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Published online before print July 3, 2008
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From the Department of Geriatrics,* Division of Experimental Diabetes and Aging, and the Department of Medicine, Division of Nephrology, Mount Sinai School of Medicine, New York, New York; and the Departments of Medicine and Surgery, Miller School of Medicine, University of Miami, Miami, Florida
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Abstract |
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Although the formation of oxidants is a part of normal metabolism, their excess causes inflammation and organ injury, compromises healthspan, and shortens lifespan.14,15 Oxidants may be derived from endogenous sources, ie, hyperglycemia16,17 or exogenous sources, namely the diet.18-20 Heat exposure of animal and human food is an important generator of variable amounts of glycoxidants, or advanced glycation end products (AGEs).19,21 The levels of carboxy-methyl-lysine (CML), a common AGE,16,22 directly correlate with the levels of other protein or lipid AGEs, ie, methyl-glyoxal (MG)23 or lipid oxidation malonyl-dialdehyde derivatives in food.21 Thus, CML has been used as a surrogate AGE marker in foods, as well as in tissues and fluid spaces of man and animals.18,19,21,24 Because AGEs increase cellular OS, AGEs are recognized as pro-oxidant substances.16,17,25 Dietary AGEs contribute to increased systemic levels of AGEs and inflammatory modulators in humans and experimental animals.18,26-28 Lowering the intake of AGEs by restricting the heat-processing of standard chow fed to normal mice reduces circulating and tissue AGE levels and systemic OS, resulting in preservation of antioxidant reserves and decreased levels of inflammatory cytokines.26,29-32 These findings also apply to patients with chronic kidney disease or diabetes.24,27,28 Other studies of aging-related diseases in experimental animal models show that an AGE-reduced, isocaloric diet provides protection from insulin resistance and diabetes as well as cardiovascular and chronic kidney disease.26,29-32 The mechanisms that may underlie these effects are thought to include decreased levels of OS regulatory components, ie, p66shc10 and RAGE,25 and increased anti-OS AGE-receptor-1 (AGER1),33,34 resulting in an improved net anti-oxidant homeostasis.26
Others have shown that the restriction of calories (CR) in rats results in a reduced consumption of dietary AGEs as an obligate accompaniment of reduced food intake.35
This unintended consequence may be of clinical relevance, because an 
50% decrease in dietary oxidants led to a reduction in oxidant load and tissue damage as well as an increase in lifespan.26
However, none of the previous studies demonstrated 1) a direct role in CR for specific AGEs, 2) that feeding a CR diet results in lower levels of AGE consumption, or 3) that a CR diet containing AGE levels similar to those of a regular diet may significantly modify the benefits of CR. We addressed these questions in normal aging mice that were pair-fed a CR diet or an identical CR diet in which the content of AGEs was increased by brief heat treatment, similar to that used in preparation of the regular diet.
We report that AGEs in food are a significant contributor to OS, by showing that addition of a chemically purified AGE to mouse chow markedly increases OS in normal mice. In addition, we show that the reduction of food consumption in a CR diet implicitly restricts the consumption of AGEs, leading to lower OS and increased healthspan. Finally, we demonstrate that a CR diet that is AGE-supplemented, so that consumption of AGEs is no longer restricted, results in high OS, accelerated aging-related cardiovascular/renal disease, and a shorted lifespan. The findings demonstrate that the oxidant content of the calorie-restricted diet, as well as the normal mouse chow diet, may be a major determinant of oxidant status in both young and aged animals.
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Materials and Methods |
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AGE-Supplemented Diet: 6-Month Study
Previous studies in animals on the effect of dietary AGEs were based on correlations between measurements of CML-like AGEs in a standard chow diet (Table 1)
, and the corresponding levels of CML in serum and tissues. In the current study a chemically defined AGE, MG-BSA prepared as previously described,21
was directly added to a low-AGE diet, which had been prepared with reduced exposure to heat.26,30,31
The levels of protein and lipid-associated MG-derivatives, as well as CML, a terminal glycoxidation product, were increased (Table 1)
. Dams fed the Low diet were maintained on the same diet during gestation, and groups of their pups (n = 15 per group, 7 males and 8 females) were pair-fed one of three diets at weaning (Table 1)
: 1) regular mouse chow (Reg), 2) a low-AGE mouse chow (Low),26
or 3) Low diet to which MG-BSA (1 mg/g food) had been added (Low + MG), followed by air-drying. All food was consumed between feedings.
