Oral Amino Acid Nutrition to Improve Glucose Excursions in PCOS
Phase: NA
Conditions: Polycystic Ovarian Syndrome, Obesity, Hepatic Steatosis
Sponsor: University of Colorado, Denver

Study Title: 
 
Oral Amino Acid Nutrition to Improve Glucose 
Excursions in PCOS (ORANGE Study) 
 
Protocol Version Date: 06/09/2020 
 
NCT03717935 
 
 

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w 
COMIRB Protocol 
 
PROTOCOL #: 
18-0803  
 
COLORADO MULTIPLE INSTITUTIONAL REVIEW BOARD 
CAMPUS BOX F-490    TELEPHONE:  303-724-1055  Fax:  303-724-0990 
 
 
 
Project Title: 
Oral Amino Acid Nutrition to Improve Glucose Excursions in PCOS  
 
Version Date: 06/09/2020  
 
Principal Investigator:  Melanie Cree Green, MD, PhD  
 
 
 
 
  
 
I. Hypotheses and Specific Aims:   
 
Polycystic Ovarian Syndrome (PCOS), characterized by excess testosterone and irregular menses, is a 
common endocrinopathy, affecting 10-15% of all women and up to 20% of obese women1. Obese adult 
women with PCOS  
have a 4-fold increased risk of developing type 2 diabetes (T2D), higher rates of non-alcoholic fatty liver 
disease (NAFLD) and risk markers for cardiovascular disease (CVD). We have found that obese girls with 
PCOS have severe insulin resistance (IR) a precursor to T2D, high rates 
of hepatic steatosis (HS) (early NAFLD) and early risk markers for 
CVD2,3. The presence of HS is the strongest predictor for development of 
T2D and CVD4. It appears that the combination of obesity and PCOS are 
synergistic for increasing the risk of metabolic disease (Figure 1).  Youth 
onset metabolic disease confers an early and more severe morbidity and 
earlier mortality than adult onset disease, emphasizing the urgency of 
treating this population5. Unfortunately, current therapeutic options for 
PCOS and HS are inadequate. Obese girls with PCOS already have 
metabolic disease, are at high risk for severe disease as they age and 
therapeutic options are limited. Development of new interventions should be focused during this 
vulnerable yet reversible stage. 
HS in PCOS appears to be due primarily to increased synthesis of new fat via de novo 
lipogenesis (DNL) and also relates to abnormalities in glucose metabolism including insulin resistance 
and hyperglycemia2,3,6-10. Beyond glucose, non-essential amino acids (NEAA) glutamine and alanine are 
substrates for increased gluconeogenesis and DNL11,12. NEAA and glucose are increased with excess 
caloric intake and muscle and hepatic IR13,14. Further, redundant cycling through the tricarboxylic acid 
cycle (TCA cycle) also leads to excess carbons available for DNL9. A potential target to reduce HS is to 
reduce the substrates available for DNL via modulating TCA cycle, glucose and NEAA excess. 
Essential amino acids (EAA) are critical for multiple physiologic processes, most notably protein 
synthesis, and can only be obtained from the diet. A full complement of dietary EAA is required for 
adequate protein synthesis15-17. If protein breakdown is greater than protein synthesis, as seen in weight 
loss or increased metabolic stress such as sepsis, resulting excess NEAA are available as substrates for 
DNL and gluconeogenesis. Further, excess branched chain amino acids (BCAA), a subset of EAA, are 
increased when all of the EAA aren’t available for protein synthesis. Excess BCAA relate to IR in T2D and 
obesity since by-products of their oxidation may cause muscle IR18,19. Women with PCOS have higher 
testosterone and thus an increased hormonal drive for protein synthesis, but do not have increased  
Abbreviations used in this protocol: AHI=apnea hypopnea index; BCAA=branched chain amino acid; BMI=body 
mass index; CAP=controlled attenuation parameter; CVD=cardiovascular disease; DHEAS=dehydroepiandrosterone 
sulfate; DNL=de novo lipogenesis; EAA= essential amino acids; EGP= endogenous glucose production; FFA=free 
fatty acids; GIR=glucose infusion rate; GLP-1=glucagon-like peptide-1; GLP-1 RA=glucagon-like peptide-1 receptor 
agonist; HOMA=homeostasis model assessment; HS=hepatic steatosis; IHTG=intrahepatic triglyceride; IR=insulin 
resistance; MRI=magnetic resonance imaging; MRS=magnetic resonance spectroscopy; NAFLD=non-alcoholic fatty 
liver disease; NEAA=non-essential amino acids NMR=nuclear magnetic resonance; OGTT=oral glucose tolerance 
test; OSA=obstructive sleep apnea; OSTT=oral sugar tolerance test; PCOS=polycystic ovarian syndrome; 
TCA=tricarboxcylic acid cycle; TG=serum triglycerides; T2D= type 2 diabetes; VAT=visceral adipose tissue; 
Figure 1: Role of excess liver 
fat  in metabolic disease 
 
 
 
Obesit
PCO
Liver 
T2D 
CVD
NAFLD 

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3DPAR=3-day physical activity recall 
 
 
muscle mass, indicating that they may have increased protein turnover, resulting in excess NEAA and 
BCAA, placing them at unique risk2,3. Of importance, in the setting of weight loss, higher protein diets 
moderately improve IR and decrease HS in obesity20,21, NAFLD22, T2D23,24 and PCOS25. We have found 
that balanced EAA supplements decrease HS in adults with IR or NAFLD15,26,27 but the role of EAA 
supplementation to improve clinical outcomes via driving efficient protein synthesis and decrease 
substrates for DNL and gluconeogenesis has not been investigated in PCOS. The goal of this project is to 
intervene with a well-tolerated and safe EAA dietary supplement to decrease HS and related IR in obese 
girls with PCOS at high risk for T2D, CVD and progressive liver disease via a blinded placebo controlled 
cross-over study.   
 
Specific Aim 1: In obese girls with PCOS and HS, determine if 4 weeks of EAA supplementation 
decreases HS via lower rates of hepatic DNL. 
Strong preliminary data shape our working hypothesis that in obese girls with PCOS and HS, intrahepatic 
triglycerides will decrease due to decreased DNL following 4 weeks of EAA, as compared to placebo. 
 
Specific Aim 2: In obese girls with PCOS and HS, determine if 4 weeks of EAA supplementation lowers 
T2D risk via decreased gluconeogenesis/gluconeogenic precursors and BCAA oxidation products/IR. 
 
HYPOTHESIS:  
We hypothesize, again based on preliminary data, that in obese girls with PCOS and HS, IR and post-
prandial hyperglycemia will decrease after 4 weeks of EAA supplementation, as compared to placebo. 
EAA supplementation will improve postprandial metabolism by correcting EAA imbalances to allow for 
increased net protein synthesis. This will decrease NEAA substrates available for futile hepatic TCA 
cycling that creates gluconeogenesis precursors and decreases BCAA oxidation products that influence 
muscle IR.  
 
II. Background and Significance:  
PCOS affects 10-15% of U.S. women, and 15-20% of obese women, with an estimated economic 
burden of $4 billion and is increasing in prevalence in parallel with the increase in obesity28,29.  PCOS 
develops in late puberty, and the phenotype of PCOS includes high androgens, irregular/infrequent 
menses and dermatologic manifestations including facial hair, balding and severe acne1,30. Metabolic 
disease is common in PCOS and in obese women with PCOS, IR is almost universal and is a major 
contributor to the earlier onset and rising incidence of inter-related NAFLD, T2D and CVD30-35. The 
primary therapy for PCOS with obesity is lifestyle modification which is unfortunately very challenging for 
youth due to peer, family and personal motivational issues. Current PCOS medical therapies include 
metformin, which only works in approximately half of patients and oral contraceptives which may actually 
increase CVD risk1,30,36-41. Thus, PCOS is a common and challenging disease for youth, and an important 
gap exists in effective therapeutic options for PCOS.  
An estimated 50-70% of obese women with PCOS have elevated HS, compared to 20-30% of similarly 
obese women without PCOS 32,42. Our previous data show that half of obese girls with PCOS have HS2. 
These findings indicate that hepatic pathology occurs early in the course of PCOS, and less severe 
abnormalities are even seen in normal weight girls with PCOS3.  Furthermore, obese women with PCOS 
and HS have more IR than those without HS, indicating a link between HS and worsening IR 33. IR in 
muscle, liver and adipose tissue is reported to be worse in PCOS adults than in adult women of similar 
BMI and thought to be related to increased androgens43-46.  We also found worse adipose, liver and 
muscle IR in girls with PCOS,regardless of BMI,than BMI-similar non-PCOS girls, despite their young age 
47. The synergy between obesity and hyperandrogenism in PCOS likely has unique influence on fat 
metabolism, inflammation, NAFLD and IR 48-50. Limited studies in adults with NAFLD indicate that 
upregulated rates of hepatic DNL and increased delivery of free fatty acids (FFA) are the primary 
contributors to the development of HS 6,51,52. However, the unique mechanisms behind increased HS 
have not been well studied, as the majority of mechanistic NAFLD studies have focused on men.        
Understanding and treating the high rates of HS in girls with PCOS at the critical preclinical stages 
may be the best strategy to prevent future NAFLD, T2D and CVD. HS may contribute to worsening IR and 

