Single-nucleus transcriptomics reveal the cytological mechanism of conjugated linoleic acids in regulating intramuscular fat deposition

  1. Liyi Wang
  2. Shiqi Liu
  3. Shu Zhang
  4. Yizhen Wang
  5. Yanbing Zhou
  6. Tizhong Shan

  • Curated by eLife

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    eLife Assessment

    This revised study provides valuable information on the single nucleus RNA sequencing transcriptome, pathways, and cell types in pig skeletal muscle in response to conjugated linoleic acid (CLA) supplementation. Based on the comprehensive data analyses, the data are considered compelling and provide new insight into the mechanisms underlying intramuscular fat deposition and muscle fiber remodeling. The revised study clarifies major aspects of its methodology and analysis, addresses previous reviewer concerns, and contributes significantly to the understanding of nutritional strategies for fat infiltration in pig muscle.

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Abstract

Conjugated linoleic acids (CLAs) can serve as a nutritional intervention to regulate quality, function and fat infiltration in skeletal muscles but the specific cytological mechanisms are still unknown. Here, we applied single-nucleus RNA-sequencing (snRNA-seq) to characterize the cytological mechanism of CLAs regulates fat infiltration in skeletal muscles based on pig models. We investigated the regulatory effects of CLAs on cell populations and molecular characteristics in pig muscles and found CLAs could promote the transformation of fast glycolytic myofibers into slow oxidative myofibers. We also observed three subpopulations including SCD + /DGAT2 + , FABP5 + /SIAH1 + , and PDE4D + /PDE7B + subclusters in adipocytes and CLAs could increase the percentage of SCD + /DGAT2 + adipocytes. RNA velocity analysis showed FABP5 + /SIAH1 + and PDE4D + /PDE7B + adipocytes could differentiate into SCD + /DGAT2 + adipocytes. We further verified the differentiated trajectory of mature adipocytes and identified PDE4D + /PDE7B + adipocytes could differentiate into SCD + /DGAT2 + and FABP5 + /SIAH1 + adipocytes by using high IMF content Laiwu pig models. The cell-cell communication analysis identified the interaction network between adipocytes and other subclusters such as fibro/adipogenic progenitors (FAPs). Pseudotemporal trajectory analysis and RNA velocity analysis also showed FAPs could differentiate into PDE4D + /PDE7B + preadipocytes and we discovered the differentiated trajectory of preadipocytes into mature adipocytes. Besides, we found CLAs could promote FAPs differentiate into SCD + /DGAT2 + adipocytes via inhibiting c-Jun N-terminal kinase (JNK) signalling pathway in vitro . This study provides a foundation for regulating fat infiltration in skeletal muscles by using nutritional strategies and provides potential opportunities to serve pig as an animal model to study human fat infiltrated diseases.

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  1. Version published to 10.7554/elife.99790.2 on eLife
  2. Version published to 10.7554/elife.99790 on eLife
  3. eLife

    eLife Assessment

    This revised study provides valuable information on the single nucleus RNA sequencing transcriptome, pathways, and cell types in pig skeletal muscle in response to conjugated linoleic acid (CLA) supplementation. Based on the comprehensive data analyses, the data are considered compelling and provide new insight into the mechanisms underlying intramuscular fat deposition and muscle fiber remodeling. The revised study clarifies major aspects of its methodology and analysis, addresses previous reviewer concerns, and contributes significantly to the understanding of nutritional strategies for fat infiltration in pig muscle.

  4. eLife

    Reviewer #1 (Public review):

    In this revised manuscript, the authors aim to elucidate the cytological mechanisms by which conjugated linoleic acids (CLAs) influence intramuscular fat deposition and muscle fiber transformation in pig models. They have utilized single-nucleus RNA sequencing (snRNA-seq) to explore the effects of CLA supplementation on cell populations, muscle fiber types, and adipocyte differentiation pathways in pig skeletal muscles. Notably, the authors have made significant efforts in addressing the previous concerns raised by the reviewers, clarifying key aspects of their methodology and data analysis.

