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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online August 21, 2002 as doi:10.1096/fj.02-0275fje. |
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Department of Human Biology, Maastricht University, 6200 MD Maastricht, The Netherlands;
* Department of Movement Sciences, Maastricht University, 6200 MD Maastricht, The Netherlands; and
Institute of Animal Sciences, Swiss Federal Institute of Technology, 8092 Zürich, Switzerland
2Correspondence: Nutrition and Toxicology Research Institute Maastricht (NUTRIM), Department of Human Biology, Maastricht University, P.O. Box 616, 6200 MD Maastricht, The Netherlands. E-mail: p.schrauwen{at}hb.unimaas.nl
SPECIFIC AIMS
Human UCP3 protein content is increased during fasting, acute exercise, and high-fat intake, all situations in which fatty acid delivery to skeletal muscle exceeds the muscle’s fat oxidative capacity, and is decreased when fat oxidative capacity is improved, like after endurance training and after weight reduction. Here we tested our hypothesis that the physiological function of UCP3 is the transport of nonmetabolizable fatty acids. For this goal, we blocked the entry of fatty acyl-CoA into the mitochondria, thereby creating a situation in which the concentration of nonmetabolizable fatty acids will increase, and we hypothesized that UCP3 would be increased in this situation.
PRINCIPAL FINDINGS
1. Etomoxir administration reduces fatty acid oxidation at rest and during exercise by 
14–19% in healthy human subjects
Etomoxir, a blocker of CPT1, was used to inhibit fatty acid oxidation in healthy male subjects (n=10, age=25.6±1.7 year, BMI=21.8±0.3 kg/m2). To create a situation in which fat oxidative capacity was maximally used, subjects were fed with high-fat diets (60 en% as fat) for 3 days at home and then during a 36 h stay in a respiration chamber. Etomoxir administration significantly reduced 24 h fat oxidation by 14% (158±6 g/24 h with placebo vs. 136±5 g/24 h with Etomoxir, P<0.05, Fig. 1
a). During the first night in the respiration chamber, only a few hours after administration of the first dosage of Etomoxir or placebo, fat oxidation tended to be reduced after Etomoxir (142±7 g/24 h with placebo vs. 130±6 g/24 h with Etomoxir, P=0.12, Fig. 1b
); this reduction increased up to 19% during the second night (139±5 g/24 h with placebo vs. 113±4 g/24 h with Etomoxir, P<0.005, Fig. 1b
), illustrating a cumulative effect of repeated Etomoxir administration. During exercise (2 h at 50% Wmax), when the relative and absolute contribution of fatty acid oxidation is high, Etomoxir attenuated the normally observed increase in fat oxidation (Fig. 1c
, P<0.05). Together, these data show that Etomoxir was effective in lowering fat oxidative capacity by 14–19%, indicating that Etomoxir reduced the entry of esterified fatty acids into the mitochondria.
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2. Etomoxir administration affects plasma fatty acid and glucose levels
Fasting blood samples were collected at rest and every 30 min during the exercise test. Resting plasma FFA levels were not significantly affected by Etomoxir administration (487±76 µmol/l after Etomoxir vs. 382±44 µmol/after placebo, NS), but during exercise FFA levels tended to increase more pronounced after Etomoxir (P=0.08). Fasting plasma glucose levels at rest were significantly lower after Etomoxir (4.60±0.14 mmol/l after Etomoxir vs. 4.84±0.13 mmol/after placebo, P<0.01). During exercise, glucose levels decreased in both treatments, but the decrease was much more pronounced after Etomoxir (from 4.6±0.1 mmol/l at t=30 to 4.0±0.1 mmol/l at t=120 with placebo and from 4.4±0.2 mmol/l at t=30 to 3.5±0.1 mmol/l at t=120 with Etomoxir, P<0.05), which further illustrates that Etomoxir inhibited fat oxidation and promoted carbohydrate utilization. Plasma ß-hydroxybutyrate (BHB) concentration, both at rest and during exercise, was not affected by Etomoxir.
3. The Etomoxir-induced reduction in fat oxidation leads to a rapid and pronounced increase in UCP3 protein content
Using Western blot, we determined UCP3 protein content in skeletal muscle biopsies from M. vastus lateralis taken in the fasted state after leaving the respiration chamber. Etomoxir administration increased UCP3 protein content in all subjects, on average by 67%, compared to placebo (72±8 AU after Etomoxir vs. 43±11 AU after placebo, P<0.0005). The increase in UCP3 protein content induced by Etomoxir was negatively correlated with the Etomoxir-induced decrease in fat oxidation during the first (r=-0.77, P<0.05) and second night (r=-0.77, P<0.05, Fig. 2
) in the respiration chamber and with 24 h fat oxidation (r=-0.71, P<0.05). Furthermore, we observed that the difference in fasting BHB concentration, an indirect indicator of the efficacy of CPT1-blockade, between Etomoxir and placebo correlated very strongly and negatively (r=-0.89, P<0.005) with the Etomoxir-induced increase in UCP3 protein. These correlations indicate that inhibition of the entry of esterified fatty acids via CPT1 into the mitochondria is crucial for the increase in UCP3 protein content.
