C2C12 Mouse Muscle Cells Research Articles

Recombinant lactate-assimilating cyanobacteria reduce high-concentration culture-associated cytotoxicity in mammalian cells.

In the fields of cultured meat, biopharmaceuticals, cell therapy, and tissue engineering, large numbers of mammalian cells are required; thus, highly-concentrated cell cultures are widely adopted. In general, such cultures can lead to cell damage caused by waste product accumulation and nutritional inadequacy. In this study, a novel co-culture system where the recombinant lactate-assimilating cyanobacterial strain, KC0110, derived from euryhaline Picosynechococcus sp. PCC 7002, and mammalian muscle cells cultured across porous membranes been developed. By using the KC0110 strain, the amount of ammonium and lactate excreted from C2C12 mouse muscle cells into the culture significantly decreased. Importantly, pyruvate and some amino acids, including pyruvate-derived amino acids, also increased significantly compared to those in monoculture of C2C12 cells. It is believed that the organic acids secreted by the KC0110 strain enhance the growth of mammalian cells, leading to a reduction in high-concentration culture-induced mammalian cell damage [lactate dehydrogenase (LDH) release] through cyanobacterial co-culture. These results show that, through co-cultivation with cyanobacteria, it is possible to culture mammalian cells, alleviating cell damage, even in highly-concentrated cultures. This study demonstrated an in vitro "symbiotic circular system" that can interchange metabolites produced by phototrophs and mammalian cells.

Postprandial fatty acid-binding protein4 is associated with muscle insulin resistance.

Fatty acid-binding protein4 (FABP4) has been reported to act as a hepatic insulin resistance factor. We previously reported that fasting FABP4 was correlated with insulin resistance measurements derived from the glucose clamp, and another study reported that postprandial FABP4 levels were decreased in healthy volunteers but were not reported (or known) in participants with type 2 diabetes. We have limited knowledge about the direct effect of FABP4 on muscle cells. We investigated the postprandial FABP4 levels in participants with type 2 diabetes, and the basic mechanism of muscle insulin resistance and FABP4. We performed a meal tolerance test and hyperinsulinaemic-euglycaemic clamp in 22 participants with type 2 diabetes and 26 participants without diabetes. We measured fasting and postprandial serum FABP4. We cultured mouse C2C12 muscle cells, and investigated the effect of FABP4 on glucose uptake. We analysed insulin signalling by western blot and insulin binding assay. The postprandial FABP4 level in participants with type 2 diabetes was higher than that in participants without diabetes. Participants without diabetes had lower postprandial FABP4 than fasting except for one participant, whereas one-third of participants with type 2 diabetes had higher postprandial FABP4 than fasting. Postprandial FABP4 was correlated with the muscle insulin resistance M/I value from a glucose clamp in participants without diabetes (r=-0.42, p<0.05). The increase in FABP4 after a meal correlated with the muscle insulin resistance M/I value (r=-0.44, p<0.05) and the difference between fasting and postprandial glucagon in participants with type 2 diabetes (r=0.36, p<0.05). FABP4 alone appears to increase glucose uptake, and the combination of FABP4 and insulin decreases glucose uptake when compared with insulin alone. FABP4 inhibits insulin signalling of muscle cells through decreases in phosphorylation of insulin receptor substrate1 and Akt. The physiological concentration of FABP4 did not inhibit insulin binding to muscle cells. These results suggested that the postprandial FABP4 level is associated with insulin resistance, and FABP4 may suppress insulin signals.

Screening for new inhibitors of the human Mitochondrial Pyruvate Carrier and their effects on hepatic glucose production and diabetes

BackgroundThe mitochondrial pyruvate carrier (MPC) is a protein complex composed of two subunits, MPC1 and MPC2. This carrier is at the interface between glycolysis and mitochondrial metabolism and plays an essential role in hepatic glucose production. MethodsHere we describe an in vitro screen for small molecule inhibitors of the MPC using a strain of Lactococcus lactis that has been engineered to co-express the two subunits of the human MPC and is able to import exogenous 14C-pyruvate. We then tested the top candidates for potential antidiabetic effects through the repression of gluconeogenesis. ResultsBy screening the Prestwick compound library of 1′200 drugs approved by the Food and Drug Administration for inhibitors of pyruvate uptake, twelve hit molecules were identified. In a secondary screen, the most potent inhibitors were found to inhibit pyruvate-driven oxygen consumption in mouse C2C12 muscle cells. Assessment of gluconeogenesis showed that Zaprinast, as well as the established MPC inhibitor UK5099, inhibited in vitro and in vivo hepatic glucose production. However, when tested acutely in mice without the administration of gluconeogenic substrates, MPC inhibitors raised blood glucose levels, pointing to liver-independent effects. Furthermore, chronic treatment with Zaprinast failed to correct hyperglycemia in both lean and obese diabetic mouse models. ConclusionsNew MPC inhibitors have been identified, showing inhibitory effects on hepatic glucose production. General significanceFor potential antidiabetic applications, MPC inhibitors should target the liver without undesired inhibition of mitochondrial pyruvate metabolism in the skeletal muscles or pancreatic beta-cells in order to avoid dual effects on glycemia.

