Intracellular toxic advanced glycation end-products (TAGE) in myoblasts may cause sarcopenia: Research article of a non-clinical study

Background: Sarcopenia is a progressive disease that is characterized by decreases in skeletal muscle mass and function. Skeletal muscle consists of myotubes that differentiated from myoblasts. Although sarcopenia is associated with non-alcoholic steatohepatitis (NASH) and type 2 diabetes mellitus (T2DM), the underlying mechanisms remain unclear. We considered that glyceraldehyde (GA), a glucose/fructose metabolism intermediate, plays a crucial role in sarcopenia. We previously designated GA-derived advanced glycation end-products (AGEs) as toxic AGEs (TAGE) because of their cytotoxicity and involvement in lifestyle-related diseases (LSRD), such as NASH and T2DM. We hypothesized that TAGE induce cytotoxicity in myoblasts. Methods: C2C12 cells, which are murine myoblasts, were treated with 0, 0.5, 1, 1.5, and 2 mM GA for 24 h, and cell viability and intracellular TAGE were measured using WST-8 and slot blot assays. Cells were pretreated with 8 mM aminoguanidine (AG), an inhibitor of AGE production, for 2 h followed by 0, 1.5, and 2 mM GA for 24 h. Cell viability and intracellular TAGE were then measured. Serum TAGE levels in STAM mice, in which there were four stages (pre-simple steatosis, simple steatosis, steatohepatitis, and brosis), were measured using an enzyme-linked immunosorbent assay. Results were expressed as TAGE units (U) per milliliter of serum, with 1 U corresponding to 1.0 μg of GA-derived AGE-bovine serum albumin (BSA) (TAGE-BSA). The viabilities of cells treated with 20 μg/mL non-glycated BSA (NG-BSA) and TAGE-BSA for 24 h were assessed using the WST-8 assay. Results: In C2C12 cells treated with 1.5 and 2 mM GA, cell viability decreased to 47.7 and 5.0% and intracellular TAGE increased to 6.0 and 15.9 μg/mg protein, respectively. Decreases in cell viability and TAGE production were completely inhibited by 8 mM AG. Serum TAGE levels at the steatohepatitis and brosis stages were 10.51 ±1.16 and 10.44±0.95 U/mL, respectively, and increased from the pre-simple steatosis stage. The viabilities of C2C12 cells treated with 20 μg/mL NG-BSA and TAGE-BSA were 99.7 and 88.3%, respectively. Conclusion: Intracellular TAGE were generated in C2C12 cells and induced cell death more strongly than extracellular TAGE. Intracellular TAGE in myoblasts may cause sarcopenia in patients with LSRD.

TAGE production were completely inhibited by 8 mM AG. Serum TAGE levels at the steatohepatitis and brosis stages were 10.51 ±1.16 and 10.44±0.95 U/mL, respectively, and increased from the pre-simple steatosis stage. The viabilities of C2C12 cells treated with 20 μg/mL NG-BSA and TAGE-BSA were 99.7 and 88.3%, respectively. Conclusion: Intracellular TAGE were generated in C2C12 cells and induced cell death more strongly than extracellular TAGE. Intracellular TAGE in myoblasts may cause sarcopenia in patients with LSRD.

Background
Skeletal muscle requires exercise, the synthesis of glycogen, and interactions with other organs, such as the liver and adipose tissue [1]. Sarcopenia is a progressive disease that is characterized by decreases in skeletal muscle mass and function, resulting in the deterioration of activities of daily living and quality of life as well as increases in fall risk and mortality [2]. One of the mechanisms contributing to the loss of skeletal muscle is the death of or dysfunctions in myoblasts because skeletal muscle consists of myotubes that differentiated from myoblasts [2][3][4][5][6]. Although accumulating evidence has shown that patients with non-alcoholic steatohepatitis (NASH) and type 2 diabetes mellitus (T2DM) have an increased risk of sarcopenia [2,[7][8][9][10], the relationships between myoblast damage and NASH and T2DM remain unclear. We hypothesized that glyceraldehyde (GA), a glucose/fructose metabolism intermediate, plays a crucial role in sarcopenia. We previously designated GA-derived advanced glycation end-products (AGEs) as toxic AGEs (TAGE) because of their cytotoxicity, and involvement in lifestyle-related diseases (LSRD), such as NASH, T2DM, cardiovascular diseases, dementia, and cancer [11][12][13][14][15]. In a clinical study, we revealed that intracellular TAGE were generated in the livers of NASH patients, and also that serum TAGE levels were elevated in patients with LSRD, such as NASH and T2DM, as well as in healthy individuals at risk of LSRD [11][12][13][14][15]. Therefore, we speculated that TAGE contribute to the development/progression of LSRD. We hypothesized that intracellular TAGE may be generated in myoblasts and induce cytotoxicity based on previous ndings showing their production in neuroblastoma cells [16], hepatic cells [17][18][19][20], pancreatic cells [21], and cardiac cells [22] as well as their induction of cell death and dysfunction. We focused on extracellular TAGE, such as TAGE in blood, which may be secreted or released from organs that generate intracellular TAGE, because they may induce cytotoxicity, such as in ammation and oxidative stress [11-13, 15, 23]. , and an anti-TAGE antibody were prepared as described previously [24].
