Can Increased Dietary Protein Prevent the Suppression of Metabolism and Maintain Anabolic Sensitivity of Bone and Skeletal Muscle During Caloric Restriction?
Phase: NA
Conditions: Weight Loss, Starvation
Sponsor: University of Nebraska Lincoln

 
 
 
 
 
 
 
 
 
 
 
 
 
 
Effect of Caloric Restriction and Protein Intake on Metabolism and Anabolic Sensitivity 
 
NCT02945410 
 
10 May 2019 
 

1. What is the significance/purpose of the project? 
Successful weight loss requires the induction of an energy deficit [1], which can be attained through 
caloric restriction, exercise, or a combination of both [2]. However, weight loss is not only associated 
with the favorable loss of adipose tissue, but also with the loss of lean mass [2]. On average, 
approximately 25% of weight loss consist of lean mass [3], but lean mass loss is even more 
pronounced in normal-weight individuals [4]. The loss of lean mass during energy deficiency is a 
result of the degradation of body protein to provide additional energy and substrates to meet the 
high glucose demand of the brain. Protein degradation takes place primarily in expendable cellular 
tissues such as the skeletal muscle, which is emphasized by recent findings of reduced muscle protein 
synthesis [5] and increased muscle protein breakdown [6] during energy deficiency. As a result, 
energy deficiency and the concomitant loss of lean mass could be associated with undesired health 
effects such as impaired blood glucose regulation [7] and the loss of functional capacity throughout 
the life span [8]. 
Energy deficiency is associated with metabolic and anti-anabolic adaptations. Parallel with the loss of 
lean mass, resting metabolic rate (RMR) is also suppressed during energy deficiency. Because this 
reduction in RMR conserves energy, it has been linked to attenuated weight loss and an increased 
propensity for weight (re)gain [9]. The reduction in RMR is a consequence of the suppression of 
expendable functions, such as growth and reproduction, in order to conserve energy for life-
sustaining functions [10]. As a result, the secretion and bioavailability of anabolic hormones is 
strongly impaired during energy deficiency [11]. This suppression of anabolic hormones is not only 
linked to the loss of skeletal muscle, but has also detrimental effects on bone [12-14]. This is 
particularly concerning because the accrual and maintenance of peak bone mass during adolescence 
and adulthood is a strong predictor of osteoporosis risk later in life [15]. 
The profound metabolic consequences of caloric restriction may negate some of the benefits of 
weight loss. Considering that calorie restriction is the preferred and most-effective strategy for the 
treatment and prevention of overweight and obesity and their associated comorbidities [2], 
strategies are needed to preserve the beneficial effects of calorie restriction while attenuating its 
potentially detrimental effects on metabolism, skeletal muscle, and bone. Lean mass can be 
preserved through increased dietary protein intake. A high dietary protein intake during energy 
deficiency attenuates the loss of lean mass and improves protein balance [16, 17]. 
Further, it is known that a high protein intake, particularly when coupled with exercise, is associated 
with improved bone strength [18], and effect that is likely modulated by the stimulating effects of 
dietary protein on the secretion of IGF-1 [15]. These findings lend to the idea that increased protein 
intake during calorie restriction may preserve the anabolic sensitivity of both skeletal muscle and 
bone. 
However, only little is known about the underlying mechanisms, particularly pertaining to the effects 
of an increased protein during caloric restriction on circulating concentrations of anabolic hormones  
and the downstream effects on bone turnover. Therefore, the purpose of our study is to determine 
whether a strategy of increased dietary protein during energy deficiency can attenuate the 
suppression of RMR and reductions in hormones with anabolic and anti-catabolic effects on bone. 
For this purpose, we will conduct a prospective, randomized controlled study involving three 
experimental conditions: a) calorie restriction with normal protein intake (0.8 g/kg; CR-NP); b) calorie 
restriction with high protein intake (1.7 g/kg; CR-HP); and c) a control period of balanced caloric 
intake and matched protein intake (1.7 g/kg; CON). 
Aim 1: To determine whether increased protein intake can preserve metabolic function and maintain 
anabolic sensitivity during calorie restriction 

Hypothesis 1A: Weight loss will be similar following CR-HP and CR-LP, but the loss of lean mass and 
the suppression of resting metabolic rate will be reduced in CR-HP when compared to CR-NP. 
Hypothesis 1B: The reduction in circulating concentrations of the anabolic hormones IGF-1, insulin, 
and testosterone will be attenuated in CR-HP when compared to CR-NP. 
Aim 2: To determine whether increased protein intake can attenuate the increase in bone turnover, 
and particularly the increase in bone resorption, during calorie restriction. 
Hypothesis 2A: The increase in markers of bone resorption (NTx, CTx) will be attenuated in CR-HP 
when compared to CR-NP; whereas, the reduction in markers in bone formation (P1NP, AP) will be 
similar in CR-HP and CR-NP when compared to CON. 
 
