Research ArticlePHYSIOLOGY

Acute sleep loss results in tissue-specific alterations in genome-wide DNA methylation state and metabolic fuel utilization in humans

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Science Advances  22 Aug 2018:
Vol. 4, no. 8, eaar8590
DOI: 10.1126/sciadv.aar8590
  • Fig. 1 Acute sleep loss induces changes in DNA methylation in adipose tissue in healthy humans.

    (A) Participants were investigated both after a night of sleep loss (that is, overnight wakefulness) and after a night of normal sleep, in each condition after an in-lab baseline day and night (26 hours in total, with an 8.5-hour baseline sleep opportunity) with standardized physical activity levels and isocaloric meals. Biopsies from the vastus lateralis muscle (VLM) and subcutaneous adipose tissue (SAT), as well as fasting blood sampling, preceded an oral glucose tolerance test (OGTT) and subsequent blood sampling. This was followed by a pipeline of omic analyses across tissues. (B) Differentially methylated regions (DMRs; FDR < 0.05) in adipose tissue showing DNA methylation (beta levels) after sleep and sleep loss (wake) across the 15 participants, with hierarchical clustering of DMR beta levels (z scores). (C) Significant gene ontology (GO) annotations based on hypermethylated (top) and hypomethylated DMRs (bottom) in adipose tissue in response to sleep loss, showing the ratio of differentially expressed gene-associated DMRs (DE) to the total number (N) of genes in a given pathway (“DE-to-N”) and adjusted P values (q values, FDR< 0.05). (D) Beta levels across some of the most significant DMRs in adipose tissue, in proximity to the specified genes, following sleep and sleep loss.

  • Fig. 2 Tissue-specific transcriptomic alterations in response to acute sleep loss in healthy humans.

    (A) Relative expression levels of differentially expressed genes (FDR < 0.05) in VLM showing levels across both VLM and SAT in both sleep and wake states (left; normalized by row, that is, all rows share the same mean and the same variance; the scale is truncated at −1 and 1). The fold changes for each tissue in response to sleep loss (that is, overnight wakefulness, wake) are also shown (right). (B) Corresponding analysis as shown in (A) for genes differentially expressed in adipose tissue in response to sleep loss. (C) Venn diagram displaying the number and overlap for significantly up- and down-regulated genes in each tissue following sleep loss. (D) GSEA using the R package GAGE against the KEGG ontology showing significant pathways (q values, with FDR < 0.05; scale shown to the right) that are down-regulated in VLM compared with pathways up-regulated in SAT in response to sleep loss (see table S4, A to D, for a complete list of all up- and down-regulated pathways in each tissue). fc, fold change.

  • Fig. 3 Acute sleep loss down-regulates protein levels in the glycolysis pathway in skeletal muscle of healthy young men.

    (A) KEGG pathway analysis of significantly altered VLM proteins (via mass spectrometry) in the morning following sleep loss compared with after a night of normal sleep (n = 15 pairs; see also Table 1 and table S5). Shown as ratio of differentially expressed proteins in relation to total number of proteins in pathway (DE-to-N), and as adjusted P values (q values; FDR < 0.05) for pathways based on up-regulated (top) and down-regulated (bottom) proteins. (B) Immunoblot analysis of PFK1 in VLM (P = 0.009), normalized to loading control (loading control shown in fig. S4A; showing 8 representative pairs out of a total of 13 analyzed pairs); quantified in the bottom for sleep loss [wake (w)] compared with normal sleep (s). qPCR analyses of significant proteomic hits in response to sleep loss in (C) VLM and in (D) SAT (P = 0.027 for FBP2 in VLM; P = 0.031 for PGK1 in adipose tissue for hypothesized contrasts between sleep versus sleep loss). Solid black bars represent values after sleep (set to 1); white bars indicate values obtained after sleep loss (n = 15 pairs for both tissues). FBP2, fructose-bisphosphatase 2; LTF, lactotransferrin; PFKM, 6-phosphofructokinase, muscle type; PKM, pyruvate kinase muscle isozyme. *P < 0.05 and **P < 0.01; two-sided t tests. TH17, T helper 17; IL-17, interleukin 17.

  • Fig. 4 Acute sleep loss induces tissue-specific changes in clock genes and downstream pathways in healthy young men.

