Mitochondrial dysfunction results from oxidative stress in the skeletal muscle of diet-induced insulin-resistant mice

Abstract

Mitochondrial dysfunction in skeletal muscle has been implicated in the development of type 2 diabetes. However, whether these changes are a cause or a consequence of insulin resistance is not clear. We investigated the structure and function of muscle mitochondria during the development of insulin resistance and progression to diabetes in mice fed a high-fat, high-sucrose diet. Although 1 month of high-fat, high-sucrose diet feeding was sufficient to induce glucose intolerance, mice showed no evidence of mitochondrial dysfunction at this stage. However, an extended diet intervention induced a diabetic state in which we observed altered mitochondrial biogenesis, structure, and function in muscle tissue. We assessed the role of oxidative stress in the development of these mitochondrial abnormalities and found that diet-induced diabetic mice had an increase in ROS production in skeletal muscle. In addition, ROS production was associated with mitochondrial alterations in the muscle of hyperglycemic streptozotocin-treated mice, and normalization of glycemia or antioxidant treatment decreased muscle ROS production and restored mitochondrial integrity. Glucose- or lipid-induced ROS production resulted in mitochondrial alterations in muscle cells in vitro, and these effects were blocked by antioxidant treatment. These data suggest that mitochondrial alterations do not precede the onset of insulin resistance and result from increased ROS production in muscle in diet-induced diabetic mice.

Figure 1. Decreased mitochondrial density in the skeletal muscle of mice fed the HFHSD for 16 weeks.

(A) mtDNA quantity calculated as the ratio of COX1 to cyclophilin A DNA levels determined by real-time PCR in the skeletal muscle of mice after 4 and 16 weeks of the SD and HFHSD (n = 6). Note that the scale of the y axis is between 0.5 and 0.8. (B) mRNA expression of mitochondria-encoded COX1 and COX3 genes determined by quantitative RT-PCR in the skeletal muscle of mice fed the HFHSD for 16 weeks (n = 6). Results were normalized by the mean value for the SD mice at 16 weeks set to 1 unit. (C) Mitochondrial density assessed by electron microscopy in the skeletal muscle of mice after 4 and 16 weeks of the SD or HFHSD. Original magnification, ×25,000. (D) Quantification of subsarcolemmal and intermyofibrillar mitochondria number per image area in the gastrocnemius muscle of mice after 16 weeks of the HFHSD (analysis of 5 images in 3 mice per group). Results were normalized by the mean value for the SD mice at 16 weeks set to 1 unit. (E) CS activity in mitochondria isolated from the gastrocnemius muscle after 4 and 16 weeks of the SD or HFHSD (n = 6). *P < 0.05, **P < 0.01 vs. SD; ^$P < 0.05 vs. SD at 4 weeks.

Figure 2. Expression of genes implicated in mitochondrial biogenesis and in mtDNA replication.

mRNA levels of mitochondrial biogenesis (A) and mtDNA replication (B) genes, determined by quantitative RT-PCR, in the gastrocnemius muscle of the mice after 4 and 16 weeks of the SD or HFHSD (n = 6). Results are expressed as fold change versus the SD diet set to 1 unit (dotted line). *P < 0.05.

Figure 3. Alterations in the mitochondrial structure of skeletal muscle of mice fed the HFHSD for 16 weeks.

(A and B) Transmission electron microscopy images at original magnifications of ×25,000 (A) and ×100,000 (B) in subsarcolemmal and intermyofibrillar mitochondria from the gastrocnemius muscle of mice after 16 weeks of the SD or HFHSD. (C) Quantification of subsarcolemmal and intermyofibrillar mitochondria area in the gastrocnemius muscle of mice fed the HFHSD for 16 weeks (analysis of 5 images in 3 mice per group). Results were normalized by the mean value for the SD mice at 16 weeks set to 1 unit. *P < 0.05, **P < 0.01 vs. SD.

Figure 4. Chronic HFHSD feeding induces oxidative stress in skeletal muscle.

