The Paradoxical Lean Phenotype of Hypothyroid Mice Is Marked by Increased Adaptive Thermogenesis in the Skeletal Muscle Rachel R

The paradoxical lean phenotype of hypothyroid mice is marked by increased adaptive thermogenesis in the skeletal muscle Rachel R. Kasparia, Andrea Reyna-Neyraa , Lara Junga,1 , Alejandra Paola Torres-Manzob, Sandro M. Hirabaraa,c,2 , and Nancy Carrascoa,b,3

aDepartment of Cellular and Molecular Physiology, Yale School of Medicine, New Haven, CT 06510; bDepartment of Molecular Physiology and Biophysics, Vanderbilt University, Nashville, TN 37203; and cDepartment of Internal Medicine, Yale School of Medicine, New Haven, CT 06520

Contributed by Nancy Carrasco, July 22, 2020 (sent for review May 7, 2020; reviewed by Antonio C. Bianco and Gregory A. Brent)

Obesity is a major health problem worldwide, given its grow- Obesity, the result of an imbalance between energy input ing incidence and its association with a variety of comorbidities. and energy expenditure (11, 12), is a serious health issue in Weight gain results from an increase in energy intake with- the United States, as over 42% of US adults were considered out a concomitant increase in energy expenditure. To combat obese in 2018, and this number is expected to continue to rise the obesity epidemic, many studies have focused on the path- (13). Many studies have focused on trying to promote energy ways underlying satiety and hunger signaling, while other stud- expenditure to combat obesity by stimulating adaptive thermo- ies have concentrated on the mechanisms involved in energy genesis (14–16). In humans, hypothyroidism is often associated expenditure, most notably adaptive thermogenesis. Hypothy- with weight gain (17). In contrast, hypothyroid mice—whether roidism in humans is typically associated with a decreased basal their hypothyroidism is induced by drug treatment or genetically metabolic rate, lower energy expenditure, and weight gain. How- engineered—are consistently leaner than euthyroid mice (18– ever, hypothyroid mouse models have been reported to have a 21). However, the mechanisms responsible for this lower body leaner phenotype than euthyroid controls. To elucidate the mech- weight remain largely unexplored. anism underlying this phenomenon, we used a drug-free mouse We have generated knockout (KO) mice for Slc5a5, the gene model of hypothyroidism: mice lacking the sodium/iodide sym- encoding NIS (22), a mouse model that makes it possible to study porter (NIS), the plasma membrane protein that mediates active the metabolic effects of hypothyroidism without the potentially iodide uptake in the thyroid. In addition to being leaner than confounding effects of the drugs used to induce hypothyroidism euthyroid mice, owing in part to reduced food intake, these in other models. Using Slc5a5 KO mice, we have demonstrated hypothyroid mice show signs of compensatory up-regulation that hypothyroid mice are leaner than controls, but the rea- of the skeletal-muscle adaptive thermogenic marker sarcolipin, son for their failure to gain weight remains unknown (20). As with an associated increase in fatty acid oxidation (FAO). Nei- mentioned above, stimulation of adaptive thermogenesis has ther catecholamines nor thyroid-stimulating hormone (TSH) are attracted a great deal of attention as a potential treatment responsible for sarcolipin expression or FAO stimulation; rather, for obesity. THs are generally thought to be pro-thermogenic thyroid hormones are likely to negatively regulate both processes in skeletal muscle. Our findings indicate that hypothyroidism in mice results in a variety of metabolic changes, which collectively Significance lead to a leaner phenotype. A deeper understanding of these changes may make it possible to develop new strategies against The rate of obesity is steadily increasing in the United States obesity. and around the world, which is a major health concern, as weight gain is strongly associated with many leading causes hypothyroidism | sodium/iodide symporter (NIS) | food intake | of death. Although recent findings have deepened our under- adaptive thermogenesis | sarcolipin standing of the mechanisms underlying obesity, there is still a lack of therapeutic targets for inducing weight loss. We have found that a drug-free mouse model of hypothyroidism is he thyroid hormones (THs; tri-iodothyronine [T3] and thy- paradoxically leaner than euthyroid controls. Here, we show Troxine [T4]) are crucial for the development of the cen- that hypothyroid mice consume less food and display strik- tral nervous system (CNS), lungs, and skeletal muscle. They ingly higher levels of skeletal-muscle adaptive thermogenesis. also regulate basal metabolic rate (BMR) throughout life (1– Our findings yield insights into the physiological adaptations 4). TH biosynthesis relies on the active transport of iodide that occur in hypothyroidism and may eventually help bring into the thyroid by the sodium/iodide symporter (NIS) (5), as to light new targets for promoting weight loss. iodine is an essential constituent of the THs. In a negative feedback loop, TH biosynthesis and release are promoted by Author contributions: R.R.K. and N.C. designed research; R.R.K., A.R.-N., L.J., A.P.T.-M., thyroid-stimulating hormone (TSH), which is, in turn, nega- and S.M.H. performed research; R.R.K., A.R.-N., L.J., A.P.T.-M., and N.C. analyzed data; tively regulated by the THs (2–4). Once in the bloodstream, and R.R.K. and N.C. wrote the paper.y the THs reach virtually all tissues throughout the body, where Reviewers: A.C.B., University of Chicago; and G.A.B., David Geffen School of Medicine at the University of California Los Angeles.y the more biologically active form, T3, predominantly interacts with nuclear TH receptors to both positively and negatively reg- The authors declare no competing interest.y ulate gene transcription, which ultimately leads to the effects Published under the PNAS license.y of these hormones (2). Sufficient consumption of iodide and 1 Present address: School of Medicine, Heidelberg University, Heidelberg 69120, Germany.y adequate NIS function are required to maintain a euthyroid con- 2 Present address: Interdisciplinary Post-Graduate Program in Health Science, Cruzeiro do dition. Thus, patients with iodide-deficiency disorders or muta- Sul University, Sao Paulo 08060-070, Brazil.y tions in NIS that cause iodide transport defects, if untreated, 3 To whom correspondence may be addressed. Email: [email protected] develop hypothyroidism (a state defined by reduced levels of THs This article contains supporting information online at https://www.pnas.org/lookup/suppl/ and increased levels of TSH in the serum) and even cognitive doi:10.1073/pnas.2008919117/-/DCSupplemental.y impairment (6–10). First published August 21, 2020.

