Peroxisome proliferator-activated receptor γ coactivator 1α regulates downstream of tyrosine kinase-7 (Dok-7) expression important for neuromuscular junction formation.

The neuromuscular junction (NMJ)-formed between a motor nerve terminal and skeletal muscle fiber-plays an important role in muscle contraction and other muscle functions. Aging and neurodegeneration worsen NMJ formation and impair muscle function. Downstream of tyrosine kinase-7 (Dok-7), expressed in skeletal muscle fibers, is essential for the formation of NMJ. Exercise increases the expression of the transcriptional coactivator peroxisome proliferator-activated receptor γ coactivator 1α (PGC1α) in skeletal muscles and restores NMJ formation. In this study, we used skeletal muscle-specific PGC1α knockout or overexpression mice to examine the role of PGC1α in regulating Dok-7 expression and NMJ formation. Our findings revealed that Dok-7 expression is regulated by PGC1α, and luciferase activity of the Dok-7 promoter is greatly increased by coexpressing PGC1α and estrogen receptor-related receptor α. Thus, we suggest PGC1α is involved in exercise-mediated restoration of NMJ formation.

LPGAT1/LPLAT7 regulates acyl chain profiles at the sn-1 position of phospholipids in murine skeletal muscles.

Skeletal muscle consists of both fast- and slow-twitch fibers. Phospholipids are important structural components of cellular membranes, and the diversity of their fatty acid composition affects membrane characteristics. Although some studies have shown that acyl chain species in phospholipids differ among various muscle fiber types, the mechanisms underlying these differences are unclear. To investigate this, we analyzed phosphatidylcholine (PC) and phosphatidylethanolamine (PE) molecules in the murine extensor digitorum longus (EDL; fast-twitch) and soleus (slow-twitch) muscles. In the EDL muscle, the vast majority (93.6%) of PC molecules was palmitate-containing PC (16:0-PC), whereas in the soleus muscle, in addition to 16:0-PC, 27.9% of PC molecules was stearate-containing PC (18:0-PC). Most palmitate and stearate were bound at the sn-1 position of 16:0- and 18:0-PC, respectively, and 18:0-PC was found in type I and IIa fibers. The amount of 18:0-PE was higher in the soleus than in the EDL muscle. Peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) increased the amount of 18:0-PC in the EDL. Lysophosphatidylglycerol acyltransferase 1 (LPGAT1) was highly expressed in the soleus compared with that in the EDL muscle and was upregulated by PGC-1α. LPGAT1 knockout decreased the incorporation of stearate into PC and PE in vitro and ex vivo and the amount of 18:0-PC and 18:0-PE in murine skeletal muscle with an increase in the level of 16:0-PC and 16:0-PE. Moreover, knocking out LPGAT1 decreased the amount of stearate-containing phosphatidylserine (18:0-PS), suggesting that LPGAT1 regulated the acyl chain profiles of phospholipids, namely, PC, PE, and PS, in the skeletal muscle.

Regulation of Skeletal Muscle Function by Amino Acids, Especially Non-Proteinogenic Amino Acids.

Amino acids are compounds that contain an amino group (-NH2) and a carboxyl group (-COOH) and are components of proteins and materials for various bioactive molecules. 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, especially non-proteinogenic amino acids, in the skeletal muscle. Leucine, a BCAA, and its metabolite, ß-hydroxy-ß-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), ß-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. ß-alanine levels are decreased in the skeletal muscles of aged mice. ß-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.

A combination of exercise and calorie restriction improves the development of obesity-related type 2 diabetes mellitus in KKAy mice.

We observed that exercise and calorie restriction reduced the body weight and blood glucose levels, concurrently improving insulin resistance and glucose tolerance in obese/diabetic model KKAy mice. Analysis of gene expression in the skeletal muscle showed enhanced mRNA levels of GLUT4 (glucose uptake), ATGL (lipolytic enzyme), and slow-twitch myosin heavy chain, which may contribute to the antiobesity and antidiabetic effects.

Slc12a8 in the lateral hypothalamus maintains energy metabolism and skeletal muscle functions during aging.

