Role of Fatty Acid Oxidation Defects in Insulin Sensitivity
Phase: NA
Conditions: Very Long-chain Acyl-CoA Dehydrogenase Deficiency, Trifunctional Protein Deficiency, Long-chain 3-hydroxyacyl-CoA Dehydrogenase Deficiency, Medium-chain Acyl-CoA Dehydrogenase Deficiency, Normal Volunteers, Carnitine Palmitoyltransferase II Deficiency, Myopathic
Sponsor: Oregon Health and Science University

1 
 
 
 
 
Clinical Trials Identifier: NCT02517307 
OHSU eIRB identifier: 11258 
Brief Title: Fatty acid Oxidation Defects and Insulin Sensitivity 
 
 
Protocol Title: Role of Fatty Acid Oxidation Defects in Insulin Sensitivity 
 
 
March 23, 2020 
 
 
 

2 
 
 
 
OCTRI Protocol # _4444_ 
eIRB # 11258 
 
 
OREGON CLINICAL AND TRANSLATIONAL RESEARCH INSTITUTE 
Oregon Health & Science University 
RESEARCH PROTOCOL 
 
 
Protocol Title: Role of Fatty Acid Oxidation Defects in Insulin Sensitivity 
 
Investigators: 
 
Name  
Degree 
Department & 
Mailcode 
Phone 
E-Mail 
Oregon Health & Science University 
Principal 
Investig
ator: 
Melanie B. 
Gillingham 
PhD 
Genetics L103 
4-1682 
gillingm@ohsu.
edu 
Principal 
Investig
ator: 
Jonathan 
Q.Purnell 
MD 
Endocrine 
UNH62 
4-1056 
purnellj@ohsu.
edu 
Co-
Investig
ator: 
Cary O. 
Harding 
MD 
Genetics L103 
4-7608 
hardingc@ohsu
.edu 
Co-
Investig
ator: 
Charles 
Roberts 
PhD 
ONPRC L584 
503-690-
5259 
robertsc@ohsu.
edu 
Co-
Investig
ator: 
Oleg 
Varlamov 
PhD 
ONPRC L584 
503-614-
3712 
varlamov@ohs
u.edu 
 
Protocol Date: November 4, 2014 
Amendment 1 Date: December 11, 2014 
Amendment 2 Date: February 13, 2015 
Amendment 3 Date: June 29, 2015 
Amendment 4 Date: November 23, 2015 
Amendment 5 Date: May 23, 2016 
Amendment 6 Date: September 6, 2016 
Amendment 7 Date: May 26, 2017 
Amendment 8 Date: April 25, 2018 
Amendment 9 Date: March 23, 2020 
 
 

3 
 
I. 
BACKGROUND AND SPECIFIC AIMS 
 
The overall goal of this proposal is to investigate the effects of disordered mitochondrial fatty acid oxidation 
(FAO) on insulin resistance in humans.  Mitochondrial dysfunction has been implicated in the development 
of insulin resistance and type 2 diabetes during excess dietary fat intake and from increased release of 
endogenous free fatty acids (FFA’s), such as occurs in obesity.(49, 70, 75, 88)  Controversy exists, 
however, as to whether this insulin resistance results from intrinsic defects in mitochondrial energy 
utilization or from abnormalities resulting from excess FFA flux, as well as the role that subsequent 
accumulation of cellular metabolic intermediates play in impaired insulin signaling. 
 
To address these controversies, we will study a unique population of patients with inherited defects in one  
of four mitochondrial enzymes in the FAO pathway: 1) carnitine palmitoyltransferase 2 (CPT2);  2) very 
long-chain acyl-CoA dehydrogenase (VLCAD); 3) trifunctional protein (TFP, which includes long-chain 3-
hydroxyacyl-CoA dehydrogenase (LCHAD)); and 4) medium-chain acyl-CoA dehydrogenase (MCAD). 
These proteins are required for the oxidation of sequentially shorter fatty acids (long-chain and medium-
chain), respectively.  We will use these patients to formally test the hypothesis that intrinsic defects in 
mitochondrial function involving oxidation of long-chain, but not medium-chain, fatty acids are sufficient to 
prevent intralipid-induced insulin resistance. 
 
In a randomized, crossover study design, patients and a healthy control group will receive infusions of 
either intralipids or a control solution during hyperinsulinemic-euglycemic clamps.  Glucose appearance 
(primarily due to hepatic glucose production) and glucose disposal (primarily by muscle tissue) will be 
measured, as will intraheptatic and intramyocellular lipid content (ectopic fat) by magnetic resonance 
spectroscopy, metabolite accumulation in muscle and fat using advanced metabolomics methodology, and 
insulin signaling pathway component expression and activation in the same biopsy samples. 
 
In the first studies to test the relationships between impaired FAO, accumulation of intracellular lipid and 
lipid oxidation intermediates, and insulin resistance in humans, we propose the following specific aims: 
 
A. Specific Aim 1: To measure the effect of intralipid infusions on ectopic fat and systemic glucose 
metabolism in FAO patients and matched controls.  We will measure ectopic lipid deposition in muscle 
and liver by 1H magnetic resonance spectroscopy (MRS) as well as glucose appearance (Ra) in the fasting 
state and insulin-mediated glucose disposal (Rd) during hyerinsulinemic-euglycemic clamps and co-
infusion of either intralipids or saline/glycerol in patients with a FAO disorder and matched controls.   
 
Hypotheses:  During saline/glycerol (control) infusions, patients with CPT2, VLCAD, and LCHAD/TFP 
deficiency will have similar ectopic lipid stores, and similar (or better) Rd and lower fasting Ra compared to 
control subjects and patients with MCAD deficiency.  In contrast, during the intralipid infusion ectopic lipids 
will increase in the FAO patients and controls alike, but insulin sensitivity will worsen (decreased Rd) in 
controls and patients with MCAD deficiency, but not in those with CPT2, VLCAD, or LCHAD/TFP 
deficiency. 
 
B. Specific Aim 2: To determine the effect of distinct FAO disorders on tissue-specific lipid 
metabolism and insulin resistance compared to matched controls.  We will measure blood, skeletal 
muscle, and adipose levels of lipids, intermediates of FAO (including organic acids) and amino acids by 
metabolomics profiling, and adipose and muscle insulin signaling in FAO patients and matched controls 
participating in aim 1.   
 
Hypotheses:  In parallel with our hypothesized outcomes in Aim 1, we expect that CPT2, VLCAD, and 
LCHAD/TFP-deficient patients, despite intracellular accumulation of lipids and long-chain FAO 
intermediates in muscle and adipose tissue, will be “protected” from intralipid-induced insulin resistance by 
exhibiting greater insulin signaling pathway activation and glucose and FFA uptake than tissue from 
controls.  On the other hand, accompanying the accumulation of medium chain FAO intermediates, 
subjects with MCAD deficiency will have reduced fat and muscle insulin action (similar to controls). 

4 
 
C. SIGNIFICANCE 
Type 2 diabetes (T2DM) affects over 18 million Americans, but the pathogenesis of insulin 
resistance underlying T2DM remains incompletely understood,(14, 19, 47, 48, 93, 101) and novel 
therapeutics are still needed.  The proposed studies in this application will advance our understanding of 
the cellular basis for insulin resistance and potentially resolve a major controversy in this field: i.e., whether 
impaired insulin signaling results from defective mitochondrial function and subsequent accumulation of 
cytosolic lipid and byproducts of FAO (for review, see ref (62) and below) or from increased flux through 
the FAO cycle and subsequent generation of metabolic intermediates (for review, see ref (69) and below).  
In addition to providing a unique opportunity to better understand the interface between FFA metabolism 
and the expression of insulin resistance in humans, the results of these studies will also help inform the on-
going development of potential pharmaceutical targets for the treatment of insulin resistance and T2DM (for 
review see refs (86, 104)). 
 
D. INNOVATION 
 
Previous investigators have studied the interactions of lipid and glucose metabolism in patients with 
existing insulin resistance or a familial predisposition for diabetes.  While these studies have yielded 
important new insights into the importance of mitochondrial dysfunction in insulin resistance, we will recruit 
and study a novel group of patients with inherited FAO defects.  By taking advantage of our current clinical 
trials program in patients with FAO disorders, autosomal-recessive genetic disorders that severely limit 
specific enzymatic steps in the FAO cascade, these studies will allow us to pinpoint a specific level of 
mitochondrial FAO dysfunction that results in either induction of, or protection from, insulin resistance 
under conditions of high-fat exposure characteristic of developed societies consuming Western diets and 
exhibiting high rates of obesity.  These studies will, therefore, have relevance well beyond the rare patient 
with a FAO disorder and indeed, by using advanced metabolomics and muscle and adipose tissue 
analyses to assess tissue-specific metabolite signatures associated with cellular lipid accumulation, 
impaired insulin signaling, and FAO products, will allow for identification of potentially novel mechanistic 
candidates of insulin resistance that would not otherwise be 
possible. 
 
E. APPROACH 
 
1. Specific Aim 1: To measure the effect of intralipid infusions on 
ectopic fat and systemic glucose metabolism in FAO patients 
and matched controls. 
 
Overview and Rationale: 
Normal Mitochondrial Function and FAO Disorders (Figure 1) 
 
Long-chain fatty acids are transported into mitochondria via the 
carnitine palmitoyltransferase (CPT) system including CPT-1, 
carnitine acylcarnitine translocase (CACT) and CPT-2.  Long-chain 
ß-oxidation occurs in a repeating, four-enzyme cycle catalyzed by 
very-long-chain acyl-CoA dehydrogenase (VLCAD) and 
trifunctional protein (TFP), resulting in sequential removal of 2-
carbon units that become acetyl-CoA and generating both shorter 
chain-length fatty acids and reducing equivalents (NADH), and 
releasing electrons directly into the ubiquinone pool via the electron 
flavin transfer protein (ETF).   
Medium-chain fatty acids are oxidized by a separate set of ß-oxidation enzymes, the first of which is 
medium-chain acyl-CoA dehydrogenase (MCAD), generating acetyl-CoA, a shorter chain-length fatty acid, 
and more reducing equivalents (FADH2 and NADH).  Acetyl-CoA then enters the tricarboxylic acid (TCA) 
cycle, where it is further oxidized to CO2 and reducing equivalents (NADH, FADH2), which enter the 
electron transport chain to generate ATP. 
 
In humans, CPT2, VLCAD, LCHAD/TFP and MCAD deficiency are inherited in an autosomal recessive 
manner where each parent carries one mutant and one normal allele.  All affected patients carry two 
Figure 1: Fatty Acid Oxidation 
Pathway 
 
Long and medium chain FAO provides 
acetyl-coA and reducing equivalents 
for the TCA and the electron transport 
chain. 

