Treating PCOS With Semaglutide vs Active Lifestyle Intervention
Phase: PHASE2
Conditions: PCOS, Adolescent Obesity, NAFLD
Sponsor: University of Colorado, Denver

Study Title: 
 
Treating PCOS with Semaglutide vs Active 
Lifestyle Intervention (TEAL Study) 
 
Protocol Version Date: 11/4/2022 
 
NCT03919929  
Garcia Reyes, Yesenia 
[Email address] 
Abstract 
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When you’re ready to add your content, just click here and start typing.] 

COMIRB Protocol # 19-0636 
 
 
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COMIRB Protocol  
COLORADO MULTIPLE INSTITUTIONAL REVIEW BOARD 
CAMPUS BOX F-490    TELEPHONE:  303-724-1055  Fax:  303-724-0990 
 
 
 
 
 
 
 
  
 
 
Project Title: Treating PCOS with Semaglutide vs Active Lifestyle Intervention (TEAL Study) 
 
Version Date: 11/4/2022  
Principal Investigator: Melanie Cree-Green 
 
Financial Sponsor: National Institutes of Health (NIH) 
 
Investigational Product name(s): Rybelsus® (semaglutide)  
 
IND Sponsor listed with FDA: Melanie Cree-Green (IND# 146652) 
 
Name and address of protocol sponsor:  
Melanie Cree-Green  
13123 E 16th Ave, B265 
Aurora, Co 80045 
Office 720 777-5743 
Fax: 720 777-7301 
Melanie.Green@childrenscolorado.org  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

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PROTOCOL SYNOPSYS 
Title: 
Treating PCOS with Semaglutide vs Active Lifestyle Intervention (TEAL Study) 
Sponsor: 
National Institutes of Health 
Phase: 
Phase 3 Clinical Trial 
Description of 
Sites/Facilities Enrolling 
Participants: 
 
Children’s Hospital Colorado  
Description of Study 
Intervention: 
Rybelsus (semaglutide) oral tablets vs dietary intervention 
Study Duration: 
60 months 
Participant Duration: 
5-6 months 
Rationale: 
In obese girls with polycystic ovary syndrome, testosterone and obesity combine to create 
unique pathology to increase metabolic disease including fatty liver and insulin resistance, 
which may be mediated by altered glucagon like peptide-1 activity. The investigators will 
treat girls with obesity and polycystic ovary syndrome for 4 months with a glucagon like 
peptide-1 receptor agonist compared to dietary intervention to primarily lower hepatic fat 
and secondarily improve whole body and adipose insulin sensitivity. Mechanisms of 
hepatic metabolism, including rates of de novo lipogenesis and relative mitochondrial flux 
will also be assessed.  
 
The objective of this clinical trial is to define the metabolic impact of 4-months of 
treatment with Rybelsus vs. diet therapy in girls with Polycystic Ovarian Syndrome 
(PCOS). Our previous research findings have shown that girls with PCOS have a higher 
degree of metabolic disease when compared to BMI matched controls. This includes an 
excess of non-alcoholic-fatty liver disease (NAFLD), dysglycemia, upregulated de-novo 
lipogenesis (DNL), hepatic, adipose and peripheral insulin resistance (IR) and decreased 
GLP-1 secretion. Our pilot data demonstrated that there were metabolic improvements with 
short-duration GLP-1 RA exenatide administration, with no side effects observed. The 
long-term consequences of NAFLD in girls with PCOS are of great clinical importance. 
This study will define the metabolic impact of a currently available medication on this 
common high-risk disease in youth thereby changing health outcomes. 
Study Design: 
This study is a randomized (2:1) semaglutide (Rybelsus®) vs dietary counseling. After 
completing a screening visit, participants will complete a baseline metabolic study and then 
be randomized to either semaglutide oral tablets or dietary counseling for a period of 4 
months. There will be a check-in visit halfway through the study (mid-treatment visit), and 
then they will complete a final metabolic study at the completion of their 4 month 
intervention.  
Primary Objective: 
 
Aim 1:  Quantitate change in IHTG in obese girls with PCOS after 4 months of GLP-1 RA 
therapy, compared to girls undergoing intensive dietary counseling and similar weight loss. 
Hypothesis:  IHTG will decrease after GLP-1 RA therapy in PCOS girls and will be greater 
than that seen from dietary counseling alone. Methods:  IHTG will be measured with MRI 
before and after interventions.  
Secondary Objectives: 
 
Exploratory Aim 2: Measure changes in substrate delivery and utilization after GLP-1 RA 
or intensive diet. 
 
 
A. Measure changes in rates of DNL and adipose IR, ie insulin suppression of FFA release  
Hypothesis: GLP-1 RA therapy will significantly improve adipose IR and decrease DNL, 
resulting in reduced IHTG. Changes will be greater than those seen from dietary 

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counseling alone. Methods: Deuterated water will be utilized to measure DNL. Whole 
body and adipose insulin sensitivity will be modeled from FFA and glucose changes 
relative to insulin concentrations during oral sugar tolerance test (OSTT). 
B. Assess rates of hepatic TCA cycling and NAFLD severity  
Hypothesis: Excess cataplerotic TCA cycling provides substrate for DNL and contributes 
to hepatic damage via the production of reactive oxygen species. Methods:  Oral U-13C3 
glycerol will be consumed and serum glucose and VLDL undergo isotopomer analysis to 
determine relative TCA cycle flux. Hepatic 31Phosphorus MR spectroscopy and MR 
elastography will be utilized to document NAFLD severity and ATP concentrations. 
xploratory Aim 3: Determine the extent to which diet or GLP-1 treatment alters neuronal 
response within, and functional connectivity between, homeostatic and non-homeostatic 
brain regions in adolescents with PCOS and obesity. 
Hypothesis: Connectivity between the homeostatic and non-homeostatic brain regions will 
be significantly greater after treatment with semaglutide compared to diet. 
Number of Patients: 
Up to 60, with the goal of completing 50  
Enrollment Criteria: 
Inclusion Criteria 
1) Female 
2) Ages 12-21 years 
3) Sedentary- less than 2 hours of moderate (jogging, swimming etc) 
exercise a week. 
4) BMI equal or greater than the 90th percentile for age and gender 
5) PCOS per the most stringent NIH criteria adapted for adolescents 
(irregular menses >12 months post-menarche and clinical or biochemical 
hypertestosteronemia)8,34 
6) Participants cannot be on hormonal contraception, so participants should 
remain abstinent or use reliable non-hormonal contraception (e.g. 
copper IUD) for the entire study period. For participants who 
receive semaglutide, they should avoid pregnancy for at least   2 
months after stopping medication to avoid fetal exposure to the 
medication. 
Exclusion Criteria: 
1) Diagnosed with or have a family history of medullary thyroid carcinoma 
(MTC) or Multiple Endocrine Neoplasia syndrome type 2 (MEN 
2). Family history of medullary thyroid cancer or thyroid nodule 
palpated by endocrinologist at screening. 
2) Use of medications known to affect insulin sensitivity: metformin (cannot 
have been used in the 3 months prior to screening), oral 
glucocorticoids within 10 days, atypical antipsychotics, 
immunosuppressant agents, HIV medications, hormonal 
contraception (cannot have been used in the 6 months prior to 
screening). Dermal patch or vaginal ring contraception methods. 
Weight loss medications or stimulants. Use of other products 
containing other GLP-1 agonists. 
3) Currently pregnant or breastfeeding women. Development of pregnancy 
during the study period will necessitate withdrawal from the study. 
4) Severe illness requiring hospitalization within 60 days. 
5) Diabetes, defined as Hemoglobin A1C > 6.4% 
6) BMI percentile less than the 90th percentile for age and sex. Weight >325 lbs. or <84 lbs. 
7) Anemia, defined as Hemoglobin < 11 mg/dL 

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8) Diagnosed major psychiatric or developmental disorder limiting informed consent. 
9) Implanted metal devices that are not compatible with MRI 
10) Use of blood pressure medications. 
11) Known liver disease other than NAFLD or AST or ALT >100 IU/L. 
12) Personal history of pancreatitis 
13) Known renal disease of any severity or an eGFR at screening of <45ml/min/1.73m2 
14) History of severe GI disease (e.g. gastroparesis) 
15) History of gallstones 
16) Untreated thyroid disease 
17) History of hypersensitivity to semaglutide  
18) Other causes of hyperandrogenism (example: tumor, CAH) or amenorrhea (untreated 
thyroid disease, tumor, primary ovarian failure, prolactinoma). 
19) Active symptoms or undergoing treatment for anorexia nervosa or binging/purging 
disorder 
Dose and Route of 
Administration: 
Rybelsus® (semaglutide) is available in 3mg, 7mg, and 14mg oral tablets. Patients will be 
on the 3mg dose for the first 30 days, and then switch to the 7mg dose for the remainder of 
the study. Since our patient population will not have diabetes, we will not utilize the 14mg 
dose, as per packaging instructions, the 14mg dose is only used if additional glycemic 
control is needed. 
Safety Assessments: 
Patients will be instructed to contact study staff if they experience any adverse events. In 
addition, they will be assessed for adverse events when they come for their study visits and 
these will be graded based on the Common Terminology Criteria for Adverse Events 
(CTCAE) as described in the protocol.  
Data Monitoring 
Committee:  
A Data Monitoring Committee (DMC) will established by the investigator to review data 
relating to safety and efficacy, to conduct and review interim analyses, and to ensure the 
continued scientific validity and merit of the study. 
Endpoints: 
Primary Endpoint:  
1. Change in Hepatic Fat Fraction 
Change from baseline in presence/severity of hepatic fat fraction will be measured with 
MRI, and calculated via the Dixon method as the proton density hepatic fat fraction, which 
ranges from 0-75%. [Time Frame: Baseline and 12 weeks] 
Secondary Endpoints:  
2. Change in Rate of De Novo Lipogenesis 
Change from baseline of the rate of overnight de novo lipogenesis will be measured 
utilizing stable isotope methods with deuterated water, and expressed as the rate of newly 
synthesized lipids in the serum triglyceride fraction. [Time Frame: Baseline and 12 weeks] 
3. Change in Whole Body Insulin Sensitivity 
Participants will undergo a 75 gram oral glucose tolerance test, and the change from 
baseline in whole body insulin sensitivity will be expressed as Si, calculated via the oral 
minimal model. [Time Frame: Baseline and 12 weeks] 
4. Change in Adipose Insulin Sensitivity 
Change from baseline of adipose insulin sensitivity will be calculated as the percent 
suppression of free fatty acids, and the nadir of free fatty acids during the oral glucose 
tolerance test. [Time Frame: Baseline and 12 weeks] 
 
Other Pre-specified Outcome Measures: 
5. Change in Relative Hepatic Mitochondrial Flux 

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Change from baseline of relative hepatic mitochondrial flux will be assessed with an oral 
glycerol stable isotope tracer and subsequent blood draws while fasting. The outcome will 
be the relative proportion of hepaticly secreted glucose and glycerol that has undergone 
label rearrangement representative of excess mitochondrial metabolism - termed % 
indirect. [Time Frame: Baseline and 12 weeks] 
6. Change in Functional Neuroimaging Measures 
Change from baseline of functional neuroimaging measures, specifically, the blood oxygen 
level-dependent (BOLD) signal in the hypothalamus, insula, anterior cingulate cortex, 
nucleus accumbens, amygdala, and superior and inferior frontal gyri, and connectivity 
between these brain regions. [Time Frame: Baseline and 12 weeks] 
Statistical Analysis Plan 
and Rationale for 
Number of Patients 
SA1 Statistical Plan 
Sample size justification.  Sample size is based on the power to detect a difference between 
the standard diet + GLP-1 RA group and the intensive dietary counseling group in the 
primary outcome of change in IHTG. Estimates for power calculations were obtained from 
a recent randomized controlled trial of liraglutide in adult women with PCOS68 and our 
baseline data on IHTG in obese girls with PCOS (Table 2). Assuming a standard deviation 
(SD) of 0.7% and a two-sided significance level of 0.05, a final sample size of 17 
participants in the intensive diet group and 33 participants in the standard diet + GLP-1 RA 
group provides 90% power to detect a difference of -0.7% between group change. This 
difference is 50% smaller than the difference of -1.34% observed by Frøssing et al.68 as 
their results are confounded by significant weight loss and therefore our study is powered 
conservatively. We anticipate a 1% decrease in hepatic fat.  We estimate an approximately 
5-10% drop out rate based on our group’s experience from similar participant 
populations9,10,111,116,118,182,183 and we will enroll up to 60 girls with the goal of 50 
completers.   
 
Analysis plan.  Data will be collected and managed in a REDCap database, a secure web-
based application designed to support data capture for research studies, including validated 
data entry, audit trails, and automated export to statistical packages. Prior to the start of any 
formal analyses, variables will be examined for unusual values that need to be queried, 
patterns of missing values, and whether their distributions are non-Gaussian. Demographic 
and clinical characteristics will be summarized with descriptive statistics. Primary analyses 
will follow the “intent-to-treat” principle so that study hypotheses are tested under realistic 
conditions in which not all participants adhere to the intervention. This will include those 
who discontinue therapy whose data will be censored at the time of discontinuation, so that 
all data we have on the non-completers up until the time they leave the study will be 
included in this primary analysis.  However, in order to fully evaluate the  physiology and 
mechanism of action of semaglutide in this population, which is an important aim of this 
investigator-initiated study, we will also perform secondary analyses in participants who 
demonstrated at least 80% adherence (i.e., at least 80% medication taken in the GLP-1 arm 
and at least 80% of targeted weight loss achieved in the intensive lifestyle arm). For the 
primary outcome, we will compare the change in % IHTG with and without GLP-1 RA 
treatment using a t-test or Mann-Whitney U test. We expect the weight loss will be similar 
in the two groups; however, if there is a clinically significant difference between the 
groups, these comparisons will also be performed while adjusting for the amount of weight 
lost. Similarly, if there are differences in activity or obstructive sleep apnea severity, 
appropriate adjustments will be made. Similar analyses will be used for secondary 
outcomes (2 hour OGTT glucose, HOMA-IR, metabolomics markers, proportion of 

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direct/indirect glucose, EAA concentrations).  Linear models will be used to identify the 
variables that are associated with change in IHTG, e.g., 2 hour OGTT glucose, HOMA-IR, 
metabolomics markers.           
SA2 Statistics 
 Sample size justification. The primary outcomes for Aim 2 are (1) change in hepatic de 
novo lipogenesis (DNL) and (2) change in FFA nadir in the standard diet + GLP-1 group 
compared to the intensive diet group.  Estimates of variability were obtained from the PI’s 
pilot data (Figure 4b). We assumed a cross-sectional DNL SD of 3.36%, a pre-post 
correlation of 0.8, and a two-sided significance level of 0.05.  With 50 participants, we will 
have 90% power to detect a between-group DNL difference of 2.1%, which is smaller than 
the difference of 2.6% reported by Armstrong et al. in adults14. Using the same 
assumptions and a cross-sectional SD of 23 nmol/L, we will have 80% power to detect a 
change of 22% in FFA nadir (Figure 4c), which is conservative given data in adults 
showing a decrease of 31% with liraglutide67.  
Analysis plan: Analyses for the primary outcomes of change in DNL and FFA nadir will be 
the same as Aim 1, as will the analyses for secondary measures of DNL (% suppression of 
triglyceride) and secondary outcomes (2-hour OSTT glucose, % TCA cycling, peripheral 
IR per OMM, metabolomics markers).  From the study day 1 cross sectional data, we will 
use regression models to evaluate the relationships between markers of hepatic disease 
(IHTG and PDE/ATP) and DNL, excess substrate (FFA nadir, mean OSTT glucose) and 
measures of TCA anapleurotic cycling (% indirect glucose).  Table 7 provides details on 
the power calculations for these models of primary interest, using estimates of variability 
obtained from the PI’s pilot data.  If the relationship between % TCA cycling and IHTG is 
significant, we will use mediation modeling to test if this effect is mediated by DNL.  The 
distribution-of-the-product method will be used to construct 95% confidence intervals for 
the mediated effects, using the RMediation software package184.  After investigating 
associations between variables in the cross-sectional data from study day 1, we will 
evaluate if baseline characteristics predict changes in the study primary outcomes, in order 
to further understand the pathophysiology and to identify patients benefit from particular 
interventions.  
 

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1. BACKGROUND AND SIGNIFICANCE 
     Polycystic ovary syndrome (PCOS) affects 6-10% of women, is defined by elevated testosterone and 
encompasses many co-morbidities including insulin resistance (IR), type 2 diabetes (T2D), nonalcoholic fatty liver 
disease (NAFLD) and decreased quality of life1,2. The testosterone excess in PCOS confers a unique additional risk 
for metabolic disease beyond obesity, with 3-4 fold higher rates of NAFLD and T2D relative to women with normal 
androgens3-8. In fact, our data in PCOS girls show a 50% prevalence of impaired glucose tolerance and hepatic 
steatosis (HS). Despite the high prevalence and gravity of co-morbidities seen in PCOS, widely effective therapeutic 
options are lacking. There is a critical need to understand the metabolic pathology underlying PCOS to inform new 
therapeutics. HS is one of the best predictors of future T2D and is already common in youth with PCOS9,10.  Thus, 
our long-term goal is to understand hepatic substrate energetics as a gateway for prevention and treatment of 
metabolic and hormonal disease in obese youth with PCOS.   
     Based on our extensive preliminary data in adolescent girls9-11, our hypothesis is that testosterone-induced 
alterations in hepatic energy metabolism, combined with excess substrate from poor diet and peripheral IR, are 
central to metabolic disease in PCOS (Figure 1). These effects are worsened by 
abnormalities in the incretin glucagon-like peptide (GLP-1), which are common 
in obesity12-22. PCOS girls in our studies have a high prevalence (50%) of 
NAFLD, defined as excess intrahepatic triglyceride (IHTG), relative to BMI-
similar controls (15%)9,10. Obese PCOS youth in our cohort have increased rates 
of hepatic de novo lipogenesis (DNL) and increased free fatty acids (FFA) despite 
hyperinsulinemia. These PCOS girls also have significant muscle and hepatic IR 
relative to BMI-similar controls, which may provide substrates for DNL9-11. 
Androgens contribute to a unique disease pathology, increasing DNL gene 
expression and fatty acid synthesis rates23,24. Studies in adults with NAFLD have 
suggested that excess anapleurotic cycling of carbons through the TCA cycle 
provide excess acyl CoA for DNL and generate reactive oxygen species that 
contribute to hepatic fibrosis25-28. 
     Inadequate GLP-1 secretion/activity relate to HS in adults with diabetes, 
PCOS and NAFLD and animal studies have identified key roles for GLP-1 in preventing HS12-22. Our preliminary 
data demonstrate that GLP is similarly pathologic in PCOS girls. Limited studies show that GLP-1 receptor agonist 
(RA) therapy decreases IHTG in T2D adults and improves menstrual regularity in PCOS women13,14,29-33. Two doses 
of a short acting GLP-1 RA exenatide significantly improved glucose metabolism in our obese youth with PCOS 
regardless of HS status, showing the potential for clinical and metabolic improvements in PCOS youth.  
 
