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* NMPA, Université Paris-Sud, 91405 Orsay, Cedex, France; INSERM U465, Centre Biomédical des Cordeliers, 75270 Paris, Cedex, France; ** UMR INSERM 476/ INRA 1260, Faculté de Médecine La Timone, 13380 Marseille, France
3 To whom correspondence should be addressed. E-mail: jean-charles.martin{at}medecine.univ-mrs.fr.
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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KEY WORDS: • milk fat • trans-fatty acid • rumenic acid • hamsters • atherosclerosis
Milk fat is usually considered to be proatherogenic in both epidemiologic (1,2) and nutritional studies (3). The presence of a large amount of saturated fatty acids (SFA;4 mainly lauric, myristic, and palmitic), together with cholesterol and trans-fatty acids (mainly vaccenic) was reported to be responsible for these deleterious effects (4–6). Nevertheless, the composition of milk fat, and especially of the fatty acid moiety, varies greatly according to different factors, especially farming conditions (7). For example, milk fat is relatively rich in PUFA and trans fatty acids and poor in SFA at the end of the summer (due to pasture feeding); conversely, it is poor in PUFA and trans fatty acids, and rich in SFA at the end of winter (barn feeding conditions) (7,8). Conjugated linoleic acid (CLA) levels closely follow that of 18:1, trans (9). Thus, the relative proportion of fatty acids having pro- (saturated and trans-monoene fatty acids) and antiatherogenic effects (PUFA, CLA) appears to vary in milk fat, thus having atherogenic consequences that might change accordingly.
Therefore, we evaluated the atherogenic potential of 2 milk fats with respect to their fatty acid composition in the Syrian hamster. The effect of the milk fats on the atherogenic process was evaluated by measuring lipid deposition in the aortic wall. These observations were linked to the changes observed in the expression of several genes involved in early arteriosclerotic events, in the plasma lipoproteins profile, and the LDL fatty acid composition.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Animals. All of the experiments were conducted according to the French Regulations for Animal Experimentation (Art 19.Oct 1987, Ministry of Agriculture) after approval of the referee for animal care at all of the institutions. Male Golden Syrian hamsters (n = 30) from the Janvier strain, 9 wk old and weighing 110 ± 10 g, were purchased from the Breeding Center Janvier. They were housed in colony cages (n = 6/cage) and fed the commercial basal diet (25/18 standard diet, from Mucedola, Settimo) for a 2-wk adaptation period. The hamsters were then caged in pairs and fed the experimental diets (n = 10/group) for the next 12 wk, in a controlled environment (22°C, 14-h:10-h light:dark cycle). Diet and water, consumed ad libitum, were provided 4 times/wk. Body weight and food intake were measured weekly.
Composition and preparation of experimental diets.
To compare the atherogenic properties of 2 milk fats, we analyzed the fatty acid composition of several batches of commercial butters and Comté cheeses. The cheeses were produced from milk collected at different times of the year. Samples were extracted for lipids, and fatty acids were analyzed by a combination of HPLC and GLC (10), using a 120-m capillary column of BPX70 (0.34 mm i.d., 25µm film thickness, SGE). This allowed the complete resolution of all of the 18:1 monoenes, trans- and cis, as well as the CLA isomers (11). CLA quantification and identification were also performed using GC-MS on 4-methyl-1,2,4-triazoline-3,5-dione derivatives (12). The 2 milk fats (a commercial butterfat and a Comté cheese fat made from milk collected in October in the Jura Mountains) that contrasted the most in their fatty acid composition were finally chosen, as shown in Table 1 (and supplemental Table 1). The butter oil (HSF, higher in saturated fat) was extracted by gravimetry. The cheese oil (LSF, lower in saturated fat) was extracted on a pilot scale using the hexane:isopropanol method (10). The same batch of a commercial basal diet was used throughout the experiment. The detailed composition of this basal diet, including the vitamin and mineral mixture, is available in supplemental Table 3. A complementary analysis indicated that the diet contained 
1.4 g of sterols (21% cholesterol, 79% phytosterols) per kilogram, as assayed by GLC determination of the unsaponifiable material (13). This basal diet was ground into powder and mixed with 200 g/kg of the butter or cheese fat to make the HSF- or LSF-diet, respectively. The stereospecific positioning of fatty acids in TG is reported elsewhere (14). Another diet was prepared by supplementing the LSF-diet with up to 9 g/kg by weight of rumenic acid (in the TG form) (LSF-R diet). The vitamin E concentration was 7 and 117 mg/kg in the HSF-butter and HSF-diet, respectively, and 11 mg/kg and 121 mg/kg in the LSF-butter and LSF- diets, respectively.
