International Journal of Molecular Sciences

Article Overexpression of 1 Inhibits Brown and Beige Adipose Tissue Activity in Mice

Yu-Jen Chen 1,2,*, Chiao-Wei Lin 1,2, Yu-Ju Peng 2, Chao-Wei Huang 2 , Yi-Shan Chien 2, Tzu-Hsuan Huang 2, Pei-Xin Liao 2, Wen-Yuan Yang 2, Mei-Hui Wang 3, Harry J. Mersmann 2, Shinn-Chih Wu 1,2, Tai-Yuan Chuang 4, Yuan-Yu Lin 2,* , Wen-Hung Kuo 5,* and Shih-Torng Ding 1,2,*

1 Institute of Biotechnology, National Taiwan University, Taipei 10617, Taiwan; [email protected] (C.-W.L.); [email protected] (S.-C.W.) 2 Department of Animal Science and Technology, National Taiwan University, Taipei 10617, Taiwan; [email protected] (Y.-J.P.); [email protected] (C.-W.H.); [email protected] (Y.-S.C.); [email protected] (T.-H.H.); [email protected] (P.-X.L.); [email protected] (W.-Y.Y.); [email protected] (H.J.M.) 3 Institute of Nuclear Energy Research, Taoyuan 325, Taiwan; [email protected] 4 Department of Athletics, National Taiwan University, Taipei 10617, Taiwan; [email protected] 5 Department of Surgery, National Taiwan University Hospital and College of Medicine, National Taiwan University, Taipei 10617, Taiwan * Correspondence: [email protected] (Y.-J.C.); [email protected] (Y.-Y.L.); [email protected] (W.-H.K.); [email protected] (S.-T.D.); Tel.: +886-2-3366-4175 (S.-T.D.)

Abstract: Adult humans and mice possess significant classical brown adipose tissues (BAT) and, upon cold-induction, acquire brown-like adipocytes in certain depots of white adipose tissues (WAT),

 known as beige adipose tissues or WAT browning/beiging. Activating thermogenic classical BAT or  WAT beiging to generate heat limits diet-induced obesity or type-2 diabetes in mice. Adiponectin is a

Citation: Chen, Y.-J.; Lin, C.-W.; beneficial adipokine resisting diabetes, and causing “healthy obese” by increasing WAT expansion to Peng, Y.-J.; Huang, C.-W.; Chien, Y.-S.; limit lipotoxicity in other metabolic tissues during high-fat feeding. However, the role of its receptors, Huang, T.-H.; Liao, P.-X.; Yang, W.-Y.; especially 1 (AdipoR1), on cold-induced thermogenesis in vivo in BAT and in Wang, M.-H.; Mersmann, H.J.; et al. WAT beiging is still elusive. Here, we established a cold-induction procedure in transgenic mice over- Overexpression of Adiponectin expressing AdipoR1 and applied a live 3-D [18F] fluorodeoxyglucose-PET/CT (18F-FDG PET/CT) Receptor 1 Inhibits Brown and Beige scanning to measure BAT activity by determining glucose uptake in cold-acclimated transgenic Adipose Tissue Activity mice. Results showed that cold-acclimated mice over-expressing AdipoR1 had diminished cold- in Mice. Int. J. Mol. Sci. 2021, 22, 906. induced glucose uptake, enlarged adipocyte size in BAT and in browned WAT, and reduced surface https://doi.org/10.3390/ijms22020906 BAT/body temperature in vivo. Furthermore, decreased expression, related to thermogenic Ucp1, BAT-specific markers, BAT-enriched mitochondrial markers, lipolysis and fatty acid oxidation, Received: 8 December 2020 and increased expression of whitening in BAT or in browned subcutaneous inguinal WAT of Accepted: 15 January 2021 AdipoR1 mice are congruent with results of PET/CT scanning and surface body temperature in vivo. Published: 18 January 2021 Moreover, differentiated brown-like beige adipocytes isolated from pre-adipocytes in subcutaneous

Publisher’s Note: MDPI stays neutral WAT of transgenic AdipoR1 mice also had similar effects of lowered expression of thermogenic Ucp1, with regard to jurisdictional claims in BAT selective markers, and BAT mitochondrial markers. Therefore, this study combines in vitro and published maps and institutional affil- in vivo results with live 3-D scanning and reveals one of the many facets of the adiponectin receptors iations. in regulating energy homeostasis, especially in the involvement of cold-induced thermogenesis.

Keywords: adiponectin; ; beige adipose tissue; brown adipose tissue; cold-induced thermogenesis; PET/CT scintigraphy; uncoupling 1 (UCP1)

Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and 1. Introduction conditions of the Creative Commons There are two distinct types of adipose tissues, brown (BAT) and white (WAT), in Attribution (CC BY) license (https:// mammals including humans and mice. Contrary to the primary role of white adipocytes creativecommons.org/licenses/by/ in lipid storage, brown adipocytes are responsible for increasing energy expenditure 4.0/).

Int. J. Mol. Sci. 2021, 22, 906. https://doi.org/10.3390/ijms22020906 https://www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2021, 22, 906 2 of 20

by maintaining thermogenesis [1]. Mice or rats constantly possess surgically obvious significant amounts of BAT, particularly around the interscapular region, even in adulthood. Given BAT weights relative to total body weights, mouse BAT is quite large. Previously, it is believed that, unlike mice, human BAT only exists in infants and recedes in the growing period [2]. Until 2009, metabolically active BAT was clearly mapped within the supraclavicular region, correlated with cold stimulation and inversely correlated with body max index (BMI), obesity, or aging [3–5]. Therefore, recent interests about BAT development and methods to activate human and mouse BAT reignite adipose researchers. BAT is mainly responsible for non-shivering thermogenesis that increases energy expenditure by uncoupling adenosine triphosphate (ATP) generation in mitochondrial respiration and dissipating chemical energy in the form of heat to maintain core body tem- perature. The mechanism is primarily through thermogenic uncoupling protein 1 (UCP1). Moreover, this thermogenic process is even more pronounced under cold- or diet-induced thermogenesis [6,7]. Recently, under certain stimuli such as cold exposure [8], peroxisome proliferator- activated receptor γ (PPARγ) agonists [9] or β-adrenergic agonists [10], subcutaneous white adipose tissues also acquire brown-like adipocytes in white adipose depots with emerging UCP1-positive adipocytes, enhanced mitochondrial biogenesis and enriched BAT-specific mitochondrial markers. Processes of developing recruitable brown-like adipocytes in white adipose depots are termed beiging/browning of white adipocytes or simply beige/brite (brown-in-white) adipocytes [11]. Therefore, activating thermogenesis solely in classical BAT or browned WAT, or in both tissues together becomes a combined interest in obesity and diabetes research. Adiponectin, with molecular weights of 30 kDa, is one of the early identified adipocyte- secreted protein hormones (adipokines) circulating into the blood in mice [12,13]. Adiponectin belongs to the complement 1q (C1q) family with a C-terminal globular domain homologous to C1q [14]. Multimer complexes (bound through a -like domain) of adiponectin, include trimer, hexamer and high-molecular-weight species are naturally formed and found in the circulation [15,16]. Receptors for adiponectin were first cloned as adiponectin recep- tors 1 and 2 (AdipoR1 and AdipoR2) from humans and mice [17]. We first cloned porcine counterparts corresponding to mouse/human AdipoR1 and AdipoR2 [18]. AdipoR1 and AdipoR2 each possesses seven transmembrane domains. However, structure, topology and function of AdipoR are distinct from common G-protein-coupled receptors [17]. AdipoR1 and AdipoR2 serve as main receptors of adiponectin for many metabolic functions in various tissues with AdipoR1 expressed universally, including in the skeletal muscle, liver, and adipose, and with AdipoR2 mostly expressed in the mouse liver [19]. Our previous studies generated transgenic AdipoR1 mice and found these mice defend against obesity, hepatosteatosis, and resistance when fed a high-fat high-sucrose diet [20]. These AdipoR1-derived mouse mesenchymal stem cell transplantations amelio- rate obesity-induced hepatosteatosis [21]. AdipoR1 also regulates mouse bone formation and osteoblast differentiation through the GSK-3β and β-catenin signaling [22]. These mice resist the decline of serum and GPRC6A expression in ovariectomized mice [23]. However, we also showed administering adiponectin or over-expressing Adi- poR1 in mice enhances the inflammatory bowel disease [24]. Therefore, adiponectin and adiponectin receptors appear to play a complicated role and participate in an array of metabolic processes. The role of adiponectin on BAT or cold-stimulated thermogenesis in vivo is conflicted and related research about adiponectin receptors on BAT is limited. Here, the purpose of this study is to establish a cold challenge procedure for FVB mice and apply it to the transgenic mouse model over-expressing porcine AdipoR1. Unravel the role of AdipoR1 in the hypothermia condition to activate thermogenesis with cold-induced glucose uptake in vivo by live positron emission tomography/computed tomography (PET/CT) scanning. This was accompanied by in vitro analyses and in vivo supporting results such as histo- logical examination of adipocyte size, surface body, and surface BAT depot temperature, Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 3 of 22

