Since the pioneering experiments by McCay et al. 36
, it has been known that calorie restriction (CR) extends lifespan in rodents and other lower species. However, little is known about the long-term effects of CR in humans. In the present study, we examined the effects of 6-months CR on biomarkers of CR, energy expenditure, and oxidative stress in humans. Our results indicate that prolonged CR caused: 1) a reversal of two out of three previously reported robust biomarkers of longevity (fasting insulin and core body temperature); 2) a metabolic adaptation (decrease in EE larger than expected on the basis of loss of metabolic mass) associated with lower thyroid hormone concentrations and 3) a reduction in DNA fragmentation as a result of less damage to DNA.
Numerous biomarkers of CR have been identified in rodents including temperature, DHEAS, glucose, and insulin. Roth et al. 26
recently observed that body temperature, insulin, and DHEAS were also altered in CR monkeys, 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 and body core temperature in CR and CREX groups. However, DHEAS and fasting glucose were unchanged by intervention. Most likely, this study was of insufficient duration to detect changes in DHEAS, which have been calculated to fall 2-4% per year in humans. Fasting glucose is not consistently altered by prolonged CR in primates and thus we question whether fasting glucose is useful as a biomarker in longer-lived species. On the other hand, Fontana et al. 27
observed that fasting glucose and insulin were substantially reduced in CR subjects who had been following self-prescribed nutritionally adequate CR diets for 6 years.
Previous studies are inconclusive regarding whether metabolic rate is reduced following prolonged CR. In rodents, adjusted resting EE was not different from controls after restricting energy intake for six months 11
or for the entire life span 12
. In monkeys, adjusted resting EE was reduced by 60kcal/d after 11 years of CR 7
, but previously these authors had reported no metabolic adaptation after 42 months 38
. Indeed, there are numerous reports in the literature showing either reduced or unchanged adjusted EE after prolonged CR in monkeys 8, 25
. In humans, the effects of prolonged, nutrient dense, calorie restricted diets on non-obese subjects have not been formally investigated. In the Keys starvation study 39
, adjusted resting EE was decreased and this coincided with a reduction in temperature indicating a real metabolic adaptation 40
. Adjusted 24h-EE was also lower in 5 subjects after 2-years CR in the Biosphere 2 experiment as compared to 152 control subjects 41
. However, resting-EE was not altered in overweight women following liquid calorie diet to normal body weight and 10 days of weight stabilization 42
. In this study, we observed a metabolic adaptation over 24-hour in sedentary conditions and during sleep following 6-months of CR. The metabolic adaptation in the CREX group was similar to that observed in CR group, suggesting that energy deficit rather than CR 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 metabolism43
. Of significance, the metabolic adaptation occurred in the first 3-months after intervention with no further adaptation at 6 months, even though weight loss continued in CR and CREX groups. Metabolic adaptation was also observed over 24 hours but not during sleep in LCD subjects who were weight stable when measured at M3 and M6. Possible explanations for the lack of significant adaptation during sleep in this group include a smaller sample size and that two men were regaining weight at M6. Interestingly, core temperature and fasting insulin at M3 were also not changed in this group, despite the largest amount of weight loss. Whether metabolic adaptation persists during weight loss 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 two factors can account for some of the metabolic adaptation, it should be remembered that the thermic effect of food account 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 lifespan has been demonstrated in numerous studies 45, 46
. Caloric restriction in mice down regulates genes involved in oxidative stress and reduces oxidative damage (8oxodG), lipid peroxidation and protein carbonyls 18, 20, 21, 23
. In non-human primates, genes involved in protection against oxidative stress are not altered by CR, although protein carbonylation is reduced 22
. In obese humans, protein carbonylation is also reduced after 4 weeks of CR 47
. Whilst we observed no change in protein carbonylation, we are the first to report a significant decline in DNA damage following six months of CR in nonobese humans. Contrary to our hypothesis, the reduction in DNA damage was not associated with reduced total or adjusted O2
consumption in the metabolic chamber. Considering the lack of correlation between these parameters and the lack of response in protein carbonylation in response to CR, we are hesitant to conclude that CR reduces oxidative stress overall. Clearly, more studies investigating different measures of oxidative stress, such as 24-h urinary samples of 8oxodG are required. Furthermore, other factors (such as mitochondrial function) may play an important role in the accumulation of oxidative stress. Indeed, the importance of uncoupling proteins in protection against ROS production, independent of changes in proton kinetics and mitochondrial respiration has recently been demonstrated48
The results of this study show that prolonged CR by diet or by a combination of diet and exercise was successfully implemented as evidenced by reduced weight, fat mass, fasting serum insulin and body core temperature. This study is unique in that individual energy requirements were carefully measured at baseline allowing us to feed and prescribe individual diet goals for each subject. Furthermore, we observed that “metabolic adaptation” develops in response to energy deficit in non-obese humans at 3 and 6 months leading to reduced oxygen consumption per unit of fat-free mass, even after weight stability is achieved. Finally, this study confirms previous findings that calorie restriction results in a decline in DNA damage. However, longer studies are required to determine if these effects are sustained and impact the aging process.