The Impact of Acipimox and Salbutamol Supplementation on the Development of Insulin Resistance and Anabolic Resistance During Forearm Immobilization in Healthy, Young Volunteers
Phase: NA
Conditions: Healthy
Sponsor: University of Exeter

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The impact of Acipimox and Salbutamol supplementation on 
the development of insulin resistance and anabolic resistance 
during forearm immobilization in healthy, young volunteers 
 
 
Forearm immobilization, metabolic health, and muscle loss 
 
 
Version 3.4 
09-03-2021 
 
 
NCT03866512 
 
 
 
This protocol has regard for the HRA guidance and order of content 
 
 

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RESEARCH REFERENCE NUMBERS 
 
IRAS Number: 
250839 
 
Clinical trials.gov Number: 
 
NCT03866512 
SPONSORS Number: 
1718/16 
 
FUNDERS Number: 
209198/Z/17/Z 
 
 

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SIGNATURE PAGE 
The undersigned confirm that the following protocol has been agreed and accepted and that the Chief 
Investigator agrees to conduct the trial in compliance with the approved protocol and will adhere to 
the principles outlined in the Medicines for Human Use (Clinical Trials) Regulations 2004 (SI 2004/1031), 
amended regulations (SI 2006/1928) and any subsequent amendments of the clinical trial regulations, 
GCP guidelines, the Sponsor’s (and any other relevant) SOPs, and other regulatory requirements as 
amended. 
I agree to ensure that the confidential information contained in this document will not be used for any 
other purpose other than the evaluation or conduct of the clinical investigation without the prior 
written consent of the Sponsor 
I also confirm that I will make the findings of the trial publically available through publication or other 
dissemination tools without any unnecessary delay and that an honest accurate and transparent 
account of the trial will be given; and that any discrepancies and serious breaches of GCP from the trial 
as planned in this protocol will be explained. 
 
For and on behalf of the Trial Sponsor: 
Signature:  
 
 
Date: 09-03-2021 
Name (please print): Ms Pam Baxter 
 
 
 
Position: Senior Research Governance Officer 
University of Exeter 
 
 
 
 
Chief Investigator: 
Signature: 
 
 
Date: 09-03-2021 
Name: 
Dr Marlou Dirks 
 
 
 
 
 

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KEY TRIAL CONTACTS 
Chief Investigator 
Dr Marlou Dirks 
M.Dirks@exeter.ac.uk 
+44 (0) 1392 725496 
University of Exeter 
Richard's Building, Sport and Health Sciences 
St Luke's Campus, Heavitree Road, Exeter, EX1 2LU 
Sponsor 
University of Exeter 
Sponsor Representative 
Pam Baxter 
Senior Research Governance Officer 
P.R.Baxter2@exeter.ac.uk 
+44 (0) 1392 723588 
Research Ethics and Governance Office 
University of Exeter 
Lafrowda House, St Germans Road, Exeter, EX4 6TL  
Funder 
Wellcome Trust 
215 Euston Road, London, NW1 2BE 
 
Dr Rachael Fowler 
Grants Adviser 
Cellular, Developmental and Physiological Science 
R.Fowler@wellcome.ac.uk 
+44 (0) 207 611 8888 (8318) 
Key Protocol Contributors 
Dr Francis Stephens 
Associate Professor 
F.B.Stephens@exeter.ac.uk 
+44 (0) 1392 722157 
University of Exeter 
Richard's Building, Sport and Health Sciences 
St Luke's Campus, Heavitree Road, Exeter, EX1 2LU 
 
Dr Rob Andrews 
R.C.Andrews@exeter.ac.uk 
+44 (0) 1392 408327 
Associate Professor of Diabetes/Honorary Consultant in Diabetes 
University of Exeter Medical School 
RILD Level 3 
Barrack Road, Exeter, EX2 5DW 
Statistician 
Dr Brad Metcalf  
B.Metcalf@exeter.ac.uk  
+44 (0) 1392 724896 
University of Exeter 
Richard's Building, Sport and Health Sciences 
St Luke's Campus, Heavitree Road, Exeter, EX1 2LU 
 
 
 

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i. LIST of CONTENTS 
GENERAL INFORMATION 
Page No. 
TITLE PAGE 
 
1 
RESEARCH REFERENCE NUMBERS 
 
2 
SIGNATURE PAGE 
3 
KEY TRIAL CONTACTS 
 
4 
i. LIST of CONTENTS 
5 
ii. LIST OF ABBREVIATIONS 
7 
iii. TRIAL SUMMARY 
 
8 
iv. FUNDING 
 
10 
v. ROLE OF TRIAL SPONSOR AND FUNDER 
10 
vii. PROTOCOL CONTRIBUTERS 
10 
viii. KEYWORDS 
10 
ix. TRIAL FLOW CHART  
11 
SECTION  
1. BACKGROUND 
 
12 
2. RATIONALE  
13 
3. ASSESSMENT AND MANAGEMENT OF RISK 
14 
4. OBJECTIVES AND OUTCOME MEASURES/ENDPOINTS  
16 
   4.1 Primary objective 
 
   4.2 Secondary objectives 
 
   4.3 Primary outcome 
 
   4.4 Secondary outcomes 
 
   4.5 Table of endpoints/outcomes 
 
5. TRIAL SETTING, DESIGN, AND PROCEDURES 
17 
   5.1 Recruitment 
 
   5.2 Screening 
 
   5.3 Payment 
 
   5.4 Consent 
 
   5.5 Trial procedures 
 
   5.6 Randomisation 
 
   5.7 Blinding 
 
   5.8 Storage and analysis of clinical samples 
 
6. PARTICIPANT ELIGIBILITY CRITERIA 
21 
   6.1 Inclusion criteria 
 

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   6.2 Exclusion criteria 
 
7. TRIAL TREATMENTS 
22 
8. ADVERSE EVENT RECORDING AND REPORTING 
23 
   8.1 Definition 
 
   8.2 Recording and reporting of adverse events 
 
   8.3 Emergency unblinding 
 
9. STATISTICS AND DATA ANALYSIS 
25 
   9.1 Sample size calculation 
 
   9.2 Planned recruitment rate 
 
   9.3 Statistical analysis plan 
 
      9.3.1. Summary of baseline data and flow of participants 
 
      9.3.2. Primary outcome analysis 
 
      9.3.3. Secondary outcome analysis 
 
      9.3.4. Procedure(s) to account for missing or spurious data 
 
10. DATA MANAGEMENT  
27 
   10.1 Source data and documents 
 
   10.2 Data areas and data types 
 
   10.3 Standards and metadata 
 
   10.4 Methods for data sharing 
 
   10.5 Proprietary data 
 
11. ETHICAL AND REGULATORY CONSIDERATIONS 
29 
   11.1 Research Ethics Committee (REC) review and reports 
 
   11.2 Peer review 
 
   11.3 Indemnity 
 
12. DISSEMINATION POLICY 
30 
13. REFERENCES 
31 
 
 
 

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ii. LIST OF ABBREVIATIONS 
BCAA 
Branched-chain amino acid 
BCKDH 
Branched-chain alpha-keto acid dehydrogenase 
CI 
Chief Investigator 
CRF 
Case Report Form 
CSA 
Cross-sectional area 
CTIMP  
Clinical Trial of Investigational Medicinal Product 
 
