Conjugated linoleic acid (CLA) is the general name of a mixture of positional and geometric isomers of linoleic acid with conjugated unsaturated double bonds. The major natural sources of CLA are meat and dairy products from ruminant animals, where CLA is synthesised as an intermediate fatty acid (FA) during the biohydrogenation of linoleic acid to stearic acid, but it can also be synthetically produced(Reference Pariza, Park and Cook1,Reference Jelińska2) for commercial purposes by different techniques(Reference Salamon, Vargáné-Visi and András3). Many investigations have shown that CLA may have beneficial effects on cancer, obesity, the inflammatory response, atherosclerosis and glucose and lipid metabolism(Reference Reynolds, Loscher and Moloney4,Reference Kennedy, Martinez and Schmidt5) . However, controversial and detrimental health effects have also been reported(Reference Reynolds, Draper and Keogh6), probably due to, among other differences, the different levels, proportions and biochemical properties of the two major isomers c9,t11-CLA and t10,c12-CLA of natural and commercial CLA and to the species-specific effects(Reference Scalerandi, Gonzalez and Saín7,Reference Ferramosca, Savy and Conte8) .
On the other hand, maternal obesity in humans is associated with an increased risk of obesity and metabolic-related disorders in adult offspring(Reference Gaillard, Felix and Duijts9,Reference Li, Sloboda and Vickers10) . Maternal overnutrition generates changes in the uterine milieu during pregnancy, leading to developmental alterations and defects in organ function and metabolism in offspring(Reference Alfaradhi, Kusinski and Fernandez-Twinn11). Thus, maternal nutritional programming by exposure to hyperenergetic diets, such as a high-fat (HF) diet, is closely related to hepatic lipid accumulation, insulin resistance(Reference Benatti, Melo and Borges12), CVD, non-alcoholic liver diseases and steatohepatitis in offspring(Reference Taylor, Samuelsson and Poston13,Reference Frihauf, Fekete and Nagy14) . Both overnutrition and undernutrition during pregnancy and infancy result in higher susceptibility to obesity and poor metabolic health in adult life(Reference Desai and Ross15). Regardless of the amount of energetic nutrients, FA composition has been suggested to be a crucial factor in the nutritional programming of metabolic phenotypes in adults(Reference Oosting, Kegler and van de Heijning16). Fetal exposure to various indicators of maternal diet, such as increased pre- or early pregnancy BMI, gestational weight gain, or gestational diabetes, increases the risk of offspring obesity and the metabolic syndrome in humans and animals(Reference Desai, Jellyman and Han17,Reference Schack-Nielsen, Michaelsen and Gamborg18) . Different studies have demonstrated the importance of the type of dietary FA consumption in rats on the reproductive performance and metabolic parameters of their offspring. Thus, CLA supplementation during critical windows of development may represent a therapeutic strategy in the prevention of early-life programming of metabolic and reproductive dysfunction(Reference Veaute, Andreoli and Racca19–Reference Segovia, Vickers and Gray21). Previously, in adult offspring rats fed normo-energetic diets, we demonstrated that a programming effect of CLA on lipid metabolic pathways leads to a preventive effect on TAG accretion in adipose tissue and liver(Reference Lavandera, Gerstner and Sain22). Therefore, the aim of this study was to investigate the preventive effect of maternal CLA consumption on TAG metabolism alterations induced by HF diets in adult male offspring receiving or not receiving CLA.
Methods
Chemicals and reagents
Nutrients for diet preparation were of analytical grade, with the exception of soyabean oil (Cada Día), sucrose and maize starch, which were food grade and obtained from local sources. CLA oil was obtained from Lipid Nutrition B.V. and consisted of an equimolecular mixture of c9,t11-CLA (38·99 %, w/w) and t10,c12-CLA (38·76 %, w/w). All solvents and reagents used for the FA quantification were of chromatography grade and were purchased from Merck. GLC-463 standards containing fifty-two fatty acid methyl esters (FAME) mixtures (purity > 99 %) were purchased from Nu-Chek Prep, Inc.. CLA, cis/trans mix (catalogue no. 05507) was purchased from Sigma-Aldrich, Inc.. The International CYTED Net (208RT0343) provided other FAME standards. Primers used for PCR analysis were synthesised by Invitrogen. For the enzyme assays, the materials were from Sigma-Aldrich, Inc.. All the other chemicals and reagents utilised were at least American Chemical Society degree or molecular grade and were acquired from Merck, Invitrogen and Applied Biosystems. The TAG test kit was obtained from the Sociedad de Bioquímicos.
Animals
Male and virgin mature female Wistar rats were provided by the facilities of our university. The animals were housed in collective cages, with the exception of mating, pregnancy and lactation periods, under controlled conditions (23 (sd 2) °C and 12 h light–12 h dark) with free access to standard food and water. All the experiments were conducted in compliance with the Guide for the Care and Use of Experimental Laboratory Animals(Reference Bayne23) and were approved by the Animal Ethics Committee of the School of Biochemistry (Universidad Nacional del Litoral).
