Nutriomic Effects of Precision Lipids on Murine Hepatic Triacylglycerol Alterations Induced by High-Fat Diets Open Access

Obesity is considered a major risk factor for non-communicable diseases such as cardiovascular heart disease, hypertension, type 2 diabetes, and metabolic associated fatty liver disease. The remarkable recent increase rates in obesity and overweight, as well as metabolic-related diseases, makes it essential to understand the factors that contribute to the development and/or prevention of such conditions [1].

Dietary fat and functional lipids (FLs) intake has been recognized in precision nutrition as important factors responsible for tailoring nutritional interventions that modulate body lipid accumulation and cardiometabolic alterations [2‒4]. The nutritional management outcome is related to the type and amount of dietary fat [5], as well as to the presence of bioactive lipid compounds [6], among other factors.

Human studies have shown that high-fat (HF) diets can easily induce obesity associated with insulin resistance, dyslipidemia, and metabolic/cardiovascular diseases [7, 8]. Animal models have greatly contributed to elucidating the mechanisms involved, but only some of them are representative of dietary-induced human lipid accretion. For instance, previous studies conducted by our group and others in rats and mice have shown that 30–60 days of HF diet consumption induces significant fat accretion in adipose tissue (AT), muscle, and liver, associated with impaired glucose tolerance, lipid dysregulation, oxidative stress, and inflammation processes [9‒14].

On the other hand, epidemiological data support that a high intake of natural bioactive lipids like n-3 fatty acids (FAs), conjugated linoleic acids (CLAs), tocopherols (TPs), and phytosterols (PSs) is associated with a decreased risk of chronic diseases including obesity, metabolic syndrome, and type 2 diabetes [15, 16]. Although CLA may adversely impact health and cause fatty liver, insulin resistance, and lipodystrophy in some animal models, they are distinguished because they could reduce body fat and improve immune function, among other effects [17]. TP and PS also elicit beneficial effects; they have specially demonstrated hypocholesterolemic, antioxidant, and anti-inflammatory properties [18, 19].

Thus, this study aimed to investigate the putative beneficial effect of an FL mixture containing CLA, TP, and PS on the prevention of some lipid alterations induced by HF diets in an experimental animal model and to analyze its potential impact on the gene expression involved in the regulation of lipid metabolism. We hypothesized that TP and PS can potentially mitigate the detrimental effects of the CLA-mediated lipodystrophic syndrome in mice, while enhancing and complementing its anti-obesogenic effect.

High-quality nutrients, vitamins, and minerals were utilized in dietary preparations, sourced as chemical grade or better, excluding locally acquired soybean oil, sucrose, cellulose, and corn starch. Commercially available CLA (Tonalin, Tampa, FL, USA) with equimolecular c9,t11-CLA and t10,c12-CLA was used, in agreement with the methodology previously employed by our group [9]. TP and PS were extracted from sunflower oil deodorizer distillate employing the technique reported by Moreira and Baltanás [20].

External standards GLC-463 (52 FA methyl ester [FAME] mixture) were purchased from Nu-Chek-Prep (Elysian, MN, USA). Linoleic acid methyl esters and CLA mix were sourced from Supelco (PA, USA) and Sigma Chemical Co. (MO, USA), respectively. Other FAME standards were provided by the International CYTED Net (208RT0343). Solvents and reagents for FA quantification were of chromatography grade, and all the other chemicals used were, at least, of American Chemical Society (ACS) purity standards. Enzyme assay materials were obtained from Sigma (MO, USA), and the triacylglycerol (TAG) test kit was supplied by Sociedad de Bioquímicos (Santa Fe, Argentina).

