HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Full Text (PDF) All Versions of this Article: 19/8/977 04-2765fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Bezaire, V. Articles by Harper, M.-E. Search for Related Content PubMed PubMed Citation Articles by Bezaire, V. Articles by Harper, M.-E.

Constitutive UCP3 overexpression at physiological levels increases mouse skeletal muscle capacity for fatty acid transport and oxidation

V. Bezaire^*, L. L. Spriet^*, S. Campbell^*, N. Sabet, M. Gerrits, A. Bonen^* and M.-E. Harper^,1

^* Department of Human Biology and Nutritional Sciences, University of Guelph, Ontario, Canada; and
Biochemistry, Immunology and Microbiology, Faculty of Medicine, University of Ottawa, Ontario, Canada

¹ Correspondence: Biochemistry, Immunology and Microbiology, Faculty of Medicine, University of Ottawa, Ottawa K1H 8M5, Canada. Email: maryellen.harper{at}uottawa.ca

SPECIFIC AIMS

The physiological function of uncoupling protein 3 (UCP3) is unresolved. UCP3 expression in skeletal muscle closely correlates to fatty acid (FA) oxidation in skeletal muscle. It has been hypothesized that it assists in facilitating high rates of fatty acid oxidation, but evidence is lacking. Our aim was to conduct the first quantitative assessments of the effects of UCP3 overexpression at physiological levels (just over 2-fold) and UCP3 ablation on key transport proteins and enzymes that are central to cytosolic and mitochondrial lipid handling in muscle of mice with congenic backgrounds.

PRINCIPAL FINDINGS

1. Whole body 24 h energy expenditure (VO₂) was unchanged, but the respiratory exchange ratio (RER) decreased with UCP3 overexpression. Serum nonesterifed fatty acids (NEFA) were lower in the UCP3-tg genotype, consistent with increased fatty acid uptake with UCP3 overexpression
UCP3 overexpressing [just over 2-fold, 230% of wild-type (WT), at the protein level; UCP3-tg], and UCP3 ablated [UCP3 (–/–)] mice backcrossed 10 generations into the C57BL6 background were studied in comparison to C57BL6 WT mice. Statistical significance was assessed by 1-way ANOVA with a post hoc Dunnett’s test using WT as control. Significance was accepted at P < 0.05. 24 h VO₂ and respiratory exchange ratio (RER) were studied under ad libitum feeding conditions at 23°C to assess metabolic rate and fuel preference across the genotypes. Additional studies showed no differences in daily energy intake of rodent chow (4.5% fat by weight; Charles River-5075). Although VO₂ was similar across the genotypes, RER was significantly lower in UCP3-tg mice than in WT mice (0.90±0.01 vs. 0.95±0.02). RER of UCP3 (–/–) mice (0.97±0.02) did not differ significantly from WT mice.

Serum analyses were conducted in the fed state. NEFA levels with UCP3 overexpression were 35% lower than those found in WT mice; no change was found in serum glucose and ketone levels. Serum profiles of UCP3 (–/–) did not differ from WT mice. Decreased serum NEFA in UCP3-tg mice is consistent with increased uptake of FA at the muscle cell level and may suggest a shift toward increased FA oxidation.

2. UCP3 overexpression results in increased muscle FA transport protein content, indicative of increased FA binding capacity in muscle
Total content of FA transport proteins on the sarcolemma and in the cytosol was assessed in muscle homogenates. Fatty acid translocase (FAT/CD36) protein content did not differ between WT controls and UCP3-tg or UCP3 (–/–). Plasma membrane fatty acid binding protein (FABPpm) was 36% greater in UCP3-tg than WT and UCP3 (–/–) mice (P<0.05). The cytosolic fraction of FABP (FABPc) was 28% greater in UCP3-tg mice than in WT mice, but did not reach statistical significance (P=0.09). The results indicate increased fatty acid binding capacity at the plasma membrane level with UCP3 overexpression but no apparent impairment of UCP3 ablation.

