INTRODUCTION

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CIRCULATING ALBUMIN-BOUND fatty acids are a major oxidizable fuel source for skeletal muscle metabolism. The metabolism of albumin-bound ligands, such as long-chain fatty acids, is a complex process that involves many steps, one of which is the permeation of free fatty acids (FFA) across the cell membranes and interstitium. Because of their lipid nature, FFA flux across plasma membranes has long been considered a passive diffusional process. This notion was reinforced by the existence of a linear relationship between total plasma FFA concentration and FFA utilization in humans and dogs at rest and during exercise (4, 13). However, >99% of the FFA carried in plasma are bound to albumin, and, according to the conventional theory of cellular uptake for protein-bound ligand, only unbound ligand participates in the uptake process (5). Within the past decade, experimental evidence has emerged to show that FFA uptake across the plasma membranes of cells with high FFA fluxes is indeed dependent on the concentration of unbound plasma FFA and that the initial uptake rate of FFA saturates with an increase in the unbound plasma FFA concentration (2, 3, 15, 19, 22, 24). In perfused rat skeletal muscle at rest, we have shown that the uptake of palmitate saturates with an increase in unbound perfusate plasma palmitate concentration (27). The existence of a saturable relationship between the cellular uptake of plasma FFA and the concentration of unbound plasma FFA suggests that FFA uptake across plasma membranes of muscle cells may also be mediated in part by a facilitated transport system (15).

At a given plasma FFA concentration, the rate of FFA utilization is dependent on the metabolic rate (7, 10). In humans, muscle contractions have been shown to increase plasma FFA uptake and utilization at a specific plasma FFA concentration (7, 10, 14, 28). In perfused rat hindquarters, plasma palmitate uptake at a plasma palmitate concentration of 2,000 µmol/l was found to be 55% higher in contracting skeletal muscles compared with muscles at rest (26). However, it is not known whether during muscle contractions plasma FFA uptake and oxidation would still follow saturation kinetics and, if so, whether the increases in plasma FFA uptake and oxidation would be associated with an increase in maximal velocity (V_max) or a decrease in Michaelis-Menten constant (K_m) or both.

Therefore, the purpose of this study was to determine the kinetics of the uptake and oxidation of plasma palmitate in the perfused isolated rat hindquarter at rest and during electrically induced muscle contractions. The perfused rat hindquarter was used to provide an intact physiological in vitro muscle preparation.

	MATERIALS AND METHODS

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Animal preparation. Male Wistar rats were housed in pairs, maintained on a 12:12-h light-dark cycle, and received regular rat chow and water ad libitum. On the day of the experiment, fed rats weighing between 250 and 300 g were anesthetized with pentobarbital sodium (5 mg/100 g body wt ip) and prepared surgically for hindquarter perfusion as previously described (18). Before insertion of the perfusion catheters, heparin (150 IU) was administered into the inferior vena cava. The rats were killed with an intracardial injection of pentobarbital sodium immediately before the catheters were inserted, and the preparation was placed in a perfusion apparatus, essentially as described by Ruderman et al. (18).

Experimental procedures. The initial perfusate (300 ml) consisted of Krebs-Henseleit solution, 1- to 3-day-old washed bovine erythrocytes (hematocrit, 30%), 5% bovine serum albumin (Cohn fraction V, Sigma Chemical, St. Louis, MO), 6 mmol/l glucose, varying amounts of albumin-bound palmitate (final perfusate concentration = 200-2,200 µmol/l), and 1 µCi of albumin-bound [1-¹⁴C]palmitate (New England Nuclear, Boston, MA). No insulin was added. The perfusate (37°C) was continuously gassed with a mixture of 35% O₂-3% CO₂ in N₂, which yielded an arterial pH value of 7.3-7.4 and an arterial PCO₂ and PO₂ of typically 30-35 Torr and 150-200 Torr, respectively. Mean perfusion pressures were 72 ± 2 and 104 ± 13 mmHg during unilateral hindquarter perfusion at rest and during electrical stimulation, respectively.