22544–22551 | PNAS | September 8, 2020 | vol. 117 | no. 36 www.pnas.org/cgi/doi/10.1073/pnas.2008919117 Downloaded by guest on October 1, 2021 Downloaded by guest on October 1, 2021 apr tal. et Kaspari (D–F correction Bonferroni a Eu: by line. followed dotted ANOVA the Eu: by two-way controls. depicted a littermate as ( assay, Eu Hypo. the and mice of Hypo limit KO of detection and the Eu below mice are samples WT (E Eu rendering three age, measurements, of TSH the wk n For 8 diet. at special starting on wk wk 10 10 for respectively, iodide), ppm 1. Fig. Their to whole-body our Contributes using hypothyroidism, Mice in occur Hypothyroid of Phenotype. Intake Lean Caloric Low The combating adapta- Results for these strategies novel Identifying develop phenotype. to us obesity. leaner enable cumula- a may which tions to hypothyroidism, lead phys- in of tively series occur a are adaptations that they indicate iological mechanism—although results mass, These compensatory cold-intolerant. a fat still as and likely hypothy- thermogenesis— our mass, most adaptive Strikingly, skeletal-muscle intake. lean up-regulate caloric mice reduced weight, roid to body part parame- in metabolic reduced owing controls several euthyroid exhibit than on leaner THs and are of mice hypothyroid levels These con- low ters. of in our effects Here, on the capitalized (23–27). have hyperthyroidism we trast, central generate to the mice or to on obtained administered peripheral were been information THs has which available THs in studies the the from by of regulation thermogenesis much of result, topic a as (23–27); .2pmidd) hc edr uhmc yohri,and hypothyroid, mice such their wk, renders for 8 which iodide) After iodide), development. [ppm] ppm normal million promote 0.02 per to kept life parts were of Slc5a5 (6 partially mice wk it which diet 8 all that first NIS, chow Therefore, excess than a (22). great other on of biosynthesis such routes amount TH in via is rescues daily thyroid iodide recommended the (28), enters the rodents that times for shown iodide 40 previously supplies have which hypothy- We their drug-induced. that when not advantage key is the roidism offer mice These model. .For 4. = and F Slc5a5 yohri Hp)mc a esadaelae hnetyod(u controls. (Eu) euthyroid than leaner are and less eat mice (Hypo) Hypothyroid R opsto fftms (E mass fat of composition MRI ) Omc eesice oa oiedfiin it(IDD; diet iodide-deficient an to switched were mice KO D–F and Omc r e tnadrdn-hwdiet, rodent-chow standard a fed are mice KO ekybd egto yoadE itraecontrols. littermate Eu and Hypo of weight body Weekly (D) switch. diet the before performed measurement baseline a is zero week H, eivsiae h eaoi leain that alterations metabolic the investigated We EF DE ABC n Slc5a5 n enms (F mass lean and ) 0 Hypo: 10; = GH Omc odetermine to mice KO n Slc5a5 .Dt r xrse stemean the as expressed are Data 9. = vrg a asadla asgie vr1 ko pca it (H diet. special of wk 10 over gained mass lean and mass fat Average (G) ). and Omouse KO H .**P ). < .1 ***P 0.01; h yohri tt asdmc oetls n,tu,b leaner be thus, 1H but and, (Fig. less mice. stable, eat euthyroid monitoring to than remained of mice caused that mo state 2 level hypothyroid the entire a the at for less the mice lower, significantly after euthyroid consumed week mice than One hypothyroid food weekly mice. IDD, the of an groups to monitoring mice, switch both by hypothyroid of intake consumption of nutrient might food phenotype investigated factor(s) leaner first what the we determine to To contributing output. be energy and intake a promote physiology indicate hypothyroid thus phenotype. mass of results leaner lean component(s) These whereas some S1 D). time, Fig. that over (SI , Appendix groups (SI mass both decreased lean in two increased of the weight whereas amounts fat S1 mice, similar weight, Fig. hypothyroid Appendix, body proportionally in to had hypothy- normalized reduced 1G). the groups were still (Fig. of mass mass was lean weight lean mass in body and differences the to fat due in When IDD likely changes were an mice the 1F roid to of (Fig. mass switched most fat mass less lean that being had and mice after 1E) hypothyroid that (Fig. weight showed MRI body 1D). (Fig. gain to failed than ISD. 1C) an (Fig. levels find- TSH previous our (T with controls THs agreement euthyroid as In (20), used S1A). ings were Fig. as mice Appendix, composition these nutrient (SI that same such the IDD, with iodide) the ppm 6 (ISD; diet Slc5a5 oywih sdtrie yteblnebtennutrient between balance the by determined is weight Body mice hypothyroid isocaloric, are IDD and ISD our Although Slc5a5 < idtp W)mc eesice oa iodide-sufficient an to switched were mice (WT) wild-type 3 .0;****P 0.001; n T and 1A] [Fig. Slc5a5 ± TadK iewr e nID( p oie n D (0.02 IDD and iodide) ppm (6 ISD an fed were mice KO and WT E n eeaaye ya unpaired an by analyzed were and SEM PNAS Omc na D a oe eu eesof levels serum lower had IDD an on mice KO B < and | 0.0001. A–C etme ,2020 8, September .Oealftms omlzdt body to normalized mass fat Overall C). 4 eu T Serum ) )admrel ihrserum higher markedly and 1B]) [Fig. Slc5a5 3 hnetyodcnrl and controls euthyroid than ) ,T (A), Tltemt otosfed controls littermate WT | 4 ,adTH(C TSH and (B), o.117 vol. ekyfo intake food Weekly ) t et(A–C test | o 36 no. .Therefore, ). eesafter levels ) n ;Hypo: 5; = and | 22545 or G)

