Anabolic signaling deficits underlie amino acid ... - The FASEB Journal

©2004 FASEB

The FASEB Journal express article10.1096/fj.04-2640fje. Published online December 13, 2004.

Anabolic signaling deficits underlie amino acid resistance of wasting, aging muscle Daniel Cuthbertson,*,† Kenneth Smith,*,§ John Babraj,* Graham Leese,† Tom Waddell,* Philip Atherton,*,‡ Henning Wackerhage,* Peter M. Taylor,* and Michael J. Rennie*,§ *Division of Molecular Physiology, School of Life Sciences, University of Dundee, Dundee, Scotland, UK DD1 4HN; †Department of Medicine, Ninewells Hospital and Medical School, Tayside NHS Trust Dundee DD1 9ST; ‡Department of Biological Sciences, University of Central Lancashire, Preston, PR1 2HE; §University of Nottingham, School of Biomedical Sciences, Division of Clinical Physiology, Graduate Entry Medical School, City Hospital, Derby, DE22 3DT, United Kingdom Corresponding author: Michael J. Rennie, University of Nottingham, School of Biomedical Sciences, Division of Clinical Physiology, Graduate Entry Medical School, City Hospital, Derby DE22 3DT. E-mail: [email protected] ABSTRACT The nature of the deficit underlying age-related muscle wasting remains controversial. To test whether it could be due to a poor anabolic response to dietary amino acids, we measured the rates of myofibrillar and sarcoplasmic muscle protein synthesis (MPS) in 44 healthy young and old men, of similar body build, after ingesting different amounts of essential amino acids (EAA). Basal rates of MPS were indistinguishable, but the elderly showed less anabolic sensitivity and responsiveness of MPS to EAA, possibly due to decreased intramuscular expression, and activation (phosphorylation) after EAA, of amino acid sensing/signaling proteins (mammalian target of rapamycin, mTOR; p70 S6 kinase, or p70S6k; eukaryotic initiation factor [eIF]4BP-1; and eIF2B). The effects were independent of insulin signaling since plasma insulin was clamped at basal values. Associated with the anabolic deficits were marked increases in NFκB, the inflammation-associated transcription factor. These results demonstrate first, EAA stimulate MPS independently of increased insulin availability; second, in the elderly, a deficit in MPS in the basal state is unlikely; and third, the decreased sensitivity and responsiveness of MPS to EAA, associated with decrements in the expression and activation of components of anabolic signaling pathways, are probably major contributors to the failure of muscle maintenance in the elderly. Countermeasures to maximize muscle maintenance should target these deficits. Key words: muscle protein synthesis • essential amino acids • sarcopenia • mTOR • p70S6k

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rom 50 years onward, aging is accompanied by loss of skeletal muscle, that is, sarcopenia (1), characterized by losses of both muscle fibers, particularly of type 2 and fiber area, (see ref 2 for recent review). Sarcopenia is probably a consequence of old age itself, but chronic illness, poor diet, and inactivity all exacerbate it; it is a major cause of disability, loss of independence, and frailty resulting in an increased risk of death. The need to limit sarcopenia is

