Mouse C2C12 Research Articles

Skeletal muscle ribosome analysis: A comparison of common assay methods and utilization of a novel RiboAb antibody cocktail.

While total RNA concentrations putatively represent ribosome content, there is a need to homologize various quantification approaches. Thus, total RNA concentrations ([RNA]) provided through UV-Vis spectroscopy (UV), fluorometry-only (Fluor), and fluorometry-based microfluidic chip electrophoresis (MFGE) were examined in C2C12 myotubes and mouse skeletal muscle to determine if values aligned with [18S + 28S rRNA] (i.e., criterion ribosome metric). A novel antibody cocktail (termed RiboAb) was also tested and compared to [18S + 28S rRNA] in these models. In myotubes, 24-h IGF-1 treatments increased [18S + 28S rRNA] (~2.0-fold) and [RNA] based on UV (~1.9-fold), Fluor (~2.3 fold), and MFGE (~2.1-fold). In C57BL/6 mice, 10 days of mechanical overload (MOV) elevated plantaris [18S + 28S rRNA] (~1.7-fold) and [RNA] according to UV (~1.5-fold), Fluor (~1.6-fold), and MFGE (~1.8-fold). Myotube and mouse plantaris RiboAb levels were significantly higher with IGF-1 treatments and MOV, respectively, versus controls (1.3-fold and 1.7-fold, respectively), and values correlated with [18S + 28S rRNA] (r = 0.637 and r = 0.853, respectively, p ≤ 0.005). UV, Fluor, and MFGE [RNA] are seemingly valid surrogates of cell/tissue ribosome content, although each method has advantages (e.g., ease of use) and disadvantages (e.g., magnitudes of bias) discussed herein. Finally, the RiboAb cocktail may also represent ribosome content, although this should be further explored in other models.

The mitochondrial lactate oxidation complex: endpoint for carbohydrate carbon disposal.

The lactate shuttle concept has revolutionized our understanding and study of metabolism in physiology, biochemistry, intermediary metabolism, nutrition, and medicine. Seminal findings of the mitochondrial lactate oxidation complex (mLOC) elucidated the architectural structure of its components. Here, we report that the mitochondrial pyruvate carrier (mPC) is an additional member of the mLOC in mouse muscle and C2C12 myoblasts and myotubes. Immunoblots, mass spectrometry, and co-immunoprecipitation experiments of mitochondrial preparations revealed abundant amounts of mitochondrial lactate dehydrogenase (mLDH), monocarboxylate transporter (mMCT), basigin (CD147), cytochrome oxidase (COx), and pyruvate carriers 1 and 2 (mPC1 and 2). In addition, using confocal laser scanning microscopy (CLSM) and in situ proximity ligation, we also demonstrated planar and three-dimensional (3-D) colocalization of pyruvate and lactate transporters with COx in fixed mouse skeletal muscle sections and C2C12 myoblasts and myotubes skeletal muscle sections, mouse muscle and C2C12 myoblasts and myotubes myotubes, and C2C12 myoblasts. This work serves as a landmark for configuring the final pathway of carbohydrate oxidation.NEW & NOTEWORTHY We expand on knowledge of the architectural design of the mitochondrial lactate oxidation complex (mLOC); key members are: mitochondrial lactate dehydrogenase (mLDH), monocarboxylate transporter 1 (mMCT1), cytochrome oxidase (COx), basigin scaffolding protein (CD147), and the mitochondrial pyruvate carrier (mPC). The mLOC is key in creating the lower end of the concentration gradient for disposal of lactate and pyruvate.