    Representative blots for protein abundance of BMAL1 in (A) skeletal muscle (VLM; P = 0.017; showing 8 representative pairs out of a total of 13 analyzed pairs) and in (B) SAT (P = 0.51; 6 representative pairs out of 11 analyzed pairs shown), (C) with quantification, after a night of sleep (s) and a night of sleep loss (wake or w). Western blots were normalized to loading control (see fig. S4, B and C; expression shown relative to controls that were set to 1). (D) Transcriptomic changes in core circadian clock genes, with log2 fold change for each of the investigated tissues (VLM and SAT, n = 15 pairs for each tissue), after sleep loss (wake) compared with after normal sleep (all FDR > 0.05). (E and F) Relative gene expression of targeted genes based on qPCR (PDK4: P = 0.007; all other P > 0.10, n = 15 pairs for each tissue). BMAL1, brain and muscle Arnt-like protein-1; GLUT4, glucose transporter 4; PDK4, pyruvate dehydrogenase kinase isozyme 4; PPARD/PPARG, peroxisome proliferator–activated receptor delta (PPARD)/gamma (PPARG); s, sleep; w, wake (sleep loss). *P < 0.05 and **P < 0.05; two-sided t tests.

  • Fig. 5 Hierarchical clustering analyses reveal close relationship between changes in serum and skeletal muscle metabolite levels in response to acute sleep loss.

    (A) Shared metabolites across subcutaneous adipose tissue, skeletal muscle, and fasting serum. Rows indicate metabolites—based on gas chromatography mass spectrometry (GCMS) metabolomic data—and have been ranked according to relatedness in terms of (i) changes across tissues (column-wise ranking) and (ii) fold changes across metabolites, following sleep loss (wake) (using log2 values for the sleep loss/sleep ratio). The degree of changes in metabolites following sleep loss is color-coded, with red indicating increased levels and blue indicating decreased levels (sleep loss/sleep). Metabolite set enrichment analysis for (B) skeletal muscle and serum metabolites and for (C) subcutaneous adipose tissue metabolites. n = 13 for each tissue.

  • Table 1 Proteins with significantly changed abundance in skeletal muscle and adipose tissue in response to acute sleep loss in humans.

    Proteins (A) up-regulated and (B) down-regulated in skeletal muscle (vastus lateralis muscle) and up-regulated in (C) subcutaneous adipose tissue in response to sleep loss compared with normal sleep. No proteins were found to be down-regulated in subcutaneous adipose tissue. Protein IDs shown are limited to 3. n = 15 pairs for each tissue; two-sided t tests.

    Protein IDsProtein names (gene name in parentheses)Log2 ratio
    (sleep loss/sleep)
    SEMP
    A. Up-regulated proteins in skeletal muscle
    O14558Heat shock protein beta-6 (HSPB6)0.330.430.012
    P07195l-Lactate dehydrogenase B chain; l-lactate dehydrogenase (LDHB)0.370.510.017
    P08238Heat shock protein HSP 90-beta (HSP90AB1)0.120.160.018
    P35609; P35609-2Alpha-actinin-2 (ACTN2)0.090.130.033
    Q2TBA0; Q2TBA0-2Kelch-like protein 40 (KLHL40)0.380.550.035
    Q9GZV1Ankyrin repeat domain-containing protein 2 (ANKRD2)0.440.720.038
    Q5XKP0Protein QIL1 (QIL1)0.220.260.040
    C9JFR7; P99999Cytochrome c (CYCS)0.210.320.042
    O14949Cytochrome b-c1 complex subunit 8 (UQCRQ)0.230.370.049
    B. Down-regulated proteins in skeletal muscle
    O00757Fructose-1.6-bisphosphatase isozyme 2 (FBP2)−0.270.280.003
    Q9NR12-6PDZ and LIM domain protein 7 (PDLIM7)−0.310.350.005
    P00558; P00558-2Phosphoglycerate kinase 1 (PGK1)−0.220.220.006
    Q9UKS6Protein kinase C and casein kinase substrate in neurons protein 3 (PACSIN3)−0.180.190.011
    P02585Troponin C, skeletal muscle (TNNC2)−0.270.320.012
    P14543-2; P14543Nidogen-1 (NID1)−0.310.350.013
    P14618-2Pyruvate kinase (PKM)−0.210.260.015
    Q08043Alpha-actinin-3 (ACTN3)−0.400.470.017
    Q5T7C4; Q5T7C6; P09429High mobility group protein B1 (HMGB1)−0.190.230.019
    Q8N142; Q8N142-2Adenylosuccinate synthetase isozyme 1 (ADSSL1)−0.140.200.022
    P61586; Q5JR08; P08134Transforming protein RhoA; Rho-related guanosine
    5′-triphosphate–binding protein RhoC (RHOA; RHOC)
    −0.170.220.024
    P55786; P55786-2Puromycin-sensitive aminopeptidase (NPEPPS)−0.080.110.026
    Q14324Myosin-binding protein C, fast-type (MYBPC2)−0.370.560.026
    P04075Fructose-bisphosphate aldolase A; Fructose-bisphosphate aldolase (ALDOA)−0.140.200.027
    P50995-2; P50995Annexin A11 (ANXA11)−0.330.280.028
    P62942; Q5W0X3Peptidyl-prolyl cis-trans isomerase FKBP1A; peptidyl-prolyl
    cis-trans isomerase (FKBP1A; FKBP12-Exip2)
    −0.120.170.030
    P17612; P17612-2; P22694-4cAMP-dependent protein kinase catalytic subunit alpha; cAMP-dependent
    protein kinase catalytic subunit beta (PRKACA; PRKACB; KIN27)
    −0.130.190.032
    P00338; P00338-3; P00338-4l-Lactate dehydrogenase A chain (LDHA)−0.210.310.036
    P07108; P07108-3; P07108-2Acyl-CoA–binding protein (DBI)−0.170.260.037
    P12882Myosin-1 (MYH1)−0.931.380.038
    P08237; P08237-3; P08237-26-Phosphofructokinase, muscle type (PFKM)−0.160.260.040
    P05976Myosin light chain 1/3, skeletal muscle isoform (MYL1)−0.160.240.041
    Q0VAK6; Q0VAK6-2Leiomodin-3 (LMOD3)−0.230.310.043
    C. Up-regulated proteins in subcutaneous adipose tissue
    P09211Glutathione S-transferase (GSTP)0.230.060.004
    P02788Lactotransferrin (LTF)0.950.260.004
    P00558; P00558-2Phosphoglycerate kinase 1 (PGK1)0.230.080.011

