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Supplemental Digital Content 1
Measures
Reagents and Chemicals. 2,5-dihydro-2,4,5-trimethylthiazoline (TMT) was purchased from
Contech Enterprises (Delta, B.C., Canada). Butyric acid (BA) was purchased from SigmaAldrich (Saint Louis, MO). ELISA Kits for rat brain natriuretic peptide (BNP) and interleukin-6 (IL6) were obtained from Novatein Biosciences (Cambridge, MA). Troponin-I was measured using
the Rat Cardiac Tn-I ELISA kit from Life Diagnostics, Inc. (West Chester, PA). EIA Kit for
Angiotensin-II was purchased from RayBiotech, Inc. (Norcross, GA). QuantiChrom™
thiobarbituric acid reactive substances (TBARS) Assay Kit was from BioAssay Systems
(Hayward, CA). The BNP ELISA Kit has a detection range of 31.2 pg/ml-1000pg/ml, a sensitivity
of 5.0 pg/ml and an overall variation coefficient of precision between assays and precision within
an assay < 10%. The IL-6 ELISA kit has a detection range of 6.25 pg/ml-200 pg/ml, a sensitivity
of 1.0 pg/ml and an overall variation coefficient of precision between assays and precision within
an assay < 10%. The EIA Angiotensin-II kit has a detection range of 0.1-1,000 pg/ml, a
sensitivity of 2.62 pg/ml, and an intra-assay coefficient of variation < 10% and inter-assay
coefficient of variation < 15%. The fluorimetric assay of TBARS Assay Kit has a lower limit of
detection of 0.1 µM MDA; the intra-assay variation is below 5%. BCA protein assay was from
Pierce (Rockford, IL). Infinity Triacylglycerols colorimetric kit was from Thermo Fisher Scientific,
Inc. (Rockford, IL). [2H4]-OEA, [2H4]-PEA, [2H4]-anandamide and 1,2-dipalmitoyl-sn-glycero-3phosphoethanolamine-N-heptadecenoyl were prepared as described previously using the
appropriate fatty acid chlorides purchased from Nu-Chek Prep, Inc. (Elysian, MN). [2H8]-2-AG
was from Cayman Chemical (Ann Arbor, MI). C17:0 heptadecanoic acid, C17:1 heptadecenoic
acid, C17:1 diheptadecenoinglycerol and C17:1 monoheptadecenoinglycerol, were from NuCheck Prep, Inc. (Elysian, MN). C12 ceramide (d18:1/12:0) and 10-cisheptadecenoylethanolamide were from Avanti® Polar Lipids, Inc. (Alabaster, AL).
Holman et al., Acute stress, endocannabinoids, and the heart: Supplemental Digital Content 1
Pg. 2
Anandamide[ethanolamine-3H] was purchased from American Radiolabeled Chemicals (Saint
Louis, MO).
Hemodynamic parameters. Systolic (SBP), diastolic (DBP) and mean blood pressure (MBP)
were measured with a tail cuff method by volume pressure recording technology using the NonInvasive Blood Pressure System CODA-6 (Kent Scientific Corporation, CODA, Torrington, CT).
Elevated plus maze (EPM). The EPM was used to evaluate anxiety-like behavior. The EPM
has two open arms, 50 x 10 cm, and two enclosed arms, 50 x 10 x 40cm that intersect to form a
plus sign; it is elevated to 60 cm. White lighting provided 35-45 lux in the open arms and 3-9 lux
in the closed arms. Rats were placed individually in the center facing an open arm; behavior
was scored for 5 minutes. A video camera was mounted vertically above the EPM and
connected to a monitor located in an adjacent room. A trained observer counted open and
closed arm entries and measured time spent in open and closed arms. Arm entry was defined
as having all four feet in one arm (1). Three indices were calculated from these observations:
Ratio time is the amount of time spent in the open arms divided by the total time spent in the
open and closed arms; ratio entry is the number of entries into the open arms divided by the
total number of entries into the open and closed arms. The third is the “anxiety index” which is
calculated from measurements of exploratory behavior as:
time spent in the open arms number of entries into the open arms
+
total number of entries into the arms
𝐴𝑛𝑥𝑖𝑒𝑡𝑦 𝑖𝑛𝑑𝑒𝑥 = 1 − total time on the maze
2
Higher scores indicate increased anxiety-like behavior (2). Total distance moved was
measured using the EthoVision 3.0 software (Leesburg, VA). After each session the EPM was
wiped with 10% ethanol and dried thoroughly.
