Mechanisms of Cerebrovascular Control

Introduction It is widely accepted that insulin resistance in MetSyn increases the risk of
cerebrovascular disease (CVD) and stroke. Additionally, MetSyn is associated with cognitive
impairments and increased risk of neurodegenerative diseases. Despite strong association of
poor cerebrovascular health in MetSyn, the cerebral blood flow (CBF) response to acute
insulin surges in humans remains largely unexplored. Cerebrovascular dysfunction in response
to insulin and potential mechanism(s) that attenuate CBF may not only highlight the central
underpinnings of the pathophysiology of stroke and CVD, it may provide key insights to
understanding cognitive decline strongly linked to older individuals with MetSyn. Therefore,
establishing the mechanisms of insulin-mediated cerebrovascular control are not only
important for decreasing mortality in MetSyn, it may be profoundly important to maintaining
brain health. Insulin-meditated vasodilation is largely a vascular endothelium-dependent
process. Elegant experiments using animal models suggests insulin resistance disrupts six
endothelial signaling pathways, with the strongest evidence implicating a loss of nitric
oxide (NO) vasodilation, and exaggerated endothelin (ET-1) constriction. The literature
indicates that in MetSyn, a spike in insulin leads to vasoconstriction and cerebral
hypoperfusion. These findings are directionally opposite from no change or insulin-mediated
vasodilation and increase in CBF reported in the literature. Evidence suggests that uncoupled
function of nitric oxide synthase (NOS) leads to unfavorable balance between NO, reactive
oxygen species, and ET-1. Normally, stimulation of insulin receptor on the vascular
endothelium should provide an environment in which NOS manufactures NO leading to
vasodilation and inhibition of ET-1. Instead, in MetSyn insulin stimulates the
vasoconstrictor ET-1 to a greater extent while, NOS is uncoupled generating more reactive
oxygen species that scavenges the NO that is generated leading to a greater vasoconstrictor
signal rather than vasodilation.

Specific Aims/Study Objectives

Specific Aims:

The central hypothesis is that vasodilator actions of insulin are already impaired in
metabolic syndrome due to loss of dilator and gain of constrictor signals. Cerebral
vasodilation to insulin is largely endothelium-dependent. Emerging evidence in animal models
suggests insulin resistance disrupts at least six endothelial signaling pathways that could
be potential targets, and this proposal focuses on two that most likely reduce CBF in MetSyn:
1) Loss of nitric oxide (NO) vasodilation, and 2) Exaggerated endothelin (ET-1) constriction.
These experiments will address three specific aims:

Aim 1: To test the hypothesis that physiologic surges of insulin acutely increase CBF in
young adults, but adults with MetSyn exhibit paradoxical insulin-mediated vasoconstriction.

Aim 2: To test the hypotheses that key mechanisms responsible for poor CBF in MetSyn are
shifts in NO and ET-1 signaling. Specifically, in healthy controls, NO mediates robust
dilation, with little to no ET-1 constriction. In contrast, adults with MetSyn exhibit
uncoupled NO synthase (NOS) and exaggerated ET-1 constriction.

Aim 3: To test the hypothesis that insulin regulation of CBF is regionally distinct (e.g. MCA
reactive than ACA or basilar), and the negative effects of IR are similarly regionally
specific.

Hypotheses:

- Adults with metabolic syndrome will exhibit reduced cerebral vasodilation in response to
an OGTT

- NOS inhibition will reduce the CBF responses (Δ CVC/Δ insulin) in lean controls to
levels of Metabolic syndrome whereas it will increase CBF responses in Metabolic
syndrome, suggesting that Metabolic syndrome have uncoupled NOS function

- ET-1 inhibition will increase CBF responses (Δ Cerebral Vascular Conductance Index
(CVCi)/Δ insulin) in lean controls and metabolic syndrome. However, it will increase
significantly more in in metabolic syndrome.

- The MCA will be more reactive to insulin surges than other cerebral arteries

Study Endpoints: Change in Cerebral vascular conductance (CVC, ml/min/100mmHg) relative to
the change in concentration of insulin in blood after consumption of glucose.

Subjects Sixty participants (30 per group) will be recruited. Determination of eligibility
will be a two-step process. The first step is all participants will complete an initial
screen to determine if they qualify for the screening visit. Preliminary eligible subjects
will be invited to the research lab for informed consent and formal screening.

