Determinants of Alpha-aminoadipic Acid (2-AAA) and Relationship to Diabetes: Study 3
Phase: PHASE1
Conditions: Diabetes Mellitus, Type 2
Sponsor: Vanderbilt University Medical Center

Sponsor: NIH, 9000 Rockville Pike, Bethesda, MD 200892 
Protocol Version #1.1 
 
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Determinants of alpha-aminoadipic acid (2-AAA) and relationship to diabetes: 
 
Study 3 
 
 
 
Principal Investigator 
 
Jane F. Ferguson, PhD 
 
Division of Cardiovascular Medicine 
Vanderbilt University Medical Center 
 
2220 Pierce Ave, PRB 354 
Nashville TN 37232 
 
 
Co-Investigator 
 
Jonathan D. Mosley, MD PhD 
 
Division of Clinical Pharmacology 
Vanderbilt University Medical Center 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

Sponsor: NIH, 9000 Rockville Pike, Bethesda, MD 200892 
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Table of Contents 
 
 
Contents 
1.0 Background ............................................................................................................... 3 
2.0 Rationale and Specific Aim ...................................................................................... 4 
3.0 Animal Studies and Previous Human Studies .......................................................... 4 
4.0 Inclusion/Exclusion Criteria ..................................................................................... 7 
5.0 Enrollment................................................................................................................. 7 
6.0 Study Procedures ...................................................................................................... 7 
7.0 Risks .......................................................................................................................... 8 
8.0 Reporting of Adverse Events or Unanticipated Problems involving Risk to 
Participants or Others ...................................................................................................... 9 
9.0 Study Withdrawal/Discontinuation ........................................................................... 9 
10.0 Statistical Considerations ........................................................................................ 9 
11.0 Privacy/Confidentiality Issues ................................................................................ 9 
12.0 Follow-up and Record Retention .......................................................................... 10 
13.0 References ............................................................................................................. 10 
 
             

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1.0 Background 
 
Diabetes is a major global health concern, associated with significantly increased 
mortality and high incidence of co-morbidities1. Given the high rate of cardiovascular and 
other diseases in individuals with diabetes, diabetes-related mortality may be under-
estimated and is estimated to be the third largest cause of death in the US2. Treatment 
efficacy and successful disease management varies between patients. This is in part 
attributable to the fact that diabetes is a heterogeneous disease, with multiple underlying 
causes. In a small proportion of cases of monogenic diabetes, the defects have been 
identified by genetic testing, sometimes leading to vastly improved treatment 
strategies3,4. However, the vast majority of cases of diabetes are polygenic and 
multifactorial. Despite some understanding of disease pathophysiology, and a variety of 
treatment options, the underlying causes of diabetes are unknown in most cases. A 
broader understanding of the variety of paths that lead to diabetes could identify 
alternative therapeutic targets. This may be crucial to preventing or managing disease in 
specific subsets of patients. Large-scale metabolomics studies have successfully 
identified novel candidates for diabetes, including a novel diabetes biomarker, α-
aminoadipic acid (2-AAA), which predicts the development of diabetes in humans5. In 
matched diabetes cases and controls from the Framingham Heart Study (FHS), higher 
plasma 2-AAA (at baseline) was associated with future diabetes, independent of fasting 
glucose and other known risk markers (age, sex, BMI, family history, diet)5.  
 
Source of 2-AAA: 2-AAA is generated from the catabolism of the amino acid lysine 
through the saccharopine (mitochondrial) and/or the pipecolic acid (peroxisomal) 
pathways6,7 (Figure 1). Both pathways converge in the generation of 2-aminoadipate-6-
semialdehyde, which is then further metabolized to 2-AAA in the cytosol8. 2-AAA is 
subsequently metabolized to 2-oxoadipate (α-
ketoadipic acid), and further to acetyl-coA, 
entering the TCA cycle. Lysine is an essential 
amino acid which is acquired from dietary 
sources, with a recommended daily intake of 
30mg/kg/day9. A portion of lysine in humans 
may be derived from the microbiome, with 
bacteria and fungi capable of de novo 
synthesis, in addition to modulation of 
bioavailability of dietary sources10.  
 
