Nutritional therapy for non-alcoholic fatty liver disease Paola Dongiovanni, Claudia Lanti, Patrizia Riso, Luca Valenti PII: DOI: Reference:

S0955-2863(15)00225-9 doi: 10.1016/j.jnutbio.2015.08.024 JNB 7438

To appear in:

The Journal of Nutritional Biochemistry

Received date: Revised date: Accepted date:

21 July 2015 26 August 2015 26 August 2015

Please cite this article as: Dongiovanni Paola, Lanti Claudia, Riso Patrizia, Valenti Luca, Nutritional therapy for non-alcoholic fatty liver disease, The Journal of Nutritional Biochemistry (2015), doi: 10.1016/j.jnutbio.2015.08.024

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ACCEPTED MANUSCRIPT Nutritional therapy for non-alcoholic fatty liver disease Paola Dongiovanni1, Claudia Lanti2, Patrizia Riso2*, Luca Valenti1,3 Internal Medicine and Metabolic Diseases, Fondazione IRCCS Ca’ Granda Ospedale

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Maggiore Policlinico, 20122 Milano, Italy

Department of Food, Environmental and Nutritional Sciences (DeFENS), Division of

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Human Nutrition, Università degli Studi di Milano, 20133 Milano, Italy Department of Pathophysiology and Transplantation (DEPT), Università degli Studi di

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Milano, 20122 Milano, Italy

Corresponding author

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Patrizia Riso - Division of Human Nutrition, Department of Food, Environmental and Nutritional Sciences (DeFENS), Università degli Studi di Milano, 20133 Milano, Italy

tel: + 39 02 50316726

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fax: + 39 02 50316721

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e-mail: [email protected]

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ACCEPTED MANUSCRIPT Abstract Following the epidemics of obesity, nonalcoholic fatty liver disease (NAFLD) has become the leading cause of liver disease in Western countries. NAFLD is the hepatic

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manifestation of metabolic syndrome and may progress to cirrhosis and hepatocellular

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carcinoma. To date, there are no approved drugs for the treatment of NAFLD and the main clinical recommendation is lifestyle modification, including increase of physical

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activity and the adoption of a healthy eating behavior. In this regard, studies aimed to elucidate the effect of dietary interventions and the mechanisms of action of specific food

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bioactives are urgently needed.

The present review try to summarize the most recent data evidencing the effects of

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nutrients and dietary bioactive compounds intake (i.e. long-chain PUFA, vitamin E, vitamin D, minerals and polyphenols) on the modulation of molecular mechanisms leading to fat accumulation, oxidative stress, inflammation and liver fibrosis in NAFLD

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patients.

Keywords: Nonalcoholic fatty liver disease (NAFLD), food bioactives, molecular

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mechanisms, in vitro studies, animal models, clinical trials

Running title: Mechanisms of food bioactives in NAFLD

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ACCEPTED MANUSCRIPT 1. Introduction Nonalcoholic fatty liver disease (NAFLD), also known as hepatic steatosis, is defined by liver fat deposition in the absence of excessive alcohol intake [1]. Following the

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epidemics of obesity, NAFLD has become the leading cause of liver disease (prevalence

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20-34%) [2, 3] and it is epidemiologically associated with the metabolic syndrome and insulin resistance (IR) [4-6]. NAFLD is an umbrella term used to described a histological

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spectrum ranging from simple steatosis, defined by a concentration of hepatic triglycerides (TG) exceeding 5% of liver weight, to nonalcoholic steatohepatitis (NASH)

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characterized by hepatocellular damage, lobular necroinflammation and fibrogenesis [7, 8]. NASH may evolve to cirrhosis and then to end stage liver failure or hepatocellular

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carcinoma [9, 10]. Genetic variants plays a major role in disease predisposition [11] by interacting with nutritional and other environmental factors, typically hypercaloric diet and lack of physical activity. To date, there are no approved drugs for the treatment of

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NAFLD and the main clinical recommendation as an initial step is lifestyle modification. Systematic reviews on the role of specific nutrients and phytochemicals on NAFLD and

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related outcomes have recently been published [12, 13]. In this review, we will specifically focus on the mechanism by which selected macro- / micro-nutrients and food

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bioactives exert a beneficial effect on the hepatic outcomes of NAFLD.

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2. Pathophysiology of NAFLD Fatty liver results from an unbalance between TG accumulation and removal and represents the safest way to store free fatty acids (FFAs) in the liver [6, 14]. Excess hepatocellular TG derives from several sources including dietary fatty acids, increased peripheral lipolysis due to adipose tissue IR and elevated hepatic de novo lipogenesis due to hyperinsulinemia. Indeed, the major determinant of NAFLD is systemic insulin resistance [4, 15]. Reduction of lipid secretion through very low-density lipoproteins (VLDL) and a decreased fatty acids oxidation are also involved in hepatic fat accumulation [5]. The development of NASH has been explained by the occurrence of multiple so-called “second-hits” leading to the activation of inflammation in the context of hepatic steatosis [16, 17]. The initial hit leading to the development of fatty liver, renders hepatocytes

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ACCEPTED MANUSCRIPT susceptible to other multiple hepatotoxic insults including: a) peroxidation, b) oxidative stress secondary to free radicals produced during - and omega- oxidation of FFAs, c) inflammation triggered by endotoxin engaging Toll-like receptor-4 in Kupffer cells and

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hepatocytes due to increased intestinal permeability, d) qualitative and quantitative

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changes in gut microbiota [18, 19], e) hepatic stellate cells activation, f) mitochondrial dysfunction. All these conditions lead in the end to inflammation, cellular damage, and

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activation of fibrogenesis [20].

