Clinical trial NCT01950520
Conditions: Healthy Volunteers
Keywords: Adipose Tissue Adult Resting Energy Expenditure RESPIRATORY QUOTIENT BASAL METABOLIC RATE
Sponsor: National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Start date: 2014-02-07; Completion date: 2024-06-11
Enrollment: 47 participants; Allocation: RANDOMIZED; Intervention model: CROSSOVER

Brief summary:
Background:

\- Changes in how a person's body burns energy or calories can affect their weight over time. The lowest level of energy the body needs to function is called basal metabolic rate. In the cold, we burn extra energy, even before we start to shiver. This is called non-shivering thermogenesis and it occurs in different types of tissue such as muscle and fat. Researchers want to learn more about this type of energy burning and how it is regulated. They hope this will help treat obesity in the future.

Objectives:

* Sub-study 1: to better understand how non-shivering thermogenesis works.
* Sub-study 2: to measure the effects of anti-obesity drugs on basal metabolic rate.
* Sub-study 3: to better understand the effects of mirabegron, a beta-3 adrenergic receptor agonist, on brown fat activity.

Eligibility:

\- Healthy, lean adult males ages 18 to 35.

Design:

* Participants will be screened with medical history, physical exam, blood test, and EKG.
* For sub-studies 1 and 2:

  * Participants will receive one X-ray scan.
  * Each day, all participants will:
* Have height and weight measured, and have urine collected.
* Spend 4 hours in a temperature-controlled room with furniture, toilet area, phone, and computer. They will wear small non-invasive devices to monitor activity, heart rate, temperature, and shivering.
* Walk for 30 minutes.
* For sub-study 3:

  * Participants will receive one DXA scan and up to 4 PET/CT scans and 4 MRIs
  * Each stay, all participants will:
* Have height and weight measured, and have urine collected.
* Spend 6 hours in a temperature-controlled room with furniture, toilet area, phone, and computer. They will wear small non-invasive devices to monitor activity, heart rate, temperature, and shivering.
* Participants will be compensated for their time and participation at the end of the study

Detailed description:
The balance between energy expenditure (EE) and energy intake ultimately determines body weight. Resting EE is the major component (60-75%) of total EE in an adult human being. Resting EE dynamically adapts to environmental changes such as ambient temperature. In our on-going study of environmental temperature changes within and around the thermoneutral zone, we observed that healthy young men can increase EE by 17 % of the basal metabolic rate through the process of non-shivering thermogenesis (NST). This capacity for NST is unexpectedly large as compared to prior reports of mild cold-induced thermogenesis (3 to 11%) and suggests that increasing NST could be explored as an intervention to combat obesity.

The aim of this study is to better understand the physiology of NST and to develop improved assays for evaluating the effect of drugs that alter EE. For example, only recently has it been realized that brown adipose tissue (BAT) is functional in adult humans and that white adipose tissue can be converted to brown-adipose-like tissue to increase heat production during cold exposures. Moreover, skeletal muscle likely also plays a role in cold-induced thermogenesis even before overt shivering occurs. It is plausible that the mechanisms governing heat production for NST contribute to regulation of body weight and thus may be contributing to the current obesity epidemic: even small changes in EE, if not compensated by changes in food intake, can have long-term effects on body weight.

This protocol has two phases. The first uses a pharmacologic approach to investigate the mechanism of NST in young healthy lean males. Since the principal physiologic stimulus to BAT (and possibly muscle for NST) is via the sympathetic nervous system (SNS), beta-adrenergic receptors may hold key roles in regulating human EE. We hypothesize that, by careful measurements of NST (at an individually-titrated cool environmental temperature, between 18 21 degrees C vs. at thermoneutrality of 27 degrees C) and using beta-adrenergic drugs that differ in receptor specificity and agonist/antagonist properties, we will gain better understanding of the regulation of human NST.

The second phase of the study focuses on measuring of FDA-approved drugs (such as anti-obesity drugs) potential effect on basal metabolic rate (BMR) under thermoneutral conditions. The rationale is that previous studies of drug effect on EE in humans have not always rigorously enforced the use of thermoneutral conditions, thus may have increased variability and underestimated the effect, contributing to inconclusive findings.

It is envisioned that this study will further our knowledge of the mechanisms that regulate the acute adaptive changes in resting energy expenditure and the effects of drug therapy targeting obesity in humans.

Arms: arm 1: Chamber temperature will be block randomized with low temperature and 27°C (Cohort 1 only)

Within each block, the following five interventions will be repeated:

Propranolol: Propanolol 160mg, oral, by mouth (Cohort 1 only)

Pindolol: Pindolol 20mg, oral, by mouth (Cohort 1 only)

Dantrolene: Dantrolene 100mg, oral, by mouth (Cohort 1 only)

Magnesium Sulfate: Magnesium Sulfate, infusion, 50 mg/kg bolus followed by maintenance infusion at 2g/h (Cohort 1 only)

Placebo Cohort 1: Placebo, oral, by mouth (Cohort 1 only) arm 2: Interventions, in random order, will be administered during one of the six one-day stays

Caffeine: Caffeine 200mg, oral, by mouth (Cohort 2 only)

Qsymia: Qsymia (topiramate 92mg CR, phentermine 15mg PO), oral, by mouth (Cohort 2 only)

Topiramate: Topiramate 200mg, oral, by mouth (Cohort 2 only)

Phentermine: Phentermine 37.5mg, oral, by mouth (Cohort 2 only)

Naltrexone: Naltrexone 100mg, oral, by mouth (Cohort 2 only)

Placebo Cohort 2: Placebo, oral, by mouth (Cohort 2 only) arm 3: Interventions, in random order, will be administered during one of the four overnight inpatient stays

Mirabegron 50mg: Mirabegron 50mg, oral, by mouth (Cohort 3 only)

Mirabegron 200mg: Mirabegron 200mg, oral, by mouth (Cohort 3 only)

Placebo for Mirabegron: Placebo for Mirabegron, oral, by mouth (Cohort 3 only)
Interventions: intervention 1: Propranolol intervention 2: Pindolol intervention 3: Dantrolene intervention 4: Magnesium Sulfate intervention 5: Caffeine intervention 6: Qsymia intervention 7: Topiramate intervention 8: Phentermine intervention 9: Naltrexone intervention 10: Mirabegron 50mg intervention 11: Mirabegron 200mg intervention 12: Placebo Cohort 1 intervention 13: Placebo Cohort 2 intervention 14: Placebo for Mirabegron
Intervention descriptions: intervention 1: Propanolol 160mg, oral, by mouth (Cohort 1 only) intervention 2: Pindolol 20mg, oral, by mouth (Cohort 1 only) intervention 3: Dantrolene 100mg, oral, by mouth (Cohort 1 only) intervention 4: Magnesium Sulfate, infusion, 50 mg/kg bolus followed by maintenance infusion at 2g/h (Cohort 1 only) intervention 5: Caffeine 200mg, oral, by mouth (Cohort 2 only) intervention 6: Qsymia (topiramate 92mg CR, phentermine 15mg PO), oral, by mouth (Cohort 2 only) intervention 7: Topiramate 200mg, oral, by mouth (Cohort 2 only) intervention 8: Phentermine 37.5mg, oral, by mouth (Cohort 2 only) intervention 9: Naltrexone 100mg, oral, by mouth (Cohort 2 only) intervention 10: Mirabegron 50mg, oral, by mouth (Cohort 3 only) intervention 11: Mirabegron 200mg, oral, by mouth (Cohort 3 only) intervention 12: Placebo, oral, by mouth (Cohort 1 only) intervention 13: Placebo, oral, by mouth (Cohort 2 only) intervention 14: Placebo for Mirabegron, oral, by mouth (Cohort 3 only)

Protocol documents:
Prot_SAP_000.pdf: 
Abbreviated Title: NST 
Version Date: 2021 July 22 
 
Page 1 
National Institute of Diabetes and Digestive and Kidney Diseases 
Abbreviated Title: NST 
NIH IRB#: 13-DK-0200 
        
Version Date: 2021 July 22 
IND NUMBER:  IND 116246 
IND NAME: Mirabegron for the indication of Thermogenesis 
IND HOLDER: NIDDK 
IND MFG: Astellas 
 
 
 
 
TITLE:  The Mechanism of Human Non-shivering Thermogenesis and Basal Metabolic 
Rate 
SHORT TITLE: Pharmacologic Perturbations of Resting Energy Expenditure  
 
IDENTIFYING WORDS:  Non-shivering thermogenesis, basal metabolic rate, adaptive 
thermogenesis, obesity 
 
TYPE OF PROTOCOL:  Interventional (Physiology) 
 
Estimated Duration of Study: 10 years 
 
START DATE:   8/28/2013   
 
 
    END DATE: 02/02/2023 
 
NUMBER AND TYPE OF PATIENTS:    Accrual Ceiling:  _134__ 
 
 
 
 
 
PRINCIPAL INVESTIGATOR:  
Kong Y. Chen, PhD, MSCI * 
Diabetes, Endocrinology, and Obesity Branch (DEOB) 
National Institutes of Diabetes and Digestive and Kidney Diseases (NIDDK) 
BLDG 10, CRC, RM 5-5750 
Phone 301-451-1636 
Email: chenkong@niddk.nih.gov                                                                            
 
 
 
 
 


Abbreviated Title: NST 
Version Date: 2021 July 22 
 
Page 2 
 
Table of Contents 
 
 
 
 
 
 
 
 
 
       Starting Page # 
Statement of Compliance  
 
 
 
 
 
 
 
3 
 
Précis  
 
 
 
 
 
 
 
 
 
4 
 
Abbreviations  
 
 
 
 
 
 
 
 
5 
 
I.  Introduction and Background 
 
 
 
 
 
 
6 
 
I.1  Scientific and Clinical Significance 
 
 
 
 
8 
 
I.2  Mechanistic basis of NST 
 
 
 
 
 
9 
 
I.3  Assaying anti-obesity drugs for their effects on EE 
 
 
13 
 
 
II.  Research Objectives 
 
 
 
 
 
 
 
15 
 
II.1  Primary objectives 
 
 
 
 
 
 
16 
 
 
Cohort 1 
 
 
 
 
 
 
 
16 
 
 
Cohort 2 
 
 
 
 
 
 
 
16 
 
 
Cohort 3 
 
 
 
 
 
 
 
17 
 
II.2  Secondary objectives 
 
 
 
 
 
 
17 
 
II.3  Exploratory objectives  
 
 
 
 
 
18 
 
III. Study Protocol 
 
 
 
 
 
 
 
 
19 
 
III.1 Subjects and recruitment 
 
 
 
 
 
19 
 
III.2-1 Protocol design, Cohort 1 
 
 
 
 
 
20 
 
III.2-2 Protocol design, Cohort 2 
 
 
 
 
 
24 
 
III.2.3 Protocol design, Cohort 3 
 
 
 
 
 
25 
III.3 Inclusion criteria  
 
 
 
 
 
 
27 
 
III.4 Exclusion criteria 
 
 
 
 
 
 
27 
 
III.5 Methods  
 
 
 
 
 
 
 
28 
 
III.6 Possible risks and hazards 
 
 
 
 
 
35 
 
III.7 Adverse events  
 
 
 
 
 
 
41 
 
III.8 Recruitment of women, children and minority individuals 
 
42 
 
III.9 Remuneration 
 
 
 
 
 
 
 
43 
 
III.10 Research Use, Storage and Disposition of Human Subject’s  
 
           Samples and Data 
 
 
 
 
 
 
43 
 
References 
 
 
 
 
 
 
 
 
 
46 
 
Appendix 
 
 
 
 
 
 
 
 
 
53 
 
 


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Version Date: 2021 July 22 
 
Page 3 
 
STATEMENT OF COMPLIANCE 
The protocol will be carried out in accordance with International Conference on 
Harmonisation Good Clinical Practice (ICH GCP) and the following:  
• United States (US) Code of Federal Regulations (CFR) applicable to clinical 
studies (45 CFR Part 46, 21 CFR Part 50, 21 CFR Part 56, 21 CFR Part 312, 
and/or 21 CFR Part 812)  
 
National Institutes of Health (NIH)-funded investigators and trial site staff who are 
responsible for the conduct, management, or oversight of NIH-funded trials have 
completed Human Subjects Protection and ICH GCP Training. 
The protocol, informed consent form(s), recruitment materials, and all participant 
materials will be submitted to the Institutional Review Board (IRB) for review and 
approval.  Approval of both the protocol and the consent form must be obtained before 
any participant is enrolled.  Any amendment to the protocol will require review and 
approval by the IRB before the changes are implemented to the study.  In addition, all 
changes to the consent form will be IRB-approved; an IRB determination will be made 
regarding whether a new consent needs to be obtained from participants who provided 
consent, using a previously approved consent form. 


Abbreviated Title: NST 
Version Date: 2021 July 22 
 
Page 4 
 
Précis 
 
The balance between energy expenditure (EE) and energy intake ultimately 
determines body weight. Resting EE is the major component (60-75%) of total EE in an 
adult human being. Resting EE dynamically adapts to environmental changes such as 
ambient temperature. In our on-going study of environmental temperature changes within 
and around the thermoneutral zone, we observed that healthy young men can increase EE 
by 17 % of the basal metabolic rate through the process of non-shivering thermogenesis 
(NST).  This capacity for NST is unexpectedly large as compared to prior reports of mild 
cold-induced thermogenesis (3 to 11%) and suggests that increasing NST could be 
explored as an intervention to combat obesity. 
 The aim of this study is to better understand the physiology of NST and to 
develop improved assays for evaluating the effect of drugs that alter EE. For example, 
only recently has it been realized that brown adipose tissue is functional in adult humans 
and that white adipose tissue can be converted to brown-adipose-like tissue to increase 
heat production during cold exposures. Moreover, skeletal muscle likely also plays a role 
in cold-induced thermogenesis even before overt shivering occurs. It is plausible that the 
mechanisms governing heat production for NST contribute to regulation of body weight 
and thus may be contributing to the current obesity epidemic:  even small changes in EE, 
if not compensated by changes in food intake, can have long-term effects on body weight.   
This protocol has two phases.  The first uses a pharmacologic approach to 
investigate the mechanism of NST in young healthy lean males. Since the principal 
physiologic stimulus to BAT (and possibly muscle for NST) is via the sympathetic 
nervous system (SNS), -adrenergic receptors may hold key roles in regulating human 
EE. We hypothesize that, by careful measurements of NST (at an individually-titrated 
cool environmental temperature, between 18-21ºC vs. at thermoneutrality of 27ºC) and 
using -adrenergic drugs that differ in receptor specificity and agonist/antagonist 
properties, we will gain better understanding of the regulation of human NST.   
The second phase of the study focuses on measuring of FDA-approved drugs’ 
(such as anti-obesity drugs) potential effect on basal metabolic rate (BMR) under 
thermoneutral conditions. The rationale is that previous studies of drug effect on EE in 
humans have not always rigorously enforced the use of thermoneutral conditions, thus 
may have increased variability and underestimated the effect, contributing to 
inconclusive findings.  
It is envisioned that this study will further our knowledge of the mechanisms that 
regulate the acute adaptive changes in resting energy expenditure and the effects of drug 
therapy targeting obesity in humans.   
 
