Impaired expression of BCAT1 relates to muscle atrophy of mouse model of sarcopenia

Background The underlying mechanism of muscle atrophy in sarcopenia is still not fully understood; branched chain aminotransferase 1(BCAT1) isocitrate dehydrogenase-1 encodes an evolutionarily conserved cytoplasmic aminotransferase for glutamate and branched-chain amino acids (BCAAs), thus constituting a regulatory component of cytoplasmic amino and keto acid metabolism. In human gliomas carrying wild-type isocitrate dehydrogenase-1, BCAT1 promotes cell proliferation through amino acid catabolism. Hence, the goals of this study were to unravel the potential role of BCAT1 expression in muscle atrophy and to explore the mechanisms underlying this process. Methods We first measured Bcat1 expression by RT-qPCR and western blotting in murine and cellular models of muscle atrophy. To understand how the Bcat1-driven changes sustained muscle cell growth, we analyzed reactive oxygen species (ROS) levels and activation of the mTORC1/S6K1 pathway in muscle cells. Furthermore, we performed Cell Counting Kit-8(CCK8) assays and fluorescence staining to evaluate growth rate of cells and ROS levels. Finally, we verified that depletion of Bcat1 impairs the growth rate of muscle cells and increases ROS levels, indicating that muscle atrophy resulted from the downregulation of the mTORC1/S6K1 pathway. Data were analyzed by two-tailed unpaired Student’s t-test or Mann-Whitney U test for two groups to determine statistical significance. Statistical analyses were performed using GraphPad Prism version 6.0 and SPSS 16.0 software. Results Bcat1 expression level in skeletal muscles was lower in murine and cellular models of sarcopenia than in the control groups. Bcat1 knockdown not only suppressed the growth of muscle cells but also increased the production of ROS. Impaired cell growth and increased ROS production was rescued by co-introduction of an shRNA-resistant Bcat1 cDNA or addition of the mTORC1 stimulator MYH1485. Muscle cells with Bcat1 knockdown featured lower mTORC1 and S6K1 phosphorylation (pS6K1) than NT muscle cells. Addition of either shRNA-resistant Bcat1 cDNA or MYH1485 rescued the suppression of cell growth, increase in ROS production, and decrease in pS6K1. Conclusions The branched chain amino acids catabolic enzyme BCAT1 is essential for the growth of muscle cells. BCAT1 expression contributes to sustained growth of muscle cells by activating mTOR signaling and reducing ROS production. Supplementary Information The online version contains supplementary material available at 10.1186/s12891-022-05332-7.

