Immunometabolism in Pediatric Obesity
Conditions: Obesity, Type 2 Diabetes Mellitus, Insulin Resistance
Sponsor: Arkansas Children's Hospital Research Institute

Version #:  5 
Date:  19APR2019  
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Study Title: 
Immunometabolism in Pediatric Obesity 
 
Principal Investigator:  
Shannon Rose, Ph.D. 
UAMS Department of Pediatrics 
Arkansas Children’s Research Institute 
13 Children’s Way, Slot 512-41B 
Little Rock, AR 72202 
Telephone:  (501) 364-4083 
Email:  SROSE@uams.edu 
 
 
Sub-Investigator (s): 
Jon D. Oden, M.D. 
  
UAMS Department of Pediatrics 
  
Arkansas Children’s Hospital 
 
1 Children’s Way, Slot 512-6 
 
Telephone: (501)-364-1430 
 
E-Mail: JDOden@uams.edu 
 
 
Emir Tas, M.D. 
  
UAMS Department of Pediatrics 
  
Arkansas Children’s Hospital 
 
1 Children’s Way, Slot 512-6 
 
Telephone: (501)-364-1430 
 
E-Mail: ETas@uams.edu 
 
 
Shipra Bansal, M.D. 
  
UAMS Department of Pediatrics 
  
Arkansas Children’s Hospital 
 
1 Children’s Way, Slot 512-6 
 
Telephone: (501)-364-1184 
 
E-Mail: SBansal2@uams.edu 
 
 
Vildan Tas, M.D. 
  
UAMS Department of Pediatrics 
  
Arkansas Children’s Hospital 
 
1 Children’s Way 
 
Telephone: (501)-364-8368 
 
E-Mail: VTas@uams.edu  
 
Study location: 
Arkansas Children’s 
 
1 Children’s Way 
 
Little Rock, AR 72202 
 
 
 
 

Title:  Immunometabolism in Pediatric Obesity 
PI:  Shannon Rose, Ph.D.  
 
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Date:  19APR2019  
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Background and Rationale 
Obesity is an evolving pediatric health crisis in the United States. Approximately 20% of 
children and adolescents in the US are obese1 and consequently at increased risk for 
type 2 diabetes (T2D), hypertension and several cancers2. Previously considered as adult 
onset conditions, the incidence of T2D and cardiovascular dysfunction is increasing 
among adolescents likely due to high prevalence of obesity in children3. In the state of 
Arkansas, more than 30% of children meet the criteria for being overweight or obese4. 
The dramatic increase in the prevalence of childhood obesity5 has enormous public health 
implications and, for the first time in history, may result in a reduction of the life expectancy 
of the current generation of children6-9. A better understanding of the pathophysiology of 
obesity and associated health complications in children is imperative for the development 
of effective preventative and treatment strategies, to prevent the progression into more 
serious health concerns, and to reduce morbidity and mortality.  
 
Obesity is strongly linked to systemic inflammation with increased circulating pro-
inflammatory cytokines and adipokines and elevated C-reactive protein (CRP) reported 
in obese children as young as age three10-15. Examining NHANES data from over sixteen 
thousand children, Skinner et al15 found that prevalence of C-reactive protein (CRP) levels 
>1mg/L was greater in very obese children beginning at age 3 and continued through 
adolescence, with greater than 80% of very obese adolescents having CRP >1mg/L as 
compared to 18% of lean adolescents. While the dataset did not permit controlling for 
pubertal age, similar results were found even when very narrow age groups were 
compared. In a smaller study of children ages 7-14 years, Codoner-Franch et al16 
classified obese children as metabolically healthy or at risk based on well-defined criteria, 
and they reported CRP, Tumor necrosis factor α (TNFα) and Interleukin (IL) 6 were all 
increased in obese children with or without metabolic risk factors, suggesting that 
inflammation may appear early in obesity before metabolic alterations. Inflammation is 
considered pivotal to the development of obesity-related metabolic disorders including 
insulin resistance, T2D17-19 and cardiovascular dysfunction20, 
21. Modulation of 
inflammation in obese children provides a putative “target” for therapeutic intervention.   
 
Suppression of inflammation is a function of regulatory T cells (Tregs), a subset of CD4+ T 
cells also responsible for maintenance of peripheral tolerance and suppression of 
antigen-specific immune responses. However, decreased circulating Tregs have been 
reported in T2D22 and obese23, 24 adults. Wagner et al24 found that reduced Tregs predicted 
a CRP level greater than 3mg/L and may therefore predict those at increased 
cardiovascular risk; however, a separate group reported no differences in Tregs between 
severely obese subjects with and without hypertension23.  As in humans, Tregs are reduced 
in several mouse models of obesity, especially VAT-resident Tregs, with a concomitant 
increase in VAT-resident T effector cells25, 26. A critical role for Tregs in the development 
of insulin resistance has been demonstrated in these models. Winer et al25 elegantly 
demonstrated that adoptive transfer of CD4+T cells into lymphocyte deficient DIO Rag1-/- 
mice reduced weight gain and improved glucose tolerance. In situ induction of Tregs in 
HFD obese wild-type mice with anti-CD3 antibody also resulted in improved glucose 
tolerance and insulin sensitivity, which persisted for months despite being maintained on 

Title:  Immunometabolism in Pediatric Obesity 
PI:  Shannon Rose, Ph.D.  
 
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Date:  19APR2019  
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HFD. Feuerer et al26 demonstrated that depletion of adipose Tregs in transgenic mice 
expressing the diphtheria toxin receptor in Tregs induces insulin resistance and increased 
inflammatory mediators in the adipose tissue. Conversely, in situ induction of Tregs with 
recombinant IL-2 and IL-2 monoclonal antibody increased VAT-resident Tregs and 
improved glucose tolerance in HFD obese mice. Findings from these two landmark 
studies have been replicated in other mouse models of obesity27, 28. Targeting Tregs may 
modulate inflammation in obesity; however, whether Tregs are altered in early stages in 
obesity (e.g. overweight/obese children) remains unknown. In this study, we will fill an 
important research gap by testing whether obese children exhibit decreased circulating 
Tregs as compared to healthy lean children.   
 
