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# Glucocorticoid-Induced Obesity Develops Independently of UCP1

Ineke H N Luijten, Katie Brooks, Nathalie Boulet, Irina G Shabalina, Ankita

Jaiprakash, Bo Carlsson, Alexander W Fischer, Barbara Cannon, Jan

Nedergaard

► To cite this version:

Ineke H N Luijten, Katie Brooks, Nathalie Boulet, Irina G Shabalina, Ankita Jaiprakash, et al. Glucocorticoid-Induced Obesity Develops Independently of UCP1. Cell Reports, 2019, 27 (6), pp.1686-1698.e5. 10.1016/j.celrep.2019.04.041. hal-04780387

HAL Id: hal-04780387

https://ut3-toulouseinp.hal.science/hal-04780387v1

Submitted on 13 Nov 2024

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# Cell Reports

## Glucocorticoid-Induced Obesity Develops Independently of UCP1

### Graphical Abstract

<figure><img src="image_3.png" /></figure>

### Authors

Ineke H.N. Luijten, Katie Brooks, Nathalie Boulet, ..., Alexander W. Fischer, Barbara Cannon, Jan Nedergaard

### Correspondence

jan@metabol.su.se

### In Brief

Glucocorticoid treatment induces obesity, and reduced brown fat thermogenesis has been suggested to be the cause. However, Luijten et al. show here that this is not the case, nor is it fully explainable by overeating. Thus, glucocorticoids channel food energy into lipid storage through a currently unexplained pathway.

### Highlights

* Glucocorticoid-induced obesity is thought to be due to decreased UCP1 expression

* Only at thermoneutrality do glucocorticoids lower UCP1 amounts

* Glucocorticoid-induced obesity develops to the same extent with or without UCP1

* Enhanced food intake cannot fully explain glucocorticoid-induced obesity

<figure><img src="image_4.png" /></figure>
# Glucocorticoid-Induced Obesity Develops Independently of UCP1

Ineke H.N. Luijten,<fnref n="1" /> Katie Brooks,<fnref n="1" />,<fnref n="3" /> Nathalie Boulet,<fnref n="1" />,<fnref n="4" /> Irina G. Shabalina,<fnref n="1" /> Ankita Jaiprakash,<fnref n="1" />,<fnref n="3" /> Bo Carlsson,<fnref n="1" />,<fnref n="5" /> Alexander W. Fischer,<fnref n="1" />,<fnref n="2" /> Barbara Cannon,<fnref n="1" /> and Jan Nedergaard<fnref n="1" />,<fnref n="6" />,*

## SUMMARY

An excess of glucocorticoids leads to the development of obesity in both mice and humans, but the mechanism for this is unknown. Here, we determine the extent to which decreased BAT thermogenic capacity (as a result of glucocorticoid treatment) contributes to the development of obesity. Contrary to previous suggestions, we show that only in mice housed at thermoneutrality (30°C) does corticosterone treatment reduce total BAT UCP1 protein. This reduction is reflected in reduced brown adipocyte cellular and mitochondrial UCP1-dependent respiration. However, glucocorticoid-induced obesity develops to the same extent in animals housed at 21°C and 30°C, whereas total BAT UCP1 protein levels differ 100-fold between the two groups. In corticosterone-treated wild-type and UCP1 knockout mice housed at 30°C, obesity also develops to the same extent. Thus, our results demonstrate that the development of glucocorticoid-induced obesity is not caused by a decreased UCP1-dependent thermogenic capacity.

## INTRODUCTION

High glucocorticoid levels promote the development of obesity (Lee et al., 2014). In humans, symptoms of diseases involving hypercortisolism, such as Cushing's syndrome, include a redistribution of adipose depots, that is, an accumulation of visceral white adipose tissue (WAT) depots and a reduction of subcutaneous WAT depots (Rockall et al., 2003). Glucocorticoid-induced central obesity often goes hand in hand with symptoms of the metabolic syndrome, such as insulin resistance and hyperlipidemia (Pasieka and Rafacho, 2016). Also in mice, systemic high levels of endogenous corticosterone lead to WAT growth, hyperglycemia, and reduced insulin tolerance, as demonstrated

in a recently developed mouse model for Cushing's syndrome (Dumontet et al., 2018).

The exact mechanisms behind the development of obesity and obesity-related diseases in response to high levels of glucocorticoids are not known. However, an involvement of brown adipose tissue (BAT), the main site for non-shivering thermogenesis in most mammals, has been discussed (van den Beukel et al., 2015; Liu et al., 2013; Lv et al., 2018; Strack et al., 1995; Viengchareun et al., 2001). Normally, upon activation by the sympathetic nervous system in response to cold exposure or certain diets, BAT produces heat through the tissue-specific mitochondrial uncoupling protein 1 (UCP1) that uncouples substrate oxidation from ATP production and dissipates the proton gradient across the inner mitochondrial membrane as heat (Cannon and Nedergaard, 2004). Therefore, by increasing energy expenditure, activated BAT may significantly reduce the percentage of ingested energy that is stored in the body (i.e., decrease metabolic efficiency). Accordingly, a reduction of BAT activity may be linked to the obesity-promoting effect of glucocorticoids.

Indeed, glucocorticoids are generally thought to suppress BAT thermogenesis. In rodents, glucocorticoid administration has been suggested to reduce BAT UCP1 mRNA and protein levels (van den Beukel et al., 2015; Poggioli et al., 2013; Strack et al., 1995), and it has therefore been hypothesized that the implied reduction in BAT thermogenesis is at least partially what causes glucocorticoid-induced obesity. Although there may be species differences between rodents and humans with respect to the acute effects of glucocorticoids (Ramage et al., 2016), there is agreement that chronic glucocorticoid excess suppresses BAT activity in humans as well as in mice (van den Beukel et al., 2015; Poggioli et al., 2013; Ramage et al., 2016; Strack et al., 1995; Thuzar et al., 2018). However, the extent to which reduced BAT activity actually contributes to glucocorticoid-induced obesity has not been determined. In this study, we have therefore examined the precise effects of corticosterone administration on BAT in mice and determined to which extent these effects cause glucocorticoid-induced obesity. We show that glucocorticoid-induced obesity develops fully independently of the presence, amounts or absence of UCP1.

<footnote n="1">¹Department of Molecular Biosciences, the Wenner-Gren Institute, Stockholm University, 106 91 Stockholm, Sweden</footnote>

<footnote n="2">²Department of Biochemistry and Molecular Cell Biology, University Medical Center Hamburg Eppendorf, Hamburg, Germany</footnote>

<footnote n="3">³Present address: School of Biosciences and Medicine, University of Surrey, Guildford GU2 7XH, UK</footnote>

<footnote n="4">⁴Present address: INSERM, UMR 1048, Team 1, I2MC, Institute of Metabolic and Cardiovascular Diseases, Université de Toulouse, BP84225, 31432 Toulouse Cedex 4, France</footnote>

<footnote n="5">⁵Present address: Karolinska Innovations AB, Berzelius Väg 3, 171 56 Stockholm, Sweden</footnote>

<footnote n="6">⁶Lead Contact</footnote>

<footnote>*Correspondence: jan@metabol.su.se
https://doi.org/10.1016/j.celrep.2019.04.041</footnote>
<figure><img src="image_5.png" /><figcaption>**Figure 1.** Under Standard Housing Conditions, 2-Week Corticosterone Treatment Increases Metabolic Efficiency but Does Not Reduce Total IBAT UCP1 Protein Levels

Mice were kept at 21°C for the duration of the study.</figcaption></figure>

(A) Fat mass and lean mass before and after veh or CORT.

(B) Food intake (kJ/day).

(C) Metabolic efficiency calculated from (A) and (B).

(D) NE-induced (1 mg/kg) O₂ consumption in anaesthetized mice after veh or CORT.

(E) Quantification of (D): NE-induced O₂ consumption = peak NE O₂ consumption minus basal O₂ consumption.

(F) Representative immunoblot showing UCP1 protein in IBAT homogenates with β-tubulin as a loading control.

(G) Quantification of (F) showing UCP1 per microgram protein, total protein per depot, and total UCP1 per depot.

(H) Representative immunoblot showing UCP1 protein in ingWAT homogenates with 50× diluted IBAT from (F) as a positive control and Ponceau staining as a loading control.

Data are represented as mean ± SEM; n = 9. \*p < 0.05, \*\*p < 0.01, and \*\*\*p < 0.001; two-way ANOVA with Bonferroni post-test, significance within each time point between treatments (A and B); Student's t test (C, E, and G).

See also Figures S1, S2, S6, and S11.

## RESULTS

### In Mice Living under Standard Housing Conditions, Corticosterone Induces Obesity but Does Not Affect Total IBAT UCP1 Levels

As indicated above, it has been reported that glucocorticoid administration induces obesity and reduces interscapular BAT (IBAT) UCP1 protein levels in rodents. To examine whether the observed downregulation in UCP1 corresponds to a decrease in whole-body thermogenic capacity, we determined the metabolic and thermogenic effects of glucocorticoid supplementation in male C57Bl6/J mice housed at 21°C. We treated the mice with 50 µg/mL corticosterone (CORT) or corresponding vehicle (veh; 0.25% EtOH) in their drinking water for 2 weeks (Auvinen et al., 2013; Karatsoreos et al., 2010). CORT mice ingested an average of 5 mg corticosterone in total during the treatment period (Figures S1A and S1B). Provided that all the supplemented corticosterone was absorbed by the animals, CORT led to an approximate doubling in physiological corticosterone concentrations, as determined by fecal corticosterone excretion (Figure S1C). There was a significant reduction in thymus weight in CORT mice, a well-known effect of glucocorticoid treatment (Cohen, 1992) (Figure S1D). CORT significantly increased body weight (Figure S1E) and fat mass and reduced lean mass

(Figure 1A). The increases in body weight and fat mass were in accordance with the fact that CORT mice increased their food intake during the second week of treatment with an average of 8 kJ/day (Figure 1B) and did not show any changes in energy expenditure or substrate use (Figures S1F–S1I). However, the mice receiving CORT showed a marked increase in metabolic efficiency (determined as the percentage of ingested energy that is stored in the body) (Figure 1C). The additional energy that the CORT mice consumed compared with the veh-treated mice was in total ~70 kJ. The thermic effect of the extra food (the obligatory physiological handling cost) would be 11.7% of the energy consumed (see STAR Methods), thus leaving ~62 kJ for possible storage. Because the energy content of the total “extra” body mass change (increase in fat minus decrease in lean) for the CORT mice over the veh mice during the treatment period was ~105 kJ, we conclude that CORT animals not only stored 100% of their “extra” energy intake but also stored a greater percentage of the “basal” food intake that they would have had they not been treated with CORT. Thus, CORT induced the development of obesity in mice not solely due to the extra food intake but also due to a reduction in the underlying metabolism.