Abstract 3493: Drug targeting of KRAS accumulation on the cilium inhibits its stemness driving activity

Abstract Most RAS drug targeting strategies focus on KRAS, due to the high KRAS mutation frequency in cancer and its high expression level. Moreover, KRAS4B (hereafter KRAS) but not HRAS is a potent driver of stemness traits in cancer cells. Exactly how KRAS promotes stemness and how this activity can be targeted with drugs is not understood. Here we studied the subcellular localization of KRAS mutants to stemness mediating centriolar organelles and the effect of these mutants on the differentiation of mouse muscle C2C12 cells. This cell line contains a pool of ciliated stem/progenitor cells, which differentiate under low serum conditions, concomitant with a drop in RAS-MAPK activity. Intriguingly, expression of oncogenic KRAS retains the stem-like state, even under differentiating conditions. We hypothesized that KRAS localization at the primary cilium of C2C12 progenitor cells is propagated via the mother centriole to the mother centrosome, which is known to asymmetrically partition into the stemness retaining cell. Using confocal microscopy, we show that the trafficking chaperone PDE6D (or PDEdelta) facilitates trafficking of KRAS not only to the plasma membrane, but also to the primary cilium. Cellular BRET-interaction experiments in HEK cells demonstrate that decreasing the affinity between KRAS and PDE6D by the phosphomimetic S181D-mutation in KRAS, correlates with a reduced KRAS localization in the cilium of C2C12 cells. Likewise, treatment with Sildenafil, which increases the phosphorylation of Ser181 of KRAS, disrupts its binding to PDE6D. We then established a flow-cytometry based C2C12 cell differentiation assay to quantify accurately that mutations and pharmacological manipulations, which decrease ciliary localization of KRAS, indeed promote differentiation. These data lend a new rationale to PDE6D inhibition. By combining computational modelling and in vitro testing, we therefore developed a new, low nanomolar inhibitor, Deltaflexin-3 (structure will be disclosed), which targets the hydrophobic pocket of PDE6D. This compound is highly soluble and displays improved on-target activity as compared to previous PDE6D inhibitors, which is demonstrated by a panel of cellular BRET- and proliferation-assay data. Our results suggest that blocking of the ciliary localization of KRAS by synergistically combining PDE6D inhibitors and Sildenafil could specifically inhibit its stemness driving activity. Citation Format: Pelin Kaya, Rohan Chippalkatti, Elisabeth Schaffner-Reckinger, Bianca Parisi, Ganesh B. Manoharan, Yashar Rouzbahani, Christian Eggeling, Daniel Abankwa. Drug targeting of KRAS accumulation on the cilium inhibits its stemness driving activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3493.

Extracellular vesicles secreted from mouse muscle cells improve delayed bone repair in diabetic mice.

Humoral factors that are secreted from skeletal muscles can regulate bone metabolism and contribute to muscle-bone relationships. Although extracellular vesicles (EVs) play important roles in physiological and pathophysiological processes, the roles of EVs that are secreted from skeletal muscles in bone repair have remained unclear. In the present study, we investigated the effects of the local administration of muscle cell-derived EVs on bone repair in control and streptozotocin-treated diabetic female mice. Muscle cell-derived EVs (Myo-EVs) were isolated from the conditioned medium from mouse muscle C2C12 cells by ultracentrifugation, after which Myo-EVs and gelatin hydrogel sheets were transplanted on femoral bone defect sites. The local administration of Myo-EVs significantly improved delayed bone repair that was induced by the diabetic state in mice 9 days after surgery. Moreover, this administration significantly enhanced the ratio of bone volume to tissue volume at the damaged sites 9 days after surgery in the control mice. Moreover, the local administration of Myo-EVs significantly blunted the number of Osterix-positive cells that were suppressed by the diabetic state at the damage sites after bone injury in mice. Additionally, Myo-EVs significantly blunted the mRNA levels of Osterix and alkaline phosphatase (ALP), and ALP activity was suppressed by advanced glycation end product 3 in ST2 cells that were treated with bone morphogenetic protein-2. In conclusion, we have shown for the first time that the local administration of Myo-EVs improves delayed bone repair that is induced by the diabetic state through an enhancement of osteoblastic differentiation in female mice.

