Discovery of FAHFA-Containing Triacylglycerols and Their Metabolic Regulation

Article abstract of DOI:10.1021/jacs.9b00045

FAHFAs are a class of bioactive lipids, which show great promise for treating diabetes and inflammatory diseases. Deciphering the metabolic pathways that regulate endogenous FAHFA levels is critical for developing diagnostic and therapeutic strategies. However, it remains unclear how FAHFAs are metabolized in cells or tissues. Here, we investigate whether FAHFAs can be incorporated into other lipid classes and identify a novel class of endogenous lipids, FAHFA-containing triacylglycerols (FAHFA-TGs), which contain a FAHFA group esterified to the glycerol backbone. Isotope-labeled FAHFAs are incorporated into FAHFA-TGs when added to differentiated adipocytes, which implies the existence of enzymes and metabolic pathways capable of synthesizing these lipids. Induction of lipolysis (i.e., triacylglycerol hydrolysis) in adipocytes is associated with marked increases in nonesterified FAHFA levels, demonstrating that FAHFA-TGs breakdown is a regulator of cellular FAHFA levels. To quantify FAHFA levels in FAHFA-TGs and determine their regioisomeric distributions, we developed a mild alkaline hydrolysis method that liberates FAHFAs from triacylglycerols for easier detection. FAHFA-TG concentrations are greater than 100-fold than that of nonesterified FAHFAs, indicating that FAHFA-TGs are a major reservoir of FAHFAs in cells and tissues. The discovery of FAHFA-TGs reveals a new branch of TG and FAHFA metabolism with potential roles in metabolic health and regulation of inflammation.

Full text of DOI:10.1021/jacs.9b00045

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Article
Discovery of FAHFA-containing
Triacylglycerols and Their Metabolic Regulation
Dan Tan, Meric Erikci Ertunc, Srihari Konduri, Justin Zhang, Antonio Michel
Pinto, Qian Chu, Barbara B. Kahn, Dionicio Siegel, and Alan Saghatelian
J. Am. Chem. Soc., Just Accepted Manuscript • Publication Date (Web): 06 May 2019
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Discovery of FAHFA-containing Triacylglycerols and Their
Metabolic Regulation
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1,‡
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Dan Tan, Meric Erikci Ertunc, Srihari Konduri, Justin Zhang, Antonio M. Pinto, Qian
1
3
2,*
1,*
Chu, Barbara B. Kahn, Dionicio Siegel, Alan Saghatelian
1
Clayton Foundation Laboratories for Peptide Biology, Salk Institute for Biological Studies, 10010 North
Torrey Pines Road, La Jolla, CA 92037-1002, United States
2
Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California-San Diego, 9500 Gilman
Drive, La Jolla, California 92093-0934, United States
3
Division of Endocrinology, Diabetes and Metabolism, Department of Medicine, Beth Israel Deaconess
Medical Center, Harvard Medical School, Boston, Massachusetts 02215, United States
ABSTRACT: FAHFAs are a class of bioactive lipids
which show great promise for treating diabetes and
inflammatory diseases. Deciphering the metabolic
pathways that regulate endogenous FAHFA levels is
critical for developing diagnostic and therapeutic
strategies. However, it remains unclear how FAHFAs
are metabolized in cells or tissues. Here, we
investigate whether FAHFAs can be incorporated into
other lipid classes and identify a novel class of
endogenous
lipids,
FAHFA-containing
triacylglycerols (FAHFA-TGs), which contain
a
FAHFA group esterified to the glycerol backbone.
Isotope-labeled FAHFAs are incorporated into
FAHFA-TGs when added to differentiated adipocytes
which implies the existence of enzymes and
metabolic pathways capable of synthesizing these
lipids. Induction of lipolysis (i.e., triacylglycerol
hydrolysis) in adipocytes is associated with marked
increases
in
nonesterified
FAHFA
levels
demonstrating that FAHFA-TGs breakdown is a
regulator of cellular FAHFA levels. To quantify
FAHFA levels in FAHFA-TGs and determine their
regioisomeric distributions, we developed a mild
alkaline hydrolysis method that liberates FAHFAs
from triacylglycerols for easier detection. FAHFA-TG
concentrations are greater than 100-fold than that of
nonesterified FAHFAs, indicating that FAHFA-TGs
are a major reservoir of FAHFAs in cells and tissues.
The discovery of FAHFA-TGs reveals a new branch of
TG and FAHFA metabolism with potential roles in
metabolic health and regulation of inflammation.
1
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INTRODUCTION
being metabolized through their carboxylates in
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FAHFAs (Fatty Acid esters of Hydroxy Fatty Acids) are a
recently discovered class of lipids that are highly elevated
in the adipose tissue of transgenic mice overexpressing
glucose transporter 4 (GLUT4) selectively in their adipose
tissue (AG4OX mice) . FAHFAs are grouped into families
based on the composition of their acyl chains, and each
family contains multiple regioisomers that differ in
position of the ester bond that links the acyl chains . Since
their initial discovery, many FAHFAs have been identified
in plants and animals
Scheme 1. Syntheses of FAHFA-based standards using
a
iterative, modified Steglich esterification reactions
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. Oral administration of 5- and 9-
PAHSA (palmitic acid ester of hydroxy stearic acid), two of
the most up-regulated FAHFA species in AG4OX mice,
reduces blood glucose levels and improves insulin
2, 8
sensitivity in mice . Analysis of serum and adipose tissue
PAHSA levels in people that differ in their insulin
sensitivity revealed that multiple PAHSA regioisomers
2
correlate highly with insulin sensitivity , with lower
FAHFA levels observed in insulin resistant individuals.
FAHFAs also exhibit anti-inflammatory effects. For
example, 9-PAHSA and 13-DHAHLA (docosahexaenoic
acid ester of 13-hydroxy linoleic acid, a polyunsaturated
FAHFA), inhibit LPS-induced proinflammatory cytokine
production in macrophages and dendritic cells
Furthermore, administration of 9-PAHSA to mice on a
high-fat diet results in lower levels of inflammatory
macrophages in their adipose tissue , and 9-PAHSA
treatment in a mouse model of colitis reduces the clinical
and pathological disease severity , highlighting a role for
FAHFAs as anti-inflammatory lipids.