5 
 
mutations in the gene which leads to partial loss of function of these enzymes (see red “X”’s, Figure 1) (42, 
44, 99) and the accumulation of long-chain acyl-CoA, long-chain hydroxyacyl-CoA, or medium-chain acyl-
CoA moieties, respectively, in the mitochondria.(64, 85)  These fatty acids exit the mitochondria via the 
reversal of the CPT system and appear in patient’s plasma as long-chain or medium-chain acylcarnitines 
and their FFAs. As a result of these defects, during periods of fasting and exercise, when demand for FAO 
increases, circulating and tissue levels of acylcarnitines increase.(31, 32, 80)  Infants and children with 
CPT2, VLCAD, LCHAD/TFP and MCAD deficiency present with fasting or illness induced hypoketotic 
hypoglycemia, and potentially, sudden infant death, but VLCAD and LCHAD/TFP can also be complicated 
by cardiomyopathy. Adult patients with CPT2, VLCAD and LCHAD/TFP have recurrent rhabdomoyolysis, 
especially after a prolonged fast or exercise that is not observed in adults with MCAD deficiency.(6, 15, 41) 
Early diagnosis and treatment leads to improved survival and normal development through prevention of 
catastrophic hypoglycemia in infancy and later-onset cardiomyopathy. Under conditions of adequate 
glucose supply, such as a low-fat, high carbohydrate diet, patients with CPT2, VLCAD, LCHAD/TFP and 
MCAD deficiencies are medically stable with no symptoms of metabolic decompensation.  
 
FAO and Insulin Sensitivity: Too Little or Too Much? 
 
Several lines of evidence have led to two current theories linking impaired mitochondrial FAO with the 
development of insulin resistance.(104)  One theory suggests that defective mitochondrial function is 
associated with accumulation of cytosolic lipid by-products, such as diacylglycerols (DAGs), ceramides, 
and triglycerides in skeletal muscle and liver, referred to as ectopic fat.(5, 58, 67, 76)  In turn, these 
cytosolic lipid intermediates have been shown to activate serine stress-kinases and signaling pathways that 
impair normal insulin signaling and are the basis of the "lipotoxicity" hypothesis for insulin resistance.(67, 
77)  Evidence in support of this theory comes from studies that show that ectopic fat in muscle and liver are 
closely associated with insulin resistance(33, 45, 54, 58, 74) and that lipid infusions lead to accumulation of 
intramyocellular lipid (IMCL)(4, 9, 11) and insulin resistance mediated, in part, through activation of protein 
kinase C ϴ (PKC-ϴ) and subsequent impairment of glucose transport.(34, 50, 51, 81) In this model, insulin 
sensitivity would be improved by increasing FAO and reducing ectopic fat, as supported by animal and 
clinical studies involving drug and genetic manipulations.(1, 12, 13, 43, 77, 102) 
 
An alternative theory, first proposed by Randle,(79) posits that increased flux through the FAO cycle 
induces insulin resistance.(69) In this model, “metabolic overload” of the mitochondria leads to a mismatch 
between FAO and TCA oxidation that leads to cellular accumulation of intermediates of FAO and 
subsequent impaired insulin signaling.(69) (57, 68)  This is supported by studies showing that during high-
fat feeding, increased production of partially oxidized substrates, such as intermediate metabolites from 
FAO in the form of fatty acylcarnitines and increased reactive oxygen species (ROS), have been 
associated with insulin resistance.(2, 40, 57, 59, 65, 66, 84)  This model predicts insulin sensitivity will be 
improved by reducing FAO, thereby alleviating the “metabolic stress” that accompanies increased FFA flux.  
Indeed, animal studies have shown that blocking FFA entry into mitochondria can alleviate high-fat-diet-
induced insulin resistance.(56, 57). 
 
Medium-chain Acylcarnitines in Expression of Insulin Resistance during Fat Overload 
 
Plasma elevations in both long- and medium-chain acylcarnitines have been observed in animal and 
human studies of obesity (2, 52, 57, 66). However, medium-chain acylcarnitine species are reported to be 
specifically elevated in T2DM and diet-induced insulin resistant animal models,(2, 52, 66) which suggests 
that long-chain acylcarnitines may be a non-specific marker for obesity and that accumulation of medium-
chain acylcarnitines may be more important for expression of insulin resistance.   This includes a recent 
study in which levels of medium-chain acylcarnitines, but not long-chain acylcarnitines or ceramides, were 
elevated in muscle tissue and inversely correlated (in plasma) with insulin sensitivity in lean men and 
women fed a high-fat diet.(52)  In addition, anecdotal reports of diabetes in patients with MCAD deficiency, 
but not those with CPT2, VLCAD or LCHAD/TFP deficiencies, have appeared in support groups of FAO 
patients (personal communication, Deb Gould, Director Parent Support Group for FOD, 
http://www.fodsupport.org).  Although these data and reports do not distinguish whether medium-chain 
acylcarnitines are causative of, or simply a marker for, insulin resistance and diabetes, in-vitro evidence 
has linked medium-chain acylcarnitines with insulin resistance in primary skeletal myocytes(55) and with 
NF-KB activation, thought to be a key mediator of inflammation induced insulin resistance, in primary cell 

6  
 
c ult ur e s .(2 ) I n t h e st u di e s pl a n n e d h er e, w e will t e st t h e r ol e of d ef e cti v e M C A D a n d a c c u m ul ati o n of 
u p str e a m F O A m et a b olit e s i n e x pr e s si o n of hi g h -
f at i n d u c e d i n s uli n r e si st a n c e. 
 
 
  
Pr eli mi n ar y D at a:  
I n e x pl or at or y st u di e s e x a mi ni n g t h e 
r el ati o n s hi p s b et w e e n d ef e cti v e F A O a n d gl u c o s e 
m et a b oli s m, w e m e a s ur e d e n er g y e x p e n dit ur e  a n d 
b o d y c o m p o siti o n  a n d p erf or m e d or al gl u c o s e 
t ol er a n c e t e st s ( O G T T s) i n 1 2 s u bj e ct s wit h  F A O 
di s or d er s  a n d c o m p ar e d t h e m  t o 1 2 a g e -, s e x-, 
a n d B MI -m at c h e d c o ntr ol s ( T a bl e 1 ).(3 0 )  T w o 
s u bj e ct s h a d C P T -2 a n d 1 0 s u bj e ct s h a d 
T F P/ L C H A D d efi ci e n c y.  B MI s r a n g e d fr o m 1 5. 3 
k g/ m 2  ( u n d er w ei g ht) t o 2 9. 9 k g/ m2  ( o v er w ei g ht), a n d  n o n e m et 
crit eri a f or o b e s it y.  It s h o ul d b e n ot e d t h at t h e s e F A O s u bj e ct s a n d 
c o ntr ol s w er e st u di e d d uri n g l o w -f at di et s a n d d o n ot r e pr e s e nt tr u e 
“ m et a b oli c str e s s” c o n diti o n s t h at w e pr o p o s e f or o ur st u di e s i n t hi s 
gr a nt a p pli c ati o n.  
L o n g -c h ai n F A O  ( L C F A O)-d efi ci e nt s u bj e ct s e x hi bit e d 
hi g h er % f at m a s s a n d e xtr a m y o c ell ul ar li pi d c o nt e nt ( E M C L) i n 
s ol e u s m u s cl e.   P er c e nt  f at m a s s w a s gr e at er a n d % f at -fr e e m a s s 
l o w er i n L C F A O s u bj e ct s t h a n c o ntr ol s (Fi g ur e 2 A ).(3 0 )   
I ntr a h e p ati c li pi d (I H L) a n d I M C L c o nt e nt w er e  n ot diff er e nt 
b et w e e n  F A O p ati e nt s a n d c o ntr ol s ( Fi g ur e 3 A a n d B ).(3 0 )  
H o w e v er, s u bj e ct s wit h a F A O di s or d er di d h a v e gr e at er  E M C L 
d e p o siti o n c o m p ar e d t o c o ntr o l s (Fi g ur e 3 A a n d B ), w hi c h w e 
s u s p e ct p ar all el s t h eir hi g h er % b o d y f at ( Fi g ur e 2 ), w hi c h, i n t ur n, 
li k el y r efl e ct s t h eir r e d u c e d c a p a cit y t o o xi di z e f at a n d t h eir m or e 
s e d e nt ar y lif e st yl e.   
R e s p o n s e s t o or al gl u c o s e l o a d s w er e i d e nti c al i n L C F A O -
d efi ci e nt s u bj e ct s a n d m at c h e d c o ntr ol s.   D uri n g a st a n d ar d O G T T, 
n eit h er f a sti n g n or p o st -gl u c o s e l o a d c o n c e ntr ati o n s of gl u c o s e or 
i n s uli n w er e diff er e nt b et w e e n F A O-d efi ci e nt s u bj e ct s a n d c o ntr ol s 
(Fi g ur e 2 B a n d C ).  E sti m at e s of i n s uli n s e n siti vi t y w er e al s o 
si mil ar b et w e e n gr o u p s ( T a bl e 1 ).  W hil e o ur fi n di n g t h at, d e s pit e 
m ar k e dl y l o w er F A O a n d gr e at er  b o d y f at c o m p ar e d t o c o ntr ol s, 
p ati e nt s wit h C P T -2, V L C A D a n d T F P d efi ci e n c y h a v e n or m al 
gl u c o s e t ol er a n c e  (a n d e sti m at e d i n s uli n s e n siti vit y a n d  s e cr eti o n ) 
i s s u p p orti v e of t h e t h e or y t h at r e d u ci n g F A O c y cl e fl u x i s 
pr ot e cti v e a g ai n st i n s uli n r e si st a n c e, t h e s e p ati e nt s di d n ot i n cl u d e 
s u bj e ct s wit h M C A D d efi ci e n c y, w er e n ot tr ul y “ m et a b oli c all y 
str e s s e d” b y virt u e of b ei n g o n a l o w -f at di et ( w hi c h c o ul d e x pl ai n 
si mil ar I M C L l e v el s t o c o ntr ol s), a n d di d n ot u n d er g o a dir e ct 
m e a s ur e of i n s uli n s e n siti vit y.   T h er ef or e, t hi s ai m will a d dr e s s 
t h e s e li mit ati o n s i n o ur pil ot d at a b y p erf or mi n g  h y p eri n s uli n e mi c -
e u gl y c e mi c cl a m p s ( H E C s) a n d c o -i nf u si o n s of i ntr ali pi d s (or a c o ntr ol s ol uti o n ) a n d b y i n cl u di n g s u bj e ct s 
wit h M C A D d ef e ct s .  
 
V L C A D -d efi ci e nt mi c e ar e pr ot e ct e d fr o m i n s uli n r e si st a n c e o n  a hi g h -f at di et.  V L C A D/ - mi c e wit h a 
t ar g et e d del eti o n of e x o n s 7 -1 9 h a v e a p h e n ot y p e si mil ar t o h u m a n s wit h  l o n g-c h ai n F A O di s or d er s a n d 
h a v e pr e vi o u sl y b e e n r e p ort e d t o b e pr ot e ct e d fr o m  hi g h -f at-di et -i n d u c e d i n s uli n r e si st a n c e.(2 1 , 1 0 7 )  W e , 
t o o, h a v e s h o w n t hi s w h e n w e m e a s ur e d i n s uli n s e n siti vit y b y H E C  i n m al e V L C A D-/- mi c e ( n = 6) c o m p ar e d 
Fi g ur e 2 : B o d y c o m p o siti o n a n d 
p l a s m a gl u c o s e a n d i n s uli n l e v el s 
d uri n g  a n O G T T.  
 