Identification of Agents That Ameliorate Hyperphosphatemia-Suppressed Myogenin Expression Involved in the Nrf2/p62 Pathway in C2C12 Skeletal Muscle Cells.

Hyperphosphatemia can occur as a result of reduced phosphate (Pi) excretion in cases of kidney dysfunction, which can induce muscle wasting and suppress myogenic differentiation. Higher Pi suppresses myogenic differentiation and promotes muscle atrophy through canonical (oxidative stress-mediated) and noncanonical (p62-mediated) activation of nuclear factor erythroid 2-related factor 2 (Nrf2) signaling. However, the crosstalk between myogenin and Nrf2/p62 and potential drug(s) for the regulation of myogenin expression needed to be addressed. In this study, we further identified that myogenin may negatively regulate Nrf2 and p62 protein levels in the mouse C2C12 muscle cell line. In the drug screening analysis, we identified N-acetylcysteine, metformin, phenformin, berberine, 4-chloro-3-ethylphenol, cilostazol, and cilomilast as ameliorating the induction of Nrf2 and p62 expression and reduction in myogenin expression that occur due to high Pi. We further elucidated that doxorubicin and hydrogen peroxide reduced the amount of myogenin protein mediated through the Kelch-like ECH-associated protein 1/Nrf2 pathway, differently from the mechanism of high Pi. The dual functional roles of L-ascorbic acid (L-AA) were found to be dependent on the working concentration, where concentrations below 1 mM L-AA reversed the effect of high Pi on myogenin and those above 1 mM L-AA had a similar effect of high Pi on myogenin when used alone. L-AA exacerbated the effect of hydrogen peroxide on myogenin protein and had no further effect of doxorubicin on myogenin protein. In summary, our results further our understanding of the crosstalk between myogenin and Nrf2, with the identification and verification of several potential drugs that can be applied in rescuing the decline of myogenin due to high Pi in muscle cells.

Upregulation of IL-8, osteonectin, and myonectin mRNAs by intermittent hypoxia via OCT1- and NRF2-mediated mechanisms in skeletal muscle cells.

Sleep apnoea syndrome is characterized by recurrent episodes of oxygen desaturation and reoxygenation (intermittent hypoxia [IH]) and is a risk factor for insulin resistance/Type 2 diabetes. The induction of insulin resistance in skeletal muscle is a key phenomenon to develop diabetes. However, the mechanisms linking IH stress and insulin resistance remain elusive. We exposed human RD and mouse C2C12 muscle cells to normoxia or IH and measured their mRNA levels by real-time RT-PCR. We found that IH significantly increased the mRNA and protein levels of muscle-derived insulin resistance-factors (myokines) such as IL-8, osteonectin (ON), and myonectin (MN) in muscle cells. We further analysed the IH-induced expression mechanisms of IL-8, ON, and MN genes in muscle cells. Deletion analyses of the human myokine promoter(s) revealed that the regions -152 to -151 in IL-8, -105 to -99 in ON, and - 3741 to -3738 in MN promoters were responsible for the activation by IH in RD cells. The promoters contain consensus transcription factor binding sequences for OCT1 in IL-8 and MN promoters, and for NRF2 in ON promoter, respectively. The introduction of siRNA for OCT1 abolished the IH-induced expression(s) of IL-8 and MN and siRNA for NRF2 abolished the IH-induced expression of ON.

