# Cold-induced thermogenesis requires neutral-lipase-mediated intracellular lipolysis in brown adipocytes

Etienne Mouisel, Anaïs Bodon, Christophe Noll, Stéphanie Cassant-Sourdy, Marie-Adeline Marques, Rémy Flores-Flores, Elodie Riant, Camille Bergoglio, Pierre Vezin, Sylvie Caspar-Bauguil, et al.

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## To cite this version:

Etienne Mouisel, Anaïs Bodon, Christophe Noll, Stéphanie Cassant-Sourdy, Marie-Adeline Marques, et al.. Cold-induced thermogenesis requires neutral-lipase-mediated intracellular lipolysis in brown adipocytes. Cell Metabolism, In press, 10.1016/j.cmet.2024.10.018. hal-04815954

HAL Id: hal-04815954

https://ut3-toulouseinp.hal.science/hal-04815954v1

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# Cell Metabolism

## Cold-induced thermogenesis requires neutral-lipase-mediated intracellular lipolysis in brown adipocytes

### Graphical abstract

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

### Authors

Etienne Mouisel, Anaïs Bodon, Christophe Noll, ..., Pierre-Damien Denechaud, André C. Carpentier, Dominique Langin

### Correspondence

etienne.mouisel@inserm.fr (E.M.), dominique.langin@inserm.fr (D.L.)

### In brief

This study shows that intracellular lipolysis in brown adipose tissue (BAT) is essential for cold-induced thermogenesis. By using a mouse model with ATGL/HSL deletion in BAT, researchers revealed impaired cold tolerance and abolished oxidative activity. These findings highlight lipolysis as a potential drug target for enhancing BAT function and treating metabolic disorders.

### Highlights

* Deletion of ATGL and HSL in BAT compromised cold tolerance in the fasted state

* Cold-induced BAT oxidative activity is abolished in mice lacking ATGL and HSL in BAT

* BAT lipolysis is crucial for cold-induced thermogenesis
## Article

# Cold-induced thermogenesis requires neutral-lipase-mediated intracellular lipolysis in brown adipocytes

Etienne Mouisel,<fnref n="1" />,<fnref n="8" />,* Anaïs Bodon,<fnref n="1" />,<fnref n="8" /> Christophe Noll,<fnref n="2" />,<fnref n="3" /> Stéphanie Cassant-Sourdy,<fnref n="1" /> Marie-Adeline Marques,<fnref n="1" />

Remy Flores-Flores,<fnref n="1" /> Elodie Riant,<fnref n="1" /> Camille Bergoglio,<fnref n="1" /> Pierre Vezin,<fnref n="1" /> Sylvie Caspar-Bauguil,<fnref n="1" />,<fnref n="4" />

Camille Fournes-Fraresso,<fnref n="1" /> Geneviève Tavernier,<fnref n="1" /> Khalil Acheikh Ibn Oumar,<fnref n="1" /> Pierre Gourdy,<fnref n="1" />,<fnref n="4" /> Denis P. Blondin,<fnref n="5" />,<fnref n="6" />

Pierre-Damien Denechaud,<fnref n="1" />,<fnref n="8" /> André C. Carpentier,<fnref n="2" />,<fnref n="3" />,<fnref n="8" /> and Dominique Langin<fnref n="1" />,<fnref n="4" />,<fnref n="7" />,<fnref n="9" />,*

## SUMMARY

Long-chain fatty acids (FAs) are the major substrates fueling brown adipose tissue (BAT) thermogenesis. Investigation of mouse models has previously called into question the contribution of brown adipocyte intracellular lipolysis to cold-induced non-shivering thermogenesis. Here, we determined the role of the lipolytic enzymes, adipose triglyceride lipase (ATGL) and hormone-sensitive lipase (HSL), in BAT thermogenesis. Brown fat from mice with inducible brown-adipocyte-specific deletion of ATGL and HSL (BAHKO) is hypertrophied with increased lipid droplet size and preserved mitochondria area and density. Maintenance of body temperature during cold exposure is compromised in BAHKO mice in the fasted but not in the fed state. This altered response to cold is observed in various thermal and nutritional conditions. Positron emission tomography-computed tomography using [¹¹C]-acetate and [¹¹C]-palmitate shows abolished cold-induced BAT oxidative activity and impaired FA metabolism in BAHKO mice. Our findings show that brown adipocyte intracellular lipolysis is required for BAT thermogenesis.

## INTRODUCTION

Brown adipose tissue (BAT) displays the unique capacity to dissipate chemical energy as heat, conferring to mammals the ability to survive in the cold without relying on shivering.<fnref n="1" /> Environmental and pharmacological stimuli, notably acute cold exposure and β-adrenergic agonists, activate BAT non-shivering thermogenesis. At the biochemical level, uncoupling mitochondrial oxidation of substrates from ATP synthesis mediated by uncoupling protein 1 (UCP1) is a major contributor to adaptive thermogenesis.

Different substrates have been shown to fuel these recruitable forms of thermogenesis.<fnref n="2" />,<fnref n="3" /> Long-chain fatty acids (FAs) occupy a critical position. They are a major energy source for oxidative metabolism. Moreover, FAs act as essential activators of

UCP1 proton transport activity.<fnref n="4" /> Triacylglycerol (TG) hydrolysis into FAs and glycerol has long been considered to be essential for cold-induced BAT thermogenesis. In adipocytes, the rate-limiting steps in lipolysis are mediated by adipose triglyceride lipase (ATGL) and hormone-sensitive lipase (HSL).<fnref n="5" />,<fnref n="6" /> Following stimulation of the sympathetic nervous system during cold exposure, binding of catecholamines to β-adrenergic receptors leads to an increase in intracellular cyclic AMP levels. Cyclic-AMP-dependent activation of protein kinase A allows phosphorylation of lipid-droplet-associated proteins and induces ATGL and HSL hydrolytic activities.

Pharmacological studies support the essential role of intracellular lipolysis in the thermogenic response. Inhibition of intracellular lipolysis showed that this catabolic pathway is critical for the acute stimulation of brown adipocyte thermogenesis

<footnote n="1">¹Institut des Maladies Métaboliques et Cardiovasculaires, I2MC, Université de Toulouse, Inserm, Université Toulouse III—Paul Sabatier (UPS), Toulouse, France</footnote>

<footnote n="2">²Centre de recherche du Centre Hospitalier Universitaire de Sherbrooke, Sherbrooke, QC, Canada</footnote>

<footnote n="3">³Department of Medicine, Division of Endocrinology, Faculty of Medicine and Health Sciences, Université de Sherbrooke, Sherbrooke, QC, Canada</footnote>

<footnote n="4">⁴Centre Hospitalier Universitaire de Toulouse, Toulouse, France</footnote>

<footnote n="5">⁵Centre de recherche du Centre Hospitalier Universitaire de Sherbrooke, Sherbrooke, QC, Canada</footnote>

<footnote n="6">⁶Department of Medicine, Division of Neurology, Faculty of Medicine and Health Sciences, Université de Sherbrooke, Sherbrooke, QC, Canada</footnote>

<footnote n="7">⁷Institut Universitaire de France (IUF), Paris, France</footnote>

<footnote n="8">⁸These authors contributed equally</footnote>

<footnote n="9">⁹Lead contact</footnote>

<footnote>*Correspondence: etienne.mouisel@inserm.fr (E.M.), dominique.langin@inserm.fr (D.L.)</footnote>

<footnote>https://doi.org/10.1016/j.cmet.2024.10.018</footnote>
in vitro<sup>7</sup> as well as *in vivo* in rats<sup>8</sup> and humans.<sup>9</sup> However, mouse genetic approaches challenged the importance of BAT lipolysis. Mice with selective knockout of ATGL or its coactivator CGI-58, also known as ABHD5, in brown adipocytes maintain body temperature in the cold.<sup>10,11</sup> The same phenotype is observed in mice with absence of lipid droplets in brown adipocytes due to deficiency in the two acylCoA:diacylglycerol acyl transferase (DGAT) enzymes.<sup>12</sup> These mouse studies suggested that BAT lipolysis-derived FAs are dispensable, whereas blood-derived substrates are essential for cold-induced thermogenesis. However, mice with blunted ATGL activity show intact HSL expression that may in part compensate the lack of ATGL-mediated intracellular lipolysis activity. HSL has a more restricted pattern of tissue expression than ATGL and is highly expressed in BAT and in white adipose tissues (WATs).<sup>13</sup> Furthermore, *in vivo* thermogenic activity of BAT was not directly reported in these investigations.<sup>10-12</sup>

Here, we sought to determine the role of the main neutral li-
pases, ATGL and HSL, and intracellular lipolysis in BAT thermo-
genesis. We generated mice with inducible brown-adipocyte-
specific deletion of ATGL and HSL and studied cold-induced
thermogenesis under various environmental and nutritional chal-
lenges, resulting in BAT with different lipid droplet sizes. We also
specifically measured *in vivo* BAT thermogenic activity at ther-
moneutrality and during cold exposure. We show that expres-
sion of ATGL and HSL in brown adipocytes is required for
cold-induced BAT thermogenesis in fasted mice.

