DOI 10.1007/s00424-002-0964-0 S K E L E T A L M U S C L E
Susanna Iossa · Maria Pia Mollica · Lill Lionetti · Raffaella Crescenzo · Monica Botta · Sonia Samec · Giovanni Solinas · Davide Mainieri · Abdul G. Dulloo · Giovanna Liverini
Skeletal muscle mitochondrial efficiency and uncoupling protein 3
in overeating rats with increased thermogenesis
Received: 24 July 2002 / Accepted: 26 September 2002 / Published online: 6 November 2002 Springer-Verlag 2002
Abstract To establish whether changes in skeletal mus-cle mitochondrial efficiency contribute to increased energy expenditure and decreased metabolic efficiency of overeating rats with increased thermogenesis, we measured basal proton leak, fatty acid-induced uncou-pling and uncouuncou-pling protein 3 (UCP3) content in subsarcolemmal and intermyofibrillar skeletal muscle mitochondria. Intermyofibrillar, but not subsarcolemmal, mitochondria from rats with increased thermogenesis exhibited a lower proton leak compared with controls. In both mitochondrial populations from rats with increased thermogenesis, fatty acid-induced uncoupling was in-creased significantly and a small recoupling effect of GDP was detected. In addition, intermyofibrillar and subsarcolemmal mitochondria from rats with increased thermogenesis showed higher UCP3 contents than con-trols. These results point out that metabolic efficiency in subsarcolemmal and intermyofibrillar mitochondria from rats with increased thermogenesis is differently regulated. In fact, in intermyofibrillar mitochondria both basal proton leak and fatty acid-induced uncoupling are altered, while in subsarcolemmal mitochondria only fatty acid-induced uncoupling increases. Both mitochondrial popu-lations in skeletal muscle cells from rats with increased thermogenesis display an increased fatty acid-induced uncoupling and UCP3 content, which could contribute to avoiding obesity.
Keywords Uncoupling protein 3 · Mitochondrial efficiency · Uncoupling · Obesity resistance
Introduction
Previous work from our laboratory has shown that post-weaning rats fed a palatable high-fat diet for 15 days increase their metabolizable energy and lipid intake by about 40 and 200%, respectively [12, 13, 20], but resist the development of obesity by markedly increasing energy expenditure [12, 13, 20]. Such a change in metabolic efficiency at the whole body level, most likely via the activation of diet-induced thermogenesis, is the outcome of changes in metabolic efficiency occurring at the level of organs and tissues, which largely contribute to whole body energy expenditure. Of the candidate sites for dissipating surplus energy as heat, skeletal muscle is particularly relevant since its metabolism accounts for about 30% of resting energy expenditure in the rat [25].
At the cellular level, the major determinants of the efficiency of energy transduction are the mitochondria, where energy coming from the oxidation of metabolic fuels is converted to ATP. It is now accepted that the efficiency of the mitochondrial machinery depends on the presence of a proton leak pathway in the inner mitochon-drial membrane [2, 31], which reportedly accounts for about 20% of resting metabolic rate in rats [24]. In addition, it is well known that free fatty acids (FFA) are responsible for so-called “mild uncoupling” in mitochon-dria [10, 16, 26, 27, 29, 30, 33], due to non-ionic diffusion coupled with the transport of the fatty acid anion out of the mitochondria through various mitochondrial carriers, such as the adenine nucleotide translocase (ANT) and the glutamate-aspartate carrier and possibly uncoupling pro-teins (UCP) 2 and 3. The combination of basal leak and fatty acid-induced uncoupling could thus determine the efficiency of the mitochondrial system.
