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Am J Physiol Endocrinol Metab 294: E416-E424, 2008. First published November 27, 2007; doi:10.1152/ajpendo.00573.2007
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Efficiency of autoregulatory homeostatic responses to imposed caloric excess in lean men

Mario Siervo,1 Gema Frühbeck,2 Adrian Dixon,3 Gail R. Goldberg,1 W. Andy Coward,1 Peter R. Murgatroyd,4 Andrew M. Prentice,5 and Susan A. Jebb1

1Medical Research Council Human Nutrition Research, Elsie Widdowson Laboratory, Cambridge, United Kingdom; 2Department of Endocrinology, Clínica Universitaria de Navarra, Pamplona, Spain; 3Department of Radiology and 4Wellcome Trust Clinical Research Facility, University of Cambridge, Addenbrooke's Hospital, Cambridge; and 5Medical Research Council International Nutrition Group, London School of Hygiene and Tropical Medicine, London, United Kingdom

Submitted 5 September 2007 ; accepted in final form 23 November 2007


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Obesity implies a failure of autoregulatory homeostatic responses to caloric excess. We studied the mechanisms, effectiveness, and limits of such responses in six lean (21.9 ± 1.3 kg/m2), healthy men based in a metabolic suite for 17 wk of progressive intermittent overfeeding (OF) (3 wk, baseline; 3 wk, 20% OF; 1 wk, ad libitum; 3 wk, 40% OF; 1 wk, ad libitum; 3 wk, 60% OF; 3 wk, ad libitum). Body composition was assessed by a four-compartment model using dual X-ray absorptiometry, deuterium dilution, and plethysmography. Magnetic resonance imaging assessed subcutaneous/visceral fat at abdominal level at baseline and at the end of 60% OF. Energy intake was assessed throughout, energy expenditure (EE) and substrate oxidation rates were measured repeatedly by whole body calorimetry (calEE), and free-living EE (TEE) was measured by doubly labeled water at baseline and after 60% OF. At the end of 60% OF, calEE and TEE had increased by just 11.4% (P = 0.001) and 16.2% (P = 0.001), respectively. Weight and body fat (fat mass) had increased by 5.98 kg (8.8%, P = 0.001) and 3.31 kg (22.6%, P = 0.01), respectively. The relative increase in visceral fat (32.6%, P = 0.02) exceeded that of subcutaneous fat (13.3%, P = 0.002) in the abdominal region. The computed energy cost of tissue accretion differed from the excess ingested by only 13.1% (using calEE) and 11.6% (using TEE), indicating an absence of effective dissipative mechanisms. We conclude that elevations in EE provide very limited autoregulatory capacity in body weight regulation, and that regulation must be dominated by hypothalamic modulation of energy intake. This result supports present conclusions from genetic studies in which all known causes of human obesity are related to defects in the regulation of appetite.

energy balance; body composition; whole body calorimetry; doubly labeled water; overfeeding; dissipative mechanisms

OVEREATING, WHETHER CHRONIC or episodic, produces a positive energy balance and favors the accretion of new tissues, particularly fat (35, 49). The efficiency of innate autoregulatory mechanisms that attempt to maintain body weight homeostasis has been explored extensively during the last century since the earliest experiments published by Neumann in 1902 (30) and Gulick in 1922 (14). These suggested the existence of energy dissipative mechanisms (termed luxuskonsumption) able to dispose of part of the energy excess as heat and decrease the storage of energy as fat. These two overfeeding studies were conducted on only one subject, and the authors claimed that an increase in energy expenditure (EE) dissipated the extra energy available and prevented weight gain. Forbes (11) reexamined the relationship between weight gain and excess energy intake in these two studies and observed that the slope of the regression line between weight gain and excess energy intake was close to the predicted cost of weight gain. Since then, numerous studies performed under experimental (3, 8, 17, 22, 24–26, 37, 41) and nonexperimental settings (5, 28, 33, 45) have failed to reach a consensus (40, 47, 51, 53). The controversy was mainly focused on the identification of the mechanisms disposing of the excess energy (futile cycles, nonexercise activity thermogenesis, mitochondrial uncoupling proteins) and on the partitioning and storage of the available energy (fat mass, glycogen, lean mass). The study of such energy dissipating mechanisms is a methodological challenge, as they are extremely sensitive to external confounding by various environmental influences (e.g., dieting, physical activity, eating behavior, psychological and physical well-being, drugs) (20) and because measurement techniques of body composition have lacked sufficient precision. The most accurate and replicable conditions to study energy balance in humans require highly controlled experimental protocols where the manipulation of energy intake and macronutrient composition can be made with standardization, and in which precise measurement of physical activity level, energy metabolism, and body composition is possible.