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C57BL/6 mice (n = 66, male, 4 months of age) from the National Institutes on Aging CR colonies were individually caged and provided free access to water. Mice were assigned to one of three diets (n = 22 per group): A) a modified version of NIH-31 calorie-deficient (40% calorie reduction) diet, which was balanced in minerals and vitamins (CR)1,2
; B) the same CR diet, in which the content of AGEs was increased by extending heat exposure (by 15 minutes. at 120°C) (CR-high); and C) The NIH-31 open formula diet (Reg) (Harlan Teklad, Madison, WI) (Tables 1 and 2)
. Diets were purchased in small amounts (<5 kg) and kept at 4°C. Food consumption was monitored daily for the first 4 weeks, and weekly thereafter (Table 2)
. After establishing the daily intake in the CR and Reg mice, the CR and CR-high were pair-fed. Food was completely consumed by the mice between feedings throughout the study. Body weight was monitored biweekly during the first 3 months and then monthly. These mice were used for survival curves.
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Measurement of AGEs
The concentrations of AGEs in sera, heart, kidney, urine, and diets were determined by enzyme-linked immunosorbent assay, using well characterized monoclonal antibodies for CML-like epitopes (4G9; Alteon, Northvale, NJ) and for MG derivatives (3D11).18,19,21,24,26 We used three reference standards, characterized by HPLC/GC-MS, a glucose-modified BSA standard containing 36 modified lys/mol, a synthetic CML-BSA containing 23 modified lys/mol, and a MG-BSA standard containing 22 MG-modified arginines/mol. CML immunoreactivity (using the 4G9 mAb) correlated with MG-immunoreactivity (based on 3D11 mAb) in vitro21 and in vivo.27 Unless otherwise stated, AGE values in the chronic study are based on CML measurements, consistent with our previous studies.18,19,24,26-32
Metabolic Studies
At 16 and 112 weeks of age, an intraperitoneal glucose tolerance test (5% dextrose solution; 2 mg/g body wt) was performed in the serial sacrifice cohort from each dietary group (n = 5), after an overnight fast.26,29,30 Blood was sampled before and at intervals between 5 and 120 minutes after glucose infusion. Glucose levels were determined with an Elite glucometer (Bayer, Mishawaka, IN) and serum insulin levels were determined by enzyme-linked immunosorbent assay (Ultra-Sensitive mouse insulin kit; Alpco Diagnostics, Windham, NH).
Glutathione (GSH), Oxidized Glutathione (GSSG), and 8-Isoprostane Studies
Blood was collected via cardiac puncture at 16 and 112 weeks of age from five animals in each diet group from the serial sacrifice cohort. GSH and GSSG were analyzed colorimetrically (OxisResearch, Portland, OR) using the manufacturer’s recommendations and quantified by an enzyme-linked immunosorbent assay reader (412 nm).26,29
Endogenous lipid peroxidation products (8-isoprostanes or 8-epi-PGF2
) were determined in fresh plasma samples, using an enzyme immunoassay kit, which correlates with values determined by GC-MS (Cayman Chemical, Ann Arbor, MI).26,29
Urinary AGEs, Creatinine, and Albumin
Urinary AGEs, albumin, and creatinine excretion were evaluated at 16 and 112 weeks of age. Urinary creatinine and albumin were measured using a DCA 2000 microalbumin/creatinine reagent cartridge with DCA 2000 analyzer (Bayer Corp., Elkhart, IN). The urinary albumin:creatinine ratio and the excretion of AGEs were calculated from aliquots of 24-hour urine samples collected from each group (n = 5).32
Western Blot Analysis
Equal amounts of protein extracts from heart and kidney tissues were separated on 10% or 8% sodium dodecyl sulfate-polyacrylamide gels and transferred to nitrocellulose membranes.26,34 The membranes were blocked in TTBS (Tris-buffered saline with 0.1% Tween 20) containing 5% powdered milk for 1 hour. Primary antibody incubations were performed in TTBS with 5% powdered milk overnight at 4°C. After washing, the membranes were incubated with the appropriate secondary peroxidase-conjugated antibody for 1 hour in TTBS. Proteins were visualized using the enhanced chemiluminescence system from Roche, Nutley, NJ. For reprobing, blots were stripped with a buffer containing 50 mmol/L Tris-HCl, pH 6.8, 2% sodium dodecyl sulfate, and 0.1 mol/L β-mercaptoethanol and probed with a second primary antibody. Antibodies: anti-Shc from BD Biosciences Transduction Laboratories (San Jose, CA), anti-MnSOD from Stressgen Biotechnologies (Victoria, Canada), and anti-phospho-Thr-32-FKHRL1 from Upstate Biotechnology, Inc. (Lake Placid, NY).
