Adipocyte PHLPP2 inhibition prevents obesity-induced fatty liver.

Increased adiposity confers risk for systemic insulin resistance and type 2 diabetes (T2D), but mechanisms underlying this pathogenic inter-organ crosstalk are incompletely understood. We find PHLPP2 (PH domain and leucine rich repeat protein phosphatase 2), recently identified as the Akt Ser473 phosphatase, to be increased in adipocytes from obese mice. To identify the functional consequence of increased adipocyte PHLPP2 in obese mice, we generated adipocyte-specific PHLPP2 knockout (A-PHLPP2) mice. A-PHLPP2 mice show normal adiposity and glucose metabolism when fed a normal chow diet, but reduced adiposity and improved whole-body glucose tolerance as compared to Cre- controls with high-fat diet (HFD) feeding. Notably, HFD-fed A-PHLPP2 mice show increased HSL phosphorylation, leading to increased lipolysis in vitro and in vivo. Mobilized adipocyte fatty acids are oxidized, leading to increased peroxisome proliferator-activated receptor alpha (PPARα)-dependent adiponectin secretion, which in turn increases hepatic fatty acid oxidation to ameliorate obesity-induced fatty liver. Consistently, adipose PHLPP2 expression is negatively correlated with serum adiponectin levels in obese humans. Overall, these data implicate an adipocyte PHLPP2-HSL-PPARα signaling axis to regulate systemic glucose and lipid homeostasis, and suggest that excess adipocyte PHLPP2 explains decreased adiponectin secretion and downstream metabolic consequence in obesity.

Ceruloplasmin gene variants are associated with hyperferritinemia and increased liver iron in patients with NAFLD.

BACKGROUND & AIMS: Non-alcoholic fatty liver disease (NAFLD) is a multifactorial disorder resulting from genetic and environmental factors. Hyperferritinemia has been associated with increased hepatic iron stores and worse outcomes in patients with NAFLD. The aim of this study was to evaluate the prevalence of variants of iron-related genes and their association with hyperferritinemia, hepatic iron stores and liver disease severity in patients with NAFLD. METHODS: From a cohort of 328 individuals with histological NAFLD, 23 patients with ferritin >750 ng/ml and positive iron staining, and 25 controls with normal ferritin and negative iron staining, were selected. Patients with increased transferrin saturation, anemia, inflammation, ß-thalassemia trait, HFE genotype at risk of iron overload and ferroportin mutations were excluded. A panel of 32 iron genes was re-sequenced. Literature and in silico predictions were employed for prioritization of pathogenic mutations. RESULTS: Patients with hyperferritinemia had a higher prevalence of potentially pathogenic rare variants (73.9% vs. 20%, p = 0.0002) associated with higher iron stores and more severe liver fibrosis (p <0.05). Ceruloplasmin was the most mutated gene and its variants were independently associated with hyperferritinemia, hepatic siderosis, and more severe liver fibrosis (p <0.05). In the overall cohort, ceruloplasmin variants were independently associated with hyperferritinemia (adjusted odds ratio 5.99; 95% CI 1.83-19.60; p = 0.0009). CONCLUSIONS: Variants in non-HFE iron genes, particularly ceruloplasmin, are associated with hyperferritinemia and increased hepatic iron stores in patients with NAFLD. Carriers of such variants have more severe liver fibrosis, suggesting that genetic predisposition to hepatic iron deposition may translate into liver disease. LAY SUMMARY: Non-alcoholic fatty liver disease (NAFLD) is a common disease which can progress to cirrhosis and liver cancer. Increased levels of serum ferritin are often detected in patients with NAFLD and have been associated with altered iron metabolism and worse patient outcomes. We found that variants of genes related to iron metabolism, particularly ceruloplasmin, are associated with high ferritin levels, hepatic iron deposition and more severe liver disease in an Italian cohort of patients with NAFLD.

PCSK9 rs11591147 R46L loss-of-function variant protects against liver damage in individuals with NAFLD.

