C2C12 Myotube Cells Research Articles

801-P: SGLT2 Inhibitor, Luseogliflozin, Prevent Sarcopenia by Improving Extracellular Lipidome

Aims: It has been reported that there is a close relationship between sarcopenia obesity and metabolic disorder such as lipid metabolism. Here we investigated the effect on lipid metabolism of the sodium glucose cotransporter-2 inhibitor (SGLT-2i) luseogliflozin in skeletal muscle. We preformed microarray analysis of mRNA in skeletal muscle and investigated the lipid profile in skeletal muscle and serum. Methods: Eight-week-old mice were fed a standard diet or a standard diet supplemented with luseogliflozin (0.01% w/w chow) for 8 weeks. Mice were divided into the following three genotype/diet groups: Db/m mice without (w/o) SGLT-2i, Db/Db w/o SGLT-2i, and Db/Db with SGLT-2i. After 8 weeks of diet, in each group, body weight, glucose tolerance, muscle weight and cross-sectional area, free fatty acid concentration, muscle and gene expression in serum were investigated. Results: The ratio of soleus muscle to body weight and the cross-sectional area of soleus muscle were significantly higher in Db/Db mice with SGLT-2i than in Db/Db mice w/o SGLT-2i. In addition, the concentration of saturated fatty acids in skeletal muscle and serum of Db/Db mice with SGLT-2i was significantly lower than that w/o SGLT2i, and the expression of Fasn, Elovl6, and Scd1, which are involved in fatty acid synthesis, in skeletal muscle was significantly increased in Db/Db w/o SGLT-2i compared to Db/m mice with SGLT-2i, and significantly decreased in Db/Db with SGLT-2i compared to mice w/o SGLT-2i. Furthermore, in cell experiments using C2C12 myotube cells, the administration of saturated fatty acid increased the expression of muscle atrophy-related genes. Conclusion: The administration of luseogliflozin suppressed skeletal muscle atrophy and reduced the concentration of saturated fatty acids in skeletal muscle. Based on these findings, we hypothesized that secondary effects, other than the hypoglycemic effects of SGLT2i, might lead to the alleviation of obesity-dependent sarcopenia. Disclosure R. Bamba: None. T. Okamura: None. Y. Hashimoto: None. T. Senmaru: None. M. Hamaguchi: Research Support; Spouse/Partner; AstraZeneca K. K. M. Fukui: None.

Identifying a New Mechanism of Sarcopenia by Autophagy

Sarcopenia is one of the critical factors in reducing Activity of Daily Life and associated with morbidity and mortality. Sarcopenia has also been linked to metabolic syndrome. In recent years, it has been reported that autophagy is one of the mechanisms as a cause of sarcopenia. Therefore, we focused on autophagy as a system that can regulate both sarcopenia and metabolic syndrome in skeletal muscle and revealed that non-receptor tyrosine kinase Fyn not only participates in metabolic syndrome but also regulates autophagy regulating sarcopenia through STAT3 regulation, mainly using transgenic mice (Cell metabolism 2010, Cell Rep. 2012). However, since these were non-physiological studies, we proceeded with further studies and demonstrating that Fyn dependent STAT3 phosphorylation by IL6, which is involved in chronic inflammation and metabolic syndrome, was observed in mouse C2C12 myotube cells. Autophagy was decreased in those cells by both IL6 dependent and Fyn dependent mechanisms. Furthermore, in the denervated mouse model, not only both Fyn and IL6 gene expressions as well as the key muscle specific E3 ubiquitin ligases, Atrogin1 and MuRf1 were increased but the expression and phosphorylation levels of STAT3 were also augmented, while the autophagy activity was decreased. We believe that a denervated mouse model alone is not enough as a model for sarcopenia, thus we next introduced a hind limb suspension mouse model that promotes disuse atrophy by suspending the hind limb. Using this model, we found that muscle atrophy was observed mainly in the soleus muscle, tibialis anterior muscle, and the gastrocnemius muscle with Atrogin1 and MuRf1 increased. Increase of both IL6 and STAT3 expression/phosphorylation were also observed in the muscles of hind limb suspension mice. Autophagy activity, examined by intraperitoneal administration of colchicine, was decreased. These results strongly suggest that Fyn is involved not only in the metabolic syndrome but also in the pathogenesis of sarcopenia, and may lead to a better understanding of the pathology of sarcopenia obesity and the development of therapeutic methods.

Discovery and optimization of novel 3-benzyl-N-phenyl-1H-pyrazole-5-carboxamides as bifunctional antidiabetic agents stimulating both insulin secretion and glucose uptake.

