initiated with a 10-min warm-up walk at 4 km/h. mnal (16) and substrate (29) cntrl and is stimulated Speed and incline were then increased every minute until in fasting (lo, 13,30) as well as in numerus pathlgical the subjects were exhausted. Oxygen cnsumptin C'V02) cnditins (22, 27). and carbn dixide prductin (VC02) were measured It has been hypthesized that substrate cycling ampli- breath by breath thrughut the exercise but with a E /90 $1.50 Cpyright@ 1990 the American Physilgical Sciety

2 TRIGLYCERIOE-FATTY ACIO CYCLE AND EXERCISE E383 metablic cart (Hrizn 4400, Beckman Instruments, Anaheim, CA). In all cases the rate f V02 reached a plateau and the respiratry qutient (RQ) was greater than ne befre exhaustin. Mean values fr age, height, bdy mass, and V02 maxare givenin Table 1.The subjects ate a regular dinner at 6 P.M. and a light snack at 8:30 P.M.. N fd was cnsumed frm 9 P.M. until the end f the study n the next day. Water was available ad libitum at all times. lstpe lnfusin and Exercise Prtcl At 7 A.M. n day 2, Tefln catheters were placed percutaneusly. The infusin catheter was intrduced in the antecubital vein and the sampling catheter was placed in the drsal hand vein f the cntralateral side. The heated-hand technique (14) was used t btain arterialized bld samples. The subject sat n a chair and rested unti! 9 A.M. when cnstant infusins f [1-13C]palmitate and -5-g1ycerl (~0.03 and 0.05 /lml. kg-l. min-l), respectively, were started with calibrated syringe pumps (Harvard Apparatus, Natick, MA). The exact infusin rates were determined fr each subject separately by measuring palmitate and glycerl cncentratins in the infusates. Bth istpes (99% atm % excess) were purchased frm Tracer Technlgies (Newtn, MA). Palmitate was bund t albumin (Cutter Bilgical, Berkeley, CA) by fllwing previusly described prcedures (26). After 1 h f infusin, with the subject at rest, treadmill exercise was initiated and the rate f istpe administratin was dubled fr palmitate and tripled fr glycerl t minimize changes in substrate istpic enrichment. The exercise prtcl cnsisted f a 4-h walk at 40% V02 max' The istpe infusin was cntinued fr 2 h after the end f exercise t determine metablite kinetics during recvery. V02 and Vcz were quantified repeatedly fr 5-10 min at regular intervals thrughut the study, using the metablic cart with nse clip and muthpiece system. Bld Sampling The first bld samples were withdrawn 1 h after catheterizatin and befre starting the istpe infusin t determine base-line cncentratins and backgrund enrichment. Bld was als taken 45, 50, 55, and 60 min after the beginning f infusin t measure resting kinetics. Mre samples were withdrawn after lo, 20, 30, 60, 90, 120, 150, 180, 210, and 240 min f exercise, and 5, lo, 20, 30, 60, 90, and 120 min f recvery. All samples were cllected in lo-mi vacutainers cntaining lithium heparin and were placed n ice. Plasma was separated by TABLE 1. Physical characteristics and maximal xygen uptake f subjects Age, yr Height, cm Bdy mass, kg Percent ideai bdy mass Va'm." ml.kg-1.min-l Values are means :!: SE; n = 5 subjects. 26.6:!: :!: :!: :!: :!:2.1 centrifugatin within 5 min f sampling and then frzen until further prcessing. Sample Analysis Plasma glucse cncentratin was measured n a glucse analyzer (Beckman Instruments) by use f the glucse xidase methd. Plasma lactate was measured n a Yellw Springs Institute Lactate Analyzer (Yellw Springs, OH). Free fatty acids (FFA) frm plasma were extracted, islated by thin-layer chrmatgraphy, and derivatized t their methyl esters. Palmitate and ttal FF A cncentratins were determined by gas chrmatgraphy (Hewlett- Packard 5890) using hepatadecanic acid as an internai standard (26). Istpic enrichment f palmitate was measured by gas chrmatgraphy mass spectrmetry (GCMS) analysis fthe methyl ester derivatives n a Hewlett-Packard 5992 (25). Ins f mass-tcharge ratis (m/e) 270 and 271 were selectively mnitred. Istpic enrichment and cncentratin f glycerl were determined by GCMS (Hewlett- Packard 5985B) by fllwing previusly described prcedures (30). Ins f m/e 205, 206, and 208 were mnitred, and a crrectin was made fr the cntributin f the enrichment at m/e 206 t the apparent