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Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods and Procedures
  5. Results
  6. Discussion
  7. SUPPLEMENTARY MATERIAL
  8. Acknowledgments
  9. Disclosure
  10. REFERENCES
  11. Supporting Information

This study assessed the effects of resistance training (RT) on energy restriction–induced changes in body composition, protein metabolism, and the fractional synthesis rate of mixed muscle proteins (FSRm) in postmenopausal, overweight women. Sixteen women (age 68 ± 1 years, BMI 29 ± 1 kg/m2, mean ± s.e.m.) completed a 16-week controlled diet study. Each woman consumed 1.0 g protein/kg/day. At baseline (weeks B1–B3) and poststudy (weeks RT12–RT13), energy intake matched each subject's need and during weeks RT1–RT11 was hypoenergetic by 2,092 kJ/day (500 kcal/day). From weeks RT1 to RT13, eight women performed RT 3 day/week (RT group) and eight women remained sedentary (SED group). RT did not influence the energy restriction–induced decrease in body mass (SED −5.8 ± 0.6 kg; RT −5.0 ± 0.2 kg) and fat mass (SED −4.1 ± 0.9 kg; RT −4.7 ± 0.5 kg). Fat free mass (FFM) and total body water decreased in SED (−1.6 ± 0.4 and −2.1 ± 0.5 kg) and were unchanged in RT (−0.3 ± 0.4 and −0.4 ± 0.7 kg) (group-by-time, P ≤ 0.05 and P = 0.07, respectively). Protein–mineral mass did not change in either group (SED 0.4 ± 0.2 kg; RT 0.1 ± 0.4 kg). Nitrogen balance, positive at baseline (2.2 ± 0.3 g N/day), was unchanged poststudy. After body mass loss, postabsorptive (PA) and postprandial (PP) leucine turnover, synthesis, and breakdown decreased. Leucine oxidation and balance were not changed. PA and total (PA + PP) FSRm in the vastus lateralis were higher after weight loss. RT did not influence these protein metabolism responses. In summary, RT helps older women preserve FFM during body mass loss. The comparable whole-body nitrogen retentions, leucine kinetics, and FSRm between groups are consistent with the lack of differential protein–mineral mass change.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods and Procedures
  5. Results
  6. Discussion
  7. SUPPLEMENTARY MATERIAL
  8. Acknowledgments
  9. Disclosure
  10. REFERENCES
  11. Supporting Information

Aging is associated with progressive body composition changes, including an increase in body mass and fat mass, contributing to the onset of obesity, and a reduction in fat-free mass (FFM), primarily muscle mass, leading to sarcopenia (1). Obesity exacerbates sarcopenia and worsens physical functioning status and frailty among older persons (2). These conditions also increase the risks of having comorbid medical conditions, including those associated with the metabolic syndrome. Older women may either desire or be instructed by a physician or other health-care provider to reduce excess body mass and fat mass in the hope of improving health status. However, body mass loss by reduced dietary energy intake alone also leads to reductions in FFM, muscle mass, muscle strength, and resting energy expenditure (3). There is concern that these adverse consequences will place an older woman closer to the critical threshold of physical frailty (4,5) and should be considered when evaluating the efficacy of diet-induced body mass loss.

It is well established that nitrogen balance is influenced by both protein (nitrogen) and energy intake, with potentially complex interactions between the two (6,7). Using data primarily from young and middle-aged adults over a wide range of nitrogen (∼30–220 mg N/kg BW/day) and energy (∼5–75 kcal/kg BW/day) intakes, it was estimated that these parameters explained 33 and 36% of the variation in nitrogen balance, respectively (6). When protein intake is held constant at “normal” amounts, nitrogen retention is thought to change by 1–2 mg N per kcal change in energy intake (8,9), with larger changes in nitrogen retention observed when the energy imbalance (deficit or surfeit) is greater (6). However, this expected outcome appears to last only 2–3 weeks, after which time a baseline level of nitrogen balance is re-established (10,11). Most published evaluations of the effects of energy restriction on nitrogen balance in normal and overweight adults are compromised by the lack of preintervention measurements to directly compare with observations during and after energy restriction (12,13,14,15), and data are lacking in older adults.

In young- and middle-aged obese subjects, energy restriction or fasting is reported to not influence (16) or decrease (15,17) the rate of amino acid (protein) turnover, oxidation, synthesis, and breakdown, with the magnitude of change related to the severity of the restriction and protein content of the diets (11,18). Regarding postmenopausal women, Gallagher et al. (4) reported that fasting-state leucine turnover, oxidation, and synthesis (expressed per kg FFM) were not significantly changed in a group of 10 obese women (age range 56–76 years) at the end of a 16-week period of energy restriction that resulted in ∼10% body mass loss. These observations were made while the subjects were in a fasting state, and comparable assessments are needed in the postprandial (PP) state. We are unaware of any published data evaluating the effects of body mass loss on muscle protein synthesis. Strictly controlled feeding studies to assess the effects of energy restriction and body mass loss on nitrogen balance and whole-body and skeletal muscle protein metabolism in older adults are lacking and needed.

Resistance training (RT) is an effective way for older people to increase muscle strength and mass (19,20) and to preserve FFM during energy restriction–induced body mass loss (21,22). The impact of RT during diet-induced body mass loss on protein metabolism, including nitrogen balance, postabsorptive (PA; fasting) and PP leucine kinetics, and the fractional synthesis rate of mixed muscle proteins (FSRm) is largely unknown in older people.

The purpose of this study was to assess the effect of RT on energy restriction–induced changes in body composition, nitrogen balance, whole-body protein metabolism, and FSRm in overweight, postmenopausal women.

