Selective Reduction of Dietary Carbohydrate Versus Fat: Effects on Metabolism, Endocrine Physiology, Brain Activity and Reward Circuitry
Phase: PHASE2
Conditions: Obesity
Sponsor: National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)

1 
Clinical Research Protocol 
National Institute of Diabetes and Digestive and Kidney Diseases 
DATE: January 24, 2017  
 
 
 
 
 
 
PROTOCOL NUMBER: 09-DK-0081 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
IND NO: 70,046 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
IND NAME: [F-18] Fallypride 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
IND SPONSOR: Clinical Center 
 
TITLE: Selective Reduction of Dietary Carbohydrate versus Fat: Effects on Metabolism, 
Endocrine Physiology, Brain Activity and Reward Circuitry 
 
SHORT TITLE: Reduced Carbohydrate vs. Fat  
 
IDENTIFYING WORDS: Obesity, Weight Loss, Carbohydrate, Fat, Macronutrient Balance, 
Brain Imaging 
 
TYPE OF PROTOCOL: Natural History – Disease Progression/Physiology 
 
ESTIMATED DURATION OF STUDY: 9 Years 
 
START DATE: January, 2009  
 
 
 
 
END DATE: January, 2018 
 
NUMBER AND TYPE OF PATIENTS: 
 
 
Number 
Sex 
Age Range 
Volunteers 
20 Control Subjects 
and 20 Obese 
Subjects 
Male and female 
18 to 45 
 
PRINCIPAL INVESTIGATOR: 
Kevin Hall, Ph.D. 
Laboratory of Biological Modeling (LBM) 
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) 
Building 12A, Room 4007 
12 South Drive, (MSC 5621) 
Phone: 301 402-8248 
Fax: 301 402-0535 
E-mail: kevinh@niddk.nih.gov 
 
MEDICAL ADVISORY INVESTIGATOR: 
 
Ranganath Muniyappa, M.D. 
NIDDK, DEOB 
Building 10, CRC Room 5-3671 

2 
10 Center Drive 
Bethesda, MD 20892-1613 
Phone: 301-451-7702 
Fax: 301-480-4517 
E-mail: muniyapr@niddk.nih.gov 
 
ASSOCIATE INVESTIGATORS: 
 
Kong Y. Chen, Ph.D.  
 
 
 
 
 
 
 
 
 
Peter Herscovitch, M.D. 
Clinical Endocrinology Branch (CEB)  
 
 
 
 
Chief, PET Department 
NIDDK  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
NIH Clinical Research Center    
Building 10, CRC, Room 5-3671 
 
 
 
 
 
 
Building 10, Room 1C401 
10 Center Drive, (MSC 1613) 
 
 
 
 
 
 
 
10 Center Drive, (MSC 1180)  
Phone: 301 451-1636  
 
 
 
 
 
 
 
 
 
Phone: 301 451-4248  
E-mail: chenkong@niddk.nih.gov  
 
 
 
 
 
E-mail: herscovitch@nih.gov  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Amber Courville, Ph.D., R.D. 
 
 
 
 
 
 
 
Lilian V. Howard, C.R.N.P. 
Nutrition Department   
 
 
 
 
 
 
 
 
 
Bldg 10 Rm 3-1604 
Building 10, CRC, Room B2-2426  
 
 
 
 
 
10 Center Drive 
10 Center Drive, (MSC 1078) 
 
 
 
 
 
 
 
Phone: 301-594-0298 
Phone: 301 594-8051  
 
 
 
 
 
 
 
 
 
E-mail: howardlv@mail.nih.gov 
E-mail: courvillea@mail.nih.gov 
 
 
 
 
 
 
 
 
Juen Guo, Ph.D.  
 
 
 
 
 
 
 
 
 
 
 
 
NIDDK  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Building 12A, Room 4059  
 
 
 
 
 
 
 
 
 
12 South Drive 
 
 
 
 
 
 
 
 
 
 
 
 
 
Phone: 301 496-5436  
 
 
 
 
 
 
 
 
 
 
E-mail: juen.guo@mail.nih.gov  
 
 
 
 
 
 
 
 
Nora D. Volkow, M.D 
National Institute on Drug Abuse (NIDA) 
NSC - Neuro Science Ctr, 5274  
6001 Exec Blvd   
Rockville, MD 
Phone: 301 443-6480 
E-mail: nvolkow@nida.nih.gov 
 
NON-INVESTIGATOR RESEARCH STAFF: 
 
John E. Ingeholm, Ph.D.  
Laboratory of Brain and Cognition, NIMH 
Building 10, Room 4C-104 
10 Center Drive, (MSC 1366) 
Phone: 301 435-4940 
E-mail: ingeholm@nih.gov 

3 
 
 
PROJECT USES IONIZING RADIATION:  
No longer uses ionizing radiation since this study is data analysis only. 
PROJECT USES “DURABLE POWER OF ATTORNEY”: No 
 
OFF-SITE PROJECT: No 
 
MULTI-INSTITUTIONAL PROJECT: No 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

4 
 
Précis 
 
 
Popular weight loss strategies often prescribe a targeted reduction of dietary carbohydrate or 
fat.  But surprisingly, no controlled human feeding study has ever investigated the effects of a 
selective reduction of dietary carbohydrate versus fat while keeping the other dietary 
macronutrients at their baseline weight-maintenance values.  The present study was designed to 
address this knowledge gap and improve our understanding of how selective reduction of dietary 
fat versus carbohydrate may differentially impact the many feedback control processes that act to 
resist weight loss.  
 
After several days of eating a weight-maintenance diet, 20 obese adult volunteers (BMI above 
30 kg/m2) will be admitted to the metabolic clinical research unit (MCRU) and, after 5 additional 
days of the baseline diet, their diets will be modified to result in either 85% reduction of the 
baseline dietary fat or a 60% reduction of the baseline dietary carbohydrate for the next 6 days.  
These diet modifications produce an equivalent caloric reduction.  The primary outcome 
measurements will be changes of metabolism, brain reward circuitry and regional brain activity 
in response to food stimuli measured during the baseline and reduced calorie diet phases.  
Immediately following each controlled diet, we will measure 3 days of ad-libitum food intake 
using a computerized vending machine system.  The subjects will return to the MCRU after a 2-
10 week washout period to receive the opposite reduced calorie diet.  Twenty control subjects 
with normal body weight (BMI between 18.5 - 25 kg/m2) will have measurements of brain 
reward circuitry and regional brain activity in response to food stimuli while on a balanced, 
weight-maintenance diet. 
 
 
Immediately following the second in-patient visit, all of the obese subjects will be assigned 
to a 12 week out-patient weight loss program with the goal of achieving at least 5% weight loss.  
We will investigate the relationship between short-term fat imbalances measured during the in-
patient phases, and the body weight and fat changes during the weight loss program.  We will 
evaluate the effects of weight loss on metabolism, brain reward circuitry, and regional brain 
activity in response to food stimuli.  Finally, if the subjects are available for long-term follow-up, 
then we will investigate their metabolic phenotype, brain reward circuitry, and regional brain 
activity in response to food stimuli yearly over the subsequent 5 years following the weight loss 
intervention.  This study will result in an improved understanding of the physiological 
mechanisms that sense and respond to negative energy balance acutely, after several weeks, and 
after several years, and may eventually lead to increased long-term success of obesity treatment. 
 
 

5 
Introduction and Background 
 
1. Dietary macronutrients and the treatment of obesity  
 
The prevalence of obesity has reached epidemic proportions and the incidence and economic 
burden of obesity-related complications are rapidly increasing (1, 2).  Any treatment of obesity 
must result in an imbalance between the energy expended by the body over the amount of energy 
derived from the intake of food.  Popular weight loss strategies often achieve this negative 
energy balance by prescribing a targeted reduction of a dietary macronutrient such as 
carbohydrate or fat.  Recent clinical trials in obese subjects have compared these strategies and 
determined that low-carbohydrate diets result in greater short-term weight loss compared with 
low-fat diets (3-5).  The mechanisms underlying this result have not been determined.  One 
hypothesis is that the macronutrient composition of a diet may significantly influence energy 
expenditure and fuel partitioning, and that low-carbohydrate diets may offer a “metabolic 
advantage” (6) in apparent violation of the long-lived principle that “a calorie is a calorie” (7).  
Nevertheless, proponents of the “metabolic advantage” concept base their arguments on sound 
biochemical mechanisms that may underlie the hypothesized alterations of metabolic efficiency 
and fuel partitioning with changes of diet composition (6).  An alternative hypothesis is that the 
macronutrient content of the diet affects hunger and satiety possibly through altered 
hypothalamic signaling or brain reward pathways.   
 
Given the importance of these topics and the intense public interest in how dietary 
macronutrients affect metabolism and weight loss, it is surprising that no human feeding study 
has ever investigated a controlled selective reduction of dietary carbohydrate versus fat while 
keeping the other dietary components at their balanced values.  The present study was designed 
to address this knowledge gap and determine how a selective reduction of dietary fat versus 
carbohydrate affects whole-body metabolism, endocrine signals, hunger, satiety, regional brain 
activity and reward circuitry, and gene expression in skeletal muscle and adipose tissue. The 
general hypothesis of the present study is that a selective reduction of dietary carbohydrate, but 
not dietary fat, will result in significant changes of these variables with respect to the baseline 
diet.   
 
2. Dietary macronutrients and their effect on fuel selection 
 
Maintenance of a stable body weight requires a state of macronutrient balance where dietary 
carbohydrate, fat, and protein intake rates are balanced by their utilization rates. This 
fundamental concept of macronutrient balance and the relationship between metabolism and the 
chemical composition of the body (e.g., body fat and lean masses) is an extension of the energy 
balance concept (8, 9).  While nothing is presently known about the effects of selective reduction 
of dietary carbohydrate versus fat on human metabolism, several overfeeding studies have 
investigated the effects of selective overfeeding of dietary carbohydrate versus fat (10-12).  
These studies found that carbohydrate overfeeding induced profound adaptations of metabolism 
by increasing carbohydrate utilization and decreasing fat utilization as indicated by an increase 
of the 24-hour respiratory quotient.  However, fat overfeeding produced no significant changes 
of macronutrient utilization.  We anticipate a similar dichotomy in the present study where we 
examine the effects of selective underfeeding of dietary carbohydrate versus fat for a 6-day 

6 
period in a controlled in-patient setting.  In particular, we expect that a selective restriction of 
dietary carbohydrate will result in significant changes of 24-hour whole-body respiratory 
quotient and macronutrient utilization rates whereas selective restriction of dietary fat will cause 
no such changes. 
 
3. Endocrine signals of dietary macronutrient intake 
 
Diet composition has a profound effect on the concentration of a wide variety of circulating 
hormones involved in regulating metabolism, appetite and satiety.  For example, dietary 
carbohydrates stimulate the secretion of insulin which orchestrates a complex sequence of events 
to promote the storage of fat as well as the uptake and oxidation of glucose (13).  Dietary fat, on 
the other hand, does not directly impact insulin secretion but can stimulate the secretion of 
cholecystokinin (CCK) which is believed to play a role in satiety (14). Thus, dietary 
carbohydrate and fat alone are known to result in distinct endocrine signals. However, most 
previous studies have focused on the changes of circulating metabolites and hormones in 
response to single meals of varying macronutrient composition (15-20).  Less is known about 24 
hour changes of circulating metabolites and hormones after a period of adaptation to diets of 
differing macronutrient content, but it is clear that diet composition can significantly change the 
24 hour circulating concentrations of key metabolites and hormones (21, 22).  
 
For the first time, we will study the 24 hour circulating metabolite and hormone levels in 
response to selective reductions of dietary carbohydrate and fat. We expect that carbohydrate 
restriction, but not fat restriction, will significantly alter the 24 hour profiles of glucose, insulin, 
glucagon, ghrelin, PYY3-36, and leptin. This will be assessed by collecting frequent blood 
samples over 24 hour periods before and on the 4th day of selective reduction of dietary 
carbohydrate and fat. These expected changes of circulating metabolites and hormones mediate 
the anticipated changes of 24-hour fuel selection during the reduced carbohydrate diet. By 
measuring both the fuel selection and the metabolite and hormone profiles over the same 24-hour 
period, we can directly relate the measurements. 
 
4. Influence of diet and weight loss on regional brain activity 
 
Functional neuroimaging has revealed several areas of the human brain that play an important 
role in the neural response to feeding and have abnormal responses in obesity (23-25).  Even 
short-term changes of diet can cause detectable changes of neural activity as was highlighted by 
a recent 3-day moderate overfeeding study where fMRI detected significant alterations of neural 
activity in the inferior temporal visual cortex, posterior parietal cortex, premotor cortex, 
hippocampus and hypothalamus in the response to visual food stimuli (26). We plan to measure 
the effects of diet and weight loss on regional brain activity through a series of four fMRI 
experiments.  The first experiment will map the brain regions underlying gustatory perception of 
fundamental tastes. The second experiment will identify the brain regions responsible for 
perception of visual food stimuli. The third experiment will examine the neural basis of food 
preference and the final experiment will map functional connectivity among brain regions 
supporting gustation and perception of food stimuli. For a detailed description of the fMRI study 
design, see the Analytical Procedures section. 
 

7 
We hypothesize that obese subjects will show differences in their neural bases of food 
perception and preference versus normal weight control subjects. Furthermore, we hypothesize 
that after 4 days of selective restriction of dietary carbohydrate, but not fat, will induce changes 
of regional brain activity in the obese subjects and that these changes will be further enhanced 
following a 12 week out-patient weight loss program in the same subjects 
 
5. Influence of diet and weight loss on brain reward circuitry 
 
The response of the brain’s reward system to the eating of food is believed to have played a 
fundamental role in evolution to ensure that an organism obtains sufficient energy reserves for 
reproduction (27).  It has been hypothesized that the central reward pathways that evolved for 
this purpose can be hijacked and exploited to result in addiction to substances such as cocaine or 
methamphetamine (27).  Similar to drug addiction, obesity is correlated with a decrease of 
dopamine D2 receptor availability in the striatum suggesting that increased food intake may be 
playing a compensatory role for the reduction of normal signaling through this reward pathway 
(28).  
 
A recent study in mice with a functional knockout of the sweet taste receptor showed that 
post-ingestive signals from dietary carbohydrate can induce a central dopamine response that is 
independent of palatability (29).  Thus, the metabolic and endocrine changes associated with 
altering dietary carbohydrate are expected to impact the dopamine reward system.  Furthermore, 
food restriction in rats has been shown to increases the dopamine D2 receptor availability (30).  
The present study will investigate whether dopamine D2 receptor availability is altered during a 
reduced carbohydrate diet versus a reduced fat diet, and following weight lost.  
 
Dopamine D2 receptor availability is measured by positron emission tomography (PET) using 
the positron emitting compound [18F]fallypride which binds competitively with dopamine to the 
D2 receptor.  We hypothesize that obese subjects will have increased [18F]fallypride binding in 
the striatum following 4 days of a reduced carbohydrate diet, but not following a reduced fat diet.  
Furthermore, we expect that D2 receptor availability will be further enhanced following a 12 
week out-patient weight loss program in the same subjects.  In addition, a recent study has shown 
a negative relationship between the presence of a polymorphism associated with the dopamine 
D2 receptor gene and BMI, therefore genetic data from blood samples will be obtained for 
determination of the presence of the polymorphism in our study subjects (31).  The presence of 
the polymorphism will be correlated with the measured dopamine D2 receptor availability, as 
well as other measured variables such as BMI.   
 
6. Determinants of food intake following controlled diet restriction 
 
A differential central response to the selective reduction of dietary carbohydrate versus fat 
may result in different degrees of hunger and satiety and may possibly lead to different food 
intake behaviors after the diet control is released.  Certainly, the diets will result in different 
macronutrient oxidation rates which may independently affect food intake patterns and 
subsequent energy balance.  For example, a high respiratory quotient has been correlated with 
subsequent gain of body weight (32, 33) and the degree of positive carbohydrate balance during 

8 
a high carbohydrate diet has been associated with protection from long-term gain of body fat 
(34).   
 
Selective reduction of dietary carbohydrate versus fat may influence subsequent food intake 
patterns.  Therefore, at the end of each dietary restriction period all subjects will be provided 
with ad libitum access to food for three days.  We will investigate the correlations between the 
intake of food during the ad libitum diet period with a variety of measurements during the 
preceding diet-restriction period.  In particular, 24-hour indirect calorimetry will be performed 
immediately prior to the ad libitum phase since previous studies have found correlations between 
carbohydrate oxidation rate and subsequent ad libitum food intake (35).  Our study will further 
explore the relationships between macronutrient utilization and subsequent food intake since the 
two diet groups are expected to induce substantially different carbohydrate and fat oxidation 
rates prior to the ad libitum feeding period.  Furthermore, we will relate the ad libitum feeding to 
the measured 24-hour metabolite and hormone levels, as well as hunger and satiety, measured 
during the reduced carbohydrate and reduced fat diets.  
 
 
7. Macronutrient imbalance and body composition change 
 
In the same obese subjects, we plan to assess the longitudinal dynamics of body composition 
change over the course of a 12 week out-patient weight loss program that will take place 
immediately following the final in-patient phase.  We will investigate the relationship between 
short-term measurements of fat imbalance measured by indirect calorimetry during the in-patient 
diets and the long-term body weight and composition changes during the out-patient weight loss 
program.  We have developed mathematical models that predict how short-term indirect 
calorimetry measurements obtained during the in-patient phase relate to the composition of 
weight loss during a more prolonged period of negative energy balance (8, 9), and these model 
predictions do not depend on the subjects adhering to a specific diet composition during the 
weight loss phase.  We will test these mathematical models and investigate the factors that 
determine the relative loss of lean versus fat mass.  Furthermore, we will investigate whether 
other measurements made during the in-patient phase can help explain the expected variability of 
weight loss and body composition change during the out-patient phase.    
 
8. Long-term follow-up of obese subjects following the weight loss intervention 
 
Because body weight change occurs over extended time scales it is important to assess 
changes of metabolism, body composition and brain activity and reward circuitry that occur over 
many years. Therefore, obese subjects that complete the out-patient weight loss phase of the 
study will be provided with the option to participate in follow-up assessments to investigate 
long-term changes of metabolism, body composition, brain reward circuitry, and regional brain 
activity. The follow-up assessments will occur on an approximately yearly basis for 5 years 
following the weight loss program and will repeat the set of procedures and timeline of the final 
2 weeks of the weight loss phase. These data will allow for an improved understanding of body 
weight regulation on the characteristic time scale of natural weight change and will provide 
insights regarding factors that influence the relative success of weight loss and weight loss 
maintenance. 
   

9 
9. Summary 
 
The importance of understanding the individual effects of selective dietary restriction of fat 
versus carbohydrate derives from the possibility that these macronutrients may differentially 
impact the many feedback control processes that sense whole-body energy imbalance and 
thereby act to resist weight loss.  To further address the issue of adaptive responses to weight 
loss, we will evaluate changes of whole-body metabolism, body composition, regional brain 
activity and reward circuitry in the same obese subjects following a 12-week weight loss 
program.  Therefore, this study will result in an improved understanding of the physiological 
mechanisms that sense and respond to negative energy balance acutely, after several weeks, and 
after several years, and may eventually lead to increased long-term success of obesity treatment.  
 

