Serum ferritin levels can predict long-term outcomes in patients with metabolic dysfunction-associated steatotic liver disease.

OBJECTIVE: Hyperferritinaemia is associated with liver fibrosis severity in patients with metabolic dysfunction-associated steatotic liver disease (MASLD), but the longitudinal implications have not been thoroughly investigated. We assessed the role of serum ferritin in predicting long-term outcomes or death. DESIGN: We evaluated the relationship between baseline serum ferritin and longitudinal events in a multicentre cohort of 1342 patients. Four survival models considering ferritin with confounders or non-invasive scoring systems were applied with repeated five-fold cross-validation schema. Prediction performance was evaluated in terms of Harrell's C-index and its improvement by including ferritin as a covariate. RESULTS: Median follow-up time was 96 months. Liver-related events occurred in 7.7%, hepatocellular carcinoma in 1.9%, cardiovascular events in 10.9%, extrahepatic cancers in 8.3% and all-cause mortality in 5.8%. Hyperferritinaemia was associated with a 50% increased risk of liver-related events and 27% of all-cause mortality. A stepwise increase in baseline ferritin thresholds was associated with a statistical increase in C-index, ranging between 0.02 (lasso-penalised Cox regression) and 0.03 (ridge-penalised Cox regression); the risk of developing liver-related events mainly increased from threshold 215.5 µg/L (median HR=1.71 and C-index=0.71) and the risk of overall mortality from threshold 272 µg/L (median HR=1.49 and C-index=0.70). The inclusion of serum ferritin thresholds (215.5 µg/L and 272 µg/L) in predictive models increased the performance of Fibrosis-4 and Non-Alcoholic Fatty Liver Disease Fibrosis Score in the longitudinal risk assessment of liver-related events (C-indices>0.71) and overall mortality (C-indices>0.65). CONCLUSIONS: This study supports the potential use of serum ferritin values for predicting the long-term prognosis of patients with MASLD.

Prevalence and Determinants of Liver Disease in Relatives of Italian Patients With Advanced MASLD.

BACKGROUND & AIMS: Metabolic dysfunction associated steatotic liver disease (MASLD) has a strong genetic component. The aim of this study was to examine noninvasively the prevalence of MASLD and of advanced fibrosis in relatives of patients with advanced MASLD and the risk factors for liver involvement, with a focus on the contribution of common genetic risk variants. METHODS: We prospectively enrolled 98 consecutive probands with advanced fibrosis and/or hepatocellular carcinoma caused by MASLD and 160 nontwin first-degree relatives noninvasively screened for MASLD and advanced fibrosis at 4 Italian centers. We evaluated common genetic determinants and polygenic risk scores of liver disease. RESULTS: Among relatives, prevalence of MASLD was 56.8% overall, whereas advanced fibrosis was observed in 14.4%. At multivariable analysis in relatives, MASLD was associated with body mass index (odds ratio [OR], 1.31 [1.18-1.46]) and tended to be associated with diabetes (OR, 5.21 [0.97-28.10]), alcohol intake (OR, 1.32 [0.98-1.78]), and with female sex (OR, 0.54 [0.23-1.15]), whereas advanced fibrosis was associated with diabetes (OR, 3.13 [1.16-8.45]) and nearly with body mass index (OR, 1.09 [1.00-1.19]). Despite that the PNPLA3 risk variant was enriched in probands (P = .003) and overtransmitted to relatives with MASLD (P = .045), evaluation of genetic risk variants and polygenic risk scores was not useful to guide noninvasive screening of advanced fibrosis in relatives. CONCLUSIONS: We confirmed that about 1 in 7 relatives of patients with advanced MASLD has advanced fibrosis, supporting clinical recommendations to perform family screening in this setting. Genetic risk variants contributed to liver disease within families but did not meaningfully improve fibrosis risk stratification.

Impact of PNPLA3 rs738409 Polymorphism on the Development of Liver-Related Events in Patients With Nonalcoholic Fatty Liver Disease.

BACKGROUND AND AIMS: Nonalcoholic fatty liver disease (NAFLD) is a complex disease, resulting from the interplay between environmental determinants and genetic variations. Single nucleotide polymorphism rs738409 C>G in the PNPLA3 gene is associated with hepatic fibrosis and with higher risk of developing hepatocellular carcinoma. Here, we analyzed a longitudinal cohort of biopsy-proven NAFLD subjects with the aim to identify individuals in whom genetics may have a stronger impact on disease progression. METHODS: We retrospectively analyzed 756 consecutive, prospectively enrolled biopsy-proven NAFLD subjects from Italy, United Kingdom, and Spain who were followed for a median of 84 months (interquartile range, 65-109 months). We stratified the study cohort according to sex, body mass index (BMI)

Oncostatin M is overexpressed in NASH-related hepatocellular carcinoma and promotes cancer cell invasiveness and angiogenesis.

