Effects of Nitrate on Liver Perfusion and Sugar Control
Condition: Type 2 Diabetes; Ageing
Study type: Interventional

Summary: 
      Rationale:

      Mediterranean style diets and diets rich in green leafy vegetables protect against the risk
      of developing type 2 diabetes and a wide range of cardiovascular disease. These diets are
      rich in nitrate. Numerous studies have shown that nitrate from the diet can have a wide range
      of beneficial effects. These include relaxing blood vessels and improving their function. It
      has been shown that following a meal with added nitrate, blood flow to the stomach increases
      more than would be expected if the same meal is given without nitrate. This is because when
      we eat nitrate the body concentrates it and recycles it through the digestive system. As it
      cycles through it is converted into nitrite and nitric oxide which cause blood vessels to
      relax. The nitrite and nitric oxide also seem to protect against infection from food sources
      such as E.coli.

      What we do not know is whether this nitrite and nitric oxide has any effect on the small
      intestine and the liver. Some nitrite reaches the small intestine and may have the same
      effect on blood flow there as it does in the stomach. This could be very important because
      the small intestine releases hormones called incretins which we now know play a very
      important role in controlling blood sugar every time we eat. These incretin hormones regulate
      insulin release and the body's sensitivity to insulin. When we eat blood containing the
      substances we have absorbed from the gut, such as sugars and fats, goes to the liver for
      processing. The blood then leaves the liver and enters the circulation. This means the blood
      supply to the liver will have much higher concentrations of nitrite than the blood
      circulating in the rest of the body. High concentrations of nitrite appear to cause blood
      vessels to open up. This means more blood vessels in the liver should be opened after a
      nitrate rich meal. It seems likely that this will help the liver to control blood sugar more
      effectively.

      Purpose To find out if supplementation by inorganic nitrate as found in beetroot or green
      leafy vegetables increases liver (hepatic) microvascular perfusion and increases incretin
      secretion.

      Plan of investigations:

      We will recruit 16 individuals for each of the three groups (Young adults, older adults and
      individuals with type 2 diabetes). Participants will be recruited from a database of
      volunteers who have consented to being contacted for research studies which are held by the
      NIHR Exeter Clinical Research Facility. This is a double blind, placebo controlled crossover
      design study (nitrate rich beetroot juice vs a placebo, nitrate depleted beetroot juice).
      Three visits will be required for participants to complete this study.

      Visit 1. Screening and consent. The experimenter will explain to the participant what the
      study is designed to test. If the participant is completely clear on the study and understand
      what they are agreeing to, they will sign a consent form. In addition a standard medical
      history and clinical examination will be undertaken by a research nurse and or Anthony
      Shepherd. A venous blood sample will be taken using standard aseptic procedures. Following
      consent participants will be assigned a study number. Study numbers will be previously
      assigned (by a research statistician) to a randomisation order to begin either the beetroot
      juice or placebo arm of the study first.

      Visit 2. Visit 2 will require the participant to fast over night from 10pm. Only water will
      be admissible from this time. The following morning participants will arrive at the
      laboratory in a fully hydrated and rested state at ~ 7.30am. This visit will take ~ 5 hours
      and will require 4 MRI scans. Participants will have the first MRI scan after a short
      acclimatisation period. Participants will then be provided with a concentrated 140 mL nitrate
      drink or placebo with a standardised breakfast (2 slices of toast with butter). Three
      subsequent MRI scans will be required (one per hour for three hours). Venous blood samples
      taken from cannulas will be sampled, in order to assess glucose, insulin, incretins and
      nitrate/nitrite prior to each scan. Visit 3. Visit 3 will take place after a minimum washout
      period of 7 days from Visit 2. Visit 3 will be identical in nature to visit 2; however, it
      will be with the opposite supplement (either nitrate rich or placebo beetroot juice).

      Impact:

      Dietary nitrate appears to offer a simple, low cost means of modifying cardiovascular risk.
      This study will deepen our understanding of the role of the nitrate/nitrite/nitric oxide
      pathway in normal physiology. By understanding what effect inorganic nitrate from the diet
      has on hepatic perfusion and other pathways involved in glucose homeostasis this may lead to
      a range of simple, low cost therapeutic strategies to prevent and treat type 2 diabetes.
    

Detailed description: 
      Hypothesis

      Inorganic nitrate from the diet, following conversion to nitrite and nitric oxide via the
      pathways described above will:

        1. Increase hepatic microvascular perfusion

        2. Increase GLP-1 secretion

        3. Increase pancreatic insulin secretion

        4. Reduce the area under the curve for glucose post meal

      Purpose To find out if inorganic nitrate found in green leafy vegetables protect against the
      risk of developing type 2 diabetes and cardiovascular disease by increasing hepatic
      microvascular perfusion and increasing incretin secretion.

      Aim The purpose of this study is to see if inorganic nitrate from the diet modulates hepatic
      perfusion and other pathways involved in post-prandial glucose homeostasis.

      MRI Hepatic micro vessel perfusion of the liver and portal vein diameter will be measured by
      Dr. Jon Fulford at the Peninsula Magnetic Resonance Research Centre, St Luke's Campus.
      Following initial survey images to obtain anatomical information apparent diffusion
      coefficients (ADC) are calculated in the posterior right lobe of the liver. Diffusion images
      are acquired with a single shot echo-planar imaging (EPI) sequence with 15 directions and b
      values of 200 and 800 s mm-2. Images are acquired during a single breathe-hold at an
      axial-oblique orientation with a repetition time (TR) of 1500 ms an echo time of 57 ms, an
      in-plane resolution of 4 x 4 mm and a slice thickness of 10 mm with fat suppression and 3
      signal averages.

      ADC is given by:

      ADC = 1/(b200-b800) Ln (S800/S200) where S800 is the signal with a diffusion b-value of 800 s
      mm-2 (b800) and S200 is the signal with a diffusion b-value of 200 s mm-2 (b200).

      Statistical analysis Statistical analysis will be performed by an experienced and trained
      researcher and statistical advice will be available from Prof. Angela Shore from within the
      team and Beverly Shields from the NIHR Exeter Clinical Research Facility. Data will be
      assessed for normality. Depending on the normality of data the differences between the groups
      will be assessed by either unpaired t-test or Mann-Whitney test.

      Sample Size Sixteen participants would need to be recruited per group in order to detect a
      1SD difference between groups data with 80% power and an a of 0.05. Twenty participants would
      be recruited in order to account for drop outs.
    

Intervention name(s): Beetroot juice; Placebo
Intervention type(s): Dietary Supplement; Dietary Supplement
Intervention description(s): 140mL concentrated Beetroot juice or placebo juice
Other intervention name(s): James Whites Drinks Ltd
Allocation: Randomized
Intervention model: Crossover Assignment
Masking: Double (Participant, Investigator)
Primary purpose: Prevention
Keywords: Diabetes; Nitrate; Nitrite; Hepatic; MRI; Incretin; Insulin; Glucose