Metabolic Effects of Sleep Extension in People With Obesity
Phase: NA
Conditions: Insulin Resistance, Obesity
Sponsor: Washington University School of Medicine

 
 
 
 
Project title: 
 
 
Metabolic Effects of Sleep Extension in People with Obesity 
  
 
 
 
 
 
Principal Investigator: 
Samuel Klein, MD 
 
 
 
 
 
Department of Medicine 
Washington Univ. School of Medicine 
 
 
 
 
 
St. Louis, MO 
  
 
 
Co-investigators: 
 
Gordon Smith, PhD 
 
 
 
 
Jun Yoshino, MD, PhD 
 
 
 
 
Joseph Beals, PhD 
Washington Univ. School of Medicine 
 
Josie Broussard, PhD 
Kenneth Wright, PhD 
University of Colorado Boulder 
 
Mohit Jain, MD 
University of California, San Diego 
 
 
 
 
 
October 8, 2025 
 
 
 
 
NCT03594994 
 
 
 
 
 
 
 
 
 

A. SPECIFIC AIMS 
Insufficient sleep causes metabolic dysfunction and is associated with an increased risk of developing 
obesity and cardiometabolic diseases, such as type 2 diabetes (T2D). The mechanism(s) responsible for the 
link between insufficient sleep and adverse metabolic outcomes is not known, but it has been proposed that 
disruption of circadian rhythms, oxidative stress, and/or inflammation are involved. The potential therapeutic 
effects of sleep countermeasures have not been evaluated in metabolically unhealthy people who are at 
increased risk of developing T2D. Therefore, the overall goals of this proposal are to assess sleep extension 
as a metabolic health countermeasure in people are metabolically unhealthy (MU) who habitually maintain 
chronic short sleep schedules.  
Accordingly, we will conduct a randomized controlled trial to assess the effect of a 4-6 wk sleep 
extension intervention on key metabolic outcomes in MU people (BMI=25.0-50.0 kg/m2, prediabetes [impaired 
fasting glucose, HOMA-IR ≥ 2.0, glucose intolerance or an elevated HbA1c]), who habitually sleep <7.0h/night 
and do not have clinically significant sleep disorders (e.g., sleep apnea, insomnia, narcolepsy). Participants will 
be studied at baseline during their habitually short sleeping pattern and then 4-6-wk after being randomized to 
i) continue their usual short sleep pattern (attention control) or ii) sleep extension, achieved by adding 1-2h of 
time in bed per night, objectively determined by using wrist actigraphy. Sleep will be assessed during the 
inpatient admissions by using polysomnography. Total energy intake will be adjusted as needed to ensure 
participants maintain a constant body weight.  
 
The following specific aims will be evaluated: 
 
Aim 1. Determine the effect of sleep extension on whole-body, multi-organ and cellular metabolic 
function in MU adults. We hypothesize that sleep extension implemented at home in people who chronically 
maintain short sleep schedules will: i) improve in vivo insulin sensitivity in the liver, adipose tissue and muscle; 
ii) have beneficial effects on 24h plasma insulin (concentration, secretion and clearance) glucose, and FFA 
profiles; iii) decrease intrahepatic triglyceride content; and iv) alter specific plasma metabolites (assessed 
every 6h for 24h) that are known to be associated with insulin resistance and the development of T2D.  
In addition, untargeted metabolomics analyses will be performed to help identify novel metabolites associated 
with sleep extension-induced improvement in metabolic function that will serve as the basis for future 
investigation of these specific metabolites/bioactives. 
Aim 2. Determine the effect of sleep extension on purported mechanisms responsible for metabolic 
dysfunction induced by obesity or sleep restriction: i) misalignment between the master clock of the 
suprachiasmatic nucleus (assessed by 24h plasma melatonin rhythm) and peripheral circadian clocks 
(assessed by serial measurements of peripheral blood mononuclear cell [PBMC] gene expression); ii) 
oxidative stress (urinary 8-iso-prostane F2α, plasma oxysterols and glutathione-to-oxidized glutathione 
ratio, and adipose tissue protein carbonylation); and iii) systemic and adipose tissue inflammation 
(proinflammatory cytokines and prostanoids). We hypothesize that sleep extension will: i) improve 
alignment between central and peripheral rhythms; ii) reduce oxidative stress; and iii) decrease plasma and 
adipose tissue markers of inflammation.  
We will test these hypotheses by: i) a one-stage hyperinsulinemic-euglycemic clamp (HEC) procedure plus 
stable isotope tracer infusions; ii) 24h urine collection iii) hourly blood sampling for 24h; iv) muscle and adipose 
tissue biopsies; and v) magnetic resonance imaging. 
 
This study represents a transdisciplinary collaboration between investigators at Washington University School 
of Medicine, the University of Colorado Boulder, and the University of California San Diego who have 
synergistic expertise in conducting complex metabolism and sleep studies in people, including sleep extension, 
required for this proposal. The results from this study will provide new information to assess whether sleep 
extension should be considered as a countermeasure to improve metabolic health in people at high risk for 
developing metabolic diseases. 
 
 

B. SIGNIFICANCE, SCIENTIFIC PREMISE, AND INNOVATION 
B1. Significance and Scientific Premise  
The average daily sleep duration in American adults has decreased by nearly 2 hours in the last 
century from ~8.3h/night1-3 to ~6.8h/night4. Sleep loss is often unavoidable in a busy modern society because 
of social activities, work-related deadlines, and work schedules of certain occupations, such as emergency 
responders, physicians, military personnel, and shift workers. There is a considerable body of evidence 
demonstrating that insufficient sleep has adverse effects on body weight and metabolic function. Data from 
epidemiological studies have found inadequate sleep is associated with an increased risk of obesity and type 2 
diabetes (T2D)5-7. For example, a 6-yr longitudinal study of 1,455 people without diabetes found a three-fold 
greater risk of developing impaired fasting glucose in those sleeping less than 6h/night than those sleeping 
8h/night7. A recent meta-analysis of prospective studies with a median follow-up of 7.5 years found the 
incidence of T2D was 37% greater in people sleeping ≤5h/night than those sleeping 7-8h/night8. Shorter sleep 
duration is also linked with increased energy intake, weight gain, and increased total body fat, abdominal fat 
and intrahepatic triglyceride content9-16. Every 1h/night reduction in sleep is associated with a 0.35 kg/m2 
increase in body mass index (BMI), and short sleepers (<5h/night) have a 55% greater risk of becoming obese 
than people who get adequate sleep (>5h/night)6.  
Although weight gain associated with inadequate sleep is likely an important contributor to its adverse 
effects on health, inadequate sleep per se can cause metabolic dysfunction independent of changes in body 
weight. Data from experimentally-induced sleep restriction studies conducted in people have demonstrated 
inadequate sleep alters glucose homeostasis, primarily by decreasing whole-body insulin sensitivity during 
hyperinsulinemic conditions17-19, without an adaptive increase in insulin secretion when controlling for food 
intake20,21. These results demonstrate inadequate sleep impairs muscle insulin sensitivity, which is a key tissue 
for postprandial glucose disposal22,23. However, the effect of inadequate sleep on insulin action in other key 
metabolic organs, such as adipose tissue and liver, is not clear. The metabolic effects of sleep restriction on 
adipose tissue might be particularly important because we and others have found sleep loss induces an 
increase in plasma FFA concentration21,24, which can affect insulin sensitivity in the liver (insulin-mediated 
suppression of glucose production) and skeletal muscle (insulin-stimulated increase in glucose uptake)25,26. 
However, we are not aware of any studies that have directly evaluated the effect of sleep restriction on in vivo 
adipose tissue insulin sensitivity during controlled energy intake. The effect of sleep restriction on liver 
metabolism is also unclear because of conflicting results from different studies that have found sleep restriction 
does17 and does not24 decrease hepatic insulin sensitivity.  
The mechanisms responsible for the adverse effect of sleep restriction on glycemic control are not 
known, but several hypotheses have been proposed, including circadian misalignment13,27, increased oxidative 
stress28,29, and altered pro-inflammatory and anti-inflammatory cytokines30,31. Studies conducted in animal 
models have consistently demonstrated an interaction among sleep, circadian biology and metabolic 
physiology32,33. We and others have found that restricting sleep decreased whole-body insulin sensitivity13 and 
resulted in high plasma melatonin levels for many hours after awakening (i.e., morning circadian 
misalignment)13,27. Moreover, the duration of high plasma melatonin correlated with the reduction in insulin 
sensitivity (see Preliminary Studies C2.3). These data suggest that sleep loss-induced circadian misalignment 
causes insulin resistance, which is a risk factor for subsequent T2D. An increase in markers of oxidative stress 
in blood and peripheral tissues are also associated with insulin resistance and the development of T2D in 
people34-36. We are aware of only one study that evaluated the effect of sleep restriction on oxidative stress in 
people, which found greater myeloperoxidase-modified oxidized low-density lipoprotein concentration after 
sleep restriction (5h/night for 5 nights) in healthy men37. Data obtained from rodent models have shown that 
sleep restriction causes oxidative stress in several regions of the brain, assessed as an increased ratio of 
oxidized-to-reduced glutathione, a decrease in reduced glutathione content, and diminished superoxide 
dismutase activity38,39. Chronic low-grade systemic and adipose tissue inflammation are associated with insulin 
resistance40. The effect of inadequate sleep on markers of inflammation is unclear, because of conflicting 
results among studies showing short sleep caused an increase, no change, or decrease in circulating markers 
of inflammation41-44. In rodents, sleep restriction increases adipose tissue macrophage infiltration and TNFα 
and IL-6 protein content45. The effect of sleep extension on diurnal rhythm, oxidative stress, or systemic and 
adipose tissue inflammation in people who are short sleepers is not known. 
Data from several epidemiological studies show that people with obesity and metabolic dysfunction are 
short sleepers (sleep <7h/night)46,47, supporting the notion that insufficient sleep can contribute to the 
metabolically unhealthy phenotype. Therefore, increasing sleep duration in this population could provide a 