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receiving increasing attention, as the numbers of older members of our society grow, but the links between causes and effects are not obvious, making effective strategies elusive. Any loss of muscle protein results from an imbalance between the rates of MPS and muscle protein breakdown (MPB), but the proximal cause of the imbalance in sarcopenia is unknown. In our experience, most gradual changes (increases or decreases) in muscle protein mass are mainly due to alterations in MPS (the facilitating process), with alterations of MPB being adaptive to them, effectively lessening their effect (3, 4). Although many authors (cited in our review; see ref 5) have suggested that the major reason for age-related sarcopenia is a reduction in basal (i.e., postabsorptive) rates of MPS, others (6, 7) disagree, having reported that neither basal MPS nor measured (or deduced) MPB was altered with healthy aging. Some of the discrepancies between earlier reports may have been due to the failure to adequately distinguish the healthy elderly from more rapidly wasting, frail persons. However, it seems to us and others (6) unlikely that the rates of basal, postabsorptive MPS are diminished by the 20–30% sometimes reported (see ref 5), or MPB elevated (by as much as 50% [8, 9]) in the elderly (even the frail elderly) compared with the young, because the rates of muscle wasting should otherwise be greater than observed (i.e., 0.5–2% per year between 50–80 years old [10, 11]). We propose, instead, that defects exist in the ability of the elderly to make adequate use of their dietary protein, which would be consistent with the gradual losses of muscle mass and quality observed. We previously showed that the EAA themselves powerfully stimulate human MPS in a dosedependent, saturable manner (12, 13), whereas the nonessential amino acids (NEAA) do not (14). Furthermore, the anabolic effect of EAA is probably independent of any but a permissive influence of insulin (12, 13). Our hypothesis centers on the possible existence of a defect in elderly muscle itself, because the availability of free amino acids to muscle after amino acid ingestion (either of small, frequent, or a large single bolus) is not diminished in the elderly, despite a higher first-pass capture of amino acids by the gut (15, 16). Certainly, there is evidence that the elderly can show an anabolic response to amino acids delivered both intravenously and orally (7, 15, 17, 18), but current evidence that the response is altered with age is poor. In recent reports by Volpi and colleagues describing studies using small and large oral doses and i.v infusions of EAA, the responses of MPS in the elderly compared with the young have included 1) a 25% lesser response to a large dose (15 g) of EAA (7), 2) a 53% greater rise after small boluses (15), and 3) an apparent 40% fall from basal after a glucose-EAA infusion, which caused a rise in the young (18) but none of which were statistically significant between young and elderly, presumably because of the small sample sizes, the variable basal rates of MPS (ranging from mean values of 0.043–0.064%.h–1 in the young and 0.049–0.061%.h–1 in elderly subjects), and the large variances (up to 30% of mean values) in measured MPS. No other researchers ave carried out studies on human MPS using oral EAA, but measurements made on isolated muscles from old rats show that the sensitivity and responsiveness of MPS to the EAA leucine are both diminished (19). To test our specific hypothesis that a defect of muscle maintenance in the elderly would be found not in alterations of basal rates of MPS, but in decrements in the extent of its stimulation after increasing the blood availability of EAA, we deliberately studied physically mobile, active (though untrained), healthy, elderly men, rather than frail, inactive subjects because we suspected that any casually detectable sarcopenia is heralded by subtle presarcopenic changes without signs


of gross dysfunction. We chose to study men only because they show a faster rate of wasting than women and we wished to maximize power in our study. MATERIALS AND METHODS Subject characteristics Twenty young and 24 elderly men, all healthy, were recruited after examination by an experienced physician who excluded any potential subject with any previous or current disease, with muscle wasting of >20%, or who was overweight (BMI >28 kg.m–2). All subjects gave informed consent, and the studies were approved by the Tayside Ethics Committee. Dual energy photon X-ray absorptiometry (DEXA; HOLOGIC Discovery W, Bedford, MA) was used to measure whole-body and appendicular lean soft tissue and fat masses, from which we calculate skeletal muscle mass according to the model of Kim et al. (20). Conduct of the study After they were screened and recruited, the subjects presented themselves, individually, at the Clinical Investigation Unit, Ninewells Hospital at 08:00, having not eaten for 12 h. We did not otherwise control the subjects’ previous diet, since we wished the results to be as representative of free-living subjects as possible. Cannulae were introduced into forearm veins for infusion of the somatostatin analog octreotide (Sandoz, Basel, Switzerland) at 1.8 mg·kg–1·h–1 (which we know from previous experience to be sufficient to inhibit secretion, in response to amino acids, of both insulin and growth hormone). Insulin (Actrapid, NovoNordisk Copenhagen, Denmark) was replaced by infusing at 360 m IU·m–2 body surface area ·h–1 throughout the investigation to maintain plasma insulin concentration at ~10 m IU·l–1. The octreotide and insulin infusions were started 30 min before the subject drank the EAA solution. At the same time, a primed, constant infusion of [1-13C] ketoisocaproic acid (KIC, 8.8 µmol·kg–1 and 13.2 µmol·kg–1·h–1, Cambridge Isotope Laboratories, Cambridge, MA) was started and continued throughout the remainder of the study to label the intramuscular leucine pools via transamination, with the aim of thereby delivering labeled leucine directly to the muscle protein synthetic apparatus (21). The young and elderly subjects were studied in groups of four; they took 500 ml water containing 0, 2.5, 5, 10, 20, and (for the elderly only) 40 g of EAA (arginine, histidine, isoleucine, methionine, leucine, phenylalanine, threonine, tryptophan, and valine) in a composition representative of muscle protein (see ref 22). [1-13C]leucine (99 Atoms %, Cambridge Isotope Laboratories) was added to the oral EAA solution at 5% of the leucine content to maintain a steady state of leucine labeling. Blood samples were taken at 20–30 min intervals for 3.5 h (with intermittent monitoring of plasma glucose) starting 30 min before EAA ingestion. Briefly, biopsies (150–300 mg) of vastus lateralis were taken before and 3 h after taking the EAA or water using the conchotome technique (23); the skin and fascia incisions were made under local anesthesia (1% lignocaine). The biopsies were from different sites. The biopsy was snap-frozen in liquid nitrogen and stored at –80°C until further analysis.


