Peripheral Vasodilation in Obese Humans

This study initially started in June 2011(IND 122211). The original Aim 1 was completed and
showed lean and obese groups did not differ in endothelial dependent dilation (EDD) and
exercise dilation responses. The original Aim 2 was contingent upon there being a difference
between lean and obese groups in Aim 1 and therefore was not pursued.

The investigators now plan to continue this study by assessing 2 additional aims that are
consistent with the original purpose of the study and are discussed below:

The growing population of obese adults is predicted to create a large public health burden in
the next few decades. Obese humans also exhibit reduced exercise capacity and lower muscle
blood flow. The overall goal of this research program is to investigate the obesity-related
changes in microvascular function contributing to impaired muscle blood flow. This proposal
seeks to directly test vascular control in human skeletal muscle resistance arteries in
exercising humans. The investigators propose to study younger obese adults, without
confounding effects of age, metabolic syndrome, or diabetes-before the negative effects of
obesity can exert their full negative impact. The general hypothesis is that endothelium
dependent dilation (EDD) and functional (exercise) vasodilation is impaired via shift away
from nitric oxide bioavailability and a shift toward increased reliance on potassium (K+)
mediated vasodilation.

Primary objective To determine whether role of K+ mediated vasodilation differs between lean
and obese adults.

Aims Aim 1: To test the hypothesis that obesity induces an endothelial phenotype change that
creates an environment for both EDD and exercise dysfunction. The investigators propose that
obesity alters endothelial enzymes, which help explain the mechanistic changes in
vasodilation.

Aim 2: To test the hypothesis that obesity elicits altered endothelial-dependent (EDD) and
functional vasodilation due to altered functional potassium channel signaling. The
investigators propose that potassium (K+) channel function accounts for unexplained
differences in EDD and functional vasodilation. More specifically, the investigators
hypothesize that inhibition of K+ mediated hyperpolarization (Inward Rectifying K+
channels-KIR) will identify differential vasodilator mechanisms between lean and obese
adults.

Study Design and Overview of Procedures This study is designed to test cardiovascular control
in clinically healthy humans. There will be 2 separate, scientifically distinct experimental
trials that will be conducted in lean and obese subject: 1) EDD or 2) Rapid Onset Vasodilator
(ROV), which studies the immediate increase in blood flow at the onset of exercise.

Common procedures between protocols: After screening to determine eligibility, subjects visit
the lab for either EDD or Rapid Onset Vasodilator (ROV) study visit. All pre-screening and
pre-visit procedures will be identical. A physician will place a brachial arterial catheter
in the non-dominant arm for local drug infusion. Changes in blood flow will be quantified by
Doppler ultrasound at the brachial artery. Monitoring of subject hemodynamics (Heart Rate,
Blood Pressure, blood oxygen) is identical.

EDD experiments: EDD agonists are drugs than cause vasodilation to increase blood flow
temporarily. The investigators will use 4 different agonists to test EDD function from
several perspectives. After control EDD agonist infusions, subjects will repeat EDD agonists
under conditions of K+ channel inhibition, and again under K+ channels plus nitric oxide and
prostaglandin inhibition. This last phase of the study is aimed at testing acute
compensatory/redundant vasodilator mechanisms. These trials will involve infusion of
endothelial agonists, with and without inhibition of specific vasodilatory mechanisms.
Agonists include: ATP, Bradykinin (BK), Isoproterenol (ISO), and Acetylcholine (Ach), will be
infused 3 times each (order randomized). Agonists will be infused under the following
conditions: 1) Alone (control) 2) in combination with Barium Chloride (BaCl2) 3) in
combination with BaCl2, L-N-monomethyl Arginine Acetate (L-NMMA), and Ketorolac. EDD agonists
each cause a temporary increase in forearm blood flow, which returns to baseline within
minutes after infusion has stopped. Antagonists (BaCl2, L-NMMA and ketorolac) will likely
reduce EDD responses to agonists, and may also reduce resting forearm blood flow or increase
blood pressure (e.g. L-NMMA may decrease forearm blood flow 30-50% an increase BP 5-10 mmHg).
The entire EDD study visit will be ~5 hours (1 hour setup and 4 hours experimental
procedures). The EDD protocol is further described here:

Drugs (IND approval) are used to test basic science vascular function & associated mechanisms
in healthy volunteers. Drugs not used to improve a health condition.

Drugs: acetylcholine, isoproterenol, bradykinin, adenosine triphosphate, barium chloride,
L-N-monomethyl Arginine Acetate, ketorolac.

1. 5 minute saline infusion along with 2 minutes of ATP infusion,

2. 10 minute washout,

3. 5 minutes of saline solution along with 2 minutes of BK infusion,

4. 10 minute washout,

5. 5 minutes of saline solution along with 2 minutes of ISO infusion,

6. 10 minute washout,

7. 5 minutes of saline infusion along with 2 minutes of ACh infusion,

8. 10 minute washout,

9. 5 minutes of BaCl2 infusion and 2 minute of ATP infusion,

10. 10 minute washout,

11. 5 minutes of BaCl2 infusion along with 2 minutes of ACh infusion,

12. 10 minute washout,

13. 5 minutes of BaCl2 infusion along with 2 minutes of ISO infusion,

14. 10 minute washout,

15. 5 minutes of BaCl2 infusion along with 2 minutes of BK infusion,

16. 10 minute washout,

17. 10 minute L-NMMA and ketorolac infusion (continuous infusion throughout remainder of
protocol) along with 5 minutes of BaCl2 infusion, and 2 minutes of ACh infusion,

18. 10 minute washout,

19. 5 minutes of BaCl2 infusion along with 2 minutes of ISO infusion,

20. 10 minute washout,

21. 5 minutes of BaCl2 infusion along with 2 minutes of ATP infusion,

22. 10 minute washout,

23. 5 minutes of BaCl2 infusion along with 2 minutes of BK infusion.