As mentioned above, corticosterone has been reported to downregulate BAT UCP1 protein levels (van den Beukel et al., 2015; Strack et al., 1995). Therefore, a reduction in underlying BAT thermogenesis could be the cause of the increase in metabolic efficiency observed in the CORT mice. BAT thermogenesis
is activated by the sympathetic neurotransmitter norepinephrine (NE), and BAT thermogenic capacity can be evaluated in sedated mice after a bolus injection of NE. The response to NE consists of a BAT-mediated response, the magnitude of which varies with acclimation temperature, plus a non-adaptive “pharmacological” response (Golozoubova et al., 2006).

Because of the inability of sedated mice to regulate their body temperature, basal and NE-induced O₂ consumption was measured at thermoneutrality (30°C). Under these conditions, CORT mice had significantly increased basal oxygen consumption (Figures 1D and 1E). As we observed no differences in energy expenditure between veh and CORT mice at 21°C (Figures S1F and S1G), this reveals an influence of environmental temperature on experimental outcomes. Unexpectedly, we did not observe a reduction but rather a trend to an upregulation of NE-induced oxygen consumption after CORT (Figures 1D and 1E). Because this indicated that there was no suppressive effect of corticosterone on BAT thermogenesis, we isolated IBAT and inguinal WAT (ingWAT) tissues from these mice and determined UCP1 protein levels. Although UCP1 protein per microgram protein in IBAT was significantly reduced in CORT mice (Figures 1F and 1G), the total amount of protein in IBAT was significantly increased, leading to no change in the total amount of UCP1 protein per IBAT depot (Figure 1G). This is a more physiologically relevant parameter to evaluate thermogenic capacity than UCP1 levels per microgram protein and is thus in accordance with the absence of a decrease in NE-induced oxygen consumption. In ingWAT, total protein content did not change (Figure S1J). No UCP1 protein was detected in the ingWAT of either veh or CORT mice (Figure 1H).

Histological analysis of adipose tissues showed an increase in the number of larger, more lipid-filled adipocytes in CORT mice, confirmed by analysis of the pictures with Adiposoft software: the mean adipocyte area was markedly right shifted (Figures S2A and S2B). Total adipose tissue weights were significantly increased by CORT (Figure S2C). In parallel with the increase in IBAT total protein levels seen in CORT mice, also IBAT total RNA levels were increased in this treatment group (Figure S2D). Calculation of total IBAT *Ucp1* mRNA levels revealed no differences between groups, again corresponding to total IBAT UCP1 protein levels being unchanged (Figure S2E). Although we did measure a significant decrease in total ingWAT *Ucp1* mRNA in response to CORT, the total ingWAT *Ucp1* mRNA levels were ~350-fold lower than total IBAT *Ucp1* mRNA levels and were therefore most likely of little physiological significance (Figures S2E and S2F).

Thus, under standard housing conditions, total BAT thermogenic capacity is undiminished in CORT mice.

## At Thermoneutrality, Corticosterone Significantly Lowers IBAT UCP1 Protein Levels

Similarly to the case in previous studies, the experiments above were performed on mice housed at standard housing temperatures of about 21°C. This is significantly below their thermoneutral zone (about 30°C). These housing conditions influence experimental metabolic outcomes, because animals must continuously expend extra energy to defend their body temperature (Cannon and Nedergaard, 2011). With regard to the exper-

iments reported above (Figures 1, S1, and S2), there is thus a concern that the cold exposure may have masked any effects of corticosterone on UCP1 protein levels. Additionally, housing mice at temperatures within their thermoneutral zone more closely mimics the thermal environment of humans, making experimental results more applicable to the human condition (Fischer et al., 2018; Ganeshan and Chawla, 2017; Reitman, 2018). For these reasons, we investigated the effects of CORT on energy metabolism and IBAT UCP1 protein levels in animals housed at 30°C.

To first establish the appropriate corticosterone dose for supplementation at 30°C, we evaluated the effects of different corticosterone levels in the drinking water on several metabolic parameters (Figure S3). Maximal effects on food intake, fat accumulation, and metabolic efficiency were reached in mice receiving 50 µg/mL corticosterone (Figures S3F-S3H). We therefore decided, also at 30°C, to supplement the animals with 50 µg/mL corticosterone in the drinking water (denoted CORT).

At 30°C, CORT mice doubled their water intake compared with veh mice and ingested an average of 6.7 mg corticosterone in total during the treatment period (Figures S4A and S4B). Similarly to mice housed at 21°C, CORT mice at 30°C showed increased fecal corticosterone excretion as well as thymus involution (Figures S4C and S4D). Serum corticosterone levels were increased in CORT mice at the start of the light period (after the mice had been awake and drinking during the night) and were unchanged compared with veh mice at the start of the dark period (Figure S4E), indicating an altered circadian rhythmicity. Also at 30°C, body weight and fat mass were increased, and lean mass was reduced, in CORT mice (Figures S4F and 2A). Food intake in both treatment groups was lower compared with the food intake of mice housed at 21°C, which is expected in the absence of a need for thermoregulatory energy expenditure (Figures 1B and 2B). CORT treatment at 30°C increased energy intake by 5 kJ/day during the first week and 10 kJ/day during the second week of treatment compared with veh treatment (Figure 2B). Metabolic efficiency was also significantly increased (Figure 2C). In contrast to the mice housed and studied under standard conditions (Figures S1F and S1G), CORT increased average energy expenditure in the light phase (but did not affect respiratory quotient) (Figures S4G-S4I). This thus revealed that standard housing conditions masked important metabolic effects of glucocorticoids (which were, however, observable even in these mice when they were studied at 30°C; Figures 1D and 1E). The increase in average expenditure in CORT mice at 30°C may be related to the increase in food intake seen in these mice while in the metabolic chambers and was also observed in anaesthetized mice (Figures 2D, 2E, and S4J). The NE-induced oxygen consumption was, as expected, markedly lower than in the mice that had been housed at 21°C (Figures 1D and 2D). However, we found no differences between the experimental groups at 30°C (Figures 2D and 2E). It was therefore surprising that western blot analyses of IBAT proteins revealed that there was a decrease of ~4-fold in total IBAT UCP1 protein amount per depot in CORT mice (Figures 2F and 2G). However, when we directly compared the total IBAT UCP1 protein levels in mice housed at 30°C with the levels in
<figure><img src="image_6.png" /><figcaption>Figure 2. CORT Increases Metabolic Efficiency and Reduces Total IBAT UCP1 Protein Amount at Thermoneutrality</figcaption></figure>

Mice were kept at 30°C for the duration of the study.

(A) Fat mass and lean mass before and after veh or CORT.

(B) Food intake (kJ/day).

(C) Metabolic efficiency calculated from (A) and (B).

(D) NE-induced (1 mg/kg) O₂ consumption in anesthetized mice after veh or CORT.

(E) Quantification of (D): NE-induced O₂ consumption = peak NE O₂ consumption minus basal O₂ consumption.

(F) Representative immunoblot showing UCP1 protein in IBAT homogenates with Ponceau staining as a loading control.

(G) Quantification of (F) showing UCP1 per microgram protein, total protein per depot, and total UCP1 per depot.

(H) Representative immunoblot comparing UCP1 protein in IBAT homogenates from mice housed at 21°C (samples as in Figure 1F, but 10× diluted) and 30°C (samples as in Figure 2F) with Ponceau staining as a loading control. Quantification of blot calculated as total UCP1 protein per depot.

(I) Representative immunoblot showing UCP1 protein in ingWAT homogenates with 2x diluted IBAT from (F) as a positive control and Ponceau staining as a loading control.

Data are represented as mean ± SEM; n = 9. \*p < 0.05, \*\*p < 0.01, and \*\*\*p < 0.001; two-way ANOVA with Bonferroni post-test, significance within each time point between treatments (A and B); Student's t test (C, E, and G); one-way ANOVA with Tukey post-test (H).

See also Figures S3, S4, S6, S10, and S11.

mice housed at 21°C (Figure 2H), it became evident that merely housing the animals at thermoneutrality decreased total IBAT UCP1 protein levels ~100-fold. Most likely, the remaining amount of UCP1 at 30°C is too small to be visible as an increase in oxygen consumption, and therefore the ~4-fold CORT-induced decrease in total IBAT UCP1 protein levels at 30°C was unable to visibly influence the NE response. Nonetheless, housing mice at thermoneutrality revealed an ability of CORT to downregulate total IBAT UCP1 protein levels, whereas these effects were masked when housing the animals below their thermoneutral zone (Figures 1D-1G versus Figures 2D-2G). UCP1 protein levels in ingWAT were again non-detectable (Figures 2I and S4K).

## The CORT-Induced Reduction in IBAT UCP1 Protein Levels Is Reflected Functionally in Cellular and Mitochondrial Respiration

We determined whether the reduction in IBAT UCP1 protein levels in CORT mice had functional consequences at the level of the individual cells or mitochondria.

