Constitutively active receptor ADGRA3 signaling induces adipose thermogenesis

Reviewer #1 (Public review):

Summary:

This article identifies ADGR3 as a candidate GPCR for mediating beige fat development. The authors use human expression data from Human Protein Atlas and Gtex databases and combine this with experiments performed in mice and a murine cell line. They refer to a GPCR bioactivity screening tool PRESTO-Salsa, with which it was found that Hesperetin activates ADGR3. From their experiments, authors conclude that Hesperetin activates ADGR3, inducing a Gs-PKA-CREB axis resulting in adipose thermogenesis.

Strengths:

The authors analyze human data from public databases and perform functional studies in mouse models. They identify a new GPCR with a role in thermogenic activation of adipocytes.

Considerations:

Selection of ADGRA3 as a candidate GPCR relevant for mediating beiging in humans:

The authors identify GPCRs that are expressed more highly in murine iBAT compared to iWAT in response to cold and assess which of these GPCRs are expressed in human subcutaneous or visceral adipocytes. Although this strategy will identify GPCRs that are expressed at higher levels in brown fat compared to beige and thus possibly more active in thermogenic function, the relevance in choosing GPCRs that also are expressed in unstimulated human white adipocytes should be considered. Thermogenic activity is not normally present in human white adipocytes. It would have strengthened the GPCR selection if the authors instead had assessed the intersection with human brown adipocytes that were activated with norepinephrine.

Strategy to investigate the role of ADGRA3 in WAT beiging:

Having identified ADGRA3 as their candidate receptor, the authors investigated the receptor in mouse models, the murine inguinal adipocyte cell line 3T3 and in human subcutaneous adipose progenitors (HAdsc) differentiated in vitro. Calling the human cells "beige" is a stretch as these cells are derived from a white adipose depot. The authors do observe regulation in UCP1 and abundance of mitochondria following modification of ADGRA3 in the cells. However, in future studies, it should be considered if the receptor rather plays a role in differentiation per se, and perhaps not specifically in thermogenic differentiation/activity.

According to the Human Protein Atlas and Gtex databases, ADGRA3 is not only expressed in adipocytes, but also in other tissues and cell types. The authors address this by measuring the expression in a panel of these tissues, demonstrating a knockdown not only in the adipose tissue, but also in the liver and less pronounced in the muscle (Figure S2). It should thus be emphasized that the decreased TG levels in serum and liver in the mice might in fact depend on Adgra3 overexpression in the liver. Even though this might not have been the purpose of the experiment, it is important to highlight this as it could serve as hypothesis building for future studies of the function of this receptor.

Reviewer #2 (Public review):

Based on bioinformatics and expression analysis using mouse and human samples, the authors claim that the adhesion G-protein coupled receptor ADGRA3 may be a valuable target for increasing thermogenic activity and metabolic health. Genetic approaches to deplete ADGRA3 expression in vitro resulted in reduced expression of thermogenic genes including Ucp1, reduced basal respiration and metabolic activity as reflected by reduced glucose uptake and triglyceride accumulation. In line, nanoparticle delivery of shAdgra3 constructs is associated with increased body weight, reduced thermogenic gene expression in white and brown adipose tissue (WAT, BAT), and impaired glucose and insulin tolerance. On the other hand, ADGRA3 overexpression is associated with an improved metabolic profile in vitro and in vivo, which can be explained by increasing the activity of the well-established Gs-PKA-CREB axis. Notably, a computational screen suggested that ADGRA3 is activated by hesperetin. This metabolite is a derivative of the major citrus flavonoid hesperidin and has been described to promote metabolic health. Using appropriate in vitro and in vivo studies, the authors show that hesperitin supplementation is associated with increased thermogenesis, UCP1 levels in WAT and BAT, and improved glucose tolerance, an effect that was attenuated in the absence of ADGRA3 expression.

Comments on revised version:
In my opinion, the critical points I raised were not adequately addressed, neither in the revision nor in the response to the reviewer. Therefore, my initial assessment has not changed, the main claims are only partially supported by the data presented.

Reviewer #3 (Public review):

Summary:

The manuscript by Zhao et al. explored the function of adhesion G protein-coupled receptor A3 (ADGRA3) in thermogenic fat biology.

