Linoleic and α-linolenic fatty acid consumption over three generations exert cumulative regulation of hepatic expression of genes related to lipid metabolism
© Springer-Verlag Berlin Heidelberg 2014
Received: 22 January 2014
Accepted: 5 May 2014
Published: 20 May 2014
The essential fatty acids, omega-3 and omega-6, consumed during pregnancy can benefit maternal and offspring health. For instance, they could activate a network of genes related to the nuclear receptor peroxisome proliferator-activated receptor α (Ppara) and sterol regulatory element binding transcription factor 1 (Srebf1), which play a role in fatty acid oxidation and lipogenesis. The present study aimed to investigate the effects of diets with different omega-3/omega-6 ratio consumed over three generations on blood biochemical parameters and hepatic expression of Ppara- and Srebf1-related genes. During three consecutive generations adult Wistar rats were evaluated in the postpartum period (21 days after parturition). Regardless of prenatal dietary omega-3/omega-6 ratio, an upregulation in liver tissue was observed for Rxra, Lxra and Srebf1 and a downregulation for Fasn in all the evaluated generations. The diet with higher omega-3/omega-6 ratio decreased triacylglycerol serum levels and resulted in a constant non-esterified fatty acid level. Our results indicated that the PUFAs effect on the modulation of genes related to fatty acid oxidation and lipogenesis is cumulative through generations.
The programming of health begins before birth and is influenced by maternal health and nutrition. Alterations in the structure and function of maternal tissues during pregnancy can cause changes in fetal metabolic status (Newnham et al. 2002) leading to greater susceptibility of the offspring to some metabolic syndromes such as obesity, hypertension, diabetes and cardiovascular diseases (Langley-Evans 2006). The mechanisms by which a metabolic disorder or unbalanced nutrition during pregnancy influence gene expression and the phenotype of the offspring are mediated by epigenetics (Simmons 2011).
Recently, it has been proposed that nutrients such as long-chain fatty acids could influence offspring development and heath (Cetin et al. 2009). They are major sources of energy, acting to control the fluidity, permeability and conformation of cellular membranes. The polyunsaturated fatty acids (PUFAs) are especially important because they serve as precursors of important bioactive compounds such as prostacyclins, prostaglandins, thromboxanes and leukotrienes besides their role in structural functions (Haggarty 2010). During pregnancy in mammals, PUFAs are required for embryo (Haggarty et al. 2006) and oocyte development (Matorras et al. 1998), cell growth (Sellmayer et al. 1996) and differentiation (Ding et al. 2002). In this regard, studies have suggested the need for feeding different ratios of omega-6 (ω-6) and omega-3 (ω-3) during pregnancy (Simopoulos 2002; Simopoulos et al. 1999; Kris-Etherton et al. 2000). For instance, offspring of dams fed with a high-lard (i.e., saturated fat) diet during pregnancy and lactation were more susceptible to metabolic syndrome, including dyslipidemia, increased blood pressure and adiposity (Armitage et al. 2005). Recently, it was demonstrated that a high omega-3 diet reversed the hypertension, hyperlipidemia and adiposity that was induced by consumption of a high fat diet from weaning in 6-month-old male Wistar rats (Zulkafli et al. 2013).
A number of studies have demonstrated the potential of the ω-3 family of PUFAs to regulate key transcription factors controlling hepatic lipid metabolism (Jump 2008; Di Minno et al. 2012). The main target nuclear receptors for this fatty acids are the peroxisome proliferator-activated receptors (Ppar α, β, γ1 and γ2), sterol regulatory element binding transcription factor 1 (Srebf1), retinoid X receptor alpha (Rxrα) and nuclear receptor, subfamily 1, group H, member 3 (Nr1h3), also known as liver X receptor alpha (Lxrα) (Jump et al. 2005; Jump 2008).
