Uncoupling of protein‐3 induces an uncontrolled uncoupling of mitochondria after expression in muscle derived L6 cells

Abstract

The uncoupling proteins (UCPs) are thought to uncouple oxidative phosphorylation in the mitochondria and thus generate heat. One of the UCP isoforms, UCP3, is abundantly expressed in skeletal muscle, the major thermogenic tissue in humans. UCP3 has been overexpressed at high levels in yeast systems, where it leads to the uncoupling of cell respiration, suggesting that UCP3 may indeed be capable of dissipating the mitochondrial proton gradient. This effect, however, was recently shown to be a consequence of the high level of expression and incorrect folding of the protein and not to its intrinsic uncoupling activity. In the present study, we investigated the properties of UCP3 overexpressed in a relevant mammalian host system such as the rat myoblast L6 cell line. UCP3 was expressed in relatively low levels (< 1 lgAEmg )1 membrane protein) with the help of an adenovirus vector. Immunofluorescence microscopy of transduced L6 cells showed that UCP3 was expressed in more than 90% of the cells and that its staining pattern was characteristic for mitochondrial localization. The oxygen consumption of L6 cells under nonphosphorylating conditions increased concomitantly with the levels of UCP3 expression. However, uncoupling was associated with an inhibition of the maximal respiratory capacity of mitochondria and was not affected by purine nucleotides and free fatty acids. Moreover, recombinant UCP3 was resistant to Triton X-100 extraction under conditions that fully solubilize membrane bound proteins.Thus, UCP3 can be uniformly overexpressed in the mitochondria of a relevant muscle-derived cell line resulting in the expected increase of mitochondrial uncoupling. However, our data suggest that the protein is present in an incompetent conformation.

Targeted expression of uncoupling protein 2 to mouse liver increases the susceptibility to lipopolysaccharide/galactosamine-induced acute liver injury

“…It is argued that the uncoupling effect of UCP may be due to a gain of function of artifacts under overexpression. 44 We have different lines of founder mice that express different levels of UCP2. Two lines of mice with significant differences in UCP2 show similar phenotypes.…”

Fatty acids do not activate UCP2 in pancreatic beta cells: comparison with UCP1

“…This artifact results in an activity of the protein in the basal state (i.e., without activation by fatty acids) that is not inhibited by purine nucleotides when studied in isolated To see all the spots of endogenous UCP2, the blot had to be exposed for 10 min instead of 2 min for the other data mitochondria [32]. In the present study, the lack of uncoupling in the basal conditions, indicated by similar mitochondrial membrane potential, oxygen consumption, and ATP/ADP ratio is an indirect indication that the insertion of UCP1 and UCP2 in the mitochondrial membrane is not causing nonspecific proton leaks.…”

Overexpression of short heterodimer partner recovers impaired glucose-stimulated insulin secretion of pancreatic β-cells overexpressing UCP2

“…Interestingly, UCP2 expression induced by 50 MOI of Ad-UCP2 was quantitatively similar to the endogenously expressed level of UCP2 induced by 15 mM glucose (data not shown). It was previously suggested that if overexpressed UCP3 protein in the mitochondria could be eluted by high concentrations of nonionic detergents, such as Triton X-100, the protein would be functionally active (Guerini et al 2002). Therefore, we applied this concept in our system where UCP2 is overexpressed in INS-1 or native -cells.…”