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/4/8/eaar8590/DC1

    Fig. S1. Correlations between methylation and gene expression levels in subcutaneous adipose tissue and skeletal muscle at baseline and in response to acute sleep loss in humans.

    Fig. S2. Insulin sensitivity is adversely affected, and cortisol levels are significantly elevated following acute sleep loss in healthy young men without changes to protein levels of mitochondrial complexes.

    Fig. S3. Gene expression and protein levels correlate at baseline but not in response to sleep loss in subcutaneous adipose tissue and skeletal muscle in humans.

    Fig. S4. Loading controls for Western blots used throughout the manuscript.

    Table S1. Data for the 15 participants that were included in the study.

    Table S2. DMRs and enriched biological pathways based on methylation changes in subcutaneous adipose tissue in response to acute sleep loss.

    Table S3. Differentially expressed genes in skeletal muscle and subcutaneous adipose tissue in response to acute sleep loss.

    Table S4. Altered pathways and transcription factors based on RNA-seq data from skeletal muscle and subcutaneous adipose tissue in response to acute sleep loss compared with normal sleep.

    Table S5. Enriched pathways based on proteomic analyses of skeletal muscle tissue in response to acute sleep loss compared with normal sleep.

    Table S6. Changes in serum, skeletal muscle, and subcutaneous adipose tissue metabolites in response to acute sleep loss.

    Supplementary Methods

    References (6282)

  • Supplementary Materials

    This PDF file includes:

    • Fig. S1. Correlations between methylation and gene expression levels in subcutaneous adipose tissue and skeletal muscle at baseline and in response to acute sleep loss in humans.
    • Fig. S2. Insulin sensitivity is adversely affected, and cortisol levels are significantly elevated following acute sleep loss in healthy young men without changes to protein levels of mitochondrial complexes.
    • Fig. S3. Gene expression and protein levels correlate at baseline but not in response to sleep loss in subcutaneous adipose tissue and skeletal muscle in humans.
    • Fig. S4. Loading controls for Western blots used throughout the manuscript.
    • Table S1. Data for the 15 participants that were included in the study.
    • Table S2. DMRs and enriched biological pathways based on methylation changes in subcutaneous adipose tissue in response to acute sleep loss.
    • Table S3. Differentially expressed genes in skeletal muscle and subcutaneous adipose tissue in response to acute sleep loss.
    • Table S4. Altered pathways and transcription factors based on RNA-seq data from skeletal muscle and subcutaneous adipose tissue in response to acute sleep loss compared with normal sleep.
    • Table S5. Enriched pathways based on proteomic analyses of skeletal muscle tissue in response to acute sleep loss compared with normal sleep.
    • Table S6. Changes in serum, skeletal muscle, and subcutaneous adipose tissue metabolites in response to acute sleep loss.
    • Supplementary Methods
    • References (6282)

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