Clinical trial NCT01935791
Conditions: Metabolic Diseases
Keywords: Metabolic Diseases
Sponsor: Imperial College London
Start date: 2013-07-01; Completion date: 2018-11-05
Enrollment: 11 participants; Allocation: RANDOMIZED; Intervention model: CROSSOVER

Brief summary:
The purpose of this study is to determine what can activate brown adipose tissue (BAT).

Detailed description:
We will investigate different stimuli (i.e. hormones) to determine which can stimulate BAT.

Arms: arm 1: Visit 1. Participants underwent F-fluorodeoxyglucose (18F-FDG) positron emission tomography, computerised tomography-(PET)CT, whilst wearing a cooling vest and receiving an infusion of gelofusine.

Visit 2 Participants underwent F-fluorodeoxyglucose (18F-FDG) positron emission tomography, computerised tomography-(PET)CT, whilst receiving an infusion of gelofusine without a cooling vest. arm 2: Visit 1. Participants underwent F-fluorodeoxyglucose (18F-FDG) positron emission tomography, computerised tomography-(PET)CT, whilst wearing a cooling vest and receiving an infusion of gelofusine Visit 2 Participants underwent F-fluorodeoxyglucose (18F-FDG) positron emission tomography, computerised tomography-(PET)CT, whilst receiving an infusion of Glucagon at a dose of 50ng/kg/min without a cooling vest. arm 3: Visit 1. Participants underwent F-fluorodeoxyglucose (18F-FDG) positron emission tomography, computerised tomography-(PET)CT, whilst wearing a cooling vest and receiving an infusion of gelofusine.

No brown adipose tissue (BAT) identified on visit 1, therefore no visit 2. arm 4: Visit 1- Each participant had calorimetry testing and thermal imaging whilst receiving an infusion of gelofusine. They were situated in an ambient temperature of 22-25 degrees celsius.

Visit 2 - Each participant had calorimetry testing and thermal imaging whilst receiving an infusion of Glucagon at a dose of 50ng/kg/min. They were situated in an ambient temperature of 22-25 degrees celsius. Visit 3 - Each participant had calorimetry testing and thermal imaging whilst receiving an infusion of gelofusine and wearing a cooling vest. arm 5: Visit 1- Each participant had calorimetry testing and thermal imaging whilst receiving an infusion of Glucagon at a dose of 50ng/kg/min. They were situated in an ambient temperature of 22-25 degrees celsius.

Visit 2 - Each participant had calorimetry testing and thermal imaging whilst receiving an infusion of gelofusine and wearing a cooling vest.

Visit 3 Each participant had calorimetry testing and thermal imaging whilst receiving an infusion of gelofusine. They were situated in an ambient temperature of 22-25 degrees celsius. arm 6: Visit 1 - Each participant had calorimetry testing and thermal imaging whilst receiving an infusion of gelofusine and wearing a cooling vest.

Visit 2 - Each participant had calorimetry testing and thermal imaging whilst receiving an infusion of gelofusine. They were situated in an ambient temperature of 22-25 degrees celsius.

Visit 3 - Each participant had calorimetry testing and thermal imaging whilst receiving an infusion of Glucagon at a dose of 50ng/kg/min. They were situated in an ambient temperature of 22-25 degrees celsius. arm 7: Visit 1 - Each participant had calorimetry testing and thermal imaging whilst receiving an infusion of gelofusine and wearing a cooling vest.

Visit 2- Each participant had calorimetry testing and thermal imaging whilst receiving an infusion of Glucagon at a dose of 50ng/kg/min. They were situated in an ambient temperature of 22-25 degrees Celsius.

Visit 3 - Each participant had calorimetry testing and thermal imaging whilst receiving an infusion of gelofusine. They were situated in an ambient temperature of 22-25 degrees Celsius. arm 8: Visit 1- Each participant had calorimetry testing and thermal imaging whilst receiving an infusion of Glucagon at a dose of 50ng/kg/min. They were situated in an ambient temperature of 22-25 degrees Celsius.

Visit 2 Each participant had calorimetry testing and thermal imaging whilst receiving an infusion of gelofusine. They were situated in an ambient temperature of 22-25 degrees Celsius.

Visit 3- Each participant had calorimetry testing and thermal imaging whilst receiving an infusion of gelofusine and wearing a cooling vest. arm 9: Visit 1 Each participant had calorimetry testing and thermal imaging whilst receiving an infusion of gelofusine. They were situated in an ambient temperature of 22-25 degrees Celsius.

Visit 2- Each participant had calorimetry testing and thermal imaging whilst receiving an infusion of gelofusine and wearing a cooling vest.

Visit 3 Each participant had calorimetry testing and thermal imaging whilst receiving an infusion of Glucagon at a dose of 50ng/kg/min. They were situated in an ambient temperature of 22-25 degrees Celsius
Interventions: intervention 1: Period 1 Visit 1 - Cold PET-CT Vehicle, Visit 2 -Warm PET-CT Vehicle intervention 2: Period 1 Visit 1 - Cold PET-CT Vehicle, Visit 2 -Warm PET-CT Glucagon intervention 3: Period 1 Visit 1 - Cold PET-CT Vehicle, no visit 2 as BAT negative intervention 4: Period 2 - Visit 1 Warm Control vehicle, Visit 2 Warm Glucagon, Visit 3 Cold control intervention 5: Period 2 - Visit 1 Warm Glucagon, Visit 2 Cold control , Visit 3 Warm control intervention 6: Period 2 -Visit 1 cold control, Visit 2 warm control, Visit 3 Warm glucagon intervention 7: Period 2 -Visit 1 cold control, visit 2 warm glucagon, visit 3 warm control intervention 8: Period 2- Visit 1 Warm glucagon, visit 2 warm control, visit 3 cold control intervention 9: Period 2 - Visit 1 Warm control, visit 2 cold control, visit 3 warm glucagon
Intervention descriptions: intervention 1: Temperature intervention 2: Hormone intervention 3: Temperature intervention 4: Hormone intervention 5: Hormone intervention 6: hormone intervention 7: hormone intervention 8: hormone intervention 9: Hormone

