APOC3 and PNPLA3 in non-alcoholic fatty liver disease: Need to clear the air

Authors


  • Conflict of interest statement: Neither author has any conflict of interest to report.

Professor Rakesh Aggarwal, Department of Gastroenterology, Sanjay Gandhi Postgraduate Institute of Medical Sciences, Lucknow 226014, India. Email: aggarwal.ra@gmail.com

Abstract

See article in J. Gastroenterol. Hepatol. 2012; 27: 951–956.

The term “non-alcoholic fatty liver disease” (NAFLD) encompasses two conditions characterized by deposition of excess fat in the liver in the absence of excessive alcohol intake, namely hepatic steatosis and non-alcoholic steatohepatitis (NASH). Hepatic steatosis in the absence of alcohol intake occurs mostly in people with metabolic syndrome or any of its components and/or insulin resistance. It is often related to increased lipolysis in the peripheral adipose tissues resulting in an increased delivery of fatty acids to the liver. The development of NASH needs an additional “hit,” namely lipotoxicity from accumulation of injurious lipid molecules (such as free fatty acids [FFA], lysophosphatidyl choline or free cholesterol), which in turn is associated with hepatic oxidative stress and recruitment of various cytokines that lead to hepatic inflammation and fibrosis. Hepatic steatosis alone is usually non-progressive; in contrast, liver injury in persons with NASH may progress to cirrhosis and/or hepatocellular carcinoma.

NAFLD is currently believed to affect around one-quarter of many populations (as high as 45% in some) and to cause a significant proportion of the total burden of liver disease in both the Western and the Asian regions.1–3 Differences in prevalence, clinical profile, histological severity and outcome of NAFLD in different ethnic groups suggest a genetic contribution.1 This has prompted investigation of polymorphisms of several genes, including those involved in lipid handling (lipolysis, triglyceride synthesis), insulin signaling, oxidative stress and hepatic fibrosis. Of these, gene polymorphisms of apolipoprotein C3 (APOC3) and patatin-like phospholipase domain-containing protein 3 (PNPLA3) have attracted the most interest.

Apolipoproteins are proteins that bind to lipids to form lipoproteins. Lipid molecules, essential for all animal cells, are by themselves not miscible with water. Their binding to apolipoproteins, which have amphipathic properties, results in lipoprotein particles, which are water-soluble and thus can be easily transported across the body in body fluids. In addition, lipoproteins also help target the lipids to particular body tissues through their affinity to specific receptors, and act as coenzymes for some body enzymes.

Apolipoproteins belong to six major classes, namely A, B, C, D, E and H, with several sub-classes for some of them. Of these, apolipoprotein C is the most abundant. It is a constituent of high density lipoprotein, very low density lipoprotein and chylomicrons. Among four subtypes (C1, C2, C3 and C4), apolipoprotein C3 (APOC3) is the most abundant. The APOC3 gene, located along with genes for some other apolipoproteins on the long arm of chromosome 11, encodes a 99-amino acid (aa) protein; this protein undergoes removal of a 20-aa signal peptide in the endoplasmic reticulum to produce a 79-aa mature APOC3 protein. The APOC3 protein inhibits lipoprotein lipase, which hydrolyses triglycerides to generate FFA (i.e. unesterified fatty acids) before their uptake by muscle and adipose tissue.

Two single nucleotide polymorphisms (SNPs) in the promoter region of the APOC3 gene (rs2854117 [–482C>T] and rs2854116 [–455T>C]), which are in strong linkage disequilibrium with each other, have been reported to be associated with hypertriglyceridemia, metabolic syndrome and coronary artery disease.4 More recently, these variants have been shown to be associated with the occurrence of NAFLD. Petersen et al.5 studied these APOC3 polymorphisms in 95 Indian and 163 non-Indian healthy men (108 white, 26 Asian, 15 Hispanic and 14 black) residing in the United States. They found NAFLD in 38% of the 76 Indian men with variant APOC3 alleles at one or both of these loci but none of the 19 with only wild-type alleles. In the non-Indian men too, NAFLD was more frequent among those with variant alleles than those without (9% vs 0%; P = 0.02). The carriers of APOC3 variants also had 30% higher fasting plasma APOC3 levels, 60% higher fasting plasma triglyceride concentrations, and nearly twofold higher post-prandial plasma triglyceride and retinyl fatty acid ester concentrations after oral fat ingestion.5 It was proposed that the variant alleles lead to increased amounts of APOC3, and inhibition of lipoprotein lipase activity and triglyceride clearance, resulting in hypertriglyceridemia due to increase in chylomicron remnants, which are taken up by the liver resulting in NAFLD.