Histology
Kidney
Specimens obtained at 16 and 112 weeks (n = 5) in the serial sacrifice cohort were fixed in 10% buffered formalin and embedded in paraffin. Sections, stained by periodic acid-Schiff were used to assess glomerulosclerosis.26,31,32
Heart
The heart was immersion-fixed in 10% buffered formalin and cross-sections taken at the mid-ventricular level were embedded in paraffin. Sections were stained with Sirius Red to reveal connective tissue.31,32
Statistics
Survival curves were calculated by the Kaplan-Meier method and statistical differences between these curves were evaluated by the log rank test. Analysis of weight and serum AGEs at indicated time points were performed by one-way analysis of variance followed by Bonferroni’s multiple comparisons test. Data are shown as mean ± SEM. P values <0.05 were defined as significant.
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Results |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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The MG-supplemented diet (Low + MG) was identical to the Low diet, except for the anticipated higher content in MG-derived AGEs (Table 1)
. As previously noted,36
litters from mothers fed a Low diet had lower baseline serum AGE levels than litters from dams fed the Reg diet during gestation (Reg-Fo, serum MG 1.2 ± 0.4 nmol/ml; Low-Fo, MG 0.6 ± 0.2 nmol/ml). Serum MG derivatives in the Low + MG and the Reg mice were three- to fourfold increased above baseline levels at 6 months, whereas the levels in the Low group did not differ from baseline values (Figure 1A)
. The levels of plasma 8-isoprostanes were also twofold elevated in the Reg and Low + MG groups (Figure 1B)
, whereas serum CML levels increased by threefold (baseline: 11 ± 2 U/ml, and at 6 months: Reg: 37 ± 7 U/ml, Low + MG: 43 ± 13 U/ml, Low: 23 ± 12 U/ml; Reg and Low + MG versus Low, P < 0.01, respectively) reflecting the higher systemic oxidants in these mice. Thus, AGEs in the diet are a direct contributor to systemic levels of AGEs and OS in young mice. There were no differences in body weight during this short-term follow-up study.
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The nutrient and micronutrient composition between the two CR diets and the regular diet was identical. The sole difference between the CR diets was the brief heating step in the CR-high diet, which increased CML and MG levels (Table 1)
.26,30-32,37
Calorie intake in both CR groups was 12 kcal/day (40% restriction) compared to that in the Reg group (19 kcal/day) and remained stable throughout the study in all groups (Table 2)
. The reduced food intake resulted in an 45% lower consumption of protein, and fat-associated dietary AGEs by the CR mice than the Reg mice. In contrast, the total intake of AGEs by the CR-high mice was 2-fold greater than in the CR mice, and 1.8-fold greater than in the Reg mice. The levels in this diet, although high, are less than in the usual Western diet.
Body weight in both CR groups remained lower than in the Reg mice throughout the study (Figure 2)
and continued to decline after 53 weeks, whereas the Reg mice maintained their highest achieved adult weight throughout the study (>24 months). After 56 weeks of age, the CR-high mice tended to have a higher body weight throughout much of their adult life than did pair-fed CR mice, despite an identical energy intake in the two CR groups.