    Strengths:

    (1) Thorough validation of key findings: The authors have addressed the need for further validation by including qPCR, immunofluorescence staining, and western blotting to verify changes in muscle fiber types and adipocyte populations, which strengthens their conclusions.

    (2) Improved figure presentation: The authors have enhanced figure quality, particularly for the Oil Red O and Nile Red staining images, which now better depict the organization of lipid droplets (Figure 7A). Statistical significance markers have also been clarified (Figure 7I and 7K).

    Weaknesses:

    (1) Cross-species analysis and generalizability of the results: Although the authors could not perform a comparative analysis across species due to data limitations, they acknowledged this gap and focused on analyzing regulatory mechanisms specific to pigs. Their explanation is reasonable given the current availability of snRNA-seq datasets on muscle fat deposition in other human and mouse.

    (2) Mechanistic depth in JNK signaling pathway: While the inclusion of additional experiments is a positive step, the exploration of the JNK signaling pathway could still benefit from deeper analysis of downstream transcriptional regulators. The current discussion acknowledges this limitation, but future studies should aim to address this gap fully.

    (3) Limited exploration of other muscle groups: The authors did not expand their analysis to additional muscle groups, leaving some uncertainty regarding whether other muscle groups might respond differently to CLA supplementation. Further studies in this direction could enhance the understanding of muscle fiber dynamics across the organism.

  5. eLife

    Reviewer #2 (Public review):

    Summary:

    This study comprehensively presents data from single nuclei sequencing of Heigai pig skeletal muscle in response to conjugated linoleic acid supplementation. The authors identify changes in myofiber type and adipocyte subpopulations induced by linoleic acid at depth previously unobserved. The authors show that linoleic acid supplementation decreased the total myofiber count, specifically reducing type II muscle fiber types (IIB), myotendinous junctions, and neuromuscular junctions, whereas type I muscle fibers are increased. Moreover, the authors identify changes in adipocyte pools, specifically in a population marked by SCD1/DGAT2. To validate the skeletal muscle remodeling in response to linoleic acid supplementation, the authors compare transcriptomics data from Laiwu pigs, a model of high intramuscular fat, to Heigai pigs. The results verify changes in adipocyte subpopulations when pigs have higher intramuscular fat, either genetically or diet-induced. Targeted examination using cell-cell communication network analysis revealed associations with high intramuscular fat with fibro-adipogenic progenitors (FAPs). The authors then conclude that conjugated linoleic acid induces FAPs towards adipogenic commitment. Specifically, they show that linoleic acid stimulates FAPs to become SCD1/DGAT2+ adipocytes via JNK signaling. The authors conclude that their findings demonstrate the effects of conjugated linoleic acid on skeletal muscle fat formation in pigs, which could serve as a model for studying human skeletal muscle diseases.

    Strengths:

    The comprehensive data analysis provides information on conjugated linoleic acid effects on pig skeletal muscle and organ function. The notion that linoleic acid induces skeletal muscle composition and fat accumulation is considered a strength and demonstrates the effect of dietary interactions on organ remodeling. This could have implications for the pig farming industry to promote muscle marbling. Additionally, these data may inform the remodeling of human skeletal muscle under dietary behaviors, such as elimination and supplementation diets and chronic overnutrition of nutrient-poor diets. However, the biggest strength resides in thorough data collection at the single nuclei level, which was extrapolated to other types of Chinese pigs.

