Fatty liver disease

Jose Luis Olcoz GoĂąi / Francisco Jorquera Plaza

Fatty liver disease A reality with many questions

Fatty liver disease A reality with many questions Jose Luis Olcoz GoĂąi Francisco Jorquera Plaza

Any form of reproduction, distribution, public communication or transformation of this work can only be done with the authorization of its owners, except as provided by law. Go to CEDRO (Spanish Center for reprographic rights) if you need to photograph or scan any fragment of this work (www.conlicencia.com; 917021970/932720447). © Eolas Ediciones Directors: José Luis Olcoz Goñi and Francisco Jorquera Plaza Design direction and layout: Mikel Mandon - www.contactovisual.es Cover photography: © 2009 Michael Bonert (https://commons.wikimedia.org/wiki/User:Nephron) I.S.B.N.: 978-84-16613-97-7 D.L.: LE 274-2018 Printed in Spain

Fatty liver disease A reality with many questions

Jose Luis Olcoz Goñi Francisco Jorquera Plaza

Research Collaboration Index Aller, Rocio Álvarez Cañas, Concepción Álvarez Cuenllas, Begoña Aransay, Ana M. Arias Loste, María Teresa Azkargorta, Mikel Ballesteros Pomar, María D. Cabezas, Joaquín Cardoso Delgado, Teresa Conde de la Rosa, Laura Cortez-Pinto H Crespo, Javier Díez Rodríguez, Rubén Elortza, Felix Fernández-Checa, Jose C. Fernández Fernández, Nereida Iruzubieta, Paula

Jiménez Palacios, Marcos João Matias, Diana Jorquera, Francisco Jover, Ramiro García-Carrasco, Almudena García-Mediavilla, María Victoria García-Monzón, Carmelo García-Ruiz, Carmen Gonzalez, Monika González-Gallego, Javier González-Rodríguez, Águeda Lavín-Trueba, Jose Luis Linares Torres, Pedro López-Riera, Mireia Machado MV Martínez-Chantar, María-Luz Mato, José M.

Molina Arriero, Gema Monteserín Ron, Luzdivina Nistal González, Esther Pisonero Vaquero, Sandra Reyes Campos, Nelson Sánchez Campos, Sonia Varela-Rey, Marta Viso Vidal, David

Intro Hepatology is a young discipline and despite this it is having substantial changes in a very short time. We are witnesses and protagonists at the same time of the beginning of the path towards the elimination of the hepatitis C only 28 years after the clonation of the virus that has been the protagonist along with the alcohol of the major part of the morbimortality by liver diseases in the world western. But while this is happening, non-alcoholic fatty liver (NAFLD) becomes the most common cause of chronic liver disease in children and adults in relation to the global epidemic of obesity and metabolic syndrome. We still do not know if NAFLD is a benign liver state or a silent killer. At the origin of this syndrome may be defects in diverse metabolic pathways that involve hepatic accumulation of triglycerides. This might determine whether NAFLD progresses to nonalcoholic steatohepatitis (NASH).

The cases of advanced liver disease, liver cirrhosis and hepatocarcinoma secondary to nonalcoholic fatty liver grow exponentially in Europe and the United States where patients on the liver transplant waiting list have increased by 170% because of this.

We are unaware of many aspects of the disease and we are far from having adequate treatments for it, since weight loss and the change in lifestyles of people who suffer from it remain the basis of treatment. There are numerous clinical trials underway at different stages of research with a few molecules from which we will surely get treatments to help control the disease.

Therefore, research in all aspects of the disease is critical to address the future that is already present. Clinical research, basic research, translational research.

Throughout the pages of this book are collected different facets of the disease, some closely related to basic research. Your reading I am sure will help us to better understand this process and give us ideas to start new lines of research.

Francisco Jorquera Jose Luis Olcoz

9

Index 1.- Natural history and appropriate categorisation of patients with NAFLD/NASH. . . . . . . . . 11 Dr. María Teresa Arias, Joaquín Cabezas, Paula Iruzubieta, Javier Crespo.

2.- Epidemiology of NAFLD/NASH and potential impact in our health area . . . . . . . . . . . . . . . 45 Diana João Matias, Nereida Fernández-Fernández, Luzdivina Monteserín, Marcos JiménezPalacios, Gema Molina, Nelson Reyes; David Viso.

3.- Pathogenic mechanisms in non-alcoholic fatty liver disease (NAFLD). . . . . . . . . . . . . . . . . . 63 María Victoria García-Mediavilla, Sonia Sánchez-Campos, Javier González-Gallego.

4.- Liver biosy in NAFLD. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85 Dra. Concepción Alvarez-Cañas.

