Transgenic Mice Overexpressing Renin Exhibit Glucose Intolerance and Diet-Genotype Interactions.

1.4 MB·English

by  Fletcher, Sarah J.

ORIGINALRESEARCHARTICLE
published:07January2013
doi:10.3389/fendo.2012.00166
Transgenic mice overexpressing renin exhibit glucose

intolerance and diet-genotype interactions

SarahJ.Fletcher1,NishanS.Kalupahana2,MorvaridSoltani-Bejnood3,JungHanKim4,ArnoldM.Saxton5,
DavidH.Wasserman6,BartDeTaeye6,BrynnH.Voy5,AnnieQuignard-Boulange7 and
NaimaMoustaid-Moussa8*

1GenomeScienceandTechnologyProgram,UniversityofTennessee,Knoxville,TN,USA
2DepartmentofPhysiology,FacultyofMedicine,UniversityofPeradeniya,Peradeniya,SriLanka

3PellissippiState,Knoxville,TN,USA

4DepartmentofPharmacology,PhysiologyandToxicology,SchoolofMedicine,MarshallUniversity,Huntington,WV,USA
5DepartmentofAnimalScience,UniversityofTennessee,Knoxville,TN,USA
6DepartmentofMolecularPhysiologyandBiophysics,SchoolofMedicineandMouseMetabolicPhenotypingCenter,VanderbiltUniversity,Nashville,TN,USA
7INRA-AgroParisTechUMR914,Paris,France
8NutritionalSciences,TexasTechUniversity,Lubbock,TX,USA

Editedby: Numerous animal and clinical investigations have pointed to a potential role of the renin-
TsuguhitoOta,KanazawaUniversity, angiotensinsystem(RAS)inthedevelopmentofinsulinresistanceanddiabetesincondi-
Japan
tionsofexpandedfatmass.However,themechanismsunderlyingthisassociationremain
Reviewedby: unclear.Weusedatransgenicmousemodeloverexpressingreninintheliver(RenTgMK)
UndurtiNarasimhaDas,UNDLife
Sciences,USA to examine the effects of chronic activation of RAS on adiposity and insulin sensitivity.

ToruAizawa,AizawaHospital,Japan Hepatic overexpression of renin resulted in constitutively elevated plasma angiotensin II

*Correspondence: (four- to six-fold increase vs. wild-type,WT). Surprisingly, RenTgMK mice developed glu-
NaimaMoustaid-Moussa,Nutritional coseintolerancedespitelowlevelsofadiposityandinsulinemia.Thetransgenicsalsohad
Sciences,TexasTechUniversity,1301,
lowerplasmatriglyceridelevels.Glucoseintoleranceintransgenicmicefedalow-fatdiet
AkronStreet,Lubbock,TX79423,
USA. wascomparabletothatobservedinhigh-fatfedWTmice.Thesestudiesdemonstratethat

e-mail:naima.moustaid-moussa@ overexpressionofreninandassociatedhyperangiotensinemiaimpairglucosetolerancein

ttu.edu adiet-dependentmannerandfurthersupportaconsistentroleofRASinthepathogenesis

ofdiabetesandinsulinresistance,independentofchangesinfatmass.

Keywords:adiposetissue,renin-angiotensinsystem,insulinresistance,angiotensinII

INTRODUCTION AGT,ACE,renin,AT1,orAT2protectsrodentsfromdiet-induced

The renin-angiotensin system (RAS) plays an important role in obesityandinsulinresistance(Massieraetal.,2001b;Yvan-Charvet

the regulation of blood pressure, fluid, and electrolyte balance etal.,2005;Takahashietal.,2007;Jayasooriyaetal.,2008).Con-

(Schmieder et al.,2007).Angiotensinogen (AGT),the precursor versely,chronicRASoveractivationviaAngIIinfusion(Ogihara

peptide of this system, undergoes successive enzymatic cleav- etal.,2002)leadstoglucoseintoleranceandinsulinresistancein

agesbyreninandangiotensinconvertingenzyme(ACE)toyield rodents, further supporting a role of RAS overactivation in the

angiotensin I (Ang I) and angiotensin II (Ang II) respectively. pathogenesisofinsulinresistance.

Thelatteristhemainbioactivepeptideofthissystem,whichacts Although obesity and increased adiposity are associated with

via two G-protein coupled receptors, namely angiotensin Type- RAS overactivation, it is not clear whether systemic RAS over-

1 (AT1) and Type-2 (AT2) receptors, to exert its physiological activation can lead to both obesity and insulin resistance. It is

effects.BecauseAT1activationbyAngIIleadstoelevationofblood important to test this because studies have documented differ-

pressure,ACEinhibitors(ACEI)andAT1blockers(ARB)arephar- ences in RAS activity in humans,which have been attributed to

macologicallyusedasanti-hypertensiveagents(Schmiederetal., polymorphisms in RAS coding (Jeunemaitre et al.,1999; Jeune-

