Effect of increased plasma free fatty acid concentrations on muscle metabolism in exercising men MARK HARGREAVES, August Krogh Institute,
BENTE University
HARGREAVES,MARK,BENTEKIENS,ANDERIK Effect of increased muscle metabolism
KIENS, AND ERIK A. RICHTER of Copenhagen, DK-2100 Copenhagen 0, Denmar% A.
RICHTER.
plasma
free fatty acid concentrations on in exercising men. J. Appl. Physiol. 70(l):
194-201, 1991.-The effect of increasing plasma concentrations of free fatty acids on substrate utilization in muscle during exercise was investigated in 11 healthy young males. One hour of dynamic knee extension at 80% of knee-extensor maximal work capacity was performed first with one leg and then with the other leg during infusion of Intralipid and heparin. Substrate utilization was assessed from arterial and femoral venous blood sampling as well as from muscle biopsies. Intralipid infusion increased mean plasma free fatty acid concentrations from 0.54 rt 0.08 to 1.12 t 0.09 (SE) mM. Thigh glucose uptake during rest, exercise, and recovery was decreased by 64, 33, and 42%, respectively, by Intralipid, whereas muscle glycogen breakdown and release of lactate, pyruvate, and citrate were unaffected. Concentrations of glucose, glucose 6phosphate, and lactate in muscle before and at termination of exercise were unaffected by Intralipid. During exercise, net leg uptake of plasma free fatty acids was not measurably increased by Intralipid, whereas uptake of ketone bodies was. Local respiratory quotient across the leg was not changed by Intralipid (control 0.87 t 0.02, Intralipid 0.86 t, 0.02). Arterial concentrations of insulin, norepinephrine, and epinephrine were similar in the two trials. It is concluded that at rest and during exercise at a moderate intensity (requiring approximately equal contributions from fat and carbohydrate metabolism), muscle carbohydrate metabolism is affected only with regard to uptake of glucose when plasma concentrations of lipid and lipid metabolites are increased. This effect may be by direct inhibition of glucose transport rather than by the classic glucose-fatty acid cycle. glycogen; glucose transport
CONSIDERABLE INTEREST has focused on the interaction between carbohydrate and lipid metabolism in the 25 years since Randle and co-workers (24,25) proposed the glucose-fatty acid cycle. Despite this, doubt persists as to whether this cycle operates in human skeletal muscle both at rest and during exercise. Elevation of plasma free fatty acids (FFA) at rest has been shown to inhibit glucose utilization (4,12), although the physiological significance of plasma FFA in the control of plasma glucose oxidation in humans has recently been questioned (35). During exercise, elevation of plasma FFA has been shown to decrease muscle glycogen use during treadmill running (9), whereas inhibition of FFA mobilization by nicotinic acid increases glycogen use (7). In contrast, carbohydrate oxidation during prolonged moderate-in194
0161~7567/91$1.50 Copyright
tensity exercise was unaffected by physiological elevations in plasma FFA (26). Two studies in perfused rat muscle have been unable to show significant alterations in muscle glucose uptake and glycogen metabolism during rest and electrical stimulation when palmitate or ketone bodies are added to the perfusion medium (5,31). In contrast, there is a report of diminished muscle glucose utilization and glycogenolysis during contraction, associated with increased muscle concentrations of citrate, glucose 6-phosphate, and glucose, when oleate was added to the perfusion medium (28). These investigators have also observed higher blood glucose levels and slower muscle glycogen utilization in exercising rats when plasma FFA are elevated (17, 29). It has also been suggested that in rat muscle the cycle operates only in red muscle and only after exercise (36). There are few studies in humans in which muscle metabolism has been examined in detail during exercise at different arterial plasma FFA levels. Furthermore, previous studies on the role of FFA in regulating carbohydrate metabolism during exercise have often used starvation (21) or fat feeding (18). These techniques have limitations because substrate and hormone levels change, as do concentrations of FFA. Thus it is difficult to isolate the effect of different FFA levels on carbohydrate metabolism. In view of the controversy surrounding the role of the glucose-fatty acid cycle for muscle metabolism in humans and the paucity of well-controlled detailed studies, the present study was undertaken. Substrate utilization was measured through arterial and femoral venous catheterization, femoral venous blood flow measurement, local respiratory quotient (RQ) determination, and muscle biopsies. As the exercise model we chose to use the one-legged knee extension model (2), because exercise with this model permits the study of a well-defined muscle group (the quadriceps femoris muscle) and results in small systemic hormonal and metabolic changes. Thus, in a study of subjects exercising first with one leg and then with the other during infusion of triglycerides, virtually the only difference between exercise bouts would be arterial concentrations of lipid and lipid metabolites. Finally, we chose subjects covering a wide range of physical fitness to elucidate whether a possible effect of fatty acids might depend on the oxidative capacity of the muscle studied. MATERIALS AND METHODS
Subjects. Eleven healthy male students, mean age 24 (range Z-28) yr and weight 73.1 (64.4-87.7) kg, were
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Physiological
Society
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CARBOHYDRATE
AND LIPID
fully informed of the risks and stresses associated with the study and agreed to participate. The study was approved by the Copenhagen Ethics Committee. Two subjects competed in endura rice events (bicycling and triath lon), whereas six subj ects participated in leisuretime physical activity to various degrees. Three subjects were quite sedentary. Maximal oxygen uptake, measured during two-legged cycling exercise, averaged 4.0 (2.8-5.5) l/min. To characterize the potential for oxidative metabolism and fatty acid P-oxidation, respectively, vastus lateralis citrate synthase (CS) and 3-hydroxyacyl-CoA dehydrogenase (HAD) activities were assayed. CS and HAD activities averaged 39.6 t 2.7 (30.2-59.3) and 39.5 t 1.7 (31.3-49.0) pmol . g dry wt-l. min-l, respectively. Preexperiment testing. Exercise was performed on a modified Krogh ergometer and limited to the knee extensors as described previously (2). Before performing the experiment, subjects attended three or four training sessions to become familiarized with dynamic knee extension exercise. Equal time was spent on each leg. In addition, on separate days the maximal work load the knee extensors could perform was determined. This maximum was gauged from a sudden steeper increase in whole body oxygen uptake and heart rate, which, until that time, had increased linearly with work load. The sudden steeper increase in oxygen uptake and heart rate is due to recruitment of additional muscle to stabilize the body. The maximal work load was identical for the two legs and averaged 48 (30-75) W. ExperimentaL protocol On the morning of an experiment, subjects reported to the laboratory at 8:00 A.M. after fasting overnight and abstaining from strenuous exert ise fo r 224 h. Catheters were introduced percutaneously into both femoral vei ns and one femoral artery and were advanced centrally so that the tips of the arterial and venous catheters were positioned ~2 cm proximal and distal to the inguinal ligament, respectively. Thermistors (Edslab TD probe 94-03002.5F) for blood flow measurements were inserted through both venous catheters and were advanced 6-8 cm proximal to the catheter tips. A catheter was also inserted into an antecubital vein for Intralipid infusion. The subjects then rested in the supine position for 45-60 min. After a further 5- to lo-min rest in the seated position, resting blood saPmples were obtained simultaneously from the femoral artery . and the femoral vein of the leg to be exe rcised fi.rst. A biopsy from th .e vastu s lateralis muscle of this 1,eg was then obtained with a Bergstrijm needle (including suction) under local anesthesia. Subjects then performed knee extension exercise for 1 h with one leg at a load requiring 80% of the maximal work load of the knee extensors. This work load was chosen so that carboh .ydrate and fat contribu ted abo lut equa lly to substrate oxidation as evidenced by RQ values (Table 1). Bet #ause of technical considerations (see DISCUSSION), the control exercise bout was always conducted first; however, the leg performing this exercise (left or right) was randomized. Immediately at the termination of exercise, another muscle biopsy was obtained. Recovery samples were obtained 10 min after completion of the first exercise bout. Then an infusion of a 20% triglycer-
METABOLISM
195
IN MUSCLE
TABLE 1. Physiological extension exercise
responses to knee Control
Heart rate, beats/min Pulmonary oxygen uptake, l/min Leg oxygen uptake, l/min RER Leg RQ Leg blood flow, l/min
105t6 0.93t0.06 0.62kO.06 0.85+0.01 0.87kO.02 4.75t0.31
In tralipid
104t5 0.92t0.05 0.59t0.06 0.82t0.01* 0.86~10.03 4.67t0.32
Values are means t SE; n = 11 subjects. Because values did not change systematically with time, values are means of measurements at 10, 20, 40, and 60 min of exercise, except for pulmonary oxygen uptake and RER, which are means of measurements at 20,40, and 60 min. Leg RQ was measured only at 40 min of exercise. * P c 0.05 from control.
