1
Regula ion o adipogenesis by ce amide 1-phospha e
Ma a O doñez, Na alia P esa, Asie Dominguez-He e a, Miguel T ueba, and An onio
Gomez-Muñoz*
Depa men o Biochemis y and Molecula Biology, Facul y o Science and
Technology, Uni e si y o he Basque Coun y (UPV/EHU), 48080 Bilbao, Spain.
*Co esponding au ho : Telephone: 34-94-601 2455; FAX: 34-94-601 3500 E-mail:
[email p o ec ed]s
ABSTRACT
We showed p e iously ha ce amide kinase (Ce K) exp ession inc eases du ing
adipogenesis poin ing o a ele an ole o in acellula C1P in his p ocess. In he
p esen wo k we demons a e ha adminis a ion o exogenous C1P inhibi s he
di e en ia ion o 3T3-L1 p e-adipocy es in o ma u e adipocy es h ough a mechanism
in ol ing ac i a ion o ex acellula ly egula ed kinases (ERK) 1-2. Exogenous C1P
educed he accumula ion o lipid d ople s and he con en o iacylglyce ol in hese
cells, and po en ly inhibi ed he exp ession o he ea ly and la e adipogenic ma ke s
C/EBP and PPAR, espec i ely. C1P also educed he sec e ion o lep in, which is a
c ucial egula o o ene gy balance and appe i e in he o ganism, and is conside ed o be
a la e ma ke o adipogenesis. In e es ingly, all o hese C1P ac ions we e e e sed by
pe ussis oxin, sugges ing he in e en ion o a Gi p o ein-coupled ecep o p e iously
iden i ied o C1P, in his p ocess. Also, exogenous C1P signi ican ly educed Ce K
ac i i y. Al oge he , he da a p esen ed in his wo k sugges ha exogenous C1P may
balance adipogenesis, and ha a ge ing Ce K may be a no el way o po en ial
applica ions in he ea men o obesi y o o he in lamma ion-associa ed diseases.
This is he accep ed manusc ip o he a icle ha appea ed in inal o m in Expe imen al Cell Resea ch 372(2) : 150-157
(2018), which has been published in inal o m a h ps://doi.o g/10.1016/j.yexc .2018.09.021. © 2018 Else ie unde CC BY-
NC-ND license (h p://c ea i ecommons.o g/licenses/by-nc-nd/4.0/)
2
G aphical abs ac :
ADIPOGENESIS
C1P
GPCR
(+)
(-)
Ce amide
G aphical Abs ac
Abb e ia ions:
AIM, adipogenic induc ion medium; BSA, bo ine se um albumin; Ce K, ce amide
kinase; C1P, ce amide 1-phospha e; EBP, enhance binding p o ein ; FBS, e al
bo ine se um; GAPDH, Glyce aldehide 3-phospha e dehyd ogenase; GM, g ow h
medium; IBMX, 3-isobu yl-1-me hylxan hine; PPAR, pe oxisome p oli e a o -
ac i a ed ecep o gamma; P x, pe ussis oxin; TG, iacylglyce ol
Keywo ds:
Adipogenesis; ce amides; ce amide kinase; ce amide 1-phospha e; sphingolipids
1. In oduc ion
Adipogenesis is he p ocess by which undi e en ia ed p e-adipocy es a e
con e ed o di e en ia ed adipocy es. The whole p ocess is complex and is
3
accompanied by changes in cell mo phology and gene exp ession o ansc ip ion
ac o s such as CCAAT/enhance binding p o eins (C/EBPs) and pe oxisome
p oli e a o -ac i a ed ecep o g (PPAR) [1]. Ea ly s ages o adipogenesis a e
con olled by membe s o he EBP ansc ip ion ac o s, such as C/EBP, whe eas la e
s ages o adipocy e di e en ia ion a e mainly con olled by PPAR, which is conside ed
he mas e egula o o adipogenesis [2]. Hype plasia caused by adipocy e p oli e a ion
and hype ophy caused by adipogenesis a e he main easons o a deposi ion in i o,
he majo i y o i being s o ed as iacylglyce ol (TG). Howe e , he adipose issue is
also an endoc ine o gan ha egula es c ucial pa hophysiological p ocesses h ough
sec e ion o speci ic ho mones such as lep in [3], o adiponec in [4]. Adipose cells can
also sec e e p o- and an i-in lamma o y cy okines, ac ions ha when uncon olled, can
lead o insulin esis ance, ype II diabe es and obesi y. Hence, deciphe ing he
mechanisms ha a e in ol ed in adipocy e di e en ia ion is essen ial o unde s anding
he p ocesses ha a e implica ed in he onse and de elopmen o obesi y and obesi y-
associa ed diseases.
