Clinical trial NCT02960659
Conditions: Type 2 Diabetes
Keywords: Diabetes Metformin Glucose
Sponsor: National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Start date: 2017-05-09; Completion date: 2022-05-20
Enrollment: 24 participants; Allocation: RANDOMIZED; Intervention model: PARALLEL

Brief summary:
Background:

The pill metformin treats diabetes. But it does not work for all youth, especially African-Americans. The injectable Liraglutide treats type 2 diabetes in adults. Researchers want to understand how these drugs work and if they decrease excess sugar made by the liver in youth with type 2 diabetes.

Objective:

To test if using liraglutide and metformin are better than just metformin for decreasing excess sugar produced by the liver in African-American youth with type 2 diabetes.

Eligibility:

African-Americans ages 12-25 with type 2 diabetes

Design:

Visit 1: Participants will be screened with medical history, physical exam, and blood and urine tests. Participants will stop taking diabetes medicines for 1 week. They will learn how to check blood sugars at home twice a day.

Visit 2: Overnight at the clinic. Participants will have:

Vital signs taken.

Pregnancy test.

A thin plastic tube (IV catheter) be inserted in each forearm by needle.

Blood drawn several times after drinking a sweet drink.

X-ray of total body fat.

Urine and stool collected.

Breath tests while wearing a clear hood for up to 45 minutes.

For several hours, participants can have only water. At 4 a.m. they will get sugar and fat with nonradioactive isotopes in one IV. Blood will be collected. Every 30 minutes from 9 a.m. to 2 p.m., they will drink small amounts of a shake and have blood drawn.

Participants will be randomly assigned to take either both study drugs daily or just metformin daily.

Visits 3-4: Participants will bring their blood sugar records and have blood tests.

Visit 5, after 3 months: Repeat of visit 2....

Detailed description:
Type 2 Diabetes in youth is an emerging public health concern that disproportionately affects minority children. Among minority youth, African-Americans have the highest complication rates, yet the reasons underlying this health disparity are not fully understood. Furthermore, current treatment options are limited, and African-American youth have high treatment failure rates. Metformin therapy is the only oral diabetes drug approved for use in youth with type 2 diabetes. However, metformin works less than 50% of the time in African-American youth and there is marked variability among individuals. Improving outcomes in youth requires understanding the way that drugs such as metformin work in youth and why it does not work in some individuals. New evidence suggests that the ability of metformin to work effectively may be influenced by certain genes or differences in gut bacteria. However, little is known about how genes or gut bacteria may affect youth, especially African-Americans.

To treat this aggressive disease, it is also necessary to simultaneously evaluate new therapeutic options, such as combination therapy of metformin with liraglutide in youth at highest risk for complications. Liraglutide is approved to treat type 2 diabetes in patient 10 years and older as an adjunct to diet and exercise. Liraglutide may be a useful early treatment in youth with type 2 diabetes because it may decrease glucose produced by the liver (an early prominent feature of type 2 diabetes in youth). This study is designed to examine the mechanism of action in the liver of these 2 agents and explore how genetic and gut factors may influence this action.

The primary objective of this pilot study is to compare the ability of two anti-diabetic regimens (metformin and liraglutide versus metformin alone) to lower gluconeogenesis (glucose produced by the liver) in African-American youth with type 2 diabetes. The secondary objectives are to evaluate the effect of these regimens on the following: (1) hepatic glucose production, and insulin sensitivity and (2) insulin and gut hormones concentrations (e.g. incretins). In addition, we will examine the relationship of known differences in genes associated with metformin transport and action with changes in gluconeogenesis and begin to explore the role of gut bacteria to metformin s glucose-lowering effect.

The study design is a parallel-randomized intervention trial of African-American youth with type 2 diabetes who are not on insulin therapy and who are within 5 years of diagnosis. Patients aged 12- 25 years with type 2 diabetes will be enrolled. Participants will be randomized into two intervention arms (16 in each group): metformin and liraglutide versus metformin alone. The study will consist of 5 visits. At Visit 1, a medical history, physical examination and screening labs will be done. Then the eligible participants will undergo a one-week drug-free run-in. At Visit 2 there will be an overnight inpatient stay to perform metabolic testing prior to starting the study drug(s). Participants will start the study drug(s) immediately after Visit 2 and remain on the study drug(s) for 12 weeks. Follow-up monitoring will be performed at 4-week intervals (Visit 3 and 4). The final visit (Visit 5) will occur after 12 weeks.

The ultimate goal of this multi-site project is to begin to address diabetes disparities in African-American youth by understanding the mechanism of action of these diabetes agents to inform precision medicine initiatives. This project brings together the skills and expertise of investigators within the National Institute of Diabetes and Digestive Disorders and Kidney Diseases (NIDDK), the National Human Genome Research Institute (NHGRI), and the Children s National Medical Center (CNMC). Patient recruitment and data collection will occur at NIH Clinical Center. Eligible patients may be identified through CNMC but no enrollment, informed consent or study visits will occur at CNMC.

Arms: arm 1: Standard release metformin (500 mg tablets) was initiated and titrated to maximum dose of metformin 1000 mg twice daily over 3 weeks and continued for 12 weeks arm 2: Standard release metformin (500 mg tablets) and liraglutide (0.6mg) were initiated and titrated to maximum dose of metformin 1000 mg twice daily, and liraglutide 1.8 mg daily over 3 weeks and continued for 12 weeks
Interventions: intervention 1: Liraglutide intervention 2: Metformin
Intervention descriptions: intervention 1: Liraglutide (6mg/ml, 3ml ) solution for subcutaneous injection, pre-filled, multi-dose pen that delivers doses of 0.6mg, 1.2mg or 1.8mg. intervention 2: Metformin 500mg oral tablet

Protocol documents:
Prot_SAP_000.pdf: 
Principal Investigator: Chung, Stephanie T.  
Last updated: October 5, 2021
Page 1
Clinical Research Protocol
National Institute of Diabetes and Digestive and Kidney Diseases
Protocol Number: 17-DK-0013
Protocol Version: October 5, 2021
[IND] [IDE] NUMBER:  N/A 
[IND] [IDE] NAME:  N/A IND exemption
[IND] [IDE] HOLDER:  N/A IND exemption
[IND] [IDE] MFG:  N/A IND exemption
Title: Therapeutic targets in African-American youth with type 2 diabetes
Short Title: Type 2 Diabetes in AA youth
Identifying Words: gluconeogenesis, pharmacogenetics, metformin, liraglutide
Principal Investigator:           Stephanie T. Chung, M.B.B.S. 
          Diabetes, Endocrinology, and Obesity Branch (DEOB)
          National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
          Building 10, Room 5-5942
          10 Center Drive
          Bethesda, MD 20814
          Phone: 301-402-2122
          Email: Stephanie.chung@nih.gov
Estimated Duration of Study: 6 years
Start Date: Fall 2016
End Date: Fall 2022
Number and Type of Patients:  Accrual Ceiling: 92
Number
Sex
Age Range
Ethnic Group
Patients:
92
Male/Female
12- 25 years
African-American only
Volunteers
0
-
-
-
Project Uses Ionizing Radiation: 
_X__Research indicated:      RSC Approval Number:
2581
 Expiration Date: 
May 2019
Project Uses “Durable Power of Attorney”: No                        Off-site Project: No
Multi-site enrollment: Yes
Multi-institutional project: Yes; Patient recruitment and data collection will occur at NIH Clinical Center and 
Pennington Biomedical Research Center (PBRC).  
Patient recruitment only will be conducted at Children’s National Medical Center.  
Pennington Biomedical Research Center (PBRC) site is CLOSED effective 10/05/2021. While a reliance agreement was 
executed, research was never initiated at this site.


Principal Investigator: Chung, Stephanie T.  
Last updated: October 5, 2021
Page 2
Table of Contents
Specific Aims--------------------------------------------------------------------------------------------------------------------4
Background ---------------------------------------------------------------------------------------------------------------------5
Metformin ------------------------------------------------------------------------------------------------------------------------------------5
Metformin Pharmacogenomics ------------------------------------------------------------------------------------------------------------7
Metformin Response and Pharmacokinetics---------------------------------------------------------------------------------------------8
Combination therapy of metformin and liraglutide -------------------------------------------------------------------------------------9
Study Objectives--------------------------------------------------------------------------------------------------------------10
Study Design and Methods -------------------------------------------------------------------------------------------------11
Study Design--------------------------------------------------------------------------------------------------------------------------------11
Randomization -----------------------------------------------------------------------------------------------------------------------------11
Study Protocol------------------------------------------------------------------------------------------------------------------------------11
Study Agents/ Intervention-------------------------------------------------------------------------------------------------15
Follow-up -----------------------------------------------------------------------------------------------------------------------------------16
Inclusion and Exclusion Criteria------------------------------------------------------------------------------------------17
Inclusion Criteria---------------------------------------------------------------------------------------------------------------------------17
Primary randomized protocol ------------------------------------------------------------------------------------------------------------17
Exclusion Criteria--------------------------------------------------------------------------------------------------------------------------17
Clinical and Laboratory Methods ----------------------------------------------------------------------------------------19
Metformin Pharmacokinetics----------------------------------------------------------------------------------------------21
Collection and Storage of Human Specimens or Data----------------------------------------------------------------21
Research Use, Storage and Disposition of Human Subjects’ Samples -------------------------------------------------------------21
Genomic Data Sharing Plan --------------------------------------------------------------------------------------------------------------22
Materials Transfer Information ----------------------------------------------------------------------------------------------------------22
Statistical and Power Analyses --------------------------------------------------------------------------------------------22
Multiple-site Studies ---------------------------------------------------------------------------------------------------------24
Human Subject Protection -------------------------------------------------------------------------------------------------24
Informed Consent/Assent Procedures---------------------------------------------------------------------------------------------------24
Rationale for Subject Selection ----------------------------------------------------------------------------------------------------------25
Rationale for Exclusion of Vulnerable Populations -----------------------------------------------------------------------------------25
Recruitment Strategies --------------------------------------------------------------------------------------------------------------------25
Risks/Benefits Analysis including Considerations of Alternatives to Participation ----------------------------------------------26
Adverse Event Reporting ---------------------------------------------------------------------------------------------------31
Event Characterization and Reporting to the IRB -------------------------------------------------------------------------------------31
Investigational New Drug Application/ Exemption-----------------------------------------------------------------------------------31
Data and Safety Monitoring Plan -----------------------------------------------------------------------------------------31
Monitoring Subjects and Criteria for Withdrawal of Subjects from the study-----------------------------------------------------32
Protection of Participant’s Privacy and Confidentiality -------------------------------------------------------------32


Principal Investigator: Chung, Stephanie T.  
Last updated: October 5, 2021
Page 3
Compensation -----------------------------------------------------------------------------------------------------------------32
Conflict of Interest -----------------------------------------------------------------------------------------------------------33
Appendix A: Sample gastrointestinal questionnaire------------------------------------------------------------------34
Appendix B: Continuous glucose monitoring (CGM) and blood glucose management instructions--------35
References----------------------------------------------------------------------------------------------------------------------35
Précis 
Type 2 Diabetes in youth is an emerging public health concern that disproportionately affects minority children. Among 
minority youth, African-Americans have the highest complication rates, yet the reasons underlying this health disparity 
are not fully understood.  Furthermore, current treatment options are limited, and African-American youth have high 
treatment failure rates.  Metformin therapy is the only oral diabetes drug approved for use in youth with type 2 diabetes.  
However, metformin works less than 50% of the time in African-American youth and there is marked variability among 
individuals.  Improving outcomes in youth requires understanding the way that drugs such as metformin work in youth 
and why it does not work in some individuals.  New evidence suggests that the ability of metformin to work effectively 
may be influenced by certain genes or differences in gut bacteria.  However, little is known about how genes or gut 
bacteria may affect youth, especially African-Americans.  
To treat this aggressive disease, it is also necessary to simultaneously evaluate new therapeutic options, such as 
combination therapy of metformin with liraglutide in youth at highest risk for complications.  Liraglutide is approved to 
treat type 2 diabetes in patient 10 years and older as an adjunct to diet and exercise.  Liraglutide may be a useful early 
treatment in youth with type 2 diabetes because it may decrease glucose produced by the liver (an early prominent feature 
of type 2 diabetes in youth).  This study is designed to examine the mechanism of action in the liver of these 2 agents and 
explore how genetic and gut factors may influence this action.  
The primary objective of this pilot study is to compare the ability of two anti-diabetic regimens (metformin and liraglutide 
versus metformin alone) to lower gluconeogenesis (glucose produced by the liver) in African-American youth with type 2 
diabetes. The secondary objectives are to evaluate the effect of these regimens on the following: (1) hepatic glucose 
production, and insulin sensitivity and (2) insulin and gut hormones concentrations (e.g. incretins).  In addition, we will 
examine the relationship of known differences in genes associated with metformin transport and action with changes in 
gluconeogenesis and begin to explore the role of gut bacteria to metformin’s glucose-lowering effect.
The study design is a parallel-randomized intervention trial of African-American youth with type 2 diabetes who are not 
on insulin therapy and who are within 5 years of diagnosis.  Patients aged 12- 25 years with type 2 diabetes will be 
enrolled.  Participants will be randomized into two intervention arms (16 in each group): metformin and liraglutide versus 
metformin alone. The study will consist of 5 visits.  At Visit 1, a medical history, physical examination and screening labs 
will be done. Then the eligible participants will undergo a one-week drug-free run-in.  At Visit 2 there will be an 
overnight inpatient stay to perform metabolic testing prior to starting the study drug(s).  Participants will start the study 
drug(s) immediately after Visit 2 and remain on the study drug(s) for 12 weeks. Follow-up monitoring will be performed 
at 4-week intervals (Visit 3 and 4).  The final visit (Visit 5) will occur after 12 weeks.  
The ultimate goal of this multi-site project is to begin to address diabetes disparities in African-American youth by 
understanding the mechanism of action of these diabetes agents to inform precision medicine initiatives.  This project 
brings together the skills and expertise of investigators within the National Institute of Diabetes and Digestive Disorders 