Experimental methods We measured the concentrations of glucose, insulin, insulin-like growth factor (IGF)-1, IGF-1 binding protein (BP)-3, and amino acids, and in muscle, we measured concentrations of total protein, total RNA and DNA, and amino acids. Plasma was separated from whole blood by centrifugation (300g) immediately after collection. Plasma glucose was measured with a YSI Stat2300 (Yellow Spring Instruments, Yellow Spring, OH) immediately after collection of each sample; the remainder of the plasma sample was frozen until further analysis. Plasma insulin and total IGF-1 and its binding protein, IGF-1 BP-3, were measured using commercial kits (IBL, Hamburg, Germany) and the Abbott IMX Immuno analyser (Abbott, Deerfield, IL). The labeling of leucine in plasma and free muscle water was determined by GC-MS after conversion to the tBDMS derivative; the 13C/12C ratio of leucine was determined by selected-ion monitoring MS by our standard methods as described previously (24, 25). Plasma amino acids were quantified as their t-BDMS derivative after the addition of norleucine as internal standard. Briefly, 200 µl of plasma were deproteinized with 1 ml of 100% ethanol; the supernatant was dried, redissolved in 0.5 M HCl, and extracted with ethyl acetate; and the aqueous layer was dried and derivatized. The concentrations were calculated with reference to a standard curve run with each batch. Intracellular amino acids were extracted into 0.2 N perchloric acid, containing norleucine, and the supernatant was neutralized and then purified on Dowex 50W-X8, H+ resin. Amino acids were eluted with 2 M NH4OH, dried, and converted to t-BDMS derivatives. Their concentrations were calculated with reference to a standard curve and expressed in µmol l–1 of tissue water, assuming values of 720 µl H2O/g of muscle, 220 µl of which was extracellular containing amino acids with a concentration similar to that of plasma (26). In addition, we also quantified plasma concentrations of EAA and NEAA by HPLC on basal samples and also those taken at peak leucine concentrations. Free amino acid concentrations were determined by reversed-phase highperformance liquid chromatography (HPLC). Plasma samples were deproteinized in 10% perchloric acid, neutralized (4M KOH), and then clarified by centrifugation (15,000g, 4°C, 30 min) before derivatization with phenylisothiocyanate. Samples and identically treated standards were injected onto a HAISIL 100 C18 column (Higgins Analytical, Mountain View, CA) and resolved according to the manufacturer’s standard protocols, with postcolumn UV (254 nm) detection of phenylthiocarbamyl amino acids. Cys and Lys were not readily quantifiable under the conditions applied. The processing of muscle has previously been described in detail (12). Frozen muscle was processed in buffer containing inhibitors (e.g., vanadate and microcystine) required to block phosphatase and protease activity, thus maintaining the phosphorylation state of signaling proteins to be analyzed in the sarcoplasmic fraction. Samples (60–80 mg) were processed to separate myofibrillar and sarcoplasmic protein as described previously (12). Protein from the fractions was hydrolyzed using 6 N HCl at 110°C overnight, and the liberated amino acids were separated using Dowex W-X8 H+ ion-exchange resin before being derivatized as their N-acetyln-propyl esters and measurement of the previously protein-bound leucine 13C/12C ratios by gas chromatography combustion isotope-ratio mass spectrometry (Finnigan DeltaPlus XL, Finnigan GMBH, Bremen, Germany). The fractional synthetic rate (FSR) of protein was calculated as the rate of increase of labeling of muscle protein-bound leucine compared with the intramuscular free leucine labeling, assuming a tracer steady state. We were justified in doing this because 1) the 13C-KIC infusion method produced values of the ratios of total muscle free/plasma 13C leucine labeling, which were identical between young and old at each dose of amino acids, and