The past decade has seen a surge in understanding the metabolic control of immune 
cells, unveiling metabolic signaling pathways that may be exploited to target specific 
immune cell subsets. Pro-inflammatory immune cells preferentially produce ATP via 
glycolysis while anti-inflammatory cells primarily utilize mitochondrial oxidative 
phosphorylation (OXPHOS)29-31. Nutrient and energy-sensing signaling pathways are 
master regulators of cellular bioenergetics with mammalian target of rapamycin (mTOR) 
driving glycolysis and adenosine monophosphate activated protein kinase (AMPK) driving 
OXPHOS. The mTOR pathway promotes glycolysis by upregulating Glut1 trafficking to 
the cell surface32, increasing glucose uptake and glycolysis. AMPK opposes the mTOR 
pathway and promotes glucose and lipid oxidation33. As depicted in Figure 1, Teff depend 
upon mTOR-driven glycolysis while 
Tregs 
require 
AMPK-driven 
OXPHOS. 
Targeting 
these 
metabolic control pathways can 
simultaneously 
inhibit 
effector 
responses 
and 
promote 
the 
generation of Tregs, opening up a 
new 
approach 
for 
treating 
inflammation 
in 
obesity 
and 
preventing the progression into 
more serious health conditions. We 
will determine, for the first time, 
whether 
this 
immunometabolic 
paradigm is altered in pediatric 
obesity by measuring activation of 
AMPK and mTOR in isolated CD4+ 
T cells. These data are necessary before the approach of immunomodulation by targeting 
metabolic control pathways to treat inflammation can be applied in this population. 
 
 
The AMPK activator, metformin, is the only anti-diabetic drug FDA-approved for use in 
children34, 35. In an open-label trial, metformin increased both the number and regulatory 
functions of Tregs in obese multiple sclerosis patients with metabolic syndrome36. In a 
placebo-controlled trial carried out by de Kreutzenberg et al37, two months of metformin 
Figure 1: Distinct metabolic pathways control regulatory T 
cells (Treg) and effector T cells (Teff). Tregs are characterized by 
high AMPK and low mTOR activation, and they primarily utilize 
mitochondrial oxidative phosphorylation (OXPHOS) to produce 
ATP. Teff are characterized by low AMPK and high mTOR 
activation, and they primary utilize glycolysis to produce ATP.  

Title:  Immunometabolism in Pediatric Obesity 
PI:  Shannon Rose, Ph.D.  
 
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Date:  19APR2019  
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treatment in pre-diabetic adults reduced mTOR activity in PBMC despite increases at the 
gene expression level; however, AMPK activity was not changed by metformin. 
Metformin’s potential to induce Tregs has otherwise not been studied in obese individuals. 
We will take advantage of the fact that this AMPK activator is commonly prescribed in 
pediatrics and compare the immune and metabolic phenotypes of CD4+ T in obese insulin 
resistant/T2D children before and after metformin treatment. As such, we will explore 
whether a potential mechanism of action of metformin is via direct modulation of CD4+T 
cell metabolism, induction of Tregs, and curbing inflammation.  
 
The main purpose of this proposed study is to determine whether the immunometabolic 
phenotypes of CD4+T cells from obese children is skewed towards Teff with mTOR-
driven glycolysis and away from Tregs with AMPK-driven OXPHOS and whether 
metformin can reverse the immunometabolic phenotypes.  These studies are imperative 
to begin testing and implementing strategies targeting the AMPK and mTOR pathways 
to combat obesity-associated inflammation and prevent progression into serious health 
complications. This translational research will move the field forward in several ways. 
First, we will gain insight into the pathophysiology of obesity-associated inflammation 
and the progression into insulin resistance and T2D. Second, we have the opportunity 
to uncover novel therapeutic avenues for obesity-associate inflammation and prevention 
of metabolic dysfunction that target immune cell metabolism. Third, we may identify an 
immunometabolic phenotype that correlates to risk for developing T2D and/or 
cardiovascular dysfunction and could be used to monitor treatment response in future 
treatment trials.  The findings from this study will form the preliminary data for a planned 
R01 application to the NIDDK to be submitted in Year 2. The goal of that proposal will 
be to conduct additional translational studies to expand upon this study and begin 
testing the ability to modifying bioenergetic control pathways to combat obesity-
associated inflammation.   

Title:  Immunometabolism in Pediatric Obesity 
PI:  Shannon Rose, Ph.D.  
 
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Date:  19APR2019  
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Preliminary Data  
 
