Combined α- and β-adrenergic receptor activation triggers thermogenesis by the futile creatine cycle

Noradrenaline is the primary physiological regulator of adipocyte thermogenesis in response to decreased environmental temperature 1 . However, the molecular factors and effector pathways that lie downstream of noradrenaline-stimulated thermogenesis are still not fully understood but are purportedly driven by cAMP downstream of β-adrenergic receptor (βAR) activation. Furthermore, while the transcriptional mechanisms regulating Ucp1 are well-characterized 2 , the transcriptional regulation of UCP1-independent thermogenesis is largely unknown. Here, we show that brown adipose tissue (BAT) is primed to respond to environmental cold by triggering coordinated α-adrenergic receptor (αAR) and βAR signaling to induce the expression of thermogenic genes of the futile creatine cycle 3,4 . Using fat-specic loss-of-function models, we reveal that EBFs, ERRs, and PGC1α are required for the cold-stimulated transcriptional induction of the futile creatine cycle in vivo. Through the application of chemogenetics, we demonstrate that combined fat-selective Gαs (activated by βARs) and Gαq (activated by αARs) signaling elevates whole-body energy expenditure to a greater extent than either signaling pathway alone in a manner that is dependent on the key effector protein of the futile creatine cycle, CKB 3 . Moreover, genetic and pharmacological studies reveal that CKB is necessary for nearly all of the α 1 AR-stimulated component of brown adipocyte-intrinsic respiration and is thus critical for the full activation of noradrenaline-stimulated thermogenesis. Thus, the futile creatine cycle is integrated into facultative and adaptive thermogenesis through coordinated α 1 AR and β 3 AR signaling. ATAC-sequencing.

