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The Journal of Nutrition Vol. 128 No. 12 December 1998,
pp. 2498-2504
,,
, and, 2
* Department of Animal Science, Texas A&M University, College Station, TX, USDA/ARS Children's Nutrition Research Center, Houston, TX, ** Department of Pathology, College of Veterinary Medicine, Cornell University, Ithaca, NY and College of Agriculture, Home Economics, and Allied Programs, Fort Valley State University, Fort Valley, GA
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ABSTRACT |
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Abstract
Introduction
Methods
Results
Discussion
References
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Sixty-eight female neonatal pigs selected for seven (Experiment 1) or eight (Experiment 2) generations for high (HG) or low (LG) plasma cholesterol were used to test the hypothesis that neonatal dietary cholesterol fed during the first 4 or 8 wk of postnatal life increases the cholesterol content of the cerebrum in young adulthood following free access to a high-fat (15%), high-cholesterol (0.5%) diet from 8 to 20 or 24 wk of age. Pigs were removed from their dams at 1 d of age and given free access to a sow-milk replacer diet containing 9.5% coconut fat and 0 or 0.5 % cholesterol. All pigs (except four HG and four LG pigs in Experiment 2, which were deprived of cholesterol throughout the study) were fed the high-fat, high-cholesterol diet from 8 wk to termination at 20 or 24 wk of age. Cerebrum weight and cholesterol concentration were higher in pigs fed cholesterol neonatally than in those deprived of cholesterol neonatally in both experiments, but weight and cholesterol concentration were unaffected by genetic line. Cholesterol concentrations in longissimus and semitendinosus muscles and in subcutaneous fat were unaffected by diet or genetic line. We conclude that dietary cholesterol deprivation during the first 4 to 8 wk of life in piglets is associated with lower cholesterol concentration and total content in the young adult cerebrum than in pigs supplemented with cholesterol in early life. These data support previous observations and suggest the possibility of a metabolic need for neonatal dietary cholesterol in normal brain development.
KEY WORDS: brain development · neonatal pigs · cerebrum cholesterol · gene x diet interaction · cholesterol deprivation
The developing brain accretes substantial amounts of cholesterol, an essential constituent of all animal cells. The myelin sheath surrounding neurons in the central nervous system and the nerve growth cones, responsible for establishing brain neuroarchitecture, contain (dry basis) 25-30% cholesterol (Norton and Cammer 1984
We report here the results of two experiments designed to test the hypothesis that the increased cerebrum cholesterol content observed in 8-wk-old pigs fed cholesterol neonatally (Schoknecht et al. 1994 Animals and diets.
Details of animals and diets used were cited in a previous report (Graf et al. 1998 Blood sampling and tissue collection.
Blood was sampled from the anterior vena cava of each pig at 4-wk intervals in each experiment, beginning at 4 wk of age, for determination of plasma total cholesterol, high-density lipoprotein cholesterol, and triglycerides. Results are reported elsewhere (Graf et al. 1998 Lipid extraction, saponification, and cholesterol determination.
Minced tissue samples were homogenized and lipids were extracted (Folch et al. 1957 Statistical analysis.
Tissue cholesterol and final body weight data were subjected to two-way ANOVA (BMDP 1993) to test for main effects of diet and genetic line and diet × genetic line interaction.
Experiment 1.