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progression to T2D and can progress to cirrhosis and liver failure 4,34,35. Elevated liver transaminases (a 
marker of NAFLD) predict hyperglycemia and T2D onset53.  Conversely, treatment of NAFLD with an 
effector of bile acid metabolism improves glycemia54, arguing for a tight connection between glycemia and 
liver health. IR in adipose, muscle and the liver increases as the degree of HS increases55. Unfortunately, 
youth onset NAFLD appears to be more severe at presentation and progress more rapidly than adult 
onset disease5,56. Thus treating NAFLD in youth has the potential of significant long term health benefits. 
Both dietary and pharmacologic strategies have been employed to treat NAFLD57,58. Diets with 
decreased calories and simple carbohydrates have been shown to decrease rates of HS across the 
lifespan22,23. Fructose is preferentially used by the liver for DNL, and just 9 days of a low fructose diet 
reduced hepatic fat by 50% in youth with HS15. Unfortunately, this diet is nearly impossible to follow for 
most families. Diabetes medications that lower glucose and thus DNL precursors such as metformin have 
had variable success59-61. However, there are currently no standard of care medications to treat HS57. 
 The interplay between protein, glucose and fat metabolism is dynamic and tightly regulated. However, 
the majority of research involving alterations in glucose and fat metabolism as they relate to T2D and 
NAFLD does not account for the 
role of protein. The balance of 
protein 
synthesis 
and 
breakdown 
is 
influenced 
by 
nutritional state, exercise, insulin 
and in particular testosterone. In 
men, 
testosterone 
concentrations of 400-700 ng/dL 
lead to increased muscle mass 
and 
absolute 
increases 
in 
protein synthesis62,63. In women 
with 
PCOS, 
testosterone 
concentrations are 40-125 ng/dL 
yet 
muscle 
mass 
is 
not 
increased, 
suggesting 
no 
increase 
in 
net 
protein 
synthesis2,64,65. 
States 
of 
decreased net protein synthesis 
secondary to increased protein 
turnover or lack of quality protein 
from starvation, lipodystrophy 
and surgical weight loss are 
known causes of HS11,57,66,67. 
The US dietary recommendation 
of protein is inadequate for 
obesity and weight loss68. In 
obese youth, excess calories 
are typically carbohydrates and 
fat and food may be deficient in 
quality protein and EAA69-71. This 
high calorie poor diet can lead to 
excess formation of NEAA and 
precursors for gluconeogenesis and DNL8,9. Additionally, BCAA not utilized for protein synthesis can be 
oxidized into acyl CoA products that induce IR and further provide glucose for DNL72. Indeed, alterations 
in BCAA metabolism relate to IR in obese adolescents18, women with PCOS73 and obese adults with 
T2D74-76.  Thus, obese youth with PCOS have poor diet quality and likely have increased protein flux from 
androgens without increased synthesis, creating a unique pathology for development of HS (Figure 2). 
 
Figure 2: Inadequate EAA from poor nutrition and high protein flux 
from testosterone in PCOS causes hepatic steatosis and hyperglycemia 
 
EAA: essential amino acids,  NEAA: non essential amino acids, BCAA: 
branched chain amino acids, DNL: de novo lipogenesis, GNG: 
gluconeogensis, Acyl C3 and C4: acyl Co-A breakdown brokducts of 
BCAA oxidation, IHTG: intrahepatic tryglycerides 

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Multiple studies have shown that dietary intervention with a high protein hypocaloric diet induces 
weight loss, improvements in insulin sensitivity or lowering HS77-84. However, the majority of these studies 
are by default low in carbohydrates and it is unclear if metabolic improvement is from the protein affect, a 
decrease in carbohydrate or decrease in overall calories85-88. 
In women with PCOS, either a high protein diet or whey 
protein supplementation improved insulin sensitivity, but all 
participants also experienced weight loss86,89-91. Yet not all 
studies with a high protein diet or BCAA supplementation 
show improvements in metabolic disease87,92-94. These 
conflicting results are likely attributable to the failure to 
improve the pathology listed in Figure 2, i.e. not enough EAA 
to stimulate net protein synthesis or supplementation with a 
protein that is high in gluconeogenic precursors alanine and 
glutamine which can increase glycemia95. Indeed, we have 
shown that the ideal composition for increasing muscle protein 
synthesis appears to be a 15 gram dose of EAA, with more 
modest effects from isolated EAA’s or intact proteins16,96-99 
(Figure 3). 
    EAA supplementation is therefore a promising treatment for HS and IR, as opposed to complete 
protein source94,100,101.  Just weeks of EAA supplementation decreases HS and IR in at-risk populations 
even in the absence of weight loss and metabolic studies in healthy men demonstrate a blunting of 
intrahepatic triglycerides (IHTG) synthesis with 3 days of EAA15,26,27,102,103. We previously found that 
ingestion of a mixture of EAAs stimulates more efficient reutilization of non-essential amino acids 
(NEAAs), and as a result reduces the plasma concentrations of NEAAs and substrate available for 
gluconeogenesis and DNL and that this is not disturbed in the presence of elevated free fatty acids17,104.  
Finally, the EAA leucine has metabolic signaling effects in addition to being crucial for protein synthesis 
and supplementation EAA higher in leucine have increased beneficial metabolic effects in those with pre-
existing disease. EAA oral supplements are readily available, acceptable to patients and have minimal 
side effects15,26,103. EAA supplementation treatment for metabolic disease in obese girls with PCOS is a 
simple intervention with a potential to reduce significant long-term morbidity and perhaps mortality. 
 
A. Preliminary Studies/Progress Report:   
We have been increasing our knowledge of the pathophysiology of IR and HS in obese 
youth though 4 distinct studies 
(Table 1, * indicates Dr. Cree-
Green as PI). All studies included 
stable 
isotope 
tracers, 
MRI 
measured IHTG and measurements 
targeted 
to 
better 
understand 
pathophysiologic mechanisms. The 
results of these studies which have 
informed our proposed study are 
shown below. 
 
 
Figure 3: 15 g of EAA stimulates the 
most net protein synthesis in adults 
 
Table 1: Youth studies used for preliminary data 
Study, N 
Participant population 
Relevant methods 
Resistant 
N=80 
Lean and obese youth  
With and without T2D 
Tissue specific clamp assessed IR  
Airs* 
N=73 
Lean and obese girls  
With and without PCOS 
Tissue specific clamp assessed IR 
Indirect measure of hepatic DNL 
Metabolomics 
Apple* 
N=92 
Obese girls 
With and without PCOS 
Tissue specific OSTT assessed IR 
Direct measure of hepatic DNL 
Plum* 
N=11 
Obese girls  
With PCOS 
Prolonged OSTT assessed IR 
Hepatic TCA cycle activity 

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• 
 Obese girls with PCOS have severe IR and high rates of HS and hyperglycemia.   
In our Resistant and APPLE studies, we examined the differential tissue-specific response to insulin in 
female youth with PCOS compared to my mentor’s (Dr. Nadeau) 
studies of T2D, utilizing a 4 phase hyperinsulinemic euglycemic clamp 
with glucose and glycerol stable isotope tracers.  By contrasting 
patterns of pathology between these disorders, we uncovered subtle 
differences that suggest unique mechanisms of metabolic change in 
PCOS.  PCOS girls in AIRS study had mildly increased hepatically-
derived serum triglycerides (TG) (Table 2), liver transaminase ALT as 
elevated as in T2D, and MRI-assessed rates of IHTG even higher than 
in T2D girls (Figure 4).  Most notably, we found that ≈50% of obese 
girls with PCOS had >5.5% liver fat (i.e. clinically significant HS), 
whereas 10% obese girls without PCOS had HS. These findings a 
have been duplicated in the APPLE cohort. Obese girls with PCOS had 
multi-tissue IR relative to similarly obese controls, but are not as IR as 
T2D girls47. The concentration of insulin required to suppress hepatic 
glucose release was double in girls with PCOS (p=0.04), indicating 
hepatic IR (Figure 4, middle), and may be reflective of dysregulated 
gluconeogenesis. Muscle IR (Figure 4, bottom), measured as insulin 
stimulated glucose uptake (GIR) was much lower (p<0.001) in PCOS. 
Simple clinical scores such as the fasting HOMA-IR as proposed as a 
secondary 
endpoint 
are 
also 
different 
between 
groups and 
correlate 
well 
with 
clamp IR in 
these 
girls 
(r=0.75, p<0.01; Table 2). Additionally, obese 
girls 
with 
PCOS 
have 
an 
increased 
hemoglobin A1c, as an indicator of blood sugar over the previous 3 months and following a (75g glucose 
+25g fructose) oral sugar tolerance test (OSTT), nearly half of girls had impaired glucose tolerance, 
defined as a blood glucose > 140 mg/dl at 2 hours. 
 
• 
 Excess hepatic triglyceride is from increased de novo lipogenesis in obese girls with PCOS. In 
our Resistant and APPLE studies, we examined the differential tissue-specific response to insulin in 
female youth with PCOS compared to my mentor’s (Dr. Nadeau) studies of T2D, utilizing a 4 phase 
Obese girls with PCOS in the Airs cohort had higher serum 
concentrations of markers of DNL (i.e. long chain fatty acids 
with a double bond in the 7th carbon position; N7 FFA) (29±13 
nmol/g obese vs. 45±15 PCOS, p<0.01)64.  IHTG in PCOS 
girls weakly correlated with markers of overall lipolysis, such 
as FFA, but was closely related to N7 FFA (r=0.53, p=0.004). 
To follow-up on these findings, in the APPLE study, we are 
directly measuring the rate of hepatic DNL with an IV infusion 
of a 1,2C13 acetate tracer. We are performing studies during 
overnight fasting and then following a glucose ingestion, to 
better understand what happens during a normal physiologic 
state. The incorporation of this acetate tracer, expressed as tracer enrichment, is greatest in the newly 
secreted hepatic VLDL-TG in girls with HS and PCOS (Figure 5). Further, DNL is even increased in 
PCOS girls without HS compared to controls, demonstrating an independent role of PCOS. These 
changes are more evident following the glucose load, rather than fasting, again emphasizing the need to 
Figure 4:  Obese PCOS girls have 
high IHTG, liver and muscle IR 
Table 2: Serum markers in obese girls 
 
Obese 
PCOS 
T2D 
Number 
of 
Participants 
19 
38 
40 
ALT (IU/L) 
29±3 
38±3 
40±2 
Triglycerides (mg/dl) 
78±8 
129±7 
207±7 
HbA1C (%) 
5.1±0.2 
5.4±0.3 
7.2±0.4 
2h OSTT glucose 
>140 mg/dL 
20% 
50% 
100% 
HOMA-IR 
3.6±1.3 
5.9±0.4 
- 
Figure 5: Increased DNL in PCOS+HS 
 
PCOS         Obese 

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study this metabolism in the fed state and the negative additive effect of PCOS and obesity.  
 