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Blood and muscle sample analyses. Arterial and venous perfusate samples were analyzed for glucose, lactate, and FFA concentrations as well as for [¹⁴C]FFA and ¹⁴CO₂ radioactivities. Samples for glucose and lactate were kept on ice and analyzed by using YSI 23 glucose and lactate analyzers (Yellow Springs Instruments, Yellow Springs, OH) within 30 min of collection. Samples for FFA were put in 200 µmol/l of EGTA (pH 7) and centrifuged, and the supernatant was frozen until analyzed fluorometrically (14). Because the FFA concentration was low in the absence of added palmitate (<90 µmol/l) and because palmitate was the only fatty acid added, measured FFA concentrations were taken to equal palmitate concentrations.

Calculations and statistics. The equilibrium concentration of unbound palmitate was calculated by the stepwise equilibrium constant method (31) by using the dissociation constants for the palmitate-albumin complex reported by both Spector et al. (23) and Richieri et al. (16). The fractional uptake was calculated as the difference in radioactivity between the arterial and venous perfusate samples divided by the radioactivity in the arterial sample (7). Palmitate delivery was calculated by multiplying perfusate plasma flow by the arterial perfusate plasma palmitate concentration. Palmitate uptake was calculated by multiplying plasma delivery by the fractional uptake (7). Percent palmitate oxidation was calculated by dividing the total amount of radioactivity recovered as ¹⁴CO₂ by the total amount of radioactivity that was taken up by the muscles (11). Total palmitate oxidation was calculated by multiplying palmitate uptake by the percent oxidation. Glucose and oxygen uptake as well as lactate release across the hindquarter were calculated by multiplying perfusate flow by the arteriovenous difference in concentration and were expressed per gram of perfused muscle, which was measured to be 7.8 ± 0.4% of body weight for unilateral hindquarter perfusion.

	RESULTS

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Palmitate metabolism. When plotted as a function of the unbound perfusate plasma palmitate concentration, calculated by using the association constants of Richieri et al. (16), the rates of palmitate uptake and palmitate oxidation displayed saturation kinetics and could be fitted to a Michaelis-Menten relationship both at rest and during electrical stimulation (Fig. 1). Analysis of the data yielded V_max and K_m estimates of 42.8 ± 3.8 nmol · min¹ · g¹ and 13.4 ± 3.4 nmol/l for palmitate uptake and of 3.8 ± 0.4 nmol · min¹ · g¹ and 8.1 ± 2.9 nmol/l for palmitate oxidation, respectively, at rest. The V_max for palmitate uptake and oxidation increased significantly during electrical stimulation to reach values of 91.6 ± 10.1 and 16.5 ± 2.3 nmol · min¹ · g¹, respectively. The K_m values for palmitate uptake and oxidation were unchanged by electrical stimulation. Hanes-Woolf plots, [S]/v vs. [S], generated straight lines with the slope equal to 1/V_max, the x-intercept equal to K_m, and the y-intercept equal to K_m/V_max (Fig. 2). The Hanes-Woolf plots for both palmitate uptake and oxidation demonstrated significantly different slopes between rest and electrical stimulation, whereas the x-intercepts remained unchanged. Thus, by both methods of estimation, electrical stimulation significantly increased the V_max for both palmitate uptake (110-115%) and oxidation (330-390%) but did not change the K_m value. When plotted as a function of the total plasma palmitate concentration, the rates of palmitate uptake and oxidation displayed straight-line relationships (Fig. 3). Electrical stimulation was associated with an increase in the slopes of the lines.

View larger version (16K): [in this window] [in a new window]   Fig. 1.   Palmitate uptake (B) and oxidation (A) in perfused rat hindquarters at rest and during electrical stimulation as a function of unbound perfusate plasma palmitate concentration calculated by using association constants provided by Richieri et al. (16). Each point represents average uptake or oxidation value of 1 rat. Data points (n = 23) were fitted to a rectangular hyperbolic function and analyzed by PRISM program (GraphPad, San Diego, CA). R2 = 0.88 and 0.92 for palmitate uptake during rest and electrical stimulation, and r2 = 0.68 and 0.89 for palmitate oxidation during rest and electrical stimulation, respectively.