enrichment at m/e 208. Calculatins Indirect calrimetry. The rate f triglyceride xidatin was calculated frm the indirect calrimetry data (11). Nitrgen excretin rate was assumed t be 7.7 /lg.kg-l. min-l. This average value was taken frm the measured values determined in anther study perfrmed in ur labratry (7) in which 16 nrmal subjects perfrmed the same exercise prtcl. A 30% errr in this assumed value (which exceeds the ttal range f values in the previus study) wuld have had n significant effect n the calculated values f fat and glucse xidatin in exercise in the current study. Fatty acid xidatin was determined by cnverting the rate f triglyceride xidatin (g.kg-l.min-l) t its mlar equivalent (/lml.kg-l. min-l), assuming the average mlecular weight f triglyceride is 860 g/ml (11) and multiplying the mlar rate f triglyceride xidatin by three because each mle f triglyceride cntains three mles f fatty acids. Rates f appearance (RJ. Ra f glycerl and palmitate were calculated using the equatin f Steele (21), as mdified fr use with stable istpes (18). In the transitin frm rest t exercise, minimal changes in istpe enrichment were bserved because f the changes in istpe infusin rate (e.g., see Fig. 2). Because f the relativelycnstant istpic enrichments in the transitin frm rest t exercise and thrughut exercise as well as because f the rapid rate f equilibratin within the palmitate (15) and glycerl (4) pls (caused by a rapid turnver rate relative t pl size), istpic steady-state equatins were used t calculate Ra at each time pint during exercise. In the early part f recvery, istpic enrichment changed rapidly, and the nn-steady-state apprximatin f Steele (21) was used. In this case, the effective vlume f distributin (V) was assumed t be 270 ml/kg fr glycerl (4) and 40 ml/kg fr palmitate.

3 E384 TRIGLYCERIDE-FATTY ACID CYCLE AND EXERCISE After the first 30 min f recvery the change ver time in istpic enrichment f glycerl was s small that the specific value fr V had n significant effect n the calculated value. The value fr V fr palmitate was chsen because acute changes in palmitate cncentratin are essentially restricted t plasma (being bund t albumin). The Ra f FFA was then calculated by dividing the Ra f palmitate by the fractinal cntributin f palmitate t the ttal FF A cncentratin, as determined by. gas chrmatgraphy. TG-FA cycling. The ttal rate f liplysis was determined by measuring Ra f glycerl. Glycerl appears in the bld nly as a prduct f liplysis (9, 24, 29) and cannt be directly reincrprated int triglyceride within the adipcyte because f the absence f glycerl kinase (6). Every mle cuie f glycerl released int the bld represents the cmplete degradatin f a triglyceride mlecule, meaning that three fatty acids were als released. Thus, in steady-state cnditins, the difference between three times the Ra f glycerl (ttal fatty acids released) and the rate f ttal fatty acid xidatin (determined by indirect calrimetry) will give the ttal rate f reesterificatin, since reesterificatin is ultimately the nly ther fate f fatty acids released by liplysis. When plasma FFA cncentratin increases, the rate f rise (times the plasma vlume) must als be subtracted frm three times the Ra f glycerl t determine reesterificatin. the rate f Ttal whle bdy reesterificatin cnsists f intracellular recycling, in which the fatty acids never leave the cell where liplysis ccurred befre reesterificatin, and extracellular recycling, in which the fatty acid passes thrugh the plasma befre reesterificatin. The mst cmmn examples f extracellular recycling are the release f FF A int plasma frm adipcytes and clearance and reesterificatin f fatty acids within the liver. The difference between three times the Ra f glycerl (intracellular release f fatty acids) and the Ra f FFA int plasma shuld prvide an indicatr f intracellular recycling. Extracellular recycling is calculated as the difference between ttal recycling and intracellular recycling. The energy cst f TG- FA cycling was estimated frm the number f high-energy phsphate bnds (ATP --> ADP) required fr reesterificatin. It was assumed that eight high-energy phsphate bnds were required per mle f triglyceride recycled (lo). Because ~ 18 kcal f