Total-body Glucose Utilization in Obesity
Phase: PHASE4
Conditions: Obesity and Overweight
Sponsor: Turku University Hospital

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Turku PET Centre 
Turku University Central Hospital 
 
 
 
CLINICAL STUDY PROTOCOL 
 
 
 
Date: 
 
24.5.2023 
 
 
Version: 
 
1.3 
 
Study code:  
TOTAL 
 
Study title: 
Total-body glucose utilization in obesity 
 
 
 
 
Principal investigators: 
 
 
Prof. Lauri Nummenmaa, PhD 
Prof. Pirjo Nuutila, MD, PhD 
Turku PET Centre 
 
Turku PET Centre  
University of Turku 
 
University of Turku and Turku University hospital 
Kiinamyllynkatu 4-8 
 
Kiinamyllynkatu 4-8, 
20520 Turku, Finland 
 
20520 Turku, Finland 
Phone: +358 50 574 7933  
Phone: +358 40 162 6834 
Email:latanu@utu.fi 
 
Email:pirjo.nuutila@utu.fi  
 
 
Responsible Physician:  
Pirjo Nuutila 
Turku PET Centre, University of Turku 
Kiinamyllynkatu 4-8, 
20520 Turku, Finland 
Email:pirjo.nuutila@utu.fi 
 
LIST OF INVESTIGATORS 
 
University of Turku 
Prof. Pirjo Nuutila, MD, MD, PhD  
Prof. Lauri Nummenmaa, PhD 
Assoc. Prof Kirsi Virtanen, MD, PhD 
Prof. Jussi Hirvonen, MD, PhD 
 
 
 
 

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1 
SUMMARY .............................................................................................................................................. 3 
2 
INTRODUCTION ................................................................................................................................. 3 
3 
OBJECTIVES AND PURPOSE ............................................................................................................ 4 
4 
STUDY DESIGN .................................................................................................................................... 4 
5 
PATIENT/SUBJECT SELECTION .................................................................................................... 4 
5.1 
SOURCE POPULATION .............................................................................................................................................4 
5.2 
INCLUSION CRITERIA FOR THE OBESE GROUP ...................................................................................................4 
5.3 
INCLUSION CRITERIA FOR THE CONTROL GROUP .............................................................................................4 
5.4 
EXCLUSION CRITERIA..............................................................................................................................................5 
6 
ASSESSMENTS ...................................................................................................................................... 5 
6.1 
GENERAL STUDY OUTLINE ....................................................................................................................................5 
6.2 
MEASURING TOTAL-BODY GLUCOSE UTILIZATION WITH PET ......................................................................6 
6.3 
PERFUSION IMAGING WITH H2[15]O ...................................................................................................................5 
6.4 
EMOTIONAL REACTIVITY MEASUREMENTS WITH H2[15]O .............................................................................7 
6.5 
LABORATORY MEASUREMENTS .............................................................................................................................7 
6.6 
QUESTIONNAIRES AND COMPUTER-BASED TASKS ............................................................................................8 
7 
ADVERSE EVENTS .............................................................................................................................. 8 
7.1 
DEFINITION OF ADVERSE EVENTS (AES) ..........................................................................................................8 
7.2 
REPORTING OF ADVERSE EVENTS (AES) ............................................................................................................8 
7.3 
EXPEDITED REPORTING ........................................................................................................................................8 
7.4 
EMERGENCIES ..........................................................................................................................................................8 
8 
ETHICS ................................................................................................................................................... 9 
8.1 
ETHICAL CONSIDERATIONS ...................................................................................................................................9 
8.2 
ETHICAL REVIEW .....................................................................................................................................................9 
8.3 
SUBJECT INFORMATION AND INFORMED CONSENT .........................................................................................9 
9 
DATA COLLECTION AND MANAGEMENT .................................................................................. 9 
9.1 
CASE REPORT FORMS ..............................................................................................................................................9 
9.2 
ELECTRONIC DATA COLLECTION .........................................................................................................................9 
9.3 
DATA MANAGEMENT ..............................................................................................................................................9 
10 
DATA ANALYSIS ................................................................................................................................ 9 
11 
STATISTICS ...................................................................................................................................... 10 
11.1 
SAMPLE SIZE....................................................................................................................................................... 10 
11.2 
STATISTICAL ANALYSES ................................................................................................................................... 10 
11.3 
POSSIBLE INTERIM ANALYSES AND STOPPING RULES................................................................................ 10 
12 
QUALITY ASSURANCE .................................................................................................................. 10 
12.1 
INFORMATION OF STUDY PERSONNEL AND TRAINING ............................................................................ 10 
12.2 
PROTOCOL AMENDMENTS .............................................................................................................................. 10 
13 
STUDY SCHEDULE ........................................................................................................................ 10 
14 
CRITERIA FOR PREMATURE STUDY TERMINATION ........................................................ 10 
15 
FINANCING ..................................................................................................................................... 10 
16 
INSURANCE ......................................................................................................................................11 
17 
STUDY REPORT AND PUBLICATION(S) ...................................................................................11 
18 
ARCHIVING ......................................................................................................................................11 
19 
REFERENCES ...................................................................................................................................11 

