Expression of adiponectin and its receptors in livers of morbidly obese patients with non-alcoholic fatty liver disease

Background and Aim:  Obesity is one of the risk factors for non-alcoholic fatty liver disease (NAFLD) and a common disease that comprises simple steatosis and non-alcoholic steatohepatitis (NASH), and can eventually lead to liver cirrhosis. Adiponectin is an adipocyte-derived protein that has anti-obesity, antidiabetic and anti-inflammatory properties, and is considered to possess a hepatoprotective function. Its role in the development and progression of NAFLD in morbidly obese patients is unknown. In this study, we examined the expression levels of adiponectin and its receptors in liver biopsies of morbidly obese patients and then determined whether there was an association with the disease severity.
Methods:  Liver biopsies from 30 morbidly obese patients (18 NASH vs 12 steatosis) were analyzed. The needle core biopsies were subjected to routine histological examination and stained immunohistochemically for adiponectin, adiponectin receptor I (adipoRI) and receptor II (adipoRII).
Results:  The two groups were comparable with respect to body mass index, age and gender distribution. The expression of adiponectin decreased in liver biopsies with NASH as compared to those with simple steatosis (1.61 ± 0.70 vs 2.25 ± 0.75, P  = 0.028). Spearman's rank correlation coefficient analysis showed that the staining intensity of adiponectin negatively correlated with the grade of inflammation ( r  = −0.368, P  = 0.045) and stage of fibrosis ( r  = −0.380, P  = 0.038). There was no significant difference in expression of adipoRI and adipoRII between the two groups.
Conclusion:  These findings indicate that decreased liver adiponectin expression may play a role in the development and progression of NAFLD, from simple steatosis to NASH, in morbidly obese patients.     

Beyond insulin resistance in NASH: TNF-alpha or adiponectin?

Adiponectin has antilipogenic and anti-inflammatory effects, while tumor necrosis factor alpha (TNF-alpha) reduces insulin sensitivity and has proinflammatory effects. We examined (1) the extent to which hypoadiponectinemia and TNF-alpha activation are features of nonalcoholic steatohepatitis (NASH) and (2) whether serum levels of these markers correlate with the severity of histological changes in 109 subjects with nonalcoholic fatty liver disease (NAFLD), including 80 with NASH and 29 with simple steatosis. By multivariate analysis, subjects with NASH had reduced adiponectin level and increased TNF-alpha and soluble TNF receptor 2 (sTNFR2)-but not leptin levels, compared with controls matched by age, sex, and body mass index; these differences were independent of the increased insulin resistance (by homeostasis model [HOMA-IR]) in NASH. When compared with simple steatosis, NASH was associated with lower adiponectin levels and higher HOMA-IR, but there were no significant differences in the levels of TNF-alpha and sTNFR2. The majority of subjects with steatohepatitis (77%) had adiponectin levels less than 10 microg/mL and HOMA-IR greater than 3 units, but only 33% of those with pure steatosis had these findings. HOMA-IR and low serum adiponectin were also independently associated with increased grades of hepatic necroinflammation. In conclusion, hypoadiponectinemia is a feature of NASH independent of insulin resistance. Reduced adiponectin level is associated with more extensive necroinflammation and may contribute to the development of necroinflammatory forms of NAFLD.     

PNPLA3 I148M and response to treatment for hepatic steatosis: A systematic review

Background

It is unclear whether the patatin-like phospholipase domain-containing protein 3 (PNPLA3) rs738409 C-to-G single nucleotide polymorphism, resulting in the substitution of isoleucine to methionine at position 148 (I148M), impedes regression of hepatic steatosis when treating non-alcoholic fatty liver disease (NAFLD).

Objectives

Investigate if carriage of the PNPLA3 148M allele affects the anti-steatotic efficacy of all possible anti-NAFLD interventions, identify gaps in current knowledge and provide guidance for individual treatment.

Methods

Research available in public databases was searched up to 13 November 2022. Studies were included if a treatment in NAFLD patients decreased hepatic steatosis in the pooled patient group or a PNPLA3 I148M polymorphism subgroup (II/IM/MM). The risk of bias was assessed using the Cochrane Risk-Of-Bias 2 Tool and the Newcastle–Ottawa Scale.

Results

Moderate evidence indicates that NAFLD patients homozygous for the PNPLA3 148M allele benefit less or not at all from omega-3 carboxylic acids to decrease liver fat, while the PNPLA3 148I allele shows moderate benefit. Low evidence suggests that interventions employing lifestyle changes are more effective to reduce liver fat in NAFLD patients homozygous for the PNPLA3 148M allele compared to patients with wild-type PNPLA3.

