N-acetyltaurine and Acetylcarnitine Production for the Mitochondrial Acetyl-CoA Regulation in Skeletal Muscles during Endurance Exercises

During endurance exercises, a large amount of mitochondrial acetyl-CoA is produced in skeletal muscles from lipids, and the excess acetyl-CoA suppresses the metabolic flux from glycolysis to the TCA cycle. This study evaluated the hypothesis that taurine and carnitine act as a buffer of the acetyl moiety of mitochondrial acetyl-CoA derived from the short- and long-chain fatty acids of skeletal muscles during endurance exercises. In human subjects, the serum concentrations of acetylated forms of taurine (NAT) and carnitine (ACT), which are the metabolites of acetyl-CoA buffering, significantly increased after a full marathon. In the culture medium of primary human skeletal muscle cells, NAT and ACT concentrations significantly increased when they were cultured with taurine and acetate or with carnitine and palmitic acid, respectively. The increase in the mitochondrial acetyl-CoA/free CoA ratio induced by acetate and palmitic acid was suppressed by taurine and carnitine, respectively. Elevations of NAT and ACT in the blood of humans during endurance exercises might serve the buffering of the acetyl-moiety in mitochondria by taurine and carnitine, respectively. The results suggest that blood levels of NAT and ACT indicate energy production status from fatty acids in the skeletal muscles of humans undergoing endurance exercise

Identification of Nedd9 as a TGF-β-Smad2/3 Target Gene Involved in RANKL-Induced Osteoclastogenesis by Comprehensive Analysis.

TGF-ß is a multifunctional cytokine that is involved in cell proliferation, differentiation and function. We previously reported an essential role of the TGF-ß -Smad2/3 pathways in RANKL-induced osteoclastogenesis. Using chromatin immunoprecipitation followed by sequencing, we comprehensively identified Smad2/3 target genes in bone marrow macrophages. These genes were enriched in the gene population upregulated by TGF-ß and downregulated by RANKL. Recent studies have revealed that histone modifications, such as trimethylation of histone H3 lysine 4 (H3K4me3) and lysine 27 (H3K27me3), critically regulate key developmental steps. We identified Nedd9 as a Smad2/3 target gene whose histone modification pattern was converted from H3K4me3(+)/H3K4me27(+) to H3K4me3(+)/H3K4me27(-) by TGF-ß. Nedd9 expression was increased by TGF-ß and suppressed by RANKL. Overexpression of Nedd9 partially rescued an inhibitory effect of a TGF-ß inhibitor, while gene silencing of Nedd9 suppressed RANKL-induced osteoclastogenesis. RANKL-induced osteoclastogenesis were reduced and stimulatory effects of TGF-ß on RANKL-induced osteoclastogenesis were partially abrogated in cells from Nedd9-deficient mice although knockout mice did not show abnormal skeletal phenotypes. These results suggest that Nedd9 is a Smad2/3 target gene implicated in RANKL-induced osteoclastogenesis

Identification of Smad2/3 binding sites.

<p>(A) BMMs were treated with 2 ng/ml TGF-ß for 1.5 h and cells were subjected to ChIP-seq analysis using anti-Smad2/3 antibody. Three known TGF-ß target genes (<i>Cdkn1a</i>, <i>Serpine1</i>, <i>and Smad7</i>) and a negative control gene (<i>Hprt1</i>) were analyzed as representative examples. Smad2/3-binding regions (SBRs; peak signal ratio ≥8) and the peak position of each SBR are shown by black bars. (B) Eight positive regions and two negative regions for Smad2/3 binding were selected from ChIP-seq data and validated by realtime PCR. ChIP using mouse IgG was used as control. Values are presented as n-fold enrichment over Hprt1. (C) Average Smad2/3 signal profile around transcriptional start site (TSS) in ChIP-seq analysis. Smad2/3 binding was enriched around TSS.</p

Impaired osteoclastogenesis in Nedd9-/- BMMs.

<p>(A) BMMs were isolated from <i>Nedd9</i> knockout (KO) and wild-type (WT) mice and cultured in the presence of RANKL with or without TGF-ß. Osteoclastogenesis was evaluated by TRAP staining (A), the number of multi-nuclear osteoclasts (B) and the expression of Cathepsin K protein (C).</p

Nedd9 is critical for osteoclast differentiation.

<p>(A) Realtime PCR analysis of effects of retroviral overexpression of <i>Nedd9</i> gene on RANKL-induced osteoclastogenesis, as indicated by expression of <i>Nedd9</i> and <i>Cathepsin K</i> mRNAs. (B) Effects of SB431542 (SB) treatment on osteoclastogenesis, as evaluated by TRAP staining, in <i>Nedd9</i>-overexpressing cells treated with RANKL. Overexpression of <i>Nedd9</i> increased RANKL-induced osteoclastogenesis. SB431542 suppressed osteoclastogenesis, which was partly recovered by <i>Nedd9</i> overexpression. Cultures were stained by TRAP. Bars = 100 μm. (C) The expression of <i>Nedd9</i> gene as determined by realtime PCR. (D) The number of TRAP positive osteoclasts was significantly increased by <i>Nedd9</i> overexpression, and the suppression of osteoclastogenesis by SB431542 was partly suppressed by <i>Nedd9</i> overexpression. *<i>P</i> < 0.05.</p

Epigenetic regulation of Nedd9 gene during osteoclastogenesis.

<p>(A) Smad2/3 binding and histone modification changes of <i>Nedd9</i> gene during osteoclastogenesis. Histone modification patterns in BMMs changed from K4(+)K27(+) to K4(+)K27(-) patterns by TGF-ß and returned to K4(+)K27(+) patterns after RANKL treatment. (B) <i>Nedd9</i> mRNA expression after TGF-ß or RANKL stimulation. The expression increased by TGF-ß stimulation and was reduced after RANKL treatment. The expression remained at low levels in the presence of SB431542.</p