- 1 5
0
3 0
6 0
9 0 
1 2 0
0
5 0
1 0 0
1 5 0
2 0 0
2 5 0
o ra l g lu c o s e
S u b je c ts
C o n tro ls
M in u te s
G lu c o s e
(m g /d l)
- 1 5
0
3 0
6 0
9 0 
1 2 0
0
5 0
1 0 0
1 5 0
2 0 0
o ra l 
g lu c o s e
M in u te s
In s u lin  
(m u n its /m l)
F at Fr e e M a s s
F at M a s s
0
2 0
4 0
6 0
8 0
1 0 0
S u b je c ts  w ith  F A O  d is o rd e r
C o n tro ls
* p  =  0 .0 3 8
* p  =  0 .0 3 7
%  b o d y  c o m p o s itio n
B o d y  C o m p o s itio n
A
B
C
T a bl e 1: Gl u c o s e, i n s uli n a n d e sti m at e s of i n s uli n 
s e n siti vit y i n C P T 2 a n d L C H A D -d efi ci e nt s u bj e ct s  
( n = 1 2) c o m p ar e d t o c o ntr ol s  ( n = 1 2). 
 
S u bj e ct s  
C o ntr ol s  
F a sti n g gl u c o s e ( m g/ dl)  
9 7 ± 1 4  
1 0 5 ± 1 9  
F a sti n g i n s uli n ( μ U/ ml)  
1 7 ± 8  
1 3 ± 5  
H O M A -I R 
4. 1 8 ± 2. 3 9  
3. 5 0 ± 1. 5 0  
I n s uli n s e n siti vit y i n d e x 
2. 8 0 ± 1. 8 0  
3. 1 3 ± 1. 5 7  
I n s uli n o g e ni c i n d e x 
0. 6 8 ± 0. 2 9  
0. 5 9 ± 0. 2 7  
D at a ar e m e a n ± st a n d ar d d e vi ati o n.  H O M A -I R = 
h o m e o st ati c m o d el a s s e s s m e nt of i n s uli n r e si st a n c e.  
 
 

7 
 
to wild-type mice (n=4) fed a high-fat diet for 2 months.  The wild-type mice required very little exogenous 
glucose to maintain euglycemia during the clamp, suggesting insulin resistance, while VLCAD-/- mice 
required greater  
glucose infusion 
rates to maintain 
euglycemia, 
indicating better 
insulin sensitivity 
(Figure 4A).  
Glucose production 
(Ra) was lower and 
glucose utilization 
(Rd) higher in 
VLCAD-/- mice 
compared to wild-
type mice (Figure 
4B, and C).  This data and previous reports of VLCAD-/- mice suggest they are protected from high-fat-
diet-induced insulin resistance.  The studies we propose here in humans will determine if FAO-deficient 
subjects are 
similarly 
protected from 
intralipid 
infusion-induced 
insulin 
resistance. 
 
 
Anticipated 
Results:  
 
We anticipate 
that intralipid 
Figure 3: Lipid deposition in long-chain FAO-deficient subjects and controls. 
IMCL
EMCL
Liver
0
2
4
6
*p = 0.047
Lipid Deposition
p=0.123
p=0.114
% of water peak
Control
Subject with 
FAO disorder
Muscle Lipid Spectra
water
IMCL
EMCL
water
IMCL
EMCL
A
B
Figure 4: Hyperinsulinemic-euglycemic clamps in VLCAD-/- and VLCAD+/+ mice. 
 
VLCAD-/- mice fed a high-fat diet for 2 months required greater glucose infusion rates (GIR) 
to maintain euglycemia and had lower glucose production with increased glucose utilization 
compared to wild-type mice during a hyperinsulinemic-euglycemic clamp. * p≤0.05 
GIR
0
10
20
30
40
50
+/+
-/-
30
60
90
120
Clamp
*
Minutes
mg/min*kg
Glucose Production - HFD
+/+
-/-
0
10
20
30
***
*
mg/min*kg
Glucose Utilization - HFD
+/+
-/-
0
10
20
30
40
50
60
70
*
mg/min*kg
A
B
C
Figure 5: Model of protection from FFA-induced insulin resistance by long-chain, but not medium-chain, FAO 
disorder.  
    A: VLCAD, LCHAD/TFP deficiency 
   
 
B: Control/MCAD deficiency 
A.  During fatty acid excess, long-chain acyl-CoAs and IMCL will accumulate, but decreased FAO will prevent 
an increase of redox equivalents and ROS generation resulting in normal insulin signaling and glucose 
uptake.  B. With increased FAO upstream from MCAD, IMCL will still accumulate, but excess redox 
equivalents will lead to an oxidized mitochondrial environment with ROS generation, activation of serine 
kinases, inhibition of insulin signaling and decreased glucose uptake. IMCL=intramyocellular lipid.  LCFAO = 
long-chain fatty acid oxidation.  MCFAO = medium chain fatty acid oxidation.  TCA = tricarboxylic acid cycle.  
ETC = electron transport chain.  ROS= reactive oxygen species.  PKC = protein kinase C. 

8 
 
infusion will increase IMCL in FAO patients and controls alike (Figure 5A and B).  After the intralipid 
infusion, respiratory quotients will reflect FAO capacity and will be highest (indicating greater carbohydrate 
oxidation) in subjects with CPT2, VLCAD or LCHAD/TFP deficiency and lower (indicating greater fat 
oxidation) in MCAD-deficient subjects and controls. Based on our preliminary data of normal OGTTs in 
patients with inherited defects in the FAO defects, our findings of protection from high-fat-diet-induced 
insulin resistance in VLCAD -/- mice, as well as studies that have shown that blocking FFA entry into 
mitochondria can alleviate high-fat-diet-induced insulin resistance in animal models,(56, 57) we anticipate 
that subjects with defects in CPT2, VLCAD, and LCHAD/TFP enzymes will be protected from high-fat-diet-
induced insulin resistance, essentially "blocking" long-chain FA oxidation (flux) at a level that avoids 
accumulation of metabolic intermediates that adversely affect insulin signaling (Figure 5A).  On the other 
hand, we anticipate that increased oxidative flux of fatty acids from long-chain fatty acids to medium chain 
metabolites will not provide the same protection from excess fatty acid substrate, impairing insulin signaling 
in patients with MCAD deficiency (Figure 5B) who will exhibit insulin resistance (compared to control 
infusion days) in muscle (reduced glucose disappearance, reduced insulin signaling ex-vivo), and adipose 
tissue (reduced suppression of FFA during the clamp) similar to controls.  We predict that the degree of 
FFA-induced insulin resistance in these subjects will be similar to control subjects (Figure 5B).  
 
 
2. Specific Aim 2: To determine the effect of distinct FAO disorders on tissue-specific lipid 
metabolism and insulin resistance compared to matched controls. 
 
Overview and Rationale: 
Skeletal muscle and adipose tissue account for most of whole-body glucose disposal in humans.  
While skeletal muscle is estimated to be responsible for up to 70% of total glucose uptake,(23) adipose 
tissue also contributes to whole-body glucose metabolism 
through Glut-4 glucose transporter translocation and induction of 
carbohydrate responsive-element-binding protein (ChREBP)- 
and  isoforms.(26, 37)  Adipose tissue also acts as a sink for 
dietary FFAs and is the source of lipolysis-mediated substrates 
for the FAO pathway in muscle.  In the resting state, skeletal 
muscle FAO can account for up to 90% of the total energy 
utilization.(22, 51) Both glucose and FFA uptake in muscle and 
adipose tissue are under the positive control of insulin through 
insulin-stimulated translocation of Glut-4(100) and the FATP-1 
fatty acid transporter.(61, 91)  These actions of insulin are 
mediated through tyrosine autophosphorylation of the insulin 
receptor and phosphorylation of insulin receptor substrates (IRS-
1/2), followed by activation of phosphoinositol 3-kinase (PI3-K) 
and the subsequent activation of the downstream protein kinase 
B/Akt(91, 106) (Figure 5).  Defects in insulin signaling in skeletal 
muscle are associated with the development of systemic insulin 
resistance. Specifically, acute lipid infusion in mice results in 
intramuscular accumulation of fat metabolites, including fatty acid 
acyl-CoA and DAG, and increased activity of PKC-ϴ.(103)  PKC-
ϴ activation is associated with the reduced tyrosine 
phosphorylation of IRS-1 and reduced skeletal muscle glucose 
uptake.(53) The effects of a lipid infusion on adipose insulin 
sensitivity are largely unknown, although exposure to a high-fat 
diet or elevated FFA’s of obesity are associated with the 
development of low-grade inflammation and adipose tissue 
microphage activation.(3, 16, 78)  Determination of insulin 
sensitivity and resistance in adipose and muscle can, therefore, 
be directly assessed through functional assays of insulin-
Figure 6: Plasma FFAs and 
acylcarnitines. 
 
0
30
60
90
120
-0.5
0.0
0.5
1.0
1.5
Subjects 
Controls
*p = 0.01
oral glucose
Minutes
Free Fatty Acids
mmol/L
0
30
60
90
120
0
1
2
3
4
5
Long-chain Acylcarnitines
*p<0.001
Minutes
umol/L
0
30
60
90
120
0.6
0.8
1.0
1.2
1.4
Short-chain Acylcarnitines
*p<0.001
Minutes
umol/L
A
B
C

9 
 
stimulated glucose uptake in muscle and fat biopsies and FFA uptake in fat biopsies, as well as analysis of 
insulin receptor, Akt, and (in muscle) PKC-ϴ phosphorylation. 
 
Preliminary Data: 
Plasma FFAs and long- and short-chain acylcarnitines were higher, medium-chain acylcarnitines were not 
different, and adiponectin levels were lower among patients with a LCFAO disorder. FAO-deficient patients 
had higher fasting FFA levels compared to controls(30) (Figure 6A), which decreased after an oral glucose 
load similarly to controls, suggesting no impairment of post-prandial, insulin-mediated FFA release from 
adipose tissue. The sum of the long-chain acylcarnitines (LC AC) (C12:1, C12:0, C14:2, C14:1, C14:0, 
C16:1, C16:0, C18:2, C18:1, C18:0) were significantly increased both before and after oral glucose load in 
the patients with FAO deficiency, as expected (Figure 6B).  Levels of short-chain acylcarnitines (SC AC) 
(C3:0, C4:0, C5:1, C5:0, C4-OH and C5-OH) were also higher before and after the oral glucose load, 
primarily due to an increase in proprionylcarnitine and C5-OH carnitine in the FAO-deficient patients, that 
was most pronounced during fasting (Figure 6C).  These species are derived from branched-chain amino 
acid (BCAA) oxidation, potentially suggesting increased protein oxidation during fasting.  We measured 
fasting plasma amino acid profiles and found there was no difference in fasting plasma branched-chain or 
aromatic amino acids between subjects and controls.(30) However, medium-chain acylcarnitine (MC AC) 
levels were not different between the FAO patients and controls(30) and fasting plasma high-molecular-
weight adiponectin levels were approximately 50% lower among FAO-deficient subjects compared to 
controls.(30) In summary, despite elevated fat mass, fasting FFA, and long-chain acylcarnitines, and lower 
adiponectin levels, under conditions of chronic low-fat diet feeding, FAO-deficient subjects had normal 
glucose tolerance compared to healthy controls. That long-chain, but not medium-chain, acylcarnitine 

10 
 
levels, were elevated comparable to controls is compatible with our model that elevations in FAO products 
upstream from MCAD are not sufficient to induce insulin resistance.  Whether this is true during conditions 
of high-fat metabolic stress, and whether MCAD deficiency and accumulation of metabolic intermediates 
resulting from this defect are associated with insulin resistance will be addressed in the studies proposed in 
this application. 
 
