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Non-alcoholic fatty liver disease: An update with special focus on the role of gut microbiota
Non-alcoholic fatty liver disease: An update with special focus on the role of gut microbiota Michael Doulberis, Georgios Kotronis, Dimitra Gialamprinou, Jannis Kountouras, Panagiotis Katsinelos PII: DOI: Reference:
S0026-0495(17)30100-2 doi: 10.1016/j.metabol.2017.03.013 YMETA 53579
To appear in:
Metabolism
Received date: Accepted date:
23 February 2017 27 March 2017
Please cite this article as: Doulberis Michael, Kotronis Georgios, Gialamprinou Dimitra, Kountouras Jannis, Katsinelos Panagiotis, Non-alcoholic fatty liver disease: An update with special focus on the role of gut microbiota, Metabolism (2017), doi: 10.1016/j.metabol.2017.03.013
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ACCEPTED MANUSCRIPT Non-alcoholic fatty liver disease: An update with special focus on
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the role of gut microbiota
Michael Doulberisa, *, Georgios Kotronisb, Dimitra Gialamprinouc, Jannis Kountourasd
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and Panagiotis Katsinelosd
Bürgerspital Hospital, Department of Internal Medicine, Solothurn 4500, Switzerland
b
Agios Pavlos Hospital, Department of Internal Medicine, Thessaloniki, Macedonia, 55134,
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a
Greece
Papageorgiou General Hospital, Department of Pediatrics, Aristotle University of
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c
Thessaloniki, Macedonia, 56403, Greece d
Ippokration Hospital, Department of Internal Medicine, Second Medical Clinic, Aristotle
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University of Thessaloniki, Thessaloniki, Macedonia, 54642, Greece. *Corresponding author at:
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Department of Internal Medicine Bürgerspital Hospital, Schöngrünstrasse 38, Solothurn 4500, Switzerland Tel: +41(0)32 627 4036, Fax: +41(0)32 627 3079 E-mail: [email protected], [email protected]
ACCEPTED MANUSCRIPT Abbreviations: CDAA: choline-deficient/L-amino acid-defined, CK-18: Cytokeratin-18, CKD: chronic
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kidney disease, CR: calorie restriction, CRTC1: cAMP-responsive element binding protein (CREB)-regulated transcriptional coactivator, CVD: cardiovascular disease, EDC: endocrinedisrupting chemicals, FFA: (plasma) free fatty acids, FXR: farnesoid X receptor, GCKR:
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glucokinase regulator, HCC: hepatocellular carcinoma, HDL: high density lipoprotein, HF: hepatic fibrosis, HP: helicobacter pylori, HSC: hepatic stellate cells, IR: insulin resistance,
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LPS: lipopolysaccharide, MBOAT7: membrane bound O-acyltransferase domain containing
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7, MetS: metabolic syndrome, NAFLD: non-alcoholic fatty liver disease, NASH: nonalcoholic steatohepatitis, PNPLA3: palatine- like phospholipase 3, SCFA: short chain fatty acids, SNP: single nucleotide polymorphism, SS: simple steatosis, T2DM: type 2
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diabetes mellitus, TLR-4: Toll Like Receptor 4, TM6SF2: Transmembrane 6 Superfamily
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member 2 gene, UDCA: Ursodeoxycholic acid, VLDL: very low-density lipoproteins
ACCEPTED MANUSCRIPT Abstract Non-alcoholic fatty liver disease (NAFLD) is a significant global health burden in children,
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adolescents and adults with substantial rise in prevalence over the last decades. Accumulating data from manifold studies support the idea of NAFLD as a hepatic manifestation of metabolic syndrome, being rather a systemic metabolic disease than a liver confined
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pathology. Emerging data support that the gut microbiome represents a significant environmental factor contributing to NAFLD development and progression. Apart from other
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regimens, probiotics may have a positive role in the management of NAFLD through a
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plethora of possible mechanisms. The current review focuses on the NAFLD multifactorial pathogenesis, including mainly the role of intestinal microbiome and all relevant issues are raised. Furthermore, the clinical manifestations and appropriate diagnostic approach of the
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disease are discussed, with all possible therapeutic measures that can be taken, also including
Keywords
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the potential beneficial effect of probiotics.
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NAFLD, gut microbiota, probiotics, gastrointestinal bacteria, metabolic syndrome, obesity.
ACCEPTED MANUSCRIPT 1. Introduction Nonalcoholic fatty liver disease (NAFLD) has been recognized as a global public health
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problem, affecting 6-45% of the general population, rising up to 70% in patients with type 2 diabetes mellitus (T2DM) or 90% in morbidly obese patients [1,2]. NAFLD is among the leading causes of liver cirrhosis, hepatocellular carcinoma (HCC) and liver transplantation
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[3]. The definition of NAFLD includes the excessive accumulation of triglycerides in the hepatocytes in absence of parallel alcohol consumption (less than 20 and 30g per day in
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women and men, respectively) or other secondary etiological factors [4,5]. Multiple factors
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(“hits”) contribute to the pathogenesis of NAFLD, considered to be a multiple-hit disorder [6,7]. Pathogenetic factors are divided into those with an established association with NAFLD, including genetic [e.g., polymorphisms of patatine-like phospholipase domain-
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containing protein 3 (PNPLA3) gene] [8], dietary factors (e.g., fructose), insulin resistance (IR) [9] and adipokines [10], as well as those with a potential association needing validation, including endocrine disruptors [11] and dysbiosis of the gut microbiota [12].
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NAFLD encompasses a range of histological phenotypes. At the initial step, lipids are
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accumulated in the hepatocytes resulting in simple steatosis (SS). In a proportion of patients, SS progresses to nonalcoholic steatohepatitis (NASH), characterized by the addition of hepatic inflammation and/or fibrosis [9]. NASH may progresses to NASH-related cirrhosis and HCC more often than SS, which is regarded as a benign entity [9,13,14]. Currently, it is believed, that NAFLD is not only a liver confined pathology, but it is regarded as a multisystem disease with a hepatic component as well as extra-hepatic manifestations [1,4,15]; except for the liver-related morbidity, NAFLD is also related to extra-hepatic morbidity, including systemic metabolic complications (e.g. T2DM), thyroid dysfunction and osteoporosis [16], chronic kidney and cardiovascular disease (CVD), and malignancies, presumably leading to higher morbidity and mortality rates [4,5,17,18]. Important to note that IR has been proposed as the common pathogenetic mechanism in
ACCEPTED MANUSCRIPT conditions clustered under the term of metabolic syndrome (MetS), including obesity, T2DM, dyslipidemia, hypertension, and NAFLD, all of which increase the risk for the CVD and
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mortality, the endpoints of MetS [19,20]. Specifically, a connection between gastrointestinal bacteria and liver has long been suspected. The first observation linking liver and gut microbiota dates back to 1921, when
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Hoefert described patients suffering from chronic liver disease with altered gut flora [21]. Since then, emerging data support that the gut microbiome represents a significant
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environmental factor contributing to NAFLD development and progression [22–24]. In this
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review, we consider mainly the growing evidence that highlights the relationship between gut microbiome and its association with the entire spectrum of NAFLD from etiology to
2. Review Criteria
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treatment.
We performed a computerized literature search using the PubMed database, to identify
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relevant published articles regardless of the publication year, until January 2017. Only full-
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text papers written in the English language were reviewed. The following terms were used: “NAFLD”, “nonalcoholic fatty liver disease” or “nonalcoholic steatohepatitis” in combination with “gut microbiota”, “gastrointestinal bacteria” and “metabolic syndrome”. Further articles were identified via relevant paper analysis obtained from the initial literature search. Overall selected papers were used for the purposes of this review.
ACCEPTED MANUSCRIPT 3. Epidemiology An estimation of the world prevalence varies from 6 to 45% in the general population, rising
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up to 70% in patients with T2DM or 90% in morbidly obese patients with concurrent IR and hyperlipidemia [1,2,25]. NAFLD remains the most common cause of abnormal liver function in western countries [26–29] and is among the leading causes of liver cirrhosis, HCC and liver
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transplantation [3]. SS progresses to cirrhosis in less than 5% of cases, whereas NASH progresses to cirrhosis in 10-15% of cases over 10 years and in 25-30% of cases in the
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presence of advanced fibrosis [30].
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Age appears to play a significant role [1,4,5,18]. Highest prevalence is reported in adults with ages ranging from 40 to 49 years while age exceeding 60 years is associated with prevalence twice as much compared to 20 years of age [4,5]. The prevalence of NAFLD in children
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varies widely from 13% to 80% and the strongest risk factor affecting this discrepancy is obesity [27,31]. NAFLD affects more male gender and this preference also includes children [4,5,18,32]. Moreover, the prevalence of NAFLD varies noticeably when comparing
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individuals of different races and ethnicities. Rates are highest in Hispanic patient populations
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compared with non-Hispanic whites and African Americans, despite similar rates of the MetS and risk factors. [1,5,33]. This observation remains poorly characterized; variations in genes that effect lipid metabolism may play a role [4,33]. 4. Pathogenesis Multiple factors (“hits”) contribute to the pathogenesis of NAFLD, considered to be a multiple-hit disorder [6,7]. NAFLD is a complex disease mediated by several metabolic, environmental, genetic and microbiological mechanisms (Fig. 1). Pathogenetic factors are divided into those with an established association with NAFLD, including genetic (e.g., polymorphisms of PNPLA3 gene) [8], dietary factors (e.g., fructose), IR [9] and adipokines [10], as well as those with a potential association needing validation, including endocrine disruptors [11] and dysbiosis of the gut microbiota [12].
ACCEPTED MANUSCRIPT Human epidemiologic studies describing polymorphisms in a number of genes involved in metabolic dysfunctions have contributed to clarify the causes leading to the
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NAFLD evolution; family studies and inter-ethnic differences in susceptibility suggest that multiple genetic factors may be important risk determinants for progressive NAFLD. Polymorphisms in genes affecting lipid metabolism, adipocytokines, fibrotic mediators and
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oxidative stress may be associated with NASH and/or fibrosis, but most current findings require replication [6,34].
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A single nucleotide polymorphism (SNP), which gathers increasing evidence, and
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considered a key genetic factor in NAFLD pathogenesis, is the I148M (rs738409 C/G) of the mentioned PNPLA3, also known as adiponutrin [35]. The PNPLA3 gene represents the main and more acknowledged determinant of interindividual differences in hepatic steatosis and
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susceptibility to NASH [36]; PNPLA3 protein is found to act as a triglycerides hydrolase agent. Specifically, the I148M variant (isoleucine to methionine substitution, position 148) results in an altered PNPLA3 protein with an impaired activity in lipid catabolism, hence
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allowing the gathering of triglycerides to hepatic cells [37] by inducing disturbance in hepatic
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very low-density lipoproteins (VLDL) secretion [38]. Furthermore, this variant affects the hepatocyte triacylglycerol remodeling [39]. In a relative meta-analysis of 16 studies, I148M exerted a strong influence not only on liver fat accumulation, but also on the susceptibility of a more aggressive disease; GG homozygous have a 3.2-fold greater risk for necroinflammation and a 3.2-fold greater risk for fibrosis when compared with CC homozygous and, therefore, NASH is more frequently observed in GG than CC homozygous genotype [40]. Furthermore, carrying two G alleles does not increase the risk of severe disease, meaning that the influence of this variant on disease severity follows a dominant model. The observed effect of I148M SNP on the natural history of NAFLD is perhaps one of the strongest ever reported for a common variant modifying the genetic susceptibility of a complex disease [40]. The humans who are carriers of the PNPLA3 (I148M) allele seem to be
ACCEPTED MANUSCRIPT at greater risk for developing cirrhosis and HCC regardless of the prepossession for steatosis [41,42].
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Other SNPs have also been associated with NAFLD characteristics. A genome-wide associated study (GWAS) analyzed 324,623 SNPs from the 22 autosomal chromosomes; NAFLD activity score (NAS) was independently associated with the SNP rs2645424 on
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chromosome 8 in farnesyl diphosphate farnesyltransferase 1, the degree of fibrosis was associated with the SNP rs343062 on chromosome 7, and lobular inflammation was
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associated with the SNP rs1227756 on chromosome 10 in COL13A1, rs6591182 on
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chromosome 11 and rs887304 on chromosome 12 in EFCAB4B [34]. In addition, a genetic variant of TM6SF2 (Transmembrane 6 Superfamily member 2 gene), E167K variant, is regarded as another crucial determinant of liver triglyceride content, thereby associating with
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the hepatic damage attributing to fat accumulation in this organ [43,44]. GCKR (glucokinase regulator) and LYPLAL1 (agent of triglycerides catabolism) variants are also associated with liver damage through inducing increased hepatic glucose uptake [45,46]. Likewise, a variant
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of MBOAT7 (membrane bound O-acyltransferase domain containing 7) gene was recently
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found to be a locus that provides susceptibility for the development and progression of NAFLD [47]. In contrast, the cAMP-responsive element binding protein (CREB)-regulated transcriptional coactivator (CRTC1) (also known as transducer of regulated CREB, TORC), confers wide spectrum protection against the development of hepatic steatosis. CRTC1 directly interferes with the expression of genes regulated by lipogenic transcription factors, most prominently liver x receptor α. As a cyclic AMP effector, CRTC1 mediates anti-steatotic effects of calorie restriction (CR). Likewise, CRTC1 mediates anti-lipogenic effects of bile acid signaling, while it is negatively regulated by miR-34a, a pathogenic microRNA upregulated in a broad spectrum of NAFLD. These patterns of gene function and regulation of CRTC1 are distinct from other CR-responsive proteins, emphasizing critical protective roles
ACCEPTED MANUSCRIPT that CRTC1 selectively plays against the development of NAFLD, which in turn offers novel opportunities for selectively targeting beneficial therapeutic CR effects [48].
