(*p? ?0.05, versus control group (?) using one-way ANOVA). metabolites1. In particular, the gut microbiota generates the short chain fatty acids (SCFAs) acetate, propionate and butyrate2. Butyrate is usually a SCFA known to act as a histone deacetylase inhibitor (HDACi), favoring histone acetylation and thus remodeling of chromatin towards an open and transcriptionally qualified state3. Microbiota-produced butyrate is also a main energy source for intestinal cells, Rabbit polyclonal to ZAK in particular colonocytes, and its absence promotes colonocyte autophagy4. A key metabolite bringing a strong chemical similarity to butyrate is usually R–hydroxybutyrate, the major ketone body of the organism, produced within the mitochondria, mainly by liver. Under physiological conditions, hepatic R–hydroxybutyrate production is enhanced by fasting or intense exercise and provides an alternative energy source replacing the low availability of glucose5. Butyrate supplementation in the diet induced an improvement in insulin sensitivity linked to augmented energy expenditure in mice6. The metabolic improvements brought by butyrate were due to (i) an increase of PGC1 and CPT1b (carnitine palmitoyltransferase Ib) mRNA expression in Tetrodotoxin skeletal muscle mass, two genes involved in mitochondrial biogenesis and fatty acid metabolism respectively6 and (ii) improved hepatic mitochondrial efficiency7. In rat myotubes, we have shown that butyrate induced histone hyperacetylation, in accordance to its HDACi activity, and alleviated palmitate-induced insulin resistance hyperacetylation in the proximity of IRS1 transcriptional start site, resulting in the overexpression of IRS1 mRNA and protein levels8. Based on dietary administration in mice, cell culture studies and enzymatic assays, it was proposed that R–hydroxybutyrate is an HDACi9. The HDACi activity of R–hydroxybutyrate has been linked to protection against oxidative stress, up-regulation of FOXO3a, catalase and mitochondrial MnSOD29; as well as to anti-inflammatory effects inhibition of the NLRP3 inflammasome10 and a promotion of autophagic flux protecting neurons from cell death11. At the same time, the identification of a novel transcriptionally-activating histone post-translational modification, lysine -hydroxybutyrylation12, suggest that regulation of gene expression by R–hydroxybutyrate might also occur lysine -hydroxybutyrylation12. Based on these prior findings, we made a side-by-side comparison to investigate whether both butyrate and R–hydroxybutyrate, due to their reported HDACi activities, have a comparable biochemical and transcriptional effects in multiple cell types and HDAC activity Cells lysates were prepared by extracting cells in a lysis buffer (20?mM Tris-HCl, 138?mM NaCl, 2.7?mM KCl, 5% (v/v), glycerol, 1?mM sodium-o-vanadate, 1% (v/v) Nonidet P-40, 5?mM EDTA, 20?mM NaF, 1:1000 proteases inhibitors cocktail (Sigma-Aldrich, P2714) pH 8.0) and centrifugation (13,000?g, 15?min, 4?C). Histone-containing residual pellets were incubated overnight at 4?C with 0.2?M HCl to solubilize total histones. Solubilized histones were centrifuged (13,000?g, 15?min at 4?C). Supernatants were collected and neutralized with 1?M Tris prior to protein quantification with the Bradford reagent (BioRad). Histones were separated by 15% SDS-PAGE. Standard immunoblotting procedures and ECL detection were employed. The primary antibodies used in this study are outlined in Table?1. HPR-conjugated anti-rabbit and anti-mouse secondary antibodies were from BioRad. Chemiluminescence was detected on a BioRad ChemidocTM XRS+ apparatus and images were processed using Image Lab 3.0 (BioRad). Table 1 list of main antibodies used in this study. Primary antibodies were diluted at 1:1000 to 1 1:2000. histone deacetylase activity was performed using the HDAC activity fluorometric assay kit (Abcam, cat# ab156064) by following the manufacturers instructions. RNA extraction, reverse transcription and real-time quantitative PCR Total RNA was isolated with TriPure Isolation Reagent Tetrodotoxin (Roche) according to the manufacturers instructions. RNA concentration and purity was verified by optical density (OD) measurement on Tetrodotoxin a Nanodrop 2000 (Thermo Fisher Scientific). cDNA synthesis was performed using the PrimescriptTM RT reverse transcription kit (Takara) according to the manufacturers instructions using 1 g of total RNA in a 20 l reaction volume. Synthesized cDNA was then brought to a 1.2?ml final volume with water. Quantitative PCR amplification was performed using a Rotor-Gene Real-Time PCR System. 5 l of cDNA template, 5 pmoles of forward and reverse primers and 15 l of ABsoluteTM QPCR SYBR Green Mix (ABgene) were added in each reaction. Reactions were incubated at 95C for 10?min, followed by 40 cycles of denaturation (95C for 10?sec), annealing (at gene-specific temperatures for 30?sec, see Table?2 for primer sequences and annealing temperatures) and elongation (72?C for 30?sec). As a quality control, qPCR amplicons.