Intracellular conversion of thyroxine to triiodothyronine is required for the optimal thermogenic function of brown adipose tissue

Amendment history:

Correction

(February 1987)

Intracellular conversion of thyroxine to

triiodothyronine is required for the optimal

thermogenic function of brown adipose tissue.

A C Bianco, J E Silva

J Clin Invest.

1987;

79(1)

:295-300.

https://doi.org/10.1172/JCI112798

.

The effect of thyroxine (T4) and triiodothyronine (T3) on the expression of uncoupling

protein (UCP) in rat brown adipose tissue (BAT) has been examined. Thyroidectomized rats

have a threefold reduction in basal UCP levels. When exposed to cold, they become

hypothermic and show a fivefold lower response of UCP than euthyroid controls. T3

augments the basal levels and the response of UCP and its mRNA to cold in a

dose-dependent manner. However, to normalize the response of UCP, T3 has to be given in a

dosage that produces systemic hyperthyroidism. Mere T3 replacement corrects the systemic

hypothyroidism but not the hypothermia or the low levels of UCP. In contrast, replacement

doses of T4 prevent the hypothermia and correct the UCP level. Both effects of T4 are

blocked by preventing T4 to T3 conversion in BAT. Thus, the optimal UCP response to cold

and protection against hypothermia require a high BAT T3 concentration, which is attained

from euthyroid levels of T4 via the activation of the BAT T4 5'deiodinase during cold

exposure, but not from euthyroid plasma T3 levels.

Research Article

Rapid

Publication

Intracellular Conversion

of Thyroxine

to

Triiodothyronine

Is

Required

for

the Optimal Thermogenic Function

of Brown

Adipose

Tissue

Antonio C. Bianco and J. Enrique Silva

Howard Hughes MedicalInstituteLaboratory, Brigham andWomen'sHospital,DepartmentofMedicine,

Harvard Medical School, Boston, Massachusetts02115

Abstract

The effect of thyroxine

(Tf)

and triiodothyronine (T3) on the expression of uncoupling protein (UCP) in rat brown adipose

tissue(BAT)hasbeenexamined.Thyroidectomizedratshavea

threefold reductioninbasalUCP levels.Whenexposedtocold,

they becomehypothermic and showafivefoldlowerresponseof

UCP than euthyroid controls. T3augmentsthe basal levels and

theresponseof UCP and itsmRNA to cold inadose-dependent manner.However,tonormalizetheresponseofUCP,T3 hasto begiven inadosage that prodpces systemic hyperthyroidism.

MereT3 replacementcorrects thesystemic hypothyroidismbut notthe hypothermiaorthelowlevels of UCP.In contrast,

re-placementdosesofT4prevent thehyppthermiaand correctthe

UCP level. Both effectsoff TXare

Olocked

by preventing T4to

T3conversionin BAT. Thus, theoptimal UCPresponsetocold

andprotection'against hypothermia requireahigh BAT T3

con-centration,which isattainedfromeuthyroidlevels ofT4viathe

activationofthe BATT45'deiodinase duringcoldexposure,but notfromeuthyroid plasma T3 levels.

Introduction

Brownadiposetissue(BAT)'isasite offacultativenonshivering

thermogenesis whereheat productionincreasesmarkedlyin

re-sponse to low ambienttemperature and overfeeding

(diet-in-duced thermogenesis)bymechanismstriggered bythe

sympa-theticnervoussystem(1-3).The cornerstoneofthese responses

is the capacityof BATto increase the oxidation offattyacids

and,atthesametime,touncoupleoxidativephosphorylation.

This latteris accomplished bya'32-kD protein located inthe

inner membrane ofmitochondria called thermogenin or un-coupling protein (UCP). This protein is unique toBAT and

AddresscorrespondencetoDr.Silva,Brighamand Women'sHospital, Department ofMedicine,HowardHughesMedical Institute Laboratory, George W. Thorn Building, 20 Shattuck Street, Room 1009,Boston,

MA02115.

Receivedfor publication20August1986.