ide emulsion (Intralipid, KabiVitrum, Stockholm) was started into the forearm venous catheter at a rate of 1.7 ml/min. This infusion was maintained through 30 min of rest and the second exercise bout with the contralatera1 leg. Subjects received an intravenous 1,000-U heparin bolus at the start of the infusion and a further 1,000 U over the 1 h of exercise. The second exercise bout was preceded by resting blood samples and a muscle biopsy as in the first bout. During blood sampling and femoral venous blood flow measurements, a cuff positioned just below the knee joint was inflated to 230 mm Hg. Analytic procedures. Blood was rapidly analyzed for glucose (YSI 23 AM glucose analyzer, Yellow Springs Instruments, Yellow Springs, OH) and hemoglobin and oxygen saturation (OSM 2 Hemoximeter, Radiometer, Copenhagen). A portion of the blood was deproteinized in 0.6 M perchloric acid for subsequent lactate, pyruvate, glycerol, ,&hydroxybutyrate, and citrate determination by enzymatic fluorometric techniques (6,22). Hematocrit was measured by use of a microcentrifuge. The remaining blood was quickly centrifuged, and the plasma was stored at -20 or -80°C for later analysis. Blood for FFA determination was sampled in a heparinized syringe and immediately transferred to an Eppendorf tube (1.5 ml) containing 30 ~1 of 0.2 M ethylene glycol-bis(P-aminoethyl ether)-N,N,N’,N’-tetraacetic acid. On high-speed centrifugation for 1 min, the plasma was frozen at -20°C. The efficacy of this procedure to prevent in vitro lipolysis of plasma triacylglycerols when subjects were given heparin was proven when this sampling method was compared with a method inactivating lipoprotein lipase by adding 0.2 ml 5 M NaCl to 0.8 ml plasma and heating the mixture for 30 min at 56OC (23). FFA were measured fluorometrically (32) as modified by Kiens (20). Triacylglycerols in plasma were measured without free glycerol (Boehringer Mannheim Diagnostica). Insulin was measured by radioimmunoassay (Novo, Copenhagen), and norepinephrine and epinephrine were measured by a radioenzymatic technique (8). Oxygen and carbon dioxide contents of whole blood were determined by the Van Slyke method (34). Expired gas was collected in Douglas bags for pulmonary oxygen uptake determination. Oxygen and carbon dioxide concentrations in the bags were measured by paramagnetic and infrared analyzers, respectively. Volume was ob-
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196
CARBOHYDRATE
AND LIPID
tained by use of a Tissot spirometer. Leg blood flow during exercise was measured by constant saline infusion thermodilution in the femoral vein (3); for measurements at rest and during recovery this method was modified (30). Muscle samples were rapidly (within lo-15 s) frozen in liquid nitrogen and stored at -8O*C for later biochemical analysis. Samples were freeze-dried and dissected free of visible connective tissue, fat, and blood. Glycogen was measured as glucose residues after hydrolysis of the muscle sample in 1 M HCl at 100°C for 2 h (22). Glucose, glucose 6-phosphate, and lactate were measured fluorometrically on neutralized perchloric acid extracts (22). Muscle CS and HAD activities were measured fluorometrically (11). Cakulations and statistics. Uptake or release of substrates, metabolites, and oxygen was calculated as arteriovenous differences times blood or plasma flow as appropriate. The data from the two exercise bouts were compared by two-way analysis of variance and Newman-Keuls post hoc testing. Resting and recovery data were compared by paired t tests. The significance for all comparisons was set at P < 0.05. RESULTS
The exercise work loads were identical for the two legs, averaging 38 (24-60) W; accordingly, there were no differences between trials in pulmonary and leg oxygen uptake, heart rate, or leg blood flow (Table 1). As a result of the Intralipid infusion, arterial FFA concentration during exercise was twofold higher than during control (Table 2), averaging 1.12 $- 0.09 and 0.54 t 0.08 mM, respectively, over the entire exercise periods. Despite this difference in arterial concentrations, net FFA uptake surprisingly was similar over the two exercise periods. However, as discussed in detail below, net FFA uptake may not accurately reflect FFA utilization because FFA is simultaneously taken up and released by the leg (16). Arterial concentrations of glycerol and ,&hydroxybutyrate were markedly increased by Intralipid infusion (Table 2). Glycerol release was similar during both exercise bouts (Table 3), whereas /3-hydroxybutyrate uptake was markedly higher when Intralipid was infused (Table 3). Still, in absolute terms P-hydroxybutyrate uptake was small. Concentrations of triacylglycerols in arterial plasma were roughly doubled by Intralipid infusion (Table 2), but during neither control exercise nor Intralipid infusion could an uptake of triacylglycerols across the thigh be detected (Table 3). Arterial blood glucose levels were similar during both exercise bouts except for small but significant differences at rest and during the first 20 min of exercise (Table 2, Fig. 1). Resting muscle glucose uptake was reduced, on average, 64% when arterial FFA were elevated. Glucose uptake during exercise was decreased by 33% (Table 3, Fig. l), averaging 0.82 t 0.13 mmol/min during control and 0.55 t 0.12 mmol/min during Intralipid infusion. After 10 min of recovery, the decrease in glucose uptake during Intralipid infusion averaged 42%. These decreases in glucose uptake were not associated
METABOLISM
IN MUSCLE
with accumulation of glucose or glucose 6-phosphate in muscle at rest or immediately after exercise (Table 4). This speaks against the possibility that the decrease in glucose uptake was caused by glucose 6-phosphate-induced inhibition of glucose phosphorylation. Arterial lactate concentrations were slightly lower at 10 and 20 min in the FFA trial compared with the control trial (Table 2). No differences between trials were seen in arteri .a1 levels of pyruvate or rele ase of lactate and pyr luvate from the leg before, dur ling, or after exercise (Tables 2 and 3). Similarly, muscle lactate both at rest and immediately after exercise was not changed by infusion of Intralipid. Muscle glycogen was similar in the two legs before exercise (Fig. 2). The amount of glycogen degraded during exercise was similar under both conditions, averaging 249 t 38 mmol/kg dry muscle during the control exercise bout and 226 t 30 mmol/kg dry muscle during exercise with Intralipid infusion. Arterial citrate was higher at rest and after 10 and 20 min of exercise when FFA were elevated (Table 2), but leg citrate release was similar in both conditions (Table 3). No differences were observed between conditions in the arterial concentrations of insulin, norepinephrine, and epinephrine (Table 5). After 40 min of exercise, the respiratory exchange ratio (RER) was significantly lower during Intralipid infusion than during control but was similar in both conditions at the 200 and 600min time points. However, when the values at 20, 40, and 60 min were averaged, RER was slightly lower (P < 0.05) during the Intralipid exercise bout than during control exercise (Table 1). No significant difference between conditions was seen in RQ measured across the leg after 40 min of exercise (Table 1). DISCUSSION
The present study has demonstrated that infusion of Intralipid and heparin resulting in a physiological elevation of plasma FFA is associated with an inhibition of thigh glucose uptake at rest, during exercise, and during recovery from exercise. In contrast, there was no measurable effect of Intralipid infusion on muscle glycogen breakdown or lactate, pyruvate, and citrate release or on leg RQ during exercise. These observations were made in a group of subjects with a fairly wide range of maximal aerobic capacities and muscle oxidative enzyme activities. No correlation was detected between muscle CS or HAD activity and the degree of inhibition of muscle glucose uptake with increased plasma FFA, indicating that the effect was not dependent on the level of enzyme activity involved in P-oxidation or the tricarboxylic acid cycle. The knee-extension model enables us to study “isolated” working muscle without major changes in the internal body milieu because the mass of working muscle is relatively small. As such, it offered the possibility to examine substrate metabolism during exercise under two conditions that ideally differed only in the arterial concentrations of plasma FFA. In reality, Intralipid infusion and heparin administration also produced large
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CARBOHYDRATE TABLE
2. Arterial
concentrations
AND LIPID
METABOLISM
197
IN MUSCLE
of substrates in blood and plasma Exercise
Preexercise
Glucose, mM Control Intralipid Lactate, mM Control Intralipid Pyruvate, PM Control Intralipid Free fatty acids, mM Control Intralipid Glycerol, PM Control Intralipid @-Hydroxybutyrate, PM Control Intralipid Citrate, PM Control Intralipid Triacylglycerol, mM Control Intralipid
10 min
20 min
40 min
60 min
Recovery
4.74t0.10 4.54tO.15*
4.72t0.15 4.48t0.13*
4.61t0.11 4.48t0.13”
4.43-+0.10 4.38tOJ3
4.44tO.11 4.34tO.14
4.50t0.13 4.48t0.14
0.54kO.05 0.5WO.05
1.65t0.21 1.37-t-0.17*
1.65-t-0.24 1.37&0.22*
1.33-t-0.20 1.29-t-0.28
1.08t0.18 1.01-1-0.21
0.72t0.09 0.74t0.11
17-c4 16_+2
22t6 22t3
26t4 24t3
2123 2224
25t3 21t4
2124 23t4
0.60t0.07 0.92t0.07”
0.48t0.06 0.91t0.06*
0.50t0.07 1.03t0.07*
0.55t0.09 1.22t0.09*
0.65t0.11 1.40t0.12”
0.71~0.10 1.44-t-0.12*
53t,5 168t8*
56t5 205t7*
64t7 224t8”
72t9 241t12*
92zk12 257,tl4*
59t9 230+17*
65t14 192t37*
45-+11 176-t39*
35tlO 165-t38*
38+14 185t40”
46tl8 215t39”
99+29 308+51*
4922 62+4*
53t2 65t3*
56-t-3 66+3*
6325 68+3
64t4 68~3
63*4 67?4
NM NM
NM NM
0.54kO.04 0.93+0.09*
0.53t0.05 1.06t0.11*
0.54t0.05 l.l9cO*12*
0.52+0.05 1.18t0.12*
Values are means _+SE; n = 11 subjects except n = 8 for triacylglycerol. NM, not measured. Glucose, lactate, pyruvate, glycerol, ,&hydroxybutyrate, and citrate concentrations were measured in blood; FFA and triacylglycerol concentrations were measured in plasma. * P < 0.05 from control.