Abstract 3852: The chemokine C-C motif ligand 2 (CCL2) plays an important role in skeletal muscle wasting associated with breast cancer progression

Abstract Up to 45% of breast cancer patients experience a condition called Skeletal Muscle Wasting (SMW). SMW causes significant progressive muscle loss, weakness, and fatigue. SMW in patients adversely affects quality of life during and results in a severely reduced ability of patients to tolerate chemotherapeutic drugs. Chemotherapy itself also exacerbates SMW in patients. There are currently no effective therapies for SMW. This condition is rarely reported in patient records and is often overlooked in patients with a high body mass index. Although this condition is associated with significantly lower survival rates compared to patients with relatively normal muscle mass, we do not yet understand the molecular mechanism by which it occurs in breast cancer patients. In this study, we investigate the role that CCL2 plays in breast cancer and chemotherapy induced SMW. CCL2 is a chemokine that regulates recruitment of immune cells to sites of injury and inflammation. CCL2 is overexpressed in breast cancer and SMW has been linked to elevated levels of CCL2. CCL2 overexpression is also associated with high mortality rates in breast cancer patients. To investigate the role of CCL2 in muscle wasting we treated C2C12 mouse muscle cells with increasing concentrations of CCL2. This led to increased expression of markers of muscle degradation, MuRF-1 and Atrogin-1, with reduced muscle cell proliferation and muscle cell size. This indicates that high levels of CCL2 cause muscle degradation. Using an MDA-MB-231 model of breast cancer, we show that CCL2 knockdown in the primary breast tumor decreases the expression of muscle degradation markers. Further experiments using the 4T1 mammary tumor model show that mice in which CCL2 was knocked down exhibited greater grip strength than those mice in which CCL2 levels remained unaltered. Therefore, CCL2 knockdown reduces muscle degradation and increases grip strength in a mouse model of breast cancer. We also investigated the effect of chemotherapy on SMW. Preliminary in vitro data shows increased expression of CCL2 in response to chemotherapy treatment, while in vivo experiments using the 4T1 breast tumor model indicate that chemotherapeutic treatment also significantly reduces grip strength in mice. Our findings suggest that we can improve patient survival rates and quality of life by developing therapies to combat muscle loss during and after treatment. Understanding the mechanism of muscle wasting is necessary to provide insight into novel, targeted therapeutics to reduce SMW and enhance effectiveness of anti-cancer drugs. Citation Format: Nadia Alissa, Wei Bin Fang, Gage Brummer, Marcela Mitchell, John Thyfault, Paige Geiger, Nikki Cheng. The chemokine C-C motif ligand 2 (CCL2) plays an important role in skeletal muscle wasting associated with breast cancer progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3852.

Identification and Characterization TSSK6 Activating Co‐chaperone (TSACC) in Skeletal Muscle

Skeletal muscle atrophy is a physiological condition that is caused by a range of conditions, including immobilization, denervation, spinal cord injury, and aging. To better characterize the molecular genetic events of neurogenic atrophy, a previous study isolated the gastrocnemius muscle from mice following 3 and 14 days of sciatic nerve transection. The gene expression profile in the denervated muscle tissue was then analyzed by microarray and compared to control muscle tissue to identify novel neurogenic atrophy‐induced genes. The microarray data revealed for the first time that TSSK6 Activating Co‐chaperone (TSACC) is expressed in skeletal muscle and is significantly induced in response to denervation. To confirm that TSACC is expressed in muscle, the predicted TSACC cDNA was cloned from cultured muscle cells, as was a novel TSACC splice variant that contains an additional exon. Quantitative PCR (qPCR) was performed to assess TSACC expression levels of the full‐length and novel splice variants in proliferating and differentiated muscle cells. The results demonstrate that TSACC expression levels are relatively low in proliferating myoblasts but show significantly elevated expression in differentiated myotubes. In addition, characterization of the transcriptional regulation of TSACC was assessed by fusing fragments of the proximal promoter located immediately upstream of the start of transcription with a reporter gene. The reporter plasmids were then transfected into C2C12 mouse muscle cells in combination with myogenic regulatory factor (MRF) expression plasmids, which resulted in significant activation of reporter gene activity. Interestingly, there are several predicted E‐box elements in the proximal promoter region of the TSACC gene further suggesting that TSACC may be regulated by muscle‐specific transcription factors. To identify a possible role for TSACC in skeletal muscle, sub‐cellular localization was assessed by fusing TSACC with GFP and expressing the fusion protein in cultured muscle cells, revealing that TSACC is localized predominantly to the cytoplasm. The discovery that TSACC is induced in response to neurogenic atrophy helps further our understanding of the molecular genetic events of muscle wasting and may eventually lead to the identification of new therapeutic targets for the treatment of muscle atrophy.

Role of peripheral myelin protein 22 in chronic exercise-induced interactions of muscle and bone in mice.

