Gene expression in the skeletal muscle of PGC-1α and ALT2, but not ALT1, is increased by fasting.

<p>Mice (12-week-old males) were either allowed ad libitum access to food or subjected to fasting for 8 h or 24 h (fed, n = 4; 8 h fasted, n = 4; and 24 h fasted, n = 4). The expression of PGC-1α-b, ALT1, and ALT2 in the skeletal muscle (gastrocnemius) is shown. Quantitative real-time RT-PCR data from fed mice were set at 100 arbitrary units. mRNA levels were normalized to those of 36B4 mRNA. Each value represents mean ± SE (n = 4). ***P < 0.001 and *P < 0.05, relative to fed mice.</p

PGC-1α regulates alanine metabolism in muscle cells

<div><p>The skeletal muscle is the largest organ in the human body, depositing energy as protein/amino acids, which are degraded in catabolic conditions such as fasting. Alanine is synthesized and secreted from the skeletal muscle that is used as substrates of gluconeogenesis in the liver. During fasting, the expression of PGC-1α, a transcriptional coactivator of nuclear receptors, is increased in the liver and regulates gluconeogenesis. In the present study, we observed increased mRNA expression of PGC-1α and alanine aminotransferase 2 (ALT2) in the skeletal muscle during fasting. In C2C12 myoblast cells overexpressing PGC-1α, ALT2 expression was increased concomitant with an increased alanine level in the cells and medium. In addition, PGC-1α, along with nuclear receptor ERR, dose-dependently enhanced the ALT2 promoter activity in reporter assay using C2C12 cells. In the absence of glucose in the culture medium, mRNA levels of PGC-1α and ALT2 increased. Endogenous PGC-1α knockdown in C2C12 cells reduced ALT2 gene expression level, induced by the no-glucose medium. Taken together, in the skeletal muscle, PGC-1α activates ALT2 gene expression, and alanine production may play roles in adaptation to fasting.</p></div

Transient transfection reporter assay of the effect of PGC-1α on the ALT2 promoter.

<p>The effect of increasing PGC-1α expression was examined by cotransfection with a reporter plasmid in C2C12 cells. A) The constructs include a 2.0-kb genomic promoter region and the first exon of the ALT2 gene (−2009 to +101, from the transcription start site), the luciferase reporter gene. B) The left panel shows the reporter construct containing ALT2 promoter. Transcription start site (+1) is shown in the panel (arrow). The constructs include 1.4-kb, 2.0-kb, and 4.9-kb genomic promoter regions and the first exon, the luciferase reporter gene. Each value represents mean ± SE (n = 3). ***P < 0.001, **P < 0.01, and *P < 0.05 compared with the samples in the absence of PGC-1α expression vector (open bar).</p

Gene and protein expression of ALT2 and medium alanine level in C2C12 cells overexpressing PGC-1α.

<p>A) Gene expression of PGC-1α, ALT1, ALT2 and BCKDH in cultured C2C12 cells overexpressing PGC-1α. Total RNA was isolated from the cells and analyzed by quantitative real-time RT-PCR. Open bars represent mock cells (n = 3), and filled bars represent PGC-1α-overexpressed cells (n = 3). Each value represents mean ± SE (n = 3). The relative values are shown (the mock control is set as 100). For PGC-1α expression, the value was set as 100 in the PGC-1α-overexpressed cells. mRNA levels were normalized to those of 36B4 mRNA. ***P < 0.001. B) Western blot analysis of ALT2 protein in mock cells (control, n = 3) and PGC-1α-overexpressed cells (n = 3). C) Alanine level of the culture medium in C2C12 cells overexpressing PGC-1α. C2C12 cells overexpressing PGC-1α are cultured in DMEM supplemented with 10% FBS until the cells reached confluence. The cells were cultured in DMEM without serum for 48 h, and the culture medium was examined for alanine content. Open bars represent mock cells (n = 3), and filled bars represent PGC-1α-overexpressed cells (n = 3). Each value represents mean ± SE. ***P < 0.001.</p

Increased ALT2 expression in no-glucose medium is suppressed by knockdown of PGC-1α in C2C12 cells.

<p>C2C12 cells were transfected with control siRNA (open bars) or siRNA of PGC-1α (filled bars) and cultured in control medium (containing 4.5g/L glucose, left) and no-glucose medium (right). After 48 h, total RNA was isolated from the cells and analyzed by quantitative real-time RT-PCR with primers for PGC-1α, ALT1, and ALT2. Endogenous PGC-1α was knocked down by siRNA, and increased ALT2 in no-glucose medium was suppressed by PGC-1α knockdown. Each value represents mean ± SE (n = 3). The relative values are shown (the control; control medium with control siRNA, is set as 100). mRNA levels were normalized to those of 36B4 mRNA. ***P < 0.001, **P < 0.01, and *P < 0.05.</p

Transient transfection reporter assay of the effect of PGC-1α on the ALT2 promoter.

