Am J Physiol Endocrinol Metab 294: E551-E557, 2008.
First published December 18, 2007; doi:10.1152/ajpendo.00467.2007
0193-1849/08 $8.00
Acute responses of muscle protein metabolism to reduced blood flow reflect metabolic priorities for homeostasis
Xiao-jun Zhang,1,2
Oivind Irtun,1,2
David L. Chinkes,1,2 and
Robert R. Wolfe1,2
1Metabolism Unit, Shriners Hospital for Children and 2Department of Surgery, The University of Texas Medical Branch, Galveston, Texas
Submitted 19 July 2007
; accepted in final form 12 December 2007
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ABSTRACT
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The present experiment was designed to measure the synthetic and breakdown rates of muscle protein in the hindlimb of rabbits with or without clamping the femoral artery. L-[ring-13C6]phenylalanine was infused as a tracer for measurement of muscle protein kinetics by means of an arteriovenous model, tracer incorporation, and tracee release methods. The ultrasonic flowmeter, dye dilution, and microsphere methods were used to determine the flow rates in the femoral artery, in the leg, and in muscle capillary, respectively. The femoral artery flow accounted for 65% of leg flow. A 50% reduction in the femoral artery flow reduced leg flow by 28% and nutritive flow by 26%, which did not change protein synthetic or breakdown rate in leg muscle. Full clamp of the femoral artery reduced leg flow by 42% and nutritive flow by 59%, which decreased (P < 0.05) both the fractional synthetic rate from 0.19 ± 0.05 to 0.14 ± 0.03%/day and fractional breakdown rate from 0.28 ± 0.07 to 0.23 ± 0.09%/day of muscle protein. Neither the partial nor full clamp reduced (P = 0.27–0.39) the intracellular phenylalanine concentration or net protein balance in leg muscle. We conclude that the flow threshold to cause a fall of protein turnover rate in leg muscle was a reduction of 30–40% of the leg flow. The acute responses of muscle protein kinetics to the reductions in blood flow reflected the metabolic priorities to maintain muscle homeostasis. These findings cannot be extrapolated to more chronic conditions without experimental validation.
stable isotope; gas chromatograph-mass spectrometer; arteriovenous balance; fractional synthetic rate; fractional breakdown rate
PROTEIN KINETICS in the skeletal muscle have been studied intensively under various conditions, including fasting, spaceflight, exercise, and catabolic illness (5, 8, 18). The importance of muscle protein metabolism is not only because of its contractile function but also because muscle is the largest mobilizable source of protein. Muscle catabolism provides free amino acids under catabolic states, thereby supporting the continual synthesis of essential proteins in other parts of the body (9, 10, 19, 21). The synthesis of muscle protein, as well as breakdown, depends on blood flow, since the metabolic precursors and products have to be transported into, and removed from, the muscle through the blood stream.
The muscle is proposed to have two vascular flow routes: nutritive and nonnutritive (4). Nutritive flow is able to exchange nutrients and hormones with the skeletal muscle myocytes. Although the nonnutritive flow does not have direct nutritional function, it carries a flow reserve and redistributes to nutrition routes in response to increased metabolic demand. It has been demonstrated that increased rates of muscle protein synthesis are often accompanied with higher flow rates, such as in the case of exercise and insulin administration (7). However, the effect of reduced flow below the normal basal rate on muscle protein metabolism is less clear. Short et al. (16) found that a reduction in leg blood flow by 25% by administration of prednisone did not change either the synthetic rate or net balance of muscle protein in healthy young people. Killewich et al. (11) reported that, despite reduction in leg blood flow by 40% in elderly peripheral arterial disease patients, the synthetic rate of calf muscle protein was similar to that in healthy elderly people. Whereas these studies suggest that the skeletal muscle could compensate for a certain magnitude of reduction in blood flow without impairing its protein synthesis, the concomitant factors (i.e., prednisone and peripheral arterial disease) could have complicated the muscle responses to reductions in flow. Thus there is limited information with respect to the role of blood flow as an independent factor in regulating muscle protein metabolism.
