Effect of Antioxidant Diets on Mitochondrial Gene Expression in Rat Brain During Aging

Age-related increase of reactive oxygen species (ROS) is particularly detrimental in postmitotic tissues. Calorie restriction (CR) has been shown to exert beneficial effects, consistent with reduced ROS generation by mitochondria. Many antioxidant compounds also mimic such effects. N-acetyl cysteine (NAC) provides thiol groups to glutathione and to mitochondrial respiratory chain proteins; thus, it may counteract both ROS generation and effects. In the present study we investigated, in different rat brain areas during aging (6, 12, and 28 months), the effect of 1-year treatment with CR and dietary supplementation with NAC on the expression of subunit 39 kDa and ND-1 (mitochondrial respiratory complex I), subunit IV (complex IV), subunit α of F₀F₁-ATP synthase (complex V) and of adenine nucleotide translocator, isoform 1 (ANT-1). The observed age-related changes of expression were prevented by the dietary treatments. The present study provides further evidence for the critical role of mitochondria in the aging process.     

Lifelong calorie restriction affects indicators of colonic health in aging C57Bl/6J mice

Diminished colonic health is associated with various age-related pathologies. Calorie restriction (CR) is an effective strategy to increase healthy lifespan, although underlying mechanisms are not fully elucidated. Here, we report the effects of lifelong CR on indicators of colonic health in aging C57Bl/6J mice. Compared to an ad libitum control and moderate-fat diet, 30% energy reduction was associated with attenuated immune- and inflammation-related gene expression in the colon. Furthermore, expression of genes involved in lipid metabolism was higher upon CR, which may point towards efficient regulation of energy metabolism. The relative abundance of bacteria considered beneficial to colonic health, such as Bifidobacterium and Lactobacillus, increased in the mice exposed to CR for 28 months as compared to the other diet groups. We found lower plasma levels of interleukin-6 and lower levels of various metabolites, among which are bile acids, in the colonic luminal content of CR-exposed mice as compared to the other diet groups. Switching from CR to an ad libitum moderate-fat diet at old age (24 months) revealed remarkable phenotypic plasticity in terms of gene expression, microbiota composition and metabolite levels, although expression of a subset of genes remained CR-associated. This study demonstrated in a comprehensive way that CR affects indicators of colonic health in aging mice. Our findings provide unique leads for further studies that need to address optimal and feasible strategies for prolonged energy deprivation, which may contribute to healthy aging.     

Oxidative stress in the aging rat heart is reversed by dietary supplementation with (R)-(alpha)-lipoic acid.

Oxidative stress has been implicated as a causal factor in the aging process of the heart and other tissues. To determine the extent of age-related myocardial oxidative stress, oxidant production, antioxidant status, and oxidative DNA damage were measured in hearts of young (2 months) and old (28 months) male Fischer 344 rats. Cardiac myocytes isolated from old rats showed a nearly threefold increase in the rate of oxidant production compared to young rats, as measured by the rates of 2,7-dichlorofluorescin diacetate oxidation. Determination of myocardial antioxidant status revealed a significant twofold decline in the levels of ascorbic acid (P = 0.03), but not alpha-tocopherol. A significant age-related increase (P = 0.05) in steady-state levels of oxidative DNA damage was observed, as monitored by 8-oxo-2'-deoxyguanosine levels. To investigate whether dietary supplementation with (R)-alpha-lipoic acid (LA) was effective at reducing oxidative stress, young and old rats were fed an AIN-93M diet with or without 0.2% (w/w) LA for 2 wk before death. Cardiac myocytes from old, LA-supplemented rats exhibited a markedly lower rate of oxidant production that was no longer significantly different from that in cells from unsupplemented, young rats. Lipoic acid supplementation also restored myocardial ascorbic acid levels and reduced oxidative DNA damage. Our data indicate that the aging rat heart is under increased mitochondrial-induced oxidative stress, which is significantly attenuated by lipoic acid supplementation.     

mRNA-Seq reveals complex patterns of gene regulation and expression in the mouse skeletal muscle transcriptome associated with calorie restriction

