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A novel cellular defect in diabetes: membrane repair failure.

Howard AC, McNeil AK, Xiong F, Xiong WC, McNeil PL - Diabetes (2011)

Bottom Line: Skeletal muscle myopathy is a common diabetes complication.Downhill running also resulted in a higher level of repair failure in diabetic mice.However, a repair defect could be induced, in the absence of high glucose, by enhancing AGE binding to RAGE, or simply by increasing cell exposure to AGE.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular Medicine and Genetics, Georgia Health Sciences University, Augusta, Georgia, USA.

ABSTRACT

Objective: Skeletal muscle myopathy is a common diabetes complication. One possible cause of myopathy is myocyte failure to repair contraction-generated plasma membrane injuries. Here, we test the hypothesis that diabetes induces a repair defect in skeletal muscle myocytes.

Research design and methods: Myocytes in intact muscle from type 1 (INS2(Akita+/-)) and type 2 (db/db) diabetic mice were injured with a laser and dye uptake imaged confocally to test repair efficiency. Membrane repair defects were also assessed in diabetic mice after downhill running, which induces myocyte plasma membrane disruption injuries in vivo. A cell culture model was used to investigate the role of advanced glycation end products (AGEs) and the receptor for AGE (RAGE) in development of this repair defect.

Results: Diabetic myocytes displayed significantly more dye influx after laser injury than controls, indicating a repair deficiency. Downhill running also resulted in a higher level of repair failure in diabetic mice. This repair defect was mimicked in cultured cells by prolonged exposure to high glucose. Inhibition of the formation of AGE eliminated this glucose-induced repair defect. However, a repair defect could be induced, in the absence of high glucose, by enhancing AGE binding to RAGE, or simply by increasing cell exposure to AGE.

Conclusions: Because one consequence of repair failure is rapid cell death (via necrosis), our demonstration that repair fails in diabetes suggests a new mechanism by which myopathy develops in diabetes.

