2. Transcriptional regulation of muscle mitochondrial oxidative capacity
Muscle oxidative capacity is mainly determined by the mitochondrial density that depends on mitochondrial biogenesis (i.e. the cellular processes involved in the synthesis of the organelles), and the mitochondrial oxidative capacity, which relies on the oxidative enzyme content and activity. A number of transcriptional modulators have been implicated in the regulation of muscle mitochondrial biogenesis and OXPHOS activity. They include PPAR gamma coactivator 1 alpha (PGC-1α), in cooperation with several factors such as the peroxisome proliferator-activated receptors (PPAR), the nuclear respiratory factors 1 and 2 (NRF-1 and NRF-2) [
1–
9], or the specificity protein 1 (Sp1), an ubiquitous transcription factor known to regulate the constitutive expression of oxidative OXPHOS genes [
10]. Of note, Sp1 can function as both a positive (e.g. cytochrome c1 and mitochondrial transcription factor A, TFAM) and a negative (e.g. adenine nucleotide translocator 2 and F1-ATPase beta subunit) regulator of transcription [
11]. PGC-1α is a master modulator of gene expression in skeletal muscle [
12]. It was found to drive the formation of oxidative type I fibres and to activate the expression of genes involved in mitochondrial oxidative capacity, through associated changes in the expression of NRF dependent genes [
13]. These combined data have therefore suggested that decreased PGC-1α gene expression could be one of the primary contributors to decreased mitochondrial oxidative capacity. However, PPARs are also good candidates. When bound to their ligand (e.g. fatty acids for PPARα), PPARs form a heterodimeric complex with the retinoid X receptor (RXR) to regulate gene transcription involved in fatty acid metabolism. PGC-1α is also known to enhance the activity of the isoforms PPARα and PPARβ/δ in skeletal muscle, which may result in the enhanced expression of genes involved in mitochondrial fatty acid oxidation [
14]. Major transcriptional modulators involved in the regulation of mitochondrial activity in skeletal muscle are illustrated in
Figure 1 (see also [
14–
16] for reviews). All factors mentioned above co-regulate the transcriptional activity of a variety of genes involved in mitochondrial biogenesis, OXPHOS activity and fatty acid oxidation. For example, in muscle cells, overexpression of PGC-1α was shown to induce the gene expression of NRF-1, NRF-2, TFAM and mitochondrial-encoded cytochrome c oxidase (COX) subunits [
15]. Likewise, muscle–specific overexpression of PPARβ/δ in mice was shown to increase oxidative enzyme activities such as citrate synthase or b-hydroxyacyl-CoA dehydrogenase, and to enhance expression of genes implicated in fatty acid catabolism [
17].
4. Potential mechanisms: intrinsic factors
As previously described, the transcriptional coactivator PGC-1α is a potent stimulator of mitochondrial biogenesis and OXPHOS activity (c.f. part 1). A decrease in the expression of mitochondrial genes associated with a concomitant reduction in NRF-1 and PGC-1α gene expression has been reported in the skeletal muscle of insulin resistant and diabetic patients [
7,
48]. However the intrinsic factors responsible for depressed NRF-1 and PGC-1α gene expression remain to be fully elucidated. There are multiple processes by which environmental or physiologic factors might play a critical role in the control of mitochondrial biogenesis and function. Among these mechanisms, we selected lipotoxicity, inflammation and glucotoxicity.
First,
lipotoxicity is the overall damage caused to tissues secondary to prolonged exposure to high levels of plasma non-esterified free fatty acids (NEFA). Excess NEFA and the accumulation of intramyocellular lipid metabolites (e.g., diacylglycerol, fatty acid acylcoA or ceramides) consecutive to increased NEFA availability, have been demonstrated to trigger insulin resistance [
49,
50] but may also disturb mitochondrial activity. Recent findings on cultured skeletal muscle cells have brought evidence that fatty acids play a significant role in the regulation of muscle oxidative metabolism. First, a study on human myotubes reported that PGC-1α gene expression was increased two- to three-fold by unsaturated fatty acids but was unchanged with saturated fatty acids [
51]. Mitochondrial activity was concomitantly enhanced by unsaturated fatty acids, but was impaired with the saturated fatty acid stearate [
51]. Second, data obtained on C2C12 showed that the saturated fatty acid palmitate, by contrast to the monounsaturated fatty acid oleate, reduces PGC-1α gene expression through a mechanism involving mitogen-activated protein kinase (MAPK), extracellular signal-related kinase [
52] and NF-κB activation [
53]. Another study showed that saturated fatty acids decrease PGC-1α and mitochondrial gene expression and function via p38 MAPK-dependent transcriptional pathways [
54]. Interestingly, Benton
et al. [
55] showed in two animal models in which muscle fatty acid accumulation was either increased (Zucker obese rats) or decreased (FAT/CD36 null mice) that PGC-1α protein expression was inversely correlated to the cellular ability to uptake and store lipids. Similarly, adult rats fed a high-fat diet for two weeks showed lower muscle mitochondrial respiration rates than low-fat fed rats [
46].
