Influence of High-fat Overfeeding on Circulating Hepatokine Concentrations: a Randomised Crossover Study
Phase: NA
Conditions: Insulin Resistance, Type2 Diabetes Mellitus, Non-Alcoholic Fatty Liver Disease, Obesity
Sponsor: Loughborough University

 
 
School of Sport, Exercise and Health Sciences 
 
 
 
Influence of High-fat Overfeeding on Circulating 
Hepatokine Concentrations: A Randomised 
Crossover Study 
 
 
 
20th November 2017 
 
 
 
 

OVEREAT Study Protocol 
 
Objective(s) 
The objectives of the present study are as follows: 
 
 To examine the effects of high-fat overfeeding across a 7-day period, on circulating 
concentrations of the hepatokines LECT2, FGF21 and Fetuin-A. 
 
 To examine the effects of high-fat overfeeding across a 7-day period, on subjective 
appetite, food preference and circulating concentrations of the appetite regulatory 
hormones Ghrelin and PYY. 
 
 To observe whether changes in these variables occur alongside impairments in 
insulin sensitivity and glucose tolerance following seven days of high fat overfeeding.  
 
Design 
The present study is a randomised, controlled, crossover design in which 12 recreationally 
active, healthy males will complete two week-long dietary conditions in a randomised order. 
The two dietary conditions consist of a high-fat condition and a control condition. In the 
high-fat condition, participants will consume a diet comprising of 65% of total energy as fat 
and a total kilocalorie content equal to 150% of their estimated daily requirement. In the 

control condition, participants’ will consume their habitual diet as confirmed by a three-day 
food diary both before and during the control condition. The two diets will last seven days 
each with a three-week washout period separating the two conditions. An oral glucose 
tolerance test will be performed before and after the two diets to assess glycaemic control 
and hepatokine and appetite hormone responses will be measured across the seven days. 
 
Methods 
Protocol 
The participants will firstly undergo a prescreening visit to the laboratory in which written 
informed consent will be obtained. Further to this, participants will complete a health 
screen questionnaire, an international physical activity questionnaire and a food preference 
questionnaire in order to ensure compliance with the eligibility criteria and that the foods 
provided during the trial will be palatable. Anthropometrical measures will be then be taken 
including height, weight, waist circumference and body fat percentage (measured via 
bioelectrical impedance analysis). A finger prick blood sample will also be taken and fasting 
blood glucose concentrations will be analysed using a Cardiochek device in order to confirm 
normal fasting blood glucose levels as stated in the eligibility criteria. Prior to completing the 
prescreening session, participants will be set up with hip-based physical activity monitor 
(Actigraph) and a thigh-based sedentary behaviour monitor (ActivPAL) and instructed to 
wear the devices for seven days. Participants will then be provided with an activity diary to 
record periods where the devices are removed as well as a 3-day food diary (including two 

week days and one weekend day) to complete. This will allow for a baseline assessment of 
the participants’ habitual eating and activity habits.  
After the completion of the 7-day physical activity and sedentary behaviour monitoring, 
participants will be randomised to either the high-fat condition or the control condition. 
Participants will attend the laboratory at 8:00 am for a pre-diet assessment following a 10-
hour overnight fast and having refrained from caffeine, exercise and alcohol in the prior 24 
hours. Upon arrival, repeated written, informed consent will then be obtained and body 
weight, body fat percentage and blood pressure will then be measured. Following this, 
participants will be fitted with a mouth piece and their resting metabolic rate and fat 
oxidation will be measured via indirect calorimetry using Douglas bags for expired gas 
collection. Subjective ratings of appetite will then be measured using visual analogue scales 
and food preference will be assessed using computer-based software (Leeds Food 
Preference Questionnaire). A cannula will then be inserted into an antecubital vein for blood 
sampling. A baseline blood sample (0 h) will be collected, followed by the commencement 
of the oral glucose tolerance test. During this, participants will consume a solution 
containing 75 g of glucose dissolved in 300 ml of water and further blood samples will be 
taken at 0.5, 1, 1.5 and 2 h. The cannula will then be removed and the dietary condition will 
commence upon leaving the laboratory. During the 7-day dietary period, participants will 
undergo further monitoring of physical activity and sedentary behaviour, while during the 
control condition; participants will also complete a 3-day food diary in order to confirm 
maintenance of their habitual diet.  
Both one day and three days after commencing the diets, participants will return to the 
laboratory at 8:00am following a 10-hour overnight fast and having refrained from caffeine, 

exercise and alcohol. Further repeat, written informed consent will be and measurements of 
body weight, blood pressure, subjective appetite and food preference will be obtained. A 
venous blood sample will then be collected via venepuncture to allow for the assessment of 
the time-course of changes in fasting circulating hepatokine and appetite hormone 
concentrations.  
Upon completion of the 7-day dietary condition, participants will attend the lab the 
following morning at 8:00am following a 10-hour overnight fast and having refrained from 
caffeine, exercise and alcohol. Participants will undergo a post-diet assessment using the 
same procedures as the pre-diet assessment. A washout period of three weeks will then 
separate the two dietary conditions. 
 
Diet 
During the high-fat dietary condition, all foods will be provided to the participants and will 
contain an energy content of 50% extra kilocalories above the participants’ daily energy 
requirement and a dietary fat composition of 65%. The participants’ daily energy 
requirement will be estimated using the Mifflin equations and subsequently multiplied by a 
physical activity correction factor of 1.7 and a thermogenic effect of feeding correction 
factor of 1.1. The foods provided will be primarily high in saturated fats and participants will 
be told to consume all foods provided within the study and no other calorie-containing 
foods or drink. In the event of any leftover food, participants will be told to bring in the food 
for weighing and subsequent subtraction from the calculated intake. Furthermore, 
participants will be told to fry foods where applicable and to minimise fat wastage during 

the cooking process. During the control condition, participants will be told to consume their 
habitual diet, which will be confirmed using the 3-day food diaries. In addition to this, 
participants will be told on both diets to maintain their normal activity habits which will be 
confirmed by the physical activity and sedentary behaviour monitoring.  
 
Blood Sampling 
Blood samples will be collected into pre-chilled 2.7 mL EDTA, 4.9 mL EDTA, 9 mL EDTA, 2.6 
mL Heparin and 2.7 mL Fluoride monovettes. All monovettes will be centrifuged at 3500 
rpm for 10 min at 4˚C and the resultant plasma supernatant will be aliquoted into 2 mL 
micro tubes. The 2.7 mL EDTA monovettes will be pre-treated with PHMB, PBS and NaOH 
and after centrifugation, HCl will be added to prevent the degradation of acylated Ghrelin 
and the sample will be spun for a further 5 minutes. All samples will then be stored at -80˚C 
until analysis.