2007). maitre,2008)orpromoterregions(Xiaoetal.,2006).Therefore,

Interestingly,severalclinicalstudieshaveshownthatpatients understandingtheimplicationsofchronicelevationofRASmay

onRASblockershavealowerriskofdevelopingType-2diabetes helpprovideinsightintometabolicconsequencesof chronically

when compared to patients on other anti-hypertensive medica- elevatedRASinhumans.

tions (Vermes et al., 2003; Bosch et al., 2006). Moreover, RAS While overexpression of RAS is consistently associated with

blockadepreventsandreversesinsulinresistanceinducedbyhigh- insulin resistance and glucose intolerance, the effect of chronic

fat feeding in rodents (Lee et al.,2008). Given that plasma and RASoveractivationonadiposityisnotclear.Thisisfurthercom-

tissuelevelsof severalRAScomponentspositivelycorrelatewith plicatedbyexistenceoflocalRASinseveraltissueswiththelocal

bodymassindex(Schorretal.,1998;VanHarmelenetal.,2000), effectscomplicatingtheunderstandingofsystemiceffectsofRAS

itispossiblethatoveractivationoftheRASislinkedtothepatho- (KalupahanaandMoustaid-Moussa,2012b).Forexample,over-

genesisofinsulinresistanceinobesity.Indeed,geneticdeletionof expressionofAGTinadiposetissueincreasesadiposityandblood

