Mitochondrial decay in hepatocytes from old rats: Membrane potential declines, heterogeneity and oxidants increase

Abstract

Mitochondrial function during aging was assessed in isolated rat hepatocytes to avoid the problem of differential lysis when old, fragile mitochondria are isolated. Rhodamine 123, a f luorescent dye that accumulates in mitochondria on the basis of their membrane potential, was used as a probe to determine whether this key function is affected by aging. A marked f luorescent heterogeneity was observed in hepatocytes from old (20-28 months) but not young (3-5 months) rats, suggesting age-associated alterations in mitochondrial membrane potential, the driving force for ATP synthesis. Three distinct cell subpopulations were separated by centrifugal elutriation; each exhibited a unique rhodamine 123 f luorescence pattern, with the largest population from old rats having significantly lower f luorescence than that seen in young rats. This apparent age-associated alteration in mitochondrial membrane potential was confirmed by measurements with radioactive tetraphenylphosphonium bromide. Cells from young rats had a calculated membrane potential of ؊154 mV, in contrast to that of the three subpopulations from old rats of ؊70 mV (the largest population), ؊93 mV, and ؊154 mV. Production of oxidants was examined using 2,7di-chlorof luorescin, a dye that forms a f luorescent product upon oxidation. The largest cell subpopulation and a minor one from old animals produced significantly more oxidants than cells from young rats. To investigate the molecular cause(s) for the heterogeneity, we determined the levels of an ageassociated mtDNA deletion. No significant differences were seen in the three subpopulations, indicating that the mitochondrial decay is due to other mutations, epigenetic changes, or both.

Changes in mitochondrial energy utilization in young and old worker honeybees (Apis mellifera)

“…In all cases, asterisks indicate statistically significant difference ( * P<0.05, ** P<0.01; two-sample t test) advancing age in the trophocytes and fat cells of workers. This result is in accord with previous studies showing that ΔΨm decreases with advancing age in the hepatocytes of rats and mice (Hagen et al 1997;Kokoszka et al 2001), in the lymphocytes of mice (Wikowski and Micklem 1985;Rottenberg and Wu 1997), and hearts of rats (Savitha and Panneerselvam 2006). This phenomenon is the result of mitochondrial dysfunction during the aging process (Ames et al 1995;Trifunovic and Larsson 2008;Artal-Sanz and Tavernarakis 2008;López-Lluch et al 2008;Chanséaume and Morio 2009).…”

Age associated low mitochondrial biogenesis may be explained by lack of response of PGC-1α to exercise training

“…The role of mitochondria as key generator of oxidative stress and also target of the damage associated with reactive oxygen species (ROS) production was postulated by Miquel in the 1970s (Johnson et al 1975). Independent work from our laboratory (Sastre et al 1996) and that from Bruce Ames' (Hagen et al 1997) using both metabolic and flow cytometric approaches provided such evidence. We showed that mitochondria are damaged within cells instead of being frail and damaged during the isolation procedure.…”

“…Later, in the 1990s, we reported that mitochondrial damage is an early event in cellular aging (Sastre et al 1996). This was independently confirmed by the group of Bruce Ames (Hagen et al 1997). In 2003, it was shown in skeletal muscle that age causes a decrease in ATP content and production by approximately 50% in isolated rat mitochondria (Drew et al 2003).…”

Failure of stress-induced downregulation of Bcl-2 contributes to apoptosis resistance in senescent human diploid fibroblasts

“…Dissipation of mitochondrial membrane potential (DC m ) has been detected during the early stage of the apoptotic process, 15 and DC m has been reported to decrease in aged cell. [16][17][18] We therefore compared the changes of DC m in young and senescent HDFs in response to H 2 O 2 , staurosporine and thapsigargin treatments by analyzing DiOC 6 fluorescence by flow cytometry. As shown in Figure 2, we observed that DC m of senescent HDFs decreased about two-fold compared with that of young HDFs, but dissipation of DC m was significantly increased in young HDFs but not in the senescent HDFs undergoing H 2 O 2 , staurosporine or thapsigargin-induced apoptosis.…”

“…(Supplementary Figure 1). Although several studies suggest that DC m is reduced in aged hepatocytes, fibroblasts, lymphocytes and cardiomyocytes, [16][17][18] the underlying molecular mechanism is not clear. Whether a decrease in Bak, Bok, Bik and Puma levels confers the decrease of DC m in senescent HDFs requires further study.…”