Adaptive Thermogenesis in a Mouse Model Lacking Selenoprotein Biosynthesis in Brown Adipocytes

International Journal of Molecular Sciences

Article Adaptive Thermogenesis in a Mouse Model Lacking Selenoprotein Biosynthesis in Brown Adipocytes

Lucia A. Seale 1,2,* , Ashley N. Ogawa-Wong 1, Ligia M. Watanabe 1,†, Vedbar S. Khadka 3 , Mark Menor 3, Daniel J. Torres 1,2 , Bradley A. Carlson 4 , Dolph L. Hatﬁeld 4 and Marla J. Berry 2

1 Department of Cell and Molecular Biology, John A. Burns School of Medicine, University of Hawaii at Manoa, Honolulu, HI 96813, USA; [email protected] (A.N.O.-W.); [email protected] (L.M.W.); [email protected] (D.J.T.) 2 Paciﬁc Biomedical Research Center, School of Ocean and Earth Science and Technology, University of Hawaii at Manoa, Honolulu, HI 96822, USA; [email protected] 3 Department of Quantitative Health Sciences, John A. Burns School of Medicine, University of Hawaii at Manoa, Honolulu, HI 96822, USA; [email protected] (V.S.K.); [email protected] (M.M.) 4 Molecular Biology of Selenium Section, Mouse Genetics Program, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA; [email protected] (B.A.C.); hatﬁ[email protected] (D.L.H.) * Correspondence: [email protected] † Current address: Department of Internal Medicine, Ribeirão Preto Medical School, University of São Paulo—FMRP/USP, São Paulo 14049-900, Brazil.

Abstract: Selenoproteins are a class of proteins with the selenium-containing amino acid selenocysteine (Sec) in their primary structure. Sec is incorporated into selenoproteins via recoding of the stop codon UGA, with specific cis and trans factors required during translation to avoid UGA [Ser]Sec recognition as a stop codon, including a Sec-specific tRNA, tRNA , encoded in mice by the gene Trsp. Whole-body deletion of Trsp in mouse is embryonically lethal, while targeted deletion Trsp Citation: Seale, L.A.; Ogawa-Wong, of in mice has been used to understand the role of selenoproteins in the health and physiol- A.N.; Watanabe, L.M.; Khadka, V.S.; ogy of various tissues. We developed a mouse model with the targeted deletion of Trsp in brown f/f +/− Menor, M.; Torres, D.J.; Carlson, B.A.; adipocytes (Trsp -Ucp1-Cre ), a cell type predominant in brown adipose tissue (BAT) controlling Hatfield, D.L.; Berry, M.J. Adaptive energy expenditure via activation of adaptive thermogenesis, mostly using uncoupling protein 1 − Thermogenesis in a Mouse Model (Ucp1). At room temperature, Trspf/f-Ucp1-Cre+/ mice maintain oxygen consumption and Ucp1 Lacking Selenoprotein Biosynthesis in expression, with male Trspf/f-Ucp1-Cre+/− mice accumulating more triglycerides in BAT than both Brown Adipocytes. Int. J. Mol. Sci. female Trspf/f-Ucp1-Cre+/− mice or Trspf/f controls. Acute cold exposure neither reduced core body 2021, 22, 611. https://doi.org/ temperature nor changed the expression of selenoprotein iodothyronine deiodinase type II (Dio2), 10.3390/ijms22020611 a marker of adaptive thermogenesis, in Trspf/f-Ucp1-Cre+/− mice. Microarray analysis of BAT from Trspf/f-Ucp1-Cre+/− mice revealed glutathione S-transferase alpha 3 (Gsta3) and ELMO domain con- Received: 8 November 2020 taining 2 (Elmod2) as the transcripts most affected by the loss of Trsp. Male Trspf/f-Ucp1-Cre+/− mice Accepted: 6 January 2021 showed mild hypothyroidism while downregulating thyroid hormone-responsive genes Thrsp and Published: 9 January 2021 Tshr in their BATs. In summary, modest changes in the BAT of Trspf/f-Ucp1-Cre +/− mice implicate a

Publisher’s Note: MDPI stays neu- mild thyroid hormone dysfunction in brown adipocytes. tral with regard to jurisdictional clai- [Ser]Sec ms in published maps and institutio- Keywords: Sec-tRNA ; selenoproteins; brown adipose tissue nal afﬁliations.

1. Introduction Copyright: © 2021 by the authors. Li- censee MDPI, Basel, Switzerland. Selenoproteins are a small group of proteins containing the micronutrient selenium in This article is an open access article their molecule. These proteins are involved in redox reactions that can reduce oxidative distributed under the terms and con- stress, activate thyroid hormones, and act on endoplasmic reticulum-associated degrada- ditions of the Creative Commons At- tion of misfolded proteins. There are only 24 and 25 selenoproteins in mice and humans, tribution (CC BY) license (https:// respectively. Nevertheless, selenoproteins exert a profound effect in several aspects of creativecommons.org/licenses/by/ health, including the pathophysiology of different types of cancer [1–3], autoimmune 4.0/). diseases [4], irritable bowel syndrome [5], type 2 diabetes [6,7], and thyroid disorders [8,9].

Int. J. Mol. Sci. 2021, 22, 611. https://doi.org/10.3390/ijms22020611 https://www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2021, 22, 611 2 of 20

A hallmark feature of selenoproteins is the incorporation of selenium as the amino acid selenocysteine (Sec) in their primary structure. Sec is encoded by the UGA codon, also recognized as a stop codon. Circumvention of the stop recognition occurs due to a combination of cis and trans factors that have been extensively reviewed elsewhere [10–18]. Notably, one of the key factors is the specific tRNA[Ser]Sec, required for successful insertion of Sec during translation of selenoproteins, and encoded by the gene Trsp. Total loss of Trsp in mice is embryonically lethal [19], a finding that highlights how essential selenoproteins are for life. To demonstrate the role of selenoproteins in various organs, tissues and cell types, targeted deletion of Trsp in specific cell types has been carried out for decades, with surprising consequences [10,20,21]. However, the targeted deletion of Trsp in brown adipocytes, a cell type critical for thermoregulation and energy expenditure in mammals, had not been attempted. Brown adipocytes are the most abundant cell type found in the brown adipose tissue (BAT), the main site for adaptive thermogenesis in rodents, and subpopulations of brown adipocytes present distinct thermogenic potential [22,23]. Mammalian adaptive thermogenesis is classically activated upon exposure to cold or after caloric overload. BAT cold-induced adaptive thermogenesis relies primarily on the actions of uncoupling protein 1 (Ucp1), a proton pump localized in the inner membrane of mitochondria of brown adipocytes that dissipates energy from ATP synthesis as heat [24,25]. Ucp1 expression is synergistically upregulated after activation of beta-adrenergic signaling and by thyroid hormone 3,30,5-triiodothyronine (T3) [26]. Interestingly, T3 is mostly produced locally in cells, after the removal of one iodine from thyroxine (T4), the main prohormone from the thyroid gland. Conversion of T4 into active T3 in brown adipocytes is catalyzed by iodothyronine deiodinase type 2 (Dio2). Dio2 is a selenoprotein highly expressed in activated brown adipocytes and essential for cold-induced thermogenesis [27], allowing for the increased Ucp1 expression [28,29]. Mice with a disruption of Dio2 cannot properly carry out adaptive thermogenesis, either by cold exposure or caloric overload at room temperature [28,30,31]. Besides Dio2, glutathione peroxidases 1, 3, and 4 (Gpx1, Gpx3, and Gpx4, respectively), thioredoxin reductases 1, 2 and 3 (Txnrd1, Txnrd2, and Txnrd3, respectively), selenoproteins H, O, T and P (SelenoH, SelenoO, SelenoT, and SelenoP, respectively), and selenophosphate synthetase 2 (Sephs2) have also been found in BAT using a proteomics analysis [32], but the role of these selenoproteins in BAT function is unknown. We report the generation of a mouse model with a targeted deletion of the gene for Trsp in brown adipocytes, the Trspf/f-Ucp1-Cre+/− mice, to characterize the influence of selenoproteins in BAT function. These mice thermoregulate without changes in energy expenditure or Ucp1 expression, and surprisingly respond to acute cold exposure adequately. We also observed novel genes that respond to the lack of Trsp in brown adipocytes both at room temperature and after acute cold exposure, particularly those involved in methylation pathways and response to thyroid hormone levels.

2. Results 2.1. Generation of the Trspf/f-Ucp1-Cre+/− Mice We developed a transgenic mouse lacking the Trsp gene in brown adipocytes, Trspf/f- Ucp1-Cre+/−. At room temperature, the Ucp1-Cre system has been previously shown to specifically express Cre recombinase only in brown adipocytes of the interscapular BAT [33], which assured specific Trsp deletion in this cell type. Deletion of the Trsp gene was confirmed by genotyping for both the floxed gene and for the presence of the Ucp1-Cre sequence (Figure1a). BAT expression of the Trsp tRNA was reduced by ~60% (Figure1b), with remaining Trsp expression possibly reflecting its presence in other cell types within the tissue. Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 3 of 20