The present experiment was therefore designed to investigate the relation between reduction in flow and muscle protein metabolism. We hypothesized that a reduction in blood flow to a leg would affect muscle protein metabolism in the leg only when the reduction reaches the threshold of insufficient flow. We used the hindlimb of rabbits as the target tissue. Because the hindlimb is predominately nourished by the femoral artery, reductions of blood flow to leg muscle can be accomplished by mechanically clamping the femoral artery. This approach enabled us to investigate the blood flow as a single factor on muscle protein metabolism, and comparison could be made between the clamped and contralateral unclamped legs.
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MATERIALS AND METHODS
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Animals.
Male New Zealand White rabbits (Myrtle's Rabbitry, Thompson Station, TN), weighing 
4.5 kg, were used. The rabbits were housed in individual cages and consumed a standard high-fiber laboratory rabbit chow for weight maintenance. This protocol complied with National Institutes of Health guidelines and was approved by the Animal Care and Use Committee of The University of Texas Medical Branch at Galveston.
Isotopes.
L-[ring-13C6]phenylalanine (Phe, 99% enriched) and L-[ring-2H5]Phe (98% enriched) were purchased from Cambridge Isotope Laboratories (Woburn, MA). L-[ring-13C6]Phe was used as the tracer, and L-[ring-2H5]Phe was used to prepare internal standard solutions for calculation of Phe concentrations in the blood and muscle.
Experimental design.
There were three groups of a total of 20 rabbits. The rabbits in group 1 (n = 9) were used to compare the femoral artery flow (measured from the ultrasonic flowmeter), total leg flow (measured from the dye dilution method), and muscle capillary flow (measured from the microsphere method). The rabbits in groups 2 (n = 5) and 3 (n = 6) were used to investigate the responses of muscle protein kinetics to reduced blood flow by either partial clamp (group 2) or full clamp (group 3) of the femoral artery. In group 2, the arteriovenous (A-V) model (1) was used to determine the protein kinetics in leg muscle, and the tracer incorporation (20) and tracee release (23) methods were used to measure protein fractional synthetic rate (FSR) and fractional breakdown rate (FBR) in the adductor femoris. In group 3, the tracer incorporation and tracee release methods were used to measure protein FSR and FBR in the gastrocnemius muscle as a representative tissue; the A-V model was not used because of full clamp on one femoral artery.
Experimental procedures.
After an overnight fast with free access to water, the rabbits were anesthetized with intramuscular injection of ketamine (35 mg/kg) and xylazine (5 mg/kg) followed by continuous infusion at various rates to maintain a sufficient depth of anesthesia (25). A tracheal tube was placed via tracheotomy. Further procedures varied to complete the measurements in each group. In four rabbits in group 1, catheters were inserted in a carotid artery and a jugular vein. The arterial line was used for blood collection and monitoring of blood pressure and heart rate; the venous line was used for infusion of anesthetics. The femoral artery was isolated in the experimental leg (either side) at the inguinal level. A flow probe (model 1.5RB; Transonic Systems, Ithaca, NY) was placed on the artery, which was connected to an ultrasonic small animal blood flowmeter (T106; Transonic Systems) for measurement of femoral artery flow rate. A 4-0 silk tie was placed with four loops around the femoral artery at the level above the flow probe, so that when the tie was gently pulled tight the blood flow in the artery was reduced to desired rates. Intravascular Over-the-Needle catheters (24G; Baxter Health Care, Deerfield, IL) were inserted in the femoral artery and vein below the level where the flow probe was placed. The femoral artery line was used for infusion of a dye solution, and the venous line was used for blood withdrawal to determine the dye dilution.
After blood (4 ml) was collected for background measurement, indocyanine green (Akorn, Decatur, IL) in 0.9% saline (0.5 mg/ml) was infused in the femoral artery at 15 ml/h. During the dye infusion, the femoral artery was either not clamped or clamped at 50, 33, or 0% (complete occlusion) of the basal rate. After the start of the dye infusion (10 min), blood samples (1.2 ml each) were drawn simultaneously from the femoral vein and jugular vein. Additional 0.2 ml of blood was drawn from the arterial line for measurement of hematocrit. During the blood collection, blood flow rate in the femoral artery was recorded from the flowmeter. After two to three times blood collection at intervals of 10 min, the dye infusion was discontinued, and the rabbit was allowed 30–60 min to recover from blood loss. The femoral artery flow rate was then changed by adjusting the silk loops on the artery, and the dye solution was infused again. In each rabbit, the total blood collection was limited to <30 ml, which was estimated to be <10% of total blood volume, and hematocrit was kept >20%.