Strengths:

Through both in vivo and in vitro studies, the authors found that the gain function of ADGRA3 leads to browning of white fat and ameliorates insulin resistance.

Comments on revised version:

The revised manuscript by Zhao et al. has limited improvement. The authors refused to perform revised experiments using primary cultures even though two reviewers pointed out the same weakness (3T3-L1 adipocytes are unsuitable). Using infrared thermography to measure body temperature is also problematic.

Author response:

The following is the authors’ response to the original reviews.

Public Reviews:

Reviewer #1 (Public Review):

Summary:

This article identifies ADGR3 as a candidate GPCR for mediating beige fat development. The authors use human expression data from the Human protein atlas and Gtex databases and combine this with experiments performed in mice and a murine cell line. They refer to a GPCR bioactivity screening tool PRESTO-Salsa, with which it was found that Hesperetin activates ADGR3. From their experiments, authors conclude that Hesperetin activates ADGR3, inducing a Gs-PKA-CREB axis resulting in adipose thermogenesis.

Strengths:

The authors analyze human data from public databases and perform functional studies in mouse models. They identify a new GPCR with a role in the thermogenic activation of adipocytes.

Weaknesses:

(1) Selection of ADGRA3 as a candidate GPCR relevant for mediating beiging in humans:

The authors identify genes upregulated in iBAT compared to iWAT in response to cold, and among these differentially expressed genes, they identify highly expressed GPCRs in human white adipocytes (visceral or subcutaneous). Finally, among these genes, they select a GPCR not previously studied in the literature.

If the authors are interested in beiging, why do they not focus on genes upregulated in iWAT (the depot where beiging is described to occur in mice), comparing thermoneutral to cold-induced genes? I would expect that genes induced in iWAT in response to cold would be extremely relevant targets for beiging. With their strategy, the authors exclude receptors that are induced in the tissue where beiging is actually described to occur.

Furthermore, the authors are comparing genes upregulated in cold in BAT (but not WAT) to highly expressed genes in human white adipocytes during thermoneutrality. Overall, the authors fail to discuss the logic behind their strategy and the obvious limitations of it.

Thanks for your valuable advice. In this study, we focus on genes that exhibited higher expression in BAT compared to iWAT under cold stimulation conditions, as these genes might play a role in adipose thermogenesis. Regarding the genes you mentioned that iWAT upregulates following cold stimulation, we did identify other intriguing targets in these genes in another ongoing study, albeit not encompassed within the scope of this study. Moreover, instead of making a comparison, we intersected 27 GPCR coding genes that were highly expressed in BAT compared to iWAT with genes that were highly expressed in human adipocytes (Figure 1C).

With your suggestions, we realized that the description of the screening strategy in the manuscript was not clear enough, so we made the following supplement:

“…dataset obtained from the Gene Expression Omnibus (GEO) database. Additionally, we utilized the human subcutaneous adipocytes dataset (Figure 1C, red) and human visceral adipocytes dataset (Figure 1C, purple) from the human protein atlas database to obtain genes that are highly expressed in human white adipocytes. The GSE118849 dataset comprises samples of brown adipose tissue (BAT) and inguinal white adipose tissue (iWAT) obtained from mice subjected to a 72-hour cold exposure at a temperature of 4℃.

A total of 1134 differentially expressed genes (DEGs) that exhibited up-regulation in BAT compared to iWAT under cold stimulation were identified in the analysis, which might play a role in adipose thermogenesis. These DEGs were further screened to identify highly…”

(2) Relevance of ADGRA3 and comparison to established literature:

There has been a lot of literature and discussion about which receptor should be targeted in humans to recruit thermogenic fat. The current article unfortunately does not discuss this literature nor explain how it relates to their findings. For example, O'Mara et al (PMID: 31961826) demonstrated that chronic stimulation with the B3 adrenergic agonist, Mirabegron, resulted in the recruitment of thermogenic fat and improvement in insulin sensitivity and cholesterol. Later, Blondin et al (PMID: 32755608), highlighted the B2 adrenergic receptor as the main activation path of thermogenic fat in humans. There is also a recent report on an agonist activating B2 and B3 simultaneously (PMID: 38796310). Thus, to bring the literature forward, it would be beneficial if the current manuscript compared their identified activation path with the activation of these already established receptors and discussed their findings in relation to previous studies.

