A Possible Link Between Skeletal Muscle Mitochondrial Efficiency and Age-Induced Insulin Resistance

Two groups of eight male Wistar rats (Charles River, Calco, Como, Italy) (aged 60 [young] and 180 [adult] days) were used for this study. They were kept at 24°C under an artificial circadian 12-h light/12-h dark cycle, with ad libitum access to water and a standard stock diet (Mucedola 4RF21; Settimo Milanese, Milan, Italy). Treatment, housing, and killing met the guidelines of the Italian Health Ministry.

On the day they were killed and without any previous food deprivation, the rats were anesthetized by an injection of chloral hydrate (40 mg/100 g body wt i.p.) and blood was collected from the inferior cava vein. Immediately after blood collection, hind leg muscles were rapidly removed and used for the preparation of SS and IMF mitochondria (18), while the rat carcasses were used for determination of body composition (18).

Oxygen consumption was measured polarographically with a Clark-type electrode (Yellow Springs Instruments, Yellow Springs, OH) at 30°C in a medium containing 30 mmol/l KCl, 6 mmol/l MgCl₂, 75 mmol/l sucrose, 1 mmol/l EDTA, 20 mmol/l KH₂PO₄, pH 7.0, and 0.1% (wt/vol) fatty acid-free BSA. Substrates used were 10 mmol/l succinate plus 3.75 μmol/l rotenone, 10 mmol/l glutamate plus 2.5 mmol/l malate, or 40 μmol/l palmitoylCoA plus 2 mmol/l carnitine plus 2.5 mmol/l malate. Measurements were performed in the absence (state 4) and presence (state 3) of 0.6 mmol/l ADP. Respiratory control ratio (RCR) was calculated as the ratio between states 3 and 4.

The degree of thermodynamic coupling (q) was obtained by applying equation 11 by Cairns et al. (23).

where J_o represents oxygen consumption. (J_o)_sh and (J_o)_unc were measured as above using succinate plus rotenone in the presence of 2 μg/ml oligomycin or 1 μmol/l FCCP [carbonyl cyanide 4-(trifluoro-methoxy)phenylhydrazone], respectively. The optimal thermodynamic efficiency of oxidative phosphorylation was calculated by using equation 13 by Cairns et al. (23).

The adenine nucleotide translocase (ANT) content of SS and IMF mitochondria was determined by titrating state 3 respiration with increasing concentrations of carboxyatractyloside (24) in a medium containing 30 mmol/l KCl, 6 mmol/l MgCl₂, 75 mmol/l sucrose, 1 mmol/l EDTA, 20 mmol/l KH₂PO₄, 0.1% (wt/vol) fatty acid-free BSA, 10 mmol/l succinate, and 3.75 μmol/l rotenone (pH 7.0). Cytochrome oxidase (COX) specific activity was measured polarographically with a Clark-type electrode (Yellow Springs Instruments) at 30°C in a medium containing 30 μmol/l cytochrome c, 4 μmol/l rotenone, 0.5 mmol/l dinitrophenol, 10 mmol/l Na-malonate, and 75 mmol/l Hepes (pH 7.4) (25).

Oxygen consumption was measured polarographically with a Clark-type electrode (Yellow Springs Instruments) maintained at 30°C in a medium containing 30 mmol/l LiCl, 6 mmol/l MgCl₂, 75 mmol/l sucrose, 1 mmol/l EDTA, 20 mmol/l Tris-PO₄, pH 7.0, and 0.1% (wt/vol) fatty acid-free BSA. Titration of state 4 respiration was carried out by sequential additions of up to 5 mmol/l malonate in the presence of 10 mmol/l succinate, 3.75 μmol/l rotenone, 2 μg/ml oligomycin, 83.3 nmol/mg safranin O, and 80 ng/ml nigericin. Mitochondrial membrane potential recordings were performed in parallel with safranin O using a JASCO dual-wavelength spectrophotometer (511–533 nm) (26). The absorbance readings were transformed into mV membrane potential using the Nernst equation: Δψ = 61 mV × log ([K⁺]_in/[K⁺]_out). Calibration curves made for each preparation were obtained from traces in which the extramitochondrial K⁺ level ([K⁺]_out) was altered in the 0.1- to 20-mmol/l range. The change in absorbance caused by the addition of 3 μmol/l valinomycin was plotted against [K⁺]_out. Then, [K⁺]_in was estimated by extrapolation of the line to the zero uptake point.