heat are released per mle f ATP hydrlyzed and synthesized (17), the ttal energy cst is ~ 144 kcal/ml f triglyceride recycled. The impact f changing the percentage reesterificatin f released fatty acids n the rate f fat xidatin during exercise was evaluated by cmparing the actual rate f fat xidatin during exercise with the theretical rate that wuld have ccurred had the percent reesterificatin nt changed frm the resting values. Similarly, the effect f the change in the percent reesterificatin that ccurred in the recvery perid was determined by cmparing the actual FF A cncentratin with the value that wuld have ccurred if the percent reesterificatin had nt changed frm the exercise value. It was assumed that all fatty acids nt xidized r reesterified wuld remain in a plasma vlume f 3 liters. Statistics Mean values measured at each time pint during and after exercise were cmpared with resting levels by use f the Dunnett's multiple range test (32). P < 0.05 was cnsidered indicative f statistically significant differences. RESULTS Substrate Cncentratins The plasma cncentratins f FF A and glycerl rse prgressively thrughut exercise,reaching levels f ~ 1.6 and 0.4 mm fr FF A and glycerl, respectively, by the end f exercise (Fig. 1). An versht in FF A cncentratin was bserved during the first lo min f recvery, after which values drpped rapidly fr the next 45 min befre stabilizing ver the 2nd h f recvery at a value significantly higher (P < 0.05) than the resting cncentratin. N such versht ccurred in glycerl cncentratin, which decreased thrughut the recvery perid, but nnetheless was significantly higher after 2 h f recvery than during rest (P < 0.05) (Fig. 2). Plasma glucse cncentratin decreased steadily frm 5 t 4 mm during exercise and stayed lw thrughut the 2-h recvery perid Plasma lactate cncentratin remained at the basai cncentratin thrughut exercise and recvery. In all cases a gd equilibrium in glycerl and palmitate enrichment was achieved at rest, and the enrichment levels were relatively cnstant thrughut exercise because f changing the istpe infusin rate (see Fig. 2 fr representative example). During the first 30 min after exercise, enrichment rse rapidly but was again relatively cnstant during the last hur f recvery :2 E ~ 0.3 w ~ O :2 1.6 E <t u u FIG. 1. Plasma cncentratins f glycerl and free fatty acids (FF A) in untrained subjects befre, during, and after 4 h f treadmill exercise at 40% \T02 max' Values are means i: SE fr n = 5 persns.

4 TRIGLYCERIDE-FATTY ACID CYCLE AND EXERCISE E385 4 w ~ 3...J g 2 W U >-...J 0 4 ResI Exerclse Recvery..'... li> re W c.. I. «31 W I-...J. I:....J... I. «:j I.... c.... O -1 O FIG. 2. Representative examples f effect f prlnged treadmill exercise n plasma glycerl (MPE, mle percent excess) and palmitate enrichment (APE, atm percent excess). Istpe infusin was started l h befre beginning exercise. Resting infusin rates were and l'ml. kg-l min-1 fr glycerl and palmitate, respectively. At time O, exercise was started, and infusin rate was tripled fr glycerl and dubled fr palmitate. O,... I 30 c «'E LI.."": LI.. I 20 <U Jl' CI:..:.. E 10.3 O I Exerclse xercise I Recver O FIG. 3. Rate f appearance (R.) f glycerl and FFA at rest, in exercise, and in recvery (n = 5). Ra f glycerl increased frm a resting value f 2.1 :t 0.3 t 6.0 :t 1.2 /lml. kg-1. min -I after 30 min f exercise and prgressively increased thereafter t a value f 10.5 :t 0.8 /lml.kg-1.min-1 after 4 h (Fig. 3). Ra f glycerl drpped rapidly during the first 20 min f recvery, fhwed by a slwer decrease t 4.7 :t 0.9 /-Lml.kg-l. min-1 at 2 h f recvery, which was still significantly higher than at rest (P < 0.05). The generai pattern f respnse f Ra f FF A during exercise and recvery was I cmparable t that f glycerl. It was significantly increased at 30 min, then rse thrughut exercise, and feh in recvery (Fig. 3). In cntrast t the versht in plasma cncentratin f FF A during the early part f recvery (Fig. 2), Ra f FF A declined rapidly immediately at the cessatin f exercise. As with glycerl, Ra f FF A remained significantly (P < 0.05) elevated abve the resting value after 2 h f recvery. lndirect Calrimetry The indirect calrimetry data indicated a stable V02 