1.Abbreviations usedinthispaper:5D, 5'deiodinase;aGPD,aglycero

phosphate dehydrogenase;BAT, brownadiposetissue;BW,body weight;

IOP, iopanoicacid; PAGE,polyacrylamide gel electrophoresis;T3, triio-dothyronine; T4, thyroxine;Tx, thyroidectomized; UCP, uncoupling protein.

consideredto be arate-limitingfactor for thethermogenic

func-tionofthistissue (3).Thyroidhormone has also a well recognized

thermogenic effect,butthisisbelieved to result largely from

the

widespread metabolic stimulation in other tissues rather than in BAT, where it would just play apermissiverole

(4,

5). How-ever, BATcontainsathyroxine(T4)

5'deiodinase (5D),

which

catalyzestheconversionof T4 to

triiodothyronine (T3),

10times more potent than T4 (6). This enzyme is markedly activated by

sympathetic stimulation (7, 8), which results in a several-fold incrementin the concentration of T3 in BAT with'no major change in plasma levels ofT4 or T3 (8). Moreover, we have recentlyfound that BAT has thepotentialto respond to T3 in thatit containsnuclearT3receptors in number comparable with thepituitary and liver,bothhighly sensitivetothyroidhormone. Over50% ofthenuclear-bound T3is generated via BAT

5D,

resultingin anoverall receptorsaturationof

"-70%

(9). These

findingssuggest a critical role for thyroid hormone and hence

forBAT

SD

in thefunction ofthe brownadipocyte. Accordingly,

we have examined in rats the effect of thyroid hormone, specif-ically the

intracellularly

generated T3, on the response of UCP

tocold stress. Wehave found that T3

is

critical for the

full

re-sponse

of

UCP

tocold and that BAT

5D

is

essential

in providing theappropriate BATT3 content ateuthyroidplasma levelsof T4 and

T3.

Methods

Animals.Theexperimentswereperformed in Sprague-Dawleyrats (Zivic-Miller,AllisonPark, PA).Thyro-parathyroidectomy withparathyroid

reimplant was performed by the supplier. Experimentswereperformed 3-4 wk after surgery.

Analytical procedures. Mitochondria were isolated from BAT by

standard techniquesof subcellularfractionation.UCPwaspurified from cold-acclimated ratsfollowingmethodspreviously published (10, 11).

Theidentity ofpurifiedUCPwasconfirmedbyits abilitytobind gua-nosine diphosphate. Proteins were analyzed slab polyacrylamidegel

electrophoresisin the presence of sodiumdodecylsulfate(SDS PAGE)

(12). Rabbits

wereimmunized withpurifiedUCP.Antiserawereanalyzed

byimmunoblottingafterelectrotransferontonitrocellulose filter in 200

mM

glycine,20% methanol, and 25 mM Tris buffer(pH 8.3). Parallel gelswerestained withCoomassieBlue.

Immunobl6tting

wasperformed

with

1/100

UCP antisera overnightat

4VC

followed bya2-hincubation with goatanti-rabbitgammaglobulin conjugatedtohorseradish per-oxidase. Immunocomplexeswerevisualized withdiaminobenzidine and

H202.

A solid-phase immunoassay was setup innitrocellulose wells

(MillititerR

plates, Millipore Corp., Bedford, MA).Theantigen,either

purifiedUCPorcrude mitochondrialprotein,wasdiluted with 150mM

NaCl,

10_mMEDTA, and 50 mM Tris buffer(pH 8.6)and immobilized

overnightat

4VC

inmultiplenitrocellulose wellplates

(Milhititer'-,

Mil-liporeCorp.) inavolume of 20,l/well.The wellswerewashed andthe

nonspecificbindingblockedwith 2% gelatin/0.1% Tween 20 for 2 h. Afterrinsing, the wellswereincubatedovernightwitha1/200dilution

J.Clin.Invest.

©TheAmerican Society for Clinical Investigation, Inc.