    Weaknesses:

    Although the authors compiled a substantial and comprehensive dataset, the scope of cellular and molecular-level validation still needs to be expanded. For instance, the single nuclei data suggest changes in myofiber type after linoleic acid supplementation, but these findings need more thorough validation. Further histological and physiological assessments are necessary to address fiber types and oxidative potential. Similarly, the authors propose that linoleic acid alters adipocyte populations, FAPs, and preadipocytes; however, there are limited cellular and molecular analyses to confirm these findings. The identified JNK signaling pathways require additional follow-ups on the molecular mechanism or transcriptional regulation. However, these issues are discussed as potential areas for future exploration. While various individual studies have been conducted on mouse/human skeletal muscle and adipose tissues, these have only been briefly discussed, and further investigation is warranted. Additionally, the authors incorporate two pig models into their results, but they only examine one muscle group. Exploring whether other muscle groups respond similarly or differently to linoleic acid supplementation would be valuable. Furthermore, the authors should discuss how their results translate to human and pig nutrition, such as the desirability and cost-effectiveness for pig farmers and human diets high in linoleic acid. Notably, while the single nuclei data is comprehensive, there needs to be a statement on data deposition and code availability, allowing others access to these datasets.

  6. eLife

    Author response:

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

    Reviewer #1 (Public Review):

    Summary:

    The authors aimed to elucidate the cytological mechanisms by which conjugated linoleic acids (CLAs) influence intramuscular fat deposition and muscle fiber transformation in pig models. Utilizing single-nucleus RNA sequencing (snRNA-seq), the study explores how CLA supplementation alters cell populations, muscle fiber types, and adipocyte differentiation pathways in pig skeletal muscles.

    Thanks!

    Strengths:

    Innovative approach: The use of snRNA-seq provides a high-resolution insight into the cellular heterogeneity of pig skeletal muscle, enhancing our understanding of the intricate cellular dynamics influenced by nutritional regulation strategy.

    Robust validation: The study utilizes multiple pig models, including Heigai and Laiwu pigs, to validate the differentiation trajectories of adipocytes and the effects of CLA on muscle fiber type transformation. The reproducibility of these findings across different (nutritional vs genetic) models enhances the reliability of the results.

    Advanced data analysis: The integration of pseudotemporal trajectory analysis and cell-cell communication analysis allows for a comprehensive understanding of the functional implications of the cellular changes observed.

    Practical relevance: The findings have significant implications for improving meat quality, which is valuable for both the agricultural and food industry.

    Thanks!

    Weaknesses:

    Model generalizability: While pigs are excellent models for human physiology, the translation of these findings to human health, especially in diverse populations, needs careful consideration.

    Thanks!

    Reviewer #2 (Public Review):

    Summary:

    This study comprehensively presents data from single nuclei sequencing of Heigai pig skeletal muscle in response to conjugated linoleic acid supplementation. The authors identify changes in myofiber type and adipocyte subpopulations induced by linoleic acid at depth previously unobserved. The authors show that linoleic acid supplementation decreased the total myofiber count, specifically reducing type II muscle fiber types (IIB), myotendinous junctions, and neuromuscular junctions, whereas type I muscle fibers are increased. Moreover, the authors identify changes in adipocyte pools, specifically in a population marked by SCD1/DGAT2. To validate the skeletal muscle remodeling in response to linoleic acid supplementation, the authors compare transcriptomics data from Laiwu pigs, a model of high intramuscular fat, to Heigai pigs. The results verify changes in adipocyte subpopulations when pigs have higher intramuscular fat, either genetically or diet-induced. Targeted examination using cell-cell communication network analysis revealed associations with high intramuscular fat with fibro-adipogenic progenitors (FAPs). The authors then conclude that conjugated linoleic acid induces FAPs towards adipogenic commitment. Specifically, they show that linoleic acid stimulates FAPs to become SCD1/DGAT2+ adipocytes via JNK signaling. The authors conclude that their findings demonstrate the effects of conjugated linoleic acid on skeletal muscle fat formation in pigs, which could serve as a model for studying human skeletal muscle diseases.

    Thanks!

    Strengths:

    The comprehensive data analysis provides information on conjugated linoleic acid effects on pig skeletal muscle and organ function. The notion that linoleic acid induces skeletal muscle composition and fat accumulation is considered a strength and demonstrates the effect of dietary interactions on organ remodeling. This could have implications for the pig farming industry to promote muscle marbling. Additionally, these data may inform the remodeling of human skeletal muscle under dietary behaviors, such as elimination and supplementation diets and chronic overnutrition of nutrient-poor diets. However, the biggest strength resides in thorough data collection at the single nuclei level, which was extrapolated to other types of Chinese pigs.