10 
Study Objectives 
We will study obese adult volunteers in an in-patient setting to assess the changes of whole-
body metabolism, endocrine physiology, psychology, and regional brain activity and reward 
circuitry in response to a 6-day isocaloric selective reduction of dietary fat versus carbohydrate 
by cross-over design.  Following completion of both arms of the in-patient study, separated by a 
2-10 week washout period, the same subjects will participate in a 12 week out-patient weight 
loss phase to assess changes of whole-body metabolism, body composition, and regional brain 
activity and reward circuitry following weight loss.  To assess whether brain activity and reward 
pathways are altered in the obese subjects and, if so, whether the diet interventions normalize 
these variables, a group of normal weight control subjects will undergo the PET and fMRI 
procedures following three days of an out-patient balanced diet.  
 
Primary Aims and Objectives:   
1. To determine the effects of two isocaloric, reduced-energy diets for 6 days, selectively 
restrictive in either fat or carbohydrate calories, on 24-hour respiratory quotient, 
macronutrient utilization and energy balance in obese subjects. 
2. To determine whether obese subjects have altered dopamine D2 receptor availability in 
comparison to lean subjects, and to measure changes of dopamine D2 receptor 
availability following 4 days of selective reduction of dietary fat versus carbohydrate, as 
well as after a 12 week out-patient weight loss program in the same obese subjects. 
3. To determine whether obese subjects have altered regional brain activity in response to 
food stimuli and to measure changes of regional brain activity following 4 days of 
selective reduction of dietary fat versus carbohydrate, as well as after a 12 week out-
patient weight loss program in the same obese subjects.                                                                                
4. To measure the loss of fat and lean body mass in obese subjects during a 12 week out-
patient weight loss program and use a mathematical model to relate the measured body 
composition changes to the indirect calorimetry measurements obtained during the in-
patient diet perturbations.  
 
Secondary Aims and Objectives: 
1. In obese subjects, to assess the impact of two reduced-energy diets selectively restrictive 
in either fat or carbohydrate on: 
a. Nitrogen balance (i.e. urinary nitrogen excretion) 
b. 24-hour plasma concentration of hormones and peptides related to feeding and 
energy balance 
c. Whole-body lipolysis, proteolysis, and glucose turnover rates.  
d. Expression of pro-inflammatory markers and of genes involved in macronutrient 
utilization in skeletal muscle and adipose tissue 
e. Changes in psychological measurements such as affective state, mood, hunger, 
satiety, as well as implicit and explicit measures of liking and wanting of food 
2. In obese subjects, to evaluate 3-day ad libitum food intake following 6 days of selective 
restriction of dietary carbohydrate versus fat and investigate correlations between the 
intake of food during the ad libitum diet day with a variety of measurements during the 
previous controlled diet restriction period.  
 
 

11 
Study Design and Methods 
 
This is a randomized, cross-over study of adult, obese, non-diabetic individuals to determine 
the metabolic, endocrine, psychological, and brain reward circuitry responses to controlled, 
selective reductions of dietary carbohydrate versus fat.  To assess whether brain activity and 
reward pathways are altered in the obese subjects, a group of control subjects with normal BMIs 
will undergo the PET and fMRI procedures following three days of an out-patient balanced diet.   
 
Subjects 
Human subjects (18-45 years of age) will be recruited to the study via inquires received from 
interested study subjects through the NIH Patient Recruitment and Public Liaison Office.  Pre-
screening by this office will exclude subjects with a BMI less than 30 k/m2 for obese subjects, 
and greater than 25 kg/m2 or less than 18.5 k/m2 for lean control subjects, those with diabetes or 
any other metabolic disorders, and those who require assistance to complete activities of daily 
living.  All others will be contacted by the protocol team to review exclusion criteria.  All 
subjects will be fully informed of the aims, nature, risks, and potential benefits of the study prior 
to giving written consent.  
 
Inclusion and Exclusion Criteria 
Obese Subjects 
Inclusion criteria:   
 
 Age 18-45 years, male or female 
 Body mass < 350 lbs. (max. weight dictated by table limit for fMRI scanner) 
- when acquisition of large bore fMRI is complete, max. wt. limit will increase to 400 lbs. 
 Weight stable (< ± 5 kg over past 6 months) 
 Body mass index > 30.0 kg/m2 
 Premenopausal (women only)  
 Healthy, as determined by medical history and laboratory tests 
 Able to complete daily bouts of walking at a moderate rate 
 Written informed consent 
 
Exclusion criteria: 
 
 Body mass > 350 lbs. (max. weight dictated by table limit for fMRI scanner) 
- when acquisition of large bore fMRI is complete, max. wt. limit will increase to 400 lbs. 
 BMI  < 30.0 kg/m2 
 Evidence of metabolic or cardiovascular disease, or disease that may influence 
metabolism (e.g. cancer, diabetes, thyroid disease)  
 Taking any prescription medication (except birth control) or other drug that may 
influence metabolism (e.g. diet/weight-loss medication) 
 Hematocrit < 34% (women only) 
 Hematocrit < 40% (men only) 
 Pregnancy, lactation (women only) 
 Allergy to lidocaine or ethanol 
 Participating in a regular exercise program (> 2h/week of vigorous activity) 
 Caffeine consumption > 150 mg/day (will be clamped at baseline intake during study) 

12 
 Regular use of alcohol (> 2 drinks per day), tobacco (smoking or chewing) 
amphetamines, cocaine, heroin, or marijuana over past 6 months 
 Past or present history of eating disorder (including binge eating) or psychiatric disease, 
including claustrophobia since part of the protocol will involve being confined to a small 
room for whole-body indirect calorimetry 
 Volunteers with strict dietary concerns (e.g. vegetarian or kosher diet, multiple food 
allergies) 
 Are claustrophobic to a degree that they would feel uncomfortable in the MRI machine.  
 Having any metal in their body (for example, pacemakers, metallic prostheses such as 
cochlear implants or heart valves, shrapnel fragments, etc.). 
 Left-handedness 
 Non-native English speakers 
 Volunteers unwilling or unable to give informed consent 
 
Control Subjects 
Inclusion criteria:   
 
 Age 18-45 years, male or female 
 18.5 kg/m2 < BMI  < 25.0 kg/m2 
 Weight stable (< ± 5 kg over past 6 months) 
 Premenopausal (women only)  
 Healthy, as determined by medical history and laboratory tests 
 Written informed consent 
 
Exclusion criteria: 
 
 BMI  < 18.5 or > 25.0 kg/m2 
 Evidence of metabolic or cardiovascular disease, or disease that may influence 
metabolism (e.g. cancer or diabetes)  
 Taking any prescription medication (except birth control) or other drug that may 
influence metabolism (e.g. diet/weight-loss medication) 
 Hyperlipidemia (fasting plasma triglyceride concentration > 150 mg/dl) 
 Hematocrit < 34% (women only) 
 Hematocrit < 40% (men only) 
 Pregnancy, lactation (women only) 
 Participating in a regular exercise program (> 2h/week of vigorous activity) 
 Caffeine consumption > 150 mg/day  
 Regular use of alcohol (> 2 drinks per day), tobacco (smoking or chewing), 
amphetamines, cocaine, heroin, or marijuana over the past 6 months 
 Past or present history of eating disorder (including binge eating) or psychiatric disease 
 Volunteers with strict dietary concerns (e.g. vegetarian or kosher diet, multiple food 
allergies) 
 Are claustrophobic to a degree that they would feel uncomfortable in the MRI machine.  
 Having any metal in their body (for example, pacemakers, metallic prostheses such as 
cochlear implants or heart valves, shrapnel fragments, etc.). 
 Left-handedness 

13 
 Non-native English speakers 
 Volunteers unwilling or unable to give informed consent 
 
Screening Procedures 
Eligible volunteers will be invited to the Clinical Research Center for a screening visit, and 
after signing the informed consent, will undergo a series of screening tests, including a history 
and physical examination.  Subjects will be required to fast for at least 12 hours before the 
screening tests begin, which will include a blood draw for assessment of blood lipid profile, liver 
panel, electrolytes, and blood count.  A urine pregnancy test will be performed on all female 
subjects.  Body weight and height will be measured and body mass index will be determined.  
All subjects will undergo a resting electrocardiogram (EKG), and resting metabolic rate will be 
measured by indirect calorimetry.  Obese subjects, but not control subjects, will then walk on a 
treadmill at a self-selected pace for 15 minutes.  This walking speed will be repeated on days of 
scheduled physical activity during the obese subjects’ in-patient visit.  All subjects will also 
undergo psychiatric evaluation by Structured Clinical Interview for DSM-IV (SCID) during the 
screening process.  The results of the interview will be evaluated during the screening phase by a 
clinical psychologist and any clinically significant findings will be communicated to the subject 
by a qualified member of the research team and appropriate follow-up with their primary care 
physician will be planned.  During the screening procedures, subjects will also briefly visit the 
MRI mock scanner.  The mock scanner simulates the noise and dimensions of the MRI scanner 
that will be used for this study and will allow us to screen for subjects that may be unwilling to 
participate in the scanning procedures due to noise level, claustrophobia, or overall fit within the 
scanner.  Following the screening visit, volunteers will be asked to continuously wear physical 
activity monitors and keep food diary and activity records for 3 days.  The monitors and records 
will be returned and reviewed after completion of the three days to ensure adequate 
macronutrient and energy intake in the habitual diet, as well as to estimate the average daily 
caloric requirements for use during the baseline period. Along with the 3-day dietary record, 
volunteers will also be asked to provide a list of any dietary restrictions or allergies.  
 
Study Overview (see Appendix for a detailed description of the study procedures) 
Control Subjects 
A total of 20 normal control subjects will undergo PET and fMRI procedures.  For 2 days 
before admission to the NIH Metabolic Clinical Research Unit (MCRU), each subject will be 
provided with a weight-maintenance diet using a standard diet composition of 50% carbohydrate, 
35% fat, and 15% protein on an out-patient basis.  The food will be supplied by the Clinical 
Center Metabolic Kitchen and subjects will eat one meal per day on the metabolic unit.  The 
daily caloric content of the diet during the out-patient segment will be based on the 3-day diet 
and activity records, as well as resting metabolic rate and body size measurements made during 
screening.  On the morning after the second day of the out-patient diet, subjects will arrive at the 
MCRU at 0800 h and be admitted to the MCRU.  During the first day of their in-patient visit, 
subjects will undergo a series of procedures including measurement of body composition by dual 
energy x-ray absorptiometry (DEXA) and air-displacement plethysmography (BodPod), 
measurement of total body water by bioelectrical impedance spectroscopy (BIA), assessment of 
taste sensitivity (including PROP taste test), three-factor eating questionnaire and socioeconomic 
questionnaire, assessment of liking and wanting of food, and a gustatory, blood draw for 
measurement of plasma hormones and substrate, and food perception fMRI scan session.  During 

14 
the second day of their in-patient visit, subjects will undergo a baseline blood draw for 
measurement of plasma hormones and substrate, an PET [18F]fallypride imaging session, 
assessment of delay discounting of monetary versus food rewards, an additional blood draw for 
measurement of plasma hormones and substrate, and a separate food rating fMRI scan session.  
Hunger will be assessed by visual analog scale periodically throughout the two days.  The 
subjects will be fed the standard baseline diet during this in-patient period.  These measurements 
on the control subjects will be used for comparison with similar measurements on obese subjects 
as described below.   
 
Obese Subjects 
A total of 20 obese subjects will undergo and complete this cross-over study.  The outline of 
the study is displayed in Figure 1.  Subjects will undergo two separate in-patient visits to the 
research unit followed by a 12 week out-patient weight loss program.  For all female subjects, 
both in-patient visits will be carried out during the follicular phase of the menstruation cycle.  On 
the first visit, subjects will be required to stay 14 consecutive days in the MCRU, beginning with 
a 5-day weight maintenance diet phase, followed by a 6-day reduced calorie diet phase, and 
ending with a 3-day ad libitum diet phase.  For 2 days before admission to the NIH Metabolic 
Clinical Research Unit (MCRU), each subject will be provided with a weight-maintenance diet 
using a standard diet composition of 50% carbohydrate, 35% fat, and 15% protein on an out-
patient basis.  The food will be supplied by the Clinical Center Metabolic Kitchen and subjects 
will eat one meal per day on the metabolic unit.   
 
 
 

15 
Figure 1.  General time line of in-patient and out-patient weight loss phases followed by yearly 
follow-ups for 5 years.  
 
 
 
The daily caloric content of the diet during the initial out-patient segment will be based on the 
3-day diet and activity records, as well as resting metabolic rate and body size measurements 
made during screening.  Upon admission, the subjects will continue to be fed the standard diet.  
The subjects will spend the second day in the metabolic chamber where the standard diet will be 
fine-tuned to achieve energy balance. The standard diet will then be continued for the next 3 
days during the first in-patient stay.  
 
Over the first 5 days following admission to the MCRU, subjects will undergo baseline 
metabolic studies consisting of a daily blood draw, a 24-hour blood sampling, indirect 
calorimetry, measurement of body composition (twice) by dual energy x-ray absorptiometry 
(DEXA) and air-displacement plethysmography (BodPod), measurement of total body water by 
bioelectrical impedance spectroscopy (BIA) (twice) and by administration of sodium bromide, 
administration of doubly labeled water (DLW) for measurement of average total energy 
expenditure, isotopically-labeled substrate infusions (optional), muscle and adipose tissue 
biopsies (optional), frequently sampled intravenous glucose tolerance test (FSIVGTT) for 
measurement of insulin sensitivity, positron emission tomography (PET) imaging, magnetic 
resonance imaging (MRI) scanning sessions of food perception and food rating, Profile of Mood 
States (POMS) questionnaire, three-factor eating questionnaire socioeconomic questionnaire, 
First 
Visit 
Out-patient (12 wks) 
Intervention diet 
Body Comp 
Metabolic study 
PET/fMRI imaging 
Metabolic chamber 
Activity monitors 
Bite counter/Ingestion device 
Weight-loss 
Intervention 
Baseline 
Standard diet 
Body comp 
Metabolic Study 
PET/fMRI 
imaging 
Metabolic 
Restrictive Diet 
Reduced fat or carbohydrate diet 
Body Comp 
Metabolic study 
PET/fMRI imaging 
Metabolic chamber 
Ad libitum 
Food intake 
Hunger/sati
ety 
 
 
 
2 - 10 week wash out period 
5 Days 
6 Days 
3 Days 
Second 
Visit 
Baseline 
Standard diet 
Body comp 
Metabolic 
chamber 
Restrictive Diet 
Reduced carbohydrate or fat diet 
Body Comp 
Metabolic study 
PET/fMRI imaging 
Metabolic chamber 
Ad libitum 
Food intake 
Hunger/sati
ety 
 
 
 
5 Days 
6 Days 
3 Days 
long term  
follow-up 

16 
hunger and satiety assessment, assessment of taste sensitivity (including PROP taste test), 
assessment of delay discounting of monetary versus food rewards, assessment of liking and 
wanting of food, as well as two 24h stays in a metabolic chamber.  Physical activity monitors 
will be worn continuously, and urine will be collected daily for measurement of 24h urinary 
nitrogen excretion.  
 
The purpose of these measurements is to provide a comprehensive assessment of baseline 
whole-body metabolism and body composition in the obese subjects as well as baseline measures 
of brain reward circuitry and regional brain activity in response to food stimuli. The baseline 
measurements will then be compared with similar measurements during the in-patient diet 
interventions and after the outpatient weight loss program as described below. 
 
Directly following the baseline period on the standard diet, subjects will continue their stay at 
the MCRU for the next 6 days and will be randomly assigned to one of the following reduced 
calorie diets: 
 
1. Red-FAT (selective reduction of 85% of baseline fat calories per day) 
2. Red-CHO (selective reduction of 60% of baseline carbohydrate calories per day) 
 
Both diet perturbations were designed to produce identical caloric deficits when compared to 
the standard diet (see Table 1).  The two reduced calorie periods will have dietary protein, 
sodium, and water intakes maintained corresponding to the baseline diet and all subjects will 
receive a daily multivitamin.   
 
To address primary aim #1 of the study, the subjects will spend the second and final baseline 
diet days and the first, fourth, and sixth reduced-calorie diet days in the metabolic chamber to 
measure 24-hour respiratory quotient, substrate utilization rates, as well as macronutrient and 
energy balances.  Primary aims #2 and #3 will be addressed by repeat PET and fMRI studies 
after 4 days of the reduced-calorie diets. 
 
 
Table 1.  Diet composition for hypothetical subject with estimated total daily energy intake of 
2000 kcal/day and estimated total daily energy intake of 3000 kcal/day. 
 
Macronutrient Ingestion, 
g/day (%  from std diet) 
Total Energy Intake 
kcal/day 
(%  from std diet) 
 
Carbohydrate 
Fat 
Protein 
Baseline diet 
250g 
78g 
75g 
2000 
Red-Fat 
250g (0%) 
12g (-85%) 
75g (0%) 
1405 (-30%) 
Red-Carb 
101g (-60%) 
78g (0%) 
75g (0%) 
1405 (-30%) 
Baseline diet 
375g 
117g 
113g 
3000 
Red-Fat 
375g (0%) 
18g (-85%) 
113g (0%) 
2100 (-30%) 
Red-Carb 
152g (-60%) 
117g (0%) 
113g (0%) 
2100 (-30%) 
 
To address the secondary aims listed above, after several days on the reduced-calorie diets we 
will repeat the daily blood draw, the 24-hour blood sampling, indirect calorimetry, measurement 

17 
of body composition by dual energy x-ray absorptiometry (DEXA) and air-displacement 
plethysmography (BodPod), measurement of total body water by bioelectrical impedance 
spectroscopy (BIA), isotopically-labeled substrate infusions (optional), muscle and adipose 
tissue biopsies (optional), positron emission tomography (PET) imaging, magnetic resonance 
imaging (MRI) scanning session of food rating, Profile of Mood States (POMS) questionnaire, 
hunger and satiety assessment, assessment of delay discounting of monetary versus food 
rewards, and assessment of liking and wanting of food.  Immediately after the conclusion of the 
6-day calorie reduction period, the subjects will remain at the MCRU for an additional 3 days 
during which they will have ad libitum food access and the food intake will be measured.   
 
After discharge from the MCRU, a wash-out period of at least 2 weeks will be required before 
subjects return for the second portion of the study (i.e., the alternate reduced calorie diet than 
what was consumed during their first visit).  This period of time will allow for sufficient wash-
out of any effects the diet manipulation may have on subsequent measurements during the 
second stay at the MCRU.  Subjects will be required to begin completion of the second reduced 
calorie portion of the study within 10 weeks after discharge in order to avoid large changes in 
physiological state.  During the wash-out period, subjects will be encouraged to return to normal 
living patterns and consume a diet as similar to the standard diet as possible and maintain weight 
stability (within 2 kg of their baseline weight). 
 
After the wash-out period, subjects will return to the MCRU for the second segment of 
selective reduced calorie intake.  Before being admitted to the MCRU, subjects will again 
consume the standard diet for 2 days on an out-patient basis, however, after subjects are admitted 
to the MCRU, they will undergo a reduced number of baseline metabolic studies in comparison 
with their first in-patient stay.  Of the baseline metabolic studies that were conducted during the 
first in-patient stay, only the following measurements will be repeated: body composition by dual 
energy x-ray absorptiometry (DEXA) and air-displacement plethysmography (BodPod), total 
body water by bioelectrical impedance spectroscopy (BIA), average total energy expenditure by 
administration of doubly labeled water (DLW), hunger and satiety assessment, and two 24h stays 
in a metabolic chamber.  Physical activity monitors will be worn continuously, and urine will be 
collected daily for measurement of 24h urinary nitrogen excretion.  Once the 6-day reduced 
calorie diet has begun, the timeline and measurements will be the same as during the reduced 
calorie portion of the first visit.  
 
