Mitochondrial dysfunction results from oxidative stress in the skeletal muscle of diet-induced insulin-resistant mice (original) (raw)
Metabolic characteristics of mice under HFHSD feeding. The metabolic characteristics of the mice are summarized in Table 1. After 4 weeks of the diets, body weight was significantly greater (20%, P < 0.001) and epidydimal adipose tissue weight was significantly greater (336%, P < 0.001) in the HFHSD mice than in the mice fed standard diet (SD). Plasma glucose, FFA, and triglyceride levels were similar in both groups of mice, whereas plasma insulin (72%, P < 0.05) and leptin (79%, P < 0.05) levels were greater in the HFHSD mice than in the SD mice. After 16 weeks of the diets, body weight and epidydimal fat weight gains in the HFHSD mice were more marked than those in the SD mice, and the HFHSD mice were clearly hyperglycemic (P < 0.001) and hyperinsulinemic (P < 0.001) compared with the SD mice. In the HFHSD mice, plasma glucose levels were significantly greater at 16 weeks (P < 0.001) than at 4 weeks. At 16 weeks, plasma leptin (P < 0.001), FFA (P < 0.05), and triglyceride (P < 0.001) levels were all greater in the HFHSD mice than in the SD mice.
Characteristics of the mice
Glucose and insulin tolerance tests showed that HFHSD mice were glucose intolerant at 4 week, whereas their response to insulin injection remained unaltered compared with SD mice (Supplemental Figure 1, A and B; supplemental material available online with this article; doi:10.1172/JCI32601DS1). In contrast, after 16 weeks of feeding, the HFHSD mice presented an altered response to both glucose and insulin injection compared with the SD mice, which indicated that the HFHSD mice were insulin resistant (Supplemental Figure 1, A and B). Decreased insulin responsiveness in HFHSD mice at 16 weeks was associated with intramyocellular lipid accumulation (Supplemental Figure 2A), increased basal IRS1 serine phosphorylation at Ser632 (45%, P < 0.05; Supplemental Figure 2B), and a decrease in ex vivo insulin-stimulated Akt phosphorylation at Ser473 (80%, P < 0.01; Supplemental Figure 2C) in gastrocnemius muscle. In contrast, at 4 weeks, insulin-stimulated Ser473 phosphorylation of Akt was not significantly different between the HFHSD and SD mice (Supplemental Figure 2C).
Mitochondrial biogenesis is reduced in the skeletal muscle of HFHSD mice. Next, we investigated the impact of HFHSD on muscle mitochondrial density. As shown in Figure 1A, the ratio of mitochondrial DNA (mtDNA) to nuclear DNA in the skeletal muscle was significantly lower in the HFHSD mice than in the SD mice (3%, P < 0.05) at 16 weeks, whereas no change was observed after 4 weeks of the diets. In agreement, the mRNA levels of the subunits 1 and 3 of cytochrome c oxidase (COX), 2 mitochondria-encoded genes, were significantly lower in the skeletal muscle of the HFHSD mice than of the SD mice at 16 weeks (Figure 1B). Using transmission electron microscopy, we found that the amounts of both subsarcolemmal (31%, P < 0.05) and intermyofibrillar (41%, P < 0.01) mitochondria in oxidative fibers were lower in the HFHSD mice than in the SD mice at 16 weeks (Figure 1, C and D). These alterations were not observed after 4 weeks of HFHSD diet (Figure 1C). As shown in Figure 1E, citrate synthase (CS) activity was slightly lower in the mitochondria isolated from the HFHSD mice than in that from the SD mice after both 4 weeks (13%, P < 0.05) and 16 weeks (16%, P < 0.05) of feeding.
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.
To clarify the mechanisms involved in the reduction of mitochondrial density in the muscle of the HFHSD mice at 16 weeks, we measured the mRNA levels of genes implicated in mitochondrial biogenesis, such as _PGC1_α, _PGC1_β, NRF1, NRF2, the mitochondrial transcription factor (mtTFA), estrogen-related receptor α (ERRα), and mitofusin 2 (Mfn2). Only the mRNA levels of _PGC1_α and Mfn2 were lower in the skeletal muscle of the HFHSD mice than of the SD mice at 16 weeks (~50% for both transcripts; Figure 2A). This difference was not seen after 4 weeks of the diets (Figure 2A). The protein levels of PGC1α were also significantly decreased in the skeletal muscle of HFHSD mice compared with SD mice at 16 weeks (data not shown). Concerning mtDNA replication and repair, we investigated both gamma DNA polymerase (POLG1, the catalytic subunit, and POLG2, the accessory subunit) and the single-strand DNA binding protein 1 (SSBP1), which play a key role in this process (16). As illustrated in Figure 2B, 16 weeks of HFHSD feeding induced a decrease in POLG2 and SSBP1 mRNA levels in skeletal muscle, whereas no effect was observed after 4 weeks of feeding. POLG1 expression was not affected by HFHSD feeding.
