Inorganic nitrate, nitric oxide and energy metabolism in humans

Lead Research Organisation: Newcastle University
Department Name: Institute of Cellular Medicine

Abstract

This research will conducted by a team of scientists with expertise in the conduction of detailed clinical studies and measurement of small molecules in circulating blood or bodily tissues. The overall aim of this programme of research is to understand how nutritional factors and the ageing process influence functions involved in the regulation of body weight and vascular function.
The research will include two independent projects which will have in common the study of a small molecule called Nitric Oxide (NO). This molecule is continuously produced by the body in small amounts and it has several and important effects including regulation of blood pressure, inflammation, exercise performance and memory. Since its official discovery in 1987, research in this area has made important advancements but, for example, we are still far from a full understanding of the nutritional and lifestyle factors that can affect the concentrations of NO in the body.
Our research will try to advance this knowledge by studying how the ageing process and nutritional, dietary and lifestyle factors can influence NO activity. This research will use one of the best methods currently available to measure NO concentrations and some of these methods have been developed by Dr Siervo.
The first project will essentially recruit 72 subjects with an age range between 18 and 75 years and they will be asked to attend the research unit for one visit. During the visit we will perform a series of detailed measurements and collect blood, urine and saliva samples which we will utilise to measure a series of molecules involved in the regulation of blood pressure, blood glucose and inflammation. We will also try to understand how certain genes influence NO concentrations.
The second project will involve the recruitment of 16 healthy older subjects. They will be asked to take a dose every day of a small molecule, called inorganic nitrate, for 14 days. Inorganic nitrate seems to have beneficial effects on the concentrations of NO in the body. Inorganic nitrate is commonly present in difference concentrations in water and food; example of foods with high nitrate content are beetroot, spinach, lettuce. During the study participants will return to the research centre in Newcastle on a few occasions. During these visits we will perform detailed measurements of vascular and metabolic health and we will collect biological (blood, urine, saliva) and tissue samples (muscle, adipose tissue) to understand the precise mechanisms that may explain the effects of inorganic nitrate on bodily functions.
Ultimately, these studies will provide novel and important information on the role of NO in the regulation of vascular and metabolic functions. We will understand better how the ageing process determine changes in physiological function and we will test whether inorganic nitrate could be a potential nutritional intervention that may protect the arteries and increase the efficiency of energy metabolism in older people. This will not only have implications for the research and advance knowledge but it can open potentially a new window for nutritional interventions that may be effective in reducing the risk of metabolic and vascular diseases such as type 2 diabetes, angina or stroke.

Technical Summary

The concepts of metabolic and vascular ageing as opposed to chronological ageing form a novel research paradigm useful to ascertain the observed between-subject variability in the association between ageing and cardiovascular and metabolic health outcomes. Older subjects show a greater impairment of endothelial function and control of vascular tone, which are both closely linked to the bio-availability and activity of nitric oxide (NO). The study of genetic, bio-molecular and physiological mechanisms linking ageing and NO metabolism as well as the identification of novel nutritional and lifestyle factors influencing NO synthesis is topical to maintain or restore a healthy ageing phenotype.
The overall aims of the proposed research programme will determine:
a. The association of ageing with NO synthesis and whether it is modified by eNOS polymorphisms
b. The effects of inorganic nitrate supplementation on resting energy expenditure, dietary-induced thermogenesis and absorptive fuel oxidation in the elderly
c. The effects of inorganic nitrate supplementation on mitochondrial respiration in muscular and adipose tissue and changes in core pathways involved in glucose and fat oxidation.

The projects will use advanced, state of the arts methods for the measurement of endothelial and metabolic functions. These include a non invasive measurement of NO production using stable isotopic tracers, and, for the first time, a measurement of the effects of inorganic nitrate on dietary induced thermogenesis and substrate oxidation using indirect calorimetry. These physiological measurements will be coupled to detailed biomolecular investigations of mitochondrial efficiency in muscular and adipose tissue.

