Single-Cell Landscape and a Macrophage Subset Enhancing Brown Adipocyte Function in Diabetes (Diabetes Metab J 2024;48:885-900)

Junfei Gu; Xinjie Zhang; Qunye Zhang; Zhe Wang

doi:10.4093/dmj.2024.0785

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Response Single-Cell Landscape and a Macrophage Subset Enhancing Brown Adipocyte Function in Diabetes (Diabetes Metab J 2024;48:885-900): Junfei Gu^1,2, Xinjie Zhang³, Qunye Zhang^4,5, Zhe Wang¹; Diabetes & Metabolism Journal 2025;49(1):162-164.
DOI: https://doi.org/10.4093/dmj.2024.0785
Published online: January 1, 2025

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¹Department of Geriatrics, Shandong Provincial Hospital Affiliated to Shandong First Medical University, Jinan, China

²Department of Endocrinology, The Second Affiliated Hospital of Wannan Medical College, Wuhu, China

³Department of Biology, University College London, London, UK

⁴Key Laboratory of Cardiovascular Remodeling and Function Research, Chinese Ministry of Education, Chinese National Health Commission, Shandong University, Jinan, China

⁵Department of Cardiology, Qilu Hospital of Shandong University, Jinan, China

Corresponding authors: Qunye Zhang

Department of Cardiology, Qilu Hospital of Shandong University, 107# Wenhuaxi Road, Jinan 250021, China E-mail: wz.zhangqy@sdu.edu.cn

Zhe Wang

Department of Endocrinology & Geriatrics, Shandong Provincial Hospital, 324# Jingwu Road, Jinan 250021, China E-mail: wangzhe.zqy@email.sdu.edu.cn

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

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See the letter "Single-Cell Landscape and a Macrophage Subset Enhancing Brown Adipocyte Function in Diabetes (Diabetes Metab J 2024;48:885-900)" on page 160.

We sincerely thank Dr. Lee and Dr. Kang for their thoughtful comments on our recently published article, “Single-cell landscape and a macrophage subset enhancing brown adipocyte function in diabetes” [1]. We also appreciate the editor for providing the opportunity to further discuss our findings.

Dr. Lee and Dr. Kang pointed out that we identified a type 2 diabetes mellitus (T2DM)-specific adipose stem/progenitor cell 2 (ASPC2) cluster, which was further subdivided into distinct subsets, including Cd163+ASPC2-s1, involved in neutrophil chemotaxis and myeloid differentiation, and S100 calcium-binding protein A9 (S100a9)+ASPC2-s2, associated with chronic inflammation and neutrophil chemotaxis. However, our study did not utilize neutrophil populations in the single-cell RNA sequencing analysis, leaving the connection between ASPCs and neutrophils unexplored. We fully agree with the viewpoint of Dr. Lee and Dr. Kang. Indeed, descriptive content constitutes a substantial proportion of this article. Among the identified ASPC clusters, we observed significant changes in the T2DM-specific ASPC2 cluster and its subclusters. The potential role of the Cd163+ASPC2-s1 subcluster in neutrophil chemotaxis and myeloid differentiation indeed warrants further study. Similarly, the association of S100a9+ASPC2-s2 subcluster with chronic inflammation and neutrophil chemotaxis also provides important clues for understanding the regulatory mechanism of chronic inflammation in T2DM. Several studies have explored the roles of white adipose tissue (WAT)-derived ASPCs in neutrophil chemotaxis and inflammation. For instance, Shang et al. [2] demonstrated that ASPCs promote neutrophil chemotaxis in the cornea, thereby contributing to wound healing; Jia et al. [3] reported that ASPCs regulate neutrophil chemotaxis, facilitating wound healing and local inflammation resolution. Gao et al. [4] found that ASPCs improve lung injury by regulating various inflammatory cells, including neutrophils, in lung tissue. However, the interactions and regulatory mechanisms between brown adipose tissue (BAT)-derived ASPCs and neutrophils, as well as other inflammatory cells, remain unclear. Only a few studies have identified inflammatory cell types in BAT using single-cell sequencing technology [5,6], and their interactions with ASPCs, along with the underlying mechanisms have not been further explored. As suggested by Dr. Lee and Dr. Kang, exploring cell-to-cell interactions between neutrophils and ASPCs is a promising research direction. We are currently investigating the role of neutrophil chemotaxis in chronic inflammation in the context of T2DM, which may provide valuable insights into the disease’s underlying mechanisms.