Sarcopenia is an age-associated loss of skeletal muscle mass and strength that increases the risk of disability. Calorie restriction (CR), the consumption of fewer calories while maintaining adequate nutrition, mitigates sarcopenia and many other age-related diseases. To identify potential mechanisms by which CR preserves skeletal muscle integrity during aging, we used mRNA-Seq for deep characterization of gene regulation and mRNA abundance in skeletal muscle of old mice compared with old mice subjected to CR. mRNA-Seq revealed complex CR-associated changes in expression of mRNA isoforms, many of which occur without a change in total message abundance and thus would not be detected by methods other than mRNA-Seq. Functional annotation of differentially expressed genes reveals CR-associated upregulation of pathways involved in energy metabolism and lipid biosynthesis, and downregulation of pathways mediating protein breakdown and oxidative stress, consistent with earlier microarray-based studies. CR-associated changes not noted in previous studies involved downregulation of genes controlling actin cytoskeletal structures and muscle development. These CR-associated changes reflect generally healthier muscle, consistent with CR's mitigation of sarcopenia. mRNA-Seq generates a rich picture of the changes in gene expression associated with CR, and may facilitate identification of genes that are primary mediators of CR's effects.     

Fructose metabolizing enzymes from mouse liver: influence of age and caloric restriction

The influence of caloric restriction (CR) on the activities of liver fructose metabolizing enzymes and metabolite levels were studied in young (3 months) and old (30 months) mice. Fructokinase activity was increased (P<0.05) in both young and old CR mice when compared to controls while triokinase activity was increased (P<0.05) only in old CR versus control mice. Aldolase was not altered by CR in either old or young mice. No age-related differences in activities were observed in controls although a trend towards an increase was observed for triokinase, while significant age-related increases were observed for fructokinase and triokinase, but not aldolase, in CR mice. Both young and old mice on CR showed significant decreases in fructose and fructose-1-phosphate, however, no age-related changes in metabolite levels were observed for either control or CR mice. A fructose-1-phosphate kinase activity was also measured and found to be unchanged in both young and old mice on CR, but the activity was significantly lower in the old mice compared with young. We show here that the enzymes involved in fructose metabolism are influenced by CR and that this could contribute to alterations in gluconeogenesis and glycolysis observed with CR.     

Anti-inflammatory action of dietary fish oil and calorie restriction

Inflammation, inflammatory mediators, cyclooxygenase (COX)-2, and inducible nitric oxide (iNOS) are all influenced by age-related oxidative status. To investigate the effect of dietary fish oil (FO) and calorie restriction (CR) on oxidative stress-related inflammatory status with age, (NZB/NZW) F1 (B/W) mice were fed for 4 and 9 months either ad libitum or calorie-restricted (60% of ad libitum intake) diets containing 5% corn oil or 5% FO. We measured several key oxidative and inflammatory markers: TBARS, xanthine oxidase (XOD)-derived superoxide generation, and PGE2 and LTB4 production. Expressions of renal COX-1, COX-2, and iNOS mRNA were analyzed by RT-PCR; additionally, COX-2 protein was estimated by Western-blot method. Results show that FO intake and CR individually and together suppressed age-related increases in lipid peroxidation and superoxide generation. The inhibitory effects of dietary FO and CR were also found for iNOS expression, COX-2 expression, which subsequently led to the suppression of PGE2 and LTB4. We conclude that the beneficial effects of FO feeding and CR are synergistic in ameliorating the age-related nephritis of B/W mice by suppressing COX-2 and iNOS, reactive species generation, and pro-inflammatory mediators.     

Dietary fat composition influences glomerular and proximal convoluted tubule cell structure and autophagic processes in kidneys from calorie‐restricted mice

Calorie restriction (CR) has been repeatedly shown to prevent cancer, diabetes, hypertension, and other age‐related diseases in a wide range of animals, including non‐human primates and humans. In rodents, CR also increases lifespan and is a powerful tool for studying the aging process. Recently, it has been reported in mice that dietary fat plays an important role in determining lifespan extension with 40% CR. In these conditions, animals fed lard as dietary fat showed an increased longevity compared with mice fed soybean or fish oils. In this paper, we study the effect of these dietary fats on structural and physiological parameters of kidney from mice maintained on 40% CR for 6 and 18 months. Analyses were performed using quantitative electron microcopy techniques and protein expression in Western blots. CR mitigated most of the analyzed age‐related parameters in kidney, such as glomerular basement membrane thickness, mitochondrial mass in convoluted proximal tubules and autophagic markers in renal homogenates. The lard group showed improved preservation of several renal structures with aging when compared to the other CR diet groups. These results indicate that dietary fat modulates renal structure and function in CR mice and plays an essential role in the determination of health span in rodents.     