Exercise is important for the prevention and treatment of sarcopenia and osteoporosis. Although the interactions between skeletal muscles and bone have recently been reported, the myokines linking muscle to bone during exercise remain unknown. We previously revealed that chronic exercise using treadmill running blunts ovariectomy-induced osteopenia in mice. We herein performed an RNA sequence analysis of the gastrocnemius and soleus muscles of male mice with or without chronic exercise to identify the myokines responsible for the effects of chronic exercise on the muscle/bone relationship. We extracted peripheral myelin protein 22 (PMP22) as a humoral factor that was putatively induced by chronic exercise in the soleus and gastrocnemius muscles of mice from the RNA sequence analysis. Chronic exercise significantly enhanced the expression of PMP22 in the gastrocnemius and soleus muscles of female mice. PMP22 suppressed macrophage-colony stimulating factor and receptor activator factor κB ligand-induced increases in the expression of osteoclast-related genes and osteoclast formation from mouse bone marrow cells. Moreover, PMP22 significantly inhibited osteoblast differentiation, alkaline phosphatase activity, and mineralization in mouse osteoblast cultures; however, the overexpression of PMP22 did not affect muscle phenotypes in mouse muscle C2C12 cells. A simple regression analysis revealed that PMP22 mRNA levels in the gastrocnemius and soleus muscles were positively related to cortical bone mineral density at the femurs of mice with or without chronic exercise. In conclusion, we identified PMP22 as a novel myokine induced by chronic exercise in mice. We first showed that PMP22 suppresses osteoclast formation and the osteoblast phenotype in vitro.

Knockdown of CNN3 Impairs Myoblast Proliferation, Differentiation, and Protein Synthesis via the mTOR Pathway.

BackgroundMyogenesis is a complex process that requires optimal outside–in substrate–cell signaling. Calponin 3 (CNN3) plays an important role in regulating myogenic differentiation and muscle regeneration; however, the precise function of CNN3 in myogenesis regulation remains poorly understood. Here, we investigated the role of CNN3 in a knockdown model in the mouse muscle cell line C2C12.MethodsMyoblast proliferation, migration, differentiation, fusion, and protein synthesis were examined in CNN3 knockdown C2C12 mouse muscle cells. Involvement of the mTOR pathway in CNN3 signaling was explored by treating cells with the mTOR activator MHY1485. The regulatory mechanisms of CNN3 in myogenesis were further examined by RNA sequencing and subsequent gene ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) and gene set enrichment analysis (GSEA).ResultsDuring proliferation, CNN3 knockdown caused a decrease in cell proliferation and migration. During differentiation, CNN3 knockdown inhibited myogenic differentiation, fusion, and protein synthesis in C2C12 cells via the AKT/mTOR and AMPK/mTOR pathways; this effect was reversed by MHY1485 treatment. Finally, KEGG and GSEA indicated that the NOD-like receptor signaling pathway is affected in CNN3 knockdown cell lines.ConclusionCNN3 may promote C2C12 cell growth by regulating AKT/mTOR and AMPK/mTOR signaling. The KEGG and GSEA indicated that inhibiting CNN3 may activate several pathways, including the NOD-like receptor pathway and pathways involved in necroptosis, apoptosis, and inflammation.

Identification of the conserved long non-coding RNAs in myogenesis

BackgroundOur understanding of genome regulation is ever-evolving with the continuous discovery of new modes of gene regulation, and transcriptomic studies of mammalian genomes have revealed the presence of a considerable population of non-coding RNA molecules among the transcripts expressed. One such non-coding RNA molecule is long non-coding RNA (lncRNA). However, the function of lncRNAs in gene regulation is not well understood; moreover, finding conserved lncRNA across species is a challenging task. Therefore, we propose a novel approach to identify conserved lncRNAs and functionally annotate these molecules.ResultsIn this study, we exploited existing myogenic transcriptome data and identified conserved lncRNAs in mice and humans. We identified the lncRNAs expressing differentially between the early and later stages of muscle development. Differential expression of these lncRNAs was confirmed experimentally in cultured mouse muscle C2C12 cells. We utilized the three-dimensional architecture of the genome and identified topologically associated domains for these lncRNAs. Additionally, we correlated the expression of genes in domains for functional annotation of these trans-lncRNAs in myogenesis. Using this approach, we identified conserved lncRNAs in myogenesis and functionally annotated them.ConclusionsWith this novel approach, we identified the conserved lncRNAs in myogenesis in humans and mice and functionally annotated them. The method identified a large number of lncRNAs are involved in myogenesis. Further studies are required to investigate the reason for the conservation of the lncRNAs in human and mouse while their sequences are dissimilar. Our approach can be used to identify novel lncRNAs conserved in different species and functionally annotated them.