Conclusions

NAFLD patients homozygous for the PNPLA3 148M allele might not benefit from omega-3 carboxylic acids to reduce hepatic steatosis in contrast to patients with wild-type PNPLA3. Instead, patients with two PNPLA3 148M alleles should be especially advised to adopt lifestyle changes. Genotyping for PNPLA3 I148M should be encouraged in therapeutic studies for NAFLD.

Registration Number (Prospero)

CRD42022375028.     

Adiponectin and its receptors in non-alcoholic steatohepatitis

BACKGROUND: Adiponectin, an adipocyte derived polypeptide, has been shown to alleviate steatosis and inflammation in mice with non-alcoholic fatty liver disease. AIM: In the present study, we wished to define liver expression of adiponectin and its receptors in morbidly obese patients undergoing bariatric surgery. Patients with non-alcoholic steatohepatitis (NASH) or simple steatosis were investigated to test whether dysregulation of this system might be involved in these disorders. PATIENTS AND METHODS: Liver mRNA expression of adiponectin and its recently cloned receptors RI and RII (adipoRI and adipoRII) were analysed by fluorescence based real time polymerase chain reaction in 13 patients with NASH and nine with simple steatosis. Adiponectin and adipoRII protein expression were assessed by immunohistochemistry in a subgroup of patients. RESULTS: Adiponectin and adipoRII mRNA expression were significantly reduced in liver biopsies of patients with NASH compared with simple steatosis while no difference was found in adipoRI mRNA expression. In NASH, adipoRII mRNA expression was negatively correlated with serum aspartate aminotransferase levels, serum alanine aminotransferase levels, and grade of fibrosis. Liver adiponectin protein expression was mainly found in endothelial cells of portal vessels and liver sinusoids whereas adipoRII expression was seen in hepatocytes only. Adiponectin and adipoRII staining were lower in biopsies of subjects with NASH compared with simple steatosis. CONCLUSION: Reduced hepatic expression of adiponectin and adipoRII might be of pathophysiological relevance in non-alcoholic fatty liver diseases.     

More Evidence for the Genetic Susceptibility of Mexican Population to Nonalcoholic Fatty Liver Disease through PNPLA3

Background. The gene for patatin-like phospholipase domain containing 3 (PNPLA3) is associated with non-alcoholic fatty liver disease (NAFLD) development. We previously found that Mexican indigenous population had the highest frequency reported of the PNPLA3 148M risk allele. Further, we observed a relationship between M148M genotype with elevated ALT levels in individuals with normal weight, overweight and obese. We sought to investigate whether PNPLA3 polymorphism is associated with NAFLD development in Mexicans.Material and methods. We enrolled 189 Mexican patients with NAFLD and 201 healthy controls. Anthropometric, metabolic, and biochemical variables were measured, and rs738409 (Ile148Met substitution) polymorphism was genotyped by sequencing.Results. Logistic regression analysis, using a recessive model, suggested that PNPLA3 polymorphism in Mexican population is significantly associated (OR = 1.711, 95% CI: 1.014-2.886; P = 0.044) with NAFLD.Conclusions: The PNPLA3 gene is associated with NAFLD in Mexican population. More studies are required to explain the high prevalence of PNPLA3 polymorphism in Mexican-Americans, Mexican-Indians, and Mexican-Mestizos.     

The Expression of PNPLA3 Polymorphism could be the Key for Severe Liver Disease in NAFLD in Hispanic Population

BackgroundNonalcoholic fatty liver disease (NAFLD) encompasses: fatty liver (SS), steatohepatitis (NASH) with or without fi-brosis and cirrhosis. Patatine-like phosphatas in domain 3 (PNPLA3; adiponutrin; SNP rs738409 C/G, M148I) shows anabolic and catabolic activities on lipid metabolism and significant association to fatty liver content; however, I148M demographics and ethnics, as its role with NAFLD have not been fully elucidated.Material and methodsPNPLA3 genotyping from peripheral blood DNA by polymerase chain reaction (PCR) and direct sequencing, 211 patients diagnosed with NAFLD including SS, NASH and fibrosis spectrum.ResultsEighty nine per cent showed the G risk allele [CC: 23 (10.5%), GC: 73 (34.7%), GG 115 (54.7%)], the allele frequency was 77%, NASH (71%), SS (80%) and fibrosis (73%). GG genotype carriers showed 3.8 times (CI 95%: 3.03 - 4.79) of increased risk of steatohepatitis and 2.3 times more (CI 95%: 1.77 ~ 3.23) risk of having liver fibrosis (CC). PNPLA3 (GC, GG) conditioned higher probability of low levels of HDL cholesterol (p < 0.010), SS even in normal weight (p < 0.007), insulin resistance by HOMA (p < 0.029), NAFLD fibrosis score showed > 0.675 (p < 0.001) and altered serum alanine aminotransferase (p < 0.05).ConclusionPNPLA3 expression in Hispanics could be decisive in NAFLD pathogenesis, it's highly prevalent and it's a key to condition and determine the spectrum associated, SS, NASH and fibrosis.     