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1 
SUMMARY 
 
Background: Brain has a central role in controlling glucose metabolism, but the central-peripheral link 
in energy metabolism and homeostasis have remained poorly understood. The goal of this project is to 
characterize the neural, psychological, and physiological mechanisms that contribute to aberrant reward 
processing and glucose metabolism in obesity using total-body PET imaging.   
 
Aims: We address the central-peripheral axis in metabolic control by imaging glucose utilization and 
perfusion. Brain and peripheral organs (heart, kidneys, liver) will be studied simultaneously to allow 
network-level analysis of the metabolic integration. Functional neural and peripheral responses to 
emotional stimuli will be measured separately for mapping the role of the brain’s reward circuit in 
metabolic regulation.  These studies will improve the understanding of the neural and psychological 
mechanisms obesity. This knowledge will translate into crucial knowledge for developing novel risk factor 
screening procedures, and novel pharmacological and psychological treatments for common obesity.  
 
Methods: A total of 120 male and female subjects aged 20 to 60 years are recruited into this prospective 
study. The subjects will undergo physical examination, body tissue composition measurement as well as 
PET imaging.  The study will start in January 2021 and end in the beginning of 2026. 
 
2 
INTRODUCTION 
Brain has a central role in controlling glucose metabolism, and glucose tolerance, insulin secretion and 
insulin sensitivity, are in part modulated by the brain (Chen et al., 1975; Obici et al., 2002).  Whereas it is 
well known that insulin sensitivity and secretion are tightly coupled (Kahn et al., 1993; Utzschneider et 
al., 2009), the mechanisms leading to this tight coupling have not been fully elucidated, with some 
researchers proposing an important role of the brain (Morton et al., 2017). Central insulin administration 
suppresses endogenous glucose production (EGP) in mice (Obici et al., 2002).  Similarly, in humans 
intranasal insulin suppresses EGP in lean but not in overweight subjects (Heni et al., 2017).  Insulin-
stimulated brain glucose uptake (BGU) associates positively with EGP in obese and overweight subjects, 
but not in lean controls (Latva-Rasku et al., 2017; Rebelos et al., 2018) suggesting that the brain 
differentially controls peripheral insulin sensitivity by degree of adiposity.  
 
The organ-specific insulin sensitivity has been associated with subtypes of metabolic conditions, for 
example insulin resistance being localized to skeletal but not heart muscle in type 1 diabetes (Nuutila et 
al., 1993).  Insulin is also a vasodilator and enhances tissue perfusion in skeletal muscle (Clark et al., 2003) 
and the heart in healthy subjects (Sundell & Knuuti, 2003) but these effects are poorly understood in 
obesity. Studies comparing insulin effects on perfusion and glucose extraction in the whole body are 
lacking. In line with this, the role of perfusion to insulin resistance remains unresolved. In addition, brown 
adipose tissue has emerged as a potential therapeutic target. Brown adipose tissue (BAT) activity has large 
catabolic potential, and its activity is conserved in human adults (Virtanen et al., 2009). BAT activity is 
negatively associated with insulin resistance and obesity (Cypess et al., 2009; Lichtenbelt et al., 2009) and 
furthermore BAT thermogenesis might be impaired in eating disorders (Bredella et al., 2012). These data 
suggest that BAT dysfunction could be an important pathway contributing to the development of obesity.  
Accordingly, it is imperative to quantify the links between peripheral and central glucose utilization in 
normal-weight and obese subjects, and their association with the potentially disrupted BAT 
thermogenesis.  
 