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3. NAFLD management

The usual management of NAFLD includes lifestyle counseling to achieve a gradual

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weight reduction and an increase in physical activity. Patients are encouraged to lose ≥8% of their body weight. An intensive lifestyle intervention focused on diet, exercise and behavior modification with a goal of 7-10% weight reduction leads to significant

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improvement in liver histology in patients with NASH [21]. Indeed, weight loss improves steatosis [22], reduces hepatic inflammation and hepatocellular injury [21, 23], and

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improves cardiovascular risk profile. However, weight loss through energy restriction is difficult to achieve and sustain [24]. Physical activity and exercise also effectively

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decrease steatosis. Cross-sectional and prospective studies have shown that physical activity decreases intrahepatic lipids [25, 26]. Both aerobic and resistant exercises have

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been shown to improve liver function, independently of weight loss [27-29]. In addition to total energy intake, the composition of the diet also affects the metabolic and endocrine functions and overall energy balance [30]. Most recommendations encourage the consumption of diets rich in fruits and vegetables for prevention of chronic disease, and NAFLD is not exception. Such diets would provide significant amount of bioactive components with known beneficial effects due in part to their antiinflammatory properties [31]. General recommendations include a reduction in the intakes of total fat, saturated fatty acids (SFAs), trans fatty acids, and fructose. Indeed, high fructose intake has been associated with increased risk of NAFLD and liver damage [32-34]. Dietary fructose (consumed in the form of soft drinks) has been implicated in the pathogenesis of NAFLD [35]. Mice with ad libitum access to fructose solution showed significantly higher levels 4

ACCEPTED MANUSCRIPT of hepatic lipid accumulation, lipid peroxidation and endotoxin levels in the portal blood compared to controls and mice fed with glucose solution [36]. Conversely, an increase in the intakes of polyunsaturated fatty acids (PUFAs) and

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include long-chain n-3 fatty acids to reduce the risk of NAFLD.

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monounsaturated fatty acids (MUFAs) is advised. Moreover, there is recommendation to

A few trials were conducted to evaluate the impact of specific dietary patterns on liver

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damage in patients with NAFLD. In this regard, Mediterranean diet led to similar weight loss, but induced a more marked reduction of liver enzymes and of insulin resistance

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compared to a low-fat high carbohydrate diet [30]. Indeed, the diet of patients with NASH is usually enriched in saturated fat and cholesterol, whereas it is poor in

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polyunsaturated fat, fibers and antioxidant vitamins C and E [31]. In addition to an imbalance in fat intake, higher odds of inflammation were associated with higher carbohydrate intake in NASH patients [37].

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Apart from lifestyle modification, statins (lipid-lowering drugs), glitazones (insulin sensitizers), antioxidants and metformin have been used as therapies for NAFLD [38,

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39]. Glitazones improve steatosis at the expense of an increase of weight and the longterm safety of their utilization is still not clear. Randomized clinical trials with

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antioxidants (Vitamin E and N-acetylcysteine) have given conflicting results, suggesting that their effect may be different depending on age, dosage and lifestyle modifications

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[39]. A few studies have tested metformin in nondiabetic patients, but with inconsistent results [40, 41].

All these findings emphasize the difficulties to achieve success in NAFLD clinical setting and attract attention to the importance of alternative approaches for the prevention of liver damage progression in NAFLD.

4. Promising food bioactives In this review, we will focus our attention on the most promising bioactive compounds studied in the last years for their possible beneficial effects on the prevention and treatment of NAFLD. We have selected the compounds that have been most investigated in in vitro and in vivo studies, especially if there is accompanying evidence of efficacy clinical trials. Evaluated bioactives and their putative mechanisms of action are listed in

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ACCEPTED MANUSCRIPT table 1. In the following paragraphs, we will review the most important evidence supporting their activity, and discuss the evidence supporting the mechanisms of their

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beneficial effects.

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4.1 Omega-3 PUFAs

Long-chain omega-3 (n-3) fatty acids have been proposed as potential treatment for

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NAFLD. These fatty acids are present in large quantities in fish oil, flaxseed and some nuts. They can be synthesized in vivo by the human body from -linolenic acid and

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mainly occur as eicosapentaenoic acid (EPA) and decosahexaenoic acid (DHA), which are both anti-inflammatory. Omega-3 PUFA supplementation ameliorates hepatic

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steatosis in animal models and in human studies [42, 43]. Clinical trials, investigating the therapeutic effect of omega-3 in patients with NAFLD suggested beneficial effects on hepatic fat accumulation, liver function tests, fasting

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blood glucose and serum triglycerides [44-46]. A randomized, double-blind, placebocontrolled trial of DHA, EPA, or DHA+EPA supplementation has shown that EPA

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enrichment in the peripheral blood is linearly associated with decreased liver fat percentage in patients with NAFLD [47]. Two controlled clinical trials performed in

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NAFLD children also demonstrated that omega-3 supplementation for 6-24 months reduced hepatic steatosis, IR, circulating TG and ALT levels [48, 49]. Moreover, a recent

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meta-analysis confirmed the beneficial effect of omega-3 on steatosis [50]. Conversely, evidence about necroinflammation and fibrosis progression in NASH after omega-3 supplementation is still lacking [51]. However, clinical studies are ongoing, and there is a strong mechanistic rationale for supporting such an effect. Indeed, DHA specifically binds with high affinity to the G protein-coupled receptor 120 (GPR120) that mediates potent insulin sensitizing effects in vivo by repressing macrophage-induced tissue inflammation [52]. In pediatric NAFLD, DHA treatment reduced liver damage, the number of inflammatory macrophages and increased GPR120 expression in hepatocytes. Modulation of GPR120 plays a key role in the regulation of the cell-to-cell cross-talk that drives inflammatory response, hepatic progenitor cell activation and hepatocyte survival [53, 54].

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ACCEPTED MANUSCRIPT In HepG2 hepatoma cells, the expression of fatty acid synthase (FAS) and sterol regulatory element binding protein 1c (SREBPC1) involved in de novo lipogenesis, were suppressed by DHA or EPA supplementation [55]. Moreover, PUFA supplementation

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modulated the antioxidant defense increasing SOD, GST and GPX activity [56].