All the experimental procedures were conducted in compliance with the regulations of the Ethics Committee of the School of Biochemistry (UNL) and compiled according to the Guide for the Care and Use of Laboratory Animals [21]. Thirty-six male CF1 mice (4 weeks), divided in 2 sets of experiments, were obtained from the University of Buenos Aires and housed in collective cages (3 mice/cage) under climate-controlled conditions (23 ± 2°C and 12 h light-12 h dark cycle) with ad libitum access to standard food and water. After 1 week of acclimation, eighteen mice weighing ∼ 30 g were randomly assigned to either a control (C), HF, or HF + FL groups (n = 6/group, 3 mice/cage). The animals were fed their respective diet for 4 weeks. The C diet was based on the American Institute of Nutrition Ad Hoc Committee recommendation (AIN-93 G) formulated for growth, pregnancy, and lactation phases of rodents [22] and contained 7% of soybean oil as fats source. The HF diet contained 30% of soybean oil and the HF + FL contained 28% of soybean oil and 2% of FL mixture. The FL mixture was prepared by a combination of commercial CLA (50%) and TP + PS mix (50%) obtained in our laboratory. The FL mixture contained (%, w/w) c9,t11-CLA: 19.5, t10,c12-CLA: 19.4, α-TP: 15.2, and β-sitosterol: 13.9. The diets were freshly prepared every 3 days, gassed with N₂, and stored at 0–4°C. The diets composition is detailed in Table 1. The relative FA contents of the diets (Table 1) were determined by gas chromatography as previously reported [12, 23].

In the first set of mice, 3 times a week throughout the study, body weight (BW) and food intake were recorded, and feces were collected and dried. At the end of the experimental assay, animals (9 weeks old) were fasted overnight and sacrificed in a CO₂ chamber, followed by cardiac puncture. Blood was collected and centrifuged for serum obtention. Liver, gastrocnemius muscle, retroperitoneal AT, and epididymal AT (EAT) were dissected, weighed, and immediately frozen. All samples were stored at −80°C until analysis. A second set of male mice were (n = 6/group) subjected to the same dietary treatments to estimate the in vivo hepatic TAG-secretion rate (TAG-SR) according to the procedure explained below.

Total fat in the dried samples of foods and feces was extracted using light petroleum ether and gravimetrically measured [24]. Energy intake was calculated from dietary energy intake and was expressed as kJ/day. Feed efficiency was determined dividing the BW gained by total energy intake and was expressed as mg/kJ [25]. Furthermore, the apparent fat absorption, as a bioavailability index, was calculated as the percentage of fat intake not excreted in the feces [26].

The extraction of total lipids from samples of liver, EAT, and experimental diets were performed by Bligh and Dyer’s method [27], followed by the transesterification with a methanolic potassium hydroxide solution (ISO 5509:2000, Point 5 IUPAC method 2.301). FAMEs obtained were separated on a capillary column CP Sil 88 (100 m, 0.25 mm film thickness) [23]. FAMEs were identified by comparison of their retention times relative to those of commercial standards using GC Solution Postrun software.

TAG levels in serum were determined by a spectrophotometric method using a commercially available test kit (Sociedad de Bioquímicos, Santa Fe, Argentina). Liver TAG levels were extracted by Bligh and Dyer’s method [27] and then quantified by a spectrophotometric method using the test kit mentioned above.

To estimate the hepatic TAG-SR, an in vivo assay was conducted with the second set of male mice (n = 6/group) after an overnight fast and under anesthesia (1 mg of acepromazine + 100 mg of ketamine/kg of BW). The TAG-SR was determined using the methodology proposed by Otway et al. [28] with some modifications [29]. Briefly, Triton WR 1339 (600 mg/kg), an agent known to inhibit the peripheral removal of TAG-rich lipoproteins, was intravenously injected. Blood samples were collected before and 120 min post-injection to assess TAG accumulation in serum. As demonstrated in previous studies from our group [29], the measurement showed linearity up to 150 min. TAG-SR was estimated based on serum TAG concentrations at 0 and 120 min, plasma volume, and BW. Results were expressed as µmol TAG per 100 g per minute.