Many previous studies have employed very short-term protocols to probe possible metabolic responses, but these would not detect slowly inducible mechanisms. Most of the previous longer-term studies have used a single (generally severe) level of overfeeding. Such conditions may overwhelm the available homeostatic processes and obscure more subtle changes that may be effective in modulating energy balance during naturally occurring episodic periods of marginal excess consumption. To overcome these limitations, we used state-of-the-art measurement methods in a highly standardized 17-wk protocol involving progressive overfeeding from 20 to 60% energy excess. Lean, healthy men were challenged with three 3-wk periods of stepwise overfeeding (OF) separated by 1 wk of ad libitum energy intake. Our objectives were as follows: 1) to study the limits of any putative energy dissipating autoregulatory mechanisms (the luxuskonsumption hypothesis); 2) to assess their importance relative to alterations in appetite and food intake during the subsequent ad libitum periods; and 3) to assess the induced changes in whole body and segmental body composition, in particular the balance between abdominal subcutaneous and visceral fat, to describe patterns of fat accretion during medium-term caloric excess.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Changes in energy metabolism and body composition induced by experimental OF were assessed in six healthy, weight-stable, habitually lean men. All subjects lived and worked in the Cambridge area and were recruited through the MRC Dunn Nutrition Unit's register of volunteers. The study was approved by the Unit's Ethics Committee. Subjects gave their written consent to participate in this study. The study was conducted at the former MRC Dunn Clinical Nutrition Centre (DCNC).

The selection criteria were good health, weight stability, habitual alcohol consumption <21 units/wk, not vegetarian, nonsmoker, no food intolerances, and willingness to complete the study procedures. Throughout the study, subjects were provided with food, accommodation, and a small honorarium. The subjects lived for the entire period in the DCNC metabolic facility and were allowed to leave only for short periods of time. The volunteers were instructed to maintain their usual level of physical activity, and, except for the exercise performed in the metabolic chamber, deliberate additional exercise was not allowed. Medical conditions and potential side effects were regularly monitored, and any health problem was reported and assessed by a medically qualified researcher. No subjects were excluded from the study for intercurrent adverse events. One subject did not complete the final OF period and ad libitum phase.

The study started with a baseline period of 3 wk during which the dietary intake provided was adjusted to maintain body weight. Subjects were then challenged with 3-wk stepwise OF phases (+20, +40, and +60% increases above the baseline energy intakes) separated by intermittent ad libitum phases.

Three meals per day (breakfast, lunch, supper) were provided on a 4-day rotating menu. Meals were prepared in the DCNC metabolic kitchen. The diets were carefully designed both to include foods eaten in the United Kingdom and to avoid excess palatability. The calculation of the energy content of the diet was based on United Kingdom food composition tables (19). As OF progressed, the portion size of meals increased, and snacks were introduced to increase total energy intake. Subjects were required to consume all the food provided. During the intercurrent ad libitum periods, subjects ate the same preweighed diets provided at similar levels of excess as in the preceding OF periods. All uneaten food was measured and total intake calculated. Water, tea, and coffee (decaffeinated) were freely available.

During the baseline period, subjects received a diet designed to meet their energy requirements (calculated as 1.5 x predicted basal metabolic rate), composed of 13% energy from protein (P), 40% from fat (F), and 47% from carbohydrate (C), and the amount of energy provided was then adjusted to maintain weight stability. The subjects then received a fixed diet providing +20% (P = 13%, F = 43%, C = 44%) of the baseline intake for 3 wk. One-half of the increase was achieved by an increase in portion size and the remainder by an increase in the proportion of fat. This was followed by a week of ad libitum food consumption in which subjects continued to be offered +20% of their baseline energy intake but were allowed to eat only as much as desired. In week 6, the subjects progressed to the second stage of OF, composed of +40% (P = 12%, F = 46%, C = 42%) of baseline energy intake for 3 wk followed again by a week of standardized ad libitum consumption with the same +40% diet offered. The final step of OF was composed of a 3-wk period of +60% (P = 12%, F = 48, C = 40%) of baseline energy intake followed by 3 wk of ad libitum energy intake from the +60% diet. The energy density of the diets (MJ/100 g) increased by only 10% between the baseline and +60% OF. Energy intake, energy density, and nutrient content of the diets and snacks provided during baseline and OF are shown in Table 1. No assessment of metabolizable energy was performed because, in previous analogous experiments, we found excellent agreement between metabolizable energy directly measured by bomb calorimetry of diet, feces, and urine and that calculated from food composition tables (r = 0.99) as described elsewhere (8).