• 
EAA metabolism is altered in girls with PCOS. We performed untargeted metabolomics analysis 
in a pilot subset of girls enrolled in the AIRS cohort. We found that fasting BCAA are higher in girls with 
PCOS compared to controls, as were NEAA alanine 
(p=0.011) and glutamine (p=0.037) concentrations. 
Glutamine during the OSTT related to HbA1C 
(p=0.0091), 
demonstrating 
that 
an 
increased 
gluconeogenic precursor related to increased serum 
glucose concentrations. Further, higher BCAA valine 
related to worse IR as assessed with the clamp 
(p=0.045). 
During 
hyperinsulinemia, 
the 
concentrations of serum BCAA trended to not 
suppress nearly as much in the PCOS compared to 
the controls, and end products of BCAA oxidation, 
acyl-CoA C3 and C4, did not decrease (Figure 6). 
This suggests that protein synthesis as a depot for 
EAA was less, shunting the excess BCAA towards 
oxidation and muscle IR in PCOS. In contrast, EAA’s (phenylalanine, methionine and tryptophan) did 
decrease similarly in PCOS as controls, suggesting that these may limit protein synthesis during 
hyperinsulinemia. These trends need to be confirmed in a physiologic setting, such as proposed. 
 
• 
EAA supplementation in older adults with IR decreased HS and improved insulin sensitivity. Older 
adults are similar to girls with PCOS in that 
they have worse IR and higher rates of pre-
diabetes, distinct form changes in obesity. 
We have shown that when adults aged 65 
years and older, without diabetes or hepatic 
steatosis, were given 11 grams of EAA 
twice a day for 16 weeks, serum lipids had 
improved by 4 weeks (Figure 7), and the 
concentration of IHTG decreased by 50%, 
as measured with 1H-MR spectroscopy26,103. 
The supplements were well tolerated, with 
no adverse effects noted. Insulin sensitivity 
as assessed with fasting HOMA-IR was 
improved. Participants with the lowest insulin sensitivity and the highest IHTG experienced the greatest 
change with EAA supplementation. 
 
• 
Intrahepatic metabolism can be measured non-invasively with an U-C13 oral glycerol tracer, in obese 
adolescent girls. Glycerol is nearly universally taken up by the liver, and when labeled with a carbon 
isotope can be used to trace hepatic metabolism. Our collaborator Dr. Malloy first described this 
methodology in adult men105,106.  In our PLUM 
study, we have implemented this protocol in 
obese girls and made adaptations to account for 
their severe IR and prolonged post oral glucose 
response. We have determined that giving the 
glycerol tracer 3 hours after the sugar tolerance 
drink and sampling for an additional 3.5 hours 
provides the best data. We have also confirmed 
that the isotopomer outcomes of interest for Aim 
2, including the percent of carbons which have 
come from hepatic anapleurotic cycling in the 
TCA cycle and then contribute to fatty acid 
synthesis and gluconeogenesis can be measured 
with this methodology in our patient population (Figure 8). The proportion of carbon TCA cycling in 
glucose trends to be related to worse HOMA-IR (R=-0.9, p=0.08 with 5 participants) and higher ALT, 
Figure 6: Hyperinsulinemia induces BCAA 
oxidation not protein synthesis PCOS girls 
 
Figure 7: EAA supplementation lowers serum and hepatic 
lipids in older adults with IR but not HS 
 
 Figure 8: Percent of carbons from excess 
TCA cycle activity in hepatically secreted 
glucose and lipid in girls with PCOS 

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suggesting that alternate sources of carbon such as from NEAA are producing glucose. This data 
demonstrates that this methodology has been optimized for our patient population, and when 
combined with the clinical outcomes, rates of DNL and gluconeogenesis and the metabolomic amino 
acid data, can yield powerful insights into underlying mechanisms of pathology. 
 
III. Research Methods 
 
A.  Outcome Measure(s):   
Primary Outcome Measure(s): Change in hepatic fat content per treatment type 
Secondary Outcome Measure(s):  Activity ratios of the hepatic pentose phosphate pathway, TCA cycle 
and TG synthesis, change in peripheral IR as assessed with an OSTT, measures of sympathetic nervous 
system activity, hepatic phosphate profile. Indirect measure of DNL (change in TG concentration with 
OSTT) Targeted glucose and fat metabolism metabolomics, and untargeted exploratory analysis, gut 
microbiota profile. 
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 assess changes with 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 MRI107-110), Whole body fat oxidation at rest and following 
glucose ingestion as measured with a metabolic cart; Physical activity/ diet:(accelerometer, 3-day 
physical activity recall questionnaire (3DPAR); Food frequency questionnaire).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:   
Study staff aims to complete 20 obese girls with PCOS (untreated) and HS. This is the number of 
participants needed to be completed statistically, thus more participants may be enrolled, to allow for 
screen failures and dropouts. Total enrollment will be up to 50 participants as they will have a screening, 
then those without hepatic steatosis will be removed from the study. 
 
Ethnic Categories 
Gender 
Females 
Males 
Total 
Hispanic or Latino 
20 
0 
20 
Not Hispanic or Latino 
30 
0 
30 
Ethnic Categories: Total of All Participants 
50 
0 
50 
  
Racial Categories 
 
 
 
American Indian/Alaska Native 
2 
0 
2 
Asian 
5 
0 
5 
Native Hawaiian or Other Pacific Islander 
5 
0 
5 
Black or African American 
15 
0 
15 
White 
23 
0 
23 
Racial Categories: Total of All Participants * 
50 
0 
50 
 
Inclusion Criteria: 
1) Female 
2) Ages 12-21 
3) Sedentary- less than 2 hours of moderate (jogging, swimming etc) exercise a week. 
4) BMI equal or greater than the 90th percentile for age and gender 
 

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5) PCOS per the most stringent NIH criteria adapted for adolescents (irregular menses >24 
months post-menarche and clinical or biochemical hypertestosteronemia)1,30 
 
6) HS per FibroScan ultrasound, with CAP score of >225111,112.   
 
Exclusion Criteria: 
1. Use of medications known to affect insulin sensitivity: metformin, oral 
glucocorticoids within 10 days, atypical antipsychotics, immunosuppressant agents, 
HIV medications, hormonal contraception. Dermal patch or vaginal ring 
contraception methods.  
2. Currently pregnant or breastfeeding women. Development of pregnancy during the 
study period will necessitate withdrawal from the study. 
3. Severe illness requiring hospitalization within 60 days 
4. Diabetes, defined as Hemoglobin A1C > 6.4% 
5. BMI percentile less than the 90th percentile for age and sex. Weight >325 lbs or <84 
lbs. 
6. Anemia, defined as Hemoglobin < 11 mg/dL 
7. Diagnosed major psychiatric or developmental disorder limiting informed consent 
8. Implanted metal devices that are not compatible with MRI 
9. Use of blood pressure medications 
10. Known liver disease other than NAFLD or AST or ALT >125 IU/L 
 
 
C. Study Design and Research Methods   
 
Overall Study Design:  
Both aims with be accomplished from a cohort of 20 obese 
girls with PCOS and HS. Girls will complete a 12 week blinded 
placebo controlled cross-over study with 4 weeks each of EAA 
and placebo, with metabolic studies after each intervention and 
approximately 4 week wash out in-between (Figure 9). 4 
weeks of EAA supplementation is adequate as it decreased IR 
and serum lipids in obese adults and IHTG can be changed 
within 3-9 days with more extreme dietary changes15. 
Participants will have a total of 5 visits, a Screening visit (Visit 
1) will ensure eligibility, Study 1 (Visits 2 and 3), and Study 2 
(Visits 4 and 5) (see Study Calendar). The study days will 
include an abdominal MRI, OSTT with isotope tracers and 
assessment of other contributors to HS and IR. The study days (Figure 10) will start in the afternoon on 
day 1 with MRI measures, with at least a 4 hour fast prior, followed by admission to the hospital. 
Participants will receive deuterated water overnight, and in the morning, they will be given U-C13 glycerol 
drink while participants 
 are fasting. 
 
Figure 9: Overall Study Design 

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Blood will be drawn for 
DNL 
and 
gluoneogensis. 
The 
oral sugar tolerance 
test (75g glucose +25g 
fructose) drink will be 
given a few hours later. 
A metabolic cart will be 
used to measure the 
rate of fat oxidation 
fasting, and throughout 
the 
OSTT. 
This 
is 
similar to our current 
PLUM study and it is 
well tolerated in youth. 
 
 
 
 
 
 EAA Supplement: The formulation of EAA (Table 3) is similar to our 
previous studies with an increased proportion of leucine, although we will 
give 10 grams of EAA twice a day, as this stimulates maximal protein 
synthesis15,26,103. The increased leucine influences IR and also maximizes 
protein synthesis94. Supplements will be given twice a day, once during the 
morning and the second in the evening, as balancing EAA intake across the 
day has a greater effect on protein synthesis113 and adolescents most 
frequently skip morning meals100. The supplement is a powder that is mixed 
in 8 ounces of water. Placebo will be matched by the pharmacy to match 
the EAA supplement in flavor and appearance. Despite the dogma that 
excess protein challenges the kidney, long-term high protein diets do not 
affect kidney function in healthy individuals114 and we do not have safety 
concerns.  
 