Our study observed significant changes in information flow. The information flow dominated by fibroblasts (FBs) in normal rat BAT stromal vascular fraction (SVF) expanded to multiple cell types, including ASPCs, smooth muscle cells (SMCs), and FBs in T2DM rat BAT SVF. Specific signaling pathways also significantly changed, particularly the angiopoietin-like proteins (ANGPTLs) pathways, which play important roles in lipid metabolism and energy homeostasis [7]. Previous studies have reported that ANGPTL3 and ANGPTL4 regulate lipid metabolism by inhibiting lipase activity [8,9]. ANGPTL4 also reduces the activity of the insulin signaling pathway, thereby contributing to the development of T2DM [10,11]. Targeting the ANGPTL signaling pathway may improve insulin sensitivity and reduce inflammation, offering a potential therapeutic approach for T2DM and other metabolic diseases. Our findings indicate that the ANGPTL signaling pathway is altered in multiple cell types in T2DM BAT. Specifically, ANGPTL signaling was significantly enhanced between adipose-derived stem cell (ADSC) and endothelial cell, as well as between ADSC and SMC. In the normal rat BAT SVF, FBs are the primary sender, receiver, regulator and influencer of intercellular ANGPTL information flow; however, in T2DM rats, ADSC take on this role. These findings underscore the importance of dissecting complex intercellular interactions. Future research should explore the regulatory mechanisms of the ANGPTL signaling pathway in BAT and assess the potential for targeted interventions in inflammatory signals of specific cell types to enhance BAT function. These research directions may not only elucidate the pathogenesis of T2DM but also provide novel therapeutic targets for diabetes management.

Dr. Lee and Dr. Kang also pointed out an important finding about the retinoic acid receptor responder 2 (Rarres2)+ macrophage subset in our study. Our scRNA-seq sequencing analysis revealed that the Rarres2+ macrophage subset was decreased in the BAT-derived SVF of T2DM rats. Interestingly, the relative expression level of the Rarres2 gene was higher in T2DM compared to controls. This seemingly contradictory result may be due to the fact that the Rarres2+ macrophage subset was determined not only based on the Rarres2 gene but also including other marker genes. In fact, the algorithm for identifying cell subsets using scRNA-seq data in Seurat software considered several marker genes. In addition to Rarres2, the marker genes for Rarres2+ macrophage subset include Cd74, chemokine C-C motif ligand 2 (Ccl2), bone morphogenetic protein 2 (Bmp2), and other genes, which collectively define this macrophage subset. To simplify terminology, we referred to this subset as Rarres2+ macrophages. Therefore, changes in the expression level of Rarres2 gene may not accurately reflect alterations in the overall number of Rarres2+ macrophages [12,13]. This highlights the complexity and heterogeneity of the composition and regulation of macrophage subsets. The limitations of the study pointed out by Dr. Lee and Dr. Kang are very pertinent. In this article, we confirmed the existence of Rarres2+ macrophage subset in BAT at the protein level by immunofluorescence but did not further validate the change in the Rarres2+ macrophage population observed in normal versus T2DM rat BAT. Therefore, future studies should employ immunofluorescence and other methods to investigate more markers of Rarres2+ macrophage subset, clarifying its changes at the tissue or cellular level. We also believe that it is important to explore the biological significance of the observed reduction in Rarres2+ macrophages and the increase in Rarres2 mRNA expression, and to reveal the genes and molecular mechanisms through which Rarres2+ macrophages promote the differentiation and function of brown adipocytes. These are promising areas for future research.

We acknowledge the concerns raised by Dr. Lee and Dr. Kang regarding the limitations related to sample size and sequencing replicates. We fully agree that the explicit number of mouse samples used for sequencing is crucial for the data reliability. In designing this study, we referred to sample sizes reported in previous single-cell sequencing studies of mouse adipose tissue [14-16]. At the same time, we also considered experimental costs. Therefore, we isolated SVF from the interscapular BAT of 9–10 normal or diabetic rats per group and separately pooled them for sequencing. Sample pooling can effectively reduce individual variability, enhance result reliability, and reduce experimental costs. Nevertheless, further studies involving larger sample sizes from both mouse and T2DM patient tissues are very necessary.

As Dr. Lee and Dr. Kang noted, current treatments for T2DM with obesity largely focus on reducing energy intake, whereas effective strategies for enhancing energy expenditure remain elusive. Many previous studies have primarily attempted to increase BAT content by browning WAT [17], while overlooking the potential of BAT itself. Our study reveals complex changes in intercellular interactions and signaling networks within BAT in T2DM, particularly those affecting the differentiation potential of BAT ASPCs. Furthermore, our findings preliminarily elucidate the mechanism by which macrophage subsets regulate the differentiation and metabolism of ASPCs, highlighting their central role. Research on these issues may facilitate the development of cell-type-specific therapeutic strategies targeting inflammation and energy metabolism.

In summary, we are very grateful for the constructive feedback on our research. Your professional insights have greatly helped us better understand the limitations of our research and have highlighted valuable directions for future investigation. Thank you again for your interest in our article.

NOTES

CONFLICTS OF INTEREST

No potential conflict of interest relevant to this article was reported.

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Single-Cell Landscape and a Macrophage Subset Enhancing Brown Adipocyte Function in Diabetes (Diabetes Metab J 2024;48:885-900)

Diabetes Metab J. 2025;49(1):162-164. Published online January 1, 2025

DOI: https://doi.org/10.4093/dmj.2024.0785

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Single-Cell Landscape and a Macrophage Subset Enhancing Brown Adipocyte Function in Diabetes (Diabetes Metab J 2024;48:885-900)

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Gu J, Zhang X, Zhang Q, Wang Z. Single-Cell Landscape and a Macrophage Subset Enhancing Brown Adipocyte Function in Diabetes (Diabetes Metab J 2024;48:885-900). Diabetes Metab J. 2025;49(1):162-164.

DOI: https://doi.org/10.4093/dmj.2024.0785.

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