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In Vitro Oxidation of Low-Density Lipoprotein in Two Species of Nonhuman Primates Subjected to Caloric Restriction

^a Department of Medicine, University of Vermont College of Medicine, Burlington
^b Department of Medicine, Comparative Medicine, and Public Health Sciences, Wake Forest University School of Medicine, Winston-Salem, North Carolina
^c Wisconsin Regional Primate Center, Madison
^d Department of Medicine, University of Wisconsin–Madison
^e GRECC, William S. Middleton VA Medical Center, Madison, Wisconsin

William T. Cefalu, Endocrine Unit, Given C331, University of Vermont College of Medicine, Burlington, VT 05405 E-mail: wcefalu{at}zoo.uvm.edu.

Decision Editor: Jay Roberts, PhD

	Abstract

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Caloric restriction (CR), which increases longevity and retards age-associated diseases in laboratory rodents, is being evaluated in nonhuman primate trials. CR reduces oxidative stress in rodents and appears to improve risk factors for cardiovascular disease in nonhuman primates. We tested the hypothesis that low-density lipoprotein (LDL) oxidizability is reduced in two monkey species (rhesus and cynomolgus) subjected to chronic CR. In both species, no significant differences occurred between CR and control animals on total, LDL, or high-density lipoprotein (HDL) cholesterol. In rhesus monkeys, triglycerides were higher in controls than CR (139 ± 23 vs 66 ± 8 mg/dl, p < .01, respectively). LDL from CR rhesus monkeys was reduced in triglyceride content and molecular weight compared to controls, whereas LDL composition in cynomolgus monkeys was similar in CR and control animals. In keeping with minor deviations in lipids, antioxidants, and LDL composition, no consistent differences in in vitro LDL oxidizability were apparent between CR and controls in either species.

CALORIC restriction (CR) has been demonstrated to consistently extend the life span and retard age-related diseases in lower species. Although it is not yet known whether CR will increase longevity in higher species, ongoing studies in monkeys suggest that risk factors associated with chronic human diseases are ameliorated with CR ⁽¹⁾⁽²⁾. By modifying risk factors, CR could delay degenerative physiological changes and chronic diseases associated with aging.

Studies in four different primate colonies have shown reduction in plasma glucose and insulin levels, increased insulin sensitivity, and improvement in body composition with chronic CR ^{(3)(4)(5)(6)(7)}. Recent studies also suggest that CR may favorably affect lipoprotein metabolism in nonhuman primates ⁽⁸⁾⁽⁹⁾. Specifically, Verdery and colleagues ⁽⁹⁾ found that CR modified high-density lipoprotein (HDL) particle distributions with increased levels of atherosclerosis-protective HDL₂ in rhesus monkeys. Further, we reported that reduced triglyceride (TG) levels in CR animals are associated with decreased interaction of low-density lipoprotein (LDL) with arterial proteoglycans in vitro ⁽⁸⁾. In both studies, cholesterol levels were relatively unaffected by CR; yet, potentially beneficial changes to lipoprotein composition and metabolism accompanied this dietary intervention ⁽⁸⁾⁽⁹⁾.

LDL particle composition strongly influences its metabolism, in part, by making it more or less susceptible to modification and possible entrapment within the artery wall. As such, modifications to circulating LDL particles, such as oxidation or glycation, are hypothesized to be highly atherogenic and contribute greatly to the development of cardiovascular disease ⁽¹⁰⁾. In vitro studies have shown that LDL oxidizability is related to LDL antioxidant content ^(11)(12), the LDL particle size ⁽¹²⁾, and the fatty acid composition of the LDL particle ⁽¹³⁾. Further, the relevance of LDL oxidizability to cardiovascular disease is suggested by studies of small, dense LDL subfractions, which are relatively poor in antioxidant content, highly susceptible to in vitro oxidation ^(11)(12), and are thought to be atherogenic.