Protocol documents:
Prot_000.pdf: 
1 
 
Ref number 13/LO/0925  Version2Date08/11/2013 
 
 
 
 
 
 
 
 
Investigating Brown Adipose Tissue Activation in Humans 
 
 
 
 
Study Protocol 
 
Ref number 13/LO/0925 
 
Date29/05/2014 
 
Version 3 
 
 
NCT ref: NCT01935791 
 
 
 
 


 
 
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Ref number 13/LO/0925  Version3Date27/05/2014 
 
Section of Investigative Medicine 
Division  of  Diabetes,  Endocrinology  and 
Metabolism 
Imperial College London 
6th Floor Commonwealth Building 
Imperial College London at Hammersmith 
Campus 
Du Cane Road, London W12 ONN, UK 
Tel: +44 (0)20 8383 3242   
Fax: +44 (0)20 8383 8320 
 
 
 
 
Study Protocol 
Ref number 13/LO/0925 
Date29/05/2014 
Version 3 
 
 
TITLE 
 
Investigating Brown Adipose Tissue Activation in Humans 
 
 
BACKGROUND 
 
Obesity is a leading cause of premature morbidity and mortality in the UK and worldwide.  Recent UK 
Government figures state that 65% of men, 58% of women and 28% of children in the England are 
overweight or obese1.   Calorie restriction and increased exercise are effective but difficult to sustain.  
The only therapeutic options currently available are anti-lipid absorption medication (which is largely 
ineffective) and bariatric surgery (known as the “gastric bypass” andassociated with significant risks).  
There is therefore an urgent need for other anti-obesity therapies to be investigated and developed. 
 
Gut hormones hold great promise as potential agents to treat obesity.  Studies of gut hormones infused 
into  human  volunteers  have  demonstrated  that  they  reduce  appetite,  induce  early  satiety  (the 
sensation of feeling full) and increase energy expenditure (the quantity of energy used by the body)2,3.  
We have also shown that the sustained weight loss achieved following bariatric surgery is mediated in 
part through an elevation in the patients’ own gut hormones
4. There is emerging evidence that the 
appetite-altering effects of gut hormones are mediated in the brain5.  However, whilst the elevation in 
energy expenditure induced by gut hormones can be reliably and safely measured in humans using 
indirect calorimetry3, the mechanism by which they actually exert this effect in humans remains unclear. 
 
Depots of functioning brown adipose tissue (BAT) have recently been identified in adult humans
6-8.  BAT 
is likely to be an importanteffector by which circulating hormones increase energy expenditure, due to 
its  unique  ability  to  dissipate  metabolic  energy  (from  ingested  food  and  fat  stores)  as  heat  (BAT 
thermogenesis).  Studies in rodents indicate that gut hormone administration increases BAT mass and 
thermogenesis9,10.  However, the effect of gut hormones on BAT activity in humans is unknown. 
 
Combined positron emission tomography (PET) and computed tomography (CT) scanning is currently 
the standard technique for assessing BAT activity in humans.  Using 18-flurodeoxyglucose ( 18F-FDG, a 
radioactive glucose analogue which is avidly taken up by thermogenically active brown adipocytes), PET-
CT has identified BAT activation in response to both cold temperature and insulin infusion 7.There are 
two weaknesses with PET-CT for BAT imaging.  The first is that it is unable to visualise BAT unless it is 


 
 
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stimulated.  This is because on a CT scan, brown and white fat stores look identical.  Only when brown 
fat is stimulated (usually by exposing the volunteer to cold temperatures) does brown fat take up more 
radioactive tracer and become identifiable on a PET-CT scan. Secondly, these scans involve exposre to 
ionising radiation. 
 
For these reasons other imaging modalities are being investigated.  The first is magnetic resonance 
imaging. By taking advantage of small differences in water content between the two types of fat, MRI 
is being developed to detect BAT deposits within WAT, even in the unstimulated state 13,14.   Secondly, 
infrared thermal imaging cameras can be used to safely and non-invasively measure the difference in 
temperature of BAT compared with surrounding tissues.  Activated BAT releases energy (in the form of 
heat) and thus activated BAT will be at a higher temperature than surrounding non-activated BAT tissue.  
Recent  data  suggests  that  thermal  imaging  can  be  used  to  assess  BAT  activation  in  humans  aged 
between  3-58  years12  in  response  to  cold  temperature  stimulation.  However  there  are  no  studies 
validating the technique of thermal imaging or Magnetic Resonance Imaging (MRI)for measurement of 
BAT with the gold standard technique of PET-CT. 
 
 
OBJECTIVES 
 
This project aims to investigatebrown adipose tissue activation in humans. To do this we will: 
 
1. 
assess  BAT  activation  in  response  to  glucagon  infusion  in  healthy  volunteers  using  PET-CT,  
thermal imagingand MRI; 
2. 
utilise  thermal  imaging  to  assess  BAT  activation  in  clinical  scenarios  and  conditions  where 
increased BAT activation is anticipated. 
 