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Pikosky, M.A., et al., Increased protein maintains nitrogen balance during exercise-induced 
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Pasiakos, S.M., et al., Whole-body protein turnover response to short-term high-protein diets 
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2. Describe the data collection procedures and what participants will be asked to complete. 
1. Initial Screening Questionnaire 
Participants will be asked for information pertaining to inclusion and exclusion criteria in order to 
establish qualification for study participation. Information provided in the Initial Screening 
Questionnaire will only be used for screening purposes and not for research purposes. 
2. Informed Consent and Screening Visit 
Informed Consent: The researcher (or designee) obtaining informed consent will thoroughly review 
the consent form with each participant and confirm that the participant understands all study 
procedures and potential risks and benefits of study participation.  
Initial Anthropometric Measurements: After obtaining informed consent, participants will be 
weighed to the nearest 0.1 kg on a digital scale in the laboratory with a standard outfit of t-shirt and 
gym shorts. Participants also will have their body fat percentage determined using calipermetry. A 
skinfold caliper will be used to take measurements of skinfold thickness at 7 sites (abdomen, triceps, 
chest, midaxillary, subscapular, suprailliac, and thigh). The skinfold measurements will be used to 
calculate body density and body fat percentage (ACSM's Guidelines for Exercise Testing and 
Prescription, Ninth Edition).  
Questionnaires: Participant will then fill out the following questionnaires: 

o 
The Eating Disorder Inventory a self-report questionnaire used to assess the presence of 
eating disorders (D. M. Garner & Olmstead, 1991; David M. Garner, Olmstead, & Polivy, 1983). 
o 
The Health Exercise and Nutrition Questionnaire, which includes questions regarding 
demographics, medical history, current and past exercise, bone health, stress fracture history, 
current medication and supplement use, and dietary habits. 
o 
The Brief Calcium Assessment Tool, a brief questionnaire used to assess habitual dietary 
calcium intake (Yang, Martin, & Boushey, 2010). 
o 
The Bone-Specific Physical Activity Questionnaire, a questionnaire used to predict. The BPAQ 
predicted indices of bone strength at clinically relevant sites in both men and women (Weeks & Beck, 
2008) 
o 
Absolute and Relative Contraindications to Exercise Testing, a questionnaire pertaining to 
absolute and relative contraindications to exercise testing to determine if the participant is able to 
exercise adequately (ACSM's Guidelines for Exercise Testing and Prescription, Ninth Edition). 
After admittance into the study, participants will be provided with an over-the-counter calcium and 
vitamin D supplement. Supplementation of calcium and vitamin D will be initiated at least 2 weeks 
prior to the start of the first intervention to maintain the participants intake at levels required for 
optimal bone health. 
3. Preliminary testing 
Prior to study start, body composition will be assessed with a bioimpedance analyser (Bodystat 
Quadscan 4000). Peak oxygen uptake (VO2peak) will be assessed using an incremental exercise test 
on a bicycle ergometer (Monark LC6). 
4. Randomization 
Each participant will be randomly assigned to one of the six possible permutations of the order of the 
study conditions (CON/CR-LP/CR-HP). Randomization of study condition order will occur in blocks of 
6. 
5. Nutritional intervention: 
After randomization of study condition order, participants will proceed through each or the following 
conditions. Each condition will last 5 days with a post-test on day 6: 
CR-LP: Participants will consume 30 kcal/kg FFM/day and 0.8 g protein/kg BW/day. 
CR-HP: Participants will consume 30 kcal/kg FFM/day and 1.7 g protein/kg BW/day. 
CON: Participants will consume 55 kcal/kg FFM/day and 1.7 g protein/kg BW/day. 
Dietary energy intake prescriptions will be controlled individually utilizing a combination of two 
clinical products (Ensure Original and Ensure High Protein, both Therapeutic Nutrition, Abbot) and 
maltodextrin (Tate & Lyle) such that the target energy intake of 30 kcal/kg FFM/day (CR-LP and CR-
HP) or 55 kcal/kg FFM/day (CON) is reached. Participants will be provided clear plastic bottles each 
day with the prescribed amount of the clinical products and will be asked to consume all of it during 
the day. 
6. Prescribed Exercise 