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Blood and tissue sampling. After 12 wk of dietary treatment, and 18 h of food deprivation, the hamsters were anesthetized between 0900 and 1100 by an i.m. injection of Zoletil 50 at a dose of 250 mg/kg. Blood for lipid analysis (3–4 mL) was collected after cardiac puncture. Plasma aliquots were stored at –80°C. The liver was removed, rinsed in cold saline, and weighed. Two portions (0.5 g) were stored at –20°C for lipid analysis. The epididymal adipose tissue was carefully dissected and weighed. The aorta was flash frozen in liquid nitrogen and stored at –80°C for further RNA isolation and lipid analysis.
Chemical and biochemical assays. Lipids were extracted from liver samples (0.3 g) by the method of Bligh and Dyer (15). A part of the extract was dried completely and dissolved in isopropyl-alcohol for cholesterol, TG, and phospholipid assays, as described above for plasma lipids. Fatty acids from total lipids were derivatized by transmethylation following the two-step method (16), which allowed preservation of CLA. The resulting fatty acid methyl esters were then analyzed by GLC (model 3800, Varian) using a polar column (BPX70, 60 m length, 0.32 mm i.d., 0.25-µm film thickness, SGE) fitted with a split/splitless injector (temperature program: 60°C hold 1 min, then 170°C at 20°C/min, hold 35 min). Liver fatty acid composition data are available in supplemental Table 2.
The cholesterol concentration in the aorta was measured by GLC as published (17) from the initial mixture prepared for RNA isolation (see below).
Plasma lipids were measured by enzymatic procedures using commercial kits (Biomerieux), using an automatic analyzer (Abbott VP): total cholesterol (TC; RTU method), TG, and phospholipids (PAP 150 method). FC was measured by a manual enzymatic procedure adapted from that used for TC but without cholesterol esterase.
Lipoproteins were fractionated by density gradient ultracentrifugation (200,000 x g; 15 x min) of plasma samples (0.4 mL), using an SW41 rotor in an L8-70 ultracentrifuge (Beckman Coulter) (17,18).
Gene expression analysis by semiquantitative and quantitative RT-PCR. Isolation of total RNA from the aorta was performed using RNAplus2 (Q.Biogene) according to the manufacturer's protocol. The amount of RNA in the sample was quantified by spectrophotometry at 260 nm.
Gene expression for ATP-binding cassette, subfamily A, type 1 (ABCA1), vascular cell adhesion molecule-1 (VCAM-1), fatty acid transporter/CD36 scavenger receptor (FAT/CD36) was determined by semiquantitative RT-PCR. Others such as peroxisome proliferator activated receptor (PPAR)
and 
, liver X receptor (LXR), IL-1ß, tumor necrosis factor-, cyclooxygenase (COX)-2, and inducible nitric oxide synthase mRNA were determined by quantitative RT-PCR using the SYBR Green method. The choice of the oligonucleotide sequences was based on available hamster data, or on data from mice (17). BLAST analysis indicated that there was no similarity with other known hamster sequences when mice were used to obtain primers for unpublished hamster gene sequences.
Statistics. Results are expressed as means ± SEM. Comparisons were made using 1-way ANOVA, followed by Fisher's protected least significant difference test. Linear and polynomial regression were calculated using Statview. Differences were considered significant at P < 0.05.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Liver fat composition. Compared with the HSF diet, consumption of LSF diet and LSF-R diet increased the liver weight and fat deposition in the adipose tissue (Table 2), whereas the food intake was smaller. The LSF diet and the LSF-R diet produced a similar elevation in the liver concentration of free cholesterol. The graded dietary intake of CLA from the HSF to LSF and LSF-R diets was accompanied by a commensurate elevation of this fatty acid in liver lipids (0.3, 1.8, and 3.0 g/100 g total fatty acids respectively) (Table 2).