this study is to establish a cold challenge procedure for FVB mice and apply it to the trans- Int. J. Mol. Sci. 2021, 22, 906 genic mouse model over-expressing porcine AdipoR1. Unravel the role of AdipoR1 in the 3 of 20 hypothermia condition to activate thermogenesis with cold-induced glucose uptake in vivo by live positron emission tomography/computed tomography (PET/CT) scanning. This was accompanied by in vitro analyses and in vivo supporting results such as histo- thermographiclogical examination imaging, of adipocyte and measurements size, surface body of thermogenesis-related, and surface BAT depot genestemperature, (thermogenic Ucp1thermographic, brown adipose imaging, specific and markers, measurements and of brown thermogenesis adipose-related selected genes mitochondrial (thermogenic markers) in brownUcp1, andbrown browned/beiged adipose specific markers, white adiposeand brown tissues. adipose selected mitochondrial mark- ers) in brown and browned/beiged white adipose tissues. 2. Results 2. Results After a pre-test confirming temperature and time length described in Materials and Methods,After wild-type a pre-test (WT) confirming and transgenic temperature porcine and time AdipoR1 length described mice werein Materials housed and in a cold- Methods,◦ wild-type (WT) and transgenic porcine AdipoR1 mice were housed in a cold- roomroom (10 (10C) °C) for for two two weeks. weeks. TheThe workflow workflow is isshown shown in Figure in Figure 1A. 1A.

Figure 1. FigureBody 1. profiles Body profiles of AdipoR1 of AdipoR1 mice mice upon upon cold cold exposure. exposure. (A (A) Wild) Wild-type-type (WT) (WT) and transgenic and transgenic adiponectin adiponectin receptor 1 receptor (AdipoR1) mice of both sexes, male (M) and female (F), were housed in a cold-room (8–10 °C) for two weeks, i.e., 14 d. (B) 1 (AdipoR1) mice of both sexes, male (M) and female (F), were housed in a cold-room (8–10 ◦C) for two weeks, i.e., 14 d. Percentage of adipose tissue weights was expressed per unit body weights in WT or AdipoR1 mice. Subcutaneous inguinal (B) Percentagewhite adipose of adipose tissues tissue (iWAT), weights gonadal/epididymal was expressed white per adipose unit bodytissues weights(gWAT), classical in WT orinterscapular AdipoR1 brown mice. adipose Subcutaneous inguinal whitetissues adipose(BAT), and tissues suprascapular (iWAT), white gonadal/epididymal adipose tissues (supWAT) white were adipose harvested tissues and (gWAT), weighted. classical (C) AdipoR1 interscapular mice had brown adipose tissuesincreased (BAT), food andintake suprascapular (averaged g of feed white per adipose mouse per tissues d/g of(supWAT) mouse body were weights) harvested while losing and more weighted. body weight (C) AdipoR1 than mice WT mice. (D) AdipoR1 mice had significant body weight changes with enhanced body weight losses. Body weight changes had increasedwere calculated food intake and (averaged compared with g of each feed previous per mouse time perpoint d/g as percentage of mouse (%). body Data weights) were presented while losingas means more ± SEM body (n weight than WT mice.= 3–8 for (D each) AdipoR1 group). *mice p ≤ 0.05; had ** p significant ≤ 0.01; *** p body≤ 0.001. weight changes with enhanced body weight losses. Body weight changes were calculated and compared with each previous time point as percentage (%). Data were presented as means 2.1. Enhanced Body-Weight Losses in AdipoR1 Mice during Cold Exposure ± SEM (n = 3–8 for each group). * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001. After cold induction, distinct white and brown adipose depots including subcutane- ous inguinal white adipose tissues (iWAT), gonadal/epididymal white adipose tissues 2.1. Enhanced(gWAT), interscapular Body-Weight brown Losses adipose in AdipoR1 tissues (BAT), Miceduring and suprascapular Cold Exposure white adipose tis- sues (supWAT) were collected. Transgenic AdipoR1 male mice had lower gWAT weights andAfter greater cold iBAT induction, weights distinctcompared white with tissues and brownin WT male adipose mice. depotsTransgenic including AdipoR1 subcuta- neousfemale inguinal mice whitehad lower adipose iWATtissues and gWAT (iWAT), weights gonadal/epididymal compared with tissues white in WT adipose female tissues (gWAT),mice interscapular(Figure 1B). These brown results adiposeshowed different tissues fat (BAT), distribution and suprascapular under cold induction white in adipose tissuesWT (supWAT) or AdipoR1 were mice collected.in both sexes. Transgenic AdipoR1 male mice had lower gWAT weights and greater iBAT weights compared with tissues in WT male mice. Transgenic AdipoR1 female mice had lower iWAT and gWAT weights compared with tissues in WT female mice (Figure1B). These results showed different fat distribution under cold induction in WT or AdipoR1 mice in both sexes. Regarding food intake and body weight changes, overall, during the whole period of cold-induction, AdipoR1 male or female mice had greater accumulated food intake compared with WT mice, especially during periods of D0 to D4 and D0 to D7 (g per mouse per d / body weights) (Figure1C). However, examination of body weight changes under cold environments indicated that both AdipoR1 male and female mice showed significant enormous body weight losses compared to WT mice, especially during periods of D0 to D4 and D0 to D7. WT female mice even gained body weights under cold exposure in D0-D7 (Figure1D). These results showed that although transgenic AdipoR1 mice had increased Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 4 of 22

Regarding food intake and body weight changes, overall, during the whole period of cold-induction, AdipoR1 male or female mice had greater accumulated food intake com- pared with WT mice, especially during periods of D0 to D4 and D0 to D7 (g per mouse per d / body weights) (Figure 1C). However, examination of body weight changes under Int. J. Mol. Sci. 2021, 22, 906 cold environments indicated that both AdipoR1 male and female mice showed significant 4 of 20 enormous body weight losses compared to WT mice, especially during periods of D0 to D4 and D0 to D7. WT female mice even gained body weights under cold exposure in D0- D7 (Figurefood intake 1D). These per unit results body showed weight, that AdipoR1 although micetransgenic still had AdipoR1 negative mice body had in- weight changes creasedduring food coldintake stimulation, per unit body indicating weight, AdipoR1 signs of mice cold-intolerance still had negative symptoms. body weight changes during cold stimulation, indicating signs of cold-intolerance symptoms. 2.2. Diminished Cold-Induced Glucose Uptake In Vivo by PET/CT Scanning in Classical BAT and 2.2. DiminishedBrowned SupWAT Cold-Induced of AdipoR1 Glucose Uptake Mice In Vivo by PET/CT Scanning in Classical BAT and BrownedTo SupWAT record live of AdipoR1 glucose Mice uptake in vivo for BAT activity during cold stimulation in BAT and brownedTo record live WAT, glucose a PET/CT uptake scanning in vivo for with BAT 2-deoxy-2-[ activity during18F]fluoro-D-glucose cold stimulation in BAT (18F-FDG) injection 18 18 and tobrowned the WT WAT, and transgenica PET/CT scanning AdipoR1 with mice 2-deoxy was- applied2-[ F]fluoro after-D cold-stimulation.-glucose ( F-FDG) AdipoR1 mice injection to the WT and transgenic AdipoR1 mice was applied after cold-stimulation. Ad- showed significant reduction of cold-induced radio-labeled glucose uptake in vivo in classical ipoR1 mice showed significant reduction of cold-induced radio-labeled glucose uptake in vivo iBAT,in classical browned iBAT, supWAT browned and supWAT browned and browned iWAT compared iWAT compared to WT mice to WT (Figure mice (Fig-2). Constructed 3D ure 2).videos Constructed showed 3D panoramic videos showed views panoramic of inhibited views cold-induced of inhibited cold glucose-induced uptake glucose in AdipoR1 mice uptake(Supplementary in AdipoR1 mice Materials). (Supplementary Both maleMaterials). and female Both male AdipoR1 and female mice AdipoR1 showed mice strong diminution, showedespecially strong diminution, pronounced especially in AdipoR1 pronounced female in mice. AdipoR1 These female results mice. demonstrated These results AdipoR1 mice demonstratedclearly exhibit AdipoR1 decreased mice clearly cold-stimulated exhibit decreased glucose cold-stimulated uptake in glucose BAT, browneduptake in supWAT, and BAT,browned browned iWAT.supWAT, and browned iWAT.