The remaining five rabbits in group 1 were used to determine muscle capillary flow with or without complete clamp of the femoral artery. In these rabbits, catheters were inserted in the femoral artery and vein of one leg (either side) through a groin incision that completely occluded the blood flow in the artery. The arterial line was used for blood collection, and the venous line was used for infusion of anesthetics. The left carotid artery was dissected through an incision on the neck. A 4.0-Fr polyurethane catheter (Cook Critical Care, Bloomington, IN) was inserted in the carotid artery and advanced retrograde in the left ventricle for injection of microspheres. The location of the tip was confirmed by monitoring the blood pressure waveform on the screen of a pressure monitor (model 7830A; Hewlett Packard, Palo Alto, CA). To prevent coagulation in the catheter placed in the left ventricle, a dose of heparin at 50 U/kg was injected intravenously. Colored microspheres (15 µm DIA Fluorescent Microspheres; Interactive Medical Technology, Los Angeles, CA) were injected in the left ventricle as we described in our previous publication (24). Each injection contained 1.25 x 106 spheres given in randomized order. Reference withdrawal from the femoral artery was at a rate of 2.0 ml/min. After completion of the microsphere injection, the rabbits were killed by intravenous injection of 5 ml saturated KCl solution under general anesthesia to stop the heart beat instantaneously. The adductor and gastrocnemius muscle samples were taken from both the clamp and nonclamp legs. The muscle samples were shipped to the Interactive Medical Technology for analysis.
In group 2, catheters were placed in a carotid artery and a jugular vein. The flow probes were placed on both femoral arteries at the inguinal level. A 4-0 silk tie was placed with four loops around one of the femoral arteries to control the flow in the artery. When the left femoral artery was clamped, the right femoral artery served as the control, and vice versa. The Intravascular Over-the-Needle catheters were inserted in both femoral veins, which were used for withdrawal of femoral venous blood. After a blood sample and a muscle specimen were taken for measurement of background enrichment, a priming dose of L-[ring-13C6]Phe (6 µmol/kg) was injected, which was immediately followed by continuous infusion (0.15 µmol·kg–1·min–1). During the isotope infusion, the blood flow in one femoral artery (clamp leg) was reduced to 50% of the contralateral femoral artery flow. During the 120–180 min of isotope infusion, four paired arterial and femoral-venous blood samples were collected at an interval of 10–15 min. The blood flow rates in both femoral arteries were recorded from the flowmeter. At 190 min, muscle samples were taken from the adductor femoris in both legs to measure protein kinetics using a three-pool model and to measure FSR using the tracer incorporation method. Thereafter, the tracer infusion was stopped, and arterial blood was drawn at 0, 5, 10, 20, 40, and 60 min. At 60 min after stopping the tracer infusion, the final muscle samples were taken from the adductor muscle of both legs. These samples were used to determine protein FBR using the tracee release method.
In group 3, catheters were inserted in the femoral artery and vein of one leg that completely occluded the femoral artery flow. The arterial catheter was used for blood collection and monitoring of arterial blood pressure and heart rate, and the venous catheter was used for infusion of the tracer and anesthetics. Because we completely clamped the femoral artery in one leg, the A-V model was not applied. This is because, if the dye dilution method had been used for the A-V model, it would have required blood withdrawal for both the blood flow measurement and A-V Phe enrichment and concentration measurement so that the blood loss would be too much for a rabbit. Thus protein FSR and FBR were measured in the gastrocnemius muscle in both legs using the sampling schedule described for group 2.
