Adipocyte microRNA-802 promotes adipose tissue inflammation and insulin resistance by modulating macrophages in obesity

  1. Yue Yang
  2. Bin Huang
  3. Yimeng Qin
  4. Danwei Wang
  5. Yinuo Jin
  6. Linmin Su
  7. Yi Pan
  8. Yanfeng Zhang
  9. Yumeng Shen
  10. Wenjun Hu
  11. Zhengyu Cao
  12. Liang Jin
  13. Fangfang Zhang

  • Curated by eLife

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    eLife assessment

    This important study utilizes a comprehensive array of animal and cellular models, alongside various techniques, to elucidate the mechanism by which adipose tissue miR-802 contributes to inflammation and metabolic dysfunction in obesity. The data is solid, with clear, reproducible changes showing low variability among biological replicates and consistency across different models. However, some conclusions should be further substantiated with additional data to enhance the scope and strength of the manuscript.

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Abstract

Adipose tissue inflammation is now considered to be a key process underlying metabolic diseases in obese individuals. However, it remains unclear how adipose inflammation is initiated and maintained or the mechanism by which inflammation develops. We found that microRNA-802 ( miR-802 ) expression in adipose tissue is progressively increased with the development of dietary obesity in obese mice and humans. The increasing trend of miR-802 preceded the accumulation of macrophages. Adipose tissue-specific knockout of miR-802 lowered macrophage infiltration and ameliorated systemic insulin resistance. Conversely, the specific overexpression of miR-802 in adipose tissue aggravated adipose inflammation in mice fed a high-fat diet. Mechanistically, miR-802 activates noncanonical and canonical NF-κB pathways by targeting its negative regulator, TRAF3. Next, NF-κB orchestrated the expression of chemokine and SREBP1, which translated into strong recruitment and M1-like polarization of macrophages. Our findings indicate that miR-802 endows adipose tissue with the ability to recruit and polarize macrophages, which underscores miR-802 as an innovative and attractive candidate for miRNA-based immune therapy for adipose inflammation.

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  1. eLife

    Author response:

    Reviewer #1 (Public Review):

    In this manuscript, Yang et al. conduct a comprehensive investigation to demonstrate the role of adipose tissue Mir802 in obesity-associated inflammation and metabolic dysfunction. Using multiple models and techniques, they propose a mechanism where elevated levels of Mir802 in adipose tissue (both in mouse models and humans) trigger fat accumulation and inflammation, leading to increased adiposity and insulin resistance. They suggest that increased Mir802 levels in adipocytes during obesity result in the downregulation of TRAF3, a negative regulator of canonical and non-canonical NF-κB pathways. This downregulation induces inflammation through the production of cytokines/chemokines that attract and polarize macrophages. Concurrently, the NF-κB pathway induces the lipogenic transcriptional factor SREBP1, which promotes fat accumulation and further recruits pro-inflammatory macrophages. While the proposed model is supported by multiple experiments and consistent data, there are areas where the manuscript could be improved. Some improvements can be addressed in the text, while others require additional controls, experiments, or analyses.

    1. The manuscript should provide measurements of lipid droplet/adipocyte size for all models, both in vitro and in vivo. In vivo studies should also include fat weight measurements. This is crucial to determine whether Mir802, TRAF3, and SREBP1 promote adiposity/fat accumulation across all models.

    Thank you for your careful reviewing. As suggested, we have measured the size of lipid droplet and adipocyte (1J, 2A, S2I, 3F, 3L, S3L, 5I), this modification can make you and other readers understand our manuscript more clearly. In vivo studies have included fat weight measurements (Figure 2K, L; Figure 3C, D; Figure 5N). Our results determined that adipose-selective overexpression Mir802 induced adipogenesis during high fat diet induced.

    1. The rationale for co-culture experiments using WAT SVF is unclear, given that Mir802 is upregulated by obesity in adipocytes, not in the stromal-vascular fraction. These experiments would be more relevant if performed using isolated adipocytes or differentiated WAT SVF.

    Thank you for this important point. We are sorry for our inaccurate expression. In our study, we used differentiated WAT SVF to co-culture with primary macrophage, we illustrated it in the methods of Migration and invasion assays. We have revised it in the Flowchart of the co-culture experiments (Figure 4A). We hope that this modification will enhance readers' comprehension of our manuscript.

