Author Response
eLife assessment
This study presents a valuable finding on the distinct subpopulation of adipocytes during brown-to-white conversion in perirenal adipose tissue (PRAT) at different ages. The evidence supporting the claims of the authors is convincing, although specific lineage tracing of this subpopulation of cells and mechanistic studies would expand the work. The work will be of interest to scientists working on adipose and kidney biology.
Public Reviews:
Reviewer #1 (Public Review):
Summary:
In this manuscript, the authors performed single nucleus RNA-seq for perirenal adipose tissue (PRAT) at different ages. They concluded a distinct subpopulation of adipocytes arises through brown-to-white conversion and can convert to a thermogenic phenotype upon cold exposure.
Strengths:
PRAT adipose tissue has been reported as an adipose tissue that undergoes browning. This study confirms that brown-to-white and white-to-beige conversions also exist in PRAT, as previously reported in the subcutaneous adipose tissue.
We did not observe any white-to-beige conversion in PRAT under regular condition. The adipocyte population that arises from brown-to-white conversion (mPRAT-ad2) can respond to cold and restore their UCP1 expression. However, brown adipocytes that arise from the mPRAT-ad2 subpopulation after cold exposure have a distinct transcriptome to that of cold-induced beige adipocyte in iWAT (Figure S6K) and are more related to iBAT brown adipocytes (Figure 6E).
Weaknesses:
- There is overall a disconnection between single nucleus RNA-seq data and the lineage chasing data. No specific markers of this population have been validated by staining.
We are not sure what “this population” refers to. We suspect it is the Ucp1-&Cidea+ mPRAT-ad2 adipocyte subpopulation. If so, we did not identify specific markers for these adipocytes as shown in Figure 1H and statement in the Discussion. mPRAT-ad2 is negative for Ucp1 and Cyp2e1, which are markers for mPRAT-ad1 and mPRAT-ad3&4, respectively. Therefore, we plan to stain the mPRAT with Ucp1, Cyp2e1 and Perilipin (a pan adipocyte marker) antibodies. Cells that are Perilipin+&Ucp1-&Cyp2e1- will represent the mPRAT-ad2 subpopulation.
- It would be nice to provide more evidence to support the conclusion shown in lines 243 to 245 "These results indicated that new BAs induced by cold exposure were mainly derived from UCP1- adipocytes rather than de novo ASPC differentiation in puPRAT". Pdgfra-negative progenitor cells may also contribute to these new beige adipocytes.
Our sequencing data and many previous studies (Angueira et al., 2021; Burl et al., 2022; Dong et al., 2022) have shown that Pdgfra is a marker for all ASPCs. We will also check adipocyte labelling pattern of mPRAT in the PdgfraCre;Ai14 mice. If all adipocytes are Tomato+, it suggests that adipocytes in mPRAT are all derived from Pdgfra-expressing cells. Also, the cold-induced adipocytes in mPRAT resemble more to the brown adipocytes of iBAT than the beige adipocytes of iWAT (Figure 6E and S6K).
Angueira, A.R., Sakers, A.P., Holman, C.D., Cheng, L., Arbocco, M.N., Shamsi, F., Lynes, M.D., Shrestha, R., Okada, C., Batmanov, K., et al. (2021). Defining the lineage of thermogenic perivascular adipose tissue. Nat Metab 3, 469-484. 10.1038/s42255-021-00380-0.
Burl, R.B., Rondini, E.A., Wei, H., Pique-Regi, R., and Granneman, J.G. (2022). Deconstructing cold-induced brown adipocyte neogenesis in mice. Elife 11. 10.7554/eLife.80167.
Dong, H., Sun, W., Shen, Y., Balaz, M., Balazova, L., Ding, L., Loffler, M., Hamilton, B., Kloting, N., Bluher, M., et al. (2022). Identification of a regulatory pathway inhibiting adipogenesis via RSPO2. Nat Metab 4, 90-105. 10.1038/s42255-021-00509-1.
- The UCP1Cre-ERT2; Ai14 system should be validated by showing Tomato and UCP1 co-staining right after the Tamoxifen treatment.
We will inject Ucp1CreERT2;Ai14 mice at 1- and 6-month-old of age with tamoxifen and collect one day after the last injection to check the overlap between the Tomato signal and UCP1 immunofluorescent staining.
Reviewer #2 (Public Review):
Summary:
In the present manuscript, Zhang et al utilize single-nuclei RNA-Seq to investigate the heterogeneity of perirenal adipose tissue. The perirenal depot is interesting because it contains both brown and white adipocytes, a subset of which undergo functional "whitening" during early development. While adipocyte thermogenic transdifferentiation has been previously reported, there remain many unanswered questions regarding this phenomenon and the mechanisms by which it is regulated.
Strengths:
The combination of UCP1-lineage tracing with the single nuclei analysis allowed the authors to identify four populations of adipocytes with differing thermogenic potential, including a "whitened" adipocyte (mPRAT-ad2) that retains the capacity to rapidly revert to a brown phenotype upon cold exposure. They also identify two populations of white adipocytes that do not undergo browning with acute cold exposure.
Anatomically distinct adipose depots display interesting functional differences, and this work contributes to our understanding of one of the few brown depots present in humans.
Weaknesses:
The most interesting aspect of this work is the identification of a highly plastic mature adipocyte population with the capacity to switch between a white and brown phenotype. The authors attempt to identify the transcriptional signature of this ad2 subpopulation, however, the limited sequencing depth of single nuclei somewhat lessens the impact of these findings. Furthermore, the lack of any form of mechanistic investigation into the regulation of mPRAT whitening limits the utility of this manuscript. However, the combination of well-executed lineage tracing with comprehensive cross-depot single-nuclei presented in this manuscript could still serve as a useful reference for the field.
The sequencing depth of our data is comparable, if not better than previously published snRNA-seq studies on adipose tissue (Burl et al., 2022; Sarvari et al., 2021; Sun et al., 2020). Therefore, the depth of our data has reached the limit of the 3’ sequencing methods. Unfortunately, due to size limitation of the adipocytes, it is also not feasible to sort them for Smart-seq.
Burl, R.B., Rondini, E.A., Wei, H., Pique-Regi, R., and Granneman, J.G. (2022). Deconstructing cold-induced brown adipocyte neogenesis in mice. Elife 11. 10.7554/eLife.80167.
Sarvari, A.K., Van Hauwaert, E.L., Markussen, L.K., Gammelmark, E., Marcher, A.B., Ebbesen, M.F., Nielsen, R., Brewer, J.R., Madsen, J.G.S., and Mandrup, S. (2021). Plasticity of Epididymal Adipose Tissue in Response to Diet-Induced Obesity at Single-Nucleus Resolution. Cell Metab 33, 437-453 e435. 10.1016/j.cmet.2020.12.004.
Sun, W., Dong, H., Balaz, M., Slyper, M., Drokhlyansky, E., Colleluori, G., Giordano, A., Kovanicova, Z., Stefanicka, P., Balazova, L., et al. (2020). snRNA-seq reveals a subpopulation of adipocytes that regulates thermogenesis. Nature 587, 98-102. 10.1038/s41586-020-2856-x.
