Investigating the Relationship Between Circadian Phase and Insulin Resistance in Obese Adolescents
Conditions: Obesity
Sponsor: University of Colorado, Denver

 
 
 
 
NCT# 02585830  
 
Protocol #: 15-0739 
Project Title: Investigating the relationship between circadian phase and insulin 
resistance in obese adolescents 
 
 
 
Principal Investigator:  Stacey L. Simon, PhD 
Version Date 22Sept2016 
Version Number 2 
 
 
 
 
 
 
I. 
Hypotheses and Specific Aims:   
HYPOTHESIS: In obese adolescents, delayed melatonin onset and offset will correlate with insulin 
resistance (IR), and may be affected by sex. 
SPECIFIC AIMS:  
Aim 1:  Establish a method to measure and model the melatonin profile, including melatonin onset and 
offset, as a marker of circadian rhythm in obese adolescents. 
Aim 2: Develop a physiologically-based mathematical model of adolescent human sleep/wake and 
circadian interactions, and apply the model to investigate the mechanisms associated with published 
changes in sleep/wake behavior observed in adolescent sleep as well as in individual subjects. 
Aim 3:  Relate the timing of melatonin onset and offset to measures of insulin resistance in obese 
adolescents and examine sex differences among these variables. 
 
Research Methods 
 
Outcome Measure(s):   
Primary Outcomes: Melatonin phase assessment and IR 
Melatonin phase assessment: During the study visit, subjects will be admitted to the Children’s Hospital 
Clinical research center inpatient unit at 4PM on Wednesday, Thursday or Friday.  The profile of 
melatonin release will be assessed at the study visit using a serial saliva sampling method from 5PM on 
Thursday or Friday to 12PM the following day. Saliva (~2mL) will be collected in 30 or 60 minute intervals 
in dim light (<10 lux in the room; ~1.9 lux in the angle of gaze, confirmed by light meter) using polystyrene 
tubes throughout the night. Individualized melatonin sampling schedules will be determined to optimize 
calculation of DLMOn and DLMOff times by introducing 30 minute sampling at the times at which DLMOn 
and DLMOff are predicted to occur based on actigraphy-reported sleep, wake, and midsleep times1.   
Assessment of IR:  During the screening visit, a 3-hour frequently sampled OGTT will be performed in the 
outpatient CTRC. The OGTT will take place in the AM, after a fast of at least 10 hours. A 20-gauge iv 
catheter will be inserted in the antecubital vein for blood sampling. After baseline samples are collected, a 
3-hour OGTT with 75 mg dextrose drink will be performed. Glucose and insulin samples will be collected 
every 30 minutes. In addition, we will assess fasting labs to check for other components of metabolic 
syndrome including hemoglobin A1C for dysglycemia, ALT/AST for liver inflammation, sex hormone 
binding globulin as a marker of IR, cholesterol panel, CRP as a marker of inflammation, leptin as a gut 
hormone known to influence hepatic IR, and adiponectin, thought to influence adipose IR. Additionally, 
blood pressure will be taken to assess for hypertension and waist/hip ratio will be measured as a marker 
of metabolic disease.  
Additional Measures: Questionnaires assessing additional sleep parameters, mood/behavior, and 
diet/activity will be completed by participants to identify variables to control for in analyses (e.g, presence 
of symptoms of sleep-disordered breathing) and/or utilize in exploratory analyses (e.g., relationship 
between sleep and diet/activity). 
i. The Morningness/Eveningness Scale for Children 2 and the Munich Chronotype 
Questionnaire for Children 3 will be used to determine chronotype (diurnal preference).  

