Chronic -hydroxyisocaproic acid treatment improves muscle recovery ...

Am J Physiol Endocrinol Metab 305: E416–E428, 2013. First published June 11, 2013; doi:10.1152/ajpendo.00618.2012.

Chronic ␣-hydroxyisocaproic acid treatment improves muscle recovery after immobilization-induced atrophy Charles H. Lang,1 Anne Pruznak,1 Maithili Navaratnarajah,1 Kristina A. Rankine,1 Gina Deiter,1 Hugues Magne,2 Elizabeth A. Offord,2 and Denis Breuillé2 1

Department of Cellular and Molecular Physiology, Pennsylvania State University College of Medicine, Hershey, Pennsylvania; and 2Nestlé Research Center, Lausanne, Switzerland Submitted 6 December 2012; accepted in final form 9 June 2013

Lang CH, Pruznak A, Navaratnarajah M, Rankine KA, Deiter G, Magne H, Offord EA, Breuillé D. Chronic ␣-hydroxyisocaproic acid treatment improves muscle recovery after immobilization-induced atrophy. Am J Physiol Endocrinol Metab 305: E416 –E428, 2013. First published June 11, 2013; doi:10.1152/ajpendo.00618.2012.—Muscle disuse atrophy is observed routinely in patients recovering from traumatic injury and can be either generalized resulting from extended bed rest or localized resulting from single-limb immobilization. The present study addressed the hypothesis that a diet containing 5% ␣-hydroxyisocaproic acid (␣-HICA), a leucine (Leu) metabolite, will slow the loss and/or improve recovery of muscle mass in response to disuse. Adult 14-wk-old male Wistar rats were provided a control diet or an isonitrogenous isocaloric diet containing either 5% ␣-HICA or Leu. Disuse atrophy was produced by unilateral hindlimb immobilization (“casting”) for 7 days and the contralateral muscle used as control. Rats were also casted for 7 days and permitted to recover for 7 or 14 days. Casting decreased gastrocnemius mass, which was associated with both a reduction in protein synthesis and S6K1 phosphorylation as well as enhanced proteasome activity and increased atrogin-1 and MuRF1 mRNA. Although neither ␣-HICA nor Leu prevented the casting-induced muscle atrophy, the decreased muscle protein synthesis was not observed in ␣-HICA-treated rats. Neither ␣-HICA nor Leu altered the increased proteasome activity and atrogene expression observed with immobilization. After 14 days of recovery, muscle mass had returned to control values only in the rats fed ␣-HICA, and this was associated with a sustained increase in protein synthesis and phosphorylation of S6K1 and 4E-BP1 of previously immobilized muscle. Proteasome activity and atrogene mRNA content were at control levels after 14 days and not affected by either treatment. These data suggest that whereas ␣-HICA does not slow the loss of muscle produced by disuse, it does speed recovery at least in part by maintaining an increased rate of protein synthesis. disuse; leucine; protein synthesis; protein degradation; mammalian target of rapamycin SKELETAL MUSCLE IS A HIGHLY PLASTIC TISSUE with muscle hypertrophy observed during strength training and, conversely, atrophy occurring during numerous catabolic conditions (e.g., inflammatory diseases, cancer, aging) (22, 30, 32, 56). Bed rest and/or immobilization cause degenerative changes that, when prolonged, lead to atrophy (18, 19, 44, 63). This muscle wasting results from an imbalance between rates of protein synthesis and breakdown (13, 35, 40, 56). Muscle protein can be degraded into free amino acids, which are subsequently used to support the immune response and select anabolic processes (e.g., gluconeogenesis and acute-phase protein synthesis). Thus, skeletal muscle not only provides power and

Address for reprint requests and other correspondence: C. H. Lang, Pennsylvania State University College of Medicine, Cellular and Molecular Physiology, Hershey, PA 17033 (e-mail: [email protected]). E416

strength for locomotion and posture but also serves as a major reservoir of proteins and amino acids. Consequently, an uncontrolled and sustained muscle wasting impairs human movement, leads to difficulties in performing daily activities, and has detrimental metabolic consequences. In some catabolic states, the extent of muscle wasting increases not only morbidity but also mortality (36, 39, 47, 64). Finally, it has been reported that preventing muscle wasting per se can improve survival in preclinical models of cancer cachexia (80). The recovery of muscle mass following disuse and its maintenance during aging are critical to maintain autonomy (49, 50, 53). Although physical intervention is the optimal method to maintain and/or accrete muscle mass, exercise is not always feasible, particularly when disuse results from trauma. Therefore, an unmet clinical need is the identification of new approaches to limit skeletal muscle atrophy in catabolic conditions and/or improve subsequent muscle recovery. Specific nutrition formulations have effectively improved muscle anabolism in select conditions (30, 62). Leucine (Leu) alone or in combination with other branched-chain amino acids acutely promotes protein synthesis via a mammalian target of rapamycin (mTOR)-mediated mechanism (15, 21) and may also have antiproteolytic properties (5, 29, 52, 57, 58, 78). However, whether these varied and numerous effects of Leu are mediated directly or in part indirectly by one of its metabolites has not been clearly defined (52). Therefore, selective addition of the Leu metabolite to a complete diet may provide insight into mechanisms of action and circumvent the inability of longterm Leu treatment to increase muscle mass (73). The efficacy of ␣-hydroxyisocaproic acid (␣-HICA; a.k.a. leucic acid or DL-2-hydroxy-4-methylvaleric acid), an end product of leucine metabolism, to modulate protein balance in skeletal muscle has not been investigated thoroughly. ␣-HICA is found in muscle (31) and is generally considered to have anticatabolic actions (8, 31, 67). Because a dietary substitution of ␣-HICA for Leu supports normal growth (14, 77), it is possible that at least part of the anabolic effect of Leu is mediated via this metabolite and that a complete diet containing relatively high amounts of ␣-HICA may have a greater anabolic effect than one containing an equal percentage of Leu alone. In this regard, a single report indicates that, compared with placebo, a 4-wk treatment with ␣-HICA combined with a complete diet increased lean body mass in humans (51). Hence, the aim of this proof-of-concept study was to investigate the ability of a nutritionally complete diet containing a relatively high amount of ␣-HICA (5%) in maintaining muscle mass during a period of immobilization and recovery. The effect of the enhanced diet was examined during the period of disuse (unilateral immobilization, i.e., “casting”) but also during a

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␣-HICA AND MUSCLE PROTEIN BALANCE

Table 1. Diet composition Casein L-Cystine Alanine Leucine Valine Isoleucine ␣-HICA Citrulline Rapeseed oil Sunflower oil Groundnut oil Sucrose Lactose Wheat starch Cellulose AIN 93M mineral mix AIN 93M vitamin mix Total

Control

␣-HICA

Leucine

166 1.8 45.3 0 0 0 0 0 30 3 27 100 134 412.9 35 35 10 1,000

166 1.8 0 0 6 10 50.4 0 30 3 27 100 134 391.8 35 35 10 1,000

166 1.8 0 50 6 10 0 0 30 3 27 100 134 392.2 35 35 10 1,000

All values are in g/kg diet. ␣-HICA, ␣-hydroxyisocaproic acid.

recovery period when muscle was reloaded following cast removal. The primary end points of efficacy assessed were muscle mass and muscle protein synthesis rate, but surrogate markers of muscle protein degradation were also quantified. MATERIALS AND METHODS