    Thanks!

    Weaknesses:

    While the authors generated a sizeable comprehensive dataset, cellular and molecular validation needed to be improved. For example, the single nuclei data suggest changes in myofiber type after linoleic acid supplementation, yet these data are not validated by other methodologies. Similarly, the authors suggest that linoleic acid alters adipocyte populations, FAPs, and preadipocytes; however, no cellular and molecular analysis was performed to reveal if these trajectories indeed apply. Attempts to identify JNK signaling pathways appear superficial and do not delve deeper into mechanistic action or transcriptional regulation. Notably, a variety of single cell studies have been performed on mouse/human skeletal muscle and adipose tissues. Yet, the authors need to discuss how the populations they have identified support the existing literature on cell-type populations in skeletal muscle.Moreover, the authors nicely incorporate the two pig models into their results, but the authors only examine one muscle group. It would be interesting if other muscle groups respond similarly or differently in response to linoleic acid supplementation.Further, it was unclear whether Heigai and Laiwu pigs were both fed conjugated linoleic acid or whether the comparison between Heigai-fed linoleic acid and Laiwu pigs (as a model of high intramuscular fat). With this in mind, the authors do not discuss how their results could be implicated in human and pig nutrition, such as desirability and cost-effectiveness for pig farmers and human diets high in linoleic acid. Notably, while single nuclei data is comprehensive, there needs to be a statement on data deposition and code availability, allowing others access to these datasets. Moreover, the experimental designs do not denote the conjugated linoleic acid supplementation duration. Several immunostainings performed could be quantified to validate statements. This reviewer also found the Nile Red staining hard to interpret visually and did not appear to support the conclusions convincingly. Within Figure 7, several letters (assuming they represent statistical significance) are present on the graphs but are not denoted within the figure legend.

    Thanks for your suggestions! We accepted your suggestion to revised our manuscript.

    For changes in myofiber type, we performed qPCR to verify the changes of muscle fiber type related gene expression after CLA treatment (Figure 2E); for changes of adipocyte and preadipocyte populations, we also performed immunofluorescence staining, qPCR, and western blotting in LDM tissues and FAPs to verify the alterations of cell types after feeding with CLA (Figure 3D, 3E, 6G, 7C, and 7D). Hence, we think these cellular and molecular results could support our conclusions.

    For JNK signaling pathway, we selected this signaling pathway based on snRNA-seq dataset and verified by activator in vitro experiment. However, we did not explore the mechanistic action and the downstream transcriptional regulators need to be further discussed. We have added these in the discussion part (line 443-448).

    We have added the comparation between different cell-type populations in skeletal muscles (line 362-368 and 385-390).

    For changes in myofiber type of Laiwu pigs, we have discussed in our previous study(Wang et al., 2023). Interestingly, we also found in high IMF content Laiwu pigs, the percentage of type IIa myofibers had an increased tendency (29.37% vs. 23.95%) while the percentage of type IIb myofibers had a decreased tendency (38.56% vs. 43.75%) in this study. We also added this discussion in the discussion part (line 392-395).

    We have supplied the information of treatment in the materials and methods part (line 469-478). We also added the discussion about significance of our study for human and pig nutrition in the discussion part (line 375-376 and 446-447).

    Our data will be made available on reasonable request (line 574-576).

    We have supplied the information of the CLA supplementation duration in the materials and methods part (line 465).

    Porcine FAPs have little lipid droplets and we improved the image quality (Figure 7A). In Figure 7, the Nile Red staining could be quantified and we have the quantification of Oil Red O staining (Figure 7B and 7J). We also added the statistical significance in figure legend.