ii. The Cleveland Adolescent Sleepiness Questionnaire 4, Sleep Disturbances Scale for 
Children/Adolescents 5, and the Adolescent Sleep Hygiene Scale 6 will be completed by 
participants to obtain information on behavioral sleep variables, including daytime sleepiness 
and sleep-promoting and inhibiting factors.  
iii. The Strengths & Difficulties Questionnaire 7,8, an adolescent self-report tool, will be used 
to assess symptoms of mood and behavior problems. 
iv. The Profile of Mood States 2 Short Version9, an adolescent self-report measure of mood, 
completed on the computer. 
v. The Three Day Physical Activity Recall (3DPAR10), a questionnaire asking youth to recall 
their physical activity levels from the previous three days, will be completed. Physical activity 
can directly affect insulin sensitivity, our primary outcome measure.  
vi. Macronutrient intake pattern will be ascertained using the SEARCH Food Frequency 
Questionnaire, devised to include common food choices among ethnically and regionally 
diverse youth aged 10-19 years. 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.  
vii. Psychomotor Vigilance Task11,12, a computerized test of sustained attention with particular 
sensitivity to sleepiness. 
viii. 
Description of Population to be Enrolled:   
Participants: This pilot study would allow for preliminary data collection on up to 30 obese adolescents 
(15 male, 15 female).  
Recruitment: Participants will be recruited from adolescent, obesity and community clinics. 
Subject Criteria:  
Inclusion criteria:  
1) High school students between the age of 15-19 years (the peak developmental period for delayed 
circadian phase13,14);  
2) BMI >90th percentile for age and sex;  
3) Tanner stage 2 or greater.  
Exclusion criteria:  
1) Any medications that affect IR or sleep (e.g., metformin, hormonal contraception, stimulants, 
atypical antipsychotics);  
2) regular use of melatonin or other sleep aids;  
3) a prior diagnosis of obstructive sleep apnea, diabetes (HbA1c > 6.5), liver disease other than 
non-alcoholic fatty liver disease, pregnancy or breastfeeding;  
4) IQ<70 or severe mental illness that may impact sleep (e.g., schizophrenia, psychotic episodes), 
verified through chart review;  
5) teens not enrolled in a traditional high school academic program (e.g., home school students);  
6) night shift employment;  
7) travel across more than two time zones in the month prior to the study.     
Informed Consent Plan: Appropriately qualified and informed personnel who have completed the 
COMIRB and HIPPAA course requirements will fully explain the study protocol and consent form to the 
subject and guardian verbally in the language they understand.  The explanation will be conducted in a 
quiet environment with adequate time given for the subject and guardian to review the study procedure 
before the commencement of the study.  Asking the subject to explain the study in their own words will 
assess the subject’s understanding.  If non-English speaking subjects are enrolled in the study, the 
investigators will adhere to Section 10C of the COMIRB Instructions for Clinical Investigators regarding 
the consent of these subjects.  The qualified personnel mentioned above will then obtain written consent 
from the guardian and assent from the subject, co-signed on the consent form, or in subjects who are 18 
years or older, direct consent. The PI will make a good faith effort to obtain both parent signatures.  The 
subject and guardian will be provided a copy of the consent form for better understanding and record 
purposes.   
Special Consent/Assent Plan; Consent will be obtained from all participants in the study.  Following 
explanation, all subjects below 18 years old will co-sign the consent form in addition to the parents 
signing the consent form.  All subjects age 18 or older will sign the standard consent form. 
Subject Compensation, Incentives & Rewards 

Subjects will be compensated with Greenphire debit card payments during each scheduled visit. 
Participants will receive $5 for the initial visit that includes signing consent and receiving the actigraphy 
watch. After completing the screening visit, consisting of OGTT, and questionnaires, participants will 
receive $50 loaded on to a debit card. Participants will receive $100 on the debit card following the 
overnight study visit. Compensation for all completed visits will total $155.  
 