Lipid extraction and liquid chromatography/mass spectrometry (LC/MS). Triacylglycerols
(TAG), free fatty acids (FFA), OEA, PEA, anandamide, 2-AG, 2-palmitoylglycerol (2-PG), 2oleoylglycerol (2-OG) and ceramides were extracted from tissues and serum. Frozen tissues
Holman et al., Acute stress, endocannabinoids, and the heart: Supplemental Digital Content 1
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were weighed and homogenized in methanol (1 ml/100 mg of tissue) containing [2H4]-OEA,
[2H4]-PEA, [2H4]-anandamide, [2H8]-2-AG, C12 ceramide (d18:1/12:0) and heptadecanoic acid as
internal standards. Lipids were extracted using 2 volumes of chloroform and washed with 1
volume of water. After centrifugation at 2000gx at 4˚C, organic phases were collected and dried
under nitrogen. The extracts were dissolved in 2 ml chloroform and fractionated onto small glass
columns packed with Silica Gel G (60-Å 230-400 Mesh ASTM; Whatman, Clifton, NJ) (3). The
first eluate was dried under nitrogen and reconstituted in 60 µl of Tert-butanol:Triton X100:Methanol (3:1:1) to determine the TAG content using a colorimetric kit (4). Lipids were
eluted with 2 ml chloroform/methanol (9:1, vol/vol), reconstituted in 60 µl of methanol and
quantified by liquid chromatography/mass spectrometry (LC/MS) (5,6).
To extract lipids from serum, 1 ml of ice-cold acetone containing [2H4]-OEA, [2H4]-PEA,
[2H4]-anandamide, [2H8]-2-AG, heptadecanoic acid and C12 ceramide (d18:1/12:0) as internal
standards was added to 1 ml of serum. Proteins were precipitated by centrifugation at 1000xg
for 20 min at 4C. The supernatant was collected and excess acetone was dried under nitrogen.
Tissue levels of OEA, PEA, anandamide, 2-AG, 2-PG, 2-OG and FFA were quantified by
LC/MS (7-9). A 1100-LC system coupled to a 1946A-MS detector (Agilent Technologies, Inc.,
Palo Alto, CA) equipped with an electrospray ionization interface was used. Fatty acid
ethanolamides and monoacylglycerols were eluted on a XDB Eclipse C18 column (50 x 4.6 mm,
inner diameter 1.8 µm, Zorbax, Agilent Technologies, Inc.) using a linear gradient of methanol in
water (from 85% to 90% methanol in 2.5 min) at a flow rate of 1 ml/min. Column temperature
was kept at 40C. MS detection was in positive ionization mode for OEA, PEA, anandamide, 2AG, 2-PG and 2-OG, capillary voltage was set at 3000 V, and fragmentor voltage was varied
from 120 to 140 V. Nitrogen was used as drying gas at a flow rate of 13 L/min and a
temperature of 350C. Nebulizer pressure was set at 60 psig. For quantification purposes we
monitored, in the selective ion-monitoring mode (SIM), the Na+ adducts ([M+Na+]) of [2H4]-OEA
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(mass-to-charge ratio, m/z = 352), OEA (m/z = 348), [2H4]-anandamide (m/z = 374),
anandamide (m/z = 370), [2H4]-PEA (m/z = 326), PEA (m/z = 322), [2H8]-2-AG (m/z =409) and
2-AG (m/z = 401). FFA were eluted on a XDB Eclipse C18 column (50 x 4.6–mm inner
diameter, 1.8 µm particle size, 80 Å of porous diameter, Zorbax, Agilent Technologies, Inc.).