RESULTS

Mice lacking ATGL and HSL in brown adipocytes show
defective BAT intracellular lipolysis without changes in
systemic levels of glycerol and NEFAs

Brown-adipocyte-specific *Pnpla2* (encoding ATGL) and *Lipe* (encoding HSL) double-knockout (BAHKO) mice were generated through crosses of 3 genetically modified mouse models: *Pnpla2*-floxed mice,¹⁴ *Lipe*-floxed mice,¹⁵ and transgenic mice expressing tamoxifen-inducible Cre recombinase (CRE-ER<sup>T2</sup>) under the control of the murine *Ucp1* promoter.¹⁶ The invalida- tion of ATGL and HSL in BAT was obtained by tamoxifen gavage. *Pnpla2Lipe*-floxed mice treated with tamoxifen were used as control (Ctrl). BAHKO mice yielded offspring with the expected Mendelian ratio of genotypes and were healthy.

Interscapular BAT from BAHKO female and male mice at stan-
dard housing temperature (21°C) showed decreased ATGL and
HSL mRNA and protein levels (Figures 1A, 1B, S1A, and S1B).
There was no difference in ATGL and HSL protein content in
inguinal subcutaneous and perigonadal WAT (Figures 1C and
S1C). BAT lipolysis was measured in mice after 2 weeks of
cold acclimation (4°C), a condition associated with activation
of the lipolytic pathway. The lower expression of lipases resulted
in blunted non-esterified FA (NEFA) and glycerol release from
BAT explants (Figures 1D, S1D, and S1E). We then look at the
metabolic parameters in BAHKO and Ctrl mice. In fasted mice
at standard housing temperature (21°C), circulating non-esteri-
fied fatty acid (NEFA), glycerol, and ketone body levels were
not different in the two genotypes (Figures 1E and S1F). In the
fed state, circulating TG, glucose, and insulin levels were not
modified (Figures 1F and S1G). There was no difference in

food intake at 21°C between the two genotypes (Figure 1G). The data show that the lack of ATGL and HSL in BAT of female and male mice results in diminished intracellular brown adipocyte lipolysis with no impairment of fasting-induced systemic lipid mobilization.

Brown fat is hypertrophied in BAHKO mice

Female and male BAHKO mice showed similar body weight and fat mass as Ctrl mice (Figures 2A, 2B, S2A, and S2B). Weights of inguinal and perigonadal white fat depots were not different in the two genotypes (Figures 2C and S2C). However, BAT was heavier and lighter in color in BAHKO than in Ctrl mice (Figures 2C, 2D, and S2C). Histological examination revealed larger lipid droplets, with the presence of seemingly unilocular adipocytes (Figures 2E and S2D). Accordingly, the distribution in lipid droplet size was profoundly altered in BAHKO mouse BAT (Figures 2F and S2E). An increase in BAT TG content was observed in BAHKO mice (Figure 2G). The store of glycogen, which has recently been shown as an alternate source of substrate in mice without BAT lipid droplets,¹² was not different between the genotypes (Figure 2H). Transmission electron microscopy showed the presence of large areas occupied by lipid droplets in brown adipocytes (Figures 2I and 2J). Despite the change in adipocyte morphology, the surface and density of mitochondria were similar in BAHKO and Ctrl mice (Figure 2K).

Transition from brown-like to white-like AT morphology was associated with an increased gene expression of *Itgax* and *Emr1* coding, respectively, Cd11c and F4/80, two markers of macrophages/dendritic cells, and of inflammation-associated *Tnf* and *Il6* (Figures 2L and S2F). The latter data prompted us to analyze the composition of the stromal vascular fraction of BAT. In line with increased macrophage-related gene mRNA levels, the percentage of CD45<sup>+</sup>/CD11b<sup>+</sup>/F4-80<sup>+</sup> macrophages was increased in BAHKO mice without difference in CD45<sup>+</sup>/(CD3<sup>+</sup>) or (CD19<sup>+</sup>) or (CD335<sup>+</sup>) lymphocytes (Figures 2M and S2G). Other modifications of cell composition were observed in the stromal vascular fraction. An increase in CD45<sup>-</sup>/Sca-1<sup>+</sup>/CD31<sup>-</sup> cells suggested a higher percentage of progenitor cells, whereas the decrease in CD45<sup>-</sup>/Sca-1<sup>+</sup>/CD31<sup>+</sup> cells pointed at a reduced proportion of endothelial cells (Figures 2M and S2H). The data show that the enlarged BAT depot in BAHKO mice shows features of WAT, with larger lipid droplets in hypertrophied adipocytes and signs of tissue remodeling.

BAHKO and Ctrl mice exhibit different brown fat
characteristics according to housing temperatures

We further investigated BAT morphology during two thermal
challenges, 2 weeks in the cold (4°C) and 4 weeks at thermoneu-
trality (30°C). During prolonged cold exposure, Ctrl mouse adi-
pocytes originating from BAT presented a uniform multilocular
appearance (Figure 3A). However, large lipid droplets persisted
in BAT from BAHKO mice (Figure 3B). The percentage of pauci-
locular large adipocytes¹⁷ remained high in these mice. Accord-
ingly, BAT weight was higher in BAHKO than in Ctrl mice (Fig-
ure 3C). At molecular level, gene expression of markers of
multilocular brown adipocytes was markedly lower in BAHKO
mice (Figure 3D). In the opposite direction, thermoneutrality
<figure><img src="image_4.png" /></figure>

<figure><img src="image_5.png" /><figcaption>**Figure 1.** Mice lacking ATGL and HSL in brown adipocytes show defective interscapular BAT intracellular lipolysis</figcaption></figure>

(A) *Lipe* and *Pnpla2* mRNA levels in brown adipose tissue (BAT) from control (Ctrl) and brown-adipocyte-specific ATGL and HSL knockout (BAHKO) female mice (n = 6/genotype) housed at 21°C.

(B) Representative immunoblot and quantitation of HSL and ATGL in BAT from Ctrl (n = 7) and BAHKO (n = 6) female mice housed at 21°C.

(C) Representative immunoblot and quantitation of HSL and ATGL in subcutaneous (s.c.) and perigonadal (pg) white adipose tissue (WAT) from Ctrl and BAHKO female mice (n = 6/genotype) housed at 21°C.

(D) Non-esterified fatty acid (NEFA) and glycerol (nmol/mg of lipid) release in culture medium of BAT explants from Ctrl (n = 8) and BAHKO (n = 7) female mice acclimated for 2 weeks at 4°C.

(E) Circulating NEFA, glycerol, and ketone body levels during fasting in Ctrl (n = 7) and BAHKO (n = 6) female mice housed at 21°C.

(F) Circulating glucose, insulin, and triacylglycerol (TG) levels in fed Ctrl (n = 7) and BAHKO (n = 6) female mice housed at 21°C.

(G) Total 48 h food intake from Ctrl (n = 7) and BAHKO (n = 8) female mice housed in individual cages at 21°C.

Values represent means ± SEM. Data were analyzed using two-way ANOVA with Sidak's multiple comparison test (A–D) and unpaired Mann-Whitney test (E–G).
**p < 0.01, ***p < 0.001, ****p < 0.0001.

See also Figure S1.

was accompanied by whitening of Ctrl BAT, with the appearance of numerous unilocular adipocytes (Figure 3E). BAT of the two genotypes became similar. The weight of the fat depot, the histological appearance, and the distribution of lipid droplet size were also not different (Figures 3F and 3G). *Clstn3b*, *Ucp1*, and *Cidea* showed comparable mRNA levels in the two genotypes (Figure 3H) at thermoneutrality. The data reveal that Ctrl mice show pronounced changes in the morphological and gene expression parameters of BAT according to housing temperatures, which are markedly blunted in BAHKO mice.

**BAHKO mice exposed to cold show impaired maintenance of body temperature in the fasting state**

To determine the contribution of BAT TG hydrolysis on body temperature, we exposed at 4°C fasted or fed BAHKO mice previously housed at different temperatures. Male and female BAHKO and Ctrl mice housed at 21°C showed, in the fed state, similar and maintained rectal body temperatures throughout the cold challenge at 4°C (Figures 4A and S3A). In the fasted state, the drop in body temperature was more pronounced in female than in male Ctrl or BAHKO mice (p < 0.05), potentially due to
<figure><img src="image_6.png" /><figcaption>**Figure 2. Deficiency of ATGL and HSL in interscapular BAT promotes tissue remodeling**</figcaption></figure>

(A–C) Body weight (A), body composition (B), and fat pad weights (C) from Ctrl (n = 7) and BAHKO (n = 6) female mice housed at 21°C.