View larger version (19K): [in this window] [in a new window]   Fig. 2.   Hanes-Woolf plots, substrate concentration ([S])/velocity vs. [S], showing saturation kinetics of palmitate uptake (B) and oxidation (A) in perfused rat hindquarters at rest (y = 0.02x + 0.34, r2 = 0.85 for palmitate uptake and y = 0.27x + 2.45, r2 = 0.73 for palmitate oxidation) and during electrical stimulation (y = 0.01x + 0.29, r2 = 0.81 for palmitate uptake and y = 0.05x + 1.81, r2 = 0.68, for palmitate oxidation). Slope is 1/Vmax, y-intercept is Km/Vmax, and x-intercept is Km, where Vmax and Km are maximal velocity and Michaelis-Menten constant, respectively. Each point represents average value of 1 rat. Data points (n = 23) were fitted to a straight line and analyzed by linear regression.

View larger version (17K): [in this window] [in a new window]   Fig. 3.   Palmitate uptake (B) and oxidation (A) in perfused rat hindquarters as a function of total palmitate concentration at rest (y = 0.014x + 4.6, r2 = 0.88 for palmitate uptake and y = 0.001x + 0.8, r2 = 0.66 for palmitate oxidation) and during electrical stimulation (y = 0.028x + 1.8, r2 = 0.92 for palmitate uptake and y = 0.005x + 0.2, r2 = 0.89 for palmitate oxidation). Data points (n = 23) were fitted to a straight line and analyzed by linear regression.

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Effect of flow rate on palmitate metabolism. To evaluate the effect of flow rate on palmitate metabolism, perfusate flow rate was increased from 9 to 15 ml/min during unilateral perfusion of the resting hindquarter at high perfusate plasma palmitate concentration (Table 1). Perfusate plasma palmitate concentration was similar in both groups and averaged 1,712 ± 57 µmol/l. The increase in perfusate flow rate from 9 to 15 ml/min was associated with a 53% increase in the rate of plasma palmitate delivery to the hindquarter and a 37% decrease in the fractional uptake rate of palmitate. Because of the corresponding decrease in the fractional uptake of palmitate, the total rate of uptake of palmitate was not changed by the increase in perfusate flow rate (P > 0.05). Similarly, the percentage and total amount of palmitate oxidized were not affected by the increase in perfusate flow rate (P > 0.05). Glucose uptake was 38% higher (P < 0.05) at the higher flow rate, whereas both lactate release and oxygen uptake remained unchanged by the increase in perfusate flow rate.

	DISCUSSION

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Our results show that in intact perfused skeletal muscle the rates of uptake and oxidation of palmitate display saturation kinetics when plotted against the unbound perfusate plasma palmitate concentration and that muscle contractions increase the V_max of the uptake and oxidation of palmitate, whereas the K_m value remains unchanged. These results are consistent with the notion that the uptake of long-chain FFA across the plasma membranes of cells with high FFA fluxes is mediated in part by a saturable membrane-associated transport mechanism (3, 15, 19). These results further suggest that the increase in the rate of FFA uptake induced by muscle contractions may be modulated in part by changes in the transport of FFA across the plasma membranes.

In isolated cell systems, the uptake of long-chain fatty acids has been shown to exhibit many of the kinetic properties of a facilitated transport process (2, 3, 21, 22, 24). Thus the initial uptake rate of long-chain fatty acids into these cells is rapid, saturable, and reduced by prior protease treatment of the cells (2, 3, 24). Although several proteins have been proposed as candidate long-chain fatty acid transporters (1, 8, 9, 15, 19), the plasma membrane fatty acid-binding protein (FABP_PM), first isolated from liver tissue, has been well characterized and has been shown to exist in all cell types studied to date (21, 22, 24). FABP_PM has a molecular mass between 40 and 43 kDa, possesses a high affinity for long-chain FFA, and is structurally and immunologically distinct from the 12- to 14-kDa cytoplasmic fatty acid-binding proteins described in many tissues (15). In each cell type studied to date, antibodies raised against the rat hepatic FABP_PM selectively inhibit both FFA binding to plasma membranes and cellular flux of FFA in a dose-dependent fashion (21, 22, 24), suggesting that FABP_PM may constitute a functional FFA transporter and be part of the structural basis behind the apparent saturation type of kinetics for FFA uptake. Other proteins have been proposed as possible candidates for the transporter in adipocytes (1, 8, 9, 19). The fatty acid translocase (FAT) was first identified in rat adipocytes by labeling plasma membrane proteins with nonpermeable sulfo-N-succinimidyl derivatives of fatty acids and measuring the initial rate of FFA uptake (8, 9). By using an expression cloning strategy and a cDNA library from 3T3-L1 adipocytes, another plasma membrane-associated protein was independently identified and called the fatty acid transport protein, FATP (19). The expression of this protein in cultured cells was found to be related to the rate of long-chain fatty acid transport (19). Skeletal muscle has been shown to express both the FATP and FAT protein (19, 30). Thus it would appear that a family of fatty acid transporters may exist.

