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Impaired BCAA catabolism in adipose tissues promotes age-associated metabolic derangement

Nature Aging volume 3, pages 982–1000 (2023)Cite this article

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Abstract

Adipose tissues are central in controlling metabolic homeostasis and failure in their preservation is associated with age-related metabolic disorders. The exact role of mature adipocytes in this phenomenon remains elusive. Here we describe the role of adipose branched-chain amino acid (BCAA) catabolism in this process. We found that adipocyte-specific Crtc2 knockout protected mice from age-associated metabolic decline. Multiomics analysis revealed that BCAA catabolism was impaired in aged visceral adipose tissues, leading to the activation of mechanistic target of rapamycin complex (mTORC1) signaling and the resultant cellular senescence, which was restored by Crtc2 knockout in adipocytes. Using single-cell RNA sequencing analysis, we found that age-associated decline in adipogenic potential of visceral adipose tissues was reinstated by Crtc2 knockout, via the reduction of BCAA–mTORC1 senescence-associated secretory phenotype axis. Collectively, we propose that perturbation of BCAA catabolism by CRTC2 is critical in instigating age-associated remodeling of adipose tissue and the resultant metabolic decline in vivo.

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Fig. 1: Adipocyte-specific depletion of Crtc2 suppresses the age-onset metabolic disorder in mice.
Fig. 2: CRTC2 is linked to the age-associated changes in lipid profiling of VAT.
Fig. 3: CRTC2–Hes-1 axis is critical in age-associated increase in plasma BCAA levels.
Fig. 4: Adipocyte-specific Crtc2 knockout reduced cellular senescence in adipose tissues.
Fig. 5: Age-associated decline in adipogenic potential of VAT is restored by Crtc2 deficiency.
Fig. 6: CRTC2-dependent regulation of secretome is critical in age-associated remodeling of VAT.
Fig. 7: Effects of SASP factors on cellular senescence and adipogenic potential of AP cells.
Fig. 8: A proposed model showing the role of CRTC2 in age-associated remodeling of VAT.

Data availability

All the data supporting this work are available as Source Data or Supplementary Information. The data for the mRNA-seq and scRNA-seq were deposited in the National Cancer Center for Biotechnology Information (accession nos. GSE207433 (for mRNA-seq) and PRJNA852570 (for scRNA-seq)). Processed data for mRNA-seq were also deposited in the same location. Processed data for the scRNA-seq are available at https://doi.org/10.5281/zenodo.7949695. MS/MS patterns from features were identified by comparing the experimental data against the METLIN (metlin.scripps.edu) database and the KEGG (https://www.genome.jp/kegg/) database was used to integrate multiomics profiles.

Code availability

Analysis scripts for scRNA-seq data are available at https://github.com/CB-postech/NATURE-AGING-adipose-CRTC2.

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Acknowledgements

This research was supported by a National Research Foundation of Korea grant funded by the Korean Government (MSIT) (NRF-2019M3A9D5A01102794 and NRF-2021R1A2C3003435 (to S.H.K.), NRF-2020R1A2C2007835 (to G.S.H.), NRF-2021M3H9A1030158 (to J.K.K.) and NRF-2021R1A2C2003171 (to M.H.M.)). G.S.H. was supported by the Korea Basic Science Institute (C370000). H.S.H. was supported by NRF-2018R1A6A3A11043165. S.H.K. was supported by a grant from Korea University. The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.