 


Abbreviated Title: NST 
Version Date: 2021 July 22 
 
Page 5 
 
Abbreviations: 
 
BAT 
brown adipose tissue 
BMI 
body mass index 
BMR 
basal metabolic rate 
Cmax 
maximum plasma concentration 
CT 
computed tomography 
DEOB 
Diabetes, Endocrinology, and Obesity Branch 
DXA 
dual energy X-ray absorptiometry 
EE 
energy expenditure 
EKG 
electrocardiograph 
FDG 
[18F]-fluorodeoxyglucose 
GWAS 
genome wide association study 
IRB 
institution review board 
Ki 
binding receptor affinity 
MRI 
magnetic resonance imaging 
NST 
Non-shivering thermogenesis 
PET 
Positron emission tomography 
REE 
resting energy expenditure 
RQ 
respiratory quotient 
SNS 
sympathetic nervous system 
Ucp 
uncoupling protein 
VAS 
visual analog scale 
TNZ 
thermoneutral zone 


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Page 6 
 
 
I.  Introduction and Background:  Energy expenditure and environmental 
temperature 
Resting energy expenditure (EE) comprises 60-75% of the total EE in an average 
adult human. Resting EE is not static. Like other mammals, humans adjust EE (or heat 
production) to balance heat loss in order to maintain constant body temperature. In 
rodents, the relationship between EE and environmental temperature is shown graphically 
in figure 1. In an ongoing protocol (12-DK-0097), we are mapping this response in men 
and women to determine the effect of adiposity, age, and race on the response to a short-
term and tolerable temperature manipulation.   
Animals invoke physiological adaptations such as vasoconstriction and 
piloerection in response to the changing ambient temperature. The temperature range at 
which such adaptations are sufficient to maintain a minimum heat production to balance 
the heat loss is called a thermoneutral zone (TNZ). 1 2 Within the TNZ, resting EE is 
known as basal metabolic rate (BMR). When these responses cannot sufficiently 
compensate for the increased heat loss associated with the further decrease in ambient 
temperature, additional EE is needed. Figure 1 shows this thermogenic response process 
is gradual which reflects the net heat balance of the animal. Moreover, when exposed to 
extreme cold, muscle shivering is recruited to increase heat production, but cannot be 
maintained for a prolonged period of time. In contrast, exposure to mild cold induces 
non-shivering thermogenesis (NST),3 which may be sustainable and tolerable. NST has 
been studied to a limited extent in adult humans, with high variability in its estimates due 
to several potential factors (Table 1). 
  
 
 
 
One source of variability is the actual definition of NST used in the studies. NST 
is the % change in resting EE at a defined cold temperature without shivering as 
compared to the BMR measured at thermoneutrality (within the TNZ). Individual TNZ is 
Figure 1. Graphs of the metabolic rate (oxygen consumption) vs environmental 
temperature relationships, Figures 1 and 4 in Cannon and Nedergaard 1.  BMR is 
basal metabolic rate (also known as resting EE in humans). 


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Version Date: 2021 July 22 
 
Page 7 
variable, for which traditional measurements of resting EE (not the true BMR) often do 
not carefully control for this factor. The second cause may be the differences in testing 
conditions, including the ambient temperature used for cold exposure and the insulating 
properties of the clothing worn during testing (quantified by a commonly used term clo 
for measuring the thermal insulation of clothes, where zero [0] Clo corresponds to a 
naked person and one [1] Clo corresponds to a person wearing a typical business suit.)   
Other sources of variability in NST include food intake, posture and physical activity 
(Table 1) during the testing period.   
 
It was recently rediscovered that brown adipose tissue (BAT) is functional in 
adult humans 4-7 and its presence and activity appear to be highly variable.  The primary 
function of BAT is to maintain body temperature and may be an important contributor to 
NST.  BAT releases the energy from substrate oxidation as heat via uncoupling protein 1 
(UCP1), rather than using the energy to generate ATP 8.  Moreover, non-shivering heat 
generation may also occur in skeletal muscle, through browning of white adipose tissue, 
and by other factors that contribute to the high variability of the cold-induced 
thermogenesis in the literature. Taken together, there is great interest in understanding the 
underlying mechanisms governing NST contribution to total body energy homeostasis 
and body weight regulation.  Understanding these control mechanisms will provide 
insight into the pathophysiology underlying the current epidemic of obesity and should 
provide insight into potential treatments.   
 
Table 1. A brief summary of literature review of cold-induced thermogenesis. 
% Increase Metric 
Temp range, 
C 
Clo 
Reference 
7.0 
24h resting 22-28 
NA 
Dauncey MJ (1981) 9  
11.0 
day resting 22-28 
NA 
Dauncey MJ (1981) 9 
6.0 
24h resting 16-22 
0.71 
van Marken Lichtenbelt (2002) 10 
4.9 
24h resting 16-22 
0.71-0.8 Wijers SL (2007) 11 
6.0 
day resting 19-24 
0.55 
Celi FS (2010) 12  
3.5 
day resting 16-22 
0.8 
Wijers SL (2011) 13 
For 
comparison: 
  
17.4 
day resting 18-27 
0.36 
12-DK-0097, preliminary analysis 
 
 
  
Within the scope of protocol 12-DK-0097, while defining the detailed relationship 
between resting EE and environmental temperature, we are defining the maximum 
amplitude or the capacity of NST.  In brief, in that protocol the range of environmental 
temperatures being studied (16-31°C) is that routinely encountered during daily activity, 
including the comfort zone 14 and slightly lower.  Importantly, the protocol is designed to 
minimize the contributions of physical activity, thermic effect of food, and behavioral 
changes (such as changes in body position) and standardizes the insulating properties of 
clothing worn for testing (clo=0.36).  Thus the measured EE is expected to reflect NST in 
response to the environment.  The protocol determines the following parameters 


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Page 8 
experimentally (see Figure 1 1,15):  1) the thermoneutral zone (TNZ), defined as the 
environmental temperature range in which EE is at a minimum and does not change with 
environmental temperature, 2) the shivering threshold, defined as the lowest 
environmental temperature before shivering occurs, and 3) the capacity for non-shivering 
thermogenesis, defined as the difference in EE measured at the shivering threshold and at 
thermoneutrality.   
To our knowledge, 12-DK-0097 is providing the first rigorous measurement of 
the capacity for non-shivering thermogenesis in humans.  In a preliminary analysis of the 
first subjects, a lean Caucasian cohort, EE at the shivering threshold was 17.4 3.7% 
(n=6) greater than BMR.  We were initially surprised by this unexpectedly large capacity 
for non-shivering thermogenesis, as prior publications of mild cold-induced 
thermogenesis found increases of only 3 to 11% (Table 1). However, we believe that the 
17% value as seen in our current study is plausible for the following reasons:  1) our 
measurements are in the fasting state, removing any masking of facultative thermogenesis 
by the thermic effect of food (a phenomenon already recognized by Rubner in 1902 9), 2) 
strict enforcement of no physical activity, 3) strict prohibition of postural and behavioral 
changes, and 4) actual measurement of the TNZ and shivering threshold, ensuring that 
the full span of NST is captured, compared to two fixed temperature points which were 
used by most previous studies.  We note that a crucial innovation is the use of a relatively 
short time period for the EE measurements, allowing stricter control of the above 
variables, enabled by specialized analysis of the chamber data 16.   
The closest estimation to our magnitude of NST was found by Claessens-can 
Ooijen et al., which a 17% increase in EE was detected in lean male subjects.17 It is worth 
noting that the testing conditions were quite different in this study, where all 
measurements were conducted at 15°C. The thermoneutral state was define when the 
subjects wore standard clothing consisted of sweat shirts and pants (clo =0.71) and 
covered with a duvet (clo=6.8). When the duvet was removed, the 1-hr exposure was 
defined as mild-cold. It is possible that a steady-state EE was not achieved by this design. 
Davis 18 reported a 34% increase over resting following 31 days of exposure to 12 °C for 
8 h per day where significant shivering at the first 20+days. While this is believed to be 
an acclimation effect but demonstrates a very significant potential capacity in healthy 
lean men. It was not understood at that time that brown fat was heat-producing,  
In summary, the preliminary results from 12-DK-0097 demonstrate that 1) a 
suitable experimental system can be devised to measure the capacity for NST in humans 
and 2) the NST contribution to EE is greater than previously recognized.   
 
The two major questions asked in this protocol are:   
• What are the mechanisms driving human NST? 
• What are the effects of drugs on human BMR? 
 
I.1  Scientific and Clinical Significance 
 
The high prevalence of obesity is a major public health concern. Small differences 
in energy expenditure, if not compensated by changes in food intake, would have long-
term effects on body weight.19 20  Mild cold environmental temperatures elevate energy 
expenditure in humans, likely via  BAT activation 4.  It is plausible that an increasingly 
thermoneutral environment is contributing to the obesity epidemic.  Conversely, 


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Page 9 
increasing heat production via lowering of environmental temperature could be a 
potential approach to combat obesity. This study will expand our understanding of the 
mechanisms underlying human thermal physiology in a temperature range that is close to 
those encountered in daily indoor living. Moreover, several FDA-approved drugs’ (such 
as anti-obesity drugs) currently on the market may act through mechanism(s) that 
modulate BMR and/or NST, however, no previous studies have rigorously quantified 
their impact with precise measurements as we plan to accomplish in this protocol.   
 
I.2  Mechanistic basis for NST 
What are the physiologic mechanisms responsible for human NST? To answer 
this question, the contributions of adipose tissue and muscle must be considered as well 
as shared and unique signaling pathways, neurotransmitters and hormones. Experiments 
in mice suggest that non-shivering facultative thermogenesis occurs in at least two 
tissues.  BAT is believed to be the major source of facultative thermogenesis via 
uncoupling protein 1 (Ucp1) 8,21.  (In this protocol, ‘BAT’ is used in the generic sense and 
includes both classic (typically interscapular in location) BAT and inducible BAT, also 
known as beige or brite adipose tissue 22).  Indeed, some data suggest that Ucp1 ablation 
cannot be compensated for by other mechanisms in sufficient magnitude to prevent 
shivering 23.  However, clinical data 13 and recent experiments in mice suggest that 
muscle can also contribute significantly to NST, likely via sarcolipin, SERCA, and/or 
RyR1 24.   
Assuming BAT is the chief source of facultative thermogenesis, one would like to 
quantitatively measure BAT activation (both tissue volume and metabolic activity per 
unit volume).  However, the current standard technique for measuring BAT activity is 
FDG-PET/CT, which has two major limitations: (1) it is not suitable for multiple serial 
measurements due to radiation dose considerations, and (2) it only measures glucose 
uptake which is about 10% of the total substrates that is utilized by BAT 3.  We therefore 
have chosen a primarily pharmacologic approach to investigate the drivers of NST.     
The principal physiologic stimulus to BAT (and possibly muscle for NST) is via 
the sympathetic nervous system (SNS). When activated, sympathetic nerves release 
norepinepherine which binds to - and -adrenergic receptors on adipocytes to stimulate 
heat production, with the -adrenergic receptors driving thermogenesis.  Indeed there are 
clinical reports that the -adrenergic receptor blocker propranolol reduces the uptake into 
BAT during FDG-PET studies by 55 to 75% 25,26.  While adipose tissue shares 1- and 
-adrenergic receptors with many tissues, the tissue distribution of -adrenergic 
receptors is restricted and thus 3-adrenergic agonists could be used to selectively target 
adipose tissue, as is done in preclinical studies (eg 27).  However, the -adrenergic 
receptors on adipose tissue do not appear to be interchangeable. While 3-receptors are 
typically assumed to be the major driver, -adrenergic receptors alone are sufficient for 
normal BAT morphology 28 and the -adrenergic receptor has been reported to be able 
to mediate most of the sympathetic stimulation of cold-induced thermogenesis 29.  -
adrenergic agonists can also augment BAT stimulation, but do not contribute in the 
absence of -adrenergic stimulation 30.     
While the molecular details of muscle NST are not clear, it appears that in mice, 
dantrolene, via inhibition of the ryanodine receptor calcium channel, Ryr1, can inhibit 


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Page 10 
muscle NST 24.  Thus, dantrolene is a unique tool that may allow distinguishing of 
muscle vs BAT sources of NST.    
In this protocol we will focus on the regulatory pathways of human NST via 
pharmacological modulations of the SNS receptors.  The results of these studies will 
allow us to elucidate the receptors controlling NST in humans, and by inference, the 
contribution of BAT.   
  
Propranolol 
This study will target the -adrenergic system contribution to NST.  A non-
specific -adrenergic antagonist might be expected to inhibit NST via abrogation of 
sympathetic signaling to BAT.  Propranolol is a  blocker approved for multiple 
conditions including hypertension, angina, atrial fibrillation, and others (Inderal label: 
http://www.accessdata.fda.gov/drugsatfda_docs/label/2011/016418s080,016762s017,017
683s008lbl.pdf).  While propranolol is reported to inhibit the cold-stimulated uptake of 
glucose into BAT 25,26, it has also been reported that propranolol does not inhibit cold-
induced thermogenesis 13. There are several possible explanations to these previous 
findings.  Propranolol is 1/-selective, with binding receptor affinity (Ki) for human 
1-, -, and -adrenergic receptors of 1.8, 0.8, and 186 nM, respectively 31.  Based on 
the pharmacokinetics of propranolol, with a maximum plasma concentration (Cmax) at 
~1-4 hours of ~150 ng/ml and t1/2 of ~4 hours (both parent and active metabolite) 32,33, 
the plasma concentration at Cmax is calculated as ~580 nM.  For a 160 mg dose of 
regular propranolol (not sustained release),  and after correcting for protein binding (90% 
bound 34), the free plasma concentrations at Cmax are 32, 73, and 0.3 times the Ki of the 
1-, -, and -adrenergic receptors, respectively. Thus, while this concentration should 
completely inhibit signaling via the 1- and -adrenergic receptors, its inhibition on the 
-adrenergic receptor is incomplete at best, with the level of inhibition dependent on the 
actual endogenous norepinepherine concentration.  Since the actions of a 160 mg 
propranolol dose on suppression of the exercise-induce heart rate increase (a 1 effect) 
lasts 36 hours 33, a suitable wash-out interval is 48-72 hours.  Note that based on the 
pharmacokinetics and receptor selectivity of propranolol, it is not surprising that 160 mg 
of sustained-release propranolol 13 is predicted to have a Cmax of of ~80 nM, which is 
4.4-, 10- and 0.43-fold of the Ki of the 1-, -, and -adrenergic receptors, 
respectively.  The inhibition at Cmax of glucose uptake into BAT by 20 mg 26 or 80 mg 25 
of regular propranolol may suggest that BAT glucose uptake is largely via 1-and/or -, 
and not - adrenergic receptors.  
The effect of 160 mg propranolol at thermoneutrality is expected to be slight. 
Propranolol’s effect on EE has been studied multiple times, with three examples being a 
reduction of 4.1% (non-significant 35), 1.6% (non-significant 36), and 5.0% (p=0.002 11.  
At the low temperature, 160 mg should have a proportionally greater effect on reducing 
EE, although any prediction of the magnitude is largely an estimate.   
 