In a current study (UAMS IRB #206164; Co-I: Rose), we have enrolled lean and obese 
subjects in a very narrow age range (5-9 years of age) in order to control for pubertal 
state. Bioenergetic profiling using 
the Seahorse XF96 Analyzer 
(Seahorse Bioscience, Inc., 
North Billerica, MA), a core ACRI 
equipment, is routinely 
performed by Dr. Rose38-40. The 
Seahorse measures oxygen 
consumption rate (OCR) and 
extracellular acidification rate 
(ECAR) in a 96-well plate of 
intact living cells. The sequential 
injection of up to four compounds 
into each well enables the 
interrogation of bioenergetics in 
real-time. Figure 2A and B 
presents the ATP Production 
Rate Assay on PBMC from lean, 
obese and T2D children enrolled 
in an ongoing study (UAMS IRB 
#206164; Co-I: Rose). In this 
assay, basal OCR and ECAR 
are initially measured, followed 
by the injection of oligomycin 
(oligo), an ATP synthase 
inhibitor, to block ATP production 
by the mitochondrial electron 
transport chain (ETC) and induce 
compensatory glycolysis. Next, 
antimycin A (AMA) and rotenone 
(ROT), inhibitors of ETC complex 
III & I, respectively, are injected 
together. Shutting down the ETC allows for calculating the amount of media acidification 
(ECAR) that was due to CO2 production during OXPHOS. Proton efflux rate (PER) is 
calculated post-assay from ECAR and the buffering capacity of the assay media, and 
finally, validated calculations derive ATP generated by OXPHOS (mitoATP) and by 
glycolysis (glycoATP)41, which is presented in Figure 2C. Figure 2D shows that the % 
ATP derived from glycolysis is significantly greater in PBMC in the T2D group as 
compared to the OB group (p=0.032). While no differences between the lean and obese 
group are observed in this preliminary data, the increased % glycolysis in the T2D group 
supports the notion that obese children with metabolic dysfunction have more pro-
inflammatory cells and fewer Tregs in circulation. There are several important points to 
Figure 2: The ATP production rate assay demonstrates 
increased % glycolysis-derived ATP production in PBMC from 
T2D children. Oxygen consumption rate (OCR) (A) and Proton 
Efflux Rate (PER) (B) are plotted over time in PBMCs from lean, 
obese (OB) and type 2 diabetic (T2D) children. Injections of 
Oligomycin (oligo) and Antimycin A (AMA) and Rotenone (ROT) are 
indicated. (C) Glycolytic ATP production rate (glycoATP) and 
mitochondrial ATP production rate (mitoATP) are calculated from 
OCR and PER. (D) % glycolysis and % OXPHOS-derived ATP 
production are calculated. * p=0.032 
Methods: Freshly isolated PBMC from lean (n=8), obese (OB; n=11), and 
treatment-naïve type 2 diabetic (T2D; n=2) children were plated at 2.5x106 
cells/well in RMPI 1640 with 1mM HEPES, 2mM glutamine, 1mM pyruvate 
and 11mM glucose on poly-D-lysine coated XF96 plates. Plates were 
centrifuged at 100xg with no brake to adhere cells to well bottoms. After a 30 
min incubation in a 37°C non-CO2 incubator, cells were assayed in a 
Seahorse XF96.  

Title:  Immunometabolism in Pediatric Obesity 
PI:  Shannon Rose, Ph.D.  
 
Version #:  5 
Date:  19APR2019  
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note regarding this data: 1) these data are from PBMC, a mixed population of cells 
including monocytes and lymphocytes of various subtypes rather than purified CD4+ T 
cells as proposed in this study; 2) the obese subjects have not been classified as 
metabolically healthy or unhealthy; 3) the age of the T2D subjects is greater (16.5± 0.1 
years; mean±SD) than the other groups (lean: 6.8±1.1 years; obese: 7.9±1.4 years). In 
the studies proposed herein, we will match groups on age and subgroup obese subjects 
as metabolically healthy and unhealthy based on insulin sensitivity. Further, after 
measuring high sensitivity CRP (hsCRP), we will compare subjects with hsCRP values 
in the top quartile to the other groups to determine whether the % glycolysis is higher in 
CD4+T cells from those obese subjects with inflammation.   
 
Activation of AMPK or mTOR have not yet been evaluated in PBMC presented in Figure 
2. In another study ongoing study of mitochondrial dysfunction in children with autism 
spectrum disorder (UAMS IRB #137162; PI: Rose), we measured PBMC bioenergetics 
and mTOR activation in 43 children including 38 children with autism spectrum disorder 
and 5 unaffected siblings. At the time of this study, the ATP production rate assay was 
not yet developed, and PBMC were bioenergetically phenotyped using the mitochondrial 
stress test39. mTOR activation was measured in lysates from approximately 2x106 PBMC 
using the 11-Plex AKT/mTOR Phosphoprotein magnetic bead Kit (Millipore). As 
demonstrated in Figure 3, phosphorylated mTOR (P-mTOR) in PBMC is significantly 
associated with basal (Figure 3A) and maximal OCR (Figure 3B) as well as the ratio of 
Figure 3: Activation of mTOR is associated with reduced mitochondrial respiration in PBMC from children. 
Activation of mTOR (p-mTOR) is associated with reduced basal respiration (A), maximal respiration (B), and
OXPHOS/Glycolysis (OCR/ECAR) (C).  
Methods: For determination of mTOR activation, phosphorylated mTOR (Ser2448) was measured using the 11-Plex 
Akt/mTOR phosphoprotein magnetic bead kit (Millipore) in PBMC from 43 children ages 2-18 years. Lysates were 
prepared from 2x106 PBMC from as directed by manufacturer. Median fluorescent intensity (MFI) was measured on a 
Luminex 200 and an average of 2 replicate wells is presented. For measures of Basal Respiration, Maximal Respiration 
and Maximal OCR/ECAR, freshly isolated PBMC were plated at 4x105 cells/well of a poly-D-lysine coated XF96 plate in 
RPMI supplemented with 1mM pyruvate, 2mM glutamine and 25mM glucose. Plates were centrifuged at 100xg with no 
brake to adhere cells to well bottoms. After a 30 min incubation in a 37°C non-CO2 incubator, cells were assayed in a 
Seahorse XF96 using the mitochondrial stress test (Agilent). Maximal respiration was measured after the addition of 
oligomycin and the uncoupler, carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP). Maximal OCR/ECAR 
represents maximal (FCCP-stimulated) respiration divided by maximal (oligomycin-stimulated) ECAR.  An average of at 
least 3 replicate wells from each subject is presented.  

Title:  Immunometabolism in Pediatric Obesity 
PI:  Shannon Rose, Ph.D.  
 