simple, low-cost therapeutic intervention. However, the potential beneficial effect of sleep extension on glucose 
homeostasis in habitual short sleepers who are metabolically unhealthy is not known. The data from one study 
conducted in healthy non-obese participants found 6 wk of sleep extension, achieved by increasing time-in-bed 
by 1h/night (resulting in an average increase of 44 min in sleep time/day), caused a decrease in fasting plasma 
insulin concentrations48. Another study, reported as an abstract only49, found 3 weeks of sleep extension in 
people with impaired glucose tolerance or T2D produced a trend (P = 0.06) toward a decrease in glucose area 
under the curve (AUC) during a 3h oral glucose tolerance test. Moreover, the increase in sleep duration 
correlated with the decrease in glucose AUC (r=0.81, P < 0.01). Finally, another study found sleep extension to 
10h/night for only 3 nights in healthy young men who regularly curtail their sleep to ~6h during the week 
resulted in an improvement in glucose tolerance during a 2h oral glucose tolerance test50. These data suggest 
chronic sleep loss is a potentially modifiable diabetes risk factor. However, we are not aware of any studies 
that have evaluated the effect of sleep extension on multi-organ system metabolic function in metabolically-
abnormal, habitual short sleepers. 
 
A non-targeted assessment of the metabolome (low molecular weight products of metabolism) could 
provide novel insights into the biological mechanisms involved in sleep-related alterations in metabolic 
function. Data from several studies that used a metabolomics approach have found that sleep restriction has a 
significant impact on the metabolomes in the blood, urine, and breath51-56, which could be involved in the 
pathogenesis of the metabolic alterations induced by sleep restriction. However, we are not aware of any 
studies that have evaluated the effect of sleep extension on the plasma metabolome in people. The 
comprehensive mass spectrometry-based analysis of the systemic metabolome proposed in this application 
could provide insight into systemic metabolic alterations that influence whole body physiology and 
homeostasis, and identify novel metabolic targets for future studies of sleep related dysfunction. 
Summary. Data from both epidemiological and sleep intervention studies have shown disrupted sleep 
has adverse effects on diabetes-related metabolism. However, the potential therapeutic benefits of sleep 
extension are not known. This proposal will help fill this important gap in our knowledge. The hypotheses being 
tested in this proposal are based on a sound foundation of data from our own studies, and studies conducted 
by other investigators in animal models and people. The assessment of sleep on metabolic function is difficult, 
because it requires careful manipulation of sleep time and a comprehensive cellular and in vivo assessment of 
multiple organ systems in controlled sleep/circadian protocols. We have assembled a transdisciplinary team of 
metabolic, sleep and circadian experts to conduct these complex studies in people.  
 
B2. Innovation 
Our proposal is innovative for several reasons: i) it involves a novel transdisciplinary collaboration 
among three research groups from different institutions, who have the combined expertise needed to conduct 
the complex studies proposed in this application (J. Broussard, K. Wright and B. Lucey [Co-Is] are experts in 
sleep interventions and sleep evaluation, S. Klein [PI] and G. Smith and J. Yoshino (Co-Is) are experts in 
evaluating in vivo and ex vivo metabolic function in people, and M. Jain [subcontract] is an expert in 
metabolomics/bioinformatics); ii) it combines a controlled sleep intervention with sophisticated, state-of-the-art, 
whole-body, multi-organ and cellular assessments of postabsorptive and 24h metabolic function in people, and 
iii) it provides a comprehensive assessment of potential therapeutic effects of sleep extension in people which 
has considerable physiological and potential clinical implications. 
 
 
 

C. FEASIBILITY – INVESTIGATOR EXPERTISE AND PRELIMINARY DATA 
 
C1. Transdisciplinary collaboration.  
This application represents a new collaboration between the University of Colorado Boulder (UCB; Co-
Is: J. Broussard and K. Wright) and Washington University School of Medicine (WUSM; PI: S. Klein; Co-Is: G. 
Smith, J. Yoshino and B. Lucey), in conjunction with specific additional support from M. Jain at the University 
of California San Diego (UCSD) (assessment of plasma metabolomics; see letter of support). The interaction 
between the PI (Klein) and Co-Is at UCB (Broussard, Wright) developed during a workshop, held by NIDDK in 
February, 2015, that focused on the “Impact of Sleep and Circadian Disruption on Energy Balance and 
Diabetes.” This meeting led to a collaborative publication57, frequent teleconference and face-to-face 
communications to design the current research proposal, and a construction of a dedicated room for sleep 
studies within the new Clinical Research Unit at WUSM. The involvement of M. Jain in this proposal developed 
from an ongoing collaboration between Drs. Klein and Jain that is evaluating the metabolome in blood samples 
from people with obesity undergoing different weight loss diets.  
 
C2. Preliminary Studies.  
The following preliminary data support the hypotheses being tested in this proposal, and demonstrate 
we have the experience needed to perform the precise sleep interventions and complex metabolic studies 
proposed in this application.  
C2.1. Sleep loss impairs insulin sensitivity. We 
recently completed a 15 d inpatient protocol in 16 healthy 
volunteers that involved a 3 d baseline condition (9h 
sleep/night), followed by 5 d each of 5h and 9h of 
sleep/night in randomized order13. Insufficient sleep 
increased ad libitum energy intake and decreased 
whole-body insulin sensitivity, assessed by the Matsuda 
Index derived from an oral glucose tolerance test (Fig. 
1A). Matsuda Index values returned to baseline when 9h 
sleep/night was instituted after the 5h sleep/night 
condition (Fig. 1B).  
C2.2. 24h blood sampling without disturbing participant sleep. We have implemented and routinely use a 
protocol for collecting frequent blood samples over 24h without awakening participants from sleep that is 
needed for this proposal. This protocol involves an indwelling intravenous catheter to a 12-ft long, small-lumen, 
extension tube passed through a wall portal for blood sampling13,30,31,58-60.  
C2.3. Sleep loss induces circadian misalignment. We have found that decreasing sleep to 5h/night leads to 
plasma melatonin levels being high for several hours after wake-time, indicating morning wakefulness during the 
biological night, or “morning circadian misalignment”58. Moreover, the duration of high plasma melatonin levels 
was negatively associated with insulin sensitivity, assessed by using the Matsuda Index, suggesting circadian 
misalignment contributed to decreased insulin action after insufficient sleep. 
C2.4. Circadian misalignment increases plasma markers of 
inflammation. We have found that circadian misalignment, 
induced by extending day length by 36 min/d (to 24.6h) for 25 d, 
increased plasma C-reactive protein (CRP) concentrations (Fig. 
2)31. In this proposal we will assess the effect of extended sleep on 
plasma and adipose tissue markers of inflammation. 
 