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  		Table 1. Energy and macronutrient content of diets and snacks provided during the baseline and overfeeding phases

 
Body Composition

Body weight (±10 g) was measured weekly after voiding and before breakfast using a digital integrating scale (Sauter E1210, Suffolk, UK). Height was measured to the nearest 5 mm using a wall-mounted stadiometer (Holtain, Dyfed, Wales, UK) at the start of the study. Body mass index (BMI) was calculated as weight (kg)/height (m)2. Body composition measurements were performed at the end of baseline and each OF phase (20% OF, 40% OF, 60% OF) and at the end of the last ad libitum phase.

Air displacement plethysmography. Measurements were performed in duplicate using an air displacement plethysmograph (BODPOD, Life Measurement Instruments, Concord, CA) according to manufacturer's instructions. Thoracic gas volume was predicted by estimation of functional residual capacity and tidal volume (27). Siri's two-compartment formula was used to calculate percent fat mass (FM%) from body density (46). From FM% and body weight, fat mass (FM) and fat-free mass (FFM) in kilograms were calculated.

Total body water. Total body water (TBW) was measured by isotope dilution. After collection of a predose saliva sample, subjects received an oral dose of deuterium oxide (0.7 g/kg body wt), and saliva samples were collected at 4, 5, and 6 h after the dose. The subjects refrained from eating or drinking for 30 min before saliva samples were taken. The concentration of deuterium in each sample was measured using isotope ratio mass spectrometry as described elsewhere (18), and the pool size was calculated. The measured pool size was reduced by 4% to account for the exchange of deuterium with nonaqueous hydrogen. The hydration fraction of FFM was assumed to be 0.7194, and FM was calculated as the difference between FFM and body weight. This method was used to measure TBW at the end of 20% OF, 40% OF, and the last ad libitum phase. TBW was measured using the doubly labeled water protocol at the end of baseline and 60% OF. The influence of repeated doses of deuterium on the background level of deuterium enrichment and the effects on the measurement of total EE (TEE) have been taken into account and corrected for as described elsewhere (38, 39).

Dual-energy X-ray absorptiometry. Whole body dual-energy X-ray absorptiometry (DXA) scans were performed using a Hologic QDR-1000W scanner (Hologic, Waltham, MA) and analyzed using an enhanced version of the software to estimate bone mineral mass (subsequently used to derive "ash"), bone mineral content (BMC), FM, and FFM. Subjects were measured wearing a standard light cotton gown to minimize clothing absorption.

Total body scanning area was divided into anatomic segments: the arms were separated from the trunk by a line passing through the humeral head and the apex of the axilla. The trunk was separated from the legs by a line passing from the iliac crest to the perineum. The head was excluded from the trunk by a horizontal line passing just below the mandible.

Four-compartment model. The four-compartment (4-C) model divides the body into fat, water, protein, and mineral, thereby avoiding the assumption that the ratio between mineral and protein in FFM is constant. The body composition data were combined to yield an estimation of FM. FM (kg) = (2.747 x body volume) + (0.710 x TBW) + (1.460 x BMC) + (2.050 x body weight), where body volume is in liters, TBW is in kilograms, BMC is in kilograms, and body weight is in kilograms (31). The protein plus carbohydrate (P+C) compartment was derived by difference (P+C = body weight – TBW – total mineral mass – FM). The precision of the 3- and 4-compartment models to assess body fat was ±0.49 and ±0.54 kg, respectively, when a 1% precision for water estimation was assumed. The precision for estimates of TBW was based on sequential measurements of the isotopic enrichment of water in saliva samples taken at 4, 5, and 6 h after oral administration of the isotope. Precision for the measurement of water calculated from this study was 0.45 kg (~1%) (13).

Magnetic resonance imaging. Abdominal adipose tissue distribution was assessed by T1-weighted magnetic resonance imaging (MRI) with a single cross-sectional image at the level of the umbilicus. The area of the cross section of the torso (total cross-sectional area) was measured manually using an electronic cursor on the MRI work station (Advantage Windows, GE, Milwaukee, WI) as a region of interest. Then the cross-sectional area of the intra-abdominal cavity was measured (IAC) just internal to the rectus abdominis, trasversus muscle, iliacus muscle, and aortic biforcation. It was assumed that any change in these areas was due to an alteration of fat. Visceral abdominal adipose tissue was assumed to be equivalent to IAC. Subcutaneous abdominal adipose tissue was calculated as the difference between the total cross-sectional area and IAC.

EE

Twenty-four-hour whole body indirect calorimetry measurements (24-h EE) were performed at the end of each phase (baseline, OF phases, ad libitum phases). Free-living TEE, using doubly labeled water (DLW), was measured only at baseline and during the last OF phase (+60%).