Mature brown adipocytes were isolated from veh or CORT mice housed at 30°C, and aliquots of the preparations were

analyzed by western blot. We observed a reduction in UCP1 protein in the cells from the CORT mice (Figures 3A and 3B). Oxygen consumption of the isolated mature brown adipocytes (Figures 3C and 3D) was measured in the presence of oligomycin to block all ATP synthase-linked respiration. Subsequently, NE was added to induce thermogenesis. The increase in brown adipocyte oxygen consumption following NE addition is fully UCP1 dependent, as no such increase is visible in brown adipocytes isolated from UCP1-knockout (KO) mice (Matthias et al., 2000). The lower levels of UCP1 protein in isolated mature brown adipocytes from CORT mice were reflected in lower NE-induced oxygen consumption (Figures 3D and 3E). This decrease was not due to a limitation in respiratory chain capacity, as brown adipocytes from CORT mice responded to the artificial uncoupler FCCP with an increase in oxygen consumption (Figures 3D and 3E).

Isolated mitochondria were prepared from IBAT homogenates from veh or CORT mice housed at 30°C, and respiration was measured with pyruvate as substrate. The initial respiratory rate, which is limited by UCP1 capacity, was lower in the IBAT mitochondria from CORT mice (Figures 3F and 3G). We found no differences between groups in basal proton leak or in
Mature brown adipocytes isolated from treated mice

<figure><img src="image_7.png" /></figure>

<figure><img src="image_8.png" /></figure>

<figure><img src="image_9.png" /></figure>

<figure><img src="image_10.png" /></figure>

IBAT mitochondria isolated from treated mice

<figure><img src="image_11.png" /></figure>

<figure><img src="image_12.png" /></figure>

<figure><img src="image_13.png" /><figcaption>Figure 3. CORT Reduces Cellular NE-Induced Respiration and Mitochondrial UCP1 Activity</figcaption></figure>

Mice were kept at 30°C for the duration of the study. (A) Immunoblot showing UCP1 protein in isolated mature brown adipocytes from mice that received veh or CORT, with Ponceau staining as a loading control.

(B) Quantification of (A) (n = 3).

(C) Total amount of mature adipocytes added to the respiratory chamber for (D) and (E) as determined by DNA extraction (n = 3).

(D) Representative oxygen trace of mature brown adipocytes after addition of oligomycin (2 µg/mL), NE (1 µM), and FCCP (titration with 40 µM).

(E) Quantification of mature brown adipocyte oxygen consumption traces (n = 6).

(F) Representative oxygen trace of mitochondria (Mit) isolated from IBAT from mice that received veh or CORT. Respiratory activity measured in the presence of pyruvate (5 mM) and after addition of GDP (2 mM), ADP (0.5 mM), CATR (3 µM), and FCCP (titration to 2.2-2.8 µM). White boxes represent re-oxygenation of the chambers.

(G) Quantification of mitochondrial oxygen consumption traces (veh, n = 4; CORT, n = 3).

(H) Quantification of UCP1 activity (pyr - GDP) and oxidative phosphorylation-linked respiration (ADP - CATR) (veh, n = 4; CORT, n = 3).

Data are represented as mean ± SEM. **p < 0.01 and ***p < 0.001; Student's t test (B, C, and H); two-way ANOVA with Bonferroni post-test, significance within each addition between treatments (E and G).

oxidative phosphorylation capacity, but the maximal respiratory capacity was decreased in mitochondria from CORT mice (Figures 3F-3H). The activity of UCP1, which is the respiration inhibited by the addition of GDP (pyr minus GDP) was reduced by approximately 80% in IBAT mitochondria from CORT mice (Figure 3H), demonstrating that UCP1 protein had a functionally lower content in these mitochondria compared with IBAT mitochondria isolated from veh mice.

CORT Alters Adipose Tissue Histology Independently of the Downregulation of IBAT UCP1

In accordance with the increase in body weight of the CORT mice housed at 30°C, total IBAT, ingWAT and epididymal WAT (eWAT) depot weights were increased compared with veh mice (Figures 4A, S5A, and S5B). Individual adipocyte size was also increased in all adipose depots of the CORT mice (Figures 4B, 4C, S5C, and S5D). The fact that these increases were comparable with those seen in mice housed at 21°C (Figures S2A and S2B) indicates that the effects of CORT on adipose tissue expansion happen independently of variations in the total amount of IBAT UCP1.

We next determined whether the downregulation of IBAT UCP1 by effect of CORT supplementation at 30°C was visible at the transcriptional level. We did not find any CORT-induced difference in total IBAT, ingWAT and eWAT RNA levels (Figures 4D and S5E). This is in parallel with total adipose tissue protein levels in these mice (Figures 2G and S4K). The mRNA levels of thermogenic and adipogenic genes in adipose tissues were

normalized to the reference gene *Tbp* (TATA-box binding protein), the expression of which was rather similar across adipose tissues and treatments (Figures 4E, S5F, and S5G). In IBAT, total *Ucp1* mRNA levels were reduced by approximately 70%, indicating that the suppression of IBAT UCP1 protein levels by CORT probably occurs at the transcriptional level (Figure 4E). No physiologically relevant *Ucp1* mRNA levels were detected in ingWAT and eWAT (Figures S5F and S5G).

We thus conclude that although the expression of IBAT *Ucp1* mRNA is suppressed by CORT in mice housed at thermoneutral-ity, the total amount of UCP1 protein present in IBAT does not influence the extent to which CORT induces adipose tissue expansion.

CORT Alters Adipose Tissue Metabolic Phenotype

In both IBAT and eWAT, the expression of the glucocorticoid receptor (Nr3c1) was downregulated by CORT, while it was upregulated in ingWAT. The expression of *Hsd11b1*, the gene that codes for 11β-hydroxysteroid dehydrogenase type 1, the enzyme that catalyzes the conversion of inactive 11-dehydrocorticosterone to active corticosterone, was upregulated in all adipose tissues (Figures 4E, S5F, and S5G).

We found no difference in the expression of *Fabp4* (fatty acid-binding protein 4; aP2), a gene that is routinely used as a marker for adipocyte differentiation, in IBAT, whereas its expression was significantly increased in ingWAT and eWAT of CORT mice. The expression of the lipogenic genes *Fasn* (fatty acid synthase) and *Elov13* (elongation of very long chain fatty acids protein 3) was
<figure><img src="image_14.png" /><figcaption>Figure 4. The Downregulation of UCP1 by CORT at Thermoneutrality Occurs at the Transcriptional Level</figcaption></figure>

Mice were kept at 30°C for the duration of the study.

(A) Total IBAT weights (n = 6).

(B) Representative H&E stainings of IBAT depots.

(C) Frequency distribution of mean adipocyte area as analyzed using Adiposoft (n = 3 mice per treatment, four pictures per mouse).

(D) Total RNA in IBAT homogenates (n = 7).

(E) Expression of thermogenic and adipogenic genes in IBAT: uncoupling protein 1 (Ucp1), glucocorticoid receptor (Nr3c1), 11β-hydroxysteroid dehydrogenase (Hsd11b1), fatty acid-binding protein 4 (Fabp4), fatty acid synthase (Fasn), elongation of very long chain fatty acids protein 3 (Elovl3), adipose triglyceride lipase (Atgl), hormone-sensitive lipase (Hsl), and TATA-box binding protein (Tbp, reference gene) (n = 7).

Data are represented as mean ± SEM. \*p < 0.05, \*\*p < 0.01, and \*\*\*p < 0.001; Student's t test (A, D, and E); two-way ANOVA with Bonferroni post-test, significance within each bin size between treatments (C).

See also Figures S5 and S6.

upregulated by CORT in all adipose tissues, whereas the expression of the lipolytic genes *Atgl* (adipose triglyceride lipase) and *Hsl* (hormone-sensitive lipase) was upregulated only in ingWAT and eWAT, perhaps indicating increased lipid turnover (Figures 4E, S5F, and S5G). Concerning glucose metabolism, we found that the mice were not diabetic under any of the experimental conditions examined here (Figure S6). Despite elevated insulin levels, they remained glucose tolerant.

## The Complete Absence of IBAT UCP1 Protein Does Not Affect the Development of Glucocorticoid-Induced Obesity

Suppression of BAT thermogenic capacity has previously been hypothesized to contribute to the development of obesity and obesity-related diseases. In mice housed at thermoneutrality, the ablation of UCP1 promotes the development of obesity, presumably by eliminating BAT-mediated diet-induced thermogenesis (Feldmann et al., 2009; Luijten et al., 2019; Rowland et al., 2016). The above data raised the question of the significance of the presence of IBAT UCP1 protein for the development of CORT-induced obesity. To determine the extent to which the total absence of UCP1 would influence the development of CORT-induced obesity, we housed a separate cohort of wild-type and UCP1-KO mice at 30°C and treated them with either veh or CORT.

During CORT treatment, UCP1-KO mice drank slightly but not significantly less water than wild-type mice and thus ingested a similar amount of corticosterone (Figures S7A and S7B). Wild-

type and UCP1-KO mice receiving CORT both increased body weight, gained similar amounts of fat mass, and lost similar amounts of lean mass compared with the veh controls (Figures S7C and 5A). Food intake in mice of both genotypes was significantly increased because of CORT treatment (Figure 5B). No statistically significant differences were found in energy expenditure, respiratory quotient, or daily food intake in the metabolic chambers between treatment groups or between genotypes (Figures S7D–S7G), somewhat different from the results shown in Figures S4G–S4J. The presence or complete absence of UCP1 did not affect the ability of CORT to increase metabolic efficiency (Figure 5C). CORT mice of both genotypes displayed a higher level of basal respiration than veh mice under anesthesia, as was also visible in our other cohorts (Figures 5D and 5E). Again, the NE-induced oxygen consumption was unchanged in wild-type CORT mice compared with wild-type veh mice (Figures 5D and 5E). In UCP1-KO mice, the NE-induced oxygen consumption was significantly lower than in the wild-type mice, but no significant differences were found between KO veh and KO CORT mice (Figures 5D and 5E). The generally lower response in the UCP1-KO mice compared with the wild-type mice may imply an additional effect of the complete lack of UCP1 in these animals. Total IBAT UCP1 protein levels were significantly downregulated in response to CORT in the wild-type mice and were undetectable in UCP1-KO mice (Figures 5F and 5G). Again, no UCP1 protein was detected in ingWAT (Figures 5H and S7H). Thus, although CORT at thermoneutrality
<figure><img src="image_15.png" /><figcaption>Figure 5. CORT-Induced Increases in Fat Mass and Metabolic Efficiency Develop Independent of the Presence of UCP1</figcaption></figure>

Wild-type and UCP1-KO mice were kept at 30°C for the duration of the study.

