Investigating the molecular biology of aging and extended longevity: Proteomic and genomic analysis of mouse liver

Abstract

Recent advances in molecular gerontology have provided important clues about the fundamental biology of the aging process including the role of oxidative stress and the genetic basis of longevity. Progressive accumulation of oxidative damage to macromolecules is thought to underlie the aging-associated decline in physiologic function characteristic of the senescent phenotype. Mitochondria are a major intracellular source of reactive oxygen species (ROS); however, other organelles are also endogenous sources of oxyradicals and oxidants that can damage macromolecules. This investigation examines the relationship between aging and oxidative damage to ER resident proteins, which exist in a strongly oxidizing environment necessary for disulfide bond formation. In these studies, young and aged mouse liver homogenates were separated into enriched sub-cellular fractions, and the ER/mitochondrial fraction was resolved by 2-dimensional gel electrophoresis and then assayed for oxidative damage as indicated by protein carbonylation. MALDI/TOF analysis and N-terminal sequencing of these proteins identified BiP/Grp78, protein disulfide isomerase (PDI), and calreticulin as exhibiting a specific age-associated increase in carbonyl content. This increase in oxidative damage to critical ER proteins in aged liver strongly indicates an impairment in protein folding, disulfide cross-linking, and glycosylation which may significantly contribute to the functional decline observed in aging liver.\r\nProviding evidence for the genetic basis of aging, several murine models demonstrate that longevity can be increased by mutations affecting endocrine signaling, particularly via the GH/IGF-1 axis. In this investigation of long-lived GH/IGF-1-deficient mice, characteristic patterns of hepatic gene expression in Pit1dw/dwJ dwarf mice were revealed. Comparative microarray analysis of young and aged male livers was utilized to identify specific genes differentially expressed in Pit1dw/dwJ mice. Further examination of both male and female livers by real-time RT-PCR demonstrated striking transcriptional differences in Pit1dw/dwJ mice comprised of genes regulating cholesterol biosynthesis, fatty acid utilization, and lipoprotein metabolism. Affecting global energy homeostasis, this programmatic shift in hepatic expression may contribute to longevity by influencing bioenergetic and oxidative reactions occurring within mitochondria, ER, and peroxisomes. Intriguingly, these long-term patterns in metabolic gene expression in Pit1dw/dwJ livers mirror many transcriptional changes induced by caloric restriction and fasting, further implicating energy metabolism in longevity.\r\n