GCP 
Good Clinical Practice 
GLP 
Good laboratory practice 
MHRA 
Medicines and Healthcare products Regulatory Agency 
PDC 
Pyruvate dehydrogenase complex 
PI 
Principal Investigator 
REC 
Research Ethics Committee 
SAE 
Serious Adverse Event 
SOP 
Standard Operating Procedure 
TMF 
Trial Master File 
 
 

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iii. TRIAL SUMMARY 
Trial Title 
The impact of Acipimox and Salbutamol supplementation on the 
development of insulin resistance and anabolic resistance during 
forearm immobilization in healthy, young volunteers 
Short title 
Forearm immobilization, metabolic health, and muscle loss 
Trial Design 
Randomized, double-blind, placebo-controlled trial with three 
parallel groups 
Study category 
Basic science study involving procedures with human participants 
Trial Participants 
Young healthy recreationally active males and females 
Planned Sample Size 
45 
Treatment duration 
2 days of forearm immobilization plus drug or placebo ingestion 
Planned Trial Period 
Minimally 12 days (screening, baseline metabolic test day, 
approximate 7 days waiting period, 2 days forearm immobilization), 
maximally 33 days (waiting period can be up to 4 weeks, depending 
on availability of the participant). 
Study sites 
Royal Devon & Exeter NHS Foundation Trust 
Clinical Research Facility (CRF) and Clinical Trials Pharmacy 
Activities: screening and consenting (CRF), forearm cast application 
and removal (CRF), metabolic testing (CRF), drug dispensing to study 
participants (Pharmacy) 
Nutritional Physiology research laboratories 
St Luke’s Campus, University of Exeter 
Activities: preparation of a standardised diet, plasma analyses. If 
required: screening and consenting, forearm cast application 
 
Objectives 
Outcome Measures 
Primary 
Determine the impact of reduced 
muscle lipid availability and 
increased glycolytic flux on forearm 
glucose uptake during short-term 
forearm immobilization in healthy 
volunteers 
Forearm glucose uptake 
Secondary 
 
Determine the impact of reduced 
muscle lipid availability and 
increased glycolytic flux on muscle 
protein synthesis, and forearm 
nutrient balance during short-term 
forearm immobilization in healthy 
volunteers 
Postabsorptive and 
postprandial muscle protein 
synthesis 
Forearm branched-chain 
amino acid and fatty acid 
balance 
Forearm volume 
Medication to be 
administered 
Administered during the 2-day forearm immobilization period: 
Acipimox OR 
Salbutamol OR 
Placebo 
Formulation, Dose, Route 
of Administration 
Acipimox, 250 mg, 4 times daily, oral administration 
Salbutamol, 4 mg, 4 times daily, oral administration 

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Placebo, no active ingredients, 4 times daily, oral administration 
CTIMP? (yes/no) 
No (confirmation from MHRA is included in this application) 
 
 
 

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iv. FUNDING AND SUPPORT IN KIND 
FUNDER(S) 
FINANCIAL AND NON FINANCIALSUPPORT GIVEN 
Wellcome Trust 
215 Euston Road 
London 
NW1 2BE 
 
Sir Henry Wellcome Postdoctoral Fellowship to 
Dr Marlou Dirks 
Sport and Health Sciences 
College of Life and Environmental Sciences  
University of Exeter 
St Luke's Campus 
Heavitree Road 
Exeter, EX1 2LU 
 
 
 
v. ROLE OF TRIAL SPONSOR AND FUNDER 
This study is funded by a 4-year Sir Henry Wellcome Postdoctoral Fellowship to Dr Marlou Dirks. Dr 
Marlou Dirks and Dr Francis Stephens were responsible for the study design. The University of Exeter 
acts as a sponsor for this study. Dr Marlou Dirks will be responsible for data collection and analysis, 
decision to publish, and preparation of manuscript(s). 
The UK Policy Framework for Health & Social Care and Medicines for Human Use (Clinical Trials) 
Regulations and Amendment Regulations 2006 require that all health-related research has a sponsor 
and sets out the responsibilities of the sponsor. The sponsor takes overall responsibility for the 
initiation, management and financing (or arranging the financing) of the research and must satisfy itself 
that appropriate arrangements are in place for management, monitoring and reporting of research 
activity. It is recognised that the sponsor can delegate specific responsibilities to any other individual 
or organisation that is willing and able to accept them. However, any delegation of responsibilities to 
another party should be formally agreed and documented by the sponsor. 
 
vii. Protocol contributors 
Dr Marlou Dirks – protocol development and scientific critique 
Dr Francis Stephens – protocol development and scientific critique 
Dr Rob Andrews (medical collaborator) – protocol development and risk assessment 
Dr Brad Metcalf – Lead statistician 
Dr Jonathan Fulford – MRI Imaging Lead 
 
viii. KEY WORDS: 
Immobilization, disuse, inactivity, atrophy, insulin resistance, muscle protein synthesis 
 
 

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ix. TRIAL FLOW CHART 
 
 
 
 

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1 
BACKGROUND 
 
During the course of life, individuals experience multiple bouts of physical inactivity, i.e. muscle disuse. 
Such periods of for instance illness or injury, which necessitate a period of bed rest or limb 
immobilisation, lead to a decline in muscle mass of 0.5-0.6% per day [1]. Importantly, older individuals 
have been shown to have an impaired recovery following such periods of disuse, such that even 
following a progressive resistance training programme muscle fibre size remains ~11% lower than prior 
to disuse [2]. As such, the accumulation of such bouts of physical inactivity is thought to contribute 
substantially to the observed loss of muscle mass with ageing [3, 4]. The occurring muscle atrophy is 
for a large part due to an impaired muscle protein synthetic response to food ingestion, termed 
anabolic resistance, a decline which can be as much as 30-55% following short-term immobilisation [5, 
6]. In parallel, periods of muscle disuse lead to a deterioration of metabolic health by inducing 
substantial insulin resistance of glucose metabolism in skeletal muscle tissue. Indeed, following 7 days 
of muscle disuse, peripheral insulin sensitivity has been shown to decrease by 30-40% [7, 8]. By means 
of comparison, such a decline in insulin sensitivity is similar to the difference between a normal glucose-
tolerant individual and a patient with type 2 diabetes [9], and is equivalent to a decline that is observed 
following 30–40 years of aging [10, 11]. Importantly, as the decline in muscle mass, strength, and insulin 
resistance have been shown to be good proxy markers for patient outcomes following hospitalization 
[12], it is highly important to attenuate or prevent this functional and metabolic decline. To design 
targeted intervention strategies, insight in the mechanisms underlying muscle disuse atrophy needs to 
be gained. 
 
Previous work has shown that aberrant muscle fat metabolism is associated with insulin resistance [13]. 
More specifically, Dr Stephens recently showed that acute lipid infusion, known to induce insulin 
resistance, simultaneously induces anabolic resistance [14]. Indeed, there are changes in muscle lipid 
metabolism following 5-7 days of muscle disuse [7, 15], with overt muscle lipid accumulation after 28 
days of bed-rest [16] or limb immobilisation [17], suggesting perturbed IMCL turnover may also play a 
major part of the physiological dysregulation associated with decreased physical activity levels. 
However, as the development of insulin resistance and anabolic resistance precedes overt skeletal 
muscle lipid accumulation, it is unclear if perturbed fat metabolism is involved in the development of 
insulin resistance and anabolic resistance during disuse. 
 
