DIVERSE INHIBITION OF FORKHEAD BOX O1 ACTIVITY BY LINOLEIC ACID ISOMERS - POTENTIAL ROLE IN LIPID METABOLISM IN HEPG2 CELLS AND LIVERS OF C57BL/6J MICE

A frequent way to cope with obesity is the use of dietary supplements that reduce amount of adipose tissue. These include e.g. conjugated dienes of linoleic acid (CLA). CLA are constitutional and geometric isomers of linoleic acid (1). Dietary supplements that potentially aid in weight loss usually contain a mixture of two linoleic acid isomers: trans-10,cis-12 (t10,c12) CLA and cis-9,trans-11(c9,t11) CLA. Both compounds are present in the mixture in a ratio of 1:1, but only one isomer (trans-10, cis-12) exhibits fat-reducing properties (2). Cis-9,trans-11 CLA, which is called rumenic acid, is the most common CLA present in food. Studies of Canadian researchers revealed that amongst dairy products, the biggest content of c9,t11 CLA was in processed cheese (6.2 mg/g fat) and cottage cheese (5.9 mg/g fat). Beef fat (1 g) contains approx. 3 mg of CLA (3). The data based on available literature indicate potential adverse effects of the use of supplements containing CLA such as: promotion of insulin resistance (4) and stimulation of atherogenic low density lipoproteins (5). In addition, CLA stimulate bile acid synthesis, thereby potentially increasing the risk of choledocholithiasis (6). The exact interaction mechanism of CLA in building up reserves of energy in the human body is still unknown. Elucidating this role is of the utmost importance for obese patients with type 2 diabetes, individuals with a metabolic syndrome, and also for professional athletes, many of whom take CLA in order to reduce fat mass in the period prior to competition.

Recent studies point out the crucial role of the forkhead box O1 (FoxO1) protein in the regulation of systemic energy transformations (7, 8). In mammalian cells a number of proteins belonging to the subclass FoxO were identified. These include: FoxO1 (FKHR), FoxO3 (FKHRL) FoxO4 (AFX) and FoxO6 (9). Posttranslational modifications, which determine the activity of FoxO1 processes, are: i) phosphorylation, ii) acetylation and iii) methylation. All these processes may lead either to activation or inactivation of the protein or both (10). FoxO perform their function by modulating the expression of genes, whose products are involved in a number of vital processes: i) control of metabolic processes, ii) coordination of the cell cycle, iii) DNA damage repair processes, iv) apoptotic response to oxidative stress (7, 9).

FoxO1 activity in the liver is regulated by insulin. It inactivates FoxO1 which contributes to repression of genes controlled by this protein (11). The same effect of insulin on FoxO1 was observed in the kidney cells (12). Proteins whose activation depends on FoxO1 and peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC-1a) are regulatory enzymes of gluconeogenesis: phosphoenolpyruvate carboxykinase (PEPCK, EC 4.1.1.49) and glucose-6-phosphatase (G-6-Pase, EC 3.1.3.9) (13). It should be noted that the activity of PGC-1a, in the context of gene expression of G-6-Pase and PEPCK is strongly dependent on the active form of FoxO1 (14). Furthermore, FoxO1 induces expression of genes whose products are involved in providing substrates for the production of glucose (15).

FoxO1 regulates the secretion of VLDL (very low density lipoproteins) in the liver. After eating, the level of VLDL decreases due to increased insulin levels. This is necessary to maintain normal postprandial lipemia. Unfortunately, in obese people with type 2 diabetes, due to insulin resistance developed in hepatocytes, the ability of insulin to regulate the synthesis of VLDL is significantly reduced. This further leads to continuous synthesis and exclusion of VLDL from the liver and accumulation of VLDL remnants in the blood, resulting in hypertriacylglycerolemia (9).

FoxO1 is a transcription factor of apolipoprotein CIII (apoCIII). With the increased level of FoxO1, an increase in the expression of apoCIII was also observed (14). ApoCIII inhibits the activity of lipases, resulting in the accumulation of triacylglycerol (VLDL). On the contrary, decreased level of ApoCIII contributes to hypertriacylglycerolemia. This is particularly evident in states of insulin resistance (16).