In groups 2 and 3, the blood samples were collected in tubes with heparin and put in an ice-water bath until the end of the infusion. The muscle samples were immediately frozen in liquid nitrogen and stored at –80°C for later processing. In all three groups, mean arterial blood pressure, heart rate, and rectal temperature were monitored continuously and were maintained relatively constant by adjusting the dose of anesthetics, infusion rate of saline, and heating lamps. These vital signs were recorded every 30 min.
Sample analysis.
Indocyanine green concentration in serum was measured on a spectrophotometer (Spectronic 1001; Bausch & Lomb, Rochester, NY) at 
of 805 nm. Blood hematocrit was measured on an automated Hematology Analyzer (model JT3; Coulter, Hialeah, FL).
To determine Phe enrichment and concentration in the blood, blood was transferred to tubes containing sulfosalicylic acid and L-[ring-2H5]Phe as an internal standard (30 µmol/l). After deproteinization, the supernatant was processed to make the N-acetyl,n-propyl ester (NAP) derivatives of amino acids (20). Muscle samples were homogenized in 5% (wt/vol) perchloric acid at 4°C. Except for the background sample, a tissue internal standard solution (6 µmol/l of L-[ring-2H5]Phe) was added to the muscle samples (1, 23). Muscle supernatant was processed to make the t-butyldimethylsilyl (TBDMS) derivative of Phe (14). The protein pellets were washed thoroughly to remove free amino acids and lipids and dried in an oven at 80°C (24). The wet and dry weights were recorded to calculate water content in the muscle. The dry protein was hydrolyzed in 6 N HCl at 110°C for 24 h and was prepared for the N-heptafluorobutyryl-n-propyl ester (HFBPr) derivatives (13).
Mass spectrometry analysis.
Isotopic enrichment in the blood prepared as the NAP derivatives was measured on a Hewlett-Packard 5985 gas chromatograph-mass spectrometer (GC-MS) (Hewelett-Packard) with chemical ionization. Ions were selectively monitored at mass-to-charge (m/z) ratios of 250, 251, 255, and 256 for Phe. Isotopic enrichment in the muscle supernatant prepared as the TBDMS derivatives was determined on a Fison MD 800 GC-MS (Beverly, MA); ions were selectively monitored at m/z ratios of 234, 235, 239, and 240 for Phe. The protein hydrolysate in HFBPr derivatives was analyzed for L-[ring-13C6]Phe enrichment using the method previously described (13). Thus the ratio of m + 6/m + 4 ions was measured on the Fison MD 800 GC-MS and converted to the true enrichment of L-[ring-13C6]Phe by means of a standard curve.
The isotopic enrichment was expressed as mole percent excess for the A-V model and FSR calculations and as tracer-to-tracee ratio for FBR calculations, which were required by the methods. The L-[ring-13C6]Phe enrichment was corrected for the contribution of the abundance of isotopomers of lower weight to the apparent enrichment of isotopomers with larger weight, and also a skew correction factor (15).
Calculations and statistics.
The rate of leg blood flow was calculated from the dye dilution method: rate = (infusate OD x 15 ml/h x 51)/[
serum OD x (1 – Hct)], where infusate OD is the optical absorption in the indocyanine green infusion solution, which was infused at 15 ml/h, and 51 is the times of dilution; serum OD is the difference of optical absorption between femoral venous and jugular venous serum. The measured blood flow rate (ml·leg–1·min–1) was divided by the weight of muscle in the leg to convert to the unit of milliliters per.100 g per minute, where the weight of leg muscle was calculated by 4.8% x body weight since the data of leg dissection in our previous experiment indicated that the muscle weight in a hindlimb was 4.8% of the body weight (22). The flow rate in the femoral artery was directly measured by the flowmeter and converted to the same unit as for the leg flow.
The rate of capillary flow was calculated from the microsphere data as follows: capillary flow rate in ml·min–1·g–1 = (total tissue spheres)/[(tissue wt in grams) x (reference spheres in ml–1/min)] (24).
