Understanding the relationship between PNPLA3, NAFLD and insulin resistance: do ethnic differences bring more questions or more answers?


Nonalcoholic fatty liver disease (NAFLD) is emerging as a problem that affects individuals of varying ethnicities across all regions of the world. Although NAFLD is linked commonly with obesity and Western diet trends, recent studies demonstrate that even nonobese individuals in developing countries are affected [1]. The burgeoning number of studies of NAFLD around the world presents an opportunity to explore how ethnic variations in disease risk and disease phenotype can shed light on its pathogenesis.

A genetic basis behind disease risk in NAFLD was suggested by early studies that showed striking ethnic differences in the prevalence of NAFLD [2]. A genetic basis for this finding became clear in 2008 following the discovery by Romeo et al.[3] of a single nucleotide polymorphism in the gene encoding PNPLA3, also known as adiponutrin, a 481 amino acid protein first described in 2001 that belongs to the patatin-like phospholipase domain containing family [4]. The I148M allele (rs738409) variant describes a cytosine to guanine substitution that leads to an isoleucine to methionine substitution at amino acid residue 148. Structural modelling suggests that this substitution results in spatial hindrance of the catalytic domain of the protein, resulting in loss of function [5]. The exact function of adiponutrin is unknown; however, it may be involved in modifying triglyceride storage in response to different nutritional states.

Several groups have independently confirmed an association between the 148M allele variant and hepatic steatosis across individuals from different ethnicities and geographical regions (Table 1). An interesting finding that has been consistent among studies of the 148M allele variant is the dissociation between the mutation and features of metabolic syndrome including obesity, hypertriglyceridemia and insulin resistance. This was confirmed in a recent meta-analysis [7].

Table 1. Frequency of PNPLA3 G allele in different ethnic populations with nonalcoholic fatty liver disease (NAFLD), association with insulin resistance, ALT and fibrosis
Author (year)PopulationnFrequency of G alleleCorrelation with Insulin ResistanceCorrelation with ALTCorrelation with fibrosis
  1. Selected population-based and case control studies, all of which show a significant association between the 148M allele mutation of the PNPLA3 (adiponutrin) gene and hepatic steatosis. G allele frequencies in patients with NAFLD and controls are shown, highlighting ethnic variation in genetic susceptibility to NAFLD.

Romeo3 (2008)Hispanic Americans3830.49NoYesn/a
Romeo3 (2008)European Americans6960.23NoNon/a
Romeo3 (2008)African Americans1,0320.17NoNon/a
Sookoian12 (2009)Argentinians with NAFLD
Kantartzis14 (2009)Whites from Southern Germany with predisposition to diabetes3300.26NoNon/a
Rotman15 (2010)US Adults (NASH CRN Network)
Caucasian controls
8940.51 (NASH), 0.23(controls)NoYesYes
Valenti16 (2010)Italian patients with NAFLD
UK patients with NAFLD
Italian controls
253, 321, 1790.32, 0.30, 0.17NoYesYes
Romeo13 (2010)Obese Italians6780.26NoYesn/a
Hotta8 (2011)Japanese NAFLD, controls253, 5780.6, 0.44NoYesYes
Wang18 (2011)Taiwan (normoglycemics with NAFLD on ultrasound)1560.51YesYesn/a
Petit19 (2011)French diabetics2340.29 NoYes

What do we know about the role of adiponutrin that might explain this dissociation? Adiponutrin is a membrane-associated protein with both lipogenic and lipolytic properties that is expressed in both liver and adipose tissue. Evidence suggests that adiponutrin is found in lipid droplets and plays a role in the hydrolysis of triglycerides [5]. Hepatic expression of adiponutrin in humans is controlled by nutritional status; expression is downregulated by fasting and upregulated by feeding in response to insulin in a glucose-dependent fashion. Studies in humans with obesity show higher mRNA expression of adiponutrin in obese compared with nonobese [6] individuals and in visceral fat compared with subcutaneous fat [6]. The 148M allele variant represents a loss of function of adiponutrin. Therefore failure to break down triglycerides in the setting of hyperinsulinemic states such as obesity might lead to hepatic steatosis and dissociation from regulation by insulin.

In this edition of Liver International, Wang et al. report an association between the 148M allele of adiponutrin and normoglycemic individuals with NAFLD in Taiwan [18]. This is one of the first studies to describe the PNPLA3 polymorphism in adult Asians with NAFLD. The investigators recruited 876 otherwise healthy individuals from a community in Taiwan and used ultrasound to identify 153 patients within this group who had NAFLD. The investigators were interested in studying patients with NAFLD in the absence of diabetes and therefore excluded diabetics.