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In high fat diet (HFD) fed mice, dietary intake of EPA reduced steatosis by reducing hepatic cholesterol, TG and FFAs [57]. Moreover, EPA intake seems to abrogate HFD-

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induced modulation in genes involved in hepatocellular lipid metabolism. These include upregulation of Srebp-1c, which induces the lipogenic program, FAS and acyl-

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coenzymeA-carboxylase-1 (ACC1) and the decrease of expression of carnytoil-palmitatetransporter-1 (CPT1), which transports FFAs to the mitochondria and promote -

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oxidation [58]. Several studies have demonstrated that EPA decreases steatosis and fibrosis progression by reducing TG synthesis and the expression of fibrogenic genes, and indeed it represents an established treatment for hypertriglyceridemia [59, 60]. EPA

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supplementation is associated with decreased hepatic ROS production and activation of AMP-activated protein kinase (AMPK) and Peroxisome-proliferator activated receptor-

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(PPARwhich stimulates lipid catabolism. In mice fed HFD and steatogenic choline deficient diets, DHA supplementation reduced hepatic steatosis, inflammation, fibrosis

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and lipid peroxidation [61, 62]. Increased activity of superoxide dismutase (SOD) and downregulation of Srebp-1c seem to account for the inhibitory effect of DHA. DHA was

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also found to ameliorate hepatic steatosis through the downregulation of other nuclear receptors involved in TG synthesis such as Peroxisome proliferator activated receptor  (PPAR and Retionid X receptor (RXR). Several unsaturated fatty acids, including DHA, have the capacity to specifically bind and activate the RXR, since PUFAs have been shown to fit into the ligand-binding pocket of the RXR crystal [63]. Finally, both DHA and EPA are able to restore adiponectin levels which contribute to improve hepatic insulin sensitivity [64]. In summary, omega-3 fatty acids may be a possible therapy for NAFLD. They have several potential mechanisms of action, the most relevant is the regulation of hepatic gene expression, thereby switching intracellular metabolism from lipogenesis and storage to fatty acid oxidation and catabolism, and activation of anti-inflammatory pathways.

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ACCEPTED MANUSCRIPT 4.2 Vitamin E Vitamin E is the generic name for eight lipophilic isoforms: four tocopherols (α, β, γ, δToc) and four tocotrienols (α, β, γ, δ-T3). Toc is present in a variety of foods as vegetable

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oil and nuts whereas T3-containing foods are limited as palm oil and cereal grains [65].

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Both Toc and T3 have a chroman ring structure with an isoprene side chain. Toc have a saturated isoprene side chain, conversely, T3 have three unsaturated bonds in the side

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chain [66]. Although all forms of Vitamin E have an antioxidant effect on lipid peroxidation, the most important for human health are α and γ-Toc due to their dietary

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abundance. Despite all the isoforms reach the liver, only α-Toc binds α-tocopherol transfer protein (α-TTP) that is located in the cytosol of hepatocytes. Afterwards, it is

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incorporated into nascent VLDLs and released into the blood circulation [67]. Since oxidative stress is a major feature of NASH [68-70], vitamin E has been investigated in this condition. Clinical trials with vitamin E supplementation in NASH patients yielded

Steatohepatitis)

trial,

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promising results. In the PIVENS (Pioglitazone, Vitamin E or Placebo for Non-alcoholic high

dose

Vitamin

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(800

IU/day)

reduced

hepatic

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necroinflammation and facilitated resolution of NASH, as compared to placebo, but there was not an improvement in fibrosis score [71]. A combination of atorvastatin, vitamin C

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and high dose vitamin E for 4 years, reduced hepatic steatosis (by 71%) in subjects with NAFLD [72]. In another study, twelve patients with NASH and 10 with NAFLD received

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300 mg/day α-toc for 1 year. Steatosis, inflammation and fibrosis were improved after αtoc treatment in NASH patient [73]. Few data are available on the effect of antioxidants/vit E in paediatric NAFLD. Diet and physical exercise in biopsy-proven NAFLD children led to a significant improvement of liver function and glucose metabolism beyond any antioxidant therapy [74]. In the TONIC (Treatment of NAFLD in Children) trial, Vitamin E improved hepatocellular ballooning and resolved NASH more frequently compared to placebo [75]. However, it should be noted that a meta-analysis of randomized controlled trials showed that high-dosage vitamin E is associated with an increase in total mortality, and vitamin E supplementation has been associated with increased risk of haemorrhagic stroke and prostate cancer [76]. Vitamin E may have pro-oxidant effect at high doses disrupting the natural balance of antioxidant systems and increasing vulnerability to oxidative damage.

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ACCEPTED MANUSCRIPT High-dose vitamin E supplements (> or =400 IU/d) have been reported to increase allcause mortality suggesting that the dose-dependent effect of vitamin E should be carefully considered [77].

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The mechanism of action is not limited to its antioxidant properties, determining reduced

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mitochondrial damage, but also to the indirect influence on other pathways as highlighted in in vitro and in vivo studies.

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Cultured human fibroblast and rat hepatic stellate cells treated with vitamin E showed a reduction of lipid peroxidation and the inhibition of collagen gene transcription [78, 79].

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In addition, T3 supplementation inhibited TG accumulation in human and mouse hepatoma cells through the down- and up-regulation of genes involved in lipogenesis and

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-oxidation respectively. In particular, γδT3 reduced Srebp2 and Apoliprotein B100 (ApoB100) enhancing VLDL efflux [80]. The uptake of fatty acids in the liver is crucial for the establishment and development of NAFLD. In a recent study, the expression of

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the fatty acid carrier CD36 was reduced by both α Toc and atorvastatin [72, 81]. In rats fed HFD, vitamin E reduced oxidative stress, protein nitrosylation and tissue TNF-

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alpha levels [82]. A 5-week dietary supplementation with either α- or γ-Toc in genetically obese Lepob/obmice decreased LPS-triggered lipid peroxidation, inflammation and hepatic

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damage [83]. Moreover, vitamin E inhibited hepatic TGF-β1 gene expression and protected against liver fibrosis in rats [84]. The amelioration of steatohepatitis and the

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reduction in lipid peroxidation were also observed in mice and rats fed a methionine choline deficient diet [85]. Conversely, in Wistar rats fed methionine choline deficient diet supplemented with vitamin E, despite the reduction of lipid peroxidation, infiltration of inflammatory cells, lipid deposition and fibrosis were not prevented [67]. In conclusion, vitamin E supplementation could be considered a therapeutic tool in NAFLD management. However, the optimal benefit–risk ratio has to be determined for the specific individual.

