Clinical trial NCT02157974
Conditions: Hepatic Steatosis Polycystic Ovarian Syndrome Obesity
Sponsor: University of Colorado, Denver
Start date: 2014-08-01; Completion date: 2022-12-01
Enrollment: 92 participants; Allocation: NON_RANDOMIZED; Intervention model: SINGLE_GROUP

Brief summary:
Women with polycystic ovarian syndrome (PCOS) have increased rates of hepatic steatosis compared to weight similar women with regular menses. It is unclear if this is related to high testosterone or insulin resistance. The investigators will assess hepatic glucose release, rates of lipolysis and hepatic de novo lipogenesis in the fasted and postprandial state to determine if alterations in the processes contribute to hepatic steatosis. Participants will be overweight, sedentary girls with or without PCOS. Those with PCOS will either be medication naive, or must be taking metformin or combined oral contraceptives (COCPs) for a period of at least 6 months prior to study procedures.

Detailed description:
Hepatic glucose release will be assessed with a stable isotope glycerol tracer, lipolysis with a glycerol tracer, and hepatic de novo lipogenesis with an acetate tracer. Data will be collected fasting and after a glucose challenge. The degree of hepatic steatosis and abdominal fat partitioning will be assessed with Magnetic Resonance Imaging (MRI), and total body composition with Dual-energy X-ray absorptiometry (DEXA).

Arms: arm 1: PCOS, medication naive; 10 girls with PCOS will receive 2 doses of Byetta. arm 2: Up to 25 girls without PCOS arm 3: Up to 45 girls with PCOS with no exposure to hormone therapy or metformin in the preceding 6 months. arm 4: Up to 10 girls with PCOS and 6 months of therapy with combined oral contraceptives (COCPs) prior to study procedures. arm 5: Up to 10 girls with PCOS and 6 months of therapy with metformin prior to study procedures
Interventions: intervention 1: Byetta 5Mcg Pen Injection
Intervention descriptions: intervention 1: 10 participants will receive 2 doses of Byetta, one at 7 PM the night prior to metabolic study and the second 30 min before ingestion of glucola

Protocol documents:
Prot_SAP_000.pdf: 
Study Title: 
 
Assessment of hepatic glucose and fat 
regulation in overweight adolescent girls 
(APPLE Study) 
 
Protocol Version Date: 11/26/2018 
 
NCT02157974 


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w 
COMIRB Protocol 
 
PROTOCOL #: 
 
14-0542 
 
COLORADO MULTIPLE INSTITUTIONAL REVIEW BOARD 
CAMPUS BOX F-490    TELEPHONE:  303-724-1055  Fax:  303-724-0990 
 
 
 
Project Title: 
Assessment of hepatic glucose and fat regulation in overweight adolescent 
girls 
 
 
Version Date: November 26th 2018 
 
 
Principal Investigator:  Melanie Cree Green, MD, PhD  
Mentor: Kristen Nadeau, MD, MS 
 
 
 
 
  
 
I. Hypotheses and Specific Aims:   
 
HYPOTHESIS:  
Obese girls with PCOS, when compared to obese controls, will have hepatic and adipose insulin 
resistance (IR) that is not overcome with endogenous insulin secretion following an oral glucose 
(OG) load. IR will be worse in girls taking oral contraceptives, and improved in girls taking 
metformin. Hepatic IR and fat will relate to a failure to suppress hepatic gluconeogenesis and utilize 
carbohydrates during feeding.  
 
Specific Aims: 
Aim 1: Optimize novel minimally-invasive physiologic methods to study liver and adipose IR.  
Rationale: Our preliminary pediatric PCOS data show high rates of NAFLD, altered interactions 
between hepatic and adipose IR during IV studies, and abnormalities during an OG load. A single 
less invasive protocol to synthesize these findings is needed, and this is a novel approach.   
Methods: 1) Optimize a combined oral glucose and stable isotope tracer model to assess hepatic 
and adipose IR, while simultaneously accounting for post-prandial hormone changes. This aim will 
be accomplished with completion of the first 15 subjects, who may or may not have PCOS. 
Aim 1A: Develop a mathematical model to describe dynamics of glucose and glycerol during an 
OG and quantify key aspects of liver & adipose metabolism 
Rationale: Tissue specific IR is detectable following an OG that includes stable isotope tracers, but 
mathematical modeling is required to correctly interpret data collected before and after an OG load.  
Methods: In the initial 6 subjects we will 1) Develop a differential equations-based mathematical 
model of glucose and glycerol dynamics and fit to data computed from stable isotope tracer data.  
2) Analyze model to characterize tissue specific IR in PCOS subjects.  3) Develop a model of 
glucose passage through the gastrointestinal tract to assess glucose absorption in PCOS subjects. 
Aim 1B: Compare the OG glucose and glycerol rates of appearance to hyperinsulinemic 
euglycemic rates of appearance 
Rationale: We need to assure that the new method produces rates of appearance (Ra) of hepatic 
glucose release and adipocyte free fatty acid release that are within range of those calculated 
following the hyperinsulinemic euglycemic clamp which is currently accepted as the gold standard 
method to assess IR using only an IV approach. 
Methods: An additional 9 subjects will be enrolled and studied with the OG model. Data from the 
raw suppressed glucose and glycerol Ra, insulin concentration required for 50 percent of peak 
suppression and percent suppression of basal Ra will be compared between OG subjects and a 
matched cohort of girls whom have already undergone hyperinsulinemic euglycemic clamps with 
identical glucose and glycerol tracer infusion rates. 
Aim 1C: Optimize timing of data collection with 31P-MRS in the liver related to a glucose load. 
Rationale: Studies in adults have shown that glycogen synthesis increases by 1 hour post glucose 
load. A preliminary step to glycogen synthesis or hepatic glucose utilization is passage of glucose 


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through glucose-6-phosphate (G-6-P). Increased concentrations of G-6-P should be detectable 45 
min after a glucose load. 
Methods: We will collect 31Phos data from the liver when subjects are fasted, and 45 min following 
an 75 gram oral glucose load. 
 
Specific Aim 2: Test the impact of oral glucose on hepatic and adipocyte insulin sensitivity in 
PCOS, and the effect of common medical therapies metformin and oral contraceptives on hepatic 
and adipose insulin sensitivity 
Rationale: Insulin sensitivity is traditionally assessed with IV only methods. However these to not 
allow for the contributions of gut hormones, and are non-physiologic. We have previously found 
significant decreases in hepatic and adipose insulin sensitivity in girls with PCOS and need to 
determine if this is also seen following a glucose load, as this would be reflective of daily life. 
Further, the effect of current therapies has not been assessed with this method. 
Methods: Perform OGTT’s with tracers in obese girls with and without PCOS, and girls with PCOS 
who have been taking oral contraceptives or metformin. 
Specific Aim 3: Determine the role of hepatic and adipose IR in the rate of hepatic de novo 
lipogenesis (HDNL) and the effect of common medical therapies metformin, oral contraceptives 
and GLP-1 receptor agonists on HDNL 
Rationale: Studies indicate that increased HDNL is the primary cause of increased liver fat. Our 
preliminary data demonstrates that hepatic fat is 3 times elevated in girls with PCOS compared to 
weight similar controls.GLP-1 concentrations are lower in girls with PCOS and hepatic steatosis. 
Methods: HDNL will be assessed in the fasted and fed state utilizing an acetate tracer in a subset 
of subjects. Rates of HDNL will be compared to measures of hepatic and adipose insulin 
sensitivity.  
 
II. Background and Significance:  
a) Overall background: 
Polycystic Ovarian Syndrome (PCOS) affects 6-10% of U.S. women, with an estimated 
economic burden of $4 billion, and is increasing in prevalence in parallel with the obesity 
epidemic1,2. PCOS includes elevated androgens, and higher rates of insulin resistance (IR), type 2 
diabetes (T2D), nonalcoholic fatty liver disease (NAFLD) and cardiovascular disease (CVD) 3-6. The 
increasing rates of obesity-related PCOS are a major contributor to the earlier onset and rising 
incidence of T2D, NAFLD and CVD3-7. Current PCOS therapies are marginally efficacious, and one 
of them, oral contraceptives, may adversely affect CVD risk 8-14. Despite the high prevalence and 
serious morbidity associated with PCOS, a gap exists in the current therapeutic options.  
b) Mechanisms of insulin resistance in PCOS 
Alterations in hepatic metabolism may be central to IR and cardiometabolic disease in PCOS. 
An estimated 50-70% of obese women 
with PCOS have NAFLD, compared to 
20-30% of obese women without PCOS 
7,15. Furthermore, obese women with 
PCOS and NAFLD are more IR than 
those without NAFLD, indicating a link 
between NAFLD and worsening IR in 
PCOS 4. Liver enzyme concentrations, as 
a marker of NAFLD, independently predict 
dysglycemia and T2D onset16, and a new 
NAFLD medication decreased 
dysglycemia17, arguing for a tight 
connection between glycemia and liver 
health. In obese non-PCOS youth, 
NAFLD correlates with adipose, hepatic 
and muscle IR, is worsened by fructose 
consumption, and reversed by weight 
Figure 1: Proposed Mechanism of NAFLD in 
PCOS 
Mechanisms influenced by hyperandrogenism and unique in 
PCOS vs. T2D are in grey. Items with an * will be studied in this 
grant. HDNL = hepatic de novo lipogenesis, VAT=visceral adipose 
tissue, FFA=free fatty acids, GLP-1=Glucagon Like Peptide 


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loss18-21. Animal models of primary hepatic IR demonstrate the causal role of hepatic dysfunction in 
the development of NAFLD, T2D and CVD 22,23. IR in muscle, liver and adipose tissue are reported 
in PCOS adults24-27, and we found the same in girls with PCOS, despite their young age. The 
synergy between obesity and hyperandrogenism relate to alterations in fat metabolism, 
inflammation, NAFLD and IR in PCOS adults28-30. Our proposed mechanism for development of 
excess liver fat from adipose and hepatic IR is shown in Figure 1. An understanding of the complex 
physiology between tissue-specific IR, hepatic fat and androgens in PCOS is lacking. Addressing 
this gap is my intermediate goal and is an aim this protocol. 
 
c) Current research methods to assess IR      
Research methods to assess early tissue-specific IR require complex protocols and few 
models exist which incorporate oral feedings31-33. Current data for evaluating both hepatic and 
adipose IR have employed intravenous (IV) approaches, which fail to account for contributions of 
hormones such as glucagon, glucagon like peptide (GLP-1) or leptin, which may play a crucial role 
in hepatic and adipose signaling20. Further, the maximum exogenous insulin doses that can be 
administered safely IV often produce lower serum insulin concentrations than generated 
endogenously following an oral glucose load, and the prolonged fasting required for IV studies is at 
the limit of tolerability in youth. Thus, a gap exists in the methodology to assess hepatic glucose 
metabolism and adipose IR in a comprehensive, minimally-invasive, yet physiologic setting. 
Addressing this gap is the first step towards addressing my long terms goals and is a focus of this 
protocol. 
 
