Aging is associated with a decline in strength, endurance, balance, and mobility. Obesity worsens the age-related impairment in physical function and often leads to frailty. The American College of Sports Medicine recommends a multicomponent (strength, endurance, flexibility, and balance) exercise program to maintain physical fitness. However, the effect of such an exercise program on physical fitness in frail, obese older adults is not known. We therefore determined the effect of a 3-month long multicomponent exercise training program, on endurance (peak aerobic capacity (VO2 peak)), muscle strength, muscle mass, and the rate of muscle protein synthesis (basal rate and anabolic response to feeding) in nine 65- to 80-year-old, moderately frail, obese older adults. After 3 months of training, fat mass decreased (P < 0.05) whereas fat-free mass (FFM), appendicular lean body mass, strength, and VO2 peak increased (all P < 0.05). Regular strength and endurance exercise increased the mixed muscle protein fractional synthesis rate (FSR) but had no effect on the feeding-induced increase in muscle protein FSR (∼0.02%/h increase from basal values both before and after exercise training; effect of feeding: P = 0.02; effect of training: P = 0.047; no interaction: P = 0.84). We conclude that: (i) a multicomponent exercise training program has beneficial effects on muscle mass and physical function and should therefore be recommended to frail, obese older adults, and (ii) regular multicomponent exercise increases the basal rate of muscle protein synthesis without affecting the magnitude of the muscle protein anabolic response to feeding.
Loss of muscle mass is a normal consequence of aging (1), which can lead to frailty (2), impaired quality of life (3), and admission to a chronic care facility (4). Obesity worsens the age-related decline in physical function (5), most likely because of excess fat mass in relation to muscle mass despite a seemingly normal or even increased absolute muscle mass (5,6). Nevertheless, diet-induced weight-loss is often not recommended to alleviate the obesity-associated decline in muscle function because it causes loss of muscle mass and is therefore feared to exacerbate the deficits in physical function (7). The American College of Sports Medicine recommends a multicomponent (i.e., strength, endurance, balance, and flexibility) exercise training program to improve and maintain physical function in older adults (8). There is good evidence that resistance training alone (9,10,11) increases the rate of muscle protein synthesis and muscle mass and strength whereas endurance training alone improves aerobic capacity (12,13) in nonobese older adults. However, it is thought that concurrent strength and endurance training inhibits strength development when compared with strength training alone (14) and may negatively affect endurance adaptation (15,16). In addition, there is some evidence that the resistant exercise-induced increases in muscle mass and strength are blunted in young, obese compared with young, lean individuals (17,18). A multicomponent exercise program, as recommended by the American College of Sports Medicine, may therefore not bring about the expected benefits in older, obese adults.
The mechanisms responsible for an increase in muscle mass with resistance exercise include and increase in the rate of muscle protein synthesis both during fasted and fed conditions (19,20,21,22). However, it is not known whether the anabolic effects of exercise and feeding are additive or possibly even synergistic (i.e., greater increase from basal values in response to exercise and feeding than either exercise or feeding alone). Fujita et al. (23) have shown that a single bout of aerobic exercise improves the anabolic response to local (leg) insulin infusion, which suggests that the effects of exercise and hyperinsulinemia were synergistic.
The purpose of our study therefore was to determine the effect of a multicomponent exercise training program on physical function, muscle mass, and the rate of muscle protein synthesis in frail, obese, older adults. We hypothesized that regular physical activity including strength, flexibility, balance, and endurance exercises would increase in the basal rate of muscle protein synthesis, augment the anabolic response of muscle to feeding, and increase muscle mass, strength, and endurance. To test these hypotheses, we evaluated: (i) the rate of skeletal muscle protein synthesis during basal, postabsorptive conditions, and feeding, (ii) muscle mass, (iii) upper- and lower-body muscle strength, (iv) peak aerobic exercise capacity (VO2 peak), and (v) indexes of general physical function (e.g., gait speed, chair rise, range of motion) in nine moderately frail, obese adults before and 3 months after participation in a multicomponent exercise training program. In addition, because both aging and obesity are accompanied by increased concentrations of inflammatory cytokines in the circulation (24,25) and circulating cytokines have been found to be negatively associated with muscle protein synthesis rates (26) and may contribute to skeletal muscle atrophy and reduced functional capacity (27,28,29) we also measured the plasma concentrations of C-reactive protein (CRP) and tumor necrosis factor (TNF-α) before and after exercise training.