Se e al lines o e idence sugges ha sphingolipids o he enzymes in ol ed in
hei me abolism egula e i al cellula unc ions including cell p oli e a ion, apop osis,
au ophagy, o cell di e en ia ion [5-11]. No ewo hy, some sphingolipids a e implica ed
in in lamma o y esponses [12-14] and in lamma ion-associa ed diseases such as
a he oscle osis, cance , diabe es, o obesi y. In pa icula , ce amides ha e been
associa ed wi h insulin esis ance and he de elopmen o ype II diabe es, and
sphingosine and sphingosine 1-phospha e (S1P) le els a e al e ed in he obese s a e [15,
16]. Ano he impo an ce amide me aboli e is ce amide 1-phospha e (C1P), which is
o med by he ac ion o ce amide kinase (Ce K) on ce amide. Ini ially, C1P was
demons a ed o be a ele an egula o o cell p oli e a ion and su i al [17-24], and o
4
ac i ely pa icipa e in in lamma o y esponses [12-14, 25-28]. Howe e , he
mechanisms o signaling pa hways by which C1P exe s i s biological ac ions ha e only
been pa ially desc ibed. I is known ha in acellula ly gene a ed C1P can ac di ec ly
on in acellula a ge s, such as acidic sphingomyelinase (A-SMase) [23], se ine
palmi oyl ans e ase (SPT) [29], o ype IV phospholipase A2 [13, 14] o modula e cell
su i al o in lamma ion, whe eas exogenous C1P, which is p esen in plasma, can
in e ac wi h a pu a i e Gi p o ein-coupled ecep o o p omo e cell mig a ion [30-35]
and glucose up ake [36].
The p esen wo k was unde aken o elucida e whe he C1P migh be able o
egula e adipogenesis, and o s udy he mechanisms in ol ed in his ac ion.
2. Ma e ials and me hods
2.1. Ma e ials
The Dulbecco´s modi ied Eagle´s cul u e medium (DMEM) used in expe imen s
was pu chased om Lonza. The iacylglyce ol assay ki was ob ained om Abno a.
NBD-Ce amide (NBD-N-hexanoyl-D-e y h o-sphingosine) was om Cayman
Chemicals. Dexame hasone, pe ussis oxin, osigli azone, insulin, 3-Isobu yl-1-
me hylxan hine (IBMX) and he Oil Red O dye we e pu chased om Sigma-Ald ich.
Ni ocellulose memb anes, p o ein ma ke s, and BCA assay eagen s we e ob ained
om Bio-Rad. Fe al bo ine se um (FBS) and newbo n cal se um (NCS) we e om
GIBCO. The ELISA ki o de e mina ion o lep in was pu chased om P ep o ech. The
PPARγ and EBP an ibodies we e supplied by Cell Signaling. The GAPDH an ibody,
non- a ge ing (nega i e) siRNA and ce amide kinase (Ce K) siRNA we e pu chased
om San a C uz Bio echnology. The Ce K an ibody was om Calbiochem o Abgen .
5
The es o chemicals and eagen s used in his wo k we e o he highes g ade
a ailable.
2.2. Cell cul u e
The mouse p e-adipocy e ( ib oblas ) 3T3-L1 cell line was pu chased om
Ame ican Type Cul u e Collec ion (ATCC) (Manassas, VA, USA) and was cul u ed
ollowing he manu ac u e ’s indica ions. The cells we e g own in 175 cm2 plas ic
lasks in DMEM supplemen ed wi h 10% hea -inac i a ed newbo n cal se um (NCS),
50 mg/l gen amicin, 200 µM L-glu amine and 4.5 g/l o glucose. Cells we e incuba ed
in a humidi ied 5% CO2 incuba o a 37 °C and we e subcul u ed e e y 3-4 days. Cells
we e used in expe imen s a abou 80-100% con luence.
2.3. 3T3-L1 p e-adipocy e di e en ia ion p o ocol
The 3T3-L1 p e-adipocy es we e seeded in 6-well pla es a 1.2 x 105cells/well,
o in 24-well pla es a 6 x 104 cells/well, o in 96-well pla es a 9 x 103 cells/well
depending on he kind o expe imen o be pe o med. Cells we e hen cul u ed in
DMEM supplemen ed wi h 10% NCS un il hey we e abou 90-100% con luen . The
cells we e u he incuba ed o 2 days. P e-adipocy es we e ea ed wi h adipogenic
induc ion medium (AIM), which is a medium consis ing o DMEM 10% FBS
supplemen ed wi h an adipogenic cock ail con aining 0.5 mM 3-isobu yl-1-
me hylxan hine (IBMX), 1 g/ml insulin, 0.25 M dexame hasone and 2 M
osigli azone. Two days la e , he medium was emo ed and cells we e incuba ed
u he in main enance medium (DMEM 10% FBS + 1 g/ml insulin) o wo addi ional
days. The cells we e hen ed e e y wo days wi h DMEM supplemen ed wi h 10% FBS
and 1 g/ml insulin.
6
2.4. Wes e n blo ing
P e-adipocy es we e ha es ed and lysed in ice-cold homogeniza ion bu e o
analyze p o eins by Wes e n-blo ing, essen ially as desc ibed in [37]. Speci ically, 3T3-
L1 cells we e seeded a 1.2 x 105 cells/well in 6-well pla es and we e di e en ia ed
ollowing he abo e desc ibed p e-adipocy e di e en ia ion p o ocol (sec ion 2.3).
