This cross-sectional study will compare fasting and overfeeding effects on 24-hour EE and its components in pre- and postmenopausal women. The investigators will study participants under three conditions in the room calorimeter: 1) eucaloric feeding (energy intake = EE); 2) acute, 24-hour fasting; and 3) acute, 24-hour overfeeding of a low-protein diet (i.e., 200% of energy requirements). Participants will complete the eucaloric study and then complete the overfeeding and fasting studies in random order. For 3 days before each condition, the investigators will provide participants with an outpatient diet designed to meet free-living energy requirements (energy intake = 1.5 x REE) and stabilize macronutrient intake (15% protein, 30% fat, 55% carbohydrate). There will be a 1-4-week washout between conditions. Studies in premenopausal women will be performed during the mid-luteal phase (i.e., days 16-24) when both E2 and progesterone are elevated and, thus, when the potential hormonal effects are maximal.
Pre-study assessments - Once enrolled, participants will complete a pre-study assessment visit in the Clinical and Translational Research Center (CTRC) Outpatient Clinic, and the following assessments will be performed:
1. Resting energy expenditure (REE) - will be measured by indirect calorimetry using a metabolic cart coupled with a canopy system (Parvo Medics TrueOne) and used to estimate energy requirements during the eucaloric condition. REE in premenopausal women will be measured in the luteal phase. Participants will arrive after an overnight fast (\>10 hours) between 7 and 9 AM. They will rest while remaining awake for 30 minutes in a temperature-controlled room (\~70⁰ F). Oxygen uptake (VO2) and carbon dioxide production (VCO2) will be measured for 20 minutes and averaged over the final 15 minutes.
2. Body composition - Body composition will be measured using dual X-ray absorptiometry (DXA, Hologic Discovery, Waltham, MA).
3. Visceral adipose tissue (VAT) volume - VAT volume and subcutaneous fat area (SFA) will be measured using computed tomography (CT). An instrument dedicated to research activity will be used for all scans (Philips Gemini 64TF). Axial CT images will be obtained through the center of the L2-L3 and L4-L5 inter-vertebral disc spaces.
Study Procedures: Once the pre-screening assessments are completed, participants will be studied under three conditions in the room calorimeter.
Room calorimeter - 24-hour EE and substrate oxidation will be measured using whole-room indirect calorimetry.For three days before each calorimeter study, participants will be instructed to refrain from exercise and provided with all food to stabilize energy and macronutrient intake. The energy content of the diet will be REE x 1.5 (30% fat, 55% carbohydrate, 15% protein). Participants will arrive at 0700h after an overnight fast on the study day. An IV catheter will be inserted in an antecubital vein. Fasting blood samples will be obtained before breakfast and at the end of the 24-hour study and analyzed for leptin, FGF21, and thyroid hormones (TSH, total T3, free T4). The investigators will also measure sex steroids (E2, progesterone) in premenopausal women to confirm that participants are studied in the luteal phase.
Participants will enter the calorimeter at 0800, exit at 0700 the following day, and be instructed to engage in primary sedentary behaviors (e.g., reading, watching TV, using the computer). During the eucaloric and overfeeding studies, meals will be provided at 0900, 1300, and 1800, and a light snack will be provided at 2000. Participants will be instructed to turn out the lights and prepare for bed no later than 2300. Participants will record their time in bed during the first visit and be instructed to prepare for bed during the subsequent two visits. The CTRC nursing staff will confirm bedtimes. Participants will be awoken at 0600, and a 30-minute measurement of REE will be obtained.
Participants will be monitored during this time to ensure that they remain awake and motionless.
The energy content of the diet during the calorimeter study will vary according to the study condition:
During the eucaloric study visit, the energy content of the diet will be 1.3 x REE (\~30% fat, 55% carbohydrate, and 15% protein at each meal). Breakfast will account for \~25% and lunch and dinner \~30-35% of total energy needs. During the overfeeding study, macronutrient intake will be 3% protein, 51% carbohydrate, and 46% fat. During the fasting study visit, participants will be permitted to consume non-caloric beverages (i.e., water, non-caloric and non-caffeinated soft drinks, caffeine-free tea, or decaffeinated coffee) but refrain from consuming any caloric food or beverages. Upon exiting the calorimeter, participants will be provided a meal from the University of Colorado Hospital Patient Kitchen.
Blood samples will also be obtained 30 minutes after each meal and snack during the eucaloric and overfeeding conditions; blood samples will be obtained at the same time during the fasting study. Blood samples during the calorimeter studies are obtained by asking participants to extend an arm through an airlock in the side of the calorimeter. The investigators will store these samples for potential future analysis (e.g., proteomics, metabolomics, and hunger and satiety hormones; see Future Directions). During each condition, a 24-hour urine sample will be collected and analyzed for urine nitrogen and urea nitrogen, which are needed to perform substrate oxidation calculations, as well as for 24-hour urinary catecholamines.