Insulin signaling cascade and FFA and glucose uptake in muscle and white adipose tissue (WAT).  
Glucose and FFA are the two major substrates used for the synthesis of triglycerides in WAT.  We have 
developed a fluorescent-based approach for simultaneous measurement of glucose and FFA uptake using 
green fluorescent 2-NBD-glucose and red fluorescent BODIPY-C12(97) (Figure 7). This approach will 
allow determination of insulin sensitivity and lipid and glucose accumulation of primary WAT explants. In 
addition, small soleus muscle biopsies will be analyzed for insulin sensitivity using 2-NBD-glucose (Figure 
8). 
 
Anticipated results: 
Mirroring increases in ectopic fat we expect to see in Aim 
1, we anticipate that intralipid infusion, but not infusion of the 
control solution, will increase plasma FFA’s associated with 
increased muscle and adipose triglycerides and DAG’s in all 
FAO patients and controls (Figure 5).  Plasma and tissue 
long-chain acylcarnitines will be greatly elevated in CPT2, 
VLCAD or LCHAD/TFP-deficient subjects; medium-chain 
acylcarnitines will be greatly elevated among subjects with 
MCAD deficiency; and both long-chain and medium-chain 
acylcarnitines will be only modestly elevated in normal 
controls.  Based on our preliminary data, we expect that 
short-chain acylcarnitines may be elevated in subjects with 
CPT2, VLCAD, LCHAD/TFP, and MCAD deficiency 
compared to controls. 
We predict that these cellular changes will be associated 
with a decrease in phospho-(p)IR and pAKT and an increase 
in pPKC-ϴ in subjects with MCAD deficiency and control 
subjects, but not in CPT2, VLCAD, and LCHAD/TFP-deficient 
subjects, paralleling the expected outcomes of system insulin 
sensitivity measurements obtained by the HEC’s in Aim 1. 
These expected decreases in insulin signaling pathway 
component expression and activation will also be reflected by 
decreased glucose uptake into muscle and adipose tissue 
and decreased FFA uptake into adipose in controls and 
subjects with MCAD deficiency.  Decreased glucose uptake 
will be correlated with medium-chain acylcarnitines, as has 
been reported in human studies(52) and in cultured 
myocytes.(52)  We anticipate no change in glucose and FFA 
uptake in muscle or adipose tissue from the glycerol/saline to 
the intralipid co-infusion samples in CPT2, VLCAD and 
LCHAD/TFP-deficient subjects.  
Figure 8: Fluorescence analysis of glucose 
uptake in monkey skeletal muscle. 
 
Skeletal muscle (vastus lateralis) explants 
obtained at necropsy from a female rhesus 
macaque were treated with 10 nM insulin at 
370C for 30 min., followed by 0.05 mM 2-
NBD-glucose for an additional 30 min., and 
analyzed by confocal microscopy.  A) 
Upper panels are images of longitudinal 
muscle fibers and lower panels are cross-
sectional images of muscle fiber bundles.  
Bars are means ± SEM of 36 individual 
muscle fibers.  Scale bar=50 µm; 
f.u.=fluorescent units.  2-NBD-glucose 
incorporation into individual fibers was 
quantified using image J.  Mean 
fluorescence per fiber was determined 
using a randomly positioned circular region 
of interest of cross-sectional images. 
Insulin  
longitudinal
cross-sectional
Basal
0
200
400
600
800
2-NBD-G uptake, f.u.
Bas                      Ins

11 
 
Metabolites will be measured 
by metabolomics, including the 
acylcarnitines, amino acids, 
organic acids and ketone bodies 
(Figure 5. Based on previous 
observations, we anticipate 
controls will have a decrease in 
plasma BCAAs,(72) but we 
anticipate normal amino acid 
profiles among subjects with a 
FAO disorder.(30) Tissue long-
chain ayclcarnitines will be much 
higher and ketones such as 
acetoacetate will be lower in 
CPT2, VLCAD and LCHAD/TFP-
deficient subjects compared to 
controls due to the decreased 
long-chain FAO. Subjects with 
MCAD deficiency will have higher 
medium-chain acylcarnitines 
compared to subjects with long-
chain FAO disorders and ketone 
concentrations similar to controls.   
 
 
II. 
Study Population 
Subjects with a fatty acid oxidation disorder: 
Inclusion Criteria: 32 subjects with a confirmed diagnosis of VLCAD (n=8), CPT2 (n=8) LCHAD/TFP (n=8), 
or MCAD (n=8) deficiency will be recruited for this study. Subjects must be > 18 years of age, and be 
willing to complete 2 CRC admissions for hyperinsulinemic euglycemic clamp studies and muscle and 
adipose tissue biopsies.  Subjects with disorders in FAO will be recruited to participate in this study through 
announcements on the FAO family support network website, through OHSU’s clinical population, and 
through letters sent to metabolic specialists across the US.  Inclusion and exclusion criteria are in Table 2.   
  
Confirming the Diagnosis: Each subject must have a confirmed diagnosis of carnitine palmitoyltransferase 
2 (CPT2), very long-chain acylCoA dehydrogenase (VLCAD), long-chain 3-hydroxyacyl CoA 
dehydrogenase (LCHAD), trifunctional protein (TFP), or medium-chain acyl-CoA dehydrogenase (MCAD) 
deficiency. Diagnosis of a disorder in fatty acid oxidation will be confirmed by obtaining medical record 
results of acylcarnitine profiles, fatty acid oxidation probe studies in cultured fibroblasts and/or mutation 
analysis (44, 71). Establishing the diagnosis of these disorders is a complex process that varies across 
metabolic centers in the US. Not all potential subjects will have had a skin biopsy and FAO probe studies in 
cultured fibroblasts. In many cases mutation analysis can identify only one recognized mutation in the 
sequenced exons of the gene. Thus, a combination of methods is most often used to establish the 
diagnosis. If two of the three diagnostic tests suggest a diagnosis of CPT2, VLCAD, LCHAD/TFP, or 
MCAD, the subject will be eligible to participate in the trial (Table 3).  
Figure 7: Concurrent measurement of FFA and glucose uptake in 
WAT explants. 
 
Subcutaneous biopsy-derived WAT explants from a female rhesus 
macaque were pretreated for 1 hour with basal or 10 nM insulin-
containing media.  Explants were co-labeled with 2-NBD-glucose and 
BODIPY-C12, imaged using confocal microscopy, and processed as 
described. Green patches enriched in 2-NBD-glucose represent 
adipocyte cytoplasm.  BODIPY-C12 accumulates in large and small 
lipid droplets (red). Scale =10 µm. 

12 
 
 
Exclusion Criteria: 
Subjects with 
CPT2, VLCAD, 
LCHAD/TFP, or 
MCAD deficiency 
may not be actively 
participating in 
another research 
project that 
prohibits their 
participation in this 
study such as a 
project that requires 
a specific diet or 
supplement that 
must be consumed 
daily. All subjects 
will be screened for 
anemia prior to 
study participation. 
Female subjects of 
childbearing 
potential will be 
screened for 
pregnancy. Anemic (Hgb < 10 g/dl) or pregnant subjects will be excluded from the study. Subjects regularly 
taking medications that strongly affect bleeding, bruising, or platelets such as Coumadin, Plavix, Aggrenox, 
Ticlid, Agrylin, Xagrid, Aricept, Namenda, Exelon, Razadyne, or aspirin will be excluded from the study.  
Subjects may not have diabetes or be on medications to treat diabetes. We do not anticipate any subjects 
with CPT2, VLCAD, or LCHAD/TFP will have diabetes, as it has never been reported. Several cases of 
diabetes have been mentioned on disease specific list serves among subjects with MCAD deficiency.  
 
Matched 
Control 
Subjects: 
Inclusion 
criteria: In 
addition, 32 
age-, sex-, and 
BMI-matched 
controls will be recruited to participate in the study through local newspaper and OHSU web site 
announcements. Control subjects must be a similar age, sex and BMI (± 2 kg/m2) as a study subject with a 
FAO disorder, be able to complete and comply with the protocol, and be in good health. Subjects must be 
weight stable (+ 5 lbs) for the past 3 months and exercise < 30 minutes, three times a week (low activity) 
for two weeks prior to each study visit. 
 
Prior to the starting the study in subjects with FAO disorders, individual study procedures such as the 
muscle and fat biopsies, the HEC and/or the MRS may be performed to practice the procedures and 
perfect them in up to 3 healthy volunteers.  These control subjects may not specifically match data from 
FAO disorders.  A separate consent form has been developed to consent these subjects. 
 
Exclusion Criteria: Control subjects may not be participating in another study that alters their macronutrient 
intake such as low carbohydrate diet. All subjects will be screened for anemia prior to study participation. 
Female subjects of childbearing potential will be screened for pregnancy. Anemic (Hgb < 10 g/dl) or 
Table 2: Participant Selection Criteria 
Subjects with an FAO disorder 
Matched controls 
Inclusion criteria 
Exclusion criteria 
Inclusion 
criteria 
Exclusion criteria 
Confirmed CPT2, 
VLCAD, LCHAD, 
TFP or MCAD 
Hgb <10g/dl 
INR>1.20 
PTT>36 seconds 
Platelets<150 K/mm3 
Same gender 
and similar 
age/BMI as 
subject with FAO 
disorder 
Hgb <10g/dl 
INR>1.20 
PTT>36 seconds 
Platelets<150 K/mm3 
≥18 years 
Pregnant /lactating 
females 
≥18 years 
Endocrine disorder 
such as diabetes, or 
untreated thyroid 
disease 
Ability to travel to 
OHSU 
Endocrine disorder 
such as diabetes, or 
untreated thyroid 
disease 
Good health, 
exercise < 30 
min, 3 x per week 
for 2 weeks prior 
to study visits. 
Cardiovascular 
disease or elevated 
plasma lipids 
Ability and 
willingness to 
complete protocol 
Cardiovascular 
disease or elevated 
plasma lipids 
Ability and 
willingness to 
complete protocol 
Taking lipid lowering 
meds 
 
Regularly taking 
meds that strongly 
affect bleeding, 
bruising or platelets. 
 