GA and AG treatment of C2C12 cells. GA was dissolved in phosphate-buffered saline (PBS) without Ca ++ and Mg ++ ((PBS)(-)), and then ltrated before being added to C2C12 cells. All experiments were performed 24 h after the treatment with 0, 0.5, 1, 1.5, and 2 mM GA. The cell culture method before the treatment with AG, an inhibitor of AGE production, was the same as that described above. Cells were pretreated with 0 or 8 mM AG for 2 h followed by 0, 1.5, and 2 mM GA for 24 h.
Cell viability of C2C12 cells treated with GA and AG. Cell viability was assessed using the WST-8 assay.
Ten microliters of WST-8 reagent was added to 96-well microplates in which C2C12 cells were cultured in medium (100 µL), and this was followed by an incubation at 37 °C for 2 h in a CO 2 incubator. Absorbance was measured at 450 and 655 nm using a microplate reader (Bio-Rad, CA, USA). Background absorbance was measured in medium without cells and subtracted from experimental values.
Slot blot (SB) analysis. This analysis was performed as described previously with some modi cations [20][21][22]. Cells were washed with (PBS)(-), and lysed in buffer [a solution of 2 M thiourea, 7 M urea, 4% CHAPS, and 30 mM Tris, and a solution of EDTA-free protease inhibitor cocktail (9:1)]. Cell extracts were then incubated on ice for 20 min, centrifuged at 10,000 × g at 4 °C for 15 min, and the supernatant was collected as cell extracts. Protein concentrations were measured using the protein assay kit for the Bradford method with BSA as a standard. Regarding the detection of TAGE, equal amounts of cell extracts, the HRP-linked molecular marker, and TAGE-BSA were loaded onto polyvinylidene di uoride (PVDF) membranes (0.45 µm; Millipore, MA, USA) xed in the SB apparatus (Bio-Rad). PVDF membranes were cut to prepare two membranes and then blocked at room temperature (r.t.) for 1 h using 5% skimmed milk in PBS(-) containing 0.05% Tween 20 (SM-PBS-T). After this step, we used 0.5% of SM-PBS-T for washing or as the solvent of antibodies. After washing twice, membranes were incubated with (1) the anti-TAGE-antibody (1:1,000) or (2) neutralized anti-TAGE-antibody (a mixture of the anti-TAGEantibody (1:1,000) and 250 µg/mL of TAGE-BSA) at 4 °C overnight. Membranes were then washed four times. Proteins on the membrane were incubated with the HRP-linked goat anti-rabbit IgG antibody (1:2,000) at r.t. for 1 h. After washing three times with PBS-T, membranes were moved into PBS(-). Immunoreactive proteins were detected with the ImmunoStar LD kit and band densities on the membranes were measured using the Fusion FX uorescence imager (M&S Instruments Inc., Osaka, Japan). The densities of HRP-linked molecular marker bands were used to correct for differences in densities between membranes. The amount of TAGE in cell extracts was calculated based on a calibration curve for TAGE-BSA.