Diet preparation
For all the experiments, three diets (Table 1) were used: control (C), HF and HF+CLA diets. The composition of the C diet was based on the American Institute of Nutrition Ad Hoc Committee recommendation (AIN-93G) formulated for the growth, pregnancy and lactation phases of rodents(Reference Reeves, Nielsen and Fahey24), using 7 % soyabean oil as a source of fat. The HF diet was enriched in fat by substituting 13 g of carbohydrate/100 g of diet with an equal amount of soyabean oil, reaching 20 g of fat/100 g of diet. The percentage of energy provided by fat (38 5 %) in the HF and HF+CLA diets slightly exceeds the recommended amount of dietary fat for the healthy adult population (20–35 % of energy)(Reference Vannice and Rasmussen25); however, it is reasonable for the human population(Reference Astorg, Arnault and Czernichow26). The amount of CLA used reflects the frequently consumed levels of nutritional supplementation. The HF+CLA diet was obtained by substituting 2·86 % (w/w) of soyabean oil in the HF diet with the same amount of CLA oil. The diets were freshly prepared, gassed with N2 and stored at 0–4°C. The FA composition of the experimental diets (Table 2) was determined by GC as indicated below.
Table 1. Composition of experimental diets (g/100 g dry diet)*
C, control diet; HF, high-fat diet; HF+CLA, HF diet supplemented with conjugated linoleic acid.
* C: based on the American Institute of Nutrition Ad Hoc Committee recommendation (AIN-93 G) formulated for growth, pregnancy and lactation phases of rodents(Reference Reeves, Nielsen and Fahey24). Vitamin mixture (per kg of diet): nicotinic acid, 30·0 mg; pantothenate, 15·0 mg; pyridoxine, 6·0 mg; thiamin, 5·0 mg; riboflavin, 6·0 mg; folic acid, 2·0 mg; vitamin K, 750·0 μg; d-biotin, 200·0 μg; vitamin B12, 24·0 μg; retinyl acetate, 1·2 mg; cholecalciferol, 0·025 mg; dl-α-tocopheryl acetate, 50 mg. Mineral mixture (mg/kg of diet): Ca, 5000·0; P, 1561·0; K, 3600·0; S, 300·0; Na 1019·0; Cl, 1571·0; Mg, 507·0; Fe, 35·0; Zn, 30·0; Mn, 10·0; Cu, 6·0; iodine, 0·2; Mo, 0·15; Se, 0·15; Si, 5·0; Cr, 1·0; F, 1·0; Ni, 0·5; B, 0·5; Li, 0·1 and V, 0·1.
Table 2. Fatty acid composition of experimental diets*
HF, high-fat diet; HF+CLA, HF diet supplemented with conjugated linoleic acid; ND, not detected.
* Values are expressed as the mean (percentage of total fatty acid methyl esters). A mixture of CLA was used as the source of CLA isomers and had an equimolecular amount of c9,t11-CLA (38·99 %) and t10,c12-CLA (38·76 %).
Experimental design
After a 1-week adaptation period (Fig. 1), eighteen female rats (274·1 (sd 8·6) g) were randomly divided into three experimental dietary groups (n 6/group), considering that one female rat was housed with one male from the same treatment group. After mating, each female was placed in an individual cage. The rats were fed the C, HF or HF+CLA diet for 4 weeks before mating and throughout pregnancy and lactation. At parturition, litter size was standardised to eight pups per dam in the C, HF and HF+CLA diets. The pups were maintained with their own mothers until weaning. The post-weaning male offspring of mothers fed with the C or HF diet continued with the same diets as their mothers, leading to the formation of the C/C and HF/HF groups (initial weights 98·6 (sd 5·3) and 97·8 (sd 6·1) g, respectively) (n 6 per group); the offspring of mothers fed HF+CLA were divided into two groups fed with HF diet (HF+CLA/HF; initial weights 98·3 (sd 4·2) g) or HF+CLA diet (HF+CLA/HF+CLA; initial weights 101·2 (sd 2·3) g) (n 6 per group). Based on the preliminary studies, the time of experimental feeding of the offspring was 9 weeks after weaning. At the end of the dietary treatment, one set of twenty-four animals (n 6 per group) was fasted overnight and killed (09.00–11.00 hours) under anaesthesia (1 mg acepromazine + 100 mg ketamine/kg body weight) by cardiac exsanguination. Blood was collected, and serum was obtained after centrifugation (1000 g for 10 min at 4°C). The liver, gastrocnemius muscle and epididymal white adipose tissue (EWAT) were dissected, weighed and immediately frozen. All samples were stored at –80°C until analysis. The same experimental protocol of feeding was followed with a second set of twenty-four male offspring rats divided into four experimental groups (n 6 per group) for the hepatic TAG secretion rate (TAG-SR) experiments, as explained below.
Fig. 1. Experimental design. C, control diet; HF, high-fat diet; HF+CLA, HF diet supplemented with conjugated linoleic acid. Maternal diet: for 4 weeks before mating and throughout pregnancy and lactation; post-weaning male offspring diet: for 9 weeks after weaning. Name of offspring groups: C/C, HF/HF, HF+CLA/HF and HF+CLA/HF+CLA. In all cases, the denomination to the left of the dash corresponds to the mother’s feeding, and the denomination to the right of the dash corresponds to the offspring’s feeding.