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The differences in the dietary content of AGEs were reflected in the serum and tissue AGE levels of the three cohorts (Figure 3, A–C)
. By 50 weeks of age, serum AGE levels in the CR-high mice were significantly higher than in the CR group from 48 weeks to the end of the study despite a restricted calorie intake (Figure 3A)
. A similar pattern was observed in tissue AGE levels in cohorts sacrificed at 16 and 112 weeks of age (Figure 3, B and C)
. Tissue AGE levels were three- to sixfold higher in old CR-high than in CR mice (Figure 3, B and C)
. Thus, the increased content of AGEs in a CR-high diet resulted in high AGE levels, similar to those found in Reg old mice fed an unrestricted diet.26
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We previously showed that aging mice pair-fed the Reg diet were insulin resistant and retained more body mass than aging mice fed a diet with a low AGE content.26
In the current study, 112-week-old CR-high and Reg mice had higher fasting insulin levels than CR mice (Table 3)
, and these changes were associated with an impaired insulin response to a glucose challenge and a blunted glucose:insulin ratio (GIR) (Figure 4, A and B)
. In contrast, 112-week-old CR mice had nearly normal insulin levels and response to glucose challenge. Insulin levels returned to baseline levels within 30 minutes, and the GIR was normal (twofold higher than in CR-high or Reg mice).
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A reduction of dietary AGEs has been shown to prevent increased OS and age-related kidney lesions.26,32
However, the effect of CR, with or without an elevated intake of AGEs, on serum and cellular markers of OS in aging mice has not been explored. Endogenous lipid oxidants, ie, 8-isoprostanes, were more than twofold higher in CR-high and Reg mice than in CR mice at 112 weeks (Figure 5A)
. Although plasma 8-isoprostanes were slightly increased with age in the Reg group (Figure 5A
, inset), in the CR-high mice they were three- to fourfold increased above the 16-week levels. Therefore, we examined intracellular GSH/GSSG in freshly isolated blood cells from each group to assess the handling of excess OS in these groups with aging. GSH:GSSH ratios at 112 weeks were approximately twofold higher in CR mice than in age-matched CR-high or Reg mice (Figure 5, A and B)
. In addition, this ratio did not diminish with age in CR mice, but was 30% higher at 112 weeks than at the 16-week baseline level (Figure 5B
, inset). In contrast, the GSH:GSSG ratio in 112-week CR-high and Reg mice was reduced by 25 to 40% below their respective baseline (16 weeks) levels. The levels of GSH was lower in the CR-high mice (103.4 ± 28.1 mmol/L) (P < 0.01) than in the Reg (152.7 ± 5.2 mmol/L) or the CR (199.5 ± 16.4 mmol/L). There were no differences in the levels of GSSG between the dietary groups at 28 months of age. These data indicated that there was significant depletion of cellular antioxidant reserves in old mice fed a diet restricted in calories but not restricted in AGEs.
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The levels of some of the AGE receptors involved in the regulation of AGEs and OS, AGER1 and RAGE, were found to be altered during aging in mice fed an AGE-restricted diet,26
but this has not been previously explored in CR. AGER1 protein levels increased by twofold in heart (Figure 6A)
and kidney tissue (data not shown) from 112-week-old CR mice, compared to 16-week-old mice, but no change was found in old CR-high or Reg mice. RAGE expression in CR mice, although higher at 112 weeks relative to 16 weeks, was significantly lower than in the Reg (by 50%) and CR-high (by 60%). RAGE expression in CR-high, as well as in Reg mice was 3.5-fold higher at 112 weeks, than at 16 weeks of age (Figure 6B
, inset). Thus, AGER1 expression in aging mice increased under CR, but decreased under CR-high and Reg diet. In contrast, RAGE expression followed a pattern opposite to AGER1 under the same diets.
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, lane 2), but was increased in CR-high, as was in Reg mice, with the highest levels being in the former. Compared to the 16 weeks CR, p66shc levels in 112-week-old CR mice were 25% lower (Figure 7B)
. This contrasted with the 15 to 20% increase above baseline (16 weeks) in p66shc levels of 112-week-old CR-high and Reg mice (Figure 7B)
.
