Docosahexaenoic acid-derived fatty acid esters of hydroxy fatty acids (FAHFAs)

40 Pages·2016·1.76 MB·English

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Docosahexaenoic acid-derived fatty acid esters of hydroxy fatty acids (FAHFAs)
with anti-inflammatory properties
Running title: Novel members of FAHFA lipid class derived from DHA

Ondrej Kuda1#, Marie Brezinova1#, Martina Rombaldova1, Barbora Slavikova2, Martin Posta2,
Petr Beier2, Petra Janovska1, Jiri Veleba3, Jan Kopecky Jr3., Eva Kudova2, Terezie Pelikanova3,
Jan Kopecky1*

1Department of Adipose Tissue Biology, Institute of Physiology of the Czech Academy of
Sciences, Videnska 1083, 14220 Praha 4, Czech Republic
2Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic
v.v.i., Flemingovo nam 2, 16610 Prague 6-Dejvice, Czech Republic
3Diabetes Centre, Institute for Clinical and Experimental Medicine, Videnska 1958/9, 140 21,
Prague, Czech Republic
# These authors contributed equally.

*Author for correspondence:
Jan Kopecky, Department of Adipose Tissue Biology, Institute of Physiology of the Czech
Academy of Sciences, Videnska 1083, 142 20 Prague, Czech Republic, E-mail:
[email protected], Telephone: [phone], Fax: [phone]

Word count: 3958
Tables: 1
Figures: 6
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Diabetes Publish Ahead of Print, published online July 5, 2016
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Abstract
White adipose tissue (WAT) is a complex organ with both metabolic and endocrine
functions. Dysregulation of all of these functions of WAT, together with low-grade inflammation
of the tissue in obesity, contributes to the development of insulin resistance and type 2 diabetes.
Omega-3 polyunsaturated fatty acids (PUFA) of marine origin play an important role in
resolution of inflammation and exert beneficial metabolic effects. Using experiments in mice and
overweight/obese type 2 diabetic patients we elucidated the structures of novel members of fatty
acid esters of hydroxy fatty acids (FAHFA) - lipokines derived from docosahexaenoic (DHA)
and linoleic acid, which were present in serum and WAT after omega-3 PUFA supplementation.
These compounds contained DHA esterified to 9- and 13-hydroxyoctadecadienoic acid (HLA) or
14-hydroxydocosahexaenoic acid (HDHA), termed 9-DHAHLA, 13-DHAHLA, and 14-
DHAHDHA, and were synthesized by adipocytes at concentrations comparable to those of
protectins and resolvins derived from DHA in WAT. 13-DHAHLA exerted anti-inflammatory
and pro-resolving properties while reducing macrophage activation by lipopolysaccharide and
enhancing the phagocytosis of zymosan particles. Our results document the existence of novel
lipid mediators, which are involved in the beneficial anti-inflammatory effects attributed to
omega-3 PUFA, in both mice and humans.

