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Calorie Restriction and Gene Expression in Aging Organisms
Posted on: September 3, 2003

Caloric restriction (CR) is the only intervention shown to extend lifespan in mammals. It is also the most effective means known of reducing cancer incidence and increasing the mean age of onset of age-related diseases and tumors. Beside this, caloric restriction retards the development of a broad spectrum of other pathophysiological changes. It is well known that aging is associated with specific transcriptional alterations in the gastrocnemius muscle, cerebral cortex, liver, cerebellum tissues, etc. CR can prevent or delay most of the largest age-related transcriptional alterations. The effects of life-span-extending caloric restriction (CR) on gene expression in the liver were examined by DNA micro array analysis — a useful tool capable to distinguish between age-related and CR-related genomic expression pattern. Mice were subjected to long-term CR (LT-CR) and short-term CR (ST-CR).

The liver is the central organ for the regulation of glucose homeostasis, xenobiotic metabolism and detoxification, and steroid hormone biosynthesis and degradation. This organ also has a major impact on health and homeostasis through its control of serum protein composition. While differentiated hepatic functions are generally well maintained with age, changes do occur. Serum and biliary cholesterol rise, liver regeneration declines, hepatic drug clearance decreases, and liver volume and blood flow decrease. The resilience of the liver to aging and its central role in the maintenance of whole body health and homeostasis make it an intriguing target for genome-wide expression analysis of aging.

Age-Related Changes in Gene Expression.

Of the genes that increased expression with age, 40% were associated with inflammation. These results suggest that inflammation may be a component of the aging process common to liver, neocortex and cerebellum. Induction of lysozyme (Lyzs) gene is normally associated with macrophage activation. Macrophages, among other inflammatory cells, are involved in a large number of liver diseases, including cirrhosis, hepatitis, and sepsis- and endotoxin-induced liver injury.

Lyzs also was up regulated with age in neocortex and cerebellum, but neither gene was affected in muscle. Changes in the expression of these genes are among the few that were common to liver, muscle, and brain.

Normal liver aging was associated with other gene expression changes consistent with pathogenesis. Old mice over expressed the mRNA for biglycan (Bgn), a proteoglycan of the hepatic extra cellular matrix, serum amyloid P-component (Sap), a glycoprotein present in all amyloid deposits, and cystatin B (Cstb), an inhibitor of cysteine proteinases. In areas of inflammation, fibrogenic myofibroblasts express Bgn and other proteoglycans, leading to hepatic fibrosis. Sap is one of the major acute-phase reactants induced by inflammation in hepatocytes. Cystatins and their target enzymes play a role in many pathological events, including inflammatory disease. In the liver, an imbalance between cystatins and their targets can disregulate matrix degradation and accumulation, leading to hepatic fibrosis.

Twenty-five percent of the genes that increased expression with age were stress response proteins/chaperones. Diverse physiological stresses, including oxidative injury, may be causally involved in aging. In mammals, the oxidative processes centered in the liver are major sources of free radicals. The induction of chaperone gene expression in the livers of aged mice may be a physiological adaptation to increased oxidative or possibly other stress during aging. Chaperones provide cytoprotective functions, including prevention of protein denaturation and aggregation, the repair of damaged structural and functional proteins, and promotion of the ubiquitination and proteasomal degradation of potentially toxic, damaged proteins produced by oxidative or glycoxidative processes.

Genes Whose Expression Decreased with Age.

Of the 26 genes that decreased expression with age in control mice, 23% are involved in DNA replication and the cell cycle. Most of these have a negative effect on cell growth and division. Among these, the product of phosphatase and tensin homolog (Pten) gene is a tumor suppressor that induces cell-cycle arrest through inhibition of the phosphoinositide 3-kinase pathway. B cell translocation gene 2 (Btg2) is a tumor suppressor that increases expression in response to DNA damage. The murine gene product of the amino-terminal enhancer of split (Aes) is a potent corepressor of gene expression and cellular proliferation. Calcium-binding protein A11 binds to and regulates the activity of annexin II, which is involved in the transduction of calcium-related mitogenic signals. Insulin-like growth factor (IGF) binding protein 1 plays an important role in the negative regulation of the IGF-1 system, a stimulator of mitogenesis.

Reduced expression of genes discussed above indicates that there is a general loss of negative cell growth control with age. Seventy-eight percent of the mice of this strain and sex fed the control diet used here die of some form of neoplasia, and the death rate from neoplasia accelerates dramatically with age. Approximately 21% of these mice die of hepatoma, mostly late in life. Decreased expression of the negative growth regulators and overexpression of the chaperone genes with age also are consistent with this higher incidence of hepatoma in aged mice.

Aging decreased expression of a second group of genes with antineoplastic potential, xenobiotic metabolism genes. These genes were negatively regulated by age. Decreased expression of such genes is likely responsible in part for the age-related decline in the xenobiotic-metabolizing capacity of the liver. This decline is a recognized source of adverse drug reactions in aged mammals. It may contribute to the increase in neoplasms with age in mice.