Based on the above considerations, the objective of the present study was to assess whether changes in mito-chondrial efficiency in the skeletal muscle could con-tribute to the elevated energy expenditure and decreased metabolic efficiency of rats fed high-fat diet. To address this issue, we measured the basal proton leak and determined the recoupling effects of carboxyatractyloside
S. Iossa ()) · M.P. Mollica · L. Lionetti · R. Crescenzo · M. Botta · G. Liverini
Department of General and Environmental Physiology, University of Naples “Federico II”, Via Mezzocannone 8, I-80134, Naples, Italy
e-mail: susiossa@unina.it Tel.: +39-81-2535086 Fax: +39-81-2535090
R. Crescenzo · S. Samec · G. Solinas · D. Mainieri · A.G. Dulloo Institute of Physiology, University of Fribourg, Fribourg, Switzerland
(CAT), glutamate, GDP and ADP to assess the contribu-tion of ANT, the glutamate-aspartate carrier and the UCP homologues to fatty acid-induced uncoupling. In addition, we measured mitochondrial UCP3 content in skeletal muscle from control rats and rats with increased thermo-genesis to determine whether changes in mitochondrial efficiency could be linked with changes in UCP3 content. These measurements were made in both subsarcolemmal and intermyofibrillar mitochondria, since we have shown previously that these two mitochondrial populations – which are functionally distinct and differently regulated under various physiological conditions [1, 4, 18, 34] – also have different basal proton leak [14] and fatty acid-induced uncoupling [15].
Materials and methods
Animals and diets
Male Wistar rats of about 25 days of age (Charles River Italia, Calco, Como, Italy) were housed individually in metabolic cages in a temperature-controlled room (24€1 C) under a 12 h light-dark cycle (0700–1900 hours). Rats were allowed free access to water and a low-fat (10.6 kJ/100 kJ, control rats) or high-fat (50 kJ/100 kJ, overeating rats) diet [12, 13, 20] for 15 days. Treatment, housing, and killing met the guidelines of Italian Health Ministry.
During the diet treatment, food intake and weight gain were monitored daily. Faeces and urine were also collected on a daily basis for the measurement of metabolizable energy (ME) intake. At the end of the experimental period, rats were killed by decapitation and hind leg muscles removed rapidly for the preparation of subsarcolemmal and intermyofibrillar mitochondria, as described previously [14], while the carcass was used for the determination of energy, lipid, protein and water body content, as reported previ-ously [12, 13, 20]. Immediately after isolation, an aliquot of mitochondria was stored at –80 C for assay of UCP3, while the remainder was used for measurement of basal and fatty acid-induced uncoupling and ANT content.
Measurements of basal proton leak kinetics
Oxygen consumption was measured polarographically with a Clark-type electrode (Yellow Springs Instruments, Yellow Springs, Ohio, USA) maintained at 30 C in a medium containing (in mM) 30 LiCl, 6 MgCl2, 75 sucrose, 1 EDTA, 20 TRIS-PO4(pH 7.0) and
0.1% (w/v) fatty acid-free bovine serum albumin (BSA). Titration of State 4 respiration was carried out by sequential additions of malonate up to 5 mM in the presence of succinate (10 mM), rotenone (3.75 M), oligomycin (2 g/ml), safranin O (83.3 nmol/ mg) and nigericin (80 ng/ml, to collapse the pH difference across the mitochondrial inner membrane and allow the whole of the proton-motive force to be represented by mitochondrial membrane potential). Mitochondrial membrane potential recordings were performed in parallel with safranin O using a JASCO dual-wavelength spectrophotometer (511–533 nm) [21]. The absorbance readings were transformed into membrane potential (Dy, in millivolt) using the Nernst equation: Dy=61·log ([K+]
in/[K+]out).
Calibration curves made for each preparation were obtained from traces in which the extramitochondrial [K+] ([K+]
out) was altered in
the range 0.1–20 mM. The change in absorbance caused by the addition of 3 M valinomycin was plotted against [K+]out. [K+]in
was then estimated by extrapolation of the line to the zero-uptake point.
Measurement of fatty acid-induced uncoupling
Mitochondrial membrane potential was measured in the same medium and with the same procedure as above, before and after addition of palmitate. Once a steady state was reached with palmitate, the recoupling effect of various substances was tested, namely CAT (10 nmol/mg protein), glutamate (7 mM), GDP (0.2 mM) and ADP (36 M), which were added in sequence.