Mobile phase A consisted of methanol containing 0.25% acetic acid and 5 mM ammonium
acetate; mobile phase B consisted of water containing 0.25% acetic acid and 5 mM ammonium
acetate. Lipids were eluted using a linear gradient from 90% A to 100% B in 2.5 min at a flow
rate of 1.5 ml/min with column temperature at 40C. ESI was in the negative mode, capillary
voltage was set at -4 kV and fragmentor voltage was 120 V. Nitrogen was used as drying gas at
a flow rate of 13 L/min and a temperature of 350C. Nebulizer pressure was set at 60 psig. FFA
were analyzed monitoring the m/z of the deprotonated molecular ions [M-H]- in SIM mode.
Detection and analysis were controlled by Agilent Chemstation. The following FFA were
monitored: palmitic acid (C16:0, m/z = 255), heptadecanoic acid (C17:0, m/z = 269), stearic acid
(C18:0, m/z = 283), arachidic acid (C20:0, m/z = 311), docosanoic acid (C22:0, m/z = 339),
tetracosanoic (C24:0, m/z = 367), hexacosanoic (C26:0, m/z = 395), palmitoleic acid (C16:1∆9,
m/z = 253), oleic acid (C18:1∆9, m/z = 281), eicosenoic acid (C20:1∆9, m/z = 309), erucic acid
(C22:1∆9, m/z = 337), nervonic acid (C24:1∆9, m/z = 365), linoleic acid (C18:2∆9,12, m/z = 279),
linolenic acid (C18:3∆9,12,15, m/z = 277), eicosatrienoic acid (20:3∆8,11,14, m/z = 305),
eicosatrienoic acid (20:3∆5,8,11, m/z = 305), arachidonic acid (C20:4∆5,8,11,14, m/z = 303),
eicosapentaenoic acid (C20:5∆5,8,11,14,17, m/z = 301), docosatetraenoic acid (22:4∆7,10,13,16, m/z =
331), docosapentaenoic acid (C22:5∆7,10,13,16,19, m/z = 329), docosapentaenoic acid
(C22:5∆4,7,10,13,16, m/z = 329), docosahexaenoic acid (C22:6∆4,7,10,13,16,19, m/z = 327).
Total FFA were calculated by summing the concentration of all FFA measured. Total
saturated FFA, monounsaturated FFA (MUFA) and polyunsaturated FFA (PUFA) were
calculated by summing the concentrations of individual FFA if detectable. Estimated desaturase
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Pg. 5
and elongase activities and de novo lipogenesis were calculated by product/substrate ratios as
follows: Steaoryl delta-9 desaturase activities were calculated by the ratios of palmitoleic acid
and palmitic acid or oleic and stearic acid respectively (16:1ω3/16:0, SCD-16; 18:1ω9/18:0,
SCD-18). Delta-5 (D5D) and delta-6 (D6D) desaturase activities were estimated by the ratios
20:4ω6/20:3ω6 and 18:3ω6/18:2ω6, respectively (10). The ratio of palmitic and linoleic acid
(16:0/18:2ω6) was used for estimation of de novo lipogenesis (11). Finally, elongase activity
was estimated by the ratio of stearic to palmitic acid (18:0/16:0) and erucic acid to nervonic acid
(22:1ω9/24:1ω9) (12).
Tissue and serum ceramides were analyzed by tandem mass spectrometry (13), using
an Agilent 1100 liquid chromatograph coupled to an ESI-ion-trap XCT mass detector.