Aging was associated with decreased expression of other genes responsible for differentiated liver functions. Apolipoprotein E (Apoe) decreased expression with age. The liver mainly secretes this protein, and it is required for clearance of lipoproteins from the blood. Disruption of Apoe is associated with severe atherosclerosis in mice. Thus, decreased Apoe expression with age may be related to an increase in atherosclerotic lesions in mice. There is a similar association between impaired or absent Apoe expression and hyperlipidemia in humans, where the expression of a specific Apoe subtype is associated with extreme longevity.

LT-CR Opposed Age-Associated Changes in Gene Expression.

LT-CR opposed the age-related increase in expression of 14 of the 20 genes that increased expression in control mice (70%). LT-CR suppressed the increase in 75% (6 of 8 genes) of the inflammatory response genes. Consistent with decreased inflammatory response gene expression, CR delays the onset and diminishes the severity of autoimmune and inflammatory diseases in mice.

LT-CR opposed the age-related increase in the expression of 3 of the 5 stress response proteins. Expression of at least 7 chaperones is negatively regulated in response to LT-CR. These data suggest that LT-CR reduces physiological stress on the liver. Further, as discussed above, reduced chaperone expression is proapoptotic and antineoplastic. Consistent with these results, LT-CR decreased expression of Api6. Together, these effects may explain the delayed onset of hepatoma in LT-CR mice. LT-CR opposed the age-associated decrease in the expression of 13 of the 26 genes that decreased expression in control mice (50%). Many of these genes are responsible for key differentiated functions of the liver. Partial restoration of the hepatic drug metabolizing and detoxifying functions of the liver may be a source of the anti-aging and anticancer effects of LT-CR.

ST-CR Reproduced the Majority of the Effects of LT-CR on Age-Responsive Gene Expression.

Overall, ST-CR reproduced nearly 70% of the effects of LT-CR on genes that changed expression with age (Table). The effects of ST- and LT-CR were highly homologous in both direction and fold-change for these genes. Thus, CR rapidly reversed, rather than prevented, many age-related changes in gene expression. ST-CR induced a more youthful gene expression profile associated with longer life and health span. Expression profiling of these genes should prove useful in rapidly identifying CR-mimetic drugs and treatments.

Categories of ST- and LT-CR-regulated genes
Gene category ST-CR / LT-CR
% * Ratio **
Major urinary problems 100 4 / 4
Intracellular signalling 75 3 / 4
Xenobiotic metabolism 75 6 / 8
Inflammatory response 67 4 / 6
Stress response / Chaperones 60 3 / 5
Cell cycle / DNA replication 50 1 / 2
Apoptosis / Cell growth and survival 44 4 / 9
Energy metabolism / Biosynthesis 45 5 / 11
Miscellaneous 27 3 / 11
Total 55 33 / 60
*The genes that changed expression with ST-CR as percentage of those that changed with LT-CR.
**The ratio between number of genes that change expression with ST-CR and LT-CR.

ST-CR reproduced 100% of the LT-CR effects on xenobiotic metabolism, stress response/chaperone, and major urinary protein gene expression. It also reproduced 67% (4 of 6) of the effects of LT-CR on inflammatory response gene expression. These results suggest that CR may rapidly ameliorate inflammation and other stresses, even in very old animals. ST-CR also reproduced the effects of LT-CR on the expression of 50% of the cell-cycle/DNA replication and apoptosis genes. The combination of these effects on gene expression suggests that ST-CR may be capable of rapidly reproducing the antineoplastic effects of LT-CR in very old animals.

This conclusion is consistent with studies showing that short-term fasting increased apoptosis of preneoplastic cells and preneoplastic lesions, and reduced rates of chemical carcinogenesis. Thus, CR mimetics might be useful anticancer therapies. The effects of ST-CR on the expression of genes associated with xenobiotic metabolism suggest that ST-CR may rapidly restore some differentiated functions in tissues of older animals.

Tissue Specificity of Expression Profiles.

Gene expression profiles found here with those reported for muscle and brain were compared. Aging induced the expression of a number of stress response genes in liver, skeletal muscle, neocortex, and cerebellum. However, in the cortex and hypothalamus, aging reduced the expression of other stress response genes. Likewise, aging induced expression of inflammatory genes in the liver, neocortex, and cerebellum, but not in skeletal muscle, cortex, or hypothalamus. These results clearly show that aging is tissue-specific in its effects. They also suggest that tissues are subjected to different stresses during aging.

CR opposed the age-related induction of stress response genes in muscle, and stress response and inflammatory genes in liver, neocortex, and cerebellum. Thus, the amelioration of physiological stress appears to be a common anti-aging effect of CR. Aging and CR also altered the expression of genes involved in cell growth in liver and brain. However, aging and CR changed the expression of apoptosis-related genes only in the liver, which is consistent with the mitotic potential of this tissue. CR altered expression of genes involved with energy metabolism and biosynthesis in liver and brain, but not in muscle. Again, these results highlight the tissue specificity of both aging and CR. As more tissues in more species are studied, characteristic tissue-specific patterns of gene regulation by aging and CR may emerge.

Source: Shelley X. Cao, Joseph M. Dhahbi, Patricia L. Mote, and Stephen R. Spindler. Genomic profiling of short- and long-term caloric restriction effects in the liver of aging mice. PNAS, September 11, 2001, vol. 98 no. 19, 10630-10635.
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