Whole body indirect calorimetry. The calorimeter chambers were comfortably furnished with a divan bed, armchair, and entertainment facilities (television, radio, telephone). All urine samples were collected for analysis. While in the calorimeter, subjects followed an identical protocol on each occasion with sleep, rest, meals, and exercise (cycle ergometer and stepping). Subjects entered the chamber at 2000 (24-h clock) on day 0. A total of 37 h were spent inside, ending at 0900 on day 2. Exchange rates for oxygen and carbon dioxide were calculated using the expressions of Brown et al. (4) for pressure-ventilated systems. A detailed description of the calorimeters is given elsewhere (29). Briefly, measures of TEE (24-h EE), basal metabolic rate (BMR), and activity plus thermogenesis (A+T) were obtained. BMR was measured for 1 h immediately on waking, between 12.5 and 13.5 h postabsorption, at thermoneutrality, and at complete physical rest and was measured twice during each 37-h period. Two 30-min periods of weight-dependent exercise (10 step ups/min on a 20-cm block) and two 30-min periods of weight-independent exercise (cycle ergometer at 25 W) were performed, and the exercise periods were closely supervised. The value for A+T was calculated by subtracting BMR from 24-h EE (24-h EE – BMR). Calculation of macronutrient balance took into account the measurements made in the calorimeter at the end of each phase. Carbohydrate and fat oxidation were calculated according to the method of Elia and Livesey (10), assuming an energy content of 39.33 kJ/g for fat, 15.76 kJ/g for carbohydrate, and 18.56 kJ/g for urinary protein.

DLW. TEE under free-living conditions was assessed by DLW during baseline and 60% OF. Doses of deuterium (0.07 g/kg) and 18O (0.174 g/kg) were given orally 14 days before the end of each period. After collection of a predose urine sample, samples were collected on the dosing day and daily for 14 days thereafter. Theoretical considerations concerning analysis, propagation of error, and calculations of EE have been described elsewhere (6, 36). Isotope enrichment of the urine samples was analyzed using continuous-flow isotope ratio mass spectrometry (Sira 10 Dual Inlet Mass Spectrometer, Micromass).

Cost of weight gain. The calculation of changes in energy stores and the agreement with excess energy intake was calculated according to Tremblay et al. (48). The calculations for each OF phase are based on the body composition (4-C model) and 24-h EE measurements in the calorimeter at the end of each OF phase. The calculations for the whole study used the body composition measurements (4-C model) and free-living TEE (DLW) performed at baseline and at the end of the 60% OF. The preceding ad libitum phases have been included in the calculations, with the assumption that body composition and EE changes from the start of each ad libitum phase to the end of OF were linear. The calculated intrinsic energetic costs associated with tissue accretion (fat, protein) were added to the increase in TEE, which includes any increase in thermic effect of food and energetic cost of physical activity associated with weight gain. The energy deposited as fat and protein was assumed to be 38.9 and 23.5 MJ/kg, respectively. The synthesis costs of fat and protein have already been accounted for in the measurement of TEE. Possible changes in glycogen stores were ignored.

Statistics

Data are expressed as means ± SD or means ± SE. The absolute (x – y) and relative [(x – y/y) * 100] changes ({Delta}) from baseline were calculated for each variable (weight, body composition, energy intake, EE). Analysis of variance (ANOVA) for repeated measures and post hoc analysis (least square method) were used to detect significant changes between phases. Pearson correlation coefficient was used to test associations among variables. Commercial software packages (SPSS 14, SPSS, and Sigmaplot 10, Systat Software) were used. The significance level was set at P < 0.05.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
The characteristics of the six subjects are shown in Table 2. Mean age was 43.3 ± 10.6 yr (range: 32–58 yr), and baseline values for weight, height, and BMI were, respectively, 68.9 ± 8.8 kg (range: 56.0–80.8 kg), 1.77 ± 0.07 m (range: 1.63–1.83 m), and 21.9 ± 1.3 kg/m2 (range: 20.8–24.1 kg/m2).