Animal protocols. The experiments described herein were broadly organized into two experimental series. Both studies used male Wistar rats (Charles River Breeding Laboratories, Cambridge, MA) that were acclimated in a controlled environment for 1 wk. Rats were shipped at 350 –375 g and were ⬃12 wk of age. Rats were housed in solidbottom cages with corncob bedding and provided water and standard chow (no. 2012, 18% protein; Harlan, Madison, WI) ad libitum. All experiments were approved by the Institutional Animal Care and Use Committee of The Pennsylvania State University College of Medicine and adhered to the National Institutes of Health guidelines for the use of experimental animals. Study 1. This study examined the ability of a diet containing 5% (wt/wt) ␣-HICA to ameliorate or prevent the normal atrophic response in skeletal muscle produced by disuse. The following custom diets were commercially prepared (Dytes, Bethlehem, PA): control diet (AIN-93M) or isocaloric isonitrogenous diets with added ␣-HICA or Leu (Table 1). A preliminary study indicated that the rats had variable consumption of the different diets when they were first introduced. Therefore, animals were randomized among three groups and provided the diet for a 6-day period prior to hindlimb immobilization. To improve data interpretation by eliminating differences in total caloric intake, after the 1st day all animals were pair-fed the same amount as the ␣-HICA-treated group. For all studies, body weight and food consumption were determined daily, and fresh food was provided to the pair-fed groups between 0800 and 0900; the time of day during which rats consumed their food was not controlled. On day 7, all rats were anesthetized with isoflurane (3% induction ⫹ 1.5–2% maintenance) and subjected to unilateral hindlimb immobilization via a fiberglass cast exactly as described (40, 70). The foot was positioned in plantar flexion to induce maximal atrophy of the gastrocnemius, and rats received 10 ml of 0.9% warmed (37°C) sterile saline for resuscitation. Previous studies demonstrated that unilateral immobilization has no effect on muscle mass or various parameters of interest in skeletal muscle from the contralateral noncasted leg (40, 72). Consequently, the contralateral limb served as the control in all subsequent experiments. However, we determined simultaneously gastrocnemius weight and protein synthesis on a cohort (n ⫽ 7) of

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pair-fed naive rats provided the control diet for the same period of time. After casting, rats were housed individually, and pair-feeding continued for a period of 7 days. For the final 48-h period, rats were housed in metabolic cages to collect urine for subsequent analysis. Water was provided ad libitum. On day 7 after casting, rats were anesthetized with pentobarbital sodium, and the cast was atraumatically and rapidly (⬍2 min) removed (Stryker Instruments, Kalamazoo, MI). Study 2. This study was performed exactly as study 1 described above, except rats were permitted a 7- or 14-day recovery period after cast removal. To minimize the duration of anesthesia, animals in this second study were anesthetized with isoflurane instead of pentobarbital sodium for cast removal. At the conclusion of the specified recovery period, rats in this group were anesthetized with pentobarbital sodium for subsequent determination of tissue protein synthesis. Protein synthesis. Food was removed at the start of the light cycle (0600), and 3– 6 h thereafter the in vivo rate of protein synthesis in gastrocnemius, liver, and heart (ventricle only) was determined using the flooding dose technique exactly as described (71). A P-50 catheter was placed in the left carotid artery for blood withdrawal. Rats were injected intravenously (iv) with L-[3H]phenylalanine (Phe; 150 mM, 30 ␮Ci/ml; 1 ml/100 g body wt), and arterial blood was collected 10 min later for determining the plasma Phe concentration and radioactivity. Thereafter, tissues were excised rapidly, and a portion was freeze-clamped and stored at ⫺70°C. The rate of protein synthesis was calculated by dividing the amount of radioactivity incorporated into protein by the plasma Phe-specific radioactivity. The advantages and disadvantages of this method have been reviewed (23). The specific radioactivity of the plasma Phe was measured by highperformance liquid chromatography (HPLC) analysis of supernatant from trichloroacetic acid extracts of plasma. Samples of fresh muscle were homogenized for Western blot analysis of selected proteins, and another piece was used for qRT-PCR, as described below. Western blotting. Fresh muscle was homogenized (Kinematic Polytron; Brinkmann, Westbury, NY) in ice-cold buffer consisting of (in mmol/l): 20 HEPES (pH 7.4), 2 EGTA, 50 sodium fluoride, 100 potassium chloride, 0.2 EDTA, 50 ␤-glycerophosphate, 1 DTT, 0.1 phenylmethane-sulphonylfluoride, 1 benzamidine, and 0.5 sodium vanadate. Protein was determined after centrifugation, and equal amounts of protein (50 –90 ␮g) per sample were subjected to standard SDS-PAGE. Specifically, Western analysis was performed for total and phosphorylated eukaryotic initiation factor 4E-binding protein-1 (4E-BP1) (Thr37/46; Bethyl Laboratories, Montgomery, TX) and S6K1 (Thr389; Cell Signaling Technology, Beverly, MA). Blots were developed with enhanced chemiluminescence Western blotting reagents (Supersignal Pico; Pierce Chemical, Rockford, IL). Dried blots were exposed to X-ray film to achieve a signal within the linear range, and film was then scanned (Microtek ScanMaker IV) and quantified using Scion Image 3b software (Scion, Frederick, MD). Protein degradation. In the current study, we used several methods for the assessment of whole body and muscle protein breakdown. First, the urinary 3-methylhistidine (3-MH)/creatinine (Cr) ratio was determined to estimate whole body myofibrillar breakdown (1). Urine was collected for a 24-h period immediately following a 24-h acclimation period of rats to the metabolic cages. Quantitation of urine 3-MH was by gas chromatography-mass spectrometry (GC-MS) was as described (1, 60). Briefly, the internal standard 3-methyl-[methyl2 H3]-histidine was added to urine and hydrolyzed for 15 h at 110°C and then isolated on cation exchange columns. 3-MH was eluted from columns, dried, and derivatized with N-methyl-N-(t-butyldimethylsilyl)-trifluoroacetamide (Regis Technologies, Morton Grove, IL) for GC-MS analysis (6890/5973; Agilent, Palo Alto, CA). Determination of Cr was as described below. Second, we determined proteasome activity on both control and casted muscle from each animal. For this analysis, gastrocnemius was homogenized in cell lysis buffer containing (in mM) 25 HEPES, 5 MgCl2, 5 EDTA, and 5 DTT, pH 7.5, at 4°C followed by centrifu-

AJP-Endocrinol Metab • doi:10.1152/ajpendo.00618.2012 • www.ajpendo.org

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gation at 14,000 rpm for 2 min at 4°C. The protein concentration in the supernatant was determined using the BCA Protein Assay Kit (Pierce). The proteasome enzymatic activity was measured using a proteasome 20S assay kit (Enzo Life Sciences, Farmingdale, NY), following the manufacturer’s instructions. Briefly, the protein extract from muscle was used to assess proteasome 20S chymotrypsin-like activity by measuring the hydrolysis of a fluorogenic peptidyl substrate Suc-Leu-Leu-Val-Tyr-AMC (AMC; 7-amino-4-methylcoumarin). This substrate was cleaved by the proteasome activity, and the subsequently released free AMC was detected by a fluorometer with an excitation wavelength of 380 nm and emission wavelength of 460 nm. The fluorescence signal was monitored before and 1 h after incubation at 37°C. Each sample/substrate combination was measured in both the presence and absence of the proteasome inhibitor MG132 (Boston Biochem, Cambridge, MA) to account for nonproteasomal degradation of the substrate, and these fluorescence units were subtracted from each measurement. Data were recorded at 2-min intervals over 60 min, and activity was plotted as abritrary fluorescence units/min over the linear range of the curves. Finally, real-time quantitative PCR was used to assess the mRNA content for the muscle-specific ubiquitin E3 ligases atrogin-1/muscle atrophy F-box and muscle RING finger protein 1 (MuRF1), which is increased in a number of catabolic conditions (7, 28). Total RNA was extracted using TRI Reagent (Molecular Research Center, Cincinnati, OH) and RNeasy mini kit (Qiagen, Valencia, CA) protocols. Muscle (50 – 80 mg) was homogenized in TRI Reagent, followed by chloroform extraction according to the manufacturer’s instruction. Equal volume of 70% ethanol was added to the aqueous phase, and the mixture was loaded on a Qiagen minispin column. The Qiagen minikit protocol was followed from this step onward, including the on-column DNase I treatment to remove residual DNA contamination. RNA was eluted from the column with RNase-free water and quantified using a NanoDrop 2000 (Thermo Fisher Scientific, Waltham, MA) spectrophotometer. Quality of the RNA was analyzed on a 1% agarose gel. Total RNA (1 ␮g) was reverse transcribed using superscript III reverse transcriptase (Invitrogen, Carlsbad, CA), following the manufacturer’s instruction. Real-time quantitative PCR was performed using 25 ng of cDNA in a StepOnePlus system using TaqMan gene expression assays for atrogin (Rn00591730_m1), murf (Rn00590197_m1), ubiquitin b (Rn03062801_gH), and gapdh (Rn01775763_g1) and the gene expression master mix according to the manufacturer’s instructions (Applied Biosystems, Foster City, CA). The comparative quantitation method 2⫺⌬⌬CT was used in presenting gene expression of target genes in reference to the endogenous control (46). Blood biochemistry and hematology. Because there are few data in the literature relative to the effect of ␣-HICA on whole body metabolic homeostasis, we also determined standard hematological and biochemical end points. For hematology analyses, arterial blood was dispensed to tubes containing EDTA (BD No. 365974; Fisher Scientific). Hematology analysis (Heska CBC-Diff Hematology Analyzer, Loveland, CO) included red and white blood cell counts, hematocrit, hemoglobin, platelets, and differential leukocyte counts. Reticulocytes were counted manually using methylene blue staining. Blood was also dispensed to a silicone-coated collection tube (BD No. 366381; Fisher Scientific) and allowed to clot. Clotted blood samples were centrifuged in a Beckman Coulter Allegra X-12R centrifuge at 3,500 rpm for 5 min at 4°C, and the serum was collected and stored. Biochemical analyses on serum were performed on a Cobas Mira Plus Chemistry Analyzer (Diamond Diagnostics, Holliston, MA) and included total bilirubin, glucose, alkaline phosphatase, aspartate aminotransferase (AST), alanine aminotransferase (ALT), lactate dehydrogenase, albumin, calcium, Cr, blood urea nitrogen (BUN), phosphate, total cholesterol, chloride, and total protein. Serum sodium and potassium were analyzed by flame photometer (IL940; Instrumentation Laboratory, Lexington, MA). Triglycerides (Abcam, Cambridge, MA) and free fatty acids (FFAs; Wako Diagnostics, Richmond, VA, respectively) were determined colorimetrically. Insulin was determined by ELISA