PHYSIOLOGY Hypothyroidism Reduces Energy Expenditure and Activity. To quan- body temperature (i.e., used for thermogenesis). Total move- titate energy expenditure, we performed a metabolic cage anal- ment (Fig. 2 C and D) was drastically reduced in hypothyroid ysis on both groups of mice 10 wk after switching them to mice during the active cycle. In summary, our drug-free hypothy- their respective diets. A striking pattern that emerged from the roid mice had a leaner phenotype than euthyroid mice, even metabolic cage analysis is that energy expenditure by hypothy- though they exhibited classic signs of hypothyroidism, such as roid mice was shifted in time relative to that of euthyroid controls reduced energy expenditure and activity (2, 17, 21, 31). (Fig. 2A). These results suggest that the circadian rhythms of hypothyroid mice are altered. When we analyzed the energy- Markers of Skeletal-Muscle Adaptive Thermogenesis Are Markedly expenditure data using a nonlinear regression fit to a sine wave Up-Regulated in Hypothyroid Mice. In addition to activity, we also to quantitate rhythmicity, it emerged that euthyroid mice have a investigated thermogenesis, the other main component of energy peak activity offset of approximately 2.3 h prior to the dark-cycle expenditure. The two groups of mice had very similar core body midpoint, whereas the circadian rhythms of hypothyroid mice temperatures pre-diet—i.e., before the diet switch. However, were shifted by a remarkable 6.9 h. Such a large shift indicates after the Slc5a5 KO mice were switched to an IDD to render that the active period is split evenly between the light and dark them hypothyroid, their core body temperature decreased signif- cycles in our hypothyroid mice. To better capture active and inac- icantly, whereas that of the euthyroid controls did not (Fig. 3A). tive periods in both groups of mice, we instead have analyzed the To differentiate more finely between the thermogenesis that metabolic data synchronized with the active/inactive periodicity occurs in euthyroid and hypothyroid mice, we investigated the in each group. Overall, hypothyroid mice appear to expend less main sites of adaptive thermogenesis: brown adipose tissue energy than euthyroid controls, with the most pronounced differ- (BAT) and skeletal muscle. In BAT, the key mediator of adap- ence occurring during the active cycle (Fig. 2 A and B). Of note, tive thermogenesis is the inner mitochondrial membrane protein the average resting energy expenditure, which is representative known as uncoupling protein 1 (UCP1) (32, 33). Ucp1 messen- of BMR, was lower in hypothyroid mice during both the active ger RNA (mRNA) levels were slightly higher in the BAT of and inactive cycles (SI Appendix, Fig. S2A). Consistent with a hypothyroid than euthyroid mice (SI Appendix, Fig. S3A), and reduced energy expenditure, hypothyroid mice consumed less there was a trend toward higher UCP1 protein levels, but the oxygen during both the active and inactive cycles (SI Appendix, difference was not statistically significant (SI Appendix, Fig. S3 Fig. S2B), but both groups had very similar respiratory quotients B and C). Hematoxylin and eosin (H&E) staining of BAT from (SI Appendix, Fig. S2C). hypothyroid mice was more multilocular and showed fewer lipids We expect that the difference in body weight between the two than H&E staining of BAT from euthyroid mice, suggesting that groups (Fig. 1D) is a substantial contributor to the difference in there was moderate activation of thermogenesis in the BAT of energy expenditure between them (Fig. 2 A and B). To quantitate hypothyroid mice (SI Appendix, Fig. S3D). In skeletal muscle, energy expenditure and adjust for differences in body weight, the small proteolipid sarcolipin, which uncouples calcium uptake we intended to perform an analysis of covariance (ANCOVA). into the sarcoplasmic reticulum from ATP hydrolysis by the However, our two experimental groups did not overlap in body sarco/endoplasmic reticulum calcium ATPase (SERCA), plays weight, and the regression slopes were very different between an important role in mediating adaptive thermogenesis (15, 34). groups (euthyroid: 0.0316; hypothyroid: −0.0389; SI Appendix, Both sarcolipin mRNA and protein levels were markedly Fig. S2D), so an ANCOVA analysis could not be performed higher in the soleus muscle of hypothyroid mice (Fig. 3 B– (29, 30). Thus, although some of the difference in energy expen- D). In agreement with previous results (35, 36), up-regulation diture between euthyroid and hypothyroid mice is probably of sarcolipin was limited to slow-twitch skeletal muscles: We related to body mass differences, we cannot quantitatively adjust detected increased expression in the diaphragms of hypothyroid for the body-mass variable. The regression analysis revealed mice (SI Appendix, Fig. S3 E and F), but no expression in either that euthyroid mice exhibit an increase in energy expenditure their gastrocnemius or their quadriceps (SI Appendix, Fig. S3 at higher body weights—the expected phenotype—but, surpris- 14 ingly, hypothyroid mice showed a decrease in energy expendi- G and H).Using [ C]-palmitate as a tracer, we found that the 14 ture with increased weight, which further supports the notion soleus muscles of hypothyroid mice produced more [ C]-CO2 that there are fundamental differences in energy expenditure (Fig. 3E) and [14C]-acid-soluble metabolites (Fig. 3F), such as between hypothyroid mice and euthyroid controls. Krebs cycle intermediates that represent incomplete lipid oxida- To gain a better understanding of whether—and, if so, how— tion, indicative of increased fatty acid oxidation (FAO). These energy expenditure is altered in hypothyroid mice, we split it results suggest that there is a pronounced stimulation of skeletal- into its components: Total energy expenditure is the sum of the muscle adaptive thermogenic mechanisms in hypothyroid mice, energy used for movement and the energy used to maintain core which may contribute to their leaner phenotype.

ABCD

Fig. 2. Hypothyroid (Hypo) mice exhibit reduced energy expenditure and activity. Mice were fed their respective diets for 10 wk prior to a metabolic cage analysis. (A and B) Energy expenditure across the entire metabolic cage analysis (A) and averaged across the active/inactive cycles (B). (C and D) Total movement across the entire metabolic cage analysis (C) and averaged across the active/inactive cycles (D). n = 7 for both groups. Data in A and C represent the average across each group of metabolic readings collected at 5-min intervals that then underwent a nonlinear regression with a sine-function ﬁt. Data in B and D represent the mean ± SEM and were analyzed by an unpaired t test. *P < 0.05; **P < 0.01. NS, not signiﬁcant. Avg., average; Eu, euthyroid.