Wes e n-blo ing was pe o med as de ailed in [33]. Abou 20-40 μg o p o ein om
each sample was loaded and sepa a ed by sodium dodecyl sul a e polyac ylamide gel
elec opho esis (SDS-PAGE), using 12% sepa a ing gels. P o eins we e ans e ed on o
ni ocellulose memb anes and blocked wi h 5% skim milk o 1 h in T is-bu e ed saline
(TBS) con aining 0.1% Tween 20, and hen incuba ed o e nigh wi h he p ima y
an ibody in TBS-0.1% Tween 20 a 4 ºC. A e h ee washes wi h TBS-0.1% Tween 20,
memb anes we e incuba ed wi h ho se adish pe oxidase-conjuga ed seconda y an ibody
a 1:4000 dilu ion o 1 h. Bands we e isualized by enhanced chemiluminescence and
exposed ilms we e analyzed wi h an ImageJ so wa e.
2.5. Oil Red O s aining p o ocol
3T3-L1 p e-adipocy es we e seeded a 6 x 104 cells/well in 24-well pla es and
di e en ia ed ollowing he abo e desc ibed p e-adipocy e di e en ia ion p o ocol
(sec ion 2.3). In acellula lipid accumula ion was de e mined using a solu ion o Oil
Red O. B ie ly, cells we e washed wi h PBS and ixed in 3.8% o maldehyde o 10
min. They we e hen washed and s ained wi h a solu ion con aining Oil Red O (3
mg/ml) and isop opanol in wa e (60/40, / ) o 20 min a oom empe a u e. S ained
cells we e washed wice wi h wa e and pho og aphed wi h a Nikon Eclipse TS100
mic oscope. To assess he deg ee o di e en ia ion, 200 μl isop opanol was added and
incuba ed o 30 min in a pla e shake . Then, 50 μl o Oil Red O ex ac ed dye was
7
ans e ed in o 96-well pla es and quan i ied by eading he abso bance a a wa eleng h
o 510 nm. The dye ex ac ed om he emp y wells ep esen ed he non-speci ic binding
o he dye o he pla e. The non-speci ic binding alue was sub ac ed om he
abso bance o each expe imen al condi ion o ob ain accu a e measu emen s o speci ic
s aining.
2.6. Measu emen o iacylglyce ol concen a ion
3T3-L1 p e-adipocy es we e seeded a 9 x 103 cells/well in 96-well pla es and
di e en ia ed ollowing he abo e desc ibed p e-adipocy e di e en ia ion p o ocol
(sec ion 2.3). T iacylglyce ol (TG) con en in cells was de e mined using an ELISA ki
( om Abno a) ollowing he manu ac u e ’s ins uc ions. B ie ly, cells we e washed
wi h PBS and 100 μl o he lipid ex ac ion solu ion was added o each well. Then, he
pla es we e incuba ed in a hea ing block a 90-100ºC o 30 min. A e his ime, 50
l/well o s anda d dilu ions o TG o 5-50 μl o he lipid ex ac s we e ans e ed in o
96-well pla es. Assay bu e was added whe e necessa y o b ing he olume up o 50 μl
in all wells. Then, 2 μl o lipase solu ion was added o each well con aining ei he
sample o s anda d, and he whole pla e was incuba ed o 10 min a oom empe a u e.
Subsequen ly, 50 μl o he eac ion mix u e (46 μl adipogenesis assay bu e + 2 μl
P obe + 2 μl Enzyme mix) was added o each well and incuba ed a 37 ºC o 30 min in
he da k. The abso bance was ead a 570 nm in a pla e eade . The p o ein
concen a ion o he lipid ex ac s was de e mined using a comme cial ki con aining
bicinchonic acid (BCA), om BioRad, and he alues we e used as in e nal con ols o
no malize he lipid concen a ion in he samples.
2.7. De e mina ion o ce amide kinase ac i i y
8
Ce amide kinase (Ce K) ac i i y was essen ially de e mined as desc ibed by Don
and Rosen [38]. 3T3-L1 cells we e seeded in 6-well pla es a 1.2 x 105 cells/well and
di e en ia ed in he absence o in he p esence o Ce K siRNA, ollowing he p e-
adipocy e di e en ia ion p o ocol desc ibed abo e (sec ion 2.3). Cell lysa es (50-100 μg
o p o ein) we e mixed wi h eac ion bu e (100 μl, 20 mM Hepes (pH 7.4), 10 mM
KCl, 15 mM MgCl2, 15 mM CaCl2, 10% glyce ol, 1 mM DTT, 1 mM ATP) con aining
10 μM o NBD-C6-ce amide (N-hexanoyl-D-e y h o-sphingosine). The eac ions we e
allowed o p oceed o 20 min in he da k a 35ºC. Then, 250 μl aliquo s o
chlo o o m:me hanol (2:1) we e added o e mina e he eac ions. Samples we e hen
cen i uged a 21,800 g o 30 s and 100 μl aliquo s o he uppe aqueous phase we e
ans e ed in o 96-well pla es. Subsequen ly, 100 µl aliquo s o dime hyl o mamide
(DMF) we e added be o e eading he NBD luo escence in an app op ia e pla e eade .