    1. Figures 1G and 1H lack a control group (time 0 or NCD). Without this control, it is impossible to determine if inflammation precedes Mir802 upregulation.

    Thank you for this insightful comment. In the previous study, we have tested the 0 weeks high fed diet treatment group of the Figures 1I and 1J, now we have added this data in the manuscript, we hope this modification can enhance our conclusion that inflammation precedes Mir802 upregulation.

    1. The statement, "The knockout of Mir802 in adipose tissue did not alter food intake, body weight, glucose level, and adiposity (data not shown)," needs more detail regarding the age and sex of the animals. These data are important and should be reported, perhaps in a supplementary figure.

    Thank you for your careful reviewing. To enhance our conclusions, we have added the data of food intake, body weight, glucose level, and adiposity about Mir802 KO mice treated with normal chow diet (NCD, Supplementary Figure 3E-I).

    ….The knockout of Mir802 in adipose tissue did not alter food intake, body weight, glucose levels, and adiposity compared with their WT littermates in both males and females when they were fed with NCD (Figure S3E-I)……

    1. The terms "KO" (knockout) and "KI" (knock-in) are misleading for AAV models, as they do not modify the genome. "KD" (knockdown) and "OE" (overexpression) are more accurate.

    Thank you for your good advice. We are sorry for our inaccurate expression. According to your advice, we have rewritten it. AAV models for Mir802 knockdown (Figure 3) and Traf3 overexpression (Figure 5) have changed to KD and OE respectively.

    1. The statement, "Mir802 expression was unaffected in other organs (Figure S3O)," should clarify that this is except for BAT.

    We appreciate the you for this insightful comment. We have clarified that Mir802 expression was unaffected in other organs except for BAT (Figure S3T, revised manuscript).

    By addressing these points, the manuscript would present a more robust and clear demonstration of the role of Mir802 in obesity-associated inflammation and metabolic dysfunction.

    Thanks for your positive comments. As suggested, we have modified all point.

    Reviewer #2 (Public Review):

    Yang et al. investigated the role of Mir802 in the development of adipose tissue (AT) inflammation during obesity. The authors found Mir802 levels are up-regulated in the AT of mouse models of obesity and insulin resistance as well as in the AT of humans. They further demonstrated that Mir802 regulates the intracellular levels of TRAF3 and downstream activation of the NF-kB pathway. Ultimately, controlling AT inflammation by manipulating Mir802 affected whole-body glucose homeostasis, highlighting the role of AT inflammatory status in whole-body metabolism. The study provides solid evidence on the role of adipocyte Mir802 in controlling inflammation and macrophage recruitment. However, how lipid mobilization from adipocytes and how engulfment of lipid droplets by macrophages control inflammatory phenotype in these cells could be better explored. The findings of this study will have a great impact in the field, contributing to the growing body of evidence on how microRNAs control the inflammatory microenvironment of AT and whole-body metabolism in obesity.

    Thanks for your positive comments.

    Reviewer #3 (Public Review):

    Mir802 appears to accumulate before macrophage numbers increase in adipose tissue in both mice and humans. The phenotype of Mir802 overexpression and deletion in vivo is sticking and novel. Deletion of Mir802 in adipose tissue after obesity onset also attenuated Adipose inflammation and improved systemic glucose homeostasis. Understanding how Mir802 affects the crosstalk between macrophage and adipocyte is a major point. For example, does Mir802 change the inflammatory of macrophages as it increases Traf3 expression in adipocytes? This is important because macrophages are the input if inflammatory mediators that will activate the TNFR receptor signaling pathway, potentially Traf3, resulting in impaired insulin stimulated Glut4 translocation and glucose uptake. Also, modulation of Mir802 levels in vivo leads to alterations in adiposity. Here, what is a direct effect of Mir802 and what is a result of simply reduced adiposity? One point that os ket is what triggers Mir802 expression, especially in obesity.

    Thanks for your important suggestions. According to your suggestions, we have addressed additional data in the revised manuscript to enhance our conclusion.

  2. eLife

    eLife assessment

    This important study utilizes a comprehensive array of animal and cellular models, alongside various techniques, to elucidate the mechanism by which adipose tissue miR-802 contributes to inflammation and metabolic dysfunction in obesity. The data is solid, with clear, reproducible changes showing low variability among biological replicates and consistency across different models. However, some conclusions should be further substantiated with additional data to enhance the scope and strength of the manuscript.