Based on the above, we decided to assess, whether, depending on the fatty acid added to the culture medium/mouse feed, we would be able to observe the following: i) a change of gene expression and activity of FoxO1 protein; ii) a diversified accumulation of lipids, and iii) a change in the concentration of apolipoprotein B100 (apoB100) protein in the culture medium due to secretion of VLDL in the cells. Of importance, research on influence of specific isomers (c9,t11 and t10,c12) on activity of FoxO1 and apoB100 concentration has never been conducted, which makes our findings novel in the field.

MATERIAL AND METHODS

HepG2 cell culture

All of the studies were performed on human hepatocarcinoma cell line HepG2 (ATCC, USA). The cells were cultured in Eagle’s minimal essential medium (EMEM, ATCC, USA) containing 10% fetal bovine serum (FBS, Gibco, USA) and antibiotics (penicillin + streptomycin, Sigma-Aldrich, Germany). The HepG2 cells were cultured on 6-well plates and incubated with various fatty acids for 48 hours at 37°C in a 5% carbon dioxide atmosphere. Linoleic acid and its isomers were delivered as complexes with bovine serum albumin (BSA): first the fatty acids were dissolved in ethanol, then sodium hydroxide was added in order to obtain fatty acids salts. The mixtures were dried under nitrogen and the salts were dissolved in double distilled water. To prevent oxidation butylated hydroxytoluene (BHT, Sigma-Aldrich, Germany) was added to each solution. At the end delipidated BSA (Sigma-Aldrich, Germany) was added. The concentration of fatty acid salts and BSA in reserve solutions and the wells were 8.1 mM, 2.7 mM and 30 µM, 10 µM, respectively. The variants of the experiment were as follows:

1. BSA (10 µM BSA),
2. CLA (15 µM c9,t11 CLA + 15 µM t10,c12 CLA + 10 µM BSA),
3. c9,t11 (30 µM c9,t11 CLA + 10 µM BSA),
4. t10,c12 (30 µM t10,c12 CLA + 10 µM BSA),
5. LA (30 µM linoleic acid + 10 µM BSA).

The results were compared to BSA and linoleic acid.

CLA isomers were purchased from Nu-Chek Prep (USA) and linoleic acid from Sigma-Aldrich (Germany).

Experimental animals

All procedures performed in studies involving animals were in accordance with the ethical standards of the institution or practice at which the studies were conducted. The Local Ethics Committee for Experiments on Animals (Szczecin, Poland) approved the animal studies (Agreement No 27/09).

The C57BL/6J mice (female, n = 25) were purchased from the animal facility of the Medical University in Bialystok (Poland). CLA isomers and linoleic acid were added to soybean oil (Clearspring, UK), and then mixed with an appropriate amount of ground feed and water, forming a homogeneous mixture. A standard feed for rodents ‘MURIGRAN’ (Agropol, Poland) was used in the experiment. Supplementation started in 6 weeks old mice, which were housed in groups:

1. SBO - a diet containing 6.5% soybean oil, n = 5;
2. c9,t11 - a diet containing 5% soybean oil and 1.5% c9,t11 CLA isomer, n = 5;
3. t10,c12 - a diet containing 5% soybean oil and 1.5% t10,c12 CLA isomer, n = 5;
4. CLA - a diet containing 5% soybean oil, 0.75% c9,t11 CLA isomer and 0.75% t10,c12CLA isomer, n = 5;
5. LA - a diet containing 5% soybean oil and 1.5% linoleic acid, n = 5.

The supplementation in this study was sustained for 4 weeks. The portions prepared each experimental day were ~3 g/mouse/day. The amount of feed consumed by the animals was recorded on a daily basis. The method used was counting the total amount of the feed leftovers in the cages. Water was freely available for the mice. Each experimental group ate a similar amount of feed. The animals were weighed before and after the experiment. There was no significant weight change observed between the mouse groups: prior to 18 – 20 g and 20 – 22 g after 4 weeks, when the study was completed and the animals were sacrificed. Rodents` organs were kept in –80°C.

Lipids staining in HepG2 cells by Oil Red O

The HepG2 cells were washed three times with PBS and then incubated with 4% formalin for 15 min in order to fix the material. After washing with 70% ethanol, the cells were stained for 10 min. in the dark with previously prepared Oil Red O (ORO) solution - powdered ORO (Sigma-Aldrich, Germany) reagent dissolved in 98% isopropanol. The HepG2 cells were again washed in 70% ethanol followed by distilled water. The next step was staining with Mayer’s hematoxylin (Aqua-med, Poland) for 2 minutes. The cells were washed three times with tap water, once with distilled water, dried and mounted with Kaiser’s glycerol gelatine (Merck, Germany). Quantification of relative lipid content was performed by image processing program ImageJ. The program counted red/orange pixels of every stained lipid droplet in 30 cells in one photograph. The pixels were counted in 5 photographs for every HepG2 group, then the mean was estimated.