 
GENERAL METHODOLOGY STATEMENT 
 
Intravenous  infusions  by  the  Section  of  Investigative  Medicine  follow  protocols  established  after 
extensive experience investigating the physiological role of numerous peptide hormones in humans. 
Single  and  combinationinfusion  of  gut  hormones  have  been  performed  on  large  cohorts  of  human 
volunteers within this department without adverse event (ethics refs: 2002/6261 and 09/H0707/76).  
 
This healthy human volunteer investigation will comply in full with the standard procedures that were 
developed  at  the  behest of  the  RPMS  ethics  committee.    This  followed  extensive  discussion  with a 
number  of  experts  and  their  purpose  is  to  ensure  safety  and  efficacy  in  peptide  preparation  and 
administration.  The procedures used in this study will be the same as those used in previous studies 
carried out by our group (ethics refs: 2002/6261 and 09/H0707/76). 
 
Indirect calorimetry is a well-established technique in our laboratory.  The calorimeter captures expired 
air from a large, transparent canopy, worn over the head and thorax, and is typically very well tolerated.  
Thermal imaging  remotely measures the emission of infrared radiation from the surface of the skin 
using a small camera, does not involve ionizing radiation, and therefore does not expose the subjects 
to any additional risks.    
 
18F-FDG  PET-CT  imaging  is  a  well-established  methodology  and    theImanovagroup  has  conducted  a 
number of similar studies over the last 6 years. While PET studies involve additional ionizing radiation 
exposure for the participants, the levels of exposure from this study are in line with other experimental 
protocols conducted in healthy volunteers. We are proposing a T2* (echo planar) MR scanning protocol 
in a 3T scanner to perform fat extraction and MRI thermography.  Magnetic resonance imaging is safe 
and widely used in clinical practice.  It does not involve ionizing radiation. 


 
 
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METHODFOR OBJECTIVE 1 
 
To assess BAT activation in response to glucagon infusion in healthy volunteers using PET-CT, thermal 
imagingand MRI. 
 
We have chosen one gut hormone, glucagon, to investigate using both thermal imaging, PET-CTand MRI.  
We  have  previously  shown  that  glucagon  induces  a  large  rise  in energy expenditure  (EE)  in  healthy 
volunteers  without  any  side  effects3.    To  minimise  radiation  exposure,  only  volunteers  who  have  a 
demonstrable rise in energy expenditure (EE) in response to glucagon infusion will  undergo PET-CT 
scans. 
 
Recruitment 
 
Healthy  male  volunteers  will  be  recruited  via  posters,  newspaper  advertisements and/or  e-mail 
distribution  lists.    Initially,  the  volunteer  will  be  asked  to  contact  the  investigators.    After  a  brief 
telephone  call  to  assess  that  the  volunteer  broadly  fits  the  inclusion  criteria  (age,  weight,  current 
illnesses), they will be invited to attend a full screening visit. Based on data from previous studies of gut 
hormone infusions carried out by our group, and the existing literature on 18F-FDG PET-CT imaging of 
BAT,  we estimate that  we will need to recruit n=20 volunteers (with n=16 needed to complete this arm 
of the study). 
 
 
Screening Visit 
 
All volunteers will undergo medical history taking and examination.  Inclusion and exclusion criteria are 
detailed  below.    Basic  investigations  performed  prior  to  inclusion  will  comprise  height  and  weight 
measurement, blood tests (full blood count, urea and electrolytes, liver function tests, thyroid function 
tests and plasma glucose) and an electrocardiogram.   
 
Informed consent is a crucial part of the study.  All of the research doctors undertaking this process are 
MRCP qualified and experienced in taking informed consent for human research studies according to 
GCP guidelines.  They will also have undertaken radiation protection courses, and will have sufficient 
expertise to discuss the ionising radiation aspects of the study with volunteers. 
 
Inclusion criteria for healthy volunteers for Objective 1 
 
Age 18-40 years* 
 
Male  
 
Healthy as determined by screening history and investigations detailed above. 
 
Body mass index 18-25 kg/m2 and a stable weight for 3 months. 
 
Exclusion criteria for healthy volunteers for Objective 1 
 
History of alcoholism or substance abuse within the last 5 years. 
 
History of major medical or psychiatric disease.  
 
Any other condition, including social circumstances, deemed likely to interfere with the ability to 
participate reliably in the trial. 
 
History of hypersensitivity to any of the components of the infusions. 
 
Treatment with an investigational drug within the preceding 2 months. 
 
Treatment with any drug that is known to stimulate or inhibit BAT activation. 
 
Volunteers who have donated, or intend to donate, blood within three months before or following 
study completion. 
 
Claustrophobia. 


 
 
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 
Pacemaker, metal implant, clips, implanted device, shrapnel or bullet, metal in eyes that precludes 
magnetic resonance imaging. 
 
Significant structural abnormality on brain scan. 
 
Cumulative radiation exposure in the preceding 12 months totalling 10mSv or more. 
 
*The lifetime risk for a healthy adult of developing a fatal malignancy following exposure to ionising 
radiation similar to that expected in our study is approximately 1:1800. This is low compared with the 
average UK cancer mortality rate of about 1 in 4, leading to a marginal added risk due to the study 
participation. However, higher BAT activation is expected in young adults
11.  We have therefore decided 
to enrol young adults in our study, in order to be able to demonstrate the principle of glucagon induced 
BAT activation in a small pilot study. Thus, while the risk e risk remains very small but is slightly greater 
in younger subjects.  Given that only those subjects who have demonstrated significant rises in EE in 
response to glucagon would be put forward for PET-CT scanning, we expect to be able to achieve the 
aims of our study with a minimal number of subjects exposed to additional ionising radiation.   
 