BACKGROUND AND AIMS: The proprotein convertase subtilisin/kexin type 9 (PCSK9) plays a key role in cholesterol homeostasis, and its inhibition represents an effective therapy to lower low-density lipoprotein cholesterol (LDL-C) levels. In this study, we examined the impact of the PCSK9 rs11591147 loss-of-function (LOF) variant on liver damage in a multicenter collection of patients at risk of nonalcoholic steatohepatitis (NASH), in clinical samples and experimental models. METHODS: We considered 1874 consecutive individuals at risk of NASH as determined by histology. The SNP rs11591147, encoding for the p.R46L variant of PCSK9, was genotyped by TaqMan assays. We also evaluated 1) PCSK9 mRNA hepatic expression in human liver, and 2) the impact of a NASH-inducing diet in mice with hepatic overexpression of human PCSK9. RESULTS: Carriers of PCSK9 rs11591147 had lower circulating LDL-C levels and were protected against nonalcoholic fatty liver disease (NAFLD) (OR: 0.42; 95% CI: 0.22-0.81; P = .01), NASH (OR: 0.48; 95% CI: 0.26-0.87; P = .01) and more severe fibrosis (OR: 0.55; 95% CI: 0.32-0.94; P = .03) independently of clinical, metabolic and genetic confounding factors. PCSK9 hepatic expression was directly correlated with liver steatosis (P = .03). Finally, liver-specific overexpression of human PCSK9 in male mice drives NAFLD and fibrosis upon a dietary challenge. CONCLUSIONS: In individuals at risk of NASH, PCSK9 was induced with hepatic fat accumulation and PCSK9 rs11591147 LOF variant was protective against liver steatosis, NASH and fibrosis, suggesting that PCSK9 inhibition may be a new therapeutic strategy to treat NASH.

From Environment to Genome and Back: A Lesson from HFE Mutations.

The environment and the human genome are closely entangled and many genetic variations that occur in human populations are the result of adaptive selection to ancestral environmental (mainly dietary) conditions. However, the selected mutations may become maladaptive when environmental conditions change, thus becoming candidates for diseases. Hereditary hemochromatosis (HH) is a potentially lethal disease leading to iron accumulation mostly due to mutations in the HFE gene. Indeed, homozygosity for the C282Y HFE mutation is associated with the primary iron overload phenotype. However, both penetrance of the C282Y variant and the clinical manifestation of the disease are extremely variable, suggesting that other genetic, epigenetic and environmental factors play a role in the development of HH, as well as, and in its progression to end-stage liver diseases. Alcohol consumption and dietary habits may impact on the phenotypic expression of HFE-related hemochromatosis. Indeed, dietary components and bioactive molecules can affect iron status both directly by modulating its absorption during digestion and indirectly by the epigenetic modification of genes involved in its uptake, storage and recycling. Thus, the premise of this review is to discuss how environmental pressures led to the selection of HFE mutations and whether nutritional and lifestyle interventions may exert beneficial effects on HH outcomes and comorbidities.

Mboat7 down-regulation by hyper-insulinemia induces fat accumulation in hepatocytes.

BACKGROUND: Naturally occurring variation in Membrane-bound O-acyltransferase domain-containing 7 (MBOAT7), encoding for an enzyme involved in phosphatidylinositol acyl-chain remodelling, has been associated with fatty liver and hepatic disorders. Here, we examined the relationship between hepatic Mboat7 down-regulation and fat accumulation. METHODS: Hepatic MBOAT7 expression was surveyed in 119 obese individuals and in experimental models. MBOAT7 was acutely silenced by antisense oligonucleotides in C57Bl/6 mice, and by CRISPR/Cas9 in HepG2 hepatocytes. FINDINGS: In obese individuals, hepatic MBOAT7 mRNA decreased from normal liver to steatohepatitis, independently of diabetes, inflammation and MBOAT7 genotype. Hepatic MBOAT7 levels were reduced in murine models of fatty liver, and by hyper-insulinemia. In wild-type mice, Mboat7 was down-regulated by refeeding and insulin, concomitantly with insulin signalling activation. Acute hepatic Mboat7 silencing promoted hepatic steatosis in vivo and enhanced expression of fatty acid transporter Fatp1. MBOAT7 deletion in hepatocytes reduced the incorporation of arachidonic acid into phosphatidylinositol, consistently with decreased enzymatic activity, determining the accumulation of saturated triglycerides, enhanced lipogenesis and FATP1 expression, while FATP1 deletion rescued the phenotype. INTERPRETATION: MBOAT7 down-regulation by hyper-insulinemia contributes to hepatic fat accumulation, impairing phosphatidylinositol remodelling and up-regulating FATP1. FUNDING: LV was supported by MyFirst Grant AIRC n.16888, Ricerca Finalizzata Ministero della Salute RF-2016-02,364,358, Ricerca corrente Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico; LV and AG received funding from the European Union Programme Horizon 2020 (No. 777,377) for the project LITMUS-"Liver Investigation: Testing Marker Utility in Steatohepatitis". MM was supported by Fondazione Italiana per lo Studio del Fegato (AISF) 'Mario Coppo' fellowship.