Participants: Recruitment: Girls will be recruited from several Children’s Hospital Colorado clinics and 
from the community. Based on a 50% prevalence of HS in this patient population, up to 50 girls will be 
screened. Based on an approximately 5% drop out rate in my cross-sectional trials and my mentors 
clinical trials, we will aim to complete 20 girls with PCOS with HS.  Inclusion criteria: PCOS per the most 
stringent NIH criteria adapted for adolescents (irregular menses >24 months post-menarche and clinical 
or biochemical hypertestosteronemia)1,30; age 12-21 years, sedentary status as exercise training affects 
IR (<2 hours of physical activity/week), BMI>90%ile for age and sex, HS per FibroScan ultrasound, with 
CAP score of >225111,112.  Exclusion criteria: diabetes, liver disease other than NAFLD, liver 
transaminases >125 IU/L, medications affecting IR including metformin or hormonal contraception, 
hemoglobin <11 mg/dl, and MRI-incompatible metal.  
Aim 1: Hepatic steatosis and DNL: IHTG percent will be assessed with MRI utilizing the Dixon method 
as in our previous studies2,47,64. The Dixon method has excellent correlation with liver biopsy and is 
preferable to spectroscopy in obese participants, where spectroscopy coil depth of penetration is limiting. 
Visceral and subcutaneous fat will also be calculated from MRI as in our previous studies. Rates of 
hepatic DNL will be measured from the tracer incorporation of deuterated water into VLDL secreted 
palmitate with samples analyzed with GCMS by collaborator Dr. Bergman, as previously described in 
youth51,115,116. We will utilize deuterated water rather than U-C13 acetate as in the APPLE study to reduce 
participant burden and blood volume draws. Further, both tracers have a carbon label and cannot be 
combined in a single protocol. 
Aim 2: Insulin resistance, hyperglycemia and hepatic glucose metabolism. After 3 doses of overnight 
deuterated water (approximate times shown in Figure 10), participants will be given a U-C13 glycerol 
isotope drink per Figure 10105. The glycerol drink is given in the morning while participant is fasting and 
Figure 10: Study day procedure timeline 
 
Table 3: Composition of 15 
g EAA nutritional therapy 
Histidine 
6% 
Isoleucine 
11% 
Leucine 
23% 
Lysine 
16% 
Methionine 
5% 
Phenylalanine 
14% 
Threonine 
8% 
Tryptophan 
3% 
Valine 
14% 

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samples are collected at frequent intervals per Figure 10 for positional NMR isotopomer analysis by 
collaborator Eunsook Jin at UT Southwestern105. Analyses will include modeling metabolic flux utilizing 
custom designed software and endpoints will include rates of excess TCA cycling contributing to 
triglyceride synthesis and gluconeogenesis as shown in Figure 8. This will be followed by a OSTT (75-
gram glucose and 25-gram fructose) with frequent sampling of glucose, insulin, C-peptide. Fasting insulin 
sensitivity will be calculated with HOMA-IR (fasting glucose* fasting insulin/22.4). The Matsuda117 index 
will be used to estimate whole body post-prandial insulin sensitivity and indices of hepatic [Glucose0–
30(AUC)/insulin0–30(AUC)] and muscle insulin sensitivity [dGlucose/dtime ÷ insulin] with calculations 
starting at the downslope of glucose concentration118. Blood glucose will be measured at the 2 hour time 
point and >140 mg/dL is defined as impaired glucose tolerance. Rates of fasting gluconeogenesis will be 
measured from the tracer incorporation of deuterated water into glucose, with samples analyzed with 
GCMS by collaborator Dr. Bergman, as previously described in youth51,115,116. Metabolomics: Targeted 
amino acid119  and amino acid breakdown product metabolomics profiles will be measured by the 
University of Colorado metabolomics core on fasting and 120 minute post glucose drink samples 
(https://www.dalessandrolab.com/home). 
 
 
Timeline: The time between the initial screening visit and completion of the study will be less than 6 
months. After confirmation of eligibility and randomization, participants will be on 4 weeks of EAA or 
placebo and then Phase I study visits will be completed (Visits 2 and 3). There will be approximately 4-
Study Calendar 
Screen 
Phase I 
Phase 2 
 
Visit 1  
Visit 2 –MRI 
Visit 3 – 
Overnight 
Visit 4 –MRI 
Visit 5 – 
Overnight 
Consenting and Eligibility 
Assessment 
X 
 
 
 
 
History & Physical 
X 
 
 
 
 
Intravenous Blood Draw 
X 
 
X 
 
X 
Fibroscan -Liver ultrasound  
X 
 
X 
 
X 
Randomization- Placebo vs EAA 
X 
 
 
 
 
Crossover- Placebo vs EAA 
 
 
X 
 
 
Accelerometer teaching 
X 
 
 
 
 
Gut Bacteria Collection 
 
 
X 
 
X 
Urine Pregnancy Test 
 
 
X 
 
X 
Questionnaires- SEARCH Food 
frequency, Activity 3DPAR, 
Strengths and Difficulties and 
Sleep assessments 
 
 
X 
 
X 
DEXA Scan (located in Leprino 
building) 
 
 
X 
 
X 
Oral Glycerol Tracer with D20 
 
 
X 
 
X 
Oral Sugar Tolerance Test (OSTT) 
 
 
X 
 
X 
WatchPAT sleep study 
 
 
X 
 
X 
Metabolic Cart 
 
 
X 
 
X 
EndoPat and Dynapulse 
 
X 
 
X 
 
MRI of abdomen and liver  
 
X 
 
X 
 
P MRS of Liver  
 
X 
 
X 
 
Total Time of visit (approximately) 
2 Hours 
2 Hours 
22 Hours 
2 Hours 
22 Hours 
Location of Visit 
CHCO 
CTRC 
Outpatient 
UCD 
Outpatient, 
Brain Imaging 
Center 
CHCO or 
University 
Hospital 
Inpatient  
UCD 
Outpatient, 
Brain Imaging 
Center 
CHCO or 
University 
Hospital 
Inpatient 

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week washout period and then participants will crossover treatment, and complete another 4 weeks of 
EAA or placebo. Phase II study visits (Visits 4 and 5) will be completed.  
 
 
Overall enrollment procedure plan per group 
VISIT 1 (SCREEN VISIT) 
Pediatric CTRC Outpatient unit:  Participants will begin with a medical screening and physical exam for 
inclusion/exclusion criteria evaluation.  During this visit, patients will review and complete consent 
documents, have demographics and medical history confirmed, assess allergies and further evaluation of 
inclusion/exclusion criteria, have blood samples drawn, and have anthropometrics completed.  HbA1c, 
ALT, AST, CBC panel and testosterone panel will be drawn at the beginning of the visit after consent in 
all participants. PCOS labs may be performed for confirmation of PCOS status, if not performed 
previously. Whole blood for DNA samples for future genetic analysis associated with hepatic steatosis will 
also be drawn and stored at this time. Participants will also have a Fibroscan (liver ultrasound) to check 
for HS (CAP score of >225).  
After eligibility assessment, participants who qualify for the study will be randomized to placebo or EAA by 
the pharmacy. Both study staff and participant will be blinded. The participant will be scheduled for Visits 
2 and 3 after being on placebo or EAA supplements for 4 weeks.  
 
Screening lab test 
Purpose for test 
HbA1C 
Rule out type 2 diabetes, if > 6.4% participant to 
be excluded 
ALT, AST 
Ensure no severe liver disease, if >125 IU/L 
participant to be excluded normal participant is 
excluded 
Hemoglobin & Hematocrit (part of CBC) 
If participant is Anemic, they will be excluded 
Testosterone  
Test for hyperandrogenism – required to meet 
NIH criteria for PCOS 
Optional PCOS labs 
PCOS status must be confirmed prior to enrollment. Referring physicians often do not perform the 
entire recommend work-up for oligomenorrhea (per 2013 Endocrine Society Clinical Guidelines for 
PCOS). The values are typically expected in PCOS publications 
TSH, total T4 
Ensure no hypo or hyperthyroidism causing 
amenorrhea 
LH, FSH 
Rule out primary ovarian failure 
17-hydroxyprogesterone 
Rule out late onset congenital adrenal 
hyperplasia 
DHEAS 
Rule out adrenal tumor 
Prolactin 
Rule out prolactin secreting brain tumor 
 
 
 
VISIT 2 (MRI VISIT) 
Overall Plan: UCD Research MRI (Brain Imaging Center): Participants 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 is optional pending scheduling. It will be 31P spectroscopy to measure 
the concentrations of phosphate molecules including PDE, PME and PCr. Vascular Endothelial function 

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and Heart Rate Variability will be assessed with Endopat and Dynapulse, which take approximately 30 
minutes. 
 
31P MRS of the Liver and Abdominal Imaging (pending scheduling) 
       MRS Data acquisition: Imaging and MRS will be performed on a Seimens 3 Tesla MRI magnet. A 
custom 1H/31P abdominal coil will be used for imaging and MRS (Clinical MR Solutions, Brookfield, WI) as 
in our previous 31P work120. 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 studies109. 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. 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 studies121. Hepatic fibrosis will be measured with a fibroscan sequence. 
 
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 122,123, utilizing AMARES (A Method of Accurate, Robust and 
Efficient Spectral fitting), a nonlinear least-square-fitting algorithm using our previously built prior 
knowledge files 124. We have utilized this method for muscle 31P analysis for the previous 6 years, and 
have extensive experience with this analysis.  The adipose data will be analyzed by radiology MRI 
research imaging, supervised by Ann Scherzinger, PhD, as in previous protocols (COMIRB #’s 10-1288 
and 14-0542). 
 
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]. It is possible that gut microbiota 
contribute to the development of NAFLD, and this has not been explored in PCOS. We will collect a 
sample to define the microbiota in girls who are already undergoing extensive metabolic profiling.  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 being collected for present and 
future research markers in the mircrobiome. Fecal samples are routinely collected in research and pose 
little risk to participants. 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 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. 
 
Accelerometer: One week prior to the overnight visit, the participant will be provided two accelerometers 
(GT3X BT by Actigraph and ActiWatch by Philips Respironics) to be worn for seven days to measure level 
of habitual physical activity, which affects insulin sensitivity, and sleep patterns. Accelerometers are 
effective tools for the objective measurement of physical activity 125 because they have the ability to 
continuously record physical activity data and such data can be used to estimate METs of activity. They 
provide more detailed information than pedometers, which only measure walking steps, and help get 
around the recall bias of questionnaires. We are currently using the GT3X BT Actigraph in adolescents in 
our other diabetes studies; therefore, we are familiar with their use in this population and have the 
necessary computer software and interpretation skills. The Actiwatch is being used as a tool for objective 
measurement of sleep patterns. The Actiwatch is fitted with a LED monitor that detects multiple 
spectrums of light to better assess sleep patterns in this population. 