22546 | www.pnas.org/cgi/doi/10.1073/pnas.2008919117 Kaspari et al. Downloaded by guest on October 1, 2021 Downloaded by guest on October 1, 2021 apr tal. et Kaspari xrsin oee,i spsil httehg eeso TSH of sarcolipin levels of high in the regulation that Role the possible Key is concerning it a known However, expression. FAO. is Play Increased little Signaling and Very Expression Catecholamine Sarcolipin nor Hypothyroidism-Induced TSH Neither impaired. be may the shivering from their removed quickly, be so to challenge had body cold mice core hypothyroid trig- our maintain are As to (33). mechanisms gered used thermogenic defense adaptive of before line temperature Furthermore, coun- first be thermogenesis. the may is adaptive BMR, shivering increased reduced suggesting a the expression, thermo- as teracting sarcolipin such adaptive mechanisms, increased for other the that “primed” to was due 4 muscle controls genesis at skeletal euthyroid to placed their similar when was and expression temperature UCP1 main- their body to challenge. though unable core cold were their mice a hypothyroid tain underwent that temper- show that room results at mice These kept hypothyroid mice and hypothyroid in ature similar very were D) unpaired an by analyzed were aclpnepeso eesi h oesmsl Fg 4 (Fig. notion, muscle this soleus with that the Consistent F in likely exposure. levels is temper- cold expression room it the sarcolipin at min), to occurred (135 prior expression time ature, protein short in very changes (SI a these only similar lasted very lenge remained S4 mice cold Fig. hypothyroid the Appendix, and after euthyroid mice of euthyroid 4 than (Fig. hypothyroid challenge of soleus the stress. cold extreme an controls body to core response euthyroid their in maintain temperature lack to than mice necessary hypothyroid mechanisms colder 4 that metabolic the demonstrate much at results These h clearly 4B). 2 (Fig. were after mice that, roid confirmed imaging 4 therefore Infrared were the they and decreased from challenge, mice the removed of hypothyroid result the a of as dramatically temperature body at core mice The hypothyroid and euthyroid to placed mice 4 We the challenge. subjected we cold temperature, them a help body to core mechanism their compensatory maintain a as mice hypothyroid in (Post-diet). diet special on wk 10 after then and (Pre-diet) switch diet the before probe [ rectal a (B using groups. measured both (temp.) temperature body Core 3. Fig. 14 i.S4 Fig. Appendix, (SI BAT the in levels UCP1 and ) ◦ C]-CO neetnl,sroii xrsinwssilmc ihrin higher much still was expression sarcolipin Interestingly, stimulated is thermogenesis adaptive whether investigate To n esrdtercr oytmeaueeey1 min. 15 every temperature body core their measured and C yohri Hp)mc xii in fsiuae kltlmsl dpietemgnss iewr e hi epciedesfr1 k (A) wk. 10 for diets respective their fed were Mice thermogenesis. adaptive skeletal-muscle stimulated of signs exhibit mice (Hypo) Hypothyroid 2 (E n [ and ) and 14 ]ai-oul eaoie (F metabolites C]-acid-soluble C aclpnmN (n mRNA Sarcolipin ) C A and and ◦ niomn fe 3 i Fg 4A). (Fig. min 135 after environment C ABC DEF .Hwvr eas h odchal- cold the because However, B). t ,wieUP eesi h BAT the in levels UCP1 while D), et **P test. < n rti xrsin(n expression protein and (B) 4) or 3 = .1 ***P 0.01; rdcdb oe nuae n[ in incubated solei by produced ) < .0;****P 0.001; ◦ ,hypothy- C, ◦ ,even C, C E < and and .01 S o infiat u euthyroid. Eu, significant. not NS, 0.0001. ee tta on.W aepeiul hw that trig- shown been previously the have have with We yet point, point. not that time may at earlier compensation gered an cat- SNS at plasma that groups of rationale levels both increased the in their quantitated echolamines for we responsible expression, directly sarcolipin our were in found 6 mice catecholamines of (Fig. hypothyroid after levels elevated diets mice the respective not euthyroid or their whether in on than wk hypothyroid 10 in higher catecholamine were (42–44), 40, findings levels previous 33, with 27, stim- agreement in 16, In plays (1, 41). it thermogenesis role adaptive the adipose-tissue to ulating owing thermogenesis (39), expression adaptive sarcolipin skeletal-muscle and regulate may signaling sarcolipin either stimulate not FAO. does signaling or expression receptor that show TSH results the These through controls. euthyroid from solei CO by (Tshr (measured models muscle mouse soleus their of in [ correlated FAO using solei of mice levels the hypothyroid higher in in with up-regulation higher sarcolipin was more, expression Sarcolipin Tshr S5D). Fig. S5C (T Fig. THs Appendix, of (SI els hypothyroidism expected, of As model S5A). a Fig. as used development. were normal and while promote IDD controls, to euthyroid life of as wk wk, 8 8 first After whereas iodide), the ppm for (6 diet diet chow a on Tshr kept hypothyroid. were markedly mice them syn- control TH making stimulate release, to unable or are thesis they directly receptor, TSH are the lack TSH mice hypothyroidism: of another of used model we levels mouse expression, elevated receptor sarcolipin increased TSH not for responsible of or BAT whether the ascertain to receptor restoring TSH [Tshr (specifically, the thermogenesis of BAT expression has in TSH as implicated expression, been sarcolipin stimulate mice hypothyroid in )(C 5) = thsbe rpsdta yptei evu ytm(SNS) system nervous sympathetic that proposed been has It 2 14 n [ and 5C) (Fig. Omc eekp na6pmidd,TH-supplemented iodide, ppm 6 a on kept were mice KO Omc atal ece hroeei;rf 7.To 37). ref. thermogenesis; rescued partially mice KO ] Omc than mice KO ]pliaea rcr aaaeepesda h mean the as expressed are Data tracer. a as C]-palmitate 14 unicto of Quantification (D) muscle. soleus the in ) ]pliaea rcr.Slifo ohhypothyroid both from Solei tracer). a as C]-palmitate , Appendix (SI levels TSH serum increased and ]) Tshr 3 PNAS i.S5B Fig. Appendix, [SI Tmc eepae na S n eeused were and ISD an on placed were mice WT 14 | Tshr Oand KO than 5D) (Fig. metabolites C]-acid-soluble etme ,2020 8, September Tshr Tmc Fg 5 (Fig. mice WT Tshr Slc5a5 Omc a eue eu lev- serum reduced had mice KO Tshr Omc eepae nan on placed were mice KO O rdcdmr [ more produced KO) | Omc 3) sthese As (38). mice KO A n T and ] o.117 vol. and A (E C. and .T ascertain To B). 4 | and IAppendix, [SI o 36 no. .Further- B). F Slc5a5 mutof Amount ) ± Tshr n E and SEM | for 4 = 14 22547 WT KO C]-

PHYSIOLOGY AB soleus muscle of hypothyroid mice under these conditions (Fig. 6 C and D). In addition, hypothyroid mice exhibited increased 14 14 levels of [ C]-CO2 (Fig. 6E) and [ C]-acid-soluble metabolites (Fig. 6F), strongly suggesting that hypothyroidism-induced sarcolipin expression and increased FAO do not depend on catecholamine signaling.

Discussion Here, we show that hypothyroidism in mice triggers compensatory mechanisms that promote weight loss. We have reported CDthat Slc5a5 KO mice fed an IDD fail to gain body weight (20), a result that is in agreement with findings from drug-generated and genetically engineered mouse models of hypothyroidism (18, 19, 21). However, it is not well understood why they do not gain weight. Here, we have shown that hypothyroid mice are leaner than euthyroid controls, at least in part because they consume less food (Fig. 1H). In a centrally hyperthyroid rat model, activation of mammalian target of rapamycin (mTOR) signaling in the arcuate nucleus (ARC) of the hypothalamus was reported EFto increase feeding (45), and Coppola et al. (46) have shown that fasting stimulates local production of T3 in the ARC to promote feeding through increased UCP2-mediated mitochondrial uncoupling. It can be inferred that the opposite occurs in hypothyroidism, as Coppola et al. (46) have reported decreased levels of hypothalamic Ucp2 mRNA under hypothyroid conditions. Furthermore, increased SNS signaling has been associated with reduced food intake (47–49), suggesting that the elevated levels of catecholamines in our hypothyroid mice may also con- Fig. 4. Hypothyroid (Hypo) mice are severely cold-intolerant. Mice were tribute to their decreased food consumption. Although the THs housed at room temperature (22 ◦C) and fed their respective diets for and SNS signal synergistically in other tissues (41), it has yet to be 10 wk. They were then moved to 4 ◦C for a cold challenge. (A) Core body determined whether they signal together in the CNS to regulate temperature (temp.) taken via temperature transponders. n = 5 for both food intake. groups. (B) Representative infrared images of a euthyroid (Eu) and a Hypo THs are key regulators of BMR throughout life (1–3), and mouse before the cold challenge and after 2 h at 4 ◦C. (C) Sarcolipin pro- hypothyroidism is often associated with a decreased BMR and tein expression in the soleus muscle of Eu and Hypo mice that underwent a lower energy expenditure. Our hypothyroid mice expend less cold challenge. (D) Quantification of C. (E) Sarcolipin protein expression in ◦ energy and display less activity than euthyroid controls (Fig. 2 the soleus muscle of Hypo mice kept at room temperature (22 C) or that and SI Appendix, Fig. S2A)—typical manifestations of hypothy- underwent a cold challenge (4 ◦C). (F) Quantification of E. n = 5 for both groups. Data are expressed as the mean ± SEM. Data in A were analyzed roidism. Our findings are consistent with results showing that by a two-way ANOVA followed by a Bonferroni correction. Data in D and F hypothyroid mice and rats exhibit reduced running-wheel activity were analyzed by an unpaired t test. *P < 0.05; **P < 0.01; ***P < 0.001; and energy expenditure (21, 31). These findings seem paradox- ****P < 0.0001. NS, not significant. ical, because they show that hypothyroid mice have a reduced body weight, despite phenotypic changes that conserve energy. However, our results differ from those authors’ in that our mice are hypothyroid after only 3 wk on an IDD (20). As hypothyroid mice, unlike theirs, displayed shifted circadian expected, after 4 wk on the IDD, Slc5a5KO mice were hypothy- rhythms (Fig. 2 A and C). Cao et al. (50) showed that mTOR sig- roid (SI Appendix, Fig. S5 E–G), but their plasma catecholamine nals in the master circadian clock located in the suprachiasmatic levels (Fig. 6 A and B) were similar to those of the euthyroid con- nucleus (SCN) of the hypothalamus to regulate the expression trols, indicating that SNS compensation had not yet occurred. of the clock proteins Period 1 and Period 2 to alter circadian Sarcolipin expression, remarkably, was still up-regulated in the rhythms. As mentioned above, THs regulate mTOR signaling in