TABLE
3. Leg exchange of substrates Exercise Preexercise
Glucose uptake, mmol/min Control Intralipid Lactate release, mmol/min Control Intralipid Pyruvate release, pmol/min Control Intralipid FFA uptake, mmol/min Control Intralipid Glycerol release, pmol/min Control Intralipid ,&Hydroxybutyrate uptake, pmol/min Control In tralipid Citrate release, pmol/min Control Intrahpid Triacylglycerol uptake, pmol/min Control Intralipid
10 min
20 min
40 min
60 min
Recovery
0.11&0.02 0.04~0.01*
0.43+0.19 0.23t0.11
0.69t0.10 0.34_+0.09*
0.87t0.12 0.53t0.10*
1.27t0.16 0.92t0.17*
0.45t0.08 0.26t0.05*
0.04t0.01 0.06t0.01
2.78kO.50 2.32t0.50
1.90t0.30 2.11kO.52
1.22t0.36 1.06kO.36
0.80t0.23 0.7320.23
0.10t0.01 0.08t0.02
176*43 151t26
147+28 129zt25
142t22 102t23
86t22 98t25
2t2 lt2
0.07+0.04 0*04t0.04
0.09+_0.04 O.ll,tO.O5
0.15t0.05 0.12t0.02
0.14-40.04 0.16,tO.O4
0.01 to.01 -0.03_tO.O2
39+9 4tlO
35t_9 33t22
21211 33t9
16st3 22t8
14t12 89t15*
1825 36t9*
Ot,l Otl -0.01~0.01 -0.02t0.01 IO+3 -8t_3
36t9 -22tl5
10*3 29t6*
12t6 36t26*
-324 40*14*
8*6 76+20*
5.0t0.7 7.1t0.5
8.9k6.0 6.6t3.5
4.5k5.4 6.5t3.6
-1.8k4.5 0.6t7.0
-0.7t7.3 -3.6t4.3
9.1t1.9 8.4kl.6
NM NM
NM NM
45-t46 -236+110
-16tl6 60t104
-8t9 -87t119
-1lt19 76t135
Values are means t SE; n = 11 subjects except n = 8 for triacylglycerol
increases in the arterial concentrations of glycerol, ketones, and plasma triacylglycerols. Glycerol has no effeet on glucose utilization in the resting state (12), and
uptake. NM, not measured. * P < 0.05 from control.
leg glucose uptake during exercise is unaffected by heparin (1). The possibility that other components of Intralipid (e.g., phospholipid) influence exercise metabolism is
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CARBOHYDRATE
AND LIPID
METABOLISM
IN MUSCLE
FIG. 1. Arterial concentrations of glucose and glucose uptake before, during, and after I h of l-legged knee extensions. Subjects exercised with 1 leg during control conditions and with the other leg during infusion of Intralipid. *‘P < 0 .05 compared with values during control conditions.
0
20
60
LO lime
(min)
difficult to assess but cannot be excluded. Nevertheless, arterial plasma insulin, catecholamines, preexercise muscle glycogen levels, and, at most time points, arterial glucose concentrations were similar in the control and Intralipid conditions. In this respect, the knee-extension model provides advantages over previous methods (e.g., starvation, fat feeding, prolonged exercise) that have been used to examine the influence of plasma FFA on carbohydrate metabolism during exercise. Such methods usually produce a number of hormonal and metabolic changes that make investigation of the direct effect of elevated FFA difficult. It was technically impossible to conduct the control and the Intralipid trial in a randomized order because it
4. Muscle metabolites after exercise
TABLE
80
is impossible to achieve low FFA concentrations after Intralipid infusion without administering agents blocking lipolysis, such as nicotinic acid and insulin, and these substances might have physiological effects unre-
before and immediately
Total glucose, mmol/kg dry wt Control Intralipid Glucose 6-phosphate, mmol/kg dry wt Control Intralipid Lactate, mmol/kg dry wt Control Intralipid Values are means t SE; n = 11 subjects.
Rest
Exercise
1.39kO.21 l/74+0.31
2.98t0.48 2.75t0.21
0.27t0.03
0.29+0.04
0.28-tO.05
0.26t0.05
7.0tl.4
13.8-1-3.5
9.022.3
ll.O_t1.9
Pfe Control
Pte Post Inttalipid
2. Concentrations of glycogen in vastus lateralis muscle before and after 1 h of l-legged knee extensions during control conditions and during infusion of Intralir>id. * dw.I”drs weight. Y FIG.
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CARBOHYDRATE TABLE
5. Arterial
plasma conentrations
AND LIPID
of hormones
Exercise
Insulin, pU/ml Control Intralipid Norepinephrine, rig/ml Control Intralipid Epinephrine, rig/ml Control Intralipid
Rest
20 min
60 min
Recovery
9.3tU.8 8.9t0.8
9.0t0.7 8.1kO.9
7.4t0.8 7.6t0.8
9.1t1.2 9.1t0.9
0.38t0.07 0.36t0.07
0.54t0.12 0.47t0.08
0.45t0.11 0.51~0.09
0.40t0.06 0.25t0.08
0.19t0.03 0.20+0.03
0.22tO.03 0.24+_0.03
0.30t0.03 0.27t0.04
0.20t0.03 0.20t0.04
Values are means t SE; n = 11 subjects for insulin, n = 8 for catecholamines.
lated to their ability to lower FFA. Therefore the only alternative to the present design is to do the two trials on separate days. However, this would mean day-to-day variation in substrate stores and hormone concentrations in the blood and thus make detection of small differences impossible. Although we cannot exclude an order effect in the present study, it is unlikely to be significant because preexercise substrate stores and plasma concentrations of insulin, epinephrine, and norepinephrine during knee extensions were identical in the two trials. Thus a biochemical mechanism responsible for a possible order effect independent of the effects of Intralipid infusion is not apparent. Resting muscle glucose uptake was reduced 64% when plasma FFA were elevated. Studies in rat muscle have produced confiicting results that demonstrate either no effect of FFA on muscle glucose uptake (5, 27, 31) or reduced glucose utilization (19,28) and, in fact, also increased glucose utilization at very high FFA levels (19). In resting human subjects, increases in plasma FFA levels have been shown to decrease whole body glucose utilization (4, 12), although a recent study found no effect of Intralipid and heparin infusion on glucose uptake and oxidation during hyperglycemic hyperinsulinemia (35). In the present study, muscle glucose uptake during exercise was, on average, 33% lower when plasma FFA were increased (Fig. 1). The mechanism underlying the decrease in muscle glucose uptake observed in the present study is not readily apparent. According to the original hypothesis (24), an increase in fat oxidation in muscle, as a consequence of higher plasma FFA levels, results in citrate-mediated inhibition of phosphofructokinase activity and accumulation of glucose 6phosphate and glucose, thereby inhibiting glucose uptake. Furthermore, carbohydrate oxidation may be reduced because pyruvate dehydrogenase activity may be diminished by increased oxidation of FFA and ketones (15, 25). In the rat hindlimb, addition of FFA to the perfusion medium has been shown to increase muscle citrate and free glucose levels during contractile activity in one study (28) but not in another (31). Muscle citrate was not measured in the present study, but arterial citrate levels were higher only at rest and
METABOLISM
IN MUSCLE
199
during the first 20 min of exercise with elevated FFA. This may simply reflect a time effect, however, because leg citrate release was similar during both exercise bouts (Table 3). Furthermore, muscle glucose and glucose 6-phosphate levels were similar in the two trials (Table 4), suggesting that the inhibition of glucose uptake by Intralipid was not due to accumulation of glucose within the cell but rather due to a direct effect of FFA on glucose transport. This is in accordance with Randle et al. (25), who demonstrated that L-arabinose transport in perfused rat heart was inhibited by FFA and ketones. However, the possibility exists that increases of glucose in a small compartment of the muscle may be physiologically important but impossible to detect in a tissue homogenate. Intralipid consists mainly of soybean oil, which upon hydrolysis liberates mainly linoleic acid (l&2) and oleic acid (l&l). It cannot be excluded that different results might have been obtained had the increase in FFA concentration been with saturated fatty acids. For instance, direct membrane effects are conceivably different with different fatty acids. Furthermore it is not inconceivable that the elevated plasma triacylglycerols during Intralipid infusion might directly affect glucose transport. Studies in exercising humans have shown an inverse relationship between plasma FFA and the rate of muscle glycogen utilization (7,9), although in a more recent study there was no effect of increased FFA on carbohydrate oxidation during prolonged moderate-intensity exercise (26). In exercising rats, muscle glycogen breakdown during exercise was decreased in the presence of increased FFA (10, 17, 29). In the present study there was no effect of increased FFA on muscle glycogen breakdown during exercise (Fig. 2). Thus the major effect of FFA on carbohydrate metabolism may be on glucose uptake rather than on glycogen metabolism. However, in the present study biopsies were obtained only before and