Bio011544 858..864

Saponin permeabilization of tissue slices is increasingly popular for characterizing mitochondrial function largely because it is fast, easy, requires little tissue and leaves much of the cell intact. This technique is well described for mammalian muscle and brain, but not for liver. We sought to evaluate how saponin permeabilization reflects aspects of liverenergymetabolism typicallyassessed in isolatedmitochondria.We studied the ground squirrel (Ictidomys tridecemlineatus Mitchell), a hibernating mammal that shows profound and acute whole-animal metabolic suppression in the transition from winter euthermia to torpor. This reversiblemetabolicsuppression isalso reflected in themetabolism of isolated liver mitochondria. In this study we compared euthermic and torpid animals using saponin permeabilized tissue and mitochondria isolated from the same livers. As previously demonstrated, isolated mitochondria have state 3 respiration rates, fueled by succinate, that are suppressed by 60-70% during torpor. This result holds whether respiration is standardized to mitochondrial protein, cytochrome a content or citrate synthase activity. In contrast, saponin-permeabilized liver tissue, showno such suppression in torpor.Neithercitrate synthase activity nor VDAC content differ between torpor and euthermia, indicating that mitochondrial content remains constant in both permeabilized tissue and isolated mitochondria. In contrast succinate dehydrogenase activity is suppressed during torpor in isolated mitochondria, but not in permeabilized tissue. Mechanisms underlying metabolic suppression in torpor may have been reversed by the permeabilization process. As a result we cannot recommend saponin permeabilization for assessing liver mitochondrial function under conditions where acute changes in metabolism are known to occur.