C2.5. Effects of increased sleep opportunity on sleep duration, 
sleep architecture, and circadian timing. We conducted a study 
in 26 healthy, normal-weight, habitual short sleepers (<6.5h 
sleep/night), who were randomized to one of two sleep schedules 
for 2 wk: i) maintenance of habitual short sleep schedule (n=12); or 
ii) extend sleep opportunity by ≥2h/night (n=14). We used the same outpatient assessments (wrist actigraphy, 
participant call-in at bed-time and wake-time, and sleep diaries) and inpatient polysomonography (PSG) 
Baseline 
9-h 
5-h
|
P=0.06
*
*
0
2
4
6
8
9-h First
B
Matsuda Index
5-h First
0
2
4
6
8
Matsuda Index
|
A
Condition
   Fig. 1 Insulin sensitivity after insufficient sleep 
Fig. 2. Circadian misalignment and inflammation
0.0
0.2
0.4
0.6
0.8
hsCRP (mg/L)
**
*
*
*
*
*
*
*
*
*
Relative Clock Hour
Sleep
2400
2400
0400 0800 1200 1600 2000
Aligned
Misaligned

proposed in the current study. The increase in sleep opportunity increased total sleep time by 2.4h/night (from 
~5.8h/night to 8.2h/night) (Table 1). In addition, duration of N1, N2 and REM sleep significantly increased (data 
not shown) without altering sleep latency (time it takes to fall asleep) or sleep efficiency (sleep time divided by 
total time in bed). These findings demonstrate our ability to increase sleep duration in people with short sleep 
schedules.  
 
Table 1. Effect of 2-week sleep extension intervention on PSG measured sleep  
 
Maintain habitual insufficient sleep 
Extension from habitual insufficient   
to sufficient sleep 
 
    Before 
    After 
   Before 
    After 
Time in bed (min) 
336.1  ± 15.5 
361.4 ± 8.7 
348.8 ± 10.5 
492.0 ± 10.4a,b 
Total sleep Time (min) 
322.2 ± 14.9 
346.8 ± 9.5 
329.3 ± 10.6 
464.5 ± 10.0a,b 
Sleep latency (min) 
  2.9 
±  1.8 
  2.1 
± 1.2 
  3.8 
±  2.0 
  7.6 
±  1.2 
 Sleep efficiency (%) 
 95.7 ±  0.8 
 96.0 ± 0.5 
 94.5 ±  1.2 
 94.5 ±  0.9 
Values are means±SEM. aValue significantly different from baseline value; p<0.01;  
bValue significantly different from effect of insufficient sleep condition, p<0.01.  
 
C2.6. Use of high throughout liquid chromatography mass spectrometry (LC-MS) for untargeted, 
discovery metabolomics analysis. The reliable analysis of thousands of metabolites in biological samples, 
and an insightful interpretation of these complex datasets is not trivial. For this reason, we are collaborating with 
Dr. Mohit Jain at UCSD, who has a productive track record in using mass spectrometry-based approaches for 
targeted and untargeted discovery metabolomics61-64, and in analyzing 
complex datasets61-66. We will use these innovative LC-MS and 
computational approaches to assay the human metabolome in plasma to 
provide insights into the effects of sleep restriction/extension on metabolite 
determinants of metabolic function. 
 
C2.7. Use of peripheral blood mononuclear cells (PBMCs) to evaluate 
diurnal rhythm of peripheral clock genes expression. We will use 
circulating PBMCs to evaluate peripheral clock genes because of the burden 
of repeated tissue biopsies on study participants. We recently found PBMCs 
obtained every 3h for 24h from metabolically-healthy people who were 
overweight exhibit clear diurnal expression patterns of clock genes (Fig. 3, 
manuscript submitted). Diurnal expression patterns of clock genes in PBMCs 
are similar to those in skeletal muscle67 and adipose tissue68. Data from 
previous studies that evaluated blood samples in people have shown that 
diurnal expression patterns of clock genes are affected by insufficient or 
mistimed sleep69-72. In addition, obesity is associated with the alterations in 
clock gene expression in human PBMCs73. Taken together, these findings 
suggest inter-relationship among metabolism, sleep homeostasis, and blood 
circadian clocks. Accordingly, we hypothesize that expression of PBMC clock 
genes will be affected by sleep extension in MU people who are short 
sleepers. 
 
D. RESEARCH APPROACH 
 
D1. Study Participants 
Participants will consist of 50 MU men and women who habitually sleep ≤7.0h/night, and meet the 
following criteria: i) age: 21-65 years old; ii) BMI 25.0-50.0 kg/m2; iii) prediabetes (fasting plasma glucose 
concentration between 100 and 125 mg/dl, or 2-h OGTT plasma glucose between 140 and 199 mg/dl, or 
HbA1c between 5.7 and 6.4%, or HOMA-IR ≥ 2.0); and iv) no evidence of clinically significant sleep disorders 
other than mild sleep apnea [apneas and/or hypopneas per hour of sleep (AHI) of 15 or fewer]. 
 
 
Screening visit 1. Written informed consent will be obtained from each subject before study 
participation. All participants will arrive in the Clinical Research Unit (CRU) in the morning after they have 
fasted for 10h-12h overnight at home, and will undergo a comprehensive screening, including a medical 
history, physical examination, standard blood tests, an oral glucose tolerance test and a urine toxicology 
screen. A clinical interview by a trained clinician will screen for psychiatric and sleep disorders and daytime 
Fig. 3. PBMC clock gene expression  

functioning. Screen for potential psychiatric and sleep problems will also include the following validated 
questionnaires: i) Beck Depression Inventory-II (BDI-II); ii) Beck Anxiety Inventory (BAI); iii) Pittsburgh Sleep 
Quality Index (PSQI); iv) Epworth Sleepiness Score (ESS); v) Insomnia Severity Index; vi) Berlin Questionnaire 
(indicates sleep apnea); and vii) Short form quality of life questionnaire. Prior to discharge, body composition 
including total body lean and fat mass will be determined by using dual energy X-ray absorptiometry (DXA). 
Screening visit 2. After consuming a standard evening meal, a PSG recording90 conducted during the 
night of admission (e.g. ~2300h until ~0500h, depending on the participant’s actual habitual schedule) will be 
used as a clinical sleep disorders screen. If evidence of a clinically significant sleep disorder (e.g. obstructive 
sleep apnea, periodic limb movement disorder) is detected, the participant will be excluded from the study and 
discharged. PSG recordings will be obtained with Siesta digital sleep recorders (Compumedics). Sleep studies 
will be performed and scored by a polysomnographic technologist and reviewed by a board-certified sleep 
medicine physician according to American Academy of Sleep Medicine guidelines. Records will be scored for 
sleep staging91 and we will perform quantitative EEG (e.g., delta power) analyses92. 
All potential participants will also be carefully screened to assess their ability to incorporate the sleep 
extension protocol into their daily lifestyle. This screen will include questions about sleep duration on workdays, 
free days and vacation to determine sleep need and whether participants will be able to extend sleep. In 
addition, we will extensively discuss any lifestyle modifications necessary to implement sleep extension, and 
examine evening and morning work, family and other obligations and commitments that make adherence to 
sleep extension very difficult or impossible.  
 
 
The following criteria will be exclusionary: i) history or evidence of abnormalities that affect sleep (e.g. 
moderate and severe obstructive sleep apnea [OSA] defined as AHI >15, periodic limb movements of sleep 
[PLMS], insomnia, narcolepsy, shift work in previous 6 months, or travel more than 1 time zone in 3 wk before 
the study); ii) significant organ system dysfunction/disease (e.g. diabetes, severe pulmonary, kidney or 
cardiovascular disease) and any evidence of active illness (e.g., fever); iii) history of seizure disorder; iv) 
history or current significant psychiatric disorder (e.g., prior or current diagnosis); v) pregnant/nursing; vi) use 
dietary supplements and/or medications known to affect sleep, circadian rhythms or metabolic function; vii) 
smoke >10 cigarettes/week or illegal drugs determined by medical history or urine screening; viii) consume 
excessive alcohol (>7 drinks/wk); ix) consume excessive amounts of caffeine daily (>500 mg; ~5 servings of 
coffee/espresso or ~8 12 oz caffeinated soft drinks per day) or consume less amounts of caffeine but believe 
withdrawal symptoms (e.g. headache) are likely if caffeine is stopped; x) participate in intense exercise (activity 
that causes heavy breathing and sweating, such as jogging) >70 min/wk or moderate exercise (e.g., brisk 
walking) for >150 min/wk; xi) unstable weight (>4% change during the last 2 months before entering the study); 
xii) have conditions that render subject unable to complete all testing procedures [e.g., metal implants that 
interfere with imaging procedures]; and xiii) unwilling/unable to provide informed consent.  
 
 
The PI has an experienced participant recruitment core, a strong track record in recruiting subjects who 
meet specific requirements and excellent participant compliance and retention in studies as rigorous or more 
rigorous than the current proposal e.g.,36,67,74-88.  
 