3. Overexpression of UCP3 results in increased activity of several key mitochondrial enzymes associated with fatty acid oxidation and in lower intramuscular triglyceride (IMTG) content
We assessed characteristics of intramuscular lipid handling. Measurements included assays of hormone sensitive lipase (HSL) activity and IMTG content. No differences were observed in HSL activity across genotypes, but IMTG content was 33% lower in UCP3-tg mice than WT and UCP3 (–/–) mice levels, consistent with increased lipid oxidation and/or decreased lipid deposition (Fig. 1 ).

View larger version (22K): [in this window] [in a new window]   Figure 1. Intramuscular triglyceride content (IMTG) of WT, UCP3-tg, and UCP3 (–/–) mice measured after chloroform-methanol lipid extraction. Results are presented as the mean ± SE. Determinations were conducted on 6–8 mice. Comparisons between genotypes were analyzed by 1-way ANOVA, with a Dunnett’s post hoc test using results of the WT group as the control. Statistical significance was accepted at P < 0.05.

We analyzed mitochondrial FA flux in an isolated mitochondrial preparation and FA oxidative capacity by measuring the maximal activity of ß-oxidation and Krebs cycle enzymes in muscle homogenate. Rate-limiting carnitine palmitoyltransferase I (CPTI) activity was found to be 46% greater in UCP3-tg mice than in WT mice whereas 3-hydroxyacyl-CoA dehydrogenase (ß-HAD) and citrate synthase (CS) activities were 83% and 33% greater, respectively. UCP3 (–/–) activity levels did not differ from WT. The increased activities in UCP3-tg mice occurred without a change in mitochondrial protein content. The results confirm the increased capacity for FA oxidation in UCP3-tg mice.

4. Elevated levels of high energy phosphagens and altered concentrations of muscle metabolites in UCP3-tg mice argue against a role for UCP3 in uncoupling and support the hypothesized role for UCP3 in facilitating fatty acid oxidation capacity
To determine whether the increased capacity for fatty acid oxidation affected levels of high energy phosphagens, we measured muscle levels of total creatine, ATP, and ADP. Samples were collected after an i.p. injection of pentobarbital sodium (30 µg/kg body wt), freeze-dried, and powdered. Contrary to what one might expect if UCP3 were fulfilling a role for mitochondrial uncoupling, total creatine, ATP, and ADP levels were 19, 32, and 17% higher in UCP3-tg than WT mice. ATP levels were significantly higher in UCP3 (–/–) mice than in WT. These findings demonstrate a marked and consistent increase in high energy phosphagens and clearly argue against a role for UCP3 in uncoupling oxidative phosphorylation.

Several relevant muscle metabolites were examined, and findings corroborate the increased oxidative capacity of UCP3-tg mice. UCP3-tg mice had increased levels of free carnitine (47%) and total carnitine (72%) compared with WT mice (Table 1 ). Free carnitine levels in muscle of UCP3 (–/–) mice were greater (24%) than levels in muscle of WT mice. UCP3 overexpression led to increased free CoA (50%) and total CoA (48%) levels compared with WT mice, whereas UCP3 (–/–) mice levels did not differ. Altogether, these results demonstrate increased levels of key cofactors to support increased mitochondrial FA oxidation in UCP3-tg mice.

CONCLUSIONS AND SIGNIFICANCE

The exact role of UCP3 in fatty acid handling is unclear. Our goal was to examine the key steps involved in the transport of FA into the muscle, intramuscular FA handling, transport of FA into the mitochondria, and oxidation of FA in order to further understand the role of UCP3 in fatty acid metabolism in muscle. Correlations between UCP3 expression and FA levels and oxidation were documented shortly after the identification of UCP3 in 1997, but mechanistic hypotheses were lacking. Our findings demonstrate that with > 2-fold increase in UCP3 expression in congenic mice, there is enhanced whole body fat oxidation and increased activity of FA-related enzymes and levels of related metabolites in muscle. Moreover, we demonstrate decreased IMTG content with increased UCP3 expression.

