Effect of 6-Month Calorie Restriction on Biomarkers of Longevity, Metabolic Adaptation, and Oxidative Stress in Overweight Individuals
A Randomized Controlled Trial
Leonie K. Heilbronn, PhD; Lilian de Jonge, PhD; Madlyn I. Frisard, PhD; James P. DeLany, PhD; D. Enette Larson-Meyer, PhD; Jennifer Rood, PhD; Tuong Nguyen, BSE; Corby K. Martin, PhD; Julia Volaufova, PhD; Marlene M. Most, PhD; Frank L. Greenway, PhD; Steven R. Smith, MD; Walter A. Deutsch, PhD; Donald A. Williamson, PhD; Eric Ravussin, PhD; for the Pennington CALERIE Team
JAMA. 2006;295:1539-1548.
ABSTRACT
Context Prolonged calorie restriction increases life span in rodents. Whether prolonged calorie restriction affects biomarkers of longevity or markers of oxidative stress, or reduces metabolic rate beyond that expected from reduced metabolic mass, has not been investigated in humans.
Objective To examine the effects of 6 months of calorie restriction, with or without exercise, in overweight, nonobese (body mass index, 25 to <30) men and women.
Design, Setting, and Participants Randomized controlled trial of healthy, sedentary men and women (N = 48) conducted between March 2002 and August 2004 at a research center in Baton Rouge, La.
Intervention Participants were randomized to 1 of 4 groups for 6 months: control (weight maintenance diet); calorie restriction (25% calorie restriction of baseline energy requirements); calorie restriction with exercise (12.5% calorie restriction plus 12.5% increase in energy expenditure by structured exercise); very low-calorie diet (890 kcal/d until 15% weight reduction, followed by a weight maintenance diet).
Main Outcome Measures Body composition; dehydroepiandrosterone sulfate (DHEAS), glucose, and insulin levels; protein carbonyls; DNA damage; 24-hour energy expenditure; and core body temperature.
Results Mean (SEM) weight change at 6 months in the 4 groups was as follows: controls, –1.0% (1.1%); calorie restriction, –10.4% (0.9%); calorie restriction with exercise, –10.0% (0.8%); and very low-calorie diet, –13.9% (0.7%). At 6 months, fasting insulin levels were significantly reduced from baseline in the intervention groups (all P<.01), whereas DHEAS and glucose levels were unchanged. Core body temperature was reduced in the calorie restriction and calorie restriction with exercise groups (both P<.05). After adjustment for changes in body composition, sedentary 24-hour energy expenditure was unchanged in controls, but decreased in the calorie restriction (–135 kcal/d [42 kcal/d]), calorie restriction with exercise (–117 kcal/d [52 kcal/d]), and very low-calorie diet (–125 kcal/d [35 kcal/d]) groups (all P<.008). These "metabolic adaptations" (~ 6% more than expected based on loss of metabolic mass) were statistically different from controls (P<.05). Protein carbonyl concentrations were not changed from baseline to month 6 in any group, whereas DNA damage was also reduced from baseline in all intervention groups (P <.005).
Conclusions Our findings suggest that 2 biomarkers of longevity (fasting insulin level and body temperature) are decreased by prolonged calorie restriction in humans and support the theory that metabolic rate is reduced beyond the level expected from reduced metabolic body mass. Studies of longer duration are required to determine if calorie restriction attenuates the aging process in humans.
Trial Registration ClinicalTrials.gov Identifier: NCT00099151
Prolonged calorie restriction increases life span in rodents and other shorter-lived species.1 Whether this occurs in longer-lived species is unknown, although the effect of prolonged calorie restriction in nonhuman primates is under investigation. One hypothesis to explain the antiaging effects of calorie restriction is reduced energy expenditure with a consequent reduction in the production of reactive oxygen species (ROS).2-3 However, other metabolic effects associated with calorie restriction, including alterations in insulin sensitivity and signaling, neuroendocrine function, stress response, or a combination of these, may retard aging.4
Total energy expenditure is made up of resting energy expenditure (50%-80% of energy), the thermic effect of feeding (~10%), and nonresting energy expenditure (10%-40%).5 Whether total energy expenditure is reduced beyond the level expected for a given reduction in the size of the metabolizing mass following calorie restriction is debated. Leibel et al6 showed that a 10% weight loss reduced sedentary 24-hour energy intake for weight maintenance between 15% and 20% in obese patients, suggesting that metabolic adaptation occurs in humans. However, the weight loss was achieved quickly with a liquid diet and, with the exception of several normal-weight patients in the study by Leibel et al, the effects of prolonged calorie restriction on energy expenditure in nonobese humans have not been assessed. In rhesus monkeys, resting energy expenditure adjusted for fat-free mass (FFM) and fat mass was lower after 11 years of calorie restriction.7 Similarly, total energy expenditure was lower in monkeys following 10 years of weight clamping.8 Studies in rodents have proven more controversial with reports of decreased, no change, or increased adjusted energy expenditure in calorie restriction vs ad libitum fed–animals.9-13