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Date:  19APR2019  
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OXPHOS/Glycolysis (OCR/ECAR; Figure 3C). These data demonstrate our ability to 
measure mTOR activation in small numbers of immune cells from children.  
 
Hypothesis, Specific Aims, and Objectives  
In this study, we hypothesize that compared to lean children, circulating CD4+T cells from 
obese children with elevated biomarkers of inflammation will have fewer Tregs and will 
exhibit increased mTOR-driven glycolysis. Metformin treatment will increase Tregs and 
shift the metabolic phenotypes of CD4+T cells towards AMPK-driven OXPHOS. We will 
test these hypotheses by executing the following Specific Aim: 
 
Specific Aim 1. Compare the immune and metabolic phenotypes of circulating CD4+T 
cells between lean children and overweight/obese children.   
 
Hypothesis: circulating CD4+T cells from obese children with elevated biomarkers of 
inflammation will have fewer Tregs and will exhibit increased mTOR activation and % 
glycolysis-derived ATP production as compared to CD4+T cells from lean children.  
 
Specific Aim 2. Compare the immune and metabolic phenotypes of circulating CD4+T 
cells between T2D children before and after metformin.   
 
Hypothesis: In obese T2D patients, metformin treatment will increase circulating Tregs, 
as well as AMPK activation and % OXPHOS-derived ATP production in CD4+ T cells.   
 
Study Design and Procedures 
This study consists of: 
1. An observational cross sectional immune and metabolic analysis of several groups 
of children including lean, overweight/obese, and T2D.   
2. A prospective immune and metabolic analysis of newly diagnosed children with 
T2D or insulin resistance who will be or were recently prescribed metformin as part 
of their clinical care.   
Children with T2D or insulin resistance who will be or were recently prescribed metformin, 
will be asked to complete two study visits. If completed, the second visit will occur 6 
months (+/- 2 weeks) after beginning metformin as part of their clinical care. All other 
children, will be asked to complete only the first visit. 
 
A prospective subject and his/her parent or legally authorized representative will come 
for the study visit that will last up to 2.5 hours depending on the duration of the consenting 
process and blood draw. A subject (and/or his/her parent) will be provided a questionnaire 
regarding the subject’s medical history that will be reviewed by study staff to identify 
patients who fulfill inclusion/exclusion criteria. In addition, study staff may review medical 
records from ACH or other healthcare practitioners (such as physicians or pharmacists) 
outside ACH in order to confirm the inclusion/exclusion criteria. Information from medical 

Title:  Immunometabolism in Pediatric Obesity 
PI:  Shannon Rose, Ph.D.  
 
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Date:  19APR2019  
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records may be collected from the time of consent up until the time that data analysis for 
this study is complete and results have been published. Study staff will ask parents about 
demographic information including biologic gender, date of birth, race/ethnicity, and 
parental occupations as well as grade in school. 
 
Prior to the study visit, study staff may contact the parent and ask questions relating to 
inclusion/exclusion criteria, for medications that the participant is currently taking and may 
ask of any new health diagnoses. In the event that the participant is taking medications 
or has a new health diagnosis that may cause him/her to be ineligible to participate in the 
study visit, the study visit may be rescheduled or canceled. Study staff may contact a 
parent and send the IRB approved consent form and information regarding the scheduled 
visit to the parent(s) prior to the scheduled date. If the consent form is revised prior to the 
scheduled visit, the new IRB approved consent form may be sent to parents. If applicable, 
the parent will be informed of the blood draw and advised that if the parent wishes their 
child to complete the blood draw at the visit, the child will need to have not taken seasonal 
allergy medication (e.g. Claritin) in the prior 72 hours and will need to be fasting including 
no food or liquid after 12 am, except for water and medications the day of the visit. 
 
At the initial study visit eligible participants will be met by study staff at the designated 
study location. Participants will arrive after not taking seasonal allergy medication in the 
previous 72 hours and an overnight fast of no food or liquid after 12 am, except for water 
and medications the day of the visit. The study visit may include collection of 
anthropometric measurements, tanner stage assessment, physical activity questionnaire, 
depression scale, body composition by bioelectrical impedance, blood, stool, and urine 
collection. Attempts to repeat anthropometric measurements, and body composition by 
bioelectrical impedance, will occur if a reading is not given the first time or if the child is 
unable to comply during the first measurement. For optional sample collection (i.e., urine 
and stool), the parent and participant will not have to stay at the research location for the 
entire visit in order for the samples to be obtained. Instructions will be given to the parents 
for collection of samples if the parent and child decide not to remain at the research 
location for the entire visit. 
 
If a procedure(s) (see below) is not successfully completed, the parent and, if warranted, 
the participant may be asked to return to the study site for an additional visit to re-attempt 
the procedure. The decision to ask the parent/participant, to return for another attempt 
will be made by the principal investigator. When a decision is made to re-attempt the 
procedure, study staff will contact the parent and explain what procedure they would like 
to re-attempt and if the parent is willing, schedule the visit. If a blood draw will be re-
attempted, the parent will be re-advised that if the parent wishes their child to complete 
the procedure, the child will need to not have taken seasonal allergy medication in the 
previous 72 hours and be fasting including no food or liquid after 12 am, except for water 
and medications the day of the visit. The study visit will be conducted in the same manner 
as the initial visit, with only the desired procedure(s) being re-attempted. 
 

Title:  Immunometabolism in Pediatric Obesity 
PI:  Shannon Rose, Ph.D.  
 
Version #:  5 
Date:  19APR2019  
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The collection of urine and stool samples is optional, and these samples may not be 
collected if the subject or parent does not want the sample(s) to be used for future 
research.  
 
Study Procedures to be conducted at each visit:  
 
1. 
Anthropometrics: Anthropometric measurements including height, weight, 
waist circumference, hip circumference, blood pressure, and pulse will be 
obtained using standardized techniques. BMI-for-age will be calculated and 
plotted on CDC growth charts to confirm lean/obese stratification. 
 
