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Published as doi: 10.1096/fj.07-099523.
 (The FASEB Journal. 2008;22:2037-2044.)
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Maternal diet influences DNA damage, aortic telomere length, oxidative stress, and antioxidant defense capacity in rats

J. L. Tarry-Adkins1, M. S. Martin-Gronert, J.-H. Chen, R. L. Cripps and S. E. Ozanne

Department of Clinical Biochemistry, University of Cambridge, Addenbrookes Hospital, Cambridge, Cambridgeshire, UK

1Correspondence: Department of Clinical Biochemistry, University of Cambridge, Addenbrookes Hospital, Box 232, Hills Rd., Cambridge, Cambridgeshire CB2 2QR, UK. E-mail: janeadkins{at}googlemail.com


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Low birth weight is associated with increased cardiovascular disease (CVD) in humans. Detrimental effects of low birth weight are amplified by rapid catch-up growth. Conversely, slow growth during lactation reduces CVD risk. Gestational protein restriction causes low birth weight, vascular dysfunction, and accelerated aging in rats. Atherosclerotic aortic tissue has shortened telomeres, and oxidative stress accelerates telomere shortening through generation of DNA single-strand breaks (ssbs). This study tested the hypothesis that maternal diet influences aortic telomere length through changes in DNA ssbs, antioxidant capacity, and oxidative stress. We used our models of gestational protein restriction followed by rapid catch-up growth (the recuperated group) and protein restriction during lactation (the postnatal low-protein [PLP] group). Southern blotting revealed fewer aortic DNA ssbs and subsequently fewer short telomeres (P<0.05) in the PLP group. This result was associated with reduced (P<0.01) 8-hydroxy-2-deoxyguanosine, a marker of oxidative stress. PLP animals expressed increased (P<0.01) manganese superoxide-dismutase, copper-zinc superoxide-dismutase, catalase, and glutathione-reductase. Age-dependent changes in antioxidant defense enzymes indicated more protection to oxidative stress in the PLP animals; conversely, recuperated animals demonstrated age-associated impairment of antioxidant defenses. We conclude that maternal diet has a major influence on aortic telomere length. This finding may provide a mechanistic link between early growth patterns and CVD.—Tarry-Adkins, J. L., Martin-Gronert, M. S., Chen, J.-H., Cripps, R. L., Ozanne, S. E. Maternal diet influences DNA damage, aortic telomere length, oxidative stress, and antioxidant defense capacity in rats.


Key Words: developmental programming • aging • aorta


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
EPIDEMIOLOGICAL STUDIES HAVE demonstrated strong correlations between low birth weight and increased susceptibility to cardiovascular disease (CVD) in later life (1 , 2) ; moreover, rapid postnatal weight gain increases this risk (3 , 4) . Maternal malnutrition can lead to low-birth-weight offspring with reduced numbers of cardiomyocytes in newborn rat pups (5) , which can amplify age-related vascular and structural changes (6) and increase cardiac fibrosis and capillarization in adulthood (7) . Conversely, studies in human and animal models have demonstrated beneficial effects of reduced nutrition and slower growth during lactation. Studies comparing long-term effects of formula feeding to bottled breast milk feeding in premature babies showed those who were given breast milk had reduced atherosclerotic risk factors later in childhood (8) . In addition, reduced CVD risk was suggested in full-term breast-fed babies compared with those who were bottle-fed (9) . Breast-feeding in general is associated with a lower nutritional plane and consequently slower growth during suckling. Slower growth during the first 2 weeks of life has been suggested to be particularly beneficial in preterm infants (10) . Studies have also shown that full-term formula-fed infants are more likely to be overweight later in childhood than breast-fed infants (11) .

Reactive oxygen species (ROS) production increases with age, causing oxidative damage to cellular macromolecules, including DNA (12) . Oxidative stress is associated with the pathogenesis of many age-associated disorders, including CVD (13) . Moreover, intrauterine malnutrition in female rats has been shown to increase production of the superoxide free radical (O2–), which may contribute to the associated endothelial dysfunction (14) .