Methods and Procedures
We studied nine 65- to 80-year-old, sedentary, moderately frail, obese men and women (Table 1). To be considered for the study, subjects had to meet at least two of three of the following criteria for mild-to-moderate frailty (5,30,31): (i) Physical Performance Test score ≥18 but ≤32 (maximum score = 36); (ii) VO2 peak ≥10 but ≤18 ml/kg/min; and (iii) self-reported difficulty and/or assistance with up to two instrumental activities of daily living and/or one basic activity of daily living. In addition, subjects had to be weight-stable (no more than ± 2 kg change in body weight during the past year) and on a stable medication regimen. Exclusion criteria were severe cardiopulmonary disease, diabetes mellitus, musculoskeletal or neuromuscular impairments that prevented participation in the exercise program, sensory or cognitive deficits, cancer diagnosis within the last 5 years, smoking, and the use of corticosteroids, or androgen- or estrogen-containing compounds within the last year. Subjects taking medications to control certain medical conditions (e.g., hypertension) were included if the drug regimen has been stable for at least 6 months before entering the study and did not change during the study. All participants were considered to be in good health and fit for the prescribed exercise after completion of a comprehensive medical evaluation, including a medical history and physical examination, standard blood and urine tests, an oral glucose tolerance test, and a graded treadmill exercise stress test. The study was approved by the Human Research Protection Office and the General Clinical Research Center Scientific Advisory Committee at Washington University School of Medicine. Written informed consent was obtained from each subject before participation in the study.
Table 1. Body weight, body composition, and physical function
Each subject completed a 3-month long multicomponent exercise training program; body composition, physical function, endurance, and skeletal muscle protein synthesis rates during basal, postabsorptive conditions and feeding were evaluated before and at the end of the training period.
Body composition analysis. Total body mass, fat mass, and fat-free mass (FFM) were measured by using DXA (Delphi 4500/w; Hologic, Waltham, MA). Body regions (head, trunk, and limbs) were isolated by using computer-generated default lines, with manual adjustment, on the anterior view planogram. Legs and arms were defined as the soft tissue extending from a line drawn through and perpendicular to the axis of the femoral neck and angled with the pelvic brim to the phalangeal tips (legs) and the soft tissue extending from the center of the arm socket to the phalangeal tips (arms). The bone mineral-free portion of the appendicular, upper and lower extremity lean mass represents primarily skeletal muscle in the extremities (32).
Evaluation of physical function. Strength was evaluated by determining each person's one repetition maximum (1-RM) on a squat rack and Hoist machine for the following nine exercises: squats, leg press, knee extension, knee flexion, seated row, upright row, seated chest press, biceps curl, and triceps extension. Peak aerobic exercise capacity was assessed during graded treadmill walking (5). Range of motion, a measure of flexibility, was assessed by using a goniometer (31). Chair rise was assessed by the time to stand-up fully without using the hands as quickly as possible (31). Gait speed was measured as the time to complete a 25-feet distance (5). Static balance was assessed by measuring the single-leg stand time (5).
Protein metabolism study. Subjects were instructed to adhere to their regular diet and to refrain from vigorous exercise (before training only) for 3 days before the study. They were admitted to the General Clinical Research Center the evening before study. At 2000 hours, they consumed a standard meal that provided 12 kcal/kg body weight; 55% of the total meal energy was provided as carbohydrates (i.e., 133–234 g of carbohydrates), 30% as fat (i.e., 32–57 g of fat), and 15% as protein (i.e., 36–64 g of protein). Subjects then rested in bed and fasted (except for water) until completion of the study the next day. At ∼0600 hours on the following morning, a cannula was inserted into an antecubital vein for the infusion of a stable isotope labeled leucine tracer; another cannula was inserted into a vein of the contralateral hand for blood sampling. At ∼0800 hours, a blood sample and a muscle biopsy from the quadriceps femoris were obtained to determine the background leucine enrichment in plasma, muscle tissue fluid and muscle protein (33). Muscle tissue was obtained