Caloric restriction and the aging process: a critique.

The main objective of this review is to provide an appraisal of the current status of the relationship between energy intake and the life span of animals. The concept that a reduction in food intake, or caloric restriction (CR), retards the aging process, delays the age-associated decline in physiological fitness, and extends the life span of organisms of diverse phylogenetic groups is one of the leading paradigms in gerontology. However, emerging evidence disputes some of the primary tenets of this conception.

Decrease in cytochrome c oxidase reserve capacity diminishes robustness of Drosophila melanogaster and shortens lifespan.

The phenotypic effects of under- and over-expression of CcO (cytochrome c oxidase) regulatory subunits IV and Vb were examined in Drosophila melanogaster in order to test further the hypothesis that suppression of the activities of mitochondrial ETC (electron-transport chain) oxidoreductases retards the aging process and extends lifespan. Underexpression of both CcO subunits, induced by RNAi, resulted in decreases in the respective mRNA and protein levels, CcO holoenzyme activity, rate of mitochondrial respiration, walking speed and the lifespan of fruitflies. Overexpression of CcO IV or Vb in young fruitflies increased the amount of mRNA, but had no effect on the protein level or CcO catalytic activity.

Biomarkers of oxidative stress study V: ozone exposure of rats and its effect on lipids, proteins, and DNA in plasma and urine.

Ozone exposure effect on free radical-catalyzed oxidation products of lipids, proteins, and DNA in the plasma and urine of rats was studied as a continuation of the international Biomarker of Oxidative Stress Study (BOSS) sponsored by NIEHS/NIH. The goal was to identify a biomarker for ozone-induced oxidative stress and to assess whether inconsistent results often reported in the literature might be due to the limitations of the available methods for measuring the various types of oxidative products. The time- and dose-dependent effects of ozone exposure on rat plasma lipid hydroperoxides, malondialdehyde, F2-isoprostanes, protein carbonyls, methionine oxidation, and tyrosine- and phenylalanine oxidation products, as well as urinary malondialdehyde and F2-isoprostanes were investigated with various techniques.

Involvement of redox state in the aging of Drosophila melanogaster.

Significance: The main objective of this review was to provide an exposition of investigations, conducted in Drosophila melanogaster, on the role of reactive oxygen species and redox state in the aging process. While early transgenic studies did not clearly support the validity of the oxidative stress hypothesis of aging, predicated on the accumulation of structural damage, they spawned a broader search for redox-related effects that might impact the aging process.

Recent Advances: Initial evidence implicating the thiol redox state as a possible causative factor in aging has been obtained in Drosophila.

Effects of age and caloric restriction on mitochondrial protein oxidative damage in mice.

The hypothesis that life-span extension by caloric restriction (CR) is contingent upon the attenuation of macromolecular oxidative damage was tested in two different strains of mice: the C57BL/6, whose life span is extended by CR, and the DBA/2, in which CR has relatively minor or no impact on longevity. Mice were fed ad libitum (AL) or restricted to 40% lesser food, starting at 4 months of age. Protein damage was measured as protein-linked adducts of 4-hydroxy-2-nonenal (HNE) and malondialdehyde (MDA) in skeletal muscle mitochondria at 6 and 23 months of age.

The redox stress hypothesis of aging.

The main objective of this review is to examine the role of endogenous reactive oxygen/nitrogen species (ROS) in the aging process. Until relatively recently, ROS were considered to be potentially toxic by-products of aerobic metabolism, which, if not eliminated, may inflict structural damage on various macromolecules. Accrual of such damage over time was postulated to be responsible for the physiological deterioration in the postreproductive phase of life and eventually the death of the organism.

Association between life-span extension by caloric restriction and thiol redox state in two different strains of mice.

The hypothesis that the life-extending effect of caloric restriction (CR) is associated with an attenuation of the age-related pro-oxidant shift in the thiol redox state was tested employing a novel experimental design. Amounts of GSH, GSSG, and protein mixed disulfides (Pr-SSG) in the skeletal muscle and liver were compared between two strains of mice that have similar life spans when fed ad libitum (AL), but different life spans under the standard CR regimen. The life span of one strain, C57BL/6, is extended under CR, whereas it remains unaffected in the other strain, DBA/2.

Expression of multiple copies of mitochondrially targeted catalase or genomic Mn superoxide dismutase transgenes does not extend the life span of Drosophila melanogaster.

The simultaneous overexpression of multiple copies of Mn superoxide dismutase (SOD) and ectopic catalase (mtCat) transgenes in the mitochondria of the fruit fly, Drosophila melanogaster, was shown previously to diminish the life span. The hypothesis tested in this study was that this effect was due primarily to the presence of one or the other transgene. An alternative hypothesis was that both transgenes have additive, negative effects.

Mitochondrial peroxiredoxins are critical for the maintenance of redox state and the survival of adult Drosophila.

Drosophila mitochondria contain two peroxidases, peroxiredoxin 3 (dPrx3) and peroxiredoxin 5 (dPrx5), which together constitute the sole known intramitochondrial mechanism for the catalytic removal of hydrogen and organic peroxides. dPrx3 exists exclusively within mitochondria, whereas dPrx5 is also present in some other intracellular compartments. Levels of these two peroxiredoxins were genetically manipulated, singly and together, in D.