thrughut exercise at precisely 40% V02 max' The average value fr V02 in exercise was 20.8 :t 0.07 mi. kg-l. min -\ whereas the resting value f 3.28 :t 0.35 mi. kg-l. min-1 VC02 decreased slightly thrughut exercise, reflecting a RQ that feh prgressively frm 0.92 :t t 0.83 :t during exercise. The RQ remained arund 0.83 during recvery. Ttal energy expenditure feh rapidly ver the first 20 min f recvery and then remained ~20% abve the preexercise resting value thrughut the remainder f recvery. The rate f ttal fatty acid xidatin during exercise is shwn in Fig. 4. Crrespnding t the fah in RQ, fat xidatin rse thrughut exercise frm a resting value f 1.9 :t 0.2 t a value f -20 /lml. kg-l. min-1 after 4 h. The largest change in fat xidatin ccurred during the first 30 min f exercise when the value rse t 13.7 :t 1.01 /lml.kg-1.min-l. Fat xidatin feh t between 3 and 3.5 /-Lml.kg-l. min-1 thrughut recvery. This was significantly higher than the resting value. TG-FA Cycle Ttal cycling. The greater rate f fat xidatin in exercise resulted nt nly frm an increased rate f liplysis, as reflected by Ra f glycerl (Fig. 3), but als 121 n 10...J- 01 CI: c 8 W.- U >-,... ç:...ji 6 0.>< C1 <U - 4 CI: z Q 15. c -X - c: 12. ; c - -" '" :::: «9, >- f- f- «LI Hurs 01 Exercise FIG. 4. Rate f ttal fatty acid xidatin (e) determined by indirect calrimetry and calculated rate f fatty acid xidatin () had there been n change in percent reesterificatin frm resting value (70%). Shaded area represents increased xidatin in exercise accunted fr by changes in percent reesterificatin, and it is >50% f ttal fat xidatin.

5 E386 TRIGLYCERIDE-FATTY ACID CYCLE AND EXERCISE because f a dramatic reductin in the percentage f fatty acids reesterified at the nset f exercise (Fig. 5). At rest 70% f released fatty acids were reesterified. This percentage fell t ~25% at 30 min f exercise and remained belw 35% thrughut exercise. As sn as exercise stpped, reesterificatin jumped t ~90% f released fatty acids. The percent had fallen back t 75% 2 h after exercise. The energy requirement f the TG- FA cycle was -0.8 kcaljh at rest, which was 1.2% f ttal energy expenditure (TEE; Table 2). Althugh the value rse slightly in exercise, it was nly 0.4% f TEE because f the large increase in TEE. In recvery the high rate f cycling accunted fr 4.7 kcaljh, which was 5% f the ttal. Mre significantly, the energy required fr the increase in cycling during recvery abve the resting value (3.9 kcaljh) accunted fr 13.7% f the increase in ttal energy expenditure in recvery. lntracellular and extracellular cycling. The rate f intracellular cycling was 1.3 }.Lml.kg-l. min-1 at rest, which was 20% f the ttal rate f fatty acid released (3 x Ra f glycerl). Thrughut exercise, intracellular cycling rse t -3 f.lml.kg-l. min-\ but this was nly ~ 12% f the ttal fatty acids released ver the last 2 h f exercise because f the increased rate f liplysis. In recvery the rate f intracellular cycling was similar t the value at rest. Thus the percent f intracehular reesterificatin feh in exercise and was als lwer than the resting value in recvery. The rate f extracehular cycling was 3.1 f.lml.kg-1.min-l at rest This value was 50% f released fatty acids (3 x Ra f glycerl) and 62% f the rate frelease fffa int plasma (Ra fffa). The ttal rate f extracellular cycling ranged frm 5 t 8 f.lml. kg-l. min-1 in exercise (20-25% f Ra f FF A). In recv- 100,' 80. () «>- f- w f «IL IL Ci: IL W f- f- 40- z w w a: () a: w 20. Il Rest I Exerclse I Recvery l! FIG. 6. Percent reesterificatin f fatty acids made available via triglyceride hydrlysis. TABLE 2. Energy cst f TG-FA cycle keal/h % Energy % Inerease Expenditure AbveRest Rest Exercise Recvery ery the extracellular recycling averaged 20 f.lml.kg-l. min-1 (72% f Ra f FFA) during the first 30 min and was at 9 f.lml.kg-1.min-l (65% f Ra FFA) at the end f 2 h. Thus mst f the change in the percentage f released fatty acids that were reesterified during exercise and recvery culd be attributed t changes in extracellular cycling. The impact f the decrease in percent reesterificatin n the rate f fat xidatin in exercise can be seen in Fig. 4. The shaded area f the graph, indicating the amunt f fat xidatin