Differentiated C2C12 Myotubes Research Articles

Black raspberry extract stimulates glucose uptake via adenosine monophosphate‐activated protein kinase and phosphatidylinositol‐3 kinase pathways in skeletal muscle cells

AbstractBlack raspberry (Rubus occidentalis) shows beneficial health effects, such as antioxidant, anti‐inflammatory, chemopreventive, antiproliferative, and antihyperglycemic activities. The present study investigated the antihyperglycemic activity of black raspberry extract (BRE) and potential mechanisms in skeletal muscle cells. Differentiated C2C12 myotubes were treated with the BRE (1–1000 μg/mL) or metformin (1 mM). The protein levels of various insulin signaling targets were measured by western blot analysis. BRE significantly increased glucose uptake by upregulated insulin receptor and phosphatidylinositol‐3 kinase (PI3K) in C2C12 cells, leading to enhanced translocation of glucose transporter 4 (GLUT4) to the plasma membrane. The expression of phosphorylated (p)‐adenosine monophosphate‐activated protein kinase (AMPK) was also enhanced by treatment with BRE, this resulting in stimulation of GLUT4 translocation to the plasma membrane and hence glucose uptake in skeletal muscles. These results suggest that BRE has an antihyperglycemic effect by stimulating GLUT4 translocation to the plasma membrane via activating the PI3K and AMPK pathways in skeletal muscle cells, thereby upregulating glucose uptake. Black raspberry is a potential functional food and has important implications for preventing and treating type‐2 diabetes. In addition, our study found that the flavonoids fraction is the main contributor fraction to promote glucose uptake in C2C12 cells.

Comparative transcriptomics of broad-spectrum and synthetic cannabidiol treated C2C12 skeletal myotubes.

Cannabidiol (CBD) is widely used in sports for recovery, pain management, and sleep improvement, yet its effects on muscle are not well understood. This study aimed to determine the transcriptional response of murine skeletal muscle myotubes to broad-spectrum CBD and synthetic CBD (sCBD). Differentiated C2C12 myotubes were treated with 10 μM CBD, sCBD, or vehicle control (DMSO) for 24 h before RNA extraction. Poly-A tail-enriched mRNA libraries were constructed and sequenced using 2 × 50 bp paired-end sequencing. CBD and sCBD treatment induced 4489 and 1979 differentially expressed genes (DEGs; p < 0.001, FDR step-up <0.05), respectively, with common upregulation of 857 genes and common downregulation of 648 genes. Common upregulated DEGs were associated with "response to unfolded protein," "cell redox homeostasis," "endoplasmic reticulum stress," "oxidative stress," and "cellular response to hypoxia." Common downregulated DEGs were linked to "sarcomere organization," "skeletal muscle tissue development," "regulation of muscle contraction," and "muscle contraction." CBD treatment induced unique DEGs compared to sCBD. The data indicate CBD may induce mild cellular stress, activating pathways associated with altered redox balance, unfolded protein response, and endoplasmic reticulum stress. We hypothesize that CBD interacts with muscle and may elicit a "mitohormetic" effect that warrants further investigation.

Endothelin-1 impairs skeletal muscle myogenesis and development via ETB receptors and p38 MAPK signaling pathway.

Myopenia is a condition marked by progressive decline of muscle mass and strength and is associated with aging or obesity. It poses the risk of falling, with potential bone fractures, thereby also increasing the burden on family and society. Skeletal muscle wasting is characterized by a reduced number of myoblasts, impaired muscle regeneration and increased muscle atrophy markers (Atrogin-1, MuRF-1). Endotheline-1 (ET-1) is a potent vasoconstrictor peptide. Increased circulating levels of ET-1 is noted with aging and is associated with muscular fibrosis and decline of strength. However, the regulatory mechanism controlling its effect on myogenesis and atrophy remains unknown. In the present study, the effects of ET-1 on myoblast proliferation, differentiation and development were investigated in C2C12 cells and in ET-1-infused mice. The results show that ET-1, acting via ETB receptors, reduced insulin-stimulated cell proliferation, and also reduced MyoD, MyoG and MyHC expression in the differentiation processes of C2C12 myoblasts. ET-1 inhibited myoblast differentiation through ETB receptors and the p38 mitogen-activated protein kinase (MAPK)-dependent pathway. Additionally, ET-1 decreased MyHC expression in differentiated myotubes. Inhibition of proteasome activity by MG132 ameliorated the ET-1-stimulated protein degradation in differentiated C2C12 myotubes. Furthermore, chronic ET-1 infusion caused skeletal muscle atrophy and impaired exercise performance in mice. In conclusion, ET-1 inhibits insulin-induced cell proliferation, impairs myogenesis and induces muscle atrophy via ETB receptors and the p38 MAPK-dependent pathway.