Methods and Procedures

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods and Procedures
  5. Results
  6. Discussion
  7. SUPPLEMENTARY MATERIAL
  8. Acknowledgments
  9. Disclosure
  10. REFERENCES
  11. Supporting Information

Subjects

Twenty healthy white women, age range 61–78 years, BMI range 24–34 kg/m2, volunteered to participate in this study. Before starting the study, each woman completed a medical evaluation that included a written medical history, a physical examination, a resting electrocardiogram, a 75-g oral glucose tolerance test, and routine blood and urine chemistries. All of the women had clinically normal heart, liver, kidney, and thyroid functions. None of the women had type 2 diabetes mellitus. None of the women smoked, used hormone replacements, or had participated in a RT program within the past 12 months. The Human Research Advisory Committee at the University of Arkansas for Medical Sciences, Little Rock, AR, and the Committee on the Use of Human Research Subjects, Purdue University, West Lafayette, IN, reviewed and approved the protocol. Each woman signed the written informed consent agreement and received a monetary stipend for participation. Three women discontinued participation for personal reasons unrelated to the study. Data from one woman were excluded from analyses because she was deemed noncompliant based on body mass gain during the study and self-reported consumption of nonprotocol foods. Thus, 16 women successfully completed the protocol. A clinical trials registration number is not provided because the study was conducted before the requirement for registration was established.

Experimental design

This 16-week controlled diet and exercise study was conducted on an in-patient basis during weeks 2, 3, 15, and 16 and on an outpatient basis during the other 12 weeks. During the in-patient weeks, each woman was required to consume all of her meals at a metabolic research kitchen facility, to be available for all testing, and to reside overnight. During the outpatient weeks, each woman came to the laboratory each weekday morning to be weighed and eat breakfast, but otherwise was encouraged to maintain her daily activities as much as possible while living at home. Before starting the protocol, each woman was randomly assigned to one of two groups, either sedentary (SED; n = 8) or RT (n = 8). For descriptive purposes, the methods and results of the study are presented in relation to the week of RT, i.e., baseline (study weeks B1–B3), mid-intervention (week RT7, study week 10), and postintervention (weeks RT12 and RT13, study weeks 15–16) (Figure 1).

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Figure 1. Experimental design and approximate study schedule of the 16-week protocol that included a 3-week baseline period (B1–B3) and an 11-week period of intervention (dietary energy restriction with (n = 8) or without (n = 8) resistance training (RT1–RT11), and a 2-week postintervention period (RT12–RT13)).

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Diet

Throughout the 16-week period of study, each woman completely consumed a controlled diet provided on a rotating cycle of three daily menus. The menus were designed to contain 1.0 g protein/kg/day, a nonprotein energy ratio of 65% carbohydrate and 35% fat. Each woman's energy requirement was estimated based on the Harris–Benedict equation of resting energy expenditure for women (23) plus an energy expenditure of physical activity allowance of 0.7 times of predicted resting energy requirement. During baseline and weeks RT12–RT13, each woman's menus contained sufficient energy to meet their estimated need and included a 2,092 kJ protein-free beverage daily. The protein-free beverage was precisely formulated to contain 198.0 g nondairy creamer, 103.0 g glucose polymers (Polycose; Ross Laboratories, Columbus, OH), 10.0 g sucrose, and 7.0 g vanilla extract. During weeks RT1–RT11, each subject's energy intake was reduced 2,092 kJ/day by omitting the protein-free beverage from the menus. The re-introduction of the protein-free beverage at weeks RT12–RT13 was done to help re-establish energy balance before and during the postintervention testing period. Because the RT program used for this study is documented to not influence the total energy requirement of older persons (24), no dietary adjustments were made for the RT group to account for the energy cost of the resistance exercise. Body mass loss was expected to occur at a rate of 0.45 kg/week (1.0 lb/week). A mismatch between a woman's expected and actual body mass loss during the first 4 weeks of energy restriction (weeks RT1–RT4) was assumed to be due to an inaccurate estimation of her baseline energy requirement. The energy content of the basal menus was adjusted during weeks RT4–RT7 if a woman's body mass loss from weeks RT1–RT4 was not within ±418 kJ (100 kcal) of the expected body mass loss over a minimum of three consecutive weeks (i.e., to attain a 2,092 kJ/day energy deficit) (25). Any adjustment to the basal menus was done by either adding or subtracting protein-free or very low–protein foods from the woman's daily menu.

The procedures used to aid in the complete consumption of the foods provided are published (25). Water, decaffeinated coffee, and decaffeinated tea were allowed ad libitum, and each woman consumed one multivitamin-mineral tablet daily during the study (Advanced Formula Centrum; Lederle Laboratories, Pearl River, NY). The macronutrient and metabolizable energy contents of the foods and beverages used for this study were calculated using Nutritionist V software (N-Squared Computing; First Data Bank, San Bruno, CA).

Strength assessments and RT protocol

All women completed assessments of maximum strength (one- repetition maximum) at weeks B1 (familiarization), RT1 (preintervention), RT7 (mid-study), and RT13 (postintervention). One-repetition maximum testing was done for the muscle groups involved with the bilateral leg extension, bilateral leg curl, bilateral leg press, bilateral chest press, and bilateral arm pull exercises on Keiser pneumatic resistance exercise equipment (Keiser Sports Health Equipment, Fresno, CA). During weeks RT1–RT13, each woman assigned to the RT group performed RT using these exercises on three nonconsecutive days per week. For each exercise, three sets of lifts were performed at a training intensity of 80% of the one-repetition maximum, with 1–2 min rest between sets. For the first two sets, eight repetitions were completed, and for the third set, repetitions were continued up to a maximum of 12. If 12 repetitions were performed, the training load was adjusted upward (typically in a 5% increment) to maintain the training intensity at ∼80% of one-repetition maximum throughout the 13-week RT period. Before and after each strength testing or resistance exercise session, each woman performed 5–10 min of easy cycling and 5–10 min of stretching exercises.

