Conditions: Menopause Obesity
Allocation: NON_RANDOMIZED | Intervention model: PARALLEL
Enrollment: 69
Healthy volunteers: yes

Brief summary:
The physiological relevance of brown adipose tissue (BAT) in humans is largely unknown. The investigators have shown that suppressing ovarian function in premenopausal women reduces resting energy expenditure (REE), and this is prevented by adding back estradiol (E2). The investigators preliminary data suggest that this may be due, in part, to reduced brown adipose tissue (BAT) activity. The overarching hypothesis is that BAT activity in humans is modulated by E2. To determine if natural declines in endogenous E2 contribute to changes in BAT activity, we will compare BAT activity in pre-and post-menopausal women. The investigators will also explore whether suppression of ovarian hormones in pre-menopausal women impairs BAT activity. BAT activity will be quantified using dynamic positron emission topography/computed tomography (PET/CT) imaging combined with 11C-acetate tracers. We will assess the thermogenic response of BAT by measuring cold-induced changes in REE, shivering, and skin and core temperature.

Detailed description:
Participants will be asked to perform studies to measure resting metabolic rate and activity of brown adipose tissue or brown fat. Brown fat is different from regular "white fat" in that it produces heat and burns calories. The investigators will measure how active brown fat is at normal room temperature and after participants have been exposed to cold.

The study will last 5-6 hours. It will consist of measurements made in warm temperatures and then after a period of cold exposure. For the cold exposure part of the study, participants will be asked to wear a cooling suit for 3 hrs. Skin temperature is normally about 90º Fahrenheit. The cooling suit will adjust the water temperature until your skin temperature is about 82º Fahrenheit. The suit will cover the entire body except for the face, hands, and feet. Participants may feel cold while wearing the suit and you may shiver some, but based on past studies, any shivering should be minor.

Before and after the cooling suit is turned on, body scans will be performed using a procedure called positron emission tomography (PET). These scans do not use radiation. Before the PET scans, the investigators will infuse radioactive tracer into an arm. These tracers have a low dose of radiation and will show us where the brown fat is and how active it is. One PET scan will be performed at room temperature, and the second scan will be performed after the cooling suit has been worn for 3 hours. Two additional scans using computed tomography (CT) will be performed at end of the study. CT scans are like an x-ray, and use radiation. The radiation dose in the CT scan is much higher than the tracers. When the CT scans are combined with the PET scans, we can separate brown fat from white fat.

15 pre-menopausal women will be asked to volunteer for a follow up study. For 5 months, pre-menopausal women in this study will receive monthly injections that contain a study drug (leuprolide) that reduces estrogen to postmenopausal levels. This drug is approved by the FDA to reduce estrogen for the treatment of uterine fibroids and endometriosis, but is not approved for use in healthy women. In this study, the drug is being used to decrease hormones to determine if this reduces brown fat activity. 6 months after the first injection, these women will be asked to repeat the PET/CT studies.

Arms:
arm 1: To determine if natural declines in endogenous E2 contribute to changes in BAT activity, the investigators will compare BAT activity in pre-and post-menopausal women. We will also explore whether suppression of ovarian hormones in pre-menopausal women (using leuprolide acetate) impairs BAT activity. arm 2: To determine if natural declines in endogenous E2 contribute to changes in BAT activity, the investigators will compare BAT activity in pre-and post-menopausal women.

Interventions: intervention 1: Leuprolide acetate
Intervention descriptions: intervention 1: A subset of premenopausal women will receive GnRHAG (leuprolide acetate) 3.75 mg/mo by intramuscular injection every 4 weeks for 24 weeks (6 doses).

Protocol document:
Prot_SAP_000.pdf: 
 
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_w 
COMIRB Protocol 
COLORADO MULTIPLE INSTITUTIONAL REVIEW BOARD 
CAMPUS BOX F-490    TELEPHONE:  303-724-1055  Fax:  303-724-0990 
 
 
 
Protocol #: 
16-1479 
Project Title: 
The impact of estrogen status on the biological function of brown adipose 
tissue in women measured using quantitative PET/CT  
 
Principal Investigator:  Edward Melanson, Ph.D. 
Version Date: 
2/7/2022 
 
 
 
 
  
 
I. Hypotheses and Specific Aims: The physiological relevance of brown adipose tissue (BAT) in 
humans is largely unknown. We have shown that suppressing ovarian function in premenopausal 
women reduces resting energy expenditure (REE), and this is prevented by adding back estradiol (E2). 
Our preliminary data suggest that this may be due, in part, to reduced brown adipose tissue (BAT) 
activity. Our overarching hypothesis is that BAT activity in humans is modulated by E2. To determine if 
natural declines in endogenous E2 contribute to changes in BAT activity, we will compare BAT activity in 
pre-and post-menopausal women. We will also explore whether suppression of ovarian hormones in 
pre-menopausal women impairs BAT activity. BAT activity will be quantified using dynamic positron 
emission topography/computed tomography (PET/CT) imaging combined with 11C-acetate tracers. We 
will assess the thermogenic response of BAT by measuring cold-induced changes in REE, shivering, 
and skin and core temperature. 
SA1: To determine if menopausal state influences constitutive BAT oxidative metabolism and 
activity volume under thermoneutral conditions (room temperature).  
H1a: BAT oxidative metabolism and activity volume will be greater in pre- than postmenopausal 
women 
H1b: REE will be associated with BAT oxidative metabolism and activity volume. 
SA2: To determine if menopausal state influences stimulated BAT oxidative metabolism and 
activity volume in response to very mild cold exposure.  
H2a: In response to a mild cold exposure, BAT oxidative metabolism and activity volume will be 
greater in pre- than postmenopausal women  
SA3 (exploratory): To determine whether 6 mo. GnRHAG in premenopausal women attenuates 
basal and stimulated BAT oxidative metabolism and activity volume. 
H3a: Basal and cold-stimulated BAT oxidative metabolism and activity volume will be reduced after 
GnRHAG. 
H3b: The decrease in REE after GnRHAG will be associated with the decrease in BAT oxidative 
metabolism and activity volume. 
II. Background and Significance: In rodents, the loss of ovarian function disrupts energy balance 
resulting in excess fat gain, and that this is prevented or reversed by estradiol (E2).71,75,89 In 
premenopausal women, we have shown that suppressing ovarian function decreases in REE (~50 
kcal/d),22,49 independent of changes in body composition,22  which may contribute to the risk for weight 
gain and obesity-related diseases that occurs with menopause.35,45,88 We have also shown that replacing 
E2 prevents this decrease in REE.49 However, the mechanisms by which the loss of E2 suppresses 
REE in women remain unknown.  
In humans, assessing the metabolic activity of BAT in vivo is challenging. Most studies have assessed 18F 
fluoro-2-deoxyglucose (18FDG) uptake using PET/CT.4,38,39 This approach provides an indication of the 
volume of active BAT, but is not a quantitative marker of BAT metabolic activity.4 In contrast, dynamic 
PET imaging (i.e., series of frames over contiguous time intervals) combined with metabolic tracers (e.g., 
11C-acetate) provides a quantitative measure of in vivo BAT metabolic activity. Using this approach, we 
have confirmed that BAT metabolic activity (clearance rate of 11C in BAT after 11C-acetate administration) 
increases in men exposed to a mild cold stimulus.6,58 We have also demonstrated that BAT is 


