Mechanisms of Cerebrovascular Control
Condition: Metabolic Syndrome X
MeSH terms: Metabolic Syndrome; Syndrome
Study type: Interventional
Phase: Phase 1

Summary: 
      The objective of this study is to identify insulin-specific cerebral blood flow (CBF) control
      mechanisms, and establish cerebrovascular responsive baseline in younger (18-45 yrs)
      metabolic syndrome adults (MetSyn) who are at substantial risk of stroke and other types of
      cardiovascular mortality even if they never develop diabetes. The central hypothesis is that
      vasodilator actions of insulin are impaired in MetSyn due to loss of dilator and gain of
      constrictor signals. This study will focus on 2 mechanisms that likely limit CBF in MetSyn:
      1) Disruption of nitric oxide (NO) vasodilation, and 2) Exaggerated endothelin (ET-1)
      constriction. Three specific aims will be addressed: 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. Middle Cerebral Artery (MCA) reactive than Anterior Cerebral
      Artery (ACA) or basilar), and the negative effects of insulin resistance (IR) are similarly
      regionally specific.
    

Detailed description: 
      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.
    

Intervention name(s): NOS Inhibition; ET-1 Inhibition; NOS Inhibition Placebo; 3 Tesla MRI; Intravenous Catheter; Oral Glucose Tolerance Test; ET-1 Inhibition Placebo
Intervention type(s): Drug; Drug; Drug; Device; Device; Other; Drug
Intervention description(s): 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.; 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. 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.; Saline will be infused at the same rate and quantity as the corresponding drug visit.; 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).; A 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.; The subject will drink distilled deionized water (300ml) containing 75 grams glucose within 5 minutes.; Placebo pill similar in size and shape to Ambrisentan will be used as placebo control.
Other intervention name(s): L-NMMA; Ambrisentan; Saline; MRI; Cath; OGTT; Lactose Capsules
Intervention MeSH terms: Ambrisentan
Allocation: Randomized
Intervention model: Parallel Assignment
Masking: None (Open Label)
Primary purpose: Basic Science
Keywords: blood flow; magnetic resonance imaging; insulin; vasodilation