Body composition

Fasting state, nude body mass (total body mass minus robe mass), was measured to the nearest 0.1 kg (scale model 15S; Ohaus, Florham Park, NJ) each weekday morning throughout the study. Body height (without shoes) was measured using a wall-mounted stadiometer. BMI was calculated as weight/height2 (kg/m2).

Body composition was measured at weeks B2 and RT13. Fasting-state body density was measured using an air-displacement plethysmographer (BOD POD; Life Measurement Instruments, Concord, CA). Total body water was determined by the deuterium oxide dilution technique (26,27), using the sample preparation procedures described by Campbell (28), and a Fourier transform infrared spectrophotometer (Avatar 360; Nicolet Instrument, Madison, WI). Percentage body fat was calculated from body density (kg/l) and total body water (expressed as a decimal fraction of body mass) from the three-compartment model equation of Siri (29). FFM was calculated as body mass minus fat mass, and protein–mineral mass was calculated as FFM minus total body water mass. Skinfold thickness at eight sites (biceps, triceps, chest, subscapula, mid-axillary, abdomen, suprailiac, and mid-thigh) was obtained from the right side of the body with Lange calipers (Cambridge Scientific Industries, Cambridge, MA) using standard techniques (30). The sum of skinfold thickness at these eight sites is reported. Body circumference measurements were made at the waist (in a horizontal plane at the level of the umbilicus) and the hips (in a horizontal plane at the greatest protrusion of the buttocks). All skinfold thickness and body circumference measurements were made to the nearest 1.0 mm, and the same investigator made repeated measurements of a given subject.

Food, urine, and stool collections

At weeks B3 and RT13 one composite of each of the three basal menus, formula beverage, and 4-day stool collection was made and aliquots frozen and stored for subsequent analyses. Each subject orally consumed encapsulated food coloring (either FD&C Blue No. 1 Alum Lake 11–13% or Carmine Red) at the start and end of the stool collection periods. The presence of the coloring in a sample (visual inspection) was used to identify the end points of the collection. At weeks B3, RT7, RT11, and RT13, aliquots were obtained from 24-h urine collections made during three (week RT11) or four (weeks B3, RT7, and RT13) consecutive days.

Nitrogen analyses and calculations

Aliquots of the food, beverage, stool, and urine samples were analyzed for total nitrogen content using a nitrogen analyzer (Leco model FP-528; Leco, St. Joseph, MI). Nitrogen balance was calculated as: IN − (UN + FN + MN), where IN = daily dietary nitrogen intake; UN = daily urinary nitrogen excretion; FN = daily fecal excretion; and MN = daily miscellaneous nitrogen losses, assumed to be 5 mg nitrogen/kg/day (9,31).

Infusion procedures and calculations

At the end of weeks B2 and RT12 (48 h postexercise for the RT group at week RT12), PA and PP whole-body leucine kinetics and the FSRm were determined using primed constant infusions of l-[1-13C]leucine and l-[ring-2H5]phenylalanine, respectively (Figure 2). The infusions were conducted 3 days after the subjects had resumed consuming the 2,092 kJ/day protein-free beverage to re-establish dietary energy balance. Details of the infusion procedures and calculations of whole-body leucine kinetics are published (20). Briefly, after triplicate baseline blood and expired breath samples were obtained, the infusion of the isotopes was started with the intravenous administration of priming doses of NaH13CO3 (2.35 µmol/kg), l-[1-13C]leucine (7.6 µmol/kg), and l-[ring-2H5]phenylalanine (2.0 µmol/kg). Immediately following these priming doses, continuous infusions of l-[1-13C]leucine (7.6 µmol/kg/h) and l-[ring-2H5]phenylalanine (3.0 µmol/kg/h) were started and maintained constant using calibrated syringe pumps (model 55-222; Harvard Apparatus, Natick, MA). For the 8-h infusion, the subjects remained in the PA state during the first 4 h and were in a PP state during the last 4 h. The PP state was achieved by having each subject orally consume within 5 min a formula beverage that contained 8.33% of her daily protein and energy intakes at minutes 240, 300, 360, and 420. The nonprotein energy of the beverage was 65% carbohydrate and 35% fat. The beverages contained portioned quantities of Ensure Plus, Polycose (Abbott Laboratories, Columbus, OH), nondairy liquid (Coffee Rich; Rich Products, Buffalo, NY), and water. The mean PP dietary leucine intake of all subjects was 4.31 ± 0.14 mmol leucine/min at both baseline and RT12. This intake was equivalent to 55.8 ± 0.6 and 60.2 ± 0.7 µmol leucine/kg/min at baseline and RT12, respectively. The components of leucine turnover are represented by the equation: turnover = oxidation + synthesis = dietary intake + infusate intake + breakdown. Leucine balances were calculated from intake (diet + infusate) minus oxidation. Plasma α-[13C]ketoisocaproate enrichment was used to estimate the intracellular leucine pool; this choice negated the need to correct the dietary leucine intakes for the ∼50% removal during the first pass through the splanchnic tissues (32,33).

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Figure 2. Protocol for l-[1-13C]leucine and l-[ring-2H5]phenylalanine infusion used to measure whole-body protein turnover and mixed muscle fractional synthesis rate, respectively. X indicates the time that a beverage was provided, resting energy expenditure (REE) measurement completed, blood draw and breath samples were taken, and muscle biopsy procedure performed. [DOWNWARDS ARROW] indicates the administration of the priming doses of NaH13CO3, l-[1-13C]leucine and l-[ring-2H5]phenylalanine. Beverages contained 8.33% of each subject's estimated daily energy and dietary protein needs.