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.
Alteration of mitochondrial ultrastructure in the skeletal muscle of HFHSD mice. In addition to the observed reduction in mitochondrial content, the transmission electron microscopy study demonstrated marked alterations in mitochondrial morphology in the gastrocnemius muscle of the HFHSD mice at 16 weeks. Areas of both subsarcolemmal and intramyofibrillar mitochondria were lower (45% and 35%, respectively; P < 0.05) in the skeletal muscle of the HFHSD mice than of the SD mice at 16 weeks (Figure 3, A and C). Higher magnification (×100,000) showed swelling of both types of mitochondria associated with an increased number of disarrayed cristae and a reduced electron density of the matrix (Figure 3B). No alterations in mitochondrial morphology were observed after 4 weeks of the HFHSD (data not shown).
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.
Altered mitochondrial function in the skeletal muscle of HFHSD mice. To investigate whether alterations in mitochondrial density and ultrastructure were associated with mitochondrial dysfunction in the skeletal muscle of HFHSD mice, we measured substrate-driven oxygen consumption in saponin-skinned skeletal muscle fibers. The respiration rates of mice fed an HFHSD for 4 weeks were not significantly different from those of the SD mice, regardless of the tested substrates (Table 2). Compared with the SD mice, respiration in muscle fibers with complex 1-linked substrates (glutamate/malate), but not with complex 2-linked substrates (succinate/rotenone), was significantly reduced in HFHSD mice at 16 weeks, both during state 3 and state 4 (Table 2). In addition, we observed a significant decrease in oxidation capacities at 16 weeks when using octanoyl- or palmitoyl-carnitine as substrates in fibers of HFHSD mice. Taken together, these data demonstrate that complex 1–linked respiration and β-oxidation were decreased specifically in diet-induced diabetic mice. Reduced oxidation of fatty acids was probably not related to altered availability of the substrates because genes involved in muscle fatty acid uptake (FAT/CD36) and entry in the mitochondria (CPT1) were significantly upregulated in the skeletal muscle of HFHSD mice at 16 weeks (Supplemental Figure 3A). In further support of a reduction in mitochondrial functions, a decrease in the activity of succinate dehydrogenase was evidenced by succinate dehydrogenase staining in histological sections of gastrocnemius muscle from HFHSD mice at 16 weeks but not at 4 weeks (Supplemental Figure 3B).
Respiration rates and respiration control ratio in permeabilized muscle fibers of mice after 4 and 16 weeks of the SD and HFHSD
Increased oxidative stress in the skeletal muscle of HFHSD mice. Because mitochondrial alterations were observed in HFHSD mice only when they were hyperglycemic and hyperlipidemic, and because both glucose and lipids are known to induce oxidative stress, we tested whether ROS levels were increased during HFHSD feeding. Plasma H2O2 levels (Table 1) and muscular protein carbonylation levels (a marker of protein oxidation; Figure 4A) were elevated in HFHSD mice compared with SD mice at 16 weeks. No differences were observed after 4 weeks of the diets (Figure 4A).
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.
In addition, mRNA levels of uncoupling proteins 2 and 3 (markers of increased mitochondrial ROS production in conditions of lipid oversupply; ref. 17) and of 4 subunits of NAD(P)H oxidase (gp91, p67, p40, and p47) were induced in the skeletal muscle of HFHSD mice at 16 weeks, which suggests an increase in both mitochondrial and cytoplasmic ROS production (Figure 4B). Only the p67 subunit of NAD(P)H oxidase was significantly induced after 4 weeks of HFHSD feeding. Concerning the antioxidant system, the mRNA levels of glutathione reductase and catalase were increased in the skeletal muscle of HFHSD mice at 16 weeks, whereas expression of other antioxidant enzymes, including glutathione peroxidase, superoxide dismutase 2, peroxiredoxin 3, and peroxiredoxin 5, were not modified (Figure 4B). None of these genes showed a modification of expression after 4 weeks of the HFHSD.
Exposure to ROS leads to apoptosis and cell damage in a variety of experimental systems. As shown in Figure 4C, we observed an increase in cytochrome c levels in the cytosol and a concomitant decrease in the mitochondria fraction of skeletal muscle in HFHSD mice at 4 weeks, which indicated the release of cytochrome c from mitochondria. Because of the strong alterations in the number and functions of mitochondria in the muscle of HFHSD-fed mice at 16 weeks, this phenomenon was difficult to observe after 16 weeks of feeding (Figure 4C). Nevertheless, the activity of caspase 3, another index of apoptosis, was markedly increased in the skeletal muscle of HFHSD mice at 16 weeks (90%, P < 0.05), whereas no difference was observed after 4 weeks of the diet (Figure 4D).