Cytochrome c oxidase loses catalytic activity and structural integrity during the aging process in Drosophila melanogaster.

The hypothesis, that structural deterioration of cytochrome c oxidase (CcO) is a causal factor in the age-related decline in mitochondrial respiratory activity and an increase in H₂O₂ generation, was tested in Drosophila melanogaster. CcO activity and the levels of seven different nuclear DNA-encoded CcO subunits were determined at three different stages of adult life, namely, young-, middle-, and old-age. CcO activity declined progressively with age by 33%.

Effects of age and calorie restriction on tryptophan nitration, protein content, and activity of succinyl-CoA:3-ketoacid CoA transferase in rat kidney mitochondria.

This study examined the protein targets of nitration and the consequent impact on protein function in rat kidney mitochondria at 4, 13, 19, and 24 months of age. Succinyl-CoA transferase (SCOT), a rate-limiting enzyme in the degradation of ketone bodies, was the most intensely reactive protein against anti-3-nitrotyrosine antibody in rat kidney mitochondria. However, subsequent mass spectrometric and amino acid analyses of purified SCOT indicated that tryptophan 372, rather than a tyrosine residue, was the actual site of simultaneous additions of nitro and hydroxy groups.

Prolonged intake of coenzyme Q10 impairs cognitive functions in mice.

Coenzyme Q(10) (CoQ(10)) is widely consumed as a dietary supplement to enhance bioenergetic capacity and to ameliorate the debilitative effects of the aging process or certain pathological conditions. Our main purpose in this study was to determine whether CoQ(10) intake does indeed attenuate the age-associated losses in motor, sensory, and cognitive functions or decrease the rate of mortality in mice. Mice were fed a control nonpurified diet or that diet containing 0.

Life span extension in mice by food restriction depends on an energy imbalance.

In this study, our main objective was to determine whether energy restriction (ER) affects the rate of oxygen consumption of mice transiently or lastingly and whether metabolic rate plays a role in the ER-related extension of life span. We compared rates of resting oxygen consumption between C57BL/6 mice, whose life span is prolonged by ER, and the DBA/2 mice where it is not, at 6 and 23 mo of age, following 40% ER for 2 and 19 mo, respectively. Mice of the 2 strains that consumed food ad libitum (AL) had a similar body mass at the age of 4 mo and consumed similar amounts of food throughout the experiment; however, the body weight subsequently significantly increased (20%) in the C57BL/6 mice but did not increase significantly in the DBA/2 mice.

Peroxiredoxin 5 confers protection against oxidative stress and apoptosis and also promotes longevity in Drosophila.

Peroxiredoxin 5 is a distinct isoform of the peroxiredoxin gene family. The antioxidative and anti-apoptotic functions of peroxiredoxin 5 have been extensively demonstrated in cell culture experiments. In the present paper, we provide the first functional analysis of peroxiredoxin 5 in a multicellular organism, Drosophila melanogaster.

The catalytic subunit of Drosophila glutamate-cysteine ligase is a nucleocytoplasmic shuttling protein.

GSH concentration is considerably lower in the nucleus than in the cytoplasm; however, it is significantly elevated during active cell proliferation. The main purpose of this study was to understand the mechanism underlying these variations in nuclear/cytoplasmic distribution of GSH. The rate-limiting step in the de novo GSH biosynthesis pathway is catalyzed by glutamate cysteine ligase (GCL), a heterodimer, composed of a catalytic subunit (GCLc) and a modulatory subunit (GCLm).

Overexpression of glucose-6-phosphate dehydrogenase extends the life span of Drosophila melanogaster.

The redox state of tissues tends to become progressively more prooxidizing during the aging process. The hypothesis tested in this study was that enhancement of reductive capacity by overexpression of glucose-6-phosphate dehydrogenase (G6PD), a key enzyme for NADPH biosynthesis, could protect against oxidative stress and extend the life span of transgenic Drosophila melanogaster. Overexpression of G6PD was achieved by combining a UAS-G6PD responder transgene at one of four independent loci with either a broad expression (armadillo-GAL4, Tubulin-GAL4, C23-GAL4, and da-GAL4) or a neuronal driver (D42-GAL4 and Appl-GAL4).

Pro-oxidant shift in glutathione redox state during aging.

The GSH:GSSG ratio, which is the primary determinant of the cellular redox state, becomes progressively more pro-oxidizing during the aging process due to an elevation in the GSSG content and a decline in the ability for de novo GSH biosynthesis. The K(m) of glutamate-cysteine ligase (GCL), the rate-limiting enzyme in de novo GSH biosynthesis, significantly increases during aging, which would adversely affect the ability for rapid GSH biosynthesis, especially under stressful conditions. Experimental studies suggest that age-related accumulation of homocysteine, an intermediate in the trans-sulfuration pathway, may be responsible for causing the loss of affinity between GCL and its substrates.

Detection and characterization of in vivo nitration and oxidation of tryptophan residues in proteins.

