Multi-layered transcriptional control of glycogen metabolism coordinates thermogenic remodeling of white adipocytes in male mice

Domain: biology
Published: 2025-12-16
Editorial outcome: major_revision

Abstract
Thermogenic activation of subcutaneous white adipocytes requires glycogen synthesis and turnover. Here we show that β-adrenergic stimulation induces a distinct glycogen metabolism gene program in inguinal white adipose tissue in a cell-autonomous and adipocyte-specific manner. Among these, Gys2 and Ppp1r3c are rapidly induced following acute β3-adrenergic receptor activation. We identify Gys2 as a direct transcriptional target of PKA-CREB signaling. In contrast, sustained expression of glycogen metabolism genes under chronic β3-adrenergic activation requires the coactivator PGC1α, whose loss blunts glycogen accumulation and thermogenic capacity. Mechanistically, PGC1α cooperates with estrogen-related receptors (ERRs) to regulate chromatin accessibility and gene transcription. Although deletion of ERRα is compensated by ERRγ, combined deletion of ERRα/β/γ abolishes expression of glycogen metabolism and thermogenic genes. Chromatin profiling confirm that ERRs directly control the glycogen metabolic program in beige adipocytes. Together, our results identify a multilayered transcriptional axis that sustains glycogen metabolism during β-adrenergic activation in male mice.

Introduction
Thermogenic adipocytes contribute to systemic energy expenditure by converting chemical energy into heat, a process predominantly regulated by sympathetic nervous system activation and β-adrenergic signaling1,2. Upon cold exposure or chronic adrenergic stimulation, white adipocytes can acquire beige characteristics, including mitochondrial biogenesis and expression of uncoupling protein 1 (UCP1)3–5, enabling them to participate in non-shivering thermogenesis6. While this transformation is classically driven by the cAMP–PKA–CREB–PGC1α axis7,8, emerging evidence suggests that additional metabolic pathways are integrated into the thermogenic program9–12.

Our recent work identified a role for glycogen metabolism in the beigeing of subcutaneous adipocytes13. Chronic β-adrenergic stimulation promotes both glycogen accumulation and turnover in these adipocytes, and this dynamic flux is necessary for beige adipogenesis by generating reactive oxygen species (ROS) necessary for activation of the p38 MAPK signaling pathway and subsequent expression of UCP1 and other thermogenic genes. Disruption of glycogen synthesis or turnover impairs this pathway, reducing thermogenic gene expression and cold-induced energy metabolism13,14. These findings indicate that glycogen is not merely a passive energy reservoir in adipose tissue, but an active metabolic node linking glucose utilization to thermogenic signaling.

Although these data implicated glycogen metabolism in white adipose tissue beiging, the transcriptional regulation of genes encoding the enzymes of glycogen metabolism in adipocytes remains poorly understood. In particular, how β3-adrenergic signaling induces this gene program and how this regulation is maintained during chronic thermogenic activation is uncertain. PGC1α (PPARGC1A) is a central transcriptional coactivator that regulates energy metabolism in adipocytes15–18. It is upregulated in response to cold exposure or β-adrenergic stimulation, promoting mitochondrial biogenesis and oxidative metabolism2. In brown and beige adipocytes, PGC1α drives the expression of thermogenic genes such as UCP1, enhancing heat production15,19–21. In white adipocytes, it also supports fatty acid oxidation and contributes to overall energy expenditure, playing a crucial role in maintaining metabolic health22. Estrogen-related receptors α and γ (ERRα and ERRγ) are orphan nuclear receptors that play crucial roles in regulating mitochondrial function and energy metabolism in adipose tissue23,24. Both ERRα and ERRγ act as key transcriptional regulators of oxidative metabolism by directly controlling the expression of genes involved in mitochondrial biogenesis25,26, fatty acid oxidation27,28, and thermogenesis24,29–35. In adipocytes, ERRα and ERRγ are activated through their interaction with PGC1α, forming a transcriptional complex that enhances the expression of metabolic and thermogenic genes, including Ucp124,36–40. ERRα is particularly important for maintaining mitochondrial function and oxidative capacity in white and beige fat, while ERRγ is highly expressed in brown adipose tissue and contributes to its constitutively high oxidative and thermogenic activity40,41. Thus, the PGC1α – ERR axis is known to control oxidative and mitochondrial pathways42, although its role in modulating glycogen metabolism in adipocytes is unknown.

Here, we dissect the regulation of a cassette of glycogen metabolism genes as a cell-autonomous component of the thermogenic program. We demonstrate that acute β-adrenergic stimulation of adipocytes selectively induces Gys2 and Ppp1r3c via PKA/CREB signaling, while the sustained expression of a subset of glycogen metabolic genes during chronic sympathetic activation requires PGC1α and ERRα/γ. Through chromatin accessibility profiling, loss-of-function genetics, and overexpression studies, we uncover a transcriptional circuit in which the PGC1α–ERR complex maintains glycogen turnover during prolonged adrenergic activation to support thermogenesis. These findings expand the known regulatory network of adipose thermogenesis and establish glycogen metabolism as a PGC1α–ERR–coordinated transcriptional module.

Results
β3 adrenergic receptor activation regulates glycogen metabolism genes in a cell autonomous fashion
To investigate the transcriptional regulation of glycogen metabolism during the browning of white adipose tissue, we examined RNA sequencing (RNA-seq) datasets that revealed a marked induction of genes involved in glycogen synthesis and degradation, including Gys1, Gys2, Pygl, Ppp1r3b, Ppp1r3c, and Agl, in inguinal white adipose tissue (iWAT) following 7 days of cold exposure or β3-adrenergic stimulation43 (Fig. 1a). To determine the temporal dynamics of this response, we treated C57BL/6J mice with the β3-adrenergic agonist CL-316,243 (CL) for 1, 3, 5, or 7 consecutive days. CL administration resulted in a progressive upregulation of Ucp1, consistent with the emergence of beige adipocytes. Concomitantly, we observed a significant increase in the expression of muscle-enriched isoforms of glycogen synthase (Gys1), liver-enriched isoforms of glycogen synthase (Gys2) and liver glycogen phosphorylase (Pygl), as well as the glycogen debranching enzyme Agl (Fig. 1b, c). Notably, Gys2 was robustly induced at both the mRNA and protein levels within 24 hours of a single CL injection, whereas the upregulation of Pygl was delayed, becoming apparent only after 3 days of treatment (Fig. 1b, c, Supplementary Fig. 1a). Consistent with these molecular changes, PAS staining revealed progressive glycogen accumulation in parallel with beige adipocyte formation in iWAT of mice treated with CL for 1, 3, 5, or 7 consecutive days (Supplementary Fig. 1d).

To further define the early transcriptional response, we performed a high-resolution time-course analysis and confirmed that Gys2, as well as Ppp1r3c and Agl mRNAs are selectively and rapidly induced within the first 24 hours, alongside the thermogenic gene Ucp1 (Fig. 1e). To test whether this response is cell autonomous, we treated differentiated adipocytes in vitro with CL for 6 h. This acute stimulation was sufficient to induce Gys2 and Ppp1r3c, but not other glycogen metabolic genes, suggesting selective and intrinsic regulation of glycogen synthesis in adipocytes (Supplementary Fig. 1b). Consistently, treatment with a cell permeable analog of cAMP for 6 h produced a similar induction of Gys2 and Ppp1r3c (Supplementary Fig. 1c). In contrast, primary mouse hepatocytes treated with cAMP or glucagon failed to induce these genes (Supplementary Fig. 1d, e), indicating that this regulation is adipocyte specific.

CREB directly regulates Gys2 expression downstream of PKA signaling
To determine whether Gys2 and Ppp1r3c are direct transcriptional targets of protein kinase A (PKA) signaling, we treated primary adipocytes with the protein synthesis inhibitor cycloheximide prior to β3-adrenergic stimulation. Inhibition of de novo protein synthesis did not impair CL-induced expression of Gys2 and Ppp1r3c, indicating that both genes are primary transcriptional targets of PKA signaling and do not require intermediary protein synthesis (Fig. 2a). Pharmacological inhibition of PKA (H89), p38 MAPK (SB203580), or CREB (666-15) attenuated Gys2 mRNA induction in response to CL treatment in vitro (Fig. 2b), and CREB inhibition also suppressed CL-induced glycogen accumulation (Fig. 2c), suggesting a functional role for CREB in regulating glycogen metabolism.

To test directly the requirement for CREB in Gys2 regulation, we employed CRISPR–Cas9-mediated gene editing in primary pre-adipocytes differentiated in vitro. Using an adeno-associated virus (AAV) to deliver Creb-targeting sgRNAs into fully differentiated pre-adipocytes from Cas9 knock-in mice44 (Supplementary Fig. 2a), we achieved efficient knockdown (KD) of Creb mRNA and protein levels without affecting Atf2 mRNA expression (Fig. 2d, e). Consistent with this, CL-induced phosphorylation of CREB at serine 133 was markedly reduced (Fig. 2e, f). Loss of Creb abolished the induction of Gys2 by CL and reduced its basal expression (Fig. 2g). Glycogen accumulation was also diminished in Creb KD adipocytes in response to CL treatment (Fig. 2h). In contrast, Ppp1r3c expression was unaltered, suggesting that its regulation under PKA signaling is mediated by transcription factors other than CREB. Luciferase reporter assays showed that CREB significantly enhanced Gys2 promoter activity through a half CRE-binding site, and mutation of this site decreased the response (Supplementary Fig. 2b). These results indicate that CREB is essential for the transcriptional activation of Gys2 in adipocytes, but dispensable for the regulation of Ppp1r3c (Fig. 2g). Together, these findings identify Gys2 as a CREB-dependent gene downstream of β3-adrenergic signaling.

In humans, the expression levels of GYS2 in adipose tissue were negatively correlated with BMI (Supplementary Fig. 3b), GYS1 and GYS2 were negatively correlated with HOMA-IR, and Waist/hip ratio (Supplementary Fig. 3a, b). Higher expression of glycogen synthase genes thus associated with lower body weight, higher insulin sensitivity, lower triglyceride, lower blood sugar and a non-obese phenotype (Supplementary Fig. 3a, b). These findings suggest a potential protective role of adipocyte glycogen synthase genes in maintaining metabolic health and preventing obesity45–52.

PGC1α sustains glycogen metabolism gene expression during chronic β3-adrenergic activation
Previous studies have shown that acute cold exposure or CL-316,243 treatment leads to desensitization of the β3-adrenergic receptor53, prompting us to investigate how the expression of glycogen metabolism genes and glycogen turnover are sustained under chronic β3-adrenergic stimulation. We thus analyzed gene expression correlations using a transcriptomic dataset from diversity outbred mice. This analysis revealed a strong positive correlation between glycogen metabolism genes and Ppargc1a (PGC1α) (Fig. 3a). Also, PPARGC1A expression shows a strong positive correlation with glycogen synthase and AGL in a human dataset45–52 (Supplementary Fig. 4a), These findings suggest that PGC1α may coordinately regulate both glycogen synthesis (via GS) and breakdown (via PYGL and AGL) in adipose tissue, supporting metabolic flexibility under energy-demanding conditions.

To test the functional requirement of PGC1α in regulating glycogen metabolism in vivo, we generated adipocyte-specific PGC1α knockout mice (PGC1α-AKO) by crossing Ppargc1a-floxed mice with AdipoQ-Cre drivers. PGC1α expression was significantly decreased in PGC1α-AKO mice, without compensatory upregulation of Ppargc1b at either the RNA or protein level (Fig. 3b, Supplementary Fig 4b, c, 5a). In iWAT from PGC1α-AKO mice treated with CL for 7 days, the expression of glycogen metabolism genes, including Gys1, Gys2, Ppp1r3c, and Agl was markedly reduced, as was the canonical PGC1α downstream target Ucp1 (Fig. 3b–d, Supplementary Fig 4b). Glycogen accumulation in iWAT was diminished in PGC1α-deficient mice following CL treatment (Fig. 3e). Furthermore, histology of iWAT from PGC1α-AKO mice revealed a marked reduction in CL-316,243-induced glycogen levels and UCP1 expression (Fig. 3f).

