Tatiana L. Fonseca,1 Joao Pedro Werneck-De-Castro,1,2 Melany Castillo,1 Barbara M.L.C. Bocco,1 Gustavo W. Fernandes,1

Elizabeth A. McAninch,1 Daniele L. Ignacio,1,2 Caio C.S. Moises,1 Alexander R. Ferreira,1 Balázs Gereben,3

and Antonio C. Bianco1

Tissue-Specific Inactivation ofType 2 Deiodinase RevealsMultilevel Control of Fatty AcidOxidation by Thyroid Hormonein the MouseDiabetes 2014;63:1594–1604 | DOI: 10.2337/db13-1768

Type 2 deiodinase (D2) converts the prohormonethyroxine (T4) to the metabolically active molecule3,5,39-triiodothyronine (T3), but its global inactivation unex-pectedly lowers the respiratory exchange rate (respiratoryquotient [RQ]) and decreases food intake. Here we usedFloxD2 mice to generate systemically euthyroid fat-specific (FAT), astrocyte-specific (ASTRO), or skeletal-muscle-specific (SKM) D2 knockout (D2KO) mice thatwere monitored continuously. The ASTRO-D2KO micealso exhibited lower diurnal RQ and greater contribu-tion of fatty acid oxidation to energy expenditure, butno differences in food intake were observed. In con-trast, the FAT-D2KO mouse exhibited sustained (24 h)increase in RQ values, increased food intake, toleranceto glucose, and sensitivity to insulin, all supportinggreater contribution of carbohydrate oxidation to energyexpenditure. Furthermore, FAT-D2KO animals that werekept on a high-fat diet for 8 weeks gained more bodyweight and fat, indicating impaired brown adipose tissue(BAT) thermogenesis and/or inability to oxidize the fatexcess. Acclimatization of FAT-D2KO mice at thermo-neutrality dissipated both features of this phenotype.Muscle D2 does not seem to play a significant meta-bolic role given that SKM-D2KO animals exhibited nophenotype. The present findings are unique in that theywere obtained in systemically euthyroid animals, reveal-ing that brain D2 plays a dominant albeit indirect role infatty acid oxidation via its sympathetic control of BAT

activity. D2-generated T3 in BAT accelerates fatty acidoxidation and protects against diet-induced obesity.

Thyroid hormone signaling is initiated by entry of 3,5,39-triiodothyronine (T3) into target cells and binding withnuclear T3 receptors, modulating the expression of T3-responsive genes (1). Signaling through this pathway isalso affected by local events, with target cells playing a rolethrough controlled expression of the activating or inacti-vating deiodinases (2). These enzymes are thyroredoxinfold-containing selenoproteins that can activate the pro-hormone thyroxine (T4) to its active form T3 (type 2deiodinase [D2]) or inactivate both T4 and T3 and T3 toT2 (type 3 deiodinase [D3]) (3–5). As a result, cells thatexpress D2 have higher T3 levels and thus exhibit anenhanced T3-dependent mRNA footprint, and the oppo-site is observed in type 3 deiodinase–expressing cells. Thuscustomizing thyroid hormone signaling via deiodinases is animportant mechanism in energy homeostasis (2), brain(6), brown adipose tissue (BAT) (7), pancreatic b-cell (8),heart (9), and skeletal muscle (10,11).

Inactivation of the D2 gene (Dio2) affects the expres-sion of T3-dependent genes in D2-expressing tissues suchas BAT and brain, essentially dampening thyroid hormonesignaling in a tissue-specific fashion (12). Accordingly, a mousewith global inactivation of the Dio2 gene (GLOB-D2KO)

1Division of Endocrinology, Diabetes, and Metabolism, Miller School of Medicine,University of Miami, Miami, FL2Biophysics Institute and School of Physical Education and Sports, Federal Uni-versity of Rio de Janeiro, Rio de Janeiro, Brazil3Department of Endocrine Neurobiology, Institute of Experimental Medicine, Hun-garian Academy of Sciences, Budapest, Hungary

Corresponding author: Antonio C. Bianco, abianco@deiodinase.org.

Received 19 November 2013 and accepted 26 January 2014.

This article contains Supplementary Data online at http://diabetes.diabetesjournals.org/lookup/suppl/doi:10.2337/db13-1768/-/DC1.

T.L.F. and J.P.W.-D.-C. contributed equally to the manuscript.

© 2014 by the American Diabetes Association. See http://creativecommons.org/licenses/by-nc-nd/3.0/ for details.

1594 Diabetes Volume 63, May 2014

METABOLISM

exhibits a significant metabolic phenotype characterizedby decreased respiratory quotient (RQ), resistance to diet-induced obesity, and super tolerance to glucose, some ofwhich is reversed upon acclimatization at thermoneutral-ity (13). A similar phenomenon was observed in theuncoupling protein 1 knockout mouse (14) and in system-ically hypothyroid mice (15). These findings indicate thatan increase in sympathetic activity at room temperatureplays a role in defining the metabolic phenotype of animalmodels exhibiting a disruption in thyroid hormone signal-ing, either localized (GLOB-D2KO) or systemic (13,16).