Final body weight, cerebrum weight, and liver weight are summarized in Table 3. Final body weight was higher in pigs fed cholesterol neonatally than in those deprived of cholesterol (81.1 vs.. 71.0 kg, P < 0.01) and was higher in HG than in LG pigs (79.9 vs. 72.3 kg, P < 0.05). The difference in body weight between neonatally cholesterol-deprived and cholesterol-fed pigs appeared by Day 28 in both genetic lines and persisted throughout the growth period. Body weight of HG pigs was greater than that of LG pigs initially and at 28 and 56 d and 16 and 24 wk of age. Liver weight was higher in pigs fed cholesterol than in those deprived of cholesterol (1530 vs. 1318 g, respectively, P < 0.01), but was not affected by genetic line (1463 vs. 1386 g, respectively, P = 0.19). Cerebrum weight was higher in pigs fed cholesterol than in those deprived of cholesterol (94.4 vs. 82.2 g, respectively, P < 0.03), but was unaffected by genetic line ( 91.6 vs. 85.0 g for HG and LG, respectively, P = 0.20). There was no interaction between diet and genetic line for any of the three traits. Liver weight and cerebrum weight expressed as percentages of body weight were unaffected by genetic line.
Experiment 2.
Final body weight, liver weight, cerebrum weight, cholesterol concentration and total cholesterol content data are summarized in Table 5. Final body weight of pigs deprived of dietary cholesterol throughout life (64.8 ± 9.8 kg) was numerically less than that of all other pigs (79.1 ± 18.4 kg); the difference was not significant (P = 0.18). Genetic line did not affect final body weight. Liver weight of pigs deprived of cholesterol throughout life was similar to that of all other pigs (1070 ± 75 g vs. 1186 ± 248 g, respectively, P = 0.57). However, expression of liver weight as a percentage of body weight revealed an effect of diet (1.512 g/100 g for pigs fed cholesterol neonatally vs. 1.677 g/100 g body weight for those deprived of cholesterol neonatally (P = 0.05), and a trend toward an effect of genetic line (1.591 vs. 1.493 g/100 g body weight for LG and HG, respectively, P = 0.08). There was no diet x genetic line interaction.
Dietary cholesterol deprivation compared with dietary cholesterol supplementation of pigs during the neonatal period from 1 to 28 or 56 d of age was shown in both experiments to result in a smaller cerebrum and a lower concentration of cerebrum cholesterol in young adulthood. These results extend our earlier observation (Schoknecht et al. 1994
INTRODUCTION
Abstract
Introduction
Methods
Results
Discussion
References
). The brain has the highest cholesterol concentration of any organ; therefore, its normal development depends on adequate synthesis and/or an exogenous source of cholesterol. Some evidence suggests that cholesterol crosses the blood-brain barrier (Davison et al. 1958, Dobbing 1963, Partridge and Meitus 1979), whereas other investigators have concluded there is no such transfer (Edmond et al. 1991, Jurevics and Morell 1995, Morris and Chaikoff 1961). The pattern of brain growth in the pig resembles that of humans more closely than that of most other mammals (Dobbing 1972, Dobbing and Sands 1979) in that the brain growth spurt occurs perinatally in both species. Human infants fed breast-milk receive a substantial amount of dietary cholesterol (Boersma et al. 1991) and may have a higher intelligence quotient in later life than formula-fed infants (Horwood and Ferusson 1998, Lucas et al. 1992). Milk contains 0.2-0.5% cholesterol, in contrast to most infant formulas, which contain much lower concentrations (0.02-0.05% cholesterol). Observations (Miller and Wehner 1994) of DBA/2Ibg mice (impaired in several learning and memory tasks) indicated that learning performance was improved by subcutaneous implantation of cholesterol pellets. We (Schoknecht et al. 1994) observed increased body weight gain and brain cholesterol content and improved indices of exploratory behavior in neonatal pigs fed a formula containing 0.5% cholesterol compared with values obtained for pigs deprived of cholesterol. On the basis of the similar pattern of brain growth and comparable lipid digestion, absorption and transport, and central nervous system cell membrane fatty acid metabolism in humans and pigs (Innis 1993), the pig is an accepted animal model for studies of the effect of dietary cholesterol deprivation on brain development.
View this table:
Table 1.
Composition of dry diets fed to 28 or 56 d of age, Experiments 1 and 21.
View this table:
Table 2.