We have designed this protocol to avoid the confounding influence of the menstrual cycle 
phase on measurements of energy balance in our female subjects (36).  The metabolic studies of 
the baseline period and calorie reduction period of the first in-patient visit, as well as the second 
in-patient visit, will be carried out during the follicular phase for all female subjects.   
 
At the end of the second visit, subjects will be discharged from the MCRU and begin a 12 
week out-patient weight loss program to address our final primary aims.  During this period, all 
subjects will be prescribed a lifestyle modification consisting of a reduced-calorie diet and 
increased physical activity with a goal to lose at least 5% of body weight by the end of 12 weeks.  
Subject’s will be given a scale to take home with them and their body weight will be monitored 
daily (by phone or email) and subjects will meet with registered dietitians biweekly to receive 
nutrition education and goal assessment.  Subjects will also return periodically (at 2, 6, 10, and 

18 
12 weeks after discharge) to the MCRU for metabolic studies consisting of body composition, 
indirect calorimetry, and 24h stay in the metabolic chamber.  At the end of the 12 week period, 
subjects will undergo similar metabolic studies as those performed during the calorie reduction 
periods.  
 
The obese subjects that complete the out-patient weight loss phase will be provided with the 
option to participate in follow-up assessments to investigate long-term changes of metabolism, 
body composition, brain reward circuitry, and regional brain activity. The follow-up assessments 
will occur on an approximately yearly basis for 5 years following the weight loss program and 
will repeat the procedures and timeline of the final 2 weeks of the weight loss phase.  
 
Analytical Procedures 
Resting energy expenditure 
The measurement will be performed in the morning after a 12 h fast and overnight rest with 
the subject in a supine position.  An indirect calorimeter with the ventilated hood technique will 
be used for the 30 min measurement (the first 5 – 10 mins will be excluded from analysis).  The 
respiratory quotient will be calculated as the ratio of carbon dioxide production to oxygen 
consumption.  Resting metabolic rate will be calculated from VO2 and VCO2 measurements 
using the Weir equation (37) and whole body fat oxidation will be calculated using the equations 
of Frayn (38).  
 
MedGem Handheld Indirect Calorimeter 
This measurement will be performed at home every morning after an overnight fast during the 
first two weeks and the last two weeks of the 12-week weight-loss program.  The device is light-
weight and equipped with a disposable mouth piece that the subject breathes through for 5 to 10 
minutes while seated and resting comfortably.  Subjects will also be wearing a disposable nose 
clip.  This device reports 24hour resting energy expenditure in kcals and VO2 in mL/min.  
 
Respiratory chamber: 24h energy expenditure 
The respiratory chamber is a specially constructed room to assess the metabolism of subjects 
for a period of 24 hours.  Designed as a walk-in “pull” calorimeter, it is an open circuit unit that 
draws conditioned room air into the chamber at the same flow rate as it is extracted into the gas 
analysis system.  Each of the three rooms is equipped with a toilet and sink with privacy screen, 
treadmill, bed, desk, and computer with access to television and other forms of entertainment.  
General physical activity level is measured continuously through a wall mounted monitoring 
device (microwave sensor).  Food and fresh water is passed through an air-lock drawer system.  
Telemetry and a nurse call are available to enhance subject safety.  In the chamber, 24h energy 
expenditure (EE), sleeping EE, respiratory quotient (RQ), and dietary induced thermogenesis 
(DIT) will be assessed.  
 
Plasma substrate and hormone concentrations 
Blood samples will be collected into chilled test tubes containing EDTA and aprotinin as 
preservatives.  Those sample for which analysis of gastrointestinal peptides will take place will 
also contain dipeptidyl peptidase IV inhibitor (10 L•mL-1 blood) as preservative and be 
collected into chilled glass tubes.  All samples will be kept on ice and then centrifuged (1600 g 
for 15 min at 4°C) within 30 min of collection for isolation of plasma.  After centrifugation, the 

19 
plasma will immediately be frozen and stored at -80°C for later analysis.  Plasma substrate and 
hormone concentrations will be assessed using the procedures outlined in Table 2.  Genomic 
DNA will be isolated from peripheral mononuclear cells using the QIAmp® system (QIAGEN). 
 
Table 2. List of procedures for measuring plasma substrates and hormones 
 
Metabolite/hormone 
Method 
Metabolites 
triglycerides 
fatty acids 
-hydroxybuterate 
glucose 
colorimetric assay 
colorimetric assay 
fluorometric assay 
colorimetric assay 
Hormones 
insulin 
glucagon 
leptin 
c-peptide 
cortisol 
epinephrine 
growth hormone 
leptin 
adiponectin 
GLP-1 (active form) 
PYY3-36 
ghrelin 
CCK 
GIP 
RIA 
RIA 
RIA 
RIA 
chemiluminescence 
HPLC 
chemiluminescence 
RIA 
RIA 
RIA 
RIA 
RIA 
RIA 
RIA 
Markers of 
Inflammation 
CRP 
IL-6 
TNF-alpha 
ELISA 
ELISA 
ELISA 
RIA = radioimmunoassay; HPLC = high performance liquid chromatography, ELISA = enzyme-
linked immunosorbent assay 
 
Isotope tracer enrichment (optional) 
The tracer:tracee ratio (TTR) of plasma phenylalanine, glucose, and glycerol will be measured 
by electron impact ionization gas chromatography/mass spectroscopy (GC/MS).  For plasma 
phenylalanine, GC/MS analysis will be performed after conversion to the tertiary-
butyldimethylsilyl derivative of phenylalanine in deproteinized plasma (39).  Heptafluorobutyric 
(HFB) anhydride will be used to form a heptafluorobutyric derivative of glycerol and glucose for 
GC/MS analysis (40).  The TTR in plasma will be used to calculate phenylalanine, glycerol, and 
glucose rate of appearance (Ra) into plasma.  Whole-body plasma phenylalanine Ra, glycerol Ra, 
and glucose Ra will be determined from the steady-state isotope dilution equation Ra = I/TTRA 
(41), where I is the tracer infusion rate (mol•kg-1•min-1) and TTRA is the TTR of [ring-2H5]-
phenylalanine, [6,6-2H2]-glucose, or [2H5]-glycerol in the arterialized sample.  
 
Muscle biopsy (optional) 
About 100 – 150 mg of muscle will be taken from the thigh during each biopsy.  The muscle 
biopsy procedure involves numbing a nickel-sized portion of the skin of the thigh with a local 
anesthetic (lidocaine), making a small incision (1/4 inch), and removing a small piece of muscle.  

20 
The muscle will quickly be partitioned into small pieces (~25-50 mg), rinsed with saline, and 
frozen in liquid nitrogen.  The incision will then be closed with a piece of sterile tape.  
Intramuscular triglyceride (IMTG) concentration will be measured from the liberation of free 
glycerol (42).  Muscle glycogen will be determined from the measurement of glucose after acid 
hydrolysis of muscle glycogen with hydrochloric acid (43).  Gene expression micorarrays will be 
conducted on muscle biopsy samples for measurement of skeletal muscle gene expression of 
proteins involved in substrate utilization within the muscle (e.g. GLUT4, PDK-4, PDH, ACC, 
IRS-1, PI3-K, PDK-1, MAPK, CPT1, FATP, LPL, CD36/FBP). 
 
Subcutaneous Adipose Tissue Biopsy (optional) 
     Subcutaneous adipose tissue (5-10 g) will be removed from the abdominal region by 
aspiration with a 16-gauge needle under local anesthesia (2% xylocaine).  The relatively large 
sample of fat from each subject will ensure extraction of sufficient RNA for microarrays, follow 
up, and quantitative real time-PCR validation.  A representative portion of the tissues will be 
formalin-fixed, paraffin-embedded, sectioned, and stained with hematoxylin and eosin using 
standard histological methods.  The remaining tissue will be separated into aliquots of 
approximately 500 mg for RNA isolation and protein extraction, flash frozen in liquid nitrogen, 
and stored at -80 ºC.  Gene expression micorarrays will be conducted on adipose tissue biopsy 
samples for measurement of adipose tissue gene expression of proteins involved in substrate 
utilization within the adipose tissue (e.g. leptin, adiponectin, HSL, LPL, CD36/FBP, Il-6, PAI-
1,TNF-). 
 
Frequently-sampled intravenous glucose tolerance test (IVGTT) 
We will use the IVGTT for measurement of insulin sensitivity using the well-established 
sensitivity index (SI) derived from Bergman’s minimal model (44).  Two intravenous catheters 
will be placed: one will be inserted in an antecubital vein for infusion of glucose and insulin and 
a second retrograde catheter will be inserted into a dorsal hand of wrist vein (the hand will be 
enclosed in a warming chamber (65°C) to “arterialize” the blood obtained from all samples).  At 
time 0, an intravenous bolus of glucose (300 mg/kg body weight) will be administered over 1 – 2 
minutes.  At 20 min, a bolus of insulin (0.03 U/kg body weight) will be given.  Blood samples 
(~2 mL) for glucose and insulin will be taken at -10, -5, 0, 2, 3, 4, 5, 6, 8, 10, 12, 14, 16, 19, 22, 
23, 24, 25, 27, 30, 40, 50, 60, 70, 80, 90, 100, and 120 minutes.  Plasma glucose and insulin will 
be measured by the methods indicated in Table 1. 
 
Dual energy x-ray absorptiometry (DEXA) 
This technique has found extensive clinical and research applications in addition to the 
assessment of bone mineralization status.  With this technique, one can determine total and 
regional body fat and fat-free masses and can estimate appendicular muscle.  This procedure will 
be performed in the MCRU. 
 
Air-displacement plethysmography 
 
Assessment of body composition will be derived from an estimate of body density obtained 
using an air displacement plethsymography chamber (BOD POD).  Subjects will be weighed and 
then evaluated in the chamber while clothed in a tight-fitting bathing suit or underwear and 
acrylic bathing cap.  Final body volume will be calculated based on initial body volume 
corrected for the subject’s thoracic gas volume as measured by the breathing circuit housed in 

21 
the rear of the chamber through a disposable tube.  The two compartment model of Siri will be 
used to estimate body fatness from the body density measurements obtained using the BOD 
POD. 
 
Physical activity monitors 
Free-living physical activity will be quantified with activity monitors using high sampling 
frequencies (32 samples per second in the chamber – minute-to-minute sampling other times) 
during all waking periods using small, portable pager-type and watch accelerometers (Mini 
Mitter / Respironics Co, Bend OR) at the subjects’ hip, dominant wrist, and non-dominant ankle. 
Overall physical activity levels, daily changes, amount of time spent in sedentary, moderate, 
vigorous intensity categories and activity-associated energy expenditures will be extracted (45).  
In addition, a portable accelerometer + heart rate combination monitor (Actiheart, also by Mini 
Mitter) will be used.  These monitors will be worn throughout both in-patient studies and 
periodically (i.e. from weeks 2 – 4, weeks 6 – 8, and weeks 10 – 12) during the 12-week out-
patient period.  
 
Bite Counter 
This method detects eating behavior and provides real-time information concerning bites taken 
during a meal by counting the total number of bites the user has taken, and providing the bites-
taken rate (bites per minute) with a 91% sensitivity (46). This method use an orientation sensor 
placed on the wrist of a user, and analyze the rolling motion of the wrist in order to detect a 
pattern related to biting behavior. 
 
Automatic Ingestion Monitor  
The automatic ingestion monitor is a device capable of automatically detecting food intake 
episodes; estimating the number of episodes in a period of time and the frequency, duration, rate 
and mass of ingestion. The principle of operation is based on non-invasive sensing of jaw motion 
characteristic of food intake, hand-to-mouth gestures during food intake, and (optionally) 
swallowing events. Automatic computer algorithms process the sensor signals to recognize and 
characterize food intake episodes. AIM consists of a jaw motion sensor connected by a wire to a 
wireless module worn as a pendant around the neck, a small radio transmitter worn on a wrist 
(used for detection of hand-to-mouth gestures), and a smart phone used for logging of the sensor 
data.  The jaw motion sensor is attached to the jaw.  This sensor is connected to the pendant 
(worn around the neck) by a wire.  The pendant is wireless and will send data about the jaw 
sensor to the smart phone.  A picture of an AIM is shown below.  
 
 
 

22 
 
 
 
 
A) Strain sensor (jaw sensor) that will attach by medical tape to the area immediately below 
the outer ear.  B)   Wireless module worn as a pendant around the neck.  It is connected to 
the jaw sensor via wire.  C)  Smart phone that will log the sensor data via a wireless 
transmission. 
There are no electrical risks associated with the automatic ingestion monitor, but subjects may 
occasionally find wearing the jaw motion sensor inconvenient. The automatic ingestion monitor 
also involves a neck band which utilizes a breakaway clasp that uses either a magnetic clasp or a 
breakaway buckle that pops apart under tension and prevents suffocation if a pulling force is 
applied to the band. 
 
 
Earlier models of this device have been approved by the Institutional Review Board at 
Clarkson University for Dr. Sazonov.  This device has previously been studied and is included in 
the following references: 
 
Detection of food intake from swallowing sequences by supervised and unsupervised methods. 
Lopez-Meyer P, Makeyev O, Schuckers S, Melanson EL, Neuman MR, Sazonov E. Ann Biomed 
Eng. 2010 Aug;38(8):2766-74. Epub 2010 Mar 30. 
 
Automatic detection of swallowing events by acoustical means for applications of monitoring of 
ingestive behavior. Sazonov ES, Makeyev O, Schuckers S, Lopez-Meyer P, Melanson EL, 
Neuman MR. IEEE Trans Biomed Eng. 2010 Mar;57(3):626-33. Epub 2009 Sep 29. 
 
Non-invasive monitoring of chewing and swallowing for objective quantification of ingestive 
behavior. Sazonov E, Schuckers S, Lopez-Meyer P, Makeyev O, Sazonova N, Melanson EL, 
Neuman M. Physiol Meas. 2008 May;29(5):525-41. Epub 2008 Apr 22. 
 
A 
B 
C 

23 
Toward objective monitoring of ingestive behavior in free-living population. Sazonov ES, 
Schuckers SA, Lopez-Meyer P, Makeyev O, Melanson EL, Neuman MR, Hill JO. Obesity 
(Silver Spring). 2009 Oct;17(10):1971-5. Epub 2009 May 14. 
 
In addition, Dr. Sazonov has been awarded the following NIDDK grant to investigate the use 
of this device with the following information: 
 
Project Number:   5R21DK085462       
 
Contact PI / Project Leader: SAZONOV, EDWARD S 
 
Title: OBJECTIVE MONITORING OF ENERGY INTAKE AND INGESTIVE BEHAVIOR 
IN A FREE LIVING POP  
 
Awardee Organization:   UNIVERSITY OF ALABAMA IN TUSCALOOSA 
 
Doubly Labeled Water 
In the morning on the first day of both in-patient periods, as well as at the beginning and end 
of the 12-week out-patient period (i.e. the beginning of week 1 and week 10) we will make a 
collection of a baseline (predose) urine sample.  Subjects will then drink from a stock solution of 
a mixture of 1.5 g of 10% enriched H218O and 0.08 g of 99% enriched 2H2O per kg of body 
weight followed by 100-200 mL tap water to rinse the dose container.  Throughout both in-
patient periods, urine samples will be collected each morning in the fasted states.  During the 
out-patient period, after giving the initial dose, urine samples will be collected on three occasions 
each week for two weeks (i.e. from weeks 1 – 2and 10 – 12).  Isotopic enrichments of urine 
samples will be measured by isotope ratio mass spectrometry.  CO2 production rate will be 
estimated from the differential disappearance of the 2 isotopes with use of the equation 
developed by Schoeller et al (47).   
 
Bioelectric Impedance 
We will use bioelectrical impedance spectroscopy (BIS) to measure the impedance of the 
body.  BIS spectra will be obtained with a HYDRA bio-impedance spectrum analyzer (model 
4200, Xitron Technologies, San Diego, CA) using a spectrum of 50 programmed frequencies 
spaced throughout a 5KHz to 1MHz range.  From this we can estimate total body water (TBW) 
and extracellular water (ECW), as tissue impedance at low frequencies is controlled by the 
electrical properties of ECW and at high frequencies by the electrical properties of TBW.  
Subjects are measured by two sets of electrodes placed on the hand/wrist and foot/ankle while 
lying supine on a nonconductive surface.  This is a non-invasive test. 
 
Bromide dilution 
 
Bromide dilution method will be used to measure ECW.  Blood samples will be taken before 
and 10 h  and 11 h after a dose of sodium bromide (60 mg bromide/liter of estimated TBW) (48).  
The bromide concentration in serum ultrafiltrate will be determined by high performance liquid 
chromatography (49).   
 
 
 

24 
Profile of Mood States (POMS) Questionnaire 
The Profile of Mood States (POMS) is a widely used questionnaire for measuring distinct 
mood states. The POMS has 65 items, each with a 5-point adjective rating scale, that measures 
six identifiable moods or feelings: Tension-Anxiety (T), Depression-Dejection (D), Anger-
Hostility (A), Vigor-Activity (V), Fatigue-Inertia (F), and Confusion-Bewilderment (C). 
Completion of the POMS takes 5-10 minutes. 
 
Structured Clinical Interview for DSM-IV (SCID) 
The SCID is a widely used semi-structured interview for making most of the major 
psychiatric diagnoses.  The interview will be conducted by a trained mental health professional.  
 
Three-Factor Eating Questionnaire 
The three-factor eating questionnaire (TEFQ) is a self-assessment questionnaire developed to 
measure dietary restraint, disinhibition and hunger (50).  The questionnaire contains 36 items 
with a yes/no response, 14 items with a 1-4 response scale, and 1 item with a 1-5 response scale.  
 
MacArthur Socioeconomic Status (SES) Questionnaire 
 
The MacArthur SES questionnaire is a widely used self-assessment questionnaire.  It begins 
with subjective social status questions, followed by questions assessing educational attainment, 
occupational status, income and assets. 
   
Hunger & Satiety Assessment 
At multiple times during their in-patient visit, subjects will be asked to complete a survey to 
identify their perception of hunger (i.e., visual analog scale [VAS]) (51).  More specifically, the 
VAS survey will consist of four questions: 1) "How hungry do you feel?" 2) "How much do you 
think you can eat?" 3) "How satisfied do you feel?" and 4) "How full do you feel?"  Below each 
question on the survey there is a horizontal 100mm line with qualifying statements on the 
extreme left and right side of this line.  In response to each question, subjects will be asked to 
draw a vertical mark on the horizontal line to represent the magnitude of their response to the 
question.  A value for each response is quantified by measuring the distance of their mark (in 
mm) relative to the left end of the line.  Therefore, the values (or "scores") for each question 
range from 0 to 100.  To ensure consistency, all VAS ratings will be measured by the same 
member of the research team. 
 
Taste Intensity Assessment 
The perception of taste intensity in response to standard taste stimuli will be assessed.  The 
standard stimuli will be sweet, sour, salty, bitter, savory (umami), and fat.  The subjects will be 
asked to take a sip of each tastant solution, hold it in their mouth for ~5 sec, and then swallow.  
A minimum 60 sec interval will be required between each taste stimuli during which subjects 
will rate their perceived intensity and rinse their mouth with a tasteless rinsing solution.  The 
rating of perceived intensity of each tastant will be recorded by VAS (anchored with “not at all” 
and “extremely”).   
In addition, the gustatory response to the bitter tasting agent, 6-n-propylthiouracil (PROP) 
will be assessed.  A difference in the perception of 6-n-propylthiouracil has been linked to 
greater perceived bitterness of some bitter compounds and divides the population into nontasters 

25 
and tasters (52).  Subjects will place a PROP filter paper on their tongue, allow it to moisten, and 
then rate the bitterness intensity using VAS.  
 