Pindolol 
A complementary test of the drivers of NST would be use of a selective -
adrenergic agonist 37, but none is currently approved for this use in humans.  However, 
pindolol (Visken, label: 
http://www.accessdata.fda.gov/drugsatfda_docs/label/2007/018285s034lbl.pdf) is an 


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approved drug with properties suitable for ‘clamping’ the -adrenergic tone.  Pindolol is 
a mixed partial agonist and antagonist.  At complete receptor occupancy, a partial agonist 
gives less response than that of the native ligand.  In the presence of a full agonist, 
saturating partial agonist will reduce the receptor response.   
Pindolol has pharmacokinetic and binding properties indicating that full receptor 
occupancy can be achieved with single oral dosing in humans.  Specifically, pindolol has 
binding Kis of 2.6, 4.8, and 44.1 nM for the human 1-, -, and -adrenergic receptors, 
respectively 31.   The pharmacokinetics of a 20 mg dose of pindolol include a Cmax at 
~1-2 hours of ~96 ng/ml, t1/2 of ~2.9 hours, and 38% plasma protein binding 38.  From 
the total plasma concentration at the Cmax of ~387 nM, one can estimate that the Cmax 
after a 20 mg dose of pindolol will yield free plasma concentrations that are 92-, 50-, and 
5.4-fold the Kis of the 1-, -, and -adrenergic receptors, respectively.  These data 
indicate that 20 mg of pindolol should saturate all three endogenous -adrenergic 
receptors.   
Pindolol is an antagonist at 1-adrenergic receptors 31,39 and a partial agonist at 
-adrenergic receptors 31,40.  It may either be a partial agonist 39 or antagonist 31 at the 
-adrenergic receptor.  Interpreting reports of -adrenergic receptor selectivity that 
antedate discovery of the -adrenergic receptor and of partial agonism that do not 
consider biased agonism 41 add some uncertainty.  However these are moot, assuming 
that pindolol saturates all three -adrenergic receptors.   
If the predominant signaling to human NST mechanisms is via -adrenergic 
receptors, one expects that by clamping -adrenergic tone with pindolol, the same level 
of change in REELow and REE27°C will be observed.  Since pindolol is a partial agonist, 
the prediction is that this clamped EE will be elevated moderately at a level between 
REE27°C and REELow with placebo.     
   
Dantrolene 
To differentiate NST from skeletal muscles, we will use the following two 
pharmacologic agents. Dantrolene (Dantrium, label:  
http://www.accessdata.fda.gov/drugsatfda_docs/label/2012/017443s043s046s048s049lbl.
pdf ) is clinically indicated for treatment of muscle spasticity 42 and of malignant 
hyperthermia 43.  Dantrolene binds and inhibits the muscle ryanodine receptor (RyR1), 
which releases calcium from the sarcoplasmic reticulum 44,45.  A recent study in mice 
suggests that dantrolene blocks muscle NST, but has no effect on BAT NST 24.   
Clinical dosing of dantrolene for spasticity is via titration from 25 mg QD to up to 
100 mg TID, or even 100 mg QID (label).  The pharmacokinetics of a single 100 mg oral 
dose of dantrolene include a Cmax at ~4.5 hours of 0.7-1.45 ug/ml (2.2-4.6 uM; Mr is 
314), and t1/2 of 9-12 hours 46-48.  Single oral doses as high as 150 mg have been reported 
47.   
It is not clear what level of dantrolene is needed to maximally inhibit RyR1.  
Dantrolene is hydrophobic and highly bound to albumin 49, but we are not aware of any 
clinical measures of free drug levels.  Thus, how an in vitro IC50 of 300 nM 45 
corresponds to the exposure achieved with a single 100 mg oral dose of dantrolene is 
unknown.   


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We are not aware of prior studies of the effect of dantrolene on EE under 
conditions similar to those in this protocol.  The prediction is that a single oral 100 mg 
oral dose of dantrolene will significantly inhibit muscle NST in humans.   
Magnesium sulfate 
 
Magnesium, a non-competitive inhibitor of inositol 1,4,5-triphosphate (IP3)-gated 
calcium channel and RyR1receptor is a mild muscle relaxant 50-53. Although, the exact 
cellular mechanisms are unclear, intravenous magnesium in randomized controlled trials 
suppresses post-operative shivering (relative risk 0.38, 95% CI: 0.17-0.88) 54-56. In 
healthy individuals, magnesium sulfate (80 mg/kg bolus followed by a maintenance 
infusion at 2 g/h) reduces shivering threshold, albeit minimally 57. Nevertheless, 
intravenous magnesium sulfate is included in pharmacological anti-shivering regimens 
during therapeutic hypothermia 58. In these clinical trials, serum magnesium 
concentrations increased by 31-84% (mean: 2.3 mmol) following a median cumulative 
intravenous magnesium dose of ~ 8 g 54.  Bolus intravenous magnesium (2 g) results in 
an acute but transient increase in serum magnesium (~ 2 mmol) with a t1/2 of ~ 4 h in 
individuals with normal renal function. However, steady state serum concentrations of ~ 
2.2 mmol can be achieved by a maintenance infusion dose of 2 g/h 59. Possible adverse 
reactions associated with intravenous magnesium include, nausea, flushing, mild 
hypotension, bradycardia, and minimal irritation at the infusion site 54.   
Whether intravenous magnesium affects NST is not known. Given the inhibitory 
effects of magnesium on skeletal muscle excitation-thermogenic coupling and calcium 
handling, we predict that increasing magnesium concentrations significantly inhibits 
muscle NST in humans. 
 
Mirabegron 
 
Mirabegron is a selective β3-AR agonist approved by the FDA in June 2012 for 
treatment of overactive bladder at a dose of 50mg once daily. (Myrbetriq label: 
http://www.accessdata.fda.gov/drugsatfda_docs/label/2012/202611s000lbl.pdf )  
According to the advisory committee report (FDA 2012), the peak mean concentration 
was achieved between 3 and 4.3 hours (p. A-81).  At 200mg, the maximal mean increase 
in pulse was 10.3 -11.8 bpm (p. A-80) and in SBP and DBP, 11.6 and 7.7 mm Hg, 
respectively, as compared to placebo at hours 3 to 6 post-dose (p.A-81).  Of note, the 
subgroup analysis by gender revealed that at a dose of 200mg, in females, mirabegron 
prolonged the QTc interval by a maximum of 10.42 msec, but there was no QTc 
prolongation in males (p. A-85).  For this reason, the present study will recruit only 
healthy male volunteers with normal baseline QTc.  No clinical effects of mirabegron 
atrial fibrillation or heart failure were detected (p. A87-A88). 
 
Mirabegron has binding Kis for the human β1-, β2-, and β3-adrenergic receptors 
of 4200, 1300, and 40 nM, respectively.  These values demonstrate a much greater β3-AR 
selectivity compared with pindolol , making mirabegron a promising candidate for 
adipose tissue-specific activation.  In males, for 50 mg, the Cmax and Tmax were 23.7 
ng/mL and 3.50 h, respectively, and for 200 mg, the Cmax and Tmax were 158 ng/mL and 
2.68 h, respectively (FDA 2012).  In our experience, as will be described in detail below, 
the Cmax for a 200 mg dose was 274 ± 64 nM based on 65% protein binding, which is 
6.9x the Ki of mirabegron for the β3-AR (40 nM).  If the 50 mg dose achieves 18% of 


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200 mg, then the Cmax would be 49 nM, which is 1.2x the Ki and 2.2x the in vitro EC50 of 
22.4 nM (Takasu et al., 2007).    
We have recently completed a pilot study of the effectiveness of a one-time dose 
mirabegron 200 mg to stimulate human BAT and increase energy expenditure in young 
(22.2±0.6 years), lean (22.7±0.5 kg/m2), healthy men who were already known to have 
detectable BAT via PET/CT.  The pilot study achieved its primary endpoint by 
demonstrating that mirabegron at 200mg significantly stimulated BAT metabolic activity 
and led to a 13% increase in RMR, demonstrating that a β3-AR agonist can stimulate 
human BAT thermogenesis and may be a promising treatment for metabolic disease. 
 
The principal limitation of the pilot study is that the dose used (200mg) was 
higher than the approved dose of 50mg, and this difference was associated with clinically 
relevant adverse effects.  At 3.5 hours after dosing with 200mg mirabegron, we observed 
increases in mean HR (14 ± 3 bpm) and systolic BP (11 ± 2 mmHg), but not diastolic BP 
(2 ± 1 mmHg).  This is consistent with information provided in the Background 
Document for Meeting of Advisory Committee for Reproductive Health Drugs (FDA 
2012); although, a slightly higher increase in diastolic blood pressure (+7.7 mm Hg) was 
reported by the FDA, which is greater than our previous experience with a 200 mg dose 
of the drug. These treatment-emergent adverse effects are thought to be a result of off-
target binding of the drug to β1-ARs (Cypess 2015).  Although very promising, these 
preliminary results indicate that mirabegron will not be useful clinically unless the 
cardiovascular stimulation can be limited while still stimulating NST and BAT.   
 
To conduct future studies of mirabegron-induced NST, we will need to determine 
the optimal dosing strategy for mirabegron.  In the first phase of this sub-study, we will 
perform a dose-response analysis of mirabegron at 0mg, 50mg, and 200mg, using mild 
cold exposure as a positive control.  Should the 50mg dosage be sufficiently effective, 
this dose will be used in subsequent studies.  If not, we will consider a second sub-study 
in which mirabegron will be combined with a selective β1-AR blocker, likely choosing 
pindolol or nebivolol. 
 
Of note, the study will continue the use of pindolol in Cohort 1 (as described 
above). There are at least two reasons we have chosen to do so. First, its mechanism of 
action is quite different from mirabegron.  It can serve as a negative control for β3-AR 
binding while having β1 and β2 binding. Second, 8 subjects (as of January, 2015) have 
already taken pindolol as part of Cohort 1, so it would be important to continue until the 
planned interim/final analysis before deciding to terminate its use early. 
 
 
I.3  Assaying anti-obesity drugs for their effects on EE 
 
Significance of improving assay of drugs that stimulate EE 
 
When studied in detail in mice, it appears that the vast majority of centrally-acting 
drugs have dual beneficial effects of decreasing food intake and increasing metabolic rate 
60,61.  Despite of the huge magnitude of facultative thermogenesis in mice, care is not 
always being taken to test drugs for metabolic effects at thermoneutrality.62  While the 
effect of environmental temperature on metabolic rate was first described by Lavoisier in 
1790 63, studies of drug effect on EE in humans have not always rigorously enforced the 


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use of thermoneutral conditions.  We propose to first explicitly demonstrate the need for 
measuring drug effect under thermoneutral conditions and then re-evaluate the effect of 
anti-obesity drugs on BMR employing our new knowledge of human capacity for NST 
under our optimized experimental conditions.  
Caffeine 
Caffeine has been chosen as the reference drug (positive control) for studying an 
increase in EE.  Caffeine has multiple pharmacologic actions, with its effects at modest 
doses in humans being attributed to antagonism of the A1R and A2AR adenosine receptors 
64.  Caffeine was first shown to increase EE by Hoppe in 1857 (see 65) and has been 
studied many times since.  In an analysis of 12 studies using doses from 50 to 536 mg 
(published between 1915 and 2008 66-77) there was an approximately linear increase in EE 
with caffeine dose, with ~10% increase at a dose of 300 mg.   
Caffeine is completely absorbed, with a Cmax of ~0.5 hours and a t1/2 of ~4-6 
hours with multiple active metabolites 64.  Notably, there is attenuation of 
pharmacodynamic responses to caffeine with repeated dosing, which is avoided by 
keeping at least 72 hours between doses 78,79.   Possible adverse reactions are: frequent 
passing of urine, nervousness, restlessness, diarrhea, headache, and stomach upset.  We 
propose dosing 300 mg caffeine, which is the same amount as in the Starbucks Grande 
size (http://www.cspinet.org/new/cafchart.htm).  Actions of caffeine include some 
increase in blood pressure and heart rate 78 and increased alertness and reduced fatigue 80.   
300 mg caffeine is predicted to increase EE at thermoneutrality by at least 10% 
(the effect will be larger if the prior studies were underestimations due to not using 
thermoneutral conditions).  The effect on BMR by caffeine will serve as a positive 
control for the potential effects by other anti-obesity drugs. 
 
 
Phentermine  
 
Phentermine is a sympathomimetic drug approved for the short-term treatment of 
obesity (Adipex-P label: 
http://www.accessdata.fda.gov/drugsatfda_docs/label/2012/085128s065lbl.pdf).  The 
dose of 37.5 mg daily (30 mg free base) is twice the amount in the recently approved 
drug Qsymia (see below).  The only publication known to us reporting the effect of 
phentermine on EE was of a 1.4% non-significant increase in resting metabolic rate with 
8 mg TID x 5 days 81.  Phentermine has a Cmax at 2-4 hours and a t½ of 19 4 hours.  
These pharmacokinetics suggest that 4 days should be allowed after dosing for drug to be 
adequately cleared.     
We propose dosing 37.5 mg phentermine (regular, not sustained release), the 
recommended daily dose. The effect of a single dose of 37.5 mg phentermine will be 
studied only at thermoneutrality.  There may be attenuation of any EE increase with 
continued dosing, so it is difficult to know what to predict from a single dose compared 
to the prior experience with 5 days dosing.   
 
Topiramate 
 
Topiramate is a drug with multiple pharmacologic actions, including blockage of 
voltage-dependent sodium channels, augmentation at some GABA-A receptor subtypes, 
antagonism of AMPA/kainate subtype of the glutamate receptor, and inhibition of 
carbonic anhydrase (particularly isozymes II and IV) 82.  Topiramate is a component of 


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Qsymia (see below) and is also indicated for epilepsy, migraine prophylaxis (Topamax 
label: 
http://www.accessdata.fda.gov/drugsatfda_docs/label/2011/020505s042,020844s036lbl.p
df).   
After topiramate 100 mg orally, the Cmax is 1.73ug/ml (5.1uM; Mr=339) and 
after 200 mg it is 10.9uM 83,84.  For single oral doses of 100-1200 mg, Tmax is 1.4-4.3 h; 
elimination t1/2 ~21 hours.  The pharmacokinetics suggest that 4 days should be allowed 
after dosing for drug to be adequately cleared and that the Cmax after a single dose of 
200 mg models the steady state of 100 mg daily dosing.  We have not located any studies 
of the effect of a single dose of topiramate on EE.  The interpretation of EE in the setting 
of chronic dosing 85,86 is complicated by changes in body weight.       
 
Qsymia 
Qsymia (label: 
http://www.accessdata.fda.gov/drugsatfda_docs/label/2012/022580s000lbl.pdf) was 
approved by the FDA in July 2012 and marketed in September 2012.  Qsymia is a 
combination of phentermine and topiramate, with the highest dose being 15 mg/92 mg.  
The most common adverse reactions are: paraesthesia, dizziness, dysgeusia, insomnia, 
constipation, and dry mouth.  There are no studies of the effect of Qsymia on EE.  Details 
of the constituent agents are in the preceding sections.  
 
