Subjects

We studied 24 sedentary young men (mean [±SD] age, 21±2 years) living in the province of Quebec. Each man gave written consent for participation in this study, which was approved by the Laval University Medical Ethics Committee and the Office for Protection from Research Risks of the National Institutes of Health, Bethesda, Md. These men constituted 12 pairs of identical twins. The youngest pair was 19 years of age, and the oldest pair was 27. The twins had been reared together and had been living together before this study.

The monozygosity of the twins was established on the basis of a questionnaire, their physical appearance, and the similarity of 12 polymorphic red-cell antigens and enzymes, the A, B, and C loci of the HLA-antigen system, and 10 polymorphic adipose-tissue proteins visualized by two-dimensional gel electrophoresis. None of the men had a history of recent illness, obesity, hypertension, diabetes, hyperlipidemia, or endocrinopathy, and each had a normal physical examination and normal fasting serum glucose, triglyceride, and cholesterol concentrations. Men whose parents were obese or had diabetes or lipid disorders were not accepted into the study.

Four pairs of twins were studied at a time over a period of 18 months: one subgroup starting in early August, a second in February, and the third the following August. The results were similar in all three subgroups. The men were housed in a closed section of a dormitory on the campus of Laval University and were under 24-hour supervision by members of the project staff living with them. Each man stayed in the unit for 120 consecutive days — 14 days for base-line testing, 3 days for testing before the period of overfeeding, 100 days for the period of overfeeding, and 3 days for testing after the period of overfeeding. During the base-line testing, body weight was measured daily, skin-fold thickness was measured at 10 sites five times, and body density was measured three times by underwater weighing.

The men were not allowed to drink alcoholic beverages during the study. Five of the pairs of twins were light smokers, but smoking was not permitted during the study. We believe that these men reduced their frequency of smoking to a few cigarettes a day, but did not stop entirely.

Base-Line Test Period

During the base-line period, the men ate freely in a special dining room. The food was prepared by university personnel but was put aside for the men by the dietitians involved in the study. All foods selected by the men were recorded and weighed. All foods not eaten were weighed separately, and their weight was subtracted from that of the food selected so that the net intake could be calculated. The nutrient composition and energy content of the food were derived from a computerized version of the Canadian food-composition tables.15 , 16 Each man's habitual daily energy intake under conditions of stable body weight and body composition was calculated from the 14-day record of food intake and was generally based on the entries for the last 12 days, the variations in body weight, and the changes in fat mass or fat-free mass, if any, determined from the body-density measurements. The energy content of body fat was assumed to be 37.8 MJ (9300 kcal) per kilogram, whereas that of lean tissue was assumed to be 4.2 MJ (1020 kcal) per kilogram.17 The body energy content was the sum of these two components. The average nutrient composition of the food eaten during the baseline period was 52±6 percent carbohydrate, 34±6 percent lipid, and 14±6 percent protein.

Testing before and after the Overfeeding Period

On the first day of the three-day test period before the overfeeding period, we measured the resting metabolic rate and the thermic effect of food. On the third day, underwater weighing, biopsies of adipose tissue, and exercise-tolerance testing were performed. About five days before the overfeeding period, a CT scan of the abdomen was performed. The same studies were repeated during the three-day test period after the overfeeding period.

Overfeeding Period

After the base-line period, the men were fed a diet containing 4.2 MJ ( 1000 kcal) per day more than their established base-line energy intake from food, 6 days a week for 100 days. On the seventh day of each week they consumed the base-line number of calories. They were thus overfed for 84 of the 100 days, the total excess energy intake being 353 MJ or 84,000 kcal. The food consumed each day had the following prescribed nutrient composition: 50 percent carbohydrate, 35 percent fat, and 15 percent protein. The men ate three meals per day plus an evening snack tailored to complete the daily prescription for energy intake.

Physical activity was limited during the entire study; the daily schedule included such activities as reading, playing video games, playing cards, and watching television, as well as an outdoor walk for 30 minutes per day. The men were occasionally taken as a group to a play or a movie. They were carefully supervised at all times during all activities, and as far as we could ascertain their compliance with the regimen of dietary intake and the limitations on their activities was perfect. During this period, body weight was measured daily, skin-fold thickness every 5 days, and waist and hip circumference every 25 days.

