Responses of skeletal muscle lipid metabolism in rat gastrocnemius to hypothyroidism and iodothyronine administration: a putative role for FAT/CD36

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1 Am J Physiol Endocrinol Met 33: E1222 E1233, 212. First pulished Septemer 11, 212; doi:1.1152/jpendo Responses of skeletl muscle lipid metolism in rt gstrocnemius to hypothyroidism nd iodothyronine dministrtion: puttive role for FAT/CD36 Assunt Lomrdi, 1 Rit De Mtteis, 2 Mri Moreno, 3 Lur Npolitno, 1 Ros Ann Busiello, 1 Rosl Senese, 4 Pieter de Lnge, 4 Antoni Lnni, 4 nd Fernndo Gogli 3 1 Diprtimento delle Scienze Biologiche, Università di Npoli Federico II, Nples, Itly; 2 Diprtimento di Scienze Biomolecolri, Università degli Studi di Urino Crlo Bo, Urino, Itly; 3 Diprtimento di Scienze per l Biologi, l Geologi e l Amiente, Università degli Studi del Snnio, Benevento, Itly; nd 4 Diprtimento di Scienze dell Vit, Second Università degli Studi di Npoli, Csert, Itly Sumitted 2 Jnury 212; ccepted in finl form 5 Septemer 212 Lomrdi A, De Mtteis R, Moreno M, Npolitno L, Busiello RA, Senese R, de Lnge P, Lnni A, Gogli F. Responses of skeletl muscle lipid metolism in rt gstrocnemius to hypothyroidism nd iodothyronine dministrtion: puttive role for FAT/CD36. Am J Physiol Endocrinol Met 33: E1222 E1233, 212. First pulished Septemer 11, 212; doi:1.1152/jpendo Iodothyronines such s triiodothyronine (T 3 ) nd 3,5-diiodothyronine (T 2 ) influence energy expenditure nd lipid metolism. Skeletl muscle contriutes significntly to energy homeostsis, nd the ove iodothyronines re known to ct on this tissue. However, little is known out the cellulr/moleculr events underlying the effects of T 3 nd T 2 on skeletl muscle lipid hndling. Since FAT/CD36 is involved in the utiliztion of free ftty cids y skeletl muscle, specificlly in their import into tht tissue nd presumly their oxidtion t the mitochondril level, we hypothesized tht relted chnges in lipid hndling nd in FAT/CD36 expression nd sucellulr redistriution would occur due to hypothyroidism nd to T 3 or T 2 dministrtion to hypothyroid rts. In gstrocnemius muscles isolted from hypothyroid rts, FAT/CD36 ws upregulted (mrna levels nd totl tissue, srcolemml, nd mitochondril protein levels). Administrtion of either T 3 or T 2 to hypothyroid rts resulted in 1) little or no chnge in FAT/CD36 mrna level, 2) decresed totl FAT/CD36 protein level, nd 3) further increses in FAT/CD36 protein level in srcolemm nd mitochondri. Thus, the min effect of ech iodothyronine seemed to e exerted t the level of FAT/CD36 cellulr distriution. The effect of further increses in FAT/CD36 protein level in srcolemm nd mitochondri ws lredy evident t 1 h fter iodothyronine dministrtion. Ech iodothyronine incresed the mitochondril ftty cid oxidtion rte. However, the mechnisms underlying their rpid effects seem to differ; T 2 nd T 3 ech induce FAT/CD36 trnsloction to mitochondri, ut only T 2 induces increses in crnitine plmitoyl trnsferse system ctivity nd in the mitochondril sustrte oxidtion rte. thyroid hormones; 3,5-diiodo-L-thyronine; ftty cid trnslocse; lipid hndling THE ROLE OF THYROID HORMONE [3,5,3=-triiodothyronine (T 3 )] s mjor endocrine regultor of metolic rte is widely recognized (33). In fct, in dults T 3 is unique in its ility to stimulte thermogenesis nd to ffect nolic nd ctolic iochemicl processes. In lipid metolism, T 3 ffects the synthesis, moiliztion, nd degrdtion of lipids, lthough degrdtion is influenced more rpidly thn synthesis (33, 36). Address for reprint requests nd other correspondence: F. Gogli, Diprtimento di Scienze per l Biologi, l Geologi e l Amiente, Università degli Studi del Snnio, Vi Port Ars 11, 821 Benevento, Itly (e-mil: Indeed, T 3 increses 1) the moiliztion of the triglycerides stored in dipose tissue, 2) the serum concentrtions of free ftty cids (FFA), 3) lipoprotein lipse ctivity, nd 4) the utiliztion of lipid sustrtes in metoliclly ctive tissues (22, 39). Under some conditions, these effects re ccompnied y reduction in ody weight (2, 24). As result, in the pst T 3 hs een proposed nd tenttively used s n ntioesity nd lipid-lowering gent. However, its eneficil weight reduction effects were ssocited with the simultneous induction of thyrotoxic stte nd with undesirle side effects t the crdiovsculr level, nd so its use for such purposes hs een stopped (2, 4). Now, in the hope of countercting dyslipidemi nd oesity, ttention is eing focused on the development of compounds tht hve the eneficil effects of excess thyroid hormone, ut with miniml deleterious effects (2, 4). In prticulr, new perspectives in this field hve een opened y the introduction of potent thyromimetics with thyroid hormone receptor- isoform (TR- ) sutype-selective ctivities (4), nd few of them hve recently entered clinicl trils (25, 4). Importntly, TR- receptors re scrcely expressed t ll in crdiomyocytes nd re involved minly in the thyroid hormone-medited regultion of lipid metolism (5, 3, 34). There is ccumulting evidence to show tht 3,5-diiodothyronine (T 2 ), thyroid hormone derivtive, hs iologicl effects oth in vivo nd in vitro (13). T 2 is le to ffect oth energy nd lipid metolism without inducing the side effects ssocited with T 3 (7, 3). Indeed, T 2 dministrtion to rts increses their resting metolic rte (31) nd prevents dietinduced oesity s well s liver stetosis, hypertriglyceridemi, hypercholesterolemi (7, 26), nd insulin resistnce (7, 32). The ctions of T 2 re medited y mechnisms not involving TRs, nd therefore, they re referred to s non-nuclermedited (7, 14). Studies of the control exerted y T 3 over ftty cid metolism hve focused principlly on liver, dipose tissue, nd hert (16, 42). Thorough investigtions of the regultory mechnisms involved in muscle ftty cid uptke nd utiliztion in vrious thyroid sttes nd of the effects of T 3 nd/or T 2 dministrtion would e expected to revel in detil how these hormonl sttes nd chnges modulte the levels of ftty cid uptke nd utiliztion in muscle tissue. Ftty cid trnslocse (FAT/CD36) seems to e the protein involved not only predominntly in the import of FFA into the tissue ut lso intrcellulrly (for review, see Refs. 12, 18, nd 39). Reportedly, the functionl presence of FAT/CD36 is not restricted to the plsm memrne, since it is lso found within mitochon- E /12 Copyright 212 the Americn Physiologicl Society