Study Design and Research Methods   
Study Timeline: Participants will have 3 visits:  an initial visit, a screening visit, and an overnight study 
visit. The initial visit will include informed consent and assent and receiving an actigraphy watch and 
instructions. The screening visit will include questionnaires, 3-hour frequently sampled oral glucose 
tolerance test (OGTT), and fasting labs to check for other components of metabolic syndrome.  During the 
overnight study visit subjects will be admitted to the Children’s Hospital Clinical research center inpatient 
unit from 4PM Thursday or Friday to 12PM the following day for salivary melatonin collection.  During the 
entire protocol, teens will be asked to abstain from caffeine, non-steroidal anti-inflammatory drugs, 
marijuana, high carbohydrate meals, and alcohol. 
Initial visit and sleep/wake monitoring: The initial visit will last approximately 30 minutes and will include 
consent and assent. Participants will be provided an actigraphy watch to monitor typical home sleep 
patterns15,16. The Spectrum sleep/wake monitor (Phillips Resperonics, Bend, OR) will be worn on the non-
dominant wrist and will collect data on movements in 1-min bins. The watch will be worn for the 7 days 
preceding the initial visit and participants will complete a concurrent sleep diary emailed to them each 
morning via RedCap to allow for removal of artifacts (e.g., failure to wear the actigraphy watch during a 
portion of the night). If participants have not completed the sleep diary by mid-morning, a RedCap email 
and text message reminder will be sent. The text messages will be generic so as not to identify the 
participant or that they are part of a research study. If participants do not have email or computer access, 
they can complete a paper/pencil sleep diary. 
Screening visit and sleep/wake monitoring:  The screening visit will last approximately 4 hours and will 
ideally occur on Thursdays or Fridays in order to be consistent with the amount of accumulated sleep 
debt incurred over the week. Menstruating girls will be scheduled in the follicular phase when possible, as 
menstrual timing can influence measures of IR. Participants will receive or be mailed an actigraphy watch 
to wear for 7 days prior to the overnight study visit while filling out a concurrent sleep diary. 
Teens will follow a self-selected sleep schedule during the pre-study week, but data collection will 
take place during the academic year to capture participants’ typical sleep schedule. Data will not be 
collected during the week following daylight savings changes or in a week following travel across one or 
two time zones. Average sleep start and end time, sleep duration, and sleep efficiency will be measured.   
 
Description, Risks and Justification of Procedures and Data Collection Tools: 
1. Blood Sampling 
Description: Blood will be drawn using a peripheral IV for HbA1c, ALT/AST, sex hormone binding 
globulin, a cholesterol panel, CRP, leptin, and adiponectin, and OGTT.  
Risk: Minimal. Risk of pain, bruising at site of blood draw, excessive amount of blood 
Minimizing Risk: The study involves sampling blood at multiple time points, thus an IV is needed to avoid 
multiple needle sticks. Proper sterile techniques will be used with blood draws and IV placement to 
decrease infection risk. EMLA cream will be used if subject desires to minimize pain of IV. The routine 
guidelines in our Pediatric CTRC are 2.5ml/kg for a single draw and no more than 5ml/kg over a 4 week 
period.  For this study, the baseline labs will include 13 ml of blood and the OGTT includes 29ml of blood. 
Thus, our study visit is within the NIH Clinical Center guidelines of 9ml/kg in 6-8 weeks and within 
Children’s Hospital Colorado’s institutional guidelines of 5 ml/kg.  We will use a minimum weight cutoff of 
38 kg to remain below the most conservative pediatric CTRC blood drawing guidelines. Finally, our CTRC 
has a system to track other studies subjects might enroll in, and we ask during our consent process if the 
subject has been involved in any other studies in the past 6 weeks to avoid excessive blood drawing. 
Justification: These labs are to measure for components of the metabolic syndrome, and to assess the 
primary study endpoint, IR. A hemoglobin A1c can be used to rule out diabetes.  
2. Oral Glucose Tolerance Test (OGTT) 
Description: An OGTT will be performed with multiple blood draws over 3 hours. The purpose of the 
OGTT is to provide a controlled oral stimulus to effect changes in lipolysis and hepatic glucose release.  