0021-9738/87/01/0295/06 $1.00

of UCP antiserumat4VC. Theamountofantibodyboundtothe wells was quantitated with 25I-labeledStaphylococcus proteinAina2-h in-cubation(13). The resultsareexpressedasmicrograms of UCP per mil-ligram ofmitochondrial protein. Immunoprecipitation of 35S-labeled UCP labeled from in vivo injections of[35S]methionineor in vitro translated mRNA was carried as described (14) with minor modifications. Equal

aliquots of mitochondria (after in vivo labeling)orof TCA-insoluble3S

counts (in vitro translation)wereincubatedovernight witha 1/100 di-lution of UCP antiserum in 10 mM phosphate buffer (pH 7.5)containing

150 mMNaCl, 1% deoxycholate, and 1%Triton X-100(14). The im-munocomplexes were precipitated with goat anti-rabbit IgG for 2 hat 4VC and recovered bycentrifugationfor 20 minat2,000gthrougha

cushionof 1 Msucrose.Thepelletswerewashed, countedoranalyzed

by SDS PAGE, andautoradiographed. PolysomalRNAwasprepared

asdescribedpreviously (15)inthe presence of 10 mMvanadyl ribonu-cleosides complexes. Polysomeswereresuspendedin4 Mguanidinium

isothiocyanate and RNAwasrecovered bycentrifugationthrough 5.7 MCsCl (15). Poly A RNAwasisolated with acommercial Poly-U-coated paper, HybondR-mAP (AmershamCorp., Arlington Heights, IL)

asdescribed elsewhere(16). Theyield was -10Isg/gtissue. 1 ,g of polysomalmRNA wastranslated inarabbitreticulocyte lysate (Bethesda

ResearchLaboratories, Gaithersburg, MD) in the presence of

carrier-free[35Sjmethionine,asdescribed by Pelham and Jackson (17) and with themodifications introduced by the manufacturer of the kit. BAT 5D anda-GDPactivitiesweremeasuredasdescribed elsewhere(6, 18, 19). Experiments. Details pertaining to specific protocols are givenwith the results.Animalswereplacedat4°Cfor 19or48 h withwaterand food adlibitum, with thesamelight/dark cycleasthe controlsat25°C.

T3 or T4 were injected subcutaneously in 10%rat serumin sterilesaline. Iopanoic acid(IOP),5mg/ 100 g bodyweight (BW)perd,wasinjected

intraperitoneally dissolved in alkalinized saline(pH10-11)(20);the first injection preceeded the T4treatment by 12 h.Controls received the

appropriate vehicles. Core body temperature was measured with a

thermistor introduced -5cmin therectum.Inoneexperiment'25I-T4

was injectedto assesstheeffectiveness oflOPtoinhibit T4toT3 conversion invivo; the injection, isolation ofBATcellnuclei,andextractiopand

analysisof the 1251-T3wereperformedasdescribedpreviously(9). Sta-tisticalanalysisof the results includedone-wayanalysisof variance and multiple comparisonusingtheRS/Iprograms of theBBNInstruments Corp.(Cambridge, MA).

Results

Purification ofUCP, antisera, and immunoassay. The yield of UCPwas -70

;g/g

ofBAT.Oneoutof four rabbits immunized

withtwoinjections of50 ,ugofUCP gave a highly specific

an-tibodythat has been used in the present experiments. Fig. 1 A illustrates the degreeof purificationof UCP and the presence of

aprominent 32-kD bandinmitochondria (lanes 4 and 5), but

not in cytosol. The immunoblotting of these proteins, after

transfertonitrocellulose paper(Fig. 1 B), identifies the 32-kD

band asUCP andillustrates the specificity of the antibody. The assaywashighly specificandsensitive; it could detect 50 ng of

UCP,which allowedus toassay as little as 1-2

,g

of mitochon-drial protein (see Fig. 2).

Effect of thyroidhormones on body temperature and UCP levels in rats maintained in the cold for 48 h. Intact rats main-tained their coretemperature after 48 h at 4°C, whereas the

hypothyroidanimals had severe hypothermia (Table 1). At 25°C, the UCP levelin hypothyroid animals was about one-third of thatof intact rats (10vs.32

ag/mg)

and, although in both groups increased significantly with the cold exposure, the level was four

to five times higherin the euthyroid than in the hypothyroid rats(76 vs. 17 -g/mg;TableI).Groups ofthyroidectomized (Tx)

a _{1 2} 3 4

92 _{_}

66

qy1

30- .

30- _{_i3* #i}

5

_b

_l

_{2 3 4}

Po 92

-w

6

MOF

aa..