Planned Impact

This research project has been primarily designed to advance scientific knowledge in order to shed some light on the biological mechanisms that control age-related decline in NO production cardio-metabolic functions and test whether dietary nitrate supplementation could be considered as a potential nutrient with a significant impact on metabolic function and beyond the established effects on vascular health. The observation of beneficial effects of nitrate on energy metabolism could open a new therapeutic area of research for chronic metabolic disorders such as type 2 diabetes and dyslipidaemia. We also believe that the results will have an impact beyond its immediate academic environment (i.e., nutrition and dietetic research), have an influence on metabolic and pharmacology research as well as may influence decision-making of funding and advisory clinical bodies. Its influence, while intended to be significant within academia and clinical practice, may also extend to food industry.
This is an active area of research and research groups in the UK (Exeter, Queen Mary, Southampton, Reading) and internationally (Stockholm, Dussedlorf) are investigating the effects of dietary nitrate on health outcomes. However, our group in Newcastle has developed a unique niche in studying the modulating role of ageing on the effects of nitrate administration; in addition, there is currently a small number of groups with a research interest in the effects of nitrate on energy metabolism in humans (Exeter, Stockholm).
We will make sure that the research will be effectively disseminated through scientific channels and public engagements activities. The details of these activities have already been discussed in our dissemination plans. However, we would like to emphasize that the evidence in this exciting and promising area of research is still limited to support a consensus for nutritional recommendations; therefore, our engagement activities will primarily focused on scientific dissemination, through publications and conferences, and public engagement events within the university and Newcastle local area to promote nutrition research for the promotion of healthy ageing. We also envisage that this three-year research programme will contribute to the nutritional taught programmes at Newcastle University and offer to undergraduate and post-graduate students projects embedded within the various clinical and analytical activities involved in the projects.
Ultimately, the scientists involved in the study will benefit from the multi-disciplinary approach and opportunity to learn and develop new skills in the application of stable isotopic models and bio-molecular analyses.

Publications

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Babateen AM (2018) Assessment of dietary nitrate intake in humans: a systematic review. in The American journal of clinical nutrition

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Houghton D (2016) The effect of age on the relationship between cardiac and vascular function. in Mechanisms of ageing and development

 
Description Alzheimer's Research UK Prevention and Risk Reduction Fund
Amount £795,000 (GBP)
Organisation Alzheimer's Research UK 
Department Alzheimers Research UK, Cambridge
Sector Charity/Non Profit
Country United Kingdom
Start 01/2018 
End 06/2020
 
Description CIMA Small Equipment Grant
Amount £8,000 (GBP)
Organisation Centre for Integrated research into Musculoskeletal Ageing 
Sector Charity/Non Profit
Country United Kingdom
Start 05/2016 
End 12/2016
 
Description MRC Confidence in Concept
Amount £49,500 (GBP)
Organisation Medical Research Council (MRC) 
Sector Academic/University
Country United Kingdom
Start 09/2017 
End 10/2018
 
Description NIHR Global Challenges
Amount £1,900,000 (GBP)
Organisation National Institute for Health Research 
Sector Public
Country United Kingdom
Start 08/2017 
End 12/2020
 
Title Assessment of Whole-Body NO production using stable isotopes 
Description I developed initially at MRC Human Nutrition Research in Cambridge and we have now implemented the full protocol including analytical capacity in our lab. This is a non-invasive method using a single dose of oral labelled nitrate and measurement of isotopic enrichments in saliva samples. GCMS is used to assess isotopic enrichments 
Type Of Material Physiological assessment or outcome measure 
Year Produced 2015 
Provided To Others? Yes  
Impact Completed study in Tanzania in children with Sickle Cell Disease Completed study in high altitude during nitrate supplementation 
 
Title Automated toolkit for assessment of body composition phenotypes 
Description Novel method to define nutritional status across the life course using DXA data. Developed automated toolkit in collaboration with MRC Human Nutrition Research Centre which is covered by MRC copyright 
Type Of Material Physiological assessment or outcome measure 
Year Produced 2015 
Provided To Others? Yes  
Impact Improvement of risk prediction from simple assessment of body composition using DXA. 
 
Description Cambridge - Newcastle Collaboration 
Organisation Medical Research Council (MRC)
Department MRC Human Nutrition Research Group
Country United Kingdom 
Sector Public 
PI Contribution I collaborate with Dr Jules Griffin and his group to investigate the role of inorganic nitrate on mitochondrial function. Specifically, our group will run the studies and the Griffin's group will perform some of the biomolecular analyses on adipose and muscle tissues.
Collaborator Contribution See above
Impact One paper under review in PloS BIOLOGY. collaboration is multi-disciplinary and involves disciplines such as nutrition, physiology, biochemistry and metabolomics
Start Year 2015
 
Description Diet, physical activity and risk of dementia in UK adults: Epidemiological analyses and the MedEx feasibility study 
Organisation University of East Anglia
Country United Kingdom 
Sector Academic/University 
PI Contribution Multi-centre study funded by Alzheimer's Research UK and led by University of East Anglia (PI: Prof Minihane). I am the lead for the Newcastle Site and coordinate the funding, staff and logistics for the study.
Collaborator Contribution Large study with several collaborators involved across the UK with expertise in nutrition, cognition, vascular health, geriatrics and epidemiology
Impact None yet
Start Year 2018
 
