EVOO and Metabolic Liver Health in MASLD

Overview

MASLD is currently one of the most common chronic non-communicable diseases and the leading cause of liver-related mortality and morbidity, with a rising prevalence worldwide, especially in the presence of obesity, diabetes, and other cardio-metabolic risk factors. Lifestyle modification, particularly through the Mediterranean Diet, is the first-line intervention, and extra virgin olive oil is a key component thanks to its monounsaturated fatty acids and bioactive compounds with anti-inflammatory and antioxidant effects. Several studies indicate that extra virgin olive oil supplementation, especially within a Mediterranean Diet pattern, reduces hepatic steatosis, improves inflammation, oxidative stress, and glucose and lipid profiles, and may promote weight loss and reduction of fat mass, also through potential effects on the gut microbiota. EFSA recognizes protective effects with a daily intake of at least 20 g of extra virgin olive oil, but it is still unclear whether this amount is optimal for individuals with MASLD, particularly those who are overweight or obese

Metabolic dysfunction-associated steatotic liver disease (MASLD) is one of the most widespread non-communicable chronic diseases worldwide and the leading cause of liver-related mortality and morbidity. It currently affects about 38% of the global adult population and between 7% and 14% of the pediatric population, and projections indicate that its prevalence will continue to rise, exceeding 44% by 2040. The term MASLD was recently introduced to replace the former nomenclature for steatotic liver disease (non-alcoholic fatty liver disease, NAFLD) and to emphasize the close relationship between "fatty liver" and metabolic syndrome, as reported in the 2023 multisociety Delphi consensus statement. MASLD is defined by the accumulation of triglycerides in the liver in the absence of significant alcohol intake or other causes of steatosis, together with at least one of the following cardiometabolic risk factors: obesity, type 2 diabetes mellitus, dyslipidemia, or arterial hypertension. Its pathophysiology is multifactorial and involves both modifiable and non-modifiable factors: liver damage results from the combined effect of metabolic dysfunction, unhealthy diet, physical inactivity, and genetic predisposition. At present, there is no strong consensus on pharmacological therapy for the management of MASLD. For this reason, lifestyle modification, with a focus on diet and physical activity, still represents the first-line intervention for the prevention and management of MASLD. Recent studies identify diet as a primary modifiable factor in the clinical approach to MASLD, highlighting the key role of diet quality and eating habits in modulating various metabolic processes. Most of the scientific evidence agrees in identifying the Mediterranean diet as the dietary pattern most strongly associated with health benefits, with protective effects against several non-communicable chronic diseases. The Mediterranean diet is a traditional dietary pattern followed by populations living in countries bordering the Mediterranean Sea. Its benefits are mainly attributable to the characteristic foods it includes, which are rich in nutrients, antioxidant compounds, and dietary fiber. The Mediterranean diet is based on high consumption of fruits, vegetables, whole grains, legumes, nuts, and extra virgin olive oil, moderate intake of fish and poultry, and low intake of red meat and processed products. Extra virgin olive oil (EVOO) is the main culinary fat used in the Mediterranean diet. It is composed of 97-99% triglycerides and a small fraction of bioactive components responsible for its biological activity and organoleptic properties. The health effects of EVOO are due to its high content of monounsaturated fatty acids, mainly oleic acid, and to a series of minor but valuable components such as polyphenols, phytosterols, tocopherols, squalene, and phenolic compounds, all known for their anti-inflammatory and antioxidant effects. Numerous clinical and interventional studies have shown that EVOO supplementation, especially when included within a Mediterranean diet regimen, can reduce hepatic steatosis in subjects with MASLD. Its hepatoprotective effect is related to improvements in inflammation, oxidative stress, and insulin resistance, which are all key mechanisms in the pathogenesis of MASLD. EVOO polyphenols have been shown to reduce levels of interleukin-6 (IL-6) and C-reactive protein (CRP), thereby exerting an anti-inflammatory effect. Oleocanthal, the compound responsible for the pungent taste of EVOO, exerts an ibuprofen-like action by inhibiting cyclooxygenase-1 and -2 (COX-1/COX-2), further reducing inflammation. In addition to its anti-inflammatory properties, EVOO is also recognized as a powerful antioxidant. A Mediterranean diet enriched with EVOO has been shown to reduce levels of 8-hydroxy-2-deoxyguanosine (a known marker of oxidative DNA damage), decrease lipid peroxidation, lower tumor necrosis factor-α (TNF-α) and myeloperoxidase levels, and increase adiponectin and interleukin-10 (IL-10) levels in both obese and normal-weight adults. Several studies have also reported postprandial antioxidant activity of EVOO, linked to reduced plasma lipid peroxide levels after meals. Improved liver function associated with EVOO intake is also reflected by reduced levels of liver enzymes alanine aminotransferase (AST), aspartate aminotransferase (ALT), and gamma-glutamyl transferase (GGT), which are important markers of liver inflammation