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Fletcheretal. Metabolicphenotypingofrenintransgenicmice
pressure and leads to insulin resistance (Massiera et al., 2001a; GLUCOSETOLERANCETEST
Kalupahanaetal.,2012).However,acuteorchronicsystemicRAS Mice were fasted overnight prior to the glucose tolerance test
overactivation leads to decreased fat mass despite the develop- (GTT). Blood was collected in heparinized capillary tubes from
mentofinsulinresistance(Brinketal.,1996).Thissuggeststhat theorbitalsinuspriortointra-peritonealinjectionwithglucose
increased fat mass in the case of adipose RAS overexpression (1g/kg body weight),and then 15,30,60,90,and 120min after
maybeduetolocaleffectsof AngIIproductionwithinadipose injection.Plasmaglucoseconcentrationswerecalculatedusinga
tissue. One Touch ultra-monitoring system (Johnson & Johnson, Co.,
TofurtherdissecteffectsofelevatedsystemicAngIIoninsulin New Brunswick, NJ, USA). The GTT was performed on mice
sensitivity and adiposity, we used a unique mouse model in 10weeks old and repeated when the mice reached 20weeks of
whichAngIIischronicallyelevatedthroughoutlifetimethrough ageandtheareaunderthecurve(AUC)forglucoseandinsulin
genetic manipulation. This mouse model is a unique genetic werecalculated.
minipump model in which renin is overexpressed in the liver.
Given that renin release is the rate-limiting step in the sys- PLASMAMEASUREMENTS
temic RAS, this model offers the advantage of constant renin Serum was separated from blood samples collected during the
overexpression independent of homeostatic control and a life- GTT by centrifugation at 3000rpm for 15min at 4˚C and then
longelevatedlevelof AngII.Asexpected,thesetransgenicmice stored in aliquots at −80˚C until assayed. Serum insulin,leptin,
(RenTgMK; Caron et al., 2002) exhibit elevated levels of circu- andadiponectinlevelsweremeasuredinduplicateusingcommer-
lating renin and Ang I and develop chronic hypertension along ciallyavailableELISAkitsfollowingthemanufacturer’sprotocol
withotherpathologicalmanifestations(Caronetal.,2002,2004). (insulin cat# 90060 and leptin cat# 90030, Crystal Chem, Inc.,
The RenTgMK mice thus allow us also to study the effects of Downers Grove, IL, USA; adiponectin cat# EZMADP-60, Linco
systemic chronic elevations of Ang II on adiposity and glucose Research,Billerica,MA,USA).Absorbancewasreadat450nmon
homeostasis,sothatwecandissecttheeffectsofsystemicvs.adi- aPackardSpectraCountmicroplatereader(PackardInstrument,
pose RAS by comparing these results with the ones previously Co.,Meriden,CT,USA).
reportedforlocaladiposeoverexpressionofRAS(Massieraetal.,
2001a). DIETSTUDY
We report here that elevated circulating Ang II due to renin Maleheterozygous(RenTgMK+/−)miceandtheirWTlittermates
overexpressionleadstoglucoseintolerance,whichisfurtherexac- wererandomlyassignedtoeitherahigh-fatdiet(60%kcalfrom
erbated by high-fat feeding. Unexpectedly, these mice exhibit fatcat#D12492,ResearchDiets,Inc.,NewBrunswick,NJ,USA)
otherwise normal glucose metabolism and a transgene dose- or a low-fat diet (10% kcal from fat cat# D12450B, Research
dependentdecreaseinfatmassandinsulinemia. Diets, Inc., New Brunswick, NJ, USA) for 18weeks. Each diet
group(n=6/group)wascomprisedofthreemaleRenTgMK+/−
MATERIALSANDMETHODS miceandthreemaleWTmice.Bodyweightmeasurementswere
ANIMALS acquiredweeklyforthedurationof thestudy.Attheconclusion
RenTgMK transgenic mice were kindly provided by Dr. Oliver of the 18-week diet study, a GTT was performed and plasma
Smithies, University of North Carolina, Chapel Hill, NC, USA insulin, leptin, and adiponectin concentrations were measured,
(Hatadaetal.,1999).Briefly,arenintransgeneconsistingofpor- asdescribedabove.Miceweresacrificed1weekaftertheGTT.
tionsoftheRen2andRen-1dgenes(Ren2/1d)wasinsertedintothe
genomeattheApoA1/ApoC3locusviahomologousrecombina- METABOLICSTUDIES
tionandplacedundercontrolofanalbuminpromoter/enhancer Metabolic studies of the RenTgMK mice were performed at the
(AlbP/E)toachieveliver-specificexpression. Mouse Metabolic Phenotyping Center (MMPC) at Vanderbilt
MaleheterozygousRenTgMK(RenTgMK−/+)miceonaniso- University,Nashville,TN,USA.Glucoseinfiltrationrate,glucose
genicSvEv129/6backgroundwerecrossedwithwild-type(WT) turnover rate, endogenous glucose turnover rate, and clearance
SvEvfemales.SubsequentheterozygousF1progenywerematedto were measured. Whole-body insulin activity in vivo was exam-
generatetheF2offspringthatwereusedinthisstudy.Miceused inedviaeuglycemichyperinsulinemicclamp.Detailedprocedure
inthisstudywerebredandmaintainedattheUniversityofTen- has been previously reported (Ayala et al., 2006). Briefly, to
nesseeaccreditedanimalfacility,ona12h:12hlight-darkcycleat assessinsulinsensitivityandglucosemetabolism,insulinwascon-
22˚C and fed a standard rodent chow and water ad libitum. All tinuously administered via euglycemic hyperinsulinemic clamp.
experimentswereapprovedbytheInstitutionalAnimalCareand Catheters were chronically implanted in the jugular vein and
UseCommitteeattheUniversityofTennessee. carotid artery. Arterial glucose levels were measured every 5–
10min during 120min and glucose infusion rates were deter-
GENOTYPING minedbasedonthearterialglucosemeasurements.Plasmaglucose
DNA was extracted from tail tips as previously described turnoverwasmeasuredinRenTgMK+/− andWTmales(n=8–
(Truett et al., 2000). PCR-based genotyping was performed 12/group).Micewerecontinuouslyinfusedwith[3-3H]glucoseat
using three primers: p1, 5(cid:48)-TGGGATTCTAACCCTGAGGACC- a rate of 0.4µCi/min. Glucose appearance (Ra) and disappear-
3(cid:48); p2, 5(cid:48)-CACAGATTGTAACTGCAAATCTGTCG-3(cid:48); p3, 5(cid:48)- ance(Rd)rateswereestimatedastheratiooftherateofinfusion
GTTCTTCTGAGGGGATC-GGC-3(cid:48)(SigmaGenosys,TheWood- of[3-3H]glucoseandthesteady-stateplasma[3H]glucosespecific
lands,TX,USA)aspreviouslydescribed(Caronetal.,2002). activity (dpm/mg),and the glucose disappearance was assumed
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Fletcheretal. Metabolicphenotypingofrenintransgenicmice
to be equal to the steady-state Ra rate. Glucose clearance was 4,6-diamidino-2-phenylindole(VectorLaboratories,Burlingame,
calculated by dividing the Rd by the arterial glucose concentra- CA,USA)fornuclearstaining.
tion.Tomeasuretissue-specificglucoseuptake,micewereinjected
with12µCiof[3H]-labeled2-deoxyglucose([2-3H]DG).Arterial STATISTICALANALYSIS
plasmasampleswerecollectedinintervalsfor40minbeforemice Data were analyzed in SAS (SAS Institute,Inc.,Cary,NC,USA)
wereanesthetizedandtissueswereextractedandfrozeninliquid using a mixed model analysis of variance (http://dawg.utk.edu).
nitrogenuntilfurtheranalysis. Fisher’stestfollowedbyTukey’sposthoc testwasusedformean
separation.P<0.05wasconsideredstatisticallysignificant.Data

PANCREASHISTOLOGYANDIMMUNOSTAINING arereportedasthemeans±SE.