Measurement of ANT content
The ANT content of subsarcolemmal and intermyofibrillar mito-chondria was determined by titrating State 3 respiration with increasing concentrations of CAT [32] in a medium containing (in mM) 30 KCl, 6 MgCl2, 75 sucrose, 1 EDTA, 20 KH2PO4, 0.1% (w/
v) fatty acid free BSA, 10 succinate and 3.75 M rotenone (pH 7.0). Mitochondria were preincubated with CAT in the respiratory medium for 1 min before ADP (0.3 mM) was added to initiate State 3 respiration. The mitochondrial content of ANT was determined by the extrapolation of the linear part of the titration curve to obtain the amount of CAT required to completely inhibit State 3 respiration. The turnover number of ANT was calculated from the slope of the inhibitory region, after conversion of respiration rate into phosphorylation rate with the ADP/O ratio [8].
Measurement of UCP3 protein content
Mitochondrial protein (40 g) was denatured in 3sample buffer (60.0 mM TRIS pH 6.8, 10% saccharose, 2% SDS, 4% b-mercaptoethanol) and loaded onto a 12% SDS-polyacrylamide gel together with a prestained protein marker (Benchmark, Life Technologies). After a 2-h run in electrode buffer (50 mM TRIS pH 8.3, 384 mM glycine, 0.1% SDS) gels were transferred onto polyvinylidene difluoride (PVDF) membranes (Immobilon-P, Mil-lipore) at 0.8 mA/cm2for 90 min. The membranes were preblocked in blocking buffer (1PBS; 5% milk powder; 0.5% Tween 20) for 1 h and then incubated overnight at 4 C with UCP3 polyclonal antibodies (Chemicon) diluted 1:3,000 in blocking buffer; the specificity of the antibody for UCP3 has been validated [3]. Membranes were washed (315 min) in 1PBS/0.5% Tween 20, 315 min in 1PBS and then incubated for 1 h at room temperature with alkaline phosphatase-labelled antibodies (Promega). The membranes were washed as described above, rinsed in distilled water and incubated at room temperature for 30 min with a 1:500 dilution of a chemiluminescence substrate (CDP star, Roche). Signals were detected by exposing autoradiographic films (Kodak) to the membranes, acquired by densitometry (Bio-Rad, model GS-700 Imaging Densitometer) and quantified using Molecular Analyst software (Bio-Rad).
Statistical analysis
Data are given as means€SEM for six rats. Statistical analyses were performed using two-tailed unpaired Student’s t-test, two-way analysis of variance for main effects and interactions, or non-linear regression curve fit. All analyses were performed using GraphPad Prism (GraphPad Software, San Diego, Calif., USA).
Materials
ADP, succinate, rotenone, nigericin, nagarse, oligomycin, safranin O were purchased from Sigma (St. Louis, Mo., USA). CAT was purchased from Calbiochem (San Diego, Calif., USA). All other reagents were of the highest purity commercially available.
Results
Young rats fed our high-fat diet increased, as expected, their ME intake (control 3,010€93 kJ, overeating 3,910€101 kJ, P<0.05), while body energy gain was not affected by high-fat feeding (control 842€54 kJ, overeat-ing 899€50 kJ) and energy expenditure significantly increased (control 2,160€80 kJ, overeating 3,011€85 kJ, P<0.05). These findings are reflected in the data for metabolic efficiency (energy gain/ME intake) which was significantly lower (0.23€0.01, P<0.05) in overeating rats with increased thermogenesis than in controls (0.28€0.02).
Figure 1 shows the titration of steady state respiration rate as a function of mitochondrial membrane potential in intermyofibrillar and subsarcolemmal skeletal muscle mitochondria. The titration curves are an indirect mea-surement of mitochondrial proton leak, since steady-state oxygen consumption rate (i.e. proton efflux rate) in non-phosphorylating mitochondria is equivalent to proton influx rate due to proton leak. Non-linear regression analysis showed that experimental data fitted an expo-nential growth equation. Comparison of non-linear re-gression curve fits revealed a significant (P<0.05) difference only in intermyofibrillar mitochondria from overeating rats with increased thermogenesis, compared with controls.