During the screening visit at the research laboratory, subjects will complete informed
consent, a health history questionnaire, an MRI Safety screening form, and a physical
activity questionnaire. Height, weight, blood pressure, and waist and hip circumference will
also be taken. Additionally, a venous blood sample will be drawn for glucose and lipid
testing to determine eligibility. If the screening visit reveals that there is an abnormality
or contraindication to participation, which is covered or not covered in the summarized
exclusion criteria, the potential subject will not be allowed to participate. As a summary,
subjects will experience the following activities during the in-lab screening visit:

Methods Following initial screening and a screening visit, participation in this study will
involve two separate MRI study visits (Saline infusion as placebo, versus L-NMMA infusion to
inhibit NOS OR Ambrisentan orally (male participants only), versus Lactose placebo orally to
inhibit ET-1). Subjects will be directed into an experimental condition (placebo/L-NMMA or
placebo/Ambrisentan) based on number of subjects need to maintain statistical power. For the
Ambrisentan trial, subjects must be male, due to potential harmful effects of drug on fetus.
Once assigned an experimental condition group, the drug order (placebo/drug) will be
performed in a randomly assigned, counter-balanced single blind fashion. The experimental
procedures will be identical on all testing days with the exception of which drug will be
infused. Throughout each visit, subjects will be monitored for heart rate, blood pressure,
end-tidal carbon dioxide, and verbal communication of adverse symptoms (how is the subject
feeling) while in the MRI scanner.

Pregnancy Test: All female subjects must have a negative urine pregnancy test prior to either
study visits (placebo or drug) to ensure they are not pregnant at the time of each study
visit. Females will not be assigned to the Ambrisentan trial, even if their pregnancy test is
negative.

Venipuncture Blood Sample: During the screening visit, blood (up to 20 ml) will be drawn
using venipuncture to be analyzed for fasting glucose, serum lipids.

Subjects will abstain from exercise, caffeine, and NSAIDS, as well as fast for ≥ 10 hours
prior to all experimental trials.

Intravenous Catheter: Trained staff will place two intravenous catheters. Placement of the
catheters will be in any arm veins deemed most suitable for ease of access. However, one will
be placed in the antecubital fossa (infusion), and one in the antecubital, hand, or wrist
vein (blood sample) of the opposite arm for each of the two study visits. The blood sampling
IV catheter will be used to draw up to 15 mL blood samples at specific time points throughout
each study visit to measure concentrations of glucose and insulin as well as for markers of
inflammation and oxidative stress. All procedures are identical during each trial
(placebo/L-NMMA/BQ123). The specific time points, which are approximate due to small
variations in MRI scan times, are as follows:

- Baseline, prior to baseline PC VIPR scan

- Before PC VIPR scan 1 after OGTT

- Before PC VIPR scan 2 after OGTT

- Before PC VIPR scan 3 after OGTT

- Before PC VIPR scan 4 after OGTT

- Before PC VIPR scan 5 after OGTT

- Before PC VIPR scan 6 after OGTT

- Before PC VIPR scan 7 after OGTT

- Before PC VIPR scan 8 after OGTT

- Before PC VIPR scan 9 after OGTT

Intra-venous infusion:

I. NOS inhibition: L - NMMA is a potent non-selective NOS inhibitor used to study vascular
physiology. L-NMMA will be infused at 3 mg/kg body weight/hr bolus (over 10min ) followed by
a maintenance infusion of 1 mg/kg/hr for the duration of the experiment. Systemic delivery of
L - NMMA has been shown to elevate blood pressure ~8-15 mmHg that is within the range
observed during exercise.

II. Placebo: Placebo infusion rates will be determined using the same formulas for L-NMMA
infusion, using the subject's most recent screening weight. Ideally, saline will be infused
at the same rate and quantity as the corresponding drug visit. Subject's that are rescreened
and have a weight change, however, may have a saline infusion rate that differs slightly from
the L-NMMA infusion rate.

A small discrepancy in saline and drug infusion rate is insignificant from a scientific
perspective as there is already variability in the amount of saline that the subject receives
since it is used to flush the IV (1-5 mL) with every blood draw (up to 10 blood draws).