One of the most widely accepted theories of aging is the oxidative stress theory, which hypothesizes that oxidative damage produced by ROS accumulates over time, leading to the development of disease such as cancer, aging, and ultimately death.14 Reactive oxygen species are byproducts of energy metabolism, with 0.2% to 2.0% of oxygen consumption (O2) resulting in ROS formation.15-16 Reactive oxygen species attack lipids, proteins, and DNA, generating a number of products that affect normal cell functioning.17 Studies in rodents subjected to calorie restriction demonstrate a 30% decrease in 8-oxo-7,8-dihydroguanine (8-oxodG) in brain, skeletal muscle, and heart; similar reductions in carbonyl content in brain and muscle18-22; and transcriptional patterns that suggest decreased oxidative stress in response to calorie restriction.23 Rhesus monkeys subjected to calorie restriction exhibit divergent responses in the expression of genes involved in oxidative stress.24
Core body temperature and levels of dehydroepiandrosterone sulfate (DHEAS) and insulin are proposed biomarkers of calorie restriction and longevity in rodents and monkeys.25 Data from the Baltimore Longitudinal Study of Aging support the association between longevity and temperature and insulin and DHEAS levels; men with plasma insulin concentration or oral temperature below the median, and DHEAS levels above the median, live longer.26 Furthermore, in a cross-sectional study that compared individuals following self-imposed nutritionally adequate calorie restriction for 6 years with normal-weight controls, Fontana et al27 found that participants in the calorie restriction group had lower levels of serum glucose, insulin, and markers of atherosclerosis.
The aims of this study were to establish whether prolonged calorie restriction by diet alone or in conjunction with exercise can be successfully implemented in nonobese individuals and to determine the effects of the interventions on established biomarkers of calorie restriction, sedentary energy expenditure, and oxidative damage to DNA and proteins.
The Comprehensive Assessment of the Long Term Effects of Reducing Intake of Energy (CALERIE) study is a randomized clinical trial conducted at the Pennington Biomedical Research Center, Baton Rouge, La. The study protocol was approved by the center institutional review board and an independent data and safety monitoring board, and participants provided written informed consent. The study was conducted between March 2002 and August 2004.
Participants
Potential participants (aged <50 years for men and <45 years for women) completed 3 screening visits to ensure physical and psychological health. Assessments of height, weight, and blood pressure were made, and all participants had a chemistry 15 panel, complete blood cell count, and an electrocardiogram. A total of 599 individuals were screened and 551 were excluded (460 were ineligible; 91 withdrew during screening) (Figure 1). Race and ethnicity were self-reported. Participants were provided significant monetary compensation both during (at set time points) and on completion of the study. Compensation was calculated and provided in accordance with our institutional review board rules for time and inconvenience. Substantial compensation, along with frequent contact with the study investigators, likely facilitated the excellent retention rate.
Figure 1. Participant Flow in the Trial
Abbreviation: BMI, body mass index.
Baseline Assessments
Total energy expenditure was measured twice over a 2-week period using doubly labeled water: once while participants followed their usual diet at home, and once while provided a weight maintenance diet. Briefly, participants provided 2 urine samples before being dosed (2.0 g of 10% enriched H218O and 0.12 g of 99.9% enriched 2H2O per kg of estimated total body water), and additional timed samples were taken at 4.5 and 6 hours and 7 and 14 days after dosing. Carbon dioxide output (CO2) and energy expenditure were calculated as previously described.28-29 After the second doubly labeled water period, participants attended a 5-day inpatient stay (baseline) where numerous metabolic tests were conducted. Participants repeated the inpatient stay at months 3 and 6.
Intervention
Participants (N = 48) were sequentially randomized into 1 of 4 groups for 6 months: (1) control (weight maintenance diet); (2) calorie restriction (25% calorie restriction of baseline energy requirements); (3) calorie restriction with exercise (12.5% calorie restriction plus 12.5% increase in energy expenditure by structured exercise); and (4) very low-calorie diet (very low-calorie diet [890 kcal/d] until 15% reduction in body weight, followed by a weight maintenance diet). Two factors were balanced in study group allocation: sex and 2 categories of body mass index (BMI, calculated as weight in kilograms divided by height in meters squared) (25 to 27.9 and 28 to <30 at screening) according to Pocock and Simon.30 Except for the intervention team, all personnel involved in data collection were blinded to participant information including treatment assignment.