these criteria (Extended Data Fig. 1a). Analysis of an independent mouse ribosomal pro ling dataset 24 con rmed BAT-enrichment of Adra1a, Adrb1 and Cxcr7 (Ptger1 was not identi ed) (Extended Data Fig.  1b). Adra1a was the most abundant a 1 AR subtype in murine brown adipocytes, followed by Adra1d, whereas Adra1b and all the a 2 AR subtypes were poorly expressed (Extended Data Fig. 1c). Of the GPCR candidates, ADRA1A was the most enriched in human deep BAT (proximal to the carotid sheath) over paired white subcutaneous adipose tissue (SAT) (Fig. 1a), followed by ADRB1 (Extended Data Fig. 1d), while PTGER1 and CXCR7 did not exhibit BAT enrichment (Extended Data Fig. 1e, f). RNA sequencing (RNA-seq) analysis of an independent human cohort revealed that ADRA1A displayed the highest expression levels in supraclavicular adipose tissue (primary location of human BAT) compared to all aARs and bARs (Extended Data Fig. 1g). Together, these data prompted us to focus our attention on aAR signaling in BAT.
aAR signaling regulates futile creatine cycling gene expression in BAT To explore the cold-stimulated transcriptional program regulated by aAR signaling, we generated RNA-seq transcriptomes from BAT of mice pre-treated with either a single intraperitoneal (i.p.) injection of the aAR antagonist phenoxybenzamine (PBZ) or saline control and then housed at 30°C or 6°C (Fig. 1b). Principal component analysis of gene expression showed that ~65% of variation (PC1) was explained by temperature and ~15% of variation (PC2) was sensitive to aAR antagonism (Extended Data Fig. 2a). PBZ treatment essentially had no effect on gene expression at 30°C (Fig. 1b and Extended Data Fig. 2b, c). We identi ed four gene clusters strati ed by differential expression pro les ( Fig. 1b and Extended Data Table   1). Cluster 4 was de ned by cold-induced genes that were reduced in abundance by PBZ treatment (Fig.  1b). Analysis of this gene set revealed signi cant GO term pathway enrichment of protein transmembrane import into organelle and protein translocation to mitochondria, among others (Extended Data Fig. 2d). Notably, mRNAs encoding the effector proteins of the futile creatine cycle: creatine kinase b (Ckb) 3 and tissue-nonspeci c alkaline phosphatase (Alpl, encoding TNAP) 4 were cold-inducible in a PBZdependent manner. Similarly, peroxisome proliferator-activated receptor gamma co-activator 1 (Ppargc1a) mRNA, encoding for PGC1α, a key transcriptional co-activator of mitochondrial and thermogenic genes 25,26 , was a cold-stimulated PBZ target (Extended Data Table 1). In a separate mouse cohort, both PBZ and a structurally distinct a 1 AR-speci c antagonist, prazosin (PZS) inhibited the coldstimulated induction of Ckb, Alpl and Ppargc1a in BAT (Extended Data Fig. 2e). Both PBZ and PZS blunted the cold-stimulated induction of CKB protein in BAT ( Fig. 1c and Extended Data Fig. 2f). In contrast, uncoupling protein 1 (Ucp1) mRNA and protein levels were unchanged by pharmacological aAR antagonism ( Fig. 1c and Extended Data Fig. 2e, f). Next, we carried out unilateral denervation of the interscapular BAT depot in which the right lobe was surgically denervated while the left lobe remained intact 22 . Strikingly, the cold-mediated elevation of Ckb, Alpl and Ppargc1a was blocked in the sympathetically denervated BAT lobes (Fig. 1d) demonstrating that innervation of BAT by the sympathetic nervous system is required to elevate futile creatine cycling genes in response to cold. Of note, even though Adrb1 was BAT-enriched (Extended Data Fig. 1a, d), ADRB1-dependent regulation of futile creatine cycling gene expression was ruled out because either genetic ablation of Adrb1 or pharmacological inhibition with propranolol did not block cold-stimulated Ckb, Alpl and Ppargc1a mRNA induction or CKB protein induction in BAT (Extended Data Fig. 3a-d). Finally, ADRA1A was positively correlated with CKB in multiple independent human BAT cohorts ( Fig. 1e and Extended Data Fig. 3e-g). In contrast, CKB did not display a positive correlation with ADRA1A in SAT (Extended Data Fig. 3h, i) or consistent associations with ADRB1, PTGER1 or CXCR7 in BAT ( Extended Data Fig. 3j). Collectively, these data suggest that the cold-stimulated elevation of futile creatine cycling genes is regulated, at least in part, by the sympathetic nervous system through aAR signaling in BAT.
BAT is primed to induce CKB by aAR and b 3 AR signaling Like ADRA1A, ADRB3 was positively correlated with CKB in human BAT (Extended Data Fig. 4a, b). This was consistent with the capacity for pharmacological b 3 AR activation (by CL 316,243) to increase CKB protein in murine BAT (Extended Data Fig. 4c). b 3 AR-stimulated induction of Ckb, Alpl, Ppargc1a, and Ucp1 mRNA and CKB, TNAP and UCP1 protein levels were not blocked by PBZ (Extended Data Fig. 4c, d), indicating that PBZ did not indirectly effect b 3 AR-stimulated thermogenic gene induction. We did not detect any difference in the amount of Ckb (or Alpl and Ucp1) induction if CL 316,243 was administered by daily i.p. injection or by continuous release through an osmotic pump (Extended Data Fig. 4e), suggesting that the induction of Ckb by individual b 3 AR stimulation occurs similarly whether stimulated transiently or continuously. However, the cold-stimulated induction of Ckb in BAT was higher (about 12fold) than b 3 AR agonism (about 4.5-fold), whereas the induction of Ucp1 mRNA expression by these interventions was comparable (Extended Data Fig. 4g). Finally, Ckb levels in BAT following b 3 AR activation (Extended Data Fig. 4d) mirrored the remaining levels of Ckb in aAR-inhibited cold-activated BAT (Extended Data Fig. 4f), suggesting that the residual cold-stimulated induction of Ckb during aAR blockade was mediated by the b 3 AR pathway. Cold exposure or b 3 AR agonism both elicited a greater relative induction of Ckb mRNA and protein in BAT compared to SAT ( Fig. 1h and Extended Data Fig. 4gj), even though b 3 AR expression was comparable between these tissues (Extended Data Fig. 4k) 23 . These data indicate that in addition to BAT-selective aAR signaling, intracellular factors contribute towards the priming of brown adipocytes to trigger CKB expression downstream of b 3 AR signaling. Of note, CKB abundance was substantially induced in SAT following 1 week of cold exposure (Extended Data Fig.  4l), suggesting that its expression was commensurate with beige adipogenesis. In aggregate, our data suggest that BAT is equipped to rapidly induce CKB expression in response to external adrenergic stimuli through combined a 1 AR and b 3 AR signaling.
Transcriptional regulation of the futile creatine cycle Using ATAC (assay for transposase-accessible chromatin) sequencing of BAT nuclei, we identi ed differentially accessible regions (DARs) proximal to the differentially expressed genes of our BAT transcriptomes (Fig. 2a). We next identi ed transcription factor motifs which were: (1) statistically enriched in DARs proximal to Cluster 4 genes and (2) present in cold-stimulated DARs proximal to both Ckb and Alpl (Fig. 2b). We found ERR (estrogen-related receptor) and EBF (early B-cell factor) response elements to be most enriched (Fig. 2c). ERRα and its co-activator partner PGC1α are known transcriptional regulators of the cold response 26,27 , and EBF2 facilitates the binding of ERRα and PGC1α on target thermogenic genes 26,28 . Thus, we explored the chromatin occupancy of ERRα at the coldresponsive DARs of Ckb and Alpl in BAT (Fig. 2b) using chromatin immunoprecipitation coupled to qPCR (ChIP-qPCR). At 30ºC, ERRα binding to DARs proximal to both Ckb and Alpl was enriched (by about 4fold) over a control region that is not bound by ERRα (Fig. 2d, e and Extended Data Fig. 5). 