ET-1 - Inhibition studies (15 control, 15 MetSyn) (Ambrisentan Arm):

I. ET-1 inhibition: Ambrisentan is an antagonist of the ETA receptor. Subjects will receive
10 mg of Ambrisentan in a pill form 90-150 minutes prior to the OGTT. This class of drugs
have been used to research ET-1 signaling in diabetes, obesity, and hypertension (Bruno,
Sudano, Ghiadoni, Masi, & Taddei, 2010; Lteif, Vaishnava, Baron, & Mather, 2007; Mather,
Mirzamohammadi, Lteif, Steinberg, & Baron, 2002). ETA receptors are the most likely to
mediate excessive constriction in MetSyn patients. Ambrisentan will be taken orally. Systemic
delivery of endothelin antagonist have been shown at chronic dosing to decrease systolic
blood pressure 4.5±10 mmHg and diastolic 3 ± 7.5 mmHg in hypertensives, and not alter mean
arterial pressure in obesity (Bruno et al., 2010; Mather et al., 2010; Spratt et al., 2001).

II. Placebo: Placebo pill similar in size and shape to Ambrisentan will be used as placebo
control.

Magnetic Resonance Imaging (MRI): A 3 Tesla MRI will be used to quantify cerebral blood flow
and capture cerebral vessel structure at designated time points throughout the study visits
(at baseline and during treatment conditions). No contrast agent will be used at any time.
The subject will be in the scanner for an initial baseline scan and during the experimental
trials. All scanning will be accomplished in the proposed timeline of experimental trials.
While in the scanner, subjects will be monitored for heart rate, ECG, end-tidal carbon
dioxide (CO2), and blood pressure. The scanner has the capabilities to collect this data,
except for end-tidal CO2, which will be collected with a subject monitor. In addition to
standard pulse sequences commonly obtained for clinical purposes (localizers, standard MR
Angiography for vessel anatomy (without a contrast agent), 2D phase contrast MRI for velocity
encoded measurements), a.s.o.; 'basic sequences'), an acquisition scheme developed at the
University of Wisconsin-Madison will be used. This scheme, PC VIPR (phase contrast vastly
under-sampled isotropic projection reconstruction) is unique in its capability to acquire
volumetric data sets with three-directional velocity encoding and high spatial resolution in
fairly short scan times. All pulse sequences used in this study are designed to stay within
the current guidelines for dB/dt established by the FDA.

Oral Glucose Tolerance Test (OGTT): The subject will drink distilled deionized water (300ml)
containing 75 grams glucose within 5 minutes. This is the standard administration of OGTT.

Re-enrollment: Male subjects that fully complete the study will have the choice to re-enroll
and complete the wing of the study they did not participate in. Subjects will be re-screened
to determine eligibility and will be separated by at least 1 week from completion of the
study. Furthermore, they only participate in the L-NMMA or Ambrisentan (which ever they have
not completed in the other wing of the study), they will not need to repeat the
control/saline visit.

Inclusion Criteria:

- Controls: Subjects will be healthy, sedentary individuals. They will be normotensive,
and without any cardiovascular co-morbidities.

- Metabolic Syndrome: Subjects must qualify under the National Cholesterol Education
Program-Adult Treatment Panel III (NCEP-ATP III) definition of metabolic syndrome as
modified by the American Heart Association and International Diabetes Federation.
Subjects must meet 3 or more of the following 5 criteria:

1. fasting glucose ≥ 100 mg/dL,

2. fasting triglycerides ≥ 150 mg/dL,

3. HDL-cholesterol < 40 mg/dL (men) or < 50 mg/dL (women),

4. elevated blood pressure (≥ 130 systolic and/or ≥ 85 diastolic mmHg),

5. waist circumference at the iliac crest > 102 cm (men) or ≥ 88 cm (women).

Exclusion Criteria:

- Subjects with ≥126 mg * dL-1 fasting glucose and having 1-2 of the above criteria for
MetSyn

- A personal medical history of coronary artery disease, stroke, heart attack, heart
valve disease, congestive heart failure, previous heart surgery, history of lung
disease or peripheral artery disease, or history of renal/kidney and liver/hepatic
disease

- Subjects that take insulin or any medications for cardiovascular-related issues

- Subjects who report sleep apnea

- Subjects who report current or past tobacco use (> 1 year in duration). Tobacco use
will include smoking and all forms of smoke-less tobacco (e-cigarettes and chewing
tobacco).

- Females that are pregnant, lactating, or postmenopausal

- Contraindications of having an MRI (such as claustrophobia, metallic implant, etc.)