 
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metabolically active in humans under thermoneutral conditions,6 but have not yet quantified the 
contribution of this activity to REE.  
Our overarching hypothesis is that BAT activity in humans is modulated by E2. Specifically, we will 
determine if BAT metabolic activity contributes to resting energy expenditure (REE), and whether BAT 
metabolic activity under basal and stimulated conditions is modulated by estrogen status. We will assess 
the contribution of BAT metabolic activity to REE under both basal (thermoneutral) and stimulated (cold 
exposure) conditions. We will compare BAT oxidative metabolism (11C clearance rate in BAT) and BAT 
activity volume (volume of 18FDG uptake) in pre- and postmenopausal women, and in a subset of 
premenopausal women after 6 mo. of ovarian hormone suppression using gonadotropin releasing 
hormone agonist (GnRHAG) treatment. 
III. Preliminary Studies/Progress Report:   
Estrogen and fat gain: Basic and preclinical studies 
provide evidence that the loss of E2 or E2 signaling 
causes excess fat gain32,37,72,89,90 which is prevented 
by E2 treatment.50 In humans, suppression of 
gonadal function by gonadotropin releasing 
hormone agonist (GnRHAG) therapy causes excess 
fat gain24-26,65,90 and abdominal fat accrual.75 In both 
women75 and men,26 evidence suggests that such 
changes are specifically related to E2 deficiency. A 
system-level mechanism that contributes to excess 
fat gain in response to E2 suppression is a reduction 
in total energy expenditure (TEE).13,90  We have 
demonstrated that, like OVX and disruption of E2 
signaling in animals, suppression of ovarian function 
in women causes a decrease in REE and TEE. We 
have used a pharmacological approach 
(gonadotropin releasing hormone agonist, GnRHAG) 
to suppress ovarian function to explore the role of E2 in modulating REE. In our first study in 
premenopausal women (N=14), REE was significantly lower after 6 days of sex hormone suppression 
(1334±36 kcal/d, mean±SE) when compared with the mid-luteal phase (1405±42 kcal/d) or the early 
follicular phase (1376±43 kcal/d).22 This also reduced SNS support of REE.21 In our recently completed 
study,49,75 70 premenopausal women were randomized to 5 months of GnRHAG with (GnRHAG+E2) or 
without (GnRHAG+PL) E2 add back. Our major findings were that GnRHAG+PL caused a decrease in REE 
(-57±105 kcal/d), which was prevented by E2 (-4±75 kcal/d). Total daily energy expenditure (TDEE) was 
also decreased in response to GnRHAG+PL (-128±42 kcal/d), but this was not fully prevented by 
GnRHAG+E2 (-96±30 kcal/d) (Figure 1).  
Evidence for BAT in adult humans: The primary function of BAT is to produce heat. BAT is highly 
vascularized and richly innervated by the SNS.66 When activated, BAT generates heat through 
mitochondrial uncoupling of oxidation and phosphorylation, mediated by UCP1 that is uniquely and 
abundantly expressed in BAT mitochondria. BAT activity is stimulated by factors that increase SNS 
activity, such as acute cold exposure.91,92 Even exposure to room temperature (~22 °C) may be sufficient 
to stimulate BAT activity.5,6,15,16,19,58,83,86,91,92  BAT is present in rodents and other mammals throughout the 
lifecycle.14 Until recently, BAT in humans was thought to be present in significant volume and activity only 
in infants. This was despite the existence of necropsy studies demonstrating that BAT persisted in adults, 
albeit in fewer sites and smaller volumes.31 This view began to shift in the early 2000’s, as radiological 
reports documented non-tumor related 18FDG uptake in tissues with low radiodensity (indicative of an 
adipose tissue).57 In these studies, bilateral, symmetrical 18FDG uptake was often observed in the 
cervical, clavicular, and paraspinal regions. Because these areas were within fat depots (based on CT 
Hounsfield units), and were more pronounced when patients were not kept warm, many nuclear imaging 
departments began referring to these as BAT. Subsequent studies established that 18FDG uptake in 
these areas was stimulated by cold exposure,73,83 and biopsy studies confirmed these areas as BAT via 
morphological assessment and identification of UCP1.20,86 The reported prevalence of adults with 
detectable BAT varies markedly. Retrospective analysis of large cohorts who had undergone clinical 