Principal Investigator: Chung, Stephanie T.  
Last updated: October 5, 2021
Page 4
and Kidney Diseases (NIDDK), the National Human Genome Research Institute (NHGRI), and the Children’s National 
Medical Center (CNMC). Patient recruitment and data collection will occur at NIH Clinical Center.  Eligible patients may 
be identified through CNMC but no enrollment, informed consent or study visits will occur at CNMC.
Introduction and Significance
Type 2 diabetes is a leading cause of non-communicable disease worldwide and afflicts over 29 million individuals in the 
United States.  Rates of type 2 diabetes in youth are expected to precipitously rise over the next 20 years and African-
American youth have a 2 fold increased risk for type 2 diabetes and a 4 fold increased risk for complications compared to 
white youth1-3.  Race/ ethnic differences in biological characteristics and/ or pharmacogenomics may contribute to 
variations in risk for diabetes and/ or medication response.  While declining insulin secretion and increasing insulin 
resistance are the pathophysiological hallmarks of type 2 diabetes, African-American youth may also have additional 
predisposing factors. Specifically, lower glucagon-like peptide 1 (GLP-1) concentrations in African-American than white 
youth may contribute to a rapid deterioration in insulin secretion and progression to diabetes4,5.  Currently, metformin 
therapy is the only oral agent approved for treating type 2 diabetes in youth6.  However, African-American youth with 
type 2 diabetes have the highest metformin failure rates, and more effective treatment options are urgently needed to 
reduce the physical, psychological and economic burden of this disease7.  
Optimizing initial therapy could dramatically improve outcomes because type 2 diabetes in youth is associated with 
increased risk for morbidity and mortality within 5-10 years of diagnosis8.  Therapeutic regimens should target the key 
pathophysiologic mechanisms of type 2 diabetes in youth (β-cell failure9 and increased gluconeogenesis10).  At present, 
metformin is the first line agent recommended, but metformin alone may not be sufficient to address the severe and 
accelerated metabolic decompensation observed in youth 6.  We propose that combination therapy with metformin and 
liraglutide (a glucagon like-1 receptor agonist) would target two key pathophysiologic targets in youth: higher 
gluconeogenesis and impaired insulin secretion.  This pilot study will compare the ability of combination therapy with 
metformin and liraglutide with metformin alone to lower gluconeogenesis in African-American youth with type 2 
diabetes.  In an effort to better understand the pleiotropic effects of metformin and liraglutide, we will simultaneously 
evaluate measures of glucose metabolism (hepatic and peripheral insulin sensitivity, insulin and incretin concentrations), 
lipid metabolism (whole body lipolysis and fatty acid turnover) and gut microbial community.  This clinical study will 
provide the mechanistic framework and foundation for the exploratory pharmacogenomic evaluation.  The exploratory 
pharmacogenomic study will determine whether metformin-induced changes in gluconeogenesis are associated with 
known pharmacogenomics markers in youth. We anticipate that this integrative approach to therapeutic development will 
help to define current and future drug targets and focus future pharmacogenomics studies, which are usually expensive, 
time-consuming and under-represent minority groups11. 
Specific Aims
Aim 1: To quantify and compare rates of gluconeogenesis in African-American youth with type 2 diabetes after short-
term combination therapy (metformin and liraglutide) vs. metformin therapy alone, after an overnight fast and during 
meal absorption.
Hypothesis: Youth treated with combination therapy will have lower rates of gluconeogenesis after an overnight fast and 
during meal absorption compared to youth treated with metformin alone. 
Aim 2: To measure the change in GLP-1 and insulin concentrations in African-American youth with type 2 diabetes after 
short-term combination therapy (metformin and liraglutide) vs. metformin therapy alone. 


Principal Investigator: Chung, Stephanie T.  
Last updated: October 5, 2021
Page 5
Hypothesis:  During meal absorption, combination therapy with liraglutide and metformin will be associated with higher 
GLP-1 and insulin concentrations than metformin alone.
Exploratory Aim 1: To identify in African-American youth with type 2 diabetes, known pharmacogenomic variants 
previously associated with metformin response in adults and determine the relationship of these variants with change in 
gluconeogenesis and metformin concentrations. 
Hypothesis:  The 9 known variants of pharmacogenomics response identified in adults (Table 1) will be detectable in 
African-American youth.  A higher genetic risk score (worse risk) of known metformin pharmacogenomic loci will be 
associated with (a) lower serum and urine metformin concentrations and (b) a smaller delta change in rates of 
gluconeogenesis.
Exploratory Aim 2: To compare the composition and distribution of gut microbiota before and after metformin. 
Hypothesis: The composition and distribution of gut microbiota will change after metformin intervention.
Exploratory Aim 2: Construct a pharmacokinetic-pharmacodynamic model based on plasma and RBC metformin 
concentrations, and treatment outcome response (including change in glucose production, gluconeogenesis, incretin 
concentrations).
Hypothesis: Metformin concentrations will correlate with treatment outcomes over the 3-month study period.
Background
Metformin
Metformin treatment alone is suboptimal in 50% of all youth and ~ 65% of African-American youth with type 2 diabetes12 
13.  This low efficacy in children was unexpected, not only because of greater metformin efficacy in adults14, but also 
because during the 8 week run-in phase of the TODAY study (Treatment Options for type 2 Diabetes in Adolescents and 
Youth), more than 85% of youth, regardless of race, were able to maintain glycemic control on metformin therapy15.  
Consequently, the reasons underlying this secondary treatment failure with metformin in children remain to be elucidated 
but do not appear to be related to either stressor scores or poor compliance16.  We postulate that metformin’s durable 
treatment efficacy is suboptimal in youth, especially African-Americans, because it has only a modest effect on lowering 
gluconeogenesis (a prominent feature of type 2 diabetes in youth). 
Targeting gluconeogenesis with metformin
To address the therapeutic conundrum of apparent lower metformin efficacy in youth, it is useful to briefly review the 
literature on the pharmacodynamics of metformin.  Although it is widely accepted that metformin’s primary mechanism 
of action is to reduce hepatic glucose production, many questions still remain regarding metformin’s target and 
downstream signaling pathways17,18.  In vitro assays of metformin action do not accurately reflect the gluconeogenic 
pathways or whether the drug ultimately influences net glucose turnover 19-22.  In vivo tracer studies provide more useful 
information on how metformin affects glucose turnover, but the few studies on this topic disagree on whether metformin’s 
primary physiological target is to inhibit gluconeogenesis (a primary contributor to increased glucose production in youth) 
or glycogenolysis23-27.  Moreover, metformin’s ability to inhibit gluconeogenesis may be an indirect consequence of 
reducing hepatic fat and energy stores28,29.  Since African-Americans have lower hepatic steatosis at the same body mass 
index when compared to white youth30, the lower hepatic fat stores could impede a durable metformin effect.  However, 
there are no studies which have examined the effect of metformin on free fatty acid flux in relation to its glucose lowering 
abilities. 


Principal Investigator: Chung, Stephanie T.  
Last updated: October 5, 2021
Page 6
We have previously demonstrated in drug naïve youth with newly diagnosed type 2 diabetes that increased rates of 
gluconeogenesis are an important contributor to mild fasting hyperglycemia (glucose ~ 7mmol/l)10.  This finding contrasts 
with prior adult studies, in which high rates of gluconeogenesis were only observed in individuals with severe 
hyperglycemia (glucose >8.5 mmol/l) and long-standing disease31.  This early and severe dysregulation in 
gluconeogenesis is consistent with the aggressive nature of type 2 diabetes in youth32 and our preliminary results suggest 
that metformin may not be a potent first-line agent for this disease.  Preliminary Results: In a pilot study of 5 multi-
ethnic youth with newly diagnosed type 2 diabetes conducted at Baylor College of Medicine, we demonstrated that 3 
months of metformin reduced HbA1c (6.8±0.1 vs. 6.0±0.2%, P<0.01) without a change in gluconeogenesis (1.78 ± 0.41 
vs. 1.72 ± 0.31 mg●kglbm●min, P=ns)33.  The participant characteristics for the pilot study conducted at Baylor were: 5 
adolescent girls, age 13.7±1.0 years; 3 African-American and 2 Hispanic.  All individuals were maintained on metformin 
1g twice daily x 12 weeks without side effects.  The participants were instructed on basic education for diet and lifestyle 
at the start of the study, but there were no ongoing organized or supervised lifestyle regimens.  Although not powered to 
detect weight loss, there was no significant detectable weight loss post metformin treatment.  If attenuation of 
gluconeogenesis is not a primary driver of metformin’s initial anti-hyperglycemic effect, metformin alone may be 
suboptimal in youth with type 2 diabetes.  These preliminary results support a multi-mechanistic role of metformin and 
add credence to the mounting evidence that metformin may have pleiotropic effects.
Targeting incretin secretion with metformin
We hypothesize that modulation of the incretin axis may also be an early mediator in metformin therapy, which could be 
an important mechanism of action for African-American youth who have lower GLP-1 concentrations4.  Novel research 
identifies the gut as an important mediator of the mechanism of metformin action34, with the most recent publication 
evaluating a delayed release metformin formulation designed to target the distal ileum and reduce metformin serum 
absorption.  In comparison to immediate release preparations, delayed release metformin optimally lowered overall 
glucose concentrations, despite lower serum metformin concentrations35.  Although the exact target of delayed release 
metformin is not yet clear, enhancing the incretin response could be a potential mediator36.  
Preliminary data: In our pilot study of 5 youth with type 2 diabetes treated with 3 months of metformin, improved 
overall glycemia occurred independent of changes in gluconeogenesis (see above) or serum metformin concentrations.  
The improved glycemia was associated with significantly greater early intact GLP-1 area under the curve (AUC) 
concentrations and a trend for increased insulin during an oral glucose tolerance test (OGTT) (Figure 1)33. 
Figure 1. (A) Early intact GLP-1 and (B) insulin area under the curve concentrations after an OGTT
Baseline
Metformin 
0
100
200
300
400
 iGLP-1 AUC0-30 min
(pgmin/ml)
P<0.01
A
Baseline
Metformin
0
2000
4000
6000
8000
Insulin AUC0-30
(Umin/l)
P=0.1
B


Principal Investigator: Chung, Stephanie T.  
Last updated: October 5, 2021
Page 7
Notably, although metformin therapy may improve GLP-1 concentrations, the magnitude of the GLP-1 increase by 
metformin alone would be relatively small and may not sufficiently improve insulin secretion to compensate for the rapid 
deterioration in β-cell function seen in youth with type 2 diabetes.  
Understanding the role of gut microbiome and metformin in African-American youth 
Modulation of gut microbiota by metformin may also be an important mechanism of action, especially because 
intravenous administration of metformin has no direct glycemic effect37.  The mounting evidence in murine models38 and 
humans34,39 indicate that metformin induced changes in gut microbiota genetic signatures are related to overall drug 
efficacy.  In an elegant study in 14 adults with type 2 diabetes on and off metformin treatment, Napolitano et al. showed 
that metformin’s glucose lowering effect was related to changes in gut microbiota and the entero-endocrine hormone 
secretion34.  As new evidence emerges, there is a conspicuous absence of data examining metformin and its effect on the 
microbiome in populations of African descent and youth.  To the best of our knowledge, there is no evidence regarding 
differences in microbiome by race/ ethnicity and this study will be the first to evaluate the relationship (or lack thereof) of 
gut microbiota, change in gluconeogenesis and incretin secretion after metformin therapy in African American youth. We 
hope that this information will provide data to design a more comprehensive evaluation of the gut as a target of metformin 
action.   
Metformin Pharmacogenomics 
Inter-individual variability in metformin response
There is marked variability in metformin clinical response in adults (35% non-responders); this may be more prominent in 
children (50% non-responders), especially African-American youth (~65% non-responders)6,40.  In the context of these 
observations, adult pharmacogenomic studies have identified variations in several genes that may be associated with 
differences in metformin response.  Since metformin is not metabolized within the body41, much of this research has 
focused on genetic polymorphisms associated with metformin transport, with the strongest evidence for an association 
with the solute carriers SLC22A1 (OCT1), SLC22A2 (OCT2), and SLC22A3 (OCT3) involved in metformin uptake into 
the bloodstream, and SLC47A1 (MATE1), and SLC47A2 (MATE2) associated with metformin excretion (Table 1).  In 
addition to these candidate genes, a hypothesis-free analysis reported an association of the ATM (ataxia telangiectasia 
mutated) gene with metformin response42, which was subsequently replicated43.  The underlying biological mechanism for 
this association has not been established42,44 but alteration of AMP kinase activation has been reported with activation or 
inhibition of ATM45-47.  Moreover genetic sequencing for integrating physiologic metformin response with a 
pharmacogenomics approach should also include variants that may be African ancestry-specific or in higher frequency 
among African ancestry individuals because of the evidence for a greater degree of metformin non-response among 
African-American youth. 
To date, the clinical utility of these genetic polymorphisms of metformin response has not been established11.  It is also 
not known whether these genetic variations are associated with metformin’s presumed primary mechanism of action, 
reducing gluconeogenesis.  In this pilot study we will provide novel information on the association between change in 
gluconeogenesis and genetic polymorphisms of metformin response.  It will be of particular interest to investigate 
individuals who do not respond to treatment for genetic variants that might be contributing to that lack of response. 
This will be the first study in African-American youth to categorize known genetic variants of metformin transport that 
are common in African ancestry individuals.  
Metformin pharmacogenomic targets
There are 3 reasons for integrating physiologic metformin response with a pharmacogenomic approach.  First, the 
frequency of reported variants (Table 1) is sufficiently high that we expect to have carriers of the effect alleles in the 
studied individuals. For example, we anticipate 13-15 of 32 participants will carry the C allele for rs622342, which has 