D2-generated T3 has been shown to play a critical rolein BAT uncoupling protein 1 expression (17,18) and BATthermogenesis (19). Thus the metabolic phenotype dis-played by the GLOB-D2KO mouse has been largely attrib-uted to the lack of D2-generated T3 in BAT (13,20,21). Infact, BAT D2 is induced by sympathetic activity and rap-idly increases thyroid hormone signaling during cold ex-posure (22,23). However, a number of other metabolicallyrelevant tissues in the rodent express D2, e.g., brain (24),skeletal muscle (25), and bone (26), and could also playa role in defining the metabolic phenotype of the GLOB-D2KO mouse. For example, D2 is expressed in the medio-basal hypothalamus (27,28) where it has been implicated inthe orexigenic response during refeeding (29) and in low-ering thyrotropin-releasing hormone/thyroid-stimulatinghormone (TSH) secretion in disease states (30). At thistime, it is still not clear what, if any, metabolic roles D2plays in skeletal muscle and bone.

The lower RQ observed in the GLOB-D2KO animals isunexpected because thyroid hormone administration isknown for accelerating fatty acid oxidation (31) and loweringRQ values (32). However, given that the lower RQ values aredissipated once the GLOB-D2KO animals are acclimatized tothermoneutrality (13), it is likely that a compensatory in-crease in BAT sympathetic activity plays a role (13,16). Ofnote, a subsequent study did not find a lower RQ in theGLOB-D2KO mouse studied briefly by indirect calorimetry(33), despite reduced liver glycogen and elevated serumbOH-butyrate levels, which is commonly associated with in-creased sympathetic activity and fatty acid oxidation (34).

The present studies were performed to define themechanism(s) and anatomical site(s) whereby D2-mediateT3 production affects fatty acid oxidation as reflected inthe RQ. To this aim, we compared the metabolic pheno-type of the GLOB-D2KO mouse with that of threeother systemically euthyroid mouse strains that exhibit1) astrocyte-specific (ASTRO-D2KO), 2) fat-specific (FAT-D2KO), or 3) skeletal-muscle-specific (SKM-D2KO) inacti-vation of Dio2 after they were admitted to a comprehensivelaboratory animal monitoring system (CLAMS) and moni-tored continuously through indirect calorimetry. Our find-ings indicate that brain D2 indirectly inhibits BAT fattyacid oxidation via dampening of sympathetic activity inBAT. In contrast, D2-generated T3 in BAT accelerates fattyacid oxidation given that selective disruption of this path-way in BAT increases RQ and predisposes to diet-induced

obesity. No significant metabolic phenotype resulting fromD2 inactivation in skeletal muscle was identified. The evi-dence obtained through the use of these animal models isunique in its physiological relevance because it reflects theresult of tissue-specific D2 inactivation in an otherwisesystemically euthyroid animal.

RESEARCH DESIGN AND METHODS

AnimalsAll experimental procedures were planned following theAmerican Thyroid Association guide to investigating thyroidhormone economy and action in rodent and cell models (35)and approved by the local Institutional Animal Care and UseCommittee. For the studies including the GLOB-D2KOmouse, ;10-week-old C57BL/6J and GLOB-D2KO micethat had been backcrossed in the same background (21)were used from our established colonies, kept at roomtemperature (22°C) with a 12-h dark/light cycle startingat 0600 h, and housed in standard plastic cages with fourmale mice per cage.

For the tissue-specific deletion of dio2 in adipose tissue,astrocytes, and skeletal muscle cells, we establish an in vivomouse model for cell-type–specific deletion of D2-generatingfloxed D2 (dio2flx) mice as described previously (36). Thedio2flx mice were crossed with transgenic mice express-ing Cre recombinase under the fatty acid binding pro-tein 4 promoter (FABP4; B6.Cg-Tg[FABP4-Cre]1Rev/J;The Jackson Laboratory, Bar Harbor, ME) (37) or glialfibrillary acidic protein promoter (GFAP; FVB-Tg[GFAP-Cre]25Mes/J; The Jackson Laboratory) (38) or myosin lightchain 1f (MLC) (39). This strategy was used in order toeliminate D2 activity in the FABP4-expressing adipocytes(FAT-D2KO mice), GFAP-expressing astrocytes (ASTRO-D2KO mice), and MLC-expressing myocytes (SKM-D2KO).In all experiments, only Cre littermates were used ascontrols. The generation of the ASTRO-D2KO mice wasrecently described (36). At the time of the studies, allmice were between 9 and 14 weeks of age.

Only male animals were used. Animals were kept onstandard chow diet (3.1 kcal/g; 2918 Teklad Global ProteinRodent Diet; Harlan Laboratories, Madison, WI) or a high-fatdiet (HFD; 4.5 kcal/g; TD 95121; Harlan Teklad, India-napolis, IN) as indicated. In the experiments involving high-fat feeding, 3-month-old littermate controls and FAT-D2KOor SKM-D2KO mice (six per group) that had been fed chowdiet were switched to a HFD for 8 weeks and kept at roomtemperature (22°C) or thermoneutrality (30°C) as indicated.