Risk: The subjects rarely experience nausea within 15 min of consuming the drink, however, the amount 
of carbohydrate is very similar to a large soda, which is regularly consumed by this patient population.  
Justification/Minimization: A standard oral challenge is needed to study insulin resistance in order to 
examine the relationship with sleep. Our team of investigators, CTRC pediatric research nursing staff and 
physicians are well experienced with the OGTT blood draw procedure. A floor nurse located on the 9th 
floor of CHC will be available during our inpatient visits and patients will be distracted by TV or other 
similar means during the OGTT, to minimize queasiness. 
3: Actigraphy  
Description: Each subject will wear an accelerometer for 14 days (Spectrum, by Philips Resperonics) to 
measure habitual sleep/wake patterns. The accelerometer will be worn on the non-dominant wrist, similar 
to a wristwatch. 
Risk: There is no risk involved with the actigraphy device. 
Justification/Minimization: Actigraphy is an effective tool for the objective measurement of sleep because 
they have the ability to continuously record sleep and activity data and such data can be used to estimate 
sleep/wake patterns. 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 Spectrum 
Actigraphy watches in adolescents in our other research studies and in routine clinical care; therefore, we 
are familiar with their use in this population and have the necessary computer software and interpretation 
skills.   
4. Questionnaires 
Description: Participants will complete questionnaires regarding their sleep, physical activity and eating 
habits, and mood and behavior.  
Risk: There is a small possibility that participants will be uncomfortable answering a particular question. 
Justification/Minimization: The questionnaires used in the study are widely used in both research and 
clinical practice, and are not of a particularly sensitive nature. Participants will be able to skip any items 
that they find uncomfortable. None of the items assess for suicidality.  
5. Overnight stay with salivary melatonin sampling  
Description: Saliva will be collected every 30-60 minutes from 5pm-noon.  
Risks: Discomforts include unsettling to sleep away from home in a strange place. Providing saliva 
samples may make the mouth feel dry. Participants may obtain inadequate sleep.   
Justification: The melatonin will provide data on the chemical signaling involved in falling asleep and waking 
up, as they relate to changes seen with the sleep study. Saliva is good alternative to blood sampling in 
pediatric populations. Short term risks are associated with inadequate sleep, including daytime sleepiness 
and inattention. Inadequate sleep is readily reversed with 1-2 nights of good sleep. As will be indicated on the 
consent form, subjects will be asked not to drive, operate heavy machinery, or engage in jobs that require a 
high degree of coordination, balance, or attention (e.g., roofing) for the day subsequent to the overnight visit. 
When reviewing the consent form, we will call attention to these prohibitions and exclude participation if they 
report being unable to follow them (e.g., the participant has a job that requires driving).   
6. Violation of Privacy and Loss of Confidentiality  
Description: These are both risks to which research participants are exposed. The possibility of these 
risks increases when protected health information is collected. Every effort will be made to decrease this 
risk by limiting access to protected health information, storing this information in a password protected 
database, and identifying subjects only by a unique identifier that is kept in a separate location in a locked 
container, traceable only by study personnel. All of the tests involve the risk of identifying asymptomatic 
abnormalities. The study may include risks that are unknown at this time. 
Justification/Minimization: Every effort will be made to decrease the risk of loss of confidentiality by 
limiting access to protected health information, storing this information in a password protected database, 
and de-identifying study specimens.   
Data Analysis Plan:   
 
