Background
Insulin resistance is a major risk factor for developing type 2 diabetes, which is caused by the inability of insulin-target tissues to respond properly to insulin [
1], and in whose aetiology mitochondrial dysfunction is thought to play a crucial role [
2,
3]. Skeletal muscle is the main tissue responsible for the insulin-stimulated disposal of glucose and is the main contributor to the development of insulin resistance in type 2 diabetes [
4].
Skeletal muscle is a heterogeneous tissue made up of different contractile fibre types, in which the relative importance of glycolysis and mitochondrial oxidative phosphorylation for energy production varies. Glycolytic muscles are mainly composed of fast twitch or fast glycolytic fibres and generate energy by means of anaerobic metabolic processes, whereas oxidative muscles have a high proportion of slow twitch or slow oxidative fibres, are very resistant to fatigue and obtain energy through oxidative metabolic processes [
5,
6]. Under normal feeding conditions, glycolytic muscles use mainly glucose metabolism, whereas oxidative muscles are highly dependent upon lipids [
7]. Because of the differences between muscle types in energy demand and reliance on mitochondrial oxidative activity, differences in mitochondrial function can not be ruled out.
Skeletal muscle oxidative capacity is mainly determined by mitochondrial function and biogenesis [
8]. Mitochondrial biogenesis involves both proliferation and differentiation processes, which imply an increase in mitochondrial content and an improvement of the functional capabilities of pre-existing mitochondria, respectively [
9]. Mitochondrial biogenesis requires the coordinate participation of both mitochondrial and nuclear genomes [
10] through numerous transcription factors. Peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) coactivates different transcription factors in response to energy requirements resulting in the activation of nuclear genes involved in mitochondrial biogenesis. Among them, mitochondrial transcription factor A (TFAM) is one of the regulatory factors needed for proper transcription of mitochondrial DNA and of the genes encoding subunits of respiratory complexes [
11,
12]. Mitochondrial dysfunction has been proposed to be involved in the alteration of oxidative metabolism associated to insulin resistance. However, the cause-and-effect relationship between mitochondrial dysfunction and the development of insulin resistance remains unclear [
2,
3,
13].
High-fat diet feeding (HFD) leads to obesity and to an impairment of insulin sensitivity [
14]. Women seem to be more protected from obesity-associated insulin resistance than men [
15]. This protection has been attributed to the sex hormone milieu and has also been associated to differences in body fat distribution and adipokine levels. In this sense, women have been found to have significantly higher adiponectin plasma concentrations than men [
15,
16]. Adiponectin is a hormone secreted by adipocytes that circulates in high concentrations in serum and plays an important role in the regulation of mitochondrial biogenesis and insulin sensitivity [
17,
18]. Adiponectin binds to its receptors (AdipoR1, the most abundantly expressed in skeletal muscle, and AdipoR2) activating 5'-AMP-activated protein kinase (AMPK), which finally leads to the stimulation of glucose uptake and fatty acid oxidation. AMPK has also been implicated in the regulation of PGC-1α, the master regulator of mitochondrial biogenesis [
17,
18].
Sex differences have been previously described in mitochondrial biogenesis of skeletal muscle [
19] and of other tissues, such as liver [
20,
21] brain [
22], heart [
23] and brown adipose tissue [
24,
25]. Moreover, we have also reported a higher skeletal muscle antioxidant capacity and a better insulin sensitivity profile in response to high fat diet (HFD) feeding in female rats compared to males [
26]. Taking this background into account, the aim of the present study was to elucidate whether sex differences in the effects of HFD feeding on insulin sensitivity might be associated to differences in muscle mitochondrial biogenesis and the adiponectin signaling pathway, and whether these effects are dependent on muscle type.
Discussion
HFD feeding induces skeletal muscle mitochondrial biogenesis in both sexes, as the increase of PGC-1α and TFAM protein levels and mtDNA values suggest. Increased levels of PGC-1α, a master regulator of mitochondrial biogenesis [
32], would involve the enhancement of skeletal muscle oxidative capacity, whereas mtDNA levels point to an increase of the mitochondrial content. Mitochondrial biogenesis could be understood as an adaptation aimed to counteract the elevated amount of substrate available. The induction by HFD feeding of skeletal muscle oxidative capacity by increasing mitochondrial PGC-1α and respiratory chain units or mitochondria number has been previously reported in male rats [
3,
33], and is here reported also in female rats. Although the HFD-associated increase of mitochondrial biogenesis is observed in both muscle types, the effect seems more marked in the glycolytic one.
In gastrocnemius muscle, the effect of HFD feeding on mitochondrial biogenesis could be sex-dependent, since male rats, compared to females, show a more patent increase in mtDNA content (80 vs 22%) and PGC-1α (194 vs 71%) and TFAM (204 vs 92%) levels, which only reach statistical significance in the latter. These results suggest a more marked HFD-feeding-induced mitochondrial biogenesis in male rats. Mitochondrial biogenesis is considered a mechanism to counteract the impairment of mitochondrial function that could be consequence of oxidative stress and of the accumulation of toxic lipids, among others [
3]. The higher adiposity index that HFD female rats show compared to their male counterparts [
34] points to a greater lipid storage capacity of adipose tissue that would protect skeletal muscle from lipid toxic derivates that could impair its function [
35]. The lower skeletal muscle TBARS levels shown by HFD female rats compared to males further supports this idea. Thus, the greater adipose tissue expandability of female rats would make the development of strategies to avoid the detrimental effects of lipotoxicity less necessary. In this sense, during evolution, mammalian females have developed mechanisms to handle their energy resources more efficiently than males to facilitate the survival of their progeny and their own [
36].