We also assessed glycogen metabolism in primary adipocytes differentiated in vitro from PGC1α fl/fl and PGC1α-AKO mice. Interestingly, there were no significant differences in expression of glycogen-related genes or Ucp1 following 6 h of CL stimulation (Supplementary Fig. 5a), suggesting that PGC1α is dispensable for acute transcriptional responses, but required to sustain glycogen metabolic programs during chronic β3-adrenergic-induced browning of white adipocytes. Our previous study revealed that attenuation of glycogen synthesis or turnover reduced the activation of p3813. The activation of p38 by CL-316,243 treatment was not different between WT and PGC1α-AKO adipocytes (Supplementary Fig. 5b, c). The phosphorylation of HSL at Ser660/563 was also unchanged, indicating that loss of PGC1α does not impair adrenergic lipolysis (Supplementary Fig. 5b, c). Furthermore, beige adipocytes exhibited higher basal levels of glycogen metabolism genes compared to white adipocytes, and treatment with the selective PGC1α inhibitor SR-18292 significantly reduced these genes at both the mRNA and protein levels54,55, reinforcing a role for PGC1α in maintaining their expression (Supplementary Fig. 5d–f).

To investigate whether PGC1α regulates chromatin accessibility at glycogen-related gene loci, we performed the assay for transposase-accessible chromatin with sequencing (ATAC-Seq) on iWAT from control and PGC1α-AKO mice following 7 days of CL treatment. Promoter regions of key glycogen metabolism genes exhibited reduced chromatin accessibility in the absence of PGC1α, suggesting impaired transcriptional competency (Fig. 3g). To test for direct PGC1α binding, we conducted chromatin immunoprecipitation (ChIP) assays and found that enrichment of PGC1α at the promoters of Gys2, Ppp1r3c, and Agl was abolished in PGC1α-AKO iWAT following CL treatment (Fig. 3h). These results collectively identify PGC1α as a critical regulator that maintains expression of glycogen metabolism genes during prolonged β3-adrenergic stimulation through direct chromatin engagement. Thus, PGC1α is a master regulator for glycogen metabolism genes in chronic β3 activation.

PGC1α and ERRα/γ cooperatively maintain glycogen metabolism gene expression during chronic β3-adrenergic signaling
PGC1α is a well-established coactivator of multiple nuclear receptors in adipocytes, among which estrogen-related receptor α (ERRα) exhibits the highest affinity23. We first analyzed the RNA-seq dataset from brown adipose tissue derived from wild-type and ERRα/γ double knockout (ERRα/γ -AKO) mice treated with or without CL-316,24339, and observed that the induction of glycogen metabolism genes was completely abolished in ERRα/γ -AKO mice (Supplementary Fig. 6a). Additionally, ChIP-seq analysis of ERRα and ERRγ revealed direct binding at the promoter regions of glycogen metabolism genes in brown fat38,40 (Supplementary Fig. 6b).

To isolate the role of ERRα, we generated adipocyte-specific ERRα knockout (ERRα -AKO) mice by crossing Esrra-flox/flox mice with AdipoQ-Cre. Surprisingly, glycogen metabolism genes and thermogenic markers such as Ucp1 remained unchanged in ERRα -AKO iWAT after CL treatment (Supplementary Fig. 7a–c), and glycogen accumulation was unaffected (Supplementary Fig. 7d). We observed a robust upregulation of Esrrg (ERRγ) expression in ERRα-AKO adipose tissue under both basal and CL-treated conditions (Supplementary Fig. 7a–c), consistent with prior reports that ERRγ not only increases in expression but can also functionally compensate for the loss of ERRα29. These findings suggested compensatory activation by ERRα homologs, particularly ERRγ, which shares 70–80% sequence similarity with ERRα in the DNA- and ligand-binding domains32,56,57. We also performed ERRγ ChIP–qPCR in ERRα-AKO mice, which revealed increased binding of ERRγ at promoter regions normally occupied by PGC1α and ERRα, indicating that enhanced ERRγ occupancy contributes to the redundant regulation of these target genes (Supplementary Fig. 8a).

To eliminate potential redundancy among ERR family members, we generated adipocyte-specific ERRα/β/γ triple knockout mice (ERRs-AKO). In these mice, chronic CL treatment failed to induce glycogen metabolism genes and thermogenic programs, including Ucp1, and glycogen content in iWAT was markedly reduced (Fig. 4a–e). Histological analysis confirmed a substantial reduction in both UCP1 protein and glycogen deposition in iWAT of ERRs-AKO mice following prolonged β3-adrenergic stimulation (Fig. 4f). To investigate whether ERRs directly modulate chromatin accessibility at glycogen-related gene loci, we performed ATAC-seq on iWAT from CL-treated control and ERRs-AKO mice. Chromatin accessibility at promoters of Gys1, Gys2, Pygl, Ppp1r3c, and Agl was significantly diminished in the absence of ERRs (Fig. 4g, Supplementary Fig. 6c). Furthermore, ChIP-seq analysis revealed that ERRα binding to these promoter regions was abolished in ERRs-AKO mice (Fig. 4g, h). Genome-wide profiling of ERRα binding revealed that occupancy is not limited to thermogenic and glycogen metabolic genes, but also includes loci associated with mitochondrial function and fatty acid metabolism, suggesting a broad regulatory role for ERRα in coordinating the transcriptional program of adipose browning (Fig. 4h). To functionally validate the role of each component, we overexpressed PGC1α, ERRα, or ERRγ individually in beige adipocytes. Overexpression of each factor led to the induction of Gys1 and Gys2 (Supplementary Fig. 8b). Together, these data demonstrate that during chronic β-adrenergic activation, PGC1α cooperates with ERRα and ERRγ to maintain glycogen metabolism gene expression through direct engagement with chromatin, ensuring sustained glycogen turnover in thermogenic adipocytes (Supplementary Fig. 9).

Methods
Animals
C57BL/6J (strain no. 000664), PGC1α fl/fl (strain no. 009666), and Adipoq-Cre (strain no. 028020), Rosa26-Cas9 knockin on B6J (strain no. 026179) mice were obtained from The Jackson Laboratory. PGC1α fl/fl mice were crossed with Adipoq-Cre mice to generate adipocyte-specific PGC1α knockout mice. Esrra (ERRα) single knockout (ERRα-AKO) and Esrra/Esrrb/Esrrg triple knockout (ERRs-AKO) mice were provided by Dr. Ronald Evans; all mice were maintained on a C57BL/6J background67. Male mice aged 8−12 weeks were maintained on standard chow (7912, Teklad) under specific pathogen-free (SPF) conditions with 12 h:12 h light–dark cycles and ad libitum access to food and water. For in vivo studies, CL-316,243 (1 mg/kg) was administered intraperitoneally. All animal procedures were approved by the Institutional Animal Care and Use Committee (IACUC) at the University of California, San Diego.

Cell culture
Preadipocyte Isolation. Preadipocytes were isolated from the inguinal white adipose tissue (WAT) of 8-to 10-week-old mice. Inguinal fat was finely minced and digested in 10 mL of serum-free DMEM/F12 containing 1 mg/mL collagenase (C6885, Sigma) for 30 min at 37 °C with gentle agitation. Digestion was stopped by adding DMEM/F12 containing 10% FBS. The tissue suspension was filtered through a 100-μm strainer and centrifuged at 750 × g for 10 min at room temperature. The supernatant was discarded, and the pellet was resuspended in DMEM/F12 with 10% FBS, then filtered through a 40 μm strainer and centrifuged again at 750 × g for 10 min. The final pellet was resuspended in DMEM/F12 containing 10% FBS and plated onto 10 cm tissue culture dishes in DMEM/F12 with 15% FBS. After 2 days, the medium was removed, and non-adherent cells were washed away with PBS. Fresh culture medium containing 10% FBS was added. Once the cells reached ~70% confluence, they were replated for experiments and maintained in DMEM/F12 with 10% FBS, with medium changes every 2–3 days.

Differentiation was initiated 1–2 days after cultures reached full confluence by adding 500 μM 3-isobutyl-1-methylxanthine, 250 nM dexamethasone, 1 μg/mL insulin, and 1 μM troglitazone for 4 days. The medium was then changed to DMEM/F12 with 10% FBS containing 1 μg/mL insulin for 3 days, followed by maintenance in DMEM/F12 with 10% FBS. Cells were used for experiments on day 8 or 9 after the initiation of differentiation. AAV8-sgControl and AAV8-sgCreb, both carrying Cre, were introduced into differentiated adipocytes derived from Rosa26-Cas9 knock-in B6J mice. Protein and mRNA levels were measured 48–72 h after transduction. All treatments were conducted in serum-free DMEM, with 2% FFA-free BSA as indicated.

For beige adipocyte differentiation, stromal vascular fraction (SVF) cells were cultured in DMEM containing 10% FBS, 5 μg/mL insulin, 1 nM T3, 1 μM rosiglitazone, 0.5 mM isobutylmethylxanthine, 250 nM indomethacin, and 250 nM dexamethasone. After 48 hours, cells were cultured in medium containing 10% FBS, 5 μg/mL insulin, 1 nM T3, and 1 μM rosiglitazone for another 5–7 days. Only cultures in which >90% of cells displayed mature adipocyte morphology were used. All media were supplemented with 10 U/mL penicillin and 10 U/mL streptomycin.

Primary Hepatocyte Isolation
Mice were anesthetized and subjected to liver perfusion with 15 ml of calcium-free HEPES–phosphate buffer (pH 7.4), followed by 25 ml of HEPES–phosphate buffer (pH 7.4) containing 40 µg/ml Liberase TM (Roche). A final perfusion step was performed using 25 ml of calcium-free HEPES–phosphate buffer (pH 7.4). The liver was then excised, and primary hepatocytes were released by mechanical dissociation in 30 ml of cold HEPES–phosphate buffer (pH 7.4). The resulting cell suspension was filtered through a 70-μm nylon mesh (Corning) and centrifuged at 50 × g for 5 min. The supernatant was discarded, and the pellet was resuspended in 50 ml of HEPES–phosphate buffer (pH 7.4) containing 36% Percoll, followed by centrifugation at 100 × g for 10 minutes. The enriched hepatocyte pellet was resuspended in 10 ml of pre-warmed William’s E medium (Life Technologies) supplemented with 10% FBS, 10 mM HEPES, 2 mM L-glutamine, 8 mg/L gentamicin, SPA, 1 μM dexamethasone, 4 μg/ml insulin, and 1 mM glucose. Cells were seeded onto collagen-coated plates at a density of 4 × 10⁵ cells/ml. Four hours post-plating, the medium was replaced to remove non-adherent and dead cells. For glucagon and cAMP treatments, hepatocytes were pre-incubated for 1 hour in FBS-free William’s E medium supplemented with 10 mM HEPES, 2 mM L-glutamine, 8 mg/L gentamicin, SPA, 1 μM dexamethasone, and 1 mM glucose. All treatments were conducted in this FBS-free medium.

Glycogen extraction
Glycogen extraction was performed as previously described with slight modifications13,68. iWAT samples were defatted by incubation in 1 mL of a methanol:chloroform solution (1:2 ratio) for 30 min at room temperature. Samples were centrifuged at 6000 × g for 5 min, the solution was aspirated, and the process was repeated once more. After the second centrifugation, samples were allowed to dry completely before proceeding. This defatting step was not applied to cells, liver, or BAT tissues.