6ºC exposure further enhanced the occupancy of ERRα (by about 2-fold over 30ºC) on all cold-responsive DARs proximal to Ckb containing ERR motifs (Fig. 2d, e). In contrast, ERRα binding to cold-triggered DARs proximal to Alpl was not cold-inducible (Extended Data Fig. 5). PBZ did not alter chromatin accessibility (Fig. 2a, b) or ERRα occupancy on DARs proximal to Ckb and Alpl (Fig. 2d, e and Extended Data Fig. 5 27 or Ebf1/Ebf2 (Ebf1/2 AdipoqCre ) 26 . Strikingly, upon loss of either Esrra/Esrrg (Extended Data Fig. 6a) or Ebf1/Ebf2 (Extended Data Fig. 6b) the cold-induced increase of Ckb mRNA in BAT was fully blocked (Fig. 2f, g). The cold-stimulated induction of CKB protein was also fully dependent on Esrra/Esrrg (Extended Data Fig. 6c). The induction of Alpl mRNA by cold was completely inhibited upon loss of Ebf1/Ebf2 (Fig. 2g); however, the cold-stimulated increase of Alpl mRNA was Esrra/Esrrgindependent (Fig. 2f), fully consistent with our ChIP-qPCR analysis (Extended Data Fig. 5). Surprisingly, the elevation of Ucp1 mRNA and protein by cold was also independent of Esrra/Esrrg ( Fig. 2f and Extended Data Fig. 6c). We discovered that Ppargc1a and Alpl were both induced to a higher level in BAT of cold-exposed Esrra/g AdipoqCre compared to control mice ( Fig. 2f and Extended Data Fig. 6a). Thus, because Ppargc1a and Alpl mirrored one another upon cold exposure in Esrra/g AdipoqCre mice, and because Ckb, Alpl, and Ppargc1a levels were all similarly regulated by aAR signaling (Fig. 1c, d), we hypothesized that futile creatine cycling gene expression is regulated by PGC1a. To test this hypothesis, we constructed mice with selective and inducible deletion of Ppargc1a in Ucp1 + cells (Ppargc1a Ucp1CreERT2 ) to avoid possible developmental effects associated with chronic Ppargc1a deletion. Tamoxifen-mediated reduction of Ppargc1a in this model exhibited a similar level of diminution as with aAR antagonism, while Ebf1, Ebf2, Esrra, and Esrrg levels were not reduced (Extended Data Fig.  6d). Strikingly, genetic depletion of Ppargc1a signi cantly diminished the cold-stimulated induction of both Ckb (by about 65%) and Alpl (by about 45%) mRNA (Fig. 2h) and CKB and TNAP protein ( Fig. 2i and Extended Data Fig. 6e). Similarly, the b 3 AR-stimulated induction of both CKB and TNAP was reduced in BAT of Ppargc1a Ucp1CreERT2 compared to Ppargc1a / mice (Extended Data Fig. 6f). Surprisingly, Ppargc1a was dispensable for the induction of Ucp1 mRNA and protein by cold exposure (Fig. 2h and Extended Data Fig. 6f). Together, our ndings show that the expression of both Ckb and Alpl is dependent on Ebf1/Ebf2 and Ppargc1a in brown adipocytes in response to cold. However, while Ckb expression by cold depends on Esrra/Esrrg, cold-stimulated Alpl expression is Esrra/Esrrg-independent and transcriptionally regulated by factors that remain to be de ned. Thus, our data is consistent with a model where PGC1a abundance is regulated by environmental cold through combined a 1 AR and b 3 AR signaling to control futile creatine cycling gene expression in brown adipocytes (Fig. 2j).
NA-stimulated thermogenesis requires a 1 AR signaling and CKB GPCR signaling regulates both the acute activation of thermogenesis and the recon guring of transcriptional networks to support enhanced catabolic demand 22,27,[29][30][31] . Since our data suggested that cold-stimulated aAR stimulation is required for maximal induction of Ckb expression, we explored the possibility that facultative thermogenesis downstream of NA-stimulated aAR signaling requires Ckb. First, we injected mice with NA which stimulated an increase in whole-body energy expenditure above the stress response elicited by saline injections, and notably was signi cantly decreased in fat-selective Ckb knockout mice (Ckb AdipoqCre ) compared to control Ckb / mice ( Fig. 3a, b). These data are consistent with the recently established role for CKB in thermogenesis by the futile creatine cycle 3 . However, given that activation of adrenergic signaling by NA is not con ned to fat, it was critical to delineate the brown adipocyte-intrinsic regulation of NA-stimulated respiration by Ckb and aAR signaling (Fig. 3c).
Importantly, the NA-stimulated rise in respiration of freshly isolated Ckb AdipoqCre interscapular brown adipocytes was signi cantly impaired (by about 45%) compared to Ckb / brown adipocytes (Fig. 3d, e), recapitulating our in vivo data. Moreover, inhibition of aAR signaling reduced NA-dependent respiration of Ckb / control brown adipocytes, without statistically altering Ckb AdipoqCre brown adipocyte respiration (Fig. 3d, e). Ckb de ciency or aAR inhibition had no effect on unstimulated (basal) respiration (Extended Data Fig. 7a), consistent with a role for CKB in facultative thermogenesis. CKB de ciency or aAR antagonism had no effect on NA-stimulated lipolysis (Extended Data Fig. 7b), indicating that the diminished capacity of Ckb AdipoqCre brown adipocytes to trigger adrenergic-stimulated thermogenesis could not be explained by reduced substrate delivery. Repeating the experiments with PZS ( Fig. 3f) also blunted NA-stimulated respiration in Ckb / control, but not Ckb AdipoqCre brown adipocytes (Fig. 3g, h) with no impact on basal respiration (Extended Data Fig. 7c). Together, these data suggest that both CKB and a 1 AR signaling are essential components of NA-stimulated thermogenesis.
Coordinated Ga q and Ga s signaling promotes energy expenditure through CKB in vivo Although our data, in addition to the work of others 32,33 independently suggested a key role of aAR signaling in adipocyte thermogenesis, the precise G protein class that couples aAR activation to signaling in the interior of brown adipocytes has never been de ned. Thus, we rst examined the G protein coupling pro le of a 1A AR by measuring agonist-induced BRET between ADRA1A-tagged Nano Luciferase (ADRA1A-Nluc) and Venus-tagged miniG protein probes in immortalized brown adipocytes (Fig. 4a). Upon agonist stimulation by the a 1A AR selective ligand A61603, the a 1 AR agonist cirazoline, or NA, ADRA1A-Nluc rapidly and primarily coupled to Ga q (Fig. 4b), partially to Ga s and Ga i (Extended Data Fig. 8a, b), and minimally to Ga o or Ga 12 (Extended Data Fig. 8c, d). Next, to determine if aAR signaling regulates energy expenditure in vivo, we employed a chemogenetic approach using DREADDs (designer receptors exclusively activated by designer drugs) 34,35 . As ADRA1A is primarily coupled to Ga q in brown adipocytes ( Fig. 4b), we constructed mice that conditionally express a HA-tagged modi ed muscarinic receptor (HA-hM3Dq) selectively in adipocytes (hM3Dq AdipoqCre ) (Fig. 4c, d). Fat-selective hM3Dq expression renders these mice capable of activating Ga q signaling upon binding the inert molecule deschloroclozapine (DCZ) 36 . Then, to determine if Ga q signaling functions through Ckb, we crossed hM3Dq AdipoqCre mice to Ckb / mice to construct mice where Ga q signaling could be activated selectively in Ckb-expressing (hM3Dq AdipoqCre :Ckb /+ ) or Ckb-de cient (hM3Dq AdipoqCre :Ckb / ) adipocytes. All mice were single-housed at 30ºC and injected i.p. with either CL 316,243 (to activate Ga s through b 3 AR stimulation) or DCZ + CL 316,243 (to activate both Ga q and Ga s through hM3Dq and b 3 AR stimulation, respectively) (Fig. 4e). In both hM3Dq AdipoqCre :Ckb /+ and hM3Dq AdipoqCre :Ckb / mice, saline treatment caused a transient spike in energy expenditure that rapidly subsided within one hour (Fig. 4f, g). Notably, combined treatment of hM3Dq AdipoqCre :Ckb /+ mice with DCZ and CL 316,243 elevated sustained energy expenditure to a signi cantly higher (by about 30%) level compared to CL 316,243 treatment alone (Fig 4h). In contrast, adipocyte-selective loss of Ckb impeded the capacity for combined DCZ and CL 316,243 treatment to stimulate whole body energy expenditure above CL 316,243 ( Fig. 4i). DCZ was not su cient on its own to trigger sustained energy expenditure (Extended Data Fig. 9a), further indicating that acute activation of Ga q signaling needs to be superimposed on Ga s activation to in uence energy expenditure. Collectively, these data imply that Ckb is genetically required for the stimulation of adipocyte energy expenditure through combined Ga s and Ga q activation in vivo.
Overlaying a 1A AR activation on cAMP signaling promotes CKB-dependent thermogenesis Ga q and Ga s signaling are engaged in all adipose depots (white and brown) of hM3Dq AdipoqCre mice treated with DCZ and CL 316,243. Thus, we next probed the su ciency of a 1 AR signaling to enhance cAMP-stimulated brown adipocyte-intrinsic thermogenesis and whether CKB is required for this effect (Fig. 4j). Mimicking bAR signaling (by augmenting intracellular cAMP levels through direct stimulation of adenylyl cyclase with forskolin) did not match the respiratory response achieved with NA in Ckb / brown adipocytes (Fig. 4k, l). We con rmed that the maximal rate of forskolin-stimulated respiration had been reached because doubling the forskolin concentration did not further elevate respiration (Extended Data Fig. 9b). Similar to individual application of DCZ, a 1 AR agonism (cirazoline) was not su cient on its own to stimulate oxygen consumption (Fig. 4k, l). Thus, separately elevating either cAMP levels or engaging a 1 AR signaling does not recapitulate NA-stimulated thermogenesis. Notably, combined treatment with forskolin and cirazoline elicited a thermogenic response similar to NA in Ckb / brown adipocytes (Fig. 4k, l), whereas the a 1 AR-mediated component of respiration was blocked (by 90%) in Ckb AdipoqCre compared to Ckb / brown adipocytes (Fig. 4k, l). Next, we tested the su ciency of selective a 1A AR agonism (A61603) to induce respiration cooperatively with cAMP ( Fig. 4m ). When combined with cAMP signaling, A61603 induced respiration in control Ckb / brown adipocytes to the same level achieved with NA; however, this response was signi cantly impaired (by about 80%) in Ckb AdipoqCre brown adipocytes (Fig. 4n, o). Together, these data demonstrate that the coordinated activation of a 1A AR and cAMP signaling converges upon CKB to promote thermogenesis by brown adipocytes.

