Paola Dongiovanni

Homozygosity for the patatin-like phospholipase-3/adiponutrin i148m polymorphism influences liver fibrosis in patients with nonalcoholic fatty liver disease

Inherited factors play a major role in the predisposition to nonalcoholic fatty liver disease (NAFLD), and the rs738409 C→G polymorphism of PNPLA3/adiponutrin, encoding for the isoleucine‐to‐methionine substitution at residue 148 (I148M) protein variant, has recently been recognized as a major determinant of liver fat content. However, the effect of the rs738409 polymorphism on the severity of liver fibrosis in patients with NAFLD is still unknown. In this study, we considered 253 Italian patients, 179 healthy controls, and 71 family trios with an affected child with NAFLD. Analyses were replicated in 321 patients from the United Kingdom. The rs738409 polymorphism was determined by TaqMan assays. Liver histology was scored according to Kleiner et al. Hepatic expression of genes regulating liver damage was assessed by real‐time polymerase chain reaction in 52 patients. The rs738409 GG genotype was more prevalent in patients than in controls (14% versus 3%, adjusted odds ratio [OR] = 3.29, 95% confidence interval [CI] = 1.8‐6.9), and in the family study, the G allele was overtransmitted to affected children (P = 0.001). In Italian and United Kingdom patients, adiponutrin genotype influenced alanine aminotransferase levels and the severity of steatosis. Adiponutrin genotype was associated with the expression of genes involved in the steatosis‐related liver damage, including the proapoptotic molecule Fas ligand. In the whole series combined, adiponutrin genotype was associated with steatosis grade >1 (OR = 1.35, 95% CI = 1.04‐1.76), nonalcoholic steatohepatitis (OR = 1.5, 95% CI = 1.12‐2.04), and fibrosis stage >1 (OR = 1.5, 95% CI = 1.09‐2.12), independent of age, body mass index, and diabetes. Adiponutrin genotype demonstrated a dose effect with heterozygote risk intermediate between CC and GG homozygotes. Conclusion: In patients with NAFLD, adiponutrin rs738409 C→G genotype, encoding for I148M, is associated with the severity of steatosis and fibrosis and the presence of nonalcoholic steatohepatitis. (Hepatology 2010;51:1209–1217)

Iron Depletion by Phlebotomy Improves Insulin Resistance in Patients With Nonalcoholic Fatty Liver Disease and Hyperferritinemia: Evidence from a Case-Control Study

OBJECTIVES:Hyperferritinemia is frequently observed in nonalcoholic fatty liver disease (NAFLD), the hepatic manifestation of the metabolic syndrome characterized by hepatic insulin resistance and considered high cardiovascular risk. Iron depletion by phlebotomy has been reported to decrease insulin resistance in NAFLD in small, uncontrolled studies. Aims of this study were to define the relationship between ferritin and iron stores in patients with NAFLD, the effect of iron depletion on insulin resistance, and whether basal ferritin levels influence treatment outcome.METHODS:Subjects were included if ferritin and/or ALT were persistently elevated after 4 months of standard therapy. Sixty-four phlebotomized subjects were matched 1:1 for age, sex, ferritin, obesity, and ALT levels with patients who underwent lifestyle modifications only. Insulin resistance was evaluated by insulin levels, determined by RIA and the HOMA-R index, at baseline and after 8 months.RESULTS:Baseline ferritin levels were associated with body iron stores (P < 0.0001). Iron depletion produced a significantly larger decrease in insulin resistance (P = 0.0016 for insulin, P = 0.0042 for HOMA-R) compared with nutritional counseling alone, independent of changes in BMI, baseline HOMA-R, and the presence of the metabolic syndrome. Iron depletion was more effective in reducing HOMA-R in patients in the top two tertiles of ferritin concentrations (P < 0.05 vs controls), and in carriers of the mutations in the HFE gene of hereditary hemochromatosis (P < 0.05 vs noncarriers).CONCLUSIONS:Given that phlebotomy reduces insulin resistance, which is associated with liver tissue damage, future studies should evaluate the effect of iron depletion on liver histology and cardiovascular end points.