Protein and Phe kinetics in leg muscle were calculated by the three-pool model (Fig. 1) (1). The equations are as follows.
 | (1) |
 | (2) |
 | (3) |
 | (4) |
 | (5) |
 | (6) |
 | (7) |
 | (8) |
where EA, EV, and EM are Phe enrichment in the arterial blood, femoral venous blood, and muscle free amino acid pool, respectively. CA and CV are Phe concentration in the arterial blood and femoral venous blood, respectively. BF is the blood flow rate in the hindlimb. NB is net Phe balance. Inflow is the rate of Phe entering the limb via artery; inward transport is the rate of delivery from artery to the muscle free pool; A-V shunting is the rate of delivery directly from artery to vein; outward transport is the rate of delivery from the muscle free pool to vein; and outflow is the rate of exit via vein. Because Phe is neither synthesized nor degraded in muscle, its appearance represents protein breakdown and its disappearance represents protein synthesis.
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Fig. 1. The three-compartment model of phenylalanine (Phe) kinetics. FV,A indicates direct flow from artery to vein without entering intracellular pool; FM,A and FV,M refer to inward and outward transport from artery to muscle and from muscle to vein, respectively. FM,O indicates intracellular rate of appearance from proteolysis; FO,M is the rate of disappearance via protein synthesis. Free Phe pools in artery (A), vein (V), and muscle (M) are connected by arrows indicating unidirectional Phe flow between compartments. Phe enters the arteriovenous system via artery (Fin) and leaves the system via vein (Fout). Because Phe is neither synthesized nor degraded in muscle, its disappearance reflects protein synthesis, and its appearance reflects protein breakdown.
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The FSR of muscle protein was calculated by the tracer incorporation method (20), and the equation is
 | (9) |
where (E
–E
) is the increment of Phe enrichment in the muscle protein-bound pool from time period 1 (t1) to time period 2 (t2) and EP(t2 – t1) is the average free Phe enrichment in muscle from t1 to t2.
FBR of muscle protein was calculated from the tracee release method (23). The equation used to calculate FBR is
 | (10) |
where EM(t) is the muscle free Phe enrichment at time t, EA(t) is the arterial Phe enrichment at time t, QM/T is the ratio of free to protein-bound Phe content in muscle, and P = EM/(EA – EM), where EM and EA are the free Phe enrichments in the muscle and arterial blood at isotopic steady state.
Data are expressed as means ± SD in the text and Tables 1–5 and as means ± SE in Figs. 1–4. Paired t-test was used to test the differences between the clamped and nonclamped legs in the same rabbits, and nonpaired t-test was used to test the differences between different groups. A P value <0.05 was considered as statistically significant.
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Fig. 4. Net protein balance (fractional synthetic rate – FBR) in the adductor muscle in group 2 and in the gastrocnemius muscle in group 3. The differences in net balance were not only insignificant (P = 0.27 in group 2 and P = 0.39 in group 3) but also minor (i.e., 0.01%/h), indicating maintenance of muscle protein mass.
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RESULTS
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There were no significant differences in the body weight (average 4.5–4.8 kg), mean arterial blood pressure (average 70–76 mmHg), heart rate (average 187–195 beats/min), or rectal temperature (average 38.6–38.9°C) among groups. In group 1, four rabbits were used to estimate the leg flow (measured by dye dilution) from the femoral artery flow (measured by the flowmeter). The paired values of femoral artery and leg flow were pooled according to the clamping of the femoral artery. Thirteen paired measurements were completed without clamping; the femoral artery flow and leg flow were 5.9 ± 1.2 and 9.1 ± 1.6 ml·100 g–1·min–1, respectively. Thus, without clamping, the femoral artery flow accounted for 65 ± 4% of the leg flow. When the flow in the femoral artery was clamped at 3.55 ± 0.20 (4 measurements) and 1.95 ± 0.13 (9 measurements) ml·leg–1·min–1, which were 40 and 67% reductions in the femoral artery flow, respectively, the percent contributions of femoral artery flow to leg flow were 53 ± 7 and 33 ± 2%, respectively (Table 1). There was a linear correlation (r = 0.93; P < 0.001) between the femoral artery flow and the ratio of femoral flow to leg flow: (ratio of femoral flow to leg flow) = 0.1230 x (femoral flow) + 0.0892, where the flow is expressed in milliliters per 100 grams per minute. When the femoral artery was fully clamped (2 measurements), the leg flow was 5.3 ml·100 g–1·min–1 (5.0 and 5.6 ml·100 g–1·min–1), which was a 42% reduction in basal leg flow when compared with the contralateral nonclamp leg.