A.1 Significance  
   Obese girls with polycystic ovarian syndrome (PCOS) already have evidence of metabolic disease, are at high risk 
for metabolic decline as they age and therapeutic options are limited. New interventions focused during this 
vulnerable yet reversible stage are thus critical. PCOS affects 6-10% of U.S. women and 10-15% of obese women, 
with an estimated economic burden of $4 billion and is increasing in prevalence in parallel with the increase in 
obesity1,2. PCOS can first be diagnosed in adolescence and its phenotype includes high androgens, 
irregular/infrequent menses and dermatologic manifestations including facial hair, balding and severe acne8,34. 
Metabolic disease is more common in PCOS and worsens with obesity. Insulin resistance (IR) is nearly universal in 
PCOS and contributes to the earlier onset and rising incidence of non-alcoholic fatty liver disease (NAFLD), type 2 
diabetes (T2D) and cardiovascular disease (CVD)3-8. We found that >50% of obese girls with PCOS have hepatic 
steatosis (HS; ie early NAFLD) as well as severe IR and frequent dysglycemia, and CVD risk markers9,10. The 
primary therapy for PCOS with obesity is lifestyle modification, which can be very challenging for youth due to 
peer, family and socioeconomic issues. Unfortunately, current therapeutic options for PCOS and HS are inadequate. 
Approved medical therapies for PCOS include metformin, which only reduces PCOS severity in approximately half 
of patients and oral contraceptives which may actually worsen IR8,34-41.  
 
Frequent abbreviations used in this protocol: ALT= alanine aminotransferase; BMI=body mass index; CVD=cardiovascular disease; 
DNL=de novo lipogenesis; D20=deuterated water; FFA=free fatty acids; GLP-1=glucagon-like peptide-1; GLP-1 RA=glucagon-like 
peptide-1 receptor agonist; HOMA=homeostasis model assessment; HS=hepatic steatosis; IHTG=intrahepatic triglyceride; IR=insulin 
resistance; MRI=magnetic resonance imaging; MRS=magnetic resonance spectroscopy NAFLD=non-alcoholic fatty liver disease; 
NMR=nuclear magnetic resonance; OGTT=oral glucose tolerance test; OMM=oral minimal model; OSTT=oral sugar tolerance test; 
PCOS=polycystic ovarian syndrome; TCA=tricarboxcylic acid cycle; TG=triglycerides; T2D= type 2 diabetes; VAT=visceral adipose 
tissue 
Fig 1: ↑ IHTG in PCOS is 
distinct from simple 
obesity and multi-
factorial 

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   Treating NAFLD in PCOS youth may confer significant long-term health benefits. Obesity and PCOS are 
synergistic for increasing the risk of NAFLD (Figure 2).  Understanding and treating HS in girls with PCOS at the 
critical preclinical stages may be an important strategy to prevent future NAFLD, T2D and CVD as HS strongly 
and, independently predicts T2D and CVD development42-44. Elevated liver 
transaminases (a marker of NAFLD) predict dysglycemia and T2D onset45.  
Conversely, treatment of NAFLD with an effector of bile acid metabolism improves 
glycemia46, arguing for a tight connection between glycemia and liver health. IR in 
adipose, muscle and hepatic tissue increases as the degree of HS increases47. HS can 
also progress to cirrhosis and liver failure6,7,42. Unfortunately, youth onset NAFLD is 
more severe at presentation and progresses more rapidly than adult disease48,49.  
 
A.2 Scientific Premise 
 
   The unique mechanisms underlying HS in obese women with PCOS have not been well-studied, as the majority of 
mechanistic NAFLD studies have focused on men. Increased androgens in PCOS confer a pathologic metabolic 
milieu beyond that seen in obesity alone (Figure 3), and this process develops with disease onset or earlier, 
regardless of BMI53-57.  Studies by our team 
in PCOS and others in adults with NAFLD 
indicate that upregulated hepatic de novo 
lipogenesis (DNL) and increased delivery of 
free fatty acids (FFA) to the liver are the two 
most important contributors to intrahepatic 
triglyceride (IHTG)28,58-61. NAFLD also 
relates to dysglycemia and IR27,28,47,60,62,63. 
Excess carbon availability (anaplerosis) to 
the TCA cycle leads to excess carbon 
availability 
for 
metabolic 
processes 
(cataplerosis) 
such 
as 
DNL 
and 
the 
production of reactive oxygen species which 
contribute to hepatic IR and progressive 
NAFLD25,26. Strategies to lower IHTG thus 
include decreasing DNL and FFA60,64,65. 
   Glucagon-like peptide-1 receptor agonists 
(GLP-1 RA) are a promising choice to treat NAFLD in PCOS. GLP-1 RA lower serum glucose and thus substrates 
for DNL and increase insulin secretion, leading to greater suppression of FFA following a meal13,18-20,66,67. However, 
GLP-1 RA cause weight loss and it is unclear if the improvements in HS seen in adults with T2D or NAFLD are 
from direct GLP-1 activity or due to weight loss13,66. Limited GLP-1 trials in women with PCOS are promising29-
32,68. We will use GLP-1 RA in PCOS to probe hepatic metabolism while also assessing if GLP-1 RA is a viable 
therapeutic option to improve metabolic disease in PCOS, beyond its weight loss affect. 
 
A.3 Scientific Premise 
   Disinhibited eating behaviors (DEB) are associated with obesity and weight gain. Non-homeostatic brain 
systems promote DEB and are altered in obesity. Homeostatic brain systems control whole-body energy 
homeostasis, contribute to eating behavior and are altered in obesity. Despite the proven role of the 
hypothalamus in controlling eating behavior to maintain whole-body energy homeostasis, few studies in 
humans have focused on how the hypothalamus responds to weight-loss interventions. Limited data are 
available on functional connectivity between brain non-homeostatic and homeostatic systems and their 
coordinated role in DEB. 
   Semaglutide and other GLP-1 receptor agonists are thought to decrease appetite through direct 
interactions with the brain in the hypothalamus. Whereas this has been extensively studied in animals, 
there is extremely limited data in humans and almost none in adolescents. With the likely future marketing 
of semaglutide for obesity in youth, it is thus critical to start to understand how these medications function 
in the brain as opposed to dietary behavior weight loss.  
   The goal of this sub-aim is to better understand the function and interaction of both homeostatic and 
non-homeostatic systems in the context of DEB in obesity. Understanding communication between 
homeostatic and non-homeostatic brain regions and their relationship to DEB among adolescents with 
Fig 2. IHTG ↑morbidity 
Fig 3. Androgens and obesity synergize to ↑ IHTG in PCOS 
We hypothesize that the synergy between obesity (orange) and 
excess testosterone (blue) in PCOS (purple) has unique influences 
on IR, DNL and IHTG50-52. Specific Aims in red. 

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PCOS and obesity undergoing weight-loss therapy will help to elucidate specific neural mechanisms that 
contribute to overeating, and ultimately to weight regulation. 
 
2. HYPOTHESES AND SPECIFIC AIMS 
Primary Specific Aim 
Aim 1:  Quantitate change in IHTG in obese girls with PCOS after 4 months of GLP-1 RA therapy, 
compared to girls undergoing intensive dietary counseling and similar weight loss. 
Hypothesis:  IHTG will decrease after GLP-1 RA therapy in PCOS girls and will be greater than that seen from 
dietary counseling alone. Methods:  IHTG will be measured with MRI before and after interventions.  
Secondary Specific Aim 
Exploratory Aim 2: Measure changes in substrate delivery and utilization after GLP-1 RA or intensive diet. 
A. Measure changes in rates of DNL and adipose IR, ie insulin suppression of FFA release  
Hypothesis: GLP-1 RA therapy will significantly improve adipose IR and decrease DNL, resulting in reduced 
IHTG. Changes will be greater than those seen from dietary counseling alone. Methods: Deuterated water will be 
utilized to measure DNL. Whole body and adipose insulin sensitivity will be modeled from FFA and glucose 
changes relative to insulin concentrations during oral sugar tolerance test (OSTT). 
B. Assess rates of hepatic TCA cycling and NAFLD severity  
Hypothesis: Excess cataplerotic TCA cycling provides substrate for DNL and contributes to hepatic damage via the 
production of reactive oxygen species. Methods:  Oral U-13C3 glycerol will be consumed and serum glucose and 
VLDL undergo isotopomer analysis to determine relative TCA cycle flux. Hepatic 31Phosphorus MR spectroscopy 
and MR elastography will be utilized to document NAFLD severity and ATP concentrations. 
 
Exploratory Aim 3: Determine the extent to which diet or GLP-1 treatment alters neuronal response within, 
and functional connectivity between, homeostatic and non-homeostatic brain regions in adolescents with 
PCOS and obesity. 
Hypothesis: Connectivity between the homeostatic and non-homeostatic brain regions will be significantly greater 
after treatment with semaglutide compared to diet. 
 
Overview of Clinical Studies 
SA1 Review of Relevant Literature 
Women with PCOS have a 2-3 fold increase in rates of HS and NAFLD. An estimated 50-70% of obese women 
with PCOS have HS, compared to 20-30% of similarly obese women without PCOS 4,69-75. Increased IHTG in non-
obese women with PCOS is also more common but not always found, likely due to varied definitions of 
PCOS4,70,72,76,77. In PCOS youth, only 2 published studies exist beyond ours and all three studies indicate increased 
rates of HS9,57,78. Many of these studies found associations between androgen concentrations and severity of HS, in 
addition to associations with PCOS status4,69-72,79, suggesting a critical role of testosterone. 
 
Decreases in HS are associated with improvements in metabolic disease. Both lifestyle interventions and 
pharmacotherapy have been utilized to acutely lower IHTG and decreases in IHTG are uniformly accompanied by 
improvements in insulin sensitivity and/or dysglycemia in animals and humans46,80-85. Stains induce mild 
improvements in NAFLD and decrease CVD risk more in those with existing NAFLD than those without, 
suggesting that the improvement in NALFD is additive to the direct lowering serum lipids86. 
 
Short-term dietary interventions can decrease HS but are difficult to implement long-term in a clinical setting. Diets 
with decreased calories and simple carbohydrates have been shown to decrease rates of HS across the 
lifespan61,81,84,87-89. Fructose is preferentially used by the liver for DNL and just 9 days of a low fructose diet reduced 
hepatic fat by 50% in youth with HS90. Unfortunately, this diet is difficult for most families to follow long-term and 
compliance with lifestyle recommendations is higher in a research setting than in the clinical setting91,84,92. The 
American Association of Clinical Endocrinologists’ guidelines for obesity state that reducing total caloric intake is 

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key for weight loss, with 4-10% weight loss recommended long-term to reduce NAFLD93. Pediatric NAFLD 
guidelines state that intensive weekly contact is needed to improve NAFLD in youth, which is beyond what most 
clinics and families can manage94.  
 
Effective pharmaceutical therapies to treat HS are lacking. Multiple pharmacologic strategies have been tested to 
treat NAFLD with no to moderate success94-96. Diabetes medications that lower glucose and thus DNL precursors 
such as metformin have had variable success96-99. Recent evidence indicates that SGLT-2 inhibitors, which increase 
glycosuria, decrease HS in adults with T2D, but may not be effective with the milder degree of dysglycemia seen in 
nondiabetic patients with PCOS100. Thiazolidinediones work via the peroxisome proliferator-activated receptor-γ 
and appear to decrease IHTG and glucose concentrations by increasing fat oxidation and redirecting TG storage to 
adipose tissue85,101-105. This class of medication is effective for treating metabolic and hormonal disease in women 
with PCOS, but the associated weight gain and possible serious side effects has reduced enthusiasm for 
thiazolidinedione use in youth106,107. Bile acid sequestrants alter bile acid metabolism and can lower IHTG and may 
do so through increasing incretins such as GLP-1108. Thus, there are no routinely recommended medications for HS 
in youth, but new classes of medications are being tested93,94. 
 
GLP-1 RA decrease HS in adults with NAFLD or T2D, although results are confounded by weight loss. GLP-1 RA 
therapy is currently used to improve glycaemia in T2D and to induce weight loss in obesity, and is a promising 
therapy for treating NAFLD13. GLP-1 is an incretin released in response to meals that increases insulin and 
decreases glucagon secretion12-14, 15. Individuals with obesity, NAFLD and T2D have both GLP-1 resistance and/or 
deficiency21,22, and inadequate meal responses were reported in obese youth109. A recent secondary analysis of T2D 
patients receiving GLP-1 RA for glucose control demonstrated a decrease in IHTG and markers of NAFLD66,110. 
Further, decreases in HS induced by GLP-1 RA were related to improvements in glucose concentrations66. Studies 
in multiple animal models demonstrated the role of GLP-1 in the development and reversal of genetic or diet-
induced IHTG accumulation18-20.   
 
Limited data indicates that GLP-1 treatment accompanied with weight loss decreases metabolic disease in women 
with PCOS. A small number of studies examined the role of GLP-1 RA in women with PCOS29-32.  Obese women 
with PCOS had lower baseline and post-meal GLP-1 concentrations compared to obese controls. GLP-1 
concentrations did not improve with oral contraceptives but did with metformin29,30. 6 months of GLP-1 RA reduced 
markers of liver fibrosis in obese PCOS women31 and 3 months of GLP-1 RA therapy reduced visceral adipose 
tissue32 or hepatic fat, although results are confounded by unequal weight loss between groups68. Improvements in 
ovulation rates, menstrual regularity and decreases in testosterone were also observed33. 
 
SA1 
Preliminary 
Data 
We 
have 
been 
increasing 
our 
knowledge 
of 
the 
physiology of HS, IR 
and dysglycemia in 
obese youth though 6 
distinct 
studies, 
including 
2 
NIH-
sponsored 
multi-
center trials: RISE111 
and 
TODAY112-114 
(Table 1). The 4 
local studies included 
stable isotope tracers, 
MR-measured IHTG 
to better understand pathophysiologic hepatic mechanisms9,10,115-119. Results from these studies that informed our 
proposed study are shown below. Our proposed team includes investigators from these trials, demonstrating our 
expertise in these type of trials in youth, as well as their tolerability by our adolescent participants and feasibility of 
the measures. 
 
Table 1: Youth studies used for preliminary data 
Study, N 
Participant population 
Team Role 
Relevant methods 
RESISTANT 
N=80 
Lean and obese youth  
With or without T2D 
Cree-Green Co-I  
Nadeau PI 
Multi-tissue clamp-assessed IR 
31P MRS of muscle   
AIRS 
N=73 
Lean and obese girls  
With or without PCOS 
Cree-Green PI  
Nadeau mentor 
Multi-tissue clamp-assessed IR 
Markers of hepatic DNL 
2 hour 75 gm OGTT 
APPLE 
N=92 
Obese girls 
With or without PCOS 
Cree-Green PI 
Nadeau mentor 
Multi-tissue OSTT-assessed IR 
Acetate tracer measured DNL 
2 dose GLP-1 RA pilot trial 
PLUM 
N=18 
Obese girls  
With PCOS 
Cree-Green PI 
Nadeau mentor 
OSTT-assessed IR 
Isotopomer TCA cycle analysis 
31P MRS of liver 
TODAY 
SITE N=45 
Obese youth 
With T2D 
Zeitler PI  
 
Intensive lifestyle intervention 
Stop light diet counseling 
RISE 
SITE N=34  
Obese youth 
With T2D 
Cree-Green Co-I 
Nadeau PI  
Zeitler Co-I 
Standard lifestyle counseling 
Stop light diet counseling 
Multiple metabolic studies 

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Obese girls with PCOS have excess IHTG and >50% have HS. Our data from more than 120 obese girls with PCOS, 
when compared to normal weight or similarly obese girls with and without T2D shows that PCOS girls have 
evidence of liver disease including 1) mildly increased 
fasting serum TG 2) liver transaminase (ALT) elevation 
severity similar to T2D girls 3) MRI-assessed IHTG 
even higher than in T2D girls (Table 2).  Most notably, 
≈50% of obese girls with PCOS had clinically 
significant HS (defined as >5.5% liver fat), whereas 
only 15% of obese girls without 
PCOS had HS, and less severe abnormalities were also seen in normal-weight girls with PCOS10. These findings 
indicate that hepatic pathology occurs early in the course of PCOS. IHTG also strongly relates to elevations in free 
testosterone (r=0.437, p<0.001) and 75% of girls had HS if their free testosterone was >50% above the upper limit 
of normal. 
 
GLP-1 and glucagon meal responses are abnormal in obese girls with PCOS. Serum GLP-1 and glucagon 
concentrations were assessed prior to and in response to an OSTT. Girls with PCOS had less GLP-1 release to an 
OSTT than obese controls, 
becoming 
progressively 
worse with greater degrees 
of HS (Figure 4A). GLP-1 
decreases 
glucagon 
and 
accordingly, 
glucagon 
concentrations 10 min into 
the OSTT were higher in 
girls with HS and PCOS 
(Figure 4B).  
 
Our current team can induce short-term, modest weight loss in obese youth utilizing the proposed methods.  The PI, 
CO-I’s and research study team have extensive research and clinical experience with lifestyle interventions in this 
and similar populations (Table 3). In the RISE study, in the first 3 months youth achieved a 1.9±0.25 kg weight loss 
utilizing the stop light diet as taught by our current staff. At completion of  
the PI’s PCOS studies with identical inclusion 
criteria to that proposed (cross-sectional AIRS, 
APPLE and PLUM studies), girls with PCOS 
receive intensive lifestyle counseling utilizing the 
stop light diet and an individualized daily calorie 
target to induce weight loss based on metabolic cart 
determined basal metabolic rate and typical activity 
from the accelerometer. At clinical follow-up ~ 3 
months later and with no additional coaching, the average weight loss is 1.8 kg. Based on the literature, our weight 
loss goals for the diet group are modest and similar to this last group of PCOS girls, with a goal of 2 kg. This goal 
will be tailored to match that seen in the GLP-1 RA group, but clearly there is room for further weight loss if 
needed, as we will be adding extensive follow-up and weight monitoring. 
 
SA2 Review of Relevant Literature 
Women with PCOS have IR in both adipose and peripheral IR in excess of that seen from obesity alone. Obese 
women with PCOS and HS have more IR than those without HS, indicating a link between HS and worsening IR5. 
IR in muscle, hepatic and adipose tissue is reported to be worse in PCOS adults than in adult women of similar BMI 
and thought to be related to increased androgens53-56.  Similar findings are seen in PCOS girls57. 
 