Effects of fluid flow shear stress to mouse muscle cells on the bone actions of muscle cell-derived extracellular vesicless.

The interactions between skeletal muscle and bone have been recently noted, and muscle-derived humoral factors related to bone metabolism play crucial roles in the muscle/bone relationships. We previously reported that extracellular vesicles from mouse muscle C2C12 cells (Myo-EVs) suppress osteoclast formation in mice. Although mechanical stress is included in extrinsic factors which are important for both muscle and bone, the detailed roles of mechanical stress in the muscle/bone interactions have still remained unknown. In present study, we examined the effects of fluid flow shear stress (FFSS) to C2C12 cells on the physiological actions of muscle cell-derived EV. Applying FFSS to C2C12 cells significantly enhanced muscle cell-derived EV-suppressed osteoclast formation and several osteoclast-related gene levels in mouse bone marrow cells in the presence of receptor activator nuclear factor κB ligand (RANKL). Moreover, FFSS to C2C12 cells significantly enhanced muscle cell-derived EV-suppressed mitochondria biogenesis genes during osteoclast formation with RANKL treatment. In addition, FFSS to C2C12 cells significantly enhanced muscle cell-derived EV-suppressed osteoclast formation and several osteoclast-related gene levels in Raw264.7 cells in the presence of RANKL. Small RNA-seq-analysis showed that FFSS elevated the expression of miR196a-5p and miR155-5p with the suppressive actions of osteoclast formation and low expression in mouse bone cells. On the other hand, muscle cell-derived EVs with or without FFSS to C2C12 cells did not affect the expression of osteogenic genes, alkaline phosphatase activity and mineralization in mouse osteoblasts. In conclusion, we first showed that FFSS to C2C12 cells enhances the suppressive effects of muscle cell-derived EVs on osteoclast formation in mouse cells. Muscle cell-derived EVs might be partly involved in the effects of mechanical stress on the muscle/bone relationships.

Role of Dkk2 in the Muscle/bone Interaction of Androgen-Deficient Mice

AbstractAndrogen deficiency is known to cause both osteoporosis and sarcopenia. Myokines, humoral factors secreted from the skeletal muscles, have recently been getting attention as the key factors related to the interactions between muscle and bone. Dickkopf (Dkk) 2 is known as an inhibitor of canonical Wnt/β-catenin signaling, and Wnt/β-catenin signaling is crucial for the maintenance of muscle and bone. The present study was therefore performed to investigate the roles of Dkk2 in the alterations of muscle and bone of androgen-deficient mice with orchidectomy (ORX). ORX significantly enhanced Dkk2 mRNA levels, but not other Dkks and secreted frizzled related proteins, in the soleus muscles of mice. Moreover, ORX enhanced serum Dkk2 levels, but not Dkk2 mRNA levels in the tibial bone tissues, the white adipose tissues and liver of mice. In simple regression analyses, serum Dkk2 levels were negatively related to trabecular bone mineral density at the tibias in mice employed in the experiments. In vitro experiments, testosterone suppressed Dkk2 mRNA levels in mouse muscle C2C12 cells. In conclusion, we showed that androgen deficiency enhances Dkk2 expression and secretion in the muscles of mice. Dkk2 might be involved in androgen deficiency-induced muscle wasting and osteopenia as a myokine linking muscle to bone.

Identification and characterization of Fbxl22, a novel skeletal muscle atrophy-promoting E3 ubiquitin ligase.

Muscle-specific E3 ubiquitin ligases have been identified in muscle atrophy-inducing conditions. The purpose of the current study was to explore the functional role of F-box and leucine-rich protein 22 (Fbxl22), and a newly identified splice variant (Fbxl22-193), in skeletal muscle homeostasis and neurogenic muscle atrophy. In mouse C2C12 muscle cells, promoter fragments of the Fbxl22 gene were cloned and fused with the secreted alkaline phosphatase reporter gene to assess the transcriptional regulation of Fbxl22. The tibialis anterior muscles of male C57/BL6 mice (12-16 wk old) were electroporated with expression plasmids containing the cDNA of two Fbxl22 splice variants and tissues collected after 7, 14, and 28 days. Gastrocnemius muscles of wild-type and muscle-specific RING finger 1 knockout (MuRF1 KO) mice were electroporated with an Fbxl22 RNAi or empty plasmid and denervated 3 days posttransfection, and tissues were collected 7 days postdenervation. The full-length gene and novel splice variant are transcriptionally induced early (after 3 days) during neurogenic muscle atrophy. In vivo overexpression of Fbxl22 isoforms in mouse skeletal muscle leads to evidence of myopathy/atrophy, suggesting that both are involved in the process of neurogenic muscle atrophy. Knockdown of Fbxl22 in the muscles of MuRF1 KO mice resulted in significant additive muscle sparing 7 days after denervation. Targeting two E3 ubiquitin ligases appears to have a strong additive effect on protecting muscle mass loss with denervation, and these findings have important implications in the development of therapeutic strategies to treat muscle atrophy.