(Alpco Diagnostics, Salem, NH). Finally, plasma amino acid concentrations were determined using a Hitachi High-Technologies Model L-8900 HPLC, using post-column derivatization with ninhydrin. The samples were deproteinized with sulfosalicylic acid, centrifuged, and filtered (0.2 ␮m) prior to adding the internal standard and injecting into the column. Statistical analysis. Data for each group are summarized as means ⫾ SE, where the number of rats per treatment group is indicated in the figure and table legends. Statistical evaluation of the data was performed using 2-way ANOVA with post hoc Student-Neuman-Keuls test when the interaction was significant. To compare the immobilization-induced decrease in muscle protein synthesis between the right and left gastrocnemius in the same rat, a two-tailed paired t-test was performed. Differences between groups were considered significant at P ⬍ 0.05. RESULTS

Food consumption, body weight, and organ weights. Animals from all studies were combined so as to present an overall pattern for food intake and change in body weight during the basal period (e.g., precasting), during immobilization, and during the recovery period. On day 1, food intake was higher in the control group than in the two experimental groups (Fig. 1A). Food intake did not differ among the three groups during days 2– 6. On day 6, rats had one hindlimb casted, and food intake dropped ⬃25%; the reduction was comparable in all groups. Despite our attempt to pair-feed rats the amount of food consumed by the ␣-HICA group, the average food consumption of the ␣-HICA group was lower than the other two groups on days 4 – 6 of immobilization. Upon cast removal, food consumption increased gradually over the remainder of the study, and there was no difference in food consumption among the three groups at any time during the recovery phase (Fig. 1A). Figure 1B illustrates the absolute change in body weight normalized to each animal’s own starting weight. There was an initial drop in body weight for the first 24 h for all rats upon introduction of the defined diet. Thereafter, body weight increased in all groups and was comparable with starting values. As a result of casting, all rats lost body weight, and the decrement was comparable in all groups. Upon cast removal, body weight initially increased and then appeared to plateau during the recovery period. Overall, there were no sustained differences in either daily food consumption or body weight among rats receiving ␣-HICA or Leu treatment compared with control rats. There was no difference in the total organ weight for liver, heart (ventricle only), adrenal gland, spleen, kidney, or testes among the different treatment groups (data not shown). The in vivo-determined rate of organ protein synthesis was determined 3– 6 h after food was removed (i.e., 0900 –1200). Protein synthesis in heart and liver did not differ among the three groups (Table 2). Alterations in muscle protein synthesis will be described later. General metabolic and hematological characteristics. Various biochemical end points were determined on serum, and data from all studies were combined because there was no statistical difference detected within groups having the same treatment for various durations. Alkaline phosphatase, an enzyme used clinically to determine liver disease and bone disorders, was elevated ⬃20% in rats treated with ␣-HICA (Table 3) compared with control values. However, other sur-



Table 3. Blood chemistry profile, metabolic substrates, and hormones

A Food Consumption (g/day)

24 21

*

18 15 12

Control HICA Leucine

†

9 6 3 Immobilization Recovery

7d

14d

0 0

3

6

9

12

15

18

21

24

27

Time (days)

Change in Body Weight (g)

B

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Creatinine, mg/dl BUN, mg/dl Bilirubin (total), mg/dl Alkaline phosphatase, U/l LDH, U/l AST, U/l ALT, U/l Protein (total), g/dl Albumin, g/dl Cholesterol, mg/dl Triglycerides, ␮mol/l FFA, ␮mol/l Glucose, mg/dl Insulin, pmol/l

Control

␣-HICA

Leucine

0.3 ⫾ 0.02 16.1 ⫾ 0.6 0.5 ⫾ 0.04 74 ⫾ 5a 292 ⫾ 30 76 ⫾ 5 25 ⫾ 2 4.9 ⫾ 0.1 3.38 ⫾ 0.04 66 ⫾ 3 678 ⫾ 45 420 ⫾ 29a 215 ⫾ 9 321 ⫾ 24a

0.3 ⫾ 0.02 16.2 ⫾ 0.6 0.5 ⫾ 0.04 89 ⫾ 6b 232 ⫾ 23 74 ⫾ 4 24 ⫾ 1 5.0 ⫾ 0.1 3.31 ⫾ 0.04 62 ⫾ 3 609 ⫾ 51 259 ⫾ 25b 223 ⫾ 11 326 ⫾ 26a

0.3 ⫾ 0.01 16.2 ⫾ 0.5 0.3 ⫾ 0.02 69 ⫾ 4a 169 ⫾ 21 68 ⫾ 4 24 ⫾ 1 5.0 ⫾ 0.1 3.42 ⫾ 0.03 63 ⫾ 4 667 ⫾ 52 520 ⫾ 35c 214 ⫾ 8 626 ⫾ 55b

Values are means ⫾ SE; n ⫽ 26, 22, and 23 in the control, ␣-HICA, and leucine groups, respectively. BUN, blood urea nitrogen; LDH, lactate dehydrogenase; AST, aspartate aminotransferase; ALT, alanine aminotransferase; FFA, free fatty acids. Means with different letters are statistically different (P ⬍ 0.05; ANOVA-SNK) within the same row.

30 20 10

*

0 -10

Control HICA Leucine

† -20 -30 Immobilization Recovery

7d

14d

-40 0

3

6

9

12

15

18

21

24

27

Time (days) Fig. 1. Daily food consumption and change in body weight for rats fed a diet containing ␣-hydroxyisocaproic acid (␣-HICA) or leucine (Leu) compared with pair-fed control rats. The initial absolute body weights for rats in the control, ␣-HICA, and Leu groups are 398 ⫾ 5, 398 ⫾ 5, and 397 ⫾ 6 g, respectively. Values are means ⫾ SE; n ⫽ 27, 25, and 23, respectively, during days 1–13; n ⫽ 19, 15, and 15, respectively, during the 1st 7 days of recovery; and n ⫽ 8/group for the 14-day recovery period. *P ⬍ 0.05, control vs. ␣-HICA- and Leu-fed rats; †P ⬍ 0.05, ␣-HICA vs. control and Leu-fed rats.

rogate markers of liver function (AST, ALT, total bilirubin) did not differ among groups. Likewise, the concentrations of Cr and BUN, which assess renal function, did not differ among the three groups. Finally, because urinary flow and the urinary Cr concentration were also determined, we calculated the rate of Table 2. In vivo-determined tissue protein synthesis Heart Liver Gastrocnemius (nonimmobilized)