<p>The effect of increasing PGC-1α expression was examined by cotransfection with a reporter plasmid in C2C12 cells. A) The constructs include a 2.0-kb genomic promoter region and the first exon of the ALT2 gene (−2009 to +101, from the transcription start site), the luciferase reporter gene. B) The left panel shows the reporter construct containing ALT2 promoter. Transcription start site (+1) is shown in the panel (arrow). The constructs include 1.4-kb, 2.0-kb, and 4.9-kb genomic promoter regions and the first exon, the luciferase reporter gene. Each value represents mean ± SE (n = 3). ***P < 0.001, **P < 0.01, and *P < 0.05 compared with the samples in the absence of PGC-1α expression vector (open bar).</p

Effect of PGC-1α and ERR on the ALT2 gene.

<p>A) Gene expression of PGC-1α, ALT1, ALT2, ERRα and ERRγ in cultured C2C12 cells overexpressing ERRα and ERRγ. Total RNA was isolated from the cells and analyzed by quantitative real-time RT-PCR. Each value represents mean ± SE (n = 3). The relative values are shown (the mock control is set as 100). mRNA levels were normalized to those of 36B4 mRNA. B) The effect of the expression of PGC-1α and ERR (ERRα, left and ERRγ, right) was examined by cotransfection with a reporter plasmid in C2C12 cells. The constructs include a 2.0-kb genomic promoter region and the first exon of the ALT2 gene (−2009 to +101, from the transcription start site), the luciferase reporter gene. Each value represents mean ± SE (n = 3). ***P < 0.001, **P < 0.01, and * P < 0.05.</p

Insulin-stimulated glucose uptake in differentiated 3T3-L1 adipocytes.

<p>Differentiated 3T3-L1 adipocytes were treated with or without TNF-α (3 ng/ml) for 72 h in the presence or absence of Igf2 (400 ng/ml). *<i>P</i>≤0.05.</p

Observed metabolite changes mapped onto the pathways involved in the pentose phosphate pathway.

<p>Changes in the metabolite levels in the skeletal muscle of PGC-1α-Tg mice and WT mice are shown. Relative metabolite changes shown in the graphs were obtained by CE-TOFMS (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129084#pone.0129084.s002" target="_blank">S1 Table</a>). Open bars, WT and filled bars, PGC-1α-Tg (N = 3). Data are expressed as the mean ± SD. Asterisks indicate statistically significant differences (**p < 0.01, *p < 0.05). Microarray data of gene expression change of enzymes in the related metabolic process are shown in the scheme.</p

Metabolomic Analysis of the Skeletal Muscle of Mice Overexpressing PGC-1α

<div><p>Peroxisome proliferator-activated receptor (PPAR) γ coactivator 1α (PGC-1α) is a coactivator of various nuclear receptors and other transcription factors whose expression increases in the skeletal muscle during exercise. We have previously made transgenic mice overexpressing PGC-1α in the skeletal muscle (PGC-1α-Tg mice). PGC-1α upregulates the expression of genes associated with red fibers, mitochondrial function, fatty acid oxidation, and branched chain amino acid (BCAA) degradation. However, global analyses of the actual metabolic products have not been investigated. In this study, we conducted metabolomic analysis of the skeletal muscle in PGC-1α-Tg mice by capillary electrophoresis with electrospray ionization time-of-flight mass spectrometry. Principal component analysis and hierarchical cluster analysis showed clearly distinguishable changes in the metabolites between PGC-1α-Tg and wild-type control mice. Changes were observed in metabolite levels of various metabolic pathways such as the TCA cycle, pentose phosphate pathway, nucleotide synthesis, purine nucleotide cycle, and amino acid metabolism, including BCAA and β-alanine. Namely, metabolic products of the TCA cycle increased in PGC-1α-Tg mice, with increased levels of citrate (2.3-fold), succinate (2.2-fold), fumarate (2.8-fold), and malate (2.3-fold) observed. Metabolic products associated with the pentose phosphate pathway and nucleotide biosynthesis also increased in PGC-1α-Tg mice. Meanwhile, BCAA levels decreased (Val, 0.7-fold; Leu, 0.8-fold; and Ile, 0.7-fold), and Glu (3.1-fold) and Asp (2.2-fold) levels increased. Levels of β-alanine and related metabolites were markedly decreased in PGC-1α-Tg mice. Coordinated regulation of the TCA cycle and amino acid metabolism, including BCAA, suggests that PGC-1α plays important roles in energy metabolism. Moreover, our metabolomics data showing the activation of the purine nucleotide pathway, malate–aspartate shuttle, as well as creatine metabolism, which are known to be active during exercise, further suggests that PGC-1α regulates metabolism in exercise. Thus, we demonstrated the roles of PGC-1α in the skeletal muscle at the metabolite level.</p></div