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Dietary factors are playing an extra role in hepatic lipogenesis and therefore in NAFLD and later on in NASH. Overconsumption of high-fat diet and increased intake of sugar-sweetened beverages are major risk factors for development of NAFLD [49]. Dietary
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fat directly supplies free fatty acids (FFA) to the liver. Furthermore, the metabolism of carbohydrate results in FFA synthesis from acetyl CoA. Consumption of excessive glucose
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also promotes liver lipogenesis through the activation of a carbohydrate responsive binding
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protein [13,14]. Moreover, it is recognized that saturated fatty acids appear to be more toxic than the unsaturated ones, through the promotion of apoptosis and liver injury as well as endoplasmic reticulum stress [50–52]. In contrast, treatment with n-3 long-chain
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polyunsaturated fatty acids induces variable success in improving NAFLD [53]. NAFLD appears to be the hepatic component of Mets. In this regard, IR has been proposed as the common pathogenetic mechanism in MetS, including obesity, T2DM,
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dyslipidemia, hypertension, and NAFLD, all of which increase the risk for CVD and
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mortality, the endpoints of MetS [19,20]. Mets is defined according to NCEP AT III (2005 revision) as the presence of at least three of the five following criteria: Obesity (waist circumference >40 inches ♂, >35 inches ♀), hypertension (>130 mmHg systolic, or
>85 mmHg diastolic or even pharmacologic
treatment), fasting glucose ≥100 mg/dl or pharmacologic treatment, hypertriglyceridemia (>150 mg/dl or pharmacologic treatment) and low high density lipoprotein (HDL) (<40 mg/dl ♂,
<50 mg/dl ♀ or pharmacologic treatment) [54]. The main pathogenetic mechanism
leading to steatosis is the continuing lipolysis and therefore the increased amount of plasma FFA due to IR in the adipose tissue [14]; insulin, when present and active, is an anabolic hormone and prevents from lipolysis. The elevated plasma FFA flow to the liver and this ectopic fat accumulation is associated with increased secretion of hepatokines [55], increased
ACCEPTED MANUSCRIPT gluconeogenesis, inhibition of insulin signaling and reduced glycogen production [56,57]. A reduction of hepatocellular triglycerides secretion through VLDL and an impaired fatty acids
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oxidation are also associated with hepatic fat accumulation [58]. In this respect, it has been estimated with an isotope technique that, in the presence of steatosis, approximately 60% of the hepatic triglycerides comes from the adipose tissue, 25% from de novo lipogenesis and
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15% from dietary lipids [59]. This study clarified the impact of adipose tissue lipolysis on hepatic steatosis. It is highlighted that the percentage of hepatic triglyceride content derived
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from de novo lipogenesis in individuals without steatosis is lower, which is mainly attributed
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to lower IR [9]. Furthermore, excessive intraperitoneal fat can cause FFA reflux into the liver directly via the portal vein [60]. The excessive liver lipid accumulation causes further worsening of IR and chronic inflammation driving to liver disease. This is caused by
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metabolites of FFA that relocate cytoplasmic protein kinase C to the membrane, resulting in phosphorylation of intracellular insulin receptors, which constitutes the main defect in IR [14].
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The adipose tissue inflammation plays a key role in the pathogenesis of NAFLD and
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the intestinal dysbiosis seems to be an additional crucial factor leading to and sustaining the disease. Apart from fats, bacterial endotoxins are absorbed by the intestine leading to further regulation of liver inflammation [61]. As the disease progresses, the hepatocyte injury drives to the development of steatohepatitis, hepatocyte necrosis and apoptosis with the contribution of oxidative stress and peroxidation as well as mitochondrial dysfunction and endoplasmic reticulum stress [14,17,62]. Mechanisms involved in lipotoxicity and glucotoxicity include endoplasmic reticulum stress, oxidative stress and mitochondrial impairment, which all promote the progression of SS to NASH [7,63].The procedure of further NASH progression is mediated by activation of hepatic stellate cells (HSCs), collagen deposition to the liver and consequently fibrosis and cirrhosis. HCC is the final stage of all these mechanisms [17].
ACCEPTED MANUSCRIPT The progression to NASH also involves increased infiltration of different subtypes of immune cells, such as macrophages, monocytes, T-lymphocytes and neutrophils, to the liver,
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as well as activation and expansion of liver immune cells, including Kupffer cells and the mentioned HSCs [64]. When counterbalancing mechanisms do not successfully limit hepatic steatosis, and even more when obesity deteriorates, intra-hepatic inflammation process starts,
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perhaps as a counterbalancing mechanism to limit steatosis [7]. During the inflammatory process, the immune cells produce cytokines and other mediators contributing to
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inflammation [9]. It also appears that a cross-talk exists between infiltrating immune cells
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and liver-residing ones (Kupffer cells and HSCs) [65]. When the inflammation prolongs, the process of hepatic fibrosis take place. The fibrosis is a major prognostic and a hard therapeutic histological end-point, because the severity of fibrosis predicts the severity of
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advanced disease, i.e. liver cirrhosis and HCC [6]. Since the discovery of adipose tissue as a highly active endocrine tissue, the adipokines produced by adipose tissue and exerting autocrine, paracrine and endocrine
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function, have been involved in the pathogenesis of various obesity-related diseases,
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including NAFLD. Data concerning the association between NAFLD and circulating leptin and adiponectin levels are generally well documented: leptin levels increase, whereas adiponectin levels decrease, by increasing the severity of NAFLD. Data concerning other adipokines in histologically confirmed NAFLD populations are inconclusive (e.g., resistin, visfatin, retinol-binding protein-4, chemerin) or limited (e.g., adipsin, obestatin, omentin, vaspin etc.). A thorough insight into the pathophysiologic mechanisms linking adipokines with NAFLD might lead to the design of studies investigating the combined adipokine peptides use as noninvasive diagnostic markers of NAFLD and novel clinical trials targeting the NAFLD treatment [10]. Endocrine disruptors or endocrine-disrupting chemicals (EDCs) constitute a highly heterogeneous group of molecules found in the environment or in consumer products.
ACCEPTED MANUSCRIPT Relative studies have suggested the diverse EDCs involvement in an increasing number of metabolic disorders, IR and IR-related co-morbidities, including obesity, T2DM, polycystic
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ovary syndrome and NAFLD [11,66]. Certain EDCs, including dioxins, bisphenol A, phthalates and other persistent organic pollutants may induce hepatic histological alterations similar to those observed in NAFLD, directly through a hepatotoxic effect and/or indirectly
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by triggering hepatic and systematic IR [11]. As a consequence, EDCs, acting in concert with other pathogenetic factors (“hits”), may play a role in the pathogenesis of fatty liver, thereby
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affecting the prevalence of obesity and NAFLD [7].
4.1 The role of gut microbiota in NAFLD
The human microbiota consists of 100 trillion microorganisms that provide essential
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metabolic and biological functions benefiting the host. However, the presence in host plasma of a gut-derived bacteria component, the lipopolysaccharide (LPS) has been recognized as a causal or complicating factor in several diseases such as obesity-associated metabolic
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disorders including NAFLD [67]. The term gut microbiota is used to define the whole
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population of bacteria, viruses, parasites as well as fungi, which colonize almost the entire gastrointestinal tract from mouth to colon [68]. There are more than 2,000 species of bacteria, including anaerobes, within the adult human gut. The density of bacterial cells increases generally from stomach to colon with a number varying from 10 – 103 (per gram of content) in the gastric – duodenal mucosa and reaching a maximal number of up to about 1014 in the colon, and weigh about 1.5 kg [22,69]. Despite the wide variety of aforementioned microorganisms, the vast majority of them (up to 95%) in human and generally mammalian intestine consists of only three bacterial phyla: Bacteroidetes, Actinobacteria and Firmicutes [70,71]. In healthy organisms gut microbiota contribute to maintenance of homeostasis, via a symbiotic mechanism, where both parties, bacteria and host benefit. A disturbance of this relationship leads to changes of the balance between beneficial and pathogen bacteria, a
ACCEPTED MANUSCRIPT condition termed as “dysbiosis” [61,68,72]. Dysbiosis may adversely impact metabolism and immune responses, favoring obesity and obesity-related comorbidity, including IR and
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NAFLD [12]. Specifically, the existence of the so-called “liver – intestine axis” has been identified and described for several decades. A plausible explanation of this connection is that liver
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receives directly about 70% of the venous intestinal outflow via portal vein, thereby serving as a first filter with a great -among others- detoxifying role [22,68,73]. Moreover, studies
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with animal models have shown strong evidence that indicates a role for bacteria in the
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development of NAFLD (Fig. 1). Germ free mice did not develop obesity, which as previously showed is linked to NAFLD, despite being fed with high- fat and sugar diet. The colonization of their guts with microbiota taken from conventionally raised mice, resulted in
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an increase of IR and body fat content, although food intake was also reduced [22,71]. In this respect, there are two mutually antagonistic phyla, the Bacteroidetes (Gram -) and the Firmicutes (Gram +) of microbiota [7]. While exceptions exist, Firmicutes are considered as
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“obese” microbiomes, whereas the bacteroidetes as the “lean” microbiomes, because the
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former were found to contain more genes associated with lipid and carbohydrate metabolism and the breakdown of otherwise indigestible polysaccharides [74]. In this regard, initial studies showed that ob/ob mice have a 50% reduction in the Bacteroidetes and a proportional increase in Firmicutes compared with lean mice [75]. Apart from genetic models of obesity, wild-type mice had a higher proportion of Bacteroidetes and lower of Firmicutes, when they were lean (on a chow diet) than obese (on a high fat diet) [76]. Possible mechanisms connecting gut microbiota with obesity and NAFLD are: a) Augmented energy extraction from food nutrients which initially are not digested because of the host limited capability to digest complex dietary polysaccharides [7,12].
ACCEPTED MANUSCRIPT b) Fermentation of polysaccharides by microbiota into monosaccharides and shortchain fatty acids (SCFAs), such as acetate, propionate, butyrate and ethanol, linked with
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NAFLD; [9] c) Increased intestinal permeability, thereby more microbiota-produced endotoxins and SCFAs accessing the liver; [7]
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d) Decreased choline metabolism leading to decreased VLDL export from the liver;[7] e) Modulation of bile acid synthesis, which are crucial for fat absorption, but also
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affect metabolism of glucose and lipoproteins by linking farsenoid X receptor (FXR)
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[12].
More specifically, a hallmark of gut microbiota with a substantial contribution to energy production is their ability to anaerobically ferment the non-digestible plant
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polysaccharides to SCFA such as butyrate, acetate and propionate, which can then be absorbed by enterocytes [77,78]. SCFA has been shown to reduce the permeability of tight junctions and increase mucus production, thereby enhancing the function of gut as barrier
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[79]. Additionally, SCFA seem to be key-players for the intestinal hormonal regulation, since
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they induce an increase of peptide YY and glucagon-like peptide, both of which influence satiety and improve oral glucose tolerance, insulin sensitivity as well as leptin levels [68,70]. Besides, intestinal microbiota are known for their ability to shape energy demands of the host and thus contribute to development of obesity and obesity- associated diseases, such as NAFLD. Germ free mice gained less body weight compared to their controls with normal gut flora, although both groups were fed with identical diet. This property of body weight gain by microbiota was transferable after bacteria transplantation and was pathogenetically mediated by enhancement of absorption of monosaccharides by small intestine epithelial cells (and thus fatty acids optimization synthesis) and parallel by suppression of fasting-induced adipocyte factor in aforementioned cells, which results in fat accumulation and development of obesity [80]. Gut microbiota have been also connected with NAFLD through the water-soluble
ACCEPTED MANUSCRIPT nutrient choline. The latter can both be ingested by animal products such as eggs and red meat, but also biosynthesized by intestinal microbiota [79].
In colon takes place the
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hydrolysis of choline to dimethylamine and to trimethylamine, both of which regarded as precursors of dimethynitrosamine, a potent hepatotoxin and liver-carcinogen [22]. Chemical catalysis is mediated by bacterially produced enzymes [73]. Moreover, choline seems to play
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a critical role in the synthesis of VLDL, which are needed for the export of lipids from liver [24]. Choline deficiency has been also shown that leads to chronic liver disease [71].
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Bile acids, along with phospholipids, biliverdin and mucous are the main components of bile,
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which multifactorially contribute to homeostasis in the small bowel through the regulation of metabolism of cholesterol, release of fat-soluble vitamins, as well as through emulsification of fats. Moreover, certain bile acids are known to act as pathway activators/suppressors for
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several host receptors, which are primarily found in intestinal cells or in cells of distant tissues and organs such as fat and liver, respectively [70,79]. The so-called nuclear bile acids receptor, FXR, is implicated in the pathogenesis of microbiota-related NAFLD. Activation of
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FXR by bile acids, which serve as signaling molecules, induces a cascade of biochemical
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reactions with end-result the suppression of bile production [77]. A relative study [81] by using a murine model of NAFLD, showed that the administration of antibiotics to mice fed with high fat diet, was able to alter the composition of bile acids, which in turn led to more conjugated bile acids and resulted in inhibition of intestinal FXR signaling as well as to less accumulation of triglycerides in liver. Similar results were observed in another relevant study [82], where microbiota-associated bile acids’ deconjugation induced a promotion of NAFLD in a murine model of obesity. The pathogenetic mechanism involved acceleration of lipid synthesis through activation of intestinal FXR signaling pathway. Furthermore, a recently published study [83], showed that the modulation of gut microbiota, mediated through bile acid binding resin, could significantly reduce adiposity in mice fed with high high-fat diet. This beneficial effect was associated with an increase of Clostridium
ACCEPTED MANUSCRIPT leptum as well as of Bacteroides-Prevotella group in mice fed high-fat diet and treated with bile acid binding protein. Moreover, a statistically significant elevation of fecal propionate
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was also observed in the aforementioned group. Another well-documented mechanism, which is known to be involved in development and progression of NAFLD by intestinal microbiota, is IR and especially the so-called LPS– Toll
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Like Receptor-(TLR)-4–monocyte differentiation antigen CD14 system [73,84]. Although this knowledge is largely derived from animal model studies [73], there is one study
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mentioning a substantial elevation of LPS plasma levels in patients suffering from T2DM,
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compared to healthy matched controls [85]. Furthermore, a reduction of fasting insulin as well as of IR can be seen in modification or suppression of small intestine bacterial overgrowth, which also results in a decrease of pro-inflammatory cytokines [73].
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Additionally, small intestinal bacterial overgrowth and bacterial translocation represent two rather common pathologies in cirrhotic patients and their prevalence has been directly correlated with the severity of hepatic disease [70,73].
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The few human studies also reported gut microbiota association with NAFLD [12].
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There are certain general lines derived from human studies: [7,61] a) As in the animal studies, obese individuals have a greater prevalence of Firmicutes and lower prevalence of Bacteroidetes than lean individuals; b) NASH patients have a lower prevalence of Bacteroidetes than obese individuals without NASH; c) the environment appears to determine most of the interindividual variability in the gut microbiota [61]. Although human studies are limited by their observational nature, small sample size and the diversity of gut microbiota identified, the indicated associations provide the basis for future large-scale studies. Apart from the classic gut microbiota, Helicobacter pylori (Hp) may also affect the pathogenesis of NAFLD, since it may colonize gut itself, but also might affect the diversity of gut microbiota [20]. Some clinical observational studies and animal studies support the
ACCEPTED MANUSCRIPT relationship of Hp infection (Hp-I) and dysbiosis of gastrointestinal tract (GIT) microbiota to metabolic disorders like IR and diabetes [86,87]. Dysbiosis of GIT microbiota has been
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involved in the development of the mentioned several human MetS disorders like obesity, NAFLD, and IR in T2DM; certain microbes, such as butyrate-producing bacteria, are reduced in T2DM patients; gastric and fecal microbiota may have been changed in Hp-infected people
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and mice to promote gastric inflammation and specific diseases; and Hp-I also induced IR, defined as a predisposing factor to T2DM development [88]. Moreover, higher rates of anti-
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Hp IgG were reported in NAFLD patients compared to controls in a cross-sectional study
pathogenetic basis of NAFLD [90].