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FigureF 2.ig Inhibitedure 2 cold-induced thermogenesis in vivo in classical brown and beige adipose de- Figure 2. Inhibited cold-induced thermogenesis in vivo in classical brown and beige adipose depots of pots of AdipoR1 mice. After two-week cold acclimation, WT and transgenic AdipoR1 mice were AdipoR1 mice. After two-week cold acclimation, WT and transgenic AdipoR1 mice were anesthetized and evaluated for cold-induced thermogenesis by live positron emission tomography/computed to- mography (PET/CT) scanning. Timeline and procedures were described in Figure1. Radiotracer of 18F-fluorodeoxyglucose (18F-FDG) was injected into the tail-vein to evaluate cold-enhanced glucose uptake in vivo. Asterisks (*) indicate classical iBAT and hashmarks (#) and plus signs (+) indicate supWAT and iWAT (potential depots for WAT browning or beiging), respectively. Procedures in different groups of mice were repeated at least twice. 3D-constructed videos are displayed in the Supplementary Materials. 2.3. Enlarged Adipocyte Size in Classical BAT and Browned iWAT of AdipoR1 Mice To confirm the inhibited cold-induced thermogenesis by PET/CT scanning in AdipoR1 mice, the structure of BAT and browned iWAT (the primary WAT browning depot) were examined by hematoxylin and eosin (H&E) staining. Higher BAT activity was revealed Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 5 of 22

anesthetized and evaluated for cold-induced thermogenesis by live positron emission tomogra- phy/computed tomography (PET/CT) scanning. Timeline and procedures were described in Fig- ure 1. Radiotracer of 18F-fluorodeoxyglucose (18F-FDG) was injected into the tail-vein to evaluate cold-enhanced glucose uptake in vivo. Asterisks (*) indicate classical iBAT and hashmarks (#) and plus signs (+) indicate supWAT and iWAT (potential depots for WAT browning or beiging), re- spectively. Procedures in different groups of mice were repeated at least twice. 3D-constructed videos are displayed in the Supplementary Materials.

Int. J. Mol. Sci. 2021, 22, 906 2.3. Enlarged Adipocyte Size in Classical BAT and Browned iWAT of AdipoR1 Mice 5 of 20 To confirm the inhibited cold-induced thermogenesis by PET/CT scanning in Adi- poR1 mice, the structure of BAT and browned iWAT (the primary WAT browning depot) in WTwere female examined mice by versushematoxylin WT maleand eo micesin (H&E) by PET/CT staining. scanningHigher BAT (Figure activity2 ).was Therefore, re- we focusvealed on in comparing WT female mice WT versus female WT mice male with mice AdipoR1by PET/CT scanning female mice.(Figure AdipoR1 2). Therefore, female mice we focus on comparing WT female mice with AdipoR1 female mice. AdipoR1 female mice both at room temperature (RT, Figure3A) and under cold exposure (Figure3B) showed both at room temperature (RT, Figure 3A) and under cold exposure (Figure 3B) showed increasedincreased adipocyte adipocyte size with with larger larger lipid lipid droplets droplets both in both BAT in and BAT browned and browned iWAT than iWAT than WTWT female female mice. mice. TheseThese results results indicated indicated AdipoR1 AdipoR1 mice miceacquire acquire lower browning/beiging lower browning/beiging effectseffects and and maintain maintain relative relative “whitening” “whitening” phenotypes phenotypes in in classical classical BAT BAT and and browned browned iWAT thaniWAT tissues than in tissues WT micein WT both mice atboth RT at and RT and under under cold cold environments. environments.

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Figure 3. Increased adipocyte size in classical brown and beige adipose depots of AdipoR1 mice. Cold-acclimated WT and Figure 3. IncreasedF adipocyteigure 3 size in classical brown and beige adipose depots of AdipoR1 mice. Cold-acclimated WT and transgenic AdipoR1 mice were evaluated for the morphology of brown and beige adipose tissues. Timeline and proce- transgenicdures AdipoR1 were described mice were in Figure evaluated 1. Classical for theiBAT morphology and iWAT (the of potential brown and white beige adipose adipose depot tissues.for browning Timeline or beiging) and procedures were described in Figure1. Classical iBAT and iWAT (the potential white adipose depot for browning or beiging) in WT and AdipoR1 female mice (A) at room temperature or (B) in cold environments were harvested, fixed, sectioned, and evaluated for histology by hematoxylin and eosin (H&E) staining. Scale bar: 100 µm for 20×, 50 µm for 40× microscope.

2.4. Decreased Surface Depot Temperature and Surface Body Temperature in Cold-Induced AdipoR1 Mice To confirm the diminished cold-induced thermogenesis by PET/CT scanning in Adi- poR1 mice, the surface brown adipose depot temperature and surface body temperature was examined by infrared thermometer and camera. AdipoR1 female mice under cold stimulation showed reduced surface BAT temperature and surface body temperature both before (RT) and after cold exposure (especially, 6 h) than WT mice (Figure4A). Thermo- graphic imaging also showed temperature distribution of surface body temperature in WT female mice was warmer than in AdipoR1 female mice (Figure4B). These results, in line with PET/CT scanning and H&E staining, indicated AdipoR1 mice have reduced BAT and browned WAT activity. Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 6 of 22

in WT and AdipoR1 female mice (A) at room temperature or (B) in cold environments were harvested, fixed, sectioned, and evaluated for histology by hematoxylin and eosin (H&E) staining. Scale bar: 100 μm for 20×, 50 μm for 40× microscope.

2.4. Decreased Surface Depot Temperature and Surface Body Temperature in Cold-Induced AdipoR1 Mice To confirm the diminished cold-induced thermogenesis by PET/CT scanning in Ad- ipoR1 mice, the surface brown adipose depot temperature and surface body temperature was examined by infrared thermometer and camera. AdipoR1 female mice under cold stimulation showed reduced surface BAT temperature and surface body temperature both before (RT) and after cold exposure (especially, 6 h) than WT mice (Figure 4A). Ther- mographic imaging also showed temperature distribution of surface body temperature in WT female mice was warmer than in AdipoR1 female mice (Figure 4B). These results, in Int. J. Mol. Sci. 2021, 22, 906 line with PET/CT scanning and H&E staining, indicated AdipoR1 mice have reduced BAT 6 of 20 and browned WAT activity.

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2.5. Increased Adipose Expression of Adiponectin, Adiponectin Receptors, and Brown Adipocyte Markers in wt Female Mice Versus wt Male Mice under Cold Exposure Toevaluate expression of thermogenesis related genes in BAT and WAT beiging/browning for energy expenditure, multiple adipose tissues were assessed. White adipose tissues in certain depots possess the ability to be browned, termed beige adipocytes, according to the guide of an adipose tissue atlas [25]. Therefore, iBAT, supWAT, iWAT (two potential WAT browning depots), and gWAT (minimal potential for browning) were sampled. First, to uncover the role of adiponectin receptors and browning (beiging) effects between different sexes in cold environments, adiponectin, adiponectin receptors (AdipoR1, AdipoR2 and T-cadhrin), and brown adipocyte markers in BAT and in browned WAT were determined in WT male and female mice. Surprisingly, Tcad (mainly in hearts) was also detectable with Ct values of ~25 by qPCR detection. However, expression of Adipor1 and Adipor2 was still dominant in BAT, iWAT, supWAT, and gWAT under cold exposure compared to adipose Tcad expression (Figure5A). In cold environments, WT female mice had greater Adipor1 expression in gWAT, increased Adipor2 expression in iWAT Int. J. Mol. Sci. 2021, 22, 906 7 of 20

and in gWAT, enhanced Tcad expression in four adipose depots (BAT, iWAT, supWAT, and gWAT) and higher Adipoq expression in BAT and in gWAT compared with tissues of WT male mice (Figure5B). Furthermore, WT female mice showed increased brown adipocyte markers, thermogenic Ucp1 in BAT and in gWAT and Prdm16, Cidea, Cox7a, and Cox8b in gWAT (Figure5C). These data indicated that WT female mice possess higher expression Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 8 of 22 of adiponectin receptors and increased brown adipocyte markers than WT male mice in cold environments.