2,
4
.
2
9
These findings open up new opportunities of treatment
for diabetes and inflammatory diseases, but a deeper
understanding of the metabolic pathways regulating
endogenous FAHFA levels are needed to proceed with
novel diagnostic and therapeutic strategies. Biochemical
studies have identified three hydrolases—CEL, AIG1 and
ADTRP—as putative FAHFA hydrolases that cleave the
aDMAP: 4-dimethylaminopyridine, EDC: 1-ethyl-3-(3-
dimethylaminopropyl)carbodiimide, PAHSA: palmitic acid
ester of hydroxy stearic acid, POHSA: palmitoleic acid ester
of hydroxy stearic acid, OAHSA: oleic acid ester of hydroxy
stearic acid.
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FAHFA ester , but the role of these enzymes in FAHFA
regulation in vivo must still be verified. Recent work has
linked antioxidant pathways regulated by nuclear factor
(
(
erythroid-derived 2)-like 2 (Nrf2) and peroxiredoxin-6
PRDX6) to changes in endogenous FAHFA levels , though
5
mammals. FAHFA metabolism appears to be specific, as we
did not observe any FAHFAs incorporated into CEs. TGs
are depots for fatty acid storage during nutritional rich
times and are hydrolyzed to release fatty acids under
fasting conditions. The regulated balance of fatty acid
esterification and TG hydrolysis provides a buffer system
the exact biochemical mechanism linking these genes to
FAHFAs remains enigmatic.
In addition to the synthesis and degradation of FAHFA
itself, the presence of a carboxylic acid in FAHFAs raises
the possibility that these lipids can be incorporated into
other lipid classes, where FAHFA replaces a fatty acid in
these structures. We set out to address this question by
synthesizing FAHFA-containing lipids, developing assays
to detect these lipids, and testing whether these lipids are
present in cells and tissues. We focused on FAHFA-
containing triacylglycerols (FAHFA-TGs) and cholesteryl
esters (FAHFA-CEs) because TGs are major storage forms
15
to maintain the physiological fatty acid levels . Our data
suggests that FAHFA-TGs may serve a similar role in
regulating FAHFA levels, and reveals a pathway with
potential relevance in the metabolic and inflammatory
roles of FAHFAs.
12-13
of fatty acids in mammalian tissues and TGs and CEs are
transported throughout the body in lipoprotein particles .
RESULTS AND DISCUSSION
14
Syntheses of FAHFA-containing triacylglycerols and
cholesterol esters
By developing mass spectrometry methods, we detected
FAHFAs in TGs, providing the only example of FAHFAs
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To test our hypothesis, we synthesized a variety of
monoacylglycerols, but such species were not detected.
Despite our best efforts, we did not detect any FAHFA-CEs
in either adipose tissue or liver where CEs are chiefly
present, suggesting that FAHFA-CEs are not present or
below the detection limit of our methodology. The
detection of FAHFA-TGs suggests that FAHFAs can be
selectively metabolized into other lipid classes.
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FAHFA-TGs and a FAHFA-CE. The syntheses of the
FAHFA-TGs were achieved through iterative Steglich
1
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esterification reactions (Scheme 1). The use of 1-ethyl-3-
(3-dimethylaminopropyl)carbodiimide (EDC) over N,N’-
dicyclohexylcarbodiimide (DCC) greatly simplified
purification. The expected reactivity of glycerol allowed
sequential esterification of the primary alcohols and lastly
coupling of the third acid onto the secondary alcohol.
Similar reaction with a single esterification between
cholesterol and FAHFA provide the FAHFA-CE standard
Since this is the first report of a FAHFA-TG, we felt it
necessary to validate the existence of FAHFA-TGs using an
orthogonal method. We used a biochemical approach to
isolate the adipose neutral lipid fraction, which should
include FAHFA-TGs, from FAHFAs by solid phase
extraction. We then hydrolyzed this neutral lipid fraction
under mild basic conditions that preferably cleave the ester
bonds on the glycerol backbone rather than the ester
within the FAHFA moiety. The chemical synthesis of
FAHFAs revealed that the FAHFA backbone ester is
relatively resistant to hydrolysis, and this knowledge
enabled us to use selective hydrolysis to liberate FAHFAs
under conditions that should not break down FAHFAs.
Using this approach, we detected FAHFAs in the neutral
lipid fraction after hydrolysis, but not in unhydrolyzed
samples, confirming the existence of FAHFA-TGs in
adipose tissue (Figure 1C).
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(
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Supporting Information). The use of glycerol-D or 9-
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PAHSA assembled using palmitic acid (U- C16) provided
heavy standards using the same synthetic strategy.
Discovery of endogenous FAHFA-TGs
In order to develop workflow for analysis of potential
endogenous FAHFA-TGs and FAHFA-CEs, we initially
examined the fragmentation rules of synthesized FAHFA-
TGs and FAHFA-CEs with high resolution mass
spectrometry. A representative MS/MS spectrum of one
FAHFA-TG species (16:0/PAHSA/18:1-TG) (Figure 1A) in
positive ionization mode has an ion for the ammonium
adduct of the intact FAHFA-TG (m/z 1133.0421) and ions
corresponding to the neutral loss of each fatty acyl chain.
The fragmentation pattern we observed for FAHFA-TGs is
1
7-18
similar to that of triacylglycerols except that the FAHFA
moiety provides two product ions, one resulting from the
entire FAHFA and a second from the cleavage of the
FAHFA ester bond (Figure 1A). For example, the fragment
ion at m/z 577.5189 is generated from the loss of the
PAHSA moiety, while the ion at m/z 603.5344 corresponds
to the simultaneous loss of two palmitic acid groups, one
from the acyl chain esterified to the glycerol backbone and
the other from the PAHSA moiety (Figure 1A). FAHFA-CEs
also form ammonium adducts in positive ionization mode.