4.3 Vitamin D Vitamin D refers to a group of fat-soluble secosteroid hormones which are involved in the regulation of mineral and skeletal homeostasis. Vitamin D derives from both dietary sources, such as oily fish (vitamin D2) and from dermal synthesis (Vitamin D3). In the

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ACCEPTED MANUSCRIPT skin, ultraviolet radiation converts 7-dehydrocholesterol to pre-vitamin D3 and then to vitamin D3. In the liver, vitamin D is metabolized to 25-hydroxyvitamin D [86] or calcidiol. Calcidiol is then transported to the kidney where is further hydroxylated to the

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active form (1α,25-dihydroxyvitamin D) or calcitriol [87]. The production of calcitriol is

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regulated by hormonal cues (such as parathyroid hormone and FGF-19), and serum calcium and phosphate levels [88].

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Besides calcium and bone homeostasis, Vitamin D also regulates cell proliferation and differentiation, and has immunomodulatory, anti-inflammatory and anti-fibrotic

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properties. Evidence is accumulating that vitamin D deficiency contributes to the development of IR and NAFLD [86]. This is relevant since up to 55% of adolescents in

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the U.S. were reported to be vitamin D deficient [89]. Obese children are more likely to be sedentary with reduced sunlight exposure and often they consume caloric foods low in vitamin content [90, 91]. Moreover, vitamin D levels are related to the histological

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severity of steatosis, necroinflammation and fibrosis [87, 92-95]. However, short-term vitamin D supplementation did not consistently improve NAFLD

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histological features or dyslipidemia in affected subjects [96], while longer-term supplementation was associated with reduced inflammation and lipid peroxidation [97].

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The biological activity of calcitriol is mediated by binding and transactivation of the nuclear Vitamin D receptor (VDR) [98, 99]. In hepatoma cells, VDR directly induces

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detoxifying enzymes and regulate bile acid homeostasis [100]. Vitamin D could avoid excessive inflammatory response in hepatic macrophages since these immune cells express both VDR and 1α hydroxylase [101]. Hepatic stellate cells are the main producers of extracellular matrix components playing a pivotal role in liver fibrosis. VDR is expressed in HSCs and orchestrates myofibroblast trans-differentiation and the fibrogenic program. VDR ligands inhibit HSC activation by TGF and abrogate fibrotic gene expression whereas VDR knockout mice spontaneously develop hepatic fibrosis. Mechanistic studies revealed that activation of VDR signalling antagonizes a wide range of TGFβ/SMAD-dependent transcriptional responses on profibrotic genes in HSCs, suggesting that the dynamic VDR/SMAD circuit could represents a possible target for anti-fibrotic therapy [102]. In vivo studies tried to investigate further applications of vitamin D as therapy in NAFLD. In rodents, vitamin D signaling was

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ACCEPTED MANUSCRIPT involved in maintaining hepatic lipid homeostasis and vitamin D depletion promoted NASH development [103, 104]. On the other hand, Vitamin D supplementation attenuated HFD-induced hepatic steatosis in a dose-dependent manner along with

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improvement in serum lipid profile, by decreasing lipogenesis and promoting FFAs

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oxidation [105].

Although the potential use of vitamin D in NAFLD has become more intriguing with

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preclinical model of steatosis, further studies are necessary to better clarify the biological

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activity of vitamin D and the clinical impact of supplementation.

4.4 Polyphenols

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Fruits, vegetable and beverages including fruit juices, wine, tea, coffee and chocolate are important sources of bioactive compounds such as polyphenols. Polyphenols are a group of phytochemicals mostly investigated for their potential role in

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the prevention and treatment of oxidative stress and inflammation. Polyphenols are secondary metabolites of plants; they are characterized by the presence of at least one

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aromatic ring in their structure, linked to different chemical group as phenolic, hydroxyl or carbon groups. They can be classified based on their source, biological function, and

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chemical structure.

Polyphenols are generally subdivided in flavonoids and non flavonoids depending on

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their chemical structure. Flavonoids are the most abundant in the diet and include flavonols (e.g. quercetin and kaempferol, flavones (eg. luteolin, apigenin), flavan-3-ols (eg. catechins), flavanones (eg. hesperetin, naringenin), isoflavones (eg. genistein), anthocyanidins (i.e. cyanidin, malvidin, pelargonidin, delphinidin, peonidin, petunidin) and proanthocyanidins (i.e. condensed tannins). Non flavonoids are represented by stilbenes, phenolic acids and hydroxycinnamates [106, 107].

Several in vitro and in vivo studies investigated polyphenols properties related to NAFLD despite there are few clinical evidences of the beneficial effect in NAFLD treatment. In a randomized, placebo-controlled, double-blind trial, participants received 250 mL of bayberry juice twice daily for 4 weeks. The consumption of polyphenols-rich bayberry juice reduced the levels of oxidative (i.e. protein carbonyl groups), inflammatory (i.e.

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ACCEPTED MANUSCRIPT TNFα and interleukin-8) and apoptotic (i.e. tissue polypeptide-specific antigen and cytokeratin-18 fragment M30) biomarkers in young individuals with NAFLD [108]. Another study demonstrated that an Hibiscus sabdariffa L. extract rich in polyphenols

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(1.43% of flavonoids, 2.5% anthocyanins and 1.7% phenolic acid), administered in

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capsules, was able to decrease body weight, serum FFAs and to improve liver steatosis in overweight subject [109].