Patatin-Like Phospholipase Domain-Containing 3 I148M Polymorphism, Steatosis, and Liver Damage in Chronic Hepatitis C

Steatosis has been reported to negatively influence the natural history of chronic hepatitis C (CHC), but controversy remains over its causal role due to the confounding effect of adiposity, insulin resistance, and diabetes. The rs738409 C>G patatin‐like phospholipase domain‐containing 3 (PNPLA3) single nucleotide polymorphism (SNP), encoding for the I148M protein variant, influences liver fat without affecting insulin resistance and body composition. The aim of this study was to evaluate the effect of the rs738409 CG genotype on liver fat and fibrosis in CHC patients. We also explored the possible effect of PNPLA3 genotype on other steatosis‐related complications, namely, treatment failure and hepatocellular carcinoma (HCC) development. To this end we considered two independent series of 325 and 494 CHC patients with available DNA and liver biopsy followed at tertiary referral centers in northern Italy. The rs738409 genotype was determined by a Taqman assay. The rs738409 GG genotype, observed in 10% of patients, was associated with steatosis independently of age, sex, body mass index (BMI), diabetes, alcohol intake, and viral genotype (odds ratio [OR] 1.90, 95% confidence interval [CI] 1.4‐2.7; P < 0.001). The association with rs738409 genotype was confirmed for severe steatosis, was independent of alanine aminotransferase (ALT) and gamma‐glutamyl transferase (GGT) values, and was observed in all viral genotypes but the 3. The rs738409 GG genotype was associated with fibrosis stage and cirrhosis (OR 1.47, 95% CI 1.2‐1.9; P = 0.002), treatment response (n = 470; OR 0.63, 95% CI 0.4‐0.8; P = 0.006), and HCC occurrence (n = 325; OR 2.16, 95% CI 1.3‐3.6; P = 0.002), independently of confounders. Conclusion: The rs738409 PNPLA3 genotype influences steatosis development in CHC and is independently associated with cirrhosis and other steatosis‐related clinical outcomes, such as lack of response to antiviral treatment and possibly HCC. (HEPATOLOGY 2011)

I148M patatin‐like phospholipase domain‐containing 3 gene variant and severity of pediatric nonalcoholic fatty liver disease

Nonalcoholic fatty liver disease (NAFLD) is one of the most common causes of chronic liver disease in children. Genetic variability, which is a main player in NAFLD, is especially characterized by polymorphisms in genes involved in the development and progression of the disease to nonalcoholic steatohepatitis (NASH). Recently, the rs738409 C>G adiponutrin/patatin‐like phospholipase domain‐containing 3 (PNPLA3) polymorphism, which encodes the I148M protein variant in the catalytic domain, has been associated with severe steatosis, NASH, and liver fibrosis in adults. In this study, we investigated the association between the rs738409 PNPLA3 gene polymorphism and NAFLD in 149 consecutive children and adolescents (age = 6‐13 years) with biopsy‐proven NAFLD. We analyzed the rs738409 polymorphism by a 5′‐nuclease TaqMan assay and assessed its association with NASH: 41% of the subjects with NAFLD showed heterozygosity and 15% showed homozygosity for the at‐risk G allele. The rs738409 genotype did not influence the body mass, adiposity, lipid levels, or insulin resistance and was not associated with alanine aminotransferase levels. Interestingly, the rs738409 G allele was strongly associated with the severity of steatosis (P < 0.0001), the presence of NASH (P < 0.0001), hepatocellular ballooning (P < 0.0001), lobular inflammation (P < 0.0001), and the presence of fibrosis (P = 0.01) independently of confounders. Individuals carrying two minor G alleles almost always had severe steatosis and NASH, heterozygotes were at intermediate risk, and patients negative for G alleles had milder and often uncomplicated steatosis. Conclusion: The PNPLA3 rs738409 polymorphism is associated with steatosis severity, hepatocellular ballooning, lobular inflammation, and perivenular fibrosis in pediatric NAFLD. (HEPATOLOGY 2010)

HFE Genotype, Parenchymal Iron Accumulation, and Liver Fibrosis in Patients With Nonalcoholic Fatty Liver Disease

BACKGROUND & AIMS Mutations in the hemochromatosis gene (HFE) (C282Y and H63D) lead to parenchymal iron accumulation, hemochromatosis, and liver damage. We investigated whether these factors also contribute to the progression of fibrosis in patients with nonalcoholic fatty liver disease (NAFLD). METHODS We studied clinical, histologic (liver biopsy samples for hepatocellular iron accumulation), serologic (iron and enzyme levels), and genetic (HFE genotype) data from 587 patients from Italy with NAFLD and 184 control subjects. RESULTS Iron accumulation predominantly in hepatocyes was associated with a 1.7-fold higher risk of a fibrosis stage greater than 1 (95% confidence interval [CI]: 1.2-2.3), compared with the absence of siderosis (after adjustment for age, body mass index, glucose tolerance status, and alanine aminotransferase level). Nonparenchymal/mixed siderosis was not associated with moderate/severe fibrosis (odds ratio, 0.72; 95% CI: 0.50-1.01). Hepatocellular siderosis was more prevalent in patients with HFE mutations than in those without; approximately one third of patients with HFE mutations had parenchymal iron accumulation (range, 29.8%-35.7%, depending on HFE genotype). Predominantly hepatocellular iron accumulation occurred in 52.7% of cases of patients with HFE mutations. There was no significant association between either the presence of HFE mutations or specific HFE genotypes and the severity of liver fibrosis. CONCLUSIONS Iron deposition predominantly in hepatocyes is associated with more severe liver damage in patients with NAFLD. However, HFE mutations cannot be used to identify patients with hepatocellular iron accumulation.