PPARβ/δ upregulates the insulin receptor β subunit in skeletal muscle by reducing lysosomal activity and EphB4 levels

BackgroundThe increased degradation of the insulin receptor β subunit (InsRβ) in lysosomes contributes to the development of insulin resistance and type 2 diabetes mellitus. Endoplasmic reticulum (ER) stress contributes to insulin resistance through several mechanisms, including the reduction of InsRβ levels. Here, we examined how peroxisome proliferator-activated receptor (PPAR)β/δ regulates InsRβ levels in mouse skeletal muscle and C2C12 myotubes exposed to the ER stressor tunicamycin.MethodsWild-type (WT) and Ppard−/− mice, WT mice treated with vehicle or the PPARβ/δ agonist GW501516, and C2C12 myotubes treated with the ER stressor tunicamycin or different activators or inhibitors were used.ResultsPpard−/− mice displayed reduced InsRβ protein levels in their skeletal muscle compared to wild-type (WT) mice, while the PPARβ/δ agonist GW501516 increased its levels in WT mice. Co-incubation of tunicamycin-exposed C2C12 myotubes with GW501516 partially reversed the decrease in InsRβ protein levels, attenuating both ER stress and the increase in lysosomal activity. In addition, the protein levels of the tyrosine kinase ephrin receptor B4 (EphB4), which binds to the InsRβ and facilitates its endocytosis and degradation in lysosomes, were increased in the skeletal muscle of Ppard−/− mice, with GW501516 reducing its levels in the skeletal muscle of WT mice.ConclusionsOverall, these findings reveal that PPARβ/δ activation increases InsRβ levels by alleviating ER stress and lysosomal degradation.

Aerobic plus resistance exercise attenuates skeletal muscle atrophy induced by dexamethasone through the HDAC4/FoxO3a pathway.

This study aimed to investigate the underlying mechanisms by which physical exercise mitigates muscle atrophy induced by Dexamethasone (Dex). A muscle atrophy model was established in the mouse C2C12 cell line and 8-week-old mice treated with Dex, with subsequent verification of phenotype and atrogene expression. The potential benefits of combined aerobic and resistance exercise in mitigating muscle atrophy were then examined. To elucidate the involvement of Histone deacetylase 4 (HDAC4) in the protective effects of exercise against muscle loss, a combination of RT-PCR, Western blotting, immunoprecipitation, and immunofluorescence staining techniques were employed. The upregulation of HDAC4 was observed following Dex-induced muscle atrophy in vitro and in vivo. Inhibition of HDAC4 in C2C12 cells resulted in an increase in myotube diameter and fusion index, along with a decrease in the expression of Atrogin-1 and MuRF1. Treatment with Tasquinimod, an HDAC4 inhibitor, effectively prevented muscle wasting and dysfunction in mice induced by Dex. After a 6-week exercise intervention, the Dex-Exercise group exhibited significant improvements in body fat level, hyperinsulinemia, muscle mass and function in comparison to the Dex-Sedentary group. Mechanistically, we discovered that HDAC4 bound to and deacetylated Forkhead box protein O 3a (FoxO3a) within the nucleus, leading to decreased phosphorylation of FoxO3a at Ser 253. This interaction subsequently facilitated the expression of downstream atrogene Atrogin-1 and MuRF1, resulting in muscle atrophy. Conversely, exercise was found to potentially mitigate muscle atrophy by inhibiting the HDAC4/FoxO3a pathway. These findings suggest that HDAC4 may be a potential therapeutic target for exercise to combat Dex-induced muscle atrophy.

Dihydroartemisinin ameliorates skeletal muscle atrophy in the lung cancer cachexia mouse model.

Cancer cachexia (CC) is characterized by weight loss with specifically reduced skeletal muscles and adipose tissues in patients with late-stage cancer. Dihydroartemisinin (DHA), an effective antimalarial derivative of artemisinin, has been demonstrated to have anti-inflammatory and antitumor properties. This study examined the effects of DHA on the Lewis lung carcinoma (LLC)-induced CC mouse model. DHA treatment significantly increases tumor-free body weight and food intake but decreases serum interleukin-6 level and tumor weight in CC mice. In addition, DHA treatment relieves muscle atrophy and decreases muscle ring finger 1 (MuRF1) and F-box-only protein 32 (Fbx32) expressions in CC mice. In vitro, DHA reverses the reduction in myotube formation induced by an LLC-conditioned medium and increases Fbx32 expression in C2C12 mouse myotubular cells. Our study demonstrated that DHA ameliorates the cachectic state and skeletal muscle atrophy in LLC-induced cachectic mouse models, suggesting its therapeutic potential for CC.