ThepancreaswascollectedfromWTandtransgenicmice.Tissues

forimmunohistochemistrywerefixedin10%neutral,phosphate- RESULTS

bufferedformalinfor24handparaffin-embedded.Subsequently, BODYWEIGHT,FATPADWEIGHT,ANDMETABOLICPARAMETERS
theparaffin-embeddedtissueswereprocessedin4-µmsections. Body weights were comparable between mice with either one

Sectionswerestainedusingrabbitanti-glucagonpolyclonalanti- or two copies of the renin transgene and WT control mice

body and guinea pig anti-insulin serum (both from Millipore, (Figure 1A). Gonadal fat pad weight (Figure 1B) and adi-

Billerica,MA,USA).Forfluorescencedetection,goatanti-guinea posity index (gonadal fat pad weight divided by body weight;

pig IgG coupled to Texas Red and donkey anti-rabbit IgG cou- Figure 1C) were significantly lower in homozygous mice com-
pledtoCy3wereused(bothfromJacksonImmunoResearch,West paredtoWTlittermates(P<0.05).Fastingserumglucose,leptin,

Grove,PA,USA)followedbyVectashieldMountingMediumwith and adiponectin levels were comparable between all genotypes

FIGURE1|Bodyandfatpadweightandglucosetoleranceinmale glucoselevelsweremeasuredat0,15,30,90,and120minandplotted
RenTgMKmice.(A)Bodyweightattheageof20weeks.(B)Mice onagraph.(E)AreaUndertheCurve(AUC)wascalculatedas
weresacrificedattheendofweek20andgonadalfatpadswere describedintheexperimentalprocedures.Valuesaremeans±SE.
collectedandweighed.(C)Theadiposityindexwasdeterminedby n=6ForWT;n=5forRenTgMK+/−;n=4forRenTgMK+/+.Different
dividinggonadalfatpadweightbyfinalbodyweight.Aglucose lettersindicateasignificantdifference(P<0.05).*Significantly

tolerancetest(GTT)wasadministeredafterovernightfasting.(D)Blood different(P<0.05)fromWT.

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Fletcheretal. Metabolicphenotypingofrenintransgenicmice
Table1|Serummetabolicmarkersinmalewild-typeandRenTgMK utilizationinthismodel.Accordingly,metabolicstudiesattheNIH
mice. MMPCatVanderbiltUniversitywereconducted.Maleheterozy-
gous mice were compared to WT mice because males exhibited
WT RenTgMK+/− RenTgMK+/+ Pvalue
glucose intolerance and sufficient numbers could be obtained
Glucose,mg/dl 81.2±6.4 89.8±3.2 93.3±5.1 0.292 from a few litters. Steady-state glucose infusion rate (Figure 2),
Insulin,ng/ml 0.62±0.07a 0.42±0.05b 0.36±0.07b 0.033 overall tissue-specific glucose uptake, glucose metabolism, and
Leptin,ng/ml 2.1±0.7 2.3±0.6 1.3±0.5 0.535 endogenous glucose production (Table 2) did not significantly
Adiponectin, 8.7±1.4 10.8±2.5 7.6±0.9 0.479 differbetweenRenTgMKandWTmice,indicatingnormalinsulin
µg/ml sensitivityinthetransgenics.
C-peptide, 1.4±0.1b 1.9±0.2a 2.0±0.1a 0.007
ng/ml EFFECTOFHIGH-FATDIETONBODYWEIGHT,ADIPOSITY,CIRCULATING
FFA,mM 0.84±0.10 0.93±0.06 0.91±0.11 0.791 ADIPOKINES,ANDGLUCOSETOLERANCE
Triglycerides, 60.1±6.4a 44.5±8.6a,b 23.7±8.7b 0.018 Asdescribedabove,renintransgeneoverexpressionledtoimpaired
mg/dL glucose tolerance compared to WT mice when mice were fed a
low-fatchowdiet.Totestwhetherthegeneticdifferenceswouldbe
Valuesaremeans±SE.Animalswere21weeksold.Initialbodyweightmea- exacerbatedbyhigh-fatfeeding,wefedmaleheterozygousandWT
surementsweretakenat10weeks.Bloodwascollectedafterfastingovernight micealow-orhigh-fatdiettoinvestigatediet-geneinteractions.
andmetabolicparametersweremeasuredfromserum.n=6ForWT;n=5for Bodyweightswerenotsignificantlydifferentbetweengroupsat
RenTgMK+/−; n=4 for RenTgMK+/+. C-peptide, connecting peptide; FFA, free thestartoftherandomizeddietstudy(Table3).High-fatfeeding
fattyacid. increased body weight only in the wild-type mice (Figure 3A).
Meansinarowwithsuperscriptswithoutacommonletterdiffer,P<0.05. Mice of both genotypes showed a trend for increased fat pad