Samples were then boiled for 30 min in 500 μL of 30% KOH solution. After cooling, 100 μL of 1 M Na₂SO₄ was added, followed by 1.2 mL of pure ethanol. Samples were boiled for 5 min and centrifuged at 16,000 × g for 5 min. Pellets were washed twice by resuspension in 500 μL of double-distilled water, followed by the addition of 1 mL of pure ethanol and a brief boil. After the final wash, pellets were completely air-dried and resuspended in 150 μL of 50 mM sodium acetate buffer (pH 4.8) containing 0.3 mg/mL amyloglucosidase and incubated at 37 °C overnight.

Glycogen content was quantified using the Autokit Glucose assay (997-03001, Fujifilm), with comparison to a standard curve.

RNA extraction, RT–PCR and qPCR
For RNA isolations from in vivo experiments, following dissection, tissues were immediately snap frozen in liquid nitrogen and stored at −80 °C until processing. Tissues were directly homogenized in TRIzol (15596018, Life Technologies) according to the manufacturer’s instructions. One micrograms of RNA were used for cDNA synthesis using HiScript III RT SuperMix for qPCR (+gDNA wiper) (R323-01, Vazyme). Quantitative PCR was performed in quadruplicate using the Taq Pro Universal SYBR qPCR Master Mix (Q712-03, Vazyme). The Applied Biosystems QS5 real-time PCR System was used with the standard curve settings.

Protein Extraction and Immunoblotting
Tissues or cells were lysed or homogenized in denature buffer (50 mM Tris-HCl, pH 7.5, 0.5 M EDTA, 1% SDS, 1 mM DTT) supplemented with freshly added Halt Protease and Phosphatase Inhibitor Cocktail (Thermo Fisher). Lysates were rotated at 4 °C for 30 min, and then centrifuged at 17,000 × g for 15–20 minutes at 4 °C. The cleared supernatants were collected, and protein concentrations were determined using a BCA Protein Assay Kit. Proteins were separated by electrophoresis on Tris-Glycine gels (Novex, Invitrogen) and transferred to PVDF membranes. The blots were blocked with 5% BSA in TBST (0.1% Tween-20 in TBS) at room temperature for 1 h and incubated with primary antibodies diluted in blocking buffer overnight at 4 °C. Membranes were incubated with the appropriate secondary antibodies conjugated with horseradish peroxidase for 2 h at room temperature SuperFemto ECL Chemiluminescence Kit (E423-02, Vazyme) and Li-Cor detection system (Fisher Scientific) were used for Western blot detection.

Antibodies
GYS1 (10566-1-AP), GYS2 (22371-1-AP), PYGL (15851-1-AP), PYGM (19716-1-AP), AGL (16582-1-AP), DIO2 (26513-1-AP), GFP (50430-2-AP) were obtained from Proteintech. pHSL-563 (4139S), pHSL-660 (45804S), HSL (4107S), p38 (9218S), p-p38 (9211), Tubulin (3873S), Actin (4970S), ERRα (13826S) were obtained from Cell Signaling Technology. PGC1α (AB3242, Sigma). UCP1 (AB209483) and ERRγ (Ab219547) were obtained from Abcam. ERRγ antibody was generated by Dr. Ronald Evans laboratory.

Histology
Tissues were collected and fixed in 10% formalin for 48 h and then washed and stored in 70% ethanol until processing. All the following steps such as paraffin embedding, sectioning, hematoxylin and eosin staining and immunohistochemistry were done at the UCSD Tissue Technology Core. For Periodic Acid–Schiff (PAS) Staining, paraffin or frozen sections (4–5 µm) were baked at 60 °C for 1 h, deparaffinized in xylene (two changes, 3 min each), and rehydrated through graded ethanol (100%, 95%, 70%) into distilled water. Sections were incubated in 0.5% periodic acid for 5 min, rinsed in distilled water, and stained with Schiff’s reagent at room temperature for 15 min until a deep magenta color developed. After washing in running tap water for 5 min, sections were counterstained with hematoxylin for 1 min, clarified, and blued. Slides were dehydrated through graded ethanol, cleared in xylene, and mounted with a coverslip. PAS staining highlights glycogen, basement membranes, and mucosubstances in bright magenta. For immunohistochemistry (IHC), paraffin-embedded sections were baked at 60 °C for 1 h, deparaffinized in xylene, and rehydrated through graded ethanol to distilled water. Antigen retrieval was performed using citrate-based antigen unmasking solution (pH 6.0, Vector) at 95 °C for 30 min. Staining was performed on an Intellipath Automated IHC Stainer (Biocare). Endogenous peroxidase activity was blocked with Bloxall (Vector) for 10 min, followed by washing in TBST and blocking with Blotto (Thermo) for 10 min. Sections were incubated with primary antibody for 1 h, washed, and incubated with an anti-species HRP polymer secondary reagent for 30 min. DAB chromogen was applied for 5 min, and slides were rinsed in water, counterstained with Mayer’s hematoxylin for 5 min, dehydrated, cleared, and mounted with a xylene-based medium.

Chromatin Immunoprecipitation (ChIP)
ChIP was performed using the SimpleChIP® Enzymatic Chromatin IP Kit (#9003, Cell Signaling Technology) with slight modifications. Briefly, immediately after euthanasia, inguinal white adipose tissue (iWAT) depots from ten mice treated with CL 316,243 for 7 days were snap-frozen. Frozen tissues were later thawed and finely minced in 10 ml of cross-linking buffer (1× PBS with 1% formaldehyde), followed by incubation on a rocker for 20 min at room temperature. Cross-linking was quenched by adding 0.5 ml of 2.5 M glycine and incubating for 5 min with mixing. Tissues were then washed three times with ice-cold 1× PBS. Cross-linked tissues were resuspended in ChIP dilution buffer containing protease inhibitors (cOmplete™, Roche) and kept on ice. Nuclei were pelleted by centrifugation at 2000 × g for 5 min at 4 °C. The supernatant was discarded, and the pellet was resuspended in 1 ml of ice-cold 1× Buffer B with DTT per IP preparation. After another centrifugation step, the pellet was resuspended in 100 µl of 1× Buffer B+DTT and transferred to a 1.5 ml microcentrifuge tube. For chromatin digestion, 0.5 µl of Micrococcal Nuclease was added per IP, mixed by inversion, and incubated at 37 °C for 20 min with gentle mixing every 3–5 min to achieve DNA fragments of ~150–900 bp. The reaction was stopped by adding 10 µl of 0.5 M EDTA and cooling on ice. Nuclei were pelleted at 16,000 × g for 1 min at 4 °C. The pellet was then resuspended in 100 µl of 1× ChIP buffer with protease inhibitors and incubated on ice for 10 minutes. Lysates (up to 500 µl) were sonicated with short pulses, placing the samples on wet ice for 30 s between pulses to disrupt nuclear membranes. Lysates were clarified by centrifugation at 9,400 × g for 10 minutes at 4 °C. For each ChIP, 500 µl of diluted chromatin was incubated with specific antibodies at 4 °C for 4 hours to overnight with rotation. Protein G magnetic beads (30 µl per IP) were added and incubated for an additional 2 hours at 4 °C with rotation. Beads were pelleted using a magnetic rack, and the supernatant was carefully removed. Beads were washed three times with low-salt wash buffer (1 ml, 5 min each at 4 °C with rotation), followed by one high-salt wash under the same conditions. DNA was eluted and purified according to the kit protocol. Sequencing libraries were generating using the KAPA Hyper Prep Kit (KAPA Biosystems, Wilmington, MA, USA) following manufacturer’s instructions using 13 cycles of amplification. The quality of the library was assessed using High Sensitivity D1000 kit on a 4200 TapeStation instrument (Agilent Technologies, Santa Clara, CA, USA). Sequencing was performed using the NovaSeq X Plus Sequencing System (Illumina, San Diego, CA, USA), generating 100 bp paired-end reads to obtain ~40 M reads per sample.

Sequencing reads were aligned to the mouse reference genome (mm10) using Bowtie2 (v2.4.5) with local alignment mode and the --no-unal option. The resulting SAM files were converted to BAM format using samtools view (v1.15) and subsequently sorted with samtools sort. Duplicated reads were removed using Picard (v2.26.10)‘s MarkDuplicates function.

ChIP-seq promoter enrichment analysis
To compare enrichment of ChIP-seq signal at gene promoters of interest, first RPKM normalized bigwig files were generated using bamCoverage from deeptools version 3.5.2. The ComputeMatrix reference-point function from deeptools version 3.5.2 was used to compute ChIP-seq signal in 10 bp bins −2000 bp to +2000 bp relative to transcription start sites annotated in mm10 Gencode Release 23. To visualize, heatmaps and average profile plots were generated.

ATAC-seq
Permeabilized nuclei were obtained by resuspending grinding tissue in 1 mL Nuclear Permeabilization Buffer (0.2% IGEPAL-CA630 (I8896, Sigma), 1 mM DTT (D9779, Sigma), Protease inhibitor (05056489001, Roche), 5% BSA (A7906, Sigma) in PBS (10010-23, Thermo Fisher Scientific)), and incubating for 10 min on a rotator at 4 °C. Nuclei were then pelleted by centrifugation for 5 min at 500 xg at 4 °C. The pellet was resuspended in 25 µL ice-cold Tagmentation Buffer [33 mM Tris-acetate (pH = 7.8) (BP-152, Thermo Fisher Scientific), 66 mM K-acetate (P5708, Sigma), 11 mM Mg-acetate (M2545, Sigma), 16% DMF (DX1730, EMD Millipore) in Molecular biology water (46000-CM, Corning)]. An aliquot was then taken and counted by hemocytometer to determine nuclei concentration. Approximately 50,000 nuclei were resuspended in 20 µL ice-cold Tagmentation Buffer, and incubated with 1 µL Tagmentation enzyme (FC-121-1030; Illumina) at 37 °C for 60 min with shaking 500 rpm. The tagmentated DNA was purified using MinElute PCR purification kit (28004, Qiagen). The libraries were amplified using NEBNext High-Fidelity 2X PCR Master Mix (M0541, NEB) with primer extension at 72 °C for 5 min, denaturation at 98 °C for 30 s, followed by 8 cycles of denaturation at 98 °C for 10 s, annealing at 63 °C for 30 s and extension at 72 °C for 60 s. Amplified libraries were then purified using MinElute PCR purification kit (28004, Qiagen), and two size selection steps were performed using SPRIselect bead (B23317, Beckman Coulter) at 0.55X and 1.5X bead-to-sample volume ratios, respectively. Libraries were sequenced to the dept of 80 M reads.

