scholarly journals Impact of intracellular toxic advanced glycation end-products (TAGE) on murine myoblast cell death: A non-clinical study

2020 ◽  
Author(s):  
Takanobu Takata ◽  
Akiko Sakasai-Sakai ◽  
Masayoshi Takeuchi
Keyword(s):  
Cell Death ◽  
Cell Viability ◽  
Advanced Glycation End Products ◽  
C2c12 Cells ◽  
Enzyme Linked Immunosorbent Assay ◽  
Advanced Glycation ◽  
Glycation End Products ◽  
End Products ◽  
Underlying Mechanisms ◽  
Myoblast Cell

Abstract Background: Sarcopenia is a progressive condition that is characterized by decreases in skeletal muscle mass and function. Although previous studies reported that sarcopenia is associated with non-alcoholic steatohepatitis (NASH), the underlying mechanisms remain unclear. We herein focused on glyceraldehyde, an intermediate of glucose/fructose metabolism. We previously designated glyceraldehyde-derived advanced glycation end-products (AGEs) as toxic AGEs (TAGE) because of their cytotoxicity and involvement in lifestyle-related diseases, such as NASH. We hypothesized that TAGE is associated with sarcopenia. 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 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 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 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 that 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 induce myoblast cell death and may cause sarcopenia. Therefore, TAGE may play a crucial role in sarcopenia associated with NASH .

scholarly journals Impact of intracellular toxic advanced glycation end-products (TAGE) on murine myoblast cell death:A non-clinical study

2020 ◽  
Author(s):  
Takanobu Takata ◽  
Akiko Sakasai-Sakai ◽  
Masayoshi Takeuchi
Keyword(s):  
Cell Death ◽  
Cell Viability ◽  
Advanced Glycation End Products ◽  
C2c12 Cells ◽  
Enzyme Linked Immunosorbent Assay ◽  
Advanced Glycation ◽  
Glycation End Products ◽  
End Products ◽  
Myoblast Cell ◽  
And Function

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.

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

2020 ◽  
Author(s):  
Takanobu Takata ◽  
Akiko Sakasai-Sakai ◽  
Masayoshi Takeuchi
Keyword(s):  
Skeletal Muscle ◽  
Cell Viability ◽  
Advanced Glycation End Products ◽  
C2c12 Cells ◽  
Enzyme Linked Immunosorbent Assay ◽  
Advanced Glycation ◽  
Simple Steatosis ◽  
Glycation End Products ◽  
End Products ◽  
Underlying Mechanisms

Abstract 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 fibrosis), 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 fibrosis 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.

scholarly journals Endogenous Secretory Receptor for Advanced Glycation End Products Protects Endothelial Cells from AGEs Induced Apoptosis

2018 ◽  
Vol 2018 ◽  
pp. 1-8 ◽  
Author(s):  
Guomin Yang ◽  
Yinqiong Huang ◽  
Xiaohong Wu ◽  
Xiahong Lin ◽  
Jinting Xu ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Advanced Glycation End Products ◽  
Proinflammatory Cytokine ◽  
Enzyme Linked Immunosorbent Assay ◽  
Advanced Glycation ◽  
Expression Levels ◽  
Qrt Pcr ◽  
Glycation End Products ◽  
End Products

Endogenous secretory receptor for advanced glycation end products (esRAGE) binds extracellular RAGE ligands and blocks RAGE activation on the cell surface, protecting endothelial cell function. However, the underlying mechanism remains unclear. Endothelial cells overexpressing the esRAGE gene were generated using a lentiviral vector. Then, quantitative real-time polymerase chain reaction (qRT-PCR) and enzyme-linked immunosorbent assay (ELISA) were used to assess esRAGE mRNA and protein levels, respectively. Hoechst-PI double staining was used to assess apoptosis. Western blot and qRT-PCR were used to assess the expression levels of apoptosis-related factors and the proinflammatory cytokine NF-кB. Compared with the control group, AGEs significantly induced endothelial cell apoptosis, which was significantly reduced by esRAGE overexpression. Incubation with AGEs upregulated the proapoptotic factor Bax and downregulated the antiapoptotic factor Bcl-2. Overexpression of esRAGE reduced Bax expression induced by AGEs and increased Bcl-2 levels. Furthermore, AGEs increased the expression levels of proinflammatory cytokine NF-кB, which were reduced after esRAGE overexpression. esRAGE protects endothelial cells from AGEs associated apoptosis, by downregulating proapoptotic (Bax) and inflammatory (NF-кB) factors and upregulating the antiapoptotic factor Bcl-2.