Sarcopenia and frailty are urgent socio-economic problems worldwide. Here we demonstrate a functional connection between the lateral hypothalamus (LH) and skeletal muscle through Slc12a8, a recently identified nicotinamide mononucleotide transporter, and its relationship to sarcopenia and frailty. Slc12a8-expressing cells are mainly localized in the LH. LH-specific knockdown of Slc12a8 in young mice decreases activity-dependent energy and carbohydrate expenditure and skeletal muscle functions, including muscle mass, muscle force, intramuscular glycolysis, and protein synthesis. LH-specific Slc12a8 knockdown also decreases sympathetic nerve signals at neuromuscular junctions and ß2-adrenergic receptors in skeletal muscle, indicating the importance of the LH-sympathetic nerve-ß2-adrenergic receptor axis. LH-specific overexpression of Slc12a8 in aged mice significantly ameliorates age-associated decreases in energy expenditure and skeletal muscle functions. Our results highlight an important role of Slc12a8 in the LH for regulation of whole-body metabolism and skeletal muscle functions and provide insights into the pathogenesis of sarcopenia and frailty during aging.

Vitamin D Activates Various Gene Expressions, Including Lipid Metabolism, in C2C12 Cells.

Vitamin D is a fat-soluble molecule, well known for its role in regulating calcium homeostasis in bone. It has become increasingly clear that it also has important effects in many other organs, including the skeletal muscle. In order to gain insight into the role of vitamin D in the skeletal muscle, we performed microarray analysis using C2C12 myoblasts treated with 1,25-dihydroxyvitamin D (1,25(OH)2D), active form of vitamin D. We found multiple genes upregulated by 1,25(OH)2D. Some of them, i.e., vitamin D receptor (Vdr), diacylglycerol O-acyltransferase (Dgat1 and Dgat2, the rate limiting steps of triacylglycerol acylation), and vascular endothelial growth factor A (Vegfa), were previously reported to be upregulated by 1,25(OH)2D in C2C12 cells. RT-qPCR analysis confirmed increased mRNA levels of Rarres2, Dio2, Tgm2, Lpl, Mdfi, Igfbp3, Dgat1, Crabp2, Gadd45a, Vagfa, Dgat2, C3, Ldhb, Cebpa, Igfbp5, Mrc2, Vdr. Thus, many genes, including lipid metabolism genes as well as genes related to muscle functions, appear to be upregulated by 1,25(OH)2D in muscle cells.

The endothelial Dll4-muscular Notch2 axis regulates skeletal muscle mass.

Adult skeletal muscle is a highly plastic tissue that readily reduces or gains its mass in response to mechanical and metabolic stimulation; however, the upstream mechanisms that control muscle mass remain unclear. Notch signalling is highly conserved, and regulates many cellular events, including proliferation and differentiation of various types of tissue stem cell via cell-cell contact. Here we reveal that multinucleated myofibres express Notch2, which plays a crucial role in disuse- or diabetes-induced muscle atrophy. Mechanistically, in both atrophic conditions, the microvascular endothelium upregulates and releases the Notch ligand, Dll4, which then activates muscular Notch2 without direct cell-cell contact. Inhibition of the Dll4-Notch2 axis substantively prevents these muscle atrophy and promotes mechanical overloading-induced muscle hypertrophy in mice. Our results illuminate a tissue-specific function of the endothelium in controlling tissue plasticity and highlight the endothelial Dll4-muscular Notch2 axis as a central upstream mechanism that regulates catabolic signals from mechanical and metabolic stimulation, providing a therapeutic target for muscle-wasting diseases.

The Steroidal Alkaloid Tomatidine and Tomatidine-Rich Tomato Leaf Extract Suppress the Human Gastric Cancer-Derived 85As2 Cells In Vitro and In Vivo via Modulation of Interferon-Stimulated Genes.