Discussion
NA is understood to be the predominant physiological regulator of ligand-mediated adaptive and facultative adipocyte thermogenesis 1 . bAR signaling and Ga s -coupled cAMP production has dominated the focus of sympathetic activation of BAT, leaving the aAR-dependent component a relatively underexplored area of adipocyte biology. A role for aAR signaling in adipocyte thermogenesis has been demonstrated previously 33,37,38 ; however, the particular aAR sub-type, the class of G protein that its coupled to, and the effector protein(s) that transduce aAR signaling into a thermogenic output have all remained elusive.
In thermogenic fat, creatine liberates a molar excess of mitochondrial ADP to promote thermogenic respiration through a futile creatine cycle catalyzed by CKB and TNAP 3,4 . However, the signal transduction pathways and transcriptional regulators that orchestrate futile creatine cycling gene expression with acute regulation of thermogenesis have remained mysterious. In this study we uncover that when combined with cAMP tone, activation of the a 1A AR subtype signals through Ga q to further enhance thermogenesis in brown adipocytes. Since CKB is necessary for thermogenesis by the futile creatine cycle, we used Ckb loss-of-function models to explore the role of the futile creatine cycle in a 1A AR-stimulated thermogenesis. We reveal CKB as a necessary effector for nearly all of the a 1A ARstimulated thermogenic component. The following data support this conclusion: First, NA-stimulated thermogenesis is signi cantly reduced in Ckb AdipoqCre compared to Ckb / brown adipocytes. Second, antagonism of aAR or a 1 AR signaling in Ckb / brown adipocytes decreases NA-dependent respiration to the same lower level elicited by Ckb AdipoqCre brown adipocytes, while aAR or a 1 AR antagonism has no effect on NA-stimulated respiration of Ckb AdipoqCre brown adipocytes. Third, superimposing a 1 AR or a 1A AR activation on cAMP signaling elicits thermogenesis akin to NA only in control Ckb / brown adipocytes but not Ckb AdipoqCre brown adipocytes. Fourth, CKB is necessary for fat-selective Ga q signaling to enhance whole-body energy expenditure above b 3 AR-stimulated thermogenesis. In addition to de ning the features of acute thermogenic activation by combined a 1A AR and cAMP signaling, we delineate the transcriptional control of futile creatine cycling genes to be similarly orchestrated by mutual a 1 AR and b 3 AR signaling via EBF1/2, ERRa/g, and PGC1a.
The a 1 AR family contains three subtypes (a 1A , a 1B , and a 1D ), which are all engaged by NA 39 . We demonstrate that the a 1A subtype (Adra1a) is the most abundant in brown adipocytes, but a 1D (Adra1d) is also expressed. Addressing this complexity using fat-selective knockout mouse models, possibly even double knockouts (Adra1a and Adra1d), will be a key area of future research to genetically uncover the required role of individual a 1 AR subtypes in brown fat thermogenesis in vivo. Until then, a complete understanding of the signal transduction cascade that couples ligand-mediated a 1 AR activation with thermogenesis by the futile creatine cycle remains to be determined.
Chronic Ga q activation has been reported to inhibit adipocyte differentiation and thermogenic output 40 . In marked contrast, we reveal a key role for Ga q signaling in promoting thermogenesis. These distinct results may be due to our focus on the acute (as opposed to chronic) Ga q activation of thermogenesis through the futile creatine cycle (as opposed to UCP1).
BAT is associated with cardiometabolic bene ts [41][42][43][44] . However, given the variability of existing BAT depots in humans, along with the reductions of BAT activity in obesity, a full understanding of the molecular mechanisms that promote the greatest activation of adipocyte thermogenesis is of considerable interest to uncover if BAT is a viable clinical target that can enhance cardiometabolic health [45][46][47][48][49] . Clinical relevance of aAR-triggered BAT thermogenesis is exempli ed by conditions such as hypothyroidism, where aAR signaling may contribute more towards thermogenic respiration than bAR signaling 50 , and in the context of NA-secreting paragangliomas, where aAR blockade reduces both glucose uptake by adipose tissue and whole body energy expenditure 32 .
Ckb is expressed in Ucp1 + adipocytes 3 , as is Alpl 4 , and we demonstrate that loss of Ckb decreases NAstimulated thermogenesis even in the presence of UCP1. Thus, our data imply that multiple metabolic heat generating pathways co-exist in BAT. Discrete thermogenic pathways could segregate inter-or intracellularly. The identi cation of distinct adipocyte subtypes 51-56 as well as mitochondrial heterogeneity within thermogenic fat cells 57 supports this idea. Thus, the futile creatine cycle probably operates in conjunction with UCP1-dependent thermogenesis. Of course, the fractional proportion of these two pathways will be dynamic and depend on extrinsic stimuli, regulated by important factors such as thermal history. Whether other thermogenic pathways also similarly operate in parallel with the futile creatine cycle and UCP1 in adipocytes remains to be determined.