At present, despite decades of physical inactivity research, the causes of disuse-induced insulin and 
anabolic resistance are still not known. Moreover, we simply do not know whether disturbances in 
muscle fuel availability and integration can explain the development of insulin resistance and anabolic 
resistance with muscle disuse. Importantly, if the development of insulin resistance and anabolic 
resistance can be attenuated or even prevented, this likely has a substantial positive effect on muscle 
mass retention during disuse. In other words, it will take shorter to get full recovery of muscle mass 
and muscle strength. Currently, the loss of muscle mass and strength results in protracted loss of 
function and increased cost on the NHS and Social care. This study will help us to understand how this 
atrophy occurs. This study will also determine if simple pharmacological interventions are a suitable 
intervention to prevent muscle loss over the first few days of muscle disuse. 
 
 
 
 

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2 
RATIONALE  
 
We hypothesize that muscle disuse alters intramuscular glucose and fatty acid metabolism, and their 
interaction, which consequently leads to decreases in insulin sensitivity and anabolic sensitivity. The 
primary aim of this study is therefore to determine the impact of reduced muscle lipid availability and 
increased glycolytic flux on forearm glucose uptake during short-term forearm immobilization in 
healthy volunteers. A secondary aim is to assess whether these simple pharmacological interventions 
have an impact on muscle mass loss during the first few days of immobilization; a critical period in a 
range of clinical scenarios, but one where we still do not know the mechanisms underpinning muscle 
loss. 
 
We will perform a randomized, double-blind, placebo-controlled study in healthy, young volunteers 
that will directly characterise the metabolic status of inactive muscle tissue in situations of altered 
substrate availability. In order to unravel the underlying mechanisms and capture metabolic changes, 
it is crucial to study the muscle immediately after the onset of insulin resistance. Therefore, participants 
will undergo 2 days of forearm cast immobilisation, combined with fully-controlled nutritional intake. 
Prior to and immediately following immobilisation, a metabolic test day will be performed using the 
AV-V forearm balance technique combined with stable isotope amino acid infusion to quantify muscle 
glucose uptake (i.e. insulin sensitivity, the primary outcome measure), muscle protein synthesis and 
breakdown, as well as fatty acid and BCAA balance in the postabsorptive state and during a 3h 
hyperinsulinaemic-euglycaemic-hyperaminoacidaemic clamp. This clamp will be used as the 
hyperinsulinaemic-euglycaemic clamp (golden standard for measuring insulin sensitivity) is associated 
with hypoaminoacidaemia [18], which likely impacts on muscle protein synthesis [19]. By clamping 
glucose and amino acid concentrations, glucose uptake and muscle protein synthesis can be quantified 
under steady-state conditions while insulin concentrations are controlled for. 
 
The following interventions will be used to alter muscle substrate availability in a randomised, double-
blind, placebo-controlled study with a parallel-group design: 
1. Placebo 
2. Acipimox, an anti-lipolytic agent which decreases circulating lipids, inhibits intramuscular lipid 
accumulation and thereby reduces lipid supply to the mitochondria [20]. 
3. Salbutamol, a selective β2-agonist. Beta2-agonists have been shown to stimulate glycogenolysis 
and glycolytic flux [21], and exert a hypertrophic effect [22] by increasing muscle protein 
synthesis and decreasing proteolysis [23, 24]. 
 
Our protocol combines the arteriovenous-venous forearm balance technique with stable isotope tracer 
infusion under hyperinsulinaemic-euglycaemic conditions prior to and following 2 days of forearm 
immobilization under conditions of altered substrate availability. As such, this study will simultaneously 
determine the impact of muscle disuse on insulin sensitivity and muscle protein synthesis, and thereby 
gain crucial insight in the role of these processes, and their interaction, in inactivity-induced muscle 
loss. It will also determine if simple pharmacological interventions are capable of preventing muscle 
loss over the first few days of muscle disuse; a critical period in a range of clinical scenarios, but one 
where we still do not know the mechanisms underpinning muscle loss. 
 
 

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3 
ASSESSMENT AND MANAGEMENT OF RISK 
 
A total of 6 venous cannulas will be inserted during the study; 3 on each metabolic test day (one in the 
elbow for infusion, one in the hand for blood sampling, and one in the elbow for blood sampling). This 
will involve the insertion of a small needle into the vein to place the cannula. This may cause dizziness 
and/or nausea if the subject is uncomfortable with needles or blood draws. There is also the possibility 
of bruising at the needle site. The cannulation will be conducted by trained research nurses and 
pressure will be applied to the needle site after cannula removal to prevent bruising. Trained first aiders 
will also be closely available. Per test day, 210 mL blood will be taken over the course of 5.5 hours. 
 
The hyperinsulinaemic-euglycaemic clamp is a procedure routinely used in human physiology research, 
and Dr Marlou Dirks, Dr Francis Stephens, and Dr Rob Andrews all have significant experience in 
independently performing this procedure. Co-infusion of an amino acid mixture will be performed to 
create hyperaminoacidaemic conditions that mimic the postprandial state following food ingestion. 
This amino acid mixture is a standard product used in clinical care. The main risk of running this 
procedure is uncontrollable hypoglycaemia. Blood glucose concentrations will be closely monitored 
every 5 min using a bedside glucose analyser, and the glucose infusion rate will be adjusted accordingly. 
If blood glucose cannot be maintained, insulin infusion will be stopped immediately, glucose infusion 
will be further increased, and a carbohydrate-rich drink (present in the room) will be provided to the 
participant. A minimum of two investigators, of which minimally one of them is emergency first aid-
trained, will be in the room at all times. Medical cover will be on call (present in the building) during 
the insulin infusion. 
 
The acipimox drug may cause some mild-moderate side effects in approximately 2 out-of 10 people. 
These side effects can include: skin rash; red ‘flushed’ skin; itching; sensations of hot skin. Less common 
effects include stomach pain and weakness. The salbutamol drug may cause mild-moderate side effects 
in approximately 1 out-of 10 people. These side effects van include fine tremor (‘shaking’, especially of 
the hands), headache, dizziness, and nausea. Less common side effects can include arrhythmias, low 
blood pressure, and muscle pain. These reactions usually disappear after 3 days, and failure of symptom 
reversal in a standard setting normally involves a participant being asked to stop taking the drug. Since 
participants in the current study are healthy volunteers who will only be taking the drugs for 2 days, no 
persistent side effects are expected. A clinical doctor (Dr Rob Andrews) will be available for advice 
throughout the study. 
 
Limb immobilization is a model routinely used to study muscle atrophy in humans, and our previous studies 
(Dirks et al. 2014 Acta Physiol, Dirks et al. 2016 Acta Physiol, Wall et al. 2013 J Clin Endocrinol Metab, Wall 
et al. 2014 Acta Physiol, Wall and Dirks et al. 2016 AJP Endo Metab) have used 3-14 days of forearm and 
leg immobilization in >100 healthy young volunteers without any problems. Forearm immobilization may 
lead to psychological limitations of having one arm in a cast. During the 2-day immobilization period, 
participants will likely experience difficulties in performing their daily activities due to only being able to 
use one arm. A sling will be provided to wear during the day, and a waterproof cover to protect the cast 
during showering. Any loss or muscle mass and strength that may occur will likely be smaller than 1%, and 
will restore quickly upon remobilization due to the inclusion of healthy young volunteers. 
 