ABCD

Fig. 5. TSH is not required for stimulation of sarcolipin expression and FAO. Tshr WT and KO mice were fed their respective diets for 4 wk, rendering WT mice euthyroid (EuT) and KO mice hypothyroid (HypoT). (A) Protein expression of sarcolipin in the soleus muscle. (B) Quantiﬁcation of A. n = 4 for all groups. 14 14 14 (C and D) Amount of [ C]-CO2 (C) and [ C]-acid-soluble metabolites (D) produced by solei incubated in [ C]-palmitate as a tracer. EuT: n = 4; HypoT: n = 4; Hypo: n = 3. Data are expressed as the mean ± SEM and were analyzed by a one-way ANOVA followed by a Tukey post hoc analysis. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. Hypo, hypothyroid.

22548 | www.pnas.org/cgi/doi/10.1073/pnas.2008919117 Kaspari et al. Downloaded by guest on October 1, 2021 Downloaded by guest on October 1, 2021 apr tal. that et Kaspari postulate we regulation, sarcolipin in involved are 6) (Fig. through signaling reported who mechanisms. without (26), UCP1-dependent pyrexia al. pre- et triggers results Dittner hyperthyroidism and recent (25) that with al. agreement et Johann in by is sented This more tissue. a adipose does than the play alterations maintain temperature may TH-induced to in role thermogenesis prominent mice suggest adaptive these findings skeletal-muscle our of Furthermore, that ability temperature. body the reduction core with the their interferes as such BMR, hypothyroidism, in of component some 3 4 that (Fig. (Fig. cold-intolerant thermogenesis severely adaptive mice, muscle (SI hyperthy- hypothyroid thermogenesis S3 out BAT studied Fig. 24) of Appendix, we carried activation (23, moderate contrast, been centrally displayed in which have or Here, THs (25–27) animals. as roid of systemically 51–53); 41, using studies (1, by most prothermogenic consequence, be THs to (14–16). a loss thought weight generally promoting to are approach promising a as altering by dis- changes TH rhythms. behavioral SCN. circadian how and the metabolic investigated about thoroughly in bring be orders signaling to plau- remains mTOR seems it alters it However, therefore, hypothyroidism (45); that intake food sible promote to ARC the *P r xrse stemean the as expressed are [ with incubated solei [ of of Amount Quantification (C (D) wk. muscle. 4 soleus wk for the 10 diets and respective 4 their after fed respectively. were mice IDD, (Hypo) or hypothyroid ISD either and on (Eu) euthyroid in levels (B) (A FAO. and expression 6. Fig. EF CD AB aigfudta ete S Fg )nrcatecholamines nor 5) (Fig. TSH neither that found Having emerged recently has thermogenesis adaptive of Stimulation < .5 ***P 0.05; aehlmnsaentrqie o tmlto fsarcolipin of stimulation for required not are Catecholamines 14 C]-CO < .0;****P 0.001; 2 n rnucdsiuaino skeletal- of stimulation pronounced and A–D) (E 14 n [ and ) ]pliaea tracer. a as C]-palmitate and ± n epinephrine and (A) norepinephrine Plasma B) E n eeaaye ya unpaired an by analyzed were and SEM 14 ]ai-oul eaoie (F metabolites C]-acid-soluble < n .01 S o significant. not NS, 0.0001. o9frbt rus For groups. both for 9 to 7 = A C. and aclpnpoenepeso in expression protein Sarcolipin ) n o ohgop.(E groups. both for 4 = .Orrslsindicate results Our B). n o ohgop.Data groups. both for 4 = B–F ,btwr still were but ), rdcdby produced ) mice C–F, and t test. F ) wtht pca it otde esrmnswr efre fe 10 after performed were measurements diet. Post-diet special the of to diet. Pre- body. wk prior special immediately side age a the of of to wk region switch 8 subcutaneous at the tempera- conducted were were into measurements Transponders age programmable diet of Systems). wk using Data 6 at Medic transpon- by injected Bio while (IPTT-300; performed thermometer, transponders rodent were ture TK8851 measurements BIOSEB a der using by formed Measurements. Temperature wk. 4 for diets “Eu as respective figures. to the catalog referred their are (ENVIGO fed mice Appendix, IDD hypothyroid were (SI and the hypothyroid Mice them of render S5A). access to Fig. libitum wk iodide) wk, ppm ad 8 0.02 8 to first TD.120723; After no. switched the development. were throughout for no. euthyroid mice levothyroxine) catalog them KO ppm (ENVIGO keep 0.33 catalog to diet and life (ENVIGO TH-supplemented of iodide ISD a ppm the to 6 to euthyroid. access TD.170763; them switched libitum keep being ad to before iodide) for given ppm life 2018S) 6 of no. TD.170762; catalog wk no. ENVIGO 8 iodide; first ppm the (6 diet rodent-chow standard followed: was regimen in text. respectively, the “Hypo,” and in “Eu” specified as as to figures. wk, referred the 10 are or mice hypothyroid 4 respec- their composition and either fed nutrient were for Mice same supplied. (SI diets iodide the of hypothyroid tive have amount them the IDD in and differ render only ISD to and The iodide) whereas S1A). ppm Fig. euthyroid, 0.02 Appendix, them TD.120723; keep no. to catalog iodide) ppm After 6 Slc5a5 development. TD.170762; normal catalog promote no. to ENVIGO catalog life iodide; of ppm wk (6 8 wk, diet 8 first chow the for standard 2018S) a no. to access libitum ad generate To which IDD. an routes, on placed nonspecific Slc5a5 when hypothyroid via Therefore, become in only thyroid (22). and is the biosynthesis normally iodide enters TH (28), it rescues rodents that partially for excess amount great recommended such daily more the times 40 than contains which iodide 2018S), no. catalog when (ENVIGO diet that rodent-chow shown have We (22). Metabolic Mouse University’s Vanderbilt (MMPC). by metabolic Center performed Phenotyping The experiments was epinephrine. FAO and analysis the norepinephrine cage to for prior collection overnight plasma were fasted and experiments were All 22 Mice approximately pathogen-free cycle. otherwise. at light/dark a ified housed 12-h littermates in male a housed on on performed were University Yale Mice at experiments. facility mouse all approved Animals. to Methods and avenues Materials new up sustained open eventually and obesity. may deep combating they mech- merit leaner as compensatory hypothyroidism a investigation, the in in that result triggered question ultimately anisms no intake is food There reduced phenotype. processes These a temperature. in with body thermogenesis along core adaptive maintain to stimulate attempt compensatory turn, an trigger in mice hypothyroid that, in mechanisms occur that BMR and muscle be skeletal also and (1). could expression stimulating THs metabolism. (Ryr) sarcolipin the important by regulating receptor that in an plausible muscle ryanodine involved highly play skeletal the seems it and the THs Therefore, SERCA in that of cycling decades expression calcium for in known accelerated role Finally, been mice. THs has euthyroid al. in than lower it hyperthyroid et that are of Johann levels muscles mRNA soleus and proposed the sarcolipin heart, that reported notion, the (54) recently in this (25) degradation with al. mRNA Consistent expression sarcolipin et increases. sarcolipin low, muscle Minamisawa are skeletal mus- levels the skeletal TH in in when expression Therefore, sarcolipin cle. regulate negatively THs ogenerate To Slc5a5 THs in decreases the that suggest findings our together, Taken Slc5a5 Tetyodand euthyroid WT Omc eesice oa iiu ceso nID(ENVIGO IDD an of access libitum ad to switched were mice KO h aeUiest ntttoa nmlCr n s Committee Use and Care Animal Institutional University Yale The Omc nteC7L6 akrudwr eeae sdescribed as generated were background C57BL/6J the on mice KO Tmc eesice oa iiu ceso nID(ENVIGO ISD an of access libitum ad to switched were mice WT Tshr PNAS uhri n yohri ie h olwn diet following the mice, hypothyroid and euthyroid | Tshr Slc5a5 etme ,2020 8, September etltmeauemaueet eeper- were measurements temperature Rectal Tmc eegvna iiu cest a to access libitum ad given were mice WT Ohptyodmc,almc eegiven were mice all mice, hypothyroid KO Slc5a5 Omc r lcdo standard a on placed are mice KO T n “Hypo and ” | o.117 vol. Slc5a5 Tshr | Omc develop mice KO T Slc5a5 ”rsetvl in respectively ,” o 36 no. Tshr ◦ Omc were mice KO nesspec- unless C euthyroid euthyroid | 22549 Tshr