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*One week prior to overnight visit, participant will receive via mail the accelerometers, sleep diary 
and stool sample collection kit (Gut bacteria collection) * 
 
 
VISIT 3 (ORAL GLYCEROL TRACER WITH D20 AND ORAL SUGAR TOLERANCE TEST)  
 
Children’s Hospital Colorado or University Hopsital CTRC Inpatient unit Only under extenuating 
circumstances, if no rooms available in either hospital, we may have patients go home after WatchPAT is 
placed, and drink deuterated water at home, and come back the following morning at 6am:  Participants 
will be asked not to have caffeine or exercise for 3 days prior to this visit. During admission, participant 
will be provided with a study diet dinner and snack. Patients will be admitted to the Pediatric CTRC for a 
monitored overnight fast. Participants will be questioned regarding changes in concomitant medications 
and medical history, height, weight, BP.  Participants will also have a Fibroscan (liver ultrasound) on the 
day of admission or the morning of the OSTT (pending staff availability). 
A DEXA scan will be performed to assess body composition. A urine pregnancy test will be done 
on all female participants prior to the DEXA scan.  If a female participant is confirmed to be pregnant, she 
will be withdrawn from the study and referred to her primary care physician for follow-up. 
The following morning, a blood sample for baseline metabolic labs will be drawn the modified 
OSTT will be completed (Figure 10). Additional blood will be drawn and stored for future inflammatory 
markers as well as for targeted and untargeted metabolomics analysis.  
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 watch pat data collection will not 
be ideal, due to waking the participants to drink the deuterated water. However, the primary endpoint is 
the apnea hypopnea index, and we can still get a rough estimate of this information, even with less than 
ideal sleep. 
Details of Stable Glycerol Isotope Tracer studies with an OSTT:  Participants will fast overnight, 
and drink 3 doses of D2O three times during the night with doses at 6-8PM, 10-11 PM and 2-3 AM as 
described previously in obese youth. The total D2O to be given (3 mL per kg of body water) is designed to 
raise body deuterium levels to 0.3% and was administrated in three doses to maintain the steady state 
until the end of the study126. Serum samples will be collected in the morning before the glycerol, and then 
throughout the OSTT, and VLDL TG separated and then enrichment in palmitate measured. By 
evaluating the incorporation of the D2O into palmitate, we will able to assess the DNL expressed as a 
percentage of DNL. We will also measure the rate of gluconeogenesis by measuring the enrichment in 
serial plasma glucose samples. 
The following morning at approximately 7:00AM, a 50 mg/kg of an oral UC13-glycerol tracer will be 
consumed mixed with water. Blood samples will be collected and at approximately 10AM, participants will 
consume a 75 gram glucose load with an additional 25 grams of fructose to stimulate hepatic de novo 
lipogenesis. Analysis of serum glucose isotopomers will be performed by our collaborator Eunsook Jin at 
UT Southwestern, using NMR isopomomer analysis and pathway modeling software. Lipolysis will be 
modeled using FFA concentrations and whole body IR by a time modified version of the Matsuda model. 
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 OSTT prior to the start of the OSTT, and two more times during 
the OSTT. This is required to determine what portion of ingested carbohydrates are subject to oxidative 
and non-oxidative glucose disposal127. 
 
Study diet: Variations in diet, activity and circadian rhythms affect metabolism 121. Therefore, OSTT 
studies will be performed in the AM, in the follicular phase where possible, preceded by 3  
days of no strenuous physical activity. Participants will be advised to eat a typical diet for the 3 days 
preceding the study. On the day of study, they will have a snack and dinner selected from limited choices 
on the Children’s hospital carbohydrate counting diet plan, with at least 20 grams of carbs in the snack 
and 40 grams of carbs for dinner. Participants will ingest their study drink (amino acid or placebo) in the 
morning of the day of admission, and then after the MRI with their snack. 

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Note: Participants will stop taking placebo or EAA supplements after completion of overnight visit for 
approximately  4 weeks (washout period). Then participants will crossover to EAA or placebo. The 
pharmacy will dispense the proper supplement, since study staff and participant will be blinded. The 
participant will be scheduled for Visits 4 and 5 after being on placebo or EAA supplements for 4 weeks.   
 
VISIT 4 (MRI VISIT) 
See Visit 2 Description.  
 
VISIT 5 (ORAL GLYCEROL TRACER WITH D20 AND ORAL SUGAR TOLERANCE TEST)  
See Visit 3 Description.  
 
Purpose for lab test to be drawn: 
OSTT Labs 
Purpose 
Lipids and glycerol samples for tracer analysis 
Determination of hepatic metabolism flux 
Glucose  
Determination of IR 
Insulin 
Determination IR 
FFA 
Measure of lipolysis 
Glycerol 
Measure of lipolysis 
Glucagon 
Gut hormone known to influence hepatic IR 
GLP-1 
Gut hormone known to influence hepatic IR 
CRP 
Marker of inflammation, know to effect IR 
Leptin 
Gut hormone known to influence hepatic IR 
Adiponectin 
Adipokine thought to influence adipose IR 
Estradiol 
Known to effect IR and HDNL 
Progesterone 
Demonstrate the participant is in the follicular 
phase of cycle, required for publication 
Metabolomics 
Correlation with hepatic metabolism flux 
1 sample for genetic analysis 
Measure for common polymorphisms 
associated with hepatic steatosis126,128 
Stored blood 
Future markers of glucose and fat metabolism, 
CVD or hormones related to PCOS 
 
Purpose for questionnaires being done: 
Questionnaire  
Description 
Study Validity 
Adolescent Sleep Hygiene Scale 
Measurement of 
sleep 
patterns/habits 
Cronbach’s alpha ranges from .46-.74; total 
scale alpha = .80 
LeBourgeois et al. 2005. The Relationship 
Between Reported Sleep Quality and Sleep 
Hygiene in Italian and American Adolescents 

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

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previous seven 
days. 
nutrients between the FFQ and true usual 
intake were 0·41 and 0·38, respectively, with 
57 % of food groups and 70 % of nutrients 
exhibiting correlations >0·35. Correlations 
were high for low-fat dairy (0·80), sugar-
sweetened beverages (0·54), cholesterol 
(0·59) and saturated fat (0·51), while 
correlations were poor for high-fibre bread 
and cereal (0·16) and folate (0·11). Reliability 
of FFQ intake based on two FFQ 
administrations was also reasonable, with 54 
% of Pearson correlation coefficients ≥0·5. 
Reliability was high for low-fat dairy (0·7), 
vegetables (0·6), carbohydrates, fibre, folate 
and vitamin C (all 0·5), but less than desirable 
for low-fat poultry and high-fibre bread, cereal, 
rice and pasta (0·2-0·3).  
Liese et al. 2015 Relative validity and 
reliability of an FFQ in youth with type 1 
diabetes 
First described in 2006:  
Mayer-Davis et al. 2006. Search FFQ Dietary 
Intake among Youth with Diabetes: The 
SEARCH for Diabetes in Youth Study 
 
Optional 7 Day Food Log 
Optional record of 
7-day diet to aid in 
food recall. Added 
per participant 
request to help fill 
out food frequency 
questionnaire. 
N/A 
Actigraphy Daily Sleep Diary  
 
Recording 
bedtime/wake time 
during actigraphy. 
Needed to 
corroborate watch 
collected data 
N/A 
 

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LIFESTYLE PRESCRIPTION 
1) At the time of screening, all participants will receive standard lifestyle recommendations including the 
reduction of liquid carbohydrate and portion control. We will not perform extensive counseling at this time 
as major changes in diet could affect the results. However, variability in the consumption of sugar 
sweetened beverage may affect results, and thus we will discourage these in all participants. Further this 
is the first step of standard lifestyle counseling for this population. 
2) 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 information at this visit will build on the previous education, 
and be personalized to include the basal metabolic rate calculations and specific dietary 
recommendations based on the food frequency survey. 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. 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 participant/family will be provided with copies of their study lab results, DEXA 
scan and physical activity monitoring. A results letter will be provided to the family within 6 months of 
completion of the study about clinically relevant results obtained during the study. Participants will be 
encouraged to contact study staff with questions regarding the results letter. 

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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 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: 
 
 
 
 
       
 
PARTICIPANT RECRUITMENT/CONSENT/PAYMENT 
1. Participant Recruitment Plan 
Participants 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 participants. Further, we enrolled >100 obese girls in studies in the last 60 months. 
The PI and Co-I’s have a treatment relationship with girls from clinic or participants can call from study 

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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 participant.  
 
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 participant and guardian verbally 
in the language they understand.  The explanation will be conducted in a quiet environment with 
adequate time given for the participant and guardian to review the study procedure before the 
commencement of the study.  Asking the participant to explain the study in their own words will assess 
the participant’s understanding.  If non-English speaking participants are enrolled in the study, the 
investigators will adhere to Section 10C of the COMIRB Instructions for Clinical Investigators regarding 
the consent of these participants. The consent form will also be translated into Spanish. The qualified 
personnel mentioned above will then obtain written consent from the guardian and assent from the 
participant, co-signed on the consent form, or in participants who are 18 years or older, direct consent. 
The participant and guardian will be provided a copy of the consent form for better understanding and 
record purposes.  
3. Special Consent/Assent Plan 
Consent will be obtained from all participants in the study.  Following explanation, all participants below 
18 years old will co-sign the consent form in addition to the parents signing the consent form.  All 
participants age 18 or older will sign the standard consent form. 
4. Participant Compensation, Incentives and Rewards  
Participants will be compensated with Target gift cards for completion of each study visit. The initial visit 
consisting of informed consent, lab draw, and abdominal ultrasound fibroscan will result in a $50 gift card. 
Each of the 2 MRI’s (Visits 2 and 4) will be $50 each. Each of the two overnight visits (Visits 3 and 5) will 
be $150 each. In an effort to maintain participant retention and completion of the study, the participants 
will be rewarded an additional $50 if they complete all study visits 1-5. Compensation for all completed 
visits will total $500. 
 