Liver transcriptomics highlights interleukin-32 as novel NAFLD-related cytokine and candidate biomarker.

OBJECTIVE: Efforts to manage non-alcoholic fatty liver disease (NAFLD) are limited by the incomplete understanding of the pathogenic mechanisms and the absence of accurate non-invasive biomarkers. The aim of this study was to identify novel NAFLD therapeutic targets andbiomarkers by conducting liver transcriptomic analysis in patients stratified by the presence of the PNPLA3 I148M genetic risk variant. DESIGN: We sequenced the hepatic transcriptome of 125 obese individuals. 'Severe NAFLD' was defined as the presence of steatohepatitis, NAFLD activity score ≥4 or fibrosis stage ≥2. The circulating levels of the most upregulated transcript, interleukin-32 (IL32), were measured by ELISA. RESULTS: Carriage of the PNPLA3 I148M variant correlated with the two major components of hepatic transcriptome variability and broadly influenced gene expression. In patients with severe NAFLD, there was an upregulation of inflammatory and lipid metabolism pathways. IL32 was the most robustly upregulated gene in the severe NAFLD group (adjusted p=1×10-6), and its expression correlated with steatosis severity, both in I148M variant carriers and non-carriers. In 77 severely obese, and in a replication cohort of 160 individuals evaluated at the hepatology service, circulating IL32 levels were associated with both NAFLD and severe NAFLD independently of aminotransferases (p<0.01 for both). A linear combination of IL32-ALT-AST showed a better performance than ALT-AST alone in NAFLD diagnosis (area under the curve=0.92 vs 0.81, p=5×10-5). CONCLUSION: Hepatic IL32 is overexpressed in NAFLD, correlates with hepatic fat and liver damage, and is detectable in the circulation, where it is independently associated with the presence and severity of NAFLD.

Dysmetabolic Hyperferritinemia and Dysmetabolic Iron Overload Syndrome (DIOS): Two Related Conditions or Different Entities?

Hyperferritinemia is observed in one-third of patients with non-alcoholic fatty liver disease (NAFLD) and Metabolic Syndrome (MetS). The condition characterized by increased body iron stores associated with components of MetS has been defined as Dysmetabolic Iron Overload Syndrome (DIOS). DIOS represents the most frequent iron overload condition, since it is observed in 15% of patients with MetS and in half of those with NAFLD and its clinical presentation overlaps almost completely with that of dysmetabolic hyperferritinemia (DH). The pathogenetic mechanisms linking insulin resistance (IR), NAFLD and DIOS to iron overload are still debated. Hepcidin seems to play a role in iron accumulation in DIOS and NAFLD patients who show elevated serum hepcidin levels. The iron challenge does not restrain iron absorption despite adequate hepcidin production, suggesting that an impaired hepcidin activity rather than a deficit of hormone production underlies DIOS pathogenesis. Acquired and genetic factors are recognized to contribute to iron accumulation in NAFLD whereas additional studies are required to clearly demonstrate whether the same or different genetic factors lead to iron overload in DIOS. Finally, iron depletion by phlebotomy, together with the modification of diet and life-style habits, represents the therapeutic approach to decrease metabolic alterations and liver enzymes in NAFLD and DIOS patients. In this review, we summarized the current knowledge on the dysregulation of iron homeostasis in NAFLD and DIOS in the attempt to clarify whether they are different or more likely strictly related conditions, sharing the same pathogenic cause i.e. the MetS.

Impact of natural neuromedin-B receptor variants on iron metabolism.