GC analysis
The FA composition of the experimental diets, liver, EWAT and serum was determined by GC using a Shimadzu (GC 2014) chromatograph equipped with a flame ionisation detector. Analyses were carried out with a capillary column CP Sil 88 (100 m, 0·25 µm film thickness). The carrier gas was H2 with a split ratio of 1:10. The column temperature was held at 75°C for 2 min after injection, then 5°C/min to 170°C, held for 40 min, 5°C/min to 220°C and held for 40 min. The injection volume was 0·5 µl, and the column flow was 0·8 ml/min. Total fats in diets, tissues and serum were extracted using the method described by Bligh & Dyer(Reference Bligh and Dyer27). The FAME were formed by transesterification with methanolic potassium hydroxide solution as an interim stage before saponification (ISO 5509:2000, Point 5 IUPAC method 2.301). FAME were identified by comparison of their retention times relative to those of commercial standards. Further GC details could be obtained from previous publications(Reference Saín, González and Lavandera28,Reference Masson, Alfaro and Camilo29) . The values of FA content were expressed as the percentage of total FAME (Table 2). The detection limit for the main FAME identified ranged from 0·01 to 0·03 %.
TAG levels in serum and liver
TAG levels in serum were determined by spectrophotometric methods using a commercially available test kit (Sociedad de Bioquímicos). To assess the liver TAG levels, portions of frozen tissue (0·5 g) were powdered and homogenised in saline (10 %, w/v) for TAG content quantification. Liver TAG levels were determined by the method of Laurell(Reference Laurell30).
Lipoprotein lipase activity in adipose tissue and gastrocnemius muscle
The removal capability of TAG-rich lipoproteins was evaluated by lipoprotein lipase (LPL) activities in the main tissues responsible for the uptake of TAG: adipose tissue and muscle. The LPL enzyme activity of adipose tissue was quantified in EWAT acetone powder by the fluorometric method of Del Prado et al. (Reference Del Prado, Hernandez-Montes and Villalpando31). Briefly, EWAT samples were delipidated by double extraction with cold acetone followed by double extraction with diethyl ether. The powders obtained were resuspended and incubated in a buffer (25 mm NH4Cl, pH 8·1 containing 1 IU/ml of heparin). The enzymatic reaction was carried out in a medium containing dibutyryl fluorescein as the enzyme substrate. The quantification of LPL activity was performed by measuring the increase in fluorescence (λ excitation = 490 nm; λ emission = 530 nm). In parallel, an identical assay was carried out in the same samples but in the presence of NaCl during the incubation to inhibit specific enzyme activity. LPL activity was estimated as the difference between non-specific lipolytic and total lipolytic activity. Values are expressed as pmol fluorescein/min per g of tissue and as nmol fluorescein/min per total EWAT. To assess muscle LPL activity, gastrocnemius muscle samples were homogenised in NH4Cl/NH4OH-heparin buffer. Then, the quantification of LPL activity in muscle was performed as previously described for adipose tissue(Reference Del Prado, Hernandez-Montes and Villalpando31). The measured activity was expressed as nmol fluorescein/min per mg of protein.
Hepatic TAG secretion rate
A second set of animals subjected to the same dietary treatments was fasted overnight and anaesthetised as indicated above. Then, 600 mg/kg of body weight of Triton WR 1339 in saline solution, an agent known to inhibit the peripheral removal of TAG-rich lipoproteins, was injected intravenously. Blood samples were taken immediately before and 120 min after the injection of the Triton solution for the estimation of TAG accumulation in serum. The hepatic TAG-SR was estimated based on the serum TAG concentration at 0 and 120 min, plasma volume and body weight. Further details have been previously reported(Reference Mocchiutti and Bernal32).
Liver enzyme activities
Liver samples were homogenised in buffer (pH 7·6) containing 150 mm KCl, 1 mm MgCl2, 10 mm N-acetyl-cysteine and 0·5 mm dithiothreitol. After centrifugation at 100 000 g for 40 min at 4°C, the supernatant fraction was used for the quantification of enzyme activities. Acetyl-CoA carboxylase (ACC, EC 6.4.1.2), FA synthase (FAS, EC 2.3.1.85) and glucose-6 phosphate dehydrogenase (G6PDH, EC 1.1.1.49) activities were measured following a previously used protocol(Reference Sain, Gonzalez and Lasa33). Enzyme activities were expressed as mU/mg protein, where 1 mU was 1 nmol NADH consumed (ACC), 1 nmol NADPH consumed (FAS) or 1 nmol NADPH produced (G6PDH), per min. Carnitine palmitoyltransferase-Ia (CPT-Ia; EC 1.3.99.3) activity was assessed in the mitochondrial fraction. Liver samples were homogenised in a buffer (pH 7·4) containing 0·25 m sucrose, 1 mm EDTA and 10 mm Tris-HCl. Homogenates were centrifuged at 700 g for 10 min at 4°C, and supernatant fluid was centrifuged again (12 000 g, 15 min, 4°C). Pellets were resuspended in a buffer (pH 7·4) containing 70 mm sucrose, 220 mm mannitol, 1 mm EDTA and 2 mm 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES). CPT-Ia activity was assessed in the pellet by the method of Bieber et al. (Reference Bieber, Abraham and Helmrath34). The pellet protein content was obtained as described above. The CPT-Ia activities were expressed as mU/mg protein (1 mU = 1 nmol CoA/min). The protein content was determined using bovine serum albumin as a standard(Reference Lowry, Rosebrough and Farr35).