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[89], but also an association between Hp infection and the mentioned IR, being the
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5. Clinical manifestations
The great majority of patients with NAFLD remain asymptomatic or may report nonspecific symptoms, especially fatigue [17,91]. As the disease progresses to NASH and later on to
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cirrhosis, the enlargement of the liver leads to a feeling of weight and discomfort or even pain
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in the right upper quadrant of the abdomen. But even then, these symptoms are not specific and therefore the disease is undetected until the clinician suggests an imaging study and/or blood chemistry, which reveals abnormal liver tests [91,92]. A physical examination reveals usually central obesity and sometimes hepatomegaly in NAFLD patients. Manifestations of diabetes such as diabetic dermopathy, necrobiosis and lipoidica diabeticorum or clinical manifestations of dyslipidemia such as xanthelasmata and xanthomas may also be recognized [93]. However, none of these findings are pathognomonic though should arouse the clinicians’ suspicion for the presence of NAFLD. Over time, when the disease advances to NASH, more specific clinical signs such as a dorsocervical (buffalo) hump and acanthosis nigricans (implying IR) are present in the physical examination [94]. When cirrhosis developed, the clinical findings become more apparent and include palmar
ACCEPTED MANUSCRIPT erythema, caput medusae (palm tree sign), spider angiomata (spider nevi), reduced muscular mass, gynecomastia, Dupuytren’s contracture and even jaundice, ascites, splenomegaly,
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edema [95]. Moreover, systemic clinical manifestations due to NAFLD-related CVD and/or kidney disease may also be recognized.
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6. Diagnosis
It is widely accepted that NAFLD is mostly diagnosed by exclusion of other possible causes
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of chronic liver disease. There are recognized three criteria for the appropriate establishment
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of diagnosis. i. Absence of significant consumption of alcohol (less than 20g or 30g daily in women and men, respectively), ii. Imaging or histological findings of liver steatosis and iii. Exclusion of other possible causes of hepatic inflammatory disease, including hereditary
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(such as Wilson’s disease or hemochromatosis) or acquired etiologies (such as viral hepatitis, hepatotoxic drugs, alcoholic liver disease or autoimmune diseases) [96,97]. Most clinicians suspect NAFLD if a patient presents with abnormal liver function tests,
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especially alanine transaminase (ALT), aspartate aminotransferase (AST) and gamma-
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glutamyl transferase. Although serum enzymes are often found to be normal even in advanced liver disease [98–100], the detection of mildly or moderately elevated enzymes are the most common first step for the diagnostic approach for NAFLD. Several studies on serological surrogate markers are used to distinguish NASH from the inflammation of SS, and single serological test methods to predict NASH have also been studied. Although the recommended surrogate markers TNF-α, IL-6, CRP, Pantraxin, Ferritin, SPEA, and sRAGE predict hepatic inflammation, most of these markers still require extensive external validation. The single test most studied in regard to the diagnosis of NASH is Cytokeratin-18 (CK-18). The CK-18 fragment, a marker of hepatocyte apoptosis, may predict NASH, which is significantly increased compared with normal or simple steatosis. CK-18 showed relatively good results in some precedent studies, suggesting the potential for
ACCEPTED MANUSCRIPT screening NASH. However, CK-18 in clinical application demonstrates a large variation in cut-off values compared to previous studies, and its sensitivity and specificity to predict
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NASH was not satisfactory [101]. Interestingly, a noninvasive marker, named HSENSI [acronym of homocysteine, SGOT (serum glutamic oxaloacetic transaminase), ESR (erythrocyte
sedimentation
rate),
Nonalcoholic
Steatohepatitis
Index],
consists
of
SC
aforementioned three low cost, easily measurable parameters and may accurately predict
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NASH [102].
In some cases, the clinician suspects the disease after detecting liver brightness and/or
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vascular blurring in abdominal ultrasound testing [5]. However, diagnostic sonography is a subjective tool providing only poor sensitivity for the detection of mild steatosis (sensitivity
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93% for steatosis >33%, but poor sensitivity for steatosis <30%), thereby introducing sonography only as an initial diagnostic approach [103,104]. CT scan (non-contrast and contrast enhanced) provides more sensitive information, allowing to measure the so-called
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liver-to spleen attenuation ratio (when measured below 0.9, the diagnosis of steatosis is certain), with the disadvantage of exposure to radiation [5]. The latter can be overcome with
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MR imaging but the increased cost of this method makes it not widely used yet. Of note, proton density fat fraction measurement by MRI is currently the most accurate and sensitive imaging method, simpler and more practical than magnetic resonance spectroscopy, but restricted, up to now, just to research and clinical trials. Although the histopathological evaluation of liver biopsy samples is central (gold standard) in the diagnosis of NAFLD, it remains a rather expensive invasive method hiding a possible risk of morbidity and rarely mortality; semi-quantitative histological scoring systems have been recommended for NAFLD, though they are not useful in clinical practice and each has certain limitations. Thus, it is not recommended for all patients with NAFLD but should be considered for those who are at great risk for developing NASH and advanced fibrosis (Strength – 1, Evidence – B), or those patients with suspected NAFLD in whom the clinical
ACCEPTED MANUSCRIPT and laboratory evaluation cannot safely exclude other causes of chronic liver disease (Strength – 1, Evidence – B) [97]. The histological examination offers significant prognostic
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information that can determine consequent management of the disease. Apart from liver biopsy, using the NAFLD Fibrosis Score, a non-invasive scoring system, can also offer a prediction of the severity of fibrosis. This score is based on the evaluation of six variables
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(hyperglycemia, Body Mass Index, age, platelets, AST/ALT and serum albumin) and provides assistance the clinician to identify the proportion of NAFLD patients with fibrosis,
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thus renders liver biopsy rather not necessary for the rest [105]. Furthermore, non- invasive
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screening of liver fibrosis can be obtained by elastography (US-based or transient, MRbased). This method provides assessment of degree of steatosis and the severity of fibrosis. However it is still expensive and is currently used on a limited basis [106]. Transient
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elastography appears to be an economically attractive alternative to liver biopsy and other non-invasive diagnostic tests especially for patients with a higher degree of liver fibrosis
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7. Therapy
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[107].
The therapeutic approach of NAFLD/NASH has been thoroughly discussed within the past decade and several strategies have been proposed for that purpose. However, currently, there is no pharmacological therapy globally approved due to lack of sufficient evidence. The first goal to control NAFLD/NASH is early detection of the disease, in order to apply proper and prompt interventions for the prevention of further progression as well as to achieve a possible regression of the hepatic damage. There is unanimously accordance that lifestyle interventions represent the most crucial first therapeutic approach; lifestyle interventions involving exercise and weight loss are currently the only accepted treatments for this disease [108]. Dietary modification includes reduction of sugar consumption and saturated fat as well as potential increase of omega 3 fatty acid uptake [109]. Based on available data, dietary
ACCEPTED MANUSCRIPT recommendations should consider energy restriction (500–1000 kcal/day) and elimination of NAFLD-promoting components (processed foods, and foods and beverages high in added
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fructose) [110]. While the reduction of liver fat appears to be driven mainly by calorie restriction and not the macronutrient composition of the diet, the Mediterranean diet is recommended by the recent guidelines [110]. Furthermore, excessive alcohol drinking (>30
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g/day for men and >20 g/day for women) is discouraged, whereas coffee is not restricted [7,110].
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Moreover, aerobic exercise (a moderate program 3 to 4 times per week) is recommended because of its positive effects in the metabolic parameters implicated in the development and
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progression of NAFLD/NASH, although exercise’s impact on liver histology is not well clarified yet [97,111]. However, recent guidelines do not recommend omega 3 fatty acids
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because of lack of enough evidence [112,113]. The above-mentioned interventions consist the major initial steps for the disease management. It is noteworthy that in the recent EASL-EASD-EASO Clinical Practice Guidelines, healthy
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diet and physical activity (including aerobic and resistance training) are recommended alone
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without pharmacotherapy for patients with no evidence of NASH or fibrosis [112]. In one published study, the exercise and diet together were found to be more efficient than metformin or rosiglitazone in normalizing ALT in NAFLD [114]. Both dietary changes and exercise offer the basis for weight reduction, which generally reduce hepatic steatosis. A goal of 5-10% weight loss is proposed for a period of 6 months. Steatosis improves after a 3-5 % minimum weight reduction while a greater reduction (up to 10%) can offer favorable effects on reducing necro-inflammatory process [97]. Furthermore, the liver fat reduction has been found to be related to the intensity of the lifestyle modification [115]. Recent guidelines recommend that the choice of training should be tailored based on patients’ preferences, so as to increase the possibility for long-term maintenance [110]. Although no data are available on their long-term effects on the natural history of NAFLD, a management combining dietary
ACCEPTED MANUSCRIPT counseling and a progressive increase in aerobic exercise/resistance training is preferable and
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should be individually tailored, targeting to progressive weight loss of 7-10% [7,110].
7.1 Pharmacological treatment
Although many pharmacological regimens assessed by randomized clinical trials have been
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proposed for the management of NAFLD, no drug has been tested in phase III clinical trials.
pharmacological agent for such a purpose.
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Therefore, there are no official and peremptory recommendations for any specific
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Therapeutic options for NAFLD aim to: improve IR via weight loss and exercise, or pharmacologically by using anti-obesity medications (i.e., orlistat, sibutramine) or insulinsensitizing agents (i.e., metformin, pioglitazone, rosiglitazone); decrease oxidative stress by
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the use of cytoprotective and/or antioxidants (i.e., vitamin E, vitamin C, betaine, Nacetylcysteine, ursodeoxycholic acid); decrease serum lipids by using of lipid-lowering agents (i.e., statins, fibrates, ezetimibe, probucol, omega-3 fatty acids); and act via other
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mechanisms, including angiotensin receptor blockers (i.e., losartan), TNF-α inhibition
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(pentoxifylline), endocannabinoid receptor antagonism (rimonabant), glucagon-like peptide-1 analogues (i.e., exenatide and liraglutide) and dipeptidyl peptidase-4 inhibitors [6,116]. Specifically, based on large trials, thiazolidinediones (pioglitazone) offer a significant benefit in liver histology. Therefore, they should be recommended for selected patients suffering from advanced liver fibrosis, especially those with T2DM [97,111–113] considering the possible adverse effects of that regimen (weight gain, possible worsening of heart failure, yet unknown long-term effects); pioglitazone use has been suspended in some European countries, because of possible slight increase in bladder cancer risk after long-term use [6]. Metformin use has not been proven to have beneficial effects on liver histology. Therefore, metformin is not recommended as a specific treatment for hepatic disease in individuals with NASH [14,97,111,112]
ACCEPTED MANUSCRIPT Vitamin E constitutes another agent that has been investigated for NAFLD and clinical trials have demonstrated its efficacy in improving steatosis, hepatic inflammation and decreasing
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liver enzyme levels in non-diabetic patients [117,118]. Its benefit is considered to lie on antioxidative activity in free radical reactions, reducing the oxidative stress-derived hepatocellular injury and progression of NASH [119,120]. Clinicians are encouraged to
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suggest vitamin E consumption to the group of patients diagnosed with non-diabetic, noncirrhotic NASH [14,97,112]. The long-term safety of high-dose vitamin E remains under
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assessment. Some experts suggest the combination of vitamin E with pioglitazone
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irrespectively of the presence of T2DM. Important to note that, although the effect of vitamin E on NAFLD nonalcoholic fatty liver diseases has been proven, this drug has not been approved in most countries [101].
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Pentoxifylline is also regarded as a compelling antioxidant and anti-TNF-α agent that seems to improve steatosis and lobular inflammation [121] though there is insufficient relative data and further studies are required to support or evade pentoxifylline’s formal use for
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NAFLD/NASH handling. Ursodeoxycholic acid is not recommended for the management of
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NAFLD or NASH as it has achieved to offer only slight reduction in liver function tests and steatosis without histological improvement over placebo in patients with NASH [122,123]. The effect of obeticholic acid on NAFLD has been proven, but this drug has not been approved in most countries [101]. In the large subgroup of patients with hypercholesterolemia, the use of statins (HMG-CoA reductase inhibitors) or ezetimibe is regarded to be safe with positive impact on reducing LDL cholesterol and cardiovascular risk and possible capacity in enhancing liver enzyme levels but without any evidence in improving liver histology. Both agents can be used to treat hypercholesterolemia in individuals with NAFLD/NASH, but their use specifically for NAFLD/NASH is not yet recommended [97,111,112]. However, a recent interesting review written by an expert panel recommended that certain NAFLD/NASH patients who are at a
ACCEPTED MANUSCRIPT high-risk for developing serious complications such as CVD or HCC should be treated with statins, optimally combined with ezetimibe and pioglitazone, for the primary or secondary
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prevention of CVD, the main cause of death in NAFLD/NASH patients, and the avoidance of cirrhosis, liver transplantation or HCC. Nevertheless, the authors have emphasized that additional carefully-designed randomized relative trials are warranted to substantiate the
SC
usage of the aforementioned regimens for the treatment of NAFLD/NASH [124]. Another potential therapeutic tool, Orlistat, a reversible pancreatic and intestinal lipase
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inhibitor, leads to reduction of liver function tests and steatosis, though the available data
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remain limited. Given the limited effect of orlistat on weight loss (3-5 kg more than placebo) and the need for long-term use to sustain its results, further evaluation of orlistat in NASH is debatable [125].
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GLP-1 receptor agonists, including exenatide and liraglutide, offer good glucose control providing at the same time lowering of body weight and promoting insulin sensitivity. Clinical and experimental data claim that these agents and/or DPP IV inhibitors can consist a
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new pharmacological tool for the management of NAFLD/NASH [126,127].
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Increased serum ferritin levels have been linked to liver inflammation, IR and worsening of fibrosis in patients with NAFLD/NASH [128]. There is limited evidence that reducing iron stores (through a phlebotomy programme) in certain patients has positive effects on IR, liver function markers and NAS score [129,130]. Nevertheless, iron reduction therapy is not currently recommended for NAFLD/NASH [97,111–113]. Other ongoing studies investigate variable NAFLD therapeutic drugs such as: NOX-1/4 Inhibitor (GKT137831, Genkyotex); Galectin-3 inhibitor (GR-MD-02, Galectin Therapeutics, Phase II); CCR2 and CCR5 inhibitor (Cenicriviroc, Tobira, Phase IIb); Pan-caspase inhibitor (Emricasan, Conatus Pharmaceuticals, Inc., Phase IIa); PPAR-α/δ agonist (GFT505, Genfit, Phase IIb); SCD-1 inhibitor (Aramchol, Galmed, Phase IIb); Apoptosis signal-regulating
ACCEPTED MANUSCRIPT kinase 1 inhibitor (GS-4997, Gilead, Phase II); and Lysyk oxidase-like 2 inhibitor (Simtuzumab, Gilead, Phase IIb) [101].
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Bariatric surgery might be introduced in morbidly obese individuals (BMI ≥40 or BMI ≥35 kg/m2 with obesity-related comorbidities) who are motivated to lose weight and who have not responded to lifestyle changes with or without pharmacotherapy with sufficient weight loss.
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A network meta-analysis of 31 randomized trials showed that all bariatric techniques were effective in reducing weight as compared with standard care [7]. The prospect of foregut
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bariatric surgery should be discussed as a measure aiming to decrease body weight and
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metabolic complications, mainly regarding obese and diabetic patients without adequate response to lifestyle modification and/or pharmacological management [111,112,131]. Steatosis, necroinflammation and fibrosis are likely to improve after bariatric surgery [132–
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134]. A 5-year prospective cohort study with 1236 severely obese individuals subjected to Roux-en-Y gastric bypass or adjustable gastric banding, showed improvement of liver function tests, IR and histological end-points (hepatic steatosis and NAS) at one year, with
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stable results up to five years; importantly, fibrosis regressed in six of 13 patients with
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bridging fibrosis at baseline and disappeared in two patients [133]. However, the clinician should take into account possible perioperative complications. Finally, end-stage patients with NASH (cirrhotic stage) or even HCC can benefit by liver transplantation gaining an overall survival equivalent to those who undergo liver transplantation for other causes of hepatic failure [135–137].