Naltrexone 
 
Naltrexone (label: http://www.accessdata.fda.gov/drugsatfda_docs/nda/2000/75-
434_Naltrexone%20Hydrochloride_prntlbl.pdf) is an opioid antagonist used in the 
treatment of ethanol dependence at 50 mg orally daily, which may alternatively be given 
as 100 mg every other day or 150 mg every third day.    Naltrexone is a component of 
Contrave (48 mg naltrexone SR, 400 mg bupropion SR), being developed by Orexigen 
for the treatment of obesity.   Naltrexone is completely orally absorbed and undergoes 
first-pass metabolism.  After single oral doses of 50, 100, or 200 mg, the AUC and Cmax 
were dose proportional and the Cmax occurred at ~1 hour for both naltrexone and the 
active 6-naltrexol metabolite, with elimination t1/2s of ~4 and ~14 hours, respectively 
87-90 .  Doses of up to 300 mg orally, twice daily for three days produced a non-significant 
4.6% increase in metabolic rate 91.   
 
II. Research Objectives 
This protocol builds on the experience from our current ongoing research (12-
DK-0097, titled “Energy expenditure responses to a range of environmental temperatures 
around the thermal neutral zone”.  We plan to use the same assay conditions and 
inclusion/exclusion criteria as in that protocol, minimizing changes to accommodate the 
goals of this protocol, which are to address the following questions:      
• What are the mechanisms driving human NST? 
• What are the effects of drugs on human BMR? 
 
These studies will be performed in healthy male subjects’ age 18-35 years with a BMI 
between18.5 and 25 kg/m2.   
 


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II.1 Primary Objectives 
 
Cohort 1 – This Cohort has completed enrollment  
1) To evaluate the effect of propranolol 160 mg PO on resting EE at a temperature just 
above the subject’s placebo shivering threshold. 
 
2) To evaluate the effect of pindolol 20 mg PO on the difference in resting EE 
(comparing thermoneutrality to a temperature just above the subject’s placebo shivering 
threshold). 
 
3) To evaluate the effect of dantrolene 100 mg PO on resting EE at a temperature just 
above the subject’s placebo shivering threshold. 
 
4) To evaluate the effect of magnesium sulfate (50 mg/kg bolus followed maintenance 
infusion at 2 g/h) on resting EE at a temperature just above the subject’s placebo 
shivering threshold. 
 
Hypotheses 
1) Propranolol 160 mg PO will reduce the resting EE measured at a temperature just 
above the subject’s placebo shivering threshold (REELow) by greater than or equal to 5% 
compared to placebo REELow.  If propranolol-treated subjects shiver at the low 
temperature, then measurement of non-shivering EE is not possible in this protocol.  
Propranolol will be judged to increase the shivering threshold if significantly more 
subjects shiver with propranolol than with placebo treatment.   
 
2) Pindolol 20 mg PO will lead to a similar difference in resting EE measured at 
thermoneutrality (REE27°C ) and REELow. 
 
3) Dantrolene 100 mg PO will reduce the REELow by greater than or equal to 5% 
compared to placebo REELow.  If dantrolene-treated subjects shiver at the low 
temperature, then measurement of non-shivering EE is not possible in this protocol.  
Dantrolene will be judged to increase the shivering threshold if significantly more 
subjects shiver with dantrolene than with placebo treatment.   
 
4) Magnesium sulfate (50 mg/kg bolus followed maintenance infusion at 2 g/h) will 
reduce the REELow by greater than or equal to 5% compared to REELow.   
  
Cohort 2 
1) To evaluate the effect of caffeine (300 mg PO) on BMR (=REE27°C).  
 
2)  To evaluate the effect of Qsymia (topiramate 92 mg CR, phentermine 15 mg PO) on 
BMR.   


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3) To evaluate the effect of phentermine (37.5 mg PO) on BMR.   
 
4) To evaluate the effect of topiramate (200 mg PO) on BMR.   
 
5) To evaluate the effect of naltrexone (100 mg PO) on BMR.   
 
Hypotheses 
 
1)  Caffeine 300 mg PO will increase BMR by greater than or equal to 5% vs placebo. 
 
2)  Qsymia (topiramate 92 mg CR, phentermine 15 mg) PO will increase BMR by greater 
than or equal to 5% vs placebo.   
 
3)  Phentermine 37.5 mg PO will increase BMR by greater than or equal to 5% vs 
placebo.     
 
4)  Topiramate 200 mg PO will increase BMR by greater than or equal to 5% vs placebo.   
 
5)  Naltrexone 100 mg PO will increase BMR by greater than or equal to 5% vs placebo.   
 
Cohort 3 – This Cohort has completed enrollment 
1) To compare the effects of mirabegron at 0mg, 50mg, and 200mg on BAT metabolic 
activity. 
 
2)  To compare the effects of mirabegron at 0mg, 50mg, and 200mg on BMR. 
 
Hypotheses 
 
1) Mirabegron at 50mg and 200mg PO will each increase BMR and BAT activity more 
than placebo. 
 
2) The increases in heart rate and systolic BP after a one-time dose of 50mg will be lower 
than that observed using 200mg. 
 
II.2  Secondary Objectives 
 
For all available conditions (temperature and drug/dosage), the following will be reported 
(when not already reported as a primary objective): 
 
1) resting EE using metabolic chamber (Cohorts 1, 2 and 3) 
 
2) difference in resting EE (EE) between REE27°C and REELow (Cohort 1),  
 
3) comparison between placebo EE (Cohort 1) and EE for each drug (Cohorts 1, 2 and 
3) 


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4) difference between drug and placebo resting EE (at the same temperature) (Cohorts 1, 
2 and 3) 
 
II.3  Exploratory outcomes 
1) resting respiratory quotient (RQ): although it is a short-term measure that reflects net 
macronutrient utilization. Our current data in the protocol 12-DK-0097 suggest that this 
is also correlated with ambient temperature.  
 
2) difference in resting RQ between RQ27°C and a temperature just above the subject’s 
placebo shivering threshold RQLow,  
 
3) difference between drug and placebo resting RQ (at the same temperature) 
 
4)  skin and core body temperature 
 
5) heart rate and its variability 
 
6) shivering by surface electromyography and accelerometry 
 
7)  hormonal changes that occur with NST by characterizing the effect of placebo and 
drug regimens on the following analytes, measured fasting at the end of each chamber 
session.  
 
Urine: 
norepinepherine 
epinepherine 
free cortisol 
creatinine 
volume 
 
Plasma: 
norepinepherine 
epinepherine 
glucose 
insulin 
free fatty acids 
-hydroxybutyrate 
T3 
T4 
TSH 
Glucagon 
Cortisol 
Parathyroid Hormone (PTH) 
Adiponectin 
Ghrelin 
Gastric inhibitory polypeptide (GIP) 
Peptide YY (PYY) 
Glucagon-like peptide-1 (GLP-1) 
Leptin 
Growth hormone 
fribroblast growth factor 21 (FGF21) 
irisin 92 
atrial natriuretic peptide (ANP) 93 
ventricular natriuretic peptide (BNP) 93 
Adrenocorticotropic hormone (ACTH)
 
 


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III.  Study Protocol 
 
III.1  Subjects and Recruitment 
We will limit this study to young healthy male subjects with a BMI of 18.5 to 
25.0 and age 18-35 years.  Studying females with the current protocol is not yet feasible 
due to its length which would cause inclusion of both phases of the menstrual cycle, a 
possible confounder.  However, the data from this study and from 12-DK-0097 will be 
used to design a shorter study protocol to study broader populations including women, 
older and younger subjects in the future. Subjects who have participated in 12-DK-0097 
are eligible for this protocol and individual data from that protocol may be used to inform 
choice of the low temperature.  Similarly, subjects who participate in Cohort 1 are also 
eligible for Cohort 2 and/or Cohort 3.  We will allow a wash-out time for the drugs (>48 
hours) and recovery time for the blood draws (to be determined by volume obtained and 
NIH guidelines). There is no additional risk due to increased radiation exposure for 
subjects participating in Cohorts 1 and 2. Although we will use BMI as a screening tool 
for practical concerns, we will measure body composition using DXA.  
This study is planned for 16 volunteers in each of Cohort 1 and 2, with enrollment 
to continue until 12 complete each Cohort.  No interim analysis is planned due to the 
small study size and the low probability that futility would be reached for all of the 
multiple endpoints, the criteria required for early termination.   
 
Power calculation: To estimate power, we used data from 12-DK-0097.  We first 
determined that the within-subject difference in the response in BMR within TNZ was 
normally distributed with a standard deviation of 2.54%. The maximum NST in 9 young 
healthy male subjects was 17.4±3.7%. With 12 subjects completing the protocol, if the 
true difference in the mean response to treatment is 5.0% as we hypothesized for each 
drug treatment, we will be able to reject the null hypothesis that this response difference 
is zero with probability (power) of 0.98 at an alpha of 0.05 in either Cohort. Since this 
study is designed to use the pharmacological agents only to test the mechanisms of NST 
and BMR, thus we will not compare any pharmacological interventions to each other. 
However, in the most conservative interpretation, multiple comparisons adjustments will 
only be necessary for the number of paired comparisons between drugs and placebo 
within each Cohort. The result of such a conservative estimate of power range from 0.92 
at an alpha value of 0.01 (0.05/5, the Bonferroni method) in Cohort 2 to 0.94 in Cohort 1 
(alpha = 0.05/4). Even if the true difference was 3%, the power to detect with n=12 is 
above 0.83 in all analyses. To consider in potential dropouts (about 30%), we will recruit 
16 subjects for each cohort (subjects can participate in both with a minimum of 2 weeks 
washout in between the studies), aiming to have 12 completers for each cohort. 
 
In Cohort 3, the primary endpoint is the difference in detectable BAT activity 
(units of mL*SUVmean*g/mL) between the 0 mg and 50 mg doses of mirabegron.  
Based on our previous study (Cypess et al., 2015), BAT activity is right-skewed and not 


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normally distributed, so our sample size determination is based on log BAT activity, 
which is normally distributed.  We found that log BAT activity after treatment with 0 mg 
mirabegron was 0.05 ± 0.62 and after treatment with 200 mg mirabegron was 2.00 ± 
0.79.  Since it is unknown what the effect of 50 mg mirabegron would be on BAT 
activity, we calculated a range based on lower and upper bound expectations about the 
reported PK/PD profile of mirabegron.  For the lower bound, the 50 mg dose has been 
found to have a mean plasma Cmax that is 18% of 200 mg.  If there were a linear 
relationship between BAT activity and dose, then we would predict 50 mg to produce a 
mean log BAT activity of 0.36.   
 
Using a different set of assumptions leads to a higher predicted effect, or the 
upper bound expectation.  We found that after the 200 mg dose, the estimated mean peak 
plasma concentration (Cmax) of mirabegron was 274 ± 64 nM based on 65% protein 
binding, which is 6.9x the Ki of mirabegron for the β3-AR (40 nM) (FDA 2012).  If the 
50 mg dose achieves 18% of 200 mg, then the Cmax would be 49 nM, which is 1.2x the Ki 
and 2.2x the in vitro EC50 of 22.4 nM (Takasu et al., 2007).  Therefore, 50 mg 
mirabegron could produce ≥50% of the effect seen with 200 mg, or ≥1.00 log BAT 
activity.  Therefore, for 50 mg mirabegron, the effect on log BAT activity ranges from 
0.36 to ≥1.00. 
 
For sample standard deviation, the log BAT activity after treatment with 0 mg 
mirabegron was 0.05 ± 0.62 and after treatment with 200 mg mirabegron was 2.00 ± 
0.79.  Assuming a linear relationship between dose and standard deviation of the outcome 
variable, for the 50 mg dose of mirabegron, the standard deviation for log BAT activity is 
predicted to be 0.66. 
 
In summary, we are planning a study of a continuous response variable from 
study subjects where each acts as his own paired control.  Assuming the true difference 
between 0 mg mirabegron and 50 mg mirabegron is 0.36 with a standard deviation of 
0.66, we will need to study 28 subjects to be able to reject the null hypothesis that this 
response difference is 0 with α=0.05 and power of 80%.  However, if the response is 
1.00, then we will need to study only 6 subjects.  Given this wide range (6-28 subjects), 
we will plan to complete 28 subjects with an interim analysis after 12 subjects. If the 
results of the interim analysis suggest that the response is closer to the upper bound 
estimate, then we will be able to reject our null hypothesis at that point.  A sample size of 
12 was chosen because if 12/12 subjects show higher BAT activity with the 50 mg, then 
non-parametric paired comparison yields a P value of 0.001. 
 
For these types of studies, we have experienced a screen-failure rate of 50% and a 
rate of detectable BAT of 66%.  We therefore plan to enroll up to 84 subjects, perform 
Study Day A in 42, and expect 28 to complete all four study days.  If we are able to 
complete the study after the interim analysis, then we would end up enrolling 36 subjects, 
initiate the screening protocol with 18, and have 12 complete all four study days. 
 
 
III.2-1 Protocol design, Cohort 1 – This Cohort has completed enrollment 


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Cohort 1 is a single-blind, fixed order (for drug treatment), partially randomized 
(for temperature), cross-over study, with data analysis performed in a blinded manner  
All study volunteers will undergo the screening visit evaluation as outlined in 
Table 2, including a medical and laboratory examination to determine study eligibility. 
Qualifying volunteers will be invited for the full study at the Metabolic Clinical Research 
Unit as inpatients. An initial one-day equilibration diet will be standard before metabolic 
testing.  This equilibration period will also serve as an orientation/ acclimatization to the 
metabolic chamber and procedures.    
 
