PMC

Liver sections from young, middle age, and old rhesus were stained with hematoxylin and eosin. Panels A and C are representative liver sections from 10-year-old rhesus (n=3; middle age). Panels B, D, E, F are representative sections from 20–25 year-old rhesus monkeys (n=3; old). The old individuals used for this experiment were healthy monkeys. 200X magnification. Arrow in panel D shows fibrosis. Arrow in panel E shows steatosis. Arrow in panel F shows area of inflammation.

A) Relative amplification of a 10 kb mtDNA fragment that was normalized for changes in mtDNA abundance. B) Frequency of mtDNA lesions per 10 kb per strand based on the relative levels of amplification of a 10 kb mtDNA fragment. n=11 young (6–7 year-old); n=10 middle age (middle age) and n=4 old (>22 years) monkeys. *p<0.002; **p ≤ 0.05 versus young.

A) Lipid peroxidation as malondialdehyde (MDA) levels. MDA levels were measured as nM of MDA per mg of Protein. n=5 young, n=4 middle age, and n=4 old monkeys; Old versus young **p<0.01 and middle aged versus young *p<0.001. B) Detection of carbonylated proteins in liver from rhesus monkeys. Densitometry of modified proteins using anti-DNP antibody. Protein loading was normalized using α-tubulin expression. n=7 young; n=6 middle age, and n=2 old monkeys. **p<0.005 ***p=0.0005 ^‡p=0.01. C) Detection of carbonylated proteins in PBMCs from rhesus monkeys. Densitometry of modified proteins using anti-DNP antibody. Protein loading was normalized using α-tubulin expression. n=5 young, n=2 middle age, and n=4 old monkeys. *p<0.0001 versus young.

A) Age-dependent decreases in the activity of glutathione peroxidase (GHSPx). One unit of GHSPx activity is equal to mM of NADPH oxidized/min/mg of protein. n=5 young, n=4 middle age, and n=3 old monkeys; *p<0.05 versus young. B) Age-dependant catalase activity. Catalase activity was determined as units/mg protein. One unit of activity is equal to the moles of H₂O₂ degraded min⁻¹ mg protein^−1; n=5 young, n=3 middle age, and n=3 old monkeys; *p<0.05 versus young. C) Total SOD activity. Activity was expressed as the amount of enzyme that inhibits the oxidation of epinephrine by 50%, which is equal to 1 unit. n=5 young, n=2 middle age, n=4 old monkeys. *p<0.05 versus middle age. D) Expression levels of APE1 and MnSOD in PBMCs from rhesus monkeys. Protein loading was normalized using α-tubulin expression. n=5 young, n=2 middle age, and n=4 old monkeys. *p<0.0001 and **p<0.0001 versus young; ***p<0.05 versus young.

Our results using rhesus monkeys are consistent with a model of mitochondrial aging involving the increased generation of ROS, increased mtDNA damage/lesions, decreased antioxidant activity and increased liver pathology, culminating in mitochondrial dysfunction. Dysfunctional mitochondria may enhance inflammatory responses and fibrogenesis induced by drugs and xenobiotics and negatively affect detoxification reactions. The response of hepatocytes to substrate metabolism (glycolysis, gluconeogenesis, tricarboxylic acid cycle, oxidative phosphorylation, ATP synthesis) may lead to insulin resistance and type 2 diabetes mellitus.

Total DNA was isolated from liver obtained from 0.6–2.0 year old (infant), 3–8 year old (young), 9–17 year old (middle age), and 19–24 years of age (aged) monkeys and analyzed by QPCR. A) Upper panel, representative gel showing an age-dependent decrease in the amplification of a 10 kb mtDNA amplicon from two monkeys per age group. Lower panel, bars indicate relative levels of amplification of a 10 kb mtDNA fragment after normalization for changes in mtDNA abundance. *p<0.0001 and **p<0.0001 versus young and ‡p<0.05 versus middle age. B) Upper panel, representative gel showing the amplification of a 100 bp mtDNA fragment. Lower panel, relative abundance of mtDNA molecules during aging. **p<0.0001 versus young. C) Frequency of mtDNA lesions per 10 kb per strand. *p<0.0001 and **p<0.0001 versus young and ‡p<0.05 versus middle age. n=5 infant, n=7 young; n=6 middle age, and n=3 old; n=4 QPCR analyses.