Genetics: Variation in genes encoding 
enzymes in the lysine degradation pathway 
(indicated in boxes in Figure 1) may influence 
2-AAA levels and downstream function. The 
strongest evidence thus far exists for DHTKD1, 
variation in which has been associated with 2-
Ketoadipic, 2-Aminoadipic and 2-Oxoadipic 
Aciduria8,11, and Charcot-Marie-Tooth 
Disease12. DHTKD1 expression correlates with 
ATP production in mitochondria in vitro, and 
siRNA knockdown of DHTKD1 leads to 
impaired mitochondrial biogenesis and 
increased reactive oxygen species production, resulting in apoptosis and reduced cell 
Figure 1. Multicompartmental model of L-lysine 
degradation (Adapted from Danhauser et al. 2012) 

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growth13. Variation in mouse Dhtkd1 has been found to associate with expression of the 
gene (eQTL) and levels of protein (pQTL) in liver, as well as with serum 2-AAA levels14. 
Liver Dhtkd1 expression correlated significantly with serum 2-AAA in these mice. 
Further, liver 2-AAA was negatively associated with liver mass, fasting glucose and 
serum cholesterol while higher liver 2-AAA was associated with higher insulin sensitivity.  
 
 
2.0 Rationale and Specific Aim 
 
Diabetes is a significant contributor to mortality and morbidity. The lysine-derived 
metabolite α-aminoadipic acid (2-AAA) has been identified as a novel predictor of 
diabetes development in humans, potentially identifying at-risk individuals before 
development of other known risk markers. Little is known about the function of 2-AAA. 
Our preliminary investigation of human clinical, human genetic, animal and cell 
experimental data has revealed multiple lines of evidence supporting 2-AAA and its 
pathway as biologically relevant to diabetes pathophysiology. However, it is not yet clear 
whether 2-AAA is itself causal in diabetes development, or is a biomarker for altered 
metabolic processes, and many questions remain as to mechanisms of action. The 
determinants of variation in 2-AAA levels between individuals are not known. The aim of 
the first phase of this study (2-AAA Screening study) was to measure plasma 2-AAA 
levels from healthy individuals from the general population, to identify subjects with 
extremely high or low 2-AAA (defined as top and bottom ~25% of distribution). The aim 
of the second phase of the study was to invite individuals with high or low 2-AAA (N=80) 
to participate in a dietary lysine modification study, to assess the effect of controlled 
lysine intake on plasma and urine 2-AAA. In this third phase of the study, we will invite a 
subset of individuals (N=20) from phase 2 to participate in a lysine metabolism study. 
We hypothesize that individuals differ in their rate of metabolism or excretion of lysine to 
2-AAA. This will be assessed by the rate of conversion of 13C lysine to 13C 2-AAA in 
plasma, and the rate of excretion of 13C lysine and 13C 2-AAA in urine. We expect that 
individuals with high plasma 2-AAA have an increased rate of metabolism of lysine to 2-
AAA, and a reduced rate of urinary excretion compared with low-2-AAA individuals. 
 
 
Specific Aim: To determine whether acute lysine administration leads to variability 
in increased plasma 2-AAA in humans. Catabolism of lysine leads to generation of 2-
AAA. To address the hypothesis that acute lysine ingestion leads to differences in the 
increased circulating and excreted 2-AAA in humans in vivo, we will administer isotope-
labeled lysine (5g orally) to healthy volunteers (N=20) and measure the level of 2-AAA in 
plasma and urine at baseline and serially post-ingestion. Carbon-13 is a stable naturally 
occurring heavy isotope of carbon. Inclusion of a 13C label on lysine allows for 
subsequent differentiation between endogenous and exogenous lysine and 2-AAA for 
calculation of clearance and excretion of the lysine bolus. 
 
 
3.0 Animal Studies and Previous Human Studies 
 
2-AAA has been identified as a marker of oxidative stress in C. elegans, and in 
endothelial cells in response to hyperglycemia15, highlighting a potential mitochondrial 
phenotype, and relationship between 2-AAA and reactive oxygen species (ROS)16,17. In 
animal models, administration of 2-AAA resulted in increased insulin levels and lower 
fasting plasma glucose, but no change in peripheral insulin sensitivity as measured by 

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insulin tolerance test5. In vitro 2-AAA treatment has been shown to enhance insulin 
secretion in pancreatic β cells and islets. 2-AAA is thought to have important functions in 
the brain18, as well as in other organs, including liver and pancreas.  
 
