In this study, we evaluated the effect of prolonged fasting on skeletal muscle mitochondrial functional capacity in humans to examine whether the mitochondrial dysfunction that is frequently reported in insulin resistance and type 2 diabetes can be a consequence of lipid-induced insulin resistance, rather than a cause. In contrast to the hyperglycemia and hyperinsulinaemia accompanying “energy excess”-induced insulin resistance (lipid infusion, high-fat diets), prolonged fasting-induced insulin resistance is associated with hypoglycemia and hypoinsulinemia. Moreover, prolonged fasting-induced lipid accumulation and insulin resistance are considered to be a functional physiologic response. Thus, reduced insulin sensitivity saves carbohydrates for the central nervous system, being obligate for glucose and not requiring insulin for its uptake, whereas increased lipid availability at the same time can serve as a direct available energy source for the muscles and is paralleled by an enhanced fat oxidative capacity (12
). Therefore, we anticipated that skeletal muscle mitochondrial function would not be impaired in this model unless mitochondrial function is impaired by factors that are secondary to the lipid-induced insulin-resistant state. Intriguingly, we found that only 60 h of fasting in humans was accompanied by an overall reduction in skeletal muscle mitochondrial capacity, which was not explained by changes in mitochondrial density.
We assessed mitochondrial functional capacity in detail () by using a wide variety of substrates and substrate combinations to determine the maximum ADP-stimulated respiration (state 3) fueled by a lipid substrate, by Complex I substrates, and upon parallel electron input into Complex I (NADH) and II (FADH2). Interestingly, state 3 respiration was reduced by ~20% upon all substrates, which reduces the possibility that the decline is caused by substrate-specific alterations such as substrate uptake into the mitochondria. Therefore, both a reduction in the activity of the electron transport chain and the oxidative phosphorylation system could underlie the reduced state 3 respiration. Using the chemical uncoupler FCCP, control over respiration by the oxidative phosphorylation system is bypassed; thus, FCCP-induced respiration reflects the maximal capacity of the electron transport chain. Irrespective of the intervention, FCCP was able to enhance mitochondrial respiration considerably over state 3 values, indicating that the electron transport chain is not rate-limiting in state 3. However, FCCP-induced respiration in itself was reduced by ~23% upon prolonged fasting (Fig. 5F), indicating that a combined reduction of both the capacity of the electron transport chain and oxidative phosphorylation underlies the reduced mitochondrial oxidative capacity upon fasting.
The reduced mitochondrial capacity was not accounted for by a reduction in mitochondrial density. Thus, mtDNA copy number, CS activity, and OXPHOS protein levels remained unaffected by fasting. Although this does not exclude the possibility that prolonged exposure to high FFA and/or insulin resistance may lead to a reduced mitochondrial function as observed in type 2 diabetes via reduced mitochondrial biogenesis (e.g., via PGC-1α), this finding indicates that fasting interferes with intrinsic mitochondrial capacity in skeletal muscle. Interestingly, a similar reduction in intrinsic mitochondrial capacity, without differences in mitochondrial content, was recently reported by us in type 2 diabetic patients and first degree relatives when compared with BMI- and age-matched obese control subjects (4
). Thus, the reduction in mitochondrial function upon fasting mimics the situation as observed in the diabetic state, and suggests that similar (secondary) effects are involved in causing mitochondrial dysfunction. However, it should be noted that fasting is not a direct model for type 2 diabetes. Furthermore, although the available data in the literature underpin the notion that fasting-induced lipid accumulation is responsible for reduced insulin sensitivity upon fasting, we cannot exclude the possibility that the reduced mitochondrial function upon prolonged fasting triggers the insulin resistance observed.
Resistance of skeletal muscle to insulin action per se has been suggested to explain the reduction in mitochondrial functional capacity observed in diabetes. Thus, in healthy individuals, it was shown that a 7 h insulin infusion increased mitochondrial protein synthesis, cytochrome C oxidase (COX), and citrate synthase (CS) enzyme activities and ATP production (26
). Moreover, it has been reported that exposing human primary muscle cells to insulin upregulates the expression of PGC-1α (27
). Therefore, the reduction in insulin action in type 2 diabetes may underlie the observed mitochondrial defects. In agreement with this hypothesis, it was demonstrated that at low doses of insulin (reflecting postabsorptive levels) the skeletal mitochondrial ATP synthesis rate was not different between diabetic patients and controls, indicating that there is no intrinsic muscle mitochondrial defect in type 2 diabetic patients (7
). On the other hand, high (postprandial) levels of insulin increased the mitochondrial ATP production rate in nondiabetic subjects, whereas this increase was absent in type 2 diabetic patients (7
). Furthermore, the lack of response in the diabetic patients was accompanied by a reduced expression of PGC-1α, CS, and COX.
Besides reduced insulin action, the hyperglycemia associated with insulin resistance and type 2 diabetes has also been suggested to exert harmful effects on mitochondrial functional capacity via induction of oxidative stress. Indeed, hyperglycemia has been shown to increase mitochondrial ROS production in endothelial cells (28
), as well as in other cell types (29
). In addition, it was reported that severe hyperglycemia inhibited respiration in human skeletal muscle, which was restored upon insulin treatment (13
It should be noted that the insulin-resistant state after prolonged fasting was accompanied by hypoinsulinemia and hypoglycemia. Mitochondrial function was thus assessed after exposure to low insulin and glucose concentrations. It is therefore unlikely that the reduced mitochondrial functional capacity observed here is caused by hyperinsulinemia and/or hyperglycemia associated with reduced insulin action. However, it remains possible that chronic hyperinsulinemia and hyperglycemia may negatively affect mitochondrial function in type 2 diabetes patients.