Iron overload heritability remains partly unexplained. By performing whole exome sequencing in three patients with a clinical phenotype of hemochromatosis not accounted by known genetic risk factors, we identified in all patients rare variants predicted to alter activity of Neuromedin-B receptor (NMBR). Coding NMBR mutations were enriched in 129 patients with hereditary hemochromatosis or iron overload phenotype, as compared to ethnically matched controls, including 100 local healthy blood donors and 1000Genomes project participants (15.5% vs 5%, P = .0038 at burden test), and were associated with higher transferrin saturation in regular blood donors (P = .04). Consistently, in 191 patients with nonalcoholic fatty liver, the most common low-frequency p.L390 M variant was independently associated with higher ferritin (P = .03). In 58 individuals, who underwent oral iron challenge, carriage of the p.L390 M variant was associated with higher transferrin saturation and lower hepcidin release. Furthermore, the circulating concentration of the natural NMBR ligand, Neuromedin-B, was reduced in response to iron challenge. It was also decreased in individuals carrying the p.L390 M variant and with hemochromatosis in parallel with increased transferrin saturation. In mice, Nmbr was induced by chronic dietary iron overload in the liver, gut, pancreas, spleen, and skeletal muscle, while Nmb was downregulated in gut, pancreas and spleen. Finally, Nmb amplified holo-transferrin dependent induction of hepcidin in primary mouse hepatocytes, which was associated with Jak2 induction and abolished by the NMBR antagonist PD168368. In conclusion, NMBR natural variants were enriched in patients with iron overload, and associated with facilitated iron absorption, possibly related to a defect of iron-induced hepcidin release.

The TM6SF2 E167K genetic variant induces lipid biosynthesis and reduces apolipoprotein B secretion in human hepatic 3D spheroids.

There is a high unmet need for developing treatments for nonalcoholic fatty liver disease (NAFLD), for which there are no approved drugs today. Here, we used a human in vitro disease model to understand mechanisms linked to genetic risk variants associated with NAFLD. The model is based on 3D spheroids from primary human hepatocytes from five different donors. Across these donors, we observed highly reproducible differences in the extent of steatosis induction, demonstrating that inter-donor variability is reflected in the in vitro model. Importantly, our data indicates that the genetic variant TM6SF2 E167K, previously associated with increased risk for NAFLD, induces increased hepatocyte fat content by reducing APOB particle secretion. Finally, differences in gene expression pathways involved in cholesterol, fatty acid and glucose metabolism between wild type and TM6SF2 E167K mutation carriers (N = 125) were confirmed in the in vitro model. Our data suggest that the 3D in vitro spheroids can be used to investigate the mechanisms underlying the association of human genetic variants associated with NAFLD. This model may also be suitable to discover new treatments against NAFLD.

PCSK7 gene variation bridges atherogenic dyslipidemia with hepatic inflammation in NAFLD patients.

Dyslipidemia and altered iron metabolism are typical features of nonalcoholic fatty liver disease (NAFLD). Proprotein convertase subtilisin/kexin type 7 (PCSK7) gene variation has been associated with circulating lipids and liver damage during iron overload. The aim of this study was to examine the impact of the PCSK7 rs236918 variant on NAFLD-related traits in 1,801 individuals from the Liver Biopsy Cohort (LBC), 500,000 from the UK Biobank Cohort (UKBBC), and 4,580 from the Dallas Heart Study (DHS). The minor PCSK7 rs236918 C allele was associated with higher triglycerides, aminotransferases, and hepatic inflammation in the LBC (P < 0.05) and with hypercholesterolemia and liver disease in the UKBBC. In the DHS, PCSK7 missense variants were associated with circulating lipids. PCSK7 was expressed in hepatocytes and its hepatic expression correlated with that of lipogenic genes (P < 0.05). The rs236918 C allele was associated with upregulation of a new "intra-PCSK7" long noncoding RNA predicted to interact with the protein, higher hepatic and circulating PCSK7 protein (P < 0.01), which correlated with triglycerides (P = 0.04). In HepG2 cells, PCSK7 deletion reduced lipogenesis, fat accumulation, inflammation, transforming growth factor ß pathway activation, and fibrogenesis. In conclusion, PCSK7 gene variation is associated with dyslipidemia and more severe liver disease in high risk individuals, likely by modulating PCSK7 expression/activity.

