Elevated S-Adenosylhomocysteine Alters Adipocyte Functionality With Corresponding Changes in Gene Expression and Associated Epigenetic Marks
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Figures
- Figure 1
SAH impairs adipocyte glucose uptake and lipolysis without affecting differentiation. Total cell number (A) and intracellular protein (B) were measured in preadipocytes at 90% confluence (Pre-DF), postconfluence at onset of differentiation (D0), or in adipocytes differentiated for 3 days (D3) or 7 days (D7) in the absence or presence of SAH. C: Dlk1 RNA level in preadipocytes before onset of differentiation (D0) and at day 5 (D5) after induction of differentiation in the absence or presence of SAH. D: Mature adipocytes were stimulated or not with insulin (100 nmol/L, 20 min) before assays. Basal and insulin-stimulated glucose uptake was then assessed. Adipocytes with and without SAH treatment were measured for total intracellular lipid (E) or stimulated with isoproterenol or not (basal) (F). Then, media were collected to measure cellular glycerol release. Statistical significance: ^Relative to basal cells without SAH treatment. #Relative to insulin-stimulated cells without SAH treatment. n.s., not significant. Results are mean ± SEM from three independent experiments and four independent experiments for glucose uptake.
- Figure 2
SAH selectively inhibits a subset of adipogenic genes. Adipocytes treated with the indicated SAH doses were harvested 5 days after differentiation, and RNA was extracted to measure expression of genes using RT-qPCR. Results are mean target-to-Ppia ratio ± SEM from three independent experiments.
- Figure 3
SAH exposure selectively increases DNA methylation on Rxrα gene. CpG methylation (CpG-Me) in adipocytes treated with 10 µmol/L and 100 µmol/L SAH (or not) were measured for Klf4 (A), Cebpβ (B), and Rxrα (C). Schematics of genomic regions depict measured CpG sites; position numbered relative to start of coding region (+1). ■, exon. Graphs: Genomic coordinates refer to location of CpG sites. The CpG-to-Me ratio for each experiment (○) was averaged from three independent experiments (
) rather than (■). - Figure 4
SAH exposure increases H3 modifications and H3K27me3 occupancy at target gene promoters in mature adipocytes. A: 3T3-L1 preadipocytes were undifferentiated (PreAd) or differentiated (Ad) at the indicated SAH doses. Histones were extracted and immunoblotted (top panel). Graphs: Mean band intensity ± SEM from three independent experiments. Statistical significance: relative to differentiated adipocytes without SAH treatment using Tukey multiple comparison test. Data were normalized to pan H3 band intensities. B: H3K27me3 ChIP was performed in mature adipocytes with or without 100 μmol/L SAH treatment. Data are mean ± SEM from three independent experiments, each done in duplicates. Statistical significance: relative to respective IgG ChIP using independent sample Student t test.
- Figure 5
Exogenous SAH exposure elevates intracellular SAH but not SAM and Hcy in mature adipocytes. Preadipocytes were differentiated over 7 days at the indicated (exogenous) SAH doses. Media at the end of the treatment regimen (A) and the cells (B) were harvested in parallel to measure SAH, SAM, and Hcy levels. Values are mean ± SD from two separate experiments. SAH level in the untreated controls for media (A) and cell extract (B) was below the limit of detection (<0.02 µmol/L). As such, the graphs depict upper bound values on the mean for SAH.
Tables
- Table 1
Primer sequences for RT-qPCR and ChIP-qPCR
Primers and primer ID Sequence RT-qPCR Pparγ2-F 5′-TTCTCCTGTTGACCCAGAGC-3′ Pparγ2-R 5′-CCATGGTAATTTCTTGTGAAGTGC-3′ Cebpα-F 5′-TGGACAAGAACAGCAACGAG-3′ Cebpα-R 5′-GTCATTGTCACTGGTCAACTCC-3′ Cebpβ-F 5′-GAGCGACGAGTACAAGATGC-3′ Cebpβ-R 5′-GAGCGACGAGTACAAGATGC-3′ Klf4-F 5′-ATTAATGAGGCAGCCACCTG-3′ Klf4-R 5′-ACGCAGTGTCTTCTCCCTTC-3′ Rxrα-F 5′-GTCGAGCCCAAGACTGAGAC-3′ Rxrα-R 5′-AACAGGGTCATTTGGTGAGC-3′ Ppia-F 5′-TACAGGTCCTGGCATCTTGTC-3′ Ppia-R 5′-ATCCAGCCATTCAGTCTTGG-3′ ChIP-PCR Klf4-F 5′-AGCGCGACACTCACGTTAGTCG-3′ Klf4-R 5′-TGACATGGCTGTCAGCGACG-3′ Cebpα-F 5′-ACTGGCGCCTTCGATCCGAGA-3′ Cebpα-R 5′-AGCTTCGGGTCGCGAATGGC-3′ Rxrα-F 5′-CCGGGCCTCTGACTTGCCGA-3′ Rxrα-R 5′-CCCTCTCCACGTCCCGAGCG-3′ F, forward; R, reverse.
- Table 2
Primer sequences for Sequenom EpiTyper MassArray
Primer ID Sequence (in upper case) plus Tag (in lower case) Template strand Klf4-2.8 kb-F 5′-aggaagagagTAGTTGGATAAAGTGGGTGAAGAGT-3′ + Klf4-2.8 kb-R 5′-cagtaatacgactcactatagggagaaggctAAACAAACAAACAAAAACAAACAAA-3′ + Klf4-4.5 kb-F 5′-aggaagagagTGTTTTTTTGAGTTAGGGATTTTTT-3′ + Klf4-4.5 kb-R 5′-cagtaatacgactcactatagggagaaggctTAACTCCACAAACTAAACTCAATCC-3′ + Cebpβ-4.1 kb-F 5′-aggaagagagGGGATTGTAGGAGTGATTTGAGTATT-3′ − Cebpβ-4.1 kb-R 5′-cagtaatacgactcactatagggagaaggctACTAAAAACCAAAAAAATCCCCTTA-3′ − Cebpβ-0.6 kb-F 5′-aggaagagagGGATTTTTAATTTTTGGGAAATAGA-3′ − Cebpβ-0.6 kb-R 5′-cagtaatacgactcactatagggagaaggctACAAAATAACTCACCCAAACACAAT-3′ − Rxrα_int1-F 5′-aggaagagagTTTATTAATATGGGGAGTTTGGAGA-3′ + Rxrα_int1-R 5′-cagtaatacgactcactatagggagaaggctAATACAACCTACACCAAACCACAAC-3′ + F, forward; R, reverse.
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