Thanks to your suggestion. We have included a supplementary discussion on the relevant human adipose thermogenic receptors in the discussion section, as presented below:

“The induction of beige fat has been investigated as a potentially effective therapeutic approach in combating obesity [23]. A clinical trial revealed that treatment with the chronic β3-AR agonist mirabegron leads to an increase in human brown fat, HDL cholesterol, and insulin sensitivity [24]. Subsequently, Blondin et al discovered that oral administration of mirabegron only elicits an increase in BAT thermogenesis when administered at the maximal allowable dose, indicating that human brown adipocyte thermogenesis is primarily driven by β2-adrenoceptor (β2-AR) stimulation [11]. Consistent with this finding, we found much higher levels of ADRB2 expression in human white adipose tissue than ADRB3 (Figure S1E). Furthermore, a recent study has demonstrated that simultaneous activation of β2-AR and β3-AR enhances whole-body metabolism through beneficial effects on skeletal muscle and BAT [25].”

In Figures 1d and e, the authors show the expression of ADGRA3 in comparison to the expression of ADRB3. In human brown adipocytes, ADRB2 has been shown to be the main receptor through which adrenergic activation occurs (PMID: 32755608), thus authors should show the relative expression of this gene as well.

We wholeheartedly endorse the proposal to augment the ADRB2 expression data in Figures 1D and E. However, it is regrettable to note that the pertinent databases (PRJNA66167 and PRJEB4337) are deficient in ADRB2 expression information. Fortunately, the GTEx database houses the ADRB2 expression data. Consequently, we have integrated these crucial data into Figure S1E.

(3) Strategy to investigate the role of ADGRA3 in WAT beiging:

Having identified ADGRA3 as their candidate receptor, the authors proceed with investigations of this receptor in mouse models and the murine inguinal adipocyte cell line 3T3.

First of all, in Figure 1D, the authors show a substantially lower expression of ADGRA3 compared to ADRB3. It could thus be argued that a mouse would not be the best model system for studying this receptor. It would be interesting to see data from experiments in human adipocytes.

Thanks for your helpful advice. We induced human adipose-derived mesenchymal stem cells (hADSCs) into adipocytes to evaluate the effect of ADGRA3 on human adipocytes (Figure 8).

Moreover, if the authors are interested in inducing beiging, why do they show expression in iBAT and not iWAT?

Maybe the description of this article wasn't clear enough, but we did show the expression and effects of ADGRA3 in iWAT and BAT (Author response image 1, Figure 3F-J and Figure 4F-J).

Author response image 1.

The authors perform in vivo experiments using intraperitoneal injections of shRNA or overexpression CMV-driven vectors and report effects on body temperature and glucose metabolism. It is here important to note that ADGRA3 is not uniquely expressed in adipocytes. A major advantage of databases like the Human Protein Atlas and Gtex, is that they give an overview of the gene expression across tissues and cell types. When looking up ADGRA3 in these databases, it is expressed in subcutaneous and visceral adipocytes. However, other cell types and tissues demonstrate an even higher expression. In the Human protein atlas, the enhanced cell types are astrocytes and hepatocytes. In the Gtex database tissues with the highest expression are Brain, Liver, and Thyroid.

With this information in mind, IP injections for modification of ADGRA3 receptor expression could be expected to affect any of these tissues and cells.

The manuscript report changes body temperature. However, temperature is regulated by the brain and also affected by thyroid activity. Did the authors measure the levels of circulating thyroid hormones? Gene expression changes in the brain? The authors report that Adgra3 overexpression decreased the TG level in serum and liver. The liver could be the primary targeted organ here, and the adipose effects might be secondary. The data would be easier to interpret if authors reported the effects on the liver, thyroid, and brain, and the gene expression across tissues should be discussed in the article.