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/− 1.5 ** + /− e + re -Cr C 1 - 1.0 p 1 /− p 1.5 ** + /− Uc + + - Uc / /f - /f e e f f f Cr r p p p 0.5 - C s s s Trsp/Gapdh - r r r 1.0 change) (fold 1 1 T T T p p -Uc 0.0 /+ f Uc f f f f/ - f/ f/ f +/− p -Trsp 0.5 p p p re s s s Trsp/Gapdh rs r r r change) (fold T T T T 1-C cp 0.0 f/f -U f - Ucp1-Cre f/ p f +/− -Trsp p Trs rs re T 1-C (a) cp (b) f/f -U Figure 1. Genotype- Ucp1-Cre confirmation of Trspf/f-Ucp1-Cre+/− after breeding.p (a) PCR result of genotyp- Trs ing, showing the heterozygote mouse DNA with the doublet (1.1 and 0.9 kb) for Trspf and Trsp+ in the first (lane,a) the Trspf/f-Ucp1-Cre+/− mice in the second lane, and(b) the Trspf/f used as controls in the third lane. Trspf/+-Ucp1-Cre+/− heterozygote− mice were not used in experiments. (b) Trsp expression Figure 1.Figure Genotype 1. Genotype confirmation confirmation of Trsp of Trspf/f-Ucp1-Cref/f-Ucp1-Cre+/+/− after breeding. breeding. (a) ( PCRa) PCR result result of genotyping, of genotyp- as measured by qPCR of whole brown adipose tissue (BAT). Values are means ± SD, n = 7. ** rep- ing, showingshowing the theheterozygote heterozygote mouse mouseDNA DNA with with the the doublet doublet (1.1 and (1.1 0.9 and kb) 0.9 for kb)Trsp fforand TrspTrspf+ andin the Trsp+ in resents p<0.01 after unpaired Student’s t-test. the first lane,first lane, the theTrspTrspf/f-Ucp1-Cref/f-Ucp1-Cre+/+/−− mice in in the the second second lane, lane, and theandTrsp thef/f usedTrsp asf/f used controls as incontrols the third in the − third lane.lane. TrspTrspf/+-Ucp1-Cref/+-Ucp1-Cre+/+/− heterozygoteheterozygote micemice were were not no usedt used in experiments. in experiments.(b) Trsp (b)expression Trsp expression as 2.2. Trspf/f-Ucp1-Cre+/− Mice Maintain Energy Expenditure as measuredmeasured by qPCR by qPCR of ofwhole whole brown brown adiposeadipose tissue tissue (BAT). (BAT). Values Values are means are± meansSD, n = 7.± SD, ** represents n = 7. ** rep- f/f +/− resents p<0.01p < 0.01 after after unpaired unpairedTrsp Student’s -Ucp1-CreStudent’st-test. t-test.male mice showed no significant weight gain differences over time f/f f/f +/− 2.2. Trspf/f-Ucp1-Crecompared+/− toMice Trsp Maintain littermate Energy control Expenditure males (Figure 2a), while female Trsp -Ucp1-Cre were modestly heavier (Figure 2b) at 12 weeks of age compared to Trspf/f littermate con- 2.2. Trspf/f-Ucp1-CreTrspf/f-Ucp1-Cre+/− Mice+/− Maintainmale mice Energy showed Expenditure no significant weight gain differences over time trols (pf/f=0.08). Brown adipocytes are the main site of adaptivef/f thermogenesis+/− in mice; Trspcomparedf/f-Ucp1-Cre to Trsp+/− malelittermate mice showed control males no significant (Figure2a), whileweight female gainTrsp differences-Ucp1-Cre over time were modestlyhence, heavier accounting (Figure2 b)for at energy 12 weeks expenditure, of age compared we toassessedTrspf/f littermate the VO controls2 consumption and total f/f f/f +/− compared(p = to 0.08). Trsp Brownenergy littermate adipocytes expenditure control are of the male mainmales and site (Figure female of adaptive Trsp2a), thermogenesis f/fwhile-Ucp1-Cre female+/− mice, in mice;Trsp after hence,-Ucp1-Cre placing mice in met- f/f were modestlyaccounting heavierabolic for energy chambers (Figure expenditure, 2b)for 24at h.12 we We weeks assessed determined of the age VO comparedthat2 consumption neither toVO andTrsp2 consumption total littermate energy (F con-igure 2c,d) nor trols (p=0.08).expenditure Browntheir of male totaladipocytes and energy female expenditure areTrsp thef/f-Ucp1-Cre main (Figure +/site− mice, 2e,f)of adaptive afterwere placing altered thermogenesis mice in Trsp in metabolicf/f-Ucp1-Cre in mice;+/- mice at room hence, accountingchambers fortemperature, for 24 h.energy We determined as expenditure, calculated that neitherby we the assessed VO average2 consumption ofthe the VO area (Figure2 consumption under2c,d) northe theircurve and (AUC)total for each total energy expenditure (Figure2e,f) were altered in Trspf/f-Ucp1-Cre+/- mice at room energy expendituremouse’s of male curve. and Energy female expenditures Trspf/f-Ucp1-Cre were+/ not− mice, altered after in placing either sex mice at room in met- temperature. temperature, as calculated by the average of the area under the curve (AUC) for each Interestingly, probing for impact on glucose homeostasis in the gonadal white adipose abolic chambersmouse’s curve. for 24 Energy h. We expenditures determined were that not neither altered inVO either2 consumption sex at room temperature. (Figure 2c,d) nor f/f +/− their totalInterestingly, energytissue expenditure probing (gWAT) for impact (Figurerevealed on 2e,f) glucosethat were Trsp homeostasis altered-Ucp1-Cre in in Trsp themice gonadalf/f-Ucp1-Cre had a white reduction+/- adiposemice in at the room activation of temperature,tissue (gWAT)as Akt,calculated revealedas assessed by that the byTrsp levelsaveragef/f-Ucp1-Cre of phosphorylated of+/ the− mice area had under aAkt reduction at the serine curve in 473 the (AUC)(pAkt-Ser473) activation for each residue (Fig- ure 2g). Reduced activation occurred in male but not female Trspf/f-Ucp1-Cre+/− mice, even mouse’sof curve. Akt, as Energy assessed expenditures by levels of phosphorylated were not altered Akt at serinein either 473 (pAkt-Ser473)sex at room residuetemperature. (Figure2g).though Reduced both activation sexes had occurred reduced in male total but Akt not levels. female Trspf/f-Ucp1-Cre+/− mice, Interestingly,even though probing both for sexes impact had reduced on glucose total Akt homeostasis levels. in the gonadal white adipose tissue (gWAT) revealed that Trspf/f-Ucp1-Cre+/− mice had a reduction in the activation of Akt, as assessed by MALESlevels of phosphorylated Akt at serine 473 (pAkt-Ser473)FEMALES residue (Fig- 30 ure 2g). Reduced activation occurred in male but not female20 Trspf/f-Ucp1-Cre+/− mice, even though both sexes had reduced total Akt levels. 15 20

2WA 2WA P = 0.5113 Pinteraction = 0.5475 10 interaction MALES FEMALES P = 0.0184 Pgenotype = 0.3615 genotype

body weight (g) P <0.0001 Ptime <0.0001 30 10 time body weight (g) 20 5 Trspf/f Trspf/f Trspf/f-Ucp1-Cre+/− Trspf/f-Ucp1-Cre+/− 15 20 0 0 46812 46812 Time (weeks) Time (weeks) 2WA 2WA P = 0.5113 Pinteraction = 0.5475 10 interaction P = 0.0184 Pgenotype = 0.3615 genotype

body weight (g) P <0.0001 Ptime <0.0001 10 time (a) body weight (g) (b) 5 Trspf/fFigure 2. Cont. Trspf/f Trspf/f-Ucp1-Cre+/− Trspf/f-Ucp1-Cre+/− 0 0 46812 46812 Time (weeks) Time (weeks)

(a) (b)

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ns 1500 ns FEMALES 1500 MALES 1000 40 fl/fl 1000 fl/fl Trsp (n=4) AUC Trsp (n=6) AUC fl/fl +/− 500 Trsp -Ucp1-Cre (n=4) 500 Trspfl/fl-Ucp1-Cre+/−(n=6) 0 0 35 l 30 l /f − /f − l +/ l +/ f f p e p re s r s r -C r -C T 1 T 1 p p c c fl -U U l/ /fl - f fl p p s s 30 Tr Tr

25 (ml/min/kg^75) 25 2 (ml/min/kg^75) 2 VO VO 20

Night Night 15 20 0 150 300 450 600 750 900 1050 1200 1350 0 150 300 450 600 750 900 1050 1200 1350 time (min) time (min)

10000 ns ns MALES FEMALES 10000 fl/fl Trsp (n=7) fl/fl 5000 Trsp (n=5) fl/fl +/− AUC 250 fl/fl +/− 5000 220 Trsp -Ucp1-Cre (n=7) Trsp -Ucp1-Cre (n=5) AUC

0 fl − 0 l / / l f + /f / − p e fl + s r p e r -C s r 200 T 1 r -C p T 1 c p c fl -U l/ fl -U f l/ p f s p r s T 200 Tr 180 EE (kcal/day/kg) EE 160 EE (kcal/day/kg) 150

140 Night Night

0 150 300 450 600 750 900 1050 1200 1350 0 150 300 450 600 750 900 1050 1200 1350 time (min) time (min)

(e) (f)

MALES FEMALES Trspf/f 0.6 Trspf/f-Ucp1-Cre+/−

pAkt- t

Ser473 Ak 0.4 Akt

(a.u.) 2WA P = 0.36 β 0.2 sex -actin Pgenotype = 0.009** pAkt-Ser473/ Pinteraction = 0.02* 0.0 /− /f /− /f f + f + Males Females p e p e s r s r r r C 0.25 T -C T - 1 1 p p 0.20 c c U f U 0.15 /f - / - f f -actin β 2WA p p (a. u.) 0.10 rs rs P > 0.99

Akt/ sex T T 0.05 Pgenotype = 0.01* Pinteraction = 0.75 0.00 Males Females

(g)

f/f +/− Figure 2. MetabolicFigure overview 2. Metabolic of Trsp overviewf/f-Ucp1-Cre of Trsp+/− mice.-Ucp1-Cre Body weightmice. assessment Body weight of male assessment (a) and offemale male ( (ba)) Trsp andf/f-Ucp1- f/f +/− Cre+/− mice overfemale 8 weeks (b) afterTrsp weaning.-Ucp1-Cre (c,d)mice VO2 overconsumption 8 weeksafter in male weaning. and female (c,d) VO in 2theconsumption Trspf/f-Ucp1-Cre in male+/− mice, and respec- f/f +/− tively. (e,f) Energyfemale expenditure in the Trsp calculations-Ucp1-Cre ofmice, male respectively.and female mice, (e,f) Energy respectively, expenditure considering calculations VCO2 of as male well and (VCO2 not shown). (g) Phosphorylatedfemale mice, respectively, Akt at the consideringSer473 residue VCO (pAkt-Ser2 as well (VCO473) 2andnot Akt shown). levels (g )in Phosphorylated the gonadal WAT Akt atof the male and female mice. ValuesSer473 are residue means (pAkt-Ser473) ± SD. p-values and were Akt calculated levels in the in gonadal (a,b,g) after WAT two-way of male and ANOVA female (2WA) mice. Valuesfollowed are by Bon- ferroni’s post-test,means while± SD. forp (-valuesc–f), p-values were calculated for the area in ( aunder,b,g) after the curve two-way (AUC) ANOVA were (2WA) calculated followed using by Student’s Bonferroni’s t-test. The sample size is displayedpost-test, while in graphs, for (c –exceptf), p-values in (g), for where the area n = under6/sex. the* p < curve 0.05, (AUC)** p < 0.01, were and calculated ns, non-significant. using Student’s Black bars are Trspf/f mice, and white bars are Trspf/f-Ucp1-Cre+/− mice. t-test. The sample size is displayed in graphs, except in (g), where n = 6/sex. * p < 0.05, ** p < 0.01, and ns, non-significant. Black bars are Trspf/f mice, and white bars are Trspf/f-Ucp1-Cre+/− mice.

