Essential Role for Uncoupling Protein-3 in Mitochondrial Adaptation to Fasting but Not in Fatty Acid Oxidation or Fatty Acid Anion Export

Abstract

Uncoupling protein-3 (UCP3) is a mitochondrial inner membrane protein expressed most abundantly in skeletal muscle and to a lesser extent in heart and brown adipose tissue. Evidence supports a role for UCP3 in fatty acid oxidation (FAO); however, the underlying mechanism has not been explored. In 2001 we proposed a role for UCP3 in fatty acid export, leading to higher FAO rates (Himms-Hagen, J., and Uncoupling protein-3 (UCP3)3 is a member of the family of mitochondrial inner membrane anion carrier proteins which includes uncoupling protein-1 (UCP1), expressed exclusively in brown adipose tissue where it mediates adaptive thermogenesis via an inducible proton leak (1). UCP3 shares 57% amino acid homology with UCP1, has the same predicted tertiary structure, and like UCP1, possesses a nucleotide binding domain (2, 3). UCP3 protein is most abundant in skeletal muscle and is present to a lesser extent in brown adipose tissue and heart (4). In contrast to UCP1, the physiological role and underlying mechanism of action of UCP3 are as yet unresolved. A proposed function for UCP3 is in lipid metabolism (5, 6) and supportive evidence has accrued. In Gullah African-Americans an exon 6 splice junction polymorphism resulting in a truncated form of UCP3 was associated with lower fatty acid oxidation (FAO) as assessed by indirect calorimetry (7); similar results were found in Ucp3 Ϫ/Ϫ mice (8). Early overexpression studies in mice and human muscle cells reported increased FAO (9, 10); however, these results may be confounded by nonspecifically increased basal proton leak due to supraphysiological UCP3 overexpression (11,12). Physiological overexpression is associated with greater FAO in L6 myotubes (13) and elevated maximal activity of carnitine palmitoyl transferase-1, ␤-hydroxyacyl dehydrogenase, and citrate synthase and lower lipid storage in mouse skeletal muscle (14,15). Although these studies functionally link UCP3 and increased FAO, the underlying mechanism remains unexplored, including whether the association between UCP3 and FAO may be related to reduced oxidative stress (13, 16 -19).Two hypotheses of UCP3-mediated fatty acid handling propose that UCP3 transports fatty acid anions from the mitochondrial matrix (20,21). Schrauwen et al. (21) suggest the physiological outcome to be reduced matrix lipotoxicity. We, on the other hand, proposed a UCP3 export function that leads to increased FAO (20). Specifically, when fatty acyl supply is high, UCP3 would act in concert with mitochondrial thioesterase-1 (MTE-1), which cleaves long chain acyl-CoA into fatty acid anions and CoA (22, 23). Fatty acid anions, which cannot be reactivated in the matrix, would be exported by UCP3 to the cytosol to be reactivated then oxidized or esterified (Fig. 1). * This work was supported by the Canadian Institutes of Health Research(Institute of Nutrition, Metabolism, and Diabetes) (to M.-E. H.), the Canadian Diabetes Association (to E. L. S.), and the Natural Sciences and Engineering Research Council (to V. B.). The costs of publication ...

Glutathionylation Acts as a Control Switch for Uncoupling Proteins UCP2 and UCP3

“…Isolation of Mitochondria from Mouse Skeletal Muscle and BAT-Muscle mitochondria were isolated as described previously (25). Skeletal muscle from forelimbs, hind limbs, and the pectoral region were pooled, cleaned, and placed in basic medium (140 mM KCl, 20 mM HEPES, 5 mM MgCl 2 , 1 mM EGTA, pH 7.0).…”

“…Following mincing, tissue was placed in homogenizing medium (basic medium supplemented with 1 mM ATP and 1% BSA (w/v)) and one unit of protease (subtilisin A) and homogenized using a glass/Teflon Potter-Elvehjem tissue grinder. Mitochondria were then isolated as described (25). BAT mitochondria were isolated using a standard protocol, as described (26).…”

“…1d). Hence, Cys 259 appears to be the major site for GSH conjugation in UCP3, although contributions from other sites, such as Cys 25 , cannot be ruled out. Glutathionylation Inhibits UCP3-mediated State 4 Respiration-Because the in vitro determinations above indicated that UCP3 can be conjugated to GSH, we next tested if glutathionylation of UCP3 impacts mitochondrial metabolism.…”

“…In contrast, diamide treatment inhibited UCP3-mediated state 4 respiration in cells carrying the Cys 230 mutant gene. Hence, Cys 25 and Cys 259 appear to be functionally important for the regulation of UCP3 by glutathionylation.…”

“…d, analysis of the site of glutathionylation in UCP3. Diagram depicts the putative locations of Cys 25 , Cys 230 , and Cys 259 (red asterisks). UCP3 genes carrying point mutations for either cysteine were overexpressed in HEK293 cells.…”