D.   Description, Risks and Justification of Procedures and Data Collection Tools: 
 
Please note: with the emergence of COVID-19 we must follow hospital policies for screening 
hospital patients and visitors. This may include testing for COVID-19. This may involve a nasal 
swab, and the risks of this are discomfort and potentially bleeding from nose. To limit participant 
time in our clinics, portions of study visits may be done virtually (phone or Telehealth). 
 
 
1. Blood Sampling 
Description: Blood will be drawn for Complete Blood Count (CBC), HbA1c, total and free testosterone, 
and sex hormone binding globulin. If participants have not had a full evaluation for oligomenorrhea, 
Prolactin, DHEAS, LH, FSH, TSH, total T4 or 17-OH progesterone may be drawn.  
Risk: Minimal. Risk of pain, bruising at site of blood draw, excessive amount of blood. 
Minimizing Risk: Certain studies at our institution draw over 7ml/kg in 6 weeks, or up to 7 ml/kg in a single 
draw, but include iron supplementation. Otherwise, the routine guidelines in our Pediatric CTRC are 
2.5ml/kg for a single draw and no more than 5 ml/kg over a 4 week period.  Our baseline visit will include 
11.5 ml of blood (HbA1c, Hb, Cr, AST, ALT) and 25.5 ml of blood for PCOS patients (additional draw for 
T4, TSH, prolactin, LH, FSH, 17OH progesterone and DHEAS). The OSTT visit includes up to 300 ml of 
blood which will occur within 4 weeks of the initial visit. Thus, our OSTT visit is within the NIH Clinical 
Center guidelines of 9 ml/kg in 6-8 weeks and within Children’s Hospital Colorado’s institutional 
guidelines of 5 ml/kg.  In addition, by study design, participants are screened by our baseline CBC and 
excluded if anemic, further increasing the safety of the study regarding blood draws. We will use a 
minimum weight cutoff of 38 kg to remain below the most conservative pediatric CTRC blood drawing 
guidelines. This screening also helps to increase the safety of the blood draw. In addition, the blood 
planned to be frozen and held could also be omitted if needed to reduce blood volume for a particular 
participant. Finally, our CTRC has a system to track other studies participants might enroll in, and we ask 

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during our consent process if the participant has been involved in any other studies in the past 6 weeks to 
avoid excessive blood drawing. 
Justification: Screening laboratory measurements are necessary to assure that patients meet 
inclusion/exclusion criteria before any further study is completed. A CBC is necessary as a screening lab, 
to rule out anemia. A hemoglobin A1c can be used to rule out diabetes. Hormone levels of free and total 
testosterone, and sex hormone binding globulin are needed to categorize patients as having PCOS, and 
prolactin, DHEAS, LH, FSH, TSH, total T4 or 17-OH progesterone to rule out other causes of 
oligomenorrhea, if not done previously.  
 
2. IV Risks 
Description: One peripheral IV will be placed during the OSTT for drawing blood samples. 
Risk: There is temporary discomfort when the needle goes in and 10% of the time there is a small amount 
of bleeding under the skin that may produce a bruise. Rarely, there is a risk of a blood clot forming or 
infection.  We will use a low dose of a medication called heparin to try to prevent blood clotting. 
Justification/Minimization: These studies involve sampling blood at multiple time points. Thus, an IV is 
needed, so as to avoid multiple needle sticks. These studies are focused on measured rates of change 
which necessitates the sampling of the same test over time. Proper sterile technique will be used with 
blood draws and IV placement to decrease the infection risk. EMLA cream will be used if participant 
desires to minimize pain of IV. 
 
3. Oral Sugar Tolerance Test (OSTT): 
Description: An OSTT will be performed with multiple blood draws. The purpose of the OSTT is to provide 
a controlled oral stimulus to effect changes in lipolysis and hepatic glucose release.  
Risk: The participants rarely experience nausea within 15 min of consuming the drink, however, the 
amount of carbohydrate is very similar to a large soda, which is regularly consumed by this patient 
population.  
Justification/Minimization: A standard oral challenge is needed to study lipogenesis, lipolysis and 
gluconeognesis in the fed state. We have chosen to start with a standard glucose and fructose load, to 
simplify the mathematical modeling. Dynamic carbohydrate metabolism in youth is made more relevant by 
the recently reported TODAY study, showing a decline in beta cell function in youth with newly diagnosed 
type 2 diabetes that was much more rapid than what has been reported in adults, and not prevented by 
metformin in the majority of the youth129. Our team of investigators, CTRC research nursing staff and 
physicians are well experienced with the OSTT blood draw procedure. A floor nurse located on the will be 
available during our inpatient visits and patients will be distracted by TV or other similar means during the 
OSTT, to minimize queasiness. 
 
4. Stable Isotope Studies Glycerol:  
Description: Oral stable isotope tracer of glycerol will be utilized to determine rates of intrahepatic 
substrate flux. These are substances normally present or produced in the body, and thus pose no more 
risk than typical glucose infusions. Measurements of these metabolic processes are only able to be made 
with the utilization of stable isotope tracers. 
Risk: We are utilizing an isotope which already exists in all humans, but are simply increasing the 
percentage. We are only giving this medication orally. These are NOT radioactive substances. 
Justification/Minimization: Pyrogen-free ²C13 glycerol will be obtained from the manufacturer and 
delivered to CHCO IDS. The IDS pharmacist will deliver the tracer to the 9th floor inpatient CTRC once 
ordered by the physician or outpatient if patient is admitted to University Hospital.  
 
4. Stable Isotope Studies Deuterated water:  
Description: Oral stable isotope tracer of deuterated water will be utilized to determine rates of de novo 
lipogenesis and gluconeogenesis. These are substances normally present or produced in the body, and 
thus pose no more risk than typical water unless consumed in large amounts, to increase total body 
enrichment in excess of 20%130. Measurements of these metabolic processes are only able to be made 
with the utilization of stable isotope tracers. 
Risk: We are utilizing an isotope which already exists in all humans, but are simply increasing the 
percentage. We are only giving this medication orally. These are NOT radioactive substances. The 

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planned dose is 3 ml/kg as has been used previously in obese youth126, with a goal enrichment of 0.3%, 
well below the 20% known to cause side effects. At the proposed dose, few subject experienced nausea, 
but it was extremely well tolerated (personal communication with PI’s Dr. Santoro and Parks). 
Justification/Minimization: Pyrogen-free deuterated water will be obtained from the manufacturer and 
delivered to CHCO IDS. The IDS pharmacist will deliver the tracer to the 9th floor inpatient CTRC once 
ordered by the physician or outpatient if patient is admitted to University Hospital.  
 
6. Standard Diet  
Description: A dinner and snack will be provided during the overnight visit. selected from limited choices 
on the Children’s Hospital cafeteria carbohydrate counting diet plan, with at least 20 grams of carbs in the 
snack and 40 grams of carbs for dinner. 
Risk: None 
Justification/Minimization: Variations in diet, activity and circadian rhythms affect metabolism 121. 
 
7. Magnetic Resonance Imaging (MRI) 
Description: The MRI will usually be obtained the day of admission to CHC), at the UCD Brain Imaging 
Center on the Fitzsimmons campus. A trained research radiographer who is supervised by Dr. Mark 
Brown, of UCD radiology, will perform an abdominal MRI to obtain hepatic, visceral and subcutaneous fat 
on a 3.0 T whole–body MRI scanner (Siemens MAGNETROM, Malverne, PA). Participants will lie supine 
while these measurements are obtained, need to hold reasonably still during the scan and cannot weigh 
>325 lbs. A second sequence to measure the amount of fibrosis (if any present) in the liver will be 
performed. A specialized phosphorus coil will be utilized to measure the concentration of 31P via MRS.  
Risks: Minimal. Participants may develop claustrophobia in the magnet. 
Minimizing Risk: The participant is provided with audio protection and optional television to help increase 
comfort. Some participants might feel claustrophobic while having an MRI and the scan will be stopped if 
it cannot be tolerated. In addition, any participants with implanted metal that is not cleared by the MRI 
technician may not be able to have the MRI due to the type of magnet involved. 
Justification/Minimization: MRI is a non-invasive and non-radiation method to assess body fat, and 
mitochondrial function. The risks are minimized by assuring patient comfort prior to starting the scan, 
placing eye goggle that plays movies on the participants. Further, per standard protocol, no patient will be 
placed into the scanner if they do not meet the rigorous safety standards for the MRI, including the 
absence of non-compatible implanted metal. 
 
8. Body Composition 
Description: Body composition will be measured using the DEXA technique and will be used to derive fat-
free mass and % body fat.  This technique relies on the absorption of dual electron wavelengths for the 
assessment of body fat, lean tissue, and bone mineral density.  During the procedure, the participant will 
be supine on the measurement table, and the arm of the machine will slowly pass over their body.  
Risk: Minimal. Radiation exposure 
Justification/Minimization: Body composition is best assed via DEXA, and the amount of muscle mass is 
needed to standardize the OSTT results, since body weight can vary greatly. This procedure will deliver the 
radiation exposure that is 2 times the level of background radiation in Colorado. Participants will be tested 
for pregnancy immediately prior to DEXA, to ensure that they are not pregnant. 
 
9. Endopat and Dynapulse 
Description: The Dynapulse Pathway and the EndoPat system are noninvasive portable systems that 
measure brachial artery distensibility and endothelial function, utilizing a standard sphygmomanometer 
cuff inflated in the same fashion as a sphygmomanometer to obtain blood pressure. The instrument 
derives brachial artery distensibility using the technique of pulse waveform analysis of arterial pressure 
signals obtained from the sphygmomanometer. Measurement of heart rate variability for autonomic tone 
will be performed using the Endopat 
Risk: This procedure may lead to mild discomfort due to the blood pressure cuff being inflated. 
Justification/Minimization: Endothelial function is a novel measure in PCOS and will aid in the 
determination of cardiovascular dysfunction with this population.  
 