Figure 5. FigureFemale 5. Female mice showed mice showed increased increased expression expression of of adiponectin adiponectin receptors receptors and and enhanced enhanced brown brown-adipocyte-adipocyte markers markersin in classical brownclassicaland brown beige and adiposebeige adipose depots depots after after cold cold exposure. exposure. ( (AA)) Indicated Indicated adipose adipose depots depots of male of and male female and WT female mice WT mice after cold acclimation were determined for expression of adiponectin receptors, including adiponectin receptor 1 (Adipor1), after coldadiponectin acclimation receptor were 2 determined (Adipor2), and for T-cadherin expression (Cdh13/Tcad of adiponectin). (B) Comparison receptors, of expression including of adiponectin adiponectin receptors receptor and 1 ( Adipor1), adiponectinadiponectin receptor (Adipoq 2 (Adipor2) between), and male T-cadherin and female ( Cdh13/TcadWT mice was). determined (B) Comparison in iBAT, of iWAT, expression supWAT, of adiponectinand gWAT, respec- receptors and adiponectintively. (Adipoq (C) Indicated) between adipos malee depots and femalein male WTor female mice WT was mice determined after cold acclimation in iBAT, iWAT, were determined supWAT, andfor expression gWAT, respectively. of brown adipocyte (WAT browning/beiging) markers. Timeline and procedures were described in Figure 1. Classical iBAT, (C) IndicatediWAT, adipose supWAT depots (both of in potential male or white female adipose WT de micepots for after browning cold acclimation or beiging) and were gWAT determined in male and for female expression WT mice of brown adipocytewere (WAT harvested, browning/beiging) extracted and measured markers. to Timelineevaluate adiponectin and procedures and its receptors were described and cold-induced in Figure thermogenic1. Classical related iBAT, iWAT, supWATgenes (both ( ofn = potential3–8 for each white group). adipose Data were depots presented for browning as relative or means beiging) (normalized and gWAT to β-actin in male in arbitrary and female units, WTAU) mice± were SEM. * p ≤ 0.05; **p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001. harvested, extracted and measured to evaluate adiponectin and its receptors and cold-induced thermogenic related genes (n = 3–8 for each group). Data were presented as relative means (normalized to β-actin in arbitrary units, AU) ± SEM. * p ≤ 0.05; **p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001.

Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 9 of 22 Int. J. Mol. Sci. 2021, 22, 906 8 of 20

2.6.2.6. Reduced Reduced Adiponectin Adiponectin and and Adiponectin Adiponectin ReceptorsReceptors in Adipose Tissues Tissues of of AdipoR1 AdipoR1 Female Female Mice VersusMice WTVersus Female WT MiceFemale under Mice Coldunder Exposure Cold Exposure GivenGiven that that WT WT female female mice mice possess possess enhanced enhanced cold-induced cold-induced glucose glucose uptake uptakein in vivo vivoby PET/CTby PET/CT scanning, scanning, and increasedand increased expression expression of adiponectin of adiponectin receptors receptors and and brown brown adipocyte adi- markerspocyte thanmarkers WT than male WT mice male in coldmice environments,in cold environments, we focused we focused on comparing on comparing WT female WT micefemale with mice AdipoR1 with AdipoR1 female micefemale under mice coldunder exposure. cold exposure. Compared Compared with with WT femaleWT female mice, AdipoR1mice, AdipoR1 female micefemale showed mice showed increased increased expression expression of Adipor1 of Adipor1in BAT, in iWAT, BAT, andiWAT, supWAT and whichsupWAT confirmed which thatconfirmed these transgenicthat these transgenic mice were mice over-expressing were over-expressing adiponectin adiponectin receptor 1 (Figurereceptor6). 1 (Figure Expression 6). Expression of Adipor2 of Adipor2was no was different no different in WT in and WT AdipoR1 and AdipoR1 female female mice. However,mice. However, AdipoR1 AdipoR1 female micefemale in mice cold in environments cold environments showed showed reduced reduced expression expression of Tcad inof iWAT Tcad andin iWAT gWAT and and gWAT decreased and decreasedAdipoq Adipoqin gWAT in gWAT (Figure (Figure6). These 6). These results results indicate indi- thesecate AdipoR1these AdipoR1 female female mice mice overexpressed overexpressed adiponectin adiponectin receptor receptor 11 and somewhat somewhat had had compensatorycompensatory reduced reduced expression expression of of T-cadherin T-cadherin inin certaincertain adipose tissue tissue depots. depots.

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Figure 6. Increased expression of adiponectin receptor 1 and decreased expression of T-cadherin in classical brown or beige Figure 6. Increased expression of adiponectin receptor 1 and decreased expression of T-cadherin in classical brown or adiposebeige depots adipose of depots AdipoR1 of AdipoR1 mice after mice cold after exposure. cold exposure Indicated. Indicated adipose adipose depots depots of WT of andWT AdipoR1and AdipoR1 female female mice mice after coldafter acclimation cold acclimation were determined were determined for expression for expression of adiponectin of adiponectin (Adipoq )(Adipoq and adiponectin) and adiponectin receptors, receptors, including including adiponectin adi- receptorponectin 1 (Adipor1 receptor), adiponectin 1 (Adipor1), receptor adiponectin 2 (Adipor2 receptor), and 2 ( T-cadherinAdipor2), and (Cdh13/Tcad T-cadherin). Timeline(Cdh13/Tcad and). proceduresTimeline and were procedures described in Figurewere 1described. Classical in iBAT, Figure iWAT, 1. Classical supWAT iBAT (both, iWAT of potential, supWAT white (both adipose of potential depots white for browning adipose depots or beiging), for browning and gWAT or in WTbeiging) and AdipoR1, and gWAT female in WT mice and were AdipoR1 harvested, female extracted,mice were andharvested, measured extracted to evaluate, and measured expression to evaluate of adiponectin expression and of its adiponectin and its receptors (n = 3–8 for each group). Data were presented as relative means (normalized to β-actin in receptors (n = 3–8 for each group). Data were presented as relative means (normalized to β-actin in arbitrary units, AU) arbitrary units, AU) ± SEM. * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001. ± SEM. * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001. 2.7. Decreased Brown-Adipocyte Selective Markers in Classical BAT and Browned iWAT of 2.7.AdipoR1 Decreased Female Brown-Adipocyte Mice under Cold Selective Exposure Markers in Classical BAT and Browned iWAT of AdipoR1Compared Female Mice with under male Cold mice, Exposure BAT activity by PET/CT scanning, expression of adi- ponectinCompared receptors with male and mice, expression BAT activity of brown by- PET/CTadipocyte scanning, selective expression makers in of BAT adiponectin or in receptorsbrowned and WAT expression were more of brown-adipocytepronounced in female selective mice. makers Therefore, in BAT we focused or in browned on classical WAT wereBAT more and pronouncedsubcutaneous in iWAT female in mice.female Therefore, mice for comparison we focused of on brown classical adipocyte BAT and markers subcu- taneousin WT iWATversus inAdipoR1 female mice. mice Brown for comparison adipocytes of express brown highly adipocyte distinc markerst markers in including WT versus FigurAdipoR1ethermogenic6 mice. Ucp1 Brown, Prdm16 adipocytes, and expressCidea and highly certain distinct specific markers mitochondrial including components thermogenic Ucp1Cox7a, Prdm16 and Cox8b, and Cideacomparedand certainwith white specific adipocytes. mitochondrial At RT (mild components cold-induction),Cox7a and WTCox8b fe- comparedmale mice with already white had adipocytes. greater expression At RT (mild of brown cold-induction), adipocyte markers, WT female such mice as Ucp1 already in hadBAT, greater iWAT expression, and gWAT, of brown than AdipoR1 adipocyte female markers, mice such with as otherUcp1 brownin BAT,-adipocyte iWAT, and marker gWAT, thangenes AdipoR1 in similar female patterns mice (Figure with other 7A). brown-adipocyteAfter prolonged exposure marker genesto cold in to similar activate patterns BAT (Figureand browned7A). After WAT prolonged (beiging), exposure expression to cold of thermogenic to activate BAT Ucp1 and in BAT, browned iWAT WAT, and (beiging), gWAT expressionfrom AdipoR1 of thermogenic female miceUcp1 wasin much BAT, lower iWAT, compared and gWAT to WT from female AdipoR1 mice. female BAT-enriched mice was muchmitochondrial lower compared marker to Cox7a WT femalealso had mice. decreased BAT-enriched expression mitochondrial in BAT, iWAT marker, and gWATCox7a alsoof hadAdipoR1 decreased mice expression than in tissues in BAT, of iWAT, WT mice and (Figure gWAT of7B). AdipoR1 Other brown mice thanadipocyte in tissues markers of WT micePrd (Figurem16 and7B). Cidea Other and brown BAT-mitochondrial adipocyte markers markerPrdm16 Cox8b inand AdipoR1Cidea and female BAT-mitochondrial mice showed markera resemblingCox8b in curtailed AdipoR1 fashion. female Therefore, mice showed these a resemblingresults showed curtailed that, both fashion. at RT Therefore, and in response to prolonged cold-exposure, AdipoR1 female mice had reduced expression of these results showed that, both at RT and in response to prolonged cold-exposure, AdipoR1 female mice had reduced expression of thermogenic Ucp1, general brown adipocyte markers and BAT-enriched mitochondria markers in BAT and in browned WAT.