attributable t the reductin in the percentage f fatty acids reesterified, is -50% f the ttal rate f fat xidatin. If the percent reesterificatin had remained at the exercise value, the plasma cncentratin f FFA wuld have risen t >7 mm in the recvery perid because f the vlume f distributin f FF A and the rate f fat xidatin in exercise. Ra f FFA in relatin t fatty acid xidatin. Ra f FFA and ttal fatty acid xidatin are presented tgether graphically in Fig. 6. At all times there was mre than enugh plasma FF A available t prvide all substrate fr fatty acid xidatin. DISCUSSION The results f this study establish the imprtance f the TG-FA cycle in amplifying the ability f stred triglyceride t respnd rapidly t majr changes in energy requirements caused by starting, maintaining, and stpping exercise. At rest, ~70% f all fatty acids released during liplysis were reesterified. During the first 30 min f exercise, that value drpped t 25%, whereas ttal fatty acid release via triglyceride hydrlysis tripled (Figs. 3 and 5). This crdinated respnse allwed a sixfld increase in FF A availability fr xidatin. By itself the sharp decrease in percent reesterificatin effectively dubled the number f FF A available fr energy metablism in wrking muscles during exercise and culd accunt fr mre than ne-half f ttal fatty acid xidatin (Fig. 4). Immediately at the cessatin f exercise almst 90% f fatty acids released frm liplysis were reesterified. This dramatic increase in percent reesterificatin was a majr reasn fr the rapid fah in FF A cncentratin " 'E 20 '" rn g 16 =t 12 8 Rest E..'cl,e 41 Q! FIG. 6. Cmparisn f rate f appearance f free fatty acids () with ttal fatty acid xidatin (e) as determined by indirect calrimetry.

6 TRIGLYCERIDE-FATTY ACID CYCLE AND EXERCISE E387 after exercise. Had the percentage f fatty acids reesterified in the recvery perid stayed at the value during exercise (25-30%), plasma FF A cncentratin wuld have risen t a level that wuld have far exceeded the binding capacity f albumin. The rapid changes in the percentage f released fatty acids that were reesterified at the start f bth exercise and recvery enhanced the metablic respnse t the rapid changes in energy requirements. Althugh the initial liplytic respnse t changes in energy requirements at the nset f exercise and recvery was rapid, the maximum adaptive respnse lagged behind. Glycerl Ra cntinued t rise thrughut exercise despite a cnstant rate f energy expenditure after the first lo min. At the beginning f recvery the percentage decline in Ra f glycerl was much less than the percentage decline in energy expenditure, and 2 h after stpping exercise Ra f glycerl had nt yet returned t the resting level. In calculating the ttal rate f TG- FA recycling, we have assumed that Ra f glycerl reflects the ttal rate f whle bdy liplysis. AmpIe evidence exists t supprt the validity f this assumptin. 1) Adipse tissue cntains n glycerl kinase, and therefre all glycerl released frm liplysis will appear in plasma (6). 2) GlycerI is nt prduced metablically by a prcess ther than liplysis (24, 29). 3) Underestimatin f Ra f glycerl because f first-pass clearance by the liver f glycerl released by the gut is nt likely t be a prblem, even in exercise (23). 4) Underestimatin f liplysis because f partial hydrlysis f triglyceride is unlikely (2, 6). It was als assumed that the determinatin f the rate f ttal fat xidatin by indirect calrimetry was accurate. The level f exercise was particularly chsen t maintain nrmal acid-base balance and thereby prevent excessive ventilatry lss f CO2. The cnstant (resting) levels f lactate thrughut rest, exercise, and recvery indicated that acid-base balance was maintained. It is cnceivable that calculatin f fat xidatin may have been underestimated because f an accelerated rate f glucnegenesis in exercise. Hwever, this pssibility is unlikely, because the rate f glucse prductin is nt stimulated at this level f exercise (8) and the lactate cncentratin did nt increase in ur study. The calculatin f intracellular recycling relies n the validity f Ra f FFA as a quantitative measure f the rate f release f fatty acids. The data recently reprted by Wasserman et al. (23), as well as earlier data frm