(D) Interscapular BAT macroscopic images representative of Ctrl and BAHKO female mice housed at 21°C.

(E and F) Hematoxylin/eosin staining of representative BAT sections (10×) (E) and lipid droplet (LD) diameter distribution and means (F) from Ctrl (n = 7) and BAHKO (n = 4) female mice housed at 21°C. Scale bars: 250 µm.

(G) Triacylglycerol (TG) in BAT (measured as TG-derived fatty acids [FAs] in µg/g proteins) from fed Ctrl and BAHKO male mice housed at 21°C (n = 3).

(legend continued on next page)
<figure><img src="image_7.png" /></figure>

<figure><img src="image_8.png" /><figcaption>**Figure 3.** Changes in interscapular BAT features of brown adipocyte ATGL- and HSL-deficient male mice depend on housing temperature
(A–D) Ctrl (n = 8) and BAHKO (n = 4) mice acclimated to 4°C for 2 weeks. Hematoxylin/eosin staining of representative interscapular BAT sections (10×; scale, 250 µm) (A). LD diameter distribution and means (B). BAT weight (C). *Clstn3b*, *Ucp1*, and *Cidea* BAT mRNA levels (D).</figcaption></figure>

(E–H) Ctrl and BAHKO (n = 6/genotype) mice acclimated to 30°C for 4 weeks. Hematoxylin/eosin staining of representative BAT sections (10×; scale, 250 µm) (E). LD diameter distribution and means (F). BAT weight (G). *Clstn3b*, *Ucp1*, and *Cidea* BAT mRNA levels (H).

Values represent means ± SEM. Data were analyzed using unpaired Mann-Whitney test (B, C, F, and G) and two-way ANOVA with Sidak's multiple comparison test (D and H). **p** < 0.01, ***p** < 0.0001.

differences in specific heat capacity conferred by lower body weight (Ctrl male and female mice: 35.8 ± 1.4 g, *n* = 10 and 24.9 ± 0.9 g, *n* = 12; BAHKO male and female mice: 33.7 ± 1.0 g, *n* = 18 and 24.4 ± 0.5 g, *n* = 15) and fat mass (Ctrl male and female mice: 5.1 ± 0.8 g, *n* = 10 and 2.7 ± 0.5 g, *n* = 12; BAHKO male and female mice: 4.6 ± 0.4 g, *n* = 18 and 3.0 ± 0.3 g, *n* = 15) in female mice.¹⁸ Of note, the drop in temperature was much more pronounced in BAHKO than in Ctrl fasted mice (Figures 4A, 4B, S3A, and S3B). Acclimation to thermoneutrality (30°C) induces a downregulation of mechanisms governing adaptive thermogenesis.¹⁹ Consequently, the acute cold challenge following acclimation to thermoneutrality resulted in a greater decrease in body temperature in the two genotypes during fasting compared with mice raised at 21°C. However, the drop in temperature remained more pronounced in BAHKO than in Ctrl mice, as observed at 21°C (Figures 4C and 4D). To pursue investigation of the effect of housing temperatures, we acclimated mice to 4°C for 10 days with *ad libitum* access to

food. Similar to the fed state at standard housing temperature, there was no difference in rectal temperature between the two genotypes (time 0 in Figure 4E). However, when food was withdrawn, BAHKO mice showed a drop in temperature that was not observed in Ctrl mice (Figures 4E and 4F). This set of data shows that BAT lipases are important for the maintenance of body temperature in fasted but not in fed mice exposed to cold.

**Cold-induced BAT thermogenic activity is altered in BAHKO mice**

To gain insight into the control of energy expenditure in BAHKO mice, we performed indirect calorimetry at 21°C following injection of CL316,243, a selective agonist of β₃-adrenoceptors expressed in mouse BAT and WAT. Acute treatment with CL-316,243 induced an increase in plasma glycerol, NEFA, and ketone body levels in BAHKO and Ctrl mice (Figure S4A). However, BAHKO mice exhibited a blunted CL-316,243-induced increase in metabolic rate (Figures 5A and S4B). The evolution of body

(H) Glycogen content in BAT of Ctrl and BAHKO male mice (n = 6–7/genotype) compared with liver content in Ctrl male mice (n = 5) housed at 21°C.

(I) Transmission electron microscopy representative image focusing on lipid droplets (left) and mitochondria (right) in BAT from fed Ctrl and BAHKO male mice subjected to 4°C for 4 h. Scale bar: 5 µm (left) and 1 µm (right).

(J and K) LD area (J), mitochondria area (K, left), and density (K, right) from transmission electron microscopy BAT images obtained in Ctrl (n = 4) and BAHKO (n = 4) male mice subjected to 4°C for 4 h.

(L) Inflammatory genes mRNA levels in BAT of Ctrl and BAHKO female mice housed at 21°C (n = 5–6/genotype).

(M) Stromal vascular fraction (SVF) populations in BAT of Ctrl and BAHKO female mice housed at 21°C (n = 5/genotype). Endo., CD45⁻/Sca-1⁺/CD31⁺ endothelial cells; Prog., CD45⁻/Sca-1⁺/CD31⁻ progenitor cells; Lympho., CD45⁺/(CD3⁺) or (CD19⁺) or (CD335⁺) lymphocytes; Macro., CD45⁺/CD11b⁺/F4-80⁺ macrophages.

Values represent means ± SEM. Data were analyzed using unpaired Mann-Whitney test (A and F), two-way ANOVA with Sidak's (B, C, and M) or Tukey's (L) multiple comparison tests, unpaired t test with Welch's correction (G), Kruskal-Wallis with Dunn's multiple comparison test (H), and unpaired t test (J and K).
\*p < 0.05, \*\*p < 0.01, \*\*\*p < 0.001.

See also Figure S2.
A

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

B

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

C

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

D

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

E

<figure><img src="image_13.png" /></figure>

F

<figure><img src="image_14.png" /></figure>

temperature during various challenges and energy expenditure following injection of a β₃-adrenergic agonist was indirectly suggestive of a defect in cold-induced BAT thermogenic activity of BAHKO mice. To directly assess BAT activity, we designed a crossover study in which mice of the two genotypes were sequentially exposed in random order at thermoneutral condition (30°C) and cold exposure (10°C) for 7 days in metabolic cages, with a 1-week washout period in between (Figure 5B). Positron emission tomography-computed tomography (PET/CT) was performed, using [¹¹C]-acetate to measure BAT blood flow and oxidative metabolism and [¹¹C]-palmitate to measure BAT NEFA uptake, oxidation, esterification, and release at the term of each of the 7-day thermoneutral vs. cold conditions, while indirect calorimetry measurements were performed throughout these exposures. At 10°C, a strong and expected increase in energy expenditure was observed in Ctrl and BAHKO mice (Figures S4C and S4D). In comparison with exposure at 30°C, cold acclimation induced an increase in the interscapular BAT blood flow index and oxidative metabolism index in Ctrl mice, which was fully abolished in BAHKO mice (Figures 5C and 5D). The time course of [¹¹C] activity in different fat depots illustrates the profound loss of BAT thermogenic response in interscapular BAT of BAHKO mice compared with Ctrl mice. It also reveals the absence of substantial oxidative metabolism in inguinal and perigonadal WAT despite differences in tissue perfusion between Ctrl and BAHKO mice (Figure S5). During cold exposure, rates of NEFA uptake, oxidation, and ester-

<caption>**Figure 4.** Maintenance of body temperature is impaired during cold exposure in fasted but not in fed male mice lacking ATGL and HSL in brown adipocytes</caption>

(A and B) Rectal body temperature (A) and Kaplan-Meier curve of mice with rectal body temperature (RBT) > 28°C (B) during acute cold exposure (4°C) in fed (dotted lines, *n* = 8–9/genotype pooled from 2 independent experiments) and fasted (solid lines, *n* = 10–17/genotype pooled from 3 independent experiments) Ctrl and BAHKO mice housed at 21°C.

(C and D) Rectal body temperature (C) and Kaplan-Meier curve of mice with RBT > 28°C (D) during acute cold exposure (4°C) in fasted Ctrl and BAHKO mice acclimated for 3 weeks in a 30°C thermoneutral environment (*n* = 6/genotype).

(E and F) Rectal body temperature (E) and Kaplan-Meier curve of mice with RBT > 28°C (F) in Ctrl (*n* = 8) and BAHKO (*n* = 4) fasted mice acclimated for 10 days in a 4°C cold environment.

Values represent means ± SEM. Data were analyzed using general linear model (A, C, and E) and log-rank (Mantel-Cox) test (B, D, and F).
**p** < 0.01, ***p** < 0.001.

