Introduction
Endurance exercise training has beneficial effects on mitochondrial oxidative capacity in skeletal muscle and is associated with enhanced muscle insulin sensitivity. We and others have previously shown that endurance and resistance exercise training programmes exert beneficial effects on muscle substrate metabolism in individuals with type 2 diabetes [
1‐
3], and that a high skeletal muscle oxidative capacity (due to chronic endurance exercise training) partially protects against lipid-induced insulin resistance [
4].
Interestingly, resting muscle of endurance-trained athletes is characterised by inefficient mitochondrial coupling (higher basal uncoupling rates). Using in vivo MR spectroscopy, a 54% higher basal rate of substrate oxidation through the tricarboxylic acid (TCA) cycle was observed in athletes compared with matched sedentary controls [
5]. Despite this increased basal TCA cycle flux, ATP synthesis rates were similar, thus indicating a decrease in the efficiency of mitochondrial coupling. The mechanistic basis for the decreased mitochondrial coupling efficiency in endurance-trained individuals, however, has not been established. The study [
5] concluded that increased mitochondrial uncoupling may represent an additional mechanism by which endurance exercise training enhances muscle insulin sensitivity; in accordance, muscle-specific overexpression of uncoupling protein (UCP) 1 [
6] and UCP3 [
7] in rodents protect against lipid-induced insulin resistance.
We have previously shown that UCP3 expression is lower in oxidative type I muscle fibres [
8,
9] than in the more glycolytic type II fibres, and
UCP3 mRNA expression and protein abundance are, in fact, reduced in muscle from endurance-trained humans [
9,
10]. Employing genetic and physiological mouse models for gain- and loss-of-function of skeletal muscle UCP3, we previously failed to find evidence for any involvement of UCP3 in mediating basal mitochondrial uncoupling [
11,
12]. Alternatively, it has been known for decades that fatty acids (FAs) can affect mitochondrial efficiency due to their ability to induce mitochondrial uncoupling [
13]. Although the mechanisms underlying FA-induced uncoupling are not yet fully understood, there are clear indications that the mitochondrial protein adenine nucleotide translocator 1 (ANT1)—in addition to its function in mitochondrial ADP–ATP exchange—can also transport FA anions across the inner mitochondrial membrane [
13,
14] and thus mediate FA-induced uncoupling.
Skeletal muscle from endurance-trained individuals shows enlarged intramyocellular lipid stores, high FA fluxes and increased
ANT1 (also known as
SLC25A4) mRNA and protein abundance compared with muscle from matched sedentary individuals [
4,
10,
15,
16]. We tested the hypothesis that endurance-trained muscle is more sensitive to the uncoupling effect of FAs and that FA-induced uncoupling plays a significant role in insulin sensitivity. To further study the role of ANT1 in FA-induced uncoupling in relation to insulin responsiveness, we also examined isolated skeletal muscle mitochondria obtained from a rodent model of type 2 diabetes (Zucker diabetic fatty [ZDF] rats) and employed a small interfering RNA (siRNA)-mediated gene silencing of
Ant1 in C2C12 myotubes.
Discussion
Endurance exercise training and insulin sensitivity are inextricably linked. We have recently demonstrated that high mitochondrial oxidative capacity (due to chronic endurance exercise training)—both reflected as elevated mitochondrial density and intrinsic function—partially prevented lipid-induced insulin resistance [
4]. The underlying mechanisms for this phenomenon are not fully understood. In addition to our findings, in vivo data has demonstrated that endurance-trained muscle exhibits increased substrate oxidation, as well as increased mitochondrial uncoupling [
5], resulting in reduced mitochondrial efficiency and increased capacity to waste excess energy. In recent years, the renaissance of research in brown adipose tissue has put a spotlight on this tissue as a thermogenic weapon in the war on obesity and diabetes. However, reducing fat in humans via thermogenesis may require more than brown adipose tissue, an organ that has primarily evolved to maintain core body temperature [
32]. Exciting new evidence has revived interest around the thermogenic potential of skeletal muscle [
33] by opening the door on the possibility that muscle significantly contributes to non-shivering thermogenesis as a means to maintain substrate metabolism and glucose homeostasis in settings of excess energy (e.g. FAs) with a low demand for ATP production.