Feeding and appetite are modulated by a complex set of brain networks involving hypothalamus, 
amygdala and ventral striatum. Multi-level pathophysiology of this circuit is involved in numerous 
conditions involved altered food intake. Several studies have reported mesoscopic alterations of this 
circuit in common obesity and eating disorders despite vastly different endophenotypes (Karlsson et al., 

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2013; King et al., 2018; Phillipou et al., 2014), thus the underlying neurobiological mechanisms leading 
to these similar cerebral alterations across different phenotypes likely differ. Bodily signals influence 
cognition, emotion, and behaviour at multiple scales and vice versa, and electrical stimulation studies 
have confirmed the coupling between visceral responses and neocortex (Penfield & Faulk, 1955). 
Emotions promote survival by automating prompt detection beneficial harmful events, and triggering of 
approach versus safety-seeking behaviour (Mobbs et al., 2015). Bodily signals influence cognition, 
emotion, and behaviour at multiple scales and vice versa, and electrical stimulation studies have 
confirmed the coupling between visceral responses and neocortex. Distinct emotions are supported by 
discernible neural circuits (Nummenmaa & Saarimäki, 2017) and dysfunction of the emotion circuit has 
dramatic impact on individuals’ well-being. However, the interplay between brain’s emotional systems 
and peripheral components of the emotional circuit in allostatic control of feeding and appetite have 
remained poorly understood as simultaneous imaging of the central-peripheral axis has not been possible 
3 
OBJECTIVES AND PURPOSE 
The goal of this project is to establish the biological links between brain-periphery axis in human 
metabolism and test how their specific alterations are linked with obesity.  Using cross-sectional design, 
we characterise individuals with obesity using total-body PET imaging and behavioural assessment. We 
test three  hypotheses derived from human and animal studies: 
 
1. Insulin-stimulated  increases  in tissue  specific perfusions are generally blunted in obesity and 
contribute to the insulin resistance   
2. Insulin-stimulated brain glucose uptake (BGU) associates positively with EGP in obese and 
subjects  
3. Central emotional reactivity is linked with myocardial perfusion and metabolism as well as 
subjective experience of emotion 
4 
STUDY DESIGN 
This is a cross-sectional study investigating the relationship between the brain and physiological functions 
in obesity nervosa. Subjects (n= 100) will undergo a screening visit, body composition measurement and 
calorimetry, and PET with radioligands H2[15]O and [18F]FDG on two separate visits. A subset of the 
subjects (Branch B) will only undergo the H2[15]O activation scans. 
 
5 
PATIENT/SUBJECT SELECTION 
5.1 
Source population 
We recruit 50 adults (males and females) with obesity from the Eating Disorder Unit at the Turku 
University Hospital. Their data are compared with 50 age and sex-matched healthy controls with normal 
body weight and no reported history of an eating disorder  
5.2 
Inclusion criteria for the obese group 
1) Age 20-60 years 
2) BMI > 30 kg/m2 
5.3 
Inclusion criteria for the control group 
1) Age 20-60 years 
2) BMI 18.5-25 kg/m2 
3) No lifetime history of obesity, eating disorders or type 2 diabetes 

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5.4 
Exclusion criteria 
1) Any chronic disease or medication that could affect glucose metabolism or neurotransmission, 
with the exception of previously diagnosed type 2 diabetes and use of metformin, DPP-IV-
inhibitors or SGLT2-inhibitors in the obese group 
2) Any current psychiatric disorder 
3) History of severe psychiatric disorders 
4) Smoking of tobacco, taking of snuffs, or use of narcotics 
5) Abusive use of alcohol  
6) Any other condition that in the opinion of the investigator could create a hazard to the subject 
safety, endanger the study procedures or interfere with the interpretation of study results 
6 
ASSESSMENTS 
6.1 
General study outline 
The baseline measurements will be performed on three separate visits (a screening visit and a two PET 
scanning visit). The interval between the screening visit and the first scanning visit is 2-28 days, scanning 
visits are done at maximum 28 days apart.  
 