Association of single nucleotide polymorphism at PNPLA3 with fatty liver,steatohepatitis, and cirrhosis of liver

Background/Purpose

The aim of this study was to determine the association of single nucleotide polymorphism (SNP) in patatin-like phospholipase domain-containing 3 (PNPLA3) at I148 with histological severity of non-alcoholic fatty liver disease (NAFLD).

Methods

Patients were selected for the study if they had histological evidence of NAFLD and clinical evidence of non-alcoholic steatohepatits (NASH) cirrhosis. We included 50 NASH cirrhosis, 99 patients of NAFLD including 36 non-NASH fatty liver (NNFL) along with 63 NASH and 75 healthy controls. PNPLA3 genotyping was done by real-time PCR using a Taqman assay for rs738409.

Results

CC, CG, and GG frequencies were 45 (60.0%)/27 (36.0%)/3 (4.0%) in healthy control, 19 (52.8%)/14 (38.9%)/ 3 (8.3%) in NNFL, 18 (28.6%)/29 (46.0%)/16 (25.4%) in NASH, and 7 (14.6%), 25 (52.1%), 16 (33.3%) in cirrhosis. The frequency of G allele was significantly higher (62.6%) in NAFLD than in healthy control. The GG genotype had 20.25 times odds of NAFLD. The GG genotype had 6.53 times odds of having NASH. HOMA-IR > 1.6 had 3.81 times odds of having NASH. Regression analysis revealed that G allele odds of having cirrhosis was 3.9 times compared to C. The G allele was also significantly associated with steatosis, lobular inflammation, NAFLD activity score, and fibrosis.

Conclusion

PNPLA3 genotype showed an association with NAFLD, NASH, fibrosis, and cirrhosis.

     

PNPLA3 and TM6SF2 polymorphisms in Brazilian patients with nonalcoholic fatty liver disease

BACKGROUND Nonalcoholic fatty liver disease(NAFLD) is becoming the most common chronic liver disease worldwide, with significant morbidity associated with nonalcoholic steatohepatitis(NASH). Genome-wide association studies demonstrated that the variants rs738409 C/G in the PNPLA3 and rs58542926 C/T in the TM6 SF2 genes are determinants of inter-individual and ethnicity-related differences in hepatic fat content and NAFLD progression.AIM To investigate PNPLA3 and TM6 SF2 genotype frequency and their association with NAFLD development and progression in Brazilian patients.METHODS This cross-sectional case-control study enrolled 285 individuals from the Gastroenterology and Hepatology clinics at a university hospital in Brazil. The case patients(n = 148) were confirmed to have NAFLD by the identification of hepatic steatosis on ultrasonography and exclusion of other causes of liver disease. According to the clinical protocol, patients underwent liver biopsy when at high risk for NASH and/or advanced fibrosis(n = 65). Steatohepatitis was confirmed in 54 patients. Individuals who did not have biopsy indication or NASH on histology were considered to have simple steatosis(n = 94). The control group(n = 137) was selected among patients that attended the Intestinal Disease clinic and was composed of subjects without abnormalities on abdominal ultrasonography and normal liver biochemical tests. All individuals underwentPNPLA3 and TM6 SF2 genotype analysis.RESULTS PNPLA3 CC, CG and GG genotype frequencies were 37%, 44% and 19%, respectively, in NAFLD patients and were 58%, 31% and 10% in controls(P 0.001). In a model adjusted for gender, age, body mass index and type 2 diabetes mellitus, the G allele increased the chance of NAFLD(OR = 1.69, 95%CI: 1.21-2.36, P = 0.002) and NASH(OR = 3.50, 95%CI: 1.84-6.64, P 0.001). The chance of NASH was even higher with GG homozygosis(OR = 5.53, 95%CI: 2.04-14.92, P = 0.001). No association was found between G allele and the features of metabolic syndrome. In histological assessment, PNPLA3 genotype was not associated with steatosis grade, although GG homozygosis increased the chance of significant NASH activity(OR = 17.11, 95%CI: 1.87-156.25, P = 0.01) and fibrosis(OR = 7.42, 95%CI: 1.55-34.47, P = 0.01) in the same adjusted model. TM6 SF2 CC, CT and TT genotype frequencies were 83%, 15% and 0.7%, respectively, in NAFLD patients and were 84%, 16% and 0.7% in controls(P = 0.78). The T allele presence was not associated with NAFLD or NASH, and was not associated with histological features.CONCLUSION PNPLA3 may be involved in susceptibility and progression of NAFLD and NASH in the Brazilian population. More advanced histological liver disease was associated with the G allele. The TM6 SF2 genetic variants were not associated with NAFLD susceptibility and progressive histological forms in the population studied, but further studies are required to confirm these findings.     