At the end of experimental period, the animals werekilled by asphyxiation in a CO2 chamber. Blood wascollected and serum levels of TSH, T4, and T3 measuredusing a MILLIPLEX rat thyroid hormone panel kit followingthe instructions of the manufacturer (Millipore Corp., Bill-erica, MA) and read on a BioPlex (Bio-Rad, Hercules, CA).

Body CompositionLean body mass and fat mass were determined by dual-energy X-ray absorptiometry (DEXA; Lunar Pixi, Janesville,WI) as described previously (13).

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Indirect CalorimetryAnimals were admitted to a CLAMS (OXYMAX System4.93; Columbus Instruments, Columbus, OH) (13) with freeaccess to food and water. Studies were performed at 22, 18,15, 11, and 5°C for the indicated times. Animals wereallowed to acclimatize in individual metabolic cages for48 h before any measurements. Subsequently, metabolicprofiles were generated as indicated and the data collectedin the next 36 h. This system allows for continuous mea-surement of oxygen consumption (VO2; mL/kg BW/h) andcarbon dioxide production (VCO2) in successive 14- or 26-min cycles, using a sensor calibrated against a standardgas mix containing defined quantities of O2 and CO2. Thesedata were used to calculate the respiratory exchange ratio(RQ; VCO2/VO2) and the energy expenditure (EE) ([3.815 +1.232 $ RQ] $ VO2). Contribution of fat acid oxidation to EEwas calculated as described using the following equation:percentage of fat contribution = [468.6 $ (1 2 RQ)] /[5.047 $ (RQ 2 0.707) + 4.686 $ (1 2 RQ)] (40). The 24 hcaloric intake was measured at the indicated times whilethe animals were admitted to the CLAMS.

Glucose and Insulin Tolerance TestsFor glucose tolerance tests, mice were fasted overnightand the glucose measurement was made as describedpreviously (8). For insulin tolerance tests, mice withfood ad libitum were injected with insulin, and the glucosemeasurement was made as described previously (8).

Deiodinase AssaysD2 activity assay was performed in BAT sonicates as describedpreviously (41,42).

Statistical AnalysisAll data are expressed as mean 6 SEM and were analyzedusing PRISM software (GraphPad Software, San Diego,CA). One-way ANOVA was used to compare more thantwo groups, followed by the Student–Newman–Keuls testto detect differences between groups. The Student t testwas used only when two groups were part of the experi-ment. P , 0.05 was used to reject the null hypothesis.

RESULTS

GLOB-D2KO Mice Exhibit Increased Diurnal Fatty AcidOxidationUnder the mild thermal stress conditions of room tem-perature (22°C), all animals exhibited the expected circa-dian rhythmicity in VO2 and EE, with higher valuesobserved nocturnally when animals are awake and eatingas opposed to the day hours when the animals are restingand eating much less (Fig. 1A and Supplementary Fig. 1A).In this setting, no differences were observed betweenGLOB-D2KO and wild-type (WT) animals (Fig. 1A). TheRQ profile also exhibited circadian rhythmicity with lowervalues observed diurnally (Fig. 1B). Notably, the diurnal de-crease in RQ values was more pronounced in the GLOB-D2KO animals (Fig. 1B), resulting in greater contribution offatty acid oxidation to the diurnal EE (;50 vs. ;35%) (Fig.1C). This diurnal drop in RQ was associated with a ;50%

reduction in diurnal food intake in the GLOB-D2KO ani-mals, with no differences observed nocturnally (Fig. 1D).

The association between reduced food intake andincreased fatty acid oxidation in the GLOB-D2KO animalssuggest an involvement of the central nervous system,presumably the hypothalamus. Thus we next testedwhether the differences between GLOB-D2KO and WTanimals would be dissipated in the setting of intensehypothalamic-driven fatty acid mobilization and oxida-tion, i.e., cold exposure. Housing for 24 h at 4°C resultedin dramatic increase in VO2 and EE in all animals thatblurred circadian rhythmicity, similar in GLOB-D2KO andWT controls (Fig. 1E and Supplementary Fig. 1B). In con-trast, the RQ profile exhibited the expected circadianrhythmicity in both groups of animals, but the diurnalRQ difference between GLOB-D2KO and WT controlswas dissipated (Fig. 1F). This equalized the contributionof fatty acid oxidation to diurnal EE (Fig. 1C). In thissetting, both diurnal and nocturnal food intake were sim-ilar in all animals (Fig. 1G). Similar findings were obtainedwhen the environment temperature was gradually andprogressively decreased every 3 days while the animalsremained in the CLAMS for a 2-week period, exceptthat in this case, the VO2 circadian rhythmicity was pre-served throughout the experiment (Fig. 1H and I).