Aging effect on myeloperoxidase in rat kidney and its modulation by calorie restriction

Myeloperoxidase (MPO), a heme protein existing in neutrophil and monocyte, is implicated in various stages of inflammatory conditions with the production of a variety of potent oxidants. To investigate the extent of the involvement of MPO in aging, we measured MPO activities in kidney of rats at different ages maintained with an ad libitum (AL) or a calorie restriction (CR) dietary regimen. Results showed that the MPO activities increased during aging in AL rats, but were significantly attenuated by CR. This result was consistent with altered protein level of MPO during aging. In addition, we were able to detect dityrosine that is a stable end MPO-oxidation product. The amount of dityrosine increased in old AL, but not in old CR rats. To examine the source responsible for increased MPO activity during aging for leukocyte recruitment and infiltration, the levels of vascular cell adhesion molecule (VCAM-1) protein were measured. The level of VCAM-1 showed age-dependent increase in AL rats, which was correlated with higher activity of MPO in old AL rats. Furthermore, we have found that LPS-induced inflammation increased the activity and protein levels of MPO, and VCAM-1 expression in young rat kidneys. These findings suggest that increased MPO activity with aging may related to increased recruitment of inflammatory cells, contributing to protein oxidation accumulation in the aging process. We propose that age-related alterations of MPO, dityrosine, and VCAM were modulated by CR through its anti-inflammatory action.     

Conserved and differential effects of dietary energy intake on the hippocampal transcriptomes of females and males

The level of dietary energy intake influences metabolism, reproductive function, the development of age-related diseases, and even cognitive behavior. Because males and females typically play different roles in the acquisition and allocation of energy resources, we reasoned that dietary energy intake might differentially affect the brains of males and females at the molecular level. To test this hypothesis, we performed a gene array analysis of the hippocampus in male and female rats that had been maintained for 6 months on either ad libitum (control), 20% caloric restriction (CR), 40% CR, intermittent fasting (IF) or high fat/high glucose (HFG) diets. These diets resulted in expected changes in body weight, and circulating levels of glucose, insulin and leptin. However, the CR diets significantly increased the size of the hippocampus of females, but not males. Multiple genes were regulated coherently in response to energy restriction diets in females, but not in males. Functional physiological pathway analyses showed that the 20% CR diet down-regulated genes involved in glycolysis and mitochondrial ATP production in males, whereas these metabolic pathways were up-regulated in females. The 40% CR diet up-regulated genes involved in glycolysis, protein deacetylation, PGC-1alpha and mTor pathways in both sexes. IF down-regulated many genes in males including those involved in protein degradation and apoptosis, but up-regulated many genes in females including those involved in cellular energy metabolism, cell cycle regulation and protein deacetylation. Genes involved in energy metabolism, oxidative stress responses and cell death were affected by the HFG diet in both males and females. The gender-specific molecular genetic responses of hippocampal cells to variations in dietary energy intake identified in this study may mediate differential behavioral responses of males and females to differences in energy availability.     

Aging differentially alters the expression of angiogenic genes in a tissue-dependent manner

Organ functions are altered and impaired during aging, thereby resulting in increased morbidity of age-related diseases such as Alzheimer’s disease, diabetes, and heart failure in the elderly. Angiogenesis plays a crucial role in the maintenance of tissue homeostasis, and aging is known to reduce the angiogenic capacity in many tissues. Here, we report the differential effects of aging on the expression of angiogenic factors in different tissues, representing a potentially causes for age-related metabolic disorders. PCR-array analysis revealed that many of angiogenic genes were down-regulated in the white adipose tissue (WAT) of aged mice, whereas they were largely up-regulated in the skeletal muscle (SM) of aged mice compared to that in young mice. Consistently, blood vessel density was substantially reduced and hypoxia was exacerbated in WAT of aged mice compared to that in young mice. In contrast, blood vessel density in SM of aged mice was well preserved and was not different from that in young mice. Moreover, we identified that endoplasmic reticulum (ER) stress was strongly induced in both WAT and SM during aging in vivo. We also found that ER stress significantly reduced the expression of angiogenic genes in 3T3-L1 adipocytes, whereas it increased their expression in C2C12 myotubes in vitro. These results collectively indicate that aging differentially affects the expression of angiogenic genes in different tissues, and that aging-associated down-regulation of angiogenic genes in WAT, at least in part through ER stress, is potentially involved in the age-related adipose tissue dysfunction.