 
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PET/CT scanning suggests that the prevalence of spontaneously detectable BAT is 5-10%, and identified 
a number of factors that increase the prevalence of spontaneously detectable BAT including colder 
outdoor temperature, female sex, younger age, and lower BMI and body fat content.1 Studies of cold 
exposed individuals have since shown that the prevalence of metabolically active BAT using 18FDG is 
much higher, ranging from 30 to 100%.5-7,58 Cold-induced BAT activity decreases with age,47,71,93 but has 
been observed even in humans up to the age of 64 yrs.5 Cold-induced BAT activity is inversely 
associated with body fatness,47,83,85 suggesting that BAT plays a role in body weight regulation, but 
whether a decline BAT activity contributes to the development of human obesity is unclear.2  
Most BAT studies in humans have used static PET/CT acquisitions to measure whole-body 18FDG 
uptake, expressed as the standardized uptake value (SUV).4,38 SUV is calculated as the average 
radioactivity concentration in a region of interest, relative to the injected dose and normalized to body 
weight. Traditionally, total volume of activity of 18FDG uptake is used to provide estimates of BAT volume. 
However, there are several limitations to this approach: 1) SUV is subject to many sources of variability 
including, but not limited to body composition, length of uptake period, and endogenous plasma glucose 
levels; 2) 18FDG uptake is influenced by the energy status of BAT (e.g. when BAT triglyceride (TG) levels 
are high, 18FDG uptake is reduced); and 3) BAT glucose uptake is associated with BAT blood flow but not 
thermogenesis.4,36,40 Thus, static 18FDG PET/CT acquisition helps determine the biodistribution of glucose 
to different organs under a given physiological condition (e.g. cold-exposure), but SUV is a semi-
quantitative unit that does not characterize physiological functions. In contrast, quantitative dynamic 
PET/CT imaging combined with metabolic tracers involves the collection of a series of frames over 
contiguous time intervals, which are used to model physiological parameters such as rate of tracer uptake 
or clearance in different tissues.55 For example, 18FDG is used to quantify facilitated transport and 
hexokinase-mediated phosphorylation of glucose, whereas the rapid conversion of 11C-acetate to acetyl-
CoA and ultimate entry into the TCA cycle can provide a measure of tissue oxidative metabolism.41 
Combining static and dynamic PET/CT methods is essential to provide accurate and valid assessments of 
changes in BAT metabolic rate and glucose uptake. 
The contribution of BAT to EE in humans: Our collaborators, Drs. Haman and Blondin, have shown that 
BAT oxidative metabolism contributes to the increased EE during acute cold exposure in humans. In one 
study, healthy young men (N=12, 23-42 yrs, BMI=24-31 
kg.m2) were studied during 3 hr. of mild cold exposure (18º 
C). Significant uptake of 18FDG was noted in areas 
identified as BAT as well as in deep muscles of the 
thoracic region. Conversely, BAT oxidative metabolism 
(quantified based on the fast monoexponenital decay of 
11C-acetate in BAT, kmono) increased significantly (two-fold) 
in BAT during cold-exposure, but not in adjacent skeletal 
muscles or subcutaneous adipose tissue (Figure 2).6 This 
indicates a cold-induced activation of oxidative 
metabolism in BAT, but not skeletal muscle or 
subcutaneous adipose tissue. Interestingly, an 
increased 11C clearance rate was also observed in 
BAT during measurements obtained at room 
temperature, suggesting that BAT oxidative 
metabolism was active even under ambient conditions 
(Figure 3). There was an inverse association between 
BAT activity volume and shivering, suggesting those with 
greater BAT volumes produced more heat via non-shivering thermogenesis (NST). In another study, 25 
men (22-64 yrs, BMI=23-35 kg.m2) were studied using a similar protocol.5 Even in older, overweight men 
with type 2 diabetes (T2DM) who demonstrate a lower 18FDG uptake in BAT relative to young, healthy 
men, BAT oxidative metabolism was increased during cold exposure to similar levels as their young, 
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Sex differences in BAT: In rodents, BAT activity is 
typically higher in females than males.69,81 In humans, 
retrospective studies have reported that the prevalence 
of BAT is greater in women than men but this sex 
difference diminishes with age, suggesting a potential 
role of female sex hormones.1,18,19,59,63,64  Conversely, 
acute cold exposure studies in humans have 
suggested that the volume of metabolically active BAT 
and BAT glucose uptake are similar in men and 
women.16 However, this may be attributed to limitations 
in the cold exposure protocols used. For example, one 
particular study recently reported that glucose uptake in BAT during a mild cold exposure was 50% lower 
in women compared to men. However, cold-induced thermogenesis (CIT, the increase in energy 
expenditure upon cold exposure) was also lower in women compared to men (0.3 vs. 0.9 kJ/min), 
suggesting the cold stimulus was not adequate to stimulate BAT in the women.30  
Evidence for regulation of BAT by estrogens: Several lines of evidence suggest that sex hormones 
regulate BAT activity.67,68,84,87 Androgens and estrogens exert opposing effects in brown adipocytes.51,70 
Testosterone reduces UCP1 expression and 
thermogenic activity, whereas E2 has the opposite 
effects.67,68,70 BAT function is compromised when ER 
signaling is disrupted.13,90 Additionally, OVX rats have 
decreased BAT thermogenic activity and UCP1 
expression in brown adipocytes,32,56,62,94 but these 
effects are reversed by E2 treatment.3,27,42,46,87  
CIT varies with E2: We recently performed a pilot study 
in healthy, premenopausal women (n=5; 31±6 yr.; BMI 
24.3±4.5 kg/m2) to determine the effects of ovarian 
hormone suppression on REE, CIT, and BAT activity. 
Before and after 3 mo of GnRHAG, participants 
underwent a 2-hr cooling protocol using a surgical 
cooling vest filled with water (~16-18º C). Following 
GnRHAG, REE decreased from 1382±218 kcal/d to 
1341±209 kcal/d; the decrease (-41±160 kcal/d) was 
similar in magnitude to that observed in response to 5 
mo of GnRHAG+PL in the study described above (-
57±105 kcal/d). CIT was also attenuated (350±228 vs. 
302±139 kcal/d, Figure 4). We also performed 18FDG 
PET/CT measurements to demonstrate the feasibility of 
measuring BAT activity with this protocol. After 
GnRHAG, no significant differences in 18FDG uptake 
were observed, but 18FDG uptake in BAT decreased in 
3 of the 5 individuals studied (Figure 5). There are two 
limitations to consider in this pilot study. Firstly, the 
period of hormone suppression may have been too 
short to invoke substantial changes in BAT activity and 
CIT, because a single injection of GnRHAG produces an 
initial sex hormone flare (for up to 3 wk) followed by continuous suppression with additional doses of 
GnRHAG. In the proposed study, we will use a longer period of ovarian suppression (6 mo) (SA3, 
exploratory). Secondly, the use of 18FDG PET/CT to study BAT metabolism is influenced by the energy 
status of the BAT. Because FM increased following GnRHAG, BAT TG content may have also increased, 
which will reduce 18FDG uptake independent of any changes in BAT metabolism.4 In the proposed 
study, in addition to 18FDG, we will use PET to measure clearance of the tracer 11C-acetate in BAT, 
which is not dependent on the energy status of the tissue and provides a direct measure of BAT 
thermogenesis. We expect to see a more clear suppression of BAT thermogenesis with a more 
prolonged period of ovarian suppression (pre- vs. postmenopausal women, SA1 and SA2; 6 mo. 


 
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GnRHAG, SA3) in combination with the use of 11C-acetate PET methodologies.  
IV. Research Methods 
A.  Outcome Measure(s):  The primary study outcome is BAT oxidative metabolism (11C-acetate 
clearance rate) during basal and cold-stimulated conditions. Secondary outcomes are BAT activity 
volume (18FDG uptake) during basal and cold-stimulated conditions; CIT; 18FDG update into muscle; 
shivering (sEMG & 18FDG uptake in deep skeletal muscles); and changes in skin and core temperature 
during cold exposure. Serum concentrations of hormones and metabolites will be measured primarily as 
descriptive variables, but we will also explore the association of these measures with the primary study 
outcomes.  
B. Description of Population to be Enrolled:  Premenopausal volunteers will be healthy, 
eumenorrheic women, aged 18-50 yrs. Eumenorrheic status will be verified by regular menses (no missed 
cycles in previous year; cycle length 25-35 d). Postmenopausal volunteers will be healthy women who 
have no menses for at least 12 months; postmenopausal status will be confirmed based on FSH (≥50 
mIU/ml). The primary exclusion criteria are:  
• 
Pre- and Postmenopausal women 
o 
BMI>40 kg/m2; we have purposely used a wide range of BMI to facilitate recruitment.  
o 
Thyroid dysfunction, defined as an ultrasensitive thyroid stimulating hormone (TSH) <0.5 or >5.0 
mU/L (pre- and postmenopausal women); volunteers with abnormal TSH values will be re-
considered for participation in the study after follow-up evaluation by the PCP with initiation or 
adjustment of thyroid hormone replacement. 
o 
Uncontrolled hypertension defined as resting systolic BP >150 mmHg or diastolic BP>90 mmHg; 
participants who do not meet these criteria at first screening will be re-evaluated, including after 
follow-up evaluation by the PCP with initiation or adjustment of anti-hypertensive medications 
o 
Self-reported diabetes or fasting glucose >126 mg/dL 
o 
Use of tobacco products (last 6 months) 
o 
Selective serotonin reuptake inhibitors (SSRIs) or any other medication or condition that may 
affect brown fat activity.  The decision will be based on the judgement of the study physician and 
primary investigator. 
o 
Serious illness within the last 6 months 
o 
Confirmed positive COVID test within the last 6 months 
 
• 
Additional exclusion criteria for postmenopausal women 
o 
Current use of hormonal replacement therapy or use <10 yrs ago. 
o 
Women who have undergone surgical menopause.  
 