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It must be considered that flavonoids are a very heterogenic group of compounds with numerous beneficial effects, indeed they could target different pathways possibly

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involved in the pathogenesis of liver diseases [110]. First of all, flavonoids could control de novo lipogenesis, inhibiting lipogenic (ACC, SREBP-1, FAS, LXRα) and increasing

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lypolitic enzymes (AMPK, PPARα, CPT-1). Secondly, flavonoids are very effective scavengers since they can protect or enhance the endogenous antioxidant defense. Lastly, flavonoids have anti-inflammatory properties inhibiting NFκB pathway [110]. In HepG2

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hepatoma cells, polyphenols reduce the activity of hydroxymethilglutaryl-CoA (HMGCoA) lyase [111] and the activity of acyl-coenzyme A diacylglycerol acyltransferase

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(DGAT), which catalizes the final step in TG synthesis [112]. In particular, within polyphenols family, quercitin inhibits TAG accumulation and promote cell proliferation

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in HepG2, while SOD, GPX and CAT activities are upregulated [113]. Primary rat hepatocytes treated with phenolic fraction show a down-regulation of ACC and

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hydroxymethilglutaryl-CoA reductase (HMGCR), regulating cholesterol synthesis, activities [114]. Caffeic acid addition in HepG2 cells induces hepatic lipolysis and reduce hepatic lipogenesis up-regulating AMPK, PPARα and decreasing ACC, SREBP-1 and FAS protein expression [115]. Concerning in vivo studies, the investigation of polyphenols properties have been performed using different poliphenols-rich extracts in heterogeneous animal models. In genetically obese db/db mice, a polyphenol extract reduced the activity of lipogenic enzymes involved in de novo fatty acid biosynthesis [116]. Moreover, in mice fed HFDdiet enriched in polyphenols the upregulation of fatty acid and TG synthesis-related genes (FAS, SCD1) was reversed [117]. In diet-induced obese C57BL/mice a polyphenol-rich Rutgers Scarlet Lettuce improves glucose metabolism, liver lipid accumulation and reduced TNF-α [118]. In mice with fatty liver, induced by orally supplementation of

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ACCEPTED MANUSCRIPT high-fat milk, a polyphenol-rich Chrysanthemum morifolium extract decreased lipid accumulation and hepatic PPARα gene expression [119]. In the light of this promising evidence, supplementation of polyphenols may represent a useful approach for the

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management of patients with NAFLD, but additional research is required to confirm this

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initial data.

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4.4.1 Anthocyanins

Anthocyanins (ACNs), a subclass of flavonoids, have been largely investigated for their

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potential protective effect in the prevention and treatment of different diseases. ACNs are the principal components of the red, blue and purple pigments of the majority of flower

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petals, fruits and vegetables such as blueberries, blackberries, raspberries, strawberries, blackcurrants, elderberries, grapes, cranberries, red cabbage, red radishes and spinach. ACNs in plants mainly exist in glycosidic forms, a total of more than 500 ACNs are

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known depending on the hydroxylation, methoxylation patterns on the B ring, and glycosylation with different sugar units [120]. The colour of ACNs is pH-dependent, i.e.,

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red in acidic and blue in basic conditions and they are chemically stable in acidic solutions [107]. To evaluate their beneficial effects on human health it must be

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considered that ACNs are rapidly metabolized and their presence in the circulation is limited to a few hours. Despite their low absorption and rapid metabolism, regular intake

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of ACNs may ameliorate hyperglycaemia, modulate endothelial function, and decrease inflammation [121]. Moreover, it has been investigated their role in the prevention of oxidative stress by scavenging reactive oxygen species and free radicals [122] and their role in the modulation of lipid metabolism and fat deposition [108, 123] in different tissues, including the liver. As we recently reviewed [121], ACNs can reduce hepatic lipid accumulation, but their impact on NAFLD has yet to be understood. Until now, only few clinical studies on humans are available and they diverge for ACNs source, doses and clinical features of patients. Suda et al. showed an effect of ACN intake (200 mg acylated ACNs from purple sweet potato) in the reduction of liver enzymes (e.g. gammaglutamyltransferases) in subjects with borderline levels of one or more hepatic markers [122]. In a recent study, NAFLD patients received either purified ACNs (320 mg/d) derived from bilberry and black currant or placebo for 12 weeks. Individuals receiving

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ACCEPTED MANUSCRIPT ACNs showed a decrease in plasma alanine aminotransferase, cytokeratin-18 fragment and myeloperoxidase, and an overall improvement of insulin resistance [123]. Several in vitro studies, performed mainly in HepG2 cells supplemented with oleic acid

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and/or palmitic acid, highlighted three different mechanisms of action by which ACNs

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could prevent the progression of liver dysfunction/damage: inhibition of lipogenesis (i.e. reducing SREBP1c), promotion of lipolysis (i.e. inducing PPARα activity with activation

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of AMPK pathway) and reduction of oxidative stress (i.e. induction of antioxidant enzymes). Mulberry ACNs (0.1, 0.3, 0.5 mg/mL) supplementation in HepG2 reduced

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lipogenesis (SREBP-1, FAS, ACC and A-FABP), cholesterol biosynthesis (SREBP-2 and HMGCR) and TG biosynthesis, and enhanced fatty acid β-oxidation (PPARα and CPT-1) [124]. Cyanidin 3-O-glucoside reduced cellular lipid concentration in HepG2 cells by

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rewiring the expression of genes involved in lipid metabolic pathway as PPARα [125], whereas in primary mice hepatocytes it decreased intracellular ROS production acting as

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free radical scavenger and enhanced PI3K/Akt activation [126]. In vivo studies were performed in different experimental models of NAFLD and

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metabolic syndrome and evaluated different outcomes as lipid metabolism, oxidative stress, and liver damage. Obese mice supplemented with 200 mg/Kg per day of

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anthocyanin fraction extracted from purple sweet potato showed a reduced hepatic fat accumulation associated with a decreased hepatic lipogenesis [127]. Moro juice with an

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ACN content of 85 mg/L administered ad libitum to mice prevented fatty liver suppressing LXR-α expression and activity [128]. Moreover 1 g of purified ACN from bilberry and blackcurrant administered to mice ameliorated hepatic steatosis, inflammation, oxidative stress as well as fibrosis [129]. Anthocyanin-rich bilberry (Vaccinium myrtillus L.) extract was tested in E3Leiden (E3L) mice fed high-fat/highcholesterol diet. The anthocyanin extract reduced NASH development, attenuating both steatosis and inflammation and reduced hepatic fibrosis. These effects were associated with a decreased hepatic free cholesterol accumulation and cholesterol crystal formation. On the basis of these data, ACN-rich food could be useful for the prevention of liver diseases as NAFLD, even if additional studies are needed to deeply characterize the molecular mechanism of the different extracts.