However, subsequent studies in Hispanic, European American, African American and European subjects have failed to confirm the association of APOC3 variants and with NAFLD.6–9 In one of these studies that included 1228 African American, 843 European American and 426 Hispanic subjects who had participated in the Dallas Heart Study, neither of the two APOC3 mutant alleles was associated with homeostatic model of insulin resistance (HOMA-IR), or hepatic fat content.6 The variants were also not associated with HOMA-IR in another additional large cohort, namely participants in the Atherosclerosis Risk in Communities Study; liver fat was not determined in this group.6

In the current issue of the Journal, Hyysalo et al.10 report a similar lack of association between the two APOC3 gene polymorphisms and NAFLD in the Finnish population. They genotyped 417 persons for the two APOC3 SNPs, and measured the liver fat using magnetic resonance spectroscopy and plasma concentration of APOC3. Persons with and without the variant alleles (–455C, –482T or both) had similar amounts of liver fat, plasma APOC3 concentrations, serum triglycerides, high-density lipoproteins, and levels of fasting plasma glucose, insulin and transaminases.

How does one explain these discrepant results in different studies? First, the association of APOC3 polymorphisms and NAFLD may differ with ethnicity. Though Petersen et al.5 found this association in both Indian and non-Indian subjects, the association was weaker in the latter group. All other studies have been in non-Asian patients. To resolve this issue, one would need large studies across multiple ethnic groups. Alternatively, larger studies in the Asian Indian population should help. The APOC3 gene is located in a region that contains several genes involved in lipid transport and metabolism. The observed relationship between APOC3 gene variants and NAFLD may be predicated on another gene, which is in linkage disequilibrium with the APOC3 gene. The association of APOC3 variants with different isoforms of that gene in various ethnic groups could then account for the discrepant observations.

Second, the differences could be related to the anthropometric profile of subjects included in different studies. Petersen et al.5 studied persons without any risk factor of metabolic syndrome and with relatively lower body mass index (BMI) of 24.7 ± 3.6 and 24.1 ± 2.9 kg/m2 in Indian and non-Indian groups, respectively (note that a BMI of 24.7 may indicate overweight in Indians but normal body weight in European. In comparison, other studies included larger proportions of persons with overweight/obesity, dyslipidemia and even full blown metabolic syndrome. For instance, in the Finnish study under discussion,10 subjects with wild-type and variant alleles of APOC3 had mean BMI of 32 and 31.5 kg/m2, mean waist circumferences of 106 and 103 cm, and prevalence rates of metabolic syndrome of 68% and 62%, respectively. The failure to find an association between APOC3 variants and NAFLD in one large study even when only persons with BMI < 25 kg/m2 were analyzed militates against this explanation;6 however, in that study, the presence of visceral obesity, which may be present despite normal BMI, was not specifically looked for.

Several other lines of evidence suggest that the influence of APOC3 promoter variants on insulin resistance, type 2 diabetes mellitus and NAFLD varies with the level of adiposity. In a recent study, the APOC3 –482T allele appeared to increase the risk of type 2 diabetes in lean persons but not in overweight persons, and the –455C allele in fact appeared to protect overweight persons against diabetes.11 The APOC3 variants increased the risk of myocardial infarction only among lean and insulin sensitive subjects, and not in those with insulin resistance and visceral obesity.12 More importantly, in a recent study, minor allele (–482T) of APOC3 was associated with high liver fat only among persons in the lowest tertile of waist circumference.9

Hepatic expression of APOC3 is physiologically inhibited by insulin, and is increased in insulin-resistant states. The SNPs under discussion are located in the insulin-response element of the APOC3 gene promoter. In one study, the variant promoter was less responsive to the suppressive effect of insulin.13 Thus, the variant SNPs may modulate the suppressive effect of insulin on APOC3 expression in states of adequate insulin, but become irrelevant in insulin-resistant states, such as obesity and metabolic syndrome. Further studies on these aspects should help clear the air on discrepant findings on the association of APOC3 variants with NAFLD.