2. 
Tanner (Puberty) Stage Assessment: Parents will be asked to complete a form 
with drawings depicting pubertal development. This form is used to determine 
pubertal status. The questionnaire given to parents to complete is subjective 
and a parent could potentially incorrectly assess Tanner Stage. Because the 
results from the samples may be influenced by the participant’s pubertal 
development, pubertal development must be accounted for. At the beginning 
stages of puberty, insulin levels rise dramatically showing insulin resistance. 
Thus, if the study determines the child to have insulin resistance but insulin 
resistance is actually due to development of puberty instead of being 
metabolically unhealthy then this could alter the results and conclusions made 
from the study. Because of this, it is important to the study and the results to 
have the correct Tanner Stage level for each participant. In order to validate 
the parent’s assessment of Tanner Stage level, hormone tests may be 
conducted on the blood sample obtained.  
 
3. 
Physical Activity Questionnaire (Elementary School): A parent or participant will 
complete a form indicating the level of physical activity the participant had 
during the previous week in order to provide general estimates of physical 
activity levels for each subject during a 7-day period prior to the study. In 
addition, the parent or participant will be asked about the participant’s physical 
activity from the previous months leading up to the study visit. 
 
4. 
Depression Scale: With assistance from the parent, a child will complete a form 
(Center for Epidemiological Studies Depression Scale for Children (CES-DC)) 
used as a depression scale. This scale will be used to account for depression 
as a confounding factor that could affect oxidative and bioenergetics 
parameters.  
 
5. 
Body composition may be assessed by bioelectrical impedance analysis using 
the Tanita Body Composition Analyzer. A device (InBody 570 Body 
Composition Analyzer) similar to the Tanita, is routinely used in the clinic. The 
Tanita requires participants to stand still with bare feet on the weighing platform 
while a small amount of electrical current is used to measure impedance.   
 

Title:  Immunometabolism in Pediatric Obesity 
PI:  Shannon Rose, Ph.D.  
 
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Date:  19APR2019  
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6. 
Blood Collection: A pediatric nurse or trained phlebotomist will collect blood 
from the participant under fasted conditions and with no seasonal allergy 
medications taken in the previous 72 hours. Depending on the child’s weight, 
up to the maximum allowed per the guidelines provided by Arkansas Children’s 
for the maximum allowable single blood draw volumes will be collected. The 
volumes are derived using a conservative 3.0 ml/kg estimate as well as a 5% 
of total body volume estimate. Numbing cream, numbing spray, or a pain 
relieving device (Buzzy) may be used for the blood draw. Blood samples may 
be used for: 
a. Immunophenotyping: Mitogen-stimulated blood will be stained with 
combinations of fluorochrome-labeled antibodies and analyzed by flow 
cytometry. 
b. CD4+ T cell isolations: CD4+T cells will be isolated by negative selection 
using antibody-labeled magnetic beads for analysis of metabolic control 
pathway activation and bioenergetics  
i. Metabolic control pathway analysis: Using cell lysates from isolated 
CD4+T cell lysates will be analyzed for multiple analytes in metabolic 
control pathways using multiplex immunoassays, ELISA’s and/or 
western blot. 
ii. Bioenergetics analysis: CD4+T cells will be bioenergetically 
phenotyped using the Seahorse Extracellular Flux (XF)96 Analyzer, 
protocols for such have been established by Rose38, 39. Cellular 
bioenergetics may be analyzed immediately, or cells may be 
cryopreserved and analyzed at a later time. Leftover cells may be 
cryopreserved for repeated bioenergetic profiling. 
c. Quantification of plasma CRP, pro-inflammatory and anti-inflammatory 
cytokines and adipokines: We will use multiplex immunoassays to quantify 
levels of CRP as well as cytokines and adipokines.  
d. Analyses of HbA1c, insulin, glucose, lipid levels, and other analytes 
including, but not limited to, redox metabolites, and hormones.  
e. Leftover blood samples collected during this study may be stored in the PI’s 
laboratory and used for future research studies on pediatric nutrition or 
metabolism.  
 
7. 
Urine collection (Optional): Urine collection may occur at the study location or 
the sample may be collected at home and brought back to study staff. When 
possible, attempts will be made to collect fasting urine. Urine collection will be 
attempted in a sterile specimen cup or in a hat and then transferred to a 
specimen cup. Samples collected during the study visit will be placed on ice, in 
an ice bath, or in a refrigerator until they can be delivered to the research site 
laboratory. Samples collected by subjects/parents after the study visit may be 
stored in a tightly closed specimen cup in a refrigerator until they are able to 
return to the research site, to occur within 6 weeks of collection. Samples 
delivered to the research site will be stored in a refrigerator until they can be 

Title:  Immunometabolism in Pediatric Obesity 
PI:  Shannon Rose, Ph.D.  
 
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Date:  19APR2019  
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transferred to the research laboratory and stored at -80°C. This sample may be 
used for future research studies involving pediatric nutrition or metabolism. This 
sample is optional and is not required for the completion of the study visit. 
 
8. 
Stool collection (Optional): Stool collection may occur at one of the study 
locations or the sample may be collected at home and brought back to study 
staff. Attempts will be made to collect the sample using a hat and then 
transferring it to a sterile specimen cup(s). Samples collected during the study 
visit will be placed on ice, in an ice bath, or in a freezer until they can be 
delivered 
to 
the 
research 
site 
laboratory. 
Samples 
collected 
by 
subjects/parents after the study visit may be stored in a tightly closed specimen 
cup in a freezer until they are able to return to the research site, to occur within 
6 weeks of collection. Samples delivered to the research site will be stored in 
a freezer until they can be transferred to the research laboratory and stored at 
-80°C. This sample will be used in future research studies involving pediatric 
nutrition or metabolism. This sample is optional and is not required for the 
completion of the study visit. 
 