under local anesthesia (lidocaine, 2%) by using a Tilley-Henkel forceps. Immediately afterward, a primed, constant infusion of [5,5,5−2H3] l-leucine (98 Atoms % purchased from Cambridge Isotope Laboratories, Andover, MA; priming dose: 4.8 µmol/kg body wt, infusion rate: 0.08 µmol/kg body wt/min) was started and maintained until completion of the study ∼6 h later. At 210 min after the start of the leucine tracer infusion, a second muscle biopsy was obtained to determine the basal rate of muscle protein synthesis (as incorporation of [5,5,5−2H3]leucine into muscle protein). We have recently validated this approach (33). Immediately after the second biopsy, a liquid meal (Ensure; Abbott Laboratories, Abbott Park, IL, containing 15% of energy as protein, 55% as carbohydrate, and 30% as fat) was given intermittently in small boluses every 10 min for 150 min so that every subject received a priming dose of 23 mg protein/kg FFM followed by 175 mg protein/kg FFM during the 2.5-h feeding period; this feeding regimen also provided a total of 726 mg carbohydrates/kg FFM and 176 mg fat/kg FFM. We chose to evaluate the effect of feeding during a 2.5-h time period because Bohé et al. (34) have shown that muscle protein synthesis increases rapidly after increasing amino acid supply and remains elevated for ∼2–3 h but returns to basal, postabsorptive values thereafter despite increased amino acid availability. At the onset of feeding, the infusion rate of labeled leucine was increased to 0.12 µmol/kg body wt/min to adjust for the increased plasma leucine availability. A third muscle biopsy was obtained at 360 min (i.e., 150 min after the first food aliquot) to determine the muscle protein synthesis response to feeding. The second and third biopsies were obtained from the leg contralateral to that biopsied initially through the same incision, but with the forceps directed in proximal and distal directions, so that the two biopsies were collected ∼5–10 cm apart.
Blood samples (4 ml each) were obtained every 30 min during the entire study period to determine plasma leucine and α-ketoisocaproic acid (KIC) enrichments, and the concentrations of leucine, glucose, insulin, CRP, and TNF-α. One milliliter was collected in prechilled tubes containing heparin; plasma was separated immediately by centrifugation and plasma glucose concentration was measured; the remaining blood was collected in prechilled tubes containing EDTA; plasma was separated by centrifugation within 30 min of collection and then stored at −80 °C until final analyses were performed. Muscle tissue was rinsed in ice-cold saline immediately after collection, cleared off all visible fat and connective tissue, and then frozen in liquid nitrogen and later transferred to a −80 °C freezer for storage until final analyses were performed.
Exercise training. The exercise training program was started ∼1 week after the first protein metabolism study. It focused on endurance, strength, and balance to improve physical function. Each week, subjects completed three 90-min exercise-training sessions, which were supervised by a physical therapist, on three nonconsecutive days at the Washington University Applied Physiology Section exercise facility; participants performed make up sessions if they missed a regularly scheduled one. Each session consisted of 15 min of flexibility exercises, followed by 30 min of endurance exercise, 30 min of strength training, and 15 min of balance exercises. The endurance exercise component included walking on a treadmill, step-ups, stair climbing, stationary cycling, or Stairmaster exercise. Initially, subjects exercised at ∼75% of peak heart rate, and the intensity of exercise was gradually increased over several weeks to ∼80% of peak heart rate. The strength training component included nine exercises (squats, leg press, knee extension, knee flexion, seated row, upright row, seated chest press, biceps curl, and triceps extension) performed on a squat rack and Hoist machine. Initially, 1–2 sets of these exercises (8–12 repetitions each) were performed at ∼65% of each person's 1-RM; gradually this was changed to 2–3 sets (6–8 repetitions each) at ∼80% of 1-RM. Each person's 1-RM was determined monthly during the program to adjust for improvements in strength. Each participant performed the goal of 36 sessions within 3.5 ± 0.8 months of training. Throughout the training program, subjects met with a dietician on a monthly basis and were counseled on maintaining a stable and weight-maintaining diet. At the end of the training program, body composition analyses, endurance and strength assessments and the protein metabolism study were repeated as described above.