  3. eLife

    Reviewer #1 (Public Review):

    In this manuscript, Yang et al. conduct a comprehensive investigation to demonstrate the role of adipose tissue miR-802 in obesity-associated inflammation and metabolic dysfunction. Using multiple models and techniques, they propose a mechanism where elevated levels of miR-802 in adipose tissue (both in mouse models and humans) trigger fat accumulation and inflammation, leading to increased adiposity and insulin resistance. They suggest that increased miR-802 levels in adipocytes during obesity result in the downregulation of TRAF3, a negative regulator of canonical and non-canonical NF-κB pathways. This downregulation induces inflammation through the production of cytokines/chemokines that attract and polarize macrophages. Concurrently, the NF-κB pathway induces the lipogenic transcriptional factor SREBP1, which promotes fat accumulation and further recruits pro-inflammatory macrophages. While the proposed model is supported by multiple experiments and consistent data, there are areas where the manuscript could be improved. Some improvements can be addressed in the text, while others require additional controls, experiments, or analyses.

    (1) The manuscript should provide measurements of lipid droplet/adipocyte size for all models, both in vitro and in vivo. In vivo studies should also include fat weight measurements. This is crucial to determine whether miR-802, TRAF3, and SREBP1 promote adiposity/fat accumulation across all models.
    (2) The rationale for co-culture experiments using WAT SVF is unclear, given that miR-802 is upregulated by obesity in adipocytes, not in the stromal-vascular fraction. These experiments would be more relevant if performed using isolated adipocytes or differentiated WAT SVF.
    (3) Figures 1G and 1H lack a control group (time 0 or NCD). Without this control, it is impossible to determine if inflammation precedes miR-802 upregulation.
    (4) The statement, "The knockout of miR-802 in adipose tissue did not alter food intake, body weight, glucose level, and adiposity (data not shown)," needs more detail regarding the age and sex of the animals. These data are important and should be reported, perhaps in a supplementary figure.
    (5) The terms "KO" (knockout) and "KI" (knock-in) are misleading for AAV models, as they do not modify the genome. "KD" (knockdown) and "OE" (overexpression) are more accurate.
    (6) The statement, "miR-802 expression was unaffected in other organs (Figure S3O)," should clarify that this is except for BAT.

    By addressing these points, the manuscript would present a more robust and clear demonstration of the role of miR-802 in obesity-associated inflammation and metabolic dysfunction.

  4. eLife

    Reviewer #2 (Public Review):

    Yang et al. investigated the role of miR-802 in the development of adipose tissue (AT) inflammation during obesity. The authors found miR-802 levels are up-regulated in the AT of mouse models of obesity and insulin resistance as well as in the AT of humans. They further demonstrated that miR-802 regulates the intracellular levels of TRAF3 and downstream activation of the NF-kB pathway. Ultimately, controlling AT inflammation by manipulating miR-802 affected whole-body glucose homeostasis, highlighting the role of AT inflammatory status in whole-body metabolism. The study provides solid evidence on the role of adipocyte miR-802 in controlling inflammation and macrophage recruitment. However, how lipid mobilization from adipocytes and how engulfment of lipid droplets by macrophages control inflammatory phenotype in these cells could be better explored. The findings of this study will have a great impact in the field, contributing to the growing body of evidence on how microRNAs control the inflammatory microenvironment of AT and whole-body metabolism in obesity.

  5. eLife

    Reviewer #3 (Public Review):

    MiR-802 appears to accumulate before macrophage numbers increase in adipose tissue in both mice and humans. The phenotype of miR-802 overexpression and deletion in vivo is sticking and novel. Deletion of miR-802 in adipose tissue after obesity onset also attenuated Adipose inflammation and improved systemic glucose homeostasis. Understanding how miR-802 affects the crosstalk between macrophage and adipocyte is a major point. For example, does miR-802 change the inflammatory of macrophages as it increases Traf3 expression in adipocytes? This is important because macrophages are the input if inflammatory mediators that will activate the TNFR receptor signaling pathway, potentially Traf3, resulting in impaired insulin stimulated Glut4 translocation and glucose uptake. Also, modulation of miR-802 levels in vivo leads to alterations in adiposity. Here, what is a direct effect of miR-802 and what is a result of simply reduced adiposity? One point that os ket is what triggers miR-802 expression, especially in obesity.

  6. Version published to 10.1101/2024.05.13.593999v1 on bioRxiv