Extraction and LC-MS analysis of acyl-CoAs has been described in detail previously. (2,3) A novel method using N-tert-butyldimethylsilyl-N-methyltrifluoroacetamide (MTBSTFA), a common derivatization reagent for GC-MS, has been converted to allow for the analysis of the derivatives of citrate, isocitrate, and alpha ketoglutarate on a C18 reversed phase LC-MS. Derivatization with pentafluororbenzyl bromide (PFBBr) coupled to liquid chromatography-electron capture atmospheric pressure chemical ionization/mass spectrometry (LC-ECAPCI/MS) allows for the analysis of succinate, fumarate, and malate via normal phase LC-MS.
Rotenone induces alterations to lipid and glutamine utilization in SH-SY5Y cells Andrew J. Worth, Sankha S. Basu, Nathaniel W. Snyder, Clementina A. Mesaros and Ian A. Blair
Centers for Cancer Pharmacology and Excellence in Environmental Toxicology University of Pennsylvania, Philadelphia, PA
Methods Rotenone is a naturally occurring mitochondrial complex I inhibitor
with a known association to Parkinsonian phenotypes in both human populations and rodent models. Despite this finding, a clear mechanistic link between rotenone exposure and neuronal damage remains to be determined. Here, we report alterations to lipid and glutamine metabolism in SH-SY5Y cells exposed to rotenone. The intracellular concentrations of acetyl-coenzyme A (CoA) were found to be maintained in spite of a significant decrease in glucose derived acetyl-CoA (1). Furthermore, palmitoyl-CoA levels were maintained, whereas, levels of many of the medium chain acyl-Coenzyme A species were significantly reduced. Additionally, using isotopolog analysis it was found that β-oxidation of fatty acids of varying chain lengths helped maintain acetyl-CoA levels. Rotenone also induced increased glutamine utilization for lipogenesis, in part through reductive carboxylation. Taken together, these findings show alterations to lipid and glutamine metabolism play an important compensatory role in response to complex I inhibition by rotenone.
Introduction
Abstract
Epidemiological studies have suggested a positive correlation between rotenone exposure and Parkinson’s Disease (PD) in human populations. Despite a known association between rotenone and dopaminergic neuronal damage, the mechanistic cause of neuronal cell death remains unknown. There is substantial evidence to suggest that mitochondrial dysfunction plays an important role in the development of PD. Therefore, defects in mitochondrial metabolism could be important to the pathogenesis of PD. Here we report compensatory metabolic alterations to lipid and glutamine metabolism in response to complex I inhibition by rotenone in SH-SY5Y cells.
• Carbon labeling from fatty acids and glutamine
NADH
FADFMN
succinateH+ H+H+
H+
H+ H+
α-‐Keto-‐glutarate
Glucose
PyruvateGlutamate
Lactate
Kreb’s cycle(complex I)
Glycolysis
CytC
UQ
O2
Innermitochondrial
membrane
Inter-membranespace
2H2O
Matrix
ComplexIII
ComplexI
ComplexIV
ComplexII
Outer mitochondrial membrane
fumarate
oxaloacetate
malate
Reverse electrontransport
Glutamine
Glutaminase
ATPSynthase
citrate
ATP
O O O
O
OCH3H3CO
H
H
rotenone
SH-SY5Y cells were treated with 2 mM [13C5 15N2]-glutamine and either