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  		Table 2. Individual and mean changes in body weight and fat mass during the OF and ad libitum phases

 
Weight Change

Table 2 also shows individual and mean changes in body weight and FM throughout the study. The periods of imposed energy excess led to a consistent weight gain, but weight changes were more variable during the ad libitum phases. Three subjects gained weight during the first and second ad libitum phases, and three subjects lost weight. Body weight decreased in all subjects during the last ad libitum phase. During the 20% OF, there was a cumulative weight gain of 0.70 kg (not significant; NS), which was substantially unchanged (+0.73 kg vs. baseline) by the end of the subsequent ad libitum phase. Weight continued to increase during the 40% OF (+2.54 kg vs. baseline, P = 0.001) followed by a loss of 0.44 kg by the end of the second ad libitum phase. Weight gain reached its peak at the end of the 60% OF (+5.98 kg vs. baseline, P = 0.001), a cumulative weight increase of 8.8% from baseline. Despite a weight loss of 2.71 kg during the last ad libitum phase, subjects gained a net 3.27 kg (5%, P = 0.03) by the end of the study, ranging from 0.93 to 5.83 kg (1.4 to 9.8%).

Analysis of the weekly rate of weight change highlighted the responses to overeating and ad libitum intake. The rate of weight gain during OF phases was greater than the rate of weight loss achieved during the subsequent ad libitum phases. Weight continued to increase during the first ad libitum phase (0.03 kg/wk), and the weight gain during the 40% OF (0.73 kg/wk) was greater than the weight decrease during the second ad libitum phase (–0.44 kg/wk). The rate of weight gain during the 60% OF (1.12 kg/wk) was almost counterbalanced (–0.90 kg/wk) during the final ad libitum phase.

Body Composition

Changes in body composition are shown in Figs. 1 and 2. The change in body fat (FM) was proportional to the degree of OF, and by the end of the 60% OF phase, there was a significant increase in FM (3.31 kg, P = 0.01) followed by a decrease of 1.61 kg during the last ad libitum phase. FFM also increased by 2.67 kg (P = 0.07) by the end of 60% OF. Most of the change in FFM was due to changes in TBW, which explained, on average, 87% of the FFM gain and 86% of FFM loss during the OF and ad libitum phases, respectively. The contribution of protein to FFM change increased during the first two OF phases (20% OF = 21%; 40% OF = 39%) but declined to 8% during the 60% OF. There were no significant changes in total mineral content.


Figure 1
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  		Fig. 1. Cumulative changes in body composition using a 4-compartment (4-C) model (weight, fat mass, total body water, and protein) in the 5 subjects who completed the study. Overfeeding (OF) phases = +20%, +40%, and +60%. AL, ad libitum. Baseline values were as follows: weight = 68.69 ± 4.39 kg; fat mass (FM) = 14.62 ± 3.26 kg; protein = 11.11 ± 0.59 kg; total body water = 39.62 ± 1.92 kg. Data are shown as means ± SE. *P < 0.05 (relative to baseline).

 

Figure 2
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  		Fig. 2. Absolute values and changes ({Delta}) in body composition at the end of +60% OF in 5 subjects. Total body fat was assessed by a 4-C model. FFM, fat-free mass; SC FM, subcutaneous FM; VIFM, visceral FM; L, left; R, right. FMBL and FFMBL are baseline (BL) values. Segmental measures of body composition were assessed by dual-energy X-ray absorptiometry (trunk, arms, legs) and MRI (SC FM, VIFM). {Delta}% = [(60% OF – baseline)/baseline] * 100.

 
The exclusion of the first subject from the analysis did not significantly alter the results. In the remaining five subjects, the segmental body composition analysis using DXA showed that truncal FM increased by ~60% at the peak of weight gain compared with only 28 and 17% increases in upper and lower limbs, respectively (Fig. 2). The substantial increase in truncal FM was confirmed by MRI. At the end of the +60% OF, the relative increase in visceral fat was 32.6% (38.6 cm2, P = 0.02) and the increase in subcutaneous fat 13.3% (47.7 cm2, P = 0.002) (Fig. 2).

EE

Table 3 shows BMR and 24-h EE measured with whole body calorimeter (calEE) and TEE measured by DLW.


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  		Table 3. Measurement of energy expenditure using indirect calorimetry and doubly labeled water

 
Whole body indirect calorimetry. On average, calEE increased during the OF phases and decreased during the ad libitum phases, although it remained above the baseline values. calEE at the end of 20, 40, and 60% OF was 11.7, 11.9, and 12.7 MJ/day, respectively, and the cumulative difference relative to baseline was statistically significant during each OF phase: 20% OF (+0.39 MJ/day, P = 0.03), 40% OF (+0.51 MJ/day, P = 0.001), and 60% OF (+1.32 MJ/day, P = 0.001). BMR increased significantly during the 40% OF (+0.39 MJ/day, P = 0.02) and 60% OF (+0.96 MJ/day, P = 0.007). The energy expended in A+T showed a significant increase during 60% OF (8.2%), reflecting increased thermogenesis, followed by a decrease during the last ad libitum phase (–6.4% vs. baseline) when subjects were all in negative energy balance (data not shown). Baseline calEE/FFM and BMR/FFM were 209.1 kJ/kg FFM and 127.6 kJ/kg FFM, respectively. At the end of 60% OF, calEE/FFM was increased by 6% (NS) and BMR/FFM by 8% (NS). The absolute changes in calEE measured at the end of each OF phase ({Delta}calEE) were significantly associated with {Delta}FM (r = 0.53, P = 0.02) and {Delta}weight (r = 0.72, P = 0.001) but surprisingly not with {Delta}FFM (r = 0.30, NS). At the end of 60% OF, an indirect association was observed between percent change ({Delta}%) in calEE and {Delta}% in body weight (n = 5; r = –0.71, P = 0.17), indicating that subjects with a greater increase in calEE experienced a lower weight gain.