_ 45

-

30-

22-14-_4.E

14-Figure1.(a)SDS PAGE ofpurifiedUCP(lane 2),BATcytosol (lane

3),andBATmitochondria fraction either treated(lane 4)ornot(lane

5)

with 3.2% lubrol.(b) Immunoblottingof thegelsshown inAwith

ourrabbitanti-ratUCP antiserum after electrotransferonto nitrocel-lulose paper.Lane1, purified

UCP;

lane2,cytosol;lane3, lubrol-treatedmitochondria; andlane4,crudemitochondria.

ratsweretreated with

T3,

150

ng/

100 gofBW

subcutaneously

twice

daily

for 5 d. This

dosage replaces

the

_daily

production

rate of T3, normalizes serum

T3

concentration,

and restores

growth

rateandliverenzymes in Tx rats

(21).

This

_regimen

of

T3 didnotprevent thecold-induced

hypothermia,

and didnot correctthe UCPconcentration eitheratroom_temperature

(in-creased

only

to 17

_pg/mg)

orin the cold

_{(increased only}

to27

pg/mg,

one-third ofthe response of intact

animals).

Notethat

T3 normalizedthemitochondrialliver enzyme

aGPD

(Table I),

a

sensitive

markerof

thyroid

hormoneactionin this organ

(18),

lb-7

0 200 400 600 800 _,000

UCP(ng)

0 5 1 15 20 25

Mitochondrial ProteinNug)

Figure2.Solid-phaseimmunoassayofUCP. Theordinaterepresents theamountof'25I-labeledproteinAboundtothe wells.PurifiedUCP (opencircles)and crude

mitochondrial

protein (squares) generated

parallellines;preadsorptionoftheantiserumwith 100 gg of UCP (tri-angles) preventedthebinding.

*....

~

::,

.y.

-C

t> 0

Table I. Effect of48h ofExposure to40C

onMitochondrial Levels ofUCP in IntactandTx Rats TreatedwithT3 orT4, withorwithoutIOP

Body

Treatment groups Temperature UCP temperature Liver aGPD

'C jg/mg protein 'C AOD/min

X102

Intact 25 31.8±2.1 37.9±0.1 8.3±0.7

Tx 25 10.0±0.5 37.7±0.2 3.1±0.4

Tx+T3 25 17.0±0.9 - 9.3±0.4

Intact 4 75.6±8.7 37.6±0.1 8.7±0.5

Tx 4 17.2±0.8 29.0±3.5" 2.3±0.3

Tx +T3 4 27.4±1.3* 35.6±0.5" 8.9±0.8

Tx +T4 4 63.4±6.9 37.0±0.3 5.2±0.6

Tx+T4+lop 4 26.0±2.3* 34.9±0.3" 3.0±0.5

Tx+T4+T3 4 58.4±4.3 - 9.2±0.5

Tx +T4+T3

+lOP 4 32.7+2.5§ - 8.6±0.7

F 379.4 106.0 215.4

Values are the means±SDof four to five animals. Tx rats were treated with T3 as described in Fig. 2. T4, 400ng/100g BW, wasgiven every 12hwhile the rats wereinthecold. lOP, 5 mg/ 100 gBWperd,was injected intraperitoneally starting12 hbefore the firstinjection of T4. Cold exposure lasted 48 h.Body temperaturewasmeasuredwithan electronicprobe inserted -5cminthe rectum.Liver aGPDwas mea-suredasdescribedpreviously and expressedasODunits/minper mg of protein. Intact,Tx+T4, andTx+T4+T3at40Cwere not differ-ent.UCPandaGPD inTx at25'Cwassignificantlylower than in the intact controls (P<0.001);coretemperaturewas notdifferent. One-wayanalysis of variance:*P<0.01vs. Tx at40C;*P<0.01vs. Tx +T4at40C; P <0.01 vs. Tx +T4+ T3at4

C;Q

P<0.01vs. intact rats.