See also Figure S3.

ification tended to be lower in BAHKO than in Ctrl mice (Figures 5E–5G). Circulating TG level was lower at 10°C than at 30°C in Ctrl mice (Figure S4E). The reduction in TG levels tended to be less pronounced in BAHKO mice. In Ctrl mice, BAT NEFA release was low and tended to diminish

during cold exposure, which is in line with high intracellular utilization of FAs (Figure 5H). In BAHKO mice, BAT NEFA release was very low at 10°C and 30°C, with no significant differences between genotypes. These results suggest that the absence of intracellular lipolysis in brown adipocytes profoundly impacts the thermogenic activity of BAT in the cold.

### Obese mice lacking ATGL and HSL in brown adipocytes exhibit altered BAT morphology and cold-induced hypothermia

To determine the importance of BAT neutral lipases in the context of obesity and impaired glucose tolerance, we fed male mice with a high-fat diet for 10 weeks at 21°C. The increase in body weight and fat mass was similar in the two genotypes (Figures 6A and 6B). As observed in chow-diet-fed animals, plasma metabolic parameters were not different between the genotypes in the fed and fasted states (Figures S6A and S6B). Glucose tolerance test was performed on mice fed the high-fat diet (Figure 6C). The area of the curve was not different in the two genotypes²⁰ (Figure 6D). At the time of euthanasia, weights of white fat pads were similar, whereas interscapular BAT was heavier in BAHKO compared with Ctrl mice (Figure 6E). Histological analyses of brown fat showed a pattern that was intermediate between those of chow-diet-fed mice raised at 21°C and at 30°C (Figures 3E, 6F, and S2D). In Ctrl mice, areas of enlarged brown adipocytes were dispersed within areas of smaller
<figure><img src="image_15.png" /></figure>

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<figure><img src="image_23.png" /><figcaption>**Figure 5.** Indirect calorimetry and PET/CT imaging reveals altered thermogenic activity in interscapular BAT from brown adipocyte ATGL- and HSL-deficient female mice</figcaption></figure>

(A) Evolution of oxygen consumption rate (VO₂) in Ctrl (n = 7) and BAHKO (n = 9) mice housed at 21°C before and during injection of the β₃-adrenergic agonist, CL-316,243.

(B) Randomized crossover design of PET/CT imaging experiments with infusion of [¹¹C]-acetate and [¹¹C]-palmitate in Ctrl and BAHKO mice (n = 8-12/genotype).

(C and D) Blood flow (C) and oxidative metabolism (D) indexes derived from [¹¹C]-acetate acquisition in Ctrl and BAHKO mice (n = 8-12/genotype).

(legend continued on next page)
<figure><img src="image_24.png" /><figcaption>Figure 6. Defect in BAT response is maintained in brown adipocyte ATGL- and HSL-deficient male mice with diet-induced obesity</figcaption></figure>

(A and B) Body weight (A) and fat mass (B) in Ctrl and BAHKO mice fed high-fat diet at 21°C (n = 6/genotype).

(C and D) Intraperitoneal glucose tolerance test in Ctrl and BAHKO mice (n = 6/genotype) fed high-fat diet. Blood glucose (C) and derived area of the curve (AOC, D).

(E–G) Fat pad weights (E), hematoxylin/eosin staining of representative interscapular BAT sections (10×; scale, 250 µm) (F), and LD diameter distribution and means (G) in Ctrl and BAHKO mice fed high-fat diet (n = 4–6/genotype).

(H–J) Rectal body temperature (H), Kaplan-Meier curve of mice with rectal body temperature (RBT) > 28°C (I), and blood glucose (J) upon acute cold exposure (4°C) in fasted Ctrl and BAHKO mice fed high-fat diet (n = 6/genotype).

Values represent means ± SEM. Data were analyzed using two-way ANOVA with Sidak's (A, B, and C) or Tukey's (J) multiple comparison tests, unpaired Mann-Whitney test (D, E, and G), general linear model (H), and log-rank (Mantel-Cox) test (I). \*p < 0.05, \*\*p < 0.01.

See also Figure S6.

multilocular cells (Figures 6F and 6G). The patches of enlarged brown adipocytes were more pronounced in BAHKO mice, as reflected by a right-shift in lipid droplet size distribution. Similar to all other tested conditions, BAHKO mice showed a more pronounced drop in body temperature than Ctrl mice during a cold test in fasting conditions (Figures 6H and 6I). Interestingly, BAHKO mice maintained a stable plasma glucose level during acute cold exposure, whereas it was decreased in Ctrl mice (Figure 6J). These data show that white-like morphology of BAT and failure to maintain body temperature is also present in BAHKO mice with diet-induced obesity.

DISCUSSION

FAs play an important role in cold-induced thermogenesis. As the main source of energy stored as TG in mammals, they are essential substrates for BAT robust oxidative metabolism in the fasted state.<sup>2,21</sup> Moreover, brown adipocyte mitochondria are equipped with a unique machinery allowing non-shivering thermogenesis. In the inner mitochondrial membrane, UCP1 dissipates the mitochondrial H<sup>+</sup> gradient, which results in the conversion of the energy of substrate oxidation into heat.<sup>22</sup> UCP1 operates as a H<sup>+</sup> carrier activated by long-chain FAs.<sup>4</sup> There

(E–H) Palmitate uptake (E), oxidation (F), esterification (G), and NEFA release (H) rates derived from [¹¹C]-palmitate acquisition in Ctrl and BAHKO mice (n = 8–12/genotype).

Values represent means ± SEM. Data were analyzed using two-way ANOVA with Sidak's multiple comparison test (A, C, and D) or uncorrected Fisher's LSD (E–H). \*p < 0.05, \*\*p < 0.01, \*\*\*p < 0.001, \*\*\*\*p < 0.0001.

See also Figures S4 and S5.
are several potential sources of FAs for β-oxidation and UCP1 activation. As a first endogenous source, intracellular lipolysis in brown adipocytes generates FAs. A second endogenous source may derive from *de novo* lipogenesis, i.e., glucose carbon incorporation into FA.²³ The third source is exogenous circulating NEFA. Finally, another exogenous source of FAs is generated by the hydrolysis of circulating TG-rich lipoproteins.²⁴ Intracellular lipolysis in brown adipocytes has long been considered as an essential pathway for the provision of FAs to brown fat thermogenesis. Although supported by a series of *in vitro* and *in vivo* data,⁷⁻⁹,²⁵ the importance of intracellular lipolysis has been challenged by mouse models with deficient ATGL activity.¹⁰,¹¹ In the current study, we show that the lack of the two rate-limiting adipose neutral lipases, ATGL and HSL, in brown adipocytes impairs cold-induced thermogenesis in the fasting state when lipolysis is maximally stimulated. The resulting hypothermia of BAHKO mice is observed in various environmental conditions. The importance of brown adipocyte ATGL and HSL is unequivocally demonstrated by direct *in vivo* measurement of BAT thermogenic activity with PET/CT imaging. In the fed state, maintenance of body temperature is not altered by the defect in brown adipocyte intracellular lipolysis of BAHKO mice.

In fasted BAHKO mice, we explored the response to cold under different housing temperature conditions, with a similar outcome: the drop in body temperature was more pronounced in mice lacking the two lipases. We obtained similar results in obese mice with diet-induced obesity. In contrast to previous mouse studies reporting no influence of partial genetic inhibition of intracellular lipolysis,¹⁰,¹¹ BAT thermogenesis was directly assessed *in vivo* using the [¹¹C]-acetate PET method in the present study. The induction of BAT oxidative metabolism by cold was abrogated in fasted BAHKO mice. Our data bring direct *in vivo* evidence of the importance of brown adipocyte lipolysis in response to cold. Accordingly, combined inhibition of ATGL and HSL in differentiated brown adipocytes totally blunts β-adrenergic mitochondrial respiration.⁷ Moreover, administration of the antilipolytic drug, nicotinic acid, in humans or rats results in impaired cold-induced thermogenesis.⁸,⁹ However, WAT intracellular lipolysis was also suppressed in these latter *in vivo* approaches. In the present study, *in vivo* inhibition of intracellular lipolysis occurred only in BAT, not in WAT. The alteration in FA oxidation seen using [¹¹C]-palmitate PET also supported the defect in cold-induced BAT thermogenic activity. Interestingly, NEFA uptake and esterification were also blunted in BAHKO mice during cold exposure. The tendency to a lower cold-induced reduction of circulating TG in BAHKO compared with Ctrl mice between 30°C and 10°C suggests that this thermogenic energy source is not used to a larger level in fasted BAHKO mice. Therefore, greater utilization of circulating NEFA and TG by BAT did not occur with suppression of BAT intracellular lipolysis. This response is consistent with a role for intracellular TG lipolysis *in vivo* in the cold-induced metabolic activation of BAT. We previously reported that activated brown-like human adipocytes show enhanced FA catabolic metabolism, i.e., lipolysis and FA oxidation, and also FA anabolic metabolism, i.e., TG synthesis, compared with white adipocytes.²⁶ In these cells, there is a tight coupling between lipolysis and esterification.²⁷