In the present study, we demonstrated that elevated sensitivity to FA-induced uncoupling (lower EC50) at baseline was associated with higher insulin sensitivity in endurance-trained compared with sedentary young men. This increased sensitivity to FA-induced uncoupling was further associated with retention of insulin-stimulated glycogen storage (higher non-oxidative glucose disposal [NOGD]) after a lipid infusion. In other words, our data suggest that enhanced sensitivity to FA-induced uncoupling (in the context of chronic endurance exercise training) partially protects skeletal muscle from lipid-induced development of insulin resistance.
In line with our findings, another study demonstrated that a short-term lipid infusion significantly reduced inner mitochondrial membrane potential in healthy (normal glucose tolerance) individuals [
34]; this was presumably linked to an increase in uncoupling by the excess FAs. Also in agreement with our data was the fact that mitochondrial content and morphology did not change after lipid infusion and there were no significant changes in citrate synthase activity or total ATP content [
34]. In parallel with the positive association between insulin sensitivity and FA-induced uncoupling observed in our human cohort, isolated muscle mitochondria from insulin-resistant ZDF rats displayed a right shift in the FA titration curve, indicating a decreased sensitivity to FA-induced uncoupling.
Although mitochondrial ANT1 protein levels were not statistically different in trained vs sedentary humans and ZDF vs wild-type control rats, the difference in sensitivity to FA-induced uncoupling between mitochondria from ZDF and wild-type controls was abolished upon chemical inhibition of ANT1 activity. This finding suggests that the difference between genotypes originates from FA-induced uncoupling due to ANT1 activity. In this context, almost a decade ago it was demonstrated that ~50% of mitochondrial proton leak can be attributed to ANT1 in murine muscle and that ANT1 can be activated by FAs [
31]. Recent work has also demonstrated that acetylation of ANT1 could have dramatic physiological effects on ANT activity and that dysregulation of acetylation of mitochondrial proteins such as ANT1 could therefore be related to changes in mitochondrial function that are associated with insulin resistance [
35].
While these in vivo experiments are useful for investigating whole-body physiology, an in vitro loss-of-function model serves to isolate downstream effects of reduction of a singular gene. Thus, we next reduced
Ant1 expression in C2C12 myotubes and studied the sensitivity to FA-induced uncoupling, as well as insulin-stimulated glucose uptake. Since ANT1 plays a crucial role in ADP/ATP exchange across the inner mitochondrial membrane, complete ablation of
Ant1 would lead to serious alterations in oxidative phosphorylation and ATP synthesis rates. Indeed, mice lacking ANT1 are characterised by cardiomyopathy and mitochondrial myopathy of limb muscles [
36]. Therefore, we specifically aimed for a partial knockdown of ANT1. By adapting the transfection protocol, we ultimately achieved a 38% reduction at the protein level, which proved not to be rate limiting for maximal ADP-stimulated respiration.
As anticipated, congruent with our findings using the chemical ANT inhibitor CATR in isolated mitochondria to decrease ANT activity, partial knockdown of ANT1 in C2C12 myotubes shifted the titration curve for FA-induced uncoupling to the right (increased EC50), thereby demonstrating reduced sensitivity. The reduction in ANT1 also resulted in a diminished insulin-stimulated glucose uptake compared with controls.
The mechanistic link between FA-induced uncoupling and insulin sensitivity may be related to the production of reactive oxygen species, which was previously identified as the common denominator and causal factor in several cellular models of insulin resistance [
37] and which is also associated with insulin resistance in humans [
38,
39]. Hence, at the expense of a slight inefficiency in ATP production, mild mitochondrial (FA-induced) uncoupling may be a tool to control the formation of reactive oxygen species when facing a high supply of fatty acids, thereby preserving insulin sensitivity. It should be noted that opening of the mitochondrial permeability transition pore (MPTP) has also been linked to insulin resistance in skeletal muscle [
40]. Since ANT is an important regulatory component of the MPTP, knockdown of ANT1 in C2C12 muscle cells may also promote MPTP opening thereby decreasing insulin sensitivity. However, since the partial knockdown of ANT1 that we achieved in the C2C12 muscle cells did not affect oxidative phosphorylation and ATP synthesis in the current study, opening of the MPTP is less likely.
Taken together, these data demonstrate the importance of ANT1 activity in maintaining insulin responsiveness via its role in FA-induced mitochondrial uncoupling. Endurance-trained athletes have superior insulin sensitivity (vs matched sedentary controls) in a setting of elevated muscle lipid content, high basal TCA cycle flux and low basal mitochondrial efficiency (elevated uncoupling) [
5]. These findings highlight the potential of FA-induced uncoupling via ANT1 as a target for improving myocellular insulin sensitivity in settings of energy excess.
Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.