The aforementioned induction of mitochondrial biogenesis in gastrocnemius muscle of both sexes in response to HFD feeding is accompanied by a marked increase of UCP3 levels and could be aimed at compensating the decreased levels of antioxidant enzymes. UCP3 has been proposed to play an important role in the protection of mitochondria against increased ROS production derived from enhanced fat oxidation [
37]. Since PGC-1α regulates the expression of UCP3 [
32], the increased levels of this coactivator found in obese animals would be aimed, at least in part, to contribute to attenuate oxidative damage in skeletal muscle. A similar effect of HFD feeding on gastrocnemius muscle enhancing UCP3 expression has been previously reported in 6 month-old male and female rats [
38] and in 18-month-old female rats, but interestingly not in their male counterparts [
26]. Taken together, both the present (performed in 9-month-old rats) and the aforementioned studies suggest the existence of age-dependent sex differences in the capacity to induce UCP3 expression in response to HFD feeding. Our results also suggest that, given the proposed role of UCP3 in the regulation of insulin sensitivity [
39], male rats would decrease their capacity to induce gastrocnemius UCP3 expression in response to HFD feeding between 9 and 18 months of age, in accordance with their higher oxidative damage and the earlier impairment of insulin sensitivity that male rats undergo with age compared to females [
40].
In soleus muscle, the increase of UCP3 levels in response to HFD feeding turns out to be more attenuated than in the gastrocnemius one, in agreement with previous studies performed only in male rats [
41], which showed a higher induction of UCP3 in glycolytic muscles than in oxidative ones. Once more, sex differences in the capacity to induce UCP3 expression are found. The response of male rats to HFD feeding increasing soleus UCP3 levels is accompanied by an increase of Cu-SOD levels that may not be enough to compensate the increase of oxidative stress, as the enhanced oxidative damage indicated. However, in soleus muscle of HFD female rats, the lack of changes in antioxidant enzymes and UCP3 protein levels, as well as in oxidative damage, suggest that UCP3 induction would not be a mechanism to protect soleus muscle from oxidative stress.
All in all, these results point to a more detrimental effect of HFD feeding on both skeletal muscles of male rats, which show a weaker capacity of response in front of an oxidative stress stimulus.
The impairment of insulin sensitivity induced by HFD feeding is also sex-dependent. Thus, although the HFD-induced increase in the levels of insulin resistance markers is more marked in female rats than in males, HFD obese female rats still maintain a better serum profile of insulin sensitivity. This less detrimental profile of obese females is reflected by lower insulin levels and HOMA-IR index values, as well as by the unchanged serum HMW adiponectin to total adiponectin ratio, a marker of the insulin-sensitizing action of this adipokine [
18]. The more marked insulin resistance status of HFD male rats is suggested by decreased serum HMW adiponectin levels and HMW adiponectin to total adiponectin ratio values. Moreover, oral glucose tolerance is more altered in HFD male rats [
34], which further reinforces this idea.
In spite of the sex dimorphism found in serum adiponectin levels, HFD feeding resulted in an impaired adiponectin response or "adiponectin resistance" in both sexes, as the increase of AdipoR1 protein levels and the decrease of p-AMPK/AMPK ratio indicate. This HFD-associated dysregulation of adiponectin-AMPK signaling has been proposed to contribute to the impairment of insulin sensitivity, through an alteration in fatty acid metabolism that increases lipid accumulation in skeletal muscle which, ultimately, leads to the development of insulin resistance [
42‐
44]. Increased levels of AdipoR1 would reflect a defective compensatory mechanism to overcome this adiponectin resistance, in agreement with previous studies showing a similar response in animal models with features of the metabolic syndrome [
42,
44].
As regards the involvement of muscle type in the effect of HFD feeding on adiponectin signaling pathway, gastrocnemius muscle shows a more marked response than soleus. In fact, the decreased activation of AMPK in gastrocnemius points to a greater adiponectin resistance than in soleus, which, otherwise, maintains the activation of AMPK unaltered. Given these findings and the above mentioned relationship between adiponectin and insulin sensitivity, our results suggest that gastrocnemius muscle may contribute to the obesity-associated onset of insulin resistance to a greater extent than soleus, despite having a metabolism less dependent on insulin [
45].
Although previous studies have reported that adiponectin resistance contributes to the impairment of skeletal muscle oxidative metabolism in HFD fed rodents [
42,
43], we found that, in response to chronic HFD feeding, adiponectin resistance is accompanied by an enhanced oxidative capacity, which is reflected by an increase of mitochondrial biogenesis. Our results are in agreement with a previous study that reports an increase of mitochondrial content as a consequence of HFD feeding to maintain normal oxidative capacity during later stages of insulin resistance [
3]. We suggest that this enhancement of mitochondrial biogenesis may be an adaptation to chronic HFD feeding as an attempt to compensate the deleterious consequences of insulin and adiponectin resistance on skeletal muscle oxidative metabolism.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
YGP has contributed in the conduct of the study, data collection and analysis, data interpretation and manuscript writing. GCA has contributed in the conduct of the study, data collection and analysis. MG has contributed in the design of the study, data interpretation and manuscript writing. IL and AMP have contributed in the design of the study, data collection and analysis, data interpretation and manuscript writing. All authors read and approved the final manuscript.