PHYSIOLOGY Cold Challenge. Euthyroid and hypothyroid littermates were maintained at Western Blot Analysis. Tissues for Western blotting were immediately flash- 22 ◦C for the first 18 wk of life. After 10 wk of either ISD or IDD, they were frozen in liquid nitrogen upon dissection and stored at −80 ◦C until use. moved to 4 ◦C for a cold challenge. Core body-temperature measurements Both skeletal muscle and BAT tissue were homogenized in buffer containing were made every 15 min by using programmable temperature transpon- 50 mM Tris·HCl (pH 7.5), 150 mM NaCl, 5 mM Na3VO4, 10 mM NaF, 2 mM pro- ders (IPTT-300; Bio Medic Data Systems), which were injected at 6 wk of tease inhibitor cocktail tablets cOmplete Mini, ethylenediaminetetraacetic age into the subcutaneous region of the side body. Mice were immedi- acid-free (Roche catalog no. 11836170001), and 0.5% Nonidet P-40. Pro- ately removed from the cold challenge if their core body temperature fell teins were electrophoresed on an Invitrogen NuPAGE 4 to 12% Bis-Tris gel below 30 ◦C. Infrared imaging was performed by using a FLIR T460 Thermal (Thermo Fisher Scientific) and transferred to a 0.2-µm nitrocellulose mem- Imaging Camera with UltraMax Technology, and the FLIR Tools+ software brane (Bio-Rad catalog no. 1620112) using a Transblot SD semidry transfer was used for image analysis. cell (Bio-Rad catalog no. 221B). The membrane was probed with primary antibodies diluted 1:1,000 against sarcolipin (Millipore Sigma catalog no. FAO. The soleus muscle was isolated from anesthetized mice and attached ABT13) and 1:10,000 against UCP1 (Abcam catalog no. ab10983). Either a to a stainless-steel clip to keep the muscle under constant tension. Muscles 1:10,000 dilution against GAPDH (Abcam catalog no. ab8245) or 1:1,000 were acclimated in an oxygenated Krebs–Ringer Bicarbonate Buffer (Sigma; dilution against cytochrome c oxidase subunit IV (COXIV). (Abcam catalog catalog no. K4002) for 30 to 60 min in a shaking water bath at 35 ◦C. After no. ab33985) was used as a loading control. acclimation, muscles were moved to a Krebs–Ringer Bicarbonate Buffer containing 0.1 mM total palmitate and 0.1 Ci/mL [1–14C] palmitate for up to 6 h, Statistical Analysis. Results were analyzed by using an unpaired t test, one- followed by addition of HCl. NaOH at a concentration of 2 M was added into way ANOVA followed by a Tukey post hoc analysis, or a two-way ANOVA 14 an isolated compartment in the incubation vial for CO2 absorption. Finally, followed by a Bonferroni correction on GraphPad Prism 7 software. In 14 14 levels of [ C]-CO2 and [ C]-acid soluble metabolites were determined by Fig. 1C and SI Appendix, Fig. S5G, three and five of the TSH values for the using a scintillation counter. euthyroid mice, respectively, were below the detection limit of the assay. For these analyses, the detection limit of the assay (180.74 pg/mL) was used Hormone Measurements. The preservative ethylene glycol tetraacetic acid– as a substitute for the missing values. Data in all figures are expressed as glutathione was added to plasma for norepinephrine and epinephrine means ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. NS measurements immediately following collection. Samples were quickly spun indicates not significant. down and stored at −80 ◦C until analysis. Norepinephrine and epinephrine To assess rhythmicity, we performed a nonlinear regression with a sine levels were measured by high-performance liquid chromatography via elec- function fit using GraphPad Prism 7 software, obtaining 2.3- and 6.9-h trochemical detection by the Vanderbilt University Medical Center (VUMC) phase shifts for euthyroid and hypothyroid mice, respectively. Active and Hormone Assay and Analytical Services Core. Similarly, T3 was measured by inactive cycles were defined as 12-h periods accordingly phase-shifted from using a radioimmunoassay by the VUMC Hormone Assay and Analytical Ser- the dark/light cycle. The same active/inactive cycles were used for evalua- vices Core. The Milliplex MAP Mouse Pituitary Magnetic Bead Panel was tion of all other metabolic parameters to permit comparison of parameters. used to measure plasma T4 (Millipore catalog no. RTHYMAG-30K) and TSH (Independently determined phase shifts for the other parameters were very (Millipore catalog no. MPTMAG-49K). Both kits were used according to the similar to those calculated for energy expenditure—total movement: 2.3 h manufacturer’s instructions. for euthyroid and 6.3 h for hypothyroid; VO2: 2.2 h for euthyroid and 6.8 h for hypothyroid; and respiratory quotient: 3.4 h for euthyroid and 7.9 h for Real-Time PCR Analysis. Tissues for RNA extraction were immediately flash- hypothyroid). frozen in liquid nitrogen upon dissection and stored at −80 ◦C until use. RNA was extracted by using TRIzol (Ambion catalog no. 15596018) and Data Availability. All study data are included in the article and SI Appendix. the RNeasy Mini Kit (Qiagen catalog no. 74104). Complementary DNA (cDNA) was synthesized by using the Quantabio qScript cDNA SuperMix ACKNOWLEDGMENTS. We thank the members of the N.C. laboratory, kit (Quantabio catalog no. 95048) in accordance with the manufacturer’s Dr. Owen P. McGuinness, Dr. Biff Forbush, Dr. Louise Lantier, and Dr. instructions. Amplification of cDNA was performed by using the Quantabio Michael J. Caplan for their insightful comments and for helpful discussion. The Tshr KO mice were a generous gift from Dr. Terry Davies (Mount PerfeCTa SYBR Green FastMix (Quantabio catalog no. 95072) in accor- Sinai Medical Center, New York). This work was supported by NIH Grants dance with the manufacturer’s instructions and was performed on an DK041544 (to N.C.) and 1F31 DK118814-01A1 (to R.R.K.). The Vanderbilt Eco Real-Time PCR System (Illumina catalog no. 1010180). Gene expres- MMPC performed the metabolic cage analysis. The MMPC is supported by sion was normalized to expression of the housekeeping gene 18S run NIH Grants DK059637 and 1S10RR028101-01. The VUMC Hormone Assay under the same conditions. Primer sequences were generated by using and Analytical Services Core is supported by NIH Grants DK059637 and Primer-BLAST. DK020593.