• 
Additional exclusion criteria for premenopausal women 
o 
Current hormonal contraceptive use (past 6 mo).  
o 
Pregnant, lactating or intention to become pregnant during the period of study 
o 
FSH and estrogen levels that suggest an individual may be approaching perimenopause 
(reviewed by PI and study MD) 
o 
Because of the risks associated with GnRHAG treatment, women who meet any of the following 
criteria will be excluded from the GnRHAG intervention study, but will be permitted to participate in 
the cross sectional study 
▪ 
History of fragility fracture 
▪ 
Low BMD (i.e., proximal femur or lumbar spine z scores < -2.0)  
▪ 
Abnormal vaginal bleeding  
▪ 
History of breast cancer or other estrogen-dependent neoplasms  
▪ 
History of venous thromboembolic events  
▪ 
Hypersensitivity to leuprolide acetate or benzyl alcohol (the vehicle for injection of leuprolide 
acetate) 
▪ 
Evidence for depressive symptoms (Score > 16 on the Center for Epidemiologic Studies 
Depression Scale, CESD)  
▪ 
Moderate or severe renal impairment defined as a calculated creatinine clearance <50 
mL/min based on the equation of Cockcroft and Gault91 


 
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▪ 
Chronic hepatobiliary disease, conservatively defined as liver function tests (AST, ALT, 
alkaline phosphatase, Total Bilirubin) >1.5 times the upper limit of normal (if such values are 
obtained on initial screening and thought to be transient in nature, repeated testing will be 
allowed) 
C. Study Design and Research Methods   
Our hypothesis is that in humans, BAT activity is modulated by E2. To test this hypothesis, we will 
compare BAT oxidative metabolism (rate of 11C clearance in BAT) and BAT activity volume (volume of 
18FDG uptake) in healthy pre- and postmenopausal women (SA1 and SA2) under basal (thermoneutral) 
and stimulated (cold exposure) conditions. After providing informed consent, volunteers will undergo 
screening procedures. Volunteers who meet the inclusion/exclusion criteria (Section 2.3.5) will be invited 
to participate in the cross-sectional study (Table 1) to measure basal and cold-stimulated BAT activity 
(PET/CT). A subset of premenopausal women will also be studied after 6 mo. of GnRHAG treatment (SA3, 
exploratory). 
Table 1. Schedule of Procedures. 
 
Cross Sectional Study (SA1-2) 
Pre- (N=32) vs. Post-menopausal 
(N=32) 
Intervention Study (SA3) 
Pre-menopausal (N=15) 
Measurement 
Screening 
(~2 hrs) 
Visit #1 
Thermoneutral 
(~5 hrs) 
Vist #2 
Cold  
(~6 hrs) 
Visits (#3-8) 
(~1hr) 
Visit #8a 
(~1 hr) 
Visit #9 
Themoneutral 
(~5 hrs) 
Visit #10 
Cold  
(~6 hrs) 
Health history and 
physical exam 
X 
 
 
 
 
 
 
Screening labs 
X 
 
 
 
 
 
 
Urine Pregnancy 
Test 
X1 
X1 
X1 
X 
X1 
X1 
X1 
Blood chemistries 
X*5 
 
X4 
 
X4 
 
X4 
DXA (body 
composition, bone 
mineral density) 
and waist 
circumference 
X 
 
 
 
X 
 
 
PET/CT studies 
 
X2 
X3 
 
 
X2 
X3 
Monthly GnRHAG 
injections  
 
 
 
X 
 
 
 
1 –  Performed by the PRA 
2 -  A single dose of 11C-acetate (185 MBq) will be administered, followed by a regional CT and dynamic 
PET scan (nose to aortic arch) 
3 –  A dose of 11C-acetate (185 MBq) will be administered, followed by a regional CT and dynamic PET 
scan (nose to aortic arch).  A dose of 18F-FDG (185 MBq) will then be administered, followed by 
another dynamic PET scan, and then whole body static PET and whole body CT scan (eye level to 
knee caps) 
4 – blood samples will be obtained before and after the cold exposure study 
*5 – FMD/CIMT/blood draw procedures will be obtained at the screening visit or Visit #1 
+ – Waist circumference obtained) 
D.   Description, Risks and Justification of Procedures and Data Collection Tools: 
 
 
Pre-enrollment screening - The initial interaction is initiated by individuals who read or hear an 
advertisement and contact staff to learn more about the study. A brief phone screen will be conducted. 
Callers who meet general study criteria and are interested in learning more about the study will be 
scheduled for an orientation session. Volunteers will be sent a copy of the consent form in advance.  
Individuals may also be recruited through the VA database.  An initial letter and informational flyer may be 
sent by U.S. mail inviting individuals to participate.   
Orientation and informed consent – Interested volunteers will meet with a member of the research team 
to a) have the study and what is expected of them explained in detail; b) discuss any practical problems 
(e.g., scheduling conflicts, vacations) that could interfere with participation; c) have their questions 