An original study recently showed that resveratrol, a phytoalexin found in particular in the skin of red grapes, can reverse the deleterious effects of high-fat diet on muscle mitochondrial function in mice [
56]. Resveratrol treatment greatly enhanced mitochondrial oxidative capacity, by induction of genes involved in oxidative phosphorylation and mitochondrial biogenesis. These adaptations were principally explained by a resveratrol-mediated activation of the protein deacetylase Sirt1, the result being a decrease in PGC-1α acetylation and an increase in PGC-1α activity [
56]. Additional evidence came from research performed in humans. A study conducted in young men has demonstrated that 3-day high-fat diet decreases the expression of genes involved in mitochondrial oxidative capacity in skeletal muscle [
8]. To strengthen the lipotoxic theory, infusion of intralipid for 48 hours in healthy humans decreases the muscle expression of both PGC-1α and several genes involved in oxidative phosphorylation [
57]. Thus, consistent data obtained on various models, from cell to human, show that intramuscular lipid sensing may be involved in regulating the muscle PGC-1α expression and activity, and consequently muscle mitochondrial oxidative capacity, although further investigations are required to determine quality, dose and time-dependent effects of fatty acids on muscle mitochondrial OXPHOS activity. This single mechanism could be suitable to explain most of the muscle mitochondrial adaptations with metabolic disorders such as obesity and type 2 diabetes. However, additional complex interactions and pathways are likely to occur.
In that respect, a number of
adipocyte-derived factors may be responsible for the reduced mitochondrial oxidative capacity. Adipose tissue makes up ~ 15–25% of body mass in men and women with normal values of body mass index (BMI = 18–25 kg/m
2) but can vary from 4–10% in athletes to 50% in obese patients (BMI > 30 kg/m
2). Adipose tissue cells comprise adipocytes and non-adipose cells that constitute the stroma-vascular fraction, mainly endothelial cells, leucocytes, monocytes and macrophages. Recent studies support the hypothesis that obesity is associated with a state of chronic low-grade inflammation [
56–
61]. Tumor necrosis alpha (TNF-α) and interleukin 6 (IL-6), pro-inflammatory cytokines produced mainly by adipocytes and macrophages but also by muscle cells, are up-regulated in obesity [
58–
60]. Recently, it has been shown that TNF-α might positively autoregulate its own synthesis in adipose tissue [
61], which might contribute to the maintenance of the elevated TNF-α observed in obesity [
62,
63]. One should also keep in mind that the saturated fatty acid palmitate enhances TNF-α expression in skeletal muscle [
64]. TNF-α signaling through TNF receptor has been implicated in the pathogenesis of insulin resistance [
58–
60,
62–
63]. In vitro and in vivo data have shown that it suppresses AMPK activity via transcriptional upregulation of protein phosphatase 2C (PP2C). This in turn reduces the phosphorylation of the enzyme acetyl-CoA carboxylase, suppressing fatty-acid oxidation, increasing intramuscular diacylglycerol accumulation, and causing insulin resistance in skeletal muscle [
65]. Importantly, TNF-α has been shown to increase the expression of the inducible isoform of the nitric oxide synthase (iNOS) [
66] and to downregulate that of the endothelial isoform (eNOS) [
67]. Decreased expression of the neuronal isoform (nNOS) has been also reported in skeletal muscle of streptozotocin-induced diabetic rats [
68]. These enzymes catalyze the biosynthesis of NO (a short-lived highly diffusible hydrophobic free radical) from
L-arginine and molecular oxygen utilizing NADPH as an electron donor and heme, FMN, FAD and tetrahydrobiopterin (H4B) as cofactors [
69]. It is now demonstrated that NO generated by eNOS increases mitochondrial biogenesis, oxidative metabolism and ATP levels in several cell types, including muscle [
70,
71]. Hickner
et al. [
72] have shown that in young women skeletal muscle eNOS protein content and activity are inversely related to body fat percentage. In addition recent evidences have demonstrated that eNOS expression and mitochondrial biogenesis are downregulated in adipose and muscle tissues of genetically and diet-induced obese mice and rats whereas iNOS is upregulated [
67]. This process has been shown to be partly mediated by cGMP, resulting from NO-dependent activation of “soluble” guanylate cyclase, and involves the increased expression of PGC-1α, NRF-1 and TFAM [
70]. Thus, in vitro and in vivo data support that TNFα may be involved in regulating muscle PGC-1a expression, and consequently muscle mitochondrial oxidative capacity.