A novel series of 3-benzyl-N-phenyl-1H-pyrazole-5-carboxamides was designed, synthesized and evaluated for their biological activities on glucose-stimulated insulin secretion (GSIS). The cytotoxicity of all 41 novel compounds was screened to assess their pharmacological safety in pancreatic β-cells. A two-step optimization process was carried out to establish the structure-activity relationship for this class and subsequently we identified the most active analogue 26. Further modification study of 26 evidenced the necessity of N-hydrogens in the core architecture. Protein expression analysis suggested that 26 increases insulin secretion via the activation of the upstream effector of pancreatic and duodenal homeobox 1 (PDX-1), which is an important factor promoting GSIS. Moreover, the administration of 26 effectively augmented glucose uptake in C2C12 myotube cells via the suppression of Mitsugumin 53 (MG53), an insulin receptor substrate 1 (IRS-1) ubiquitination E3 ligase.

Increase in PPARγ inhibitory phosphorylation by Fetuin—A through the activation of Ras-MEK-ERK pathway causes insulin resistance

Obesity induced insulin resistance is primarily regulated by the inhibitory phosphorylation of peroxisome proliferator-activated receptor γ at serine 273 (PPARγS273) which has been shown to be regulated by MEK and ERK. An upstream regulatory molecule of this pathway could be a therapeutic option. Here we analyzed the involvement of Fetuin-A (FetA), a key hepato-adipokine implicated in insulin resistance, as an upstream regulator molecule for the regulation of PPARγ inhibitory phosphorylation. Mice fed with standard diet (SD), high fat diet (HFD) and HFD with FetA knockdown (HFD-FetAKD) were used to examine the role of FetA on PPARγS273 phosphorylation in adipocytes. The mechanism of regulation and its effect on skeletal muscle were studied using primary adipocytes, 3T3-L1 (preadipocyte) and C2C12 (myotube) cell lines. Increased FetA in HFD mice strongly correlated with augmentation of PPARγS273 phosphorylation in inflamed adipocytes while knockdown of FetA suppressed it. This effect of FetA was mediated through the activation of Ras which in turn activated MEK and ERK. On addressing how FetA could stimulate activation of Ras, we found that FetA triggered TNFα in inflamed adipocytes which induced Ras activation. The ensuing sharp fall in adiponectin level attenuated AMPK activation in skeletal muscle cells affecting mitochondrial ATP production. Our data reveal the essential role of FetA induced activation of Ras in regulating PPARγ inhibitory phosphorylation through Ras-MEK-ERK pathway which downregulates adiponectin disrupting skeletal muscle mitochondrial bioenergetics. Thus, FetA mediated PPARγ inactivation has adverse consequences upon adipocyte-myocyte crosstalk leading to disruption of energy homeostasis and loss of insulin sensitivity.

Linalool Prevents Cisplatin Induced Muscle Atrophy by Regulating IGF-1/Akt/FoxO Pathway.

Skeletal muscle atrophy is an important feature of cancer cachexia, which can be induced by chemotherapy, and affects the survival and quality of life of cancer patients seriously. No specific drugs for cancer cachexia have been applied in clinical practice. This study explored the therapeutic effect of linalool (LIN) on cisplatin (DDP) induced skeletal muscle atrophy. In vivo, LIN can improve skeletal muscle weight loss, anorexia, muscle strength decline and other cachexia symptoms caused by cisplatin treatment in a Lewis lung cancer tumor bearing mouse model, and cause no adverse effects on the anti-tumour effect. LIN treatment decreased the expression of muscle RING-finger protein-1 (MuRF1) and Atrogin1(MAFbx) in muscle, and the activation of insulin-like growth factor-1 (IGF-1)/protein kinase B (Akt)/forkhead box O (FoxO) pathway was observed. In vitro, LIN alleviated DDP induced C2C12 myotube atrophy, and IGF-1 receptor inhibitor Picropodophyllin (PIC), which had no adverse effect on C2C12 myotube cells, could reverse the protective effect of LIN. These results indicate that LIN down-regulates the expression of Atrogin1 and MuRF1 through the IGF-1/Akt/FoxO pathway, alleviating DDP-induced muscle atrophy and improving cachexia symptoms. LIN has the potential to be developed as a drug against cancer cachexia.

Abstract 17084: Nuclear Smooth Muscle α-actin Participates in Chromatin Remodeling at Smooth Muscle Contractile Gene Promoters