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Despite unaltered energy expenditure, observation of brown adipocytes from the male Trspf/f-Ucp1-Cre+/− mice revealed increased lipid deposition compared to either control orDespite female unaltered Trspf/f-Ucp1-Cre energy+/ expenditure,− mice as indicated observation by hemato of brownxylin-stained adipocytes histology from the male(Fig- f/f +/− Trspure 3a)-Ucp1-Cre and measurementmice revealed of triglyceride increased cont lipident deposition in BAT (Figure compared 3b). BAT to either adaptive control ther- or f/f +/− femalemogenesisTrsp is-Ucp1-Cre conducted bymice the actions as indicated of Ucp1 by, therefore hematoxylin-stained we examined histology its gene (Figureexpression3a) and(Figure measurement 3c) and protein of triglyceride levels using content both in immunohistochemistry BAT (Figure3b). BAT adaptive (Figurethermogenesis 3d) and Western is conductedblotting (Figure by the 3e). actions In all of analyses, Ucp1, therefore Ucp1 expression we examined and its levels gene were expression unchanged (Figure in3c) Trsp andf/f- proteinUcp1-Cre levels+/− mice. using both immunohistochemistry (Figure3d) and Western blotting (Figure3e). In all analyses, Ucp1 expression and levels were unchanged in Trspf/f-Ucp1-Cre+/− mice.

MALE FEMALE

f/f Trsp -Ucp1-Cre+/−

f/f Trsp

(a) (b)

ns 1.0 ns ns 1.5 50 0.8 40 -Ucp1 30 1.0 0.6

-actin (a.u.) -actin 20 β β 0.4 - -actin 10

Ucp1/18s 0.5 Ucp1/ Ucp1 OD /cell

(fold change) 0 0.2 f/f − +/ f/f p Trsp -Ucp1- f/f rs re 0.0 Trsp T -C +/− 1 f/f − 0.0 Cre cp +/ U f/f − fl - re +/ fl/ Trsp C p rsp rs T -Cre T 1

f -Ucp1- l Ucp f/ /f - fl rsp sp T Tr (e)

f/f +/− FigureFigure 3.3.BAT BAT morphology morphology and and Ucp1 Ucp1 expression expression in inTrsp Trsp-Ucp1-Cref/f-Ucp1-Cre+/− mice. ( a) Representative images ofof BATBAT histologyhistology − afterafter hematoxylinhematoxylin staining, nn == 11. 11. (b ()b BAT) BAT triglyceride triglyceride content content of Trsp of Trspf/f-Ucp1-Cref/f-Ucp1-Cre+/−- mice+/ mice(n = 7 ( nper= 7sex). per (c sex).) Ucp1 (c )mRNAUcp1 mRNAexpression expression in male inTrsp malef/f-Ucp1-CreTrspf/f-Ucp1-Cre+/− mice. (+/d−) Ucp1mice. levels (d) Ucp1as assessed levels by as optical assessed densitometry by optical densitometryafter Ucp1 immunohisto- after Ucp1 immunohistochemistrychemistry of male samples of malein (a). samples (e) Western in ( ablot). ( e(n) = Western 11) image blot of( Ucp1n = 11) levels image and of gr Ucp1aph with levels band and densitometry graph with calcu- band densitometrylation. Values calculation.are mean ± SD, Values and are p-values mean were± SD, calculated and p-values using were unpaired calculated Student’s using t-test. unpaired ns, non-significant; Student’s t-test. * pns<0.05., non- signiﬁcant; * p < 0.05. 2.3. Mitochondrial Content in BAT of Trspf/f-Ucp1-Cre+/− Mice 2.3. MitochondrialWe next investigated Content in whether BAT of Trspunchangedf/f-Ucp1-Cre Ucp1+/− reflectedMice similar mitochondrial con- f/f +/− tent Wein BAT next investigatedof Trsp -Ucp1-Cre whether mice. unchanged We employed Ucp1 reﬂected levels similar of peroxisome mitochondrial proliferator- content inactivated BAT of Trspreceptorf/f-Ucp1-Cre gamma-coactivator+/− mice. We employed 1-alpha (PGC-1 levels ofα peroxisome) and ATP synthase proliferator-activated subunit beta receptor(ATPB) as gamma-coactivator proxies for mitochondrial 1-alpha (PGC-1 contentα) and and function. ATP synthase PGC-1 subunitα is a betatranscriptional (ATPB) as proxiescoactivator for mitochondrial and central inducer content of and mitochondria function. PGC-1l biogenesisα is a transcriptional [34], while ATPB coactivator is a member and centralof the complex inducer ofV mitochondrialof the electron biogenesis transport ch [34ain], while in the ATPB inner is membrane a member ofof thethe complexmitochon- V ofdria the and electron commonly transport used chain as a inmitochondria the inner membranel marker. ofInterestingly, the mitochondria BAT of and female commonly Trspf/f- used as a mitochondrial marker. Interestingly, BAT of female Trspf/f-Ucp1-Cre+/− mice

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presentedUcp1-Cre+/ increased− mice presented levels of increased both PGC-1 levelsα (Figure of both4 a)PGC-1 andα ATPB (Figure (Figure 4a) and4b), ATPB while male(Figure f/f +/− Trsp4b), while-Ucp1-Cre male Trspmicef/f-Ucp1-Cre kept similar+/− mice expression kept similar levels expression of both markers. levels of both markers.

MALE FEMALE MALE FEMALE -PGC-1α -PGC-1α -ATPB β -ATPB - -actin - β-actin - β-actin - β-actin f/f f/f Trsp Trsp - f/f Trspf/f- +/− Trsp Ucp1-Cre +/− Trspf/f Trspf/f- Trspf/f Trspf/f- Ucp1-Cre Ucp1-Cre+/− Ucp1-Cre+/− ns 5 ns ns 5 ** 3 2.5 ** 4 4 2.0 2 3 3 1.5 -actin (a.u.) -actin -actin (a.u.) -actin -actin (a.u.) -actin (a.u.) -actin β β 2 / β

β 2 / α 1 1.0 α 1 1 0.5 ATPB/ ATPB/ PGC-1 0 0 0 PGC-1 /f − 0.0 l − l fl +/ /f / /f − /f − fl + fl +/ p e fl / s r + p e re Tr -C p e s r 1 s r r -C -C p Tr -C T 1 Tr s p c 1 p l U p c /f - c fl U fl -U cp1 l U fl - / p /f - l/ fl s fl f Tr p p sp s s Tr Tr Tr

(a) (b)

f/f +/− FigureFigure 4. 4.BAT BAT mitochondrial mitochondrial markers markers in inTrsp Trsp-Ucp1-Cref/f-Ucp1-Cre+/−mice. mice. LevelsLevels ofof mitochondrialmitochondrial markers markers ( a(a)) PGC-1 PGC-1ααand and ( b(b)) ATPB ATPB − inin BAT BAT of of male male and and female femaleTrsp Trspf/f-Ucp1-Cref/f-Ucp1-Cre+/ +/−-mice mice were were assessed assessed by by Western Western blot blot (n (n= = 8 8 per per sex). sex). Values Values are are mean mean± ±SD SD andandp -valuesp-values were were calculated calculated using using unpaired unpaired Student’s Student’st-test. t-test.ns ns, non-signiﬁcant,, non-significant, ** **p

2.4. Changes in Gene Expression and Pathways in BAT of Trspf/f-Ucp1-Cre+/− Mice at Room 2.4. Changes in Gene Expression and Pathways in BAT of Trspf/f-Ucp1-Cre+/− Mice at Temperature Room Temperature Maintenance of Ucp1 expression in Trspf/f-Ucp1-Cre+/− mice led us to seek genes and Maintenance of Ucp1 expression in Trspf/f-Ucp1-Cre+/− mice led us to seek genes and f/f +/−- pathwayspathways that that were were either either up- up- or or down-regulated down-regulated in in BAT BAT of of male maleTrsp Trspf/f-Ucp1-Cre-Ucp1-Cre+/− mice mice atat room room temperature. temperature. We We performed performed microarray microarray analysis analysis and foundand found 183 genes 183 genes with atwith least at aleast 2-fold a 2-fold of change of change in expression in expression between between the two the genotypes two genotypes (Supplementary (Supplementary Table Table S1). TableS1). 1Table shows 1 shows the top the 10 differentiallytop 10 differentially expressed expressed genes in genes these mice.in these At roommice. temperature,At room tem- noperature, genes for no selenoproteins genes for selenoprotei were affectedns were by affected the loss by of Trspthe .loss of Trsp.

Table 1. Top differentially expressed genes in BAT of Trspf/f-Ucp1-Cre+/−- mice at room temperature. Table 1. Top differentially expressed genes in BAT of Trspf/f-Ucp1-Cre+/− mice at room temperature. Top Upregulated Top Upregulated Gene Symbol Gene Name Fold Change p-Value Gene Symbol Gene Name Fold Change p-Value Plekhm2 pleckstrin homology and RUN domain containing M2 4.59 0.0361 Sntb2 syntrophinpleckstrin beta homology2 4.02 0.0008 Plekhm2 and RUN domain 4.59 0.0361 Phf20 PHD finger containingprotein 20 M2 3.78 0.0271 Vps4a vacuolarSntb2 protein sortingsyntrophin 4 homolog beta 2 A 4.02 3.33 0.0008 0.0296 Fads3 Phf20 fatty acidPHD desaturase ﬁnger protein 3 20 3.78 3.3 0.0271 0.0119 vacuolar protein S100pbp Vps4a S100P binding protein 3.33 3.26 0.0296 0.0005 sorting 4 homolog A Lcp1 lymphocyte cytosolic protein 1 3.17 0.0453 fatty acid desaturase Fads3 3.3 0.0119 Fxyd5 FXYD domain containing ion transport3 regulator 5 3.07 0.0334 Elmod2 S100pbpELMO domainS100P containing binding protein 2 3.26 2.79 0.0005 0.003 lymphocyte cytosolic Kpna2 Lcp1karyopherin subunit alpha 2 3.17 2.72 0.0453 0.0061 protein 1 Top Downregulated FXYD domain Gene Symbol Fxyd5 Gene Namecontaining ion 3.07 Fold Change 0.0334 p-Value Tmsb4x thymosin, betatransport 4, X chromosome regulator 5 −4.24 0.0172 ELMO domain Trappc4 traffickingElmod2 protein particle complex 4 2.79−3.63 0.003 0.0183 Hscb HscB mitochondrial iron-sulfurcontaining cluster 2 cochaperone −3.12 0.0196 karyopherin subunit Kpna2 2.72 0.0061 Ccdc85a coiled-coil domain containingalpha 2 85A −2.98 0.003 Skil SKI-like proto-oncogene −2.87 0.0297 Sorl1 sortilin related receptor 1 −2.8 0.0299 Gk glycerol kinase −2.66 0.0094

Int. J. Mol. Sci. 2021, 22, 611 7 of 20

Table 1. Cont.