1. Screening visit 
- 
Physical examination 
- 
Anthropometry 
- 
ECG 
- 
Blood samples, including OGTT 
- 
Body fat mass measurement with bioimpedance 
- 
Body fat and composition measurement with BodPod 
- 
Questionnaire measurements 
 
2. PETscanning visit 1 
- 
Branch A: Sensitivity of brain’s emotion circuits will be measured using activation protocol with 
H2[15]O 
- 
Branch B: Total-body perfusion changes during cold exposure will be measured using 
activation protocol with H2[15]O. Subjects participating in Branch B will not participate in the 
glucose utilization scan (visit 2) 
- 
 
 
3. PET Scanning visit 2 
- 
Central and peripheral perfusion will be measured with radioligand H2[15]O 
- 
Skeletal muscle microvascular perfusion will be measured with contrast-enhanced ultrasound 
- 
Carotid artery intima media thickness will be measured with ultrasound 
- 
Central and peripheral glucose utilization measured with radioligand [18F]FDG. 
- 
Whole-body insulin resistance will be measured with euglycemic hyperinsulinemic clamp 
technique 
6.2 
Perfusion imaging with H2[15]O 
Total-body PET imaging is performed with Siemens Biograph Vision Quadra scanner (Siemens 
Healthineers) at Turku PET Centre. The system consists of four consecutive digital detector rings and in 
comparison with conventional PET-CT, it provides four times wider FOV and 40 times higher SNR in 
whole-body imaging and 10 times higher SNR for single-organ (e.g. brain) imaging. Spatial resolution is 
3mm3 and the FOV (106 cm) covers brain and all internal organs and skeletal muscles down to the 
quadriceps. Total-body perfusion scans are taken three times (Figure 1B): At the beginning of the 

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session, at baseline, after intravenous insulin priming (at and after 50 minutes of the insulin clamp. 
Additionally, we measure cerebral and peripheral responses to audiovisual emotional stimuli using 
ultrafast perfusion imaging. For each scan, H2[15]O (400 ± 10 MBq) is injected intravenously, and 
radioactivity is followed for 5 minutes. In the brain, regions of interest (ROIs) will be delineated using 
the AAL atlas (Tzourio-Mazoyer et al., 2002) after spatially normalizing the brain data to functional PET 
template image in MNI space.  For peripheral data, ROIs will be defined for myocardium, lungs, skeletal 
muscle (upper thigh / biceps), liver, pancreas, kidneys, and gut.  
 
6.3 
Measuring total-body glucose utilization with PET 
We measure whole-body glucose utilization with radiotracer [18F]FDG. Whole-body low-dose CT scan 
and MRIs are acquired for anatomical reference. Euglycemic hyperinsulinemic clamp technique is used 
as previously described (DeFronzo et al., 1979). Briefly, plasma glucose concentration is kept constant at 
5 mmol/l by a variable glucose infusion using a negative feedback principle. Under steady-state 
euglycemic condition, the glucose infusion rate equals glucose uptake by all the tissues in the body and is 
therefore a measure of tissue sensitivity to exogenous insulin. The rate of insulin infusion is set at 40 mU 
m-2 min-1. During hyperinsulinemia, euglycemia is maintained by infusing 20 % glucose intravenously. The 
rate of glucose infusion is adjusted according to plasma glucose concentrations measured every 5-10 min 
from arterialized blood. At the time point 100+-10 minutes of euglycemic hyperinsulinemic clamp, 
[18F]FDG (100 ± 9 MBq) is injected intravenously over 40 second and PET data is acquired from the 
106 cm of the field of view including the brain, neck area (BAT), heart, liver, abdomen and skeletal muscle 
regions. Arterialized venous blood samples are drawn for radioactivity analysis. A GE Advance PET 
scanner (General Electric Medical Systems, Milwaukee, WI, USA) with resolution of 4.25 mm was used 
for PET studies as previously described  (Kaisti et al., 2003; Kaisti et al., 2002). [18F]FDG is synthesized 
as previously described (Hamacher et al., 1986). Plasma radioactivity is measured with an automatic 
gamma counter (Wizard 1480 3", Wallac, Turku, Finland). Cerebral glucose uptake rate is measured for 
each voxel separately from dynamic PET scans as described previously (Kaisti et al., 2003; Kaisti et al., 
2002). Urine samples are collected following the PET scan and the amount of radiotracer lost into urine 
is measured using isotope dose calibrator (Model VDC-205, Comecer Netherlands, Joure, Netherlands). 
Endogenous glucose production (EGP) is assessed by subtracting glucose infusion rate from rate of 
glucose disposal derived from 18F-FDG consumption (Iozzo et al., 2007). After PET, insulin infusion is 
stopped. Glucose infusion is continued until a stable level of plasma glucose is achieved (healthy 
individuals: 6.5 mmol/l).  
 