The steroidal alkaloid tomatidine is an aglycone of α-tomatine, which is abundant in tomato leaves and has several biological activities. Tomatidine has been reported to inhibit the growth of cultured cancer cells in vitro, but its anti-cancer activity in vivo and inhibitory effect against gastric cancer cells remain unknown. We investigated the efficacy of tomatidine using human gastric cancer-derived 85As2 cells and its tumor-bearing mouse model and evaluated the effect of tomatidine-rich tomato leaf extract (TRTLE) obtained from tomato leaves. In the tumor-bearing mouse model, tumor growth was significantly inhibited by feeding a diet containing tomatidine and TRTLE for 3 weeks. Tomatidine and TRTLE also inhibited the proliferation of cultured 85As2 cells. Microarray data of gene expression analysis in mouse tumors revealed that the expression levels of mRNAs belonging to the type I interferon signaling pathway were altered in the mice fed the diet containing tomatidine and TRTLE. Moreover, the knockdown of one of the type I interferon-stimulated genes (ISGs), interferon α-inducible protein 27 (IFI27), inhibited the proliferation of cultured 85As2 cells. This study demonstrates that tomatidine and TRTLE inhibit the tumor growth in vivo and the proliferation of human gastric cancer-derived 85As2 cells in vitro, which could be due to the downregulation of ISG expression.

FOXO1 cooperates with C/EBPδ and ATF4 to regulate skeletal muscle atrophy transcriptional program during fasting.

Catabolic conditions, such as starvation, inactivity, and cancer cachexia, induce Forkhead box O (FOXO) transcription factor(s) expression and severe muscle atrophy via the induction of ubiquitin-proteasome system-mediated muscle proteolysis, resulting in frailty and poor quality of life. Although FOXOs are clearly essential for the induction of muscle atrophy, it is unclear whether there are other factors involved in the FOXO-mediated transcriptional regulation. As such, we identified FOXO-CCAAT/enhancer-binding protein δ (C/EBPδ) signaling pathway as a novel proteolytic pathway. By comparing the gene expression profiles of FOXO1-transgenic (gain-of-function model) and FOXO1,3a,4-/- (loss-of-function model) mice, we identified several novel FOXO1-target genes in skeletal muscle including Redd1, Sestrin1, Castor2, Chac1, Depp1, Lat3, as well as C/EBPδ. During starvation, C/EBPδ abundance was increased in a FOXOs-dependent manner. Notably, knockdown of C/EBPδ prevented the induction of the ubiquitin-proteasome system and decrease of myofibers in FOXO1-activated myotubes. Conversely, C/EBPδ overexpression in primary myotubes induced myotube atrophy. Furthermore, we demonstrated that FOXO1 enhances the promoter activity of target genes in cooperation with C/EBPδ and ATF4. This research comprehensively identifies novel FOXO1 target genes in skeletal muscle and clarifies the pathophysiological role of FOXO1, a master regulator of skeletal muscle atrophy.

Hoxa10 mediates positional memory to govern stem cell function in adult skeletal muscle.

Muscle stem cells (satellite cells) are distributed throughout the body and have heterogeneous properties among muscles. However, functional topographical genes in satellite cells of adult muscle remain unidentified. Here, we show that expression of Homeobox-A (Hox-A) cluster genes accompanied with DNA hypermethylation of the Hox-A locus was robustly maintained in both somite-derived muscles and their associated satellite cells in adult mice, which recapitulates their embryonic origin. Somite-derived satellite cells were clearly separated from cells derived from cranial mesoderm in Hoxa10 expression. Hoxa10 inactivation led to genomic instability and mitotic catastrophe in somite-derived satellite cells in mice and human. Satellite cell-specific Hoxa10 ablation in mice resulted in a decline in the regenerative ability of somite-derived muscles, which were unobserved in cranial mesoderm-derived muscles. Thus, our results show that Hox gene expression profiles instill the embryonic history in satellite cells as positional memory, potentially modulating region-specific pathophysiology in adult muscles.

Citrus hassaku Extract Powder Increases Mitochondrial Content and Oxidative Muscle Fibers by Upregulation of PGC-1α in Skeletal Muscle.

Peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) is expressed in skeletal muscles and regulates systemic metabolism. Thus, nutraceuticals targeting skeletal muscle PGC-1α have attracted attention to modulate systemic metabolism. As auraptene contained in citrus fruits promotes lipid metabolism and improves mitochondrial respiration, it could increase mitochondrial function through PGC-1α. Therefore, we hypothesized that PGC-1α is activated by auraptene and investigated its effect using Citrus hassaku extract powder (CHEP) containing >80% of auraptene. C2C12 myotubes were incubated with vehicle or CHEP for 24 h; C57BL/6J mice were fed a control diet or a 0.25% (w/w) CHEP-containing diet for 5 weeks. PGC-1α protein level and mitochondrial content increased following CHEP treatment in cultured myotubes and skeletal muscles. In addition, the number of oxidative fibers increased in CHEP-fed mice. These findings suggest that CHEP-mediated PGC-1α upregulation induced mitochondrial biogenesis and fiber transformation to oxidative fibers. Furthermore, as CHEP increased the expression of the protein sirtuin 3 and of phosphorylated AMP-activated protein kinase (AMPK) and the transcriptional activity of PGC-1α, these molecules might be involved in CHEP-induced effects in skeletal muscles. Collectively, our findings indicate that CHEP mediates PGC-1α expression in skeletal muscles and may serve as a dietary supplement to prevent metabolic disorders.