Principal Investigator: Chung, Stephanie T.  
Last updated: October 5, 2021
Page 8
been associated with reduced HbA1c response to metformin48.  Second, since the course of type 2 diabetes and metformin 
response may differ between youth and adults, it is important to understand if the relationship of these variants and 
clinical outcomes is the same as has been observed in adults.  Third, variation in these genes among African-Americans 
has not been comprehensively described in relation to metformin response.  Previous studies of metformin 
pharmacogenomics that included African-Americans have focused on select variants or on common variation49-51.  For 
example, Choi et al. began their study by sequencing ethnically diverse individuals, but this study was limited to 
SLC47A2 (MATE2) 49-51. Given the evidence for a greater degree of metformin non-response among African-American 
youth, it is imperative to include variants that may be African ancestry-specific or in higher frequency among African 
ancestry individuals; our sequencing strategy will allow us to detect these variants (Table 1). 
Table 1: Single Nucleotide Polymorphisms related to Metformin Response 
SNP
Gene
Minor/
Major
Alleles
AFR 
MAFa
Chr:bpb
Reported Association(s)c
rs622342
SLC22A1 (OCT1)
C/A
0.27
6:160151834
Increased HbA1c48
rs628031
SLC22A1 (OCT1)
A/G
0.25
6:160139813
Metformin Intolerance52
rs461473
SLC22A1 (OCT1)
G/A
0.05
6:160543562
Increased HbA1c53
rs662301
SLC22A2 (OCT2)
T/C
0.01
6:160696919
Reduced T2D incidence50
rs316019
SLC22A2 (OCT2)
C/A
0.16
6:160670282
Reduced HbA1c54 
rs8065082
SLC47A1 (MATE1)
T/C
0.37
17:19561878
Reduced T2D incidence50
rs2252281
SLC47A1 (MATE1)
C/T
0.39
17:19533874
Reduced HbA1c in T2D, reduced 
glucose in non-T2D 51
rs12943590
SLC47A2 (MATE2)
A/G
0.19
17:19716685
Increased metformin clearance 51, 
higher HbA1c49
rs11212617
ATM
A/C
0.24
11:108412434
Less HbA1c control42,43
SNP: single nucleotide polymorphism; AFR: African Ancestry; BP: Base Pair; Chr: Chromosome; MAF: Minor Allele 
Frequency; HbA1c: hemoglobin A1c; T2D: type 2 diabetes. aMAF among the African Ancestry samples in the 1000 
Genomes data; bHuman Genome build 37 chromosome and base pair position; cReported association for the individuals 
with the minor vs. major allele after metformin administration.
We hypothesize that the above 9 variants of pharmacogenomics response will be present in these youth.  These variants 
have been associated with overall glycemic measures (e.g. HbA1c, incidence of type 2 diabetes) and markers of 
medication tolerance.  However, there are no studies examining the association of these variants to the key physiologic 
target of metformin, the liver.  Therefore, despite not having a large sample size, this pilot study will provide new 
information on the relationship of these variants with changes in gluconeogenesis, and enable us to begin to explore 
whether known pharmacogenomics variants of metformin response may be useful for predicting changes at the tissue 
specific level. It is anticipated that the expansion of this study (or other related studies) will be undertaken in due course, 
depending on the results of the pilot protocol currently being proposed. 
Metformin Response and Pharmacokinetics
To elucidate the discrepancy in metformin clinical response in youth and children, a detailed analysis of pharmacokinetic-
pharmacodynamic response is needed in youth with type 2 diabetes who are receiving therapeutic metformin doses.  
Currently, metformin plasma concentrations are not used for routine clinical management and the available literature is 
sparse and inconsistent.  In a recent systematic review, Kajbaf et. Al 55 identified 120 publications with pharmacokinetic 
and therapeutic metformin values.  The majority of the articles cited a therapeutic range between 0.1-4mg/dl although 
cited individual values varied from 0.129 to 90 mg/dl. The 40-fold variation in these reported therapeutic range is 


Principal Investigator: Chung, Stephanie T.  
Last updated: October 5, 2021
Page 9
problematic because it provides no additional information to guide clinical management if levels fall within that wide 
range.  
Interestingly, only 3 of the 120 articles measured “therapeutic” metformin concentrations defined as trough concentrations 
12 hours after drug administration and during continual therapy.  This is in contrast to the multiple articles that cite 
maximum concentration (Cmax) as the pharmacokinetic parameter of interest.  Therefore, establishing a database or 
algorithm to define more accurately the therapeutic window of metformin, especially in adolescents is urgently needed.  
To our knowledge there is only one database of metformin plasma concentrations which was collected from 467 adult 
patients in France56. Mean metformin concentrations were 2.7+/-7.3mg/L with range in plasma levels between 0-61mg/L.  
This range is markedly greater than the currently published toxic levels of 45mg/L assigned by the International 
Association of Forensic Toxicologists.
We will assess metformin pharmacokinetics in youth with type 2 diabetes who are receiving metformin daily for 3 
months.  This information will be used to construct a pharmacokinetic-pharmacodynamic model that may be useful for 
elucidating mechanisms of metformin responsiveness and non-responsiveness in youth and identifying target drug 
exposures in this population.
Combination therapy of metformin and liraglutide
Combination therapy is likely to be an ideal therapeutic strategy to address the accelerated trajectory towards metabolic 
decompensation in youth with type 2 diabetes57.  We propose that a combination regimen of metformin and liraglutide 
will be complementary in their glucose lowering effect because of their individual ability to lower gluconeogenesis and 
enhance the incretin response. This proposal will directly test this hypothesis by comparing African-American youth on 
combination therapy with metformin and liraglutide to youth treated with metformin alone under basal fasting conditions 
and during meal absorption.
The secondary aim of this study is to evaluate the between group differences in GLP-1 and GIP area under the curve 
concentrations.  We hypothesize that combination therapy will result in higher GLP-1 and GIP AUC concentrations 
compared to metformin alone. This hypothesis was based on 2 studies which used a long-acting GLP-1 analog, liraglutide.  
A preclinical study of liraglutide in mice resulted in 1.4 higher GLP-1 concentrations58.  In a more recent randomized 
double blind placebo-controlled cross-over study of liraglutide in 20 individuals with type 2 diabetes, liraglutide 
significantly increased GLP-1 and GIP concentrations after 17 days59.  We have also identified 2 additional studies using a 
short-acting GLP-1 analog (exenatide) which had the opposite effect on GLP-1 response.  In a pilot study of patients with 
Prader Willi Syndrome, exenatide was associated with a reduced GLP-1 response60.  Similarly, Rother et al. showed 
exenatide use was associated with lower post-meal GLP-1 concentrations in long-standing patients with type 1 diabetes61 .  
Therefore, the effect of GLP-1 analogs on gut hormones (GLP-1 and GIP) is unclear but could be related to type of drug 
(long vs. short acting), patient characteristics or unknown factors.  Our study will help to clarify the effect of a long-acting 
GLP-1 analog, liraglutide, on incretin hormone secretion in youth with type 2 diabetes.
Liraglutide
Liraglutide is an acylated human GLP-1 receptor agonist that has 97% amino acid sequence homology to endogenous 
GLP-1 (7-37).  Liraglutide directly activates the GLP-1 receptor, leading to glucose-dependent insulin release and 
glucagon suppression, resulting in improved postprandial glucose homeostasis.  A long acting GLP-1 agonist, such as 
liraglutide, would be a suitable add-on agent to metformin because of its pharmacodynamics, potency and safety62.  
Liraglutide’s pharmacokinetic and safety profile in children appears to be similar to that in adults, and it was associated 
with improved glycemia in a small randomized double-blind placebo-controlled trial of 19 youth62.  Victoza® (liraglutide) 
therapy is approved by the Federal Drug Administration (FDA) as an adjunct to diet and exercise to improve glycemic 
control in patients 10 years and older with type 2 diabetes mellitus (approval on June 17, 2019). The FDA approval was 


Principal Investigator: Chung, Stephanie T.  
Last updated: October 5, 2021
Page 10
based on data submitted from the ELLIPSE clinical trial, which was published in the New England Journal of Medicine in 
April 201963.  This trial showed superior efficacy of liraglutide with metformin compared to placebo + metformin for 
lowering HbA1c.  Liraglutide improves overall glucose homeostasis by enhancing glucose-dependent insulin secretion, 
reducing glucagon secretion and delaying gastric emptying64.  In addition, liraglutide also lowers fasting glucose 
concentrations64,65 but the exact mechanism of action has not been extensively investigated.  It is presumed that liraglutide 
lowers fasting glucose primarily by increasing the endogenous insulin: glucagon ratio that subsequently inhibits hepatic 
gluconeogenesis and glycogenolysis66.  However, GLP-1 agonists may also directly lower hepatic glucose production: an 
extra-pancreatic effect demonstrated in murine models67, as well as in elegant experiments using prolonged GLP-1 
infusion during pancreatic clamps in healthy individuals68.  If liraglutide decreases glucose production specifically by 
reducing gluconeogenesis, it would also be important for targeting this early pathological feature of type 2 diabetes in 
youth.  
Study Objectives
The primary objective of this study is to compare the change in gluconeogenesis between the two intervention arms 
(combination therapy of metformin and liraglutide and metformin only) in African-American youth. 
Primary outcome: The change in gluconeogenesis from baseline (prior to study drugs) to 12 weeks.
Secondary outcomes: The change from baseline to 12 weeks in:
1.
Glucose production
2.
GLP-1 and GIP area under the curve concentrations (AUC) during OGTT and meal absorption
3.
Insulin sensitivity
a.
Hepatic insulin sensitivity 
b.
Whole body insulin sensitivity
4.
Insulin area under the curve concentrations during an OGTT and meal absorption
5.
Glycerol and palmitate turnover as an indicator of lipid metabolism.  
Exploratory outcomes: 
Pharmacogenomic variants of metformin response:
1.
Identify the 9 known variants of metformin response (Table 1) in African-American youth with type 2 diabetes
2.
Generate a composite genetic risk score based on these 9 variants and determine the relationship of this composite 
risk score with:
a.
Serum and urine metformin concentrations 
b.
Change in gluconeogenesis from baseline to 12 weeks. 
3.
Screen for unknown genomic variants in metformin response in specific genes of interest.
Gut microbiome and metformin
a)
The change in the gut microbial composition and distribution in African-American youth with type 2 diabetes 
after 12 weeks of metformin.
Metformin response and pharmacokinetic-pharmacodynamic model
b)
Construct a pharmacokinetic-pharmacodynamic model based on plasma and RBC metformin concentrations, and 
treatment outcome response (including change in glucose production, gluconeogenesis, incretin concentrations).


Principal Investigator: Chung, Stephanie T.  
Last updated: October 5, 2021
Page 11
Study Design and Methods
Study Design
This is a pilot randomized parallel design study of African-American youth with type 2 diabetes who are not on insulin 
therapy, with two intervention arms (combination therapy with metformin and liraglutide, and metformin alone).  
Participants enter 5-7 day drug-free run-in period prior to initial metabolic evaluation.  Participants will be evaluated 
before and after 12 weeks of study drug(s). 
Randomization
The NIH Clinical Center (CC) Pharmacy will perform block randomization stratified by sex.  Randomization will be 
stratified by sex with a block size of 6.  The randomization schedules will be constructed by an independent statistician 
and shared with the NIH CC Pharmacy.  The PI and study staff will not view the randomization schedule a priori.  
Subjects will be randomized as they enter the protocol as follows: The PI or study staff will notify the NIH CC pharmacy 
of the eligible enrolled participant and the pharmacy will assign the intervention arm according to the predetermined 
randomization schedule.  All participant data will be entered into a NIDDK RedCap® database and the participant will be 
randomized consecutively using the single randomization list maintained by the NIH CC Pharmacy. 
Study Protocol
Figure 1 illustrates the study design in which there will be 5 study visits: Visit 1 (screening outpatient), Visit 2 (tracer 
inpatient), Visit 3 and 4 (follow-up outpatient) and Visit 5 (tracer inpatient). Eligible patients may be identified at CNMC 
but no study visits will occur at CNMC.   
Visit 1: Screening Outpatient Visit 
This visit is designed to explain the protocol to the participant and their parents, determine participant eligibility, provide 
study protocol details to the family, and obtain informed consent and assent. Each participant will undergo a history and 
physical examination, routine blood work (including lipase and amylase) and pregnancy test to determine eligibility.  
Youth will meet with a member of the nutrition team to discuss your diet and to be educated on keeping 3-day food 
records before Visit 2 and 5 and will discuss metabolic diet selection during the inpatient stay. In addition, the dietician 
will perform body measurements including waist, hip and neck circumferences, those measurements will take only few 
minutes and are not painful. 


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Education and Blood glucose monitoring during the run-in period
All participants will be seen by a nutritionist and counseled on standard diabetes management, diet and lifestyle 
management.  Participants will be asked to discontinue all anti-diabetic medications for the 5-7 days preceding Visit 2.  
This drug-free period is necessary to assess the participant’s metabolic response off of anti-diabetic medications.  During 
the drug-free period and throughout the study, youth will monitor their blood glucose either by fingerstick blood glucose 
(FSBG) at least twice daily or they will wear a continuous glucose monitoring (CGM) device. Participants who do not 
have their own glucometer will be provided with glucose-monitoring devices and supplies and educated on their use.  The 
CGM supplies will be provided by the study team and all participants will receive instruction on how to wear and use the 
CGM device. All individuals will be closely monitored during the drug-free run-in period with 2-3 times daily blood 
glucose checks via home glucose monitoring and daily check-in reports by study staff.  If severe hyperglycemia, FSBG is 
>200mg/dl fasting or 300mg/dl pre-dinner, is noted during the run-in period, they will notify the study PI immediately.  
To monitor participants with elevated FSBG the physician may recommend admission to the NIH metabolic unit prior to 
Visit 2. Individuals with persistently elevated FSBG and/ or signs and symptoms of metabolic decompensation (e.g. 
vomiting, dehydration, lethargy, abdominal pain, ketonuria and acidosis) will be withdrawn from the study and restarted 
on his/ her medication.  During the drug-free run-in period, individuals who cannot wear the CGM device or complete 
fingerstick blood glucose measures or who cannot be reached for ≥3 days during this period, will be withdrawn from the 
study.  
Visit 2: Baseline Tracer Inpatient Visit 
Visit 2 will take place ~ 1-2 weeks after the screening visit.  All participants will be admitted to the Metabolic Research 
Unit at the NIH for an overnight tracer visit to measure indices of glucose and lipid metabolism, energy expenditure and 
obtain samples for genetic analysis prior to initiation of the study drugs. The tracer protocol is illustrated in Figure 2 and 
outlined below:
a.
Admission, oral glucose tolerance test (OGTT) and Dual x-ray absorptiometry (DXA) scan
b.
Stable isotope administration and steady state labs
c.
Urine collection
d.
Indirect calorimetry


Principal Investigator: Chung, Stephanie T.  
Last updated: October 5, 2021
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a.
Admission, OGTT and DXA scan 
Youth will be admitted in the morning (~7am) on Day 1 and blood samples for hormones, genetic analysis, 
inflammatory markers and metabolites (including glucose, insulin, C-peptide, glucagon, and isotopic enrichments of 
glucose, glycerol and body water) will be collected at baseline. One intravenous catheter will be placed in the arm for 
blood draws. Blood and urine collection for genetic samples for future analysis will occur on admission for Visit 2 
only.  

