Effect of Melatonin on Cardiometabolic Risk- FULL

All participants will be recruited locally in the Boston area, and all study visits and
procedures will be conducted through Partners and the Harvard Catalyst.

Briefly, the trial-phase will consist of four visits, two as outpatient, and two as
inpatient. The cohort-phase will consist of two additional outpatient visits. The screening
outpatient visit will involve history taking, physical examinations (when appropriate),
phlebotomy to measure circulating factors and urine pregnancy testing for female subjects.
The 6-week outpatient check-up visit will be with a study coordinator. In addition to
evaluating for adverse effects, the coordinator will perform a pill-count. Phlebotomy will be
performed to measure circulating factors. The two inpatient visits will require two overnight
stays each; the procedures performed for these two inpatient stays will be identical, and
will include: timed urine collection, measurement of brachial artery flow mediated dilation,
phlebotomy (including an IV protocol using low dose heparin), hyperinsulinemic euglycemic
clamp, hyperglycemic clamp, central blood pressure, and placement of an ambulatory blood
pressure monitor that the participant will wear home for 24 hours and return by pre-paid
FedEx mailer. We will take blood samples at each visit, collecting about 54 tablespoons
(798.5 mL) of blood over the 36-week course of the study.

Outpatient visits: A screening visit will be scheduled when a participant expresses interest
in the study and is deemed potentially eligible by the study coordinator after a telephone
interview. At the screening visit, a study physician will obtain informed consent.
Thereafter, a study physician will conduct a history and physical examination, and phlebotomy
will be performed. Female subjects will provide urine for pregnancy testing. The purpose of
the screening visit is to evaluate potential volunteers based upon the inclusion and
exclusion criteria listed above, and to explain the purpose and procedures of the study to
the volunteers. If someone is eligible and willing, a baseline inpatient visit will be
scheduled.

A 6-week check up will be scheduled after the subject completes the initial inpatient visit.
In addition to evaluating for adverse effects, the coordinator will perform a pill-count.
Phlebotomy will be performed to measure circulating factors.

For the cohort phase, 24- and 36-week check up visits will be scheduled after the subject
completes the final inpatient visit. At these visits, in addition to evaluating for adverse
effects, the coordinator will perform a pill-count. Phlebotomy will be performed to measure
circulating factors.

Inpatient visits: Prior to each inpatient visit, participants will be mailed 9 bouillon
packets (containing 50 mmol of sodium each) and instructed to consume 3 packets each day for
three days prior to admission to the Clinical Trials Center; the purpose of this is to
achieve high sodium balance, which is necessary for our measurements of the renin-angiotensin
system. The high sodium diet will be continued during the inpatient setting. Female subjects
will have a urine pregnancy test done at admission. The following measurements will be
performed during the two inpatient visits, and the changes in these measurements (comparing
the baseline value with the 8-week value) are the endpoints for this study:

1. A hyperinsulinemic euglycemic clamp will be performed to measure peripheral insulin
sensitivity. Intravenous catheters are placed within each arm (one for infusion, and one
for blood collection). Then, a compound called insulin (supplied by the Investigational
Drug Service (IDS)) is infused as a loading dose followed by a continuous infusion to
achieve a steady state condition. Blood samples are collected every 5 minutes, and
glucose levels are measured. Blood glucose levels will be controlled by the infusion of
a 20% dextrose solution. The mean dextrose infusion rate (M) over the period between 120
and 180 minutes will be used to measure insulin sensitivity. At 180 minutes, the study
will conclude, and both infusions will be stopped.

2. A hyperglycemic clamp will be performed to measure pancreatic β-cell function.
Intravenous catheters are placed within each arm (one for infusion, and one for blood
collection). After a baseline period of 30 minutes, the blood glucose level is increased
with a loading dose of dextrose followed by a continuous infusion to achieve a steady
state condition. Blood samples are collected every 5 minutes, and glucose levels are
measured. Insulin levels are measured at 0, 2.5, 5.0, 7.5, 10, 20, 40, 60, 80, 100, 120,
140, 160, 180 minutes after the infusion. The 1st-phase insulin release is the sum of
the plasma insulin concentrations at 2.5, 5.0, 7.5 and 10 minutes. The 2nd-phase is the
average plasma insulin concentration during the final hour.

