[Obesity Discussion]

Figure 6. Fasting Plasma Protein Carbonyls and DNA Damage Measured by the Comet Assay
DNA damage was significantly reduced from baseline in the calorie restriction, calorie restriction with exercise, and very low-calorie diet groups at month 6 (all P<.005).

Since the pioneering experiments by McCay and Maynard,37 it has been known that calorie restriction extends life span in rodents and other lower species. However, little is known about the long-term effects of calorie restriction in humans. In the current study, we examined the effects of 6-month calorie restriction on biomarkers of calorie restriction, energy expenditure, and oxidative stress in humans. Our results indicate that prolonged calorie restriction caused: (1) a reversal in 2 of 3 previously reported biomarkers of longevity (fasting insulin level and core body temperature); (2) a metabolic adaptation (decrease in energy expenditure larger than expected on the basis of loss of metabolic mass) associated with lower thyroid hormone concentrations; and (3) a reduction in DNA fragmentation, reflecting less DNA damage.

Numerous biomarkers of calorie restriction have been identified in rodents including temperature, and DHEAS, glucose, and insulin levels. Roth et al26 recently observed that body temperature and insulin and DHEAS levels were also altered in monkeys subjected to calorie restriction, validating their usefulness as biomarkers in longer-lived species. Importantly, they also showed that these parameters were altered in longer-lived men. These findings support the role of these factors as biomarkers of longevity in humans. Similar to the primate model, we observed significantly reduced fasting insulin levels and core body temperatures in the calorie restriction and calorie restriction with exercise groups. However, DHEAS and fasting glucose levels were unchanged by the interventions. Most likely, this study was of insufficient duration to detect changes in DHEAS level, which has been calculated to fall 2% to 4% per year in humans. Fasting glucose level is not consistently altered by prolonged calorie restriction in primates, and thus we question whether fasting glucose level is useful as a biomarker in longer-lived species. On the other hand, Fontana et al27 observed that fasting glucose and insulin levels were substantially reduced in calorie restriction participants who had been following self-prescribed nutritionally adequate calorie restriction diets for 6 years.

Previous studies are inconclusive regarding reductions in metabolic rate following prolonged calorie restriction. In rodents receiving a restricted energy diet for 6 months11 or the entire life span,12 adjusted resting energy expenditure was not different from controls. In monkeys, adjusted resting energy expenditure was reduced by 60 kcal/d after 11 years of calorie restriction,7 but in previous work, these authors reported no metabolic adaptation after 42 months of calorie restriction.38 Indeed, there are numerous reports in the literature showing either reduced or unchanged adjusted energy expenditure after prolonged calorie restriction in monkeys.8, 25 In humans, the effects of prolonged, nutrient-dense, calorie-restricted diets in nonobese patients have not been formally investigated. In a starvation study by Keys et al,39 adjusted resting energy expenditure was decreased, which coincided with a reduction in body temperature indicating a real metabolic adaptation.40 In the Biosphere 2 experiment, adjusted 24-hour energy expenditure was lower in 5 participants after 2-year calorie restriction, compared with 152 controls.41 In a study of weight-stable women who had achieved normal body weight using a low-calorie liquid diet, Weinsier et al found that after adjustment for reduced body size, there was no change in resting energy expenditure.42

In this study, we observed a metabolic adaptation over 24 hours in sedentary conditions and during sleep following 6 months of calorie restriction. The metabolic adaptation in the calorie restriction with exercise group was similar to that observed in the calorie restriction group, suggesting that energy deficit rather than calorie restriction itself is driving the decrease in energy expenditure. Importantly, the metabolic adaptations were closely paralleled by a drop in thyroid hormone plasma concentrations confirming the importance of the thyroid pathway as a determinant of energy metabolism.43 Of significance, the metabolic adaptation occurred in the first 3 months of the intervention, with no further adaptation at 6 months, even though weight loss continued in the calorie restriction and calorie restriction with exercise groups.

Metabolic adaptation was also observed over 24 hours but not during sleep in participants in the very low-calorie diet group who were weight stable when measured at months 3 and 6. Possible explanations for the lack of significant adaptation during sleep in this group include a smaller sample size and the fact that 2 men were regaining weight at month 6. Interestingly, core body temperature and fasting insulin level at month 3 were not changed in this group, despite their having the largest weight loss. Whether metabolic adaptation following calorie restriction persists during weight maintenance remains to be determined in humans.