Antioxidant enzyme defense is critical in reducing oxidative stress and maintaining redox homeostasis within the cell. Accumulated O2– is converted to H2O2 by superoxide dismutase (SOD). Manganese (Mn)SOD and copper- and zinc-containing (Cu/Zn)SOD are 2 major isoforms of SOD. MnSOD is mitochondrially localized and has been shown to be essential to life, particularly with respect to cardiac dysfunction. MnSOD knockout mice have a neonatal lethal phenotype and were shown to die of dilated cardiomyopathy (15) . In contrast, overexpression of MnSOD reduced diabetic cardiomyopathy (16) . However, the association between MnSOD, increased oxidative stress, and life span has not been shown in all studies (17) . Cytoplasmically localized Cu/ZnSOD has also been shown to have important roles in CVD; heterozygous Cu/ZnSOD-deficient mice express an increased vascular phenotype with age compared with wild-type mice (18) . However, other studies have reported that Cu/ZnSOD is not essential for life (19) . H2O2 is broken down further into H2O and O2 by glutathione peroxidase (Gpx) and catalase. A murine model of catalase overexpression demonstrated delayed cardiac pathology, reduced oxidative damage, decreased H2O2 production, and increased longevity (20 , 21) . A murine model of heterozygous cellular Gpx-1 deficiency caused abnormalities in vascular and cardiac function and structure (22) . Gpx requires glutathione (GSH) for this reaction, and the antioxidant enzyme glutathione reductase (GR) mediates the conversion of oxidized GSH to GSH. GR activity has been demonstrated to be reduced in the blood of patients with myocardial infarction (23) .

Telomere length has been implicated in aging processes for many years (24) , and it has been suggested that telomere shortening may be linked to many age-associated pathologies, including heart disease in humans (25) . Indeed, increased rates of telomere loss have been associated with sites of hemodynamic stress (26) . In addition, aortic telomere length has been shown to be negatively correlated with age and atherosclerotic grade in humans (27 , 28) . Telomere shortening has been suggested to be an in vivo marker of myocyte replication and aging in the rat (29) . Our previous studies (30 , 31) in rats have demonstrated that maternal diet can influence telomere length in the offspring. Maternal protein restriction during lactation increased longevity and reduced renal telomere shortening compared with offspring that were maternally protein restricted in utero and then suckled by normally fed dams.

Accumulation of oxidative stress is also known to accelerate telomere shortening, and antioxidants are known to decelerate it, because oxidative damage repairs less efficiently in telomeres (32) . Moreover, oxidative stress is known to induce single-strand breaks (ssbs) in the telomeres of human epithelial cells (33) , and ssb accumulation has been shown to be the major cause of telomere shortening in human fibroblasts (34) . Other factors have also been shown to be important in telomere length modulation, including changes in DNA repair mechanisms. In particular, Ku70 and Ku80, which promote the nonhomologous end-joining of DNA breaks and fusion of dysfunctional telomeres, have been shown to play a role in telomere protection (35) .

The aims of this study were therefore to investigate the effects of maternal diet on the frequencies of aortic DNA ssbs and resultant aortic telomere length in both young (3 month) and old (12 month) male rats, focusing on DNA repair mechanisms, antioxidant enzyme defense capacity, and 8-oxo-dG, a biomarker of oxidative stress.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
All procedures involving animals were conducted under the Animals (Scientific Procedures) Act (1986). Wistar rat dams were placed on ad libitum standard laboratory chow diet (20% protein) and water until pregnancy was confirmed through observation of vaginal plugs. Pregnant animals were maintained on a 20% normal protein diet (control) or an isocaloric low-protein (LP; 8%) diet, as described (36) . Both diets were purchased from Arie Blok (Woerden, The Netherlands). Cross-fostering techniques were used to generate protein-restricted offspring during gestation or lactation. The postnatal low-protein (PLP) group were offspring of rat dams fed the control diet during pregnancy and then nursed by rat dams fed the low-protein diet. The recuperated group were offspring of mothers fed the low-protein diet during gestation and then cross-fostered to control diet-fed rat dams. The control group were control diet-fed offspring that were suckled by control diet-fed rat dams. All animals were weaned onto a standard diet containing 20% protein (SDS, Witton, Essex, UK) at 21 days of age and remained on this diet until the end of the study. Body weights were recorded at days 3, 7, 14, and 21 and at 3 and 12 months. All groups consisted of 15 offspring at each time point, each of which came from a different litter.

