ABSTRACT
AIMS: The purpose of the study was to establish if enzyme activities from key metabolic pathways and levels of markers of oxidative damage to proteins and lipids differed between distinct liver mitochondrial sub-populations, and which specific sub-populations contributed to these differences. MAIN METHODS: Male C57BL/6J mice were fed non-purified diet for one month then separated into two groups, control and calorie-restricted (CR). The two groups were fed semi-purified diet (AIN93G), with the CR group receiving 40% less calories than controls. After two months, enzyme activities and markers of oxidative damage in mitochondria were determined. KEY FINDINGS: In all mitochondrial sub-populations, enzyme activities and markers of oxidative damage, from control and CR groups, showed a pattern of M1>M3>M10. Higher acyl-CoA dehydrogenase (ß-oxidation) and ß-hydroxybutyrate dehydrogenase (ketogenesis) activities and lower carbonyl and TBARS levels were observed in M1 and M3 fractions from CR mice. ETC enzyme activities did not show a consistent pattern. In the Krebs cycle, citrate synthase and aconitase activities decreased while succinate dehydrogenase and malate dehydrogenase activities increased in the M1 mitochondria from the CR versus control mice. SIGNIFICANCE: CR does not produce uniform changes in enzyme activities or markers of oxidative damage in mitochondrial sub-populations, with changes occurring primarily in the heavy mitochondrial populations. Centrifugation at 10,000 g to isolate mitochondria likely dilutes the mitochondrial populations which show the greatest response to CR. Use of lower centrifugal force (3000 g or lower) may be beneficial for some studies.
Subject(s)
Caloric Restriction , Mitochondria, Liver/enzymology , Oxidative Stress/physiology , Aconitate Hydratase/metabolism , Animals , Citrate (si)-Synthase/metabolism , Citric Acid Cycle/drug effects , Electron Transport Chain Complex Proteins/metabolism , L-Lactate Dehydrogenase/metabolism , Lipid Peroxidation/drug effects , Malate Dehydrogenase/metabolism , Male , Mice , Mice, Inbred C57BL , Mitochondria, Liver/physiology , Protein Carbonylation/drug effects , Succinate Dehydrogenase/metabolism , Thiobarbituric Acid Reactive Substances/metabolismABSTRACT
Calorie restriction (CR) has been shown to decrease H(2)O(2) production in liver mitochondria, although it is not known if this is due to uniform changes in all mitochondria or changes in particular mitochondrial sub-populations. To address this issue, liver mitochondria from control and CR mice were fractionated using differential centrifugation at 1,000 g, 3,000 g and 10,000 g into distinct populations labeled as M1, M3 and M10, respectively. Mitochondrial protein levels, respiration and H(2)O(2) production were measured in each fraction. CR resulted in a decrease in total protein (mg) in each fraction, although this difference disappeared when adjusted for liver weight (mg protein/g liver weight). No differences in respiration (State 3 or 4) were observed between control and CR mice in any of the mitochondrial fractions. CR decreased H(2)O(2) production in all fractions when mitochondria respired on succinate (Succ), succ+antimycin A (Succ+AA) or pyruvate/malate+rotenone (P/M+ROT). Thus, CR decreased reactive oxygen species (ROS) production under conditions which stimulate mitochondrial complex I ROS production under both forward (P/M+ROT) and backward (Succ & Succ+AA) electron flow. The results indicate that CR decreases H(2)O(2) production in all liver mitochondrial fractions due to a decrease in capacity for ROS production by complex I of the electron transport chain.