Study Visitsfor Objective 1 
 
After the screening visit, volunteers will be invited to attend 7 study visits.  There will be a time interval 
of at least two days between visits.    
 
During Visit 1, 2 and 3, volunteers will receive an intravenous infusion of (A) saline under thermoneutral 
conditions,  (B)  saline  with  cold  stimulationor(C)  glucagon,  allocated  in  a  randomised  single-blinded 
fashion.      Resting  and  stimulated  energy  expenditure  will  be  measured  with  a  standard  indirect 
calorimeter (as previously used successfully in this laboratory3). Supraclavicular deposits of BAT will be 
imaged using infrared thermography (i.e. thermal imaging). 
 
 
If the volunteers have demonstrated a measurable rise in EE or BAT activation during these first 3 study 
visits, they will be asked to complete Visits4to 7.  During  Visit 4 and Visit 5, volunteers will receive an 
intravenous infusion of (A) saline or(B) glucagon, allocated in a randomised single-blinded fashion, and 
undergo a 60-minute dynamic FDG PET-CT scan.During Visit 6 and Visit 7, volunteers will receive an 
intravenous infusion of (A) saline  (with cold stimulation)  or (B) glucagon, allocated in a randomised 
single-blinded fashion, and undergo a 60-minute MRI scan. 
 
 
Details of Study Visit Preparation, Blood Sampling, GlucagonInfusion, and Cold Stimulation. 
 
To minimise the usual hepatic glycogen depletion that occurs during an overnight fast, participants will 
be asked to avoid alcohol and strenuous exercise for 24 hours prior to each study visit, and to consume 
a high energy snack as late as possible on the evening before each visit, in addition to their normal 
dietary intake.  When they arrive on the ward in the morning, two pink (20G) intravenous cannulaewill 
be inserted – one in each arm. 
 
The infusion rate will be calculated according to body surface area, with the dose of glucagon based on 
our previous published experience of administering the hormone 3.  The expected maximum glucagon 
dose will be up to an equivalent of 100 ng/kg/min.  To minimise peptide adsorption to syringes and 
infusion lines, the vehicle for intravenous infusions will be Haemaccel, or an equivalent safe 
colloid,diluted with 0.9% saline.   The infusion will last 90 minutes.  During the course of each study visit 
the volunteer will undergo serial venous blood samples.  There will be a maximum of ten 8 ml samples 
taken per visit.  These will be tested for circulating hormones, glucose and other metabolites. Over the 
course of the seven studyvolunteers will donate no more than a total of 560ml of blood.  At regular time 
points, the volunteers will also be asked about their general wellbeing.  Heart rate and blood pressure 
will be measured periodically using conventional ward equipment. 


 
 
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BAT in humans has been reliably demonstrated to be activatedby exposure to cold temperatures.  Thus, 
cold exposure can be used as a positive control method.  We will do this by placing the volunteers’ 
hands in a bowl of cool water or using a cooling vestfor the duration of the infusion.  We will ensure 
that the volunteer does not become uncomfortably cold, since we specifically want to avoid 
shivering.The cooling vest will be a commercially available unit that can be comfortably worn on the 
torso (as pictured below).  The vest has integrated tubing through which cold water can be pumped to 
cool the volunteer.  Temperatures can be accurately controlled using a thermostat on the water pump, 
and we are aiming to cool  the volunteer comfortably.   If  the volunteer starts to shiver  or is 
uncomfortable will will raise the temperature or remove the vest immediately. 
 
 
 
 
Details of Indirect Calorimetry 
 
On Visits 1, 2 and 3, volunteers will undergo indirect calorimetry to measure energy expenditure in 
response to glucagon or saline infusion.  After arrival and insertion of the intravenous cannulae (but 
prior to the start of the intravenous infusion), participants will be placed inside the comfortable canopy 
of thecalorimeter, whilst reclining on a bed.  
 
The calorimeter captures expired air from a large, transparent canopy, worn over the head and thorax, 
and is typically very well tolerated.  Each participant will be asked to lie in a semi-recumbent position 
under the canopy.  Once the carbon dioxide content of the air entering the chamber has stabilised, 
measurements will be taken for 20 minutes in order to assess the participants’ basal metabolic rate 
(BMR) and respiratory quotient (RQ).A second set of measurements will be taken 60 minutes after the 
start of the intravenous infusion (for a period of 20 minutes) to assess the effect of the infusion on the 
participants’ metabolic rate.  
 
 
 
 
Details of Thermal Imaging 
 
Thermal images will be obtained during Visits 1, 2 and 3 after the periods of indirect calorimetryhave 
been completed. The area of the body that will be imaged (in a single frame) will include the lower 
head, neck and upper thorax. Volunteers will be asked to wear a vest style T-shirt on the day of the 
study in order to facilitate this, and an additional wrap will be provided for modesty. The volunteer will 
sit facing the thermal imaging camera (FLIR T440bx) mounted on a tripod. Participants will sit still in an 
upright position with arms adducted, shoulders relaxed and horizontal for the duration of the imaging 
to ensure image quality. They will be seated away from direct heat sources and open windows.  Room 
temperature will be maintained between 19-23OC. 


 
 
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The thermal imaging camera will be positioned 1.0 – 1.6 m from the chair on which the volunteer is 
seated.  The  optimal  distance  for  image  acquisition  will  be  the  distance  at  which  the  volunteer’s 
shoulders fill the camera-viewing screen. The distance from the chair to the camera will be recorded. 
Three  consecutive  images  will  be  taken  of  a  temperature  controlled  block  in  order  to  calibrate  the 
images (i.e. these will be control images).  
After  the  acclimatisation  period  and  acquisition  of  control  images,  study  images  will  be  acquired 
continuously for 10 minutes prior to the start of the infusion.  End-infusion study images will be acquired 
continuously during the last 10 minutes of the infusion. The visit protocol for Visits 1, 2 and 3 is outlined 
in Figure 1. 
From each thermal image stream, using existing methodology12, the maximal temperature over defined 
regions of interest in the neck region will be recorded. Data will be analysed using Prism. 
 