Extraction and analysis of RNA and quantification by RT-PCR
Total RNA was isolated from the liver using TRIzol (Invitrogen), according to the manufacturer’s instructions. RNA samples were then treated with a DNA-free kit (Applied Biosystems) to remove any contamination with genomic DNA. The yield and quality of the RNA were assessed by measuring the absorbance at 260, 270, 280 and 310 nm and by electrophoresis on 1·3 % agarose gels. A quantity of 1 μg of total RNA from each sample was reverse-transcribed to first-strand complementary DNA (cDNA) using Moloney murine leukaemia virus reverse transcriptase (M-MLV RT). Relative mRNA levels were quantified using real-time PCR with a StepOne 18TM Real-Time PCR Detection System (Applied Biosystems). Sequence-specific commercially synthesised primers (Invitrogen Custom Primers) were used (Genbank: ACC, NM_022193.1; FAS, NM_017332.1; sterol regulatory element-binding protein-1c (SREBP-1c), NM_0012 76708.1; stearoyl-CoA desaturase-1 (SCD-1), NM_139192.2; CPT-Ia, NM_031559.2; PPARα, NM_013196.1; β-actin, NM_031144.3; ubiquitin C (UBC), NM_017314.1 and hypoxanthine phosphoribosyltransferase 1 (HPRT1), NM_012583.2), and the sequences were as follows: ACC: 5′-AAC AGT GTA CAG CAT CGC CA-3′ (forward), 5′-CAT GCC GTA GTAG GTT GAG GT-3′ (reverse); FAS, 5′-CAG AAC TCT TCC AGG ATAG TCA ACA-3′ (forward), 5′-GTC GCC CTAG TCA AGG TTC AG-3′ (reverse); SREBP-1c, 5′-GGA GCC ATAG GAT TAGC ACA TT-3′ (forward), 5′-GCT TCC AGA GAG GAG CCC AG-3′ (reverse); SCD-1, 5′-CAC ACG CCG ACC CTC ACA ACT-3′ (forward), 5′-TCC GCC CTT CTC TTT GAC AGC C-3′ (reverse); CPT-Ia, 5′-ACG TAGA GTAG ACT GGT GGG AAG AAT-3′ (forward), 5′-TCT CCA TAGG CGT AGT AGT TAGC TAGT-3′ (reverse); PPARα, 5′-CCC CAC TTAG AAG CAG ATAG ACC-3′ (forward), 5′-CCC TAA GTA CTAG GTA GTC CGC-3′ (reverse); β-actin 5′-CAT GAA GAT CAA GAT CAT TAGC TCC T-3′ (forward), 5′-CTAG CTT GCT GAT CCA CAT CTAG-3′ (reverse); UBC 5′-ACACCAAGAAGGTCAAACAGGA-3′ (forward), 5′-CA CCTCCCCATCAAACCCAA-3′ (reverse); HPRT1 5′-TCCTCCTCA GACCGCTT TTC-3′ (forward), 5′-ATCACTAATCACGACGCT GGG-3′ (reverse). Standard curves for each primer were generated on separate runs using several serial dilutions (1/10–1/1000) of pooled cDNA samples. The corresponding primer efficiency (E) of one cycle in the exponential phase was calculated according to the equation E = 10 (−1/slope)(Reference Rasmussen, Meuer, Wittwer and Nakagawara36). All the efficiencies of the primers were 100 % (sem 10 %). Target genes were standardised with the geometric mean of three housekeeping genes: β-actin, UBC and HPRT1. Relative expression ratios were calculated using the recommended 2–ΔΔCt method(Reference Vandesompele, De Preter and Pattyn37).
Statistical analysis
Values in Tables 3–6 are expressed as the mean values with their standard errors of six animals per group. The sample size was calculated taking into account a minimum test power of 0·80, at a level of significance of α = 0·05, and a maximum difference between the response of each variable of the control group with respect to the average of the differences with the experimental groups. The combined standard deviation was estimated from the data to generate the power curve. In all cases, these conditions were met with six(Reference Cohen38), which did not require further adjustment. The R software package pwr was used.
Table 3. Body and tissue weights of offspring*
(Mean values with their standard errors; six animals per group)
C, control diet; HF, high-fat diet; HF+CLA, HF diet supplemented with conjugated linoleic acid; BW, body weight; EWAT, epididymal white adipose tissue.
a,b Mean values within a row with unlike superscript letters were significantly different (P < 0·05).
* C/C, HF/HF, HF+CLA/HF, HF+CLA/HF+CLA: in all cases, the denomination to the left of the dash corresponds to the mother feeding, and the denomination to the right of the dash corresponds to the offspring feeding. Statistical analyses between groups were established by one-way ANOVA, followed by Tukey’s post hoc test to determine the critical differences between the groups.