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At minutes 60, 240, and 480 of each infusion, muscle samples were obtained from the vastus lateralis using a 6-mm Bergstrom needle and the procedures previously described (34). The samples were immediately rinsed, blotted, and flash frozen and stored in liquid nitrogen. To start the analysis, the samples were thawed and weighed. Protein was precipitated out of the muscle sample with 0.5 ml of 10% trichloroacetic acid. An internal standard, 22.8 µmol/l of l-[ring-13C6] phenylalanine, was added to the protein. The standard and precipitated proteins were homogenized and centrifuged. The phenylalanine was extracted from the supernatant by cation exchange chromatography (Dowex AG 50W-8X, 100–200 mesh H+ form; Bio-Rad Laboratories, Richmond, CA) and dried using a vacuum pump evaporator (Savant Instruments, Farmingdale, NY). Intracellular enrichment of phenylalanine was determined using the tert-butyldimethylsilyl derivative (35,36). The muscle pellet was rinsed and dried. During the next 36 h, the proteins were hydrolyzed in 6 N HCl at 110 °C. The protein bound with l-[ring-2H5] phenylalanine enrichment was determined using a gas chromatograph mass spectrometer (GC-MS, HP model 5989; Hewlett Packard, Palo Alto, CA.)

The one pool venous model was used to determine the FSRm via phenylalanine amino acid kinetics (37). Phenylalanine was the amino acid chosen because it is not synthesized or metabolized in the muscle. The equation uses the precursor—product model from the incorporation on the l-[ring-2H5] phenylalanine to determine FSRm. The FSRm was calculated from the following equation (38):

  • image

EP1 and EP2 are the enrichments of attached l-[ring-2H5] phenylalanine in two consecutive skeletal muscle biopsies. EM is the l-[ring-2H5] phenylalanine enrichment within the biopsied skeletal muscle–free pool. t is the time between skeletal muscle biopsies.

Statistical analyses

Values are reported as means ± s.e.m. At baseline, differences between the two groups in mean values for all dependent variables were assessed using Student's unpaired t-tests. The main effects and interactions of group and time were assessed using two-factor repeated measures ANOVA. Statistical significance was assigned at P ≤ 0.05 (two sided). When a statistically significant group-by-time effect was established using the two-factor ANOVA, the change over time in the variable was calculated and separate comparisons (Student's unpaired t-tests) were then done. Microsoft Excel 5.0 (Microsoft, Redmond, WA) software was used for data processing, and JMP software (version 3.2.2; SAS Institute, Cary NC) was used for statistical evaluations.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods and Procedures
  5. Results
  6. Discussion
  7. SUPPLEMENTARY MATERIAL
  8. Acknowledgments
  9. Disclosure
  10. REFERENCES
  11. Supporting Information

Dietary intake and energy expenditure

At baseline, dietary energy, protein, carbohydrate, and fat intakes were not different between the SED and RT groups (Table 1). By design, protein intake remained unchanged throughout the study, while energy, carbohydrate, and fat intakes were lower at week RT10 (reflective of the 11-week period of energy restriction). To achieve the desired rate of body mass loss during the first 4 weeks of energy restriction (RT1–RT4), the energy, carbohydrate, and fat contents of the basal diet needed to be decreased by an average of 444 kJ/day, 19 g/day and 4 g/day, respectively (in addition to omitting the 2,092 kJ/day protein-free beverage). Re-introduction of the beverage during week RT12 and RT13 increased energy, carbohydrate, and fat intakes, albeit to slightly lower levels than at baseline, due to the 444 kJ/day adjustment. The performance of RT did not influence the need to adjust the basal menus to achieve the desired rate of body mass loss. During baseline and week RT12 and RT13, the macronutrient distribution of the menus was 15:55:30% energy from protein, carbohydrate, and fat, respectively. During the weeks of energy restriction (RT4–RT11), the macronutrient distribution was 18:54:28%. The dietary energy and macronutrient intakes expressed as kJ/kg/day and g/kg/day, respectively, are presented in Supplementary Table S1 online.

Table 1.  Dietary intakes of older women before, during, and after body mass loss with or without resistance training
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Resting energy expenditure was not different between the SED and RT groups at baseline. It decreased from baseline to week RT13 by 5.1% (P ≤ 0.05), with no difference in response between the two groups. The baseline and week RT13 values for the SED group were 4.91 ± 0.15 and 4.68 ± 0.21* MJ/day, and were 5.07 ± 0.22 and 4.79 ± 0.19* MJ/day for the RT group (*within group difference from baseline, P ≤ 0.05).

Body composition

At baseline, the women in the SED and RT groups were not different in age, height, body mass, BMI, % body fat, fat mass, FFM, total body water, protein–mineral mass, sum of skinfold thicknesses, or waist-to-hip ratio (Supplementary Table S2 online). For all subjects combined (n = 16), there were significant changes over time in body mass, BMI, % body fat, fat mass, and sum of skinfold thicknesses. These changes over time in body mass and body composition were not different between the women in the SED and RT groups (Figure 3). There was no change in waist-to-hip ratio. FFM was not changed in the RT group (P = 0.55) and decreased in the SED group (P ≤ 0.01) (group-by-time, P ≤ 0.05; Figure 3). A similar trend (group-by-time, P = 0.07) was observed for total body water. It was unchanged in the RT group (P = 0.59) and decreased in the SED group (P ≤ 0.01; Figure 3). Protein–mineral mass did not change after body mass loss in either group (Figure 3).

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Figure 3. Body mass and composition changes after an 11-week period of energy restriction in overweight postmenopausal women who were sedentary (n = 8) or resistance training (n = 8). FFM, fat-free mass; PMM, protein–mineral mass; TBW, total body water. Differential response between the sedentary and resistance training groups, group-by-time interaction, *P ≤ 0.05; †P = 0.07.

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Muscle strength

At baseline, maximal strengths of the muscle groups used to perform the leg extension, leg curl, leg press, and chest press exercises were not different between the SED and RT groups, while maximal arm pull strength was lower in the RT group compared with the SED group (P ≤ 0.05) (Supplementary Table S3 online). Over time, maximal strength increased 12–34% in the RT group and was unchanged in the SED group (group-by-time, P ≤ 0.01), for the exercises performed.