D2. Experimental Design  
 
D2.1. Study flow and overview. A randomized, controlled trial will be conducted in MU people to evaluate the 
effect of 4-6 wk of sleep extension (to account for the time needed to complete follow-up testing) on metabolic 
function, circadian biology, inflammation and oxidative stress (Fig. 4). A 4-6 wk duration of sleep extension was 
chosen because a decrease in fasting plasma insulin concentration has been reported after 6 wk of sleep 
extension in healthy, non-obese subjects48. A randomized, cross-over study design (i.e. participants 
randomized to habitual short sleep or to extended sleep and then cross-over to the other condition) is not being 
done because of the concern that subjects would find that extending their sleep was beneficial and would not 
go back to their previous short sleep pattern. Indeed, in our previous sleep extension study (see preliminary 
data in section C2.5), subjects who extended their sleep completed a 3-wk post-study sleep log which showed 
they continued to sleep more than 7h per night by their own choice. 

 
  
D2.2. Baseline testing. Throughout baseline testing participants will be instructed to maintain their habitual 
sleep patterns. Participants will perform body composition analysis (magnetic resonance imaging will be used 
to determine intra-abdominal adipose tissue volume77 and intrahepatic triglyceride content89) in the Center for 
Clinical Imaging Research (CCIR) after a 4 h fast. Participants who experience anxiety associated with the MR 
scans will have the option of receiving a single dose of Ativan or Xanax prior to the scans. In addition, 
participants will be admitted to the CRU on 2 occasions separated by 7-10 d. Before admission to the CRU for 
all inpatient visits, participants will maintain their habitual short sleep of ≤7.0h/night for 7 d, verified by 
actigraphy, sleep logs and bedtime and wake up time call-ins to a time stamped recorder to ensure subjects do 
not acutely or chronically sleep extend before the inpatient studies. Wrist actigraphy with concurrent light 
exposure (Actiwatch Spectrum Pro; Philips Respironics) will be used to assess sleep timing and total sleep 
time (TST) during the 7-10 d period prior to all inpatient testing visits, during the 4-6 wk sleep 
extension/habitual sleep intervention and throughout repeat testing. A daily sleep log will be maintained to 
assess subjective sleep onset latency, TST, sleep quality, number of awakenings, factors that might have 
disturbed sleep, and daily caffeine and alcohol intake. Bedtime and wake-up time call-ins to a time-stamped 
recorder will be used to help evaluate sleep and wake up times. Participants will be instructed to avoid alcohol 
for 7 d, avoid moderate or intense exercise for 3 d, and avoid caffeinated drinks for 3 d before admission. For 3 
days before their in-patient visits, participants will consume packed-out meals produced by the metabolic 
kitchen, which will provide 50% of total energy as carbohydrate, 35% as fat and 15% as protein. Total energy 
Screening Visit 1
Medical history and exam
Oral glucose tolerance test
Questionnaires
Routine blood tests
Body composition/DEXA scans
Screening Visit 2
Sleep study
Baseline Visit 1
24 h Blood and urine sampling
Sleep Study
Baseline Visit 2
Insulin infusion study
Fat and muscle biopsies
Baseline Visit 3
Magnetic resonance scans
Final Visit 1
Routine blood tests
24 h Blood and urine sampling
Sleep Study
Final Visit 2
Insulin infusion study
Fat and muscle biopsies
Final Visit 3
Magnetic resonance scans
Group 1
4-6 wks Sleep extension only
Group 2
4-6 wks Sleep maintenance
Final Visit 1
Routine blood tests
24 h Blood and urine sampling
Sleep Study
Final Visit 2
Insulin infusion study
Fat and muscle biopsies
Final Visit 3
Magnetic resonance scans
Fig. 4. Study overview                             
 
 
 
 
 

content will be estimated to meet individual daily energy requirements, calculated as 1.4 times resting energy 
expenditure (REE). Subjects will be informed that they should make every attempt to consume meals at 
specific times determined based on their habitual wake time (e.g. at ~0900h, 1500h, 2100h if wake time is 
0800h [meals will be +1h, +7h and +13h after awakening]). 
D2.2.1. Inpatient CRU Visit 1 (24h metabolic profile, and assessment of PSG, circadian misalignment, 
oxidative stress and systemic inflammation). Participants will be admitted at ~1800h and stay in the CRU 
for ~36h and will continue their usual ≤7.0h/night sleep schedule. 
 
24h blood sampling and urine collection. Approximately 30 min after awaking on Day 2, after subjects 
fast for ~11.5h overnight, a catheter will be inserted into an antecubital vein. This catheter will be connected to 
a 12-ft long, small-lumen extension tubing that will be passed through a wall portal to allow blood sampling 
without disturbing the participant. Blood samples will be obtained hourly for 24h starting 1h after awaking (i.e., 
immediately prior to breakfast) to assess glucose, FFA, C-peptide, insulin, and melatonin concentrations [a 
total of 25 blood samples (3 mL each)]. Blood samples obtained every 3h will be used to assess: i) markers of 
inflammation (C-reactive protein, IL-6, TNFα, MCP-1, and selected prostanoids [prostaglandin [PG]E2, PGD2 
PGF2α and thromboxane A2]); ii) markers of oxidative stress (plasma oxysterols and glutathione-to-oxidized 
glutathione ratio), and markers of insulin resistance (branched-chain amino acids, aromatic amino acids, 
glycine, and glutamine, and acetylcarnitine C2)93. An additional 10 ml of blood will be obtained every 3h to 
isolate PBMCs, which will be used to evaluate the expression of clock genes. PBMCs will be immediately 
isolated through a density gradient centrifugation by using the Histopaque-1077 (#10771; Sigma, St. Louis, 
MO). Subjects will be seated for at least 15 min before each blood sample because posture can impact 
melatonin levels94. Light exposure during 24h sampling will be <8 lux max (~1.9 lux angle of gaze), because 
bright light supresses melatonin, our primary marker of circadian phase90,95. At other times during the study 
subjects will be exposed to normal room lighting (e.g., 200 lux). Meals will be supervised to ensure all food is 
consumed within 30 min. Urine will be collected throughout the 24h blood sampling protocol to determine 
urinary 8-iso-prostane F2α content as a marker of oxidative stress.  
Sleep study. Baseline PSG recordings will be obtained from ~2300h on Day 2 until ~0500h on Day 3. 
PSG recordings will be obtained with Siesta digital sleep recorders (Compumedics). Sleep studies will be 
performed and scored by a polysomnographic technologist and reviewed by a board-certified sleep medicine 
physician according to American Academy of Sleep Medicine guidelines. Records will be scored for sleep 
staging91 and we will perform quantitative EEG (e.g., delta power) analyses92. 
Inpatient activity and diet. During free time, subjects will be permitted to ambulate (e.g., read, walk, 
watch movies, computer work) in their room. Additionally, participants will perform a standardized bout of 
exercise (5 min stepping at 60 steps/min) 2 h after each meal. Dietary energy content will be designed to meet 
individual total daily energy requirements, determined as 1.2 x REE. Meals will be provided 3 times/day, given 
every 6h beginning with breakfast 1h after awakening (e.g. if participant awakes at 0530h, breakfast, lunch and 
dinner will be given at 0630h, 1230h, and 1830h). The macronutrient content of the diet will be the same as the 
outpatient diet and will not contain any caffeine. Subjects will be instructed to finish meals completely within 30 
min.  
D2.2.2. Inpatient CRU Visit 2 (Hyperinsulinemic-euglycemic clamp (HEC) procedure in conjunction with 
stable isotopically labeled tracer infusion and muscle and adipose tissue biopsies). At ~1800h, 7-10 d 
after Inpatient CRU Visit 1, participants will be readmitted to the CRU. A standardized meal will be provided at 
1900h. The macronutrient content of the diet will be the same as the outpatient diet and will not contain any 
caffeine. Subjects will be instructed to finish the meal completely within 30 min. At ~0700h in the morning after 
admission, after subjects have fasted for ~11.5 hours overnight, an intravenous catheter will be inserted into an 
antecubital vein to infuse stable isotope tracers, insulin and dextrose. A second catheter will be inserted into a 
hand vein for blood sampling, which will be heated using a thermostatically controlled box, to obtain arterialized 
venous samples. A primed-constant infusion of [6,6-2H2]glucose and a constant infusion of [U-13C]palmitate will 
be started at ~0800h and maintained for 3.5 h to obtain basal glucose and fatty acid kinetics. At ~1130h, a one-
stage HEC procedure will be initiated and continued for an additional 3.5 h. During the clamp insulin will be 
infused at a rate of 50 mU/m2 body surface area [BSA]/min after initiation with a two-step priming dose: 200 
mU/m2 BSA/min for 5 min followed by 100 mU/m2 BSA/min for 5 min. Euglycemia (~100 mg/dl) will be 
maintained by variable rate infusion of 20% dextrose enriched to 2.5% with [6,6-2H2]glucose. Adding glucose 
tracer to the dextrose infusion provides a more accurate measure of glucose kinetics by minimizing changes in 

plasma glucose enrichment96. The infusion rates of [6,6-2H2]glucose and [U-13C]palmitate will be stopped during 
the clamp because of the expected decreases in endogenous glucose production and palmitate release into the 
circulation. We have found this approach helps maintain steady tracer-to-tracee ratio (TTR) values throughout 
the HEC procedure. Blood samples will be obtained immediately before starting the tracer infusion and every 
10 min during the final 20 min (4 samples) of the basal period and HEC procedure, to determine glucose and 
insulin concentrations and substrate kinetics. Additional blood samples will be obtained every 10 min during the 
HEC procedure to monitor blood glucose concentration. The insulin concentration achieved during the clamp 
reflects postprandial plasma insulin concentrations97,98 and is ideal to evaluate insulin’s effect on glucose 
disposal99. 
 