One male from each litter was maintained until 3 months and then killed with carbon dioxide and decapitation. A second male from the same litter was maintained on ad libitum chow diet and water until 12 months and killed as above. At postmortem, aortas were removed, snap-frozen in liquid nitrogen, and stored at –80°C until required for analysis.

Telomere length and DNA ssb measurements
Nonsheared, high-molecular-size DNA was isolated from finely powdered aorta samples using the phenol/chloroform/isoamylalcohol DNA extraction protocol (37) . DNA quantity and integrity was determined using a spectrophotometer (Nanodrop; Nanodrop Technologies, Wilmington, DE, USA). DNA (1.2 µg) was digested with HinfI and RsaI restriction enzymes (38) . DNA for telomere length analysis was quenched with 5x SDS loading buffer (Roche Diagnostics, Mannheim, Germany). Samples for DNA ssb analysis were precipitated using 95% ethanol and sodium acetate (3M; Sigma, Poole, Dorset, UK), then centrifuged at 4°C for 5 min at 13,000 revolutions per minute. After washing the DNA pellets in 75% ethanol, the pellets were dissolved in TE buffer, and 1 U/µg S1 nuclease (Promega, Madison, WI, USA) was added and incubated for 1 h at 37°C. The reaction was quenched using 5x loading buffer (Roche Diagnostics). All digested DNA was then separated using pulsed-field gel electrophoresis (38) . After electrophoresis, the gels were checked for nonspecific degradation of the undigested DNA and complete digestion of the digested DNA by staining with ethidium bromide. The gels were visualized using an Alpha Imager UV light source (Alpha Innotech Corporation, San Leandro, CA, USA). The separated DNA fragments were transferred by Southern blotting onto nylon-positive membranes (Roche Diagnostics), and telomeric repeat length was determined using a commercial method of chemiluminescent detection (38) . Molecular weight markers on each gel were a midrange pulsed-field gel marker (New England Biolabs, Ipswich, MA, USA) and dioxygenin (low range) molecular weight marker (Roche Diagnostics). Standard undigested and digested genomic sample DNA from the same 3-month-old control animal were run on each gel to demonstrate digestion efficiency and to minimize any intergel differences. As a negative control for the DNA ssb analysis, the gels were repeated in the absence of S1 nuclease. Telomere signals were analyzed using Adobe Photoshop (Adobe Systems Inc., San Jose, CA, USA) and MacBas computer software (Fujifilm UK Ltd., Bedford, Bedfordshire, UK). Telomere length was measured as previously described (38) . Each gel was accepted on the criteria that the percent of telomere length of the control DNA in any of the 4 telomeric regions analyzed, (1.3–4.2, 4.2–8.6, 8.6–48.5, and 48.5–145 kb) was <1.5 SD from the mean.

Urinary 8-oxo-dG measurements
Urinary 8-oxo-dG levels were determined using a commercially available ELISA kit (Trevigen, Gaithersburg, MD, USA).