5.- Biomarkers in NAFLD. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101 Teresa Cardoso Delgado, Mikel Azkargorta, Jose Luis Lavín-Trueba, Marta Varela-Rey, Monika Gonzalez, José M. Mato, Felix Elortza, Ana M. Aransay, María-Luz Martínez-Chantar.

6.- NASH and Hepatocellular carcinoma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 135 Pedro Linares, Begoña Alvarez-Cuenllas, Francisco Jorquera.

7.- Extrahepatic conditions and cardiovascular risk in NAFLD. . . . . . . . . . . . . . . . . . . . . . . . . . . 167 Maria Ballesteros, Ruben Díez.

8.- Hypoxia and Non-alcoholic Steatohepatitis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 185 Carmelo García-Monzón, Almudena García-Carrasco and Águeda González- Rodríguez.

9.- Gut microbiota and dysbiosis in the pathogenesis of non-alcoholic fatty liver disease. 199 Esther Nistal, Sonia Sánchez-Campos.

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Fatty liver disease. A reality with many questions

10.- Cholesterol and sphingolipids in NAFLD. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 225 Laura Conde, Carmen García-Ruiz, Jose C. Fernández-Checa 11.- Transcription factors and microRNAs involved in liver lipid metabolism and their influence in non-alcoholic fatty liver disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 267 Mireia López-Riera, Ramiro Jover.

12.- Hepatitis C virus-induced steatosis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 299 Sandra Pisonero, María Victoria Garcia-Mediavilla, Sonia Sánchez-Campos.

13.- Dietary and nutritional recommendations in nonalcoholic steatohepatitis . . . . . . . . . . 319 Rocío Aller.

14.- Treatment of NAFLD/NASH . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 335 Mariana V Machado, Helena Cortez-Pinto.

11

1

Natural history and appropriate categorisation of patients with NAFLD/NASH

María Teresa Arias Loste, Joaquín Cabezas, Paula Iruzubieta, Javier Crespo Department of Gastroenterology and Hepatology, Marqués de Valdecilla University Hospital.

Infection, Immunity and Digestive Pathology Group, Research Institute Marqués de Valdecilla (IDIVAL). Santander, Spain.

Hepatic disease due to fat deposition encompasses a broad spectrum of hepatic lesions whose common denominator is an ectopic accumulation of fat in the liver known as steatosis.1 While the association between obesity and diabetes mellitus and the presence of steatosis has been well known since the beginning of the last century,2–4 it was not until 1980 that the first references were made to a form of steatohepatitis designated non-alcoholic steatohepatitis (NASH). Although this form mimics that of the alcoholic patient, it appears in subjects without this history.5 It is characterised by the presence of a variable degree of steatosis accompanied by lobular inflammatory infiltrate and hepatocellular damage with or without fibrosis.

Subsequently, the broader term hepatic disease was accepted for non-alcoholic fatty liver disease (NAFLD), which more accurately encompass the entire spectrum of lesions that appear in these patients.6

1. EPIDEMIOLOGY NAFLD has become the commonest cause of liver disease in developed countries7,8 due to the current epidemic of obesity and metabolic syndrome.9

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Fatty liver disease. A reality with many questions

According to the WHO, more than 1900 million adults aged 18 and over were overweight by 2014, and more than 600 million were obese. However, there are few studies on the incidence of NAFLD, and their conclusions may be difficult to extract because of the differences in the populations studied, the follow-up time, and the criteria used to diagnose NAFLD. An incidence of between 31 and 86 new cases per 1,000 people/year has been reported in Japan,10,11 while in Europe, incidences of between 4 and 29 per 1,000 people/year12,13 and 19 per 1,000 people/year14 have been reported.

Regarding the prevalence of NAFLD, estimates vary according to the diagnostic method used and the population studied (age, sex, ethnicity, and comorbidities).

Most of the data come from studies in which steatosis has been evaluated by noninvasive techniques, especially ultrasound and magnetic resonance imaging, and the prevalence of NAFLD evaluated only by ultrasound varies from 17 to 46%.7 The results of a recent meta-analysis that analysed a total of 86 studies with a sample of more than 8 million individuals from 22 countries show a global prevalence of NAFLD of 25%.15 Regarding sex and age, NAFLD is more frequent in men, and its presence increases with age, as well as the probability of progression of the disease and outcomes derived from it.16–18 With respect to racial differences, NAFLD is more frequent among white Hispanics, whereas the incidence is especially low among the African American population.19,20 NAFLD is strongly associated with the factors that constitute metabolic syndrome, considerably increasing the prevalence in this group of patients,7,21,22 and this relationship is especially close in the case of morbid obesity (a BMI greater than 40 kg/m2). In this group of patients, more than 90% have NAFLD, NASH is present in 1/3 of the cases, and the liver disease has progressed to cirrhosis in up to 5-10% of the subjects.23,24 Among patients with diabetes mellitus, the prevalence of NAFLD is estimated to be 40–70% and that of NASH is approximately 22%.7,23,25 In addition, the insulin resistance is related to the severity of the hepatic injury, the presence of steatohepatitis, cirrhosis, and even the development of complications such as hepatocellular carcinoma (HCC).26,27 Dyslipidaemia is also frequent in these patients, mainly in the form of hypertriglyceridaemia and low levels of HDL-cholesterol.28,29 Overall, 80% of the patients have any of the cardiovascular risk factors that constitute metabolic syndrome (insulin resistance, obesity, dyslipidaemia, and hypertension),30 and the prevalence of NAFLD increases directly with the number of these factors.31