Analysis of serum TAGE levels in STAM mice. Brie y, each well of the 96-well microplate was coated with 1.0 µg/mL TAGE-BSA and incubated overnight in a cold room. Wells were washed three times with 0.3 mL of PBS containing 0.05% Tween 20 (PBS-T). Wells were then blocked by an incubation for 1 h with 0.2 mL of a solution of PBS containing 1% BSA. After washing with PBS-T, test samples (50 µL) were added to each well as a competitor for 50 µL of the anti-TAGE antibody (1:1,000), followed by an incubation at r.t. for 2 h with gentle shaking on a horizontal rotary shaker. Wells were then washed with PBS-T and developed with alkaline phosphatase-linked anti-rabbit IgG utilizing p-nitrophenyl phosphate as the colorimetric substrate. Results were expressed as TAGE units (U) per milliliter of serum, with 1 U corresponding to 1.0 µg of a TAGE-BSA standard as described previously [24]. Sensitivity and intra-and interassay coe cients of variation were 0.01 U/mL and 6.2 and 8.8%, respectively [25].
NG-BSA and TAGE-BSA treatment of C2C12 cells and measure of cell viability. In total, 20 µg/mL of NG-BSA and TAGE-BSA were added to C2C12 cells, which were then incubated for 24 h. Cell viability was measured using the WST-8 assay. The ratio of cell viability was calculated based on the viability of cells treated with TAGE-BSA versus those treated with NG-BSA.
Statistical analysis. Stat Flex (ver. 6) software (Artech Co., Ltd., Osaka, Japan) was used for statistical analyses. Data were expressed as means ± S.D. When statistical analyses were performed on data, signi cant differences in the means of each group were assessed by a one-way analysis of variance (ANOVA). We then used Tukey's test for an analysis of variance. P-values < 0.05 were considered to be signi cant.

Results
Viability of C2C12 cells treated with GA. The viability of C2C12 cells treated with 0.5 and 1 mM GA did not decrease, whereas dose-dependent decreases to 47.7 and 5.0% were observed in those treated with 1.5 and 2 mM GA, respectively (Fig. 1a).
Quantity of intracellular TAGE in C2C12 cells treated with GA. Intracellular TAGE were not generated in C2C12 cells treated with 0, 0.5, and 1 mM GA (Fig. 1b). Intracellular TAGE dose-dependently increased to 6.0 and 15.9 µg/mg protein in C2C12 cells treated with 1.5 and 2 mM GA, respectively (Fig. 1b).
Effects of the AG pretreatment on the viability of C2C12 cells treated with GA. The viability of C2C12 cells treated with 1.5 and 2 mM GA without AG dose-dependently decreased to 35.0 and 3.0%, respectively (Fig. 1c). In C2C12 cells pretreated with 8 mM AG, cell viabilities were 71.7, 71.3, and 74.3% in those subsequently treated with 0, 1.5, and 2 mM GA, respectively. No signi cant differences were observed between each treatment (Fig. 1c). The AG pretreatment completely inhibited decreases in the viability of C2C12 cells treated with 1.5 and 2 mM GA.
Effects of the AG pretreatment on the quantity of intracellular TAGE in C2C12 cells treated with GA. GA concentrations of 1.5 and 2 mM without AG dose-dependently increased intracellular TAGE to 7.9 and 13.4 µg/mg protein, respectively (Fig. 1d). Intracellular TAGE levels in C2C12 cells pretreated with 0 mM AG followed by 0 mM GA and in those pretreated with 8 mM AG followed by 0, 1.5, and 2 mM GA were not signi cantly different. The AG pretreatment completely inhibited the generation of intracellular TAGE in C2C12 cells treated with 1.5 and 2 mM GA.
Serum TAGE levels in STAM mice. Serum TAGE levels in the pre-simple steatosis and simple steatosis stage groups were 7.27 ± 0.18 and 8.69 ± 1.01 U/mL, respectively (Fig. 2a). Serum TAGE levels in the steatohepatitis and brosis stage groups increased to 10.51 ± 1.16 and 10.44 ± 0.95 U/mL, which were higher than that in the pre-simple steatosis stage group.
Viability of C2C12 cells treated with NG-BSA and TAGE-BSA. The viabilities of C2C12 cells treated with 20 µg/mL NG-BSA and TAGE-BSA were 99.7 and 88.3%, respectively, and the ratio of cell viability was 88.6% (Fig. 2b).
GA, which is a precursor of TAGE, is generated by the liver via three pathways [11,15,23]. (1) Glucose is metabolized to GA via glycolysis. (2) Fructose is metabolized to GA via the pathway involving fructokinase and aldolase B (fructolysis). (3) Glucose is metabolized to fructose via the sorbitol pathway, which regulates aldose reductase and sorbitol dehydrogenase, and this fructose is metabolized to GA via fructolysis. Since skeletal muscle uses glycolysis and has fructokinase [26], aldolase B [26], aldose reductase [27], and sorbitol dehydrogenase [28], we considered the three pathways of GA metabolism to occur in this tissue, similar to the liver.