1887-P: Intermittent Hypoxia Increased the Expressions of Interleukin (IL)-8, Osteonectin, and Myonectin via OCT1 and Nrf2 Binding to the Promoters in Muscle Cells

Sleep apnea syndrome (SAS) is characterized by recurrent episodes of oxygen desaturation and reoxygenation (intermittent hypoxia [IH]) and is a risk factor for insulin resistance/type 2 diabetes. However, the mechanisms linking IH stress and insulin resistance remain elusive. We exposed human RD and mouse C2C12 muscle cells either to 70 cycles/24 h of IH (5 min hypoxia [1% O2]/10 min normoxia [21% O2]), mimicking SAS patients, or normoxia for 24 h, measured mRNA levels by real-time reverse transcription polymerase chain reaction, and found that IH significantly increased the mRNA levels of interleukin-8 (IL-8), osteonectin (ON), and myonectin (MN). IL-8, ON, and MN in IH-treated RD cell medium were also increased (P&amp;lt;0.0001, P&amp;lt;0.0001, and P=0.0079, respectively). We further analyzed the IH-induced expression mechanisms of IL-8, ON, and MN genes in RD muscle cells. Deletion analyses revealed that -152 to -151 region in IL-8, -105∼-99 region in ON, and -3,741∼-3,738 region in MN promoter(s) were responsible for the activation by IH. The promoters contain consensus transcription factor binding sequences for octamer binding transcription factor 1 (OCT1) in IL-8 and MN, and for nuclear factor erythroid 2-related factor 2 (NRF2) in ON, respectively. The introduction of siRNA for OCT1, and NRF2 abolished the increase of mRNAs for IL-8 and MN, and ON, respectively. As IL-8, ON, and MN are reported to be myokines and associated with glucose intolerance, insulin resistance, and diabetes, the results could well explain a mechanism of glucose intolerance, insulin resistance, and diabetes in SAS patients. Disclosure S. Takasawa: None. R. Shobatake: None. A. Itaya-Hironaka: None. M. Makino: None. S. Sakuramoto-Tsuchida: None. T. Uchiyama: None. H. Ota: None. A. Yamauchi: None.

Extracellular vesicles secreted from mouse muscle cells suppress osteoclast formation: Roles of mitochondrial energy metabolism.

Recent reports have described the interactions of muscle and bone. Various muscle-derived humoral factors, known as myokines, affect bone. Although extracellular vesicles (EVs) play a vital role in physiological and pathophysiological processes by transferring their contents to distant tissues during bone metabolism, the roles of EVs in the muscle-bone interactions remain unknown. In the present study, we investigated the effects of EVs secreted from mouse muscle C2C12 cells on mouse bone cells and mitochondrial biogenesis. EVs secreted from C2C12 cells (Myo-EVs) were isolated from the conditioned medium of C2C12 cells by ultracentrifugation. Myo-EVs suppressed osteoclast formation as well as the expression of tartrate-resistant acid phosphatase, cathepsin K, nuclear factor of activated T-cells cytoplasmic 1 and dendritic cell-specific transmembrane protein induced by receptor activator of nuclear factor κB ligand (RANKL) in mouse bone marrow cells and preosteoclastic Raw264.7 cells. Moreover, Myo-EVs suppressed oxygen consumption and mRNA expression of the mitochondrial biogenesis markers enhanced by RANKL in these cells. However, Myo-EVs did not affect the phenotypes or mitochondrial biogenesis of mouse primary osteoblasts. In conclusion, the present study showed for the first time that Myo-EVs suppress osteoclast formation and mitochondrial energy metabolism in mouse bone marrow and Raw264.7 cells. EVs secreted from skeletal muscles might be a crucial mediator of muscle-bone interactions.