As exemplified by the MS/MS spectrum of OAHSA-CE
(
Figure S1), the most intense fragment corresponds to the
1
8-19
characteristic cholestene cation at m/z 369.3505 . Also
observed are acylium ions from hydroxy stearic acid (m/z
2
5
83.2622), oleic acid (m/z 265.2508), and OAHSA (m/z
47.5053). The ion at m/z 565.5176 corresponds to the
water adduct of the acylium ion of OAHSA.
We next analyzed the lipid extract from adipose tissue of
WT mouse, where highest levels of nonesterified FAHFAs
2
were detected . Using the combination of accurate masses,
retention time, and fragmentation behavior, various
endogenous FAHFA-TGs were detected, which differ in the
FAHFA moiety and the two other fatty acid groups
attached to the glycerol backbone. For example, the
MS/MS spectrum of the ion at m/z 1133.0417 from the
adipose tissue is in agreement with the spectrum of the
authentic standard (Figure 1B). The additional fragment
ion at m/z 551.5023 is probably derived from the loss of the
OAHSA moiety from 16:0/OAHSA/16:0-TG which shares
the same mass with 16:0/PAHSA/18:1-TG. In addition, we
examined FAHFA-TGs with two FAHFA moieties, FAHFA-
containing diacylglycerols, and FAHFA-containing
Figure 1. Discovery of FAHFA-TGs from adipose tissue of
WT mice. High energy collisional dissociation (HCD)
spectrum of m/z 1133.0417 corresponding to the ammonium
adduct for 16:0/PAHSA/18:1-TG from (A) synthetic standard
and (B) lipid extract of mouse adipose tissue. The shorthand
notation of FAHFA-TGs is described in the method. (C)
FAHFAs were detected after hydrolyzing a pool of
triacylglycerols from adipose tissue under basic conditions
that can cleave the ester bond to release the FAHFA from
the FAHFA-TG. Shown are extracted ion chromatograms of
three most highly upregulated FAHFA members in AG4OX
mice—PAHSAs, POHSAs, and OAHSAs. The numbers
above the peaks denote different regioisomers.
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FAHFA-TGs are synthesized from FAHFAs in
differentiated adipocytes
To determine whether FAHFA-TGs can be synthesized
from FAHFAs in cells, we incubated differentiated 3T3-L1
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adipocytes with a C isotope labeled FAHFA and measured
its incorporation into triacylglycerols. The isotope labeled
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C-PAHSA provides a unique signal in mass spectra which
allows us to measure its incorporation with minimal
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background noise. We detected various C-PAHSA-
containing triacylglycerol species in lipid extracts from
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cells treated with C-PAHSA but not from cells treated
with vehicle (Figure 2, Figure S2). Furthermore, we
observed an increase in FAHFA-TG levels between 5 and
4 hours (Figure S3), suggesting that once produced, these
lipids are fairly stable, providing further support that
FAHFA-TGs might be a storage form of FAHFAs.
2
Figure 3. FAHFA levels are regulated by lipolysis. FAHFA
levels were increased when lipolysis was stimulated by FSK
or IBMX in 3T3-L1 adipocytes. The ATGL inhibitor
Atglistatin and the HSL-MAGL dual inhibitor CAY10499
reduced the effect of IBMX on FAHFA levels. N = 3, *p < 0.05
versus vehicle, #p < 0.05 versus IBMX, data are means ± SEM.
The acyl CoA:diacylglycerol acyltransferases, DGAT1 and
20
DGAT2, are two key enzymes of triacylglycerol synthesis .
To examine whether they are responsible for FAHFA-TGs
13
synthesis, we co-treated 3T3-L1 adipocytes with C-PAHSA
and DGAT1 or/and DGAT2 inhibitor(s). As shown in Figure
2
, inhibiting DGAT1 or DGAT2 alone did not affect the
13
incorporation of
C-PAHSA into triacylglycerols
nM) to treat the cells because higher doses led to cell death
during extended periods (24 hours) in our experiment.
significantly, whereas simultaneous inhibition of both
enzymes significantly reduced the incorporation,
suggesting that DGAT1 and DGAT2 may compensate for
each other to catalyze FAHFA-TG synthesis, requiring the
loss of both activities to observe a change in FAHFA-TG
production. We observed that the synthesis was not
completely abolished by DGAT1 and DGAT2 inhibition
under our experimental conditions. We suspect that the
reason for this is our use of low doses of inhibitors (200
To figure out whether higher doses could have a stronger
impact on FAHFA-TG levels, we treated the cells with
higher dosage of inhibitors (5 µM) but shortened the
incubation time (3 hours) and avoided killing the cells.
Despite the higher doses of inhibitors, FAHFA-TG
synthesis was still observed (Figure S4). As a comparison,
we examined the effect of DGAT1 and DGAT2 inhibitors
under the same conditions on TG synthesis by co-treating
1
3
the cells with
incorporation of
C16-palmitic acid and measuring the
1
3
C
16-palmitic acid into TGs. TG
production was inhibited in a dose-dependent manner
with 2-8 fold decrease with 5 µM of inhibitors (Figure S5).
13
To illustrate the difference, if we compare C16-PA_18:1_18:1
13
to C
4
-PAHSA_18:1_18:1, we observe a ~4-fold reduction in
the TG production but only a 1.7-fold reduction in FAHFA-
TG levels (Figures S4 and S5). Though more work is
necessary to understand the reason that DGAT1/2
inhibition is not completely blocking FAHFA-TG levels,
one possible explanation is the existence of additional
enzymes that catalyze FAHFA-TG synthesis.