Oral Glucose Tolerance Test (OGTT)
Youth will undergo a 75gram oral glucose tolerance test with blood samples collection at 0, 15, 30, 60, 90 and 
120 minutes for hormones and metabolites (including glucose, insulin, glucagon-like-1 peptide and 
gastrointestinal inhibitory peptide) and metabolites.  To measure blood (plasma and RBC) plasma samples for 
metformin at baseline, 3ml of blood will be collected at 0-time point.  

DXA Scan
Dual x-ray absorptiometry scan will be performed during admission Day 1 to evaluate body composition and 
determine fat mass and lean body mass.  
After the OGTT, youth will eat a standard breakfast, a lunch meal (~ 1pm) and a snack (~3pm) to provide ~20%, 30% 
and 10% of total energy needs respectively.  Youth will eat a standard dinner meal at ~ 6pm to provide 40% of total 
energy needs (50% carbohydrate, 33% fat and 17% protein).  Total energy expenditure will be estimated from the 
from the Mifflin St. Jeor equation 69 with a standardized activity factor.  Youth will then be placed NPO at 8pm with 
ad libitum access to water.  Another intravenous catheter will be placed in the evening of Day 1, such that the 
participant will have two intravenous catheters placed, one in each forearm, for stable isotope infusion and blood 
draws. 
On the first day of admission youth will be asked to review their 3 day food records that they recorded prior to 
admission with a member of the nutrition team. If youth do not bring these records to this appointment, the nutrition 
team will perform a 24-hour recall to capture the foods consumed the day prior to admission.  This will help the 
investigators to identify if there are differences in dietary intake prior to admission that may impact the results of the 
testing.  The nutrition staff will also obtain body circumference measures, such as neck, waist and hip circumference. 
These measurements will be done over minimal clothing with a non-stretch measuring tape.
b.
Stable isotope administration and steady state labs
Stable isotope administration will begin the night of the admission; glucose appearance, fractional 
gluconeogenesis and glycerol turnover will be measured with [6,62H2]glucose, deuterated water [2H2O], 
[U13C16]potassium palmitate and [2H5]glycerol respectively. Lean body mass (LBM) measurements from dual x-
ray absorptiometry scans will be used to calculate the isotope administration rates. Deuterated water (3 
gram●kgLBM) will be administered in 3 divided doses at 9pm, 12am, and 3am. Primed (60×the minute infusion 
rate) and continuous infusions of [6,62H2]glucose and [2H5]glycerol will start at 4am (t= -240m) and continue until 
9am (t= 0m). At 730am, an unprimed [U-13C16] palmitate infusion will be started.  At 9am (t= 0m), the glycerol 
and palmitate infusions will be discontinued.  The [6,62H2]glucose infusion will continue until 2pm (300m) to 
quantify gluconeogenesis during meal absorption using a continuous feeding protocol with a nutrient drink and 
[6,62H2]glucose intravenous infusion will be used to mimic the steady-state prandial state70,71.  At 9am (t= 0m), 
participants will be fed a lactose-free nutrient drink to provide ~50% of estimated total energy needs and 
macronutrient composition that matches the previous days’ meals in equal portions every 30 minutes over the 5h 


Principal Investigator: Chung, Stephanie T.  
Last updated: October 5, 2021
Page 14
of feeding. The purpose of this feeding strategy is to evaluate the acute response to refeeding, and not to provide 
full caloric requirements71.  Isotope enrichment will be measured over a period of 30 minutes during steady state.  
Therefore, blood samples for steady-state conditions will be obtained at the following time periods: (1) fasting 
between 830-9am (t= -30 to 0m) and (2) prandially between 130-200pm (t=270 to 300m).   Blood samples for 
hormones and metabolites (including glucose, insulin, incretins in protease inhibitor treated tubes) will be 
collected every 30 minutes between 9am and 2pm.  Whole blood will be centrifuged and plasma transferred and 
frozen at -80C until analyses.  
c.
Urine collection
A timed 24-hour urine collection to measure urinary nitrogen will start at noon on admission and continue until noon 
on the study day.  Total daily urinary nitrogen excretion will be used to determine the total body glucose and lipid 
oxidation rates as previously described 72. 
d.
Indirect calorimetry
To calculate substrate oxidation, indirect calorimetry (hood) will be performed for 30-45 minutes between 7-8am, and 
1-2pm to measure oxygen consumption and carbon dioxide production rates.  
e.
Stool Sample
A research stool sample will be collected at home and brought to the visit or collected during the admission. The stool 
sample will be stored at 4C in a sterile plastic vial and processed within 24 hours of collection.  
f.
Side-effect Questionnaire
Questionnaires will be completed by the participants at the study visit (Appendix A)
g.
Blood glucose monitoring
Blood glucose will be monitored with either fingerstick blood glucose twice daily (fasting and bedtime) or by using an 
FDA- approved continuous glucose monitoring device (CGM) (for example, the Dexcom® CGM System). The 
device will be used to monitor glucose in real-time, approximately every 5 minutes.  The system consists of a small 
sensor, transmitter, and hand-held receiver. The small sensor, with a small needle attached, will be inserted 
subcutaneously. The transmitter, which is attached to the sensor, will send the measured glucose to the receiver. The 
sensor is changed per manufacturer recommendations. Participants will receive training on removing and replacing 
the sensor and education on the signs and symptoms of hyperglycemia see Appendix B. All CGM or glucometer 
supplies will be provided by the study team.  
Visit 3 and 4: Follow-up Outpatient Visit
Participants will return to the NIH CC clinic for an outpatient follow-up visit.  Visit 3 will occur between 4-5 weeks and 
Visit 4 will occur between 8-9 weeks.  The procedures at Visit 3 and 4 will be identical, and are outlined as follows:
1.
Blood glucose records and glucometers will be reviewed. 
2.
Pill and pen counts performed by the study staff.
3.
Blood and urine samples will be collected for glucose and metformin monitoring (including hemoglobin A1c, 
metformin concentrations). 
4.
We will review medication adherence and tolerance and emphasize compliance with study visits.
5.
A research stool sample will be collected at home and brought to the visit or collected during the admission. The 
stool sample will be stored at 4C in a sterile plastic vial and processed within 24 hours of receipt.  
6.
We will collect questionnaire to assess side effects after starting the medication or changing dose. The sample 
questionnaire is in Appendix A. 


Principal Investigator: Chung, Stephanie T.  
Last updated: October 5, 2021
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Visit 5: Final Tracer Study
Participants will return for an inpatient tracer visit protocol after 12 weeks ± 14 days on study medication. Participants 
will receive their last dose of study medication at supper in the evening of admission Day 1 during Visit 5.  
Visit 5 procedures will be identical to Visit 2 above with the following additional blood sampling for metformin 
concentrations. To measure blood (plasma and RBC) plasma samples for metformin for participants within the study 
receiving metformin at doses 1000-2000mg daily.  Samples for metformin analysis (3ml of whole blood) will be 
collected, processed for isolation of plasma and RBC and frozen at -80C until analysis during the OGTT as follows:
a)
Day 1 during the OGTT at time 0, 0.5h, 1h, 1.5h, 2h, 3h 4h, 6h, 8h and 8pm (pre-second dose).
b) Day 2 at 8am and at 2pm
After completion of Visit 5, participants will be instructed to resume their home medication regimen. 
Study Agents/ Intervention 
Two study agents will be used: metformin oral 500mg oral tablet and liraglutide (6mg/ml, 3ml ) solution for subcutaneous 
injection, pre-filled, multi-dose pen that delivers doses of 0.6mg, 1.2mg or 1.8mg.  Both metformin and liraglutide will be 
used within the approved dosing regimens.  Neither drug will be altered from the approved dosage formulation.  Table 3 
illustrates the titration schedule for each study drug. 
Table 3: Titration Schedule for Metformin and Liraglutide
Study schedule
Week
Liraglutide
(subcutaneous injection)
Metformin
(oral tablet)
Run-in
-1 to 0
-
-
After baseline visit
0-1
0.6mg once daily
500mg once daily
1-2
1.2mg once daily
500mg  twice daily
Target study dose
3-12
1.8mg once daily
1000mg twice daily
The participant will start the study drug(s) on Day 2 of Visit 2, after the tracer protocol is completed.  The PI will review 
blood glucose logs and/ or CGM readings weekly and ensure that the study drug(s) titrated to the highest tolerable dose as 
guided by Table 3 above.  For patients on combination therapy, the dose of liraglutide will not be increased if the fasting 
blood glucose is <75mg/dl on 2 or more consecutive days.  If the fasting blood glucose subsequently rises to ≥90mg/dl on 
2 or more consecutive days, the dose of liraglutide will increased by 0.6mg once daily every 5-7 days to achieve 
maximum tolerable dose.    
Dose titration for gastrointestinal intolerance
For participants on the combination therapy arm, who experience gastrointestinal side effects, the dose of liraglutide will 
be decreased first.  Liraglutide dosage will be decreased by 0.6mg once daily.  If gastrointestinal symptoms persist after 2-
3 days, metformin dose will be decreased by 500mg.  If symptoms persist, dose adjustments will be made every 2-3 days, 
alternating between liraglutide (0.6mg) and metformin (500mg) dose reductions.  If the highest tolerable metformin dose 
is < 1000mg daily or liraglutide <0.6mg daily, the subject will be withdrawn from the study. 
For participants on metformin alone, metformin will be decreased by 500mg every 2-3 days until symptoms resolve. If the 
highest tolerable dose of metformin is <1000mg daily, the subject will be withdrawn from the study.


Principal Investigator: Chung, Stephanie T.  
Last updated: October 5, 2021
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If gastrointestinal symptoms resolve, and at the discretion of the PI, the dose of metformin and/or liraglutide may be 
increased every 2-3 days to the maximum tolerable dose.  Incremental dose increases will be as follows: metformin 
500mg every 2-3 days and liraglutide 0.6mg every 2-3 days.
Dispensing and Storing Study Medications
The NIH CC Pharmacy will acquire and store metformin and liraglutide medications for the study.  The study 
medication(s) will be coded and dispensed by the NIH CC Pharmacy.  The pharmacy will dispense the study 
medication(s) according to the predetermined randomization table.  The Randomization section contains additional details 
on the processes we will use to assign randomization.  The storage of medications will be according to the package insert 
for each study drug. 
Study medications will be dispensed to the patient by the NIH CC pharmacy.  At the end of Visit 2, 3, and 4, patients will 
receive 1 month supply of medications with 20% overflow to improve compliance and accountability as indicated below.  
Medication dispensing schedule: 
Visit 2: Metformin (144 tablets) +/- Liraglutide (3 pens)
Visit 3: Metformin (138 tablets) +/- Liraglutide (3 pens)
Visit 4: Metformin (142 tablets) +/- Liraglutide (3 pens)
Compliance and Adherence
When potential participants are considering participation, the importance of compliance with the intervention assignment 
will be stressed.  The ability to adhere to the protocol of the study, and take medication daily, as instructed is one of the 
inclusion criteria of the study and will be underscored.  One month supplies of medication will be dispensed by the NIH 
CC pharmacy.   Medication adherence will be determined by study personnel counting of pills and pens that were 
dispensed at each medical visit and returned at the next medical visit (Visit, 3, 4 and 5).  The percent of pills taken over 
the 3-month study period will be assessed. We will employ the following strategies:
1.
Twenty-percent extra of metformin tablets will be added to improve compliance, as the participants will not know 
how many extra pills are sent (although they will be told that there are more than the exact amount of pills so that 
we can be sure they are taking it).  Participants will return the remaining doses at the next visit and receive 
another month supply with 20% extra. The number of unused tablets will be recorded.
2.
Adherence to scheduled blood glucose monitoring will be assessed by reviewing the participant’s glucose meter 
or CGM device. 
3.
The participants will be contacted every 1-2 weeks by phone, email and/or text message to encourage and assess 
compliance. Text and email-based reminders are an important adherence tool which can be tailored to the needs of 
the individual youth and family.  Text messaging has been shown to improve outcomes in African-American 
children with other chronic diseases, such as asthma 73 and sickle cell anemia 74 and in adults with type 2 diabetes 
75. 
Follow-up
Post-Study Treatment
The patient’s clinically relevant data, available at the end of the study, will be shared with their diabetes provider with the 
consent of the participant and/or the parent. At the family’s request, it will also be provided to his or her primary care 
provider.  After completion of this study, participants and families will discuss their diabetes control with their providers 
and it will be up to the clinical diabetes provider and the patient/ family to decide whether they would like to continue 
liraglutide, after reviewing the potential risks and benefits.  