Int. J. Mol. Sci. 2021, 22, 906 9 of 20

Figure 7. ReducedFigureBAT 7. thermogenic and mitochondrial markers in classical brown and beige adipose depots of AdipoR1 mice before and after cold-stimulation. Expression of brown adipocyte markers and BAT-enriched mitochondrial markers was evaluated in indicated adipose tissues of WT and AdipoR1 female mice, both (A) at room temperature (RT) before and (B) following the cold exposure at 10 ◦C. Timeline and procedures were described in Figure1. Classical iBAT, iWAT, supWAT (both of potential white adipose depots for browning or beiging), and gWAT in WT and AdipoR1 female mice were harvested, extracted, and measured to evaluate cold-induced thermogenic related genes at RT or after cold acclimation (n = 3–8 for each1 group). Data were presented as relative means (normalized to β-actin in arbitrary units, AU) ± SEM. * p ≤ 0.05; ** p ≤ 0.01.

2.8. Decreased Expression of Glucose Uptake, Lipolysis, and Fatty Acid Oxidation Genes in Classical BAT and Browned iWAT of AdipoR1 Mice under Cold Exposure To confirm inhibited cold-induced glucose uptake in vivo and reduced brown adipocyte markers in BAT and in browned iWAT of AdipoR1 female mice, gene regulation related Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 11 of 22

Int. J. Mol. Sci. 2021, 22, 906 2.8. Decreased Expression of Glucose Uptake, Lipolysis, and Fatty Acid Oxidation Genes in 10 of 20 Classical BAT and Browned iWAT of AdipoR1 Mice under Cold Exposure To confirm inhibited cold-induced glucose uptake in vivo and reduced brown adi- pocyteto glucose markers uptake, in BAT to and lipolysis, in browned and toiWAT fatty of acidAdipoR1 transport, female oxidation,mice, gene regulation and synthesis was relateddetermined. to glucose AdipoR1 uptake, female to lipolysis, mice and compared to fatty acid to WT transport, female oxidation, mice, upon and cold synthesis exposure, had wasreduced determined. expression AdipoR1 of Glut4 femalein mice BAT compared (related to to glucose WT femal uptake),e mice, uponHsl in cold BAT, exposure,Atgl in BAT and hadin iWAT reduced (both expression related of to Glut4 lipolysis), in BATCpt1a (relatedin BAT,to glucosePpara uptake),in BAT Hsl and in in BAT, iWAT Atgl (both in related BATto fatty and acidin iWAT oxidation), (both related and Srebpf1to lipolysis),in BAT Cpt1a and in inBAT iWAT, Ppara (related in BAT to and fatty in acidiWAT synthesis) (both related to fatty acid oxidation), and Srebpf1 in BAT and in iWAT (related to fatty (Figure8). However, expression of Fabp4, Cd36, Lpl, Acox1, Acc1, Acc2, or Fasn was not acid synthesis) (Figure 8). However, expression of Fabp4, Cd36, Lpl, Acox1, Acc1, Acc2, or different between WT and AdipoR1 female mice (Figure8). These results indicate AdipoR1 Fasn was not different between WT and AdipoR1 female mice (Figure 8). These results indicatemice had AdipoR1 decreased mice had gene decreased expression gene relatedexpression to glucoserelated to uptake,glucose uptake, lipolysis, lipolysis and, fatty acid andoxidation fatty acid that oxidation is congruent that is congruent with in vivo withresults in vivo of results lowered of lowered BAT activity BAT activity of cold-induced of coldglucose-induced uptake glucose and uptake diminished and diminished brown adipocyte brown adipocyte markers markers in AdipoR1 in AdipoR1 mice. mice.

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to (A) glucose uptake, (B) lipolysis, (C) fatty acid transport, (D) fatty acid oxidation, and (E) fatty acid synthesis was evaluated in indicated adipose tissues of WT and AdipoR1 female mice follow- ing cold exposure. Timeline and procedures were described in Figure 1. Classical iBAT and iWAT (the potential white adipose depot for browning or beiging) in WT and AdipoR1 female mice were harvested, extracted, and measured to evaluate adipose metabolism genes (n = 3–8 for each group). Data were presented as relative means (normalized to β-actin in arbitrary units, AU) ± Int. J. Mol. Sci. 2021, 22, 906 SEM. * p ≤ 0.05; ** p ≤ 0.01. 11 of 20

2.9. Decreased Expression of Browning Genes and Enhanced Whitening Genes in Classical BAT and2.9. Browned Decreased iWAT Expression of AdipoR1 of Browning Mice After Genes Cold and Exposure Enhanced Whitening Genes in Classical BAT and BrownedInhibited iWAT cold- ofinduced AdipoR1 glucose Mice After uptake Cold in Exposure vivo, reduced brown adipocyte markers and enlargedInhibited adipocyte cold-induced size revealed glucose uptakeby H&Ein staining, vivo, reduced in BAT brown and in adipocyte browned WAT markers of AdipoR1and enlarged mice, adipocyte all implicate size diminished revealed by BAT H&E function staining, in BAT in BAT and and browned in browned WAT. WATTo con- of firmAdipoR1 this mice, phenotype, all implicate additional diminished gene BAT expression function [26,27] in BAT related and browned to adipose WAT. To browning confirm (highlythis phenotype, expressed additional in BAT vs gene WAT) expression and adipose [26 ,27whitening] related (highly to adipose expressed browning in WAT (highly vs BAT)expressed was selected in BAT and vs WAT)determined. and adipose AdipoR1 whitening mice showed (highly reduced expressed expression in WAT of vsbrown- BAT) ingwas genes, selected Cpt1b and, Dio2 determined., Elovl3, and AdipoR1 Pgc1a in mice BAT showed and in reduced iWAT and expression increased of whitening browning genesgenes, Hmgn3Cpt1b, Dio2in iWAT, Elovl3 than, and WTPgc1a micein (Figure BAT and 9). in These iWAT r andesults increased indicate whitening AdipoR1 genes mice maintainHmgn3 in a iWAT relative than adipose WT mice whitening (Figure 9phenotype). These results in BAT indicate and browned AdipoR1 iWAT mice maintaineven after a prolongedrelative adipose cold exposure. whitening phenotype in BAT and browned iWAT even after prolonged cold exposure.

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2.10. Decreased Brown Adipocytes Markers in Differentiated Beige Adipocytes In Vitro Isolated from iWAT of AdipoR1 Mice Subcutaneous inguinal WAT is the largest white adipose depot in mice for isolating pre-adipocytes and also meets with retaining great potentials for differentiating into either traditional white or beige (brown-like) adipocytes, especially under PPARγ-agonist stimu- lation during differentiation process [9]. To further confirm the in vivo results of abated cold-inducedFigure glucose9 uptake and inhibited expression of brown adipocyte markers in BAT and browned WAT, we isolated pre-adipocytes from iWAT and differentiated them into beige adipocytes with or without synergistic browning agents of PPARγ-agonists, rosiglita- Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 13 of 22