Liking and Wanting Computer Procedure 
The liking and wanting procedure comprises two tasks designed to assess (1) explicit liking 
and wanting, followed immediately by (2) implicit wanting for the same target food stimuli (53, 
54).  The separate task elements will be integrated to fully randomize explicit and implicit trials.  
Experiment generator software (E-prime v1.2) is used to integrate the single stimulus trials for 
the liking task with the paired stimuli trials for the wanting task.  The integrated software is also 
programmed to center the cursor between each trial to produce more consistent response times, 
and different question prompts will be presented in contrasting colors to encourage 
discrimination. 
Food stimuli presented in the procedure are selected based on two key dimensions associated 
with loss of appetite control and over-consumption: the fat/carbohydrate content and 
taste/sensory properties of foods.  Stimuli will be presented on a 17-in flat-screen monitor.  The 
food items are selected from a database of photographic stimuli and sorted according to their fat 
content and taste properties into one of four separate categories: high-fat savory (HFSA); low-fat 
savory (LFSA); high-fat sweet (HFSW); and low-fat sweet (LFSW).  Each category is 
represented by four different foods; hence a total of 16 different food stimuli will be presented in 
the procedure (see Table 3). 
The aim of the explicit task is to obtain introspective hedonic measures for the same stimuli 
used in the implicit wanting task.  Therefore, each food stimulus is assessed independently using 
visual analogue scales (VAS).  The explicit computer task trials consist of 16 food stimuli 
presented one at a time and rated according to a 100-mm VAS anchored at each end by the 
statements “not at all” and “extremely”.  Subjects will be prompted with the statements “How 
pleasant would it be to taste some of this food now?” and “How much do you want some of this 
food now?”.  In particular, the liking question is constructed to reflect the pleasure of the 
experience of tasting some of the given food to avoid eliciting a more general evaluation to do 
with properties inherent to the food itself (53). 
 
Table 3. Photographic food stimuli used in liking and wanting computer task (grouped by 
food category). 
HFSA 
LFSA 
HFSW 
LFSW 
Salted peanuts 
Savory biscuits 
Blueberry muffin 
Jelly (jello) 
Salted crisps 
Pilau rice 
Milk chocolate 
Popcorn 
Swiss cheese 
Boiled potatoes 
Jam doughnut 
Jelly sweets (candies) 
French fries 
Bread roll 
Cream cake 
Fruit salad 
 HFSA: high-fat savory; LFSA: low-fat savory; HFSW: high-fat sweet; LFSW: low-fat sweet  
 
The VAS will be presented on-screen beneath each food stimulus and subjects use the 
computer mouse to move a centered cursor along the line to indicate their response.  When a 
rating is made, the procedure automatically cycles to the next stimulus trial.  Responses on the 
software will be recorded online and mean ratings for each food category (HFSA, LFSA, etc.) 
are automatically computed. 
Implicit wanting is measured by a behavioral “forced choice” methodology.  In this task, a 
food stimulus from one of the four food categories is paired with one stimulus from the 

26 
remaining categories to form a series of 96 trials in which the subjects will be given the 
standardized instruction to select the food they “most want to eat now”.  In addition to recording 
the frequency of selections made in each category (with a possible range of 0–48) which may 
reveal a relative preference, reaction time (in milliseconds) of each choice is also measured.  By 
covertly recording reaction time, subjects will be unaware of implicit changes in their behavior 
on the task, while remaining free to determine the direction of their choices.  In this measure, the 
motivated behavioral response independent of the explicit awareness of its incentive value is the 
key variable.  Data from the forced-choice task—including frequency of choice for each food 
category and reaction time of choice—will be recorded online for later calculation of the means. 
 
Implicit Liking and Wanting Procedure using the ISCAN® ETL-300 Eye Tracker 
 
We will perform pilot testing of this commercially available device to measure eye gaze 
duration, direction, and pupil dilation during the assessment of liking and wanting of food 
procedure. The ISCAN® ETL-300 Eye Tracking device shown in the figure below provides 
detailed measurements about what people look at when they are presented with a visual stimulus 
such as a picture of food. We will use the eye tracker to measure various quantities like pupil 
dilation and gaze time when people are presented with various food images. These measurements 
will help us better understand the psychological factors underlying food choices. 
 
The ISCAN® ETL-300 Eye Tracker is a lightweight (< 150 g or 1/3 of a pound) head-
mounted device with an adjustable headband, thus allowing freedom of head movement (See 
Figure). This device can be used with contact lens or glasses. The subject’s eyes are viewed 
using a specialized miniature infrared video camera system embedded in the headgear. A second 
video imaging system captures the scene that the subject is viewing. A 25 foot tether will 
connect the head-mounted device to a computer for data storage and analysis. 
 
The measurements obtained from the device will be used in conjunction with food liking and 
wanting measures to provide an implicit measure of food wanting. The liking and wanting 
procedure lasts about 45 min.  The eye tracker device will only be worn during the final liking 
and wanting procedure of each in-patient visit for pilot testing. 
 
 
 
 
 
Figure - ISCAN® ETL-300 Eye Tracker 
 

27 
Delay Discounting Computer Procedure 
People generally prefer immediate rewards in comparison to delayed rewards, even when the 
delayed reward has a higher value. The degree to which delayed rewards are discounted in 
comparison to immediate rewards may provide an index of impulsive decision making. It has 
been hypothesized that obese individuals may have difficulty adhering to diets because the long-
term rewards of weight loss are strongly discounted in comparison to the immediate rewards of 
palatable food (55).   
We will implement a delay discounting computer procedure using an image of a food that 
scored highly on each individual’s liking scale. This food image will be presented along with a 
forced choice statement such as “Would you rather eat this food now or receive $10 tomorrow?” 
The subject must select either the “food” or “money” option. We will also measure how long it 
takes to make each selection. The same food image will always be used as the hypothetical 
immediate reward, but hypothetical monetary choices will vary in the amounts of: $1, $5, $10, 
$20, $50, $100 and the associated delay times will vary from: now, 4 hours, tomorrow, 1 week, 2 
weeks, and 1 month. Experiment generator software (E-prime v1.2) will be used to present the 
paired stimuli trials and will be programmed to center the cursor between each trial to produce 
more consistent response times. Stimuli will be presented on a 17-in flat-screen monitor. 
 
PET imaging 
PET scans will be performed on a dedicated head scanner, the High Resolution Research 
Tomograph (HRRT) with ~2.5 mm resolution.  Prior to the scan, a swimming cap with small 
light reflectors will be put on the subject’s head.  It is used to monitor the position of the head 
during the scan.  Information about head movement is used in the image reconstruction process 
to reduce the blurring effect of head movement on the PET images.  Scanning will be carried out 
for 3.5 hours, with short breaks to allow the subject to get up and to void.  A transmission scan 
will be obtained with a 137Cs rotating pin source before radiotracer injection and before the 
emission scan to correct for attenuation.  Scans will start immediately after intravenous injection 
of 5 mCi of [18F]fallypride using a Harvard® pump and will be carried out for a total of 3.5 
hours.  After completion of the scan, subjects will be asked to void to minimize radiation 
exposure to the bladder.  We will control the environmental stimulation to the subjects to ensure 
that it is standardized for all subjects (noise kept to a minimum and room dimly light). 
To estimate D2 receptor availability we will obtain the time-activity curves for tissue 
concentration in striatum (ST) and in cerebellum (CB) along with the time activity curves for 
[18F]fallypride, which we will use to calculate the distribution volume (DV).  The DV 
corresponds to the equilibrium measurement of the ratio of tissue concentration to plasma 
concentration and is estimated using a graphical analysis technique for reversible systems (56).  
The ratio of the DV in ST to CB, which corresponds to (Bmax/Kd) +1 and is insensitive to 
changes in cerebral blood flow, will be used as model parameter for determination of specific 
binding (56).  
 
Functional Neuroimaging 
All MRI studies will be performed at the High-Field MRI Laboratory on a 3.0T whole body 
instrument.  A 27 cm tube resonator will be used for radio frequency transmission and detection.  
Structural images will be obtained with a T1-weighted axial 3D modified driven equilibrium 
Fourier transform (57), and a T2-weighted coronal hyper-echo pulse sequences (58).  fMRI will 

28 
be performed using a coronal single-shot gradient-echo echo-planar imaging (EPI) sequence.  
Padding will be used to minimize motion.   
 
Neural bases of gustatory perception.  As part of a larger battery of fMRI scans examining the 
neural representation of food perception in obese and lean subjects, we will localize in each 
subject the brain regions underlying perception of gustatory stimuli.  The gustatory mapping task 
will involve three types of events: tastant delivery events, cue-catch events, and wash/swallow 
events.   
 
During tastant delivery events, the subject will see a word-cue for five seconds indicating the 
imminent delivery of either a sweet (pleasant) tastant, or a tasteless solution.  Next the cue word 
will be replaced with the word “TASTE” for 5 seconds, during which time the subject will 
receive 0.4 ml of either a sweet tastant, or a tasteless (neutral) solution.  The sweet tastant will be 
composed of apple juice sweetened with sucrose, and the tasteless solution will be composed of 
12.5 mM KCL + 1.24 mM NaHCO3.  The neutral solution closely matches the properties of 
human saliva, and thus serves as a good control stimulus because its delivery closely mimics all 
properties of the sweet solution, yet has no taste.  After delivery of the tastant, the subject will 
next see a fixation mark appear on the screen for between 2.5 and 12.5 seconds, after which the 
word “WASH” will appear on the screen for 2.5 seconds and the subject will receive .8ml of the 
tasteless control solution to wash away the preceding tastant.  Immediately after the wash period, 
the word “SWALLOW” will appear, at which time the subject will swallow the fluid in the 
mouth.  Tastant delivery events will occur eighteen times in each scanning run, 9 for the sweet 
tastant, and 9 times for the neutral tastant. 
 
During cue-catch events, a subject will see a word-cue for five seconds.  Importantly, these 
events only involve the presentation of the cue-words, and not the delivery of fluid.  Cue-catch 
events will be followed by a variable duration fixation period.  These free-standing “catch-trial” 
events, which will occur 12 times in each scanning run (6 for the sweet cue, 6 for the neutral 
cue), and will be modeled in the design matrix along with the cues in the tastant delivery events, 
thus allowing us mathematically deconvolve the response to the cues from the tastants. 
 
Finally, during wash/swallow events, a subject will see the word “WASH” appear on the screen 
for 2.5 seconds and the subject will receive .8ml of the tasteless control solution.  Immediately 
after the wash period, the word “SWALLOW” will appear, at which time the subject will 
swallow the tasteless solution.  Wash/swallow events will be followed by a variable duration 
fixation period.  Freestanding wash/swallow events will occur 9 times in each scanning run, and 
will be modeled in the design matrix along with the 18 wash/swallow events during the tastant 
delivery events, again allowing us to mathematically deconvolve the response associated with 
receiving a tastant from receiving a wash solution and swallowing. 
 
By comparing activations following administration of tastant and neutral control stimuli, we will 
be able to functionally localize in each subject the brain regions underlying gustatory perception.  
Additionally, by comparing patterns of activity in response to sweet and neutral cues 
independent of the delivery of tastants into the mouth, we will be able to map regions engaged 
during anticipation of rewarding tastes.  The brain regions identified in this study will be used as 
functional regions of interest (ROIs) in the fMRI studies of food perception performed with these 

29 
same subjects.  In addition, the dataset will allow us to examine whether there are group 
differences in the neural bases of gustatory perception for obese and lean subjects.  Finally, we 
will also be able to examine whether there are group differences between obese and lean subjects 
in their anticipatory responses for rewarding tastes. 
 
Neural bases of food stimulus perception.  To evaluate the neural bases of food stimulus 
perception, subjects will be presented with photographs of four different types of foods:  high-fat 
savory foods, low-fat savory foods, high-fat sweet foods, and low-fat sweet foods.  Presentation 
of the food stimuli will be blocked by food category.  Each block will contain 5 photographs of 
food stimuli from a given category, each presented for 2.5 seconds with a 500 ms interstimulus 
interval.  In addition to food picture blocks, subjects will also be shown blocks of non-food 
object photographs that will serve as an object perception control condition.  The participants’ 
task while viewing the stimuli will be simply to press a button on a hand-held response box 
anytime they see the same picture presented twice in a row.  Picture presentation blocks will be 
separated by 10-second ‘null-stimulus’ blocks in which only a small fixation mark will be 
presented on the screen.   These null periods will be included to allow the blood oxygenation-
related fMRI signal to return to baseline after each stimulus block.  By comparing the activity 
associated with viewing food- and non-food stimuli, we will localize in each subject the brain 
regions supporting food perception in general.  In addition, by comparing activity among the four 
food stimulus categories, we will localize ROIs supporting perception of each food type.  
Finally, by comparing activity between obese and lean individuals, this research design will also 
allow us to identify group differences in the perception of food stimuli in general, as well as 
among the four food stimulus categories 
 
Relationship between diet and activation in brain regions supporting food preference judgments.  
To investigate the effects of dietary changes on neural responses to food stimuli and food 
preference, obese subjects will undergo multiple fMRI scan sessions in which they will provide 
preference ratings in response to photographs of high-fat savory foods, low-fat savory foods, 
high-fat sweet foods, and low-fat sweet foods.  Lean subjects will also undergo this procedure, 
though only in a single scan.  Food stimuli will be presented in a fast event-related design, with 
individual stimuli appearing in a pseudo-random order.  Each stimulus will be presented for 5-
seconds during which subjects will provide one of two different ratings:  
 “if given the opportunity right now, how pleasant it would be to eat this food?” 
AND 
“if given the opportunity right now, how much self-control would it take to NOT eat this food?” 
by manipulating a handheld MR-compatible scroll wheel to select a location on a number line. 
To allow mathematical deconvolution of the responses to stimulus presentations, each picture 
presentation will be followed by the appearance of a simple ‘null-stimulus’ fixation mark for 
between 2 and 12 seconds.  Analyses will proceed in the following manner.  First, subjects’ 
ratings on the “liking” question will be used as covariates on the imaging data to identify brain 
regions supporting food preference judgments for each of the four classes of food stimuli.  
Second, activity during the liking task will be subtracted from the activity during the food 
impulse control question to identify regions involved in behavioral inhibition in response to food 
stimuli.  A corollary prediction is that these regions should be modulated by subjects’ responses 
on the food impulse control question.  To explore whether diet affects brain regions supporting 
food preference judgments and impulse control, we will compare activity measured in the first 

30 
scan session collected during the baseline diet phase, with activity measured in subsequent scan 
sessions when obese subjects are in either the dietary carbohydrate- or fat-reduction phase of the 
study.  In addition to addressing these two questions using whole-brain imaging analyses, we 
will also explore how diet affects activity during food-preference judgments and impuse control 
within the functionally-defined ROIs identified in the gustatory stimulation and food perception 
functional localizer experiments described above. 
 
Functional connectivity among brain regions supporting gustation and perception of food 
stimuli.  Resting-state functional connectivity analyses will be used to examine group differences 
in connectivity between gustatory and food perception areas, as well as possible changes in the 
connectivity among these regions as a function of diet.  During the initial baseline scan session, 
both obese and lean subjects will undergo an 8-minute “resting state” fMRI scan.  During this 
scan subjects will be asked to stare at a fixation mark located in the center of the display, and to 
do their best to clear their minds and not think about anything in particular.  In the subsequent 
scan sessions, obese subjects will again undergo this 8-minute resting state scan.  Seed voxels for 
the resting-state connectivity analyses will be selected using data from gustatory perception ROIs 
and food perception ROIs.  Group differences in functional connectivity will be explored by 
comparing connectivity profiles in the data collected during the baseline scan sessions for obese 
and non-obese individuals.  Diet-related changes in obese subjects’ functional connectivity 
profiles will be explored by comparing data collected during the first session (baseline diet) with 
the data collected in the subsequent scan sessions (low-fat or low-carbohydrate diets). 
 
Statistical Analysis 
We performed our power/sample size calculations based on predicted measurements 
described in our primary aims.  No direct preliminary data is available for our power/sample size 
calculations for this study. However, we have performed computer simulations of the proposed 
study design using a mathematical model of human macronutrient metabolism developed by our 
group (9) and the predicted changes and standard deviations are consistent with the literature as 
will be described.  
 
Sample size for Primary Aim #1.   
Figure 2 shows the simulated 24-
hour respiratory quotient (RQ) 
values for the two in-patient 
phases using a mathematical 
model of human macronutrient 
metabolism (9).  The computer 
simulations were obtained by 
generating a population of 100 
“virtual study subjects” (50 male 
and 50 female) with a range of 
initial body weights matching our 
inclusion criteria (e.g., body 
weight = 110 ± 20 kg).  The two 
in-patient phases of the protocol 
were 
simulated 
with 
an 
Figure 2. Simulated RQ Changes
0.78
0.80
0.82
0.84
0.86
0.88
0.90
0.92
0
1
2
3
4
5
6
Time on Diet (days)
24h RQ
Red-FAT
Red-CHO

31 
intervening 2 week washout period when the standard baseline diet was reinstated.  We also 
simulated realistic variations of physical activity, body water, as well as random errors in our 
assessment of the baseline diet such that the simulated average energy balance before the 
reduced calorie diets had a realistically wide uncertainty range of ± 340 kcal/d.  The simulated 
standard deviations of the 24-hour RQ values were 0.02 which are similar to those observed in 
other feeding studies (59, 60).  As shown in Figure 2, the 24-hour RQ was predicted to change 
from 0.87 ± 0.02 to 0.85 ± 0.02 after the first day of the reduced carbohydrate diet which is 
consistent with data where dietary carbohydrate was reduced but replaced isocalorically with 
dietary fat (59, 60).  
 
From these estimates, we calculated that detection of a significant decrease of 24-hour RQ 
on day 1 of the reduced carbohydrate diet requires sample size of N = 4 for one sided test at 
80% power and α = 0.05.  We expect an attrition rate of roughly 50%, therefore 
achievement of adequate statistical power for this outcome measurement will require a 
recruitment sample size of N = 8.  
 
Sample size for Primary Aims #2 and #3.   
Our power analysis is based on the primary analysis of the main hypothesis, that is, to assess 
the effects of diet on D2 receptor availability and on reactivity of brain limbic regions when 
exposed to food stimuli. We will compare the D2 receptor availability before and after diet using 
the paired samples t-test or its nonparametric counterpart.  In order to detect a meaningful 
difference of effect size 1 (the effect size is the ratio of the expected mean and the standard 
deviation of the paired differences), with 20 subjects measured, we will achieve a power of 98% 
via the paired samples t-test ( = 0.05, 2-sided).  The normality assumption will be examined via 
the Shapiro-Wilk test.  In the event of non-normality, the paired samples t-test will be replaced 
by the Wilcoxon signed-rank test, which will result in an estimated 5% loss in test power and 
thus we estimate a power of 93% for the Wilcoxon signed-rank test (61).  For fMRI scanning, we 
base our power analysis on the number of subjects needed to detect significant differences in 
fMRI activation between two randomized conditions in normal control subjects (62).  As a result 
of the preliminary data we know that: for control subjects the inter-individual variability of fMRI 
signals in cognitive activation tasks is be approximately 0.7%.  We aim to detect larger signal 
changes than 0.5%. 
 