Natural history and appropriate categorisation of patients with NAFLD/NASH

13

In recent years, an association between NAFLD and other risk factors independent of the classic cardiovascular risk factors has been described. These include the sleep apnoea-hypopnoea syndrome,32 polycystic ovary syndrome,33 and endocrinopathies such as hypothyroidism,34,35 hypopituitarism,36 hypogonadism,37 and psoriasis,38 particularly when psoriasis involves elevated inflammatory activity.

Although all of these entities occur frequently in obese patients, their associations with NAFLD have been demonstrated even independent in the presence of obesity and insulin resistance. Finally, a recent study by our group has demonstrated that psychotic patients undergoing pharmacological treatment are at a high risk of developing NAFLD in their early years.39 Regarding the prevalence NASH-related cirrhosis, it is estimated to be approximately 2% in the United States,40,41 taking into account that cirrhosis related to NAFLD may be underestimated since the histological signs of steatohepatitis may no longer be present in the cirrhotic phase of the disease. Cryptogenic cirrhosis related to NASH has been clearly established by different population and retrospective studies, with NASH being a prevalent cause.42 It has been noted that the number of NASH cirrhosis patients on liver transplant waiting list has tripled in the last decade, and it is now the second most common indication for transplant in the United States.41 It is anticipated that by 2020 with advances in the treatment of hepatitis C, it will be the most common cause of liver transplantation in the United States.40 Between 5–10% of NASH patients will have decompensated cirrhosis and between 1–2% will have HCC.43

2. NATURAL HISTORY NAFLD includes a broad spectrum of diseases ranging from simple steatosis to NASH, fibrosis, and cirrhosis. Of the total number of patients diagnosed with NAFLD, it is estimated that approximately 10–20% have a histology that is compatible with the diagnosis of NASH.44 From the viewpoint of liver histology, NAFLD has a slow progression to advanced fibrosis. While hepatic steatosis is considered practically to be a static entity with a very low risk of developing fibrosis within 15 to 20 years, NASH patients are at risk of progressing to fibrosis and cirrhosis. In these cases, the expected progression of fibrosis is also slow, progressing from one stage of fibrosis

14

FATTY LIVER DISEASE. A REALITY WITH MANY qUESTIONS

to another over a period of 6 to 15 years, but approximately 20% of NASH cases have a more rapid progression of fibrosis over the course of 5 years. Figure 1 shows a summary of the natural history of NAFLD.

5-18 % over 3-7 years (rapid progression)

SIMPLE STEATOSIS

ADVANCED FIBROSIS / CIRRHOSIS

10-25 % over 8-15 years

20% over 2-14 years

2-13 % over 3-7 years

44-65 % over 3-7 years

NASH 0,16% over 6 years

HCC

Figure 1. Summary of the natural history of non-alcoholic fatty liver disease.

For years simple steatosis has been considered a benign condition but recent studies have challenged this view. Several studies with paired biopsies showed progression from simple steatosis to NASH and fibrosis.45–58 A first retrospective study of 70 patients with NAFLD showed that 65% of patients with simple steatosis progressed to NASH, while 24% developed advanced fibrosis in less than 5 years.45 McPherson et al.46 studied 108 patients and observed that 44% of the patients with simple steatosis on the initial biopsy progressed to NASH, with 37% progressing to fibrosis and 22% progressing to advanced fibrosis over a median follow up of 6.6 years. Recently, Singh et al.47 performed a systematic review and meta-analysis of 11 studies that included 411 NAFLD patients and paired biopsies. They found that 33.6% of the patients with steatosis and NASH progressed to fibrosis and observed an improvement in 22.3% of the patients. However, all of these studies suggest that 1/3 of patients with simple steatosis and NASH will have progressive fibrosis,

Natural history and appropriate categorisation of patients with NAFLD/NASH

15

and 20% will have some regression over a follow-up averaging between 2.2–13.8 years. The published rates of progression of fibrosis among NASH patients vary between 32–53%.47,50–54,57,59 In general, within a period of 15 years, 13% of patients with a fibrosis grade of F2 and 25% of those with F3 will develop cirrhosis.16,45,46 However, this lack of homogenisation in the rate of progression of fibrosis is largely due to the presence of certain patient characteristics, as will be described later.