 
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answered; and d) demonstrate their ability to provide informed consent by describing their understanding 
of the major study goals and what is expected of them if they choose to participate. Written, signed 
informed consent and HIPAA authorization will be obtained from volunteers willing to participate in the 
study.  
Screening - After providing informed consent, volunteers will undergo screening procedures, including a 
review of medical history (including menstrual cycle history), and a clinical laboratory evaluation including 
fasting glucose, creatinine, liver function tests, thyroid function tests, and a complete blood count. TSH 
will be measured in the Clinical and Translational Research Center (CTRC) core lab; other chemistries 
(metabolic panel, CBC) will be performed by the University of Colorado Hospital laboratory. This will be 
followed by measurement of bone mineral density (BMD). Premenopausal volunteers with a history of 
fragility fracture or low BMD (lumbar spine or proximal femur z-scores ≤ –2.0) will not be enrolled in the 
intervention study because of the possibility that BMD will decrease during GnRHAG. 
Serum hormones and metabolites - At study entry and at the end of the intervention (on the day of the 
cold exposure study), fasted blood samples will be obtained for the measurement of estrone (E1), E2, 
progesterone, testosterone, sex hormone binding globulin (SHBG), follicular stimulating hormone (FSH), 
leptin, cortisol, free fatty acids, glycerol, and thyroid hormones (T3, T4). An additional sample will be 
obtained at the end of the cooling protocol (at minute 120, before the 11C acetate infusion) for 
measurement of T3, T4, TSH, free fatty acids and glycerol.  Changes in these parameters will be 
evaluated for correlation with the changes in the primary outcomes. In rodents, BAT activation improves 
glucose tolerance.76,82 In humans, BAT activity is inversely associated with fasting blood glucose and 
hemoglobin A1c (HbA1c),47 and cold acclimation induces improvements in insulin sensitivity.43 Thus, 
samples will also be obtained for the measurement of glucose and insulin for the assessment of insulin 
resistance by the homeostasis model.48 Assays will be performed in the Clinical and Translational 
Research Center (CTRC) core lab. Samples will be stored at -80º C.  
Dual-energy X-ray Absorptiometry (DXA) – Body composition and BMD will be measured using a Hologic 
Discovery DXA instrument (Waltham, MA).  
Waist Circumference (WC) – will be measured using a spring-loaded tape measure.  Circumference will 
be measured at baseline, Visit 3-8, and after 6 months of suppression.  
Experimental procedures – PET/CT imaging studies will be performed on two separate days.  These 
studies will be performed as close as possible to each other (e.g., within 1-2 weeks) and within the luteal 
phase for pre-menopausal women).  Studies will be performed in the morning (~7 am). Participants will be 
asked to fast overnight (12 h) and refrain from strenuous physical activity for 48 h prior to the study day. A 
urine pregnancy test will be performed 
(premenopausal women) prior to initiating study 
procedures. Clothing will be standardized, with 
participants clad only in slippers socks, shorts, and a 
short sleeve t-shirt. During Visit #1, BAT activity 
under thermoneutral (room temperature) conditions 
will be determined by administering a single dose of 
11C-acetate and quantifying the decay using 
dynamic PET.  A regional CT scan will also be 
performed to define the regions of interest.  During 
Visit #2, BAT activity following cold-exposure will be 
determined by administering a single dose of 11C-
acetate and quantifying the decay using dynamic 
PET.  BAT volume and activity will then be 
determined by administering a single dose of 18F-
FDG followed by another dynamic PET scan, and 
then whole body static PET and whole body CT 
scan (eye level to knee caps) 
Visit #1 (Thermoneutral conditions, Figure 6a) – 
Participants will report to the Colorado Research 
Imaging Center at 8:00 AM.  Upon arrival, a urine pregnancy test will be performed in pre-menopausal 


 
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volunteers.  A fasted blood sample will be obtained for the measurement respiration and protein 
measures of mitochondrial markers, then FMD and C-IMT will be performed. 
Participants will be escorted to the CTRIC imaging center and be instrumented with wireless skin 
temperature sensors (Thermochron iButton model DS1922H; Maxim integrated, San Jose, CA, USA) and 
sEMG electrodes (Delsys, EMG System, Natick, MA, USA). Indwelling catheters will then be placed in an 
antecubital vein for tracer infusions.  Participants rest in a supine position under thermoneutral conditions 
(∼25°C) for 120 min.  After 90 min, REE will be measured using indirect calorimetry with a ventilated 
hood.  At 120 min, BAT oxidative metabolism will be determined by administering a ∼185 MBq bolus of 
11C-acetate followed by a 30 min list-mode dynamic PET acquisition and a regional CT scan (40 mA.s) 
centered at the cervico-thoracic junction to correct for attenuation and to define PET regions of interest 
(ROI).  
Visit #2 (Cold exposure, Figure 6b) – Participants will 
report to the Colorado Research Imaging Center at 
8:00 AM.    Upon arrival, a urine pregnancy test will be 
performed in premenopausal women.  A fasted blood 
sample will then be obtained for the measurement of 
estrone (E1), E2, progesterone, testosterone, sex 
hormone binding globulin (SHBG), FSH, leptin, cortisol, 
free fatty acids, glycerol, and thyroid hormones (total 
T3, free T4). Changes in these parameters will be 
evaluated for correlation with the changes in the 
primary outcomes. In rodents, BAT activation improves 
glucose tolerance.76,82 In humans, BAT activity is 
inversely associated with fasting blood glucose and 
hemoglobin A1c (HbA1c),47 and cold acclimation 
induces improvements in insulin sensitivity.43 Thus, 
samples will also be obtained for the measurement of 
glucose and insulin for the assessment of insulin 
resistance by the homeostasis model.48 Assays will be 
performed in the CTRC core lab. Samples will be 
stored at -80º C.  Participants will be escorted to the 
CTRIC imaging center and then be instrumented with wireless temperature sensors (Thermochron 
iButton model DS1922H; Maxim integrated, San Jose, CA, USA) and sEMG electrodes (Delsys, EMG 
System, Natick, MA, USA). Temperature sensors will be placed on the forehead, chest, forearm, back of 
the hand, lower back and quadriceps to measure mean skin temperature.60 sEMG electrodes will be 
placed on the belly of 8 large muscles known to contribute significantly to shivering during cold exposure 
(m. trapezius, m. deltoideus, m. sternoclastomastoid, m. pectoralis major, m. rectus abdominis, m. vastus 
lateralis, m. vastus medialis, m. rectus femoris).29 Participants will then be fitted with the liquid-conditioned 
suit, swallow a telemetric thermometry capsule to measure core temperature (Vital Sense monitor and 
Jonah temperature capsule, Mini Mitter Co., Inc., Bend, OR, USA) and will perform a series of maximal 
voluntary contractions (MVCs) of each of the eight muscles being recorded by sEMG for normalization of 
the shivering measures. Indwelling catheters will then be placed in an antecubital vein for tracer infusions. 
The liquid-conditioned suit will be perfused with 18° C water for 210 min (min 0-210). This is expected to 
induce a thermogenic response (~75% increase in REE) but minimize shivering. In our previous study 
using the same cold stimulus, mean skin temperature was 33.1±0.3º C at room temperature, and 
decreased to 28.6±0.3 ºC during cold exposure; shivering, measured using sEMG was minimal (1.6% of 
MVC); and REE increased from 6.7±0.3 to 12.2±0.8 kj.min-1 (~2300 to 4170 kcal.d-1).6 This suggests that 
the contribution of NST to CIT was substantial and at its highest level.9 Participants will be monitored 
continuously for shivering using sEMG collected using a digital recorder (Trigno Wireless EMG System, 
Delsys, Inc., Boston, MA).  REE will then be measured at minute 90 to determine CIT (cold-simulated 
REE – baseline REE). At time 120 min, a fasted blood sample will be obtained for the measurement of 
thyroid hormones (TSH, total T3, free T4) and markers of lyploysis (free fatty acids and glycerol). 
Following the blood sample, a ∼185 MBq bolus of 11C-acetate will be injected intravenously, followed by a 
30 min list-mode dynamic PET acquisition. This will be immediately followed by a regional CT scan 