Top Downregulated Gene Symbol Gene Name Fold Change p-Value thymosin, beta 4, X Tmsb4x −4.24 0.0172 chromosome trafﬁcking protein Trappc4 −3.63 0.0183 particle complex 4 HscB mitochondrial Hscb iron-sulfur cluster −3.12 0.0196 cochaperone coiled-coil domain Ccdc85a −2.98 0.003 containing 85A SKI-like Skil −2.87 0.0297 proto-oncogene sortilin related Sorl1 −2.8 0.0299 receptor 1 Gk glycerol kinase −2.66 0.0094 Nbea neurobeachin −2.46 0.0228 thioredoxin related Tmx4 transmembrane −2.43 0.0234 protein 4 period circadian Per2 −2.42 0.0481 regulator 2

Pathway analysis revealed that, at room temperature, nicotine degradation, antigen presentation, glycerol degradation, LPS/IL-1 mediated inhibition of RXR function, and myo-inositol biosynthesis were the pathways most affected in BAT of Trspf/f-Ucp1-Cre+/− mice (Table2 and Supplementary Table S1 for the full list).

Table 2. Top 5 pathways affected in BAT of Trspf/f-Ucp1-Cre+/− mice at room temperature.

Canonical Pathways −log(p-Value) Molecules Nicotine Degradation II 1.85 FMO2,FMO5 Antigen Presentation Pathway 1.78 HLA-DMB,MR1 Glycerol Degradation I 1.59 Gk LPS/IL-1 Mediated Inhibition 1.58 GSTA3,FMO2,SCARB1,FMO5 of RXR Function Myo-inositol Biosynthesis 1.47 IMPAD1

2.5. Body Temperature in the Trspf/f-Ucp1-Cre+/− Mice after Acute Cold Exposure The phenotype observed in the Trspf/f-Ucp1-Cre+/− mice at room temperature led us to consider that the effects of Trsp loss in brown adipocytes might be revealed to a greater extent after activation of adaptive thermogenesis. Hence, we acutely exposed Trspf/f-Ucp1- Cre+/− and Trspf/f mice to 4 ◦C for 4 h. Both male and female Trspf/f-Ucp1-Cre+/− mice reduced their core body temperature at the same rate as corresponding controls (Figure5). An interaction effect of genotype and time was observed in males (Figure5a), but not in females (Figure5b). Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 7 of 20

Nbea neurobeachin −2.46 0.0228 Tmx4 thioredoxin related transmembrane protein 4 −2.43 0.0234 Per2 period circadian regulator 2 −2.42 0.0481

Table 2. Top 5 pathways affected in BAT of Trspf/f-Ucp1-Cre+/−- mice at room temperature.

Canonical Pathways −log(p-Value) Molecules Nicotine Degradation II 1.85 FMO2,FMO5 Antigen Presentation Pathway 1.78 HLA-DMB,MR1 Glycerol Degradation I 1.59 Gk LPS/IL-1 Mediated Inhibition of RXR Function 1.58 GSTA3,FMO2,SCARB1,FMO5 Myo-inositol Biosynthesis 1.47 IMPAD1

2.5. Body Temperature in the Trspf/f-Ucp1-Cre+/− Mice after Acute Cold Exposure The phenotype observed in the Trspf/f-Ucp1-Cre+/− mice at room temperature led us to consider that the effects of Trsp loss in brown adipocytes might be revealed to a greater extent after activation of adaptive thermogenesis. Hence, we acutely exposed Trspf/f-Ucp1- Cre+/− and Trspf/f mice to 4 °C for 4 h. Both male and female Trspf/f-Ucp1-Cre+/−- mice reduced their core body temperature at the same rate as corresponding controls (Figure 5). An in- Int. J. Mol. Sci. 2021, 22, 611 teraction effect of genotype and time was observed in males (Figure 5a), but not in females8 of 20 (Figure 5b).

MALES FEMALES

38 Trspfl/fl (n=4) 38 Trspfl/fl-Ucp1-Cre+/- (n=4)

37 37

36 36 Trspfl/fl (n=4) fl/fl +/- 2WA Trsp -Ucp1-Cre (n=4) 2WA Body temperature (ºC) 35 Body temperature (ºC) temperature Body 35 P = 0.1039 Pinteraction = 0.0012 interaction P = 0.7592 Pgenotype = 0.853 genotype P < 0.0001 Ptime < 0.0001 time 34 34 0 60 120 180 240 0 60 120 180 240 time (min) time (min)

(a) (b)

Figure 5. Trspf/f Trspf/f-Ucp1-Cre+/− a b Figure 5.Core Core body body temperature temperature in in Trspf/fand and Trspf/f-Ucp1-Cre+/−- male (a) and female (b) mice duringduring acuteacute coldcold exposure.exposure. DataData are are mean mean± ± SD;SD; two-waytwo-way ANOVAANOVA (2WA)(2WA) withwithBonferroni’s Bonferroni’spost-test post-testwas wasperformed, performed,and andp p-values-values are are displayed displayed in in graphs,graphs, as as well well as as sample sample sizes. sizes.

2.6. Changes in Gene Expression and Pathways in BAT of Trspf/f-Ucp1-Cre+/− Mice after Acute 2.6. Changes in Gene Expression and Pathways in BAT of Trspf/f-Ucp1-Cre+/− Mice after Acute ColdCold ExposureExposure SlightSlight differencesdifferences in in core core body body temperature temperature during during acute acute cold exposurecold exposure in male inTrsp malef/f- Ucp1-CreTrspf/f-Ucp1-Cre+/− mice+/−- ledmice us led to investigateus to investigate whether whether male Trsp malef/f and TrspTrspf/f andf/f-Ucp1-Cre Trspf/f-Ucp1-Cre+/− mice+/− showedmice showed similar similar changes changes in gene in expressiongene expression in BAT in asBAT when as when the mice the weremice atwere room at room temperature.temperature. Interestingly, Interestingly, microarray microarray analyses analyses of BATof BAT from fromTrsp Trspf/f andf/f andTrsp Trspf/f-Ucp1-Cref/f-Ucp1-Cre+/−+/− micemice exposedexposed toto coldcold revealedrevealed onlyonly 6565 differentiallydifferentially expressedexpressed genes genes comparedcompared to to the the 183 183 differentiallydifferentially expressedexpressed genesgenes observedobserved atat roomroom temperaturetemperature (Supplementary(Supplementary TableTable S2),S2), i.e., one-third of the number of gene expression changes. Table3 shows the top up- and down-regulated genes in the BAT of Trspf/f-Ucp1-Cre+/− mice after cold exposure.

Table 3. Top differentially expressed genes in BAT of Trspf/f-Ucp1-Cre+/− mice acutely exposed to cold.

Top Upregulated Gene Symbol Gene Name Fold Change p-Value Nnmt nicotinamide N-methyltransferase 24.07 0.0012 Elmod2 ELMO domain containing 2 4.1 0.0018 Gsta3 glutathione S-transferase alpha 3 4.05 0.0038 Laptm5 lysosomal protein transmembrane 5 3.15 0.0029 Lyz lysozyme 3.15 0.0087 eosinophil-associated, ribonuclease A family, Ear2 2.74 0.0013 member 2 methylenetetrahydrofolate dehydrogenase Mthfd2 2.69 0.0126 (NADP+ dependent) 2 Rab3a RAB3A, member RAS oncogene family 2.6 0.0131 Sorl1 sortilin related receptor 1 2.44 0.0467 Ctss cathepsin S 2.41 0.0376 Top Downregulated Gene Symbol Gene Name Fold Change p-Value Mbp myelin basic protein −3.17 0.0408 Gucd1 guanylyl cyclase domain containing 1 −2.86 0.0375 Rnf149 ring ﬁnger protein 149 −2.56 0.0272 Rnf125 ring ﬁnger protein 125 −2.54 0.0276 Thrsp thyroid hormone responsive (Spot14) −2.47 0.0044 Tshr thyroid stimulating hormone receptor −2.46 0.0266 Insig1 insulin induced gene 1 −2.43 0.0413 Art3 ADP-ribosyltransferase 3 −2.39 0.0255 Il18 interleukin 18 −2.36 0.0035 Akap12 A-kinase anchoring protein 12 −2.35 0.0085 Int. J. Mol. Sci. 2021, 22, 611 9 of 20

Pathway analysis revealed that the autophagy pathway was the most affected pathway of those examined by cold exposure in the Trspf/f-Ucp1-Cre+/− mice compared to their controls, followed by histidine degradation III (Table4 and Supplementary Table S2 for the full list). Nevertheless, levels of scaffold protein p62 (sequestosome 1; p62-SQSTM1), an autophagy marker implicated in the control of BAT thermogenesis [35], remained unchanged in the Trspf/f-Ucp1-Cre+/− mice, as revealed by Western blot analysis (Figure6a). Quantitation of the Western blot is shown in Figure6b.

Table 4. Top 5 pathways affected in BAT of Trspf/f-Ucp1-Cre+/− mice after acute cold exposure.

Canonical Pathways −log(p-Value) Molecules autophagy 2.13 WDFY3,CTSS Histidine Degradation III 1.94 MTHFD2 Tetrahydrofolate Salvage from 1.81 MTHFD2 5,10-methenyltetrahydrofolate Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEWFolate Transformations I 1.72 MTHFD2 9 of 20 LXR/RXR Activation 1.58 IL18,LYZ

0.25 ns

- p62-SQSTM1 -actin 0.20 β 0.15

- β-actin (a.u.) 0.10 0.05

p62-SQSTM1/ 0.00 f/f f/f /f − Trsp Trsp -Ucp1- fl +/ p +/− s re Tr -C Cre 1 p c U /fl - fl p s Tr (a) (b)

FigureFigure 6. 6.Autophagy Autophagy in in the the cold-exposed cold-exposed male maleTrsp Trspf/f-Ucp1-Cref/f-Ucp1-Cre+/−+/−mice.- mice. Levels Levels of of autophagy autophagy marker f/f +/− p62-SQSTM1marker p62-SQSTM1 in the BAT in of the cold-exposed BAT of cold-exposed male Trspf/f male-Ucp1-Cre Trsp +/-Ucp1-Cre− mice were mice obtained were byobtained (a) Western by (a) Western blot; (b) graph displays quantification analysis. Values are expressed as mean + SD, n = blot; (b) graph displays quantiﬁcation analysis. Values are expressed as mean + SD, n = 11. Student’s 11. Student’s unpaired t-test was performed; ns, non-significant. unpaired t-test was performed; ns, non-signiﬁcant.

