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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online April 8, 2003 as doi:10.1096/fj.02-0839fje. |
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,2
aDipartimento di Scienze Biologiche ed Ambientali, Università degli Studi del Sannio, 82100 Benevento, Italy;
Dipartimento di Fisiologia Generale ed Ambientale, Università degli Studi di Napoli "Federico II," 80134 Napoli, Italy; and
Dipartimento di Scienze della Vita, Seconda Università degli Studi di Napoli, 81100 Caserta, Italy
2Correspondence: Dipartimento di Scienze Biologiche ed Ambientali, Università degli Studi del Sannio, Via Port’Arsa 11, 82100 Benevento, Italia. E-mail: goglia{at}unisannio.it; A.L., Dipartimento di Scienze della Vita, Seconda Università degli Studi di Napoli, Via Vivaldi 43, 81100 Caserta, Italy. E-mail: antonia.lanni{at}unina2.it
SPECIFIC AIMS
Uncertainty surrounds the physiological role of uncoupling protein (UCP3), a recently discovered member of a family of mitochondrial carriers almost exclusively expressed in skeletal muscle. An uncoupling function of this protein should be associated with an increased energy expenditure, but a puzzling situation is observed during fasting, in which there is 1) an up-regulation of UCP3, 2) no change in mitochondrial uncoupling, and 3) a decreased energy expenditure.
During fasting, skeletal muscle switches to a reliance on lipids, rather than glucose, as energy substrate. Little is known about 1) the involvement of UCP3 in such fatty acid utilization, 2) the possible interrelated role with the activity of mitochondrial thioesterase I (MTE I), a lipid handling-linked enzyme, and 3) whether the lack of UCP3-mediated uncoupling during fasting is due to low levels of coenzyme Q (CoQ), which may be in vivo a cofactor for UCP3-mediated uncoupling.
PRINCIPAL FINDINGS
1. In skeletal muscle in fasting rats, UCP3 mRNA and MTE I mRNA, and the corresponding proteins, were increased. Injection of T3 further increased these parameters, with the exception of MTE I protein
In fasting, there is UCP3 overexpression, increased lipid utilization, a decreased level of thyroid hormone (T3), and a lack of UCP3-mediated uncoupling. T3, on the other hand, induces an up-regulation of UCP3 with a concomitant uncoupling activity that might be related to the stimulation of some cofactors involved in the regulation of UCP3-mediated uncoupling, such as CoQ and MTE I. Thus, an appropriate experimental design enabling us to study the function of UCP3 might involve fasting rats receiving or not a single dose of T3. Using just such a model, we measured expression levels of UCP3 and MTE I (mRNAs and proteins) in gastrocnemius muscle from normal (N), fasting (exposed to 48 h of fasting, referred to as Fa), and T3-injected fasting rats (exposed to 48 h of fasting and injected i.p. with a single dose of T3 (25 µg/100 g b.w.) at the start of the fast, referred to as Fa+T3). mRNAs for UCP3 and MTE I were increased during fasting by 
2.5- and 2.6-fold, respectively, and further increased in Fa + T3 rats (to 3.6- and 4.3-fold the level seen in N rats, respectively). Protein densities followed the mRNA levels fairly closely in the case of UCP3: protein was 2.6-fold normal in Fa and 4.3-fold normal in Fa+T3 rats. For MTE I, protein was 2.5-fold normal in Fa rats, but T3 administration did not increase it further.
2. Fatty acid oxidation and MTE I activity were increased in fasting rats and further increased after T3 treatment. The same trend was observed for a system involved in fatty acid uptake [carnitine-palmitoyl transferase (CPT)]
To better evaluate the link between the expressions of UCP3 and MTE I and lipid handling, we measured fatty acid oxidation, MTE I activity, and CPT activity. MTE I is involved in intramitochondrial hydrolysis of AcylCoA, resulting in the liberation of free fatty acid (FFA) anions and CoA, in high demand when lipid utilization is elevated. An increase in MTE I activity should always be observed if MTE I activity is linked to UCP3 activity, whose function should be to export excess FFA anions outside the mitochondrial matrix to prevent lipid-induced oxidative damage. Mitochondrial fatty acid oxidation capacity increased in both Fa and Fa+T3 rats: expressed in nAtoms oxygen/min mg protein, values were 110.8 ± 10 in N rats, 160 ± 11 in Fa rats (P<0.05 vs. N rats), and 360 ± 20 in Fa+T3 rats (P<0.05 vs. N rats; P<0.05 vs. Fa rats). Total CPT activity was significantly increased in Fa and Fa+T3 rats. MTE I activity was increased in Fa rats and further increased in Fa+T3 rats, indicating a good correlation between fatty acid oxidative capacity and MTE I activity under such conditions.