If agreed to, the second visit for children with T2D or insulin resistance who will be or 
were recently prescribed metformin will occur 6 months (+/- 2 weeks) after beginning 
metformin as part of their clinical care. This additional study visit is optional as we do not 
want to discourage potential enrollment due to the possibility of multiple visits or lengthy 
visits. We anticipate 50% of the participants in this group will decline a 2nd study visit with 
a goal of 10 subjects completing a 2nd visit at 6 months (± 2 weeks).The second study 
visit will include the same procedures as the first study visit. In addition, 
participants/parents will be asked to verify their compliance with metformin by bringing 
their medication to the visit to show study staff and asking the parent/participant to report 
their compliance. As done for the first study visit, if a procedure(s) is not successfully 
completed, the parent and, if warranted, the participant may be asked to return to the 
study site for an additional visit to re-attempt the procedure. The decision to re-attempt 
the procedure(s) will be made by the principal investigator and the same steps as 
described for the re-attempt of a procedure from the initial visit followed. 
 
A participant/parent pair will be compensated for each study visit completed. For the first 
and, if applicable, second study visit, a $50 gift card (or gift cards totaling $50) will be 
provided to the study participants/parents after completion of the procedures to be 
completed at the research site (e.g., anthropometric measurements, Tanner Stage 
Assessment, physical activity questionnaire, depression scale, body composition, and 
blood collection). Parents/participants with T2D or insulin resistance who are prescribed 
metformin therapy will be asked to complete two study visits and will be paid up to $100.00 
in gift cards ($50.00 in gift cards at the first study visit completed and $50.00 in gift cards 
at the second visit completed). Other parents/participants will be asked to complete one 
visit and will be paid up to $50 in gift cards for this completed visit. Those 
participants/parents who are asked to return to re-attempt procedure(s) not successfully 
completed at a study visit (initial or second) will be given a $25 gift card after re-attempting 

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to complete the procedure(s). At the first, second, or reattempt visits, participants/parents 
may be given up to $20 in ACRI merchandise and/or child-friendly stickers. Food and 
drinks may be provided during study visits following the fasting blood draw. 
 
Study Population 
 
110 children ages 5-17 years old will be recruited with the goal that approximately 80 
subjects stratified across the following groups will complete the study: i) healthy lean 
(approximately n=20); ii) overweight/obese (approximately n=40, with the anticipation that 
approximately 20 of these children will be insulin resistant); iii) overweight/obese with T2D 
and prescribed Metformin (approximately n=20). Every attempt will be made to ensure 
that the desired number for each group is achieved and groups are balanced with respect 
to age, sex and ethnicity.  
Inclusion Criteria:  
• Age 5-17 years, inclusive 
• Either healthy lean (BMI≥ 5th percentile and <85th percentile for age/sex) or 
overweight (BMI ≥ 85th percentile and <95th) or obese (BMI ≥ 95th percentile for 
age/sex) 
• For those with BMI≥ 85th percentile for age/sex, parental verbal confirmation that 
the child had a history of BMI≥ 85th percentile for age/sex for at least six months 
prior to study enrollment 
OR 
• Age 5 years - 17 years 5 months, inclusive 
• Either overweight or obese (BMI≥ 85th percentile for age/sex)  
• Parental verbal confirmation that the child had a history of BMI≥ 85th percentile for 
age/sex for at least six months prior to study enrollment 
• Diagnosed with type 2 diabetes mellitus or insulin resistance  
• Prescribed metformin (either not yet taking or began taking within 3 weeks of 
enrollment) 
Exclusion criteria: 
• Having an infection (viral, respiratory, gastrointestinal) in the previous 4 weeks 
• Genetic or physical conditions impacting mobility over past year as determined by 
the PI  
• Having known chronic illnesses/disorders that may independently affect study 
outcome measures: type 1 diabetes mellitus, neurologic (e.g. epilepsy), 
developmental (developmental delay, autism spectrum disorder), endocrine 
(thyroid, Cushing’s), hepatic, autoimmune, cardiac and renal disorders.  Also, 
chronic lung disorders except well controlled asthma that does not require 
permanent use of inhaled/oral steroids 

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• Taking any of the following medications that can affect study outcome: 
antipsychotics, thyroid hormone replacement therapy, inhaled/oral steroids, 
insulin, anabolic drugs (growth hormone replacement therapy and oxandrolone) 
and stimulants 
• Taking metformin prescribed as part of their clinical care for longer than 3 weeks 
at the time of enrollment (may begin metformin therapy prescribed as part of their 
clinical care while enrolled in the study) 
• BMI<5th percentile for age/sex (classified as underweight based on CDC growth 
charts) 
• Subjects determined ineligible by the PI or delegated staff. 
Recruitment:  
 
Study investigators, research staff, or any qualified personnel will conduct recruitment of 
study participants using IRB approved advertisements and methods.  
 
We will recruit some subjects who agreed to be contacted for future research from an 
ongoing human research study, Bioenergetics and Metabolism in Pediatric Populations 
(BMPP; UAMS IRB #206164; Rose and Carvalho, Co-PIs). In the BMPP study, we have 
enrolled 55 children in a period of 1 year and thus, we do not anticipate difficulty meeting 
this recruitment goal as the recruitment criteria are relaxed, with a wider age range, for 
the proposed study. 
 