Thank you for your valuable advice. We supplemented the results of the effect of local BAT injection of Adgra3 OE on thermogenic genes (Figures S5G-H), the levels of circulating thyroid hormones (Figures S2H, S4F and S5B) and the effects of Adgra3 overexpression/knockdown on Adgra3 expression levels (Figures S2A-B and S4B-C) in multiple tissues as well as discussed in the article, as follows:

“Given the consideration that the non-targeted nanoparticle approach utilized in this study for modulating Adgra3 expression levels in vivo alter Adgra3 expression in tissues beyond adipose tissue (Figures S2A-B and S4B-C), notably the liver and skeletal muscle, the construction of Adgra3 adipose tissue-specific knockout/overexpression mouse models is imperative for a more nuanced understanding of the precise mechanisms underlying the influence of on adipose thermogenesis. We will employ more sophisticated models in subsequent studies to further elucidate the effects of ADGRA3 on adipose thermogenesis and metabolic homeostasis. Nevertheless, our findings underlie a potential therapeutic feature of…”

Finally, the identification of Hesperetin using the PRESTO-Salsa tool, and how specific the effect of Hesperetin is on ADGRA3, is currently unclear. This should be better discussed, and authors should consider measuring the established effects of Hesperetin in their model systems, including apoptosis.

Thanks for your suggestion. We have further discussed the relevant content and added it in the discussion section as follows:

“Previously, the influence of hesperetin on ADGRA3 has remained unreported. In this study, we screened hesperetin as a potential agonist for ADGRA3 by using the PRESTO-Salsa tool as well as discovered that hesperetin has an agonist effect on ADGRA3 through a series of experiments. This study focuses on the regulatory effect of hesperetin on adipose thermogenesis and explores whether this effect is dependent upon ADGRA3. As such, we refrained from conducting further investigations into other potential effects of hesperidin, including its potential role in antioxidant and in apoptosis.”

Reviewer #2 (Public Review):

Based on bioinformatics and expression analysis using mouse and human samples, the authors claim that the adhesion G-protein coupled receptor ADGRA3 may be a valuable target for increasing thermogenic activity and metabolic health. Genetic approaches to deplete ADGRA3 expression in vitro resulted in reduced expression of thermogenic genes including Ucp1, reduced basal respiration, and metabolic activity as reflected by reduced glucose uptake and triglyceride accumulation. In line, nanoparticle delivery of shAdgra3 constructs is associated with increased body weight, reduced thermogenic gene expression in white and brown adipose tissue (WAT, BAT), and impaired glucose and insulin tolerance. On the other hand, ADGRA3 overexpression is associated with an improved metabolic profile in vitro and in vivo, which can be explained by increasing the activity of the well-established Gs-PKA-CREB axis. Notably, a computational screen suggested that ADGRA3 is activated by hesperetin. This metabolite is a derivative of the major citrus flavonoid hesperidin and has been described to promote metabolic health. Using appropriate in vitro and in vivo studies, the authors show that hesperetin supplementation is associated with increased thermogenesis, UCP1 levels in WAT and BAT, and improved glucose tolerance, an effect that was attenuated in the absence of ADGRA3 expression.

Overall, the data suggest that ADGRA3 is a constitutively active Gs-coupled receptor that improves metabolism by activating adaptive thermogenesis in WAT and BAT. The conclusions of the paper are partly supported by the data, but some experimental approaches need further clarification.

(1) The in vivo approaches to modulate Adgra3 expression in mice are carried out using non-targeted nanoparticle-based approaches. The authors do not provide details of the composition of the nanomaterials, but it is highly likely that other metabolically active organs such as the liver are targeted. This is critical because Adgre3 is expressed in many organs, including the liver, adrenal glands, and gastrointestinal system. Therefore, many of the observed metabolic effects could be indirect, for example by modulating bile acids or corticosterone levels. Consistent with this, after digestion in the gastrointestinal tract, hesperetin is rapidly metabolized in intestinal and liver cells. Thus, hesperetin levels in the systemic circulation are likely to be insufficient to activate Adgra3 in thermogenic adipocytes/precursors. Overall, the authors need to repeat the key metabolic experiments in adipose-specific Adgra3 knockout/overexpression models to validate the reliability of the in vivo results. In addition, to validate the relevance of hesperetin supplementation for adaptive thermogenesis in BAT and WAT vivo, the levels of hesperetin present in the systemic circulation should be quantified.