Ppara is the most abundant PPAR isotype in the liver (Pegorier et al. 2004), and when activated by PUFAs it enhances the expression of genes related to fatty acid oxidation (Jump 2008) such as carnitine palmitoyltransferase 1a (Cpt1a), which is the hepatic Cpt isoform that catalyzes the transport of long-chain acyl-CoA from cytoplasm to the mitochondrial matrix by the conversion of long-chain acyl-CoA to acylcarnitines in order to go through β-beta oxidation (Sadana et al. 2007). Acyl-CoA oxidase 1 (Acox1) is another enzyme that takes part in the catabolism of straight-chain acyl-CoAs through peroxisomal β-oxidation (Kassam et al. 2001), and acyl-CoA dehydrogenase very long chain (Acadvl) is a target gene also involved in the mitochondrial β-oxidation pathway (Spiekerkoetter and Wood 2010). The modulation of Ppara target gene transcription is due to the binding of the activated heterodimer Ppar/Rxr to a specific DNA sequence, the peroxisome proliferator responsive element (PPRE) (Olefsky 2001).
While PUFAs promote Ppar activation, enhancing fatty acid oxidation, they suppress Srebf1, inhibiting lipogenesis (Jump 2008). Studies demonstrated that a decrease in Srebf1 transcription leads to hepatic inhibition of critical enzyme for lipogenesis (Teran-Garcia et al. 2007), as fatty acid synthase (Fasn) and acetyl-CoA carboxylase alpha (Acaca) (Ronnebaum et al. 2008).
Some investigations have evaluated the effect of prenatal nutrition with PUFAs on metabolic pathways and gene regulation of lipid metabolism, and whether alterations could be transmitted between generations (Armitage et al. 2005; Burdge et al. 2007; Moon et al. 2013). Apart from previously established evidence that dietary PUFAs have an effect on the regulation of hepatic gene expression related to lipid metabolism, the multigenerational effect of the ω-3/ω-6 ratio during pregnancy on the offspring metabolism has not yet been established. Therefore, our objective was to evaluate the effect of diets with different ω-3/ω-6 ratio in successive generations on blood parameters and hepatic expression level of some nuclear receptors and target genes involved in lipid metabolism.
Materials and methods
Animals, diets and experimental procedures
Ingredient composition of the experimental diets (g/kg diet)
AIN-93G (g/kg diet)
Every week a portion of the diet was randomly sampled for chemical analyses and fatty acid profile. The fat was isolated by the Bligh and Dyer method (Bligh and Dyer 1959) and the fatty acid profile analyzed by gas chromatography.
Blood sample and biochemical analysis
At the weaning day, females (with approximately 110 days old) were fasted for 12 h overnight, and then they were anaesthetized and euthanized according to the protocol approved by the University Animal Care and Use Committee. Blood was collected by intracardiac puncture in clean and EDTA-FK containing tubes. The samples were collected for measurement of plasma concentration of glucose, triacylglycerol (TAG) and non-esterified fatty acids (NEFA). Upon collection, samples were centrifuged at 3,000×g for 15 min. Plasma was harvested and stored at −80 °C until analyzed. The metabolites were analyzed using commercial biochemical assay kits, Glucose PAP Liquiform and Triacylglycerol (Labtest Diagnostica, Brazil) in a visible light spectrophotometer (Biospectro SP 220) and NEFA (Wako, USA) in a microplate reader (Thermo Plate Reader). All samples were analyzed in a single batch and intra-assay coefficient of variation was below 10 %.