1. J. E. Silva, Thermogenic mechanisms and their hormonal regulation. Physiol. Rev. 86, 12. J. O. Hill, H. R. Wyatt, J. C. Peters, Energy balance and obesity. Circulation 126, 126– 435–464 (2006). 132 (2012). 2. R. Mullur, Y. Y. Liu, G. A. Brent, Thyroid hormone regulation of metabolism. Physiol. 13. C. M. Hales, M. D. Carroll, C. D. Fryar, C. L. Ogden, Prevalence of obesity and severe Rev. 94, 355–382 (2014). obesity among adults: United States, 2017-2018 (NCHS Data Brief 360, National 3. C. Portulano, M. Paroder-Belenitsky, N. Carrasco, The Na+/I– symporter (NIS): Center for Health Statistics, Hyattsville, MD, 2020). Mechanism and medical impact. Endocr. Rev. 35, 106–149 (2014). 14. H. M. Feldmann, V. Golozoubova, B. Cannon, J. Nedergaard, UCP1 ablation induces 4. S. Ravera, A. Reyna-Neyra, G. Ferrandino, L. M. Amzel, N. Carrasco, The sodium/iodide obesity and abolishes diet-induced thermogenesis in mice exempt from thermal stress symporter (NIS): Molecular physiology and preclinical and clinical applications. Annu. by living at thermoneutrality. Cell Metab. 9, 203–209 (2009). Rev. Physiol. 79, 261–289 (2017). 15. N. C. Bal et al., Sarcolipin is a newly identified regulator of muscle-based 5. G. Dai, O. Levy, N. Carrasco, Cloning and characterization of the thyroid iodide thermogenesis in mammals. Nat. Med. 18, 1575–1579 (2012). transporter. Nature 379, 458–460 (1996). 16. E. T. Chouchani, L. Kazak, B. M. Spiegelman, New advances in adaptive thermogene- 6. O. Levy, C. Ginter, A. De La Vieja, D. Levy, N. Carrasco, Identification of a structural sis: UCP1 and beyond. Cell Metab. 29, 27–37 (2019). requirement for thyroid Na+/I– symporter (NIS) function from analysis of a mutation 17. L. Chaker, A. C. Bianco, J. Jonklaas, R. P. Peeters, Hypothyroidism. Lancet 390, 1550– that causes human congenital hypothyroidism. FEBS Lett. 429, 36–40 (1998). 1562 (2017). 7. J. Pohlenz, S. Refetoff, Mutations in the sodium/iodide symporter (NIS) gene as a 18. C. B. Ueta, E. L. Olivares, A. C. Bianco, Responsiveness to thyroid hormone and to cause for iodide transport defects and congenital hypothyroidism. Biochimie 81, 469– ambient temperature underlies differences between brown adipose tissue and skele- 476 (1999). tal muscle thermogenesis in a mouse model of diet-induced obesity. Endocrinology 8. J. P. Nicola et al., Iodide transport defect: Functional characterization of a novel muta- 152, 3571–3581 (2011). tion in the Na+/I– symporter 5’-untranslated region in a patient with congenital 19. J. Weiner et al., Thyroid hormone status defines brown adipose tissue activity and hypothyroidism. J. Clin. Endocrinol. Metab. 96, E1100–E1107 (2011). browning of white adipose tissues in mice. Sci. Rep. 6, 38124 (2016). 9. M. D. Reed-Tsur, A. De la Vieja, C. S. Ginter, N. Carrasco, Molecular characterization 20. G. Ferrandino et al., Pathogenesis of hypothyroidism-induced NAFLD is driven by of V59E NIS, a Na+/I– symporter mutant that causes congenital I– transport defect. intra- and extrahepatic mechanisms. Proc. Natl. Acad. Sci. U.S.A. 114, E9172–E9180 Endocrinology 149, 3077–3084 (2008). (2017). 10. Y. Watanabe, R. S. Ebrhim, M. A. Abdullah, R. E. Weiss, A novel missense mutation in 21. K. Patyra et al., Partial thyrocyte-specific Gαs deficiency leads to rapid-onset hypothy- the SLC5A5 gene in a Sudanese family with congenital hypothyroidism. Thyroid 28, roidism, hyperplasia, and papillary thyroid carcinoma-like lesions in mice. FASEB J. 32, 1068–1070 (2018). 6239–6251 (2018). 11. B. M. Spiegelman, J. S. Flier, Obesity and the regulation of energy balance. Cell 104, 22. G. Ferrandino et al., An extremely high dietary iodide supply forestalls severe 531–543 (2001). hypothyroidism in Na+/I– symporter (NIS) knockout mice. Sci. Rep. 7, 5329 (2017).