Regularly taking 
meds that strongly 
affect bleeding, 
bruising or platelets 
Table 3: Medical Records to Confirm the Diagnosis 
Disorder 
Acylcarnitine Profiles 
Fatty acid oxidation 
Probe studies 
Mutation analysis 
VLCAD/CPT2 
 C14:0 or C14:1  
 FAO flux with       
 disease specific 
acylcarnitines 
1 or 2 known 
mutations in 
specific gene 
LCHAD/TFP 
 OH-acylcarnitines  
MCAD 
 C8:0 or C10:0 

13 
 
pregnant subjects will be excluded from the study. Subjects regularly taking medications that strongly affect 
bleeding, bruising, or platelets such as Coumadin, Plavix, Aggrenox, Ticlid, Agrylin, Xagrid, Aricept, 
Namenda, Exelon, Razadyne or aspirin will be excluded from the study. Subjects may not have diabetes or 
be on medications to treat diabetes. Control subjects may not have a history of an endocrine disorder such 
as thyroid disease, renal disease or cardiovascular disease. 
 
Inclusion of Children, Women and Minorities:  All participants will be adults able to provide informed 
consent. We believe the complex full-day testing schedule planned for Day 2 of each inpatient admission, 
including two biopsies, is best conducted in 
the adult population. Both genders will be 
included in this research project. FAO 
disorders appear to occur equally in males 
and females so we expect to include 
similar numbers of males and females. 
Women of child-bearing potential are 
included in this research.  Pregnant woman 
will be excluded because the DEXA scan 
would increase risk to the fetus and 
pregnancy may affect insulin sensitivity. 
Recruitment should result in study 
demographics similar to that of the national 
disease demographics.  Caucasians will 
comprise the majority of subjects in this 
study.  To date, the majority of patients with fatty acid oxidation disorders have been of Caucasian 
ancestry.  We will welcome subjects from other racial backgrounds but anticipate that more, if not all, 
subjects will be white.  
  
III. 
Study Design 
This is a randomized cross-over study in which all 
participants will complete 2 inpatient admissions with a 4 
month washout period in between visits (Figure 9). 
Participants will be consented prior to the first admission and 
medical records will be reviewed to document the participants 
meet the inclusion criteria. Most if not all of the subjects with 
an FAO disorder will be traveling from other parts of the US to 
participate. Travel arrangements will be made and the 1st 
inpatient visit scheduled once the consent is complete, and 
the participant meets all the inclusion criteria. The outline for 
each inpatient visit is provided in figure 10. Subjects living 
locally will be consented in person at an outpatient screening 
visit while the others will be consented by mail. 
All subjects (those with an FAO disorder and controls) 
will be counseled to consume a standard diet consisting of 20% of total calories from fat, 20% from protein, 
and 60% from carbohydrates for the three days before the study by a registered dietitian. The dietitian will 
provide written and oral instructions to the participants on the phone. Subjects will keep a record of foods 
consumed during these 3 days (3-day diet record) prior to the inpatient admission.  
Figure 9: Randomized cross-over study design 
 
Participants will complete 2 inpatient admissions with a 4 month 
wash-out period in between. The order of the clamp procedures, 
with or without intralipid, will be randomly assigned.  
1stAdmission
2ndAdmission
Wash-out
4months
Figure 10: Inpatient visit schedule 
 
Subjects will travel to OHSU if necessary 
prior to day 1. Day 1 they will be fed a 
standardized diet and have a DEXA scan. 
On day 2, a hyperinsulinemic, euglycemic 
clamp with MRS, indirect calorimetry and 
tissue biopsies will be completed. The 
following day, subjects will consume a 
standard breakfast and then be discharged 
to home.  
Day1
Day2
Day3
TraveltoOHSU
Dischargetohome

14 
 
Participation will include two 
admissions to the OHSU CTRC 
inpatient unit.  Subjects will be 
admitted the day before the clamp for 
measurement of body composition by 
DEXA scans and to be fed the 
standardized diet (20% of total 
calories from fat, 20% from protein, 
and 60% from carbohydrates) prior to 
the clamp procedure (Day 1).  The 
two intravenous lines needed for the 
clamp will be placed the evening 
before the procedure or in the 
morning as determined by nursing 
assessment. 
 
DEXA: Whole body and regional fat, 
lean, and bone mass will be 
measured by DEXA (Lunar iDXA, GE 
Healthcare Lunar, Madison, WI) 
through the OHSU Clinical and 
Translational Research Center (CTRC) Bionutrition Unit.   
The following day, the clamp study will begin at approximately 6:00 AM and end at about 4:00 PM 
(Day 2; Figure 11).  Indirect calorimetry to measure energy expenditure and respiratory quotient for 
estimation of fat oxidation will be performed in the last hour of the clamp. The order of the intralipid vs. 
glycerol/saline infusion during HEC will be randomly assigned. Subjects will rest overnight and consume a 
standardized breakfast the following day (Day 3) and be discharged to home.  Subjects will return 4 months 
later and repeat the admission with the alternative clamp procedure.  If accurate clamp results are not 
achieved at Visit 1 or 2, the subject may be asked to return for another study visit to repeat the clamp 
procedure, DEXA, indirect calorimetry and/or biopsies. During periods of hospital recommendations for 
decreased in-person contact, study visits will be postponed until study staff are notified of return to normal 
research visit operations. 
 
1H and 13C MRS Procedure: of the liver and muscle: will be performed as previously described(18, 30) prior 
to (-100 min) and following both clamp procedures.  Each subject will complete 4 MRS measurements of 
liver and muscle lipid content (Figure 11).  Image-guided, 1H localized, MRS following high-resolution T-
weighted spin-echo imaging will be performed on a Siemens 3T whole-body system.  Radiofrequency 
surface coils will be used for region-of-interest (ROI) voxel localization and spectroscopy data acquisition. 
Depending on the schedule for the MRS instruments and to allow for more time for a meal before the 
testing, the MRS measurements may not be completed until approximately 5:30 PM.  
 
MRI of the abdomen to quantify visceral and subcutaneous fat: Although not a primary outcome, these 
measures will be acquired using a Siemens 3T system as previously described (18, 30). 
 
HEC with co-infusion of [6,6-2H2] glucose and intralipids will be used to determine whole-body [SI], liver [SI-
H], and skeletal muscle [SI-M] insulin sensitivity.  Following a 10-hour overnight fast, at approximately 0600 
hr, the  intravenous catheters placed the evening before in one arm for infusions and in the contralateral 
hand for blood withdrawal will be warmed to 70°C using a warming mitt for sampling of arterialized venous 
blood.  A primed (200 mg/m2) IV bolus followed by 2.0 mg/m2 body surface area per minute constant 
infusion of [6,6-2H2] glucose (Cambridge Isotope Laboratories, Andover, MA) will be infused to achieve 
~1.0 mol percent excess for all subjects.  Plasma samples will be obtained at -125, -120, -20, -15, -10, and 
-5 minutes to estimate basal endogenous glucose production and/or fasting insulin concentration.  At the 
completion of the 2-hr infusion with glucose isotope, a primed, constant infusion of regular insulin at 40 
mU/m2/min will be started and continued for 5 hours.  Concurrent with the insulin infusion, either a 
Figure 11: Study Protocol for Aims 1 and 2. 
 
Participants will start a dideuterated glucose infusion 2 hours prior to 
the clamp.  Lipid deposition will be measured by 1H spectroscopy.  The 
insulin infusion with either co-infusion of glycerol/saline or intralipid will 
begin at time 0 and last for 5 hours.  Glucose will be infused at a 
variable rate to maintain euglycemia.  At the end of the clamp, a muscle 
and adipose biopsy will be collected and lipid deposition re-measured. 

15 
 
saline/2.5% glycerol or 20% intralipid infusion of 1.5 ml/min will be started and continued until the 5-hour 
clamp has been completed and the muscle and fat biopsy have been performed.  Blood glucose will be 
monitored every 5-10 min during the insulin infusion using a Hemocue Glucose 201(HemoCue America, 
Brea, CA), or an i-STAT analyzer (Abbott Point of Care Inc, Abbott Park, IL) and euglycemia will be 
maintained throughout the clamp by infusing 20% dextrose at a variable rate. To maintain a constant 
enrichment of [6,6-2H2] glucose in blood during the clamp, [6,6-2H2] glucose infusion will be continued 
throughout the clamp and at 2% enrichment in the 20% dextrose solution.  Additional blood samples will be 
collected every every 60 minutes for insulin, ketones,and FFA, and at the beginning and end of the clamp 
for 2H2-glucose and metabolomics. Using the blood samples collected during the 30-minute period just 
preceding and at the end of the glucose clamp, glucose disposal rates (Rd) and basal endogenous glucose 
production (Ra) will be calculated according to the steady-state equations of Steele.(92) At the end of the 5 
hour infusion and completion of the muscle and fat biopsies, the insulin and intralipid or glycerol/saline 
infusions will be stopped. After 10 minutes, the subject will be fed a low-fat meal. Blood glucose will 
continue to be monitored for an additional hour after the insulin infusion is stopped with adjustment to the 
glucose infusion as indicated to ensure subjects reestablish endogenous glucose regulation. 
 
Rationale for HEC approach: Patients will undergo insulin sensitivity measurement using HECs along with 
determination of ectopic fat accumulation by 1H magnetic resonance  spectroscopy (MRS).  If the defective 
mitochondrial function and “lipotoxicity” hypothesis is true, then insulin resistance should be common 
among patients with long-chain FAO disorders in whom FAO capacity is greatly reduced.  However, insulin 
resistance and diabetes have not been previously reported in case reports of patients with CPT2, VLCAD, 
or LCHAD/TFP deficiency(35) and, in our preliminary data(30) (see also above), these patients have 
comparable levels of IMCL, IHL, and glucose tolerance to controls while consuming a low-fat diet.   
 
 
Because a low-fat diet is standard therapy for these patients, previous clinical reports and our preliminary 
data could be argued to lack the proper conditions to permit accumulation of insulin resistance-inducing 
cytosolic lipid byproducts. However, prolonged high-fat feeding is contraindicated in FAO patients due to 
increased risk of hypoglycemia and rhabdomyolysis (see also Human Subjects).  Instead, we propose 
creating a "fat challenge" by infusing intralipids (20% IL, 1.5 mL/min+heparin) during a HEC, which has 
been shown to increase IMCL and induce insulin resistance within 4 to 6 hours.(4, 9, 11, 94)  From studies 
using an identical intralipid infusion protocol, levels of non-esterified free fatty acids in the blood can range 
between 0.3 to 4.5 mmol/L (mean 1.49 mmol/L).(4, 8, 9, 11, 17, 27, 28, 36, 39, 60, 73, 81, 82, 89, 90)  In 
comparison, non-esterified fatty acid (NEFA) levels have been reported to rise up to 0.8 - 2 mmol/L in 
adults and children after an overnight fast(7, 10, 83, 95) and up to 0.8 - 1.0 mmol/L after a high-fat 
meal.(87) 
  
 
Safety: By performing a HEC during the intralipid infusion, hypoglycemia and rhabdomyolysis are avoided 
through the variable glucose infusion to maintain glucose levels in a safe range and NEFA levels remain 
closer to physiological levels.  Therefore, we feel the intralipid + HEC protocol proposed can be conducted 
safely among FAOD subjects while achieving high insulin and high NEFA levels.  By using a co-infusion of 
dideuterated glucose, we will be able to separately measure glucose appearance in the fasting state (Ra; 
mostly hepatic glucose output) as well as insulin-induced glucose disposal (Rd; mostly muscle), as there is 
reason to predict that FAO defects may affect these two organs separately.(104, 105)  Using a 
randomized, crossover design, the change in insulin sensitivity with co-infusion of intralipid will be 
compared between FAO patients and matched controls to a control infusion (glycerol / saline).  
 