Tissue collection, RNA isolation and qRT-PCR analyses
Six female per group, with 21 days postpartum, were euthanized in each of the three generations (G0, F1 and F2). Immediately after euthanasia the liver was collected, stored in cryotubes and snap-frozen by submerging in liquid nitrogen. Total RNA was isolated from the liver samples using TRIzol reagent (Invitrogen, USA) according to the manufacturer’s instructions. Total RNA was purified using RNeasy columns and on-column RNase-free DNase treatment (Qiagen, Germany), following the manufacturer’s protocol. The absence of RNA degradation was checked on a 1 % Tris–borate-EDTA buffer/agarose gel analyzing 28S and 18S rRNA subunits, after electrophoresis in 80 V during 1.5 h. The purity and concentration were measured with an UV spectrophotometer (UV Mini 1240, Shimadzu). The A260/A280 ratio was used as an indication of RNA quality. Reverse transcription was carried out with 1 μg of RNA using the High Capacity cDNA Reverse Transcription Kit with RNase Inhibitor (Applied Biosystems, USA) in a 10 μL volume. The reaction was performed in a thermo cycler (MyCycleTM Thermo Cycler, Bio Rad) using the following temperatures: 25 °C for 10 min, 37 °C for 120 min and 85 °C for 5 min. The cDNA was then diluted to 5 ng/μL.
Primers sequence used for qRT-PCR, source and accession number at NCBI site
Forward primer (5′–3′)
Reverse primer (5′–3′)
Somm et al. (2009)
Gillio-Meina et al. (2009)
Cha and Repa (2007)
Cha and Repa (2007)
Baarine et al. (2012)
Yang et al. (2011)
Buettner et al. (2006)
Hansmannel et al. (2006)
Ronnebaum et al. (2008)
Malik et al. (2010)
Bonefeld et al. (2008)
Nelissen et al. (2010)
Bonefeld et al. (2008)
The results are presented as mean ± SEM. All the statistical analyses were performed using SAS (SAS Institute Inc, Cary, NC, USA). Data (mRNA abundance) were log transformed prior to statistical analysis. The pregnancy rate was compared among groups and generations by Chi-square. Diet consumption, diet fatty acid profile, number and weight of pups were compared by one-way ANOVA and Tukey test. For biochemical parameters and mRNA abundance the Mixed Model procedure was used, considering the effects of diet, generation, and its interactions. A value of P < 0.05 was considered statistically significant. To the groups H/C (F1) and H/C/C (F2), the H group (G0) was taken as the founder generation, as they were derived from it (see Fig. 1).
Fatty acid oxidation genes
The generational effect in Ppara expression was observed in the C group, with a reduction from G0 to F1 (P = 0.05) and G0 to F2 (P = 0.03) (Fig. 2a). For Rxra, all groups did not have difference from G0 to F1, but had a significant increase in the F2 expression, H (G0–F2: P < 0.001 and F1–F2: P = 0.044), H/C (G0–F2: P < 0.001 and F1–F2: P = 0.009) and C group (G0–F2: P < 0.001 and F1–F2: P = 0.011) (Fig. 2d). Along generations the expression of Acadvl was opposite to Cpt1a, while for Cpt1a there was a decrease from G0 to F1(H, P = 0.003; H/C, P < 0.001; C, P < 0.001) followed by an increase from F1 to F2 (H/C, P < 0.001; C, P < 0.001) (Fig. 2b), for Acadvl there was an increase from G0 to F1 (H, P = 0.020; H/C, P = 0.003; C, P = 0.034) and then a decrease from F1 to F2 (P < 0.001 for all groups) (Fig. 2c). No generational effect was observed in Acox1 expression (P > 0.05—see supplemental Fig. 1).
A difference of Srebf1 mRNA expression between groups can be observed only in the F1 generation, where C/C group had a greater expression than the H/H group (P = 0.019) (Fig. 2g). Regarding the target genes, differences between groups were observed only for Fasn, in the F1 generation, where the C/C group had greater (P = 0.006) expression than the H/H group (Fig. 2h).
The generational effect could be observed in the increasing expression of Lxra in all groups, H (G0–F1: P < 0.001, G0–F2: P < 0.001 and F1–F2: P = 0.023), H/C (G0–F1: P < 0.001, G0–F2: P < 0.001 and F1–F2: P = 0.001) and C (G0–F1: P < 0.001, G0–F2: P < 0.001 and F1–F2: P = 0.002) (Fig. 2e). The hepatic expression of Srebf1 of the H group decreased between G0 to F1 (P = 0.035) followed by an increase between G0 to F2 (P < 0.001) and F1 to F2 (P < 0.001). The groups H/C and C had no differences between G0 to F1; however, the expression of Srebf1 increased significantly in F2 generation compared to F1 and G0 generations (P < 0.001 for all comparisons) (Fig. 2g).