MTORC1 and BMP-Smad1/5 regulation of serum-stimulated myotube hypertrophy: a role for autophagy.

Protein synthesis regulation is critical for skeletal muscle hypertrophy, yet other established cellular processes are necessary for growth-related cellular remodeling. Autophagy has a well-acknowledged role in muscle quality control, but evidence for its role in myofiber hypertrophy remains equivocal. Both mammalian target of rapamycin complex I (mTORC1) and bone morphogenetic protein (BMP)-Smad1/5 (Sma and Mad proteins from Caenorhabditis elegans and Drosophila, respectively) signaling are reported regulators of myofiber hypertrophy; however, gaps remain in our understanding of how this regulation is integrated with growth processes and autophagy regulation. Therefore, we investigated the mTORC1 and Smad1/5 regulation of protein synthesis and autophagy flux during serum-stimulated myotube growth. Chronic serum stimulation experiments were performed on day 5 differentiated C2C12 myotubes incubated in differentiation medium [2% horse serum (HS)] or growth medium [5% fetal bovine serum (FBS)] for 48 h. Rapamycin or LDN193189 was dosed for 48 h to inhibit mTORC1 and BMP-Smad1/5 signaling, respectively. Acute serum stimulation was examined in day 7 differentiated myotubes. Protein synthesis was measured by puromycin incorporation. Bafilomycin A1 and immunoblotting for LC3B were used to assess autophagy flux. Chronic serum stimulation increased myotube diameter 22%, total protein 21%, total RNA 100%, and Smad1/5 phosphorylation 404% and suppressed autophagy flux. Rapamycin, but not LDN193189, blocked serum-induced myotube hypertrophy and the increase in total RNA. Acute serum stimulation increased protein synthesis 111%, Smad1/5 phosphorylation 559%, and rpS6 phosphorylation 117% and suppressed autophagy flux. Rapamycin increased autophagy flux during acute serum stimulation. These results provide evidence for mTORC1, but not BMP-Smad1/5, signaling being required for serum-induced myotube hypertrophy and autophagy flux by measuring LC3BII/I expression. Further investigation is warranted to examine the role of autophagy flux in myotube hypertrophy.NEW & NOTEWORTHY The present study demonstrates that myotube hypertrophy caused by chronic serum stimulation requires mammalian target of rapamycin complex 1 (mTORC1) signaling but not bone morphogenetic protein (BMP)-Smad1/5 signaling. The suppression of autophagy flux was associated with serum-induced myotube hypertrophy and mTORC1 regulation of autophagy flux by measuring LC3BII/I expression. Rapamycin is widely investigated for beneficial effects in aging skeletal muscle and sarcopenia; our results provide evidence that rapamycin can regulate autophagy-related signaling during myotube growth, which could benefit skeletal muscle functional and metabolic health.

Abrus precatorius Leaf Extract Stimulates Insulin-mediated Muscle Glucose Uptake: In vitro Studies and Phytochemical Analysis.

Diabetes mellitus, linked with insulin resistance and hyperglycaemia, is a leading cause of mortality. Glucose uptake through glucose transporter type 4, especially in skeletal muscle, is crucial for maintaining euglycaemia and is a key pathway targeted by antidiabetic medication. Abrus precatorius is a medicinal plant with demonstrated antihyperglycaemic activity in animal models, but its mechanisms are unclear.This study evaluated the effect of a 50% ethanolic (v/v) A.precatorius leaf extract on (1) insulin-stimulated glucose uptake and (2) related gene expression in differentiated C2C12 myotubes using rosiglitazone as a positive control, and (3) generated a comprehensive phytochemical profile of A.precatorius leaf extract using liquid chromatography-high resolution mass spectrometry to elucidate its antidiabetic compounds. A.precatorius leaf extract significantly increased insulin-stimulated glucose uptake, and insulin receptor substrate 1 and Akt substrate of 160 kDa gene expression; however, it had no effect on glucose transporter type 4 gene expression. At 250 µg/mL A.precatorius leaf extract, the increase in glucose uptake was significantly higher than 1 µM rosiglitazone. Fifty-five phytochemicals (primarily polyphenols, triterpenoids, saponins, and alkaloids) were putatively identified, including 24 that have not previously been reported from A.precatorius leaves. Abrusin, precatorin I, glycyrrhizin, hemiphloin, isohemiphloin, hispidulin 4'-O-β-D-glucopyranoside, homoplantaginin, and cirsimaritin were putatively identified as known major compounds previously reported from A.precatorius leaf extract. A.precatorius leaves contain antidiabetic phytochemicals and enhance insulin-stimulated glucose uptake in myotubes via the protein kinase B/phosphoinositide 3-kinase pathway by regulating insulin receptor substrate 1 and Akt substrate of 160 kDa gene expression. Therefore, A.precatorius leaves may improve skeletal muscle insulin sensitivity and hyperglycaemia. Additionally, it is a valuable source of bioactive phytochemicals with potential therapeutic use for diabetes.