The removal capacity of TAG-rich lipoproteins was assessed through lipoprotein lipase (LPL) activity in key tissues responsible for TAG uptake: AT and skeletal muscle, using the fluorometric method described by Del Prado et al. [30]. EAT samples underwent delipidation, and gastrocnemius muscle samples were homogenized in an NH₄Cl/NH₄OH-heparin buffer. Dibutyryl fluorescein served as the substrate, and the released fluorescein was measured to quantify LPL activity. Parallel assays with NaCl were conducted to inhibit specific enzyme activity. LPL activity was expressed as nmol fluorescein/min per total tissue. Extensive methodological details have been previously published [12].

Carnitine palmitoyl transferase-1a (CPT-1a) (EC 1.3.99.3) activity was assessed in the mitochondrial fraction [31]. Briefly, liver samples were homogenized, and the mitochondria were isolated by differential cold centrifugation. The activity was evaluated by CoA liberation at 412 ηm. In parallel and under similar conditions, unspecific activity was assessed in the absence of L(-) carnitine. The results were expressed as the difference between activity in the presence and absence of L(-) carnitine. The pellet protein content was determined as described above. The CPT-1a activity was expressed as mU/mg of protein (1 mU = 1 ηmol CoA/min).

Total RNA was isolated from the liver and EAT samples using TRIzol. RNA samples were then treated with a DNA-free kit (Applied Biosystems, Foster City, CA, USA). A total of 1.0 μg of RNA from each sample was reverse-transcribed to first-strand complementary DNA using an M-MLV Reverse Transcriptase. Relative mRNA levels were quantified using Real-Time PCR with a StepOne 18 TM Real-Time PCR Detection System (Applied Biosystems). Sequence-specific primers (Table 2) were designed from GenBank database: acetyl-CoA carboxylase (ACC); sterol regulatory element-binding protein-1c (SREBP-1c); diacylglycerol O-acyltransferase 2 (DGAT2); carnitine palmitoyltransferase-1a (CPT-1a); peroxisome proliferator-activated receptor alpha (PPAR-α); adipose triglyceride lipase (ATGL); hormone-sensitive lipase (HSL); uncoupling protein 2 (UCP2). Target genes were normalized with the geometrical mean of three accepted housekeeping genes: β-actin (ACT), ubiquitin C (UBC), and hypoxanthine phosphoribosyltransferase 1 (HPRT1). All primers were commercially synthesized and supplied by Invitrogen Custom Primers. Relative expression ratios were calculated using the recommended 2^−ΔΔCt method [32].

The statistical analysis was conducted with SPSS software (version 17.0; SPSS Inc., Chicago, IL, USA) and values were presented as means ± standard errors (n = 6) [33]. Sample size calculation determined a minimum of six for 80% power (p < 0.05). One-way ANOVA followed by Tukey post hoc test was used to establish differences between means. The unpaired Student’s t test was employed to compare levels of c9,t11-CLA and t10,c12-CLA or to analyze the results when there were fewer than three groups. Statistical significance was defined at p < 0.05.

The experimental diets were well received, maintaining animal health. While the daily energy intake increased in both HF groups, an increase in BW gain was observed in the HF group but not the in the HF + FL group and this outcome was inversely related to the feed efficiency (Table 3). Both HF groups exhibited increased fat intake and fecal fat excretion, as well as decreased apparent fat absorption. EAT weight was enhanced in the HF group and highly decreased in the HF + FL group. While retroperitoneal AT weight was not changed in the HF group, this tissue showed a drastic reduction in the HF + FL group.

Lipid modifications in the diet led to changes in the relative FA profile of liver and EAT (Table 4). Both CLA isomers were incorporated into the liver and EAT of mice fed the HF + FL diet, exhibiting higher levels in EAT than in liver. The c9,t11-CLA isomer predominated over the t10,c12-CLA in both tissues, despite the equimolecular isomer contents in commercial CLA.