Advertisements may be distributed in the form of postcards or flyers via direct mail, at 
information booths or events, or to various locations for posting or distributing, including 
pediatricians’ offices, health fairs, daycare centers, schools/universities, recreational 
centers, grocery stores, supermarkets, child retail stores, websites (ACH, ACRI, ACNC, 
UAMS, and others as applicable), and churches. Also, print or digital ads may appear in 
newspapers, magazines, social media, newsletters, and circulars. On-hold phone 
messages, BoomText messages, screensavers, and radio/television advertisements may 
be used. In addition, research staff may contact parents who expressed an interest in our 
studies or who previously agreed to be contacted regarding future studies. Research staff 
may also contact parents who have signed up for the AC research registry. The research 
staff will educate the parents and children about the study and if interested determine 
potential eligibility. At the visit, study staff would obtain informed consent in person. 
 
Study staff will also work with healthcare providers at ACH and outside of ACH to identify 
subjects that may qualify. Study staff may assist healthcare providers at ACH with 
reviewing patients who have upcoming appointments with them to identify those who may 
qualify based on their electronic medical records at Arkansas Children’s. When a patient 
is identified by the study staff or healthcare provider, the healthcare providers will 
introduce the study to their patients (e.g., provide letter/flyer and/or briefly describe the 
study); if the patient is interested, the physician may obtain contact information so that 
the study staff can follow up with the parent or encourage the patient to contact study 

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staff. Alternatively, the healthcare provider may introduce their patient directly to study 
staff if study staff is available to meet the patient during the visit. Healthcare providers 
outside of ACH may provide their patient with a letter/flyer or, if the patient is interested, 
may use the recruitment HIPAA authorization form to obtain permission from the parent 
and use this form to provide their information to study staff. This form can then be sent to 
study staff so that they can follow-up with the parent. The only contact by study staff with 
those identified by healthcare providers would be to explain the study, determine potential 
eligibility, and schedule a visit.  At the visit, study staff would obtain informed consent in 
person.  
 
Study staff will also use the electronic medical records (EMR) at Arkansas Children’s to 
identify children who may be eligible to participate. When a child is identified who has a 
MyChart account we will post this study’s information in their MyChart account under the 
available studies page. This page lists all studies the child may be eligible for that have 
chosen to recruit using MyChart. On this page, the parent can indicate if they are 
interested or not. If they are interested, the system will notify the research team so they 
can follow-up with the parent. In addition, when a child is identified, study staff may work 
with the child’s health care providers to inform the parent about the study. This may be 
done by providing/mailing the parent a letter/flyer to let the parent know about the 
research opportunity their child may be eligible for. If the parent is interested, the 
physician may obtain contact information so that the study staff can follow up with the 
parent or encourage the patient to contact study staff. If the parent indicates an interest 
in the study, study staff will proceed to determine potential eligibility and scheduled a visit 
where informed consent would be obtained in person.  
 
We will protect all personal information obtained during recruitment, enrollment, and 
testing processes, and maintain this in a locked drawer or cabinet. 
 
Risks and Benefits 
There is the potential risk of loss of confidentiality to study participants. Measures to 
protect the confidentiality of study participants will be implemented by applying the 
appropriate steps to secure the collected data as described in the Data Handling and 
Recordkeeping section below. There is a small risk that participants will encounter 
bruising and/or infection after having blood taken, however, the use of well-established 
blood taking techniques, and sterile procedures, by trained phlebotomists or nurses, will 
ensure the risk is minimal. Also, numbing cream, numbing spray, and/or a pain relieving 
device (Buzzy) may be used to minimize the pain associated with the blood draw. There 
is the risk that researchers could develop ways in the future to link genetic information 
back to the subjects even though the stored samples do not include any personal 
identifiers, except for the assigned unique study code, study visit number, and the study 
acronym. Any adverse effects or unanticipated problems will be reported to the study PI, 
the IRB, and the study sponsor in accordance with IRB Policy 10.2. There will be no direct 
benefits to the study participants; however, knowledge gained from the study could 
potentially benefit patients in the future. 

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Data Handling and Recordkeeping 
The Principal Investigator will carefully monitor study procedures to protect the safety of 
research subjects, the quality of the data and the integrity of the study.  All study subject 
material will be assigned a unique identifying code or number.  The electronic key to the 
code will be kept in a secure, access-restricted, pass-code required, location created 
specifically for the study. Only the PI and research personnel will have access to the code 
and information that identifies the subject in this study. 
The PI and research study personnel will complete and maintain appropriate CITI training. 
Source documents and CRFs will be stored in a locked location. Documents will be 
archived according to UAMS/ACH/ACRI policies regarding destruction of research 
records.  
Data will be entered into a research database. Access to the study database(s) is 
password protected and will be limited to study personnel and regulatory authorities. 
At the time of collection, samples will be kept on ice or refrigerated (with the exception of 
stool which will be kept in a freezer) in the pediatric clinical research unit or in Dr. Rose’s 
laboratory (Rm 4015 of ACRI) until they can be transported to the research or hospital 
laboratory.  
 
For testing to be completed by the Arkansas Children’s hospital laboratory (e.g., HbA1c), 
sample(s) will be labeled with the participant’s medical label generated through EPIC 
which includes the participant’s name, date of birth, gender, and medical record number. 
This sample will be provided to the hospital laboratory where they will complete the 
testing, storage, and disposal of the sample per their standard operating procedures. 
Results will be reported through the participant’s medical record.  
 
For testing to be completed by Dr. Rose’s laboratory, samples will be kept at -80°C once 
the initial processing and analyses have taken place. These samples will be stored in the 
Arkansas Children’s Research Institute, Room R4017 Freezers 4017FT12 or R4017FT8 
or R2108 Freezer R2108FT16. Cryopreserved samples may also be kept under liquid 
nitrogen in the Arkansas Children’s Research Institute, Room R4017. These are 
monitored continuously for proper temperature and working condition. None of a subject’s 
personal identifiers will be present on any biological sample, except the unique subject 
ID, study visit number, and the study acronym. The samples may be used until they are 
used up for research on pediatric nutrition or metabolism.  
 