High baseline plasma 2-AAA in healthy individuals was associated with increased future 
risk of diabetes (12-year follow-up) in FHS participants and validation samples 
(N~2,000)5. Insulin sensitizing therapy leads to reduction in 2-AAA, but not other putative 
diabetes-biomarkers, including branched-chain amino acids (BCAAs)19. Increased 2-
AAA is found in skin in the setting of diabetes, as well as in chronic renal failure and 
sepsis, and increases with age20. Further, decreased urine 2-AAA has been reported in 
diabetes patients compared with non-diabetic14, highlighting a likely complex feedback 
relationship between 2-AAA levels and early diabetes development vs. the setting of 
established disease.  Treatment of overweight/obese adults with impaired fasting 
glucose or untreated diabetes with pioglitazone (45 mg/day) and metformin (1000 mg 
twice/day) led to a significant reduction in plasma 2-AAA and lysine, concurrent with an 
increase in insulin sensitivity19. Acute insulin infusion (7 hrs) in these subjects also led to 
a decrease in both lysine and 2-AAA19,21. The epilepsy drug vigabatrin, which acts 
through GABA transaminase inhibition, leads to increased 2-AAA in plasma and urine, to 
levels similar to those seen in alpha-aminoadipic aciduria22. Apart from diabetes, 2-AAA 
has been found to be elevated in the setting of other cardiometabolic or inflammatory 
diseases including atherosclerosis23, chronic renal failure and sepsis20, non-alcoholic 
fatty liver disease (NAFLD)24 and polycystic ovary syndrome (PCOS)25. There is likely 
some reciprocal regulation between insulin and 2-AAA: insulin infusion in humans leads 
to decreased 2-AAA19,21,26, while insulin deprivation and hyperglycemia in the setting of 
type 1 diabetes leads to increased 2-AAA27. 
 
Human clinical: We tested our ability to use lysine to 
modulate 2-AAA in 
the acute setting 
(human pilot 
study, N=10, 
NCT02756117). 
The rationale to 
use lysine as a 
probe to increase 
2-AAA was based 
in our hypothesis 
that exogenous 
lysine is a 
contributor to 2-
AAA levels, and 
that individuals 
with altered 2-AAA 
have alterations in 
the lysine-2-AAA 
metabolic 
pathway. We 
demonstrated that 
acute oral lysine 
(10g) resulted in 
an increase in plasma lysine (P<0.001, Fig.2A) and 
Figure 3. The effect of an oral lysine 
bolus (10g) on plasma insulin (A); and 
glucose (B) in 10 healthy subjects. 
Closed shapes denote male, open 
shapes denote female subjects. 
A 
 
 
 
 
 
 
 
 
B 
 
 
 
 
Figure 2. The effect of an oral lysine 
bolus (10g) on plasma lysine (A); and 
2-AAA (B) in 10 healthy subjects. 
Closed shapes denote male, open 
shapes denote female subjects. 
A 
 
 
 
 
 
 
 
 
B 
 
 
 

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2-AAA (P<0.0001, Fig.2B). There was an 
increase in plasma insulin (P<0.0001, 
Fig.3A) with no change in glucose 
(Fig.3B). Amino acids are known to 
stimulate insulin secretion, so the effect on 
insulin is not necessarily attributable to 2-
AAA. There were no changes in other 
amino acids. We detected a significant 
increase in urinary lysine and 2-AAA, and 
importantly, observed variation in the 
fractions excreted as lysine (Fig.4A), or as 
the 2-AAA metabolite (Fig.4B). We thus 
confirmed the hypothesis that plasma 2-AAA is modulated in part by oral lysine intake. 
However, we demonstrated that there is considerable inter-individual variability in the 2-
AAA response to lysine (Fig.2B), supporting the premise that factors other than diet (e.g. 
genetics) contribute to modulation of 2-AAA.  
Human lysine tracer 
study: The stable 
isotope tracer 13C-lysine 
replaces 12C with 13C on 
the lysine molecule. 
Previous studies have 
used isotope labeled 
lysine to calculate amino 
acid flux28,29. We invited 
two subjects from the 
pilot study to return for a 
labeled lysine challenge 
to distinguish between 
endogenous and 
exogenous metabolism 
(NCT03063476). We 
administered 5g of 
stable isotope-labeled 
lysine (U-13C lysine, 
Cambridge Isotope 
Laboratories) as an oral bolus, with repeated 
sampling of blood and urine over 6 hours for 
measurement of 12C and 13C lysine and 2-AAA. 
We observed the expected increase in 13C 
isotope-labeled lysine and 2-AAA in plasma in 
both subjects (Figure 5). Interestingly, we also 
observed a marked increase in endogenous 
(unlabeled) lysine and 2-AAA in Subject 02, not 
observed in Subject 01, suggesting secretion of 
stored intracellular lysine and 2-AAA into the 
circulation, potentially as a result of catabolism 
of lysine from endogenous proteins26.  
Figure 4. Estimates of urinary lysine fraction 
excreted (Fe) and 2-AAA fraction metabolized 
(Fm) over 4 hours post lysine bolus. 
A 
 
 
B 
Figure 6. Estimates of urinary lysine 
fraction excreted (Fe) and 2-AAA fraction 
metabolized (Fm) over 6 hours post 13C 
lysine bolus. Closed shapes denote male, 
open shapes denote female subject. 
Figure 5. The effect of an oral isotope-labeled 13C lysine bolus (5g) 
on plasma lysine and 2-AAA in 2 healthy subjects.  