Ku70, Ku80, and antioxidant enzyme protein expression
Western blot analysis was used to determine protein expression of MnSOD, Cu/ZnSOD, catalase, GR, Gpx-1, and Ku70 and Ku80 in aorta samples. Protein was extracted and assayed as previously described (37) , and 20 µg protein was loaded onto 15% polyacrylamide gels, electrophoresed, and transferred to polyvinylidene difluoride membrane (37) . MnSOD was detected using anti-MnSOD (type II)-specific rabbit IgG (Upstate Biochemicals, Watford, Hertfordshire, UK). Cu/ZnSOD was measured using anti-Cu/ZnSOD-specific goat IgG (R&D Systems, Abingdon, Oxfordshire, UK). Catalase protein expression was evaluated using anti-catalase-specific rabbit IgG (Abcam, Cambridge, Cambridgeshire, UK). Gpx-1 protein expression was analyzed using anti-Gpx-1-specific rabbit IgG (Abcam). GR protein expression was analyzed using anti-GR-specific rabbit IgG (Abcam). Ku70 and Ku80 protein expression was determined using anti-Ku70- and anti-Ku80-specific rabbit IgG (Abcam). To check linearity and reproducibility, a standardization gel was run (38) . Each gel was then loaded with 20 µg protein per sample plus the same 2 samples as used on the standard gel (S1 and S2) as well as 10 µg of S1. Gels were accepted on the criteria that the ratios of S1:S2 were less than the mean integrated density value (arbitrary units) of the standardization gel ± 1.5 SD. In addition, linearity was ensured by accepting blots where the signal obtained by 10 µg of S1 was half of that of 20 µg of S1.

Soluble cellular adhesion molecule expression
ELISA was used to determine serum expression levels of soluble intercellular adhesion molecule (sICAM)-1 (R&D Systems).

Statistical analysis
All data were analyzed using a factorial 2-way ANOVA with maternal diet and age as the independent variables, followed by Duncan’s post hoc testing where appropriate. All data were represented as mean ± SE values. A value of P < 0.05 was considered statistically significant. All statistical analysis was performed using Statistica 7 software (Statsoft Inc., Milton Keynes, Buckinghamshire, UK).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Preweaning body weights
The PLP group had similar birth weights to control animals and became significantly (P<0.001) smaller than the control group by day 7. This PLP group also remained smaller throughout lactation. The recuperated group, which were born smaller than control animals, rapidly caught up in weight so that they were of weights similar to controls by day 14 (Table 1 ).


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  		Table 1. Body weight from birth until weaning

Postweaning body weights
The PLP group, at both 3 months (362.9±5.7 g) and 12 months (636.8±19.2 g) were significantly (P<0.001) lighter compared with both control (3 months: 435.0±10.2 g; 12 months: 750.4±39.8 g) and recuperated groups (3 months: 443.3±17.2 g; 12 months: 767.9±30.1 g). No significant difference in postweaning body weights were observed between the recuperated and control groups at either time point.

DNA ssb data
In the 48.5–145 kb size fragment, the region which can be most confidently quantified, in relation to ssbs, there were more telomeres from this region at both ages in the PLP group compared with both control (P<0.01; 3 months and P=0.05; 12 months) and recuperated rats (P<0.05; both ages). The increase in telomere length in the PLP group indicates reduced susceptibility to S1 nuclease and therefore reduced frequency of DNA ssbs in the PLP group. Conversely, the greater loss of telomere length in both the recuperated and control groups indicates increased frequency of DNA ssb damage in these groups. Significantly fewer telomeres in the 48.5–145 kb region were observed in all S1 nuclease-treated samples, confirming efficiency of the S1 nuclease enzyme (Fig. 1 A).


Figure 1
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  		Figure 1. A) The effect of gestational or lactation protein restriction on aortic SSB frequency in 3- and 12-month-old rats, including negative controls: DNA samples from all three groups, without S1 nuclease treatment. Results are expressed as means ± SE; n = 8 per group. *P < 0.05, PLP vs. recuperated; **P < 0.01, PLP vs. control. B) The effect of gestational or lactation protein restriction on aortic telomere length in 3- and 12-month-old male rats. Results are expressed as means ± SE. *P < 0.05, PLP vs. recuperated; **P < 0.01, recuperated vs. control and PLP.