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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: Participant will be provided two accelerometers (GT3X BT by Actigraph and ActiWatch by Philips 
Respironics) to be worn for seven days to measure level of habitual physical activity, which affects insulin 
sensitivity, and sleep patterns. The accelerometers will be worn on the participant’s wrist. The wrist position has 
been validated to hip position actigraphy in this population. 
Risk: There is no risk involved with the accelerometer. 
Justification/Minimization: Accelerometers are effective tools for the objective measurement of physical 
activity 125 because they have the ability to continuously record physical activity data and such data can 
be used to estimate METs of activity. They provide more detailed information than pedometers, which 
only measure walking steps, and help get around the recall bias of questionnaires. We are currently using 
the GT3X BT Actigraph in adolescents in our other diabetes studies; therefore, we are familiar with their 
use in this population and have the necessary computer software and interpretation skills.  The Actiwatch 
is being used as a tool for objective measurement of sleep patterns. The Actiwatch is fitted with a LED 
monitor that records multiple spectrums of light to better assess sleep patterns in this population. 
 
12: Metabolic Cart: 
Description: The metabolic cart measures the amount of air that the participant breathes in and out. The 
machine attaches to the participant’s mouth through a tube, or a plastic bubble that is placed over the 
participant’s head. There is the potential for experiencing claustrophobia from having the plastic bubble 
over the participant’s head. A metabolic cart will be utilized multiple times during the OSTT study day to 
measure rates of oxygen consumption and carbon dioxide release. These rates can be utilized to 
calculate rates of carbohydrate and fat oxidation and resting energy expenditure. 
Risk: Minimal risk of claustrophobia. 
Justification/Minimization: These studies are well tolerated by youth, and involve placing a clear plastic hood 
over the participant’s head for approximately 20 minutes. The data collected from the baseline study is also very 
useful for assisting obese participants in determining their true caloric needs, and useful in setting dietary goals 
for weight loss. This piece of information is thus utilized in post-study nutritional counseling. 
 
13. Food Frequency Questionnaire (SEARCH FFQ) 
Description: Customary macronutrient pattern will be ascertained by diet interview at the time of admission 
using a SEARCH FFQ, modified to incorporate common food choices among ethnically and regionally 
diverse youth aged 10-19 participating in another large childhood diabetes study, SEARCH (48).  The 
instrument is self-administered with staff support to provide instructions, answer questions, and to review 
the form after completion, and captures the last week of dietary intake.   
Risk: None 
Justification/Minimization: Several of the measurements being assed are affected by prior nutritional intake. 
Furthermore, participants will receive dietary counseling at the end of the study, and by knowing what their 
previous dietary pattern is, suggestions for improvement can be tailored to their specific dietary habits. 
 
14. 3DPar Questionnaire  
Description: A questionnaire (3DPAR) recalling the physical activity levels of the three previous days will be 
completed at visit 3 and visit 5. 
Risk: None 
Justification/Minimization: Physical activity can directly affect insulin sensitivity, our primary outcome 
measure. The 3DPar is a well validated measure to asses 3 days of physical activity in youth, and includes a 
variety of youth centric activities. 

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15. Strengths and Difficulties Questionnaire: 
Description: This is a survey which identifies areas in a youth’s life that they believe they are strong or weak 
in dealing with, as a measure of coping skills. Low coping skills have been associated with the development 
of depression. 
Risk: None 
Justification/Minimization: This survey can help identify youth at risk for depression or anxiety, and identify 
poor coping skills. It does not directly assess depression or suicidality. 
 
16. WatchPAT and Questionnaires to assess for Obstructive Sleep Apnea 
Description: The WatchPAT is a noninvasive portable system that measures the oxygen saturation and 
apnea hypopnea index. Three surveys querying signs and symptoms of obstructive sleep apnea. 
Risk: No risk associated with the questionnaires and the WathchPAT,other than a mild discomfort from 
having to wear the watch and cuff around finger during sleep. It is possible that we will discover that the 
participant has obstructive sleep apnea, and will need to be referred for further clinical care. 
Justification: Obstructive sleep apnea is associated with obesity, and can worsen both fatty liver and insulin 
resistance. Thus the presence of OSA must be accounted for when measuring either of these outcomes. 
These surveys selected are currently being utilized by the NIH multiple center study in obese youth at risk for 
diabetes, and are well validated in youth from multi-ethnic populations.  If OSA is suspected during the 
course of the screen, the participants will be referred for further evaluation and treatment. The WatchPAT is 
an FDA approved device that can be used specifically for oxygen saturation and apnea hypopnea index and 
is approved in children as young as 12 years of age, within the age range of our study population. 
 
17: Gut Bacteria Collection: 
Description: A week prior to visit three, participants will be provided with stool collection swabs to collect a 
small sample of stool from the toilet paper they use after having a bowel movement. 
Risk: Although the risk is minimal, participants may feel uncomfortable taking a sample of stool from the 
toilet paper following a bowel movement. All participants will be instructed to follow proper bathroom 
etiquette as fecal matter can transmit diseases. 
Justification: Studies in obese individuals with type 2 diabetes have alterations in the gut microbiota that 
may be related to NAFLD. These studies have not been performed in PCOS. 
 
18. Violation of Privacy and Loss of Confidentiality  
Description: These are both risks to which research participants are exposed. The possibility of these 
risks increases when protected health information is collected. Every effort will be made to decrease this 
risk by limiting access to protected health information, storing this information in a password protected 
database, and identifying participants only by a unique identifier that is kept in a separate location in a 
locked container, traceable only by study personnel. All of the tests involve the risk of identifying 
asymptomatic abnormalities. The study may include risks that are unknown at this time.  
Justification/Minimization: Every effort will be made to decrease the risk of loss of confidentiality by 
limiting access to protected health information, storing this information in a password protected database, 
and de-identifying study specimens.   
 
E. Benefits of the study: 
Benefits to Society: 
PCOS affects 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: 

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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. The data to be generated from this project 
will be utilized to inform R01 grant applications of treatment trials with new medications. Baseline and 
placebo phase data will add to our knowledge of NALFD in girls with PCOS. This study will also test the 
efficacy of a food based supplement to decrease hepatic steatosis and lead to a new treatment option. 
Individual: Participant 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 participants who completed related protocols (10-1288 and 14-0542) 
and received this counseling have higher rates of weight loss upon clinical follow-up than those children 
being seen in obesity clinics alone. Further, participants may benefit from the amino acid supplement and 
all participants will receive this therapy. 
Benefits to participant: 
1) All participants 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 participants 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 participants have signs of 
obstructive sleep apnea, they will be referred to the sleep clinic for appropriate evaluation and treatment. 
2) All of the participants enrolled must be sedentary, with less than 2 hours a week of physical activity. 
This is less than the time recommended 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 participants will receive counseling on how to increase their activity levels by trained study staff. 
Increased activity has been shown to reverse the risk for diseases later in life. 
3) All participants will complete a 7 day food questionnaire. This will be reviewed with them, and healthier 
food choices and meal planning will be discussed with both the participant and their parent by the PI or 
PRA, all of who are trained in providing diet prescriptions. Additionally, obese participants 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. 
5) All participants will receive the amino acid supplement, which may decrease hepatic steatosis and 
improved glucose metabolism. 
Evidence of Direct Benefit 
We believe that this protocol is in the 405 risk category for pediatric research. Participant s can benefit 
from the above study measurements. Further, at the conclusion of the study, all participants in the 
protocol will also be given counseling on the benefits of exercise as discussed above, and given an 
exercise prescription. The participants may also be re-contacted once by phone call to follow-up on their 
study results and exercise recommendations. They may also be followed up in a clinical setting if PI notes 
further benefit of clinical follow up. Sedentary participants 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 participants 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 or fibrosis. The blood tests 

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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 participants, 
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 
participant. 
Inclusion criteria is less than 2 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. 
The primary benefit from the OSTT 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.  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, and diabetes. 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. Fifty percent of obese girls with PCOS, a hormonal disorder affecting 
15-20% of obese females, have hepatic steatosis (HS), which causes premature mortality due to an 
elevated risk for type 2 diabetes (T2D), cardiovascular disease (CVD) and progressive liver disease in 
early adulthood. The proposed project will combine a tolerable and relatively inexpensive therapeutic 
option with both clinical outcomes and innovative methods to quantify intrahepatic metabolism. Treatment 
options for PCOS are limited and implementation of effective lifestyle changes are very difficult for obese 
adolescents. This work would contribute to the development of a dietary intervention that could treat 
NAFLD in PCOS thus decreasing current and long term risks for T2D, CVD and advanced NAFLD and 
prolonging life for these high risk girls. 
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.  
 
F. Alternative Treatment 
The alternative is for participants to not participate in the study 
G. Consideration of Specific Participant Categories 
1. Inclusion of Women 
All participants will be women, as PCOS only occurs in females. 
2. Inclusion of Minorities 

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Every effort will be made to include a diverse participant distribution. PCOS affects Caucasians, 
Hispanics and African Americans equally. 
3. Inclusion of Children 
All participants will be between ages 12 and 21. Insulin sensitivity needs to be studied in the adolescent 
age group as available is scarce in this age group and it is critical to understand the pathophysiology of 
PCOS in its developing stages.  
IV. Potential Scientific Problems:   
Limitation of Method Development: 
The protocol as described includes an OSTT with an oral tracer will be conducted in all participants. The 
specific points that are to be evaluated are listed below: 
1) Measurement of Hepatic Substrate flux in youth during fasting. 
a. Purpose: Asses hepatic substrate flux in youth looking at shifts from pentose phosphate 
pathway (PEP), TCA cycle and fatty acid synthesis 
i. Rational: utilizing and oral glycerol model with NMR isotopmer analysis is a newer technique. 
We have validated the timing with an OSTT in youth. However, this relies on consistent OSTT 
responses. When we anticipate that the response to the OSTT may change, the timing and 
thus results would vary, and we are thus better off using a fasting model. This has been done 
in adults, but not in our youth. 
ii. Measurement: NMR isotopomer analysis with pathway flux calculations and modeling in the 
fasting state 
iii. Revision plan if model not correct: 1) increase the amount of tracer given 2) increase the 
frequency of blood draws in the later sampling period. 
 