2 dri, where it hs puttive role in the modultion of ftty cid oxidtion (17, 39). However, conflicting results re present in the literture. Indeed, regrding oth the locliztion nd the role plyed y FAT/CD36 t the mitochondril level, the existing disgreements re sed principlly on 1) the sence of cler evidence of coimmunolocliztion of FAT/CD36 with mitochondri (2 22) nd 2) contrsting dt concerning the reduced ility of skeletl muscle mitochondri isolted from FAT/CD36-null mice to oxidize lipid sustrtes (i.e., plmitte, plmitoyl-coa, plmitoyl crnitine) (17, 22, 39). However, recent reports my resolve some of the ove discrepncies insofr s functionl dt suggest tht FAT/CD36 is locted on the outer mitochondril memrne upstrem of long-chin cyl-coa synthetse (39), nd thus it my fcilitte plmittesupported nd not plmitoyl-coa- or plmitoyl crnitine-supported respirtion. Moreover, evidence tht, in skeletl muscle mitochondri from FAT/CD36-null mice, ftty cid oxidtion ws reduced ut not olished suggested tht ltertion of FAT/CD36 content t the mitochondril level provides dditionl regultion tht my e importnt for ugmenting FFA oxidtion rtes (17, 39). This my e prticulrly importnt when the energy demnds from FFA oxidtion re sustntilly incresed (17). In fct, in cses of incresed energy demnd, the cpcity of skeletl muscle to rpidly modulte the uptke of FFAs nd their successive oxidtion cn e incresed y rpid trnsloctions of FAT/CD36 from its endosoml comprtment to the plsm memrne (for FFA uptke) nd mitochondri (for FFA oxidtion) (3, 18). In the present study, we tested our hypothesis tht relted chnges in lipid hndling nd FAT/CD36 expression nd sucellulr distriution would occur 1) in hypothyroidism nd 2) upon T 3 or T 2 tretment of hypothyroidism. To this end, we crried out rod nlysis of the metolic prmeters ssocited with skeletl muscle lipid hndling in hypothyroidism with or without iodothyronine (T 3 or T 2 ) dministrtion. We lso directed our ttention towrd chnges in FAT/CD36 protein expression/locliztion. For n niml model we used rts in which hypothyroidism hd een induced y comined tretment with propylthiourcil nd iopnoic cid. This tretment induces severe hypothyroidism nd t the sme time inhiits ll of the three known types of deiodinse enzyme (31). This llows us to ttriute the oserved effects to the iodothyronines ctully injected rther thn to ny of their deiodinted products. In ddition, in view of the occurrence of rpid trnsloctions of FAT/CD36 from its endosoml comprtment to the plsm memrne nd mitochondri, we thought it would e interesting to investigte whether T 3 nd/or T 2 might influence FAT/ CD36 levels in the short term (viz. within 1 h fter their injection into hypothyroid nimls). To gin further insight into the sucellulr distriution of FAT/CD36, we performed immunohistochemistry on gstrocnemius skeletl muscle (GSkM), nd in ddition we used n in vitro model of skeletl muscle fiers (C 2 C 12 cell line) to exmine the intrcellulr nd srcolemml locliztions of FAT/CD36, nd lso the T 3 - nd T 2 -induced chnges in its distriution, y the ppliction of immunocytochemistry nd confocl miscroscopy. METHODS E1223 Animls. Mle Wistr rts (275 3 g) were otined from Hrln Lortories (Itly). They were kept one per cge in temperturecontrolled room t 28 C under 12:12-h light-drk cycle. A commercil msh nd wter were ville d liitum. Six groups of rts were used throughout, with ech one comprising six nimls. Group N (N) consisted of euthyroid rts tht received vehicle. Group H (H) consisted of hypothyroid rts [hypothyroidism eing induced y the intrperitonel (ip) dministrtion of propylthiourcil (1 mg/1 g ody wt) for 4 wk together with weekly ip injection of iopnoic cid (6 mg/1 g ody wt) (31)]. H T 3 1 wk nd H T 2 1 wk contined hypothyroid rts (treted like those in group H) tht in the lst week of tretment received dily injection of either T 3 (15 g/1 g ody wt) or T 2 (25 g/1 g ody wt). H T 3 for 1 h nd H T 2 for 1 h contined hypothyroid rts (treted like those in group H) tht received T 3 (25 g/1 g ody wt) or T 2 (25 g/1 g ody wt) 1 h efore eing euthnized (see elow). At the end of the tretments, rts were nesthetized y n ip injection of chlorl hydrte (4 mg/1 ody wt) nd euthnized y decpittion. This ws done t 6 h fter the end of the nocturnl periods. Blood ws collected, nd then gstrocnemius muscles were excised, weighed, nd immeditely processed for mitochondril isoltion or frozen in liquid nitrogen. Authoriztion to perform these experiments on rts ws given y the Itlin Ministero dell Snità (decree no. 176/25-A). Metolic prmeters. Oxygen consumption (V O 2), cron dioxide production (V CO 2), nd respirtory quotient (V CO 2/V O 2) mesurements were mde using four-chmer, indirect, open-circuit clorimeter Oxymx system (Pn L, Cornell, Brcelon, Spin) with one rt per chmer. After 1-h period of dpttion to the metolic chmer, V O 2 nd V CO 2 were mesured in individul rts t 15-min intervls for 4 h. Isoltion of mitochondri for exmintion of plmitte nd succinte oxidtions. Skeletl muscle mitochondri were isolted s descried previously, with slight modifiction (28). Briefly, tissue frgments were immersed in ice-cold uffer consisting of 22 mm mnnitol, 7 mm sucrose, 2 mm Tris HCl, 1 mm EDTA, nd 5 mm EGTA, ph 7.4, nd then homogenized in Potter-Elvehjem homogenizer. Nuclei nd cell deris were removed y centrifugtion t 5 g for 1 min, with the resulting superntnt eing centrifuged t 3, g. The mitochondril pellet ws wshed twice nd resuspended in miniml volume of isoltion medium nd kept on ice. The protein concentrtion ws determined y the method of Hrtree (15), using ovine serum lumin (BSA) s stndrd. Mesurements of ftty cid nd succinte oxidtion rtes. Mitochondril ftty cid nd succinte oxidtion rtes were ssessed polrogrphiclly using Clrk-type electrode t 3 C in finl volume of.5 ml of 8 mm KCl, 5 mm HEPES (ph 7.), 1 mm EGTA, 5 mm K 2HPO 4, 1% BSA (wt/vol), nd ADP (12 g/ml). To detect succinte oxidtion, mitochondri (.25 mg) were incuted for 3 min in the respirtory medium, nd the rection ws strted y ddition of succinte (5 mm). To detect plmitte oxidtion, the respirtory medium ws supplemented with 2.5 mm mlte, 1 mm L-crnitine, 2 mm ATP,.5 mm CoA, nd 1 mm DTT. Mitochondri (.5 mg) were incuted for 3 min in the respirtory medium, nd the rection ws strted y ddition of plmitte (12 M). Gstrocnemius muscle totl lyste, plsm memrnes, nd mitochondril isoltion for Western lotting nlysis. For Western lotting nlysis, gstrocnemius muscle ws homogenized in RIPA uffer (15 mm sodium chloride, 1.% Triton X-1,.5% sodium deoxycholte,.1% SDS, nd 5 mm Tris, ph 8.; ll from Sigm-Aldrich, St. Louis, MO) using n Ultr-turrx. The homogente ws left on ice for 1 h, during which it ws shken every 1 min. The lyste ws then ultrcentrifuged t 86. g for 1 min t 4 C. The superntnts of the ultrcentrifuged clered lystes were used for detection of FAT/CD36.