Principal Investigator: Chung, Stephanie T.  
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Post-Study Obligations
There are no anticipated post-study obligations.
The study drugs (metformin and liraglutide) will not be made available to participants through this study protocol either at 
the end of the study or if they are withdrawn from the study prior to completion.  However, the patient’s clinically 
relevant data will be shared with their diabetes provider with the consent of the participant and the parent in order to help 
them decide whether they would like to continue the medication(s) outside of this study protocol. 
Inclusion and Exclusion Criteria
Inclusion Criteria 
Primary randomized protocol
1.
Youth must self-identify as African-American and identify both parents as African-American
2.
Age 12- 25 years
3.
Pubertal or post-pubertal: Girls – Tanner stage IV-V breast; Boys – Testicular volume 11-25cc
4.
Diagnosis of type 2 diabetes of ≤5 years duration, as per American Diabetes Association Criteria76
5.
Hemoglobin A1C <9% at study initiation
6.
Negative to mild ketonuria without acidosis (negative or 1+ ketones on urinalysis)
7.
Negative test for diabetes-related autoantibodies (glutamic acid decarboxylase 65 and tyrosine phosphatase-
related islet antigen 2 (IA-2))
8.
Willing and able to take daily medications and check blood glucose levels at least twice per day or wear a 
continuous glucose monitoring device (CGM).
Exclusion Criteria
1.
Pregnancy or breastfeeding
2.
Allergy to study medications
3.
Allergy to milk protein
4.
Chronic insulin therapy 
5.
Treatment with other medication which are known to affect the parameters under study (for example sodium-
glucose transporter 2 (SGLT-2) inhibitors, dipeptidyl peptidase-4 (DPP-IV) inhibitors, non-selective beta 
blockers).
6.
Metabolic derangement such as metabolic acidosis, severe hyperglycemia (fasting blood glucose ≥ 200mg/dL), 
and/or liver enzymes > three times the upper limit of normal. 
7.
Personal or family history of medullary thyroid cancer or Multiple Endocrine Neoplasia syndrome type 2
8.
Any other condition that, in the opinion of the investigators, will increase risk to the subject, or impede the 
accurate collection of study-related data.
9.
Body weight ≥ 450lbs 
10. Body weight ≤58kg
11. Serum triglyceride concentrations ≥500mg/dl
12.  Hemoglobin concentration <10g/dL
Rationale for Inclusion Criteria
African-American Youth
Individuals will be considered to be African-American if they self-identify as African-American and describe both parents 
as being African-American.  In brief, African-American youth are disproportionately affected by type 2 diabetes and have 
lower treatment efficacy and higher morbidity rates compared to other ethnic groups. This project is designed to 


Principal Investigator: Chung, Stephanie T.  
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Page 18
investigate the underlying biological determinants (including genetic) in African-American youth with type 2 diabetes and 
provide information that will help to reduce the burden of disease in this vulnerable population. 
Age Range 12- 25 years and pubertal status
The age range of 12- 25years was chosen to maximize recruitment and includes a wide range of youth afflicted with type 
2 diabetes.  However, using a wide age range increases the potential to recruit youth across the pubertal spectrum and 
puberty is associated with changes in insulin resistance.  Therefore, to minimize clinical variability, we will only recruit 
pubertal or post-pubertal youth (defined as Tanner stage IV-V for girls and testicular size 11-25cc for boys).  We believe 
this approach is reasonable because we will have a well-defined cohort (by pubertal status) and a balanced distribution in 
intervention arm by sex (randomization stratified by sex).  In addition, the primary outcome for this study (change in 
gluconeogenesis) varies minimally with age or pubertal status among pre-pubertal and pubertal youth10,77. 
Pubertal status will be defined based on breast examination (Tanner stage IV-V) in girls and testicular examination (11-
25cc) in boys.  Pubic hair and bone age will not be used to characterize pubertal stage.
Diabetes diagnosis criteria and HbA1c <9% 
Type 2 diabetes in youth will be diagnosed according to ADA criteria76 and eligibility criteria were based on the 
Treatment Options for Type 2 Diabetes in Adolescents and Youth (TODAY) study design6.  Our overall aim is to help 
identify early therapeutic targets in youth with type 2 diabetes.  In order to achieve this goal, we want to evaluate drugs 
that could be used early in the course of diabetes, before the onset of severe hyperglycemia, and ones that will work 
effectively at physiologic targets to help stem the rapid progression of type 2 diabetes in youth.  Therefore, we are 
recruiting youth who are relatively early in the disease course as defined as having a diagnosis of diabetes ≤5 years 
duration with mild to moderate hyperglycemia as defined by HbA1c <9%.  Youth with HbA1c ≥9% often have severe 
hyperglycemia and glucotoxicity which is associated with increased risk for dehydration and ketoacidosis and these 
conditions would interfere with measurements of insulin sensitivity and glucose production.
Rationale for Exclusion Criteria
Pregnancy and Breastfeeding
Pregnancy and breastfeeding are associated with physiologic increases in insulin resistance, which would confound data 
interpretation.  In addition, the DXA scan cannot safely be performed in pregnant women.  Female participants of 
childbearing age who meet other eligibility requirements and wish to participate in the study will be informed of the 
potential risks to a pregnancy conceived while on any study medication.  Participants will also be informed of the 
potential risks of hyperglycemia to a pregnancy including fetal malformations, pre-term delivery and increase maternal 
complications. Participants who consent to participate will be asked to practice reliable birth control including systemic 
hormones and/or barrier methods.  Patients who are pregnant and/or sexually active and not using adequate birth control 
will be excluded from enrollment in the trial.  Pregnancy tests will be obtained from all female participants before 
inpatient visits and right before DXA scans. 
Chronic insulin therapy
Participants who are currently taking insulin therapy or who are within 3 months of insulin treatment will be excluded 
from the study.  Insulin therapy will directly affect hepatic glucose production and insulin sensitivity. 
Any condition which may increase risk to the subject or impede accurate data collection
Examples include participants with a history of active thyroid disease, liver disease, pancreatitis, nephrotic syndrome or 
lupus.  Liraglutide may be associated with an increased risk for pancreatitis and C-cell carcinoma.  Therefore, any 
individual with a history of acute or chronic pancreatitis, or increased risk for pancreatitis as indicated by elevated serum 
triglycerides≥500mg/dl at baseline, will be excluded.  Hypertriglyceridemia is a well-known common cause of 


Principal Investigator: Chung, Stephanie T.  
Last updated: October 5, 2021
Page 19
pancreatitis.  In general, the risk for pancreatitis occurs in patients with severe hypertriglyceridemia >1000mg/dl78.  
However, the exact lower threshold of risk is not well defined.  Although African-Americans have lower triglyceride 
concentrations compared to other US ethnic groups, there are no studies that would suggest that African-Americans have 
a higher population risk for pancreatitis at lower triglyceride concentrations.  Therefore, in accordance with National 
Cholesterol Education Program ATPIII guidelines, we propose using the current recommended triglyceride threshold of 
≥500mg/dl for denoting increased risk for pancreatitis78.  In addition, any individuals with a personal or family history of 
medullary thyroid cancer or Multiple Endocrine Neoplasia syndrome type 2 will be excluded.
Weight ≥ 450lbs
Individuals with weight ≥ 450lbs will be excluded because the maximum weight for the dual energy absorptiometry 
(DXA) scan is 450lbs. 
Clinical and Laboratory Methods
Glucose Turnover
During the stable isotope tracer protocol, hepatic glucose production will be partitioned to evaluate rates of 
gluconeogenesis and glycogenolysis overnight and during a meal. Rates of glucose and glycerol turnover will be 
calculated as described below. 
Rates of glucose appearance (Raglucose) will be calculated under near-steady-state conditions using the average enrichment 
of [6, 6-2H2]glucose into the systemic circulation using conventional isotope dilution calculations 77.  
    
𝑅𝑎𝑔𝑙𝑢𝑐𝑜𝑠𝑒=
𝐼𝑛𝑓𝑢𝑠𝑎𝑡𝑒 𝑒𝑛𝑟𝑖𝑐ℎ𝑚𝑒𝑛𝑡
𝑃𝑙𝑎𝑠𝑚𝑎 𝑒𝑛𝑟𝑖𝑐ℎ𝑚𝑒𝑛𝑡×   𝑅𝑎𝑡𝑒 𝑜𝑓 𝑖𝑛𝑓𝑢𝑠𝑖𝑜𝑛 𝑜𝑓 𝑙𝑎𝑏𝑒𝑙𝑒𝑑 𝑔𝑙𝑢𝑐𝑜𝑠𝑒
Under fasting and steady state conditions, rate of glucose appearance reflects hepatic glucose production. 
Glucose production rate is the sum of the rates of gluconeogenesis and glycogenolysis [20]:
𝐺𝑙𝑢𝑐𝑜𝑠𝑒 𝑝𝑟𝑜𝑑𝑢𝑐𝑡𝑖𝑜𝑛 𝑟𝑎𝑡𝑒= 𝑅𝑎 𝑡𝑜𝑡𝑎𝑙―rate of infusion of labeled glucose
Fractional gluconeogenesis will be determined using 2H2O and the average enrichment of 2H enrichments of carbon 
1,3,4,5,6 of glucose as previously described 79.  
𝐹𝑟𝑎𝑐𝑡𝑖𝑜𝑛𝑎𝑙 𝑔𝑙𝑢𝑐𝑜𝑛𝑒𝑜𝑔𝑒𝑛𝑒𝑠𝑖𝑠=
[
(𝑀+ 1)2𝐻(𝑚/𝑧169)
6
]
2𝐻2𝑂 
where (M+1)2H(m/z169) is the M+1 enrichment of 2H glucose measured using the mass-to-charge ratio (m/z) 170/169 
fragment of glucose, 6 is the number of 2H labeling sites on the m/z 169 fragment of glucose, and 2H2O is the enrichment 
in body water. 
Absolute rates of gluconeogenesis and glycogenolysis will be calculated as follows:
 
𝑅𝑎𝑡𝑒 𝑜𝑓 𝑔𝑙𝑢𝑐𝑜𝑛𝑒𝑜𝑔𝑒𝑛𝑒𝑠𝑖𝑠 = 𝑅𝑎 𝑡𝑜𝑡𝑎𝑙×  𝑓𝑟𝑎𝑐𝑡𝑖𝑜𝑛𝑎𝑙 𝑔𝑙𝑢𝑐𝑜𝑛𝑒𝑜𝑔𝑒𝑛𝑒𝑠𝑖𝑠
𝑅𝑎𝑡𝑒 𝑜𝑓 𝑔𝑙𝑦𝑐𝑜𝑔𝑒𝑛𝑜𝑙𝑦𝑠𝑖𝑠= 𝑔𝑙𝑢𝑐𝑜𝑠𝑒 𝑝𝑟𝑜𝑑𝑢𝑐𝑡𝑖𝑜𝑛 𝑟𝑎𝑡𝑒― 𝑟𝑎𝑡𝑒 𝑜𝑓 𝑔𝑙𝑢𝑐𝑜𝑛𝑒𝑜𝑔𝑒𝑛𝑒𝑠𝑖𝑠


Principal Investigator: Chung, Stephanie T.  
Last updated: October 5, 2021
Page 20
During meal ingestion, the Ra of dietary glucose into the plasma pool (Ra meal) will be estimated from the carbohydrate 
content in the liquid meal.  We assumed that ~85% of orally ingested glucose enters the systemic circulation in the normal 
volunteers 80.
The rate of glycogenolysis during meal absorption (Glycogenolysisfeed) will be estimated as: 
𝐺𝑙𝑦𝑐𝑜𝑔𝑒𝑛𝑜𝑙𝑦𝑠𝑖𝑠𝑓𝑒𝑒𝑑= 𝑅𝑎 𝑡𝑜𝑡𝑎𝑙―(𝐺𝑙𝑢𝑐𝑜𝑛𝑒𝑜𝑔𝑒𝑛𝑒𝑠𝑖𝑠+ 𝑅𝑎 𝑚𝑒𝑎𝑙+ 𝑖𝑛𝑓𝑢𝑠𝑖𝑜𝑛 𝑟𝑎𝑡𝑒 𝑜𝑓 𝑙𝑎𝑏𝑒𝑙𝑒𝑑 𝑔𝑙𝑢𝑐𝑜𝑠𝑒) 
Glycerol and Palmitate Turnover
Rates of appearance (Ra) will be calculated under near-steady-state conditions using the average enrichment of 
[2H5]glycerol or  [U-13C16] palmitate into the systemic circulation using conventional isotope dilution calculations 77.  
𝑅𝑎 =
𝐼𝑛𝑓𝑢𝑠𝑎𝑡𝑒 𝑒𝑛𝑟𝑖𝑐ℎ𝑚𝑒𝑛𝑡
𝑃𝑙𝑎𝑠𝑚𝑎 𝑒𝑛𝑟𝑖𝑐ℎ𝑚𝑒𝑛𝑡×   𝑅𝑎𝑡𝑒 𝑜𝑓 𝑖𝑛𝑓𝑢𝑠𝑖𝑜𝑛 𝑜𝑓 𝑙𝑎𝑏𝑒𝑙𝑒𝑑 𝑔𝑙𝑦𝑐𝑒𝑟𝑜𝑙 𝑜𝑟 𝑝𝑎𝑙𝑚𝑖𝑡𝑎𝑡𝑒
Insulin Resistance    
Hepatic insulin sensitivity we will be calculated using the hepatic insulin sensitivity index by measuring hepatic glucose 
production in relation to the fasting insulin concentration 81.  Since acute changes in insulin concentration decrease 
glycogenolysis but gluconeogenesis remains constant, glycogenolysis is the component of hepatic glucose production that 
is most sensitively regulated by insulin 70,82.  Indices of hepatic sensitivity that include gluconeogenesis may 
underestimate the effect of hepatic insulin action and could blunt the assessment of insulin sensitivity.  Therefore, we will 
also calculate a hepatic insulin sensitivity index using glycogenolysis and fasting insulin concentration83.
Whole body insulin sensitivity will be calculated with the Matsuda index during the oral glucose tolerance test 81. 
Pharmacogenomic Analysis of Metformin 
Blood collection for genetic samples for future analysis will occur on admission for Visit 2.  We will take two approaches 
to address this exploratory aim.  First, we will assay all participants using a genome-wide genotyping chip. The chip used 
will be selected based on the best option for this purpose at the time of assay, though one likely option is Illumina’s Multi-
Ethnic Genotyping Array (MegaChip), which included population genetic studies of African-Americans in its design and 
allows for some customizable content.  The Chip data will give us genome-wide genotyping of common variants that will 
include (by design or customization) variants previously associated with metformin response (Table 1).  In addition, chip 
data will be used to adjust the analyses for genome-wide proportion of African ancestry 84, an important covariate in the 
data analysis of admixed African-Americans.  Variants were selected for analysis based on evidence for an interaction 
between the variant and glucose-related metformin response.  These variants were further limited to those with evidence 
from more than one study or in genes with strong biological plausibility.  Additionally, we will sequence the entire gene 
and promoter region of genes with the strongest evidence for an association with metformin response: SLC22A1, 
SLC22A2, SLC22A3, SLC47A1, SLC47A2, and ATM.  As most of the study of these pharmacogenomic loci has been 
conducted in non-African ancestry samples, genetic variation relevant for African-Americans youth may have been 
missed.
Secondary genetic findings: A “secondary genetic finding” describes a finding that is not related to the primary reason 
that the study is being done. While identifying secondary genetic findings is usually the realm of genome- or exome-wide 
sequencing studies, common genome-wide genotyping chips, such as the MegaChip, have added a set of rare variants that 
are considered by the American College of Medical Genetics and Genomics (ACMG) to be actionable.  An individual’s 
knowledge of carrying these rare variants can have significant implications for medical decisions in terms of treatment or 
monitoring in individuals and their families. It is expected that whole exome sequencing would identify a clinically 
actionable variant among 1.2 - 3.4% of individuals sequenced 85. With the sample size of the current study and 
considering that chip array is substantially less comprehensive than whole exome sequencing, this analysis is likely to 
identify one or no participants with actionable variants. Given the potential medical value of this information, however, 
we will evaluate the ACMG variants on the chip and will be prepared to return these findings to participants using the 
following procedures.  First, the ACMG variants will be extracted from the chip data, and the genotypes for all 