Overall Project Limitations: 
We expect to complete 20 participants in 2.5 years. As with any clinical trial, recruitment and retention 
can be challenging. We have successfully recruited over 120 obese girls with untreated PCOS, and thus 
our recruitment goals are in line with previous work. We have never needed to recruit in the community or 
other endocrinology practices, but can if needed. Within our department we have been successful with 3 
month, 21 month and 3 year interventional protocols and we have designed the study with techniques 
found to be successful in similarly obese adolescent populations. Adherence to treatment is also a 
challenge, and we will use texting and emails as preferred contacts in youth to send supplement 
reminders. Major changes to diet and exercise could alter results, and we will request participants not to 
initiate major lifestyle changes during the study. An alternative would be to recommend lifestyle changes 
for all girls, although from our clinical experience these are poorly followed. 
 
Aim 1: We anticipate that after EAA, absolute IHTG will decrease by 3% compared to placebo, secondary 
to decreased DNL. Based the mean of 11±3.5% IHTG in PCOS girls with HS, this is less than the relative 
50% decrease we found in most of older adult participants. Further, 9 days of low fructose dietary 
modification reduced IHTG by 50% via decreased DNL in obese youth with HS, demonstrating that IHTG 
can be rapidly changed with dietary modification and relates to decreased DNL132. However, this patient 
population does have a very poor diet, and it is possible that those with excessive soda intake will not 
benefit as anticipated if EAA cannot overcome the excess glucose. An alternative would be to provide the 
diet for participants for 3 days prior to studies. We will be using a FibroScan ultrasound based method to 
determine the presence of HS at screening. MRI is the most sensitive and repeatable measure for IHTG. 
Ideally we would also perform an MRI for IHTG at screening to determine eligibility, however the cost and 
time of this for the 50% of girls to be excluded is excessive and the processing time for the results is not 
conducive for a screening test. Whereas our lab has not used deuterated water to measure DNL51, this 
method has been used successfully in other youth populations by our collaborator Dr. Parks, and we thus 
are confident that we will be able to perform this analysis. 
 

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Aim 2: We expect that insulin sensitivity will improve following EAA and that the glucose concentration at 
2 hours of the OSTT will decrease. The reproducibility of glucose measurement from an OSTT in any 
individual is only 75%, however, this is when the effects of exercise, diet, sleep and menstrual cycle are 
not controlled. An alternate approach for assessing insulin sensitivity would be a hyperinsulnemic 
euglycemic clamp, for which we have extensive experience. We have not elected to use this as it is not 
physiologic and does not allow for isotopomer assessments. We expect to measure hepatic metabolism 
with metabolomics and isotopomer analysis and to integrate these results with clinical outcomes. We 
hypothesize that those with worse HS will have increased futile TCA cycling and increase BCAA which 
will improve with improved insulin sensitivity and decreases in liver fat after EAA. If our hypotheses are 
incorrect, the data may suggest additional mechanistic pathways unique to PCOS. Interpretation of the 
resulting data requires extensive knowledge of biochemistry and I have the unique skills, time, 
institutional support, collaborators, and mentoring to be successful.  
 
 
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 
(Redcap).  
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 study coordinator will monitor the protocol and the safety of the research 
participants.  The PI will review all laboratory data and report any abnormal values to the patient and 
guardian and instruct the participant to follow-up with their PCP.  If an abnormal result from a research 
procedure exists, the PI will notify the family and their PCP and refer the participant to the appropriate 
clinic for further evaluation. The PI may also share research results in a reasonable and prudent manner 
with appropriate medical professionals if the participant was seriously injured as a result of a procedure or 
if follow-up of the result of the procedure is in the best interest of the participant’s health as determined by 
a medical professional. If immediate medical follow-up of participant required, the PI will share the 
research results via EPIC when clinically relevant. The PI will report adverse events, and any decision to 
suspend or halt the protocol to CTRC and COMIRB immediately.  The PI will also prepare a written report 
for the yearly continuing review required by COMIRB and the CTRC.  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 (DOB, sex, ethnicity, address, etc.), diagnosis (es), history and physical, laboratory or tissue 
studies, radiology studies, procedure results, survey/questionnaire results, research visit records, and 
portions of previous Medical Records that are relevant to this study. This information will be accessible 
only by the study investigators, Federal agencies overseeing human participant research, the Colorado 

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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 OSTT 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 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. If follow-up or referral of abnormal research results or procedure 
is required, the PI will place a referral in EPIC, update the PCP on patient condition and reason for 
referral and contact family to discuss follow-up treatment options. 
a) Adverse Event Reporting 
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 study enrollment) are not considered or 
recorded as AEs or SAEs unless the condition worsens in intensity or frequency after enrollment.  
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 as 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 
COMIRB and any other required institutional monitoring committee.   
            

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C. Participant Discontinuation Criteria 
If a participant experiences any of the following, the participant will be withdrawn from the study. 
1. Inability to complete study procedures 
2. Abnormal screening labs (LFT’s >125 IU, HbA1C>6.4%, Hg <11 mg/dL) 
3. Participant becomes pregnant during study 
 
D. Protocol Stopping Criteria 
If one or more participants experience any of the SAE’s listed above, the PI will consult with the study 
staff prior to continuing study visits with participants.  The PI and RSA will consult about the significance 
of the SAE’s and make a recommendation about participant continuation in the study.  
 
VII. Data Analysis Plan:   
 
Overall Project Statistical Plan: 
   Sample size analysis: Sample size is based on the power to detect a change in IHTG with treatment 
with EAA as compared to placebo (primary endpoint).  Assuming a 2-sided α=0.05, 20 participants 
provides 86% power to detect a change of 2.8% in IHTG, assuming a 3.9% standard deviation of change, 
which corresponds to a correlation of 0.8 between repeated measures. For Aim secondary measures, a 2 
sample size of 20 gives 99% power to detect a change of 14.4 mg/dL in 2 hour OSTT glucose and 81% 
power to detect a change of 1.8 in HOMA-IR, assuming a standard deviation of 14.4 mg/dL and 2.7, 
respectively.  Estimates of effect size and variability were conservatively estimated from the PI’s 
extensive pilot data.  
    
Analysis plan: No interim analyses are planned. Prior to the start of any formal analyses, unusual values 
that need to be queried, patterns of missing values, and whether their distributions are non-Gaussian will 
be identified.  Demographic and clinical characteristics will be summarized with descriptive statistics.  
Analyses will follow the “intent-to-treat” principle so that study hypotheses are tested under realistic 
conditions in which not all participants adhere to the intervention.  For the primary outcome, we will 
compare IHTG with and without EAA treatment using a paired t-test or a Wilcoxon signed rank test. 
Similar analyses will be used for secondary outcomes (2 hour OSTT glucose, HOMA-IR, metabolomics 
markers, proportion of direct/indirect glucose, EAA concentrations).  Linear models will be used to identify 
the variables that are associated with change in IHTG, such 2 hour OSTT glucose, HOMA-IR, 
metabolomics markers, etc.   
 
All measures will be adjusted for OSA status (yes/no) and/or severity of AHI. 
 
Secondary outcomes include a Pearson correlation test of potentially related variables followed by 
targeted multiple regression to assess the relationship between hepatic fat and adipose IR, PEP, TCA, 
TG synthesis pathways.  
 
Likely contributors to endpoints: Variables known to affect IHTG and IR will be measured: lipid/glucose 
markers (lipid panel, HbA1c); inflammatory markers (C-reactive protein, AST, ALT, GGT); glucose-related 
hormones (glucagon, GLP-1 and leptin), sex-steroids (DHEA-S, free and total testosterone, sex hormone 
binding globulin, progesterone, estradiol); body composition (BMI, waist:hip ratio, lean and fat mass by 
DEXA); Variations in diet, activity, menstrual phase and circadian rhythms affect metabolism and will be 
assessed: dietary intake (7 day food diary), habitual activity (3 day questionnaire and accelerometer for 7 
days prior to study) and sleep timing and quality (sleep questionnaires and sleep and light watch). OSTT 
studies will be performed in the morning fasting, in the follicular phase of the menstrual cycle where 
possible, preceded by 3 days of no strenuous physical activity and a typical diet for the participant. 
 
Non-Isotope OSTT calculations: The Matsuda model of IR will be used but adapted for the 6.5 hour 
OSTT, 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 calculated109,129,133,134. Differences 
between groups will be evaluated with and without controlling for activity and sleep status. 
      

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Fitting glucose and glycerol concentrations curves:  Using computed data points, glycerol and glucose 
concentrations will be fitted with an appropriate model.  In control participants, concentration typically 
shows 3 phases in OSTT 135. Cubic polynomial, piecewise-linear, spline, and dynamic models have been 
used for fitting Ra in previous work130,136. 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 (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 concentration changes. 
 
Metabolomics data: Differences in targeted metabolic concentrations will be compared between groups, 
after appropriate cleaning of the data, as performed by the processing site. Changes in metabolites over 
time will be compared between groups, to determine the times that demonstrate the largest differences. 
Results will also be compared to isotopomer results, to see if a metabolomics endpoint can yield similar 
information to the isotopomer analysis. 
 
Gut Microbiota: The microbiota profile is entirely exploratory, and will be related to status of hepatic 
steatosis and 2 hour glucose status of impaired glucose tolerance or not. 
          
VIII.  Summarize Knowledge to be Gained:  
Overall Expected Results:  We anticipate that we will determine which specific areas of hepatic 
metabolism are upregulated in girls with hepatic steatosis and can these be changed with EAA 
supplementation. This data would then provide the background evidence for future larger therapeutic 
trials. We will also continue to refine the isotopomer protocol with extension into fasting. 
 
Significance: Recent evidence in adults with PCOS indicates that 60-70% have NAFLD32,42. 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 protocols. 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 failure34,35. Thus, 
understanding the hepatic metabolism associated with 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. Further, we 
will be testing a well-tolerated oral nutritional supplement. 
 
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