(A) Fat mass and lean mass before and after veh or CORT.

(B) Food intake (kJ/day).

(C) Metabolic efficiency calculated from (A) and (B).

(D) NE-induced (1 mg/kg) O₂ consumption in anesthetized mice after veh or CORT. Left: wild-type; right: UCP1-KO.

(E) Quantification of (D): NE-induced O₂ consumption = peak NE O₂ consumption – basal O₂ consumption.

(F) Representative immunoblot showing UCP1 protein in IBAT homogenates with Ponceau staining as a loading control.

(G) Quantification of (F) showing UCP1 per microgram protein, total protein per depot, and total UCP1 per depot. ND, non-detectable.

(H) Representative immunoblot showing UCP1 protein in ingWAT homogenates with 2x diluted IBAT from (F) as a positive control and Ponceau staining as a loading control.

Data are represented as mean ± SEM; n = 6. \*p < 0.05, \*\*p < 0.01, and \*\*\*p < 0.001; two-way ANOVA with Bonferroni post-test, significance within each time point between treatments (A and B); one-way ANOVA with Tukey post-test (C, E, and G). See also Figures S7, S8, and S11.

reduces total IBAT UCP1 protein levels in mice, it does not alter the NE response, and, importantly, the presence or complete absence of UCP1 does not affect the development of CORT-induced obesity.

The complete absence of UCP1 may be expected to have only minor physiological effects in mice housed at thermoneutrality, as BAT exhibits UCP1-dependent diet-induced thermogenesis, but not thermoregulatory thermogenesis, at this temperature. However, also in mice housed under mild cold exposure (21°C), UCP1 ablation does not alter the extent to which CORT increases body weight, fat mass, or metabolic efficiency (Figure S8).

Thus, glucocorticoid-induced obesity develops independently of UCP1, regardless of the recruitment state of BAT.

## Long-Term Corticosterone Treatment Leads to a Progressive Increase in Fat Mass but a Normalization of Total IBAT UCP1 Protein Levels

Previous studies have indicated that in certain animal models, the effects of glucocorticoid treatment on adipose tissue biology may differ depending on the length of treatment (Auvinen et al., 2013). As shown above, short-term glucocorticoid treatment at thermoneutrality led to development of an obese phenotype independent of the presence of UCP1. We investigated whether long-term glucocorticoid treatment led to similar results. For this purpose, we housed male C57Bl6/J mice at 30°C from 10 weeks of age and treated them as described above from 12 until 24 weeks of age (denoted veh12 or CORT12). During the entire treatment

period, CORT12 mice drank significantly more than veh12 mice (Figure S9A). In total, they ingested an average of 28 mg corticosterone (Figure S9B). The body weights of CORT12 mice increased progressively throughout the treatment period (Figure S9C), which was due to rapid, massive increases in fat mass after week 2 of treatment, when the difference in lean mass between both treatment groups stabilized (Figures 6A and S9D). Total food intake over the whole treatment period was significantly higher in CORT12 mice (Figure 6B). CORT12 also led to an increase in energy expenditure in the light phase (Figures S9E–S9H). Interestingly, exposure to corticosterone for 12 weeks reduced the effects on metabolic efficiency seen in mice receiving corticosterone for 2–7 weeks (Figure 6C). In addition, long-term exposure to corticosterone led to a small trend toward an increase in NE-induced oxygen consumption (Figures 6D and 6E) and normalized total IBAT UCP1 protein levels (Figures 6F, 6G, and 2G). Again, no UCP1 protein was detected in ingWAT (Figures 6H and S9I).

Thus, the effects on metabolic efficiency and UCP1 protein levels seen after short-term (2 weeks) exposure to glucocorticoids were not maintained during long-term (12 weeks) exposure to glucocorticoids, and obesity stabilized at a body weight increase of ~15 g.

## Corticosterone Has No Acute Thermogenic Effect, and CORT Does Not Alter Insulation

At thermoneutrality, CORT-induced obesity develops not only independently of the presence of UCP1 but paradoxically also
<figure><img src="image_16.png" /><figcaption>**Figure 6. Severe Obesity but No Difference in Total IBAT UCP1 Protein after Long-Term CORT Treatment at Thermoneutrality**</figcaption></figure>

Mice were kept at 30°C for the duration of the study, receiving veh (0.25% EtOH) or corticosterone (50 µg/mL) in their drinking water for 12 weeks (Veh12, CORT12).

(A) Fat mass and lean mass before and after 2, 7, or 12 weeks of treatment.

(B) Food intake (kJ/day) (week 6 excluded because of housing in metabolic chambers). Total food intake at the end of the 12-week treatment calculated in kilojoules.

(C) Metabolic efficiency calculated from (A) and (B).

(D) NE-induced (1 mg/kg) O₂ consumption in anaesthetized mice after Veh12 or CORT12.

(E) Quantification of (D): NE-induced O₂ consumption = peak NE O₂ consumption – basal O₂ consumption.

(F) Representative immunoblot showing UCP1 protein in IBAT homogenates with Ponceau staining as a loading control.

(G) Quantification of (F) showing UCP1 per microgram protein, total protein per depot, and total UCP1 per depot.

(H) Representative immunoblot showing UCP1 protein in ingWAT homogenates with 1.5× diluted IBAT from (F) as a positive control and Ponceau staining as a loading control.

Data are represented as mean ± SEM; n = 7. \*p < 0.05, \*\*p < 0.01, and \*\*\*p < 0.001; two-way ANOVA with Bonferroni post-test, significance within each time point between treatments (A–C); Student's t test (B, E, and G).

See also Figures S6 and S9.

despite an increase in basal metabolism (Figures 2D, S4G, and S4H). To determine the reason behind this increase in basal energy expenditure, we first investigated whether corticosterone has any acute thermogenic effects. Male C57Bl6/J mice were housed at 30°C for 2 weeks, after which they were sedated with pentobarbital and successively injected intraperitoneally (i.p.) with 0.3, 3, or 30 mg/kg corticosterone, veh (5% EtOH in saline), or NE (1 mg/kg) as a positive control. As shown in Figure S10, none of the injected doses of corticosterone induced an acute thermogenic response.

Hypercortisolism, whether in the form of endogenous Cushing's syndrome or induced by therapeutic glucocorticoid administration, affects skin physiology in a variety of ways (Sevilla and Pérez, 2018; Stratakis, 2016). In addition, glucocorticoids have been hypothesized to induce hypertension by direct interaction with vascular smooth muscle cells, inducing vasoconstriction (Goodwin, 2015). Because skin and vascular function are major factors in mouse insulation, we investigated to which extent CORT affects the animal's heat retention capacity and thereby influences the environmental temperature range over which the animal must expend the least amount of energy to defend its body temperature (i.e., the thermoneutral zone). Thus, we analyzed whether the animals could have increased their metabolism to compensate for reduced insulation. For this purpose, we implanted transponders i.p. into 8-week-old male C57Bl6/J mice, allowed the animals to recover for over 3 weeks while

housed at 30°C, and started veh or CORT treatment at 12 weeks of age. After 2 weeks of treatment, we performed a modified Scholander experiment (Fischer et al., 2016a; Scholander et al., 1950). At ambient temperatures ranging from 30°C–33°C, CORT mice again expended a higher amount of energy than veh mice (Figures S11A and S11B), in agreement with data shown in Figures 2, 5, and S4. At temperatures below 25°C, we found no change in insulation between the treatment groups, determined by the slopes of the Scholander curves (Figure S11A). Thus, we found no indication that CORT-treated animals needed to increase their energy expenditure because of decreased insulation.

The tails of rodents are major thermoregulatory organs. To specifically determine if the animals lost more heat through their tails and therefore had to increase their basal metabolic rate to defend body temperature, we performed thermal imaging. We found no differences in tail temperatures measured at the base or middle of the tail, or in body temperature measured in the ear, between treatment groups housed at 30°C, further indicating that the increase in energy expenditure was not a result of a reduction in insulation (Figures S11C and S11D). CORT mice exposed acutely to 21°C for 2 h showed significantly lower tail temperatures than veh mice, indicating an increased vasoconstriction (Figures S11C and S11D), while at 4°C all animals demonstrated extensive vasoconstriction and very cold tails.
<figure><img src="image_17.png" /><figcaption>Figure 7. CORT-Induced Increases in Fat Mass and Metabolic Efficiency Develop Independent of Food Intake</figcaption></figure>

Mice were kept at 30°C for the duration of the study, and they received veh and food ad lib (Veh), CORT and food ad lib (CORT), or CORT and food pair-fed to weight-matched veh mice (CORT pf).

(A) Food intake (kJ/day).

(B) Fat mass and lean mass before and after veh, CORT, or CORT pf.

(C) Metabolic efficiency calculated from (A) and (B).

(D) NE-induced (1 mg/kg) O₂ consumption in anesthetized mice after veh or CORT.

(E) Quantification of (D): NE-induced O₂ consumption = peak NE O₂ consumption – basal O₂ consumption.

(F) Representative immunoblot showing UCP1 protein in IBAT homogenates with Ponceau staining as a loading control.

(G) Quantification of (F) showing UCP1 per microgram protein, total protein per depot, and total UCP1 per depot.

(H) Representative immunoblot showing UCP1 protein in ingWAT homogenates with 2x diluted IBAT from (F) as a positive control and Ponceau staining as a loading control.