For Fasn, all the groups had a very similar profile, a decrease between G0 to F1 (P < 0.001 for all groups) and also G0 to F2 (H, P < 0.001; H/C, P < 0.001; C, P = 0.004). Only in the H group expression increased (P = 0.002) from F1 to F2 (Fig. 2h). For Acaca, an increase (H, P = 0.001; H/C, P = 0.002; C, P < 0.001) was observed in all groups between F1 to F2 (Fig. 2i).
Nuclear co-repressor expression
Dietary fatty acid profile and intake
Fatty acid profile of the experimental diets (percentage of each fatty acid)
H (flaxseed oil)
C (soybean oil)
61.11 ± 0.742a
49.52 ± 0.791b
17.75 ± 1.180b
45.99 ± 0.540a
43.26 ± 1.849a
3.39 ± 0.384b
0.04 ± 0.005b
0.07 ± 0.013a
0.04 ± 0.003
0.04 ± 0.010
0.02 ± 0.011
0.03 ± 0.021
20.68 ± 0.486b
27.74 ± 0.582a
0.18 ± 0.003a
0.16 ± 0.004b
0.03 ± 0.006
0.04 ± 0.006
20.33 ± 0.477b
27.14 ± 0.571a
0.14 ± 0.022b
0.41 ± 0.061a
14.87 ± 0.299b
17.83 ± 0.298a
0.08 ± 0.054
0.03 ± 0.009
0.04 ± 0.015
0.02 ± 0.003
0.06 ± 0.002a
0.05 ± 0.002b
0.06 ± 0.002a
0.05 ± 0.002b
0.37 ± 0.006a
0.33 ± 0.010b
0.08 ± 0.001a
0.06 ± 0.001b
7.69 ± 0.185b
11.00 ± 0.060a
0.10 ± 0.001b
0.11 ± 0.001a
5.58 ± 0.123a
4.57 ± 0.162b
0.40 ± 0.105b
0.80 ± 0.256a
0.24 ± 0.017b
0.56 ± 0.012a
0.05 ± 0.002b
0.06 ± 0.007a
0.12 ± 0.015
0.16 ± 0.024
0.01 ± 0.002b
0.03 ± 0.003a
Blood serum concentration (mmol/L) of glucose, triacylglycerol (TAG) and non-esterified fatty acids (NEFA)
129.15 ± 9.47
71.75 ± 7.03
0.325 ± 0.021
138.75 ± 8.63
80.21 ± 13.39B
0.343 ± 0.031B
128.68 ± 2.98
49.89 ± 16.02
0.327 ± 0.025
119.96 ± 2.34
89.04 ± 21.79
0.268 ± 0.026
130.85 ± 10.98
60.41 ± 11.78C
0.258 ± 0035B
141.054 ± 4.51
57.93 ± 8.56b
0.329 ± 0.035b
166.83 ± 12.11
86.80 ± 11.60ab
0.220 ± 0.007c
144.18 ± 4.43
109.25 ± 16.01Aa
0.417 ± 0.034Aa
Reproductive parameters and body weight
The pregnancy rate was similar between the groups throughout the generations (G0, F1 and F2—P > 0.05) and were in the acceptable range of 62.5–87.5 %. There was no effect of dietary treatment on the number of pups per litter (P > 0.05), but in the F1 generation the pup mean weight at birth was greater in the H/H group (7.33 ± 0.21 g) than the C/C group (6.55 ± 0.13 g) (P = 0.01). Considering the C group throughout the generations, we observed a reduction (P = 0.01) in the pup mean weight from G0 (7.39 ± 0.22 g) to F1 (6.55 ± 0.13 g) that was maintained in the F2 (6.88 ± 0.41 g).