A. Preliminary Studies/Progress Report:   
SA1: Optimize novel minimally-invasive physiologic methods to study liver metabolism  
     SA 1 Pilot Data on Liver fat and Insulin Resistance in youth with T2D: In our recent adolescent 
studies,       
we examined the differential tissue-specific 
expression of IR in youth with T2D and PCOS 
utilizing a sophisticated 3 stage hyperinsulinemic 
euglycemic clamp with multiple stable isotope 
tracers. By contrasting patterns of pathology 
between PCOS, obese controls and T2D we are 
uncovering subtle differences suggesting unique 
mechanisms of hepatic IR in PCOS. T2D youth 
appear similar to T2D adults in having adipose, 
hepatic and muscle IR. However, the IR is more 
severe than in adults, with unexpectedly progressed 
markers of CVD and NAFLD at diagnosis despite 
their young age 34. Adipose IR in our T2D youth is 
exhibited by a persistent glycerol rate of 
appearance (Ra) despite very high induced insulin 
concentrations (200 mU/L) and hepatic IR is 
demonstrated by a doubling in the insulin 
concentration required for 50% suppression of 
glucose Ra (IC50 Ra)(Figure 2A). This IR translates 
to persistent elevations in serum free fatty acids 
(FFA) and glucose in these individuals. Elevated 
hepatic fat content (Figure 2B) correlates with both 
elevated serum FFA concentrations and glycerol Ra 
in T2D, indicating that hepatic steatosis is related to adipocyte IR. Muscle IR, assessed by glucose 
clearance rate, is very tightly correlated with both glycerol Ra and serum FFA concentrations 
during hyperinsulinemia, indicating that adipocyte IR may impact muscle IR as well. Finally, our 
T2D youth have muscle mitochondrial dysfunction as assessed with 31P magnetic resonance 
spectroscopy (MRS), which relates to muscle IR. 
Figure 2: Hepatic Measures in Girls 
A   Hepatic Insulin Resistance 
B    Hepatic Fat Percentage 


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Pilot Data on Liver fat and Insulin 
Resistance in youth with PCOS:       
Obese girls with PCOS have 
moderate adipose, hepatic (Figure 
2A) and muscle IR relative to obese 
controls, but are not as IR as T2D 
girls. Adipokines such as adiponectin 
are similarly low in PCOS and T2D 
girls, whereas leptin is higher in 
PCOS than even T2D (Table 1). 
Unlike T2D, muscle mitochondrial 
function is not impaired in PCOS 
compared to obese controls. PCOS 
girls have increased hepatically-
derived serum triglycerides, liver 
enzymes as elevated as in T2D, and 
MRI-assessed rates of hepatic steatosis even higher than in T2D girls (Figure 2B). Hepatic fat in 
PCOS girls is weakly associated to markers of overall lipolysis, but is more closely related to 
visceral adipose tissue (VAT) and plasma markers of hepatic de novo lipogenesis (FFA n7, Table 
1). Further, during IV-induced hyperinsulinemia during the clamp, girls with PCOS have lower rates 
of carbohydrate oxidation than obese controls (0.013±0.0001 vs. 0.017±0.002 mmol/min/kg), 
indicating preferential storing of glucose as evidence of metabolic inflexibility. Finally, markers of 
CVD, including carotid plaque development and exercise tolerance are similar between PCOS and 
T2D, indicating that PCOS status may be a significant risk factor for CVD even in youth. In 
summary, in youth with PCOS, excess liver fat may relate to adipose IR, hepatic IR and metabolic 
inflexibility with increased de novo lipogenesis from carbohydrates. Targeting improved 
understanding of these pathologic processes in a physiologic model is a logical step for reducing 
progression of T2D, CVD and NAFLD in PCOS youth. 
      Preliminary data from oral glucose tolerance 
tests in girls with PCOS: 
Obese girls with PCOS have high 2 hour 
oral glucose tolerance test (OGTT) glucose and 
insulin concentrations (Figure 3 A and B). Of note, 
the PCOS OGTT insulin concentrations are higher 
than those achieved during the clamp 245±40IU/ml). 
Despite the relative hyperinsulinemia, they have 
poor insulin secretion relative to IR, i.e. a lower 
OGTT-derived oral disposition index (oDI) vs. obese 
controls (Table 1) indicating the presence of both β-
cell dysfunction and IR. Girls with PCOS and an oDI 
of <1, an established risk for T2D35, have half the 
insulin sensitivity vs. those with an oDI of >1 
(5.3±2.1 vs. 10.8±0.8 mg/kg lean/min). Thus it may 
be that inadequate insulin secretion during an OGTT 
allows for persistent lipolysis and gluconeogenesis, 
which increases hepatic fat content and worsens 
muscle IR. GLP-1 and leptin, both hormones 
affected by oral ingestion, may be involved in this 
physiologic interplay, which is completely ignored 
with hyperinsulinemic clamp IV glucose delivery. 
The combination of isotope tracers and oral nutrient 
delivery is required to answer this critical question. 
This data is one of the primary reasons that we need 
to move to an oral model.  
 
 
Table 1: Serum Markers in Obese Girls 
 
Control 
PCOS 
T2D 
Number 
19 
38 
40 
AST (IU/L) 
19±2 
36±2 
32±2 
ALT (IU/L) 
29±3 
38±3 
40±2 
Triglycerides (mg/dl) 
78±8 
129±7 
207±7 
Adiponectin (ng/dL) 
8.4±0.9 
5.8±0.4 
5.7±0.1 
Leptin (ng/dL) 
37±6 
43±3 
36±1 
CRP (ng/dL) 
1.7±1.4 
4.3±0.6 
4.1±0.1 
HbA1C (%) 
5.1±0.2 
5.4±0.3 
7.2±0.4 
Oral Disposition Index (oDI) 
5.2±2.2 
3.2±0.5 
N/A 
Fasting FFA (mmol/l) 
523±37 
629±22 
N/A 
FFA n7 (nmol/g) 
29±13  
45±15 
N/A 
Figure 3: OGTT Plasma Results 
A Serum glucose concentrations 
B Serum Insulin Concentrations 
 
 


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Data and methods to study hepatic glucose use: 
Hepatic glucose utilization may relate to hepatic de novo lipogenesis (HDNL). Liver glucose uptake 
occurs via the Glut-2 glucose receptor and is concentration dependant. Thus girls with increased 
glucose following an oral glucose load may preferentially have increased HDNL from increased 
glucose supply. It is unknown if there is a decrease in gluconeogenesis with a shift towards 
lipogenesis as another possible contributor to increased hepatic fat in PCOS. Hepatic glucose 
uptake can be demonstrated with either increased glycogen synthesis, as assessed with carbon 
MRS, or via increased glucose-6-phosphate. Increased glycogen synthesis has been demonstrated 
in subjects with insulin resistance. Hepatic phosphomonoesters (PME) largely represent G-6-P,and 
are detectable with 31Phosphorus spectroscopy. PME differentiate between patients with and 
without liver disease, change with nutritional manipulation and can be measured with 31P MRS 36,37. 
Specifically, increased G-6-P activity has been shown fasted in adults with NAFLD. Increased G-6-
P change following a nutrient load was demonstrated in healthy patients relative to no change in 
subjects with cancer related liver disease, but has not been examined n NALFD or obesity37. We 
thus propose to study gluconeogenesis non-invasively by combining an oral glucose (OG) load with 
31P MRS, an entirely novel approach. 
 
Preliminary data from polysomnograms in girls with PCOS:                   
Results from the inpatient polysomnograms that were performed in this protocol indicate the there 
is a strong relationship between obstructive sleep apnea (OSA) defined as an apnea hypopnea 
index (AHI) of 5 events per hour or greater and fatty liver disease. Girls with OSA also had higher 
serum triglycerides and glucoses 2 hours after the glucola drink, despite similar BMI’s (Table 1).  
 
Mean AHI 
(apnea/hour) 
BMI 
%ile 
Hepatic fat 
fraction 
(>5.5%= fatty 
liver) 
Serum TG, 
mg/dL (>150 
abnormal) 
2 hour glucose, 
mg/dL (>140 pre-
diabetes) 
AHI 
>5, 
N=13 
14.6±3.5 
98±1 
10.5±2.6 
162±19 
153±6 
AHI< 
5, 
N=13 
1.3±0.4 
97±1 
5.3±1 
110±12 
131±5 
 
Further, at least 50% of the girls studied had mild to moderate obstructive sleep apnea, and a few 
had severe sleep apnea, although there was a bias to performing sleep studies in girls with 
symptoms suggestive of OSA, so this is not a true prevalence estimate from this obese female 
population. Clinically, from the PI’s PCOS clinic, approximate 1/3 of obese girls with PCOS are 
requiring continuous positive airway pressure therapy for OSA. Thus, it may be that the presence 
and severity of sleep apnea is a confounder for our primary outcome of altered hepatic metabolism. 
Accordingly, we need to quantify the presence and severity of OSA in all of our subjects to be 
enrolled in 16-2399. 
Traditionally, inpatient overnight polysomnograms are performed in youth, to determine 
OSA. However, through our experience in performing these in this protocol, these can only be 
performed at Children’s Hospital Colorado on Thursday nights, which greatly limits scheduling and 
enrollment. Additionally, whereas the price for these was $500 a piece, this has now been 
changed, and is currently nearly $2,000 per patient, as a research price. Due to these limitations, 
we have sought alternative means of quantifying the degree and severity of OSA.  
A wearable sleep device, WatchPAT, designed for home use has been utilized for the last 
5 years clinically in adults. In July of 2016, the FDA approved the use of this device down to the 
age of 12 specifically for the oxygen saturation and apnea hypopnea index, but not the respiratory 
disturbance index (>17 years only). We have included the device company provided general 
description of the device, as well as the descriptions of limitation and use with individuals 12-17 
years. As many of the limitations of pediatric use pertain to placement and utilization of the device, 


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we will perform the sleep study while the patients are having their already planned and approved 
overnight stay in the hospital, and will have the device placed by our study personnel, who will be 
trained in correct placement and interpretation. Our study team also has extensive experience with 
similar devices from this company, and they are a supportive and easy to work with company. 
Known obstructive sleep apnea or treatment with CPAP will not be an exclusion criteria, 
however, those individuals already utilizing CPAP at home will be requested to bring it for 
admission and use it during the WatchPAT study, so that we capture their typical home sleep 
patterns. 
 