Thank you for your valuable advice. Unfortunately, we could not perform quantitative determination of hesperetin concentration in the systemic circulation because we had used the serum of hesperetin-treated mice for the quantitative determination of serum insulin, fT4 and TG. According to your other suggestions, we supplemented the results of the effect of local BAT injection of Adgra3 OE on thermogenic genes (Figures S5G-H), the levels of circulating thyroid hormones (Figures S2H, S4F and S5B) and the effects of Adgra3 overexpression/knockdown on Adgra3 expression levels (Figures S2A-B and S4B-C) in multiple tissues as well as discussed in the article, as follows:

“Given the consideration that the non-targeted nanoparticle approach utilized in this study for modulating Adgra3 expression levels in vivo alter Adgra3 expression in tissues beyond adipose tissue (Figures S2A-B and S4B-C), notably the liver and skeletal muscle, the construction of Adgra3 adipose tissue-specific knockout/overexpression mouse models is imperative for a more nuanced understanding of the precise mechanisms underlying the influence of on adipose thermogenesis. We will employ more sophisticated models in subsequent studies to further elucidate the effects of ADGRA3 on adipose thermogenesis and metabolic homeostasis. Nevertheless, our findings underlie a potential therapeutic feature of…”

(2) Standard measurements for energy balance are not presented. Quantitative data on energy expenditure, e.g. by indirect calorimetry, and food intake are missing and need to be included to validate the authors' claims.

We are in full agreement with your proposal. Regrettably, owing to the constraints of experimental facilities, we are presently unable to access quantitative data pertaining to the energy expenditure of animals. However, we believe that the present results can also partially support the idea that ADGRA3 promotes energy metabolism and the results of the effect of ADGRA3 on food intake were shown in Figure S2C and Figure S5A respectively.

(3) The thermographic images used to determine the BAT temperature are not very convincing. The distance and angle between the thermal camera and the BAT have a significant effect on the determination of the temperature, which is not taken into account, at least in the images presented.

Thank you very much for pointing out the lack of our method description. According to the methods of literatures (Xia, Bo et al. PLoS biology. 2020. doi:10.1371/journal.pbio.3000688) and (Warner, Amy et al. PNAS. 2013. doi:10.1073/pnas.1310300110), the same batch of representative infrared images of mice were all captured using a thermal imaging camera (FLIR ONE PRO), measured at the same distance perpendicular to the plane on which the mice were located. We have supplemented this description in the Materials and Methods section, as shown below:

“2.20. Infrared Thermography.

BAT temperature was measured at room temperature by infrared thermography according to previous publications [22, 23]. The same batch of representative infrared images of mice were all captured using a thermal imaging camera (FLIR ONE PRO), measured at the same distance perpendicular to the plane on which the mice were located. To quantify interscapular region temperature, the average surface temperature from a region of the interscapular BAT was taken with FLIR Tools software.”

(4) The 3T3-L1 cell line is not an adequate cell culture model to study thermogenic adipocyte differentiation. To validate their results, the key experiments showing that ADGRA3 expression modulates thermogenic marker expression in a hesperetin-dependent manner need to be performed in a reliable model, e.g. primary murine adipocytes.

Induction of 3T3L1 cell line into white adipocytes is indeed not suitable for studying thermogenic adipocyte differentiation. However, with reference to previous studies (Wei, Gang et al. Cell metabolism. 2021. doi: 10.1016/j.cmet.2021.08.012 ) and (Bae IS, Kim SH. Int J Mol Sci. 2019. doi: 10.3390/ijms20246128), 3T3-L1 cell line was used to differentiate into beige-like adipocytes in this study, and many studies believe that this method is suitable for studying the thermogenic effect of adipocytes in vitro. Meanwhile, we provided a more detailed description of the induction of beige-like adipocytes by 3T3-L1 in the Materials and Methods section and induced human adipose-derived stem cells (hADSC) into adipocytes to evaluate the effect of ADGRA3 on human adipocytes (Figure 8).

“…supplemented with 10% FBS. Confluent 3T3-L1 pre-adipocytes were induced into mature beige-like adipocytes with 0.5 mM isobutyl methylxanthine (IBMX), 1 μM dexamethasone, 5 μg/ml insulin, 1 nM 3, 3', 5-Triiodo-L-thyronine (T3), 125 μM indomethacin and 1 μM rosiglitazone in high-glucose DMEM containing 10% FBS for 2 days, then treated with high-glucose DMEM containing 5 μg/ml insulin, 1 nM T3, 1 μM rosiglitazone and 10% FBS for 6 days and cultured with high-glucose DMEM containing 10% FBS for 2 days. hADSCs were seeded on plates coated with 0.1% gelatin and culture and grown to confluence in human mesenchymal stem cells (hMSCs) specialized culture medium (ZQ-1320). Confluent hADSCs were induced into mature human adipocytes with adipogenic induction medium (PCM-I-004) according to the manufacturer’s instructions.”