Oxidative stress is hypothesized to contribute to aging processes as well as to chronic diseases including cardiovascular disease, Parkinson's disease, and cancer. Although CR reduces some forms of age-associated oxidative damage by lowering steady-state levels of reactive oxygen species ⁽¹⁴⁾, the effect of CR on resistance of LDL to in vitro oxidation has not been studied. One report describes less plasma lipid peroxidation products in CR rats compared to ad lib-fed controls ⁽¹⁵⁾; however, this does not directly address the susceptibility of specific lipoprotein particles to oxidation. Therefore, to directly test the effect of chronic CR on LDL oxidizability in higher species, we measured in vitro LDL oxidation in two species of nonhuman primates that were part of two long-term trials evaluating the effects of CR versus control diets.

	Methods

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Animals and Diets
This study evaluated rhesus monkeys (Macaca mulatta) as part of the University of Wisconsin (UW) Caloric Restriction Project and cynomolgus monkeys (Macaca fascicularis) from the Wake Forest University Caloric Restriction and Cardiovascular Aging Project. The study designs for both trials have been previously described ⁽³⁾⁽⁷⁾.

At Wake Forest, 32 male cynomolgus monkeys were randomized to control or CR in 1994. The Wisconsin Regional Primate Research Center (WRPRC) randomized three separate groups of rhesus monkeys: 30 male monkeys entered the UW study in 1989; a second group of female animals (15 control, 15 CR) and a final group of male animals (8 control, 8 CR) were randomized into the study in 1994. A total of 40 male (20 CR) and 25 female (12 CR) rhesus monkeys from the UW cohort were included in the present report. Animals for both studies were "run in" on a control diet; half then were randomly assigned to receive 30% fewer calories than they were regularly consuming. In the Wisconsin study, adult rhesus monkeys were randomly assigned to either the control or CR group. Control cynomolgus monkeys at Wake Forest University School of Medicine received a high-fat, high-cholesterol "Western" diet, whereas CR animals received 30% fewer calories of the same diet; cholesterol intake for the Wake Forest animal cohort was kept equal for both control and CR groups by supplementing the CR diet with crystalline cholesterol ⁽⁷⁾. The composition of each diet is outlined in Table 1 and has been previously described ⁽³⁾⁽⁷⁾.

Total cholesterol (TC), TG, and HDL cholesterol (HDL-C) were measured in a Centers for Disease Control standardized Lipid Laboratory ⁽¹⁶⁾. LDL cholesterol (LDL-C) was estimated by the Friedewald formula when triglycerides were <400 mg/dl ⁽¹⁷⁾.

LDL Isolation
LDL isolation was performed as described by Edwards and colleagues ⁽⁸⁾. LDL was isolated by sequential density gradient ultracentrifugation. Approximately 4–5 ml plasma (density 1.006 g/ml) was ultracentrifuged in a Beckman 50.3Ti rotor for 20 hrs at 40,000 rpm at 4°C. The density of the infranate (density > 1.006) was adjusted with KBr to 1.063 g/ml, and ultracentrifugation was repeated for 43 hrs at 40,000 rpm at 4°C. The supernate (density < 1.063) containing LDL was collected and kept at 4°C under Argon until analysis.

LDL Characterization
LDL molecular weight (MW) was determined by high-pressure liquid chromatography (HPLC) ⁽⁸⁾. LDL lipid composition was determined after HPLC isolation using microtiter plate assays as previously described ⁽⁸⁾. LDL fatty acids were determined after lipid extraction, saponification, and methylation as previously described ⁽¹⁸⁾. Determination of the fatty acid methyl esters was quantitated by gas chromatography using a DB225 .25mm x 30m column (J&W) with FID detection on a Hewlett Packard 5890 gas chromatograph with autoinjection. Vitamin E (-tocopherol) was determined using HPLC as described ⁽¹⁹⁾.

LDL Oxidation
LDL oxidation studies were started within two days of LDL isolation and were determined as previously described ^(13)(20). Isolated LDL was dialyzed against multiple changes of 10 µM diethylene-triaminepentaacetic acid-phosphate buffered saline continuously sparged with nitrogen for 48 hrs at 4°C prior to oxidation studies. An aliquot of dialyzed LDL containing 30 µg protein was used for all studies. LDL oxidation was studied at 37°C in air-saturated 25 mM phosphate-buffer saline with 60 mM NaCl (pH 7.2). In separate studies, oxidation was initiated using both 666 µM 2,2'-azobis(amidinopropane·2HCl) or 3.6 µM free Cu⁺². LDL protein was determined by the Lowry method ⁽²¹⁾.