Bass and Havel (3), indicate that n mre than 10% f FF A flux riginates frm mesenteric liplysis and that nly 25% f that is cleared in the first pass f the liver. Therefre, <3 % f released fatty acids wuld be missed using istpic tracers t measure Ra f FF A. It is pssible t verestimate intracellular recycling if fatty acids released by liplysis were t be directly xidized by adjacent tissues withut appearing in plasma. Because the rate f plasma FFA xidatin, determined istpically, is nly abut ne-half the ttal rate f fat xidatin, it has been suggested that ne-half f fat xidatin ccurs by such "direct" rutes (20). Hwever, the rate f plasma FF A xidatin determined by traditinal tracer techniques must be an underestimate f the true value, because the intracellular enrichment at the site f xidatin is nt measured (25). Furthermre, retentin f the label in 14C-labeled fatty acids that are xidized can ccur via istpic exchange frm the xalacetate and a-ketglutarate pls f the tricarbxylic acid cycle (28). Figure 6 shws that at all times the Ra f FFA was well in excess f the ttal rate f fatty acid xidatin, which means that ampie fatty acids were available frm plasma t satisfy xidative requirements. In additin the difference between Ra FF A and fat xidatin (Fig. 6) is cnsistent with expected values f hepatic fatty acid uptake and very lw density lipprtein release (31). Furthermre, the arterivenus difference f plasma FF A acrss resting and exercising muscle is cnsistent with the ntin that plasma FF A supplies all f the fat used fr energy (1). The bserved rati f Ra f FFA t Ra f glycerl in this and ther studies (e.g., see Re. 12) f ~3:1 als supprts the idea that there is little xidatin f fatty acids released by liplysis that d nt enter the plasm pl, because this wuld result in a lwer rati. Fr al] these reasns, we believe that ur calculated values fol ttal and intracellular recycling, and (by deductin) ex. tracellular recycling, are reasnably accurate. The valu( fr ttal recycling is nt dependent n the site f xida. tin r Ra f FF A and thus reliable. is likely t be extremel~ The primary factrs determining the extent f reester ificatin within the adipcyte are the ability f th! plasma t carry away released FF A (i.e., bld flw an< adequate albumin binding sites) and the availability glucse t prduce glycerl 3-phsphate fr reesterifica tin. The lw rates f intracellular recycling in all phase: f this experiment indicate that adipse tissue bld flv was always adequate t clear away fatty acids release( by liplysis. Glycerl 3-phsphate levels were nt at : high level because f the lwer glucse levels thrughu the experiment. Thus the ttal rate f flux via the extra cellular rute (Ra f FF A) was nt greatly influenced b: the extent f intracellular reesterificatin. In cntrast t the situatin with intracellular reester ificatin, changes in the extent f extracellular reesteri ficatin f FF A that entered the plasma were imprtan in determining the verall respnse t exercise. HweveI it is unlikely that the reductin in the percent reesteri ficatin that ccurred in exercise was accmplished b: direct inhibitin f reesterificatin, (i.e., active regula tin f recycling). In fact, althugh in exercise the frac tin f flux recycled decreased, the abslute amunt reesterificatin via the extracellular rute dubled, ani this amunt f reesterificatin crrespnded with th expected change in FF A delivery t the liver. Althug hepatic bld flw prbably decreased by 10-20% i exercise (19), the FFA cncentratin increased b slightly mre than twfld. A stimulatin f fatty aci xidatin in muscle, cupled with a redistributin ( bld flw favring muscle ver the liver, undubted cntributed t the reductin in the fractin f flux th; was reesterified during exercise. Thus, in this circur stance, the rate f recycling appears t be predminant