Quantitative determination of apoB100 concentration in culture medium by ELISA

Measurement of apoB100 concentration was performed by Human Apoprotein B100 (apo-B100) ELISA Kit (TSZ ELISA, USA) according to the manufacturer’s procedure.

Quantification of gene expression using real-time PCR (qPCR)

Isolation of total RNA and reverse transcription were performed using an RNeasy Mini Kit (Qiagen, Netherlands) and SuperScript® First Strand Synthesis System for RT-PCR (Invitrogen, USA) respectively, according to the manufacturer’s procedures. Reagents for PCR reactions were: TaqMan® Gene Expression PCR Master Mix and TaqMan® Gene Expression Assays (human and mouse probes for FoxO1, carnitine palmitoyltransferase 1 (CPT1), fatty acid synthase (FAS), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), ApoCIII and 18S - reference gene for ApoCIII) were purchased from Applied Biosystems, USA. PCR was performed in a thermocycler 7500 fast real-time PCR system (Applied Biosystems, USA). The time-temperature profile was: 50°C/2 min, 95°C/10 min.; 40 cycles 95°C/15 s and 60°C/60 s. Each sample was analyzed in two replications. Data was compiled with 7500 Software v2.0.2. The relative expression of selected genes was determined by ΔΔCt method - double normalization: first in relation to a reference gene of constitutive expression (GAPDH or 18S) and then to the control samples (BSA and LA). The final results were calculated using the 2–ΔΔCt formula.

Western blot analysis

The sample preparation and further procedures were previously described by Stachowska et al. (17). A few modifications made in that procedure are presented in Tables 1 and 2. Primary and secondary antibodies were purchased from Santa Cruz Biotechnology, USA: FKHR antibody (H-128): sc-11350, p-FKHR antibody (Ser 256): sc-101681, β-actin antibody (C4): sc-47778, GAPDH antibody (FL-335): sc-25778, goat anti-rabbit IgG-HRP: sc-2004, goat anti-mouse IgG-HRP: sc-2005). Densitometric analysis was performed using the imaging system GelScan v6.0. (BioSciTech, Germany) and TotalLab Quant 2.2 (Totallab, UK).

Statistical analysis

Statistical analysis was performed using Statistica 10.0 (StatSoft, Poland). Nonparametric tests Wilcoxon matched-pairs test for the cells and U Mann-Whitney test for the mice were used for both, in vitro and in vivo studies. P-value < 0.05 was considered as statistically significant. All the results are expressed as mean values ± standard deviation (SD).

RESULTS

Evaluation of the lipid content in HepG2 cells after Oil Red O staining

The lowest accumulation of lipids was noticed in the cells cultured with BSA only (Fig. 1A). The lipids content of HepG2 cells incubated with t10,c12 CLA was about twice that of the BSA control (Fig. 1D and 1F). Greater accumulation of lipids (approx. 3-fold increase, statistically significant) was observed in the cells cultured with the mixture of CLA, the cells supplemented c9,t11 CLA isomer and the HepG2 cells incubated with linoleic acid (Fig. 1B, 1C, 1E and 1F). Comparing the results with the linoleic acid control, a statistically significant decrease of lipid content was observed in the cells cultured with t10,c12 CLA (Fig. 1F).

Fig. 1. Effect of fatty acids added to the cultured hepatocytes on lipid content. The hepatocytes were incubated with acids for 48 hours, then they were stained by ORO method and the relative lipid content was estimated. Confocal microscope pictures show the hepatocytes incubated in medium containing: BSA (A); CLA (B); c9,t11 CLA (C); t10,c12 CLA (D) and linoleic acid (E). Lipids are stained from orange to red. The quantitative results are expressed as arithmetic means + SD. Statistically significant results were considered those with P < 0.05 (Wilcoxon matched-pairs test, n = 5); *P < 0.05 versus BSA; #P < 0.05 versus LA (F).