 
Abdominal subcutaneous adipose tissue sampling will be performed during the basal period of the HEC 
procedure. The periumbilical area on one side of the body will be cleaned and anesthetized with 1% lidocaine, 
then a small skin incision (~0.5 cm) will be made in the skin and a small liposuction cannula will be inserted 
into the incision to aspirate ~5 grams of subcutaneous adipose tissue under sterile conditions. The sample will 
be rinsed immediately in ice-cold saline, cleaned of blood, and submerged in liquid nitrogen and stored at -80 
°C until further processing.  
 
 
 
Thigh muscle sampling will be performed during the basal period of the HEC procedure. The site will be 
cleaned and anesthetized with 2% lidocaine, a small incision (~0.5 cm) will be made in the skin, with a muscle 
sample (~100 mg) obtained under sterile conditions by using conchotome forceps. The sample will be rinsed 
immediately in ice-cold saline, cleaned of blood and connective tissue, and submerged in liquid nitrogen and 
stored at -80 °C until further processing. 
 
D2.3. Sleep extension intervention. After completing baseline testing, subjects will be randomized, stratified 
by sex, to either: 1) sleep extension for 4-6 wk (n=25) or 2) continued habitual short sleep for 4-6 wk followed 
by 4-6 wk sleep extension (n=25). Randomization will be accomplished using the online randomization module 
of REDCap, a secure, web-based application designed to support data capture for research studies100. 
Permuted blocks with varying block sizes will be used to ensure that there is no temporal bias in group 
assignment. When randomization occurs, the study coordinator will enter the REDCap system and will be 
asked to respond to a series of questions establishing the eligibility of the subject. The randomization 
assignment will only be revealed if all eligibility criteria are satisfied. We chose a 6-wk sleep extension 
intervention, because the only published study that showed a potential benefit of sleep extension on metabolic 
function used a 6-wk sleep extension protocol48. Sleep and circadian expert members of the research team will 
use the baseline assessments to develop an individualized targeted intervention for each subject randomized 
to the sleep extension group with the goal of increasing each subject’s sleep schedule by ~2h/night to reach a 
minimum of 7h/night. We chose 7.0h/night as a cut-off for short sleepers, since it is the minimum 
recommendation from the American Academy of Sleep Medicine recommendation for our targeted age-range 
(i.e., 18-65 years old). Our preliminary data on sleep extension in this age-group demonstrate our ability to 
extend sleep by over 2h/night (Table 1, section C2.5).  
 
 
The intervention will be designed to fit the lifestyle/school/work schedule of each subject (e.g. some 
subjects might add 1h to each end of the sleep opportunity whereas others may add 2h in the evening before 
usual bedtime). After setting the desired sleep extension timing with participants, they will be instructed to 
maintain a consistent bedtime (±30 min) and wake time (±15 min) 7 d/wk for 4-6 wk, with exceptions allowed 
for special occasions. We will also work with participants to arrange their bedrooms for optimal sleep hygiene, 
and instruct them to reduce exposure to light at night and increase light exposure in the morning to help 
achieve an earlier timed circadian clock that helps participant’s go to bed at an earlier hour, thereby increasing 
the sleep opportunity. Maintaining a consistent wake time is the most important factor, because morning 
sunlight exposure is a strong cue for synchronizing the circadian clock101-109, whereas increased flexibility with 
bedtime improves adherence. K. Wright (Co-I) has successfully implemented the proposed sleep extension 
strategy in adults who, by choice, maintain short insufficient sleep schedules (see preliminary data in section 
C2.5). He is also currently conducting similar sleep extension trials (NIH R01 HL131458 and R01 HL135598) 
to improve vascular function in patients with HIV infection and in people with high blood pressure. 
 
 
To help ensure compliance with maintaining appropriate habitual or extended sleep patterns and body 
weight, participants will be seen weekly by the research team to review progress and problems, and obtain 
body weight. Participants’ sleep and activity behavior will also be monitored by: i) wrist actigraphy (Actiwatch) 
worn for the duration of the study; ii) participant call-in at bed-time and wake-time to a time stamped recorder 
that will be reviewed daily; and iii) maintaining careful sleep logs. We will review call-ins daily, download the 

Actiwatch weekly, and provide participants weekly feedback about maintaining a consistent schedule. When 
daily call-ins are reviewed, subjects will be sent a text message letting them know to keep on track or remind 
them to maintain their extended sleep schedule if they report deviations from the planned schedule (i.e. 
bedtime ±30 min from scheduled bed time and/or wake-time ±15 min away from scheduled wake-time). In our 
preliminary data extending sleep in this age group, 14 of 15 subjects were able to extend sleep. If participants 
report 3 consecutive unscheduled deviations of ±30 min for bedtime or ±15 min for waketime they will be asked 
to come to the laboratory and their sleep schedule will be verified by downloading the Actiwatch data. If 
participants are unable to adhere to the sleep extension protocol they will be withdrawn from the study. Lastly, 
a urinary toxicology screen will be performed at week 2 to ensure subjects continue to abstain from 
recreational drug use. 
 
D2.4. Repeat testing. After subjects complete 4-6 wk of sleep extension or habitual sleep, all visits and 
procedures performed during baseline testing will be repeated. In addition, the DXA scan and questionnaires 
completed at screening will be repeated. Subjects will continue their assigned sleep duration until all follow-up 
tests are completed. Subjects randomized to the continued habitual short sleep group will have the opportunity 
to receive a personalized sleep treatment plan provided by our Sleep Medicine Center after the study is 
completed. 
 
D2.5. Sample analyses and calculations 
 
 
Blood samples. All blood sample analyses are routinely performed in our laboratories. Blood samples 
for inflammatory markers (C-reactive protein, IL-6, TNFα and MCP-1 concentrations) and estrogen and 
progesterone concentrations will be measured by using high sensitivity ELISA kits. Plasma C-peptide and 
insulin concentrations will be measured by using an Elecsys assay. Plasma melatonin will be analysed by 
Buhlmann RIA. Total, reduced and oxidized glutathione will be assessed in plasma by using a commercially 
available colorimetric assay (Cayman Chemicals, Ann Arbor, MI). Plasma FFA will be quantified by using 
gas chromatography110. Plasma glucose and palmitate TTRs will be determined by using gas 
chromatography-mass spectroscopy86,110.  
Metabolomics. State-of-the-art mass spectrometry techniques will be used to measure specific 
circulating metabolites that have been linked with metabolic dysfunction and/or sleep restriction, including 
markers of inflammation, oxidative stress, and insulin resistance (see section D2.2.1). These metabolites will 
be evaluated in blood samples obtained every 6h for 24h to provide an integrated assessment of plasma 
concentrations throughout the day. Additionally, we will measure a key marker of oxidative stress in a 24h 
urine collection (urinary 8-iso-prostane F2α)62,63. To capture these metabolites of interest, multiple 
complementary LC methods will be performed as previously described62,63, each optimized to capture a 
chemical subset of the metabolome, and coupled to a high resolution Thermo Orbitrap QExactive mass 
spectrometer. This orthogonal chromatography will include hydrophilic interaction liquid chromatography 
(HILIC) using a Sequant Zic-pHILIC HPLC column to capture polar metabolites, reverse phase 
chromatography using a superficially porous Phenomenex Kinetex C18 UPLC column to capture lipids and 
peptides, and weak ion exchange chromatography using a Phenomenex Luna aminopropyl HPLC column to 
capture sugars, nucleotides, and phosphates. Metabolites of interest are definitively identified by using MS/MS 
analysis, with matching of LC measures and MS/MS fingerprint to commercial metabolite standards. In addition 
to targeted measures, simultaneous untargeted metabolomics analyses will be performed to comprehensively 
identify novel metabolites that may be associated with sleep related improvement in metabolic function. These 
metabolite markers will serve as the basis for future investigation and potentially enable discovery of novel 
metabolite biomarkers and bioactives involved in improved metabolic function induced by sleep extension in 
persons with MUO.  
Molecular analyses. Total RNA will be isolated from PBMC, muscle and adipose tissue biopsy samples 
in Trizol reagent (Invitrogen, Carlsbad, CA) or RNeasy kit (Qiagen, Valencia, CA). Real time-PCR will be used 
to determine gene expression36,67,87,88. Expression of clock genes (e.g. CLOCK, BMAL1, PER1, PER2, CRY1, 
CRY2, REV-ERBα, DBP) in PBMC and markers of inflammation (e.g. IL6, TNF, CCL2, CD68) and oxidative 
stress (e.g. NQO1, DHCR24, UCHL1)36 in muscle and adipose tissue will be determined. Adipose tissue 
protein carbonylation will be evaluated with an OxyBlot Protein Oxidation Detection Kit (Millipore)111. 
 