 
III. Research Methods 
 
A.  Outcome Measure(s):   
Primary Outcome Measure(s): Insulin concentration suppressing 50% glucose Ra (IC50 glucose 
Ra) during OGTT. 
Secondary Outcome Measure(s):  Hepatic PME/ATP ratio change after OG load, Glycerol Ra 
during OGTT, Hepatic fat fraction from MRI, Rate of hepatic de novo lipogenesis. 
Likely contributors to above measures: Lipid/glucose markers:(fasting C-peptide and lipid panel, 
HbA1c); hepatic markers:(c-reactive protein, glucagon, GLP-1 and leptin at baseline, 5 min and 30 
min post glucose load to asses change with OG, adiponectin, AST, ALT, GGT), sex-
steroids:(DHEAS, free and total testosterone, sex hormone binding globulin, progesterone, 
estradiol); Body size and composition: (BMI, waist/hip ratio, DEXA, hepatic visceral fat via MRI38-
41), Whole body fat oxidation at rest and following glucose ingestion as measured with a metabolic 
cart; metformin, OCP, Physical activity/ diet:(accelerometer, activity survey (3DPAR); Food 
frequency survey).Questionnaires for presence of obstructive sleep apnea. Questionnaires for 
perceived mental strengths and difficulty (note there is no assessment of suicidality on this tool). 
Obstructive Sleep Apnea, to be assessed with an overnight sleep study via WatchPAT 
 
B. Description of Population to be Enrolled:   
60 obese adolescent females with PCOS will be studied compared to 20 obese non-PCOS 
controls.  This is number of studies needed to be completed statistically, thus more 
subjects may be enrolled, to allow for screen failures and dropouts. Total enrollment will be 
up to 105 subjects. 
 
Ethnic Categories 
Gender 
Females 
Males 
Total 
Hispanic or Latino 
27 
0 
27 
Not Hispanic or Latino 
78 
0 
78 
Ethnic Categories: Total of All Subjects 
105 
0 
105 
  
Racial Categories 
 
 
 
American Indian/Alaska Native 
3 
0 
3 
Asian 
4 
0 
4 
Native Hawaiian or Other Pacific Islander 
3 
0 
3 
Black or African American 
20 
0 
20 
White 
75 
0 
75 
Racial Categories: Total of All Subjects* 
105 
0 
105 
Inclusion Criteria: 
1) Female 
2) Ages 12-21 


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3) Sedentary- less than 3 hours of moderate (jogging, swimming etc) exercise a 
week. 
4) For PCOS groups: (NIH definition) irregular menstrual cycles at least 1.5 years 
after menarche and either clinical evidence of hyperandogenism or elevated 
Testosterone (above the norms for age/tanner stage) at time of screening or 
documented prior to initiation of therapy for OCP and metformin groups. 
5) For PCOS groups: patients un-treated or currently treated with either Metformin or 
OCP’s for at least 6 months. 
6) For non-PCOS groups: regular menstrual cycles and no clinical evidence of hyperandrogenism 
7) BMI equal or greater than the 90th percentile for age and gender 
 
Exclusion Criteria: 
1. For non-treated PCOS and non PCOS group: Use of medications known to affect 
insulin sensitivity: oral glucocorticoids within 10 days, atypical antipsychotics, 
immunosuppressant agents, HIV medications, oral contraceptives- Subjects may 
withdraw from oral contraceptive (OCP's) to meet the inclusion criteria only if a good 
pregnancy prevention plan can be created. If they are CHC patients, and have 
documented abnormal testosterone concentrations in our computer system prior to 
starting OCP's, they may screen while on OCP's.  They must have been off the 
medication for at least 6 months prior to doing the MRI.  
2. For Treated PCOS groups: Use of medications known to affect insulin sensitivity 
other than Metformin or OCP’s: oral glucocorticoids within 10 days, atypical 
antipsychotics, immunosuppressant agents, HIV medications 
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, sex and weight > 300 lbs 
7. Anemia, defined as Hemoglobin < 9 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. Liver disease other than NAFLD or AST or ALT >150 mg/mL  
12. History of renal disease 
13. History of clotting disorders or Warfarin use 
 
Rationale for Inclusion of Non-PCOS Subjects  
Obese adolescents are at risk of T2D and its complications, reduced exercise capacity, 
increased liver and visceral fat, muscle dysfunction and cardiovascular dysfunction therefore the 
tests performed screening for each of these problems provide useful information to these subjects. 
Likewise, sedentary subjects of any weight are at increased risk of reduced bone mineral density 
(BMD), T2D, reduced exercise capacity, increased liver and visceral fat, muscle dysfunction and 
cardiovascular dysfunction. Therefore, all of the subjects may benefit from the results of DEXA, 
glucose testing and exercise prescription and dietary counseling.  
 


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C. Study Design and Research Methods   
 
 
 
The study consists of 3 visits - 2 outpatient visits and one overnight inpatient visit. Inpatient visit will 
last approximately 24 hours, outpatient screening visit approximately 3 hours, outpatient MRI visit 
approximately 2 hours. The 3 visits would be completed within 4 months time. 
 
Overall enrollment procedure plan per group 
 
Test 
Group 
Screening 
MRI/MRS 
Overnight 
stay 
Glucose and 
Glycerol 
tracers  
DEXA 
Overnight 
acetate tracer 
GLP-1 
Injection 
(Byetta) 
PCOS 
40-50 
50 
50 
50 
50 
30 
10 
PCOS w/ 
Metformin 
10-13 
10 
10 
10 
10 
5 
0 
PCOS w/  
OCP’s 
10-12 
10 
10 
10 
10 
5 
0 
Obese 
20-30 
20 
20 
20 
20 
10 
0 
Total 
Up to 105 
90 
90 
90 
90 
50 
10 
 
VISIT 1 (SCREEN VISIT) 
Pediatric CTRC Outpatient unit:  Participants will begin with a medical screening and physical 
exam.  During this visit, patients will review and complete consent documents, have demographics 
Study Calendar 
Visit 1 – 
Screen 
Visit 2 –MRI 
Visit 3 – 
Overnight 
Consenting and Eligibility Assessment 
X 
 
 
History & Physical 
X 
 
 
Intravenous Blood Draw 
X 
 
X 
Finger Stick Blood Draw 
 
X 
 
Urine Pregnancy Test 
 
 
X 
Accelerometer Teaching  
X 
 
 
Gut Bacteria Collection 
 
 
X 
Questionnaires- SEARCH Food frequency, Activity 
3DPAR, Strengths and Difficulties, a sleep diary, 
and Sleep assessments 
 
 
X 
Sleep Study with salivary melatonin collection 
(pending scheduling) 
 
 
X 
WatchPAT sleep study 
 
 
X 
DEXA Scan  
 
 
X 
Glucose, Glycerol and Acetate Tracers 
 
 
X 
Study Drug (Byetta) GLP-1 Injection 
 
 
X 
EndoPat and Dynapulse 
 
X 
 
MRI of abdomen and liver, P MRS of Liver  
 
X 
 
Metabolic Cart 
 
 
X 
Total Time of visit (approximately) 
3 Hours 
2 Hours 
24 Hours 
Location of Visit 
CHCO 
CTRC 
Outpatient 
UCD Outpatient 
Brain Imaging 
Center 
CHCO Inpatient 
Hospital 


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and medical history confirmed, assess allergies and inclusion/exclusion criteria, have blood 
samples drawn, and have anthropometrics completed.  HbA1c, ALT, AST, hemoglobin and 
testosterone samples will be drawn at the beginning of the visit after consent in all subjects.  PCOS 
subjects will have more labs performed for confirmation of PCOS status, if not performed 
previously.  
Screening lab test 
Purpose for test 
HbA1C 
Rule out type 2 diabetes, if > 6.5% subject to be 
excluded 
ALT, AST 
Ensure no severe liver disease, if >4x subject to 
be excluded normal subject is excluded 
Hemoglobin & Hematocrit (part of CBC) 
If subject is Anemic, they will be excluded 
Testosterone  
Test for hyperandrogenism – required to meet 
NIH citeria for PCOS 
Optional PCOS labs 
PCOS status must be confirmed prior to enrollment in PCOS group. 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 
17hydroprogesterone 
Rule out late onset congenital adrenal 
hyperplasia 
DHEAS 
Rule out adrenal tumor 
Prolactin 
Rule out prolactin secreting brain tumor 
 
Accelerometer: At the completion of the screening visit, the subject will be given an accelerometer 
to be worn for the following seven days to measure level of habitual physical activity (MTI Actigraph 
by Actigraph), which affects insulin sensitivity. Accelerometers are effective tools for the objective 
measurement of physical activity 42 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 MTI 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.   
 
VISIT 2 (MRI/CV VISIT) 
Overall Plan: UCD Research MRI (Brain Imaging Center): Subjects will be asked to fast for 4-6 
hours prior to this visit. The imaging will be of the liver. Two different studies may be conducted. 
One will be standard MRI of the mid abdomen to assess the amount of subcutaneous fat, visceral 
fat and percent liver fat. The second type of scan will be 31P spectroscopy to measure the 
concentrations of phosphate molecules including PDE, PME and PCr. This will be done twice, 
before and 30-45 min following a standard 75 gram glucose load. The MRI time is approximately 
an hour in a 2 hour visit. Vascular Endothelial function will be assessed with Endopat and 
Dynapulse, which take approximately 30 minutes. 
 
31P MRS of the Liver and Abdominal Imaging 


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       MRS Data acquisition: Imaging and MRS will be performed on a General Electric (GE) 3 Tesla 
MRI magnet (GE, Milwaukee, WI), upgraded with GE MRS research software. A custom 1H/31P 
abdominal coil will be used for imaging and MRS (Clinical MR Solutions, Brookfield, WI) as in our 
previous 31P work43. The coil will be a concentric probe with an inner coil 16 cm in diameter (for 31P) 
and a 20 cm outer coil (for 1H scout imaging and shimming).  A 2 cm x 2 cm x 2 cm area of focus is 
found in the liver in homogenous tissue for MRS, similar to our previous studies40. A 31P MRS scan 
will then be performed for baseline measurements. We will continue to work with our current 
collaborators Mark Brown, PhD, Assistant Professor, Department of Radiology, UC Denver 
Anschutz and Bradley Newcomer, PhD, Professor, Department of Radiography, University of 
Alabama at Birmingham to optimize the MR signal collection. Subjects will then consume 75 grams 
of glucose and the scan will be repeated every 10 min for an hour. Visceral adiposity will be 
measured using the gold standard of an MRI slice at L4-L5. Hepatic fat fraction will be performed 
using modification of the Dixon method as in our previous studies34. 
 
MRS Data Analysis: For the 31P data, peak positions and areas of interest [phosphocreatine (PCr), 
inorganic free phosphate (Pi), β-ATP(3 peaks), α-ATP(2 peaks), γ-ATP(2 peaks), and PME] will be 
determined by time domain fitting with jMRUi 44,45, utilizing AMARES (A Method of Accurate, 
Robust and Efficient Spectral fitting), a nonlinear least-square-fitting algorithm using our previously 
built prior knowledge files 46. We have utilized this method for muscle 31P analysis for the previous 
6 years, and have extensive experience with this analysis.  Percent PME relative to all other 
phosphate peaks will be calculated before and every 10 min following a glucose load, and percent 
suppression calculated.  The adipose data will be analyzed by Collaborate Ann Scherzinger, PhD, 
as in previous protocols (COMIRB 10-1288). 
 
