Genetics of Nonalcoholic Fatty Liver Disease

Genetics of Nonalcoholic Fatty Liver Disease

Non-alcoholic fatty liver disease (NAFLD) and its more severe form non-alcoholic steatohepatitis (NASH) are associated with several diseases (obesity, type 2 diabetes, dyslipidemia and hypertension), having insulin resistance as the common factor. These conditions cluster to form the insulin resistance syndrome or, according to a recent proposal, the metabolic syndrome, carrying a high risk for cardiovascular complications. NASH itself, as well as pure fatty liver, is an insulin-resistant state, not only in subjects with additional metabolic disorders, but also in lean normoglycemic patients. The prevalence of the metabolic syndrome, according to well-defined criteria, is higher in NAFLD patients compared with the general population. NAFLD patients with metabolic syndrome have a higher prevalence and severity of fibrosis and necroinflammatory activity, compared to subjects with pure fatty liver. The presence of the metabolic syndrome is associated with a high risk of NASH among NAFLD subjects, after correction for gender, age and body mass. In particular, it is associated with a high risk of severe fibrosis. The increasing prevalence of obesity, coupled with diabetes, dyslipidemia, hypertension and metabolic syndrome puts a very large population at risk for succumbing to liver failure in the next decades.

The adipocyte as a source and target for inflammation

The adipocyte is a remarkable cell type in several respects. It stores excess energy in the form of lipids and is thus able to dramatically change its size in accordance with changing metabolic needs. This ability gives adipose tissue an almost unlimited capacity for growth, making it perhaps the only tissue in the body with the ability to so drastically increase in its size without an underlying transformed cellular phenotype. Adipose tissue is responsive to both central and peripheral metabolic signals and is itself capable of secreting a number of proteins. These adipocyte-specific or enriched proteins, termed adipokines, have been shown to have a variety of local, peripheral, and central effects. Adipose tissue is therefore able to integrate signals from other organs and respond by regulating secretion of multiple proteins. As an active participant in whole body energy homeostasis, adipose tissue can negatively influence other systems when dysregulated. Although adipocytes are capable of increasing in size, the cellular homeostasis and the secretory profile of larger adipocytes become altered and increasingly dysregulated compared with adipocytes of smaller size.

Cytokines and NASH

Because the histopathology of NASH resembles that of alcohol-induced steatohepatitis (ASH), common pathogenic mechanisms may mediate both of these diseases. Immunological mechanisms have a pivotal role in the pathogenesis of ASH. This has been remarkably well demonstrated by studies of patients and experimental animals. In hospitalized patients with severe ASH, serum levels of several pro-inflammatory cytokines, including TNF-α, are increased significantly. Cytokine levels correlate well with liver disease severity, generally decreasing in those who recover but remaining elevated in those who do not. These seminal observations stimulated subsequent studies in small animal models for ASH to determine if inflammatory cytokines directly mediate alcohol-related hepatotoxicity. The results strongly support this concept. For example, various therapies that inhibit gut-derived lipopolysaccharide (LPS) endotoxemia or that block the activity of TNF-α, an LPS-induced cytokine, provide mice and rats nearly complete protection from ASH.

NASH is strongly associated with obesity in humans, mice and rats. Once considered to be a relatively inert storage depot for fat, adipose tissue is now known to produce many different hormones and cytokines, including TNF-α. Thus, the increased adipose tissue mass of obese individuals provides a major source of serum TNF- α. Recent evidence suggests that resident immune cells in organs such as the liver may also contribute to obesity-related increases in proinflammatory cytokine production.

Exposure to excessive levels of adipocyte-derived TNF- α relative to its antagonist, adiponectin, favors increased biologic activity of TNF, which further inhibits the actions of adiponectin. Reduced adiponectin activity promotes hepatocyte steatosis by enhancing fatty acid uptake, inhibiting fatty acid oxidation and reducing lipid export. Faced with excessive TNF- α and fatty acids but little adiponectin, hepatocytes store lipids. The retention of fatty acids in turn, unleashes signals that activate NF-κB within hepatocytes, inducing NF-κB-sensitive genes, and thereby increases the generation of various mediators, including IL-6, TNF- α, and IL-8. The pro-inflammatory hepatic milieu is perpetuated by the relative death of local anti-inflammatory (Th-2) cytokines that result from liver NKT cell depletion. Increased sustained release of IL-6 from the liver causes systemic insulin resistance. Local increases in TNF- α and IL-8 promote hepatocye oxidative stress and eventual apoptosis, and recruit inflammatory cells into the liver. As antioxidant and antiapoptotic defenses are overwhelmed hepatocyte death increases and inflammatory cells accumulate, signifying the emergence of NASH. Animal studies have proved that the unbalanced production of fat derived cytokines promotes the early stages of NAFLD.