Circadian Melatonin Phase Assessment. Blood samples from the 24h blood sampling studies will be 
used to measure melatonin concentrations to determine the Dim Light Melatonin Onset (DLMO) and the Dim 
Light Melatonin Offset (DLMOff) (linear interpolation of 25% of the fitted peak to trough amplitude), as we have 

done previously108,112-114. We will also calculate the timing of the midpoint between the DLMO and DLMOff. We 
considered other circadian phase markers (e.g. core body temperature and cortisol), but will use melatonin 
because it is the most precise and accurate marker of circadian phase in people115. 
 
Calculation of substrate and hormone kinetics. Endogenous glucose and palmitate Ra into the 
circulation and glucose Rd will be calculated as we have previously described36,87. The hepatic insulin 
sensitivity index will be calculated as the reciprocal of the product of endogenous glucose Ra and basal 
plasma insulin concentration116. The adipose tissue insulin resistance index will be calculated as the product of 
endogenous palmitate Ra and basal plasma insulin concentration117. Plasma substrate and hormone 
concentration areas-under-the-curve for 5 h after initiating meal consumption and for the entire 24h will be 
calculated by using the trapezoid method118. Insulin secretion will be estimated by C-peptide deconvolution 
during the 24h blood sampling study119 and insulin clearance rates by dividing the ISR AUC by the insulin AUC 
over each meal period and over the full 24h and by compartmental modelling during the HEC procedure120. 
 
D3. Retention and Recidivism Plan. Several strategies will be used to enhance adherence with the study 
protocols and reduce drop-outs: i) a philosophy of partnership and collaboration will be encouraged between 
research site personnel and study participants; ii) reimbursement for time required for study visits will be 
provided; iii) participants will be seen weekly by the research coordinator and research nurse, sleep duration 
will be tracked weekly, and participants will be contacted frequently via text message and phone calls to 
encourage continued success with the sleep schedule or discuss deviations; and iv) participants who are not 
adhering to the study protocol will receive additional support by the study team with an intervention plan 
established after reviewing the participant’s specific barriers for compliance.  
 
D4. Statistical analyses 
D4.1. Data analyses. Statistical evaluation of the study data will be conducted in collaboration with Dr. 
Kenneth Schechtman, a biostatistician in the Washington University Institute of Clinical and Translational 
Sciences (ICTS) Research Design & Biostatistics Group. We will use a modified intention-to-treat (mITT) 
analysis, in which all subjects that complete the study will be analyzed regardless of their adherence to the 
assigned sleep intervention (i.e., continued short sleeping pattern in the control group and successful sleep 
extension [adding ~2 h of time-in-bed per night ensuring at least 7h/night] in the sleep extension group). 
Analysis of covariance (ANCOVA) with the post-treatment value as the dependent variable will be used to 
determine whether there are between group differences in our metabolic outcomes assessed during the HEC 
procedure and 24-h blood sampling study with the pre-intervention value used as a covariate. Characteristics 
at baseline between groups will be compared by using t-tests or Wilcoxon’s test for continuous variables and 
chi square tests for dichotomous variables; any significant differences between groups will be used to adjust 
subsequent statistical analyses when necessary. Violations of the assumption of homogeneity of variance 
and/or sphericity will be corrected, if necessary, via transformation of data to produce data that satisfy 
normality and equal variance assumptions90,121-123. As this protocol incorporates a 2-time point, pre-post study 
design there is no basis to input follow-up data when only baseline data are available. Because of this 
limitation, we will compare characteristics between completers and non-completers by using t-tests or 
Wilcoxon’s test for continuous variables and chi square tests for dichotomous variables; these results will be 
reported in addition to the ANCOVA analysis. Metabolite analyses will be performed by using an ANOVA and 
Tukey post-hoc testing for statistical significance between conditions and across time. For discovery based 
untargeted metabolomics analysis, a conservative statistical threshold will be applied at the outset (Bonferroni 
corrected P value of <10-5 to account for multiple hypothesis testing). To further minimize false discovery, we 
will also use principal component analysis as well as sparse partial least squares and LASSO regression with 
internal cross-validation. Metabolites identified using these methods will be prioritized for future investigation as 
potential mechanisms underlying sleep related metabolic improvements. 
D4.2. Sample size considerations. Using G*Power 3.1.9.2124 we estimate that 15 participants/group will be 
required to detect realistic and clinically meaningful effects of sleep extension (based on data from our own 
studies evaluating weight loss or weight gain36,87,88 and to data from our own and other groups assessing the 
metabolic effect of sleep restriction17-19,24) with >90% power using two-sided tests at the α=0.05 level of 
significance in all primary and secondary outcomes listed below.  
 

D4.2.1 Primary outcome 
Insulin-mediated glucose Rd. We have found the average insulin-mediated glucose Rd during high-dose 
insulin infusion to be 59.1 µmol/kg FFM/min in people with obesity with a day-to-day variability of 6.4 µmol/kg 
FFM/min (mean ± SD) 76. Using this SD and assuming similar variance in the sleep restriction and extension 
groups, we estimate we will be able to detect a ~7.9 µmol/kg FFM/min (~13%) difference between groups. This 
difference is smaller than the ~20% reduction in insulin-mediated glucose Rd observed after acute (1 to 7 d) 
sleep restriction17-19,24. 
D4.2.2 Secondary outcomes 
24-h glucose area-under-the curve (AUC). We have found average 24h glucose AUC is 2650 mg/dl/24h 
in people with obesity with a day-to-day variability of 80 ± 130 mg/dl/24h (mean ± SD). Using this SD and 
assuming similar variability in both groups, we estimate we will be able to detect a between group difference of 
~137 mg/dl/24h (~5.2%). Five nights of sleep restriction increases average plasma glucose concentration by 
11% over 10 h during the day125. Two nights of sleep restriction increases postprandial breakfast plasma 
glucose AUC by 7.4% after breakfast126.  
24-h insulin AUC. We have found average 24-h insulin AUC is 818 ± 333 µU/ml/24h in people with 
obesity with a day-to-day variability of 26 ± 85 µU/ml/24h (mean ± SD). Using this SD and assuming similar 
variability in both groups, we estimate we will be able to detect a between group difference of ~90 µU/ml/24h 
(~11%). Five nights of sleep restriction increases average plasma insulin concentration by 29% over 10 h125. 
Two nights of sleep restriction increases postprandial breakfast plasma insulin AUC by 52%126.  
Tissue gene expression and markers of inflammation and oxidative stress. It is difficult to make robust 
estimates of our study power to detect changes in molecular outcomes in adipose tissue and PBMCs, because 
of limited data evaluating these variables and the unknown effects of extending sleep. However, in our own 
experience, the population variance in PBMC and adipose tissue gene expression is ~30%36,87,88. In those 
studies, we used real-time PCR methods and detected transcriptional changes after diet-induced weight gain or 
weight loss36,87,88. Accordingly, we estimate that we have ≥80% power to detect ~35% difference in gene 
expression outcomes between groups, with a two-sided p-value of <0.05. 
 
D5. Scientific Rigor.  
We have made every effort to ensure we obtain robust results: i) the hypotheses being tested in this 
proposal are based on a sound foundation of preliminary data from our own studies and results from other 
studies; ii) we will use a randomized controlled study design, and well-established and validated techniques to 
obtain the study outcomes; iii) a carefully-defined population (metabolically unhealthy with strict exclusion 
criteria to remove factors known to influence outcomes) will be enrolled in the study; iv) we provide packed-out 
meals for 3 days before each inpatient study and provide standardized meals during admission to reduce 
variability; v) treatment groups will be balanced with regards to sex; and vi) the sample size has the statistical 
power needed to detect meaningful treatment effects. 
 
