A STUDY OF SERUM ACID SPHINGOMYELINASE ACTIVITY AND CLINICAL SEVERITY IN INFANTS WITH RESPIRATORY SYNCYTIAL VIRUS BRONCHIOLITIS

Respiratory syncytial virus (RSV) bronchiolitis is the leading cause of hospitalization in infants without any effective treatment strategies. Identification of biomarkers associated with disease severity may be significant in improving management. However, several studies have failed to identify specific biomarkers for bronchiolitis. Serum secretory acid sphingomyelinase (S-ASM) activity has been considered a biomarker of cytokine release, inflammation, and oxidative stress in various diseases. This study aimed to evaluate whether serum S-ASM activity increases and correlates with disease severity in infants with RSV bronchiolitis. Serum S-ASM activity was measured in 31 infants with RSV bronchiolitis, 9 infants with RSV-negative febrile infection, and 8 healthy infants. Laboratory data and clinical observational findings were analyzed for correlation with serum S-ASM activity. Serum S-ASM activity was significantly higher in the 31 infants with RSV bronchiolitis (9.5±5.4 nmol/mL/h) than individuals in the control groups (RSV-negative febrile infection patients : 4.3±1.9 mol/mL/h, p<0.005 ; healthy infants : 4.0±1.4 nmol/mL/h, p<0.005). Serum S-ASM activity was negatively correlated with interferon-γ levels (rho=−0.448, p=0.012) but not with any other outcomes. Serum S-ASM activity was significantly higher in infants with RSV bronchiolitis than in individuals in the control groups ; however, its clinical significance requires further investigation

PGC-1α-Mediated Branched-Chain Amino Acid Metabolism in the Skeletal Muscle

<div><p>Peroxisome proliferator-activated receptor (PPAR) γ coactivator 1α (PGC-1α) is a coactivator of various nuclear receptors and other transcription factors, which is involved in the regulation of energy metabolism, thermogenesis, and other biological processes that control phenotypic characteristics of various organ systems including skeletal muscle. PGC-1α in skeletal muscle is considered to be involved in contractile protein function, mitochondrial function, metabolic regulation, intracellular signaling, and transcriptional responses. Branched-chain amino acid (BCAA) metabolism mainly occurs in skeletal muscle mitochondria, and enzymes related to BCAA metabolism are increased by exercise. Using murine skeletal muscle overexpressing PGC-1α and cultured cells, we investigated whether PGC-1α stimulates BCAA metabolism by increasing the expression of enzymes involved in BCAA metabolism. Transgenic mice overexpressing PGC-1α specifically in the skeletal muscle had increased the expression of branched-chain aminotransferase (BCAT) 2, branched-chain α-keto acid dehydrogenase (BCKDH), which catabolize BCAA. The expression of BCKDH kinase (BCKDK), which phosphorylates BCKDH and suppresses its enzymatic activity, was unchanged. The amount of BCAA in the skeletal muscle was significantly decreased in the transgenic mice compared with that in the wild-type mice. The amount of glutamic acid, a metabolite of BCAA catabolism, was increased in the transgenic mice, suggesting the activation of muscle BCAA metabolism by PGC-1α. In C2C12 cells, the overexpression of PGC-1α significantly increased the expression of BCAT2 and BCKDH but not BCKDK. Thus, PGC-1α in the skeletal muscle is considered to significantly contribute to BCAA metabolism.</p></div

Gene expression of BCAA metabolic enzyme in skeletal muscle of PGC-1α Tg mice.

<p>Expression of A) BCAT2, B) BCKDH, and C) BCKDK genes in WT (control; open columns, N = 9) and PGC-1α Tg (filled columns, N = 7) mice by quantitative real-time RT-PCR. RNA was obtained from mice with feeding condition. These samples were as used in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0091006#pone.0091006-Tadaishi1" target="_blank">[4]</a>. In the sample, PGC-1α expression was 30 fold higher in Tg mice than in WT mice (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0091006#pone-0091006-g001" target="_blank">Fig. 1</a> of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0091006#pone.0091006-Tadaishi1" target="_blank">[4]</a>). The relative values are shown (the control is set as 100). ***P<0.001.</p

BCAA content in skeletal muscle and blood of PGC-1α Tg mice.

