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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

Myoblasts cultured in high glucose medium for prolonged periods display impaired membrane repair. A: C2C12 myoblasts were cultured in medium with 7.5 mmol/L glucose (N Gluc), 30 mmol/L glucose (H Gluc), or iso-osmolar control 30 mmol/L mannitol (Man) and then subject to laser analysis of repair (arrows indicate injury site) at 1 or 8 weeks of exposure. The interval (seconds) postinjury is indicated. Only cells grown in 30 mmol/L glucose for 8 weeks (H Gluc +Ca) were observed to fill with dye after laser injury in the presence of calcium. B: Dye uptake, fluorescence, measured within cells over time after injury. After 1 week of exposure to high glucose (left panel), all cells displayed calcium-dependent repair with no significant difference in dye uptake between groups. By contrast, cells cultured for 8 weeks in high glucose (right panel) showed a significant increase in dye uptake compared with normal glucose and mannitol controls. Data are presented as the mean ± SEM. *P < 0.05; 1 week, n = 15 for N Gluc +Ca, n = 14 for H Gluc +Ca, n = 13 for Man +Ca, and n = 17 for N Gluc –Ca; 8 weeks, n = 26 for N Gluc +Ca, n = 30 for H Gluc +Ca, n = 28 for Man +Ca, and n = 15 for N Gluc –Ca. Scale bar, 50 μm and 40× magnification. (A high-quality digital representation of this figure is available in the online issue.)
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Figure 3: Myoblasts cultured in high glucose medium for prolonged periods display impaired membrane repair. A: C2C12 myoblasts were cultured in medium with 7.5 mmol/L glucose (N Gluc), 30 mmol/L glucose (H Gluc), or iso-osmolar control 30 mmol/L mannitol (Man) and then subject to laser analysis of repair (arrows indicate injury site) at 1 or 8 weeks of exposure. The interval (seconds) postinjury is indicated. Only cells grown in 30 mmol/L glucose for 8 weeks (H Gluc +Ca) were observed to fill with dye after laser injury in the presence of calcium. B: Dye uptake, fluorescence, measured within cells over time after injury. After 1 week of exposure to high glucose (left panel), all cells displayed calcium-dependent repair with no significant difference in dye uptake between groups. By contrast, cells cultured for 8 weeks in high glucose (right panel) showed a significant increase in dye uptake compared with normal glucose and mannitol controls. Data are presented as the mean ± SEM. *P < 0.05; 1 week, n = 15 for N Gluc +Ca, n = 14 for H Gluc +Ca, n = 13 for Man +Ca, and n = 17 for N Gluc –Ca; 8 weeks, n = 26 for N Gluc +Ca, n = 30 for H Gluc +Ca, n = 28 for Man +Ca, and n = 15 for N Gluc –Ca. Scale bar, 50 μm and 40× magnification. (A high-quality digital representation of this figure is available in the online issue.)

Mentions: C2C12 cells, a skeletal muscle myocyte culture model, were assessed for membrane repair after growth for 1–8 weeks in medium containing normal glucose (7.5 mmol/L), elevated glucose (30 mmol/L), or mannitol (30 mmol/L). After 1 week of elevated glucose exposure, there was no qualitative change in repair (for example, dye entry pattern) (Fig. 3A, 1 week “+Ca” all conditions; and Supplementary Movie 6). However, after 8 weeks of high glucose exposure, dye entry after laser injury was clearly elevated, relative to the normal glucose and mannitol controls (Fig. 3A, 8 weeks N Gluc +Ca and Man +Ca), indicating an inability to repair (Fig. 3A, 8 weeks H Gluc +Ca; and Supplementary Movie 7). These observations are confirmed by quantitative analysis (Fig. 3B). Thus, prolonged exposure to high glucose is required for inducing a repair defect, a finding that is not compatible with other short-term responses, such as induction of insulin resistance.