3. Palmitoyl-carnitine induced uncoupling only in T3-injected fasting rats
To study mitochondrial UCP3-mediated uncoupling in a situation that involves MTE I, we adopted a novel experimental system in which palmitoyl-carnitine was given as a source of endogenous acyl-CoA. This permits us to 1) avoid exogenous addition of FFA, which could induce a non specific fatty acid-dependent uncoupling and 2) supply a substrate for the formation of free acyls in the matrix by the action of MTE I. In this system mitochondria were energized using succinate and the membrane potential was titrated by sequential addition of palmitoyl-carnitine in the presence of BSA (to chelate the endogenous FFA).
Figure 1
shows the effect of palmitoyl-carnitine on respiration rate (panel A) and membrane potential (panel B) in succinate-energized skeletal muscle mitochondria from Fa and Fa+T3 rats in the presence and absence of 10 µM CoQ. Palmitoyl-carnitine induced an increase in respiration rate and a concomitant decrease in membrane potential (diagnostic of uncoupling) only in mitochondria from Fa+T3 rats, not in those from Fa or N (data not shown) rats. The uncoupling effect induced by palmitoyl-carnitine in Fa+T3 and Fa+CoQ mitochondria was abolished when GDP or superoxide dismutase (SOD) was added to the incubation medium.
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The lack of an effect of palmitoyl-carnitine on membrane potential in mitochondria from Fa rats is unlikely to be due to a poor release of FFA in the mitochondrial matrix since MTE I and CPT activities were increased in these rats (vs. N rats), but can be due to a low level of some endogenous cofactor such as CoQ.
4. Mitochondrial CoQ content, very low in fasting rats, was strongly increased by T3 injection
As shown in Fig. 1
, addition of CoQ (10 µM) to the incubation medium did not further increase the uncoupling effect exerted by palmitoyl-carnitine on mitochondria from Fa+T3 rats but did induce uncoupling in mitochondria from Fa rats. This difference between Fa and Fa+T3 rats in the effect of exogenous CoQ on mitochondria could have been due to different endogenous levels of CoQ. This would explain why although the mitochondria from Fa rats contain a higher level of UCP3 and greater MTE I activity, they not did exhibit uncoupling activity under non-T3-stimulated conditions. To test this possibility, we next measured the mitochondrial CoQ content.
As shown in Fig. 2
, the total amount of mitochondrial CoQ in gastrocnemius muscle from Fa rats was about one-seventh that seen in N animals. In fasting rats, a single injection of T3 induced a strong increase in CoQ levels; values reached were not different from those of N animals. Finally, the above uncoupling effects induced by palmitoyl-carnitine were abolished when SOD was added to the medium, indicating the involvement of reactive oxygen species (ROS) in the UCP3-mediated uncoupling.
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CONCLUSIONS AND SIGNIFICANCE
Current research on UCPs focused on the functions and molecular mechanisms of the novel candidate UCPs identified by the sequence homologies they share with UCP1. It is important both to establish the extent to which they actually possess intrinsic or inducible uncoupling activity in vivo (at physiological levels of expression) and identify the cofactors regulating or influencing that activity. It was recently suggested that CoQ may play a role in activating UCP3 uncoupling activity and that, at least in vitro, the effect is mediated by ROS. We thought that a physiological demonstration of this in an in vivo or ex vivo system would help promote a better understanding of the physiological role of UCP3. We were also unsure as to the physiological association between MTE I protein density/activity (not just mRNA expression) and the presence/activity of UCP3 in a situation involving enhanced lipid metabolism.
By injecting fasting rats with T3, we have demonstrated that 1) the association between UCP3 and MTE I [in terms of their expressions (at mRNA and protein levels) and their activities] holds good in physiological states in which fatty acid oxidation is increased, pointing to a functional link between these factors, 2) the lack of UCP3-mediated uncoupling in skeletal muscle mitochondria from Fa rats is related to a very low level of endogenous CoQ, 3) acute administration of T3 to fasting rats can restore UCP3-mediated uncoupling by regulating the levels of a crucial cofactor for UCP3 such as CoQ, and 4) the functions attributed to UCP3 (i.e., uncoupling and lipid handling) are not mutually exclusive and may coexist in vivo.
Finally, the results reported here (see Fig. 3
) allow us to suggest that in vivo CoQ may represent the factor that, in concert with ROS formation, switches the UCP3-mediated uncoupling on and off. The question remains, Is this "switching on and off" of UCP3 activity directed toward the control of energy expenditure? The present observations favoring such a possibility are that 1) in the fasting state, the increases in UCP3 protein density and MTE I activity are associated with low levels of CoQ and an absence of UCP3-mediated uncoupling whereas 2) in T3-treated animals, increases in UCP3 protein density and MTE I activity are associated with high levels of CoQ and a UCP3-mediated uncoupling.
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FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0839fje; to cite this article, use FASEB J. (April 8, 2003) 10.1096/fj.02-0839fje 
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