after exercise, and it cannot be excluded that differences in glycogen breakdown rate might have been found at other time points. It is generally believed that the rate of FFA uptake by muscle during exercise is directly related to the arterial concentration or inflow of FFA (13); however, a doubling of the arterial FFA concentration in the present study had no measurable effect on net FFA uptake during exercise. Part of the explanation for this difference may lie in technical aspects of the study. Because FFA are simultaneously released and taken up by the exercising leg (14, 16), FFA arteriovenous differences are small, and hence it is difficult to detect small changes in FFA uptake without isotopically labeled FFA. In addition, it is likely that some of the infused triacylglycerol was hydrolyzed as it passed through exercising muscle, resulting in a higher femoral release of FFA than in control exercise. This would decrease the arteriovenous concentration difference of FFA (if the FFA were not taken up by the muscle) and hence the net uptake. If this were the case, however, one might also expect an increase in apparent glycerol release and an apparent uptake of triacylglycerols during Intralipid infusion, and this was not observed. In any case, in this particular
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200
CARBOHYDRATE
AND LIPID
setting of Intralipid infusion, net FFA uptake may be a poor indicator of FFA uptake. An alternative explanation is that the capacity of the muscle to take up FFA was fully taxed already during control exercise. Recent in vitro studies in cardiac myocytes (33) and in perfused rat muscle (L. P. Turcotte, B. Kiens, and E. A. Richter, unpublished observations) have demonstrated that FFA uptake is a saturable process. If this is also the case in contracting skeletal muscle, it might explain why a doubling of the arterial FFA concentration did not measurably increase net FFA uptake, because FFA uptake may have been already close to maximal in the control situation. Finally, it cannot be excluded that different results might have been obtained at a lower work load. However, the present load was moderate because it could easily be sustained for 1 h and because fat and carbohydrate combustion contributed about equally to substrate oxidation. It remains to be explained that Intralipid caused a 33% reduction in glucose uptake during exercise but did not change leg RQ. Because glycogen breakdown was not changed by Intralipid, a compensatory increase in oxidation of other fuels must have occurred. The source of such fuels remains unknown, however. In any event, in terms of energy delivery, the reduction in glucose uptake amounts to only 6% of energy consumption, which, if compensated for by lipid combustion, would decrease the RQ across the leg by 0.02. Thus the failure to see a significant reduction in RQ across the working leg may be due to the fact that the expected decrease in RQ is too small to detect. In fact, pulmonary RER was slightly decreased when averaged over the entire exercise period (Table I), supporting a very small shift in fuel combustion, but RER represents metabolism in the entire body; it is likely that metabolism in organs other than contracting leg muscle, e.g., in the heart, was changed by Intralipid. This might explain the lower RER but unchanged leg RQ during Intralipid infusion. In summary, the results of the present study demonstrate that, at rest and during moderate-intensity exercise, muscle carbohydrate metabolism is affected only with regard to uptake of glucose when plasma concentrations of lipid and lipid metabolites are increased. It is proposed that this effect may be by direct inhibition of glucose transport rather than by the classical glucosefatty acid cycle. The authors thank Betina Bolmgreen, Ingelise Kring, Hanne Sjoholm, Hanne Thomsen, and Merete Vanby for excellent technical assistance. This study was supported by the Danish Medical Research Council, Fonden af 1870, and the Danish Diabetes Association. M. Hargreaves was supported by the Danish Ministry of Education and the National Health and Medical Research Council of Australia. Present address of M. Hargreaves: Footscray Institute of Technology, Footscray, Victoria 3011, Australia. Address for reprint requests: E. A. Richter, August Krogh Institute, University of Copenhagen, 13 Universitetsparken, DK-2100 Copenhagen 0, Denmark. Received 29 January 1990; accepted in final form 8 August 1990. REFERENCES 1. AHLBORG, G., AND L. HACENFELDT. Effect of heparin on the substrate utilization during prolonged exercise. &and. J. C&n. Lab. Invest. 37: 619-624, 1977.
METABOLISM
IN
MUSCLE
2. ANDERSEN, P., R. P. ADAMS, G. SJBGAARD, A. THORBOE, AND B. SALTIN. Dynamic knee extension as a model for study of isolated exercising muscle in humans. J. AppZ. Physiol. 59: 1647-1653,1985. 3. ANDERSEN, P., AND B. SALTIN. Maximal perfusion of skeletal muscle in man. J. Physiol. Lond. 366: 233-249,1985. 4. BALASSE, E. O., AND M. A. NEEF. Operation of the “glucose-fatty
acid cycle” during experimental elevation of plasma free fatty acid levels in man. Eur. J. Clin. Invest. 4: 247-252, 1974. 5. BERGER, M., S. A. HAGG, M. N. GOODMAN, AND N. B. RUDERMAN. Glucose metabolism in perfused skeletal muscle: effects of starvation, diabetes, fatty acids, acetoacetate, insulin and exercise on glucose uptake and disposition. Biochem. J. 158: 191-202,1976. 6. BERGMEYER, H. V. (Editor). Methods of Enzymatic Analysis. New York: Academic, 1974, p. 1836-1839 and 1404-1406. 7. BERGSTROM, J., E. HULTMAN, L. JORFELDT, B. PERNOW, AND J. WAHREN, Effect of nicotinic acid on physical working capacity and on metabolism of muscle glycogen in man. J. Appl. Physiol. 26: 170-176,1969. 8. CHRISTENSEN, N. J., P. VESTERGAARD, T. SQRENSEN, AND 0. J. RAFAELSEN. Cerebrospinal fluid adrenaline and noradrenaline in depressed patients. Acta Psychiatr. Stand. 61: 178-182,198O. 9. COSTILL, D. L., E. COME, G. DALSKY, W. EVANS, W. FINK, AND D.
HOOPES. Effects of elevated plasma FFA and insulin on muscle glycogen usage during exercise. J. Apph Physiol. 43: 695-699,1977. 10. DOHM, G. L., E. P. TAPSCOTT, H. A. BARAKAT, AND G. J. KASPEREK. Influence of fasting in rats on glycogen depletion during exercise. J. Appl. 11. ESSEN,
Physiol.
55: 830-833,
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B., A. LINDHOLM, AND J. THORNTON. Histochemical properties of muscle fibre types and enzyme activities in skeletal muscle of standard bred trotters of different ages. Equine Vet. J 12: 175-
180,198O. 12. FERRANNINI, E., FRONZO. Effect of
E. J. BARRETT, S. BEVILACQUA, AND R. A. DEfatty acids on glucose production and utilization in man. J. CZin. invest. 72: 1737-1747, 1983. 13. HAGENFELDT, L. Turnover of individual free fatty acids in man. Federatim Proc. 34: 2236-2240,1975. 14. HAGENFELDT, L., AND J. WAHREN.
15.
16. 17. 18. 19.
20.
21.
Human forearm muscle metabolism during exercise. II. Uptake, release and oxidation of individual FFA and glycerol. Stand. J. CZk. Lab. Invest. 21: 263-2’76,1968. HAGG, S. A., S. I. TAYLOR, AND N. B. RUDERMAN. Glucose metabolism in perfused skeletal muscle: pyruvate dehydrogenase activity in starvation, diabetes and exercise. Biochem. J 158: 203-210,1976. HAVEL, R. J., B. PERNOW, AND N. L. JONES. Uptake and release of free fatty acids and other metabolites in the legs of exercising men. J Apph PhysioZ. 23: 90-99, 1967. HICKSON, R. C., M. J. RENNIE, R. K. CONLEE, W. W. WINDER, AND J. 0. HOLLOSZY. Effects of increased plasma fatty acids on glycogen utilization and endurance. J. AppZ. Physiol. 43: 829-833, 1977. JANSSON, E., AND L. KAIJSER. Effect of diet on the utilization of blood-borne and intramuscular substrates during exercise in man. Acta Physiol. Stand. 115: 19-30, 1982. JENKINS, A. B., L. H. STORLIEN, D. J, CHISHOLM, AND E. W. KRAEGEN. Effects of nonesterified fatty acid availability on tissue-specific glucose utilization in rats in vivo. J. CZin. Invest. 82: 293-299, 1988. KIENS, B. Metabolisame in Skeletmuskelen under Arbejde hos Mennesket. Efekten af TrGning (PhD thesis). Copenhagen: Univ. of Copenhagen, 1984. KNAPIK, J. J., C. N. MEREDITH, B. H. JONES, L. SUEK, V. R. YOUNG, AND W. J. EVANS. Influence of fasting on carbohydrate and fat metabolism during rest and exercise in men. J. AppZ. Physiol. 64:
1923-1929,1988. 22. LOWRY, 0. H., AND J. V. PASSONNEAU. A Flexible System matic Analysis. New York: Academic, 1972. J., B. E. BIHAIN, G. BENGTSSON-OLIVECRONA, 23. PETERSON, DECKELBAUM, Y. A. CARPENTIER, AND T. OLIVEGRONA.