3 E1224 To isolte plsm memrnes nd mitochondri for Western lotting nlysis, gstrocnemius frgments were homogenized in isoltion uffer (consisting of 1 mm NHCO 3,.25 M sucrose, 5 mm NN 3, nd 1 M PMSF, ph 7.). The homogente ws centrifuged t 1,3 g for 1 min. The superntnt ws centrifuged t 3, g, nd the pellet, consisting of the mitochondril frction, ws wshed nd resuspended in RIPA uffer. The superntnt ws then centrifuged t 1, g for 1 min. The resulting pellet ws discrded nd the supernntnt centrifuged t 19, g for 1 h. The resulting pellet, consisting of the memrne-rich frction, ws resuspended in RIPA uffer. Determintion of skeletl muscle TG-hydrolse ctivity. Dissectedout, freeze-dried GSkMs were homogenized on ice in uffer contining.25 M sucrose, 1 mm EDTA, 1 mm dithiothreitol, 4 mm -glycerophosphte, 1 mm N pyrophosphte, 2 g/ml leupeptin, 2 g/ml ntipin, 6.25 g/ml pepsttin A, nd.31 M okdic cid. The homogente ws centrifuged t 18,5 g t 4 C for 45 s; the resulting superntnt ws used for TG-hydrolse ctivity mesurements. The sustrte, contining triolein nd tri-[9,1(n)- 3 H]-olein (GE Helthcre), ws emulsified with phosphtidylcholine-phosphtidylinositol (3:1) in 3 ml of.1 M potssium phosphte uffer (ph 7.) nd 1 ml of 2% ftty cid-free BSA in.1 M potssium uffer (ph 7) y soniction. To detect TG-hydrolse ctivities, 1 g of lyste protein ws incuted in shking wter th for 6 min t 37 C with 1 l of[ 3 H]triolein sustrte (167 nmol, cpm) nd n enzyme dilution uffer (2 mm potssium phosphte, ph 7., 1 mm EDTA, 1 mm dithioerythreitol, nd.2% ftty cid-free BSA) in totl volume of 2 l. Incutions were ech performed in triplicte. The rection ws stopped y ddition of 3.25 ml of methnol-chloroform-heptne (1:9:7) nd 1 ml of.1 M potssium cronte nd.1 M oric cid, ph 1.5. The mixture ws vortexed for 1 s nd then centrifuged t 1,1 g for 2 min. Finlly, 1 ml of the upper phse ws removed, nd the rdioctivity of the relesed 3 H-leled ftty cids ws determined y liquid scintilltion counting (Tri-Cr; Pckrd Instruments). Gsrocnemius muscle lipolysis ssy. In vitro relese of glycerol from GSkM under sl conditions ws determined s reported y Enoksson et l. (9), with slight modifictions. Briefly, three to four GSkM strips deprived of visile ft nd weighing 5 mg ech were incuted for 9 min in 2 ml of Kres-Ringer icronte uffer supplemented with 2 mg/ml BSA (ph 7.4, BSA frction V; Sigm), 1 mg/ml glucose, nd.1 mg/ml scoric cid in shking wter th (37 C) nd gssed with 95% O 2-5% CO 2. Strips were incuted in duplicte, nd fter incution they were removed from the medium. Then, n liquot of the medium ws used for nlysis of glycerol. A commercilly ville sornce-sed enzyme ssy (Free Glycerol Regent; Sigm) for glycerol ws converted to fluorescencesed detection y the inclusion of the hydrogen peroxide-sensitive dye Amplex UltrRed, s reported y Clrk et l. (6). Detection of sl nd ftty cid-induced proton conductnce. Mitochondril inner memrne (MIM) proton conductnce is evluted y mesuring the rtio of the flux of protons tht cross the inner memrne nd re not ssocited with synthesis of ATP (proton lek) to the MIM potentil. The respirtion rte of mitochondri mesured in the presence of oligomycin is proportionl to proton lek; indeed, in stedy stte, proton flux cn e determined y multiplying oxygen consumption y the H /O rtio, which is considered to e 6 when succinte is used s sustrte. Becuse the reltionship etween proton conductnce nd memrne potentil is not liner, the kinetic response of the proton conductnce pthwy to its driving force (memrne potentil) ws mesured. To compre proton conductnce vlues mong the vrious mitochondril preprtions otined from different groups of nimls, proton conductnce ws evluted t fixed memrne potentil (viz. the highest common memrne potentil detected). To determine respirtion rte nd mitochondril memrne potentil, electrodes sensitive to oxygen nd to the potentil-dependent proe TPMP were used. With the im of detecting the kinetic response of the proton conductnce pthwy to its driving force (memrne potentil), mitochondri (.5 mg/ml protein) were incuted in ssy medium contining 8 mm KCl, 5 mm HEPES (ph 7), 1 mm EGTA, 5 mm K 2HPO 4,5mMMgCl 2,1 g/ml oligomycin, 8 ng/ml nigericin (to collpse the difference in ph cross the MIM nd to llow the whole proton motive force to e expressed s memrne potentil), nd.5% BSA. The electrode ws clirted y mking sequentil dditions of TPMP 2 M, nd then 6 mm succinte ws dded to strt the rection. Respirtion rte nd MIM potentil were progressively inhiited through successive stedy sttes y dditions of mlonte 2 mm. At the end of ech run,.2 M FCCP ws dded to dissipte the memrne potentil nd relese ll TPMP ck to the medium, llowing correction for ny smll electrode drift. The TPMP-inding correction for skeletl muscle ws tken to e.4 l/mg protein. To exmine the effect of FFA in inducing proton conductnce, the incution medium ws supplemented with 3 M rchidonic cid (AA). The proton conductnce detected in the presence of AA represents the sum of the sl nd AA-induced ones ( totl proton conductnce). The proton conductnce detected in the sence of AA represents sl proton conductnce. The difference etween these totl nd sl proton conductnce vlues represents the AAinduced proton conductnce. Western immunolot nlysis. Mitochondril memrnes nd srcolemm from gstrocnemius muscle were resuspended in SDS loding uffer, followed y heting for 1 min t 8 C. Mitochondril or skeletl muscle plsm memrnes contining 6 g of protein from single rt were loded in ech lne nd electrophoresed on n 8% SDS-PAGE gel. A monoclonl ntiody (mouse monoclonl 1744; ACm, Cmridge, UK) nd nti-rit ntiody were used s primry nd secondry ntiodies, respectively, in chemiluminescence protein detection method (NEN Life Science Products, Boston, MA). Equl loding ws verified y Ponceu S stining or, where stted, y detecting -ctin levels. To evlute the purities of the mitochondril nd srcolemml frctions, N/K ATPse, uncoupling protein 3 (UCP3), nd steroyl- CoA desturse 1 (SCD1) were detected y immunolotting with the use of the following ntiodies: nti- 1 sodium potssium ATPse ntiody (mouse monoclonl A 7671 from ACm), nti-humn UCP3 ntiody (rit polyclonl A346 from Chemicon Interntionl), nd nti-scd1 (got polyclonl sc-1472 from Snt Cruz Biotechnology). RNA isoltion. Totl gstrocnemius muscle or liver RNA ws isolted using the Trizol stndrd protocol (Invitrogen Life Technologies, Miln, Itly). Tissue/Trizol mixtures were homogenized using polytron, keeping the viscosity of the solution to minimum to ensure effective inctivtion of endogenous RNAse ctivity. Quntittive rel-time PCR. PCR primers were designed using Primer Express version 2. (Invitrogen). The expression levels of cyclophilin F were used for normliztion. The primers used were cyclophilin F sense 5=-AGGCAGATGTCGTGCCAAAGAC-3=, cyclophilin F ntisense 5=- ACATGAAGGCTGGGATGACC-3=, FAT/CD36 sense 5=-GGAACCCA- GACAACCACT-3=, nd FAT/CD36 ntisense 5=-ATGTCCAGCA- CACCATACGA-3=. A totl of 1 g of totl RNA ws used to generte cdna strnds in 2- l rection volume using Cloned AMV First Strnd Synthesis Kit (Invitrogen). The equivlent of 25 ng of totl RNA ws used susequently in the mplifiction step, with 5 nm gene-specific primers nd 4 ml of SYBR green mix (Applied Biosystems) in totl volume of 8 l, using stndrd cycle prmeters in n Applied Biosystems Model 75.