NumbersinboldindicateasignificanceofP<0.05. weight and adiposity with high-fat feeding,although the differ-
encewasonlysignificantforadiposityintheRenTgMK+/− mice
(Table1).Fastingseruminsulin,however,wassignificantlylower (Figures3B,C).
inthetransgenicmice(bothhomozygousandheterozygous)com- Changesinadiposityareknowntoalterhormonalandmetabo-
pared to WT littermates (P<0.05). Serum C-peptide levels,on litelevels.Asexpected,high-fatfeedingincreasedserumglucose
the other hand, were higher in the transgenics than in the WT andleptinlevelsinbothmaleWTandtransgenicmice(P<0.05
mice(Table1).Interestingly,serumtriglyceridesweresignificantly for diet effect – Table 3). Interestingly, high-fat feeding also
lowerinthehomozygousmicecomparedtoWTlittermates.On increasedserumresistinlevelsinWT,butnotintransgenicmales
thislow-fatchowdietcondition,theoverallmetabolicphenotype (Table3).IntheWTmales,serumtriglycerideconcentrationwas
waslesspronouncedinfemaletransgenicmicecomparedtoWT higherinthelow-fatfedmicewhencomparedtohigh-fatfedones
femalelittermates(datanotshown). (Table3).Thiseffectwasminimalinthetransgenics.
Low-fatfedmaleheterozygousmiceexhibitedahigherglucose
GLUCOSEINTOLERANCEINRenTgMICE excursion and area under the glucose curve compared to their
ToassessglucosetoleranceintheRenTgmice,anintra-peritoneal WTcounterparts,indicatingglucoseintolerance(Figures3D,E).
GTTwasadministered.Baselinefastingglucoselevelswerecom- High-fatfeedingdidnotexacerbateglucoseintoleranceinRenT-
parable between WT, RenTgMK+/− and RenTgMK+/+ mice gMKmice.
(81.17±15.68, 89.80±7.16, and 93.25±10.28mg/dl, respec-
tively).Heterozygousmicemaintainedsignificantlyhigherlevels PANCREASHISTOLOGYANDIMMUNOSTAININGOFRenTgMICE
of glycemia compared to WT within 60min and remained ele- Because of the consistently lower insulin levels in heterozygous
vated throughout the GTT (Figure 1D). These differences were micecomparedtoWTmice,weperformedimmunohistological
observedasearlyas10weeksofage(datanotshown)andbecame studiesinthepancreastoassessisletmorphologyandhormone
more pronounced with age by 20weeks. Glucose intolerance in content.Inbothgenotypes,isletsappearednormalandexhibited
maleRenTgMKmicewasalsoevidentfromacomparisonofthe comparablestainingforglucagonandinsulin(Figure4).
glucoseAUC(Figure1E).TheAUCvaluesforbothheterozygous
and homozygous mice were higher (P<0.05) than that of WT DISCUSSION
mice implying greater glucose intolerance in the transgenics. In Manylinesof evidencehavelinkedactivationof theRAStothe
females,nosignificantdifferencesinGTTwereobservedbetween developmentofobesityandinsulinresistance(Schorretal.,1998;
the three genotypes at 20weeks of age in these low-fat feeding Van Harmelen et al., 2000), but the effects of increased circu-
conditions(datanotshown). lating levels of angiotensins on adiposity remain controversial.
WehypothesizedthatchronicsystemicRASactivationviatrans-
METABOLICPHENOTYPINGOFRenTgMICE genic renin overexpression in the liver would lead to glucose
Insulin resistance is commonly associated with high adiposity. intolerance and systemic insulin resistance. We also predicted
Theparadoxicalglucoseintolerancedespitelowadiposityandlow that increased systemic Ang II would increase adiposity, based
insulinemia in the renin transgenic male mice vs. control litter- onpreviousworkbyusandothersshowingthatAngIIincreases
matesledustofurtherinvestigatewhetherthesedifferenceswere adipocytelipogenesisandtriglyceridestorage.Ourresultsdemon-
due to altered insulin sensitivity and/or glucose production or stratethatelevatedcirculatingAngIIduetoreninoverexpression
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Fletcheretal. Metabolicphenotypingofrenintransgenicmice