ACCEPTED MANUSCRIPT 7.2 Microbiome and treatment of NAFLD Probiotics represent a relatively new and promising therapeutic option for patients suffering
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from NAFLD. According to WHO, probiotics, defined as “ live microorganisms, when administered in adequate amounts, confer a health benefit on the host” [79]. Prebiotics are called the non-digestible food substances, like oligofructose or lactulose, which can promote
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the growth of beneficial bacteria [24,73]. Synbiotics is a term that is commonly used in the literature to describe the combined administration of both categories [70].
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Probiotics are postulated to be beneficial for the treatment of NAFLD and generally of
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chronic hepatic damage through a plethora of suggested mechanisms: they inhibit bacterial translocation as well as epithelial invasion by maintaining the intestinal barrier integrity; and they induce the production of antimicrobial peptides, reducing inflammation and thereby
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shaping immune system of host. Further, they enhance the hyperdynamic circulation state of cirrhosis and avert the onset of hepatic encephalopathy [79,84]. Commonly known bacteria serving as probiotics include spore forming and lactic acid bacteria, such as Bifidobacteria or
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Clostridium/Bacillus bacteria [4].
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An early meta-analysis of four randomized trials revealed that probiotics improved liver function tests, IR, TNF-α, total cholesterol and HDL cholesterol (HDL-C) in NAFLD patients, but did not affect BMI [138]. A more recent meta-analysis of nine randomized trials confirmed that probiotics improve liver function tests (especially in children), IR, TNF-α, triglycerides, total cholesterol and HDL-C in NAFLD patients, without affecting BMI [139]. Specifically, while more than one type of probiotics were studied for NAFLD
treatment,
most
of
these
regimens
included
combinations
of Bifidobacteria and Lactobacilli [140]. The administration of Lactobacilli alone or combined to Streptococci led to reduction of liver function tests [68]. Moreover, the administration of Lactobacilli species was able to attenuate TLR-4 inflammatory pathway and endotoxemia. In a clinical trial, Bifidobacterium has been demonstrated to ameliorate liver
ACCEPTED MANUSCRIPT steatosis and endotoxemia, but only when coupled to fructo-oligosaccharides [68]. The same effect could be observed in animal models, where the restoration of Bifidobacteria or
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Akkermansia muciniphila levels along with oligofructose supplementation in mice fed with high-fat nutrition, reduced hepatic fatty accumulation and rest metabolic syndrome hallmarks as well as endotoxemia [24]. Besides, in an interesting recent meta-analysis, it has been found
SC
that patients who received probiotics prior to liver transplantation had substantially decreased rates of infections and hospital accommodation [141]. The administration in mice fed with a
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high-fat diet of a multi-strain probiotic preparation commercially named as VSL#3 and
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composed of Streptococcus thermophiles, Lactobacillus delbreckii subsp. Bulgaricus, L. acidophilus, L. plantarum, L. casei, Bifidobacterium infants, B. breve and B. longum, led to multiple favorable effects: reduction of serum ALT levels; enhancement of insulin sensitivity;
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hepatic inflammation and steatosis reductions, which were mediated by modulation of hepatic natural killer T cells, suppression of the TNFα/IKK-β signaling pathway and reducing activity of Jun N-terminal kinase as well as by decreasing DNA binding activity of NF-κΒ in ob/ob
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mice [24]. The same beneficial effect of VSL#3 was seen in a randomized controlled, double-
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blinded clinical study, where obese children with NAFLD were supplemented with the aforementioned probiotics preparation for four months [142]. Likewise, the administration of a preparation with Lactobacillus acidophilus, L. helveticus and Bifidobacterium spp. plus a prebiotic had a protective effect regarding alcohol-induced hepatic injury and associated endotoxemia as well as abnormal liver enzyme levels in the Sprange-Dawley rat model of acute pancreatitis [73]. Lactobacillus rhamnosus and paracasei in mouse and rat models have been both managed to improve hepatic inflammation and steatosis [24]. In another study [70], Lactobacillus rhamnosus, paracasei and as well as Bifidobacterium breve were given to Zucker obese rats. L. paracasei was the only one that could not reduce hepatic steatosis. Nevertheless, all these prebiotics could reduce with a variable extent the level of blood inflammatory cytokines,
ACCEPTED MANUSCRIPT including TNF-α, LPS and IL-6. Another probiotic, Clostridium butyricum strain MIYAIRI 588 prevented the progression of NAFLD and associated tumorigenesis in a rat model of
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choline-deficient/L-amino acid-defined (CDAA)-diet-induced NAFLD [143]. Interestingly, probiotic Lactobacillus johnsonii La1 when given to rats, (served as cirrhotic animal model) alone or combined to antioxidants, could reduce bacterial translocation and endotoxemia
SC
[144]. Lactobacillus reuteri GMLN-263 represents another very promising therapeutic agent since has been shown to exhibit multiple beneficial effects: in high fructose-fed rats improved
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liver steatosis and ΙR and, moreover, remarkably reduced TNF-α and IL-6 concentrations in
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adipose tissue [145]. Likewise, Ting et al. also demonstrated, that heat killed Lactobacillus reuteri GMLN-263 reduced fibrosis effects on the liver as well as on the heart of hamsters fed with high-fat diet. This action was associated with a reduction of the expressed fibrotic factor
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TGFβ [146]. Finally, in young male albino rats, an ingestible probiotic mixture showed promise as a treatment for NAFLD, by improving high fat and sucrose diet-induced steatosis
markers [147].
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through its effects on leptin, resistin, pro-inflammatory biomarkers, and hepatic function
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A limitation of the current review is the confined randomized large human trials, which would substantially further strengthen the results of animal models on the role of microbiome. Moreover, the available literature included numerous different compositions and formulas of probiotics and prebiotics, which limit the interpretation of the results of clinical trials and add an heterogeneity of relevant meta-analyses [7]. Because, there is as yet no consensus on the role of probiotics in the management of NALFD, future relative information is warranted to validate the usage of probiotics as therapeutic option for NAFLD/NASH patients. Except of the aforementioned limitations, an important strength of our paper is the assiduous analysis in multiple levels of all possible mechanisms, which contribute to the development of NAFLD and its complications. Besides, we discuss and propose a plethora of other pharmacological agents that are currently being implemented in the multifactorial
ACCEPTED MANUSCRIPT therapy of NAFLD. The potential combination of such pharmacological agents with probiotics might further benefit NAFLD patients, though future relative studies are needed to
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elucidate this consideration.
8. Prognosis and course of disease
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The expected survival of NAFLD patients is reduced compared to general population and although some of these cases remain stable and asymptomatic [1], the majority of patients
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will die due to cardiovascular events and to a lesser extend due to malignancy and cirrhosis
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[17,148]. Moreover, approximately one fifth of the patients can be presented with a cirrhotic liver, as the result of the disease progression from the reversible benign steatosis to NASH, cirrhosis and HCC sequence [149]. The epigenetics, an inheritable phenomenon that affects
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gene expression without altering the DNA sequence, may provide novel molecular indicators that can determine not only the initial risk but also the disease progression and prognosis [149]. The microscopic hepatic lesions and especially the presence of hepatic fibrosis are
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directly correlated to prognosis [4,17]; the long-term hepatic prognosis mostly depends on the
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histologic stage at initial liver biopsy, but multiple risk factors might concur [150]. Diabetic obese patients are at a greater risk for such a progression and likewise patients with sonographic findings suggestive of steatosis are likely to develop diabetes [1]. Furthermore, there are also some predictive parameters, such as hepatic venous pressure gradient, serum albumin level and MELD-Score (Model for End-stage Disease), which can seem helpful for the estimation of hepatic exacerbation and even the development of a malignancy [17]. Additionally, whereas the disease at the early stages is potentially reversible by following the aforementioned management regimens, liver transplantation remains a promising solution for patients with advanced disease [1]. Further clarification of the natural history is essential to identify the predictors of long-term outcomes of NAFLD; these predictors should be used to direct development of clinical trial endpoints in NAFLD [151].
ACCEPTED MANUSCRIPT 9. Conclusion NAFLD is regarded as a systemic pathology with fundamental metabolic components, where
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liver is one centrally affected organ and intestine with its flora constitute a key-regulator that distantly orchestrates and shapes the evolution of disease. Patients die frequently due to extrahepatic complications and to a lesser extent due to NAFLD per se.
The clinical
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appearance of patients with NAFLD is not specific and is often observed with delay, thereby raising clinical suspicion is mandatory. Regarding therapeutic options, there is no evidence-
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based medication, which seems to be unanimously approved of the medical society for the
MA
treatment of NAFLD. An alteration of gut microbiome with the administration of probiotics and/prebiotics has yielded many positive and encouraging results. A new area, introducing more “sophisticated” ways for the treatment of the “old” diseases has been inaugurated and
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the sequel seems to be exciting.
Funding
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The work was not supported by any funds
Conflict of interest
The authors declare no conflict of interest
Author contributions: MD, GK and DG contributed equally in the design and conduct of the study as well as in the data collection, analysis and manuscript writing. JK and PK revised the draft critically for important intellectual content
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Engl J Med 2014;370:2002–13. doi:10.1056/NEJMoa1401329. [132] Mummadi RR, Kasturi KS, Chennareddygari S, Sood GK. Effect of bariatric surgery on nonalcoholic fatty liver disease: systematic review and meta-analysis. Clin
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Gastroenterol Hepatol 2008;6:1396–402. doi:10.1016/j.cgh.2008.08.012. [133] Caiazzo R, Lassailly G, Leteurtre E, Baud G, Verkindt H, Raverdy V, et al. Roux-en-Y
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gastric bypass versus adjustable gastric banding to reduce nonalcoholic fatty liver
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disease: a 5-year controlled longitudinal study. Ann Surg 2014;260:893-8-9. doi:10.1097/SLA.0000000000000945.
[134] Lassailly G, Caiazzo R, Buob D, Pigeyre M, Verkindt H, Labreuche J, et al. Bariatric
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Surgery Reduces Features of Nonalcoholic Steatohepatitis in Morbidly Obese Patients. Gastroenterology 2015;149:379-88-6. doi:10.1053/j.gastro.2015.04.014. [135] Malik SM, DeVera ME, Fontes P, Shaikh O, Ahmad J. Outcome after liver
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transplantation for NASH cirrhosis. Am J Transplant 2009;9:782–93.
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doi:10.1111/j.1600-6143.2009.02590.x. [136] Charlton MR, Burns JM, Pedersen RA, Watt KD, Heimbach JK, Dierkhising RA. Frequency and outcomes of liver transplantation for nonalcoholic steatohepatitis in the United States. Gastroenterology 2011;141:1249–53. doi:10.1053/j.gastro.2011.06.061. [137] Bhagat V, Mindikoglu AL, Nudo CG, Schiff ER, Tzakis A, Regev A. Outcomes of liver transplantation in patients with cirrhosis due to nonalcoholic steatohepatitis versus patients with cirrhosis due to alcoholic liver disease. Liver Transpl 2009;15:1814–20. doi:10.1002/lt.21927. [138] Ma Y-Y, Li L, Yu C-H, Shen Z, Chen L-H, Li Y-M. Effects of probiotics on nonalcoholic fatty liver disease: a meta-analysis. World J Gastroenterol 2013;19:6911– 8. doi:10.3748/wjg.v19.i40.6911.
ACCEPTED MANUSCRIPT [139] Gao X, Zhu Y, Wen Y, Liu G, Wan C. Efficacy of probiotics in non-alcoholic fatty liver disease in adult and children: A meta-analysis of randomized controlled trials.
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Hepatol Res 2016;46:1226–33. doi:10.1111/hepr.12671. [140] Bashiardes S, Shapiro H, Rozin S, Shibolet O, Elinav E. Non-alcoholic fatty liver and the gut microbiota. Mol Metab 2016;5:782–94. doi:10.1016/j.molmet.2016.06.003.
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[141] Sawas T, Al Halabi S, Hernaez R, Carey WD, Cho WK. Patients Receiving Prebiotics and Probiotics Before Liver Transplantation Develop Fewer Infections Than Controls:
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74-4. doi:10.1016/j.cgh.2015.05.027.
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A Systematic Review and Meta-Analysis. Clin Gastroenterol Hepatol 2015;13:1567-
[142] Alisi A, Bedogni G, Baviera G, Giorgio V, Porro E, Paris C, et al. Randomised clinical trial: The beneficial effects of VSL#3 in obese children with non-alcoholic
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steatohepatitis. Aliment Pharmacol Ther 2014;39:1276–85. doi:10.1111/apt.12758. [143] Endo H, Niioka M, Kobayashi N, Tanaka M, Watanabe T. Butyrate-Producing Probiotics Reduce Nonalcoholic Fatty Liver Disease Progression in Rats: New Insight
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into the Probiotics for the Gut-Liver Axis. PLoS One 2013;8:e63388.
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doi:10.1371/journal.pone.0063388. [144] Chiva M, Soriano G, Rochat I, Peralta C, Rochat F, Llovet T, et al. Effect of Lactobacillus johnsonii La1 and antioxidants on intestinal flora and bacterial translocation in rats with experimental cirrhosis. J Hepatol 2002;37:456–62. [145] Hsieh F-C, Lee C-L, Chai C-Y, Chen W-T, Lu Y-C, Wu C-S. Oral administration of Lactobacillus reuteri GMNL-263 improves insulin resistance and ameliorates hepatic steatosis in high fructose-fed rats. Nutr Metab (Lond) 2013;10:35. doi:10.1186/17437075-10-35. [146] Ting W-J, Kuo W-W, Hsieh DJ-Y, Yeh Y-L, Day C-H, Chen Y-H, et al. Heat Killed Lactobacillus reuteri GMNL-263 Reduces Fibrosis Effects on the Liver and Heart in High Fat Diet-Hamsters via TGF-β Suppression. Int J Mol Sci 2015;16:25881–96.
ACCEPTED MANUSCRIPT doi:10.3390/ijms161025881. [147] Al-Muzafar HM, Amin KA. Probiotic mixture improves fatty liver disease by virtue of
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its action on lipid profiles, leptin, and inflammatory biomarkers. BMC Complement Altern Med 2017;17:43. doi:10.1186/s12906-016-1540-z.
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[149] Lee J, Kim Y, Friso S, Choi S-W. Epigenetics in non-alcoholic fatty liver disease. Mol
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Aspects Med 2017;54:78–88. doi:10.1016/j.mam.2016.11.008. [150] Marengo A, Jouness RIK, Bugianesi E. Progression and Natural History of Nonalcoholic Fatty Liver Disease in Adults. Clin Liver Dis 2016;20:313–24.
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doi:10.1016/j.cld.2015.10.010.
[151] Goh GB-B, McCullough AJ. Natural History of Nonalcoholic Fatty Liver Disease. Dig
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Dis Sci 2016;61:1226–33. doi:10.1007/s10620-016-4095-4.
ACCEPTED MANUSCRIPT Legend Fig. 1: Some fundamental mechanisms involved in the pathogenesis of non-alcoholic fatty liver disease (NAFLD), which can explain the complex pathophysiology of the disease.
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The significant contribution of gut microbiota in the development of the same pathology is separately depicted in light blue labels.