(A) Immunoblots showing total protein carbonylation in the gastrocnemius muscle of mice after 4 and 16 weeks of the SD and HFHSD. (B) mRNA levels of oxidant stress–related genes determined by real-time RT-PCR in the gastrocnemius muscle of mice after 4 and 16 weeks of the SD and HFHSD (n = 6). Results are expressed as fold change versus the SD diet set to 1 unit (dotted line). *P < 0.05. (C) Immunoblot showing cytochrome c protein in the mitochondrial fraction (MF) and cytosolic fraction (CF) of the gastrocnemius muscle of mice after 4 and 16 weeks of the SD and HFHSD. (D) Caspase 3 activity measured in the gastrocnemius muscle of mice after 4 and 16 weeks of the HFHSD (n = 6). Results were normalized by the mean value for the SD mice at 4 and 16 weeks. *P < 0.05. UCP, uncoupling protein; GSR, glutathione reductase; GPx, glutathione peroxidase; CAT, catalase; SOD, superoxide dismutase; Prdx, peroxiredoxin.

Figure 5. STZ-induced oxidative stress alters mitochondria density and structure in skeletal muscle.

(A) Immunoblots showing total protein carbonylation in the gastrocnemius muscle of control (Co), STZ, and insulin-treated STZ (STZ+INS) mice. (B) Immunoblot showing cytochrome c protein in the MF and CF of the gastrocnemius muscle of control, STZ, and insulin-treated STZ mice. (C) mtDNA copy number was calculated as the ratio of COX1 to cyclophilin A DNA levels, determined by real-time PCR, in the skeletal muscle of control, STZ, insulin-treatd STZ, and phlorizin-treated STZ (STZ+PHL) mice (n = 6). Note that the y axis scale is between 0.5 and 1. Results were normalized by the mean value for the control mice set to 1 unit. *P < 0.01 vs. control; ^#P < 0.05 vs. STZ. (D) Transmission electron microscopy images (original magnification, ×25,000) of subsarcolemmal and intermyofibrillar mitochondria from the gastrocnemius muscle of control, STZ, insulin-treated STZ, and phlorizin-treated STZ mice.

Figure 6. Antioxidant treatment restores mitochondrial alterations in STZ mice.

(A) Plasma H₂O₂ levels in Co and STZ mice treated or not with NAC (10 mM in drinking water). (B) Immunoblots showing total protein carbonylation in the gastrocnemius muscle of control, STZ, and NAC-treated STZ mice. (C) mtDNA copy number was calculated as the ratio of COX1 to cyclophilin A DNA levels, determined by real-time PCR, in the skeletal muscle of control, STZ, and NAC-treated STZ mice. Note that the y axis scale is between 0.5 and 1. Results were normalized by the mean value of the control condition set to 1 unit. *P < 0.01 vs. control; ^#P < 0.05 vs. STZ. (D) Transmission electron microscopy images (original magnification, ×25,000) of subsarcolemmal and intermyofibrillar mitochondria from the gastrocnemius muscle of STZ and NAC-treated STZ mice.

Figure 7. ROS-induced mitochondrial alterations in C₂C₁₂ muscle cells.

(A) ROS production, measured by NBT reduction, in differentiated C₂C₁₂ myotubes incubated with glucose (25 mM) or palmitate (200 μM) in the presence or absence of NAC (10 mM) for 96 hours (n = 4). Data are expressed relative to the respective control (dotted line). (B) Effect of H₂O₂ (0.1 mM), glucose (25 mM), and palmitate (200 μM) on mtDNA levels in differentiated C₂C₁₂ myotubes. Myotubes were treated for 96 hours in the presence or absence of 10 mM NAC (n = 4). Data are expressed relative to the control condition (dotted line). (C) CS activity measured in total lysates of myotubes treated for 96 hours with H₂O₂ (0.1 mM), glucose (25 mM), or palmitate (200 μM) in the presence or absence of NAC (10 mM) (n = 4). (D) mRNA levels of POLG2, SSBP1, and PGC1α genes determined by quantitative RT-PCR in H₂O₂-, glucose-, or palmitate-treated myotubes in the presence or absence of NAC for 96 hours (n = 4). All results are expressed as fold change relative to the values of untreated cells set to 1 unit (dotted line). *P < 0.05, **P < 0.01 vs. respective control; ^#P < 0.05 vs. without NAC.