asitnormalized serum T3 (not shown but see Table III for a similarexperiment). Incontrast,anamount of T4 equal to the production rate (7), 400 ng/100 g BW subcutaneously twice daily,

justduring the 48 hofthe cold stress, resulted in a UCP level

of63 ug/mg, statisticallynotdifferent fromthe response in the

euthyroid animals,andprevented the hypothermia. This regimen

ofT4didnot correctthesystemic hypothyroidismbecauseit did notnormalizeeither serum T4, T3, or liver aGPD. 1OP, a potent

competitive inhibitor of 51D (20), abolished thecorrection by T4 ofboth, the UCP levels andhypothermia. Theeffectiveness

of1OPtoblock T4toT3 conversionwasconfirmed with

125I-T4

injectedto aparallel group of animals. 1OP decreased nuclear

1251-T3

by >90%withoutaffectingplasma orBAT

125I-T4

con-centrations,and abolished theactivity oftheBAT

51D.

ResponseofUCP to variousdosesofT3.To understand better thequantitative relationshipsbetween theUCP responses and

BATT3,Txrats were treated for5d with0.15,ug T3/100 gBW

subcutaneously twice adayand then placed them inthecold for 48 h. Atthistime,they weresegregated in groups of four ratswhich wereinjected subcutaneously with various doses of T3at - 12-h intervals. The resultsareshown in Table II.The

highestdose,4.7

_gtg/l

00 gBWperd, elevated thepost-coldlevel of UCP from -8,in the untreated rats,to 41 Ag/mg of

mito-chondrialprotein,aresponse still lower than ineuthyroidanimals

orinTx ratstreatedwith T4for48 h where UCP level reached

~-70

gg/mg

(Table I). Incontrast, the lowest dose of T3

nor-TableII.Effects ofVarious DosesofT3

onMitochondrial LevelsofUCP inThyroidectomized(Tx)

RatsAfter48hofCold Exposure

DoseT3 UCP Serum T3 LiveraGPD

ig/1i00gBWper d ug/mgprotein ng/ml AOD/minX102

0.0 8.0±0.4 0.08±0.02 3.3±0.5

0.3 10.6±0.5 0.59±0.04 8.9±0.4

0.5 11.7±0.9 1.13±0.08 10.7±1.3

0.8 12.4±1.0 1.74±0.10 12.4±0.9

1.3 15.3±1.1 2.77±0.40 14.1±0.3

2.1 23.4±1.5 4.66±0.66 16.4±1.0

4.7 41.6±3.7 10.72±2.10 19.6±2.3

Intact 75.6±8.7 0.67±0.08 8.7±0.5

F 202.9 256.6 63.9

Valuesarethemeans±SD of four animals.Tx rats wereinjected twice

dailywith either 150,ggT3/100g BW orvehicle (top group) for 5 d;

theywerethensegregatedinsevengroupsandgiventhedailydosesof T3 indicated in the table divided intwosubcutaneousinjections. This

treatmentlastedtwoconsecutive days while theratsweremaintained

at40C.Serum T3wasmeasured in unextractedserumby radioimmu-noassay(14). For all three variables theresponsetothe lowest dose of T3wassignificant(P<0.01).

malizedtheliveraGPD andserumT3concentration,whereas

higher doses resulted in

hyperthyroid

serum levels ofT3 and aGPD activity. Actually, the highest dose of T3provided an

average nuclear receptor saturation of>90%,as canbecalculated

from thehalf-saturating plasma T3 concentration (9) and the

integrated plasmaT3concentration obtainedfrom theserumT3

level in TableIIand the half-time of

disappearance

ofT3from

plasma incold-exposedrats(8).

Effects of

acutecold exposure and

thyroid

hormonesonthe

endogenous labeling of

UCPwith

[35Sjmethionine

in

_hypothyroid

rats. Todeterminewhether theeffect of

thyroid

hormonewas

duetoincreased

synthesis

of

UCP,

Txrats weretreated with T3

asabove and placed in the cold. Other animals were

injected

withasingleintraperitonealdose of 800 ng T4/100 gBW,either aloneorin addition tothe T3

regimen,

atthetime of

starting

the coldstress. The total duration of the coldstresswas 19h.

After 16 h in thecold,therats were

injected intravenously

with -300

,uCi

of

[35S]methionine

and killed 3 hlater. The

immu-noprecipitates

of mitochondrial

proteins

were

analyzed by

SDS PAGE and

autoradiography.