ToTo pinpoint pinpoint BAT BAT genes genes that that were were most most heavily heavily affected affected by by the the loss loss of ofTrsp Trspin in brown brown adipocytes,adipocytes, we we combined combined and and compared compared the the microarray microarray results results from from mice mice maintained maintained at at roomroom temperature temperature and and mice mice acutely acutely exposed exposed to to cold. cold. Figure Figure7 a7a shows shows a Venna Venn diagram diagram builtbuilt with with the the differentially differentially expressed expressed genes genes obtained obtained from from the the microarray microarray analyses. analyses. Our Our resultsresults showed showed two two genes genes at at the the main main intersection intersection of theof the diagram, diagram, glutathione glutathione S-transferase S-transfer- alpha-3ase alpha-3 (Gsta3 (Gsta3) and) and ELMO ELMO domain domain containing containing 2 (Elmod2 2 (Elmod2). Gsta3). Gsta3is is a genea gene regulated regulated by by thethe thyroid thyroid hormonehormone inin the liver liver [36] [36] that that catalyzes catalyzes the the conjugation conjugation of ofglutathione glutathione to sev- to severaleral electrophiles, electrophiles, and and it itwas was upregulated upregulated in in the the TrspTrspf/f-Ucp1-Cref/f-Ucp1-Cre+/−+/- mice,− mice, regardless regardless of ofen- environmentalvironmental temperature temperature (Figure (Figure 7b).7b). On On the the other other hand, hand, Elmod2Elmod2, a GTPase, a GTPase activating activating pro- proteintein which which regulates regulates adipocyte adipocyte triglyceride triglyceride lipase lipase recruitment recruitment [37], [37 was], was found found to be to besig- signiﬁcantlynificantly upregulated upregulated after after cold cold exposure exposure in both genotypes.genotypes. However,However, the the upregulation upregulation occurredoccurred with with a a further further ~40% ~40% increase increase in in BAT BAT in inTrsp Trspf/f-Ucp1-Cref/f-Ucp1-Cre+/−+/−mice mice (Figure (Figure7c). 7c). Loss Loss ofofTrsp Trspalso also affected affected the the transcription transcription of Trappc4of Trappc4(trafﬁcking (trafficking protein protein particle particle complex complex 4), an 4), interactoran interactor of ERK2 of ERK2 implicated implicated in vesicle in vesicle transport transport in colorectal in colorectal carcinoma carcinoma cells, whichcells, which was diminishedwas diminished in Trsp inf/f -Ucp1-CreTrspf/f-Ucp1-Cre+/− mice+/− mice (Figure (Figure7d) [ 387d)]. [38].

** * 20 Trspf/f f/f +/− 15 Trsp -Ucp1-Cre

10 2WA Ptemperature = 0.63 Gsta3/18s Gsta3/18s 5 Pgenotype < 0.0001*** (fold change) P = 0.59 0 interaction d RT l Co (b)

(a)

** 1.5 Trspf/f f/f +/− 5 * Trspf/f Trsp -Ucp1-Cre − 4 Trspf/f-Ucp1-Cre+/ 1.0 2WA 3 2WA Ptemperature = 0.72 2 0.5 Ptemperature = 0.0005*** P = 0.016* Trappc4/18s Trappc4/18s

(fold change) genotype

Elmod2/18s P = 0.014* (fold change) 1 genotype P = 0.38 P = 0.09 0.0 interaction 0 interaction T ld T d R o R C Col (c) (d) Figure 7. Gene expression changes in Trspf/f-Ucp1-Cre+/−- mice at room temperature and after acute cold exposure. (a) Venn diagram of microarray data sets, showing the number of genes differentially expressed between Trspf/f-Ucp1-Cre+/− and

Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 9 of 20

0.25 ns

- p62-SQSTM1 -actin 0.20 β 0.15

- β-actin (a.u.) 0.10 0.05

p62-SQSTM1/ 0.00 f/f f/f /f − Trsp Trsp -Ucp1- fl +/ p +/− s re Tr -C Cre 1 p c U /fl - fl p s Tr (a) (b) Figure 6. Autophagy in the cold-exposed male Trspf/f-Ucp1-Cre+/−- mice. Levels of autophagy marker p62-SQSTM1 in the BAT of cold-exposed male Trspf/f-Ucp1-Cre+/− mice were obtained by (a) Western blot; (b) graph displays quantification analysis. Values are expressed as mean + SD, n = 11. Student’s unpaired t-test was performed; ns, non-significant.

To pinpoint BAT genes that were most heavily affected by the loss of Trsp in brown adipocytes, we combined and compared the microarray results from mice maintained at room temperature and mice acutely exposed to cold. Figure 7a shows a Venn diagram built with the differentially expressed genes obtained from the microarray analyses. Our results showed two genes at the main intersection of the diagram, glutathione S-transferase alpha-3 (Gsta3) and ELMO domain containing 2 (Elmod2). Gsta3 is a gene regulated by the thyroid hormone in the liver [36] that catalyzes the conjugation of glutathione to several electrophiles, and it was upregulated in the Trspf/f-Ucp1-Cre+/−- mice, regardless of en- vironmental temperature (Figure 7b). On the other hand, Elmod2, a GTPase activating protein which regulates adipocyte triglyceride lipase recruitment [37], was found to be sig- nificantly upregulated after cold exposure in both genotypes. However, the upregulation occurred with a further ~40% increase in BAT in Trspf/f-Ucp1-Cre+/− mice (Figure 7c). Loss Int. J. Mol. Sci. 2021, 22, 611 of Trsp also affected the transcription of Trappc4 (trafficking protein particle complex10 of 20 4), an interactor of ERK2 implicated in vesicle transport in colorectal carcinoma cells, which was diminished in Trspf/f-Ucp1-Cre+/− mice (Figure 7d) [38].

** * 20 Trspf/f f/f +/− 15 Trsp -Ucp1-Cre

10 2WA Ptemperature = 0.63 Gsta3/18s Gsta3/18s 5 Pgenotype < 0.0001*** (fold change) P = 0.59 0 interaction d RT l Co (b)

(a)

** 1.5 Trspf/f f/f +/− 5 * Trspf/f Trsp -Ucp1-Cre − 4 Trspf/f-Ucp1-Cre+/ 1.0 2WA 3 2WA Ptemperature = 0.72 2 0.5 Ptemperature = 0.0005*** P = 0.016* Trappc4/18s Trappc4/18s

(fold change) genotype

Elmod2/18s P = 0.014* (fold change) 1 genotype P = 0.38 P = 0.09 0.0 interaction 0 interaction T ld T d R o R C Col (c) (d)

FigureFigure 7. Gene 7. Gene expression expression changes changes in in TrspTrspf/ff/f-Ucp1-Cre-Ucp1-Cre+/−−- micemice at at room room temperature andand after after acute acute cold cold exposure. exposure. (a) ( Venna) Venn diagramdiagram of ofmicroarray microarray data data sets, sets, showing showing the numbernumber ofof genes genes differentially differentially expressed expressed between betweenTrsp Trspf/f-Ucp1-Cref/f-Ucp1-Cre+/− and+/− and Trspf/f mice at room temperature and after cold exposure. (b–d) Validation of microarray results by qPCR analysis of the expression of Gsta3, Elmod2 and Trappc4, respectively. Data are mean ± SD. p-values are shown in graphs and were p p p calculated by two-way ANOVA (2WA) followed by Bonferroni’s post test analysis. * < 0.05,** < 0.01, and *** < 0.001. RT, room temperature.

2.7. Thyroid Function in the Trspf/f-Ucp1-Cre+/− Mice Activation of adaptive thermogenesis is critically dependent on thyroid hormone availability. We assessed thyroid function in Trspf/f-Ucp1-Cre+/− mice at room temperature and after acute cold exposure. We found male mice to have unchanged circulating total T4 levels but diminished total T3 levels. Cold exposure reduced total T4 levels but increased total T3 levels in female mice of both genotypes. Interestingly, male Trspf/f-Ucp1-Cre+/− mice at room temperature had modest elevation of circulating thyroid-stimulating hormone (TSH), a sign of mild hypothyroidism, in comparison to their Trspf/f counterparts. Upon acute cold exposure, male mice of both genotypes reduced circulating TSH levels, however a greater reduction was observed in the Trspf/f-Ucp1-Cre+/− mice (Table5). In BAT, thyroid hormone activation is catalyzed by selenoprotein Dio2, and upregulation of Dio2 is considered a classic marker of BAT activation. Interestingly, we observed that in BAT of Trspf/f-Ucp1-Cre+/− mice, which had histological changes suggestive of physiological impairment, Dio2 expression was higher after cold exposure. However, this increase occurred at the same levels in both genotypes (Figure8a). Using our microarray dataset, we next validated by qPCR other thyroid hormone- regulated genes that were differentially expressed in BAT of Trspf/f-Ucp1-Cre+/− mice. Thrsp (thyroid hormone-inducible hepatic protein, also known as Spot14), a classic T3-responsive gene that regulates lipogenesis [39,40] and acts as a transcriptional coactivator with thyroid hormone receptor beta [41], was downregulated in Trspf/f-Ucp1-Cre+/− mice according to both temperature and genotype (Figure8b). Moreover, the TSH receptor gene, Tshr, also presented a non-signiﬁcant trend (p = 0.057) towards downregulation in the Trspf/f-Ucp1- Int. J. Mol. Sci. 2021, 22, 611 11 of 20

Cre+/− mice. However, when acutely exposed to cold, both genotypes showed the same expression levels (Figure8c).

Table 5. Thyroid function of the Trspf/f-Ucp1-Cre+/- mice.

Room Temperature Cold Exposure Two-Way ANOVA f/f f/f +/− f/f f/f +/− Trsp Trsp -Ucp1-Cre Trsp Trsp -Ucp1-Cre Pg Pt Pi TSH (pg/mL) Males 1895.2 ± 86.0 2036 ± 46.05 a 1312 ± 112.9 1161 ± 211.7 b 0.861 <0.0001 0.031 Females 710.3 ± 75.4 541.1 ± 282.8 363.7 ± 107.2 503.8 ± 149.4 0.043 0.868 0.094 T4 (µg/dl) Males 3.768 ± 1.18 4.06 ± 1.01 3.447 ± 0.46 3.16 ± 0.61 0.994 0.154 0.487 Females 3.661 ± 0.65 3.421 ± 0.42 3.082 ± 0.58 3.009 ± 0.19 0.488 0.038 0.712 T3 (ng/mL) Males 1.02 ±0.19 0.79 ± 0.34 1.08 ± 0.24 0.87 ± 0.21 0.01 0.42 0.9 Females 0.62 ± 0.24 0.58 ± 0.1 0.86 ± 0.22 0.75 ± 0.04 0.36 0.01 0.62 Results are expressed as mean ± SD, n = 9–10 per genotype. Bold indicates signiﬁcant p-values calculated by two-way ANOVA. a p < 0.05 Trspf/f Trspf/f-Ucp1-Cre+/− b p Trspf/f-Ucp1-Cre+/− Int. J. Mol. Sci.vs. 2021, 22, x FOR PEERmice REVIEW at room temperature; and < 0.05 room temperature vs. cold exposure for mice, after11 of 20 Bonferroni’s post-test. Pg, p-value for the genotype factor; Pt, p-value for the temperature factor; Pi, p-value for interaction of both factors; TSH, thyroid-stimulating hormone.