risk of physical weakness and death. Applying the European Working Group on Sarcopenia (EWGSOP) definition of sarcopenia in a community-dwelling population (the UK-based Hertfordshire Cohort Study), researchers reported that sarcopenia was present in 4.6% of men and 7.9% of women at a mean age of 67 years [1].
The etiologies of sarcopenia include aging, drug abuse, lack of activity, disease, and malnutrition, among others, many of which often coexist. However, the underlying mechanisms of muscle atrophy in sarcopenia are not fully understood [2].
Branched-chain amino acids (BCAAs) are essential amino acids that play an important regulatory role in nutrient signaling, body nitrogen metabolism, and protein synthesis [3]. The first enzymatic step is the reversible transamination catalyzed by branched chain amino acid transaminase (BCAT) isozymes (mitochondrial BCATm and cytosolic BCATc) [4]. The second step is the oxidative decarboxylation of branched-chain α-keto acids (BCKAs) to their respective branched chain acyl-CoA derivatives, which is coupled with the formation of nicotinamide adenine dinucleotide (NADH) [5].
Branched chain amino acid transaminase 1(BCAT1) is an enzyme that initiates the catabolism of BCAAs. BCAT1 encodes an evolutionarily conserved cytoplasmic transaminase that acts on glutamate and BCAAs and thus constitutes a regulatory component of cytoplasmic amino and keto acid metabolism. Although skeletal muscle represents the most important site for BCAA transamination, the role of BCAT1 in human sarcopenia remains unknown. BCAT1 is well studied in immune cells like macrophages, and BCAT1 influence cell proliferation through mammalian target of rapamycin (mTOR) signaling [6]. However, whether BCAT1 affects the muscle cells though mTOR pathway have not been studied yet.
Low expression levels of BCATm and BCKDC were found to be associated with elevated plasma BCAAs [7], which may affect the mTOR pathway, more specifically, mammalian target of rapamycin complex 1 (mTORC1), in peripheral tissues and influence protein synthesis [8], it is a conserved serine/threonine kinase that plays a key role in cell growth and metabolism [9,10]. The researches above suggested that BCAT1 may influence protein synthesis and cell growth through mTOR pathway. The typical characteristic of mTOR activity is the decrease in phosphorylation of the downstream substrate ribosomal S6 kinase 1(S6K1). Activation of the mTOR pathway (through mTORC1) can stimulate protein synthesis [11]. Furthermore, mTOR activity regulates protein synthesis by integrating and transforming various extracellular cues from growth factors and nutrients, as well as those related to cellular energy status and environmental pressure [12]. Among the two different intracellular mTOR complexes (mTORC1 and mTORC2), mTORC1 contains regulatory proteins related to mTOR, and is sensitive to rapamycin. Reportedly, mTORC1 is the most important regulator of protein synthesis [13], as it phosphorylates its downstream effectors S6K1 and 4E-binding protein (4E-BP) in a rapamycin-sensitive manner [14,15]. Additionally, mTORC1 regulates protein translation, cell proliferation, apoptosis, and autophagy. The mTORC1/S6K1 axis transmits and integrates important signals that are essential for biological processes involving nutrients, growth factors, and energy metabolites.
Previous studies have shown that mTORC1 activation was associated with muscle hypertrophy [16]. In addition, muscle-specific raptor gene knockout mice showed severe muscle atrophy and weight loss, leading to early death [17].
Therefore, the aims of this study were to investigate the potential role of BCAT1 expression in muscle atrophy, and to further explore the underlying mechanism. We first measured the expression of Bcat1 in murine and cellular models of muscle atrophy. To understand how Bcat1-driven changes affected muscle cell growth, we analyzed reactive oxygen species (ROS) levels in muscle cells and activation of the mTORC1/S6K1 pathway. Finally, we validated our hypothesis that Bcat1 depletion could induce muscle atrophy by downregulating the mTORC1/S6K1 pathway and increasing ROS levels.

Mice
Ten Male C57BL/6 mice were randomly assigned to the following two groups of 5 mice each: (1) the control group, (2) dexamethasone (DEX)-induced sarcopenia group. Dead or ill mice that could not cooperate were removed from the experiment, and no mice were removed.
The mice were obtained from the Health Science Center of Peking University. In this study, the average age of the animals was 10 weeks. All mice were sacrificed by cervical dislocation after the experiment. Fresh muscle samples were stored at − 80 °C. All animal experiments were approved by the Animal Care and Use Committee of Peking University People's Hospital.

Generation of a mouse model of sarcopenia
The control group was intraperitoneally injected with sterile normal saline over 10 days, and the sarcopenia group was intraperitoneally injected with 25 mg/kg/day of water-soluble DEX (Sigma Aldrich) for 10 days [18]. The animals were euthanized by cervical dislocation on day 11. Body weights were measured, and total muscle mass, tibial anterior muscles, and gastrocnemius muscles were removed, weighed, and quickly frozen for analysis of mRNA and protein levels.

Cell culture
Murine C2C12 myoblasts were cultured as recommended by the supplier (Cell Resource Bank of the Chinese Academy of Sciences). In short, C2C12 myoblasts were maintained in DMEM supplemented with 10% FBS, 100 U/mL penicillin, and 100 U/mL streptomycin. For routine differentiation, the cells were grown to approximately 80% confluence.

Immunofluorescence staining
C2C12 cells were cultured in multiwells, treated with DEX (1 μM) or sterile saline for 24 h, fixed with 4% paraformaldehyde at 4 °C, washed three times with phosphate-buffered saline (PBS), permeabilized with 0.3% Triton X-100, washed three times with PBS, blocked with 10% goat serum in PBS, and incubated with primary antibody diluted in PBS (1:200) at 4 °C. After three sequential washes with PBS, the cells were incubated with fluorescently labeled donkey anti-rabbit IgG (H + L) as secondary antibody (594) diluted in PBS (1:300) at 4 °C, and washed three times with PBS afterwards. The nuclei were counterstained with DAPI and subsequently washed with PBS. Digital images of fluorescently labeled cells were obtained using a fluorescence microscope (Olympus IX51, Japan).