Over the last three decades, research on fatty acid metabolism, specially PUFAs, have exposed the benefits of these molecules on normal health and chronic diseases, through the regulation of lipid metabolism (Soulimane-Mokhtari et al. 2005; Kagohashi et al. 2010; Makni et al. 2011), cardiovascular (Adkins and Kelley 2010; Tousoulis et al. 2014; de Oliveira Otto et al. 2013) and immune function (Tapia et al. 2014; Yates et al. 2014; Liu et al. 2013). All fetal supply of ω-3 and ω-6 comes from the mother, crossing the placenta, either in the form of the essential fatty acid, or their long-chain PUFAs derivatives (Herrera 2002). The experimental diets offered to the animals in this study contained high amounts of PUFAs, almost 50 % in the C diet, and more than 60 % in the H diet. The consumption of food was not affected by the fatty acid profile in accordance with Rice and Corwin (Rice and Corwin 2002). The essential fatty acids offered through the diet were converted to other metabolites and incorporated in the liver in different proportions. The H group had more ALA and its metabolites, eicosapentaenoic acid (EPA) and docosapentaenoic acid (DHA) than the C group; while the animals that received the C diet had greater concentrations of LA and its metabolite arachidonic acid (AA) (data not shown), indicating activity of desaturases and elongases.
Studies demonstrated the existence of an interrelationship between Ppars, Srebf1 and Lxr. More than 10 years ago, in vitro and in vivo experiments proved that PUFAs suppressed Srebf1 activity by inhibition the Lxr/Rxr binding to LXREs (Yoshikawa et al. 2002, 2003). In the present study, only the C group decreased Ppara mRNA abundance from G0 to F1 and F2 generations, the other groups maintained their expression levels (Fig. 2a). In contrast, the expression of Srebf1 increased from G0 through F2 regardless of experimental group. A similar profile was observed for Lxra and Rxra expression, concerning with the studies cited above, providing evidence of the regulation of Srebf1 via Lxra activation. As the level of Ppara did not increase significantly, and Srebf1 did not decrease with a high PUFA diet, we suggest the existence of a post-transcriptional regulatory mechanism controlling fatty acid oxidation and lipogenic function potentially through proteolytic processing and autoloop regulatory circuit (Takeuchi et al. 2010).
In the liver, Ppara activation represses Srebf1 expression (Fernandez-Alvarez et al. 2011) and the decrease in Ppara coupled with the increase in Srebf1 expression across generations seems to support the mechanistic regulation between these transcription factors. Other factors, such as mammalian target of rapamycin complex 1 (mTOC1) (Lewis et al. 2011; Bakan and Laplante 2012; Takashima et al. 2009), protein kinase Akt (Yecies et al. 2011), fibroblast growth factor 21 (FGF21) (Zhang et al. 2011), S6 kinase 1 (S6K1) (Li et al. 2011) and adiponectin (Awazawa et al. 2009) could regulate Srebf1 transcription. Although the insulin response was not evaluated in this study, a higher insulin sensitivity across generations could be acting on the activation of Srebf1 promoter, primarily by increasing the activity of Lxrs or their heterodimerizing partner Rxrs (Chen et al. 2004). In accordance with this, a recent study demonstrated the increase in insulin sensitivity in the second generation of rats exposed to diets with high PUFAs content (Hirabara et al. 2013). The Rxra is intimately linked with Ppara, since its heterodimerization is necessary to activate and to promote the binding to PPRE in target gene promoters (Pegorier et al. 2004), and its increase during the experimental period might have been a response to Lxra and Srebf1.
The observed reduction in Fasn expression over the three generations, even with enhanced Srebf1 expression, seems opposite to the well-known effect of Srebf1 on Fasn expression (Teran-Garcia et al. 2007), suggesting that other factors may be regulating Fasn. One study with canola oil as PUFA source did not report a suppression of lipogenic gene expression, suggesting that diet did not induce liver lipid catabolism nor suppressed lipid synthesis, while a diet with highly unsaturated fatty acids from a fish/fungal source strongly suppressed both pathways (Sealls et al. 2008). Other factors such as growth hormone (GH), prolactin (PRL), STAT5, glucagon and insulin may also participate in the regulation of Fasn expression (Hogan and Stephens 2005; Moon et al. 2002).