Data are represented as mean ± SEM; n = 6. \*p < 0.05, \*\*p < 0.01, and \*\*\*p < 0.001; two-way ANOVA with Bonferroni post-test, significance within each time point between treatments (A and B); one-way ANOVA with Tukey post-test (C, E, and G).

See also Figure S12.

Glucocorticoid-Induced Obesity Develops Independently of an Increase in Food Intake

Despite the increase in energy expenditure that we observed in CORT mice, we found no change in insulation, and an absence of UCP1 could not explain the development of obesity. We therefore investigated whether the obese phenotype could be fully explained by the increase in food intake seen in CORT mice. We performed a pair-feeding experiment in which *ad libitum* (ad lib) food intake was measured daily in the veh mice, and weight-matched CORT mice received the same amount of food in two equal portions per day. An ad lib-fed CORT group was included to track the usual development of obesity. Veh and pair-fed CORT mice thus had a similar food intake during the entire treatment period (Figure 7A). In week 2 of the treatment, food intake was significantly higher in ad lib CORT mice compared with the other two groups (Figure 7A). Compared with ad lib CORT mice, pair-fed CORT mice significantly reduced their water intake, although it was still significantly higher than in veh mice (Figure S12A). Because of the lower water intake, pair-fed CORT mice ingested a significantly lower total amount of corticosterone during the treatment period than the ad lib-fed CORT mice (Figure S12B). This could be expected to affect the extent to which obesity developed in pair-fed CORT mice, as the effects of CORT are dose dependent (Figure S3). Nonetheless, pair-fed CORT mice significantly increased fat mass (although to a lesser extent than ad lib CORT mice) and significantly decreased lean mass (Figure 7B). In addition, both ad lib- and pair-fed CORT mice significantly increased metabolic efficiency compared with veh mice (i.e., independently of food

intake) (Figure 7C). Thus, even in the absence of a higher food intake, a greater percentage of ingested energy was stored in CORT animals. In this cohort of mice, we did not find any significant differences in energy expenditure in the light phase between groups, but energy expenditure in the dark phase was significantly decreased in the pair-fed CORT mice compared with the ad lib CORT mice, partly highlighting the contribution of obligatory diet-induced thermogenesis (the thermic effect of food) to whole-body energy expenditure (Figures S12D–S12F). Pair feeding normalized the higher basal energy expenditure (measured under pentobarbital anesthesia) seen in ad lib CORT mice, indicating that this higher basal respiration seen consistently in all experiments was dependent on the higher food intake (Figures 7D and 7E). Again, no differences in NE-induced oxygen consumption were found between groups, but total IBAT UCP1 protein levels were significantly lower in both CORT groups compared with veh mice, indicating that the downregulation of UCP1 occurs independently of food intake (Figures 7D–7G). No UCP1 protein was found in ingWAT (Figures 7H and S12G). From these data, we conclude that the increase in basal energy expenditure seen in CORT mice is dependent upon food intake but that the downregulation of total IBAT UCP1 protein and the development of CORT-induced obesity are not.

DISCUSSION

Earlier studies have indicated that glucocorticoid-induced obesity is secondary to a repressed expression of the brown fat-specific protein UCP1 and an ensuing decrease in
thermogenesis (van den Beukel et al., 2015; Lv et al., 2018; Strack et al., 1995; Viengchareun et al., 2001). In the present investigation, we have examined the significance of UCP1 for the development of glucocorticoid-induced obesity. We find that under standard housing conditions (21°C), glucocorticoids do induce obesity but do not affect total BAT UCP1 protein levels. Although UCP1 levels are significantly decreased by glucocorticoid treatment in mice housed at thermoneutrality, obesity develops qualitatively and quantitatively unaltered by the amounts of UCP1 present. Glucocorticoids increase energy expenditure, but this can be understood as being secondary to the increased food intake, including the increased obligatory diet-induced thermogenesis that follows. Strikingly, glucocorticoid-induced obesity develops even in the absence of enhanced food intake and with no measurable decline in energy expenditure. Translationally, these experiments, performed with thermally humanized mice, imply that also in humans, the obesity developing because of chronically endogenously high circulating glucocorticoid levels or exogenous glucocorticoid administration may not be a result of the BAT inhibition ensuing prolonged hypercortisolism.

## Glucocorticoids Lower UCP1 Levels Only at Thermoneutrality

Glucocorticoids have previously been reported to lower BAT UCP1 protein levels, but these levels have been expressed per microgram protein (van den Beukel et al., 2015). We found that in mice housed at standard conditions (~21°C), glucocorticoids increased total protein amounts in BAT (Figure 1). This is most likely due to an enhanced gene transcription, as total BAT RNA levels were also increased, but the regulatory purpose of this is not evident. However, the outcome is that the apparent decrease in BAT UCP1 protein per microgram protein in mice housed at 21°C is chimeric: an unchanged amount of UCP1 present is diluted by the glucocorticoid-induced increase in total BAT protein levels (Figure 1). This is in accordance with the general tenet that in mice below thermoneutrality, the degree of recruitment of BAT is dominated by the cold experienced (Cannon and Nedergaard, 2011). Also in accordance with this tenet, no effect of glucocorticoids on energy expenditure can be observed under these conditions, as the energy expenditure is determined by the heat loss experienced by the mice (Figure S1). However, when these mice are examined at thermoneutrality, an increased basal metabolism is revealed (Figure S4).

Mice housed at thermoneutrality did show a functional decrease in total BAT UCP1 protein levels upon short-term glucocorticoid treatment (Figures 2 and 3). This decrease was due to a downregulation of *Ucp1* transcription, probably in a cell-autonomous manner (Figure 4) (van den Beukel et al., 2014; Soumano et al., 2000; Viengchareun et al., 2001). Whether the liganded glucocorticoid receptor directly downregulates *Ucp1* expression through binding to a negative glucocorticoid response element in the *Ucp1* promoter region, or by binding to other transcription factors that regulate *Ucp1* expression, is not known.

Thus, glucocorticoids indeed suppress BAT *Ucp1* expression at thermoneutrality, where there is an absence of a marked sympathetic drive, but as understood from Figures 1 and 2, this suppression is overcome by cold exposure that enhances sympa-

thetic drive when UCP1 is needed for thermoregulation. These findings are in agreement with observations that corticosterone-treated mice that were moved acutely from 21°C to 4°C were able to upregulate BAT *Ucp1* expression to similar levels as were vehicle-treated animals (van den Beukel et al., 2015). Thus, glucocorticoids do not irreversibly inhibit *Ucp1* gene expression.

## Glucocorticoids Induce Obesity Independently of UCP1

Although glucocorticoid treatment induced a significant downregulation of BAT UCP1 protein levels in animals housed at 30°C, glucocorticoid-induced obesity developed to the same extent in wild-type mice housed at 30°C or at 21°C, as well as in UCP1-KO mice housed at either temperature (Figures 1, 2, 4, 5, S1, S2, S4, S5, S7, and S8). Thus, the expansion of WAT in response to glucocorticoid treatment is not influenced by BAT UCP1 protein levels. Similarly, low doses of corticosterone are sufficient to lower UCP1 levels, whereas higher doses are required to induce obesity (Figure S3), also indicating that these two effects happen independently of each other.

## There Are No Physiologically Relevant Effects of Glucocorticoids on Brite/Beige Adipose Tissue Thermogenesis

Glucocorticoids have been indicated to not only lower BAT thermogenic capacity but also to lower the ability of WAT to develop brown-like characteristics (Kong et al., 2015). Thus, it may be hypothesized that glucocorticoids induce the development of obesity through decreasing thermogenesis in WAT. We found that although glucocorticoids induced a decrease in total *Ucp1* mRNA levels in the ingWAT of mice housed at 21°C, these levels were ~350 times lower than those in IBAT and are therefore unlikely to be physiologically relevant (Figure S2). Correspondingly, we were unable to detect any UCP1 protein in the WAT of these vehicle- or glucocorticoid-treated mice (Figure 1). In mice housed at thermoneutrality, we found no effect of corticosterone on *Ucp1* mRNA levels in ingWAT, and UCP1 protein levels were also undetectable (Figures 2 and S5). Thus, we conclude that as there is no UCP1 in ingWAT, a glucocorticoid-induced decrease in UCP1-dependent thermogenic activity in ingWAT cannot contribute to glucocorticoid-induced obesity.

## BAT-Independent Factors Contributing to the Development of Glucocorticoid-Induced Obesity

On the basis of our data showing that glucocorticoid-treated mice store more than 100% of their extra energy intake, but that obesity develops independent of BAT UCP1 levels, we conclude that glucocorticoids must induce a BAT-independent reduction in the underlying catabolic metabolism and thereby promote the development of obesity. Paradoxically, the development of glucocorticoid-induced obesity under thermally humanized conditions coincides with a glucocorticoid-induced increase in energy expenditure (Figure S4). This observation at first sight implied an unrecognized direct thermogenic effect of glucocorticoids. However, glucocorticoids are not directly thermogenic (Figure S10), and the increased energy expenditure is not due to increased heat loss (Figure S11). Rather, the increase in energy expenditure can be understood as being
related to the extra food intake and the associated extra handling costs (Figures 7 and S12).

Indeed, glucocorticoid treatment is known to increase food intake in both humans and mice (Berthon et al., 2014; Dallman et al., 2004). This glucocorticoid-induced increase in food intake may have been sufficient to fully explain the development of glucocorticoid-induced obesity. However, glucocorticoid treatment led to an expansion of WAT even in the absence of higher food intake (Figure 7). We thus conclude that there must be additional effects of glucocorticoid treatment that channel a fraction of the ongoing catabolic energy into anabolic processes and thus induce the development of obesity, independent of a reduction in BAT-mediated thermogenesis and independent of an increase in food intake. What these effects are remains to be determined.