There will be a time burden on the participant, for which they will receive recompense. The time burden 
will be spread over a period of around 1.5 weeks, and they will only be required to visit the laboratory 
for a total of 4 visits. The largest cumulative time burden will be that required to complete the two 
metabolic test days, approximately 6.5 hours per day. 
 
During the two days of forearm immobilization, participants will be asked to consume a controlled diet 
and nothing else. Because this diet will be calculated per participant to ensure energy-balanced 

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conditions, and participants are asked to indicate any food products they cannot or will not eat, the 
controlled diet should provide adequate dietary intake and not feel as too much of a burden for 
participants. Of note, this approach has been used in 4 separate studies from the Nutritional Physiology 
research group, and has proven successful in maintaining energy balance during the studies. 
 
The hyperinsulinaemic-euglycaemic-hyperaminoacidaemic clamp is occasionally associated with 
gastro-intestinal issues in the last hour of the 3-hour clamp, including vomiting and diarrhoea. To 
prevent these issues from occurring, we will administer 10 mg metoclopramide hydrochloride 
intravenously in an already present elbow cannula after 2 hours of the clamp. 
 
Subjects can contact the CI at any time (24/7) if they have concerns or need help. Dr Rob Andrews will 
be available for advice throughout the supplementation period. 
 
 
 

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4 
OBJECTIVES AND OUTCOME MEASURES/ENDPOINTS 
 
4.1 
Primary objective 
Determine the impact of reduced muscle lipid availability and increased glycolytic flux on forearm 
glucose uptake during short-term forearm immobilization in healthy volunteers. 
 
Research question: Does lowering muscle lipid availability and increasing glycolytic flux attenuate the 
immobilization-induced decline in forearm glucose uptake? 
Null hypothesis: Lowering muscle lipid availability and increasing glycolytic flux does not attenuate the 
immobilization-induced decline in forearm glucose uptake. 
 
3.2 
Secondary objectives 
Determine the impact of reduced muscle lipid availability and increased glycolytic flux on muscle 
protein synthesis, and forearm nutrient balance during short-term forearm immobilization in healthy 
volunteers 
 
3.3 
Primary outcome 
The primary outcome measure of this study is forearm glucose uptake, as a measure of insulin 
sensitivity, measured using the arteriovenous-venous forearm balance technique. This will be 
measured under hyperinsulinaemic-euglycaemic conditions, which is the gold standard for assessing 
insulin sensitivity in vivo. 
 
3.4 
Secondary outcomes 
Secondary outcome measures include postabsorptive and postprandial muscle protein synthesis, 
forearm branched-chain amino acid and fatty acid balance, and forearm volume. 
 
3.5  
Table of endpoints/outcomes 
Objectives 
Outcome Measures  
Timepoint(s) of evaluation of 
this outcome measure (if 
applicable) 
Primary Objective 
Determine the impact of 
reduced muscle lipid 
availability and increased 
glycolytic flux on forearm 
glucose uptake during short-
term forearm immobilization in 
healthy volunteers. 
Forearm glucose uptake 
During the steady-state phase 
of the hyperinsulinaemic-
euglycaemic clamp, during the 
metabolic test days prior to 
and immediately following 2 
days forearm immobilization 
Secondary Objectives 
Determine the impact of 
reduced muscle lipid 
availability and increased 
glycolytic flux on muscle 
protein synthesis, and forearm 
nutrient balance during short-
term forearm immobilization in 
healthy volunteers 
Postabsorptive and 
postprandial muscle protein 
synthesis 
Forearm branched-chain 
amino acid and fatty acid 
balance 
Forearm volume 
 
During the metabolic test days 
 
 
 

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5 
TRIAL SETTING, DESIGN, AND PROCEDURES 
 
This study is a randomized, double-blind, placebo-controlled trial with a parallel group design in healthy 
young volunteers. This study will determine the impact of pharmacological manipulation of substrate 
availability on insulin sensitivity, and anabolic sensitivity during 2 days of forearm immobilization. This 
study comprises of four study visits, depicted in the study schematic on Page 11 and described in detail 
in paragraph 5.5 below. 
 
This study is a single-centre study, with all visits performed in the Clinical Research Facility at the Royal 
Devon & Exeter NHS Foundation Trust. Dispensing of the drugs will be performed by the Clinical Trials 
Pharmacy at the Royal Devon & Exeter hospital in Exeter. 
 
5.1 
Recruitment 
Participants will be recruited via convenience sampling. Participants will be recruited via posters, flyers, 
social media, and word of mouth, from the general public of Exeter and the surrounding areas. 
 
5.2 
Screening 
Participants will be asked to meet the investigator so that the study can be explained to them in detail, 
and it is clear what they can expect from the research team and what will be expected of them. 
Assuming they are happy with what has been discussed, they will then provide written consent. 
Subsequently, they will complete a medical screening questionnaire and have their height, weight, body 
composition, and blood pressure measured. All in-and exclusion criteria (please see Section 6) will be 
assessed. The CI will discuss any issues about eligibility to take part with the medical collaborator. 
 
5.3  
Payment  
The participant will receive an inconvenience allowance of £125, which will be transferred to their bank 
account of choice by the University of Exeter finance department. Payment for participants who withdraw 
from the study or are removed from the study will be dealt with on case-by-case basis. 
 
5.4 
Consent  
After registering interest, the study will be explained in detail to the participant via email, and they will 
be given the participant information sheet. They will be asked to carefully read the document, so that 
they can generate any initial questions. This will occur at least one week in advance of screening. Upon 
meeting the participant face-to-face the study will be verbally explained in detail. This will include 
explaining to the participant what they can expect were they to participate, including details of all the 
procedures involved and time commitments. It will also be made clear what is expected of them. 
Potential participants will be encouraged to ask any questions they may have. It will be explicitly stated 
that participants can withdraw at any point during the study without giving a reason, and that they can 
be afforded time to decide on whether they wish to take part. If they are satisfied, and want to 
participate in the study, they will subsequently sign the informed consent form (all done under the 
supervision of an investigator who has undergone informed consent training). 
Consent will be sought for surplus muscle samples to be used for ancillary studies subject to additional 
ethical approval, with the participant having the choice of whether to place restrictions on sample use 
or not (see informed consent form). 
 
 
 
 

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5.5 
Trial procedures 
 
Please refer to page 11 for trial flow chart and schematic of the metabolic test days. The trial procedures 
are described below: 
 
Screening visit (visit 1): 
• 
Firstly, participants will be asked to meet the investigator so that the study can be explained to 
them in detail, and it is clear what they can expect from the research team and what will be 
expected of them. Assuming they are happy with what has been discussed, they will then 
provide written consent. Subsequently, they will complete a medical screening questionnaire 
and have their height, weight, body composition (Bodpod), and blood pressure measured. 
• 
Participant’s caloric requirements will be calculated by estimating Resting Metabolic Rate via 
the Henry equation, and then multiplying this value by an activity factor derived from the 
International Physical Activity Questionnaire (IPAQ) questionnaire. 
• 
Forearm volume will be measured via anthropometry. 
• 
Participants will be asked for any allergies or intolerances to ensure they can eat everything 
they will receive as part of the standardized diet. 
• 
Participants will be provided with a food diary to record their habitual dietary intake for 3 
consecutive days, including 2 week days and 1 weekend day. 
 