Assuming a Type I error of 0.05 and 80% power, we need a minimum of 20 obese subjects 
and 20 control subjects.  We expect an attrition rate of roughly 50% with the obese 
subjects. Therefore, achievement of adequate statistical power for these outcome 
measurements will require a recruitment sample size of N = 40 obese subjects.  
 
 
 

32 
Sample size for Primary Aim #4.   
The goal for primary aim #4 is to measure the change of body fat mass and fat-free mass 
during the 12 week out-patient weight loss period and relate those measured body composition 
changes to the macronutrient imbalances measured during the in-patient diet perturbations.  
Using our computational model, we simulated a prescribed decrease of 750 kcal/d from the 
standard baseline diet.  The predicted changes in body composition are depicted in Figure 3. The 
overall body weight decreased by an average of 7.9 ± 4.6 kg with a fat mass loss of 4.2 ± 2.6 kg 
and a lean mass loss of 3.6 ± 3.7 kg.  These estimates are similar to data from previous studies 
reporting changes in body composition after 12 weeks of a prescribed 750 kcal/d reduction in 
dietary intake leading to a reduction of 
body weight by 9.5 ± 5 kg, a fat mass 
decrease of 6.6 ± 3 kg, and a fat-free 
mass decrease of 2.8 ± 2.5 kg (63).   
 
From these estimates, we calculated 
that 
detection 
of 
a 
significant 
difference in fat mass requires sample 
size of N = 4 for two sided test at 80% 
power and α = 0.05.  We expect an 
attrition 
rate 
of 
roughly 
50%, 
therefore achievement of adequate 
statistical power for this outcome 
measurement 
will 
require 
a 
recruitment sample size of N = 8.  
 
We have previously described a 
simple equation for using short-term 
measurements 
of 
 
macronutrient 
imbalance to predict the fraction of body 
weight loss resulting from loss of body 
fat (8).  The predicted fat fraction of weight loss is calculated as follows: 
 
(
)
(
)
(
)
24h Fat Imbalance
24h Fat Imbalance
24h Energy Imbalance -  24h Fat Imbalance
L
L
F
FM
BW




=

+
 
 
Figure 3. Simulated Body 
Composition Changes
-9
-8
-7
-6
-5
-4
-3
-2
-1
0
Mass (kg)
Fat Mass Change
Lean Mass Change

33 
where ρF = 9.4 kcal/g and 
ρL = 1.8 kcal/g are the 
energy densities of body 
fat 
and 
lean 
mass 
changes, 
respectively.  
Using 
the 
average 
imbalances 
measured 
during the last days of the 
two reduced calorie diet 
periods, 
this 
simple 
equation was used to 
predict the fat fraction of 
weight 
loss 
in 
our 
simulated 
weight 
loss 
intervention 
and 
the 
results 
are 
shown 
in 
Figure 
4. 
 
These 
simulations suggest that 
the predictions from the 
simple equation can relate 
short-term 
energy 
imbalances to long-term body composition changes.  We estimate that an N = 20 will be 
sufficient to detect significant correlations between the predicted fat fraction of weight loss and 
the body composition changes following the 12 week weight loss period.  With an expected 
attrition rate of 50%, we estimate that we will need to recruit 40 subjects to detect significant 
correlations between the predicted fat fraction of weight loss and the body composition changes.  
 
Safety Considerations 
Possible Risks and Hazards 
 
Research-related risks in this study include those associated with study procedures, namely 
blood drawing, indirect calorimetry, measurement of body composition by dual energy x-ray 
absorptiometry (DEXA) and air-displacement plethysmography (BodPod), measurement of total 
body water by bioelectrical impedance spectroscopy (BIA) and by administration of sodium 
bromide, administration of doubly labeled water (DLW) for measurement of average total energy 
expenditure, isotopically-labeled substrate infusions (optional), muscle and adipose tissue 
biopsies (optional), frequently samples intravenous glucose tolerance test (FSIVGTT) for 
measurement of insulin sensitivity, positron emission tomography (PET) imaging, magnetic 
resonance imaging (MRI) scanning sessions of food perception and food rating, Profile of Mood 
States (POMS) questionnaire, hunger and satiety assessment, assessment of liking and wanting 
of food, wearing physical activity monitors, handheld indirect calorimeters, as well as 24h stays 
in the metabolic chamber.  There are no study medications and we do not expect any diet 
intervention-related risks since the study will be under close follow-up with trained 
dietitians/nutrition experts throughout the study. 
 
Blood drawing.  The placement of intravenous needles may cause transient pain, and may also 
result in bruising, bleeding, and/or clotting at the site of needle insertion.  The application of 
Figure 4. Simulated Correlations of Short-term 
Imbalances with Body Composition Change
y = 0.9916x + 0.0269
R2 = 0.693
0
0.5
1
1.5
0
0.5
1
1.5
Predicted Fat Fraction
Simulated Fat Fraction

34 
direct pressure at the catheterization site will be used to help prevent these symptoms.  There is a 
possibility that a catheter placement would be unsuccessful or need to be removed.  If this should 
occur, another catheter would be placed.  It is possible that this may occur more than once during 
the subject’s participation in the protocol. The daily blood draw schedule for the obese subjects 
is as follows: 
 
BLOOD DRAWN – 1ST IN-PATIENT VISIT 
Day 
1 
Day 
2 
Day 
3 
Day 
4 
Day 
5 
Day 
6 
Day 
7 
Day 
8 
Day 
9 
Day 
10 
Day 
11 
Day 
12 
Day 
13 
Day 
14 
Day 
15 
Total 
0 
mL 
12 
mL 
10 
mL 
85 
mL 
60 
mL 
15 
mL 
4 
mL 
19 
mL 
57 
mL 
15 
mL 
8 
mL 
3 
mL 
3 
mL 
3 
mL 
3 
mL 
297 
mL 
 
BLOOD DRAWN – 2ND IN-PATIENT VISIT 
Day 
1 
Day 
2 
Day 
3 
Day 
4 
Day 
5 
Day 
6 
Day 
7 
Day 
8 
Day 
9 
Day 
10 
Day 
11 
Day 
12 
Day 
13 
Day 
13 
Day 
15 
Total 
0 
mL 
12 
mL 
0 
mL 
0 
mL 
5 
mL 
11 
mL 
4 
mL 
19 
mL 
57 
mL 
15 
mL 
8  
mL 
3  
mL 
3  
mL 
3  
mL 
3  
mL 
143 
mL 
 
 
Buccal mucosa sampling.  During the in-patient visit, an oral (buccal) mucosa sample will be 
obtained for DNA.  This is a non-invasive procedure and there are no known risks with 
performing this procedure. 
 
Indirect calorimetry.  The use of the ventilation hood may cause some minimal discomfort in 
claustrophobic subjects.  
 
DEXA.  The amount of radiation during the DEXA scan is less than one mRem to the whole 
body.  This amount is less than the radiation exposure received by anyone living in the United 
States in one day.  The use of the DEXA scan apparatus may cause some minimal discomfort in 
claustrophobic subjects and may cause some minimal back pain in a small minority of the 
individuals.  
 
Air-displacement plethysmography.  This procedure may cause discomfort in claustrophobic 
subjects.  
 
Bioelectrical impedance spectroscopy.  There are no risks involved with this measurement and 
subjects will not feel the electric current.  
 
Sodium bromide dilution.  At the dose administered, there are no known toxicities, although the 
salty taste may be unpleasant.   
 
Doubly labeled water.  The doubly labeled water procedure has no known risks.  
 
Intravenous glucose tolerance test.  Minor discomforts include arm tenderness and transient 
flushing and nausea resulting from the glucose infusion.  The major risk of this procedure is 
hypoglycemia due to the administration of insulin.  To allow for early detection of this potential, 

35 
though infrequent complication, study participants will be closely monitored throughout the test.  
Glucose levels will be assessed at the beside throughout the test.  Should chemical (plasma 
glucose < 60 mg/dL) or symptomatic hypoglycemia (hunger, sweating, palpitations, or mental 
status change) occur, the test will be stopped immediately and the study volunteer will be treated 
with oral glucose or with 50% glucose IV, if medically indicated.  
 
Profile of mood states questionnaire. There is no known risk associated with the POMS 
questionnaire, however, there is the potential of discovering clinically relevant information 
requiring further follow-up.  If this is the case, subjects will be notified by a qualified member of 
the research team and appropriate follow-up with the primary care physician will be planned.  
 
Structured clinical interview for DSM-IV. There is no known risk associated with the SCID 
interview, however, there is the potential of discovering clinically relevant information requiring 
further follow-up.  If this is the case, subjects will be notified by a qualified member of the 
research team and appropriate follow-up with the primary care physician will be planned.  
 
Three-factor eating questionnaire.  There is no known risk associated with the TFEQ. 
 
MacArthur Socioeconomic Status (SES) Questionnaire.  There are no known risks associated with the 
MacArthur SES questionnaire although some subjects may find completing the task tedious or some 
may find the questions probing and too personal in nature to comfortably answer.  Subjects will be 
informed that they do not have to respond to all the questions if they have reservations about sharing 
such personal information. 
 
Hunger and satiety assessment. There are no known risks associated with hunger and satiety 
assessment 
 
Taste intensity assessment.  There is no known risk associated with assessment of taste intensity, 
however an unpleasant sensation from tasting the stimuli may result in some individuals, 
specifically in response to propylthiouracil (PROP).  
 
Delay discounting computer procedure.  There is no known risk associated with the assessment 
of delay discounting by computer.  
 
Assessment of liking and wanting of food.  There is no known risk associated with assessment of 
liking and wanting of food. 
 
Assessment of implicit liking and wanting of food using the ISCAN® ETL-300 Eye Tracker.  
There are no known risks associated with assessment of liking and wanting of food or the eye 
tracking device. It may be temporarily inconvenient to wear the head-mounted eye tracking 
device. The device will not be used to diagnose, treat or prevent disease during the procedure and 
does not significantly or permanently affect any function or structure of the body. The device can 
be used with contact lens or glasses. 
 

36 
Metabolic chamber.  Besides inconveniences that can reasonably be expected as a result of 
spending an extensive time (24h) in the live-in room calorimeter, the serious risk to subjects’ 
health is minimum.  
 
Physical activity monitors.  There are no risks associated with the monitors, but subjects may 
find them to occasionally be inconvenient.  
 
MedGem handheld indirect calorimeters.  The test is non-invasive, but subjects may find the 
mouth piece and nose clip to be slightly uncomfortable. 
 
Tracer infusion (optional).  The tracer-labeled solutions infused are not commercially prepared 
and will be prepared by the pharmacy using proper compounding procedures.  Although 
unlikely, the infusions may cause discomfort at the catheter site, fever, and/or infection. 
 
Subcutaneous fat and skeletal muscle biopsy (optional).  The muscle biopsy may cause transient 
pain, and may also result in bruising, bleeding, clotting, infection, and/or scaring at the site of 
needle insertion.  The application of direct pressure at the biopsy site will be used to help prevent 
these symptoms.  This procedure may also result in dizziness or fainting.  To reduce the chance 
of feeling faint or dizzy, this procedure will be performed while the subject is in bed.  The 
procedure will be performed under sterile technique to minimize the chances of infection.  Pain 
will be reduced during the muscle biopsy by using a local anesthetic (lidocaine).  Injection of 
lidocaine itself can result in brief discomfort (a “stinging” sensation).  A very low dose of 
lidocaine will be used, as doses several times greater that what will be administered in this 
protocol can affect the central nervous system and the cardiovascular system.  Post-biopsy, the 
needle site will be cleaned and covered with gauze and translucent dressing tape.  Study 
participants will be instructed to report to the clinical staff any changes at the biopsy site 
including bleeding, secretion, erythema, pain, and signs and symptoms of infection.  Study 
participants will be instructed to self-monitor the biopsy site after discharge from the Clinical 
Center.  
 
Positron emission tomography.  This research study involves exposure to radiation from the 
radioactive tracer [18F]fallypride and the transmission scans.  The total amount of radiation obese 
subjects will receive in this study is from four injection of 5 millicuries of [18F]fallypride (control 
subjects will receive just one injection).  The NIH Radiation Safety Committee has reviewed the 
use of radiation in this research study and has approved this use as necessary to obtain the 
research information desired.   
Using the standard way of describing radiation dose, from participating in this study, obese 
subjects will receive a total of 8.80 rem to their gallbladder wall, 6.60 rem to their urinary 
bladder wall, and 5.20 rem to their liver.  All other organs will receive smaller amounts of 
radiation.  Control subjects will receive a fourth of this radiation to their respective organs. 
Although each organ will receive a different dose, the amount of radiation exposure obese 
subjects will receive from these procedures is equal to a uniform whole-body exposure of 1.56 
rem (0.39 rem for control subjects).  The amount of radiation received in this study is within the 
dose guideline established by the NIH Radiation Safety Committee for research subjects.  The 
guideline is an effective dose of 5 rem (or 5,000 mrem) received per year.   

37 
One possible effect that could occur at these doses is a slight increase in the risk of cancer.   
The natural chance of a person getting a fatal cancer during his/her lifetime is about 1 out of 4 
(or 25 percent).  The increase in the chance of getting a fatal cancer, as a result of the radiation 
exposure received from this research study, is 0.06 percent for obese subjects (0.02 percent for 
control subjects).  Therefore, the total risk of fatal cancer may be estimated to increase from 25 
percent to 25.06 percent in obese subjects (25.02 for control subject).  This change in risk is 
small and cannot be measured directly.  Compared with other everyday risks, such as flying in an 
airplane or driving a car, this increase is considered slight.   
 
If subjects are pregnant or breast feeding, they may not participate in this research study.  It 
is best to avoid radiation exposure to unborn or nursing children since they are more sensitive to 
radiation than adults.  
 
Another unlikely risk associated with this procedure is intravenous infiltration and radioactive 
ligand extravasation into the tissues in which case standard care will include warm compresses to 
the area of infiltration, administration of intravenous fluids, and use of a survey meter to take 
recordings over the infiltration site. The appearance of radioactivity increase in the brain will be 
closely monitored to enable early detection of intravenous infiltration in which case the scan will 
be terminated. In order to minimize risks of intravenous line infiltration, attempts may be made 
to use wrist or hand rather than antecubital veins for intravenous placement. 
 
Magnetic resonance imaging.  People are at risk for injury from the MRI magnet if they have 
pacemakers or other implanted electrical devices, brain stimulators, some types of dental 
implants, aneurysm clips (metal clips on the wall of a large artery), metallic prostheses 
(including metal pins and rods, heart valves, and cochlear implants), permanent eyeliner, 
implanted delivery pump, or shrapnel fragments.  Welders and metal workers are also at risk for 
injury because of possible small metal fragments in the eye of which they may be unaware.  
Subjects will be screened for these conditions before having the scan, and if they have any, they 
will not receive an MRI scan.  In addition, all magnetic objects (for example, watches, coins, 
jewelry, and credit cards) will be removed before entering the MRI scan room.  
 
It is not known if MRI is completely safe for a developing fetus.  Therefore, all women of 
childbearing potential will have a pregnancy test performed on the morning of the scanning visit 
before any scans are performed.  The scan will not be done if the pregnancy test is positive.  
People with fear of confined spaces may become anxious during an MRI.  Those with back 
problems may have back pain or discomfort from lying in the scanner.  The noise from the 
scanner is loud enough to damage hearing.  Subjects should not have a research MRI scan if they 
know that they have hearing loss.  Everyone having a research MRI scan will be fitted with 
hearing protection.  If it is difficult for subjects to tolerate the confinement or the noise within the 
scanner they will be taken out immediately if they so request.  There are no known long-term 
risks of MRI scans. 
 
DNA and tissues containing genetic material. Some of the blood drawn for this study and some 
of the adipose and muscle tissue collected at the biopsy will be used to analyze genes that may be 
important in obesity and its related traits.  Family studies are not conducted under this protocol.  
The results of the testing are likely not to have direct clinical relevance and thus, we will not 

38 
provide participants with the results of such testing.  By agreeing to participate in this study, 
participants do not waive any rights regarding access to and disclosure of medical records.  If our 
testing discovers information important to the health of the participant or the health of the 
participant’s offspring, we will share it with the participant and recommend appropriate follow-
up which may include genetic counseling.  Possible risks of knowing such results include: 
anxiety or other psychological distress; and the possibility of insurance and job discrimination.  
A possible risk of not knowing includes being unaware of the need for treatment.  These risks 
can change depending on the results of the research and whether there is a treatment or cure for a 
particular disease.  Sometimes patients have been required to furnish information from genetic 
testing for health insurance, life insurance, and/or a job. A Federal law, called the Genetic 
Information Nondiscrimination Act (GINA), generally makes it illegal for health insurance 
companies, group health plans, and most employers to discriminate against employees based on 
genetic information. This law generally will protect participants in the following ways:  
• 
Health insurance companies and group health plans may not request genetic information 
that we get from this research. 
• 
Health insurance companies and group health plans may not use genetic information 
when making decisions regarding patient eligibility or premiums.  
• 
Employers with 15 or more employees may not use genetic information that we get from 
this research when making a decision to hire, promote, or fire employees or when setting 
the terms of employment. 
All health insurance companies and group health plans must follow this law by May 21, 2010. 
All employers with 15 or more employees must follow this law as of November 21, 2009.  This 
Federal law does not protect against genetic discrimination by companies that sell life insurance, 
disability insurance, or long-term care insurance.  Donation of tissues (blood, fat, and muscle) for 
these research purposes will not be included the participant’s medical records. 
   
Risks Related to Clinical Relevance of Test Results 
If any lab tests, questionnaires, PET or MRI scans, or any other measurements made during the 
screening or procedures of this protocol show clinically significant abnormalities that may 
impact on the health and well-being of the subjects, they will be notified by a qualified member 
of the research team and appropriate follow-up with their primary care physician will be planned. 
 
Safety and Event Reporting and Data Monitoring 
 
Adverse events, protocol deviations, unanticipated problems (UP), serious adverse events, 
sponsor and serious, are defined in NIH HRPP SOP 16 (“Reporting Requirements for 
Unanticipated Problems, Adverse Events and Protocol Deviations.”). All adverse events 
occurring during the study either observed by the physicians, nurses, or dietitians or reported by 
the subject will be recorded.  
 

39 
Serious unanticipated problems and serious protocol deviations will be reported to the IRB and 
CD (Clinical Director) as soon as possible, but not more than 7 days after the PI first learns of 
the event. Unanticipated problems that are not serious will be reported to the IRB and CD as 
soon as possible, but not more than 14 days after the PI first learns of the event. Protocol 
deviations that are not serious will be reported to the IRB as soon as possible, but not more than 
14 days after the PI first learns of the event. 
 
SAE’s and AE’s that are at least possibly related to the research interventions and are not UPs 
will be reported only as aggregated summaries at Continuing Review (CR). If the rate and/or 
severity of these events exceeds those of comparable events previously observed in this or 
similar studies, the events will be classified and reported as though they are Unanticipated 
Problems.  
 
All deaths will be reported to the Clinical Director and the IRB within 7 days after the PI first 
learns of the event.  
 
Data and Safety Monitoring Plan 
 
No DSMB will be instituted to record adverse events given the modest level of risk involved 
in the research and the involvement of a multidisciplinary team in the study.  Adverse events will 
be recorded and monitored by the Principal Investigator and Medical Advisory Investigator: Dr. 
Kevin Hall and Dr. Ranganath Muniyappa, respectively.  
 