PNPLA3 I148M variant in nonalcoholic fatty liver disease:Demographic and ethnic characteristics and the role of the variant in nonalcoholic fatty liver fibrosis

Patatin-like phospholipase domain-containing 3(PNPLA3 or adiponutrin) displays anabolic and catabolic activities in lipid metabolism,and has been reported to be significantly associated with liver fat content.Variousstudies have established a strong link between the 148 isoleucine to methionine protein variant(I148M) of PNPLA3 and liver diseases,including nonalcoholic fatty liver disease(NAFLD).However,detailed demographic and ethnic characteristics of the I148 M variant and its role in the development of nonalcoholic fatty liver fibrosis have not been fully elucidated.The present review summarizes the current knowledge on the association between the PNPLA3 I148 M variant and NAFLD,and especially its role in the development of nonalcoholic fatty liver fibrosis.First,we analyze the impact of demographic and ethnic characteristics of the PNPLA3 I148 M variant and the presence of metabolic syndrome on the association between PNPLA3 I148 M and NAFLD.Then,we explore the role of the PNPLA3 I148 M in the development of nonalcoholic fatty liver fibrosis,and hypothesize the underlying mechanisms by speculating a pro-fibrogenic network.Finally,we briefly highlight future research that may elucidate the specific mechanisms of the PNPLA3 I148 M variant in fibrogenesis,which,in turn,provides a theoretical foundation and valuable experimental data for the clinical management of nonalcoholic fatty liver fibrosis.     

Abdominal fat interacts with PNPLA3 I148M,but not with the APOC3 variant in the pathogenesis of liver steatosis in chronic hepatitis C

The patatin‐like phospholipase domain‐containing 3 gene (PNPLA3) and the apolipoprotein C3 gene (APOC3) have been studied in relation to liver steatosis and liver disease outcome. The aim of this study was to evaluate the influence of PNPLA3 p.I148M and APOC3 rs2854116 and rs2854117 polymorphisms on the clinical and histological presentation of chronic hepatitis C in an Italian population and their relationship with viral and anthropometric parameters. Patients with hepatitis C (n = 166) entered the study receiving a clinical, histological, virological and biochemical evaluation. APOC3 (rs2854116 and rs2854117) and PNPLA3 (p.I148M) variants were genotyped. PNPLA3 polymorphisms were associated with liver steatosis, which was significantly higher in patients with p.148I/M (P = 0.034) and p.148M/M (P = 0.004) variants than those homozygous for the PNPLA3 wild type. Excluding patients with HCV genotype 3, the association with liver steatosis and PNPLA3 variants was more marked (p.148I/I genotype vs p.148I/M, P = 0.02, and vs p.148M/M, P = 0.005). The APOC3 polymorphism was not associated with any of the evaluated parameters. Among the interacting factors, BMI and waist circumference correlated with liver steatosis (P = 0.008 and 0.004, respectively). Relationship between waist circumference and liver steatosis was analysed for the different PNPLA3 genotypes. Homozygous 148M patients showed a stronger correlation between waist circumference and steatosis than those carrying the other genotypes (P = 0.0047). In our hepatitis C‐infected population, the PNPLA3 polymorphism influenced the development of liver steatosis, but not fibrosis progression. APOC3 polymorphisms had no effect on the development of steatosis and no influence on the PNPLA3 polymorphism. The amount of abdominal fat can increase the association of PNPLA3 p.I148M with liver steatosis.     

