The Relationship Between Sleep and Glucose Tolerance in Prediabetes: the Role of GLP-1 in Short Sleepers
Phase: NA
Conditions: Prediabetes
Sponsor: Ramathibodi Hospital

Protocol Title: The Relationship between Sleep and Glucose 
Tolerance in Prediabetes: the Role of GLP-1 in Short Sleepers  
 
NCT02229487 
 Date: September 5, 2014 

  
 
Protocol and Planned Statistical Analysis 
     
 Background and Rationale 
 
            Humans spend approximately a third of their lifetime sleeping. Sleep is viewed as a state of energy 
conservation and replenishment of energy storage. Human behaviors and certain pathological conditions 
may affect this important physiological process, resulting in sleep disturbances, including insufficient sleep, 
poor sleep quality and obstructive sleep apnea (OSA). There is a large body of evidence from human 
experimental as well as observational studies linking sleep disturbances to abnormal glucose metabolism 
and diabetes risk. 
 
           Insufficient sleep has been linked to reduced insulin sensitivity and increased risk of type 2 diabetes, 
both in laboratory studies in healthy humans and in epidemiologic studies. A causative role of partial sleep 
restriction in promoting alterations in glucose metabolism was first established in 1999(1). Intravenous 
glucose tolerance testing (IVGTT) following sleep restriction to 4 hours per night(h/night) for 5 nights 
resulted in a 24% decrease in insulin sensitivity as well as a 30% decrease in the acute insulin response 
to intravenous glucose(1,2).  Multiple cross sectional epidemiologic studies have indicated that short sleep 
duration (usually < 6 h/night) is associated with increased impaired glucose tolerance and diabetes risk(3).  
A recent meta-analysis including 7 of these 10 studies (total 107,756 participants concluded that short 
-6 h/night) predicts the development of type 2 diabetes with a pooled relative risk (RR) of 1.28(4). 
 There are only a few studies examining the impact of insufficient sleep on glycemic control in patients with 
established type 2 diabetes. A questionnaire survey study of 161 African Americans with type 2 diabetes 
found that 3 hours of perceived sleep debt per day (i.e. a self-report of insufficient sleep duration) predicted 
a HbA1c level of 1.1% above the median(5). The magnitude of this difference is comparable to the effect 
size of several FDA-approved diabetes medications. Recently, a large cross sectional study of 4,870 
Japanese participants revealed a U-shaped relationship between sleep duration and glycemic control, with 
higher HbA1c levels in patients with self-reported sleep duration <
to those with 6.5-7.4 h/night (6). These data support a role of sleep duration on glycemic control in patients 
with type 2 diabetes. However, the assessments of sleep duration in these studies were done subjectively 
i.e. by questionnaires. The only study which assessed the relationship between glycemic control (HbA1c) 
utilizing objective sleep measurement by actigraphy revealed that only sleep fragmentation, but not sleep 
duration, was associated with higher HbA1c level (7).  
            In addition, other aspects of sleep disturbances have also been shown to affect glucose control in 
patients with type 2 diabetes, including poor sleep quality(8) and the severity of untreated OSA (9). 
Therefore, the independent role of sleep duration and glycemic control requires further investigation. 
Whether the relationship extends to those with prediabetes is unknown. We hypothesized that prediabetes 
patients with short sleep duration, as measured objectively, will have worse glucose tolerance than those 
with normal sleep duration.  
            GLP-1 is an important incretin hormone which plays a role in regulating postprandial glucose 
excursion and appetite regulation. The role of sleep in GLP-1 regulation was explored in a few experimental 
studies in healthy human. Sleep restriction to 4 hours per night in healthy participants resulted in lower 
GLP-1 levels in the afternoon in women (10). In addition, total sleep deprivation in healthy participants 
resulted in a delayed GLP-1 response to breakfast (11). Sitagliptin treatment for 12 months in patients with 
type 2 diabetes resulted in 0.6% decrease in HbA1c as well as improved sleep quality as assessed by 
questionnaire (12). The role of GLP-1 response in patients with prediabetes who are short sleepers has 
not been explored. We hypothesize that those with chronic short sleep will have a lower or delayed GLP-
1 response to meal compare to those with normal sleep duration. If this is proven, GLP-1 therapy may 
especially be effective in improving glycemic control in patients with abnormal sleep. Since the current 
proposed study is exploratory in nature by means of association, future interventional trial will be needed 
to show the causal relationship between GLP-1 and sleep.  
             Therefore, the aims of the current study are to explore the relationship of sleep duration and 
glucose tolerance in patients with prediabetes, as well as the effects of chronic short sleep duration on 
GLP-1 response to meals in these patients 

 
     Objectives & Hypotheses:  
            1. To determine the independent role of sleep duration on glucose tolerance in patients with 
prediabetes. 
            2. To determine if patients with prediabetes with short sleep have delayed or reduced GLP-1 
response to  
                meal 
 
     Hypothesis 
           1.  Prediabetes patients who have insufficient sleep will have worse glucose tolerance than those 
with normal  
                sleep duration. 
           2.  Prediabetes patients with short sleep will have a delayed or reduced GLP-1 response to a 
standardized meal      
 
     Study Design/Population/Method 
The study has three components; 
 
Step 1: Questionnaire study of 600 patients with prediabetes. The questionnaires will capture 
subjective sleep measurements and eating habits. 
Step 2: Participants ages 40-65 reporting short sleep duration (≤6 h/night) and normal sleep 
duration (7-8 h/night) will undergo objective sleep measurements including sleep duration, 
sleep quality and obstructive sleep apnea. This step will serve as a screening for step 3 
(expected N= 80). 
Step 3: Study to compare GLP-1 response to standard mixed meal test in patients with 
chronic short sleep and those with normal sleep duration. (n= 31 in each group).  
 