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Related in: MedlinePlus

AGE/RAGE interaction is responsible for the repair defect. A: BS-C-1 cells were cultured in 30 mmol/L glucose supplemented with or without 1 mmol/L AMG (H Gluc AMG or H Gluc), or 30 mmol/L mannitol (Man) for 8 weeks. Cells were laser wounded in the presence of physiological saline containing calcium. Cells exposed to high glucose alone showed significantly increased (*) dye uptake when compared with mannitol controls. However, cells treated with high glucose and AMG did not exhibit this increase. B: RAGE-deficient fibroblasts (RAGE−/−) were cultured for 12 weeks in 30 mmol/L glucose (H Gluc), 5.5 mmol/L glucose (L Gluc), or 30 mmol/L mannitol (Man). Cells were laser wounded in the presence or absence of calcium. RAGE−/− cells exposed to high glucose and injured in the presence of calcium (H Gluc +Ca) did not exhibit a significant increase in dye uptake compared with controls (L Gluc +Ca and Man +Ca). C: BS-C-1 cells were transfected with a RAGE-EGFP expression vector (pEGFP-N1-RAGE). Cells displaying EGFP fluorescence (FL RAGE), and as controls, nonfluorescent cells in the same culture (NF), were selected for laser analysis of repair. One culture was exposed to 5 mmol/L AGE-BSA, and the other was not. Laser analysis showed membrane repair of fluorescent cells (FL RAGE) to be indistinguishable from nonfluorescent controls (NF) in cultures not exposed to AGE-BSA. However, fluorescent cells treated with AGE (FL RAGE AGE) showed a significant (**) elevation in dye uptake when compared with cells not treated with AGE, comparable to the dye uptake seen in cells wounded without calcium (–Ca). D: BS-C-1 cells were treated or not with 1 or 5 mmol/L AGE-BSA for 3 days before laser injury. Dye uptake was found to be significantly (*) greater for cells treated with AGE-BSA than cells left untreated and resembled the lack of repair for cells injured in the absence of Ca2+ (–Ca). Data are presented as the mean ± SEM. *P < 0.001 and **P < 0.05. BS-C-1: n = 14 cells for Man, n = 12 for H Gluc, and n = 12 for H Gluc AMG. Fibroblasts: n = 23 cells for L Gluc +Ca, n = 23 for H Gluc +Ca, n = 24 for Man +Ca, and n = 19 for L Gluc –Ca. Transfected: n = 12 cells for NF, n = 9 for NF AGE, n = 10 for FL RAGE, n = 10 for FL RAGE AGE, and n = 15 for –Ca. AGE treated: n = 21 cells for +Ca, n = 18 for –Ca, n = 22 for 1 mmol/L AGE, and n = 17 for 5 mmol/L AGE.
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Figure 6: AGE/RAGE interaction is responsible for the repair defect. A: BS-C-1 cells were cultured in 30 mmol/L glucose supplemented with or without 1 mmol/L AMG (H Gluc AMG or H Gluc), or 30 mmol/L mannitol (Man) for 8 weeks. Cells were laser wounded in the presence of physiological saline containing calcium. Cells exposed to high glucose alone showed significantly increased (*) dye uptake when compared with mannitol controls. However, cells treated with high glucose and AMG did not exhibit this increase. B: RAGE-deficient fibroblasts (RAGE−/−) were cultured for 12 weeks in 30 mmol/L glucose (H Gluc), 5.5 mmol/L glucose (L Gluc), or 30 mmol/L mannitol (Man). Cells were laser wounded in the presence or absence of calcium. RAGE−/− cells exposed to high glucose and injured in the presence of calcium (H Gluc +Ca) did not exhibit a significant increase in dye uptake compared with controls (L Gluc +Ca and Man +Ca). C: BS-C-1 cells were transfected with a RAGE-EGFP expression vector (pEGFP-N1-RAGE). Cells displaying EGFP fluorescence (FL RAGE), and as controls, nonfluorescent cells in the same culture (NF), were selected for laser analysis of repair. One culture was exposed to 5 mmol/L AGE-BSA, and the other was not. Laser analysis showed membrane repair of fluorescent cells (FL RAGE) to be indistinguishable from nonfluorescent controls (NF) in cultures not exposed to AGE-BSA. However, fluorescent cells treated with AGE (FL RAGE AGE) showed a significant (**) elevation in dye uptake when compared with cells not treated with AGE, comparable to the dye uptake seen in cells wounded without calcium (–Ca). D: BS-C-1 cells were treated or not with 1 or 5 mmol/L AGE-BSA for 3 days before laser injury. Dye uptake was found to be significantly (*) greater for cells treated with AGE-BSA than cells left untreated and resembled the lack of repair for cells injured in the absence of Ca2+ (–Ca). Data are presented as the mean ± SEM. *P < 0.001 and **P < 0.05. BS-C-1: n = 14 cells for Man, n = 12 for H Gluc, and n = 12 for H Gluc AMG. Fibroblasts: n = 23 cells for L Gluc +Ca, n = 23 for H Gluc +Ca, n = 24 for Man +Ca, and n = 19 for L Gluc –Ca. Transfected: n = 12 cells for NF, n = 9 for NF AGE, n = 10 for FL RAGE, n = 10 for FL RAGE AGE, and n = 15 for –Ca. AGE treated: n = 21 cells for +Ca, n = 18 for –Ca, n = 22 for 1 mmol/L AGE, and n = 17 for 5 mmol/L AGE.

Mentions: One well-established effect of elevated blood glucose is the production of AGEs, thought to contribute to the development of chronic diabetes complications (27,28). Numerous studies (29,30) have detected AGE generation in vitro as soon as 48 h after initiating high glucose incubation. To test if this AGE production is required for the development of the membrane repair defect, we simultaneously exposed BS-C-1 cells to elevated glucose and AMG, a potent inhibitor of AGE formation and accumulation (31). As expected, after 8 weeks of high glucose treatment in the absence of AMG, membrane repair was defective (Fig. 6A, H Gluc). However, this high glucose repair defect was completely eliminated by cotreatment with AMG (Fig. 6A, H Gluc AMG). Thus, glycation is implicated in the development of the membrane repair defect.