Aim 1 Analysis Plan: Demographic and clinical characteristics will be summarized by group with 
descriptive statistics. Prior to analysis, variables will be examined for unusual values for query, missing 
data, and whether distributions are non-normal, in which case a transformation may be applied.   
Melatonin modeling:  The timing of melatonin onset and offset will be computed according to standard 
procedures that have been validated in adolescents 13,17. We will define DLMOn phase as the clock time 

at which evening salivary melatonin concentrations increase and remain above a 4pg/mL threshold using 
linear interpolation between successive samples 17,18. Similarly, we will define DLMOff phase as the clock 
time at which salivary melatonin concentrations fall below this threshold. Phase angles of entrainment will 
be computed as the time interval between DLMO and average actigraphic estimates of sleep variables, 
including bedtime, mid-sleep time, and wake time.  Additional characterization of the melatonin profile, 
including assessment of melatonin amplitude, will also be conducted. 
Aim 2 Data Modeling Plan: To investigate the relationship between sleep/wake behavior and circadian 
phase in obese adolescents, we will develop a physiologically-based mathematical model of sleep/wake 
and circadian interactions that simulates adolescent sleep patterns.  Model equations will be implemented 
in MATLAB and solved numerically using an appropriate solver.  Initially, we will fit model behavior to 
published characterizations of adolescent sleep/wake behavior.  After establishing a representative model 
of adolescent sleep, we will assess the role of key model parameters in the production of key elements of 
adolescent sleep, and we will investigate the role of these parameters in inter-individual differences 
observed in sleep/wake behavior in study participants as characterized by actigraphy data.  
 Aim 3 Calculations and Data Analysis Plan:  
Power calculation: A sample size of 30 participants will provide 80% power to detect a correlation 
coefficient equal to 0.47, given a 2-sided α=0.05. Because this is a pilot study, even if the null hypothesis 
is not rejected, the information gained regarding possible effect sizes will be useful in obtaining 
subsequent extramural funding for additional participants and in planning a larger study.  
Calculation of insulin resistance: IR will be calculated with the Matsuda Index, a composite model for 
insulin sensitivity which accounts for changes in both the insulin and glucose concentrations over time 19. 
The value derived from this equation is an M value of glucose uptake in mg/m2/min, which is 
approximated to results that would likely have been obtained if a more invasive hyperinsulinemic-
euglycemic clamp test had been performed 19. The range of values is 0–14, with 14 being the highest 
level of insulin sensitivity and zero the lowest. Additional exploratory measures including B-cell function 
[insulinogenic index (ΔI30/ΔG30) and (ΔC30/ΔG30) and disposition index (1/IFasting × ΔI30/ΔG30) and 
(1/CFasting ×ΔC30/ΔG30) will also be calculated 20-22. 
Relationship between circadian phase and IR:  DLMOn and DMLOff will be determined from the 
measures of salivary melatonin concentrations as described above.  Each subject will have one DMLOn 
time and one DLMOff time.  The relationship between DLMOn and DLMOff and IR will be assessed by 
comparing DLMOn and DLMOff to the whole body Matsuda index using correlation and linear models. All 
models will have melatonin onset or offset as the outcome, and gender effects will be considered.  The 
relationship between average actigraphy-estimated sleep duration (from sleep watch) and IR will also be 
tested using correlation and linear models.   
 
 
1. 
Crowley SJ, Acebo C, Fallone G, Carskadon MA. Estimating dim light melatonin onset (DLMO) 
phase in adolescents using summer or school-year sleep/wake schedules. Sleep. 
2006;29(12):1632-1641. 
2. 
Carskadon MA, Vieira C, Acebo C. Association between puberty and delayed phase preference. 
Sleep. 1993;16(3):258-262. 
3. 
Roenneberg T. Munich Chronotype Questionnaire. n.d.; http://www.imp.med.uni-
muenchen.de/index.html. Accessed February 18, 2015. 
4. 
Spilsbury JC, Drotar D, Rosen CL, Redline S. The Cleveland adolescent sleepiness 
questionnaire: a new measure to assess excessive daytime sleepiness in adolescents. Journal of 
clinical sleep medicine : JCSM : official publication of the American Academy of Sleep Medicine. 
2007;3(6):603-612. 
5. 
Bruni O, Ottaviano S, Guidetti V, et al. The Sleep Disturbance Scale for Children (SDSC). 
Construction and validation of an instrument to evaluate sleep disturbances in childhood and 
adolescence. Journal of sleep research. 1996;5(4):251-261. 
6. 
Storfer-Isser A, Lebourgeois MK, Harsh J, Tompsett CJ, Redline S. Psychometric properties of 
the Adolescent Sleep Hygiene Scale. Journal of sleep research. 2013;22(6):707-716. 
7. 
Goodman R. The Strengths and Difficulties Questionnaire: a research note. Journal of child 
psychology and psychiatry, and allied disciplines. 1997;38(5):581-586. 
8. 
Goodman R, Meltzer H, Bailey V. The Strengths and Difficulties Questionnaire: a pilot study on 
the validity of the self-report version. European child & adolescent psychiatry. 1998;7(3):125-130. 

