Journal of Hepatology
Volume 40, Issue 5 , Pages 853-856, May 2004

NASH: are genes blowing the hits?

  • Fabio Marra

      Affiliations

    • Corresponding Author InformationTel.: +39-055-429-6475; fax: +39-055-417-123

Dipartimento di Medicina Interna, University of Florence, Viale Morgagni 85, I-50134 Florence, Italy

See Article, pages 781–786

Article Outline

 

In the last few years, active investigation has focused on the group of non-alcoholic fatty liver diseases (NAFLD) because these entities, once considered benign and of little interest to the clinician, include non-alcoholic steatohepatitis (NASH), a relatively aggressive form of chronic liver disease [1]. Despite some confusion in the nomenclature, it is now accepted that besides steatosis, the conditio sine qua non for these forms, evidence of necro-inflammation and/or fibrosis is necessary for the diagnosis of NASH. NASH represents a potentially evolving form of NAFLD, and in a substantial proportion of cases may lead to end-stage liver disease, requiring transplantation, and hepatocellular carcinoma [2], [3]. One of the main reasons for the explosion of information provided by clinical and basic science studies is the very high prevalence of NAFLD in developed countries, related to the widespread presence of risk factors, such as obesity, type II diabetes mellitus, and dyslipidemia. The dimension of the problem is impressive, because as many as 20–25% of the general population are believed to be affected by NAFLD, and about one-fourth to one-fifth of these may have NASH [4]. In light of these figures, the high burden of advanced chronic liver disease, with major impact on health care and the need for liver transplantation, is easily understood. Since currently only liver biopsy may reliably differentiate between simple NAFLD and NASH, any methods allowing to identify those at higher risk to develop the more aggressive form would result in more rationale use of the available resources.

A better understanding of the pathogenetic mechanisms leading to NAFLD and NASH is extremely helpful not only to devise more effective therapeutic options, but also to identify the patients at higher risk to progress to cirrhosis. To explain the mechanisms underlying the pathogenesis of NAFLD, Day and James have elaborated an elegant working model known as the ‘two-hit’ hypothesis, in relation to a two-step process leading to the development of NASH [5]. The first ‘hit’ is represented by fatty infiltration of the liver, an alteration common to several liver diseases such as those caused by alcohol and by certain drugs. In the case of NAFLD, work from several groups indicates that steatosis is dependent on the presence of insulin resistance, a condition representing the pathogenetic basis for the so-called ‘metabolic syndrome’ [6]. Insulin resistance leads to the accumulation of fat within the hepatocytes through a number of mechanisms including increased lipolysis and fatty acid uptake, increased fatty acid synthesis, and reduced triglyceride output via secretion of VLDL [7]. This first hit is present in almost all patients with the metabolic syndrome, but is not sufficient to cause NASH, despite the fact that it renders the liver more susceptible to different insults, including ischemic injury or viral hepatitis [8]. Progression to NASH occurs when additional damaging factors act on the predisposed liver, causing hepatocellular damage and fibrosis. Oxidative stress, due to excessive production of reactive oxygen species (ROS) within the cells, has been indicated as a possible factor responsible for the ‘second hit’ leading to steatohepatitis, in analogy with the proposed mechanisms of severe damage in alcoholic liver disease. Not only this theory fits well with the available data from humans and experimental models, but is compatible with the involvement of other factors that have been shown to participate in the development of NASH, such as excessive availability of free fatty acids, and overexpression of cytokines such as tumor necrosis factor-alpha, which lead to inflammation and reduced insulin action [9].

The interesting study by Namikawa et al. published in the current issue of the Journal [10] is the first attempt to correlate a genetic background with the ‘two-hit hypothesis’, in order to identify factors accounting for the development of NASH in a group of Japanese patients. The intriguing objective of the study was in fact to evaluate the polymorphisms of two genes encoding for molecules regulating fatty infiltration of the liver and production of ROS, representing the two hits, respectively. The microsomal triglyceride transfer protein (MTP) regulates the incorporation of triglycerides into apolipoprotein B and is a key enzyme for the assembly and secretion of VLDL from hepatocytes [11]. The role of this pathway as a possible mechanism to generate fatty liver is clearly demonstrated by the massive steatosis observed in patients with abetalipoprotenemia, a rare autosomal recessive disease caused by mutations in the coding region of MTP, and by the induction of fatty liver after tissue-specific inactivation of MTP [12]. A G/T polymorphism in the 5′ promoter region has been associated with reduced transcription of this gene, possibly resulting in lower MTP levels and therefore in a predisposition to accumulate triglycerides within the liver. Remarkably, the NASH patients studied by Namikawa et al. had both an increased frequency of the G allele and of the G/G genotype, indicating a genetic background favorable for the development of steatosis. More important, comparing the group of NASH patients with the G/G with those with the G/T phenotype, a more severe steatosis was observed in the group expected to have lower MTP levels. This latter group also had a more advanced stage, indicating that the progression of fibrosis was somehow accelerated in the presence of a ‘low MTP’ genotype. This represents the first study in which MTP genetic polymorphism was evaluated in patients with biopsy-proven NASH. This approach also allowed the Authors to quantify the amount of liver fat not only using radiologic techniques—in this case CT scan—but also morphometrically measuring steatosis in the biopsy specimen.