 
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(40 mA.s) centered at the cervico-thoracic junction to correct for attenuation and to define PET ROI. At 
time 150 minute, a 2 minute PET frame will be performed to account for any residual 11C-acetate. A 
∼185 MBq i.v. bolus of 18FDG will then be administered followed by a 30 minute dynamic mode PET 
scan.  At time 180 min, participants will be instructed to void to minimize radiation exposure (30 min. after 
18FDG administration). A whole body CT scan (16 mA.s) and whole body static PET acquisition will then 
be performed to determine whole body 18FDG organ distribution and tissue SUV. To minimize radiation 
exposure, participants will be instructed to void a second time as soon as the study is completed (2 hrs. 
after 18FDG administration). 
Pre-menopausal women participating in the intervention study will repeat the PET/CT studies after 6 
month of ovarian hormone suppression (Visits #9 and 10). 
PET/CT image analysis - Dr. Scherzinger, a co-I in the Department of Radiology, will oversee data 
acquisition of all PET images and analysis of whole-body FDG PET acquisitions, with the guidance of our 
collaborator Dr. Denis Blondin. Drs. Blondin and Dr. Carpentier will provide additional guidance in the 
analysis and interpretation of the 11C-acetate data and the 18FDG dynamic PET data. 
The volume of supraclavicular BAT will be determined according to the following criteria: tissue radio 
density between −30 and −150 Hounsfield units and 18FDG uptake during cold exposure of more than 1.5 
SUV units.7,44 BAT volume of 18FDG will be quantified by from the voxel size of the image (i.e., in-plane 
length x width of the pixel x slice thickness), and BAT mass and activity will be quantified from volume 
using the assumption of a density of fat of 0.90 g/mL.58,59 The maximal and mean SUV, defined as the 
radioactivity per mm within the regions of interest, divided by the injected dose in MBq/kg BW, will be 
determined. Tissue-specific BAT oxidative metabolism index (the rapid fractional tissue clearance of 11C-
acetate, k, in s–1) will be estimated from BAT 11C activity over time using a monoexponential fit from the 
time of peak tissue activity.11 Total BAT oxidative metabolism index will be calculated as the product of 
the total volume of supraclavicular BAT and BAT oxidative metabolism index.7 BAT fractional glucose 
uptake (Ki) will be determined by Patlak linearization analysis of the 18FDG data.61 BAT glucose uptake 
(Km) will be calculated by multiplying Ki glucose by plasma glucose levels, which assumes a lump 
constant value of 1.77 Drs. Blondin and Carpentier will oversee data analysis of 11C-acetate dynamic PET 
acquisitions.  
sEMG - Raw EMG signals (collected at 1000 Hz) will be filtered to remove spectral components <20 Hz 
and >500 Hz, as well as 60 Hz contamination and related harmonics, and analyzed using custom-
designed MATLAB algorithms (Mathworks, Natick, MA, USA). Shivering intensity of individual muscles 
will be determined from root-mean-square (RMS) values. Baseline RMS values (RMSbaseline: 5 min RMS 
average measured prior to cold exposure) will be subtracted from shivering RMS (RMSshiv) values and 
RMS values obtained from the maximal voluntary contractions of individual muscles (RMSmvc). Shivering 
intensity will be normalized to RMSmvc, as previously described.5  sEMG data will be analyzed in Dr. 
Haman’s laboratory. 
GnRHAG therapy – Fifteen premenopausal women will be recruited from the cross-sectional study to 
participate in the longitudinal study. Following the next menses after baseline testing, participants will 
initiate 6 mo of GnRHAG therapy (intramuscular injection of leuprolide acetate 3.75 mg for depot 
suspension; Lupron; TAP Pharmaceutical Products, Inc.; Lake Forest, IL) to chronically suppress ovarian 
hormones. A single injection of leuprolide acetate produces an initial stimulation (for up to 3 wk) followed 
by a prolonged suppression of pituitary gonadotropins and ovarian hormones. Repeated monthly dosing 
suppresses ovarian hormone secretion. GnRHAG therapy has been used clinically for up to 1 year without 
serious adverse events, although there is a decrease in BMD that recovers following cessation of 
treatment.34,79 The drug is used for the management of endometriosis and for pre-operative management 
of anemia caused by uterine fibroids. Side effects are those typical of hypoestrogenemia. In studies of 
women with uterine fibroids, GnRHAG 3.75 mg/mo. induced amenorrhea in 61%, 86%, and 90% of women 
after 1, 2, and 3 mo of treatment, respectively.28,74 Return of menses typically occurs within 1 to 3 mo of 
cessation of therapy.  
Absence of pregnancy (urine test) will be confirmed before each dosing. Participants will be observed for 
30 min after drug injection to monitor for hypersensitivity reactions. Women who do not tolerate GnRHAG 
therapy in the proposed study can refuse further treatment after each monthly dosing. Among the first 94 
women treated with GnRHAG in our previous and ongoing studies (R01 AG018198, P50 HD073063), this 


 
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occurred only once. Participants will also meet with the study research nurse practitioner, providing a 
regular mechanism by which adverse events will be queried. A health status questionnaire will be used to 
track changes in use of medications (prescription and over-the-counter) or health (e.g., doctor visits, 
hospitalizations), as well as any study-related problems/concerns over the past 4 weeks. Participants will 
also complete a Menopausal Symptom List (MSL) to monitor the frequency and severity of menopause-
related symptoms (e.g., hot flashes, depressed feelings); the Eating Behavior Questionnaire (EBQ) to 
monitor dietary restraint, disinhibition, and hunger;78 and the Pittsburgh Sleep Quality Index (PSQI) to 
monitor sleeping behavior.12  
Potential risks 
• 
Leuprolide acetate – A subset of premenopausal women will receive GnRHAG (leuprolide acetate) 
3.75 mg/mo by intramuscular injection every 4 weeks for 24 weeks (6 doses). 
o 
Contraindications for use include: hypersensitivity to GnRH, GnRH agonist analogs or any of the 
excipients in Lupron Depot; undiagnosed vaginal bleeding; known or suspected pregnancy; 
lactation. 
o 
In women who are pregnant, use of leuprolide acetate may cause fetal abnormalities. 
o 
Leuprolide acetate may cause an anaphylactic reaction in volunteers with hypersensitivity to 
GnRH.  
o 
Regular menstruation should stop during leuprolide acetate therapy but spotting or breakthrough 
bleeding may occur. Cessation of menses does not ensure that pregnancy will not occur. Normal 
menstrual function is usually restored within 2-3 months after therapy is discontinued. 
o 
Leuprolide acetate may cause symptoms related to hypoestrogenism including hot flashes, 
headaches, nausea, emotional lability, decreased libido, acne, myalgia, sleep disorders, 
reduction in breast size, hypertension, and vaginal dryness. Bone loss may occur, although the 
amount lost over the 24-week intervention should be recovered slowly after discontinuation of 
leuprolide acetate treatment. There may be a development or worsening of depression and the 
occurrence of memory disorders 
o 
In rare cases, leuprolide acetate may cause seizures (post-marketing concern). 
o 
Leuprolide acetate therapy for 24 weeks may cause an increase in FM (~1.0-1.5 kg), a decrease 
in fat-free mass (1-1.5 kg), an increase in total, LDL, and HDL cholesterol (5-10%), and an 
increase in triglycerides (15-20%). 
o 
Local injection site reactions including induration and abscess may occur.  
• 
Risk of radiation exposure: Participants will be exposed to radiation during the PET/CT and DXA 
scans. During the PET/CT scans, participants are exposed to radiation from the CT and from the 
radiopharmaceuticals.  The primary risk of this level of radiation exposure is to the unborn fetus of 
pregnant women. All participants will perform one DXA and PET/CT study, and a subset of 
premenopausal women will perform these tests twice. The estimated radiation exposure during each 
study visit is listed in the table below 
 
Table 2.  Estimated radiation exposure (mSv) 
 
o 
During the DXA scans, the radiation exposure is approximately 0.015 mSv per total body 


 
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scan. 
o 
During study visit #1 (room temperature), participants will be exposed to approximately 1.87 
mSv of radiation.   
o 
During study visit #2 (cold exposure), participants will be exposed to approximately 7.92 mSv 
of radiation.   
o 
After allowing for 20% extra exposure from the procedures, women who complete study visits 
#1 and #2 will be exposed to a total of up to 11 mSv or radiation (~3.6 cross-country flights).  
Pre-menopausal women who complete study visits #8 and 9 will be exposed to a total of up 
to 22 mSv of radiation (7.3 cross-country flights).   This is below the annual amount of 
exposure for radiation workers (50 mSv).    
 