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The rate of urinary clearance of 13C lysine and excretion of metabolized 13C 2-AAA also 
differed between the 2 subjects (Figure 6). Subject 01 excreted a greater proportion of 
the lysine and 2-AAA, with concurrently lower plasma levels of both. While the goal of 
this pilot study was to demonstrate feasibility, and was not powered to detect 
differences, these data further highlight inter-individual differences in the metabolic 
response to an identical lysine load. We hypothesize that there are genetic differences, 
between individuals with high or low 2-AAA, which control the rate of lysine and 2-AAA 
metabolism.  
 
 
4.0 Inclusion/Exclusion Criteria 
 
Inclusion Criteria:  
• 
Prior participant in 2-AAA study. 
Exclusion Criteria:  
• 
Newly diagnosed disease, including cardiovascular, renal, liver disease, bleeding 
disorder, or Diabetes mellitus. 
• 
Individuals who are pregnant or lactating. 
• 
Inability to provide electronic informed consent. 
• 
Inability to fast for 8 hours. 
 
5.0 Enrollment 
 
Our goal is to obtain study visit data for 20 individuals. To allow for expected drop-outs, 
we will enroll up to 30 participants who meet the inclusion and exclusion criteria. We will 
identify potential participants based on their participation in the previous phases of the 
study. We will contact eligible participants directly, by email or phone, and invite them to 
participate. 
 
6.0 Study Procedures 
 
Screening 
 
Potential participants will be identified based on their participation in the previous phases 
of the study. Informed consent will be obtained electronically. Inclusion/exclusion criteria 
will be reviewed to confirm that the individual meets study eligibility requirements. 
Individuals who meet the inclusion criteria, and do not meet any of the exclusion criteria 
will be invited to participate in the study.  
 
Study Visit 
Participants will be requested to arrive at the Vanderbilt Clinical Research Center on the 
morning of the study visit (~8:00 AM) in a fasting state (at least 8 hours, with no food or 
drink, excluding water). After informed consent has been reviewed and documented, 
inclusion/exclusion criteria will be reviewed to confirm that the subject meets study 
eligibility requirements. The participant’s medical history and medications will be 
discussed and documented by study personnel to assess for any changes that may 
potentially exclude the subject from participating. Study personnel will collect the 

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participant’s vital signs, height, weight, waist and hip circumference prior to study 
interventions. All participants will be asked to provide a urine sample for baseline 2-AAA 
measurement and will undergo a urine pregnancy test (if the woman is of child-bearing 
potential).  
 
Following the baseline urine sample, an intravenous line will be inserted into a superficial 
peripheral vein to perform blood draws. Participants will be asked to remain in a seated 
position for the duration of the study, but will be permitted to walk for brief periods 
throughout the study (e.g. to use the restroom). A baseline blood draw will be taken 
(time = 0 hr) for measurement of baseline plasma 2-AAA levels and related biomarkers. 
Immediately following the baseline blood draw, subjects will be given an oral bolus of 13C 
L-lysine (5g) (Cambridge Isotope Laboratories, Tewksbury, MA) in 50ml water. This is an 
amount of lysine equivalent to that which is found in a 5oz serving of beef. A similar dose 
of lysine has previously been shown to alter the response to glucose17. Blood samples 
will be taken at time = 30 minutes 1, 2, 3, 4, 5 and 6 hrs post-lysine administration. 
Normal (0.9%) Saline (NS) will be infused at a rate of 10 ml/hr to flush the canula prior to 
each blood draw, and a 3-5ml blood discard will be performed prior to each collection of 
samples. Each blood draw (including baseline) will collect about 20cc of blood, for a total 
collection of 160cc, or approximately 11 tablespoons of blood. Blood will be collected 
into one tube for serum (5ml) and 2 tubes for plasma (4ml each). Urine samples will be 
collected throughout the visit, in 2-hour increments (0-2, 2-4 hrs and 4-6 post-lysine). 
Peak post-prandial plasma 2-AAA levels are expected to be observed ~3hours post-
lysine27. Plasma and serum will be prepared from blood samples, and both plasma and 
urine aliquots will be frozen at -80°C and stored prior to analysis. 2-AAA and other 
metabolites and biomarkers (e.g. lysine, creatinine) will be measured at Vanderbilt core 
facilities (e.g. Mass Spectrometry core).  
 
Compensation 
Participants will receive $150 following successful completion of the study visit.  
 