Actin genes encode for cytoskeletal proteins that polymerize to function in cellular motility, adhesion, and contraction. In mammalian cells, ubiquitously expressed β-actin also moves into the nucleus and associates with chromatin remodeling complexes, however a nuclear function of muscle-specific α-actins has not been previously assessed. We hypothesized that smooth muscle α-actin (SMA) plays a role in chromatin remodeling during the differentiation of smooth muscle cells (SMCs) to enable cell fate specification of SMCs. In explanted SMCs from human and mouse ascending aortas, cell fractionation and 2D gel electrophoresis identify both SMA and β-actin in the nuclear lysates. Nuclear SMA but not β-actin accumulates with SMC differentiation driven by serum starvation and transforming growth factor-β1 treatment. SMA accumulates into the nucleus early in the differentiation of SMCs from neural crest progenitor cells, prior to cytosolic accumulation. Immunoprecipitation studies show that SMA binds specifically to the INO80 and the SWI/SNF chromatin remodeling complexes, and this binding increases with SMC differentiation. Chromatin immunoprecipitation reveals that SMA is bound to the promoters of SMC-specific genes, including Acta2 , Cnn1, and Myh11 and that SMA is enriched over β-actin at these promoters with SMC differentiation. Finally, overexpression of SMA tagged with a nuclear localization sequence (NLS) in multiple cell types increases expression of SMC markers, whereas NLS-tagged β-actin localizes to the nucleus to the same extent but does not increase SMC marker expression in any cell type. Finally, we assessed whether skeletal muscle α-actin (SKA) and cardiac muscle α-actin (CMA) may play a similar role in skeletal and cardiac muscle cells. Both SKA and CMA translocate into the nucleus. CMA accumulates into the nucleus early in the differentiation of cardiomyocytes from pluripotent stem cells. Immunoprecipitation reveals that SKA binds to the SWI/SNF complex in differentiated C2C12 myotube cell cultures. These data support that nuclear SMA enriches with and participates in SMC differentiation, and suggest a potential nuclear role for other muscle specific α-actins in developing muscle cells.

1885-P: Fyn Regulates Sarcopenia by Autophagy

Sarcopenia is an important factor in reducing Activity of Daily Life. Sarcopenia has also been linked to insulin resistance. It has been reported that autophagy is one of the mechanisms as a cause of sarcopenia. We focused on autophagy as a system that can regulate both sarcopenia and metabolic syndrome in skeletal muscle and revealed that non-receptor tyrosine kinase Fyn not only participates in metabolic syndrome but also regulates autophagy regulating sarcopenia through STAT3 regulation (Cell metabolism 2010, Cell Rep. 2012). We proceeded with further studies and demonstrating that Fyn-dependent STAT3 phosphorylation by IL6, which is involved in chronic inflammation and metabolic syndrome, was observed in mouse C2C12 myotube cells. Autophagy was decreased in those cells by both IL6-dependent and Fyn-dependent mechanisms. The binding of STAT3 and Fyn was confirmed by immunoprecipitation in an in vitro system. Furthermore, in the denervated mouse model, not only both Fyn and IL6 gene expressions were increased but the expression and phosphorylation levels of STAT3 were also augmented, while the autophagy activity was decreased. We believe that a denervated mouse model alone is not sufficient as a model for sarcopenia, thus we next introduced a hind limb suspension mouse model that promotes disuse atrophy by suspending the hind limb. Using this model, we found that muscle atrophy was observed mainly in the soleus muscle, tibialis anterior muscle, and the gastrocnemius muscle. In addition, the findings are consistent with autophagy failure, and accumulation of glycogen (PAS staining) was observe. Increase of both IL6 and STAT3 expression/phosphorylation were observed in the muscles of those mice. Autophagy activity, examined by intraperitoneal administration of colchicine, was decreased. These results strongly suggest that Fyn is involved not only in the metabolic syndrome but also in the pathogenesis of sarcopenia, and may lead to a better understanding of the pathology of sarcopenia obesity and the development of therapeutic methods. Disclosure E. Yamada: None. R. Uehara: None. Y. Nakajima: None. K. Horiguchi: None. E. Ishida: None. S. Matsumoto: None. S. Okada: None. C.C. Bastie: None. M. Yamada: None.

MiR-23b-3p acts as a counter-response against skeletal muscle atrophy.

To investigate the role of microRNA (miRNA) in muscle atrophy, we performed microarray analysis of miRNA expression in skeletal muscles of Sham, orchiectomized (ORX) mice, and ORX mice treated with androgen and identified that the expression of miR-23b-3p in ORX mice was significantly higher than that in Sham mice (P = 0.007); however, miR-23b-3p expression in ORX mice treated with androgen was lower (P = 0.001). We also investigated the mechanism by which overexpression or knockdown of miR-23b-3p influences the expression of myosin heavy chain, muscle protein synthesis, ATP activity, and glucose uptake in C2C12 myotube cells. Moreover, we examined the serum miR-23b-3p levels among male subjects with type 2 diabetes and whether the serum miR-23b-3p levels could be a biomarker for muscle atrophy. The overexpression of miR-23b-3p in C2C12 myotube cells significantly upregulated the expression of myosin heavy chain, protein synthesis, ATP activity, and glucose uptake. Reporter assays raised a possible direct post-transcriptional regulation involving miR-23b-3p and the 3'-UTR of PTEN mRNA. Among subjects with type 2 diabetes, serum miR-23b-3p levels in the subjects with decreased muscle mass were significantly higher compared to the levels in the subjects without. Our results indicate that miR-23b-3p downregulates the expression of PTEN in myotube cells and induces the growth of myosin heavy chain. In addition, the serum level of miR-23b-3p can be used as a diagnostic marker for muscle atrophy.