Having dealt appropriately with the first hit, the Authors went on analyzing another gene that could possibly confer predisposition to develop a second insult, such as oxidative stress. The choice fell on the polymorphism of manganese superoxide dismutase (MnSOD). Superoxide dismutase catalyzes the conversion of two molecules of superoxide anion, a highly unstable ROS, into hydrogen peroxide and molecular oxygen, a more stable ROS that is neutralized by the action of catalase. Eukariotes contain two forms of superoxide dismutase, a copper–zinc dependent cytosolic form and a manganese containing version located in mitochondria, critical organelles for the production of ROS within the cell [13]. A T to C polymorphism in the first exon of MnSOD determines the substitution of valine with alanine in the mitochondrial targeting region of the enzyme. The resulting structural change brought about by the presence of alanine is associated with an increased localization of the active MnSOD within the mitochondrial matrix, as elegantly confirmed in a recent study [14]. This localization pattern may increase the ability of MnSOD to process superoxide anion produced in the mitochondria, and on the contrary, the presence of valine would be associated with increased generation of ROS. In agreement with this hypothesis, a higher frequency of the T/T (valine/valine) genotype was observed in the NASH patients in comparison to the control group. Thus, this group of patients has an increased frequency of genotypes predisposing not only to fatty liver, but also to oxidative stress, one of the factors believed to be involved in the causation of more severe injury and the development of NASH.

The fact that NASH is observed only in a fraction of patients with NAFLD clearly suggests genetic predisposition to this disease, and together with other studies that have been undertaken to explore different genetic factors, sometimes with conflicting results [15], [16], the study by Namikawa and coworkers contributes to increase our knowledge on one of the hottest issues in the field. A particular merit of this study is that of having investigated genes potentially regulating two sequential pathogenetic mechanisms in the same group of well-characterized patients. In fact, while the possible contribution of the −493 G/T polymorphism of MTP to the occurrence of fatty liver had been reported in a group of patients with type II diabetes, in that study only elevated transaminase levels were used as a surrogate marker of NAFLD [17]. Nevertheless, the data provided by the work of Namikawa et al. should be interpreted with some caution, due to the characteristics of this type of studies in general and of the present one in particular. A low sample size may limit the statistical power and preclude the discovery of relevant relationships. For example, an unexpected finding in the study by Namikawa et al. is the obervation of a significantly higher stage of the disease (i.e. fibrosis) in the group with a low MTP genotype, despite the fact that the grade was not different. This implies that the severity of necro-inflammation, that has been indicated as a predictor of the development of fibrosis [18], was not increased in the group with the G/G phenotype, because steatosis, another determinant of grading in NASH [19], was actually increased in this group. Along these lines, a study on a higher number of patients would have enabled the Authors to analyse the distribution of the different genotypes of each of the two genes or of their combination according to the grades and stages of the disease. For example, one would expect to find a higher frequency of the combination of the ‘detrimental’ MTP and MnSOD genotypes in the more severely ill patients.

Another factor that is of critical relevance for the correct understanding of genetic epidemiology studies is the interaction of the genotype with other factors, including the environment, unmodifiable factors (e.g. sex and age) or other genes that could affect the expression of a given gene. In this Japanese study, one-fourth of the subjects in the control group were found to have fatty liver at abdominal ultrasound, a technique that detects moderate to severe steatosis [20]. Although it cannot be ruled out that some of these controls had NASH themselves, despite the presumable normality of liver function tests [21], these subjects are likely to represent a subgroup with ‘simple’ NAFLD. Remarkably, the frequency of the G allele or of the G/G genotype for MTP was not different from that of controls without fatty liver. This might appear in contrast with the initial hypothesis, because one would expect low levels of MTP, as a determinant of the ‘first hit’, to be implicated in the increased deposition of fat also in the ‘uncomplicated’ NAFLD. However, in vitro studies have shown that insulin downregulates the expression of MTP in HepG2 cells, and in those patients with a higher degree of insulin resistance, such as those with NASH, the resulting hyperinsulinemia could lead to a more pronounced decrease in MTP in the subjects with a G/G genetic background. Similarly, MTP is also regulated by endotoxin and pro-inflammatory cytokines [22], factors that have been implicated in the pathogenesis of steatohepatitis and may represent an additional mechanism for the association between NASH and MTP polymorphisms. Therefore, it would have been interesting to correlate the distribution of the MTP polymorphisms with the severity of insulin resistance, the sex of the patients, or the presence of type II diabetes. Further studies on larger series are warranted to clarify these issues.