7 E388 TRIGLYCERIDE-FATTY ACID CYCLE AND EXERCISE passively regulated, reflecting the balance between the actively regulated prcesses f liplysis and xidatin. The physilgical significance f TG- FA cycling is nt diminished by what appears t be the passive nature f its regulatin in respnse t exercise. This is appreciated by cntrasting the extent f increased fat metablism in exercise, which results abut equally as a cnsequence f changes in release and recycling, t the situatin fr glucse, the ther majr circulating energy substrate. At rest almst ali f the glucse released frm the liver is metablized [<15% recycling (16)], s that changes in availability f glucse during exercise must entirely result frm changes in the rate f prductin. In cntrast t the situatin described fr rest and exercise in which the liver may passively reesterify a cnstant fractin f delivered FF A, during recvery bth the abslute and relative rate f reesterificatin f released fatty acids increased dramatically, despite a decrease in the delivery f FFA t the liver as plasma cncentratin fell. One plausible explanatin is that there was accelerated clearance and reesterificatin f FF A in peripheral adipse tissue, but the signal fr this type f peripheral clearance and reesterificatin is unclear. It is als unclear why clearance and reesterificatin f plasma FF A by adipse tissue wuld ccur at an accelerated rate in the absence f a change in the rate f intracellular TG- FA recycling. Thus an increased efficiency f reesterificatin cluded. within the liver cannt be ex- The unique aspect f the extracellular TG-FA cycle, as cmpared with ther previusly described substrate cycles, is that the rate f net flux is nt simply passively determined by the difference between the ttal rate f flux and the amunt that is recycled. Rather, the rate f net flux, which is actually the rate f fat xidatin, can be directly regulated. In respnse t exercise, the large increase in the energy requirement f the exercising muscles was undubtedly respnsible fr the accelerated rate f fat xidatin, and thus the decreased availability f fatty acids fr reesterificatin. This is reflected by an increased percent f FF A flux that was xidized (Fig. 6). As expected, the reverse situatin ccurred in recvery. The energy cst f TG- FA cycling required <2 % and 0.5% f ttal energy expenditure at rest and in exercise, respectively. The benefit affrded by a high rate f TG- FA cycling at rest in terms f regulatin f substrate availability is thus accmplished ecnmically in terms f verall energy expenditure. In cntrast, during recvery frm exercise, the high rate f cycling accunted fr a cnsiderable percentage (14%) fthe increase in energy expenditure abve the resting value befre exercise. The ptential physilgical significance f this is evident when it is cnsidered that the cycling persisted at an increased rate fr at least 2 h after stpping exercise. Cnsequently, TG-FA cycling may be imprtant nt nly in the cntrl f substrate flux, but als in the effect f exercise n verall energy balance. The authrs acknwledge the assistance f Marta Wlfe, Susan Fns, and Antnella Arrar fr mass spectrmetry analysis, LeAnne Rman fr analytical assistance, Tm Rutan fr assistance in perfrming the indirect calrimetry, and Judy Chadwick fr manuscript preparatin. This research was supprted by Natinal Institutes f Health Grants CRC RR and DK Present addresses: J.-M. Weber, Cncrd Field Statin, Harvard University, Old Causeway Rad, Bedfrd, MA 01730; F. Carrar, Istitut di Fisilgia Generale, Universita degli Studi di Siena, via Laterina 8, Siena, Italy. Address fr reprint requests: R. R. Wlfe, Shriners Burns Institute, 610 Texas Avenue, Galvestn, TX Received 12 Octber 1988; accepted in final frm 31 Octber REFERENCES l. AHLBORG, G., P. FELIG, L. HAGENFELOT, R. HENOLER, ANO J. W AHREN. Substrate turnver during prlnged exercise in man Splanchnic and leg metablism f glucse, free fatty acids, and amin acids. J. Clin. Invest. 53: , ARNER, P., ANO J. OSTMAN. Mn and di-acyl glycerls in human adipse tissue. Bichim. Biphys. Acta 369: , BASSO, L. V., ANO R. HAVEL. Hepatic metablism fffa in nrmal and diabetic dgs. J. Clin. Invest. 49: , BEYLOT, M., C. MARTlN, B. BEAUFRERE, J. P. RIOU, ANO R. MORNEX. Determinatin f steady-state and nn-steady state glyceri kinetics in humans using deuterium-iabeled tracer. J. Lipid Res. 28: , BULOW, J. Subcutaneus adipse tissue bld flw and triacylglycerl-mbilizatin during prlnged exercise in dgs. Pfluegers Arch. 392: , BROOKS, B., J. R. S. ARCH, ANO E. A. NEWSHOLME. Effects f hrmnes n the rate f the triacylglycerljfatty acid substrate cycle in adipcytes and epididymal fat pads. FEBS Lett. 146: , CARRARO, F., S. PERNIA, D. K. LAYMAN, F. JAHOOR, C. STUART, ANO R. R. W OLFE. Effect f exercise n prtein synthesis in nrmal vlunteers (Abstract). Federatin Prc. 2: 1090, COOPER, D. M., T. J. BARSTOW, W.