Statistics
Univariate comparisons of plasma lipids, vitamin E, and LDL composition and oxidation between control and CR monkeys were performed using t tests. Plasma TG concentrations were log transformed prior to analysis. Comparisons between cynomolgus and rhesus monkeys and among rhesus monkey groups were performed using general linear models (SAS procedure GLM, Cary, NC) with treatment allocation (CR or control) as a covariate. LDL composition and fatty acids in CR and control animals were compared using von Mises distribution to analyze continuous proportions ⁽²²⁾. This method detects differences in distributions of continuous proportions between treatment groups. All analyses were performed using SAS version 6.1.

	Results

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Plasma Lipids
Plasma lipids for both studies are presented in Fig. 1. In rhesus monkeys, plasma TG was approximately 50% lower in CR compared to control animals (Fig. 1). In addition, TC, along with LDL-C and HDL-C, were similar between rhesus monkeys randomized to receive CR or control diets in the UW cohort (Fig. 1). No significant differences in plasma lipid levels were apparent between cynomolgus monkeys randomized to CR versus control diets in the Wake Forest cohort (Fig. 1). Plasma TG levels were again 50% lower in cynomolgus monkeys randomized to CR versus control; however, this did not attain statistical significance because the values in the cynomolgus monkeys were very low. LDL-C was elevated in the two groups of cynomolgus monkeys regardless of treatment allocation to CR or control (Fig. 1).

View larger version (16K): [in this window] [in a new window]   Figure 1. Levels of plasma lipids observed for both control (closed bars) and CR (open bars) groups for rhesus monkeys as part of the WRPRC cohort, A, and cynomolgus monkeys from the Wake Forest cohort, B. Data are mean ± SE. . p < .01.

LDL Composition
LDL chemical composition is presented for both species in Fig. 2. As shown, LDL levels from control rhesus monkeys were enriched in TG compared to CR animals (13% vs 8%, respectively, p < .02); statistically significant differences were also found in protein, phospholipid (PL), and cholesterol ester (CE) content (Fig. 2). In cynomolgus monkeys, no statistically significant differences in LDL chemical composition were apparent between CR and control (Fig. 2). LDL MW was significantly lower in rhesus monkeys randomized to CR versus control (2.9 ± 0.1 vs 3.2 ± 0.1 g/mol, p < .05). In the cynomolgus monkeys fed cholesterol-enriched diets, LDL particles were larger than those for chow-fed rhesus monkeys, but there were no significant differences in LDL MW between control cynomolgus monkeys and those randomized to CR (3.9 ± 0.1 vs 4.2 ± 0.2 g/mol).

View larger version (16K): [in this window] [in a new window]   Figure 2. LDL chemical composition for the rhesus monkeys, A, and cynomolgus monkeys, B, for both control (closed bars) and CR (open bars) groups. Data are mean ± SE. . *p < .02; **p < .001.

	Discussion

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This report bridges two areas of current research into mechanisms of CR: (a) the effect of CR on lipids, and (b) changes in oxidative damage with CR. Recent studies suggest that changes in lipoproteins ⁽⁸⁾⁽⁹⁾ and lowering of oxidative damage associated with aging are possible with CR ⁽²⁾. However, the present data suggest that resistance of LDL to in vitro oxidation using two different methods of initiation is relatively unaffected by CR in both species of nonhuman primates. These findings are consistent with small differences in plasma lipids and LDL composition measured in CR compared to control animals of both species.

In the present study, plasma cholesterol concentrations were relatively unaffected by CR regardless of monkey species. Verdery and coworkers ⁽⁹⁾ also found that cholesterol levels per se are not influenced by CR. Specifically, the cynomolgus monkeys, as part of an atherosclerosis endpoint study at Wake Forest, were fed a high-saturated fat, high-cholesterol diet, and this resulted in LDL-C levels for both control and CR animals of approximately 350 mg/dl and uniformly low HDL-C concentrations. Thus, a diet restricted by 30% in calories, but still rich in SFA and cholesterol, produced an atherogenic lipid profile in cynomolgus monkeys. In rhesus monkeys, the CR diet, containing corn oil (10% of the diet weight) as the main source of fat, resulted in a 50% reduction in plasma TG compared to controls, although this purified diet produced high HDL/low LDL profiles regardless of treatment regimen. Decreased TG levels were previously reported with CR in another study of rhesus monkeys that included juvenile, adult, and older animals, but only in adult animals were differences in TG apparent with CR ⁽⁹⁾.