An alternative candidate to explain the observed reduction in mitochondrial capacity upon fasting is prolonged exposure to elevated plasma FFA levels. This is underscored by previous findings in isolated mouse and human skeletal muscle mitochondria showing a dosage-dependent inhibition of ATP synthesis upon incubation with high but physiologic levels of FFA metabolites (30
). Furthermore, it was shown in mice that prolonged consumption of a high-fat diet for the duration of 16 weeks reduced mitochondrial function (31
). Also in human in vivo studies, negative associations between high fatty acid availability and markers for mitochondrial function have been reported. Thus, PGC-1α expression (9
) and the insulin-stimulated increase in skeletal muscle ATP synthesis (10
) were reduced upon lipid infusion. Furthermore, it was recently shown that mitochondrial membrane potential was impaired upon short-term lipid infusion in healthy individuals, although several other markers of mitochondrial function remained unaffected (32
). Despite the reported negative associations between mitochondrial function and (plasma) FFA, there are also several lines of evidence suggesting the opposite. Thus, raising plasma FFA by high-fat feeding combined with daily heparin injections for 4 weeks in rats increased skeletal muscle mitochondrial biogenesis and mitochondrial enzymes involved in fat oxidation, the citric acid cycle, and the respiratory chain (33
). Furthermore, we previously showed that high-fat feeding for 8 weeks in rats resulted in a twofold increase of PGC-1α protein levels (34
Despite the obvious species differences between these studies, the explanation for the discrepancy in these results remains unclear. Adding to the complexity is the fact that several approaches (high-fat feeding and lipid infusion combined with a hyperinsulinemic-euglycemic clamp) to elevate plasma FFA levels are accompanied by hyperinsulinemia and/or hyperglycemia and insulin resistance, all factors that have also been suggested to interfere with mitochondrial capacity (26
). Finally, differences in absolute levels of plasma FFA achieved in the different studies may contribute to (part of) the variation.
Within the context of mitochondrial lipotoxicity, we and others have previously postulated that mitochondrial UCP3 may be involved in protecting mitochondria against (lipid-induced) oxidative damage (35
). Therefore, we determined protein levels of UCP3 and found that UCP3 content was similar between the fed and the fasted condition. This is a surprising finding since fasting has been quite convincingly shown to increase UCP3 protein levels in animal studies (36
). Moreover, UCP3 mRNA levels were also elevated after 15 h (~5-fold) and 40 h (~10-fold) of fasting in humans (38
). It should be noted, however, that this is the first study to evaluate UCP3 protein content upon prolonged fasting in humans.
The impressive increase in plasma FFA upon prolonged fasting is in line with previous findings in humans (12
), although the absolute values achieved in this study (~2.0 mol/l) are high. Also, the ~2.7-fold increase in IMTG levels after 60 h of fasting is slightly higher in comparison with previous reports (12
). The high plasma FFA and IMTG levels might be caused by complete compliance to the fasting regimen in the present study since, in contrast to other studies, the subjects stayed in a respiration chamber throughout the whole period.
The reduction in insulin-stimulated glucose uptake observed in the present study confirms previous observations showing that prolonged fasting reduced glucose Rd, which was accounted for by a reduction in both insulin-stimulated glucose oxidation and nonoxidative glucose disposal (40
). In agreement with previous reports (40
), we also detected a decreased metabolic flexibility (i.e., the ability to switch from predominantly fat oxidation to glucose oxidation upon insulin stimulation) upon prolonged fasting (). However, not all studies show this effect (41
As anticipated, whole-body fat oxidation increased significantly upon prolonged fasting. Therefore, the decrease in mitochondrial capacity in skeletal muscle is counterintuitive, especially since this decrease was substrate-independent and also apparent upon a lipid substrate.
These results indicate that the reduced mitochondrial capacity is secondary to the fatty acid surplus associated with the insulin-resistant state. It is important to note however, that the reduction in muscle mitochondrial capacity does not (yet) affect the capability of the body to enhance fat oxidation. This is an important finding since it has generally been assumed that a reduction in muscle mitochondrial function will result in reductions in whole-body fat oxidative capacity (3
). Here we show that this extrapolation may not be justified, although we cannot exclude that the fasting-induced reduced mitochondrial function in muscle may decrease muscle-specific fat oxidation compensated by increased fat oxidation in other organs, or may have an impact on the capacity to switch from carbohydrate to fat oxidation (metabolic flexibility).
In conclusion, 60 h of fasting in humans lowered insulin-stimulated glucose uptake down to ~50% along with drastically elevated plasma FFA and IMTG levels. This was accompanied by an overall reduction in intrinsic mitochondrial functional capacity in skeletal muscle, despite a pronounced increase in whole-body fat oxidation. Since prolonged fasting is a physiologic condition in which increased fat oxidation becomes very important, a reduced mitochondrial function seems unbeneficial from a physiologic point of view. Our findings suggest that the elevated plasma FFA and/or intramuscular lipid levels associated with the insulin-resistant state are responsible for the secondary negative effects on mitochondrial function.