Apoptosis, mastocytosis, and diminished adipocytokine gene expression accompany reduced epididymal fat mass in long-standing diet-induced obese mice

Altintas, Rossetti, Nayer Behzad, Puig Alvaro, Zagallo Patricia, Ortega, Johnson, Mcnamara George, Reiser Jochen, Mendez, Nayer Ali

Description

Obesity is characterized by increased cell death and inflammatory reactions in the adipose tissue. Here, we explored pathophysiological alterations taking place in the adipose tissue in long-standing obesity. In the epididymal fat of C57BL/6 mice fed a high-fat diet for 20 weeks, the prevalence and distribution of dead adipocytes (crown-like structures), mast cells (toluidine blue, mMCP6), macrophages (F4/80), and apoptotic cells (cleaved caspase-3) were measured. Moreover, gene and/or protein expression of several adipocytokines (leptin, adiponectin, TNF-α, IL-10, IL-6, MCP-1), F4/80, mMCP6, cleaved caspase-3 were determined. Results We observed that the epididymal fat mass was lower in obese than in lean mice. In obese mice, the epididymal fat mass correlated inversely with body weight and liver mass. Dead adipocytes, mast cells, macrophages, and apoptotic cells were abundant in the epididymal fat of obese mice, especially in the rostral vs. caudal zone. Accordingly, mMCP6, F4/80, and cleaved caspase-3 gene and/or protein expression was increased. Conversely, adiponectin, leptin, IL-6, and MCP-1 gene expression levels were lower in the epididymal fat of obese than lean mice. Although TNF-α and IL-10 gene expression was higher in the epididymal fat of obese mice, their expression relative to F4/80 and mMCP6 expression were lower in the heavily infiltrated rostral than caudal zone. Conclusions This study demonstrates that in mice with long-standing obesity diminished gene expression of several adipocytokines accompany apoptosis and reduced mass of the epididymal fat. Our findings suggest that this is due to both increased prevalence of dead adipocytes and altered immune cell activity. Differential distribution of metabolically challenged adipocytes is indicative of the presence of biologically diverse zones within the epididymal fat.

Sujets

• Obesity
• Adipose tissue
• Inflammation
• Mast cell
• Macrophage
• Apoptosis
• Adipokine

Informations

• Publié par: biomed
• Publié le: 01 janvier 2011
• Nombre de lectures: 12
• Langue: English
• Poids de l'ouvrage: 1 Mo

Extrait

Altintas
etal
.
LipidsinHealthandDisease
2011,
10
:198
http://www.lipidworld.com/content/10/1/198

RESEARCH

OpenAccess

Apoptosis,mastocytosis,anddiminished
adipocytokinegeneexpressionaccompany
reducedepididymalfatmassinlong-standing
diet-inducedobesemice
MehmetMAltintas
1
†
,MariaARossetti
2
†
,BehzadNayer
3
,AlvaroPuig
1
,PatriciaZagallo
1
,LuisMOrtega
1
,
KevinBJohnson
2
,GeorgeMcNamara
4
,JochenReiser
1
,ArmandoJMendez
2
andAliNayer
1*

Abstract
Background:
Obesityischaracterizedbyincreasedcelldeathandinflammatoryreactionsintheadiposetissue.
Here,weexploredpathophysiologicalalterationstakingplaceintheadiposetissueinlong-standingobesity.Inthe
epididymalfatofC57BL/6micefedahigh-fatdietfor20weeks,theprevalenceanddistributionofdead
adipocytes(crown-likestructures),mastcells(toluidineblue,mMCP6),macrophages(F4/80),andapoptoticcells
(cleavedcaspase-3)weremeasured.Moreover,geneand/orproteinexpressionofseveraladipocytokines(leptin,
adiponectin,TNF-
a
,IL-10,IL-6,MCP-1),F4/80,mMCP6,cleavedcaspase-3weredetermined.
Results:
Weobservedthattheepididymalfatmasswaslowerinobesethaninleanmice.Inobesemice,the
epididymalfatmasscorrelatedinverselywithbodyweightandlivermass.Deadadipocytes,mastcells,
macrophages,andapoptoticcellswereabundantintheepididymalfatofobesemice,especiallyintherostralvs.
caudalzone.Accordingly,mMCP6,F4/80,andcleavedcaspase-3geneand/orproteinexpressionwasincreased.
Conversely,adiponectin,leptin,IL-6,andMCP-1geneexpressionlevelswerelowerintheepididymalfatofobese
thanleanmice.AlthoughTNF-
a
andIL-10geneexpressionwashigherintheepididymalfatofobesemice,their
expressionrelativetoF4/80andmMCP6expressionwerelowerintheheavilyinfiltratedrostralthancaudalzone.
Conclusions:
Thisstudydemonstratesthatinmicewithlong-standingobesitydiminishedgeneexpressionof
severaladipocytokinesaccompanyapoptosisandreducedmassoftheepididymalfat.Ourfindingssuggestthat
thisisduetobothincreasedprevalenceofdeadadipocytesandalteredimmunecellactivity.Differential
distributionofmetabolicallychallengedadipocytesisindicativeofthepresenceofbiologicallydiversezoneswithin
theepididymalfat.
Keywords:
Obesity,adiposetissue,inflammation,mastcells,macrophages,crown-likestructures,apoptosis,
adipokines