The past few years have seen the emergence of microarray analysis as a method of assessing the expression of a large number of genes simultaneously in a tissue sample. Gene expression profiling can be the source of a large amount of information that can be used to better the understanding of disease pathogenesis, bring forward hypotheses and predictions, and open new avenues for investigation and management of disease. [Deaciuc IV, Arteel GE, Peng X, et al. Gene expression in the liver of rats fed alcohol by means of intragastric infusion. Alcohol 2004;33:17 30 ] Moreover, these techniques are well suited to investigate the molecular basis of complex diseases such as chronic liver disease, diabetes, and obesity.[ Baranova A, Schlauch K, Gowder S, et al. Microarray technology in the study of obesity and non alcoholic fatty liver disease. Liver Int 2005; 25:1091 1096]. Microarray analysis has been applied to several different chronic liver diseases including primary biliary cirrhosis[ Shackel NA, McGuiness PH, Abbot CA, et al. Identi cation of novel molecules and pathogenic pathways in primary biliary cirrhosis. Gut 2001;49:565 576], hepatitis C virus [Shackel NA, McGuiness PH, Abbot CA, et al. Insights into the pathobiology of Hepatitis C virus associated cirrhosis: analysis of intrahepatic differential gene expression. Am J Pathol 2002; 160:641 654], ALD, and NAFLD [Seth D, Gorrell MD, Cordoba S, et al. Intrahepatic gene expression in human alcoholic hepatitis. J Hepatol 2006; 45: 306 320 , Younossi ZM, Gorreta F, Ong JP, et al. Hepatic gene expression in patients with obesity-related non-alcoholic steatohepatitis. Liver Int 2005;25:760 771 ] and has already contributed to a greater understanding of disease pathogenesis and identified several candidate genes worthy of examining in association studies.

Characterization of the entire sequence of proteins in a biological sample with methods such as surface-enhanced laser desorption/ionization time-of- ight (SELDI-TOF) has been described. These studies can be used to compare patterns of gene expression in organs such as the liver with protein pro les in blood and serum in large cohorts of patients in relatively little time. The rst description of this technique in patients with NAFLD by Younossi and colleagues in 91 patients included 12 with steatosis alone, 52 with steatosis and nonspecific inflammation, and 27 with de nite NASH. [Younossi ZM, Baranova A, Ziegler K, et al. A genomic and proteomic study of the spectrum of nonalcoholic fatty liver disease. Hepatology 2005;42:665 674] Seven obese patients among this cohort who were undergoing bariatric surgery served as controls. Each group with NAFLD was compared with the obese controls. Twenty-two genes with more than twofold differences in gene expression were identified and the proteomic analysis for the same groups showed 12 significantly different protein peaks in the three subtypes of NAFLD. The population of patients in this study, however, was not representative of a general NAFLD population because they were all morbidly obese.

Genes Influencing The Severity of Steatosis

Recognition of the role played by steatosis in the pathogenesis of progressive liver disease [Day CP, James OF. Hepatic steatosis: innocent bystander or guilty party? Hepatology 1998;27:1463 1466] suggests that factors determining its severity may influence the risk of advanced ALD and NAFLD. Clearly, genetic and environmental factors determining the degree of obesity would fall into this category. Polymorphisms in genes involved in the synthesis, storage, and export of hepatic triglyceride clearly influence the magnitude of steatosis. MTP is critical for the synthesis and secretion of very low density lipoprotein (VLDL) in the liver and intestine, and a frameshift mutation in the gene is associated with abetalipoproteinemia. A G/T single nucleotide polymorphism (SNP) at position 493 in the 50 promoter region has been associated with lower levels of transcription resulting in lower MTP levels and failure to excrete triacylglycerol from the liver. Evidence has been presented that patients with NAFLD homozygous for the G allele have increased steatosis and histological NASH grade compared with heterozygous patients or patients homozygous for the high activity allele, and the same SNP has been associated with advanced ALD in a preliminary study. Unfortunately, the study that phenotyped the NAFLD patients with liver biopsy included only 63 patients, and therefore its results should be interpreted with caution. More recently, an even smaller study reported an association between NAFLD and a loss-of-function SNP in the gene encoding phosphatidylethanolamine methyltransferase (PEMT), which is involved in phosphatidylcholine synthesis required for VLDL synthesis. [Song J, da Costa KA, Fischer LM, et al. Polymorphism of the PEMT gene and susceptibility to nonalcoholic fatty liver disease (NAFLD). FASEB J 2005;19:1266 1271] Clearly, more studies are needed, not only of MTP and PEMT polymorphisms but also of polymorphisms of other genes encoding proteins involved in hepatic lipid metabolism as susceptibility factors for progressive ALD and NAFLD.