Another interesting finding of this study was the opposite behavior of the Cpt1a and Acadvl genes. We speculate that the decrease from G0 to F1 observed in Cpt1a expression, when there was an increase at the same time point in Acadvl, was a cellular mechanism of regulation in the equilibrium between fatty acid transfer from cytoplasm to the mitochondria and β-oxidation, a manner to increase fatty acid entrance to supply the reduction in fatty acid oxidation. Related to the expression control exerted by co-repressors, PPAR and the evaluated target genes must be under influence of others main co-regulators, as SRC-1 (Dasgupta et al. 2014) and SMRT (Watson et al. 2012), since NCor1 expression level was almost constant. As what was observed with Acox1, a lack of diet and/generational effect on the regulation of their expression.
DNA methylation is the major epigenetic modification that controls gene expression in physiologic and pathologic states (Barres and Zierath 2011). As reviewed by Holness et al., nutrients can cause acute and long-term modifications at the gene expression level and lead to epigenetic modifications (Holness et al. 2010) and a recent study has already indicated epigenetic regulation induced by ALA (Niculescu et al. 2013). In this context, the dietary PUFA may act at the “end” of the process, or by controlling the activity of the nuclear receptors, not their abundance. This phenomenon must be evaluated in the future, so the observed changes in mRNA abundance from this study can be better understood.
The experimental diets did not alter the plasma glucose concentration, in accordance with other recent studies and indicating that chronic feeding of diets rich in PUFAs do not alter the glucose metabolism and its plasma concentration (Rice and Corwin 2002; Mellouk et al. 2012). It has already been shown that feeding animals with ω-3 and ω-6 diets is effective in lowering plasma triacylglycerol levels (de Assis et al. 2012; Harris 1989), as we observed in this experiment. We must highlight the apparent regulation of gene expression and blood metabolites across generations induced in the C group, indicating that diets with a high concentration of PUFAs, independently of the ω-3/ω-6 ratio, can induce metabolic regulation across generations.
About the reproductive parameters, the lack of difference in pregnancy rate between the groups and generations suggests that the ratio between ω-6 and ω-3 did not influence female fertility, similar to a study feeding flaxseed (Leal Soares et al. 2010). The greater pup mean weight of the F1 generation in the group fed with the omega-3 rich diet than controls, which were fed with the omega-6 rich diet, demonstrates that maternal consumption of omega-3 PUFAs during pregnancy had an effect on fetal and offspring growth and development. The reduction in pup mean weight in the control group between G0 to F1, and its maintenance in F2, indicated that more attention should be given to the ω-3/ω-6 ratio during the perinatal period, especially to ω-3 excess and its deleterious effect (Church et al. 2008). Again these results suggest the effects of maternal diet on offspring development.
In conclusion, feeding animals with PUFAs allowed for an initial study of the transgenerational control of hepatic lipogenesis and metabolism. There is clear evidence of cumulative effects throughout generations, independent of the PUFAs source, at least in part via up and down regulation of lipid metabolism-related genes.
We thank Dra. Leonor Almeida de Souza Soares for help with the experimental diets and animal handling training. This research was funded by the PNPD Program (2278/2009) from the Coordination for the Improvement of Higher Level- or Education-Personnel (CAPES).
Conflict of interest
Carolina B. Jacometo, Eduardo Schmitt, Luiz F. M. Pfeifer, Augusto Schneider, Francielle Bado, Fernanda T. da Rosa, Simone Halfen, Francisco A. B. Del Pino, Juan J. Loor, Marcio N. Corrêa and Nelson J. L. Dionello declare that they have no conflict of interest.
All institutional and national guidelines for care and use of laboratory animals were followed and the experimental protocol was approved by the Animal Welfare Committee from Federal University of Pelotas (Permit number: 0976).
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