The Mediterranean diet has been proven to be highly effective in the prevention of cardiovascular diseases and cancer and in decreasing overall mortality. Nowadays, transcriptomics is gaining particular relevance due to the existence of non-coding RNAs capable of regulating many biological processes. The present work describes a systematic review of current evidence supporting the influence of the Mediterranean diet on transcriptomes of different tissues in various experimental models. While information on regulatory RNA is very limited, they seem to contribute to the effect. Special attention has been given to the oily matrix of virgin olive oil. In this regard, monounsaturated fatty acid-rich diets prevented the expression of inflammatory genes in different tissues, an action also observed after the administration of olive oil phenolic compounds. Among these, tyrosol, hydroxytyrosol, and secoiridoids have been found to be particularly effective in cell cycle expression. Less explored terpenes, such as oleanolic acid, are important modulators of circadian clock genes. The wide range of studied tissues and organisms indicate that response to these compounds is universal and poses an important level of complexity considering the different genes expressed in each tissue and the number of different tissues in an organism.

Squalene is a natural bioactive triterpene and an important intermediate in the biosynthesis of sterols. To assess the effect of this compound on the hepatic transcriptome, RNA-sequencing was carried out in two groups of male New Zealand rabbits fed either a diet enriched with 1% sunflower oil or the same diet with 0.5% squalene for 4 weeks. Hepatic lipids, lipid droplet area, squalene, and sterols were also monitored. The Squalene administration downregulated 9 transcripts and upregulated 13 transcripts. The gene ontology of transcripts fitted into the following main categories: transporter of proteins and sterols, lipid metabolism, lipogenesis, anti-inflammatory and anti-cancer properties. When the results were confirmed by RT-qPCR, rabbits receiving squalene displayed significant hepatic expression changes of LOC100344884 (PNPLA3), GCK, TFCP2L1, ASCL1, ACSS2, OST4, FAM91A1, MYH6, LRRC39, LOC108176846, GLT1D1 and TREH. A squalene-enriched diet increased hepatic levels of squalene, lanosterol, dihydrolanosterol, lathosterol, zymostenol and desmosterol. Strong correlations were found among specific sterols and some squalene-changed transcripts. Incubation of the murine AML12 hepatic cell line in the presence of lanosterol, dihydrolanosterol, zymostenol and desmosterol reproduced the observed changes in the expressions of Acss2, Fam91a1 and Pnpla3. In conclusion, these findings indicate that the squalene and post-squalene metabolites play important roles in hepatic transcriptional changes required to protect the liver against malfunction.

Olive oil is the main source of lipid energy in the Mediterranean diet and there is strong evidence of its health benefits. The effect of extra virgin olive oil (EVOO) in the form of a preparation of spreadable virgin olive oil (S-VO) on the progression of atheroma plaques was investigated in Apoe-deficient mice, a model of accelerated atherosclerosis. Methods: Two isocaloric Western purified diets containing 20% fat, either as S-VO or as dairy butter, were used to feed 28 males and 16 females of two-month-old Apoe-deficient mice for 12 weeks. S-VO was prepared by blending more than 75% virgin olive oil with other vegetal natural fat to obtain a solid fat. Plasma total cholesterol, triglycerides and HDL cholesterol were measured. Hepatic lipid droplets were analyzed. Areas of atherosclerotic aortic lesions were quantified in cross-sectional images of the proximal aorta and en face analysis of the whole aorta. Results: Total plasma cholesterol was increased in mice on the butter-supplemented diet in both female and male mice compared to S-VO, and the ratio of TC/HDL-cholesterol was significantly lower in S-VO than in the butter diet, although only in males, and no differences in plasma triglycerides were observed. No significant differences in hepatic lipid droplets were observed between diets in either sex. Aortic lesion areas were significantly higher in mice consuming the butter versus the S-VO diet in both sexes. Conclusion: Extra virgin olive oil prepared in spreadable form maintained the delay in atheroma plaque progression compared to butter.