Sample processing and analyses
Plasma glucose concentration was determined on an automated glucose analyzer (Yellow Spring Instruments, Yellow Springs, OH). Plasma insulin concentration was determined by radioimmunoassay (Linco Research, St Louis, MO). Commercially available ELISA kits were used to measure the plasma concentrations of CRP (ALPCO Diagnostics, Salem, NH) and TNF-α (Biosource Europe, Nivelles, Belgium). To determine plasma leucine concentration and plasma leucine and KIC enrichments, a known amount of norleucine was added to the plasma, proteins were precipitated, and the supernatant, containing free amino acids and their keto-analogs, was collected to prepare the t-butyldimethylsilyl and O-t-butyldimethylsilyl quinoxalinols derivatives of leucine and KIC, respectively; their tracer-to-tracee ratios (TTR) were determined by gas-chromatography/mass-spectrometry (MSD 5973 System; Hewlett-Packard, Palo Alto, CA) as previously described (35,36,37). To determine leucine enrichments in muscle proteins and muscle tissue fluid, muscle samples (∼20 mg) were homogenized in 1 ml trichloroacetic acid solution (3% wt/vol), proteins were precipitated by centrifugation, and the supernatant, containing free amino acids, was collected. The pellet containing muscle proteins was washed and then hydrolyzed in 6 N HCl at 110 °C for 24 h. Amino acids in the protein hydrolysate and supernatant samples were then purified on cation-exchange columns (Dowex 50W-X8-200; Bio-Rad Laboratories, Richmond, CA), and leucine converted to its t-butyldimethylsilyl derivative to determine the TTR by gas-chromatography/mass-spectrometry (MSD 5973 System; Hewlett-Packard) (35,36,37). All gas-chromatography/mass-spectrometry analyses were carried out in conjunction with standards of known isotope enrichment (36,38); the following mass-to-charge ratios were monitored: 200 (M+0), 202 (M+2), and 203 (M+3) for leucine and 259 (M+0) and 262 (M+3) KIC.
The homeostasis model assessment of insulin resistance score was calculated as the product of fasting plasma insulin (in mU/l) and glucose (in mmol/l) concentrations divided by 22.5 (39).
The fractional synthesis rate (FSR) of mixed muscle protein was calculated based on the incorporation rate of [5,5,5−2H3]leucine into muscle proteins by using a standard precursor-product model as follows: FSR = ΔEp/Eic × 1/t × 100; where ΔEp is the change in enrichment (TTR) of protein-bound leucine in two subsequent biopsies (i.e., the first and second and the second and third, respectively), Eic is the enrichment of the precursor for protein synthesis and t is the time between biopsies (33). We used both the average plasma KIC and the free leucine enrichment in muscle tissue fluid as surrogates for the immediate precursor for muscle protein synthesis (i.e., aminoacyl-tRNA) (40).
All data sets were tested for normality. Differences between pre- and post-training body composition, physical function tests, and the plasma concentrations of CRP and TNF-α were evaluated by the Student t-test or the Mann-Whitney U test as appropriate. Two-way repeated measures ANOVA was used to evaluate differences in plasma glucose, insulin, and leucine concentrations, and mixed muscle protein FSR during basal, postabsorptive, and fed conditions, before and after training. In no instance was a significant interaction found; therefore, we did not perform post hoc analyses. A P value of ≤0.05 was considered statistically significant. Data in the text are presented as mean ± s.e.m. (normally distributed data) or median with 25th and 75th percentiles in brackets (not normally distributed data); data in tables and figures are presented as indicated in the corresponding legends.
Body composition and physical function
Total body mass was maintained from the beginning until the end of the exercise training program whereas fat mass significantly decreased and FFM and appendicular lean body mass significantly increased (in eight of the nine subjects) in response to exercise training (Table 1). On average, lower-body strength (knee extension, knee flexion, and leg press 1-RM) increased by ∼20% (Table 1) and upper body strength (seated row, bench press, and bicep curl 1-RM) increased by 10–20% as a result of training; although the difference did not reach statistical significance (P = 0.10) for bicep curl (Table 1). All nine subjects increased their bench press and leg flexion strength; eight of the nine subjects increased their leg press and leg extension performances, seven out of eight increased their seated row performance, and five out of eight increased their biceps curl performance. One subject could not complete the final evaluation for seated row and biceps curl due to shoulder pain at the end of the study. Peak aerobic exercise capacity improved by ∼15% from the beginning until the end of the exercise training program (Table 1). In addition, we observed improvements in flexibility (range of motion), chair rise, gait speed, one-legged stand (Table 1).
Plasma CRP, TNF-α, glucose, insulin, and leucine concentrations
Regular exercise had no statistically significant effect on the plasma concentrations of CRP (3.54 (2.70; 7.92) vs. 3.88 (1.70; 6.53) mg/l before and after training, respectively; P = 0.77) and TNF-α (1.03 ± 0.22 vs. 0.61 ± 0.13 pg/ml before and after training, respectively; P = 0.10).