Decreasing BAT’s contribution to metabolism by accli-matization to thermoneutrality normalized the RQ in theGLOB-D2KO mouse (13). Thus, in the next set of experi-ments, we used an alternative strategy to decrease BAT’scontribution to metabolism, i.e., fasting. In fact, a 48 hfasting dramatically reduced VO2 and EE while it mini-mized their circadian rhythmicity (Fig. 1J and Supplemen-tary Fig. 1C). Furthermore, fasted GLOB-D2KO and WTcontrols markedly lowered their RQ values, and the 24-profile lost circadian rhythmicity (Fig. 1K). These changesin RQ elevated the contribution of fatty acid oxidation todiurnal EE to almost 80% in all animals (Fig. 1C). Moreimportantly, such as with cold exposure, fasting also dissi-pated the diurnal differences in RQ between GLOB-D2KOanimals and WT controls (Fig. 1K).

ASTRO-D2KO Mice Partially Recapitulate theMetabolic Phenotype of the GLOB-D2KO AnimalsIt has been proposed that D2-generated T3 in glial cellsnear the arcuate nucleus play an orexigenic role in mice(43,44). Thus, to test whether the lower diurnal RQ ob-served in GLOB-D2KO mouse is caused by D2 inactivationin the brain, we studied the ASTRO-D2KO mouse thatlacks D2 activity in the glia but preserves D2 expressionin other tissues/cells, including the ependymal tanycytes(36). These animals have normal serum levels of T4, T3,and TSH and are considered systemically euthyroid (36).The ASTRO-D2KO animals exhibit a normal growth curve(Fig. 2A) and, during adulthood, have similar body weight(Fig. 2B) and body composition (Fig. 2C) compared withWT littermate controls. The CLAMS studies revealed thatASTRO-D2KO animals have VO2 and EE profiles that

1596 Thyroid Hormone Regulates Fatty Acid Oxidation Diabetes Volume 63, May 2014

were unremarkable (Fig. 2D and Supplementary Fig. 1D)but a RQ profile that resembled that of the GLOB-D2KOmouse, i.e., lower diurnal values (Fig. 2E), with greater con-tribution of fatty acid oxidation to the diurnal EE (Fig. 2F);food intake, however, was not different when comparedwith WT littermate controls (Fig. 2G).

FAT-D2KO Mice Exhibit Around-the-Clock Reductionin Fatty Acid OxidationD2 is expressed in BAT, and D2-generated T3 has beenshown to regulate local fatty acid synthesis (16,45,46). Totest whether D2-generated T3 in BAT affects fatty acid

oxidation, we next studied FAT-D2KO mice that lack BATD2 expression (Supplementary Fig. 2A) and activity (Sup-plementary Fig. 2B) while expressing D2 normally in othertissues (Supplementary Fig. 2C). These animals have nogross abnormalities and develop and grow normally upuntil age 5 months, when they exhibit a slightly reducedrate of weight gain (Fig. 3A). These animals also exhibitnormal serum levels of T4, T3, and TSH (SupplementaryFig. 2D–F) and are considered systemically euthyroid.

Adult FAT-D2KO mice exhibit similar body weight (Fig.3B) and body composition (Fig. 3C) when compared withlittermate controls. Their VO2 and EE profiles showed no

Figure 1—Metabolic phenotype of the GLOB-D2KO mouse. GLOB-D2KO and WT controls were acclimated to individual metabolic cages inthe CLAMS for 48 h before measurements were recorded. (A) Oxygen consumption (VO2) during 12-h light and dark cycles recorded at thesecond day after acclimatization. (B) Same as in A, except that what is shown is RQ. (C) Contribution of fat oxidation to daily EE duringthe light cycle. (D) Food intake during light and dark cycles of animals kept on regular chow diet at room temperature (22°C). (E) VO2 during12-h light and dark cycles recorded during acute cold (4°C) exposure. (F) Same as in E, except that what is shown is RQ. (G) Food intakeduring light and dark cycles of animals kept on regular chow diet during the period of cold (4°C) exposure. (H) VO2 register during chronically(15 days) cold exposed, where the environment temperature was gradually and progressively decreased every 3–4 days. (I) Same as in H,except that what is shown is RQ. (J) VO2 during 12-h light and dark cycles recorded on the first day of 48 h of fasting. (K) Same as in J, exceptthat what is shown is RQ. Entries are mean6 SEM of 3–7 animals. Area under the curve was calculated during light and dark cycles for eachindividual animal. Statistical significance is shown in each graph and was set as P< 0.05. Student t test was used to compare WT and GLOB-D2KO groups within treatment conditions. Black horizontal bars denote the dark period of the day (12 h). AUC, area under the curve.

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Figure 2—Metabolic phenotype of the ASTRO-D2KO mouse kept on regular chow diet at room temperature (22°C). (A) Body weightevolution during the 2-month period. (B) Body weight of the animals right before admission to CLAMS. (C ) Body composition measured byDEXA 48 h before the animals were admitted to CLAMS. (D) VO2 during 12-h light and dark cycles. ASTRO-D2KO mouse and controls wereacclimated to individual metabolic cages in the CLAMS for 48 h before measurements were recorded. Data shown are from the second dayafter acclimatization. (E) Same as in D, except that what is shown is RQ. (F) Contribution of fat oxidation to daily EE during the light cycle.(G) Food intake of the same animals during light and dark cycles. Entries are mean 6 SEM of 4–5 animals. Area under the curve wascalculated during light and dark cycles for each individual animal. Statistical significance is shown in each graph and was set as P < 0.05.Student t test was used to compare controls and ASTRO-D2KO groups within treatment conditions. Black horizontal bars denote the darkperiod of the day (12 h). AUC, area under the curve.