9. 
Curran SL, Andrykowski MA, Studts JL. Short form of the Profile of Mood States (POMS-SF): 
Psychometric information. Psychological Assessment. 1995;7(1):80-83. 
10. 
McMurray RG, Ring KB, Treuth MS, et al. Comparison of two approaches to structured physical 
activity surveys for adolescents. Medicine and science in sports and exercise. 2004;36(12):2135-
2143. 
11. 
Basner M, Dinges DF. Maximizing sensitivity of the psychomotor vigilance test (PVT) to sleep 
loss. Sleep. 2011;34(5):581-591. 
12. 
Drummond SP, Bischoff-Grethe A, Dinges DF, Ayalon L, Mednick SC, Meloy MJ. The neural 
basis of the psychomotor vigilance task. Sleep. 2005;28(9):1059-1068. 
13. 
Carskadon MA, Acebo C, Richardson GS, Tate BA, Seifer R. An approach to studying circadian 
rhythms of adolescent humans. Journal of biological rhythms. 1997;12(3):278-289. 
14. 
Roenneberg T, Kuehnle T, Pramstaller PP, et al. A marker for the end of adolescence. Current 
biology : CB. 2004;14(24):R1038-1039. 
15. 
Morgenthaler TI, Lee-Chiong T, Alessi C, et al. Practice parameters for the clinical evaluation and 
treatment of circadian rhythm sleep disorders. An American Academy of Sleep Medicine report. 
Sleep. 2007;30(11):1445-1459. 
16. 
Meltzer LJ, Montgomery-Downs HE, Insana SP, Walsh CM. Use of actigraphy for assessment in 
pediatric sleep research. Sleep medicine reviews. 2012;16(5):463-475. 
17. 
Wright KP, Jr., Drake CL, Lockley SW. Diagnostic Tools for Circadian Rhythm Sleep Disorders. 
In: Kushida C, ed. Handbook of Sleep Disorders. Taylor & Francis Group; 2008:147-173. 
18. 
LeBourgeois MK, Carskadon MA, Akacem LD, et al. Circadian phase and its relationship to 
nighttime sleep in toddlers. Journal of biological rhythms. 2013;28(5):322-331. 
19. 
Matsuda M, DeFronzo RA. Insulin sensitivity indices obtained from oral glucose tolerance testing: 
comparison with the euglycemic insulin clamp. Diabetes care. 1999;22(9):1462-1470. 
20. 
Bacha F, Pyle L, Nadeau K, et al. Determinants of glycemic control in youth with type 2 diabetes 
at randomization in the TODAY study. Pediatric diabetes. 2012;13(5):376-383. 
21. 
Cree MG, Fram RY, Barr D, Chinkes D, Wolfe RR, Herndon DN. Insulin resistance, secretion and 
breakdown are increased 9 months following severe burn injury. Burns : journal of the 
International Society for Burn Injuries. 2009;35(1):63-69. 
22. 
Cree MG, Newcomer BR, Katsanos CS, et al. Intramuscular and liver triglycerides are increased 
in the elderly. The Journal of clinical endocrinology and metabolism. 2004;89(8):3864-3871.