Protection from palmitate-induced mitochondrial DNA damage prevents from mitochondrial oxidative stress, mitochondrial dysfunction, apoptosis, and impaired insulin signaling in rat L6 skeletal muscle cells - PubMed (original) (raw)

Protection from palmitate-induced mitochondrial DNA damage prevents from mitochondrial oxidative stress, mitochondrial dysfunction, apoptosis, and impaired insulin signaling in rat L6 skeletal muscle cells

Larysa V Yuzefovych et al. Endocrinology. 2012 Jan.

Abstract

Saturated free fatty acids have been implicated in the increase of oxidative stress, mitochondrial dysfunction, apoptosis, and insulin resistance seen in type 2 diabetes. The purpose of this study was to determine whether palmitate-induced mitochondrial DNA (mtDNA) damage contributed to increased oxidative stress, mitochondrial dysfunction, apoptosis, impaired insulin signaling, and reduced glucose uptake in skeletal muscle cells. Adenoviral vectors were used to deliver the DNA repair enzyme human 8-oxoguanine DNA glycosylase/(apurinic/apyrimidinic) lyase (hOGG1) to mitochondria in L6 myotubes. After palmitate exposure, we evaluated mtDNA damage, mitochondrial function, production of mitochondrial reactive oxygen species, apoptosis, insulin signaling pathways, and glucose uptake. Protection of mtDNA from palmitate-induced damage by overexpression of hOGG1 targeted to mitochondria significantly diminished palmitate-induced mitochondrial superoxide production, restored the decline in ATP levels, reduced activation of c-Jun N-terminal kinase (JNK) kinase, prevented cells from entering apoptosis, increased insulin-stimulated phosphorylation of serine-threonine kinase (Akt) (Ser473) and tyrosine phosphorylation of insulin receptor substrate-1, and thereby enhanced glucose transporter 4 translocation to plasma membrane, and restored insulin signaling. Addition of a specific inhibitor of JNK mimicked the effect of mitochondrial overexpression of hOGG1 and partially restored insulin sensitivity, thus confirming the involvement of mtDNA damage and subsequent increase of oxidative stress and JNK activation in insulin signaling in L6 myotubes. Our results are the first to report that mtDNA damage is the proximal cause in palmitate-induced mitochondrial dysfunction and impaired insulin signaling and provide strong evidence that targeting DNA repair enzymes into mitochondria in skeletal muscles could be a potential therapeutic treatment for insulin resistance.

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Figures

Fig. 1.

Fig. 1.

Targeting of hOGG1 to mitochondria from L6 myotubes. A, Adenoviral vectors (GFP or MTS-hOGG1) were added to cells in differentiation media at the indicated MOI. After a 48-h transduction, mitochondrial fractions were isolated from the transduced L6 myotubes, and Western blot analysis was performed using hOGG1 antiserum. Immunodetection of cytochrome c was performed to assure mitochondrial localization. B, Nuclear and cytosolic fractions were isolated from L6 myotubes transduced with GFP or MTS-hOGG1 viruses at a MOI of 70. Equal loading was confirmed using Ponceau staining of the membrane. Lamin A and actin were used to indicate nuclear and cytosolic localization, respectively. To exclude possible contamination from mitochondrial fractions, a separate portion of the same blot was probed with cytochrome c.

Fig. 2.

Fig. 2.

Overexpression of hOGG1 in mitochondria from L6 myotubes prevents palmitate-induced mtDNA damage and increased mitochondrial function. A, Break frequency per 10.8-kb fragment of nuclear and mtDNA after 6 h of treatment with the indicated concentrations of palmitate (n ≥ 3). *, P < 0.05 vs. all other groups. B, ATP levels were increased in L6 myotubes transduced with MTS-hOGG1 adenoviruses after palmitate treatment. Cells were transduced with the adenoviruses for 48 h and then treated with the indicated concentration of palmitate, and ATP production was measured. The mean results ±

se

are shown (n ≥ 3). *, P < 0.05 vs. GFP-transduced cells treated with the same concentration of palmitate. C, Mitochondrial function is increased in MTS-hOGG1-expressing myotubes 24 h after exposure to indicated concentrations of palmitate. The average results ±

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are shown (n = 3). *, P < 0.05 vs. GFP-transduced cells treated with the same concentration of palmitate.

Fig. 3.

Fig. 3.