of Enxy-
R. J. Fatty acid control of lipoprotein lipase: a link between energy metabolism and lipid transport. Proc. Natl. Acad. Sci. USA 87: 909-913,199O. 24. RANDLE, P. J., R. B. GARLAND, C. N. HALES, AND E. A. NEWSMOLME. The glucose-fatty acid cycle: its role in insulin sensitivity and the metabolic disturbances of diabetes mellitus. Lancet 1: 785-789, 1963. 25. RANDLE,
P. J., E. A. NEWSHOLME, AND P. B. GARLAND. Regulation of glucose uptake by muscle. Biochem. J. 93: 65%665,1964. 26. RAVUSSIN, E., C. BOGARDUS, K. SCHEIDEGGER, B. LAGRANGE, E. D.
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CARBOHYDRATE
27.
28. 29. 30. 31.
AND LIPID
HORTON, AND E. S. HORTON. Effect of elevated FFA on carbohydrate and lipid oxidation during prolonged exercise in humans. J. Appl. PhysioZ. 60: 893-900, 1986. REIMER, F.,G. LOFFLER,G.HENNING,ANDO.WIELAND. Theinfluence of insulin on glucose and fatty acid metabolism in the isolated perfused rat hindquarter. Hoppe-Segler’s 2. PhysioZ. Chem. 356:1055-1066,19?5. RENNIE, M. J., AND J. 0. HOLLOSZY. Inhibition of glucose uptake and glycogenolysis by availability of oleate in well-oxygenated perfused skeletal muscle. Biochem. J. 168: 161-170,1977. RENNIE, M. J., W. W. WINDER, AND J. 0. HOLLOSZY. A sparing effect of increased plasma fatty acids on muscle and liver glycogen content in the exercising rat. Biochem J. 156: 647-655,1976. RICHTER, E. A., K.J. MIKINES, H. GALBO, AND B. KIENS. Effect of exercise on insulin action in human skeletal muscle. J. AppZ. PhysioZ. 66: 876-885, 1989. RICHTER, E.A.,N.B. RUDERMAN, H. GAVRAS, E.R. BELUR,ANDH. GALBO. Muscle glycogenolysis during exercise: dual control by epinephrine and contractions. Am. J. Physiol. 242 (Endocrinol Metab. 5): E25-E32, 1982.
METABOLISM
IN MUSCLE
201
32. SHIMIZU,~., K. INOUE,Y.TANI,AND H. YAMADA. Enzymaticmicrodetermination of serum free fatty acids. Anal. Biochem. 98: 341345,1979. 33. SORRENTINO, D., D. STUMP, B. J. POTTER, R. B. ROBINSON, R. WHITE, C.-L. KIANG, AND P. D. BERK, Oleate uptake by cardiac myocytes is carrier mediated and involves a 40-kD plasma membrane fatty acid binding protein similar to that in liver, adipose tissue and gut. J. Clin. Invest. 82: 928-935, 1988. 34. VAN SLYKE, D. D., AND J. M. NEIL. The determination of gases in blood and other solutions by vacuum extraction and manometric measurement. J. BioC Chem. 61: 523~573,1924. 35. WOLFE, B. M., S. KLEIN, E. J. PETERS, B. F. SCHMIDT, AND R. R. WOLFE. Effect of elevated free fatty acids on glucose oxidation in normal humans. Metabolism 37: 323-329,1988. 36. ZORZANO,A.,T.W. BALON, L.J. BRADY, P. RIVERA, L.P. GARETTO, J. C. YOUNG, M. N. GOODMAN, AND N. B. RUDERMAN. Effects of starvation and exercise on concentrations of citrate, hexose phosphates and glycogen in skeletal muscle and heart: evidence for selective operation of the glucose-fatty acid cycle. Biochem. J. 232: 585-591,1985.
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BENTE University
HARGREAVES,MARK,BENTEKIENS,ANDERIK Effect of increased muscle metabolism
KIENS, AND ERIK A. RICHTER of Copenhagen, DK-2100 Copenhagen 0, Denmar% A.
RICHTER.
plasma
free fatty acid concentrations on in exercising men. J. Appl. Physiol. 70(l):
194-201, 1991.-The effect of increasing plasma concentrations of free fatty acids on substrate utilization in muscle during exercise was investigated in 11 healthy young males. One hour of dynamic knee extension at 80% of knee-extensor maximal work capacity was performed first with one leg and then with the other leg during infusion of Intralipid and heparin. Substrate utilization was assessed from arterial and femoral venous blood sampling as well as from muscle biopsies. Intralipid infusion increased mean plasma free fatty acid concentrations from 0.54 rt 0.08 to 1.12 t 0.09 (SE) mM. Thigh glucose uptake during rest, exercise, and recovery was decreased by 64, 33, and 42%, respectively, by Intralipid, whereas muscle glycogen breakdown and release of lactate, pyruvate, and citrate were unaffected. Concentrations of glucose, glucose 6phosphate, and lactate in muscle before and at termination of exercise were unaffected by Intralipid. During exercise, net leg uptake of plasma free fatty acids was not measurably increased by Intralipid, whereas uptake of ketone bodies was. Local respiratory quotient across the leg was not changed by Intralipid (control 0.87 t 0.02, Intralipid 0.86 t, 0.02). Arterial concentrations of insulin, norepinephrine, and epinephrine were similar in the two trials. It is concluded that at rest and during exercise at a moderate intensity (requiring approximately equal contributions from fat and carbohydrate metabolism), muscle carbohydrate metabolism is affected only with regard to uptake of glucose when plasma concentrations of lipid and lipid metabolites are increased. This effect may be by direct inhibition of glucose transport rather than by the classic glucose-fatty acid cycle. glycogen; glucose transport
CONSIDERABLE INTEREST has focused on the interaction between carbohydrate and lipid metabolism in the 25 years since Randle and co-workers (24,25) proposed the glucose-fatty acid cycle. Despite this, doubt persists as to whether this cycle operates in human skeletal muscle both at rest and during exercise. Elevation of plasma free fatty acids (FFA) at rest has been shown to inhibit glucose utilization (4,12), although the physiological significance of plasma FFA in the control of plasma glucose oxidation in humans has recently been questioned (35). During exercise, elevation of plasma FFA has been shown to decrease muscle glycogen use during treadmill running (9), whereas inhibition of FFA mobilization by nicotinic acid increases glycogen use (7). In contrast, carbohydrate oxidation during prolonged moderate-in194
0161~7567/91$1.50 Copyright
tensity exercise was unaffected by physiological elevations in plasma FFA (26). Two studies in perfused rat muscle have been unable to show significant alterations in muscle glucose uptake and glycogen metabolism during rest and electrical stimulation when palmitate or ketone bodies are added to the perfusion medium (5,31). In contrast, there is a report of diminished muscle glucose utilization and glycogenolysis during contraction, associated with increased muscle concentrations of citrate, glucose 6-phosphate, and glucose, when oleate was added to the perfusion medium (28). These investigators have also observed higher blood glucose levels and slower muscle glycogen utilization in exercising rats when plasma FFA are elevated (17, 29). It has also been suggested that in rat muscle the cycle operates only in red muscle and only after exercise (36). There are few studies in humans in which muscle metabolism has been examined in detail during exercise at different arterial plasma FFA levels. Furthermore, previous studies on the role of FFA in regulating carbohydrate metabolism during exercise have often used starvation (21) or fat feeding (18). These techniques have limitations because substrate and hormone levels change, as do concentrations of FFA. Thus it is difficult to isolate the effect of different FFA levels on carbohydrate metabolism. In view of the controversy surrounding the role of the glucose-fatty acid cycle for muscle metabolism in humans and the paucity of well-controlled detailed studies, the present study was undertaken. Substrate utilization was measured through arterial and femoral venous catheterization, femoral venous blood flow measurement, local respiratory quotient (RQ) determination, and muscle biopsies. As the exercise model we chose to use the one-legged knee extension model (2), because exercise with this model permits the study of a well-defined muscle group (the quadriceps femoris muscle) and results in small systemic hormonal and metabolic changes. Thus, in a study of subjects exercising first with one leg and then with the other during infusion of triglycerides, virtually the only difference between exercise bouts would be arterial concentrations of lipid and lipid metabolites. Finally, we chose subjects covering a wide range of physical fitness to elucidate whether a possible effect of fatty acids might depend on the oxidative capacity of the muscle studied. MATERIALS AND METHODS
Subjects. Eleven healthy male students, mean age 24 (range Z-28) yr and weight 73.1 (64.4-87.7) kg, were
0 1991 the American
Physiological
Society
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CARBOHYDRATE
AND LIPID