Excess IHTG is secondary to excess DNL and substrate delivery from peripheral and adipose IR. Several studies in 
adults with NAFLD have linked increased IHTG to increases in DNL28,60,63. In obese adolescents and adults, 
increased IHTG was related to the degree of excess FFA from adipose IR42,47,120. Importantly, following weight loss 
or weight gain, DNL and FFA dynamically change in accordance with increases or decreases in IHTG60,81. NAFLD 
also relates to dysglycemia and peripheral IR27,28,47,60,62. Adipose IR and peripheral IR are tightly related in obese 
youth62,121. In obese women with PCOS, peripheral and adipose IR is closely related to increased IHTG4,70,77,122 with 
similar results in two existing studies in youth57,123. 
Table 2: Obese PCOS girls have ↑IHTG, serum TG 
and markers of liver inflammation 
Population 
IHTG % 
TG (mg/dL) 
ALT (IU/L) 
Lean Control 
1.0±0.1 
75±6 
23±4 
Obese Control 
2.3±1.5 
78±8 
29±3 
Obese PCOS 
6.2±2.3 
129±7 
38±3 
Obese T2D 
4.5±1.8 
207±7 
40±2 
Fig 4: GLP-1 and glucagon release are altered with PCOS and HS 
Table 3: Study team’s success with short term 
weight loss in obese youth 
Study 
RISE 
Clinical follow-up 
after PI’s studies 
Participants 
Obese, both sexes 
T2D, pre-diabetes 
Obese, girls 
PCOS 
Weight loss (kg) 
1.9±0.25 
1.8 ± 0.4 
Follow-up time 
3 months 
3.3 months 

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Reductions in HS improve IR and clinical symptoms of PCOS. In youth, a combination of a thiazolidinedione, 
metformin and an androgen receptor blocker flutamide lowered IHTG and improved insulin sensitivity76. Further, 
change in IHTG was the best predictor of improved ovulation and lowered free testosterone76. However, flutamide is 
not available in the US and TZD’s cause weight gain, precluding widespread use of this combination. A year of 
metformin reduced NAFLD and increased ovulation in overweight women with PCOS124. However, metformin does 
not consistently improve PCOS severity or NAFLD34,125-127. 
 
Hepatic 31P MRS can be utilized to understand hepatic metabolism and document progressive NAFLD. In addition 
to the three ATP peaks and free phosphate (Pi), the liver contains phosphodiester peaks (PDE), thought to represent 
endoplasmic reticulum stress128. PDE’s are higher in progressive NALFD128-132 and this finding is consistent with 
findings in animals of increased hepatic stress and NAFLD128,133,134. ATP and Pi concentrations also relate to hepatic 
function134. MRS performed during an OSTT demonstrated increased mitochondrial flux thought to be secondary to 
excess FFA delivery to the liver120. 
 
GLP-1 RA may alter intrahepatic energy metabolism. Mechanistic data suggest that all of the pathways related to 
increased IHTG as listed in figure 3 can be impacted by a GLP-1 RA. GLP-1  increases insulin and decreases 
glucagon secretion which lowers postprandial FFA and glucose,12-15 potentially decreasing substrates for IHTG 
production135. Moreover, hepatocytes express GLP-1 receptors, thus GLP-1 can directly impact hepatic insulin 
signaling, leading to decreased gluconeogenesis and DNL through decreased carbohydrate uptake16-18. GLP-1 also 
suppresses genes directly involved in the formation of IHTG, particularly stearoyl-coA desaturase, a key enzyme in 
DNL 18-20. Mechanistic studies on the role of GLP-1 RA in NAFLD are limited in humans. In healthy men, GLP-1 
RA lowers post-prandial FFA, secondary to increased insulin67. GLP-1 RA decreased glucose release and DNL in 
adults with NAFLD14. Thus, GLP-1 affects both drivers of increased IHTG: DNL and FFA delivery to the liver and 
is a target for reducing IHTG. 
 
SA2 Preliminary Data 
Obese girls with PCOS have significant multi-tissue IR and increased rates of dysglycemia. We examined the 
differential tissue-specific response to insulin in female youth with PCOS compared to Dr. Nadeau’s studies of T2D, 
utilizing a 4 phase hyper-insulinemic euglycemic clamp with glucose and glycerol stable isotope tracers. By 
contrasting patterns of pathology between these disorders, we uncovered subtle differences that suggest unique  
mechanisms of metabolic 
change in PCOS. Obese 
girls with PCOS (N=38) 
had multi-tissue IR relative 
to similarly obese controls 
(N=19), but were not as IR as T2D girls (N=40)136. Whereas the concentration of insulin required to suppresses 
lipolysis assessed with glycerol Ra  
trended to be higher in PCOS compared to obese controls (p=0.06), FFA were higher during hyperinsulinemia and 
the amount of insulin required to suppress 50% of fasting FFA was significantly higher, indicating adipose IR 
(Table 4). The concentration of insulin required to suppress 50% of fasting glucose release was 2-fold higher in girls 
with PCOS (p=0.04), indicating hepatic IR and may be reflective of dysregulated gluconeogenesis. Muscle IR, 
measured as glucose rate of disappearance was much worse (p<0.001) in PCOS. Following a 75-gram oral glucose 
tolerance test (OGTT), nearly half of PCOS girls had impaired glucose tolerance compared to 20% of obese 
controls. We found that OGTT insulin concentrations were different when girls were divided into tertiles of muscle 
IR (Table 5). We thus modelled OGTT glucose and insulin with the oral minimal model (OMM)137-139 and found 
that 2 hour OGTT OMM data correlated well with the 
clamp muscle IR (r=0.65, P<0.001). Further, the OMM 
was more precise when extended to 6 hours, when insulin 
returns to baseline post-OGTT in this population.  
 
HS in obese girls with PCOS relates to DNL, adipose IR 
and peripheral IR. Obese girls with PCOS undergoing 
clamps had higher serum concentrations of markers of DNL (i.e. long chain fatty acids with a double bond in the 7th 
carbon position140; N7 FFA; 29±13 nmol/g obese vs. 45±15 PCOS, p<0.01)141 and the N7 FFA concentrations 
correlated with IHTG% (r=0.53, p=0.004)9. IHTG% also correlated with serum FFA nadir during the highest dose of 
the clamp (r=0.362, p=0.043) indicating a potential contribution of excess hepatic FFA delivery, secondary to 
adipose IR. Muscle IR correlated with IHTG (r=-0.374, p=0.021). 
Table 4:  Obese PCOS girls have adipose, liver and muscle IR  
 
PCOS 
OB CONT 
Adipose IR, insulin (mIU/L) to suppress 50% of fasting FFA 
60±14 
39±6 
Hepatic IR, insulin (mIU/L)  to suppress 50% of fasting EGP 
100±25 
50±15 
Muscle IR, glucose rate of disappearance, mg•kgFFM•min 
10±3 
17±5 
Table 5:  OGTT insulin and OMM by muscle IR 
Muscle IR 
(mg•kgFFM•min) 
2 hr insulin 
(mIU/L) 
2 hr OMM  
(Si) 
<12 
294±52 
1.21±0.21 
12-18 
185 ± 48 
2.6±0.38 
>18 
118 ± 19 
5.6±0.51 

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   We next assessed the 
integrated glucose and fat 
response to an OSTT with 
75 grams of glucose and 
25 mg of fructose. With 
Co-I 
Dr. 
Parks 
and 
consultant 
Metabolomic 
Solutions, 
we 
are 
measuring the rate of 
hepatic 
DNL 
with 
an 
intravenous infusion of a 
1,2-C13 acetate tracer in 
obese girls with PCOS 
fasting and following the 
OSTT, 
to 
better 
understand what happens 
in the post-prandial state. 
In girls with PCOS, fasting 
and 
post 
OSTT 
DNL 
increased with the degree 
of IHTG (Figure 5A). In 
the entire cohort of 62 girls, serum TG decreased as a reflection of glucose stimulated DNL and TG secretion, in 
obese and PCOS without HS, whereas girls with PCOS and HS had a minimal change or increase in TG depending 
on the severity of HS (Figure 5B). Increases in serum TG following an OSTT have been shown to directly reflect 
changes in DNL142. The contribution of FFA to HS is again evident with this study design, as the OSTT nadir of 
FFA suppression (Figure 5C) was higher in both PCOS and increasing IHTG%, and OSTT FFA nadir correlated 
with IHTG% (r=0.045, p<0.001). Finally, peak insulin during the OSTT (Figure 5D), as a marker of overall IR, 
increased with PCOS and degree of HS. The FFA nadir appears to be related to IR, as the higher the FFA nadir, the 
higher the insulin (r=0.504, p<0.0001). This combined data demonstrates that obese girls with PCOS have high 
rates of HS which relate to adipose IR (manifested as increased OSTT or clamp FFA), higher rates of DNL and 
whole body IR, when assessed with either a hyperinsulinemic euglycemic clamp or the simpler OSTT. 
 
 In obese girls with PCOS, regardless of HS status, GLP-1 RA therapy lowers the OSTT glucose response, FFA 
nadir, TG release and glucagon, and increases first-phase insulin secretion. We gave a subgroup of 10 obese PCOS 
participants 2 doses of fast acting exenatide, 1 prior to dinner the night of admission and 1 thirty minutes prior to the 
OSTT the next day. As shown in Figure 6A and B, regardless of HS status, GLP-1 RA therapy decreased serum 
glucose concentrations significantly, below those seen in controls without PCOS. As expected, insulin secretion 
increased with GLP-1 RA (Figure 6C and D). Of note, the large second-phase insulin peak in  
Fig 5. HS in PCOS relates to post-OSTT alterations in direct and indirect 
measures of DNL, FFA release and insulin peak  
 
 
 

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PCOS girls with HS (Figure  6D, purple dashed line) was completely mitigated with GLP-1 RA (green dotted 
line). GLP-1 RA also decreased fasting and post-prandial glucagon responses.                                           
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
In terms of markers of DNL, post-OSTT TG suppressed 24±5% in PCOS girls without HS and 8±3% in girls with 
PCOS and HS which is greatly improved compared to without GLP-1 RA (8±5% and 3±3%, respectively Figure 
5B). The OSTT FFA nadir was also lower with GLP-1 RA in PCOS girls without HS 45±6 nmol/L, similar to 
controls in Figure 5C, and was also lower in girls with PCOS and HS at 74±4 nmol/L. Thus, a short exposure of 
GLP-1 RA treatment induces multiple metabolic changes in obese PCOS girls regardless of HS status and 
prolonged therapy has the potential to cause even greater changes. 
 
Excess hepatic TCA cycle activity may provide substrate for DNL and cause inflammation that promotes NAFLD 
progression. In adults with NAFLD, tracer-assessed rates of mitochondrial oxidation and anaplerotic/cataplerotic 
flux were 2-fold increased and related tightly to IHTG content25. Excess mitochondrial activity can produce reactive 
oxygen species and NAFLD severity per biopsy related to oxidative stress26. In animals, excess FFA provided the 
substrate for anaplerosis/cataplerosis and increased oxidative stress, and these processes could be prevented with 
fatty-acid oxidation knockout animals or metformin26. Our proposed project would confirm that similar metabolic 
processes are occurring in obese youth with PCOS. 
Intrahepatic TCA cycle metabolism can be measured non-invasively with a U-C13 oral glycerol tracer in obese 
adolescent girls. Our team has demonstrated that TCA cycle anaplerosis/cataplerosis is up-regulated in NAFLD, yet 
the method is patient intensive and requires multiple isotopes25. Glycerol is nearly universally taken up by the liver, 
and when labeled with a carbon isotope can be used to trace hepatic metabolism. Co-I’s Drs. Malloy and Jin first 
described this methodology in adult men143,144. The premise behind this methodology and sample spectra from our 
obese girls with PCOS are shown in Figure 7143,144. We successfully implemented this protocol in obese girls with  
 
 
 
 
 
 
 
 
 
 
Fig 6. GLP-1 RA improves OSTT glucose via increased first phase insulin 
secretion  in PCOS regardless of HS status 
 
 
Fig 7. Carbon isotopomers to assess percent hepatic anaplerosis in obese PCOS girls 
 
Glycerol-derived carbons can 
go 
to 
gluconeogenesis 
directly, 
via 
pentose 
phosphate pathway (PPP, 
blue) or the indirect pathway 
(red) where the carbons go 
via the TCA cycle.    
 A similar process measures glycerol contribution to TG synthesis.    

COMIRB Protocol # 19-0636 
 
 
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PCOS and confirmed that the isotopomer outcomes of interest for Aim 2B, including the percent of carbons 
originating from hepatic anapleurotic cycling in the TCA cycle and then contributing to triglyceride synthesis and 
gluconeogenesis, can be measured with this methodology in our patient population. Our preliminary data indicates 
that an increased indirect/direct ratio in glucose, indicative of higher proportions of cataplerosis, trends to be related 
to worse HOMA (R=-0.75, p=0.05 with 7 participants) and higher ALT. This data demonstrates that this 
methodology has been optimized for our patient population and when combined with the clinical outcomes, rates of 
DNL and gluconeogenesis can yield powerful insights into underlying mechanisms of pathology. 
 
Hepatic 31P-MRS demonstrates alterations in intrahepatic phosphate concentrations associated with IR and 
isotopomer-assessed TCA cycle activity. We implemented a 31P-MRS protocol with consultant Dr. Befroy of Peak 
Analysts and Co-I Dr. Brown to measure fasting hepatic phosphate concentrations with a custom built 18 cm H/P 
dual tuned coil utilized on a large bore Siemens 3.0 Tesla dedicated research magnet, which is large enough to 
collect data in obese individuals. Data shown (Figure 8) are from 14 obese girls with and without PCOS who had an 
OSTT, of whom 6 also had the U-C13 oral glycerol drink with isotopomer analysis of serum. 
 
 
 
 
 
 
Deuterated water is well tolerated in youth and overnight dosing is adequate to measure DNL. The assessment of 
DNL with deuterated water method has been used successfully in obese youth by our Co-I Dr. Parks, and we thus 
are confident in our ability to perform this analysis58 (NCT02960659). Previous protocols utilized a prolonged water 
loading protocol to increase the precursor enrichment60. Dr. Parks adapted the protocol to a more feasible multi-
bolus overnight protocol with adequate precursor enrichment in obese individuals28,58.  
 
Fig 8: Hepatic phosphate compounds are measuresurable with 31P MRS in obese 
girls weighing up to 325 pounds and releate to measures of whole body IR and 
hepatic TCA cycle activity 
 
 
 
A typical pre-spectra image of the liver with studied tissue outlined in red is in 9A. A sample 
spectra is in 9B. A low phosphocreatine (PCr) peak confirms capture of liver tissue without 
muscle 
in 
the 
area 
sampled. 
Outcome 
measures, 
include: 
ATP/total 
phosphate 
(ATP/TP)(marker of overall energy state) and PDE/ATP (normalized marker of oxidative 
stress).  9C  Those with a higher PDE/total phosphate ratio, ie greater oxidative stress have 
more severe IR with a lower Matsuda score. The ATP/TP ratio and TCA cataplerotic cycling, 
represented as glucose % TCA, correlate strongly (9D), validating ATP/TP as a non-invasive 
measure of hepatic TCA activity.   
A
B
D
C
r=-0.75 
P=0.003 
N=14 
r=0.94 
P<0.001 
N=6 

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3.  STUDY RATIONALE 
    Here we propose a 4-month randomized study of GLP-1 RA semaglutide oral tablets vs. diet therapy in 50 drug-
naive obese girls with PCOS. The rationale for this research is our observation of excess NAFLD, dysglycemia, 
upregulated DNL, hepatic, adipose and peripheral IR and decreased GLP-1 secretion in PCOS girls combined with 
pilot data demonstrating metabolic improvements with short-duration GLP-1 RA administration. The long-term 
consequences of NAFLD in girls with PCOS are of great clinical importance. This study will define the metabolic 
impact of a currently available medication on this common high-risk disease in youth thereby changing health 
outcomes. As such, this is a paradigm-changing approach to the management of this life-long disease. In addition, 
the innovative methodology employed will allow for non-invasive determination of specific mechanisms involved in 
metabolic disease in PCOS.  
4. STUDY DESIGN 
Overall Study Design  
All aims will be accomplished from a 
cohort of 50 obese girls with PCOS. Girls 
will complete a 16-week randomized, open-
label 
intervention 
study 
(Figure 
9). 
Treatments will consist of 1) a GLP-1 RA, 
semaglutide (Rybelsus®), with standard 
dietary counseling (n=33 girls) or 2) 
intensive 
nutritional 
counseling 
(n=17 
girls). 16 semaglutide will be enrolled first, 
to determine target weight loss of the 
intervention group, then the remainder will 
be randomized 1:1. Metabolic studies will 
be performed prior to and after 4 months of 
intervention. A mid-point visit at 2 months 
to assess compliance, side effects, HbA1c, 
BMI and PCOS severity will occur.  
Participants: PCOS as defined as in our 
previous work by the most stringent NIH 
criteria, with adaptation for adolescents 
(oligomenorrhea 
>12 
months 
post-
menarche or primary amenorrhea after age 
15 
years 
and 
clinical/ 
biochemical 
hypertestosteronemia)8,34,145. NIH criteria 
were chosen as they identify women with a 
higher metabolic disease risk34,146 than the Rotterdam PCOS criteria77.  Rotterdam criteria also include ovarian 
ultrasound, which cannot be applied in teens as ovarian norms do not exist for youth145,147,148. Other 
inclusion/exclusion criteria are in Figure 9 and include HbA1c to exclude diabetes, per guidelines, since non-
controlled OGTT’s have 20% discordance in youth149. Girls will be instructed not to change physical activity, as 
increased activity can independently improve HS80,82.  
Recruitment plan: Participants will be recruited from our adolescent obesity and PCOS specialty clinics. The PI has 
enrolled >120 obese girls with PCOS for research studies in the last 6 years and finished every study ahead of 
schedule. As the director of our growing multi-disciplinary PCOS clinic with a 7-state referral area, we do not 
anticipate a change in this pattern. If needed, we can add community gynecology and endocrinology clinics.  
Incentives: Participants will be reimbursed for their time for each visit. To aid in retention, additional monetary 
incentives are given for completing the 2 month and final study visit, as in our previous studies111,114. 
Randomization: The first 16 participants who are enrolled in the study will automatically be randomized to GLP-1 
RA (~50% of the group). These 16 participants must complete the 4-month study treatment and complete their final 
study visits. From these participants we will get their typical weight loss by measuring changes in weight from 
baseline to mid- and final-treatment. From this we will attempt to match the same amount of weight loss in the 
participants who are randomized to the lifestyle group. After the first 16 participants complete the study, the 
Figure 9: Overall study design 
Participants will have 4 visits: A screening to ensure eligibility, 2 
identical study visits before and after therapy and a mid-point check-
in visit (black arrows). 16 GLP-1 will be completed to determine 
typical weight loss, then remaining girls will be randomized 1:1. 
During treatment, all participants will communicate with study staff 
weekly and have biweekly weights (blue dashed arrows). Extra 
visits will occur in diet participants not meeting weight loss goals. 
Inclusion criteria 
Exclusion criteria 
Female 
PCOS 
Age 12-21 years 
BMI >90 %ile for age/sex 
Diabetes (HbA1c ≥6.5%) 
Liver disease other than NAFLD 
Liver transaminases >100 IU/L  
Medications affecting IR  
Anemia (hemoglobin <11 mg/dL)  
Implanted MRI-incompatible metal 

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remaining participants will be randomized 1:1 to intensive lifestyle or GLP-1 RA by the study team statistician, with 
the randomization schedule created prior to any enrollment. 
 
SA1 Detailed Methodology 
MRI for IHTG. No MRI studies utilize contrast. IHTG percent will be assessed with MRI using multiple hepatic 
slices through the entirety of the liver and a modification of the Dixon technique as in our previous studies9,136,141 on 
a Siemens 3.0 Tesla research-dedicated magnet (Siemens Skyra large bore magnet with full research and multi-
nuclear capabilities). The Dixon method has excellent correlation with IHTG from liver biopsy and measures fat 
from the entirety of the liver which is important as IHTG is not homogenously distributed. Whereas the PI and Co-I 
Dr. Brown have extensive experience with proton MR spectroscopy150-156, MRI is preferable to proton MRS in obese 
participants because the signal depth of penetration can be reduced by abdominal obesity and only small area of the 
liver tissue is sampled. Visceral and subcutaneous fat will also be calculated from MRI as in our previous 
studies9,136,141. Our research MRI has in-MRI movie-viewing and thus is very tolerable to youth. 
 