NASH, non-alcoholic steatohepatitis; FFA, free fatty acids; PNPLA3-I148M, palatine- like
Superfamily
member
2
gene;
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phospholipase 3- isoleucine to methionine at residue 148; TM6SF2, Transmembrane 6 GCKR-LYPLAL,
glucokinase
regulator
protein-
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lysophospholipase like protein; MBOAT7, membrane bound O-acyltransferase domain
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containing 7; FIAF, fasting-induced adipocyte factor; SCFA, short chain fatty acids; VLDL, very low-density lipoproteins; FXR, farnesoid X receptor; LPS, lipopolysaccharide; TLR-4,
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Toll Like Receptor 4.
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ACCEPTED MANUSCRIPT
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Fig. 1
ACCEPTED MANUSCRIPT Table 1. Probiotics and their beneficial effects in non-alcoholic fatty liver disease (NAFLD) Special effects according to observations from studies Probiotic Action Lactobacilli & Streptococci spp. Reduction of aminotransferases
a. Inhibition of bacterial translocation
Lactobacilli spp.
Attenuation of TLR4 inflammatory pathway and endotoxemia
b. Inhibition of epithelial invasion Bifidobacterium spp.
c. Maintenance of intestinal barrier integrity
Amelioration of liver steatosis and endotoxemia
Bifidobacterium spp.& Akkermansia muciniphila + oligofructose
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d. Induction of production of antimicrobial peptides
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General effects
Decrease of hepatic fatty accumulation and rest metabolic syndrome hallmarks as well as endotoxemia
Lactobacillus acidophilus, L. helveticus and Bifidobacterium spp. + prebiotic
f. Enhancement of hyperdynamic circulation state of cirrhosis
Improvement of alcohol-induced hepatic injury, endotoxemia and increased liver enzymes
Lactobacillus rhamnosus and paracasei, Bifidobacterium breve
Reduction of hepatic steatosis and TNF-α, LPS and IL-6
Clostridium butyricum strain MIYAIRI 588
Prevention of NAFLD progression and tumorigenesis
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e. Reduction of inflammation Shaping of host’s immune system
TLR-4,
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g. Aversion of the onset of hepatic encephalopathy
Toll
Like
Lactobacillus johnsonii La1
Reduction of bacterial translocation and attenuation of endotoxemia
Lactobacillus reuteri GMLN-263
Improvement of liver steatosis and insulin resistance & reduction of TNFα, IL-6 (adipose tissue) and HF (TGFβ depended)
VSL#3 Probiotics cocktail
Reduction of ALT, enhancement of insulin sensitivity, hepatic inflammation/steatosis, suppression of the TNFα/IKK-β signaling pathway, reduction of activity of Jun N-terminal kinase, decrease DNA binding activity of NF-κB
Receptor
4;
ΤΝFα,
Tumor
Necrosis
Factor-α;
LPS,
lipopolysaccharide; IL-6, Interleukin 6; HF, Hepatic Fibrosis; TGFβ, Transforming Growth Factor beta; ALT, Alanine Aminotransferase; IKK-β, inhibitor of κΒ (ΙκΒ) kinase-β; NF-κB, Nuclear Factor-kappa B
S0026-0495(17)30100-2 doi: 10.1016/j.metabol.2017.03.013 YMETA 53579
To appear in:
Metabolism
Received date: Accepted date:
23 February 2017 27 March 2017
Please cite this article as: Doulberis Michael, Kotronis Georgios, Gialamprinou Dimitra, Kountouras Jannis, Katsinelos Panagiotis, Non-alcoholic fatty liver disease: An update with special focus on the role of gut microbiota, Metabolism (2017), doi: 10.1016/j.metabol.2017.03.013
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ACCEPTED MANUSCRIPT Non-alcoholic fatty liver disease: An update with special focus on
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the role of gut microbiota
Michael Doulberisa, *, Georgios Kotronisb, Dimitra Gialamprinouc, Jannis Kountourasd
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and Panagiotis Katsinelosd
Bürgerspital Hospital, Department of Internal Medicine, Solothurn 4500, Switzerland
b
Agios Pavlos Hospital, Department of Internal Medicine, Thessaloniki, Macedonia, 55134,
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a
Greece
Papageorgiou General Hospital, Department of Pediatrics, Aristotle University of
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c
Thessaloniki, Macedonia, 56403, Greece d
Ippokration Hospital, Department of Internal Medicine, Second Medical Clinic, Aristotle
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University of Thessaloniki, Thessaloniki, Macedonia, 54642, Greece. *Corresponding author at:
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Department of Internal Medicine Bürgerspital Hospital, Schöngrünstrasse 38, Solothurn 4500, Switzerland Tel: +41(0)32 627 4036, Fax: +41(0)32 627 3079 E-mail: [email protected], [email protected]
ACCEPTED MANUSCRIPT Abbreviations: CDAA: choline-deficient/L-amino acid-defined, CK-18: Cytokeratin-18, CKD: chronic
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kidney disease, CR: calorie restriction, CRTC1: cAMP-responsive element binding protein (CREB)-regulated transcriptional coactivator, CVD: cardiovascular disease, EDC: endocrinedisrupting chemicals, FFA: (plasma) free fatty acids, FXR: farnesoid X receptor, GCKR:
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glucokinase regulator, HCC: hepatocellular carcinoma, HDL: high density lipoprotein, HF: hepatic fibrosis, HP: helicobacter pylori, HSC: hepatic stellate cells, IR: insulin resistance,
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LPS: lipopolysaccharide, MBOAT7: membrane bound O-acyltransferase domain containing
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7, MetS: metabolic syndrome, NAFLD: non-alcoholic fatty liver disease, NASH: nonalcoholic steatohepatitis, PNPLA3: palatine- like phospholipase 3, SCFA: short chain fatty acids, SNP: single nucleotide polymorphism, SS: simple steatosis, T2DM: type 2
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diabetes mellitus, TLR-4: Toll Like Receptor 4, TM6SF2: Transmembrane 6 Superfamily
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member 2 gene, UDCA: Ursodeoxycholic acid, VLDL: very low-density lipoproteins
ACCEPTED MANUSCRIPT Abstract Non-alcoholic fatty liver disease (NAFLD) is a significant global health burden in children,
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adolescents and adults with substantial rise in prevalence over the last decades. Accumulating data from manifold studies support the idea of NAFLD as a hepatic manifestation of metabolic syndrome, being rather a systemic metabolic disease than a liver confined
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pathology. Emerging data support that the gut microbiome represents a significant environmental factor contributing to NAFLD development and progression. Apart from other
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regimens, probiotics may have a positive role in the management of NAFLD through a
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plethora of possible mechanisms. The current review focuses on the NAFLD multifactorial pathogenesis, including mainly the role of intestinal microbiome and all relevant issues are raised. Furthermore, the clinical manifestations and appropriate diagnostic approach of the
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disease are discussed, with all possible therapeutic measures that can be taken, also including
Keywords
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the potential beneficial effect of probiotics.
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NAFLD, gut microbiota, probiotics, gastrointestinal bacteria, metabolic syndrome, obesity.
ACCEPTED MANUSCRIPT 1. Introduction Nonalcoholic fatty liver disease (NAFLD) has been recognized as a global public health
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problem, affecting 6-45% of the general population, rising up to 70% in patients with type 2 diabetes mellitus (T2DM) or 90% in morbidly obese patients [1,2]. NAFLD is among the leading causes of liver cirrhosis, hepatocellular carcinoma (HCC) and liver transplantation
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[3]. The definition of NAFLD includes the excessive accumulation of triglycerides in the hepatocytes in absence of parallel alcohol consumption (less than 20 and 30g per day in
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women and men, respectively) or other secondary etiological factors [4,5]. Multiple factors
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(“hits”) contribute to the pathogenesis of NAFLD, considered to be a multiple-hit disorder [6,7]. Pathogenetic factors are divided into those with an established association with NAFLD, including genetic [e.g., polymorphisms of patatine-like phospholipase domain-
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containing protein 3 (PNPLA3) gene] [8], dietary factors (e.g., fructose), insulin resistance (IR) [9] and adipokines [10], as well as those with a potential association needing validation, including endocrine disruptors [11] and dysbiosis of the gut microbiota [12].
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NAFLD encompasses a range of histological phenotypes. At the initial step, lipids are
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accumulated in the hepatocytes resulting in simple steatosis (SS). In a proportion of patients, SS progresses to nonalcoholic steatohepatitis (NASH), characterized by the addition of hepatic inflammation and/or fibrosis [9]. NASH may progresses to NASH-related cirrhosis and HCC more often than SS, which is regarded as a benign entity [9,13,14]. Currently, it is believed, that NAFLD is not only a liver confined pathology, but it is regarded as a multisystem disease with a hepatic component as well as extra-hepatic manifestations [1,4,15]; except for the liver-related morbidity, NAFLD is also related to extra-hepatic morbidity, including systemic metabolic complications (e.g. T2DM), thyroid dysfunction and osteoporosis [16], chronic kidney and cardiovascular disease (CVD), and malignancies, presumably leading to higher morbidity and mortality rates [4,5,17,18]. Important to note that IR has been proposed as the common pathogenetic mechanism in
ACCEPTED MANUSCRIPT conditions clustered under the term of metabolic syndrome (MetS), including obesity, T2DM, dyslipidemia, hypertension, and NAFLD, all of which increase the risk for the CVD and
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mortality, the endpoints of MetS [19,20]. Specifically, a connection between gastrointestinal bacteria and liver has long been suspected. The first observation linking liver and gut microbiota dates back to 1921, when
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Hoefert described patients suffering from chronic liver disease with altered gut flora [21]. Since then, emerging data support that the gut microbiome represents a significant
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environmental factor contributing to NAFLD development and progression [22–24]. In this
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review, we consider mainly the growing evidence that highlights the relationship between gut microbiome and its association with the entire spectrum of NAFLD from etiology to
2. Review Criteria
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treatment.
We performed a computerized literature search using the PubMed database, to identify
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relevant published articles regardless of the publication year, until January 2017. Only full-
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text papers written in the English language were reviewed. The following terms were used: “NAFLD”, “nonalcoholic fatty liver disease” or “nonalcoholic steatohepatitis” in combination with “gut microbiota”, “gastrointestinal bacteria” and “metabolic syndrome”. Further articles were identified via relevant paper analysis obtained from the initial literature search. Overall selected papers were used for the purposes of this review.
ACCEPTED MANUSCRIPT 3. Epidemiology An estimation of the world prevalence varies from 6 to 45% in the general population, rising
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up to 70% in patients with T2DM or 90% in morbidly obese patients with concurrent IR and hyperlipidemia [1,2,25]. NAFLD remains the most common cause of abnormal liver function in western countries [26–29] and is among the leading causes of liver cirrhosis, HCC and liver
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transplantation [3]. SS progresses to cirrhosis in less than 5% of cases, whereas NASH progresses to cirrhosis in 10-15% of cases over 10 years and in 25-30% of cases in the
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presence of advanced fibrosis [30].
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Age appears to play a significant role [1,4,5,18]. Highest prevalence is reported in adults with ages ranging from 40 to 49 years while age exceeding 60 years is associated with prevalence twice as much compared to 20 years of age [4,5]. The prevalence of NAFLD in children
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varies widely from 13% to 80% and the strongest risk factor affecting this discrepancy is obesity [27,31]. NAFLD affects more male gender and this preference also includes children [4,5,18,32]. Moreover, the prevalence of NAFLD varies noticeably when comparing
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individuals of different races and ethnicities. Rates are highest in Hispanic patient populations
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compared with non-Hispanic whites and African Americans, despite similar rates of the MetS and risk factors. [1,5,33]. This observation remains poorly characterized; variations in genes that effect lipid metabolism may play a role [4,33]. 4. Pathogenesis Multiple factors (“hits”) contribute to the pathogenesis of NAFLD, considered to be a multiple-hit disorder [6,7]. NAFLD is a complex disease mediated by several metabolic, environmental, genetic and microbiological mechanisms (Fig. 1). Pathogenetic factors are divided into those with an established association with NAFLD, including genetic (e.g., polymorphisms of PNPLA3 gene) [8], dietary factors (e.g., fructose), IR [9] and adipokines [10], as well as those with a potential association needing validation, including endocrine disruptors [11] and dysbiosis of the gut microbiota [12].
ACCEPTED MANUSCRIPT Human epidemiologic studies describing polymorphisms in a number of genes involved in metabolic dysfunctions have contributed to clarify the causes leading to the
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NAFLD evolution; family studies and inter-ethnic differences in susceptibility suggest that multiple genetic factors may be important risk determinants for progressive NAFLD. Polymorphisms in genes affecting lipid metabolism, adipocytokines, fibrotic mediators and
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oxidative stress may be associated with NASH and/or fibrosis, but most current findings require replication [6,34].
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A single nucleotide polymorphism (SNP), which gathers increasing evidence, and
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considered a key genetic factor in NAFLD pathogenesis, is the I148M (rs738409 C/G) of the mentioned PNPLA3, also known as adiponutrin [35]. The PNPLA3 gene represents the main and more acknowledged determinant of interindividual differences in hepatic steatosis and
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susceptibility to NASH [36]; PNPLA3 protein is found to act as a triglycerides hydrolase agent. Specifically, the I148M variant (isoleucine to methionine substitution, position 148) results in an altered PNPLA3 protein with an impaired activity in lipid catabolism, hence
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allowing the gathering of triglycerides to hepatic cells [37] by inducing disturbance in hepatic
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very low-density lipoproteins (VLDL) secretion [38]. Furthermore, this variant affects the hepatocyte triacylglycerol remodeling [39]. In a relative meta-analysis of 16 studies, I148M exerted a strong influence not only on liver fat accumulation, but also on the susceptibility of a more aggressive disease; GG homozygous have a 3.2-fold greater risk for necroinflammation and a 3.2-fold greater risk for fibrosis when compared with CC homozygous and, therefore, NASH is more frequently observed in GG than CC homozygous genotype [40]. Furthermore, carrying two G alleles does not increase the risk of severe disease, meaning that the influence of this variant on disease severity follows a dominant model. The observed effect of I148M SNP on the natural history of NAFLD is perhaps one of the strongest ever reported for a common variant modifying the genetic susceptibility of a complex disease [40]. The humans who are carriers of the PNPLA3 (I148M) allele seem to be
ACCEPTED MANUSCRIPT at greater risk for developing cirrhosis and HCC regardless of the prepossession for steatosis [41,42].