Telomere length analysis
At 3 months, no significant effect of maternal diet was observed in aortic telomere length; however, by 12 months, significantly (P<0.05) more long telomeres (in the 48.5–145 kb region) were seen in the PLP group compared with the recuperated group. In addition, significantly (P<0.01) more telomeres from one of the shortest telomere regions (4.2–8.6 kb) were observed in the recuperated group compared with both control and PLP groups (Fig. 1B ). Also, significantly (P<0.01) more 4.2–8.6 kb telomeres were observed in the recuperated group between 3 and 12 months.

Ku70 and Ku80 protein expression data
There was no significant effect of maternal diet on protein expression of Ku70 or Ku80 at either 3 or 12 months of age. A highly significant (P<0.001) effect of aging was observed in Ku80 protein expression; however, no significant changes in Ku70 protein expression were seen between 3 and 12 months of age (Fig. 2 A, B).


Figure 2
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  		Figure 2. A) The effect of gestational or lactation protein restriction on Ku80 protein expression. P < 0.05 vs. control; P < 0.001, PLP and recuperated, 3 months vs. 12 months). B) The effect of gestational or lactation protein restriction on Ku70 protein expression. Results are expressed as means ± SE.

Urinary 8-oxo-dG levels
Urinary levels of 8-oxo-dG were similar at 3 months; however, by 12 months, significantly increased urinary 8-oxo-dG levels were observed in the recuperated group compared with the control (P<0.05) and PLP (P<0.01) groups (Fig. 3 ). There was also a significant effect of aging on urinary 8-oxo-dG, with levels increasing between 3 and 12 months. This result was most pronounced in the control (P<0.05) and recuperated (P<0.001) groups, with the effect of age in the PLP group attaining just borderline statistical significance (P=0.05; Fig. 3 ).


Figure 3
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  		Figure 3. The effect of gestational or lactation protein restriction on urinary 8-oxo-dG levels. 3-month groups: n = 11 per group. 12-month groups: control, n = 7; PLP, n = 9; recuperated, n = 7. Results are expressed as means ± SE. *P < 0.05, recuperated vs. control; **P < 0.01, PLP vs. recuperated.

Antioxidant enzyme data
MnSOD
There was a significant effect (P<0.001) of maternal diet on MnSOD protein expression. The PLP group demonstrated significantly increased MnSOD protein expression at 3 months (P<0.05) compared with control and at 12 months compared with both control (P<0.05) and recuperated (P<0.01) groups (Fig. 4 A). In addition, there was a significant (P<0.05) aging effect on MnSOD protein expression, with a significant (P<0.05) reduction in MnSOD protein levels seen in the recuperated group at 3 months compared with 12 months. No significant difference in MnSOD protein expression was observed between 3 and 12 months in either the control or PLP groups (Fig. 4A ).


Figure 4
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  		Figure 4. The effect of gestational or lactation protein restriction on expression of proteins: A) MnSOD, B) Cu/ZnSOD, C) catalase, D) Gpx-1, E) GR. Western blotting was performed on aorta homogenates at 3 and 12 months; n = 8 per group. Results are expressed as means ± SE. *P < 0.05; **P < 0.01, PLP vs. control and/or recuperated.

Cu/ZnSOD
At 3 months, Cu/ZnSOD protein expression was not different between groups. However, by 12 months, the PLP group demonstrated significantly increased Cu/ZnSOD protein expression compared with both control (P<0.01) and recuperated (P<0.05) groups (Fig. 4B ). Furthermore, there was a significant (P<0.01) effect of aging on Cu/ZnSOD protein expression with a significant (P<0.05) increase in Cu/ZnSOD protein expression observed in the PLP group with age. No significant difference in Cu/ZnSOD protein expression was seen in either the control or recuperated groups with aging (Fig. 4B ).