Description Dietary nitrate and sarcopenia in older age 
Organisation University of Liverpool
Department School of Psychological Sciences
Country United Kingdom 
Sector Academic/University 
PI Contribution Developed grant proposal submitted to MRC CIMA studentship call. responsible for dietary advice on grant and laboratory analysis
Collaborator Contribution leading the grant and responsible for conduction of studies
Impact None yet
Start Year 2017
 
Description Dietary nitrate availability and cognition in older age 
Organisation G's Fresh Beetroot
Country United Kingdom 
Sector Private 
PI Contribution Co-supervision of PhD student funded by G's Fresh company. Advisory role on study design and conduction of laboratory experiments for analysis of nitric oxide biomarkers
Collaborator Contribution Provision of funding (G's Fresh) and conduction and coordination of studies
Impact None yet
Start Year 2016
 
Description Dietary nitrate availability and cognition in older age 
Organisation Newcastle University
Country United Kingdom 
Sector Academic/University 
PI Contribution Co-supervision of PhD student funded by G's Fresh company. Advisory role on study design and conduction of laboratory experiments for analysis of nitric oxide biomarkers
Collaborator Contribution Provision of funding (G's Fresh) and conduction and coordination of studies
Impact None yet
Start Year 2016
 
Description Effects of dietary nitrate and folate supplementation on blood pressure in hypertensive Tanzanians: a feasibility trial 
Organisation Kilimanjaro Clinical Research Institute
Country Tanzania, United Republic of 
Sector Academic/University 
PI Contribution Developed the study, applied for funding and coordinating trial
Collaborator Contribution PI is Prof Richard Walker from NHS Northumbria Trust and Prof Blandina Ngoma in Tanzania. We designed study together and they are co-coordinating staff and logistics of the trial.
Impact None yet. But three students have been employed to work on the study for 6 months
Start Year 2017
 
Description Exercise, muscular nitrate and metabolic health 
Organisation National Institutes of Health (NIH)
Country United States 
Sector Public 
PI Contribution My group and collaborators at Plymouth University have conceived the project and establish the link between the university of Victoria in Australia and NIH. Project is looking at the effects of dietary nitrate on muscular nitrate depot and modulation of exercise performance in humans. My group has covered the costs for the shipping of samples from Australia to the NIH where analyses will be performed. My group will lead the analyses and writing of publications.
Collaborator Contribution Prof McDonnell from University of Victoria in Melbourne Australia has conducted the study and provided the samples for the analyses. dr Piknova at the NIH will conduct the measurements of nitrate and nitrite in muscle and plasma. Dr Raul Bescos from Plymouth University has contributed to design the study and will contribute to analyses and drafting of manuscript.
Impact None yet
Start Year 2019
 
Description Exercise, muscular nitrate and metabolic health 
Organisation University of Plymouth
Country United Kingdom 
Sector Academic/University 
PI Contribution My group and collaborators at Plymouth University have conceived the project and establish the link between the university of Victoria in Australia and NIH. Project is looking at the effects of dietary nitrate on muscular nitrate depot and modulation of exercise performance in humans. My group has covered the costs for the shipping of samples from Australia to the NIH where analyses will be performed. My group will lead the analyses and writing of publications.
Collaborator Contribution Prof McDonnell from University of Victoria in Melbourne Australia has conducted the study and provided the samples for the analyses. dr Piknova at the NIH will conduct the measurements of nitrate and nitrite in muscle and plasma. Dr Raul Bescos from Plymouth University has contributed to design the study and will contribute to analyses and drafting of manuscript.
Impact None yet
Start Year 2019
 
Description Exercise, muscular nitrate and metabolic health 
Organisation Victoria University
PI Contribution My group and collaborators at Plymouth University have conceived the project and establish the link between the university of Victoria in Australia and NIH. Project is looking at the effects of dietary nitrate on muscular nitrate depot and modulation of exercise performance in humans. My group has covered the costs for the shipping of samples from Australia to the NIH where analyses will be performed. My group will lead the analyses and writing of publications.
Collaborator Contribution Prof McDonnell from University of Victoria in Melbourne Australia has conducted the study and provided the samples for the analyses. dr Piknova at the NIH will conduct the measurements of nitrate and nitrite in muscle and plasma. Dr Raul Bescos from Plymouth University has contributed to design the study and will contribute to analyses and drafting of manuscript.
Impact None yet
Start Year 2019
 