Sleep Study:  (Based on sleep study technician availability, participant may or may not have the 
sleep study). If subjects have had a previous clinical sleep study, we will collect that appointment’s 
data for analysis. Pediatric CTRC Inpatient unit: Polysomnographic (PSG) Methods and Analysis 
will be performed overnight into visit 3 using pediatric criteria as suggested by the American 
Thoracic Society and standard sleep scoring techniques for evaluating sleep structure and 
respiratory patterns. A polysomnogram will be performed overnight on all subjects, by a trained 
pediatric polysomnographic technician. A sleep study is a routine, noninvasive clinical test. A 
parent will remain with the child throughout testing. During the sleep study, surface electrodes and 
monitoring devices (acquired digitally by Somnoligica, Broomfield, CO) will measure signals from: 
the central EEG, right and left electro-oculogram, surface EMG, ECG, chest and abdominal wall 
motion, pulse oximetry (Masimo, Irvine, California), and end-tidal PCO2 (Novametrix, 
Wallingford, CT). Airflow will be measured by oro-nasal thermistor and by nasal pressure in order 
to obtain a quantitative signal (Protech, Mukilteo, Washington). All studies are monitored with real-
time video for motion analysis and snoring recording. Prior to falling asleep and from 5 AN to noon, 
salivary melatonin samples will be collected once an hour to determine how melatonin offset 
relates to PSG abnormalities. 
 
Gut Bacteria Collection: The gut microbiome consists of the microorganisms, predominantly 
bacteria, that inhabit the gastrointestinal tract and are estimated to outnumber mammalian cells by 
up to a factor of 10, and their genes outnumber human genes by a factor of over 100 [69]. The gut 
microbiota will be collected with BBL culture swabs (Becton, Dickinson and Company, Sparks, 
Maryland) one week prior to visit 3. Fecal samples will be collected from the first bowel movement 
of any day the week before visit 3 and stored in the freezer. Fecal samples are routinely collected 
in research and pose little risk to subjects. For all samples, bacterial DNA will be extracted from the 
swab using established methods and the V4 region of 16S bacterial rRNA will be amplified using 
previously published primers and PCR conditions. [66, 67, 68] To provide a full picture of microbial 
diversity in the gut, we have combined phylogenetic and Operation Taxonomic Units (OTC)-based 
methods for comparing communities. Grouping bacterial rRNA sequences by similarity is important 
for asking questions about which particular species, genera, phyla, etc, contribute to differences 
between samples. To choose OTUs, groups of similar 16S bacterial rRNA sequences are 


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identified, and candidate OTUs are identified as sets of sequences connected to each other. 
Candidate OTUs are considered valid if the average density of connection is above 70% (i.e., if 
70% of the possible pairwise connections between sequences in the set exist. 
 
VISIT 3 (ORAL GLUCOSE TOLERANCE TEST WITH ISOTOPE TRACERS)  
Pediatric CTRC Inpatient unit:  Subjects will be asked not to have caffeine or exercise for 3 days 
prior to this visit. During admission, subjects will be provided with a study diet dinner and snack. 
Patients will be admitted to the Pediatric CTRC for a monitored overnight fast and in select subjects 
an overnight stable isotope tracer infusion. Subjects will be questioned regarding compliance, 
adverse events, changes in concomitant medications and medical history, height, weight, BP, and 
blood sugars.  
A DEXA scan will be performed to assess body composition. A urine pregnancy test will be 
done on all female subjects prior to the DEXA scan.  If a female subject is confirmed to be 
pregnant, she will be withdrawn from the study and referred to her primary diabetes physician for 
follow-up. 
WatchPAT: During the hospital overnight stay, trained study staff will place the WatchPAT 
sleep monitor. The primary measures will be for oxygen saturation and apnea hypopnea index 
(AHI). Each participant will wear the watch with a one-time use finger cuff as recommended by the 
FDA..The watch will be placed by 8 PM, and will be removed the following morning. 
 
The following morning, a blood sample for baseline metabolic labs will be drawn and 
infusion studies with the modified OGTT will be completed.  
Study Drug Injections Exenatide (Byetta: 
A subset of 10 untreated girls with PCOS who are receiving the overnight Acetate infusion will be 
administered two injections of Exenatide (Byetta) (5 mcg) during the in-patient admission for the 
OGTT. The first injection will be administered the night of admission at 6:30-7 PM. 30 minutes prior 
to the study diet containing a fixed macronutrient distribution listed below. The subject  will be 
monitored throughout the night by nursing staff. The second injection will be administered at 
approximately 8 AM, 30 minutes prior to the OGTT load of 75 grams glucose. Following the second 
injection, study staff and CTRC nursing staff will be bedside for the reminder of the patient’s 
inpatient admission at CHCO. Blood glucose measurements will be taken regularly for 6 hours post 
OGTT.  
 
Details of Stable Isotope Tracer studies with an OGTT: After the overnight fast, 
subjects will consume a 75 grams glucose load with an additional 25 grams of fructose to stimulate 
hepatic de novo lipogenesis, and a 1C13 glucose tracer (40 mg/kg) to calculate the rate of 
appearance of exogenous glucose21. 
The stable isotope method to be utilized is an OGTT with IV glucose and glycerol stable 
isotope tracers in all subjects, and acetate tracers in select subjects, based on power analysis and 
cost. For the glucose and glycerol tracers, we will utilize the tracer doses as in our previous 
hyperinsulinemic clamp studies, as these rates successfully detected basal and stimulated glucose 
and glycerol Ra. Whole-body lipolysis will be measured under basal conditions and during the 
OGTT (75 grams glucose + 25 grams fructose + 1C13 tracer), using a primed (1.6 µmol/kg), 
constant (0.11 µmol/kg/min) infusion of 2H5glycerol47. Hepatic IR will be measured under basal 
conditions and during the OGTT, using a primed (4.5 mg/kg), constant (0.03 mg/kg/min) infusion of 
6,6-2H2glucose48. Blood sampling will continue for 6 hours post-OG to allow return to baseline, 
based on our pilot data shown in Figure 2 that 2 hours is inadequate. Blood for tracer analysis and 
glucose and insulin concentrations will be drawn at baseline, every 10 min during the last 40 min of 
the basal period, and every 15 min post OG load. 
The rate of the acetate tracer will be 2 umol/kg/min with no prime, based on previous 
studies 49. The oral challenge study design is shown in Figure 4. Hepatic De Novo lipogenesis will 
be measured in the fasting state, and in the fed state following the glucose load, by analyzing the 
incorporation of the C13 tracer into hepatically secreted lipids such as 16 and 18 carbon long chain 


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fatty acids in VLDL49.  Blood will be drawn at baseline and for the last 4 hours of the basal state 
and the postprandial state.  
 A total of 40 girls will receive the overnight acetate infusion. This total will be 
broken down into 20 un-treated PCOS patients (10 without Byetta and 10 with Byetta), 10 
obese control patients and 10 from PCOS patients treated with Metformin or OCP (5 from 
each group).Analysis of 2H5glycerol,  6,6-2H2  and long chain fatty acid incorporation of 2C13 
Acetate, will be performed by our collaborator Bryan Bergman, PhD,  using a modification of the 
negative ion chemical ionization gas chromatography mass spectrometry as in our previous studies 
49,50.  
Using a metabolic cart and hood, resting VO2 (ml/kg/min) and VCO2 (ml/kg/min) 
measurements (REE) will be collected the morning of the OGTT prior to the start of the OGTT, as 
well as, 30 min after the start of the OGTT and 3 hours and 30 min after the start of the OGTT. An 
additional test may be administered if subject’s glucose levels have not returned to baseline by the 
third metabolic cart test. This is required to determine what portion of ingested carbohydrates are 
being oxidized as opposed to stored, or utilized for HDNL as well as help distinguish between rates 
of oxidative and non-oxidative glucose disposal51. 
 
Study diet: Variations in diet, activity and circadian rhythms affect metabolism 34. Therefore, OGTT 
studies will be performed in the AM fasting, in the follicular phase where possible, preceded by 3  
Figure 4: Proposed Oral Glucose Challenge 
 
The study will have 2 physiologic states. 1. A basal fasted state, and 2. A post-prandial state following a 
glucose load. Hepatic IR is assessed glucose tracers, adipose IR with the glycerol tracer, and Hepatic De 
Novo Lipogenesis with the acetate tracer. 


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days of no strenuous physical activity and a fixed macronutrient, high carbohydrate (65% 
carbohydrate, 20% fat, 15% protein), fixed grams of fructose, Calculated as Females: ([8.365 
 (weight in kg) + 465 (height in m) + 200] X Activity Factor x 1.25), dinner and snack provided by 
the Colorado CCTSI metabolic kitchen (similar to our previous studies34). 1.25 x weight 
maintenance was chosen as this as most similar to our subjects food consumption based on pilot 
subject’s food frequency questionnaires and optimal for detection of hepatic glucose Ra 52. 
Purpose for lab test to be drawn: 
OGTT Labs 
Purpose 
Glucose Tracer 
Determination of hepatic IR 
Glycerol Tracer 
Determination of adipose IR 
Glucose Concentrations 
Determination of hepatic IR 
Insulin 
Determination of hepatic, adipose IR 
FFA 
Measure of lipolysis 
Glucagon 
Gut hormone known to influence hepatic IR 
GLP-1 
Gut hormone known to influence hepatic IR 
Acetate Tracer 
Measurement of HDNL 
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 subject is in the follicular 
phase of cycle, required for publication 
Stored samples for future metabolic studies  
(not DNA) 
Derminiation of potential other contributors to 
altered glucose and fat metabolism 
 
LIFESTYLE PRESCRIPTION 
The final visit will conclude with education regarding the importance of physical activity, diet and 
lifestyle modification to mediate the risks associated with sedentary lifestyle and an exercise 
prescription designed to increase physical activity. The exercise information and prescription are 
the standard of care used in our Children’s Hospital Colorado Pediatric Metabolic Syndrome Clinic, 
designed by the Children’s Hospital Colorado Pediatric Exercise Physiologist. This Exercise 
Physiologist will also be available to the study for consultation as needed.  Families will be provided 
with standard information about follow up care with their primary care provider and contact 
information for Children’s Hospital Colorado Diabetes and Child Health Clinics if needed regarding 
any abnormal study findings. In addition, the subject/family will be provided with copies of their 
study lab results, DEXA scan, physical activity monitoring. Study staff will also call the family within 
6 months of after completion of the study to check-in on the recommended follow up care and 
answer any questions about test results. 
Follow-up from Sleep study results: 
As discussed above, 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 


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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: 
 
 
 
 
       
       
SUBJECT RECRUITMENT/CONSENT/PAYMENT 
1. Subject Recruitment Plan 
Subjects will be recruited from pediatric endocrine, PCOS, Lifestyle, adolescent and gynecology 
clinics, and from the community. We receive 4-8 new PCOS referrals a month, showing the 
feasibility of recruiting the required subjects. Further, we enrolled >100 obese girls in studies in the 
last 30 months. The PI and Co-I’s have a treatment relationship with girls from clinic or subjects 