Quantifying Biomass and Visualizing Cell Coverage on Fibrous Scaffolds for Cultivated Meat Production.

Cultivated meat represents a transformative solution to environmental and ethical concerns of traditional meat industries, replicating livestock meat's texture and sensory attributes in vitro with a focus on cost, safety, and nutritional quality. Central to this process are biomaterial scaffolds that support tissue development from isolated animal cells grown in or on these matrices. Understanding scaffold interactions with cells, including scaffold degradation and biomass production, is crucial for process design and for scaling-up goals. In this article, we outline comprehensive methods to quantify scaffold-cell interactions for such scenarios, focusing on biomaterial scaffold degradation and changes in cell biomass [measured by cell weight, extracellular matrix (ECM) deposition, and cell coverage] during cell culture. We introduce two methodologies for assessing cell coverage: fixation and staining for detailed imaging analysis, and non-invasive, real-time evaluation across scaffolds. Here we focus on fiber-based scaffolds, while the assessments can be extrapolated to 2-dimensional (2D; films), and in part to 3-dimensional (3D; sponge) systems. Utilizing the C2C12 mouse myoblast cell line as a gold standard, the protocols deliver precise, step-by-step instructions for preparing fiber scaffolds (using silk proteins here), seeding cells, and monitoring key parameters for cultivated meat production, providing a framework for advancing cellular agriculture techniques. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Fabrication and preparation of silk fiber scaffolds for cell seeding Support Protocol 1: Cultivation of C2C12 cells and seeding onto fibrous scaffolds Basic Protocol 2: Quantification of decellularized yarn scaffold degradation during cell culture Basic Protocol 3: Quantification of biomass variation and ECM deposition on yarn scaffolds during C2C12 cell culture Basic Protocol 4: Visualization of cell-laden yarn scaffolds and determination of cell coverage ratio using confocal microscopy Support Protocol 2: Real-time imaging of cell-laden yarn scaffolds using Celigo system Support Protocol 3: Applying green CellTracker fluorescent probes to C2C12 cells seeded on yarn scaffolds.

Fibrillar Hydrogel Inducing Cell Mechanotransduction for Tissue Engineering.

One of the key strategies for tissue engineering is to design multifunctional bioinks that balance printability with cytocompatibility. Here, we describe fibrillar hydrogels produced by Schiff base formation between B-type gelatin and oxidized sodium alginate, followed by the incorporation of type I collagen, yielding a new gel (MyoColl). The resulting hydrogel exhibits a temperature- and mass-ratio-dependent sol-gel transition, showing variability of hydrogel properties depending on the component ratio. MyoColl composition provides a convenient platform for biofabrication in terms of shear thinning, yielding, Young's modulus, and shape accuracy. Metabolic activity tests and fluorescent microscopy of 2D hydrogel-based mouse C2C12 myoblast cell culture show significant cytocompatibility of the developed carriers. In addition, primary signs of cell mechanotransduction and myofilament formation of 3D printed MyoColl-based cell cultures were detected and described. Due to these promising results, the described hydrogel composition has shown itself as a convenient platform for muscle tissue engineering.