The principal class of genes that influences the oxidant load in patients with obesity, insulin resistance, and the metabolic syndrome are those encoding proteins involved in the oxidation of FFA. The role of FFA oxidation in the pathogenesis of NAFLD is complex. On the one hand, appropriate fat oxidation is required to prevent fat accumulation in the liver, but on the other, excessive fatty acid oxidation leads to oxidative stress. [Stewart SF, Leathart JB, Chen Y, et al. The valine-alanine manganese superoxide dismutase polymorphism is not associated with increased oxidative stress or susceptibility to advanced alcoholic liver disease. Hepatology 2002;36:1355 1360 , Sanyal AJ, Campbell-Sargent C, Mirshahi F, et al. Non-alcoholic steatohepatitis: association of insulin resistance and mitochondrial abnormalities]. Children with inherited defects in mitochondrial β-oxidation develop steatosis but they do not get NASH, strongly suggesting that intact mitochondrial oxidation of FFA is required for progression to inflammation and fibrosis. With respect to peroxisomal and microsomal fat oxidation, because both are capable of generating ROS, it might be predicted that gain-of-function polymorphisms in genes encoding proteins involved in these processes would predispose to NASH. However, these pathways play a role in limiting mitochondrial overload during times of excessive FFA supply and therefore it may be that loss-of function polymorphisms affecting these pathways would predispose to NASH. The latter hypothesis is supported by a study showing that mice lacking the gene encoding fatty acyl-CoA oxidase (AOX), the initial enzyme of the peroxisomal β-oxidation system, develop severe micro-vesicular NASH. [Fan CY, Pan J, Usuda N, et al. Steatohepatitis, spontaneous peroxisome proliferation and liver tumors in mice lacking peroxisomal fatty acyl-CoA oxidase: implications for peroxisome proliferator-activated receptor alpha natural ligand metabolism. J Biol Chem 1998;273:15639 15645] Similar difficulties apply to interpreting a preliminary report that a mutation (PPARA*3) in the gene encoding peroxisome proliferator activated receptor (PPARa) is associated with NASH. [Merriman R, Aouizerat B, Molloy M, et al. A genetic mutation in the peroxisome proliferator-activated receptor alpha gene in patients with non-alcoholic steatohepatitis. Hepatology 2001; 34:441A] PPARa regulates the transcription of a variety of genes encoding enzymes involved in mitochondrial, peroxisomal b-oxidation and microsomal -oxidation of fatty acids. [Berger J, Moller D. The mechanism of action of PPARs. Annu Rev Med 2002; 53:409 435] Functional data on the mutation are somewhat contradictory at present; however, studies in PPARa knockout mice and the fact that adiponectin activates PPARa and protects against steatosis [Xu A, Wang Y, Keshaw H, et al. The fat-derived hormone adiponectin alleviates alcoholic and non-alcoholic fatty liver disease in mice. J Clin Invest 2003; 112:91 100 , Yamauchi T, Kamon J, Minokoshi Y, et al. Adiponectin stimulates glucose utilization and fatty-acid oxidation by activating AMP-activated protein kinase. Nat Med 2002;8: 1288 1295] suggest that any PPARA mutation associated with NASH should be associated either with loss of function or reduced gene expression.

Genetic factors are important to the development of NAFLD, and recent advantages in SNP genotyping methods have enabled the detection of genetic variations associated with increased susceptibility to NAFLD. There have been a few reports of genetic variations that are associated with NAFLD, and they have been in the genes for TNF receptor 2 (TNFR-2), TNF-α, microsomal triglyceride transfer protein (MTP), and methylenetetrahydrofolate reductase (MTHFR) genes. [Gambino R, Cassader M, Pagano G, Furazzo M, Musso G: Polymorphism in microsomal triglyceride transfer protein: a link between liver disease and atherogenic postprandial lipid profile in NASH? Hepatology 2007, 45:1097-107] , Tokushige K, Takakura M, Tsuchiya-Matsushita N, Taniai M, Hashimoto E, Shiratori K: Influence of TNF gene polymorphism in Japanese patients with NASH and simple steatosis. J Hepatol 2007, 46:I 104-10]. All of these genes are related to inflammation, lipid metabolism, and oxidation.