 
Tissue Biopsies: Blood, muscle, and adipose tissue samples will be collected near the end of each clamp 
to allow determination of both changes in serum and intracellular energy metabolites and tissue-specific 
insulin sensitivity ex vivo.   
 
Muscle Biopsy Technique:  Biopsies of the vastus lateralis muscle from study subjects will be obtained by 
percutaneous puncture using a Bergstrom side-cut needle(142) (144). Briefly, after local infiltration with 
lidocaine (2%), a small incision is made with a scalpel on the overlying skin. The needle is then advanced 
and the fascia traversed.  Once in the muscle, the tip of the needle is advanced 3 cm and aspiration 

16 
 
applied through the back of the needle to enhance the amount of tissue protruding into the side-port cutting 
window.  A sharp and quick cut is made and the specimen extracted. Usually more than 100 mg of wet-
weight tissue is obtained.  This tissue is dabbed dry, weighed, and cut into pieces that are immediately 
frozen in liquid nitrogen.  The incision is closed with a simple steristrip; a pad dressing is applied at the end 
of the visit.  Complications can include pain/soreness and superficial hematoma.  
 
Adipose Biopsy Techniques: At the time of biopsy, ~ 1.0 gram of fat tissue will be extracted from just lateral 
to the umbilicus on the abdomen by aspiration under local anesthesia.  We have successfully and safely 
used this technique to harvest samples in more than 100 patients to date participating in ongoing studies at 
our institution.  Immediately after harvest, the sample is divided two ways by the lab technician.  The first 
sample is placed in buffer and taken for size and insulin sensitivity measures.  The second sample is then 
stored at –80 C for later analysis. 
 
Biopsy Anticipated adverse events: Prior to the aspiration fat and muscle biopsies, a local anesthetic is 
injected into the skin.  An allergic reaction to the anesthetic is rare (less than 1 in 10,000).  Significant 
bleeding from the fat or muscle biopsy is rare.  Baseline lab work will include measurement of platelets, 
INR and PTT to assure these levels are normal prior to the biopsies.  Potential subjects taking regular 
medications that may have an anticoagulant affect will also be excluded from participation in the study. 
Infection of a fat biopsy may occur in up to 1 in 10 cases.  A small scar will result at the biopsy site. The 
scar is usually smaller than the biopsy incision and is frequently almost invisible. 
 
Blood and tissue metabolites collected from subjects with a FAO disorder and controls during clamps with 
and without co-infusion of intralipid will be measured by the West Coast Metabolomics Center (UC Davis, 
see specific methods below).  Activation and expression of the insulin-signaling cascade components IR, 
AKT, PKC-ϴ, will be measured in muscle and WAT samples as described in detail below.  Basal and 
insulin-stimulated glucose and FFA uptake into muscle and WAT explants from biopsies will be measured 
by the novel florescence technique described above. Changes in insulin signaling and tissue fatty acid and 
glucose uptake between the intra-lipid and glycerol infusions will be compared between CPT2, VLCAD, 
and LCHAD/TFP-deficient subjects, subjects with MCAD deficiency, and normal controls. 
 

17 
 
IV. 
Study Procedures 
 
Laboratory Methods: 
Lab Assays: Insulin by RIA, leptin and total and high-molecular-weight adiponectin by ELISA, and FFA 
levels by enzymatic colorimetric kit will be determined by the OHSU CTRC Laboratory Core. 
Metabolomics: Plasma, muscle, and subcutaneous adipose tissue aliquots will be extracted, derivatized, 
centrifuged, decanted and dried as previously described.42 A set of 13 C8-C30 fatty acid methyl ester 
internal standards will be added, samples derivatized, and analytes separated using an Agilent 6890 gas 
chromatograph (Santa Clara, CA).  Chromatography will be performed followed by mass spectrometry by a 
Leco Pegasus IV time-of-flight mass spectrometer (St. Joseph, MI) with 280°C transfer line temperature, 
electron ionization at 270eV and an ion source temperature of 250°C.  Mass spectra will be acquired from 
m/z 85–500 at 17 spectra s21 and 1850 V detector voltage.  Result files will be exported to our servers and 
further processed by the metabolomics BinBase database. All database entries in BinBase will be matched 
against the Fiehn mass spectral library of 1,200 authentic metabolite spectra.  Identified metabolites will be 
reported if present within at least 50% of the samples per study design group.  Peak heights of quantifier 
ions defined for each metabolite in BinBase will be normalized to the sum intensities of all known 
metabolites and used for statistical investigation.  Compiled data sets will be analyzed using an appropriate 
array of multivariate data analyses techniques including principal components analysis, hierarchical cluster 
analyses, and projection to latent structures discriminate analyses using the an R-based Microsoft Excel 
Add-in developed in the laboratory of Dr. Newman and SIMPCA-P v.12.0. 
 
Laboratory analysis of Tissue Biopsies: Subcutaneous WAT and muscle biopsies will be processed for 
explant assays of glucose and FFA uptake as well as for the preparation of lysates for Western immunoblot 
 Table 4. Schedule of Study Procedures 
 
 
Screening 
Visit 
(Local 
Subjects) 
Prior 
to 
Admit 
At 
Admit 
or on 
Day 1 
Day 2 
Day 3 
Informed Consent/HIPAA Authorization 
X 
X 
 
 
 
Inclusion/Exclusion Assessment 
X 
X 
X 
 
 
Demographics and Medical History 
X 
X 
 
 
 
CBC with platelets, INR, PTT 
 
 
X 
 
 
Pregnancy Test (if applicable) 
 
 
X 
 
 
Vital Signs and Weight 
X 
 
X 
X 
X 
Height 
X 
 
X 
 
 
Physical Exam 
 
 
X 
 
 
Standard FAO Low Fat Diet  
 
X 
X 
X 
X 
Low Fat Diet Instruction 
X 
X 
 
 
 
3-Day Diet Diary 
 
X 
 
 
 
DEXA Scan 
 
 
X 
 
 
Intravenous (IV) Catheter Placement 
 
 
X 
 
 
Indirect Calorimetry 
 
 
 
X 
 
Magnetic Resonance Spectroscopy (MRS) 
 
 
 
X 
 
Hyperinsulinemic-Euglycemic Clamp  
(Figure 11) 
 
 
 
X 
 
Muscle Biopsy 
 
 
 
X 
 
Fat Biopsy 
 
 
 
X 
 
Discharge from CTRC 
 
 
 
 
X 

18 
 
analysis of insulin signaling.  Insulin-stimulated FFA and glucose uptake in WAT explants from umbilical 
biopsies will be performed as previously established for non-human primate tissue and as shown in our 
previous publications and in Figure 8.(96-98)  Glucose uptake in skeletal muscle biopsies will be evaluated 
in samples treated alone or with 10 nM insulin for 30 min and then exposed to 50 M 2-NBD-glucose for an 
additional 30 min.  Samples are then washed, fixed in 4% paraformaldehyde, washed in HBSS with 50 mM 
CoCl2, and stored in HBSS with 25 mM CoCl2 until analyzed by confocal microscopy.  2-NBD-glucose 
incorporation into individual fibers is quantified using image J.  Mean fluorescence per fiber is determined 
using a randomly positioned circular region of interest of cross-sectional images.  Insulin signaling pathway 
expression and activation will be analyzed by preparing total protein lysates in RIPA buffer after 
homogenization in a motor-driven glass-glass homogenizer for muscle and in X buffer for WAT.  Lysates 
are then subjected to standard Western immunoblotting for phosphorylated (activated) IR, Akt, and (in 
muscle) PKC-ϴ, followed by stripping and probing with pan-IR, Akt, and PKC-ϴ antibodies to determine 
general expression levels and for normalization of phospho-antibody signals as previously described.(24, 
25, 63)  We acknowledge that insulin receptor substrates (IRS)-1 and 2 are important components of the 
insulin signaling pathway in fat and muscle and are involved in insulin resistance, but it is our experience 
that analysis of IRS-1/2 activation requires significant amounts of lysate and immunoprecipitation prior to 
Western immunoblotting, which we are unlikely to acquire from the biopsies that will be obtained.  The 
proposed analysis of IR, Akt, and PKC-ϴ should, however, provide sufficient data to ascertain the status of 
the insulin-signaling pathway in samples from the different treatment groups. 
 
V. 
Statistical Analysis: 
The primary analyses to compare disease groups with healthy controls will use mixed models with a 
random effect for matched pair to allow for correlation between the observations on the matched 
observations.  In addition to the random pair effect, a disease effect (disease versus healthy), and a 
diagnostic group effect (CPT2, VLCAD, LCHAD/TFP, MCAD), we will also incorporate a fixed effect for the 
order of testing (interlipid infusion tested first or second).  The hypotheses focus on the comparisons 
between diseased individuals and their matched controls with regard to the difference in outcomes under 
the two conditions (glycerol vs interlipid infusion).  For these analyses, we will use the difference in a given 
outcome (e.g., Rd uptake) between infusion conditions as the dependent variable and compare these 
differences between diseased and matched controls, separately for the four diagnostic groups (CPT2, 
VLCAD, LCHAD/TFP, MCAD).  Although we will conduct a formal test of interaction of disease (disease vs 
healthy) and diagnostic group, we are not powered for such a test and, hence, will focus on separate 
inferences for the four disease groups.  Note that, as indicated in the hypotheses, we expect to find 
differences between the controls and the CPT2, VLCAD, and LCHAD/TFP subjects, but not between the 
controls and the MCAD subjects.  For the latter comparison, our emphasis will be on estimating the 
magnitude of the mean differences via 95% confidence intervals rather than formal hypothesis testing.  We 
will use a significance level of 0.05 for each outcome comparison, with no adjustment for multiple 
comparisons due to the limited sample sizes. We are unable to increase sample sizes due to the small 
study populations and budgetary constraints, so cannot power the study to take into account the multiple 
comparisons. However, we will present for each outcome, both unadjusted p-values as well as p-values 
adjusted for the two multiple comparisons (one for each disease group) using the Holm procedure.(38)  
 
Power Considerations: 
For each outcome, we have two measurements on every subject, one for each infusion condition.  
Our analyses will focus on comparisons between patients and controls of the differences between 
outcomes under the two conditions.  We expect to see meaningful differences between controls CPT2, 
LCHAD/TFP, and VLCAD-deficient participants.  These are the groups of interest for power calculations, 
whereas for MCAD, our aim is to provide useful descriptive information.  There are two sets of correlations 