Nitrogen balance

At baseline, dietary total nitrogen intake of all subjects averaged 12.8 ± 0.4 g N/day, with no differences between groups, and was purposefully maintained constant throughout the study period (Table 2). Urinary total nitrogen excretion averaged 9.1 ± 0.4 g N/day at baseline, with no difference between groups. At baseline, nitrogen balance was positive (2.2 ± 0.3 g N/day) and did not differ between the two groups. Urinary total nitrogen excretion and nitrogen balance were not changed during the imposed energy restriction (weeks RT7 and RT 11) or the re-establishment of energy balance (week RT13), in either the SED group or the RT group. Expression of the nitrogen balance data as mg N/kg BW/day resulted in a modest progressive increase over time (P ≤ 0.01) in relative dietary nitrogen intake (167 ± 2, 174 ± 3, 179 ± 4, and 182 ± 4 mg N/kg BW/day at baseline and weeks RT7, RT11, and RT13, respectively), but no change in the overall urinary total nitrogen excretion and nitrogen balance results.

Table 2.  Nitrogen balance of older women before and after body mass loss with or without resistance training
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Whole-body leucine kinetics

There were no differences between the SED group and RT group for any of the leucine kinetic parameters at baseline or in the responses over time from baseline to week RT13 (Figure 4 and Supplementary Table S4 online). For all subjects combined, leucine turnover (P < 0.0001), oxidation (P < 0.0001), synthesis (P ≤ 0.05), and balance (P < 0.0001) were higher, and breakdown (P < 0.0001) was lower, in the PP state vs. the PA state. Leucine turnover (P < 0.0001), synthesis (P < 0.001), and breakdown (P < 0.001) were decreased, and oxidation and balance were unchanged at week RT13, compared with baseline. The PA and PP 13C enrichments of plasma α-ketoisocaproate are shown in Supplementary Figure S1 online.

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Figure 4. Changes in the rates of whole-body leucine turnover (Flux), synthesis (Syn), and oxidation (Ox) after an 11-week period of energy restriction–induced body mass loss in overweight postmenopausal women who were sedentary (n = 8) or who were resistance training (n = 8). The rates were measured during an 8-h primed, constant infusion of l-[1-13C]leucine while the subjects were in postabsorptive and postprandial states, and the changes calculated as postintervention − baseline. Values are mean ± s.e.m. *Significant main effect of time, P ≤ 0.05.

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Muscle protein synthesis

At baseline, PA, PP, and total FSRm were not different between the SED and RT groups, and FSRm was lower in the PA state vs. the PP state (0.041 ± 0.006 and 0.083 ± 0.009%/h, respectively, P ≤ 0.05; Figure 5). Poststudy (week RT12), PA, and total FSRm had increased (P ≤ 0.05) and PP FSRm was not changed, compared to baseline. RT did not influence these responses and the PA, PP, and total FSRm were not different between the RT and SED groups postintervention. The l-[ring-2H5] phenylalanine enrichment of the vastus lateralis–free phenylalanine pool from the muscle samples obtained at min 60, 240, and 480 of the infusions are shown in Supplementary Table S5 online.

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Figure 5. Fractional synthesis rate of mixed muscle proteins (FSRm) in the vastus lateralis before (baseline) and after (resistance training week 12, RT12) an 11-week period of energy restriction–induced body mass loss in overweight postmenopausal women who were sedentary (n = 8) or resistance training (n = 8). The FSRm was measured during an 8-h primed, constant infusion of l-[ring-2H5]phenylalanine while the subjects were in postabsorptive and postprandial states. Total FSRm was measured from the incorporation of isotope during the postabsorptive and postprandial periods combined. Values are mean ± s.e.m. *Significant main effect of metabolic state (postabsorptive vs. postprandial) at baseline, P ≤ 0.05. †Significant main effect of time (baseline vs. week R12), P ≤ 0.05.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods and Procedures
  5. Results
  6. Discussion
  7. SUPPLEMENTARY MATERIAL
  8. Acknowledgments
  9. Disclosure
  10. REFERENCES
  11. Supporting Information

Recommendations for safe and effective body mass loss by overweight and obese older persons include moderate energy restriction (500–1,000 kcal/day energy deficit) and the regular performance of physical activities (2,39). RT is emphasized to help preserve FFM, including muscle and bone, and improve muscle strength and physical functioning. Several findings from the current study were expected and support the effectiveness of moderate energy restriction with adequate dietary protein intake and RT as therapeutic tools available to overweight older women. These findings include the energy restriction–induced losses of body mass and fat mass (40) and the RT-induced increase in muscle strength and preservation of FFM (41). Our body composition results indicated that the SED group lost 1.6 kg FFM (28% of body mass loss), but all of the FFM loss was apparently body water; as a change in protein–mineral mass was not detected. Although these results do not exactly match theoretical outcomes (4,41,42,43), they are generally consistent with the fact that changes in FFM are primarily attributable to changes in body water.

The preservation of FFM, which includes muscle mass, is considered an important component of an effective body mass loss program for overweight and obese older persons (2). It would seem reasonable to expect that changes in nitrogen balance and protein metabolism that favor anabolism should help achieve the FFM preservation. Theoretically, a 500 kcal/day energy deficit would result in a loss of 500–1,000 mg N/day (3.1–6.2 g protein/day), whereas the complete preservation of FFM with RT would result in a differential nitrogen balance response of 0.50 g N/day. The 500 mg N/day change in nitrogen excretion is quantitatively the same as would be expected from a loss of 0.26 g protein mass from a person who loses 5.45 kg after a 12-week period of consuming a 500 kcal/day energy deficit diet. However, this expected outcome only occurs during an initial 2- to 3-week period of energy restriction, and then nitrogen balance returns to baseline levels (10,11). The lack of change in nitrogen balance during and after the period of energy restriction among the older women in the current study is consistent with findings in adolescents (44) and young- and middle-aged obese subjects (10,11). The theoretical 0.50 g N/day loss in SED subjects represents ∼4% of protein intake provided to the women in the current study (1.0 g/kg/day), and is within the inherent variability of the nitrogen balance method (11). These calculations underscore the subtlety of nitrogen balance responses that over time could result in changes in FFM, as well as the limitations of this method as a tool to critically evaluate changes in whole-body protein.