Oncostatin M (OSM) is a pleiotropic cytokine of the interleukin (IL)-6 family that contributes to the progression of chronic liver disease. Here we investigated the role of OSM in the development and progression of hepatocellular carcinoma (HCC) in non-alcoholic fatty liver disease (NAFLD)/non-alcoholic steatohepatitis (NASH). The role of OSM was investigated in (1) selected cohorts of NAFLD/NASH HCC patients, (2) liver cancer cells exposed to human recombinant OSM or stably transfected to overexpress human OSM, (3) murine HCC xenografts, and (4) a murine NASH-related model of hepatic carcinogenesis. OSM was found to be selectively overexpressed in HCC cells of NAFLD/NASH patients, depending on tumor grade. OSM serum levels, barely detectable in patients with simple steatosis or NASH, were increased in patients with cirrhosis and more evident in those carrying HCC. In this latter group, OSM serum levels were significantly higher in the subjects with intermediate/advanced HCCs and correlated with poor survival. Cell culture experiments indicated that OSM upregulation in hepatic cancer cells contributes to HCC progression by inducing epithelial-to-mesenchymal transition and increased invasiveness of cancer cells as well as by inducing angiogenesis, which is of critical relevance. In murine xenografts, OSM overexpression was associated with slower tumor growth but an increased rate of lung metastases. Overexpression of OSM and its positive correlation with the angiogenic switch were also confirmed in a murine model of NAFLD/NASH-related hepatocarcinogenesis. Consistent with this, analysis of liver specimens from human NASH-related HCCs with vascular invasion showed that OSM was expressed by liver cancer cells invading hepatic vessels. In conclusion, OSM upregulation appears to be a specific feature of HCC arising on a NAFLD/NASH background, and it correlates with clinical parameters and disease outcome. Our data highlight a novel pro-carcinogenic contribution for OSM in NAFLD/NASH, suggesting a role of this factor as a prognostic marker and a putative potential target for therapy. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

The Predictive Role of Extracellular NAPRT for the Detection of Advanced Fibrosis in Biopsy-Proven Non-Alcoholic Fatty Liver Disease.

Intrahepatic oxidative stress is a key driver of inflammation and fibrogenesis in non-alcoholic fatty liver disease (NAFLD). We aimed to investigate the role of extracellular Nicotinamide phosphoribosyltransferase (eNAMPT) and extracellular nicotinic acid phosphoribosyltransferase (eNAPRT) for the detection of advanced fibrosis. eNAMPT and eNAPRT were tested in 180 consecutive biopsy-proven NAFLD patients and compared with liver stiffness (LS) and the FIB-4 score. eNAMPT was similarly distributed across fibrosis stages, whereas eNAPRT was increased in patients with advanced fibrosis (p = 0.036) and was associated with advanced fibrosis (OR 1.08, p = 0.016). A multiple stepwise logistic regression model containing significant variables for advanced fibrosis (eNAPRT, type 2 diabetes, age, male sex, ALT) had an area under the curve (AUC) of 0.82 (Se 89.6%, Sp 67.3%, PPV 46.7%, NPV 93.8%) when compared to that of LS (0.79; Se 63.5%, Sp 86.2%, PPV 66.0%, NPV 84.8%) and to that of the FIB-4 score (0.73; Se 80.0%, Sp 56.8%, PPV 44.9%, NPV 86.6%). The use of eNAPRT in clinical practice might allow for the better characterization of NAFLD patients at higher risk of disease progression.

Caucasian lean subjects with non-alcoholic fatty liver disease share long-term prognosis of non-lean: time for reappraisal of BMI-driven approach?

OBJECTIVE: The full phenotypic expression of non-alcoholic fatty liver disease (NAFLD) in lean subjects is incompletely characterised. We aimed to investigate prevalence, characteristics and long-term prognosis of Caucasian lean subjects with NAFLD. DESIGN: The study cohort comprises 1339 biopsy-proven NAFLD subjects from four countries (Italy, UK, Spain and Australia), stratified into lean and non-lean (body mass index (BMI) 10 483 person-years), 4.7% of lean vs 7.7% of non-lean patients reported liver-related events (p=0.37). No difference in survival was observed compared with non-lean NAFLD (p=0.069). CONCLUSIONS: Caucasian lean subjects with NAFLD may progress to advanced liver disease, develop metabolic comorbidities and experience cardiovascular disease (CVD) as well as liver-related mortality, independent of longitudinal progression to obesity and PNPLA3 genotype. These patients represent one end of a wide spectrum of phenotypic expression of NAFLD where the disease manifests at lower overall BMI thresholds. LAY SUMMARY: NAFLD may affect and progress in both obese and lean individuals. Lean subjects are predominantly males, have a younger age at diagnosis and are more prevalent in some geographic areas. During the follow-up, lean subjects can develop hepatic and extrahepatic disease, including metabolic comorbidities, in the absence of weight gain. These patients represent one end of a wide spectrum of phenotypic expression of NAFLD.