A novel cellular defect in diabetes: membrane repair failure.

Howard AC, McNeil AK, Xiong F, Xiong WC, McNeil PL - Diabetes (2011)

AGE/RAGE interaction is responsible for the repair defect. A: BS-C-1 cells were cultured in 30 mmol/L glucose supplemented with or without 1 mmol/L AMG (H Gluc AMG or H Gluc), or 30 mmol/L mannitol (Man) for 8 weeks. Cells were laser wounded in the presence of physiological saline containing calcium. Cells exposed to high glucose alone showed significantly increased (*) dye uptake when compared with mannitol controls. However, cells treated with high glucose and AMG did not exhibit this increase. B: RAGE-deficient fibroblasts (RAGE−/−) were cultured for 12 weeks in 30 mmol/L glucose (H Gluc), 5.5 mmol/L glucose (L Gluc), or 30 mmol/L mannitol (Man). Cells were laser wounded in the presence or absence of calcium. RAGE−/− cells exposed to high glucose and injured in the presence of calcium (H Gluc +Ca) did not exhibit a significant increase in dye uptake compared with controls (L Gluc +Ca and Man +Ca). C: BS-C-1 cells were transfected with a RAGE-EGFP expression vector (pEGFP-N1-RAGE). Cells displaying EGFP fluorescence (FL RAGE), and as controls, nonfluorescent cells in the same culture (NF), were selected for laser analysis of repair. One culture was exposed to 5 mmol/L AGE-BSA, and the other was not. Laser analysis showed membrane repair of fluorescent cells (FL RAGE) to be indistinguishable from nonfluorescent controls (NF) in cultures not exposed to AGE-BSA. However, fluorescent cells treated with AGE (FL RAGE AGE) showed a significant (**) elevation in dye uptake when compared with cells not treated with AGE, comparable to the dye uptake seen in cells wounded without calcium (–Ca). D: BS-C-1 cells were treated or not with 1 or 5 mmol/L AGE-BSA for 3 days before laser injury. Dye uptake was found to be significantly (*) greater for cells treated with AGE-BSA than cells left untreated and resembled the lack of repair for cells injured in the absence of Ca2+ (–Ca). Data are presented as the mean ± SEM. *P < 0.001 and **P < 0.05. BS-C-1: n = 14 cells for Man, n = 12 for H Gluc, and n = 12 for H Gluc AMG. Fibroblasts: n = 23 cells for L Gluc +Ca, n = 23 for H Gluc +Ca, n = 24 for Man +Ca, and n = 19 for L Gluc –Ca. Transfected: n = 12 cells for NF, n = 9 for NF AGE, n = 10 for FL RAGE, n = 10 for FL RAGE AGE, and n = 15 for –Ca. AGE treated: n = 21 cells for +Ca, n = 18 for –Ca, n = 22 for 1 mmol/L AGE, and n = 17 for 5 mmol/L AGE.
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Figure 6: AGE/RAGE interaction is responsible for the repair defect. A: BS-C-1 cells were cultured in 30 mmol/L glucose supplemented with or without 1 mmol/L AMG (H Gluc AMG or H Gluc), or 30 mmol/L mannitol (Man) for 8 weeks. Cells were laser wounded in the presence of physiological saline containing calcium. Cells exposed to high glucose alone showed significantly increased (*) dye uptake when compared with mannitol controls. However, cells treated with high glucose and AMG did not exhibit this increase. B: RAGE-deficient fibroblasts (RAGE−/−) were cultured for 12 weeks in 30 mmol/L glucose (H Gluc), 5.5 mmol/L glucose (L Gluc), or 30 mmol/L mannitol (Man). Cells were laser wounded in the presence or absence of calcium. RAGE−/− cells exposed to high glucose and injured in the presence of calcium (H Gluc +Ca) did not exhibit a significant increase in dye uptake compared with controls (L Gluc +Ca and Man +Ca). C: BS-C-1 cells were transfected with a RAGE-EGFP expression vector (pEGFP-N1-RAGE). Cells displaying EGFP fluorescence (FL RAGE), and as controls, nonfluorescent cells in the same culture (NF), were selected for laser analysis of repair. One culture was exposed to 5 mmol/L AGE-BSA, and the other was not. Laser analysis showed membrane repair of fluorescent cells (FL RAGE) to be indistinguishable from nonfluorescent controls (NF) in cultures not exposed to AGE-BSA. However, fluorescent cells treated with AGE (FL RAGE AGE) showed a significant (**) elevation in dye uptake when compared with cells not treated with AGE, comparable to the dye uptake seen in cells wounded without calcium (–Ca). D: BS-C-1 cells were treated or not with 1 or 5 mmol/L AGE-BSA for 3 days before laser injury. Dye uptake was found to be significantly (*) greater for cells treated with AGE-BSA than cells left untreated and resembled the lack of repair for cells injured in the absence of Ca2+ (–Ca). Data are presented as the mean ± SEM. *P < 0.001 and **P < 0.05. BS-C-1: n = 14 cells for Man, n = 12 for H Gluc, and n = 12 for H Gluc AMG. Fibroblasts: n = 23 cells for L Gluc +Ca, n = 23 for H Gluc +Ca, n = 24 for Man +Ca, and n = 19 for L Gluc –Ca. Transfected: n = 12 cells for NF, n = 9 for NF AGE, n = 10 for FL RAGE, n = 10 for FL RAGE AGE, and n = 15 for –Ca. AGE treated: n = 21 cells for +Ca, n = 18 for –Ca, n = 22 for 1 mmol/L AGE, and n = 17 for 5 mmol/L AGE.
Mentions: One well-established effect of elevated blood glucose is the production of AGEs, thought to contribute to the development of chronic diabetes complications (27,28). Numerous studies (29,30) have detected AGE generation in vitro as soon as 48 h after initiating high glucose incubation. To test if this AGE production is required for the development of the membrane repair defect, we simultaneously exposed BS-C-1 cells to elevated glucose and AMG, a potent inhibitor of AGE formation and accumulation (31). As expected, after 8 weeks of high glucose treatment in the absence of AMG, membrane repair was defective (Fig. 6A, H Gluc). However, this high glucose repair defect was completely eliminated by cotreatment with AMG (Fig. 6A, H Gluc AMG). Thus, glycation is implicated in the development of the membrane repair defect.