Transmembrane 6 superfamily member 2 gene variant disentangles nonalcoholic steatohepatitis from cardiovascular disease

Excess hepatic storage of triglycerides is considered a benign condition, but nonalcoholic steatohepatitis (NASH) may progress to fibrosis and promote atherosclerosis. Carriers of the TM6SF2 E167K variant have fatty liver as a result of reduced secretion of very‐low‐density lipoproteins (VLDLs). As a result, they have lower circulating lipids and reduced risk of myocardial infarction. In this study, we aimed to assess whether TM6SF2 E167K affects liver damage and cardiovascular outcomes in subjects at risk of NASH. Liver damage was evaluated in 1,201 patients who underwent liver biopsy for suspected NASH; 427 were evaluated for carotid atherosclerosis. Cardiovascular outcomes were assessed in 1,819 controls from the Swedish Obese Subjects (SOS) cohort. Presence of the inherited TM6SF2 E167K variant was determined by TaqMan assays. In the liver biopsy cohort, 188 subjects (13%) were carriers of the E167K variant. They had lower serum lipid levels than noncarriers (P < 0.05), had more‐severe steatosis, necroinflammation, ballooning, and fibrosis (P < 0.05), and were more likely to have NASH (odds ratio [OR]: 1.84; 95% confidence interval [CI]: 1.23‐2.79) and advanced fibrosis (OR, 2.08; 95% CI: 1.20‐3.55), after adjustment for age, sex, body mass index, fasting hyperglycemia, and the I148M PNPLA3 risk variant. However, E167K carriers had lower risk of developing carotid plaques (OR, 0.49; 95% CI: 0.25‐0.94). In the SOS cohort, E167K carriers had higher alanine aminotransferase ALT and lower lipid levels (P < 0.05), as well as a lower incidence of cardiovascular events (hazard ratio: 0.61; 95% CI: 0.39‐0.95). Conclusions: Carriers of the TM6SF2 E167K variant are more susceptible to progressive NASH, but are protected against cardiovascular disease. Our findings suggest that reduced ability to export VLDLs is deleterious for the liver. (Hepatology 2015;61:506‐514)

Iron in fatty liver and in the metabolic syndrome: A promising therapeutic target

The dysmetabolic iron overload syndrome (DIOS) is now a frequent finding in the general population, as is detected in about one third of patients with nonalcoholic fatty liver disease (NAFLD) and the metabolic syndrome. The pathogenesis is related to altered regulation of iron transport associated with steatosis, insulin resistance, and subclinical inflammation, often in the presence of predisposing genetic factors. Evidence is accumulating that excessive body iron plays a causal role in insulin resistance through still undefined mechanisms that probably involve a reduced ability to burn carbohydrates and altered function of adipose tissue. Furthermore, DIOS may facilitate the evolution to type 2 diabetes by altering beta-cell function, the progression of cardiovascular disease by contributing to the recruitment and activation of macrophages within arterial lesions, and the natural history of liver disease by inducing oxidative stress in hepatocytes, activation of hepatic stellate cells, and malignant transformation by promotion of cell growth and DNA damage. Based on these premises, the association among DIOS, metabolic syndrome, and NAFLD is being investigated as a new risk factor to predict the development of overt cardiovascular and hepatic diseases, and possibly hepatocellular carcinoma, but most importantly, represents also a treatable condition. Indeed, iron depletion, most frequently achieved by phlebotomy, has been shown to decrease metabolic alterations and liver enzymes in controlled studies in NAFLD. Additional studies are warranted to evaluate the potential of iron reductive therapy on hard clinical outcomes in patients with DIOS.