Author information

Author notes

  1. These authors contributed equally: Hye-Sook Han, Eunyong Ahn, Eun Seo Park.

Authors and Affiliations

  1. Division of Life Sciences, Korea University, Seoul, Korea

    Hye-Sook Han, Tom Huh, Seri Choi, Yongmin Kwon, Byeong Hun Choi & Seung-Hoi Koo

  2. Integrated Metabolomics Research Group, Western Seoul Center, Korea Basic Science Institute, Seoul, Korea

    Eunyong Ahn, Jueun Lee & Geum-Sook Hwang

  3. Department of New Biology, DGIST, Daegu, Korea

    Eun Seo Park & Jong Kyoung Kim

  4. Department of Life Sciences, POSTECH, Pohang, Korea

    Yoon Ha Choi, Yujin L. Jeong & Jong Kyoung Kim

  5. Department of Chemistry, Yonsei University, Seoul, Korea

    Gwang Bin Lee & Myeong Hee Moon

  6. Department of Anatomy & Cell Biology, Seoul National University College of Medicine, Seoul, Korea

    Minji Kim & Hang-Rae Kim

  7. Department of Biomedical Sciences, Seoul National University College of Medicine, Seoul, Korea

    Minji Kim, Hyun Mu Shin & Hang-Rae Kim

  8. BK21 FOUR Biomedical Science Project, Seoul National University College of Medicine, Seoul, Korea

    Minji Kim, Hyun Mu Shin & Hang-Rae Kim

  9. Korea Mouse Phenotyping Center, Seoul National University, Seoul, Korea

    Je Kyung Seong

  10. Wide River Institute of Immunology, Seoul National University, Hongcheon, Korea

    Hyun Mu Shin & Hang-Rae Kim

  11. College of Pharmacy, Chung-Ang University, Seoul, Korea

    Geum-Sook Hwang

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  1. Hye-Sook Han
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Contributions

S.H.K., G.S.H. and J.K.K. conceived the idea and developed the study design. H.S.H., E.A., E.S.P., T.H., S.C., Y.K., B.H.C., J.L., Y.H.C., Y.L.J., G.B.L. and M.K. performed experiments and analyzed the data. H.S.H., E.A., E.S.P., J.K.S., H.M.S., H.R.K., M.H.M., J.K.S., G.S.H., K.W.C. and S.H.K. interpreted data and H.S.H., E.A., E.S.P., J.K.K., G.S.H. and S.H.K wrote the manuscript.

Corresponding authors

Correspondence to Jong Kyoung Kim, Geum-Sook Hwang or Seung-Hoi Koo.

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Extended data

Extended Data Fig. 1 Age-induced expression of CRTC2 in VAT affects energy homeostasis.

a. A representative western blot analysis showing relative expression of CRTC2 among the adipose tissues (3-month-old mice) (top). *: non-specific band. The quantitation of CRTC2 bands was also shown (n = 3 mice per group). A representative western blot analysis showing effects of aging on CRTC2 expression in mature adipocytes from VAT, SAT, and BAT (3-month-old mice and 18-month-old mice, n = 4 mice per group for VAT and SAT, and n = 1 mouse for BAT) (bottom). The quantitation of CRTC2 bands from mature adipocytes of VAT and SAT was also shown. b. Schematic diagram showing the targeting strategy for generating adipocyte-specific Crtc2 knockout mice. c and d. Confirmation of adipocyte-specific depletion of Crtc2 in Crtc2 AKO mice. mRNA levels (c, QPCR) and protein levels (d, a representative western blot analysis) of Crtc2 in metabolic tissues showing the specificity of adipocyte-specific depletion of Crtc2 in 4 h-fasted, 3-month-old mice under normal chow diet (NCD). N = 4 mice per group for mRNA analysis and n = 3 mice per group for protein analysis. Data in a and c represent mean ± SEM. P values were determined using one-way ANOVA with Tukey’s multiple comparisons test (a, top) or student’s t-test (a, bottom and c).

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Extended Data Fig. 2 Adipocyte-specific depletion of Crtc2 restores age-associated changes in insulin signaling.

a and b. Effects of aging and adipocyte-specific depletion of Crtc2 on insulin signaling. A representative western blot analysis showing changes in insulin signaling in the liver (a) and VAT (b) of male young Crtc2 f/f mice (3-month-old), old Crtc2 f/f mice (18-month-old), and old Crtc2 AKO mice (18-month-old). N = 2 mice per group for PBS-injected samples, and n = 4 mice per group for insulin-injected samples. Quantitation of p-AKT(S)/AKT, p-AKT(T)/AKT, and p-IR(Y)/IR were also shown. c and d. Effects of adipocyte-specific depletion of Crtc2 on insulin signaling. A representative western blot analysis was shown to measure changes in insulin signaling in the liver (c) and VAT (d) of male young Crtc2 f/f mice (3-month-old), and young Crtc2 AKO mice (3-month-old). N = 2 mice per group for PBS-injected samples, and n = 4 mice per group for insulin-injected samples. Quantitation of p-AKT(S)/AKT, p-AKT(T)/AKT, and p-IR(Y)/IR were also shown. For a-d, statistical analysis was performed only in insulin-injected samples. Data represent mean ± SEM. P values were determined using student’s t-test.

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Extended Data Fig. 3 Adipocyte-specific depletion of Crtc2 restores age-associated changes in lipid profiles of VAT.