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Other SNPs have also been associated with NAFLD characteristics. A genome-wide associated study (GWAS) analyzed 324,623 SNPs from the 22 autosomal chromosomes; NAFLD activity score (NAS) was independently associated with the SNP rs2645424 on
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chromosome 8 in farnesyl diphosphate farnesyltransferase 1, the degree of fibrosis was associated with the SNP rs343062 on chromosome 7, and lobular inflammation was
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associated with the SNP rs1227756 on chromosome 10 in COL13A1, rs6591182 on
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chromosome 11 and rs887304 on chromosome 12 in EFCAB4B [34]. In addition, a genetic variant of TM6SF2 (Transmembrane 6 Superfamily member 2 gene), E167K variant, is regarded as another crucial determinant of liver triglyceride content, thereby associating with
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the hepatic damage attributing to fat accumulation in this organ [43,44]. GCKR (glucokinase regulator) and LYPLAL1 (agent of triglycerides catabolism) variants are also associated with liver damage through inducing increased hepatic glucose uptake [45,46]. Likewise, a variant
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of MBOAT7 (membrane bound O-acyltransferase domain containing 7) gene was recently
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found to be a locus that provides susceptibility for the development and progression of NAFLD [47]. In contrast, the cAMP-responsive element binding protein (CREB)-regulated transcriptional coactivator (CRTC1) (also known as transducer of regulated CREB, TORC), confers wide spectrum protection against the development of hepatic steatosis. CRTC1 directly interferes with the expression of genes regulated by lipogenic transcription factors, most prominently liver x receptor α. As a cyclic AMP effector, CRTC1 mediates anti-steatotic effects of calorie restriction (CR). Likewise, CRTC1 mediates anti-lipogenic effects of bile acid signaling, while it is negatively regulated by miR-34a, a pathogenic microRNA upregulated in a broad spectrum of NAFLD. These patterns of gene function and regulation of CRTC1 are distinct from other CR-responsive proteins, emphasizing critical protective roles
ACCEPTED MANUSCRIPT that CRTC1 selectively plays against the development of NAFLD, which in turn offers novel opportunities for selectively targeting beneficial therapeutic CR effects [48].
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Dietary factors are playing an extra role in hepatic lipogenesis and therefore in NAFLD and later on in NASH. Overconsumption of high-fat diet and increased intake of sugar-sweetened beverages are major risk factors for development of NAFLD [49]. Dietary
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fat directly supplies free fatty acids (FFA) to the liver. Furthermore, the metabolism of carbohydrate results in FFA synthesis from acetyl CoA. Consumption of excessive glucose
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also promotes liver lipogenesis through the activation of a carbohydrate responsive binding
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protein [13,14]. Moreover, it is recognized that saturated fatty acids appear to be more toxic than the unsaturated ones, through the promotion of apoptosis and liver injury as well as endoplasmic reticulum stress [50–52]. In contrast, treatment with n-3 long-chain
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polyunsaturated fatty acids induces variable success in improving NAFLD [53]. NAFLD appears to be the hepatic component of Mets. In this regard, IR has been proposed as the common pathogenetic mechanism in MetS, including obesity, T2DM,
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dyslipidemia, hypertension, and NAFLD, all of which increase the risk for CVD and
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mortality, the endpoints of MetS [19,20]. Mets is defined according to NCEP AT III (2005 revision) as the presence of at least three of the five following criteria: Obesity (waist circumference >40 inches ♂, >35 inches ♀), hypertension (>130 mmHg systolic, or
>85 mmHg diastolic or even pharmacologic
treatment), fasting glucose ≥100 mg/dl or pharmacologic treatment, hypertriglyceridemia (>150 mg/dl or pharmacologic treatment) and low high density lipoprotein (HDL) (<40 mg/dl ♂,
<50 mg/dl ♀ or pharmacologic treatment) [54]. The main pathogenetic mechanism
leading to steatosis is the continuing lipolysis and therefore the increased amount of plasma FFA due to IR in the adipose tissue [14]; insulin, when present and active, is an anabolic hormone and prevents from lipolysis. The elevated plasma FFA flow to the liver and this ectopic fat accumulation is associated with increased secretion of hepatokines [55], increased
ACCEPTED MANUSCRIPT gluconeogenesis, inhibition of insulin signaling and reduced glycogen production [56,57]. A reduction of hepatocellular triglycerides secretion through VLDL and an impaired fatty acids
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oxidation are also associated with hepatic fat accumulation [58]. In this respect, it has been estimated with an isotope technique that, in the presence of steatosis, approximately 60% of the hepatic triglycerides comes from the adipose tissue, 25% from de novo lipogenesis and
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15% from dietary lipids [59]. This study clarified the impact of adipose tissue lipolysis on hepatic steatosis. It is highlighted that the percentage of hepatic triglyceride content derived
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from de novo lipogenesis in individuals without steatosis is lower, which is mainly attributed
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to lower IR [9]. Furthermore, excessive intraperitoneal fat can cause FFA reflux into the liver directly via the portal vein [60]. The excessive liver lipid accumulation causes further worsening of IR and chronic inflammation driving to liver disease. This is caused by
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metabolites of FFA that relocate cytoplasmic protein kinase C to the membrane, resulting in phosphorylation of intracellular insulin receptors, which constitutes the main defect in IR [14].
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The adipose tissue inflammation plays a key role in the pathogenesis of NAFLD and
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the intestinal dysbiosis seems to be an additional crucial factor leading to and sustaining the disease. Apart from fats, bacterial endotoxins are absorbed by the intestine leading to further regulation of liver inflammation [61]. As the disease progresses, the hepatocyte injury drives to the development of steatohepatitis, hepatocyte necrosis and apoptosis with the contribution of oxidative stress and peroxidation as well as mitochondrial dysfunction and endoplasmic reticulum stress [14,17,62]. Mechanisms involved in lipotoxicity and glucotoxicity include endoplasmic reticulum stress, oxidative stress and mitochondrial impairment, which all promote the progression of SS to NASH [7,63].The procedure of further NASH progression is mediated by activation of hepatic stellate cells (HSCs), collagen deposition to the liver and consequently fibrosis and cirrhosis. HCC is the final stage of all these mechanisms [17].
ACCEPTED MANUSCRIPT The progression to NASH also involves increased infiltration of different subtypes of immune cells, such as macrophages, monocytes, T-lymphocytes and neutrophils, to the liver,
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as well as activation and expansion of liver immune cells, including Kupffer cells and the mentioned HSCs [64]. When counterbalancing mechanisms do not successfully limit hepatic steatosis, and even more when obesity deteriorates, intra-hepatic inflammation process starts,
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perhaps as a counterbalancing mechanism to limit steatosis [7]. During the inflammatory process, the immune cells produce cytokines and other mediators contributing to
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inflammation [9]. It also appears that a cross-talk exists between infiltrating immune cells
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and liver-residing ones (Kupffer cells and HSCs) [65]. When the inflammation prolongs, the process of hepatic fibrosis take place. The fibrosis is a major prognostic and a hard therapeutic histological end-point, because the severity of fibrosis predicts the severity of
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advanced disease, i.e. liver cirrhosis and HCC [6]. Since the discovery of adipose tissue as a highly active endocrine tissue, the adipokines produced by adipose tissue and exerting autocrine, paracrine and endocrine
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function, have been involved in the pathogenesis of various obesity-related diseases,
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including NAFLD. Data concerning the association between NAFLD and circulating leptin and adiponectin levels are generally well documented: leptin levels increase, whereas adiponectin levels decrease, by increasing the severity of NAFLD. Data concerning other adipokines in histologically confirmed NAFLD populations are inconclusive (e.g., resistin, visfatin, retinol-binding protein-4, chemerin) or limited (e.g., adipsin, obestatin, omentin, vaspin etc.). A thorough insight into the pathophysiologic mechanisms linking adipokines with NAFLD might lead to the design of studies investigating the combined adipokine peptides use as noninvasive diagnostic markers of NAFLD and novel clinical trials targeting the NAFLD treatment [10]. Endocrine disruptors or endocrine-disrupting chemicals (EDCs) constitute a highly heterogeneous group of molecules found in the environment or in consumer products.
ACCEPTED MANUSCRIPT Relative studies have suggested the diverse EDCs involvement in an increasing number of metabolic disorders, IR and IR-related co-morbidities, including obesity, T2DM, polycystic
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ovary syndrome and NAFLD [11,66]. Certain EDCs, including dioxins, bisphenol A, phthalates and other persistent organic pollutants may induce hepatic histological alterations similar to those observed in NAFLD, directly through a hepatotoxic effect and/or indirectly
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by triggering hepatic and systematic IR [11]. As a consequence, EDCs, acting in concert with other pathogenetic factors (“hits”), may play a role in the pathogenesis of fatty liver, thereby
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affecting the prevalence of obesity and NAFLD [7].
4.1 The role of gut microbiota in NAFLD
The human microbiota consists of 100 trillion microorganisms that provide essential
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metabolic and biological functions benefiting the host. However, the presence in host plasma of a gut-derived bacteria component, the lipopolysaccharide (LPS) has been recognized as a causal or complicating factor in several diseases such as obesity-associated metabolic
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disorders including NAFLD [67]. The term gut microbiota is used to define the whole
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population of bacteria, viruses, parasites as well as fungi, which colonize almost the entire gastrointestinal tract from mouth to colon [68]. There are more than 2,000 species of bacteria, including anaerobes, within the adult human gut. The density of bacterial cells increases generally from stomach to colon with a number varying from 10 – 103 (per gram of content) in the gastric – duodenal mucosa and reaching a maximal number of up to about 1014 in the colon, and weigh about 1.5 kg [22,69]. Despite the wide variety of aforementioned microorganisms, the vast majority of them (up to 95%) in human and generally mammalian intestine consists of only three bacterial phyla: Bacteroidetes, Actinobacteria and Firmicutes [70,71]. In healthy organisms gut microbiota contribute to maintenance of homeostasis, via a symbiotic mechanism, where both parties, bacteria and host benefit. A disturbance of this relationship leads to changes of the balance between beneficial and pathogen bacteria, a
ACCEPTED MANUSCRIPT condition termed as “dysbiosis” [61,68,72]. Dysbiosis may adversely impact metabolism and immune responses, favoring obesity and obesity-related comorbidity, including IR and
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NAFLD [12]. Specifically, the existence of the so-called “liver – intestine axis” has been identified and described for several decades. A plausible explanation of this connection is that liver
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receives directly about 70% of the venous intestinal outflow via portal vein, thereby serving as a first filter with a great -among others- detoxifying role [22,68,73]. Moreover, studies
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with animal models have shown strong evidence that indicates a role for bacteria in the
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development of NAFLD (Fig. 1). Germ free mice did not develop obesity, which as previously showed is linked to NAFLD, despite being fed with high- fat and sugar diet. The colonization of their guts with microbiota taken from conventionally raised mice, resulted in
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an increase of IR and body fat content, although food intake was also reduced [22,71]. In this respect, there are two mutually antagonistic phyla, the Bacteroidetes (Gram -) and the Firmicutes (Gram +) of microbiota [7]. While exceptions exist, Firmicutes are considered as
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“obese” microbiomes, whereas the bacteroidetes as the “lean” microbiomes, because the
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former were found to contain more genes associated with lipid and carbohydrate metabolism and the breakdown of otherwise indigestible polysaccharides [74]. In this regard, initial studies showed that ob/ob mice have a 50% reduction in the Bacteroidetes and a proportional increase in Firmicutes compared with lean mice [75]. Apart from genetic models of obesity, wild-type mice had a higher proportion of Bacteroidetes and lower of Firmicutes, when they were lean (on a chow diet) than obese (on a high fat diet) [76]. Possible mechanisms connecting gut microbiota with obesity and NAFLD are: a) Augmented energy extraction from food nutrients which initially are not digested because of the host limited capability to digest complex dietary polysaccharides [7,12].
ACCEPTED MANUSCRIPT b) Fermentation of polysaccharides by microbiota into monosaccharides and shortchain fatty acids (SCFAs), such as acetate, propionate, butyrate and ethanol, linked with
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NAFLD; [9] c) Increased intestinal permeability, thereby more microbiota-produced endotoxins and SCFAs accessing the liver; [7]
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d) Decreased choline metabolism leading to decreased VLDL export from the liver;[7] e) Modulation of bile acid synthesis, which are crucial for fat absorption, but also
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affect metabolism of glucose and lipoproteins by linking farsenoid X receptor (FXR)
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[12].
More specifically, a hallmark of gut microbiota with a substantial contribution to energy production is their ability to anaerobically ferment the non-digestible plant
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polysaccharides to SCFA such as butyrate, acetate and propionate, which can then be absorbed by enterocytes [77,78]. SCFA has been shown to reduce the permeability of tight junctions and increase mucus production, thereby enhancing the function of gut as barrier
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[79]. Additionally, SCFA seem to be key-players for the intestinal hormonal regulation, since
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they induce an increase of peptide YY and glucagon-like peptide, both of which influence satiety and improve oral glucose tolerance, insulin sensitivity as well as leptin levels [68,70]. Besides, intestinal microbiota are known for their ability to shape energy demands of the host and thus contribute to development of obesity and obesity- associated diseases, such as NAFLD. Germ free mice gained less body weight compared to their controls with normal gut flora, although both groups were fed with identical diet. This property of body weight gain by microbiota was transferable after bacteria transplantation and was pathogenetically mediated by enhancement of absorption of monosaccharides by small intestine epithelial cells (and thus fatty acids optimization synthesis) and parallel by suppression of fasting-induced adipocyte factor in aforementioned cells, which results in fat accumulation and development of obesity [80]. Gut microbiota have been also connected with NAFLD through the water-soluble
ACCEPTED MANUSCRIPT nutrient choline. The latter can both be ingested by animal products such as eggs and red meat, but also biosynthesized by intestinal microbiota [79].
In colon takes place the
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hydrolysis of choline to dimethylamine and to trimethylamine, both of which regarded as precursors of dimethynitrosamine, a potent hepatotoxin and liver-carcinogen [22]. Chemical catalysis is mediated by bacterially produced enzymes [73]. Moreover, choline seems to play
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a critical role in the synthesis of VLDL, which are needed for the export of lipids from liver [24]. Choline deficiency has been also shown that leads to chronic liver disease [71].
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Bile acids, along with phospholipids, biliverdin and mucous are the main components of bile,
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which multifactorially contribute to homeostasis in the small bowel through the regulation of metabolism of cholesterol, release of fat-soluble vitamins, as well as through emulsification of fats. Moreover, certain bile acids are known to act as pathway activators/suppressors for
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several host receptors, which are primarily found in intestinal cells or in cells of distant tissues and organs such as fat and liver, respectively [70,79]. The so-called nuclear bile acids receptor, FXR, is implicated in the pathogenesis of microbiota-related NAFLD. Activation of
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FXR by bile acids, which serve as signaling molecules, induces a cascade of biochemical
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reactions with end-result the suppression of bile production [77]. A relative study [81] by using a murine model of NAFLD, showed that the administration of antibiotics to mice fed with high fat diet, was able to alter the composition of bile acids, which in turn led to more conjugated bile acids and resulted in inhibition of intestinal FXR signaling as well as to less accumulation of triglycerides in liver. Similar results were observed in another relevant study [82], where microbiota-associated bile acids’ deconjugation induced a promotion of NAFLD in a murine model of obesity. The pathogenetic mechanism involved acceleration of lipid synthesis through activation of intestinal FXR signaling pathway. Furthermore, a recently published study [83], showed that the modulation of gut microbiota, mediated through bile acid binding resin, could significantly reduce adiposity in mice fed with high high-fat diet. This beneficial effect was associated with an increase of Clostridium
ACCEPTED MANUSCRIPT leptum as well as of Bacteroides-Prevotella group in mice fed high-fat diet and treated with bile acid binding protein. Moreover, a statistically significant elevation of fecal propionate
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was also observed in the aforementioned group. Another well-documented mechanism, which is known to be involved in development and progression of NAFLD by intestinal microbiota, is IR and especially the so-called LPS– Toll
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Like Receptor-(TLR)-4–monocyte differentiation antigen CD14 system [73,84]. Although this knowledge is largely derived from animal model studies [73], there is one study
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mentioning a substantial elevation of LPS plasma levels in patients suffering from T2DM,
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compared to healthy matched controls [85]. Furthermore, a reduction of fasting insulin as well as of IR can be seen in modification or suppression of small intestine bacterial overgrowth, which also results in a decrease of pro-inflammatory cytokines [73].