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can call from study advertisements. Protected health information will only be accessible by study 
investigators. The initial patient contact will be made by personnel who have a treatment 
relationship with the subject. 
2. Informed Consent Plan 
Appropriately qualified and informed personnel who have completed the COMIRB and HIPPAA 
course requirements will fully explain the study protocol and consent form to the subject and 
guardian verbally in the language they understand.  The explanation will be conducted in a quiet 
environment with adequate time given for the subject and guardian to review the study procedure 
before the commencement of the study.  Asking the subject to explain the study in their own words 
will assess the subject’s understanding.  If non-English speaking subjects are enrolled in the study, 
the investigators will adhere to Section 10C of the COMIRB Instructions for Clinical Investigators 
regarding the consent of these subjects.  The qualified personnel mentioned above will then obtain 
written consent from the guardian and assent from the subject, co-signed on the consent form, or in 
subjects who are 18 years or older, direct consent. The PI will make a good faith effort to obtain 
both parent signatures.  The subject and guardian will be provided a copy of the consent form for 
better understanding and record purposes.   
3. Special Consent/Assent Plan 
Consent will be obtained from all participants in the study.  Following explanation, all subjects 
below 18 years old will co-sign the consent form in addition to the parents signing the consent form.  
All subjects age 18 or older will sign the standard consent form. 
4. Subject Compensation, Incentives and Rewards  
Subjects will be compensated with Target gift cards during each scheduled visit. The initial visit 
consisting of informed consent, lab draw and questionnaires and the 2nd for the MRI will result in a 
$50 gift card. The 3rd and final visit consisting of the overnight portion will reward a $100 gift card. 
Compensation for all completed visits will total $200. 
 
D.   Description, Risks and Justification of Procedures and Data Collection 
Tools: 
 
 
1. Blood Sampling 
Description: Blood will be drawn for Complete Blood Count, HbA1c, total and free testosterone, and 
sex hormone binding globulin. If subjects 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 5ml/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 
OGTT visit includes 207.5 ml of blood which will occur within 4 weeks of the initial visit. Thus, our 
OGTT visit is within the NIH Clinical Center guidelines of 9ml/kg in 6-8 weeks and within Children’s 
Hospital Colorado’s institutional guidelines of 5 ml/kg.  In addition, by study design, subjects 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. In addition, we pre-screen subjects with a 
hemoglobin and hematocrit at their baseline visit, and exclude subjects with anemia. 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 subject. 
Finally, our CTRC has a system to track other studies subjects might enroll in, and we ask during 
our consent process if the subject 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 


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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: Two peripheral IV’s will be placed during the OGTT. One will be to infuse the isotopes, 
and the other will be 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.   
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 
subject desires to minimize pain of IV. 
 
3. Oral Glucose Tolerance Test (OGTT): 
Description: An OGTT will be performed with multiple blood draws over 6 hours. The purpose of 
the OGTT is to provide a controlled oral stimulus to effect changes in lipolysis and hepatic glucose 
release. Subjects will also drink a glucose drink as part of the liver spectroscopy. 
Risk: The subjects 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 youth53. Our team of 
investigators, CTRC pediatric research nursing staff and physicians are well experienced with the 
OGTT blood draw procedure. A floor nurse located on the 9th floor of CHC will be available during 
our inpatient visits and patients will be distracted by TV or other similar means during the OGTT, to 
minimize queasiness. 
 
4. Stable Isotope Studies:  
Description: Stable isotope traces of glycerol, glucose and acetate will be utilized to determine 
rates of lipolysis, glucose release and hepatic de novo lipogenesis. 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 isotopes which already exist in all humans, but are simply increasing the 
percentage. The risk would be if these infusions were to become contaminated during preparation. 
These are NOT radioactive substances. 
Justification/Minimization: These isotopes are specially produced to be sterile and pyrogen-free by 
the manufacturer, and in addition, after being reconstituted by the Children’s Hospital Colorado 
Investigational Drug Pharmacy, they are retested for pyrogens and sterility prior to use at the 
University of Colorado CTRC laboratory and the University of Colorado Clinical Laboratory, 
respectively, and discarded if not used in the standard expiration period. This is the standard 
procedure used by numerous studies at the University of Colorado. The stable glucose and 
glycerol isotopes have previously been determined to be usable for research at our institution 
(currently being used in our protocols 06-0665, 07-0988, 10-1288) and others, and an IND is not 
required. To date, we have had no complications from the stable isotope infusions. However, as 
with any infusion, there is the possibility of infection.  


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Sterile and pyrogen-free 2H5glycerol, 6,6-2H2 glucose and  1,2 -2C13 Acetate will be obtained in 
powder form the manufacturer. The CTRC pharmacist will reconstitute the glucose with sterile 
technique, filter (0.22 micron) for additional sterilization, aliquot, and freeze at –20 C in the CTRC 
investigational pharmacy freezer. One aliquot is then sent to the CTRC laboratory for confirmatory 
quantitative pyrogenicity testing and a second aliquot to the Children’s Hospital laboratory for 
anaerobic and aerobic culture to confirm sterility. 
5: Finger stick for glucose measurement during MRI: 
Description: A glucometer will be utilized to measure fasting and 1 hour blood sugars following the 
glucose drink in the MRI. 
Risk: There is a small amount of pain with the finger poke and risk of infection. 
Justification/Minimization: The change in blood sugar needs to be assessed to correlate with changes in 
the glucose-6-phosphate concentrations. The finger will be well cleaned and dried with an alcohol pad. 1 
time use Lancet’s will be utilized to avoid the potential for blood exposure to other patients that has 
occurred with multi-use Lancet devices. 
 
6. Standard Diet  
Description: A dinner and snack will be provided from the CTRC the night prior to the OGTT with 
tracers. The diet will be composed of 65% carbohydrates, 25% fat, and 10% protein at 1.25 daily 
needs and will be will be provided by the CTRC. Risk: None 
Justification/Minimization: 1.25 x weight maintenance was chosen as this as most similar to our 
subjects food consumption based on pilot subject’s food frequency questionnaires and optimal for 
detection of hepatic Ra 52.  
 
7. Magnetic Resonance Imaging (MRI) 
Description: The MRI will usually be obtained the day of admission to CHC, at the UCHSC brain 
imaging center on the Fitzsimmons campus. Debra Singel, a trained research radiographer who is 
supervised by Dr. Mark Brown, of UCHSC radiology, will perform an abdominal MRI to obtain 
hepatic, visceral and subcutaneous fat on a 3.0 T whole–body MRI scanner (GE Medical Systems, 
Waukesha, WI). When this machine is being replaced, the imaging only will be performed on a 
clinical magnet on the Anshutz Campus. Subjects will lie supine while these measurements are 
obtained, need to hold reasonably still during the scan and cannot weigh >275 lbs. In some 
subjects, a specialized phosphorus coil will be utilized to measure the concentration of 31P via MRS 
to calculate glucose-6-Phosphate concentrations before and after the glucose drink. 
Risks: Minimal. Subjects may develop claustrophobia in the magnet. 
Minimizing Risk: The subject is provided with audio protection and optional television to help 
increase comfort. Some subjects might feel claustrophobic while having an MRI and the scan will 
be stopped if it cannot be tolerated. In addition, any subjects with implanted metal cannot have an 
MRI due to the magnet involved. 
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 place movies on the subjects. 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 on the pediatric CTRC 
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 subject 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 OGTT 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.   


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Subjects will be tested for pregnancy immediately prior to DEXA, to ensure that they are not 
pregnant. 
 
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. 
Risk: This procedure may lead to mild discomfort due to the blood pressure cuff being inflated. 
Justification/Minimization: Endothelial function is an novel measure in PCOS and will aid in the 
determination of cardiovascular dysfunction with this population. A trained cardiovascular 
technician will be on hand to witness the procedure.  
 
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:  
Description: Each subject will wear an accelerometer once (MTI Actigraph by Actigraph) to measure 
habitual level of physical activity, which affects insulin sensitivity. The accelerometer will be worn in a 
pouch that rests on the subject’s hip and is positioned upright against the body to measure movement, 
similar to a pedometer. 
Risk: There is no risk involved with the accelerometer. 
Justification/Minimization: Accelerometers are effective tools for the objective measurement of physical 
activity 42 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 
MTI 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.   
 
12: Metabolic Cart: 
Description: The metabolic cart measures the amount of air that the subject breathes in and out. 
The machine attaches to the subject’s mouth through a tube, or a plastic bubble that is placed over 
the subject’s head. There is the potential for experiencing claustrophobia from having the plastic 
bubble over the subject’s head. A metabolic cart will be utilized multiple times during the OGTT 
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 hood over the 
subjects head for approximately 20 minutes. The data collected from the baseline study is also very useful 
for assisting obese subjects 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, 


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answer questions, and to review the form after completion, and captures the last week of dietary 
intake.   
Risk: None 
Justification/Minimization: Several of the measurements being assed are affected by prior nutritional 
intake. Further, subjects 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 screening. 
Risk: None 
Justification/Minimization: Physical activity can directly effect insulin sensitivity, our primary outcome 
measure. The 3DPar is a well validated measure to asses 3 days of physical activity in youth, an 
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 you at risk for depression or anxiety, but 
identify poor coping skills. It does not directly assess depression or suicidality. 
 
16. WatchPAT and Questionnaire to assess for Obstructive Sleep Apnea 
Description: The WatchPAT is a noninvasive portable system that measures  the oxygen saturation 
and apnea hypopnea index. This is a 1 page survey 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. The survey selected is currently being utilized by the NIH multiple center study in obese 
youth at risk for diabetes, and is well validate in youth from multi-ethnic populations.  If OSA is 
suspected during the course of the screen, the subjects 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. Overnight sleep study with salivary melatonin sampling (Pending scheduling availability) 
Description: Stickers will be attached to the head and chest for approximately 11 hours to  measure 
brain activity, oxygen status and breathing overnight. Saliva will be collected hourly while awake. 
Risks: Discomforts include unsettling to sleep away from home in a strange place, and the risk of 
mild irritation from the sticker adhesive. The swab to collect spit can make your mouth feel dry 
when it is first placed. 
Justification: Obstructive sleep apnea is associated with obesity, and can worsen both fatty liver and 
insulin resistance. The presence of OSA is often missed by questionnaires, and can only be assessed 
with a sleep study. The melatonin will provide data on the chemical signaling involved in falling asleep 
and waking up, as they relate to changes seen with the sleep study. Saliva is good alternative to blood 
sampling in pediatric populations. 
 
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. 


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Risk: Although the risk is minimal, subjects 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 sickness. 
 