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4. The increase in UCP3 protein content is accompanied by an enhanced translocation of GLUT 4 to the plasma membrane
Since Etomoxir administration promoted carbohydrate utilization (illustrated by lowered fasting glucose levels), we examined whether up-regulation of UCP3 after Etomoxir administration, as a secondary effect, promoted translocation of GLUT4 in human skeletal muscle. Quantitative image analysis of all fasting (pre-exercise) samples revealed that the ratio of intensity of the green fluorescent GLUT4 signal in sarcolemma: sarcoplasm was significantly higher after Etomoxir compared to placebo (1.18±0.02 vs. 1.12±0.02, P<0.05), indicating increased recruitment of GLUT4 to the cell surface (representative pictures are shown in the full paper).
CONCLUSIONS AND SIGNIFICANCE
Soon after the discovery of human UCP3, indications were found that the primary physiological role of UCP3 is not the regulation of energy turnover, as would be predicted from its homology to UCP1. Thus, mice lacking UCP3 have a normal metabolic rate and body weight, and fasting, an energy-preserving condition, rapidly up-regulates the expression of UCP3. Rather, several groups suggested a role for UCP3 in fatty acid handling. Based on the finding that UCP3 content is high in situations where fatty acid delivery to the muscle exceeds the oxidative capacity, we hypothesized that UCP3, which had been shown to be able to transport fatty acid anions, functions as an outward transporter of non-esterified fatty acid anions from the mitochondrial matrix, thereby protecting mitochondria against accumulation of non-esterified fatty acids that enter the mitochondrial matrix by "flip-flop" across the mitochondrial inner membrane (Fig. 3
). To test this hypothesis, we used Etomoxir to inhibit CPT1, thereby blocking the entry of fatty acids into the mitochondria in a form (fatty acyl-CoA) in which they are available for oxidation. We observed that Etomoxir was effective in lowering fat oxidative capacity by 14–19. For comparison, the complete absence of acetyl-CoA carboxylase, which indirectly inhibits CPT1, increases fat oxidation by 30%, indicating that a 14–19% reduction in fat oxidation undeniably reflects an effective blockade of CPT1. As a consequence, non-esterified (and thus nonmetabolizable) fatty acids accumulate in the cytoplasm and the entry of these non-esterified fatty acids into the mitochondrial matrix by flip-flop is increased (Fig. 3)
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In accordance with our hypothesis, we found that the Etomoxir-induced reduction in fat oxidative capacity was accompanied by a rapid and pronounced increase in UCP3 protein content by 67%. This up-regulation of UCP3 after Etomoxir was related to the reduction of fat oxidative capacity. These data provide support for our hypothesis that UCP3 functions as an outward transporter of non-esterified fatty acid anions from the mitochondrial matrix (Fig. 3)
. Outward transport of non-esterified fatty acid anions from the mitochondrial matrix by UCP3 has also been hypothesized by Himms-Hagen et al. However, they suggested that UCP3 exports fatty acid anions from the matrix that are delivered by hydrolysis of fatty acyl-CoA by mitochondrial thioesterases, when fatty acids are the principal substrate used. Clearly, the presently observed up-regulation of UCP3 on inhibition of fat oxidation by CPT1 blockade is not in line with their hypothesis, though we cannot exclude that under certain conditions the fatty acid anions exported by UCP3 are derived from hydrolysis of fatty acyl-CoA.
As a result of the reduction in fat oxidative capacity, carbohydrate oxidation needs to increase to supply sufficient energy. Indeed, Etomoxir increased carbohydrate oxidation and reduced plasma glucose concentration both at rest and during exercise. Mice overexpressing UCP3 are also characterized by lower plasma glucose levels and by an improved glucose clearance rate and overexpression of UCP3 in L6 myotubes increases glucose uptake through an increased recruitment of glucose transporter-4 (GLUT4) to the cell surface. In the present study, sarcolemmal staining was increased after Etomoxir, indicating increased recruitment of GLUT4 to the cell surface. Whether the up-regulation of UCP3 was responsible for GLUT4 translocation cannot be deduced from the present study; therefore, further studies are needed to reveal whether the up-regulation of UCP3 and increased GLUT4 translocation are related events.
In conclusion, we present support here for a novel physiological function of the human uncoupling protein 3 as an exporter of fatty acid anions. This physiological function of UCP3 can explain the majority of the findings on UCP3 regulation under different (physiological) conditions. Still, UCP3 could act as an uncoupling protein, since the outward transport of fatty acid anions actually decreases the proton gradient across the mitochondrial membrane, thereby uncoupling mitochondrial respiration, explaining the previously observed association of UCP3 with energy metabolism. As a secondary effect, up-regulation of UCP3 was accompanied by GLUT4 translocation. Given that UCP3 protein content is decreased in type 2 diabetic subjects, UCP3 might be an important pharmaceutical target in the treatment of type 2 diabetes mellitus.
FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0275fje; to cite this article, use FASEB J. (August 19, 2002) 10.1096/fj.02-0275fje 
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