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 fluorescent 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 fluorescent 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 fluorescence pattern, with the largest population from old rats having significantly lower fluorescence 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',7'dichlorofluorescin, a dye that forms a fluorescent 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 age-associated 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.

Figure 1

Hepatocytes isolated from old rats exhibit marked R123 fluorescent heterogeneity. Cells, from either young or old rats, were incubated with R123 30 min before analysis by flow cytometry. Hepatocytes from old rats contained mitochondria that have significantly altered average membrane potential. Shown is a fluorogram typical of that seen in at least six experiments.

Figure 2

R123 fluorescence profiles of cells from young and old rats after centrifugal cell elutriation. Elutriation of cells from young rats resulted in only one population of cells, while elutriation of cells from old rats yielded one major and two smaller cell subpopulations. (A) The cells that had the lowest R123 fluorescence (fraction A). (B) Cells that had average R123 staining patterns similar to hepatocytes from young rats (fraction B). (C) Cells of this fraction had mitochondria with membrane potential equivalent to or higher than hepatocytes from young rats (fraction C). (D) Cells from young rats. Shown is a typical fluorogram after cell separation. The vertical lines indicate the mean fluorescence value for the majority of cells in the isolated fraction.

Figure 3

Hepatocytes from old rats produce significantly higher rates of oxidant production as measured by DCFH. Results show that fractions A and C from old rats had significantly higher rates of oxidant production (P ≤ 0.01) than cells from young rats. U denotes unelutriated cells.

Figure 4

Detection and quantification of the 16-nt direct repeat-associated mtDNA deletion in rat hepatocytes. (A) PCR was performed on total DNA isolated from two young (lanes 1–4 and 5–8) and two old (lanes 9–12 and 13–16) rats. Unelutriated (lanes 1, 5, 9, and 13) or cells from fractions A (2, 6, 10, and 14), B (3, 7, 11, and 15), and C (lanes 4, 8, 12, and 16) were used. Lane 17, negative PCR control; lane 18, molecular weight markers of HaeIII-digested φX174 DNA. (B) Quantification of the mtDNA deletion in unelutriated hepatocytes (U) or hepatocyte subpopulations A–C from old rats (n = 3 or 4).