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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

Calcium-dependent repair fails in diabetic myocytes. A: A myocyte within the soleus muscle of a wild-type (Control B6, top two rows) and type 1 diabetic (Type 1 INS2, bottom two rows) mouse was imaged before (0 s) and after membrane irradiation with an infrared laser (arrow indicates injury site) in the presence of Ca2+ or its absence. FM 1–43 dye enters the myocyte through the injury site. Repair (Control B6 +Ca) impedes further dye uptake and confines the resultant fluorescence to a hot spot at the site of injury. Failed repair (Control B6 –Ca, Type 1 INS2 –Ca, and Type 1 INS2 +Ca) results in a sustained filling of the entire fiber with dye. B: Fluorescence signal was monitored over time to determine uptake of dye by myocytes from diabetic and control mice. The data presented represent five fiber measurements per soleus (Supplementary Table 1). The red circle indicates the time point where the first significant difference of P < 0.05 is observed (compared with all conditions). Data are presented as mean ± SEM. *P < 0.01; myofibers injured, n = 25 for B6 +Ca, n = 25 for B6 -Ca, n = 35 for INS2 +Ca, n = 30 for INS –Ca, n = 47 for BLKS +C, n = 45 for BLKS –Ca, n = 50 for db/db +Ca, and n = 49 for db/db –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 1: Calcium-dependent repair fails in diabetic myocytes. A: A myocyte within the soleus muscle of a wild-type (Control B6, top two rows) and type 1 diabetic (Type 1 INS2, bottom two rows) mouse was imaged before (0 s) and after membrane irradiation with an infrared laser (arrow indicates injury site) in the presence of Ca2+ or its absence. FM 1–43 dye enters the myocyte through the injury site. Repair (Control B6 +Ca) impedes further dye uptake and confines the resultant fluorescence to a hot spot at the site of injury. Failed repair (Control B6 –Ca, Type 1 INS2 –Ca, and Type 1 INS2 +Ca) results in a sustained filling of the entire fiber with dye. B: Fluorescence signal was monitored over time to determine uptake of dye by myocytes from diabetic and control mice. The data presented represent five fiber measurements per soleus (Supplementary Table 1). The red circle indicates the time point where the first significant difference of P < 0.05 is observed (compared with all conditions). Data are presented as mean ± SEM. *P < 0.01; myofibers injured, n = 25 for B6 +Ca, n = 25 for B6 -Ca, n = 35 for INS2 +Ca, n = 30 for INS –Ca, n = 47 for BLKS +C, n = 45 for BLKS –Ca, n = 50 for db/db +Ca, and n = 49 for db/db –Ca. Scale bar, 50 μm and 40× magnification. (A high-quality digital representation of this figure is available in the online issue.)

Mentions: Two diabetic mouse models, C57BL/J INS2Akita+/− (INS2Akita+/−) with type 1 diabetes and BKS Cg-Dock7m+/+ (db/db) with type 2 diabetes, were assessed using this in situ laser assay. In muscle from C57BL/6 (B6) and C57BLKS/J (BLKS) control mice, laser injured in the presence of Ca2+, a hot spot of FM 1–43 appeared at the membrane disruption site, but widespread staining of internal membrane was not observed, indicating repair had occurred (Fig. 1A, Control B6 + Ca, 445 s; and Supplementary Movie 3). However, when Ca2+ was absent, control fibers displayed continuous cytosolic filling with FM 1–43, characteristic of failed membrane repair (Fig. 1A, Control B6 – Ca). Strikingly, in both diabetic models, dye entered continuously into diabetic fibers, even when Ca2+ was present, indicating repair failed to occur (Fig. 1A, Type 1 INS2 + Ca; and Supplementary Movies 4 and 5). These results are illustrated quantitatively in Fig. 1B: all fibers initially displayed similar dye uptake kinetics in the presence and absence of Ca2+, indicating the size of the disruption made was equivalent. However, at time points thereafter (375 s for the INS2 and 175 s for the db/db), significantly more dye uptake, compared with wild-type controls, in the presence of Ca2+, was recorded in solei from both diabetic models. In fact, measured dye uptake in diabetic myocytes was indistinguishable, whether Ca2+ was present or absent. Thus, using a highly sensitive in situ assay, we show that Ca2+-dependent muscle membrane repair is deficient in both type 1 and type 2 diabetes mouse models.