and damage. Studies have reported reductions in liver enzyme levels following EVOO supplementation lasting 2 to 12 months. Consistent with its metabolic benefits, EVOO consumption, particularly in the context of the Mediterranean diet, has shown significant improvements in glycemic control and insulin sensitivity. Studies have reported lower fasting glucose, insulin, and HOMA-IR values, suggesting that extra virgin olive oil contributes to improved glucose metabolism, which is a key component of MASLD and closely linked to insulin resistance. Positive effects of EVOO consumption have also been observed on the lipid profile. Studies have demonstrated reductions in LDL cholesterol and triglyceride levels, as well as improvements in the activity and composition of high-density lipoproteins (HDL). Some authors suggest that the beneficial effects of EVOO on metabolic diseases and health in general may be mediated by changes in the gut microbiota. In rats, a diet enriched with EVOO modified the gut microbiota profile, increasing its biodiversity and subsequently improving metabolic parameters such as insulin resistance and body weight. In addition, it has been hypothesized that tyrosol, one of the phenolic components of EVOO, may induce weight loss in mice by modulating the gut microbiota and triggering adipose tissue thermogenesis through increased gene expression of factors such as uncoupling protein 1 (UCP1). A diet that includes daily consumption of extra virgin olive oil is also considered a valid and effective option for weight loss and weight maintenance. It has been observed that diets including three or four tablespoons of EVOO per day lead to greater weight reduction than low-fat diets. Moreover, for the same amount of weight loss, daily EVOO consumption can produce greater fat mass reduction compared with the daily intake of generic seed oils. According to in vitro and animal studies, the potential mechanisms underlying this additional reduction in fat mass are attributable to EVOO phenols, which decrease adipocyte proliferation and increase thermogenesis via activation of brown adipose tissue. At present, the European Food Safety Authority (EFSA) states that the protective effects of extra virgin olive oil can be obtained with a daily intake of at least 20 g of EVOO or 5.0 mg of hydroxytyrosol. However, it is still unclear whether these amounts are equally effective in individuals with MASLD, particularly those who are overweight or obese. The study in question will be interventional in design and will include two intervention arms: 60 consecutive subjects with MASLD and with 20 ≤ BMI \< 30 will be recruited from the "Centro di Nutrizione Clinica per la Ricerca e la Cura dell'Obesità e delle Malattie del Metabolismo" of our Institute. Recruitment will be supported by general practitioners, through voluntary participation and by drawing on already ongoing cohort studies. The intervention will be nutritional: subjects enrolled in both arms will receive an isocaloric Mediterranean-style diet, designed taking into account age, sex and lifestyle, in accordance with the LARN, 5th Revision. Subjects who meet the study inclusion criteria, assessed during a Screening Visit (V0), will be invited, for the first 14 days, to consume a predefined amount of EVOO (3 tablespoons - approximately 30 g), in order to reduce selection bias. At the end of the 14 days, enrolled subjects will be randomized into two intervention arms. * Group A: Subjects enrolled in this group will be asked to consume, in addition to the foods indicated in the diet, 3 tablespoons (approximately 30 g) of EVOO per day, corresponding to 270 kcal, included in the total calories of the prescribed diet. The oil must be used raw at any time of the day. * Group B: Subjects enrolled in this group will be asked to consume, in addition to the foods indicated in the diet, 9 tablespoons (approximately 90 g) of EVOO per day, corresponding to 810 kcal, included in the total calories of the prescribed diet. The oil must be used raw at any time of the day. The treatment will last approximately 74 days and, in addition to V0 (Screening Visit), 3 further visits are planned: * V1: V0 + 14 days * V2: 30 days after delivery of the diet * V3: V2 + 30 days At V0, potentially eligible subjects, after signing the informed consent form, will undergo: * Fibroscan, to assess the degree of hepatic steatosis * Measurement of anthropometric characteristics (height, weight and BMI) If the inclusion criteria are met, recruited subjects will be asked to consume a predefined amount of EVOO (3 tablespoons - approximately 30 g), in order to reduce selection bias. Recruited subjects will be invited to return for V1 after 14 days (V0 + 14 days), fasting for at least 12 hours, to undergo a Fibroscan to confirm inclusion criteria. If the inclusion criteria are confirmed, subjects will be enrolled in the study and will undergo: * Medical history (family, physiological, past and recent pathological, and pharmacological) * Measurement of anthropometric characteristics (weight and BMI, waist circumference and neck circumference) * Bioimpedance analysis * DEXA * Blood sampling by venipuncture * Online dietary questionnaire Subjects will be asked to bring: * A stool sample * A urine sample (N.B.: If the subject no longer meets the inclusion criteria, the biological samples will be immediately discarded.) Subjects who meet the inclusion criteria at V1 will be randomized to one of the two intervention arms. Subjects will receive the isocaloric diet by email, prepared by the Institution's Dietitian. After 30 days