Diets
Energy requirements at baseline were individually calculated from measured energy expenditure. Menus were then prescribed for each participant within 100 kcal of his/her daily target intake. Menus were designed using Moore's Extended Nutrient Database (MENu 2000, PBRC, Baton Rouge, La) and ProNutra 3.0 (Viocare, Princeton, NJ). Participants were provided with all their food from the last 2 weeks of baseline through week 12. Participants ate 2 meals at the center each weekday, with 1 meal plus snacks packaged for take-out. During weeks 13 through 22, participants self-selected their diet based on individual calorie targets. During weeks 22 through 24, 2 meals per day were provided at the center, with 1 meal and snacks for take-out. All diets (except the very low-calorie diet) were based on American Heart Association recommendations (30% fat). The very low-calorie diet was 890 kcal/d (HealthOne, Health and Nutrition Technology, Carmel, Calif) given as 5 shakes containing 75 g of protein, 110 g of carbohydrate, 5 g of fat plus a 10-g bolus of fat per day. Once target weight loss (–15%) was achieved, participants in the very low-calorie diet group were slowly refed to an energy level that maintained body weight. Generally, target weight was achieved by week 8 in men and by week 11 in women.
Behavioral and Exercise Strategies
Participants attended weekly group meetings and initiated a midweek telephone call to report energy intake so that any problems adhering to the protocol were quickly addressed. Cognitive-behavioral techniques were used to foster adherence to diet and exercise prescriptions, including self-monitoring and stimulus control. The Health Management Resources Calorie System (HMR, Boston, Mass) was used to train participants to estimate the caloric content of food.
Participants in the calorie restriction with exercise group increased energy expenditure by 12.5% above resting by undergoing structured exercise (walking, running, cycling) 5 days per week. The mean (SD) target energy cost was 403 (63) kcal per session for women and 569 (118) kcal per session for men. Individual exercise prescriptions were calculated by measuring the oxygen cost (V-Max29 Series, SensorMedics, Yorba Linda, Calif) at 3 levels of the prescribed activity and an equation for estimating energy expenditure was generated. Mean (SD) exercise duration per session was 53(11) minutes in women and 45 (14) minutes in men. Participants were required to participate in 3 sessions per week under supervision and wore portable heart rate monitors (Polar S-610, Polar Beat, Port Washington, NY) to assess adherence during unsupervised sessions.
Biochemical Analyses
Fasting serum insulin, DHEAS, thyroxine (T4), and triiodothyronine (T3) levels were measured using immunoassays (DPC 2000, Diagnostic Product Corporation, Los Angeles, Calif). Glucose was analyzed using a glucose oxidase electrode (Syncron CX7, Beckman, Brea, Calif). The carbonyl content in proteins was determined using a modified 2,4-dinitrophenylhydrazine assay according to the method of Mates et al.31
Metabolic Tests
Weight was measured weekly in a hospital gown following a 12-hour fast after participants had voided. All other metabolic tests were conducted while participants were inpatients at baseline, month 3, and month 6. Fasting blood samples were taken. Body composition was measured by dual-energy x-ray absorptiometry (QDA 4500A, Hologics, Bedford, Mass). Sedentary energy expenditure (24-hour energy expenditure) was measured over 23 hours in a whole room indirect calorimeter as previously described.32 Three meals and 1 snack were provided at scheduled intervals, and participants were instructed to eat all their food within 30 minutes. Energy expenditure was calculated from O2, CO2, and 24-hour urinary nitrogen excretion33 and extrapolated to 24 hours. Sleeping energy expenditure was calculated between 2 AM and 5 AM, when motion detectors were reading zero activity.
At baseline, energy intake was matched to measured energy expenditure. However, in keeping with the assigned protocols at months 3 and 6, participants in the calorie restriction group were fed 25% less and participants in the calorie restriction with exercise group were fed 12.5% less than baseline energy expenditure, whereas the participants in the very low-calorie diet group were fed at a level that matched energy expenditure.
During the metabolic chamber study at baseline and month 6, core body temperature was measured every minute using telemetry pills (CorTemp, HQ Inc, Palmetto, Fla).34 Mean 24-hour, daytime (8 AM-10:30 PM), and nighttime (2 AM-5 AM) temperatures were computed. Due to malfunctions with the monitor or participants passing the pill, complete data were only obtained in 7 of 11 controls, 11 of 12 participants in the calorie restriction group, 8 of 12 participants in the calorie restriction with exercise group, and 9 of 11 participants in the very low-calorie diet group.
DNA Fragmentation
Single cell gel electrophoresis (Comet assay) was conducted according to Deutsch et al.35 Briefly, whole blood cells were suspended in low melting point agarose on commercially available slides (Trevigen, Gaithersburg, Md). The slides were viewed under an ultraviolet microscope (Nikon Microphot FXA, Hamamatsu, Japan [high-resolution 512 lines, Image I AT software, FITC 3 filter]). The extent of DNA damage was determined by calculating the comet tail moment, which is the integrated density in the comet tail multiplied by the distance from the center of the nucleus to the center of mass of the tail, for 25 cells using freely available software (Herbert M. Geller;
http://www2.umdnj.edu/~geller/lab/comet.htm). In 20 individuals measured on 2 consecutive days, the intraclass correlation coefficient of the method was 0.95.