Once cirrhosis has developed, the risk of developing complications of portal hypertension is 17%, 23%, and 52%, at 1, 3, and 10 years, respectively.60 The survival of patients with NASH cirrhosis declines markedly after the first decompensation occurs, with a mean survival of approximately 2 years.61 Several studies have observed complication and survival rates of cirrhosis that are similar to those observed in patients with cirrhosis due to the hepatitis C virus (HCV),62,63 although this finding is controversial.64,65 For another complication of cirrhosis, HCC, several recent studies of patients with NASH cirrhosis have demonstrated a progression to HCC in approximately 7% of patients in 6.5 years.63–65 Compared with individuals in the general population of the same age and gender, those with NAFLD have a lower-than-expected survival rate, with a standardised mortality rate of 1.34–1.69 according to American and Swedish studies.16,17,66 The prognosis of an individual patient with NAFLD is highly variable, and an increased likelihood of progressive disease was initially described in patients with NASH,44 which is often defined according to the NAFLD activity score (NAS). The NAS score is the sum of the different degrees of steatosis, balloonisation, and lobular inflammation,67 and it was originally developed as a tool for assessing efficacy in clinical trials. Recently, it has been more widely applied to define NASH and evaluate histological activity, but undoubtedly, fibrosis is a key factor in the likelihood of death related to liver disease and even non-hepatic disease. Several prospective cohort studies have suggested that liver fibrosis predicts liver-related and non-liver-related mortality more reliably than NASH or its individual components.68–70 In a study of 209 patients with a follow-up of 12 years, it was observed that advanced fibrosis was the only histological lesion that was independently associated with liver-related mortality (hazard ratio = 5.68; 95% confidence interval (1.5–21.4)).68 These findings have been most recently reproduced in a series of 229 patients with a follow-up of 26.4 years.71 In this study, the overall mortality did not increase in patients with

16

Fatty liver disease. A reality with many questions

high NAS scores (5–8) without severe fibrosis (0–2) (HR 1.41, CI 0.97–2.06, p = 0.07).

However, patients with a fibrosis stage of 3 or 4, regardless of the NAS score, had an increase in mortality compared with a reference population (HR 3.3, CI 2.27–4.76, p < 0.001). Finally, Angulo et al.69 conducted a longitudinal study with an international cohort to determine the long-term prognostic significance of the histological characteristics of NAFLD. This study confirmed that the stage of fibrosis rather than NASH was the most important histological feature associated with overall survival and liver-related complications. Even patients with mild (stage 1) fibrosis were at a higher overall risk compared to those without fibrosis, although only those with moderate fibrosis (stage 3 and 4) had an increased risk of liver complications such as ascites, encephalopathy, or esophageal varices. These studies emphasise the need to routinely evaluate fibrosis in all patients with NAFLD to assess their prognosis and, therefore, the need for monitoring and treatment directed at the liver.

The main cause of death in NAFLD, however, is not liver disease, which is relegated to the third position, but rather cardiovascular disease (CVD) followed by different types of cancer.16,17 In this aspect, the mortality from CVD of patients with NAFLD is double that of the general population, and there are no differences in cardiovascular mortality between patients with simple steatosis and steatohepatitis.

However, liver-related mortality is up to 10 times higher for steatohepatitis.66,72 Population-based cross-sectional studies and meta-analyses have demonstrated that NAFLD is an independent factor of cardiovascular mortality that increases the predictive value of some parameters that assess the severity of CVD such as endothelial dysfunction, arterial stiffness, and myocardial dysfunction.73–76 On the other hand, NAFLD patients frequently have insulin resistance, hyperglycaemia, atherogenic dyslipidaemia, and elevated levels of proinflammatory cytokines and plasmatic procoagulant factors, as well as markers associated with metabolic syndrome and involved in the pathogenesis of atherosclerosis such as CD36 in its soluble form (sCD36), which is a membrane receptor responsible for, among other things, the transport of fatty acids.77 Several cohort studies have suggested that other subgroups of patients with NAFLD, such as those with type 2 diabetes mellitus or men with elevated levels of gamma-glutamyl transpeptidase, may have an increased risk of cardiovascular events compared with subjects without NAFLD.45,46,78 Finally, the severity of hepatic histology can stratify the risk of cardiovascular mortality, as shown by Ekstedt et al. 55, who demonstrated that

Natural history and appropriate categorisation of patients with NAFLD/NASH

17

subjects with simple steatosis did not have an increased risk of all-cause mortality or CVD-related mortality. However, those with NASH were twice as likely to die from CVD compared with the general reference population (15.5% versus 7.5%) during a mean follow-up period of 13.7 years.