Analysis of apoB100 protein concentration measurement by ELISA

Studies have shown that in relation to the BSA control group any acid added to the culture resulted in a statistically significant increase in the concentration of apoB100 in the medium. The largest (more than 3-fold) increase was observed in the medium with t10,c12 CLA and approx. 2.5-fold in the environment of the mixture of the two isomers. Addition of isomer c9,t11 CLA and linoleic acid resulted in an increase in the concentration of the protein in the medium of approx. 2 times (Fig. 2). When the results were compared to linoleic acid, a significant statistical increase in the apoB100 concentration was observed in culture with isomer t10,c12 CLA (1.75-fold) and the mixture of both isomers (approx. 1.4-fold) (Fig. 2).

Fig. 2. Effect of fatty acids added to the cell culture on apoB100. The hepatocytes were incubated with acids for 48 hours, where upon the concentration of protein apoB100 was measured in the culture medium. The results are expressed as arithmetic means + SD. Statistically significant results were considered those with P < 0.05 (Wilcoxon matched-pairs test, n = 5); *P < 0.03, **P < 0.02, ***P < 0.01 versus BSA; #P < 0.03, ##P < 0.02 versus LA.

Table 1. Western blot analysis of FoxO1and phosphorylated form of FoxO1 (p-FoxO1) in HepG2 cells.

Analysis of gene expression by real-time PCR

Comparing the results to the BSA control, they showed a statistically significant decrease in the relative levels of FoxO1 mRNA expression in all options of the experiment. The highest decrease (61.5%) was observed in the cells cultured with isomer t10,c12 CLA. For the HepG2 cells cultured in the environment of both isomers; only the isomer c9,t11 CLA; and only the isomer t10,c12 CLA, gene expression decreased sequentially by 35.4%, 30.4% and 28.7% (Fig. 3A). When the results were compared with respect to linoleic acid, calculations indicated statistical significance only for the cells cultured with isomer t10,c12 CLA, wherein the relative level of gene expression decreased by 44.1% (Fig. 3A). Comparing both isomers to each other, t10,c12 CLA caused a 51.7% greater decrease than c9,t11 CLA.

Table 2. Western blot analysis of FoxO1and phosphorylated form of FoxO1 (p-FoxO1) in C57BL/6J mice.

Fig. 3.Effect of fatty acids added to the cell culture and mouse feed on FoxO1 (A), apoCIII (B), FAS (C)and CPT1 (D) gene expression. The relative levels of the mRNA expression were determined by real time PCR. The results were determined by ΔΔCt method - double normalization: first in relation to reference gene of constitutive expression (GAPDH or 18S) and then to the control samples (BSA and LA). The final results were calculated using 2 - ΔΔCt formula. The results are expressed as arithmetic means + SD. Statistically significant results were considered those with P < 0.05: Wilcoxon matched-pairs test, n = 5; *P < 0.05 versus BSA; #P < 0.05 versus LA (HepG2 cells); U Mann-Whitney test, n = 5; *P < 0.05 and ***P < 0.01 versus SBO; #P < 0.05 and ###P < 0.01 versus LA (C57BL/6J mice).

Statistical significances were also observed in CPT1 gene expression: a 2,68 fold and a 2,78 fold increase in the cells cultured with CLA and t10,c12 CLA, respectively (versus BSA, Fig. 3B). FAS gene expression decreased by 44% (versus BSA) and 67% (versus LA) in the cells cultured with t10,c12 CLA (Fig. 3C). The same cells showed increased ApoCIII expression versus BSA - 26% and versus LA - 19%. We found a 57% increase in gene expression of ApoCIII in the cells cultured with CLA mix (Fig. 3D).

In vivo studies confirmed the highest decrease of FoxO1 gene expression in the livers of mice that consumed t10,c12 CLA (73.4% versus SBO and 65.4% versus LA). A statistically significant decrease was also observed in the livers of c9,t11 CLA mice (54.4% versus SBO and 40.7% versus LA) (Fig. 3A). The decrease caused by t10,c12 CLA was 37.5% greater than c9,t11 CLA.

Statistical significances were also observed in CPT1 gene expression: a 10% decrease in the t10,c12 CLA mice, versus BSA, and a 15% increase in the same group versus LA (Fig. 3B). FAS gene expression increased by 82% and 14 % (versus BSA) in the CLA and t10,c12 CLA group, respectively. In comparison to LA, the t10,c12 CLA mice showed a decrease in FAS gene expression by 31% (Fig. 3C). The CLA mice also showed increased ApoCIII expression versus LA 28% (Fig. 3D).