The finding that RT-induced preservation of FFM was due to body water retention brings into question the importance of hydration status for muscle mass and function. Hypo-hydration compromises muscle strength, power, and high-intensity endurance (45). The SED group did not show clinical signs of dehydration (urinary specific gravity of the subjects below threshold of dehydration; data not shown) and maintained muscle strength postweight loss. Measurements of muscle hydration and muscle function are needed to improve upon the tentative conclusion that the loss of body water due to moderate energy restriction in older women did not contribute to sarcopenia.

The experimental design of this study, i.e., conducting the baseline and postintervention testing with the subjects in an energy balance state, provided the opportunity to make some unique observations without the confounding effect of an acute energy deficit. The decision to re-establish energy balance postintervention was made after considerable discussion among the research team and reflects the recommendation of the National Institutes of Health study panel that reviewed our grant proposal. We knew this design feature would be questioned in whichever way the experiment was conducted, but resources prevented us from conducting tests during both the period of energy restriction and postenergy restriction. Our purpose was to evaluate the longer-term effects of weight loss, not the acute effects of energy restriction. It is noted that one-half of the leucine kinetic assessments were done in an acute negative energy balance, which is standard to all studies that include measurements in the PA state. The ∼10% reduction in fasting-state leucine turnover following body mass loss is consistent with the 10–17% decrease measured in obese premenopausal women who lost ∼10% body mass as body fat (no change in FFM) by consuming a 500 kcal/day energy deficit diet for 16 weeks (46). The reductions in leucine turnover, synthesis, and breakdown observed postbody mass loss might reflect the influence of reduced body fat on protein metabolism, i.e., a reversal of increased protein turnover associated with increasing body fat and obesity (46,47). In contrast, no significant change in fasting-state leucine turnover (−3.5%) was measured in obese postmenopausal women after a 10% body mass loss (19% of loss as FFM) over a 16-week period (4).

To our knowledge, the current study is the first to evaluate the effects of body mass loss on PP leucine kinetics. The body mass loss–induced reductions in PP leucine turnover, synthesis, and breakdown were comparable to those observed in the fasting state. These results also indicate that while body mass loss causes a reduction in the rates of protein synthesis and breakdown, the magnitude of these changes is not different. The resulting lack of change in net leucine balance is consistent with a metabolic adaptation to preserve whole-body protein mass, and is consistent with the body composition and nitrogen balance responses.

Regarding FSRm, the feeding-induced increase in FSRm measured at baseline is consistent with findings in healthy young adults (48). Smith et al. (49) recently reported that muscle protein synthesis was not stimulated by feeding in older women, possibly related to an absence of a feeding-induced stimulation of protein translation initiation. Our baseline FSRm data contrast this finding and support previous research (50) that older women retain the capacity to stimulate FSRm with feeding. The observation that FSRm in the PA state was more than twice as high postbody mass loss, compared to baseline, was unexpected. We are not aware of other published research addressing this issue. Further research is required to evaluate possible explanations, including the impact of increased insulin sensitivity due to the body mass loss, and whether the re-introduction of the 500 kcal/day beverage several days before the poststudy measurement stimulated basal FSRm. Perhaps following the period of energy restriction, the body is primed to efficiently store nutrients when they become more abundant (51). Further research is also warranted to evaluate the independent effects of energy restriction vs. body mass reduction on FSRm. Lastly, the apparent lack of effect of RT on FSRm is generally consistent with the notion that any acute increase in FSRm following resistance exercise is diminished and approaches the preexercise rate when measurements were taken at 36–48 h after exercise (52,53).

The choice of time points used for the FSRm determination may be considered a study limitation. Ideally, the FSRm should be calculated when the precursor pool is in steady state (i.e., a constant muscle-free l-[ring-2H5]phenylalanine enrichment). To accomplish both PA and PP FSRm assessments within the structure of an 8-h infusion protocol, we chose to quantify the incorporation of l-[ring-2H5]phenylalanine into mixed muscle proteins from 60 to 240 min (PA) and from 240 to 480 min, and to use the muscle-free l-[ring-2H5]phenylalanine enrichments at 240 (PA) and 480 (PP) as the steady-state precursor pool values. The higher muscle-free l-[ring-2H5]phenylalanine enrichments measured at 240 min vs. 60 min suggest modest underpriming of the precursor pool, while the lower enrichments at 480 min vs. 240 min are consistent with increased unlabeled phenylalanine due to feeding. Additional biopsies would have been needed to better establish the time course of steady state in both the PA and PP periods, but this was not deemed appropriate to do in these older women who had already consented to three biopsies each testing day. The lack of steady state could result in the underestimation of PA and overestimation of PP FSRm, respectively.

In conclusion, these results show that RT during a period of moderate energy restriction effectively helps older women increase muscle strength and preserve FFM. They also indicate that modest body mass loss does not significantly impact whole-body protein balance, as reflected by nitrogen balance and net leucine balance measurements, and the apparent retention of whole-body protein–mineral mass. These findings are consistent with the fact that the predominant component of FFM is water.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods and Procedures
  5. Results
  6. Discussion
  7. SUPPLEMENTARY MATERIAL
  8. Acknowledgments
  9. Disclosure
  10. REFERENCES
  11. Supporting Information

We thank the staff members of Prof. Campbell's research staff and the General Clinical Research Center nursing and metabolic kitchen staff for providing expert support for this study. We are grateful to the participants in this study for their cooperation and dedication. This research was supported by grants from National Institutes of Health 1 R29 AG13409 and 1 R01 AG15750, US Department of Agriculture 98-35200-6151, and General Clinical Research Center MO1 RR14288.