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Aortic cholesterol concentration and relation with plasma lipids and gene expression. The cholesterol deposition in the aortic wall was decreased by LSF feeding compared with HSF feeding, although only the change in the esterified fraction was significant (P < 0.05) (Table 2). Providing twice as much rumenic acid in the LSF-diet further reduced the FC and TC concentrations in the aorta, compared with the value in the HSF-fed hamsters (Table 2). The plasma LDL-C level as well as the LDL-C:HDL-C ratio were predictive of the cholesteryl-ester deposition in the aorta (r = 0.70, P < 0.05, and r = 0.62, P < 0.05, respectively) (Fig. 1). In addition, this lipid loading was positively associated with the LDL P:S (r = 0.57, P < 0.05) (Fig. 1). When these 3 variables (LDL-C, LDL:HDL-C ratio, and LDL P:S) were taken into consideration in a stepwise regression model, the overall coefficient of regression was increased to 0.76 (r2 = 0.58, P < 0.05).
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and , and LXR) were not affected by the dietary treatments (data not shown).
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Hamsters that ate a hyperlipidic diet based on summer-type milk fat (LSF) for 12 wk had less aortic cholesteryl ester loading (–25%) than those consuming a diet closer in fatty acid composition to a winter-type fat (HSF). This beneficial result can be attributed to the following: improvement in blood cholesterol (decrease of both LDL-C and LDL-C:HDL-C ratio) and local inflammatory status (decrease in transcripts of IL1ß, and COX-2), and a reduced potential of monocyte recruitment in the arterial wall (decrease in VCAM gene expression). The slight increase in FAT/CD36 transcription, a receptor involved in lipid uptake and foam cell formation, appears to be inconsequential. The differences in the SFA and PUFA concentration in the 2 types of fat appear too small to explain satisfactorily the marked variation in atherogenic outcome (19,20).Vitamin E, a potent antiatherogenic compound (21) present in dairy fat, cannot be responsible for the observed results because its levels did not differ in hamsters fed HSF and LSF diets (see Methods section). Phytosterols and oxysterols, other potentially anti- and proatherogenic compounds present in the diet, respectively, could offer an alternative explanation, but their daily intake did not markedly differ in the 3 groups of hamsters (data not shown). Thus, among the other micronutrients of milk fat that could modulate the atherogenic risk (22), rumenic acid is likely the best candidate for explaining the beneficial effect of the LSF vs. the HSF diet. In a previous study, we demonstrated that adding up to 10 g/kg rumenic acid to a butter-based diet with a low background level in that CLA isomer clearly reduced the atherogenic outcome in hyperlipidemic hamsters (17). Lock et al. (23) recently found that manipulating the cattle diet to achieve a high rumenic acid concentration in milk fat (3.6 g/100 g total fatty acids, 5.4 g/kg in the diet) improved the atherogenic lipoprotein profile in hyperlipidemic hamsters. Accordingly, the same effect was present in this study, which used a milk fat (LSF) with naturally high concentrations of rumenic acid (2.4 g/100 g of total fatty acids, 4.5g/kg in the diet) (LSF-diet). Furthermore, we found that this was associated with a reduction in cholesterol loading in the aorta, and with an improvement in the expression pattern of selected genes. This result suggests that an attenuation of atherogenesis can be obtained through consumption of a milk fat produced under natural conditions that favor high levels of rumenic acid.
We then analyzed whether the early signs of atherogenesis could be further ameliorated by providing twice as much rumenic acid in the diet. The LSF diet was thus supplemented with up to 9 g synthetic rumenic acid/kg diet in the TG form (LSF-R diet). Several risk factors that were not modified by consumption of the LSF-diet then clearly improved compared with the HSF-fed hamsters, such as an increase in HDL-C as well as the gene expression of ABCA1 (both involved in the reverse-cholesterol transport), a lowering of the LDL P:S ratio (an index of lipid peroxidability), and a decrease in the gene expression of IL-1ß (a local inflammatory mediator) and of FAT/CD36 (a receptor involved in lipid uptake). In addition, the correlations depicted in Figure 1 also suggest a dose-dependent relation between the intake of rumenic acid and antiatherogenic effects.
Hence, compared with the HSF diet, the LSF-R diet, even more than the LSF-diet, slightly but significantly improved key atherogenic risk factors, thereby attenuating cholesterol deposition in the aorta. This highlights the central role of dietary rumenic acid in retarding the development of atherosclerosis (17),(24),(25).
Many epidemiologic studies have agreed on the hazardous effect of trans-fatty acids in cardiovascular diseases (26), although in those studies, the roles of the various positional 18:1, trans-isomers were largely ignored. Paradoxically, in our study, hamsters from the 2 LSF groups consumed almost twice as much 18:1, trans (mainly in the form of trans-vaccenic acid, 18:1, trans-11) than those fed the HSF-diet, but they had fewer early signs of atherogenesis. In fact, trans-vaccenic acid would be an effective precursor of rumenic acid through 
-9 desaturase conversion (27,28). On the basis of the fatty acid composition and the lipid concentration in the liver of hamsters (data not shown), 30% of rumenic acid in the LSF-fed hamsters may have originated via this pathway in our study. In addition, our results demonstrated that the level of trans-monoene fatty acids (trans-vaccenic acid in particular) per se is not necessarily a determining factor in the proatherogenic character of milk fat. This is in agreement with the observations of Lock et al. (23) in which hamsters were fed a butter-based diet containing 15.4 g/100 g trans-vaccenic acid and 3.6 g/100 g rumenic acid. The similar benefit of adding rumenic acid either to a low trans [see (17)] or to a high trans milk fat (present report), strongly suggests that the beneficial effects of the CLA isomer prevail over the detrimental effects of trans-fatty acids from dairy fat, if any.
Finally, despite similar food intake, hamsters fed the LSF-diets displayed slightly more body fat deposition than those fed the HSF diet, as demonstrated by the enlargement of the epididymal adipose tissue taken as an index of body fat (Table 2). This is consistent with a role for rumenic acid in promoting body fat accumulation in hamsters, as reported elsewhere (17,29). Nevertheless, this body fat accumulation does not seem detrimental because the atherogenic character was less severe in these groups, and was not accompanied by an impairment of the insulin resistance index (HOMA index, data not shown). Similarly, plasma TG levels were higher in hamsters fed the LSF diet only, but this appeared insufficient to have deleterious consequences in this group. It should be noted that supplemental rumenic acid (LSF-R diet) inhibited this effect.
In conclusion, this study clearly shows that all milk fats are not identical in promoting atherosclerosis in hamsters. The milk fat produced from pasture-fed cattle is less atherogenic than the milk fat closer in fatty acid composition to that produced under barn-feeding conditions. A plausible explanation could be the differences in the concentration of rumenic acid, a CLA isomer now described as having antiatherogenic properties. Our study thus suggests that feeding practices that improve the rumenic acid concentration in milk fat should be encouraged. The presence of large amount of trans-vaccenic acid that invariably accompanies such an increase in rumenic acid (9) seems to be inconsequential in this range of concentration. Nevertheless, further validation of our observations is necessary and should be undertaken in another animal model.
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ACKNOWLEDGMENTS |
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-tocopherol measurements. Jean-Jacques Bret, from the CIGC, is also acknowledged for providing the Comté cheese. |
FOOTNOTES |
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2 K.V. was a recipient of a French ministry of research fellowship (grant 727/2000).
4 Abbreviations used: ABCA1, ATP binding cassette transporter 1; CLA, conjugated linoleic acid; COX, cyclooxygenase; FAT/CD36, fatty acid transporter/CD36 scavenger receptor; FC, free cholesterol; HSF, dairy fat with a high content of saturated fatty acids; LDL P:S, polyunsaturated:saturated fatty acid ratio in LDL particle; LSF, dairy fat with a low content of saturated fatty acids; LSF-R, lower in saturated fat with supplemental rumenic acid; LXR, liver X receptor; PPAR, peroxisome proliferator activated receptor; P:S, polyunsaturated:saturated fatty acid ratio; SFA, saturated fatty acid; TC, total cholesterol; TG, triacylglycerol/triglyceride; VCAM-1, vascular cell adhesion molecule-1.
Manuscript received 6 November 2005. Initial review completed 7 December 2005. Revision accepted 9 February 2006.
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