Composition of the low-fat, zero-cholesterol diet fed from 57 d to 20 wk of age in Experiment 2 and the high-fat, high-cholesterol diet fed from 57 d to 24 wk of age in Experiment 1 or 20 wk of age in Experiment 2.
View this table:
Table 3.
Body weight, liver weight and cerebrum weight at 24 wk of age in pigs selected for high (HG) or low (LG) plasma total cholesterol and fed diets containing 0% or 0.5% cholesterol from 1 to 28 d of age (Experiment 1).
View this table:
Table 4.
Tissue cholesterol concentrations of pigs selected for high (HG) or low (LG) plasma total cholesterol and fed diets containing 0 or 0.5% cholesterol from 1 to 28 d of age (Experiment 1).
View this table:
Table 5.
Cerebrum weight, cholesterol concentration, and total cholesterol content of pigs selected for high (HG) or low (LG) plasma cholesterol and fed diets containing 0 or 0.5% cholesterol from 1 to 28 d of age (Experiment 2).
) persists into young adulthood.
MATERIALS AND METHODS
Abstract
Introduction
Methods
Results
Discussion
References
) describing the effects of genetic background and neonatal dietary cholesterol deprivation on plasma lipids and early atherogenesis during the period from birth to 5 or 6 mo of age. Briefly, a total of 68 neonatal female crossbred pigs (Chester White, Landrace, Large White, and Yorkshire) selected for seven (Experiment 1, 24 pigs) or eight (Experiment 2, 44 pigs) generations for high (HG)3 or low (LG) plasma total cholesterol were used. Piglets were weaned from their dams at 1 d of age and assigned to a sow-milk replacer formula containing 0 or 0.5% USP grade cholesterol (Table 1). The modified sow-milk replacer diets were used previously in this laboratory (Graf et al. 1998, Schoknecht et al. 1994), and the 0% cholesterol formula is similar to those widely used commercially for early-weaned pigs. It contains ~11% fat, compared with 35-40% fat in mature sow milk (dry weight basis). The experimental diet was modified by replacing all animal fat sources with coconut fat to provide a diet essentially devoid of cholesterol. All pigs were given their respective formulas in liquid form for the first 3 d, then given free access to the diets in dry form. Pigs were housed individually in stainless steel wire-bottom cages in an environmentally controlled animal room (29°C, 12 h light/dark cycle) and provided supplemental heat during the first week to maintain a cage floor temperature of 33°C. All pigs (except four HG and four LG pigs deprived of cholesterol throughout Experiment 2) were given free access to a corn-soy-based high-fat (15.7%), high-cholesterol (0.5%) diet (Table 2) beginning at 57 d of age and were moved to concrete-floor pens, where they remained throughout the remainder of each experiment.
). Pigs were killed by electrical stunning and exsanguination at the end of each experiment at the Texas A&M University, Rosenthal Meat Science and Technology Center.
). Lipid extracts were saponified with KOH and cholesterol concentration was determined (Rhee et al. 1982, modified from Searcy and Bergquist 1960) in a Beckman DU-7 spectrophotometer (Beckman Instruments, Inc., Irvine, CA).
RESULTS
Abstract
Introduction
Methods
Results
Discussion
References
). There was no effect of diet or genetic line on plasma triglycerides (overall mean = 0.49 ± 0.38 mmol/L). Overall plasma total cholesterol was higher (P < 0.01) in pigs fed cholesterol neonatally than in those deprived of cholesterol (4.05 vs. 2.96 mmol/L respectively,). Final plasma total cholesterol (24 wk) was not significantly different in pigs fed or deprived of cholesterol neonatally (3.01 vs. 3.21 ± 0.66 mmol/L respectively,).
). There was no effect of diet or genetic line on plasma triglyceride concentration (overall mean .56 ± 0.35 mmol/L). Final plasma total cholesterol concentrations (20 wk) were 4.98 and 5.56 ± 1.75 mmol/L (P < 0.01) for the 12 and 24 pigs, respectively, fed or deprived of neonatal cholesterol and fed the high cholesterol-high fat diet from 57 d to 20 wk. Final plasma total cholesterol was 2.52 mmol/L for the 8 pigs deprived of cholesterol from 2 d to 20 wk of age.