A novel cellular defect in diabetes: membrane repair failure.

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

Myoblasts cultured in high glucose medium for prolonged periods display impaired membrane repair. A: C2C12 myoblasts were cultured in medium with 7.5 mmol/L glucose (N Gluc), 30 mmol/L glucose (H Gluc), or iso-osmolar control 30 mmol/L mannitol (Man) and then subject to laser analysis of repair (arrows indicate injury site) at 1 or 8 weeks of exposure. The interval (seconds) postinjury is indicated. Only cells grown in 30 mmol/L glucose for 8 weeks (H Gluc +Ca) were observed to fill with dye after laser injury in the presence of calcium. B: Dye uptake, fluorescence, measured within cells over time after injury. After 1 week of exposure to high glucose (left panel), all cells displayed calcium-dependent repair with no significant difference in dye uptake between groups. By contrast, cells cultured for 8 weeks in high glucose (right panel) showed a significant increase in dye uptake compared with normal glucose and mannitol controls. Data are presented as the mean ± SEM. *P < 0.05; 1 week, n = 15 for N Gluc +Ca, n = 14 for H Gluc +Ca, n = 13 for Man +Ca, and n = 17 for N Gluc –Ca; 8 weeks, n = 26 for N Gluc +Ca, n = 30 for H Gluc +Ca, n = 28 for Man +Ca, and n = 15 for N Gluc –Ca. Scale bar, 50 μm and 40× magnification. (A high-quality digital representation of this figure is available in the online issue.)
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Figure 3: Myoblasts cultured in high glucose medium for prolonged periods display impaired membrane repair. A: C2C12 myoblasts were cultured in medium with 7.5 mmol/L glucose (N Gluc), 30 mmol/L glucose (H Gluc), or iso-osmolar control 30 mmol/L mannitol (Man) and then subject to laser analysis of repair (arrows indicate injury site) at 1 or 8 weeks of exposure. The interval (seconds) postinjury is indicated. Only cells grown in 30 mmol/L glucose for 8 weeks (H Gluc +Ca) were observed to fill with dye after laser injury in the presence of calcium. B: Dye uptake, fluorescence, measured within cells over time after injury. After 1 week of exposure to high glucose (left panel), all cells displayed calcium-dependent repair with no significant difference in dye uptake between groups. By contrast, cells cultured for 8 weeks in high glucose (right panel) showed a significant increase in dye uptake compared with normal glucose and mannitol controls. Data are presented as the mean ± SEM. *P < 0.05; 1 week, n = 15 for N Gluc +Ca, n = 14 for H Gluc +Ca, n = 13 for Man +Ca, and n = 17 for N Gluc –Ca; 8 weeks, n = 26 for N Gluc +Ca, n = 30 for H Gluc +Ca, n = 28 for Man +Ca, and n = 15 for N Gluc –Ca. Scale bar, 50 μm and 40× magnification. (A high-quality digital representation of this figure is available in the online issue.)
Mentions: C2C12 cells, a skeletal muscle myocyte culture model, were assessed for membrane repair after growth for 1–8 weeks in medium containing normal glucose (7.5 mmol/L), elevated glucose (30 mmol/L), or mannitol (30 mmol/L). After 1 week of elevated glucose exposure, there was no qualitative change in repair (for example, dye entry pattern) (Fig. 3A, 1 week “+Ca” all conditions; and Supplementary Movie 6). However, after 8 weeks of high glucose exposure, dye entry after laser injury was clearly elevated, relative to the normal glucose and mannitol controls (Fig. 3A, 8 weeks N Gluc +Ca and Man +Ca), indicating an inability to repair (Fig. 3A, 8 weeks H Gluc +Ca; and Supplementary Movie 7). These observations are confirmed by quantitative analysis (Fig. 3B). Thus, prolonged exposure to high glucose is required for inducing a repair defect, a finding that is not compatible with other short-term responses, such as induction of insulin resistance.

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