1937-P: miR-23b-3p Upregulates Glucose Uptake in C2C12 Myotube Cells by Regulating the Expression of PTEN

Sarcopenia, i.e., the loss of muscle mass and degradation of muscle that can occur with aging, increases an individual's risk of death. MicroRNAs are negative regulators of gene expression that have been implicated in many biological processes, including metabolism. Here we investigated the role of microRNA in sarcopenia. Orchiectomized (ORX) mice have decreased body weight and muscle mass, a smaller cross-sectional area of muscle, and impaired glucose tolerance. In our microarray analysis of the microRNA expression in skeletal muscle, the expression of miR-23b-3p in ORX mice was significantly higher than that in Sham mice; the expression in ORX mice treated with androgen was conversely lower. The overexpression of miR-23b-3p in C2C12 myotube cells significantly upregulated the expression of myosin heavy chain, protein synthesis, ATP activity, and glucose uptake. Reporter assays revealed direct post-transcriptional regulation involving miR-23b-3p and the 3'-untranslated region of PTEN mRNA. Among male subjects with type 2 diabetes, the serum miR-23b-3p levels in the subjects with sarcopenia was significantly higher compared to the levels of the subjects without sarcopenia. Our results indicate that miR-23b-3p negatively regulates the expression of PTEN in myotube cells and induces the upregulation of glucose uptake. In addition, the serum level of miR-23b-3p can be a surrogate marker of sarcopenia. Disclosure T. Okamura: None. Y. Hashimoto: Research Support; Self; Asahi Kasei Pharma. T. Osaka: None. T. Senmaru: None. M. Hamaguchi: None. M. Fukui: Research Support; Self; Astellas Pharma Inc., Boehringer Ingelheim Pharmaceuticals, Inc., Daiichi Sankyo Company, Limited, Eli Lilly and Company, Johnson & Johnson, Kyowa Hakko Kirin Co., Ltd., Merck & Co., Inc., Mitsubishi Tanabe Pharma Corporation, Novo Nordisk Inc., Ono Pharmaceutical Co., Ltd., Sanofi, Sanwa Kagaku Kenkyusho, Takeda Pharmaceutical Company Limited. Speaker's Bureau; Self; AstraZeneca, Eli Lilly and Company, Kowa Pharmaceutical Europe Co. Ltd., Kyowa Hakko Kirin Co., Ltd., Merck & Co., Inc., Mitsubishi Tanabe Pharma Corporation, Novo Nordisk Inc., Ono Pharmaceutical Co., Ltd., Sanofi, Sanwa Kagaku Kenkyusho, Taisho Pharmaceutical Co., Ltd., Takeda Pharmaceutical Company Limited.

Myristic acid specifically stabilizes diacylglycerol kinase δ protein in C2C12 skeletal muscle cells

Decreased levels of the δ isozyme of diacylglycerol kinase (DGK) in skeletal muscle attenuate glucose uptake and, consequently, are critical for the pathogenesis of type 2 diabetes. We recently found that free myristic acid (14:0), but not free palmitic acid (16:0), increased the DGKδ protein levels and enhanced glucose uptake in C2C12 myotube cells. However, it has been unclear how myristic acid regulates the level of DGKδ2 protein. In the present study, we characterized the myristic acid-dependent increase of DGKδ protein. A cycloheximide chase assay demonstrated that myristic acid, but not palmitic acid, markedly stabilized DGKδ protein. Moreover, other DGK isozymes, DGKη and ζ, as well as glucose uptake-related proteins, such as protein kinase C (PKC) α, PKCζ, Akt and glycogen synthase kinase 3β, failed to be stabilized by myristic acid. Furthermore, DGKδ was not stabilized in cultured hepatocellular carcinoma cells, pancreas carcinoma cells or neuroblastoma cells, and only a moderate stabilizing effect was observed in embryonic kidney cells. A proteasome inhibitor and a lysosome inhibitor, MG132 and chloroquine, respectively, partly inhibited DGKδ degradation, suggesting that myristic acid prevents, at least in part, the degradation of DGKδ by the ubiquitin-proteasome system and the autophagy-lysosome pathway. Overall, these results strongly suggest that myristic acid attenuates DGKδ protein degradation in skeletal muscle cells and that this attenuation is fatty acid-, protein- and cell line-specific. These new findings provide novel insights into the molecular mechanisms of the pathogenesis of type 2 diabetes mellitus.