Dietary intervention: Stop Light Diet, data driven personalized daily calorie goals, biweekly measured weight,  
frequent dietician contact, and meal replacement therapy if needed.  The stop light diet was first described for 
improving youth dietary choices over 20 years ago157. It was adapted for use in obese adolescents for the 
TODAY113,114 
study, 
and 
all 
of 
the 
materials 
are 
available 
free-of-charge 
online 
(https://portal.bsc.gwu.edu/web/today/materials). We implemented a similar strategy with reduced intensity of 
teaching in the RISE study111. Similar to the education that our team delivers with the PI’s current cross-sectional 
studies, youth and their families will receive intensive dietary teaching by an experienced study dietician after 
randomization has been completed158. Additionally, participants will be oriented with the website Calorie King 
(www.calorieking.com) to assist them in assigning foods to the red (rarely), yellow (sometimes) or green (anytime) 
category. Individual targeted weight loss will be translated into daily calorie goals as determined from the measured 
basal metabolic rate plus the participants’ activity factor from their 7-day accelerometer data and target weight loss. 
The dietician will receive bi-weekly weights and speak to the participants and their families in the diet arm weekly 
to encourage adherence, answer questions regarding food categories and offer general support. Participants will also 
be given the option of completing a 3-day photographic food diary prior to their scheduled in-person visits to help 
direct their diet therapy. Goals will be adjusted every 1-2 weeks, based on bi-weekly weight data. Intensification 
with in-person visits will be added if needed. At clinical discretion of the study RD, if weight loss trajectory is not 
meeting intervention goals, RD will add meal replacement shakes to intervention, ordered in participant’s flavor 
preference..  All participants will receive diet counseling at the final study visit and a follow-up phone call as they 
transition back to clinical care. 
 
Rybelsus® (semaglutide). We have selected semaglutide for many reasons including:  
ease of use, oral formulation, excellent tolerability (Table 
6), less weight loss with equal glucose lowering effects 
and quantity of preliminary data available to-date in obese 
youth with the same class medication exenatide159. 
Exenatide was one of the first GLP-1 RA approved by the 
FDA, thus most studies in youth have used exenatide160-
166. Oral semaglutide is the first subsequent GLP-1 RA to 
have a similar side-effect profile to that of exenatide. This 
combined with the oral formulation, which eliminates the 
possibility of injection site reactions, means that Rybelsus has the most favorable risk/benefit ratio of any available 
GLP-1 RA.  Our team has extensive experience with encouraging adherence to daily oral study medication through 
coaching, parental involvement and additional incentives with typical adherence of 85-95%. Study staff will text the 
participant weekly reminders and encourage setting of daily reminders on participants phones, if applicable..  
   GLP-1 RA are overall well tolerated in youth and the primary side effects are nausea and hypoglycemia160-166. Co-I 
Dr. Zeitler is an investigator for a multi-center exenatide trial in youth with T2D (NCT01554618, NCT00658021) 
which is well underway. A trial of exenatide in obese youth with hypothalamic obesity secondary to brain tumor 
resection is also currently enrolling (NCT01484873). The medication has been well tolerated in both of these studies 
with no significant hypoglycemia or nausea leading to discontinuation at our local site or in the hypothalamic trial 
(personal communication with PI Dr. Ashley Shoemaker). There is a concern for increased risk of medullary thyroid 
cancer following long-term use of GLP-1 RA but recent safety results from a large multi-year study in adults 
(LEADER) showed that this risk is minimal if those with thyroid cancer risk factors are excluded 168. Finally, there 
Table 6:  GLP-1 RA effects per manufacturer 
Drug & Weekly Dose 
Nausea 
Weight loss 
Exenatide 2 mg 
8% 
1.4 kg (28 wk) 
Dulaglutide 1.5 mg 
21% 
2.3 kg (26 wk) 
Semaglutide 1 mg 
20% 
4.7 kg (30 wk) 
Semaglutide (oral) 7 mg 
11% 
2.2 kg (26 wk) 
Liraglutide 1.8 mg *daily 
20% 
3.3 kg (26 wk) 

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was concern for pancreatic disease with GLP-1 RA, but again the recent pancreatic safety data from the LEADER 
study was also very reassuring168.  
 
Physical Activity: Activity affects IR and HS. Habitual activity will be assessed objectively with a gold-standard 
waterproof thigh-worn accelerometer (ActivPal, PAL Technologies), sensitive to sedentary and light activities. This 
approach is ideal for the proposed sedentary population169 to measure sedentary, upright, and ambulatory activity 
counts, intensity, energy expenditure and METs. The participant will be mailed an accelerometer to be worn for the 
7 days prior to each study visit. We have utilized accelerometers extensively in youth and we are familiar with their 
use and have the necessary software and interpretation skills. Pre-intervention, this data will be utilized to customize 
calorie goals and post-study can be utilized to control for changes in activity, if needed. 
 
Body Composition: DXA (Hologic, Waltham, MA) will be performed for fat mass and fat-free mass 170. 
 
 
SA2 Detailed  
Methodology 
Overall Metabolic Study 
Day All of these types of 
methodologies 
are 
currently in use by the PI 
and CO-I’s in similarly 
obese youth and the study 
day plan is well tolerated 
in our patient population. 
Variations in diet, activity, 
menstrual phase and sleep 
affect metabolism and will 
be 
controlled 
and/or 
assessed prior to each 
study visit, as in our current protocols. Study visits will be performed in the follicular phase of the menstrual cycle 
where possible (most girls with PCOS are in a follicular-like non-ovulatory state), preceded by 3 days of no 
strenuous physical activity and a typical diet for the participant. A standardized afternoon snack and dinner will be 
provided the night prior to the metabolic study. Data on dietary intake (7-day food diary), habitual activity (3 day 
questionnaire and accelerometer for 7 days prior to study) and sleep quality (surveys and sleep watch for sleep 
apnea) are collected. Study day 1 will include an abdominal MRI, hepatic MR elastography (MRE) as measure of 
fibrosis, hepatic 31P-MRS, brain rs-fMRI, and DXA. Participants will be admitted for an inpatient overnight 
monitored fast followed by an OSTT with isotope tracers on day 2, assessment of other contributors to HS and IR 
and PCOS severity. For the OSTT (approximate times shown in Figure 10) participants will receive deuterated 
water overnight to assess DNL, a fasting assessment of hepatic glycerol TCA flux with a U-C13 glycerol drink 
(Figure 10143), followed by an OSTT with 75g of glucose and 25g of fructose. Indirect calorimetry will be 
performed throughout the OSTT to measure the rate of fat oxidation. The study day is complete at 2:30 PM.  
   Variables known to be closely related to IHTG and IR will also be measured: lipid panel, HbA1c, inflammatory 
markers (C-reactive protein, AST, ALT, FGF-21; metabolic hormones (glucagon, GLP-1 and adiponectin), sex-
steroids (DHEA-S, free and total testosterone, sex hormone binding globulin, progesterone, estradiol); and body 
composition (BMI, waist:hip ratio, fat and fat-free mass by DXA) and sleep apnea. Targeted metabolomic analysis 
to assess for serum markers of DNL and mitochondrial oxidation including fatty acids, acylcarnitines171 and bile 
acids will be measured by the University of Colorado metabolomics core in fasting and 120-minute post-OSTT 
drink samples to assess changes with the OSTT.  
 
 
Measures of hepatic DNL. Rates of hepatic DNL will be calculated based on tracer incorporation of deuterium from 
oral D20 into VLDL-secreted palmitate with samples analyzed via GCMS by Co-I Dr. Parks, as previously described 
in youth58,172,173. Water will be given in the amount of 3 ml/kg fat free mass (per DXA), to reach a goal enrichment 
of 0.3%. We will utilize deuterated water rather than U-C13 acetate as in our current study to reduce participant 
burden, need for multiple IV’s and blood volume. Further, the acetate tracer has a carbon label and cannot be 
combined in a single protocol with the U-C13 glycerol tracer needed for the isotopomer analysis. An alternative 
measure for DNL, the % change in TG secretion during the OSTT (TG0-TG180/TG0 *100) will also be calculated. 
Fig 10: Study days procedure timeline 

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Serum concentrations from fasting and end OSTT 16:0, 16:1 N7, 18:0, 18:1 N7 and 18:2 will be determined from 
metabolomics analysis as markers of DNL as in our preliminary data. 
 
 
Measures of adipose and peripheral IR. Prior to and during the OSTT we will perform frequent sampling of glucose, 
insulin and FFA. Whole body insulin sensitivity (Si) will be calculated with a 4 hour OMM, utilizing SAM II 
software137. Adipose IR will be calculated as the FFA nadir as in our preliminary data. 
 
 
Measures of proportion anaplerosis. Glucose and VLDL-TG are isolated from serum, as previously described143. 
Samples are then analyzed by Co-I’s Drs. Malloy and Jin utilizing high resolution 13C NMR with a Varian Inova 
14.1 T spectrometer (Agilent, Santa Clara, CA) equipped with a 3-mm broadband probe with the observe coil tuned 
to 13C (150 MHz)143. All NMR spectra are analyzed using ACD/Labs NMR spectral analysis program (Advanced 
Chemistry Development, Inc., Toronto, Canada). Endpoints include the relative proportion of carbon from TCA 
cycling contributing to TG synthesis and gluconeogenesis as shown in Figure 7. ([1,2-13C2] or [2,3-13C2]) 
glycerol in TAGs reflects the “indirect” contribution from [U-13C3] glycerol through the TCA cycle, representative 
of cataplerosis. Total gluconeogenesis from [U13C] glycerol is measured as the sum of all glucose isotopomers with 
13C in excess.  Because the incorporation of 13C tracer in position 4,5,6 is not affected by other pathways (pentose 
phosphate pathway), the labelling of [4,5-13C2]- or [5,6-13C2] glucose indicates metabolism of [U-13C3] glycerol 
through the TCA cycle prior to gluconeogenesis.  Therefore, indirect gluconeogenesis through the TCA cycle from 
glycerol is measured by [5,6 13C2]/ [4,5,6 13C3] glucose. All of the diet intervention group will get the glycerol tracer, 
and approximately half of the semaglutide group will get the glycerol tracer.   
 
 
Hepatic 31P-MRS. Imaging and MRS will be performed on a Siemens 3.0 Tesla MRI magnet. A custom 1H/31P 
abdominal coil will be used for imaging and MRS (Clinical MR Solutions, Brookfield, WI) as in our previous 31P 
work174. This does not involve contrast agents. 256 individual 31P MRS spectra are acquired with a 2.3 second echo 
time and a 2 second repetition time from the entire liver (Figure 8A). After minimal phasing corrections, the 6 
continuous spectra in the superior/inferior direction with the largest phosphocreatine peak, representing intercostal 
muscle, are identified and then 6 hepatic spectra, 2-3 voxels medial of the muscle spectra with the largest ATP 
signal but no PCr signals are identified and summed (Figure 8b).  Peak positions and areas of interest 
[phosphocreatine (PCr), inorganic free phosphate (Pi), ATP (3 peaks), PDE and PME] are determined by time 
domain fitting with jMRUi , utilizing AMARES (A Method of Accurate, Robust and Efficient Spectral fitting), a 
nonlinear least-square-fitting algorithm using our previously built prior knowledge files. We have utilized a similar 
method for muscle and liver 31P analysis for the previous 9 years10,115,118,175,176. 
 
 
Indirect Calorimetry: Resting VO2 (ml/kg/min) and VCO2 (ml/kg/min) measurements will be collected prior to the 
OSTT and twice during the OSTT to determine changes in fat oxidation and metabolic flexibility.  
 
SA3 Detailed Methodology 
rs-fMRI Image Preprocessing. rs-fMRI preprocessing will be conducted in MATLAB using Statistical Parametric 
Mapping (SPM 12, Welcome Trust Centre for Neuroimaging, London, UK). Translational movement (i.e., x, y, z) 
and rotational movement (i.e., pitch, roll, yaw) will be quantified in SPM. All volumes will undergo framewise 
displacement quality assurance checks via implementation of the MRIQC Toolbox, such that volumes exceeding 
greater than 1mm or 0.3 degrees rotation will be excluded. All functional images will be slice-time corrected to the 
slice acquired halfway through the volume collection. Volumes will be realigned to the first volume, co-registered to 
the participant’s anatomical T1, and spatially normalized to MNI152 template space. Normalized images will be 
smoothed using an 8-mm full-width half-maximum Gaussian kernel. Physiologic noise specifically, pulse and 
respiratory rate will be quantified and aligned with the BOLD time series data via the PhysIO Toolbox and removed 
through covariate adjustment in all models to maximize the temporal signal-to-noise ratio in the defined ROIs. 
Additionally, movement parameters will be used as covariates in all models. 

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5. OUTCOME MEASURES 
Primary Outcome Measure 
For the primary outcome, we will compare the change in % IHTG from baseline to end of treatment, with and 
without GLP-1 RA treatment.  
The primary outcomes for Aim 2 are (1) change in hepatic de novo lipogenesis (DNL) and (2) change in FFA nadir 
in the standard diet + GLP-1 RA group compared to the intensive diet group.  
Secondary Outcome Measure 
Variables known to be closely related to IHTG and IR will also be measured: lipid panel, HbA1c, inflammatory 
markers (C-reactive protein, AST, ALT, GGT, FGF-23); metabolic hormones (glucagon, GLP-1 and adiponectin), 
sex-steroids (DHEA-S, free and total testosterone, sex hormone binding globulin, progesterone, estradiol); and body 
composition (BMI, waist:hip ratio, fat and fat-free mass by DXA) and sleep apnea. Targeted metabolomic analysis 
to assess for serum markers of DNL and mitochondrial oxidation including fatty acids, acylcarnitines, and bile acids. 
Hepatic mitochondrial function per MR spectroscopy and isotopomer analysis. Insulin sensitivity as assessed with 
the oral minimal model. Whole blood for DNA samples for future genetic analysis associated with hepatic steatosis. 
Stool samples will be collected to look at changes in microbiome pre-post treatment. Functional neuroimaging 
measures, specifically, the blood oxygen level-dependent (BOLD) signal in the hypothalamus, insula, 
anterior cingulate cortex, nucleus accumbens, amygdala, and superior and inferior frontal gyri, and 
connectivity between these brain regions. 
6. 
PARTICIPANT SELECTION 
Study Population 
Description of Population to be Enrolled:   
Study staff aims to complete 50 obese girls with PCOS (untreated). This is the number of participants needed to be 
completed statistically, thus more participants may be enrolled to allow for screen failures and dropouts. Total 
enrollment will be up to 60 participants. 
 
Ethnic Categories 
Gender 
Females 
Males 
Total 
Hispanic or Latino 
20 
0 
20 
Not Hispanic or Latino 
30 
0 
30 
Ethnic Categories: Total of All Participants 
50 
0 
50 
  
Racial Categories 
 
 
 
American Indian/Alaska Native 
2 
0 
2 
Asian 
5 
0 
5 
Native Hawaiian or Other Pacific Islander 
5 
0 
5 
Black or African American 
15 
0 
15 
White 
23 
0 
23 
Racial Categories: Total of All Participants * 
50 
0 
50 
Inclusion Criteria 
1) Female 
2) Ages 12-21 years 
3) Sedentary- less than 2 hours of moderate (jogging, swimming etc) exercise a week. 
4) BMI equal or greater than the 90th percentile for age and gender 
5) PCOS per the most stringent NIH criteria adapted for adolescents (irregular menses >12 months 
post-menarche and clinical or biochemical hypertestosteronemia)8,34 
6) Participants cannot be on hormonal contraception, so participants should remain abstinent or use 
reliable non-hormonal contraception (e.g. copper IUD) for the entire study period. For 

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participants who receive semaglutide, they should avoid pregnancy for at least   2 months 
after stopping medication to avoid fetal exposure to the medication. 
Exclusion Criteria 
1) Diagnosed with or have a family history of medullary thyroid carcinoma (MTC) or Multiple 
Endocrine Neoplasia syndrome type 2 (MEN 2). Family history of medullary thyroid cancer 
or thyroid nodule palpated by endocrinologist at screening. 
2) Use of medications known to affect insulin sensitivity: metformin (cannot have been used in the 3 
months prior to screening), oral glucocorticoids within 10 days, atypical antipsychotics, 
immunosuppressant agents, HIV medications, hormonal contraception (cannot have been 
used in the 6 months prior to screening). Dermal patch or vaginal ring contraception 
methods. Weight loss medications or stimulants. Use of other products containing other 
GLP-1 agonists. 
3) Currently pregnant or breastfeeding women. Development of pregnancy during the study period 
will necessitate withdrawal from the study. 
4) Severe illness requiring hospitalization within 60 days. 
5) Diabetes, defined as Hemoglobin A1C > 6.4% 
6) BMI percentile less than the 90th percentile for age and sex. Weight >325 lbs. or <84 lbs. 
7) Anemia, defined as Hemoglobin < 11 mg/dL 
8) Diagnosed major psychiatric or developmental disorder limiting informed consent. 
9) Implanted metal devices that are not compatible with MRI 
10) Use of blood pressure medications. 
11) Known liver disease other than NAFLD or AST or ALT >100 IU/L. 
12) Personal history of pancreatitis 
13) Known renal disease of any severity or an eGFR at screening of <45ml/min/1.73m2 
14) History of severe GI disease (e.g. gastroparesis) 
15) History of gallstones 
16) Untreated thyroid disease 
17) History of hypersensitivity to semaglutide  
18) Other causes of hyperandrogenism (example: tumor, CAH) or amenorrhea (untreated thyroid disease, tumor, 
primary ovarian failure, prolactinoma). 
19) Active symptoms or undergoing treatment for anorexia nervosa or binging/purging disorder 
 
Participant Recruitment Plan 
Participants will be recruited from our adolescent obesity and PCOS specialty clinics. The PI has enrolled >120 
obese girls with PCOS for research studies in the last 6 years and finished every study ahead of schedule. As the 
director of our growing multi-disciplinary PCOS clinic with a 7-state referral area, we do not anticipate a change in 
this pattern. If needed, we can add community gynecology and endocrinology clinics.  
 