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INTRODUCTION
White adipose tissue (WAT) is an extremely plastic organ with important roles in energy balance,
whole body glucose homeostasis and the immune system (1-4). The systemic effects of WAT
largely reflect its role in the control of blood lipid levels, as well as the secretion of numerous
bioactive peptides (adiponectin, leptin, etc.) from WAT cells (5; 6). Adipocytes also release lipid-
based mediators such as branched fatty acid hydroxy fatty acid esters (FAHFAs) (7) and
palmitoleate (8), which could improve local and whole body glucose metabolism (9; 10).
Moreover, FAHFA administration also stimulated GLP-1 and insulin secretion and reduced
obesity-associated WAT inflammation in mice through cell surface G protein-coupled receptor
120 (GPR120)-dependent signaling (7). Both FAHFAs and palmitoleate are produced in
adipocytes via the fatty acid (FA) synthesis pathway (de novo lipogenesis). The activity of this
pathway is decreased in response to obesity-associated hyperinsulinemia and WAT inflammation
(8; 11; 12), resulting in reduced levels of the beneficial lipid mediators. Although the biosynthetic
enzymes of FAHFAs are unknown, two FAHFA specific hydrolases, AIG1 and ADTRP, were
recently identified (13).
These newly-described FAHFAs are members of a lipid class called estolides
(intermolecular esters of hydroxy fatty acids) serving mainly as biodegradable lubricants (14).
Recently, the discovery of FA estolides in humans and mice (7) and triacylglycerol estolides in
the brushtail possum (15) brought these lipids to mammalian physiology. The FAHFA
nomenclature introduced by Yore et al (7) combines abbreviations of esterified FAs and hydroxy
FAs – e.g. the combination of esterified palmitic acid (PA) and hydroxyl stearic acid (HSA) was
abbreviated as PAHSA. Although a recently published in-silico library of all potential FAHFAs
uses a nomenclature based on chemical structure (16), for practical reasons, here we use the
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shorter abbreviations, e.g. PA for palmitic acid, LA for linoleic acid (LA), DHA for
docosahexaenoic acid with the “H” prefix for hydroxy fatty acids and the position of branching.
The obesity-associated WAT inflammation imposes adverse local as well as whole-body
metabolic effects (1; 2; 11; 17-19). This inflammatory response is accompanied by macrophage
repolarization to the pro-inflammatory (classically activated) M1 state, which negatively affect
WAT functions (20) and could be counteracted using both dietary and pharmacological
interventions (21; 22). Regarding the dietary influence, namely omega-3 polyunsaturated fatty
acids (PUFA) of marine origin, which are considered to be healthy dietary constituents in
diabetics (ref. (23); see Discussion), play an important role in the resolution of WAT
inflammation (24-28) and probably potentiate beneficial functions of immune cells in WAT like
efferocytosis or autophagy (17).
Omega-3 PUFA exhibit their anti-inflammatory effects through GPR120, as well as via
several other signaling pathways, resulting in improved insulin sensitivity in obese mice (29-31).
In addition, DHA-derived lipid mediators such as resolvin D1 has been reported to decrease
WAT inflammation, shifting macrophage polarization toward the M2 form and improving insulin
sensitivity in obese mice (24-27). A wide range of lipid mediators including resolvins D1 and D2,
protectin D1, lipoxin A4, and 17-HDHA, 18-HEPE and 14-HDHA were identified in human
subcutaneous WAT, while the levels of protectin D1 and 17-HDHA decreased in the
subcutaneous WAT of patients with peripheral vascular disease (32). Protectin DX, also known
to be produced in WAT, alleviated insulin resistance in diabetic db/db mice, but did not resolve
WAT inflammation (33).
Given the beneficial effects of omega-3 PUFA on WAT inflammatory status, we
hypothesized that novel FAHFA structures derived from omega-3 PUFA, with possible anti-
inflammatory properties, could be found. To test this hypothesis, we performed lipidomic
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analysis using human and murine serum and WAT samples collected from subjects supplemented
or not with omega-3 PUFA.
MATERIALS AND METHODS
Materials and reagents
All chemicals were purchased from Sigma-Aldrich (Prague, Czech Republic) unless otherwise
stated. FAHFA standards (5-,9-,12-,13-PAHSA, POHSA, SAHSA, OAHSA, respectively, and 5-
PAHSA-2H , 9-PAHSA-13C ) were purchased from Cayman Pharma (Neratovice, Czech
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Republic).
Human samples
Serum samples were acquired within the framework of a clinical trial focused on the combined
effects of the antidiabetic drug pioglitazone and omega-3 PUFA (34). Briefly, overweight/obese
patients 40-70 years of age, diagnosed with type 2 diabetes and already treated with metformin,
were given either 5 g/day corn oil (Placebo) or 5 g/day EPA+DHA concentrate (Omega-3; EPAX
1050TG, EPAX AS, containing about 15% EPA, 40% DHA, wt/wt; i.e., ~2.8 g EPA+DHA) for
24 weeks. The serum samples and biopsies of abdominal subcutaneous WAT collected during the
final visit after an overnight fast were stored at -80 ºC until LC-MS/MS analysis.
Murine samples
Male mice (C57BL/6J; Jackson Laboratory, ME, USA) were maintained in a controlled
environment (22 ºC; 12-h light-dark cycle; light from 6.00 a.m.) and fed a corn oil-based high-fat