FDG will accumulate in the bladder.  The estimated whole body radiation exposure following the 
FDG infusion depends on when subjects void.  Participants will be instructed to void 1 hour after 
the FDG infusion.  Please note that the cold exposure will induce diuresis, and in our experience, 
subjects void immediately after the procedures.  The void volume will also be larger than what 
might be seen at room temperature, and this increases elimination of the radiopharmaceuticals 
 
• 
PET/CT: Aside from the radiation exposure, the risks associated with the PET/CT scan are the 
chance of incidental findings. PET/CT is used to diagnose a few diseases like some cancers and 
diseases of the brain. There is a very small risk that diseases like this may be found. Some 
participants may also experience claustrophobia in the scanner.  Some participants may experience a 
burning sensation in the arm during the 11C-acetate infusion.  The infusion only lasts for a few 
seconds and the burning sensation quickly diminishes after the infusion is complete.   
• 
sEMG and skin temperature sensors: The sEMG and temperature sensors may cause some minor 
discomfort and/or skin irritation due to the paste used to attach the sensors.  
• 
Core temperature pills: The temperature pill moves through the intestinal tract like food does, but on 
rare occasions, can become stuck in the intestines. 
• 
Venipuncture and IV catheters: Venipuncture will be performed to obtain blood samples for laboratory 
analyses, and an IV catheter will be used during the 11C-acetate and 18FDG infusion.  There is a small 
risk of syncope, local hematoma, infection, and thrombosis associated with intravenous blood 
sampling. 
• 
Confidentiality and privacy: The use of questionnaires, interviews, and collection of personal medical 
information poses a risk to confidentiality and privacy and may cause embarrassment. 
Plans to minimize risks 
Leuprolide acetate: The risks associated with use of the study drugs will be minimized by enrolling 
participants who do not have contraindications for their use. Volunteers will undergo a medical history and 
physical examination by a study clinician. Absence of pregnancy (urine test) will be confirmed before 
GnRHAG injections are administered. Participants will be observed for 30 min after drug injection to 
monitor for new hypersensitivity reactions. Women will be instructed that they should not become 
pregnant while taking study drugs because of risks to the fetus. This physician will discuss non-hormonal 
based contraceptive methods during the health and physical examination. They will be instructed that 
cessation of menses may or may not occur during the study and that cessation of menses does not 
provide protection against pregnancy; contraception (not hormonal) must be used.  
Women who do not tolerate GnRHAG therapy in the proposed study can refuse further treatment after 
each monthly dosing. Among the first 94 women treated with GnRHAG in our previous and ongoing 
studies (R01 AG018198, P50 HD073063), this occurred only once. Participants will also meet with the 
study research nurse practitioner, providing a regular mechanism by which adverse events will be 
queried. A health status questionnaire will be used to track changes in use of medications (prescription 
and over-the-counter) or health (e.g., doctor visits, hospitalizations), as well as any study-related 
problems/concerns over the past 4 weeks. Participants will also complete e Menopausal Symptom List 
(MSL) to monitor the frequency and severity of menopause-related symptoms (e.g., hot flashes, 
depressed feelings); the Eating Behavior Questionnaire (EBQ) to monitor dietary restraint, disinhibition, 


 
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and hunger;78 and the Pittsburgh Sleep Quality Index (PSQI) to monitor sleeping behavior.12 After 
completing the study, Participants will be asked to notify study team when menstrual cycles have 
resumed.  The study team will follow up after 2 months if no contact has been made by participant. 
Radiation exposure: All participants will perform one DXA and PET/CT study, and a subset of 
premenopausal women will perform these tests twice. Women who complete study visits #1 and #2 will 
be exposed to a total 9.91 mSv or radiation.  Pre-menopausal women who complete study visits #8 and 9 
will be exposed to 19.61 mSv of radiation. 
• 
To minimize risk to an unborn fetus, participants must have a negative pregnancy test prior to each 
measurement.  
• 
DXA: Conducting two scans before and after the intervention involves a total radiation exposure of 
approximately 0.43 mSv, which is <1% of the annual allowable exposure for radiation workers (50 
mSv/yr over a 40 year career).  
• 
PET/CT: The FDA annual allowable exposure for radiation workers for whole body or organ specific 
radiation exposure is 50 mSv/yr over a 40 year career.  Radiation exposure increases the risk of a 
fatal cancer by 4-5% per Sievert.  Radiation exposure in this study is <50 mSv, so the increased risk 
is <0.001%. The highest organ radiation exposure in this study will be in the bladder. Conducting two 
studies before and after the intervention in the subset of premenopausal women will involve a total 
radiation exposure of 38 mSv in the bladder, which is ~76% of the annual allowable exposure. To 
minimize exposure, participants will void 30 min. after the FDG infusion, and then again as soon as 
the study is completed (2 hrs. after 18FDG administration). In our experience the cold exposure 
causes all participants to void by the time the study is completed. According to the Institute for 
Science and International Security, the average annual dose equivalent of natural ionizing radiation in 
Denver is 11.8 mSv/yr. In comparison to natural background radiation, the radiation exposure in the 
proposed experiments is comparable to 1.6 years of natural radiation from living in Denver. 
Incidental findings during the PET/CT scans: All images for this study will be de-identified and reviewed 
for incidental findings. Should anything significant be found, the Nuclear Medicine physician will discuss 
these findings with the study MD. The participant will be contacted immediately by the Nuclear Medicine 
physician and advised on clinical follow-up to confirm findings. 
Venipuncture and IV catheter: The risks of hematoma and infection are minimized by having trained 
personnel perform the procedures using sterile techniques. 
Core temperature pills: Participants will be advised to avoid having an MRI test for at least a week after 
the study. Because the capsule is a radiotransmitter, participants will also be advised to avoid travel on 
commercial airlines for at least a week after the study. The recording device is electrically isolated and 
complies with hospital standards for electrical safety. 
sEMG and skin temperature sensors: Any skin irritation from the sensors is expected to be minor and 
temporary.  
Confidentiality and privacy: These risks will be minimized by not including personal identifying information 
on the forms, when possible, and by conducting interviews and collection of personal information in a 
private setting. 
Evaluation of study-related events – The subset of premenopausal women enrolled in the intervention 
study will meet with a study clinician every 4 weeks to review a Health Status Questionnaire that the 
participants will complete. The questionnaire queries about changes over the past 4 weeks in 
medications, health status, and any concerns with study medications. The clinician will specifically note 
on the form whether there are any concerns that are possibly, probably, or definitely related to the study. 
Participants will also be instructed to report concerns that may be study related when they occur to the 
research nurse practitioner, Ms. Quick, who will initiate event reports for both the programmed and 
spontaneous complaints. 
E.   Potential Scientific Problems:   
We considered studying women across the menstrual cycle, rather than a cross-sectional comparison of 
pre- vs. postmenopausal women. The strength of the cross-sectional design is that postmenopausal 