In contrast, data on the dimorphism of the MnSOD gene pose some questions on the relevance of the investigated allele and on the phenotype–genotype correlation. These genotypes have been extensively studied and reportedly modulate the risk of atherosclerosis, breast cancer, motor neuron disease and severe alcoholic liver disease [23], [24], [25], [26]. However, while in some cases it was the ‘valine’ mutation to confer the risk for the disease, as also in the case of the paper discussed herein, in others it was the ‘alanine’ genotype that was associated with an increased risk. Remarkably, this latter genotype was reported to confer a higher risk of developing severe liver disease among alcoholics, because alanine homozygosity led to a several-fold increased risk of developing alcoholic hepatitis and cirrhosis [26]. Although these data have not been confirmed by a different group [27], they are particularly striking because the ‘hits’ involved in the pathogenesis of alcoholic liver disease are believed to be very similar to those implicated in NASH. In agreement with these discrepancies, the exact significance of the dimorphism in the MnSOD gene is still unclear. It is possible, as stated by Namikawa et al., that the presence of more MnSOD in the mitochondrial matrix affords a better protection against pro-oxidant stimuli because of a more efficient conversion of superoxide anion. However, others speculate that under the same conditions, more hydrogen peroxide would be generated, resulting in more severe cell damage [26]. Thus, in many studies investigating genetic polymorphisms it would be important to provide evidence for a genotype–phenotype correlation. In the study by Namikawa et al., a phenotype characterized by increased hepatic steatosis did correlate with the G/G phenotype of MTP. It would have been interesting to establish similar correlations between MnSOD polymorphisms and some indication of the levels of oxidative stress, as done in other studies [27]. Protein adducts formed with hydroxynonenal, a non-oxidant product of oxidative stress, were detected in the hepatocytes of the patients with NASH and especially in the mitochondria. Larger studies should be aimed at evaluating a possible correlation between these parameters and the different genotypes.

Besides oxidative stress, other factors may function as ‘second hits’ that are believed to induce progressive damage to the fat-laden hepatocytes during NASH. These include excessive production of pro-inflammatory cytokines and/or chemokines or an inappropriate pattern thereof, and other factors linked to metabolism of glucose and free fatty acids [9]. Actually, insulin resistance itself has been indicated as a possible source of the second hit, because the disease occurs more frequently in those with more severe metabolic disturbances [16]. On the other hand, the fact that NASH is observed in only a subset of patients with type II diabetes and is relatively uncommon in patients with polycystic ovarian disease indicates that other factors must be sought after. The approach of the study by Namikawa et al., where more than one genotype was investigated at the same time, should be expanded in larger population studies analysing polymorphisms of several genes whose products regulate multiple and different aspects of the pathogenetic pathways leading to NASH, to identify possible patterns that more precisely reflect a predisposition to develop the disease. In addition, the importance of genetic studies should not lead us to overlook the outstanding progress recently obtained in other fields of investigation on NASH, such as that of adipokines [28]. This group of adipocyte-derived cytokines comprises known factors produced also in other tissues, such as tumor necrosis factor or interleukin-6, and molecules uniquely or predominantly produced at that level, such as leptin, resistin and adiponectin, the role of some of which has recently been highlighted [29]. These studies, together with those on genetic backgrounds, are likely to provide relevant information on the predisposition to NASH and its progression. If the study in the current issue of the Journal takes us one step forward on the road toward the understanding of the pathogenesis of NASH, there is still a lot of road to hit.

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Acknowledgements 

I am indebted to Maurizio Parola (University of Turin, Italy) for helpful discussion.

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PII: S0168-8278(04)00109-6

doi:10.1016/j.jhep.2004.03.005

Journal of Hepatology
Volume 40, Issue 5 , Pages 853-856, May 2004

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