Gromwell (Lithospermum erythrorhizon) Attenuates High-Fat-Induced Skeletal Muscle Wasting by Increasing Protein Synthesis and Mitochondrial Biogenesis.

Gromwell (Lithospermum erythrorhizon, LE) can mitigate obesity-induced skeletal muscle atrophy in C2C12 myotubes and high-fat diet (HFD)-induced obese mice. The purpose of this study was to investigate the anti-skeletal muscle atrophy effects of LE and the underlying molecular mechanism. C2C12 myotubes were pretreated with LE or shikonin, and active component of LE, for 24 h and then treated with 500 μM palmitic acid (PA) for an additional 24 h. Additionally, mice were fed a HFD for 8 weeks to induced obesity, and then fed either the same diet or a version containing 0.25% LE for 10 weeks. LE attenuated PA-induced myotubes atrophy in differentiated C2C12 myotubes. The supplementation of LE to obese mice significantly increased skeletal muscle weight, lean body mass, muscle strength, and exercise performance compared with those in the HFD group. LE supplementation not only suppressed obesity-induced skeletal muscle lipid accumulation, but also downregulated TNF-α and atrophic genes. LE increased protein synthesis in the skeletal muscle via the mTOR pathway. We observed LE induced increase of mitochondrial biogenesis and upregulation of oxidative phosphorylation related genes in the skeletal muscles. Furthermore, LE increased the expression of peroxisome proliferator-activated receptor-gamma coactivator-1 alpha and the phosphorylation of adenosine monophosphate-activated protein kinase. Collectively, LE may be useful in ameliorating the detrimental effects of obesity-induced skeletal muscle atrophy through the increase of protein synthesis and mitochondrial biogenesis of skeletal muscle.

Effects of C-Peptide on Dexamethasone-Induced In Vitro and In Vivo Models as a Potential Therapeutic Agent for Muscle Atrophy.

This study aimed to investigate the effects of C-peptide on C2C12 myotubes and a mouse model. Both in vitro and in vivo experiments were conducted to elucidate the role of C-peptide in muscle atrophy. Various concentrations (0, 0.01, 0.1, 1, 10, and 100 nM) of C-peptide were used on the differentiated C2C12 myotubes with or without dexamethasone (DEX). C57BL/6J mice were administered with C-peptide and DEX for 8 days, followed by C-peptide treatment for 12 days. Compared to the DEX group, C-peptide increased the fusion and differentiation indices and suppressed atrophic factor expression in C2C12 myotubes. However, 100 nM C-peptide decreased the fusion and differentiation indices and increased atrophic factor expression regardless of DEX treatment. In C57BL/6J mice, DEX + C-peptide co-treatment significantly attenuated the body and muscle weight loss and improved the grip strength and cross-sectional area of the gastrocnemius (Gas) and quadriceps (Quad) muscles. C-peptide downregulated the mRNA and protein levels of muscle degradation-related markers, particularly Atrogin-1, in Gas and Quad muscles. This study underscores the potential of C-peptides in mitigating muscle weight reduction and preserving muscle function during muscle atrophy via molecular regulation. In addition, the work presents basic data for future studies on the effect of C-peptide on diabetic muscular dystrophy.

Hexavalent chromium inhibits myogenic differentiation and induces myotube atrophy

Hexavalent chromium [Cr(VI)] is extensively used in many industrial processes. Previous studies reported that Cr(VI) exposures during early embryonic development reduced body weight with musculoskeletal malformations in rodents while exposures in adult mice increased serum creatine kinase activity, a marker of muscle damage. However, the impacts of Cr(VI) on muscle differentiation remain largely unknown. Here, we report that acute exposures to Cr(VI) in mouse C2C12 myoblasts inhibit myogenic differentiation in a dose-dependent manner. Exposure to 2 μM of Cr(VI) resulted in delayed myotube formation, as evidenced by a significant decrease in myotube formation and expression of muscle-specific markers, such as muscle creatine kinase (Mck), Myocyte enhancer factor 2 (Mef2), Myomaker (Mymk) and Myomixer (Mymx). Interestingly, exposure to 5 μM of Cr(VI) completely abolished myotube formation in differentiating C2C12 cells. Moreover, the expression of key myogenic regulatory factors (MRFs) including myoblast determination protein 1 (MyoD), myogenin (MyoG), myogenic factor 5 (Myf5), and myogenic factor 6 (Myf6) were significantly altered in Cr(VI)-treated cells. The inhibitory effect of Cr(VI) on myogenic differentiation was further confirmed in freshly isolated mouse satellite cells, a stem cell population essential for adult skeletal muscle regeneration. Furthermore, Cr(VI) exposure to fully differentiated C2C12 myotubes resulted in a decrease in myotube diameter, which was exacerbated upon co-treatment with dexamethasone. Together, our results demonstrate that Cr(VI) inhibits myogenic differentiation and induces myotube atrophy in vitro.