1598 Thyroid Hormone Regulates Fatty Acid Oxidation Diabetes Volume 63, May 2014

differences when compared with littermate controls (Fig. 3Dand Supplementary Fig. 1E). However, RQ values were sub-stantially higher in the FAT-D2KO mice across the 24 h cycle(Fig. 3E), indicating that the absence of D2 in BAT (and whiteadipose tissue) reduces the contribution of fatty acid oxida-tion to EE (Fig. 3F). It is notable that FAT-D2KO animals alsoexhibit greater food intake throughout the 24 h cycle (Fig.3G), although their body weight (Fig. 3B) and body composi-tion (Fig. 3C) are not different from WT littermate controls.The relatively greater contribution of glucose oxidation to EE

was further documented through intraperitoneal glucose tol-erance test and insulin tolerance test (Fig. 3H and I). FAT-D2KO animals were more tolerant to glucose (Fig. 3H) andexhibited a greater sensitivity to insulin administration(Fig. 3I) when compared with WT littermate controls.

Given that D2 activity in white adipose tissue is minimal,these differences in RQ are likely to reflect the absence of D2in BAT. To test if this was the case, we used similar strategyas with the GLOB-D2KO animals, i.e., to minimize BATcontribution to EE by acclimatization at thermoneutrality

Figure 3—Metabolic phenotype of the FAT-D2KOmouse kept on regular chow diet. (A) Body weight evolution during the 5-month period ofanimals kept at room temperature (22°C). The FAT-D2KO mouse and controls were acclimated to individual metabolic cages in the CLAMSfor 48 h before measurements were recorded. (B) Body weight of the animals right before the animals were admitted to CLAMS. (C) Bodycomposition measured by DEXA 48 h before the animals were admitted to CLAMS. (D) VO2 during 12-h light and dark cycles recorded thesecond day after acclimatization of animals kept on regular chow diet at room temperature (22°C). (E) Same as in D, except that what isshown is RQ. (F ) Contribution of fat oxidation to daily EE during the light cycle in the same animals. (G) Food intake during light and darkcycles in the same animals. (H) Blood glucose concentrations at the indicated time points after intraperitoneal glucose injection (2 g/kg) in2-month-old FAT-D2KO and control animals. (I) Blood glucose concentration at the indicated time points before and after intraperitonealinjection of regular human insulin (0.75 units/kg body weight) in 2-month-old FAT-D2KO and control animals. (J) VO2 during 12-h light anddark cycles recorded during chronic (15 days) exposure to thermoneutrality (30°C). (K) Same as in E, except that what is shown is RQ. (L)Food intake during light and dark cycles of animals kept at thermoneutrality. (M) VO2 during 12-h light and dark cycles recorded on the firstday of 48 h of fasting. (N) Same as in J, except that what is shown is RQ. Entries are mean 6 SEM of 5–12 animals. Area under the curvewas calculated during light and dark cycles for each individual animal. Statistical significance is shown in each graph and was set as P <0.05. Student t test was used to compare controls and FAT-D2KO groups within treatment conditions. Black horizontal bars denote thedark period of day (12 h). *, P < 0.05; **, P < 0.01; ***, P < 0.001 vs. control; AUC, area under the curve; IP, intraperitoneal.

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(30°C for 8 weeks) or by fasting. Acclimatization at ther-moneutrality resulted in significant reduction in VO2 andEE, with no differences between FAT-D2KO and controlanimals (Fig. 3J and Supplementary Fig. 1F). Notably, inthis setting, the RQ values for the FAT-D2KO decreasedand became indistinguishable from WT littermate con-trols (Fig. 3K), with similar observations for food intake(Fig. 3L). At the same time, fasting dramatically reducedVO2 and EE as well as their circadian rhythmicity (Fig. 3Mand N and Supplementary Fig. 1G). More importantly,fasting also dissipated the differences in RQ betweenFAT-D2KO and WT littermate controls (Fig. 3N).

FAT-D2KO Mice Are More Susceptible to Diet-InducedObesityGiven the relative reduction in fatty acid oxidation in theFAT-D2KO BAT, we hypothesized these animals would

have impaired BAT thermogenesis and therefore be moresusceptible to diet-induced obesity. To test if this was thecase, FAT-D2KO animals were placed on an HFD for 8weeks and notably gained significantly more body weightthan WT littermate controls (Fig. 4A and B) while exhib-iting similar food consumption (Fig. 4C). In addition, atthe end of the experimental period, the FAT-D2KO ani-mals had experienced a greater increase in body fat thanWT littermate controls kept on the same diet (Fig. 4D andE), but the contribution of fatty acid oxidation to EEremained unaffected (Fig. 4F), and the VO2, EE, and RQprofiles were not different in these animals (Fig. 4G and Hand Supplementary Fig. 1H). Next, to confirm that thegreater gain in body weight and body fat in the FAT-D2KO animals were due to impaired BAT thermogenesis,we repeated the HFD for 8 weeks while the animals werekept continuously at thermoneutrality. While food intake