DISCUSSION
Abstract
Introduction
Methods
Results
Discussion
References
) that cerebrum cholesterol concentration was higher at 56 d of age in pigs supplemented with dietary cholesterol from 1 to 56 d of age than in pigs fed a cholesterol-free diet. Our data reveal that the increase in cerebrum cholesterol at 56 d of age in pigs fed cholesterol neonatally persists well beyond the neonatal period despite the continuous ingestion of a high-fat, high-cholesterol diet from 57 d to 20 or 24 wk of age. The concentration of cholesterol in cerebrum was higher at 24 (Experiment 1) than at 20 wk of age (Experiment 2), indicating that cerebrum continues to accrete cholesterol during young adulthood, even as cerebrum weight continues to increase beyond puberty. Cerebrum weight was about 94 g in adult female pigs at 119 wk of age (Pond et al. 1990) compared with a mean in the present study of 88.3 g for pigs at 24 wk of age in Experiment 1 and 63.8 g for pigs 20 wk of age in Experiment 2. We are not aware of data describing genetic effects or dietary neonatal cholesterol effects on cerebrum weight or cholesterol content in older swine. The failure to detect differences in degree of myelination of cerebrum in stained sections in either experiment is similar to observations in younger pigs (Schoknecht et al. 1994) and suggests that histopathological methods at the light microscope level are not correlated with quantitative differences in cholesterol accretion, most of which is presumably concentrated in the myelin sheath of neurons.
) persists into young adulthood. The data from two, separate experiments clearly support this hypothesis. Additional information on specific membrane phospholipid composition, fatty acid composition and myelin proteins in the brain of young adult pigs deprived of neonatal dietary cholesterol is needed. Such information should provide further insight into the role of neonatal dietary cholesterol in functional development of the central nervous system.
, Partridge and Meitus 1979), although other investigators have concluded there is no such transfer (Edmond et al. 1991, Jurevics and Morell 1995, Morris and Chaikoff 1961). Dietschy et al. (1993) concluded that brain cells probably obtain most of their cholesterol by synthesis within the brain. The higher cerebrum cholesterol concentration of pigs fed cholesterol could have resulted from contamination of plasma at the time the brain was collected. Edmond et al. (1991) used perfusion to avoid brain cholesterol contamination in rats; we did not do so in our work with pigs. Final plasma cholesterol concentration did not differ in pigs fed or deprived of dietary cholesterol neonatally in Experiment 1 (3.01 vs. 3.21 ± 0.66 mmol/L, respectively, NS) and was lower in pigs fed cholesterol neonatally than in those deprived of cholesterol neonatally (4.98 vs. 5.56 ± 1.75 mmol/L, respectively, P < 0.01) in Experiment 2. Therefore, the higher cerebrum cholesterol concentration at 20 or 24 wk of age in pigs fed cholesterol neonatally could not have been due to contamination with plasma cholesterol. Our results support the suggestion that cholesterol is transferred across the blood-brain barrier. However, we cannot ignore the possibility that brain cholesterol synthesis is increased by a humoral and/or neural signal in response to cholesterol ingestion. The presence of low density lipoprotein receptor (LDLR) and 3-hydroxy-3-methylglutaryl-coenzyme A (HMG CoA) reductase mRNA in the developing brain (DeWille and Farmer 1992, Hofmann et al. 1987, Staels et al.1990, Swanson et al. 1988) of several animal species suggests that brain cholesterol accretion may be modulated by both endogenous cholesterol synthesis in, and exogenous cholesterol uptake by, the central nervous system.