Principal Investigator: Chung, Stephanie T.  
Last updated: October 5, 2021
Page 21
participants at these loci will be evaluated. If an actionable variant is identified at one of the ACMG loci, a new blood 
sample will be requested, as these results are intended for research purposes only. At this point, participants will be 
informed that they may carry a genetic variant that could have important consequences for their health, but that these 
findings will need to be confirmed. The blood sample will be sent to a commercial laboratory to obtain Clinical 
Laboratory Improvement Amendments (CLIA)-certified results.  If the finding is confirmed, a genetic consult will be 
requested at the NIH Clinical Center, and a genetic counselor will meet with the participant and/ or the participant’s 
parent. The research team will offer to pay for the cost of confirmatory findings.  
Microbiome Analysis
Fecal samples will be collected in sterile cryovials and stored at 4C for less than 24 hours.  The sample will be transferred 
to -80C and stored until further analysis.  This analysis will be performed in collaboration with Dr. Sushil Rane and Dr. 
Hariom Yadav.  After thawing of fecal sample, fecal DNA will be isolated for microbial compositional analysis using 
PowerSoil® DNA Isolation Kit.  Amplicon sequencing using next generation technology (bTEFAP®) will be used to for 
microbial sequencing using Illumina MiSeq and HiSeq platforms technologies.  In brief, the 16S universal Eubacterial 
primers 27F AGAGTTTGATYMTGGCTCAG and 519R GTNTTACNGCGGCKGCTG will be utilized to evaluate the 
microbial ecology of each sample on the Illumina MiSeq with methods via the bTEFAP® DNA analysis service.  A 
single-step 30 cycle PCR using HotStarTaq Plus Master Mix Kit (Qiagen, Valencia, CA) will be used under the following 
conditions: 94C for 3 minutes, followed by 28 cycles of 94C for 30 seconds; 53C for 40 seconds and 72C for 1 minute; 
after which a final elongation step at 72C for 5 minutes will be performed.  Following PCR, all amplicon products from 
different samples will be mixed in equal concentrations and purified using Agencourt Ampure beads (Agencourt 
Bioscience Corporation, MA, USA).  Samples will be sequenced utilizing the Illumina MiSeq chemistry following 
manufacturer’s protocols.
The Q25 sequence data derived from the sequencing process will be processed using a pipeline (MR DNA, Shallowater, 
TX).  Sequences will be depleted of barcodes and primers then short sequences < 200bp are removed, sequences with 
ambiguous base calls removed, and sequences with homopolymer runs exceeding 6bp removed.  Sequences are then 
denoised and chimeras removed.  Operational taxonomic units (OTU) will be defined after removal of singleton 
sequences, clustering at 3% divergence (97% similarity)86-89.  OTUs will be taxonomically classified using BLASTn 
against a curated GreenGenes/RDP/NCBI derived database90 and will be compiled into each taxonomic level into both 
“counts” and “percentage” files.  Count files contain the actual number of sequences while the percent files contain the 
relative (proportion) percentage of sequences within each sample that map to the designated taxonomic classification.
Metformin Pharmacokinetics
Samples will be processed for isolation of plasma and RBC and frozen at -80C until analysis.  Both plasma and RBCs will 
be processed in duplicate cryotubes (800 – 1000 µl per cryotube for each time-point collected). Cryotubes will be labeled 
with participant ID and sample time collection and the timing of metformin administration documented. Metformin is 
stable under refrigerated conditions for up to 24 hours after collection- therefore samples may be kept in the fridge for 
pickup and processing the next day (the later PK time points- 8 and 12 hours).  Analysis for metformin concentration will 
be via a validated LC-MS/MS assay.
Collection and Storage of Human Specimens or Data
Research Use, Storage and Disposition of Human Subjects’ Samples 
For future reference and potential use, we will store all samples (blood or fluids) in our locked freezers for an unlimited 
period of time. Samples will be labeled with coded identifiers linked to patient identity only via a secured database. 


Principal Investigator: Chung, Stephanie T.  
Last updated: October 5, 2021
Page 22
Research records and data with personal identifiers will be stored in our locked offices, the medical record department, 
and the electronic study database. This material will additionally be protected by medical record and computer access 
procedures.  Access to records and data associated with personal information will be restricted to the Principal 
Investigator, Co-Investigators, study support staff, and database support staff.
Subjects may request that unused samples be removed from our freezers and be destroyed.  If no such request is made, we 
will keep samples until they are completely used or no longer of scientific value, at which time they will be destroyed. We 
do not plan to destroy personal medical information or stored data. The Principal Investigator will report loss or 
destruction of data or samples to the IRB. 
Genomic Data Sharing Plan
This protocol involves the collection of human and non-human genomic DNA.  In the pharmacogenetics study of 
metformin, genomic DNA from patients will be stored for genome-wide testing and targeted gene sequencing.  Fecal 
samples will also be stored for analysis of microbial DNA.  The participants will consent for their data to be shared 
through publicly accessible databases.  De-identified data from this study will be made available by submission to dbGAP 
or a similar publicly available database, for which user registration is required.  Data will be submitted at the time of 
publication and will include the relevant genomic data and corresponding phenotypic data.  Users must agree to the 
conditions of use governing access to the public release data, including limitation of research to investigations consistent 
with the participants’ consent, restrictions against attempting to identify study participants, destruction of the data after 
analyses are completed, reporting responsibilities, restrictions on redistribution of the data to third parties and proper 
acknowledgment of the data resource. 
Materials Transfer Information
Stored samples and/or data may be sent to outside collaborating laboratories, or shared with other NIH collaborating 
investigators, to study questions related to diabetes or its complications (including, for example: glucose metabolism, 
diabetes, obesity, weight, and lipid metabolism). Samples may be sent to outside commercial laboratories for analysis.  
Samples and data sent to outside laboratories and collaborators for analysis and/or testing will contain only coded 
numbers, without personal identifiers. Materials Transfer Agreements will be completed before the exchange of samples 
and/or data with outside collaborators.
Samples for isotopic enrichment of gluconeogenesis and other metabolites will be centrifuged and plasma stored at -80C.  
The samples will be run in duplicate at the Baylor College of Medicine, Children’s Nutrition Research Center and at the 
NIDDK, Mass Spectrometry Core Laboratory.  Coded samples without personal identifiers will be sent to Dr. Morey 
Haymond (co-investigator) at the Children’s Nutrition Research Center, Baylor College of Medicine.  After IRB approval, 
a Materials Transfer Agreement will be executed between NIDDK and Baylor College of Medicine, Houston, TX.  No 
data or samples will be transferred without fully executed tech transfer agreements.
Statistical and Power Analyses
Sample Size Justification – Primary Study
Sample size estimates are based on the mean changes and standard deviations observed in our pilot study (Table 2).  
Assuming a two-sided alpha of 0.05 and standard deviations of change from baseline of 0.20 mg●kglbm●min for 
gluconeogenesis and 0.50 mg●kglbm●min for glucose production, a trial with 12 participants in each group will have 80% 
power to detect a difference between metformin and combination therapy of 0.24 mg●kglbm●min in gluconeogenesis and 
in 0.60 mg●kglbm●min in glucose production.  Analyses within each group will have 80% power to detect a change from 
zero if the true change is ≥0.18 and ≥0.44 mg●kglbm●min for gluconeogenesis and glucose production respectively. For 


Principal Investigator: Chung, Stephanie T.  
Last updated: October 5, 2021
Page 23
the secondary outcome of change in GLP-1 AUC after an OGTT, we will have > 95% power to detect the mean difference 
of 101 pg●min/ml.  Assuming a 25% attrition rate, we will recruit 16 in each group.
Table 2: Mean and Estimated Rates of Gluconeogenesis and Glucose Production and Projected Effect Sizes* 
Baseline Mean 
(SD)
Follow-up 
Mean (SD)
Difference 
Mean (SD)
15% Decrease
20% Decrease
Gluconeogenesis 
(mg●kglbm●min)
1.78 (0.41)
1.73 (0.31)
0.05 (0.19)
0.27
0.36
Glucose Production 
(mg●kglbm●min)
2.93 (0.92)
2.67 (0.54)
0.27 (0.50)
0.44
0.59
Early intact GLP-1 AUC 
(pg●min/min)
137 (113)
238 (128)
101 (32)
-
-
*Data from pilot study in 5 youth with type 2 diabetes treated with metformin 1000mg twice daily for 3 months.
This power analysis was performed with two-sided T tests to detect a between group difference of a 15% change in 
gluconeogenesis which would require 12 individuals per group.  Our primary assumption is that there will be a 15% 
difference in gluconeogenesis between the two study arms.  This assumption is underscored by the fact that metformin 
will not result in a change in gluconeogenesis in vivo 24,26,27 but liraglutide will decrease rates of gluconeogenesis by 15%.  
This sample size is in keeping with previous studies assessing metformin effect using similar techniques to measure of 
gluconeogenesis 24,25.  Nevertheless, we acknowledge that the delta difference and SD for change between two differently 
treated groups could be larger. Therefore, this pilot study will provide valuable preliminary information for the magnitude 
of the difference between intervention groups that would be crucial for a larger randomized placebo controlled trial. 
Analysis of Primary and Secondary Outcomes
The primary outcome, the change in gluconeogenesis from baseline to 12 weeks, will be analyzed using an analysis of 
variance that compares the two randomized groups adjusting for baseline levels. Similar analyses will be performed for 
the secondary outcomes, using transformations or nonparametric tests as appropriate. Secondary regression analyses will 
adjust for the effect of other variables (such as fat mass, metabolite and hormonal concentrations etc.) on these changes.
Analysis of Exploratory Outcomes
Pharmacogenomic Variants of Metformin Response
Given the limited sample size, the proposed genetic aims are exploratory in nature.  First we will use 9 previously-
identified genetic variants (Table 1) to construct a genetic risk score, calculated as the sum of the risk alleles that the 
individual has for any of the variants. Metformin response will be assigned as a 15% change in gluconeogenesis from 
baseline (minimum clinically relevant effect size) for each individual26,91. We will then compare this risk score in the 
metformin responders vs. non-responders.  The genetic risk score based on these 9 variants associated with metformin 
response in adults will be analyzed as a single predictor, and not by individual variants.  Since this approach may miss 
important variation in these genes, particularly given that the question of metformin response has been less well studied 
among African-Americans, we will also sequence the genes of interest in these individuals.  
We will use regression models to determine if this risk score predicts metformin response in terms of changes in rates of 
gluconeogenesis and metformin concentrations in serum and urine, after adjustment for genome-wide proportion of 
African ancestry (additional covariates will be evaluated for inclusion), with p<0.05 considered statistically significant.  
Rare variants identified during targeted gene sequencing will be analyzed in a composite way, collapsed by gene and 
considered with a rare variant analysis tool (e.g. SKAT 92).  While this analysis may have limited power, the identification 


Principal Investigator: Chung, Stephanie T.  
Last updated: October 5, 2021
Page 24
of additional variants in these genes among African-American youth will inform future larger studies.  Additionally, we 
will determine whether adding the sum of the risk alleles as covariates modifies the statistical analyses for the outcomes in 
Aim 1 and Aim 2.  The composite genetic risk score will be used to adjust the primary outcome analysis of change in 
gluconeogenesis to see if accounting for this variation influences our conclusions.  These results will be evaluated using a 
likelihood ratio test comparing a regression model adjusted and unadjusted for the variant.
Gut Microbiome and Metformin
Statistical analysis will be performed using a variety of computer packages including XLstat, NCSS 2007, “R” and NCSS 
2010.  Alpha and beta diversity analysis will be conducted as previously described86-88,93 using Qiime (www.qiime.org). 
Significance reported for any analysis is defined as p<0.05. 
Metformin Pharmacokinetics
Statistical analysis and non-linear mixed effect modeling will be performed using the “Phoenix WinNonlin”, an industry 
standard for non-compartmental analysis, pharmacokinetic/ pharmacodynamic modeling.  
Multiple-site Studies
Eligible patients may be identified through CNMC but no enrollment, informed consent or study visits will occur at 
CNMC.
The primary IRB on record will be NIH IRB.  A Federalwide Assurance for Human Subject’s Protection (FWA) for 
Children’s National Medical Center is FWA00004487 and for PBRC is FWA 00006218.
We will also utilize the Translational Research in Pediatrics Program (TRIPP), an established contractual agreement 
between CNMC and the National Institutes of Health Clinical Center (NIH CC).  A Reliance agreement has been executed 
between NIH and CNMC. 
Human Subject Protection
Informed Consent/Assent Procedures
Informed consent/ assent will be obtained as consistent with the requirements of SOP 301 and 402.  Written 
consent/assent will be obtained from each subject after detailed explanations of the planned procedures by the principal or 
an associated investigator. .  Prospective subjects will be given the research information in layman’s term and in language 
understandable to the subjects. The consent process will take place prior to any study procedures.  Subjects have the right 
to withdraw participation from this protocol at any time.
When a document that is in electronic format is used for the documentation of consent, this study will use the iMED 
platform, which is 21 CFR, Part 11 compliant, to obtain the required signatures. The iMED consenting process will 
happen at NIH clinical center. All patient identification will be confirmed with hospital issued wrist band.  During the 
consent process, participants and investigators will view individual copies of the approved consent document on screens at 
the NIH Clinical Center. Both the investigator and the participant will sign the document with a hand signature using a 
finger, stylus, or mouse.
Language for Minors
Written informed consent and assent will be obtained from the minor and his/her parent/guardian or (LAR) prior to any 
screening visits, study procedures or interventions. The Principal Investigator or other designated qualified protocol 
investigators will explain the study in language understandable to the parent/guardian or (LAR)).  The investigator will 