REFERENCES

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods and Procedures
  5. Results
  6. Discussion
  7. SUPPLEMENTARY MATERIAL
  8. Acknowledgments
  9. Disclosure
  10. REFERENCES
  11. Supporting Information
  • 1
    Baumgartner RN. Body composition in healthy aging. Ann NY Acad Sci 2000; 904: 437448.
  • 2
    Villareal DT, Apovian CM, Kushner RF, Klein S. Obesity in older adults: technical review and position statement of the American Society for Nutrition and NAASO, The Obesity Society. Obes Res 2005; 13: 18491863.
  • 3
    Jakicic JM, Clark K, Coleman E et al. American College of Sports Medicine position stand. Appropriate intervention strategies for weight loss and prevention of weight regain for adults. Med Sci Sports Exerc 2001; 33: 21452156.
  • 4
    Gallagher D, Kovera AJ, Clay-Williams G et al. Weight loss in postmenopausal obesity: no adverse alterations in body composition and protein metabolism. Am J Physiol Endocrinol Metab 2000; 279: E124E131.
  • 5
    Miller SL, Wolfe RR. The danger of weight loss in the elderly. J Nutr Health Aging 2008; 12: 487491.
  • 6
    Pellett PL, Young VR. The effects of different levels of energy intake on protein metabolism and of different levels of protein intake on energy metabolism: a statistical evaluation from the published literature. In: Scrimshaw NS, Schurch B (eds). Protein-Energy Interactions. I/D/E/C/G: Waterville Valley, NH, 1991, pp 81121.
  • 7
    Young VR, Yu YM, Fukagawa N. Whole body energy and nitrogen (protein) relationships. In: Kinney HM, Tucker HN (eds). Energy Metabolism: Tissue Determinants and Cellular Corollaries. Raven: New York, 1992, pp 139160.
  • 8
    Calloway DH, Spector H. Nitrogen balance as related to caloric and protein intake in active young men. Am J Clin Nutr 1954; 2: 405412.
  • 9
    FAO/WHO/UNU. Energy and protein requirements. Technical Report Series no. 724. World Health Organization: Geneva, Switzerland, 1985.
  • 10
    Gougeon R, Hoffer LJ, Pencharz PB, Marliss EB. Protein metabolism in obese subjects during a very-low-energy diet. Am J Clin Nutr 1992; 56: 249S254S.
  • 11
    Hoffer LJ, Bistrian BR, Young VR, Blackburn GL, Matthews DE. Metabolic effects of very low calorie weight reduction diets. J Clin Invest 1984; 73: 750758.
  • 12
    Davies HJ, Baird IM, Fowler J et al. Metabolic response to low- and very-low-calorie diets. Am J Clin Nutr 1989; 49: 745751.
  • 13
    Friedlander AL, Braun B, Pollack M et al. Three weeks of caloric restriction alters protein metabolism in normal-weight, young men. Am J Physiol Endocrinol Metab 2005; 289: E446E455.
  • 14
    Marliss EB, Murray FT, Nakhooda AF. The metabolic response to hypocaloric protein diets in obese man. J Clin Invest 1978; 62: 468479.
  • 15
    Stanko RT, Tietze DL, Arch JE. Body composition, nitrogen metabolism, and energy utilization with feeding of mildly restricted (4.2 MJ/d) and severely restricted (2.1 MJ/d) isonitrogenous diets. Am J Clin Nutr 1992; 56: 636640.
  • 16
    Garlick PJ, Clugston GA, Waterlow JC. Influence of low-energy diets on whole-body protein turnover in obese subjects. Am J Physiol 1980; 238: E235E244.
  • 17
    Stein TP, Rumpler WV, Leskiw MJ et al. Effect of reduced dietary intake on energy expenditure, protein turnover, and glucose cycling in man. Metabolism 1991; 40: 478483.
  • 18
    Oi Y, Okuda T, Koishi H et al. Effects of low energy diets on protein metabolism studies with [15N]glycine in obese patients. J Nutr Sci Vitaminol (Tokyo) 1987; 33: 227237.
  • 19
    Campbell WW, Crim MC, Young VR, Joseph LJ, Evans WJ. Effects of resistance training and dietary protein intake on protein metabolism in older adults. Am J Physiol 1995; 268: E1143E1153.
  • 20
    Campbell WW, Trappe TA, Jozsi AC et al. Dietary protein adequacy and lower body versus whole body resistive training in older humans. J Physiol 2002; 542. 2: 631642.
  • 21
    Ryan AS, Pratley RE, Elahi D, Goldberg AP. Resistive training increases fat-free mass and maintains RMR despite weight loss in postmenopausal women. J Appl Physiol 1995; 79: 818823.
  • 22
    Ballor DL, Katch VL, Becque MD, Marks CR. Resistance weight training during caloric restriction enhances lean body weight maintenance. Am J Clin Nutr 1988; 47: 1925.
  • 23
    Harris JA, Benedict FG. A Biometric Study of Basal Metabolism in Man. Carnegie Institute of Washington: Washington, DC, 1919.
  • 24
    Campbell WW, Kruskall LJ, Evans WJ. Lower body versus whole body resistive exercise training and energy requirements of older men and women. Metabolism 2002; 51: 989997.
  • 25
    Campbell WW, Cyr-Campbell D, Weaver JA, Evans WJ. Energy requirement for long-term body weight maintenance in older women. Metabolism 1997; 46: 884889.
  • 26
    Lukaski HC, Johnson PE. A simple, inexpensive method of determining total body water using a tracer dose of D2O and infrared absorption of biological fluids. Am J Clin Nutr 1985; 41: 363370.
  • 27
    Schloerb PR, Friis-Hansen BJ, Edelman IS, Solomon AK, Moore FD. The measurement of total body water in the human subject by deuterium oxide dilution. J Clin Invest 1950; 29: 12961310.