The statistical analyses displayed in Tables 3–6 to compare the treatments for each variable were performed using SPSS 17.0 (SPSS, Inc.) by one-way ANOVA. To determine the differences between the groups, post hoc Tukey’s test was used. When there was no homogeneity of variance, the Games–Howell non-parametric test was applied. For the significant differences between the c9,t11-CLA and t10,c12-CLA levels in serum, liver and EWAT (described in the Results section), Student’s t test was performed. For all statistical analyses, significant differences were considered at P < 0·05.
Results
During all the experiments in mothers and offspring, the diets were well accepted and the animals showed a healthy status without any pathological manifestation from the treatments received. The mean food intakes of male offspring of different experimental groups were C/C: 362·1 (sd 23·3) kJ/d; HF/HF: 373·9 (sd 14·1) kJ/d; HF+CLA/HF: 404·4 (sd 24·4) kJ/d and HF+CLA/C+CLA: 398·14 (sd 20·1) kJ/d (P = 0·08).
Nutritional parameters
The body and tissue weights of the offspring are shown in Table 3. The body weight of offspring was higher in the HF/HF (P = 0·001) and HF+CLA/HF (P < 0·001) groups than in the C/C group. However, male offspring of the HF+CLA/HF+CLA group (P = 0·173) had a body weight value similar to that of the C/C group. The absolute and relative liver weights were greater in the HF/HF (P < 0·001), HF+CLA/HF (P < 0·001) and HF+CLA/HF+CLA (P < 0·001) groups than those in the C/C group. On the other hand, a significant decrease was observed in absolute and relative EWAT weight in offspring of HF+CLA/HF (P = 0·01 and P < 0·001) and HF+CLA/HF+CLA (P < 0·001 and P < 0·001), both when compared with the C/C and HF/HF groups, respectively.
Incorporation of conjugated linoleic acid in liver, epididymal white adipose tissue and serum
The incorporation of CLA isomers in serum and tissues was expressed as the percentage of total FAME. In all tissues of HF+CLA/HF+CLA offspring, c9,t11-CLA showed higher levels v. t10,c12-CLA. The c9,t11-CLA and t10,c12-CLA levels were serum: 1·08 (sd 0·08) and 0·31 (sd 0·06), respectively (P < 0·001); liver: 1·84 (sd 0·04) and 0·72 (sd 0·05), respectively (P < 0·001) and EWAT: 4·40 (sd 0·11) and 2·80 (sd 0·14), respectively (P < 0·001). As reflected, the levels of CLA incorporation in HF+CLA/HF+CLA offspring were EWAT > liver > serum.
Serum and liver TAG regulation
The serum and liver TAG contents and parameters related to their bioregulation are shown in Table 4. Serum and liver TAG levels were increased in the HF/HF (P < 0·001 in both tissues) v. the C/C group. In the HF+CLA/HF and HF+CLA/HF+CLA groups, this parameter in both tissues reached similar values to the C/C group. Compared with the C/C group, the hepatic TAG-SR was decreased in HF/HF (P < 0·001) but did not change in the two groups of offspring whose mothers were fed the HF+CLA diet. EWAT LPL activity was greatly decreased in all groups fed HF (P < 0·001) diets with or without CLA (HF+CLA/HF, P < 0·001 and HF+CLA/HF+CLA, P = 0·001). No changes were observed in gastrocnemius muscle LPL activity among the experimental groups.
Table 4. Serum and liver TAG levels, hepatic TAG-secretion rate (SR) and lipoprotein lipase activities in epididymal white adipose tissue (EWAT) and muscle of offspring*
(Mean values with their standard errors; six animals per group)
C, control diet; HF, high-fat diet; HF+CLA, HF diet supplemented with conjugated linoleic acid; LPL, lipoprotein lipase; GM, gastrocnemius muscle.
a,b,c Mean values within a row with unlike superscript letters were significantly different (P < 0·05).
* C/C, HF/HF, HF+CLA/HF, HF+CLA/HF+CLA: in all cases, the denomination to the left of the dash corresponds to the mother feeding and the denomination to the right of the dash corresponds to the offspring feeding. Statistical analyses between groups were established by one-way ANOVA, followed by Tukey’s post hoc test to determine the critical differences between the groups.
Hepatic lipogenic and oxidative enzyme activities
Lipogenic and oxidative enzyme activities are shown in Table 5. The hepatic ACC, FAS and G6PDH activities were decreased in the HF/HF, HF+CLA/HF and HF+CLA/HF+CLA groups v. the C/C group (P < 0·001). On the other hand, CPT-1a activity was diminished in the two groups of offspring whose mothers were fed the HF+CLA diet (HF+CLA/HF, P = 0·011 and HF+CLA/HF+CLA, P = 0·026) v. C/C. This parameter was also decreased v. HF/HF in both groups (HF+CLA/HF, P = 0·009 and HF+CLA/HF+CLA, P = 0·023).