In group 1, the colored microspheres were injected in five rabbits for measurement of the capillary flow in leg muscles. The measured capillary flow rates in the right and left kidney were almost identical (4.70 ± 0.99 vs. 4.73 ± 1.22 ml·g–1·min–1; P = 0.4), indicating that the injected microspheres were uniformly distributed in the two legs. Full clamp of the femoral artery reduced (P = 0.007 and 0.018) the capillary flow by 59% in the gastrocnemius muscle from 9.99 ± 3.10 to 4.09 ± 2.71 ml·100 g–1·min–1 and in the adductor muscle from 7.83 ± 3.85 to 3.19 ± 1.03 ml·100 g–1·min–1.
Group 2 was used to investigate the effect of a 50% reduction of the femoral artery flow on protein kinetics in leg muscle. During the isotope infusion, the femoral artery flow rate in the control leg was 6.11 ± 1.41 ml·100 g–1·min–1. The leg flow rate was estimated to be 9.41 ± 2.17 ml·100 g–1·min–1, which was calculated by dividing the femoral flow rate by 65%. In the partial clamp leg, according to the results in group 1, the femoral artery flow was no longer 65% of leg flow but followed the equation: (ratio of femoral flow to leg flow) = 0.1230 x femoral flow + 0.0892. Thus the leg flow rate in the partial clamp leg was estimated to be 6.52 ± 0.29 ml·100 g–1·min–1 (Table 1). The Phe enrichment and concentration in the arterial and venous blood and Phe enrichment in the muscle free amino acid pool (data not presented here) were used to calculate protein kinetics and Phe transport rates (Table 2) using the three-pool model (1). Whereas in the partial clamp leg the rate of inward transport decreased significantly, the rates of protein synthesis, breakdown, and net balance were not significantly different from those in the control leg (Table 2). The synthetic rate of muscle protein in the partially clamped leg tended to be lower than that in the control leg (Table 2), although statistical significance was not achieved (P = 0.18). This could be explained by the fact that the A-V model calculates protein metabolism not only in the muscle but also in bone and skin. The convincing data are the identical (P = 0.50) protein FSR in the adductor muscle between the control and partial clamp legs (Table 3), which confirmed that muscle protein synthesis was not changed by the 50% reduction in femoral artery flow.
Group 3 was used to investigate the effect of full clamp of the femoral artery on protein kinetics in leg muscle. Full clamp of the femoral artery resulted in a decrease (P = 0.003) of FSR in the gastrocnemius muscle compared with that in the control leg (Table 3).
FBR were measured in both groups 2 and 3. During the 120–180 min of tracer infusion, Phe enrichment in the arterial blood reached isotopic plateau. After stopping the tracer infusion, the enrichment decay in the arterial blood followed the same pattern in the two groups (Fig. 2). The percentages of protein in wet muscle in the adductor muscle (group 2) and in the gastrocnemius muscle (group 3) were 23 ± 1 and 21 ± 2% by weight, respectively. We previously reported that 1 g of dry muscle protein contained 250 µmol Phe (22). Thus the contents of protein-bound Phe in 1 g of adductor and gastrocnemius muscle were 57.5 and 52.5 µmol, respectively. The femoral artery clamp, either partial or full, did not change (P = 0.14–0.15) muscle free Phe concentration (Fig. 3). Protein FBR in the adductor muscle of the partial clamp leg was close (P = 0.38) to that in the control leg (Table 4). Protein FBR in the gastrocnemius muscle of the full clamp leg was lower (P = 0.03) than that in the control leg. Protein net balance (FSR – FBR) was not significantly (P = 0.27 or 0.39) different either in the adductor muscle of the partial clamp leg (–0.07 ± 0.08 vs. –0.06 ± 0.07%/h) or in the gastrocnemius muscle of the full clamp leg (–0.11 ± 0.05 vs. –0.10 ± 0.05%/h) compared with their corresponding control values (Fig. 4). When comparison was made between the control legs in groups 2 and 3, both FSR and FBR were (P < 0.05 or P < 0.01) lower in the adductor muscle (group 2) than in the gastrocnemius muscle (group 3) (see Tables 3 and 4), and free Phe concentration in the adductor muscle was lower (P < 0.05) than that in the gastrocnemius muscle (0.17 ± 0.05 vs. 0.22 ± 0.04 µmol/ml).