Table 2. Screening tests: 
Eligibility phone contact pre-screen questionnaire   
O 
1st REG H+P or Protocol Screen Progress note 
O 
Temp, Pulse, BP, RR, WT, HT 
O 
Nutritional consult 
O 
Informed Consent Version: 
O 
Consent documented in CRIS 
O 
Eligibility Criteria Documentation 
O 
Clinical Labs/Procedures 
  
EKG 
O 
CBC with Differential 
O 
Acute care + hepatic + mineral panels (Chem 20) 
O 
Lipid Panel 
O 
Thyroid (TSH) 
O 
Toxicology (urine) 
O 
Ferritin 
O 
  
To match the criteria from 12-DK-0097, we will recruit subjects who do not work 
in a cold environment and are not trained athletes. Drugs will be administered exactly at 
8:00 am (+/- 15 minutes), (except dantrolene, which will be administered at 6:00 am (+/- 
15 minutes), and bolus magnesium sulfate (~ 30 min infusion) at 7:30 AM (+/- 30 
minutes) and maintenance infusion at 8:00 AM (+/- 15 minutes)), upon entering the 
metabolic chamber.  We will measure REE at times close to the Tmax of the drugs (9:30-
10:00 and 10:30-11:00).   
During a 17-day admission (with 4 off days on pass) to the NIH Hatfield Clinical 
Research Center, study volunteers will undergo daily EE measurements (up to 4 hours) in 
a metabolic chamber with temperatures set between 18.0 to 21.0 and 27.0C.  
Individualized determination of the lowest environmental temperature that does not cause 
shivering is important to maximize assay sensitivity.  This will be accomplished by using 
the mini-adaptive design approach (Table 3):  On Day 2, the first full inpatient study day, 
volunteers will be acclimatized to the metabolic chamber at 19.0C, wear multiple body 
sensors, and undergo standard  testing procedures that will be repeated throughout the 
study. If shivering occurs on Day 2 at 19.0C, the chamber temperature will be set to 
20.0C on Day 3.  If shivering occurs at 20.0C on Day 3 the low temperature will be 


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21.0C; if no shivering at 20.0C on Day 3, the low temperature will be 20.0C.  If no 
shivering occurs on Day 2, then the chamber temperature will be set to 18.0C on Day 3.  
If shivering occurs at 18.0C on Day 3 the low temperature will be 19.0C; if no 
shivering at 18.0C on Day 3 the low temperature will be 18.0C.   
Table 3 
Day 2 
chamber 
temperature 
Day 2 result 
Day 3 
chamber 
temperature 
Day 3 result 
Low 
Temperature 
19C 
Day 2 shiver 
20C 
Day 3 shiver 
21C 
  
  
  
Day 3 no shiver 
20C 
  
Day 2 no 
shiver 
18C 
Day 3 shiver 
19C 
  
  
  
Day 3 no shiver 
18C 
 
 
Table 4 Protocol flow 
day
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
Sat
Sun Mon Tue Wed Thu
Fri
Sat
Sun Mon Tue Wed Thu
Fri
Sat
Sun Mon
drug
pbo
pbo pbo
pbo
pro
pro
off
off 
dan
dan
Mg
Mg
pin
off
off 
pin
temp.
19
18 or 20 low
27
low
27
low
27
27
low
27
low
notes admit
pass
return
pass
return
d/c
blood
X,X
X,X
X
X
X
X
X
X
X
X
Where pbo=placebo, pro=propranolol (oral 160mg PD), dan=dantrolene (oral 100mg 
PD), Mg=magnesium sulfate (IV), and pin=pindolol (oral 20mg PD). 
   
On Days 4-17, chamber temperature will be varied as indicated in Table 4 and 
procedures will be repeated.  Temperatures will be block randomized (one day at the low 
temperature and one at 27.0C), with blocks being: Days 4,5; Days 6,7; Days 10,11; Days 
12,13; and Days 14,17.  Subjects will not be informed of the environmental temperature.   
Oral medications are placebo on Days 2, 3, 4, and 5; regular propranolol (not 
sustained release) 160 mg on Days 6 and 7; dantrolene 100 mg on days 10 and 11, 
pindolol 20 mg on Days 14 and 17; and magnesium sulfate on Days 12 and 13.  Since it 
is not possible for all medications to have the same appearance, drug or placebo will be 
administered by Clinical Center personnel without subjects being able to see or taste the 
medication. The off-days (weekends) serve as washouts for propranolol and pindolol. 
Since both dantralene and magnesium sulfate function as muscle NST inhibitors, 
minimum washout is needed. Finally, magnesium sulfate has a half-life of ~4 hrs, thus its 
impact on EE of the next day is minimum. This compact schedule is the result of 
balancing the rigorous study design and the feasibility for the subjects. 
For each chamber study, volunteers will be fasted after midnight and during the 
up to 4-hour study period while they are in the chamber. During this time, all subjects 
will be asked to minimize their physical activity while staying awake in a reclining 
position in the designated chair in the chamber. Between 9:30-10:00 and 10:30-11:00 
AM, the subject is required to lower the ambient lighting and recline without any 
physical activity, but remain awake.  Clothing will be standardized and limited to the 
following: one pair of undershorts, one sleeveless form-fitting cotton shirt, one pair of 


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Page 23 
form-fitting shorts, and one pair of light socks (issued by the inpatient unit), for a 
combined thermal insulation value of 0.36 (clo).94  After each chamber study, participants 
will return to the inpatient room on the 5SW inpatient unit at a comfortable range 
between 23-25C for the remainder of the day until the next morning. All meals will be 
served in the patient rooms. Each afternoon, study volunteers will have a standard 
walking session from 15:00-15:30 on a treadmill. The pace of the walking will be self-
selected but will be standard throughout the inpatient study period for each individual and 
reflect the subject’s prior activity level.   
Subjects will be allowed to go on pass on Cohort 1 Days 8/9, 15/16, but refrain 
from increased exercise, exposures to extreme temperatures, and all caffeine intake, and 
must return before dinner.  If deemed necessary, at the discretion of the Principal 
Investigator, any of the pass durations may be increased.   
During each of the stays in the metabolic chamber, we will record the following 
parameters and will perform these procedures: 
 
1) Real-time energy expenditure, respiratory quotient 
2) Continuous heart rate 
3) Surface electromyography and spontaneous movements 
4) Core (ear) and skin temperature  
5) 4-hour urine collection (8:00-12:00) for urea, cortisol, creatinine, and catecholamines 
6) Infra-red thermography (8:30, 10:00, and 12:00)  
7) Thermal comfort and hunger questionnaire (visual analog scales, Appendix I) 
 
 
 
Table 5: The detailed scheduled of events for Cohort 1.  
DAY number 
1 
2 
3 
4 
5 
6 
7 
8 
9 
1
0 
1
1 
1
2 
1
3 
1
4 
1
5 
1
6 
1
7 
ADMIT PHYSICAL/HISTORY (Wk 1) O                     
  
      
    
Weight and Height 
O                                 
DXA + Anthropometrics  
O         
            
  
      
    
Daily Progress Note 
  O O O O O O 
O O O O O  
 
O 
Daily Vital Signs  
O O O O O O O 
O O O O O  
 
O 
Metabolic Diet 
O O O O O O O 
O O O O O  
 
O 
Habitual Physical Activity by 
accelerometer 
O O O O O O O O O O O O O O O O O 
Room Temperature Log 
O O O O O O O 
O O O O O  
 
O 
Continuous recording during 
chamber study 
              
          
 
 
  
REE in Metabolic Chamber, up to 
4hr Continuous Recording (08:00-
12:00) 
  O O O O O O  
 
O O O O O  
 
O 
Skin Temp 
  O O O O O O 
O O O O O  
 
O 


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Electrocardiography (Holter) 
O O O O O O 
O O O O O  
 
O 
Surface Electromyography  
  O O O O O O 
O O O O O  
 
O 
At 08:00, 10:00, and 12:00 of 
Chamber study 
 
 
 
Tympanic Temp, IR thermography 
  O O O O O O 
O O O O O  
 
O 
Thermal comfort and hunger 
questionnaire  
  O O O O O O 
O O O O O  
 
O 
Research Labs 
              
    
  
     
 
  
4hr urine (08:00-12:00) 
  
O O O O 
O O O O O  
 
O 
Blood samples (08:00) 
O O 
 
 
 
Blood samples (12:00) 
O O O O 
O O O O O  
 
O 
 
 
III.2-2  Protocol design, Cohort 2  
Cohort 2 is a randomized, double-blind, placebo controlled, 6-period cross-over 
study, with data analysis performed in a blinded manner.  Oral medications, dosed 
exactly at 8:00 am (+/- 15 minutes) on entering the chamber, will be randomly assigned 
so that each subject receives each of the six treatments listed in Table 6.  Since it is not 
possible for all medications have the same appearance, drug or placebo will be 
administered by Clinical Center personnel without subjects or study personnel being able 
to see the medication.   
 
All Cohort 2 study volunteers will undergo the screening visit with the following 
tests which are identical to Cohort 1 (Table 2), a medical and laboratory examination to 
determine study eligibility. Qualifying volunteers will be invited for the full study at the 
Metabolic Clinical Research Unit as inpatients.  Subject criteria are the same as in Cohort 
1.   Subjects will be studied with 7-14 days between admissions and complete the entire 
study (Cohort 2) within a 12-week time window to prevent the variability in outdoor 
environmental temperatures and potentially body weight which will confound the study. 
 
During six 1-day stays at the NIH Hatfield Clinical Research Center, study 
volunteers will undergo daily BMR measurements (up to 4 hours) in a metabolic chamber 
while exposed to temperatures of 27.0C.  Chamber conditions and assays are exactly as 
described for Cohort 1 (Table 5).   
 
Table 6. Medications in Cohort 2 
Treatment 
Dose 
Route 
Minimum 
washout 
Placebo 
PO 
None 
Caffeine 
300 mg 
PO 
3 days   
Qsymia 
topiramate 92 mg CR 
phentermine 15 mg 
PO 
4 days 


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Phentermine HCl 
37.5 mg 
PO 
4 days 
Topiramate 
200 mg 
PO 
4 days 
Naltrexone 
100 mg 
PO 
? Days 
 
III.2-3  Protocol design, Cohort 3 - This Cohort has completed enrollment   
Cohort 3 is a randomized, single-blind, placebo controlled study, with the 
quantifier of BAT activity blinded to the four different interventions.  Volunteers will 
undergo up to four experiments to study the effects of mirabegron on BMR and BAT 
activity.  
 
All Cohort 3 study volunteers will undergo the screening visit with the following 
tests which are identical to Cohorts 1 and 2 (Table 2), including a medical and laboratory 
examination to determine study eligibility. Qualifying volunteers will be invited for the 
full study at the Metabolic Clinical Research Unit as inpatients.  Subject criteria are the 
same as in Cohorts 1 and 2. At least 48 hours will separate each of the study days, and all 
4 study visits will be completed within a 12-week time window to prevent the variability 
in outdoor environmental temperatures and potentially body weight which will confound 
the study. 
 
 
 
 
 
The first study visit will involve a 6-hour stay in the metabolic chamber. Each 
study volunteer will also be fitted with a cooling vest (CFA-9 vest, Polar Products, Stow, 
OH) while in the chamber. The first 240 minutes (4 hours) will be an acclimatization 
period with BMR measurement; the temperatures of the chamber and the water vest will 
be set at 20°C. At +240 minutes, the study team will reduce the temperature of the water 
Figure 2. Schematic depicting the testing parameters and procedures for Cohort 3. 


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in the cooling vest to 14.0-16.0C by turning on the vest pump which will be set up on 
the outside of the metabolic chamber. The tubing that circulates the chilled water through 
the vest will be placed through the access port of the chamber so that there is no 
disruption to the metabolic chamber data collection. Subjects may continue usual 
sedentary activities during this time. After 60 minutes of cooling, the volunteer will be 
injected with 18FDG for PET scan. Those subjects with detectable BAT (by PET/CT) will 
then proceed to the additional study days in which they will receive oral mirabegron at 
0mg, 50mg, and 200mg. If there is not sufficient detectable BAT after cold exposure, 
subjects will only complete the study day with mirabegron at 200 mg, not 0 mg or 50 mg. 
This will serve as a control for the differences in the amount of increase EE due to drug 
versus cold independent of BAT presence.  
 
The second, third and fourth study visits will be conducted similarly. Volunteers 
will enter the metabolic chamber at 8:00 am for daily BMR measurements while exposed 
to a room temperature of 27.0C.  After a 60 minute acclimatization period, the drug or 
placebo will be administered in a randomized fashion at time -0- (9:00 am ± 15 minutes). 
Blood samples will be obtained at -0-, +15, +30, +60, +120, +180, +240, +315, +360, and 
+420 minutes. At +240 minutes of mirabegron administration, the volunteer will be 
injected with 18FDG for PET scan. 
 
For each study day (A, B, C, and D), a dose of [18F]-fluorodeoxyglucose (FDG) 
will be given at 13:00 (+60 minutes of cold exposure or +240 minutes of mirabegron 
administration) and the volunteers will remain in the chamber for an additional 60 
minutes to allow for uptake of the radiotracer. Volunteers will then exit the chamber and 
be transported via wheelchair to the Clinical Center Nuclear Medicine Department for a 
positron emission tomography/computerized tomography (PET/CT) scan at 14:30. The 
scan will take no more than 60 minutes. Immediately upon completion of the PET/CT, 
subjects may be transported to the Radiology Department for a Magnetic Resonance 
Imaging (MRI) study. This scan will take no more than 60 minutes, as well. Following 
completion of the PET/CT and MR imaging, volunteers will then return to their room on 
the Metabolic Clinical Research Unit where they may have a meal and then may be 
discharged. Volunteers will be instructed to return for the next study visit until testing is 
complete.   
 
For each chamber study, volunteers will be fasted after midnight and during the 8-
hour study period they are in the chamber followed by PET/CT and MRI. During this 
time, all volunteers will be asked to minimize their physical activity while staying awake 
in a reclining position in the designated chair in the chamber. Between 08:30-09:00 
10:30-11:00, and 12:40-13:10, the volunteer is required to lower the ambient lighting and 
recline without any physical activity, but remain awake.  Clothing will be standardized 
and limited to the following: one pair of undershorts, one short-sleeve hospital scrub 
shirt, one pair of hospital scrub pants, and one pair of light socks (provided by the 
inpatient unit), for a combined thermal insulation value of 0.4 (clo).94 
 
 
 


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Page 27 
 
 
 
III.3  Inclusion Criteria: 
• Generally healthy 
• Males between the age 18-35 years 
• Written informed consent. 
 
III.4  Exclusion Criteria: 
• BMI less than 18.5 or greater than 25.0 kg/m2 
• History of cardiovascular disease such as congestive heart failure, heart block, 
clinically abnormal EKG as determined by investigators.  
• History of a cerebrovascular accident (CVA) or seizures. 
• History of liver disease or ALT serum level greater than two times the laboratory 
upper limit of normal 
• History of kidney diseases or renal insufficiency or estimated creatinine clearance 
≤ 50 mL/min (MDRD equation) 
• History of cancer or bariatric surgery 
• History of diabetes mellitus or fasting serum glucose  > 126 mg/dL 
• History of hypo- or hyper-thyroid  or TSH >5.0<0.4 miU/L   
• History of asthma, chronic obstructive pulmonary disease and glaucoma 
• Psychological conditions, such as (but not limited to) claustrophobia, clinical 
depression, bipolar disorders, that would be incompatible with safe and successful 
participation in this study 
• Weight change >5% in the past 6 months or a trained athlete  
• Blood pressure greater than 140/90 mmHg or current antihypertensive therapy 
• Iron deficiency (Hemoglobin <13.7 g/dL and Hematocrit <40.1%)  
• History of illicit drug, opioids,  or alcohol abuse within the last 5 years; current 
use of drugs (by history) or alcohol (CAGE≥2) 95 
• Current use of medications/dietary supplements/alternative therapies known to 
alter energy metabolism 
• Current medications that may have interactions with study drugs as determined by 
the investigators 
• Hypersensitivity and associated allergic reactions to mirabegron or similar drug 
substances or components of this medication 
• Daily caffeine intake >500 mg (about 4 cups) and have withdrawal symptoms 
• Current smoker or user of tobacco products 
• Cannot commit to the schedule of visits to the Clinical Research Center (CRC) as 
required by the study timeline  
• Have had previous radiation exposure within the last year (X-rays, PET scans, 
etc.) that would exceed research limits (please let us know if you have received 
radiation for research purposes) 
• Have inflexible dietary restrictions 
• Any other reason that the investigator thinks would make interpretation of the 
study results difficult. 