scholarly journals Relevance of Receptor for Advanced Glycation end Products (RAGE) in Murine Antibody-Mediated Autoimmune Diseases

2019 ◽  
Vol 20 (13) ◽  
pp. 3234
Author(s):  
Alexandra Eichhorst ◽  
Christoph Daniel ◽  
Rita Rzepka ◽  
Bettina Sehnert ◽  
Falk Nimmerjahn ◽  
...  
Keyword(s):  
B Cells ◽  
Autoimmune Diseases ◽  
Advanced Glycation End Products ◽  
Plasma Cells ◽  
Inflammatory Responses ◽  
Joint Inflammation ◽  
Enzyme Linked Immunosorbent Assay ◽  
Advanced Glycation ◽  
Glycation End Products ◽  
End Products

It is incompletely understood how self-antigens become targets of humoral immunity in antibody-mediated autoimmune diseases. In this context, alarmins are discussed as an important level of regulation. Alarmins are recognized by various receptors, such as receptor for advanced glycation end products (RAGE). As RAGE is upregulated under inflammatory conditions, strongly binds nucleic acids and mediates pro-inflammatory responses upon alarmin recognition, our aim was to examine its contribution to immune complex-mediated autoimmune diseases. This question was addressed employing RAGE−/− animals in murine models of pristane-induced lupus, collagen-induced, and serum-transfer arthritis. Autoantibodies were assessed by enzyme-linked immunosorbent assay, renal disease by quantification of proteinuria and histology, arthritis by scoring joint inflammation. The associated immune status was determined by flow cytometry. In both disease entities, we detected tendentiously decreased autoantibody levels in RAGE−/− mice, however no differences in clinical outcome. In accordance with autoantibody levels, a subgroup of the RAGE−/− animals showed a decrease in plasma cells, and germinal center B cells and an increase in follicular B cells. Based on our results, we suggest that RAGE deficiency alone does not significantly affect antibody-mediated autoimmunity. RAGE may rather exert its effects along with other receptors linking environmental factors to auto-reactive immune responses.

scholarly journals Elevated level of the soluble receptor for advanced glycation end-products involved in sarcopenia: an observational study

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Shou-En Wu ◽  
Yi-Lin Chiu ◽  
Tung-Wei Kao ◽  
Wei-Liang Chen
Keyword(s):  
Muscle Mass ◽  
Advanced Glycation End Products ◽  
Community Dwelling ◽  
Enzyme Linked Immunosorbent Assay ◽  
Health Examination ◽  
Soluble Receptor ◽  
Advanced Glycation ◽  
Srage Level ◽  
Glycation End Products ◽  
End Products

Abstract Background The soluble receptor for advanced glycation end products (sRAGE) has been proposed to serve as a marker for disease severity, but its role in sarcopenia, an age-related progressive loss of muscle mass and function, remains elusive. This study examines the association between sRAGE and sarcopenia. Methods A total of 314 community-dwelling elderly adults who had their health examination at Tri-Service General Hospital from 2017 to 2019 underwent protein analysis with enzyme-linked immunosorbent assay. The relationship with sarcopenia and its detailed information, including components and diagnosis status, were examined using linear and logistic regressions. Results As for sarcopenia components, low muscle mass (β = 162.8, p = 0.012) and strength (β = 181.31, p = 0.011) were significantly correlated with sRAGE, but not low gait speed (p = 0.066). With regard to disease status, confirmed sarcopenia (β = 436.93, p < 0.001), but not probable (p = 0.448) or severe sarcopenia (p = 0.488), was significantly correlated with sRAGE. In addition, females revealed a stronger association with sRAGE level by showing significant correlations with low muscle mass (β = 221.72, p = 0.014) and low muscle strength (β = 208.68, p = 0.043). Conclusions sRAGE level showed a positive association with sarcopenia, illustrating its involvement in the evolution of sarcopenia. This association is more evident in female groups, which may be attributed to the loss of protection from estrogen in postmenopausal women. Utilizing sRAGE level as a prospective marker for sarcopenia deserves further investigation in future studies.