3. Endothelial-dependent vasodilation using brachial artery diameter will be measured by
ultrasound. A blood pressure cuff will be inflated on the arm to a supra-systolic
pressure for 5 minutes. This results in vasodilation of the downstream blood vessels.
Following release of the cuff, there is usually a 6 to 10-fold increase in flow through
the brachial artery, i.e., reactive hyperemia. Flow is a physiologic stimulus that
releases nitric oxide and causes dilation of the brachial artery, and hence, an increase
in the diameter of the brachial artery; this can be measured using ultrasound. The
maximal increase in brachial artery diameter occurs at one minute of reactive hyperemia
and this dilation is mediated by nitric oxide. Endothelium-independent vasodilation (as
a positive control) will be assessed by measuring brachial artery diameter under basal
conditions and 3 minutes following the administration of sublingual nitroglycerin (0.4
mg). Maximal brachial artery dilation occurs 3 to 4 minutes after the administration of
sublingual nitroglycerin.

4. Central aortic blood pressure measurement: The SphygmoCor XCEL System is a portable
machine that will be used to measure central aortic blood pressure at each of the
inpatient visits (this machine is FDA approved for measurement of central blood
pressure). A thin pillow is placed under the subject's arm to align the arm with the
heart. A blood pressure cuff is placed on the patient's arm, over the brachial artery to
obtain this measurement. The entire procedure will take approximately 10 minutes to
perform. Coded data is automatically stored via the SphygmoCor XCEL System and can be
downloaded onto a secure research computer for storage and analysis.

5. Phlebotomy will be performed to measure circulating melatonin, plasma renin activity,
and angiotensin II. These assays are markers of the systemic renin angiotensin system.
Phlebotomy will also measure adiponectin, leptin, ghrelin, and HbA1c. These assays are
markers of glucose homeostasis and insulin sensitivity. In addition, intravenous access
will be placed to permit blood draws every 2 hours from 7 pm until 9 am the following
morning (ie, the morning of day 1). These nocturnal blood draws will be used to measure
nocturnal serum melatonin levels. In order for these blood draws to be completed without
entering the subject's room and possibly waking the subject, a specialized circadian IV
protocol will be used whereby an antecubital IV line will be secured with a kerlix wrap
and connected to a 12 foot IV tubing that will traverse a metal window in the wall that
was constructed especially for such purposes and that is present in patient rooms in the
BWH CCI. Patency of the IV tubing during the night will be ensured by infusing a low
dose of unfractionated heparin (approximately 200 units per hour). The heparin will be
stopped and the IV converted to a standard IV at approximately 9 am on Day 1. This is
standard protocol in the BWH CCI for measuring overnight melatonin concentrations.

6. A 24-urine will be collected to measure excretion of epinephrine, norepinephrine, and
aldosterone.

7. Ambulatory blood pressure (for 24 hours) will be measured using a standard technique
employed commonly in clinical practice. An ambulatory blood pressure monitor will be
placed on the participant's arm before the participant is discharged from the inpatient
visit; the participant will also be instructed on its appropriate use, including how to
shut the monitor off after a 24-hour period. The participant will be discharged from the
inpatient visit with the cuff in place and will be given a pre-paid FedEx mailer so that
the monitor can be returned to the study coordinator for data download and re-use.

Inclusion Criteria:

- age ≥18 years

- body mass index (BMI) >30

- HbA1c 5.4-6.4%

- if hypertensive, BP <150/90

Exclusion Criteria:

- Use of a melatonin supplement

- Current use of anticoagulants

- Use of hypoglycemic or antihypertensive meds

- Current or prior history of diabetes mellitus, or random serum glucose ≥ 200 mg/dL

- Major bleeding event in the past 60 days

- Recent (past 2 weeks) or upcoming travel across time zones

- Known obstructive sleep apnea or use of continuous positive airway pressure

- Known heparin allergy

- Hepatic impairment

- Estimated GFR <60 mL/min/1.73m2

- Pregnant

- Job requires rotating night shifts

- Preexisting lung disease requiring oxygen

- Active chronic or acute infection

- Preexisting CVD

- Known melatonin allergy

- Bariatric surgery within the prior 12 months

- Preexisting hemoglobinopathy

- Use of weekly or daily NSAID for chronic pain

- Use of cholesterol lowering medications

- Active or uncontrolled psychotic disorder

- Use of hypnotic medications

- Active or uncontrolled bipolar illness

- Active or uncontrolled depression

- Recent (within the past three months) blood donation or plan to donate blood during
study period

- Use of a phosphodiesterase inhibitor

- Active malignancy within the prior 5 years

- Hypertension (>150/90)

- Currently lactating (women)

- History of stroke

- Hematocrit <36 (women) or <41 (men)

- Thrombocytopenia or other bleeding disorders

- Surgery in the past 60 days