Acrolein induces mitochondrial dysfunction and insulin resistance in muscle and adipose tissues in vitro and in vivo.

Type 2 diabetes mellitus (DM) is a common chronic condition characterized by persistent hyperglycemia and is associated with insulin resistance (IR) in critical glucose-consuming tissues, including skeletal muscle and adipose tissue. Oxidative stress and mitochondrial dysfunction are known to play key roles in IR. Acrolein is a reactive aldehyde found in the diet and environment that is generated as a fatty acid product through the glucose autooxidation process under hyperglycemic conditions. Our previous studies have shown that acrolein impairs insulin sensitivity in normal and diabetic mice, and this effect can be reversed by scavenging acrolein. This study demonstrated that acrolein increased oxidative stress and inhibited mitochondrial respiration in differentiated C2C12 myotubes and differentiated 3T3-L1 adipocytes. As a result, insulin signaling pathways were inhibited, leading to reduced glucose uptake. Treatment with acrolein scavengers, N-acetylcysteine, or carnosine ameliorated mitochondrial dysfunction and inhibited insulin signaling. Additionally, an increase in acrolein expression correlated with mitochondrial dysfunction in the muscle and adipose tissues of diabetic mice. These findings suggest that acrolein-induced mitochondrial dysfunction contributes to IR, and scavenging acrolein is a potential therapeutic approach for treating IR.

1642-P: Deferoxamine Prevented Dexamethasone-Induced Muscle Atrophy through Inhibition of KLF15 and FOXO3a

Aim/hypothesis: Muscle atrophy is caused by various factors such as aging, a lack of exercise, and diseases. During these events, the balance between protein synthesis and degradation is disrupted and finally muscle mass is decreased. This has been reported to occur via the ubiquitin-proteasome dependent pathway, autophagy, or apoptosis. In this study, we investigated the main mechanisms of dexamethasone-induced muscle atrophy and preventing mechanisms of deferoxamine on DEX-induced muscle atrophy. Method: To measure the effects of DFO on muscle atrophy, C2C12 myotubes were treated with dexamethasone, with or without DFO, then myotubes atrophy was measured by phalloidin staining, RT-PCR, and western blotting. Results: In differentiated C2C12 myotubes, DEX induced muscle atrophy in a time- and dose-dependent manner. DEX induced expressions of muscle-specific ligases, including muscle ring finger 1 and muscle atrophy F box, and activated the ubiquitin-proteasome system leading to muscle atrophy, not autophagy or apoptosis. Interestingly, DFO administration alleviated DEX-induced myotubes atrophy. DFO inhibited DEX-activated expressions of KLF15, not REDD or FOXO3a. DFO dramatically diminished FOXO3a translocation from cytosol to nucleus through the activation of AKT. Finally, expressions of muscle-specific ligases, such as MURF1 and Atrogin-1, were reduced by treatment of DFO, and ubiquitinated myoprotein was also reduced. In addition, mice treated with DEX for 12 days had a significant decrease in grip strength and TA muscle mass. However, treatment with DFO prevented DEX-induced reduction of muscle fiber size, loss of TA muscle, and grip strength. Conclusion: DEX activated muscle specific E3 ligase and induced-muscle atrophy through ubiquitin-proteasome system. DFO ameliorates muscle atrophy by inhibition of E3 ligase expression via KLF15 and FOXO3a in DEX-treated C2C12 myotubes and C57BL6J mice. Disclosure Y.Kim: None. H.Kim: None. D.Kim: None. T.Kim: None. S.An: None. K.Lee: None. N.Lee: None. S.Choi: None. Y.Kim: None. Y.Son: None. S.Choi: None. Y.Y.Kang: None. J.Jeon: None. S.Han: None.