Catalase
At 3 months, catalase protein expression was not significantly different between groups. However, at 12 months, the PLP group showed significantly (P<0.01) increased catalase protein expression compared with controls. Also, a significant (P<0.05) effect of aging on catalase protein expression was observed with a significant (P<0.05) reduction in catalase protein expression seen in the recuperated group between 3 and 12 months. No significant difference in catalase protein expression with age was demonstrated in either control or PLP groups (Fig. 4C ).

Gpx-1
There was no significant effect of maternal diet observed on Gpx-1 protein expression at either time point (Fig. 4D ). There was a highly significant (P<0.001) effect of age on Gpx-1 protein expression with an overall increase between 3 and 12 months (Fig. 4D ).

GR
At 3 months, GR protein expression was statistically similar. However, by 12 months, the PLP group demonstrated significantly (P<0.05) higher GR protein expression compared with the recuperated group (Fig. 4E ). There was also a highly significant (P<0.001) aging effect on GR protein expression with significantly reduced GR protein expression observed in both control (P<0.05) and recuperated (P<0.01) groups between 3 and 12 months. A nonsignificant reduction in GR protein expression was seen in the PLP group between 3 and 12 months (Fig. 4E ).

Cellular adhesion molecule data
No significant differences between groups were observed at 3 months. However, by 12 months, significantly (P<0.001) reduced serum protein levels of sICAM-1 were found in the PLP group compared with the control group (Fig. 5 ). There was also a significant (P<0.001) effect of aging on sICAM-1 protein levels. Significantly elevated sICAM-1 levels were observed in both control (P<0.001) and recuperated groups (P<0.05) between 3 and 12 months. However, no significant differences in sICAM-1 protein expression was seen in the PLP group between 3 and 12 months (Fig. 5) .


Figure 5
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  		Figure 5. The effect of gestational or lactation protein restriction on sICAM-1 protein levels in serum in 3- and 12-month-old male rats. Results are expressed as means ± SE; n = 9 per group. ***P < 0.001, control vs. PLP (12 months).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In humans, a strong association exists between low birth weight, accelerated postnatal growth, and increased risk of CVD in later life (3 , 4) . Conversely, slower growth during the lactation period has been demonstrated to be beneficial. Comparisons of the long-term effects of breast feeding and formula feeding in premature babies showed that the breast-fed infants, which grow more slowly during lactation due to the reduced nutritional plane, had reduced atherosclerotic risk factors in later life (8) . In addition, breast-fed full-term babies have reduced CVD risk compared with bottle-fed infants (9) . In rats, low-birth-weight offspring, resulting from maternal protein restriction, demonstrated cardiac structural abnormalities (7) , vascular dysfunction (39) , and reduction in aortic wall thickness and elastin content (6) in adulthood. Conversely, long-term caloric restriction protected against age-related rat aorta fibrosis (40) . This study explored potential underlying mechanisms.

At 3 months, no significant effect of maternal diet on aortic telomere length was observed. Because DNA ssb damage precedes telomere shortening (34) , frequencies of aortic DNA ssbs were compared at this earlier time point and at 12 months. Only ssbs in the largest (48.5–145 kb) portion of the telomeres can be confidently assessed because the other telomeric size regions may represent the balance of telomeres gained by telomere digestion from larger fragments and those lost through S1 nuclease digestion. At 3 months of age, significantly more telomeres from this region were seen in the PLP group compared with both control and recuperated groups following S1 nuclease digestion. This result indicates reduced frequency of DNA ssb damage in this group. This difference in DNA ssb damage in the absence of a change in telomere length is consistent with such damage being an event that precedes telomere shortening, consistent with the hypothesis that, at 12 months of age, PLP animals had both reduced ssbs and significantly more long telomeres (48.5–145 kb) than did the other 2 groups. Because of lipoprotein profile differences, rodents do not develop atherosclerosis, unless transgenically modified (41) . Therefore, the observed differences in aortic telomere length cannot be attributed to differences in degrees of atherosclerosis. Taken together, these data may suggest that slow growth during lactation could exert a protective effect on the aorta, with reduced DNA damage occurring at an early age, which results in the conservation of longer aortic telomeres at 12 months in the PLP group and maintenance of reduced DNA ssb damage. Conversely, gestational protein restriction followed by rapid catch-up growth seems to increase DNA ssb damage and accelerates aortic telomere shortening in aged rats.

