Carboxyatractyloside effects on brown-fat mitochondria imply that the adenine nucleotide translocator isoforms ANT1 and ANT2 may be responsible for basal and fatty-acid-induced uncoupling respectively

In brown-fat mitochondria, fatty acids induce thermogenic uncoupling through activation of UCP1 (uncoupling protein 1). However, even in brown-fat mitochondria from UCP1−/− mice, fatty-acid-induced uncoupling exists. In the present investigation, we used the inhibitor CAtr (carboxyatractyloside) to examine the involvement of the ANT (adenine nucleotide translocator) in the mediation of this UCP1-independent fatty-acid-induced uncoupling in brown-fat mitochondria. We found that the contribution of ANT to fatty-acid-induced uncoupling in UCP1−/− brown-fat mitochondria was minimal (whereas it was responsible for nearly half the fatty-acid-induced uncoupling in liver mitochondria). As compared with liver mitochondria, brown-fat mitochondria exhibit a relatively high (UCP1-independent) basal respiration (‘proton leak’). Unexpectedly, a large fraction of this high basal respiration was sensitive to CAtr, whereas in liver mitochondria, basal respiration was CAtr-insensitive. Total ANT protein levels were similar in brown-fat mitochondria from wild-type mice and in liver mitochondria, but the level was increased in brown-fat mitochondria from UCP1−/− mice. However, in liver, only Ant2 mRNA was found, whereas in brown adipose tissue, Ant1 and Ant2 mRNA levels were equal. The data are therefore compatible with a tentative model in which the ANT2 isoform mediates fatty-acid-induced uncoupling, whereas the ANT1 isoform may mediate a significant part of the high basal proton leak in brown-fat mitochondria.

Life without UCPI: mitochondrial, cellular and organismal characteristics of the UCPI-ablated mice

Mice devoid of the original uncoupling protein UCP1 have provided opportunities to delineate UCP1 function in a series of biochemical and physiological contexts. The isolated brown-fat mitochondria from such mice are fully coupled (without the addition of GDP), but still exhibit a depressed capacity for ATP synthesis. However, they only show a 2-fold decrease in sensitivity to the de-energizing effect of free fatty acids, compared with UCP1-containing mitochondria, whereas they possess a (UCP1-independent) 50-fold higher sensitivity than liver mitochondria; the fatty acid sensitivities in wild-type and UCP1-deficient mitochondria may, however, be of different natures. Despite the fact that brown-fat cells from UCP1-ablated mice cannot produce heat when stimulated by noradrenaline (‘norepinephrine’) or fatty acids, UCP1-ablated mice can be induced to tolerate extended cold exposure, but the heat then fully results from shivering thermogenesis. Recruitable or adaptive (by cold acclimation or adaptation to a cafeteria diet) adrenergically-stimulated thermogenesis does not exist in the UCP1-ablated animals, demonstrating the unique ability of UCP1 to mediate recruitable non-shivering thermogenesis. In addition to information on the function of UCP1, the UCP1 -ablated mice can be used to gain information concerning the function of the UCP1 homologues. Thus whereas an uncoupling function of the UCP1 homologues cannot be excluded, UCP1-ablated animals clearly lack any ability to recruit any UCP1 homologue to functionally replace the loss of thermogenesis resulting from UCP1. UCP1 (thermogenin) thus remains the only protein the activity of which can be recruited for the purpose of facultative thermogenesis.