Figure 4—Effect of HFD in the FAT-D2KO mice at room temperature and thermoneutrality. The FAT-D2KO and controls were fed with HFDfor 8 weeks. (A) Body weight evolution during 8 weeks of treatment of animals kept at room temperature (22°C). *P < 0.05 vs. control. (B)Area under the curve from the body weight shown in A was calculated from each individual animal. *P < 0.05 vs. control. (C) Food intakeduring light and dark cycles in the same animals. (D) Body composition measured by DEXA in the animals kept at room temperature before(day 1) and after (day 60) HFD. *P < 0.05 vs. day 1. (E ) D% of fat measured by DEXA in D, calculated by the difference between days 1 and60. *P< 0.05 vs. control. (F ) Contribution of fat oxidation to daily EE during the light cycle in the same animals. (G) VO2 during 12-h light anddark cycles in the animals kept at room temperature after 8 weeks on HFD. The FAT-D2KO mouse and controls were acclimated to individualmetabolic cages in the CLAMS for 48 h before measurements were recorded. Data shown are from the second day after acclimatization.(H) Same as in G, except that what is shown is RQ. (I) Body weight evolution during 8 weeks of treatment of animals kept at thermoneutrality(30°C). (J) Area under the curve from the body weight represented in I, calculated from each individual animal. (K) Food intake during light anddark cycles in the same animals. (L) Body composition measured by DEXA in the animals kept at thermoneutrality before (day 1) and after(day 60) HFD. *P< 0.05 vs. day 1. (M) D%of fat measured by DEXA in L, calculated by the difference between days 1 and 60. (N) Contributionof fat oxidation to daily EE during the light cycle in the same animals. (O) VO2 during 12-h light and dark cycles in the animals kept atthermoneutrality after 8 weeks on the HFD. (P) Same as in O, except that what is shown is RQ. Entries are mean 6 SEM of 5–6 animals.Statistical significance is shown in each graph and was set as P < 0.05. Student t test was used to compare controls and FAT-D2KO groupswithin treatment conditions. Black horizontal bars denote the dark period of the day (12 h). AUC, area under the curve.

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remained largely unaffected, both groups of animals gainedbody weight and body fat in the same proportion onceBAT’s contribution to EE was minimized (Fig. 4I–M). Fur-thermore, CLAMS studies indicated that VO2, EE, andRQ profiles as well as fatty acid contribution to EE wereindistinguishable between both groups of animals (Fig.4N–P and Supplementary Fig. 1I ).

SKM-D2KO Mice Have Neutral Metabolic PhenotypeSkeletal muscle expresses low levels of D2 activity, but itsrelatively large mass suggests that D2-generated T3 in thisorgan may contribute significantly to the overall metabo-lism. We tested if this was the case by using a mouse modelthat lacks D2 in the skeletal muscle but preserves D2activity in all other tissues, i.e., the SKM-D2KO mouse (47).These animals have normal serum levels of T4, T3, and TSHand are considered systemically euthyroid (47). SKM-D2KOmice exhibit a similar growth curve as littermate controls

(Fig. 5A) and, during adulthood, also exhibit similar bodyweight (Fig. 5B) and body composition (Fig. 5C). The VO2

(Fig. 5D), EE (Supplementary Fig. 1J), and RQ (Fig. 5E)profiles as well as the contribution of fatty acid oxidationto EE (Fig. 5F) showed no differences when compared withWT littermate controls; food intake was also similar betweengroups (Fig. 5G). Next, SKM-D2KO animals were placed onan HFD for 8 weeks to test their ability to handle an excessof calories. Remarkably, these animals exhibited similargain of body weight (Fig. 5H and I) and fat (Fig. 5J andK) as compared with WT littermate controls, with nodifferences observed in the VO2 (Fig. 5L), EE (Supple-mentary Fig. 1L), and RQ (Fig. 5M) profiles.

DISCUSSION

The utilization of systemically hypothyroid or hyperthy-roid animals has been the traditional approach to studythyroid hormone action (35). However, almost all tissues

Figure 5—Metabolic phenotype of the SKM-D2KO mouse kept on regular chow diet and HFD at room temperature (22°C). (A) Body weightevolution during the 2-month period. (B) Body weight of the animals right before admission to CLAMS. (C) Body composition measured byDEXA 48 h before the animals were admitted to CLAMS. (D) VO2 during 12-h light and dark cycles. SKM-D2KO mouse and controls wereacclimated to individual metabolic cages in the CLAMS for 48 h before measurements were recorded. Data shown are from the second dayafter acclimatization. (E) Same as in D, except that what is shown is RQ. (F ) Contribution of fat oxidation to daily EE during the light cycle.(G) Food intake of the same animals during light and dark cycles. (H) Body weight evolution during 8 weeks of treatment with HFD inanimals kept at room temperature (22°C). (I) Area under the curve from the body weight represented in H, calculated from each individualanimal. (J) Body composition measured by DEXA in the animals kept at room temperature before (day 1) and after (day 60) HFD. (K) D% offat measured by DEXA in J, calculated by the difference between days 1 and 60. (L) VO2 during 12-h light and dark cycles in the animalskept at room temperature after 8 weeks on HFD. The SKM-D2KO mouse and controls were acclimated to individual metabolic cages in theCLAMS for 48 h before measurements were recorded. Data shown are from the second day after acclimatization. (M) Same as in E, exceptthat what is shown is RQ. Entries are mean6 SEM of 3–6 animals. Statistical significance is shown in each graph and was set as P < 0.05.Student t test was used to compare controls and SKM-D2KO groups within treatment conditions. Black horizontal bars denote the darkperiod of the day (12 h). AUC, area under the curve.