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The albumin excretion rate was only slightly increased in CR mice as they aged (Figure 8A)
. The albumin excretion rate increased at an early time point (24 weeks) in the CR-high and Reg mice, and continued to increase with aging. The urinary AGE excretion increased at an early time point and up to 8 months in the CR-high and Reg mice, consistent with their higher AGE intake (Figure 8B)
. However, between 16 and 24 months AGE excretion was markedly decreased in these two groups of mice, suggesting that the ability of the kidney to excrete AGEs was already impaired and progressively worsened. In contrast, AGE excretion in CR mice rose less rapidly, consistent with the lower AGE intake, but continued to rise for up to 16 months, and remained higher than CR-high and Reg mice thereafter. This suggests that renal AGE-excretory capacity was less impaired in CR, compared to CR-high and Reg mice, with aging.
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The histological changes in the kidney paralleled the proteinuria and kidney AGE levels in all three diet groups (Figure 9, A–C)
. The glomerular changes in 112-week-old CR mice were modest consisting of scattered areas of tubulointerstitial fibrosis, associated with tubular atrophy (Figure 9B)
. Both glomerular and tubulointerstitial sclerosis were moderately severe in old Reg mice (Figure 9A)
, and although the lesions were more pronounced in the CR-high mice (Figure 9C)
, these two groups were otherwise similar.
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. Namely the myocytes were normal in appearance, uniform in size, and there was no increase in interstitial connective tissue (Figure 9E)
.40
However, CR-high mice had diffuse, fine filamentous subendocardial bundles of collagen fibrils, visualized with Sirius Red staining, that isolated and compartmentalized the cardiomyocytes (Figure 9F)
. There was also an increase in the size of individual myocytes, and hyaline changes in the cytoplasm. These changes involved nearly one-third of the thickness of the left ventricle of aged CR-high mice, and were similar, but less marked, in the old Reg mice (Figure 9D)
. These changes were in line with the significant increase in tissue AGEs in these two groups of mice (Figure 3, B and C)
. Survival
Lifespan in CR mice was longer (median and maximal survival 13.2% and 6%, respectively) than in Reg or CR-high mice (P < 0.001) (Figure 10
and Table 4
). In addition, survival in CR-high mice was shorter than in Reg (P < 0.02) mice. At the median survival of CR-high mice, 65% of CR mice were alive, and at the maximal survival level of CR-high mice, 30% of CR mice were still alive (Figure 10
and Table 4
).
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Discussion |
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Recent studies have indicated that mouse diets, although well balanced nutritionally, expose mice to a 5- to 10-fold higher AGE load throughout their life time, on a body weight basis, than are present in the human diet.18,21,24,26-32 We searched for a link between calorie and AGE restriction by feeding mice a typical CR diet (which resulted in a low AGE intake) or with a modified CR diet (CR-high) that resulted in an AGE intake that contained 50% higher amounts of CML and MG derivatives than the Reg diet. We compared mice fed these diets to mice fed regular mouse chow on an ad libitum basis throughout adult life. Serum and tissue levels of AGEs and 8-isoprostanes in the old mice fed the CR diet were reduced, and GSH levels were maintained, consistent with reduced AGE intake in these mice. However, old mice given the CR-high diet developed the typical pattern of increased serum AGEs, lipid peroxides, and loss of anti-oxidant reserves found in old mice fed the regular diet.1-3,26 These findings suggest that increasing the AGE intake in calorie-restricted mice to amounts similar to or moderately higher than that consumed by mice fed the regular ad libitum diet, reproduces the pattern of high oxidant load and anti-oxidant depletion associated with aging under an energy replete diet. Thus, increasing the intake of AGEs in the diet erases the benefits of CR.
High circulating levels of AGEs are linked to diabetes and its complications in both humans and animal models.16,24 In addition, both CR and AGE restriction reduce the risk of diabetes and prolong lifespan.26,29-32 Mice fed regular mouse chow maintained their maximal weight throughout normal life.1-3,26 As expected, body weight was lower in CR mice and they maintained normal insulin and GIR levels with aging.1-7 In contrast, old CR-high mice had elevated fasting insulin levels and GIRs, consistent with insulin resistance, and they tended to have a higher body weight. Age-related IR in these mice was likely attributable to the increased consumption of oxidants, because their energy intake was restricted, matching that of the CR diet. This conclusion is consistent with earlier findings whereby mice pair-fed an AGE-restricted diet, otherwise identical in calories to Reg diet, did not develop IR and showed a trend of lower body weight with age.26 The amount of AGEs consumed by the CR mice in the present study was similar to that of mice fed a Low-AGE diet. Because AGEs are known to promote irreversible protein-protein and protein-lipid cross links that resist degradation,16,17,22,44 the higher intake of AGEs in CR-high versus the CR mice may account for their trend toward higher body mass retention after the initial weight gain.29,30
The development of insulin resistance in the old CR-high mice, coupled with the loss of systemic anti-oxidant reserves and higher tissue deposits of AGEs, may have contributed to the cardiovascular fibrosis and kidney disease of aging noted in the old CR-high mice, but not in the old CR mice.4,5,40 The protection from interstitial fibrosis in the kidneys of CR mice parallels that previously seen in mice fed a low AGE diet.26 Consistent with that study, the contrast between the CR and CR-high mice indicates that the overall amount of oxidants in the diet may be one of the principal determinants of age-related cardiovascular and renal disease.