 
Figure 1 – Visit Protocol for Study Visits 1, 2 and 3 for Healthy Volunteers 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Details of PET-CT 
Volunteers who have demonstrated rises in EE or BAT activation in response to glucagon infusion over 
Visits 1-3 will be invited to attend Visits 4 and 5, to measure BAT activation using PET-CT at the Imanova 
Imaging Unit in Hammersmith Hospital. The study timetable for Objective 1 is shown in Figure 2. 
 
Positron emission tomography (PET),has become a routine research tool over the past 25 years.  We 
will use 18-flurodeoxyglucose (18F-FDG), an analogue of glucose to measure the metabolic rate of BAT. 
Metabolically  active  brown  adipocytes  avidly  take  up  glucose,  and  are  expected  to  increase  their 
metabolic rate, and therefore the uptake of both glucose and 18F-FDG following stimulation with cold 
or glucagon.  18F-FDG has been previously used to study BAT activation with PET6-8.  Dynamic PET data 
will be collected from  the upper torso and neck will be acquired, areas known to contain significant 
amounts of BAT. In order to quantify the PET data adequately, a low-dose CT scan of the upper torso 
and chest will be conducted prior to each PET scan, in order to estimate tissue attenuation of the PET 
signal.  The  ionizing  radiation  exposure  from  the  two 
18F-FDG  PET  scans  (maximal  administration  of 
185MBq  per scan)  and  two  low-dose  CT  scans of  the  upper  torso,  is expected  to  be  approximately 
9.5mSv.  
 
 At each PET-CT visit two intravenous cannulae will be inserted into the forearm or cubital veins in order 
to administer the 18F-FDG and glucagon or saline infusion (via one cannula), and collect blood samples 
during the course of the PET-CT scans (via the second cannula). The glucagon or saline infusion will be 
initiated and then the volunteers will be then be led into the scanner room where the 18F-FDG bolus 
will be is administered immediately prior to the scan.  Subjects will undergo a brief low dose CT followed 
by  a  60-minute  dynamic  PET  scan.    Volunteers  will  be  lying  on  a  bed  throughout  the  scan  and  can 
communicate with (and will be reassured by) the radiographer and researchersas necessary. 
Indirect 
calorimetry 
Indirect 
calorimetry 
Volunteer arrives & 
insertion of 2 
intravenous cannulae 
End of infusion  
± cold 
stimulation  
Volunteer 
departs 
Time  relative  to  start 
of infusion(min) 
-40 
-20 
0 
60 
 
80 
 
 
Acclimatisation 
period 
-10 
Acquisition  
of thermal 
images 
Start of 
infusion ± cold 
stimultation 
90 
Acquisition  
of thermal 
 images 


 
 
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Images will be quantified by deriving standard outcome parameters for the defined regions of interest, 
using established methodology.  All of the PET-CT scans will also be formally reported by a consultant 
radiologist. Although the PET-CT scans are not performed with the aim of clinical diagnosis, and this 
may  not  be  considered  of  diagnostic  quality,  they  may  reveal  an  incidental  finding  relevant  to  the 
subjects health status.  In this situation the report would be communicated to the volunteer and the 
volunteer’s GP, who would arrange the appropriate specialist referral.  A significant abnormality would 
exclude that volunteer from further participation in the study.   
 
Figure 2 – Study Timetable for Objective 1 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Details of MRI 
Volunteers who have successfully completed visits 1-5 will also be invited to attend Visits 6 and 7.  The 
objective  of  these  visits  is  to  measure  BAT  activation  using  MRI  at  the  Imanova  Imaging  Unit, 
Hammersmith Hospital. 
 
Magnetic Resonance Imaging (MRI), has become a routine clinical and research tool
13,14.  Our 60 minute 
scanning  protocol  is  designed  to  firstly  differentiate  BAT  from  WAT  in  the  resting  (baseline)  state 
(utilizing differences in their relative water contents) and then to measure BAT temperature change 
following stimulation with either cold or glucagon infusion.  MRI data will be collected from the upper 
torso and neck, areas known to contain significant amounts of BAT. MRI does not involve the use of 
ionizing radiation.  
 
 At each MRI visit two intravenous cannulae will be inserted into the forearm or cubital veins.  One will 
be used to administer the glucagon or saline infusion and the other for blood sampling (as in all previous 
study visits). Volunteers will be lying on a bed throughout the scan and can communicate with (and will 
be reassured by) the radiographer and researchers as necessary. 
 
All of the MRI scans will also be formally reported by a consultant radiologist. Although the MRI scans 
are not performed with the aim of clinical diagnosis, and thus may not be considered of diagnostic 
quality, they may reveal an incidental finding (not previously picked up on the CT scan) relevant to the 
subjects health status.  In this situation the report would be communicated to the volunteer and the 
volunteer’s GP, who would arrange the appropriate specialist referral.  A significant abnormality would 
exclude that volunteer from further participation in the study. 
 
OBJECTIVE 1:  To assess BAT activation in response to glucagon infusion in healthy volunteers using 
PET-CT and thermal imaging. 
 