Plasma metabolomic profile in nonalcoholic fatty liver disease

The plasma profile of subjects with nonalcoholic fatty liver disease (NAFLD), steatosis, and steatohepatitis (NASH) was examined using an untargeted global metabolomic analysis to identify specific disease-related patterns and to identify potential noninvasive biomarkers. Plasma samples were obtained after an overnight fast from histologically confirmed nondiabetic subjects with hepatic steatosis (n = 11) or NASH (n = 24) and were compared with healthy, age- and sex-matched controls (n = 25). Subjects with NAFLD were obese, were insulin resistant, and had higher plasma concentrations of homocysteine and total cysteine and lower plasma concentrations of total glutathione. Metabolomic analysis showed markedly higher levels of glycocholate, taurocholate, and glycochenodeoxycholate in subjects with NAFLD. Plasma concentrations of long-chain fatty acids were lower and concentrations of free carnitine, butyrylcarnitine, and methylbutyrylcarnitine were higher in NASH. Several glutamyl dipeptides were higher whereas cysteine-glutathione levels were lower in NASH and steatosis. Other changes included higher branched-chain amino acids, phosphocholine, carbohydrates (glucose, mannose), lactate, pyruvate, and several unknown metabolites. Random forest analysis and recursive partitioning of the metabolomic data could separate healthy subjects from NAFLD with an error rate of approximately 8% and separate NASH from healthy controls with an error rate of 4%. Hepatic steatosis and steatohepatitis could not be separated using the metabolomic profile. Plasma metabolomic analysis revealed marked changes in bile salts and in biochemicals related to glutathione in subjects with NAFLD. Statistical analysis identified a panel of biomarkers that could effectively separate healthy controls from NAFLD and healthy controls from NASH. These biomarkers can potentially be used to follow response to therapeutic interventions.     

The PNPLA3 I148M polymorphism is associated with insulin resistance and nonalcoholic fatty liver disease in a normoglycaemic population

Background and Aims: The adiponutrin/patatin‐like phospholipase‐3 (PNPLA3) I148M polymorphism has recently been found to contribute to differences in hepatic lipid content. Nonalcoholic fatty liver disease (NAFLD) has recently been considered a hepatic component of insulin resistance and a risk factor in the emergence of type 2 diabetes. However, whether there is an association between PNPLA3 I148M and insulin resistance and NAFLD in a normoglycaemic population is still unknown. Methods: This study enrolled 156 normoglycaemic individuals with NAFLD and 723 controls. All participants received complete biochemical and clinical workups including liver ultrasonography. They were then genotyped for the PNPLA3 I148M polymorphism. Results: We found significant differences in the genotype and the dominant model of the PNPLA3 I148M polymorphism between the NAFLD groups and the controls (P=0.018 and P=0.01 respectively). Furthermore, there was a dose effect of the PNPLA3 I148M genotype, in that CG heterozygotes had a risk of NAFLD between CC and GG homozygotes [adjusted odds ratio (OR)=2.03, 95% confidence interval (CI)=1.23–3.375 for the GG genotype and adjusted OR=1.55, 95% CI=1.02–2.35 for the CG genotype]. The dominant model of the PNPLA3 I148M polymorphism showed higher waist circumference, fasting insulin, Homeostasis Model Assessment (HOMA‐IR), alanine aminotransferase concentrations and ferritin level. Multivariate analysis showed the PNPLA3 I148M polymorphism to be independently and significantly associated with NAFLD in our normoglycaemic participants. Conclusion: This study reports an association between the PNPLA3‐I148M polymorphism and insulin resistance and NAFLD in a normoglycaemic population.     

Understanding the pathophysiological mechanisms in the pediatric non-alcoholic fatty liver disease: The role of genetics

Classically, the non-alcoholic fatty liver disease(NAFLD) physiopathology and progression has been summarized in the two hits hypothesis. The first hit is represented by the action of hyperinsulinemia and insulin resistance, accompanying obesity, that leads to liver steatosis increasing the absolute non esterified fatty acids uptake in the liver and the esterification to form triacylglycerol. The oxidative stress is involved in the second hit leading to the progression to nonalcoholic steatohepatitis(NASH) because of its harmful action on steatosic hepatocytes. However, at the present time, the two hits hypothesis needs to be updated because of the discover of genetic polymorphisms involved both in the liver fat accumulation and progression to NASH that make more intriguing understanding the NAFLD pathophysiological mechanisms. In this editorial, we want to underline the role of PNPLA3 I148 M, GPR120 R270 H and TM6SF2 E167 K in the pediatric NAFLD development because they add new pieces to the comprehension of the NAFLD pathophysiological puzzle. The PNPLA3 I148 M polymorphism encodes for an abnormal protein which predisposes to intrahepatic triglycerides accumulation both for a loss-of-function of its triglyceride hydrolase activity and for a gain-of-function of its lipogenic activity.Therefore, it is involved in the first hit, such as TM6SF2 E167 K polymorphisms that lead to intrahepatic fat accumulation through a reduced very low density lipoprotein secretion. On the other hand, the GPR120 R270 H variant, reducing the anti-inflammatory action of the GPR120 receptor expressed by Kuppfer cells, is involved in the second hit leading to the liver injury.     