scholarly journals Advanced glycation end products increased placental vascular permeability of human BeWo cells via RAGE/NF-kB signaling pathway

2020 ◽  
Author(s):  
Yuehua Shi ◽  
Jie Qian ◽  
Qinfen Zhang ◽  
Yan Hu ◽  
Dongdong Sun ◽  
...  
Keyword(s):  
Vascular Permeability ◽  
Signaling Pathway ◽  
Advanced Glycation End Products ◽  
Cell Junction ◽  
Mrna Levels ◽  
Advanced Glycation ◽  
Bewo Cells ◽  
Glycation End Products ◽  
End Products ◽  
Underlying Mechanisms

Abstract Background: The placental barrier plays an important role in protecting the fetus from the maternal environment. Gestational diabetes mellitus (GDM), caused by placental permeability, is a serious pregnancy complication with a high incidence worldwide. This study aimed to investigate the effects of advanced glycation end products (AGEs) on cell tight conjunction, and explore underlying mechanisms of the dys-regulation of placental vascular permeability in BeWo cells. Methods: Monolayer permeability assay was employed to reveal the transformation of the placental vascular permeability in BeWo cells under different administrations. Transmission electron microscopy was utilized to evaluate cell tight conjunction of BeWo cells. Immunofluorescence, wetern blot and RT-qPCR were adopted to determine the protein and mRNA levels of ZO-1, Occludin, RAGE and NF-kB. In addition, anti-RAGE and NF-kB inhibitor (PDTC) were used to inactivate the RAGE/NF-kB signaling pathway. Results: AGEs significantly decreased trans-epithelial electrical resistance (TEER), while increased paracellular permeability (P < 0.05). TEM results showed that AGEs made the cell junction loose. Immunofluorescence, wetern blot and RT-qPCR analyses showed that AGEs significantly inhibited the expressions of ZO-1 and Occludin, while anti-RAGE or PDTC partially restored their levels. Besides, AGEs also significantly increased the expressions of mRNA RAGE and NF-kB in BeWo cells (P < 0.05), and their expressions were inhibited by anti-RAGEy or PDTC. Conclusion: AGEs could reduce the expressions of ZO-1 and Occludin by activating RAGE/NF-kB signaling pathway, thus increasing placental vascular permeability of BeWos cells. These findings would provide a novel sight for improving the development and prognosis of GDM.

scholarly journals An Inducible Model for Unraveling the Effects of Advanced Glycation End-Products in Collagen with Age and Diabetes

2020 ◽  
Author(s):  
Austin G. Gouldin ◽  
Jennifer L. Puetzer
Keyword(s):  
Cell Viability ◽  
Blue Light ◽  
Advanced Glycation End Products ◽  
Advanced Glycation ◽  
Cell Matrix ◽  
Matrix Interactions ◽  
Glycation End Products ◽  
End Products ◽  
Cell Matrix Interactions

AbstractIn connective tissues there is a clear link between increasing age and degeneration. It is believed advanced glycation end-products (AGEs) play a central role in this degeneration. AGEs are sugar induced non-enzymatic crosslinks which accumulate in collagen with age and diabetes, altering tissue mechanics and cellular function. Despite ample correlative evidence linking collagen glycation to degeneration, little is known how AGEs impact cell-matrix interactions, limiting therapeutic options. One reason for this limited understanding is AGEs are typically induced in vitro using high concentrations of ribose which decrease cell viability and make it impossible to investigate cell-matrix interactions. The objective of this study was to develop a system to trigger AGE accumulation while maintaining cell viability. Using cell-seeded high density collagen gels, we investigated the effect of two different systems for AGE induction, ribose at low concentrations (30, 100, and 200 mM) over 15 days of culture and riboflavin (0.25 mM and 0.75mM) induced with blue light for 40 seconds. We found ribose and riboflavin with blue light are capable of producing a wide range of AGE crosslinks which match and/or exceed reported human AGE levels for various tissues, ages, and diseases, without affecting cell viability and metabolism. Interestingly, a single 40 second treatment of riboflavin and blue light produced similar levels of AGEs as 3 days of 100 mM ribose treatment and matched aged mouse tendon AGE levels. This riboflavin treatment option is an exciting means to trigger AGE crosslinks on demand in vivo or in vitro without impacting cell metabolism or viability and holds great promise for further unraveling the mechanism of AGEs in age and diabetes related tissue degeneration.

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