Fatty Liver in Obesity: Long-lifestyle Follow-up (FLiO) (FLiO)

Non-alcoholic Fatty Liver Disease (NAFLD) in Overweight and Obese People Under Nutritional and Lifestyle Follow-up: a Randomized Controlled Trial

Non-alcoholic fatty liver disease (NAFLD) is a condition of excessive hepatic lipid accumulation in subjects that consume less than 20g ethanol per day, without other known causes as drugs consumption or toxins exposure. In Western countries, the rate of this disease lies about 30% in the general adult population. The process of developing NAFLD can start from simple steatosis to non-alcoholic steatohepatitis (NASH), which eventually can lead to cirrhosis and hepatocellular carcinoma in the absence of alcohol abuse. Liver biopsy is considered the "gold standard" of steatosis, fibrosis and cirrhosis. However, it is rarely performed because it is an invasive procedure and investigators are focusing in the application of non-invasive liver damage scores for diagnosis. The pathogenesis of NAFLD is multifactorial and triggered by environmental factors such as unbalanced diets and overnutrition as well as by lack of physical activity in the context of a genetic predisposition. Nowadays, the treatment of NAFLD is based on diet and lifestyle modifications. Weight loss, exercise and healthy eating habits are the main tools to fight NAFLD. Nevertheless, there is no a well characterized dietary pattern and further studies are necessary. With this background, the general aim of this project is to increase the knowledge on the influence of nutritional/lifestyle interventions in obese patients with NAFLD, as well as contribute to identify non-invasive biomarkers/scores to early diagnosis of this pathology in future obese people.

This project is framed within the promotion of health and lifestyles and, specifically, in liver disorder linked to obesity (FLiO: Fatty Liver in Obesity). The investigation addresses a randomized, parallel, long-term personalized nutritional intervention with two strategies: 1) Control diet based on American Heart Association (AHA); 2) Fatty Liver in Obesity (FLiO) diet based on previous results (RESMENA project).The diet is based on macronutrient distribution, quality and quantity, and is characterized by a low glycemic load, high adherence to the Mediterranean diet and a high antioxidant capacity, with the inclusion of anti-inflammatory foods. It also takes into account the distribution of food throughout the day, number of meals, portion sizes, timing of meal, individual needs, dietary behavior (behavioral therapy: eat slowly, teach what to buy, what to eat, when to eat). The participants are instructed to follow this strategy. This strategy (RESMENA) was even more effective than AHA after 6 months follow-up, in terms of significant reduction of abdominal fat and blood glucose level. In addition, this diet had beneficial effects for participants who were obese and had values of altered glucose, reducing significantly in RESMENA participants LDL-oxidized marker. These results are very important to apply in the present investigation since that patients with NAFLD are commonly insulin resistant. Both strategies were designed within a hypocaloric dietary pattern (-30%) in order to achieve the American Association for the Study of Liver Diseases (AASLD) recommendations for the management of non-alcoholic liver disease (loss of at least 3-5% of body weight appears necessary to improve steatosis, but a greater weight loss, up to 10%, may be needed to improve necroinflammation). At this time the participants are individually supervised and encouraged to follow with the dietary planning instructions assigned. Furthermore, at baseline, 6, 12 and 24 months anticipated variables are obtained. Both dietary groups receive routine control (weight, body composition, strategy adherence) and dietary advice daily by phone (if they need help) and face to face at the time of routine control. In order to get a integral lifestyle intervention, all participants will be encouraged to follow a healthy lifestyle. Thus, physical activity will be recorded in each dietary group. The specific tasks: To recruit and select patients with the adequate characteristics to validate the conclusions reached. To develop and adequately transmit to each patient a personalized strategy according to the group randomly assigned ( AASLD vs FLiO strategy). To check the degree of adherence to the strategy set by regular monitoring: semiquantitative questionnaires of food consumption frequency, pedometers, accelerometers, weight control, satiety. To assess the effect of each strategy on body composition (weight, waist circumference, body fat, muscle mass, bone mineral density), physical status, general biochemistry (lipid profile, glycaemic profile, albumin, blood count, transaminases), specific biomarkers/metabolites in blood or urine (inflammation, oxidative stress, liver damage, appetite, psychological status), quality of life and related factors (anxiety, depression and sleep). To check the evolution of the liver damage, using non-invasive techniques (ultrasound, elastography and magnetic resonance imaging (MRI), metabolomics analysis) and calculating different validated liver scores from the data obtained with each strategy. To compare the effectiveness of strategies, considering not only the ability to decrease body fat, but also other risk factors present in the NAFLD patient such as insulin resistance and cardiovascular risk, which will result in improvement of liver damage. To analyze SNPs (DNA from oral epithelial cells) and the association with NAFLD (diagnosis and response to the strategies). To study gene expression (mRNAs) and microRNAs in white blood cells for identifying biomarkers of diagnosis and response to dietary strategy. To analyze gene DNA methylation patterns in white blood cells for identifying biomarkers of diagnosis and response to dietary strategy. To describe the intestinal microbiota composition by 16s sequencing at baseline and after nutritional intervention for diagnosis and response.