In contrast to our model with combined ATGL and HSL deficiency, mice with BAT lacking ATGL or its coactivator CGI-58

were reported to exhibit no cold-induced hypothermia.¹⁰,¹¹ Although ATGL plays a predominant role in initiating the sequential reactions leading to complete hydrolysis of TG in AT, HSL possesses high TG hydrolase activity.²⁷⁻²⁹ Moreover, there is functional redundancy between HSL and ATGL.³⁰ HSL may partially replace ATGL and ensure sufficient intracellular lipolysis in BAT in mice with deficient ATGL activity. In the heart, HSL overexpression restores TG hydrolase activity and rescues the lethal cardiac phenotype observed in ATGL knockout mice.³¹ Similar to ATGL and CGI-58 BAT-specific knockout mice, mice lacking lipid droplets in brown adipocytes due to deficiency in DGAT1, and 2 catalyzing the final committed step in TG synthesis, are tolerant to cold.¹² These mice show increased glucose uptake and glycogen BAT content as well as enhanced *de novo* lipogenesis. Confirming earlier studies,³²,³³ use of *in situ* microdialysis in humans suggests that the main fate of glucose is *de novo* synthesis of FAs that are esterified into TG prior to release during lipolysis for oxidation and heat production.³⁴ In BAT-specific DGAT knockout mice, glucose from the circulation and glycogen store may be metabolized to produce FAs fueling thermogenesis. Another route for glucose in brown adipocytes is the synthesis of glycerol-3-phosphate that constitutes the backbone for FA esterification.²⁶ In obese mice, we observed preserved plasma glucose levels during cold in BAHKO but not in Ctrl mice. Moreover, there was no accumulation of glycogen in BAT from BAHKO mice. Together, the data obtained in mice deficient in brown adipocyte ATGL and HSL are consistent with the use of glucose for FA synthesis and esterification into TG during cold.³⁴

At standard housing temperature (21°C), BAT from BAHKO mice show larger lipid droplets than Ctrl mice. BAT hypertrophy is observed in mice with deficiency in ATGL or HSL, two models with reported cold tolerance.¹⁰,³⁵ Thermoneutral housing temperature attenuated the morphological differences between Ctrl and BAHKO mice. Expression of *C1stn3b*, a selective marker of multilocular adipocytes, followed a similar pattern.³⁶ At thermoneutrality, BAT from the two genotypes were indistinguishable. However, cold intolerance persists in that condition, further suggesting that acute BAT intracellular TG lipolysis is essential for cold-induced BAT thermogenesis and that brown adipocyte lipid droplet morphology is not per se a determinant of cold-induced thermogenesis.

In conclusion, we postulate that, during fasting, intracellular TG hydrolysis in brown adipocytes is an obligatory step of the BAT thermogenic response, which, in turn, appears necessary to sustain whole-body cold-induced thermogenesis. From a therapeutic perspective, activation of BAT lipolysis using agonists of G-protein-coupled receptors (GPCRs) was identified as a means to activate the thermogenic program.³⁷⁻³⁹ Recent work revealed that BAT intracellular lipolysis itself may also control expression of GPCRs promoting thermogenesis.⁴⁰ Selectively targeting BAT intracellular TG lipolysis therefore constitutes a promising way in which to activate adaptive thermogenesis.

## Limitations of the study

Our study aims to determine the importance of the combined actions of the two lipases, ATGL and HSL, responsible for most of adipocyte TG hydrolytic activities, in cold-induced BAT thermogenesis.²⁷,²⁹ The individual contributions of ATGL and
HSL have not been investigated here. Comparison of individual knockout models in the same experimental settings may prove interesting. However, ATGL and HSL have interchangeable functions in intracellular TG hydrolysis but also transacylation of acyl-glycerols.<sup>30,31,41</sup> Redundancy may complicate interpretation in the context of the lack of one of the two lipases. Metabolic fluxes have recently been quantified during acute cold exposure in wild-type mice.<sup>21</sup> How these fluxes and other sources of BAT thermogenesis, such as intracellular *de novo* lipogenesis, circulating lipoprotein-TG, and circulating NEFAs, are modulated in mice with abolished brown adipocyte intracellular lipolysis is not known. Specifically, whether, as suggested by the data, glucose is used for FA synthesis and esterification into TG during cold in BAHKO mice has not been experimentally shown. Moreover, longer adaptations to cold may prove different from the relatively short and midterm exposures used in the present study. Direct measurement of BAT thermogenic activity was obtained using PET/CT imaging, a method that requires anesthesia. Although anesthesia has been shown to diminish the BAT thermogenic response,<sup>42</sup> the pronounced increase in BAT thermogenic response following 7 days of acclimation at 10°C was markedly blunted in BAHKO mice. Therefore, the anesthetic cannot explain the near-total disappearance of BAT thermogenesis in BAHKO mice. The data also show that BAT lipases are important for the maintenance of body temperature in fasted mice exposed to cold in the two sexes. The difference in amplitude of the change in body temperature may be related to differences in body composition in male vs. female mice. Whether this difference is related to other factors warrants further studies. Finally, the reason for the restriction of cold intolerance to the fasting state in BAHKO mice is not known. Future studies need to be performed to directly assess *in vivo* oxidative metabolism in BAT and other tissues of these animals during the fed state.

## RESOURCE AVAILABILITY

### Lead contact

Dominique Langin (dominique.langin@inserm.fr) is the lead contact for this manuscript and should be contacted for further information and requests for resources and reagents.

### Materials availability

No unique reagents or materials were generated in these studies.

### Data and code availability

* Uncropped scans and source data are available as Data S1.

* No proprietary data or code is associated with this manuscript.

* Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.

## ACKNOWLEDGMENTS

We thank Maryne Araujo (Centre Hospitalier Universitaire de Toulouse) for technical assistance on lipid analyses; Mélanie Archambault and Véronique Dumulon-Perreault (Université de Sherbrooke) for experimental assistance with animal care, treatments, and metabolic measurements; Jean-François Beaudoin and Tommy Galarneau (CRCHUS and Université de Sherbrooke) for technical assistance with μTEP; and Dr. Serge Phoenix, Sophie Beauchemin, and René Ouellet (Université de Sherbrooke) for the synthesis of radiotracers. We are grateful to Dr. Ouhida Benrezzak (Université de Sherbrooke) for assistance with protocols related to animal care and ethics committee. The following technological facilities contributed to the work: GenoToul Anex-

plo (Sophie Le Gonidec, Morgane Rousseau, CREFRE UMS06, Toulouse), GenoToul TRI (Christèle Segura and Alexia Zakaroff, I2MC, Toulouse, and Vanessa Soldan and Stéphanie Balor, METI imaging facility, CBI, Toulouse), GenoToul GeT Health (Emeline Lhuillier, I2MC, Toulouse), and WeMet (Alexandre Lucas, I2MC, Toulouse). This work was supported by Inserm, Université Toulouse III, European Foundation for the Study of Diabetes (EFSD/Novo Nordisk Programme for Diabetes Research in Europe 2019 to D.L.), Société Francophone du Diabète (to E.M.), Fondation pour la Recherche Médicale (DEQ20170336720 (to D.L.), AstraZeneca France (to D.L.), European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme (SPHERES, ERC Synergy Grant agreement no 856404 to D.L.), and a grant from the Canadian Institutes of Health Research (#299962 to A.C.C.). GenoToul TRI is a member of the national infrastructure France-Biolmaging supported by the French National Research Agency (ANR-10-INBS-04).

## AUTHOR CONTRIBUTIONS

Conceptualization, E.M., P.-D.D., A.C.C., and D.L.; methodology, E.M., R.F.-F., E.R., D.P.B., and D.L.; investigation, E.M., A.B., C.N., S.C.-S., M.-A.M., E.R., C.B., P.V., S.C.-B., C.F.-F., and P.-D.D.; writing—original draft, E.M. and D.L.; writing—review & editing, E.M., A.B., C.N., G.T., P.G., D.P.B., P.-D.D., A.C.C., and D.L.; funding acquisition, E.M. and D.L.; resources, G.T. and K.A.I.O.; supervision, E.M., P.-D.D., A.C.C., and D.L.

## DECLARATION OF INTERESTS

The authors declare no competing interests.