Human Subject Consideration 
Recruitment Strategies 
 
Subjects will be recruited from the community through advertisements in clinicaltrials.gov 
and possibly, local flyers, newspapers, or magazines.  We will utilize the Patient Recruitment 
and Public Liaison Office in the Clinical Research Center in establishing and implementing a 
recruitment strategy.  
The age range for inclusion in this protocol is 18-45 years.  The rational for excluding 
subjects older than 45 years is multi-fold.  First, obese and lean subjects will not be matched for 
age; therefore, in order to diminish the chance of a type 1 error in our results we chose to limit 
the age range to provide us with a more homogenous subject population.  Second, a number of 
the major metabolic outcome variables of this protocol have been shown to change significantly 
with aging (e.g., metabolic rate and body composition).  Third, much of the current information 
on our primary outcome measurements was gathered from subjects within the age range 
specified by this protocol; therefore, we believe that this age range is appropriate as it allows us 
to put our results into direct context with the current knowledge base. Finally, a more 
homogeneous population of obese subjects with respect to age will allow us to randomize the 
first in-patient diet intervention to match for gender and BMI without large variations of age. 
This is important because we anticipate a relatively high attrition rate following the first in-
patient stay and the crossover study design may therefore be compromised. In that case, our 
backup plan is to compare the diet interventions across subjects and such a comparison is 
facilitated when the variance of age is not too great.  
 
Recruitment of Women, Children, and Minority Individuals 

40 
 
We will actively encourage the participation of women and minorities.  Considering the 
prevalence of obesity in the general population, we expect that women will represent 
approximately half of the study population.  Moreover, given the increased incidence and 
morbidity of obesity among minorities and the social demographics of the greater Washington, 
DC area, we anticipate that African-Americans and Hispanics will constitute at least 20% of 
enrollees.  
 
On the other hand, we have deliberately chosen not to study children and adolescents at this 
time pending the outcome of the findings of this protocol.  At present, there is not information 
suggesting that the metabolic responses to selective reductions of dietary macronutrients are 
likely different in adults and children and so we see no compelling reason to expose children to 
the rigors and inconvenience of this type of study.  We fully recognize, however, that the insights 
derived from the data analysis in this study may very well direct our attention to other potentially 
fruitful areas of clinical research involving childhood obesity, given the early onset and alarming 
risks associated with obesity and other major public health issues in this population. 
 
Subject Withdrawal 
 
Participants may voluntarily withdraw from this study at any given time.  Also, there are 
several conditions that require the Principal/Co-Investigator to drop a study volunteer from this 
protocol, but not limited to the following: 
 Development of any new medical condition or start of medications that would have 
prevented enrollment in this study as it pertains to the exclusion criteria.  
 Inability or unwillingness to comply with study requirements 
 The Principal/Co-Investigator, Dr. Kevin Hall or Dr. Nick Knuth, deems it unsafe to 
remain in the study 
 Pregnancy 
 The study is terminated 
 
Research Use, Storage, and Disposition of Human Subjects’ Samples and Data 
As with all clinical data, the findings will be kept confidential. Volunteer clinical data will be 
protected and tracked using standard operating procedures in the medical record department.  All 
research charts and records will be kept in a secure place in a locked file cabinet in the office of 
the Principal Investigator.  All research samples and data will be identified by a study code 
linked to the subject’s name and the code and the results of all analyses will be kept strictly 
confidential.  All research samples (blood, fluids, tissue, DNA, mRNA) will be coded for storage 
in refrigerators and freezers in a locked laboratory.  The plan is to store the samples until they are 
analyzed and a sample of serum, tissue, and genetic material will be safeguarded for future 
analysis of other factors related to obesity and its phenotype.  These samples will be stored 
indefinitely.  The IRB will be notified in the event these samples are accidentally destroyed, lost 
or are anonymized.  Studies on these samples will always be related to the scope of this proposal.   
Data from the assessment of liking and wanting of food will be sent to Graham Finlayson, 
PhD, a collaborator on this study, at the University of Leeds.  These data will be coded and will 
include the measured rating, selection, and reaction time to visual food stimuli.  No identifying 
information will be sent.  The Investigators will retain the link to all coded samples.  It is 
necessary to retain the link to the volunteer’s identity to correlate study data with the individual’s 

41 
clinical information for this collaboration and all other collaborations where data will be shared 
in a coded manner.   
Dr. Edward Sazonov from the University of Alabama will be a collaborator on this study but 
will not have any direct contact with the study subjects.  Data files from the automated ingestion 
monitor will be coded by subject ID and the coded data will be sent to Dr. Sazonov for further 
processing to identify periods of food intake. The data will remain in a protected storage in Dr. 
Sazonov's lab until the study is complete. The data collected from the device will include the 
following: 
 
A. Data stream from the chewing sensor, which is a single channel sampled at 1000Hz.  
B. Data stream from the hand gesture sensor, single channel sampled at 10Hz.  
C. Data stream from the motion sensor, three channels sampled at 100Hz 
D. Data stream from self report buttons, two channels samples at 10Hz 
 
All of these sensor streams carry no personal data that could be used to identify subjects. The 
smartphone portion of the device serves as the data logger storing sensor information transmitted 
in real time via Bluetooth on its SD card. The wireless transmission is protected by the 
encryption mechanism of Bluetooth standard. The data on the smartphone is deprived of any 
personal information on the subject. The data is stored in a binary format which is impossible to 
decipher without knowledge of its internal structure. The subjects will not have access to the data 
stored on the cell phone as initiation, completion of data collection and downloading on the data 
will performed by the investigators with phone being password locked at other time. The data 
from the phone will be retrieved after every 24-hr period.   
Dr. Eric Muth from Clemson University will be a collaborator on the study but will not have 
any direct contact with the study subjects.  Data files from the bite counter will be coded by 
subject ID and the coded data will be sent to Dr. Muth for further processing to quantify food 
intake.   
Dr. Carla Prado from Florida State will be a collaborator on the study, but will not have any 
further direct contact with the study subjects.  Data files from diet, hunger and satiety will be 
coded by subject ID and the coded data will be sent to Dr. Prado for further processing.    
Dr. Siervo from Newcastle University will be a collaborator on the study, but will not have 
any further direct contact with the study subjects.  Data files from the indirect calorimetry, 
urinary nitrogen, diet, and body composition data will be coded by subject ID and the coded data 
will be sent to Dr. Siervo for further processing. 
 
Some of the blood drawn for this study and some of the muscle and fat tissue collected at the 
biopsy (for obese subjects only) will be used to analyze certain genes that are important in the 
development of obesity.  We will be looking for changes in the expression of different genes 
comparing obese subjects with control subjects and studying the obese subjects before and after 
weight loss.  We will also search for changes (mutations) in genes directly associated with 

42 
obesity, but not test for mutations that cause known genetic diseases or conduct a study on 
family history of health or illness.   
Some clinically relevant research data will be stored indefinitely in the medical record and 
will be accessible to the subject for review by others of their choosing (doctors, insurance 
companies etc.) after executing a release of information.  This and other data will be maintained 
in databases in the Clinical Endocrine Section of the CEB, password protected and secure.  
 
Collaborations on Stored Tissue and Blood Samples  
 
Collaborations requiring transfer of stored tissue and blood samples will be done in a coded 
fashion after informing the IRB and obtaining necessary assurances from the outside institution.  
The protocol will be amended and IRB approval will be sought when such collaborations are 
established.     
 
Informed Consent 
The investigational nature of the study and its risks will be carefully explained to the subject 
and a signed consent will be obtained prior to performing any protocol described testing.  The 
informed consent process will be documented on a progress note and a copy of the note and the 
original informed consent will be filed in the subject’s record.   
As we have amended our protocol to include the MacArthur Socioeconomic Status (SES) 
Questionnaire recently, we are lacking data for those subjects recruited prior to amendment 
approval. These patients will be contacted by the principal investigator or one of the study co-
investigators. The investigational nature of the questionnaire and its attendant risks will be 
carefully explained to the subject and a signed consent will be obtained prior to collecting the 
data. For those subjects who are unable or unwilling to travel to the NIH the consent will be 
executed by telephone. The subject will receive a copy of the protocol consent in the mail prior 
to being consented. After they have had an opportunity to review the consent the investigator 
will contact the subject by telephone. The investigator will review the investigational nature of 
the protocol with the subject and answer questions in the presence of a witness. If the subject 
chooses to participate, the subject and the witness will sign and date the consent. The informed 
consent document will be mailed to the principal or associate investigator who led the 
discussion, who will sign and date and mail back a fully executed copy for the subject’s records.  
The informed consent process will be documented on a progress note and a copy of the note and 
the original informed consent will be filed in the subject’s record. We will ask the subject to also 
mail a copy of the completed SES questionnaire to the study team. 
 
We have also amended our protocol to include buccal cell DNA collection. We are lacking 
data for those subjects recruited prior to amendment approval. These patients will be contacted 
by the principal investigator or one of the study co-investigators. The investigational nature of 
this DNA sample and its attendant risks will be carefully explained to the subject and a signed 
consent will be obtained prior to collecting the data. For those subjects who are unable or 
unwilling to travel to the NIH the consent will be executed by telephone. The subject will receive 
a copy of this protocol telephone consent in the mail prior to being consented. After they have 
had an opportunity to review the consent the investigator will contact the subject by telephone. 
The investigator will review the investigational nature of the protocol with the subject and 
answer questions in the presence of a witness. If the subject chooses to participate, the subject 
and the witness will sign and date the consent. The informed consent document will be mailed to 
the principal or associate investigator who led the discussion, who will sign, date and mail back a 

43 
fully executed copy for the subject’s records.  The informed consent process will be documented 
on a progress note and a copy of the note and the original informed consent will be filed in the 
subject’s record. We will ask the subject to also mail the DNA sample to the study team. 
 
Benefits to Study Participants 
 
This study will enroll both lean (control) and obese individuals.  All volunteers will receive 
a full history and physical and information about their health prior to beginning their individual 
study.  For the obese subjects, this study has a goal of a achieving a 5% weight loss.  As noted in 
the introduction, as weight loss is difficult, this study will offer a supervised outpatient program 
designed to achieve weight loss.  This may lead to favorable future metabolic outcomes in 
individuals who are successful in losing and maintaining weight loss (such as decreased risk of 
diabetes or hypertension).  There will be no other direct benefits from participation in this study 
aside from the knowledge that they are contributing to advancing our understanding of the brain 
regions involved with food intake and our understanding of obesity, and that these insights may 
lead to new treatment options in the future.  Abnormal values will be discussed with the study 
volunteers and forwarded to their primary care physicians.   
 
Remuneration 
Subjects will receive payments for time and effort connected with the outpatient visits, 
hospital stays, and procedures according to the following schedule: 
 
Control Subjects 
EVENT 
COMPENSATION OCCURANCE 
TOTAL 
out-patient food record 
$20 
1 
$20 
in-patient per diem  
$40 
2 
$80 
body composition (DEXA, Bod-Pod, 
BIA) (2 units) 
$50 
1 
$50 
Hunger and satiety assessments 
$25 
1 
$25 
POMS questionnaires (1 unit) 
$25 
1 
$25 
SES and TFE questionnaires (1 unit) 
$25 
1 
$25 
taste sensitivity assessment (1 unit) 
$25 
1 
$25 
delay discounting procedure (1 unit) 
$25 
1 
$25 
food preference assessment (1 unit) 
$25 
1 
$25 
PET (4 units) 
$100 
1 
$100 
fMRI (gustatory and food perception 
scans) (4 units) 
$100 
1 
$100 
fMRI (resting-state and food rating 
scans) (4 units) 
$100 
1 
$100 
 
 
TOTAL: 
$600 
 
 
 

44 
Obese Subjects, Main Study 
EVENT 
COMPENSATION OCCURANCE 
TOTAL 
out-patient food record 
$20 
1 
$20 
in-patient per diem 
$40 
32 
$1280 
body composition (DEXA, Bod-Pod, 
BIA) (2 units) 
$50 
10 
$500 
sodium bromide dose 
$25 
2 
$50 
doubly labeled water (DLW) (1 unit) 
$25 
4 
$100 
24-h blood sampling (2 units) 
$50 
3 
$150 
tracer labeld substrate infusions (2 units) 
(optional) 
$50 
4 
$200 
muscle & fat biopsy (4 units) (optional) 
$100 
4 
$400 
indirect calorimetry (1unit)  
$25 
7 
$175 
IVGTT (1 unit) 
$25 
1 
$25 
PET (4 units) 
$100 
4 
$400 
fMRI (gustatory and food perception 
scans) (4 units) 
$100 
1 
$100 
 
fMRI (resting-state and food rating 
scans) (4 units) 
$100 
4 
$400 
 
24-hour respiratory chamber (4 units) 
$100 
12 
$1200 
physical activity monitors (1 unit) 
$25 
5 
$125 
bite counter (1 unit) 
$25 
3 
$75 
automatic ingestion monitor (1 unit) 
$25 
3 
$75 
hunger questionnaires (1 unit) 
$25 
17 
$425 
POMS questionnaire (1 unit) 
$25 
4 
$100 
SES & TFE questionnaires (1 unit) 
$25 
1 
$25 
taste sensitivity assessment (1 unit) 
$25 
1 
$25 
delay discounting procedure (1 unit) 
$25 
4 
$100 
food preference assessment (1 unit) 
$25 
4 
$100 
Attendance at bi-weekly counseling 
$20 
5 
$100 
At-home weights & indirect calorimetry 
$10 
28 
$280 
out-patient meal days  
$10  
4 
$40 
bonus for completing study 
$200 
1 
$200 
 
 
TOTAL REMUNERATION: 
$6,670 
 
 
 

45 
Obese Subjects, Each Yearly Follow-up  
EVENT 
COMPENSATION 
OCCURANCE 
TOTAL 
out-patient food record 
$20 
1 
$20 
in-patient per diem 
$40 
2 
$80 
body composition (DEXA, Bod-Pod, 
BIA) (2 units) 
$50 
2 
$100 
sodium bromide dose 
$25 
1 
$25 
doubly labeled water (DLW) (1 unit) 
$25 
1 
$25 
tracer labeld substrate infusions (2 units) 
(optional) 
$50 
1 
$50 
muscle & fat biopsy (4 units) (optional) 
$100 
1 
$100 
indirect calorimetry (1unit)  
$25 
1 
$25 
IVGTT (1 unit) 
$25 
1 
$25 
PET (4 units) 
$100 
1 
$100 
fMRI (resting-state and food rating 
scans) (4 units) 
$100 
1 
$100 
 
24-hour respiratory chamber (4 units) 
$100 
1 
$100 
physical activity monitors (1 unit) 
$25 
1 
$25 
bite counter (1 unit) 
$25 
1 
$25 
automatic ingestion monitor (1 unit) 
$25 
1 
$25 
hunger questionnaires (1 unit) 
$25 
1 
$25 
POMS questionnaire (1 unit) 
$25 
1 
$25 
SES & TFE questionnaires (1 unit) 
$25 
1 
$25 
delay discounting procedure (1 unit) 
$25 
1 
$25 
food preference assessment (1 unit) 
$25 
1 
$25 
At-home weight 
$10 
1 
$10 
 
 
REMUNERATION: 
$960 

46 
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50 
Appendix 
 
Detailed Study Design and Methods 
 
Screening Visit 
At the initial screening visit, study volunteers will be asked questions about their general 
health status and undergo a routine blood test to measure basic metabolic functions in an effort to 
address the various inclusion/exclusion criteria.  All subjects who are interested in participating 
in the full study protocol will have the following procedures and test performed after an 
overnight fast and after signing the standard informed consent document: 
 
 History and physical examination 
 Fasting blood tests [complete blood count (CBC), lipid profile, glucose, insulin] 
 Body weight and body mass index (BMI) determination 
 Resting electrocardiogram (EKG) 
 Resting metabolic rate (measured by indirect calorimetry) 
 15 minute treadmill walking at self-selected speed  – (obese subjects only) 
 Psychiatric evaluation by Structured Clinical Interview for DSM-IV (SCID) 
 Visit to the MRI mock scanner 
 Pregnancy test (women only) 
 
Volunteers will be asked to continuously wear physical activity monitors and keep food diary 
and activity records for the subsequent 3 days. All subjects will be given directions to record 
their dietary intake for three days.  The physical activity monitors and food records will be 
returned and reviewed after 3 days to ensure adequate macronutrient and energy intake in the 
habitual diet, as well as to gage the average daily caloric requirements for use during the baseline 
period.  Food records will be analyzed using Nutrition Data Systems for Research (NDS-R) 
software, developed by the Nutrition Coordinating Center, University of Minnesota, 
Minneapolis, MN.  If qualified for entry into the study on the basis of the determinations listed 
above, discussions will take place as to the scheduling of the beginning of active participation in 
the study (i.e. beginning of the baseline period for obese subjects, or scanning day for control 
subjects). 
 
Control Subjects 
If the results from the screening visit permit their continued participation in this research 
study control subjects will be invited to the Clinical Research Center for a second visit.  During 
the 2 days before their second visit, control subjects will receive a standard diet with a relative 
macronutrient content of 50% carbohydrate, 35% fat, and 15% protein on an out-patient basis.  
Subjects will be required to consume one meal per day of the standard diet at the hospital, and 
will take away with them the remainder of the day’s meals.  The daily caloric content of the diet 
during the out-patient segment will be based on the 3-day dietary record provided during 
screening.  On the morning after the second day of the out-patient diet, subjects will arrive at the 
MCRU at 0800 h and be admitted to the MCRU.  At 0815h, body weight and body composition 
(by DEXA and air-displacement plethysmography) will be measured, as well as measurement of 
total body water content by bioelectrical impedance.  At 1100 h subjects will undergo assessment 
of taste sensitivity (including PROP) and complete the three-factor eating, profile of mood state, 

51 
and MacArthur socioeconomic questionnaires.  At 1345 h, a blood sample will be taken and 
hunger will be assessed by VAS, and then at 1400 h, subjects will undergo an fMRI scanning 
session comprised of gustatory functional localizer scans, food perception functional localizer 
scans, and an anatomical scan (see Analytical Procedures).  Finally, at 1700 h hunger will again 
be assessed by VAS before subjects undergo assessment of liking and wanting of food by 
computer presentation of four separate food categories: high-fat savory (HFSA), low-fat savory 
(LFSA), high-fat sweet (HFSW), and low-fat sweet (LFSW).  Subjects will be fed the 
standardized diet at 0900h, 1200 h, and 1900 h during their first in-patient day. 
The following morning, a blood sample will be taken at 0715 h.  Between 0930 and 1130h 
(dependent on available scan time), subjects will undergo a positron emission tomography (PET) 
scan.  Briefly, after placement of an intravenous catheter and positioning, subjects will be given 
an intravenous injection of ~5 mCi of [18F]fallypride.  For the next 3.5 hours, scanning will be 
carried out in sets with periodic breaks to allow the subjects to get up and to void.  At 5.5h after 
the scheduled PET scan subjects will undergo a delay discounting computer procedure where 
they will be required to make decisions between hypothetical immediate food rewards in 
comparison to various delayed monetary rewards.  Finally, at 1745 h  subjects will have a final 
blood draw followed by, at 1800 h, a second fMRI scanning session, comprised of the required 
clinical scans, a resting-state scan, food rating scans, and a second anatomical scan (see 
Analytical Procedures).  Hunger will be assessed by VAS before the PET scan, before the 
delayed discounting procedure, and before the final fMRI scan.  Subjects will be fed the 
standardized diet during their second in-patient day at 1200 h, 1500 h, and 2000 h.  The subjects 
will then be discharged following completion of their evening meal.  
 