<p>Val, Leu, and Ile levels in (A) skeletal muscle and (B) blood. Open columns represent for WT (N = 4) and filled columns represent Tg (N = 4). ***P<0.001, **P<0.01. T.R., trace level.</p

Amino acid content in blood of PGC-1α Tg mice.

<p>The samples were used as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0091006#pone-0091006-g004" target="_blank">Figure 4</a>.</p><p>*P<0.05.</p

Pathway map of Val, Leu, and Ile degradation.

<p>Individual gene name of the KEGG pathway of Val, Leu, and Ile degradation extracted by pathway analysis is shown as a metabolic map. Red asterisks indicate increased gene expression by microarray of Tg mice. Gene names corresponding to enzyme numbers with red asterisks are as follows: 2.8.3.5, 3-oxoacid CoA transferase 1; 2.3.1.16, acetyl-Coenzyme A acyltransferase 2 (mitochondrial 3-oxoacyl-Coenzyme A thiolase); 1.3.8.1, acyl-Coenzyme A dehydrogenase, short chain; 2.6.1.42, branched chain aminotransferase 2, mitochondrial; 1.2.4.4, branched chain ketoacid dehydrogenase E1, alpha polypeptide; 1.8.1.4, dihydrolipoamide dehydrogenase; 1.1.1.35, hydroxyacyl-Coenzyme A dehydrogenase; 4.2.1.17, hydroxyacyl-Coenzyme A dehydrogenase/3-ketoacyl-Coenzyme A thiolase/enoyl-Coenzyme A hydratase (trifunctional protein), alpha subunit; 5.1.99.1, methylmalonyl CoA epimerase; 1.1.1.31, 3-hydroxyisobutyrate dehydrogenase.</p

Gene expression of BCAA metabolic enzymes in cultured C2C12 cells overexpressing PGC-1α.

<p>Total RNA was isolated from the cells and analyzed by quantitative real-time RT-PCR with primers for A) PGC-1α, B) BCAT2, C) BCKDHa, and D) BCKDK. Open columns represent mock cells (N = 3), and filled columns represent PGC-1α-overexpressed cells (N = 3). Each value represents mean ± SE (N = 3). The relative values are shown (the control is set as 100). For PGC-1α expression, the value was set as 100 in the PGC-1α overexpressed cells. ***P<0.001, **P<0.01.</p

Bioinformatics analysis of transcription factors enriched in the BCAA metabolic pathway genes up-regulated in PGC-1α Tg mice.

<p>List of transcription factors, which are statistically identified as ones that can be recruited to the BCAA metabolic genes, up-regulated in PGC-1α Tg mice. Target genes were previously found in ChIP assay for interacting with indicated transcription factors in the literature <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0091006#pone.0091006-Lachmann1" target="_blank">[27]</a>. Abbreviations of the transcription factors are as follows, KLF4, Krueppel-like factor 4; PPARG, Constitutive coactivator of peroxisome proliferator-activated receptor gamma (Constitutive coactivator of PPAR-gamma) (Constitutive coactivator of PPARG); EKLF, Krueppel-like factor 1 (Erythroid krueppel-like transcription factor); ESRRB, Steroid hormone receptor ERR2 (Estrogen receptor-like 2) (Estrogen-related receptor beta) (ERR-beta); PPARD, Peroxisome proliferator-activated receptor delta (PPAR-delta); ZFP42, Zinc finger protein 42; WT1, Wilms tumor protein; NR0B1, Nuclear receptor subfamily 0 group B member 1 (Nuclear receptor DAX-1); TET1, Methylcytosine dioxygenase TET1 (EC 1.14.11.n2) (CXXC-type zinc finger protein 6) (Ten-eleven translocation 1 gene protein homolog); GATA4, Transcription factor GATA-4 (GATA-binding factor 4).</p

Pathway analysis.

<p>Compared with WT mice, 315 genes were found to be up-regulated in PGC-1α Tg mice by microarray and classified into KEGG pathway analysis as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0091006#s2" target="_blank">Methods</a>.</p