Impact of intracellular toxic advanced glycation end-products (TAGE) on murine myoblast cell death

Abstract

Background: Sarcopenia is a progressive condition that is characterized by decreases in skeletal muscle mass and function. Although sarcopenia is associated with lifestyle-related diseases (LSRD), the mechanisms underlying cell death in myoblasts, which differentiate to myotubes, remain unclear. We previously designated glyceraldehyde (an intermediate of glucose/fructose metabolism)-derived advanced glycation end-products (AGEs) as toxic AGEs (TAGE) because of their cytotoxicity and involvement in LSRD, and hypothesized that TAGE contribute to cell death in myoblasts. Methods: C2C12 cells, which are murine myoblasts, were treated with 0, 0.5, 1, 1.5, and 2 mM glyceraldehyde for 24 h. Cell viability and intracellular TAGE were then assessed using 5-[2,4,-bis(sodioxysulfonyl)phenyl]-3-(2-methoxy-4-nitrophenyl)-2-(4-nitrophenyl)-2H-tetrazole-3-ium (WST-8) and slot blot assays. Cells were pretreated with 8 mM aminoguanidine, an inhibitor of AGE production, for 2 h, followed by 0, 1.5, and 2 mM glyceraldehyde for 24 h. Cell viability and intracellular TAGE levels were then assessed. Serum TAGE levels in STAM mice, in which there were four stages (no steatosis, simple steatosis, steatohepatitis, and fibrosis), were measured using a competitive enzyme-linked immunosorbent assay. Results were expressed as TAGE units (U) per milliliter of serum, with 1 U corresponding to 1.0 μg of glyceraldehyde-derived AGE-bovine serum albumin (BSA) (TAGE-BSA). The viability of cells treated with 20, 50, and 100 μg/mL non-glycated BSA and TAGE-BSA for 24 h was assessed using the WST-8 assay. Results: In C2C12 cells treated with 1.5 and 2 mM glyceraldehyde, cell viability decreased to 47.7% (p = 0.0021) and 5.0% (p = 0.0001) and intracellular TAGE levels increased to 6.0 and 15.9 μg/mg protein, respectively. Changes in cell viability and TAGE production were completely inhibited by 8 mM aminoguanidine. Serum TAGE levels at the steatohepatitis and fibrosis stages were 10.51 ± 1.16 and 10.44 ± 0.95 U/mL, respectively, and were higher than those at the no steatosis stage (7.27 ± 0.18 U/mL). Cell death was not induced by 20 or 50 μg/mL TAGE-BSA. The viabilities of C2C12 cells treated with 100 μg/mL non-glycated BSA and TAGE-BSA were 105.0% (p = 0.2890) and 85.3% (p = 0.0217), respectively. Conclusion: Intracellular TAGE strongly induced cell death in C2C12 cells and may also induce myoblast cell death in LSRD model mice.

Potential of an Interorgan Network Mediated by Toxic Advanced Glycation End-Products in a Rat Model

“…Therefore, effects of intracellular TAGE in the kidney was not analyzed. However, the amount of intracellular TAGE which can induce cell death was more than 6.0 μg/mg protein in the hepatic cells, pancreatic cells, cardiomyocytes, and myoblasts cells [ 17 , 18 , 20 , 21 ]. Since if intracellular TAGE were generated in the kidney, they might not induce cell death of dramatically dysfunction.…”

“…Then, we focused the blood TAGE which may be secreted/released from organs which generated intracellular TAGE [ 15 , 21 ]. One study reported that TAGE in the blood promoted the apoptosis of human renal mesangial cells in vitro [ 11 , 39 ].…”

“…It has been shown that TAGE in the liver of patients with non-alcoholic steatohepatitis (NASH) are higher than those in the liver of patients with simple steatosis [ 14 , 15 ]. Intracellular TAGE are produced in hepatocytes [ 16 , 17 ], pancreatic cells [ 18 ], neuroblasts [ 19 ], and cardiomyocytes [ 20 ] and induce cell death in vitro [ 21 ]. Similarly, serum levels of TAGE increased in patients with LSRD, including NASH; arteriosclerosis; diabetes; CVD; and infertility [ 13 ].…”

Toxic AGEs (TAGE) theory: a new concept for preventing the development of diseases related to lifestyle

“…We postulate that non-toxic AGE structures may be physiologically relevant for preventing the potentially damaging consequences of the advanced glycation process. CEL: Nε-(carboxyethyl)lysine; MG-H1: methylglyoxal-hydroimidazolone; MOLD: methylglyoxyl-derived lysine dimer; GOLD: glyoxyl-derived lysine dimer Takeuchi Diabetol Metab Syndr (2020) 12:105 neuronal cell damage [32], but also hepatocellular damage [33][34][35] and pancreatic ductal epithelial cell damage [36], cardiomyocyte pulsation arrest and cell death [37], and myoblast cell death [38]. Therefore, TAGE are accumulate in cells, cause cell damage, and leak extracellularly into the blood, thereby increasing TAGE levels in circulating fluids.…”

“…More recently, we demonstrated that intracellular TAGE formation and accumulation induced not only neuronal cell damage [ 32 ], but also hepatocellular damage [ 33 – 35 ] and pancreatic ductal epithelial cell damage [ 36 ], cardiomyocyte pulsation arrest and cell death [ 37 ], and myoblast cell death [ 38 ]. Therefore, TAGE are accumulate in cells, cause cell damage, and leak extracellularly into the blood, thereby increasing TAGE levels in circulating fluids.…”