## STAR★METHODS

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

* KEY RESOURCES TABLE

* EXPERIMENTAL MODEL AND STUDY PARTICIPANT DETAILS

* Mouse models

* METHOD DETAILS

* Glucose tolerance test

* Blood analyses

* Cold tolerance test

* Measurement of energy expenditure following injection of the β₃-adrenergic agonist, CL-316,243

* Indirect calorimetry and PET/CT imaging and analyses

* Histology and lipid droplet size determination

* Transmission electron microscopy (TEM)

* Flow cytometry

* Lipolysis in BAT explants

* Determination of mRNA levels

* Protein analyses

* Quantitation of tissue TG and glycogen levels

* Statistical analyses

## SUPPLEMENTAL INFORMATION

Supplemental information can be found online at https://doi.org/10.1016/j.cmet.2024.10.018.

Received: February 2, 2024

Revised: July 1, 2024

Accepted: October 21, 2024

Published: November 19, 2024

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46. Ye, R.Z., Montastier, É., Noll, C., Frisch, F., Fortin, M., Bouffard, L., Phoenix, S., Guérin, B., Turcotte, É.E., and Carpentier, A.C. (2022). Total Postprandial Hepatic Nonesterified and Dietary Fatty Acid Uptake Is Increased and Insufficiently Curbed by Adipose Tissue Fatty Acid Trapping in Prediabetes With Overweight. Diabetes 71, 1891-1901. https://doi.org/10.2337/db21-1097.

47. Bankhead, P., Loughrey, M.B., Fernández, J.A., Dombrowski, Y., McArt, D.G., Dunne, P.D., McQuaid, S., Gray, R.T., Murray, L.J., Coleman, H.G., et al. (2017). QuPath: open source software for digital pathology image analysis. Sci. Rep. 7, 16878. https://doi.org/10.1038/s41598-017-17204-5.

48. Kluyver, T., Ragan-Kelley, B., Perez, F., Granger, B., Bussonnier, M., Frederic, J., Kelley, K., Hamrick, J., Grout, J., Corlay, S., et al. (2016). Jupyter Notebooks-a publishing format for reproducible computational workflows. In Positioning and Power in Academic Publishing: Players, Agents and Agendas (IOS Press), pp. 87-90. https://doi.org/10.3233/978-1-61499-649-1-87.

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53. Schindelin, J., Arganda-Carreras, I., Frise, E., Kaynig, V., Longair, M., Pietzsch, T., Preibisch, S., Rueden, C., Saalfeld, S., Schmid, B., et al. (2012). Fiji: an open-source platform for biological-image analysis. Nat. Methods 9, 676-682. https://doi.org/10.1038/nmeth.2019.

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<figure><img src="image_26.png" /></figure>

## 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">Antibodies</td></tr><tr><td>Rabbit polyclonal ATGL</td><td>Cell Signaling Technology</td><td>Cat# 2138, lot 6; RRID:AB_2167955</td></tr><tr><td>Rabbit monoclonal HSL</td><td>Cell Signaling Technology</td><td>Cat# 18381, clone D6W5S, lot 1; RRID:AB_2798800</td></tr><tr><td>Rabbit monoclonal GAPDH</td><td>Cell Signaling Technology</td><td>Cat# 2118, clone 14C10, lot 14; RRID:AB_561053</td></tr><tr><td>Anti-rabbit horse radish peroxidase-labelled secondary antibody</td><td>Cell Signaling Technology</td><td>Cat# 7074, lot 32</td></tr><tr><td>CD45-VioBlue</td><td>REAfinity, Miltenyi</td><td>130-110-802; RRID:AB_2658222</td></tr><tr><td>F4/80 APC</td><td>REAfinity, Miltenyi</td><td>130-116-525; RRID:AB_2733417</td></tr><tr><td>CD31-PE</td><td>REAfinity, Miltenyi</td><td>130-111-540</td></tr><tr><td>Rat anti-mouse CD16/32</td><td>BD Biosciences</td><td>Cat# 553141, clone 2.4G2; RRID:AB_394656</td></tr><tr><td colspan="3">Chemicals, peptides, and recombinant proteins</td></tr><tr><td>CL-316,243</td><td>Sigma-Aldrich</td><td>Cat# C5976</td></tr><tr><td>Tamoxifen Free Base</td><td>Sigma-Aldrich</td><td>Cat# T5648</td></tr><tr><td>Multiscribe Reverse Transcriptase</td><td>ThermoFischer Scientific</td><td>Cat# 4368813</td></tr><tr><td>Fast SYBR Green Master Mix</td><td>ThermoFischer Scientific</td><td>Cat# 4385614</td></tr><tr><td>Taqman Fast Advanced Master Mix</td><td>ThermoFischer Scientific</td><td>Cat# 4444558</td></tr><tr><td>Clostridium histolyticum collagenase</td><td>Sigma</td><td>C6885</td></tr><tr><td>LiberaseTM Research Grade</td><td>Roche</td><td>05401127001</td></tr><tr><td>Streptomyces hyalurolyticus hyaluronydase</td><td>Sigma</td><td>H1136</td></tr><tr><td>SDS-PAGE gel (4-20% gradient)</td><td>Biorad</td><td>Cat# 5678094 ou 5678095</td></tr><tr><td>Trans-Blot turbo RTA transfert kit, nitrocellulose membrane</td><td>Biorad</td><td>Cat# 1704271</td></tr><tr><td>Clarity Western ECL Substrate</td><td>Biorad</td><td>Cat# 170-5060</td></tr><tr><td>Aspergillus niger amyloglucosidase</td><td>Sigma</td><td>Cat# 10113-1G, lot BCBD1453V</td></tr><tr><td>D-(+) glucose</td><td>Sigma</td><td>G8270</td></tr><tr><td>Bovine serum albumin</td><td>Sigma</td><td>A7030</td></tr><tr><td>Formalin solution</td><td>Sigma</td><td>HT501128</td></tr><tr><td>Eosin G o Y 0.5% alcoholic</td><td>Diapath</td><td>C0353</td></tr><tr><td>Mayer hematoxylin</td><td>Diapath</td><td>C0303</td></tr><tr><td>Cacodylate buffer</td><td>EMS</td><td>11655</td></tr><tr><td>EMbed812 resin</td><td>EMS</td><td>14120</td></tr><tr><td colspan="3">Critical commercial assays</td></tr><tr><td>NEFA C kit</td><td>Fujifilm Wako</td><td>Cat# SBDW1W434-91795; SBDW1W436-91995</td></tr><tr><td>Free Glycerol reagent</td><td>Sigma</td><td>Cat# F6428</td></tr><tr><td>Glucose GOD FS kit</td><td>DiaSys</td><td>Cat# 1 2500 99 10 026, lot 24781</td></tr><tr><td>Insulin</td><td>ALPCO Diagnostics</td><td>Cat# 80-INSMS-E1</td></tr><tr><td>Ketone bodies</td><td>Abbott</td><td>Cat# 71302-75</td></tr><tr><td>Qiazol buffer</td><td>Qiagen</td><td>Cat# 79306</td></tr><tr><td>RNeasy kit</td><td>Qiagen</td><td>Cat# 74106</td></tr><tr><td>DNasel</td><td>Invitrogen</td><td>Cat# 18068-015</td></tr><tr><td>BCA Protein Assay kit</td><td>ThermoFisher</td><td>Cat #23225</td></tr><tr><td>Zombie UV</td><td>BioLegend</td><td>423107</td></tr><tr><td>Brilliant Stain Buffer</td><td>BD Biosciences</td><td>563794</td></tr><tr><td colspan="3">Experimental models: Organisms/strains</td></tr><tr><td>C57BL/6J</td><td>Janvier Laboratories</td><td>N/A</td></tr></tbody></table>

(Continued on next page)
Continued

<table><thead><tr><td>REAGENT or RESOURCE</td><td>SOURCE</td><td>IDENTIFIER</td></tr></thead><tbody><tr><td>Mouse: B6 N.129S-Pnpla2<sup>tm1Eek</sup>/J (Pnpla2<sup>flox/flox</sup>)</td><td>Jackson Laboratory</td><td>N/A</td></tr><tr><td>Mouse: B6.129P2-Lipe<sup>tm2Rze</sup>/J (Lipe<sup>flox/flox</sup>)</td><td>Rudolf Zecher, University of Graz</td><td>N/A</td></tr><tr><td>Mouse: C57BL/6-Tg(Ucp1-icre/ERT2)Cw/J</td><td>Christian Wolfrum, ETH, Zürich</td><td>N/A</td></tr><tr><td>Mouse: Double-Pnpla2Lipe-floxed</td><td>This paper</td><td>N/A</td></tr><tr><td>Mouse: BAHKO</td><td>This paper</td><td>N/A</td></tr></tbody></table>

Oligonucleotides

<table><tr><td>Oligonucleotide sequences are listed in Table S1</td><td>N/A</td></tr></table>