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and cells respond to thyroid hormone and set off waves ofsecondary and tertiary effects that are not directly regulatedby thyroid hormone. Furthermore, that thyroid hormonemembrane transporters, deiodinases, and T3-receptor sub-types can customize thyroid hormone effects on targettissues makes the systemic approach unsuitable for thestudy of cell-specific thyroid hormone actions. The presentstudies used a series of systemically euthyroid animal mod-els that exhibit tissue-specific disruption of the D2 pathwayand identified a multilevel physiological control of fattyacid oxidation by thyroid hormone. The present findingsare unique in that the animal models used remained sys-temically euthyroid throughout the experiments, revealingthe physiological multilevel roles played by D2-generatedT3 in fatty acid oxidation.

Selective inactivation of the D2 pathway in glial cells(ASTRO-D2KO) accelerates the diurnal rate of fatty acidoxidation in the BAT (Fig. 2E). These findings are in linewith the concept that localized thyroid hormone signalingin the hypothalamus and other brain areas (e.g., brainstem) regulate appetite, BAT sympathetic activity (48,49),and downstream metabolic and cardiovascular targets (50).In contrast, inactivation of D2 in a peripheral tissue, i.e.,BAT (FAT-D2KO), led to the opposite metabolic pheno-type, including a slower rate of fatty acid oxidation (Fig.3E), and increased the contribution of carbohydrate oxida-tion to the overall EE (Fig. 3F). FAT-D2KO mice were alsosusceptible to diet-induced obesity and gained more weightand body fat on a HFD (Fig. 4C and D). Notably, disruptionof the D2 pathway in a second peripheral tissue, i.e., skel-etal muscle, was metabolically neutral (Fig. 5A–I), an in-dication that D2 activity in this tissue is unlikely to playany significant metabolic role.

The RQ reflects the relative contribution of fatty acid,glucose, and protein oxidation to daily EE. Given that VO2

(Fig. 1A) and total daily EE (Supplementary Fig. 1A) re-mained stable in the GLOB-D2KO animals, the lower di-urnal RQ values (Fig. 1B) indicate an acceleration of fattyacid oxidation (Fig. 1C). Liver, heart, skeletal muscle, andBAT all exhibit high rates of fatty acid oxidation and couldplay a role in this process (51). That the difference in RQ isdissipated by BAT inactivation, i.e., thermoneutrality (13)or fasting (Fig. 1J and K), identifies BAT as the most likelytissue underlying this phenotype. However, is D2-generatedT3 playing a direct role in BAT by controlling local fatty acidoxidation or playing an indirect role in BAT via the centralnervous system and sympathetic activity or both?

The fact that a diurnal reduction in food intake is partof this phenotype (Fig. 1D) suggests an involvement ofthe central nervous system. In addition, the fact that thedifference in RQ is diurnal and not across the 24 h cycledoes not support a “defect” in BAT but rather an adjust-ment made by the sympathetic nervous system with thepossible involvement of higher control areas in the brain.Accordingly, maximal sympathetic stimulation of BAT dur-ing cold exposure also dissipated the differences in RQbetween GLOB-D2KO and WT controls (Fig. 1F and I).

That the mechanism leading to lower RQ values in theGLOB-D2KO mouse is centrally mediated is supported bythe previous observation that BAT norepinephrine turn-over is accelerated in GLOB-D2KO mice maintained atroom temperature (21). For example, it has been pro-posed that D2-generated T3 in the glial cells surroundingthe arcuate nucleus is important for the orexigenic re-sponse during fasting by increasing appetite and decreas-ing BAT activity in order to preserve energy (29). It isconceivable that a similar mechanism in the hypothala-mus or another brain area is taking place as illustratedwith the ASTRO-D2KO mouse that lacks D2 in glial cells.Notably, the disruption of the glia D2 pathway partiallyrecapitulated the phenotype of the GLOB-D2KO mouse,including a lower diurnal RQ (Fig. 2E) but not a reducedfood intake (Fig. 2G).