Statistical analysis
Statistical analyses were conducted using GraphPad Prism 9. Data are presented as mean ± s.e.m. Comparisons between two groups were performed using unpaired Student’s t-tests. For comparisons involving more than two groups, one-way or two-way ANOVA was used, as specified in figure legends. P-values are indicated in the figures. No data were excluded from analysis unless due to technical issues or failure of control conditions. During tissue processing for RNA extraction, RT–PCR, and lysis, sample blinding was achieved by random allocation without treatment knowledge. Investigators were otherwise not blinded. Variance was not formally estimated between groups, and sample sizes were not predetermined, but instead based on experimental feasibility and animal or cell availability. When applicable, mice were randomly assigned to treatment or control groups, with treatments randomized across cages. All in vivo experiments were replicated in at least two independent cohorts. In vitro experiments were not randomized.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "This manuscript investigates the regulation of glycogen in adipocytes during the process of β3AR-induced beiging. The roles of PGC1α and ERRs are elucidated using knockout mouse models. Overall, the results are supportive of the conclusions drawn. In addition, this is an understudied area and there are interesting implications for the interactive roles of adipose carbohydrate and lipid metabolism, metabolic flexibility and the ability of a tissue to sustain thermogenesis. There are some issues with the Figures described below. One persistent criticism is the lack of quantification and statistical analysis of the western blots throughout the manuscript.\nMajor\nThroughout the paper, the changes in RNA are analyzed statistically whereas the changes in protein levels are not. In some cases, the changes in the blots appear to be convincing, whereas in others it is not very clear. In some cases, n=2.\nFig 1b. The changes in ERRγ protein are hard to interpret. The protein seems to decrease and then increase again. At day 7, it is not clear that the ERRγ protein is higher than baseline. As with other figures, protein levels not quantified or analyzed statistically. This is important for PGC1α and ERRγ as it is questionable whether the induction at later timepoints (5d, 7d) is statistically significant.\nIt is stated that there are “significant increases in the expression ..“ in GYs1, Gys2, Pygl, and Agl (Fig. 1b, c), but there are no statistics performed on the protein levels in Fig 1b. Some description of statistics or repeat experiments are needed.\nSimilar issues are noted with Extended Fig 4C, 4A, 6A, C and Fig 7.\nFig 2 and extended 4b are done in preadipocytes. Given the focus on beiging, why are these signaling pathways not evaluated in differentiated beige adipocytes (as done in extended figure 4c,d)? Extended Figure 8 shows acute versus chronic effects, although it does not mention the specific cell type. Is the rationale that they are determining acute priming in Fig. 2 and extended 4b?\nFig 3b same issue as Figure 1. It is not clear that PYGL and AGL protein reduction by PGC1α knockout is significant.\nFigure 3F needs a scale bar. IHC methods are not described in detail. Why is PAS + UCP1 zoomed out so much? It is impossible to discriminate between UCP1 (presumably brown?) and PAS at this scale. Perhaps this panel could be eliminated since UCP1 and PAS are assessed earlier in the figure.\nMinor\nThe mouse strain background of ERR mouse model and refs are not provided.", "reviewer_2": "In this study, the authors aim to elucidate the mechanism underlying the regulation of gene expression in adrenergic stimulation-induced beige fat cell activation. They observed that adrenergic stimulation upregulates the expression of glycogen metabolism genes in white adipocytes (WAT) and promotes WAT browning. Additionally, they proposed several mediators of this effect, including CREB, PGC1α, and ERRs. Using multiple gene knockout mouse models, the authors demonstrated the essential role of PGC1α and ERRs in mediating the effect of CL (clenbuterol) on promoting glycogen gene expression and WAT browning. This study is a follow-up to their previous research highlighting the importance of glycogen metabolism in beige fat. However, the current study lacks novel insights, fails to clarify significant gene regulatory mechanisms, and lacks rigorous validation of the proposed transcriptional regulatory pathways.\nMajor Concerns:\nLimitation of adrenergic agonists and mechanistic clarity of CL action\nOnly CL was used as an agonist for adrenergic stimulation of WAT; other adrenergic agonists should be included for validation. The mechanism by which CL acts on white adipocytes in vivo and in vitro remains unclear:\nIs there involvement of adrenergic receptors?\nAre there mechanistic differences in glycogen metabolism gene expression between in vivo and in vitro models, given the distinct time scales observed post-CL treatment?\nInsufficient evidence for \"autonomous gene regulation\"\nThe authors claim that CL treatment induces autonomous gene regulation, but this conclusion is not supported, as it was only tested in in vitro-cultured preadipocytes. For claims of chronic CL action via autonomous regulation, indirect effects (e.g., via other cell types in the stromal vascular fraction) in in vivo models must be excluded. To conclusively demonstrate the requirement for adipocyte-intrinsic expression of key genes (e.g., Gys2) in mediating browning/glycogen metabolic responses, adipocyte-specific knockout or knockdown models (e.g., using Adipoq-Cre) targeting Gys2 should be employed. These models should be used to assess the impact on CL-induced glycogen gene expression, UCP1 induction, and actual glycogen metabolism in inguinal WAT (iWAT).\nIncomplete analysis of signaling pathways and post-translational regulation\nThe authors showed that inhibitors of PKA (PKAi), p38 (p38i), and CREB (CREBi) block CL-induced Ucp1 and Gys2 expression, but data for other genes are missing. Additionally:\nWhen CREB is knocked down via sgRNA, how do global gene expression profiles change?\nGiven the claim of autonomous regulation, is there a change in GSK3 phosphorylation status?\nAre glycogen synthase or other related enzymes regulated via post-translational modifications (e.g., phosphorylation)?\nUnclear role and regulation of PGC1α\nPGC1α is a key regulator of multiple intracellular metabolic pathways. Critical questions remain:\nIs PGC1α responsive to CL treatment in white adipocytes? If so, is this response via changes in expression or post-translational modification?\nHow does PGC1α respond to CL or other chronic adrenergic stimuli? Is this response independent of PKA, p38, or CREB?\nIn adipocytes, does PGC1α knockout reduce mitochondrial content, and how would this affect lipidolysis in response to CL?\nATAC-seq and gene expression analyses suggest PGC1α directly binds and regulates glycogen genes, but this is not reproducible in in vitro-cultured adipocytes (Extended Data Fig. 4a), in contrast to CREB. This discrepancy must be explained, along with how PGC1α and CREB cooperate, which acts as the primary mediator of CL effects, and the underlying mechanism.\nMechanistic basis of ERRs’ role\nThe authors show that ERRs are required to mediate CL effects, but key questions persist:\nHow do ERRs respond to CL? Is this response autonomous?\nIs ERR activation dependent on PKA, p38, CREB, or PGC1α? If dependencies exist, what is the underlying mechanism?\nLack of detailed analysis of transcriptional regulation\nTo support claims of direct regulation of glycogen metabolism genes by CREB, PGC1α, or ERRs, detailed investigations are needed, including:\nBinding motifs and nuclear translocation of these factors.\nMutational assays of binding motifs.\nKinetics (acute vs. chronic) of regulation.\nCooperative interactions between factors and their physical associations.\nInvolvement of epigenetic markers.\nThese analyses should be validated using multiple stimuli combined with pathway inhibitors.\nIncomplete validation of ERR redundancy\nTriple-knockout (ERRs-AKO) data support functional redundancy of ERRs, but overexpression studies (Extended Data Fig. 7) show individual ERRs can induce gene expression. To rigorously test redundancy, rescue experiments in ERRs-AKO adipocytes should be performed: re-express ERRα, ERRγ, or PGC1α individually or in combination, and determine if this restores CL-induced glycogen gene expression and glycogen accumulation.\nMissing evidence for PGC1α-ERR complex formation\nThe model proposes that PGC1α cooperates with ERRα/γ, implying physical interaction. Direct evidence of complex formation is required, such as co-immunoprecipitation (Co-IP) experiments in CL-treated primary adipocytes or iWAT lysates using antibodies against PGC1α, ERRα, and ERRγ. Reciprocal Co-IPs (e.g., immunoprecipitating PGC1α followed by immunoblotting for ERRγ, and vice versa) are essential to confirm these interactions in vivo under relevant stimuli.\nAbsence of a working model\nThe authors should provide a diagrammatic working model to summarize their conclusions.\nMinor Concerns:\nFig. 1a: Poor consistency between duplicate samples, particularly under the \"Cold\" condition.\nFig. 1c: The dramatic induction of Agl at day 1 contradicts the authors’ description.\nFig. 1c and Fig. 1d: Inconsistent trends/quantification (y-axis scales) for Gys1, Pygl, and Ppp1r3c expression.\nExtended Data Fig. 1a: To claim \"cell autonomy,\" additional treatments (e.g., glucagon, cAMP) should be tested. In the right panel, many glycogen metabolism genes do not respond to CL, conflicting with mouse data; extend in vitro CL treatment time courses in differentiated adipocytes to check if Pygl and Agl induction is delayed but intrinsic.\nInconsistencies between Extended Data Fig. 1a and Fig. 2b: Gys2 shows mild upregulation at 2 h, 3-fold at 4 h (Extended Data Fig. 1a), but ~4-fold in Fig. 2b; similar issues exist for Ppp1r3c. These inconsistencies, combined with the mild response at 3 h, challenge the claim that de novo protein synthesis is unnecessary. A cycloheximide (CHX) treatment time course (2–8 h) is needed to validate this.\nExtended Data Fig. 1: Adipocyte specificity is claimed via comparison to hepatocytes, but other tissues (e.g., skeletal muscle, heart) also express glycogen metabolism genes. Analyze gene expression kinetics in these tissues from the same CL-treated mice used for the iWAT time course to establish uniqueness.\nFig. 2c: UCP1 shows a 30-fold increase at 6 h CL treatment, inconsistent with the >100-fold increase in Extended Data Fig. 1a; a similar discrepancy exists for Gys2.\nFig. 3g: Negative controls (without CL treatment) are missing.\nFig. 3h: Enrichment fold changes are overly modest.\nExtended Data Fig. 4b: The authors claim CL induces acute (p38-dependent) and chronic (PGC1α-dependent) effects. For the chronic effect, test in vitro models and determine if de novo protein synthesis is required.\nExtended Data Fig. 4: PGC1α is dispensable for acute (6 h) induction but essential for sustained expression during chronic stimulation. Define the kinetics of PGC1α mRNA and protein induction in iWAT/primary adipocytes to correlate with its functional requirement.\nExtended Data Fig. 6a: ERRα-AKO mice show no increase in ERRγ under vehicle conditions, conflicting with Extended Data Fig. 6c (0 day); GS shows the opposite trend.\nCompensatory upregulation of ERRγ in mouse ERRα-KO models: Test if this mechanism exists in human primary adipocytes.", "reviewer_3": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.", "reviewer_4": "This manuscript described important new findings of a role for catecholamine-stimulated glycogen metabolism during the process of increasing uncoupled respiration and nonshivering thermogenesis. The work employs mouse models and cells in culture and there is a nice addition (Ext data 2) and connection to human physiology. The findings show the major roles of transcription factors and coregulators in the control of a range of genes involved in glycogen synthesis and metabolism. For example, Fig 3 shows the role of PGC1α in the transcription of glycogen synthase genes; but this is complemented by experiments showing increased glycogen production both biochemically (e) and histologically (f). This new finding is a significant addition to the literature so that we continue to develop a better physiological understanding of this important adaptive metabolic process. The manuscript is generally well written with a very nice Discussion. There are some unexplained as well as discordant findings that require further experimentation and discussion and these are noted below.