SDS-PAGE and western blot analysis
The cell samples were directly dissolved in cryolysis buffer [RIPA buffer (Beyotime, China), PMSF (Amresco, China), cocktail (Shanghai Yuanye, China), Triton X-100 (China Amresco), and NaVO3] for 15 min. The lysates were clarified by centrifugation (12,000×g, 15 min at 4 °C). The supernatant was collected and stored at − 80 °C. Protein concentration was determined using the Beyond Time ™ BCA Test Kit (Shanghai Beiotim Biotechnology Company, China). For western blotting, SDS-PAGE was used to separate the same amount of protein per sample, which was then transferred to a PVDF membrane (Millipore, USA). Afterwards, the membrane was incubated with 5% skimmed milk in Tris-buffered saline with Tween20 (TBS-T buffer) for 2 h, followed by addition of the primary antibodies against the indicator proteins at 4 °C. Then, the appropriate horseradish peroxidase-coupled secondary antibody was added, and the membrane was washed with TBS-T at room temperature for 2 h. Immune response bands were detected by an enhanced chemiluminescence system and analyzed by Quantity One software (Pierce ECL, western blotting substrate; Thermo Fisher Scientific, Pierce, Rockford, IL, US). ImageJ software [19] was used to quantify each protein band and standardize it to a loading control (GAPDH).

Cell viability analysis
Cell viability was determined by the Cell Counting Kit-8(CCK8) assay according to the manufacturer's instructions (China). Cells were used at a density of 2 × 10 3 cells/well. After treatment with DEX or sterile normal saline, the monolayer was washed three times with PBS, and 10 μL of CCK8 reagent-containing DMEM solution was added and incubated at 37 °C for 2 h. Absorbance was measured using a microplate reader at 450 nm and expressed as percentage of the control value. The cell viability(% of control) was calculated by the following formula [20]: [OD]test/[OD] control X 100.

ROS production
ROS production was measured by using a Cellular Reactive Oxygen Species Detection Assay (Abcam, England). Cells were seeded on 20-well plates and incubated with 5 μM dichlorodihydrofluorescein diacetate (DCFH-DA) for 20-30 min and subsequently washed twice with PBS. Changes in relative fluorescence were measured using a fluorescence microscope (Olympus IX51, Japan).

RNA isolation and quantitative real-time PCR (RT-qPCR)
The gastrocnemius muscle was homogenized in Trizol reagent (Transgen Biotechnology, Beijing, China) on ice, and then separated into organic and aqueous phases by chloroform. After ethanol precipitation, total RNA was extracted from the aqueous phase. RT-qPCR was performed by Transgen Biotechnology (Beijing). Bcat1 expression was quantified using the thermal cycling dice real-time system and SYBR premix ex Taq II (Takara Bio, Shiga, Japan). All samples were measured in duplicate.

Whole muscle lysates
The gastrocnemius muscle was homogenized in RIPA buffer (25 mM Tris-HCl, pH 7.6, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, and 0.1% SDS) supplemented with a mixture of protease and phosphatase inhibitors (Thermo Fisher Scientific, US). The whole procedure was performed on ice.

Preparation of frozen sections
Fresh gastrocnemius muscles were dissected, frozen in n-hexane at − 70 °C, and cooled in liquid nitrogen for 2 min. Cross sections (10 μm) were cut at − 20 °C using a cryostat slicer and stained with nicotinamide adenine dinucleotide hydrogen (NADH) according to a previously described method [21].

Statistical analysis
Data were analyzed using Student's t-test or Mann Whitney U test. Unless specified otherwise, values are presented as mean + standard deviation, and p < 0.05 was considered statistically significant. Statistical analyses were performed using GraphPad Prism 6.0 or SPSS 16.0 software.

Data availability
All other relevant data are available from the corresponding author upon request.