-N. P. LEE, ANO A. BERGNER. Bld glucse turnver during high- and lw-intensity exercise. Am. J. Physil. 257 (Endcrinl. Metab. 20): E405-E412, DIXON, M., ANO E. L. WEBB. Enzymes. New Yrk: Academic, 1979, p lo. ELlA, M.,C. ZEO, G. NEALE, ANO G. LIVESEY. The energy cst f triglyceride-fatty acid recycling in nn-bese subjects after an vernight fast and fur days f starvatin. Metablism 36: , Il. FRAYN, K. N. Calculatin f substrate xidatin rates in viv frm gaseus exchange. J. Appl. Physil. 55: , HAVEL, R. J. Sme influences f the sympathetic nervus system and insulin n mbilizatin f fat frm adipse tissue: studies f the turnver rates f free fatty acids and glycerl. Ann. NY Acad. Sci. 131: , KLEIN, S., E. J. PETERS, O. B. HOLLANO, ANO R. R. WOLFE. Effect f shrt- and lng-term {3-adrenergic blckade n liplysis during fasting in humans. Am. J. Physil. 257 (Endcrinl. Metab. 20): E65-E73, MCGUlRE, E. A. H., J. H. HELDERMAN, J. D. TOBIN, R. ANORES, ANO M. BERMAN. Effects f arterial versus venus sampling n analysis f glucse kinetics in man J. Appl. Physil. 41: , MILES, J. M., M. G. ELLMAN, K. L. MCCLEAN, ANO M. D. JENSEN. Validatin f a new methd fr determinatin f free fatty acid turnver. Am. J. Physil. 252 (Endcrinl. Metab. 15): E431-E438, MIYOSHI, H., G. 1. SHULMAN, E. J. PETERS, D. ELAHI, ANO R. R. WOLFE. Hrmnal cntrl f substrate cycling in humans. J. Clin. Invest. 81: , NEWSHOLME, E., ANO B. CRABTREE. Substrate cycles in metablic regulatin and in heat generatin. Bichem. Sc. Symp. 41: , ROSENBLA'IT, J. L, ANO R. R. WOLFE. Calculatin f substrate flux using stable istpes. Am. J. Physil. 254 (Endcrinl. Metab. 17): E526-E531, ROWELL, L. B., J. R. BLACKMON, R. H. MARTIN, J. A. MAZZA- RELLA, ANO R. A. BRUCE. Hepatic clearance f endcyanine green ----

8 TRIGLYCERIDE-FATTY ACID CYCLE AND EXERCISE E389 in man under thermal and exercise stress. J. Appl. Physil. 20: , SHAW, J. H. F., AND R. R. WOLFE. Fatty acid and glycerl kinetics in septic patients and in patients with gastrintestinal cancer: the respnse t glucse infusin and parenteral feeding. Ann. Surg. 205: , SHAW, W. A. S., T. B. ISSEKUTZ, AND B. ISSEKUTZ, JR. Interrelatinship f FF A and glycerl turnvers in resting and exercising dgs. J. Appl. Physil. 39: 30-36, STEELE, R. Influences f glucse lading and f injected insulin n hepatic glucse utput. Ann. NY Acad. Med. Sci. 82: , WASSERMAN, D. H., D. B. LACY, R. E. GOLDSTEIN, P. E. WIL- LIAMS, AND A. P. CHERRINGTON. Exercise-induced fai! in insulin and increase in fat metablism during prlnged muscular wrk. Diabete" 38: , WINKLER, B., R. STEELE, AND N. ALTSZULER. Relatinship f glycerl uptake t plasma glycerl cncentratin in the nrmal dg. Am. J. Physil. 216: , WOLFE, R. R. Tracers in Metablic Research: Radiistpe and Stable Istpe/Mass Spectrmetry Techniques. New Yrk: Liss, 1983, p WOLFE, R. R., J. E. EVANS, C. J. MULLANY, AND J. F. BURKE. Measurement f plasma free fatty acid turnver and xidatin using 1-13C-palmitic acid Bimed. Mass. Spectrm. 7: , WOLFE, R. R., D. N. HERNDON, F. JAHOOR,H. MIYOSHI,ANDM. H. WOLFE. Effect f severe burn injury n substrate cycling by glycse and fatty acids. N. Engl. J. Med. 317: , WOLFE, R. R., ANDF. JAHOOR. Recvery f labeled CO2 during the infusin f 1- versus 2-carbn-labeled acetate: implicatins fr tracer studies f substrate xidatin. Am. J. Clin. Nutr. In press 29. WOLFE, R. R., AND E. J. PETERS. Liplytic respnse t glucse infusin in human subjects. Am. J. Physil. 252 (Endcrinl. Metab. 15): E218-E223, WOLFE, R. R., E. J. PETERS, S. KLEIN, O. B. HOLLAND,J. I. ROSENBLATT,AND H. GARY,JR. Effect f shrt-term fasting n liplytic respnsiveness in nrmal and bese human subjects. Am. J. Physil. 252 (Endcrinl. Metab. 15): E189-E196, WOLFE, R. R., J. H. F. SHAW,ANDM. J. DURKOT.Effect f sepsis n VLDL kinetics: respnses in basai state and during glucse infusin. Am. J. Physil. 248 (Endcrinl. Metab. 11): E732-E740, ZAR,J. H. Bistatistical Analysis (2nd ed.). Englewd Cliffs, NJ: Prentice-Hal!,1984.