Effects of Nitrate on Liver Perfusion and Sugar Control

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.