Skeletal muscle mass, muscle atrophy, and Bcat1 expression
Animal characteristics are shown in Table 1. The average body weight of DEX-induced sarcopenia mice was lower than that of the mice in the control group. Absolute and total masses of the gastrocnemius and tibialis anterior muscles were measured, and the sarcopenic index was calculated. The absolute muscle mass and sarcopenia index in the sarcopenic group were lower than those in the control group. Compared with the control group, muscle atrophy was more pronounced in the sarcopenia group ( Fig. 1B-C).
To investigate the role of Bcat1 in DEX-induced muscle atrophy in mice, we measured Bcat1 expression level in skeletal muscles (gastrocnemius and tibialis anterior muscles) of mice, and found that Bcat1 expression in skeletal muscle in the sarcopenic group was lower than that in the control group ( Fig. 1A and supplementary material Fig. 1).

Bcat1 expression is downregulated in DEX-treated muscle cells
To investigate the role of Bcat1 in muscle atrophy, C2C12 cells were treated with DEX to establish a cellular model of muscle atrophy. We confirmed that C2C12 cell growth was impaired upon DEX treatment (Fig. 2B). Next, we investigated Bcat1 expression in C2C12 cells. BCAT1 protein expression in growth-restricted C2C12 cells was lower than that in normal muscle cells ( Fig. 2 and supplementary material Fig. 2).

Bcat1 knockdown impairs growth and promotes ROS production in C2C12 cells
Since Bcat1 expression was downregulated in a DEXinduced cellular muscle atrophy model, we wanted to determine the role of Bcat1 in the pathogenesis of muscle atrophy. Therefore, we prepared lentivirus, which could knock down the expression of Bcat1, the first enzyme in the BCAA catabolic pathway, in C2C12 muscle cells. Bcat1 knockdown inhibited the growth rate of C2C12 cells from day 2 (Fig. 3B). Next, we studied the mechanisms by which Bcat1 contributed to muscle cell growth. Therefore, we analyzed whether loss of Bcat1 increased ROS production. Compared with the vector control group, shBcat1 (Bcat1 gene knockdown) inhibited the growth of muscle cells in vitro. Further evidence suggested that Bcat1 knockdown not only inhibited the growth of the muscle cells but also increased the production of ROS (Fig. 3A), which in turn inhibited cell growth. Quantitative analysis of cell viability according to the CCK8 assay showed that cell growth in the Bcat1 knockdown group was decreased after 24 h, which was not statistically significant. After 48 h, quantitative data showed that cell growth had decreased significantly in response to Bcat1 knockdown compared with the control group (Fig. 3B). Bcat1 knockdown also significantly promoted ROS production (Fig. 3A). Combined introduction of anti-shRNA Bcat1 cDNA or the mTORC1 stimulator MYH1485 alleviated both impaired cell growth and increased ROS production (Fig. 3A-B).

mTOR contributes to Bcat1 function in muscle cells
Finally, we asked how the metabolic regulator Bcat1 affected muscle cell growth and ROS production. To understand this, we analyzed ROS levels and activation of the mTORC1/S6K1 pathway in myocytes. Western blotting was used to determine the phosphorylation status and total protein concentration of the mTORC1 downstream target S6K1 in the gastrocnemius muscle. As shown in Fig. 4 and supplementary material Fig. 4, the ratios of p-mTOR and mTOR, as well as those of pS6K1 and S6K1 in the Bcat1 knockdown group were significantly reduced compared with the control group. To further confirm the role of the mTOR1/S6K1 axis in Bcat1 knockdown-induced muscle cell growth impairment, we used shRNA-resistant Bcat1 cDNA and mTOR activators/inhibitors. Compared with the shBcat1 plasmid, the viability of muscle cells transfected with anti-Bcat1 shRNA could be recovered significantly. In addition, myocytes treated with the mTOR activator MYH1485 showed improved cell viability, while muscle cells treated with an mTOR inhibitor were damaged. Treatment with MYH1485, an activator of mTOR, was associated with significantly reduced cell viability after shBcat1 transfection. Rapamycin, an mTOR inhibitor, blocked the effect of Bcat1 on cell viability and growth. In conclusion, compared with NT myocytes, the phosphorylation levels of mTORC1 and S6K1 in growth-impaired myocytes were lower.