Ion Transporters Involved in Dissipation of Transmembrane Na+ and K+ Gradients in C2C12 Myotubes Triggered by Electrical Pulse Stimulation

Background/Aims: Cultured skeletal muscle cells subjected to electrical pulse stimulation (EPS) are widely employed as an in vitro model of exercising skeletal muscle. Numerous studies demonstrated that sustained excitation of skeletal muscle results in the dissipation of the transmembrane gradient of monovalent cations. During exercises the impending loss of excitability has to be counterbalanced by rapid restoration of the Na+i/K+i ratio. To understand mechanisms of the maintenance of muscle contractility, it is necessary to know which transporters are participated in the dissipation of Na+i and K+i gradients and how to activate Na,K-ATPase for its regeneration. This study was aimed at the identification of ion transporters involved in the dissipation of the transmembrane gradients of Na+ and K+induced by EPS, and Na,K-ATPase isoforms involved in its restoration. Methods: The differentiated C2C12 myotubes were subjected to electrical pulse stimulation in the presence or absence of different ion transport systems inhibitors followed by measurement of intracellular monovalent cations by flame atomic absorption spectrometry. Results: Electrical pulse stimulation of C2C12 myotubes results in the dissipation of Na+i/K+i gradient, which is maintained by α2-Na,K-ATPase. Na-K-2Cl cotransporter (NKCC), voltage-gated Na+ (Nav), and large conductance Ca2+-activated K+ channels (BKCa), and Na/H exchanger (NHE) are involved in the dissipation of this gradient. Suppression of calmodulin-dependent protein kinase II (CaMKII) increases Na+ efflux in EPS-treated C2C12 myotubes. Conclusion: NKCC, Nav, BKCa, and NHE are involved in the dissipation of Na+i/K+i gradient in EPS-treated C2C12 myotubes.

Lack of Sod1 induces disruption of innervation accompanied by Schwann cell proliferation in skeletal muscle

The neuromuscular junction (NMJ) is an excitatory synapse that constitutes the primary site of communication between the nervous system and skeletal muscle and therefore any degenerative changes at the NMJ have the potential to impair muscle function. The primary cellular components of the NMJ are the motor neuron, the muscle fiber and terminal Schwann cells (tSC) that surround the NMJ. The importance of redox homeostasis for the maintenance of NMJ integrity is supported by work from our group showing degenerative changes at NMJs in mice lacking copper zinc superoxide dismutase (CuZnSOD; Sod1 -/- ) as early as 4 months of age. Prior work from others suggests that tSCs contribute trophic support for the NMJ and facilitate motor unit remodeling, but changes in tSC structure and function under conditions of elevated oxidative stress have not been thoroughly explored. Using in vitro assays, we previously showed that primary Schwann cells treated with a sublethal dose of the reactive oxygen species inducing agent Paraquat (PQ) resulted in increased Schwann cell number, ki67+ staining, and gene expression of trophic factors and the critical NMJ organizing protein Agrin. Furthermore, differentiated C2C12 myotubes treated with conditioned media from PQ treated Schwann cell cultures showed increased number of acetylcholine receptor (AChR) plaques. The goal of the current study was to characterize NMJs in young Sod1 -/- mice (2-months) to determine the early effects of elevated oxidative stress on NMJ structure in vivo with a specific focus on tSCs. We hypothesized that loss of Sod1 would lead to aberrant tSC and NMJ structure associated with decreased muscle function. We assessed maximum isometric force, NMJ morphology, and number of Schwann cells in gastrocnemius muscles of WT (n = 3) and Sod1 -/- (n = 3) mice. In Sod1 -/- mice, muscle force was reduced by 26% ( p = 2e-4) with nerve stimulation compared to direct muscle stimulation, and overlap between motor nerve terminals and AChRs was reduced by nearly 60% compared to WT controls. Post-synaptic AChR area was increased by 37% in Sod1 -/- mice, and tSC area and numbers per NMJ were increased by 60% and 155%, respectively in Sod1 -/- mice. Finally, Schwann cells proximal to NMJs showed increased Ki67+ labeling in Sod1 -/- mice compared to WT mice. This analysis demonstrates that early changes in both pre- and post-synaptic NMJ components and reduced neurotransmission due to loss of Sod1 are associated with marked increases in tSC proliferation. Our data implicate a protective role of muscle-resident Schwann cells in preserving NMJ integrity in response to alterations in redox homeostasis that may be mediated in part through activation and proliferation of these cells. These findings indicate that further investigation of the dynamics between Schwann cells and NMJ function is important to increase understanding of neuromuscular degenerativeconditions that involve altered redox homeostasis such as sarcopenia. Supported by the University of Michigan Medical School Office of Research, the American Physiological Society, and NIH AR069620. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Betaine Treatment Prevents TNF-α-Mediated Muscle Atrophy by Restoring Total Protein Synthesis Rate and Morphology in Cultured Myotubes.

