Muscle-specific deletion of BDK amplifies loss of myofibrillar protein during protein undernutrition

Ishikawa, T., Kitaura, Y., Kadota, Y. et al. Muscle-specific deletion of BDK amplifies loss of myofibrillar protein during protein undernutrition. Sci Rep 7, 39825 (2017). https://doi.org/10.1038/srep3982

Regulation of Skeletal Muscle Function by Amino Acids

Amino acids are components of proteins that also exist free-form in the body; their functions can be divided into (1) nutritional, (2) sensory, and (3) biological regulatory roles. The skeletal muscle, which is the largest organ in the human body, representing ~40% of the total body weight, plays important roles in exercise, energy expenditure, and glucose/amino acid usage—processes that are modulated by various amino acids and their metabolites. In this review, we address the metabolism and function of amino acids in the skeletal muscle. The expression of PGC1α, a transcriptional coactivator, is increased in the skeletal muscle during exercise. PGC1α activates branched-chain amino acid (BCAA) metabolism and is used for energy in the tricarboxylic acid (TCA) cycle. Leucine, a BCAA, and its metabolite, β-hydroxy-β-methylbutyrate (HMB), both activate mammalian target of rapamycin complex 1 (mTORC1) and increase protein synthesis, but the mechanisms of activation appear to be different. The metabolite of valine (another BCAA), β-aminoisobutyric acid (BAIBA), is increased by exercise, is secreted by the skeletal muscle, and acts on other tissues, such as white adipose tissue, to increase energy expenditure. In addition, several amino acid-related molecules reportedly activate skeletal muscle function. Oral 5-aminolevulinic acid (ALA) supplementation can protect against mild hyperglycemia and help prevent type 2 diabetes. β-alanine levels are decreased in the skeletal muscles of aged mice. β-alanine supplementation increased the physical performance and improved the executive function induced by endurance exercise in middle-aged individuals. Further studies focusing on the effects of amino acids and their metabolites on skeletal muscle function will provide data essential for the production of food supplements for older adults, athletes, and individuals with metabolic diseases

Screening dataset of food components that enhance transcriptional activity of PGC1-beta

PGC-1β is a transcriptional co-activator of nuclear receptors, which acts to increase energy expenditure. PGC-1β fused to GAL4 DNA-binding domain transfected in HEK293T cells showed a reporter luciferase activity. We screened food-derived and natural compounds using a reporter assay system to measure the transcriptional activity of PGC-1β.We found that soy-derived isoflavones, genistein and daidzein, and several resveratrols activated PGC-1β, see “Genistein, daidzein, and resveratrols stimulate PGC-1β-mediated gene expression” [1]. The list of 166 compounds and their reporter activity is shown here. Keywords: Screening, Reporter assay, Transcriptional activit

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

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

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

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

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