Background
signalingpathwaysandalterationsinsystemicandlocal
Asteadyincreaseintheprevalenceofobesityhasbeenlevelsofinflammatorycytokines[1].Manyofthesecyto-
observedworldwide.Increasedcaloricintakeandkines,includingtumornecrosisfactor-
a
(TNF-
a
),
decreasedphysicalactivityaremajorcontributors.Obe-interleukin-6(IL-6),interleukin-10(IL-10),andmono-
sityisoftenaccompaniedbyactivationofinflammatorycytechemotactiveprotein-1(MCP-1)haveprofound
effectsonmetabolism[1].Theadiposetissueisdirectly
involvedinthechronicinflammatorystatethatis
*Correspondence:[e-mail]
observedinobesity.Cellsofinnateimmunesystem,
†
Contributedequally
1
DepartmentofMedicine,MillerSchoolofMedicine,UniversityofMiami,
suchasmacrophages[2],mastcells[3,4],andneutro-
Miami,FL,USA
phils[5]accumulateintheadiposetissueinobesity.
Fulllistofauthorinformationisavailableattheendofthearticle
©2011Altintasetal;licenseeBioMedCentralLtd.ThisisanOpenAccessarticledistributedunderthetermsoftheCreativeCommons
AttributionLicense(http://creativecommons.org/licenses/by/2.0),whichpermitsunrestricteduse,distribution,andreproductionin
anymedium,providedtheoriginalworkisproperlycited.

Altintas
etal
.
LipidsinHealthandDisease
2011,
10
:198
http://www.lipidworld.com/content/10/1/198

Moreover,cellsofadaptiveimmunity,includingregula-
toryTcells[6,7],CD8
+
Tcells[7],andnaturalkillerT
cells[8]arealsoinvolvedinadiposetissueinflammation
inobesity.
Althoughthedetailsoftheunderlyingmechanisms
thatprovokeinflammationintheadiposetissueinobe-
sityremainlargelyelusive,adipocyteinjuryanddeath
playacentralrole[9].Basedonultrastructuraland
immunohistochemicalalterations,itwasproposedthat
manyadipocytesinwhiteadiposetissueofobese
rodentsandhumansdemonstratecharacteristicfeatures
ofnecrosis,butnotapoptosis[9].Arecentstudy,how-
ever,showedthatbothmitochondrial-anddeathrecep-
tor-mediatedcaspaseactivationandadipocyteapoptosis
wereincreasedintheadiposetissueofobesehumans
anddiet-inducedobesemice[10].Regardlessofthe
pathway(s)involved,metabolicallychallengedadipocytes
aremoreprevalentandaccompanyinginflammatory
reactionsaremoresevereinvisceralthaninsubcuta-
neousfatinobesity[3,11].Thereisalsoaremarkable
heterogeneityintheseverityofadiposetissueinflamma-
tionindifferentvisceralfatdepotsinobesesubjects
[3,12].
Althoughmuchemphasishasbeenplacedonthe
understandingofmolecular,biochemical,andimmuno-
logicalmechanismsinvolvedinadiposetissueinflamma-
tioninobesity,littleisknownaboutimmuneresponses
takingplaceintheadiposetissueduringtheadvanced
stagesofobesity.Inthepresentstudy,weexaminedthe
epididymalfatofmicewithlong-standingobesityand
determined:1)thedensityanddistributionofdeadadi-
pocytes,macrophages,andmastcellsthroughoutthefat
depot,2)geneexpressionlevelsofseveraladipocyto-
kines,3)theprevalenceofapoptosis,and4)therelation
amongapoptosis,adipocytokinegeneexpression,and
thedegreeofinflammatorycellinfiltration.Weshowed
thatreducedmassoftheepididymalfatinmicewith
long-standingobesityisaccompaniedbydivergentdis-
tributionofcrown-likestructures,apoptoticcells,mast
cells,andmacrophagesleadingtodiminishedadipocyto-
kinegeneexpression.
Methods
Experimentalanimals
Wefollowedthe
‘
Principlesoflaboratoryanimalcare
’
establishedbytheNationalInstitutesofHealth.The
InstitutionalAnimalCareandUseCommitteeofthe
UniversityofMiamispecificallyapprovedthisstudy
(IACUCprotocolnumber08-245).Bloodsamplesand
tissueswerefromarecentstudy[3].MaleC57BL/6
micewerepurchasedfromTaconic(Hudson,NY)and
acclimatedfortwoweeksbeforethebeginningofthe
study.Micewererandomizedtoeitherahigh-fatdiet
(HFD)consistingof60%caloriesfromfat(D12492)ora