Non-cytotoxic, biocompatible pyrochlore Cerium Zirconium Oxide nanoparticles for asymmetric supercapacitor applications

A novel biocompatible and non-cytotoxic Ce2Zr2O7 (CZO) nano-sized particles were developed by a straightforward hydrothermal method. Cubic structure with Fd-3m space group (227) of prepared nanoparticles has been confirmed from X-ray diffraction (XRD) patterns. Nano-sphere like particle morphology was conspicuously noticed from the FE-SEM and TEM investigations thoroughly. Particle size, inter-planar distance, SAED pattern and elemental presence were affirmatively evaluated. The elemental presence in the sample and their ionic states were systematically investigated through X-ray photoelectron spectroscopy analysis. We have investigated the cytotoxicity of the Ce2Zr2O7 nanoparticles on C2C12 (mouse myoblast cell line) cells and observed to be non-cytotoxicity on C2C12 cells which supports the eco-friendly and biocompatible nanoparticles. We have investigated the electrochemical performance of Ce2Zr2O7 nanoparticles using various electrolytes such as Na2SO4, Li2SO4 and KOH. We have obtained the predominant electrochemical performance of 250 F g−1 at 0.05 A g−1 along with appreciable cycling durability from KOH electrolyte interestingly. In addition, we fabricated asymmetric supercapacitor device and evaluated its energy and power densities systematically. We have evaluated maximum energy and power densities are in the order of 13 Wh kg−1 and 3692 W kg−1, respectively. Therefore, eco-friendly and biocompatible Ce2Zr2O7 nanoparticles could be suggested as a contender for the electrode substance for supercapacitor applications. This investigation has also been an usher hope of further technological advancement in the field of nano-biomedical along with energy storage research fields.

Nrf2 deficiency in muscle attenuates experimental autoimmune myositis-induced muscle weakness.

Idiopathic inflammatory myopathies (IIMs) are systemic autoimmune diseases characterised by muscle weakness. Although multiple physiological and pathological processes are associated with IIMs, T-lymphocyte infiltration into muscle plays a key role in the development and exacerbation of IIMs. Nuclear factor erythroid 2-related factor 2 (Nrf2) is a key transcription factor that regulates inflammatory responses; therefore, muscle Nrf2 may serve an important role in the development of IIMs. In this study, we demonstrated that experimental autoimmune myositis (EAM) causes loss of muscle mass and function in oxidative and glycolytic muscles in C57BL/6 mice. EAM increased CD4+ and CD8+ T-lymphocyte infiltration, as well as interferon-gamma (IFN-γ) and tumour necrosis factor-alpha (TNF-α) mRNA expression in oxidative soleus and glycolytic extensor digitorum longus muscles, along with elevated chemokine mRNA levels (i.e. CCL3, CCL5, CXCL9, CXCL10 and CXCL16). IFN-γ and TNF-α treatments increased the mRNA expression levels of these chemokines in C2C12 myotubes. EAM also increased phosphorylated Nrf2 at Ser40 in soleus and glycolytic white vastus lateralis muscle. Although the expression of several chemokines was affected by Nrf2 activation following tert-butylhydroquinone treatment or Keap1 knockdown, CCL5 mRNA expression significantly increased in C2C12 myotubes and mouse skeletal muscle. Moreover, muscle-specific Nrf2 knockout in mice attenuates EAM-induced loss of muscle mass and function, which was associated with the inhibition of CCL5 mRNA expression, CD8+ T-lymphocyte infiltration and IFN-γ mRNA expression. Collectively, these findings reveal that regulating Nrf2 activity is a promising therapeutic approach for treating IIM-mediated muscle weakness. KEY POINTS: Experimental autoimmune myositis (EAM) causes loss of muscle mass and function. Loss of muscle mass and function in EAM were associated with increased chemokine mRNA expression (i.e. CCL3, CCL5, CXCL9, CXCL10 and CXCL16), T-lymphocyte infiltration and inflammatory cytokine mRNA expression (i.e. IFN-γ and TNF-α) in the skeletal muscle. EAM activated Nrf2 in muscle and increased Nrf2 activity in vivo and in vitro increased CCL5 mRNA expression. Muscle-specific Nrf2 knockout in mice attenuated EAM-induced muscle weakness by inhibiting CCL5 mRNA expression, CD8+ T-lymphocyte migration and IFN-γ mRNA expression in muscles. These results provide further evidence for the potential therapeutic targeting of Nrf2 to mitigate EAM-induced muscle weakness.