    Recommendations for the authors:

    Reviewer #1 (Recommendations For The Authors):

    Suggestions for Improved or Additional Experiments, Data, or Analyses

    Cross-species analysis: To strengthen the generalizability of the results, it would be beneficial to include a comparative analysis with other species, such as human, bovine, or rodent models, using publicly available snRNA-seq datasets.

    Thanks! Our previous study has compared the conserved and unique signatures in fatty skeletal muscles between different species(Wang, Zhou, Wang, & Shan, 2024). We mainly focused on the regulatory mechanism of CLAs in regulating intramuscular fat deposition. However, there is still a blank in the snRNA-seq or scRNA-seq datasets about the effects of CLAs on regulating fat deposition in muscles across other species, including human, bovine or rodent models. Hence, we only analyze the regulatory mechanisms of CLAs influencing intramuscular fat deposition in pigs.

    Functional link: the authors should discuss in the manuscript how the muscles differ in terms of texture, flavor, aroma, etc. before and after CLA administration or between Heigai and Laiwu to provide context and help readers better understand how the observed high-resolution cellular changes relate to these functional properties of meat.

    Thanks! We have added these in the introduction part (line 90-98).

    Improve figures: some figures, particularly those involving Oil Red O and Nail Red, could be improved by including higher magnification images to assess the organization of lipid droplets of individual adipocytes (Figure 7A, I, and K).

    Thanks! Porcine FAPs have little lipid droplets and we improved the image quality (Figure 7A).

    Reviewer #2 (Recommendations For The Authors):

    All of my comments are above. However, I would recommend improving the writing as several areas throughout the results needed clarity.

    Thanks! We have revised our manuscript carefully after accepting your revisions.

    Wang, L., Zhao, X., Liu, S., You, W., Huang, Y., Zhou, Y., . . . Shan, T. (2023) Single-nucleus and bulk RNA sequencing reveal cellular and transcriptional mechanisms underlying lipid dynamics in high marbled pork NPJ Sci Food 7: 23. https://doi.org/10.1038/s41538-023-00203-4

    Wang, L., Zhou, Y., Wang, Y., & Shan, T. (2024) Integrative cross-species analysis reveals conserved and unique signatures in fatty skeletal muscles Sci Data 11: 290. https://doi.org/10.1038/s41597-024-03114-5

  7. Version published to 10.1101/2024.05.30.596608v2 on bioRxiv
  8. Version published to 10.7554/elife.99790.1 on eLife
  9. eLife

    eLife assessment

    The present study provides valuable information into the regulatory mechanisms through which conjugated linoleic acids influence intramuscular fat deposition and muscle fiber transformation in pigs. The data are analyzed comprehensively using solid and validated single nuclei methodology. Overall, the provided data are convincing and support the conclusion of the study.

  10. eLife

    Reviewer #1 (Public Review):

    Summary:

    The authors aimed to elucidate the cytological mechanisms by which conjugated linoleic acids (CLAs) influence intramuscular fat deposition and muscle fiber transformation in pig models. Utilizing single-nucleus RNA sequencing (snRNA-seq), the study explores how CLA supplementation alters cell populations, muscle fiber types, and adipocyte differentiation pathways in pig skeletal muscles.

    Strengths:

    Innovative approach: The use of snRNA-seq provides a high-resolution insight into the cellular heterogeneity of pig skeletal muscle, enhancing our understanding of the intricate cellular dynamics influenced by nutritional regulation strategy.

    Robust validation: The study utilizes multiple pig models, including Heigai and Laiwu pigs, to validate the differentiation trajectories of adipocytes and the effects of CLA on muscle fiber type transformation. The reproducibility of these findings across different (nutritional vs genetic) models enhances the reliability of the results.

    Advanced data analysis: The integration of pseudotemporal trajectory analysis and cell-cell communication analysis allows for a comprehensive understanding of the functional implications of the cellular changes observed.

    Practical relevance: The findings have significant implications for improving meat quality, which is valuable for both the agricultural and food industry.