\nSpecific Comments:\n1. Fig 1. GS and related proteins in inguinal white adipose tissue don’t increase until day 3 of CL txt, understandable in the sense that these ‘beige’ adipocytes appear in patches in the tissue, and when one homogenizes the whole tissue these proteins/genes are diluted out. So it would be nice to add some histology of these tissues at the various time points – as was done in Figure 3. It would help the reader better understand the temporal and spatial behavior.\n2. Fig 2. Please make clear in the legend that what is being treated and examined are ‘differentiated’ adipocytes from preadipocytes. As written, it says treating ‘preadipocytes’.\n3. Fig 2a: There is clearly an inhibitory effect of the cycloheximide treatment on Ucp1 mRNA, yet nothing is said about it. This implies that a (presumably) short-lived protein is required to be synthesized that is very important for the transcription of this gene. It is quite striking and at least for this reviewer, cannot find in the literature any previous mention of such a result or explanation for it. So either address this finding or just remove panels a and b: the rest of the Figure clearly stands on its own.\n4. Fig 2g: Why did the authors focus on CREB and not examine other factors previously shown to be more important for transcription of Ucp1 and Ppargc1a genes: ATF2. Is the CREB sgRNA specific to the Creb gene or might it also be affecting ATFs? Data to confirm this is important.\n5. Ext data 4 regarding Pgc1α adipose KO data: given the established literature of a primary role for PGC1α being required for Ucp1 gene transcription, which in these data is minimally affected, is PGC1β compensating for this complete loss of Pgc1α expression? This seems plausible given the more acute treatment with the inhibitor shows the role of PGC1α dependence. Please show protein levels of PGC1α and -1β in these tissues and provide some discussion for the findings.\n6. Regarding the work on the ERRs for deleting all three Err genes, this has been done previously and their redundancy when deleting one or another has been previously shown. Please cite Gantner et al. 2016 Endocrinology PMID 27763777 and note this earlier observation."}, "author_response": "Point to point response\nREVIEWER COMMENTS\nReviewer #1 (Remarks to the Author):\nThis manuscript investigates the regulation of glycogen in adipocytes during the process of β3AR-\ninduced beiging. The roles of PGC1α and ERRs are elucidated using knockout mouse models. Overall,\nthe results are supportive of the conclusions drawn. In addition, this is an understudied area and there\nare interesting implications for the interactive roles of adipose carbohydrate and lipid metabolism,\nmetabolic flexibility and the ability of a tissue to sustain thermogenesis. There are some issues with the\nFigures described below. One persistent criticism is the lack of quantification and statistical analysis of\nthe western blots throughout the manuscript.\nMajor\n1.Throughout the paper, the changes in RNA are analyzed statistically whereas the changes in\nprotein levels are not. In some cases, the changes in the blots appear to be convincing, whereas\nin others it is not very clear.\nReponse: We thank the reviewer for this helpful suggestion to improve our study. We have now\nincluded quantifications of protein levels for all in vitro and in vivo experiments.This strengthens the\nconsistency between RNA and protein data and provides statistical support for our conclusions.\n2.Fig 1b. The changes in ERRγ protein are hard to interpret. The protein seems to decrease and\nthen increase again. At day 7, it is not clear that the ERRγ protein is higher than baseline. As\nwith other figures, protein levels not quantified or analyzed statistically. This is important for\nPGC1α and ERRγ as it is questionable whether the induction at later timepoints (5d, 7d) is\nstatistically significant.\nReponse: We appreciate the reviewers’ comments and acknowledge the need to improve the strength\nand clarity of our conclusions. To directly address these concerns, we conducted the protein\nquantification to verify our findings (Extended Fig.1a). This analysis confirms the trends observed in the\nrepresentative blots and clarifies the changes at later time points.\n3.It is stated that there are “significant increases in the expression ..“ in GYs1, Gys2, Pygl, and\nAgl (Fig. 1b, c), but there are no statistics performed on the protein levels in Fig 1b. Some\ndescription of statistics or repeat experiments are needed.\nReponse: Thank you for the valuable suggestion. We have now performed statistical analyses of these\nprotein blots, and the results are presented in Extended Fig. 1a.\n4.Similar issues are noted with Extended Fig 4C, 4A, 6A, C and Fig 7.\nReponse: Thank you for the valuable suggestions. We have now added protein quantification results\nthroughout the manuscript.\n5.Fig 2 and extended 4b are done in preadipocytes. Given the focus on beiging, why are these\nsignaling pathways not evaluated in differentiated beige adipocytes (as done in extended figure\n4c,d)? Extended Figure 8 shows acute versus chronic effects, although it does not mention the\nspecific cell type. Is the rationale that they are determining acute priming in Fig. 2 and extended\n4b?\nReponse: We thank the reviewer for drawing our attention to this point and apologize for the lack of\nclarity in our original description. To clarify, the experiments in Fig. 2 and Extended Fig. 4b were\nperformed in primary adipocytes differentiated in vitro from SVF-derived preadipocytes, rather\nthan in undifferentiated precursors. We used this model because it provides a tractable system for\nshort-term manipulations (e.g., cycloheximide, pharmacological inhibitors, AAV-mediated CRISPR\ndelivery) and allowed us to define the acute, cell-autonomous signaling cascade through PKA–CREB\nthat primes Gys2 expression.\nIn contrast, the experiments in Extended Fig. 4c,d were conducted in differentiated beige adipocytes\nto address a distinct biological question—the sustained PGC1α-dependent maintenance of glycogen\nmetabolism gene expression under chronic adrenergic stimulation.\nThus, the choice of SVF-derived adipocytes in Fig. 2/Extended Fig. 4b versus beige adipocytes in\nExtended Fig. 4c,d reflects the different phases of regulation being interrogated: acute CREB-\ndependent induction (“priming”) versus chronic PGC1α–ERR–mediated maintenance. Extended Fig. 9\nsummarizes this conceptual framework, emphasizing the temporal distinction between acute and\nchronic regulation in white adipocytes browning.\n6.Fig 3b same issue as Figure 1. It is not clear that PYGL and AGL protein reduction by PGC1α\nknockout is significant.\nReponse: We thank the reviewer for the helpful comment. For protein level, AGL is significantly reduced\nin PGC1α-AKO mice, but not PYGL. Please see Extended Fig.3b.\n7.Figure 3F needs a scale bar. IHC methods are not described in detail. Why is PAS + UCP1\nzoomed out so much? It is impossible to discriminate between UCP1 (presumably brown?) and\nPAS at this scale. Perhaps this panel could be eliminated since UCP1 and PAS are assessed\nearlier in the figure.\nReponse: We thank the reviewer for these helpful suggestions. We have now included detailed IHC\nmethods in the Methods section. Scale bars have been added to Fig. 3f, as well as to Fig. 1c and Fig.\n4f for consistency. To improve clarity, we have also adjusted the PAS + UCP1 images in Fig. 3f to allow\nbetter visualization and discrimination of staining.\nMinor\n8.The mouse strain background of ERR mouse model and refs are not provided.\nReponse: Thanks for your suggestion. The detail of mouse strain background of the ERR mouse\nmodels has been added to the Methods section.\nReviewer #2 (Remarks to the Author):\nIn this study, the authors aim to elucidate the mechanism underlying the regulation of gene expression\nin adrenergic stimulation-induced beige fat cell activation. They observed that adrenergic stimulation\nupregulates the expression of glycogen metabolism genes in white adipocytes (WAT) and promotes\nWAT browning. Additionally, they proposed several mediators of this effect, including CREB, PGC1α,\nand ERRs. Using multiple gene knockout mouse models, the authors demonstrated the essential role\nof PGC1α and ERRs in mediating the effect of CL (clenbuterol) on promoting glycogen gene expression\nand WAT browning. This study is a follow-up to their previous research highlighting the importance of\nglycogen metabolism in beige fat. However, the current study lacks novel insights, fails to clarify\nsignificant gene regulatory mechanisms, and lacks rigorous validation of the proposed transcriptional\nregulatory pathways.\nMajor Concerns:\nLimitation of adrenergic agonists and mechanistic clarity of CL action\nOnly CL was used as an agonist for adrenergic stimulation of WAT; other adrenergic agonists\nshould be included for validation. The mechanism by which CL acts on white adipocytes in vivo\nand in vitro remains unclear:\n1.Is there involvement of adrenergic receptors?\nResponse: Thank you for drawing our attention to this misunderstanding. CL (CL-316,243 ) is not\nclenbuterol, but rather a b-3 specific adrenergic agonist widely used in metabolic studies to target\nadipocytes. . Clenbuterol is a non-selective β2-adrenergic receptor agonist (with some β1 activity) that\nacts broadly on skeletal muscle, heart, and airway smooth muscle. In contrast, CL-316,243 is a highly\nselective β3-adrenergic receptor agonist that has been extensively validated to act through β3 receptors\nin adipocytes. We use CL in these experiments because it is β3-specific, and these receptors are not\nexpressed on any other cells, with the possible exception of bladder, which is irrelevant. Moreover, β3-\nreceptors are expressed at levels 1000-fold higher than β1 or β2 in mouse adipocytes. Consistent with\nthis, our data show that CL rapidly activates canonical β3-adrenergic signaling, including cAMP–PKA–\nCREB phosphorylation (Fig. 2 and Extended Fig. 4b). These points are well established and justify the\nuse of CL as a fat cell specific agonist, as has been done in thousands of papers. Inhibition of PKA or\nCREB effectively blocks induction of Gys2, demonstrating that CL acts through the expected β3–\ncAMP–PKA–CREB axis. Thus, the mechanistic involvement of adrenergic receptors is well supported\nby both the pharmacology of CL and the downstream signaling responses we observe. While we did\nnot include additional agonists in this study, numerous prior reports have shown that isoproterenol and\nnorepinephrine elicit similar transcriptional responses in adipocytes via β3 adrenergic receptors.\n2.Are there mechanistic differences in glycogen metabolism gene expression between in vivo\nand in vitro models, given the distinct time scales observed post-CL treatment?\nResponse: Both in vivo and in vitro studies demonstrate that β3 adrenergic signaling cell-autonomously\ninduces glycogen metabolism genes. Our goal here was to use these systems to capture distinct\nphases of the response. In vitro, acute CL treatment rapidly induces Gys2 and Ppp1r3c within hours,\nreflecting direct PKA–CREB–dependent priming. In vivo, chronic adrenergic stimulation expands the\nprogram to include Pygl, Agl, which requires sustained activation of the PGC1α–ERR axis and\nchromatin remodeling. This layered regulation explains the different time scales: in vitro assays reveal\nacute cell-autonomous induction, whereas in vivo studies capture the prolonged, transcriptionally\nmaintained response under sustained adrenergic tone and systemic inputs. We have revised the text\nto clarify this temporal distinction (summarized in Extended Fig. 9).\n3.Insufficient evidence for \"autonomous gene regulation\"\nThe authors claim that CL treatment induces autonomous gene regulation, but this conclusion\nis not supported, as it was only tested in in vitro-cultured preadipocytes. For claims of chronic\nCL action via autonomous regulation, indirect effects (e.g., via other cell types in the stromal\nvascular fraction) in in vivo models must be excluded. To conclusively demonstrate the\nrequirement for adipocyte-intrinsic expression of key genes (e.g., Gys2) in mediating\nbrowning/glycogen metabolic responses, adipocyte-specific knockout or knockdown models\n(e.g., using Adipoq-Cre) targeting Gys2 should be employed. These models should be used to\nassess the impact on CL-induced glycogen gene expression, UCP1 induction, and actual\nglycogen metabolism in inguinal WAT (iWAT).\nResponse: We thank the reviewer for raising this important point. To clarify, our in vitro experiments\nwere performed in primary adipocytes differentiated from stromal vascular fraction (SVF) cells, rather\nthan undifferentiated preadipocytes. We have corrected this terminology throughout the manuscript.\nThe goal of these experiments was to demonstrate that CL treatment can directly induce glycogen\nmetabolism gene expression in adipocytes in the absence of other stromal vascular cell types.