A diet defined by its content of bovine milk exosomes and their RNA cargos has moderate effects on gene expression, amino acid profiles and grip strength in skeletal muscle in C57BL/6 mice

Exosomes are nanoparticles that transfer cargos from donor cells to recipient cells where they elicit changes in gene expression and metabolism. Evidence suggests that exosomes and their cargos are also absorbed from dietary sources such as bovine milk, and bovine exosomes promote the growth of myofibers in murine C2C12 myotube cell cultures. The aim of the current study was to determine whether the dietary intake of bovine milk exosomes alters strength, gene expression and amino acid profiles in murine skeletal muscles. Male and female C57BL/6 mice, age three weeks, were fed an AIN93G-based, exosome and RNA-depleted (ERD) diet for six weeks; controls were fed an exosome and RNA-sufficient (ERS) diet. Variables of feeding behavior, metabolism, grip strength, liver and kidney function, amino acid profiles, and gene expression patterns were analyzed by using metabolic cages, grip strength analyzers, clinical chemistry analyzers, targeted LC/MS–MS, and RNA sequencing analysis. The diets had no effect on food and water intake, respiratory exchange rate, physical activity, grip strength, markers of liver and kidney dysfunction, and amino acid profiles in muscle. Only twelve and nine mRNAs were differentially expressed in skeletal muscle from female and male mice, respectively, fed ERD and ERS diets. The modest effect of the ERD diet on gene expression and levels of free amino acids in skeletal muscle is consistent with observations that bovine milk exosomes and their cargos accumulate in tissues other than skeletal muscle.

Study of muscle contraction induced by electrical pulse stimulation and nitric oxide in C2C12 myotube cells.

[Purpose]This study aimed to examine the independent effect of electrical pulse stimulation(EPS) and nitric oxide(NO) on muscle contraction and their synergistic or combined effect on contraction phenomenon using C2C12 mouse skeletal muscle cells.[Methods]Some differentiated C2C12 myotube cells were untreated (control). Other cells did not receive EPS and did receive 0.5, 1.0, or 2.0 mM of the NO donor, S-nitroso-N-acetyl-penicillamine (SNAP; -E/S0.5, -E/S1.0, and -E/S2.0, respectively). For the EPS treatments (0.3 V/mm, 1.0 Hz, and 4.0 ms), differentiated C2C12 myotube cells received only EPS or both EPS and the SNAPtreatments at the same concentrations (+E/-S, +E/S0.5, +E/S1.0, and +E/S2.0, respectively). All samples were then cultured for 4 days.[Results]Differentiated C2C12 cellswere stimulated by the EPS, NO, and EPS+NO treatments. The cell length of the +E/S2.0 Group after the 4-day culture (84.2±13.2㎛) was the shortest of all the groups. The expressions of AMPK, JNK, Akt, eNOS, GLUT4, and PGC1α proteins were noticeably dominant. The results indicated synergistic effect on muscle contraction of simultaneously applied EPS and SNAP.[Conclusion]Motor skills were significantly improved when exercise was accompanied by the intake of NO precursor and/or NO, compared to that upon their independent application or treatment.

Eburicoic Acid, a Triterpenoid Compound from Antrodia camphorata, Displays Antidiabetic and Antihyperlipidemic Effects in Palmitate-Treated C2C12 Myotubes and in High-Fat Diet-Fed Mice.

This study was designed to investigate the antidiabetic and antihyperlipidemic effects and mechanisms of eburicoic acid (TRR); one component of Antrodia camphorata in vitro and in an animal model for 14 weeks. Expression levels of membrane glucose transporter type 4 (GLUT4); phospho-5′-adenosine monophosphate-activated protein kinase (AMPK)/total AMPK; and phospho-Akt/total Akt in insulin-resistant C2C12 myotube cells were significantly decreased by palmitate; and such decrease was prevented and restored by TRR at different concentrations. A group of control (CON) was on low-fat diet over a period of 14 weeks. Diabetic mice; after high-fat-diet (HFD) induction for 10 weeks; were randomly divided into six groups and were given once a day oral gavage doses of either TRR (at three dosage levels); fenofibrate (Feno) (at 0.25 g/kg body weight); metformin (Metf) (at 0.3 g/kg body weight); or vehicle (distilled water) (HF group) over a period of 4 weeks and still on HFD. Levels of glucose; triglyceride; free fatty acid (FFA); insulin; and leptin in blood were increased in 14-week HFD-fed mice as compared to the CON group; and the increases were prevented by TRR, Feno, or Metf as compared to the HF group. Moreover, HFD-induction displayed a decrease in circulating adiponectin levels, and the decrease was prevented by TRR, Feno, or Metf treatment. The overall effect of TRR is to decrease glucose and triglyceride levels and improved peripheral insulin sensitivity. Eburicoic acid, Feno, and Metf displayed both enhanced expression levels of phospho-AMPK and membrane expression levels of GLUT4 in the skeletal muscle of HFD-fed mice to facilitate glucose uptake with consequent enhanced hepatic expression levels of phospho-AMPK in the liver and phosphorylation of the transcription factor forkhead box protein O1 (FOXO1) but decreased messenger RNA (mRNA) of phosphenolpyruvate carboxykinase (PEPCK) to inhibit hepatic glucose production; resulting in lowered blood glucose levels. Moreover; TRR treatment increased hepatic expression levels of the peroxisome proliferator-activated receptor α (PPARα) to enhance fatty acid oxidation; but displayed a reduction in expressions of hepatic fatty acid synthase (FAS) but an increase in fatty acid oxidation PPARα coincident with a decrease in hepatic mRNA levels of sterol response element binding protein-1c (SREBP-1c); resulting in a decrease in blood triglycerides and amelioration of hepatic ballooning degeneration. Eburicoic acid-treated mice reduced adipose expression levels of lipogenic FAS and peroxisome proliferator-activated receptor γ (PPARγ) and led to decreased adipose lipid accumulation. The present findings demonstrated that TRR exhibits a beneficial therapeutic potential in the treatment of type 2 diabetes and hyperlipidemia.

