Overweight and obesity are major problems and their complications such as cardiovascular disease and type 2 diabetes mellitus pose great burdens on healthcare systems. There is accumulating evidence to support obesity being a chronic inflammatory disorder mediated in part by the expansion of adipose (fat) tissue.

In addition to adipocytes (fat cells), adipose tissue contains a whole range of other cell types including some immune (white blood) cells. The relative proportions of immune cell subpopulations and interactions between the different cell types within adipose tissue may be important in the development of type 2 diabetes.

Previous research by the investigators has suggested that there may be important differences in the activation status of certain immune cells located in adipose tissue with increased overweight and the production of leptin - an appetite regulating hormone. Properties of adipose tissue and inflammatory cells located within it may therefore be sensitive to changes in our nutritional status.

The purpose of the present study is to investigate changes in T cell activation in adipose tissue alongside other immunometabolic properties before and after a short period of dieting compared to changes in blood.

Participants will include males aged between 35-55 years old who meet the criteria for inclusion. After taking some preliminary measurements and monitoring of normal daily activities, participants will reduce their calorie intake to 50 % of their normal intake/requirements for 3 consecutive days and will attend 1 session of Laboratory testing before and 1 after this period which will take place in the Physiology Laboratories at the University of Bath.

It is hoped that investigating immunometabolic responses within adipose tissue following an acute nutritional challenge will help lead to a better understanding of the development of diseases associated with being overweight and ultimately help in the development of more effective methods for prevention and treatment.

The aim of this research is to investigate whether the immunometabolic properties of adipose tissue and the inflammatory cells located within it can be modified by short term changes in nutritional status.

Following advertisement of the study, interested potential participants will be asked to contact the Chief Investigator for further information via email/telephone correspondence. There will be an initial assessment of eligibility based on inclusion/exclusion criteria and, if these requirements are met, the potential participant will be invited to a meeting to further discuss the trial. After they have read the participant information sheet and seen the flowchart outlining the timeline for the study, if they would like to take part, they will be asked to sign a consent form. Dates will then be scheduled for some preliminary anthropometric measurements, 1 week of diet and physical activity monitoring and 3 days of calorie restriction with trial dates either side.

Preliminary measurements:

Preliminary testing will include anthropometric measurements of height, weight, waist and hip circumferences, sagittal waist height and blood pressure. Participants will also have their body composition precisely assessed using a dual energy X-ray absorptiometer (DEXA). These measurements will take place at the University of Bath Physiology Laboratories.

Monitoring of energy balance:

For 1 week, participants will be fitted with a physical activity monitor (Actiheart™) and asked to record a corresponding diary of their physical activity during this period to aid its interpretation and allow a more accurate calculation of average daily activity energy expenditure. Participants will also be asked to record their food and drink intake during this period so their corresponding energy intake during this period can be analysed and thus their 'energy balance' can be estimated. Participants should not make any conscious changes to their normal lifestyle habits/routines during this period. This analysis will then be used to write a 'diet prescription' for the 3 days to ensure the participant will receive 50 % of their 'normal' calorie intake using foods they would normally consume.

Main trial day 1:

In the 2 days prior to the first main trial day, participants should refrain from performing any strenuous physical activity. In the 24 hours prior, participants should also refrain from consuming any alcohol or caffeine. Participants should arrive at the Physiology Resting Laboratory in the morning following a 10 hour fast (approximately 8 am) where blood pressure will be measured and a saliva sample collected. A cannula will be inserted into a forearm vein and baseline blood sample(s) taken for analysis of metabolic/inflammatory markers and isolation of T cells. A fat sample will also be obtained using a needle aspiration technique. The participant will then be asked to consume a glucose drink consisting of 75 g anhydrous glucose (113 mL Polycal and 87 mL of water) and a cannula blood sample taken every 15 minutes for the following 2 hours.

Intervention - 3 day calorie restriction:

For the following 3 days, participants will be instructed to consume a diet calculated to be 50% of their 'normal' energy intake by reducing the proportions of the normal diet consumed. No strenuous physical activity should be performed by the participant for the last 2 days of this 3 day period and on the final day of this intervention, no caffeine or alcohol should be consumed.

Main trial day 2:

The following day after the 3 days of calorie restriction, participants will arrive at the laboratory at the same time as on main trial day 1 (to minimise variation in timing of sample collection) and will undergo exactly the same procedures.

Analysis:

On the day of the trial whole blood will be analysed for white blood cell count. Plasma and serum samples will be extracted from the whole blood via centrifugation and stored at -80 degrees C. Monocytes and T lymphocytes (populations of white blood cells) will also be isolated from whole blood and stored to later assess changes in their proportions and activation following the calorie restriction period. In each fat sample, separate portions of either adipose tissue or adipocytes (isolated from the other cells within the adipose tissue using a digestion method) will be cultured and media collected for later investigations. The remaining cells from the adipose tissue (SVF) will also be stored for later analysis of cell populations and their activation using flow cytometry, and expression/secretion analysis. mRNA expression will also be examined in adipose tissue.

Inclusion Criteria:

• Waist circumference > 94 cm
• Weight stable for more than 3 months (no change in weight +/- 3 %)
• Non-smoker

Exclusion Criteria:

• Personal history of/existing cardiovascular disease, metabolic disease or dyslipidaemia
• Taking medications that may influence lipid or carbohydrate metabolism or immune system function