\nImportantly, in our previous work pubulished in Nature (Keinan et al.,2021, PMID: 34707293), we\nreported that adipocyte-specific knockout of PTG (protein targeting to glycogen) markedly reduces\nglycogen storage and blunts thermogenic and browning responses, providing additional evidence for\nadipocyte-autonomous regulation of glycogen metabolism. Published studies have already\ndemonstrated the functional relevance of adipocyte-intrinsic glycogen metabolism in vivo. Furthermore,\nadipocyte-specific deletion of GYS1 (Adipoq-Cre; Gys1 fl/fl) has been shown to abolish CL- or cold-\ninduced glycogen accumulation and UCP1 upregulation in iWAT, confirming that glycogen synthesis in\nadipocytes is required for adaptive thermogenesis (Zhuo S et al., 2024 PMID: 38568151). And as\nmentioned above, the use of CL either in vitro or in vivo activates only adipocytes, as preadipocytes,\nimmune cells, fibroblasts or any other resident cells in adipose tissue do not express b-3 adrenergic\nreceptors. The combination of our current in vitro data together with published GYS1 and PTG knockout\nstudies collectively support the conclusion that glycogen metabolism in adipocytes is regulated in a\ncell-autonomous manner.\n4.Incomplete analysis of signaling pathways and post-translational regulation\nThe authors showed that inhibitors of PKA (PKAi), p38 (p38i), and CREB (CREBi) block CL-\ninduced Ucp1 and Gys2 expression, but data for other genes are missing. Additionally:\nWhen CREB is knocked down via sgRNA, how do global gene expression profiles change?\nResponse:\nWe thank the reviewer for these thoughtful points. In Fig. 2c, we highlighted Ucp1 and Gys2 as\nrepresentative CREB-dependent targets to illustrate the parallel regulation of a thermogenic gene and\na glycogen metabolic gene. In Extended Fig. 1b, we show that treatment of primary preadipocytes\ndifferentiated in vitro with CL selectively affected Gys2 and Ppp1r3c expression. We therefore focused\nour analyses on Gys2 and Ppp1r3c. In inhibitor-treated experiments, we observed a reduction in Gys2\nexpression but not in Ppp1r3c. For this reason, we present and conclude specifically on Gys2 rather\nthan other glycogen metabolism genes.\nOur CRISPR–Cas9–mediated CREB knockdown experiments (Fig. 2e-h) were designed to test the\nrequirement of CREB for Gys2 induction and glycogen accumulation in adipocytes. We did not perform\nRNA-seq in this study and thus cannot comprehensively report global changes. However, prior studies\nhave established CREB as a broad regulator of adipocyte differentiation and metabolic gene expression\n(Reusch et al., 2000, PMID: 10629058; Hossain et al., 2020, PMID: 31401979). Based on these reports\nand our findings, we anticipate that CREB knockdown would impair not only Gys2 but also subsets of\ngenes involved in thermogenesis, lipid metabolism, and cAMP-responsive transcription.\n5.Given the claim of autonomous regulation, is there a change in GSK3 phosphorylation status?\nResponse: We thank the reviewer for these thoughtful points. We blotted GSK3α/β Ser21/Ser9\nphosphorylation in CL treatment primary adipocyte differentiated in vitro, showing no differences of\nGSK3 and p-GSK GSK3α/β levels (For reviewer only, see below). Because GSK3 regulation is\nclassically linked to the insulin–AKT axis that modulates glycogen synthase activity post-translationally,\nwhereas our data map an insulin-independent, PKA–CREB–driven transcriptional program. Consistent\nwith this, all acute in vitro assays were performed in serum-free medium, minimizing\ninsulin/AKT inputs. Because these data are not directly related to the questions under investigation, we\nhave not included them in the paper, but can do so if requested.\n6.Are glycogen synthase or other related enzymes regulated via post-translational\nmodifications (e.g., phosphorylation)?\nResponse: We thank the reviewer for this insightful question. Yes, glycogen synthase (both Gys1 and\nGys2) is well known to be regulated by post-translational modifications, most prominently\nphosphorylation. Multiple kinases including GSK3, AMPK, and PKA phosphorylate glycogen synthase\nat distinct serine residues, leading to its inactivation. Conversely, dephosphorylation by protein\nphosphatase 1 (PP1) restores glycogen synthase activity. In addition, glycogen metabolism is tightly\ncoordinated with other enzymes through post-translational regulation: glycogen phosphorylase (PYGL)\nis activated by phosphorylation via phosphorylase kinase, while protein targeting to glycogen (PTG)\nfunctions as a regulatory subunit that recruits PP1 to glycogen particles to promote dephosphorylation\nof both glycogen synthase and glycogen phosphorylase. Together, these mechanisms ensure rapid,\nreversible control of glycogen turnover in response to hormonal and nutrient signals. These effects are\nwell established and have been the focus on our investigations going back 30 years.\nIn our current study, we have focused primarily on transcriptional regulation of glycogen metabolic\ngenes rather than post-translational regulation. However, we now include discussion of these well-\nestablished post-translational mechanisms in the revised manuscript to provide a more complete\ncontext for how glycogen metabolism is regulated in adipocytes (Line 325-333).\n7.Unclear role and regulation of PGC1α\nPGC1α is a key regulator of multiple intracellular metabolic pathways. Critical questions remain:\nIs PGC1α responsive to CL treatment in white adipocytes? If so, is this response via changes\nin expression or post-translational modification?\nResponse: In white adipocytes, PGC-1α is responsive to β3-adrenergic stimulation by CL316,243, as\nhas been widely reported by many groups. At the transcriptional level, acute CL treatment elevates\nPppargc1a mRNA in white adipose depots as shown in Extended Fig.5a. in parallel with thermogenic\ngene induction (e.g., Ucp1).\nIn addition, PGC-1α activity is enhanced by post-translational modifications downstream of\nadrenergic/cAMP signaling. Specifically, p38 MAPK and AMPK phosphorylate PGC-1α to increase its\nstability and coactivator function(Fan et al.,2004 PMID: 14744933), and SIRT1 deacetylation further\npotentiates its activity—mechanisms engaged by β-adrenergic cues and metabolic stress(Rodgers et\nal., 2007 PMID: 18036349).\nTogether, these data support a model in which CL316,243 activates PGC-1α in white adipocytes via\nboth increased expression and activation through PTMs (phosphorylation and deacetylation),\nthereby\npromoting thermogenic and oxidative programs.\n8.How does PGC1α respond to CL or other chronic adrenergic stimuli? Is this response\nindependent of PKA, p38, or CREB?\nResponse: We thank the reviewer for this thoughtful question. PGC-1α is robustly induced by chronic\nβ-adrenergic stimulation (e.g., CL316,243 or cold ) with slower kinetics than acute phosphorylation\nevents, and is required to sustain long-term expression of thermogenic and metabolic genes.\nMechanistically, PGC-1α operates downstream of canonical adrenergic pathways rather than\nindependently: CREB directly activates Ppargc1a transcription following β-adrenergic/PKA signaling\n(Herzig et al., Nature 2001; PMID: 11557984), while p38 MAPK phosphorylates and stabilizes PGC-1α\nto enhance its coactivator function (Puigserver et al., Mol Cell 2001; PMID: 11741533). Consistent with\nthis, adipose tissue exhibits delayed but sustained induction of PGC-1α in response to cold/β-\nadrenergic cues (Uldry et al., Cell Metab 2006; PMID: 16679291; Wu et al., Cell 1999; PMID:\n10412986).\nTogether with our differentiated adipocyte data, these studies support a model in which chronic\nadrenergic inputs engage PKA/CREB and p38 to elevate PGC-1α expression/activity and, in parallel,\ndrive adipocyte-intrinsic glycogen programs that sustain thermogenic gene expression.\n9.In adipocytes, does PGC1α knockout reduce mitochondrial content, and how would this affect\nlipidolysis in response to CL?\nResponse: We thank the reviewer for pointing this out. Consistent with prior studies, adipocyte-specific\ndeletion of PGC-1α results in impaired metabolic and mitochondrial gene signatures in adipose tissue\n(Kleiner et al., Cell Metab 2012; PMID: 22645355). In addition, review articles highlight the necessity\nof PGC-1α in brown and beige adipose development and oxidative capacity (Wang & Seale, Nat Rev\nMol Cell Biol 2016; PMID: 27552974). In our study, PGC-1α knockout diminished CL-induced\nexpression of mitochondrial and thermogenic genes (Ucp1, Cidea) and reduced glycogen turnover,\nconsistent with impaired oxidative remodeling (Fig. 3).\nImportantly, however, acute lipolysis in response to CL is largely preserved in PGC-1α–deficient\nadipocytes. Extended Data Fig. 4b shows that phosphorylation of hormone-sensitive lipase (HSL) at\nSer563 and Ser660 occurs normally in PGC-1α knockout cells following CL stimulation, indicating that\nadrenergic signaling to lipolysis remains intact. Thus, the immediate mobilization of triglyceride stores\nremains functional despite reduced mitochondrial biogenesis/function.\n10.ATAC-seq and gene expression analyses suggest PGC1α directly binds and regulates\nglycogen genes, but this is not reproducible in in vitro-cultured adipocytes (Extended Data Fig.\n4a), in contrast to CREB. This discrepancy must be explained, along with how PGC1α and CREB\ncooperate, which acts as the primary mediator of CL effects, and the underlying mechanism.\nResponse: We thank the reviewer for these thoughtful comments and would like to clarify a point of\ninterpretation.\nFirst, our ATAC-seq and gene expression analyses indicate that ERR shows direct binding to and\nregulation of glycogen metabolic genes. PGC-1α functions primarily as a transcriptional co-activator\nthat does not bind DNA directly, but instead interacts with sequence-specific transcription factors (\nsuch\nas ERRs and PPARs) to potentiate their transcriptional activity. Thus, the statement that “PGC-1α\ndirectly binds and regulates glycogen genes” is not accurate.\nSecond, in our dataset (Extended Data Fig. 4a), the apparent discrepancy reflects differences in\nregulatory context. CREB exhibits clear functional necessity for Gys2 induction, whereas PGC-1α\nactivity is more context-dependent, especially in beige adipocytes rather than in white adipocytes. This\nexplains why only subtle differences in glycogen gene expression are observed in primary adipocytes\ndifferentiated from PGC-1α knockout SVF. This likely reflects that PGC-1α regulation of glycogen\nmetabolism requires additional in vivo cues and is preferentially engaged in beige adipocyte programs.\nTaken together, we propose that CREB acts as the primary mediator of CL effects on glycogen\nmetabolism by directly binding and activating Gys2, while PGC-1α functions as a co-activator in specific\ncontexts, coordinating with ERRs to sustain the expression of thermogenic and glycogen metabolism\ngenes.\n11.Mechanistic basis of ERRs’ role\nThe authors show that ERRs are required to mediate CL effects, but key questions persist:\nHow do ERRs respond to CL? Is this response autonomous?\nResponse: We thank the reviewer for pointing this out. ERRs do not respond to CL in an autonomous\nmanner. In our system, we find that acute CL treatment does not alter Esrra and Esrrg expression in 6\nhours whereas chronic CL stimulation leads to increased Esrra expression, consistent with regulation\nby secondary transcriptional programs engaged downstream of β3-adrenergic signaling. CL induces\nPgc-1α, which functions as a potent co-activator of ERRs. Together, these mechanisms explain how\nERR transcriptional output is enhanced following CL treatment: ERRs are not direct receptors for CL,\nbut their expression and co-activator availability are regulated in a way that amplifies their function\nduring sustained adrenergic stimulation.\n12.Is ERR activation dependent on PKA, p38, CREB, or PGC1α? If dependencies exist, what is\nthe underlying mechanism?\nResponse: We thank the reviewer for these thoughtful points. ERR activation is largely indirect and\ndepends on upstream adrenergic signaling and co-activator availability. ERRs are constitutively DNA-\nbound but require co-activators such as PGC-1α to enhance transcriptional output. CL stimulation\nengages the β3-cAMP-PKA-CREB cascade, which acutely induces Ppargc1α PGC-1α then interacts\nwith ERRs to stimulate glycogen metabolic and thermogenic gene expression.\n13.Lack of detailed analysis of transcriptional regulation\nTo support claims of direct regulation of glycogen metabolism genes by CREB, PGC1α, or ERRs,\ndetailed investigations are needed, including:\nBinding motifs and nuclear translocation of these factors.\nMutational assays of binding motifs.\nKinetics (acute vs. chronic) of regulation.\nCooperative interactions between factors and their physical associations.\nInvolvement of epigenetic markers.\nThese analyses should be validated using multiple stimuli combined with pathway inhibitors.\nResponse: We thank the reviewer for raising this important point regarding the mechanistic depth of\ntranscriptional regulation. Our study directly addresses several of these concerns:\n1) Binding motifs and mutational assays. We performed luciferase reporter assays using the Gys2\npromoter in HEK293T cells, as differentiated adipocytes are technically challenging to transfect.