Body Composition and Regional Fat Distribution

Blood pressure, pulse rate, and body weight were measured (the latter with the men wearing light exercise shorts) each morning before breakfast. The body-mass index was calculated as the body weight (in kilograms) divided by the height (in meters squared). Body density was determined by underwater weighing,18 and fat mass and fat-free mass were calculated with a standard equation.19 The volume of air remaining in the lungs was determined while each man was in the water tank, and the pulmonary residual volume was measured by the helium-dilution technique.20 The data on body weight represent the mean measurements of three days, in each case the day on which the underwater weighing was done and the days before and after. The skin-fold thickness was measured at 10 sites, as were the waist and hip circumferences, according to the procedures recommended at the Airlie Conference.21 The ratio of the sum of the skin-fold—thickness values for the trunk (subscapular, suprailiac, abdominal, midaxillary, and chest) to the sum of the values for the limbs (biceps, triceps, front midthigh, suprapatellar, and medial calf) was used as one estimate of the regional subcutaneous fat distribution.22 The ratio of the waist to the hip circumference was used as another estimate of regional fat distribution.21 For all the anthropometric measurements, two measurements were always made, and the mean was used if the difference between the two measurements was less than 5 percent. If the difference was 5 percent or more, a third measurement was made, and the mean of the two most similar values was used.21 We also determined the mean adipose-cell mass in the abdominal (umbilicus level) and femoral (midthigh) areas,23 using data on isolated fat cells obtained by collagenase digestion and the density of triolein to convert the adipose-cell volume to mass.

CT scanning was performed before and after the overfeeding period with a Siemens Somatom DRH scanner (Erlangen, Federal Republic of Germany). The men were examined in the supine position with their arms stretched above their heads.24 The scans were obtained at the pubic symphysis and at the levels of the disks between the fourth and fifth lumbar vertebrae and between the eighth and ninth thoracic vertebrae. The attenuation interval used in the quantification of the areas of adipose tissue was −30 to −190 Hounsfield units. The total areas of abdominal and abdominal visceral fat were calculated by delineating their surfaces with a computerized pen. The abdominal visceral fat was defined by drawing a line on the inside of the muscle wall surrounding the abdominal cavity. The area of subcutaneous abdominal fat was computed as the total abdominal fat minus the visceral fat. The sum of the three trunk scans was used as an estimate of the response of the trunk fat to overfeeding.

The reproducibility of all the measurements of fat areas by anthropometry, studies of body composition, and CT scanning has been determined by several investigators and is known to be quite high when performed by the same observer, as in this study. The intraclass reliability coefficients for the variables reported were generally above 0.9.21 , 24 , 25

Statistical Analysis

The study design allowed testing for the presence of similarities within pairs of twins in the response to long-term overfeeding. The effects of overfeeding and the interactions between genotype and overfeeding were assessed with a two-way analysis of variance for repeated measures on one factor (time). The twins were considered nested within the pair, whereas the treatment effect was defined as a fixed variable. The intraclass correlation coefficient for the changes brought about by overfeeding was computed from the between-pairs and within-pair means of squares.26 This coefficient provides a quantitative estimate of the similarity within pairs in the response to overfeeding. An intra-class correlation coefficient close to 1.0 would indicate a perfect within-pair resemblance in response to overfeeding, whereas a coefficient close to zero would imply that there was no within-pair resemblance in response to the treatment. Similarly, a high F ratio indicates a high ratio of the variance between pairs to the variance within pairs in response to overfeeding, whereas a low F ratio (close to 1) indicates that the variances in response between and within pairs of twins are comparable. In some analyses, the results were adjusted for gains in total fat mass. These adjustments were performed by regression of the variable to be adjusted on the gain in fat mass after overfeeding, with the 24 subjects considered as unrelated persons. P values ≤0.05 were considered statistically significant.