Informed Consent Plan 
Appropriately qualified and informed personnel who have completed the COMIRB and HIPAA course requirements 
will fully explain the study protocol and consent form to the participant and guardian verbally in the language they 
understand.  The explanation will be conducted in a quiet environment with adequate time given for the participant 
and guardian to review the study procedure before the commencement of the study.  Asking the participant to 
explain the study in their own words will assess the participant’s understanding.  If non-English speaking 
participants are enrolled in the study, the investigators will adhere to Section 10C of the COMIRB Instructions for 
Clinical Investigators regarding the consent of these participants. The consent form will also be translated into 
Spanish. The qualified personnel mentioned above will then obtain written consent from the guardian and assent 
from the participant, co-signed on the consent form, or in participants who are 18 years or older, direct consent. The 
participant and guardian will be provided a copy of the consent form for better understanding and record purposes.  
Special Consent/Assent Plan 

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Consent will be obtained from all participants in the study.  Following explanation, all participants under 18 years 
old will co-sign the consent form in addition to the parents signing the consent form.  All participants age 18 or 
older will sign the standard consent form. 
Participant Compensation, Incentives and Rewards  
Participants will be compensated with Target gift cards for completion of each study visit. The initial Visit 1 
consisting of informed consent, and blood draw will result in a $50 gift card. Each of the imaging visits (Visits 2a 
and 4a) will be $50 each. Each of the overnight visits (Visits 2b and 4b) will be $150 each. Participants will be paid 
$50 for completing the mid-treatment visit (Visit 3). In an effort to maintain participant retention and completion of 
the study, the participants will be rewarded an additional $50 if they complete all study visits. Compensation for all 
completed visits can total up to $550, and participants will only be paid for the visits they complete. 
Alternative Treatment 
The alternative is for participants to not participate in the study. 
Consideration of Specific Participant Categories 
1. 
Inclusion of Women 
All participants will be women, as PCOS only occurs in females. 
2. 
Inclusion of Minorities 
Every effort will be made to include a diverse participant distribution. PCOS affects Caucasians, Hispanics and 
African Americans equally. 
3. 
Inclusion of Children 
All participants will be between ages 12 and 21. Insulin sensitivity needs to be studied in the adolescent age group as 
available is scarce in this age group and it is critical to understand the pathophysiology of PCOS in its developing 
stages. 
 
7. CONCURRENT MEDICATIONS 
All participants should be maintained on the same medications throughout the entire study period, as medically 
feasible, with no introduction of new chronic therapies. Study medication should be taken at least 2 hours apart from 
any regular medications. 
Allowed Medications and Treatments 
Prior to starting any new medications, participants should contact the study team. 
Individuals treated with semaglutide may have an improvement of PCOS, including improved rates of ovulation and 
therefore the possibility for pregnancy if sexually active. Non-hormonal methods of contraception including barrier 
methods (condoms), abstinence or seeking placement of a non-hormonal intrauterine device will be discussed at 
randomization, mid-study check-in and at the end of the study. Condoms will be offered to all participants at each of 
these time points. 
Prohibited Medications and Treatments 
The following medications are prohibited during the study and administration will be considered a protocol 
violation. 
Medications known to affect insulin sensitivity: metformin (cannot have been used in the 3 months prior to 
screening), oral glucocorticoids within 10 days, atypical antipsychotics, immunosuppressant agents, HIV 
medications, hormonal contraception (cannot have been used in the 6 months prior to screening). Weight loss 
medications or stimulants. Use of other products containing other GLP-1 agonists. Dermal patch or vaginal ring 
contraception methods. Blood pressure medications 

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8. STUDY TREATMENTS 
Method of Assigning Participants to Treatment Groups 
Treatment 1: Rybelsus® (semaglutide, GLP-1 RA) (n=33 girls) 
Treatment 2: Lifestyle Intervention: Intensive nutritional counseling (n=17 girls) 
The first 16 participants who are enrolled in the study will automatically be randomized to GLP-1 RA (~50% of the 
group). These 16 participants must complete the 4-month study treatment and complete their final study visits. From 
these participants we will get their typical weight loss by measuring changes in weight from baseline to mid- and 
final-treatment. From this we will attempt to match the same amount of weight loss in the participants who are 
randomized to the lifestyle group. After the first 16 participants complete the study, the remaining participants will 
be randomized 1:1 to intensive lifestyle or GLP-1 RA by the study team statistician, with the randomization 
schedule created prior to any enrollment. 
Investigational Drug Information 
Drug Name: Semaglutide  
Drug Trade Name: Rybelsus®  
Other Names: GLP-1 RA 
Dosage Form: oral tablets  
Dosage Information: Once daily 3mg dose for 30 days, then once daily 7mg dose for remainder of study. If 
participants cannot tolerate 7 mg, the dose will be backed down to 3 mg for 1-2 more weeks, then another attempt 
made to increase to 7 mg. If they still cannot tolerate 7 mg, the duration of therapy will be completed with 3 mg.  
Storage: Children’s Hospital Colorado Investigational Drug Services 
Drug Ordering and Drug Accountability: Children’s Hospital Colorado Investigational Drug Services 
Measures of Treatment Compliance 
Participants on GLP-1 RA will be asked to keep a patient diary noting any adverse events. They will be asked to 
bring their patient diary to each study visit along with all used and unused study drug containers. Study staff will 
also send reminders to help with compliance. 
Participants randomized to the intensive Lifestyle Intervention will be contacted by a Registered Dietician. 
Individual targeted weight loss will be translated into daily calorie goals as determined from the measured basal 
metabolic rate plus the participants’ activity factor from their 7-day accelerometer data and target weight loss. The 
dietician will receive bi-weekly weights and speak to the participants and their families in the diet arm weekly to 
encourage adherence, answer questions regarding food categories and offer general support. Goals will be adjusted 
every 1-2 weeks, based on bi-weekly weight data. Intensification with in-person visits will be added if needed. 
9. STUDY PROCEDURES AND GUIDELINES 
A Schedule of Events representing the required testing procedures to be performed for the duration of the study is 
diagrammed in Appendix 1 below. 
Prior to conducting any study-related activities, written informed consent and the Health Insurance Portability and 
Accountability Act (HIPAA) authorization must be signed and dated by the participant or participant’s legal 
representative. If appropriate, assent must also be obtained prior to conducting any study-related activities. 

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Clinical Assessments: 
Concomitant Medications 
All concomitant medication and concurrent therapies will be reviewed at all visits. Reminders to take study 
medication 2 hours separate from regular medications will be made at all visits.  
Demographics  
Demographic information (date of birth, gender, race) will be recorded at Screening Visit 1. 
Medical History  
Relevant medical history, including history of current disease, other pertinent history, and information regarding 
underlying diseases will be recorded at Screening Visit 1. 
Physical Examination 
A complete physical examination will be performed by either the investigator or a sub-investigator who is a 
physician at the Screening Visit 1. Qualified staff (MD, NP, and PA) may complete the abbreviated physical exam at 
all other visits.  New abnormal physical exam findings must be documented and will be followed by a physician or 
other qualified staff at the next scheduled visit.   
Adverse Events 
Information regarding occurrence of adverse events will be captured throughout the study. Duration (start and stop 
dates), severity/grade, outcome, treatment and relation to study drug will be recorded.  
Assessments for symptoms of hypoglycemia will be conducted in participants at baseline, 2 months and the end of 
the study. The family will be counseled on symptoms of hypoglycemia and counseled to contact the study staff 
immediately with any increase from baseline in symptoms of hypoglycemia. Symptoms include acute feelings of 
lightheadedness, shaking, nausea, sensations of hunger, heart racing and rarely vomiting. Treatment for this is eating 
a small carbohydrate snack. 
Clinical Laboratory Measurements  
Blood Chemistry Profile 
See Appendix 2 for blood sample analysis during the Screening Visit 1 and during the Oral Sugar Tolerance Test 
(Visits 2b and 4b). A finger stick blood draw will also be done at Mid-Treatment Visit 3 to test HbA1c. 
Pregnancy Test 
A urine pregnancy test will be obtained from all participants at Screening Visit 1, Visit 2a, Visit 3, and Visit 4a. 
Urinalysis  
Urine will be collected and stored for future analysis of hormone and metabolic related markers. 
 
EVALUATIONS BY VISIT (SEE APPENDIX 1 FOR REFERENCE) 
Visit 1 (Screening)-Outpatient Pediatric CTRC at Children’s Hospital Colorado 
1. 
Review the study with the participant (participant’s legal representative) and obtain written informed 
consent and HIPAA authorization and assent, if appropriate. 
2. 
Assign the participant a unique screening number. 
3. 
Record demographics data. 

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4. 
Record medical history 
5. 
Record concomitant medications. 
6. 
Perform a physical examination, if one wasn’t done clinically within 3-months of screening visit.  
7. 
Perform and record vital signs. 
8. 
Collect blood for clinical laboratory tests. See Appendix 2, for screening labs. 
9. 
Perform urine pregnancy test  
10. 
Schedule participant for Baseline Metabolic Study Visits 2a and 2b. 
11.  
Pregnancy prevention counseling and offer condoms 
Visit 2a (Imaging)-Leprino Building or Anschutz Health Sciences Building and UCD Brain Imaging 
Center 
1. 
Perform urine pregnancy test 
2. 
Perform DEXA scan 
3. 
Perform MRI of abdomen and liver, P-MRS of the liver, and rs-fMRI of the brain. 
4. 
Perform cardiovascular tests: EndoPAT and Dynapulse 
Visit 2b (Overnight)-Pediatric CTRC at Children’s Hospital Colorado or University Hospital 
1. 
7-days prior, mail out gut bacteria collection kit, accelerometer and sleep watch. 
2. 
Perform abbreviated physical examination. 
3. 
Perform and record vital signs. 
5. 
Perform WatchPAT sleep study. 
6. 
Give 3 doses of deuterated water oral tracer overnight. 
7. 
Nurse will collect urine and place IV first thing in the morning. 
8. 
Nurse will draw baseline labs and then participant will drink the Oral Glycerol Tracer (~7AM). This tracer 
will be given to all the diet intervention group and approximately half of the GLP-1 RA group. Samples for 
NMR analysis will be collected.  
9. 
Measure basal metabolic rate and two additional metabolic carts during the Oral Sugar Tolerance Test 
(OSTT). 
10. 
Participant will drink the glucose drink (~10AM) and complete the (OSTT) with frequent blood draws (See 
Appendix 2 for samples that will be drawn). 
11. 
Administer participant questionnaires (see list in Appendix 3).  
12. 
Participant will be randomized to GLP-1 RA or Intensive Lifestyle Intervention.  
13.  
Baseline hypoglycemia assessment 
14.  
Pregnancy prevention counseling and offer condoms 
15.  
 If randomized to Rybelsus, hypoglycemia and orthostatic hypotension symptom recognition teaching. 
Visit 3 (Mid-Treatment Visit)-Outpatient Pediatric CTRC at Children’s Hospital Colorado 
1. 
Perform physical examination. 
2. 
Perform urine pregnancy test. 
3. 
Perform and record vital signs. 
4. 
Perform venipuncture blood draw for HbA1c and complete metabolic panel. 
5. 
Administer 3DPAR and FFQ Questionnaires. 

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6.  
If randomized to Rybelsus, hypoglycemia and orthostatic hypotension symptom recognition teaching. 
Visit 4a (Imaging)-Leprino Building or Anschutz Health Sciences Building and UCD Brain Imaging 
Center 
1. 
Perform urine pregnancy test 
2. 
Perform DEXA scan 
3. 
Perform MRI of abdomen and liver, P-MRS of the liver, and rs-fMRI of the brain. 
4. 
Perform cardiovascular tests: EndoPAT and Dynapulse 
 
Visit 4b (Overnight)-Pediatric CTRC at Children’s Hospital Colorado or University Hospital 
1. 
7-days prior, mail out gut bacteria collection kit, accelerometer and sleep watch. 
2. 
Perform abbreviated physical examination. 
3. 
Perform and record vital signs. 
5. 
Perform WatchPAT sleep study. 
6. 
Give 3 doses of Deuterated Water oral tracer overnight. 
7. 
Nurse will collect urine and place IV first thing in the morning. 
8. 
Nurse will draw baseline labs and then participant will drink the Oral Glycerol Tracer (~7AM). This tracer 
will be given to all the diet intervention group and approximately half of the GLP-1 RA group. Samples for 
NMR analysis will be collected. 
9. 
Perform basal metabolic rate and two more metabolic carts during the Oral Sugar Tolerance Test (OSTT). 
10. 
Participant will drink the glucose drink (~10AM) and complete the (OSTT) with frequent blood draws (See 
Appendix 2 for samples that will be drawn). 
11. 
Administer participant questionnaires (see list in Appendix 3). 
12. 
Hypoglycemia Assessment 
13.  
Pregnancy prevention counseling and offer condoms 
14.  
Reminded to call study team in the next 4 weeks with new onset GI symptoms, worsening symptoms of 
hypoglycemia or postural hypotension. 
Risk / Benefit Assessment 
Please note: with the emergence of COVID-19 we must follow hospital policies for screening hospital 
patients and visitors. This may include testing for COVID-19. This may involve a nasal swab, and the 
risks of this are discomfort and potentially bleeding from nose. To limit participant time in our clinics, 
portions of study visits may be done virtually (phone or Telehealth). 
 
1. Blood Sampling 
Description: Blood will be drawn for Complete Blood Count (CBC), HbA1c, total and free testosterone, and sex 
hormone binding globulin. If participants have not had a full evaluation for oligomenorrhea, Prolactin, DHEAS, LH, 
FSH, TSH, total T4 or 17-OH progesterone may be drawn.  
Risk: Minimal. Risk of pain, bruising at site of blood draw, excessive amount of blood. 
Minimizing Risk: Certain studies at our institution draw over 7ml/kg in 6 weeks, or up to 7 ml/kg in a single draw, 
but include iron supplementation. Otherwise, the routine guidelines in our Pediatric CTRC are 2.5ml/kg for a single 
draw and no more than 5 ml/kg over a 4 week period.  Our baseline visit will include 11.5 ml of blood (HbA1c, Hb, 
Cr, AST, ALT) and 25.5 ml of blood for PCOS patients (additional draw for T4, TSH, prolactin, LH, FSH, 17OH 
progesterone and DHEAS). The OSTT visit includes up to 300 ml of blood which will occur within 4 weeks of the 

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initial visit. Thus, our OSTT visit is within the NIH Clinical Center guidelines of 9 ml/kg in 6-8 weeks and within 
Children’s Hospital Colorado’s institutional guidelines of 5 ml/kg.  In addition, by study design, participants are 
screened by our baseline CBC and excluded if anemic, further increasing the safety of the study regarding blood 
draws. We will use a minimum weight cutoff of 38 kg to remain below the most conservative pediatric CTRC blood 
drawing guidelines. This screening also helps to increase the safety of the blood draw. In addition, the blood planned 
to be frozen and held could also be omitted if needed to reduce blood volume for a particular participant. Finally, 
our CTRC has a system to track other studies participants might enroll in, and we ask during our consent process if 
the participant has been involved in any other studies in the past 6 weeks to avoid excessive blood drawing. 
Justification: Screening laboratory measurements are necessary to assure that patients meet inclusion/exclusion 
criteria before any further study is completed. A CBC is necessary as a screening lab, to rule out anemia. A 
hemoglobin A1c can be used to rule out diabetes. Hormone levels of free and total testosterone, and sex hormone 
binding globulin are needed to categorize patients as having PCOS, and prolactin, DHEAS, LH, FSH, TSH, total T4 
or 17-OH progesterone to rule out other causes of oligomenorrhea, if not done previously.  
 
2. IV Risks 
Description: One peripheral IV will be placed during the OSTT for drawing blood samples. 
Risk: There is temporary discomfort when the needle goes in and 10% of the time there is a small amount of 
bleeding under the skin that may produce a bruise. Rarely, there is a risk of a blood clot forming or infection.  We 
will use a low dose of a medication called heparin to try to prevent blood clotting. 
Justification/Minimization: These studies involve sampling blood at multiple time points. Thus, an IV is needed, so 
as to avoid multiple needle sticks. These studies are focused on measured rates of change which necessitates the 
sampling of the same test over time. Proper sterile technique will be used with blood draws and IV placement to 
decrease the infection risk. EMLA cream will be used if participant desires to minimize pain of IV. 
 
3. Oral Sugar Tolerance Test (OSTT): 
Description: An OSTT will be performed with multiple blood draws. The purpose of the OSTT is to provide a 
controlled oral stimulus to effect changes in lipolysis and hepatic glucose release.  
Risk: The participants rarely experience nausea within 15 min of consuming the drink, however, the amount of 
carbohydrate is very similar to a large soda, which is regularly consumed by this patient population.  
Justification/Minimization: A standard oral challenge is needed to study lipogenesis, lipolysis and gluconeognesis in 
the fed state. We have chosen to start with a standard glucose and fructose load, to simplify the mathematical 
modeling. Dynamic carbohydrate metabolism in youth is made more relevant by the recently reported TODAY 
study, showing a decline in beta cell function in youth with newly diagnosed type 2 diabetes that was much more 
rapid than what has been reported in adults, and not prevented by metformin in the majority of the youth177. Our 
team of investigators, CTRC research nursing staff and physicians are well experienced with the OSTT blood draw 
procedure. A floor nurse located on the will be available during our inpatient visits and patients will be distracted by 
TV or other similar means during the OSTT, to minimize queasiness. 
 
4. Stable Isotope Studies Glycerol:  
Description: Oral stable isotope tracer of glycerol will be utilized to determine rates of intrahepatic substrate flux. 
These are substances normally present or produced in the body, and thus pose no more risk than typical glucose 
infusions. Measurements of these metabolic processes are only able to be made with the utilization of stable isotope 
tracers. 
Risk: We are utilizing an isotope which already exists in all humans, but are simply increasing the percentage. We 
are only giving this medication orally. These are NOT radioactive substances. 
Justification/Minimization: Pyrogen-free ²C13 glycerol will be obtained from the manufacturer and delivered to 
CHCO IDS. The IDS pharmacist will deliver the tracer to the pediatric CTRC (inpatient unit or 3rd floor outpatient) 
once ordered by the physician. 
 
5. Stable Isotope Studies Deuterated water:  
Description: Oral stable isotope tracer of deuterated water will be utilized to determine rates of de novo lipogenesis 
and gluconeogenesis. These are substances normally present or produced in the body, and thus pose no more risk 
than typical water unless consumed in large amounts, to increase total body enrichment in excess of 20%178. 
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Risk: We are utilizing an isotope which already exists in all humans, but are simply increasing the percentage. We 
are only giving this medication orally. These are NOT radioactive substances. The planned dose is 3 ml/kg as has 
been used previously in obese youth179, with a goal enrichment of 0.3%, well below the 20% known to cause side 
effects. At the proposed dose, few participant experienced nausea, but it was extremely well tolerated (personal 
communication with PI’s Dr. Santoro and Parks). 
Justification/Minimization: Pyrogen-free deuterated water will be obtained from the manufacturer and delivered to 
CHCO IDS. The IDS pharmacist will deliver the tracer to the pediatric CTRC (inpatient unit or 3rd floor outpatient) 
once ordered by the physician. 
6. Standard Diet  
Description: A dinner and snack will be provided during the overnight visit. Daily calorie intake will be calculated 
using the St. Jeor Equation for females: ([10(weight in kg) + 6.25(height in m) – (5*18) – 161] X Activity Factor x 
1.25). 1.25 x weight maintenance was chosen as this as most similar to our participants food consumption based on 
pilot participant’s food frequency questionnaires and optimal for detection of hepatic glucose Ra 180. The snack will 
be calculated to be 10% of the daily calorie intake and the dinner will be calculated to be 26% of the daily calorie 
intake.   
Risk: None 
Justification/Minimization: Variations in diet, activity and circadian rhythms affect metabolism 155. 
 