fully informed of the risks and stresses associated with the study and agreed to participate. The study was approved by the Copenhagen Ethics Committee. Two subjects competed in endura rice events (bicycling and triath lon), whereas six subj ects participated in leisuretime physical activity to various degrees. Three subjects were quite sedentary. Maximal oxygen uptake, measured during two-legged cycling exercise, averaged 4.0 (2.8-5.5) l/min. To characterize the potential for oxidative metabolism and fatty acid P-oxidation, respectively, vastus lateralis citrate synthase (CS) and 3-hydroxyacyl-CoA dehydrogenase (HAD) activities were assayed. CS and HAD activities averaged 39.6 t 2.7 (30.2-59.3) and 39.5 t 1.7 (31.3-49.0) pmol . g dry wt-l. min-l, respectively. Preexperiment testing. Exercise was performed on a modified Krogh ergometer and limited to the knee extensors as described previously (2). Before performing the experiment, subjects attended three or four training sessions to become familiarized with dynamic knee extension exercise. Equal time was spent on each leg. In addition, on separate days the maximal work load the knee extensors could perform was determined. This maximum was gauged from a sudden steeper increase in whole body oxygen uptake and heart rate, which, until that time, had increased linearly with work load. The sudden steeper increase in oxygen uptake and heart rate is due to recruitment of additional muscle to stabilize the body. The maximal work load was identical for the two legs and averaged 48 (30-75) W. ExperimentaL protocol On the morning of an experiment, subjects reported to the laboratory at 8:00 A.M. after fasting overnight and abstaining from strenuous exert ise fo r 224 h. Catheters were introduced percutaneously into both femoral vei ns and one femoral artery and were advanced centrally so that the tips of the arterial and venous catheters were positioned ~2 cm proximal and distal to the inguinal ligament, respectively. Thermistors (Edslab TD probe 94-03002.5F) for blood flow measurements were inserted through both venous catheters and were advanced 6-8 cm proximal to the catheter tips. A catheter was also inserted into an antecubital vein for Intralipid infusion. The subjects then rested in the supine position for 45-60 min. After a further 5- to lo-min rest in the seated position, resting blood saPmples were obtained simultaneously from the femoral artery . and the femoral vein of the leg to be exe rcised fi.rst. A biopsy from th .e vastu s lateralis muscle of this 1,eg was then obtained with a Bergstrijm needle (including suction) under local anesthesia. Subjects then performed knee extension exercise for 1 h with one leg at a load requiring 80% of the maximal work load of the knee extensors. This work load was chosen so that carboh .ydrate and fat contribu ted abo lut equa lly to substrate oxidation as evidenced by RQ values (Table 1). Bet #ause of technical considerations (see DISCUSSION), the control exercise bout was always conducted first; however, the leg performing this exercise (left or right) was randomized. Immediately at the termination of exercise, another muscle biopsy was obtained. Recovery samples were obtained 10 min after completion of the first exercise bout. Then an infusion of a 20% triglycer-
METABOLISM
195
IN MUSCLE
TABLE 1. Physiological extension exercise
responses to knee Control
Heart rate, beats/min Pulmonary oxygen uptake, l/min Leg oxygen uptake, l/min RER Leg RQ Leg blood flow, l/min
105t6 0.93t0.06 0.62kO.06 0.85+0.01 0.87kO.02 4.75t0.31
In tralipid
104t5 0.92t0.05 0.59t0.06 0.82t0.01* 0.86~10.03 4.67t0.32
Values are means t SE; n = 11 subjects. Because values did not change systematically with time, values are means of measurements at 10, 20, 40, and 60 min of exercise, except for pulmonary oxygen uptake and RER, which are means of measurements at 20,40, and 60 min. Leg RQ was measured only at 40 min of exercise. * P c 0.05 from control.
ide emulsion (Intralipid, KabiVitrum, Stockholm) was started into the forearm venous catheter at a rate of 1.7 ml/min. This infusion was maintained through 30 min of rest and the second exercise bout with the contralatera1 leg. Subjects received an intravenous 1,000-U heparin bolus at the start of the infusion and a further 1,000 U over the 1 h of exercise. The second exercise bout was preceded by resting blood samples and a muscle biopsy as in the first bout. During blood sampling and femoral venous blood flow measurements, a cuff positioned just below the knee joint was inflated to 230 mm Hg. Analytic procedures. Blood was rapidly analyzed for glucose (YSI 23 AM glucose analyzer, Yellow Springs Instruments, Yellow Springs, OH) and hemoglobin and oxygen saturation (OSM 2 Hemoximeter, Radiometer, Copenhagen). A portion of the blood was deproteinized in 0.6 M perchloric acid for subsequent lactate, pyruvate, glycerol, ,&hydroxybutyrate, and citrate determination by enzymatic fluorometric techniques (6,22). Hematocrit was measured by use of a microcentrifuge. The remaining blood was quickly centrifuged, and the plasma was stored at -20 or -80°C for later analysis. Blood for FFA determination was sampled in a heparinized syringe and immediately transferred to an Eppendorf tube (1.5 ml) containing 30 ~1 of 0.2 M ethylene glycol-bis(P-aminoethyl ether)-N,N,N’,N’-tetraacetic acid. On high-speed centrifugation for 1 min, the plasma was frozen at -20°C. The efficacy of this procedure to prevent in vitro lipolysis of plasma triacylglycerols when subjects were given heparin was proven when this sampling method was compared with a method inactivating lipoprotein lipase by adding 0.2 ml 5 M NaCl to 0.8 ml plasma and heating the mixture for 30 min at 56OC (23). FFA were measured fluorometrically (32) as modified by Kiens (20). Triacylglycerols in plasma were measured without free glycerol (Boehringer Mannheim Diagnostica). Insulin was measured by radioimmunoassay (Novo, Copenhagen), and norepinephrine and epinephrine were measured by a radioenzymatic technique (8). Oxygen and carbon dioxide contents of whole blood were determined by the Van Slyke method (34). Expired gas was collected in Douglas bags for pulmonary oxygen uptake determination. Oxygen and carbon dioxide concentrations in the bags were measured by paramagnetic and infrared analyzers, respectively. Volume was ob-
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196
CARBOHYDRATE
AND LIPID
tained by use of a Tissot spirometer. Leg blood flow during exercise was measured by constant saline infusion thermodilution in the femoral vein (3); for measurements at rest and during recovery this method was modified (30). Muscle samples were rapidly (within lo-15 s) frozen in liquid nitrogen and stored at -8O*C for later biochemical analysis. Samples were freeze-dried and dissected free of visible connective tissue, fat, and blood. Glycogen was measured as glucose residues after hydrolysis of the muscle sample in 1 M HCl at 100°C for 2 h (22). Glucose, glucose 6-phosphate, and lactate were measured fluorometrically on neutralized perchloric acid extracts (22). Muscle CS and HAD activities were measured fluorometrically (11). Cakulations and statistics. Uptake or release of substrates, metabolites, and oxygen was calculated as arteriovenous differences times blood or plasma flow as appropriate. The data from the two exercise bouts were compared by two-way analysis of variance and Newman-Keuls post hoc testing. Resting and recovery data were compared by paired t tests. The significance for all comparisons was set at P < 0.05. RESULTS
The exercise work loads were identical for the two legs, averaging 38 (24-60) W; accordingly, there were no differences between trials in pulmonary and leg oxygen uptake, heart rate, or leg blood flow (Table 1). As a result of the Intralipid infusion, arterial FFA concentration during exercise was twofold higher than during control (Table 2), averaging 1.12 $- 0.09 and 0.54 t 0.08 mM, respectively, over the entire exercise periods. Despite this difference in arterial concentrations, net FFA uptake surprisingly was similar over the two exercise periods. However, as discussed in detail below, net FFA uptake may not accurately reflect FFA utilization because FFA is simultaneously taken up and released by the leg (16). Arterial concentrations of glycerol and ,&hydroxybutyrate were markedly increased by Intralipid infusion (Table 2). Glycerol release was similar during both exercise bouts (Table 3), whereas /3-hydroxybutyrate uptake was markedly higher when Intralipid was infused (Table 3). Still, in absolute terms P-hydroxybutyrate uptake was small. Concentrations of triacylglycerols in arterial plasma were roughly doubled by Intralipid infusion (Table 2), but during neither control exercise nor Intralipid infusion could an uptake of triacylglycerols across the thigh be detected (Table 3). Arterial blood glucose levels were similar during both exercise bouts except for small but significant differences at rest and during the first 20 min of exercise (Table 2, Fig. 1). Resting muscle glucose uptake was reduced, on average, 64% when arterial FFA were elevated. Glucose uptake during exercise was decreased by 33% (Table 3, Fig. l), averaging 0.82 t 0.13 mmol/min during control and 0.55 t 0.12 mmol/min during Intralipid infusion. After 10 min of recovery, the decrease in glucose uptake during Intralipid infusion averaged 42%. These decreases in glucose uptake were not associated