100 nM rotenone or vehicle control DMSO for 6 hours. Rotenone induces reductive glutamine metabolism by the action of isocitrate dehydrogenase (IDH). Notably, rotenone treatment results in a significant enrichment in the M+5 isotope of citrate and isocitrate. Additionally, citrate derived from reductive carboxylation undergoes cleavage by ATP citrate-lyase (ACL), yielding an enrichment in the M+3 isotope of fumarate and malate. Rotenone inhibits mitochondrial complex I
C2:0 C4:0 C6:0 C8:0C10
:0C12
:0C14
:0C16
:0C18
:00.00
0.25
0.50
0.75
1.00
1.25
1.50DMSORotenone
**
*
***
**
*** **
Acyl-CoA
Rel
ativ
e pi
com
oles
[13C5]-L-glutamine
glutaminase
[13C5]-L-glutamate
GDH
α-ketoglutarate
succinyl-CoA
succinate
fumarate D-isocitrate
citrate oxaloacetate
L-malate
acetyl-CoA
IDH
oxaloacetate
L-malate fumarate
L-aspartate acetyl-CoA
ACL
unlabeled carbon 13C from reductive metabolism 13C from oxidative metabolism
(cis-aconitate)
C-1
CS MD
FH
SD
SCAS αKGD
MD
AAT
FH
ASS ASL
[13C16]-palmitate [13C16]-palmitoyl-CoA
but-3-enoyl-CoA
D-β-hydroxy- butyryl-CoA
glucose
pyruvate
acetoacetyl-CoA
acetoacetate D-β-hydroxy- butyrate
acetyl-CoA
malonyl-CoA
ACAC
fatty acid biosynthesis
thiolase
(S)-HMG-CoA
cholesterol
HMGS
BHBD
PD
butanoyl-CoA acetyl-CoA (6 mol/ mol [13C16]-palmitate)
+
ACAD
ECAH
3-HCAD
HMGCAL
acetyl-CoA
+
H2N
O
OHNH2
OHO
O
OHNH2
O
HO
O
OHO
O
HOSCoA
O
O
HOOH
O
O
HOOOH
OH
OOHO
SCoA
O
HOOH
O
O OH
HOOH
O
O O
SCoA
O
HOOOH
OOHO
OH
SCoA
O
HOOH
O
O
HOOH
O
O NH2
SCoA
O
SCoA
OOH
OH
O
O
SCoA
OO
OH
OO
OH
OOH
SCoA
O
SCoA
OHO
HO
O
SCoA
O
HOOH
O
O
CoASH
CO2
SCoA
O
or
or
or HOOOH
OOHOaconitase
aconitase
HO O
OH OH
O O HO
HOOOH
OOHO
HOOOH
OOHO
OH
HOOH
O
O O
HOOH
O
O OHSCoA
O
HO
O
Results
• Rotenone decreases medium chain acyl-CoA species
• β-oxidation of fatty acids supports acetyl-CoA production
0
5
10
15
20
**
RotenoneDMSO
[13C
4]- O
ctan
oate
% la
belin
gin
ace
tyl-C
oA M
+2
A
0
5
10
15
20**
[13C
16]-
Palm
itate
% la
belin
gin
ace
tyl-C
oA M
+2
B
• Rotenone induces reductive glutamine metabolism Acetyl CoA
M+0 M+1 M+20
20
40
60
80
100
**
***
RotenoneDMSO
Isotopomer
[13C
515
N2]
- Glu
tam
ine
labe
ling
in A
cety
l CoA
(% o
f poo
l)
Citrate
M+0 M+1 M+2 M+3 M+4 M+5 M+60
20
40
60
80
100
***
** *
***
DMSORotenone
Isotopomer
[13C
515
N2]
- Glu
tam
ine
labe
ling
in C
itrat
e (%
of p
ool)
Isocitrate
M+0 M+1 M+2 M+3 M+4 M+5 M+60
20
40
60
80
100
***
****
*
DMSORotenone
Isotopomer
[13C
515
N2]
- Glu
tam
ine
labe
ling
in Is
ocitr
ate
(% o
f poo
l)
Alpha-Ketoglutarate
M+0 M+1 M+2 M+3 M+4 M+50
20
40
60
80
100
***
***DMSORotenone
Isotopomer
[13C
515
N2]
- Glu
tam
ine
labe
ling
inα
KG
(% o
f poo
l)
Succinyl CoA
M+0 M+1 M+2 M+3 M+40
20
40
60
80
100
***
**
DMSORotenone
Isotopomer
[13C
515
N2]
- Glu
tam
ine
labe
ling
in S
ucci
nyl C
oA (%
of p
ool)
Succinate
M+0 M+1 M+2 M+3 M+40
20
40
60
80
100
***
**
**
DMSORotenone
Isotopomer
[13C
515
N2]
- Glu
tam
ine
labe
ling
in S
ucci
nate
(% o
f poo
l)
Fumarate
M+0 M+1 M+2 M+3 M+40
20
40
60
80
100
***
*
DMSORotenone
Isotopomer
[13C
515
N2]
- Glu
tam
ine
labe
ling
in F
umar
ate
(% o
f poo
l)
Malate
M+0 M+1 M+2 M+3 M+40
20
40
60
80
100
****
DMSORotenone
Isotopomer
[13C
515
N2]
- Glu
tam
ine
labe
ling
in M
alat
e (%
of p
ool)
SH-SY5Y cells were treated with 100 µM [13C4]- octanoate (A) or [13C16]- palmitate (B) and either 100 nM rotenone or vehicle control DMSO for 6 hours.