DLW. There was a significant increase (17%) in TEE at the end of 60% OF relative to baseline (+1.89 MJ/day, P = 0.01). The physical activity level (PAL; PAL = TEE/BMR) was not significantly different from baseline (PALbaseline = 1.60 vs. PAL60% OF = 1.65; NS).

Energy Intake

On average, there was an absence of compensatory adjustments to OF. The mean energy intake (EI) during ad libitum periods was not significantly different from baseline for all the ad libitum phases as a result of the large between-subject variability. Indeed, during the first and second ad libitum periods, EI was +4.9 and +10% higher, respectively, than baseline. During the final ad libitum period, EI was decreased compared with the preceding OF period (–38%) and was comparable to baseline (–2.3%) (Fig. 3).


Figure 3
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  		Fig. 3. Change in energy intake (EI) relative to baseline during the study. Open circles show when subjects were in the calorimeters. Each circle during the ad libitum phases represents a 1-day period (some EI data during the last ad libitum phase are not available). *Each circle during the OF phases (+20% OF, +40% OF, +60% OF) represents a 1-wk period. Values are expressed as means ± SE.

 
Macronutrient Oxidation

Macronutrient balance, calculated as macronutrient intake (MJ/day) minus substrate oxidation (MJ/day), at baseline and at the end of each OF phase is shown in Fig. 4. The analysis was carried out on the five subjects who completed all phases to allow direct comparisons. Stepwise OF promoted fat accumulation by reciprocal changes in fat (decrease, P = 0.01) and carbohydrate oxidation (increase, P = 0.001). There was a significant increase in protein oxidation (P = 0.007), although the contribution to overall energy balance was minimal. The net change relative to baseline at the end of 60% OF was +0.4 MJ/day for protein, –1.3 MJ/day for fat, and +2.1 MJ/day for carbohydrate oxidation.


Figure 4
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  		Fig. 4. Macronutrient oxidation (Ox) and intake (In) (MJ/day) for carbohydrate (CHO), fat (FAT), and protein (PRO) at the end of baseline and at the end of OF phases (20% OVF, 40% OVF, 60% OVF) measured by whole body indirect calorimetry. One subject did not complete the study and was excluded from this analysis. *P < 0.05.

 
At the end of baseline, subjects were in fat balance (NS), but by the end of the +20% OF, fat intake significantly exceeded oxidation (P = 0.003) and the gap became progressively larger in subsequent OF phases (+40% OF: P = 0.003; +60% OF: P = 0.001); this was, as expected, associated with weight gain (r = 0.83, P = 0.001). It is notable that there was no detectable net change in carbohydrate balance. Despite an increase in protein oxidation, this was consistently lower than protein intake, and subjects were in positive nitrogen balance.

Cost of Weight Gain

The calculations of the cost of weight gain are shown in Table 4. The first phase (OF +20%CAL) showed a high discrepancy between energy surplus and cost of weight gain (26.98 MJ), and 71% of the energy was unaccounted for by changes in energy stores and calEE. However, the mean change in body composition at this stage was very small (FM = +0.08 kg), with substantial interindividual variability and with measurements in two subjects indicating opposite changes in FM (loss) and TBW (gain).


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  		Table 4. Calculation of cost of weight gain for each OF phase (using calorimetry TEE) and for the whole study (using doubly labeled water TEE) in 5 lean, healthy men

 
The energy cost of fat and protein deposition and increased calEE explains almost all the extra energy consumed during the 60% OF (13.1%). The measurement of calEE in calorimeters does not reproduce free-living conditions, and if this is increased by 25% as suggested by Ravussin et al. (37), the unexplained energy is only 9.3% for the 60% OF.