Targeting of hOGG1 to mitochondria in L6 myotubes protected against palmitate-induced mtROS generation, reduced activation of JNK kinase, and prevented cells from undergoing apoptosis. A, Mitochondrial superoxide production in MTS-hOGG1- and GFP-transduced L6 myotubes treated with the indicated concentrations of palmitate for 24 h. Cells were analyzed in a fluorescent plate reader, and the increase in ROS production was calculated as a percentage increase compared with control. The mean results ±

se

are shown (n ≥ 3). *, P < 0.05 vs. GFP-transduced cells treated with the same concentration of palmitate. B, Adenovirus-transduced L6 myotubes were exposed to control medium (C) (2% BSA) or medium containing 1 m

m

palmitate (P). Total cell lysates were isolated and analyzed by Western blotting with the indicated antibodies. Equal loading was confirmed using antiactin antibody. C, The values from densitometry from three (pJNK) independent experiments were normalized to the level of total JNK and expressed as fold of difference normalized to GFP control data ±

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; *, P < 0.05 vs. all other groups. D, Results of the Western blottings using caspase-3 antibodies which recognize the full-length (35-kD) and the large (17 kD) fragment of caspase-3 resulting from its cleavage. Equal loading was confirmed by loading antiactin antibody.

Fig. 4.

Fig. 4.

Targeting of hOGG1 to mitochondria in L6 myotubes ameliorated palmitate-mediated inhibition of insulin-induced. A, C tyrosine phosphorylation of IRS-1. D and E, Akt (Ser473) phosphorylation. G and F, GLUT4 translocation to the PM. MTS-hOGG1- or GFP-transduced L6 myotubes were exposed to control medium (C) (2% BSA) or to medium containing 1 m

m

palmitate (P) for 16 h and then treated with insulin as described in Materials and Methods. Total cell lysates or PM (as specified) were isolated and analyzed by Western blot analysis with the indicated antibodies. A, top panel, IRS-1 was immunoprecipitated from 200 μg of total cell lysates, and Western blottings were performed using an pTyr antibody. Bottom, Representative Western blotting of total IRS-1. B, Densitometry data for the total IRS-1 protein levels normalized to GFP control data (n ≥ 3). *, P < 0.05 vs. all other groups. C, Densitometry data for insulin dependent (pTyr-IRS-1) were normalized to GFP (control plus insulin) data and presented as means ±

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(n ≥ 3). *, P < 0.05 vs. GFP-transduced cells treated with palmitate. D, Representative blots from at least three independent experiments for phosphorylation of Akt (Ser473) are shown. Total cell lysates were used. E, The values from densitometry from at least three (pAkt) independent experiments were normalized to the level of total Akt and expressed as fold of difference after addition of insulin normalized to the GFP (control plus insulin) data. The mean results ±

se

are shown. *, P < 0.05 vs. GFP-transduced cells treated with palmitate. G, PM were isolated from adenovirus-transduced L6 myotubes, as described in Materials and Methods, and proteins were analyzed by Western blotting using GLUT4 antibody. Immunodetection of the α-subunit of Na+/K+-ATPase was performed to confirm PM localization. The values from densitometry performed on three to four independent GLUT4 translocation to PM independent experiments were normalized to the level of α-subunit of Na+/K+-ATPase and then normalized to the GFP (control plus insulin) data ±

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(n ≥ 3). *, P < 0.05 vs. GFP-transduced cells treated with palmitate.

Fig. 5.

Fig. 5.

Targeting of hOGG1 to mitochondria in L6 myotubes protected against palmitate-induced decrease in insulin-stimulated 2DG uptake. A, MTS-hOGG1- and GFP-transduced L6 myotubes were treated with 2% BSA (C, 2% BSA) or 2% BSA plus 1 m

m

palmitate (P) for 16 h. After that, cells were incubated in the absence or presence of insulin for 20 min and then for 5 min with 2DG, and uptake was measured as described in Materials and Methods. Values were normalized to the GFP control basal data and are the means ±

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(n ≥ 3). *, P < 0.05 vs. respective basal; #, P < 0.05 vs. all other groups treated with insulin. B, graph, Effect of a JNK inhibitor on palmitate-induced inhibition of insulin-stimulated 2DG uptake. L6 myotubes were incubated in the medium containing only 2% BSA (C, Control) (2% BSA) or 2% BSA plus 1 m

m

palmitate (P) in the presence or absence of the JNK inhibitor, SP-600125 for 16 h before stimulation with insulin and measurement of 2DG uptake. Values were normalized to the control basal data and are the means ±

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(n ≥ 3). Δ, Fold induction on insulin. *, P < 0.05 vs. both control and palmitate plus SP-600125. B, panel above graph, Effect of JNK inhibitor on palmitate-induced mtDNA damage. L6 myotubes were incubated with 2% BSA (C) or 2% BSA plus 1 m

m

palmitate (P) in the presence or absence of the JNK inhibitor, SP-600125 for 6 h. mtDNA damage was evaluated as described in Materials and Methods.

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