During each conditions, participants will conduct daily supervised exercise on a bicycle ergometer 
(Monark LC6) at an exercise intensity of 60% VO2peak. Exercise duration will be adjusted individually 
such that exercise energy expenditure will amount to 15 kcal/kg FFM/day. Exercise will take 
approximately 60-90 minutes. Additional exercise and intense physical activity will be prohibited. 
Compliance will be monitored using an activity monitor (Actigraph). Activity monitors will be 
returned at the end of each study period. 
7. Washout 
Once a participant completes a study condition, they will be allowed a washout period before 
beginning the next study condition. Washout periods between conditions will be 14 days to allow 
protein balance to return to baseline (Hoffer & Forse, 1990). During this time, participants will 
resume their regular diet and physical activity. Calcium and vitamin D supplementation will continue 
to maintain their intake at levels required for optimal bone health. 
8. Assessments 
All assessments will be identical during each study condition, which lasts for 5 days and includes post 
testing on day 6. On day 1 and 6, participants will arrive at the lab in the morning following an 
overnight fast and undergo the following assessments: 
o 
Bioimpedance: Body composition is assessed with a bioimpedance analyser (Bodystat 
Quadscan 4000). While lying in the supine position (with no parts of the body touching one another), 
four electrodes are attached to the wrist, hand, ankle and foot on the right side of the body. The 
cables from the bioimpedance analyzer are then attached to the electrodes and the device is turned 
on, running an electrical current through the body to estimate body composition.  
o 
Resting Metabolic Rate: Resting Metabolic Rate (RMR) will be measured following an 
overnight fast (no food or drink, except water, 12 hours before the scheduled test) between 6-10:00 
a.m. and before exercise. Participants will be asked to lie supine at rest for 30 minutes in order to 
achieve a steady state prior to measurement. Oxygen consumption and carbon dioxide production 
will then be measured with a ventilated hood system (COSMED Quark CPET Metabolic Testing 
System) for 30-45 minutes. For the determination of RMR, only data during steady state will be used, 
which is defined as variations in oxygen uptake, carbon dioxide production of less than 10%, and in 
respiratory quotient by less than 5% for at least 10 minutes. RMR will be calculated from oxygen 
uptake and carbon dioxide production using the Weir equation (Weir, 1949).  
o 
Fasting Blood Collection:  A fasting blood sample will be collected from a forearm vein by 
trained personnel.  
o 
VO2peak: Participants will be asked to complete a graded cycle ergometer test to determine 
aerobic fitness (VO2 peak). Participants will begin cycling on the bicycle ergometer at 60 W for 3 
minutes. The work rate will be increased by 35 W every 3 minutes until exhaustion (Achten & 
Jeukendrup, 2003). Exhaustion is operationally defined when at least two of the following are met: 1) 
90% age predicted heart rate max [220-age], 2) respiratory exchange ratio 1.1, 3) a rate of perceived 
exertion 19, 4) a plateau in VO2 despite an increase in workload, 5) and/or unable to maintain a 
pedal rate of 60 rpm. During the test, expired gases will be measured through indirect calorimetry 
(COSMED Quark CPET Metabolic Testing System), and heart rate will be monitored through 
telemetry (Garmin).  
o 
Questionnaires: The Three-Factor Eating Questionnaire, a questionnaire that measures three 
dimensions of human eating behavior (cognitive dietary restraint, disinihibition, hunger) (Stunkard & 
Messick, 1985), The Profile of Mood States (POMS), a questionnaire used to assess overall mood. This 

profile describes six sub-components of the overall mood construct: anger, confusion, depression, 
fatigue, tension and vigor (McNair et al., 1971); an abbreviated version of the Eating Disorder 
Inventory, a self-report questionnaire used to assess the presence of eating disorders (D. M. Garner 
& Olmstead, 1991; David M. Garner, Olmstead, & Polivy, 1983); Satiety/Hunger Questionnaire: 
Participants will complete a visual analog scale (VAS) which quantifies 1) appetite, 2) fullness, 3) 
perception of quantity of food which can be eaten, 4) nausea, 5) thirst, and 6) stress 
 
3. Describe how long, in terms of time, the procedures will take the participant to complete. The 
description should include the duration of a session, the number of sessions, over what period of 
time and the total time required to complete the procedures. 
Following screening, the study will take at least 9 weeks to complete: 
- 2 week pre-study period for preliminary testing and calcium/vitamin D equilibration 
- 6 days per study condition (3x) 
- Minimum 2 weeks between study conditions (2x) 
 
4. Statistical Analysis Plan 
Paired one-sided t tests will be used to assess whether changes from baseline are significantly 
different from 0. Where significant differences from 0 exist and differences between groups would 
be expected, additional t tests will be used to compare change scores between CR-LP and CR-HP or 
CR-HP and CON. Differences between CR-LP and CON will not be evaluated due to the existence of 
two manipulated variables (protein and kcal) causing the difference in those groups. If exploratory 
analyses are conducted, multiple comparisons will be corrected for using Holm’s sequential version 
of the Bonferroni correction (Holm S 1979).