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Additionally, small intestinal bacterial overgrowth and bacterial translocation represent two rather common pathologies in cirrhotic patients and their prevalence has been directly correlated with the severity of hepatic disease [70,73].
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The few human studies also reported gut microbiota association with NAFLD [12].
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There are certain general lines derived from human studies: [7,61] a) As in the animal studies, obese individuals have a greater prevalence of Firmicutes and lower prevalence of Bacteroidetes than lean individuals; b) NASH patients have a lower prevalence of Bacteroidetes than obese individuals without NASH; c) the environment appears to determine most of the interindividual variability in the gut microbiota [61]. Although human studies are limited by their observational nature, small sample size and the diversity of gut microbiota identified, the indicated associations provide the basis for future large-scale studies. Apart from the classic gut microbiota, Helicobacter pylori (Hp) may also affect the pathogenesis of NAFLD, since it may colonize gut itself, but also might affect the diversity of gut microbiota [20]. Some clinical observational studies and animal studies support the
ACCEPTED MANUSCRIPT relationship of Hp infection (Hp-I) and dysbiosis of gastrointestinal tract (GIT) microbiota to metabolic disorders like IR and diabetes [86,87]. Dysbiosis of GIT microbiota has been
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involved in the development of the mentioned several human MetS disorders like obesity, NAFLD, and IR in T2DM; certain microbes, such as butyrate-producing bacteria, are reduced in T2DM patients; gastric and fecal microbiota may have been changed in Hp-infected people
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and mice to promote gastric inflammation and specific diseases; and Hp-I also induced IR, defined as a predisposing factor to T2DM development [88]. Moreover, higher rates of anti-
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Hp IgG were reported in NAFLD patients compared to controls in a cross-sectional study
pathogenetic basis of NAFLD [90].
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[89], but also an association between Hp infection and the mentioned IR, being the
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5. Clinical manifestations
The great majority of patients with NAFLD remain asymptomatic or may report nonspecific symptoms, especially fatigue [17,91]. As the disease progresses to NASH and later on to
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cirrhosis, the enlargement of the liver leads to a feeling of weight and discomfort or even pain
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in the right upper quadrant of the abdomen. But even then, these symptoms are not specific and therefore the disease is undetected until the clinician suggests an imaging study and/or blood chemistry, which reveals abnormal liver tests [91,92]. A physical examination reveals usually central obesity and sometimes hepatomegaly in NAFLD patients. Manifestations of diabetes such as diabetic dermopathy, necrobiosis and lipoidica diabeticorum or clinical manifestations of dyslipidemia such as xanthelasmata and xanthomas may also be recognized [93]. However, none of these findings are pathognomonic though should arouse the clinicians’ suspicion for the presence of NAFLD. Over time, when the disease advances to NASH, more specific clinical signs such as a dorsocervical (buffalo) hump and acanthosis nigricans (implying IR) are present in the physical examination [94]. When cirrhosis developed, the clinical findings become more apparent and include palmar
ACCEPTED MANUSCRIPT erythema, caput medusae (palm tree sign), spider angiomata (spider nevi), reduced muscular mass, gynecomastia, Dupuytren’s contracture and even jaundice, ascites, splenomegaly,
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edema [95]. Moreover, systemic clinical manifestations due to NAFLD-related CVD and/or kidney disease may also be recognized.
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6. Diagnosis
It is widely accepted that NAFLD is mostly diagnosed by exclusion of other possible causes
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of chronic liver disease. There are recognized three criteria for the appropriate establishment
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of diagnosis. i. Absence of significant consumption of alcohol (less than 20g or 30g daily in women and men, respectively), ii. Imaging or histological findings of liver steatosis and iii. Exclusion of other possible causes of hepatic inflammatory disease, including hereditary
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(such as Wilson’s disease or hemochromatosis) or acquired etiologies (such as viral hepatitis, hepatotoxic drugs, alcoholic liver disease or autoimmune diseases) [96,97]. Most clinicians suspect NAFLD if a patient presents with abnormal liver function tests,
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especially alanine transaminase (ALT), aspartate aminotransferase (AST) and gamma-
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glutamyl transferase. Although serum enzymes are often found to be normal even in advanced liver disease [98–100], the detection of mildly or moderately elevated enzymes are the most common first step for the diagnostic approach for NAFLD. Several studies on serological surrogate markers are used to distinguish NASH from the inflammation of SS, and single serological test methods to predict NASH have also been studied. Although the recommended surrogate markers TNF-α, IL-6, CRP, Pantraxin, Ferritin, SPEA, and sRAGE predict hepatic inflammation, most of these markers still require extensive external validation. The single test most studied in regard to the diagnosis of NASH is Cytokeratin-18 (CK-18). The CK-18 fragment, a marker of hepatocyte apoptosis, may predict NASH, which is significantly increased compared with normal or simple steatosis. CK-18 showed relatively good results in some precedent studies, suggesting the potential for
ACCEPTED MANUSCRIPT screening NASH. However, CK-18 in clinical application demonstrates a large variation in cut-off values compared to previous studies, and its sensitivity and specificity to predict
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NASH was not satisfactory [101]. Interestingly, a noninvasive marker, named HSENSI [acronym of homocysteine, SGOT (serum glutamic oxaloacetic transaminase), ESR (erythrocyte
sedimentation
rate),
Nonalcoholic
Steatohepatitis
Index],
consists
of
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aforementioned three low cost, easily measurable parameters and may accurately predict
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NASH [102].
In some cases, the clinician suspects the disease after detecting liver brightness and/or
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vascular blurring in abdominal ultrasound testing [5]. However, diagnostic sonography is a subjective tool providing only poor sensitivity for the detection of mild steatosis (sensitivity
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93% for steatosis >33%, but poor sensitivity for steatosis <30%), thereby introducing sonography only as an initial diagnostic approach [103,104]. CT scan (non-contrast and contrast enhanced) provides more sensitive information, allowing to measure the so-called
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liver-to spleen attenuation ratio (when measured below 0.9, the diagnosis of steatosis is certain), with the disadvantage of exposure to radiation [5]. The latter can be overcome with
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MR imaging but the increased cost of this method makes it not widely used yet. Of note, proton density fat fraction measurement by MRI is currently the most accurate and sensitive imaging method, simpler and more practical than magnetic resonance spectroscopy, but restricted, up to now, just to research and clinical trials. Although the histopathological evaluation of liver biopsy samples is central (gold standard) in the diagnosis of NAFLD, it remains a rather expensive invasive method hiding a possible risk of morbidity and rarely mortality; semi-quantitative histological scoring systems have been recommended for NAFLD, though they are not useful in clinical practice and each has certain limitations. Thus, it is not recommended for all patients with NAFLD but should be considered for those who are at great risk for developing NASH and advanced fibrosis (Strength – 1, Evidence – B), or those patients with suspected NAFLD in whom the clinical
ACCEPTED MANUSCRIPT and laboratory evaluation cannot safely exclude other causes of chronic liver disease (Strength – 1, Evidence – B) [97]. The histological examination offers significant prognostic
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information that can determine consequent management of the disease. Apart from liver biopsy, using the NAFLD Fibrosis Score, a non-invasive scoring system, can also offer a prediction of the severity of fibrosis. This score is based on the evaluation of six variables
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(hyperglycemia, Body Mass Index, age, platelets, AST/ALT and serum albumin) and provides assistance the clinician to identify the proportion of NAFLD patients with fibrosis,
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thus renders liver biopsy rather not necessary for the rest [105]. Furthermore, non- invasive
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screening of liver fibrosis can be obtained by elastography (US-based or transient, MRbased). This method provides assessment of degree of steatosis and the severity of fibrosis. However it is still expensive and is currently used on a limited basis [106]. Transient
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elastography appears to be an economically attractive alternative to liver biopsy and other non-invasive diagnostic tests especially for patients with a higher degree of liver fibrosis
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7. Therapy
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[107].
The therapeutic approach of NAFLD/NASH has been thoroughly discussed within the past decade and several strategies have been proposed for that purpose. However, currently, there is no pharmacological therapy globally approved due to lack of sufficient evidence. The first goal to control NAFLD/NASH is early detection of the disease, in order to apply proper and prompt interventions for the prevention of further progression as well as to achieve a possible regression of the hepatic damage. There is unanimously accordance that lifestyle interventions represent the most crucial first therapeutic approach; lifestyle interventions involving exercise and weight loss are currently the only accepted treatments for this disease [108]. Dietary modification includes reduction of sugar consumption and saturated fat as well as potential increase of omega 3 fatty acid uptake [109]. Based on available data, dietary
ACCEPTED MANUSCRIPT recommendations should consider energy restriction (500–1000 kcal/day) and elimination of NAFLD-promoting components (processed foods, and foods and beverages high in added
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fructose) [110]. While the reduction of liver fat appears to be driven mainly by calorie restriction and not the macronutrient composition of the diet, the Mediterranean diet is recommended by the recent guidelines [110]. Furthermore, excessive alcohol drinking (>30
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g/day for men and >20 g/day for women) is discouraged, whereas coffee is not restricted [7,110].
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Moreover, aerobic exercise (a moderate program 3 to 4 times per week) is recommended because of its positive effects in the metabolic parameters implicated in the development and
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progression of NAFLD/NASH, although exercise’s impact on liver histology is not well clarified yet [97,111]. However, recent guidelines do not recommend omega 3 fatty acids
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because of lack of enough evidence [112,113]. The above-mentioned interventions consist the major initial steps for the disease management. It is noteworthy that in the recent EASL-EASD-EASO Clinical Practice Guidelines, healthy
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diet and physical activity (including aerobic and resistance training) are recommended alone
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without pharmacotherapy for patients with no evidence of NASH or fibrosis [112]. In one published study, the exercise and diet together were found to be more efficient than metformin or rosiglitazone in normalizing ALT in NAFLD [114]. Both dietary changes and exercise offer the basis for weight reduction, which generally reduce hepatic steatosis. A goal of 5-10% weight loss is proposed for a period of 6 months. Steatosis improves after a 3-5 % minimum weight reduction while a greater reduction (up to 10%) can offer favorable effects on reducing necro-inflammatory process [97]. Furthermore, the liver fat reduction has been found to be related to the intensity of the lifestyle modification [115]. Recent guidelines recommend that the choice of training should be tailored based on patients’ preferences, so as to increase the possibility for long-term maintenance [110]. Although no data are available on their long-term effects on the natural history of NAFLD, a management combining dietary
ACCEPTED MANUSCRIPT counseling and a progressive increase in aerobic exercise/resistance training is preferable and
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should be individually tailored, targeting to progressive weight loss of 7-10% [7,110].
7.1 Pharmacological treatment
Although many pharmacological regimens assessed by randomized clinical trials have been
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proposed for the management of NAFLD, no drug has been tested in phase III clinical trials.
pharmacological agent for such a purpose.
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Therefore, there are no official and peremptory recommendations for any specific
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Therapeutic options for NAFLD aim to: improve IR via weight loss and exercise, or pharmacologically by using anti-obesity medications (i.e., orlistat, sibutramine) or insulinsensitizing agents (i.e., metformin, pioglitazone, rosiglitazone); decrease oxidative stress by
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the use of cytoprotective and/or antioxidants (i.e., vitamin E, vitamin C, betaine, Nacetylcysteine, ursodeoxycholic acid); decrease serum lipids by using of lipid-lowering agents (i.e., statins, fibrates, ezetimibe, probucol, omega-3 fatty acids); and act via other
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mechanisms, including angiotensin receptor blockers (i.e., losartan), TNF-α inhibition
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(pentoxifylline), endocannabinoid receptor antagonism (rimonabant), glucagon-like peptide-1 analogues (i.e., exenatide and liraglutide) and dipeptidyl peptidase-4 inhibitors [6,116]. Specifically, based on large trials, thiazolidinediones (pioglitazone) offer a significant benefit in liver histology. Therefore, they should be recommended for selected patients suffering from advanced liver fibrosis, especially those with T2DM [97,111–113] considering the possible adverse effects of that regimen (weight gain, possible worsening of heart failure, yet unknown long-term effects); pioglitazone use has been suspended in some European countries, because of possible slight increase in bladder cancer risk after long-term use [6]. Metformin use has not been proven to have beneficial effects on liver histology. Therefore, metformin is not recommended as a specific treatment for hepatic disease in individuals with NASH [14,97,111,112]
ACCEPTED MANUSCRIPT Vitamin E constitutes another agent that has been investigated for NAFLD and clinical trials have demonstrated its efficacy in improving steatosis, hepatic inflammation and decreasing
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liver enzyme levels in non-diabetic patients [117,118]. Its benefit is considered to lie on antioxidative activity in free radical reactions, reducing the oxidative stress-derived hepatocellular injury and progression of NASH [119,120]. Clinicians are encouraged to
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suggest vitamin E consumption to the group of patients diagnosed with non-diabetic, noncirrhotic NASH [14,97,112]. The long-term safety of high-dose vitamin E remains under
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assessment. Some experts suggest the combination of vitamin E with pioglitazone
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irrespectively of the presence of T2DM. Important to note that, although the effect of vitamin E on NAFLD nonalcoholic fatty liver diseases has been proven, this drug has not been approved in most countries [101].
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Pentoxifylline is also regarded as a compelling antioxidant and anti-TNF-α agent that seems to improve steatosis and lobular inflammation [121] though there is insufficient relative data and further studies are required to support or evade pentoxifylline’s formal use for
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NAFLD/NASH handling. Ursodeoxycholic acid is not recommended for the management of
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NAFLD or NASH as it has achieved to offer only slight reduction in liver function tests and steatosis without histological improvement over placebo in patients with NASH [122,123]. The effect of obeticholic acid on NAFLD has been proven, but this drug has not been approved in most countries [101]. In the large subgroup of patients with hypercholesterolemia, the use of statins (HMG-CoA reductase inhibitors) or ezetimibe is regarded to be safe with positive impact on reducing LDL cholesterol and cardiovascular risk and possible capacity in enhancing liver enzyme levels but without any evidence in improving liver histology. Both agents can be used to treat hypercholesterolemia in individuals with NAFLD/NASH, but their use specifically for NAFLD/NASH is not yet recommended [97,111,112]. However, a recent interesting review written by an expert panel recommended that certain NAFLD/NASH patients who are at a
ACCEPTED MANUSCRIPT high-risk for developing serious complications such as CVD or HCC should be treated with statins, optimally combined with ezetimibe and pioglitazone, for the primary or secondary
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prevention of CVD, the main cause of death in NAFLD/NASH patients, and the avoidance of cirrhosis, liver transplantation or HCC. Nevertheless, the authors have emphasized that additional carefully-designed randomized relative trials are warranted to substantiate the
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usage of the aforementioned regimens for the treatment of NAFLD/NASH [124]. Another potential therapeutic tool, Orlistat, a reversible pancreatic and intestinal lipase
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inhibitor, leads to reduction of liver function tests and steatosis, though the available data
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remain limited. Given the limited effect of orlistat on weight loss (3-5 kg more than placebo) and the need for long-term use to sustain its results, further evaluation of orlistat in NASH is debatable [125].