Principal Investigator: Chung, Stephanie T.  
Last updated: October 5, 2021
Page 25
also explain the study to the minor who is of a younger age and level of understanding.  Sufficient time and opportunity 
will be given for discussion of the research as well as to answer any questions they may have, taking care to minimize or 
eliminate the perception of coercion or undue influence. In accordance with 45 CFR 46.408, [the parent/guardian or 
(LAR)], will sign the current IRB approved informed consent.  The minor will provide assent as appropriate for the 
child’s age and level of understanding, and sign the assent document if possible. The investigator will sign the assent as 
well. A copy of the consent and assent will be given to the minor and [his/her [parent/guardian or (LAR)] for future 
reference. The signed documents will be sent to the Medical Records Department for placement in the subject's permanent 
CC medical record. The consent process will additionally be documented in the electronic medical record (CRIS). 
For minors who reach the age of consent while enrolled in our protocol, the parent and the minor will be forewarned at the 
time of enrollment, that there will be a need to consent the minor on reaching the age of consent as consistent with the 
requirements of SOP-301 and 402—Requirements for Informed Consent.  This is because the prior parental permission 
and child assent are not equivalent to legally effective informed consent for the now-adult subject (consistent with NIH 
HRPP SOP 402). All individuals will be given the opportunity to ask questions. In these situations, reconsenting will be 
performed at the NIH CC.  
Rationale for Subject Selection
Type 2 diabetes in youth is a rapidly progressive and aggressive disease yet current treatment options are limited and not 
effective.  Furthermore, African-American youth are disproportionately affected by 2-fold increased risk for the disease 
and a 4-fold increased risk for complications compared to white youth.  Currently, clinical therapy with metformin in 
suboptimal and African-American youth have the highest failure rates.  To improve outcomes, we need to understand the 
pathogenesis of the disease as well (as the pharmacodynamics and pharmacogenomics) in these high risk youth.  
Therefore, this study was designed to specifically examine these parameters in African-American youth to improve 
outcomes and inform precision medicine initiatives. Volunteers eligible for this study include mature adolescents and 
young adults with type 2 diabetes.  Young adults will be included in the protocol to enhance recruitment and because the 
disease progression is similar to adolescents with type 2 diabetes.  We will not recruit pre-pubertal or peri-pubertal 
children with diabetes because this increases the risk that they have type 1 diabetes (a condition not relevant to the aims of 
this study). 
Rationale for Exclusion of Vulnerable Populations
This study will involve recruitment of children age 12-17 years as described above.  The study will not involve 
recruitment of other vulnerable populations including pregnant women, prisoners, adults who are or maybe unable to give 
consent because they do not meet the inclusion criteria for this study.  We will protect participants as described in SOP 
402.
Recruitment Strategies
Local subject recruitment documents, including listserv, flyers, social media posts, press releases, correspondence to local 
subjects will be submitted for approval to the relying IRB. 
Tools used for the recruitment of subjects will include the posting of the study description on the Clinical Center Public 
Service Announcement (NIH CC website, the NIH Record, the Clinical Center News , Clinical Center Facebook, Clinical 
Center Twitter, OPR LISTSERV, ResearchMatch), and CNMC; radio media, referrals from other protocols, outside 
physician referrals and self-referral,  newspaper advertisements and video ad. Additionally, fliers may be distributed 
throughout the NIH campus, the CNMC, George Washington University, the University of Maryland and other 
pediatrician or doctors’ office sites in the Washington DC Metropolitan and Baton Rouge areas.


Principal Investigator: Chung, Stephanie T.  
Last updated: October 5, 2021
Page 26
Strategies to address the challenges of clinical trials in youth with type 2 diabetes 
There are two major challenges to pediatric research in type 2 diabetes in youth: (1) difficulty with recruiting eligible 
participants and (2) poor retention rates94.  Multiple social (decreased recognition of importance of research within this 
field) and physical barriers (inconvenient study site locations and lack of travel reimbursement) contribute to these 
challenges. Therefore, to maximize recruitment opportunities our research approach includes:
1)
Partnering with CNMC, the largest tertiary care pediatric diabetes center in the Mid-Atlantic region, and leveraging 
that relationship to build relationships with physicians and families within the community – with a view to promoting 
health and research literacy.
2)
Collaboration between myself and Dr. Fran Cogen, the Director of Diabetes Clinic at CNMC where we care for many 
youth with type 2 diabetes (see below). 
3)
Utilizing the Transitional Research in Pediatrics (TRIPP) agreement, an agreement established in 2013 between 
CNMC and the NIH Clinical Center, which provides additional administrative and institutional support to facilitate 
transportation and organizational support.  
The Pediatric Diabetes and Endocrinology Division at CNMC, Northeast location in Washington DC cares for over 250 
youth and young adults with type 2 diabetes per year, the majority of whom are African-American.  Table 4 summarizes 
the demographics of eligible patients with type 2 diabetes at CNMC and illustrates that ~ 60-70 youth would meet our 
eligibility criteria and the majority of these youth are seen within the monthly type 2 diabetes clinics at CNMC. We 
anticipate that recruitment and enrollment will occur over a 3-year period.
Table 4: Demographics of Eligible Patients with Type 2 Diabetes seen at CNMC, 2014-2015
1.
New-onset T2D patients:
a.
# of newly diagnosed patients with T2D seen each year (within 3 mos of 
dx) 
40-50
b.
Approximate % pediatric (<18 yrs) 
89%
c.
Approximate %adult (>18 yrs) 
11%
2.
Established T2D patients:
a.
# of established patients currently seen at least once a year
195
b.
Approximate % pediatric (<18 yrs) 
86%
c.
Approximate %adult (>18 yrs) 
14%
3.
Insulin Use:
a.
Approximate % of patients using insulin
41%
4.
Race/ethnicity of T2D Patients: Indicate approximate distribution of the T2D patients you see:
a.
Approximate % Non-Hispanic White 
10%
b.
Approximate % African American
66%
c.
Approximate % Hispanic
2%
d.
Approximate % Asian
3%
e.
Approximate % American Indian / Alaska Native
0%
T2D – type 2 diabetes
To improve retention in this study we will employ the following strategies: 
1)
Promote research and health literacy through community outreach.
2)
Daily to weekly telephone and text message reminders. 
3)
Monthly follow-up visits to monitor medication and glycemia.
4)
Strategize with individual families regarding their unique socio-economic barriers and offer alternative and supportive 
strategies to encourage study adherence (e.g. finding creative solutions to transportation issues).
5)
Offer study visits on the weekends and holidays to encourage participation without interrupting the children’s 


Principal Investigator: Chung, Stephanie T.  
Last updated: October 5, 2021
Page 27
academic schedule and performance. 
Risks/Benefits Analysis including Considerations of Alternatives to Participation
Evaluation of Benefits
This research protocol is designed to elucidate the physiologic targets of metformin and liraglutide.  Both agents are anti-
diabetic agents and are expected to improve overall glycemia in individuals.  Metformin and liraglutide are approved for 
the treatment of youth with type 2 diabetes 10 years or older as an adjunct to diet and exercise..  Therefore, participants 
may benefit from participating in the protocol with the use of these 2 agents.  Participating in the protocol procedures may 
also be beneficial to the individual because it may provide specific information about the individual’s glycemic control 
and diabetes management, as well as more frequent interaction with the study team. If a secondary genetic finding is 
identified during genetic testing, a participant may learn valuable information about their health which could lead to 
treatments or monitoring that could significantly improve their health outcomes. Such a finding could also have 
implications for family members who may share the risk variant. 
Evaluation of Risks
  The specific risks and our approach to minimize risks are outlined below.  
1.
Stable Isotope Administration:  Stable isotopes are naturally occurring and non-radioactive and are not associated 
with any toxicity at the doses used in these studies.  However, rapid administration of deuterated water (2H2O) can 
be associated with mild and transient dizziness.  Administering the deuterated water in 3 divided doses overnight 
while the patient is lying down significantly minimizes this risk.   If the subject needs to get out of bed (e.g. to use 
the bathroom), the patient will first sit upright in bed for a few minutes prior to standing and a nurse will be 
available to supervise and assist. The isotopes will be prepared by Pine Pharmaceuticals and tested to certify 
sterility and freedom from bacteria and substances that may cause pyrogenicity.  The NIH Clinical Center 
Pharmacy will determine final approval of isotope products after careful review of sterility and stability records. 
2.
Blood Sampling:  Peripheral blood draws (venipuncture) performed during this study for research will not exceed 
10.5 mL/kg, or 550 mL (whichever is smaller) per 8-week period for adults.   For pediatric patients, blood draws 
will not exceed 5 mL/kg in a single day, or 9.5 mL/kg or 550 mL (whichever is smaller) per 8-week period.  The 
total blood volume for this study is 520 ml in 12-week period.  Patients may experience some discomfort at the 
site of the needle entry, and there is a risk of bruising at the site.  There is a remote risk of fainting or local 
infection.  
3.
Intravenous Catheter Placement:  The placement of intravenous catheters can be uncomfortable and pose the 
potential risk of bleeding, bruising and infection.  All catheters will be placed under sterile conditions and 
universal precautions will be observed.  Should any complications occur, they will be addressed immediately. 
4.
Timed urine collection:  Urine collection is not associated with any health risk but may be inconvenient for 
subjects.
5.
Resting Energy Expenditure: There are no significant risks associated with measurement of resting energy 
expenditure.  Some patients may feel uncomfortable from lying still with a plastic hood over their head for 
approximately 30 minutes.


Principal Investigator: Chung, Stephanie T.  
Last updated: October 5, 2021
Page 28
6.
Risk associated with metformin 
i.
Gastrointestinal upset:  Nausea, vomiting, diarrhea, flatulence, abdominal discomfort and indigestion are 
common side effects associated with metformin use.  The following actions will be taken to minimize the 
risk for gastrointestinal disturbance:
a. Metformin dosing will be gradually increased over 4-week period (Table 3). 
b. Participants who experience mild to moderate gastrointestinal symptoms will contact the study 
team and the dose of metformin decreased incrementally for 1 week.  If symptoms have resolved, 
the dose will be incrementally increased to achieve maximum dose of 1gram twice daily. 
c. Participants who experience severe and/or recurrent gastrointestinal symptoms will be withdrawn 
from the study.
ii.
Boxed Warning – Lactic acidosis (rare): The risk of lactic acidosis with metformin use is increased in 
elderly individuals with renal dysfunction or congenital heart failure. Participants with chronic renal or 
heart failure, or other chronic metabolic illnesses will not be recruited for this study. 
7.
Risks associated with liraglutide (Victoza®)
i.
Gastrointestinal upset: Nausea, vomiting and/ or diarrhea are common adverse reactions associated with 
liraglutide (Victoza®) occurring in 5-20% of patients (FDA labeling insert).  Combination therapy of 
Victoza® and metformin slightly increases the incidence rate of nausea, vomiting and diarrhea to 9-24%62.  
In a 52-week randomized double blind controlled trial of liraglutide vs. placebo in 135 youth with type 2 
diabetes treated with metformin, liraglutide was associated with a higher rate of serious adverse events 
although these were not associated with any specific finding or patter. The most common adverse event 
noted in at least 5% of patients was: nausea (29%), vomiting (26%), diarrhea (23%) and abdominal pain 
(18%) and nasopharyngitis (17%)63.  The majority of all adverse events were mild in severity and 
resolved.  There were no deaths reported. The symptoms were mild and resolved spontaneously within 1-
4 days.  The following actions will be taken to minimize the risk for gastrointestinal disturbance:
a. Liraglutide dosing will be gradually increased over 4-week period (Table 3). 
b. Participants who experience mild to moderate gastrointestinal symptoms will contact the PI and 
the dose of liraglutide decreased incrementally for 1 week.  If symptoms have resolved, the dose 
will be incrementally increased in 1-week intervals to achieve maximum dose of 1.8mg once 
daily. 
c. Participants who experience severe and/or recurrent gastrointestinal symptoms will be withdrawn 
from the study.
ii.
Hypoglycemia: Victoza® lowers glucose by increasing glucose-dependent insulin secretion, lowering 
glucagon secretion and delaying gastric emptying.  These actions result in lower fasting and postprandial 
glucose concentrations in the circulation; in adults, Victoza® can increase the risk of hypoglycemia at a 
rate of 0.24 episodes/ patient years.  In youth treated with liraglutide and metformin, minor hypoglycemia 
occurred in 24% of patients and there were no cases of severe hypoglycemia63. We will monitor the 
participants closely and the following precautions and actions will be taken to minimize the risk for 
hypoglycemia:
a. Blood glucose monitoring. Patients will monitor their blood glucose by either checking their 
fingerstick blood glucose levels at least twice daily (pre-breakfast and bedtime) or wearing an 
FDA-approved CGM device for the duration of the study. 


Principal Investigator: Chung, Stephanie T.  
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b.All participants will be educated on the signs and symptoms of hypoglycemia: dizziness or 
lightheadedness, sweating, confusion, headache, blurred vision, slurred speech, hunger, 
weakness, anxiety or irritability, fast heartbeat.  
c. Blood glucose values <70mg/dl with or without signs and symptoms of hypoglycemia (described 
above) will be immediately reported to the principal investigator and dose of liraglutide 
decreased. 
d. If a participant experiences recurrent severe hypoglycemia, defined as blood glucose <50mg/dl 
with or without symptoms, the need for a third party to resolve a hypoglycemic episode or loss of 
consciousness or seizure on 2 or more occasions, liraglutide will be stopped and they will be 
withdrawn from the study. 
iii.
Pancreatitis:  In post marketing reports, Victoza has been associated with acute pancreatitis. In a large 52- 
week randomized trial of liraglutide vs. placebo in 135 youth with type 2 diabetes on metformin, 
liraglutide was associated with higher lipase concentrations, but there were no cases of pancreatitis. The 
following measures are being taken to minimize the risk of pancreatitis in this study:
a.Subjects at increased risk of pancreatitis are excluded, as follows:
i.
Triglycerides over 500 mg/dl.  This will exclude patients with extreme 
hypertriglyceridemia at baseline, who have a higher risk for pancreatitis.
ii.
Prior history of more than one episode of pancreatitis. 
iii.
Current lipase > 60 units/L (the upper limit of normal in the NIH assay, 02/08/2014 to 
present).  While both amylase and lipase are often used clinically in the assessment of 
pancreatitis, amylase is a non-specific test that may be elevated for other reasons.  
Subjects with elevated lipase at study entry will be considered to have laboratory findings 
consistent with pancreatitis and will be excluded.
b. If pancreatitis is clinically suspected (either based on signs/symptoms consisting of moderate to 
severe abdominal pain with nausea or vomiting, or based on lipase), the following will take place:
i.
Amylase and lipase levels will be drawn.
ii.
Gastroenterology (GI) consultation will be obtained.
c. If a diagnosis of pancreatitis is likely or confirmed based on labs and/or GI consultation, the 
following additional actions will be taken:
i.
The patient will be referred to CNMC for further management and admission.
ii.
Appropriate treatment will be initiated based on GI recommendations (usually, NPO and 
pain medications).
iii.
The patient will be withdrawn from the study.
iv.
The IRB will be notified per guidelines for serious adverse effects.
iv.
Kidney problems: In individuals with kidney failure, diarrhea, nausea and vomiting may cause a loss of 
fluids and dehydration.  Subjects with impaired renal function at baseline evaluation will not be recruited 
for this study.
v.
Thyroid C-Cell Tumors: Victoza® has a black box warning for the risk of thyroid c-cell tumors. 
Liraglutide causes dose-dependent and treatment-duration-dependent thyroid C-cell tumors (adenomas 
and/or carcinomas) at clinically relevant exposures in both genders of mice. It is unknown whether 
Victoza will cause thyroid C-cell tumors and in humans, the relevance of liraglutide-induced rodent 
thyroid C-cell tumors has not been determined. There is insufficient evidence to establish or exclude a 