*** *** 1.5 Trspf/f *** Trspf/f-Ucp1-Cre+/− 25 Trspf/f 1.5 *** Trspf/f − 1.0 20 Trspf/f-Ucp1-Cre+/ f/f +/− Trsp -Ucp1-Cre 2WA 1.0 ** 15 Ptemperature = 0.059

Tshr/18s 0.5 2WA 2WA P = 0.34 10 change) (fold genotype Ptemperature < 0.0001*** Ptemperature = 0.0026** Dio2/18s Dio2/18s 0.5

Thrsp/18s Thrsp/18s P = 0.031* P = 0.1176 P < 0.00001*** interaction (fold change) 5 genotype (fold change) genotype 0.0 P = 0.117 P = 0.004** 0 interaction 0.0 interaction RT Cold T ld RT R o Cold C (a) (b) (c)

f/f f/f +/− Figure 8. Gene expression of thyroid hormonehormone responsiveresponsive transcripts transcripts in in male maleTrsp Trspf/fand andTrsp Trspf/f-Ucp1-Cre-Ucp1-Cre+/− mice. ExpressionExpression levels of ( a) Dio2,,( (b) Thrsp (i.e., Spot14),), and and ( (c)) Tshr were assessed by qPCR. RT, room temperature. Data are mean + SD; n = 8–10 per genotype. p-values-values are are displayed in graphs and calculated by two-way ANOVA followedfollowed byby Bonferroni’sBonferroni's post-test. * pp << 0.05, 0.05, ** ** pp << 0.01 0.01 and and *** *** pp << 0.001. 0.001.

2.8. Methylation Genes in the Trspf/f-Ucp1-Cre+/− Mice 2.8. Methylation Genes in the Trspf/f-Ucp1-Cre+/− Mice Surprisingly, we found that two genes involved in methylation in cancer cells were Surprisingly, we found that two genes involved in methylation in cancer cells were altered in our BAT microarray analysis and were selected for validation by qPCR. Mthfd2, altered in our BAT microarray analysis and were selected for validation by qPCR. Mthfd2, which encodes for methylenetetrahydrofolate dehydrogenase (NADP+ dependent) 2, par- which encodes for methylenetetrahydrofolate dehydrogenase (NADP+ dependent) 2, par- ticipates in one-carbon metabolism. This enzyme also controls RNA methylation, partic- ticipates in one-carbon metabolism. This enzyme also controls RNA methylation, particu- ularly N6-methyladenosine (m6a) in mRNAs, in renal cell carcinoma [42]. Mthfd2 expres- larly N6-methyladenosine (m6a) in mRNAs, in renal cell carcinoma [42]. Mthfd2 expression sion was upregulated in the Trspf/f-Ucp1-Cre+/- mice, and further enhanced after acute cold was upregulated in the Trspf/f-Ucp1-Cre+/- mice, and further enhanced after acute cold exposure (Figure 9a). On the other hand, Nnmt, which encodes nicotinamide N-methyl exposure (Figure9a). On the other hand, Nnmt, which encodes nicotinamide N-methyl transferase, is an enzyme that inhibits autophagy in breast cancer cells [43]. It showed a transferase, is an enzyme that inhibits autophagy in breast cancer cells [43]. It showed dramatic ~24-fold upregulation in BAT of Trspf/f-Ucp1-Cre+/− mice after cold exposure com- a dramatic ~24-fold upregulation in BAT of Trspf/f-Ucp1-Cre+/− mice after cold exposure paredcompared to corresponding to corresponding controls controls (Supplementary (Supplementary Table S2). Table Our S2). qPCR Our validation qPCR validation revealed anrevealed upregulation an upregulation dependent dependent solely on solely cold exposure, on cold exposure, without withouta genotype a genotype effect, in effect, BAT of in theseBAT ofmice these (Figure mice (Figure9b). 9b).

20 Trspf/f 6 * f/f Trsp Trspf/f-Ucp1-Cre+/− Trspf/f-Ucp1-Cre+/− 15 4 2WA 2WA 10 Ptemperature = 0.004** Ptemperature = 0.0028** 2 Nnmt/18s 5 P = 0.97 Mthfd2/18s Mthfd2/18s Pgenotype = 0.0072** (fold change) genotype (fold change) P = 0.58 Pinteraction = 0.94 0 interaction 0 ld T RT o R C Cold (a) (b) Figure 9. Gene expression of transcripts involved in methylation pathways in male Trspf/f and Trspf/f-Ucp1-Cre+/− mice. Expression of (a) Mthfd2 and (b) Nnmt were assessed by qPCR. RT, room temperature. Data are mean + SD; n = 8–10 per genotype. p-values are displayed in graphs and were calculated by two-way ANOVA (2WA) followed by Bonferroni's post test; * p < 0.05, ** p < 0.01, and *** p < 0.001.

3. Discussion We have reported the development of a novel transgenic mouse model, the Trspf/f- Ucp1-Cre+/− mice. Our model was characterized by targeted deletion of the gene Trsp in brown adipocytes, which are the primary cell type of BAT. The product of the Trsp gene, tRNA[Ser}Sec, is responsible for carrying out the synthesis of selenoproteins, including Dio2, a regulator of cold-induced adaptive thermogenesis in BAT. Dio2 upregulation is promoted by thyroid hormone and is considered a primary indicator of activation of adaptive thermogenesis in cold exposure [44]. In males, Dio2 expression was unchanged between Trspf/f and Trspf/f-Ucp1-Cre+/− mice, despite a reduction

Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 11 of 20

*** *** 1.5 Trspf/f *** Trspf/f-Ucp1-Cre+/− 25 Trspf/f 1.5 *** Trspf/f − 1.0 20 Trspf/f-Ucp1-Cre+/ f/f +/− Trsp -Ucp1-Cre 2WA 1.0 ** 15 Ptemperature = 0.059

Tshr/18s 0.5 2WA 2WA P = 0.34 10 change) (fold genotype Ptemperature < 0.0001*** Ptemperature = 0.0026** Dio2/18s Dio2/18s 0.5

Figure 8. Gene expression of thyroid hormone responsive transcripts in male Trspf/f and Trspf/f-Ucp1-Cre+/− mice. Expression levels of (a) Dio2, (b) Thrsp (i.e., Spot14), and (c) Tshr were assessed by qPCR. RT, room temperature. Data are mean + SD; n = 8–10 per genotype. p-values are displayed in graphs and calculated by two-way ANOVA followed by Bonferroni's post-test. *p < 0.05, ** p < 0.01 and *** p < 0.001.

2.8. Methylation Genes in the Trspf/f-Ucp1-Cre+/− Mice Surprisingly, we found that two genes involved in methylation in cancer cells were altered in our BAT microarray analysis and were selected for validation by qPCR. Mthfd2, which encodes for methylenetetrahydrofolate dehydrogenase (NADP+ dependent) 2, par- ticipates in one-carbon metabolism. This enzyme also controls RNA methylation, particularly N6-methyladenosine (m6a) in mRNAs, in renal cell carcinoma [42]. Mthfd2 expression was upregulated in the Trspf/f-Ucp1-Cre+/- mice, and further enhanced after acute cold exposure (Figure 9a). On the other hand, Nnmt, which encodes nicotinamide N-methyl transferase, is an enzyme that inhibits autophagy in breast cancer cells [43]. It showed a dramatic ~24-fold upregulation in BAT of Trspf/f-Ucp1-Cre+/− mice after cold exposure compared to corresponding controls (Supplementary Table S2). Our qPCR validation revealed Int. J. Mol. Sci.an2021 upregulation, 22, 611 dependent solely on cold exposure, without a genotype effect, in BAT of 12 of 20 these mice (Figure 9b).

FigureFigure 9. Gene 9. expressionGene expression of transcripts of transcripts involved involv in methylationed in methylation pathways pathways in male Trsp inf/f maleand Trsp Trspf/ff/f-Ucp1-Cre and +/− mice. ExpressionTrspf/f of-Ucp1-Cre (a) Mthfd2+/− andmice. (b Expression) Nnmt were of assessed (a) Mthfd2 by qPCR. and ( RT,b) Nnmt room temperature.were assessed Data by are qPCR. mean RT, + SD; roomn = 8–10 per genotype.temperature.p-values are Data displayed are mean in graphs + SD; andn = were8–10 calculatedper genotype. by two-way p-values ANOVA are displaye (2WA) followedd in graphs by Bonferroni’s and post test; * werep < 0.05, calculated ** p < 0.01. by two-way ANOVA (2WA) followed by Bonferroni's post test; * p < 0.05, ** p < 0.01, and *** p < 0.001. 3. Discussion 3. Discussion We have reported the development of a novel transgenic mouse model, the Trspf/f- f/f We have reportedUcp1-Cre the+/ −developmentmice. Our model of a wasnovel characterized transgenic by mouse targeted model, deletion the of Trsp the gene- Trsp in Ucp1-Cre+/− mice. Ourbrown model adipocytes, was characterized which are the primaryby targeted cell typedeletion of BAT. of Thethe productgene Trsp of the in Trsp gene, brown adipocytes,tRNA which[Ser}Sec are, the is responsible primary ce forll carrying type of outBAT. the The synthesis product of selenoproteins, of the Trsp gene, including Dio2, tRNA[Ser}Sec, is responsiblea regulator for of carrying cold-induced out the adaptive synthesis thermogenesis of selenoprotei in BAT.ns, including Dio2, a regulator of cold-inducedDio2 upregulation adaptive thermogenesis is promoted byin BAT. thyroid hormone and is considered a primary Dio2 upregulationindicator is promoted of activation by ofthyroid adaptive hormone thermogenesis and is considered in cold exposure a primary [44]. In in- males, Dio2 Trspf/f Trspf/f-Ucp1-Cre+/− dicator of activationexpression of adaptive was unchanged thermoge betweennesis in cold andexposure [44]. In males,mice, Dio2 despite ex- a reduction in circulating T3. In addition, circulating T4, the substrate used by Dio2 to generate T3 pression was unchanged between Trspf/f and Trspf/f-Ucp1-Cre+/− mice, despite a reduction that, in turn, activates thermogenesis, was unchanged. In BAT, Ucp1 levels were also unchanged. Nevertheless, BAT of male Trspf/f-Ucp1-Cre+/− mice had a distinct tissue morphology resembling more a white adipocyte with increased lipid accumulation, typical of under-stimulated brown adipocytes [45]. Together, these results suggest that, despite T4 levels not being affected by the loss of Trsp in male Trspf/f-Ucp1-Cre+/− mice, the physiology of their brown adipocytes are indeed impaired. This impairment in the morphology of BAT in males possibly occurs since early development, because mice lacking Dio2 show permanent defect in adaptive thermogenesis in the embryonic BAT [46]. Murine selenoproteins are expressed in a sexually dimorphic manner in other organs, such as liver and kidneys [47,48]. A possible explanation for the sexual dimorphism we observed in BAT of male Trspf/f-Ucp1-Cre+/− mice could be alterations in the sympathetic tone in our mouse model. However, one of the regulators of the sympathetic tone of BAT is T4 via central and peripheral mechanisms [49]. However, the Dio2 knockout mouse model does not present sexual dimorphism in their thermogenic responses after a caloric overload at room temperature [31]. Moreover, T4 is not altered in males, making it unlikely that the sympathetic tone is diminished in BAT of these mice. An alternative to be explored in future studies is whether another selenoprotein, another factor involved in selenium metabolism, or another selenium-related mechanism plays a role in regulating the sympathetic tone in BAT according to sex. Interestingly, elevated circulating TSH levels in male Trspf/f-Ucp1-Cre+/− mice at room temperature suggest either impaired thyroid stimulation to produce T4—which does not occur as T4 levels are maintained—or a direct effect of TSH via its receptor TSHR in brown adipocytes, which could explain the morphological changes of BAT. Such direct effect of TSH has been demonstrated in rats to positively modulate oxygen consumption and inversely regulate Dio2 mRNA [50]. However, this observation is discrepant with our results herein in the male Trspf/f-Ucp1-Cre+/− mice, as circulating TSH were higher but effects on neither oxygen consumption nor Dio2 mRNA were observed. It is possible that other Int. J. Mol. Sci. 2021, 22, 611 13 of 20