The

autoradiograms

showed

only

one32-kDband of

radioactivity

in all the

experimental

condi-tions

(Fig.

3

A).

The

intensity

of this

band,

which represents the

fraction of 35S counts

incorporated

into

protein

as

UCP,

was

enhanced

by

cold exposure. This responsewas

only

slightly

im-proved byT3 but

markedly

enhancedafteronedoseof T4.Table

III presents a more detailed and

quantitative

account ofthis

experiment.

Resultsare

expressed

asthemean±SD percent of the

protein-bound 35S

recovered in the

immunoprecipitate

in the individual rats. While T3

augmented

the response to cold

by-90%overthe untreated animals

([4.73-2.45]/[3.62-2.45]),

a

single injection

ofT4

amplified

fivefold the responseto cold

in

hypothyroid

rats

([8.37-2.45]/[3.62-2.45];

seeTable

III).

The

increments in

[35S]methionine incorporation

after these

manip-ulationswere of

comparable

magnitude

to those observed in

2 3 4

a _b 2 3 4 5 6 7

92-

66-97

45- 69

53

46

30-30

22-

14--3.1 10.6 1.6 2.7 4.6 8.6 4.8

Figure 2.(a) SDS PAGE ofimmunoprecipitatesof in vivo

[35S]methionine-labeledBATmitochondrialproteinsfromTx rats. Lane 1, Tx at roomtemperature; lane 2, untreatedTxinthecold; lane3, T3-treatedTxin the cold; and lane 4,asin lane 3 plusone dose of 800 ngT4/I 00 gBW atthestartof thecold stress.Coldstress lastedfor 19 h.[35S]methioninewasinjected after 16h at 4C.T3 dos-age, 150ng/100 gBWsubcutaneously every - 12hfor5 d.(b)SDS

PAGE ofimmunoprecipitated UCP translated in vitro inarabbit

re-ticulocytelysate.(BethesdaResearchLaboratories)incubated with 1

tgof BATcytoplasmicmRNA and carrier-free[35S]methionine.Rats weremaintainedatroomtemperatureorplaced inthe cold room at

4°Cfor 19 h; theyweregrouped and treatedasfollows;lane 1, intact rats; lane2, intactrats4°C;lane3,Txrats; lane4,Tx rats at4°C; lane 5,Txratsat4°Candtreated withT3asfor lane 3 in A; lane 6, Tx rats at4°Ctreated with T3 and T4asin A, lane 4; lane 7,same as in lane 6 plus IOP 5mg/100 gBWintraperitoneally12 hbefore T4 administration. Each lane represents the pooledmRNAfromfour rats.Thenumbers at thebottomofeachlane representthepercent of TCA-insolublecountsthatwereprecipitated with the antibody.

ofdegradationofUCP,theincrementsoverthe untreated ani-mals would have been timedependent, i.e.,much larger after 48 hofcold exposure than after 3 hofendogenous labeling.

Thus, coldinducesarapidbut modest responseofUCPtocold inhypothyroidratsandthyroidhormoneamplifies the response

byincreasingthesyntheticrateof theprotein.Aswith the im-munoreactive levels, the responseto coldisjustmodestly in-creased by 5-d replacement with T3 but markedly augmented

byonereplacementdose ofT4ina19-h interval. The T3regimen

normalized theserumconcentration of T3 and the liver aGPD,

asit did in the other experiments, whereas the dose of T4 did notimprove the low level of aGPD nor did it normalize the

serumlevelof T3 (not shown).

Effect of coldexposure and thyroidhormones onthe UCP mRNA levels.Todetermine whether the thyroid hormone-in-ducedincrease in UCP synthesis was pre- or posttranslational, weexaminedtheproducts oftranslation of BAT mRNA obtained under thesameconditionsofthe preceeding experiment. Neither the amountofmRNA recovered per gramof tissue nor the re-coveryof TCA-insoluble

35S

countsafter translation was

signif-icantly affected by the various experimental manipulations. When identical numbersof TCA-precipitable 35Scounts were

Table III. In Vivo [35S]Methionine Incorporation into BAT UCP after 19h of ColdExposure in Tx Rats Treated with T3 and/orT4