22550 | www.pnas.org/cgi/doi/10.1073/pnas.2008919117 Kaspari et al. Downloaded by guest on October 1, 2021 Downloaded by guest on October 1, 2021 8 .C Marians C. R. 38. tissue adipose brown in receptor hormone Thyroid-stimulating Kobayashi, T. Endo, T. expres- Differential 37. Periasamy, M. Billman, E. G. Carnes, A. C. Bhupathy, P. muscle Babu, in J. G. sarcolipin of 36. role The physiological Periasamy, Maurya M. and K. Maurya, S. Function K. S. 35. tissue: Sahoo, K. S. adipose Bal, Brown C. N. Nedergaard, 34. J. Cannon, B. 33. 2 .Enerback S. 32. Mina I. A. 29. 0 .R pamn esrn nrymtbls ntemuetertcl practical, mouse-theoretical, Klieverik P. the L. in 31. metabolism energy Measuring Speakman, R. J. 30. Nutrition, Animal Laboratory on Subcommittee Council Research National acute 28. thermoneutrality, At Nedergaard, Guilherme A. J. 27. Cannon, B. Lindsund, E. Dittner, C. 26. Lopez M. 23. 5 .Johann K. 25. Martinez-Sanchez N. 24. apr tal. et Kaspari odhroesnhssb sn hrtoi eetrnl mice. receptor-null thyrotropin U.S.A. using by synthesis hormone roid 54E1 (2008). thermogenesis. E514–E518 of regulation the in involved is pathophysiology. cardiac and Cardiol. development Cell. muscle Mol. during J. protein sarcolipin of sion muscle. skeletal in metabolism oxidative thermogenesis. non-shivering significance. obese. not but iseseicftyai paei vivo. in uptake acid fatty tissue-specific 173. Metab. xii, Cell pp. 1995), 4, ed. DC, Washington, Sciences, of Academy National Animals Laboratory of Requirements integration. signal hormone thyroid and adrenergic n nltclconsiderations. analytical and (2019). UCP1. 20–34 of 25, independent are pyrexia and consumption. thermogenesis thyroxine-induced glucose and thermogenesis to (2019). 3385–3400.e3 contribute not does balance. energy of regulation rlatoso hri omnso nrybalance. energy on hormones thyroid (2017). of actions tral 57–58 (2002). 15776–15781 99, aR e-ae nlssto o nietclrmtyexperiments. calorimetry indirect for tool analysis web-based A CalR: al., et yohlmcAP n at cdmtbls eit thyroid mediate metabolism acid fatty and AMPK Hypothalamic al., et 5–6.1(2018). 656–666.e1 28, hri-omn-nue rwigo ht dps tissue adipose white of browning Thyroid-hormone-induced al., et ielcigmtcodiluculn rti r cold-sensitive are protein uncoupling mitochondrial lacking Mice al., et hso.Rev. Physiol. hri omn fet nwoebd nryhmotssand homeostasis energy whole-body on effects hormone Thyroid al., et oto faioyetemgnssadlpgnssthrough lipogenesis and thermogenesis adipocyte of Control al., et enn hrtoi-eedn n idpnetseso thy- of steps -independent and thyrotropin-dependent Defining al., et aclpnsgaigpooe iohnra ignssand biogenesis mitochondrial promotes signaling Sarcolipin al., et Nature 1–2 (2007). 215–222 43, yohlmcAP-Rsrs-N1ai eitstecen- the mediates axis stress-JNK1 AMPK-ER Hypothalamic al., et 09 (1997). 90–94 387, 7–5 (2004). 277–359 84, rn.Physiol. Front. rn.Physiol. Front. a.Med. Nat. Ntin eurmnso oetcAnimals, Domestic of Requirements (Nutrient Endocrinology 0110 (2010). 1001–1008 16, elRep. Cell 27(2018). 1217 9, 4(2013). 34 4, m .Pyil nornl Metab. Endocrinol. Physiol. J. Am. 9923 (2018). 2919–2931 24, elRep. Cell 6954 (2009). 5639–5648 150, elMetab. Cell 058(2020). 107598 31, rc al cd Sci. Acad. Natl. Proc. 212–229.e12 26, elRep. Cell o.Metab. Mol. Nutrient 295, β 27, 3 - 4 .Minamisawa by by temperature S. regulation body of balance 54. regulation the Energy in concepts Dieguez, fever—novel than C. More Mittag, Nogueiras, J. R. 53. Alvarez, V. C. Lopez, M. 52. Golozoubova V. signaling rapamycin 51. of target Mammalian Obrietan, K. Lee, B. Cho, Y. H. Li, A. affective Cao, R. and 50. anorectic of Dissociation Lafrance, Mantzoros L. S. C. Cabanac, 49. M. Racotta, S. I. 48. Experimental activity: sympathetic and intake food of relation Reciprocal Bray, A. G. 47. Coppola in A. concentrations 46. catecholamine Plasma Varela Walker, L. P. 45. Dussault, H. J. the Coulombe, on P. states hypothyroid 44. and hyper- prolonged of influence The Nash, W. C. on Tu, T. hormones adrenal 43. and thyroid, pituitary, clinical of Influence and Axelrod, Physiological J. interactions: Landsberg, L. Thyroid-adrenergic Bianco, 42. D. S. metabolic Silva, and E. thermogenic J. key 41. A Sarcolipin: Bachman Periasamy, S. E. M. 40. Bal, C. N. Pant, M. 39. (2006). ytioohrnn nteara myocardium. atrial the in triiodothyronine by hormones. thyroid level. central at hormones thyroid receptors. hormone thyroid hormone-binding all Endocrinol. of devoid mice in recruitment clock. (2010). circadian suprachiasmatic 6302–6314 the of entrainment photic modulates mice. in intake food of (1996). inhibition 909–914 leptin-independent 45, a mediates and sion rats. in glucose and epinephrine to responses (2000). S8–S17 implications. clinical and observations UCP2. and T3 nucleus arcuate between interplay hyperthyroidism. in hyperphagia of rates hypothyroidism. efflux and hyperthyroidism and accumulation the on and tissues noradrenaline. tritiated rat of content noradrenaline heart. rat the in metabolism (1968). and turnover norepinephrine implications. resistance. obesity muscle. skeletal in regulator yohlmcmO aha eitstyodhormone-induced thyroid mediates pathway mTOR Hypothalamic al., et 8–0 (2004). 384–401 18, eta hroei-iemcaimi edn euain An regulation: feeding in mechanism thermogenic-like central A al., et Thyroid tal., et ciainof Activation al., et erse hroeei u optn rw dps tissue adipose brown competent but thermogenesis Depressed al., et ottasrpinldwrglto fsroii mRNA sarcolipin of downregulation Post-transcriptional al., et PNAS Science x.Ci.Edcio.Diabetes Endocrinol. Clin. Exp. 5–6 (2008). 157–165 18, Rsgaigrqie o itidcdtemgnssand thermogenesis diet-induced for required signaling βAR a.J hso.Pharmacol. Physiol. J. Can. | 4–4 (2002). 843–845 297, etme ,2020 8, September rnsEdcio.Metab. Endocrinol. Trends .Pathol. J. rnsMl Med. Mol. Trends β 3 Metabolism n.J bs ea.Mtb Disord. Metab. Relat. Obes. J. Int. deegcrcpossprse etnexpres- leptin suppresses receptors adrenergic 0–2 (2012). 209–222 227, hso.Behav. Physiol. elMetab. Cell 2–3 (2020). 428–431 128, | 7–7 (1976). 973–979 25, 48 (1975). 74–80 53, 1–2 (2013). 418–427 19, o.117 vol. ESLett. FEBS 8–9 (2016). 881–892 27, 2–3 (1995). 125–130 58, 13 (2007). 21–33 5, ic Res. Circ. | o 36 no. 2247–2252 580, .Neurosci. J. 24 559–571 22, spl 2), (suppl. | Diabetes 22551 Mol. 30,

PHYSIOLOGY