3. FACTORS THAT INCREASE THE PROGRESSION RATE OF FIBROSIS IN NAFLD PATIENTS There is no doubt about the capacity for progression of this disease from simple steatosis to steatohepatitis with inflammation and fibrosis.79 In this sense, studies involving paired biopsies provide us with very useful information. Collectively, these studies suggest that simple steatosis in general has a more indolent progression rate than NASH. However, there is considerable heterogeneity, with a quarter of patients with simple steatosis developing bridging fibrosis over a relatively short period of time. A summary of the factors that have been associated with this risk of fibrosis progression is presented in Table 1.

Established

Under discussion

Stage of fibrosis

Advanced age

Necroinflammation in liver biopsy

Arterial hypertension

Obesity

Alcohol consumption

Insulin resistance / diabetes

Vitamin D deficiency

Dyslipidemia Menopause PNPLA3 rs738409 TM6SF2 rs58542926

Table 1. Risk factors for progression of NAFLD.

The risk factors most commonly associated with this progression phenomenon are the coexistence of insulin resistance/diabetes mellitus, arterial hypertension, central obesity, and advanced age. However, the predictor of outcome in NAFLD is the presence of necroinflammation or fibrosis in the liver biopsy.68,69 The association between sex and progression of fibrosis is controversial. Two cross-sectional studies have shown that men and postmenopausal women have an increased risk of fibrosis

18

Fatty liver disease. A reality with many questions

compared with premenopausal women. In addition, early menopause is associated with an increased risk of fibrosis.80â&#x20AC;&#x201C;83 Hispanic patients have a higher prevalence of NAFLD compared to Caucasians, although the progression rate of fibrosis does not appear to be higher.19,20 Dozens of genes with multiple polymorphisms associated with NAFLD have been discovered thanks to genome-wide association studies (GWASs), but the number of genes validated with confidence in large independent cohorts has been reduced to two, patatin-like phospholipase domain-containing 3 (PNPLA3) and transmembrane 6 superfamily member 2 (TM6SF2).84 The presence of the single nucleotide polymorphisms (SNPs) rs738409 and rs58542926 of the PNPLA3 and TM6SF2 genes, respectively, have been associated with an increased risk of NAFLD, as well as a more severe form of the disease.85â&#x20AC;&#x201C;91 A study of more than 1,000 individuals with biopsies demonstrating NAFLD showed that these SNPs were associated with a 40â&#x20AC;&#x201C;88% increase in the risk of advanced fibrosis (F2-4) after adjustment for age, sex, and metabolic variables. Recently, an SNP of IL28b (also implicated in the response to interferon in patients with chronic hepatitis because of HCV) has been associated with increased fibrosis in patients with NAFLD.92 In addition, a case control study leaded by our group found that the variants rs1421085 and rs1558902 of the FTO gene confers a high risk of hepatic inflammation, particularly in patients with NAFLD and normal weights (unpublished). Several cross-sectional studies have shown that the disease is more severe in older patients, although this phenomenon may be due to the sum of the pathogenic factors and to a longer duration of the hepatic disease itself and of the associated diseases.17,18,28 In fact, longitudinal studies have failed to demonstrate that age is a factor that aggravates the disease per se.53 NAFLD tends to be more severe in patients with several factors of metabolic syndrome, particularly diabetes and obesity. In fact, increases or decreases in weight are associated with an increase or resolution of liver fibrosis. Similarly, good glycaemic control tends to decrease the progression of fibrosis.93,94 NAFLD is a common complication of morbid obesity, but its severity varies greatly from one patient to another. Recent research suggests that these differences are determined by the age at the onset of morbid obesity. If it starts very prematurely, the fat is mostly deposited in the subcutaneous cellular tissue, with the patients undergoing bariatric surgery before significant liver damage occurs. In contrast, a late onset of morbid obesity results in bariatric surgery being performed at more advanced ages, with