Liquid chromatography- mass spectrometry o The tBDMS derivatives of citrate, isocitrate and α-ketoglutarate were
separated using a reversed-phase HPLC Xbridge BEH130 C18 column (2.0 mm × 50 mm, pore size 3.5 µm) with 5 mM ammonium acetate in water/ACN/formic acid (40:60:0.1; v/v/v) as solvent A, and ACN/formic acid (100:0.1; v/v) as solvent B at a flow rate of 500 µL/min. Samples were analyzed using an API 4000 triple-quadrupole MS equipped with an electrospray source operating in positive ionization mode.
o Succinate, fumarate, and malate were separated using a Chiralpak AD-H column (250 × 4.6 mm i.d., 5 µm; Daicel Chemical Industries, Ltd., Tokyo, Japan) at a flow rate of 1 mL/min. Solvent A was hexanes and solvent B was IPA/methanol (1:1; v/v). MS analysis was conducted on a Thermo Finnigan TSQ Quantum Ultra AM mass spectrometer equipped with an atmospheric pressure chemical ionization (APCI) source in the electron capture (EC) negative ion mode.
Conclusion and future directions
• Rotenone induces compensatory metabolic alterations to maintain acetyl- and palmitoyl-CoA levels while medium chain acyl-CoAs decrease
• Oxidation of multiple fatty acids for acetyl-CoA production is upregulated in response to complex I inhibition by rotenone
• Glutamine supports both short and long acyl-CoA levels in part through reductive carboxylation in rotenone treated cells
• Other metabolic substrates may play an important compensatory roles
• Primary rat cortical neurons will provide a better model for studying the pathogenesis of PD
• Neuronal damage caused by rotenone may be due in part to previously unidentified metabolic abnormalities
SH-SY5Y cells were treated with 2 mM [13C5 15N2]-glutamine and either
100 nM rotenone or vehicle control DMSO for 6 hours. Isotopic enrichment in palmitoyl-CoA indicates an increase in glutaminolysis sopports lipogenesis in response to rotenone.
M+2 M+4 M+60
2
4
6DMSORotenone
***
***
Isotopomer
[13C
515
N 2]-
Glu
tam
ine
%la
belin
g in
Pal
mito
yl C
oA
References
(1) Basu SS.; Blair IA. Rotenone-mediated changes in intracellular coenzyme A thioester levels: implications for mitochondrial dysfunction. Chemical Research in Toxicology. (2011) (2) Snyder NW.; Basu SS.; Zhou Z.; Worth AJ.; Blair IA. Stable isotope dilution liquid chromatography-mass spectrometry analysis of cellular and tissue medium- and long-chain acyl-coenzyme A thioesters. Rapid Communications in Mass Spectrometry. (In press) (3) Basu SS.; Blair IA. SILEC: a protocol for generating and using isotopically labeled coenzyme A mass spectrometry standards. Nature Protocols. (2011) Supported by NIH grants P30ES013508, T32ES019851 & P42 ES023720
• Reductive glutamine metabolism contributes to lipogenesis