The use of TEE measured in free-living conditions using DLW allows a better estimation of the changes in EE. With the use of this method, there is near perfect agreement between the total average energy excess throughout the study (238.9 MJ) and the total energy explained by tissue accretion and increased TEE (211.16 MJ) based on the assumption of a linear increment in EE between baseline and the 60% OF measurement. The unexplained energy was only 27.7 MJ (11.6%). The average energy equivalent of weight gain calculated for the whole study, with the assumption of a linear increase in TEE, was 19.25 MJ/kg. The theoretical calculation of the cost of weight gain based on average FM and FFM changes and assuming a protein content of 20.6% for FFM was calculated as follows: (38.9 x 0.37) + (23.6 x 0.20 x 0.62) = 17.3 MJ/kg. The observed mean value of cost of weight gain differed from this by only 9%.


   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In everyday life, overeating occurs spontaneously in many individuals in response to environmental, psychological, and social cues (16). In experimental settings, the replication of free-living conditions to investigate human energy metabolism is problematic, but this study has attempted to reproduce the recurrent periods of overeating that characterize the weight history of the vast majority of overweight subjects (9). To our knowledge, this represents the first study of this kind, as previous OF studies are characterized by a continuous OF rather than stepwise increases in energy intake separated by ad libitum phases. This novel paradigm allows the measurement of both changes in EE and compensatory effects on appetite control.

The net result of this imposed OF regimen was a weight gain of 6 kg by the end of the 60% OF. Fat gain accounted for 55% of the increase and FFM gain for 45%. This ratio is comparable to that in the study of Ravussin et al. (37), where five subjects were overfed (+60%) for 9 days (FM = 57%; FFM = 43%). Diaz et al. (8) overfed nine subjects for 42 days by 50% above energy requirements; subjects gained 7.6 ± 1.6 kg body wt, and FM accounted for 58% of the change in body weight. This study showed that changes in body composition during the OF phases were in close agreement with the theoretical calculations proposed by Forbes et al. (11), who provided a detailed analysis of the relationship between cost of weight gain and OF in experimental conditions. They observed that OF always induced weight gain in experimental conditions, that weight gain is proportional to the total amount of energy excess consumed, and that the average composition of weight gain was 44% lean and 56% fat, which are nearly identical to the changes in body composition observed in this study. Total body water explained most of the variation in the change in body mass initially, but its contribution declined when energy intake and fat accretion increased. The preferential deposition or mobilization of glycogen stores in response to initial changes would cause an initial much larger displacement of glycogen than fat stores, which might explain the initial higher shifts in total body water (15, 44). Previous OF studies have not used multicompartment models to measure body composition changes, and a direct comparison with our results is not possible.

A striking observation was the high between-subject variability in weight change during the ad libitum phases, which reflects different compensatory responses to OF. Some subjects were able to control their energy intake (compensators), but others were not (noncompensators), perhaps indicating an interaction between physiological and cognitive mechanisms, the latter arising from the perception of an increased body weight and/or food portions (8, 23, 31). The inability of subjects to return to their baseline levels of energy intake points to an asymmetric regulation of appetite in humans as has been previously noted (1, 23). Diaz et al. (8) did not show data on energy intake during their 6-wk ad libitum post-OF phase. However, body weight did not return to baseline values, and subjects were able to lose only 55% of the body weight gained. The variability in weight loss was significant, ranging from 42 to 86%, and probably reflected an individual ability to reduce energy intake between subjects and compensate for the preceding OF. The compensation for overeating was also explored in 12 pairs of monozygotic twins after 4 mo from the end of an 84-day OF study (Quebec twin study), and in free-living conditions and without controlling for physical exercise, the average body weight was still above baseline by 1.4 kg (48). The same study remeasured body weight five years later to explore interindividual variability of weight change and uncover individual weight trajectories (2).

Intrapair trajectories of weight gain were collinear, whereas the interpair trajectories were divergent and associated with variable rates and amounts of weight change during both OF and free-living periods (3, 7, 48, 50). It was evident that genotype influences the metabolic responses (EE, body composition) to the imposed energy excess in standardized and nonstandardized conditions and contributes significantly to the between-subject variability.

OF in our study was associated with marked changes in visceral fat in this group of lean subjects. At the end of 60% OF, the relative increase in visceral fat was nearly twice that of subcutaneous fat, which confirms the more active energy mobilization and deposition of visceral adipocytes (12), and this demonstrates that even short bouts of overeating may induce greater metabolic effects that may initiate some of the mechanisms leading to insulin resistance (43). In contrast, CT scans in the Quebec twin OF study showed a higher proportion of fat deposited as subcutaneous abdominal tissue, which increased by 95%, while the visceral layer increased by 70% (3). The greater proportion of visceral fat gain in our shorter study might have two possible explanations that need not be mutually exclusive: either that the visceral depot acts as a short-term "buffer" that can rapidly assimilate fat before a later redistribution; or that the visceral depot has a relatively limited capacity, and once this is reached, fat is then preferentially diverted to subcutaneous regions. The dynamics of such differential storage would be complex, given that fat loading will induce the formation of new adipocytes that will gradually increase depot capacity.