Fatty acid-induced uncoupling in subsarcolemmal and intermyofibrillar skeletal muscle mitochondria was mea-sured as the palmitate concentration needed to obtain a half-maximal decrease in membrane potential (C1/2) and
is presented in Fig. 2. Fatty acid-induced uncoupling was significantly higher in subsarcolemmal than in intermy-ofibrillar mitochondria from control rats, and increased significantly in both mitochondrial populations from overeating rats with increased thermogenesis. We also determined the recoupling effects of CAT, glutamate, GDP and ADP by assessing the percentage decrease in membrane potential induced by palmitate at C1/2, which
was abolished by the above substances. In intermyofib-rillar mitochondria, fatty acid-induced uncoupling was mainly attributable to ANT (51.9€7.6%) and glutamate carrier (23.2€2.1%), with no recoupling effect of ADP (0€0%) or GDP (0€0%). In subsarcolemmal mitochon-dria, the glutamate carrier contribution was similar to that observed in intermyofibrillar mitochondria (20.5€2.9%), the ANT contribution was significantly lower (12.9€1.4%, effect of mitochondrial type, P<0.05) whilst a small recoupling effect of ADP (4.0€2.7%, effect of mitochondrial type, P<0.05) but not of GDP (0€0) was observed. In overeating rats with increased thermogene-sis, the recoupling effect of ANT was decreased signif-icantly in intermyofibrillar (26.5€4.4%, effect of treatment, P<0.05) and subsarcolemmal (3.7€0.7%, effect of treatment, P<0.05) mitochondria, while the glutamate (intermyofibrillar 22.7€2.2%; subsarcolemmal 14.9€2.9%) and ADP (intermyofibrillar 0€0%; subsarcolemmal 5.7€2.1%) recoupling effects were not affected. In addition, a recoupling effect of GDP could be detected
in intermyofibrillar (3.7€1.3%, effect of treatment, P<0.05) and subsarcolemmal (3.2€1.2%, effect of treat-ment, P<0.05) mitochondria.
The ANT contents of intermyofibrillar and subsar-colemmal mitochondria are shown in Figs. 3 and 4. In control rats, ANT content was higher in subsarcolemmal
Fig. 1 Kinetics of the basal proton leak in subsarcolemmal and intermyofibrillar skeletal muscle mitochondria from control and overeating rats. Means€SEM, n=6
Fig. 2 Fatty acid-induced uncoupling in subsarcolemmal and intermyofibrillar skeletal muscle mitochondria from control and overeating rats. Results are reported as the palmitate concentration needed to obtain a half-maximal decrease in mitochondrial membrane potential. Means€SEM, n=6. *P<0.05 for main effect of treatment;#P<0.05 for main effect of mitochondrial type
than in intermyofibrillar mitochondria. In addition, in overeating rats with increased thermogenesis, a signifi-cant increase in ANT content was found only in subsarcolemmal mitochondria. In control rats, the ANT turnover number was 835€54 and 112€10 min–1 in intermyofibrillar and subsarcolemmal mitochondria, re-spectively. No variation was found in overeating rats with increased thermogenesis (800€35 and 133€19 min–1 in intermyofibrillar and subsarcolemmal mitochondria). ANT total activity, calculated from content and turnover, was 2,472€198 and 482€33 nmol/min per mg protein in intermyofibrillar and subsarcolemmal mitochondria from
control rats and 2,384€201 and 654€48 nmol/min per mg protein in intermyofibrillar and subsarcolemmal mito-chondria from overeating rats with increased thermogen-esis.
Both the intermyofibrillar and subsarcolemmal mito-chondria from overeating rats with increased thermogen-esis showed significantly higher contents of UCP3 than those from control rats (Fig. 5).