2.10. Decreased Brown Adipocytes Markers in Differentiated Beige Adipocytes In Vitro Isolated from iWAT of AdipoR1 Mice Subcutaneous inguinal WAT is the largest white adipose depot in mice for isolating pre-adipocytes and also meets with retaining great potentials for differentiating into ei- ther traditional white or beige (brown-like) adipocytes, especially under PPARγ-agonist stimulation during differentiation process [9]. To further confirm the in vivo results of abated cold-induced glucose uptake and inhibited expression of brown adipocyte mark- Int. J. Mol. Sci. 22 2021, , 906 ers in BAT and browned WAT, we isolated pre-adipocytes from iWAT and differenti12ated of 20 them into beige adipocytes with or without synergistic browning agents of PPARγ-ago- nists, rosiglitazone. Rosiglitazone clearly augments beige (browning of white) adipocytes zone. Rosiglitazone clearly augments beige (browning of white) adipocytes with increased withgene increased expression gene of brown expression adipocyte of brown markers adipocyte and brown-adipocyte markers and brown mitochondrial-adipocyte markers mito- chondrial(Figure 10 markers). However, (Figure mature 10). However, beige adipocytes mature differentiated beige adipocytes with differentiated rosiglitazone with from rosiglitazoneAdipoR1 mice from showed AdipoR1 reduced mice expression showed reduced of brown expression adipocyte of markers, brown thermogenicadipocyte mark-Ucp1, ers,and thermogenicCidea compared Ucp1 with, and rosiglitazone-supplementedCidea compared with rosiglitazone beige- adipocytessupplemented from beige WT miceadi- pocy(Figuretes from10A). WT Brown-adipocyte mice (Figure enriched10A). Brown mitochondrial-adipocyte makers,enrichedCox7a mitochondrialand Cox8b ,makers, showed Cox7asimilar and effects Cox8b of, showed diminution similar in effects beige adipocytes of diminution from in AdipoR1beige adipocytes compared from to AdipoR1 WT mice compared(Figure 10 toB). WT Therefore, mice (Figure these results10B). Therefore, showed both thesein results vivo and showedin vitro bothresults in vivo of reduced and in vitroadipose results expression of reduced of brownadipose adipocyte expression markers of brown in AdipoR1 adipocyte mice. markers in AdipoR1 mice.

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FigureFigureFigu 10.r 10.e DecreasedDecreased10 BAT BAT thermogenic thermogenic and mitochondrial markersmarkers inindifferentiated differentiated beige beige adipocytes adipo- cytesfrom from iWAT iWAT of AdipoR1 of AdipoR1 mice. mice. Expression Expression of (Aof) ( brownA) brown adipocyte adipocyte markers markers and and (B )(B BAT-enriched) BAT-en- richedmitochondrial mitochondrial markers markers was evaluated was evaluated by qPCR. by qPCR. Pre-adipocytes Pre-adipocytes in the in stromal/vascular the stromal/vascular fraction of fractionsubcutaneous of subcutaneous iWAT (the iWAT potential (the depotpotential for depot WAT browningfor WAT browning or beiging) or were beiging) derived were from derived WT or fromtransgenic WT or AdipoR1transgenic mice AdipoR1 (n = 3) mice and ( differentiatedn = 3) and differentiated into mature into beige mature adipocytes beige adipocytes with or without with a orsynergistic without a browningsynergistic agent browning of PPAR agentγ, rosiglitazoneof PPARγ, rosiglitazone (Rosi). Data (Rosi). were presentedData were as presented relative meansas relative means (normalized to β-actin in arbitrary units, AU) ± SEM. * p ≤ 0.05. (normalized to β-actin in arbitrary units, AU) ± SEM. * p ≤ 0.05.

3.3. Discussion Discussion AdiponectinAdiponectin is is a abeneficial beneficial adi adipokinepokine defending defending against against obesity obesity-induced-induced diabetes. diabetes. However,However, the the local local effects effects of adiponectin on BAT activityactivity oror onon cold-stimulatedcold-stimulated thermoge- thermo- genesisnesis in in vivo vivoare are in in dispute. dispute. InvestigationInvestigation of adiponectin receptors on on BAT BAT is is even even more more limitedlimited and and not not conclusive. conclusive. The The current current research, research, to to our our knowledge, knowledge, is is the the first first to to show show that,that, in in live live inin vivo vivo coldcold-stimulated-stimulated glucose glucose uptake uptake scanned scanned by by PET/CT PET/CT scintigraphy, scintigraphy, after after prolongedprolonged cold cold-exposure-exposure for for two two weeks, weeks, mice mice over over-expressing-expressing AdipoR1 AdipoR1 had had diminished diminished 18F-FDG uptake and abated surface body temperature compared to WT mice. These sup- pressive results in AdipoR1 mice are supported by further analyses. Cold-acclimated AdipoR1 mice exhibited maintaining large lipid droplets of adipocyte size and reduced expression of brown adipocyte markers and of brown-adipocyte enriched mitochondrial markers, both in BAT and in browned WAT in vivo and in browned iWAT in vitro.

3.1. PET/CT and Cold-Induced Glucose Uptake PET/CT scintigraphy was first widely applied in clinical cancer research because cancer cells favor large amounts of glucose consumption. Therefore, radio-labeled glucose, Int. J. Mol. Sci. 2021, 22, 906 13 of 20

18F-FDG helps physicians spot glucose-consuming cancer cells in a tumor area [28–31]. Later, PET/CT scanning was adopted by endocrinologists searching for potential BAT in adult humans [3–5]. Nowadays, 18F-FDG PET/CT scanning is also used in preclinical mouse models for evaluating BAT activity or for brain research such as Alzheimer’s disease. For example, a recent research for adipose depot classification in the mouse adipose-tissue atlas used 18F-FDG or [123/125I]-β-methyl-p-iodophenyl-pentadecanoic acid coupled with PET/CT or SPECT/CT to image live cold-induced nutrient uptake in adipose tissues in vivo [25]. Depots of anatomically distinct brown, white, and beige (WAT browning) adipose tissues in mice are mapped using this method. Based on the above mentioned mouse brown and white adipose-tissue atlas, in the current study, we choose (1) the interscapular region of brown adipose tissues (representing classical BAT), (2) the suprascapular and (3) the subcutaneous inguinal white adipose depots (both representing potential WAT browning sites), and (4) the gonadal region of white adipose tissues (representing dull WAT browning site) to evaluate cold-induced glucose uptake in BAT and in browning of WAT. By PET/CT scanning, our results showed inhibited cold-induced glucose uptake in BAT and browned WAT of AdipoR1 mice in both sexes. Notably, this study also unveils that WT female mice possess greater amounts of cold- induced glucose uptake in vivo in BAT than WT male mice. Cold-acclimated WT female mice also had higher expression of thermogenic UCP1 in BAT and gWAT than WT male mice. These results are consistent with another study indicating that the β3-adrenergic receptor agonist CL316,243 induces enhanced brown adipocyte markers and mitochondrial respiratory chain in gWAT of female mice than in gWAT of male mice, but without effects in iWAT [32]. Furthermore, , a major female sex hormone known to increase BAT activity, known to activate hypothalamic AMP-activated protein kinase (AMPK)- related [33] and augment bone morphogenetic protein 8b (BMP8b)-related thermogenic signaling [34], may account for the sex differences in BAT activity and consequently estradiol could elevate BAT activity in female mice. Compared with WT male mice, cold-acclimated WT female mice also had elevated expression of adiponectin in BAT and gWAT, of AdipoR1 in gWAT, of AdipoR2 in iWAT and gWAT, and of T-cadherin in BAT, iWAT, supWAT, and gWAT. Female mice also had enhanced browning processes of cold-induced glucose uptake in BAT and thermogenic UCP1 in BAT and gWAT. Thus, our current study focused on female mice. Moreover, female mice with greater expression of adiponectin receptors and thermogenic UCP1 in adipose may be suitable for evaluating maximal effects of adiponectin receptor signaling on the cold-induced browning process in classical brown or browned white adipose tissues.

3.2. BAT and Cold-Induced Glucose Uptake Without reducing food intake for cutting excessive supply of calories and impacting de novo lipogenesis, an alternative way to combat obesity-induced diabetes is increasing energy expenditure, potentially through activating brown adipose or browning/beiging of white adipose depots [35]. The thermogenic ability of BAT not only oxidizes fats but also consumes significant amounts of glucose as a metabolic “glucose sink”, especially activated by cold stimulation. For example, elevating BAT activity by cold exposure consumes significant amounts of glucose, which can be assessed by radio-labeled glucose coupled with PET/CT scanning, and also dramatically promotes clearance of plasma triglycerides and uptake into BAT through membrane receptor CD36 [36]. Therefore, activating BAT or WAT browning could be a viable way to defend against diet-induced obesity or type 2 diabetes. In the clinically human translational application, β3-adrenergic receptor agonists, cold or other agents robustly activate human BAT and increase thermogenesis and energy expenditure both in healthy or obese human subjects. This cold stimulation in human studies can be achieved by staying in a cold-room or by putting on a running cooling water-filled suit with set temperature [37–42]. Therefore, application of PET/CT scanning Int. J. Mol. Sci. 2021, 22, 906 14 of 20

in these human clinical studies or in above mentioned preclinical mouse models clearly showed that human and mouse BAT can be activated by cold-stimulation, thereby resulting in elevated energy expenditure and contributing to body fat reduction. This current study also established a cold-induction procedure for mice to activate BAT or browned WAT assessed by PET/CT scanning. As opposed to a study of mice staying at 4 ◦C for C57BL/6 mice [43], we found it is much safer to perform cold-induction around 10 ◦C in a cold-room for our WT or transgenic AdipoR1 mice with FVB/NJNarl background. Therefore, strains of mouse species and transgene effects may account for different temperatures and periods of varying lengths for cold-induced thermogenesis.