4 E1225 Detection of serum levels of FFA nd glycerol. FFA nd glycerol levels were determined in serum smples y the use of kits from Wko Chemicls (Richmond, VA) nd Sigm, respectively. Determintion of skeletl muscle FFA nd triglyceride levels. Lipid ws extrcted from frozen tissues in chloroform-methnol using the method of Folch et l. (11). The levels of FFA nd triglycerides in the skeletl muscle lipid extrct were determined using kits from Wko nd Sigm, respectively. Determintion of crnitine plmitoyl trnsferse system ctivity. Mitochondril crnitine plmitoyl trnsferse (CPT) system (CPT1 plus CPT2) ctivity ws mesured spectrophotometriclly y following (t 412 m) the kinetics of crnitine-dependent CoASH production in the presence of 5,5=-dithio-is(2-nitroenzoic cid) using plmitoyl-coa s sustrte, s descried y Alexson nd Nedergrd (1). An 412 vlue of 13.6 mm/cm ws pplied for the clcultion of totl CPT ctivity. Immunohistochemistry. Prffin-emedded gstrocnemius muscle sections (5 m) were incuted with nti-fat/cd36 polyclonl ntiody ( 7854 diluted 1:2; Acm). The immunorection ws detected y the vidin-iotin-peroxidse complex (ABC) method using Vectstin ABC Elite Kit (Vector Lortories, Burlingme, CA), s descried previously (32). The muscle sections were counterstined with hemtoxylin to revel nuclei nd then mounted in Eukitt (Kindler, Freiurg, Germny). Omission of the primry ntiody served s the negtive control. Cell culture. C 2C 12 mouse myolsts were routinely cultured in growth medium consisting of DMEM supplemented with het-inctivted 1% vol/vol fetl ovine serum, 2 mm glutmine, nd ntiiotics (5 U/ml penicillin, 5 g/ml streptomycin). Medium of the sme composition ws used to promote C 2C 12 cell differentition, when 7 8% confluence ws otined, with the only exception tht het-inctived fetl ovine serum ws reduced to 1% vol/vol. The cells were mintined in humidified 5% CO 2 tmosphere t 37 C. For the immunofluorescence stining study, ll of the cells were grown on 18-mm glss coverslips in six-well pltes. The differentition medium ws chnged every 48 h. Polynucleted myotues t the lte-differentition stge (5 6 dys fter the switch to differentition medium) were used for experimentl tretments. For these tretments, the medium ws replced with fresh DMEM contining either T 2 or T 3 (ech t concentrtion of 1 6 M), nd cells were incuted for 1 h t 37 C. As control, myotues were cultured with the vehicle lone eing dded. At the end of the tretment, the cells were processed for immunolocliztion procedures. Confocl fluorescence immunocytochemistry. After the ove experimentl tretments, cells were fixed in 2% formldehyde in PBS for 15 min, wshed twice in PBS, nd then permeilized for 1 min in PBS contining.25% Triton X-1 (or wshed in PBS when permeiliztion ws not required). For mitochondril colocliztion experiments, live cells were preincuted with Mitotrcker Red CXMRos (Invitrogen) t 37 C efore fixtion. After tht, the cells were immeditely wshed in DMEM nd then in PBS. After fixtion nd permeiliztion, cells were locked with 2% norml got serum t room temperture for 2 min, nd immunostining ws crried out using rit nti-fat/cd36 polyclonl ntiody ( 7854 from Acm, diluted 1:4), with Alex fluor 488 got nti-rit IgG (H L) s secondry ntiody. Nuclei were counterstined with TO-PRO-3 Iodide (Invitrogen). Cells were mounted in Vectshield mounting medium nd nlyzed y confocl microscopy using Leic TCS SP5 II confocl microscope equipped with 63 ojective. Sttisticl nlysis. Dt were sujected to sttisticl nlysis (1-wy ANOVA followed y Student-Newmn-Keuls test) for differences mong the experimentl groups. Differences were considered sttisticlly significnt when P.5. RESULTS Serum levels of thyroid hormones nd metolic prmeters. To test the effectiveness of the tretments, the serum levels of thyroxine (T 4 ) nd T 3 nd vrious metolic prmeters were mesured in N, H, H T 3 1 wk, nd H T 2 1 wk rts. As shown in Tle 1, serum levels of totl T 4 (TT 4 ) nd totl T 3 (TT 3 ) were reduced significntly y hypothyroidism. As expected, significnt increse in TT 3 levels ws oserved following long-term dministrtion of T 3 to H rts. On the other hnd, dministrtion of T 2 to H rts did not ffect either the TT 4 or TT 3 vlues. Resting metolic rte ws reduced significntly ( 25%) y hypothyroidism. Long-term dministrtion of either T 3 or T 2 to H rts incresed resting metolic rte significntly to vlues higher thn those oserved in N rts. Concerning the respirtory quotient, no significnt chnge ws induced y hypothyroidism; on the other hnd, long-term dministrtion of either T 3 or T 2 to H rts significntly reduced it, indicting n increse in the use of lipids s sustrte for oxidtion in H T 3 1 wk rts nd lso in H T 2 1 wk rts. The rts ody weight gin with time ws reduced significntly y hypothyroidism (see Tle 1). When we looked t the whole tretment period (4 wk of tretment with propylthiourcil nd iopnoic cid with or without either T 3 or T 2 dministrtion during the lst week of tretment), the dministrtion of those iodothyronines did not ffect ody weight gin signifi- Tle 1. Effects of thyroid stte nd of long-term dministrtion of iodothyronines on serum levels of T 3 nd T 4, metolic prmeters (RMR nd RQ), ody weight gin, food intke, diposity, nd serum levels of glycerol nd free ftty cids N H H T3 1wk H T2 1wk TT 4 serum level, nm TT 3 serum level, nm c.18.7 RMR, ml O 2/min kg c RQ Serum glycerol level, mg/dl c.18.4 Serum free ftty cid level, mg/dl c Body weight gin, g Body weight gin during lst week of tretment, g c Food intke, g Food intke during lst week of tretment, g Viscerl dipose tissue/ody weight , 3..2 c 3.4.2,c Vlues represent mens SE from 6 1 different experiments. T 3, 3,5,3=-triiodothyronine; T 4, thyroxine; RMR, resting metolism; RQ respirtory quotient; N, group N (consisting of euthyroid rts tht received vehicle); H, group H (consisting of hypothyroid rts); TT 3 nd TT 4, totl T 3 nd T 4, respectively. Vlues leled with different letters re significntly different (P.5) from ech other.