Basal plasma glucose, insulin, and leucine concentrations and the homeostasis model assessment of insulin resistance score were not statistically significantly different before and after the exercise training program (Table 2). Feeding increased (P < 0.05) plasma glucose concentration by ∼35%, tripled (P < 0.05) plasma insulin concentration, and increased (P < 0.05) plasma leucine concentration by ∼10% (Table 2); exercise training had no effect on the feeding-induced changes in plasma glucose, insulin, and leucine concentrations (Table 2).
Table 2. Plasma glucose, insulin, and leucine concentrations and HOMAIR score
Plasma leucine and α-KIC and muscle tissue leucine labeling
The extent of leucine and α-KIC labeling in plasma and free leucine labeling in muscle tissue was not affected by exercise training (all P > 0.2). The average plasma leucine TTR before and after exercise training was 0.069 ± 0.004 and 0.068 ± 0.004, respectively during the basal, postabsorptive period, and 0.090 ± 0.004 and 0.090 ± 0.004 during feeding. The muscle free leucine TTR before and after exercise training was 0.041 ± 0.004 and 0.043 ± 0.003, respectively at the end of the basal, postabsorptive period and 0.062 ± 0.004 and 0.063 ± 0.002 at the end of feeding. The average plasma α-KIC TTR before and after exercise training was 0.057 ± 0.003 and 0.061 ± 0.003, respectively during the basal, postabsorptive period and 0.080 ± 0.005 and 0.081 ± 0.004 during feeding.
Muscle protein synthesis rate
Using the muscle free leucine TTR as the precursor pool enrichment in the calculation of the muscle protein synthesis rate we found that the mixed muscle protein FSR (Figure 1) was ∼50% greater after than before exercise training (P = 0.047) with no difference before and after exercise training in the anabolic response to feeding (ΔFSR before and after exercise training: 0.019 ± 0.010 and 0.024 ± 0.013%/h; feeding effect: P = 0.02; training effect: P = 0.84). When using the plasma α-KIC TTR as the precursor pool enrichment, the results were essentially the same; i.e., no difference before and after exercise training in the anabolic response to feeding.
The increasing prevalence of obesity in older adults is a major public health problem and provides a challenge to health care professionals. Obesity is associated with metabolic abnormalities (41,42) and exacerbates the age-related decline in physical function, which leads to frailty and predisposes to loss of independence in older persons (43). In this study we provide evidence that a multicomponent exercise training program in community-dwelling frail, obese older adults has beneficial effects on both muscle protein metabolism and physical function. The exercise regimen in our study, which complied with the recommendations by the American College of Sports Medicine, and included strength and endurance components increased both endurance and muscle mass and strength in frail, obese older adults even though there is evidence that the resistant exercise-induced increases in muscle mass and strength are blunted in obese compared with lean individuals (17,18); furthermore, concurrent strength and endurance training inhibits strength development when compared with strength training alone (14) and may negatively affect endurance adaptation compared with endurance exercise training alone (15,16). In addition, the exercise-induced improvements in endurance and strength were accompanied by improvements in flexibility and factors that are associated with frailty in obese older adults (5) such as standing up from a chair, the ability to maintain balance while standing, and walking speed. These activities are necessary for independent mobility and are independent predictors of the ability to perform instrumental activities of daily living such as the ability to cook, travel, and shop; furthermore, they are predictors of disability and nursing home admission (44,45). Thus, the present data suggest that regular diverse physical activity improves overall physical fitness and is therefore important in the care of frail, older, obese adults to slow or prevent the normal age-associated functional decline.
The training-induced increase in muscle mass was mediated by an increase in the basal rate of muscle protein synthesis rather than an increase in the anabolic response to feeding (i.e., greater change from baseline FSR). The stimulatory effect of regular physical activity including strength, endurance, balance, and flexibility exercises in our subjects on the rate of muscle protein synthesis the day after the last bout of exercise appears to be bigger than would be expected after endurance exercise training alone (46,47,48,49) but was somewhat less than typically observed after resistance exercise training alone (9,10,50,51,52,53,54,55); probably because concurrent endurance training interferes with the resistance training-induced adaptation of muscle protein metabolism. We measured the rate of muscle protein synthesis ∼12–14 h after the last bout of exercise at which point the rate of muscle protein synthesis was ∼50% greater than before training. A single bout of a typical resistance exercise work-out increases the rate of muscle protein synthesis by ∼100–200% within ∼24 h of exercise in both young (50,51) and old (9,10,52,53,54,55) adults after which it declines toward baseline values by ∼48–72 h (50,51). Resistance exercise training shortens the response with a return to baseline values by ∼24–30 h after the last bout of exercise; however, the relative response in the early phase of recovery is greater after than before training (56). Endurance exercise alone on the other hand increases the rate of muscle protein synthesis to a much lesser degree (<50%) both acutely and after a period of training in young and old individuals (46,47,48,49).