19: Risk of stopping oral contraceptives 
Description: OCP’s will alter study variables, and cannot be used for 6 months prior to participating 
in the study unless they are enrolled as a PCOS patient treated with OCP’s. If a non-treated 
subject is taking OCP’s and wishes to participate, they will need to stop therapy. 
Risk: 1) Subjects are at risk of having an increase in acne and hair growth, and a potential for some 
irregular menstrual bleeding if these symptoms were present prior to starting oral contraceptives. 2) 
The second risk is to those subjects who are also using the oral contraceptive for pregnancy 
prevention. 
Justification/Minimization: For acne and hair growth, the severity of these changes in the short time 
off the OCP’s is likely to be low, and have a minimal impact on the subjects. Furthermore, these 
symptoms will again subside once the subjects restart their OCP’s. OCP prescribers will be notified 
of subjects’ intent to stop OCP’s for the study. The risk of pregnancy is likely low, as many women 
with PCOS have infertility issues due to anovulation. To decrease the risk further, all subjects who 
are sexually active will be provided with barrier protection in the form of condoms, and will be 
counseled about safe sexual practices. Subjects can additionally have a IUD placed by their 
gynecologist, which is not an exclusion to the study. 
 
20. Study Medication Injection (Byetta) 
Description: When Byetta is administered as a monotherapy for a prolonged duration, the most 
significant adverse events were nausea, vomiting and Dyspepsia. Hypoglycemia was rarely 
encountered. Adverse events were most common at the maximal dose of 10 mcg, and rare at the 
introduction dose of 5 mcg. Every effort will be made to monitor for these adverse events and study 
staff will also monitor for signs of hypoglycemia via glucometer readings. 
Justification/Minimization: GLP-1 receptor agonists such as exenatide (Byetta) aid in secretion of 
insulin and suppress the action of glucagon. Our preliminary data show increased glucose 
concentrations secondary to relative inadequate insulin secretion following an oral glucose load in 
adolescent girls with PCOS compared to their obese control counterparts. Participants in this 
overnight study will only receive 2 doses of this short acting medication, and the well tolerated 
lower risk 5 mcg dose. They will be monitored at CHCO inpatient following the first injection of 5 
mcg of Byetta. Patient will be monitored for excessive vomiting and dehydration overnight. Vital 
signs will be taken overnight every 4 hours by clinical inpatient nurses per CHCO overnight nursing 
policy. The patient will be instructed to alert the night nursing staff if any nausea or dehydration 
occurs overnight and study staff will be notified. During the study day (visit 3), study staff and 
CTRC nursing staff will be bedside monitoring patient from 6AM to 2PM periodically taken vital 
signs and assessing signs of excessive vomiting and dehydration. Glucometer readings will be 
taken at regular intervals for 6 hours after the OGTT to measure blood glucose concentrations. 
Study staff and research nursing will continuously monitor patient until discharge from hospital. At 
the time of discharge, the half-life of Byetta will have expired.  
 
21. 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 subjects 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.   


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E. Benefits of the study: 
Benefits to Society: 
PCOS effects 6-15% of the female population in the US, has an estimated $4 billion economic 
burden and the associated irregular periods, obesity, fatty liver disease and excessive facial hair 
are especially socially difficult for teens. Current treatment options for PCOS are limited. Women in 
their 20's and 30's with PCOS already have evidence of cardiovascular disease and diabetes, 
making adolescent studies, when the disease starts, crucial to understanding disease 
development. Current therapies options are limited and minimally efficacious. This understanding 
will lead to the development of more effective early treatments, before diabetes and cardiovascular 
disease develop. 
Knowledge to be gained: 
A better understanding of how NAFLD develops in girls with PCOS could lead to more effective 
treatment strategies. This understanding could ameliorate development of diabetes and heart 
disease for these girls as they become adults, and may also help with many of the health and 
social difficulties teens with PCOS experience. Since PCOS is one of the most common endocrine 
diseases in the US female population, improving PCOS care could have major health implications. 
We will also begin to learn the effect of the three existing medical therapies. 
Individual: Subjects will benefit from in depth testing for pre-diabetes, fatty liver disease and sleep 
disorders that are not clinically offered. They will receive extensive counseling for both dietary and 
exercise lifestyle changes. Similar subjects who completed a related protocol (10-1288) and 
received this counseling have higher rates of weight loss upon clinical follow-up than those children 
being seen in obesity clinics alone. 
Benefits to participant: 
1) All subjects will be undergoing measures that can identify insulin resistance, hyperlipidemia, 
NAFLD or early cardiovascular disease. These measures are not typically done within the scope of 
daily pediatric practice, and subjects would likely not otherwise know this information. If they have 
one of these conditions, they will be referred for appropriate follow-up and treatment. If the subjects 
have obstructive sleep apnea, they will be referred to the sleep clinic for appropriate treatment. 
2) All of the subjects enrolled must be sedentary, with less than 3 hours a week of physical activity. 
This is less than the time recommend by the US Preventive Task Force and the American College 
of Sports Medicine for this age group. This lifestyle puts them at risk for several diseases including 
diabetes and cardiac disease later in life, even if they don't have evidence of disease at this time. 
At the end of the study, all subjects will receive counseling on how to increase their activity levels 
by either Amy Baumgartner, MS or Greg Coe, MS, both of whom have degrees in exercise 
physiology, and are experts in providing exercise prescriptions. Increased activity has been shown 
to reverse the risk for diseases later in life. 
3) All subjects will complete a 3 day food questionnaire. This will be reviewed with them, and 
healthier food choices and meal planning will be discussed with both the subject and their parent 
by the PI or PRA, all of who are trained in providing diet prescriptions. Additionally, obese subjects 
will be counseled on a weight loss diet. Over 80% of obese adolescents are obese as adults, if they 
do not change their eating habits and lose weight when they are still a teen. 
4) The participants will benefit from getting a sleep study during their overnight stay at the hospital 
and discovering if they have obstructive sleep apnea. Depending on the results they may be 
referred to sleep clinic for further evaluation of sleep apnea. 
Evidence of Direct Benefit 
We believe that this protocol is in the 405 risk category for pediatric research.  Subjects can benefit 
from the above study measurements. Further, at the conclusion of the study, all subjects in the 


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protocol will also be given counseling on the benefits of exercise as discussed above, and given an 
exercise prescription. The subjects will also be re-contacted once by phone call to follow-up on 
their study results and exercise recommendations. Sedentary subjects will thus gain direct benefit 
from the study through the benefits of specialized counseling and recommendations for increased 
physical activity that would not otherwise be available to them.  
Sedentary and obese adolescents are at increased risk for diabetes, reduced bone mineral density, 
reduced exercise capacity, cardiovascular dysfunction, and increased visceral and liver fat. 
Therefore, obese non-PCOS subjects may benefit from the results of glucose testing, insulin 
resistance assessment, DEXA, cardiovascular measures, and MRI testing. The DEXA scan can 
detect evidence of osteopenia, which is becoming more prevalent in adolescents, especially those 
who are sedentary females. The MRI of the liver can detect early evidence of fatty liver disease. 
The blood tests done for screening can detect alterations in blood glucose, pubertal sex hormones, 
as well as abnormalities in fasting lipids. The risk of all of these endpoints is increased in sedentary 
subjects, especially those who are also obese, and if left untreated can increase long term health 
risk, thus the benefit of detecting any of these would directly impact both the health and the 
longevity of the individual subject. 
Inclusion criteria is less than 3 hours of exercise per week, a level of activity well below that 
recommended by the U.S. Surgeon General for youth (Children and teenagers should exercise for 
1 hour of vigorous physical activity daily and weight-bearing activities that strengthen their bones). 
Numerous studies have linked low activity with development of diabetes, heart disease and insulin 
resistance; all of the end-points which we are studying. Recent studies have shown that sedentary 
lifestyle increases risk of cardiovascular disease and all-cause mortality. Youth in the U.S. now 
suffer from obesity in epidemic proportions, with about 32% of US adolescents currently being 
overweight and 17% being obese.  A sedentary state is an increasingly common problem in the 
U.S., especially for adolescents, as a recent study showed that the most sedentary groups in the 
United States were adolescents and adults over 60 years. Adolescents in this study spent about 
60% of their waking time in sedentary pursuits, making sedentary adolescents a critical group to 
study. The amount of time spent in sedentary behaviors has been independently associated with 
increased risk of weight gain and increased risk of metabolic syndrome, diabetes, and heart 
disease. In light of these links to adverse health outcomes and the continued increase in the 
prevalence of overweight and obesity in the United States, sedentary behaviors have emerged as 
an important target of health promotion and obesity and disease prevention efforts, complementing 
efforts to increase levels physical activity. For this reason, sedentary lifestyle can be considered a 
pre-disease state. 
  Because of their sedentary nature, there are several primary direct benefits to the non-PCOS 
subjects from the OGTT, which justify the risk. The primary benefit from the OGTT would be the 
discovery of pre-diabetes via a non-invasive measurement of glucose tolerance. As mentioned 
before, surrogate measures are unable to detect insulin resistance adequately, and insulin 
resistance is a strong predictor of NAFLD.  Thus, it is very possible that we may find evidence of 
NAFLD in the obese non-PCOS population. Discovery of insulin resistance would enable us to 
recommend education and an exercise and diet plan to treat or prevent further development of 
insulin resistance, diabetes and NAFLD. The direct benefit of the isotopic tracers would be the 
differentiation of whether the liver, and/or the muscle and/or the adipose tissue is involved in the 
insulin resistance. Again, if this were true the individual could benefit from referral to the metabolic 
syndrome clinic or the GI clinic to be followed for evidence of hepatic disease that may need to be 
treated if there is any disease progression. 
WatchPAT results: The patient population is at high risk for OSA. Untreated OSA is 
thought to contribute to worse fatty liver disease and glucose metabolism. By 
identifying that these youth have a problem, they are then in the position to work with 
their physicians to address this, and potentially improve their metabolic health long-
term. Additionally, treatment of sleep disordered breathing is associated with weight 
loss, and this is a goal for all of these participants.  