As expected, the imposed positive energy balance was associated with a progressive increase in calEE and BMR by 11.4 and 14.4%, respectively. The increase in BMR explained >70% of the total change in calEE and >55% of the total change in free-living TEE. These figures are close to the increases in calEE seen in previous OF studies. Diaz et al. (8) observed an average rise in calEE of 17%, and ~50% was due to an increase in BMR. Webb et al. (52) overfed eight subjects for 30 days, and they observed an increase in TEE of 7.4%. The twin OF study overfed 24 twins by a total of 353 MJ, which produced an increase in BMR of 9.6%, accounting for 46% of the total change in TEE (48). Conversely, during the ad libitum phases, there was a decrease in BMR and calEE; however, BMR was still above baseline values because weight was higher at the end of the last ad libitum phase, whereas calEE was similar to baseline values because of the drop in A+T.

The ability of the body to accumulate fat when in a positive energy balance and prioritize carbohydrate oxidation over protein and fat is an established mechanism that we have previously described in terms of an "oxidative hierarchy" (22, 34). Jebb et al. (22) showed in a 12-day controlled OF study that fuel selection in response to OF is dominated by carbohydrate intake, and an increase in carbohydrate oxidation produces a counterregulatory suppression of fat oxidation even in the presence of high fat intake. This study showed a progressive, significant, linear increase in carbohydrate oxidation that in each phase differed from carbohydrate intake by <0.5 MJ/day (some of which can be accounted for by measurement imprecision), reiterating the precise control of carbohydrate balance. The increase in fat intake was not tracked by fat oxidation, and fat was thus preferentially stored. The fat storage was primarily derived from the diet with no evidence of net de novo lipogenesis, because respiratory quotient values were <1, and metabolic experiments have shown that de novo lipogenesis is, under most circumstances, a minor component in humans (41). The increase in energy intake produced a state of positive nitrogen balance and a subsequent increase in lean mass as a consequence of the gain in body weight.

The cost of tissue accretion per kilogram of body weight in our calculations gives a figure of 19.2 MJ/kg, which is almost identical to the 17.6 MJ/kg obtained by Ravussin et al. (37). The theoretical cost of weight gain calculated using the gross energy content of FM and protein in FFM (20.6%) was very close to the observed cost, and they differed by only 9%. A similar difference was observed in a previous OF study conducted in our laboratory (8). These calculations are necessarily somewhat crude because, although energy intake was assessed continuously, EE was only measured on seven occasions by calorimetry and twice by DLW, necessitating an assumption of linear changes between measurements. Nonetheless, these calculations of the energy cost of weight gain provide further evidence against the existence of energy dissipative mechanisms, because most of the weight change was explained by the cost of adipose and lean tissue deposition and by the increased EE associated with weight gain. The errors were close to 10%, which is comfortably within the limits of precision of the various techniques employed.

Conclusions

This detailed experimental study has reemphasized the very limited ability of humans to compensate for episodes of OF through autoregulatory elevations in EE (23, 42). This result is teleologically predictable, given that the thermogenic dissipation of even a 20% energy excess would put humans (with their small surface area-to-volume ratio) under considerable thermal stress. Instead, the energy and substrate load is disposed of by substantial downregulation of fat oxidation and resultant fat storage. The energy cost of weight gain was very close to the excess energy provided and thus does not support the existence of significant dissipative mechanisms to offset overeating in lean men. These results support the present conclusions from genetic studies in which all known causes of human obesity are related to defects in the regulation of appetite and the intake side of the energy balance equation (32). Finally, we have shown that the available energy is preferentially directed toward abdominal visceral fat, which has important implications for the development of the metabolic complications of weight gain.


   ACKNOWLEDGMENTS
 
We are grateful to Anthony Wright for the stable isotope analysis, Elaine Collard for the preparation of the diets, and Sri Aitken for assistance with the magnetic resonance body fat analysis. We also extend thanks to the staff members, including night nurses, who supervised subjects in the calorimeter and metabolic suite.


   FOOTNOTES
 

Address for reprint requests and other correspondence: M. Siervo, MRC Human Nutrition Research, Elsie Widdowson Laboratory, Fulbourn Rd., Cambridge, UK CB1 9NL (e-mail: mario.siervo{at}mrc-hnr.cam.ac.uk)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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