Mitochondrial membrane potential and oxygen consumption were measured as above in the presence of 10 mmol/l succinate, 3.75 μmol/l rotenone, 2 μg/ml oligomycin, 83.3 nmol/mg safranin O, and palmitate at a concentration of 45 μmol/l for SS mitochondria or 75 μmol/l for IMF mitochondria. Due to the presence of 0.1% BSA in the incubation medium, the above concentrations of palmitate correspond to 17 (for SS mitochondria) and 62 (for IMF mitochondria) nmol/l free (not bound) fatty acid calculated using the equation of Richieri et al. (27). Palmitate-induced proton leak was estimated from titration of respiration by sequential additions of up to 600 μmol/l malonate for IMF and 1 mmol/l for SS mitochondria.

Mitochondrial protein content of UCP3 was estimated by Western blot analysis, as previously reported (28), by using UCP3 polyclonal antibodies (Chemicon).

Serum insulin and leptin concentrations were measured using radioimmunoassay kits in a single assay to remove interassay variations (Linco Research, St. Charles, MO). Serum glucose and NEFA concentrations were measured by colorimetric enzymatic method using commercial kits (Pokler Italia, Genova, Italy, and Roche Diagnostics, Mannheim, Germany, respectively). Insulin resistance was assessed by homeostasis model assessment (HOMA) index ([glucose {mg/dl} × insulin {mU/l}]/405) (29).

Data are summarized using means ± SE for eight rats. Statistical analyses were performed using a two-tailed unpaired Student’s t test or nonlinear regression curve fit. All analyses were performed using GraphPad Prism (version 4; GraphPad Software, San Diego, CA).

ADP, succinate, rotenone, glutamate, malate, carnitine, palmitoylCoA, ascorbate, dinitrophenol, TMPD (N, N, N′, N′-tetramethyl-p-phenylenediamine), nigericin, nagarse, oligomycin, and safranin O were purchased from Sigma Chemical (St. Louis, MO). Carboxyatractyloside was purchased from Calbiochem (San Diego, CA). All other reagents were of the highest purity commercially available.

The transition from young (60 days) to adult (180 days) age in Wistar rats was associated with a significant increase in body weight and body lipids (Table 1). In addition, significantly higher serum concentrations of leptin, insulin, and NEFAs, together with higher HOMA index, were found in 180-day-old compared with younger rats (Table 1).

Skeletal muscle mitochondrial oxidative capacity was evaluated using lipid and FAD- and NAD-linked substrates. The results obtained in SS mitochondria (Table 2) show that state 3 respiratory rates and RCR values significantly increased in 180- compared with 60-day-old rats, irrespective of the substrate used. No variation in state 4 respiratory rates with any substrate, ANT content, and COX specific activity (Table 2) due to age was found, while UCP3 protein content significantly increased in 180- compared with 60-day-old rats. As for IMF mitochondria (Table 3), a significant increase in state 3 respiratory rates was only found in 180- compared with 60-day-old rats with glutamate, a NAD-linked substrate, although the values with FAD-linked and lipid substrate tended to be higher. State 4 respiratory rates tended to be lower with all the substrates used in 180-day-old rats, although statistical significance was never reached. Consequently, a significant increase in RCR values was found in 180-day-old rats with all the substrates. In addition, ANT content significantly decreased, UCP3 protein content significantly increased, and no significant variation was found in COX specific activity in 180-day-old rats (Table 3).

The degree of thermodynamic coupling (q) and optimal thermodynamic efficiency (η) were calculated to allow evaluation of energetic efficiency of the mitochondrial populations from skeletal muscle. The results show that both SS and IMF mitochondria from 180-day-old rats exhibited higher q and η values compared with mitochondria from 60-day-old rats (Table 4).

Figures 1 and 2 illustrate titration of steady-state respiration rate as a function of mitochondrial membrane potential in SS and IMF skeletal muscle mitochondria. The above titration curves are an indirect measurement of proton leak, since steady-state oxygen consumption rate (i.e., proton efflux rate) in nonphosphorylating mitochondria is equivalent to the proton influx rate due to proton leak. The results show that no variation in basal proton leak was found in SS and IMF mitochondria from 180- compared with 60-day-old rats (Fig. 1). On the other hand, fatty acid-induced proton leak (Fig. 2) significantly decreased in SS and IMF mitochondria in 180- compared with 60-day-old rats.

This study reveals that, in adult rats, higher HOMA index is associated with an increase in oxidative capacity and energy efficiency in skeletal muscle mitochondria. Adult rats can be considered a good experimental tool for studying the onset of the aging phenomenon. In fact, we have found that at 180 days of age, rats display signs of age-induced metabolic disturbance, since they exhibit higher body weight and lipid mass, hyperinsulinemia, hyperleptinemia, and a higher HOMA index. These metabolic perturbations are in agreement with those previously found by others. In fact, Barzilai and Rossetti (3) found that an early fall in insulin responsiveness occurs in rats between 2 and 4 months of age. Our present results also show that, in adult rats, concomitantly with the above metabolic perturbations, there is an increase in skeletal muscle mitochondrial capacity and efficiency.