Software and algorithms

<table><tr><td>GraphPad Prism (version 9.5.0 for PC, version 10.01 for Mac)</td><td>GraphPad</td><td>https://www graphpad com</td></tr><tr><td>QuPath-0.3.0</td><td>QuPath-0.3.0.Ink</td><td>N/A</td></tr></table>

Other

<table><tr><td>Rectal probe for mice</td><td>BIOSEB</td><td>BIO-BRET3</td></tr><tr><td>Thermocouple Thermometer-Single Input</td><td>AZ Instrument Corp.</td><td>Single K.J.T AZ8851</td></tr><tr><td>Mouse: Chow diet</td><td>Ssniff</td><td>V1534</td></tr><tr><td>Mouse: 60% high-fat diet</td><td>Ssniff</td><td>E15742-347</td></tr></table>

Deposited data

<table><tr><td>Data S1 - Source Data</td><td>This paper</td><td>N/A</td></tr></table>

EXPERIMENTAL MODEL AND STUDY PARTICIPANT DETAILS

Mouse models

C57BL/6J mice were obtained from Janvier Laboratories. B6N.129S-Pnpla2<sup>tm1Eek</sup>/J (Pnpla2<sup>flox/flox</sup>) and B6.129P2-Lipe<sup>tm2Rze</sup>/J (Lipe<sup>flox/flox</sup>) were first crossed to obtain double transgenic mice.<sup>14,15</sup> Pnpla2Lipe-floxed mice were then crossed with C57BL/6-Tg(Ucp1-icre/ERT<sup>2</sup>)Cw/J mice.<sup>16</sup> To activate the inducible Cre recombinase and obtain double knock-out in brown adipocytes (BAHKO mice), tamoxifen (Sigma) was dissolved and solubilized in Corn Oil (Sigma), and given daily at a dose of 1.5 mg by oral gavage for 5 days. Ctrl mice were Pnpla2Lipe-floxed littermates that do not express Cre treated with tamoxifen. Mice from several litters were used in each protocol to avoid litter-to-litter variation. Mice were housed at 21°C with food and water provided ad libitum and maintained on a 12-h light (7am-7pm), 12-h dark cycle. Mice were fed chow (Ssniff V1534) or 60% high fat (Ssniff E15742-347) diets. During chronic cold (4°C) and thermoneutrality (30°C) experiments, mice were placed 2 and 4 weeks in a cold-room and a vented animal cabinet (Noroit A-box EP-20), respectively. To determine body composition, NMR analyses (Minispec, Bruker) were performed on awake mice following the manufacturer's instructions. Mice were housed and manipulated according to Inserm guidelines and European Directive 2010/63/UE in the local animal care facility (agreements A 31 555 04 and C 31 555 07). Protocols were approved by the French Ministry of Research after review by local ethical committee (comité d'éthique en expérimentation animale de l'UMS006/CREFRE, CEEA122, Toulouse, France). Ctrl and BAHKO mice were sent to the University of Sherbrooke for indirect calorimetry and PET/CT imaging and analyses as described below. Experiments were approved by the Ethical Committee for Animal Care and Experimentation of the Université de Sherbrooke.

METHOD DETAILS

Glucose tolerance test

Glucose tolerance tests were performed around 2 p.m. D-(+) glucose (Sigma) at 1 mg/g body weight was administered by intraperitoneal route to 6h fasted mice. Glycemia was checked on blood from tail vein 30 min before glucose injection and then at various times after glucose administration with a Glucotrend Accu-Chek Performa (Roche SAS).

Blood analyses

Blood was collected in EDTA-coated tubes (Sarstedt) through tail vein or retro-orbital sampling on fed or 6h- to 16h-fasted mice. For systemic assessment of lipolysis, blood was collected on fed mice before and 20 or 45min. after intraperitoneal injection of the β₃-adrenergic agonist, CL-316,243 (Sigma) at 1 mg.kg⁻¹. Plasma glycerol was measured by enzymatic assay (Free Glycerol reagent, Sigma), plasma NEFA and TG levels were measured using the NEFA C kit (Wako) and TG reagent (Sigma), respectively. Plasma insulin was measured using an ultrasensitive ELISA kit (ALPCO Diagnostics). Blood glucose and ketone bodies levels were measured using a glucometer (Accucheck, Roche) or a β-ketone bodies meter (Freestyle Optium H meter, Abbott), respectively.
# Cell Metabolism

## Article

<figure><img src="image_27.png" /></figure>

### Cold tolerance test

Animals were placed around 8 a.m. in individual cages in a cold room and exposed at 4°C with free access to water. Rectal temperature was monitored using a RET-3 rectal probe (Physitemp Instruments) connected to a thermometer (K/J/T type, Bioseb). A particular attention was brought on mouse behaviour. When rectal body temperature was below 28°C, mice were withdrawn from cold and heated upon specific lights.

### Measurement of energy expenditure following injection of the β₃-adrenergic agonist, CL-316,243

Indirect calorimetry was performed after 24 h of acclimation in individual cages placed at room temperature (around 21°C). Oxygen consumption, carbon dioxide production, ambulatory activity and food and water intakes were measured (Phenomaster: TSE Systems, Bad Homburg v.d.H., Germany) at 15-minute intervals during a 48-hour period at constant temperature (22°C). Food intake was monitored with weight sensors on feeders. On day 4 and 5 at approximately 8:30 a.m., food was removed a few minutes before mice were injected intraperitoneally with NaCl and CL-316,243 (2 mg·kg⁻¹, Sigma), respectively. The respiratory exchange ratio as the ratio of carbon dioxide production to oxygen consumption and energy expenditure (kilocalories of heat produced) were calculated.

### Indirect calorimetry and PET/CT imaging and analyses

Mice underwent a randomized, cross-over thermoneutrality (30°C) vs. cold exposure (10°C) for 7 days in metabolic cages, with a one-week washout period in between. The Promethion High-Definition Room Calorimetry System was used for the indirect calorimetry studies (GA3, Sable Systems. Las Vegas, NV) during these periods. Data acquisition and instrument control were coordinated by MetaScreen v.2.3.12.6 and the obtained raw data was processed using ExpeData v.1.9.15 (Sable Systems, Las Vegas, NV) using an analysis script detailing all aspects of data transformation. A standard 12 h light/dark cycle (6:00–18:00) was maintained throughout the calorimetry studies.

After a 6-h fasting at the end of each of the 7-day thermoneutrality and cold exposure periods, a 100 µL blood sample was obtained from the saphenous vein in order to measure plasma NEFA and TG levels. Mice were then immediately anaesthetized with isoflurane (2.0%, 1.5 L·min⁻¹) delivered through a nose cone and a cannula was inserted in the tail vein for injection of PET tracers. Animals were put on a heating table to maintain skin temperature at 31°C during scanning to avoid difference in body temperature and shivering between animals and genotypes. Mice were placed in the prone position on the scanner bed with the heart centered within the field-of-view of the scanner to include the interscapular region. All imaging experiments were performed on the avalanche photodiode-based small animal PET scanner (LabPET/Triumph, Gamma Medica, Northridge, CA) of the Sherbrooke Molecular Imaging Centre, having a 7.5 cm axial field-of-view. Then, a bolus of [¹¹C]-acetate (10 MBq, in 0.1 mL of 0.9% NaCl) was injected intravenously followed by a 15-min dynamic µPET data acquisition. In the session at the end of the 7-day 10°C exposure, an intravenous injection of the β₃-adrenergic agonist CL-316,243 (2 mg·kg⁻¹, Sigma, #C5976) was performed less than one minute prior to the injection of [¹¹C]-acetate to maintain BAT activation during the PET scanning environment while the mouse was not exposed to cold. At the end of the 15-min [¹¹C]-acetate PET acquisition, a bolus of [¹¹C]-palmitate (10 MBq, in 0.2 mL of 0.9% NaCl) was injected and another 15-min PET acquisition was performed. Residual [¹¹C]-acetate activity during [¹¹C]-palmitate acquisition was corrected by acquiring a 60 s frame prior to the injection of [¹¹C]-palmitate, to perform correction for residual [¹¹C]-acetate activity accounting for the disintegration rate of [¹¹C]. Low dose CT scan imaging was performed on a preclinical CT scanner model U-CT with MSA option (Milabs B.V., Houten, Netherlands). The CT image acquisition sequence was performed using 55 kV energy with current set to 0.33 mA using an exposure time of 40 ms and 0.5 step angle. Image reconstruction was performed with Milabs reconstruction software 11.00 with the voxel size set to 150 µm with Hann filter using the fine grid reconstruction setting. A dynamic series of 26 frames (12×10s, 8×30s and 6×90s) were used for [¹¹C]-acetate imaging and a dynamic series of 28 frames (12×5s, 6×10s, 6×30s and 4×150s) were used for [¹¹C]-palmitate imaging. 3D images were reconstructed using 20 iterations of a maximum-likelihood expectation-maximization (MLEM) algorithm incorporating physical description of the detector response functions. Regions of interest (ROIs) were drawn on short-axis images on Amide software v.1.0.5 and confirmed with the µCT scan. Input curves were extracted by means of a ROI drawn on the left ventricular cavity blood pool in summed last-frame images to seek better contrast. BAT blood flow index ($K_1$ in min⁻¹ of [¹¹C]-acetate) and oxidative metabolism index ($K_2$ in min⁻¹ of [¹¹C]-acetate) were estimated from [¹¹C]-acetate acquisition using a four-compartment pharmacokinetic model specific to BAT.⁴³ BAT non-esterified fatty acid uptake, oxidation and esterification were estimated from [¹¹C]-palmitate acquisition using a two-tissue multicompartmental model⁴⁴,⁴⁵ and calculations that were previously described.⁴⁶