Natural history and appropriate categorisation of patients with NAFLD/NASH

19

a higher degree of fatty liver infiltration. In addition, the exposure of hypertrophic adipocytes to lipolysis is greater, with the consequent release of adipokines that may aggravate liver damage.95 However, it is worth mentioning that the association between obesity and NAFLD does not follow a parallel course. In fact, despite the unquestionable evidence of an association between obesity and liver damage, it has been observed that among patients with descompensated cirrhosis admitted to a hospital, the obese cirrhotic patients have a lower mortality rate. This paradox of obesity could be due to an increase in the nutritional reserves of obese patients.96 Whether or not a third component of metabolic syndrome, arterial hypertension, is associated with the improvement of the histology of NAFLD is controversial.7,47 It has also been demonstrated that the increase in large particles of VLDL and the decrease in the concentration of smaller particles is associated with an increase in the risk of NAFLD and its severity.97 Alcohol consumption in NAFLD is a controversial point. First, the diagnosis of NAFLD itself would be placed in doubt, and second, the impact of alcohol consumption is not properly defined, providing contradictory outcomes in the literature with some favouring and some against the progression of NAFLD.98 However, despite its possible benefit (if it has been properly evaluated), it is possible that patients with any liver damage, however small it may be thought to be, should be banned from consuming any amount of alcohol, as even minimal amounts of alcohol have an impact on the mortality of patients with obesity.99 It is also worth noting that, in line with the idea that alcohol poses a comorbidity in patients with NAFLD, the implication of endogenous alcohol production in the pathogenesis of NAFLD is provoking a great deal of interest,100,101 but further studies are needed to confirm this relationship.

Finally, vitamin D deficiency is prevalent among American patients with NAFLD, and its levels have been negatively correlated with the severity of steatosis, necroinflammation, and fibrosis.102,103 The mechanism by which this association occurs may have to do with the potential anti-inflammatory role of vitamin D itself and its modulation of NF-ÎşB.104,105 The variability of the disease is so great and the difficulty in predicting fibrosis so high that some mathematical models with relatively easy applicability have been developed that are based on routine analytical determinations or the interpretation of imaging biomarkers.106,107

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Fatty liver disease. A reality with many questions

4. CANCER AND NAFLD NAFLD patients have an increased risk of HCC.108 The cumulative incidence of HCC in NASH cirrhosis ranges from 2.4–12.8% over a period of 3.2–7.2 years, and the cumulative mortality of HCC in NAFLD/NASH cohorts is 0–3% over 5.6–21 years.109 A study of 195 patients with NASH cirrhosis from the Cleveland Clinic found that the annual cumulative incidence of HCC was marginally lower than in a comparative population of patients with cirrhosis caused by hepatitis C virus (HCV) (2.6% vs. 4.0%, p = 0.09),110 and these findings have been replicated in other cohorts in America and Japan.62,111 All of these studies followed a defined protocol that excludes other aetiologies of HCC, including hepatitis C and B infection risk factors for the development of HCC included diabetes, age, any prior alcohol consumption, and the presence of intrahepatic iron.112 Once HCC has developed in cirrhotic NAFLD patients, survival seems to be greater than in patients with HCC because of HCV.113,114 This may be related to the fact that patients with NAFLD-HCC are older, have larger tumours, and are diagnosed later than HCV-HCC. However, among patients who have conserved hepatic function and tumours eligible for HCC curative treatment, the overall survival is similar or better than for patients with hepatitis C or alcohol-induced cirrhosis.111 The development of HCC is associated with NAFLD, even in the abcence of cirrhosis.115 In non-cirrhotic NAFLD patients, the development of HCC has been increasingly reported, Cross sectional studies showed that between 15–50% of HCC cases are diagnosed without cirrhosis.116,117 The cumulative mortality rate for HCC in non-cirrhotic NAFLD patients is 3% over 20 years, whereas in cirrhotic patients, it is up to 12% over a period of 3–12 years of follow-up.109 In addition, cases of HCC have been reported in subjects with only simple steatosis and no evidence of NASH or fibrosis.118 A minority of these cases may be related to the transformation of hepatic adenomas, while most appear to be related to risk factors for NAFLD and metabolic syndrome (especially obesity and diabetes). 119 Several studies have also suggested that HCC in non-cirrhotic patients with NASH and/or metabolic syndrome are more likely to occur in men.119,120 In general, NAFLD is estimated to be behind 15–30% of all cases of HCC.121 Several studies have demonstrated an association between metabolic syndrome,

Natural history and appropriate categorisation of patients with NAFLD/NASH

21

type 2 diabetes mellitus, obesity, and HCC, and their results suggest that NAFLD may also play some role in the development of HCC.113,117 The pathogenic mechanisms involved are unknown, but insulin resistance, oxidative stress, and lipotoxicity create a favourable microenvironment.122 Perhaps the intestinal dysbiosis associated with obesity promotes the release of endotoxins.123,124 Diets high in fats and fructose can worsen the cytokine patterns and promote lipoperoxidation,125 and genetics also contribute to an increased risk of HCC, mainly through the rs738409 variant of PNPLA3.126 Extrahepatic malignancy is the second most common cause of death among NAFLD patients. The risk of developing these type of cancers may be due to factors related to the underlying disease, as well as factors not related to the disease, (environmental and genetic factors or adverse effects of medication taken by patients).16,127 For this reason, risk analysis is complex, and the information published related to the increased risk of extrahepatic cancer in NAFLD is controversial.128,129