From an enzymatic point of view, the excessive lipid storage in the corticosterone-treated mice is understandable. Glucocorticoid treatment at thermoneutrality increases the transcription of the lipogenic Fasn and Elov3 genes in IBAT, ingWAT, and eWAT (Figures 4 and S5). Indeed, many genes involved in lipid homeostasis are regulated by glucocorticoid signaling in adipocytes (Yu et al., 2010). The fact that lipid accumulation in adipose tissues is regulated directly by glucocorticoid signaling through the glucocorticoid receptor is confirmed by the abolishment of adipose tissue lipid accumulation in mice lacking the glucocorticoid receptor in adipose tissues (Bose et al., 2016; Shen et al., 2017). Thus, glucocorticoid signaling induces an accumulation of lipids in BAT and WAT through an upregulation of lipogenic enzymes, but this accumulation occurs independently of the amount of UCP1 present.

## BAT and Glucocorticoid-Induced Obesity in Humans

In mice housed at 21°C, glucocorticoids do not reduce UCP1 levels (Figure 1). However, results from such (principally cold-adapted) mice are difficult to extrapolate to the human condition, as we humans spend the vast majority of our lives at thermoneutrality. We here thermally humanized mice by housing them at thermoneutrality (i.e., 30°C) (Fischer et al., 2018; Ganeshan and Chawla, 2017; Reitman, 2018). Although glucocorticoids induce a functional downregulation of UCP1 at this temperature, the glucocorticoid-induced obesity develops independent of the presence or absence of UCP1 (Figures 5 and S7).

The glucocorticoid-induced BAT inhibition observed here in mice clearly has parallels in humans, in whom long-term exogenous glucocorticoid treatment, comparable with our mouse model, reduces glucose uptake in BAT and supraclavicular skin temperature, indicating a reduction in BAT thermogenesis (Thuzar et al., 2018). Additionally, in cultured human brown adipocytes, glucocorticoids suppress the adrenergically induced transcription of *Ucp1* (Barclay et al., 2015), similarly to the implication of the experiments described here and in agreement with our unpublished observations in cultured mouse brown adipocytes. It would therefore be natural to ascribe glucocorticoid-induced obesity in humans to a decrease in BAT thermogenic capacity. However, the data presented here demonstrate that at least in mice, glucocorticoid-induced obesity develops irrespective of the levels of UCP1. It is therefore unlikely that the decrease in BAT activity caused by glucocorticoids in humans

is causative of glucocorticoid-induced obesity in humans. This does not, however, preclude the possibility that a recruitment and activation of BAT in humans could counteract glucocorticoid-induced obesity.

## STAR★METHODS

Detailed methods are provided in the online version of this paper and include the following:

* KEY RESOURCES TABLE

* CONTACT FOR REAGENT AND RESOURCE SHARING

* EXPERIMENTAL MODEL AND SUBJECT DETAILS

* Animal Models

* METHOD DETAILS

* Food Intake and Body Composition

* Acute Corticosterone Injections

* Serum Corticosterone

* Fecal Corticosterone

* Norepinephrine Injections

* Pair-Feeding

* 24 Hour Energy Expenditure

* Glucose Tolerance Test

* Scholander Experiment

* Thermal Imaging

* Histology

* RNA Isolation, cDNA Synthesis, and qPCR

* Western Blot Analyses

* Isolation of Mature Brown Adipocytes

* Mature Brown Adipocyte Oxygen Consumption

* Isolation of Mitochondria from BAT

* Mitochondrial Oxygen Consumption

* QUANTIFICATION AND STATISTICAL ANALYSIS

## SUPPLEMENTAL INFORMATION

Supplemental Information can be found online at https://doi.org/10.1016/j.celrep.2019.04.041.

## ACKNOWLEDGMENTS

We thank the members of the Cannon and Nedergaard lab at Stockholm University for discussions. We also thank Vasilios Tsarouhas and Michael Oschmann (Stockholm University) for technical assistance and Jan Kroon (Leiden University Medical Centre) and Johanna van den Beukel (Erasmus University Medical Centre) for their advice. The experimental core facility at Stockholm University is acknowledged for its technical assistance. This project was supported by grants from the Swedish Research Council.

## AUTHOR CONTRIBUTIONS

I.H.N.L., J.N., and B. Cannon conceived the project, interpreted data, and wrote the paper. I.H.N.L. designed mouse studies. I.H.N.L., K.B., and A.J. performed mouse studies and analyzed physiological data. N.B. performed mature adipocyte isolation, measurements of oxygen consumption, and analyses. I.G.S. performed mitochondrial isolation, measurements of oxygen consumption, and analyses. B. Carlsson performed transponder implantations. A.W.F. performed thermal imaging. I.H.N.L. and K.B. performed fecal corticosterone extraction and ELISA, western blot, qPCR, H&E staining, and Adiposoft analyses. All authors critically reviewed the manuscript and approved the final version.
## DECLARATION OF INTERESTS

The authors declare no competing interests.

Received: December 31, 2018

Revised: March 18, 2019

Accepted: April 8, 2019

Published: May 7, 2019

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## STAR★METHODS

### KEY RESOURCES TABLE

<table><thead><tr><th>REAGENT or RESOURCE</th><th>SOURCE</th><th>IDENTIFIER</th></tr></thead><tbody><tr><td colspan="3"><strong>Antibodies</strong></td></tr><tr><td>Rabbit polyclonal anti-UCP1 (against the C-terminal decapeptide)</td><td>Produced for the lab</td><td>N/A</td></tr><tr><td>Rabbit monoclonal anti-β-tubulin</td><td>Cell Signaling</td><td>Cat#2128; RRID: AB_823664</td></tr><tr><td>Goat anti-rabbit IgG, HRP-linked</td><td>Cell Signaling</td><td>Cat#7074; RRID: AB_8 2099233</td></tr><tr><td colspan="3"><strong>Chemicals, Peptides, and Recombinant Proteins</strong></td></tr><tr><td>Corticosterone</td><td>Sigma</td><td>Cat#27840</td></tr><tr><td>L-(-)-Norepinephrine (+)-bitartrate salt monohydrate</td><td>Sigma</td><td>Cat#A9512</td></tr><tr><td>Pentobarbital solution 60 mg/ml</td><td>APL</td><td>Cat#338327</td></tr><tr><td>Buprenorphine (Bupaq® multidose vet)</td><td>Salfarm Scandinavia</td><td>N/A</td></tr><tr><td>Eukitt® mounting medium</td><td>Sigma</td><td>Cat#03989</td></tr><tr><td>Collagenase type I</td><td>Sigma</td><td>Cat#0130</td></tr><tr><td>cOmplete™ mini protease inhibitor</td><td>Roche</td><td>Cat#04693159001</td></tr><tr><td>Ponceau S</td><td>Sigma</td><td>Cat#141194</td></tr><tr><td>Fatty acid-free BSA</td><td>Roche</td><td>Cat#10775835001</td></tr><tr><td>Oligomycin</td><td>Sigma</td><td>Cat#O4876</td></tr><tr><td>Carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP)</td><td>Sigma</td><td>Cat#C2920</td></tr><tr><td>Sodium pyruvate</td><td>Sigma</td><td>Cat#P8574</td></tr><tr><td>Guanosine 5'-diphosphate sodium salt</td><td>Sigma</td><td>Cat#G7127</td></tr><tr><td>Adenosine 5'-diphosphate sodium salt</td><td>Sigma</td><td>Cat#A2754</td></tr><tr><td>Carboxyatractyloside</td><td>Calbiochem</td><td>Cat#216200</td></tr><tr><td colspan="3"><strong>Critical Commercial Assays</strong></td></tr><tr><td>Corticosterone ELISA kit</td><td>Enzo Life Sciences</td><td>Cat#ADI-900-097</td></tr><tr><td>Insulin ELISA kit</td><td>Crystal Chem</td><td>Cat#90080</td></tr><tr><td>High-Capacity cDNA Reverse Transcription kit</td><td>ThermoFisher</td><td>Cat#4368814</td></tr><tr><td>SYBR® Green JumpStart™ Taq ReadyMix™</td><td>Sigma</td><td>Cat#S4438</td></tr><tr><td>Pierce™ ECL Plus Western Blotting Substrate</td><td>ThermoFisher</td><td>Cat#32132</td></tr><tr><td>TRI Reagent®</td><td>Sigma</td><td>Cat#T9424</td></tr><tr><td colspan="3"><strong>Experimental Models: Organisms/Strains</strong></td></tr><tr><td>Mouse: wild-type: C57Bl6/J</td><td>Scanbur</td><td>000664</td></tr><tr><td>Mouse: wild-type: C57Bl6/J<sup>Ucp1/Ucp1</sup></td><td>Own breeding</td><td>N/A</td></tr><tr><td>Mouse: UCP1-KO: C57Bl6/J<sup>-/-</sup></td><td>Own breeding</td><td>N/A</td></tr><tr><td colspan="3"><strong>Oligonucleotides</strong></td></tr><tr><td colspan="3">For primers, see Table S1</td></tr><tr><td colspan="3"><strong>Software and Algorithms</strong></td></tr><tr><td>FLIR Tools</td><td>FLIR®</td><td>https://www.flir.eu/products/flir-tools/</td></tr><tr><td>Adiposoft (Fiji plugin)</td><td>Galarraga et al., 2012</td><td>https://imagej.net/Adiposoft</td></tr><tr><td>Multi Gauge V3.0 Software</td><td>Fujifilm</td><td>http://www.fujifilm.com/support/digital_cameras/software/</td></tr><tr><td>Oroboros DatLab 6.0</td><td>Oroboros</td><td>www.oroboros.at</td></tr><tr><td colspan="3"><strong>Other</strong></td></tr><tr><td>Chow diet</td><td>Lactamin AB</td><td>R70</td></tr></tbody></table>
## CONTACT FOR REAGENT AND RESOURCE SHARING

Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Jan Nedergaard (jan@metabol.su.se).