Control

␣-HICA

Leucine

2.96 ⫾ 0.10 23.62 ⫾ 0.71 1.42 ⫾ 0.04a

2.76 ⫾ 0.11 24.41 ⫾ 0.90 1.26 ⫾ 0.04b

2.93 ⫾ 0.11 22.51 ⫾ 0.88 1.32 ⫾ 0.04a,b

Values are means ⫾ SE; n ⫽ 27, 23, and 23 rats in the control, ␣-HICA, and leucine groups, respectively. Data from each treatment group were combined for all studies (i.e., casted ⫹ no recovery, casted ⫹ 7 days recovery, and casted ⫹ 14 days recovery), as there was no difference in rates of protein synthesis between individual studies (data not shown). Means with different superscripted letters are statistically different (P ⬍ 0.05); values with the same letter are not significantly different. Units ⫽ nmol phenylalanine (Phe) incorporated per hour per milligram of tissue protein.

endogenous Cr clearance, which provides a more accurate estimate of glomerular filtration rate. Cr clearance did not differ between groups (control ⫽ 1.24 ⫾ 0.08, ␣-HICA ⫽ 1.56 ⫾ 0.25, and Leu ⫽ 1.41 ⫾ 0.09 ml·min⫺1·g kidney⫺1). Markers of nutrition and metabolism (total protein, albumin, glucose, and triglycerides) did not differ among the three groups. In contrast, the plasma FFA concentration was reduced 35% in the rats treated with ␣-HICA compared with timematched control rats (Table 3). Conversely, Leu-treated rats had a 20% increase in their circulating FFA concentration compared with control values. The serum insulin concentration did not differ between control and ␣-HICA-treated rats (Table 3) but was increased 95% in the Leu-treated group. There was no significant difference in the hematocrit, hemoglobin concentration, the number of white blood cells, red blood cells, and platelets, or the percentage of neutrophils, lymphocytes, monocytes, eosinophiles, or reticulocytes among the three groups (data not shown). Plasma amino acid concentrations. The concentration of total and individual plasma amino acids at the end of the casting period, after casting, and 7 and 14 days of recovery is presented in Tables 4, 5, and 6, respectively. To reiterate, blood for these amino acids was collected 3– 6 h after removal of food. At the end of the 7-day casting period, there were only a few modifications in amino acid concentrations. However, the plasma concentration of hydroxyproline was decreased 25%, and the concentrations of glutamine and proline were increased 30% in ␣-HICA-treated rats compared with control rats. The concentration of individual amino acids did not differ between Leu-treated and control-fed rats. The plasma leucine concentration at this time point was not elevated in Leu-treated rats compared with control values (Table 4). Overall, the concentration of total plasma amino acids did not differ between the three groups. At the conclusion of the 7-day recovery period, threonine, glutamine, proline, methionine, leucine, and phenylalanine were all increased in ␣-HICA-treated rats compared with control values (Table 5). The plasma leucine concentration at this time point was increased in Leu-treated rats compared with



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Table 6. Plasma AA concentrations after cast removal and 14 days of recovery

Table 4. Plasma AA concentrations after 7 days of immobilization Taurine Aspartate Hydroxy-L-proline Threonine Serine Asparagine Glutamate Glutamine Proline Glycine Alanine Citrulline Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Tryptophan Ornithine Lysine Histidine Arginine Total AA

Control

␣-HICA

Leucine

183 ⫾ 10 19 ⫾ 1 27 ⫾ 2a 245 ⫾ 15 235 ⫾ 7 33 ⫾ 2 77 ⫾ 7a 642 ⫾ 33 179 ⫾ 23a 218 ⫾ 15 447 ⫾ 35 55 ⫾ 2 160 ⫾ 8 49 ⫾ 3 81 ⫾ 3 130 ⫾ 8 69 ⫾ 5 53 ⫾ 2 69 ⫾ 6 90 ⫾ 18 453 ⫾ 22 54 ⫾ 2 133 ⫾ 23 3,703 ⫾ 110

187 ⫾ 12 19 ⫾ 1 20 ⫾ 1b 246 ⫾ 11 225 ⫾ 8 34 ⫾ 4 102 ⫾ 9b 613 ⫾ 22 235 ⫾ 20b 193 ⫾ 15 415 ⫾ 30 55 ⫾ 1 142 ⫾ 8 49 ⫾ 1 71 ⫾ 4 151 ⫾ 6 73 ⫾ 3 54 ⫾ 1 66 ⫾ 6 70 ⫾ 14 415 ⫾ 9 55 ⫾ 1 156 ⫾ 22 3,646 ⫾ 63

180 ⫾ 22 19 ⫾ 1 23 ⫾ 2a,b 215 ⫾ 17 237 ⫾ 12 32 ⫾ 3 68 ⫾ 5a 574 ⫾ 13 151 ⫾ 9a 217 ⫾ 20 372 ⫾ 31 56 ⫾ 2 146 ⫾ 11 43 ⫾ 2 78 ⫾ 5 143 ⫾ 16 60 ⫾ 7 55 ⫾ 3 64 ⫾ 7 58 ⫾ 6 402 ⫾ 23 50 ⫾ 2 165 ⫾ 16 3,408 ⫾ 132

Taurine Aspartate Hydroxy-L-proline Threonine Serine Asparagine Glutamate Glutamine Proline Glycine Alanine Citrulline Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Tryptophan Ornithine Lysine Histidine Arginine Total AA

Control

␣-HICA

Leucine

174 ⫾ 13 16 ⫾ 2 28 ⫾ 2a 261 ⫾ 17a 259 ⫾ 21 41 ⫾ 3 84 ⫾ 5a 661 ⫾ 23 200 ⫾ 16a 210 ⫾ 25 586 ⫾ 25 65 ⫾ 4 157 ⫾ 12 58 ⫾ 3 71 ⫾ 5 110 ⫾ 9a 84 ⫾ 4 46 ⫾ 3a 75 ⫾ 3 86 ⫾ 12 432 ⫾ 14 51 ⫾ 3 121 ⫾ 21 3,878 ⫾ 104

156 ⫾ 11 18 ⫾ 2 21 ⫾ 1b 358 ⫾ 9b 235 ⫾ 17 39 ⫾ 2 123 ⫾ 9b 650 ⫾ 35 331 ⫾ 19b 196 ⫾ 16 478 ⫾ 19 68 ⫾ 3 169 ⫾ 17 59 ⫾ 4 89 ⫾ 7 165 ⫾ 9b 78 ⫾ 5 57 ⫾ 2b 74 ⫾ 4 65 ⫾ 10 409 ⫾ 25 54 ⫾ 2 116 ⫾ 10 4,010 ⫾ 148

148 ⫾ 20 17 ⫾ 2 27 ⫾ 1a 346 ⫾ 12b 279 ⫾ 21 44 ⫾ 3 77 ⫾ 6a 683 ⫾ 34 181 ⫾ 14a 176 ⫾ 30 507 ⫾ 31 68 ⫾ 4 160 ⫾ 12 55 ⫾ 3 88 ⫾ 10 158 ⫾ 8b 72 ⫾ 4 59 ⫾ 3b 77 ⫾ 5 70 ⫾ 9 412 ⫾ 21 55 ⫾ 3 131 ⫾ 12 3,890 ⫾ 106

Values are means ⫾ SE; n ⫽ 8, 7, and 8, in the control, ␣-HICA, and leucine groups, respectively. AA, amino acids. All concentrations are in ␮mol/l. Values in the same row with a different superscripted letter are statistically different (P ⬍ 0.05).

Values are means ⫾ SE; n ⫽ 8/group. All concentrations are in ␮mol/l. Values in the same row with a different superscripted letter are statistically different (P ⬍ 0.05).

control values, although the increase was less pronounced than that seen in the ␣-HICA-treated rats (25 vs. 50%, respectively). Again, the concentration of total plasma amino acids did not differ between the three groups at this time point.