Fluo escence was quan i ied wi h a 495 nm exci a ion il e and a 520 nm emission
il e wi h a Syne gy HT (Bio ek) pla e eade equipped wi h Gen5 so wa e.
2.8. De e mina ion o lep in sec e ion
Lep in concen a ion in he cul u e medium was de e mined using a “Mouse
S anda d ELISA De elopmen Ki ” (Pep o ech) ollowing he manu ac u e ’s
indica ions.
2.9. S a is ical analyses
Resul s a e exp essed as means ± SEM o h ee o six independen expe imen s
pe o med in duplica e unless indica ed o he wise. S a is ical analyses we e pe o med
using he wo- ailed, pai ed S uden ’s - es , whe e p<0.05 was conside ed o be
signi ican (G aphPad P ism so wa e, San Diego, CA) [33].
9
3. Resul s
3.1. Exogenous C1P inhibi s p e-adipocy e di e en ia ion in o ma u e adipocy es.
We ha e ecen ly shown ha Ce K is up egula ed du ing adipogenesis [39],
sugges ing a ele an ole o in acellula C1P in his p ocess. Howe e , as men ioned
abo e, C1P is p esen in plasma and can ac h ough in e ac ion wi h a pu a i e plasma
memb ane ecep o o modula e a a ie y o cell unc ions [30, 31, 35]. To examine he
e ec s o exogenous C1P on p e-adipocy e di e en ia ion, con luen 3T3-L1 cells we e
g own in g ow h medium (GM) o adipogenic induc ion medium (AIM) o en days.
Adminis a ion o C1P a concen a ions ha a e ound in i o (0.5 – 20 µM) [40-42]
dec eased he accumula ion o lipid d ople s, which we e isualized using Oil Red O
s aining, in a ime dependen manne in hese cells (Fig. 1). In addi ion, he con en o
TG, which was inc eased in di e en ia ed cells compa ed o p e-adipocy es, was
subs an ially dec eased by adminis a ion o C1P (Fig. 2), also indica ing ha his
phosphosphingolipid inhibi s adipocy e di e en ia ion.
3.2 C1P inhibi s he exp ession o ea ly and la e adipogenic ma ke s.
The p ocess o adipocy e di e en ia ion is di ided in o ea ly, in e media e, and
la e s ages. Ea ly s ages o adipogenesis a e con olled by membe s o he EBP
ansc ip ion ac o s, such as C/EBP, whe eas la e s ages o adipocy e di e en ia ion
a e mainly con olled by PPAR. Fig. 3 shows ha ea men wi h C1P caused a
signi ican educ ion in he le els o C/EBP phospho yla ion (panels A, B) as well as a
signi ican dec ease in PPAR exp ession (panels C, D), sugges ing ha C1P p omo es
i s an i-adipogenic ac i i y by down egula ing hese ansc ip ion ac o s. C1P did no
16
concen a ion was measu ed 10 days a e inducing cell di e en ia ion as desc ibed in
sec ion 2.5. Resul s a e no malized o p o ein concen a ion o each sample and a e he
mean ± SEM o 4 independen expe imen s pe o med in iplica e (cells incuba ed in
GM s cells incuba ed in AIM in he absence o C1P, ***p<0.001; cells incuba ed in
AIM in he absence o C1P s cells incuba ed in AIM in he p esence o 20 µM C1P,
##p<0.01).
Figu e 3. C1P dec eases EBP and PPAR exp ession. 3T3-L1 cells we e seeded in
6-well pla es (1.2 x 105 cells/well) and g own in DMEM con aining 10% NCS un il
hey we e abou 90-100% con luen . The cells we e u he incuba ed o 2 days be o e
inducing cell di e en ia ion. A e his ime, cells we e cul u ed in g ow h medium
(GM) o adipogenic induc ion medium (AIM), wi h o wi hou C1P. The medium was
changed e e y 2 days and C1P o ehicle we e added each ime. A. On day 4, cell
lysa es we e p epa ed and C/EBPβ phospho yla ion was de ec ed by Wes e n blo ing
using a speci ic an ibody agains p-C/EBPβ. Equal loading o p o ein was assessed wi h
an an ibody agains GAPDH. Simila esul s we e ob ained in each o 3 independen
expe imen s. B. Resul s o he scanning densi ome y o exposed ilm. Da a a e
exp essed as a bi a y uni s o in ensi y ela i e o GAPDH and a e he mean ± SEM o
3 independen expe imen s (cells incuba ed in GM s cell incuba ed in AIM in he
absence o C1P, *p<0.05; cells incuba ed in AIM in he absence o C1P s cells
incuba ed in AIM in he p esence o 20 µM C1P, ##p<0.01). C. Cell lysa es we e
p epa ed om cells ha we e di e en ia ed o 7 days and PPARγ exp ession was
de ec ed by Wes e n blo ing using a speci ic an ibody agains PPARγ. Equal loading o
p o ein was assessed wi h an an ibody agains GAPDH. Simila esul s we e ob ained in
each o 5 eplica e expe imen s. D. Resul s o he scanning densi ome y o exposed
17
ilm. Da a a e exp essed as a bi a y uni s o in ensi y ela i e o GAPDH and a e he
mean ± SEM o 5 independen expe imen s (cells incuba ed in GM s cell incuba ed in
AIM in he absence o C1P, ***p<0.001; cells incuba ed in AIM in he absence o C1P
s cells incuba ed in AIM in he p esence o 20 µM C1P, #p<0.05).