Lipolysis increases nonesterified FAHFA levels
Figure 2. FAHFA-TGs can be synthesized in cells. 13
4
C -
In mammalian cells, lipolysis mediates the breakdown of
triacylglycerols to release nonesterified fatty acids, which
can be used as fuels to meet energy needs or serve as
PAHSA-containing triacylglycerols were synthesized after
incubating differentiated 3T3-L1 adipocytes with 20 µM
1
3
C
4
-PAHSA for 24 hours. The synthesis was inhibited by
1
5
signaling molecules . Adipose triglyceride lipase (ATGL),
hormone-sensitive lipase (HSL), and monoacylglycerol
lipase (MAGL) are the major enzymes responsible for the
three-step triacylglycerol hydrolysis cascade in adipose
co-treating the cells with both DGAT1 inhibitor (1i) and
1
3
DGAT2 inhibitor (2i). Shown is
4
C -PAHSA_16:1_18:1-TG
level. N = 3, *p < 0.05, data are means ± SEM. The bottom
panel shows the extracted ion chromatograms of two
1
3
15
transitions for
transition corresponding to the loss of
ammonia; Blue curve, transition corresponding to the loss
C
4
-PAHSA_16:1_18:1-TG. Red curve,
tissue . To test whether FAHFA levels are regulated by this
1
3
C
4
-PA plus
lipolysis pathway, we stimulated lipolysis in 3T3-L1
adipocytes with 3-isobutyl-1-methylxanthine (IBMX) or
13
4
of C -PAHSA plus ammonia.
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To circumvent this issue and develop a robust method
forskolin (FSK). Under these conditions FAHFA levels
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increased 1.5-5-fold in adipocytes treated with either of the
two lipolysis inducers. Co-culturing the cells with an ATGL
inhibitor Atglistatin or an HSL-MAGL dual inhibitor
CAY10499 reduced the FAHFA levels augmented by IBMX
(Figure 3). We also measured FAHFA levels in conditioned
media from these cells and observed similar trends for
12/13-POSHA (Figure S6). The levels of other FAHFA
isomers in the medium were too low to be quantified. Our
results in 3T3-L1 adipocytes is consistent with the
observation that nonesterified FAHFA levels are
significantly increased in PG-WAT and SQ-WAT of fasted
for quantifying FAHFA-TGs, we took advantage of the
stability of the internal FAHFA esters to hydrolysis and
utilized the aforementioned selective alkaline hydrolysis
method to quantify FAHFA-TGs (Figure 4A). We loaded
crude lipid extract from adipose tissue onto a silica
column, and washed it with 95:5 hexane:ethyl acetate.
FAHFAs were still bound to the column due to their high
polarity. Neutral lipids were eluted and then hydrolyzed
with lithium hydroxide to avoid the hydrolysis of the ester
1
3
bond within FAHFA. We added a C16-PAHSA-containing
triacylglycerol during lipid extraction, assuming that the
endogenous FAHFA-TGs have similar hydrolysis rate as
the internal standard to quantify the FAHFAs in FAHFA-
TGs. Considering that the position of FAHFA may affect
the hydrolysis rate (i.e. sn1 vs sn2), we performed the
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mice (Figure S7). These experiments indicate that FAHFA-
TG hydrolysis can regulate cellular and extracellular
FAHFA levels.
1
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experiment twice with two standards separately,
16
C -
FAHFAs are stored as triacylglycerols in adipose tissue
1
3
PAHSA/16:0/16:0-TG (sn1) or 16:0/ C16-PAHSA/16:0-TG
sn2). Using this method, we quantified the level of
We next sought to characterize the concentrations of
FAHFA-TGs in tissues to determine their potential
contribution to overall FAHFA concentrations, which
required us to develop a robust method for quantifying
FAHFA-TG concentrations. As described above, we
(
FAHFAs in the triacylglycerol fraction from SQ-WAT of
WT mice. Strikingly, the level of FAHFA-TGs, which are
represented by the level of esterified FAHFAs, is more than
1
00-fold greater that of nonesterified FAHFAs (Figure 4B),
13
utilized an MRM-based method to quantify C-PAHSA-
confirming that triacylglycerols are a storage form of
FAHFAs in adipose tissue. In addition, no obvious
difference was observed between sn1 and sn2 standards
containing triacylglycerols in adipocytes. We treated the
cells with heavy labeled PAHSA, which provides unique
signals of FAHFA-TGs in mass spectra. However, when we
applied the same LC-MS method to measure endogenous
FAHFA-TGs in mouse tissues, we observed multiple
chromatographic peaks which are not fully resolved. These
peaks overlapped with internal standards with similar
retention times, masses, and MS/MS spectra. However, the
relative intensity of the fragment ions in the MS/MS
spectra differed, suggesting a more complex mixture in
vivo.
(
Figure 4B).
By studying the various synthetic FAHFA-TGs, we
reasoned that the complexity in the LC-MS
chromatograms is at least partially derived from the
presence of many isomers which result from the numerous
possible combinations of the four fatty acyl chains in a
FAHFA-TG molecule. For example, PAHSA/18:1/18:1,
POHSA/18:1/18:0, and OAHSA/16:0/18:1 triacylglycerols
share the same exact mass, a similar set of fragments, and
close retention time on C18 reverse phase. The mixture of
the three isomers generated distorted mass traces with
different transition ratios compared with each individual
standard (Figure S8). This does not even take into account
the possibility of different FAHFA regioisomers (e.g.
FAHFA-TGs with 9-PAHSA vs 5-PAHSA). We also found
that the relative abundance of transitions depends on the
position of FAHFA on the glycerol backbone. The loss of
FAHFA at sn1 position is more favorable than FAHFA at
sn2 position (Figure S9). Therefore, the intensity change of
a transition cannot reflect the abundance change of
FAHFA-TG as this can also be caused by the compositional
change of positional isomers.
Figure 4. Quantification of FAHFA-TGs in adipose tissue of
WT mice. (A) Workflow using a selective alkaline hydrolysis
method. (B) Quantification of nonesterified and TG-
esterified FAHFAs in SQ-WAT of WT mice. N = 5, data are
means ± SEM.
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Regulation of FAHFA-TGs under high fat diet feeding
levels are markedly higher than FAHFA levels in adipose
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tissue, supporting the idea that FAHFA-TGs may serve as a
depot for FAHFAs. Whether FAHFA-TGs themselves have
additional roles in cell function remains to be elucidated.
Furthermore, we observed that FAHFA-TG levels are
reduced when DGAT1 and DGAT2 are inhibited, and
FAHFA levels are increased when lipolysis is induced.