7. Magnetic Resonance Imaging, Magnetic Resonance Spectroscopy, and resting state – functional Magnetic 
Resonance Imaging (MRI, MRS, rs-fMRI) 
Description: The MRI will usually be obtained the day of admission to CHC), at the UCD Brain Imaging Center on 
the Fitzsimmons campus. A trained research radiographer who is supervised by Dr. Mark Brown, of UCD radiology, 
will perform an abdominal MRI to obtain hepatic, visceral and subcutaneous fat on a 3.0 T whole–body MRI 
scanner (Siemens MAGNETROM, Malvern, PA). Participants will lie supine while these measurements are 
obtained, need to hold reasonably still during the scan and cannot weigh >325 lbs. A second sequence to measure 
the amount of fibrosis (if any present) in the liver will be performed. A specialized phosphorus coil will be utilized 
to measure the concentration of 31P via MRS. For the fMRI, participant’s head will be stabilized within the head coil 
using foam padding. Participants will be instructed to keep eyes open with relaxed fixation on a projected bright 
crosshair on a dark background. Anatomical scans will be collected using a T1- weighted, 3D Magnetization 
Prepared Rapid Gradient Echo sequence and will take ~5 mins to complete. Blood Oxygen Level Dependent 
(BOLD) resting-state functional MRI (rs-fMRI) data will be collected using a multiband gradient echo planar 
imaging sequence with a 32-channel head coil. The rs-fMRI sequence will take ~20 min to complete. Pulse and 
respiratory rate will be recorded during all scanning procedures via a respiratory belt and pulse sensor. 
Risks: Minimal. Participants may develop claustrophobia in the magnet. 
Minimizing Risk: The participant is provided with audio protection and optional television to help increase comfort. 
Some participants might feel claustrophobic while having an MRI and the scan will be stopped if it cannot be 
tolerated. In addition, any participants with implanted metal that is not cleared by the MRI technician may not be 
able to have the MRI due to the type of magnet involved. There is a very small risk that structural brain abnormality 
is identified during the rs-fMRI scan. If the participant is determined to have a structural abnormality, the participant 
and their primary care provider will be notified, and the participant will be encouraged to follow-up with them. This 
notification may cause the participant psychological distress. 
Justification/Minimization: MRI is a non-invasive and non-radiation method to assess body fat, and mitochondrial 
function. The risks are minimized by assuring patient comfort prior to starting the scan, placing eye goggle that 
plays movies on the participants. Further, per standard protocol, no patient will be placed into the scanner if they do 
not meet the rigorous safety standards for the MRI, including the absence of non-compatible implanted metal. 
8. Body Composition 
Description: Body composition will be measured using the DEXA technique and will be used to derive fat-free mass 
and % body fat.  This technique relies on the absorption of dual electron wavelengths for the assessment of body fat, 
lean tissue, and bone mineral density.  During the procedure, the participant will be supine on the measurement 
table, and the arm of the machine will slowly pass over their body.  
Risk: Minimal. Radiation exposure 
Justification/Minimization: Body composition is best assed via DEXA, and the amount of muscle mass is needed to 
standardize the OSTT results, since body weight can vary greatly. This procedure will deliver the radiation exposure 
that is 2 times the level of background radiation in Colorado. Participants will be tested for pregnancy immediately 
prior to DEXA, to ensure that they are not pregnant. 

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9. EndoPAT and Dynapulse 
Description: The Dynapulse Pathway and the EndoPAT system are noninvasive portable systems that measure 
brachial artery distensibility and endothelial function, utilizing a standard sphygmomanometer cuff inflated in the 
same fashion as a sphygmomanometer to obtain blood pressure. The instrument derives brachial artery distensibility 
using the technique of pulse waveform analysis of arterial pressure signals obtained from the sphygmomanometer. 
Measurement of heart rate variability for autonomic tone will be performed using the EndoPAT 
Risk: This procedure may lead to mild discomfort due to the blood pressure cuff being inflated. 
Justification/Minimization: Endothelial function is a novel measure in PCOS and will aid in the determination of 
cardiovascular dysfunction with this population.  
 
10. Body fat distribution   
Description: Height, weight, waist circumference, and hip circumference will be measured. Body fat distribution will 
be determined using the waist-to-hip ratio where the waist circumference is measured 1/2 the distance from the xiphoid 
process to the navel and the hip circumference is measured at the level of the greater trochanter. 
Risk: None 
Justification/Minimization: IR has been associated with central obesity, as has hyperandrogenism. Whereas we are 
measuring central obesity with MRI, it is important to see if this simple non-invasive measure matches the MRI results, 
as it is a much simpler measure to follow clinically. 
 
11. Accelerometer and Sleep Watch:  
Description: Participant will be provided an accelerometer and a sleep watch (GT3X BT by Actigraph and ActiWatch by 
Philips Respironics) to be worn for seven days to measure level of habitual physical activity, which affects insulin 
sensitivity, and sleep patterns. The accelerometer and sleep watch will be worn on the participant’s wrist. The wrist position 
has been validated to hip position actigraphy in this population. 
Risk: There is no risk involved with the accelerometer. 
Justification/Minimization: Accelerometers are effective tools for the objective measurement of physical activity 181 
because they have the ability to continuously record physical activity data and such data can be used to estimate 
METs of activity. They provide more detailed information than pedometers, which only measure walking steps, and 
help get around the recall bias of questionnaires. We are currently using the GT3X BT Actigraph in adolescents in 
our other diabetes studies; therefore, we are familiar with their use in this population and have the necessary 
computer software and interpretation skills.  The Actiwatch is being used as a tool for objective measurement of 
sleep patterns. The Actiwatch is fitted with a LED monitor that records multiple spectrums of light to better assess 
sleep patterns in this population. 
 
12. Metabolic Cart: 
Description: The metabolic cart measures the amount of air that the participant breathes in and out. The machine 
attaches to the participant’s mouth through a tube, or a plastic bubble that is placed over the participant’s head. 
There is the potential for experiencing claustrophobia from having the plastic bubble over the participant’s head. A 
metabolic cart will be utilized multiple times during the OSTT study day to measure rates of oxygen consumption 
and carbon dioxide release. These rates can be utilized to calculate rates of carbohydrate and fat oxidation and 
resting energy expenditure. 
Risk: Minimal risk of claustrophobia. 
Justification/Minimization: These studies are well tolerated by youth, and involve placing a clear plastic hood over the 
participant’s head for approximately 20 minutes. The data collected from the baseline study is also very useful for assisting 
obese participants in determining their true caloric needs, and useful in setting dietary goals for weight loss. This piece of 
information is thus utilized in post-study nutritional counseling. 
 
13. Food Frequency Questionnaire (SEARCH FFQ) 
Description: Customary macronutrient pattern will be ascertained by diet interview at the time of admission using a 
SEARCH FFQ, modified to incorporate common food choices among ethnically and regionally diverse youth aged 
10-19 participating in another large childhood diabetes study, SEARCH (48).  The instrument is self-administered 
with staff support to provide instructions, answer questions, and to review the form after completion, and captures 
the last week of dietary intake.   
Risk: None 

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Justification/Minimization: Several of the measurements being assed are affected by prior nutritional intake. 
Furthermore, participants will receive dietary counseling at the end of the study, and by knowing what their previous 
dietary pattern is, suggestions for improvement can be tailored to their specific dietary habits. 
 
14. 3DPAR Questionnaire  
Description: A questionnaire (3DPAR) recalling the physical activity levels of the three previous days will be completed 
at Visit 2b, Visit 3 and Visit 4b. 
Risk: None 
Justification/Minimization: Physical activity can directly affect insulin sensitivity, our primary outcome measure. The 
3DPar is a well validated measure to asses 3 days of physical activity in youth, and includes a variety of youth centric 
activities. 
 
15. Strengths and Difficulties Questionnaire: 
Description: This is a survey which identifies areas in a youth’s life that they believe they are strong or weak in dealing 
with, as a measure of coping skills. Low coping skills have been associated with the development of depression. 
Risk: None 
Justification/Minimization: This survey can help identify youth at risk for depression or anxiety, and identify poor coping 
skills. It does not directly assess depression or suicidality. 
 
16. WatchPAT and Questionnaires to assess for Obstructive Sleep Apnea 
Description: The WatchPAT is a noninvasive portable system that measures the oxygen saturation and apnea hypopnea 
index. Three surveys querying signs and symptoms of obstructive sleep apnea. 
Risk: No risk associated with the questionnaires and the WathchPAT, other than a mild discomfort from having to wear 
the watch and cuff around finger during sleep. It is possible that we will discover that the participant has obstructive 
sleep apnea, and will need to be referred for further clinical care. 
Justification: Obstructive sleep apnea is associated with obesity, and can worsen both fatty liver and insulin resistance. 
Thus the presence of OSA must be accounted for when measuring either of these outcomes. These surveys selected are 
currently being utilized by the NIH multiple center study in obese youth at risk for diabetes, and are well validated in 
youth from multi-ethnic populations.  If OSA is suspected during the course of the screen, the participants will be 
referred for further evaluation and treatment. The WatchPAT is an FDA approved device that can be used specifically 
for oxygen saturation and apnea hypopnea index and is approved in children as young as 12 years of age, within the age 
range of our study population. 
 
17: Questionnaires on Motivation for Weight Loss, Binge Eating, and Social Support 
 
Description: Four surveys asking about the participant’s motivation to lose weight, indicators of uncontrollable eating 
habits, and the participant’s social support from family, friends, and others (ie. Teachers, classmates) in achieving set 
goals. 
Risk: None 
Justification: Assistance in developing a participant-specific weight loss plan and targeting goals.  
 
18: Gut Bacteria Collection: 
Description: A week prior to visit three, participants will be provided with stool collection swabs to collect a small 
sample of stool from the toilet paper they use after having a bowel movement. 
Risk: Although the risk is minimal, participants may feel uncomfortable taking a sample of stool from the toilet 
paper following a bowel movement. All participants will be instructed to follow proper bathroom etiquette as fecal 
matter can transmit diseases. 
Justification: Studies in obese individuals with type 2 diabetes have alterations in the gut microbiota that may be 
related to NAFLD.  
 
 
19:  Rybelsus® semaglutide): 
Description: Rybelsus®  (semaglutide) is a GLP-1 receptor agonist. This medication comes in the form of daily oral 
tablets available in 3mg, and 7mg.Risk: 1) The most common side effects with Rybelsus® (semaglutide) may include 
nausea, abdominal pain, diarrhea, decreased appetite, vomiting, and constipation.. 2) Some patient can develop low 
blood sugars. Symptoms include acute feelings of lightheadedness, shaking, nausea, sensations of hunger, heart 

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racing and rarely vomiting. Treatment for this is eating a small carbohydrate snack. The family/participant should let 
the study team know if these symptoms develop. 3) From animal studies, there is a concern for increased risk of 
medullary thyroid cancer following long-term use of GLP-1 RA, but this has not been duplicated in human studies. 
4) Inflammation of the pancreas and gallstones have rarely been associated with this class of medications. 
Symptoms include serve central abdominal pain that may radiate to the back, nausea and vomiting and may be 
worse after meals, particularly fatty meals. These symptoms will be reviewed with the family/participant and the 
family/participant should let the study team know if these symptoms develop. 5) It is very important not to get 
pregnant while taking this medication. 
Justification: This class of medication is utilized for weight loss in normoglycemic patients and it is not known to 
cause hypoglycemia in patients without diabetes. Multiple clinical trials of use of medications of this class in 
pediatric patients have been published, showing good safety and efficacy data similar to that found in adults. Of this 
class of medications Rybelsus is the first oral GLP-1 RA therapy.. Finally, our pediatric providers are experienced in 
prescribing this medication in youth. From February 2017- February 2019, 65 unique pediatric patients were 
prescribed a GLP-1 receptor agonist by our pediatric endocrinology type 2 diabetes providers (Zeitler, Kelsey, 
Nadeau, Green, Williams) at the Anschutz Medical Campus.. Medullary thyroid cancer is extremely rare in humans 
and this risk is negligible with short-term use (as proposed by this study) and those with thyroid cancer risk factors 
are excluded. Patients will receive extensive education on how to properly take this medication and they will be 
encouraged to contact study staff if they experience any adverse events. The family should notify the study team for 
significant GI side effects and our team will also be routinely monitoring for this via phone contact. If a participant 
has emesis for > 24 hours or nausea limiting the ability to drink fluids, we will bring them in for hydration 
assessment that will include orthostatic blood pressures and complete metabolic panel (sodium, potassium, and renal 
function). Additionally, if a patient presents with symptoms consistent with pancreatitis during the study or follow-
up period, we will bring them in for an assessment (physical exam, vital signs, blood draw), which will can include 
an amylase, lipase, and complete metabolic panel, along with consultation with gastroenterology. 
 
20. Violation of Privacy and Loss of Confidentiality  
Description: These are both risks to which research participants are exposed. The possibility of these risks increases 
when protected health information is collected. Every effort will be made to decrease this risk by limiting access to 
protected health information, storing this information in a password protected database, and identifying participants 
only by a unique identifier that is kept in a separate location in a locked container, traceable only by study personnel. 
All of the tests involve the risk of identifying asymptomatic abnormalities. The study may include risks that are 
unknown at this time.  
Justification/Minimization: Every effort will be made to decrease the risk of loss of confidentiality by limiting 
access to protected health information, storing this information in a password protected database, and de-identifying 
study specimens.   
 
 
 
 
 
 
11. ADVERSE EVENTS 
 
a) 
Definitions of Adverse Events 
 
According to the International Conference on Harmonization (ICH) guidelines (Federal Register. 
1997;62(90):25691-25709) and 21 CFR 312.32, IND Safety Reports, and ICH E2A, Definitions and 
Standards for Expedited Reporting, an adverse event is defined as follows: 
 
An adverse event is any untoward medical occurrence in a participant or clinical investigational participant 
administered a pharmaceutical product and which does not necessarily have a causal relationship with this 
treatment. An adverse event can therefore be any untoward medical occurrence regardless of relationship to the 
medicinal (investigational) product. 
 

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Adverse events will be recorded from the time of informed consent. 
 
b) 
Documentation and Monitoring Adverse Events 
 
All baseline conditions and adverse events encountered during the treatment following participant consent 
through the last day of treatment will be recorded in the patient’s medical records. Serious adverse events that 
are considered related (i.e., determined to be possibly, probably, or definitely related) to the investigational 
product by the treating physician will be documented on the patient’s medical record. 
 
c) 
Assessment of Adverse Events 
 
For each adverse event, the start and resolution dates, severity, seriousness (i.e., whether the event meets the 
definition of an SAE, relationship of the event to the study drug, action taken regarding study drug, and 
outcome of the event will be documented on the patient’s medical record. 
 
(1) Severity 
 
Common Toxicity Criteria for Adverse Events (CTCAE) Version 4.03 will be used to assess and grade adverse 
event severity, including laboratory abnormalities judged to be clinically significant. If the event is not covered 
in the CTCAE, the guidelines shown in the following table will be used to grade severity. 
 
Adverse Event Severity Grading Scale: 
 
Severity 
Grade 
Description 
Mild 
1 
The event is of little concern to the participant and/or of no 
clinical significance. The event is not expected to have any effect 
on the participant’s health or well being 
Moderate 
2 
The participant has enough discomfort to interfere with or 
necessitate change in usual activities. The event is of some 
concern to the participant’s health or well being. The event may 
require medical intervention. 
Severe 
3 
The participant is incapacitated and unable to work or participate 
in many or all usual activities. The event is of definite concern to 
the participant and/or poses substantial risk to the participant’s 
health or well being. The event is likely to require medical 
intervention and/or close follow-up 
Life-Threatening 
4 
The participant is at risk of death due to the adverse event as it 
occurred. This does not refer to an event that, hypothetically, 
might have caused death if it were more severe. 
Death 
5 
Death related to adverse event. 
 
 
(2) Relationship to the investigational product: 
 
The relationship of an adverse event to study drug will be assessed using the guidelines presented in the table 
below.  
 
Adverse Event Relationships to Investigational Product: 
 
Relationship to Drug 
The Adverse Event: 
Related 
Exhibits previously known toxicity of agent; or 
Follows a reasonable temporal sequence from administration of the drug; 
Follows a known or expected response pattern to the suspected drug; 
Is confirmed by stopping or reducing the dosage of the drug; and 
Is not explained by any other reasonable hypothesis 

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Probably Related 
Follows a reasonable temporal sequence from the time of study drug administration; and/or 
Follows a known response pattern to the study drug; and 
Was unlikely to have been produced by other factors such as the participant’s clinical state, 
therapeutic intervention, or concomitant therapy 
Possibly Related 
Follows a reasonable temporal sequence from the time of study drug administration; and/or 
Follows a known response pattern to the study drug; but 
Could have been produced by other factors such as the participant’s clinical state, therapeutic 
intervention, or concomitant therapy. 
Unlikely Related 
Does not follow a reasonable temporal sequence from the time of study drug administration; 
and 
Was likely produced by other factors such as the participant’s clinical state, therapeutic 
intervention, or concomitant therapy, but for which relationship cannot be definitely ruled 
out. 
Not Related 
The adverse event can be determined with certainty to have no relationship to the study drug 
 
(3) Adverse Event Outcome 
 
Each adverse event will be characterized according to the outcomes described in the following table: 
 
Adverse Event Outcomes: 
Outcome 
Description 
Recovered/resolved 
The participant has fully recovered from the event with no observable residual effects. 
Recovering/resolving 
The effects of the event are improving, or events have stabilized (are constant and not 
expected to improve or worsen) but have not returned to baseline 
Not recovered/not resolved 
The effects of the event are still present and changing. The event is not considered 
stabilized or resolved. 
Recovered/resolved with 
sequelae 
The participant has fully recovered from the event with some observable residual effects. 
Fatal 
The event was the primary cause of death (may or may not be the immediate cause of 
death). 
Unknown 
The event outcome is unknown. 
 
Note: Death is an outcome of an event and not an event per se. Sudden death or death due to unexplainable 
cause(s) will be reported, but follow-up will be pursued until cause of death is determined. 
 
 
(4) Action Taken with Investigational Product 
 
Action taken with investigational product in relation to each adverse event will be characterized as 
follows: 
 
 
 
Action taken with investigational product: 
 
• 
None 
 
• 
Drug withdrawn 
 
• 
Drug interrupted 
 
• 
Not applicable 
 
• 
Other (specify in medical record) 
 

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12. SERIOUS ADVERSE EVENTS 
 
Any adverse event or abnormal laboratory test value that is serious (see definition below) and occurs after 
administration of the investigational product will be documented by the Sponsor-Investigator within 24 hours of 
discovery of the event.  
 
(a) Definitions 
 
An adverse event will be classified as a serious adverse event (SAE) if it meets one of the following criteria: 
 
Classification of Serious Adverse Events: 
 
Classification 
Description 
Fatal 
Adverse event resulted in death 
Life Threatening 
The adverse events placed the patient at immediate risk of death. This classification 
did not apply to an adverse event that hypothetically might cause death if it were 
more severe. 
 