Biomarkers 
Blood and urine samples will be coded for subject confidentiality. Plasma, serum and 
urine aliquots will be frozen at -80°C and stored for subsequent measurements. 
Peripheral blood mononuclear cells (PBMCs) may be isolated from blood for analysis.  
2-AAA and other relevant metabolites and biomarkers will be measured at Vanderbilt 
core facilities (e.g. Mass Spectrometry core, Hormone Assay core). For possible future 
investigations, excess blood, cell, and urine samples will be frozen and stored in Dr. 
Ferguson’s laboratory.  
 
 
 
7.0 Risks 
 
Venous Blood draw: This is a routine procedure that is considered standard of care in 
clinical medicine. At the study visit, subjects will undergo venous blood draws. All blood 
draws will be performed by trained personnel using universal precautions to protect both 
the subject and personnel. The risks to subjects are minimal, but may include pain, 
bruising, allergic reaction, infection or bleeding at the needle stick site. A small number 
of individuals may experience dizziness, lightheadedness or fainting. These usually 
resolve without any specific medical therapy over the course of minutes to days. 
 

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Lysine Administration: Lysine is expected to be well-tolerated by all participants. Lysine 
is used as a dietary supplement, available over the counter, with no known long-term 
risks. There are no additional risks associated with the use of stable isotope tracer 13C 
lysine. The proposed dose of lysine may cause mild gastrointestinal upset in some 
participants. This is expected to be minor and transient. 
 
Private Health Information: This information will be collected during the course of the 
study. However, only key study personnel will have access to this information, which will 
be stored in a HIPAA compliant, password protected REDCap database. No protected 
health information will be shared with employers, insurers, or non-research personnel.  
 
8.0 Reporting of Adverse Events or Unanticipated Problems involving Risk to 
Participants or Others 
 
Adverse events will be reported to the IRB per Vanderbilt University IRB policy. 
 
 
9.0 Study Withdrawal/Discontinuation 
 
Subjects may withdraw from the study at any time and should notify study personnel if 
they wish to withdraw from the study. Subjects may request their biological samples to 
be destroyed at any time. However, any data or biological samples that have already 
been used for research cannot be destroyed. Subjects may be discontinued from the 
study at the discretion of the investigator (possible reasons listed below). Subjects will 
receive financial compensation for completion of the visit. 
 
Possible reasons for withdrawal/discontinuation from study include, but are not limited 
to: 
• 
Noncompliance with treatment or procedures 
• 
Decision by participant/participant withdraws consent 
• 
Significant adverse event deemed by investigator to preclude continued 
participation 
 
 
10.0 Statistical Considerations 
 
Based on our preliminary study, we expect that some individuals will have modest 
increases in 13C 2-AAA post-lysine, estimated at 0.075µM at peak. We expect that other 
individuals will have greater response, estimated at 0.13µM peak 13C 2-AAA. Assuming 
a SD of 0.04µM, we would require a total of 16 individuals to detect significant 
differences in response by group, at α=0.05 and 80% power. We aim to recruit 20 
individuals to allow for adequate power in the event of attrition or lower than expected 
separation between groups. 
 
 
11.0 Privacy/Confidentiality Issues 
 
Strict confidentiality will be maintained to the fullest extent by the research team, 
including keeping all data in a secure location. All specimens will be coded anonymously 

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to remain confidential and identifiers will be kept in a separate, secure location.   
Samples may be shared with third parties outside of Vanderbilt for future testing but will 
remain anonymous to the recipient.  Participants may contact the principal investigator at 
any time to request that samples be destroyed.  
 
12.0 Follow-up and Record Retention 
 
Anticipated study duration is 5 years. Research data will be maintained by the PI after 
study closure. After study closure, research data will be maintained for a minimum of 6 
years and possibly indefinitely. Data will be stored on the Vanderbilt University computer 
network in a password-protected database. Only members of the study team will have 
access. Pertinent paper documentation will be kept in locked office and only study 
personnel will have access. Only personnel directly involved with the study will have 
access to source data and the electronic database. Data will be de-identified to protect 
study participants’ identities. 
 
13.0 References 
 
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Carmody D et al. GCK-MODY in the US National Monogenic Diabetes Registry: 
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Naylor RN et al. Cost-effectiveness of MODY genetic testing: translating genomic 
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8.  
Danhauser K et al. DHTKD1 mutations cause 2-aminoadipic and 2-oxoadipic 
aciduria. American journal of human genetics. 2012;91:1082–7.  
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11.  Stiles AR et al. New Cases of DHTKD1 Mutations in Patients with 2-Ketoadipic 
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