We hypothesized that intracellular TAGE may be generated in and damage myoblasts cells because they were previously shown to be produced in neuroblastoma cells [16], hepatic cells [17][18][19][20], pancreatic cells [21], and cardiac cells [22], and induced cell death.
We treated C2C12 cells with GA to rapidly generate intracellular TAGE. In the present study, C2C12 cells were treated with GA at a physiological concentration to generate TAGE within 24 h. Taniguchi et al. previously demonstrated that islets of the pancreas exposed to 20 mM glucose accumulated 0.025 pmol/islet GA, whereas exposure to 10 mM GA caused the accumulation of 0.12 pmol/islet GA [29]. Based on these ndings, Takahashi et al. used 2 mM GA in their experiments, which is a similar concentration to those using 20 mM glucose [30]. On the other hand, in NASH and T2DM model mice, their plasma levels of glucose increased by more than 25 mM [31][32][33]. The viability of C2C12 cells treated with 1.5 and 2 mM GA for 24 h dose-dependently decreased (Fig. 1a). In contrast, intracellular TAGE were generated in a dose-dependent manner. (Fig. 1b). To demonstrate that the generation of TAGE decreased cell viability, C2C12 cells were pretreated with 8 mM AG, an inhibitor of the generation of AGEs, for 2 h followed by 1.5 and 2 mM GA for 24 h. AG inhibited decreases in cell viability as well as the generation of TAGE (Fig. 1c, d). To the best of our knowledge, this is the rst study to show that intracellular TAGE were generated from GA at a physiological concentration in myoblasts, and strongly induced cell death. The death of myoblasts will lead to the loss of skeletal muscle. Living myoblasts that generate intracellular TAGE may also lead to the loss of skeletal muscle. In our previous study, when rat primary cardiomyocytes were treated with 4 mM GA for 6 h, cell viability decreased to 39.2% and intracellular TAGE were generated to 12.0 µg/mg protein [22]. Furthermore, living cardiomyocytes completely stopped beating. The viability of C2C12 cells treated with 1.5 mM GA was 47.7% and living cells generated intracellular TAGE levels of 6.0 µg/mg protein (Fig. 1a, b). The generation of skeletal muscle may be inhibited in myotubes with dysfunctional differentiation [2][3][4][5][6]. Collectively, these ndings Declarations Ethics approval and consent to particle Not applicable.

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Availability of data and materials
The datasets used and/or analyzed during the present study are available from the corresponding author upon reasonable request.
Competing interests and the present results suggest that cell death or dysfunction of myoblasts that gain excess glucose or fructose and generate high levels of intracellular TAGE may inhibit the differentiation of myoblasts.
In STAM mice, which is a NASH model, serum TAGE levels were higher in the steatohepatitis and brosis stages than in the pre-simple steatosis stage (Fig. 2a). We previously reported that serum TAGE levels were higher in NASH patients than in healthy individuals [11][12][13][14][15]. To the best of our knowledge, this is the rst study to show that serum TAGE levels in NASH model mice were similar to those in NASH patients. Since TAGE in blood induces responses, such as in ammation and oxidative stress, in some organs [11-13, 15, 23], we investigated the cytotoxicity of TAGE-BSA, a model of extracellular TAGE, in myoblasts. We applied 20 µg/mL TAGE-BSA, which is approximately 2-fold that of serum TAGE levels in STAM mice that develop steatohepatitis and brosis (Fig. 2). To examine the effects of TAGE in C2C12 cells, we assessed the viability of C2C12 cells treated with both NG-BSA and TAGE-BSA (Fig. 2b). The effects of 20 µg/mL TAGE-BSA on cell viability were markedly weaker than those of intracellular TAGE generated from GA at a physiological concentration (Figs. 1a, c, and 2b).

Conclusion
The present study demonstrated that intracellular TAGE were generated in C2C12 cells and induced cell death more strongly than extracellular TAGE. These results suggest that intracellular TAGE induce the death of myoblasts and inhibit the generation of myotubes, which leads to the loss of skeletal muscle and, ultimately, sarcopenia in individuals with LSRD, such as NASH and T2DM.