Table 5. Activities of lipogenic and oxidative enzymes in the livers of offspring*
(Mean values with their standard errors; six animals per group)
C, control diet; HF, high-fat diet; HF+CLA, HF diet supplemented with conjugated linoleic acid; ACC, acetyl-CoA carboxylase; FAS, fatty acid synthase; G6PDH, glucose-6-phosphate dehydrogenase; CPT-Ia, carnitine palmitoyltransferase-Ia.
a,b,c Mean values within a row with unlike superscript letters were significantly different (P < 0·05).
* C/C, HF/HF, HF+CLA/HF, HF+CLA/HF+CLA: in all cases, the denomination to the left of the dash corresponds to the mother feeding, and the denomination to the right of the dash corresponds to the offspring feeding. Statistical analyses between groups were established by one-way ANOVA, followed by Tukey’s post hoc test to determine the critical differences between the groups. 1 mU = 1 nmol NADH consumed/min (ACC); 1 nmol NADPH consumed/min (FAS); 1 nmol NADPH produced/min (G6PDH) and 1 nmol CoA/min (CPT-Ia).
mRNA levels of lipogenic and β-oxidative enzymes and transcription factors
The liver expression of some key enzymes and transcription factor genes related to the synthesis (Acc, Fas, Scd-1 and Srebp-1c) and β-oxidation (Cpt-1a and Pparα) of FA are shown in Table 6. Compared with C/C, the expression of the hepatic FA biosynthesis enzymes, ACC and FAS, was diminished in HF/HF (P = 0·012 and P < 0·001, respectively), HF+CLA/HF (P < 0·001 for both enzyme activities) and HF+CLA/HF+CLA (P < 0·001 for both enzyme activities). Additionally, SCD-1 mRNA expression was significantly decreased in the HF/HF, HF+CLA/HF and HF+CLA/HF+CLA diets (P < 0·001 in all cases), with a greater decrease in HF+CLA/HF (P = 0·009) than in HF/HF. The gene expression of the transcription factor Srebp-1c did not show any significant change between the experimental groups. Regarding FA oxidation, CPT-1a expression did not change in all groups v. the C/C group but was increased in the HF+CLA/HF (P = 0·021) group v. HF/HF; however, PPARα was decreased in groups fed HF diets (HF/HF, P = 0·001; HF+CLA/HF, P < 0·001 and HF+CLA/HF+CLA, P < 0·001) v. C/C. The reduction was even more accentuated in HF+CLA/HF+CLA.
Table 6. Hepatic mRNA levels of lipogenic and β-oxidative enzymes and transcription factors of offspring*
(Mean values with their standard errors; six animals per group)
C, control diet; HF, high-fat diet; HF+CLA, HF diet supplemented with conjugated linoleic acid.
a,b,c Mean values within a row with unlike superscript letters were significantly different (P < 0·05).
* C/C, HF/HF, HF+CLA/HF, HF+CLA/HF+CLA: in all cases, the denomination to the left of the dash corresponds to the mother feeding, and the denomination to the right of the dash corresponds to the offspring feeding. Statistical analyses between groups were established by one-way ANOVA, followed by Tukey’s post hoc test to determine the critical differences between the groups.
Discussion
Human and animal studies have shown that maternal consumption of a HF diet significantly impacts the fetal environment and that offspring could become susceptible to obesity, hyperphagia, hypertension, non-alcoholic fatty liver disease, insulin resistance, glucose intolerance and cardiovascular impairments during adulthood(Reference Laker, Wlodek and Connelly39). CLA has been shown to prevent certain alterations induced by nutritional imbalances(Reference Kennedy, Martinez and Schmidt5,Reference Maslak, Buczek and Szumny40) . In this sense, we have recently demonstrated a programming effect of CLA on lipid metabolic pathways leading to a preventive effect on TAG accretion in adipose tissue and liver of male adult offspring rats fed normo-energetic diets(Reference Lavandera, Gerstner and Sain22). Thus, the aim of this study was to investigate the potential preventive effect of maternal CLA consumption on TAG metabolism alterations induced by HF diets in adult male offspring receiving or not receiving CLA.
Different studies have shown significant evidence that inadequate maternal nutrition, whether undernutrition or overnutrition, results in adverse effects in offspring(Reference Taylor, Samuelsson and Poston13,Reference Frihauf, Fekete and Nagy14,Reference Reynolds20) . Moreover, it is known that there are key developmental windows during which the adverse effects associated with early-life stressors can be prevented(Reference Sullivan, Nousen and Chamlou41), and in this framework, supplementation with a lipid regulatory agent such as CLA could be a useful tool to prevent lipid abnormalities observed in non-communicable chronic diseases. Thus, to the best of our knowledge, this is the first study demonstrating the influence of maternal CLA on lipid accretion in serum, liver and adipose tissue of adult male offspring receiving or not receiving CLA with high levels of dietary fat.