Discussion
The most important finding in the present study is that the increase in energy expenditure in response to overfeeding in young rats is accompanied by changes in mitochondrial efficiency and UCP3 content. Mitochondrial efficiency is primarily the result of two cellular mechanisms, i.e. basal proton leak and fatty acid-induced uncoupling. The basal proton leak operates in a variety of cells and tissues [2, 31] and its quantitative contribution to resting energy expenditure is believed to be important [25]. To date, however, it is still not clear which mechanisms are responsible for regulating basal proton leak [22, 31]. In addition, the evidence that the basal proton leak is altered in response to physiological stimuli is scarce [9, 19]. We have shown recently that the basal proton leak increases in subsarcolemmal, but not in intermyofibrillar skeletal muscle mitochondria after 24-h fasting [14]. Here we show that basal proton conductance decreases in intermy-ofibrillar, but not in subsarcolemmal, skeletal muscle mitochondria from rats with increased thermogenesis. The variation observed in basal proton leak is in the direction opposite to that expected for its postulated thermogenic role. In addition, the present results in rats with increased thermogenesis further confirm the dissociation previously
Fig. 4 ANT content in subsarcolemmal and intermyofibrillar skeletal muscle mitochondria from control and overeating rats. Means€SEM, n=6. The individual ANT contents were determined on each mitochondrial preparation by extrapolation of the linear part of the titration curve, as shown in Fig. 3. *P<0.05 for main effect of treatment;#P<0.05 for main effect of mitochondrial type; §P<0.05 for interaction
Fig. 5 Uncoupling protein 3 (UCP3) content in subsarcolemmal and intermyofibrillar skeletal muscle mitochondria from control and overeating rats. Results are given as arbitrary units (control=1). Means€SEM, n=4. UCP3 levels in overeating rats are relative to controls. 1, 3, 5 and 7 overeating rats; 2, 4, 6 and 8 control rats. *P<0.05 vs. controls
Fig. 3 Determination of adenine nucleotide translocase (ANT) content by titration of State 3 respiration with carboxyatractyloside in subsarcolemmal and intermyofibrillar skeletal muscle mitochon-dria from control and overeating rats. Means€SEM, n=6. For statistical analysis of the means, see legend to Fig. 4
observed during fasting between basal proton leak and UCP3 protein levels [14]. In fact, the increase in UCP3 in both subsarcolemmal and intermyofibrillar skeletal mus-cle mitochondria from rats with increased thermogenesis is accompanied by unchanged or decreased basal proton leak, respectively.
It is well known that FFA serve two roles in the cell fuel metabolism: they act as substrates for mitochondrial oxidation and ATP production and have been postulated to mediate the phenomenon of “mild uncoupling” through the operation of the proton-conducting fatty acid cycle [12, 25]. This cycle is based on the passive DpH-driven influx of protonated fatty acids to the mitochondrial matrix via the phospholipid bilayer of the inner mito-chondrial membrane and the electrophoretic efflux of the deprotonated fatty acid anion mediated by mitochondrial anion carriers, namely ANT, the aspartate/glutamate antiporter and possibly UCP2 and 3 [10, 16, 26, 27, 29, 30, 33]. The result of the operation of such a cycle is the entry of protons into the mitochondrial matrix, which lowers DH+and stimulates oxygen consumption.
Previ-ously, we have shown an increase both in FFA serum levels in rats with increased thermogenesis [20] and in the fatty acid-induced uncoupling in intermyofibrillar and subsarcolemmal skeletal muscle mitochondria [15]. We were, therefore, interested in investigating the recoupling effect of CAT, glutamate, GDP and ADP to assess the role of ANT, the glutamate-aspartate carrier and the UCP homologues, respectively. In control rats, ANT was responsible for about 52 and 13% of the uncoupling in intermyofibrillar and subsarcolemmal mitochondria, re-spectively, despite the fact that subsarcolemmal mito-chondria contained a higher amount (+46%) of ANT than intermyofibrillar ones. However, if it is taken into account that the turnover number of ANT was about sixfold higher in intermyofibrillar than in subsarcolemmal mitochondria, then the total activity of ANT was about fourfold higher in intermyofibrillar than in subsarcolemmal mitochondria, thereby explaining its different contribution to fatty acid-induced uncoupling in the two mitochondrial populations. In rats with increased thermogenesis, ANT contribution to uncoupling significantly decreased in intermyofibrillar and subsarcolemmal mitochondria, respectively, despite an increase in ANT protein content in subsarcolemmal mitochondria, while no significant variation was found in the glutamate carrier contribution.