METABOLISM
IN MUSCLE
with accumulation of glucose or glucose 6-phosphate in muscle at rest or immediately after exercise (Table 4). This speaks against the possibility that the decrease in glucose uptake was caused by glucose 6-phosphate-induced inhibition of glucose phosphorylation. Arterial lactate concentrations were slightly lower at 10 and 20 min in the FFA trial compared with the control trial (Table 2). No differences between trials were seen in arteri .a1 levels of pyruvate or rele ase of lactate and pyr luvate from the leg before, dur ling, or after exercise (Tables 2 and 3). Similarly, muscle lactate both at rest and immediately after exercise was not changed by infusion of Intralipid. Muscle glycogen was similar in the two legs before exercise (Fig. 2). The amount of glycogen degraded during exercise was similar under both conditions, averaging 249 t 38 mmol/kg dry muscle during the control exercise bout and 226 t 30 mmol/kg dry muscle during exercise with Intralipid infusion. Arterial citrate was higher at rest and after 10 and 20 min of exercise when FFA were elevated (Table 2), but leg citrate release was similar in both conditions (Table 3). No differences were observed between conditions in the arterial concentrations of insulin, norepinephrine, and epinephrine (Table 5). After 40 min of exercise, the respiratory exchange ratio (RER) was significantly lower during Intralipid infusion than during control but was similar in both conditions at the 200 and 600min time points. However, when the values at 20, 40, and 60 min were averaged, RER was slightly lower (P < 0.05) during the Intralipid exercise bout than during control exercise (Table 1). No significant difference between conditions was seen in RQ measured across the leg after 40 min of exercise (Table 1). DISCUSSION
The present study has demonstrated that infusion of Intralipid and heparin resulting in a physiological elevation of plasma FFA is associated with an inhibition of thigh glucose uptake at rest, during exercise, and during recovery from exercise. In contrast, there was no measurable effect of Intralipid infusion on muscle glycogen breakdown or lactate, pyruvate, and citrate release or on leg RQ during exercise. These observations were made in a group of subjects with a fairly wide range of maximal aerobic capacities and muscle oxidative enzyme activities. No correlation was detected between muscle CS or HAD activity and the degree of inhibition of muscle glucose uptake with increased plasma FFA, indicating that the effect was not dependent on the level of enzyme activity involved in P-oxidation or the tricarboxylic acid cycle. The knee-extension model enables us to study “isolated” working muscle without major changes in the internal body milieu because the mass of working muscle is relatively small. As such, it offered the possibility to examine substrate metabolism during exercise under two conditions that ideally differed only in the arterial concentrations of plasma FFA. In reality, Intralipid infusion and heparin administration also produced large
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CARBOHYDRATE TABLE
2. Arterial
concentrations
AND LIPID
METABOLISM
197
IN MUSCLE
of substrates in blood and plasma Exercise
Preexercise
Glucose, mM Control Intralipid Lactate, mM Control Intralipid Pyruvate, PM Control Intralipid Free fatty acids, mM Control Intralipid Glycerol, PM Control Intralipid @-Hydroxybutyrate, PM Control Intralipid Citrate, PM Control Intralipid Triacylglycerol, mM Control Intralipid
10 min
20 min
40 min
60 min
Recovery
4.74t0.10 4.54tO.15*
4.72t0.15 4.48t0.13*
4.61t0.11 4.48t0.13”
4.43-+0.10 4.38tOJ3
4.44tO.11 4.34tO.14
4.50t0.13 4.48t0.14
0.54kO.05 0.5WO.05
1.65t0.21 1.37-t-0.17*
1.65-t-0.24 1.37&0.22*
1.33-t-0.20 1.29-t-0.28
1.08t0.18 1.01-1-0.21
0.72t0.09 0.74t0.11
17-c4 16_+2
22t6 22t3
26t4 24t3
2123 2224
25t3 21t4
2124 23t4
0.60t0.07 0.92t0.07”
0.48t0.06 0.91t0.06*
0.50t0.07 1.03t0.07*
0.55t0.09 1.22t0.09*
0.65t0.11 1.40t0.12”
0.71~0.10 1.44-t-0.12*
53t,5 168t8*
56t5 205t7*
64t7 224t8”
72t9 241t12*
92zk12 257,tl4*
59t9 230+17*
65t14 192t37*
45-+11 176-t39*
35tlO 165-t38*
38+14 185t40”
46tl8 215t39”
99+29 308+51*
4922 62+4*
53t2 65t3*
56-t-3 66+3*
6325 68+3
64t4 68~3
63*4 67?4
NM NM
NM NM
0.54kO.04 0.93+0.09*
0.53t0.05 1.06t0.11*
0.54t0.05 l.l9cO*12*
0.52+0.05 1.18t0.12*
Values are means _+SE; n = 11 subjects except n = 8 for triacylglycerol. NM, not measured. Glucose, lactate, pyruvate, glycerol, ,&hydroxybutyrate, and citrate concentrations were measured in blood; FFA and triacylglycerol concentrations were measured in plasma. * P < 0.05 from control.
TABLE
3. Leg exchange of substrates Exercise Preexercise
Glucose uptake, mmol/min Control Intralipid Lactate release, mmol/min Control Intralipid Pyruvate release, pmol/min Control Intralipid FFA uptake, mmol/min Control Intralipid Glycerol release, pmol/min Control Intralipid ,&Hydroxybutyrate uptake, pmol/min Control In tralipid Citrate release, pmol/min Control Intrahpid Triacylglycerol uptake, pmol/min Control Intralipid
10 min
20 min
40 min
60 min
Recovery
0.11&0.02 0.04~0.01*
0.43+0.19 0.23t0.11
0.69t0.10 0.34_+0.09*
0.87t0.12 0.53t0.10*
1.27t0.16 0.92t0.17*
0.45t0.08 0.26t0.05*
0.04t0.01 0.06t0.01
2.78kO.50 2.32t0.50
1.90t0.30 2.11kO.52
1.22t0.36 1.06kO.36
0.80t0.23 0.7320.23
0.10t0.01 0.08t0.02
176*43 151t26
147+28 129zt25
142t22 102t23
86t22 98t25
2t2 lt2
0.07+0.04 0*04t0.04
0.09+_0.04 O.ll,tO.O5
0.15t0.05 0.12t0.02
0.14-40.04 0.16,tO.O4
0.01 to.01 -0.03_tO.O2
39+9 4tlO
35t_9 33t22
21211 33t9
16st3 22t8
14t12 89t15*
1825 36t9*
Ot,l Otl -0.01~0.01 -0.02t0.01 IO+3 -8t_3
36t9 -22tl5
10*3 29t6*
12t6 36t26*
-324 40*14*
8*6 76+20*
5.0t0.7 7.1t0.5
8.9k6.0 6.6t3.5
4.5k5.4 6.5t3.6
-1.8k4.5 0.6t7.0
-0.7t7.3 -3.6t4.3
9.1t1.9 8.4kl.6
NM NM
NM NM
45-t46 -236+110
-16tl6 60t104
-8t9 -87t119
-1lt19 76t135
Values are means t SE; n = 11 subjects except n = 8 for triacylglycerol
increases in the arterial concentrations of glycerol, ketones, and plasma triacylglycerols. Glycerol has no effeet on glucose utilization in the resting state (12), and
uptake. NM, not measured. * P < 0.05 from control.
leg glucose uptake during exercise is unaffected by heparin (1). The possibility that other components of Intralipid (e.g., phospholipid) influence exercise metabolism is
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CARBOHYDRATE
AND LIPID
METABOLISM
IN MUSCLE
FIG. 1. Arterial concentrations of glucose and glucose uptake before, during, and after I h of l-legged knee extensions. Subjects exercised with 1 leg during control conditions and with the other leg during infusion of Intralipid. *‘P < 0 .05 compared with values during control conditions.
0
20
60
LO lime
(min)
difficult to assess but cannot be excluded. Nevertheless, arterial plasma insulin, catecholamines, preexercise muscle glycogen levels, and, at most time points, arterial glucose concentrations were similar in the control and Intralipid conditions. In this respect, the knee-extension model provides advantages over previous methods (e.g., starvation, fat feeding, prolonged exercise) that have been used to examine the influence of plasma FFA on carbohydrate metabolism during exercise. Such methods usually produce a number of hormonal and metabolic changes that make investigation of the direct effect of elevated FFA difficult. It was technically impossible to conduct the control and the Intralipid trial in a randomized order because it
4. Muscle metabolites after exercise
TABLE
80
is impossible to achieve low FFA concentrations after Intralipid infusion without administering agents blocking lipolysis, such as nicotinic acid and insulin, and these substances might have physiological effects unre-
before and immediately
Total glucose, mmol/kg dry wt Control Intralipid Glucose 6-phosphate, mmol/kg dry wt Control Intralipid Lactate, mmol/kg dry wt Control Intralipid Values are means t SE; n = 11 subjects.