D6. Timeline.  
We plan to complete all inpatient studies within the first 4.5 years to leave time for final sample 
processing and data analyses in the last 6 months of the grant period (Table 2). This schedule will 
accommodate the time required to initiate the study, recruit and screen subjects, perform the experimental 
protocols before and after subjects complete the sleep extension intervention, process and analyze the study 
samples, collate the data, finalize the data analyses, and write manuscripts.  
 
 Table 2. Study timetable 
 
 
Subjects complete 
Admission 1 studies 
(baseline) (n) 
Subjects complete 
Admission 2 studies 
(baseline) (n) 
Subjects complete 
Admission 3 studies 
(post-intervention) (n) 
Subjects complete 
Admission 4 studies 
(post-intervention) (n) 
Year 1 
  9 
  9 
  5 
  5 
Year 2 
 13 
 13 
 13 
 13 
Year 3 
 13 
 13 
 13 
 13 
Year 4 
 13 
 13 
 13 
 13 
Year 5 
  2 
  2 
  6 
  6 
Total  
 50 
 50 
 50 
 50 

 
D7. Special considerations 
D7.1 PI does not have experience in conducting sleep studies or sleep extension. Although the PI (S. 
Klein) has considerable experience in conducting the complex outcome measures needed for this study, he 
does not have experience in conducting sleep studies or sleep extension. Therefore, the inpatient sleep 
regulation and the outpatient sleep extension protocol will be assisted by supervision and input from 
experienced Co-Is at UCB (J. Broussard and K. Wright) and at WUSM (B. Lucey). K. Wright is Professor and 
Director of the Sleep and Chronobiology Laboratory at the University of Colorado Boulder and has 25 years of 
experience in sleep research, including oversight of multicenter research grants and in sleep extension 
proposed in this study. He will provide guidance on issues related to the inpatient studies and in implementing 
sleep extension. He will also attend the bi-weekly videoconference meetings and travel to WUSM once per 
year for in-person investigator meetings. J. Broussard is an early career investigator who has considerable 
expertise in conducting sleep studies in people. She will travel to WUSM for 2 weeks at the initiation of the 
study and then every 2 months in the first year to work with research staff and Dr. Lucey to ensure correct use 
of sleep and circadian data collection methods. During her initial 2-week visit, she will oversee initiation of the 
sleep extension protocol and the constant routine protocol used to assess circadian rhythms. After year 1, Dr. 
Broussard will visit WUSM 3 times per year to review study protocols and progress, as well as participate in 
data analyses. Finally, she will provide support for day-to-day operations by email or phone calls as needed 
and will participate in bi-weekly videoconferencing with the research group. B. Lucey is a physician-scientist 
who is a sleep neurologist and Interim Director of the Washington University Sleep Medicine Center at WUSM. 
He has considerable experience in evaluating sleep and sleep architecture, and participates in daily 
polysomnogram review at the Sleep Medicine Center. He is a member of our Nutrition Obesity Research 
Center (NORC), because of his current interest in the relationship between sleep and metabolic health. He will 
be responsible for: i) supervising the registered sleep technician in conducting the inpatient PSG studies; ii) 
reviewing the data from each study to ensure technical reliability and quality; iii) identifying participants who 
have moderate to severe sleep apnea or other exclusionary, clinically significant sleep disorders; iv) facilitating 
referral of such participants to our Sleep Medicine Center for further evaluation and therapy, if indicated, 
including providing a report to the Sleep Medicine Center physicians; and v) participating in the bi-weekly 
videoconference project meeting, which will include colleagues at UCB. 
D7.2 Facilities for conducting sleep studies. The PI (S. Klein) is Director of the Clinical Research Unit, 
which now houses a newly-constructed, state-of-the-art sleep laboratory that coordinates services (certified 
polysomnographic technicians and equipment maintenance) with the Washington University Sleep Medicine 
Center. The CRU sleep laboratory was designed with direct and comprehensive input from the Co-Is of this 
proposal, B. Lucey, K. Wright and J. Broussard. The sleep laboratory is comprised of a 170 sq. ft. temperature-
, sound- and light-controlled sleep study room which is equipped with a Compumedics Grael PSG/EEG system 
with video monitoring. A control room with a dedicated computer for PSG review and processing is located just 
outside the sleep study room. A sound-insulated, light-proof catheter portal passes from the control to the 
sleep study room which allows blood sampling without entering the room and infusion of tracers and hormones 
with the infusion pump located in the control room to eliminate infusion pump noise at the bedside. The sleep 
study room is connected via intercom to the control room and the CRU nurses station. A shared software 
license is available for off-site sleep scoring and two Alice PDx Portable sleep diagnostic systems (Philips 
Health) are available for off-site or home sleep monitoring. 
D7.3 Compliance with sleep extension and attrition. We recognize the potential risk of attrition in this long 
duration sleep extension outpatient study. Therefore, we have developed a specific strategy to help ensure 
compliance with the sleep extension protocol that is described in section D2.3. Moreover, our Co-Is at UCB 
have shown that successful sleep extension is possible (see preliminary data in section C2.5) with good 
compliance and minimal drop-outs (only 1 of 15 subjects was not compliant with the sleep extension protocol 
and was removed from the study). In addition, the research coordinators who will be involved in the present 
proposal have demonstrated considerable success in retaining study participants in previous studies (>90%) 
that involved rigorous long-term lifestyle intervention (diet-induced weight loss and weight gain, and exercise 
training) with even more complex metabolic assessments e.g., 36,87,88,127,128.  
D7.4 Ambitious project that requires highly committed subjects, considerable organizational efforts, 
and interpretation of complex datasets. We have an experienced participant recruitment core with access to 
a large pool of potential participants, a strong track record in recruiting subjects who meet specific 

requirements, excellent participant compliance and retention in studies as rigorous or more rigorous than the 
current proposal, extensive experience in performing the complex metabolic studies proposed in this 
application, and expertise in conducting all sample analyses and interpretation of data needed for this project 
e.g.,21,31,36,67,74-88,90,92,94,108,110,129,130. Dr. Kenneth Schechtman will serve as the biostatistician for this project and, 
will ensure appropriate statistical procedures and adjustments for potential confounding variables and drop-
outs are used. 
D7.5 Effect of study participant age on sleep architecture. The age of our study participants will range from 
18-65 years old. We chose this range to increase our ability to identify people with MUO, which is much less 
common in younger age groups. This age range has implications for sleep, because there is a marked 
reduction in slow wave sleep (considered to be the most physically restorative sleep stage) and a reduction in 
sleep efficiency that begins in early adulthood131,132. Accordingly, people who are 18-65 years old are likely to 
have room for improvements in sleep. However, as noted in section D1 and D2.2, participants who have 
insomnia or other clinically significant sleep disorders will be excluded and participants who are unable or 
unwilling to extend sleep will be removed from the study. 
D7.6 Menstrual cycle in women. Women who are menstruating will be selected on the basis of a history of 
regular menstrual cycle ranging in length from 25-32 days with a maximum of 3 days variation month-to-month. 
We will use plasma progesterone and estrogen levels from the HEC procedure days and use menstrual phase 
as a co-variate in our analyses. To account for menstrual phase, we will initiate the pre- and post-intervention 
studies in the early follicular phase (confirmed by plasma progesterone and estrogen). Women with Late Luteal 
Phase Dysphoric Disorder will be excluded from participation because of alterations in sleep and circadian 
function associated with mood disorders. Postmenopausal women will also be included in this study to 
increase our ability to identify subjects with prediabetes or hyperinsulinemia. Since postmenopausal women 
could have lower sleep quality when not using hormone replacement therapy133, participants who have 
clinically significant sleep disorders will be excluded. 
D7.7 Clinical relevance. This study will determine whether sleep extension in people with MUO who are short-
sleepers has therapeutic effects on a series of key metabolic and inflammatory outcomes that are associated 
with sleep restriction. Accordingly, this study will prove or disprove this specific question, but is not meant to 
establish whether sleep extension is possible in a real world setting or what specific approaches are needed to 
extend sleep in clinical practice.   
 