Erythrodiol is a terpenic compound found in a large number of plants. To test the hypotheses that its long-term administration may influence hepatic transcriptome and this could be influenced by the presence of APOA1-containing high-density lipoproteins (HDL), Western diets containing 0.01% of erythrodiol (10 mg/kg dose) were provided to Apoe- and Apoa1-deficient mice. Hepatic RNA-sequencing was carried out in male Apoe-deficient mice fed purified Western diets differing in the erythrodiol content. The administration of this compound significantly up- regulated 68 and down-regulated 124 genes at the level of 2-fold change. These genes belonged to detoxification processes, protein metabolism and nucleic acid related metabolites. Gene expression changes of 21 selected transcripts were verified by RT-qPCR. Ccl19-ps2, Cyp2b10, Rbm14-rbm4, Sec61g, Tmem81, Prtn3, Amy2a5, Cyp2b9 and Mup1 showed significant changes by erythrodiol administration. When Cyp2b10, Dmbt1, Cyp2b13, Prtn3 and Cyp2b9 were analyzed in female Apoe-deficient mice, no change was observed. Likewise, no significant variation was observed in Apoa1- or in Apoe-deficient mice receiving doses ranging from 0.5 to 5 mg/kg erythrodiol. Our results give evidence that erythrodiol exerts a hepatic transcriptional role, but this is selective in terms of sex and requires a threshold dose. Furthermore, it requires an APOA1-containing HDL.

Squalene is the major unsaponifiable component of virgin olive oil, the fat source of the Mediterranean diet. To evaluate its effect on the hepatic transcriptome, RNA sequencing was carried out in two groups of male Large White x Landrace pigs developing nonalcoholic steatohepatitis by feeding them a high fat/cholesterol/fructose and methionine and choline-deficient steatotic diet or the same diet with 0.5% squalene. Hepatic lipids, squalene content, steatosis, activity (ballooning + inflammation), and SAF (steatosis + activity + fibrosis) scores were analyzed. Pigs receiving the latter diet showed hepatic squalene accumulation and twelve significantly differentially expressed hepatic genes (log₂ fold change < 1.5 or <1.5) correlating in a gene network. These pigs also had lower hepatic triglycerides and lipid droplet areas and higher cellular ballooning. Glutamyl aminopeptidase (ENPEP) was correlated with triglyceride content, while alpha-fetoprotein (AFP), neutralized E3 ubiquitin protein ligase 3 (NEURL3), 2′-5′-oligoadenylate synthase-like protein (OASL), and protein phosphatase 1 regulatory inhibitor subunit 1B (PPP1R1B) were correlated with activity reflecting inflammation and ballooning, and NEURL3 with the SAF score. AFP, ENPEP, and PPP1R1B exhibited a remarkably strong discriminant power compared to those pathological parameters in both experimental groups. Moreover, the expression of PPP1R1B, TMEM45B, AFP, and ENPEP followed the same pattern in vitro using human hepatoma (HEPG2) and mouse liver 12 (AML12) cell lines incubated with squalene, indicating a direct effect of squalene on these expressions. These findings suggest that squalene accumulated in the liver is able to modulate gene expression changes that may influence the progression of non-alcoholic steatohepatitis.

Background and aim: The type and amount of dietary protein has become a topic of renewed interest in light of their involvement in metabolic diseases, atherosclerosis and thrombosis. However, little attention has been devoted to the effect of avian proteins despite their wide human consumption. The aim was to investigate the influence of chicken and turkey as sources of protein compared with that of soybean on atherosclerosis and fatty liver disease. Methods and results: To this purpose, male and female Apoe-deficient were fed purified Western diets differing in their protein sources for 12 weeks. After this period, blood, liver, aortic tree and heart base samples were taken for analyses of plasma lipids and atherosclerosis. Plasma triglycerides, non-esterified fatty acids, esterified cholesterol levels and radical oxygen species in lipoproteins changed depending on the diet and sex. Females consuming the turkey protein-containing diet showed decreased atherosclerotic foci, as evidenced by the en face atherosclerosis analyses. The presence of macrophages and smooth muscle cells in plaques were not modified, and no changes were observed in hepatic lipid droplets in the studied groups either. Paraoxonase activity was higher in the group consuming turkey protein without sex differences, but only in females, it was significantly associated with aortic lesion areas. Conclusions: Compared to soybean protein, the consumption of avian proteins depending on sex resulted in similar or lower atherosclerosis development and comparable hepatic steatosis.