    Weaknesses:

    Model generalizability: While pigs are excellent models for human physiology, the translation of these findings to human health, especially in diverse populations, needs careful consideration.

  11. eLife

    Reviewer #2 (Public Review):

    Summary:

    This study comprehensively presents data from single nuclei sequencing of Heigai pig skeletal muscle in response to conjugated linoleic acid supplementation. The authors identify changes in myofiber type and adipocyte subpopulations induced by linoleic acid at depth previously unobserved. The authors show that linoleic acid supplementation decreased the total myofiber count, specifically reducing type II muscle fiber types (IIB), myotendinous junctions, and neuromuscular junctions, whereas type I muscle fibers are increased. Moreover, the authors identify changes in adipocyte pools, specifically in a population marked by SCD1/DGAT2. To validate the skeletal muscle remodeling in response to linoleic acid supplementation, the authors compare transcriptomics data from Laiwu pigs, a model of high intramuscular fat, to Heigai pigs. The results verify changes in adipocyte subpopulations when pigs have higher intramuscular fat, either genetically or diet-induced. Targeted examination using cell-cell communication network analysis revealed associations with high intramuscular fat with fibro-adipogenic progenitors (FAPs). The authors then conclude that conjugated linoleic acid induces FAPs towards adipogenic commitment. Specifically, they show that linoleic acid stimulates FAPs to become SCD1/DGAT2+ adipocytes via JNK signaling. The authors conclude that their findings demonstrate the effects of conjugated linoleic acid on skeletal muscle fat formation in pigs, which could serve as a model for studying human skeletal muscle diseases.

    Strengths:

    The comprehensive data analysis provides information on conjugated linoleic acid effects on pig skeletal muscle and organ function. The notion that linoleic acid induces skeletal muscle composition and fat accumulation is considered a strength and demonstrates the effect of dietary interactions on organ remodeling. This could have implications for the pig farming industry to promote muscle marbling. Additionally, these data may inform the remodeling of human skeletal muscle under dietary behaviors, such as elimination and supplementation diets and chronic overnutrition of nutrient-poor diets. However, the biggest strength resides in thorough data collection at the single nuclei level, which was extrapolated to other types of Chinese pigs.

    Weaknesses:

    While the authors generated a sizeable comprehensive dataset, cellular and molecular validation needed to be improved. For example, the single nuclei data suggest changes in myofiber type after linoleic acid supplementation, yet these data are not validated by other methodologies. Similarly, the authors suggest that linoleic acid alters adipocyte populations, FAPs, and preadipocytes; however, no cellular and molecular analysis was performed to reveal if these trajectories indeed apply. Attempts to identify JNK signaling pathways appear superficial and do not delve deeper into mechanistic action or transcriptional regulation. Notably, a variety of single cell studies have been performed on mouse/human skeletal muscle and adipose tissues. Yet, the authors need to discuss how the populations they have identified support the existing literature on cell-type populations in skeletal muscle. Moreover, the authors nicely incorporate the two pig models into their results, but the authors only examine one muscle group. It would be interesting if other muscle groups respond similarly or differently in response to linoleic acid supplementation. Further, it was unclear whether Heigai and Laiwu pigs were both fed conjugated linoleic acid or whether the comparison between Heigai-fed linoleic acid and Laiwu pigs (as a model of high intramuscular fat). With this in mind, the authors do not discuss how their results could be implicated in human and pig nutrition, such as desirability and cost-effectiveness for pig farmers and human diets high in linoleic acid. Notably, while single nuclei data is comprehensive, there needs to be a statement on data deposition and code availability, allowing others access to these datasets. Moreover, the experimental designs do not denote the conjugated linoleic acid supplementation duration. Several immunostainings performed could be quantified to validate statements. This reviewer also found the Nile Red staining hard to interpret visually and did not appear to support the conclusions convincingly. Within Figure 7, several letters (assuming they represent statistical significance) are present on the graphs but are not denoted within the figure legend.

  12. Version published to 10.1101/2024.05.30.596608v1 on bioRxiv