3.3. Adiponectin/AdipoR1 on Modulating BAT and Browned WAT Adiponectin is a beneficial adipokine to resist diabetes and causes a “healthy obese” state by increasing white adipose expansion and by limiting lipotoxicity in other tissues during high-fat feeding. For example, a classic mouse model over-expressing adiponectin in the prone-obese ob/ob background generates a super-obese phenotype with twice the size of the controls of already obese ob/ob mice. Nevertheless, these super-obese mice over-expressing adiponectin are protected from glucose intolerance and insulin resistance [44,45]. Therefore, these studies indicated that adiponectin reduces energy expenditure and increases energy preservation in white adipose tissues. Other studies also support that adiponectin knockout mice showed a lean phenotype or fat reduction [46–48]. However, exogenous administration of adiponectin to mice decreases plasma glucose, free fatty acids and triglycerides, increases muscle fatty acid oxidation and induces weight loss [49]. One of the functions of adiponectin is carried out through the activation of AMPK [46,50]. Therefore, short-term induction versus long-term supply of adiponectin and exogenous administration versus gene overexpression/deletion may cause discrepancy and result in inconsistent results of fat reduction or expansion. In the current study, cold-acclimated AdipoR1 mice exhibited fat reduction of gWAT weights and showed whitening phenotype of iBAT assessed by H&E staining and by expression levels. AdipoR1 mice coupled with these fat alterations cannot resist acute cold exposure and flaunted lowered surface body and surface BAT depot temperature, accompanied with significant bodyweight losses. Therefore, these cold-acclimated AdipoR1 mice indicate implications of cold intolerance. Non-shivering thermogenesis primarily occurs in brown adipose tissues. BAT is enriched with abundant mitochondria identified with many brown-specific mitochondrial makers, such as Cox7a and Cox8b [51]. Thermogenic UCP1 in mitochondria is respon- sible for uncoupled respiration from ATP synthesis and generation of heat to maintain core body temperature, especially during cold-induced hypothermia and in diet-induced thermogenesis [52]. In this study, we found that these AdipoR1 female mice, after prolonged cold expo- sure, showed not only inhibited cold-induced BAT activity of glucose uptake assessed by PET/CT but also concomitant strong reduction of brown adipocyte markers, includ- ing thermogenic gene Ucp1, brown adipocyte markers Prdm16, Cidea, Dio2, Cpt1b, Elovl3, and Pgc1a, and BAT-specific mitochondrial markers Cox7a and Cox8b in classical BAT or browned iWAT. Furthermore, AdipoR1 female mice at RT already showed this diminished pattern which is even more pronounced in cold environments. In vitro gene expression analysis of brown adipocyte markers in differentiated beige adipocytes from iWAT of AdipoR1 mice also support in vivo results of PET/CT scanning and expression of brown adipocyte markers. Cold induction is known to decrease serum adiponectin concentrations through sympa- thetic activation in mice [53], especially evidenced in atherogenic conditions [54]. However, studies related to adiponectin receptors on cold-induced thermogenesis are limited. Our results of adiponectin receptor 1 on inhibition of thermogenesis are consistent with studies revealing that administering adiponectin also suppresses cold-induced thermogenesis and thermogenic UCP1 expression in mouse BAT [55]. Although the same study also showed that Int. J. Mol. Sci. 2021, 22, 906 15 of 20

ablating adiponectin in mice elevates while abating AdipoR1 or AdipoR2 in mice decreases cold-induce thermogenesis in vivo [55]. These results are confounding and inconclusive. One possible speculation is that reduced thermogenesis by adiponectin may be simultaneously through multiple adiponectin receptors or adiponectin signaling. Individually ablating each adiponectin receptor (AdipoR1, AdipoR2, or T-cadherin) separately may actually enhance adiponectin signaling by causing compensatory increases in other adiponectin receptors. Therefore, both over-expressing (in our mouse model) or ablating individual adiponectin receptor could all augment adiponectin signaling, thereby diminishing cold-induced thermo- genesis. However, detailed mechanisms await further investigation. Cold-induced thermogenesis requires fatty acids as energy fuels, largely attributed to WAT lipolysis. In response to cold exposure under hypothermia, the sympathetic nervous system sends signals to activate lipolysis and thermogenesis. Circulating liberated fatty acids from WAT lipolysis are taken up by BAT for mitochondrial oxidation and thermogenesis [56]. Mechanistically, adiponectin suppresses basal and catecholamine- induced lipolysis in white adipocytes mediated by inhibited protein-kinase-A signaling [57]. Therefore, adiponectin decreases lipolysis in WAT which could result in decreased fuel sources for other tissues including BAT. Recently, a new study also showed adiponectin or receptor agonist AdipoRon sup- presses cold-induced thermogenic gene expression and energy expenditure in vivo through limiting immune-cell ILC2s-activated thermogenesis in BAT or beige (browned WAT) adi- pose tissues [58]. Therefore, this current study showed that reduction of cold-induced thermogenesis in mice over-expressing AdipoR1 is congruent with other studies of inhib- ited thermogenesis in mice administered adiponectin and of increased thermogenesis in mice with ablated adiponectin. Therefore, the combination of above-mentioned results of inhibited BAT thermogenesis by adiponectin or receptor overexpression might be partly through fatty acid retention in WAT with diminished lipolysis in WAT, resulting in de- creased supply of fatty acids as thermogenic substrates. Moreover, we also found that over-expressing AdipoR1 suppresses WAT browning both in vivo and in vitro. Hence, in our mouse model, AdipoR1 inhibits cold-induced thermogenesis in both classical BAT and browned WAT. To conclude, the live 18F-FDG PET/CT scanning for measurements of cold-induced BAT activity in mice showed that mice over-expressing adiponectin receptor 1 exhibit reduced cold-induced glucose uptake in BAT and browned supWAT, accompanied with decreased surface body and surface BAT depot temperature and increased adipocyte size of whitening morphology in BAT and browned iWAT. Further in vitro and in vivo analyses indicated expression of thermogenic gene Ucp1, brown fat-specific markers and brown adipocyte-enriched mitochondrial markers are largely attenuated in BAT or browned WAT of AdipoR1 mice. However, white adipocyte markers in BAT or browned WAT are main- tained or elevated in AdipoR1 mice. These combined results with live PET/CT scanning reveal the role of adiponectin receptor 1 in regulating energy homeostasis, especially in cold-induced thermogenesis. Unveil detailed mechanisms underlying adiponectin signal- ing on brown adipose tissues or browning/beiging of white adipose tissues may provide insights for therapeutic application in obesity-associated metabolic diseases.

4. Materials and Methods 4.1. Animals and Diets Transgenic FVB/NJNarl mice over-expressing porcine adiponectin receptor 1 were generated through the pronuclear microinjection of fertilized eggs, and bred as previ- ously reported [23,24]. Briefly, founder mice were crossed with WT mice to generate F1 heterozygous (AdipoR1+/−) offspring. The F1 heterozygous mice were backcrossed with WT mice to generate the offsprings as F2 mice. F2 heterozygous mice were double- crossed to generate progeny of homozygous (AdipoR1+/+) littermates. These F3 littermates were checked as homozygous mice via quantitative real-time PCR analyses using DsRed2 primers (Supplementary Table S1) and GFP/RFP-fluorescent checking systems. All experi- Int. J. Mol. Sci. 2021, 22, 906 16 of 20

ments were performed on homozygous offsprings starting from the F4 or later generations housing at the mouse facility in our department. Adult age-paired male and female mice (>12 weeks old) of WT or with the transgene, from the F4 or later generations, were randomly assigned for each experimental group (n ≥ 3 for each sex per group), maintained at room temperature or at a cold-room with a light-dark cycle of 12: 12 h, and provided with feed (LabDiet 5001; LabDiet, St Louis, MO, USA) and water ad libitum. The animal experiments were approved by the Institutional Animal Care and Use Committee at National Taiwan University, Taipei and Institute of Nuclear Energy Research, Taoyuan, Taiwan (NTU-103-EL-42, NTU-106-EL-00088, NTU-108-EL-00076).

