Alteration of mitochondrial function and insulin sensitivity in primary mouse skeletal muscle cells isolated from transgenic and knockout mice: role of ogg1

Abstract

Recent evidence has linked mitochondrial dysfunction and DNA damage, increased oxidative stress in skeletal muscle, and insulin resistance (IR). The purpose of this study was to determine the role of the DNA repair enzyme, human 8-oxoguanine DNA glycosylase/apurinic/apyrimidinic lyase (hOGG1), on palmitate-induced mitochondrial dysfunction and IR in primary cultures of skeletal muscle derived from hind limb of ogg1(-/-) knockout mice and transgenic mice, which overexpress human (hOGG1) in mitochondria (transgenic [Tg]/MTS-hOGG1). Following exposure to palmitate, we evaluated mitochondrial DNA (mtDNA) damage, mitochondrial function, production of mitochondrial reactive oxygen species (mtROS), mitochondrial mass, JNK activation, insulin signaling pathways, and glucose uptake. Palmitate-induced mtDNA damage, mtROS, mitochondrial dysfunction, and activation of JNK were all diminished, whereas ATP levels, mitochondrial mass, insulin-stimulated phosphorylation of Akt (Ser 473), and insulin sensitivity were increased in primary myotubes isolated from Tg/MTS-hOGG1 mice compared to myotubes isolated from either knockout or wild-type mice. In addition, both basal and maximal respiratory rates during mitochondrial oxidation on pyruvate showed a variable response, with some animals displaying an increased respiration in muscle fibers isolated from the transgenic mice. Our results support the model that DNA repair enzyme OGG1 plays a pivotal role in repairing mtDNA damage, and consequently, in mtROS production and regulating downstream events leading to IR in skeletal muscle.

Figure 1.

OGG1 expression in skeletal muscle isolated from KO and Tg animals. A, Skeletal muscle mitochondrial and nuclear fractions were isolated from different genotype littermates of KO, WT, and Tg mice and analyzed by Western blot for the purity of fractions. Lamin A was used as a marker for nuclear proteins, and subunit IV of mitochondrial complex IV and MnSOD were used as markers for mitochondrial proteins. Equal loading was confirmed using Ponceau staining of the membrane. No detected nuclear contamination was present in the skeletal muscle mitochondria and no contamination of mitochondrial proteins was found in nuclear fraction. B, OGG1 activity in mitochondrial and nuclear fractions isolated from skeletal muscle of KO, WT, and Tg mice. Note the almost complete absence of the product in the KO and minimal product in WT animals and increased amount of incised product in mitochondrial fractions isolated from Tg mice.

Figure 2.

Analysis of KO and Tg animals, and PMC. Expression of various proteins in skeletal muscle (A) and in PMC (C) isolated from different genotypes (as indicated). B, Mitochondrial respiration in skeletal muscle fibers on pyruvate, basal (top) and maximally uncoupled (bottom) (n = 5). P values were assessed using the nonparametric Kruskal-Wallis test.

Figure 3.

Overexpression of hOGG1 in mitochondria from PMC isolated from Tg mice prevented palmitate-induced mtDNA damage and increased mitochondrial function. A, Break frequency/14-kb mtDNA fragment. ATP level (B), MTS-viability (C), and mitochondrial copy number (D) were increased in Tg as compared to both KO or WT cultures after exposure to indicated concentrations of palmitate. The average results ± SE are shown (n ≥ 3). *, P < .05 vs PMC from KO and WT mice treated with the same concentration of palmitate. E, Primary skeletal muscle myotubes were treated with 0.5 mM palmitate for 24 hours. Total cell lysates were isolated and expression of mitochondrial proteins was analyzed by Western blot with the indicated antibodies. The values from densitometry from at least four independent experiments were normalized to the level of actin and expressed as fold difference compared to the corresponding control (2% BSA) conditions (n ≥ 4). *, P < .05 vs other groups.

Figure 4.

Overexpression of hOGG1 in mitochondria from PMC isolated from Tg mice prevented palmitate-induced mtROS generation and activation of JNK kinase. A, Mitochondrial superoxide production in primary myotubes isolated from KO, WT, and Tg animals and treated with the indicated concentrations of palmitate for 24 hours. Cells were analyzed in a fluorescent plate reader, and the increase in ROS production was calculated as a percentage increase compared to control. The means ± SE are shown (n ≥ 3). *, P < .05 vs PMC isolated from KO and WT mice treated with the same concentration of palmitate. B, Primary skeletal muscle myotubes were exposed to control medium (C, 2% BSA) or medium containing 0.5 mM palmitate (P). Total cell lysates were isolated and analyzed by Western blot with the indicated antibodies. The figure shows a representative experiment, which has been repeated 3 times. Equal loading was confirmed using Ponceau staining of the membrane. 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 control data (2% BSA) ± SE. *, P < .05 vs all other groups.

Figure 5.

Targeting of hOGG1 to mitochondria of PMC from Tg mice prevented against palmitate-mediated inhibition of insulin-signaling and IR. A, Akt (Ser 473) phosphorylation in primary myotubes isolated from KO, WT, and Tg mice. PMC were exposed to control medium (C, 2% BSA) or to medium containing 0.5 mM palmitate (P) for 16 hours and then serum starved and incubated in the presence or absence of 100 nM insulin for 15 minutes as described in Materials and Methods. Total cell lysates were isolated and analyzed by Western blot analysis with the indicated antibodies. B, The values from densitometry from at least three (p-Akt) independent experiments were normalized to the level of total Akt and expressed as fold of difference after addition of insulin normalized to the control (2% BSA) plus insulin data. The mean results ± SE are shown. *, P < .05 vs both KO and WT transduced cells treated with palmitate. C, Primary myotubes cultures were treated with 2% BSA (C, 2% BSA) or 2% BSA plus 0.5 mM palmitate (P) for 16 hours. Next, cells were incubated for 20 minutes in the absence or presence of insulin and then for 5 minutes with 2DG and uptake was measured. Values were normalized to the PMC from KO mice control basal data and are the means ± SE (n ≥ 3). *, P < .05 vs respective basal, unpaired two-tailed Student t test.