Chemical synthesis, in vitro testing and in silico Nampt-based molecular docking of novel aniline aromatic ring-substituted 2-Aminothiazole analogues.

The heterocyclic 2-Aminothiazoles scaffolds are used in a wide range of therapeutic applications against various diseases for its antioxidant, anti-inflammatory, antimicrobial and anticancer actions. In the present study, we synthesized novel aniline aromatic ring-substituted 2-Aminothiazole derivatives. Molecular docking was performed using Glide module of the Schrödinger Suite to fit compounds JG-49, JG-62, and KBA-18 against the Nicotinamide phosphoribosyl transferase (Nampt) enzyme, an intracellular regulator of Nicotinamide adenine dinucleotide (NAD) redox cofactor involved in energy metabolism and epigenetics and are implicated in aging and metabolic diseases. The three compounds viz. JG-49, JG-62, and KBA-18 showed an increase in Nampt enzymatic activity in vitro. All three substituted derivatives of 2-Aminothiazole showed no cytotoxicity with the mouse C2C12 myoblasts cultures assessed with the MTT cell viability assay. Moreover, the wound closure of the mouse C2C12 myoblasts in vitro displayed no significant difference between the treatment groups of the 2-Aminothiazole derivatives compared with the control naïve and DMSO treated myoblasts cultures, except for the 2-Aminothiazole substituted derivatives JG-62 and KBA-18, which showed a significant increase in the wound closure compared with the control cells at different concentrations. Taken together, we demonstrated that 2-Aminothiazole substituted derivatives provide enhanced Nampt activity, wound closure, and no cytotoxic effects in vitro. Further studies will allow to improve the substitution of 2-Aminothiazole derivatives and test their potential therapeutic applications.

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.

Endurance exercise-induced histone methylation modification involved in skeletal muscle fiber type transition and mitochondrial biogenesis

Skeletal muscle is a highly heterogeneous tissue, and its contractile proteins are composed of different isoforms, forming various types of muscle fiber, each of which has its own metabolic characteristics. It has been demonstrated that endurance exercise induces the transition of muscle fibers from fast-twitch to slow-twitch muscle fiber type. Herein, we discover a novel epigenetic mechanism for muscle contractile property tightly coupled to its metabolic capacity during muscle fiber type transition with exercise training. Our results show that an 8-week endurance exercise induces histone methylation remodeling of PGC-1α and myosin heavy chain (MHC) isoforms in the rat gastrocnemius muscle, accompanied by increased mitochondrial biogenesis and an elevated ratio of slow-twitch to fast-twitch fibers. Furthermore, to verify the roles of reactive oxygen species (ROS) and AMPK in exercise-regulated epigenetic modifications and muscle fiber type transitions, mouse C2C12 myotubes were used. It was shown that rotenone activates ROS/AMPK pathway and histone methylation enzymes, which then promote mitochondrial biogenesis and MHC slow isoform expression. Mitoquinone (MitoQ) partially blocking rotenone-treated model confirms the role of ROS in coupling mitochondrial biogenesis with muscle fiber type. In conclusion, endurance exercise couples mitochondrial biogenesis with MHC slow isoform by remodeling histone methylation, which in turn promotes the transition of fast-twitch to slow-twitch muscle fibers. The ROS/AMPK pathway may be involved in the regulation of histone methylation enzymes by endurance exercise.

PEI/MMNs@LNA-542 nanoparticles alleviate ICU-acquired weakness through targeted autophagy inhibition and mitochondrial protection.