 
 
Figure 1. Structure of the PET scanning sessions completed on two separate visits.   
 

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6.4 
Mapping cerebral and peripheral emotion circuits with H2[15]O activation protocol 
Conventional brain perfusion imaging with H2[15]O is based on approximately 60-second activation 
blocks with bolus radiowater injections intserspreded by > 14 minute (seven half-lives) breaks allowing 
radiotracer decay (Jonides et al., 1993). Increased sensitivity of the total-body system (Tan et al., 2020). 
Coupled with recent developments in dynamic PET data modelling at our centre however allow us to 
perform functional activation studies with H2[15]O with significantly higher stimulation cycle frequencies 
(Watabe et al., submitted). The technique is based on accounting for the residual radioactivity from the 
previous scans for each subsequent bolus injection. Simulations and primate data suggest that interscan 
intervals below 5 minutes are feasible, with the prospects of bringing the interval below two minutes 
(Watabe et al., submitted). This significantly increases the rCBF signal acquired per minute and coupled 
with the high-SNR-low-dose capabilities of total-body PET, allows simultaneous high-SNR activation 
studies of the brain and the peripheral organs.  For the activation experiment in Branch A we use 
previously validated paradigm (Saarimäki et al., 2016) where emotional videos are shown to participants 
in blocks, with single block duration matching the peak of the H2[15]O activity in tissue (Figure 1A). 
Fast phasic bolus injection protocol will be used. Two different stimulus categories will be used – 
emotional and neutral.  Five blocks per emotion condition will be run. Subjects are asked to empathize 
with the movies as strongly as possible.  Central and organ-specific peripheral perfusion will be modelled, 
and network-level interactions between the central and peripheral activation will be measured. For Branch 
B, we used the previously validated cold pressor test (UDin et al., 2016; Walsh et al., 1989). Briefly, 
subjects foot is immersed into cold water for the duration of each of the PET scans. This induces 
sympathetic activation, vasoconstriction, and unpleasant feelings. Subjective responses to the cold 
stimulus are measured using VAS scales. Five blocks with cold stimulation and five blocks with neutral 
stimulation (no cold) are run; otherwise scanning is similar as in Branch A. 
 
6.4b Dosage estimation experiment 
To estimate the lowest possible dose for the new total-body PET camera, for a subset of subjects the 
H2[15]O activity study will be run without external stimulation and with variable H2[15]O doses (set 1: 
0.05, 10, 100, 300, 1000 MBq, set2: 50,100,150,200,300 MBq, set 3: 100,200,400, 800 MBq). Subjects 
participating in the dosage estimation experiment (n=20) will not undergo the FDG scan.  
6.5 
Assessment of skeletal muscle microvascular perfusion with ultrasound 
On the insulin clamp day, skeletal muscle microvascular perfusion will be assessed by using contrast 
enhanced ultrasound (CEUS). For the study, a steady flow of contrast agent (Sonovue, Bracco) will be 
administered, and after reaching a steady tissue concentration (app. 2 min), a high-intensity ultrasound 
pulse is used to destroy the contrast agent microbubbles. After this, four 30 sec cine-loop recordings are 
stored for further analysis to obtain a replenishment curve, which will represent tissue microcirculation.
 
Vascular health will also be evaluated with carotid intima media thickness measured with 
ultrasound. For the measurement, 4 cine loops of 5 cardiac cycles will be recorded from the carotid 
bifurcation area. 
 
6.6 
Laboratory measurements 
Physicians will evaluate the general physical condition of the subjects, involving weight, height, blood 
pressure, medical history, and current medication. Adiposity is determined by BMI and BodPod device 
as described earlier (Kantonen et al., 2021). Screening laboratory measurements also include oral glucose 
tolerance test (OGTT). A zero time (baseline) intravenous blood sample is drawn prior to the OGTT. 
75g glucose mixed in 2 dl of water is administered orally up to 5 minutes (WHO recommendation). 
During OGTT blood samples will be taken at time points 0 (baseline sample), 15, 30, 60, 90, 120 min for 
glucose, C-peptide and insulin analysis and calculation of AUC. Fasting blood samples include blood 
count, glucose, insulin, liver enzymes (ALT, AST, AFOS, GT), potassium, sodium, creatinine, albumin, 
magnesium, chloride, phosphate, high-sensitive CRP, TSH, free T4, free T3, troponin T and NT-