Skeletal muscle atrophy is represented by a dramatic decrease in muscle mass, and it is related to a lower life expectancy. Among the different causes, chronic inflammation and cancer promote protein loss through the effect of inflammatory cytokines, leading to muscle shrinkage. Thus, the availability of safe methods to counteract inflammation-derived atrophy is of high interest. Betaine is a methyl derivate of glycine and it is an important methyl group donor in transmethylation. Recently, some studies found that betaine could promote muscle growth, and it is also involved in anti-inflammatory mechanisms. Our hypothesis was that betaine would be able to prevent tumor necrosis factor-α (TNF-α)-mediated muscle atrophy in vitro. We treated differentiated C2C12 myotubes for 72 hr with either TNF-α, betaine, or a combination of them. After the treatment, we analyzed total protein synthesis, gene expression, and myotube morphology. Betaine treatment blunted the decrease in muscle protein synthesis rate exerted by TNF-α, and upregulated Mhy1 gene expression in both control and myotube treated with TNF-α. In addition, morphological analysis revealed that myotubes treated with both betaine and TNF-α did not show morphological features of TNF-α-mediated atrophy. We demonstrated that in vitro betaine supplementation counteracts the muscle atrophy led by inflammatory cytokines.

Antimicrobial protein REG3A regulates glucose homeostasis and insulin resistance in obese diabetic mice

Innate immune mediators of pathogen clearance, including the secreted C-type lectins REG3 of the antimicrobial peptide (AMP) family, are known to be involved in the regulation of tissue repair and homeostasis. Their role in metabolic homeostasis remains unknown. Here we show that an increase in human REG3A improves glucose and lipid homeostasis in nutritional and genetic mouse models of obesity and type 2 diabetes. Mice overexpressing REG3A in the liver show improved glucose homeostasis, which is reflected in better insulin sensitivity in normal weight and obese states. Delivery of recombinant REG3A protein to leptin-deficient ob/ob mice or wild-type mice on a high-fat diet also improves glucose homeostasis. This is accompanied by reduced oxidative protein damage, increased AMPK phosphorylation and insulin-stimulated glucose uptake in skeletal muscle tissue. Oxidative damage in differentiated C2C12 myotubes is greatly attenuated by REG3A, as is the increase in gp130-mediated AMPK activation. In contrast, Akt-mediated insulin action, which is impaired by oxidative stress, is not restored by REG3A. These data highlight the importance of REG3A in controlling oxidative protein damage involved in energy and metabolic pathways during obesity and diabetes, and provide additional insight into the dual function of host-immune defense and metabolic regulation for AMP.

Efficacy of Eisenia bicyclis phlorotannins in the treatment of diabetes and reducing inflammation

Crude phlorotannins from Eisenia bicyclis (CPEb), a common perennial brown seaweed were screened for pharmacological activities such as anti-inflammatory, antioxidant, and antidiabetic effects. Crude Phlorotannins (CPEb) studied by FT-IR, LC-MS, UHPLC, and UV chromatography showed the occurrence of dieckol, 8, 8'-bieckol, fucofuroeckol, and the carotenoid, zeaxanthin (0.5 mg/kg). LPS, an inflammatory mediator, enhanced the production of cytokines such IL-1 (interleukin-1), IL-6 (interleukin-6), IL-8 (interleukin-8), IL-10 (interleukin-10), TNF-α (tumour necrosis factor-α), chemokine CXCL10, and NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) in differentiated THP1 macrophage cells. On differentiated human monocytic cell line LPS-induced THP1 cells, CPEb was found to have anti-inflammatory effects by reducing the expression of IL-1, IL-6, IL-8 (1.2 fold), CXCL10, NF-κB, and TNF-α. CPEb promoted 2-NBDG absorption in differentiated C2C12 myotubes under both basal (16.08%) and insulin-stimulated (51.09%) conditions. CPEb increased IRS/AKT-dependent glucose absorption and also activates the AMPK pathway. Anti-α-glucosidase, anti-inflammatory and antioxidant activity of CPEb indicate their role in treating hyperglycemia by scavenging ROS and inflammatory responses.

Kampo formula hochu-ekki-to (Bu-Zhong-Yi-Qi-Tang, TJ-41) ameliorates muscle atrophy by modulating atrogenes and AMPK in vivo and in vitro