Exosomes derived from pancreatic cancer cells induce insulin resistance in C2C12 myotube cells through the PI3K/Akt/FoxO1 pathway

Prospective epidemiological studies have consistently suggested that pancreatic cancer-associated new-onset diabetes mellitus (PC-DM) represents a potential platform for early diagnose of pancreatic cancer (PC). Despite the studies performed, the mechanism behind this phenomenon remains ambiguous. In this study, we explored the effects of two types of exosomes released by murine pancreatic cancer and ductal epithelial cells on murine skeletal muscle cells. The results show that PC-derived exosomes can readily enter C2C12 myotubes, triggering lipidosis and glucose intake inhibition. We also demonstrate that PC-derived exosomes can inhibit insulin and PI3K/Akt signalling, in which insulin-induced FoxO1 nuclear exclusion is preserved and Glut4 trafficking is impaired. Microarray and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses show that exosomal microRNAs (miRNAs) probably play key roles in this process, an assumption that is corroborated by in vitro studies. These results confirm that the insulin resistance (IR) of skeletal muscle cells is governed by PC-derived exosomes through the insulin and PI3K/Akt/FoxO1 signalling pathways, where exosomal miRNAs potentially contribute to this phenomenon. These novel findings pave the way towards a comprehensive understanding of the cancer theories: “metabolic reprogramming” and “metabolic crosstalk”.

Chronic administration of myristic acid improves hyperglycaemia in the Nagoya-Shibata-Yasuda mouse model of congenital type 2 diabetes.

Previously, we demonstrated that myristic acid (14:0) increases levels of diacylglycerol kinase (DGK) δ, a key enzyme involved in type 2 diabetes exacerbation, and enhances glucose uptake in C2C12 myotube cells. Moreover, results from a population-based cohort study suggest that consumption of high-fat dairy products, which contain high amounts of myristic acid, is associated with a lower risk of developing type 2 diabetes. Taken together, we hypothesised that intake of myristic acid reduces type 2 diabetes risk in vivo. The aim of this study was to examine the glucose-lowering effect of myristic acid in Nagoya-Shibata-Yasuda (NSY) mice, a spontaneous model for studying obesity-related type 2 diabetes. Male NSY mice were orally administered vehicle (n=9), 300mg/kg of myristic acid (n=14) or 300mg/kg of palmitic acid (16:0) (n=9) every other day from 4weeks of age. Glucose and insulin tolerance tests were performed at weeks 18, 24 and 30, and weeks 20 and 26, respectively. DGKδ levels were measured in skeletal muscle from 32-36-week-old NSY mice via western blot. Chronic oral administration of myristic acid ameliorated glucose tolerance (24-28% decrease in blood glucose levels during glucose tolerance tests) and reduced insulin-responsive blood glucose levels (~20% decrease) in male NSY mice compared with vehicle and palmitic acid groups at 24-30weeks of age (the age at which the severity of type 2 diabetes is exacerbated in NSY mice). Myristic acid also attenuated the increase in body weight seen in NSY mice. Furthermore, the fatty acid increased DGKδ levels (~1.6-fold) in skeletal muscle of NSY mice. These results suggest that the chronic oral administration of myristic acid improves hyperglycaemia by decreasing insulin-responsive glucose levels and reducing body weight, and that the fatty acid accounts for the diabetes protective properties of high-fat dairy products. Myristic acid is a potential candidate for the prevention and treatment of type 2 diabetes mellitus and its related diseases.