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low-fatdiet(LFD)consistingof10%caloriesfromfat
(D12450B)attheageof6weeksandweresacrificed
when6monthsold(ResearchDietsInc.,St.Louis,MO).
MicewereweighedwithaScoutProbalanceSP202
(Ohaus,PineBrook,NJ).Organweightsweremeasured
withaSartoriusED124SAnalyticalBalance(Sartorius,
Bohemia,NY).
Glucose,insulinandcholesterolassays
Afterovernightfasting(15hours),bloodwassampled
fromthetailofunanesthetizedmice.Bloodglucosecon-
centrationsweremeasuredusingaContourglucometer
(Bayer,Tarrytown,NY).Seruminsulinconcentrations
weremeasuredbyimmunoassayfollowingmanufacturer
’
s
instructions(CrystalChem,DownerGrover,IL).Serum
cholesterolconcentrationsweredeterminedbyCholes-
terolLiquiColorenzymaticassay(StanbioLaboratory,
Boerne,TX).HomeostasisModelofAssessment-Insulin
Resistance(HOMA-IR)wascalculatedusingtheformula:
fastingglucose(mg/dl)×fastinginsulin(mU/L)/405.
Theanatomyoftheepididymalfatinthemouse
Theepididymalfatisananatomicallydistinctandmeta-
bolicallyactiveabdominalfatdepotwidelyusedtostudy
adiposetissuebiologyinrodents[2-12].Thepairedepi-
didymalfatisattachedcaudallytotheipsilateraltestis
andepididymisandextendsproximallytowardthedia-
phragm(Figure1).Theepididymalfatincorporates
spermaticbloodvesselsandaccompaniesthemmedially
totheretroperitoneum[13].Usingspermaticbloodves-
selsasalandmark,theepididymalfatcanbedivided
intothreezones:1)medialzone,locatedaroundthe
spermaticarteryanditsmainbranches,2)caudalzone,
attachedtothetestisandepididymis,and3)rostral
zone,thelooseendproximaltothemedialzone.We
quantifiedthedensityofmastcellsandcrown-like
structures(CLS)inallthreezones.Acrown-likestruc-
tureconsistsofseveralmacrophagesenvelopinganadi-
pocyte[9].Proteinandgeneexpressionwasmeasured
onlyinthecaudalandrostralzonesthatshowedthe
greatestdifferenceinthedensityofmastcellsandCLS
(seebelow).

Histology
TissueswerefixedinCarnoy
’
sfixativeandembeddedin
paraffin.Fivemicron-thicksectionswerecut,baked
overnightat60°C,deparaffinizedinxylene,rehydrated
inagradedethanolseries,andthenstained.Todemon-
stratemastcells,toluidinebluestainingwascarriedout
bybrieflysubmergingtissuesectionsin0.1%aqueous
toluidineblue(EMS,Hatfield,PA)[3].Inaddition,the
esteraseactivityofmastcellswasdetectedusing
naphtholAS-Dchloroacetateassubstrateaspreviously
described[3].

Altintas
etal
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LipidsinHealthandDisease
2011,
10
:198
http://www.lipidworld.com/content/10/1/198

Figure1
Theepididymalfatinthemalemouse
.Todemonstrate
abdominalorgans,theanteriorabdominalwallwasremovedand
theleftepididymalfat(1)wasdeflectedtotheleft.Theepididymal
fatwasdividedintoacaudal,medial,androstralzonerelativeto
thelocationofspermaticbloodvessels(2).Thelefttestis(3),the
urinarybladder(4),asmallbowelloop(5),andattachedmesenteric
fat(6)arealsoshown.
ImmunohistochemicalstainingformacrophageF4/80
Aspreviouslydescribed,followingquenchingendogen-
ousperoxidaseactivityandheat-inducedepitoperetrie-
val,deparaffinizedtissuesectionswereblockedwith
rabbitserumandsequentiallyincubatedwithamono-
clonalantibodyagain