Oxygen and nitrogen centered reactive species can cause specific structural modifications in amino acids and proteins, such as the addition of a nitro group onto aromatic residues. Heretofore, studies on protein nitration have mainly focused on the in vitro and in vivo nitro addition to tyrosine residues (3-nitrotyrosine or 3NT), whereas the formation of nitrotryptophan in proteins in vivo and/or its functional significance has remained quite obscure. A novel structural modification, involving the addition of nitro and hydroxy groups to tryptophan, has been detected in the mitochondrial protein succinyl-CoA:3-oxoacid CoA transferase (SCOT) in rat heart.

Detection and characterization of peroxynitrite-induced modifications of tyrosine, tryptophan, and methionine residues by tandem mass spectrometry.

Nitration and oxidation of tyrosine, tryptophan, and methionine residues in proteins are potential markers of their interaction with peroxynitrite. This chapter describes the procedure for the detection of these nitro-oxidative modifications by tandem mass spectrometry. The peptide YGDLANWMIPGK, shown to contain a nitrohydroxytryptophan in the mitochondrial enzyme succinyl-CoA:3-ketoacid coenzyme A transferase (SCOT) in vivo, was synthesized and exposed to peroxynitrite in order to test whether an identical tryptophan derivative could be generated in vitro.

Comparison of metabolic rate and oxidative stress between two different strains of mice with varying response to caloric restriction.

Metabolic rate and parameters associated with oxidative stress were compared in two strains of mice, one of which, C57BL/6, exhibits an extension of life span in response to caloric restriction while the other, DBA/2, shows no such effect. Metabolic rate was higher in the DBA/2 than in the C57BL/6 mice, when measured at 5-6 months of age as in vivo and in vitro rates of oxygen consumption or body temperature. There were no remarkable inter-strain differences in activities of the antioxidant enzymes, superoxide dismutase, catalase, and glutathione peroxidase or in the rates of mitochondrial superoxide anion radical generation in heart or skeletal muscles.

Age-related decrease in expression of mitochondrial DNA encoded subunits of cytochrome c oxidase in Drosophila melanogaster.

A key feature of the aging process is that the mitochondrial respiratory capacity declines and, the production of reactive oxygen species increases in the later part of life span. In previous studies, cytochrome c oxidase (CcO), the terminal component of the mitochondrial electron transport chain, was found to be the only oxidoreductase exhibiting an age-related decrease in activity in Drosophila melanogaster. The present study tested the hypothesis that decreases in the abundance of catalytic subunits of CcO, encoded in mitochondrial DNA, could underlie the age-associated loss of enzyme activity.

Analysis of oxidative modification of proteins.

Reactions between protein molecules and reactive oxygen species (ROS) often lead to the modification of certain amino acid residues such as histidine, lysine, arginine, proline, and threonine, forming carbonyl derivatives. Carbonylation of proteins has thus often been employed for the quantification of generalized protein oxidation. Besides carbonylation, other types of oxidative damage that have been investigated in depth are the modifications of cysteine, tyrosine, and aspartate, or asparagine residues.

Modulating longevity in Drosophila by over- and underexpression of glutamate-cysteine ligase.

A notable extension of life span (up to 50%) was achieved in Drosophila melanogaster when the catalytic subunit of glutamate-cysteine ligase (GCLc) was overexpressed in neuronal tissue, while a moderate increase (up to 24%) was observed when the modulatory subunit of GCL (GCLm) was overexpressed globally. We sought to identify specific tissue domains that are particularly sensitive to the beneficial effects of GCLc overexpression. Overexpression of GCLc using the mushroom body driver (OK107-GAL4) had a small but significant beneficial effect on longevity (approximately 12%) while overexpression in serotonergic (MZ360-GAL4) neurons or dopaminergic and serotonergic neurons (Ddc-GAL4) had small, nonsignificant effects on longevity.

Carbonylation of mitochondrial proteins in Drosophila melanogaster during aging.

Age-related changes in carbonylation of mitochondrial proteins were determined in mitochondria from the flight muscles of Drosophila melanogaster. Reactivity with antibodies against (i) adducts of dinitrophenyl hydrazone (DNP), commonly assumed to react broadly with derivatized carbonyl groups, (ii) malondialdehyde (MDA), or (iii) hydroxynonenal (HNE), was compared at five different ages of flies. MDA and HNE are carbonyl-containing products of lipid peroxidation, which can form covalent adducts with proteins.

Effect of long-term caloric restriction on oxygen consumption and body temperature in two different strains of mice.

The hypothesis, that a decrease in metabolic rate mediates the life span prolonging effect of caloric restriction (CR), was tested using two strains of mice, one of which, C57BL/6, exhibits life span extension as a result of CR, while the other, DBA/2, shows little or no effect. Comparisons of the rate of resting oxygen consumption and body temperature were made between the strains after they were fed ad libitum (AL) or maintained under 40% CR, from 4 to 16 months of age. Ad libitum-fed mice of the two strains weighed the same when young and consumed similar amounts of food throughout the experiment; however, the C57BL/6 mice weighed 25% more than DBA/2 mice at 15 months of age.