These data suggest that the homeostasis between FAHFAs
and FAHFA-TGs are enzymatically regulated. Even though
there are similarities between the regulation of
FAHFA/FAHFA-TG metabolism and fatty acid/regular TG
metabolism, higher nonesterified FAHFAs have
distinguishing insulin sensitizing and anti-inflammatory
functions whereas increases in nonesterified fatty acids can
be detrimental to cellular function. The differential effects
of DGAT1/2 inhibitors on the synthesis of FAHFA-TGs and
regular TGs also distinguish these two pools of TGs. Future
experiments may investigate whether additional enzymes
are involved in regulating the balance of FAHFA-TG
synthesis and breakdown, and how they respond to
different physiological and pathological metabolic status
to eventually control the net effect of FAHFAs.
We next examined the levels of FAHFA-TGs in mice fed
with high-fat-diet (HFD). We previously reported that
nonesterified PAHSA levels increase in PG-WAT while
2
they decrease in SQ-WAT . By contrast, placing mice on a
HFD results in increase in 10-PAHSA and 10-OAHSA or no
change in FAHFA-TG levels in both WAT depots. The data
support a model of FAHFA-TGs serving as a storage form
of FAHFAs under nutrient rich conditions. The relative
abundance of FAHFA regioisomers in FAHFA-TGs is
different from what we previously reported for
nonesterified FAHFAs in tissues. For example, 9-PAHSA is
the most abundant nonesterified PAHSA isomer detected
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in tissues . However, in the current study we detect 10-
PAHSA and 10-OAHSA at the highest levels in FAHFA-TGs
from mice on a HFD (Figure 5). We suspected that this is
likely due to higher 10-FAHFA levels in the HFD we are
using now and diet can drive increases in FAHFA-TGs. In
subsequent work, it will be of interest to investigate
whether FAHFAs and FAHFA-TGs are co-regulated or
differentially regulated in different metabolic states.
CONCLUSIONS
METHODS
In this work, we report the discovery of a structurally
novel class of triacylglycerols which contain a FAHFA in
place of one of the three fatty acids. The discovery and
characterization of FAHFA-TGs indicates the existence of
an uncharacterized branch of endogenous lipid
metabolism. This novel pathway is potentially involved in
metabolic and inflammatory diseases because of the
biological activities of FAHFAs. We found FAHFA-TG
Materials
Solvents for LC-MS analysis were purchased from
1
3
Honeywell Burdick & Jackson.
4
C -PAHSA, d31-PAHSA,
Atglistatin, and CAY10499 were purchased from Cayman
Chemical. Inhibitors of DGAT1 (PF 04620110) and DGAT2
(PF 06424439) were purchased from Tocris Bioscience.
Animals
HFD fed C57BL/6 male mice were obtained from the
Jackson Laboratory (Bar Harbor, ME; stock number
380050), and maintained on high fat diet (Research Diets,
D12492) until the age of 36 weeks when they were sacrificed
for tissue collection. Ad lib fed and fasted mice in C57BL/6
genetic background were bred in-house and were
maintained on chow diet (RD, PicoLab 5053 Lab Diet). 10
week-old male mice were ad lib fed or fasted for 18 hours
before they were sacrificed for tissue collection. All mice
were maintained on a 12 h light and dark cycle. All
procedures involving mice were approved by the
Institutional Animal Care and Use Committee (IACUC) of
the Salk Institute for Biological Studies.
Syntheses of FAHFA-TGs and 12-OAHSA-CE
Syntheses of FAHFA-TGs and 12-OAHSA-CE are detailed
in the Supporting Information along with the spectral
characterization data.
Lipid Nomenclature
Synthetic FAHFA-TGs are annotated as the FAHFA
moiety and the two regular fatty acyl moieties which are
represented by the number of carbon atoms:number of
double bonds in a sequence reflecting their sn positions
linked to the glycerol. The three moieties are separated by
a slash (/). Since the sn position of fatty acids cannot be
determined by the mass spectrometry analysis, an
underscore symbol (_) was used to annotate endogenous
Figure 5. Measurement of TG-esterified FAHFAs reveals
diet-induced changes in FAHFA-TG levels in WAT depots.
(
A) TG-esterified FAHFA levels in PG-WAT and SQ-WAT of
WT mice on chow (black bars) or HFD (blue bars). N= 3-4,
p < 0.05, data are means ± SEM. (B) Extracted ion
*
chromatograms of TG-esterified 9-, 10-, 11-, and 12/13-PAHSAs
in PG-WAT from chow (black) or HFD (blue) fed mice.
21
FAHFA-TGs .
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following ion source parameters: spray voltage, 3.4 kV;
Lipid Extraction
1
2
sheath gas, 15 L/min; aux gas, 7 L/min; sweep gas, 0 L/min;
ion transfer tube temperature, 329 °C; vaporizer
temperature, 297 °C. Q1 and Q3 resolution were both set to
Lipids were extracted based on the Bligh-Dyer method
22-23
unless otherwise stated . In brief, for extraction from
cells, the cell pellet was re-suspended in PBS, followed by
addition of methanol and chloroform to make 1:1:2
PBS:methanol:chloroform solvent (v/v/v). The mixture
was shaken vigorously for 30 s, vortexed for 15 s. For
extraction from tissues, the sample was Dounce
homogenized in 1:1:2 PBS:methanol:chloroform solvent on
ice. An appropriate amount of internal standards were
added to chloroform prior to extraction as indicated in
each experiment. The resulted homogenate was
centrifuged at 2200 g for 6 min at 4 °C. The bottom organic
layer was collected and dried under a gentle stream of
nitrogen.
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1
3
13
0.7.
C
16-PAHSA/16:0/16:0-TG,
C
16-PAHSA/16:1/16:0-TG,
1
3
and various
C
4
-PAHSA-containing TG species were
monitored. Their ammonium adducts were selected as the
precursor ions. Simultaneous neutral loss of a fatty acyl
1
3
13
13
13
4 4
group ( C16-PAHSA/ C -PAHSA, C16-PA/ C -PA, or each
of the other two acyls) plus ammonia were selected as the
product ions. Collision energy and RF lens voltage were
optimized using the two standards. The transition
0
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9
0
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9
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9
0
1
3
13
corresponding to the loss of
4
C16-PAHSA or C -PAHSA
1
3
was used for quantification. Absolute quantification of C
PAHSA-containing TGs was performed using
PAHSA/16:1/16:0-TG except that C -PAHSA_16:0_16:0-TG
4
4
-
-
1
3
C
16
1
3
3
T3-L1 Cell Differentiation
1
3
was quantified using C16-PAHSA/16:0/16:0-TG. The results
were normalized to protein concentration.