FIGURE2|Assessmentofinsulinsensitivityinmalewild-typeandRenTgMK+/−miceusinghyperinsulinemiceuglycemicclamp.Changesinblood
glucoseconcentration(A)andglucoseinfusionrate(B)overtimeareshown.Valuesaremeans±SE.Animalswereapproximately9monthsold.n=8–9For
WT;n=7forRenTgMK+/−.

Table2|Metaboliccharacteristicsandaccumulationof[2-3H]DG thananincreaseinadiposityinmalemice.However,thesemice

duringthehyperinsulinemic-euglycemicclampexperimentsinmale exhibit otherwise normal glucose metabolism and a transgene

wild-typeandRenTgMK+/−mice. dose-dependentdecreaseininsulinemia.

WT RenTgMK+/− Pvalue
GLUCOSEINTOLERANCEINRenTgMKMICE
GLUCOSEKINETICS ConsistentwithpreviousstudiesofAngIIinfusionandtransgenic
Bloodglucose,mg/dl 114.7±5.8 114.6±5.0 0.996 reninexpression(Leeetal.,1996),maleRenTgMKtransgenicmice
GTR,mg/kg/min 47.4±4.0 49.4±6.7 0.794 exhibited glucose intolerance, even on a low-fat diet. However,
endoGTR,mg/kg/min 4.96±4.62 −2.29±7.60 0.416 despitethisglucoseintolerance,theRenTgMKmicemaintained
Glucoseclearance,mg/kg/min 42.0±3.1 43.7±5.7 0.790 lowfastinginsulinemiaandnormalinsulinsensitivity,asindicated
GIR,mg/kg/min 42.4±4.3 51.7±4.0 0.142 bynormalsteady-stateglucoseinfusionduringthehyperinsuline-
ACCUMULATIONOF[2-3H]DG mic,euglycemic clamp studies. RenTgMK mice maintained low
Soleus,µg/min/mgtissue 0.035±0.008 0.036±0.005 0.923 insulinemiaevenunderhigh-fatfeeding.Theglucoseintolerance
Gastro,µg/min/mgtissue 0.025±0.005 0.033±0.003 0.229 inRenTgMKmiceinthepresenceofnormalfastingglucoselevels
Vastusl.,µg/min/mgtissue 0.041±0.007 0.047±0.005 0.550 and low insulinemia, a feature that is a rather typical hallmark
WAT,µg/min/mgtissue 0.004±0.001 0.006±0.001 0.067 ofincreasedinsulinsensitivity,couldbeduetodecreasedinsulin
Diaphragm,µg/min/mgtissue 0.131±0.019 0.091±0.007 0.103 production/secretion and/or increased insulin clearance. Serum
Heart,µg/min/mgtissue 0.431±0.057 0.320±0.043 0.161 C-peptidelevelwashigherinheterozygouscomparedtoWTmice
Brain,µg/min/mgtissue 0.048±0.005 0.049±0.003 0.888 arguing against decreased insulin secretion accounting for low
insulinemia in the RenTgMK mice. Immunohistochemistry of
Valuesaremeans±SE.Animalswereapproximately9monthsold.n=8–9For the pancreas indicated normal islet morphology and hormone
WT;n=7forRenTgMK+/−.GTR,glucoseturnoverrate;endoGTR,endogenous content, possibly indicating normal pancreatic function. How-
glucoseturnoverrate;GIR,glucoseinfusionrate;[2-3H]DG,2-deoxy-[3H]glucose; ever,suchstudiesareonlyqualitativeanddonotallowtodetect
Gastro,gastrocnemius;Vastusl.,vastuslateralis;WAT,whiteadiposetissue. clearquantitativedifferences.Thus,itisprobablyinsulinclearance,
ratherthaninsulinsecretionthatmaybealteredinthismodel.
leadstoglucoseintolerance,butwithconsistentlylowerlevelsof Liver is the primary site of insulin clearance (Duckworth
plasma insulin. Further,chronic elevation of systemicAng II by etal.,1998),whichcanbeaffectedbybothnutritionalandhor-
hepaticoverexpressionofthereningeneledtoareductionrather monalsignals.Insulinclearancerateisheritable(Goodarzietal.,
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Fletcheretal. Metabolicphenotypingofrenintransgenicmice
Table3|Effectsofhigh-fatdietonbodyweightandmetaboliccharacteristicsinmalewild-typeandRenTgMK+/−mice.

WT RenTgMK+/− Pvalue

LF HF LF HF Geno Diet GenoXdiet

Initialbodyweight,g 27.2±2.2 29.2±1.4 26.2±0.6 25.7±0.9 NS NS NS
Finalbodyweight,g 37.6±3.2a,b 46.9±5.6a 35.4±1.8b 37.8±2.1a,b NS NS NS
Glucose,mg/dL 92.3±3.2b 123.7±7.7a 98.3±5.2b 127.3±2.9a NS 0.001 NS
Insulin,ng/ml 1.29±0.31 2.07±0.60 0.91±0.04 1.14±0.25 NS NS NS
Leptin,pg/ml 9.5±1.6 16.2±2.9 7.6±1.1 12.7±2.3 NS 0.019 NS
Adiponectin,µg/ml 12.6±0.3 14.1±2.0 12.9±0.4 12.4±0.2 NS NS NS
Resistin,pg/ml 475.8±37.3b 702.2±19.0a 545.8±48.7a,b 493.3±83.9b NS NS 0.030
MCP-1,pg/ml 29.9±8.0 53.0±18.9 30.5±1.6 34.0±12.9 NS NS NS
PAI-1,pg/ml 3903.4±666.2 5903.4±834.8 4733.5±734.0 4526.8±961.3 NS NS NS
C-peptide,ng/ml 1.9±0.3 3.3±1.2 2.4±0.4 2.3±0.2 NS NS NS
FFA,mM 1.00±0.18 0.80±0.04 1.23±0.36 1.16±0.39 NS NS NS
Triglycerides,mg/dL 132.4±16.5a 66.9±4.1b 66.8±4.4b 39.2±5.4b 0.001 0.001 0.074

Valuesaremeans±SE.Animalswerefedahigh-fatorlow-fatdietfor19weeks.Initialbodyweightmeasurementsweretakenatthebeginningofthestudy.Mice
were3–5monthsold.Bloodwascollectedafterfastingovernightandmetabolicparametersweremeasuredfromserum.n=3Foreachgroup.LF,low-fat;HF,high-fat;
MCP-1,monocytechemoattractantprotein-1;PAI-1,plasminogenactivatorinhibitor-1;C-peptide,connectingpeptide;FFA,freefattyacid.
Meansinarowwithsuperscriptswithoutacommonletterdiffer,P<0.05.
NumbersinboldindicateasignificanceofP<0.05.