5 E1226 cntly. However, differences were oserved during the lst week when either T 3 or T 2 ws dministered. Administrtion of T 3 to H rts tended to reduce ody weight of the nimls, wheres dministrtion of T 2 incresed it significntly. Food intke ws reduced significntly y hypothyroidism, nd slight (not significnt) tendency to increse ws oserved following dministrtion of either T 3 or T 2 to H rts. To test whether the increse in metolic rte nd the enhncement in lipid utiliztion induced y dministrtion of T 3 or T 2 to H rts might ffect the rts diposity, we mesured the totl viscerl dipose tissue s percentge of ody weight. As shown in Tle 1, no significnt difference in tht prmeter ws oserved etween H nd N rts. However, dministrtion of T 3 to H rts reduced it significntly, wheres T 2 tended to reduce it only nonsignificntly. Glycerol nd FFA serum level. To evlute whether hypothyroidism, nd lso T 3 or T 2 dministrtion, might ffect ftty cid vilility to the tissues, we next determined the serum glycerol nd FFA levels in N, H, H T 3 1 wk, nd H T 2 1 wk rts. Since lood ws collected 6 h fter the end of the nocturnl period, the glycerol nd FFA detected in the serum represent the lnce mong their uptke from intestine (negligile), their uptke nd utiliztion y peripherl orgns, nd their delivery from depots. As shown in Tle 1, hypothyroidism strongly reduced ( 5% vs. N rts) the serum glycerol level. Although dministrtion of T 3 to H rts mrkedly incresed it ( 3% vs. H nd 17% vs. N), T 2 did not induce ny chnge. The euthyroid nd hypothyroid sttes did not differ in FFA serum levels (Tle 1), nd wheres T 3 incresed the FFA level ( 154% vs. oth N nd H rts), T 2 decresed it ( 6% vs. oth H nd N rts). Mitochondril cpcity to oxidize FFA. We next investigted the effect of the rts thyroid stte on mitochondril lipid uptke nd oxidtion. Since the import of ftty cids into mitochondri is key event determining the ftty cid oxidtion rte, we first evluted the ctivity of the CPT system (see Tle 2). CPT ctivity ws reduced y hypothyroidism ( 22% vs. N rts), wheres dministrtion of T 3 to H nimls incresed it significntly to vlues higher thn those oserved in N nimls ( 71% vs. H nd 32% vs. N). Notly, the effect of T 2 on CPT system ctivity ws very rpid; indeed, it ws lredy evident t 1 h fter its injection. The plmitte oxidtion rte ws significntly lower in H thn in N ( 41%) (Tle 2), nd T 3 dministrtion incresed it ( 4% vs. H rts), s did T 2 tretment ( 34% vs. H). T 3 nd T 2 ech hd very rpid effects on the plmitte oxidtion rte, with the increses eing evident t 1 h fter their dministrtion (Tle 2). To exmine whether ltertions in mitochondril respirtory pthwys might underlie some prt of the effects of iodothyronines on ftty cid oxidtion rte, we ssessed the ility of mitochondri to oxidize succinte s sustrte in experimentl conditions in which the synthesis nd export of ATP re t mximl rtes (Tle 2). The succinte oxidtion rte ws reduced significntly y hypothyroidism ( 25% vs. N). Long-term dministrtion of T 3 to H rts incresed it significntly, restoring it to the levels oserved in N nimls. However, T 3 ws ineffective t 1 h fter its cute dministrtion. Long-term dministrtion of T 2 to H rts incresed the succinte oxidtion rte significntly ( 3%), nd its effect ws very rpid since it ws lredy evident t 1 h fter single T 2 dministrtion. Lipolysis in gstrocnemius skeletl muscle nd triglyceride hydrolse ctivities. To ssess the ility of one week s iodothyronine dministrtion to H rts to ffect skeletl muscle lipolysis nd lipse ctivities, we evluted glycerol relese y muscle strips nd triglyceride hydrolse ctivity in whole homogente. As shown in Fig. 1, hypothyroidism did not ffect GSkM s ility to relese glycerol. One week s dministrtion of T 3 to H nimls incresed glycerol relese from GSkM significntly ( 7% vs. H nd N), wheres dministrtion of T 2 tended to increse glycerol relese y only 25% (not significnt). Triglyceride hydrolse ctivities displyed the sme trend s tht oserved for glycerol relese; viz. hypothyroidism cused no significnt chnge, nd dministrtion of T 3 to H rts induced significnt increse ( 7% vs. H), wheres dministrtion of T 2 led to only nonsignificnt tendency to increse ( 12%). Effect of hypothyroidism nd iodothyronine dministrtion on mitochondril oxidtive phosphoryltion efficiency. To exmine whether n ltertion in the efficiency of mitochondril oxidtive phosphoryltion might contriute to the effects of iodothyronines on the ftty cid oxidtion rte, we mesured mitochondril proton conductnce in mitochondri from N, H, H T 3 1 wk, nd H T 2 1 wk rts, nd we discriminted etween the contriutions of sl nd ftty cid-induced proton conductnce (Fig. 2). An inhiition of proton conductnce ( 4%) ws oserved in mitochondri from H rts compred with N ones, with sl proton conductnce eing reduced y 2% nd ftty cidinduced proton conductnce eing reduced y 6%. One week s dministrtion of T 3 to H rts led to n increse in proton conductnce ( 33%), which did not rech the vlues oserved in N rts. Actully, T 3 incresed oth sl nd ftty cid-induced proton conductnce ( 33% in ech cse), nd lthough sl proton conductnce reched levels not significntly different from the N ones, ftty cid-induced proton conductnce remined significntly lower ( 45% vs. N). Administrtion of T 2 to H rts incresed mitochondril proton conductnce y 27%, ut it did not rech the vlues oserved in N rts. T 2 ffected only ftty cid-induced proton Tle 2. Effects of thyroid stte nd of dministrtion of iodothyronines on mitochondril ility to import nd oxidize ftty cids N H H T3 1h H T3 1wk H T2 1h H T2 1wk Plmitte oxidtion, nmol O min 1 mg protein c d c,d c CPT ctivity, nmol CoA min 1 mg protein c Succinte oxidtion, nmol O min 1 mg protein Vlues represent mens SE for 4 6 different mitochondril preprtions for ech group. T 2, 3,5-diiodothyronine; CPT crnintine plmitoyl trnsferse. Vlues leled with different letters re significntly different (P.5) from ech other.