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Fig. 2. The decay of Phe enrichment in the arterial blood after stopping the tracer infusion. The data are used in calculation of protein fractional breakdown rate (FBR) using the tracee release method.
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Fig. 3. Free Phe concentrations in the adductor (group 2) and gastrocnemius (group 3) muscle. Neither partial nor full clamp of the femoral artery reduced the free Phe contents in muscle compared with the control leg. Basal concentration of free Phe in the gastrocnemius muscle was greater (P < 0.05) than that in the adductor muscle.
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DISCUSSION
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We found that, in the basal state, the femoral flow accounted for 65% of total leg flow, and the remaining was derived from collateral flow. A 50% reduction of femoral flow resulted in a 28% decrease in leg blood flow, which did not significantly change protein turnover rate in leg muscle. This finding indicates that the muscle protein metabolism can compensate for an 30% reduction of blood flow. When the leg blood flow was reduced by 42% by fully clamping the femoral artery, the rates of muscle protein synthesis and breakdown both decreased, indicating that the reduction of blood flow exceeded the compensation capacity. Therefore, in this animal model, the flow threshold to cause a fall of protein turnover was between 30 and 40% reduction in total leg flow.
The responses of muscle protein kinetics to the reductions in blood flow may reflect the mechanisms of muscle homeostasis. When the reduction in blood flow was <30% of the basal flow, the synthetic and breakdown rates of muscle protein were not changed (Tables 2–4). According to the definition of the three-pool model, the inflow of amino acids in the leg muscle from the artery is the sum of the inward transport and A-V shunting (Fig. 1). Thus the inward transport is analogous to the nutritive flow, and the A-V shunting is nonnutritive (1). The 28% reduction in leg flow, which resulted from 50% clamp of femoral artery flow, was associated with a 26% reduction in inward transport (Table 2). Thus the responses to the reduction in flow cannot be explained by blood redistribution between the nutritive and nonnutritive routes as was proposed to explain increases in metabolic activities induced by insulin or exercise (2, 6, 17). A reasonable explanation would be that, in the basal state, the nutritive flow delivers amino acids to the muscle at the rate that exceeds the minimal requirement for the basal rate of protein synthesis. A relatively small decrease of the nutritive flow, such as a 26% decrease of inward transport, did not cause a deficiency of precursors for protein synthesis.
When the flow in the leg was reduced by 42% by fully clamping the femoral artery, both the synthetic and breakdown rates of muscle protein were decreased significantly (Tables 3 and 4). However, the difference in net balance (FSR – FBR) was not only insignificant but also minor, indicating maintenance of muscle mass (Fig. 4). In other words, when the reduction in blood flow reached the flow threshold, the muscle homeostasis was preserved at the expense of decreasing its turnover rate. Because the A-V model was not applied with full clamp of the femoral artery, the amino acid transport data were not available. Therefore, we measured the capillary flow in five rabbits in group 1 to examine the changes in nutritive flow. Because the injected colored microspheres have a diameter of 15 µm, entrapment in the tissue reflects capillary flow that is considered to be nutritive. In the nonclamp leg, the capillary flow rates were 9.99 ± 3.10 and 7.83 ± 3.8 ml·100 g –1·min-1 in the gastrocnemius and adductor muscles. These values were close to the leg flow rates of 9.14 ± 1.57 and 9.41 ± 2.17 ml·100 g–1·min–1 in groups 1 and 2 measured from the dye dilution method. With the full clamp, the leg flow was reduced by 42%, and the capillary flow was reduced by 59%. The reduction in the capillary flow appeared to be greater than that of leg flow. Therefore, the results do not support blood redistribution from the nonnutritive to nutritive route.