In the liver, the HF group showed higher levels of n-6 polyunsaturated FA (PUFA), and lower monounsaturated FA (MUFA) and saturated FA levels, while the HF + FL group increased n-6 and n-3 PUFA and reduced the MUFA levels. However, it should be noted that the presence of FL at HF levels increased the n-3 very long chain PUFA (VLC-PUFA) biosynthesis efficiency, assessed by the EPA/ALA and DHA/ALA ratios, and to a lesser extent the n-6 VLC-PUFA biosynthesis estimated by the ARA/LA ratio. On the contrary, the HF diet depressed the biosynthesis of n-3 VLC-PUFA, while there was no significant change in the n-6 PUFA to n-6 VLC-PUFA conversion. The biomarkers of hepatic de novo FA biosynthesis, 16:1/16:0 and 18:1/18:0 ratios, were decreased at HF levels and were exacerbated by the presence of FL.

In EAT, both HF and HF + FL groups showed elevated PUFA levels, mainly at the expense of the increase in n-6 PUFA. These changes were in agreement with the reduced levels of saturated FA and MUFA. Consistent with liver observations, 16:1/16:0 and 18:1/18:0 ratios were decreased in HF, but only the 16:1/16:0 ratio was reduced in the HF + FL group.

Parameters related to the TAG metabolism in different tissues are shown in Table 5. Fasting serum TAG levels were decreased in the HF and HF + FL groups. However, elevated fasting serum glucose levels in both HF groups were found, leading to low serum TAG/glucose indices.

Hepatic TAG-SR and extrahepatic TAG removal are involved in the serum TAG regulation. In this regard, liver TAG levels were increased in the HF group but markedly reduced in the HF + FL group. This effect can be explained by the increased CPT-1a activity only in the HF + FL group. On the other hand, these changes cannot be explained by the hepatic TAG-SR that remained unchanged by both HF and HF + FL diets but might be attributed to the increased muscle LPL activity in the HF and to a lesser extent in the HF + FL group. Furthermore, EAT LPL raised activity in HF but not in HF + FL.

Hepatic TAG regulation involves lipogenesis and β-oxidation, which were assessed through the gene expression of key enzymes and transcription factors. SREBP-1c mRNA levels were significantly reduced in the HF + FL group, while ACC mRNA levels were also reduced in the HF group but to a lesser extent than in the HF + FL group (Fig. 1a). Regarding FA β-oxidation, PPARα and CPT-1a mRNA levels were lower in HF group, and only the CPT-1a mRNA levels were decreased in the HF + FL group (Fig. 1b).

In EAT, SREBP1-c and ACC mRNA levels were increased in the HF group and were restored to the control levels by the FL supplementation (Fig. 1c). Regarding lipid catabolism, mRNA levels of PPARα, CPT-1a, and UCP2 were increased, while the HSL expression decreased in the HF group. However, in the HF + FL group, PPARα and CPT-1a mRNA levels were decreased, and ATGL, HSL, and UCP2 expressions were higher compared to the HF group (Fig. 1d).

The purpose of the FL mix used in this research was to take advantage of the very well-known properties of CLA on lipid metabolism by testing and avoiding some recognized undesirable effects on mice, such as lipodystrophy [17], by the addition of TP and PE. Thus, some genes associated with the metabolic pathways involved in the potential beneficial effect of FL on TAG regulation as well as several biomarkers related to the metabolism of FAs and triacylglycerides were investigated.

In agreement with previous studies and with other researchers [12, 34], the AT showed the highest CLA incorporation. The isomer content in tissues depends on factors such as treatment duration, affinity for tissue incorporation, isomer secretion rate, and oxidation capacity [35, 36]. Considering the high metabolic activity of the liver and the function of AT as an energy reservoir for FA, the discrepancy between the CLA content in both tissues is not surprising [37]. Moreover, the higher levels of c9,t11-CLA compared to t10,c12-CLA were aligned with previous findings in various animal models [9, 12, 34] and may be attributed to the greater oxidative capacity of t10,c12-CLA.