Hospitalization 
The AE required or prolonged an existing inpatient hospitalization. Hospitalizations 
for elective medical or surgical procedures or treatments planned before the signing 
of informed consent in the study or routine check-ups are not serious adverse events 
by this criterion. Admission to a palliative unit or hospice care facility is not 
considered to be a hospitalization. Hospitalizations or prolonged hospitalizations 
for scheduled therapy of the underlying cancer or study target disease need not be 
captured as SAEs. 
Disabling/incapacitating 
Resulted in a substantial and permanent disruption of the patient’s ability to carry 
out activities of daily living 
Congenital anomaly or birth 
defect 
An adverse outcome in a child or fetus of a patient exposed to the molecule or study 
treatment regimen before conception or during pregnancy. 
Medically significant 
The adverse event did not meet any of the above criteria, but could have 
jeopardized the patient and might have required medical or surgical intervention to 
prevent one of the outcomes listed above. 
 
Death due to progressive disease (PD) will not be considered an SAE. However, if a patient requires 
hospitalization due to an AE related to PD, the specific sign or symptom leading to hospitalization will be 
reported as an SAE. If death due to PD occurs outside of hospitalization and is considered to be due solely to 
the patient's malignancy, no SAE report is required. The death will be recorded as part of the tumor response 
and patient disposition. 
 
Serious adverse events that are considered related (i.e., determined to be possibly, probably, or definitely related) 
to the investigational product by the Sponsor-Investigator will be followed until the event resolves or stabilizes. 
Any SAE that occurs after treatment completion, and is considered by the Sponsor-Investigator to be related to 
the investigational product, will be documented and reported as appropriate. 
 
 
(b) Reporting Serious Adverse Events to Regulatory Agencies 
 
Events meeting the following criteria will be submitted to the Food and Drug Administration (FDA) as expedited 
IND Safety Reports according to the following guidance and timelines: 
 
7 Calendar Day Telephone or Fax Report: 
The Sponsor-Investigator will notify the appropriate review division at the FDA of any fatal or life-threatening 
adverse event that is unexpected and assessed by the Sponsor-Investigator to be possibly related to the use of the 
investigational product. An unexpected adverse event is one that is not already described in the investigational 

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product’s Investigator Brochure. Such reports are to be telephoned or faxed to the appropriate review division at 
FDA and to the manufacturer of the investigational product within 7 calendar days of first learning of the event. 
 
15 Calendar Day Written Report 
The Sponsor-Investigator will notify the appropriate review division at the FDA, in a written IND Safety Report, of 
any serious, unexpected AE that is considered reasonably or possibly related to the use of the investigational 
product. An unexpected adverse event is one that is not already described in the investigational product’s 
investigator brochure. 
 
Written IND Safety reports will include an Analysis of Similar Events in accordance with regulation 21 CFR § 
312.32. All safety reports previously filed by the Sponsor-Investigator with the IND concerning similar events will 
be analyzed and the significance of the new report in light of the previous, similar reports commented on. 
 
Written IND safety reports with Analysis of Similar Events will be submitted to the appropriate review division at 
the FDA, and the manufacturer of the investigational product within 15 calendar days of first learning of the event, 
using FDA Form 3500A. 
 
Written IND Safety Reports will be submitted to the IRB(s) of record per IRB Guidelines. 
 
13. IND Annual Reports 
The Sponsor-Investigator will provide annual reports to the appropriate review division at the FDA within 60 days 
of the IND’s anniversary date, until the IND is withdrawn or terminated. 
14. DISCONTINUATION AND REPLACEMENT OF PARTICPANTS 
Early Discontinuation of Study Drug  
A participant may be discontinued from study treatment at any time if the participant, or the investigator, feels that it 
is not in the participant’s best interest to continue.   
 
The following is a list of possible reasons for study treatment discontinuation:  
• 
Participant withdrawal of consent (or assent) 
• 
Participant is not compliant with study procedures 
• 
Adverse event that in the opinion of the investigator would be in the best interest of the participant to 
discontinue study treatment– classified as a CTCAE grade 3 attributable to the medication or a CTCAE 
regardless of attribution to the drug. 
• 
Protocol violation requiring discontinuation of study treatment 
• 
Lost to follow-up 
• 
Positive pregnancy test 
• 
If a patient has recurrent emesis at the lowest dose (3 mg) of semaglutide they will be removed from the 
trial or if they are so nauseated as to be unable to maintain adequate hydration, they will be removed from 
the trial. 
• 
If participants cannot tolerate the study medication due to side effects, participants will be instructed to 
contact the study team immediately and they may be brought in to an additional visit, which may include a 
blood draw. Study team will evaluate their symptoms and determine if symptoms are severe enough for 
them to withdraw from the study, or if they are on the 7 mg dose, back down to 3 mg for at least a week, 
before reattempting to increase to 7 mg.  

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• 
We will monitor liver enzymes following the criteria listed below: 
o 
If the ALT at the second visit increases by > 40% and becomes > 100 IU/mL, and the participant 
is randomized to active drug, we will not start the drug immediately, but will repeat the CMP in 2-
4 weeks. If the value remains >40% and > 100 IU/ml of baseline, the participant will be removed 
from the study. 
o 
Once on therapy, if there is an increase in ALT > 5x baseline and > 100 IU/ml at any visit, ALT 
will be repeated within 3-7 days and if the elevation is persistent, medication will be stopped for 
one week, and ALT repeated after 7-10 days of discontinuation. If ALT is no longer elevated 
medication will be restarted, with another ALT 7-10 days following restart of medication will be 
performed. If ALT is not elevated, trial will continue, if ALT returned to > 5x baseline and > 100 
IU/ml, participants will be removed from trial. If removed from the trial, a repeat safety CMP will 
be performed 3-7 days after stopping drug. 
o 
Once on therapy, if participants develop persistent right upper quadrant pain or signs of jaundice, 
they will be brought in for assessment, including vital signs, physical exam and complete 
metabolic panel. The above guidance on point 2 will apply to laboratory values obtained. 
o 
It will be recommended that any participant who is removed from the trial have a follow-up 
appointment with hepatology, but this will not be part of the study. 
 
Withdrawal of Participants from the Study 
All participants are free to withdraw from participation at any time, for any reason, specified or unspecified, and 
without prejudice. 
Reasonable attempts will be made by the investigator to provide a reason for participant withdrawals.  The reason 
for the participant’s withdrawal from the study will be specified in the participant’s source documents. Refer to the 
protocol for early termination procedures. 
A participant may be withdrawn from the study at any time if the participant, the investigator feels that it is not in 
the participant’s best interest to continue.   
Replacement of Participants 
Participants who withdraw or are withdrawn from the study will be replaced.   
15. CRITERIA FOR STOPPING THE TRIAL 
 
• 
Three patients develop the same Grade 3 Common Terminology Criteria for Adverse Events (CTCAE) 
attribution to the study drug OR 
• 
Two patients develop any Grade 4 CTCAE attribution to study drug OR 
• 
One patient develops a grade 5 CTCAE attribution to study drug  
16.  PROTOCOL VIOLATIONS 
A protocol violation occurs when the participant or investigator fails to adhere to significant protocol requirements 
affecting the inclusion, exclusion, participant safety and primary endpoint criteria.  Protocol violations for this study 
include, but are not limited to, the following: 
Failure to meet inclusion/exclusion criteria 
Use of a prohibited concomitant medication 
Failure to comply with Good Clinical Practice (GCP) guidelines will also result in a protocol violation. The 
investigator will determine if a protocol violation will result in withdrawal of a participant. 

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When a protocol violation occurs, it will be discussed with the investigator and a Unanticipated Problem form will 
be completed, and submitted to COMIRB, detailing the violation. This form will be signed by the Investigator. A 
copy of the form will be filed in the site’s regulatory binder and in the COMIRB’s files. 
 
17. DATA SAFETY MONITORING 
 
The PI, Dr. Melanie Green, will establish a Data Monitoring Committee (DMC) to review data relating to safety and 
efficacy, to conduct and review interim analyses, and to ensure the continued scientific validity and merit of the 
study. The committee will consist of members from outside institutions who are experts in the field: Tania Burgert, 
pediatric endocrinologist at Mercy Children’s in Kansas City, Missouri; Ashley Shomaker, pediatric endocrinologist 
at Vanderbilt; and Lisa Chow, adult endocrinologist at the University of Minnesota Rochester, MN, and a pediatric 
gastroenterologist.. The DMC will talk for the purpose of monitoring study conduct and assessing patient safety. The 
conference calls will be held after the first three participants are complete, then approximately every 6 months, or 
with the occurrence of any acute adverse event.  
The principal investigator and will meet with the research coordinator to monitor the protocol and the safety of the 
research participants.  The PI will review all laboratory data and report any abnormal values to the patient and 
guardian and instruct the participant to follow-up with their PCP.  If an abnormal result from a research procedure 
exists, the PI will notify the family and their PCP and refer the participant to the appropriate clinic for further 
evaluation. The PI may also share research results in a reasonable and prudent manner with appropriate medical 
professionals if the participant was seriously injured as a result of a procedure or if follow-up of the result of the 
procedure is in the best interest of the participant’s health as determined by a medical professional. If immediate 
medical follow-up of participant required, the PI will share the research results via EPIC when clinically relevant. 
The PI will report adverse events, and any decision to suspend or halt the protocol to CTRC and COMIRB 
immediately.  The PI will also prepare a written report for the yearly continuing review required by COMIRB and 
the CTRC. The PI will also submit relevant reports to the FDA as required per FDA regulations.   
 
No protected health information will be collected until the appropriate HIPAA forms are completed. The protected 
health information that will be collected will include: Name and phone number, demographic information (DOB, 
sex, ethnicity, address, etc.), diagnosis (es), history and physical, laboratory or tissue studies, radiology studies, 
procedure results, survey/questionnaire results, research visit records, and portions of previous Medical Records that 
are relevant to this study. This information will be accessible only by the study investigators, Federal agencies 
overseeing human subject research, the Colorado Multiple Institutional Review Board, regulatory officials from the 
institution where the research is being conducted to monitor safety and compliance with policies. 
18. STATISTICAL METHODS AND CONSIDERATIONS 
Prior to the analysis of the final study data, a detailed Statistical Analysis Plan (SAP) will be written describing all 
analyses that will be performed.  The SAP will contain any modifications to the analysis plan described below.   
 
 
 
 
SA1 Statistical Plan 
Sample size justification.  Sample size is based on the power to detect a difference between the standard diet + GLP-
1 RA group and the intensive dietary counseling group in the primary outcome of change in IHTG. Estimates for 
power calculations were obtained from a recent randomized controlled trial of liraglutide in adult women with 
PCOS68 and our baseline data on IHTG in obese girls with PCOS (Table 2). Assuming a standard deviation (SD) of 
0.7% and a two-sided significance level of 0.05, a final sample size of 17 participants in the intensive diet group and 
33 participants in the standard diet + GLP-1 RA group provides 90% power to detect a difference of -0.7% between 
group change. This difference is 50% smaller than the difference of -1.34% observed by Frøssing et al.68 as their 
results are confounded by significant weight loss and therefore our study is powered conservatively. We anticipate a 
1% decrease in hepatic fat.  We estimate an approximately 5-10% drop out rate based on our group’s experience 
from similar participant populations9,10,111,116,118,182,183 and we will enroll up to 60 girls with the goal of 50 

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completers.   
Analysis plan.  Data will be collected and managed in a REDCap database, a secure web-based application designed 
to support data capture for research studies, including validated data entry, audit trails, and automated export to 
statistical packages. Prior to the start of any formal analyses, variables will be examined for unusual values that need 
to be queried, patterns of missing values, and whether their distributions are non-Gaussian. Demographic and 
clinical characteristics will be summarized with descriptive statistics. Primary analyses will follow the “intent-to-
treat” principle so that study hypotheses are tested under realistic conditions in which not all participants adhere to 
the intervention. This will include those who discontinue therapy whose data will be censored at the time of 
discontinuation, so that all data we have on the non-completers up until the time they leave the study will be 
included in this primary analysis.  However, in order to fully evaluate the physiology and mechanism of action of 
semaglutide in this population, which is an important aim of this investigator-initiated study, we will also perform 
secondary analyses in participants who demonstrated at least 80% adherence (i.e., at least 80% medication taken in 
the GLP-1 arm and at least 80% of targeted weight loss achieved in the intensive lifestyle arm). For the primary 
outcome, we will compare the change in % IHTG with and without GLP-1 RA treatment using a t-test or Mann-
Whitney U test. We expect the weight loss will be similar in the two groups; however, if there is a clinically 
significant difference between the groups, these comparisons will also be performed while adjusting for the amount 
of weight lost. Similarly, if there are differences in activity or obstructive sleep apnea severity, appropriate 
adjustments will be made. Similar analyses will be used for secondary outcomes (2 hour OGTT glucose, HOMA-IR, 
metabolomics markers, proportion of direct/indirect glucose, EAA concentrations).  Linear models will be used to 
identify the variables that are associated with change in IHTG, e.g., 2 hour OGTT glucose, HOMA-IR, 
metabolomics markers.    
          
SA2 Statistics 
 Sample size justification. The primary outcomes for Aim 2 are (1) change in hepatic de novo lipogenesis (DNL) and 
(2) change in FFA nadir in the standard diet + GLP-1 group compared to the intensive diet group.  Estimates of 
variability were obtained from the PI’s pilot data (Figure 4b). We assumed a cross-sectional DNL SD of 3.36%, a 
pre-post correlation of 0.8, and a two-sided significance level of 0.05.  With 50 participants, we will have 90% 
power to detect a between-group DNL difference of 2.1%, which is smaller than the difference of 2.6% reported by 
Armstrong et al. in adults14. Using the same assumptions and a cross-sectional SD of 23 nmol/L, we will have 80% 
power to detect a change of 22% in FFA nadir (Figure 4c), which is conservative given data in adults showing a 
decrease of 31% with liraglutide67.  
 Analysis plan: Analyses for the primary 
outcomes of change in DNL and FFA 
nadir will be the same as Aim 1, as will 
the analyses for secondary measures of 
DNL (% suppression of triglyceride) and 
secondary 
outcomes 
(2-hour 
OSTT 
glucose, % TCA cycling, peripheral IR 
per OMM, metabolomics markers).  From 
the study day 1 cross sectional data, we will use regression models to evaluate the relationships between markers of 
hepatic disease (IHTG and PDE/ATP) and DNL, excess substrate (FFA nadir, mean OSTT glucose) and measures of 
TCA anapleurotic cycling (% indirect glucose).  Table 7 provides details on the power calculations for these models 
of primary interest, using estimates of variability obtained from the PI’s pilot data.  If the relationship between % 
TCA cycling and IHTG is significant, we will use mediation modeling to test if this effect is mediated by DNL.  The 
distribution-of-the-product method will be used to construct 95% confidence intervals for the mediated effects, using 
the RMediation software package184.  After investigating associations between variables in the cross-sectional data 
from study day 1, we will evaluate if baseline characteristics predict changes in the study primary outcomes, in order 
to further understand the pathophysiology and to identify patients benefit from particular interventions.  
 
Summarize Knowledge to be Gained:  
SA1 Expected Results  
We anticipate that after GLP-1 RA, IHTG will decrease by at least 0.7% compared to the diet intervention. With the 
mean of 6.2±2.3% IHTG in PCOS girls, this would change the clinical classification of HS in many girls. These 
findings would substantiate the role of GLP-1 RA as a treatment to reduce HS in obese girls with PCOS and 
potentially mitigate long-terms health effects associated with HS. Alternatively, if both treatment groups had a 
reduction in IHTG, data could be utilized to best predict a favorable response to diet or GLP-1 RA. 
Table 7: Power calculations for regression models 
Outcome (SD) 
Predictor (SD) 
Slope detected 
with 80% power 
IHTG (6%) 
DNL (3.4%) 
0.66 
IHTG (6%) 
FFA nadir (23 nmol/L) 
1.44 
IHTG (6%) 
Mean OSTT glucose (23 mg/dL) 
0.10 
IHTG (6%) 
% indirect glucose (5%) 
0.45 
PDE/ATP (0.12) 
% indirect glucose (5%) 
0.01 

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SA2 Expected Results  
We expect that DNL and FFA will relate to IHTG at baseline and that changes in IHTG will relate to changes in 
DNL and FFA suppression. Further DNL will relate to glucose excursions, rates of hepatic anaplerosis and ATP 
concentrations measured with 31P MRS. These findings will provide mechanistic data relating to IHTG in PCOS and 
will contribute to the overall understanding of the unique pathology seen in PCOS. This information may lead to the 
development of alternate approaches to treat HS in PCOS or even NAFLD in simple obesity. 
 
Confirmation of our anticipated outcomes would provide critical evidence to pursue larger clinical trials with a 
GLP-1 RA. Longer trials of GLP-1 in adult women with PCOS showed an increase in ovulatory cycles and decrease 
in testosterone185. Future trials could include a longer duration to better assess changes in clinical severity in a larger 
adolescent cohort. If the DNL contribution to IHTG is significant in this population, future trials could examine the 
efficacy of glucose lowering SGLT-2 medication in PCOS girls with dysglycemia. 
 
SA3 Expected Results 
We expect that connectivity between the homeostatic and non-homeostatic brain regions will be significantly greater 
after treatment with semaglutide compared to diet. Neurobiologically, eating behavior is driven by both homeostatic 
and non-homeostatic regions in the brain. Understanding the communication between homeostatic and non-
homeostatic brain regions and their relationship to weight-loss interventions among adolescents with obesity and 
PCOS will help to elucidate specific neural mechanisms that contribute to overeating, and ultimately to weight 
regulation.  
 
Potential Scientific Problems:   
SA1 Limitations/Alternate Approaches 
1) As with any clinical trial, recruitment and retention can be challenging. We have successfully recruited over 120 
obese girls with untreated PCOS using identical enrollment criteria, and thus our recruitment goals are in line with 
our previous work. We have never needed to recruit in the community or other endocrinology practices but can if 
needed. Our overall lab has been successful with 3-month, 21-month and 3-year interventional protocols and we 
have designed the study with retention techniques that were successful in similarly obese adolescent populations. 
The PI is the only investigator recruiting from this patient population at UC Denver, and the majority of recruitment 
for our existing funding would be complete by the time the proposed project would start, thus we will not lose 
potential participants to other studies. 2) Adherence to treatment is also a challenge, and we will use texting and 
emails as preferred contacts in youth to send supplemental reminders. 3) Major changes to diet and exercise could 
alter results, thus we will request participants in the GLP-1 RA group to not to initiate major diet or exercise changes 
during the study. Those in the diet group will be asked to not change activity during the study period. As acute 
exercise effects measurements of IR, we will request no exercise in the 72 hours prior to each admission. 4) Among 
the three current sets of guidelines for care of adolescents with PCOS, the consistent initial recommendation is for 
lifestyle intervention8,34,126. Thus, those girls in the dietary arm are receiving standard of care, whereas those in the 
medication arm will be receiving a medication with a clear potential for benefit. 5) We acknowledge the fact that 
inducing weight loss in adolescents with diet is challenging and not sustainable in most youth long-term. However, 
we are experienced and have been successful in the short-term with our planned approach in an identical patient 
cohort and will be utilizing several additional strategies. We will also perform statistical analysis accounting for 
differences in weight loss, should they not be equal between the groups. Although this is not ideal, this will be 
achieved by correcting the analyses for differences in weight loss. 6) There are several other long-acting GLP-1 RA 
available that may have a similar glucose lowering effect, but a greater effect on weight loss and higher side effects 
and lower tolerability159. We choose to use Rybelsus (semaglutide)due to a)being the first GLP-1 RA therapy 
available in oral tablets instead of injection b) our preliminary data in youth with PCOS c) a small degree of weight 
loss which is realistic to obtain in a dietary intervention group.  
 