Prevalence and Risk Factors of Significant Fibrosis in Patients With Nonalcoholic Fatty Liver Without Steatohepatitis.

BACKGROUND & AIMS: In patients with nonalcoholic fatty liver disease (NAFLD), nonalcoholic steatohepatitis (NASH) is a risk factor for the development of fibrosis. However, fibrosis has been observed in livers of patients without NASH. We aimed to estimate the prevalence of fibrosis in patients without NASH and risk factors for fibrosis. METHODS: We analyzed data from 1738 subjects (44.9% with severe obesity) in a cross-sectional liver biopsy cohort enrolled at referral centers in Italy and Finland. Biopsy specimens were analyzed histologically by a blinded pathologist at each center, and a diagnosis of NASH was made based on steatosis (≥5% of hepatocytes), hepatocellular ballooning, and lobular inflammation. We also collected data on demographic features, metabolic comorbidities, and genetic factors, and performed logistic regression analyses. Findings were validated using data from 118 consecutive patients with NAFLD who underwent sequential liver biopsies at tertiary referral centers in Italy. RESULTS: In the cross-sectional cohort, 132 of 389 patients (33.9%) with significant fibrosis had no NASH and 39 patients (10.0%) had no inflammation. The dissociation between NASH and fibrosis was significantly greater in patients with severe obesity (P < .005). Steatosis, ballooning, and lobular inflammation each were associated independently with significant fibrosis (P < .001); age, adiposity, fasting hyperglycemia, and the PNPLA3 I148M variant also were associated with fibrosis. In patients without, but not in those with NASH, significant fibrosis was associated with steatosis grade and the PNPLA3 I148M variant. In patients without NASH, age, fasting hyperglycemia, ballooning, and inflammation were associated with fibrosis. In the validation cohort, 16 of 47 patients (34.0%) with clinically significant fibrosis did not have NASH at baseline. In patients with fibrosis without baseline NASH, worsening of fibrosis (based on later biopsies) was associated with fasting hyperglycemia and more severe steatosis (P = .016). CONCLUSIONS: In an analysis of biopsy specimens collected from patients with NAFLD at a single time point, one third of patients with significant fibrosis did not have NASH. We validated this finding in a separate cohort. In patients without NASH, fasting hyperglycemia, severe steatosis, mild inflammation or ballooning, and the PNPLA3 I148M variant identified those at risk of significant fibrosis.

Genetic and Epigenetic Modifiers of Alcoholic Liver Disease.

Alcoholic liver disease (ALD), a disorder caused by excessive alcohol consumption is a global health issue. More than two billion people consume alcohol in the world and about 75 million are classified as having alcohol disorders. ALD embraces a wide spectrum of hepatic lesions including steatosis, alcoholic steatohepatitis (ASH), fibrosis, cirrhosis, and hepatocellular carcinoma (HCC). ALD is a complex disease where environmental, genetic, and epigenetic factors contribute to its pathogenesis and progression. The severity of alcohol-induced liver disease depends on the amount, method of usage and duration of alcohol consumption as well as on age, gender, presence of obesity, and genetic susceptibility. Genome-wide association studies and candidate gene studies have identified genetic modifiers of ALD that can be exploited as non-invasive biomarkers, but which do not completely explain the phenotypic variability. Indeed, ALD development and progression is also modulated by epigenetic factors. The premise of this review is to discuss the role of genetic variants and epigenetic modifications, with particular attention being paid to microRNAs, as pathogenic markers, risk predictors, and therapeutic targets in ALD.

Protein phosphatase 1 regulatory subunit 3B gene variation protects against hepatic fat accumulation and fibrosis in individuals at high risk of nonalcoholic fatty liver disease.