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Page 28 
• For subjects having an MRI (Cohort 3), history of pacemaker, metallic heart 
valves, aneurysm clip, pedicle screws, metallic foreign body in eye, or other 
metallic implant 
• For subjects receiving mirabegron (Cohort 3). a diagnosis of bladder outlet 
obstruction or the use of antimuscarinic medications for the treatment of 
overactive bladder 
• For subjects receiving mirabegron (Cohort 3). a history of hypersensitivity to 
mirabegron or other β3-Adrenergic Receptor Agonist medications.  
 
 
All subjects will be fully informed of the aims, nature, and risks of the study prior 
to giving written informed consent. 
 
III.5  Methods:  
 
Pre-Screening 
Prior to the official screening appointment, prospective subjects will be interviewed in 
person or by phone. Both general and protocol-specific recruitment scripting will be used 
to guide pre-screening interviews. Limited identifiable information pertaining to 
demographics, general health status, and contact information will be collected. We are 
requesting a waiver of consent for the pre-screening interviews since, per 45 CFR 
46.116(d) (pre-2018 requirements): 
1. 
The collection of limited identifiable information for pre-screening purposes 
involves no more than minimal risk to the subjects. 
2. 
The waiver will not adversely affect subjects’ rights or welfare. Pre-screening 
interviews will not impact subjects’ patient care or ability to decline participation 
before or during the study. 
3. 
Pre-screening interviews could not practicably be carried out without a waiver of 
consent as central recruiting services, utilized in the NIH Clinical Center, perform 
pre-screening activities for multiple studies and obtaining consent for each one is 
beyond their resources. 
4. 
It is unlikely that any pertinent information about the pre-screening process will 
be discovered after subjects have completed it, but should that occur, we will 
provide subjects with that new information as appropriate. 
 
Screening visit 
 
Following prescreening interviews, all prospective subjects will be screened and 
counseled at the initial screening visit. After obtaining informed consent, the study 
volunteers will undergo history and physical examination and the following screening 
tests including Chem 20, lipid panel, CBC with differential, TSH, ferritin, EKG, and 
urinalysis. Subjects are required to fast for at least 12 hours prior to the laboratory testing. 
Study volunteers will be allowed to drink water during the fasting period. During the 
screening visit the study participants will be interviewed by a dietitian in order assess the 
calorie intake and food preferences of each study participant. Body weight, height, and 
anthropometrics (circumferences at the neck, waist, hip, arms and legs) will be 
performed.  
 
Hospital stay-Cohort 1 


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Page 29 
 
Study volunteers will be housed in the Metabolic Unit of the NIH Clinical Center 
intermittently throughout the study period. Height will be measured. Daily afternoon 
physical activity consistent with prior physical activity levels will be devised in 
consultation with the dietitian in order to avoid deconditioning during the hospital stay. 
Sleep patterns and quality may influence REE and body temperature. Thus, all of the 
subjects will be asked to keep consistent sleeping habits (time, temperature, covers, no 
drinking or eating, etc) from three days prior to and throughout the entire inpatient study 
period, including any time on pass.  
 
Hospital stay-Cohort 2 
 
Study volunteers will be housed in the Metabolic Unit of the NIH Clinical Center 
on each admission. Height will be measured. Sleep patterns and quality may influence 
REE and body temperature. Thus, all of the subjects will be asked to keep consistent 
sleeping habits (time, temperature, covers, no drinking or eating, etc) from three days 
prior to and throughout the entire inpatient study period, including any time on pass.  
 
Hospital stay-Cohort 3 
 
Study volunteers will be housed in the Metabolic Unit of the NIH Clinical Center 
on each admission, which will be from 18:00 the night before the study until the imaging 
is completed - approximately 23 hours later. Sleep patterns and quality may influence 
REE and body temperature. Thus, all of the subjects will be asked to keep consistent 
sleeping habits (time, temperature, covers, no drinking or eating, etc.) from three days 
prior to the study period.  
 
Diet- Cohort 1 
 
Throughout the stay at the Metabolic Unit study volunteers will follow an 
isocaloric diet with the following macronutrient distribution: 55% carbohydrate, 15% 
protein, and 30% fat, as recommended by the American Dietetic Association. Subjects 
will be weighed (in triplicate), post-voiding each morning.  Diet caloric content will be 
adjusted to maintain a stable body weight.   
Diet- Cohort 2 
 
Study volunteers will follow an isocaloric diet with the following macronutrient 
distribution: 55% carbohydrate, 15% protein, and 30% fat, as recommended by the 
American Dietetic Association.  
 
Diet- Cohort 3 
 
Study volunteers will be provided with an isocaloric dinner meal the evening prior 
to metabolic testing. It will consist of the following macronutrient distribution: 55% 
carbohydrate, 15% protein, and 30% fat, as recommended by the American Dietetic 
Association. A late night snack high in carbohydrates and protein will also be provided 
due to the prolonged fasting period the following day. Volunteers will be expected to 
consume their snack by midnight and then fast until testing is complete, approximately 16 
hours. Then, he may resume a regular diet. 
 
Body composition 
 
A DXA scan (iDXA, GE Healthcare, Madison WI) will be performed at either 
Day 1 or 2 of the inpatient study stage. With this technique, one can determine total and 


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Page 30 
regional body fat and lean soft tissue masses, bone mineral content and density. DXA 
produces photons at two different energy levels, 40 and 70 KeV. The photons pass 
through tissues and attenuate at rates related to elemental composition. Bone mineral, 
with highly attenuating calcium and phosphorous, is readily distinguished from soft 
tissues. The different elemental profiles of fat and bone-mineral free lean components 
allows for the analysis of soft tissue fat content, so that bone mineral, fat, and bone 
mineral fat-free lean components may be resolved.  
 
Environmental questionnaire  
 
On Day 1, each subject will answer general questions about their typical thermal 
environment, such as the number of hours/day usually spent outside, clothing typically 
worn, environmental temperature at work and at home, and level of physical activity.   
 
PET/CT (Cohort 3 only) 
 
A PET/CT scan is the current standard for measuring BAT activation in humans. 
Study volunteers in Cohort 3 will be scanned following each stay in the metabolic 
chamber. At 13:00, a 5mCi dose of 18FDG will be given intravenously to volunteers 
through the blood port of the metabolic chamber. At the end of the study (14:00), study 
volunteers exit the metabolic chamber and are then transported via a wheelchair to 
Nuclear Medicine Department of the NIH Clinical Center and a PET/CT scan will be 
performed on the Siemens mCT scanner. The PET/CT scan will expose the study 
volunteer to about 1.25 rem of total effective radiation exposure. 
 
MRI (Cohort 3 only) 
 
A MRI scan is a technology that may become extremely beneficial to measuring 
BAT activity. The MRI produces detailed images of tissues. In this study, it may also be 
useful in evaluating cell receptors associated with BAT activity. Study volunteers in 
Cohort 3 may be scanned following each stay in the metabolic chamber, depending on 
scanner availability. After completion of the PET/CT (described above), volunteers will 
be transported to the Radiology Department of the NIH Clinical Center; an MRI will be 
performed on the Siemens MRI T3 scanner. The MRI will not expose the study volunteer 
to additional radiation exposure.  
BAT mass will be assessed using water-saturation efficiency and water-to-fat 
contrast ratio and the Dixon method, which separates fat from water by acquiring two 
images in the same scan, one at an echo time when fat and water signals are in phase and 
another when fat and water signals are out of phase.  Several MRI sequences will also 
assess BAT activation by measuring changes in tissue blood flow and other physiological 
parameters. 
 
Use of Fiducial Markers (Cohort 3 only) 
 
The use of MR images to assess brown adipose tissue has not been proven as a 
useful technology to date. This study will explore this technique more thoroughly. In 
order to more effectively measure BAT, we will co-register the MR images with the 
PET/CT images, the standard method of measuring BAT activity.  We will utilize 
fiducial markers to designate landmarks for the co-registration.  
 


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Page 31 
A fiducial marker is an object placed in the field of view of an imaging system 
which appears in the image produced, for use as a point of reference. The markers in this 
study will contain 1 µCi of Na-22. A total of up to 16 of these markers will be placed on 
the skin prior to the PET/CT scan. They will remain in place during the MRI that 
immediately follows. Subjects will be able to remove them after the MRI is complete.  
 
Metabolic chamber 
 
Study volunteers will enter the metabolic chamber at 08:00 AM after a minimum 
8-hour fast and voiding. Superficial skin temperature probes will also be placed (see 
below). Drug or placebo will be administered (witnessed, blinded dosing) with 120 ml of 
water at exactly 08:00 (09:00 for Cohort 3).  Study volunteers will wear standard clothing 
and will be instructed to be minimally active while staying awake in the metabolic 
chamber after the first 30 minutes. To help with the compliance and occupying the time 
in the chamber, each subject will be allowed to watch television (sports, movies, series, 
etc), play non-active video games, read, or perform deskwork. Details of the chamber 
have been published previously. 16,96 During the period of 08:00 to 12:00 (08:00 to 13:00 
for Cohort 3), urine will be collected by the study volunteers, and then analyzed for total 
nitrogen, urea, cortisol, creatinine, and catecholamines. 
 
 
 
The metabolic chamber is a specially constructed room to assess energy 
expenditure.  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.97 The room is equipped with toilet and sink with privacy screen, 
treadmill, bed, desk, and computer with access to television and other forms of 
entertainment.  Physical activity level is measured continuously through a wall mounted 
monitoring device (microwave sensor).  Calibration tests over the past three years at 
temperature settings from 15.3 to 32.1C have demonstrated an accuracy of 98.4±2.5% 
for EE as compared to a weighed propane standard oxidized by combustion.   
 
 
The relative humidity of each chamber is controlled between 30-50% for all 
temperatures. The overhead circulation fan unit mixes the air of the chamber uniformly. 
At the bed and the recliner where the subjects will be spending the 5 hour resting periods, 
the wind speed is less than 0.4 m/s (0.8 mile/hour), making the windchill and humidex 
(heat index) factors less than 0.2C. We will measure chamber wall temperature using 
iButtons,98 and calculate the exact “operational temperature” as defined previously99. 
 
 
The following parameters will also be recorded inside the metabolic chamber: 
 
Continuous heart rate and heart rate variability 
 
Heart rate will be measured and recorded using a portable ECG Holter monitor, 
which will be downloaded and analyzed for heart-rate variability using power spectrum 
analyses.    
 
Muscle contraction and Shivering 
 
Wireless surface electromyography (EMG) electrodes (Trigno, DelSys Inc, 
Boston MA) will be placed on upper arm (biceps brachii), chest (pectoralis major), neck 


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Page 32 
(upper fibers of the trapezius), and leg (rectus femoris) muscles to objectively measure 
the intensity and overall muscle contractions. Each surface sensor will include a triaxial 
accelerometer to simultaneously measure dynamic movements.100,101  Signals will be 
transmitted to a laptop computer outside the chamber for data analysis to look for activity 
below the threshold of shivering, and for shivering. 102,103 To standardize the EMG 
measures, each EMG sensor is placed carefully in the same spot of the muscle with the 
edges clearly marked by surgical markers (water and sweat proof), and a five minute 
baseline data is collected before each chamber study at a constant temperature (23ºC) 
while the subject is reclined and rested under a blanket (no voluntary muscle contraction 
and no shivering). All EMG data during the chamber study will be normalized to this 
baseline period (as % of baseline) and compared between subjects. The normalized EMG 
data from the two resting periods (9:30-10:00 and 10:30-11:00) on the 27 ºC placebo day 
will be used as the baseline measurement. Form our preliminary data (12-Dk-0097) 
collected in the identical fashion, we found that EMG during cold exposure > mean + 2 x 
SD of the baseline period as the threshold closely compared to self-reported shivering, 
and we will use criterion of two spontaneously shivering muscle groups out of the four 
measured as the final determination of shivering.  
 
 
Tympanic and skin temperature 
 
 We will measure core temperature using a handheld infrared tympanic 
thermometer (PRO4000 by Braun) as it has been shown to have the closest agreement 
with nasopharyngeal temperature measurements 104 but without being invasive. Tympanic 
temperature will be measured every 30 minutes by the study subjects after training by our 
study staff. Two measurements in the right ear will be taken each time. If the two data 
points are different by 0.3ºC, a third measurement will be taken with the median of the 
measurements averaged as the final result. All data points will be recorded immediately 
after each measurement.  
 
Wireless probes (iButton, Maxim Inc., Sunnyvale CA) will record skin 
temperature and heat flux continuously with data reported at each minute. [48]105  The 
positions of the skin sensors at eight sites (anterior aspect of forearm, forehead, 
subscapular, triceps, pectoralis major, abdomen, anterior thigh, and calf) on the right side 
of the body will be according to the ISO standards.94  
 
Thermal comfort and hunger questionnaire 
 
Patients will be given a questionnaire to determine their level of thermal comfort 
and hunger using visual analog scales (VAS), as shown in Appendix I.  The hunger VAS 
question has been characterized previously.106  A computerized version of the 
questionnaire will be used.   
 
Infrared thermography 
 
Skin temperature can be monitored by infrared thermography (T400, FLIR, North 
Billerica MA) which has been described previously.107 The camera will be positioned 
between 1-1.5 meters away from the subject in the chamber and used to take a picture at 
defined time periods (8:30, 10:00, and 12:00).  This will be done in Cohorts 1 and 2 only. 
 
Genetic testing 


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Genomic DNA will be isolated from peripheral mononuclear cells from a blood 
sample collected during screening. The purpose of obtaining genetic material is to 
perform genotype-phenotype association studies. Specifically, samples will be initially 
analyzed for the type-2 deiodinase gene Thr92Ala and -258 A/G,108,109 and uncoupling 
protein-1 (UCP1) 110polymorphisms, i.e. candidate genes for modulation of the metabolic 
response to changes in environmental temperature and should the literature indicate novel 
candidate genes/variants, for subsequent screening of candidate genes involved in 
obesity, endocrinology, metabolism, diabetes, and related diseases and conditions, as well 
as scanning for novel mutations in genes involved in metabolic pathways. Currently, we 
believe that obesity results from small influences on body weight from many genes and 
their complex interactions with environmental factors over time (years to decades). While 
future studies may be performed to explore such areas, the results of this testing are 
unlikely to have direct clinical relevance to the subjects.  For example, whole 
genome/whole exome sequencing could be performed that may reveal novel findings in 
energy metabolism, fuel partitioning, and/or heat loss regulations in this study population. 
DNA samples may also be tested for variation that may affect drug pharmacokinetics.   
With such broad range of genetic inquiry, it is possible that future testing could 
discover information (either directly related to the metabolic pathways under 
investigation or incidental genetic findings) that may be important to the subjects’ health 
or the health of their offspring.   
Because our genetic studies will take years to complete, we may not be able to 
reach all subjects in the event we find important health information. We do not intend to 
return results of genetic tests to subjects.   
The information will not become part of the subject’s hospital medical record 
since they are not CLIA-certified assays. For this reason, the DNA results gathered in this 
study are not appropriate for immediate clinical use.  This information will be kept in a 
research data file separate from the subject’s medical records.  If the patient requests the 
results of the research files, those results will be discussed and clearly labeled as being 
research information only and not appropriate for clinical use.  This is not a family study 
therefore genetic information about paternity is not applicable.  
Other risks of participating in genetic research include anxiety about learning of 
possible disease association and insurance issues, including obtaining disability insurance 
and life insurance.  The Genetic information Non-discrimination Act (GINA) generally 
makes it illegal for health insurance companies, group health plans, and most employers 
to discriminate based on genetic information.  This Federal law does not protect against 
genetic discrimination by companies that sell life insurance, disability insurance, or long-
term care insurance.   
In order to protect the privacy of potentially sensitive individual genetic data, 
steps will be taken to de-identify and assign a code to each subject’s research sample.   
 