2005) and is reduced in obesity and Type-2 diabetes (Duck- tissue(Achardetal.,2007)andthereforemaymediatetheobserved
worth et al., 1998). Therefore, it could be an important factor adiposetissuephenotype.
in the pathogenesis of Type-2 diabetes. Conversely, there are
mousemodelswhichexhibitincreasedinsulinclearancesuchas RASOVERACTIVATIONANDINSULINRESISTANCE
themouseoverexpressingcarcinoembryonicantigen-relatedcell Renin-angiotensinsystemoveractivationviachronicAngIIinfu-
adhesionmolecule1(CEACAM1)intheliver(Najjar,2002).Addi- sion leads to the development of systemic insulin resistance
tional studies beyond the scope of this work will be required in rodents. This is, in most part, due to the Ang II-mediated
to address whether the RAS is involved in regulating insulin impairment of skeletal muscle glucose transport and utilization
clearance. (KalupahanaandMoustaid-Moussa,2012a).AngIIimpedesthe
Thefindingthattheglucoseintoleranceinmaletransgenicmice insulin-mediatedtyrosinephosphorylationoftheinsulinreceptor
didnotworsenwithhigh-fatfeedingcouldpossiblyindicatethat substrate(IRS)-1,activationofAkt,andtranslocationofglucose
RASoveractivationcouldatleastinpartplayaroleinhigh-fatdiet- transporter (Glut)-4 in the skeletal muscle in an NADPH oxi-
inducedobesity.Alongthesamelines,miceoverexpressingAGT dase,AT1,and NF-kB-dependent manner. Ang II also increases
inadiposetissuealsodevelopglucoseintoleranceonalow-fatdiet, hepaticglucoseproduction,whichalsopotentiallycontributesto
whichisnotfurtherexacerbatedbyhigh-fatfeeding(Kalupahana alteredsystemicinsulinsensitivity.Incontrast,theRenTgMKmice
etal.,2012).Femaletransgenicmiceexhibitednormalglucosetol- inthisstudyexhibitednormalsystemicinsulinsensitivity.While
eranceonalow-fatdietwhilemalesbecameglucoseintoleranton theexactunderlyingmechanismsforthisdiscrepancyof insulin
thesamedietwhencomparedtoWTlittermates.Further,female sensitivity between different models of RAS overactivation are
transgenics became glucose intolerant when fed a high-fat diet unknown, it is possible that the low insulinemia present in the
(datanotshown). RenTgMKmicecouldprotectthesemicefromthedevelopment
ItislikelythatthemetabolicphenotypeoftheRenTgMKmice ofinsulinresistance.Previousstudieshaveshownthatanincrease
is due to Ang II effects, rather than the effects of renin acting in plasma insulin by itself can induce insulin resistance. In the
on the renin/prorenin receptor. We argue this because in renin studybyShaniketal.(2008),micetransfectedwithextracopies
knockoutmice,themetabolicphenotypeofincreasedinsulinsen- of the insulin gene had a two- to four-fold increase in plasma
sitivityandresistancetohigh-fatdiet-inducedglucoseintolerance insulinandexhibitednormalbodyweight,insulinresistanceand
andinsulinresistancewasreversedbyAngIIinfusion(Takahashi hypertriglyceridemia.
et al., 2007). It is also likely that these effects are mediated via Unlike models of chronic Ang II infusion (Ran et al.,2004),
angiotensinreceptors,aspreviousstudiesontheRenTgMKmice RenTgMKmiceexhibitedlowerplasmatriglyceridelevels.Thus,
demonstratedthatAT1receptorblockadereversedrenalpathol- the hypoinsulinemia in the RenTgMK could also potentially
ogyandnormalizedbloodpressureintheRenTgMKmice(Caron explainthelowserumtriglyceridelevelsseeninthesemice.Given
etal.,2002).Alternativemechanismsmayinvolvedirecteffectsof thismetabolicphenotypeofRenTgMKmice,itwouldbeinterest-
reninmediatedbytherenin/proreninreceptoronthevasculature ingtoexplorewhethertheinsulinresistanceseeninseveralmodels
oradiposetissue.Indeed,reninreceptorsareexpressedinadipose of chronic RAS overactivation is insulin-dependent and further
FrontiersinEndocrinology|Diabetes January2013|Volume3|Article166|6
Fletcheretal. Metabolicphenotypingofrenintransgenicmice

FIGURE3|Effectofhigh-fatdietonbodyandfatpadweightand padweightbyfinalbodyweightX100.(D)Aglucosetolerancetest(GTT)was
glucosetoleranceofmalewild-typeandRenTgMK+/−mice.(A)Weight administeredafterovernightfasting.Bloodglucoselevelsweremeasuredat
gainwascalculatedasthedifferencebetweentheinitialbodyweight 0,15,30,90,and120minandplottedonagraph.(E)AreaUndertheCurve
measuredatweek1andthefinalweightmeasuredafter18weeks.(B)Mice (AUC)wascalculatedasdescribedintheexperimentalprocedures.Valuesare
weresacrificedattheendofweek19andgonadalfatpadswerecollected means±SE.n=3Foreachgroup.*Significantlydifferent(P<0.05)from

andweighed.(C)Theadiposityindexwasdeterminedbydividinggonadalfat WT-LF.Differentlettersindicateasignificantdifference(P<0.05).