Figu e 4. C1P educes lep in sec e ion. 3T3-L1 cells we e seeded in 6-well pla es (1.2
x 105 cells/well) and hey we e g own in DMEM con aining 10% NCS un il hey we e
abou 90-100% con luen . Cells we e u he incuba ed o 2 days be o e inducing cell
di e en ia ion. A. A e his ime, cells we e incuba ed in adipogenic induc ion media
(AIM) in o de o induce cell di e en ia ion, as desc ibed in sec ion 2.5. The cul u e
medium was collec ed a he indica ed ime poin s, and cen i uged. Lep in
concen a ion in supe na an s was measu ed using an ELISA ki , as indica ed in sec ion
2.5. The cells we e ha es ed in lysis bu e in o de o measu e p o ein concen a ion.
Resul s a e no malized o he p o ein concen a ion and a e he mean ± SEM o 4
independen expe imen s pe o med in duplica e (*p<0.05). B. Cells we e incuba ed in
adipogenic induc ion media (AIM) o wo days in he absence o in he p esence o 20
μM o C1P. The medium was changed e e y 2 days and C1P o ehicle we e added
each ime. On day 10, he cul u e medium was collec ed, and cen i uged. Lep in
concen a ion in supe na an s we e measu ed using an ELISA ki , as indica ed in sec ion
2.5. Cells we e ha es ed in lysis bu e in o de o measu e p o ein concen a ion.
Resul s a e no malized o he p o ein concen a ion and a e he mean ± SEM o 5
independen expe imen s pe o med in duplica e (#p<0.05).
Figu e 5. C1P induces ERK 1-2 phospho yla ion in 3T3-L1 cells. P e-adipocy es
we e seeded in 6-well pla es (1.2 X 105 cells/well) and g own in DMEM con aining
18
10% NCS un il hey we e abou 90-100% con luen . Cells we e u he incuba ed o 2
days be o e inducing cell di e en ia ion. A e his ime, cells we e incuba ed in
adipogenic induc ion medium (AIM) wi h o wi hou C1P, as indica ed. A. Cells we e
ha es ed a e he indica ed pe iods o ime, and ERK phospho yla ion was de ec ed by
Wes e n-blo ing using a speci ic an ibody agains phospho yla ed (p) ERK1-2. Cells
om day 0 we e collec ed a e 1 h o incuba ion in AIM. Equal loading o p o ein was
assessed wi h an ibodies o GAPDH o o al ERK, as indica ed. Simila esul s we e
ob ained in each o 3 eplica e expe imen s. B. Resul s o he scanning densi ome y o
he exposed ilm. Da a a e exp essed as a bi a y uni s o in ensi y ela i e o GAPDH
and a e he mean ± SEM o 3 independen expe imen s (*p<0.05). C. ERK
phospho yla ion, o al ERK and GAPDH we e de ec ed as in B. D. Resul s o he
scanning densi ome y o he exposed ilm. Da a a e exp essed as a bi a y uni s o
in ensi y ela i e o GAPDH and a e he mean ± SEM o 5 independen expe imen s
(*p<0.05; **p<0.01).
Figu e 6. Inhibi ion o ERK 1-2 phospho yla ion dec eases TG le els in 3T3-L1
cells. P e-adipocy es we e seeded in 6-well pla es (1.2 x 104 cells/well) o quan i y lipid
accumula ion, o in 96-well pla es (9 x 103 cells/well) o measu emen o TG
concen a ion. Cells we e incuba ed as indica ed in igu e 5, and we e p e ea ed wi h
ehicle o wi h he speci ic MEK inhibi o PD98059 o 1 h p io o induc ion o
adipogenesis. When p esen , C1P was a 20 µM. A. Cells we e s ained wi h Oil Red O,
and lipid accumula ion in he cells was de e mined as indica ed in sec ion 2.5. Fo
quan i a i e analysis, he dye was dissol ed in isop opanol. Resul s a e exp essed
ela i e con ol (AIM) alues and he abso bance o emp y wells (wi hou cells) was
sub ac ed om he abso bance o expe imen al wells. Resul s a e he mean ± SEM o 5
19
independen expe imen s pe o med in iplica e (cells incuba ed in GM s cells
incuba ed in AIM in he absence o C1P, ***p<0.001; cells incuba ed in adipogenic
induc ion medium (AIM) in he absence o C1P s cells incuba ed in AIM in he
p esence o 20 µM C1P, ###p<0.001; cells incuba ed in AIM in he p esence o 20 µM
C1P s cells incuba ed in AIM in he p esence o 20 µM C1P and 10 o 20 µM o
PD98059, as indica ed, *P<0.05, **P<0.01). B. TG concen a ion was measu ed 10
days a e inducing cell di e en ia ion as desc ibed in sec ion 2.5. Resul s a e
no malized o p o ein concen a ion o each sample and a e he mean ± SEM o 3
independen expe imen s pe o med in iplica e (cells incuba ed in GM s cells
incuba ed in AIM in he absence o C1P, *p<0.05; cells incuba ed in adipogenic
induc ion medium (AIM) in he absence o C1P s cells incuba ed in AIM in he
p esence o 20 µM C1P, #p<0.05; cells incuba ed in AIM in he p esence o 20 µM C1P
s cells incuba ed in AIM in he p esence o 20 µM C1P and 10 o 20 µM o PD98059,
as indica ed, *P<0.05, **P<0.01). The e was no s a is ically signi ican di e ences
be ween cells ea ed wi h C1P and PD98059 in cells g own in GM.