Lipid profiling analysis was performed by using male young Crtc2 f/f mice (3-month-old), young Crtc2 AKO mice (3-month-old), old Crtc2 f/f mice (18-month-old), and old Crtc2 AKO mice (18-month-old). a. Lipid profiling analysis of mouse VAT in vivo (n = 7 for young Crtc2 f/f mice, n = 7 for young Crtc2 AKO mice, n = 7 for old Crtc2 f/f mice, and n = 6 for old Crtc2 AKO mice). A heatmap analysis results from significantly differential metabolites (q-values from permutation analysis to test the recovery pattern < 0.05). Measured relative intensities are normalized into z-score across samples. Clustered heatmap was drawn by using R package “heatmap”. Euclidean distance and complete method were used for the clustering. b. Fractional labeling of citrate m + 5 from [U-13C] glutamine via reductive TCA cycle fluxes. Data represent mean ± SEM (n = 3 biological replicates per group). P values were determined using student’s t-test.

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Extended Data Fig. 4 Adipocyte-specific depletion of Crtc2 restores age-associated changes in polar metabolome of VAT.

Metabolomics analysis was performed by using male young Crtc2 f/f mice (3-month-old), old Crtc2 f/f mice (18-month-old), and old Crtc2 AKO mice (18-month-old). A heatmap of metabolites from mouse VAT in vivo, with recovery pattern. Permutation analysis was conducted to detect recovery pattern from metabolic profiling data. Metabolites with q value less than 0.05 were plotted and further curated according to their residing KEGG pathways (n = 7 for young Crtc2 f/f mice, n = 7 for young Crtc2 AKO mice, n = 7 for old Crtc2 f/f mice, and n = 6 for old Crtc2 AKO mice). Measured relative intensities are normalized into z-score across samples.

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Extended Data Fig. 5 Assessment the effect of aging and adipocyte-specific depletion of Crtc2 on transcriptome in mature adipocytes.

RNA sequencing analysis were performed by using male young Crtc2 f/f mice (3-month-old), old Crtc2 f/f mice (18-month-old), and old Crtc2 AKO mice (18-month-old). a. PCA plot of RNA sequencing results (n = 3 mice per group). Each cluster’s center was plotted as a bigger circle using the same colors. b. Box plot of Log2 transformed fold changes of FPKM values of transcriptomic data. 25%, 75%, and the median were presented in the boxplot with whiskers that were 1.5 times of the inerquantile region (n = 3 mice per group, young f/f vs old f/f (left), old f/f vs old AKO (middle), and old AKO vs young f/f (right)). c. GO analysis: Biological Process results of RNA sequencing results (n = 3 mice per group). d. Distribution of -log10(FDR) from GO analysis: Biological Process. e. RNA sequencing results of genes annotated to mmu00280, BCAA degradation, in KEGG pathway (n = 3 mice per group). Measured FPKM values are normalized into z-score across samples.

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Extended Data Fig. 6 Effects of aging and adipocyte-specific depletion of Crtc2 on BCAA catabolism.

a. A representative western blot analysis showing effects of aging and adipocyte-specific depletion of Crtc2 on proteins involved in BCAA catabolism in VAT of male young Crtc2 f/f mice (3-month-old), old Crtc2 f/f mice (18-month-old), and old Crtc2 AKO mice (18-month-old). N = 3 mice per group (left) or n = 4 mice per group (right). Relative intensity of specific bands were shown. Data represent mean ± SEM. b. A representative western blot analysis showing effects of Crtc2 deficiency on proteins involved in BCAA catabolism in differentiated immortalized AP cells from Crtc2 f/f mice and Crtc2 AKO mice (n = 3 biological replicates per group). Relative intensity of specific bands were shown. Data represent mean ± SEM. c. Quantification of HMB and 3-HMG, catabolic intermediates in BCAA metabolism, from VAT of young Crtc2 f/f mice (3-month-old) and young Crtc2 AKO mice (3-month-old) (n = 7 biological replicates per group). Data represent mean ± SEM. P values were determined using student’s t-test (a (left), b, c) or one-way ANOVA with Tukey’s multiple comparisons test (a (right)).