In 2001, two hypotheses were put forward proposing that UCP3 acts as a fatty acid anion exporter. Schrauwen and colleagues proposed that when fatty acid supply exceeds oxidation, a greater proportion of FA enter the mitochondria by an intramembrane FA flip-flop mechanism (bypassing CPT mechanisms), leading to an accumulation of NEFA in the matrix. They proposed that UCP3 is required for outward translocation of the NEFA. Though this hypothesis does not link UCP3 with improved capacity for FA oxidation, it does propose a mechanism for efflux of fatty acid anions that cannot be used in the matrix for fuel.

An hypothesis that does associate UCP3 function with improved capacity for FA oxidation was put forward by Himms-Hagen and Harper. They hypothesized that UCP3 functions in conjunction with a mitochondrial thioesterase (MTE-1) to export fatty acid anions when FA supply exceeds oxidation. MTE-1 functions to cleave CoA units from matrix fatty acyl-CoA and is thought to liberate CoA to support high rates of Krebs cycle and ß-oxidation pathway activities. Himms-Hagen and Harper extended this line of thinking to propose that UCP3 would export the remaining fatty acid anions, as the latter could not be oxidized until they were reactivated outside of the matrix by acyl CoA synthetases (e.g., ACS5, located on the mitochondrial outer membrane). By lowering mitochondrial membrane potential (through anion export from the matrix), reactive oxygen species production would be decreased.

Previous studies have demonstrated increased MTE-1 expression in UCP3-tg mice. The MTE1-UCP3 hypothesis is supported by a strong correlation between UCP3 and MTE-1 expression and activity levels. It has been demonstrated that palmitate oxidation is increased with UCP3 overexpression. Our data substantially extend those findings by demonstrating increased activity of FA-related enzymes and metabolites in congenic mice that constitutively overexpress UCP3 protein at levels observed during fasting. Here we document decreased serum NEFA with UCP3 overexpression and an increased potential for mitochondrial fatty acid uptake, transport, and flux through ß-oxidation and Krebs cycle pathways. Finally, we document decreased IMTG content with increased UCP3 expression (Fig. 2 ). That CoA and carnitine levels are elevated in UCP3-tg mice is consistent with enhanced rates of fat oxidation. Although this study was not designed to test mechanistic aspects of this hypothesis, our results provide additional correlative support.

View larger version (26K): [in this window] [in a new window]   Figure 2. Schematic diagram outlining skeletal muscle fatty acid oxidation and esterification pathways with and without UCP3 overexpression. Boldface arrows and shapes indicate increased flux, activity, or content. FA, fatty acid; LCFA-CoA, long-chain fatty acid CoA; LCFA–, long-chain fatty acid anion; ACS, acyl-CoA synthase; ETC, electron transport chain; CPT2, carnitine palmitoyltransferase 2.

Our results provide novel integrative findings on the effects of UCP3 overexpression and ablation in congenic mice. That genetic background can affect phenotypic characteristics is now widely recognized. Results demonstrate the potential for increased fatty acid transport at the plasma and mitochondrial membranes, less intramuscular storage, and increased mitochondrial fatty acid oxidation with UCP3 overexpression. The observed high energy phosphates and muscle metabolites clearly argue against a role for UCP3 as an uncoupler and support the proposed function of UCP3 as a fatty acid anion exporter in a cycle to support high rates of fatty acid oxidation.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-2765fje; doi: 10.1096/fj.04-2765fje

This article has been cited by other articles:

				Pieter de Lange, M. Moreno, E. Silvestri, A. Lombardi, F. Goglia, and A. Lanni Fuel economy in food-deprived skeletal muscle: signaling pathways and regulatory mechanisms FASEB J, November 1, 2007; 21(13): 3431 - 3441. [Abstract] [Full Text] [PDF]