Discussion
Although BCAT1 is expressed in skeletal muscle and plays an important role in muscle energy metabolism [22,23], the molecular basis of its involvement in the pathogenesis of muscle atrophy is still unclear.
In this study, we put forward some important suggestions. First, we found that Bcat1 expression was downregulated in a model of muscle atrophy. Second, we demonstrated for the first time that Bcat1 could maintain muscle cell growth and reduce ROS production. Third, we found that Bcat1 regulated muscle cell growth and ROS production through the mTORC1/S6K1 signaling pathway. These results suggest that downregulation of Bcat1 and mTORC1/S6K1 pathway is one of the reasons for impaired cell growth in atrophic muscle cells.
BCAAs are the cornerstone of protein synthesis and energy provision [24]. However, the relative importance of the bioenergy contribution of amino acids in muscle is still unclear under both physiological and pathological conditions. BCAT1 is the first enzyme to initiate the catabolism of BCAAs. Our study provides additional evidence that downregulation of BCAT1 had adverse effects on muscle cell growth and ROS production.
Accumulating evidence suggests that regulation of mTOR is essential for cell growth. Cardiomyocyte-specific mTOR depletion led to decreased proliferation and Diagam of experimental design and tested pathway ROS production. Rapamycin inhibited mTOR signaling and blocked the effect of BCAT1 on ROS production and subsequent cell growth impairment. Because BCAT1 depletion decreased the levels of p-mTOR and pS6K1, BCAT1 expression is presumed to positively regulate mTORC1 activation. Importantly, shRNA-resistant BCAT1 cDNA and mTOR activators effectively reversed the growth impairment induced by BCAT1 knockdown and led to increased pS6K1 levels in a rapamycin-sensitive manner. In conclusion, we assigned an important role to the BCAT1/mTOR axis with regard to the pathogenesis of muscle atrophy, and provide theoretical proof for reducing muscle atrophy by regulating the BCAT1/ mTOR pathway. However, further studies are needed to explain how the catabolic enzyme BCAT1 activates mTOR in muscle cells.
increased apoptosis, resulting in a decrease in embryonic heart size [21]. However, whether BCAT1 affected the regulation of mTOR1/S6K1 signal transduction remained unclear. This study provides an insight into the roles of the BCAT1/mTOR/S6K1 pathway. Our results indicate for the first time that BCAT1 depletion significantly reduced the activation of the mTORC1/S6K1 signaling pathway in muscle cells. Therefore, we established a link between BCAT1 and muscular atrophy/sarcopenia. BCAT1 increases the mitochondrial content by activating key regulatory factors of mitochondrial biogenesis [25]. Our results suggest that BCAT1 may promote mitochondrial function and cell growth by reducing ROS production and regulating a variety of nutritional sensors, including mTOR and S6K1. Interestingly, we found that BCAT1 could activate mTOR signaling and downregulate In conclusion, we demonstrated that BCAT1 is essential for the growth of muscle cells. BCAT1 maintains muscle cell growth by activating mTOR signaling and reducing ROS production. Therefore, stable BCAT1 expression is necessary to maintain the growth of muscle cells and reduce ROS production. Moreover, BCAT1 may be a potential target for the treatment of muscle atrophy. Our observations highlight potential therapeutic targets for nutritional or pharmaceutical interventions aimed at reducing severe sarcopenia in patients with muscular atrophy. In view of the limitations faced in the current study, we have no evidence that BCAT1 regulates the mTORC1/S6K1 signaling pathway in vivo. Moreover, we have not explored other important factors or molecules related to muscle atrophy, such as inflammation and Notch signaling, among others. To to compensate for this, we plan to use the BCAT1 knockdown animal model to verify our findings and explore other effects and mechanisms of BCAT1 in muscle atrophy. Accordingly, further experiments are needed to confirm whether BCAT1 is also involved in the development of muscle atrophy in vivo, and whether changes in BCAT1 levels are associated with the progression of muscle atrophy. Finally, the results have not been verified in sarcopenia patients. We will try to use human tissues and conducting clinical research when conditions permit in the future. In all, the BCAA catabolic enzyme BCAT1 is essential for the growth of muscle cells. BCAT1 expression contributes to sustained growth of muscle cells by activating mTOR signaling and reducing ROS production.