Detection of proteins: FoxO1 and p-FoxO1 by Western blot (p-FoxO1/total FoxO1 ratio)

The relative levels of p-FoxO1/total FoxO1 ratio increased in comparison to BSA control in all variants of the cell culture. Statistically significant results were obtained in the cells cultured in medium with isomer t10,c12 CLA (2,34-fold) alone and isomer c9t11 CLA alone (1,31-fold) (Fig. 4). Almost identical results were obtained in relation to the HepG2 cells cultured with linoleic acid: a 2,21-fold and a 1,31-fold for t10,c12 CLA and c9t11 CLA, accordingly (Fig. 4).

Fig. 4. Effect of fatty acids added to the cell culture and mouse feed on relative p-FoxO1/total FoxO1 ratio. The relative level of p-FoxO1 and total FoxO1 was determined by Western blot method with GAPDH as a reference protein, then the relative p-FoxO1/total FoxO1 ratio was calculated. The results present arithmetic means + SD. Statistically significant results were considered those with P < 0.05: Wilcoxon matched-pairs test, n = 5; *P < 0.05 versus BSA; #P < 0.05 versus LA (HepG2 cells); U Mann-Whitney test, n = 5 (C57BL/6J mice).

The animal studies did not indicate statistically significant changes of the relative levels of p-FoxO1/total FoxO1 ratio. The ratio increased slightly in relation to both control groups (BSA and LA) in all variants of the experiment (Fig. 4).

DISCUSSION

VLDL from HepG2 cells, shortly after secretion, are converted to LDL because of the very active extracellular hepatic lipase produced by these cells (18). Scientific reports indicate a correlation between serum apoB100 and cholesterol derived from LDL (19). What is more important, some scientists believe that concentration of apoB100 is a better risk factor of cardiovascular disease risk assessment than LDL cholesterol (20). In our study, c9,t11 CLA caused greater accumulation of lipids within the cells (Fig. 1C and 1F) and reduced VLDL assembly (lower concentration of apoB100), which in humans is associated with the lower risk of atherosclerosis.

The experiment conducted by Go et al. (21) compared the effect of t10,c12 CLA on lipid accumulation in HepG2 cells. Quantitative analysis revealed a higher content of neutral lipids, triglycerides and cholesterol esters within the cells cultured with isomer t10,c12 CLA in relation to linoleic acid (in contrast to the results of this work, Fig. 1F). The results of the mentioned study showed that t10,c12 CLA stimulates the activity of many enzymes involved in lipogenesis process. In HepG2 cells, the largest increase in gene expression (approx. 6-fold) was observed for acetyl-CoA carboxylase (ACC, EC 6.4.1.2) and fatty acid synthase (FAS, EC 2.3.1.85). In addition, t10 c12 isomer increased gene expression of glycerol-3-phosphate 1-O-acyltransferase (GPAT, EC 2.3.1.15) responsible for triacylglycerol accumulation in cells. The authors of these results claim that activation of mTOR (mammalian target of rapamycin) and SREBP1c (sterol regulatory element-binding protein 1) is the main pathomechanism of hepatic steatosis caused by accumulation of triacylglycerols due to activity of t10,c12 CLA (21). Comparing the results of Go et al. and the results obtained in this study, we need to stress that a greater amount of fatty acid was added to media in the Go et al. experiment (the final concentration was 100 µM versus 30 µM in this work). In addition, incubation in the presence of fatty acids was longer and lasted 72 hours comparing to 48 hours in our study. Moreover, we are surprised, that Go et al. in their experiments used ethanol in the medium as a solvent for t10,c12 CLA as it is well known that this alcohol activates the biochemical pathways associated with fatty liver disease.