The generalized increase in state 3 oxidative capacity found in SS mitochondria from 180-day-old rats suggests an increase in one of the steps below complex II. The most feasible target of the above increase should be at the level of cytochromes and/or ATP synthetase, since COX activity was not significantly altered with increasing age, although a tendency to increase was detected. On the other hand, only a stimulation of complex I activity was evident in IMF mitochondria. The determination of ANT content in the two mitochondrial populations allows us to exclude that the above increase in respiratory rates is due to ANT, which was found unchanged or decreased in SS and IMF mitochondria, respectively. The divergent effect of increasing age on respiratory rates and ANT content is not contradictory, since ANT exerts a low control on mitochondrial respiration in skeletal muscle (30). A possible explanation for the different effect of increasing age on SS and IMF mitochondria could be that these two mitochondrial populations have different cellular localization and subserve different metabolic roles in the cell. In fact, IMF mitochondria mainly meet ATP requirements of the contractile elements, while SS mitochondria supply ATP for cytoplasmic reactions.

Previous results have shown that old rats display a decrease in skeletal muscle mitochondrial activities (31–33), and when SS and IMF mitochondria have been studied separately in the heart, the above decrease was found to be restricted to IMF organelles (22). In addition, we have previously found that an aging-induced decline in hepatic mitochondrial activity of complex II is already evident in 180-day-old rats (7), indicating that the liver is affected early by the aging process. Our present results show that skeletal muscle behaves very differently from liver and that, at 180 days of age, oxidative capacity has not yet reached its maximum. In agreement, it has been found that complex II-IV activity of skeletal muscle mitochondria peaks in middle life (1 year) and then declines with increasing age (32).

As for mitochondrial efficiency, both mitochondrial populations display an increase in the degree of thermodynamic coupling and optimal thermodynamic efficiency as well as a decreased fatty acid-dependent proton leak. For IMF mitochondria, decreased fatty acid-dependent proton leak is in agreement with the decreased ANT content of this mitochondrial population, while in SS mitochondria, the reduced sensitivity to palmitate is probably due to the other carrier systems involved, since their ANT content was not affected. Our results also underline the dissociation between regulation of mitochondrial fatty acid-dependent proton leak and UCP3 levels. In fact, the increased UCP3 protein content of SS and IMF mitochondria from 180-day-old rats is in contrast with the lower fatty acid-dependent leak but is consistent with the increased serum NEFA levels, which have been shown to be closely linked to changes in UCP3 protein expression (34).

A number of recent human studies have focused the attention on the mitochondrial compartment as a cellular site involved in the pathogenesis of insulin resistance. In fact, mitochondrial dysfunction has been found in old men (9) and insulin-resistant young subjects (35). In addition, a significant direct relationship between skeletal muscle oxidative capacity and insulin sensitivity has been found (10). An alternative way to reduce mitochondrial oxidation of energy substrates is to increase the efficiency of oxidative phosphorylation. In fact, a higher efficiency implies that less substrate needs to be burned to obtain ATP. Since NEFA serum levels are significantly higher in adult rats, a deleterious consequence of reduced substrate burning could be intracellular triglyceride accumulation and lipotoxicity. The above consequence is of relevance because one of the main causes leading to insulin resistance in skeletal muscle is intramuscular triglygeride deposition (17,36). In addition, skeletal muscle is the primary site of insulin action and is thus inherently linked to the development of whole-body insulin resistance (17).

The increased mitochondrial efficiency and resulting lower thermogenesis in 180-day-old rats could be due to a reduced sensitivity of skeletal muscle cell to leptin. In fact, it has been shown that leptin directly stimulates thermogenesis in skeletal muscle (37) and that 180-day-old rats display age-induced leptin resistance (6). Therefore, leptin resistance could be regarded as one of the primary events leading to insulin resistance.

In conclusion, our present results show that although at 180 days of age no clear biochemical impairment of mitochondrial machinery can be detected, an increased efficiency in the production of ATP takes place. The impact of this modification on cellular metabolism could contribute to the onset of skeletal muscle insulin resistance.

This work was supported by a grant from the Italian Ministry of Education, University and Research (COFIN 2002).

We thank Dr. Emilia De Santis for her skilful management of the animal house.