Obese Subjects 
First In-Patient Visit 
Baseline dietary procedures 
If the results from the screening visit permit their continued participation in this research 
study obese subjects will be invited to the Clinical Research Center for their first in-patient visit, 
which will last 14 days.  During the 2 days before admission to the MCRU, subjects will receive 
a standard diet with a relative macronutrient content of 50% carbohydrate, 35% fat, and 15% 
protein on an out-patient basis.  Subjects will be required to consume one meal per day of the 
standard diet at the hospital, and will take away with them the remainder of the day’s meals.  The 
daily caloric content of the diet during the out-patient segment will be based on the 3-day dietary 
record provided during screening.  After these 2 days, subjects will be admitted to the hospital 
and will continue to consume the standard diet for an additional 5 days.  The initial daily caloric 
content of the standard diet will be equal to 1.5 x resting metabolic rate (RMR), which will be 
measured during their initial screening visit.  This caloric content has been found to approximate 
the total energy daily energy expenditure when sedentary (64).  Body weight will be monitored 
daily and the amount of food given to the subjects will be adjusted to maintain a stable body 
weight. 
 
Specific procedures during baseline period 
A schematic timeline of events during the first in-patient visit is depicted in Figure A1.  The 
first 5 days of this visit will serve as the baseline period, and during this baseline period, subjects 
will consume a standard diet supplied by the clinical center metabolic kitchen (as described 
above).  Beginning two days before admission, all food to be consumed by the subjects will also 

52 
be supplied by the clinical center metabolic kitchen.  Subjects will arrive each morning at the 
clinical center at 0800 h for measurement of body weight, after which they will eat breakfast, and 
then take the remainder of the day’s meal with them to consume later during the day. Subjects 
will be instructed to consume only and all the food provided to them and to return those portions 
that they were unable to eat the following day when they return for the next day’s meals.   
 
 
On the morning following the second day of the out-patient standard diet, subjects will arrive 
in the fasted state at the MCRU at 0800 h for admission to the MCRU (Day 1, Figure A1).  
Following admission, subjects will be fitted with accelerometers, a bite counter and an automatic 
ingestion monitor for continuous measurement of activity, which will be worn throughout their 
entire in-patient visit.  At 0845 h a baseline urine sample will be collected (24h urine specimens 
will be collected throughout the duration of the in-patient period for measurement of nitrogen 
excretion). At 0855 h subjects will drink from a stock solution of 0.15 g/kg body weight of 
H218O and 0.08 g/kg body weight of 2H2O followed by 100-200 mL tap water to rinse the dose 
container (measurement of tracer decay will be made in urine samples collected every morning 
for the remainder of the study).  At 1100 h subjects will undergo assessment of taste sensitivity 
(including PROP) and complete the three-factor eating,profile of mood state, and MacArthur 
socioeconomic questionnaires.  Subjects will be fed the standardized diet at three meals during 
the day on Day 1 at 0900 h, 1300 h, and 1800 h.  At 1500 h, subjects will begin a regular 
scheduled (i.e. daily) physical activity session of treadmill walking for a total of 60 minutes (6 x 
10-minute walking period with 10-minute rest period between each walking period) at the self 
selected speed determined during the screening process.  This session of walking will be 
repeated each day during the remainder of the in-patient period, including while in the metabolic 
chamber.  The daily caloric intake will account for the additional calories expended by the added 
physical activity.    
 
At 0700 h on the morning of Day 2 body weight and body composition (by DEXA and air-
displacement plethysmography) will be measured, as well as measurement of total body water 
content by bioelectrical impedance.  At 0745 h a blood sample will be taken, and at 0800 h, 
subjects will enter the metabolic chamber for measurement of 24h energy expenditure.  Subjects 
will be fed the standardized diet at three meals during the day on Day 1 at 0900 h, 1300 h, and 
2000 h while in the metabolic chamber, and hunger and satiety will be assessed by visual analog 
scale and questionnaire before each meal, and every 30 min for 2h and then every hour after each 
meal.  At 1500 h subjects will undergo their scheduled session of treadmill walking, and at 1800 
h, subjects will also undergo assessment of liking and wanting of food by computer presentation 
of four separate food categories: high-fat savory (HFSA), low-fat savory (LFSA), high-fat sweet 
(HFSW), and low-fat sweet (LFSW).  At 2200 h, overall energy balance will be assessed and a 
standard shake will be administered to maintain energy balance for the remainder of the stay in 
Figure A1.  Schematic timeline of first in-patient visit. 
Reduced calorie diet
Day 6
Day 7
Day 10
Day 13
Metab.
study
0800
0800
0800
Ad libitum
Day 12
Day 9
0800
PET imaging,
fMRI (food rating)
Standard diet
Day 2
Day 3
Day 8
Admit
Discharge
0800
0800
0500
Metabolic
Chamber/
24h blood
sampling
Metab.
study
Day 5
0800
1200
0800
Metabolic
Chamber/
24h blood
sampling
Metabolic
Chamber
PET imaging,
fMRI (food rating)
Metabolic
Chamber
0800
Metabolic
Chamber
Day 4
Day 11
0500
0800
1000
Day 14
Day 15
fMRI
(food perception)
Assess food
preference
Assess food
preference
0800
Day 1

53 
the metabolic chamber.  The amount of shake required to maintain energy balance will be given 
with the dinner for the remainder of the baseline days (Day 3-5).  
 
On the morning of Day 3, subjects will be allowed to leave the chamber.  At that time a urine 
sample will be collected, body weight will be measured, a blood sample will be taken, and 
subjects will be allowed to shower but not to engage in any other activities.  Between 0930 and 
1130h (dependent on available scan time), subjects will undergo a positron emission tomography 
(PET) scan.  Briefly, after placement of an intravenous catheter and positioning of the subjects, 
subjects will be given an intravenous injection of ~5 mCi of [18F]fallypride.  For the next 3.5 
hours, scanning will be carried out in sets with periodic breaks to allow the subjects to get up and 
to void.  At 5.5h after the scheduled PET scan subjects will undergo a delay discounting 
computer procedure where they will be required to make decisions between hypothetical 
immediate food rewards in comparison to various delayed monetary rewards.  At 1500 h, 
subjects will begin the regular scheduled session of treadmill walking for a total of 60 minutes (6 
x 10-minute walking period with 10-minute rest period between each walking period).  At 1730 
h, a blood sample will be taken, and then at 1800 h subjects will undergo fMRI scanning, 
comprised of the required clinical scans, a resting-state scan, food rating scans, and an 
anatomical scan (see Analytical Procedures).  On day 3, subjects will be fed their standardized 
diet at 1200 h and 1500 h and 2000 h.  At 2200 h on Day 3, two intravenous catheters will be 
inserted for infusions during the “metabolic study” the next day, during which we will assess 
protein synthesis, lipolysis, glucose uptake, and insulin sensitivity (Figure A2).  The tracer 
infusion procedures and the muscle and adipose tissue biopsies are optional procedures; however 
the indirect calorimetry and measurement of insulin sensitivity (beginning at 1000 h on Day 4) 
will not be optional.  
 
At 0500 h on the morning of Day 4, a blood sample will be obtained from the hand vein in 
order to measure baseline isotope enrichment of phenylalanine, glycerol, and glucose.  After the 
blood draw, a primed-constant infusion of [ring-2H5]-phenylalanine and [6,6-2H2]-glucose will 
be started ([ring-2H5]-phenylalanine: 2 mol/kg priming dose, 0.05 mol/kg/min continuous 
infusion; [6,6-2H2]-glucose: 35 mol/kg priming dose, 0.41 mol/kg/min continuous infusion).  
Two hours after starting the [ring-2H5]-phenylalanine and [6,6-2H2]-glucose infusions, we will 
begin a 2h constant infusion of [2H5]-glycerol (1.5 mol/kg priming dose, 0.1 mol/kg/min 
continuous infusion).  After 3h 50min of the phenylalanine and glucose infusions and 1h 50min 
Figure A2.  Schematic timeline of metabolic study. 
0500
0600
0700
0800
0900
1000
[ring-2H5]-phenylalanine infusion
[2H5]-glycerol inf.
Time of day (hours)
Blood samples
X
X
X
X
XX
Indirect
cal
Biopsy
[6,6-2H2]-glucose infusion
IVGTT
1200

54 
of the glycerol infusion (the time necessary to achieve isotopic equilibrium of phenylalanine, 
glucose, and glycerol within the body), three arterialized blood samples will be obtained from the 
heated hand vein (to obtain “arterialized” blood samples (65)) in 5 min intervals for 
determination of phenylalanine, glucose, and glycerol rate of appearance (Ra) into the 
circulation.  Substrate oxidation will be measured using indirect calorimetry from 0745-0815 h 
and all the infusions will be stopped at 0900 h.  At 0915 h, we will obtain a small muscle sample 
(~100 mg) from the muscle of the thigh for measurement of intramuscular substrates (e.g. 
glycogen, lipids), and a subcutaneous adipose tissue sample (5-10 g) will be obtained from the 
abdominal region for measurement of adipose tissue proteins.  At 1000 h, subjects will undergo 
an intravenous glucose tolerance test (IVGTT) to assess insulin sensitivity using the minimal 
model method (66).  Briefly, subjects will be injected with a bolus of glucose (300 mg/kg body 
weight) at 1000 h and a bolus of insulin (0.03 U/kg) at 1020 h and blood samples will be taken at 
frequent time points for 2h after the glucose bolus.  Subjects will be fed immediately following 
the conclusion of the IVGTT at 1200 h, as well as at 1600 h and 1800 h on Day 4.  At 1345 h, a 
blood sample will be taken, and then at 1400 h subjects will undergo an fMRI scanning session 
comprised of gustatory functional localizer scans, food perception functional localizer scans, and 
an anatomical scan (see Analytical Procedures).  At 1600 h, subjects will begin the regular 
scheduled session of treadmill walking for a total of 30 minutes (3 x 10-minute walking period 
with 10-minute rest period between each walking period).  The second 30 minutes of scheduled 
walking will occur at 1900 h (3 x 10-minute walking period with 10-minute rest period between 
each walking period).  Finally, at 2200 h, subjects will be administered a dose of a sodium 
bromide solution for measurement of extracellular water content.  Subjects will be weighed, and 
a urine and blood sample will be collected before the sodium bromide dose is given.  
 
On Day 5, at 0700 h, a urine sample will be collected, and subject’s body weight and body 
composition (by DEXA and air-displacement plethysmography) will be measured, as well as 
measurement of total body water content by bioelectrical impedance.  At 0730 h, an intravenous 
catheter will be inserted and a blood sample will be taken.  Throughout the following 24h period, 
additional blood samples will be frequently taken to allow measurement of plasma hormone 
concentrations (i.e every 30 min for 4h after breakfast and lunch, and for 2h after dinner, and 
then at 2100 h and 2200 h on Day 5 and 0300 h and 0700 h on Day 6).  While in the metabolic 
chamber, hunger and satiety will be assessed by visual analog scale and questionnaire before 
each meal, and every 30 min for 2h and then every hour after each meal.  Subjects will be fed 
their standardized diet at 0900 h, 1300 h, and 1800 h, while in the metabolic chamber and 
undergo their scheduled session of treadmill walking at 1500 h.  
 
Calorie reduction dietary procedures  
After the 5-day baseline period, subjects will be randomized into either the Red-Fat or Red-
Carb segments of the protocol: 
 
Red-Fat:  Total caloric content of the “reduced-fat” diet will be 30% per day lower than the 
calories required to maintain energy balance during the baseline period, and will be achieved 
strictly by the removal of 85% of the baseline fat calories from the diet (the absolute amount of 
fat [in grams] removed will be dependent on the initial caloric intake during the baseline period, 
see Table 1).  The absolute amount of carbohydrate and protein (in grams) will be identical to 

55 
that provided in the baseline diet.  The relative macronutrient content for this diet will be 
approximately 71% carbohydrate, 8% fat, and 21% protein. 
 
Red-Carb:  Total caloric content of the “reduced-carbohydrate” diet will also be 30% per day 
lower than the calories required to maintain energy balance during the baseline period, and will 
be achieved strictly by the removal of 60% of baseline carbohydrate calories from the diet (the 
absolute amount of carbohydrate [in grams] removed will be dependent on the initial caloric 
intake during the baseline period).  The absolute amount of fat and protein (in grams) will be 
identical to that provided in the baseline diet.  The relative macronutrient content for this diet 
will be approximately 29% carbohydrate, 50% fat, and 21% protein.  An example of real foods 
for the two calorie-restricted diets, as well as the standard diet, can be found in Appendix Table 
A1.  
 
Specific procedures for calorie reduction period 
A schematic timeline of events during the calorie reduction period (Days 6 – 11) is depicted 
in Figure A1.  On the morning of Day 6 (after the 24h metabolic chamber stay that will begin on 
Day 5) subjects will be allowed to leave the chamber.  At that time a urine sample will be 
collected, body weight will be measured, and subjects will be allowed to shower but not to 
engage in any other activities.  After collection of a basal blood sample, subjects will then re-
enter the metabolic chamber for measurement of 24h energy expenditure.  The first meal of the 
reduced calorie diet will be given in the chamber as breakfast on Day 6 at 0900 h, and the 
remainder of the meals will be fed at 1300 h and 1800 h while in the chamber.  While in the 
metabolic chamber, hunger and satiety will again be assessed by visual analog scale and 
questionnaire before each meal, and every 30 min for 2h and then every hour after each meal, 
and subjects will undergo their scheduled session of treadmill walking at 1500 h.    Subjects will 
leave the chamber on the morning of Day 7 and a basal blood sample will be collected.  Subjects 
will be fed their reduced calorie diet at 0900 h, 1300 h, and 1800 h, on Day 7 and undergo their 
scheduled session of treadmill walking at 1500 h.  The procedures during the last four days of the 
reduced calorie period (Days 8 – 11) will be similar to those during the last four days of the 
baseline period as described above with the exceptions noted below.  Briefly, at the end of Day 7 
at 2200 h two intravenous catheters will be inserted for infusions during the “metabolic study” 
(i.e. tracer infusion, indirect calorimetry, muscle and adipose tissue biopsy) that will occur on 
Day 8.  The “metabolic study” procedures will again be optional.  On Day 8, at 0500h, the 
metabolic study will begin, however the IVGTT procedure, the fMRI  (gustatory and food 
perception) scanning session, and the sodium bromide dose that will all performed following the 
metabolic study on Day 4 will be not be repeated on Day 8.  Therefore, the subjects will be fed at 
1000 h, 1300 h, and 1800 h, and at 1500 h they will undergo their regular scheduled session of 
treadmill walking for a total of 60 minutes (6 x 10-minute walking period with 10-minute rest 
period between each walking period).  On Day 9, at 0730 h, after three days on the reduced 
calorie diet, an intravenous catheter will be inserted and a blood sample will be taken.  At 0800h 
subjects will enter the metabolic chamber and undergo a 24h stay in order to measure changes in 
energy expenditure as a result of the diet.  Hunger and satiety will be assessed by visual analog 
scale and questionnaire while in the chamber.  Throughout the 24h period in the chamber, blood 
samples will be frequently taken to allow measurement of plasma hormone concentration.  On 
Day 10, after leaving the metabolic chamber, a blood sample will be taken before subjects 
undergo PET imaging, followed by the delay discounting computer procedure at 1400 h, a blood 

56 
sample at 1730 h, and at 1800 h subjects will undergo the same fMRI session as preformed on 
Day 3 of the baseline diet period.  On Day 11, at 0715 h, subject’s body weight, body 
composition (by DEXA and air-displacement plethysmography), and total body water content 
(by bioelectrical impedance) will be measured, and then at 0800 h, subjects will enter the 
metabolic chamber for measurement of 24h energy expenditure.  While in the metabolic 
chamber, hunger and satiety again will be assessed by visual analog scale, the POMS 
questionnaire will be administered, and subjects will undergo assessment of liking and wanting 
of food by computer presentation of four separate food categories.   
 
Ad libitum dietary procedures 
 
Subjects will begin an ad libitum period on the morning of Day 12 after their 24h stay in the 
metabolic chamber, and continue this period for 3 days until 0800 h on Day 15 (Figure A1).  An 
automated food-selection system made up of a refrigerated vending machine that contains 
multiple trays of food will be used.  Multiple food items will be available to the subjects during 
the ad libitum period and will accommodate the appropriateness of certain foods for breakfast, 
lunch, dinner, and evening snacks.  The subjects will have unrestricted access to the vending 
machines and will be instructed to follow their typical eating pattern as closely as possible.  The 
subjects will be instructed to eat whatever and whenever they desire and to return the food 
wrappers and unconsumed food portions to the vending machines.  Physical activity during this 
period will be unregulated, but monitored by the accelerometers that the subjects will be 
wearing. Daily energy intake and protein, fat, and carbohydrate intake will be calculated by 
weighing of the food consumed and eating behavior will also be assessed with the bite counter 
and the automatic ingestion monitor. 
 
Specific procedures during ad libitum period 
On the morning of Day 12, subjects will leave the metabolic chamber.  At 0800 h, a urine 
sample will be collected, body weight will be measured, and a blood sample taken.  Hunger and 
satiety will be assessed by visual analog scale and questionnaire hourly from 0800 h to 2200 h.  
These procedures will be repeated during each day of the 3-day ad libitum diet period.  On the 
morning of Day 15, after collection of a urine sample and body weight, at 0730 h, a blood 
sample will be taken for measurement of baseline plasma substrate and hormone levels.  At 0800 
h, subjects will be discharged. 
 
Second In-Patient Visit 
After discharge from the MCRU, a wash-out period of at least 2 weeks will be required before 
subjects return for the second portion of the study (i.e. the alternate reduced calorie diet than 
what was consumed during their first visit).  This period of time will allow for sufficient wash-
out of any effects the diet manipulation may have on subsequent measurements made during the 
second stay at the MCRU.  Subjects will be required to begin completion of the second reduced 
calorie portion of the study within 10 weeks after discharge in order to avoid large changes in 
physiological state.  During the wash-out period, subjects will be encouraged to return to normal 
activity patterns, to consume a diet as similar to the standard diet as possible (50% carbohydrate, 
35% fat, and 15% protein), and to maintain weight stability (within 2kg of the starting weight). 
 
Specific procedures during baseline period 

57 
A schematic timeline of events during the second in-patient visit is depicted in Figure A3.    
Beginning two days before admission, all food to be consumed by the subjects will again be 
supplied by the MCRU.  During this time, subjects will arrive each morning at the MCRU at 
0800 h for measurement of body weight, after which they will eat breakfast, and then take the 
remainder of the day’s meal with them to consume later during the day. Subjects will be 
instructed to consume only and all the food provided to them and to return those portions that 
they were unable to eat the following day when they return for the next day’s meals.   
 