4.2. Cold-Acclimation Procedure First, a pre-test of cold-induction protocol for our mouse strain was implemented to evaluate cold-stimulated thermogenesis in mice. Because different genetic inbred or outbred backgrounds of mice may respond very differently regarding cold tolerance at discrete low temperature and exposure times, a pre-trial with small numbers of WT and transgenic AdipoR1 mice (n = 3) in the FVB/NJNarl genetic background were housed in a cold-room at 4, 7, or 10 ◦C. Below 10 ◦C, WT or transgenic mice cannot tolerate the cold for more than 7 d. This was especially prevalent at 4 ◦C (data not shown). Therefore, in the following experiments, cold-induced thermogenesis in WT or AdipoR1 of FVB/NJNarl mice will be evaluated at about 10 ◦C. Adult WT or AdipoR1 transgenic mice were divided and maintained on a regular chow diet. For cold exposure experiments after the pre-test, mice were transferred into a cold-room (8–10 ◦C) for two weeks and provided with feed and water ad libitum, with the same light-dark cycle of 12 h–12 h. Mice were closely monitored in cold environments and checked for their health and behavior at least once per day to prevent widespread sudden death from cold intolerance. Early termination will be initiated if extensive sudden death is observed.

4.3. Sample Collection and Preparation Food intake and body weights of each group were measured on d 0, 4, 7 and 11. For tissue sampling, mice were sacrificed using carbon dioxide. Peripheral adipose tissues, including subcutaneous inguinal, gonadal, and suprascapular white adipose tissues, and interscapular brown adipose tissues, were harvested, weights recorded, snap-frozen in liquid nitrogen, and stored at −70 ◦C for future analysis.

4.4. PET/CT Scanning After two weeks of cold acclimation with at least 6 h of terminal fasting, mice were escorted to the Institute of Nuclear Energy Research, Taoyuan, Taiwan for the 18F-FDG PET/CT Scanning (Mediso NanoPET/CT, Mediso Medical Imaging Systems, Budapest, Hungary). Before radiotracer injection, mice were anesthetized with 2% isoflurane injected through a tail vein. Then, 150 µCi of locally synthesized 18F-FDG was injected into the tail vein of the mice and mice were held in cages for 1 h waiting for biodistribution before scanning. Dynamic emission scans for each WT or AdipoR1 mouse were acquired for 20 min. The mice were constantly anesthetized with 0.1% isoflurane in air during the imag- ing. Results of 18F-FDG PET/CT images were coregistrated (constructed) using a PMOD software (PMOD Technologies LLC, Zurich, Switzerland) to estimate the radioactivity concentration. Volumes of interest and the activity were expressed as %ID/g.

4.5. Histological Analysis by H&E Stain To evaluate the structure of interscapular BAT and subcutaneous iWAT, adipose depots were harvested, fixed in 10% formalin and then embedded in paraffin. The paraffin- embedded specimens were sliced at 4 µm using a microtome. The sections were stained with H&E to observe the pathological morphology of mouse adipose tissues. Int. J. Mol. Sci. 2021, 22, 906 17 of 20

4.6. Depot Surface Temperature Measurements and Thermographic Imaging WT and transgenic AdipoR1 mice, before or after housing in the cold, were as- sessed for regional surface temperature on skins above brown adipose tissue areas and on the abdomen by an infrared thermometer (HFS-1000, Thermofinder Plus, HuBDIC, Gyeonggi-do, Korea). Readings were recorded with constant values at least 3 times. Thermography was performed using an infrared camera (IRM-P384G, Ching Hsing Computer-Tech Ltd., Taipei, Taiwan) on WT and transgenic AdipoR1 mice acclimated at a cold room. No anesthesia was applied to avoid disturbing mouse body temperature with human-handling.

4.7. Gene Expression Analysis by qPCR Total RNA was extracted from mouse tissues and cells by the TRIsure reagents (Bioline, Meridian Bioscience, London, UK), with contaminating genomic DNA later removed by the TURBO-DNase free kit (AM1907; Thermo Scientific, Waltham, MA, USA), followed by reverse transcription into cDNA using the High Capacity cDNA Reverse Transcription kit (Thermo Scientific). Quantitative real-time polymerase chain reaction (qPCR) was performed using a SensiFAST SYBR Hi-ROX Kit (Bioline) on an ABI StepOnePlus Real- Time PCR System (Applied Biosystems, Thermo Scientific) or a LightCycler 480 Instrument II (Roche Diagnostics, Indianapolis, IN, USA). qPCR was performed as follows; activate enzyme at 95 ◦C for 2 min, and then 40 cycles of 95 ◦C for 5 s and 60 ◦C for 30 s with a final extension of 60 ◦C for 1 min. Primers used for amplification of qPCR reaction are listed in Supplementary Table S1. The relative value for each target gene was normalized to β-actin expression in the same sample. The threshold cycle (Ct) values were obtained, and relative gene expression was calculated using the comparative Ct method. Amplification of specific transcripts was further confirmed by melting-curve analysis and agarose-gel electrophoresis to check resulting product specificity.

4.8. Cell culture for Isolation and Differentiation of Mouse Stromal/Vascular Cells into Mature Adipocytes Mouse pre-adipocytes within the stromal/vascular fraction (SVF) from subcutaneous iWAT were isolated from adult WT or transgenic AdipoR1 mice. Detailed procedures for pre-adipocyte isolation, cell culture and differentiation into mature adipocytes were described previously with slight modification [59–61]. Briefly, the mouse subcutaneous inguinal region of WAT was surgically dissected, minced and digested with collagenase (C6885, Sigma-Aldrich, St. Louis, MO, USA) and then filtered through chiffon. Pre- adipocytes within SVF were derived through a series of centrifugation and washing steps with red-blood-cell-lysis before the final washing step. Then, pre-adipocytes were sus- pended and seeded on tissue-culture treated plates containing medium of Dulbecco’s Modified Eagle Medium/F12 (Gibco DMEM/F12, Thermo Scientific), 10% fetal bovine serum (Thermo Scientific) and 1% antibiotics (penicillin, streptomycin and amphotericin B; Biological Industries, Kibbutz Beit-Haemek, Israel). Cells were incubated at 37 ◦C in air containing 5% CO2 until confluency. Confluent pre-adipocytes were subsequently switched to induction medium with or without the PPARγ-agonist rosiglitazone for adipocyte differ- entiation. After around 7 to 9 d, mature adipocytes with at least 70% differentiation were collected for further analysis.

4.9. Statistical Analysis Data were expressed as means ± standard error of the mean (SEM). Results of two groups were compared and determined for statistical significance by the Student’s t-test (Graphpad Prism 6; Graphpad Software, San Diego, CA, USA). The p-values ≤ 0.05 were considered statistically significant.

Supplementary Materials: Supplementary materials can be found at https://www.mdpi.com/1422 -0067/22/2/906/s1. Int. J. Mol. Sci. 2021, 22, 906 18 of 20

Author Contributions: Conceptualization, Y.-J.C., S.-T.D.; Methodology, Y.-J.C., C.-W.L., Y.-J.P., C.- W.H., Y.-Y.L.; Software, M.-H.W.; Validation, Y.-J.C.; Formal Analysis, Y.-J.C.; Investigation, Y.-J.C., Y.-S.C., T.-H.H., P.-X.L., W.-Y.Y.; Resources, M.-H.W., S.-C.W., T.-Y.C., Y.-Y.L.; Data Curation, Y.-J.C., H.J.M., S.-T.D.; Writing—Original Draft Preparation, Y.-J.C.; Writing—Review & Editing, Y.-J.C., H.J.M., Y.-Y.L., S.-T.D.; Visualization, Y.-J.C.; Funding Acquisition, T.-Y.C., W.-H.K., S.-T.D.; Project Administration, S.-T.D.; Project Supervision, S.-T.D. All authors have read and agreed to the published version of the manuscript. Funding: Research performed in the lab was supported by grants from Ministry of Science and Technology, National Taiwan University Hospital and College of Medicine, and National Taiwan University, Taiwan to T.-Y.C., W.-H.K. and S.-T.D. Institutional Review Board Statement: The animal experiments were approved by the Institutional Animal Care and Use Committee at National Taiwan University, Taipei and Institute of Nuclear Energy Research, Taoyuan, Taiwan (NTU-103-EL-42, 1 August 2014; NTU-106-EL-00088, 1 May 2017; NTU-108-EL-00076, 1 August 2019). Informed Consent Statement: Not applicable. Data Availability Statement: The data that support the findings of this study are available from the corresponding author upon reasonable request. Acknowledgments: The authors would like to express gratitude to all the lab members for the extensive discussion and technique supports in this study. Conflicts of Interest: The authors declare no conflict of interest.

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