Partial LDL characterization has been reported for the rhesus monkeys ⁽⁸⁾. LDL from these animals showed small but significant differences in particle composition with CR. Most notably, TG content was reduced in rhesus LDL from CR animals compared to controls, consistent with the reduction in plasma TG described above. In addition, LDL MW was decreased in CR versus control rhesus monkeys. Although differences in LDL composition between CR and control cynomolgus monkeys mirrored those in rhesus animals, these trends did not reach statistical significance. Major differences in LDL composition were apparent between the two species (e.g., increased LDL CE content and MW in cynomolgus vs rhesus monkeys). These differences most likely resulted from the disparate diets, as previous studies have shown that rhesus monkeys also respond to atherogenic diet challenges with enlarged, CE-rich LDL particles ⁽²³⁾.

LDL from cynomolgus monkeys, comprising of about 50% CE, contained predominantly oleate-derived esters. Monkeys fed atherogenic diets preferentially incorporate cholesteryl oleate into apolipoprotein B-containing particles through increased hepatic acyl-coenzyme A:cholesterol acyltransferase (ACAT) activity ^(24)(25). Cholesteryl oleate-enriched LDL, such as those isolated from both CR and control cynomolgus monkeys, are strongly associated with coronary atherosclerosis extent in nonhuman primates ⁽²⁴⁾. Conversely, rhesus monkeys consuming the purified diets had LDL CE that were relatively enriched in 18:2 and longer chain PUFA compared to the LDL from the cynomolgus monkeys. PUFA-enriched LDL are readily oxidized in vitro ^{(13)(20)(26)(27)(28)}; yet, paradoxically, high PUFA diets are associated with reduced atherosclerosis in monkeys ⁽²⁹⁾. Control animals of both species had LDL PL that were somewhat enriched in PUFA content versus CR, whereas LDL from CR cynomolgus monkeys were enriched in 16:0 and MUFA content versus control animals. Again, while relatively large differences were apparent between the species, smaller differences were detected between CR and control animals.

In agreement with the small differences in LDL particle composition, neither the Cu⁺² nor the azo initiator suggested that LDL from CR and control animals differed greatly in its resistance to oxidation. Azo lag times were almost identical in CR compared to control animals in both species. These findings may not be surprising given that (a) vitamin E was supplemented (along with a standard vitamin mix containing other antioxidants) in both animal species, and (b) those randomized to CR received increased amounts to compensate for restricted diet intake ⁽⁵⁾⁽⁷⁾. Studies show small but significant correlations between vitamin E and resistance of LDL to in vitro oxidation in humans, and supplementing vitamin E increases lag time to LDL oxidation ⁽³⁰⁾. Although we did not measure LDL vitamin E content per se, CR had little effect on plasma vitamin E or LDL-C concentrations in either species of monkey, so it would be unlikely that vitamin E content of LDL differed greatly between CR and control animals. In cynomolgus monkeys, previous studies suggest that azo lag time is linearly dependent on vitamin E content of LDL; however, Cu⁺² oxidation is more complex, and LDL fatty acid composition may influence resistance of LDL to Cu⁺²-initiated oxidation ^(13)(20).

The azo rates are slightly faster for cynomolgus versus rhesus monkeys. If fatty acid content played a dominant role, then Cu⁺² should have shown a similar trend. However, Cu⁺²-catalyzed oxidation depends on the number of and type of Cu⁺² binding sites. These results suggest that rhesus LDL have at least twice as many prooxidant active Cu⁺² binding sites compared to cynomolgus LDL ^{(13)(20)(27)(28)}. It is also likely that other physical and chemical properties of LDL that differed between the two species of monkeys, such as LDL particle size and TG content, also contributed to differences in LDL oxidation ^{(11)(12)(13)(20)}. Previous studies in cynomolgus monkeys suggest that azo rate is correlated with LDL MW ⁽³¹⁾, an observation that is consistent with species differences in azo propagation rate and increased azo rate in cynomolgus CR compared to control animals in the present study.