E. PROTECTION OF HUMAN SUBJECTS 
 
E1. Risks to the subjects 
E1.1. Human Subjects Involvement and Characteristics. A total of 50 human subjects, aged 18-65 years 
old, will be studied. Subjects will be carefully screened with a medical history and physical examination, blood 
tests, an oral glucose tolerance test and resting electrocardiogram (see section D1. for details of 
inclusion/exclusion criteria). Mentally disabled persons, prisoners, pregnant and lactating women, and persons 
whose ability to grant voluntary informed consent is questionable will not be recruited. 
E1.2. Sources of Materials. All specimens will be obtained solely for research purposes. Although these 
generally will be obtained specifically for the purposes of the study, use will be made, where appropriate, of 
existing records and data obtained as part of routine clinical care. Measures to be obtained include: 1) detailed 
medical history; 2) physical examination; 3) clinical blood tests, including measurement of complete metabolic 
panel, complete blood count, plasma lipids, and HbA1c; 4) resting 12 lead electrocardiogram; 5) PSG 
recordings, 6) DXA scan and MRI to assess body composition and fat distribution, 7) hourly blood samples for 
24h to assess plasma melatonin, C-reactive protein, IL-6, TNF-α, MCP-1, glucose, FFA, and insulin 
concentrations; 8) urine to assess markers of inflammation and oxidative stress, 9) a HEC procedure to assess 
insulin sensitivity, 10) serial PBMC sampling to evaluate clock genes, and 11) muscle and adipose tissue 
biopsy specimens to evaluate markers of inflammation and oxidative stress. All data from each subject will be 
maintained confidentially and their names and identities will not be disclosed in any published document. 
E1.3. Potential Risks. We anticipate no psychological, social or legal risks beyond those of participation in 
health-related research in general. The potential risks associated with participation in this study are small and 
are listed below. They will be explained to all subjects who desire to participate in this research project. In 
addition, subjects will be informed that there is a possibility of unforeseeable risk, although we consider this 
unlikely. The research coordinator, research nurse and/or the PI will ensure understanding of the consent and 
study procedures, as well as laboratory results. In addition, the research nurse and/or PI will explain the 
various procedures and answer any questions the subjects may have before initiating any procedures. 
Whenever concerns arise, subjects will be informed that they are free to withdraw from the study at any time 
with no bias or prejudice. 
This research involves exposure to radiation from the dual-energy x-ray absorptiometry (DXA) for body 
composition measurements. The amount of radiation from these procedures, when averaged over the entire 
human body, is equivalent to a uniform whole-body dose of <1 mrem. This is equivalent to less than 3% of the 
amount of natural background radiation exposure all people in St. Louis receive each year. 
Possible side effects of intravenous catheter insertion are discomfort, bruising, damage to the blood 
vessel, bleeding at the site of needle insertion, and infection at the site of catheter insertion. Some people 
experience dizziness or feel faint.  
The intravenous infusion of any solution can cause infection if that solution is not sterile. The infusion of 
stable isotopically labelled tracers, per se, does not cause any additional risk because these tracers are 
infused at very small “tracer” amounts and already exist in our body and in the food we eat. In addition, only 
sterile and pyrogen-free stable isotopically labelled tracers are compounded, careful sterile technique are used 
in preparing the solutions, and all tracers are infused through a 0.22 micron filter during the study to decrease 
the risk of infection.  
Fat biopsies can cause swelling, bruising, temporary numbness, or long-term (>1 year) loss of 
sensation and tingling at the biopsy sites. Some people experience dizziness or feel faint. 
Blood sugar levels could change (become low or high) during the infusion of insulin and dextrose and 
may result in subjects feeling sweaty, shaky or nauseated. However, this risk is extremely small because blood 
sugar will be carefully monitored throughout the metabolic study. 
 
There is a possibility that potentially adverse medical conditions (e.g., diabetes mellitus, OSA) will be 
identified as a result of the research testing. Unless it is a medical emergency, we will inform the participant of 
the finding so they can follow-up with their physician for further investigation/treatment. In addition, we will 
review the information with the subject’s physician if the subject gives us permission to do so.  
 

E2. Adequacy of protection against risks 
E2.1. Recruitment and Informed Consent. Subjects will be recruited by flyers posted in the WUSM Medical 
Center and in the community, through the Volunteers for Health program at WUSM, and if necessary via radio 
and TV ads. The objectives of the project, all experimental procedures, all of the requirements for participation, 
and any possible discomforts and risks and benefits of participation will be clearly explained in writing and 
orally, in lay terms, to the subject by the principal investigator, co-investigators, or research coordinator. After 
all questions have been answered, and the subjects have been informed orally and in writing that they are free 
to withdraw from the study at any time with no bias or prejudice, and the subject agrees to participate, written 
informed consent, which has been approved by the Institutional Review Board at WUSM will be obtained. The 
consenting procedure will be conducted in the CRU approximately 1 week to 1 month (but at least 48h) before 
initiating the research protocol described in section D2. Subjects will be recruited from the general public and 
will include persons of all races and ethnic groups. We have a strong track record in recruiting subjects who 
meet specific study requirements, and we do not anticipate any problems in recruiting a sufficient number of 
subjects for timely completion of these two study protocols. 
 
E3. Protection Against Risks 
E3.1. Confidentiality. All key personnel involved in the design or conduct of the research involving the human 
subjects will receive the required education on the protection of human research participants prior to the start 
of this project. All specimens will be obtained solely for research purposes. Study samples and data sheets will 
be coded with an identification number for each subject. All data will be treated confidentially and the subjects' 
names and identities will not be disclosed in any published reports. Clinical records will be maintained in locked 
file cabinets within a locked file room. 
E3.2. Risks described in section E1.3. The potential risks of this study are small. Rigorous screening 
procedures will be used to screen out potential subjects with health problems and so to screen out subjects 
that might be at higher risk of adverse events. A pregnancy test will be conducted at screening and upon each 
admission to the CRU to ensure female subjects are not pregnant. If subjects become excessively sleepy and 
want to stop the study, they may do so and will be permitted to sleep before discharge. Subjects will be shown 
the sleep study rooms before the study, and study procedures will be described in detail to ensure there is no 
misunderstanding of the protocol. The sleep room is larger than a typical hospital room. Subjects will be 
informed about the continuous observation throughout the study. Subjects will be informed that they can stop 
the protocol at any time. All procedures will be performed by qualified and experienced personnel, and subjects 
will be carefully monitored during all procedures and during the intervention.  
Intravenous tracer solutions will be prepared in a designated, sterile mixing room by an experienced 
technician, and only sterility- and pyrogenicity- tested stable isotopically labeled tracers will be compounded. In 
addition, the tracer solutions are infuse through a 0.22 micron filter during the study as an additional protection 
to ensure no bacteria (which are usually 1.0 micron) will be infused. Careful aseptic techniques will be used 
when inserting the catheters, and when obtaining blood and adipose tissue samples to decrease the risk of 
infection. The total amount of blood collected will be <625 ml. Blood sugar will be carefully monitored 
throughout the HEC procedures. If a participant develops a health problem or potential health problem the 
PI/study physician and, if necessary a medical consultant who is not part of the research team, will decide 
whether the participant should continue in the study, and/or what further steps regarding medical 
evaluation/treatment should be performed. The CRU and hospital facilities used for the studies are equipped 
with defibrillators and all appropriate emergency medications. Both subjects and their primary care physicians 
(if consent is provided by the participant) will be made aware of any abnormal findings while participating in the 
study. The participants will be given the phone numbers of the members of the research team (including an 
emergency phone number) and told to call one of us immediately if they develop any unusual signs or 
symptoms. 
E4. Potential benefits of the proposed research to the subjects and others. Subjects participating in these 
studies will be reimbursed for their time and effort. Potential benefits to the subject, include information from 
the medical examination as well as the potential benefits of the sleep extension intervention. The benefits to 
society include a thorough evaluation of the effects of sleep extension on metabolic function in habitual short 
sleepers that may result in a low-cost intervention to improve metabolic function. 
 

E5. Importance of the knowledge to be gained. The information gained from this work has important public 
health implications regarding the effect of reduced sleep duration on metabolic health.  
 
F. DATA AND SAFETY MONITORING PLAN 
 
Data from the study will be monitored on a continuous basis by the PI. All serious adverse events (SAEs), 
adverse events (AEs), and laboratory values will be reviewed by the PI on an ongoing basis. Dr. Klein will be 
responsible for annual reports to the WUSM IRB and for reporting any adverse events (AEs) to the IRB.  
The IRB will be notified within 24 hours of any SAE occurring at that site via their online system for 
reporting SAEs and additional information will be forwarded to the IRB as it becomes available. The CRU 
will also be notified within 24 hours of any SAE by contacting the Research Subject Advocate and 
submitting to him/her the information provided to the IRB. In addition, all laboratory values outside of the 
normal range will be discussed with the study subject, and appropriate arrangements will be made for 
treatment, if necessary. 
A Data and Safety Monitoring Board, chaired by David Carr M.D. (Professor of Medicine), and 
including Dominic Reeds M.D. (Associate Professor of Medicine) and David Alpers (Professor of Medicine) 
will meet every 12 months with the PI and Co-Is to review study data, discuss any safety issues, and ensure 
compliance with the protocol. Collectively, these individuals have decades of experience performing and 
monitoring the safety of complex metabolic studies, including those employing hyperinsulinemic-euglycemic 
clamp procedures and stable isotope tracer infusions, in people.  
 
 

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