In the present study, using an equimolecular mixture of c9,t11-CLA and t10,c12-CLA isomers, we found, as expected, that CLA was incorporated in the serum, liver and EWAT of the HF+CLA/HF+CLA group but was not detected in the HF+CLA/HF group, as previously observed(Reference Lavandera, Gerstner and Sain22). In agreement with previous results from our laboratory(Reference Andreoli, Illesca and González42) and from other authors(Reference Purushotham, Shrode and Wendel43,Reference Vyas, Kadegowda and Erdman44) , the incorporation of c9,t11-CLA was higher than that of the t10,c12-CLA isomer in EWAT > liver > serum. In HF/HF offspring, we observed an increase in body weight. However, HF+CLA/HF+CLA offspring showed a lower final body weight related to reduced fat depots. The effect of CLA on body weight has been widely investigated in several animal models, including mice and rats(Reference Purushotham, Shrode and Wendel43,Reference Park and Pariza45) . Some studies have shown that CLA decreases weight gain(Reference Ryder, Portocarrero and Song46,Reference Terpstra, Beynen and Everts47) and adipose tissue depots(Reference Andreoli, Gonzalez and Martinelli48), whereas others have shown no effects on these parameters(Reference Park, Albright and Liu49,Reference Yamasaki, Ikeda and Oji50) . The potential mechanisms by which CLA reduces adipose tissue depots include its impact on energy metabolism, adipogenesis, inflammation, lipid metabolism and apoptosis(Reference Kennedy, Martinez and Schmidt5) and are mainly related to the action of the t10,c12-CLA isomer. Our results showed that in both offspring groups of CLA-fed mothers, a decrease in EWAT weight would be associated with a decrease in LPL enzyme activity. However, in the HF/HF group, even though the LPL enzyme activity decreased, the weight of the EWAT did not change. Thus, there could be other possible mechanisms mediated by CLA that would be involved in the reduction in EWAT, such as the inhibition of SCD-1 activity, enhanced apoptosis of pre-adipocytes and adipocytes and/or lipolysis modulation(Reference Park and Pariza45). In line with some of our results, it has been demonstrated that maternal c9,t11-CLA supplementation reversed HF diet-induced increases in fat mass, hyperleptinaemia and dyslipidaemia(Reference Reynolds20), impaired insulin sensitivity and adipocyte hypertrophy(Reference Segovia, Vickers and Gray21), and skeletal muscle atrophy and inflammation(Reference Pileggi, Segovia and Markworth51) in adult male rat offspring. However, there is no clear explanation of the mechanisms involved in the reduction in adipose tissue by c9,t11-CLA.
Fasting serum TAG levels are regulated by the circulating TAG removal rate from adipose tissue and muscle, as well as by the hepatic TAG-SR. Our results showed that the HF/HF group had the highest serum TAG levels despite the reduced hepatic TAG-SR. This result could be explained by the diminished EWAT LPL enzyme activity without changes in the activity of this enzyme in the muscle. CLA feeding of mothers reduced the levels of TAG in the serum of offspring fed HF diets, even when the hepatic TAG-SR remained at normal levels and was higher than that of HF/HF offspring. Similar results were found in hamsters(Reference Deckere, van Amelsvoort and McNeill52) and mice(Reference Scalerandi, Gonzalez and Saín53) fed CLA diets. Other authors found no differences in the serum TAG content after CLA supplementation in the diet(Reference Andreoli, Gonzalez and Martinelli48,Reference Kloss, Linscheid and Johnson54) . The dissimilarities in the responses could be attributed to differences in the type of diet and dose levels, meal pattern, age, isomers, species and animal model, among other causes.
Under our experimental conditions, offspring of the HF/HF group showed a significant increase in hepatic TAG levels. These results are in agreement with those reported by Ferramosca & Zara(Reference Ferramosca and Zara55), supporting that fatty liver is diet-inducible, including HF diets, in rodent animal models. The increased hepatic TAG accretion observed in the HF/HF group of the present study was associated with a lower hepatic TAG-SR. Some studies have demonstrated that a maternal HF diet during pregnancy and lactation alters liver TAG accretion in male offspring but the mechanisms involved in this alteration have not been described(Reference Reynolds20,Reference Seet, Yee and Jellyman56) . Although there is information available about the effect of CLA on lipid accretion, there are no studies on the potential preventive effects of maternal CLA consumption on liver steatosis induced in experimental animal models. In this regard, the liver is an organ that has been reported to be a key target for CLA(Reference Pariza, Park and Cook1), but the biological effects of CLA isomers on hepatic TAG metabolism differ among species, resulting in particular differences between rats and mice and between the type of isomer. Thus, while t10,c12-CLA increased the TAG concentration and induced liver steatosis in mice(Reference Arbonés-Mainar, Navarro and Acín57), data from studies on a model of insulin-resistant rats showed protective effects of t10,c12-CLA against hyperlipidaemia and increased liver weight and lipid content(Reference Stringer, Zahradka and De Clercq58). Specifically, in our study, an equimolecular mixture of c9,t11-CLA and t10,c12-CLA supplementation showed the same levels of hepatic TAG content in both the HF+CLA/HF and HF+CLA/HF+CLA groups as in the C/C group, associated with a normal liver TAG-SR. A very interesting finding of this study was related to the fact that the modulating action of CLA on liver TAG accretion remained even in the absence of CLA in the offspring diet. Thus, taking into account that the same results were observed in both offspring groups, consuming or not consuming CLA after weaning, and that we did not detect CLA in the liver of the HF+CLA/HF group, we could hypothesise that an early-life CLA programming mechanism that controls liver steatosis may be involved. Similar mechanisms of TAG regulation were previously observed in a study that investigated the influence of maternal CLA on TAG metabolism in adult offspring rats fed a normo-energetic diet(Reference Lavandera, Gerstner and Sain22).