Baseline metabolic test day (visit 2): 
• 
Approximately 1 week prior to casting, at 8 AM, participants will visit the Clinical Research 
Facility at the Royal Devon & Exeter NHS Foundation Trust in an overnight fasted state for a 
metabolic test day. 
• 
The following cannulas will be inserted by a trained research nurse: 1) retrogradely into a deep 
antecubital vein (draining the muscle bed) of the non-immobilised arm, 2) retrogradely into a 
superficial dorsal hand vein of the to-be-immobilised hand, and 3) anterogradely into an 
antecubital vein in the elbow of the to-be-immobilised arm for infusion of insulin, glucose, 
amino acids, and stable isotope amino acids. The cannulated hand will be placed into a hand-
warming unit (air temperature 55°C) to arterialize the venous drainage of the hand. 
• 
After insertion of all cannulas, a primed, continuous stable isotope infusion of L-[ring-2H5]-
phenylalanine (prime: 0.5 mg·kg-1, infusion 0.5 mg·kg-1·h-1) will be started with participants in a 
supine position. Using this tracer approach we will be able to calculate muscle protein synthesis 
and plasma leucine oxidation. 
• 
At t = 2.5 h, a hyperinsulinaemic-euglycaemic-hyperaminoacidaemic clamp will be started. For 
this, insulin will be infused at a rate of 50 mU·m-2·min-1, and a simultaneous infusion of 20% 
glucose will be started to maintain euglycaemia at 5.0 mmol·L-1. A 0.3% potassium chloride 
solution will be infused in the (to be) immobilized arm at a rate of 1 ml/kg body weight/h during 
the 3-h clamp to maintain plasma potassium concentrations [25]. Hyperaminoacidaemia will 
be induced by infusion of an amino acid solution (Primene 10%, Baxter) at BCAA concentrations 
of 700 μmol·L-1. This concentration was chosen as it is the average postprandial BCAA 
concentration following mixed meal ingestion in the pilot study. Primene will be 7% enriched 
with L-[ring-2H5]-phenylalanine to prevent a decrease in plasma enrichments with Primene 
infusion. Measurements of blood glucose (Yellow Springs Instruments; every 5 min) and BCAA 
(spectrophotometric assay, [26]; every 20 min) concentrations will be performed at the 
bedside, and the infusion rate of glucose and amino acids adjusted accordingly to maintain 
steady state. Throughout the entire 5.5 h infusion period, repeated arterialised-venous (AV) 
and deep-venous (V) blood samples will be taken in order to calculate forearm AV-V balance of 
glucose (i.e. insulin sensitivity), amino acids, and free fatty acids using the Fick principle [27]. 
Prior to every blood sample, brachial artery blood flow will be measured using Doppler 
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• 
After 2 hours of the 3h clamp, a single dose of 10 mg metoclopramide hydrochloride will be 
administered intravenously in an antecubital elbow cannula, to precent potential gastro-
intestinal issues during the final hour of the clamp. 
• 
Following the 3 h clamp, infusion of insulin, amino acids and stable isotope amino acids will be 
stopped while infusion of glucose will be decreased in a stepwise manner based on blood 
glucose concentrations. The last 30 min of the clamp will be used to calculate the mean glucose 
infusion rate (GIR) and mean amino acid infusion rate. Participants will be monitored for 
minimally 20 min following cessation of the glucose infusion before they are allowed to leave 
the laboratory. 
 
Forearm casting and drug dispensing (visit 3; to take place between 7 and 28 days after visit 2): 
• 
In the morning at 7:30 AM, participants will visit the Clinical Research Facility at the Royal Devon 
& Exeter NHS Foundation Trust in an overnight fasted state. 
• 
A fiberglass forearm cast (randomized between dominant and non-dominant arm) will be 
applied from below the elbow to approximately 2-3 cm below the finger tips, to immobilize the 
wrist. Participants will be given a sling to wear at all times, except for during sleeping and 
showering. A waterproof cover will be provided to cover the cast during showering. 
• 
Participants will then receive the food products for the controlled diet, which they will consume 
during the 2 days of forearm immobilization. Participants will be fed in energy balance, with 
energy requirements calculated as basal metabolic rate (Henry equations) times an activity 
factor (IPAQ questionnaire). The diet consists of 1.2 g/kg body weight/day protein (approx. 10-
15 en%), 50-55 en% carbohydrate, and 35 en% fat. The diet will be composed of three main 
meals (i.e. breakfast, lunch, dinner) and snacks, and will contain normal food products. A food 
diary including recipes for the evening meals will be given, and the entire diet will be explained. 
• 
Lastly, participants will visit the Clinical Trials Pharmacy at RD&E to collect their allocated 
treatment. Instructions on the times and way to ingest the treatment is provided by trained 
staff, and a treatment log will be provided. The first out of eight doses will be taken. 
• 
Participants will be sent home, and will return to the CRF two days later for visit 4. 
 
Post-immobilization metabolic test day (visit 4): 
• 
In the morning at 8 AM, participants will visit the Clinical Research Facility at the Royal Devon 
& Exeter NHS Foundation Trust in an overnight fasted state. 
• 
The metabolic test day will be repeated. 
• 
The fibreglass cast will be removed. 
 
5.6 
Randomisation 
To determine whether altering muscle substrate availability impacts upon metabolic health, participants 
will be asked to take an oral supplement 4 times a day during the 2-day forearm immobilization period.  
Participants will be randomly allocated to receive a supplement containing either acipimox, salbutamol, or 
a placebo containing only cellulose microcrystalline. 
Randomisation will be via a computer generated randomisation schedule provided by the involved 
statistician (Dr Metcalf), and held in a folder in his office in Richards Building (St Luke’s Campus). A secure 
Excel document of the randomization schedule (treatment A, B, or C) will be accessible by all members of 
the study team, including Dr Andrews and the Pharmacy contact. Supplementation compliance will be 
monitored via self-reported supplement intake diaries, and content counting from returned supplement 
containers. 
 
The arm-to-be-immobilized will be counterbalanced between the dominant and non-dominant arm. 
 

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5.7 
Blinding 
The trial products (i.e. Acipimox, Salbutamol, or placebo) will be received by the Clinical Trials Pharmacy in 
a distinguishable manner. They will be handled only by unblinded personnel until repackaged in an opaque 
container. Each container will be labelled with a unique pack ID to identify each pack and its content. The 
treatment will be prescribed by Dr Rob Andrews, and dispensed to the participant by the Pharmacy. 
Participants will be instructed not to discuss the product they receive with anyone inside or outside the 
research team. 
 