BackgroundMuscle disuse results in loss of skeletal muscle mass and function. Hochu-ekki-to (TJ-41; Bu-Zhong-Yi-Qi-Tang in Chinese) is an herbal medicinal formulation used to treat patients with frailty, fatigue and appetite loss. It has been suggested that two atrogenes, atrogin-1 and muscle Ring finger 1 (MuRF1), are ubiquitin ligases involved in disuse-induced muscle atrophy and that 5’ adenosine monophosphate-activated protein kinase (AMPK) is involved in skeletal muscle metabolism. Effects of TJ-41 on disuse-induced muscle atrophy are unclear.MethodsWe subjected differentiated C2C12 myotubes to serum starvation, then examined the effects of TJ-41 on atrogenes expression, AMPK activity and the morphology of the myotubes. Male C57BL/6J mice were subjected to tail-suspension to induce hindlimb atrophy. We administered TJ-41 by gavage to the control group and the tail-suspended group, then examined the effects of TJ-41 on atrogene expression, AMPK activity, and the muscle weight.ResultsSerum starvation induced the expression of atrogin-1 and MuRF1 in C2C12 myotubes, and TJ-41 significantly downregulated the expression of atrogin-1. Tail-suspension of the mice induced the expression of atrogin-1 and MuRF1 in skeletal muscle as well as its muscle atrophy, whereas TJ-41 treatment significantly downregulated the expression of atrogin-1 and ameliorated the loss of the muscle weight. In addition, TJ-41 also activated AMPK and inactivated Akt and mTOR in skeletal muscle in vivo.ConclusionTJ-41 inhibited atrogenes in an Akt-independent manner as well as activating AMPK in skeletal muscles in vivo, further implying the therapeutic potential of TJ-41 against disuse-induced muscle atrophy and other atrogenes-dependent atrophic conditions.

MiR21 deletion in osteocytes has direct and indirect effects on skeletal muscle in a sex-dimorphic manner in mice

BackgroundOsteocytic microRNA21 (miR21) removal alters cytokine production and bone mass by modulating osteoclast and osteoblast differentiation and activity. Removing osteocytic miR21 increases osteoclast/osteoblast numbers and bone mass in male mice, whereas it decreases osteoclasts/osteoblasts without affecting bone mass in female mice. On the other hand, it leads to sex-independent increases in bone mechanical properties. Because changes in bone remodeling and strength affect skeletal muscle through bone–muscle crosstalk, we investigated whether osteocytic miR21 deletion influences skeletal muscle.MethodsmiR21fl/fl mice and 8kbDMP1-Cre mice were mated to obtain miR21-deficient mice primarily in the osteocyte (OtmiR21Δ) and littermate controls (miR21fl/fl). Four-month-old male and female mice were analyzed. Body composition was examined by DXA/Piximus and gene expression was assessed by qPCR. Ex vivo cultures of long bones devoid of bone-marrow cells from male and female 4-month-old were maintained for 48 h. Conditioned media were collected and used for the C2C12 assays. Two-way ANOVA analyses were performed to determine the contributions of genotype and sex and their interaction to the effects of miR21 deficiency.ResultsLean body mass was increased only in female OtmiR21Δ mice, although miR21 levels in soleus muscle were similar in miR21fl/fl (0.05 ± 0.02) and OtmiR21Δ (0.09 ± 0.04) mice. Female, but not male, OtmiR21Δ mice exhibited increased soleus (42%) and gastrocnemius (21%) muscle weight compared to miR21fl/fl littermates. However, muscle strength and gastrocnemius muscle fiber cross-sectional area were unaltered for either sex. Kinase phosphorylation (phospho/total protein ratio) in soleus muscle, measured as a surrogate for kinase activity by means of multiplex analysis, was also selectively changed depending on the mouse sex. Thus, female OtmiR21Δ mice had higher T185/Y187-ERK1/2 but lower S473-Akt phosphorylation than miR21fl/fl controls, while male OtmiR21Δ mice had higher S473-Akt phosphorylation, suggesting sex-dimorphic shifts in anabolic vs. catabolic signaling. Consistently, levels of FOXO3 and MuRF-1, known to be regulated by Akt, were only increased in male OtmiR21Δ mice. Atrogin-1 mRNA levels were upregulated in female OtmiR21Δ mice, suggesting a potential shift in protein regulation. Sex-specific effects were also found by exposing myotube cultures to conditioned media from 48-h-cultured marrow-flushed bones. Thus 5-day differentiated C2C12 myotubes treated with conditioned media of female OtmiR21Δ mice exhibit 12% higher average diameter compared to cells exposed to miR21fl/fl bone conditioned media. Yet, conditioned media from male bones had no effect on myotube size.ConclusionsWe present a novel aspect of bone–muscle crosstalk in which osteocyte-derived miR21 influences skeletal muscle size, but not strength, in female but not male mice; whereas, intracellular signaling alterations resulting from loss of miR21 seem to alter protein dynamics in a sex-dimorphic fashion.