diet (HF; lipid content 35%, wt/wt) or HF diet with EPA+DHA (HFF) concentrate (EPAX
1050TG) for 8 weeks as before (21). Epididymal WAT, subcutaneous WAT, liver and
interscapular brown adipose tissue and serum samples were collected after an overnight fast and
stored in liquid nitrogen. All animal experiments were approved by the Animal Care and Use
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Committee of the Institute of Physiology, Czech Academy of Sciences (Approval Number:
172/2009) and followed the guidelines.
Cell cultures
Murine adipocyte cell line 3T3-L1 and human adipocytes hMADS (7) were grown according to
standard protocols. Differentiated adipocytes were incubated with 100 µM LA and 100 µM DHA
complexed to BSA 3:1 for 24 hours and extracted for FAHFA analysis. Adipocytes and stromal-
vascular cells (SVC) were prepared as before (26). RAW 264.7 cells and murine bone marrow-
derived macrophages (BMDM) were grown and stimulated with lipopolysaccharide (LPS) as
before (35; 36). Human peripheral blood mononuclear cells (PBMC) were isolated from buffy
coats and treated with lectin as described before (37).
FAHFA extraction
FAHFA extraction was performed based on the published method (7). Murine tissue (~300 mg)
or cells were homogenized using a MM400 bead mill (Retsch, Germany) chilled to -20 ºC in a
mixture of citric acid buffer, methanol, and further extracted with dichloromethane (1:1:2 final
ratio). Internal standards of 5-PAHSA-2H and 9-PAHSA-13C were added to the homogenate
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(100 pg/sample). Serum samples were extracted according to the same protocol, apart from the
homogenization. The organic phase was collected, dried in a Speed-vac (Savant SPD121P,
Thermo), resuspended in dichloromethane and applied on Strata SI-1 Silica SPE columns (55µm,
70Å, Sigma). FAHFA were eluted from the SPE columns with ethylacetate, concentrated in the
Speed-vac, resuspended in methanol and immediately measured using LC-MS as follows.
Liquid chromatography and mass spectrometry
Chromatographic separation was performed in an UPLC Ultimate 3000 RSLC (Thermo)
equipped with a Kinetex C18 1.7 µm 2.1x150 mm column (Phenomenex). The flow rate was 200
µl/min at 50 ºC. The gradient program used to separate FAHFA was as follows: solvent A (70%
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water, 30% acetonitrile, 0.01% acetic acid, pH 4), solvent B (50% acetonitrile, 50% isopropanol);
1 min (100% A); 5 min (20% A); 18 min (10% A); 20 min (100% A); 25 min (100% A) while a
linear gradient was maintained between the steps. Isocratic elution (20% A, 80% B, 60 minutes)
was used for structural studies. UPLC was coupled to a QTRAP 5500/SelexION, a hybrid, triple
quadrupole, linear ion trap mass spectrometer equipped with an ion mobility cell (Sciex). FAHFA
were detected in negative ESI mode with the following parameters: DP: -130; CE: -35; CXP: -15;
CUR: 25; CAD: high; IS: -4500, TEM: 400; GS1: 40; GS2: 50. For ion mobility experiments,
DMS settings were as follows: DT: high, DM: isopropanol/high; SV: 3800; DMO: -3; DR: low;
CoV: -5.0 for 13-PAHSA and -1.2 for 12-PAHSA. Multiple Reaction Monitoring (MRM) mode
with one quantifier and two qualifier transitions per FAHFA was used for quantitation (ref (7)
and Table 1). Quantifier ion MRM was used as a survey scan for information-dependent
acquisition in the linear ion trap for enhanced-resolution MS/MS and MS/MS/MS spectra (scan
rate: 1000 Da/s; scan mode: profile, step size: 0.05 Da; LIT fill time: 200 ms). Pure standards of
PAHSAs were used for quantitation. For DHAHLA and DHAHDHA compounds, the 5-PAHSA
calibration curve was used as a surrogate.
Synthesis of DHAHLA standard
Organic synthesis of 13-DHAHLA was performed according to Steglich esterification from DHA
and 13-HODE (38; 39). Details are provided in Supplemental Information.
Markers of inflammation
The anti-inflammatory properties of DHAHLA were assessed according to published methods (7;
36; 37; 40). Briefly, murine BMDM or RAW 264.7 macrophages were incubated in the presence
of LPS (100 ng/ml; E.coli 0111:B4, Sigma-Aldrich) or 13-DHAHLA (10 µM) alone or in
combination with 9-PAHSA (10 µM) or 13-DHAHLA (10 µM) for 18 hours. Control cells were
incubated with the vehicle alone. Murine IL-6 ELISA (Cayman Chemicals) and qPCR (21) were
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used to measure the markers of macrophage activation, details are provided in Supplemental
Information. BMDM were incubated in the presence of LPS alone or in combination with DHA
(10 µM), INF-γ (50 ng/ml) or 13-DHAHLA (10 µM) for 18 hours. LC-MS metabolipidomics
(26; 36; 37; 41) was used to measure macrophage metabolic activation and levels of lipid
mediators. PBMC were pre-treated with 1µM 13-DHAHLA for 30 minutes, stimulated with
lectin (phytohaemagglutitin 10 µg/ml) for 24 hours, and the levels of tryptophan and kynurenine
were measured in the media. RAW 264.7 macrophages were stimulated with LPS (10 ng/ml) and
incubated in the presence of various 13-DHAHLA concentrations for 18 hours to explore dose-
dependent inhibition of macrophage activation. The phagocytosis of fluorescein-labeled zymosan
(Life Technologies) by BMDM was measured using a Victor X4 plate reader (40).
Statistics
Statistical analysis was performed with SigmaStat and p<0.05 was considered significant.