Nevertheless, the scientific data also point towards lipid accumulation in HepG2 cells caused by the isomer t10,c12 CLA, where no ethanol was used as fatty acids’ solvent (22). In one experiment, both isomers accelerated formation of triacylglycerol, but only c9,t11 CLA also increased their secretion out of the HepG2 cells. Lipid accumulation as an effect of t10,c12 CLA was confirmed in the livers of mice (22). Studies of triacylglycerol mass (derived mainly from VLDL) in the medium supplemented with CLA showed an approx. 3-fold increase in VLDL secretion in HepG2 cells cultured with c9,t11 CLA and the HepG2 cells cultured with linoleic acid. A 1.5-fold increase was observed in the hepatocytes cultured with t10,c12 CLA (control cells - BSA) (23). Comparing the above results with the results of this study, an inverse proportion of the increase in VLDL (apoB100) concentration in the medium with c9,t11 CLA and the medium with t10,c12 CLA was noted (Fig. 2). Taken into account the fact that fatty acids in the cited study were added in a very high concentration (1 mM medium, which is 33.3 times higher than in this work), we conclude that the effect of activity of CLA isomers can be both inhibitory and a stimulatory to VLDL secretion, dependent on fatty acid concentration.

Studies of mouse adipocytes and rat hepatocytes revealed that t10,c12 CLA worked very specifically, and caused a lesser extent of lipid accumulation than c9,t11 CLA (24). Similarly, the results obtained in in vivo experiments of hamsters (25), were confirmatory to the results of our research. However, the examples of contradictory results could also be found in the literature. Experiments on mice showed an increase in the level of fatty acid transporter protein FAT/CD36 (fatty acid translocase) in rodents, whose feed was enriched in isomer t10,c12 CLA. That led to greater fat accumulation in the livers than in mice whose fed was supplemented with c9,t11 CLA (26). Also, other results indicate that isomer t10,c12 CLA can stimulate accumulation of fat in the liver in an insulin-independent way, promoting insulin resistance (27). Studies on rabbits fed with feed that contains CLA showed that both isomers and their mixture almost equally prevented formation of atherosclerotic plaque compared to animals supplemented with cholesterol (28).

Microsomal triglyceryde transfer protein (MTP) transports lipids involved in formation of lipoproteins that contain apoB100 or/and apoB48 (29, 30), which means that it plays a key role in synthesis of VLDL. The experiment on mouse hepatocytes, where MTP inhibitor was used, showed a decrease in secretion of triglycerides by 85% and a decrease of apoB100 level in microsomes. Concentrations of both compounds also decreased in media by 70% (triacylglycerols) and 90% (apoB100) (30). One of the Mttp gene transcription factors, responsible for MTP synthesis, is FoxO1. This protein is inactivated when concentration of insulin increases. Experiments performed in vitro in HepG2 cells and in vivo in mice clearly showed an inhibitory effect of insulin on FoxO1 which was followed by a decrease of MTP protein and VLDL level in the culture medium and in the rodents’ sera (30, 31).

The results of our current study illustrate a decrease in mRNA expression of FoxO1 in all culture media compared to BSA (Fig. 3A), which in theory should lead to reduced levels of VLDL and apoB100 protein. Meanwhile, in each variant of the experiment, apoB100 content increased. We were not able to find the relative information in the literature about concerning effects of particular CLA isomers (in relation to linoleic acid) on apoB100 protein content in human hepatocytes. The researchers who compared the effect of trans fatty acids on apoB100 secretion in HepG2 cells noted an increased concentration of this protein in the medium of cells cultured with the mixture of CLA isomers in comparison to linoleic acid (19), which confirms the results of our work (Fig. 2).

According to some researchers, MTP protein is necessary to form only the immature form of VLDL (pre-VLDL) consisting mainly of apoB100 protein and small amounts of lipids. MTP would, in addition to providing triacylglycerols, facilitate the formation of nascent apoB100, which if synthesized incorrectly, would experience ubiquitination and degradation. Further processes of pre-VLDL lipidation would occur via an MTP-independent way (29, 30).

This could explain why, even at a reduced level of active FoxO1, apoB100 concentration increases, as the results of this work showed (Fig. 2 and 4). It seems that the amount of MTP produced in the cells with decreased level of active FoxO1 was sufficient to initiate the synthesis of pre-VLDL. Further steps depend on available pool of triacylglycerols (29), whose quantity was sufficient in each of the cells supplemented with fatty acids.

Unfortunately, there is no compelling evidence that would explain the molecular mechanism of CLA isomers’ effect on apoB100 synthesis or transcription factors that affect gene expression of this protein. The results of our study suggest that fatty acids added at a low concentration (30 µM) lead in HepG2 cells to increased secretion of VLDL (measured by apoB100 protein concentration in medium), in comparison with BSA. The largest increase was caused by isomer t10,c12 CLA.