3
T3-L1 differentiation was performed as described
24
previously . 3T3-L1 preadipocytes were maintained in
DMEM with 10% bovine calf serum (BCS) at 37 °C in a
3
T3-L1 Cell Lipolysis
Lipolysis was stimulated by incubating 3T3-L1 adipocytes
for 2 hrs with 1 mM IBMX or 20 µM FSK in phenol-free
DMEM containing 0.2% fatty acid free BSA. For inhibition
experiments, cells were pre-treated with 10 µM Atglistatin
or 0.1 µM CAY10499 for 2 hrs. Cells were washed twice with
ice-cold PBS, scraped into PBS, and pelleted by
centrifugation at 2000 g for 10 min at 4 °C. Medium was
collected and centrifuged at 5000 rpm for 10 min at 4 °C.
2
humidified atmosphere of 10% CO in air. Two days after
the cells reach confluency (day 0), differentiation was
induced by culturing the cells in DMEM containing 10%
fetal bovine serum (FBS), 5 µg/ml insulin, 5 µM
dexamethasone, and 0.1 mM IBMX. On day 2, the media
were changed to DMEM with 10% FBS and 5 µg/ml insulin.
Cells were fed every other day with the insulin-
supplemented medium until days 8-10, when they are
ready for treatment.
1
3
Lipids were extracted from cells and media with
PAHSA spiked in as the internal standard.
4
C -
13
Incorporation of C
4
-PAHSA, DGAT inhibition and
13
Quantification of nonesterified FAHFAs and TG-
esterified FAHFAs
quantification
triacylglycerols
of
4
C -PAHSA-containing
Frozen tissues of similar size were weighed and
Differentiated 3T3-L1 adipocytes were fed with 20 µM
13
13C
subjected to lipid extraction.
4
C -PAHSA was added for
4
-PAHSA or vehicle in phenol-free DMEM with 10% FBS
quantification of nonesterified FAHFAs in the lipid extract.
for 24 hrs. For inhibition experiments, the cells were
pretreated with 200 nM PF-04620110 (DGAT1 inhibitor)
or/and PF-06424439 (DGAT2 inhibitor) for 30 minutes.
Cells were washed twice with ice-cold PBS, harvested by
scraping, and re-suspended in PBS. To determine protein
concentration, a small aliquot of cell suspension was saved
for Bradford assay. Lipid extraction was performed using
1
3
13
C
16-PAHSA/16:0/16:0-TG (sn1) or 16:0/ C16-PAHSA/16:0-
TG (sn2) was added for quantification of FAHFA-TGs. The
amount of internal standards were based on the tissue
weight. Two rounds of solid-phase extraction (SPE) were
8
performed as described previously . Briefly, the silica
cartridge (Strata SI-1, 500 mg silica, 3 mL, Phenomenex, 8B-
S012-HBJ-T) was conditioned with 6 ml of hexane. Crude
lipid extract normalized to tissue weight was loaded onto
the column. Neutral lipids were eluted with 6 ml of 95:5
hexane:ethyl acetate. FAHFAs were subsequently eluted
with 4 ml of ethyl acetate. This nonesterified FAHFA
fraction was dried, reconstituted in 60 µl of methanol, and
1
3
the protocol described above with 0.1 pmol of
16
C -
1
3
PAHSA/16:0/16:0-TG and 0.15 pmol C16-PAHSA/16:1/16:0-
TG added as internal standards. Lipid extract was
reconstituted in 100 µl of methanol: chloroform solvent
(1:2, v/v), and 10 µl was injected for LC-MS analysis on an
Acquity UPLC system interfaced with a TSQ Quantiva
mass spectrometer (Thermo Fisher Scientific). Lipids were
separated on a Kinetex (Phenomenex) C18 column (2.6 µm,
1
0 µl was subjected to LC-MS analysis. The neutral lipid
fraction was reconstituted in ethanol and treated with 20
µl of 1M LiOH at room temperature for 24 hrs with
constant shaking. At the end of the reaction, 20 µl of 2 M
HCl was added to neutralize the base. Lipids was extracted
from the solution with 1 ml of PBS, 1 ml of ethanol, and 2
1
50 x 2.1 mm) over a 68-min gradient as follows: 0–70% B
in 5ꢀmin, 70–85% B in 45ꢀmin, 85–100% B in 0.1ꢀmin, held at
00% B for 4.9ꢀmin, 100-0% B in 0.1 min, held at 0% B for
2.9 min. Solvent A was 64:40 methanol:water with 0.1%
formic acid and 5 mM ammonium formate. Solvent B was
0:10 isopropanol:methanol with 0.1% formic acid and 5
mM ammonium formate. Flow rate was set to 0.1 ml/min
at the beginning and ramped to 0.25 ml/min in 1 min.
Multiple Reaction Monitoring (MRM) experiment was
carried out in the negative ionization mode with the
1
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1
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4
ml of chloroform with C -PAHSA or d31-PAHSA. A second
SPE was performed to remove the remaining neutral lipids
as they interfere with the FAHFA measurement. The
resulting FAHFA fraction was collected for LC-MS analysis.
Hydrolysis rate was calculated by dividing the amount of
9
13
13
C
16-PAHSA that is released from the
C16-PAHSA-
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containing TG standard by the amount of the C16-PAHSA-
containing TG standard. TG-esterified FAHFAs are then
quantified by dividing the amount of FAHFA after
hydrolysis by the hydrolysis rate.
endogenous mammalian lipids with anti-diabetic and anti-
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inflammatory effects. Cell 2014, 159 (2), 318-32.
3
.