On the morning following the second day of the out-patient standard diet, subjects will arrive 
in the fasted state at the MCRU at 0800 h for admission to the MCRU (Day 1, see Figure A3).  
Following admission, subjects will be fitted with accelerometers (activity monitors), a bite 
counter to detect a pattern related to biting behavior and an automatic ingestion monitor to 
estimate frequency, duration, rate and mass of ingestion. Both will be worn continuously during 
the in-patient period.  At 0845 h a baseline urine sample will be collected (24h urine specimens 
will be collected throughout the duration of the in-patient period for measurement of nitrogen 
excretion).  At 0855 h subjects will drink from a stock solution of 0.15 g/kg body weight of 
H218O and 0.08 g/kg body weight of 2H2O followed by 100-200 mL tap water to rinse the dose 
container (measurement of tracer decay will be made in urine samples collected every morning 
for the remainder of the study).  Subjects will be fed the standardized diet at three meals during 
the day on Day 1 at 0900 h, 1300 h, and 1800 h.  At 1500 h, subjects will undergo the same 
treadmill walking session as described above. 
 
At 0700 h on the morning of Day 2 body weight and body composition (by DEXA and air-
displacement plethysmography) will be measured, as well as measurement of total body water 
content by bioelectrical impedance.  At 0745 h a blood sample will be taken, and at 0800 h, 
subjects will enter the metabolic chamber for measurement of 24h energy expenditure.  While in 
the metabolic chamber, hunger and satiety will be assessed by visual analog scale and 
questionnaire before each meal, and every 30 min for 2h and then every hour after each meal.  At 
1500 h, subjects will undergo the same treadmill walking session as described above.  This 
physical activity session will be repeated every day during the second in-patient period, and daily 
caloric intake will be adjusted to reflect the increase in caloric expenditure as a result of the 
added physical activity in order to maintain energy balance.  Subjects will be fed the 
standardized diet at 0900 h, 1300 h, and 1800 h while in the metabolic chamber.  At 2200 h, 
overall energy balance will be assessed and a standard shake will be administered to maintain 
energy balance for the remainder of the stay in the metabolic chamber.  The amount of shake 
required to maintain energy balance will be given with the dinner for the remainder of the 
baseline days (Day 3-5).  
Figure A3. Schematic timeline of second in-patient visit. 
Reduced calorie diet
Day 6
Day 7
Day 10
Day 13
Metab.
study
0800
0800
0800
Ad libitum
Day 12
Day 9
0800
PET imaging,
fMRI (food rating)
Standard diet
Day 2
Day 3
Day 8
Admit
Discharge
0800
0800
0500
Metabolic
Chamber
Day 5
0800
0800
Metabolic
Chamber/
24h blood
sampling
Metabolic
Chamber
Metabolic
Chamber
0800
Metabolic
Chamber
Day 4
Day 11
0800
1000
Day 14
Day 15
Assess food
preference
0800
Day 1

58 
 
On the morning of Day 3, subjects will be allowed to leave the chamber.  At that time a urine 
sample will be collected and body weight will be measured.  Subjects will be fed the 
standardized diet at 0900 h, 1300 h, and 1800 h, and undergo their scheduled session of treadmill 
walking at 1500 h.  The timeline of events on Day 4 will be identical to those on day 3.   
 
At 0700 h on the morning of Day 5 body weight and body composition (by DEXA and air-
displacement plethysmography) will be measured, as well as measurement of total body water 
content by bioelectrical impedance.  At 0745 h a blood sample will be taken, and at 0800 h, 
subjects will enter the metabolic chamber for measurement of 24h energy expenditure.  While in 
the metabolic chamber, hunger and satiety will be assessed by visual analog scale and 
questionnaire before each meal, and every 30 min for 2h and then every hour after each meal.  At 
1500 h, subjects will undergo the same treadmill walking session as described above.  Subjects 
will be fed the standardized diet at 0900 h, 1300 h, and 1800 h while in the metabolic chamber.   
 
Specific procedures for calorie reduction period 
A schematic timeline of events during the calorie reduction period of the second in-patient 
visit (Days 6 – 11) is depicted in Figure A3.  The procedures during this calorie reduction period 
will be identical to those during the calorie reduction period of the first in-patient visit described 
above.  Briefly, on the morning of Day 6 (after the 24h metabolic chamber stay that will begin on 
Day 5) subjects will leave the chamber, a urine sample will be collected, body weight measured, 
and a basal blood sample take.  Subjects will then be allowed to shower before re-entering the 
metabolic chamber at 0800 h for measurement of 24h energy expenditure.  The first meal of the 
reduced calorie diet will be given in the chamber as breakfast on Day 6 at 0900 h, and the 
remainder of the meals will be fed at 1300 h and 1800 h while in the chamber, and hunger and 
satiety will again be assessed throughout the day.   At 2200 h on Day 7, an intravenous catheters 
will be inserted for infusions during the “metabolic study” (i.e. tracer infusion, indirect 
calorimetry, muscle and adipose tissue biopsy) that will occur on Day 8.  The “metabolic study” 
procedures will again be optional.  At 0730 h on Day 9, after three days on the reduced calorie 
diet, an intravenous catheter will be inserted and a blood sample will be taken.  Throughout the 
following 24h period, additional blood samples will be frequently taken to allow measurement of 
plasma hormone concentrations.  At 0800h, subjects will then enter the metabolic chamber and 
undergo a 24h stay in the metabolic chamber in order to measure changes in energy expenditure 
as a result of the diet.  Hunger and satiety again will be assessed by visual analog scale and 
questionnaire while in the chamber.  On Day 10, after leaving the metabolic chamber, subjects 
will have a blood sample taken, undergo PET imaging, followed by the delay discounting 
computer procedure, a blood sample at 1730 h, and at 1800 h subjects will undergo the same 
fMRI session as preformed on Day 2 of the initial baseline diet period (first in-patient visit).  On 
Day 11, at 0715 h, subject’s body weight, body composition (by DEXA and air-displacement 
plethysmography), and total body water content (by bioelectrical impedance) will be measured, 
and then at 0800 h, subjects will enter the metabolic chamber for measurement of 24h energy 
expenditure.  While in the metabolic chamber, hunger and satiety again will be assessed by 
visual analog scale, the POMS questionnaire will be administered, and subjects will undergo 
assessment of liking and wanting of food by computer presentation of four separate food 
categories.  
 

59 
Specific procedures during ad libitum period 
Procedures during the ad libitum period will be identical to those during the first in-patient 
visit.  Briefly, on Day 12, subjects will leave the metabolic chamber.  At 0800 h, a urine sample 
will be collected, body weight will be measured, and a blood sample taken.  Hunger and satiety 
will be assessed by visual analog scale and questionnaire hourly from 0800 h to 2200 h.  These 
procedures will be repeated during each day of the 3-day ad libitum diet period.  On the morning 
of Day 15, after collection of a urine sample and body weight, at 0730 h, a blood sample will be 
taken for measurement of baseline plasma substrate and hormone levels.  At 0800 h, subjects 
will be discharged. 
 
Out-patient lifestyle intervention period 
Specific procedures during out-patient period 
At the end of the ad libitum period of the second in-patient visit, subjects will be discharged 
from the MCRU and begin a 12 week out-patient period.  During this period, all subjects will be 
encouraged to undergo lifestyle modification consisting of diet modification and increased 
physical activity with a goal to reduce body weight by at least 5% by the end of 12 weeks.  At 
the onset of the out-patient period, all volunteers will be provided with a weight-loss counseling 
session with registered dietitians to set their weight-loss goals and explain their prescribed diet.  
Based on the measurements conducted during the in-patient stay, we will calibrate a 
mathematical model of the individual subject’s metabolism and body composition and this 
mathematical model will be used to project the expected weight loss and set appropriate goals 
(9).  All subjects will be asked to report their body weights on a daily basis (by phone or email) 
using bathroom scales provided to them.  On a biweekly basis subjects will meet with registered  
 
Figure A4. Schematic timeline of out-patient period. 
 
 
 
dietitians and receive reminders about their diet and weight loss goals, along with general 
nutrition and lifestyle information, as well as additional feedback using the mathematical model 
on their average degree of energy imbalance, how this conforms to their weight loss goals, and 
their projected total weight loss.  If the subjects are not on track to meet their weight loss goals, 
then specific corrective adjustments of diet or physical activity will be advised.  In between their 
biweekly meetings, subjects will receive guidance from the registered dietitians by phone or 
email.   

60 
A number of metabolic studies will take place periodically during the 12 week out-patient 
period, with the first studies beginning at the conclusion of the subject’s second in-patient visit 
(Figure A4).  Specifically, before subjects are discharged, a collection of a baseline urine sample 
will occur, after which subjects will drink from a stock solution of 0.15 g/kg body weight of 
H218O and 0.08 g/kg body weight of 2H2O followed by 100-200 mL tap water to rinse the dose 
container (urine samples will be collected twice more during the week and three times during the 
following week for measurement of tracer decay).  Subjects will be given a handheld indirect 
calorimeter (MedGem, Dunedin, FL) to measure and record resting metabolic rate at home each 
morning for the next two weeks and will also be given physical activity monitors, a bite counter 
and an automatic ingestion monitor to wear continuously during these two weeks.  At 2 weeks 
after discharge, subjects will arrive to the MCRU at 0700 h after an overnight fast. At that time 
body composition will be measured by DEXA and air-displacement plethysmography, and total 
body water content will be measured by bioelectrical impedance.  At 0800 h, subjects will enter 
the metabolic chamber for measurement of 24h energy expenditure.  The content and calories of 
food fed in the chamber will be the same as that fed to the subjects while in the chamber during 
the baseline period and physical activity will also be identical to that in the chamber during the 
baseline period.  Subjects will continue to wear the physical activity monitors, the bite counter 
and the automatic ingestion monitor while in the chamber.  At 6 weeks after discharge subjects 
will again arrive to the MCRU at 0700 h after an overnight fast and body composition will be 
measured by DEXA and air-displacement plethysmography, and total body water content will be 
measured by bioelectrical impedance.  At 0800 h, resting energy expenditure and fat oxidation 
rate will be determined by indirect calorimetry.  At 10 weeks after discharge, subjects will again 
arrive to the MCRU at 0700 h after an overnight fast and, after collection of a baseline urine 
sample, subjects will be given a dose of DLW (urine samples will again be collected twice more 
during the week and three times during the following week for measurement of tracer decay).  
Following the consumption of the DLW dose, body composition will be measured by DEXA and 
air-displacement plethysmography, and total body water content will be measured by 
bioelectrical impedance.  At 0800 h, resting energy expenditure and fat oxidation rate will be 
determined by indirect calorimetry.  Before leaving the MCRU, subjects will again be given a 
handheld indirect calorimeter to measure and record resting metabolic rate at home each morning 
for the next two weeks and will also be given physical activity monitors, a bite counter and an 
automatic ingestion monitor to wear continuously during these two weeks.  Before each visit at 
week 2, 6, and 10, subjects will be required to consume a standard meal (i.e. 50% carbohydrate, 
35% fat, 15% protein) the night before their arrival that will be provided by the clinical center 
metabolic kitchen.  Two weeks after their visit during week 10, subjects will arrive at the MCRU 
at 1700 h and be admitted to the hospital and given a standard meal for the evening.  At 2200 h, 
subjects will be administered a dose of a sodium bromide solution for measurement of 
extracellular water content.  Subjects will be weighed, and a blood sample will be collected 
before the sodium bromide dose is given.  At 0700 h the following morning, DEXA and air-
displacement plethysmography measurement of body composition will be taken and total body 
water content will be measured by bioelectrical impedance.  Following measurement of body 
composition, a blood sample will be taken for measurement of sodium bromide dilution.  At 
0800 h, subjects will enter the metabolic chamber for measurement of 24h energy expenditure, 
and hunger and satiety will be assessed by visual analog scale and questionnaire.  Conditions 
within the chamber (i.e., food fed and physical activity) will again be identical to the chamber 
conditions during the baseline period.  At 1100, the POMS questionnaire will be administered, 

61 
and at 1800 h, subjects will again undergo assessment of liking and wanting of food by computer 
presentation of four separate food categories.  The next morning, after leaving the metabolic 
chamber, subjects will have a blood sample taken, undergo PET imaging, followed by the delay 
discounting computer procedure, a blood sample at 1730 h, and at 1800 h subjects will undergo 
an fMRI scanning comprised of the required clinical scan, a resting-state scan, food rating scans, 
and an anatomical scan.  At 2200 h on, an intravenous catheters will be inserted for infusions 
during the “metabolic study” (i.e. tracer infusion, indirect calorimetry, muscle and adipose tissue 
biopsy) that will occur the following morning.  The “metabolic study” procedures will again be 
optional and if a subject chooses not to participate, they will be discharged following the fMRI 
session the night before.  The “metabolic study” procedures and timeline will be identical to 
those outlined in Figure A2, minus the IVGTT.  Following the conclusion of these procedures, 
subjects will be discharged.  
 
Longitudinal follow-up of obese subjects that completed the outpatient weight loss phase 
Specific procedures during long-term follow-up 
On an approximately yearly basis for 5 years, subjects will be given the option to return to the 
MCRU and complete many of the procedures that were performed over the last 2 weeks of the 
outpatient weight loss phase.  Specifically, subjects will arrive at the MCRU at 0700 h after an 
overnight fast and, after collection of a baseline urine sample, subjects will be given a dose of 
DLW (urine samples will again be collected twice more during the week and three times during 
the following week for measurement of tracer decay).  Following the consumption of the DLW 
dose, body composition will be measured by DEXA and air-displacement plethysmography, and 
total body water content will be measured by bioelectrical impedance.  At 0800 h, resting energy 
expenditure and fat oxidation rate will be determined by indirect calorimetry.  Before leaving the 
MCRU, subjects will again be given physical activity monitors, a bite counter and an automatic 
ingestion monitor to wear continuously during the next 2 weeks.  Subjects will be required to 
consume a standard meal (i.e. 50% carbohydrate, 35% fat, 15% protein) the night before visiting 
the MCRU and will be provided by the clinical center metabolic kitchen. Subjects will return to 
the MCRU 2 weeks later at 1700 h and be admitted to the hospital and given a standard meal for 
the evening.  At 2200 h, subjects will be administered a dose of a sodium bromide solution for 
measurement of extracellular water content.  Subjects will be weighed, and a blood sample will 
be collected before the sodium bromide dose is given.  At 0700 h the following morning, DEXA 
and air-displacement plethysmography measurement of body composition will be taken and total 
body water content will be measured by bioelectrical impedance.  Following measurement of 
body composition, a blood sample will be taken for measurement of sodium bromide dilution.  
At 0800 h, subjects will enter the metabolic chamber for measurement of 24h energy 
expenditure, and hunger and satiety will be assessed by visual analog scale and questionnaire.  
Conditions within the chamber (i.e., food fed and physical activity) will again be identical to the 
chamber conditions during the baseline period.  At 1100, the POMS questionnaire will be 
administered, and at 1800 h, subjects will again undergo assessment of liking and wanting of 
food by computer presentation of four separate food categories.  The next morning, after leaving 
the metabolic chamber, subjects will have a blood sample taken and undergo PET imaging 
beginning at 0800 h, followed by the delay discounting computer procedure at 1400 h, a blood 
sample at 1730 h, and at 1800 h subjects will undergo an fMRI scanning comprised of the 
required clinical scan, a resting-state scan, food rating scans, and an anatomical scan.  At 2200 h 
on, an intravenous catheters will be inserted for infusions during the “metabolic study” (i.e. 

62 
tracer infusion, indirect calorimetry, muscle and adipose tissue biopsy) that will occur the 
following morning.  The “metabolic study” procedures will again be optional and if a subject 
chooses not to participate, they will be discharged following the fMRI session the night before.  
The “metabolic study” procedures and timeline will be identical to those outlined in Figure A2.  
Following the conclusion of these procedures, subjects will be discharged.  These procedures 
will be repeated on a yearly basis for 5 years following the weight loss phase of the study. 

63 
Table A1.  Example of standard and restricted diets for hypothetical subject with estimated total 
daily energy intake of 2000 kcal/day. 
 
Standard Diet 
Carbohydrate Restricted 
Diet 
Fat Restricted Diet 
Breakfast 
- 6 oz fat-free blueberry 
yogurt 
- Bagel w/ 21 g cream cheese 
- 210 g (~ ¾ cup) orange juice 
- 120 g (~ ½ cup) fruit salad 
- Scrambled eggs: 
  ∙ 70 g egg white (~ 3 egg) 
  ∙ 35 g egg yolk (~ 2 ½ egg) 
  ∙ cooked in 4 g butter  
- 3 slices bacon 
- 1 slice wheat toast w/ 4 g 
butter 
- Water 
- 35 g Corn Pops (~ ¾ oz) 
- 200 g skim milk (~ ¾ cup) 
- 1 medium banana 
- 
¼ 
cup 
scrambled 
egg 
substitute 
Lunch 
- Turkey sandwich: 
  ∙ 2 slices multigrain bread 
  ∙ 45 g turkey breast (1½ oz) 
  ∙ 30 g provolone cheese (1 
slice) 
  ∙ 1 leaf lettuce 
  ∙ 2 slices raw tomato  
  ∙ 10 g Dijon mustard 
- Salad: 
  ∙ 40 g lettuce 
  ∙ 40 g tomato 
  ∙ 30 g cucumber 
  ∙ 43 g Italian salad dressing  
- 1 oz potato chips 
- 120 g red grapes (~ ½ cup) 
- Water or diet beverage 
- Turkey wrap: 
  ∙ 1 small tortilla 
  ∙ 80 g turkey breast (~ 3 oz) 
  ∙ 30 g American cheese (1 
slice) 
  ∙ 1 lettuce leaf 
- Salad: 
  ∙ 40 g lettuce 
  ∙ 40 g tomato 
  ∙ 40 g cucumber 
  ∙ 45 g Italian salad dressing  
- 100 g red grapes (~ ½ cups) 
- Water or diet beverage 
- Turkey sandwich: 
  ∙ 2 slices multigrain bread  
  ∙ 60 g turkey breast (2 oz) 
  ∙ 1 leaf lettuce 
  ∙ 1 slice raw tomato 
  ∙ 10 g Dijon mustard 
- Salad: 
  ∙ 40 g lettuce 
  ∙ 30 g tomato 
  ∙ 40 g cucumber 
  ∙ 50 g fat-free Ranch salad 
dressing 
- 100 g red grapes (~ ½ cup) 
- 130 g lemonade (~ ½ cup) 
 
Dinner 
- 100 g salmon (~ 3½ oz) 
cooked w/ 5 g (1 tsp) olive oil 
- 150 g brown rice (~ ¾ cup) 
- 100 g broccoli (~ ¾ cup) 
- Chocolate chip cookie 
- Water or diet beverage 
- Chicken stir fry: 
  ∙ 40 g (~ 1 oz) stir fry sauce 
  ∙ 130 g stir fry vegetables  
  ∙ 75 g chicken breast (~ 2½ 
oz) 
  ∙ cooked in 13 g olive oil  
- 120 g brown rice (~ 5/8 cup) 
- 100 g peaches (~ ½ cup) 
- Water or diet beverage 
 
- 100 g chicken breast (~ 3 oz) 
- ½ cup cooked carrots 
- ¾ cup brown rice 
- Salad: 
  ∙ 50 g lettuce 
  ∙ 40 g tomato 
  ∙ 40 g cucumber 
  ∙ 50 g fat-free French salad 
dressing 
- 1 slice angel food cake 
- 100 g tropical fruit salad 
- Water or diet beverage 
Total 
2000 calories 
75 g protein (15%) 
78 g fat (35%) 
250 g carbohydrate (50%) 
1405 calories 
75 g protein (21%) 
78 g fat (50%) 
101 g carbohydrate (29%) 
1405 calories 
75 g protein (21%) 
12 g fat (8%) 
250 g carbohydrate (71%)