As it becomes apparent that D2 plays an importantmetabolic role in multiple tissues, the development of theFAT-D2KO allowed for a unique insight into the directrole played by D2-generated T3 in BAT physiology. Thefindings of increased RQ values in these animals indicatea relative state of BAT hypothyroidism despite normalserum T3 levels and provide clear evidence that D2-generated T3 in BAT accelerates fatty acid oxidation.Notably, T3 accelerates fatty acid oxidation by stimulat-ing the expression of rate-limiting elements in this process(31,52,53), lowering RQ values (32). However, these ex-periments frequently involve systemically hypothyroid ani-mals treated with large doses of T3. The evidence obtainedin the current study is unique in its physiological context,reflecting the result of D2 inactivation in BAT cells in anotherwise systemically euthyroid animal. Only through thisstrategy the physiological role played by D2-generated T3inside BAT cells could be appreciated.

The faster clearance of glucose from the circulation andthe increased insulin sensitivity (Fig. 3H and I) suggestthat an increased oxidation of glucose compensates forthe impaired fatty acid oxidation in BAT. Whereas thisseems to provide sufficient compensation under standardconditions, placing the FAT-D2KO animals on a HFDrevealed greater susceptibility to diet-induced obesity (Fig.4A–D), suggestive of impaired adaptive thermogenesis. Thisis supported by the observation that at thermoneutralitythis phenotype was neutralized (Fig. 4I–L), indicating thatthe sympathetic nervous system and BAT play an underly-ing role in this process.

Skeletal muscle expresses very low levels of D2, abouttwo orders of magnitude lower than BAT (25,54). However,given its large size, it is conceivable that D2 contributessignificantly to metabolism, similarly to the D2-generatedT3 in BAT. The present studies demonstrate that D2 in-activation in skeletal muscle is metabolically neutral, notaffecting any of the parameters analyzed, including VO2

and RQ and body weight and composition. In addition, wealso increased the metabolic demand by placing these ani-mals on a HFD, and their susceptibility to diet-induced obe-sity was not affect by disruption of D2 activity in skeletal

1602 Thyroid Hormone Regulates Fatty Acid Oxidation Diabetes Volume 63, May 2014

muscle (Fig. 5F–I). Thus D2-generated T3 in skeletal muscleis not sufficient to play any significant metabolic role.

The analysis of the multiple metabolic parameters andthe subsequent adjustments that follow disruption of theD2 pathway—under adverse conditions of environmentaltemperature and caloric intake—prompt us to speculatewhat the physiological imperative is, around which allother parameters adjust. In the present studies, VO2 andEE remained stable in all animal models of global or tissue-specific disruption of the D2-generated T3 mechanism. Thepresent data, and also previously published studies, indi-cate that localized disruption in thyroid hormone signalingis well neutralized by adjustments in the sympathetic ac-tivity while maintaining a stable rate of EE and thermo-genesis. Even in systemically hypothyroid mice, an increasein sympathetic activity neutralizes much of the expectedmetabolic phenotype (15). Thus, given the relatively highsurface-to-volume ratio in mice, EE and thermoregulationseem to be key physiological imperatives determining thedirection and intensity of the adjustments observed in thepresent investigation.

D2 is expressed in the human brain, including thehypothalamus (55), as well as BAT that is present in adultindividuals (51,56,57). This indicates that the present findingsare potentially applicable to humans. In fact, a common Dio2gene polymorphism has been associated with a number ofmetabolic conditions, including increased BMI and resistanceto insulin (51), supporting the idea that D2 could play a roleregulating fatty acid utilization in humans.

In conclusion, the present studies deconstructed themetabolic phenotype exhibited by a mouse with globalinactivation of D2, explaining the unexpected finding ofdiurnal acceleration of fatty acid oxidation. The findings inthe ASTRO-D2KO mouse indicate that D2-generated T3 inthe brain plays a dominant albeit indirect role, inhibitingBAT activity and thus slowing down fatty acid oxidation.The findings in the FAT-D2KO mouse, on the contrary,indicate that D2-generated T3 in BAT accelerate fatty acidoxidation, a mechanism that when absent, is partiallycompensated for by an increase in glucose utilization.However, the FAT-D2KO mouse remains susceptible todiet-induce obesity, illustrating how critical this mecha-nism is. In contrast, the D2 pathway in skeletal muscle isunlikely to play any significant metabolic role. The metaboliccharacterization of these animal models provides a uniqueinsight into how deiodinase-mediated thyroid hormonesignaling is critical for energy homeostasis.

Acknowledgments. The authors are grateful to Carlos Moraes for kindlyproviding the Cre-MLC mouse used in this study.

Funding. This work was supported by National Institute of Diabetes andDigestive and Kidney Diseases R01 65055, the European Community’s SeventhFramework Programme (FP7/2007–2013, no. 259772), Brazilian National Re-search Council, and American Thyroid Association.

Duality of Interest. No potential conflicts of interest relevant to this articlewere reported.

Author Contributions. T.L.F., J.P.W.-D.-C., and M.C. conducted experi-ments, data analyses, and manuscript preparation. B.M.L.C.B., G.W.F., E.A.M., D.L.I.,C.C.S.M., and A.R.F. conducted experiments and data analyses. B.G. created theFloxD2 mouse and reviewed the manuscript. A.C.B. conducted data analyses andmanuscript preparation. T.L.F. is the guarantor of this work and, as such, had fullaccess to all the data in the study and takes responsibility for the integrity of thedata and the accuracy of the data analysis.

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