The authors found that the prevalence of the PNPLA3 148M allele variant was higher in subjects with NAFLD compared with controls and observed a dose effect in GG [OR = 2.03, 95% confidence interval (CI) = 1.23–3.375] and GC (OR = 1.55, 95% CI 1.02–2.35) variants compared with individuals with the CC wild type. These findings support studies published by other investigators [3, 8, 9, 12-14] and provide further support for an association between the 148M allele variant of PNPLA3 and NAFLD. The allelic frequency of the PNPLA3 148M [G] variant in this population was 0.51, which is as high as the frequency of 0.49 reported in Hispanics in the Romeo study [3]. Hotta et al. reported a G allele frequency of 0.6 in Japanese adults with biopsy-proven NAFLD [8]. Compared with other ethnicities, Asians may therefore fall at the high end of the spectrum along with Hispanics from the standpoint of genetic risk, although further studies investigating G allele frequency in Asians are needed.

The authors found an association between the 148M variant and BMI, waist circumference, hypertriglyceridemia and importantly HOMA-IR. This is in contrast to other studies that have not shown an association between the PNPLA3 148M allele and features of metabolic syndrome including insulin resistance [3, 9, 10]. The reasons for these discrepant findings are unclear, although this study enrolled only normoglycemic individuals with ultrasound evidence of steatosis, in contrast to other studies that included diabetics and used either 1H magnetic resonance spectroscopy (MRS) or biopsy to establish a diagnosis. A Type 1 error resulting from limitations in case definition is possible. The average BMI of NAFLD cases in this study was 27; it is known that metabolic syndrome and fatty liver occurs in Asians at lower BMI thresholds than in the West [11]. Perhaps there are ethnic differences in the relative role of obesity and insulin resistance in the pathogenesis of NAFLD that explain variability in association between the PNPLA3 148M allele and insulin resistance. Further studies are needed to explore this question.

A second study published in this edition, which explores the relationship between PNPLA3 polymorphism and NAFLD comes from France by Petit et al.[19]. The authors included 234 patients with type 2 diabetes and measured liver fat content using 1H MRS and fibrosis using FibroTest®, a noninvasive panel of serum markers that includes total bilirubin, GGT, α2-macroglobulin, apolipoprotein A1 and haptoglobin. Genotyping for the rs738409 mutation of adiponutrin was performed using real time PCR. Sixty-three per cent of subjects had steatosis. 10.2% had moderate to severe (stage F2 or above) fibrosis estimated by FibroTest®. Overall frequency of the G allele of PNPLA3 was 29.2%. The number of patients with steatosis was higher in carriers of the rs738409 minor G allele than in C allele homozygote carriers (70.3% vs 57.1%, P = 0.04). The rs738409 variant was associated with liver fibrosis > F2 in multivariate analysis (OR 0.28, 95% CI 0.1–0.76 for C allele homozygote carriers). Although the use of a noninvasive serum test to estimate fibrosis in this study poses a methodological limitation, the findings are consistent with other studies that show a relationship between the 148M allele variant and fibrosis using biopsy staging [8, 15, 16] (Table 1).

Both studies published in this edition provide further support of an association between adiponutrin gene polymorphisms and hepatic steatosis in two ethnically different populations, one with diabetes and one without diabetes. The studies support the findings of previous groups (with the exception of the association between the PNPLA3 polymorphism and insulin resistance noted in the study by Wang et al.). Clearly hepatic steatosis is more prevalent among carriers of the G allele variant of PNPLA3 regardless of ethnicity. Although most studies to date dissociate the polymorphism from insulin resistance, further studies that explore this question in other ethnic groups are warranted.

There is a growing worldwide interest in NAFLD, a disease that commonly affects individuals of all ethnicities. We now have some insight into the genetic basis of ethnic differences in the prevalence of NAFLD. NAFLD is the phenotypic manifestation of a polygenic disease process with both genetic and environmental factors. Genome-wide association studies have identified other gene associations involved in NAFLD [17] and more will hopefully be identified in the future. Of interest is the relationship between adiponutrin mutations, steatosis and insulin resistance. Better understanding of the function of adiponutrin may help elucidate how these are involved in the pathophysiology of NAFLD.