Nonalcoholic fatty liver disease (NAFLD) is a major cause of liver damage and has a strong genetic component. The rs4841132 G>A variant, modulating the expression of protein phosphatase 1 regulatory subunit 3B (PPP1R3B), which is involved in glycogen synthesis, has been reported to reduce the risk of NAFLD but at the same time may favor liver disease by facilitating glycogen accumulation. The aim of this study was to assess the impact of rs4841132 on development of histologic steatosis and fibrosis in 1,388 European individuals in a liver biopsy cohort, on NAFLD hepatocellular carcinoma in a cross-sectional Italian cohort (n = 132 cases), and on liver disease at the population level in the United Kingdom Biobank cohort. We investigated the underlying mechanism by examining the impact of the variant on gene expression profiles. In the liver biopsy cohort, the rs4841132 minor A allele was associated with protection against steatosis (odds ratio [OR], 0.63; 95% confidence interval [CI], 0.42-0.95; P = 0.03) and clinically significant fibrosis (OR, 0.35; 95% CI, 0.14-0.87; P = 0.02) and with reduced circulating cholesterol (P = 0.02). This translated into protection against hepatocellular carcinoma development (OR, 0.22; 95% CI, 0.07-0.70; P = 0.01). At the population level, the rs4841132 variation was not associated with nonalcoholic or nonviral diseases of the liver but was associated with lower cholesterol (P = 1.7 × 10-8). In individuals with obesity, the A allele protecting against steatosis was associated with increased PPP1R3B messenger RNA expression and activation of lipid oxidation and with down-regulation of pathways related to lipid metabolism, inflammation, and cell cycle. Conclusion: The rs4841132 A allele is associated with protection against hepatic steatosis and fibrosis in individuals at high risk of NAFLD but not in the general population and against dyslipidemia. The mechanism may be related to modulation of PPP1R3B expression and hepatic lipid metabolism. (Hepatology Communications 2018;2:666-675).

GNPAT rs11558492 is not a Major Modifier of Iron Status: Study of Italian Hemochromatosis Patients and Blood Donors

ABSTRACT Background and Aim. HFE-related Hemochromatosis (HH) is characterized by marked phenotype heterogeneity, probably due to the combined action of acquired and genetic factors. Among them, GNPATrs11558492 was proposed as genetic modifier of iron status, but results are still controversial. To shed light on these discrepancies, we genotyped 298 Italian p.C282Y homozygotes and 169 healthy controls. Material and methods. Allele and genotype frequencies were analysed and compared with those reported in Exorne Variant Server (EVS). To explore the role of rs11558492 as a potential modifier of iron status, serum ferritin (SF), liver iron concentration (LIC) and iron removed (IR) were studied according to allele and genotype frequencies. In addition, the effect of the SNP on liver fibrosis was examined comparing patients with absent/mild-moderate fibrosis to those with severe fibrosis-cirrhosis. Results. GNPAT rs11558492 minor allele (G) frequency (MAF) was 20.3% in HFE- HH, 17.2% in controls and 20.6% in EVS database. Genotype frequencies were 64% and 69.2% (AA), 31.2% and 27.2% (AG), 4.8% and 3.6% (GG) in HFE-HH and controls, respectively. No significant differences were found comparing genotype and allele frequencies even selecting subgroups of only-males with extreme phenotypes and low alcohol intake. SF, IR and LIC levels did not significantly differ according to rs11558492 genotypes. Also, MAF did not differ between patients with absent/mild fibrosis and severe fibrosis/cirrhosis. Conclusions. Our findings indicate that GNPAT rs11558492 is not a major modifier of iron status and is not associated with liver fibrosis in HFE- HH patients.(AU)

Insulin resistance promotes Lysyl Oxidase Like 2 induction and fibrosis accumulation in non-alcoholic fatty liver disease.

In patients with non-alcoholic fatty liver disease (NAFLD), insulin resistance (IR) associates with fibrosis progression independently of the hepatic inflammation, but the mechanisms are still unclear. We modeled the independent contribution of inflammation (non-alcoholic steatohepatitis: NASH) by exploiting the methionine-choline deficient (MCD) diet, and that of IR by insulin receptor (InsR) haploinsufficiency (InsR+/-) in the pathogenesis of liver fibrosis in C57BL/6 mice. We confirmed the study findings in 96 patients with NAFLD. InsR+/- enhanced hepatic fat content and impaired hepatic insulin signaling leading to Forkhead box protein O1 (FoxO1) accumulation in MCD-fed mice. Remarkably, despite reduced inflammation and hampered transdifferentiation of hepatic stellate cells (HSCs), InsR+/- promoted hepatic fibrosis accumulation, which correlated with the induction of the Lysyl Oxidase Like 2 (Loxl2), involved in matrix stabilization. Loxl2 up-regulation was not a cell autonomous property of insulin resistant HSCs, but was dependent on microparticles (MPs) released specifically by insulin resistant hepatocytes (HEPs) exposed to fatty acids. The mechanism entailed FoxO1 up-regulation, as FoxO1 silencing normalized Loxl2 expression reversing fibrosis in InsR+/- MCD-fed mice. Loxl2 up-regulation was similarly detected during IR induced by obesity, but not by lipogenic stimuli (fructose feeding). Most importantly, LOXL2 up-regulation was observed in NAFLD patients with type 2 diabetes (T2D) and LOXL2 hepatic and circulating levels correlated with histological fibrosis progression. IR favors fibrosis deposition independently of the classic 'inflammation - HSC transdifferentiation' pathway. The mechanism entails a cross-talk between enhanced lipotoxicity in insulin resistant HEPs and Loxl2 production by HSCs, which was confirmed in patients with diabetes, thereby facilitating extracellular matrix (ECM) stabilization.