## EXPERIMENTAL MODEL AND SUBJECT DETAILS

### Animal Models

All animal experiments were approved by the North Stockholm Animal Ethics Committee (permission N196/16). Male 8-week-old C57Bl6/J mice were purchased from Scanbur (Sweden). Alternatively, as indicated, UCP1-KO mice on a C57Bl6/J background and corresponding wild-types were bred in-house. From 10 weeks of age, animals were single-caged and housed at 30°C or 21°C with free access to water and chow diet R70 (Lactamin, Sweden) on a 12 h/12 h light/dark cycle for the duration of the study. After a two-week acclimation period, the animals (then 12 weeks old) received either vehicle (0.25% EtOH) or corticosterone (50 µg/ml) via the drinking water for two weeks (for dose-determination at 21°C, see Auvinen et al., 2013; Karatsoreos et al., 2010). For determination of the optimal corticosterone dose for supplementation at 30°C, animals received either vehicle (0.25% EtOH) or 25, 50 or 100 µg/ml corticosterone in the drinking water for two weeks. Drinking water was replaced at least once per week. During week 3 of the treatment, one of the experiments described below was performed (with exclusion of the acute corticosterone injections). After this, mice were sacrificed by CO₂ and cervical dislocation, and organs were collected.

In a separate experiment, male C57Bl6/J mice (Scanbur, Sweden) were housed as described above from 10 weeks of age at 30°C and were supplemented with vehicle (0.25% EtOH) or corticosterone (50 µg/ml) in the drinking water from 12 to 24 weeks of age. Drinking water was replaced at least once per week. During the last week of treatment, one of the experiments described below was performed (with exclusion of the acute corticosterone injections). After this, mice were sacrificed by CO₂ and cervical dislocation, and organs were collected.

## METHOD DETAILS

### Food Intake and Body Composition

Weekly measurements of body weight, food intake, and water intake were made for all mice. Food intake in kJ/day was calculated by dividing weekly food intake by 7 and multiplying by the stated energy density of the chow diet (11.18 kJ/g). The theoretical thermic effect of food was calculated using the thermic effects of fat (2.5%), carbohydrates (7.5%) and protein (25%) (Trumbo et al., 2002). For mice receiving a two-week vehicle or corticosterone treatment, body composition (EchoMRI-100TM, Echo Medical Systems, USA) and fecal corticosterone concentrations (see below) were measured before the treatment and after two weeks of treatment. For mice receiving a twelve-week vehicle or corticosterone treatment, body composition, and fecal corticosterone concentrations were measured before the treatment and after 2, 7 and 12 weeks of treatment. Metabolic efficiency (% of energy intake stored in the body) was calculated as follows: (Δfat mass(kJ) + Δlean mass(kJ)) / food intake(kJ) x 100, where the energy densities for fat mass and lean mass were 39 kJ/g respectively 5 kJ/g.

### Acute Corticosterone Injections

Male 10-week-old C57Bl6/J mice were acclimated to 30°C for two weeks. When the animals were 12 weeks old, they were sedated with 90 mg/kg pentobarbital (APL, Sweden) i.p. and baseline oxygen consumption was measured at 30°C in metabolic chambers (INCA system, Somedic, Sweden). Measurements were obtained every second minute. The mice were then briefly taken out of the chambers and acutely injected i.p. with vehicle (5% EtOH in sterile saline), 1 mg/kg norepinephrine bitartrate salt monohydrate (NE, Sigma, Germany), or 0.3 mg/kg, 3 mg/kg or 30 mg/kg corticosterone (Sigma, Germany), after which O₂ consumption measurements were continued.

### Serum Corticosterone

Serum corticosterone levels in mice were determined at 7:30 AM or 6:30 PM. Trunk blood was collected in centrifuge tubes immediately after decapitation, that occurred within 90 s of first moving the cage. After clotting the blood for 30 min at room temperature, samples were centrifuged at 2500 x g for 15 min at 4°C. Serum was collected, snap-frozen in liquid nitrogen and stored at -80°C. Serum corticosterone concentrations were measured by ELISA (Enzo Life Sciences, Belgium) according to the manufacturer's instructions.

### Fecal Corticosterone

24 h bedding was collected from the cages and air-dried at room temperature for 1 day. Subsequently, feces were collected from the bedding, crushed, and homogenized with a mortar and pestle. Corticosterone extraction was performed as described by van den Beukel et al., 2014. 50 mg homogenized feces were boiled for 20 min at 80°C in 2.5 mL 99.5% EtOH (Solveco, Sweden). Samples were centrifuged at 4500 rpm for 15 min and supernatants were collected in glass vials. The remaining pellets were boiled again for 20 min at 80°C in 1.5 mL 99.5% EtOH. Samples were centrifuged at 4500 rpm for 15 min and the supernatants were combined in the
glass vials. The ethanol was evaporated by rotary evaporation at 45°C and samples were re-dissolved in 250 µL MeOH (VWR Chemicals, France). Corticosterone concentrations were measured by ELISA (Enzo Life Sciences, Belgium) according to the manufacturer's instructions.

## Norepinephrine Injections

Animals were sedated with 90 mg/kg pentobarbital (APL, Sweden) i.p. and baseline oxygen consumption was measured at 30°C in temperature-controlled metabolic chambers (INCA system, Somedic, Sweden). Subsequently, 1 mg/kg norepinephrine bitartrate salt monohydrate (NE, (Sigma, Germany)) was injected i.p. after which O₂ consumption measurements continued. The NE response was calculated by subtracting baseline oxygen consumption (average of 3 successive stable points) from peak NE-induced O₂ consumption (average of 3 successive highest points).

## Pair-Feeding

Corticosterone-treated animals were matched to vehicle-treated animals during the treatment period based on their body weight. Daily food intake was measured for vehicle-treated animals, after which paired corticosterone-treated animals received the same amount of food in two equal portions every day at 8 AM and 7 PM. The cages of all animals in the study were disturbed at these two time points.

## 24 Hour Energy Expenditure

Animals were placed in the temperature-controlled metabolic chambers at 30°C or 21°C on a 12 h/12 h light/dark cycle with free access to food and water. After overnight acclimation, measurements of O₂ consumption and CO₂ production were collected every second min for 24 h. Energy expenditure in watts was calculated using a modified Weir equation:

$$EE(W) = \left( 0.2716 \frac{W \times min}{ml} \times vO_2\text{consumed} \left[ \frac{ml}{min} \right] + 0.07616 \frac{W \times min}{ml} \times vCO_2\text{produced} \left[ \frac{ml}{min} \right] \right)$$

(Fischer et al., 2016b; Weir, 1949). Respiratory quotient was calculated by dividing CO₂ production by O₂ consumption.

## Glucose Tolerance Test

After a 6 h fast, blood glucose levels were measured with an Accu-Chek® Aviva glucometer (Roche, Sweden) by obtaining a drop of blood from the tail. Subsequently, animals received 2.5 g glucose (Merck) / kg lean weight (as measured the previous day by MRI) by i.p. injection, and blood glucose levels were measured again 15, 30, 60, 90 and 120 min after injection.

Before glucose injection and 15 min after injection, 30 µL of blood was also collected from the tail in a capillary tube containing clotting activator (Microvette® CB 300 Z, Sarstedt, Germany) and kept on ice. To obtain serum, the capillary tubes were spun at 10000 x g at 4°C for 5 min. Serum was collected and stored at -80°C until insulin levels were determined by ELISA according to the manufacturer's instructions (Crystal Chem, USA).

## Scholander Experiment

A modified Scholander experiment (Scholander et al., 1950) was performed. At 8 weeks of age, animals were implanted intraperitoneally with transponders (G2 E-Mitter®, Starr Life Sciences, USA) under sterile conditions. First, the animals were anaesthetized by inhalation of 4% isoflurane (Abbott, Sweden). Then their abdomen was shaved, sterilized with a 2% chlorhexidine solution, a 1 cm skin incision was made and the peritoneum was opened with a 0.6 cm incision. Before implantation, the transponders were sterilized with 3% hydrogen peroxide and washed with sterile saline. After placement of the transponders in the peritoneal cavity, the peritoneal incision was closed using a VicrylTM 5-0 suture (Ethicon Endo-Surgery GmbH, Germany). The skin incision was closed using an Ethilon IITM 4-0 suture (Ethicon Endo- Surgery GmbH, Germany). The mice were kept at 30°C after the surgery and for the rest of the study. The first three days after surgery, animals were injected subcutaneously with 0.1 mg/kg of the painkiller buprenorphine (Bupaq vet 0.3 mg/ml Salfarm Scandinavia) two times per day. The animals were allowed to recover for 3 more weeks after this. From 12 weeks of age, animals then received vehicle or corticosterone in the drinking water as described above. During week 3 of the treatment, animals were placed in the metabolic chambers at 30°C with free access to food and water. After overnight acclimation, the chamber temperature was first increased to 33°C and then gradually decreased in steps to 30°C, 25°C, 20°C and 15°C. Every temperature was maintained for a 2 h period. Measurements of O₂ consumption, CO₂ production and body temperature were collected every second min. O₂ consumption and body temperature values were averaged from 9 successive points per chamber temperature interval when the physical activity measured by the transponders was closest to 0. Energy expenditure was calculated as described above.

## Thermal Imaging

Animal tail and inner ear temperatures were measured after exposure to 30°C, 21°C and 4°C using a FLIR® E60 infrared camera (FLIR Instruments, Germany). Pictures were taken while animals were briefly placed on the lid of their cage and were able to move around freely. Several pictures were taken per animal at 30°C, after a 2 h exposure to 21°C and subsequently after a 2 h exposure to 4°C. Images were analyzed using FLIR Tools Software.
## Histology

For histological analysis, adipose tissues were taken from mice that had not received any extra adrenergic stimulation (by NE or cold exposure) during the experiment. Interscapular brown adipose tissue (IBAT), inguinal white adipose tissue (ingWAT) and epididymal adipose tissue (eWAT) pieces were dissected and fixed for 24 h in alcoholic formalin (4% in 95% ethanol) at 4°C. Tissues were rinsed and stored in 95% ethanol until embedding in paraffin. Paraffin blocks containing embedded tissues were trimmed and sectioned at 5 µm using a Leica RM2255 Automated Microtome (Leica Biosystems, Germany). Slides were dried overnight at 37°C, after which they were deparaffinized, rehydrated, stained with hematoxylin & eosin, dehydrated, and mounted in Eukitt® mounting medium (Sigma, Germany). Pictures were taken at 100x magnification using a Leica DM LB light microscope, the Leica DC200 imaging system (both Leica Microsystems, Germany) and Adobe® Photoshop® CS4. Adipocyte area was calculated using the Fiji software plug-in Adiposoft (Galarraga et al., 2012).