Dietary Depletion of Bovine Milk Exosomes Elicits Changes in Amino Acid Metabolism in C57BL/6 Mice

BackgroundExosomes are small, cargo‐containing vesicles that are secreted by donor cells to elicit changes in gene expression and metabolism in recipient cells. We have discovered that dietary exosomes from bovine milk can be absorbed by non‐bovine species; studies in murine C2C12 myotube cell cultures suggest that bovine exosomes promote the growth of myofibers.HypothesisThe dietary intake of bovine milk exosomes alters amino acid metabolism, and possibly muscle protein accretion, in non‐bovine species.SignificanceThe dietary intake of exosomes might promote muscle protein accretion in patients with sarcopenia and low‐birth‐weight babies.MethodsC57BL/6 mice, age 3 weeks, were fed an exosome‐depleted (Exo−) AIN93G‐based diet for four weeks; controls were fed an exosome‐sufficient (Exo+) diet. Livers were harvested. The hepatic metabolome was assessed by non‐targeted LC/MS‐MS and by peak intensity analysis; the hepatic transcriptome was assessed by RNAseq using an Illumina HiSeq2500 platform. A second cohort of mice was fed Exo− or Exo+ diets for 4–6 weeks for subsequent analysis of grip strength, respiratory exchange ratio (RER), feeding and activity patterns; skeletal muscle samples are currently analyzed by RNAseq. Statistical significance was assessed using unpaired, two‐tailed t‐test.ResultsHepatic concentrations of several amino acids were significantly higher (up to 18 times) in mice fed the Exo− diet than in Exo+ controls (Fig. 1). The mRNA expression of branched chain amino acid (BCAA) transporters 1 (cytoplasm) and 2 (mitochondria) was greater in mice fed Exo− compared to Exo+ (n.s. for BCAT2; Fig. 2). The RER was not affected by feeding, whereas grip strength was a moderate 5% higher in Exo+ vs. Exo− females after only 4 weeks of feeding (n.s.; not shown). The trend toward a greater grip strength was not caused by differences in food and water consumption or physical activity, which were not significantly different between treatment groups in females.Conclusion & future plansDiets defined by their bovine milk exosome content alter amino acid metabolism in mice. Future studies will investigate protein metabolism by using stable isotopes, and assess muscle protein accretion in response to stimuli such as muscle wasting.Support or Funding InformationNIFA 2015‐67017‐23181, NIFA 2016‐67001‐25301/NIH DK107264, NIH 1P20GM104320, the Gerber Foundation, and USDA Hatch Act and W3002.

S100B impairs glycolysis via enhanced poly(ADP-ribosyl)ation of glyceraldehyde-3-phosphate dehydrogenase in rodent muscle cells

S100 calcium-binding protein B (S100B), a multifunctional macromolecule mainly expressed in nerve tissues and adipocytes, has been suggested to contribute to the pathogenesis of obesity. To clarify the role of S100B in insulin action and glucose metabolism in peripheral tissues, we investigated the effect of S100B on glycolysis in myoblast and myotube cells. Rat myoblast L6 cells were treated with recombinant mouse S100B to examine glucose consumption, lactate production, glycogen accumulation, glycolytic metabolites and enzyme activity, insulin signaling, and poly(ADP-ribosyl)ation of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Glycolytic metabolites were investigated by enzyme assays or metabolome analysis, and insulin signaling was assessed by Western blot analysis. Enzyme activity and poly(ADP-ribosyl)ation of GAPDH was evaluated by an enzyme assay and immunoprecipitation followed by dot blot with an anti-poly(ADP-ribose) antibody, respectively. S100B significantly decreased glucose consumption, glucose analog uptake, and lactate production in L6 cells, in either the presence or absence of insulin. In contrast, S100B had no effect on glycogen accumulation and insulin signaling. Metabolome analysis revealed that S100B increased the concentration of glycolytic intermediates upstream of GAPDH. S100B impaired GAPDH activity and increased poly(ADP-ribosyl)ated GAPDH proteins. The effects of S100B on glucose metabolism were mostly canceled by a poly(ADP-ribose) polymerase inhibitor. Similar results were obtained in C2C12 myotube cells. We conclude that S100B as a humoral factor may impair glycolysis in muscle cells independent of insulin action, and the effect may be attributed to the inhibition of GAPDH activity from enhanced poly(ADP-ribosyl)ation of the enzyme.

Sialylation of Glycosylphosphatidylinositol (GPI) Anchors of Mammalian Prions Is Regulated in a Host-, Tissue-, and Cell-specific Manner

Prions or PrP(Sc) are proteinaceous infectious agents that consist of misfolded, self-replicating states of the prion protein or PrP(C) PrP(C) is posttranslationally modified with N-linked glycans and a sialylated glycosylphosphatidylinositol (GPI) anchor. Conformational conversion of PrP(C) gives rise to glycosylated and GPI-anchored PrP(Sc) The question of the sialylation status of GPIs within PrP(Sc) has been controversial. Previous studies that examined scrapie brains reported that both sialo- and asialo-GPIs were present in PrP(Sc), with the majority being asialo-GPIs. In contrast, recent work that employed cultured cells claimed that only PrP(C) with sialylo-GPIs could be recruited into PrP(Sc), whereas PrP(C) with asialo-GPIs inhibited conversion. To resolve this controversy, we analyzed the sialylation status of GPIs within PrP(Sc) generated in the brain, spleen, or cultured N2a or C2C12 myotube cells. We found that recruiting PrP(C) with both sialo- and asialo-GPIs is a common feature of PrP(Sc) The mixtures of sialo- and asialo-GPIs were observed in PrP(Sc) universally regardless of prion strain as well as host, tissue, or type of cells that produced PrP(Sc) Remarkably, the proportion of sialo- versus asialo-GPIs was found to be controlled by host, tissue, and cell type but not prion strain. In summary, this study found no strain-specific preferences for selecting PrP(C) with sialo- versus asialo-GPIs. Instead, this work suggests that the sialylation status of GPIs within PrP(Sc) is regulated in a cell-, tissue-, or host-specific manner and is likely to be determined by the specifics of GPI biosynthesis.