GNPAT rs11558492 is not a Major Modifier of Iron Status: Study of Italian Hemochromatosis Patients and Blood Donors.

BACKGROUND AND AIM: HFE-related Hemochromatosis (HH) is characterized by marked phenotype heterogeneity, probably due to the combined action of acquired and genetic factors. Among them, GNPAT rs11558492 was proposed as genetic modifier of iron status, but results are still controversial. To shed light on these discrepancies, we genotyped 298 Italian p.C282Y homozygotes and 169 healthy controls. MATERIAL AND METHODS: Allele and genotype frequencies were analysed and compared with those reported in Exome Variant Server (EVS). To explore the role of rs11558492 as a potential modifier of iron status, serum ferritin (SF), liver iron concentration (LIC) and iron removed (IR) were studied according to allele and genotype frequencies. In addition, the effect of the SNP on liver fibrosis was examined comparing patients with absent/mild-moderate fibrosis to those with severe fibrosis-cirrhosis. RESULTS: GNPAT rs11558492 minor allele (G) frequency (MAF) was 20.3% in HFE-HH, 17.2% in controls and 20.6% in EVS database. Genotype frequencies were 64% and 69.2% (AA), 31.2% and 27.2% (AG), 4.8% and 3.6% (GG) in HFE-HH and controls, respectively. No significant differences were found comparing genotype and allele frequencies even selecting subgroups of only-males with extreme phenotypes and low alcohol intake. SF, IR and LIC levels did not significantly differ according to rs11558492 genotypes. Also, MAF did not differ between patients with absent/mild fibrosis and severe fibrosis/cirrhosis. CONCLUSIONS: Our findings indicate that GNPAT rs11558492 is not a major modifier of iron status and is not associated with liver fibrosis in HFE-HH patients.

Hepcidin resistance in dysmetabolic iron overload.

BACKGROUND & AIMS: Dysmetabolic iron overload syndrome (DIOS) is a frequent condition predisposing to metabolic, cardiovascular and hepatic damage, whose pathogenesis remains poorly defined. Aim of this study was to characterize iron metabolism in DIOS. METHODS: We evaluated 18 patients with DIOS, compared to 18 with nonalcoholic fatty liver and 23 healthy individuals with normal iron status, and 10 patients with hereditary haemochromatosis by a 24-h oral iron tolerance test with hepcidin measurement and iron metabolism modelling under normal iron stores. RESULTS: Dysmetabolic iron overload syndrome patients had higher peak transferrin saturation and area under the-curve of transferrin saturation than subjects with normal iron status, but lower values than haemochromatosis patients (P < 0.05 for all). Conversely, they had higher peak circulating hepcidin levels and area under the curve of hepcidin than the other groups (P < 0.05 for all). This was independent age, sex, haemoglobin, ferritin, and transferrin saturation levels (P = 0.0002). Hepcidin increase in response to the rise in transferrin saturation (hepcidin release index) was not impaired in DIOS patients. Viceversa, the ability of the hepcidin spike to control the rise in transferrin saturation at the beginning of the test (hepcidin resistance index) was impaired in DIOS (P = 0.0002). In DIOS patients, the hepcidin resistance index was correlated with ferritin levels at diagnosis (P = 0.016). CONCLUSIONS: Dysmetabolic iron overload syndrome is associated with a subtle impairment in the ability of the iron hormone hepcidin to restrain iron absorption following an iron challenge, suggesting a hepcidin resistance state. Further studies are required to better characterize the molecular mechanism underpinning this new iron metabolism alteration.