Ceramides were eluted on a Poroshell 300 SB C18 column (2.1 x 75 mm, inner diameter 5 µm;
Agilent Technologies, Inc.) maintained at 30°C. A linear gradient of methanol in water containing
5 mM ammonium acetate and 0.25% acetic acid (from 80% to 100% of methanol in 3 min) was
used at a flow rate of 1 ml/min. Detection was in the positive mode, capillary voltage was 4.5 kV,
skim1 −40 V, and capillary exit −151 V. Nitrogen was used as drying gas at a flow rate of 12
L/min, temperature set at 350°C, and nebulizer pressure set at 80 psig. Helium was used as
collision gas. Ceramide species were identified by comparison of its MSn fragmentation pattern
and LC retention time with that of authentic standards. Extracted ion chromatograms were used
to quantify the following ceramide species: d18:0/14:0 [M+H]+ (m/z = 510.5>492.5>264.3),
d18:1/16:0 [M+H]+ (m/z = 538.5>520.5>264.3), d18:1/18:0 [M+H]+ (m/z = 566.5>548.5>264.3),
d18:1/24:1 [M+H]+ (m/z = 648.6>630.8>264.3), d18:1/24:0 [M+H]+ (m/z = 650.5>632.5>264.3),
d18:1/26:0 [M+H]+ (m/z = 678.5>660.5>264.3), using d18:1/12:0 [M+H]+ (m/z =
482.5>464.5>264.3) as an internal standard.
To determine serum levels of OEA, PEA, AEA, 2-AG, 2-PG and 2-OG lipids were eluted
on a HP ODS Hypersil column (100 x 4.6 mm inner diameter, 5 µm particle size, Thermo
Holman et al., Acute stress, endocannabinoids, and the heart: Supplemental Digital Content 1
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Scientific, West Palm Beach, FL) using a gradient of methanol in water (from 75% to 100%
methanol in 35 min) at a flow rate of 0.5 ml/min. Column temperature was kept at 20C. MS
detection was in the positive ionization mode, capillary voltage was set at 3000 V, and
fragmentor voltage was varied from 120 to 140 V. Nitrogen was used as drying gas at a flow
rate of 13 L/min and a temperature of 350C. Nebulizer pressure was set at 30 psig. FFAs were
eluted and monitored as described above.
Enzyme activities
Heart tissue was homogenized on ice-cold Tris-HCl (20 mM, pH 7.4, 10 vol) containing
0.32 M sucrose. Homogenates were centrifuged at 1,000xg for 15 min. Supernatants were
collected and protein concentration determined using a bicinchoninic acid (BCA) assay kit
(Pierce, Rockford, IL).
N-acylphosphatidylethanolamine-specific phospholipase D (NAPE-PLD) activity.
NAPE-PLD activity was measured at 37˚C for 30 min in Tris-HCl buffer (50 mM, pH 7.4)
containing 0.1% Triton X-100, phenylmethylsulphonylfluoride (PMSF, 1 mM), protein (100 µg)
and 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-heptadecenoyl (100 µM) as
substrate. The reactions were stopped by adding chloroform/methanol (1:1, vol/vol) containing
[2H4]oleoylethanolamide (100 pmol/reaction). After centrifugation at 1,500xg at 4˚C for 15 min,
the organic layers were collected and dried under nitrogen. The residues were suspended in 50
µl of methanol and analyzed by LC/MS. Fatty acid ethanolamides were eluted on a XDB Eclipse
C18 column (50 x 4.6 mm, inner diameter 1.8 µm, Zorbax, Agilent Technologies, Inc.) using a
linear gradient of methanol in water (from 85% to 90% methanol in 2.5 min) at a flow rate of 1.5
ml/min. Column temperature was kept at 40C. MS detection was in positive ionization mode,
capillary voltage was set at 4000 V, and fragmentor voltage was 150 V. Nitrogen was used as
drying gas at a flow rate of 13 L/min and a temperature of 350C. Nebulizer pressure was set at
Holman et al., Acute stress, endocannabinoids, and the heart: Supplemental Digital Content 1
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60 psig. For quantification purposes, we monitored the [M+Na]+ ions of [2H4]-oleoylethanolamide
(m/z = 352) and N-heptadecenoylethanolamide (m/z = 334) (9,14).