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proBNP. Drug screen from the urine include: amphetamine, cannabis, cocaine, opiate, methadone, 
bentsodiazepine, buprenorphine and creatinine. Lipids, lipoprotein subclasses, amino acids and 
metabolites of glucose, fatty acid and amino acid metabolism are measured using quantitative nuclear 
magnetic resonance and mass spectrometry metabolomics technique. Chronic stress responses will be 
measured using hair cortisol samples. Serum progesterone and estradiol samples will be collected on 
screening visit to determine the phase of menstrual cycle. An urine sample, a stool sample and blood 
samples (plasma and serum) will also be collected and stored at a freezer to be used in further analyses. 
All personal identification information will be removed from these samples, and they will be anonymized 
before storing. 
6.7 
Questionnaires and computer-based tasks 
All the participants will complete the following self-administered questionnaires before the study:  
UCLA-LS (Russell et al., 1978), MAIA-2 (Mehling et al., 2012), SDT (Furnham et al., 2013), DASS-21 
(Lovibond & Lovibond, 1995), TIPI (Gosling et al., 2003) and (van Strien et al., 1986). Subjects will 
also evaluate a short standardized set of emotional pictures to assess affective reactivity (Lang et al., 
2005). Emotional state will be evaluated using VAS scales at the beginning of the experiment and after 
each PET scan. Measurements will be done using the Gorilla.sc toolbox.   
 
7 
ADVERSE EVENTS 
7.1 
Definition of Adverse Events (AEs) 
An AE is the development of an undesirable medical condition or the deterioration of a pre-existing 
medical condition following or during exposure to a pharmaceutical product, whether or not considered 
causally related to the product. An undesirable medical condition can be symptoms (e.g. nausea), signs 
(e.g. tachycardia) or the abnormal results of an investigation (e.g. laboratory findings). 
 
A serious adverse event (SAE) or serious adverse drug reaction (SADR) is any event that is fatal, life-
threatening, results in hospitalisation, prolongs hospitalisation, results in persistent or significant injury 
or inability to conduct normal life functions, causes a congenital anomaly or a birth-defect or is an 
important medical event that may jeopardize the subject or may require intervention to prevent one of 
the outcomes listed above. A suspected unexpected serious adverse reaction (SUSAR) is a suspected 
serious adverse event that is also unexpected. 
7.2 
Reporting of Adverse events (AEs) 
All adverse events will be registered, and when necessary, the ethical committee and the Finnish 
Medicines Agency will be notified.  All adverse events will be fully documented and followed in order to 
determine the final outcome (until the event has subsided or the condition as stabilised).    
7.3 
Expedited Reporting 
According to Finnish national regulation, fatal or life-threatening unexpected adverse events will be 
reported as soon as possible but at least within 7 days. All other SAEs, SADRs and SUSARs will be 
reported within 15 days. 
7.4 
Emergencies 
Emergencies may occur during the experiments. A medical doctor will be available during all the 
measurements in the study. Vital functions will be monitored, and materials and drugs needed for first 
aid/resuscitation are readily available. 
 

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8 
ETHICS 
8.1 
Ethical considerations 
The studies will be performed using standard procedures that have been in clinical or research routine 
use for years in the Turku PET Centre. This study will be conducted according to Good Clinical Practice 
and the Declaration of Helsinki. Written informed consent for the study will be obtained from all 
subjects- before the beginning of the study. Subjects will be informed of their right to withdraw from the 
study at any time. At least one medical doctor will be available at all times during the study.  
 