STEP 1: 
Inclusion Criteria: 
1. Patients with prediabetes (HbA1c 5.7-6.4% or history of fasting plasma glucose 100-
125 mg/dl) who receive medical care at Ramathibodi Hospital  
2. Age 18 or older 
3. Can understand Thai (speaking, listening and reading) 
4. Agree to participate by written informed consent 
 
Exclusion criteria: 
1. Those who depend on others for feeding (such as stroke patients) 
2. Shift workers 
3. History of congestive heart failure or low ejection fraction  
4. Chronic obstructive pulmonary disease, end stage renal disease or chronic liver 
disease (AST or ALT > 3 times the upper limit of normal) 
5. Use of medications: opioids/ narcotics , alpha blockers (prazosin, doxazosin, 
terazosin), clonidine, methyldopa, nitroglycerin  
6. Patients with permanent pacemaker  
7. History of previous stroke 
 
STEP 2: 
Participants age 40-65 who completed step 1 and were not taking diabetes medications will 
be invited to participate if they reported sleep duration <6 h/day, or normal sleep duration (7-8 
h/day). They will undergo objective sleep assessments 
 
 
STEP 3: 
Participants who complete step 2 and are confirmed to have short sleep duration and normal 
sleep duration by objective assessments will undergo a mixed meal test with measurements 
of GLP-1, glucose and insulin.  

  
 
 
     
 Study Procedures/Study Flowchart 
 
 
 
 
STEP 1 
After informed consents are obtained, the participants will be interviewed regarding their medical history 
and sleep schedules on weekdays and weekends, as well as other aspects of sleep, mood and dietary 
habits. Standardized questionnaires will be utilized, including  Morningness-Eveningness questionnaire 
(13) Pittsburg Sleep Quality Index to assess sleep quality (14), Berlin Questionnaire to assess risk of 
sleep apnea(15), depressive symptoms (CES-D)(16), Epworth Sleepiness Scale to assess daytime 
sleepiness (17), and review of dietary intake in the past 24 hours (24 hour food recall). 
Medical records will be reviewed to collect data regarding medication use. 
Weight, height, hip and waist circumference as well as neck circumference will be measured. 
Blood sample will be obtained for hemoglobin A1c levels within 6 months of an interview if not done in 
the prior 6 months. Additionally, blood sample will be saved for possible further genetic and metabolic 
analyses. 
 
STEP 2  
Those between ages 40-65 years who reported short sleep (≤6h/day) and normal sleep (7-8 h/day) will 
undergo objective sleep measurements as follow. Expected participants = 80 
Participants will wear Actiwatch (Respironics, USA) at home for 1 week. Actiwatch is a small, watch-like 
device which can measure sleep timing and quality, daytime activities as well as light exposure. 

Participants will wear the device on their non-dominant wrist and instructed not to wear clothes which 
sleeves may cover the watch. The device is water resistant. 
During this week, the participants will keep a record of their sleep schedules and dietary intake.  
On the last morning of actiwatch wearing, participants will collect overnight urine for urine melatonin 
measurements. They will fast after midnight and return to the hospital to have blood sample taken for 
fasting glucose and insulin, as well as return the actiwatch device.   
The participants will bring the device, Watch PAT 200(18), to wear overnight at home. This device will 
assess the presence and the severity of sleep apnea (Apnea Hypopnea Index). This non-invasive device 
is shaped similar to a large watch which the participants will wear on their non-dominant wrist. There will 
be two probes connecting the device to their fingers to measure peripheral arterial tone and oxygen 
saturation. The participants will wear the device before going to bed, remove it upon awakening and 
return the device to the investigators in the morning. 
 
STEP 3 
After confirming the sleep duration by Actiwatch, both short and normal sleepers will undergo a 75-gram 
OGTT after an overnight fasting. Serum glucose, insulin and GLP-1 will be collected every 30 minutes for 
3 hours. Expected participants in each group = 31 
 
Statistical Methods 
 
STEP 1 and 2: 
 
Sleep variables from questionnaires (subjective) and actigrapgy/WatchPAT (objective) including sleep 
duration, sleep quality and sleep apnea will be analyzed to explore their relationship with HbA1c, and when 
available, fasting glucose and insulin levels. Bivariate analysis will be used. To identify the independent 
sleep predictor of glucose tolerance status, multivariate regression analysis will be employed. 
 
STEP 3: 
 
Area under the curve of GLP-1 and its peak response will be compared between short and normal 
sleepers. 
 
 
Power/Sample Size: 
 
STEP 1: The sample size of 600 was selected to follow a rule of 10 participants for each variable in the 
multivariate regression analysis. Predictors of HbA1c include age, sex and BMI. Sleep predictors will 
include sleep duration, quality and sleep apnea risk. In addition, the sample size was chosen to ensure 
adequate recruitment of short sleepers (based on prevalence of 20-25%) into STEP 2 and 3.  
 
STEP 2: The sample size of 80 in step 2 was chosen to ensure adequate recruitment for STEP 3 which 
requires 62 participants, with a 75% concordance rate of self-reported and objectively measured sleep 
duration 
 
 
STEP 3: Based upon a sample size of n=31 patients per group, this study has 80% power to detect a 
35% difference in the peak time of GLP-1 between groups GLP-1 level with a 10% drop out rate. This 
calculation is based on data of a previous study of acute sleep deprivation and GLP-1 response (11).     
 
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