Inducible deletion of Ppargc1a
Mice were reared at 21°C-22°C until 7 weeks of age, then injected with tamoxifen (75 mg kg−1) for 3 days and allowed to recover for 4 days until intervention.

Administration of chemicals by osmotic pump
Osmotic pumps (Alzet) were loaded with CL 316,243 in 100 μl total volume. Mice were anesthetized with iso urane. A small skin incision was made directly above the interscapular BAT depot. Filled osmotic pumps were placed above the interscapular BAT and the skin was then sewed. Tissues were harvested for gene expression analyses following intervention. The release rate from the osmotic pumps is 0.5 μl per hour, so, as an example, loading 0.5mg of CL 316,243 in 100 μl for a 24g mouse will administer approximately 0.1mg kg -1 CL 316,243 every hour.

Administration of chemicals by intraperitoneal injection
Phenoxybenzamine (PBZ) was dissolved in saline and was injected (5mg kg -1 ) once over 24 hours.

RNA extraction
Total RNA from frozen mouse tissues was extracted using QIAzol (Qiagen) and puri ed with RNeasy Mini spin columns (Qiagen) according to the manufacturer's instructions. Total RNA from human tissues, third cohort, was extracted with TRIzol (Gibco BRL, Life Technologies, Roskilde, Denmark). RNA was quanti ed using a NanoDrop 8000 Spectrophotometer (Thermo Scienti c Pierce, Waltham, Maine, USA). cDNA was synthesized using a Verso cDNA kit (cat# Ab-1453, Thermo Fischer Scienti c) with random hexamer primers.

Reverse transcription quantitative polymerase chain reaction (RT-qPCR)
Puri ed RNA was reverse-transcribed using a High-Capacity cDNA Reverse Transcription kit (Applied Biosystems). The resultant cDNA was analysed by RT-qPCR. In brief, 20 ng cDNA and 150 nmol of each primer were mixed with GoTaq qPCR Master Mix (Promega). Reactions were performed in a 384-well format using a CFX384 Real-time PCR system (Bio-rad). Normalized mRNA expression was calculated using the ΔΔCt method, using Ppib or 36b4 mRNA as the reference gene.

RNA sequencing analysis
Total RNA was quanti ed using a NanoDrop Spectrophotometer ND-1000 (NanoDrop Technologies, Inc.) and its integrity was assessed on a 2100 Bioanalyzer (Agilent Technologies). Libraries were generated from 250 ng of total RNA as following: mRNA enrichment was performed using the NEBNext Poly(A) Magnetic Isolation Module (New England BioLabs). cDNA synthesis was achieved with the NEBNext RNA First Strand Synthesis and NEBNext Ultra Directional RNA Second Strand Synthesis Modules (New England BioLabs). The remaining steps of library preparation were done using and the NEBNext Ultra II DNA Library Prep Kit for Illumina (New England BioLabs). Adapters and PCR primers were purchased from New England BioLabs. Libraries were quanti ed using the Kapa Illumina GA with Revised Primers-SYBR Fast Universal kit (Kapa Biosystems). Average size fragment was determined using a LabChip GX (PerkinElmer) instrument. The libraries were normalized and pooled and then denatured in 0.05N NaOH and neutralized using HT1 buffer. The pool was loaded at 225pM on a Illumina NovaSeq S4 lane using Xp protocol as per the manufacturer's recommendations. The run was performed for 2x100 cycles (paired-end mode). A phiX library was used as a control and mixed with libraries at 1% level. Base calling was performed with RTA v3.4.4 . Program bcl2fastq2 v2.20 was then used to demultiplex samples and generate fastq reads. Adaptor sequences and low quality score bases (Phred score < 30) were rst trimmed using Trimmomatic v.0.36 62 . The resulting reads were aligned to the GRCm38 mouse reference genome assembly, using STAR v.2.0.2 63 . Read counts were obtained using HTSeq v.0.6.0 64 with parameters -m intersection-nonempty -stranded=reverse. For all downstream analyses, we excluded lowlyexpressed genes with an average read count lower than 10 across all samples, resulting in 17,952 genes in total. Raw counts were normalized using edgeR's TMM algorithm v3.26.8 65 and were then transformed to log2-counts per million (logCPM) using the voom function implemented in the limma R package 66 . To assess differences in gene expression levels between the different conditions, we tted a linear model using limma's lm t function. Nominal p-values were corrected for multiple testing using the Benjamini-Hochberg method. To speci cally identify temperature-sensitive genes that further respond differently to PBZ treatment, we rst obtained differentially expressed genes in 6°C vs 6°C+PBZ (FDR < 0.1) and then ltered for those that change expression in comparison to 30°C (i.e., in either 6°C vs 30°C or 6°C+PBZ vs 30°C; FDR < 0.01 and |log2FC| > 1). Unsupervised hierarchical clustering of the 764 differentially expressed genes showed four distinct patterns of changes in expression (R hclust function). The complete list of differentially expressed genes and their cluster annotation is presented in Extended Data 24097267). 10% of the pre-ampli ed mixture, was used to run a qPCR to determine the number of additional cycles needed as previously described 68 . Next, the ampli cation pro les were manually assessed and the required number of additional cycles were determined as previously described 69 . The nal PCR reaction was puri ed using a Qiagen MinElute PCR Puri cation Kit and eluted in 20 μl elution buffer. A subsample of each library was diluted to 1:1,000 to fall within range of the standards to perform concentration quanti cation using the KAPA Library Quanti cation Kit (Cat No. KK4854) according to the manufacturer's instructions. Paired end, 150 bp sequencing was performed on a HiSeq instrument at the Michael Smith Genome Sciences Centre (BC Cancer Research Institute).
ATAC sequencing analysis ATAC-seq reads were rst trimmed for adapter sequences and low quality score bases using Trimmomatic 62 . The resulting reads were mapped to the mouse reference genome (mm10) using BWA-MEM 70 in paired-end mode at default parameters. Only reads that had a unique alignment (mapping quality > 20) were retained and PCR duplicates were marked using Picard tools (https://broadinstitute.github.io/picard/). Peaks were called and annotated using MACS2 71 . Peak annotation and transcription factor (TF) motif enrichment analysis were performed using the annotatePeaks and ndMotifsGenome commands, respectively, from HOMER software suite 72 . Peaks were associated to a gene if located within 20 kb of the TSS. To assess differences in chromatin accessibility, a "reference peak set" was generated by merging ATAC-seq peaks across samples, using bedtools merge with parameters: -sorted -d -125 (https://bedtools.readthedocs.io/). Read counts were obtained within these genomic regions using HOMER. Raw counts were normalized using edgeR's TMM algorithm 65 and were then transformed to log2-counts per million (logCPM) using the voom function implemented in the limma R package 66 . To test for differential occupancy, we tted a linear model that takes into account the batch effects in the experiment. Nominal p-values were corrected for multiple testing using the Benjamini-Hochberg method. Read density metagene plots and heatmaps were obtained using ngs.plot 73 with the following parameters: -G mm10 -BOX 0 -SE 0 -VLN 0 -LWD 2 -WD 9 -L 1500. Genome browser tracks were created with the HOMER makeUCSC le command and bedGraphToBigWig utility from UCSC. Tracks were normalized so that each value represents the read count per base pair per 10 million reads. UCSC Genome Browser (http://genome.ucsc.edu/) was implemented for track visualization.