19 
 
between measures that need to be considered in power calculations (and statistical analyses).  The first is 
the correlation between outcome measures on a given subject under the two infusion conditions, the 
second is the correlation of the 
differences in outcomes under the 
two infusion conditions, between 
each subject and his/her matched 
pair.  For power calculations, we 
considered various values for each 
of these two correlations and used 
standard deviation estimates for 
each infusion condition as reported 
for glucose infusion rate (GIR) and 
for PDK4 mRNA levels (81, 94).  
GIR is a primary result during the 
HEC, closely related to insulin 
resistance (specific aim 1).  PDK4 
message is associated with our hypothesized mechanisms for insulin resistance in muscle and adipose 
tissue (Specific Aim 2).  Mean and SD values for these outcomes in healthy controls are given in Table 5.  
We assume that normal controls and MCAD subjects will have mean outcomes as given in the table but 
that subjects with CPT2, VLCAD, and LCHAD/TFP disorder will have mean decreases when given the 
interlipid infusion that are less than 10% from that under the glycerol infusion condition for GIR and a mean 
increase of less than 100% for PDK4 mRNA.  That is, we expect the mean change from glycerol infusion 
condition to be less than 5.7, and 0.18 for GIR, and PDK4 mRNA, respectively, for the CPT2, VLCAD, and 
LCHAD/TFP subjects. 
In Table 6, we show 
the differences between 
groups in mean change that 
can be detected with 80% 
power at level 0.05 when the 
sample size per long-chain 
disorders is 20.  For n=10 
subjects, we have adequate 
power for detecting expected 
differences for GIR and PDK4 
mRNA with modest 
correlation between repeated 
observations on the same 
subject and between matched 
pairs.  For GIR, even with 
zero correlation, we would 
have about 97% power at 
level 0.05 to detect the 
expected difference in change 
between groups (31.5 vs 5.7) 
with n=10.  For PDK4 mRNA, we could detect the expected difference in change (-0.62 vs -0.18) with 63% 
power if zero correlation and with 78% power if the correlation is 0.25 between and within n=10 matched 
patients. 
 
Timeline: Enrollment and clamp procedures will be completed in years 1, 2, and 3. Biopsy assays and 
insulin signaling measures will be completed real time with collection of the muscle and adipose samples 
during years 1-3. In year 4, we will batch analyze stored plasma, muscle, and adipose samples for 
metabolomics to control for run variation of the metabolomics methods, prepare manuscripts and present 
the results of the project. 
Table 6: Change between glycerol infusion and interlipid infusion 
that can be detected between control and the VLCAD, LCHAD/TFP 
and CTP2 groups with 80% power at level 0.05 with n=20 subjects 
long-chain FAO disorders. 
Outcome Mean Δ 
(Glycerol 
–
Intralipid)  
Controls 
Correlation 
between 
observations 
on the same 
subjects 
Correlation 
of Δ 
between 
matched 
controls 
and 
patients 
Mean 
Detectable  
Δ (Glycerol –
Intralipid)* 
LCHAD/TFP 
subjects 
GIR  
31.5 
.25 
.25 
17.6 
31.5 
.50 
.25 
20.0 
31.5 
.50 
.50 
22.1 
PDK4 
mRNA  
0.62 
.25 
.25 
0.17 
0.62 
.50 
.25 
0.19 
0.62 
.50 
.50 
0.27 
Δ= change; * Note that changes smaller than these can be 
detected with greater power as smaller changes are further from 
the changes expected in the control groups. 
Table 5: Glucose Infusion Rate (GIR), and PDK4 mRNA/actin in 
healthy controls and hypothesized mean differences in subjects. 
Outcome 
Glycerol 
Infusion 
 
Interlipid 
Infusion 
Ha: mean Δ 
In Controls 
and MCAD 
Ha: mean Δ 
In VLCAD and 
LCHAD/TFP 
GIR 
(μmol/kg/min) 
(n=9) 
57.2 ± 9.3 
25.7 ± 7.8 
31.5 
5.7 
PDK4 
(mRNA/actin) 
(n=10) 
0.18 ± 0.06 
0.8 ± 0.38 
-0.62 
-0.18 
Data are mean ± standard deviation from Roden et al and Tsintzas et al. 
GIR = glucose infusion rate; PDK4 = pyruvate dehydrogenase kinase 4; 
Ha: mean Δ= hypothesized mean difference. 

20 
 
 
VI. 
Protection of Human Subjects 
 
A. Potential Risks  
Risks from the procedures in this study may include: pain, bleeding, bruising and infection from the 
catheter placement, the muscle biopsy and the subcutaneous fat biopsy; and exposure to a small amount 
of radiation (1.5 – 3.0 mrem) during the DEXA scan. Nonradioactive stable isotopes of 2H will be given to 
subjects. The safety of stable isotopes is well documented and does not pose an added risk to the subjects 
(46). Alterations in dietary intake associated with poor intake or prolonged fasting in subjects with CPT2, 
VLCAD, LCHAD/TFP or MCAD deficiency carry the risk of associated hypoglycemia, or rhabdomyolysis. 
The research meals during the study will be designed to provide adequate total energy and pose little risk 
to subjects. Dextrose will be infused at a variable rate during the hyperinsulinemic clamp and will prevent 
hypoglycemia.  
 
Infusion of intralipid in subjects with a fatty acid oxidation disorder could increase partial fatty acid 
oxidation metabolites in plasma and tissues such as long-chain, long-chain hydroxyl and medium-chain 
acylcarnitines. When normal amounts of nonfat energy are provided and the subject is not dependent upon 
FAO at the time, we do not believe the acylcarnitines in themselves induce metabolic decompensation. The 
chronic provision of high fat diets or intravenous lipid is not recommended clinically for this population. One 
case report of intravenous lipid infusion raised plasma acylcarnitines but did not result in any adverse event 
or metabolic decompensation (20). The effects of high fat diet feeding have been documented in only one 
case report where a child was inadvertently fed a high fat, low carbohydrate formula and developed 
lethargy, rhabdomyolysis and acidosis after several days (29). It is important to note that the clinical 
symptoms of metabolic decompensation observed in this case report and in subjects with FAO disorders 
occur in the absence of adequate glucose or energy to provide an alternative fuel. Episodes of 
rhabdomyolysis or hypoglycemia occur with prolonged fasting, exercise, or intercurrent illness. We will 
avoid metabolic decompensation in this study by providing a dextrose infusion to maintain euglycemia and 
provide substrate for energy production. 
 
B. Subject Recruitment  
Subjects with CPT2, VLCAD, LCHAD/TFP or MCAD deficiency will be recruited to participate in this 
study. Recruitment techniques will include announcements on Fatty Acid Oxidation Support group web 
pages and chat boards and through our center’s clinic population. Each subject must have a confirmed 
diagnosis of CPT2, VLCAD, LCHAD/TFP or MCAD deficiency, be 18 years of age or older, and be able to 
complete and comply with the protocol. Subjects may not be participating in another research project that 
alters macronutrient content of the diet.  
 
We will also recruit normal matched control subjects. These subjects will be recruited from the local 
Portland area via the OHSU webpage, and informational flyers on campus. Control subjects will be chosen 
to be similar in age, BMI, gender, and activity level as a subject with a fatty acid oxidation disorder, and 
must be in good health and able to complete the protocol. As many as 100 subjects (both with FAO 
disorders and normal controls) may be screened for this study. 
 
 
C. Informed Consent 
 A principal investigator or the study coordinator will explain and discuss all procedures and tests to 
be completed during the study and will be available to answer questions for the subjects, and their families. 
Informed consent will be obtained from each subject prior to beginning the study. Because these disorders 
are rare diseases, many subjects with CPT2, VLCAD, LCHAD/TFP or MCAD deficiency will live out of 
state. Local subjects will be brought in for an outpatient screening visit to consent them in person. If 
informed consent cannot be obtained in person, it will be obtained using the following process: 
 
1. A potential subject will contact the PI or study staff. 

21 
 
2. A packet of information including a cover letter, consent form, HIPAA authorization form,  medical release 
form and a stamped return envelope will be mailed to their address. 
3. The consent form and HIPAA authorization will be on colored paper and copies of the forms will be 
provided on white paper for the subject to retain for their records. 
4. The PI or authorized study staff will review the consent on the phone, confirm subject understanding and 
answer any questions. 
5. Once the investigator is convinced that the subject verbally demonstrates understanding and agrees to the 
process, the consent, HIPAA and medical release documents will be signed and mailed to the investigator. 
6. When the forms arrive at OHSU and are signed by the researcher who obtained verbal consent, the HIPAA 
authorization will be copied and mailed back to the subject. 
7. When the investigator receives the consent and medical release forms, study staff will obtain the necessary 
records to verify the diagnosis of a fatty acid oxidation disorder. 
8. If the subject is eligible to participate, a visit will be scheduled and travel arrangements to OHSU will be 
made. 
9. Individuals authorized to obtain written consent are the principal investigator, co-investigators and assigned 
research staff specifically designated by the principal investigator to work on this project. 
 
Compensation of Study Participants: 
 
Both subjects with a fatty acid oxidation disorder and controls will be compensated for their participation 
in this study and reimbursed for travel expenses. The compensation plan for each group is outlined below. 
1. Subjects with a fatty acid oxidation disorder: 
a. The majority of subjects with CPT2, VLCAD, LCHAD/TFP or MCAD will be traveling from a 
distant US city to Portland to participate. Subjects will not be compensated directly for airline 
tickets but their travel costs will be covered. Study staff will book their airfare and purchase the 
airline ticket.  In rare situations where subjects live quite a distance from the airport and need to 
take an early flight, a night in a hotel will be booked for them.  They will be compensated for 
miles traveled in their own car and airport parking.  Miles are reimbursed based on the current 
IRS rate per mile traveled for business. Baggage fees, if needed, will also be covered.  Study 
staff will pick-up most subjects at PDX and return them to the airport, but if a taxi is needed the 
fare will be reimbursed.  
b. A few subjects may be local. Local subjects may be compensated for miles traveled in their own 
car. They will travel to OHSU for 3 separate visits.  
c. If subjects decide to extend their stay in Portland before or after the study visit and stay in a 
hotel or eat food not provided by OHSU, they will do it at their own expense.  
d. If the local FAOD subjects come in for a screening visit (requires 2 hours of their time), they will 
be compensated $25. 
e. At the completion of the 1st visit (including 3 nights, 2 days at the CTRC, and muscle/fat 
biopsies), subjects will be compensated $300.  
f. 
At the completion of the 2nd visit 4 months later (including 3 nights, 2 days at the CTRC, and 
muscle/fat biopsies), subjects will be compensated $700. Maximum compensation for 1 
screening visit and 2 inpatient visits will be $1025 plus travel reimbursement.  
g. If subjects are asked to return for an extra visit to repeat the clamp procedure, DEXA, indirect 
calorimetry and/or muscle/fat biopies, they will have their airfare covered, the same travel 
reimbursements noted in (a) above and will receive additional compensation of $300. Subjects 
may return for up to two additional visits.  
h. If subjects are traveling from a country other than the United States, they will be asked to 
purchase their own airline tickets.  Travel reimbursement for airfare will be a maximum of $1000 
for each of the two inpatient visits.  They will also receive additional travel reimbursement as 
noted in (a) above and $300 compensation for Visit 1 and $700 compensation for Visit 2.  
2. Control subjects who do not have a fatty acid oxidation disorder: 
a. All of the control subjects will be recruited from the Portland metro area. Participation will require 
3 trips to OHSU: 1 screening visit and 2 separate inpatient admissions.  They will be 
compensated for miles traveled in their own car using the current IRS rate per mile.. 