Carvalho, E.; Kotani, K.; Peroni, O. D.; Kahn, B. B., Adipose-
specific overexpression of GLUT4 reverses insulin resistance
and diabetes in mice lacking GLUT4 selectively in muscle. Am
J Physiol Endocrinol Metab 2005, 289 (4), E551-61.
ASSOCIATED CONTENT
Supporting Information
4
.
Kuda, O.; Brezinova, M.; Rombaldova, M.; Slavikova, B.; Posta,
M.; Beier, P.; Janovska, P.; Veleba, J.; Kopecky, J., Jr.; Kudova,
E.; Pelikanova, T.; Kopecky, J., Docosahexaenoic acid-derived
fatty acid esters of hydroxy fatty acids (FAHFAs) with anti-
inflammatory properties. Diabetes 2016, 65 (9), 2580-90.
Kuda, O.; Brezinova, M.; Silhavy, J.; Landa, V.; Zidek, V.;
Dodia, C.; Kreuchwig, F.; Vrbacky, M.; Balas, L.; Durand, T.;
Hubner, N.; Fisher, A. B.; Kopecky, J.; Pravenec, M., Nrf2-
mediated antioxidant defense and peroxiredoxin 6 are linked
to biosynthesis of palmitic acid ester of 9-hydroxystearic acid.
Diabetes 2018, 67 (6), 1190-1199.
Zhu, Q. F.; Yan, J. W.; Zhang, T. Y.; Xiao, H. M.; Feng, Y. Q.,
comprehensive screening and identification of fatty acid
esters of hydroxy fatty acids in plant tissues by chemical
isotope labeling-assisted liquid chromatography-mass
spectrometry. Anal Chem 2018, 90 (16), 10056-10063.
Ma, Y.; Kind, T.; Vaniya, A.; Gennity, I.; Fahrmann, J. F.; Fiehn,
O., An in silico MS/MS library for automatic annotation of
novel FAHFA lipids. J Cheminform 2015, 7, 53.
Syed, I.; Lee, J.; Moraes-Vieira, P. M.; Donaldson, C. J.;
Sontheimer, A.; Aryal, P.; Wellenstein, K.; Kolar, M. J.; Nelson,
A. T.; Siegel, D.; Mokrosinski, J.; Farooqi, I. S.; Zhao, J. J.; Yore,
M. M.; Peroni, O. D.; Saghatelian, A.; Kahn, B. B., Palmitic acid
hydroxystearic acids activate gpr40, which is involved in their
beneficial effects on glucose homeostasis. Cell Metabolism
2018, 27 (2), 419-427 e4.
The Supporting Information is available free of charge on the
ACS Publications website.
Supplementary figures, detailed description of synthetic
procedures and spectral data for FAHFA-TGs and FAHFA-CE
(PDF)
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AUTHOR INFORMATION
Corresponding Author
6
.
*drsiegel@ucsd.edu and asaghatelian@salk.edu
Author Contributions
‡
M.E.E. and S.K. contributed equally.
7
.
Funding Sources
This work was supported by the National Institutes of Health
Grants R01 DK043051 and P30DK57521 (to B. B. K.) and R01
DK106210 (to B. B. K. and A. S.), a grant from the JPB
Foundation (to B. B. K.), Leona M. and Harry B. Helmsley
Charitable Trust Grant 2012-PG-MED002 (to A. S.), Pioneer
Fund (to D.T.), F32 DK111159 (M.E.E), the NCI Cancer
Center Support Grant P30 (CA014195 MASS core, to A.S.),
and Dr. Frederick Paulsen Chair/Ferring Pharmaceuticals
(to A.S.).
8
.
9
. Lee, J.; Moraes-Vieira, P. M.; Castoldi, A.; Aryal, P.; Yee, E. U.;
Vickers, C.; Parnas, O.; Donaldson, C. J.; Saghatelian, A.; Kahn,
B. B., Branched fatty acid esters of hydroxy fatty acids
Notes
(
FAHFAs) protect against colitis by regulating gut innate and
The authors declare no competing financial interest.
adaptive immune responses. Journal of Biological Chemistry
2016, 291 (42), 22207-22217.
ACKNOWLEDGMENT
10. Kolar, M. J.; Kamat, S. S.; Parsons, W. H.; Homan, E. A.; Maher,
T.; Peroni, O. D.; Syed, I.; Fjeld, K.; Molven, A.; Kahn, B. B.;
Cravatt, B. F.; Saghatelian, A., Branched fatty acid esters of
hydroxy fatty acids are preferred substrates of the mody8
protein carboxyl ester lipase. Biochemistry 2016, 55 (33), 4636-
41.
1. Parsons, W. H.; Kolar, M. J.; Kamat, S. S.; Cognetta, A. B., 3rd;
Hulce, J. J.; Saez, E.; Kahn, B. B.; Saghatelian, A.; Cravatt, B. F.,
AIG1 and ADTRP are atypical integral membrane hydrolases
that degrade bioactive FAHFAs. Nat Chem Biol 2016, 12 (5),
We thank Dr. Matthew Kolar for helpful discussions and
performing activity assay; Tina Chang for performing activity
assay; Cynthia Donaldson for technical assistance.
ABBREVIATIONS
1
FAHFA, fatty acid ester of hydroxy fatty acid; PAHSA, palmitic
acid ester of hydroxy stearic acid; POHSA, palmitoleic acid
ester of hydroxy stearic acid; OAHSA, oleic acid ester of
hydroxy stearic acid; FAHFA-TG, FAHFA-containing
triacylglycerol; FAHFA-CE, FAHFA-containing cholesteryl
ester; TG, triacylglycerol; CE, cholesteryl ester; DGAT, acyl-
CoA:diacylglycerol acyltransferase; IBMX, 3-isobutyl-1-
methylxanthine; FSK, forskolin; Atgli, Atglistatin; CAY,
CAY10499; PG-WAT, perigonadal white adipose tissue; SQ-
WAT, subcutaneous white adipose tissue.
3
67-372.