Adiponectin gene polymorphisms modulate acute adiponectin response to dietary fat: Possible pathogenetic role in NASH

Factors underlying the independent association of nonalcoholic steatohepatitis (NASH) with increased cardiovascular risk are unknown. Adiponectin polymorphisms predict cardiometabolic risk in the general population. This association is not always mediated by low fasting adiponectin levels, adipose tissue accumulation, or traditional risk factors. Adiponectin modulates lipid metabolism and liver injury in nonalcoholic fatty liver disease (NAFLD) even in the absence of obesity, dyslipidemia, and diabetes. We hypothesized adiponectin polymorphisms may predispose to NAFLD and may increase cardiovascular risk by modulating circulating lipoprotein and adiponectin response postprandially. The prevalence of adiponectin single-nucleotide polymorphisms (SNPs) 45GT and 276GT was assessed in 70 nonobese, nondiabetic, normolipidemic NAFLD patients and 70 healthy matched controls; the impact of the adiponectin SNPs was subsequently correlated to liver histology and postprandial adiponectin and lipoprotein responses to oral fat load in a subgroup of 30 biopsy-proven patients with NASH and 30 controls. The 45TT and 276GT/TT genotypes were more prevalent in NAFLD patients than in controls and independently predicted the severity of liver disease in NASH. In both patients and controls, these genotypes exhibited a blunted postprandial adiponectin response and higher postprandial triglycerides (Tg), free fatty acids (FFA), oxidized LDL (oxLDL), and VLDL levels than their counterparts, despite comparable fasting adipokines, lipids, dietary habits, adiposity, and insulin resistance. They were also independently associated, together with dietary polyunsaturated fatty acid intake, with postprandial adiponectin response. IAUC adiponectin independently predicted postprandial Tg, FFA, oxLDL, and intestinal and hepatic VLDL subfraction responses in NASH. Conclusion: The at-risk adiponectin SNPs 45TT and 276GT are significantly more prevalent in NAFLD than in the general population; they are associated with severity of liver disease, with blunted postprandial adiponectin response, and with an atherogenic postprandial lipoprotein profile in NASH independently of fasting adipokine and lipid levels.     

Genetic and epigenetic variants influencing the development of nonalcoholic fatty liver disease

Nonalcoholic fatty liver disease (NAFLD) is common worldwide. The importance of genetic and epigenetic changes in etiology and pathogenesis of NAFLD has been increasingly recognized. However, the exact mechanism is largely unknown. A large number of single nucleotide polymorphisms (SNPs) related to NAFLD has been documented by candidate gene studies (CGSs). Among these genes, peroxisome proliferatoractivated receptor-γ, adiponectin, leptin and tumor necrosis factor-α were frequently reported. Since the introduction of genome-wide association studies (GWASs), there have been significant advances in our understanding of genomic variations of NAFLD. Patatin-like phospholipase domain containing family member A3 (PNPLA3, SNP rs738409, encoding I148M), also termed adiponutrin, has caught most attention. The evidence that PNPLA3 is associated with increased hepatic fat levels and hepatic inflammation has been validated by a series of studies. Epigenetic modification refers to phenotypic changes caused by an adaptive mechanism unrelated to alteration of primary DNA sequences. Epigenetic regulation mainly includes microRNAs (miRs), DNA methylation, histone modifications and ubiquitination, among which miRs are studied most extensively. miRs are small natural single stranded RNA molecules regulating mRNA degradation or translation inhibition, subsequently altering protein expression of target genes. The miR-122, a highly abundant miR accounting for nearly 70% of all miRs in the liver, is significantly under-expressed in NAFLD subjects. Inhibition of miR-122 with an antisense oligonucleotide results in decreased mRNA expression of lipogenic genes and improvement of liver steatosis. The investigation into epigenetic involvement in NAFLD pathogenesis is just at the beginning and needs to be refined. This review summarizes the roles of genetics and epigenetics in the development of NAFLD. The progress made in this field may provide novel diagnostic biomarkers and therapeutic targets for NAFLD management.     