Underlying mechanisms, which may be responsible for the observed differences in DNA damage and telomere length, were then investigated. Oxidative stress represents a disturbance in the equilibrium status of prooxidant/antioxidant reactions in living organisms (13) and has been implicated in causing increased DNA ssbs (33) , thus accelerating telomere shortening (34) . Therefore, 8-oxo-dG, a commonly used measure of oxidative stress (42) , was analyzed. At 3 months, no significant effect of maternal diet was observed in urinary 8-oxo-dG levels; however, by 12 months of age, significantly reduced levels of urinary 8-oxo-dG were observed in the PLP group compared with the other groups. This result may be associated with the significantly reduced DNA ssb damage seen at 12 months. Conversely, increased oxidative stress levels have been observed in children born small for gestational age (43) . As oxidative damage is known to accelerate telomere shortening, the reduced oxidative stress in the PLP group may explain why this group has more long telomeres at 12 months. Moreover, urinary levels of 8-oxo-dG increased over time in all 3 groups. These data reflect the established theory that oxidative damage accumulates in cells over time (44) . However, this increase was only statistically significant in the control and recuperated groups, suggesting that a protective mechanism is present in the PLP group. Thus, antioxidative capacity was compared in our groups. The antioxidant enzyme SOD is the first line of cellular antioxidant defense and is responsible for the catalysis of the O.2– free radical into H2O2. MnSOD, the mitochondrial isoform of SOD is essential to life (15 , 16) , although not all studies have shown this association (17) . Maternal diet significantly altered protein expression of MnSOD, with the PLP group demonstrating significantly increased MnSOD levels at both ages. Moreover, the recuperated group showed an age-associated reduction of MnSOD protein expression. MnSOD has been shown to be particularly important in cardiac function, with MnSOD knockout mice demonstrating dilated cardiomyopathy and neonatal lethality (15) and murine MnSOD overexpression showing reduction of diabetic cardiomyopathy (16) . These data suggest that the PLP group may be protected against cardiac damage, and the recuperated group may have age-associated impairment of MnSOD protection, which would increase the cardiac damage risk in this group. Cu/ZnSOD, the cytoplasmically localized SOD isoform, was significantly up-regulated in the PLP group at 12 months compared with control and recuperated groups. In addition, a significant age-related increase in Cu/ZnSOD expression was observed in the PLP group. As Cu/ZnSOD-deficient mice have been shown to have greatly enhanced age-associated vascular dysfunction (18) , and endothelial dysfunction has been observed in Cu/ZnSOD knockout mice (45) , our data may suggest that our PLP group is more protected against vascular and endothelial dysfunction compared with the other groups. The peroxidases, catalase, and Gpx-1 are responsible for the conversion of H2O2 into O2 and H2O. Catalase protein expression was significantly up-regulated at 12 months in the PLP group. As catalase overexpression has been shown to delay cardiac pathology and reduce oxidative damage (20) , our data once again suggest cardioprotection to the PLP group and increased cardiac damage to the recuperated group. In addition, levels of catalase remained stable with aging in the PLP group, whereas the recuperated group exhibited significantly reduced catalase protein expression with age. Given that catalase overexpression is known to increase longevity in the mouse (21) and that our previous data have demonstrated significantly increased life span in the PLP group and significantly reduced longevity in the recuperated group compared with controls (30) , the altered catalase protein expression appears directly associated with the observed differences in longevity in our model. Gpx-1 remained unchanged between groups at either time point; however, all 3 groups showed elevation of Gpx-1 over aging. GR is responsible for the conversion of oxidized GSH back into GSH. At 12 months, significantly reduced levels of GR were observed in the recuperated group compared with the PLP group. As GR activity is reduced in the blood of patients with myocardial infarction after reperfusion (23) , this finding may suggest that the recuperated group is at more risk of cardiac dysfunction compared with the PLP group.