Age Dependent Changes in Muscle Expression of Enzymes Effecting The Production of Testosterone and Dihydrotestosterone in Young and Old Mice and the C2C12 Cell Line

BackgroundSarcopenia is a debilitating condition that exhibits a progressive decline in skeletal muscle mass and function with age. Sarcopenia can lead to an increased susceptibility to falls and can result in the loss of independent living. Despite this, the causes and mechanism(s) underlying sarcopenia are not fully understood.MethodsYoung (3 month) and aged (>22 month) male mouse extensor digitorum longus (edl; a fast‐twitch muscle), soleus (a slow‐twitch muscle), and anterior tibialis muscle bundles were rapidly isolated and subsequently prepared for use in western blotting. The expression of enzymes influencing testosterone (T) and dihydrotestosterone (DHT) production were investigated in the different age groups, with similar protein targets being investigated in C2C12 myoblast and myotube cells after treatments with either DHT (5nM) or T (30nM).ResultsOverall, all muscles investigated showed more pronounced expression of 5α‐reductase‐2 than that of 5α‐reductase‐1, both of which generate DHT from T, irrespective of age. Both reductases were more abundant in aged muscles, irrespective of muscle type except that of the anterior tibialis for reductase‐2 which was reduced with age. We report androgen receptor expression in myoblasts that increased significantly with exposure to DHT or T, whereas the expression of the androgen receptor in myotubes was only weakly detectable. In response to the DHT or T treatments, myoblast expression of; aromatase, 5α‐reductase‐1/2, 17‐β‐hydroxysteroid dehydrogenase, Myo‐D, Pax7 and the amino acid transporters SNAT2 and LAT2 were unchanged. Interestingly myotubes showed increased expression upon exposure to DHT or T of Myo‐D and trends to increased expression of 5α‐reductase‐2 and SNAT2 with no change in Pax7, aromatase, 17‐β‐hydroxysteroid dehydrogenase or LAT2.ConclusionFrom these results we suggest that a component of sarcopenia may arise from an age‐dependent reduction of the DHT or T dependant anabolic stimulus. We also suggest that muscles such as the soleus and edl attempt to resist this reduction in androgens by locally generating DHT by enhancing expression of 5α‐reductase enzymes. A possible mechanism of action to DHT exposure may involve active satellite cells and the increased expression of SNAT2 to then modify amino acid transport into muscle, where both of these may influence positively muscle viability.Support or Funding InformationFunded by the BBSRC.

Iron-induced skeletal muscle atrophy involves an Akt-forkhead box O3–E3 ubiquitin ligase-dependent pathway

Skeletal muscle wasting or sarcopenia is a critical health problem. Skeletal muscle atrophy is induced by an excess of iron, which is an essential trace metal for all living organisms. Excessive amounts of iron catalyze the formation of highly toxic hydroxyl radicals via the Fenton reaction. However, the molecular mechanism of iron-induced skeletal muscle atrophy has remained unclear. In this study, 8-weeks-old C57BL6/J mice were divided into 2 groups: vehicle-treated group and the iron-injected group (10mg ironday−1mouse−1) during 2 weeks. Mice in the iron-injected group showed an increase in the iron content of the skeletal muscle and serum and ferritin levels in the muscle, along with reduced skeletal muscle mass. The skeletal muscle showed elevated mRNA expression of the muscle atrophy-related E3 ubiquitin ligases, atrogin-1 and muscle ring finger-1(MuRF1), on days 7 and 14 of iron treatment. Moreover, iron-treated mice showed reduced phosphorylation of Akt and forkhead box O3 (FOXO3a) in skeletal muscles. Inhibition of FOXO3a using siRNA in vitro in C2C12 myotube cells inhibited iron-induced upregulation of atrogin-1 and MuRF1 and reversed the reduction in myotube diameters. Iron-load caused oxidative stress, and an oxidative stress inhibitor abrogated iron-induced muscle atrophy by reactivating the Akt–FOXO3a pathway. Iron-induced skeletal muscle atrophy is suggested to involve the E3 ubiquitin ligase mediated by the reduction of Akt-FOXO3a signaling by oxidative stress.