### Histology and lipid droplet size determination

Intrascapular BAT were fixed with 4% paraformaldehyde in PBS, dehydrated, embedded in paraffin and cut into 5 µm sections. Sections were stained with hematoxylin and eosin using standard protocols. Images of individual tissues were extracted at a 0.228 µm resolution using QuPath.⁴⁷ LD detection, measurement and distribution visualization was performed using a collection of Python Jupyter Notebooks.⁴⁸ The images were opened (https://github.com/AllenCellModeling/aicsimageio), converted in gray-scale⁴⁹ and inverted before application of Cellpose algorithm at different scales (15, 40 and 80 pixels) for lipid droplet segmentation.⁵⁰ The model was trained with transfer learning from the Cellpose’s cyto2 model with a dataset of 7 1000x1000 pixels and 7 2000x2000 pixels images extracted from the full tissue’s images and annotated with the ImageJ software. The resulting label images are filtered with a size bandwidth adapted to each scale. Then these different scales are merged by adding them successively in increasing order. Labels were added if less than 10% of their area is occupied by other labels from smaller scales. On the resulting multi-scale image labels area are measured, then converted to an equivalent diameter. From these measurements, LD
diameter distribution was estimated for each tissue with a weighted kernel density estimation using the seaborn library.⁵¹ The weight used was the area of each detection.

### Transmission electron microscopy (TEM)

Tissues were fixed with 2,5% glutaraldehyde and 2% paraformaldehyde in Cacodylate buffer (0.1 M, pH 7.2, EMS, Hatfield, PA) overnight at 4°C and post-fixed with 1% OsO4 in the same buffer and finally treated for 1 h with 2% aqueous uranyl acetate. The samples were then dehydrated in a graded acetone series and embedded in Epon (EMBed-812, EMS). After 48 h of polymerization at 60 °C, ultrathin sections (80 nm thick) were mounted on 200 mesh Formvar-carbon-coated copper grids. Finally, sections were stained with Uranylëss and lead citrate (em-grade.com). Grids were examined with a TEM (Jeol JEM-1400, JEOL Inc, Peabody, MA, USA) at 80 kV. Images were acquired using a digital camera (Gatan Orius, Gatan Inc, Pleasanton, CA, USA). About 15 TEM images were taken per animal with a total of 58-71 fields analyzed for quantitation of LD and mitochondria area.

Mitochondria density was calculated as the percentage of the surface occupied by mitochondria to the surface occupied by the cytoplasm (excluding lipid droplet areas) on the TEM images. The surface occupied by mitochondria was measured by segmenting mitochondrion individually using a trained model for Cellpose.⁵² The model was trained with the Cellpose GUI human-in-loop pipeline from the pretrained model cyto2. Subsequently mitochondrion segmentations were manually corrected using the FIJI software.⁵³ Cytoplasm surface was calculated by subtracting the LD surface from the image surface. The LD surface was measured with the pixel semantic classification workflow of the Ilastik software.⁵⁴ Beforehand the images were preprocessed using a FIJI macro (histogram normalization with ImageJ enhance contrast command).

### Flow cytometry

Interscapular brown fat pads were digested using 1mg/ml collagenase (C6885 Sigma), 0.05mg/ml liberase (05401127001 ROCHE) and 0.025mg/ml hyaluronydase (H1136 Sigma). Following filtering and centrifugation (1500 rpm, 5 min), isolated stromal vascular cells were stained in 500 µL of Zombie UV (1:1000, Ref 423107 BioLegend) in PBS and incubated on ice for 20 min covered in foil. The reaction was quenched with 1 mL of FACS Buffer (PBS supplemented with 2.5% heat-inactivated fetal calf serum and 2 mM EDTA), and cells were pelleted as above. Cells were resuspended in 100 µL of FACS Buffer with 1 µL FcBlock (rat anti-mouse CD16/32, clone 2.4G2, ref 553141 BD Biosciences) for 10 min covered in foil before addition stain cocktail comprised of various fluorophore-conjugated antibodies in Brilliant Stain Buffer (Ref 563794, BD Biosciences). The following antibodies were used: CD45-VioBlue (Ref 130-110-802, REAfinity, Miltenyi), F4/80 APC (Ref 130-116-525, REAfinity, Miltenyi), CD31-PE (ref 130-111-540, REAfinity, Miltenyi) and lymphocytes are gated according to FSC/SSC parameters (Low FSC / Low SSC). Cells were stained for 40 min on ice covered in foil and then washed in 500 µL FACS Buffer. After the final resuspension, 25 µL CountBright Absolute Counting Beads (ThermoFisher) were added for enumeration of cell numbers.

All flow cytometry was performed on a 4-laser Cytek Biosciences Aurora CS spectral cytometer. Flow cytometric analyses were performed with SpectroFlow (version 1.1, Cytek Biosciences) and OMIQ Dotmatics, and cells were gated on singlets and live cells.

### Lipolysis in BAT explants

Interscapular fat pads were minced with scissors in KRBH buffer at 37°C to obtain regular pieces of about 10 mg. Explants (40 µl) were incubated in 200 µl of KRBH buffer pH 7.4 with 2% bovine serum albumin for 90min at 37°C. Concentrations of glycerol and FA released in culture medium were determined as described above.

### Determination of mRNA levels

Tissues were homogenized in Qiazol buffer (Qiagen) using Precellys tissue homogenizer. Total RNA from tissues was extracted using RNeasy kit (Qiagen). RNA concentration and purity were assessed spectrophotometrically using NanoDrop (DigitalBio). After treatment with DNase I (Invitrogen) and reverse transcription of 1 mg of total RNA with Multiscribe Reverse Transcriptase, real-time quantitative PCR was performed with Fast SYBR Green Master Mix or Taqman Fast Advanced Master Mix and QuantStudio5 real-time PCR system (Thermofisher Scientific). HPRT mRNA was used as control to normalize gene expression.

### Protein analyses

Tissues were homogenized in lysis buffer (50 mM Tris pH8, 150 mM NaCl, 5 mM EDTA, 1% Triton) containing protease and phosphatase inhibitors (Sigma) using Precellys homogenizer and centrifuged. Supernatants were harvested for determination of total protein concentration. Equal amounts of solubilized proteins were loaded on 4-20% gradient SDS-PAGE gels (Bio-Rad), blotted onto nitrocellulose membranes and incubated overnight with primary antibodies, rabbit polyclonal anti ATGL (Cell Signaling Technology, 2138, lot 6); rabbit monoclonal anti HSL (Cell Signaling Technology, clone D6W5S, 18381, lot 1) and rabbit monoclonal anti GAPDH (Cell Signaling Technology, 2118, clone 14C10, lot 14). Subsequently, immuno-reactive proteins were blotted with anti-rabbit horseradish peroxidase-labelled secondary antibodies for 1h at room temperature and revealed by enhanced chemiluminescence reagent (Clarity Western ECL Substrate, Bio-Rad), visualized using ChemiDoc Touch Imaging System and data analyzed using the ImageLab 4.2 version software (Bio-Rad Laboratories). Full scans of uncropped blots are available in Data S1.
Cell Metabolism
Article

<figure><img src="image_28.png" /></figure>

CellPress
OPEN ACCESS

Quantitation of tissue TG and glycogen levels

Measurements of fatty acid and TG contents were performed as described.⁵⁵ Glycogen was measured using *Aspergillus niger* amyloglucosidase (Sigma) followed by measurements of glucose levels with Glucose GOD FS kit (DiaSys).

Statistical analyses

Data are expressed as mean ± SEM. Analyses were performed with GraphPad Prism 9.5.0 for PC (GraphPad, San Diego, CA) except general linear model for analyses of rectal body temperatures using Python jupyter notebook with the Pingouin library (https://github.com/raphaelvallat/pingouin). A two-tailed P value <0.05 was considered significant.