Interestingly, Cu⁺² oxidation rate in rhesus monkeys varied according to the group in which they were randomized into the study. After control for treatment effects, the female rhesus monkeys in group 2 had LDL that oxidized approximately 12% faster using the Cu⁺² initiator than LDL from either group of male monkeys. The same trend was present for the azo initiator although the differences did not reach statistical significance. Therefore, the binding site number may be an important variable. Because the first and last groups randomized (all males) did not differ in LDL oxidation rate, the group effect appears to be gender related and not due to length of time in the study. Although this gender difference in oxidation rate was not explained by differences in lipids or LDL composition, previous studies suggest male–female differences in LDL particle size and number in response to dietary cholesterol ⁽²³⁾, and it is possible that CR affects female and male monkeys differently as well.

The steady-state levels of reactive oxygen species, an unavoidable byproduct of aerobic respiration, increase with age in some tissues ⁽³²⁾, causing damage to macromolecules such as DNA, proteins, and lipids ^(15)(33)(34). Although research has shown reduction in free radical generation and resultant damage to macromolecules with CR in rodents ^{(15)(32)(33)(34)(35)}, these studies do not address the influence of CR on oxidizability of LDL. Oxidation of LDL is thought to occur within the artery wall by mechanisms that may include oxidizing enzymes, metals, or free radicals ⁽¹⁰⁾. The azo and Cu⁺² initiators have different mechanisms of action, so differences in oxidation rate and lag time are not surprising; however, because the mechanism by which LDL is oxidized within the artery is unknown, it is important to use both initiators for in vitro studies ^(13)(20). In addition, oxidatively modified LDL are rapidly taken up by macrophages in culture and are, thereby, hypothesized to contribute to foam cell formation within the artery intima ⁽¹⁰⁾. Consequently, oxidized LDL are removed from circulation, so measuring lipid peroxides in plasma may not reveal the effect of an intervention on oxidizability of LDL per se. Although we cannot rule out the possibility that LDL oxidizability is retarded by CR, the present study argues that such effects are not large enough to detect using current methods. Changes in LDL accompanying CR did not demonstrably affect LDL oxidizability in vitro, when two different methods in two species of animals with differing CR diets were used. In the cynomolgus monkeys, the rate of oxidation is proportional to the PUFA concentration. However, results from the rhesus cohort suggest that changes in distribution of fatty acid types may affect the overall rate of oxidation, and this may serve as an important observation in fully explaining the dietary and/or species differences. Other studies have shown no association of aging with in vitro LDL oxidation despite changes in fatty acid composition of LDL ⁽³⁶⁾. These studies together argue that LDL does not become more oxidizable with age nor does CR measurably protect LDL from oxidation. However, the data presented in this study do not directly assess how CR may affect the endogenous levels of lipid peroxidation. That is, by reducing oxidative stress in vivo, CR may reduce total levels of LDL oxidation while not affecting inherent susceptibility to oxidation. Given the findings of Tian and colleagues ⁽¹⁵⁾ that CR reduces plasma lipids in rodents, this is a possibility. Therefore, although other cardiovascular risk factors are well described to be reduced with chronic CR (and, therefore, one can postulate a delay in progression of atherosclerosis), LDL oxidation does not appear to be a parameter that is significantly affected.

	Acknowledgments

 
This work was supported by grants from the National Institute on Aging: K01AG00578 and R29AG10816 to Dr. William Cefalu; P01AG11915 to Dr. Richard Weindruch; R03AG14190 to Dr. Iris Edwards; P51 RR00167 to Dr. Joseph Kemnitz; and National Heart, Lung and Blood Institute Grant HL 49373 to Dr. Lawrence Rudel.

The authors thank Becky Aksdal for her skillful preparation of the manuscript and her valuable editorial assistance.

Received August 10, 1999

Accepted January 18, 2000

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