 
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women will have been estrogen deficient for several years, which should maximize the attenuation of E2 
deficiency on BAT activity. A limitation is that we will not be able to determine whether this is also partially 
due to age. However, if the exploratory aim confirms that GnRHAG reduces BAT activity, this will support 
the overall hypothesis that E2 deficiency, rather than age, is the primary factor contributing to reduced 
BAT in postmenopausal women. 
There is no known way to distinguish the energy expenditure associated with shivering and non-shivering 
thermogenesis in humans. Thus, we will employ rigorous techniques to measure shivering during cold 
exposure using both sEMG and 18FDG uptake into deep muscles. These data will be used quantify 
shivering and used to interpret differences and changes in CIT.  
Postmenopausal women will likely have higher levels of body fat, and body fat may increase in 
premenopausal women after GnRHAG. As described above, the cooling suit delivers an identical cold 
stimulus during each study. Our intent is not to determine the maximal activity of BAT, but rather, 
determine if differences in BAT activation in response to a constant stimulus is either different between 
pre- and postmenopausal women (SA2), or changes after GnRHAG (SA3). To account for differences or 
changes in body composition, REE will be expressed per kg of fat free mass. 
We acknowledge that GnRHAG causes a more abrupt withdrawal of ovarian function (i.e., weeks rather 
than years) and does not generate the same gonadotropin response of menopause (i.e., low rather than 
high). However, the GnRHAG model is also a strength because it allows us to control the sex hormone 
environment in a manner that is not possible when studying the natural menopause transition.  
F.   Data Analysis Plan:   
Sample size justification and power analyses – Sample size calculations and power analyses were 
performed using SAS 9.4 (SAS Institute, Inc., Cary, NC). For our estimate of effect size, we refer to our 
previous study that measured CIT in the follicular and luteal phases (Figure 5).8 In that study, CIT was 
higher during the luteal phase (2530±524 kcal.d-1 vs. 2225±440 kcal.d-1). Unfortunately, we did not 
measure BAT activity. Because there were no differences in shivering in this study, we will assume 
differences in CIT were due to differences in NST; we also assume that this reflects differences in BAT 
activity. Using a pooled SD of 484 kcal/d, 32 evaluable participants per group (64 total) will provide 80% 
power to detect a difference of 305 kcal/d in CIT between pre- and postmenopausal women using a 2-
sided t-test and a type I error rate at 0.05. Assuming a dropout rate of 15%, 38 participants per group will 
need to be enrolled to yield the desired sample size of 64 participants. The primary endpoint is BAT 
oxidative metabolism (11C clearance rate, expressed per mL of BAT per s-1). In our previous study of 12 
men, BAT oxidative metabolism increased during cold exposure from 0.35±0.13 to 0.74±0.19 
(mean±SEM).6 Using a pooled SD of 0.45, we estimate that we will have 80.0% power to detect a 
difference between groups of 0.32 during thermoneutral conditions. Using a pooled SD of 0.66, we will 
have 84.7% power to detect a difference between groups of 0.50 during cold exposure. In another 
study, BAT oxidative metabolism at room temperature was 0.35 in lean men vs. 0.05 in overweight men 
with T2D.10 We have also reported that BAT oxidative metabolism increased in lean mean from 0.73 to 
1.59 following a 4-week cold acclimation.7 Thus, we consider this to be a meaningful effect size to 
examine differences in BAT oxidative metabolism between groups during thermoneutral conditions and 
during cold exposure. A secondary endpoint is BAT activity volume. In our previous study, BAT activity 
volume was 57±16 ml (mean±SE).6 Using a pooled SD of 55 ml, the study will have 80.0% power to 
detect a difference of 39 mL between groups. In another study, we reported that BAT activity volume was 
48 vs. 4 ml in young healthy men compared to older men with T2DM.5 Thus, we consider this to be a 
meaningful effect size. 
Analysis strategy - Statistical analyses will be performed using SAS 9.4. Analyses will begin with 
descriptive plots of individual variables to evaluate distributional characteristics and for quality assurance. 
The effect of menopausal status on each endpoint will be estimated using a linear regression model. The 
endpoint will be the response variable and a variable indicating menopausal status will be the explanatory 
variable (equivalent to a 2-sample t-test). If a variable that is related to the response variable is not 
balanced between menopausal groups, that variable will be included in the regression model to eliminate 
the potential of the effect of menopausal status being confounded by that variable. For all multivariable 
analyses, regression diagnostics will be used to ensure that the group of explanatory variables are not 
collinear. Secondary analyses will be performed to investigate whether REE is associated with either BAT 


 
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oxidative metabolism or BAT activity volume. Exploratory analyses will investigate the changes in BAT 
oxidative metabolism and BAT volume of activity associated with GnRHAG treatment (Aim 3) Because 
these aims are exploratory, results will be reported using descriptive statistics, mean change, and 95% 
confidence intervals (CIs) for the changes in the primary and secondary endpoints.  
G.  Summarize Knowledge to be Gained:   
The importance of understanding mechanisms that contribute to risk for obesity cannot be overstated 
given the current epidemic of overweight and obesity in the U.S. and other developed countries 
worldwide. Although the loss of ovarian function is only one of a multitude of physiological, behavioral, 
and environmental factors that increase risk for fat gain, it is potentially a very important one in women. 
The strong evidence from multiple large RCTs and smaller physiologic trials indicates that estrogen-
based HRT attenuates weight gain in postmenopausal women by an average of 40%. Further, in the 
absence of HRT, weight gain in postmenopausal women is accompanied by a disproportionate increase 
in abdominal fat. Although abdominal obesity and the related metabolic dysfunction increases risk for 
coronary artery disease  in both women and men, the risk of mortality may be ~5-fold higher in women. 
Also, it has been estimated that abdominal obesity and metabolic dysfunction account for about 20% of 
coronary events in men, but 48% of events in women. Such statistics underscore the importance of 
understanding the mechanisms of abdominal fat gain in women – including both energy expenditure and 
energy intake regulation. In this context, the proposed study has the potential to reveal a consequence of 
the loss of ovarian function that has widespread health implications.  


 
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Contributions of white and brown adipose tissues and skeletal muscles to acute cold-induced 
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