The PNPLA3 rs738409 C > G polymorphism is associated with the risk of progression to cirrhosis in NAFLD patients

Background and aims: The patatin-like phospholipase domain-containing 3 (PNPLA3) rs738409 C?>?G single nucleotide polymorphism (SNP) has been associated with steatosis and fibrosis in previous NAFLD populations in which cirrhotic patients were very poorly represented. Since not all NAFLD with fibrosis evolve to cirrhosis, we investigated the specific risk of cirrhosis conferred in NAFLD patients by carrying this SNP. Methods: Three groups were studied: patients with NASH-cirrhosis; patients with biopsy-proven non-cirrhotic NAFLD; healthy subjects undergoing medicine check-ups. Epidemiological, anthropometric, and clinical data were collected, and the SNP was analyzed by pyrosequencing. Results: Sixty-one patients with NASH-cirrhosis, 60 with non-cirrhotic NAFLD, and 125 healthy controls were included. Frequency of the PNPLA3 minor (G) allele was increased in patients with NASH-cirrhosis compared with non-cirrhotic NAFLD and controls (allele frequency: 0.598 versus 0.367 versus 0.2, respectively, p?p?Conclusions: In NAFLD patients, carriage of the PNPLA3G allele, and particularly of the GG genotype, is significantly associated with the risk of cirrhotic evolution. If confirmed in larger series, these results would suggest that most of NASH cases require the contribution of an altered PNPLA3 function to progress until cirrhosis.     

Adipose tissue is inflamed in NAFLD due to obesity but not in NAFLD due to genetic variation in PNPLA3

Aims/hypothesis

The rs738409 C>G single-nucleotide polymorphism in PNPLA3 leads to a missense mutation (I148M) which increases liver fat but does not cause insulin resistance. We hypothesised that patients with non-alcoholic fatty liver disease (NAFLD) due to the PNPLA3 variant (‘PNPLA3 NAFLD’?=?PNPLA3-148MM) do not have adipose tissue (AT) inflammation in contrast with those with NAFLD due to obesity (‘obese NAFLD’).

Methods

Biopsy specimens of AT were taken, and PNPLA3 genotype and liver fat (¹H-magnetic resonance spectroscopy) were determined in 82 volunteers, who were divided into groups based on either median BMI (obese 36.2?±?0.7 kg/m²; non-obese 26.0?±?0.4 kg/m²) or PNPLA3 genotype. All groups were similar with respect to age and sex. The PNPLA3 subgroups were equally obese (PNPLA3-148MM, 31.1?±?1.3 kg/m²; PNPLA3-148II, 31.2?±?0.8 kg/m²), while the obese and non-obese subgroups had similar PNPLA3 genotype distribution. Gene expression of proinflammatory (MCP-1, CD68) and anti-inflammatory (Twist1, ADIPOQ) markers was measured using quantitative real-time RT-PCR.

Results

Liver fat was similarly increased in obese NAFLD (9.5?±?1.3% vs 5.1?±?0.9%, obese vs non-obese, p?=?0.007) and PNPLA3 NAFLD (11.4?±?1.7% vs 5.3?±?0.8%, PNPLA3-148MM vs PNPLA3-148II, p?<?0.001). Fasting serum insulin was higher in the obese than the non-obese group (76?±?6 vs 47?±?6 pmol/l, p?<?0.001), but similar in PNPLA3-148MM and PNPLA3-148II (60?±?8 vs 62?±?5 pmol/l, NS). In obese vs non-obese, MCP-1 and CD68 mRNAs were upregulated, whereas those of Twist1 and ADIPOQ were significantly downregulated. AT gene expression of MCP-1, CD68, Twist1 and ADIPOQ was similar in PNPLA3-148MM and PNPLA3-148II groups.

Conclusions/interpretation

PNPLA3 NAFLD is characterised by an increase in liver fat but no insulin resistance or AT inflammation, while obese NAFLD has all three of these features.     

The genetic backgrounds in nonalcoholic fatty liver disease

Nonalcoholic fatty liver disease (NAFLD) is the most prevalent chronic liver disease worldwide. Nonalcoholic steatohepatitis (NASH), a severe form of NAFLD, can lead to hepatocellular carcinoma (HCC) and hepatic failure. The development and progression of NAFLD are determined by environmental and genetic factors. The effect of genetic factors has been demonstrated by familial studies, twin studies and several cross-sectional studies. In the past 10 years, genome-wide association studies have revealed several single nucleotide polymorphisms (SNPs) associated with the pathology of NAFLD. Among them, the Patatin-like phospholipase domain-containing 3 (PNPLA3) gene variant I148M showed a strong relationship with the development and progression of NAFLD, NASH, and NAFLD-related HCC. The transmembrane 6 superfamily member 2 (TM6SF2) gene variant E167 K was also associated with NAFLD, and it has a relationship with cardiovascular disease. Furthermore, several genes have been proposed as candidate genes to be associated with NAFLD based on case–control studies. We conducted a comprehensive literature search and review on the genetic background of NAFLD.     