To further elucidate the mechanism involved in hepatic TAG accretion, the potential participation of lipogenesis and β-oxidation of FA was studied. The elevated hepatic TAG content in the HF/HF group was accompanied by a reduction in lipogenesis without changes in β-oxidation. Specifically, the reduced ACC, FAS and G6PDH activities and mRNA levels of Acc, Fas, Scd-1 and Srebp-1c, with normal CPT-1a activity, clearly demonstrated an imbalance between lipogenesis and β-oxidation. These effects might be explained by the fact that a HF diet suppresses de novo lipogenesis in rodents(Reference Duarte Joao, Carvalho and Pearson59). In addition, CLA has the capability of acting as a potent ligand and activator of the PPAR group of nuclear receptors that regulate the gene expression of enzymes involved in lipogenesis and lipid oxidation(Reference Reynolds, Draper and Keogh6), and these mechanisms attenuate the hepatic steatosis observed in adult Wistar rats(Reference Andreoli, Illesca and González42). In our results, the normalisation of the hepatic TAG levels in offspring whose mothers were fed HF+CLA diets was not related to an accentuated decrease in lipogenesis or increase in β-oxidation. In fact, the enzyme activities of ACC, FAS and G6PDH in HF+CLA/HF and HF+CLA/HF+CLA and the gene expression of Acc, Fas and Scd-1 remained strongly inhibited by HF diets, as shown by the similar pattern of changes observed in the HF/HF group. The expression of lipogenic enzymes is regulated by SREBP-1(Reference Sampath and Ntambi60), among other factors. However, the mRNA abundance of this protein did not show a decrease with CLA feeding. In agreement with our results, decreased FAS(Reference Azain, Hausman and Sisk61) activity and Fas and Scd-1 (Reference Maslak, Buczek and Szumny40) mRNA levels in the liver were observed in CLA-fed rats. Regarding FA oxidation, the mRNA levels of Cpt-1a and Pparα did not change in the two groups of offspring whose mothers were fed HF+CLA diets, in agreement with the results observed by Park et al. (Reference Park, Albright and Liu49) in mice. However, increased(Reference Degrace, Demizieux and Gresti62) and reduced(Reference Rasooly, Kelley and Greg63) FA oxidation induced by CLA feeding have been reported. In agreement with previous findings reported by Reynolds(Reference Reynolds20), we found that maternal consumption of a HF diet during pregnancy and lactation results in increased lipid accretion, associated with several metabolic disorders. Moreover, our results showed a similar reduction in adipose tissue lipid accretion and normal TAG levels in the plasma and liver of both offspring groups of CLA-fed mothers, indicating that CLA prevented the alterations induced by a HF diet through programming mechanisms, at least through the modulation of the gene expression and activities of key enzymes, as well as by the liver TAG-SR. These results reveal emerging evidence that early-life interventions may prevent detrimental effects of maternal diet-induced programming. Our results are in agreement with the findings of Segovia et al. (Reference Segovia, Vickers and Zhang64), who demonstrated that there are critical windows of developmental plasticity in which the effects of an adverse early-life environment can be reversed by maternal dietary interventions. Additionally, Gray et al. (Reference Gray, Vickers and Segovia65) reported that a maternal HF diet causes developmental programming of endothelial dysfunction and hypertension in male offspring, which can be partially improved by maternal CLA supplementation, independent of offspring body weight. In our study, we specifically highlighted that maternal CLA supplementation prevented alterations produced by a HF diet on TAG accretion and metabolism in adult offspring receiving or not receiving CLA. Thus, we hypothesise that an early-life CLA programming mechanism that controls liver and EWAT TAG accretion may be involved. While it might have been interesting to study the effects of CLA intake on offspring not exposed to CLA in the uterus, our goal focused on the potential preventive effect of maternal CLA consumption in the development of hepatic steatosis of adult male offspring receiving a HF diet alone or supplemented with CLA for 9 weeks after weaning. Thus, this study emphasises the importance of balanced nutrition during pregnancy and demonstrates the efficacy of nutritional supplements with CLA as novel therapeutic agents during critical windows of development. Furthermore, the data provided by this study could be very important in the development of dietary strategies that allow for reducing or preventing obesity and fatty liver disease in humans consuming HF or unbalanced diets.
Acknowledgements
We wish to thank Walter DaRú for his technical assistance.
This study was supported by Consejo Nacional de Investigaciones Científicas y Técnicas (PIP CONICET no. 112-200801-00786).
C. A. B. designed and planned the study as well as analysed the results, interpreted the findings and wrote the manuscript; M. A. G. contributed to the design and planning of the study, interpretation of the findings and writing of the manuscript; J. L. performed the sample analysis, analysed the data, interpreted findings and prepared the manuscript; C. G.; A. C. F. and J. S. carried out data collection and analytical determinations and assisted in the maintenance and sacrifice of rats.
The authors declare that there are no conflicts of interest.