PNPLA3, also known as adiponutrin, is a gene located on chrome 22q13 that encodes for a 481-aa protein with triacylglycerol lipase activity, which mediates triacylglycerol hydrolysis. Its expression is mainly on the surface of lipid droplets in hepatocytes and adipocytes, and is regulated by insulin. Two SNPs in this gene have been investigated in relation to NAFLD: (i) a G-to-C change leading to substitution of isoleucine with methionine at codon 148 (I148M; rs738409), and (ii) a G-to-T change leading to substitution of serine with isoleucine at codon 453 (S453I; rs6006460).

In contrast to the APOC3 variants, association of PNPLA3 I148M variant with NAFLD is much clearer, with most studies showing an increased risk though the strength of association has varied. In a large genome-wide association the study of subjects included in the Dallas Heart study, the mutant allele was a major determinant of increased hepatic fat content and hepatic inflammation, independent of BMI, diabetes status, ethanol use and ancestry; further, this allele was most common in Hispanics, the subgroup most susceptible to NAFLD.14 In another study from Italy and the UK, this variation was associated with severity of steatosis and liver fibrosis, independent of age, BMI, and diabetes.15 A recent meta-analysis showed that the I148M polymorphism exerts a strong influence not only on liver fat accumulation (73% higher lipid content with GG genotype than with CC genotype) but also on disease severity at histology (3.24-fold greater risk of high necro-inflammatory scores and 3.2-fold higher risk of liver fibrosis with GG than CC genotype).16 These data clearly indicate that this SNP is a strong modifier of occurrence and natural history of NAFLD irrespective of ethnic origin.16 In fact, even in the study by Petersen et al.5 that showed association of APOC3 variants with NAFLD in Indian men, the presence of GG genotype of PNPLA3 was associated with higher liver triglyceride content. Furthermore, the association of this PNPLA3 variant with NAFLD begins in early childhood, and is present in both obese and non-obese persons, and in those with and without diabetes.17,18 Hence, the finding of Hyysalo et al.10 that Finnish carriers of PNPLA3 GG genotype had a 2.7-fold higher hepatic fat than those with CC genotype is no surprise.

In contrast, the other variant (S453I) of PNPLA3 is associated with a reduced liver fat content and is common among African Americans,18 and may explain the lower prevalence of NAFLD in this ethnic group.

The PNPLA3 and APOC3 genes are by no means the only genetic players in the causation of NAFLD. A recent meta-analysis of several genome-wide association studies of hepatic steatosis revealed loci in or near the NCAN (neurocan), GCKR (glucokinase regulatory protein), LYPLAL1 (lysophospholipase-like protein 1), and PPP1R3B (protein phosphatase 1, regulatory subunit 3B) genes, that associate with glycemic traits, serum lipid levels, hepatic steatosis, hepatic inflammation/fibrosis, or a combination of these.19 Future studies on these loci would add to our knowledge on heritability of NAFLD.

What do we do with the available information? With a strong evidence base supporting it, the relationship of PNPLA3 variant with NAFLD is ripe for moving from the bench to the bedside. We need to now generate data to find out whether the determination of PNPLA3 genotype in an individual with suspected or confirmed NAFLD can add to the diagnostic algorithm, say by predicting disease severity. This may be particularly helpful in children since the effect of genotype may be additive over time and early institution of preventive measures may be important. Similarly, understanding the biology of PNPLA3 in relation to NAFLD may help in the design of novel treatment strategies. Emerging data on the effect of PNPLA3 variants on other diseases with hepatic steatosis, such as alcoholic liver disease and chronic hepatitis C, may mean that such interventions may play a role beyond NAFLD.20,21

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