, Hoefer and Hardy 1929, Horwood and Fergusson 1998, Lucas et al. 1992, Morley et al. 1988, Rodgers 1978, Taylor 1977, Uauy and De Andreca 1995). Potential causes that have been cited include the presence of higher levels of long-chain polyunsaturated fatty acids in breast-milk than in infant formulas (Arbuckle et al. 1994, Dobbing 1993, Farquharson et al. 1992, Lucas et al. 1992) and the presence of several known trophic factors in milk (Read 1988).
observed that learning performance of DBA/2Ibg mice (impaired on several learning and memory tasks) was improved by subcutaneous implantation of cholesterol pellets. Earlier, Caffrey and Patterson (1971) reported that rats fed a saturated fat or high-cholesterol diet performed better on a water maze test than those fed low-fat diets. In our laboratory, Schoknecht et al. (1994) observed improved indices of exploratory behavior in neonatal pigs fed a formula containing 0.5% cholesterol compared with values obtained in pigs deprived of cholesterol (the same diets as those fed to neonatal pigs in the present experiments).
)], probably due to polymorphism in the cholesterol 7-
-hydroxylase (CYP7) gene locus (Davis et al. 1998). Restriction fragment length polymorphism analysis of seventh-generation HG and LG pigs (Davis et al. 1994) revealed 5.0- and 2.8-kb TaqI restriction fragments. Only the 2.8-kb allele was seen in the HG pigs, whereas both the 2.8- and 5.0-kb alleles were present in some LG pigs, and only the 5.0-kb allele was seen in others. The 5.0-kb allele is associated with low plasma total cholesterol and the 2.8-kb allele with high plasma cholesterol. CYP7 is the rate-limiting enzyme in bile acid production from cholesterol, and in this way it modulates the catabolism of cholesterol and the excretion of cholesterol-produced bile acids. CYP7 has not been demonstrated in brain. The presence of HMG CoA reductase mRNA and LDLR in brain (DeWille and Farmer 1992, Hofmann et al. 1987, Staels et al. 1990, Swanson et al. 1988) of several animal species suggests these two modulators of cholesterol metabolism as possible factors in processing or transport of cholesterol in cerebrum. HG and LG pigs appear to have similar liver HMG CoA reductase activity (Schoknecht et al. 1994), but activity of this enzyme or its mRNA in cerebrum of these pigs is unknown. Although HG and LG pigs were shown to differ in allele frequencies of TaqI restriction fragments of LDLR (Davis et al. 1998), differences between HG and LG pigs in cerebrum LDLR level have not been studied. The lack of difference in cerebrum cholesterol concentration between HG and LG pigs suggests that the modulation of cholesterol metabolism by CYP7 is not operative in the observed effect of dietary cholesterol on cerebrum cholesterol accretion in our experimental pigs. Future work should examine the role of other modulators of cholesterol metabolism, including HMG CoA reductase and LDLR, in the observed difference in cerebrum cholesterol content in response to neonatal cholesterol deprivation.
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FOOTNOTES |
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Manuscript received 8 May 1998. Initial reviews completed 24 June 1998. Revision accepted 11 September 1998.
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ACKNOWLEDGMENTS |
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We thank B. Harrell and J. Nelson, Sam Houston State University, Huntsville, TX, for animal supervision and management; R. Riley and staff, Texas A&M University, College Station, TX, for animal slaughter and tissue collection; J. Cunningham, F. Biggs and H. Asberry for animal care during the neonatal period; Prairie View A&M University Cooperative Agricultural Research Center for analysis of some tissues; B. Hunter, CNRC, for final manuscript preparation, and L. Loddeke for editorial improvements in the manuscript.
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LITERATURE CITED |
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Abstract
Introduction
Methods
Results
Discussion
References
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-hydroxylase (CYP7) gene in pigs selected for high or low plasma total cholesterol.
Proc. Soc. Exp. Biol. Med.
1998;
217:466-470[Medline]-hydroxide (CYP7) locus.
J. Anim. Sci.
1994;
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