Adipose tissue dysfunction and visceral fat are associated with hepatic insulin resistance and severity of NASH even in lean individuals.

BACKGROUND & AIMS: Non-alcoholic fatty liver disease (NAFLD) is a heterogeneous disorder, but the factors that determine this heterogeneity remain poorly understood. Adipose tissue dysfunction is causally linked to NAFLD since it causes intrahepatic triglyceride (IHTG) accumulation through increased hepatic lipid flow, due to insulin resistance and pro-inflammatory adipokines release. While many studies in NAFLD have looked at total adiposity (i.e. mainly subcutaneous fat, SC-AT), it is still unclear the possible impact of visceral fat (VF). Thus, we investigated how VF versus SC-AT was related to NAFLD severity in lean, overweight and obese individuals versus lean controls. METHODS: Thirty-two non-diabetic NAFLD with liver biopsy (BMI 21.4-34.7 kg/m2 ) and eight lean individuals (BMI 19.6-22.8 kg/m2 ) were characterized for fat distribution (VF, SC-AT and IHTG by magnetic resonance imaging), lipolysis and insulin resistance by tracer infusion, free fatty acids (FFAs) and triglyceride (TAG) concentration and composition (by mass spectrometry). RESULTS: Intrahepatic triglyceride was positively associated with lipolysis, adipose tissue insulin resistance (Adipo-IR), TAG concentrations, and increased saturated/unsaturated FFA ratio. Compared to controls VF was higher in NAFLD (including lean individuals), increased with fibrosis stage and associated with insulin resistance in liver, muscle and adipose tissue, increased lipolysis and decreased adiponectin levels. Collectively, our results suggest that VF accumulation, given its location close to the liver, is one of the major risk factors for NAFLD. CONCLUSIONS: These findings propose VF as an early indicator of NAFLD progression independently of BMI, which may allow for evidence-based prevention and intervention strategies.

Long-term outcomes and predictive ability of non-invasive scoring systems in patients with non-alcoholic fatty liver disease.

BACKGROUND & AIMS: Non-invasive scoring systems (NSS) are used to identify patients with non-alcoholic fatty liver disease (NAFLD) who are at risk of advanced fibrosis, but their reliability in predicting long-term outcomes for hepatic/extrahepatic complications or death and their concordance in cross-sectional and longitudinal risk stratification remain uncertain. METHODS: The most common NSS (NFS, FIB-4, BARD, APRI) and the Hepamet fibrosis score (HFS) were assessed in 1,173 European patients with NAFLD from tertiary centres. Performance for fibrosis risk stratification and for the prediction of long-term hepatic/extrahepatic events, hepatocarcinoma (HCC) and overall mortality were evaluated in terms of AUC and Harrell's c-index. For longitudinal data, NSS-based Cox proportional hazard models were trained on the whole cohort with repeated 5-fold cross-validation, sampling for testing from the 607 patients with all NSS available. RESULTS: Cross-sectional analysis revealed HFS as the best performer for the identification of significant (F0-1 vs. F2-4, AUC = 0.758) and advanced (F0-2 vs. F3-4, AUC = 0.805) fibrosis, while NFS and FIB-4 showed the best performance for detecting histological cirrhosis (range AUCs 0.85-0.88). Considering longitudinal data (follow-up between 62 and 110 months), NFS and FIB-4 were the best at predicting liver-related events (c-indices>0.7), NFS for HCC (c-index = 0.9 on average), and FIB-4 and HFS for overall mortality (c-indices >0.8). All NSS showed limited performance (c-indices <0.7) for extrahepatic events. CONCLUSIONS: Overall, NFS, HFS and FIB-4 outperformed APRI and BARD for both cross-sectional identification of fibrosis and prediction of long-term outcomes, confirming that they are useful tools for the clinical management of patients with NAFLD at increased risk of fibrosis and liver-related complications or death. LAY SUMMARY: Non-invasive scoring systems are increasingly being used in patients with non-alcoholic fatty liver disease to identify those at risk of advanced fibrosis and hence clinical complications. Herein, we compared various non-invasive scoring systems and identified those that were best at identifying risk, as well as those that were best for the prediction of long-term outcomes, such as liver-related events, liver cancer and death.

Lean NAFLD: A Distinct Entity Shaped by Differential Metabolic Adaptation.