Plasma amino acid concentrations in Leu- and ␣-HICAtreated rats at the conclusion of the 14-day recovery period were generally comparable with those observed at 7 days (Table 6). However, at this time point, the plasma leucine concentration did not differ between the ␣-HICA- or Leutreated rats, with both groups being increased 45–50% above control values. Muscle mass and protein balance. The wet weight of the noncasted control gastrocnemius did not differ between groups (Fig. 2A). Immobilization decreased gastrocnemius mass in all groups to a similar extent (control ⫽ 0.58 ⫾ 0.06, ␣-HICA ⫽ 0.61 ⫾ 0.09, and Leu ⫽ 0.57 ⫾ 0.08 g). There was no increase in gastrocnemius mass 7 days after removal of the cast in rats consuming the control diet (Fig. 2B). Furthermore, muscle mass was also not altered by ␣-HICA or Leu treatment at this time point. However, by recovery day 14, all immobilized muscles had regained some mass (Fig. 2C). At this time, a reduction in mass of the previously immobilized muscle was still detected in the control and Leu-treated rats compared with the contralateral uncasted muscle. In contrast, the weights of the previously casted and uncasted control gastrocnemius in the ␣-HICA-treated group did not differ. These group differences were more apparent when the data were expressed as the increment in muscle mass (e.g., uncasted ⫺ casted for same animal; Fig. 2D). Protein synthesis in the uncasted muscle from control and Leu-treated rats did not differ (Table 2). In contrast, the basal rate of protein synthesis in the uncasted muscle from the ␣-HICA-treated rats was significantly lower (10%) than the control rats. Casting decreased protein synthesis to the same extent in control and Leu-treated rats. In contrast, there was no immobilization-induced decrease in muscle protein synthesis

Table 5. Plasma amino acid concentrations after cast removal and 7 days of recovery Taurine Aspartate Hydroxy-L-proline Threonine Serine Asparagine Glutamate Glutamine Proline Glycine Alanine Citrulline Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Tryptophan Ornithine Lysine Histidine Arginine Total AA

Control

␣HICA

Leucine

160 ⫾ 12 18 ⫾ 1 27 ⫾ 1a 280 ⫾ 11a 262 ⫾ 11a,b 45 ⫾ 3 82 ⫾ 5a 653 ⫾ 18 210 ⫾ 15a 232 ⫾ 15 575 ⫾ 23 64 ⫾ 3 148 ⫾ 11 48 ⫾ 1a 68 ⫾ 4 104 ⫾ 8a 80 ⫾ 3 47 ⫾ 2a 76 ⫾ 2 87 ⫾ 11 407 ⫾ 13 51 ⫾ 2 102 ⫾ 16 3,826 ⫾ 85

140 ⫾ 10 16 ⫾ 1 20 ⫾ 1b 341 ⫾ 7b 230 ⫾ 9a 45 ⫾ 3 111 ⫾ 8b 665 ⫾ 15 329 ⫾ 20b 176 ⫾ 10 541 ⫾ 18 60 ⫾ 2 170 ⫾ 10 60 ⫾ 2b 92 ⫾ 8 163 ⫾ 10b 78 ⫾ 5 56 ⫾ 1b 77 ⫾ 5 63 ⫾ 9 401 ⫾ 22 56 ⫾ 1 111 ⫾ 10 4,003 ⫾ 68

131 ⫾ 12 18 ⫾ 1 26 ⫾ 1a 324 ⫾ 16b 294 ⫾ 15b 49 ⫾ 2 82 ⫾ 5a 671 ⫾ 20 185 ⫾ 13a 232 ⫾ 17 516 ⫾ 29 68 ⫾ 4 156 ⫾ 6 51 ⫾ 2a 78 ⫾ 2 130 ⫾ 2c 67 ⫾ 4 56 ⫾ 2b 78 ⫾ 4 85 ⫾ 11 408 ⫾ 18 55 ⫾ 2 128 ⫾ 10 3,889 ⫾ 97

Values are means ⫾ SE; n ⫽ 10, 6, and 7 in the control, ␣-HICA, and leucine groups, respectively. All concentrations are in ␮mol/l. Values in the same row with a different superscripted letter are statistically different (P ⬍ 0.05).



2.5 2.0

*

1.5

0.5 0.0

HICA

2.5

D *

2.0 1.5 1.0 0.5 0.0

HICA

Leucine

Immobilized

2.5 2.0 1.5

*

*

*

1.0 0.5 0.0

Control

Immobilized

*

Control

Control

Leucine

14-day Recovery Period Control

Gastrocnemius Weight (g)

*

*

1.0

Control

7-day Recovery Period

Immobilized



Control

C

B

Immobilization Period


A

E421

HICA

Leucine

Increment in Mass after 14-day Recovery b

0.05 0.00 -0.05

Fig. 2. Weight of gastrocnemius in the uncasted (control) limb or the contralateral limb either at the end of 7 days of immobilization (A), after 7 days of immobilization ⫹ 7 days of recovery (B), or after 14 days of recovery (C). Values are means ⫾ SE. The sample sizes for the control, ␣-HICA, and Leu groups are n ⫽ 8, 10, and 9, respectively, for the immobilization period; n ⫽ 11, 7, and 7, respectively, for the 7-day recovery period; and n ⫽ 8/group for the 14-day recovery period. *P ⬍ 0.05 compared with uncasted control muscle from the same group at the same time point. The weight of the uncasted muscle did not differ among the different dietary groups for any of the 3 time points. D: change in absolute gastrocnemius weight between the noncasted control muscle and the previously immobilized muscle from the same rat after 14 days of recovery. Values with different letters are significantly different, P ⬍ 0.05.

-0.10 -0.15

a

a

-0.20

Control

in ␣-HICA-treated rats (Fig. 3A, right). After a 7-day recovery period, protein synthesis was increased in the previously immobilized muscle (Fig. 3B). Although the increment in protein synthesis tended to be greater (P ⫽ 0.16) in the ␣-HICAtreated rats compared with control rats (0.77 ⫾ 0.17 vs. 0.46 ⫾ 0.13 nmol Phe·h⫺1·mg protein⫺1, respectively), this difference did not achieve statistical significance. By day 14 of recovery, the rate of protein synthesis in the previously immobilized leg was not different from the uncasted muscle for the control and Leu-treated groups (Fig. 3C). However, protein synthesis in the previously immobilized muscle of the ␣-HICA-treated group remained increased compared with the contralateral control muscle. At this time, the increment in muscle protein synthesis was twofold greater in the ␣-HICA-treated rats compared with either of the other two groups (Fig. 3C, right). We determined gastrocnemius weight and protein synthesis simultaneously on a separate group of pair-fed naive rats (n ⫽ 7) provided the control diet for the same period of time. The weight of the left and right gastrocnemius from this control naive group (2.17 ⫾ 0.04 and 2.18 ⫾ 0.05 g, respectively) did not differ from the weight of the uncasted control muscle in the control-fed group (2.16 ⫾ 0.05 g). Furthermore, protein synthesis in the left (1.31 ⫾ 0.07 nmol Phe·h⫺1·mg protein⫺1) and right gastrocnemius (1.39 ⫾ 0.08 nmol Phe·h⫺1·mg protein⫺1) from naive control rats did not differ from the uncasted muscle in control-fed rats (1.36 ⫾ 0.07 nmol Phe·h⫺1·mg protein⫺1). Both these and previously published data (30) and suggest that the contralateral muscle in casted rats is an appropriate internal control and does not undergo significant compensatory hypertrophy. S6K1 and 4E-BP1 phosphorylation. Alterations in mTOR kinase activity typically lead to coordinate changes in the

HICA

Leucine

phosphorylation state of its downstream substrates S6K1 and 4E-BP1, which are often proportional to changes in global rates of protein synthesis (17). The basal level of S6K1 phosphorylation (Thr389) in the uncasted control muscle showed considerable variability, and hence, there were no statistical differences among the various treatment groups (Fig. 4A). Immobilization resulted in a consistent reduction in S6K1 phosphorylation that did not differ between groups. At the conclusion of 7 days of recovery, S6K1 phosphorylation was increased in the previously immobilized muscle from all rats regardless of diet (Fig. 4B). By day 14 of recovery, there was no difference in S6K1 phosphorylation between the control and previously immobilized muscle, except in the ␣-HICA-treated rats, which showed a small, albeit statistically significant, increase in the previously immobilized muscle (Fig. 4C). These casting- and reloading-induced changes in S6K1 phosphorylation were not the result of changes in total S6K1 (Fig. 4D). The basal level of 4E-BP1 phosphorylation (Thr37/Thr46) in the uncasted control muscle did not differ between control rats and animals treated with ␣-HICA or Leu (Fig. 5A). However, 4E-BP1 phosphorylation was increased in muscle from the casted limb for all groups compared with the contralateral control muscle from the same rat (Fig. 5A). Although the overall magnitude of this increase was small (⬃20%), analysis of the paired data from the same rat indicated that this change was statistically significant (P ⬍ 0.05). The phosphorylation of 4E-BP1 remained elevated after 7 days of recovery, regardless of treatment (Fig. 5B). By day 14 of recovery, 4E-BP1 phosphorylation had returned to control values in all groups, except for those rats fed ␣-HICA, where phosphorylation remained elevated (Fig. 5C). These casting- and reloading-induced