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F. Alternative Treatment 
The alternative is for subjects to not participate in the study 
 
G. Consideration of Specific Subject Categories 
1. Inclusion of Women 
All subjects will be women, as PCOS only occurs in females. 
2. Inclusion of Minorities 
Every effort will be made to include a diverse subject distribution. PCOS affects Caucasians, 
Hispanics and African Americans equally. 
3. Inclusion of Children 
All subjects will be between ages 12 and 21. Insulin sensitivity needs to be studied in the 
adolescent age group as no data is currently available in this age group and it is critical to 
understand the pathophysiology of PCOS in its developing stages.  
IV. Potential Scientific Problems:   
Limitation of Method Development: 
The protocol as described includes the entire subject set, which allow for comparisons between 
obese girls with and without PCOS. OGTT with tracers will be conducted in all subjects, and 
overnight assessment of HDNL in 20 subjects (10 with PCOS and 10 without). However, data from 
the first 6-12 subjects (both PCOS and non-PCOS) will be immediately evaluated to see if any 
changes are needed in terms of protocol details.  The specific points that are to be evaluated are 
listed below: 
1) OGTT with glucose and glycerol tracers 
a. Purpose: Develop an oral stimulus model to assess hepatic and adipose insulin 
sensitivity. 
i. Question 1.  Is 4 hours post oral load long enough to model hepatic and adipose insulin 
sensitivity?   
1. Rationale: Our preliminary data from OGTT’s in obese girls indicates that subjects’ 
insulin and glucose concentrations have not returned to baseline by 2 hours.  Data 
from adults showed that all non-diabetic adults were back to baseline by 3.5 hours.  
2. Measurement. Serum glucose and insulin will be measured post-glucose load for 4 
hours in the first 6 subjects. If 4 hours post glucose values are within 10 mg/dL for 
glucose and 10 IU/mL for insulin relative to baseline values, 4 hours will be 
considered sufficient.  
ii.  We are revising the protocol to 6 hours based on our results from our first several 
subjects.Question 2: Are glucose and glycerol Ra during the OGTT similar to those 
obtained with a hyper-insulinemic euglycemic clamp. 
1. Rational: Peak insulin concentrations are similar between an OGTT and a clamp, thus 
Ra rates should also be similar. Comparisons in adults of the glucose Ra from clamps 
and an oral meal challenge were similar. 
2. Measurement:  The peak suppressed glucose and glycerol Ra for the first 12 subjects 
will be compared to peak suppressed Ra values calculated from data from 70 girls 
during hyper-insulinemic euglycemic clamps.  
3. Revision plan if model not correct: None. Findings of either similar or different glucose 
and glycerol Ra are valuable information. 
2) Measurement of Hepatic De Novo Lipogenesis (HDNL) 


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a. Purpose: Optimize a model to asses HDNL with minimal blood draws, minimal time 
of tracer intervention and no interference with measures of glucose and glycerol 
Ra 
i. Rational: HDNL can be difficult to measure. Previous studies have used weeks of 
deuterated  water loading or large volume deuterated water overnight.  However 
Deuterated water cannot be used at that same time as other tracers methods. The use 
of 1C13 acetate requires the use of patented MIDA calculations, to which we do not have 
access.  Rate’s of HDNL are low, and glucose loading may be required for detection. 
We will be using 2C13 acetate which will allow us to avoid use of the MIDA calculations. 
We will infuse the tracer for 19 hours, and also precede the tracer study with a dinner 
and snack of 1.25 REE meal, which can increase fasting lipogenesis rates. 
ii. Measurement: Enrichment of long chain fatty acid moieties after 19 hours of infusion 
rate will be measured. 
iii. Revision plan if model not correct: 1) increase the time for overnight acetate infusion or 
2) increase rate of acetate tracer infusion either by giving a prime at that start of 
infusion, or increasing the hourly rate of acetate infusion. 
b. Purpose: Measure HDNL in the fed state concomitantly during the OGTT with 
tracers 
i. Rational: HDNL needs to be measured in the fasted and the fed state. We will be using 
the OGTT to also measure glucose and glycerol kinetics. There is a chance that the oral 
1C13 glucose needed for the glucose model may be incorporated into long chain fatty 
acids via HDNL, and falsely elevated the rate of HDNL calculated. 
ii. Measurement: Measure the enrichment of long chain fatty acids during the OGTT with 
tracers, without the acetate tracer 
iii. Revision plan if needed: Calculate the mean rate of incorporation of oral 1C13 into long 
change fatty acids and develop a correction factor to apply to overall rates of HDNL. 
3) Measurement of Glucose-6-Phosphate(G6P)  in liver before and after glucose load 
a. Purpose: Determine if glucose flux through the liver can be measured with MRS 
Phos 
i. Rational:  Increased rates of HDNL may be related to preferential uptake and utilization 
of oral glucose to the liver. Phosphorylation is the first step following hepatic uptake of 
glucose. G6P can be measured with MRS Phos technologies 
ii. Measurements: The area under the curve of G-6-P will be measured before and 45 min 
after a glucose load. Serum glucose peaks between 30 and 60 min in the majority of 
subjects, which is why this time point was selected. Further, liver studies using carbon 
MRS have demonstrated increased glycogen production 60 min following a oral glucose 
load, and G-6-P is a precursor step for this process. 
 
Overall Project Limitations: 
1) Subject recruitment is always a potential concern, but is minimized by a streamlined 
recruitment system and experience with this population; the previous, more intensive study 
is well tolerated in youth and enrolled faster than projected. In addition, we had >100 new 
PCOS referrals to our pediatric endocrinology department in 2013 alone, and the PI has a 
clinic specifically for girls with PCOS.  
2) Subject drop-out: We expect reasonably good completion rates due to the non-invasive 
nature of the two study visits and our low dropout rate in our previous studies in control 
adolescents using similar procedures. 
 


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V. Data Management and Security Plan 
Data Entry   
Data will be entered from paper forms. Once forms are completed, verified and corrected for 
inconsistencies, they will be manually entered at our site using a computerized data management 
system (Redcaps).  
Edit Checks    
Computerized data validation routines will be used to enhance data quality and verify the accuracy 
of data within predefined value ranges. These checks include, but are not limited to: (a) initial 
screening of data, using logic and range checks built into data entry screens; (b) cross-form 
functional and consistency checks; and (c) edits assessing the serial integrity of data. 
Disaster Recovery    
Routine data backup will occur on data in conjunction with the children’s hospital secure server and 
Redcaps. 
Security and Confidentiality   
All hard copy forms will be de-identified with a study number and filed in a locked cabinet, to which 
only the investigators will have access.  Standard protection against computer hackers is 
implemented. Recovery from natural disasters (water, fire, or electrical) can occur through the 
ability to reconstruct both the database management system and the data from nightly backups.   
VI. 
Data and Safety Monitoring Plan 
The principal investigator and the mentor Kristen Nadeau, MD will monitor the protocol and the 
safety of the research subjects.  The PI will review all laboratory data and report any abnormal 
values to the patient and guardian and instruct the subject to follow-up with their PCP.  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.  There are no other entities that require notification about this protocol.   
 
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 (age, 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. 
A. Adverse Events (AE)  
The hyperinsulinemic euglycemic clamp is a standard procedure used in a large number of 
research studies and settings.  Adverse events are uncommon when the procedure is done by 
experienced personnel in an appropriate setting.   
 
1. Adverse Event Definition 
For the purposes of this study, an Adverse Event (AE) is defined as any untoward medical event 
associated with the use of a drug in humans, whether or not considered drug-related. AEs also 
include any significantly abnormal physical finding identified on examination and any significantly 
abnormal laboratory result obtained on the patient between visits or at the time of the visit. 
Questions answered YES and any new abnormal physical findings are pursued by the study staff in 
order to determine the seriousness of the event and the need for further evaluation, follow-up, or 
referral.   
a) Adverse Event Reporting 


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AEs are reported on a standard form that is completed by the study staff at each regular follow-up 
visit and phone interview.  Adverse events reported or ascertained between clinic visits are 
captured and reported at the time of the next scheduled visit. 
 
Pre-existing conditions (that is, conditions present prior to randomization) are not considered or 
recorded as AEs or SAEs unless the condition worsens in intensity or frequency after 
randomization.  Likewise, continuing adverse events are not reported as AEs at subsequent visits 
unless they increase in severity or frequency between the visits, they result in criteria for an SAE, 
and/or they resolve between visits. 
B. Serious Adverse Events (SAE) 
1. Serious Adverse Event Definition 
Events are divided into those that are not serious (AE) and those that are serious (SAE).  The 
distinction between an SAE and an AE is a regulatory definition established by the FDA, not a 
clinical definition.  The definition of SAE is not always related to clinical severity of the event.  For 
the purposes of this study an AE is considered a Serious (SAE) when it satisfies any one of the 
following criteria: 
 
• 
The event results in an inpatient hospitalization (any overnight stay associated with an 
admission). 
• 
The event results in the prolongation of a hospital stay. 
• 
The event results in permanent or severe disability. 
• 
The event results in death. 
• 
The event is life-threatening. 
• 
Treatment is required to prevent a serious event. 
 
There have been no SAE’s in the research groups experience in the Pediatric CTRC.  We do not 
anticipate encountering SAE’s; however, we have identified the following as possible SAE’s for the 
purposes of monitoring: 
• 
Infection related to blood draw or IV placement 
 
a) Serious Adverse Event Reporting 
Study patients are instructed to contact the clinic with any serious adverse event meeting the 
above criteria.  Each SAE is recorded on the study form and the PI is informed soon as possible 
after they occur and preferably within 24 hours of the notification of the clinic staff.  This notification 
should occur even if data are incomplete.  Additional data and follow-up information are 
documented and sent subsequently as an update to the original report.  The PI immediately 
forwards SAE reports to the Safety Officer and COMIRB and any other required institutional 
monitoring committee.   
            
C. Subject Discontinuation Criteria 
If a subject experiences any of the following, the subject will be withdrawn from the study. 
1. Inability to complete study procedures 
2. Abnormal screening labs (LFT’s >4 times elevate, HbA1C>6.4%, Hg <9 mg/dL) 
3. Subject becomes pregnant during study 
 
D. Protocol Stopping Criteria 
If one or more subjects experience any of the SAE’s listed above, the PI will consult with the 
mentor prior to continuing study visits with subjects.  The mentor and RSA will consult about the 
significance of the SAE’s and make a recommendation to the PI.  
 
VII. Data Analysis Plan:   


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Statistical Plan for Method Development: The previously described tracer protocol and 
mathematical modeling with any refinements will be performed in an additional 9 subjects.  
Outcome measures (raw suppressed glucose and glycerol Ra, insulin concentration required for 50 
percent of peak suppression and percent suppression of basal Ra) will be compared between the 
OG subjects and a matched cohort of girls who have undergone hyperinsulinemic euglycemic 
clamps with identical glucose and glycerol tracer infusion rates.  Subjects in the two groups will be 
matched on PCOS diagnosis, race, percent body fat, percent liver fat, and time post-menarche.  An 
equivalence design will be used to compare the outcomes in the two groups and to demonstrate 
whether there are clinically significant differences in outcomes from the OG model and the 
hyperinsulinemic clamp.  For purposes of sample size calculation, we used estimates from the 
investigator’s pilot study.  Using an equivalence limit of one standard deviation for each outcome 
measure (SD=50 units for glycerol IC50 and SD=39 units for glucose IC50) and a correlation of 0.2 
between outcomes on matched subjects, 15 matched pairs gives 80% power to demonstrate 
equivalence.  70 girls have completed the hyperinsulinemic euglycemic clamps, so adequate 
matching will be possible. 
 
Overall Project Statistical Plan: 
 
Power calculation: The primary outcome is hepatic IR measured as IC50 glucose Ra. Using our 
preliminary data, a two-tailed t-test and an alpha of 0.05, completing 13 controls and 32 PCOS 
provides 86% power to detect a difference of 35 IU/L in the insulin concentration.  
 