Intensive care unit-acquired weakness (ICU-AW) is prevalent in critical care, with limited treatment options. Certain microRNAs, like miR-542, are highly expressed in ICU-AW patients. This study investigates the regulatory role and mechanisms of miR-542 in ICU-AW and explores the clinical potential of miR-542 inhibitors. ICU-AW models were established in C57BL/6 mice through cecal ligation and puncture (CLP) and in mouse C2C12 myoblasts through TNF-α treatment. In vivo experiments demonstrated decreased muscle strength, muscle fiber atrophy, widened intercellular spaces, and increased miR-542-3p/5p expression in ICU-AW mice model. In vitro experiments indicated suppressed ATG5, ATG7 and LC3II/I, elevated MDA and ROS levels, decreased SOD levels, and reduced MMP in the model group. Similar to animal experiments, the expression of miR-542-3p/5p was upregulated. Gel electrophoresis explored the binding of polyethyleneimine/mesoporous silica nanoparticles (PEI/MMNs) to locked nucleic acid (LNA) miR-542 inhibitor (LNA-542). PEI/MMNs@LNA-542 with positive charge (3.03 ± 0.363 mV) and narrow size (206.94 ± 6.19 nm) were characterized. Immunofluorescence indicated significant internalization with no apparent cytotoxicity. Biological activity, examined through intraperitoneal injection, showed that PEI/MMNs@LNA-542 alleviated muscle strength decline, restored fiber damage, and recovered mitochondrial injury in mice. In conclusion, PEI/MMNs nanoparticles effectively delivered LNA-542, targeting ATG5 to inhibit autophagy and alleviate mitochondrial damage, thereby improving ICU-AW.

Senescent cells inhibit mouse myoblast differentiation via the SASP-lipid 15d-PGJ2 mediated modification and control of HRas

Senescent cells are characterized by multiple features such as increased expression of senescence-associated β-galactosidase activity (SA β-gal) and cell cycle inhibitors such as p21 or p16. They accumulate with tissue damage and dysregulate tissue homeostasis. In the context of skeletal muscle, it is known that agents used for chemotherapy such as Doxorubicin (Doxo) cause buildup of senescent cells, leading to the inhibition of tissue regeneration. Senescent cells influence the neighboring cells via numerous secreted factors which form the senescence-associated secreted phenotype (SASP). Lipids are emerging as a key component of SASP that can control tissue homeostasis. Arachidonic acid-derived lipids have been shown to accumulate within senescent cells, specifically 15d-PGJ2, which is an electrophilic lipid produced by the non-enzymatic dehydration of the prostaglandin PGD2. This study shows that 15d-PGJ2 is also released by Doxo-induced senescent cells as an SASP factor. Treatment of skeletal muscle myoblasts with the conditioned medium from these senescent cells inhibits myoblast fusion during differentiation. Inhibition of L-PTGDS, the enzyme that synthesizes PGD2, diminishes the release of 15d-PGJ2 by senescent cells and restores muscle differentiation. We further show that this lipid post-translationally modifies Cys184 of HRas in C2C12 mouse skeletal myoblasts, causing a reduction in the localization of HRas to the Golgi, increased HRas binding to Ras Binding Domain (RBD) of RAF Kinase (RAF-RBD), and activation of cellular Mitogen Activated Protein (MAP) kinase–Extracellular Signal Regulated Kinase (Erk) signaling (but not the Akt signaling). Mutating C184 of HRas prevents the ability of 15d-PGJ2 to inhibit the differentiation of muscle cells and control the activity of HRas. This work shows that 15d-PGJ2 released from senescent cells could be targeted to restore muscle homeostasis after chemotherapy.

Senescent cells inhibit mouse myoblast differentiation via the SASP-lipid 15d-PGJ2 mediated modification and control of HRas.