At any time if the subject decides that he/she does not want to participate anymore, that 
subject’s data will be included as part of the planned analysis of study data. No more 
information will be collected after withdrawal, however all data and samples collected prior 
to withdrawal will still be used. 
 
Information pertaining to this study will be made publically available through the 
ClinicalTrials.gov website. 
Data Analysis 

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Prior to any analyses, endpoints data for each group will be closely inspected for the 
potential presence of unusual values; effort will be made to conserve all data. Analyses 
will be carried out in SAS 9.4 (SAS Institute, Cary NC) at the 5% significance level; point 
and 95% interval estimates will be obtained for relevant metabolic group comparisons. 
Parametric techniques will be first choice and nonparametric equivalents if assumptions 
are not met. Special attention will be paid to unequal variances.  
 
The study planned four groups of children: lean, healthy obese/overweight, insulin-
resistant obese/overweight, and overweight/obese with T2D. Primary comparison of 
interest are these three: the average of insulin-resistant and T2D obese children 
compared to (i) lean children and (ii) healthy obese children; and (iii) lean children 
compared to healthy obese children. Outcomes important to this aim are proportions of 
Treg cells among circulating CD4+T cells and CD4+T glycoATP and mitoATP production. 
Initially, we will use a one-factor ANOVA to analyze these outcomes (possibly with 
suitable transformations). Comparisons of interest will then be made with 0.05 
significance level t-tests conducted within the ANOVA framework.  
 
The requested sample sizes will provide about 0.80 power for detecting differences in 
means of size 0.8 SD for comparisons (i) and (ii) and size 0.9 SD for comparison (iii), 
where SD is the within-group standard deviation (a.k.a., root mean square error from the 
ANOVA). For proportions of Treg cells – a primary outcome of interest, an estimate of the 
SD is 2.3 percentage points (ref Table 1 of Arismendi et al,42). So, if the means differ by 
at least 1.85 percentage points in comparisons (i) & (ii) and by 2.10 percentage points for 
comparison (iii), we will be able to detect these differences with a reasonably high 
probability (0.80 or higher). 
 
Ethical Considerations 
This study will be conducted in accordance with all applicable government regulations 
and University of Arkansas for Medical Sciences research policies and 
procedures.  This protocol and any amendments will be submitted and approved by the 
UAMS Institutional Review Board (IRB) to conduct the study.   
The formal consent of each subject, using the IRB-approved consent form, will be 
obtained before that subject is submitted to any study procedure.  All participants for this 
study will be provided a consent form describing this study and providing sufficient 
information in language suitable for participants/parent/legally authorized guardian to 
make an informed decision about their participation in this study.  The person obtaining 
consent will thoroughly explain each element of the document and outline the risks and 
benefits, alternate treatment(s), and requirements of the study.  The consent process will 
take place in a quiet and private room, and participants/parent/legally authorized guardian 
may 
take 
as 
much 
time 
as 
needed 
to 
make 
a 
decision 
about 
their 
participation.  Participation privacy will be maintained and questions regarding 
participation will be answered.  No coercion or undue influence will be used in the consent 
process.  This consent form will be signed by the parent or legally authorized guardian. 
Written assent will be obtained from participants aged 7-17 years, inclusive. A copy of the 

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signed consent will be given to the participants/parent/legally authorized guardian, and 
the informed consent process will be documented in each subject’s research record. A 
signed copy of the consent form may be placed in patients’ medical record. We request 
a waiver of consent for subjects who turn 18 while their samples are still available for use 
and who may still have data collected from their medical records, for the purposes of this 
study and/or for future research studies if consent obtained for this purpose. If the waiver 
is approved by the institutional review board and samples are still available for use/data 
is still being collected when a participant turns the age of 18, the participant will not be re-
contacted to sign a consent form. However, these participants can contact study staff 
after they turn 18 to ask that any remaining samples be removed and data collection stop. 
In addition, participants may be re-contacted about participating in future research 
studies. 
Initially, samples will be stored at Arkansas Children’s Hospital and/or Arkansas 
Children’s Research Institute. The sample provided to the Arkansas Children’s Hospital 
laboratory will be tested, stored, and disposed of per their standard operating procedures 
and will not be used for future research studies. The information collected at the study 
visit, urine, and stool samples, and remaining blood samples may be stored indefinitely 
and may be used for future research studies on pediatric nutrition or metabolism. Prior to 
the information collected at the study visit and samples being used for future research 
studies, the PI will assess the ethics and scientific merit of the proposed research with 
the samples, and proposed future research will be reviewed by the IRB as may be 
required. The samples and health information collected for the study visit may be shared 
with researchers at the University of Arkansas for Medical Sciences, Arkansas Children’s 
Hospital, or Arkansas Children’s Research Institute. The samples may be shared with an 
outside group. The samples will only have a study ID number, study visit number, and 
study acronym to maintain confidentiality.  If participants decide they no longer want us 
to use their samples for future research studies, they may ask the study staff that the 
sample be removed. If the sample has been shared or if publication of results has 
occurred, then we may not be able to remove the sample.  
A summary of the study visit will be provided to the parent indicating the participant’s 
anthropometric and body composition measurements and the CES-DC results from the 
study visit. The parent will be directed to follow-up with their child’s PCP if they have 
concerns. If the parent has concerns and the child does not have a PCP, research staff 
will assist the parent with identifying a PCP. If participant scores a 15 or greater on the 
CES-DC, suggesting that the participant expresses symptoms of depression, research 
staff will follow-up with the parent to explain these results, discuss the need to follow-up 
with the child’s PCP, and provide the parent with the ‘Resources to Seek Help for 
Depression in Children’ form. If the child does not have a PCP, research staff will assist 
the parent with identifying a PCP.  
Dissemination of Data 
Results of this study may be used for presentations, posters, or publications. The 
publications will not contain any identifiable information that could be linked to a 
participant.   

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