Indirect calorimetry
Mice had ad libitum access to drinking water and a low fat diet (2920X, Envigo). All mice used for indirect calorimetry experiments were born and housed in groups (3-5 mice per cage) at 22°C with bedding and shredded paper strips in the cage until experimental intervention. Mice (6-8 weeks of age) were placed, single-housed, in metabolic cages (Sable Systems International, Promethion high-de nition behavioural phenotyping system) housed in thermal cabinets set to 30°C with a 12-h light/dark schedule. Mice had ad libitum access to food and water and were allowed to acclimate to 30°C for 5 days. The following morning between Zeitgeber time (ZT) ZT2-3, mice were injected with vehicle (saline) and placed back in the metabolic cages to monitor the stress response to i.p. injection. The next morning (at ZT2-3), the same volume of the β 3 -adrenoreceptor agonist CL 316,243 (0.5 mg kg− 1 ) or CL 316,243 + DCZ (0.5 mg kg− 1 each) was administered i.p. and mice were placed back in the metabolic cages. For NA experiments, NA (Sigma, Cat. No. A9512) was prepared fresh in saline and administered i.p. at 1 mg kg− 1 at 30°C. Responses to drugs were followed every 3 min. Mass-dependent variables (energy expenditure) was not normalized to body weight. Energy expenditure (kcal), physical movement (measured by infrared beam breaks), and food intake were recorded every 3 min using Sable Systems data acquisition software (IM-3 v.20.0.3). Data were analyzed using Sable Systems International MacroInterpreter software (v.2.41) using One-Click Macro (v.2.37).

Isolation of brown adipocytes
Interscapular BAT was minced and digested in a Krebs-Ringer bicarbonate modi ed buffer (KRBMB: 135 mM NaCl; 5 mM KCl; 1 mM CaCl 2 ; 1 mM MgCl 2 ; 0.4 mM K 2 HPO4; 25 mM NaHCO 3 ; 20 mM HEPES; 10 mM glucose; 4% fatty acid-free BSA), supplemented with 2 mg/ml collagenase B (Worthington) and 1 mg/ml soybean trypsin inhibitor (Worthington). Minced BAT from 10 mice was digested in 20 ml KRBMB digestion buffer with continuous shaking at 37ºC for 45 minutes. The tissue suspension was ltered through a 100 μm cell strainer. Brown adipocytes were allowed to oat for 5 minutes at room temperature before and after spinning at 100 g for 5 minutes. Half of the infranatant was removed (~10 ml) with a 20 ml syringe/18G needle, followed by the removal of the stromal vascular fraction (SVF). Adipocytes were washed with 10 ml DMEM/F12 supplemented with 10% FBS and were allowed to oat for 20 minutes at room temperature before spinning at 200 g for 5 minutes. Adipocytes were washed three times. After the nal wash, the mature adipocytes present under the fat layer were transferred to a new tube. Cell number was approximated by counting nuclei. Brie y the number of cells was counted by mixing 0.16% trypan blue diluted in nuclease lysis buffer (NLB: 250 mM Sucrose; 10 mM Hepes; 10 mM KCl; 1.5 mM MgCl 2 ; 0.1% IGEPAL) with cells diluted in NLB (1:4 ratio cells:NLB). The mixture was left at room temperature for 10 minutes before applying 10 µl to the hemocytometer.
Respirometry of puri ed adipocytes using an oxygen electrode A Clark type electrode (Rank Bros) was used to measure the oxygen consumption of adipocytes.
DMEM/F12 supplied with 10% FBS was added to the chamber and left to equilibrate with atmospheric oxygen. Approximately 300,000 cells were then added to the chamber, covered with a lid and continuously stirred. The initial rate of cellular respiration prior to the addition of a thermogenic activator was termed "basal respiration". Thermogenic drugs were added to the continuously stirring cells via a Hamilton syringe (0.1 µM noradrenaline, 3 µM forskolin, 1 µM cirazoline, 1 µM A61603). For traces containing aAR inhibitors, phenoxybenzamine (1 µM) and prazosin (1 µM) were added to the respiration buffer prior to the addition of cells. The excess of oxygen consumed upon the addition of the drugs was subtracted from the basal respiration rate to account for the drug-dependent oxygen consumption. Rank Brothers Dual Digital model 20: Picolog 6 data logging software was used for data collection.

Unilateral denervation of interscapular BAT (iBAT)
Unilateral denervation was carried out as previously described 22,74 . Brie y, 22°C-housed mice were anesthetized by inhalation of iso urane (2.5% for induction, 1.5% for maintenance) and the incision site was shaved and disinfected using rst 0.5% chlorohexidine in 85% ethanol and then 70% ethanol. Prior to surgery, mice received local anesthesia (lidocaine, 1.4 mg kg -1 ) and general analgesia (Rimadyl, 10 mg kg -1 ). The iBAT was prepared by a midline incision of the skin in the interscapular region and the detachment of the iBAT from the underlaying muscle layer. The ve nerve bers innervating the right iBAT lobe were identi ed and cut (denervated) and the nerve bers innervating the left iBAT lobe were identi ed and touched with forceps (sham). Following the procedure, the fat pads were rinsed with sterile isotonic saline and the incision was closed with suture. The mice were individually housed in clean cages at 22°C with access to a 37°C heating pad during the rst 24 h post operation. Animals were monitored daily.

Glycerol release
Freshly isolated brown adipocytes (1x10 6 cells in 0.3ml) were incubated in DMEM/F-12 (Thermo Fisher Scienti c) supplemented with 4% fatty-acid-free BSA and incubated with NA (0.1 μM) and/or PBZ (1 μM) for 1 hour at 37°C. Following incubation, released glycerol was separated from the adipocytes by spinning through a centrifugal lter (Millipore sigma, UFC30LG25) at 8,000 g x 30 seconds at room temperature. The glycerol content in the media was determined using free glycerol reagent (Sigma, F6428) and glycerol standard solution (Sigma, G7793) at an absorbance of 540 nm after incubating the sample and standards with free glycerol reagent for 15 min at room temperature in the dark.
Bioluminescence resonance energy transfer (BRET)-based miniG subtype recruitment Immortalized mouse brown preadipocytes were grown with DMEM with 10% fetal bovine serum and penicillin/streptomycin. Upon con uency, cells were differentiated with DMEM containing a differentiation cocktail of 20 nM insulin, 1 μM dexamethasone, 0.5 μM rosiglitazone, 1 nM T3 and 500 μM methyl isobutyl xanthine. After 2 days of differentiation, media was replaced with DMEM with 10% FBS containing 1 nM T3 and 20 nM insulin. The next day, the differentiated adipocytes were transfected using TransIT-X2 (Mirus) as per manufacture's protocol. Brie y, plasmid DNA encoding Adra1a-NanoLuc, as well as venus-tagged miniG protein subtypes (miniG i , miniG s , miniG q , miniG 12  For BRET experiments, 24-hours post-transfection, differentiated adipocyte media was replaced with Hank's balanced salt solution (HBSS) supplemented with 10 mM HEPES, pH 7.5, and 10 μM furimazine (NanoGlo, Promega). BRET measurements were performed at 37°C using a PHERAstar Microplate Reader (BMG Labtech) with a dual-luminescence readout BRET1 plus lter (donor wavelength: 460-490 nm bandpass, acceptor wavelength: 520-550 nm long-pass). Following four baseline measurements, the cells were treated with vehicle or agonists (NA, Cirazoline or A61603) in triplicate for each condition, with the BRET signal measured every 2 min for 1 h. The BRET ratio (acceptor 520-550nm emission over donor 460-490 nm emission) was calculated for each well over time. The resulting ligand-induced BRET ratio was calculated by subtracting the baseline vehicle read from the agonist-stimulated read for each condition.