(5) The experimental setup only allows the measurement of basal cellular respiration. More advanced approaches are needed to define the contribution of ADGRA3 versus classical adrenergic receptors to UCP1-dependent thermogenesis.

Thanks for your suggestion. The maximum oxygen consumption rate of the cells was also measured (Figures 2G and 2N) by adding FCCP, an uncoupler of oxidative phosphorylation (OXPHOS) in mitochondria.

Reviewer #3 (Public Review):

Summary:

The manuscript by Zhao et al. explored the function of adhesion G protein-coupled receptor A3 (ADGRA3) in thermogenic fat biology.

Strengths:

Through both in vivo and in vitro studies, the authors found that the gain function of ADGRA3 leads to browning of white fat and ameliorates insulin resistance.

Weaknesses:

There are several lines of weak methodologies such as using 3T3-L1 adipocytes and intraperitoneal(i.p.) injection of virus. Moreover, as the authors stated that ADGRA3 is constitutively active, how could the authors then identify a chemical ligand?

(1) Primary cultured cells should be used to perform gain and loss function analysis of ADGRA3, instead of using 3T3-L1. It is impossible to detect Ucp1 expression in 3T3-L1 cells.

Induction of 3T3L1 cell line into white adipocytes is indeed difficult for detecting UCP1 expression. However, with reference to previous studies (Wei, Gang et al. Cell metabolism. 2021. doi:10.1016/j.cmet.2021.08.012) and (Bae IS, Kim SH. Int J Mol Sci. 2019. doi:10.3390/ijms20246128), 3T3-L1 cell line was used to differentiate into beige-like adipocytes in this study, and many studies believe that this method is suitable for studying the thermogenic effect of adipocytes in vitro. Meanwhile, we provided a more detailed description of the induction of beige-like adipocytes by 3T3-L1 in the Materials and Methods section and induced human adipose-derived stem cells (hADSC) into adipocytes to evaluate the effect of ADGRA3 on human adipocytes (Figure 8).

“…supplemented with 10% FBS. Confluent 3T3-L1 pre-adipocytes were induced into mature beige-like adipocytes with 0.5 mM isobutyl methylxanthine (IBMX), 1 μM dexamethasone, 5 μg/ml insulin, 1 nM 3, 3', 5-Triiodo-L-thyronine (T3), 125 μM indomethacin and 1 μM rosiglitazone in high-glucose DMEM containing 10% FBS for 2 days, then treated with high-glucose DMEM containing 5 μg/ml insulin, 1 nM T3, 1 μM rosiglitazone and 10% FBS for 6 days and cultured with high-glucose DMEM containing 10% FBS for 2 days. hADSCs were seeded on plates coated with 0.1% gelatin and culture and grown to confluence in human mesenchymal stem cells (hMSCs) specialized culture medium (ZQ-1320). Confluent hADSCs were induced into mature human adipocytes with adipogenic induction medium (PCM-I-004) according to the manufacturer’s instructions.”

(2) For virus treatment, the authors should consider performing local tissue injection, rather than IP injection. If it is IP injection, have the authors checked other tissues to validate whether the phenotype is fat-specific?

Thank you for your valuable advice. We supplemented the results of the effect of local BAT injection of Adgra3 OE on thermogenic genes (Figures S5G-H) and the effects of Adgra3 overexpression/knockdown on Adgra3 expression levels (Figures S2A-B and S4B-C) in other tissues.

(3) The authors should clarify how constitutively active GPCR needs further ligands.

Thank you for your suggestion. In fact, we only identified hesperetin as a potential agonist of ADGRA3 rather than a ligand. The results also indicate that overexpression of ADGRA3 without additional hesperetin is sufficient to activate downstream PKA signaling pathways through constitutive activity (Figure 5). Recently, Chen et al identified oleic ethanolamine (OEA) as a potential endogenous agonist of GPR3, which is also a constitutively active GPCR. Overall, the high constitutive activity of constitutively active GPCRs arises from the combined effects of stimulation by endogenous agonists and their basal coupling with Gs.