mechanisms modulated by TSH alone, such as activation of Erk or Akt phosphorylation, are recruited [50], reducing the expected responses cited above. Acute cold exposure leads to intense lipolysis, adipocyte autophagy, and remodeling of BAT, using the lipid deposits available to increase the mitochondrial oxidative cycle. This, in turn, could produce heat, maintaining the animal’s core body temperature and avoiding hypothermia. Extensive use of lipid deposits leads to activation of a lipogenic phase with enhanced lipid uptake in brown adipocytes, guaranteeing fuel for thermogenesis maintenance as cold exposure continues [51]. Despite BAT undergoing intense remodeling in the first hours of cold exposure, it is intriguing that male Trspf/f-Ucp1-Cre+/− mice do not present alterations in the autophagy flux; levels of autophagy factor p62/SQSTM1 were sustained in male Trspf/f-Ucp1-Cre+/− mice, while autophagy genes were upregulated in our microarray analysis. This discrepancy suggests either a reduction in translational efficiency of autophagic transcripts or a temporal dissonance in autophagy activation in male Trspf/f-Ucp1-Cre+/− mice acutely exposed to cold. The varying length of cold exposure has been considered as a source of conflicting data for autophagy response in adaptive thermogenesis ([52] citing [53–55]). These studies did not expose mice for 4 h in the cold; therefore, it is also possible that we uncovered a transitional moment for autophagy. However, male Trspf/f-Ucp1-Cre+/− mice accumulated more triglycerides than control mice, despite similar levels of mitochondrial markers at room temperature. These combined findings suggest that male Trspf/f-Ucp1-Cre+/− mice maintained mitochondrial capacity with excess resources for the initial lipolytic phase of adaptive thermogenesis. It is possible that during cold exposure, as brown adipocytes switch from lipolysis to lipogenesis, the advantage provided by additional fuel is counteracted by deficiencies in thermogenic responses that are dependent on other factors, such as thyroid hormone activation or sympathetic tone [56]. It is puzzling that the loss of Trsp, i.e., selenoprotein synthesis capacity, does not lead to a more dramatic impact in gene expression, particularly of selenoproteins after acute cold exposure. As mentioned in the previous paragraph, the late lipogenic phase of chronic cold exposure depends on the proper transcriptional activation of T3-responsive genes encoding key proteins involved in lipogenesis early on, which will later allow for withstanding the constant temperature stress. Our finding that the Thrsp transcript, which encodes a coactivator of lipogenic genes via interaction with the thyroid hormone receptor [40,41], was downregulated in Trspf/f-Ucp1-Cre+/− mice suggests lower availability of thyroid hormones locally in the early stages of cold exposure. It is plausible to infer that these mice, upon chronic exposure to cold, may diminish their thermogenic capacity due to impaired lipid uptake, because the proteins required for lipogenesis could be at lower levels. Prospective studies examining the chronic activation of BAT adaptive thermogenesis, either in a more physiological manner after chronic cold exposure or in a pathological context, after a high-fat diet, may consolidate whether observed changes in thyroid hormone response of the Trspf/f-Ucp1-Cre+/− mice are sustained. Moreover, the participation of additional gut hormones in concert with the sympathetic response may mediate chronic thermogenic responses. A chronic paradigm for adaptive thermogenesis activation, particularly in a pathological context after a hypercaloric overload, may allow for display of a worsened metabolic dysfunction. We have observed a moderate impairment of Akt activation, i.e., in insulin response, in the gWAT of Trspf/f-Ucp1-Cre+/− mice. Therefore, it is likely that the feeding of a high-fat diet will accelerate the development of a metabolic dysfunction in a Trspf/f-Ucp1-Cre+/− mouse. An interesting aspect found by our microarray analysis was the upregulation of genes involved in methylation. As occurs with other tRNAs [57], a portion of the tRNA[Ser]Sec population is methylated at the uridine position 34 (Um34) yielding a specific subpopula- tion [58]. In fact, differential methylation of Um34 leads to the existence of two possible molecular conformations in relation to the anticodon sequence [59]. Consequently, two sub- sets of selenoproteins, deemed stress-related and housekeeping, occur, with tRNA[Ser]Sec with Um34 supporting primarily translation of stress-related selenoproteins. Members Int. J. Mol. Sci. 2021, 22, 611 14 of 20

of this class of selenoproteins are sensitive to selenium status [20,21], which is likely the case for Dio2 in the activated BAT. The specific methyltransferase responsible for the methylation of Um34 has not been identified, although one that is sensitive to S-adenosyl- homocysteine accumulation has been indicated as a promising candidate [60]. In our mouse model, loss of tRNA[Ser]Sec in brown adipocytes led to upregulation of Mthfd2, which encodes an enzyme from folate one-carbon metabolism. This enzyme also binds to several ribonucleoproteins and is involved in protein translation [61], controlling the methylation of N6-methyladenosine (m6a) of mRNAs [42]. It is tempting, at this point, to suggest that this enzyme may be an additional promising candidate for at least controlling the Um34 methylation of tRNA[Ser]Sec. Considering the impairment of selenoprotein synthesis in the brown adipocyte, it is intriguing that a general decline in selenoprotein gene expression, particularly Dio2, is not observed. Stress-related selenoprotein mRNAs, such as Dio2, are primary targets for nonsense-mediated decay (NMD) degradation, a mechanism that misreads the in-frame UGA codon of selenoproteins as a nonsense codon [62]. Selenium deficiency further activates the NMD machinery to target selenoprotein mRNAs for degradation [63]. Trspf/f-Ucp1-Cre+/− mice were fed diets with adequate selenium, which likely provided enough selenium to inhibit NMD machinery, allowing selenoprotein mRNAs to circumvent NMD degradation. Moderate dietary selenium intake in Trspf/f-Ucp1-Cre+/− mice also raises a striking possibility regarding the fate of selenium in brown adipocytes. With the impairment of selenoprotein synthesis, available selenium may be either released back into circulation or to neighboring cell types, or redirected within the brown adipocyte for use in additional pathways. Jedrychowski et al. [32] have demonstrated that excess selenium in brown adipocytes impacts adaptive thermogenesis via incorporation of Sec in the place of cysteine in several non-selenoproteins, i.e., facultative selenation. Notably, cysteine residue 253 of Ucp1 was identified to be selenated. Selenation of Ucp1 increases its redox sensitivity, enhancing energy expenditure, activating adaptive thermogenic responses, and protecting from high-fat diet-induced obesity. It is possible that selenium not used in selenoprotein synthesis by the Trspf/f-Ucp1-Cre+/− mice enhances facultative selenation. Enhanced facultative selenation, particularly of Ucp1, could explain the mild effects of acute cold exposure observed in our mouse model, with the lack of Trsp and consequent selenoprotein synthesis freeing selenium to be redirected towards selenation in brown adipocytes. It is unknown whether facultative selenation in brown adipocytes requires tRNA[Ser]Sec. We believe that the Trspf/f-Ucp1-Cre+/− mouse model may be fitting to test this hypothesis in the future. A limitation of this study encompasses the inability to breed or maintain mice at their thermoneutrality (~28–30 ◦C) due to the lack of this capacity at our animal facilities. Mice maintenance at temperatures slightly lower than thermoneutrality could have primed their BAT to activate thermogenic mechanisms. Such prior activation could diminish the impact of acute cold exposure, and this effect was observed in a mouse model lacking Dio2 [64]. Despite this limitation, we still observed subtle changes at the transcriptional level after acute cold exposure, particularly in genes responding to thyroid hormone and involved in methylation pathways. The fact that we unveiled changes in gene expression between the Trspf/f-Ucp1-Cre+/− and Trspf/f control mice indicates that the effects of this temperature priming were potentially minimized. In summary, we developed a novel mouse model lacking the Trsp gene in brown adipocytes and demonstrated that loss of Trsp in these cells leads to a modest impairment of acute cold exposure responses in male Trspf/f-Ucp1-Cre+/− mice, but not in females. We also observed subtle changes in thyroid hormone responsiveness and genes involved in methylation pathways. Future studies moving towards a chronic cold exposure may help refine our knowledge of the role of selenoproteins in rodent adaptive thermogenesis. Int. J. Mol. Sci. 2021, 22, 611 15 of 20

4. Materials and Methods 4.1. Chemicals and Antibodies All chemicals used in experiments were from Fisher Scientiﬁc (Hampton, NH, USA) or Sigma-Aldrich (St. Louis, MO, USA), unless speciﬁed. Primary antibodies included anti-β-actin (1:3000; cat. #A2228; Sigma-Aldrich), anti-Ucp1 (1:1000; cat. #ab10983, Abcam; Cambridge, MA, USA), anti-PGC1α (1:750; cat. #AB3242, MilliporeSigma; Burlington, MA, USA), anti-ATPB (1:1000; cat. #ab14730; Abcam), anti-Akt (1:1000; cat. #9272, Cell Signaling Technology, Beverly, MA, USA); anti-phospho-Akt-Ser473 (1:750; cat#9271, Cell Signaling Technology), and anti-p62/SQSTM1 (1:750; cat. #5114T, Cell Signaling Technology).