Human studies
In this report, we have utilized human adipose tissue biopsies collected from two independent cohorts.
First cohort -Joslin Diabetes Center adipose tissue cohort: Details on procedures of human subject adipose tissue biopsy collection and clinical characteristics of subjects have been described previously 75,76 . Brie y, ten paired human neck fat samples were obtained from super cial subcutaneous adipose tissue (SAT) depots and deep BAT tissue located proximal to the carotid sheath (n = 10 for each tissue). Tissue processing, RNA isolation, and analysis of gene expression has been previously described 75 . Brie y, analysis of gene expression using GeneChIPÒ PrimeView (Affymetrix, Santa Clara, CA) was performed on matched biopsies as previously described 76 . RNA was isolated from clonal cell lines using Direct-zol RNA MiniPrep kit (Zymo Research, Irvine, CA) according to the manufacturer's instructions. The quality of total RNA was evaluated by A260/A280 ratio, which was within the value of 1.9 to 2.0 de ned as high quality total RNA. Biotin-labeled cRNA was synthesized, puri ed and fragmented using GeneChip 3'IVT Express Kit (Affymetrix, Santa Clara, CA). Integrity and fragmented cRNA was assessed by running aliquots on the Agilent 2100 Bioanalyzer prior to proceeding further. The high quality cRNA meets the following criteria: the A260/A280 ratio should fall within the value of 1.9 to 2.0; the 28S/18S RNA bands (from the gel) should be crisp and the intensity of the 28S band should be roughly twice the intensity of the 18S band. Array hybridization and scanning were performed by the Advanced Genomics and Genetics Core of Joslin Diabetes Center according to established methods.
Microarray data were normalized using robust multi-array average (RMA), which placed it on a log-2 scale. All subjects gave informed consent prior to taking part in the study. This study followed the institutional guidelines of and was approved by the Human Studies Institutional Review Boards of Beth Israel Deaconess Medical Center and Joslin Diabetes Center Second cohort -UTMB Washington University adipose tissue cohort: Twenty-three men and women with overweight or obesity (age 41 ± 12 years, BMI 31.0 ± 3 kg/m 2 ) were enrolled in two clinical trials (NCT02786251 and NCT01791114) performed to determine the role of BAT in metabolic regulation in people. All participants completed a comprehensive screening evaluation that included a medical history and physical examination, standard blood tests, and an oral glucose tolerance test. Potential participants were excluded if they had diabetes or other serious diseases, smoked cigarettes, consumed excessive alcohol, were pregnant or lactating, or had metal implants that interfered with the imaging procedures. The studies were approved by the Institutional Review Board of the University of Texas Medical Branch (UTMB) in Galveston and the Washington University School of Medicine in St. Louis. Written informed consent was obtained from all subjects before their participation. Each participant completed a cold exposure study visit to assess BAT volume and activity and to obtain supraclavicular adipose tissue biopsies. During this visit, a standard cooling protocol was performed to maximize non-shivering thermogenesis [77][78][79] . After 6 hours of mild exposure to cold (~20 o C), an 18 F-uoro-deoxy-glucose ( 18 F-FDG)-positron emission tomography-computed tomography (PET/CT) scan was performed to determine BAT characteristics (volume and activity) 77 . Adipose tissue samples from the supraclavicular areawhere BAT is primary localized in humans -obtained using a PET/CT-guided percutaneous needle biopsy Third cohort -Danish adult neck adipose tissue cohort: Adipose tissue biopsies from the super cial (subcutaneous and subplatysmal) neck fat and deep (carotid sheath, longus colli, and prevertebral) neck fat were collected during surgery 81 . None of the subjects had diabetes nor were they administered βadrenergic antagonists. All biopsies were collected during winter and early spring and were instantly frozen in liquid nitrogen. Only paired biopsies from SAT and BAT of the same subjects were used for associations (n = 73). All study participants gave informed written consent. The study was approved by the Central Denmark Region ethics committee and was performed in accordance with the Declaration of Helsinki. CKB, ALPL, ADRA1A, and UCP1 mRNA expression was analyzed using RT-qPCR as described above.