As for why we screened and identified potential agonists of ADGRA3, we hope to find more convenient pathways for its clinical application than gene overexpression, as described in the article:

“Considering the difficulty of overexpressing ADGRA3 in clinical application, hesperetin was screened as a potential agonist of ADGRA3 by PRESTO-Salsa database (Figure 6A). The…”

Recommendations for the authors:

Reviewer #1 (Recommendations For The Authors):

Minor comments

The title appears to be overstated as no clinical trials were performed and experiments were not even performed in human brown adipocytes.

Thank you for your critical suggestion, therefore we have added the experimental results of human adipocytes (Figure 8) and revised the title to “Constitutively active receptor ADGRA3 signaling induces adipose thermogenesis”.

Please specify n-number and what are replicates or independent experiments. Please also state if any outliers were excluded and why.

Thanks for your valuable suggestion. We have added a description of the n-number in the Figure legends section, number of independent experiments and exclusion criteria for outliers in the Materials and Methods section, as follows:

“…of tissue samples. Cohorts of ≥4 mice per genotype or treatment were assembled for all in vivo studies. All in vivo studies were repeated 2-3 independent times. All procedures related to…”

“…μM H-89) was added to 3T3-L1 mature beige-like adipocytes for 48 hours. All in vitro studies were repeated 2-3 independent times.”

“All data are presented as mean ± SEM. In this study, outliers that met the three-sigma rule were excluded from analysis, with the exception of those presented in Figure S1E. Given the possibility that the outliers in Figure S1E represent extreme expressions of the inherent variability within the population sample, we have chosen to retain these specific outliers for further analysis. Student’s t-test was used to compare two groups. One-way analysis of…”

Authors use Infrared Thermography to measure body temperature. Depending on the distance between the mouse and the camera, the mouse needs to be at the same spot.

Thank you very much for pointing out the lack of our method description. According to the methods of literatures (Xia, Bo et al. PLoS biology. 2020. doi:10.1371/journal.pbio.3000688) and (Warner, Amy et al. PNAS. 2013. doi:10.1073/pnas.1310300110), the same batch of representative infrared images of mice were all captured using a thermal imaging camera (FLIR ONE PRO), measured at the same distance perpendicular to the plane on which the mice were located. We have supplemented this description in the Materials and Methods section, as shown below:

“2.20. Infrared Thermography.

BAT temperature was measured at room temperature by infrared thermography according to previous publications [22, 23]. The same batch of representative infrared images of mice were all captured using a thermal imaging camera (FLIR ONE PRO), measured at the same distance perpendicular to the plane on which the mice were located. To quantify interscapular region temperature, the average surface temperature from a region of the interscapular BAT was taken with FLIR Tools software.”

Please discuss the limitations of the experiments and discuss the relevant literature.

Thanks for your recommendations. We discussed the limitations of the experiments and the relevant literature in the discussion section, as follows:

“The induction of beige fat has been investigated as a potentially effective therapeutic approach in combating obesity [23]. A clinical trial revealed that treatment with the chronic β3-AR agonist mirabegron leads to an increase in human brown fat, HDL cholesterol, and insulin sensitivity [24]. Subsequently, Blondin et al discovered that oral administration of mirabegron only elicits an increase in BAT thermogenesis when administered at the maximal allowable dose, indicating that human brown adipocyte thermogenesis is primarily driven by β2-adrenoceptor (β2-AR) stimulation [11]. Consistent with this finding, we found much higher levels of ADRB2 expression in human white adipose tissue than ADRB3 (Figure S1E). Furthermore, a recent study has demonstrated that simultaneous activation of β2-AR and β3-AR enhances whole-body metabolism through beneficial effects on skeletal muscle and BAT [25].”

“Given the consideration that the non-targeted nanoparticle approach utilized in this study for modulating Adgra3 expression levels in vivo alter Adgra3 expression in tissues beyond adipose tissue (Figures S2A-B and S4B-C), notably the liver and skeletal muscle, the construction of Adgra3 adipose tissue-specific knockout/overexpression mouse models is imperative for a more nuanced understanding of the precise mechanisms underlying the influence of on adipose thermogenesis. We will employ more sophisticated models in subsequent studies to further elucidate the effects of ADGRA3 on adipose thermogenesis and metabolic homeostasis. Nevertheless, our findings underlie a potential therapeutic feature of…”