Principal Investigator: Chung, Stephanie T.  
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Page 30
causal relationship between medullary thyroid carcinoma and Victoza use in humans.  The following 
measures are being taken to minimize the risk of thyroid c-cell carcinoma and Victoza use in this study:
a.Patients with a personal or family history of medullary thyroid carcinoma, and patients with 
multiple endocrine neoplasia 2 will be excluded from the study because the use of Victoza in 
these patients is contraindicated. 
b.We will monitor subjects clinically for signs of thyroid nodules or neck swelling.
c.Serum calcitonin concentrations will be measured at baseline and at the end of the study.
d.If a thyroid nodule or neck swelling is observed, further evaluation will be pursued with thyroid/ 
neck imaging and measuring serum calcitonin concentrations. 
vi.
Hypersensitivity Reactions: There are no studies linking Victoza® to allergic reactions; however, in post-
marketing reports, Victoza has been associated with serious allergic reactions (e.g. swelling, hives, 
difficulty breathing).  Individuals who are known to have an allergic reaction to Victoza® will not be 
enrolled in this study.
8.
Risk associated with drug-free run-in period
i.
Severe Hyperglycemia with metabolic decompensation: During the 5-7-day period between Visit 1 and 
Visit 2, youth may be at increased risk for severe hyperglycemia since the youth will be asked to stop 
their anti-diabetic medication.  This risk is reduced because we will only enroll individuals who are in 
relatively good glycemic control (HbA1c <9%) who will be at lower risk for decompensation.  The 5-7 
day period was chosen to minimize risk, and diet and lifestyle treatment is in keeping with current 
standard of care clinical guidelines as an important treatment modality for youth with type 2 diabetes 95.  
For youth previously on metformin (the majority of eligible patients will be in this category), 5-7 days is 
sufficient to ensure complete elimination of metformin from the plasma and erythrocyte compartments; 
elimination half-life of 4-6 hours 96and 18 hours respectively 97.  Therefore, sufficient drug elimination 
will occur after ~ 5 half-lives i.e. plasma: 30hrs (1.25 days) and erythrocyte: 90 hrs (3.75 days).  
The following steps will be taken to minimize the risks of severe hyperglycemia: 
a.Participants at higher risk for severe hyperglycemia defined as those with HbA1c ≥9% will not be 
eligible to participate.
b.All participants will receive up-to-date education on diet and lifestyle management by the PI and 
study staff.
c.During the run-in drug free period all individuals will monitor their blood glucose levels with a 
FDA-approved CGM device or a home glucometer 2-3 times daily.  Study staff will review blood 
glucose logs and CGM tracings daily. For participants with persistently elevated FSBG without 
metabolic decompensation, the physician may recommend admission to the NIH metabolic unit 
for close monitoring of their blood glucose levels prior to Visit 2.
d.If FSBG is >200mg/dl fasting, or 300mg/dl bedtime is noted during the run-in period, they will 
notify the study PI immediately.  Individuals with persistently elevated FSBG and signs and 
symptoms of metabolic decompensation (e.g. vomiting, dehydration, lethargy, abdominal pain) 
will be withdrawn from the study and restarted on his/ her medication. 
e.If vomiting, abdominal pain or dehydration is noted, participant will be evaluated by a medical 
professional or PI immediately for metabolic evaluation of acidosis and ketosis. If metabolic 
decompensation (acidosis or ketosis) is noted, participants will be promptly treated and 
withdrawn from the study. 
9.
Stool sampling: Stool sampling is not associated with any health risk but may be uncomfortable for some 
participants.


Principal Investigator: Chung, Stephanie T.  
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10. Radiation Exposure: Dual energy absorptiometry (DXA) scan. The DXA scan is a reliable and reproducible 
method to measure body composition, specifically body fat and lean body mass.  The patient lies on a flat table 
with the x-ray source below the table and the detector above. Each scan takes about 3 minutes. For the DXA scan 
the effective dose of radiation is 0.00006 rem, which is below the guideline of 5 rem (or 0.5 rem in children) per 
year allowed for research subjects by the NIH Radiation Safety Committee.  The average person in the United 
States receives a radiation exposure of 0.3 rem per year from natural sources, such as the sun, outer space, and the 
earth’s air and soil.  If participants want to learn more about radiation, they will be given the pamphlet: An 
Introduction to Radiation for NIH Research Subjects. 
11. Genetic Testing: Genetic and phenotype data will be placed in a dbGaP or similar database in accordance with 
NIH policy on sharing of genetic data.  Through this database, researchers with approved proposals will be able to 
access these data; however, the data will be de-identified prior to data-sharing.  As genetic information is unique 
to an individual, there is a small chance that an individual may be identified through these data, and it is possible 
that this risk will grow over time.  Additionally, there is a small risk that genetic information could lead to 
discrimination, for instance for employment or insurance; however, there are laws in place that protect against this 
sort of discrimination.  Genetic information may lead to statements about an individual’s ethnic group that they do 
not agree with.  If a secondary genetic finding is identified, learning about that finding may be upsetting for the 
participant. Their relatives may also be upset if the variant has implications for their health. Participants who do 
not receive a report of a secondary finding may falsely interpret this lack of finding as assurance of absence of any 
medically serious genetic variants, though the genetic testing conducted is very limited and cannot be interpreted 
in this way. 
Alternatives to participation
Participation in clinical trials is completely voluntary.  Refusal to participate will not affect a subject's ability to participate 
in other studies at NIH or elsewhere.
Adverse Event Reporting 
Event Characterization and Reporting to the IRB 
Adverse events, non-compliance both serious or continuing, protocol deviations both major and minor, as well as 
unanticipated problems are defined & described by the NIH Office of Human Subjects Research Protection policy #801 
and will be reported in accordance with this policy. 
Investigational New Drug Application/ Exemption
The FDA expanded the indication for Victoza® (liraglutide) therapy as an adjunct to diet and exercise to improve 
glycemic control in patients 10 years and older with type 2 diabetes mellitus on June 17, 2019. The FDA approval was 
based on data submitted from the ELLIPSE clinical trial, which was published in the New England Journal of Medicine in 
April 201963.  Prior to, this approval, we submitted and received an IND exemption (June 2016) for the study drugs used 
in this clinical study.   This trial is not being conducted with the intention to support advertising or to submit data to a 
marketing application for metformin or liraglutide.  In addition, we plan to conduct this clinical trial in accordance with 21 
CFR Part 50 and Part 56 and the drugs are used within the labeling requirements.  
Data and Safety Monitoring Plan
Study procedures will be subject to audits and/or monitoring visits to ensure compliance with the protocol and applicable 
regulatory requirements consistent with the NIDDK quality assurance program plan.  Audit and/or monitoring visit results 


Principal Investigator: Chung, Stephanie T.  
Last updated: October 5, 2021
Page 32
will be reported to the Principal Investigator for further reporting as appropriate.   Study documents and pertinent hospital 
or clinical records will be reviewed to verify that the conduct of the study is consistent with the protocol plan.
The collection, monitoring and analysis of adverse events will be the responsibility of the Principal Investigator and the 
investigative team.  
As required by FDA 21 CFR 312.50, trial procedures will be subject to review to ensure compliance with the protocol and 
applicable regulatory requirements.  Results will be reported to the Principal Investigator/Sponsor for further reporting to 
the FDA consistent with applicable regulations.   The specific monitoring plan will be developed with the Principal 
Investigator and frequency of monitoring visits determined by such factors as study enrollment, data collection status and 
regulatory obligations.  Monitoring visit results will be reported to the Principal Investigator for further reporting as 
appropriate.  Study documents and pertinent hospital or clinical records will be reviewed to verify that the conduct of the 
study is consistent with the protocol plan.
Monitoring Subjects and Criteria for Withdrawal of Subjects from the study
Withdrawal Criteria
1.
Withdrawal of consent.  A subject wish to withdraw from the study as stated in the informed consent (all 
subjects reserve the right to withdraw from the study without prejudice).
2.
Adverse event.  A subject experience an adverse event that in the investigator’s opinion necessitates withdrawal 
from the study.  Specific examples of adverse events that would result in withdrawal are:
a)
Pancreatitis during liraglutide administration.  Pancreatitis is a clinical diagnosis and will be suspected in 
the presence of moderate to severe abdominal pain accompanied by nausea and/or vomiting.  If 
pancreatitis is suspected, amylase and lipase levels will be drawn, and GI consultation obtained.
b)
Gastrointestinal intolerance to study drug (metformin or combination therapy) characterized by persistent 
and/ or severe nausea, vomiting, diarrhea, abdominal pain or stomach upset.
c)
Diabetic ketoacidosis or severe hyperglycemia requiring insulin replacement.
d)
Severe hypoglycemia defined as: 1) severe symptomatic hypoglycemia with seizures or unconsciousness 
and blood glucose <50mg/dl or 2) fasting hypoglycemia <30mg/dl on two consecutive days with or 
without symptoms.
3.
Abnormal screening blood tests. Subjects who have abnormal blood tests and who meet exclusion criteria will 
be withdrawn from the study by the investigator. 
4.
Investigator decision.  An investigator feels it is in the subject's best interest to terminate participation. The 
detailed reasoning behind this decision will be documented.
5.
Protocol deviation.  Includes subject noncompliance with fingerstick blood monitoring during the run-in period, 
pregnancy, study entry criterion deviation, or start of a concomitant medication that would impede accurate study 
analysis.  If a female participant’s urine pregnancy test is positive, we will ask the girl’s permission to inform her 
family so that she can get optimal medical care.  During the drug-free run-in period, individuals who cannot wear 
the CGM device or complete fingerstick blood glucose measures or who cannot be reached for ≥3 days during this 
period will be withdrawn from the study.
Protection of Participant’s Privacy and Confidentiality 
At the time of enrollment each subject is given a study-specific code name. The coding system is available only to study 
staff and kept in a secure, password protected database accessible only to study staff. The actual names of the subjects will 
not be provided or ever made available in any publication.


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Compensation
All participants will receive financial compensation for their time per NIH Clinical Center guidelines for on-site visits and 
additional compensation will be provided for specific procedures based on inconvenience units, as follows:
Table 5: Compensation per Visit – to be given as $ or gift card at the end of each visit
Visit
Compensation to patient ($)
Visit: 1 Screening
$ 30.00
Visit: 2 Inpatient
$ 50.00
Visit: 3 Follow-up
$ 25.00
Visit: 4 Follow up
$ 25.00
Visit: 5 Inpatient
$ 50.00
Total
$180.00
Table 6:  Compensation per Procedure – to be given as lump sum at end of study
Visit
Compensation to patient ($)
Visit 2: Inpatient
OGTT
Genetics
DEXA
Tracer
REE
$ 50.00
$ 20.00
$ 30.00
$ 100.00
$ 20.00
Visit 5: Inpatient
OGTT
DEXA
Tracer
REE
$ 50.00
$ 30.00
$ 100.00
$ 20.00
Completion Bonus
$ 100.00
Total
$ 520.00
Total compensation to participants for entire study is $180 for visits + $520 for procedures = $700. If a participant 
were to drop out, the participant will only receive payment for whatever visits and procedures they completed up to that 
point. For example, if a participant drops out after visit 2, he/she will be compensated for Visit 1, Visit 2, 1 OGTT, 1 
REE, 1Genetics and 1 tracer = $300.
Total compensation for guardians accompanying minors for the entire study is $40 per visit x 5 visits = $200. The 
guardians will only receive payments for whatever visits were completed and will be paid as a lump sum at the 
completion of the study.  
Compensation for participating in the making of the video ad is $ 100.00. This is one-time compensation for the 
subject(s) who will participate in the making of the recruitment video ad when needed.
Conflict of Interest 
1.
The National Institutes of Health reviews NIH employees at least yearly for conflicts of interest. The following link 
contains details on this process: http://ethics.od.nih.gov/forms/Protocol-Reveiw-Guide.pdf. 


Principal Investigator: Chung, Stephanie T.  
Last updated: October 5, 2021
Page 34
2.
This protocol has investigators who are not NIH employees. They are expected to comply with their Institutions’ 
conflict of interest policies. 


Principal Investigator: Chung, Stephanie T.  
Last updated: October 5, 2021
Page 35
Appendix A: Sample gastrointestinal questionnaire
Gastrointestinal medication questionnaire. 


Principal Investigator: Chung, Stephanie T.  
Last updated: October 5, 2021
Page 36
Appendix B: Continuous glucose monitoring (CGM) and blood glucose management 
instructions
NAME: 
DATE RANGE:  
Instructions:

You will have your continuous glucose monitor (CGM) sensor placed before you go home. 

Check the receiver twice daily, fasting and before bedtime (~ 2 hours after dinner).

If your CGM sensor malfunctions and you are not able to replace the sensor yourself, we will ask you to either 
check finger stick glucose levels for the remainder of the week, or you may come in to have the sensor replaced 
by the study team.  

A member of the study team will review the glucose levels either on the secure web-based application, over the 
phone or via secure email every week.

If blood glucose is ever >300mg/dl or <70mg/dl, call study team immediately.
What to do if you have high blood sugars:

Call the study team and follow the instructions below:
Blood glucose level
Symptoms
Excessive thirst, urination or 
abdominal pain 
What to do
Greater than 250 mg/dL
NO

Drink 16 oz of water and 
recheck your blood sugar in 
30 minutes

If blood sugar is still 
elevated call study team
Greater than 250 mg/dL
YES

Call Study team

Drink 16 oz of water and 
recheck your blood 
sugar in 30 minutes
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