With the physiological system used in these studies, it is not known where in the steps from the dissociation of palmitate from albumin to cellular oxidative metabolism the saturation occurs. It may be argued that the saturating effect observed in our experiments reflects saturation of an intracellular metabolic step rather than plasma membrane transport, which, in other words, indicates that metabolism rather than plasma membrane transport of FFA is rate limiting for FFA utilization. An alternative explanation might be that sarcolemmal transport is rate limiting for FFA utilization. In resting dog muscle it has recently been shown that the intracellular concentration of FFA is extremely low and that a steep gradient exists between blood and the intracellular space (29). This finding supports the notion that sarcolemmal transport is rate limiting for FFA utilization. If so, it follows that FFA utilization can only increase during muscle contractions if FFA transport is increased compared with rest. More definitive studies using antibodies against the different putative FFA transporters and sarcolemmal membrane preparations that can more directly measure FFA transport rates are needed to answer the question of whether sarcolemmal FFA transport capacity is increased with muscle contractions and, if so, by what mechanism.

Because perfusate plasma palmitate concentration was maintained at a constant level from rest to electrical stimulation, the increase in perfusion flow rate with electrical stimulation was accompanied by an increase in the delivery of plasma palmitate to the rat hindquarter. To ensure that the measured contraction-induced increase in palmitate uptake was not due to the increases in perfusate plasma flow and plasma delivery, we perfused resting hindquarters at a rate of 15 ml/min, the same rate used for the electrical stimulation protocol. In agreement with earlier observations (26), the uptake and oxidation of palmitate in resting perfused skeletal muscle were not affected by the increase in perfusate flow rate. Thus the contraction-induced increase in palmitate uptake was not due to an increase in plasma palmitate delivery. In contrast to palmitate and in agreement with previous results (12, 20), glucose uptake was found to be dependent on glucose delivery. With this muscle preparation, the increase in glucose uptake that was attributable to the increase in perfusate flow rate was calculated to be ~24%, a value that is close to the 30% estimate calculated previously (12).

There has been some controversy regarding the use of the FFA-albumin association constants determined by Spector et al. (23). Indeed, it has been suggested that the association constants may be too low, resulting in calculated unbound FFA concentrations that are correspondingly high (17). We estimated the unbound FFA concentration by using the association constants provided by both Richieri et al. (16) and Spector et al. (23) and compared the resulting calculated V_max and K_m values. Although the absolute values of these estimates were slightly different, the shape of the curves relating unbound palmitate concentration to palmitate uptake and oxidation and the contraction-induced changes in these estimates were similar. Accordingly, whereas the quantitative estimates may be different, this does not affect the more important qualitative assessment of the effects of muscle contractions on FFA uptake and oxidation.

In summary, the present study has shown that, when plotted against the unbound plasma palmitate concentration, palmitate uptake and oxidation by resting and contracting perfused skeletal muscle display saturation kinetics. Muscle contractions, but not an increase in plasma FFA delivery, increase palmitate uptake and oxidation by an increase in V_max without changing the K_m value. These results are consistent with the traditional theory of cellular uptake for protein-bound ligand and with the hypothesis that palmitate uptake across the plasma membranes of cells with high FFA fluxes is mediated in part by a transport system.