We also investigated ROS production and mitochondrial dysfunction in KKAy mice, a genetic model of obesity and diabetes. KKAy mice were obese, hyperglycemic, hyperinsulinemic, and hypertrygliceridemic compared with age-matched control mice (Supplemental Table 1), but they had normal plasma FFA levels (Supplemental Table 1). Plasma H2O2 (Supplemental Table 1), but not skeletal muscle protein carbonylation (Supplemental Figure 4A), was greater in KKAy mice than in C57BL/6 mice. Interestingly, in contrast with the HFHSD model, mitochondrial density and structure were not altered in KKAy mice compared with age-matched control mice (Supplemental Figure 4, B and C). Taken together, these data suggest that oxidative stress in skeletal muscle is a determinant of mitochondrial alterations in diabetic mice.
Alteration of mitochondria biogenesis and structure in the skeletal muscle of streptozotocin-treated mice. To test whether ROS production is a key feature in HFHSD-induced mitochondrial dysfunction, we investigated mitochondrial structure and function in mice treated with streptozotocin (STZ), a model of hyperglycemia-associated oxidative stress with no insulin resistance and obesity. Eleven days after STZ administration, the mice were hyperglycemic (P < 0.001) and hypoinsulinemic (P < 0.001), had no changes in plasma FFA levels, and had a reduction in body weight (P < 0.005; Table 1). Insulin injection of the STZ mice rapidly decreased plasma glucose levels; 24 hours after insulin injection, plasma glucose levels were lower (P < 0.001), body weights were higher (P < 0.05), and FFA levels were undetectable in insulin-injected STZ mice compared with STZ mice (Table 1). Phlorizin injection of STZ mice reproduced the effect of insulin on glycemia: glucose decreased by 25% compared with STZ mice (P < 0.05).
In agreement with the observed hyperglycemia-induced oxidative stress, protein carbonylation levels were elevated in the skeletal muscle of STZ mice, and insulin treatment restored the extent of protein carbonylation to levels close to those observed in control mice (Figure 5A). Furthermore, STZ treatment induced a release of cytochrome c from mitochondria, and insulin treatment reversed this proapoptotic process (Figure 5B). Regarding mitochondrial density, the mtDNA/nuclear DNA ratio (Figure 5C) and amount of mitochondria per area (Figure 5D) were reduced in the muscle of STZ mice compared with control mice. The morphology of both types of mitochondria was also affected in the skeletal muscle of STZ mice; the number of cristae was reduced, and the electron density of the matrix decreased (Figure 5D). Importantly, density and structural abnormalities of mitochondria in the muscle of STZ mice were restored by insulin and phlorizin treatments (Figure 5, C and D).
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.
To verify whether mitochondrial alterations were related to ROS production, we treated STZ mice with N-acetylcysteine (NAC), a general antioxidant. NAC treatment did not modify systemic oxidative stress (Figure 6A), but did decrease muscle protein carbonylation to the levels of control mice (Figure 6B) and restored mitochondria density (Figure 6C) and structure (Figure 6D) in the gastrocnemius muscle of STZ mice.
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.
Taken together, these results demonstrate that oxidative stress in hyperglycemic mice is associated with altered mitochondrial structure and function in skeletal muscle and that both the amelioration of glycemia and antioxidant treatment restored mitochondrial structure.
ROS induce mitochondrial alterations and dysfunction in cultured myotubes. We examined the effects of high glucose and lipid levels on ROS production and mitochondria density and functions in C2C12 muscle cells. ROS production was markedly increased by glucose (25 mM) and by palmitate (200 μM) treatments for 96 hours, and the addition of NAC (10 mM) blocked these effects (Figure 7A). The addition of H2O2 (100 μM) for 96 hours decreased mtDNA levels (Figure 7B) and reduced CS activity (Figure 7C) in C2C12 cells. Incubation with glucose or with palmitate also decreased CS activity (Figure 7C), but the effects on mtDNA were not significant (Figure 7B). Furthermore, POLG2, SSBP1, and _PGC1_α mRNA levels were decreased in myotubes treated with H2O2, glucose, or palmitate for 96 hours. The addition of NAC counteracted all these effects, which indicated that ROS contributed to the observed mitochondrial alterations in cultured muscle cells (Figure 7, B–D). Finally, we also performed experiments in primary cultures of human myotubes and found similar results (Supplemental Figure 5), which suggests that these effects could also take place in human muscle cells. Transmission electron microscopy studies have nicely illustrated that the addition of both H2O2 and glucose for 96 hours altered mitochondria structure in myotubes compared with their respective control cells (Supplemental Figure 5D).
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 _PGC1_α 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.