5. SCREENING FOR NAFLD AND HCC Given the high prevalence of NAFLD and its potential progression to NASH and cirrhosis, the practice of screening for this disease in the general population may seem reasonable. In fact, some studies have shown a prevalence of significant fibrosis of 6% in general population and of 18% in the population with risk factors, with NAFLD being the most frequent cause of fibrosis in these cases.129â&#x20AC;&#x201C;131 An analysis of the usefulness of NAFLD screening by Controlled Attenuation Parameter (CAP) and transient elastography (FibroScanÂŽ) has recently been published in more than 1,900 patients with type 2 diabetes.132 In this study, an elastograph compatible with significant fibrosis was observed in 17.7% of patients, and an attenuation coefficient suggestive of hepatic steatosis was observed in 72.8% of cases. In addition, 80% of patients with above normal elastographs underwent liver biopsies, demonstrating NASH in 56% of cases and stage F3-4 fibrosis in 50% of the cases. The presence of diabetes mellitus, a high BMI, a low VLDL, elevated transaminases, and albuminuria were associated with significant fibrosis in multivariate analysis. However, costutility studies of these screening programmes are needed before putting them into practice.

22

Fatty liver disease. A reality with many questions

The high risk of HCC in NAFLD justifies the practice of screening for it, as screening tends to decrease the mortality of patients. It is recommended for patients with a risk of developing HCC equal to or greater than 1.5%/year (this criterion is met in patients with cirrhosis) and for those who are potential candidates for curative treatment if HCC is diagnosed. As for the rest of the patients, the recommended tool for HCC screening is an ultrasound every 6 months.129 However, the efficacy of this strategy for these patients may be slightly lower than that for the general population, since, as we all know, marked obesity is very common in patients with NAFLD and may limit the detection of small liver nodules. It is not surprising that HCC associated with non-cirrhotic NAFLD is less likely to be detected during surveillance and therefore is more likely to be more advanced compared with HCC in patients with cirrhosis.40,66 However, survival is equivalent or better in non-cirrhotic patients with NAFLD than in subjects with cirrhotic HCC, probably because of preserved liver function.

6. EVALUATION OF ASSOCIATED COMORBIDITIES In recent years, numerous studies have shown that the morbimortality associated with NAFLD is not only limited to its hepatic manifestations but also involves multisystemic effects on different organs.133,134 In this sense, the coexistence of the comorbidities typical of metabolic syndrome should be ruled out in these patients, with special attention paid to the presence of type 2 diabetes, as the risk doubles in this population.73,94 Hypertension, hyperlipaemia (particularly atherogenic dyslipidaemia with increased triglyceride levels and a decrease in HDL levels), and associated CVD are also common.135 However, NAFLD is not only associated with metabolic syndrome itself but is also associated with the development of chronic kidney disease,136 sleep apnoea-hypopnoea syndrome,137 osteoporosis,138 psoriasis,139 colorectal cancer,140 and different endocrinopathies such as polycystic ovarian syndrome.141 Several prospective studies have analysed the association between NAFLD and incidence of cardiovascular events. They observed a clear increase in the risk of cardiovascular events in NAFLD patients that was independent of the presence of other classic vascular risk factors such as age, sex, abdominal perimeter, arterial

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hypertension, etc.142,143 As we mentioned previously, the limited number of studies that illustrate the natural history of the disease show that the principal cause of death in these patients is cardiovascular disease, with liver-related death moving from the thirteenth most common cause of death in general population to the third in this subgroup of patients.17,144 This is due to the association between NAFLD and subclinical manifestations of atherosclerosis and endothelial dysfunction, such as increased carotid intima-media thickness, increased coronary artery calcification, altered flow-mediated vasodilation, arterial stiffness, and carotid artery inflammation.145â&#x20AC;&#x201C;148 NAFLD and, fundamentally, NASH, can be considered to be lowgrade systemic inflammatory diseases. Similarly, atherosclerosis is also considered to be a low-grade inflammatory disease, and both share multiple pathogenic mechanisms.149 The risk of accelerated atherosclerosis depends on factors such as endothelial dysfunction and the release of proatherogenic and procoagulant inflammatory molecules. In addition, multiple alterations of the lipid profile have been described. Endothelial dysfunction is an initial step in the development of atherosclerosis, and this can be defined as a deteriorated capacity of the artery to dilate in response to physical and chemical stimuli due to the reduced release or greater degradation of nitric oxide (NO). Endothelial dysfunction can be easily determined noninvasively by assessing the flow-mediated dilation (FMD) after brachial artery occlusion using high-resolution ultrasound.150 Although screening for cardiovascular risk in NAFLD is currently not included in the clinical management guidelines, and therefore, its use is not widespread in clinical practice, it is important to note that there are different techniques for the detection of subclinical atherosclerosis that are bloodless, and some of them are very simple to perform. These include the measurement of the ankle-arm index, carotid ultrasound, computed tomography, and high-resolution magnetic resonance imaging for the detection of atheromatous plaques.78 These explorations are especially useful for patients with intermediate cardiovascular risk, and these patients constitute a very significant portion of the population with NAFLD.151 Although hepatologists are often unfamiliar with the search for vascular risk factors to detect subclinical atherosclerosis, we believe that particular attention should be paid to this aspect.