Alterations in DNA damage repair capacity may also contribute to the difference in aortic telomere length observed in this study. Ku70 and Ku80 are 2 core nonhomologous end-joining factors that are localized to telomeres and play an important role in telomere maintenance (46) . The observation that there was no significant difference in protein expression of Ku70 and Ku80 between the control, PLP, and recuperated groups suggests that DNA repair mechanisms involved in telomere maintenance may not be the prime factors modulated by maternal diet in our rats. The age-associated decrease in Ku80 protein levels in all groups is consistent with a recent report showing that telomere maintenance proteins decrease with age and may contribute to cellular senescence (47) .

Taken together, these data suggest that slow growth during lactation up-regulates antioxidant defense capacity; conversely, gestational protein restriction followed by rapid catch-up growth impairs this capacity. This finding may provide a mechanistic basis for the apparent reduced oxidative stress observed in the PLP group, the reverse being the case for the recuperated group. These alterations in oxidative stress levels may be associated with the observed telomere length differences. Indeed, vascular dysfunction, microvascular rarefaction, and hypertension in low-protein rat offspring have been prevented by in utero exposure to antioxidants (48) .

ROS has also been implicated in the up-regulation of proinflammatory proteins such as soluble adhesion molecules (sAMs), which have been associated with various cardiovascular risk factors, including myocardial infarction (49) . sAMs increase during normal aging in rats, however these increases are suppressed during caloric restriction (50) . We focused on the measurement of serum sICAM-1 levels because it is known that ICAM-1 expression can be inhibited by SOD in human epithelial cells (51) . Measurement of sICAM-1 in our groups showed significantly reduced levels of sICAM-1 in the PLP group at 12 months, which is consistent with the suppression observed in caloric restricted rats. In addition, significant age-associated increases in sICAM-1 expression were not observed in the PLP group. The increased levels of MnSOD and Cu/ZnSOD in the PLP group may contribute to the reduced expression of sICAM-1 due to the reported inhibition of ICAM-1 by SOD (51) .

In summary, the time window of protein restriction and subsequent growth trajectories seems to be of critical importance for consequent DNA ssb damage and telomere length in the aorta of male rats, which is associated with altered oxidative damage, modulation of antioxidative defense capacity, and changes in sAM levels. However, maternal protein restriction does not seem to promote changes in Ku-mediated DNA repair mechanism. Taken together, these data may explain why low birth weight followed by rapid catch-up growth has deleterious effects on cardiovascular structure and function and, conversely, why slow growth during the lactation period reduces cardiovascular risk.


   ACKNOWLEDGMENTS
 
We thank A. Wayman and D. Hawkes for their technical expertise and Dr. S. Jackson for help with the Ku70/Ku80 analysis. This work was supported by the Parthenon Trust, the British Heart Foundation, the Biotechnology and Biological Sciences Research Council, and the U.S. National Institutes of Health.

Received for publication September 29, 2007. Accepted for publication January 3, 2008.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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J. L. Tarry-Adkins, J. H. Chen, N. S. Smith, R. H. Jones, H. Cherif, and S. E. Ozanne
Poor maternal nutrition followed by accelerated postnatal growth leads to telomere shortening and increased markers of cell senescence in rat islets
FASEB J, May 1, 2009; 23(5): 1521 - 1528.
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