Genetic factors as well as environmental factors are important to the development of NAFLD, and the gene for peroxisome proliferator-activated receptor γ coactivator 1α (PPARGC1A) is a candidate gene for susceptibility to NAFLD, since it is involved in insulin resistance, mitochondrial biogenesis, and oxidative phosphorylation, which are key factors in the development of NAFLD. Recent evidence also implicates PPARGC1A in the homeostatic control of systemic energy metabolism, and PPARGC1A knockout mice have been reported to develop hepatic steatosis due to a combination of reduced mitochondrial respiratory capacity and increased expression of lipogenic genes. [Leone TC, Lehman JJ, Finck BN, Schaeffer PJ, Wende AR, Boudina S, Courtois M, Wozniak DF, Sambandam N, Bernal-Mizrachi C, Chen Z, Holloszy JO, Medeiros DM, Schmidt RE, Saffitz JE, Abel ED, Semenkovich CF, Kelly DP: PGC-1alpha deficiency causes multi-system energy metabolic derangements: muscle dysfunction, abnormal weight control and hepatic steatosis. PLoS Biol 2005, 3:e101]

APOE3/3 genotype is statistically significantly associated with increased risk for NASH in the overall group of NASH patients compared with controls. The association was more remarkable in male patients with NASH than female patients with NASH, although the number of female patients were less than that of the male NASH patients and controls. The APOE3/3 genotype had a 7.941-fold increased risk for overall NASH, a 14.362-fold increased risk for male NASH and a 4.737-fold increased risk for female NASH, whereas the APOE3/4 genotype had protection against the overall NASH group. [Demirag MD, Onen HI, Karaoguz MY, Dogan I, Karakan T, Ekmekci A, Guz G (2007) Apolipoprotein E gene polymorphism in nonalcoholic fatty liver disease. Dig Dis Sci 52(12):3399-3403.

CYP2E1 enzyme is related to nonalcoholic steatohepatitis (NASH) due to its ability for reactive oxygen species production, which can be influenced by polymorphisms in he gene. In a study whose aim was to investigate hepatic levels, activity, and polymorphisms of the CYP2E1 gene to correlate it with clinical and histological features in 48 female obese NASH patients, CYP2E1 content was significantly higher in the steatohepatitis (45%; p=0.024) and steatosis (22%; p=0.032) group compared with normal group. Chorzoxazone hydroxylase activity showed significant enhancement in the steatohepatitis group (15%; p=0.027) compared with the normal group. c2 rare allele of Rsa1/Pst1 polymorphisms but no C allele of Dra1 polymorphism was positively associated with CHZ hydroxylation, which in turn is correlated with liver CYP2E1 content. c2 allele is positively associated with liver injury in NASH. This allele may determine a higher transcriptional activity of the gene, with consequent enhancement in pro-oxidant activity of CYP2E1 thus affording liver toxicity.

Microsomal triglyceride transfer protein (MTP) 493 G/T polymorphism modulates circulating lipid and lipoprotein levels in different subsets and has been linked to NAFLD. Plasma lipids, triglyceriderich lipoprotein subfractions, high-density lipoprotein-C (HDL-C), and oxidized low-density lipoprotein (LDL) after an oral fat load were cross-sectionally correlated to MTP 493 G/T polymorphism, dietary habits, adipokines, and liver histology in 29 nonobese nondiabetic patients with NASH and 27 healthy controls. The severity of liver histology, the magnitude of triglycerides (Tg), free fatty acid (FFA), and LDL-conjugated diene responses, and the fall in HDL-C and apoA1 were significantly higher in NASH G/G (66% of patients) than in the other genotypes, despite similar adipokine profile and degree of insulin resistance. Postprandial large intestinal VLDL subfraction A increases independently predicted Tg, FFA, HDL-C, and LDL-conjugated diene responses. VLDL A apoB48 response was independently associated with liver steatosis. Postprandial LDL-conjugated diene response predicted severe necro-inflammation and fibrosis; postprandial apoA1 fall predicts severe fibrosis. MTP -493 G/T polymorphism may impact NASH by modulating postprandial lipemia and lipoprotein metabolism; homozygous GG carriers have a more atherogenic postprandial lipid profile than the other genotypes, independently of adipokines and insulin resistance. [Karpe F, Lundahl B, Ehrenborg E, Eriksson P, Hamsten A. A common functional polymorphism in the promoter region of the microsomal triglyceride transfer protein gene influences plasma LDL levels. Arterioscler Thromb Vasc Biol 1998; 18:756-761 , Namikawa C, Shu-Ping Z, Vyselaar JR, Nozaki Y, Nemoto Y, Ono M, et al. Polymorphisms of microsomal triglyceride transfer protein gene and manganese superoxide dismutase gene in non-alcoholic steatohepatitis J Hepatol 2004;40:781-786 , Swift LL, Kakkad B, Boone C, Jovanovska A, Gray Jerome W, Mohler PJ, et al. Microsomal triglyceride transfer protein expression in adipocytes: a new component in fat metabolism. FEBS Lett 2005; 579:3183-3189.]