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Extended Data Fig. 7 CRTC2 in adipocytes is critical in age-associated remodeling of VAT.

a. QPCR analysis of genes involve in the fibrosis and ECM (extracellular matrix) remodeling in VAT of male young Crtc2 f/f mice (3-month-old), young Crtc2 AKO mice (3-month-old), old Crtc2 f/f mice (18-month-old), and old Crtc2 AKO mice (18-month-old). Data represent mean ± SEM. For the top figure, n = 10 mice per group for young Crtc2 f/f mice, n = 4 for old Crtc2 f/f mice, and n = 4 for old Crtc2 AKO mice. For the bottom figure, n = 4 mice per group. b. A representative Pico Sirius red staining showing effects of aging and adipocyte-specific depletion of Crtc2 on the fibrotic structure in VAT of male young Crtc2 f/f mice (3-month-old), young Crtc2 AKO mice (3-month-old), old Crtc2 f/f mice (18-month-old), and old Crtc2 AKO mice (18-month-old) (scale bars, 100 μm (top) or 150 μm (bottom)). N = 5 mice per group (top figure) or n = 6 mice per group (bottom figure). Relative intensity was also shown. Data represent mean ± SEM. P values were determined using one-way ANOVA with Tukey’s multiple comparisons test (top figures, a, b) or student’s t-test (bottom figures, a, b).

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Extended Data Fig. 8 Effects of aging and adipocyte-specific depletion of Crtc2 on AP cells in VAT.

a. Heatmap showing relative expression of AP subtype marker genes. b. Heatmap showing relative pathway activity score of AP subtype across conditions. c. Adipocyte progenitor cells (PDGFRα +) isolated from VAT of young Crtc2 f/f mice (3-month-old), young Crtc2 AKO mice (3-mont-old), old Crtc2 f/f mice (20-month-old), and old Crtc2 AKO mice (20-month-old) were analyzed. A representative image showing SA-β-gal staining (scale bars, 150 μm). The staining was performed 1 day after seeding for detecting cellular senescence. Data represent mean ± SEM (n = 7 biological replicates per group). P values were determined using one-way ANOVA with Tukey’s multiple comparisons test.

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Extended Data Fig. 9 Depletion of Crtc2 reduced cellular senescence in cultured adipocytes.

a. Experimental scheme showing treatment of senescence-inducing agents on differentiated adipocytes that were derived from AP cells of Crtc2 f/f mice or Crtc2 AKO mice. b. SA-β-gal staining was performed to assess the effect of cellular senescence. A representative image was shown (scale bars, 150 μm). Relative values for SA-β-gal positive cells were also shown. Data represent mean ± SEM (n = 3 biological replicates each control, n = 4 biological replicates each for bleomycin, and n = 5 biological replicates each for H2O2). c. A representative western blot analysis showing effects of senescence-inducing agents or genetic depletion of Crtc2 on BCAA catabolic pathway as well as cellular senescence. d. QPCR analysis showing effects of senescence-inducing agents or genetic depletion of Crtc2 on Il1b expression. Data represent mean ± SEM (n = 3 biological replicates per group, except control condition of Crtc2 AKO group, where n = 2 biological replicates were used). e. Effects of ADCM (induced by senescence-inducing agent bleomycin) on cellular senescence of AP cells from Crtc2 f/f mice or Crtc2 AKO mice were analyzed. Cells were treated with either control ADCM or ADCM from adipocytes treated with bleomycin, with or without anti-IL-1β antibody or anti-TNF-α antibody for 6 days. To induce adipogenic differentiation, cells were incubated with insulin and rosiglitazone for 5 days, and then stained with oil red O staining. A representative data was shown (scale bars, 150 μm). P values were determined using student’s t-test.

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Extended Data Fig. 10 Role of SASP receptors of adipocytes in age-mediated cellular senescence.

a. Effects of ADCM on cell proliferation of AP cells from WT mice were analyzed. Cells were transfected with control siRNA, siRNA against TNFR1, or siRNA against IL1R1, and then treated with either control ADCM or ADCM from old mice for 5 days. Cell proliferation was assessed every 2 days after plating using the CyQuant assay. Data represent mean ± SEM (n = 3 biological replicates per group). b. Effects of ADCM on cellular senescence of AP cells from WT mice were analyzed. Cells were transfected with control siRNA, siRNA against TNFR, or siRNA against IL-1R, and then treated with either control ADCM or ADCM from old mice for 5 days. SA-β-gal staining was performed for detecting cellular senescence. A representative image was shown (scale bars, 150 μm). Relative values for SA-β-gal positive cells were also shown. Data represent mean ± SEM (n = 3 biological replicates per group). P values were determined using one-way ANOVA with Tukey’s multiple comparisons test.

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Han, HS., Ahn, E., Park, E.S. et al. Impaired BCAA catabolism in adipose tissues promotes age-associated metabolic derangement. Nat Aging 3, 982–1000 (2023). https://doi.org/10.1038/s43587-023-00460-8

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