In relation to BSA control, levels of FoxO1 active form dropped in all the cells, the highest decrease was observed in the HepG2 cells cultured with t10,c12 CLA (Fig. 2 and 4). Since FoxO1 is a transcription factor of regulatory enzymes of gluconeogenesis (G-6-Pase and PEPCK) (5, 13, 16), this metabolic pathway was inhibited to the greatest extent in the cells incubated with isomer t10,c12 CLA. The results of our in vivo study differ from the in vitro ones. Namely, although the active FoxO1 level also decreased in all the livers, the drop was not as big as in the cells and it was not statistically significant (Fig. 4). A mechanism of CLA-FoxO1 crosstalk is intermediated by PPARg (peroxisome proliferator-activated receptor g) and SIRT1 protein (32, 33). Both isomers: c9,t11 CLA and t10,c12 CLA are PPAR’s ligands activating these receptors (34, 35). PPARs inactivate sirtuins and FoxO1 remains in its acetylated (inactive) form (32). Our study showed that CLA isomers cause FoxO1 phosphorylation (inactivation) in HepG2 cells. What is more, each isomer inactivates FoxO1 to a different extent. In the livers, there are no big differences in FoxO1 phosphorylation between the groups, presumably due to presence of insulin which activates PI3K-Akt (phosphatidylinositol-3-kinases - protein kinase B) pathway (36) thereby inactivating FoxO1 (10).

Gene expression of key enzymes involved in fatty acid synthesis and beta-oxidation (FAS, CPT1) and one of the apolipoproteins involved in lipolysis (ApoCIII) showed statistical significance mainly in the cells cultured with t10,c12 CLA and in the livers of mice whose fed was supplemented with the same fatty acid. Decreased FAS gene expression (Fig. 3C, versus BSA and LA) indicates that there was sufficient amount of fatty acids in the cells and their synthesis was not needed. Although there could be one possible process delivering free fatty acids - lipolysis. ApoCIII inhibits activity of lipases (37). In our study gene expression of apoCIII in t10,c12 cells was increased (Fig. 3B, versus BSA and LA) what means that lipolysis was not active. Depending on energetic demands of the cells, CLAs, as every other fatty acid, could be substrates for triglycerides synthesis (lipogenesis) in the liver cells or they could be oxidized in order to produce metabolic energy.

It is worth mentioning that different alleles of apoE (apoE3 and apoE4) in patients with NAFLD contribute to different lipid profiles, disorders of lipid metabolism and a fibrosis tendency (38). Latest research shows that higher concentration of FGF21 (fibroblast grow factor 21) in the serum of morbidly obese women with NAFLD is associated with more extent steatosis (39). Although further research is needed, an FGF21 serum level seems to be a promising non-invasive method for NAFLD patients.

CPT1 plays the main role in beta-oxidation (40) and in our study an increase of this enzyme’s gene expression was noted (Fig. 3D, versus BSA) in t10,c12 cells. Active fatty acid oxidation could also explain why the lowest accumulation of lipids was noted in these cells (Fig. 1D).

Our in vivo experiments brought a high number of statistically significant results for the t10,c12 CLA in mice. FAS and CPT1 gene expression confirmed our in vitro results in relation to LA mice. Surprisingly, in comparison to SBO control these results differ from the HepG2 cells study, namely a slightly increased FAS gene expression and a slightly decreased CPT1 gene expression was noted (Fig. 3C and 3D).

In summary, both isomers of linoleic acid have different effects on lipid and carbohydrate metabolism in the cells of human line HepG2. Isomer c9,t11 CLA accelerates lipogenesis, whereas isomer t10,c12 CLA activates secretion of lipids. Isomer t10,c12 CLA to the greatest extent inactivates the FoxO1 protein, which presumably results in the largest inhibition of gluconeogenesis in the cells cultured with this acid. Unfortunately pro-health property of t10,c12 CLA was not confirmed in the in vivo model. This casts a shadow on CLA dietary supplements that are commonly used by people with type 2 diabetes, NAFLD or a metabolic syndrome in order to reduce weight. Further research is needed to find out if CLA supplements are safe for humans.

Acknowledgements: Authors express their special thanks to Javier Chiarri, and Madeline Osborne for their invaluable help with graphics and proofreading.

Conflict of interest: Non declared.

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