studies are warranted. The issue of whether the renin recep- thesefindings,theRenTgMKmicealsoexhibitedlowerfatmass
tor may also in part modulate insulin sensitivity merits further comparedtoWTlittermates.Theadiposemasswassignificantly
investigationaswell. decreased by the renin transgene in a gene dosage-dependent
manner.Incontrast,micewithprimaryAGToverproductionin
RASOVERACTIVATIONANDADIPOSITY adipose tissue exhibit higher adiposity (Massiera et al., 2001a).
Both human and rodent studies have shown that obesity and Further, deletion of AGT and other RAS genes leads to lower
increasedadiposityareassociatedwithbothsystemicandadipose fat mass and resistance to diet-induced obesity (Massiera et al.,
RASoveractivation(KalupahanaandMoustaid-Moussa,2012a). 2001b; Takahashi et al., 2007). Thus, it appears that while sys-
However, it is not known whether primary RAS overactivation temic RAS overactivation leads to reductions in body weight,
leadstoobesity.Transgenicmousemodelsclearlydemonstratethat localincreasesinRASactivityinadiposetissueleadstoincreased
manipulatingcomponentsoftheRASaltersadiposity:miceover- adiposity.
expressingAGTinadiposetissuehaveincreasedadiposity,while Thelow-fatmassobservedfollowingAngIIinfusionisattrib-
deletionof eithertheAGTorAngIIreceptorgenesreducesfat- uted to both increased energy expenditure and reduced energy
ness.Paradoxically,previousstudiesofchronicAngIIinfusionin intake (Brink et al., 1996; Cassis et al., 1998). In the RenTgMK
rodentshaveshownthatchronicsystemicRASoveractivationleads mousemodel,wedidnotdetectanysignificantdifferencesinfood
toweightloss,ratherthanweightgain(Griffinetal.,1991;Cassis intake (data not shown).Activation of the sympathetic nervous
etal.,1998).ThetransgenicTGR(mREN2)27ratoverexpressing system may also account for changes in weight via modulation
the mouse Ren2 renin gene also has a lean phenotype (Mullins of lipid metabolism and energy expenditure by catecholamines
etal.,1990;Langheinrichetal.,1996;Leeetal.,1996).Similarto (Cassis, 2000). The differential effect of systemic vs. adipose
www.frontiersin.org January2013|Volume3|Article166|7
Fletcheretal. Metabolicphenotypingofrenintransgenicmice

FIGURE4|Isletpathology.Pancreashistologyandimmunostainingwereconductedtoassessisletmorphologyandhormonecontentinmalewild-typeand
RenTgMKmice,20weeksofage.

specific RAS overactivation on adiposity indicates that specific hypoinsulinemia,andhypotriglyceridemia,withnormalsystemic

local overproduction of AGT in adipose tissue per se, may be insulin sensitivity. The hypoinsulinemia in these mice is possi-

requiredforincreasingadiposity.Indeed,AngIIexertslocalana- bly due to increased insulin clearance, as indicated by elevated

bolic effects in the adipose tissue (Massiera et al., 2001a). Ang C-peptide levels and normal pancreatic insulin levels indicating

II also increases lipogenic gene expression and enzyme activ- normalpancreaticfunction.Whethertheunexpectedlowadipos-

ityin3T3-L1murineadipocytesandhumanadipocytesinvitro ityandnormalinsulinsensitivitydespitethepresenceofglucose

(Jonesetal.,1997).Thisisalsoinagreementwithstudiesshowing intoleranceintheRenTgMKmiceissecondarytohypoinsulinemia

differentiation-dependent increase in AGT gene expression and meritsfurtherinvestigation.

secretion in preadipocytes (Kim and Moustaid-Moussa, 2000).

Ubiquitous inactivation of AGT, on the other hand, results in ACKNOWLEDGMENTS

significant loss of fat mass. However, it is unclear whether tar- TheauthorsthankDr.OliverSmithiesforgenerouslyprovidingus

geted inactivation of AGT in adipose tissue would specifically withtheRenTgmiceweusedtoperformthisstudyandDr.K.Car-

alter fat mass and such studies would convincingly confirm ronandJ.Hagamanforprovidinghelpwithanimalbreedingand

the role of adipose AGT in modulating insulin resistance or genotyping.ThisworkwassupportedbyaUSDANIFA-NRIGrant

fatmass. 2005-35200-15224andanAHAGrantinAid(GreaterSoutheast

Insummary,ourdatademonstratethattransgenichepaticover- affiliate0755626B).TheauthorswouldliketothankJeffreyMorris

expressionofreninleadstoglucoseintolerance,decreasedfatmass, andTarynStewartfortheirtechnicalassistance.

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