PNPLA3 I148M variant affects non-alcoholic fatty liver disease in liver transplant recipients

De novo non-alcoholic fatty liver disease(NAFLD) is a common late complication for long-term survivors after liver transplantation. Genomic studies confirmed that PNPLA3 I148 M and TM6SF2 E167 K polymorphisms affected NAFLD susceptibility in the general population. However,this association was not validated in survivors after liver transplantation(LT). We performed a crosssectional survey to investigate this relationship. A comprehensive survey,including anthropometric measurements,fasting venous blood sampling,ultrasound,and questionnaires was performed in the shortterm. The clinical indications and patient's steatosis status before LT were collected from inpatient medical records. Sixty-five long-term recipients with a survival exceeding 10 years were enrolled in the final analysis. De novo NAFLD was more frequent in PNPLA3 GG carriers(0.33 vs 0.10 for GG vs CC + CG carriers,P = 0.018),while the genetic impact on NAFLD susceptibility was insignificant when categorized by the TM6SF2 polymorphism(0.19 in CC vs 0.14 in CT + TT carriers,P = 0.883). Multi-covariate analysis revealed that PNPLA3 exerted a significant genetic effect on de novoNAFLD following a recessive model(GG vs CC + CG,OR = 14.2,95%CI:1.78-113,P = 0.012). Compared to recipients with only the PNPLA3 GG allele or obesity(defined as body mass index 25 kg/m2),steatosis was highly prevalent(71.4%) in PNPLA3 GG carriers Table 1 Current status of long-term recipients surviving more than 10 years Univariate Multivariate NAFLD(n = 12)Control(n = 53)P value OR P value Age(yr) 56.5 ± 8.4 53.6 ± 10.1 0.356 1.04(0.92-1.18)0.528Gender(M/F) 10/2 47/6 0.611 1.40(0.14-14.2)0.427 Indication for LT Hepatitis/cirrhosis/ cancer/others1/8/2/17/35/9/20.889 Survival time(yr) 11.2 ± 0.9 11.5 ± 1.4 0.541 BMI(kg/m2) 25.1 ± 3.0 22.5 ± 2.6 0.003 1.47(1.03-2.08)0.032 TG(mmol/L) 1.6 ± 1.1 1.1 ± 0.6 0.038 1.34(0.38-4.71)0.652 HDL-C(mmol/L) 1.2(1.0-1.4) 1.3(1.0-1.7) 0.267 FBG(mmol/L) 7.6 ± 3.4 5.7 ± 1.9 0.013 1.49(0.93-2.37)0.095 Hypertension(Yes/no)3/9 21/32 0.343 SUA(μmol/L) 381.6 ± 75.6 342.6 ± 76.4 0.116 Met S(Yes/no) 4/8 9/44 0.201 ALT(U/L) 36.7 ± 7.0 38.8 ± 6.7 0.882 Alcohol intake(g/wk)11.6 ± 7.3 21.0 ± 8.2 0.766 Smoking(cigar/d) 4.2 ± 3.4 4.3 ± 1.2 0.969 Exercise(min/d) 18.5 ± 6.0 22.9 ± 2.9 0.513 Immunosuppression Tacrolimus/ cyclosporine/ MMF/sirolimus/ none11/1/0/0/036/12/1/2/20.575 PNPLA3(CC/CG/GG)1/3/8 16/21/16 0.018 14.2(1.78-113)0.012 TM6SF2(CC/CT/TT)11/1/0 47/5/1 0.883 2.68(0.25-28.5)0.413 Continuous variables with equal variance are presented as the mean ± SD; Continuous variables with unequal variance are presented as the median(interquartile range); Categorical variables are presented as the number of subjects. One-way ANOVA was used for the comparison between continuous variables with equal variance,Mann-Whitney U test was used for the comparison between continuous variables with unequal variance,chi-square test was used for the comparison between categorical variables in univariate analysis,and logistic regression analysis was used in the multivariate analysis. The effect of the PNPLA3 I148 M polymorphism was evaluated by a recessive genetic model(GG vs CG + CC); the effect of the TM6SF2 E167 K polymorphism was evaluated by a dominant genetic model(CC + CT vs TT) for decreased prevalence of TT carrier. ALT:Alanine aminotransferase; BMI:Body mass index; F:Female; FBG:Fasting blood glucose; HBs Ag:Hepatitis B surface antigen; HDL-C:Highdensity lipoprotein cholesterol; LT:Liver transplantation; M:Male; Met S:Metabolic syndrome; MMF:Mycophenolatemofetil; SUA:Serum uric acid; TG:Triglyceride. De novo NAFLD Controlwith obesity. In conclusion,PNPLA3 I148 M,but not TM6SF2 E167 K,affects de novo NAFLD occurrence with a prominent interaction with obesity. Weight control might be a meaningful method to reduce the genetic susceptibility to NAFLD exerted by PNPLA3 variants.