SA 2 Limitations/Alternate Approaches 
1)  The reproducibility of glucose measurement from an OSTT in any individual is only 75%, however, this is when 
the effects of exercise, diet, sleep and menstrual cycle are not controlled149. An alternative would be to provide the 
diet for participants for 3 days prior to studies, although this would then be a measure of the diet itself and not the 
participant’s home intake.  As the oligomenorrhea in PCOS best approximates the follicular phase in terms of 
estrogen and progesterone concentrations, we will attempt to perform studies in the follicular phase and confirm this 

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with a serum progesterone concentration (<2 ng/mL is consistent with the follicular phase). The OSTT results will 
remain valid if serum progesterone concentration is >2ng/mL on the day of the OSTT, but this will be disclosed in 
publications. We will record the presence or severity of sleep apnea and can adjust for typical sleep duration if 
needed. 2) An alternate approach for assessing insulin sensitivity would be a hyperinsulinemic euglycemic clamp, 
for which we have extensive experience in pediatrics9,10,150,153. We have elected not to use this approach as IHTG is 
formed primarily in the fed state and a clamp bypasses the effects of GLP-1. We have also validated that the 4 hour 
OMM approximates clamp results well in our patient population. 3) Certain genotypes have been associated with 
increased rates of NAFLD and DNL58,186,187. As we are calculating the change per individual, this affect should be 
minimized. Further, cost-efficient yet comprehensive panels for multiple relevant genes are not yet available. At the 
time of initial consent, we will seek permission for future genetic testing and save blood samples for such testing, 
should we deem this data necessary for interpretation of results and testing panels for multiple genes become more 
available. 
 
SA3 Limitations/Alternate Approahces 
1)  One limitation is that we may not see any change with treatment. In addition, we will not have enough 
participants for a fully powered study, but this is a pilot study to inform potential future studies. There is no 
alternative approach. However, even if no change is observed with treatment, it would still be an important finding. 
 
Scientific Rigor: Plan for robust and unbiased results  
Additional OSTT studies in this patient population will replicate and validate our preliminary findings, providing 
redundancy. Modulating hepatic metabolism via nutritional and pharmacologic means will help with establishing 
cause and effect for excess DNL and IHTG. We are appropriately powered to answer our hypotheses and are using 
robust statistical analyses to test our hypotheses in an unbiased manner. The protocol includes a control group and 
participants are randomized to groups once pilot data is collected. 
 
Consideration of relevant biological variables  
PCOS occurs in women only, and the metabolic consequences have been understudied, especially relative to 
investigations of NAFLD in men. However, NAFLD appears to occur differently in Blacks compared to Hispanics 
and Whites94,188.  While we are not fully powered to examine these differences, our baseline data can be utilized to 
explore racial/ethnic differences and generate preliminary data for future applications 
 
19. DATA COLLECTION, RETENTION AND MONITORING 
Data Collection Instruments 
The Investigator will prepare and maintain adequate and accurate source documents designed to record all 
observations and other pertinent data for each participant treated with the study drug.   
 
The Investigator is responsible for all information collected on participants enrolled in this study.  All data collected 
during the course of this study must be reviewed and verified for completeness and accuracy by the Investigator.  
Data Management Procedures 
The data will be entered into a validated database.  The Data Management group will be responsible for data 
processing, in accordance with procedural documentation.   
All procedures for the handling and analysis of data will be conducted using good computing practices meeting FDA 
guidelines for the handling and analysis of data for clinical trials. 
Data Quality Control and Reporting 
After data have been entered into the study database, a system of computerized data validation checks will be 
implemented and applied to the database on a regular basis.  

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Archival of Data 
The database is safeguarded against unauthorized access by established security procedures; appropriate backup 
copies of the database and related software files will be maintained.  Databases are backed up by the database 
administrator in conjunction with any updates or changes to the database.   
Availability and Retention of Investigational Records 
The Investigator must make study data accessible to authorized representatives of the Sponsor (or designee), 
COMIRB and Regulatory Agency (e.g., FDA) inspectors upon request.  A file for each participant must be 
maintained that includes the signed Informed Consent, HIPAA Authorization and Assent Form and copies of all 
source documentation related to that participant.  The Investigator must ensure the reliability and availability of 
source documents from which the information was derived. 
All study documents (patient files, signed informed consent forms, copies of CRFs, Study File Notebook, etc.) must 
be kept secured for a period of two years after centers have been notified that the IND has been discontinued.   
20. ADMINISTRATIVE, ETHICAL, REGULATORY CONSIDERATIONS 
The study will be conducted according to the Declaration of Helsinki, Protection of Human Volunteers (21 CFR 50), 
Institutional Review Boards (21 CFR 56), and Obligations of Clinical Investigators (21 CFR 312). 
To maintain confidentiality, all laboratory specimens, evaluation forms, reports and other records will be identified 
by a coded number and initials only, with the exception of HIPPA protected hospital and CTRC CORE laboratories 
which require patient identifiers.  All study records will be kept in a locked file cabinet and code sheets linking a 
patient’s name to a patient identification number will be stored separately in another locked file cabinet.  Clinical 
information will not be released without written permission of the participant, except as necessary for monitoring by 
the FDA.  The Investigator must also comply with all applicable privacy regulations (e.g., Health Insurance 
Portability and Accountability Act of 1996). 
Protocol amendments cannot be implemented without prior written COMIRB, except where it may be necessary to 
eliminate an immediate hazard to a research participant.  In such case, the deviation will be reported to COMIRB as 
soon as possible.  
The protocol and consent form will be reviewed and approved by COMIRB prior to study initiation. 
 

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APPENDIX 1. SCHEDULE OF STUDY VISITS  
Study Calendar 
Visit 1 
(Screening) 
BASELINE METABOLIC STUDY 
Visit 3  
(Mid-Treatment) 
POST-TREATMENT METABOLIC STUDY 
Visit 2a 
(Imaging) 
Visit 2b  
(Overnight) 
Visit 4a 
(Imaging) 
Visit 4b  
(Overnight) 
Consent & Eligibility Assessment 
X 
  
  
  
  
  
History & Physical 
X 
  
  
X 
  
  
Intravenous Blood Draw 
X 
  
X 
X 
  
X 
Urine Pregnancy Test 
X 
X 
  
 X  
X 
  
Gut Bacteria Collection 
 
  
X 
  
  
X 
Accelerometer and sleep watch 
 
 
7-days prior 
 
 
7-days prior 
DEXA Scan  
 
X 
  
  
X 
  
MRI of abdomen and liver  
 
X 
  
  
X 
  
P-MRS of Liver & rs-fMRI of Brain 
 
X 
  
  
X 
  
EndoPAT and Dynapulse 
 
X 
  
  
X 
  
WatchPAT sleep study 
 
  
X 
  
  
X 
Oral Glycerol and deuterated water 
tracers 
 
  
X 
  
  
X 
Oral Sugar Tolerance Test (OSTT) 
 
  
X 
  
  
X 
Metabolic Cart 
 
  
X 
  
  
X 
Questionnaires (Refer to appendix 3) 
 
  
X 
 X 
  
X 
Randomization- GLP-1 (Rybelsus) 
vs Lifestyle intervention 
  
  
X 
  
  
  
Hypoglycemia Questionnaire 
 
 
X 
X 
 
X 
Pregnancy prevention 
counseling/condoms 
X 
 
X 
X 
 
X 
Total Time of Visit (approximately) 
2 Hours 
4.5 Hours 
 21 Hours 
1.5 Hour 
4.5 Hours 
21 Hours 
Timeline: The time between the initial screening visit and completion of the study will be less than 6 months. After confirmation of eligibility participants will 
complete the Baseline Metabolic Study (Visit 2a and 2b) and they will be randomized to GLP-1 (Rybelsus) or Lifestyle. Visit 3 will be completed approximately 
2-months post baseline metabolic study. The Post-Treatment Metabolic Study (Visit 4a and 4b) will be completed after 4-months of GLP-1 or Lifestyle 
Intervention.   

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APPENDIX 2.  SCREENING AND ORAL SUGAR TOLERANCE TEST (OSTT) 
LABS 
Screening Labs 
Purpose  
HbA1C 
Rule out type 2 diabetes, if > 6.4% participant to be excluded 
ALT, AST, total bilirubin, alkaline phosphotase (Part of 
complete metabolic panel) 
Ensure no severe liver disease, if >100 IU/L participant to be excluded 
normal participant is excluded 
Hemoglobin & Hematocrit (part of CBC) 
If participant is Anemic, they will be excluded 
Sodium, Potassium, Creatinine and BUN (Part of complete 
metabolic panel) 
To check for undetected renal dysfunction 
Calcium and Albumin (Part of complete metabolic panel) 
To screen for occult hypercalcemia potentially associated with MEN 
Testosterone  
Test for hyperandrogenism – required to meet NIH criteria for PCOS 
1 sample for genetic analysis 
Measure for common polymorphisms associated with hepatic steatosis179,189 
Optional Screening PCOS labs 
PCOS status must be confirmed prior to enrollment. Referring physicians often do not perform the entire recommend work-up for 
oligomenorrhea (per 2013 Endocrine Society Clinical Guidelines for PCOS). The values are typically expected in PCOS publications 
TSH, total T4 
Ensure no hypo or hyperthyroidism causing amenorrhea 
LH, FSH 
Rule out primary ovarian failure 
17-hydroxyprogesterone 
Rule out late onset congenital adrenal hyperplasia 
DHEAS 
Rule out adrenal tumor 
Prolactin 
Rule out prolactin secreting brain tumor 
Mid-treatment 
Purpose 
Complete Metabolic Panel 
This will include measures of transaminases, renal function and 
hypercalcemia. 
HbA1c 
 
OSTT Labs 
Purpose 
Lipids and glycerol samples for tracer analysis 
Determination of hepatic metabolism flux 
Glucose  
Determination of IR 
Insulin 
Determination IR 
FFA 
Measure of lipolysis 
Glycerol 
Measure of lipolysis 
Glucagon 
Gut hormone known to influence hepatic IR 
GLP-1 
Gut hormone known to influence hepatic IR 
CRP 
Marker of inflammation, know to effect IR 
Leptin 
Gut hormone known to influence hepatic IR 
Adiponectin 
Adipokine thought to influence adipose IR 
Estradiol 
Known to effect IR and HDNL 
Progesterone 
Demonstrate the participant is in the follicular phase of cycle, required for 
publication 
Complete Metabolic Panel 
This will include measures of transaminases, renal function and 

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hypercalcemia. 
Metabolomics 
Correlation with hepatic metabolism flux 
Stored blood 
Future markers of glucose and fat metabolism, CVD or hormones related to 
PCOS 
Apolipoprotein B 
Measure of triglyceride secretion 
 

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APPENDIX 3.  QUESTIONNAIRES BEING ADMINISTERED 
Questionnaire  
Description 
Study Validity 
Adolescent Sleep Hygiene Scale 
Measurement of sleep 
patterns/habits 
Cronbach’s alpha ranges from .46-.74; total scale 
alpha = .80 
LeBourgeois et al. 2005. The Relationship Between 
Reported Sleep Quality and Sleep Hygiene in Italian 
and American Adolescents 
Center for Epidemiological Studies 
Depression (CES-D) 
Measuring for 
depression 
See Table 3 below, adapted from: 
Stockings et al. 2015. Symptom screening scales for 
detecting major depressive disorder in children and 
adolescents: A systematic review and meta-analysis 
of reliability validity and diagnostic utility 
Cleveland Adolescent Sleepiness 
Questionnaire 
Measurement of 
sleepiness during a 
typical week 
alpha = 0.89 
Spilsbury et al. 2007. The Cleveland Adolescent 
Sleepiness Questionnaire: A New Measure to Assess 
Excessive Daytime Sleepiness in Adolescents 
SDQ: Strengths and Difficulties 
Questionnaire 
Behavioral screening 
questionnaire 
The internal reliability of the various self report 
scales was assessed using Cronbach’s alpha 
coefficient. This was 0.82 for the total difficulties, 
0.75 for emotional symptoms, 0.72 for conduct 
problems, 0.69 for hyperactivity, 0.65 for prosocial 
behaviour, and 0.61 for peer problems. 
Goodman et al. 2003. The Strengths and Difficulties 
Questionnaire: a pilot study on the validity of the 
self-report version 
Sleep Disturbances Scale for Children 
Gain understanding of 
sleep-wake rhythm 
and any problems in 
sleep behavior 
Internal consistency ranged from .71-.79l test-retest 
reliability r = .71 
Bruni et al. 1996. The Sleep Disturbance Scale for 
Children (SDSC)Construct ion and validation of an 
instrument to evaluate sleep disturbances in 
childhood and adolescence 
3DPAR: Activities Scale 
Measuring activity in 
the 3 days previous as 
a typical activity score 
Interrater and test-retest reliability was 0.99 and 0.98, 
respectively (P < 0.01). The correlation between 
relative energy expenditure from the PDPAR 
(kcal.kg-1.l.d-1) and pedometer and Caltrac counts 
was 0.88 (P < 0.01) and 0.77 (P < 0.01), respectively. 
The correlation between percentage heart rate range 
(HRmax-HR-rest) and mean energy expenditure from 
the PDPAR was 0.53 (P < 0.01). The correlation 
between 1-min heart rates > 50% HRR sustained for 
20 min and the number of 30-min blocks with a 
relative energy expenditure of at least four metabolic 
equivalent tasks (MET) was 0.63 (P < 0.01). The 
PDPAR provides valid and reliable estimates of 
physical activity and also accurately identifies bouts 
of moderate to vigorous activity. 
Weston et al. 1997 Validation of an instrument for 
measurement of physical activity in youth  
 
 

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Questionnaire  
Description 
Study Validity 
Food Frequency Questionnaire 
Measuring typical 
food intake over 
previous seven days. 
The mean correlations, adjusted for measurement 
error, of food groups and nutrients between the FFQ 
and true usual intake were 0·41 and 0·38, 
respectively, with 57 % of food groups and 70 % of 
nutrients exhibiting correlations >0·35. Correlations 
were high for low-fat dairy (0·80), sugar-sweetened 
beverages (0·54), cholesterol (0·59) and saturated fat 
(0·51), while correlations were poor for high-fibre 
bread and cereal (0·16) and folate (0·11). Reliability 
of FFQ intake based on two FFQ administrations was 
also reasonable, with 54 % of Pearson correlation 
coefficients ≥0·5. Reliability was high for low-fat 
dairy (0·7), vegetables (0·6), carbohydrates, fibre, 
folate and vitamin C (all 0·5), but less than desirable 
for low-fat poultry and high-fibre bread, cereal, rice 
and pasta (0·2-0·3).  
Liese et al. 2015 Relative validity and reliability of an 
FFQ in youth with type 1 diabetes 
First described in 2006:  
Mayer-Davis et al. 2006. Search FFQ Dietary Intake 
among Youth with Diabetes: The SEARCH for 
Diabetes in Youth Study 
 
Optional 7 Day Food Log 
Optional record of 7-
day diet to aid in food 
recall. Added per 
participant request to 
help fill out food 
frequency 
questionnaire. 
N/A 
Actigraphy Daily Sleep Diary  
 
Recording 
bedtime/wake time 
during actigraphy. 
Needed to corroborate 
watch collected data 
N/A 
The Reward Based Eating Drive 
(RED) Scale 
To assess the changes 
in reward eating 
behaviors 
Epel, et al. (2014) The Reward-Based Eating Drive 
Scale: A Self-Report Index of Reward-Based Eating 
Youth Quality of Life Instrument-
Short Form (YQOL-SF) 
Self-report measure of 
quality of life in youth 
Validated in Adolescent quality of life, Part II:initial 
validation of a new instrument. (Patrick, et al 2002) 
Used in a diabetes prevention community in Latinx 
teens (Soltero et al. 2018) that used/reported on the 
short-form of the original measure 
Treatment Self-Regulation 
Questionnaire (TSRQ 
To capture the 
motivation for 
engaging in weight 
loss treatment 
Mokhtari et al. (2017)  Motivation and perceived 
competence for healthy eating and exercise among 
overweight/obese adolescents in comparison to 
normal weight adolescents 
Three-Factor Eating Questionnaire 
Measures of eating 
restraint, disinhibition, 
and hunger 
Bryant et al. (2018) Development and validation of 
the Child Three-Factor Eating Questionnaire 
(CTFEQr17) 
 

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Child and Adolescent Social Support 
Scale for Healthy Behaviors 
Survey designed to 
capture the role of 
family involvement in 
dietary habits 
Cullum et al. (2015) A Review of the Child and 
Adolescent Social Support Scale for Healthy 
Behaviors 
Adolescent Binge Eating Scale 
Assessment of 
behaviors related to 
binge eating 
Chamay-Weber et al. (2017) Screening Obese 
Adolescents for Binge Eating Disorder in Primary 
Care: The Adolescent Binge Eating Scale 
 
 

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APPENDIX 4.  WATCHPAT SLEEP RESULTS FOLLOW-UP GUIDELINES 
We anticipate that approximately 30-40% of our participants will have an abnormal apnea hyponea index 
(AHI), requiring some type of follow-up. We have worked with Drs. Ann Halbower, Stephen Hawkins and 
Ben Hughes, our primary pediatric sleep pulmonologists to develop a post-study follow-up algorithm. Of 
note, Dr. Hallbower is currently working with Kaiser to verify the accuracy of the results from the 
WatchPAT device as compared to inpatient polysomnograms in children younger than 12, and is very 
familiar with this device, it’s output and limitations. Our youth fall into a grey zone in terms of what is an 
abnormal sleep study, as pediatric criteria are defined for less than 12, and adult for 18 or older. The 
international accepted clinical criteria are listed below, as well as the American Academy of Sleep 
Medicine’s recommendations of how to handle age 12-17: 
 
Pediatrics: 
 
Mild OSA 
Moderate OSA 
Severe OSA 
1 to 4.9 
5 to 9.9 
>10 
Adults 
Mild OSA 
Moderate OSA 
Severe OSA 
5 to 14.9 + symptoms 
15-30 
>30 
 
 
Since the recommendations are not concrete for the 12-17 year old range, we sought to follow what is being 
done in clinical practice at Children’s Hospital Colorado sleep clinics, were a patient to have an inpatient 
polysomnogram. Sleep studies are read within 5 business days, and we will adhere to this same turnaround 
timeline. In terms of interpretation, currently, the pediatric guidelines are being applied for the 12-17 year old 
age group but 2 is considered normal in this age, and thus our post-study for the 12-17 year olds will follow 
this, and are shown below. Approximately half of the participants have a patient relationship with the PI Dr. 
Green, and thus she can order F/U evaluation if needed, and if not, Dr. Green will request the follow-up be 
arranged by the primary care provider. We have 2 algorithms by age. 

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For participants 12-17 years of age: 
 
 
 
 
For participants ≥18 years of age: 
 
 
 
 
       
 
 

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