The relative FA composition on tissues reflected the amount and type of dietary lipids, as well as the tissue FA uptake and metabolism. FL intake improved both, n-3 and n-6 PUFA levels, as well as the hepatic conversion efficiency of PUFA to VLC-PUFA reflected by the EPA/ALA, DHA/ALA, and ARA/LA ratios. This increased conversion rate of LA to ARA and of ALA to EPA, as well as to DHA could be associated with the availability of lower substrates for the incorporation and/or retention into tissues and organs, due to the competition with the CLA present in the FL mixture, as previously demonstrated under different conditions [34]. In addition, the FL mixture seemed to activate the VLC-PUFA biosynthesis pathways, leading to an increase of the relative amounts of ARA, EPA, and DHA. The components of the FL mixture could be acting as ligands for the nuclear receptors or transcription factors involved in the regulation of genes encoding enzymes in the VLC-PUFA biosynthesis pathways. This could lead to the increased transcription and expression of these enzymes, thereby upregulating the biosynthesis of VLC-PUFAs [38, 39].

In relation to the FA profile, Warensjö et al. [40] suggested that the changes in serum FA composition, as well as the estimation measurement of desaturase activities, using FA product-to-precursor ratios, could act as important predictors of the metabolic disorders commonly observed in the metabolic syndrome. In the present work, both hepatic ratios 16:1/16:0 and 18:1/18:0, used as estimated SCD activity [40], decreased in the HF group and were more pronounced, in the HF + FL denoting a reduced lipogenesis. Probably, this finding is the result of different situations occurring in parallel. On the one hand, dietary PUFA, such as LA, decreased SCD activity as well as the gene expression in liver [41]. Additionally, CLA has shown a decreased SCD mRNA stability and/or transcription [15]. Therefore, it is likely that the beneficial effect on lipid regulation, in the HF + FL group, could be linked to the modulation of the hepatic FA profile.

The synergistic effect of the FL mixture employed in the present work was also reflected in nutritional parameters. Therefore, the lower BW gain and reduced AT in the HF + FL group, with respect to the other groups, could be due to the lower feed efficiency, likely influenced by the combined effects of the FL components. The mechanisms proposed by other authors include CLA’s impact on lipogenesis, lipolysis, and CPT-1 activity [15]. In addition, other authors have reported that soybean PS decreased BW in a 60-day experiment [42]. Our assays suggest that the positive effects of FL on nutritional parameters may be attributed to reduced FA biosynthesis, indicated by the lower mRNA levels of SREBP1-c and ACC in AT, along with reduced flux through SCD estimated by the 16:1/16:0 ratio. In a similar manner, Burdeos et al. [43] showed in differentiated 3T3-L1 preadipocytes that unsaturated vitamin E, δ- and γ-tocotrienol significantly down-regulated the expression of crucial lipid metabolism-related genes such as FAS, SCD-1, ACC-1, SREBP-1c, LDLR, and PPARγ. Additionally, in our study, the HF + FL group seems to have a more efficient lipid breakdown. In this regard, mRNA levels of ATGL, HSL, and UCP2 in AT were increased. These data are in agreement with the findings of other authors who demonstrated that the mechanism through which vitamin E inhibits fat accumulation involves the significant upregulation of CPT-1 and UCP2 gene expression in preadipocytes by δ- and γ-tocotrienol [15, 43, 44].

The assessment of serum glucose and lipid measurements plays a crucial role in evaluating metabolic health and identifying potential risk factors associated with various metabolic disorders. For example, the HF diets are associated with the development of metabolic associated fatty liver disease, characterized by a wide spectrum of liver lipid accretion, metabolic syndrome, hyperglycemia, and impaired glucose tolerance [45]. This effect was observed in our study, in which higher serum glucose levels were found; however, it was mitigated by the FL mixture. Additionally, serum TAG levels (regulated by hepatic TAG synthesis, secretion, and removal from the tissues) decreased in both HF diets; other authors have reported similar findings in mice [9, 46] and rats [12] fed HF diets. In our work, the hepatic TAG-SR does not explain the reduction in serum TAG levels found in both HF groups, but this effect could be attributed, at least in part, to the enhanced LPL activity (mainly in muscle) generated by the HF diet.