ROV experiments: After control single forearm muscle contractions, subjects will repeat
single contractions under conditions of K+ channel inhibition, and again under K+ channel
plus nitric oxide and prostaglandin inhibition. This last phase of the study is aimed at
testing acute compensatory/redundant vasodilator mechanisms. This trial consists of three
sets of 6 singular muscle contractions (three at 30%, three at 60% of maximal effort), each
contraction lasting less than 3 seconds with ~90s rest in between. These single contractions
evoke a robust (100-600% increase) and rapid (3-6 heart beats post contraction) increase in
blood flow that returns to normal typically in about 30 seconds. This rapid and robust
response is why these single muscle contractions are termed "Rapid Onset Vasodilation". 3
minutes of rest before each trial will allow for baseline measurements and loading of
inhibitors of K+ mediated vasodilation. During this 3 min, subjects will perform a single
contraction at 15, 30, and 45s of the 3-min loading/resting period to facilitate delivery of
the drug to the active tissues (this adds an additional 18 contractions. The first
experimental set of single contractions will be completed without drugs (saline control).
Next, an ATP infusion (no exercise) will also be used to test the pharmacological efficacy of
subsequent inhibitor (BaCl2). The first ATP infusion will be performed without other drugs.
Following this, a second set of single contractions will be conducted with infusion of BaCl2.
This is then followed by a second ATP infusion to determine the efficacy of BaCl2. The final
set of single contractions will start and be performed with simultaneous infusion of all
three inhibitors (BaCl2, L-NMMA, and Ketorolac). This last phase of the study is aimed at
testing acute compensatory/redundant vasodilator mechanisms. ROV (exercise) studies will be
~4 hours (1 hour set up and 3 hours experimental procedures). The ROV protocol is further
described here:

Drugs: adenosine triphosphate, barium chloride, L-N-monomethyl Arginine Acetate, ketorolac

1. 3 muscle contractions during 3 minute saline infusion followed by 3 additional muscle
contractions,

2. Repeat 3 muscle contractions during 3 minute saline infusion followed by 3 additional
muscle contractions ,

3. 10 minute washout,

4. Two minutes of ATP infusion,

5. 10 minute washout,

6. 3 muscle contractions during 3 minute BaCl2 infusion followed by 3 additional muscle
contractions,

7. 3 muscle contractions during 3 minute BaCl2 infusion followed by 3 additional muscle
contractions,

8. 10 minute washout,

9. Infusion of BaCl2 (3 minutes) and ATP (2 minutes),

10. 10 minute washout,

11. 3 muscle contractions during Infusion of BaCl2 (3 minutes) and L-NMMA and ketorolac
infusion (5 minutes) followed by 3 additional muscle contractions,

12. L-NMMA and ketorolac will be infused throughout remainder of protocol at a lower dosage

13. 3 muscle contractions during Infusion of BaCl2 (3 minutes) and L-NMMA and ketorolac (5
minutes) followed by 3 additional muscle contractions.

Inclusion Criteria:

- Age: ≥18 - ≤ 40 years of age

- Blood pressure systolic ≤125 and diastolic ≤85 mmHg (Lean control) and <140 and
diastolic <90 mmHg (Obese subjects)

- BMI <25 kg/m2 (Lean control) and ≥30 kg/m2 (Obese subjects)

- Fasting blood glucose <100 mg/dL

- Fasting LDL <130 mg/dL

- Fasting blood triglycerides <150 mg/dL

- Females: Premenopausal with a regular menstrual cycle. Negative pregnancy test. Note:
Must be studied in the early follicular phase (day 1-5) of their menstrual cycle or
the low hormone phase of oral contraceptive use.

Exclusion Criteria:

- Specific to obese subjects only (to exclude obese subjects with metabolic syndrome):

a. If Systolic BP 130 ≤ BP < 140 mmHg or Diastolic BP 85 ≤ BP < 90 mmHg i. HDL < 40
mg/dL for men ii. HDL < 50 mg/dL for women

- Increased risk of bleeding

- Sensitivity to lidocaine

- Procoagulant or other clotting disorders

- Taking cardiovascular medications (anti-hypertensives, statins, platelet inhibitors,
etc.) or metabolic medications (insulin-sensitizing)

- Regularly (≥5 days/week) take acetylsalicylic acid and/or non-steroidal
anti-inflammatory drugs (NSAIDs)

- Self-reported current diagnosis of exhibiting obstructive sleep apnea

- Self-reported current or past diagnosis of diabetes

- Self-reported history of peripheral vascular disease

- Self-reported history of hepatic disease

- Self-reported history of renal disease

- Self-reported history of hematologic disease

- Self-reported history of stroke

- Current use of tobacco (i.e. smoke, smokeless, and vapor). Prior tobacco use with >1
year abstaining is allowed.

- Participation in vigorous aerobic exercise >90 minutes per week

- Arterial structures in the arm that are not conducive to study in the laboratory
setting

- Females: Self-reported pregnancy and/or breastfeeding, diagnosed with polycystic
ovarian syndrome, or without regular menses. Urine pregnancy test will be performed on
study day to ensure subject is not pregnant on study day.

- An abnormality or contraindication to study participation which is not covered in the
eligibility criteria.