				B. Faraut, B. Giannesini, V. Matarazzo, T. Marqueste, C. Dalmasso, G. Rougon, P. J. Cozzone, and D. Bendahan Downregulation of uncoupling protein-3 in vivo is linked to changes in muscle mitochondrial energy metabolism as a result of capsiate administration Am J Physiol Endocrinol Metab, May 1, 2007; 292(5): E1474 - E1482. [Abstract] [Full Text] [PDF]

				V. Bezaire, E. L. Seifert, and M.-E. Harper Uncoupling protein-3: clues in an ongoing mitochondrial mystery FASEB J, February 1, 2007; 21(2): 312 - 324. [Abstract] [Full Text] [PDF]

				P. de Lange, P. Farina, M. Moreno, M. Ragni, A. Lombardi, E. Silvestri, L. Burrone, A. Lanni, and F. Goglia Sequential changes in the signal transduction responses of skeletal muscle following food deprivation FASEB J, December 1, 2006; 20(14): 2579 - 2581. [Abstract] [Full Text] [PDF]

				J. L. Barger, B. M. Barnes, and B. B. Boyer Regulation of UCP1 and UCP3 in arctic ground squirrels and relation with mitochondrial proton leak J Appl Physiol, July 1, 2006; 101(1): 339 - 347. [Abstract] [Full Text] [PDF]

				S. R. Costford, S. N. Chaudhry, M. Salkhordeh, and M.-E. Harper Effects of the presence, absence, and overexpression of uncoupling protein-3 on adiposity and fuel metabolism in congenic mice Am J Physiol Endocrinol Metab, June 1, 2006; 290(6): E1304 - E1312. [Abstract] [Full Text] [PDF]

				M. Damon, I. Louveau, L. Lefaucheur, B. Lebret, A. Vincent, P. Leroy, M. P. Sanchez, P. Herpin, and F. Gondret Number of intramuscular adipocytes and fatty acid binding protein-4 content are significant indicators of intramuscular fat level in crossbred Large White x Duroc pigs J Anim Sci, May 1, 2006; 84(5): 1083 - 1092. [Abstract] [Full Text] [PDF]

				G. Solinas, S. Summermatter, D. Mainieri, M. Gubler, J. P. Montani, J. Seydoux, S. R. Smith, and A. G. Dulloo Corticotropin-Releasing Hormone Directly Stimulates Thermogenesis in Skeletal Muscle Possibly through Substrate Cycling between de Novo Lipogenesis and Lipid Oxidation Endocrinology, January 1, 2006; 147(1): 31 - 38. [Abstract] [Full Text] [PDF]

				J. Buchanan, P. K. Mazumder, P. Hu, G. Chakrabarti, M. W. Roberts, U. J. Yun, R. C. Cooksey, S. E. Litwin, and E. D. Abel Reduced Cardiac Efficiency and Altered Substrate Metabolism Precedes the Onset of Hyperglycemia and Contractile Dysfunction in Two Mouse Models of Insulin Resistance and Obesity Endocrinology, December 1, 2005; 146(12): 5341 - 5349. [Abstract] [Full Text] [PDF]

				J. D. MacLellan, M. F. Gerrits, A. Gowing, P. J.S. Smith, M. B. Wheeler, and M.-E. Harper Physiological Increases in Uncoupling Protein 3 Augment Fatty Acid Oxidation and Decrease Reactive Oxygen Species Production Without Uncoupling Respiration in Muscle Cells Diabetes, August 1, 2005; 54(8): 2343 - 2350. [Abstract] [Full Text] [PDF]

This Article Full Text (PDF) All Versions of this Article: 19/8/977 04-2765fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Bezaire, V. Articles by Harper, M.-E. Search for Related Content PubMed PubMed Citation Articles by Bezaire, V. Articles by Harper, M.-E.