Figu e 7. E ec o pe ussis oxin (P x) on C1P-induced educ ion o lipid
accumula ion, TG concen a ion and ERK phospho yla ion. Cells we e seeded and
ea ed as in igu es 1, 2 and 5 o de e mina ion o lipid accumula ion, TG
concen a ion, o ERK phospho yla ion, espec i ely. When p esen , C1P was a 20 µM.
In expe imen al condi ions con aining P x (0.1 µg/ml), he cells we e p e-incuba ed wi h
he oxin o 16 h p io o addi ion o C1P o ehicle. A. Oil Red O dye was dissol ed in
isop opanol and abso bance was measu ed o quan i y neu al lipid accumula ion. The
abso bance o emp y wells (wi hou cells) was sub ac ed om he abso bance o
expe imen al wells. Resul s a e he mean ± SEM o 5 independen expe imen s
20
pe o med in iplica e (cells incuba ed in GM s cells incuba ed in AIM in he absence
o C1P and P x, ***p<0.001; cells incuba ed in AIM in he absence o C1P s cells
incuba ed in AIM in he p esence o 20 µM C1P, ###p<0.001; cells incuba ed in AIM
in he p esence o 20 µM C1P and 0.1 µg/ml P x, *p<0.05; di e ences be ween cells
incuba ed in AIM in he absence o C1P and in he p esence o 0.1 µg/ml P x we e no
s a is ically signi ican , n.s.). B. TG concen a ion was measu ed 10 days a e inducing
cell di e en ia ion as desc ibed in sec ion 2.5. Resul s a e no malized o p o ein
concen a ion o each sample and a e he mean ± SEM o 3 independen expe imen s
pe o med in iplica e (cells incuba ed in GM s cells incuba ed in AIM in he absence
o C1P and P x, *p<0.05; cells incuba ed in AIM in he absence o C1P s cells
incuba ed in AIM in he p esence o 20 µM C1P, #p<0.05; cells incuba ed in AIM in
he p esence o 20 µM C1P s cells incuba ed in AIM in he p esence o 20 µM C1P
and 0.1 µg/ml P x, *p<0.05. The e ec o P x on TG concen a ion was no s a is ically
signi ican ). C. ERK phospho yla ion was de ec ed as in igu e 5. Simila esul s we e
ob ained in each o 5 eplica e expe imen s. D. Resul s o scanning densi ome y o
exposed ilms. Da a a e exp essed as a bi a y uni s o in ensi y ela i e o GAPDH and
a e he mean ± SEM o i e independen expe imen s (cells incuba ed in AIM in he
absence o C1P and P x s cells incuba ed in AIM in he p esence o 20 µM C1P,
**p<0.05; cells incuba ed in AIM in he p esence o 20 µM C1P s cells incuba ed in
he p esence o 20 µM C1P and 0.1 µg/ml P x, ##p<0.05).
21
Re e ences
[1] Y.J. Jang, H.J. Koo, E.H. Sohn, S.C. Kang, D.K. Rhee, S. Pyo, Theob omine
inhibi s di e en ia ion o 3T3-L1 cells du ing he ea ly s age o adipogenesis ia
AMPK and MAPK signaling pa hways, Food Func 6 (2015) 2365-2374.
[2] K. Sa jean , J.M. S ephens, Adipogenesis, Cold Sp ing Ha b Pe spec Biol 4
(2012) a008417.
[3] Y. Zhang, R. P oenca, M. Ma ei, M. Ba one, L. Leopold, J.M. F iedman,
Posi ional cloning o he mouse obese gene and i s human homologue, Na u e
372 (1994) 425-432.
[4] P.E. Sche e , S. Williams, M. Fogliano, G. Baldini, H.F. Lodish, A no el se um
p o ein simila o C1q, p oduced exclusi ely in adipocy es, J Biol Chem 270
(1995) 26746-26749.
[5] A. Gomez-Munoz, N. P esa, A. Gomez-La au i, I.G. Ri e a, M. T ueba, M.
O donez, Con ol o in lamma o y esponses by ce amide, sphingosine 1-
phospha e and ce amide 1-phospha e, P og Lipid Res 61 (2016) 51-62.
[6] Y.A. Hannun, L.M. Obeid, P inciples o bioac i e lipid signalling: lessons om
sphingolipids, Na Re Mol Cell Biol 9 (2008) 139-150.