A Novel Muscle Atrophy Mechanism: Myocyte Degeneration Due to Intracellular Iron Deprivation

Muscle atrophy is defined as the progressive degeneration or shrinkage of myocytes and is triggered by factors such as aging, cancer, injury, inflammation, and immobilization. Considering the total amount of body iron stores and its crucial role in skeletal muscle, myocytes may have their own iron regulation mechanism. Although the detrimental effects of iron overload or iron deficiency on muscle function have been studied, the molecular mechanism of iron-dependent muscle atrophy has not been elucidated. Using human muscle tissues and in the mouse rotator cuff tear model, we confirmed an association between injury-induced iron depletion in myocytes and muscle atrophy. In differentiated C2C12 myotubes, the effects of iron deficiency on myocytes and the molecular mechanism of muscle atrophy by iron deficiency were evaluated. Our study revealed that the lower iron concentration in injured muscle was associated with the upregulation of ferroportin, an iron exporter that transports iron out of cells. Ferroportin expression was increased by hypoxia-inducible factor 1α (HIF1α), which is activated by muscle injury, and its expression is controlled by HIF1 inhibitor treatment. Iron deprivation caused myocyte loss and a marked depletion of mitochondrial membrane potential leading to muscle atrophy, together with increased levels of myostatin, the upstream regulator of atrogin1 and muscle RING-finger protein-1 (MuRF1). Myostatin expression under iron deficiency was mediated by an orphan nuclear receptor, dosage-sensitive sex reversal-adrenal hypoplasia congenita critical region on the X chromosome (DAX1).

The NLRP3 inflammasome contributes to inflammation-induced morphological and metabolic alterations in skeletal muscle.

Systemic inflammation is associated with skeletal muscle atrophy and metabolic dysfunction. Although the nucleotide-binding oligomerization domain-like receptor family pyrin domain containing 3 (NLRP3) inflammasome contributes to cytokine production in immune cells, its role in skeletal muscle is poorly understood. Here, we studied the link between inflammation, NLRP3, muscle morphology, and metabolism in in vitro cultured C2C12 myotubes, independent of immune cell involvement. Differentiated C2C12 myotubes were treated with lipopolysaccharide (LPS; 0, 10, and 100-200ng/mL) to induce activation of the NLRP3 inflammasome with and without MCC950, a pharmacological inhibitor of NLRP3-induced IL-1β production. We assessed markers of the NLRP3 inflammasome, cell diameter, reactive oxygen species, and mitochondrial function. NLRP3 gene expression and protein concentrations increased in a time-dependent and dose-dependent manner. Intracellular IL-1β concentration significantly increased (P<0.0001), but significantly less with MCC950 (P=0.03), suggestive of moderate activation of the NLRP3 inflammasome in cultured myotubes upon LPS stimulation. LPS suppressed myotube growth after 24h (P=0.03), and myotubes remained smaller up to 72h (P=0.0009). Exposure of myotubes to IL-1β caused similar alterations in cell morphology, andMCC950 mitigated these LPS-induced differences in cell diameter. NLRP3 appeared to co-localize with mitochondria, more so upon exposure to LPS. Mitochondrial reactive oxygen species were higher after LPS (P=0.03), but not after addition of MCC950. Myotubes had higher glycolytic rates, and mitochondria were more fragmented upon LPS exposure, which was not altered by MCC950 supplementation. LPS-induced activation of the NLRP3 inflammasome in cultured myotubes contributes to morphological and metabolic alterations, likely due to its mitochondrial association.

Role of VEGFB in electrical pulse stimulation inhibits apoptosis in C2C12 myotubes

Skeletal muscle is the major effector organ for exercise. It has been proposed that VEGFB is significantly related to apoptosis in various cell types but not yet in skeletal muscle. We hypothesize that the decrease of VEGFB in skeletal muscle participates in the occurrence of skeletal muscle apoptosis and that exercise inhibits apoptosis by elevating the expression of VEGFB in skeletal muscle cells. Based on this hypothesis, we developed in vitro experiments to mimic the effect of exercise through electrical pulse stimulation (EPS) to observe the effect of EPS on apoptosis and the change in VEGFB expression in differentiated myotubes. In addition, we employed RNA interference to explore whether VEGFB is directly involved in the regulation of myotube apoptosis during EPS. Our results showed that exogenous VEGFB167 significantly inhibited C2C12 myotube apoptosis induced by TNF-α treatment and that endogenous VEGFB in differentiated C2C12 myotubes was significantly upregulated by EPS. In addition, EPS significantly changed the expression of the apoptotic indicators Bax and Bcl-2 at the mRNA level and downregulated the protein expression of cleaved caspase-3. The antiapoptotic effect of EPS weakened substantially as VEGFB in C2C12 myotubes was inhibited. Taken together, these results indicate that exercise-like EPS inhibits apoptosis by increasing the expression of C2C12 myotube-derived VEGFB.