\nImportantly, deletion of the half-CRE binding site within the Gys2 promoter markedly reduced luciferase\nactivity, demonstrating that CREB directly engages this motif to activate transcription.\n2) Direct factor binding. Our ChIP-seq analyses revealed strong ERRα occupancy at glycogen\nmetabolism gene promoters. In ERRα-AKO tissues, newly performed ChIP-qPCR experiments showed\nincreased ERRγ binding at the same promoter regions, providing evidence of compensatory\nrecruitment of ERRγ when ERRα is absent (Extended Data Fig. 8a).\n3) Cooperative interactions with PGC1α. The ERRγ binding sites coincide with the regions where\nPGC1α ChIP-qPCR shows enrichment. Since PGC1α lacks intrinsic DNA-binding capacity, its function\nmust be mediated through association with ERRα/ERRγ. These findings establish a cooperative\nmechanism in which ERRα/ERRγ provide DNA anchoring while PGC1α delivers co-activator function,\nexplaining the sustained regulation of glycogen metabolism genes during chronic adrenergic\nstimulation.\n4) Kinetics of regulation (acute vs. chronic). Our datasets delineate a temporal division of labor: CREB\nis required for the acute induction of glycogen metabolism genes, while the ERR/PGC1α axis is\nindispensable for maintaining their expression under chronic stimulation.\n5) Additional considerations. We agree with the reviewer that further studies, such as inhibitor-based\ndissection and involvement of epigenetic markers, could provide additional layers of mechanistic detail.\nHowever, these questions are beyond the scope of the present manuscript, which is focused on defining\nthe transcriptional architecture linking adrenergic signaling to glycogen metabolism.\nTogether, these results provide direct and functional evidence that CREB, PGC-1α, and ERRs regulate\nglycogen metabolism genes, with ERRγ compensating for ERRα loss and cooperating with PGC1α to\nsustain gene expression.\n14.Incomplete validation of ERR redundancy\nTriple-knockout (ERRs-AKO) data support functional redundancy of ERRs, but overexpression\nstudies (Extended Data Fig. 7) show individual ERRs can induce gene expression. To rigorously\ntest redundancy, rescue experiments in ERRs-AKO adipocytes should be performed: re-express\nERRα, ERRγ, or PGC1α individually or in combination, and determine if this restores CL-induced\nglycogen gene expression and glycogen accumulation.\nResponse: We thank the reviewer for raising this important point. Our triple-knockout (ERRs-AKO)\ndata indicate functional redundancy among ERR isoforms, whereas our overexpression experiments\n(Extended Data Fig. 8b) show that individual ERRs can modestly enhance glycogen metabolism gene\nexpression when expressed in isolation, although the effects are not robust (see quantifications).\nThese\noverexpression assays were performed in beige adipocytes, a context in which endogenous ERRα,\nERRγ, and PGC1α levels are already higher than in white adipocytes. Thus, additional overexpression\nis sufficient to further boost transcriptional output. Consistent with this, our Western blot analyses show\na trend toward increased glycogen metabolism protein expression. We also acknowledge that these\neffects could in part be indirect, as overexpression of ERRs or PGC1α may also influence other\ntranscriptional networks that secondarily enhance glycogen metabolism gene expression.\nTo further address redundancy, we performed additional ChIP-qPCR analyses in ERRα-AKO tissues.\nThese revealed increased ERRγ occupancy at promoters of glycogen metabolism genes, precisely at\nthe same regions where PGC1α shows enrichment. Since PGC1α lacks intrinsic DNA-binding capacity,\nthis finding supports a model in which ERRγ compensates for ERRα loss and recruits PGC1α to sustain\ntranscriptional activation (Extended Data Fig. 8a).\nWe agree that re-expression (“rescue”) studies in ERRs-AKO adipocytes—introducing ERRα, ERRγ,\nor PGC1α individually or in combination and assessing restoration of CL-induced glycogen gene\nexpression and glycogen accumulation—would provide rigorous evidence to dissect redundancy and\nspecificity. At present, these technically demanding experiments fall outside the scope of this\nmanuscript. Instead, we relied on the complementary strengths of knockout, overexpression, and ChIP-\nbased binding analyses to establish both the redundancy and sufficiency of ERR activity in regulating\nglycogen metabolism. We fully acknowledge the value of the proposed rescue strategy and view it as\nan important direction for future studies.\n15.Missing evidence for PGC1α-ERR complex formation\nThe model proposes that PGC1α cooperates with ERRα/γ, implying physical interaction. Direct\nevidence of complex formation is required, such as co-immunoprecipitation (Co-IP)\nexperiments in CL-treated primary adipocytes or iWAT lysates using antibodies against PGC1α,\nERRα, and ERRγ. Reciprocal Co-IPs (e.g., immunoprecipitating PGC1α followed by\nimmunoblotting for ERRγ, and vice versa) are essential to confirm these interactions in vivo\nunder relevant stimuli.\nResponse: We thank the reviewer for this important suggestion. Prior literature provides solid\nprecedent for physical and functional interaction between PGC-1α and ERR family members. For\nexample, Huss et al. (J Biol Chem 2002; PMID: 12181319) showed that PGC-1α physically interacts\nwith ERRα and ERRγ and identified interaction motifs. Schreiber et al. (J Biol Chem 2003; PMID:\n12522104) further demonstrate that PGC-1α regulates expression and activity of ERRα and can\ncoactivate ERR target promoters. Moreover, Gantner et al. (Endocrinology 2016; PMID: 27763777)\nprovide in vivo evidence in adipose tissue that ERRα and ERRγ have redundant roles: deletion of one\nisoform leads to compensatory changes in the other, with partial preservation of mitochondrial/adaptive\nresponses.\nIn our current work, however, we do not yet have data showing direct endogenous complex formation\nbetween PGC-1α and ERRα/γ in CL-treated primary adipocytes or iWAT via reciprocal Co-IP. We\nacknowledge this as a limitation and suggest that such experiments would strengthen the model. We\nhave revised the manuscript to explicitly reflect that our model is supported by these prior observations,\nbut that direct interaction under adrenergic stimulation in adipose remains to be demonstrated.\n16.Absence of a working model\nThe authors should provide a diagrammatic working model to summarize their conclusions.\nResponse: Thank you for pointing this out. We have made some minor changes of orignal working\nmodel, which is now included in Extended Fig.9\nMinor Concerns:\n17.Fig. 1a: Poor consistency between duplicate samples, particularly under the \"Cold\" condition.\nResponse: We thank the reviewer for this pointing this out. Fig. 1a was included as supportive data from\nan independent dataset (adapted from GSE86338), and we agree that the duplicates under the cold\ncondition show variability. However, our main conclusions are based on our own CL-treatment\nexperiments, which consistently validate the findings. We therefore view Fig. 1a as complementary\nevidence, and the observed variability does not affect the overall interpretation.\n18. Fig. 1c: The dramatic induction of Agl at day 1 contradicts the authors’ description.\nResponse: We thank the reviewer for the helpful comment. We have revised the text to remove this\npoint and to ensure consistency between the data presentation and our interpretation.\n19.Fig. 1c and Fig. 1d: Inconsistent trends/quantification (y-axis scales) for Gys1, Pygl, and\nPpp1r3c expression.\nResponse: We thank the reviewer for this comment. The variability in the apparent trends reflects both\nbiological differences among individual samples and the relative nature of the quantification. When the\nreference sample shows unusually high or low expression, this can exaggerate relative fold changes,\nalthough the underlying absolute expression values remain consistent. Importantly, these differences\ndo not affect the overall interpretation of our results.\n20.Extended Data Fig. 1a: To claim \"cell autonomy,\" additional treatments (e.g., glucagon, cAMP)\nshould be tested. In the right panel, many glycogen metabolism genes do not respond to CL,\nconflicting with mouse data; extend in vitro CL treatment time courses in differentiated\nadipocytes to check if Pygl and Agl induction is delayed but intrinsic.\nResponse: We thank the reviewer for the helpful suggestions. In this study, we focused on β3-\nadrenergic stimulation of thermogenesis using CL, as it is a well-established and specific agonist for\nadipocyte β3-adrenergic receptors and reliably elicits cell-autonomous responses. We also conduct\nexperiment for cAMP treatment in primary preadipocyte differentiated in vitro, Gys2 and Ppp1r3c, are\nstill increased (Extended Fig.1c). The lack of Pygl and Agl induction in short-term in vitro assays likely\nreflects differences from the more sustained and complex signaling in vivo. We agree that extending\ntreatment time courses may reveal delayed, cell-intrinsic responses, and we will pursue this in future\nwork.\n21.Inconsistencies between Extended Data Fig. 1a and Fig. 2b: Gys2 shows mild upregulation\nat 2 h, 3-fold at 4 h (Extended Data Fig. 1a), but ~4-fold in Fig. 2b; similar issues exist for Ppp1r3c.\nThese inconsistencies, combined with the mild response at 3 h, challenge the claim that de\nnovo protein synthesis is unnecessary. A cycloheximide (CHX) treatment time course (2–8 h) is\nneeded to validate this.\nResponse: We thank the reviewer for carefully pointing out these differences. The variation in fold\nchange for Gys2 and Ppp1r3c between Extended Data Fig. 1a (now Extended Data Fig. 1b) and Fig.\n2b (now Fig. 2a) arises from the fact that these experiments were performed in separate batches of\nprimary adipocytes, with distinct baseline expression levels and normalization references. Despite\nquantitative differences, the qualitative pattern of early induction after CL treatment is reproducible\nacross experiments. We agree with the reviewer that a cycloheximide (CHX) time-course (2–8 h) would\nprovide a more definitive test of whether de novo protein synthesis is required. While such an\nexperiment is beyond the scope of the current study, our data—showing transcriptional induction within\n2–4 h and sensitivity to signaling inhibitors—support the interpretation that Gys2 regulation occurs\nlargely independent of new protein synthesis. We have revised the text to make this point clearer and\nto acknowledge this limitation.\n22.Extended Data Fig. 1: Adipocyte specificity is claimed via comparison to hepatocytes, but\nother tissues (e.g., skeletal muscle, heart) also express glycogen metabolism genes. Analyze\ngene expression kinetics in these tissues from the same CL-treated mice used for the iWAT time\ncourse to establish uniqueness.\nResponse: We thank the reviewer for this helpful comment. In Extended Data Fig. 1, we used\nhepatocytes as a comparator because the liver is a major glycogen-metabolic organ, providing a\nphysiologically relevant reference for systemic regulation. Although skeletal muscle and heart also\nexpress glycogen metabolism genes, they are not direct targets of CL treatment. CL is a highly selective\nβ3 adrenergic receptor (β3AR) agonist, and β3AR expression is largely restricted to adipocytes, with\nminimal or absent expression in other tissues. Thus, the effects we observe following CL administration\npredominantly reflect adipocyte-intrinsic signaling rather than systemic effects on other glycogen-\nmetabolizing tissues.\n23.Fig. 2c: UCP1 shows a 30-fold increase at 6 h CL treatment, inconsistent with the >100-fold\nincrease in Extended Data Fig. 1a; a similar discrepancy exists for Gys2.\nResponse: We thank the reviewer for this thoughtful comment. The apparent discrepancy in fold\ninduction between Fig. 2c (now Fig. 2b) and Extended Data Fig. 1a (now Extended Data Fig. 1b)(e.g.,\nUcp1 and Gys2) arises because the experiments were performed in independent cohorts of primary\nadipocytes, each with distinct basal expression levels and normalization references. Since qPCR\nresults are reported as relative fold changes, variation in baseline control values can influence the\napparent magnitude of induction. Importantly, in all cases the direction and kinetics of regulation are\nconsistent, with both datasets showing robust induction of Ucp1 and Gys2 following CL treatment. We\nhave revised the text to clarify this point and to emphasize that, despite differences in fold-change\nmagnitude, the biological conclusion is unchanged.\n24.Fig. 3g: Negative controls (without CL treatment) are missing.