Despite the high serum glucose levels induced by the HF diet, the decrease in circulating TAG levels resulted in a reduction in the TAG/glucose index. This index is used to assess insulin resistance and the risk of cardiovascular diseases. However, given the behavior exhibited by TAG, removed from serum by LPL and accumulated in the liver, in this particular case, this index may not fully reflect the overall effect generated.

It is widely recognized that the consumption of commercial CLA causes lipodystrophy and fatty liver in mice [9, 17]. In the present study, to prevent these pathologies, PS and TP were added in the FL mixture. In this regard, Rideout et al. [47] suggested that PSs are protective against diet-induced hypertriglyceridemia and associated this effect with reduced hepatic mRNA PPARα levels, FAS protein abundance, and de novo lipogenesis caused by the HF diet. A similar effect was observed by Tapia et al. [19], in male mice fed the HF diet supplemented with γ- and α-TP. In the current investigation, the hepatic TAG levels were deeply decreased in the HF + FL group. This observed effect could be attributed to the enhanced activity and gene expression of CPT-1a, denoting an increased β-oxidation. Additionally, our research revealed a decrease in the mRNA levels of genes associated with FA biosynthesis, specifically SREBP-1c and ACC, in both HF groups. Thus, the FL mixture employed in this study appears to regulate hepatic TAG levels, primarily impacting oxidation through CPT-1.

In conclusion, the FL mixture composed of CLA, PS, and TP effectively prevented the lipid alterations induced by a HF diet. This was achieved through the modulation of mRNA levels associated with lipid homeostasis control. Furthermore, the FL mixture was successful avoiding the lipoatrophic syndrome induced by CLA in a HF mouse model. These results imply that the utilized mixture of FL could serve as a valuable component in precision nutrition strategies for the management of cardio-metabolic diseases.

The present investigation offers important mechanistic insights into the beneficial effects of the FL mixture evaluated, elucidating its impact on preventing lipid alterations induced by the HF diet. These findings could potentially result in a valuable tool for preventing lipid metabolism alterations commonly observed in the population, thus contributing to an improved biochemical-nutritional profile consistent with a reduction in the incidence of non-communicable chronic diseases. Nevertheless, as expected, it has some limitations. Although it assessed various parameters related to lipid metabolism and the underlying molecular mechanisms, other relevant biomarkers or physiological endpoints could have been included to provide a more comprehensive evaluation of the FL mixture effects. Moreover, the study employed a single dosage and formulation of the FL mixture. Variability in dosages or compositions of the mixture could yield different outcomes. Finally, the relatively short duration of the intervention period may not fully represent long-term effects or potential adaptations to the experimental diets. Longer-term studies, as well as different dosage and formulation, could provide a more comprehensive understanding of the sustained effects of the FL mixture.

The authors wish to acknowledge the support received from Universidad Nacional del Litoral (UNL) and Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET).

This study protocol was reviewed and approved by the Comité Asesor de Ética y Seguridad de la Investigación (CAESI), Facultad de Bioquímica y Ciencias Biológicas, and Universidad Nacional del Litoral, Approval No. #01/17.

The authors have no conflicts of interest to declare.

This study was supported by Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Proyecto de Investigación Plurianual (PIP) (1220170100316C).

C.L.S., A.C.F., and J.V.L. carried out the care and maintenance of the animals throughout the experimental period, including the preparation of the experimental diets, as well as the sacrifice of animals and the determination of nutritional parameters and some biochemical determinations. A.C.F. and J.V.L. also analyzed the data, interpreted the findings, and prepared the manuscript. L.V.C. contributed to the analytical determinations. C.A.B. designed and planned the study, analyzed the results, interpreted findings, and corrected the manuscript. All authors have read and approved the final version manuscript.

All data generated or analyzed during this study are included in this article. Further inquiries can be directed to the corresponding author.