Senescent cells are characterized by multiple features such as increased expression of senescence-associated β-galactosidase activity (SA β-gal) and cell cycle inhibitors such as p21 or p16. They accumulate with tissue damage and dysregulate tissue homeostasis. In the context of skeletal muscle, it is known that agents used for chemotherapy such as Doxorubicin (Doxo) cause buildup of senescent cells, leading to the inhibition of tissue regeneration. Senescent cells influence the neighboring cells via numerous secreted factors which form the senescence-associated secreted phenotype (SASP). Lipids are emerging as a key component of SASP that can control tissue homeostasis. Arachidonic acid-derived lipids have been shown to accumulate within senescent cells, specifically 15d-PGJ2, which is an electrophilic lipid produced by the non-enzymatic dehydration of the prostaglandin PGD2. This study shows that 15d-PGJ2 is also released by Doxo-induced senescent cells as an SASP factor. Treatment of skeletal muscle myoblasts with the conditioned medium from these senescent cells inhibits myoblast fusion during differentiation. Inhibition of L-PTGDS, the enzyme that synthesizes PGD2, diminishes the release of 15d-PGJ2 by senescent cells and restores muscle differentiation. We further show that this lipid post-translationally modifies Cys184 of HRas in C2C12 mouse skeletal myoblasts, causing a reduction in the localization of HRas to the Golgi, increased HRas binding to Ras Binding Domain (RBD) of RAF Kinase (RAF-RBD), and activation of cellular Mitogen Activated Protein (MAP) kinase-Extracellular Signal Regulated Kinase (Erk) signaling (but not the Akt signaling). Mutating C184 of HRas prevents the ability of 15d-PGJ2 to inhibit the differentiation of muscle cells and control the activity of HRas. This work shows that 15d-PGJ2 released from senescent cells could be targeted to restore muscle homeostasis after chemotherapy.

Anti-muscle atrophy effect of fermented Tenebrio molitor larvae extract by modulating the PI3K-Akt-mTOR/FoxO3α pathway in mice treated with dexamethasone

This study investigated the anti-sarcopenic effect of fermented Tenebrio molitor larvae (mealworms) extract (FME) in both dexamethasone (DEX)-treated C2C12 cells and mice. FME (100 µg/mL) increased the diameter of myotubes and inhibited the gene and protein expression of atrogin-1 compared to DEX- or non-fermented mealworms extract (ME)-treated C2C12 cells. Male C57BL/6N mice were divided into five groups: Normal Control (NC), DEX (10 mg/kg, intraperitoneal), and three groups of DEX+FME (100, 200, or 500 mg FME/kg/day, oral) for two weeks. FME at doses of 200 and 500 mg/kg effectively improved grip strength when compared to the DEX group. Histological analysis of the quadriceps muscle showed a larger muscle fiber size in the DEX+FME groups compared to DEX group. FME (200 and 500 mg/kg) significantly increased cross-sectional area of the muscle fiber compared to DEX group. FME (500 mg/kg) significantly decreased the ubiquitin, atrogin-1 and MuRF-1 protein levels, and increased levels of MHC and MyoG in DEX-treated mice. The puromycin labeling assay revealed that FME increased protein synthesis in DEX-induced muscle atrophy. The FME treatment demonstrated significant upregulation in phosphorylation levels, including mTOR, FoxO3α, Akt, and PI3K compared to DEX group. In conclusion, FME inhibited the increase in proteins associated with muscle atrophy, including, atrogin-1 and MuRF-1, by regulating the PI3K-Akt-FoxO3α pathway. FME improved the PI3K-Akt-mTOR signaling pathway, which was reduced by DEX. This study suggests that FME has the potential for use in sarcopenia therapy, possibly serving as a natural agent that counteracts the negative effects of DEX on muscle tissue.

Cell-Based Meat Scaffold Based on a 3D-Printed Starch-Based Gel.