1
2. Listenberger, L. L.; Han, X.; Lewis, S. E.; Cases, S.; Farese, R.
V., Jr.; Ory, D. S.; Schaffer, J. E., Triglyceride accumulation
protects against fatty acid-induced lipotoxicity. Proc Natl
Acad Sci U S A 2003, 100 (6), 3077-82.
13. Zimmermann, R.; Strauss, J. G.; Haemmerle, G.; Schoiswohl,
G.; Birner-Gruenberger, R.; Riederer, M.; Lass, A.; Neuberger,
G.; Eisenhaber, F.; Hermetter, A.; Zechner, R., Fat
mobilization in adipose tissue is promoted by adipose
triglyceride lipase. Science 2004, 306 (5700), 1383-6.
4. Yetukuri, L.; Soderlund, S.; Koivuniemi, A.; Seppanen-Laakso,
T.; Niemela, P. S.; Hyvonen, M.; Taskinen, M. R.; Vattulainen,
I.; Jauhiainen, M.; Oresic, M., Composition and lipid spatial
distribution of HDL particles in subjects with low and high
HDL-cholesterol. J Lipid Res 2010, 51 (8), 2341-51.
5. Zechner, R.; Zimmermann, R.; Eichmann, T. O.; Kohlwein, S.
D.; Haemmerle, G.; Lass, A.; Madeo, F., FAT SIGNALS--lipases
and lipolysis in lipid metabolism and signaling. Cell
Metabolism 2012, 15 (3), 279-91.
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triacylglycerol biosynthesis. J Lipid Res 2008, 49 (11), 2283-301.
1. Liebisch, G.; Vizcaino, J. A.; Kofeler, H.; Trotzmuller, M.;
Griffiths, W. J.; Schmitz, G.; Spener, F.; Wakelam, M. J.,
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
2
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
TOC Figure
9
ACS Paragon Plus Environment

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Figure 1
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
O
O
O
A
NH4+
O
+
[
M+H-PAHSA]
5
77.5189
z=1
O
O
O
100
m/z 1133.0417 @ HCD 12V
16:0/PAHSA/18:1-TG Standard
O
+
8
6
4
0
0
0
+
Chemical Formula: C H138NO8
[
M+H-16:0-16:0]
71
6
03.5344
z=1
Exact Mass: 1133.0417
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
+
[
M+H-16:0]
+
+
859.7744
[M+NH4]
[
M+H-18:1]
33.7589
z=1
20
0
z=1
8
1116.0103 1133.0421
z=1
z=?
1
00
200
300
400
500
600
700
800
900
1000
1100
m/z
5
77.5179
z=1
B 100
m/z 1133.0417 @ HCD 12V
16:0_PAHSA_18:1-TG Adipose Tissue
8
6
4
0
0
0
603.5333
z=1
859.7723
z=1
551.5023
z=1
833.7552
z=1
1133.0385
z=?
20
0
1115.9941
z=?
1
00
200
300
400
500
600
700
800
900
1000
1100
m/z
C
hydrolyzed
unhydrolzyed
PAHSA
POHSA
OAHSA
2
8
6
4
2
0
2
1
10
10
9
10
1
.5
0
9
9
12/13
0
12/13
0.5
12/13
1
1
11
11
20
0
16
18
20
22
24
10
12
14
16
18
16
18
22
24
Retention Time (min)
Retention Time (min)
Retention Time (min)
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Page 11 of 14
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Figure 2
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
13
C -PAHSA_16:1_18:1
4
4
00
00
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
*
*
3
O
*
O
O*
200
100
0
*
*
HO
1
3
C -PAHSA
4
13
DGAT1/2
inhibitors
C -PAHSA
+
+
+
+
+
+
+
+
4
DGAT1i
DGAT2i
+
13
+
[M+NH ] > [M+H- C -PA]
4
4
+
13
+
[M+NH ] > [M+H- C -PAHSA]
4
4
13
13
13
13
Vehicle
C -PAHSA only
C -PAHSA + 1i
C -PAHSA + 2i
C -PAHSA + 1i + 2i
4
4
4
4
2
2
1
5
0
2
1
5
0
2
1
5
0
2
1
5
0
1
5
0
35
40
45
35
40
45
35
40
45
35
40
45
35
40
45
RT (min)
RT (min)
RT (min)
RT (min)
RT (min)
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Journal of the American Chemical Society
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Figure 3
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
12/13−PAHSA
12/13−POHSA
12/13−OAHSA
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
*
3e+05
2e+05
1e+05
8e+05
1.5e+05
1.0e+05
5.0e+04
0.0e+00
*
*
*
6e+05
#
*
4e+05
2e+05
0e+00
#
#
0e+00
9−PAHSA
9−POHSA
9−OAHSA
3
e+04
e+04
*
*
5e+04
4e+04
3e+04
2e+04
1e+04
0e+00
1e+05
5e+04
0e+00
*
2
*
#
1e+04
0e+00
#
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1
2
3
4
5
6
7
8
9
Figure 4
A
95:5 Hex:EA
EA
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Neutral lipids
Nonesterified FAHFA
Released FAHFA
95:5 Hex:EA
EA
LiOH, 24 hr
13
(
C -PAHSA TG,
16
sn1/sn2)
v
B
Nonesterified FAHFA (sn1)
Nonesterified FAHFA (sn2)
TG-esterified FAHFA (sn1)
TG-esterified FAHFA (sn2)
6
4
2
0
0
.05
.04
0
.02
.00
0
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1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Figure 5
A
Esterified FAHFAs from
Esterified FAHFAs from
PG-WAT TG fraction
SQ-WAT TG fraction
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
0
8
6
4
2
0
Chow
HFD
8
6
4
2
0
*
*
*
*
*
A
A
A
A
A
A
S
S
S
9
S
S
9
S
H
H
H
H
H
H
A
A
O
O
A
A
P
P
P
P
O
O
-
-
-
-
-
-
0
9
0
0
1
1
1
B
Esterified PAHSAs from
Esterified PAHSAs from
Chow TG fraction
HFD TG fraction
5
4
3
2
1
0
5
4
3
2
1
0
10
1
0
9
12/13
9
12/13
11
1
1
10
15
RT (min)
20
10
15
RT (min)
20
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