Treatmentgroups Temperature UCP Liver aGPD

0C % vAOD/minX102

25 2.45±0.27 3.4±0.2

4 3.62±0.25* 3.5±0.5

T3 4 4.73±0.39* 8.9±0.3

T4 4 8.37±0.39§ 4.3±0.6 T3 +T4 4 8.29±0.34§ 8.8±0.2

F 154.6 194.0

Values are the means±SD of three animals. Tx rats were treatedwith T3and/or T4 as inFig. 2 A. Cold exposure lasted 19hand

[35S]methi-oninewasinjectedintravenously 3 h before the end of thisperiod.

UCP wasimmunoprecipitated as described under Fig. 2Aand ex-pressed as percent of total

_{[35S]methionine-labeled}

mitochondrial pro-tein. Liver aGPDasinTable I.

One-wayanalysis of variance:*P<0.01 vs. Tx at250C;tP<0.01 vs. Txat40C;IP<0.01 vs. Tx +T3at4-C.

immunoprecipitatedwith the UCPantiserum,theincorporation of

[35S]methionine

into UCP was foundhigherineuthyroidthan

inuntreated Tx rats at roomtemperature (3.1 vs. 1.6% of the

TCA-insoluble counts, Fig. 2 B). After 19 h of coldexposure there was a3.5-fold incrementin immunoprecipitable counts

in intact animalsto 10.6% ofthe TCA-insolublecounts,whereas

in the untreatedTx ratsthis valuewas2.7%.T3replacementfor

5dasdescribed above improvedtheresponsetocoldtoabout onehalf of thatobserved in the intactanimals,whereas asingle injection of 800 ngT4/100 gBW to Tx rats sufficed to normalize

the UCP mRNA responsetocold. Asabove,the blockade ofT3 generation fromT4withIOPpreventedthe effect of the latter.

Thus, the responsesinimmunoreactiveUCP concentration and

insyntheticrate arelikelytoreflect levels of thecorresponding

mRNA.

Discussion

UCPisratelimitingfor thethermogenicresponse ofBAT(3).

Itisthankstothisprotein that the augmented oxidation of

sub-stratesin BAT,largely fatty acids,canresult in marked incre-ments inheatproductionand energydissipationinoverfeeding

(1-3). Hypothyroid animals and humansare known to beat

riskofhypothermiain coldenvironments.Theprotective effect of thyroidhormonehasalwaysbeenconsideredtoresultfrom

the stimulationof oxidations in a variety of tissues. Wehave

confirmedthathypothyroid animalscannotmaintain theircore

temperature whenexposedtocold and that theabnormalityis

rapidlycorrectedbythyroidhormone, but theprevention of the hypothermia correlated with the correction of the UCP levels,

notwith the correction of liver aGPD.

Because ofourearlier demonstration of the activation of

BAT5Dbysympatheticstimulation and the several fold increase in BAT T3, we suspected that T3 was more important for BAT thanpreviously recognized(7, 8). On the other hand, the enzyme israpidlyinhibited by high doses ofT4 (22), and the experiments

exploring

theeffectofthyroidhormoneon BAThad been done with high doses of T4 in animals exposed to cold (4, 5). We reasoned that thecold-stimulated deiodinase activity and the magnitudeofthe T4doses, probably resulted in shallow

response curves leading to the concept that thyroid hormones

were just permissive. Wehave shown here that the UCP response

is clearly T3 dose dependent.In addition, we found that the dose

of T3 necessary to normalize UCP isinthehyperthyroidrange, which we have documented with the hyperthyroid levels of aGDP activity in the liver. Based in recentstudies (9), we cal-culated that the highest dose ofT3 used in thepresentexperiments maintained the nuclear T3 receptors -90% saturated. A cal-culation of nuclear occupancy in similar experiments (Bianco, A. C., and J. E. Silva, manuscript in preparation)indicatesthat the UCP response is not linear withnuclear occupancy but of

the amplified pattern shown by Oppenheimer etal. foraGPD and malic enzyme in rat liver (23). This, and the finding that the receptors are 70-75% occupied in euthyroid rats at 250C (9), suggest that the optimal response ofthis tissueto coldrequires ahighdegree of saturation.