The MBOAT7-TMC4 Variant rs641738 Increases Risk of Nonalcoholic Fatty Liver Disease in Individuals of European Descent.

BACKGROUND & AIMS: Nonalcoholic fatty liver disease (NAFLD) is a leading cause of liver damage and is characterized by steatosis. Genetic factors increase risk for progressive NAFLD. A genome-wide association study showed that the rs641738 C>T variant in the locus that contains the membrane bound O-acyltransferase domain-containing 7 gene (MBOAT7, also called LPIAT1) and transmembrane channel-like 4 gene (TMC4) increased the risk for cirrhosis in alcohol abusers. We investigated whether the MBOAT7-TMC4 is a susceptibility locus for the development and progression of NAFLD. METHODS: We genotyped rs641738 in DNA collected from 3854 participants from the Dallas Heart Study (a multi-ethnic population-based probability sample of Dallas County residents) and 1149 European individuals from the Liver Biopsy Cross-Sectional Cohort. Clinical and anthropometric data were collected, and biochemical and lipidomics were measured in plasma samples from participants. A total of 2736 participants from the Dallas Heart Study also underwent proton magnetic resonance spectroscopy to measure hepatic triglyceride content. In the Liver Biopsy Cross-Sectional Cohort, a total of 1149 individuals underwent liver biopsy to diagnose liver disease and disease severity. RESULTS: The genotype rs641738 at the MBOAT7-TMC4 locus associated with increased hepatic fat content in the 2 cohorts, and with more severe liver damage and increased risk of fibrosis compared with subjects without the variant. MBOAT7, but not TMC4, was found to be highly expressed in the liver. The MBOAT7 rs641738 T allele was associated with lower protein expression in the liver and changes in plasma phosphatidylinositol species consistent with decreased MBOAT7 function. CONCLUSIONS: We provide evidence for an association between the MBOAT7 rs641738 variant and the development and severity of NAFLD in individuals of European descent. This association seems to be mediated by changes in the hepatic phosphatidylinositol acyl-chain remodeling.

Proprotein convertase 7 rs236918 associated with liver fibrosis in Italian patients with HFE-related hemochromatosis.

BACKGROUND AND AIM: p.Cys282Tyr homozygosity is the prevalent genotype in (HFE)-related Hereditary Hemochromatosis with low penetrance and variable expression. However, liver cirrhosis and hepatocellular carcinoma remain the main causes of mortality in these patients. Detection of genetic modifiers identifying patients at risk for liver damage would be relevant for their clinical management. We evaluated proprotein convertase 7 (PCSK7) rs236918 as genetic marker of risk of liver fibrosis in an Italian cohort of p.Cys282Tyr homozygotes. METHODS: Liver fibrosis was histologically assessed by Ishak score. We evaluated PCSK7 alleles and genotypes frequencies according to single or grouped staging scores: absent/mild fibrosis (stage: 0-2), moderate (stage: 3-4), and severe fibrosis/cirrhosis (stage: 5-6). Single nucleotide polymorphism genotyping was performed by restriction fragment length polymorphism or Taqman 5'-nuclease assays. RESULTS: The rs236918 allele C frequency increased from stages 0-2 to 5-6 (7.1% vs 13.6%, vs 21.9%, P = 0.003). The wild-type genotype was significantly more frequent in the absent/mild fibrosis group (54.2%) compared with only 17% in patients with severe fibrosis/cirrhosis. At univariate proportional odds model, patients with GC + CC genotypes were 2.77 times (P = 0.0018) more likely to have worse liver staging scores than wild-type patients. In the adjusted analysis, odds ratio was 2.37 (P = 0.0218), and 2.56 (P = 0.0233) when the analysis was restricted to males. An exploratory mediation analysis suggested a direct effect of genotype on severe fibrosis/cirrhosis (odds ratio = 3.11, P = 0.0157), and a mild non-significant indirect effect mediated through iron accounting for 28%. CONCLUSIONS: These findings confirm that PCSK7 rs236918 C allele is a risk factor for cirrhosis development in Italian patients with HFE-Hemochromatosis.