Metabolomic analysis on blood of transgenic mice overexpressing PGC-1α in skeletal muscle.

PGC-1α expression increases in skeletal muscles during exercise and regulates the transcription of many target genes. In this study, we conducted a metabolomic analysis on the blood of transgenic mice overexpressing PGC-1α in its skeletal muscle (PGC-1α-Tg mice) using CE-TOFMS. The blood level of homovanillic acid (dopamine metabolite) and the gene expression of dopamine metabolic enzyme in the skeletal muscle of PGC-1α-Tg mice were high. The blood level of 5-methoxyindoleacetic acid was also high in PGC-1α-Tg mice. The blood levels of branched-chain α-keto acids and ß-alanine were low in PGC-1α-Tg mice. These metabolites in the skeletal muscle were present in low concentration. The changes in these metabolites may reflect the skeletal muscle condition with increasing PGC-1α, such as exercise.

Effects of fenofibrate and its combination with lovastatin on the expression of genes involved in skeletal muscle atrophy, including FoxO1 and its targets.

Fibrates and statins have been widely used to reduce triglyceride and cholesterol levels, respectively. Besides its lipid-lowering effect, the side effect of muscle atrophy after fibrate administration to humans has been demonstrated in some studies. Combination therapy with fibrates and statins also increases the risk of rhabdomyolysis. FoxO1, a member of the FoxO forkhead type transcription factor family, is markedly upregulated in skeletal muscle in energy-deprived states and induces muscle atrophy via the expression of E3-ubiquitine ligases. In this study, we investigated the changes in FoxO1 and its targets in murine skeletal muscle with fenofibrate treatment. High doses of fenofibrate (greater than 0.5% (wt/wt)) over one week increased the expression of FoxO1 and its targets in the skeletal muscles of mice and decreased skeletal muscle weight. These fenofibrate-induced changes were diminished in the PPARα knockout mice. When the effect of combination treatment with fenofibrate and lovastatin was investigated, a significant increase in FoxO1 protein levels was observed despite the lack of deterioration of muscle atrophy. Collectively, our findings suggest that a high dose of fenofibrate over one week causes skeletal muscle atrophy via enhancement of FoxO1, and combination treatment with fenofibrate and lovastatin may further increase FoxO1 protein level.

Skeletal muscle-specific forkhead box protein-O1 overexpression suppresses atherosclerosis progression in apolipoprotein E-knockout mice.

Calorie restriction (CR) reportedly prevents atherosclerotic diseases. Furthermore, CR induces forkhead box protein-O1 (FOXO-1) expression in the skeletal muscle, altering the character of the skeletal muscle. We previously reported that the change in skeletal muscle character, induced by the overexpression of peroxisome proliferator-activated receptor γ coactivator-1α, suppresses atherosclerotic progression in an atherosclerotic apolipoprotein E-knockout (ApoE-KO) mouse model. Thus, we hypothesized that skeletal muscle alternation induced by FOXO-1 may also have an anti-atherosclerotic effect in ApoE-KO mice. In this study, we investigated whether skeletal muscle-specific FOXO-1 overexpression suppresses the progression of atherosclerosis in ApoE-KO mice. We generated ApoE-KO/FOXO-1 mice, in which an ApoE-KO mouse was crossbred with a mouse presenting skeletal muscle-specific FOXO-1 overexpression (FOXO-1Tg). The mice were sacrificed at 20 weeks of age, and atherosclerotic plaque area and protein expression in the plaque were measured. Additionally, we measured the tumor necrosis factor α (TNFα)- induced mRNA expression in human umbilical vein endothelial cells (HUVECs), using serum collected from the FOXO-1Tg mice. Accordingly, ApoE-KO/FOXO-1 mice showed a 65% reduced atherosclerotic plaque area when compared with the ApoE-KO mice, with concomitantly reduced vascular cell adhesion molecule-1 (VCAM-1) and macrophage infiltration. As compared to serum from wild-type mice, the serum collected from the FOXO-1Tg mice significantly suppressed the mRNA expression of VCAM-1, an atherosclerosis initiation factor, in TNFα-treated HUVECs. Therefore, these data suggest that skeletal muscle-specific FOXO-1 overexpression suppresses the progression of atherosclerosis in ApoE-KO mice. In part, the CR-induced anti-atherosclerotic effect could be attributed to FOXO-1 upregulation in the skeletal muscle.