The radiation dose for the study will be 9.02-9.72 mSv depending on the number of whole-
body CT scans required for the studies. The higher dose, 9.72 mSv corresponds to 1 year and 8 months 
of background radiation in Finland, so the risk for radiation related adverse events is low when subjects 
with significant previous radiation burden will be excluded. For Branch B activation studies the total 
radiation dose will be 5,1 mSv, which corresponds to 11 months of background radiation. 
8.2 
Ethical Review 
Study protocol, patient information, and informed consent form will be submitted to the Ethical 
Committee of the Hospital District of the South-Western Finland (EC). Studies will not start before 
getting ethical permission. The EC will be informed by the Investigator of all subsequent protocol 
amendments and of serious or unexpected adverse experiences occurring during the study, which are 
likely to affect the safety of the subjects or the conduct of the study. 
8.3 
Subject information and informed consent 
Information about the study procedures and design will be given in both oral and written form to the 
study subjects and their parents. All necessary investigator contact information will be provided along 
with a description of the study. Written informed consent for the study will be obtained from all subjects 
before the beginning of the study. The principles of informed consent in the current edition of the 
Declaration of Helsinki will be implemented. Subjects will be informed of their right to withdraw from 
the study at any time.  
 
9 
DATA COLLECTION AND MANAGEMENT 
9.1 
Case Report Forms 
One set of case report forms will be filled during the initial clinical examination at screening visit in order 
to collect subjects’ personal information and their medical status and history relevant to the aims and 
exclusion criteria of this study. 
9.2 
Electronic data collection 
Data from the case report forms, clinical observations (such as adverse effects or changes in medical 
status) as well as results from the study (laboratory analyses and PET imaging data) will be gathered in 
electrical form. 
9.3 
Data management 
All data collected is strictly confidential. It will be archived on paper or electronically at least for fifteen 
years. 
 
10 DATA ANALYSIS 
Brain-PET data (perfusion and glucose uptake) will be analyzed with SPM12 and in-house MAGIA 
toolbox (Karjalainen et al., 2020). After motion correction and normalization to MNI space, data will 
be analyzed using conventional random effects model, where task events are modeled as boxcar 

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functions. Peripheral PET data will be analyzed as described previously (Rebelos et al., 2019). Structural 
equation modelling and network analyses will be used for quantifying the relationship between regional 
cerebral and peripheral glucose uptake and perfusion.  
 
11 STATISTICS 
11.1 Sample size 
The sample size was calculated based on comparable experiments on brain structure and obesity. In these 
experiments statistically significant differences in brain structure could be established between lean (n = 
22) and obese individuals (n = 23). A priori power calculations with the G*Power software suggest that 
the predicted effects will be observed with a power of 0.95 with sample sizes exceeding 30 in both groups. 
The design is thus sufficiently powered.  
11.2 Statistical analyses  
Appropriate statistical analyses will be performed corresponding to current standards.  
11.3 Possible interim analyses and stopping rules 
No interim analysis is intended. 
 
12 QUALITY ASSURANCE 
 
12.1 Information of study personnel and training 
All the personnel involved in the study will be informed about the aims and the practical issues of the 
study. The measurements are routinely performed at the Turku PET Centre and have been applied for 
years.  
12.2 Protocol amendments 
According to Finnish regulations, protocol amendments can be made if all investigators agree.  They are 
presented in a written form and dated. They include the original chapter of the study protocol and the 
amended chapter with and explanation of this change. All protocol amendments will be submitted for 
review to the ethical committee. 
 
13 STUDY SCHEDULE 
The study will start in September 2022 and end in beginning of 2030. Analysis and reporting of the data 
will be started as soon as possible and will be continued until all the results have been reported. 
 
14 CRITERIA FOR PREMATURE STUDY TERMINATION 
No interim analysis is intended. 
 
15 FINANCING 
The studies in PET Centre will be financed with project grants and VTR-funding of prof. Lauri 
Nummenmaa and prof. Pirjo Nuutila. The study has neither a straight nor indirect financing from the 
industry. The financing of the study is governed through the Turku University Hospital and University 
of Turku. 

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Expense 
Per subject 
Total for 100 
subjects 
OGTT  
60 
6000 
TYKSLAB analyses 
122 
12200 
PET-imaging 
300 
30000 
Pharmacy services 
30 
3000 
Travel expenses 
50 
5000 
Subject compensations 
340 
34000 
Total  
90200 
 
16 INSURANCE 
The subjects are covered by the patient insurance. No extra insurance will be taken. 
 
17 STUDY REPORT AND PUBLICATION(S) 
The results will be reported in international peer-review journals.  
 
18 ARCHIVING 
All data collected will be strictly confidential. It will be archived on paper and electronically for at least 
fifteen years.  
 
19 REFERENCES 
 
 
 
 
 
 
References 
 
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Density and Lower Pref-1 Than Women without Brown Adipose Tissue: A Study in Women with Anorexia 
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