Muscle Protein Synthesis (nmol Phe/h/mg protein)

Control

B

1.5 1.2

*

*

0.9 0.6 0.3 0.0

Control

HICA

C

Immobilized

2.5

*

*

2.0

*

1.5 1.0 0.5 0.0 HICA

Control

0.0 -0.1 -0.2 -0.3 a

-0.4 -0.5 Control

HICA

Leucine

Leucine

1.0 0.8 0.6 0.4 0.2 0.0 Control

HICA

Leucine

14-day Recovery Control


Leucine

b 0.1



Fig. 3. In vivo-determined protein synthesis in gastrocnemius in the uncasted limb or the contralateral limb at the end of 7 days of immobilization (A), after 7 days of immobilization ⫹ 7 days of recovery (B), or after 14 days of recovery (C). Values are means ⫾ SE. The sample sizes for the control, ␣-HICA, and Leu groups are n ⫽ 8, 10, and 9, respectively, for the immobilization period; n ⫽ 11, 7, and 7, respectively, for the 7-day recovery period; and n ⫽ 8/group for the 14-day recovery period. A–C, left: quantitation of absolute rates of muscle protein synthesis. A–C, right: change (increment or decrement) in muscle protein synthesis (control ⫺ casted) in the same animal. *P ⬍ 0.05 compared with uncasted control muscle from the same group. There was no difference in the weight of the uncasted muscle among the different dietary groups. Values with different letters are significantly different, P ⬍ 0.05.

Immobilized

Change in Protein Synthesis (nmol Phe/h/mg protein)



A

Immobilized

*

1.8 1.5 1.2 0.9 0.6 0.3 0.0 Control

changes in 4E-BP1 phosphorylation were not the result of changes in total 4E-BP1 (data not shown). 3-MH, atrogenes, and proteasome activity. Whole body myofibrillar degradation was estimated based on the 3-MH/Cr ratio in urine (1). The 3-MH/Cr ratio did not differ between control, ␣-HICA-treated, and Leu-treated rats at the conclusion of the 7-day period of immobilization (2.44 ⫾ 0.16, 2.36 ⫾ 0.19, and 2.22 ⫾ 0.14 ␮mol·mmol⫺1·100⫺1, respectively; P ⫽ not significant). However, at the end of the 7-day recovery period, the 3-MH/Cr ratio for each group was decreased (1.97 ⫾ 0.07, 1.78 ⫾ 0.09, and 1.97 ⫾ 0.16 ␮mol·mmol⫺1·100⫺1, respectively; P ⬍ 0.05 compared with the respective value obtained during the immobilization period for the same group), although only the decrease in the control and ␣-HICA-treated rats achieved statistical significance. The 3-MH/Cr ratio for each group at the end of the 14-day recovery period (1.95 ⫾ 0.08, 1.75 ⫾ 0.07, and 1.92 ⫾ 0.08 ␮mol·mmol⫺1·100⫺1; P ⬍ 0.05 compared with the respective value obtained during the immobilization period for the same group) did not differ from values at the 7-day recovery time point.

HICA

Leucine


E422

0.4 b 0.3 0.2

a

a

0.1 0.0 Control

HICA

Leucine

We also determined the mRNA content for the striated muscle-specific E3 ligases atrogin-1 and MuRF1, which are increased in diverse catabolic conditions (7, 26). Collectively, atrogin-1 and MuRF1 have been referred to as “atrogenes.” Immobilization markedly increased both atrogin-1 and MuRF1 mRNA compared with the contralateral muscle from the same rat (Fig. 6, A and B). The magnitude of this increase did not differ with either ␣-HICA or Leu treatment. There were no significant differences between the uncasted control muscle from the control group and that of the uncasted muscle from either of the two treatment groups (Fig. 6, A and B, left, open bars). The poly-Ub mRNA content was also increased in immobilized muscle compared with the contralateral control muscle (Fig. 6C), and this increase was comparable between groups. An in vitro proteasome activity assay yielded comparable results, with immobilization increasing proteasome activity by 30% in control rats, but this increment was not altered when rats were fed either ␣-HICA or Leu (Fig. 6D). Collectively, these markers of muscle protein breakdown were consistently elevated at the end of the immobilization



Control

S6K1 phosphorylation (T389; AU/total)

C


1.00 0.75

*

*

*

0.25 0.00 Control

Control

Immobilized

1.25

0.50

14-day Recovery


A

HICA

*

1.5 1.2 0.9 0.6 0.3 0.0 Control

Leucine

C


Immobilized


3

2

S6K1-P S6K1-T

*

HICA

Control


*

Immobilized

1.8

D B

E423

*

I

Leucine

HICA C

I

Leu C

I

Fig. 4. S6K1 phosphorylation (Thr389) in gastrocnemius in the uncasted or contralateral limb at the end of 7 days of immobilization (A), after 7 days of immobilization ⫹ 7 days of recovery (B), or after 14 days of recovery (C). Values are means ⫾ SE. The sample sizes for the control, ␣-HICA, and Leu groups are n ⫽ 8, 10, and 9, respectively, for the immobilization period; n ⫽ 11, 7, and 7, respectively, for the 7-day recovery period; and n ⫽ 8/group for the 14-day recovery period. Bar graphs are densitometric quantitation of all immunoblots, where bars represent means ⫾ SE; *P ⬍ 0.05 compared with uncasted control muscle from the same group. Values with different letters are significantly different, P ⬍ 0.05. D: representative Western blots for total and Thr389-phosphorylated S6K1 in gastrocnemius from rats in all treatment groups. C, control; I, immobilized.

S6K1-P 7-d recovery S6K1-T

1

S6K1-P

0 Control

HICA

Leucine

14-d recovery S6K1-T

period and were not altered by treatment with ␣-HICA or Leu. When examined after 7 days of recovery, the castinginduced increases in atrogin-1, MuRF1, and poly-Ub had been completely resolved (Fig. 6, A–D, right). However, there tended to be a twofold increase in atrogene and poly-Ub mRNA content in the previously immobilized muscle from control rats that was not seen in either the ␣-HICA- or Leu-treated groups. Proteasome activity did not differ between previously immobilized muscle and the contralateral control muscle at this time point. Finally, there was no casting- or treatment-induced difference in either atrogene after 14 days of recovery (data not shown). DISCUSSION

Effect of ␣-HICA and Leu treatment on basal conditions. We assessed the ability of a complete diet containing either 5% ␣-HICA or Leu to ameliorate the atrophic response produced by immobilization and/or to improve the ability of muscle to recover mass after cast removal. Treatment with ␣-HICA or Leu for up to 3 wk produced few significant differences for numerous biochemical and hematological end points compared with rats fed the isocaloric isonitrogenous fed controls. One exception might be the 20% increase in the plasma alkaline phosphatase concentration observed in ␣-HICA-treated rats. Although histopathological analysis of liver was not performed, we do not believe this increase signifies hepatic damage because the increase is considerably smaller than the severalfold increase detected in other hepatic diseases (33) and because AST and ALT levels were not coordinately increased. Furthermore, weights of various organs (e.g., liver, heart,

spleen, adrenal, kidney, testes) were unaffected, and there was no significant change in the rate of in vivo-determined protein synthesis for liver or heart. Hence, these various treatments, when superimposed on a complete diet, do not appear to have any overt organ toxicity at the relatively high doses used in the current study. Branched-chain amino acids (BCAAs) in general and Leu in particular can acutely increase protein synthesis and decrease muscle proteolysis under both in vitro and in vivo conditions (11, 45). Other studies have reported that short-term infusion of BCAAs or Leu decreases proteolysis without significantly increasing protein synthesis in skeletal muscle (48, 57). However, there is little evidence that long-term Leu treatment leads to increased muscle mass, especially under conditions that are not inherently catabolic (3, 4). In animal studies, the range of Leu treatment sufficient to stimulate muscle protein synthesis appears to be relatively narrow, ranging from 1.35 to 1.90 g/kg body wt, when added to other nutrients provided by a complete diet (15, 74, 79). Rats in the current study were treated with Leu to a comparable extent. The plasma Leu concentration determined at the end of the 7-day casting period did not differ between control and Leu-treated rats, although Leu concentrations were progressively increased throughout the entire 14day recovery period. The reason for the initial lack of increase in plasma Leu in the Leu-treated rats is not known, but we cannot exclude the possibility of a difference in the temporal pattern of food consumption over the duration of the experimental protocol in the same group or between groups. Moreover, diet-induced increases in plasma Leu are transient in nature, and we may have missed increases because of our single-time point determinations. Although temporal differ-