22 
 
b. At the completion of the screening visit (requires about 2 hours of their time), subjects will be 
compensated $25. 
c. At the completion of the 1st visit (including 3 nights, 2 days at the CTRC, and muscle/fat 
biopsies), subjects will be compensated $300.  
d. At the completion of the 2nd visit 4 months later (including 3 nights, 2 days at the CTRC, and 
muscle/fat biopsies), subjects will be compensated $700. Maximum compensation for a 
screening visit and 2 inpatient visits will be $1025 plus travel reimbursement.  
3. Control subjects participating for study procedure practice: 
a. All subjects will be recruited from the Portland metro area.  Compensation will depend on which 
practice procedures they elect to participate in.  If an HEC will be performed then there will be 
an overnight admission with discharge the next afternoon.  Compensation will be $200.  If a 
muscle and fat biopsy are performed, compensation will be $100.  If an MRS study is 
performed, compensation will be $25. Maximum compensation for all three procedures will 
be $325 plus travel reimbursement. 
 
D. Protection Against Risk 
Studies will be reviewed and approved by the OHSU Institutional Review Board (IRB). All subjects 
will be admitted to the inpatient unit of the Oregon Clinical and Translational Research Institute (OCTRI). 
Nursing staff and the investigative team will monitor subjects and care for wound or bleeding complications 
as a result of catheter placement and biopsy. To prevent radiation exposure to a fetus, a urine pregnancy 
test will be completed for females of child bearing potential prior to each DEXA Scan.  Subjects who are 
pregnant will be excluded. A stable isotope 2H2 glucose will be given to subjects. As noted above, the 
safety of stable isotopes is well documented and does not pose an added risk to the subjects (46). Special 
care will be taken to explain the use of stable isotopes to the subjects and their families. Plasma glucose 
will be monitored during the clamp procedure. The MD will discharge subjects at the end of each admission 
after a physical exam, vital signs are normal and they have no complaints of muscle pain. If they do have 
muscle pain, we will check a plasma creatine kinase (CK) level prior to discharge.  
 
E. Confidentiality 
Upon enrollment, subjects will be assigned a unique identifier that will be used instead of their name, 
medical record number or other personally identifying information.  To ensure confidentiality, all data and 
specimens collected will be kept in locked laboratory facilities. Urine, blood, muscle and adipose samples 
will be stored with a unique identifier in locked laboratory facilities at OHSU. Long-term storage of blood 
muscle and adipose will be stored at -80C and urine samples will be stored at -20 C in the laboratory of 
Dr. Gillingham. The laboratory is a locked facility in the Richard T. Jones building, room 4549 at OHSU, 
Portland, Oregon. Only the PI and study staff will have access to paper records and the key code which will 
be stored in locked filing cabinets in restricted access offices at OHSU. Computer files of collected data will 
be password protected and stored on a restricted drive behind the OHSU firewall. The study coordinator 
will code urine, blood, muscle and adipose samples with the study ID code to remove any identifiable 
information on the sample prior to sending the samples to other laboratories for analysis. Subject’s names 
or other identifiable information will not be published or shared with outside research facilities.  
 
 
F. Potential Benefits of the Proposed Research to the Subjects and Others 
The proposed studies may provide new insights into the relationship between fatty acid oxidation 
and the development of insulin resistance in humans. This knowledge will be useful in designing future 
pharmaceutical targets for patients with insulin resistance and type 2 diabetes. The participants in this 
study will not benefit from the proposed research.   
 
G. Data and Safety Monitoring Plan 
 
1. Data quality and management. The principal investigators, Jonathan Purnell, and Melanie Gillingham, will 
review the data collection forms on an annual basis for completeness and accuracy of the data as well as 

23 
 
protocol compliance. A statement reflecting the results of the review will be sent to the NIH in the annual 
report (non-competing continuation).  The data that will be reported to and monitored by the PI will include: 
• 
Subject anthropometric data including height, weight and body composition 
• 
Subject laboratory results 
• 
Indirect calorimetry analysis of energy needs 
• 
Data from the hyperinsulinemic-euglycemic clamp study 
• 
Data from the magnetic resonance spectroscopy procedure 
• 
Subject reports of adverse events or unanticipated problems 
 
2. Adverse Event Definitions and Grading: An adverse event (AE) is defined as any unfavorable or 
unintended sign (including an abnormal laboratory finding), symptom or disease temporally associated with 
the use of a medical treatment or procedure that may or may not be considered related to the medical 
treatment or procedure.  Adverse events in this study are evaluated for relationship to study intervention by 
a physician co-investigator using the Common Terminology Criteria for Adverse Events version 4.0.  This 
grading criterion is listed as follows: 
 
Grade 1: Mild; asymptomatic or mild symptoms; clinical or diagnostic observations only; intervention not 
indicated. 
Grade 2: Moderate; minimal, local, or noninvasive intervention indicated; limiting age-appropriate 
instrumental activities of daily living. 
Grade 3: Severe or medically significant but not immediately life-threatening; hospitalization indicated; 
disabling; limiting self-care activities of daily living. 
Grade 4: Life-threatening consequences; urgent intervention needed. 
Grade 5: Death related to AE. 
 
A Serious Adverse Event (SAE) is defined as an event that results in death, is life threatening, requires in-
patient hospitalization, results in persistent or significant disability or is an important medical event that 
does not meet the above criteria but may require medical or surgical intervention.  SAE’s that are 
unexpected and possibly related will be reported to the Independent Safety Monitor for the study, and the 
IRB as indicated in the OHSU Research Integrity Office policy on Reporting Unanticipated Problems and 
Adverse Events. 
 
Some risks are considered expected in this study.  They are considered mild to moderate risks and are 
addressed in the consent form.  The expected risks include: 
• 
Inserting a venous catheter needle for blood sampling and infusion can be associated with some 
discomfort, bruising, and, very rarely, with inflammation and infection of the arm veins.  
• 
Muscle and adipose biopsies can be associated with some discomfort, bleeding, bruising, and, very 
rarely, with inflammation and infection of the biopsy site. 
• 
Undergoing DEXA scans is associated with a small degree of radiation exposure. 
• 
Insulin infusion has a risk of hypoglycemia. 
• 
Heparin infusion may cause bruising or heavy bleeding, and rarely low platelet levels. 
 
3. Plan for Reporting Both Anticipated and Unanticipated Adverse Events.  
a) Each subject will be evaluated for any adverse events at study visits. 
b) Any event that is reported to either the principal investigator or his or her designated research 
associates by the subject or medical staff caring for the subject and which meets adverse event 
criteria will be documented as such.  An MD Co-Investigator will review AEs real-time.   
c) Any AE or protocol deviation that represents an unanticipated problem /will be reviewed by the PI 
and submitted to the OHSU IRB. The report will include a description of the event, when and how it 
was reported, as well as any official chart records or documentation to corroborate the event or the 
reporting of the event.  

24 
 
d) AEs will be reported as outlined by the OHSU Research Integrity Office policy on Reporting 
Unanticipated Problems and Adverse Events. 
e) AEs and unanticipated problems will be summarized annually and submitted to the IRB at 
continuing review. 
f) Any action resulting in a temporary or permanent suspension of the study (e.g. IRB actions, or 
actions by the investigators or co-investigators) will be reported to the appropriate NIH program 
official.  
4. Safety Review and Monitoring 
a) Independent Safety Monitor. The study will be monitored by the principal investigator, MD co-
investigator and an Independent Safety Monitor.  The Safety Monitor will be Penelope Hogarth MD, 
Associate Professor of Neurology and Molecular & Medical Genetics at OHSU.  Dr. Hogarth will be 
notified immediately if a significant adverse event occurs, and then she, as Safety Monitor, together 
with the PI, will notify the OHSU IRB, the NIH, and the CRC. The role of the Safety Monitor will be: 
i) The Safety Monitor will review the annual report and all adverse events prior to submission of the 
IRB review 
ii) She will prepare a memo of her findings and recommendations to be included in the submission 
to the IRB with the annual report. 
iii) If any serious unexpected adverse event occurs, the safety monitor will be notified with 24 hours 
and she will review the medical records. 
iv) The Safety Monitor, together with the PI, will notify the OHSU IRB, the NIH, and the CRC of 
serious unexpected adverse events. 
b) Annual Review. The principal investigator and MD co-investigator will review this protocol on a 
continuing basis for subject safety and include results of the review in the annual progress reports 
submitted to the IRB and NIH. 
c) Annual Report. The annual report will include a list of adverse events and the content listed below 
as well as a memo of findings and recommendations provided by the Safety Monitor. 
d) Content of the Annual Report. The annual report will address: a) Whether adverse event rates are 
consistent with pre-study assumptions; b) Reason for dropouts from the study; c) Whether all 
participants met entry criteria; d) Whether continuation of the study is justified on the basis that 
additional data are needed to accomplish the stated aims on the study; and e) Conditions whereby 
the study might be terminated prematurely.  
 
 
 
 
 

25 
 
VII. 
References Cited 
 
 
1. 
Abu-Elheiga L, Oh W, Kordari P, and Wakil SJ. Acetyl-CoA carboxylase 2 mutant mice are 
protected against obesity and diabetes induced by high-fat/high-carbohydrate diets. Proc Natl Acad Sci U S A 
100: 10207-10212, 2003. 
2. 
Adams SH, Hoppel CL, Lok KH, Zhao L, Wong SW, Minkler PE, Hwang DH, Newman JW, and 
Garvey WT. Plasma acylcarnitine profiles suggest incomplete long-chain fatty acid beta-oxidation and altered 
tricarboxylic acid cycle activity in type 2 diabetic African-American women. J Nutr 139: 1073-1081, 2009. 
3. 
Arkan MC, Hevener AL, Greten FR, Maeda S, Li ZW, Long JM, Wynshaw-Boris A, Poli G, 
Olefsky J, and Karin M. IKK-beta links inflammation to obesity-induced insulin resistance. Nat Med 11: 191-
198, 2005. 
4. 
Bachmann OP, Dahl DB, Brechtel K, Machann J, Haap M, Maier T, Loviscach M, Stumvoll M, 
Claussen CD, Schick F, Haring HU, and Jacob S. Effects of intravenous and dietary lipid challenge on 
intramyocellular lipid content and the relation with insulin sensitivity in humans. Diabetes 50: 2579-2584, 
2001. 
5. 
Befroy DE, Petersen KF, Dufour S, Mason GF, de Graaf RA, Rothman DL, and Shulman GI. 
Impaired mitochondrial substrate oxidation in muscle of insulin-resistant offspring of type 2 diabetic patients. 
Diabetes 56: 1376-1381, 2007. 
6. 
Bennett MJ, Rinaldo P, and Strauss AW. Inborn errors of mitochondrial fatty acid oxidation. Crit Rev 
Clin Lab Sci 37: 1-44., 2000. 
7. 
Blanc S, Normand S, Pachiaudi C, Fortrat JO, Laville M, and Gharib C. Fuel homeostasis during 
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