PNPLA3 has retinyl-palmitate lipase activity in human hepatic stellate cells

Retinoids are micronutrients that are stored as retinyl esters in the retina and hepatic stellate cells (HSCs). HSCs are key players in fibrogenesis in chronic liver diseases. The enzyme responsible for hydrolysis and release of retinyl esters from HSCs is unknown and the relationship between retinoid metabolism and liver disease remains unclear. We hypothesize that the patatin-like phospholipase domain-containing 3 (PNPLA3) protein is involved in retinol metabolism in HSCs. We tested our hypothesis both in primary human HSCs and in a human cohort of subjects with non-alcoholic fatty liver disease (N = 146). Here we show that PNPLA3 is highly expressed in human HSCs. Its expression is regulated by retinol availability and insulin, and increased PNPLA3 expression results in reduced lipid droplet content. PNPLA3 promotes extracellular release of retinol from HSCs in response to insulin. We also show that purified wild-type PNPLA3 hydrolyzes retinyl palmitate into retinol and palmitic acid. Conversely, this enzymatic activity is markedly reduced with purified PNPLA3 148M, a common mutation robustly associated with liver fibrosis and hepatocellular carcinoma development. We also find the PNPLA3 I148M genotype to be an independent (P = 0.009 in a multivariate analysis) determinant of circulating retinol-binding protein 4, a reliable proxy for retinol levels in humans. This study identifies PNPLA3 as a lipase responsible for retinyl-palmitate hydrolysis in HSCs in humans. Importantly, this indicates a potential novel link between HSCs, retinoid metabolism and PNPLA3 in determining the susceptibility to chronic liver disease.

Iron depletion by deferoxamine up-regulates glucose uptake and insulin signaling in hepatoma cells and in rat liver.

Iron depletion improves insulin resistance in patients with nonalcoholic fatty liver disease and diabetes and also stabilizes the hypoxia-inducible factor (HIF)-1, resulting in increased glucose uptake in vitro. This study investigated the effect of iron depletion by deferoxamine on insulin signaling and glucose uptake in HepG2 hepatocytes and in rat liver. In HepG2 cells, deferoxamine stabilized HIF-1alpha and induced the constitutive glucose transporter Glut1 and the insulin receptor. Up-regulation of insulin receptor by deferoxamine was mimicked by the intracellular iron chelator deferasirox and the hypoxia inducer CoCl2 and required the HIF-1 obligate partner ARNT/HIF-1beta. Iron depletion increased insulin receptor activity, whereas iron supplementation had the opposite effect. Deferoxamine consistently increased the phosphorylation status of Akt/PKB and its targets FoxO1 and Gsk3beta, which mediate the effect of insulin on gluconeogenesis and glycogen synthesis, and up-regulated genes involved in glucose uptake and utilization. Iron depletion of Sprague-Dawley rats increased HIF-1alpha expression, improved glucose clearance, and was associated with up-regulation of insulin receptor and Akt/PKB levels and of glucose transport in hepatic tissue. Conversely, gluconeogenic genes were not affected. In rats with fatty liver because of a high-calorie and high-fat diet, glucose clearance was increased by iron depletion and decreased by iron supplementation. Thus, iron depletion by deferoxamine up-regulates glucose uptake, and increases insulin receptor activity and signaling in hepatocytes in vitro and in vivo.

PNPLA3 I148M polymorphism and progressive liver disease

The 148 Isoleucine to Methionine protein variant (I148M) of patatin-like phospholipase domain-containing 3 (PNPLA3), a protein is expressed in the liver and is involved in lipid metabolism, has recently been identified as a major determinant of liver fat content. Several studies confirmed that the I148M variant predisposes towards the full spectrum of liver damage associated with fatty liver: from simple steatosis to steatohepatitis and progressive fibrosis. Furthermore, the I148M variant represents a major determinant of progression of alcohol related steatohepatitis to cirrhosis, and to influence fibrogenesis and related clinical outcomes in chronic hepatitis C virus hepatitis, and possibly chronic hepatitis B virus hepatitis, hereditary hemochromatosis and primary sclerosing cholangitis. All in all, studies suggest that the I148M polymorphism may represent a general modifier of fibrogenesis in liver diseases. Remarkably, the effect of the I148M variant on fibrosis was independent of that on hepatic steatosis and inflammation, suggesting that it may affect both the quantity and quality of hepatic lipids and the biology of non-parenchymal liver cells besides hepatocytes, directly promoting fibrogenesis. Therefore, PNPLA3 is a key player in liver disease progression. Assessment of the I148M polymorphism will possibly inform clinical practice in the future, whereas the determination of the effect of the 148M variant will reveal mechanisms involved in hepatic fibrogenesis.