Laboratory testing 
 
A screening test (Chem 20, lipid panel, CBC with differential, TSH, ferritin, 
EKG, urinalysis) will be performed by the Clinical Center Department of Laboratory 
Medicine (DLM).  
 
In Cohort 1, research blood samples will be drawn at 8:00 am on Days 4 and 5 
and at 12:00 on 10 different days (Days 4,5,6,7,10,11,12,13,14,17) immediately after the 


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volunteer exits the chamber, for a total of 12 samples. For safety monitoring of 
medications being given, we will add a hepatic panel on Day 10, a mineral panel and 
potassium level on Days 11-14, and a creatinine clearance level on Day 11.  On Day 17, 
one clinical sample (5 ml tube) will be added (Chem 20) to monitor any changes. The 
volume of each research blood draw will be approximately 24 ml, plus a one-time DNA 
sample of 10 ml. For the entire inpatient study, the total volume of blood sampling will 
be no more than 300 ml. 
In Cohort 2, research blood samples will be drawn each Day at 8:00 am and at 
12:00 immediately after the volunteer exits the chamber, for a total of 12 samples. The 
volume of each research blood draw will be approximately 24 ml, plus a one-time DNA 
sample of 10 ml. For the entire inpatient study, the total volume of blood sampling will 
be no more than 300 ml. 
If a subject participates in Cohorts 1 and 2, we will make sure that sufficient 
recovery time between the tests such that no more than 550 ml blood will be drawn in 
any 8-week period. 
In Cohort 3, research blood samples will be drawn each day at 8:00 am and at 
1:00 pm, immediately before the 18FDG injection. Pharmacokinetic blood samples for 
glucose, insulin and  mirabegron levels will also be collected on study days B, C and D at 
ten time points: 0, +15, +30, +60, +120, +180, +240, +315, +360, and +420 minutes. 
Samples at +315, +360, and +420 minutes will be collected in the PET Department by 
trained technologists due to the radioactivity of the blood. A one-time DNA sample of 10 
mL will also be collected. For the entire study (four visits), the total volume of blood 
sampling will be approximately 500 mL which is less than the NIH guidelines of 550 mL 
in 8 weeks for research subjects. 
Research samples will be analyzed to measure changes in leptin, adiponectin, 
glucose, insulin, free-fatty acid, thyroid hormones (T3, T4, TSH), parathyroid, growth 
hormone, cortisol, adrenocorticotropic hormone (ACTH),  catecholamines (epinephrine, 
norepinephrine), glucagon, ghrelin, peptide YY (PYY), glucagon-like peptide-1 (GLP-1), 
and gastric inhibitory polypeptide (GIP), fibroblast growth factor (FGF) 21, irisin, ANP, 
BNP, and β-hydroxybutyrate. Subjects in Cohort 3 will also have blood analyzed for 
pyruvate, lactate and glycerol. 
 
Research use, storage, and disposition of human subject’s samples and data  
 
Plasma and nucleic acid samples will be coded and stored in a -80○ C freezer. The 
volume of the 4-hr urine collected each study day will be measured and recorded, then 
aliquoted into 3 10cc tubes and stored in a -80○ C freezer. Study data will be kept in 
locked files, and subjects will be identified by codes. A registry of the samples’ location 
will be kept in the PI’s office. This will allow the PI and the AIs to analyze research data 
in relation to clinical data stored in the CRIS system. Data and samples will be retained 
for the foreseeable future in order to allow secondary analyses after the completion of the 
protocol. Such analyses include association studies for known mutations of genes 
involved in obesity, energy, carbohydrate, thyroid hormone and glucose metabolism, as 
well as gene scanning for novel mutations. Serum plasma and nucleic acids samples will 
be similarly utilized for future association studies of known and novel markers of obesity, 
energy, carbohydrate, thyroid hormone and glucose metabolism. Genomic DNA will be 
isolated from peripheral mononuclear cells using the QIAmp® system (QIAGEN) in Dr. 


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Cypess’ laboratory or in the laboratory of another qualified AI. The PI will inform the 
IRB in case of loss of data/samples secondary to intrusion in the laboratory or database. 
 
Portions of blood samples will be sent to the laboratory of Dr. Michael A. Kiebish at 
Berg, LLC for lipidomic, metabolomic, and similar screening platforms.  The purpose of 
these studies is to screen for potentially physiologically relevant lipid and metabolic 
species in human plasma from subjects with known amounts of detectable BAT.  We will 
also share samples with Evan Rosen, MD, PhD, at Beth Israel Deaconess Medical Center 
(BIDMC) to measure myostatin levels in human plasma from subjects with known 
amounts of detectable brown adipose tissue (BAT).  Dr Rosen’s lab at BIDMC has 
uncovered data that strongly suggest a direct link between BAT and muscle function. 
 
To protect the privacy and confidentiality of the human subjects, the specimens that are 
sent to these outside laboratories will be coded and not contain any personal identifiers 
that could be traced to the actual human volunteer. The PI and LAI will maintain the code 
and it will not be shared with the collaborator. No additional consent is required; the 
Informed Consent signed by the subjects explicitly stated that “Specimens and/or data 
may be shared with investigators external to NIH in the event future collaborations are  
identified. 
 
 
 
Non NIH Collaborators 
 
Name 
Institution 
Project 
Michael A. 
Kiebish, PhD 
BERG, 
LLC 
Plasma samples analyzed for lipidomic, 
metabolomic, and similar screening 
platforms.  The purpose of these studies 
is to screen for potentially physiologically 
relevant lipid and metabolic species in 
human plasma from subjects with known 
amounts of detectable BAT.  
Evan Rosen, 
MD, PhD 
 
Beth Israel 
Deaconess 
Medical 
Center 
Human Plasma samples to measure 
myostatin levels from subjects with 
known amounts of detectable brown 
adipose tissue (BAT).  Dr Rosen’s lab at 
BIDMC has uncovered data that strongly 
suggest a direct link between BAT and 
muscle function. 
 
 
 
 
 
 
III.6  Possible Risks and Hazards: 
 
Study medication-related risks are listed individually below. All these are Federal 
Drug administration (FDA)-approved medications for long term use. While we 


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summarize the possible risks and hazards as published by the FDA below, we also note 
that the general risks of taking these medications only once or twice (in two days) in 
healthy subjects and under resting conditions are considered to be low.  In Cohort 1, the 
total dose per drug is two (once per day). Other research-related risks in this study 
include those associated with study procedures, namely blood drawing. We do not expect 
any significant risk connected to the modestly low or high environmental temperatures 
for the short exposure time at resting state.  Shivering confounds measurement of resting 
metabolic rate so any subject who shivers will promptly be removed from the metabolic 
chamber and not be exposed to temperatures lower than those causing the shivering.  If 
any medically significant findings are discovered incidentally, the subject will be referred 
to their primary care provider for follow up.   
 
Study medications: 
Propranolol 
Propranolol will be administered as a pill by mouth once a day for two days at a dose of 
160 mg in this study.  
 
Side effects of Propranolol 
The most common side effects of this medication include dizziness or lightheadedness, 
unusually slow pulse, diarrhea, drowsiness, nausea, unusual tiredness or weakness and 
numbness or tingling of the fingers and/or toes. Serious skin reactions can occur during 
treatment with this medicine, however, this side effect is very rare.  
 
Black box warnings of exacerbated angina, MI, and ventricular arrhythmias are 
associated with abrupt withdrawal of medication after ongoing daily use; these reactions 
are not expected as we will be administering only two consecutive one time oral doses.   
 
Pindolol 
In this study, a single 20 mg dose of pindolol will be administered by mouth once a day 
for two days. 
 
Side effects of Pindolol 
More common potential side effects include insomnia (10%),  muscle pain (10%),  
dizziness (9%),  fatigue (8%),  nervousness (7%),  elevated liver  enzymes (7%),  joint 
pain (7%),  edema (6%).  Difficulty breathing (5%), wheezing, and tightness in chest 
(3%) are less common. 
 
There are no black box warnings specifically for pindolol.  
 
Dantrolene  
In this study, you will be taking a dantrolene 100 mg capsule by mouth once a day for 
two days.  
 
Side effects of Dantrolene  
The most common side effects of dantrolene are drowsiness, dizziness, weakness, general 
malaise, fatigue, and diarrhea. About one-third of patients may experience drowsiness, 


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about 1 in 10 experiences dizziness and muscle weakness.  Liver injury (1.8%), hepatitis 
(0.6%) and fatal hepatitis (0.3%) have been reported. These tend to occur more often at 
higher doses (800 mg and above) and longer duration of administration (greater than 
three months).  Damage that includes fatal hepatitis has occurred in less than 2% of the 
patients. This medication can cause serious side effects affecting the blood, bone marrow, 
lungs and heart are but are usually seen at high doses  or after taken for prolonged 
periods. 
 
Black box warnings for this medication are for hepatoxicity as detailed in the previous 
paragraph.  
 
Safety Monitoring 
Safety measures will be done to monitor for potential, but uncommon side effects of 
muscle weakness and drowsiness/respiratory depression associated with taking 
dantrolene.  To monitor for changes in muscle strength, we will perform a hand grip 
dynamometer measurement to be done during protocol week one (baseline) and then 
before and after chamber stays on protocol Day 10 and 11 to assess for any change after 
the subject has received dantrolene during protocol participation.  More frequent 
respiratory rate will be assessed on protocol Day 10 and 11 during four hour chamber 
stay – hourly.  
 
Mirabegron  
This medication is a β3-adrenergic receptor agonist with sympathomimetic activity.  It 
will be given one time to each volunteer at 50mg and 200mg. 
 
Side effects of Mirabegron 
The principal side effects of mirabegron are reported in chronic use at a 50 mg dose. 
These include: increased blood pressure (9-11%), headache (4%), dizziness (3%), 
increased heart rate (2%), constipation (2%-3%), diarrhea (2%), dry mouth (3%),urinary 
tract infection (3%-6%), back pain (3%), joint pain (2%), head cold (4%), sinusitis (3%), 
and flu-like symptoms (3%). In healthy adults, there are no known effects on the bladder 
or urination. We expect with a 200mg one-time dose, the effects may be the same or 
slightly more common and intense; in particular the effects on blood pressure, heart rate, 
and palpitations.  Of note, only those three adverse effects were reported in our previous 
studies.  None of the others were reported by healthy subjects with a one-time dose of 
mirabegron, including headache, dizziness, constipation, diarrhea, dry mouth, UTI, back 
pain, joint pain, head cold, sinusitis, and flu-like symptoms. It is possible that side effects 
with 200 mg may occur more frequently and become more symptomatic than those 
observed when mirabegron is prescribed at the standard dose approved for treatment, 50 
mg. 
 
Based on the effects of other related medications, high doses of mirabegron, well above 
what will be given in this study, may cause sweating, nausea or vomiting, tremor, 
weakness, dizziness, confusion, delirium, hallucinations, pallor, and difficulty breathing. 
None of these side effects were observed in healthy subjects in our previous studies with 


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mirabegron at a one-time dose of 200 mg. Screening for underlying susceptibility to mild 
catecholamine excess will be done during the medical history and ECG.  
 
There is a potential connection between mirabegron use and hypersensitivity reactions.  
Specifically, in the clinical development program it was determined that there was not a 
consistent pattern to establish an association of mirabegron with nonimmediate, primarily 
noncutaneous hypersensitivity reactions such as hemolytic anemia and thrombocytopenia 
(1 patient) and neutropenia (1 patient).  Similarly, an association could not be ruled out 
between mirabegron, particularly at doses ≥ 100 mg, with nonimmediate, primarily 
cutaneous hypersensitivity reactions such as urticaria, leukocytoclastic vasculitis, rash, 
pruritus, purpura, and lip and eyelid edema, as well as the serious adverse effects of 
cutaneous vasculitis (1 patient) and urticaria (2 patients). 
 
Magnesium Sulfate 
Magnesium will be given through an intravenous line directly into your vein for 3 hours 
in the morning for two days. (dosing: 50 mg/kg bolus followed by a maintenance infusion 
of 2g/h for 3 hours)   
 
Side effects of Magnesium Sulfate 
Magnesium can cause nausea, flushing, low blood pressure, slowing the heart rate, and 
minimal irritation at the infusion site may occur. 
 
There are no black box warnings associated with this magnesium.  
 
Caffeine 
Caffeine 300 mg in capsule form will be given once by mouth. 
 
Side effects of Caffeine 
This medication can cause insomnia, (inability to sleep) nervousness and restlessness, 
stomach irritation, nausea and vomiting, increased heart and respiration rates. 
 
There are no black box warnings associated with caffeine. 
 
Phentermine 
Phentermine will be given as a single dose of 37.5 mg as an oral pill.  
 
Side effects of Phentermine  
The common side effects are difficulty sleeping, dry mouth, and nervousness. Less 
common side effects include heart palpitations, increased blood pressure, chest pain, 
shortness of breath, dizziness, upset stomach, constipation, and itching.  
 
 
 
There are no black box warnings for phentermine. 
 
Topiramate 
Topiramate  200 mg pill by mouth will be given just for one dose in this study. 


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Side effects of Topiramate 
The most common side effects include nausea (6-10%), anxiety (2-3%), appetite loss (4-
24%), dizziness (4-25%), double vision (1-10%), drowsiness (4-25%), fatigue (9-16%), 
tingling or burning sensations (1-11%), tremors (3-9%), and “metallic” taste in mouth. 
These side effects are more frequent with higher dosages of topiramate. Other reported 
effects include abnormal vision (2-13%) and recent FDA reports of acute myopia and 
secondary angle closure glaucoma syndrome associated with decreased visual acuity 
and/or ocular pain; typically occurring within one month of initiating topiramate.  
 
There are no black box warnings for topiramate.  
 
Qsymia 
A single capsule containing 15 mg of phentermine and 92 mg of topiramate will be given 
once by mouth. 
 