5.8 
Storage and analysis of clinical samples 
Blood samples will be taken throughout the study, as outlined in the study protocol. At each sampling 
point 8 mL of blood will be taken via cannula, and immediately divided into a plasma (BD Vacutainer - 
lithium heparin) and serum tube (BD Vacutainer – SST). Plasma samples will be directly snap frozen in 
liquid nitrogen and analysed for branched-chain amino acids and non-esterified fatty acid 
concentrations. Serum will be allowed to clot for 30 minutes prior to centrifugation, before being 
aliquoted into Eppendorf’s, snap frozen in liquid nitrogen, and stored in a freezer at -80°C +/- 10°C until 
analysis for insulin concentration. Plasma L-[ring-2H5]-phenylalanine enrichments will be measured via 
gas chromatography-mass spectrometry (GC-MS; [6]). The CI will keep a record of all blood samples 
taken, of their respective aliquots, and of their location within the freezer. 
Samples will be stored for a period of 5 years, in line with HTA guidelines, for further analysis beyond the 
primary variables depending on the changing landscape of the literature. Samples will be stored in a secure 
location accessible only via security-card access. Samples will be destroyed either in the process of analysis, 
or, after the allotted storage period has passed, by being placed in clinical waste and subsequently 
incinerated. Disposal of plasma and serum will be recorded by the research team in their own archives. 
Overall responsibility for the samples will reside with Dr Marlou Dirks. Samples will be appropriately 
labelled in accordance with the trial procedures to comply with the 1998 Data Protection Act. Biological 
samples collected from participants as part of this trial will be transported, stored, accessed and processed 
in accordance with national legislation relating to the use and storage of human tissue for research 
purposes and such activities shall at least meet the requirements as set out in the 2004 Human Tissue Act 
and the 2006 Human Tissue (Scotland) Act. 
 

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6 
PARTICIPANT ELIGIBILITY CRITERIA 
 
6.1 
Inclusion criteria 
• 
Males and females 18-40 years of age 
• 
Body mass index between 18.5 and 30 kg/m2 
 
6.2 
Exclusion criteria 
• 
Any diagnosed metabolic impairment (e.g. type 1 or 2 diabetes) 
• 
Any diagnosed cardiovascular disease 
• 
Hypertension (≥140 mmHg systolic and/or ≥90 mmHg diastolic) 
• 
Chronic use of any prescribed or over the counter pharmaceuticals (excluding oral 
contraceptives and contraceptive devices) 
• 
Regular use of nutritional supplements 
• 
A personal or family history of thrombosis, epilepsy, seizures or schizophrenia 
• 
Any previous motor disorders 
• 
Any known disorders in lipid metabolism 
• 
Any known disorders in muscle metabolism 
• 
Known allergy for Acipimox, Salbutamol, or other substances in the tablets 
• 
Known sensitivity for sympathomimetic drugs 
• 
Known hypokalaemia 
• 
Presence of an ulcer in the stomach or gut and/or strong history of indigestion 
• 
Known severe kidney problems 
• 
Previous reaction to metoclopramide 
• 
Known phaeochromocytoma (tumour that secretes noradrenaline or adrenaline) or past 
history of one 
• 
Recent abdominal surgery 
• 
Pregnancy 
• 
Unable to give consent 
  
 
 
 
 

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7 
TRIAL TREATMENTS 
 
The present study is a basic physiology study, which aims to elucidate mechanisms underlying muscle 
atrophy by studying muscle metabolism in conditions of pharmacologically altered substrate 
availability. Participants will be randomized to receive one of two drugs (Acipimox and Salbutamol) or 
a placebo, to be taken orally during the 2-day forearm immobilization period. Products will be 
prescribed by Dr Rob Andrews, after thorough checking for the listed in- and exclusion criteria (Section 
6). 
 
All products will be taken 4 times daily, with or after the three main meals (i.e. breakfast, lunch, dinner), 
and before bed. 
 
Participants will be able to contact the CI 24/7, and Dr Rob Andrews will be available for advice 
throughout the supplementation period. 
 
The study products are described below: 
 
Acipimox 
The drug Acipimox is a nicotinic acid analogue that inhibits adipose tissue lipolysis, lowers intramuscular 
lipid metabolites, and improves insulin sensitivity. Acipimox will be given in 250 mg capsules, taken 4 
times daily, as this dose has been shown to improve insulin sensitivity in type 2 diabetes patients within 
a few days [20]. 
Acipimox may cause some mild-moderate side effects in approximately 2 out-of 10 people. These side 
effects can include: skin rash; red ‘flushed’ skin; itching; sensations of hot skin. Less common effects 
include stomach pain and weakness.  
 
Salbutamol 
The drug Salbutamol is a β2-adrenoceptor agonist commonly used as a bronchodilator in asthma. In 
addition, β2-agonists have been shown to stimulate glycogenolysis and glycolytic flux [21], and exert a 
hypertrophic effect [22] by increasing muscle protein synthesis and decreasing proteolysis [23, 24]. 
Salbutamol will be given in 4 mg tablets, taken 4 times daily, as this dose has been shown to lead to a 
significant increase in lean mass in  [22]. 
Salbutamol may cause mild-moderate side effects in approximately 1 out-of 10 people. These side 
effects van include fine tremor (‘shaking’, especially of the hands), headache, dizziness, and nausea. 
Less common side effects include arrhythmias, low blood pressure, and muscle pain.  
 
Placebo 
The placebo supplement will contain lactose, microcrystalline cellulose, pregelatinised starch, sodium 
starch glycolate, and magnesium stearate. This are inert substances widely used in many pill and tablet 
formulations, and are unlikely to cause toxicity when taken orally.  
 
 
 

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8 
ADVERSE EVENT RECORDING AND REPORTING 
 
8.1  
Definitions 
An Adverse Event (AE) is any unfavourable and unintended sign, symptom, syndrome or illness that 
develops or worsens during the period of observation in the study.  
 
An AE does include a / an: 
1. exacerbation of a pre-existing illness. 
2. increase in frequency or intensity of a pre-existing episodic event or condition. 
3. condition detected or diagnosed after medicinal product administration even though it may 
have been present prior to the start of the study. 
4. continuous persistent disease or symptoms present at baseline that worsen following the start 
of the study. 
 
An AE does not include a / an: 
1. medical or surgical procedure (e.g., surgery, endoscopy, tooth extraction, transfusion); but the 
condition that leads to the procedure is an AE. 
2. pre-existing disease or conditions present or detected at the start of the study that did not 
worsen. 
3. situations where an untoward medical occurrence has not occurred (e.g., hospitalisations for 
cosmetic elective surgery, social and / or convenience admissions). 
4. disease or disorder being studied or sign or symptom associated with the disease or disorder 
unless more severe than expected for the participant’s condition. 
5. overdose of concurrent medication without any signs or symptoms. 
 
A Serious Adverse Event (SAE) is any adverse event occurring following study mandated procedures, 
having received the treatment or intervention that results in any of the following outcomes: 
1. Death 
2. A life-threatening adverse event 
3. Inpatient hospitalisation or prolongation of existing hospitalisation 
4. A disability / incapacity 
5. A congenital anomaly in the offspring of a participant 
 
Important medical events that may not result in death, be life-threatening, or require hospitalisation 
may be considered a serious adverse event when, based upon appropriate medical judgment, they may 
jeopardize the patient or participant and may require medical or surgical intervention to prevent one 
of the outcomes listed in this definition 
 
All adverse events will be assessed for seriousness, expectedness and causality: 
 
A distinction is drawn between serious and severe AEs. Severity is a measure of intensity whereas 
seriousness is defined using the criteria above. Hence, a severe AE need not necessarily be serious.  
 