Description Leeds Beckett - Newcastle University Nitrate-Hypoxia Studies 
Organisation Leeds Beckett University
Country United Kingdom 
Sector Academic/University 
PI Contribution Collaboration on two projects investigating effects of dietary nitrate on muscular performance in hypoxic conditions. Our team advised on study design and performed analyses of nitric oxide biomarkers
Collaborator Contribution The studies were conducted at Leeds Beckett university in their hypoxia chambers. These studies were part of a PhD programme of research which has now been successfully submitted
Impact 1: Shannon OM, Duckworth L, Barlow MJ, Woods D, Lara J, Siervo M, O'Hara JP. Dietary nitrate supplementation enhances high-intensity running performance in moderate normobaric hypoxia, independent of aerobic fitness. Nitric Oxide. 2016 Sep 30;59:63-70. doi: 10.1016/j.niox.2016.08.001. PubMed PMID: 27553127. 2: Shannon OM, Barlow MJ, Duckworth L, Williams E, Wort G, Woods D, Siervo M, O'Hara JP. Dietary nitrate supplementation enhances short but not longer duration running time-trial performance. Eur J Appl Physiol. 2017 Mar 1. doi: 10.1007/s00421-017-3580-6. [Epub ahead of print] PubMed PMID: 28251402.
Start Year 2016
 
Description Metabolic mechanisms of dietary nitrate on insulin sensitivity and adipose tissue biology 
Organisation University of Leeds
Department School of Food Science and Nutrition Leeds
Country United Kingdom 
Sector Academic/University 
PI Contribution We developed a PhD grant proposal to MRC DTP Discovery Medicine North. We advised on study design and we are responsible for analysis of nitrate/nitrite in adipose and muscle tissues
Collaborator Contribution Leading the project in animals and conducting detailed molecular analyses
Impact None yet
Start Year 2016
 
Description NIH - Newcastle Partnership 
Organisation National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Country United States 
Sector Public 
PI Contribution We have initiated a strong link with this group to explore the mechanistic basis of the conversion of nitrate into nitrite and nitric oxide. Our group will perform the human experiments and collect the biological samples (plasma and tissue). There will be a possibility for members of our research team to have short internships in the US to acquire specific biomolecular skills
Collaborator Contribution The group will be involved in the measurement of nitrate and nitrite content in muscle and adipose tissues by using chemiluminescnece techniques
Impact None yet
Start Year 2015
 
Description NIHR Global Health Research Group on Dementia Prevention and Enhanced Care (DePEC) 
Organisation Newcastle University
Department Institute of Health and Society
Country United Kingdom 
Sector Academic/University 
PI Contribution I am leading a work package on the delivery of nutritional intervention in Malaysia for the prevention of cognitive decline. Lead for the design of the study and management of staff and resources for the trial. Management of two post-docs and one post-graduate student
Collaborator Contribution Several people involved in Newcastle and Malaysia who are contributing to the design and development of the trial
Impact Two publications currently under review
Start Year 2017
 
Title Automated Toolkit for the assessment of body composition phenotypes 
Description Body composition (BC) is fundamental to assess nutritional status and evaluate disease risk across the life course. While the research and clinical principles and applications of BC are clear, the assessment is complicated by utilization of diagnostic models and techniques with different levels of complexity and accuracy. A great advancement in BC was achieved with the introduction of Dual X Ray Absorptiometry (DXA) and multi-compartment models. Over the years, DXA has become increasingly available, more rapid and safer and it is now considered as one of the most accurate methods for BC assessment. DXA utilize a 3-compartment model and can provide information on whole-body and segmental fat mass (FM), lean body mass (LBM) and bone mineral mass (BMM). DXA has been extensively used to evaluate the association with disease risk and the efficacy of nutritional interventions. Ageing is an important factor associated with BC changes; currently there is a need for novel diagnostic models that could capture the increase in disease risk associated with age-related changes in BC. Two innovative models were proposed by Baumgartner (BC Phenotypes, BCP) and Wells (Load-Capacity, LC). However, these models have not been consistently applied, which has resulted in the proposition of modified versions of the models and consequent observation of mixed results. An important limitation of existing models is the lack of adjustment for the effects of age, body size and gender on BC. Therefore, we derived age, gender and BMI-specific reference curves for the adult population (18-85y) using the 1999-2004 NANHES DXA dataset. The complexity of the models to derive the BC phenotypes may however limit their application. Therefore, we have developed a simple and user-friendly automated toolkit that can be used to calculate BC phenotypes for both models. The toolkit allows a rapid analysis of individual data and the possibility to add repeated measurements of BC. Specific import and export functions can be used to analyze large epidemiological DXA datasets. The toolkit includes summary graphs of the individual data (Scatter Plots) and descriptive statistics (count and prevalence, Bar Charts) for each BC model. The toolkit is freely available for download and its use is protected by MRC copyright. 
IP Reference  
Protection Copyrighted (e.g. software)
Year Protection Granted 2016
Licensed Yes
Impact None yet as the toolkit is still being tested and minor technical issues are being resolved. we are expending to go live and be freely available at the end of 2017.