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ACCEPTED MANUSCRIPT 4.4.2 Silibinin Silymarin and its major costituent, Silibinin, are flavonoid compounds extracted from the medicinal plant Silybum marianum (milk thistle). Extracts have traditionally been used

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for the treatments of liver disease [130-134].

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Only few controlled randomized studies has been conducted in patients with NAFLD. These studies suggested that silymarin could reduce steatosis severity, liver ballooning

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and fibrosis, acting also as efficient insulin sensitizer and lowering aminotransferase levels in both short and long term trials [135]. In a study where patients received Vitamin

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E, L-gluthatione, L-cysteine, L-methionine and Silybum Marianum, it was observed that silymarin was effective in reducing the biochemical and ultrasonographic changes

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induced by NAFLD. These data were in agreement with those obtained by other authors [136]. Similarly, in a multicenter, double-blind clinical trial, patients with steatosis received silybinin combined with phosphatidylcholine and vitamin E for 12 months.

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Combined treatment was associated with an improvement in liver enzymes, insulin resistance and liver histology without increase in body weight [137]. In another recent

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study the intake of 210 mg/day silymarin orally for 8 weeks decreased hepatic enzymes in patients with NASH [138].

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Few in vitro study investigated silibinin properties. In hepatoma cells silibinin prevented lipid accumulation and resistin induction by fatty acids targeting the insulin signaling

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pathway [139]. In hepatic stellate cells isolated from human liver, silybin inhibited dosedependently cell proliferation, cell motility and de novo synthesis of extracellular matrix components. Silibinin was also confirmed to act as a potent anti-oxidant and antiinflammatory agent [140]. As concern in vivo studies, in obese db/db mice fed MCD silibinin decreased IR, serum ALT and NAFLD histological activity. This was associated with reduced oxidative stress and inflammation, due to lower isoprostanes, 8-OHG and TNF-α expression and restored mitochondrial reduced glutathion levels [141]. Again, in obese mice fed MCD, and in rats fed HFD, silibinin improved hepatic oxidative and nitrosative stress mediated by iNOS and inflammation modulating the expression and the activity of lipid metabolic enzymes [142]. This resulted in improvement in liver damage. In HFD rats, silibilin ameliorated IR

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ACCEPTED MANUSCRIPT mainly by reducing visceral fat, up-regulating lipolysis and inhibiting gluconeogenesis [143]. These findings are a first step in the comprehension of the plausible mechanisms of

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action of silibilinin, but further work is necessary to better characterize the possible use

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of these polyphenolic compounds.

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4.4.3 Resveratrol

Resveratrol (trans-3,4’,5-trihydroxystilbene) is a stilbene occurring naturally in several

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plants and provided in the diet by various food stuffs such as grapes, berries, red wine and nuts. Evidences have shown its health benefits, such as improvement of insulin

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sensitivity and glucose tolerance, reduction of serum lipids and suppression of inflammation and oxidative stress. Moreover, resveratrol is able to modify lipid metabolism and more specifically to induce a reduction in liver TG content [144]. Few

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studies were performed in humans with conflicting results. Recently, patients with NAFLD who received 1500-mg resveratrol capsule twice daily for 12 weeks had and

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improvement of liver enzymes, insulin resistance and inflammation [145]. Similarly, patients receiving 50 mg resveratrol twice daily for three months associated with lifestyle

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modification had a higher attenuation of inflammatory markers and hepatocellular apoptosis compared to placebo treatment [144]. Conversely, 8 weeks administration of

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3000 mg resveratrol led to increased liver enzymes in NAFLD patients [146]. Few in vitro studies investigated the potential hepatoprotective effect of resveratrol. In primary rat hepatocytes, resveratrol supplementation (25 µmol/L) reduced ACC activity [147]. In a study performed in HepG2 cells exposed to high concentration of glucose, resveratrol supplementation reduced triacylglycerol accumulation by increasing AMPK activity and down-regulating SREBP-1c and ACC activity [148]. In vivo studies revealed that resveratrol could reduce liver weight and TG content. In mice treated with 22.4 mg resveratrol/kg, histological examination revealed a reduced accumulation of large lipid droplets [149]. Recent studies in mice fed high fat diet showed that resveratrol protected the liver from fat accumulation by activating Sirt1 [150, 151]. Sirt1, a NAD dependent deacetylase, stimulates the activity of FOXO1 [152], which may in turn indirectly inhibit SREBP1 expression [153, 154], suggesting that the

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ACCEPTED MANUSCRIPT AMPK/Sirt1 axis could down-regulate genes of the lipogenic pathways (FAS, ACC) and up-regulate genes of the lypolitic pathway (PPARα, CPT-1). As concern the antioxidant activity, in Zucker rats supplemented with two different doses of resveratrol (15 and 45

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mg/kg body weight, it was able to reduce oxidative damage in liver measured as hepatic

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thiobarbituric acid and oxidized glutathione (GSSG) levels [153].

In conclusion, recent evidence demonstrated that resveratrol is effective in decreasing de

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novo lipogenesis in the liver and it could be used in the prevention of liver diseases.

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5. Minerals

It is generally recognised a progressive decay in the homeostasis of trace minerals in patients with NAFLD; this may reflects an increased oxidative stress and

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inflammation condition. In particular, minerals such as copper, selenium and iron have been investigated for their possible contribution to the development and treatment of liver

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diseases as NAFLD.