Differences between the two primary groups (PCOS, controls) will be assessed with a t test or for 
non-normally distributed data a non-parametric test such as Mann-Whitney U. Secondary 
outcomes include a Pearson correlation test of potentially related variables followed by targeted 
multiple regression to assess the relationship between hepatic IR or hepatic fat and adipose IR, 
hepatic gluconeogenesis, VAT, carbohydrate oxidation, androgens, metformin or OPC use and 
presence of OSA. As an exploratory analysis, the four groups will also be compared using an 
ANOVA. We recognized that we may not be powered for these analyses, but the data will be used 
as hypothesis generating for future studies.  
The rate of hepatic De Novo lipogenesis is a secondary outcome. Based on our preliminary 
data that plasma markers of hepatic De Novo lipogenesis are 40% greater in those with fatty liver 
disease and the use of a high carbohydrate and fructose drink to increase the differences between 
groups,  10 subjects in each group will be needed to detect a difference in hepatic De Novo 
lipogenesis21,49,52. The rate of hepatic De Novo lipogenesis will be correlated with measures of 
hepatic and adipose IR and MRI measures of NAFLD. 
 
Non-Isotope OGTT calculations: The Matsuda model of IR will be used, and B-cell function 
[insulinogenic index (ΔI30/ΔG30) and (ΔC30/ΔG30) and disposition index (1/IFasting × ΔI30/ΔG30) 
and (1/CFasting ×ΔC30/ΔG30) will be calculated40,53-55. 
 
 
     
Proposed Mathematical Model of Glucose and Glycerol Dynamics:  The differential 
equations-based isotope labeled oral glucose minimal model (OMM*) 56 will be adapted to describe 
glucose and glycerol dynamics present in the collected data.  In OMM*, the oral glucose minimal 
model (OMM) is modified to account for labeled glucose oral data obtained with the inclusion of a 
stable isotope tracer.  This refinement allows an estimate of insulin sensitivity which is unique in 
that it accounts for both stimulation of adipose and muscle glucose uptake and inhibition of hepatic 
glucose release.  In OMM*, glucose concentration G and insulin action on glucose disposal X are 
governed by the following equations: 


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where SG* is the fractional glucose effectiveness; Ra(α, t) is the glucose rate of appearance; V* is 
the distribution volume; p2*and p3* are rate constants; I is plasma insulin concentration; and Ib is 
basal plasma insulin concentration.  The Ra in this equation represents a best fit (see Fitting Ra 
below) to Ra values computed from data (see Proposed Isotope Tracer Calculations below).  This 
model will allow differentiation between exogenous and endogenous contributions to changes in 
glucose concentration.  A similar model, modified to reflect the absence of an exogenous glycerol 
contribution from OGTT, will be used to describe glycerol dynamics.  The concomitant study of 
glycerol has been shown to not interfere or contribute to glucose Ra.57 
 
     Proposed Isotope Tracer Calculations: The glycerol and glucose Ra during fasting and the 
entire post-glucose load period will be calculated using the non-steady-state equation of Steele58,59. 
The glucose rate of disappearance (Rd), and metabolic clearance rate (MCR) will be calculated for 
the same time periods 58,59:  
where F represents isotope infusion rate; IE1 and IE2 are isotopic enrichments at sampling time 
points 1 (t1) and 2 (t2), respectively; C1 and C2 are metabolite concentrations at t1 and t2; and V is 
the estimated volume distribution (glucose is 180 ml/kg and glycerol 27 ml/kg). Ra glucose from the 
glucose drink (Ra EXO) and endogenous glucose production (Ra ENDO) will be calculated:  
where ED and EP are the enrichments of the 1-13C-glucose in the drink and plasma, respectively. 
Ra, Rd and MCR will be presented with and without correction for serum insulin concentrations. 
          
 
Fitting Ra:  Using computed data points, glycerol and glucose Ra will be fitted with an 
appropriate model.  In control subjects, Ra typically shows 3 phases in OGTT 60. Cubic polynomial, 
piecewise-linear, spline, and dynamic models have been used for fitting Ra in previous work61,62. In 
a comparison of different models, the choice of model form did not significantly impact the resulting 
estimates of insulin sensitivity or glucose Ra.  Given the expected 3 phase form of Ra, we will 
assume a cubic polynomial model; we will validate estimates obtained with the cubic polynomial 
model by computing comparable estimates using at least one other model form. In previous work 
comparing different models for Ra (piecewise-linear, cubic polynomial, dynamic), results have been 
relatively unaffected by choice of model.  If we find that this is not the case for our data, then we 
would certainly consider the advantages of adopting a more complex approach, such as the state-
space, for describing Ra. 
          
 
Characterization of Liver and Adipose Metabolism through Model Analysis:  The novel 
OMM* model will be used to measure insulin sensitivity and provide a quantitative characterization 
of glucose Ra in the 5 PCOS subjects, thereby providing insight into liver metabolism in these 
subjects.  The following measures will be computed:  mean of Ra; area under the curve describing 
Ra during 4 hour period associated with OGTT; time to peak Ra suppression; and time to return to 
basal Ra post glucose load.  A similar characterization of glycerol Ra in the 5 PCOS subjects with 
be computed using the novel OMM* glycerol model, thereby providing insight into adipose 


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metabolism in these subjects.  In addition, the glucose and glycerol Ra suppression associated 
with OGTT will be compared to those already collected from hyper-insulinemic euglycemic clamp 
glycerol and glucose Ra suppression from an identical subject population to identify similarities and 
differences associated with protocol.  
          
 
Proposed Model of Glucose Absorption:  A key advantage of OGTT over IV glucose 
delivery is the physiologic aspect of oral administration.  When glucose is ingested, the physiologic 
pathway through the gastrointestinal tract contributes to the glucose rate of appearance in plasma.  
Although gastric emptying of liquids occurs exponentially in control subjects, there is evidence that 
gastric emptying may be delayed in certain populations, thereby impacting glucose absorption 63-65. 
In order to assess possible abnormalities in glucose absorption in PCOS, we will relate a model 
that describes mechanisms of glucose transit through the gastrointestinal tract 60 to Ra data 
computed for PCOS subjects.  The three-compartment model for glucose transit through the 
stomach and upper small intestine is described by the following system of differential equations: 
 
where qsto1 and qsto2 are the amounts of glucose in the stomach (solid and liquid phase, 
respectively); δ is the impulse function associated with OGTT glucose delivery; D is the amount of 
ingested glucose; qgut is the glucose mass in the intestine; k21 is the rate of grinding; and kabs is the 
rate constant of intestinal absorption.  The rate of gastric emptying, kempt(qsto), is a nonlinear 
function of the total amount of glucose in the stomach: qsto=qsto1+qsto2.  Then we compute Ra(t) =  f 
kabs qgut(t) where f is the fraction of intestinal absorption appearing in plasma.  Model parameters 
will be determined by fitting to Ra and tracer data.  Since model parameters reflect specific 
physiologic processes, they will provide additional insight into the mechanisms underlying 
abnormalities of glucose Ra in PCOS subjects.  
 
 
Analysis of the sleep study: Respiratory parameters of interest (defined below) include 
arousals, apneas, hypopneas, low oxygen saturation time (SaO2T), hypercapnia time (CO2>50), 
and oxygen saturation nadir (SaO2N). The arousal index (AI) is the number of arousals and 
awakenings measured by a shift of EEG signal to > alpha range for more than 3 seconds, divided 
by total sleep hours as previously defined(57). Apnea is defined as an absence of airflow for two or 
more breath cycles. Hypopnea is a visible decrease in airflow by nasal pressure signal (or by 
thermistor when pressure signal was unavailable) and either an EEG arousal or a drop in oxygen 
saturation of 3% or greater. Mixed apnea is an obstructive apnea combined with a central (absent 
effort) apnea. The AHI is comprised of the obstructive, mixed, and hypopnea events divided by 
total sleep hours; central apneas (seen in normal children)) are not included in the AHI. Intermittent 
hypoxemia, even brief episodes of mild drops from the normal baseline, is hypothesized to be a 
possible cause of neuropsychological dysfunction. SaO2T is the time in minutes with 
oxyhemoglobin saturation less than 95% measured by pulse oximetry in order to detect mild 
intermittent desaturation, (not seen in normal children. SaO2N is the nadir of oxyhemoglobin 
saturation. Hypercapnia time (CO2>50) is time in minutes with an end-tidal CO2 level greater than 
50 mm Hg. Carbon dioxide levels are generally not measured in adult sleep laboratories. The AHI 
in normal non-snoring children has been determined to be less than 1.0, with little hypercapnia 
(sleep time with CO2 > 50 mm Hg, reported to be < 10-20%.There are no established diagnostic 
criteria for mild, moderate, or severe sleep apnea in children. OSA in adults is defined by the AHI: 
the number of apnea or hypopnea (partial obstruction) events per hour. However, continuous 
partial obstruction is underestimated by these criteria, and is considered significant by the Pediatric 
Sleep Program. Therefore, normal participants with evidence of continuous partial obstruction not 
meeting adult criteria for OSA, an AHI >1, or hypercapnia > 10% of sleep time will be excluded. For 


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this protocol, mild OSA is defined as AHI 1–4, moderate OSA as AHI 5–10, and severe OSA as 
AHI greater than 10. 
 
All measures will be adjusted for OSA status (yes/no) and/or severity of AHI. 
 
 
Melatonin offset: Salivary melatonin swabs will be analyzed to determine salivary 
melatonin concentrations. Concentrations will be used to calculate melatonin offset, or the time that 
melatonin decreases for the overnight sleeping peak, to the day time low. Offset will be determine 
relative to arousal time, and calculated in minutes. 
 
VIII.  Summarize Knowledge to be Gained:  
Overall Expected Results:  We anticipate that we will develop 2 new (OGTT with tracers and liver 
31 MRS Phos) novel minimally invasive ways of assessing hepatic metabolism in a physiologic 
state. We will delineate the relationship between adipose lipolysis suppression, hepatic glucose 
release, and hepatic fat. We expect that PCOS girls will uniquely have glucose Ra and hepatic fat 
excess driven by hyperadrogenemia, and that IR will be improved in girls taking metformin but not 
girls taking OCP’s. In addition to our protocol, these models can be utilized in other patient 
populations with IR and/or obesity. Further, if Glucagon or GLP-1 have a role in hepatic IR or 
HDNL, there are existing available therapeutic choices that later these gut hormones. This data 
would then provide the background evidence for future therapeutic trials. 
 
Significance: Recent evidence in adults with PCOS indicates that 60-70% have NAFLD7,15. Our 
data shows that > 45% of obese girls with PCOS have NAFLD, likely an underestimate due to 
exclusion criteria of T2D and weight >250 lbs in the previous protocol. The majority of NAFLD 
studies have focused on males, and none have been performed in multi-race cohorts of girls with 
PCOS, despite this high prevalence. NAFLD has been described as the primary driver of 
worsening metabolic syndrome and CVD in obesity across populations, and can progress to 
cirrhosis and liver failure5,6. Thus, understanding the relative contributions of adipose and hepatic 
IR to NAFLD is critical to designing strategies to improve the long term health of these girls and 
reduce their risk of T2D, CVD and liver failure. 
 
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