A 4E-BP1 phosphorylation (T37/T46; AU/total)

Control

B

1.5

*

*

Control

*

0.9 0.6 0.3 0.0 Control

14-day Recovery

Immobilized

1.2

HICA

Leucine

*

1.5 1.2 0.9 0.6 0.3 0.0 Control

HICA

HICA C

I

I

Leu C

I

Immobilized

*

1.5

Leucine

Control C

Control

Immobilized

1.8

D

7-day Recovery

4E-BP1 phosphorylation (T37/T46; AU/total)

Fig. 5. Eukaryotic initiation factor (eIF)4E-binding protein-1 (4E-BP1) phosphorylation (Thr37/Thr46) in gastrocnemius in the uncasted or contralateral limb at the end of 7 days of immobilization (A), after 7 days of immobilization ⫹ 7 days of recovery (B), or after 14 days of recovery (C). Values are means ⫾ SE. The sample sizes for the control, ␣-HICA, and Leu groups are n ⫽ 8, 10, and 9, respectively, for the immobilization period; n ⫽ 11, 7, and 7, respectively, for the 7-day recovery period; and n ⫽ 8/group for the 14-day recovery period. Bar graphs are densitometric quantitation of all immunoblots, where bars represent means ⫾ SE. *P ⬍ 0.05 compared with uncasted control muscle from the same group. Values with different letters are significantly different, P ⬍ 0.05. D: representative Western blots for Thr37/Thr46-phosphorylated 4E-BP1 in gastrocnemius from rats in all treatment groups; eIF4E was used as a loading control. Total 4E-BP1 was not different between groups (data not shown).

C


4E-BP1 phosphorylation (T37/T46; AU/total)

E424

*

4EBP1-P


*

eIF4E

1.2 0.9

4EBP1-P 7-d recovery

0.6

eIF4E

0.3 0.0

4EBP1-P Control

HICA

Leucine

14-d recovery eIF4E

ences in feeding behavior are difficult to control, this limitation could be circumvented in future studies by employing methods (e.g., doubly labeled water) that have the potential to determine protein synthesis and degradation over the course of 1 or more days (6). Dietary treatment with ␣-HICA for 4 wk in humans was previously reported to increase lean body mass in response to

intensive physical training (51). However, in the current study, ␣-HICA added to the diet did not alter the weight of the uncasted gastrocnemius compared with uncasted muscle from control rats. This difference may be related to the level of physical activity that was limited in our study (e.g., cage bound) compared with the previous report (e.g., intensive training), to the manner in which ␣-HICA was provided

*

Immobilized

*

Recovery (7d)

*

600 300 0

Immobilization

Leucine

HICA

Control

Leucine

0

Recovery


3

*

* *

2 1 0

Immobilization

Recovery

Leucine

300

4

HICA

*

600

HICA

0

D

900

Control

100

Control

* *

200

Proteasome Activity (µmol/min/mg)

1200

* * *

HICA

900

300

Leucine

1200

400

Control

1500

Control

PolyUb mRNA (AU/GAPDH - % control)

C

B

MuRF1 mRNA (AU/GAPDH - % control)

Fig. 6. Content of atrogin-1, muscle RING finger 1 (MuRF1), and polyubiquitin (Ub) mRNA as well as proteasome activity in gastrocnemius in the uncasted or contralateral limb either at the end of 7 days of immobilization or immobilization ⫹ 7 day recovery. A, B, and C: atrogin-1, MuRF1, and poly-Ub mRNA, respectively, normalized to GAPDH and expressed as percentage of the uncasted muscle from the control-fed group. The GAPDH mRNA content did not differ among muscles from the 3 experimental groups (data not shown). D: 20S proteasome activity determined in immobilized and control muscle after casting. Values are means ⫾ SE. The sample size for the control, ␣-HICA, and Leu groups is n ⫽ 8, 10, and 9, respectively, for the immobilization period and n ⫽ 11, 7, and 7, respectively, for the 7-day recovery period. There were no immobilization- and/or diet-induced changes in either atrogin-1, MuRF1, or poly-Ub mRNA content at the 14-day recovery time point (data not shown). *P ⬍ 0.05 compared with uncasted control muscle from the same group.

Atrogin-1 mRNA (AU/GAPDH - % control)

A


(continuously in food vs three discrete doses each day), or to other unidentified variables. Under basal conditions, treatment with ␣-HICA did decrease the basal rate of global protein synthesis by 10% in the uncasted muscle compared with the uncasted muscle from control-fed rats. This change in muscle protein synthesis in ␣-HICA-treated rats was independent of a change in the plasma insulin and total amino acid concentrations. Treatment with ␣-HICA did not significantly alter the basal rate of proteasome activity or mRNA content for atrogin-1 or MuRF1 in the uncasted muscle compared with the uncasted muscle from either control or Leu-fed rats. Atrophic response to immobilization. Various models have been used to investigate disuse atrophy, including hindlimb suspension (e.g., unloading), extended bed rest, denervation, and hindlimb immobilization, the latter being either uni- or bilateral. Although each model has advantages and disadvantages (49) relative to the others, the present study used unilateral casting to produce disuse atrophy. This model allows comparison of immobilized muscle with control muscle in the same rat, maintains neural innervation to the hindlimb musculature, and permits recovery-type studies to be performed after cast removal. It is also arguably more clinically relevant than other models. Our study was also conducted in ⬃14-wk-old Wistar rats that were no longer in the rapid growth phase of their development. Hence, differences between the uncasted and casted muscle are more likely to represent an atrophic response as opposed to a failure of normal muscle growth. However, it is noteworthy that Wistar rats of this age regained muscle mass relatively quickly. In general, limb immobilization has been reported to decrease muscle mass and fiber diameter in mice, rats, and humans (24, 35, 37, 40, 54, 61). This disuse atrophy is caused by an imbalance between rates of protein synthesis and degradation. The majority of evidence supports a reduction in mixed or global muscle protein synthesis that commences as early as 6 h after immobilization (10) and can remain suppressed for several days to weeks (24, 35, 69). In other catabolic conditions characterized by muscle wasting (e.g., sepsis, alcohol, excess glucocorticoids, inflammatory cytokine excess), such a reduction in muscle protein synthesis is temporally associated with a suppression in mTOR activity, as evidenced by a concomitant reduction in phosphorylation of S6K1 and 4E-BP1 (35, 41, 42, 75). Our current data demonstrate a clear decrease in S6K1 phosphorylation (as well as decreased S6 phosphorylation; data not shown) in response to disuse. Such a decrease is consistent with the reduction in muscle protein synthesis observed in our study during immobilization and has been reported in some (2, 27) but not other (28, 40) studies. In contrast, we detected a small (20%) increase in 4E-BP1 phosphorylation in the immobilized muscle from all rats compared with the contralateral uncasted muscle. Again, this increase in 4E-BP1 phosphorylation is consistent with some studies (28, 40, 70) but differs from the reduced phosphorylation state of this protein observed by others (2, 27). The reason for this discordant 4E-BP1 response is unclear but emphasizes the importance of directly determining protein synthesis and not just assessing the phosphorylation state of select signaling proteins. Disuse atrophy is also mediated at least in part via increased muscle proteolysis (56, 65). The majority of the data support the conclusion that disuse upregulates Ub-proteasome-depen-