6 E1227 A B nmoles glycerol relesed /gr tissue nmoles olete/min mg prot N H H+T3 1W H+T2 1W N H H+T3 1w H+T2 1w Fig. 1. Effects of hypothyroidism nd dministrtion of iodronines to hypothyroid rts on gstrocnemius muscle lipolysis (A) nd tricylglycerol hydrolse ctivity (B). Mens SE from 6 8 independent experiments, ech one performed in duplicte. Within given pnel, rs leled with different letters re significntly different (P.5). N, group N (consisting of euthyroid rts); H, group H (consisting of hypothyroid rts); T 3, 3,5,3=-triiodothyronine; T 2, 3,5-diiodothyronine; H T 3 1W, group H tht during the lst week of tretment received dily injection of T 3;H T 2, group H tht during the lst week of tretment received dily injection of T 2. conductnce ( 67% vs. H), with sl proton conductnce eing unffected. FFA nd triglyceride levels in gstrocnemius muscle. We next investigted whether the ltertions in GSkM oxidtive cpcity nd ility to hydrolize triglycerides induced y 1) the hypothyroid stte nd 2) long-term dministrtion of either T 3 or T 2 to hypothyroid rts might lter FFA nd triglyceride levels in GSkM. GSkM FFA levels were not ffected y either hypothyroidism or long-term dministrtion of iodothyronines to H rts (Fig. 3B). In contrst, the triglyceride content of GSkM ws significntly higher (y 26%) in H rts thn in N ones. Long-term dministrtion of T 3 to H nimls decresed the triglyceride levels in GSkM ( 2% vs H), restoring them to the vlue seen in N nimls. Long-term T 2 tretment reduced the triglyceride level in GSkM y 4% (vs. H) so tht it reched vlue lower thn tht seen in N (Fig. 3A). Tissue nd sucellulr FAT/CD36 levels in GSkM. To evlute whether FAT/CD36 might e ffected y the thyroid stte of the niml, the GSkM content ws ssessed t oth tissue nmoles H+/min mg protein mv ftty cid induced sl N H H+T3 1w H+T2 1w Fig. 2. Effects of hypothyroidism nd dministrtion of iodothyronines to hypothyroid rts on mitochondril gstrocnemius proton conductnce. Totl height of ech r represents proton conductnce detected in the presence of ftty cid [i.e, the sum of sl conductnce (gry) nd ftty cid-induced proton conductnce (white)]. Mens SE from 4 independent experiments, ech one performed in duplicte. Within given pnel, rs leled with different letters re significntly different (P.5). nd sucellulr levels. Hypothyroidism gretly incresed the GSkM FAT/CD36 mrna level ( 2%; Fig. 4A), nd this enhncement ws ccompnied y increses in the totl lyste (Fig. 4B) nd srcolemml nd mitochondril (Fig. 5) FAT/ A B GSkM TG levels (mg/g tissue) GSkM FFA levels (mg/g tissue) N H H+T3 1w H+T2 1w N H H+T3 1w H+T2 1w Fig. 3. Effects of hypothyroidism nd dministrtion of iodothyronines to hypothyroid rts on gstrocnemius muscle content of triglycerides (TG; A) nd free ftty cids (FFA; B). Mens SE from 6 independent experiments, ech one performed in duplicte. Within given pnel, rs leled with different letters re significntly different (P.5). GSkM, gstrocnemius skeletl muscle. c c c

7 E1228 A B FAT/CD36 mrna level (ritrry units) GSkM lyste FAT/CD36 Bet ctin FAT/CD36 protein level (ritrry units) N H+T2 1w CD36 protein contents, with the increses detected eing 5, 6, nd 213%, respectively, vs. N (Fig. 5). Long-term dministrtion of T 3 to H rts did not lter the GSkM FAT/ CD36 mrna level, ut T 2 dministrtion reduced it slightly ( 3%; Fig. 4A). Long-term dministrtion of either T 3 or T 2 reduced the FAT/CD36 GSkM protein lyste level, with the effect of T 2 eing the greter of the two ( 6 nd 25% for T 2 nd T 3, respectively; Fig. 4B). Long-term dministrtion of T 3 to H rts incresed oth the srcolemml nd mitochondril levels of FAT/CD36 ( 7 nd 184%, respectively, vs. H; Fig. 6A), with the effects eing very rpid since they were lredy evident t 1 h fter T 3 dministrtion (Fig. 6A). Long-term dministrtion of T 2 to H rts hd effects similr to those elicited y T 3, with the increses in the FAT/CD36 srcolemml nd mitochondril contents eing 9 nd 165%, respectively (Fig. 6B). Agin, the effects were rpid, eing evident t 1 h fter T 2 dministrtion. N H H+T3 1w H+T2 1w H H+T31w N H H+T3 1w H+T2 1w Fig. 4. Effects of hypothyroidism nd of long-term dministrtion of iodothyronines to hypothyroid rts on expressions of ftty cid trnslocse (FAT)/ CD36 mrna (A) nd protein (B) in gstrocnemius lystes. A: quntittive RT-PCR nlysis of FAT/CD36 mrna levels. B: representtive Western lotting nlysis of FAT/CD36 protein levels. Ech lne contined 3 g of protein of totl tissue lyste from single rt. -Actin ws used to control for loding differences. Dividing lines indicte omissions/rerrngements of lnes from the sme gel. The rs, which show quntifiction of the signls (expressed s percentge of the vlue otined from control euthyroid rts), represent mens SE for 5 smples. Within given pnel, rs leled with different letters re significntly different (P.5). c c We could exclude the possiility of the FAT/CD36 detected in the srcolemml nd mitochondril frctions hving origins different from the frction under considertion, since they did not cross-contminte ech other. To estlish this, we evluted the presence of N-K-ATPse ( protein present in the srcolemm ut not in mitochondri), UCP3 ( protein present in the inner mitochondril memrne), nd SCD1 ( protein present t the endoplsmic reticulum level) in ech of the ove frctions. The dt shown in Fig. 7 indicte tht neither N/K ATPse nor SCD1 ws present in mitochondril memrne-rich frctions, wheres neither UCP3 nor SCD1 ws present in the srcolemm-rich frction. Immunohistochemicl nlysis. Immunohistochemicl detection of FAT/CD36 in the GSkM of rts sujected to the experimentl conditions lredy descried (N, H, H T 3 1h, nd H T 2 1 h) reveled tht T 2 nd T 3 ech rpidly induced n incresed srcolemml-ssocited FAT/CD36 immunorectivity nd lso tht there ws intense stining of susrcolemml regions (Fig. 8). The intrcellulr distriution of FAT/CD36 ws exmined y immunofluorescence detection using n in vitro model of skeletl muscle. Such fluorescence immunocytochemistry showed tht within 1 h fter ddition to C 2 C 12 cells in vitro, T 2 nd T 3 enhnced the srcolemml FAT/CD36 stining (Fig. 9), ut such stining ws lso within the cytoplsm, minly in the perinucler region (Fig. 1). To determine whether the cytoplsmic FAT/ CD36 might e loclized to mitochondri, FAT/CD36 ntiody stining nd the mitochondril trcking dye Mitotrcker Red were covisulized y confocl microscopy. In T 2 -treted nd T 3 -treted cells, ut not in control cells, FAT/CD36 ws prtilly coloclized with Mitotrcker Red (Fig. 1). Srcolemml FAT/CD36 Mitochondril FAT/CD36 FAT/CD36 protein level (ritrry units) N N H srcolemm H mitochondri Fig. 5. Effects of hypothyroidism on srcolemml nd mitochondril FAT/ CD36 protein contents. Representtive Western lotting nlysis of FAT/ CD36 protein levels in isolted srcolemm nd mitochondri. Ech lne contined 6 g of protein from single rt. The rs, which show quntifiction of the signls (expressed s percentge of the vlue otined from control euthyroid rts), represent mens SE for 5 smples. Brs leled with different letters re significntly different (P.5).

8 A Srcolemml FAT/CD36 Mitochondril FAT/CD36 B FAT/CD36 protein level ritrry units DISCUSSION Srcolemml FAT/CD36 Mitochondril FAT/CD36 FAT/CD36 protein level ritrry units H H+T 3 1h H+T 3 1w srcolemm H H+T3 1h H+T3 1w mitochondri H H+T 2 1h H+T 2 1w H H+T2 1h H+T2 1w srcolemm Despite the metolic effects of T 3 hving first een demonstrted more thn century go (29), the mechnisms y which it cuses metolic chnges re still not completely understood. The present study sought to provide detiled insight into the effects of oth hypothyroidism nd tretment with T 3 or T 2 on GSkM ftty cid uptke nd utiliztion, with prticulr focus on the role plyed y FAT/CD36. Growing evidence supports the ide tht the metolic dpttions tht occur in the hypothyroid stte re not mirror imge of those tht occur in the trnsition from the euthyroid to the hyperthyroid stte (37). The effects of hypothyroidism nd T 3 dministrtion on the FAT/CD36 mrna level nd protein sucellulr redistriution within the GSkM cells, descried for the first time in the present study, provide good exmple of this. Indeed, lthough hypothyroidism incresed mitochondri Fig. 6. Effects of dministrtion of either T 3 (A) ort 2 (B) to hypothyroid rts on srcolemml nd mitochondril FAT/CD36 protein contents; comprison etween long-term (1 wk) nd rpid (1 h) effects. Representtive Western lotting nlysis of FAT/CD36 protein levels in isolted srcolemm nd mitochondri. Ech lne contined 6 g of protein from single rt. Dividing lines indicte omissions/rerrngements of lnes from the sme gel. Brs, which show quntifiction of the signls (expressed s percentge of the vlue otined from hypothyroid rts), represent mens SE for 5 smples. Within ech pnel, rs leled with different letters re significntly different (P.5). the FAT/CD36 mrna nd protein levels (the ltter t tissue, srcolemml, nd mitochondril levels), these effects were not reversed y long-term dministrtion of T 3. The rpidity of the effect induced y T 3, eing evident t 1 h fter its dministrtion to hypothyroid rts (Fig. 4A), led us to speculte tht it is most likely non-nucler-medited effect. However, further detiled studies re needed to exmine this possiility. The utiliztion of FFA y GSkM involves mny mechnisms, including 1) lipolysis nd relese/moiliztion of FFA from dipose tissue, 2) delivery of FFA to GSkM nd trnsport cross the srcolemm, 3) lipoprotein lipse ctivity, 4) lipolysis of intrmusculr tricylglycerol, nd 5) FFA trnsport into mitochondri nd their susequent oxidtion. (18). Another importnt fctor involved in the ility of mitochondri to oxidize ftty cid is the mitochondril proton conductnce, which is known to modulte the efficiency of oxidtive phosphoryltion. Indeed, rise in the NADH/NAD rtio or cetyl-coa/coa rtio results in inhiition of ftty cid -oxidtion, wheres n increse in mitochondril proton conductnce, y promoting the oxidtion of sustrtes, mintins low NADH/NAD or cetyl-coa/coa rtio, thus enhncing ftty cid oxidtion. Aprt from lipoprotein lipse ctivity tht in strited muscle hs een reported to not e ffected y the niml s thyroid stte (23), the dt presented here indicte tht most of ove mechnisms re ffected y oth hypothyroidism nd T 3 tretment. Hypothyroidism did not chnge FFA vilility to the tissues (s indicted y the serum levels of FFAs) ut did result in n incresed srcolemml FAT/CD36 level, giving rise to n enhnced ility of GSkM to import FFA for utiliztion. Despite the increse in the uptke of FFA y GSkM, hypothyroidism decresed the tissue s ftty cid oxidtion, leding to n imlnce etween FFA supply nd oxidtion (with the ltter lso eing more efficient) nd hence, n ccumultion of intrmyocellulr triglyceride. In ddition, lthough the presence of FAT/CD36 t the mitochondril level hs een sid to e relted to the ility of mitochondri to use ftty cid s fuel sustrte (17, 39), our dt clerly indicte tht in hypothyroidism the reduced ility of mitochondri to oxidize ftty cids (Tle 1) is ssocited with n upregultion of the mitochondril FAT/CD36 content (Fig. 3). This discrepncy seems to e more pprent thn rel since, in the hypothyroid stte, structurl shift occurs in GSkM towrd slow-twitch phenotype (19, 38), which mkes predominnt use of lipid s n energy source. Thus, the N+/K+ pump UCP3 Mito Mito GSkM lyste SCD-1 PM PM Mito PM E1229 Fig. 7. Representtive Western immunolotting of -suunits of sodium/ potssium (N /K ) pump, uncoupling protein 3 (UCP3), nd steroyl-coa desturse 1 (SCD1), used s specific sucellulr protein mrkers, s detected in mitochondri (Mito) nd plsm memrne (PM). Ech lne contined 3 g of protein.