The critical level of BAT T3 needed for a full response is much more efficiently reached by in situ intracellular generation than from plasma T3. Only one replacement dose of T4 can normalize the response of UCP to cold in animalswithchronic hypothyroidism, but several days of T3 replacement were

in-sufficient. The latter, however, normalized both serum T3 and liver aGPD, documenting the adequacy of the regimen to replace

thishormone. Blockade of T4 to T3 conversion by IOP prevented theeffect of T4 but not that of T3, without affecting the plasma levels of either iodothyronine or decreasing the T4 availability in BAT. This suggests that it is the T3 generated in BAT, and notplasma T4 or T3, the relevant hormone for the UCP response

tocold. In previous studies (8),weshowed that, after a dose of T4identical to that used in the present experiments, 4 h of cold exposure resulted in - five times as high locally generated BAT

T3 as that of rats maintained at room temperature. The excess

ofT3wasdue to thesympatheticactivation of BAT 51D because it was prevented by prazosin, an

al-antiadrenergic

agent that impairs the cold- and the norepinephrine-induced activation of BAT5'D (7, 8). The present results illustrate the relevance of such activation, fora doseof T4 insufficient to normalize the serum T3 concentration or liver aGPD, normalized the UCP response to cold. To obtain the same response with exogenous T3requires ofdoses causing hyperthyroid levels of serum T3 and

liveraGPD.

Acute exposure to low ambient temperature results in a rapid increase of the mRNA levels of UCP (24). T3 also increases the rateofsynthesis of this protein both, at room temperature and in response to cold, which reflects proportional elevations of

corresponding mRNA.Whether the effect of thyroid hormone is direct, either by increasing the transcription rate of UCP mRNA or to stabilizing this mRNA, or whether the effect is indirect byamplifyingasignal triggered by the sympathetic ner-vous system, remains to be demonstrated. Whatever the mech-anism involved,thisis anothersituationwherethyroidhormone

amplifies the responseto aprimary stimulus, in thiscase the

sympatheticnervoussystem,asit does with the response of

li-pogenicenzymestocarbohydrates (25), and in that constitutes

anexcellent modeltostudythyroidhormone actionatmolecular level.Of course, theuseof UCPasamodel of thyroid hormone

actionatcellular level mandatestodemonstrate that the UCP responses observed hereare direct e.g., not mediated through

aneffectontheadrenergic pathway, which will probably need thedevelopment ofan invitro system. BecauseBAT function inhibernating species isto warmthehypothermicbody of these animalsatthe end ofhibernation(2, 3), thepossibilitythat

hy-pothermiaitself impaired the raise of UCP in the present

ex-perimentsis remote, but also must be excluded.

Two corollaries emerge from the results presented here. First, that BAT5'Dis a key element in the function of BAT and hence a variable that may affect the physiological responses of this

tissue.This conclusion is in agreement with the observation that ob/ob mice die of hypothermia when exposed to cold, for in theseanimals the BAT 5'D is not activated by the cold as in lean controls (26). Secondly, that at euthyroid levels of T4 and

T3,the thermogenic effect of T3, evidenced in its ability to pre-vent hypothermia at low ambient temperature, results largely from its action on BAT and not from its various and widespread metabolic effects in other tissues. The present data suggest that the stimulation of UCP synthesis is one of the mechanisms by which thyroid hormone exerts this physiological effect, but the possibility that T3 affects other aspects of BAT metabolism lead-ing to the same results, remains open. Because the calorigenic BATresponse may be limited by the saturation ofthe receptors, onewould expect that the increased thermogenesis observed in hyperthyroidism results largely from the direct action of thyroid hormones in other tissues, where an increase in respiration has beenwelldocumented(27, 28).

Acknowledgments

We aregrateful ofDr. Jean Himms-Hagen forthegift of antiserum against the uncoupling protein whichwashelpfultoidentify thisprotein

during the purification.Wealsoappreciatethesecretarialhelp of Janice Bridges.

Dr.Bianco isarecipient ofaFellowship fromtheFundacaode Am-paroaPesquisa do EstadodeSaoPaulo, Brazil.

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