A novel cellular defect in diabetes: membrane repair failure.

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

Calcium-dependent repair fails in diabetic myocytes. A: A myocyte within the soleus muscle of a wild-type (Control B6, top two rows) and type 1 diabetic (Type 1 INS2, bottom two rows) mouse was imaged before (0 s) and after membrane irradiation with an infrared laser (arrow indicates injury site) in the presence of Ca2+ or its absence. FM 1–43 dye enters the myocyte through the injury site. Repair (Control B6 +Ca) impedes further dye uptake and confines the resultant fluorescence to a hot spot at the site of injury. Failed repair (Control B6 –Ca, Type 1 INS2 –Ca, and Type 1 INS2 +Ca) results in a sustained filling of the entire fiber with dye. B: Fluorescence signal was monitored over time to determine uptake of dye by myocytes from diabetic and control mice. The data presented represent five fiber measurements per soleus (Supplementary Table 1). The red circle indicates the time point where the first significant difference of P < 0.05 is observed (compared with all conditions). Data are presented as mean ± SEM. *P < 0.01; myofibers injured, n = 25 for B6 +Ca, n = 25 for B6 -Ca, n = 35 for INS2 +Ca, n = 30 for INS –Ca, n = 47 for BLKS +C, n = 45 for BLKS –Ca, n = 50 for db/db +Ca, and n = 49 for db/db –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 1: Calcium-dependent repair fails in diabetic myocytes. A: A myocyte within the soleus muscle of a wild-type (Control B6, top two rows) and type 1 diabetic (Type 1 INS2, bottom two rows) mouse was imaged before (0 s) and after membrane irradiation with an infrared laser (arrow indicates injury site) in the presence of Ca2+ or its absence. FM 1–43 dye enters the myocyte through the injury site. Repair (Control B6 +Ca) impedes further dye uptake and confines the resultant fluorescence to a hot spot at the site of injury. Failed repair (Control B6 –Ca, Type 1 INS2 –Ca, and Type 1 INS2 +Ca) results in a sustained filling of the entire fiber with dye. B: Fluorescence signal was monitored over time to determine uptake of dye by myocytes from diabetic and control mice. The data presented represent five fiber measurements per soleus (Supplementary Table 1). The red circle indicates the time point where the first significant difference of P < 0.05 is observed (compared with all conditions). Data are presented as mean ± SEM. *P < 0.01; myofibers injured, n = 25 for B6 +Ca, n = 25 for B6 -Ca, n = 35 for INS2 +Ca, n = 30 for INS –Ca, n = 47 for BLKS +C, n = 45 for BLKS –Ca, n = 50 for db/db +Ca, and n = 49 for db/db –Ca. Scale bar, 50 μm and 40× magnification. (A high-quality digital representation of this figure is available in the online issue.)
Mentions: Two diabetic mouse models, C57BL/J INS2Akita+/− (INS2Akita+/−) with type 1 diabetes and BKS Cg-Dock7m+/+ (db/db) with type 2 diabetes, were assessed using this in situ laser assay. In muscle from C57BL/6 (B6) and C57BLKS/J (BLKS) control mice, laser injured in the presence of Ca2+, a hot spot of FM 1–43 appeared at the membrane disruption site, but widespread staining of internal membrane was not observed, indicating repair had occurred (Fig. 1A, Control B6 + Ca, 445 s; and Supplementary Movie 3). However, when Ca2+ was absent, control fibers displayed continuous cytosolic filling with FM 1–43, characteristic of failed membrane repair (Fig. 1A, Control B6 – Ca). Strikingly, in both diabetic models, dye entered continuously into diabetic fibers, even when Ca2+ was present, indicating repair failed to occur (Fig. 1A, Type 1 INS2 + Ca; and Supplementary Movies 4 and 5). These results are illustrated quantitatively in Fig. 1B: all fibers initially displayed similar dye uptake kinetics in the presence and absence of Ca2+, indicating the size of the disruption made was equivalent. However, at time points thereafter (375 s for the INS2 and 175 s for the db/db), significantly more dye uptake, compared with wild-type controls, in the presence of Ca2+, was recorded in solei from both diabetic models. In fact, measured dye uptake in diabetic myocytes was indistinguishable, whether Ca2+ was present or absent. Thus, using a highly sensitive in situ assay, we show that Ca2+-dependent muscle membrane repair is deficient in both type 1 and type 2 diabetes mouse models.

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