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5.1 Copper

Copper has a role in antioxidant defence, lipid peroxidation and mitochondrial function. deficiency

has

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Copper

been

linked

to

different

metabolic

disorders

as

hypercholesterolemia, increased blood pressure and glucose intolerance both in rodents

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[155, 156] and in humans [157]. Since inadequate copper availability may increase lipid accumulation in the liver it has been investigated its potential contribution to the development of NAFLD. Hepatic copper concentrations were reduced in NAFLD and inversely correlated with steatosis, NASH and IR [157]. Development of hepatic steatosis and IR in response to dietary copper restriction in rats suggests that copper availability has a causal role in the development of NAFLD [157]. Interestingly, fructose feeding exacerbates complications of copper deficiency. In rats, fructose consumption impaired copper status and precipitated copper deficiency possibly inhibiting its absorption through the intestinal epithelium. Moreover, copper deficiency and fructose appear to act together to accelerate hepatic fat accumulation and liver damage [156, 158]. According with histological findings, copper deficiency markedly suppressed CPT-1 and up-regulated FAS expression [158]. In addition, copper deficiency

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ACCEPTED MANUSCRIPT also contributed to an impaired antioxidant defence system considering that the activity of Cu/Zn superoxide dismutase depends on adequate copper availability [159].

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5.2 Selenium

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Selenium deficiency could be considered a dietary condition correlated with oxidative stress in patients with liver diseases [160]. Selenium levels have been associated with

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cardiovascular disease and its supplementation leads to decrease in total cholesterol and triglyceride levels [161]. Unfortunately, selenium status on NAFLD has not been yet

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investigated.

Human hepatoblastoma cells supplemented with selenite showed a reduced TGFβ1-

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induced collagen and IL-8 production and maximized the expression of antioxidant enzymes in response to FFAs overload [162]. In experimental models selenium supplementation decreased triglyceride levels, protected LDL from oxidation by restoring

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the antioxidant properties of the low density lipoprotein (LDL) associated enzyme Paraoxonase 1 [163].

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Considering that, in vitro and in vivo selenium supplementation has shown a potential effect in the reduction of oxidative stress, it is important to take into account the possible

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5.3 Iron

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clinical implication in subjects with liver disease who have selenium deficiency.

Iron is essential to the life of all mammalian organisms. It has a key role in oxygen transport and in enzymes involved in mitochondrial respiration, DNA biosynthesis and the citric acid cycle via the capability to change its redox state. However, this characteristic also renders excess iron detrimental, mostly via the formation of reactive oxygen species, which may lead to severe organ damage. Iron perturbations are frequently observed in patients with obesity, insulin resistance and NAFLD. The term Dysmetabolic Iron Overload Syndrome (DIOS) is now most frequently used to describe the typical association of hepatic steatosis with mild to moderate iron deposition in liver biopsies and increased serum ferritin in patients with NAFLD [164]. Hyperferritinemia is usually associated with NASH and the severity of liver damage whereas iron depletion, achieved by phlebotomy, in patients with mild iron overload

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ACCEPTED MANUSCRIPT could have beneficial effects more than lifestyle modifications alone in normalizing insulin resistance and liver enzymes [165, 166]. Moreover, iron depletion up-regulated glucose uptake and increased insulin receptor expression and signaling in hepatocytes in

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vitro and in vivo [167], whereas dietary iron supplementation induced IR and

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dyslipidemia [168]. To investigate the mechanisms underlying DIOS, we recently examined the effect of FFAs on hepatic iron metabolism. The main finding was that

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exposure of hepatocytes to FFAs, leading to steatosis, was associated with a subversion of iron metabolism characterized by increased expression of transferrin receptor, and

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facilitation of iron storage [169]. Additional studies are warranted to evaluate the

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potential of iron reductive therapy on clinical outcomes in NAFLD patients.

6. Conclusions

NAFLD, a disease caused by an unhealthy eating behaviour and lifestyle has become the

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leading cause of liver diseases in the Western countries. To date, there are no approved drugs for this indication and the main clinical recommendation as an initial step is

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lifestyle modification including both improvement of dietary pattern and increased physical activity. The identification of the molecular mechanisms leading to fat

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accumulation, oxidative balance impairment and liver fibrosis is expected to improve both diagnostic and therapeutic approaches. Food bioactive compounds, which modulate

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the activation of genes involved in lipogenesis, fibrogenesis, lipid peroxidation and inflammation represent a new attractive therapeutic approach for this condition.

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ACCEPTED MANUSCRIPT Acknowledgements The authors would like to thank Dr Cristian Del Bo’ and the Metabolic Liver Diseases Research Lab for careful reading of the manuscript and suggestions. Luca Valenti and

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Patrizia Riso designed the study and critically reviewed the paper literature and approved

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the final manuscript. Paola Dongiovanni wrote the paper draft and contributed to critical discussion of results. Claudia Lanti performed the literature search and contributed to the

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PT

ED

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writing of the paper draft. The authors declare no conflict of interest.