  • 28
    Campbell WW. Can resistance training maintain physical function? Contemp Intern Med 1994; 6: 2637.
  • 29
    Siri WE. Body composition from fluid spaces and density: analysis of methods. In: Brozek J, Henschel A (eds). Techniques for Measuring Body Composition. National Academy of Sciences: Washington, DC, 1961, pp 223244.
  • 30
    Lohman TG, Roche AF, Martorell R (eds). Anthropometric Standardization Reference Manual. Human Kinetics Books: Champaign, IL, 1988.
  • 31
    Rand WM, Pellett PL, Young VR. Meta-analysis of nitrogen balance studies for estimating protein requirements in healthy adults. Am J Clin Nutr 2003; 77: 109127.
  • 32
    Boirie Y, Gachon P, Beaufrere B. Splanchnic and whole-body leucine kinetics in young and elderly men. Am J Clin Nutr 1997; 65: 489495.
  • 33
    Matthews DE, Schwarz HP, Yang RD et al. Relationship of plasma leucine and alpha-ketoisocaproate during a L-[1-13C]leucine infusion in man: a method for measuring human intracellular leucine tracer enrichment. Metabolism 1982; 31: 11051112.
  • 34
    Evans WJ, Phinney SD, Young VR. Suction applied to a muscle biopsy maximizes sample size. Med Sci Sports Exerc 1982; 14: 101102.
  • 35
    Bergstrom J, Furst P, Noree LO, Vinnars E. Intracellular free amino acid concentration in human muscle tissue. J Appl Physiol 1974; 36: 693697.
  • 36
    Ferrando AA, Lane HW, Stuart CA, Davis-Street J, Wolfe RR. Prolonged bed rest decreases skeletal muscle and whole body protein synthesis. Am J Physiol 1996; 270: E627E633.
  • 37
    Wolfe RR. Radioactive and Stable Isotope Tracers in Biomedicine: Principles and Practice of Kinetic Analysis. Wiley: New York, 1992.
  • 38
    Baumann PQ, Stirewalt WS, O'Rourke BD, Howard D, Nair KS. Precursor pools of protein synthesis: a stable isotope study in a swine model. Am J Physiol 1994; 267: E203E209.
  • 39
    Nelson ME, Rejeski WJ, Blair SN et al. Physical activity and public health in older adults: recommendation from the American College of Sports Medicine and the American Heart Association. Med Sci Sports Exerc 2007; 39: 14351445.
  • 40
    Villareal DT, Banks M, Sinacore DR, Siener C, Klein S. Effect of weight loss and exercise on frailty in obese older adults. Arch Intern Med 2006; 166: 860866.
  • 41
    Ballor DL, Poehlman ET. Exercise-training enhances fat-free mass preservation during diet-induced weight loss: a meta-analytical finding. Int J Obes Relat Metab Disord 1994; 18: 3540.
  • 42
    Bossingham MJ, Carnell NS, Campbell WW. Water balance, hydration status, and fat-free mass hydration in younger and older adults. Am J Clin Nutr 2005; 81: 13421350.
  • 43
    Mahon AK, Flynn MG, Iglay HB et al. Measurement of body composition changes with weight loss in postmenopausal women: comparison of methods. J Nutr Health Aging 2007; 11: 203213.
  • 44
    Pencharz PB, Motil KJ, Parsons HG, Duffy BJ. The effect of an energy restricted diet on the protein metabolism of obese adolescents: nitrogen-balance and whole-body nitrogen turnover. Clin Sci (Lond) 1980; 59: 1318.
  • 45
    Judelson DA, Maresh CM, Anderson JM et al. Hydration and muscular performance: does fluid balance affect strength, power and high-intensity endurance? Sports Med 2007; 37: 907921.
  • 46
    Kanaley JA, Haymond MW, Jensen MD. Effects of exercise and weight loss on leucine turnover in different types of obesity. Am J Physiol 1993; 264: E687E692.
  • 47
    Welle S, Nair KS. Relationship of resting metabolic rate to body composition and protein turnover. Am J Physiol 1990; 258: E990E998.
  • 48
    Caso G, Garlick PJ, Ballou LM et al. The increase in human muscle protein synthesis induced by food intake is similar when assessed with the constant infusion and flooding techniques. J Nutr 2006; 136: 15041510.
  • 49
    Smith GI, Atherton P, Villareal DT et al. Differences in muscle protein synthesis and anabolic signaling in the postabsorptive state and in response to food in 65–80 year old men and women. PLoS ONE 2008; 3: e1875.
  • 50
    Symons TB, Schutzler SE, Cocke TL et al. Aging does not impair the anabolic response to a protein-rich meal. Am J Clin Nutr 2007; 86: 451456.
  • 51
    Miller BF. Human muscle protein synthesis after physical activity and feeding. Exerc Sport Sci Rev 2007; 35: 5055.
  • 52
    MacDougall JD, Gibala MJ, Tarnopolsky MA et al. The time course for elevated muscle protein synthesis following heavy resistance exercise. Can J Appl Physiol 1995; 20: 480486.
  • 53
    Phillips SM, Tipton KD, Aarsland A, Wolf SE, Wolfe RR. Mixed muscle protein synthesis and breakdown after resistive exercise in humans. Am J Physiol 1997; 273: E99E107.

Supporting Information

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods and Procedures
  5. Results
  6. Discussion
  7. SUPPLEMENTARY MATERIAL
  8. Acknowledgments
  9. Disclosure
  10. REFERENCES
  11. Supporting Information

supporting Information

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