8.2 
Recording and reporting of adverse events 
Any adverse effects noted will be recorded in the adverse event log and reported to the CI and the 
study sponsors as per standard University of Exeter sponsor protocols (please refer to Letter from 
sponsor, included in this application). The CI will have a professional obligation to closely monitor 
participants and in the event of an adverse event being identified, follow established clinical treatment 
or referral protocols. 
 
Participants will be instructed to contact the study team immediately should they feel unwell, and will 
be provided with contact numbers for contacting the team directly. Participants will be issued with a 

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card indicating their participation. This card will also identify their allocation code and identify reasons 
and who to contact should breaking the allocation code become necessary. 
 
8.3 
Emergency unblinding 
The investigators may identify the treatment for each participant through sealed envelopes in the event of 
a medical emergency or SAE when treatment is dependent on knowledge of the actual drug received. The 
CI will be notified if the treatment code for a participant is broken, and the date and the reason(s) for 
breaking the blind will be noted in the CRF in a timely manner (notification of Sponsor immediately as 
practicable by phone or fax, followed by a written narrative of the event within 24 hours, as described 
above). Emergency unblinding will be carried out by the medical collaborator (Dr Rob Andrews). He will at 
all times have access to the sealed list with participant names and treatment allocation. 
 
 

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9 
STATISTICS AND DATA ANALYSIS 
 
9.1 
Sample size calculation 
We have powered the study to detect a difference in forearm glucose uptake (primary outcome 
measure) between 1) Acipimox vs placebo, and 2) Salbutamol vs placebo. We have powered the study 
based on detecting differences in the change in forearm glucose uptake as measured during the 
hyperinsulinaemic-euglycaemic clamp, which is golden standard to measure insulin sensitivity. We have 
recently generated pilot data to demonstrate that forearm glucose uptake decreases by 13±8% 
(mean±SD) in response to mixed meal ingestion following 2 days of forearm immobilization. Based on 
our hypotheses, lowering lipid availability (via Acipimox) and stimulating glycolytic flux (via Salbutamol) 
will alleviate insulin resistance (i.e. prevent the decline in forearm glucose uptake). The following data 
were used to estimate the effect size, using α = 0.05; power = 80%; significance = 5%: 
1) Placebo (-13±8%) vs Acipimox (0±8%; preservation of forearm glucose uptake [14]. Effect size: 
1.625. Required: n=8 
2) Placebo (-13±8%) vs Salbutamol (-4.3±6%; 66% attenuation of decline in forearm glucose 
uptake by a beta2-agonist [28, 29]. Effect size: 1.23. Required: n=12 
To have enough power for both comparisons, n=12 participants will be included in all groups (i.e. 
placebo, Acipimox, Salbutamol). 
Thus, assuming a ≤20% dropout rate (in our experience, similar immobilization studies do not exceed 
this drop-out rate when using healthy adults), we will recruit 45 healthy adults 18-40 years old, from 
both sexes, who will be split across 3 parallel groups (i.e. n=15 per group, ensuring completion of n=12 
per group). 
Sample size was calculated using G*Power 3.1.9.2. 
 
9.2 
Planned recruitment rate 
The study will recruit 45 (taking into account a 20% dropout rate, see paragraph 10.1), young healthy 
males and females, primarily from the University student population. The planned recruitment rate is 
2 participants per month over a 24 month period. Participants will be recruited at a single centre, and 
we foresee no issue capturing the eligibility criteria. We expect a screening failure rate not exceeding 
50%, based on previous experience. 
 
9.3 
Statistical analysis plan 
9.3.1 
Summary of baseline data and flow of participants 
Key baseline data will include anthropometric data (height, weight, BMI, fat mass), glucose and insulin 
concentrations, forearm glucose uptake, postabsorptive and postprandial muscle protein synthesis, 
forearm muscle volume, and forearm branched-chain amino acid (BCAA) and fatty acid balance. All 
baseline variables will be analysed using One-Way ANOVA to determine whether there are differences 
between groups. 
A CONSORT flow diagram will be produced following completion of the study, and published in the 
manuscript(s) when possible. 
 
9.3.2 
Primary outcome analysis 
The primary outcome measure for this study is forearm glucose uptake, measured during the steady-
state phase of a 3-h hyperinsulinaemic-euglycaemic-hyperaminoacidaemic clamp. A two-way ANOVA, 
with and treatment (placebo vs Acipimox vs Salbutamol) as between-subjects factor and time (pre vs 
post immobilization) as within-subjects factor. A Bonferroni post-hoc test will be used to locate 
individual differences. 
 

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9.3.3 
Secondary outcome analysis 
Secondary outcome measures include postabsorptive and postprandial muscle protein synthesis, and 
forearm branched-chain amino acid and fatty acid balance. These data will be analysed in the same way 
as the primary outcome described above. 
 
9.3.4 
Procedure(s) to account for missing or spurious data  
The study will be designed to a high level of rigour, and processes will be methodically applied to 
minimise the occurrence of missing data. Where data are missed due to participant error / non-
compliance or technical error, there will be a follow up investigation carried out by the research team 
try to locate the data, or, failing that, to optimise future data collection. If data are missed due to 
researcher error or technical failure, then this will be recorded on data sheets available to the research 
team at all times. Missing data analyses will be carried out using a commercial statistical analysis 
package (GraphPad Prism 7.03). 
 

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10 
DATA MANAGEMENT  
 
10.1 Source data and documents 
All source data and source documents collected during the trial will be retained to allow subsequent 
reconstruction and evaluation of the trial. Researchers will ensure that all source data and documents 
are accurate, legible, complete and consistent to allow verification of the process for the purposes of 
confirmation, quality control and audit. Personal details will be stored up to a period of 5 years from 
the completion of the study, as we would like to store the personal details at least in line with the 
storage of blood samples to allow maximum data generation from the study performed.  
Dr Marlou Dirks will act as custodian, and only her and Dr Francis Stephens will have access to the 
source data. All members of the research team will comply with the Data Protection Act 1998 with 
regards to the collection, storage, processing and disclosure of personal information and will uphold 
the Act’s principles. 
Trial conduct may be subject to systems audit of the Trial Master File (TMF) for inclusion of essential 
documents; permissions to conduct the trial; Trial Delegation Log; CVs of trial staff and training 
received; local document control procedures; consent procedures and recruitment logs; adherence to 
procedures defined in the protocol (e.g. inclusion / exclusion criteria, correct randomisation, timeliness 
of visits); adverse event recording and reporting; accountability of trial materials and equipment 
calibration logs. 
 
10.2 Data areas and data types  
The research data to be managed are principally quantitative in nature obtained from investigation of 
healthy young male and female participants (18-40) e.g. screening data (height, weight, BMI, age, blood 
clinical chemistry, blood pressure, forearm volume etc.), habitual diet, body composition and muscle 
mass obtained from BodPod, and muscle protein synthesis rates and plasma tracer enrichments and/or 
amino acid and fatty acid concentrations of the collected blood samples. A proportion of the data will 
be qualitative (e.g. screening and physical activity questionnaires and diet diaries). 
 