Another inner membrane protein that could be in-volved in the fatty acid-induced uncoupling is UCP3, since it shares homology not only with UCP1, but also with the other mitochondrial carriers [23]. Unfortunately, no molecule displaying a clear inhibitory effect on UCP3 function is available at the moment. It is well known that UCP1 is inhibited by GDP [17] and reconstituted UCP3 is inhibited by micromolar ADP [6]. We therefore tested the efficacy of ADP and GDP as inhibitors of the fatty acid-induced uncoupling. Under our experimental conditions, ADP displayed a very weak (about 4%) inhibitory effect in subsarcolemmal mitochondria and was completely without effect in intermyofibrillar mitochondria. GDP, on
the other hand, had no effect in both mitochondrial populations from control rats, while in rats with increased thermogenesis a small recoupling effect (about 4%) of GDP became evident. The recoupling effect of GDP in mitochondria from rats with increased thermogenesis is in line with their increased UCP3 protein content. Similarly, an inhibitory effect of GDP on uncoupling effect of fatty acid is seen in heart muscle mitochondria, concomitantly with an increase in UCP2 mRNA levels, albeit in rats exposed to cold [28]. Very recently, Echtay et al. [7] have found that uncoupling through UCP3 is activated by externally added xanthine+xanthine oxidase and inhibited by GDP. However, independent of the putative uncou-pling effect of UCP3, the increased UCP3 protein levels in rats with increased thermogenesis are in line with its proposed involvement in the regulation of lipids as fuel substrates [5]. In fact, we have shown previously that skeletal muscle substrate metabolism switches to a state of enhanced lipid oxidation in rats with increased thermogenesis [15] and it has been proposed that UCP3 might be involved in the putative export of unoxidised fatty acids out of mitochondria to avoid intramitochon-drial depletion of reduced coenzyme A (CoASH) when fatty acid oxidation predominates [11], although the exact mechanism remains to be established.
In conclusion, the present studies indicate that the metabolic efficiency in subsarcolemmal and intermyofib-rillar mitochondria from rats with increased thermogen-esis is regulated differently. In intermyofibrillar mitochondria both basal leak and fatty acid-induced uncoupling are altered, while in subsarcolemmal mito-chondria only fatty acid-induced uncoupling is increased. A possible explanation for this discrepancy between both populations could be that intermyofibrillar mitochondria mainly meet ATP requirements of the contractile ele-ments [1, 4]. Therefore, an increase in fatty acid-induced uncoupling in rats with increased thermogenesis, together with increased serum FFA levels, could result in a very severe depression of the metabolic efficiency of intermy-ofibrillar mitochondria and hence in the ATP supply for muscle activity. The decrease in basal proton leak in favour of increased fatty acid-induced uncoupling would have the advantage of providing the mitochondria with a proton leak pathway regulated by cytoplasmic FFA concentrations. On the other hand, in subsarcolemmal mitochondria, which supply ATP for cytoplasmic reac-tions [1, 4], the control of metabolic efficiency is less stringent, as also shown by the fact that subsarcolemmal mitochondria are more sensitive to the uncoupling effect of palmitate than intermyofibrillar ones [15]. Therefore, in this mitochondrial population an increase in fatty acid-induced uncoupling is not counterbalanced by a decrease in basal proton leak. The net result of the above modifications is that both mitochondrial populations in skeletal muscle cells from rats with increased thermo-genesis display an increased fatty acid-induced uncou-pling, which, together with their increased lipid oxidation capacity [15] and UCP3 protein content, could contribute to resistance to obesity.
Acknowledgements This work was supported by a grant from University “Federico II” of Naples, by Grant 3200.061687 of the Swiss National Science Foundation, and by a grant from the Roche Research Foundation (Switzerland).
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