In the current study, high levels of oxidants in the CR-high diet may have competed against the benefits of CR by mechanisms that remain to be identified. A role for the AGE-associated receptors is likely, of which the best studied are AGER1 and RAGE. AGER1 blocks OS and inflammatory responses,33,34 whereas RAGE increases OS and inflammatory responses.25,45 Because AGER1 blocks RAGE and OS in vitro,33,34 the nearly twofold enhanced AGER1 expression found in old CR mice may have contributed to protection against OS in this group. Similarly, the lowered AGER1 levels in the old Reg and CR-high mice may have promoted OS and inflammatory responses. Because AGER1 also mediates the uptake and degradation of AGEs,33 the increased AGER1 levels in the kidneys of CR mice could underlie the maintenance of higher urinary AGE excretion with aging in these mice. The combination of increased turnover and higher urinary excretion of AGEs in aging CR mice may have contributed to a reduced tissue AGE burden and to long-term tissue protection against AGE toxicity.44
Recent data implicates AGER1 in a novel anti-OS mechanism involving the inhibition of the EGFR-ERK activation pathway and suppression of the OS-regulatory protein, p66shc.46 This is thought to provide protection from AGE-induced inactivation of the forkhead transcription factor, FOXO3a, and diminished MnSOD levels.11,46 The increased AGER1 expression in old CR mice may have played a role in preserving anti-oxidant defenses in these mice via this mechanism. This is supported by the fact that CR mice did not develop the age-dependent rise in p66shc protein,10,11 which has been linked to aging-related diseases such as atherosclerosis and diabetic nephropathy.38,39 This postulate is consistent with the findings in mice fed a low-AGE diet,26 and may provide an in vivo link between the sustained high AGER1 levels and extended lifespan in CR mice of the current study. In contrast to CR mice, p66shc levels were elevated in CR-high mice, as were the levels of tissue RAGE and systemic oxidants, consistent with markedly elevated OS and a depletion of GSH in these mice. Of note, RAGE levels in old CR mice were reduced, identifying CR as one means of suppressing this pro-oxidant factor in aging. These data, although not proving a causal relationship, provide the first indication that oxidant homeostasis in mice may depend on the maintenance of a positive balance between AGER1 and RAGE/OS.
AGE levels in vivo correlate with AGEs in foods19,21,37 and with the cellular effects elicited by synthetic MG and CML.17,23,37,45 This study validates these correlations by showing that specific AGEs in the food contribute directly to serum AGEs and to AGE-induced in vivo responses. It also demonstrates the importance of AGEs contained in the diet, although other oxidants may also contribute to the findings of the current study.20
The findings support evidence that age-related changes in OS and healthspan are parallel in mice, and that OS can be reduced, and healthspan increased, in mice fed a diet that is restricted in the content of AGEs. The data also open the question of normal aging to new paradigms, and warrant further studies of the molecular mechanisms involved. They also point out the necessity of considering the dietary content of oxidants an important variable in studies of aging in both humans and animals.
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Footnotes |
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Supported by the National Institutes of Health (grants AG00943 and HL73417 to H.V.).
Accepted for publication April 24, 2008.
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This article has been cited by other articles:
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  T. Buetler, T. Henle, and H. Vlassara The Effects of AGEing on Diet Am. J. Pathol., January 1, 2009; 174(1): 351 - 353. [Abstract] [Full Text] [PDF]
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P < 0.01, 