Statistical analyses
Statistical analyses were performed with GraphPad Prism 9. Data analysis was performed using Microsoft O ce Excel 2021 (version 16.56). Data were expressed as mean ± s.e.m. Unpaired two-tailed Student's t-test for pairwise comparison, one-way ANOVA and two-way ANOVA for multiple comparisons involving two independent variables, and Pearson correlation for linear regression, were used to calculate P values to determine statistical differences. Signi cance was considered as P < 0.05. Mice were  Figure 1 CKB expression is regulated by a-adrenergic receptor signaling. a, Relative mRNA expression of ADRA1A from human BAT (n = 10) and paired SAT (n = 10), rst cohort. b, Heatmap of patterns of gene expression from BAT of wild-type male mice (C57BL6/N, 6-8 weeks of age), reared at 22°C, housed at 30°C for 5 days and then subjected to 30°C or 6°C at zeitgeber time 4 (ZT4) (n = 3 per group). 1 hour prior to onset of 6°C exposure (ZT3), mice were injected intraperitoneally (i.p.) with PBZ (5 mg kg -1 ) or saline (Sal). BAT was harvested 24 hours after onset of 6°C exposure. Heatmap was constructed using unsupervised hierarchical clustering of the Differentially Expressed Genes (DEGs) in 6°C+Sal vs 6°C+PBZ (FDR < 0.1 using 6°C+Sal as baseline). DEGs were further ltered by comparing 6°C+Sal vs 30°C+Sal or 6°C+PBZ vs 30°C+Sal (log2FC > 1 and FDR < 0.01). To the right of the heatmap is the mean expression levels (scaled) of DEGs in each of the 4 clusters. Complete list of DEGs and their clusters can be found in Extended Data Table 1. c, Western blot of BAT from mice treated as in b. d, RT-qPCR analysis of sham-operated (Sham) or denervated (Dener.) BAT lobes of mice exposed to 22°C or 5°C for 24 hours (n = 4 per group). e, Pearson correlation of CKB mRNA with ADRA1A in human BAT, rst cohort (n = 10). f, Western blot from BAT (top) and SAT (bottom) of male wild-type mice (C57BL6/N, 6-8 weeks of age) reared at 22°C, housed at 30°C for 5 days and then subjected to 30°C or 6°C for 48 hours. Data are presented as mean ± s.e.m. of biologically independent samples. a, two-tailed student's t-tests; d, One-way ANOVA (Fisher's LSD); e, Pearson correlation. chr12:111670556-111670909) (n = 3 per group). f-h, RT-qPCR from BAT of (f) female Esrra/g AdipoqCre and Esrra/g / (n = 5 per group) (BAT was harvested 24 hours after onset of 6°C exposure); (g) male Ebf1/2 AdipoqCre and Ebf1/2 / (n = 3-4 per group) (BAT was harvested 7 days after onset of 4°C exposure), and (h) male Ppargc1a Ucp1CreERT2 and Ppargc1a / (n = 4 per group) mice (6-10 weeks of age) (BAT was harvested 24 hours after onset of 6°C exposure). i, Western blot of BAT from Ppargc1a Ucp1CreERT2 and Ppargc1a / female mice reared at 22°C, housed at 30°C for 5 days and then subjected to 30°C or 6°C at zeitgeber time 4 (ZT4). BAT was harvested 48 hours after onset of 6°C exposure. j, Model of transcriptional control of the futile creatine cycle. Data are presented as mean ± s.e.m. of biologically independent samples. d-h, Two-way ANOVA (Fisher's LSD). NA-stimulated thermogenesis requires a 1 AR signaling and CKB. a-b, Energy expenditure (EE) of Ckb / and Ckb AdipoqCre male mice (n = 8 per group) reared at 22°C and then housed for 5 days at 30°C prior to administration of (a) saline (Sal) or (b) noradrenaline (NA, 1 mg kg -1 ). c, f, Cartoon of approach to determine if (c) aAR or (f) a 1 AR signaling and CKB are necessary for NA-stimulated brown adipocyte thermogenesis. d, g, Representative basal and NA-stimulated (0.1 mM) oxygen consumption trace of freshly isolated Ckb / and Ckb AdipoqCre brown adipocytes, treated with (d) PBZ (1 mM) or (g) PZS (1 mM) both compared to vehicle control. The time of NA addition was normalized to 100%. e, h, NA-stimulated oxygen consumption rates (above basal) of freshly isolated Ckb / and Ckb AdipoqCre brown adipocytes, treated with (e) PBZ (n = 5).or (h) PZS (n = 4). both compared to vehicle control. Data are presented as mean ± s.e.m. of biologically independent samples. a, b, Two-way ANOVA (Fisher's LSD) from minutes 0 to 21; e, h, One-way ANOVA (Fisher's LSD).

Figure 4
Thermogenesis by combined a 1A AR and cAMP signaling genetically requires Ckb. a, Schematic of bioluminescence resonance energy transfer (BRET)-based miniG subtype recruitment assay. b, Agonistinduced BRET between ADRA1A-tagged Nano Luciferase (ADRA1A-Nluc) and Venus-tagged miniGa q protein sensor in immortalized brown adipocytes (n = 3 per group, each agonist/vehicle performed in triplicate). Noradrenaline (NA), cirazoline, and A61603 were all used at 1 mM. c, Cartoon of hM3Dq AdipoqCre mouse construction. d, Western blot of BAT, SAT, perigonadal adipose tissue (PgAT), liver (Liv) and kidney (Kid) from hM3Dq AdipoqCre mice. e, Schematic of activation of Ga s and Ga q signaling in adipocytes from hM3Dq AdipoqCre mice. f, h, Energy expenditure (EE) of hM3Dq AdipoqCre :Ckb /+ mice reared at 22°C and then housed for 5 days at 30°C prior to administration of (f) saline or (h) CL 316,243 (CL, 0.5 mg kg -1 ) (n = 24: n = 16 males, n = 8 females) or CL + DCZ (both at 0.5 mg kg -1 ) (n = 25: n = 17 males, n = 8 females). g-i, EE of hM3Dq AdipoqCre :Ckb / mice reared at 22°C and then housed for 5 days at 30°C prior to administration of (g) saline or (i) CL (0.5 mg kg -1 ) (n = 15: n = 7 males, n = 8 females) or CL + DCZ (both at 0.5 mg kg -1 ) (n = 15: n = 7 males, n = 8 females). j, Cartoon of approach to study brown adipocyte-intrinsic thermogenesis by individual and combined activation of a 1 AR and cAMP signaling. k, Representative oxygen consumption trace of freshly isolated Ckb / and Ckb AdipoqCre brown adipocytes, treated with NA (0.1 mM), cirazoline (a 1 AR agonist, Ciraz, 1 mM), forskolin (Fsk, 3 mM), or Ciraz+Fsk. The time of drug addition was normalized to 100%. l, Oxygen consumption rates from multiple experiments of brown adipocytes treated as in k (n = 3-7 per group). m, Cartoon of approach to study brown adipocyteintrinsic thermogenesis by individual and combined activation of a 1A AR and cAMP signaling. n, Representative oxygen consumption trace of freshly isolated Ckb / and Ckb AdipoqCre brown adipocytes, treated with NA, A16063 (a 1A AR agonist, 1 mM), Fsk, or A16063+Fsk. The time of drug addition was normalized to 100%. o, Oxygen consumption rates from multiple experiments of brown adipocytes treated as in n (n = 3-5 per group). Data are presented as mean ± s.e.m. of biologically independent samples. f, g, Two-way ANOVA (Fisher's LSD) from hours -1 to 5; h, i, Two-way ANOVA (Fisher's LSD) from hours 1 to 5 (1 hour after injection was chosen because the stress response had subsided); l, o, One-way ANOVA (Fisher's LSD).

Supplementary Files
This is a list of supplementary les associated with this preprint. Click to download. ExtendedDataFigures.docx