Bottom Line: Skeletal muscle myopathy is a common diabetes complication.Downhill running also resulted in a higher level of repair failure in diabetic mice.However, a repair defect could be induced, in the absence of high glucose, by enhancing AGE binding to RAGE, or simply by increasing cell exposure to AGE.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular Medicine and Genetics, Georgia Health Sciences University, Augusta, Georgia, USA.

ABSTRACT

Objective: Skeletal muscle myopathy is a common diabetes complication. One possible cause of myopathy is myocyte failure to repair contraction-generated plasma membrane injuries. Here, we test the hypothesis that diabetes induces a repair defect in skeletal muscle myocytes.

Research design and methods: Myocytes in intact muscle from type 1 (INS2(Akita+/-)) and type 2 (db/db) diabetic mice were injured with a laser and dye uptake imaged confocally to test repair efficiency. Membrane repair defects were also assessed in diabetic mice after downhill running, which induces myocyte plasma membrane disruption injuries in vivo. A cell culture model was used to investigate the role of advanced glycation end products (AGEs) and the receptor for AGE (RAGE) in development of this repair defect.

Results: Diabetic myocytes displayed significantly more dye influx after laser injury than controls, indicating a repair deficiency. Downhill running also resulted in a higher level of repair failure in diabetic mice. This repair defect was mimicked in cultured cells by prolonged exposure to high glucose. Inhibition of the formation of AGE eliminated this glucose-induced repair defect. However, a repair defect could be induced, in the absence of high glucose, by enhancing AGE binding to RAGE, or simply by increasing cell exposure to AGE.

Conclusions: Because one consequence of repair failure is rapid cell death (via necrosis), our demonstration that repair fails in diabetes suggests a new mechanism by which myopathy develops in diabetes.

Show MeSH
Related in: MedlinePlus