Side effects of Qsymia 
Qsymia can cause increased heart rate (14-78%), mild dizziness (3-9%), anxiety and 
depression (3-8%), concentration, memory and speech problems (2-8%), headaches 
(10%), feeling tired or irritable (2-4%), constipation (8-16%), insomnia (5-9.4%), 
numbness or tingling feelings (4-20%), an altered or unpleasant sense of taste (1-9%), 
and dry mouth (7-19%).   Less common (< 1%) but serious side effects include increased 
pressure in your eyes, acute angle-closure glaucoma and suicidal ideations.  The 
incidence of side effects could be observed by or after 4 weeks of taking the medication 
daily, but can potentially occur as early as one week after starting the medication.  The 
most common side effects are parasthesias, dizziness, an unpleasant sense of taste, 
insomnia, constipation and dry mouth.   
 
Precautions while taking Qsymia include refraining from alcohol intake and not driving a 
car or operating heavy machinery. Qsymia can cause sleepiness and dizziness in 
combination with alcohol, and can slow your thinking and motor skills, as well as affect 
vision.  
 
The company that manufactures Qsymia has put in place a REMS (Risk Evaluation and 
Mitigation Strategy) program since Qsymia is contraindicated in pregnancy for women 
due to an increased risk of birth defects, cleft lip and cleft palate.  This program requires 
that information on this contraindication is given to all who receive the drug.   
 
 
There are no black box warnings associated with phentermine/topiramate (Qsymia).  
 
Naltrexone 
A 100 mg tablet of Naltrexone will be given once. 
 
Side effects of Naltrexone 
This medication can cause nausea (10-33%),  headache (3-25%),  dizziness (4-13%), 
anxiety 2-12%), tiredness (4%), and trouble sleeping  (3-14%) may occur with 


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Naltrexone. In a small number of people, mild opiate withdrawal symptoms (< 1%) may 
occur, including abdominal cramps, restlessness, bone/joint pain, muscle aches, and 
runny nose. Liver injury (13%) has been reported in individuals taking this drug. 
However, this appears to occur when the dose of naltrexone is higher (more than 300 mg 
once a day) and when taken for weeks. A single dose of naltrexone does not appear to 
cause liver injury. 
 
Black box warning for hepatotoxicity with excessive doses beyond indicated dosing.  
 
Metabolic Chamber (up to 6 hours per day) 
 
Besides inconveniences that can reasonably be expected as a result of spending an 
extensive time (up to 6 hours) in the live-in room calorimeter, the serious risk to subjects' 
health is minimal.  
 
Continuous EKG (up to 6hours per day) 
 
A portable Holter monitor will be used to collect heart rate variability in the 
standard three-lead configuration (4 hypoallergenic disposable EKG electrodes). No 
known risks have been reported. 
 
Tympanic and surface temperature measurements. (up to 6 hours per day) 
 
A non-invasive tympanic thermometer (PRO4000 by Braun) will be used 
measured every 30 minutes by the study subjects after trained by our study staff. Wireless 
surface patches (VitalSense, Mini Mitter, Bend OR) will be used to measure the skin 
temperature by a portable receiver positioned close to the body.  No known side-effect 
has been reported. 
 
Surface Electromyography sensors with accelerometers (up to 6 hours per day) 
 
The solid-state sensors, each measuring 1.5x1x0.6 inch, pick up electric signals 
generated by the muscle fibers, which are transmitted wirelessly to the computer. Sensors 
will be taped with thin hypoallergenic double-sided tape to the specific skin sites, and 
pose no risk to the subjects. 
 
Infrared thermography 
 
We know of no significant risks from taking pictures with a camera in the infra-
red range.    
 
Urine collection (up to 6 hours per day) 
 
Besides the inconveniences that can reasonably be expected as a result of 4-5 hour 
urine collection during specific time periods, there is no health risk of this procedure. 
 
DXA Scan (1 test) 
 
The use of DXA scan apparatus may cause some minimal discomfort in 
claustrophobic subjects and may cause some minimal back pain in a small minority of the 
individuals. 
 
PET/CT (4 scans) 


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The PET/CT scanner apparatus may cause some discomfort in claustrophobic 
patients and from lying in a supine position for an extended period of time. 
Volunteers may also experience minor discomfort and/or bruising at the site of the 
intravenous injection of the PET radiotracer. 
 
MRI (4 scans) 
 
The MRI scanner apparatus may cause some discomfort in claustrophobic patients 
and from lying in a supine position for an extended period of time.  
 
Use of fiducial markers (4 times) 
 
The placement of these markers (up to 16) is not expected to cause any pain to the 
subjects. Volunteers may experience some skin irritation at the site(s) of placement. They 
will be adhered to the skin surface. 
 
Radiation Safety   
 
The radiation exposure of a DXA scan is less than 1 mrem per session. A normal 
individual is exposed to 300 mrem/year from natural sources (background exposure). 
 
Each PET/CT scan will use 5 mCi of 18FDG.  The radiation exposure of the 
PET/CT is an effective dose of 0.83 rem, 3.5 rem/year for 4 PET/CT scans, which is 
below the guideline of 5 rem per year allowed for research subjects by the NIH Radiation 
Safety Committee. A typical radiation dosage from a chest x-ray is 20 mrem.  
 
The fiducial markers will contain 1 µCi of Na-22. This adds 0. 000085 rem per 
PET to the current radiation dosage. The effective dose for this study remains 3.5 
rem/year. 
 
Blood drawing  
 
The placement of intravenous needles may cause transient pain, and rare infection 
or bruising at the insertion site.  
 
Fasting over time 
 
The discomfort of fasting daily from midnight until 13:00 (16:30 for Cohort 3) of 
next day can be difficult for some subjects. There is no known long term harm to the 
subjects as overall energy balance (dietary intake = energy expenditure) is preserved. 
 
III.7  Adverse events  
 
Adverse events, non-compliance both serious or continuing, protocol deviations both 
major and minor, as well as unanticipated problems are defined & described by the NIH 
Office of Human Subjects Research Protection policy #801, and will be reported in 
accordance with this policy.  
Serious adverse events 


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The PI is responsible for summarizing all serious adverse events and adverse 
events at least possibly related to the research procedure and interventions at the 
time of Continuing Review.  All deaths that have occurred among study 
participants since the previous review will be summarized at the time of 
continuing review.A serious adverse event is any experience that: 
1. Results in death; 
2. Is life threatening; 
3. Results in hospitalization or prolongs hospitalization; 
4. Results in persistent or significant disability/incapacity;  
5. Results in congenital anomaly/birth defect; or 
6. Results in a condition, which in the judgment of the investigator represents a 
significant hazard.   
Clinical Monitoring 
Study procedures will be subject to audits and/or monitoring visits to ensure 
compliance with the protocol and applicable regulatory requirements consistent with the 
NIDDK quality assurance program plan.  Audit and/or monitoring visit results will be 
reported to the Principal Investigator for further reporting as appropriate. Study 
documents and pertinent hospital or clinical records will be reviewed to verify that the 
conduct of the study is consistent with the protocol plan. 
 
Data and Safety Monitoring Plan 
 
No DSMB will be instituted to record adverse events in Cohorts 1 and 2 since this 
study administers a single dose of FDA-approved medications.  Thus the level of risk is 
minimal, and Dr. Muniyappa will serve as study monitor. 
 
In Cohort 3, no DSMB will be instituted to record adverse events since this study 
is open label, and given the modest level of risk involved. For this sub-study, LAI, Dr. 
Cypess, will serve as study monitor. 
 
Data collection is the responsibility of the clinical trial staff at the site under the 
supervision of the site manager. The investigator is responsible for ensuring the accuracy, 
completeness, legibility, and timeliness of the data reported. The PI and study team will 
review participant data on at least a monthly basis to review the data for safety and 
efficacy. Events meeting requirements for expedited reporting as described in HRPP 
Policy 801 will be submitted within the required timelines. 
 
III.8  Recruitment of Women, children and minority individuals 
 
 
We will actively encourage the participation of minorities as indicated. The study 
design precludes the recruitment of children since the differences in thermal biology and 
metabolic rate between adults and children would impede the interpretation of the data 
collected. Similarly, the durations of the study (each Cohorts) preclude studying women 


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due to the variation introduced by the phases of the menstrual cycle.  However, our aim is 
to use data from young men of this study and our experiences with 12-DK-0097 (women 
and men of different ages) to design a shorter study protocol to study broader populations 
including women, older and younger subjects in the future. 
 
Benefits 
 
 
Study subjects will receive no direct benefit from participation in this study. A 
thorough medical examination and a series of diagnostic tests will be provided as part of 
the evaluation. Abnormal values will be discussed with the study volunteers and 
forwarded to their primary care physicians.  
 
 
III.9  Remuneration 
 
Cohorts 1 and 2 
Patients will receive payments for time and discomfort connected with the 
visits/procedures according to the following scheme: 
Per unit        Cohort 1              Cohort 2 
1. Daily compensation for inpatient stay       $40      x 14  = $560         
x 6  = $240 
2. Metabolic chamber stay                          $40      x 12  = $480          x 6  = $240 
3. Body sensor measurements                    $20      x 12  = $240 
x 6  = $120         
4. Hunger and thermal questionnaires         $10      x 12  = $120  
x 6  =   $60 
5. DXA and anthropometrics                     $35        x 1  =  $35 
           x 1  =   $35 
6. Blood draws                                          $20      x 12  = $240     
x 6  = $120  
7. Bonus for completing the study  
 
       
   $100__ 
          $100 __ 
           Maximum compensation:                                               $1,775                      $ 915 
 
Cohort 3 
Patients will receive payments for time and discomfort connected with the visits/ 
procedures according to the following scheme: 
 
 
Intervention 
Per Unit 
Number Cohort 3 
Metabolic chamber stay                            
$40 
X 4 
$160 
Body sensor measurements                      
$20 
X 4 
$80 
Hunger and thermal questionnaires            $10 
X 4 
$40 
DXA 
$35 
X 1 
$35 
Anthropometrics                        
$20 
X 4 
$80 
PET/CT                                                  
$200 
X 4 
$800 
Blood draws                                             
$20 
X 4 
$80 
MRI 
$80 
X4 
$320 
Bonus for completing the study 
 
$100 
X 1 
$100 
Maximum Compensation 
 
 
$1695 
 
 
 


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Version Date: 2021 July 22 
 
Page 44 
III.10  Research Use, Storage and Disposition of Human Subject’s Samples and 
Data 
    
The following safeguards will be regularly employed to protect subject privacy and 
confidentiality: 
• All medical records will be kept in the medical records department and that 
research records will be kept in a secure place in a locked file cabinet or in a 
password protected computer in the office of the Principal Investigator. 
• Access to records and data associated with personal information will be restricted 
to the Principal, Co-, and Associate Investigators  
• Samples (blood, fluids, or tissues) sent to outside laboratories for analysis and 
testing will contain coded numbers, without personal identifiers such as name or 
address. 
• Although samples will be coded, we will retain the key to the sources in order to 
interpret the data in the context of additional tests performed after the completion 
of the active recruitment of study volunteers. 
• Data and samples will be stored for the foreseeable future; research charts will be 
kept in the PI’s office in a locked file cabinet. E-mail messages containing 
identifiers of the study participants will be encrypted, and samples will be stored 
in locked refrigerators. 
• The PI will report to the IRB the loss of data or information in case the loss could 
result in dissemination of study participants’ personal information or could 
threaten the validity of the study, i.e. inability of interpreting the data. 
 
 
Consent process and documents 
 
Before any screening or study procedures can begin, written informed consent will be 
obtained.  The process of informed decision-making by research subjects will include 
discussion about the research study with the Principal Investigator and/or designee in 
language understandable to the subject. Because there are both clinical and technical 
areas that comprise this protocol, we will ensure both areas are fully addressed to the 
subjects by the research team with a clinical and technical member present during 
screening and consenting. Sufficient time and opportunity will be given to discuss the 
research, (minimizing or eliminating coercion or undue influence) as well as to answer 
any questions the subject may have.  The current IRB-approved informed consent 
document will be signed by the subject and the Investigator. The investigator signature on 
the informed consent will be signed by the principal investigator or a designated clinical 
investigator. A copy of the consent will be given to the subject for future reference. The 
signed documents will be sent to the Medical Records Department for placement in the 
subject's permanent CC medical record.  The consent process will additionally be 
documented in the electronic medical record (CRIS). 
 
We anticipate the enrollment of  NIH staff into this study and will provide the NIH 
Information Sheet on Staff Research Participation  to NIH employees who are 


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Page 45 
considering participation on our protocol prior to obtaining consent.  In addition, we will 
discuss the following applicable safeguards to NIH staff as a vulnerable class of subjects: 
- 
Unbiased participation  for protocol integrity and participant risk assessment. 
- 
Ensure there is no perceived workplace pressure or expectation on either 
participation or deciding not to participate on the protocol in regards to a 
benefit or adverse effect on their NIH employment or staff position.   
- 
Protection of privacy and confidentiality will be maintained , but also with 
acknowledgement of the limits due to sensitive information that may be in 
their NIH file.   
- 
Discussion of time commitments of the study and compensation in accordance 
with NIH policy 2300-630-3, Leave Policy for NIH Employees Participating 
in NIH Medical Research Studies. 
 
 
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Appendix I 
 
The following survey will appear on a custom-programmed iPAD App, in the identical 
appearance as follows: 
Comfort Questionnaire 
 
 
Date of survey (MM/DD/YYYY):    _ _ / _ _ / _ _ _ _ 
 
Time of survey (HH:MM):    
_ _ : _ _  AM PM (circle AM or PM)  
 
 
 
 
 
 
 
     How do you feel about the room temperature? 
 
       Very hot  
 
 
 
 
 
 
 
 Very cold 
 
 
 
 
        How do you feel about the room humidity? 
 
 Very humid  
 
 
 
 
 
 
           Very Dry 
 
 
 
      
        How do you feel about the room air flow? 
 
Very drafty 
 
 
 
 
 
 
 
 
No air   
 
 
 
 
 
 
 
 
 
    movement at all 
 
 
 
 
 
    Are you sweating? 
 
  Profusely 
 
 
 
 
 
 
 
 
Not at all 
 
 
 
 
 
 
    Are you shivering? 
 
  Constantly 
 
 
 
 
 
 
 
 
Not at all 
 
 
 
 
 
 
How hungry do you feel? 
I am not 
 
 
 
 
 
 
 
         I have never 
hungry at all  
 
 
 
 
 
 
      been more hungry  
 


Abbreviated Title: NST 
Version Date: 2021 July 22 
 
Page 54 
 


Abbreviated Title: NST 
Version Date: 2021 July 22 
 
Page 55 
Other parameters (noted by investigator at time of VAS report; this section is not shared 
with subject): 
 
Current Chamber Temperature:  
 
 
 
Current Relative Humidity:   
 
 
 
Previous day Chamber Temperature:   
 
 
 
Current Outdoor Temperature:  
 
 
 
Previous day Outdoor temperature (high):   
 
 
 
 
Previous day Outdoor temperature (low):  
 
 
 
 
 
Patient room temperature:  
 
 
 
 
Other comments: