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© 2003 The American Society for Nutritional Sciences J. Nutr. 133:2526-2531, August 2003

Biochemical and Molecular Actions of Nutrients

Dietary Trans Fatty Acids Alter the Compositions of Microsomes and Mitochondria and the Activities of Microsome {Delta}6-Fatty Acid Desaturase and Glucose-6-Phosphatase in Livers of Pregnant Rats

Elvira Larqué*, Pedro-Antonio García-Ruiz{dagger}, Francisca Perez-Llamas*, Salvador Zamora* and Angel Gil**

* Departments of Physiology and {dagger} Organic Chemistry, University of Murcia, Murcia, Spain and ** Department of Biochemistry and Molecular Biology, University of Granada, Granada, Spain

2To whom correspondence should be addressed. E-mail: elvirada{at}um.es.


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 
This study was designed to investigate the effects of three diets with different levels of trans fatty acids and the physiologic status on the physicochemical properties and enzymatic activities of liver microsomes and mitochondria. Three groups of 10 female weaning rats each were fed for 10 wk one of three diets differing in their trans fatty acid contents (Control, 0 mol/100 mol total fatty acids; high, 14.5 mol/100 mol; very high, 30 mol/100 mol). At the onset of adult life (10 wk of age), they were mated. Six rats in each group were killed at the end of gestation (Pregnant rat groups). The four remaining pregnant rats continued to receive their experimental diets until weaning of their litters. Six pups from the litters for each group (3 males and 3 females) were selected and fed the same experimental diet as the dams from wk 3 to 10 of age (2nd generation virgin groups) and then killed. Trans fatty acid levels in liver microsomes and mitochondria rose in parallel with the dietary trans fatty acid content, whereas saturated fatty acids dropped in both organelles with increasing trans fatty acids. Pregnant and 2nd generation adult rats fed trans isomers also had lower levels of cholesterol and a lower cholesterol/phosphorus ratio in their liver microsomes compared with controls. A significant interaction between diet and pregnancy was detected in the activities of {Delta}6-desaturase and glucose-6-phosphatase in liver microsomes. Dietary trans fatty acids decreased the activities of both enzymes but only in pregnant rats. No differences in the fluorescence anisotropy of membranes or the enzymatic activities in liver mitochondria were observed. In conclusion, dietary trans fatty acids significantly lowered cholesterol and the cholesterol/phosphorus ratio in liver microsomes. This effect might contribute to low {Delta}6-desaturase and glucose-6-phosphatase activities in liver microsomes of pregnant rats.

KEY WORDS: • cholesterol • {Delta}6-fatty acid desaturase • glucose-6 phosphatase • pregnancy • trans fatty acids

Exposure to an adverse environment in utero and failure to grow well may have negative influences in childhood and adult life (1). Several authors have reported that dietary trans fatty acids (trans FA) may cross the placenta and disturb fetal growth and birth weight (2–4). Inverse associations between trans FA and arachidonic and docosahexaenoic acids (DHA) have been reported in plasma lipids of preterm and full-term infants (2,4,5), as well as in liver and heart of experimental rats (6). Although a disturbance of the maternal supply of long-chain PUFA (LC-PUFA) to the fetus could explain in part these physiologic effects, the mechanisms of action of trans FA have not yet been elucidated.

Dietary trans FA accumulate in fetal and adult tissues, but the effects of the incorporation of trans FA on the biological function of membranes are unknown. Trans FA may accumulate in substantial amounts in tissue phospholipids, modulating some variables such as the PUFA pattern; this affects membrane microviscosity and the behavior of membrane-bound receptors and enzymes (7). Some investigators have used isolated mitochondria to investigate the effects of dietary trans FA on biological membranes (8–10) but significant changes in membrane-associated enzymes have been reported only when diets were deficient in essential fatty acids (EFA). However, Alam et al. (11) detected low activity of the enzyme adenylate cyclase in membranes of heart in rats fed trans FA even though the diets contained sufficient amounts of EFA. Until now, no studies of the effects of trans FA on organelle membrane composition and specific enzyme activities during pregnancy have been reported.

Dietary trans FA also modify the PUFA profile of rat plasma and tissues. This has been related to an inhibitory effect on liver {Delta}6-fatty acid desaturase (12), which might have serious consequences for the newborn. Although {Delta}6-fatty acid desaturase may be modulated by membrane fluidity, it is unclear how trans FA might impair this key enzymatic activity in the conversion of EFA to LC-PUFA.

Thus, the objective of the present study was to evaluate the influence of three experimental diets containing very low (~0 mol/100 mol), high (15 mol/100mol) and very high (30 mol/100 mol) trans FA contents on the compositions of liver microsomes and mitochondria as well as on some physical variables and enzymatic activities of pregnant and 2nd generation virgin rats. The cholesterol/phosphorus ratio (chol/P ratio) and the FA profile of liver microsomes and mitochondria as well as some physical and functional properties of these organelles, including fluorescence anisotropy as a marker of membrane fluidity, liver microsome {Delta}6-fatty acid desaturase and glucose-6-phosphatase activities, and mitochondrial ATPase and succinate dehydrogenase activities were measured.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 LITERATURE CITED
 
Study design.

The protocol of this study was approved by the Animal Laboratory Service of the University of Murcia, and rats received humane treatment according to the regulations for Animal Research of the European Union. Female Wistar rats at weaning (21 d of age), supplied by the Animal Laboratory Service of the University of Murcia (Spain), were kept individually in metabolic stainless steel cages in a room with controlled temperature (22°C) and light (0800–2000 h) and allowed free access to food and water. Rats were randomly assigned to 3 groups of 10 rats each and were fed three isoenergetic (1743 kJ/100 g diet) purified diets (13) containing 100 g/kg diet of fat and differing in their fatty acid contents: control diet (C diet), with a total trans FA concentration of ~0 mol/100 mol total FA; a high trans diet (H diet) with a total trans FA concentration of ~14.5 mol/100 mol (3.2% energy of the diet as trans isomers); and a very high trans FA diet (VH diet) with total trans FA concentration of ~30 mol/100 mol (6.4% energy of the diet as trans isomers). The diet classification was established on the basis of the trans levels in human diets (14,15). The FA composition of the three diets is summarized in Table 1.


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  		TABLE 1 Proportions of selected fatty acids and their indices in experimental diets1,2

 
The three groups of 10 female weaning rats (21 d of age) were fed the experimental diets for 7 wk; after this period, a male rat was introduced into each cage for 1:1 mating. On d 1 of pregnancy, as indicated by the presence of a mating plug, male rats were removed and female rats remained in their cages through the pregnancy period (3 wk). On d 20 of gestation, six rats from each group were killed with sodium pentothal; they comprised the first group of "pregnant rats." The remaining four pregnant rats of each experimental group remained in their cages until delivery. Eight pups per dam were fed maternal milk until weaning (21 d of age). Then, 3 groups of six rats each (3 males and 3 females per group) were fed the same diet for another 7 wk until adulthood (2nd generation virgin group) and then killed as previously described.

Collection and preparation of samples.

On d 20 of gestation for pregnant rats, and at the end of wk 10 for the nonpregnant rats of the second generation, rats were killed as indicated above. Rat livers were rinsed in cold saline solution immediately upon removal. A part of each liver was frozen directly in liquid nitrogen and stored at -80°C until analyzed. A second part of the fresh liver was used to obtain the mitochondrial fraction, which was isolated by the method of Fleisher et al. (16). After mitochondria were isolated, the remaining supernatant was centrifuged again at 105,000 x g for 60 min to obtain the microsomal fraction as previously described (17).

Analytical methods.

Total lipids from microsomal and mitochondrial preparations were extracted according to the method of Folch et al. (18) and FAME were prepared with methanolic 3 mol/L HCl (Supelco, Bellefonte, PA) at 85°C for 1 h and dissolved in hexane. FAME were analyzed by GLC using a SP-2560 fused silica capillary column (100 m x 0.25 mm i.d., 20-µm film thickness; Supelco) in a Hewlett-Packard 5890-A gas chromatograph (Hewlett-Packard, Avondale, PA). The oven temperature was programmed for 39 min at an initial temperature of 175°C and was increased at a rate of 3°C/min to 230°C and maintained at that temperature for 14 min. The injector and detector were set at 250°C. Helium was used as the carrier gas at a pressure of 290 kPa and peaks were identified by comparison of their retention times with appropriate FAME standards purchased from Sigma Chemical, St. Louis, MO. Finally, the double bond positions of isomers were confirmed by GLS/MS analysis of their 2-alkenyl-4,4-dimethyloxazoline (DMOX) derivatives, as previously reported (13).

The mitochondrial and microsomal cholesterol concentrations were measured by an enzymatic method according to Röschlau et al. (19). The phosphorus content of membrane phospholipids was assayed according to the method of Barlett (20). The unsaturation index was calculated as the sum of the molar proportion of each FA multiplied by the number of cis double bonds. Mitochondrial and microsomal membrane suspensions were treated with the lipid-soluble fluorophore 1,6-diphenyl-1,3,5-hexatriene (DPH) supplied by Sigma Chemical, and steady-state fluorescence polarization measurements were accomplished using a Perkin Elmer MPF-4 fluorescence spectrophotometer (Perkin Elmer Analytical Instruments, Shelton, CT) (excitation wavelength 356 nm, emission wavelength 430 nm). The polarization of fluorescence was expressed in terms of the fluorescence anisotropy r as previously described (21).

Liver microsomes were also assayed for {Delta}6-fatty acid desaturase activity, as previously described by Girón et al. (22), using 0.5 mg protein of microsomal suspension and studying the conversion of 75 nmol of [1-14C] linoleic (specific activity 0.58 Ci/mol acid) in [1-14C] {gamma}-linolenic acid. FAME were extracted in hexane and separated on 10% AgNO3 and 2,7-diclorofluorescein 2 g/L methanol solution) impregnated Silica-gel 60G plates. The spots were visualized under UV light, and scraped off into counting vials; 10 mL of liquid scintillation cocktail (5 g PPO + 0.3 g POPOP in 66 mL ethyl acetate and 934 mL of toluene) was added and the radioactivity was counted in a Beckman LS 7500 (Beckman Instruments, Palo Alto, CA) liquid scintillation spectrometer. The results are expressed as pmol/(min · mg protein).

The ATPase activity (EC 3.13.1) of the mitochondrial suspension and the glucose-6-Pase (EC 3.1.3.9) activity from liver homogenates in 0.1 mmol/L pH 6.5 citrate buffer were assayed by measuring the phosphorus released in the presence of ATP and glucose-6-phosphate, respectively. Phosphorus was measured according to the method of Fiske and Subarrow (23). Succinate dehydrogenase activity (EC 1.3.99.1) of liver mitochondria was assayed using the method of Bonner (24).

Statistical methods.

Data are expressed as means ± SEM. Because FA molar percentages usually do not follow a normal distribution, data were transformed to their decimal logarithms. Two-way ANOVA was used to evaluate the two sources of variations in the study (trans FA dietary content and physiological status, i.e., pregnant vs. 2nd generation virgin rats) using the Statgraphics 7.0 (Statistical Graphics, Bitstream, Cambridge, M.A) software. Bonferroni’s multiple range test was used for comparisons between groups. Differences were considered significant at P < 0.05. When we detected a significant interaction between diet and physiologic status, we estimated by t test the specific differences among groups but using the mean-square error of the previous two-way ANOVA. Pearson’s test correlation coefficients between SFA and trans fatty acids in biological membranes were also calculated.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 LITERATURE CITED
 
Trans FA levels were higher in liver microsomes and mitochondria of rats fed H and VH diets compared with the Control group (Fig. 1 and Fig. 2). In contrast, the proportion of saturated fatty acids (SFA) in both organelles were significantly lower and paralleled the trans FA intake (Fig. 1 and Fig. 2). Similarly, significant inverse correlations between the concentration of SFA and trans FA in the membranes of liver microsomes of pregnant and virgin rats of the second generation were established (R = -0.93, P < 0.0001; R = -0.83, P < 0.0001) (Fig. 3) as well as in liver mitochondria (R = -0.63, P < 0.05; R = -0.68, P < 0.05) (Fig. 4). Cis PUFA and (n-6) LC-PUFA proportions were unaffected in those organelles by the diets. Pregnant rats had higher proportions of SFA and (n-3) LC-PUFA than 2nd generation virgin rats, whereas trans FA and cis monounsaturated fatty acids (cis MUFA) proportions were lower, in both liver microsomes and mitochondria (Fig. 1 and Fig. 2). Significant interactions were found between diet and physiologic status for cis MUFA (P = 0.0016) and (n-3) LC-PUFA (P = 0.0096) of liver microsomes, and for cis MUFA of liver mitochondria (P = 0.0130).



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  		FIGURE 1 Selected fatty acid (FA) proportions in liver microsomes of pregnant rats and 2nd generation adult virgin rats fed diets containing low (Control), high (H) or very high (VH) levels of trans FA. Panel A: Saturated fatty acids (SFA), cis monounsaturated fatty acids (cis MUFA) and cis PUFA. Panel B: trans FA, (n-6) long-chain PUFA (LC-PUFA) and (n-3) LC-PUFA. Values are means ± SEM, n = 6. Means for a variable without a common letter differ, P < 0.05. Interactions were detected between diet and physiologic status for cis MUFA (P = 0.0016) and (n-3) LC-PUFA (P = 0.0096).

 


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  		FIGURE 2 Selected fatty acid (FA) proportions in liver mitochondria of pregnant rats and 2nd generation adult virgin rats fed diets containing low (Control), high (H) or very high (VH) levels of trans FA. Panel A: Saturated fatty acids (SFA), cis monounsaturated fatty acids (cis MUFA) and cis PUFA. Panel B: trans FA, (n-6) long-chain PUFA (LC-PUFA), (n-3) LC-PUFA. Values are means ± SEM, n = 6. Means for a variable without a common letter differ, P < 0.05. Interactions were detected between diet and physiologic state for cis MUFA (P = 0.0130).

 


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  		FIGURE 3 Inverse correlations between trans fatty acids (FA) and saturated fatty acids (SFA) detected in pregnant rats and 2nd generation adult virgin rats fed diets differing in their trans FA content in (A) liver microsomes of pregnant rats (y = -0.9718x + 49.098) and (B) liver microsomes of 2nd generation adult virgin rats (y = -0.8991x + 47.46).

 


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  		FIGURE 4 Inverse correlations between trans fatty acids (FA) and saturated fatty acids (SFA) detected in pregnant rats and 2nd generation adult virgin rats fed diets differing in their trans FA content in (A) liver mitochondria of pregnant rats (y = -1.2659x + 47.9565) and (B) liver mitochondria of 2nd generation adult virgin rats (y = -0.4763x + 41.216).

 
Pregnant and 2nd generation virgin rats fed dietary trans isomers (H and VH experimental groups) had lower levels of cholesterol and cholesterol to phosphorus (chol/P) ratios in their liver microsomes than control rats (Table 2). These differences did not affect the fluorescence anisotropy of liver microsomes (Table 2). The chol/P ratio was higher in pregnant than in 2nd generation virgin rats, and the low amount of phosphorus in the liver microsomes of pregnant rats also contributed to the increase in this ratio. The unsaturation index was also significantly higher in liver microsomes of pregnant compared with virgin rats and an interaction between diet and pregnancy was detected (P = 0.0227). The fluorescence anisotropy of liver microsomes was not affected by physiologic state, although it tended to be lower (P = 0.11) in liver microsomes of pregnant rats (Table 2).


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  		TABLE 2 Cholesterol and phosphorus contents and fluorescence anisotropy of liver microsomes in pregnant and second generation adult rats fed diets containing low (Control), high (H) or very high (VH) levels of trans fatty acids1,2

 
The physicochemical variables studied in liver mitochondria were not affected by the trans FA intake or physiologic state of rats (results not shown), except that the anisotropy fluorescence of liver mitochondria of pregnant rats (0.137 ± 0.03) was lower (P < 0.05) than that in 2nd generation adult virgin rats (0.154 ± 0.01). Dietary trans FA intake did not affect the cholesterol level or the chol/P ratio in mitochondrial membranes.

Significant interactions between diet and pregnancy on the activities of {Delta}6-desaturase (P = 0.0148) and glucose-6-phosphatase (P < 0.001) were detected in liver microsomes (Table 3). Dietary trans FA decreased the activities of both enzymes, but only in pregnant rats. Glucose-6-phosphatase, ATPase and succinate dehydrogenase activities were significantly higher in pregnant than in virgin rats (Table 3); the {Delta}6-desaturase activity showed a similar trend (P = 0.16).


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  		TABLE 3 Enzymatic activities in liver microsomes and mitochondria of pregnant and second generation adult rats fed diets containing low (Control), high (H) or very high (VH) levels of trans fatty acids1,2

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
LITERATURE CITED
 
Because partially hydrogenated vegetable oils that contain relatively high levels of trans FA may represent a large part of the daily fat intake in humans, studies of the influence of trans FA intake on tissue composition and functionality in different physiologic states are of interest.

In the present study, trans FA accumulated in high proportion and in a dose-dependent manner in liver microsomes and mitochondria of rats fed relatively high doses of trans isomers. Pettersen and Opstvedt (25) reported that dietary trans FA decreased SFA, which was compensated for by increased levels of MUFA in liver mitochondria of mature female pigs. In the present study, the incorporation of trans FA into liver microsomes decreased SFA independently of the physiologic state of the rats (Figs. 1, 2), and helped keep constant the unsaturation index of the membranes (Table 2).

Although trans FA lowered the proportion of (n-3) LC-PUFA in liver microsomes of pregnant rats fed the VH diet, the proportions of (n-3) LC-PUFA were significantly higher in liver microsomes and mitochondria than in the 2nd generation virgin rats. Thus, although it has been reported that trans FA may interfere with the conversion of parent EFA into their derived LC-PUFA (2,12), especially when parent EFA concentrations are low (10), it is possible that the amount of LC-PUFA in liver organelles (especially during pregnancy) is insufficient for the adequate development of the fetus.

Cholesterol and the chol/P ratio were significantly lower in liver microsomes of rats fed trans FA diets compared with control rats. The association of cholesterol with trans phospholipids is less favorable than with isomeric cis phospholipids (26). If the function of cholesterol in biological membranes is to control hydrocarbon-chain fluidity in a reversible manner, it may be that membranes with a high incorporation of trans isomers require less cholesterol in their membranes than those containing cis FA. This mechanism might be used by the organism to maintain a relatively constant fluidity of biological membranes. Nevertheless, the reduction in the chol/P ratio by trans FA did not occur in liver mitochondria (9), whose membranes appear to be less sensitive to modification than liver microsomes.

Fluorescence anisotropy of DPH, an indirect indicator of the microviscosity of membrane lipids, was influenced only slightly by diets with different contents of trans FA. These results are in agreement with those obtained by Wolff and Entressangles (27) who did not detect differences in DPH fluorescence between phospholipids from liver mitochondria with or without elaidic acid. These authors concluded that when elaidic acid replaces SFA in phospholipids, even in a high proportion (one third), the physical state of the acyl chains in the hydrophobic core of membranes is not grossly modified. Our results support this theory in part because we observed an inverse correlation between levels of trans FA and SFA in liver microsomes and mitochondria in both pregnant and 2nd generation virgin rats (Figs. 3, 4). However, the sensitivity of the analytical method used to determine membrane fluidity is not very high, and it is likely that these structural changes might influence enzymatic activities in some organelles without being detected.

We evaluated the activity of several membrane enzymes that might be affected by changes in the membrane architecture. The two enzymes analyzed in liver microsomes, {Delta}6-fatty acid desaturase and glucose-6-phosphatase, had lower activities in pregnant rats fed the VH diet than in controls (Table 3). Koba et al. (28) showed a direct correlation between {Delta}6-fatty acid desaturase activity and the chol/P ratio in liver microsomes of rats fed different dietary proteins. In the present study, the chol/P ratio decreased in liver microsomes of pregnant rats in relation to the trans FA incorporation; moreover, the chol/P ratio was higher in pregnant than in virgin rats. Thus, we found a negative effect of trans FA on the enzymes analyzed in liver microsomes of pregnant rats. However, no adverse effects of trans FA on mitochondrial enzyme activities were observed, as also reported by others (29). The physiologic relevance of dietary trans fatty acids is controversial. The results of the present study suggest that a relatively high intake of trans fatty acids (3.2–6.4% of the total daily energy) may decrease the liver microsome chol/P ratio, thereby altering the {Delta}-6 fatty acid desaturase activity, a key enzyme in the synthesis of LC-PUFA. These fatty acids are important for growth and development, particularly in infancy and childhood.

In conclusion, dietary trans FA significantly decreased the SFA proportions, cholesterol levels and the chol/P ratio in liver microsomes of pregnant and 2nd generation virgin rats. These changes decreased the {Delta}6-fatty acid desaturase and glucose-6-phosphatase activities of liver microsomes but only in pregnant rats. ATP-ase and succinate dehydrogenase activities of liver mitochondria were unaffected by trans FA intake in both pregnant and 2nd generation virgin rats.


   FOOTNOTES
 
1 E.L. was the recipient of a fellowship (Beca de formación de personal investigador) provided by the University of Murcia. Back

3 Abbreviations used: C, control diet; DPH, 1,6-diphenyl-1,3,5-hexatriene; EFA, essential fatty acids; FA, fatty acids; H, high trans fatty acid diet; LC-PUFA, long-chain PUFA; MUFA, monounsaturated fatty acids; SFA, saturated fatty acids, trans FA, trans fatty acids; VH, very high trans fatty acid diet. Back

4 AgNO3 and 2,7-diclorofluorescein, PPO, POPOP, ethyl acetate and toluene were obtained from Sigma Chemical, St. Louis, MO. Back

Manuscript received 9 October 2002. Initial review completed 12 November 2002. Revision accepted 8 May 2003.


   LITERATURE CITED
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 

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