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dent protein degradation (40, 56). For example, disuse increases the mRNA for several components of the Ub-proteasome pathway, including polyubiquitin, atrogin-1, and MuRF1 (2, 27, 28, 40). This atrogene response is an archetypical marker of many proteolytic conditions (including cancer cachexia, denervation, glucocorticoid excess, fasting, hindlimb suspension, and sepsis), with changes coordinating with rates of ATP-dependent proteolysis (20). Our current data are consistent with these previous observations. In addition, others have corroborated the increased ubiquitin immunoreactivity in muscle homogenates of unilaterally immobilized skeletal muscle (40, 50), a result comparable with that obtained by immunoblot analysis in other well-characterized catabolic states (66, 76). Although these data do not directly assess conjugating activity, they nevertheless support the concept that unilateral immobilization enhances the ubiquitination of muscle proteins and argues for involvement of energy-dependent proteolysis by the proteasome during hindlimb casting. Such a conclusion is supported by our current data, indicating an increase in in vitro-determined proteasome activity. Finally, the injection of the proteasome inhibitor Velcade (also known as PS-341 or bortezomib) partially ameliorates the loss of gastrocnemius mass following 3 days of immobilization (40), thereby directly implicating the proteasome in catalyzing a portion of the muscle wasting occurring in this model of disuse. Collectively, these various lines of investigation provide strong physiological evidence that increased protein degradation causes gastrocnemius weight loss with immobilization, and interventions that limit proteolysis during the atrophic period may have therapeutic potential. Impact of ␣-HICA or Leu on the atrophic response. Acute administration of Leu stimulates global protein synthesis in skeletal muscle predominantly by enhancing mRNA translation initiation (17, 21). However, Leu treatment alone failed to prevent the casting-induced decrease in protein synthesis in gastrocnemius. It is possible that Leu treatment failed to increase the plasma Leu concentration to a level necessary for mTOR activation. Alternatively, an anabolic resistance to either amino acid or Leu stimulation has been reported in other catabolic conditions (21, 34, 43) as well as in aging (16, 62). Moreover, our data are consistent with those of a previous study in which the short-term infusion of BCAAs also did not reverse the casting-induced decrease in protein synthesis (or the increase in proteolysis) (38). Contrary to the lack of Leu effect, feeding rats a complete diet containing ␣-HICA alone prevented the casting-induced decrease in muscle protein synthesis. In select catabolic states, Leu decreases muscle protein breakdown and lowers the expression for some components of the Ub-proteasome pathway (5, 78). However, cachexia may also render muscle proteolysis resistant to the inhibitory effect of Leu (12). In the current study, Leu treatment did not alter surrogate markers of proteolysis, i.e., atrogin-1 and MuRF1, or proteasome activity per se, so these casting-induced increases were comparable in magnitude with those seen in control rats. In contrast to the ability of ␣-HICA to modulate the synthetic side of the protein balance equation, ␣-HICA was ineffective at preventing or ameliorating the immobilization-induced increase in atrogene mRNA content or proteasome activity. Although this is the first report of the effect of ␣-HICA on muscle proteolysis, ␣-HICA has been shown to decrease pro-


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tein degradation in liver (55). These latter data suggest that neither treatment used in the current study effectively limited the increase in proteolysis produced by immobilization. It is noteworthy that despite the ability of ␣-HICA to prevent the normal reduction in muscle protein synthesis, this metabolite failed to prevent or ameliorate the accompanying reduction in muscle mass per se. Neither treatment ameliorated the atrophic response to disuse. Impact of ␣-HICA or Leu on recovery from immobilization. Seven days after cast removal (i.e., “recovery”), protein synthesis was increased in the previously immobilized muscle in all groups. Such a compensatory increase in muscle protein synthesis has been reported to start as early as 6 –24 h after cast removal (25, 69). Moreover, the increased protein synthesis was associated with the enhanced phosphorylation of 4E-BP1 and S6K1, implicating activation of mTOR. Furthermore, the casting-induced increase in atrogin-1 and MuRF1 mRNA content and proteasome activity had returned to basal values by 7 days of recovery, which is consistent with previous reports (25, 72). Despite these changes, there was no net increase in the mass of the previously immobilized muscle in control rats after 7 days of recovery. These discordant results suggest that other pathways of protein degradation may remain activated, thereby minimizing the effectiveness of the compensatory increase in protein synthesis. Alternatively, a time lag may exist between any modification of protein synthesis or degradation and their impact in terms of muscle mass. Thus, it may be too early to detect a beneficial effect on muscle mass if the increased protein synthesis and/or decreased degradation were present for only 2–3 days before the measurement. Although partial recovery of muscle mass has been observed when the duration of immobilization is relatively limited (e.g., several days), this lack of change after cast removal has been reported in studies where the immobilization was prolonged for at least 1 wk (9, 50, 72), such as in the current investigation. Neither treatment modified the recovery-induced change in protein synthesis, proteolysis, or muscle mass after 7 days. It is noteworthy that the Leu- and ␣-HICA-treated rats had a 25 and 50% increase, respectively, in plasma leucine. The mechanism by which ␣-HICA treatment increases Leu was not determined, but the enzymes necessary for the conversion of ␣-HICA to its corresponding ␣-keto analog and finally back to Leu have been identified (14, 68). However, we cannot exclude other possibilities that might result from alterations in intestinal absorption and/or oxidation of Leu. It is unclear whether such an in vivo increase in Leu is sufficient to increase muscle protein synthesis, but in our study it did not enhance the normal anabolic response to reloading. In general, when examined across all groups at all times, the elevations in plasma Leu were poorly correlated with increases in muscle protein synthesis and indices of mTOR activity in the previously immobilized muscle. By recovery day 14, the compensatory increase in protein synthesis detected in the previously immobilized muscle at 7 days had returned to basal values in the control and Leu-fed rats. In contrast, protein synthesis was still increased in the previously immobilized muscle from rats fed ␣-HICA. The elevated protein synthesis was associated with a coordinate increase in the mass of the previously immobilized muscle back to control levels. The complete recovery of mass in the atrophic limb differed from the ⬃70% partial recovery of gastrocnemius

mass in rats treated with Leu or fed the control diet. Because the plasma Leu concentrations did not differ between Leu- and ␣-HICA-treated rats at this time, a differential increase in this BCAA does not appear to be responsible for the anabolic effect of ␣-HICA. However, we cannot exclude the potential for a direct anabolic effect of ␣-HICA on skeletal muscle. It is unclear why protein synthesis and mTOR activity remain elevated in the previously immobilized muscle in ␣-HICAtreated rats after 14 days of recovery, a time when the mass of the atrophic muscle has returned to baseline values. The improved recovery of muscle mass in the ␣-HICA-treated group also does not appear to be attributable to a differential response of protein degradation since atrogin-1, MuRF1, and poly-Ub mRNA content and proteasome activity had returned to basal values by day 7 of recovery and continued to be comparable with control values on recovery day 14. Finally, of the changes in plasma amino acid concentrations detected in ␣-HICA-treated rats, one of the most striking and consistent was the 30 – 45% increase in circulating glutamate. Although the etiology of the increased glutamate was not determined, glutamate and Leu are readily taken up by muscle and linked together through various transamination reactions involved in tricarboxylic acid cycle anaplerosis (59). In contrast, elevations in plasma glutamate were not seen in rats treated with Leu. Hence, we cannot exclude the possibility that the beneficial effect of ␣-HICA treatment during the reloading phase, at least in part, was associated with the elevated glutamate maintaining oxidative metabolism and ATP turnover. In summary, our data demonstrate that long-term treatment with ␣-HICA or Leu did not prevent the typical castinginduced increase in atrogene expression or proteasome activity. Neither ␣-HICA nor Leu prevented or ameliorated the reduction in muscle mass produced by unilateral hindlimb immobilization in adult rats. However, treatment with ␣-HICA as part of a nutritionally complete diet throughout the period of immobilization and recovery produced a sustained increase in protein synthesis and mass of the previously immobilized muscle that exceeded that seen in control-fed rats. A similar treatment effect on muscle recovery was not seen in Leutreated rats, demonstrating that a Leu metabolite can potentially be more efficient than Leu per se as a nutraceutical. Although further studies are needed to determine ␣-HICA metabolism and pharmacokinetics, the amount of ␣-HICA used herein appeared to be safe based on the lack of whole body and tissue-specific effects on numerous hematological, biochemical, and metabolic endpoints. Provision of ␣-HICA may represent a novel approach aimed at speeding recovery from disuse atrophy. ACKNOWLEDGMENTS We gratefully acknowledge the expert assistance of Fariba Roughead (protocol design) and Nicole Fernandes (diet preparation) and the technical assistance of Dr. Margaret Shumate. GRANTS This work was supported by a contract from Nestlé. DISCLOSURES H. Magne, E. A. Offord, and D. Breuillé are employed by Nestlé, and C. H. Lang was partially supported by a research contract from Nestlé.


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