N=16 healthy male volunteers to complete this arm 
 
NB. -       Visits 1-3 will be allocated in a random order. 
- 
Visits 4 to 7 will only be undertaken if there has been a demonstrable response to glucagon 
infusion over Visits 1-3. 
- 
All infusions will be single-blinded. 
Visit 1 
Thermoneutral 
Thermal imaging 
Calorimetry 
Saline infusion 
Visit 2 
Cold stimulus 
Thermal imaging 
Calorimetry 
Saline infusion 
 
Visit 3 
Thermoneutral 
Thermal imaging 
Calorimetry 
Glucagon infusion 
 
Visit 4 
Cold stimulus 
PET-CT 
Saline infusion 
(or 
glucagon 
infusion) 
 
Visit 5 
Thermoneutral 
PET-CT  
Glucagoninfusion (or 
saline infusion)   
Visit 6 
Cold stimulus 
MRI 
Visit 7 
Thermoneutral 
MRI 
Glucagon 
infusion 


 
 
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METHOD FOR OBJECTIVE 2 
 
To utilise thermal imaging to assess BAT activation in clinical scenarios  
 
Utilising  indirect  calorimetryand  thermal  imaging,  we  will  investigate  energy  expenditure  and  BAT 
activation in patients with conditions  which are rationally associated with a potential rise in energy 
expenditure and/or BAT activation for example bariatric surgery, phaeochromocytoma and 
thyrotoxocosis. 
 
Recruitment 
 
Patients  will  be  recruited  from  Outpatient  Clinics  in  Imperial  College  NHS  Healthcare  Trust  by  the 
researchers.  We  aim  to  recruit  n=30  patientsfor  each  one  of  the  following  conditions  of  interest: 
bariatric surgery, phaemochromocytoma, thyrotoxicosis and neuroendocrine tumours.  Participation in 
the study will not delay or influence the clinical management of the patient and their conditions. 
 
Screening Visit 
 
All volunteers will undergo medical history taking and examination.  Inclusion and exclusion criteria are 
detailed  below.    Basic  investigations  performed  prior  to  inclusion  will  comprise  height  and  weight 
measurement, blood tests (full blood count, urea and electrolytes, liver function tests, thyroid function 
tests and plasma glucose) and an electrocardiogram.  Where other blood tests have been performed as 
part of routine clinical care, these results may be accessed by the clinical research team. 
 
Informed consent is considered a crucial part of the study.  All of the research doctors undertaking this 
process are medicallyqualified and experienced in taking informed consent for human research studies 
according to GCP guidelines. 
 
 
Inclusion criteria for patients with conditions of interest 
 
Age >18 years 
 
Diagnosed with a condition known to be associated with prolonged exposure to endogenously 
elevated gut hormones or likely to be associated with increased energy expenditure and/or BAT 
activation. 
 
Exclusion criteria for patients with conditions of interest 
 
 
As for Objective 1, but allowing for the primary condition of interest. 
 
Pregnancy. 
 
Patients with conditions of interests visits 
 
Each patient will attend their first study visit on the same day they are recruited from an Outpatient 
Clinic.  Up to 5 subsequent follow-up visits will then be scheduled to investigate the effect of treatment 
of the underlying condition over time on EE and/or BAT activation. Therefore each patient will be invited 
to attend a maximum of 6 study visits.   
 
Each  study  visit  will  consist  of  a  single  20  ml  blood  sample,  thermal  imaging  (with  or  without  cold 
stimulation, as described above) for up to 30 minutes and indirect calorimetry (as described above) for 


 
 
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up to 60 minutes. Premenopausal females will be asked to take a urine pregnancy test at each study 
visit to exclude pregnancy. The participants with medical conditions of interest will not have intravenous 
cannulae inserted. They will not be subjected to any gut hormoneor saline infusions, and they will not 
have any PET-CT scans as part of this study.However if the patient has one or more PET-CT scans as part 
of their routine clinical care, then analysis of the scan(s) will be included in the study providing the 
region of interest (supraclavicular area and neck) is included in the scan(s).  
 
 
The venous blood sample taken at each study visit will be tested for gut hormones, pituitary hormones, 
thyroid hormones, glucose and other metabolites.  Over the course of their participation in this study, 
each patient will donate no more than a total of 150ml of blood. 
 
 
OUTCOME MEASURES 
 
1. Change in BAT signal between the baseline and stimulated state in response to glucagon infusion, 
as detected by PET-CT, thermal imagingand MRI. 
2. BAT activation in different pathological states pre- and post-treatment. 
 
 
SAFETY AND WELLBEING OF THE VOLUNTEERS 
 
Over the last 25 years, the Department of Metabolic Medicine has infused or injected synthetic human 
gut hormone peptides (singly or in combination) to a large number of volunteers with no untoward side 
effects at the doses that will be used in this study.   
 
Every effort will be made to ensure the volunteer’s comfort throughout each study visit.  At all times, 
the volunteers will be in direct communication with the investigators.  They may press an alarm bell to 
request immediate cessation of the procedure and withdrawal from the PET-CT scanner.  If the volunteer 
is unable to cope with the scan, from the point of view of lying still for the required time or because of 
claustrophobia, they will be excluded from further participation.  Subjects will be free to discontinue 
their participation at any time. 
 
The maximum number of PET-CT scans a volunteer will have in this study  is 2 PET-CT scans, and only 
those volunteers with significant response to glucagon infusion in Visits 1-3 for Objective 1 (i.e. up to 
16 volunteers) will have PET-CT scans.  Since the total radiation exposure will not exceed 9.5mSv, the 
estimated risk of developing a fatal cancer as a result of having 2 PET-CT scans in this study  is 1 in 1800 
for a healthy adult.  This is much lower than the UK cancer mortality rate of approximately 1 in 4. 
 