\nResponse: We thank the reviewer for this comment. In this experiment, our focus was to evaluate the\nrequirement of PGC-1α for CL-induced responses in beige adipocytes rather than its role in basal\nregulation in white adipocytes. As shown in our other datasets, PGC-1α deletion does not affect\nglycogen synthesis gene expression under basal conditions, and thus the critical comparison here is\nbetween control and PGC-1α–deficient cells in the presence of CL stimulation. For this reason, non–\nCL-treated samples were not included in Fig. 3g, as they do not alter the interpretation of the specific\nquestion being addressed. However, they are included elsewhere throughout the manuscript.\n25.Fig. 3h: Enrichment fold changes are overly modest.\nResponse: We thank the reviewer for this comment. It is true that the enrichment fold changes in Fig.\n3h appear modest; however, this is a common feature of ChIP–qPCR analyses, where relative fold\nchanges are typically small compared to transcriptomic readouts. In our case, this is further expected\nbecause PGC-1α functions as a cofactor rather than a sequence-specific DNA-binding factor, and\nadditional regulators also contribute to glycogen metabolic gene control. Despite the modest amplitude,\nthe enrichment is reproducible and statistically significant across biological replicates, which supports\nthe biological relevance of PGC-1α occupancy at these loci.\n26.Extended Data Fig. 4b: The authors claim CL induces acute (p38-dependent) and chronic\n(PGC1α-dependent) effects. For the chronic effect, test in vitro models and determine if de novo\nprotein synthesis is required.\nResponse: We thank the reviewer for this comment and would like to clarify a point of interpretation.\nWe did not state that the acute induction of glycogen metabolism genes by CL is p38-dependent; rather,\nour data support a rapid phase of glycogen gene regulation and a more sustained, PGC-1α–dependent\nphase. Importantly, chronic effects of CL are best captured in vivo, as long-term adrenergic stimulation\ninvolves systemic and tissue-level cues that are difficult to model accurately in vitro. Our current\ncombination of in vivo time courses and genetic perturbation models is therefore sufficient to distinguish\nacute versus chronic regulation. While cycloheximide treatment in cell culture could provide additional\ninformation, such experiments do not fully recapitulate the chronic setting, and we believe our present\ndata adequately support the conclusion.\n27.Extended Data Fig. 4: PGC1α is dispensable for acute (6 h) induction but essential for\nsustained expression during chronic stimulation. Define the kinetics of PGC1α mRNA and\nprotein induction in iWAT/primary adipocytes to correlate with its functional requirement.\nResponse: We agree with the reviewer’s interpretation that PGC-1α is dispensable for the acute (6 h)\ninduction but required for sustained expression during chronic stimulation. In our study, we prioritized\nfunctional genetics (PGC-1α loss-of-function) to separate these phases, rather than a full kinetic\nprofiling of PGC-1α abundance. Two points are important for interpreting kinetics versus function: (i)\nPGC-1α is a co-activator whose activity is strongly regulated by post-translational mechanisms (e.g.,\nphosphorylation/deacetylation); therefore, activity can change without large changes in bulk protein or\nmRNA at early time points, consistent with its acute dispensability. (ii) During chronic adrenergic\nstimulation, both co-activator activity and expression increase in adipose tissue, aligning with the\nsustained requirement we observe.\nOur existing datasets (acute vs chronic CL) already capture the functional transition that motivates the\nmodel. We believe this mechanistic rationale in the text and noted that a high-resolution kinetic series\n(iWAT and primary adipocytes, coupled to activity readouts) would be an informative future extension,\nbut is beyond the scope of the present manuscript.\n28.Extended Data Fig. 6a: ERRα-AKO mice show no increase in ERRγ under vehicle conditions,\nconflicting with Extended Data Fig. 6c (0 day); GS shows the opposite trend.\nResponse: We appreciate the reviewer’s careful attention. The differences between Extended Data\nFig. 6a and 6c (Now Extended Fig.7a and 7e) reflect technical and normalization factors rather than\ntrue biological inconsistency (Extended Fig.7b). We agree that ERRγ shows increased the mRNA\nexpression under basal conditions in ERRα-AKO mice, although the protein level differences are\nmodest. In contrast, GS protein levels do not display a clear increase after normalization and\nquantification. Importantly, our main focus is on beige adipocytes, where coactivators such as PGC1α\nare more abundantly expressed and cooperate with ERR factors to sustain glycogen metabolism gene\nregulation.\n29.Compensatory upregulation of ERRγ in mouse ERRα-KO models: Test if this mechanism\nexists in human primary adipocytes.\nResponse: We thank the reviewer for this thoughtful suggestion. Our finding of compensatory Esrrg\nupregulation in ERRα-deficient mice highlights isoform redundancy within the ERR family in adipocytes.\nWhether a similar mechanism operates in human adipocytes is indeed an important question. However,\ndirect genetic ablation models are not readily feasible in human primary adipocytes, and our study was\ndesigned to focus on the in vivo mouse system where genetic perturbation is tractable. We therefore\ncannot directly address this point here. We have clarified this limitation in the Discussion and noted that\ninvestigating isoform compensation in human adipocytes will be an important direction for future\nresearch(Line314-324).\nReviewer #4 (Remarks to the Author):\nThis manuscript described important new findings of a role for catecholamine-stimulated glycogen\nmetabolism during the process of increasing uncoupled respiration and nonshivering thermogenesis.\nThe work employs mouse models and cells in culture and there is a nice addition (Ext data 2) and\nconnection to human physiology. The findings show the major roles of transcription factors and\ncoregulators in the control of a range of genes involved in glycogen synthesis and metabolism. For\nexample, Fig 3 shows the role of PGC1α in the transcription of glycogen synthase genes; but this is\ncomplemented by experiments showing increased glycogen production both biochemically (e) and\nhistologically (f). This new finding is a significant addition to the literature so that we continue to develop\na better physiological understanding of this important adaptive metabolic process. The manuscript is\ngenerally well written with a very nice Discussion. There are some unexplained as well as discordant\nfindings that require further experimentation and discussion and these are noted below.\nSpecific Comments:\n1. Fig 1. GS and related proteins in inguinal white adipose tissue don’t increase until day 3 of\nCL txt, understandable in the sense that these ‘beige’ adipocytes appear in patches in the tissue,\nand when one homogenizes the whole tissue these proteins/genes are diluted out. So it would\nbe nice to add some histology of these tissues at the various time points – as was done in Figure\n3. It would help the reader better understand the temporal and spatial behavior.\nReponse: We thank the reviewer for this valuable suggestion. We have now added PAS staining of\niWAT across the CL treatment time course to Fig. 1, which provides histological evidence for the\ntemporal and spatial dynamics of glycogen accumulation during beiging.\n2. Fig 2. Please make clear in the legend that what is being treated and examined are\n‘differentiated’ adipocytes from preadipocytes. As written, it says treating ‘preadipocytes’.\nReponse: We thank the reviewer for pointing this out. We have corrected the terminology throughout\nthe manuscript and figure legends to specify that the experiments were performed in primary\npreadipocytes differentiated in vitro, rather than in undifferentiated preadipocytes.\n3. Fig 2a: There is clearly an inhibitory effect of the cycloheximide treatment on Ucp1 mRNA,\nyet nothing is said about it. This implies that a (presumably) short-lived protein is required to\nbe synthesized that is very important for the transcription of this gene. It is quite striking and at\nleast for this reviewer, cannot find in the literature any previous mention of such a result or\nexplanation for it. So either address this finding or just remove panels a and b: the rest of the\nFigure clearly stands on its own.\nReponse: We thank the reviewer for this valuable suggestion. To avoid potential misinterpretation, we\nhave removed the Ucp1 data from Fig. 2 and now focus on the regulation of Gys2, which more directly\nsupports our conclusions.\n4. Fig 2g: Why did the authors focus on CREB and not examine other factors previously shown\nto be more important for transcription of Ucp1 and Ppargc1a genes: ATF2. Is the CREB sgRNA\nspecific to the Creb gene or might it also be affecting ATFs? Data to confirm this is important.\nReponse: We thank the reviewer for drawing our attention to this important point. The sgRNA sequence\nwe used has no mismatch with the ATF2 genomic DNA. In addition, we assessed Atf2 expression in\nCREB sgRNA–treated adipocytes and found no obvious differences in Atf2 mRNA levels compared\nwith control, suggesting that the sgRNA does not inadvertently target the ATF2 gene. At the same time,\nwe acknowledge that ATF2 may also contribute to the regulation of glycogen metabolism genes. Indeed,\nGys2 mRNA is decreased in the presence of a p38 inhibitor during CL treatment (Fig. 2c), consistent\nwith the idea that ATF2, as a downstream effector of p38 MAPK, could participate in Gys2 transcription.\nWhile our data establish CREB as a direct regulator of Gys2, we cannot rule out a potential role for\nATF2, and we have revised the Discussion to note this as an additional regulatory pathway that\nwarrants further investigation (Line283-287).\n5. Ext data 4 regarding Pgc1α adipose KO data: given the established literature of a primary role\nfor PGC1α being required for Ucp1 gene transcription, which in these data is minimally affected,\nis PGC1β compensating for this complete loss of Pgc1α expression? This seems plausible\ngiven the more acute treatment with the inhibitor shows the role of PGC1α dependence. Please\nshow protein levels of PGC1α and -1β in these tissues and provide some discussion for the\nfindings.\nReponse: We thank the reviewer for this thoughtful suggestion. We examined PGC-1β levels in iWAT\nfrom PGC1α-AKO mice, both at the RNA and protein level, and found that PGC-1β expression was\ncomparable across genotypes (Fig.3b, Extended Fig.3b,c). This is consistent with prior reports (e.g.,\nKleiner et al., 2012 PMID: 226453550), which also observed no compensatory upregulation of PGC1β\nin the absence of PGC1α in adipocytes, but we cannot exclude the functional compensation of PGC1β\nin regulating these genes expression here.\nImportantly, we do not dispute the established role of PGC1α in regulating Ucp1 transcription. In our in\nvivo experiments, Ucp1 mRNA and protein levels were clearly reduced in PGC1α-AKO mice after\nchronic CL treatment (Fig. 3), highlighting its importance for sustained thermogenic remodeling. By\ncontrast, in acute in vitro assays (Extended Fig. 4a,b), Ucp1 induction appeared largely preserved. We\nbelieve this reflects two factors: (i) in vitro culture conditions may mask regulatory differences that are\nmore evident in vivo,(ii) basal PGC1α expression is extremely low in cultured adipocytes, which\ndiminishes its relative contribution to Ucp1 transcription in this setting, and (iii) PGC1α inhibitor\ntreatment experiments were performed in beige adipocytes, supporting our conclusion that PGC1α\nplays a strong regulatory role in controlling glycogen metabolism gene expression in this context. Thus,\nacute Ucp1 induction in vitro is predominantly driven by direct β3-adrenergic signaling and its\nimmediate downstream transcription factors (e.g., CREB, ATF2), whereas in vivo chronic stimulation\nrequires PGC-1α for transcriptional maintenance.We have included clarifying discussion in the revised\nmanuscript (Line304-313).\n6. Regarding the work on the ERRs for deleting all three Err genes, this has been done\npreviously and their redundancy when deleting one or another has been previously shown.\nPlease cite Gantner et al. 2016 Endocrinology PMID 27763777 and note this earlier observation.\nReponse: We thank the reviewer for pointing this out. We have now cited Gantner et al. in the revised\nmanuscript and explicitly noted this precedent, highlighting how our results extend the concept of ERR\nredundancy to the regulation of glycogen metabolism in white adipose tissue under chronic β3-\nadrenergic stimulation."}, {"round": 2, "reviewer_comments": {"reviewer_1": "No further comments. The authors have addressed the concerns.", "reviewer_2": "The author has addressed all my concerns. I have no more questions.", "reviewer_3": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.", "reviewer_4": "My concerns have been largely addressed satisfactorily"}, "author_response": ""}]

Decision letter