4.2. Animals Animal procedures were approved by the University of Hawaii Office of Research Compliance Animal Welfare Program, protocol #17-2521, first approved by the Institutional Animal Care and Use Committee on 19 October 2017. Mice were held in our vivarium and used for experiments in minimal numbers needed to provide significant results. B6.FVB- Tg(Ucp1-cre)1Evdr/J (MGI J:206508; shortened here as Ucp1-Cre) mice on a C57BL6/J background were purchased from The Jackson Laboratory (cat. #024670; Bar Harbor, ME, USA). Trsp-LoxP were generated as previously described [65]. To generate a strain with a targeted deletion of Trsp in brown adipocytes, the Trspf/f-Ucp1-Cre+/− mice, Trspf/+ mice were crossed with Ucp1-Cre mice. Homozygote Trspf/f littermate controls were used to compare with Trspf/f-Ucp1-Cre+/− mice after genotyping confirmation by PCR. Mice were in a 12 h light–dark cycle, fed ad libitum with chow containing ~0.25 ppm sodium selenite, and group-caged until exposure to cold. Euthanasia occurred at 12-weeks old by CO2 asphyxiation, and all mice were euthanized ~2 pm, to avoid circadian influences in results. After euthanasia, blood and interscapular BAT, which comprises approximately 60% of all BAT depots and considered the most significant in mice [66], were removed immediately, snap-frozen into liquid nitrogen, perfused with formalin, or incubated in RNALater Stabilization Solution (Thermo Fisher Scientific; Waltham, MA, USA).

4.3. Energy Expenditure Assessment

Oxygen consumption (VO2), respiratory quotient (RQ) and energy expenditure (EE) were assessed for 24 h with the PanLab OxyLetProTM System (Harvard Apparatus, Barcelona, Spain) in 11-week-old mice. Mice were habituated in individual cages for 24 h prior to running experiments, as previously described [67]. Data were analyzed using PanLab Metabolism (Prague, Czech Republic) software.

4.4. Cold Exposure and Core Body Temperature Assessment A G2 emitter thermal probe (Starr Life Sciences Corp.; Oakmont, PA, USA) was surgically inserted into the peritoneal cavity of mice one week prior to exposure to cold. Only mice that adequately recovered from survival surgery were used in experiments. On the day of experiment, 12-week-old mice were transferred to individual cages with only water and minimal bedding, transported to a cold room set at 4 ◦C between 09:00 and 10:00 a.m., and each cage placed on a receiver telemetry platform (Starr Life Sciences Corp.). Mice remained in the cold room uninterrupted for 4 h and were euthanized ~2 pm. Core body temperature was recorded every 5 min by the receiver using Vital View Legacy Version 5.1 software (Starr Life Sciences Corp.).

4.5. Triglyceride Content Triglyceride content of BAT was measured using the Triglyceride Quantiﬁcation Assay Kit (Abcam) for colorimetric detection, following the manufacturer’s protocol.

4.6. RNA Extraction Mouse interscapular BAT was collected at time of euthanasia (~2pm) either in RNALater (Thermo Fisher Scientiﬁc) or snap-frozen, according to ﬁnal use. For quantitative PCR Int. J. Mol. Sci. 2021, 22, 611 16 of 20

(qPCR), total RNA was extracted from snap-frozen tissues using a TissueRuptor (Qiagen; Germantown, MD, USA) with disposable probes, and isolated using the EZNA total RNA kit I (Omega Biotek; Norcross, GA, USA). For microarray, total RNA was extracted from tissues in RNALater, processed with TissueRuptor and isolated using the Qiagen RNA extraction kit (Qiagen).

4.7. Microarray Analysis RNA quality was assessed in an Agilent 2100 BioAnalyzer (Agilent Technologies; Santa Clara, CA, USA) and samples with RIN > 5.5 were deemed satisfactory for microarray. Mouse ClariomS Array gene hybridization (Affymetrix—Thermo Fisher Scientific) was carried out with 100 ng of total RNA, run and scanned in GeneChip Fluidics Station 450 and a GeneChip Scanner (Affymetrix—Thermo Fisher Scientific) in the Genomics and Bioinformatics Shared Resource facility at the University of Hawaii Cancer Center. The CEL files were processed and analyzed using the Transcriptome Analysis Console (TAC) 4.0 from Thermo Fisher Scientific. Genes with a fold change greater than 2 and p-value less than 0.05 were considered differentially expressed. Canonical pathway and network analysis of differentially expressed genes was performed using Qiagen’s Ingenuity Path- way Analysis (IPA; Qiagen; https://www.qiagenbioinformatics.com/products/ingenuity- pathway-analysis).

4.8. Real-Time qPCR One microgram of total RNA extracted from the interscapular BAT was reverse transcribed using the High Capacity kit (Applied Biosystems—Thermo Fisher Scientific). 10 ng of cDNA were used in real-time qPCR reactions with the PerfeCTa SYBR Green SuperMix (Quantabio; Beverly, MA) and specific primers, listed in Supplementary Table S4. Relative quantification of target gene expression was calculated based on the ∆Ct method, normalized to the expression of housekeeping genes for 18s or Gapdh, and plotted as fold change relative to Trspf/f at room temperature values.

4.9. Immunohistochemistry Mice acclimated to room temperature had their BAT extracted after perfusion with formalin and were paraffin-embedded for histological analysis. Immunohistochemistry was performed in slides using citrate buffer in a pressure cooker. Slides were labeled with primary antibody (anti-Ucp1; 1:100), Mouse on Mouse Basic kits with Vectastain ABC, and diaminobenzidine peroxidase substrate kits (Vector Labs; Burlingame, CA, USA) as previously described [68]. Slides were counter-stained with hematoxylin to visualize adipocyte nuclei. Four brightfield images were acquired from a single section of BAT from each subject at 40× magnification using the simple random sampling workflow in Stereo Investigator (MBF Bioscience; Williston, VT, USA). To compare Ucp1 immunoreactivity, the optical density of each image was quantified using ImageJ (public domain) and averaged for each subject. To calculate Ucp1 optical density, images were converted to black-and-white, inverted, and the mean pixel value for the entire image recorded. Total nuclei were counted using ImageJ Cell Counter plugin (public domain) and used to normalize the mean pixel value for each image.

4.10. Western Blot BAT and gWAT protein were extracted using RIPA Lysis and Extraction buffer containing protease and phosphatase inhibitors (Thermo Scientiﬁc) followed by sonication and two rounds of centrifugation. 10–20 µg of total protein was loaded into 4–20% TGX SDS-PAGE (Bio-Rad; Hercules, CA, USA) and wet-transferred overnight in Tris-glycine buffer containing 9% methanol to an Immobilon-FL® membrane (MilliporeSigma) using the Criterion Blotter (Bio-Rad) apparatus. Primary and secondary antibodies were incubated for 1 h at room temperature with rotation, and secondary antibodies were IRDyes (Li-Cor Int. J. Mol. Sci. 2021, 22, 611 17 of 20

Biosciences; Lincoln, NE) for use at an Odyssey CTx infra-red scanner (Li-Cor Biosciences). All blots were used only once, except the measurement of phosphorylated Akt, performed on the same membrane after conﬁrmation of complete stripping of previous antibodies using NewBlot PVDF Stripping Buffer (Li-Cor Biosciences). Analysis of the Western blot was conducted with the Image Studio Lite version 5.2.5. software (Li-Cor Biosciences).

4.11. Thyroid Function Thyroid function was inferred from analyses of total T4, total T3, and TSH levels in the serum. Twenty microliters of serum were used to assay for TSH with a Milliplex MAP Mouse Pituitary Magnetic Bead Panel (Millipore-Sigma) on a Luminex 200 platform (Luminex; Austin, TX, USA). Twenty-ﬁve microliters of serum were used to assay for total T4 and total T3 using the AccuDiagTM ELISA—T4 kit (Diagnostic Automation; Woodland Hills, CA, USA) and the T3 (Total) ELISA kit (Abnova; Taipei, Taiwan), respectively, according to the manufacturer’s instructions.

4.12. Statistical Analysis Results were plotted and graphed using Graphpad Prism v. 8.0 (San Diego, CA), and an alpha value of 0.05 was adopted. Unpaired Student’s t-test or two-way analysis of variance (2WA) with Bonferroni’s ad hoc post-test were used according to the number of variables analyzed in each experiment.

Supplementary Materials: The following are available online at https://www.mdpi.com/1422-006 7/22/2/611/s1, Table S1: Differentially expressed genes, networks, and pathways in BAT of Trspf/f- Ucp1-Cre+/− mice at room temperature obtained by microarray analysis. Supplementary Table S2: Differentially expressed genes, networks, and pathways in BAT of Trspf/f-Ucp1-Cre+/− mice after acute cold exposure obtained by microarray analysis. Supplementary Table S3: All comparisons between differentially expressed genes of microarray analysis. Supplementary Table S4: Mouse primer sequences used in this study. Original gel images are also uploaded within Supplementary Materials. Author Contributions: Conceptualization, L.A.S. and M.J.B.; methodology, L.A.S., A.N.O.-W., L.M.W., and D.J.T.; resources, B.A.C. and D.L.H.; formal analysis and data curation, L.A.S., A.N.O.-W., L.M.W., D.J.T., V.S.K. and M.M.; visualization, V.S.K. and M.M.; writing—original draft preparation, L.A.S.; writing—review and editing, all authors.; supervision, L.A.S.; project administration, L.A.S. and M.J.B.; funding acquisition, L.A.S., L.M.W., and M.J.B. All authors have read and agreed to the published version of the manuscript. Funding: Research in our laboratory is funded by the National Institutes of Health (NIH) grants R01DK047320, R01DK047320-23S1 and R01DK047320-22S2 to M.J.B., F32DK124963-01 to D.J.T., and U54MD007601—subproject 5544; Hawaii Community Foundation grant 20ADVC-102166, and start- up funds from the JABSOM Office of the Associate Dean of Research to L.A.S., and fellowship 2018/09478-4 from Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) to L.M.W. The Core Facilities used to conduct some of the assays are funded by NIH grants U54MD007601, P30CA071789, P20GM103466 and P30GM114737. Institutional Review Board Statement: Animal procedures were approved by the University of Hawaii Office of Research Compliance Animal Welfare Program, protocol #17-2521, first approved by the Institutional Animal Care and Use Committee on 19 October 2017. Informed Consent Statement: Not applicable. Data Availability Statement: The data presented in this study are available upon request to the corresponding author. Acknowledgments: We are grateful to Ann C. Hashimoto for excellent technical support in mouse husbandry; Miyoko Bellinger and Kristen Ewell from the Histopathology Core Facility for assistance with histological sample preparation; Maarit Tiirikainen and Karolina Peplowska from the Genomics and Bioinformatics Shared Resource for the microarray run; and Alexandra Gurary and Jourdan Posner from the Molecular and Cellular Immunology Core Facility, for help with the Luminex assay. Int. J. Mol. Sci. 2021, 22, 611 18 of 20

Conﬂicts of Interest: The authors declare no conﬂict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Abbreviations

BAT Brown adipose tissue Dio2 Iodothyronine deiodinase type 2 qPCR Quantitative PCR Sec Selenocysteine T3 3,30,5-triiodothyronine T4 Thyroxine TSH Thyroid-stimulating hormone Ucp1 Uncoupling protein 1

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