Rest
Exercise
1.39kO.21 l/74+0.31
2.98t0.48 2.75t0.21
0.27t0.03
0.29+0.04
0.28-tO.05
0.26t0.05
7.0tl.4
13.8-1-3.5
9.022.3
ll.O_t1.9
Pfe Control
Pte Post Inttalipid
2. Concentrations of glycogen in vastus lateralis muscle before and after 1 h of l-legged knee extensions during control conditions and during infusion of Intralir>id. * dw.I”drs weight. Y FIG.
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CARBOHYDRATE TABLE
5. Arterial
plasma conentrations
AND LIPID
of hormones
Exercise
Insulin, pU/ml Control Intralipid Norepinephrine, rig/ml Control Intralipid Epinephrine, rig/ml Control Intralipid
Rest
20 min
60 min
Recovery
9.3tU.8 8.9t0.8
9.0t0.7 8.1kO.9
7.4t0.8 7.6t0.8
9.1t1.2 9.1t0.9
0.38t0.07 0.36t0.07
0.54t0.12 0.47t0.08
0.45t0.11 0.51~0.09
0.40t0.06 0.25t0.08
0.19t0.03 0.20+0.03
0.22tO.03 0.24+_0.03
0.30t0.03 0.27t0.04
0.20t0.03 0.20t0.04
Values are means t SE; n = 11 subjects for insulin, n = 8 for catecholamines.
lated to their ability to lower FFA. Therefore the only alternative to the present design is to do the two trials on separate days. However, this would mean day-to-day variation in substrate stores and hormone concentrations in the blood and thus make detection of small differences impossible. Although we cannot exclude an order effect in the present study, it is unlikely to be significant because preexercise substrate stores and plasma concentrations of insulin, epinephrine, and norepinephrine during knee extensions were identical in the two trials. Thus a biochemical mechanism responsible for a possible order effect independent of the effects of Intralipid infusion is not apparent. Resting muscle glucose uptake was reduced 64% when plasma FFA were elevated. Studies in rat muscle have produced confiicting results that demonstrate either no effect of FFA on muscle glucose uptake (5, 27, 31) or reduced glucose utilization (19,28) and, in fact, also increased glucose utilization at very high FFA levels (19). In resting human subjects, increases in plasma FFA levels have been shown to decrease whole body glucose utilization (4, 12), although a recent study found no effect of Intralipid and heparin infusion on glucose uptake and oxidation during hyperglycemic hyperinsulinemia (35). In the present study, muscle glucose uptake during exercise was, on average, 33% lower when plasma FFA were increased (Fig. 1). The mechanism underlying the decrease in muscle glucose uptake observed in the present study is not readily apparent. According to the original hypothesis (24), an increase in fat oxidation in muscle, as a consequence of higher plasma FFA levels, results in citrate-mediated inhibition of phosphofructokinase activity and accumulation of glucose 6phosphate and glucose, thereby inhibiting glucose uptake. Furthermore, carbohydrate oxidation may be reduced because pyruvate dehydrogenase activity may be diminished by increased oxidation of FFA and ketones (15, 25). In the rat hindlimb, addition of FFA to the perfusion medium has been shown to increase muscle citrate and free glucose levels during contractile activity in one study (28) but not in another (31). Muscle citrate was not measured in the present study, but arterial citrate levels were higher only at rest and
METABOLISM
IN MUSCLE
199
during the first 20 min of exercise with elevated FFA. This may simply reflect a time effect, however, because leg citrate release was similar during both exercise bouts (Table 3). Furthermore, muscle glucose and glucose 6-phosphate levels were similar in the two trials (Table 4), suggesting that the inhibition of glucose uptake by Intralipid was not due to accumulation of glucose within the cell but rather due to a direct effect of FFA on glucose transport. This is in accordance with Randle et al. (25), who demonstrated that L-arabinose transport in perfused rat heart was inhibited by FFA and ketones. However, the possibility exists that increases of glucose in a small compartment of the muscle may be physiologically important but impossible to detect in a tissue homogenate. Intralipid consists mainly of soybean oil, which upon hydrolysis liberates mainly linoleic acid (l&2) and oleic acid (l&l). It cannot be excluded that different results might have been obtained had the increase in FFA concentration been with saturated fatty acids. For instance, direct membrane effects are conceivably different with different fatty acids. Furthermore it is not inconceivable that the elevated plasma triacylglycerols during Intralipid infusion might directly affect glucose transport. Studies in exercising humans have shown an inverse relationship between plasma FFA and the rate of muscle glycogen utilization (7,9), although in a more recent study there was no effect of increased FFA on carbohydrate oxidation during prolonged moderate-intensity exercise (26). In exercising rats, muscle glycogen breakdown during exercise was decreased in the presence of increased FFA (10, 17, 29). In the present study there was no effect of increased FFA on muscle glycogen breakdown during exercise (Fig. 2). Thus the major effect of FFA on carbohydrate metabolism may be on glucose uptake rather than on glycogen metabolism. However, in the present study biopsies were obtained only before and after exercise, and it cannot be excluded that differences in glycogen breakdown rate might have been found at other time points. It is generally believed that the rate of FFA uptake by muscle during exercise is directly related to the arterial concentration or inflow of FFA (13); however, a doubling of the arterial FFA concentration in the present study had no measurable effect on net FFA uptake during exercise. Part of the explanation for this difference may lie in technical aspects of the study. Because FFA are simultaneously released and taken up by the exercising leg (14, 16), FFA arteriovenous differences are small, and hence it is difficult to detect small changes in FFA uptake without isotopically labeled FFA. In addition, it is likely that some of the infused triacylglycerol was hydrolyzed as it passed through exercising muscle, resulting in a higher femoral release of FFA than in control exercise. This would decrease the arteriovenous concentration difference of FFA (if the FFA were not taken up by the muscle) and hence the net uptake. If this were the case, however, one might also expect an increase in apparent glycerol release and an apparent uptake of triacylglycerols during Intralipid infusion, and this was not observed. In any case, in this particular
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200
CARBOHYDRATE
AND LIPID
setting of Intralipid infusion, net FFA uptake may be a poor indicator of FFA uptake. An alternative explanation is that the capacity of the muscle to take up FFA was fully taxed already during control exercise. Recent in vitro studies in cardiac myocytes (33) and in perfused rat muscle (L. P. Turcotte, B. Kiens, and E. A. Richter, unpublished observations) have demonstrated that FFA uptake is a saturable process. If this is also the case in contracting skeletal muscle, it might explain why a doubling of the arterial FFA concentration did not measurably increase net FFA uptake, because FFA uptake may have been already close to maximal in the control situation. Finally, it cannot be excluded that different results might have been obtained at a lower work load. However, the present load was moderate because it could easily be sustained for 1 h and because fat and carbohydrate combustion contributed about equally to substrate oxidation. It remains to be explained that Intralipid caused a 33% reduction in glucose uptake during exercise but did not change leg RQ. Because glycogen breakdown was not changed by Intralipid, a compensatory increase in oxidation of other fuels must have occurred. The source of such fuels remains unknown, however. In any event, in terms of energy delivery, the reduction in glucose uptake amounts to only 6% of energy consumption, which, if compensated for by lipid combustion, would decrease the RQ across the leg by 0.02. Thus the failure to see a significant reduction in RQ across the working leg may be due to the fact that the expected decrease in RQ is too small to detect. In fact, pulmonary RER was slightly decreased when averaged over the entire exercise period (Table I), supporting a very small shift in fuel combustion, but RER represents metabolism in the entire body; it is likely that metabolism in organs other than contracting leg muscle, e.g., in the heart, was changed by Intralipid. This might explain the lower RER but unchanged leg RQ during Intralipid infusion. In summary, the results of the present study demonstrate that, at rest and during moderate-intensity exercise, muscle carbohydrate metabolism is affected only with regard to uptake of glucose when plasma concentrations of lipid and lipid metabolites are increased. It is proposed that this effect may be by direct inhibition of glucose transport rather than by the classical glucosefatty acid cycle. The authors thank Betina Bolmgreen, Ingelise Kring, Hanne Sjoholm, Hanne Thomsen, and Merete Vanby for excellent technical assistance. This study was supported by the Danish Medical Research Council, Fonden af 1870, and the Danish Diabetes Association. M. Hargreaves was supported by the Danish Ministry of Education and the National Health and Medical Research Council of Australia. Present address of M. Hargreaves: Footscray Institute of Technology, Footscray, Victoria 3011, Australia. Address for reprint requests: E. A. Richter, August Krogh Institute, University of Copenhagen, 13 Universitetsparken, DK-2100 Copenhagen 0, Denmark. Received 29 January 1990; accepted in final form 8 August 1990. REFERENCES 1. AHLBORG, G., AND L. HACENFELDT. Effect of heparin on the substrate utilization during prolonged exercise. &and. J. C&n. Lab. Invest. 37: 619-624, 1977.
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IN
MUSCLE
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