In the partial and full clamp legs, the net protein balance was 0.01%/h more or less than the corresponding control legs (Fig. 4). A reverse-power analysis indicates that 567 rabbits are needed to achieve a power of 0.80. Because the differences in net protein balance were not only insignificant but also minor, the conclusion of muscle homeostasis is considered to be valid without a further statistical analysis after increasing numbers in each group. According to our previous publication, the muscle mass in the hindlimb of rabbits is 4.8% x body weight (22), which is equivalent to 216 g of leg muscle in a 4.5-kg body weight rabbit. If the muscle contains 20% of dry protein, there is 43 g of muscle protein in a hindlimb. The difference of 0.01%/h equals 0.0043 g muscle protein/h. Even if there was such an additional loss (or gain) and the loss (or gain) lasted for 24 h, the clamp leg would have lost (or gained) 0.1 g more muscle protein than the control leg. However, such a minor change, if it turns out to be true from statistical analysis of a large number of subjects, could lead to physiological importance when it persists for a prolonged period of time. Therefore, the findings from the present study cannot be extrapolated to more chronic disturbances of blood flow without further experimental validation.
The muscle homeostasis was reflected by not only a constant protein mass but also a constant intracellular amino acid concentration (Fig. 3). This finding is consistent with a previous study in which hemodialysis significantly decreased plasma amino acid concentrations and muscle protein synthesis, but the intracellular essential amino acid concentrations in the muscle were maintained at the basal level (3, 12). The results support the notion that maintenance of intracellular essential amino acid concentrations and protein mass are the metabolic priorities superior to the maintenance of protein turnover rate. By clamping the femoral artery, the maximal reduction in leg flow was 42% of the basal rate. Therefore, it is not known how muscle would respond to a flow reduction that is greater than 42% of the basal rate. Furthermore, the present study was conducted in the basal state, so the findings may not be extrapolated to other conditions such as during exercise or insulin administration.
When comparison is made between the two control groups in groups 2 and 3, the basal rates of protein synthesis and breakdown in the gastrocnemius muscle were greater (P < 0.01–0.05) than those in the adductor muscle (Table 4). Consistent with the difference in protein turnover rate, basal concentration of free Phe in the gastrocnemius muscle was greater (P < 0.05) than that in the adductor muscle. The metabolic heterogeneity of leg muscles might be related to their physiological functions and fiber composition, but the mechanism is not clear.
In summary, with 50% clamp of the femoral artery, the nutritive flow was reduced by 26%, and the leg flow was reduced by 28%, which did not change muscle protein turnover rate in the leg. Full clamp of the femoral artery resulted in 59% reduction in nutritive flow and 42% reduction in leg flow; the maintenance of muscle mass was accomplished by parallel decreases of both synthesis and breakdown. Therefore, the flow threshold that caused a decrease in muscle protein turnover rate was a 30–40% reduction of the basal flow. The clamp of the femoral artery, either partial or full, did not decrease muscle free amino acid concentration. Thus the acute responses of leg muscle protein metabolism to the reductions in blood flow by femoral artery clamp reflected the metabolism priorities for homeostasis.
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GRANTS
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This work was supported by Shriners grants 8630 and 8490 and National Institute of Arthritis and Musculoskeletal and Skin Diseases Grant AR-49038.
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ACKNOWLEDGMENTS
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We are grateful to Yunxia Lin, Zhipin Dong, and Guy Jones for technical assistance. We thank the Animal Resource Center of the University of Texas Medical Branch for professional care of animals and the Clinical Laboratory of the Shriners Hospital for Children for measurement of hematocrit.
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FOOTNOTES
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Address for reprint requests and other correspondence: R. R. Wolfe, Dept. of Geriatrics, Center for Translational Research in Aging & Longevity, Univ. of Arkansas Medical School, 4301 Markham Slot 806, Little Rock, AR 72205 (e-mail: rwolfe2{at}uams.edu)
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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