Non‑toxic sulfur enhances growth hormone signaling through the JAK2/STAT5b/IGF‑1 pathway in C2C12 cells.

Insulin‑like growth factor‑1 (IGF‑1) regulates cell growth, glucose uptake and protein metabolism, and is required for growth hormone (GH) signaling‑mediated insulin production and secretion. IGF1 expression is associated with STAT5, which binds to a region (TTCNNNGAA) of the gene. Although sulfur is used in various fields, the toxicity of this element is a significant disadvantage as it causes indigestion, vomiting, diarrhea, pain and migraine. Therefore, it is difficult to conduct invitro experiments to directly determine the effects of dietary sulfur. Additionally, it is difficult to dissolve non‑toxic sulfur (NTS). The present study aimed to identify the role of NTS in GH signaling as a Jak2/STAT5b/IGF‑1 pathway regulator. MTT assay was used to identify an optimum NTS concentration for C2C12 mouse muscle cells. Western blotting, RT‑PCR, chromatin immunoprecipitation, overexpression and small interfering RNA analyses were performed. NTS was dissolved in 1mg/ml DMSO and could be used invitro. Therefore, the present study determined whether NTS induced mouse muscle cell growth via GH signaling. NTS notably increased STAT5b binding to the Igf1 promoter. NTS also promoted GH signaling by upregulating GH receptor expression, similar to GH treatment. NTS enhanced GH signaling by regulating Jak2/STAT5b/IGF‑1 signaling pathway factor expression in C2C12 mouse muscle cells. Thus, NTS may be used as a GH‑enhancing growth stimulator.

Trim33 (Tif1\u03b3) is not required for skeletal muscle development or regeneration but suppresses cholecystokinin expression

The expression of Trim33 (Tif1γ) increases in skeletal muscles during regeneration and decreases upon maturation. Although Trim33 is required for the normal development of other tissues, its role in skeletal muscle is unknown. The current study aimed to define the role of Trim33 in muscle development and regeneration. We generated mice with muscle-specific conditional knockout of Trim33 by combining floxed Trim33 and Cre recombinase under the Pax7 promoter. Muscle regeneration was induced by injuring mouse muscles with cardiotoxin. We studied the consequences of Trim33 knockdown on viability, body weight, skeletal muscle histology, muscle regeneration, and gene expression. We also studied the effect of Trim33 silencing in satellite cells and the C2C12 mouse muscle cell line. Although Trim33 knockdown mice weighed less than control mice, their skeletal muscles were histologically unremarkable and regenerated normally following injury. Unexpectedly, RNAseq analysis revealed dramatically increased expression of cholecystokinin (CCK) in regenerating muscle from Trim33 knockout mice, satellite cells from Trim33 knockout mice, and C2C12 cells treated with Trim33 siRNA. Trim33 knockdown had no demonstrable effect on muscle differentiation or regeneration. However, Trim33 knockdown induced CCK expression in muscle, suggesting that suppression of CCK expression requires Trim33.

4-PHENYLBUTYRATE: MOLECULAR MECHANISMS AND AGING INTERVENTION POTENTIAL

4-Phenylbutyrate (PBA) is a FDA approved drug for treating patients with urea cycle disorders. Additionally, PBA acts upon several pathways thought of as important modifiers of aging including: histone deacetylation, proteostasis as a chemical chaperone, and stress resistance by regulating expression of oxidative stress response proteins. PBA has also been shown to extend lifespan and improve markers of age-related health in Drosophila. Due to its wide range of effects PBA has been investigated for use in numerous age-related disorders including neurodegenerative and cardiovascular diseases. To better understand the effects of PBA on the molecular level, we used both in cellulo and in vivo studies. Treatment of primary mouse fibroblasts, C2C12 mouse muscle cells, and NCTC 1469 mouse liver cells with PBA demonstrated differential responses among cell lines to upregulation of oxidative stress response and histone acetylation. Specifically, upregulation of the oxidative stress response protein DJ-1 by PBA was found to have a corresponding dose response curve to histone H3 acetylation in primary fibroblasts. To study effects of PBA in vivo, four cohorts of HET3 mice were treated with PBA at different doses in drinking water for 4 weeks. PBA was well tolerated and led to different effects on body composition dependent on the sex of mice. We are currently investigating the molecular effects of PBA treatment in multiple tissues samples from these mice. The potential of PBA to alter many fundamental pathways, and specifically those related to stress responses, make it an attractive prospect for treatment of many age-related disorders.