A maternal high-fat diet modulates fetal SIRT1 histone and protein deacetylase activity in nonhuman primates

Abstract

In nonhuman primates, we previously demonstrated that a maternal high-fat diet (MHFD) induces fetal nonalcoholic fatty liver disease (NAFLD) and alters the fetal metabolome. These changes are accompanied by altered acetylation of histone H3 (H3K14ac). However, the mechanism behind this alteration in acetylation remains unknown. As SIRT1 is both a lysine deacetylase and a crucial sensor of cellular metabolism, we hypothesized that SIRT1 may be involved in fetal epigenomic alterations. Here we show that in utero exposure to a MHFD, but not maternal obesity per se, increases fetal H3K14ac with concomitant decreased SIRT1 expression and diminished in vitro protein and histone deacetylase activity. MHFD increased H3K14ac and DBC1-SIRT1 complex formation in fetal livers, both of which were abrogated with diet reversal despite persistent maternal obesity. Moreover, MHFD was associated with altered expression of known downstream effectors deregulated in NAFLD and modulated by SIRT1 (e.g., PPARΑ, PPARG, SREBF1, CYP7A1, FASN, and SCD). Finally, ex vivo purified SIRT1 retains deacetylase activity on an H3K14ac peptide substrate with preferential activity toward acetylated histone H3; mutagenesis of the catalytic domain of SIRT1 (H363Y) abrogates H3K14ac deacetylation. Our data implicate SIRT1 as a likely molecular mediator of the fetal epigenome and metabolome under MHFD conditions.

Figures

Figure 1.

Fetal hepatic histone H3K14 acetylation, HDAC activity, and HAT expression are altered by virtue of maternal diet exposure. We have previously reported that H3K14 acetylation is increased with exposure to an MHFD. A) Here we show that H3K14ac levels return to those of control-diet levels with diet reversal exposure (n=6/group). A representative Western blot is shown at bottom. B) Using a commercially available kit to measure class III HDAC activity, we found that activity is decreased with HFD exposure. Levels remain decreased in diet-reversal animals (n=6/group). C) Using qPCR, we measured GCN5 expression levels in fetal hepatic tissue. We found increased GCN5 levels that correspond with increased H3K14 acetylation levels in HFD-exposed animals. GCN5 expression is similar to controls with diet reversal (n=10 control, 6 HFD, and 6 reversal). *P < 0.05.

Figure 2.

Fetal hepatic SIRT1 expression, protein, and activity decrease with HFD exposure. A, B) SIRT1 mRNA expression levels (A) and SIRT1 protein levels (B) decrease with HFD exposure. A representative SIRT1 Western blot is included at bottom. C) SIRT1 activity was measured using a commercially available kit (see Materials and Methods) using p53 acetylated substrate (n=9 control, 6 HFD, and 7 reversal for A–C). D) DBC1-Sirt1 interaction in HFD-fed primate liver. Equivalent amounts of liver protein homogenates from primates fed either control diet or an HFD or exposed to diet reversal were subjected to immunoprecipitation with Sirt1, followed by Western blot analysis with DBC1 (see Materials and Methods). Fold change relative to control-diet-fed animals expressed relative to IgG input. E) Representative Western blot for DBC1 following anti-SIRT1 immunoprecipitation demonstrates significant increased fetal DBC1 in association with in utero HFD exposure. *P < 0.05.

Figure 3.

SIRT1-associated genes are altered with HFD exposure. A–D) We found that PPARG (A), PPARA (B), SREBF1 (C), and CYP7A1 (D) are all increased with HFD exposure in macaque fetal liver. E) FASN. F) SCD. Except for PPARA (B), whose expression levels remain elevated, all genes show levels similar to control-diet-exposed animals with diet reversal (n=10 control, 6 HFD, and 6 reversal). *P < 0.05.

Figure 4.

Catalytically active SIRT1 deacetylates H3K14ac and has preference for acetylated histone H3. A) Cos-1 cells were used for expression and purification of huSIRT1. Cos-1 cells have a low level of expression of endogenous SIRT1, as seen with empty vector (pcDNA3.1), but levels increase with expression of huSIRT1 or huSIRT1H363Y. B) Mass spectrometric analysis identifies H3K14ac as a SIRT1 substrate. Spectrum lanes 1 and 2 shows no-peptide and no-lysate controls, while lane 3 shows that H3K14ac peptide, plus commercially available purified recombinant huSIRT1, demonstrates a shift of −42 Da, indicative of complete deacetylation of K14 by SIRT1. Cos cell lysate (lane 4) without transfected Sirt1 lacks detectable H3K14 deacetylase activity (no −42 Da shift). However, with transfection of huSirt1 (lane 5) we observe again a −42 Da shift specific to H3K14 deacetylation, and addition of resveratrol (a SIRT1 agonist) to the Cos lysate (lane 6) preserves deacetylation. Transfection with dominant-negative huSirt1 H363Y into Cos cells without (lane 7) or with (lane 8) resveratrol abrogates this observed deacetylation of H3K14ac. C) Quantitative mass spectrometry shows H3K14ac and H3K9ac as preferential substrates for SIRT1 relative to a tetraacetylated H4 (K5K8K12K16) peptide.