Vitamin D and Sarcopenia: Potential of Vitamin D Supplementation in Sarcopenia Prevention and Treatment.

Skeletal muscle, the largest organ in the human body, accounting for approximately 40% of body weight, plays important roles in exercise and energy expenditure. In the elderly, there is often a progressive decline in skeletal muscle mass and function, a condition known as sarcopenia, which can lead to bedridden conditions, wheelchair confinement as well as reducing the quality of life (QOL). In developed countries with aging populations, the prevention and management of sarcopenia are important for the improvement of health and life expectancy in these populations. Recently, vitamin D, a fat-soluble vitamin, has been attracting attention due to its importance in sarcopenia. This review will focus on the effects of vitamin D deficiency and supplementation on sarcopenia.

Author Correction: A spatial similarity of stereochemical environments formed by amino acid residues defines a common epitope of two non-homologous proteins.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

FOXO1 suppresses PGC-1ß gene expression in skeletal muscles.

Peroxisome proliferator-activated receptor-gamma coactivator-1ß (PGC-1ß) is a transcriptional regulator whose increased expression activates energy expenditure-related genes in skeletal muscles. However, how PGC-1ß is regulated remains largely unclear. Here, we show that PGC-1ß gene expression is negatively correlated with the expression of a transcription factor, forkhead box protein O1 (FOXO1), whose expression is increased during muscle atrophy. In the skeletal muscles of FOXO1-overexpressing transgenic mice, PGC-1ß gene expression is decreased. Denervation or plaster cast-based unloading, as well as fasting, increases endogenous FOXO1 expression in skeletal muscles, with decreased PGC-1ß expression. In the skeletal muscles of FOXO1-knockout mice, the decrease in PGC-1ß expression caused by fasting was attenuated. Tamoxifen-inducible FOXO1 activation in C2C12 myoblasts causes a marked decrease of PGC-1ß expression. These findings together reveal that FOXO1 activation suppresses PGC-1ß expression. During atrophy with FOXO1 activation, decreased PGC-1ß may decrease energy expenditure and avoid wasting energy.

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, ß-hydroxy-ß-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), ß-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. ß-alanine levels are decreased in the skeletal muscles of aged mice. ß-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.

Health Promotion Effects of Soy Isoflavones.

Soybeans contain several physiologically active ingredients, such as soy phytosterol, soyasaponin, soy protein, and lecithin, and are therefore expected to express the functionalities of said ingredients. Among them, soy isoflavones have been studied in recent years for their various functions, including their obesity-preventing effect, blood glucose level reducing effect, osteoporosis and breast cancer risk reduction, and anti-oxidative effect, and several health promoting effects and disease preventing effects are expected. For example, it has been determined that soy isoflavones reduce body and fat weight in experiments in which mice were fed a diet containing soy isoflavones in studies on anti-obesity. Epidemiologic studies with humans have also shown that women who consume more soybeans have lower BMI than those who consume less. We previously found that soy isoflavones may have anti-obesity effects in myoblasts through the activation of transcriptional coactivator PGC-1ß, which increases energy expenditure. In recent studies, a decrease in blood glucose level due to soy isoflavone was seen in an experiment in which diabetic model mice were fed a diet containing soy isoflavone. It has also been suggested that soy isoflavone intake may increase bone mineral density in postmenopausal women and reduce the risk of breast cancer. This review focuses on the actions of soy isoflavones known to date, including their anti-obesity and anti-diabetic effects, bone loss preventing effects, and cancer risk reduction effects, and introduces reports on the health promotion and disease prevention effects of soy isoflavones.