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ACCEPTED MANUSCRIPT Table 1. Food bioactives for the prevention of nonalcoholic fatty liver: promising compounds and mechanisms Nutrient/bioactive Experimental model

Mechanism ↓lipogenesis [55]

HepG2 cells

↑antioxidant defence system [56]

In vivo

↓inflammation [52],[61],[62]

HFD fed mice,

↓hepatic cholesterol, TG, FFAs [57]

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In vitro

ricefish medaka (Oryzias latipes) ↓lipogenesis [58, 64]

Omega-3

↑lipolysis [58]

Wistar rat

PUFAs

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↓Steatosis and fibrosis [59-62] ↓lipid peroxidation [61],[62]

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Patients

↓Steatosis, FFAs, fasting glucose [44, 45]

Adults and children with

↓IR, hepatic steatosis, , ALT ,[49]

NAFLD

↓ inflammatory macrophage [53],[54] ↓lipid peroxidation [78]

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In vitro

↓collagen up-regulation [79]

stellate cells, human and mouse

↓lipogenesis [80]

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human fibroblast, rat hepatic

↑β-oxidation [80, 170]

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hepatocellular carcinoma, Hepa

In vivo

↓CD36 receptor [81]

Wistar rats, obese (ob/ob) mouse

↓oxidative stress [82]

model of NASH, guinea pigs

↓lipid peroxidation [84, 85]

Vitamin E

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1-6, HepG2 cells

↓fibrogenesis[84], inflammation [82, 83], lipid uptake [81] ↓Steatosis, inflammation, fibrosis [72]

Patients Adults with NASH and NAFLD Children with NAFLD

Vitamin D

In vitro

↑detoxifying enzyme [100]

HepG2 cells

↓inflammation [103]

Hepatic stellate cells

↓FXR and LXR [100] ↓fibrosis [102]

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ACCEPTED MANUSCRIPT In vivo

↓lipogenesis [103]

Balb/C mice

↑lipolysis [103]

D-depleted rat ↓hs-CRP,↓ serum MDA[97]

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Patients

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Adults with NAFLD

↓TG synthesis [112],[113]

HepG2 cells

↓oxidative stress [113]

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In vitro

↑lipolysis [114],[115]

Primary rat hepatocyte

↓de novo lipogenesis [116]

In vivo db/db mice fed MCD mice fed HFD-diet

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Patients

↑glucose metabolism [118]

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Polyphenol

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↓lipogenesis [115]

↓liver lipid accumulation [118],[119] ↓oxidative stress, inflammation [108] ↓body weight, FFA, steatosis [109]

In vitro

↓lipogenesis [124]

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NAFLD patients

↑lipolysis [125]

Primary rat hepatocyte

↓oxidative stress [126]

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HepG2 cells

↓ROS production [126, 128] ↓lipogenesis [127]

In vivo

Obese mice

↓steatosis [128]

ApoE3 Leiden mice

↓inflammation, oxidative stress, fibrosis

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Anthocyanins

↓inflammation [118]

[129]

Patients

↓ liver enzymes [122]

Adults with NAFLD

↓oxidative stress, apoptosis [123]

In vitro

↓lipid accumulation, resistin[139]

HepG2

↓ inflammation, fibrogenesis [140]

HSC Silybin/silybinin

In vivo

↓IR, ALT [141]

db/db mice fed MCD

↓inflammation, oxidative stress [142]

obese fed MCD-diet mice

↓visceral fat, gluconeogenesis [143] ↑lipolysis [143]

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ACCEPTED MANUSCRIPT

↑liver enzymes and histology [137, 138]

Patients Adults with NAFLD

↓lipogenesis [148]

HepG2 cells

↓TG synthesis [147]

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In vitro

Primary rat hepatocyte

↓liver weight, TG accumulation [149]

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In vivo Resveratrol

↓lipogenesis [153]

Mice fed HFD-diet

↑lipolysis [154]

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Zucker rats

↓ liver enzymes, insulin resistance,

Patients

In vitro

inflammation [144, 145]

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Adults with NAFLD

↓fibrosis, inflammation [162]

HepG2 cells

↑antioxidant enzymes[162]

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Human hepatoblastoma (C3A)

Iron

In vivo

Copper deficiency

Rabbit

↓lipolysis, antioxidant system [159]

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↑trasferrin receptor [169]

↑lipogenesis [158]

Sprague-Dawley rats

Selenium ↓TG levels [163]

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Minerals

Selenium

↓cholesterol levels [161] ↑antioxidant defenses [163] Iron ↑IR and dyslipidemia [168]

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ACCEPTED MANUSCRIPT

Abbreviations: Nonalcoholic fatty liver disease (NAFLD), nonalcoholic steatohepatitis

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(NASH), necroinflammatory score (NAS), high fat diet (HFD), free fatty acids (FFAs),

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insulin resistance (IR), sterol regulatory binding protein (Srebp), fatty acid synthase (Fas), acetyl-CoA carboxylase (ACC), carnitinepalmitoyltransferase(CPT1), radical

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oxygen species (ROS), AMP-activated proten kinase (AMPK), peroxisome proliferatoractivated receptor (PPAR), cytochrome P450 3A4 (CYP3A4), transforming growth factor

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(TGF), tumor necrosis factor (TNF), cluster of differentiation 36 (CD36), Farnesoid X receptor (FXR), liver X receptor (LXR), C-reactive protein (CRP), malondialdehyde

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(MDA), necroinflammatory score (NAS), glutathione peroxidase (GPX), thioredoxin

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reductase (TrxR1).

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ACCEPTED MANUSCRIPT Figure 1. Molecular mechanisms explaining the hepatoprotective effect of food bioactives. Development of NAFLD/NASH is induced by different risk factors, such as Western-

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type diet, physical inactivity and genetic predisposition. In the presence of obesity and

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insulin resistance (IR) there is an increased flux of FFAs to the liver. These FFAs are stored as TG in lipid droplets leading to hepatic fat accumulation, or undergo -oxidation

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increasing oxidative stress and the inflammatory pathway.

The damaged hepatocyte leads to a further increase of inflammatory signalling (IL-1,

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TNFa, IL-6) and the recruitment of circulating and residual macrophages (Kupffer cells: KC).

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All of these mechanisms can directly induce the activation of hepatic stellate cells (HSC), the major cell type involved in extracellular matrix deposition and liver fibrosis. The bioactive compounds may exert beneficial effects on NAFLD development and

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progression by inhibiting lipogenesis, -oxidation of free fatty acids, inflammation and hepatic stellate cells activation. In the cartoon, we have listed the food bioactives

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indicating the putative mechanisms by which they may improve liver damage in NAFLD.

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Nutritional therapy for nonalcoholic fatty liver disease.

Following the epidemics of obesity, nonalcoholic fatty liver disease (NAFLD) has become the leading cause of liver disease in western countries. NAFLD...
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