2) because the plasma leucine labeling remained constant, within the limits of experimental error over the entire period of the measurements, within a subject group or dose treatment (complete data not shown). Thus, for the 10 g EAA dose, the zero time, 60 min (near peak EAA), and 360 min plasma leucine tracer/tracee ratio values in % for young subjects were 6.5 ± 0.5, 6.0 ± 0.1, and 6.5 ± 0.5, respectively; the corresponding values in the elderly were 6.5 ± 1.0, 7.0 ± 1.5, and 7.0 ± 0.5. Similar results were obtained at other doses of EAA. Total protein concentration in the muscle biopsies was determined by using the Bradford reaction with a commercial kit (B6916, Sigma, St. Louis, MO). Muscle RNA and DNA concentrations were determined using the method of Fleck and Munro (27). Western analysis of signaling proteins We estimated the relative concentrations and extent of phosphorylation using Western blotting in the basal state and after stimulation by 10 g of EAA (with which there was the greatest difference of MPS between groups) of components of the mammalian target of rapamycin (mTOR) signaling pathway, known to be important for regulation of mammalian MPS (28, 29) and also eukaryotic initiation factor (eIF)2B, a downstream substrate of GSK3α. In addition, we estimated the concentrations in muscle of the inflammation-associated protein NFκB. Twenty milligrams of muscle were homogenized in 0.6 ml of 50 mM Tris-HCl; 0.1% Triton-X; 1 mM EDTA; 1 mM EGTA; 50 mM NaF; 10 mM β-glycerophosphate; 5 mM Na pyrophosphate; and 0.1% 2-mercaptoethanol, 4°C, and extracted by rotation 60 min at 4°C, before centrifugation (13,000g, 10 min). The supernatant protein concentration was adjusted to 2 mg·ml–1 in diluted sample buffer containing sodium dodecyl sulfate, bromophenol blue, and glycerol. Twenty micrograms of protein were loaded per lane onto a 10% polyacrylamide gel. Rainbow molecular weight marker (RPN800, Amersham Bioscience, Buckinghamshire, UK) was loaded at 10 µl per well. Electrophoresis Samples were electrophoresed on a 10% (7% for mTOR; 13.5% for 4E-BP1) SDS-PAGE gel at 100 V for 30 min through the stacking layer and then 200 V until the dye marker reached the bottom of the gel (~40 min). Proteins were then transferred to a polyvinylidene difluoride (PVDF) membrane. The transfer from gel to membrane was run for 2 h (4 h for mTOR) at 100 V. Blotting Membranes were blocked and exposed to the following antibodies overnight at 4°C: phosphomTOR (New England Biolabs, Beverly, MA; catalog number NEB2971; Ser2448; 1:500); phospho-p70 S6 kinase (p70S6k) (NEB9205; Thr389; 1:2000); phospho-4E-BP1 (NEB9459; Ser37/46; 1:2000); phospho-eIF2B (courtesy of C.G. Proud, University of Dundee; Ser544; 1:1000); phospho-4E-BP1 (NEB9459; Ser37/46; 1:2000); NF-κB (NEB3034; 1:2000); mTOR (NEB2971; 1:500); p70S6k (NEB9202; 1:2000); and 4E-BP1 and eIF 2Bε (both courtesy of C.G. Proud; 1:1000). On the next morning, the membrane was incubated with the appropriate secondary antibody, which was one of horse radish peroxidase (HRP)-linked anti-mouse IgG (NEB 7072; 1:2000), anti-rabbit IgG (NEB7074; 1:2000), or anti-goat IgG 1:2000 (Santa Cruz,


sc-2354; 1:1000, Santa Cruz Biotechnology, Santa Cruz, CA). Membranes were exposed to ECL chemiluminescent detection HRP reagents (RPN2106, Amersham Biosciences) mixed 1:1 in 10 ml for 1 min, exposed to X-ray film, and developed, and the film was subjected to densitometry. All results were normalized to the control values of the young subjects and calculated in arbitrary units. The results obtained by using phosphoantibodies were related to the amount of the total proteins in the samples. Statistics The results were analyzed with InStat v3.0 for Windows (GraphPad Software, San Diego, CA); we chose to apply one-way ANOVA with multiple datasets for the five groups of young subjects and six groups of elderly subjects with Bonferroni post hoc test procedures for comparison of group means. Where we tested hypotheses of greater responses in the young, we calculated P values using one-tailed procedures. All values in the text and in the tables and figures derived from quantitation of Western blots by densitometry are means ± SD, but SE is used elsewhere for clarity where results from groups overlapped. P ≤ 0.05 was considered to be significant. The studies were carried out according to the Declaration of Helsinki under the auspices of the Tayside Regional Ethics Committee (Ref 224/01). RESULTS Characteristics of the subjects The groups were well matched (Table 1) with no visible wasting in the elderly men. However, total and appendicular lean soft tissue and calculated total skeletal muscle masses were reduced slightly though significantly (by ~15%) in the elderly. There was no marked decrement in the muscle DNA unit size (i.e., the protein/DNA ratio, a measure of the amount of cytoplasm managed by each nucleus [30, 31]) in the elderly, but there were significant decrements in the total capacity of muscle for protein synthesis (i.e., the RNA/protein and RNA/DNA ratios). In addition, the efficiency (i.e., the FSR/RNA ratio) of both myofibrillar and sarcoplasmic (not shown) protein synthesis at EAA doses of ≥10 g (but not in the basal state) was less in the elderly. The lack of differences in the insulin/glucose ratios between groups indicated normal glucose tolerance and insulin sensitivity of tissue glucose metabolism. The elderly subjects showed slightly but significantly (P