The following have been described in NAFLD: a decrease in flow-mediated brachial artery reactivity, which is more intense in patients with NASH; an increase in

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Fatty liver disease. A reality with many questions

carotid intima-media thickness that is directly proportional to the stage of NASH and independent of the classic vascular risk factors; an increase in the number of both calcified and non-calcified plaques in coronary arteries; and diastolic dysfunction in patients with hypertension related to the severity of the NASH.74,148,152 In addition, an increase in serum levels of some proinflammatory and proatherogenic factors such as high-sensitivity C-reactive protein, PAI-1 and fibrinogen, or decrease in anti-inflammatory adipokines such as adiponectin have been documented.153,154 The latter outcomes were adjusted for visceral fat, suggesting that liver disease is an independent atherogenic factor. Therefore, we can conclude that NAFLD is associated with an increase in vascular risk and that this increase is primary and independent of the association with metabolic syndrome or other vascular risk factors observed in these patients. Although no definitive conclusions can be drawn so far, it is possible that NASH is not only a marker of CVD but is involved in its pathogenesis.155â&#x20AC;&#x201C;157 Studies have shown that intrahepatic fat and not visceral fat, is linked with metabolic complication associated with obesity and that the liver is key to the regulation of lipid and glucose metabolism, so it should be considered as the trigger of the metabolic syndrome rather than its target.158,159

7. ASSESSMENT OF INSULIN RESISTANCE Insulin resistance is considered to be present when a normal amount of this hormone produces a lower than expected biological response. At the cellular level, this resistance may be due to a decrease in insulin sensitivity, a normal insulin sensitivity but a decrease in maximal responsiveness, or a combination of both phenomena.160 Insulin resistance is a key factor in the pathophysiology of NAFLD and is associated with increased fat deposition and fibrosis, which translates into the need for the use of different indices as screening tools for detecting its presence in daily clinical practice.73 The reference technique for its diagnosis is the hyperinsulinemic-euglycemic clamp, although this procedure is expensive and complex and therefore not routinely used in clinical practice.161 Insulin sensitivity can also be estimated dynamically by analysing the glucose and insulin response curves following the intravenous administration of glucose.

There are several models that are based on this principle, with the minimal model of glucose metabolism being used most often.162,163 In contrast to these models,

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other means of static measurement of insulin resistance have been developed based on baseline fasting glucose and insulin levels including the Homeostatic Model Assessment for Insulin Resistance (HOMA-IR) and the Quantitative Insulin Sensitivity Check Index (QUICKI). Although they are simple, low cost techniques with great usefulness in epidemiological-type studies, they have low reproducibility, and they are not applicable for patients treated with insulin or with poorly controlled diabetes.164

8. CONCLUSIONS AND PENDING ASPECTS NAFLD is an extremely common disease in the general population, yet its natural history and therefore the impact of NAFLD on patient morbimortality are difficult to pinpoint. Although patients with histological NASH are known to progress, the prognostic implications are not entirely understood, and longitudinal studies are necessary to clearly define the risk of major clinical events such as survival free of liver transplantation, the development of major complications of liver disease, and the development of cardiovascular events and chronic kidney disease.

Metabolic factors such as diabetes and obesity, as well as the common genetic polymorphisms of the PNPLA3 and TM6SF2 genes, influence the severity of the underlying liver histology and, therefore, may have an impact on the risk of developing cirrhosis and HCC. Recent studies have demonstrated that simple steatosis, in addition to NASH, can lead to progressive fibrosis and have emphasised the importance of the stage of fibrosis in determining the future risk of mortality. A greater understanding of the factors that alter the natural history of NAFLD would allow a stratified approach to managing NAFLD patients and identifying those who would benefit most from specific treatments.

Financial Support Supported by grant from Instituto de Salud Carlos III; FIS PIE15/00079 (to JC).

The book you hold in your hands collects the experiences shared among clinicians and basic researchers related to liver disease by non-alcoholic fatty deposits. These experiences have been discussed in different meetings held in Leรณn (Spain), trying to comprehend in its entire dimension a process that is currently the most common liver disorder in developed countries.

This collection of experiences is not intended to explore all aspects of the problem but those in which the different authors have a relevant opinion.

We hope you enjoy reading while you can discover nuances that help you have a broader view of this reality that is the pathological deposit of fat in the liver.

ISBN: 978-84-16613-97-7