RESULTS
Targeted lipidomics of FAHFAs using LC-MS/MS/MS
In view of the beneficial effects of PAHSAs (7), we developed a targeted lipidomic methodology
using liquid chromatography coupled to hybrid tandem mass & linear ion trap spectrometry to
identify and quantify FAHFAs in human and murine samples. We took advantage of the ability
of MS to switch from sensitive triple quadrupole scan modes to highly sensitive full-scan ion trap
mode within one analysis to obtain both quantitative and qualitative (structural) information. This
approach enabled us to precisely quantify FAHFA levels using MRM and identify the branching
position on the backbone HFA using MS/MS/MS. For instance, 9-PAHSA can be ionized in
negative mode to [M-H]- ion 537.488 m/z. Fragmentation by collision-induced dissociation
results in 3 daughter ions: 299.3, 281.2, and 255.2 m/z, identified as fragments of hydroxystearic,
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octadecanoic and palmitic acid, respectively (Figure 1A and B) (7). Further fragmentation of ion
299.3 m/z (hydroxystearic acid) in the linear ion trap gave rise to the ions 155.144 m/z and
127.113 m/z, which are specific to the position of the hydroxyl group on the hydroxystearic acid
backbone (Figure 1A), thus enabling us to identify the branching carbon.
The quantities and structures of FAHFA isomers were analyzed in human serum samples
and with the PAHSA isomers, we were able to identify 9 positional isomers (13-, 12-, 11-, 10-, 9-
, 8-, 7-, 6-, 5-PAHSA) (Figure 1C) according to their MS/MS/MS spectra (Figure 1D). Given the
practical impossibility of separating the positional isomers of 12- and 13-PAHSA in human
samples (see (7) and Figure 1 in Supplemental Information; SF1), the 12/13-PAHSA levels are
reported together.
PAHSA levels were not altered by omega-3 PUFA supplementation in diabetic patients and
obese mice.
First, we investigated the possible effect of omega-3 PUFA supplementation on the levels of the
known FAHFAs (see Introduction) in diabetic humans, and therefore serum samples of
metformin-treated patients supplemented with either corn oil capsules (Placebo) or Omega-3
PUFA capsules (Omega-3) for 24 weeks (34) were analyzed. Serum levels of 5-, 9-, and 12/13-
PAHSA isomers were not altered by Omega-3 supplementation (Figure 2A). This observation
was also supported by animal experiments on dietary obese mice fed an omega-3 PUFA diet for 8
weeks (21) where no differences in serum PAHSA levels were detected (Figure 2B).
Identification of novel FAHFA derived from LA and DHA
Using the same MS approach as in the identification of PAHSA isomers, any combination of FAs
and the branching position could be theoretically detected. Therefore, we focused on alternative
combinations of FAs besides PAHSAs and were able to identify novel members of the FAHFA
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family derived from LA and DHA, specifically DHAHLA, LAHDHA and DHAHDHA in human
serum (see Supplemental Information SF2 for structures).
With DHAHLA, DHA esterified to a hydroxy LA, two positional isomers of the hydroxy
FA backbone were detected: 9- and 13-hydroxyoctadecadienoic acid (HLA aka HODE),
therefore 9- and 13-DHAHLA. This is in agreement with the high concentrations of 9(S)- and
13(S)-HODE, enzymatic products of 15-lipoxygenase in the organism. As shown in the 13-
DHAHLA fragmentation scheme (Figure 3A), the ion 605.457 m/z gave rise to the fragment
295.228 m/z (13-HODE), which was further fragmented to characteristic ions 179.144 and
195.139 m/z (SF3 for details).
Chromatographic separation of the murine serum sample revealed additional complexity
of DHAHLA isomers when four separated DHAHLA peaks were detected (Figure 3B). Structural
characterization in the linear ion trap revealed that 2 major peaks were 13-DHAHLA and 2 minor
peaks were 9-DHAHLA cis-trans isomers of double bonds in HLA acyl chains. Identity of the
backbone fragmentation was confirmed using synthetic standards for 9(S)-HODE and 13(S)-
HODE (Figure 3C and SF2). Of note, only 2 peaks corresponding to physiologically relevant
(9Z,11E,13S)-13-hydroxy-9,11-octadecadienoic acid- and (9S,10E,12Z)-9-hydroxy-10,12-
octadecadienoic acid-derivatives, 9(S)-HODE and 13(S)-HODE, respectively, were observed in
human WAT and serum samples as well as cultured cells (hMADS, not shown), and therefore,
only these were considered for further analyses.
Similar to PAHSA tissue distribution (7), 13-DHAHLA was detected in murine adipose
tissue depots and was upregulated after omega-3 PUFA supplementation (Figure 4A). Levels of
FAHFA-specific hydrolases (13), Aig1 and Adtrp, were downregulated in murine adipose tissue
and upregulated in the liver after omega-3 PUFA supplementation. Interestingly, while Adtrp was
exclusively associated with adipocytes, Aig1 was present also in SVC (Supplemental Figure 4).
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