from receipt of the diet, they will return for V2 where, fasting for at least 12 hours, they will undergo: * Measurement of anthropometric characteristics (weight and BMI, waist circumference and neck circumference) * Bioimpedance analysis * Blood sampling by venipuncture * After 30 days from V2, subjects will return for the final visit (V3) where, fasting for at least 12 hours, they will undergo: * Fibroscan * Measurement of anthropometric characteristics (weight and BMI, waist circumference and neck circumference) * Bioimpedance analysis * DEXA * Blood sampling by venipuncture Subjects will be asked to bring: * A stool sample * A urine sample Considering that one of the objectives of the study is to evaluate the effect of EVOO consumption exclusively on MASLD, it is necessary that there are no significant weight changes in the recruited subjects. Therefore, participants will be asked to report their body weight weekly. In the case of significant weight variation, the nutritional plan will be adjusted. Whole blood and serum samples obtained will be sent to the central laboratory for measurement of routine and nutritional, metabolic and cardiovascular risk parameters (fasting glucose, insulin, glycated hemoglobin, triglycerides, total cholesterol, HDL and LDL, transaminases (AST and ALT), γGT, creatinine, uric acid, complete blood count, ferritin, 25-OH-vitamin D, calcium, IGF-I, TSH, FT3, FT4, proBNP). On the basis of the results, the degree of insulin resistance (HOMA index) and the degree of hepatic steatosis FLI (fatty liver index), based on anthropometric (BMI, waist circumference) and biochemical parameters (triglycerides and γGT), and hepatic fibrosis (FIB-4), will be calculated. FIB-4 allows the level of liver fibrosis to be established by relating the subject's age, AST levels and platelet count. The FIB-4 formula is: (Age \[years\] × AST \[U/L\]) / ((PLT \[10(9)/L\]) × (ALT \[U/L\]\^(1/2))). A FIB-4 score \<1.45 has a negative predictive value of 90% for advanced fibrosis, whereas a FIB-4 \>3.25 has a specificity of 97% and a positive predictive value of 65% for advanced fibrosis. The main adipokines (leptin, adiponectin, RBP-4, resistin, visfatin, chemerin), the major hepatic growth factors (HGF, Fetuin-A, FGF21, FGF19, PAI-1), the main pro-inflammatory cytokines (high-sensitivity C-reactive protein, IL-6, IL-8 and TNF-α) and the anti-inflammatory cytokine IL-4 will also be measured; other aliquots will be used for lipidomic and biochemical analyses. Lipidomic analysis will be performed on the red blood cell membranes of recruited subjects. Fatty acids will be extracted, derivatized and analyzed with a gas chromatograph equipped with a 60 m polar capillary column and an FID detector. Lipid profiles will be identified by comparing them with chromatograms of fatty acid methyl esters used as standards. In serum samples collected at all visits, antioxidant activity, inflammatory status and serum levels of deglycating enzymes will be assessed by studying SOD1 and SOD2, and the enzymes Fructosamine 3-Kinase and glyoxalase. Subjects in the study will also be asked to bring, at each visit, 1 stool sample for the assessment of gut microbiota and fecal metabolome, and 1 urine sample. For the assessment of the gut microbiota, total bacterial metagenomic DNA will be extracted from stool samples using the QIAamp FAST DNA Stool Mini Kit (Qiagen, Hilden, Germany). Microbiome characterization will be carried out using a DNA metabarcoding and shotgun metatranscriptomic approach. DNA metabarcoding analysis will be conducted using the hypervariable V5-V6 regions of 16S rRNA for bacteria (MiSeq-Illumina platform). Samples that are significant on DNA metabarcoding analysis will undergo metatranscriptomic analysis (NextSeq 500 - Illumina platform). Metagenomic and metatranscriptomic data will be analyzed using bioinformatic pipelines. Metabolomic analyses will be carried out on stool and urine samples; samples will be analyzed for volatile organic compounds (VOCs), which will be thermally desorbed and immediately transferred to the heated injection port (220 °C) of a Clarus 680 gas chromatograph (Perkin Elmer, Beaconsfield, UK) equipped with an Rtx-WAX column (30 m × 0.25 mm i.d., 0.25 µm film thickness) (Restek) and coupled to a Clarus SQ8MS (Perkin Elmer), with source and transfer line temperatures maintained at 250 and 210 °C, respectively. Optional visits Enrolled subjects will be asked, on a completely optional and voluntary basis that does not affect participation in the study, whether they agree to undergo two outpatient sigmoidoscopies with cold biopsies. One sigmoidoscopy will be performed after the screening visit and before the start of the study; the second will be performed at the end of treatment. Flexible sigmoidoscopy is performed by passing an endoscope into the rectum and advancing it into the sigmoid colon. To obtain small tissue samples (1-2 mm), cold biopsy forceps are used. Biopsy samples will be placed on dry ice for research purposes. Analyses of colon mucosal samples will not provide additional clinically relevant information for participants or their family members. Subjects enrolled in the study may refuse to undergo sigmoidoscopy and the related biopsies. Sigmoidoscopy and colon mucosal biopsies are associated with some risks: Less likely: Bleeding from biopsies Abdominal cramps due to the air used during flexible sigmoidoscopy Rare but serious: Colon perforation Subjects will receive the reports of routine blood tests, Fibroscan, bioimpedance analysis, DEXA and, if performed, sigmoidoscopy \[1\](comet://newtab/)