Starch was mixed with a gel to produce a starch-based gel ink, which exhibited favorable printing characteristics. Through the optimization of infill density, 3D-printed scaffolds with 50% infill density and a highly ordered microstructure were successfully fabricated. The addition of calcium carbonate nanoparticles-glucono delta lactone (CaCO3 NPs-GDL) had notable effects on the swelling degree, in vitro digestion, water stability, and pore distribution of the scaffolds. When the amount of CaCO3 NPs in the starch-based gel was 0.075 g, the resulting 3D-printed gel scaffold with a 50% infill density proved to be the most suitable for cultivating cell-based meat. It featured pore sizes ranging from 80 to 120 μm and a compression modulus of 246.76 Pa. After 7 days of proliferation, the C2C12 mouse skeletal myoblasts exhibited an approximately 2.81-fold increase in cell numbers. The fusion index and maturation index of C2C12 cells on the scaffolds were 57.00 ± 0.45% and 34.56 ± 0.56%, respectively. The starch-based gel scaffolds demonstrated excellent water stability and in vitro degradability. Moreover, C2C12 cells exhibited successful proliferation and differentiation on the starch-based scaffolds, ultimately leading to the production of cell-based meat. This study developed a starch-based composite gel scaffold for the manufacture of cell-based meat.

Material extrusion additive manufacturing of graphene oxide reinforced 13–93B1 bioactive glass scaffolds for bone tissue engineering applications

Graphene-reinforced bioactive glass scaffolds have gained significant attention in the field of bone tissue engineering due to their unique combination of mechanical strength, bioactivity, and electrical conductivity. Additive manufacturing techniques, such as 3D printing, provide a versatile platform for fabricating these scaffolds with precise control over their architecture and composition. Consequently, in this work, we have fabricated graphene oxide (GO)-reinforced 13–93B1 bioactive glass scaffold using the material extrusion-based additive manufacturing. A Pluronic F-127-based ink was prepared for scaffold fabrication, and its rheological properties were assessed for shear thinning behaviour, structural support, and recovery. Further, the fabricated scaffolds were characterized using micro-computed tomography, scanning electron microscopy, and energy dispersive x-ray spectroscopy. Additionally, computational fluid dynamics simulations with Dulbecco’s modified eagle medium and blood were performed to evaluate the perfusion kinetics of the scaffolds. The inclusion of GO enhanced the compressive strength of the fabricated scaffolds by ∼202 %. The morphological characterization based on micro-computed tomography showed that additively manufactured scaffolds have appropriate porosity, pore size, pore throat size, and interconnectivity for bone tissue engineering. The synergy with surrounding muscle tissue is also crucial for scaffold-guided bone regeneration. A scaffold supporting the muscle growth around the bone will be able to promote bone-muscle crosstalk and, in turn, bone regeneration. The live-dead assay results showed no cytotoxicity towards C2C12 mouse myoblast cells. Also, cell adhesion and cell viability results show better cell growth on the nanocomposite scaffolds. Overall, the fabricated scaffolds are found suitable for bone tissue engineering applications.

Jie-Du-Tong-Luo formula protects C2C12 myotubes against high glucose and palmitic acid injury by activating the PI3K/Akt/PPARγ pathway in vitro

IntroductionIn prior reports, Jie-Du-Tong-Luo (JDTL) was reported to help control insulin secretion and blood glucose in patients with diabetes, while also protecting liver and pancreatic islet cells against injury caused by exposure to high glucose (HG) levels. This study was thus developed to assess the effects of JDTL on HG and palmitic acid (PA)-induced muscle injury and to explore the mechanistic basis for these effects. MethodsA model of muscle injury was established using mouse C2C12 myotubes treated with HG + PA. A proteomics approach was used to assess changes in protein levels following JDTL treatment, after which Western immunoblotting was employed to validate significantly affected pathways. ResultsJDTL was able to protect against HG + PA-induced muscle cell injury in this experimental system, altering lipid metabolism and inflammatory activity in these injured C2C12 myotubes. Western blotting suggested that JDTL is capable of activating PI3K/Akt/PPARγ signaling to control lipid metabolism without any corresponding impact on the inflammatory NF-κB pathway. ConclusionsThese data highlight the ability of JDTL to protect against HG + PA-induced injury to muscle cells, and suggest that the underlying basis for such efficacy is related to the PI3K/Akt/PPARγ pathway-mediated modulation of lipid metabolism.

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.