At least one experienced physician will be present at all times during the study visits and subjects will 
be encouraged to report any unusual or unpleasant sensations immediately.  During the entire course 
of the study, a physician will be available 24 hours on a direct line, which will be given to all volunteers.  
A second physician will be on back-up call. 
 
The most common minor side effect (although still rare) of glucagon infusion is mild nausea.  This effect 
is even less likely to occur in this study, which proposes to use doses that produce physiological post-
prandial  plasma  levels  in  fasted  subjects.    If  nausea  does  occur,  it  wears  off  very  quickly  when  the 
infusion is discontinued, because this peptide hormone is significantly metabolised within minutes.  If 
a volunteer experiences this side effect then no further infusions of glucagon will be administered to 
that volunteer.  
 
 
 


 
 
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ADVERSE EVENTS 
 
An adverse event is any untoward medical occurrence in a patient or clinical study subject. A serious 
adverse event is an adverse event that results in death, is life-threatening, requires hospitalization or 
prolongs an existing inpatient’s hospitalization, results in persistent or significant disability or incapacity, 
or is a congenital anomaly or birth defect.  All non-serious adverse events will be recorded in the Trial 
Master File. If a serious adverse event occurs, a serious adverse event form will be completed and sent 
to  the  Chief  Investigator  and  the  Sponsor  within  24  hours.  All  serious  adverse  events  that  are 
unexpected and related to the study will be reported to the Local Research Ethics Committee. Serious 
adverse events will be reported as required by their Local Research Ethics Committee, Sponsor and/or 
Research & Development Office. 
 
Although no serious adverse effects are anticipated, any such effect will terminate the arm of the study 
during which the serious adverse effect occurred. 
 
 
 
DATA HANDLING 
 
The Chief Investigator will preserve the confidentiality of participants taking part in the study and is 
registered under the Data Protection Act.Personal contact details are required for communication with 
the subjects participating in the study. This information is held solely for communication between the 
researchers and participants. Details stored on university computers will be password protected and 
will only be accessed by the researchers involved in the study.  Personal identifiers will be deleted 6 
months after the participant has completed their involvement in the study. 
 
Information held on NHS computers is solely for the purpose of hospital booking and routine sample 
collection and analysis (e.g. for medical screening). This information is password protected in a similar 
manner to that of other hospital patients.  
 
Subjects will be given a personal study code number which will be used throughout the study and in 
the analysis of data. Coded samples or images will also be treated with confidentiality when undergoing 
analysis.    Analysis  will  take  place  in  the  Imanova  Clinical  Imaging  Centre  and  the  Department  of 
Investigative Medicine at Hammersmith Hospital and be carried out by the researchers themselves. The 
data  will  be  kept  in  a  secure  environment  in  these  departments,  under  the  authority  of  Professor 
Bloom. Data will be stored for 15 years after completion of the study. Only the researchers will have 
access to this data. 
 
 
REGULATORY COMPLIANCE 
 
The Chief Investigator has obtained approval from the xxx Research Ethics Committee. The study has 
been submitted for Site Specific Assessment (SSA) at each participating NHS Trust. The Chief Investigator 
will obtain a copy of the Trust R&D approval letter before accepting participants into the study. The 
study will be conducted in accordance with the recommendations for physicians involved in research 
on human subjects adopted by the 18th World Medical Assembly, Helsinki 1964 and later revisions.  
 
Consent to enter the study will be sought from each participant only after a full explanation has been 
given, an information leaflet offered and time allowed for consideration. Signed participant consent will 
be  obtained.  The  right  of  the  participant  to  refuse  to  participate  without  giving  reasons  will  be 
respected.  After the  participant  has  entered  the  study  the  clinician  remains  free to  give  alternative 
treatment to that specified in the protocol at any stage if he/she feels it is in the participant’s best 
interest, but the reasons for doing so should be recorded. In these cases the participants remain within 


 
 
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the study for the purposes of follow-up and data analysis. All participants are free to withdraw at any 
time from the protocol treatment without giving reasons and without prejudicing further treatment.  
 
 
The  study may  be  subject  to  inspection  and  audit  by  Imperial  College  London under  their  remit  as 
sponsor and other regulatory bodies to ensure adherence to GCP and the NHS Research Governance 
Framework for Health and Social Care (2nd edition).  
 
 
 
FUNDING AND INDEMNITY 
 
The study will be funded internally by the Imanova PET Pilot Study Grant (awarded in November 2012) 
and the Section of Investigative Medicine, Division of Diabetes, Endocrinology and Metabolism. Healthy 
volunteers will be paid expenses of up to £100 per study visit and patients with conditions of interest 
will be paid expenses of up to £75 per study visit. 
 
Imperial College London will act as the main Sponsor for this study. Delegated responsibilities will be 
assigned to the NHS trust taking part in this study.Imperial College London holds negligent harm and 
non-negligent harm insurance policies which apply to this study, and Imperial College Healthcare NHS 
Trust holds standard NHS Hospital Indemnity and insurance cover with NHS Litigation Authority for NHS 
Trusts  in  England,  which  apply  to  this  study  (as  both  the  healthy  volunteers  and  the  patients  with 
conditions of interest will be registered as patients of Imperial College Healthcare NHS Trust). 
 