9 E123 C H H+T2 1h H+T3 1h Fig. 8. Immunolocliztion of FAT/CD36 protein in skeletl muscle. Gstrocnemius muscle tken from euthyroid control rts (C), hypothyroid rts (H), nd hypothyroid rts t 1 h fter dministrtion of iodothyronines to hypothyroid rts (H T 2 1 h nd H T 3 1 h, respectively). After T 2 nd T 3 dministrtion, fine punctte stining ws seen round the memrne (rrows) of some skeletl fiers ut lso in susrcolemml regions (rrowheds). FAT/CD36 is stined rown y the vidin-iotin-peroxidse complex method. Nuclei were counterstined with hemtoxylin (lue). Br, 2 m. upregultion of FAT/CD36, which would increse ftty cid supply to the mitochondri (39), could e interpreted s form of compenstion for the impired cpcity of mitochondri to import ftty cids nd to oxidize sustrtes. One week s dministrtion of T 3 to hypothyroid rts led to n increse in the vilility of FFAs to the tissues. Within 1 h of its dministrtion to hypothyroid rts, T 3 induced oth n increse in the import of FFA into GSkM nd n increse in their oxidtion therein. Although the moleculr mechnism underlying the modultion y T 3 of the redistriution of FAT/ CD36 within the gstrocnemius muscle cells remins to e unrveled, we speculte tht there is involvement of kinses such s AMPK nd Akt, ech of which hs een shown to e involved in FAT/CD36 redistriution (12). In support of this notion, we showed recently tht single dministrtion of T 3 to hypothyroid rts rpidly ctivted oth AMPK nd Akt (8). One week s T 3 dministrtion to hypothyroid rts did not ffect the mrna level of FAT/CD36 in skeletl muscle, finding in pprent contrst to the oservtion mde y Flores-Morles et l. (1) in mouse liver. They noted significnt decrese in FAT/CD36 following 1-wk T 3 dministrtion to hypothyroid mice. This my suggest tissue- or species-specific regultion of FAT/CD36 trnscription y T 3. Following the present long-term T 3 tretment, the induced increses in ftty cid uptke nd oxidtion were ssocited with n unchnged level of GSkM FFA nd reduced level of GSkM triglyceride. These dt my indicte tht following such tretment, the increse in FFA oxidtion, which lso ecme less efficient, exceeds the increse in the import of FFA into the tissue, with the consequence tht FFAs re not deposited s triglycerides. In ddition, lipolysis of triglycerides tkes plce in GSkM. The rised srcolemml FAT/CD36 levels seen upon longterm dministrtion of T 3 nd the elevted FFA serum level re in ccord with dt pulished y Klieverik et l. (23), who reported tht significnt rise in the uptke of lumin-ound ftty cids y strited muscle ws induced y n excess of thyroid hormone. The different time scles of the effects of T 3 on the mitochondril ftty cid oxidtion rte (viz. recruitment of FAT/CD36 vs. effect on the CPT system) supports FAT/ CD36 eing primrily responsile for the erly increse in lipid hndling (vs. tht in the hypothyroid condition). C T2-1h T3-1h Fig. 9. Locliztion of FAT/CD36 protein in control nd in T 2- nd T 3-treted (1 h) C 2C 12 cells. Confocl representtive imges of immunostining of nonpermeilized cells. Fine stining of the cell surfce ws seen (rrowheds) in oth T 2- nd T 3-treted cells, ut not in control cells, when n nti-fat/cd36 ntiody nd polyclonl secondry ntiody conjugted to Alex 488 (green) were employed. Nuclei were counterstined with TO-PRO-3 iodide (pink). Brs, 1 m.

10 CD36 Mitotrcker Red E1231 Merge C T2 T3 Fig. 1. Sucellulr immunolocliztion of FAT/CD36 protein in control nd in T2- nd T3-treted C2C12 cells. Mitochondril presence of FAT/CD36 ws investigted in differentited C2C12 myotues using nti-fat/cd36 ntiody (green) nd MitoTrcker Red CMXRos (red). Enlrgement of the oxed res in the merged imges reveled mitochondril locliztion of FAT/CD36 protein in the perinucler regions in T2- nd T3-treted cells ut not in control cells. Brs, 8 m. It should lso e mentioned tht, when expressed in Xenopus levis oocytes, FAT/CD36 medites the import of T3 into the cell s well s FFA import (41). Thus, we speculte tht T3, y promoting rpid trnsloction of such protein to the srcolemm, could promote its rpid uptke into muscle cells, thus fcilitting its ction t the cellulr level nd fvoring skeletl muscle metolic dpttion. However, in vivo or ex vivo functionl dt on such FAT/CD36-medited import of T3 re lcking. Unlike tht of T3, dministrtion of T2 to hypothyroid rts induces significnt reduction in the serum FFA level tht could e consequence of the T2-induced increse in lipid utiliztion y GSkM, which is reported here. However, the involvement of tissues other thn GSkM cnnot e excluded. Concerning the effect of T2 on FAT/CD36, T2 hd effects similr to those induced y T3 in promoting the trnsloction of the protein from cellulr depots to the srcolemm nd to mitochondri, ech in very rpid fshion, thus fvoring oth the import of FFAs into the tissue nd their susequent oxidtion t the mitochondril level. Although T2 nd T3 ech incresed the mitochondril ftty cid oxidtion rte, the mechnisms underlying the short-term effects of these two iodothyronines seem to differ, s indicted y the differing onsets of CPT ctivtion nd mitochondril respirtory pthwy ctivtion. Indeed, FAT/CD36 is evidently not the min fctor responsile for the rpid T2-induced ctivtion of ftty cid oxidtion, lthough it ppers to e for T3. Rther, such ctivtion y T2 depends lso on the CPT system nd the sustrte oxidtion rte (thus confirming the dt reported in Ref. 27). Both processes, in fct, were lredy ctivted t 1 h fter T2 dministrtion, wheres T3 ws ineffective t tht time point. FAT/CD36, CPT ctivity, nd the sustrte oxidtion rte ll displyed increses s elements in the long-term effect of T2 on ftty cid oxidtion rte. Different mechnisms lso seem to underlie the long-term effects of T2 nd T3 on the efficiency of oxidtive phosphor-