Fatty acid amide hydrolase (FAAH) activity. Reactions were conducted at 37°C for 30
min in Tris-HCl buffer (50 mM, pH 7.5, 0.5 ml) containing fatty acid-free bovine serum albumin
(0.05%), protein (100 µg) and anandamide[ethanolamine-3H] (10,000 dpm, specific activity 20
Ci/mmol). After stopping the reactions with chloroform/methanol (1:1, vol/vol, 1 ml) and
centrifugation at 1,500xg at 4˚C for 15 min, radioactivity was measured in the aqueous layers by
liquid scintillation counting (15,16).
Monoacylglycerol lipase (MGL) activity. MGL activity was determined at 37°C for 10
min in Tris-HCL buffer (50 mM pH 8.0, 0.5 ml) containing fatty acid-free bovine serum albumin
(0.05%), protein (10 µg/reaction) and C17:1 monoheptadecenoinglycerol (10 µM/reaction) as
substrate. The reactions were stopped by adding 1.5 ml of cold chloroform/methanol (2:1,
vol/vol) containing C17:0 heptadecanoic acid 5 µM/reaction). After centrifugation at 1,500xg at
4˚C for 5 min, the organic layers were collected and dried under nitrogen. The residues were
suspended in 100 µl of methanol and analyzed by LC/MS. FFA were eluted on a XDB Eclipse
C18 column (50 x 4.6–mm inner diameter, 1.8 µm particle size, 80 Å of porous diameter,
Zorbax, Agilent Technologies, Inc.). Mobile phase A consisted of methanol containing 0.25%
acetic acid and 5 mM ammonium acetate; mobile phase B consisted of water containing 0.25%
acetic acid and 5 mM ammonium acetate. Lipids were eluted using a linear gradient from 90% A
to 100% B in 2.5 min at a flow rate of 1.5 ml/min with column temperature at 40C. ESI was in
the negative mode, capillary voltage was set at -4 kV and fragmentor voltage was 120V.
Nitrogen was used as drying gas at a flow rate of 13 L/min and a temperature of 350C.
Nebulizer pressure was set at 60 psig. FFA were analyzed monitoring the m/z of the
deprotonated molecular ions [M-H]- in SIM mode. For quantification purposes, we monitored the
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267 (m/z) for the reaction product, C17:1 heptadecenoic acid, and 269 (m/z) for C17:0
heptadecanoic acid (17).
Diacylglycerol lipase (DGL) activity. Reactions were conducted at 37°C for 30 min in
Tris-HCL buffer (50 mM, pH 7.0, 0.2 ml) containing Triton X-100 (0.1%), protein (10 µg/reaction)
and C17:1 diheptadecenoinglycerol (50 µM/reaction) as substrate. The reactions were stopped
by adding 0.6 ml of cold chloroform/methanol (2:1, vol/vol) containing [2H8]-2-AG as internal
standard (25 pmol/reaction). After centrifugation at 1,500xg at 4˚C for 5 min, the organic layers
were collected and dried under nitrogen. The residues were suspended in 60 µl of methanol and
analyzed by LC/MS. Lipids were eluted on a XDB Eclipse C18 column (50 x 4.6 mm, inner
diameter 1.8 µm, Zorbax, Agilent Technologies, Inc.) using a linear gradient of methanol in
water (from 85% to 90% methanol in 2.5 min) at a flow rate of 1.5 ml/min. Column temperature
was kept at 40C. MS detection was in positive ionization mode, capillary voltage was set at
4000 V, and fragmentor voltage was 150 V. Nitrogen was used as drying gas at a flow rate of
13 L/min and a temperature of 350C. Nebulizer pressure was set at 60 psig. The quantification
was done by monitoring the [M+Na]+ ions for the reaction product monoheptadecanoinglycerol
(m/z = 365) and [2H8]-2-AG (m/z = 409) (18).