 
References 
 
1. 
https://catalogue.ic.nhs.uk/publications/public-health/obesity/obes-phys-acti-diet-eng-2013/obes-phys-acti-
diet-eng-2013-rep.pdf Accessed on 9 April 2013. 
2. 
Flint A, Raben A, Astrup A, Holst JJ.  Glucagon-like peptide 1 promotes satiety and suppresses energy intake in 
humans. J Clin Invest 1998;101:515-520 
3. 
Tan TM, Field BCT, McCullough KA, Troke RC, Chambers ES, Salem V, Maffe JG, Baynes KCR, De Silva A, Viardot 
A,  Alsafi  A,  Frost  GS,  Ghatei  MA,  Bloom  SR.    Coadministration  of  glucagon-like  peptide-1  during  glucagon 
infusion  in  man  results  in  increased  energy  expenditure  and  amelioration  of  hyperglycaemia.  Diabetes 
2013;62:1131-1138 
4. 
Beckman LM, Becckman TR, Sibley SD, Thomas W, Ikramuddin S, Kellogg TA, Ghatei MA, Bloom SR le Roux CW, 
Earthman  CP.  Changes  in  gastrointestinal  hormones  and  leptin  after  Roux-en-Y  gastric  bypass  surgery.  J 
Parenter Enteral Nutr 2011;35:169-180 
5. 
De Silva A, Salem V, Long CJ, Makwana A, Newbould RD, Rabiner EA, Ghatei MA, Bloom SR, Matthews PM, 
Beaver JD, Dhillo WS. The gut hormones PYY 3-36 and GLP-1 7-36 amide reduce food intake and modulate 
brain activity in appetite centers in humans. Cell Metab 2011;12:700-706 
6. 
Virtanen KA, Lidell ME, Orava J, Heglind M, Westergren R, Niemi T, Taittonen M, Laine J, Savisto N-J, Enerback 
S, Nuutila P.  Functional brown adipose tissue in healthy adults. N Engl J Med 2009;360:1518-1525 
7. 
Orava J, Nuutila P, Lidell ME, Oilonen V, Noponen T, Viljanen T, Scheinin M, Taittonen M, Niemi T, Enerback S, 
Virtanen KA. Different metabolic responses of human brown adipose tissie to activation by cold and insulin.  
Cell Metabolism 2011;14:272-279 
8. 
Cypess AM, Chen Y-C, Sze C, Wang K, English J, Chan O, Holman AR, Tal I, Palmer M, Kolodny GM, Kahn CR.  
Cold but not sympathomimetics activates human brown adipose tissue in vivo.  ProcNatlAcadSci 
2012;109:10001-10005 
9. 
Billington CJ, Bartness TJ, Briggs J, Levine AS, Morley JE. Glucagon stimulation of brown adipose tissue growth 
and thermogenesis. Am J Physiol 1987;252:R160-R165 
10. 
Billington  CJ,  Briggs  JE,  Link  JG,  Levine  AS.    Glucagon  in  physiological  concentrations  stimulates  brown  fat 
thermogenesis. Am J Physiol 1991;261:R501-R507 
11. 
Nedegaard J, Bengtsson T, Cannon B. Unexpected evidence for active brown adipose tissue in adult humans. 
Am J PhysiolEndocrinolMetab 2007;293:E444-E452 
12. 
Symonds ME, Henderson K, Elvidge L, Bosman C, Sharkey D, Perkins AC, ,Budge H.  Thermal imaging to assess 
age-related  changes  of  skin  temperature  within  the  supraclavicular  region  co-locating with  brown  adipose 
tissue in health children. J Pediatr 2012;161:892-898 


 
 
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13. 
Hu HH, Smith DL Jr, Nayak KS, Goran MI, Nagy TR. Identification of brown adipose tissue in mice with fat-water 
IDEAL-MRI. J MagnReson Imaging 2010;31:1195-1202 
14. 
Chen Y-CI, CypessAM, Chen Y-C, Palmer M, Kolodny G, Kahn CR, Kwong KK. Measurement of human brown 
adipose tissue volume and activity using anatomic MR imaging and functional MR imaging. J Nucl Med 2013; 
54:1594-1587 

SAP_001.pdf: 
 
Investigating Brown Adipose Tissue Activation in Humans 
Statistical Analysis Plan 
Date: 29/05/2014
MAIN SPONSOR: Imperial College London 
FUNDERS: Medical Research Council (MRC) 
STUDY COORDINATION CENTRE: Imperial College London 
Research Ethics Committee (REC) reference: 13/LO/0925 
NCT No. NCT01935791


Statistical Analysis
Based on previous studies of the hormonal and cold exposure effects on human BAT 
activity 1, 2, we calculated that, for an equivalent rise in EE, a sample size of 4 per 
group (glucagon versus control) would have 95% power to detect a significant 
difference between the effects of cold exposure and glucagon using 18F‐FDG PET/CT 
in confirmed BAT‐positive volunteers. Because of safety considerations, as PET/CT 
scans expose healthy patients in this study to ionising radiation, we limited this study 
to males only and to the smallest possible sample size to answer our initial question 
about the differential effects of cold exposure and glucagon administration on human 
BAT activity for a given rise in EE.
All data were analysed using GraphPad Prism 6 software (GraphPad Software, Inc., 
San Diego, CA, USA). Results are presented as means ± standard error of the 
mean. One‐way repeated measures anova with Tukey's test was used to compare 
differences between the three intervention groups. A p value < 0.05 was considered 
to indicate statistical significance.
REFERENCES
Vosselman MJ, van der Lans AA, Brans B et al.  Systemic beta‐adrenergic stimulation of 
thermogenesis is not accompanied by brown adipose tissue activity in humans. Diabetes  
2012;  61: 3106– 3113
Lahesmaa M, Orava J, Schalin‐Jantti C et al.  Hyperthyroidism increases brown fat metabolism 
in humans. J Clin Endocrinol Metab  2014;  99:  E28– E35.