Our findings are consistent with earlier reports regarding the effects of exercise and feeding; namely that, compared to rest, both in the fasted and fed state, the rate of muscle protein synthesis is greater after resistance exercise both acutely, after a single bout (19,20,21,22) and after regular exercise training (10,56,57). Furthermore, they extend these observations by demonstrating that the independent anabolic effects of regular exercise and feeding are additive but not synergistic because the muscle protein FSR was greater after than before exercise, both during basal conditions and feeding, and the feeding-induced increase in muscle protein FSR (i.e., rise above basal values) was not different before and after exercise. Only one study (23) so far has looked at this phenomenon after a single bout of exercise. In contrast to the results from our study, Fujita et al. (23) found that a single bout of treadmill walking augments the anabolic response of muscle protein synthesis (i.e., the relative increase in the rate of muscle protein synthesis from basal values) to hyperinsulinemia-euglycemia-euaminoacidemia in nonobese older adults on the morning after the exercise. Whether the difference in results between our study and that of Fujita et al. (23) is due to the type of exercise performed, the training status of the subjects, the type of nutritional anabolic stimulus (mixed-meal feeding vs. insulin alone), and/or subject characteristics remains to be determined.
There are some limitations to our study. First, we cannot discern the acute effect of exercise on muscle protein synthesis from the adaptive response to repeated exercise challenges. Second, our study provides potentially time-sensitive information, which is restricted to a short time-window after the last bout of exercise. However, repeated studies to address these issues would have required an unfeasible amount of work. We chose to evaluate muscle protein metabolism before training (in the absence of any exercise) and between 15 and 21 h after the last bout of exercise because: (i) we were interested in the effect of regular exercise on muscle protein metabolism, (ii) it is recommended (8) that people engage in exercise at least 3–5 times a week (i.e., every 24–72 h), and (iii) it has been shown that although that the acute effect of resistance exercise lasts for 48–72 h in untrained subjects (50,51) an increase in the rate of muscle protein synthesis in healthy young trained subjects is evident for only ∼24–30 h after the last bout of exercise (56). Therefore, studying our subjects after the acute effect of exercise has vanished (>24–30 h after the last bout of exercise), would provide little information of practical relevance.
Systemic low-grade inflammation is known to be associated with the development of muscle wasting (27,28,29) and muscle protein synthesis rates have been shown to be inversely correlated with circulating cytokine concentrations (26). Nonetheless, the marked exercise-induced stimulation in muscle protein synthesis rate occurred independently of significant changes in systemic inflammatory markers (i.e., CRP and TNF-α) in our frail, obese older adults. However, it is possible that we failed to detect a change in plasma TNF-α concentration due to lack of statistical power because training reduced the average plasma TNF-α concentration by ∼40% but the difference was not statistically significant. Furthermore, we cannot exclude local, muscle-specific exercise-mediated changes in inflammatory activity (9,58).
In summary, the results of this study provide evidence that a multicomponent exercise training program has beneficial effects on endurance, strength, and muscle mass and muscle protein metabolism in frail, obese older adults; it should therefore be considered in the care for frail, older, obese adults.
This research was supported by National Institutes of Health Grants AG 025501, AR 049869, AG 021164, RR 00036 (General Clinical Research Center), RR 00954 (Biomedical Mass Spectrometry Resource), and DK 56341 (Clinical Nutrition Unit) and the Longer Life Foundation. G.I.S. was supported by an Ellison Medical Foundation/American Federation for Aging Research Postdoctoral Fellowship. The study was designed by D.T.V. and B.M.; data collection was performed and supervised by D.T.V., G.I.S., D.R.S., K.S., and B.M.; data analyses and interpretation were performed by D.T.V., G.I.S., and B.M.; writing was performed by D.T.V., G.I.S., and B.M. We are grateful to Nicole Wright and the nursing staff of the General Clinical Research Center for their skilled technical assistance in performing this study and to the study subjects for their cooperation.