[7] A.H. Me ill, E.M. Schmel z, D.L. Dillehay, S. Spiegel, J.A. Shayman, J.J.
Sch oede , R.T. Riley, K.A. Voss, E. Wang, Sphingolipids-- he enigma ic lipid
class: biochemis y, physiology, and pa hophysiology, Toxycology and Applied
Pha macology 142 (1997) 208-225.
22
[8] A.H. Me ill, M.C. Sulla ds, W. E., K.A. Voss, R.T. Riley, Sphingolipid
me abolism: oles in signal ansduc ion and dis up ion by umonisins, En i on.
Heal h Pe spec . 109 (2001) 283-289.
[9] N. P esa, A. Gomez-La au i, I.G. Ri e a, M. O donez, M. T ueba, A. Gomez-
Munoz, Regula ion o cell mig a ion and in lamma ion by ce amide 1-
phospha e, Biochim Biophys Ac a 1861 (2016) 402-409.
[10] S. Spiegel, A.H. Me ill, Sphingolipid me abolism and cell g ow h egula ion,
FASEB J. 10 (1996) 1388-1397.
[11] J. S iban, R. Tidha , A.H. Fu e man, Ce amide syn hases: oles in cell
physiology and signaling, Ad Exp Med Biol 688 (2010) 60-71.
[12] C. Chal an , S. Spiegel, Sphingosine-1-phospha e and ce amide-1-phospha e:
expanding oles in cell signaling, J. Cell Sci. 118 (2005) 4605-4612.
[13] B.J. Pe us, A. Bielawska, S. Spiegel, P. Roddy, Y.A. Hannun, C.E. Chal an ,
Ce amide kinase media es cy okine- and calcium ionopho e-induced a achidonic
acid elease, J. Biol. Chem. 278 (2003) 38206-38213.
[14] B.J. Pe us, A. Bielawska, P. Sub amanian, D.S. Wijesinghe, M. Mayceka, C.C.
Leslie, J. F eibe g, P. Roddy, Y.A. Hannun, C.E. Chal an , Ce amide-1-
phospha e is a di ec ac i a o o cy osolic phospholipase A2., J. Biol. Chem.
279 (2004) 11320-11326.
[15] P.J. La sen, N. Tennagels, On ce amides, o he sphingolipids and impai ed
glucose homeos asis, Mol Me ab 3 (2014) 252-260.
[16] M.H. Moon, J.K. Jeong, S.Y. Pa k, Ac i a ion o S1P2 ecep o , a possible
mechanism o inhibi ion o adipogenic di e en ia ion by sphingosine
1phospha e, Mol Med Rep 11 (2015) 1031-1036.
23
[17] P. Gangoi i, L. A ana, A. Ou o, M.H. G anado, M. T ueba, A. Gomez-Munoz,
Ac i a ion o mTOR and RhoA is a majo mechanism by which Ce amide 1-
phospha e s imula es mac ophage p oli e a ion, Cell Signal 23 (2011) 27-34.
[18] P. Gangoi i, C. Be nacchioni, C. Dona i, F. Cence i, A. Ou o, A. Gomez-
Munoz, P. B uni, Ce amide 1-phospha e s imula es p oli e a ion o C2C12
myoblas s, Biochimie 94, 597-607 (2012).
[19] P. Gangoi i, M.H. G anado, L. A ana, A. Ou o, A. Gomez-Munoz, Ac i a ion o
p o ein kinase C-alpha is essen ial o s imula ion o cell p oli e a ion by
ce amide 1-phospha e, FEBS Le 584 (2010) 517-524.
[20] P. Gangoi i, M.H. G anado, S.W. Wang, J.Y. Kong, U.P. S einb eche , A.
Gomez-Munoz, Ce amide 1-phospha e s imula es mac ophage p oli e a ion
h ough ac i a ion o he PI3-kinase/PKB, JNK and ERK1/2 pa hways, Cell
Signal 20 (2008) 726-736.
[21] A. Gómez-Muñoz, P.A. Du y, A. Ma in, L. O'B ien, H.S. Byun, R. Bi man,
D.N. B indley, Sho -chain ce amide 1-phospha es a e no el s imula o s o
DNA syn hesis and cell di ision: an agonism by cell-pe meable ce amides, Mol.
Pha macol. 47 (1995) 883-889.
[22] A. Gómez-Muñoz, J.Y. Kong, K. Pa ha , S.W. Wang, P. Gangoi i, M. González,
S. Ei ema k, B. Salh, V. Du onio, U.P. S einb eche , Ce amide-1-phospha e
p omo es cell su i al h ough ac i a ion o he phospha idylinosi ol 3-
kinase/p o ein kinase B pa hway, FEBS Le . 579 (2005) 3744-3750.
[23] A. Gómez-Muñoz, J.Y. Kong, B. Salh, U.P. S einb eche , Ce amide-1-
phospha e blocks apop osis h ough inhibi ion o acid sphingomyelinase in
mac ophages, J. Lipid Res. 45 (2004) 99-105.
24
[24] A. Ou o, L. A ana, M. Riazy, P. Zhang, A. Gomez-La au i, U. S einb eche , V.