Spontaneous physical activity and the thermic effect of food were not changed from baseline. However, even if these 2 factors can account for some of the metabolic adaptation, the thermic effect of food accounts for only 10% of daily energy expenditure,44 and the cost of activity is already accounted for by a decrease in body weight. Therefore, these 2 factors can only account for a minor part of the metabolic adaptation.

The inverse relationship between increased free radical production, oxidative damage to DNA, and maximum life span has been demonstrated in numerous studies.45-46 Caloric restriction in mice down-regulates genes involved in oxidative stress and reduces oxidative damage (8-oxodG), lipid peroxidation, and protein carbonyls.18, 20-21,23 In nonhuman primates, genes involved in protection against oxidative stress are not altered by calorie restriction, although protein carbonylation is reduced.22 In obese humans, protein carbonylation is also reduced after 4 weeks of calorie restriction.47 While we observed no change in protein carbonylation, we are the first to report a significant decline in DNA damage following 6 months of calorie restriction in nonobese men and woman. Contrary to our hypothesis, the reduction in DNA damage was not associated with reduced total or adjusted oxygen consumption in the metabolic chamber.

Considering the lack of correlation between these parameters and the lack of response in protein carbonylation associated with calorie restriction, we are hesitant to conclude that calorie restriction reduces oxidative stress overall. Clearly, more studies investigating different measures of oxidative stress, such as 24-hour urinary excretion of 8-oxodG, are required. Furthermore, other factors (such as mitochondrial function) may play an important role in oxidative stress. For example, the role of uncoupling proteins in protection against ROS production, independent of changes in proton kinetics and mitochondrial respiration, has recently been demonstrated.48

The results of this study show that prolonged calorie restriction by diet or by a combination of diet and exercise was successfully implemented as evidenced by reduced weight, fat mass, fasting serum insulin levels, and core body temperature. This study is unique in that individual energy requirements were carefully measured at baseline and individualized diet goals were determined for each study participant. Furthermore, we observed that "metabolic adaptation" develops in response to energy deficit in nonobese humans at 3 and 6 months leading to reduced O2 per unit of FFM, even after weight stability is achieved. Finally, this study confirms previous findings that calorie restriction results in a decline in DNA damage. However, longer-term studies are required to determine if these effects are sustained and whether they have an effect on human aging.

Corresponding Author: Eric Ravussin, PhD, Pennington Biomedical Research Center, Louisiana State University, 6400 Perkins Rd, Baton Rouge, LA 70808 (ravusse@pbrc.edu).

Author Contributions: Dr Ravussin had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.

Study concept and design: DeLany, Larson-Meyer, Volaufova, Greenway, Deutsch, Williamson, Ravussin.

Acquisition of data: Heilbronn, de Jonge, Frisard, DeLany, Larson-Meyer, Nguyen, Martin, Most, Smith, Williamson, Ravussin.

Analysis and interpretation of data: Heilbronn, de Jonge, DeLany, Rood, Volaufova, Deutsch, Williamson, Ravussin.

Drafting of the manuscript: Heilbronn, Rood, Martin, Most, Deutsch, Williamson, Ravussin.

Critical revision of the manuscript for important intellectual content: de Jonge, Frisard, DeLany, Larson-Meyer, Nguyen, Martin, Volaufova, Greenway, Smith, Deutsch, Williamson, Ravussin.

Statistical analysis: Heilbronn, Volaufova, Ravussin.

Obtained funding: Williamson, Ravussin.

Administrative, technical, or material support: Heilbronn, de Jonge, DeLany, Rood, Nguyen, Martin, Greenway, Smith, Williamson, Ravussin.

Study supervision: Heilbronn, Frisard, Larson-Meyer, Most, Greenway, Deutsch, Williamson, Ravussin.

Financial Disclosures: None reported.

Funding/Support: This work was supported by research grant U01 AG20478 from the National Institutes of Health.

Role of the Sponsor: The funding agency had no role in the analysis or interpretation of the data or in the decision to submit the report for publication.

Other Members of the Pennington CALERIE Research Team: Steven Anton, PhD, Emily York-Crowe, MA, Catherine Champagne, PhD, Paula Geiselman, PhD, Michael Lefevre, PhD, Jennifer Howard, LDN, RD, Jana Ihrig, BSN, Brenda Dahmer, Anthony Alfonso, MS, Darlene Marquis, BS, Connie Murla, BS, Aimee Stewart, BS, Amanda Broussard, BS, and Vanessa Tarver, BS (all from Pennington Biomedical Research Center, Louisiana State University, Baton Rouge).