Regarding IL-6, Al-Khalili
et al. have recently established on culture cells from skeletal muscle that IL-6 regulates muscle substrate utilization, enhancing glycogen storage and lipid oxidation [
72]. Yet based on the current data, it can be assumed that IL-6 exerts different effects according to the physiological situation, i.e. in response to exercise [
73] or during low-grade inflammation in obesity [
74,
75]. By contrast to the pro-inflammatory cytokines TNF-α and IL-6, IL-15 is a cytokine highly expressed in skeletal muscle which induces fatty acid oxidation [
73] and facilitates glucose metabolism [
74]. Furthermore, leptin and adiponectin are anti-inflammatory hormones exclusively produced by adipocytes [
76,
77]. Leptin is an adipocytokine produced proportionally to adipose tissue size [
78], initially described for its action in brain regions to reduce food intake. Adiponectin, which is present at a high concentration in the plasma, is downregulated with obesity [
58]. Leptin and adiponectin have been shown to activate muscle fatty acid oxidation and this action appears to be mediated by AMP-activated protein kinase (AMPK) activation that triggers stimulation of mitochondrial function and biogenesis [
79,
80]. Indeed, activation of AMPK enhances PGC-1α gene expression [
75] and stimulates PPARα [
80,
82]. The balance between pro-inflammatory factors and adipocytokines may be therefore a connective link between adipose tissue mass and function, and metabolic disorders in skeletal muscle [
81].
Finally,
glucotoxicity is commonly defined by the overall damage caused to tissues, secondary to prolonged exposure to elevated plasma glucose concentration. The degree of mitochondrial failure has been correlated with the duration of diabetes. Complexes I, III and IV of the electron transport chain have been shown to be the main mitochondrial targets of hyperglycaemia-induced injury [
76]. The presence of chronic hyperglycaemia can cause structural alterations of proteins through the Maillard reaction, and can lead to oxidative stress, a state of imbalance between the production of reactive oxygen species (ROS) and antioxidant defences, and consequently to cellular oxidative damage [
77,
78]. In that respect, recent evidences have demonstrated that oxidative stress in skeletal muscle is probably one of the major determinants of the mitochondrial alterations in obesity and type 2 diabetes [
79]. This is supported by in vivo and in vitro data showing that 1) an increase in muscle ROS production occurs specifically after hyperglycaemia and hyperlipidemia have appeared in high fat fed mice; 2) in this model, normalization of glycaemia by insulin or phlorizin and treatment with an antioxidant (
N-acetylcysteine) decreases muscle ROS production and restores mitochondrial integrity; 3) incubation of cultured muscle cells with high glucose or lipid concentrations induces ROS production and alters mitochondrial density and functions; 4) these effects are blocked by an antioxidant treatment. Enhanced mitochondrial ROS production has been also shown to activate the redox-sensitive transcription factor NF-κB [
80], which has been associated with PGC-1α downregulation in C2C12 skeletal muscle cells [
53]. In addition, ROS overproduction is likely to enhance several metabolic pathways such as the hexosamine biosynthesis pathway (HBP) [
81]. HBP is a nutrient-sensing pathway that has been implicated in the development of insulin resistance [
81]. Obici et al. have reported that HBP activation in response to short-term overfeeding is accompanied by an inhibition of the expression of genes (e.g. malate dehydrogenase, acyl-CoA dehydrogenase, propionyl-CoA carboxylase, subunits of complexes I, III, IV and V, adenine nucleotide translocator 2, mitochondrial 2-oxoglutarate/malate carrier protein) involved in mitochondrial oxidative capacity within skeletal muscle [
47]. The molecular mechanisms may in part relate to the control of Sp1 activity via
O-linked
N-acetylglucosamine (O-GlcNAc) modification [
11].