N-acylethanolamine-hydrolyzing acid amidase (NAAA) activity. Heart tissue was
homogenized on ice and transferred into ice-cold Tris-HCl buffer (20 mM, pH 7.4) containing
0.32 M sucrose (final volume-to-weight ratio, 9:1). Samples were centrifuged at 1000xg for 10
min at 4°C, and the supernatants were ultra-centrifuged at 12,000xg for 30 min at 4°C. The
pellets were suspended in ice-cold PBS (pH 7.4), subjected to one freeze/thaw cycle at -80°C,
sonicated (5 seconds, amplitude 50%) in a digital sonifier (Branson Ultrasonics, Danbury, CT)
and frozen again. Suspensions were centrifuged at 105,000xg for 1-h at 4°C and the
supernatants were kept. Protein concentration was measured and samples stored at -80°C until
use. Proteins (50 µg/reaction) were incubated in NAAA assay buffer [0.1 M NaH2PO4, 0.1 M
Holman et al., Acute stress, endocannabinoids, and the heart: Supplemental Digital Content 1
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sodium citrate, 0.1% NP-40, 3 mM dithiothreitol (DTT), pH 4.0] for 30 min at 37°C in the
presence of substrate (10-cis-heptadecenoylethanolamide, 50 µM) at 37°C for 30 min.
Reactions were terminated by the addition of 0.6 ml ice-cold methanol/chloroform (1:2)
containing 100 pmol heptadecanoic acid as internal standard. Organic phases were collected,
dried under nitrogen and suspended in 100 µl of methanol/chloroform (3:1). Heptadecenoic
(17:1) and heptadecanoic (17:0) acids were eluted on an Eclipse XDB-C18 column (Agilent
Technologies, Santa Clara, CA) isocratically at 2.2 ml/min for 1 min with a solvent mixture of
95% methanol and 5% water, both containing 0.25% acetic acid and 5mM ammonium acetate.
The column temperature kept at was 50°C. Electrospray ionization was in the negative mode,
capillary voltage was 4 kV, and fragmentor voltage was 100 V. N2 was used as drying gas at a
flow rate of 13 L/min and a temperature of 350°C. Nebulizer pressure was set at 60 psig. We
monitored [M-H]- in the selected-ion monitoring mode using heptadecanoic acid as internal
standard. Calibration curves were generated using commercial 17:0 and 17:1 acids (m/z = 269
and m/z = 267, respectively) (14).
RNA extraction and gene expression analysis. Total RNA was extracted from heart
with Trizol® (Life Technologies, Grand Island, NY) and purified by using the RNeasy® Plus Mini
Kit (Quiagen, Valencia, CA). RNA (2 µg) was then translated into cDNA using the High Capacity
cDNA Reverse Transciption Kit (Life Technologies, Grand Island, NY). mRNA expression of
genes of interest was determined by quantitative RT-PCR with Taqman® PCR Master Mix
(Applied Biosystems-Roche, Carlsbad, CA) in a Mx3000P plate thermocycler (Stratagene, La
Jolla, CA) and normalized to β-actin expression. TaqMan gene expression assays for β-actin,
sterol regulatory element-binding protein-1 (SREBP-1, Rn01495769_m1), carnitine
palmitoyltransferase-1β (CPT-1β, Rn00682395_m1) caspase-3 (Rn00563902_m1), interleukin1β (IL-1β, Rn00580432_m1) and interleukin-6 (IL-6, Rn01410330_m1) were from Applied
Biosystems-Roche (Carlsbad, CA). Primers and probes for glyceraldehyde 3-phosphate
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dehydrogenase (GAPDH), peroxisome proliferator-activated receptor gamma (PPAR-γ), cluster
of differentiation 36 (CD36), lipoprotein lipase (LPL), fatty acid synthase (FAS), NAPE-PLD1, Nacylethanolamine-hydrolyzing acid amidase (NAAA), fatty acid amide hydrolase (FAAH),
monoacylglycerol lipase (MGL), diacylglycerol lipase-β (DGL-β) and tumor necrosis factor-α
(TNF-α) were from TIB Molbiol (Adelphia, NJ).
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