Du onio, A. Gomez-Munoz, Vascula endo helial g ow h ac o media es
ce amide 1-phospha e-s imula ed mac ophage p oli e a ion, Exp Cell Res 361
(2017) 277-283.
[25] K. Baudiss, C.K. Aya a, Z. Laza , S. Cicko, J. Becke , A. Meye , A. Zech, R.P.
Viei a, R. Bi man, A. Gomez-Munoz, I. Me o , M. Idzko, Ce amide-1-
phospha e inhibi s ciga e e smoke-induced ai way in lamma ion, Eu Respi J
45 (2015) 1669-1680.
[26] K. Baudiss, R. de Paula Viei a, S. Cicko, K. Aya a, M. Hoss eld, N. Eh a , A.
Gomez-Munoz, H.K. El zschig, M. Idzko, C1P A enua es Lipopolysaccha ide-
Induced Acu e Lung Inju y by P e en ing NF-kappaB Ac i a ion in Neu ophils,
J Immunol 196 (2016) 2319-2326.
[27] D.S. Wijesinghe, M. B en nall, J.A. Mie la, L.A. Hoe e lin, R.F. Diegelmann,
L.H. Boise, C.E. Chal an , Ce amide kinase is equi ed o a no mal eicosanoid
esponse and he subsequen o de ly mig a ion o ib oblas s, J Lipid Res 55
(2014) 1298-1309.
[28] D.S. Wijesinghe, P. Sub amanian, N.F. Lamou , L.B. Gen ile, M.H. G anado, A.
Bielawska, Z. Szulc, A. Gomez-Munoz, C.E. Chal an , Chain leng h speci ici y
o ac i a ion o cPLA2alpha by C1P: use o he dodecane deli e y sys em o
de e mine lipid-speci ic e ec s, J Lipid Res 50 (2009) 1986-1995.
[29] M.H. G anado, P. Gangoi i, A. Ou o, L. A ana, A. Gomez-Munoz, Ce amide 1-
phospha e inhibi s se ine palmi oyl ans e ase and blocks apop osis in al eola
mac ophages, Biochim Biophys Ac a 1791 (2009) 263-272.
[30] L. A ana, M. O donez, A. Ou o, I.G. Ri e a, P. Gangoi i, M. T ueba, A. Gomez-
Munoz, Ce amide 1-phospha e induces mac ophage chemoa ac an p o ein-1
25
elease: in ol emen in ce amide 1-phospha e-s imula ed cell mig a ion, Am J
Physiol Endoc inol Me ab 304 (2013) E1213-1226.
[31] M.H. G anado, P. Gangoi i, A. Ou o, L. A ana, M. Gonzalez, M. T ueba, A.
Gomez-Munoz, Ce amide 1-phospha e (C1P) p omo es cell mig a ion
In ol emen o a speci ic C1P ecep o , Cell Signal 21 (2009) 405-412.
[32] J.L. Hankins, K.E. Wa d, S.S. Lin on, B.M. Ba h, R.V. S ahelin, T.E. Fox, M.
Kes e , Ce amide 1-phospha e media es endo helial cell in asion ia he annexin
a2-p11 he e o e ame ic p o ein complex, J Biol Chem 288 (2013) 19726-19738.
[33] M. O donez, I.G. Ri e a, N. P esa, A. Gomez-Munoz, Implica ion o ma ix
me allop o einases 2 and 9 in ce amide 1-phospha e-s imula ed mac ophage
mig a ion, Cell Signal 28 (2016) 1066-1074.
[34] M.Z. Ra ajczak, M. Suszynska, S. Bo kowska, J. Ra ajczak, G. Schneide , The
ole o sphingosine-1 phospha e and ce amide-1 phospha e in a icking o
no mal s em cells and cance cells, Expe Opin The Ta ge s 18 (2014) 95-107.
[35] I.G. Ri e a, M. O donez, N. P esa, P. Gangoi i, A. Gomez-La au i, M. T ueba,
T. Fox, M. Kes e , A. Gomez-Munoz, Ce amide 1-phospha e egula es cell
mig a ion and in asion o human panc ea ic cance cells, Biochem Pha macol
102 (2016) 107-119.
[36] A. Ou o, L. A ana, P. Gangoi i, I.G. Ri e a, M. O donez, M. T ueba, R.S.
Lankalapalli, R. Bi man, A. Gomez-Munoz, Ce amide 1-phospha e s imula es
glucose up ake in mac ophages, Cell Signal 25 (2013) 786-795.
[37] J.A. Hamil on, D. Mye s, W. Jessup, F. Coch ane, R. By ne, G. Whi y, S.
Moss, Oxidized LDL can induce mac ophage su i al, DNA syn hesis, and
enhanced p oli e a i e esponse o CSF-1 and GM-CSF, A e ioscle Th omb
Vasc Biol 19 (1999) 98-105.
32
33
34
HIGHLIGHTS
Exogenous C1P inhibi s adipogenesis
ERK1-2 a e implica ed in he inhibi ion o adipogenesis by C1P
C1P inhibi ion o adipogenesis is blocked by Gi p o ein inhibi o pe ussis oxin