10.3 Standards and metadata  
In compliance with UK Data Protection Laws, subject identifiable data (screening data, consent forms 
and screening questionnaires), and all hard copies of lab notebooks and documents will be kept in a 
locked room within the Clinical Research Facility (RILD building, RD&E site), specifically dedicated to 
storage of identifiable data. Once enrolled into a study, participants are assigned a subject number and 
research data generated incorporate this subject number as an identifier, i.e. no direct link to 
participant identity is used. Encrypted and password-protected electronic copies of these data will be 
made to ensure against the risk of loss. All members of the research team have relevant expertise of 
information security; having received training within the host institution. 
All forms of experimental data will be collected electronically onto computer hard drives intrinsic to the 
equipment being used to make the measurement in question (e.g. mass spectrometer). Sufficient 
storage space is available on each system to allow adequate storage of large raw data, but all data will 
be backed up onto the project specific computer. Where possible the project will use open or widely 
used data formats and files. Electronic data will subsequently be stored on University managed network 
drives, featuring data backup and redundancy systems, with access rights to the storage area restricted 
to key personnel. Each researcher is allocated up to 100GB of secure, backed up network storage. Any 
data stored on the CI’s computer will be backed up daily to an encrypted external hard drive, stored in 
a geographically different location, before being transferred to the network drive at the earliest 
opportunity. Good laboratory practice (GLP) will be applied throughout the project in the acquisition, 
management and archiving of study data. In compliance with the ICH/GCP guidelines, regulations and 
in accordance with the University of Exeter Research Ethics, the Chief Investigator will maintain all 
records and documents regarding the conduct of the study. All data will be retained for a period of at 

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least five years after completion of the project. If the responsible investigator is no longer able to 
maintain the study records, a second person will be nominated to take over this responsibility. 
 
10.4 Methods for data sharing  
The sponsor and investigators are fully committed to share data in accordance with the HRA Policy. 
High volume data generation is not a feature of the proposed project, and so the data will be 
predominantly disseminated via publication in high-impact peer-reviewed journals (within 12 months 
of study completion) and via presentation at conferences. Publications will be in accordance with the 
University of Exeter’s Open Access policy and where possible immediately made openly available to 
adhere to the funder’s policy. All data published and/or shared will be anonymized to prevent subject 
identification. Importantly, on request we will make available our primary data to external researchers, 
subject to preservation of intellectual property and written acknowledgement that data ownership will 
reside with the research team. While it is not anticipated, data that is not included for publication but 
may be of value to the research community will be archived in the University’s Institutional Repository, 
Open Research Exeter (ORE). All data will be deposited and will be citable using a persistent identifier. 
 
10.5 Proprietary data 
The results generated from this study will give insight in the role of fuel integration during inactivity, as 
well as identify potential molecular treatment targets which could be of interest to major Pharma 
companies who currently have exciting programs around preventing muscle wasting. The University of 
Exeter Innovation, Impact and Business (IIB) team can help with industrial liaison and knowledge 
transfer, from IP protection to finding a company interested in the research outputs. 
 
 

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11 
ETHICAL AND REGULATORY CONSIDERATIONS 
 
11.1  
Research Ethics Committee (REC) review and reports 
The study will be conducted in compliance with the principles of the Declaration of Helsinki (1996), the 
principles of GCP and in accordance with all applicable regulatory requirements. Local ethical approval was 
obtained from the University of Exeter’s Sport and Health Sciences Ethics Committee (please see attached 
certificate of ethical approval). This application is now submitted for REC approval as part of the Clinical 
Research Network portfolio adoption process, so that the RD&E Pharmacy can be accessed for dispensing 
of the drugs. 
 
Before the start of the study, approval will be recognised from a REC for the study protocol, informed 
consent forms and any other relevant documents. Substantial amendments that require review by REC will 
not be implemented until the REC grants a favourable opinion for the study. All correspondence with the 
REC will be retained in the Trial Master File. The Chief Investigator will submit an annual progress report 
(APR) to the REC within 30 days of the anniversary date on which the favourable opinion was given, and 
annually until the study is declared ended. The Chief Investigator will notify the REC of the end of the study, 
and if the study is ended prematurely, the Chief Investigator will notify the REC, including the reasons for 
the premature termination. Within one year after the end of the study, the Chief Investigator will submit a 
final report with the results, including any publications/abstracts, to the REC. 
 
11.2  
Peer review 
The scientific quality of this research has been assessed within the research team and CI's host 
institution, before submitting this as part of the CI's application for a Sir Henry Wellcome Postdoctoral 
Fellowship. The Fellowship application process consisted of two rounds of written submissions (please 
find reviewers comments from 3 independent expert reviewers included in this application) and an 
interview round with the Wellcome Trust's Basic Science Panel, consisting of approximately twenty 
panel members from various UK institutions. Dr Marlou Dirks was awarded this 4-year Fellowship in 
November 2017. 
 
11.3  
Indemnity 
The University of Exeter has expanded its standard indemnity cover with an extra medical malpractice 
extension, specifically for this study (please see documents attached). This ensures that Dr Rob Andrews, 
whose activity on this study is not covered by the NHS indemnity scheme, is fully covered in his role of 
medical collaborator. This insurance covers the potential harm to participants arising from the 
management, design, and conduct of the research. 
 
 
 

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12 
DISSEMINIATION POLICY 
 
The current proposal aims to conform to the principles of the ‘European Charter for Researchers’ by 
guaranteeing that the novel knowledge generated by the study is disseminated in a way that can be 
understood by the general public. The primary goal of these efforts is to distribute the key findings to 
the society and its affected individuals, as well as to health departments, policy makers, researchers, 
and health advocacy groups. The main mediums of communication and information dissemination are: 
1) Peer-reviewed journal article(s). Data will be published in peer-reviewed journals according to 
RCUK/HEFCE guidelines, with gold or green access. The data will be published as one or more 
journal articles, depending on the outcomes of the study. 
2) A project website that will be launched to inform the general public on the objectives, funding 
body, conclusions, and general recommendations of the project.  
3) Social media such as specifically created Twitter and Facebook pages as well as my personal 
Twitter account. Data will also be shared via ResearchGate.com, where a project page for this 
study will be set up. 
4) Local events. Examples of these are Soapbox Science, which showcases research of women 
who are making significant contributions to the scientific community, and Pint of Science, a 
festival which allows scientists to discuss their research with the general public in a local pub. 
 
 

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13 
REFERENCES 
 
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Suetta, C., et al., Ageing is associated with diminished muscle re-growth and myogenic 
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3. 
English, K.L. and D. Paddon-Jones, Protecting muscle mass and function in older adults during 
bed rest. Curr Opin Clin Nutr Metab Care, 2010. 13(1): p. 34-9. 
4. 
Wall, B.T., M.L. Dirks, and L.J. van Loon, Skeletal muscle atrophy during short-term disuse: 
implications for age-related sarcopenia. Ageing Res Rev, 2013. 12(4): p. 898-906. 
5. 
Glover, E.I., et al., Immobilization induces anabolic resistance in human myofibrillar protein 
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Wall, B.T., et al., Short-term muscle disuse lowers myofibrillar protein synthesis rates and 
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p. E137-47. 
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Dirks, M.L., et al., One Week of Bed Rest Leads to Substantial Muscle Atrophy and Induces 
Whole-Body Insulin Resistance in the Absence of Skeletal Muscle Lipid Accumulation. Diabetes, 
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8. 
Stuart, C.A., et al., Bed-rest-induced insulin resistance occurs primarily in muscle. Metabolism, 
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Jocken, J.W., et al., Insulin-mediated suppression of lipolysis in adipose tissue and skeletal 
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