NAFLD, NASH, Obesity, AASLD, AHA, Energy-restricted diet, Lifestyle intervention, Mediterranean diet, Weight loss, Liver damage, Inflammation, Oxidative stress, Microbiota composition, Eating behaviour, Satiety, Physical activity, Sedentary behaviours, Sleep quality, Sleep duration, Psychological factors, Nutrigenomics, Nutrigenetics, Transcriptomics, Epigenetics, Metabolomics, miRNAs, Lipidomics

A conventional and balanced diet based on American Heart Association (AHA) guidelines and lifestyle advice to achieve the objective of American Association for the Study of Liver Diseases (AASLD): loss of at least 3-5% of the initial body weight and up to 10% needed to improve necroinflammation.

A mediterranean dietary strategy based on macronutrient distribution (quantity and quality), antioxidant capacity, meal frequency, dietary behaviour and lifestyle advice to achieve the objective of AASLD: loss of at least 3-5% of the initial body weight and up to 10% needed to improve necroinflammation.

The participants follow a conventional and balanced distribution of macronutrients (30% fat, 15% protein, 55% carbohydrates), adequate fiber (25-30 g/day) and dietary cholesterol (<250 mg/day) intake according to AHA guidelines. This strategy was included within a personalized energy-restricted diet (-30% individual needs) under healthy lifestyle advice in order to achieve the objectives of AASLD (loss of at least 3-5% of the initial body weight and up to 10% needed to improve necroinflammation).

The participants follow a strategy based on a distribution of macronutrients 30-35% lipid (extra virgin olive oil and fatty acids Ω3 in detriment of saturated, trans and cholesterol)/ protein 25% (vegetable against animal)/carbohydrates 40-45% (low glycaemic index, fiber 30-35 g/day); high adherence to the Mediterranean diet and natural antioxidants; meal frequency of 7 meals/day; size/composition of the ration suitable for each moment; including traditional foods with no additional economic cost that will allow diet adherence without abandonment; avoid inappropriate mealtimes and the eating manners as the eating rate. The participants are instructed to follow this strategy within a personalized energy-restricted diet (-30%) and under healthy lifestyle advice to achieve AASLD objectives.

Serum free fatty acids, triglycerides, total cholesterol, LDL cholesterol and HDL cholesterol concentrations will be measured in a fasting state

Serum free fatty acids, triglycerides, total cholesterol, LDL cholesterol and HDL cholesterol concentrations will be measured in a fasting state

Serum free fatty acids, triglycerides, total cholesterol, LDL cholesterol and HDL cholesterol concentrations will be measured in a fasting state

Serum aspartate aminotransferase, alanine aminotransferase, gamma-glutamyltransferase, total bilirubin, direct bilirubin, alkaline phosphatase, creatinine, total protein, albumin, prothrombin will be measured in a fasting state

Serum aspartate aminotransferase, alanine aminotransferase, gamma-glutamyltransferase, total bilirubin, direct bilirubin, alkaline phosphatase, creatinine, total protein, albumin, prothrombin will be measured in a fasting state

Serum aspartate aminotransferase, alanine aminotransferase, gamma-glutamyltransferase, total bilirubin, direct bilirubin, alkaline phosphatase, creatinine, total protein, albumin, prothrombin will be measured in a fasting state

Plasma antioxidant capacity will be assessed by measuring the ferric reducing ability of plasma (FRAP)

Plasma antioxidant capacity will be assessed by measuring the ferric reducing ability of plasma (FRAP)

Plasma antioxidant capacity will be assessed by measuring the ferric reducing ability of plasma (FRAP)

White blood cell count includes: Leucocytes, Neutrophils, Lymphocytes, Monocytes, Eosinophil, Basophils.