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GLP-1 receptor agonists, including exenatide and liraglutide, offer good glucose control providing at the same time lowering of body weight and promoting insulin sensitivity. Clinical and experimental data claim that these agents and/or DPP IV inhibitors can consist a
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new pharmacological tool for the management of NAFLD/NASH [126,127].
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Increased serum ferritin levels have been linked to liver inflammation, IR and worsening of fibrosis in patients with NAFLD/NASH [128]. There is limited evidence that reducing iron stores (through a phlebotomy programme) in certain patients has positive effects on IR, liver function markers and NAS score [129,130]. Nevertheless, iron reduction therapy is not currently recommended for NAFLD/NASH [97,111–113]. Other ongoing studies investigate variable NAFLD therapeutic drugs such as: NOX-1/4 Inhibitor (GKT137831, Genkyotex); Galectin-3 inhibitor (GR-MD-02, Galectin Therapeutics, Phase II); CCR2 and CCR5 inhibitor (Cenicriviroc, Tobira, Phase IIb); Pan-caspase inhibitor (Emricasan, Conatus Pharmaceuticals, Inc., Phase IIa); PPAR-α/δ agonist (GFT505, Genfit, Phase IIb); SCD-1 inhibitor (Aramchol, Galmed, Phase IIb); Apoptosis signal-regulating
ACCEPTED MANUSCRIPT kinase 1 inhibitor (GS-4997, Gilead, Phase II); and Lysyk oxidase-like 2 inhibitor (Simtuzumab, Gilead, Phase IIb) [101].
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Bariatric surgery might be introduced in morbidly obese individuals (BMI ≥40 or BMI ≥35 kg/m2 with obesity-related comorbidities) who are motivated to lose weight and who have not responded to lifestyle changes with or without pharmacotherapy with sufficient weight loss.
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A network meta-analysis of 31 randomized trials showed that all bariatric techniques were effective in reducing weight as compared with standard care [7]. The prospect of foregut
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bariatric surgery should be discussed as a measure aiming to decrease body weight and
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metabolic complications, mainly regarding obese and diabetic patients without adequate response to lifestyle modification and/or pharmacological management [111,112,131]. Steatosis, necroinflammation and fibrosis are likely to improve after bariatric surgery [132–
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134]. A 5-year prospective cohort study with 1236 severely obese individuals subjected to Roux-en-Y gastric bypass or adjustable gastric banding, showed improvement of liver function tests, IR and histological end-points (hepatic steatosis and NAS) at one year, with
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stable results up to five years; importantly, fibrosis regressed in six of 13 patients with
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bridging fibrosis at baseline and disappeared in two patients [133]. However, the clinician should take into account possible perioperative complications. Finally, end-stage patients with NASH (cirrhotic stage) or even HCC can benefit by liver transplantation gaining an overall survival equivalent to those who undergo liver transplantation for other causes of hepatic failure [135–137].
ACCEPTED MANUSCRIPT 7.2 Microbiome and treatment of NAFLD Probiotics represent a relatively new and promising therapeutic option for patients suffering
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from NAFLD. According to WHO, probiotics, defined as “ live microorganisms, when administered in adequate amounts, confer a health benefit on the host” [79]. Prebiotics are called the non-digestible food substances, like oligofructose or lactulose, which can promote
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the growth of beneficial bacteria [24,73]. Synbiotics is a term that is commonly used in the literature to describe the combined administration of both categories [70].
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Probiotics are postulated to be beneficial for the treatment of NAFLD and generally of
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chronic hepatic damage through a plethora of suggested mechanisms: they inhibit bacterial translocation as well as epithelial invasion by maintaining the intestinal barrier integrity; and they induce the production of antimicrobial peptides, reducing inflammation and thereby
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shaping immune system of host. Further, they enhance the hyperdynamic circulation state of cirrhosis and avert the onset of hepatic encephalopathy [79,84]. Commonly known bacteria serving as probiotics include spore forming and lactic acid bacteria, such as Bifidobacteria or
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Clostridium/Bacillus bacteria [4].
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An early meta-analysis of four randomized trials revealed that probiotics improved liver function tests, IR, TNF-α, total cholesterol and HDL cholesterol (HDL-C) in NAFLD patients, but did not affect BMI [138]. A more recent meta-analysis of nine randomized trials confirmed that probiotics improve liver function tests (especially in children), IR, TNF-α, triglycerides, total cholesterol and HDL-C in NAFLD patients, without affecting BMI [139]. Specifically, while more than one type of probiotics were studied for NAFLD
treatment,
most
of
these
regimens
included
combinations
of Bifidobacteria and Lactobacilli [140]. The administration of Lactobacilli alone or combined to Streptococci led to reduction of liver function tests [68]. Moreover, the administration of Lactobacilli species was able to attenuate TLR-4 inflammatory pathway and endotoxemia. In a clinical trial, Bifidobacterium has been demonstrated to ameliorate liver
ACCEPTED MANUSCRIPT steatosis and endotoxemia, but only when coupled to fructo-oligosaccharides [68]. The same effect could be observed in animal models, where the restoration of Bifidobacteria or
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Akkermansia muciniphila levels along with oligofructose supplementation in mice fed with high-fat nutrition, reduced hepatic fatty accumulation and rest metabolic syndrome hallmarks as well as endotoxemia [24]. Besides, in an interesting recent meta-analysis, it has been found
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that patients who received probiotics prior to liver transplantation had substantially decreased rates of infections and hospital accommodation [141]. The administration in mice fed with a
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high-fat diet of a multi-strain probiotic preparation commercially named as VSL#3 and
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composed of Streptococcus thermophiles, Lactobacillus delbreckii subsp. Bulgaricus, L. acidophilus, L. plantarum, L. casei, Bifidobacterium infants, B. breve and B. longum, led to multiple favorable effects: reduction of serum ALT levels; enhancement of insulin sensitivity;
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hepatic inflammation and steatosis reductions, which were mediated by modulation of hepatic natural killer T cells, suppression of the TNFα/IKK-β signaling pathway and reducing activity of Jun N-terminal kinase as well as by decreasing DNA binding activity of NF-κΒ in ob/ob
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mice [24]. The same beneficial effect of VSL#3 was seen in a randomized controlled, double-
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blinded clinical study, where obese children with NAFLD were supplemented with the aforementioned probiotics preparation for four months [142]. Likewise, the administration of a preparation with Lactobacillus acidophilus, L. helveticus and Bifidobacterium spp. plus a prebiotic had a protective effect regarding alcohol-induced hepatic injury and associated endotoxemia as well as abnormal liver enzyme levels in the Sprange-Dawley rat model of acute pancreatitis [73]. Lactobacillus rhamnosus and paracasei in mouse and rat models have been both managed to improve hepatic inflammation and steatosis [24]. In another study [70], Lactobacillus rhamnosus, paracasei and as well as Bifidobacterium breve were given to Zucker obese rats. L. paracasei was the only one that could not reduce hepatic steatosis. Nevertheless, all these prebiotics could reduce with a variable extent the level of blood inflammatory cytokines,
ACCEPTED MANUSCRIPT including TNF-α, LPS and IL-6. Another probiotic, Clostridium butyricum strain MIYAIRI 588 prevented the progression of NAFLD and associated tumorigenesis in a rat model of
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choline-deficient/L-amino acid-defined (CDAA)-diet-induced NAFLD [143]. Interestingly, probiotic Lactobacillus johnsonii La1 when given to rats, (served as cirrhotic animal model) alone or combined to antioxidants, could reduce bacterial translocation and endotoxemia
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[144]. Lactobacillus reuteri GMLN-263 represents another very promising therapeutic agent since has been shown to exhibit multiple beneficial effects: in high fructose-fed rats improved
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liver steatosis and ΙR and, moreover, remarkably reduced TNF-α and IL-6 concentrations in
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adipose tissue [145]. Likewise, Ting et al. also demonstrated, that heat killed Lactobacillus reuteri GMLN-263 reduced fibrosis effects on the liver as well as on the heart of hamsters fed with high-fat diet. This action was associated with a reduction of the expressed fibrotic factor
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TGFβ [146]. Finally, in young male albino rats, an ingestible probiotic mixture showed promise as a treatment for NAFLD, by improving high fat and sucrose diet-induced steatosis
markers [147].
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through its effects on leptin, resistin, pro-inflammatory biomarkers, and hepatic function
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A limitation of the current review is the confined randomized large human trials, which would substantially further strengthen the results of animal models on the role of microbiome. Moreover, the available literature included numerous different compositions and formulas of probiotics and prebiotics, which limit the interpretation of the results of clinical trials and add an heterogeneity of relevant meta-analyses [7]. Because, there is as yet no consensus on the role of probiotics in the management of NALFD, future relative information is warranted to validate the usage of probiotics as therapeutic option for NAFLD/NASH patients. Except of the aforementioned limitations, an important strength of our paper is the assiduous analysis in multiple levels of all possible mechanisms, which contribute to the development of NAFLD and its complications. Besides, we discuss and propose a plethora of other pharmacological agents that are currently being implemented in the multifactorial
ACCEPTED MANUSCRIPT therapy of NAFLD. The potential combination of such pharmacological agents with probiotics might further benefit NAFLD patients, though future relative studies are needed to
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elucidate this consideration.
8. Prognosis and course of disease
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The expected survival of NAFLD patients is reduced compared to general population and although some of these cases remain stable and asymptomatic [1], the majority of patients
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will die due to cardiovascular events and to a lesser extend due to malignancy and cirrhosis
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[17,148]. Moreover, approximately one fifth of the patients can be presented with a cirrhotic liver, as the result of the disease progression from the reversible benign steatosis to NASH, cirrhosis and HCC sequence [149]. The epigenetics, an inheritable phenomenon that affects
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gene expression without altering the DNA sequence, may provide novel molecular indicators that can determine not only the initial risk but also the disease progression and prognosis [149]. The microscopic hepatic lesions and especially the presence of hepatic fibrosis are
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directly correlated to prognosis [4,17]; the long-term hepatic prognosis mostly depends on the
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histologic stage at initial liver biopsy, but multiple risk factors might concur [150]. Diabetic obese patients are at a greater risk for such a progression and likewise patients with sonographic findings suggestive of steatosis are likely to develop diabetes [1]. Furthermore, there are also some predictive parameters, such as hepatic venous pressure gradient, serum albumin level and MELD-Score (Model for End-stage Disease), which can seem helpful for the estimation of hepatic exacerbation and even the development of a malignancy [17]. Additionally, whereas the disease at the early stages is potentially reversible by following the aforementioned management regimens, liver transplantation remains a promising solution for patients with advanced disease [1]. Further clarification of the natural history is essential to identify the predictors of long-term outcomes of NAFLD; these predictors should be used to direct development of clinical trial endpoints in NAFLD [151].
ACCEPTED MANUSCRIPT 9. Conclusion NAFLD is regarded as a systemic pathology with fundamental metabolic components, where
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liver is one centrally affected organ and intestine with its flora constitute a key-regulator that distantly orchestrates and shapes the evolution of disease. Patients die frequently due to extrahepatic complications and to a lesser extent due to NAFLD per se.
The clinical
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appearance of patients with NAFLD is not specific and is often observed with delay, thereby raising clinical suspicion is mandatory. Regarding therapeutic options, there is no evidence-
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based medication, which seems to be unanimously approved of the medical society for the
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treatment of NAFLD. An alteration of gut microbiome with the administration of probiotics and/prebiotics has yielded many positive and encouraging results. A new area, introducing more “sophisticated” ways for the treatment of the “old” diseases has been inaugurated and
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the sequel seems to be exciting.
Funding
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The work was not supported by any funds
Conflict of interest
The authors declare no conflict of interest
Author contributions: MD, GK and DG contributed equally in the design and conduct of the study as well as in the data collection, analysis and manuscript writing. JK and PK revised the draft critically for important intellectual content
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ACCEPTED MANUSCRIPT Legend Fig. 1: Some fundamental mechanisms involved in the pathogenesis of non-alcoholic fatty liver disease (NAFLD), which can explain the complex pathophysiology of the disease.
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The significant contribution of gut microbiota in the development of the same pathology is separately depicted in light blue labels.
NASH, non-alcoholic steatohepatitis; FFA, free fatty acids; PNPLA3-I148M, palatine- like
Superfamily
member
2
gene;
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phospholipase 3- isoleucine to methionine at residue 148; TM6SF2, Transmembrane 6 GCKR-LYPLAL,
glucokinase
regulator
protein-
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lysophospholipase like protein; MBOAT7, membrane bound O-acyltransferase domain
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containing 7; FIAF, fasting-induced adipocyte factor; SCFA, short chain fatty acids; VLDL, very low-density lipoproteins; FXR, farnesoid X receptor; LPS, lipopolysaccharide; TLR-4,
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Toll Like Receptor 4.
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ACCEPTED MANUSCRIPT
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Fig. 1
ACCEPTED MANUSCRIPT Table 1. Probiotics and their beneficial effects in non-alcoholic fatty liver disease (NAFLD) Special effects according to observations from studies Probiotic Action Lactobacilli & Streptococci spp. Reduction of aminotransferases
a. Inhibition of bacterial translocation
Lactobacilli spp.
Attenuation of TLR4 inflammatory pathway and endotoxemia
b. Inhibition of epithelial invasion Bifidobacterium spp.
c. Maintenance of intestinal barrier integrity
Amelioration of liver steatosis and endotoxemia
Bifidobacterium spp.& Akkermansia muciniphila + oligofructose
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d. Induction of production of antimicrobial peptides
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General effects
Decrease of hepatic fatty accumulation and rest metabolic syndrome hallmarks as well as endotoxemia
Lactobacillus acidophilus, L. helveticus and Bifidobacterium spp. + prebiotic
f. Enhancement of hyperdynamic circulation state of cirrhosis
Improvement of alcohol-induced hepatic injury, endotoxemia and increased liver enzymes
Lactobacillus rhamnosus and paracasei, Bifidobacterium breve
Reduction of hepatic steatosis and TNF-α, LPS and IL-6
Clostridium butyricum strain MIYAIRI 588
Prevention of NAFLD progression and tumorigenesis
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e. Reduction of inflammation Shaping of host’s immune system
TLR-4,
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g. Aversion of the onset of hepatic encephalopathy
Toll
Like
Lactobacillus johnsonii La1
Reduction of bacterial translocation and attenuation of endotoxemia
Lactobacillus reuteri GMLN-263
Improvement of liver steatosis and insulin resistance & reduction of TNFα, IL-6 (adipose tissue) and HF (TGFβ depended)
VSL#3 Probiotics cocktail
Reduction of ALT, enhancement of insulin sensitivity, hepatic inflammation/steatosis, suppression of the TNFα/IKK-β signaling pathway, reduction of activity of Jun N-terminal kinase, decrease DNA binding activity of NF-κB
Receptor
4;
ΤΝFα,
Tumor
Necrosis
Factor-α;
LPS,
lipopolysaccharide; IL-6, Interleukin 6; HF, Hepatic Fibrosis; TGFβ, Transforming Growth Factor beta; ALT, Alanine Aminotransferase; IKK-β, inhibitor of κΒ (ΙκΒ) kinase-β; NF-κB, Nuclear Factor-kappa B