## RNA Isolation, cDNA Synthesis, and qPCR

For determination of gene expression, adipose tissues were taken from mice that had not received any extra adrenergic stimulation (by NE or cold exposure) during the experiment. One lobe each of IBAT, ingWAT and eWAT were quantitatively dissected, weighed, snap-frozen and stored at -80°C. Tissues were homogenized in TRI Reagent® (Sigma, Germany) and RNA was isolated according to the manufacturer's instructions. 500 ng of RNA was reverse-transcribed using the High-Capacity cDNA Reverse Transcription kit (Life Technologies, the Netherlands). qPCR was performed using the CFX Connect™ Real-Time PCR Detection System (Bio-Rad) and SYBR® Green JumpStart™ Taq ReadyMix™ (Sigma, Germany). Primer sequences are found in Table S1. Thermal cycling conditions were 2 min at 50°C, 10 min at 95°C, and 40 cycles of 15 s at 95°C and 1 min at 60°C. A melting curve was included at the end to determine the specificity of amplification. Gene expression was calculated using the ΔC~T~ method (2^(-ΔC~T~)), using TATA-box binding protein (Tbp) as a reference gene to account for any variability in cDNA synthesis.

## Western Blot Analyses

For protein measurements, one lobe of IBAT, ingWAT and eWAT were quantitatively dissected, weighed, snap-frozen and stored at -80°C. Tissues were homogenized in RIPA buffer containing protease (cOmplete™ Mini, Roche, Germany) and phosphatase (5 mM Na fluoride, 1 mM Na orthovanadate) inhibitors with a Teflon pestle. Protein concentrations of the homogenates were measured using the Lowry method. Proteins were separated on a 10% SDS-polyacrylamide gel and transferred to a PVDF membrane (Biorad) using a semi-dry electrophoretic transfer machine (Transblot Turbo, Biorad). Membranes were incubated in Ponceau (0.1% (w/v) in 5% acetic acid; Sigma, Germany) to control for equal loading of proteins, and a digital image was taken with a LAS-1000 CCD camera (Fujifilm). Membranes were subsequently blocked in 5% milk (w/v; Semper, Sweden) in TBS-T for 1 h, after which they were incubated overnight at 4°C with a primary anti-UCP1 antibody (rabbit polyclonal, produced in lab, 1:5000 (for protein from mice acclimated to 30°C) 1:10000 (for protein from mice acclimated to 21°C) in 5% milk in TBS-T) or an anti-β-tubulin antibody (rabbit monoclonal, Cell Signaling 2128, 1:1000 in 5% milk in TBS-T). The following day, membranes were incubated for 1 h with a secondary goat anti-rabbit HRP-conjugated antibody (Cell Signaling 7074, 1:10000 in 5% milk in TBS-T for UCP1 measurements, 1:1000 in 5% milk in TBS-T for β-tubulin measurements). Proteins were visualized using Pierce ECL Plus western blotting substrate (ThermoFisher) and imaged with the LAS-1000 CCD camera. Multi Gauge V3.0 Software (Fujifilm) was used for quantification.

## Isolation of Mature Brown Adipocytes

Mature brown adipocytes were isolated by collagenase digestion of pooled BAT depots (interscapular, axillary, and cervical). For each experiment, 3 C57Bl6/J mice were killed by CO₂ anesthesia and decapitated during week 3 of the vehicle or corticosterone treatment. After dissection, BAT depots were placed in Krebs Ringer phosphate buffer (KRPB) (1.4 mM KH₂PO₄, 3.76 mM NaH₂PO₄, 16.74 mM Na₂HPO₄, 110.38 mM NaCl, 5.55 mM KCl, 1.4 mM MgSO₄, 1.5 mM CaCl₂, 10 mM glucose, 10 mM fructose, 4% fatty acid-free BSA, pH 7.4). Tissues were minced with scissors and incubated in KRPB containing 1.3 mg/ml collagenase type I (Sigma-Aldrich, Germany) in a shaking water bath (100 rpm, 37°C), while being vortexed every 2 min. After 7 min, the buffer was discarded through filtration, the tissues were minced again and incubated in KRPB containing 0.67 mg/ml collagenase type I for 30-45 min in a shaking water bath (100 rpm, 37°C), while being vortexed every 5 min. The buffer, containing the mature adipocytes, was filtered through a 250 µm nylon filter, after which the filtrate was centrifuged for 10 min at 2 x g. The infranatant was discarded and KRPB was added to the adipocyte suspensions. The remaining tissues were incubated for another 20-30 min in KRPB containing 0.33 mg/ml collagenase type I, while being vortexed every 5 min, and subsequently filtered. The filtrate was centrifuged 10 min at 2 x g, and the adipocytes were collected. The two adipocyte suspensions were pooled and washed by floating for 1 h.

## Mature Brown Adipocyte Oxygen Consumption

Oxygen consumption of isolated mature brown adipocytes was measured using a high-resolution oxygraph (OROBOROS Oxygraph-2K, Austria). Oxygen concentration (nmol/ml) and rate of oxygen consumption (in pmol / s * ml) were recorded continuously using DAT LAB software 6. Respiration medium (DMEM 4500 mg/l glucose (Sigma, Germany), 4% fatty acid-free BSA (Roche, Germany), 5 mM pyruvate) was pre-heated to 37°C, after which 2 mL was added to the oxygen electrode chambers, where it was magnetically stirred, and kept at 37°C. 10 µL of mature adipocyte cell suspension was added, the chamber was closed with the lid, and the cells were incubated until a stable basal respiratory rate was reached. Additions of oligomycin (2 µg/ml, Sigma-Aldrich), NE (1 µM,
Sigma-Aldrich) and FCCP (titration with 40 µM until the maximal oxygen consumption rate was obtained, Sigma-Aldrich) were successively made to the chamber with a Hamilton syringe. In parallel, 10 µL of mature adipocyte cell suspension was taken for determination of total DNA content to ensure loading of equal numbers of cells. The oxygen consumption rate values were analyzed in pmol / (sec * ml) or represented as percentage of basal oxygen consumption rates.

## Isolation of Mitochondria from BAT

Mitochondria were isolated as previously described (Cannon and Nedergaard, 2008). Mitochondria from vehicle- and corticosterone-treated mice were always isolated in parallel, and all procedures were conducted at 0-2°C. Mice were briefly anaesthetized with a mixture of 79% CO₂ and 21% O₂ and decapitated. Interscapular, axillary and cervical BAT were dissected from 2-3 mice per isolation, pooled in isolation medium (250 mM sucrose, 10 mM HEPES, 0.1 mM EGTA, 2% fatty acid-free BSA, pH 7.2) and weighed. Tissues were minced with scissors and homogenized in a glass homogenizer with a Teflon pestle. Homogenates were filtered through a cotton gauze and centrifuged at 8500 x g for 10 min in an Allegra TM 25R centrifuge (Beckman Coulter, Germany). The supernatants, containing floating fat, were discarded, and the pellets were resuspended in isolation medium and centrifuged at 800 x g for 10 min. The resulting supernatants were transferred to clean tubes and centrifuged at 8500 x g for 10 min. The resulting mitochondrial pellets were washed in KCI-TES buffer (100 mM KCI, 20 mM K⁺-TES, 1 mM EDTA, 0.6% fatty acid-free BSA, pH 7.2) by centrifugation at 8500 x g for 10 min, and then resuspended in a small volume of KCI-TES buffer by hand homogenization. Mitochondrial protein concentration was measured using fluorescamine (Udenfriend et al., 1972). Fluorescence was measured in a Shimadzu RF-540 fluorophotometer (Japan) at an excitation of 390 nm and emission of 475 nm and compared to a BSA standard curve.

## Mitochondrial Oxygen Consumption

Oxygen consumption of mitochondria isolated from brown adipose tissue was measured using a high-resolution oxygraph (OROBOROS Oxygraph-2K, Austria). Brown fat mitochondrial respiration from vehicle- and corticosterone-treated mice was always measured in parallel. 2 mL of mitochondrial incubation medium (125 mM sucrose, 20 mM K⁺-TES, 2 mM MgCl₂, 1 mM EDTA, 4 mM KPi, 0.1% fatty acid-free BSA, 3 mM malate pH 7.2) was added to the oxygen electrode chambers where it was magnetically stirred and kept at 37°C. The mitochondria (0.25 mg protein) were added to the chambers and respiratory activity was measured in the presence of 5 mM pyruvate. Thereafter, UCP1-dependent thermogenesis was determined by addition of 2 mM GDP. Next, 0.5 mM ADP was added and subsequently 3 µM carboxyatractyloside (CATR), an inhibitor of the ATP/ADP-antiporter. Oxidative phosphorylation capacity was defined as maximal oxygen consumption rate after ADP addition minus the residual oxygen consumption rate after CATR addition (basal respiration). Finally, maximal respiratory capacity was determined by titrating FCCP to a final concentration of 2.2 – 2.8 µM. Data were collected and analyzed using DAT LAB software 6 and expressed as nmol O₂ / (min * mg mitochondrial protein).

## QUANTIFICATION AND STATISTICAL ANALYSIS

Statistical analyses were done with Prism version 5.0f. Data are represented as mean ± SEM, and the number of replicates per experiment can be found in the figure legends. Differences between two groups were examined by Student's t test. Differences between more than two groups were examined by a one-way ANOVA with a Tukey post-test. Differences between two or more groups in which the same parameter was measured at different time points were examined by a two-way ANOVA, including repeated-measured (where possible) with a Bonferroni post-test. In all analyses, a p value of p < 0.05 was considered significant.