White blood cell count includes: Leucocytes, Neutrophils, Lymphocytes, Monocytes, Eosinophil, Basophils.

White blood cell count includes: Leucocytes, Neutrophils, Lymphocytes, Monocytes, Eosinophil, Basophils.

Red blood cell count, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, red cell distribution width, platelet count, platelet distribution width, mean platelet volume, plateletcrit

Red blood cell count, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, red cell distribution width, platelet count, platelet distribution width, mean platelet volume, plateletcrit

Red blood cell count, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, red cell distribution width, platelet count, platelet distribution width, mean platelet volume, plateletcrit

Peripheral Dopamine concentration will be analysed using high-performance liquid chromatography (HPLC)

Peripheral Dopamine concentration will be analysed using high-performance liquid chromatography (HPLC)

Peripheral Dopamine concentration will be analysed using high-performance liquid chromatography (HPLC)

Peripheral Serotonin concentration will be analysed using high-performance liquid chromatography (HPLC)

Peripheral Serotonin concentration will be analysed using high-performance liquid chromatography (HPLC)

Peripheral Serotonin concentration will be analysed using high-performance liquid chromatography (HPLC)

Peripheral Noradrenaline concentration will be analysed using high-performance liquid chromatography (HPLC)

Peripheral Noradrenaline concentration will be analysed using high-performance liquid chromatography (HPLC)

Peripheral Noradrenaline concentration will be analysed using high-performance liquid chromatography (HPLC)

Peripheral 5-hydroxyindoleacetic acetic concentration will be analysed using high-performance liquid chromatography (HPLC)

Peripheral 5-hydroxyindoleacetic acetic concentration will be analysed using high-performance liquid chromatography (HPLC)

Peripheral 5-hydroxyindoleacetic acetic concentration will be analysed using high-performance liquid chromatography (HPLC)

Inclusion Criteria: Overweight or obese Diagnosis of NAFLD Age: 30-80 years Female / Male Exclusion Criteria: Known liver disease (other than NAFLD) Abuse of alcohol (>21 and >14 units of alcohol a week for men and women, respectively, eg 1 unit = 125 mL of wine); Drug treatments: immunosuppressants, cytotoxic agents, systemic corticosteroids, agents potentially causing fatty liver disease or abnormal liver tests or weight modifiers Active cancer or a history of malignancy in the last 5 years Problems of massive edemas Obesity known endocrine origin (except treated hypothyroidism) Surgical procedure for weight loss ≥ 3kg weight loss in the last 3 months Severe psychiatric disorders Lack of autonomy or inability to follow the diet (including food allergies or intolerances) or/and lifestyle recommendations as well as to follow scheduled visits. Consumption of any type of food supplements (antioxidants, prebiotics, probiotics, etc.)

Centre for Nutrition Research, University of Navarra. CIBER Obesity and Physiopathology of Nutrition (CIBERobn), Institute of Health Carlos III, Madrid, Spain

Centre for Nutrition Research, University of Navarra. CIBER Obesity and Physiopathology of Nutrition (CIBERobn), Institute of Health Carlos III, Madrid, Spain

Centre for Nutrition Research, University of Navarra. CIBER Obesity and Physiopathology of Nutrition (CIBERobn), Institute of Health Carlos III, Madrid, Spain

Centre for Nutrition Research, University of Navarra. CIBER Obesity and Physiopathology of Nutrition (CIBERobn), Institute of Health Carlos III, Madrid, Spain

Centre for Nutrition Research, University of Navarra. CIBER Obesity and Physiopathology of Nutrition (CIBERobn), Institute of Health Carlos III, Madrid, Spain

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