BACKGROUND AND AIMS: Nonalcoholic fatty liver disease (NAFLD) affects a quarter of the adult population. A significant subset of patients are lean, but their underlying pathophysiology is not well understood. APPROACH AND RESULTS: We investigated the role of bile acids (BAs) and the gut microbiome in the pathogenesis of lean NAFLD. BA and fibroblast growth factor (FGF) 19 levels (a surrogate for intestinal farnesoid X receptor [FXR] activity), patatin-like phospholipase domain containing 3 (PNPLA3), and transmembrane 6 superfamily member 2 (TM6SF2) variants, and gut microbiota profiles in lean and nonlean NAFLD were investigated in a cohort of Caucasian patients with biopsy-proven NAFLD (n = 538), lean healthy controls (n = 30), and experimental murine models. Patients with lean NAFLD had a more favorable metabolic and histological profile compared with those with nonlean NAFLD (P < 0.05 for all). BA levels were significantly higher in NAFLD with advanced compared with earlier stages of liver fibrosis. Patients with lean NAFLD had higher serum secondary BA and FGF19 levels and reduced 7-alpha-hydroxy-4-cholesten-3-one (C4) levels (P < 0.05 for all). These differences were more profound in early compared with advanced stages of fibrosis (P < 0.05 for both). Lean patients demonstrated an altered gut microbiota profile. Similar findings were demonstrated in lean and nonlean murine models of NAFLD. Treating mice with an apical sodium-dependent BA transporter inhibitor (SC-435) resulted in marked increases in fgf15, a shift in the BA and microbiota profiles, and improved steatohepatitis in the lean model. CONCLUSIONS: Differences in metabolic adaptation between patients with lean and nonlean NAFLD, at least in part, explain the pathophysiology and provide options for therapy.

Presence of Serum Antinuclear Antibodies Does Not Impact Long-Term Outcomes in Nonalcoholic Fatty Liver Disease.

INTRODUCTION: We investigated the longitudinal impact of antinuclear antibody (ANA) on clinical outcomes and survival in nonalcoholic fatty liver disease (NAFLD). METHODS: ANA were found in 16.9% of 923 biopsy-proven NAFLD patients, but none of them had histologic autoimmune hepatitis (AIH) or developed AIH after a mean follow-up of 106±50 months. RESULTS: Although ANA-positive cases had a higher prevalence of nonalcoholic steatohepatitis at baseline, the occurrence of liver-related events, hepatocellula carcinoma, cardiovascular events, extrahepatic malignancy, and overall survival were similar to ANA-negative. DISCUSSION: Once AIH has been ruled out, the long-term outcomes and survival are unaffected by the presence of ANA in patients with NAFLD.

Mechanisms for increased risk of diabetes in chronic liver diseases.

OBJECTIVE: Patients with chronic liver disease (CLD), both non-alcoholic fatty liver disease (NAFLD) and chronic hepatitis C (CHC), are at high risk of diabetes (T2D), but mechanisms are still unknown. Muscle/liver insulin resistance (IR) and pancreatic dysfunction are the major metabolic defects leading to T2D. However, if the risk of T2D in CLD patients is because of reduced insulin response and/or to IR, and the impact of liver histology has not been investigated. DESIGN: We studied 220 non-T2D patients with chronic liver disease (129 NAFLD, BMI = 27.3 kg/m2 ; 91 CHC, BMI = 25.0 kg/m2 ) that received a 75-gram oral glucose tolerance test (OGTT) with the measurement of glucose and insulin concentrations for 2 hours, glucose tolerance (NGT vs IGT) and liver biopsy. The results were compared to 26 controls (CT-NGT, BMI = 25.6 kg/m2 ). We evaluated peripheral insulin sensitivity (OGIS), OGTT-insulin response (ΔAUC-I/ΔAUC-G) and disposition-index (DI = OGIS∙ΔAUC-I/ΔAUC-G) for the risk to develop T2D. RESULTS: NAFLD had increased muscle IR (associated to NASH, steatosis and fibrosis), higher than in CHC or CT-NGT (OGIS = 8.9 vs 11.3 and 10.5 mL/min kg, P < .0001). In NAFLD, OGTT-insulin response (ΔAUC-I/ΔAUC-G) was the highest while it was significantly decreased in CHC (2.2 vs 1.1 and 1.6, NAFLD vs. CHC and CT-NGT, P < .005). The highest T2D risk (low DI) was observed in CHC-IGT (7.5), CHC-NGT (13.5) and NAFLD-IGT (10.8) vs CT-NGT (14.9, all P < .0001), but not in NAFL-NGT or NASH-NGT. CONCLUSION: We observed an increased T2D risk in NAFLD-IGT, CHC-IGT and CHC-NGT mainly because of reduced OGTT-insulin response, while insulin response in NAFLD-NGT compensates the IR thus maintaining normal glycaemia.

Interplay between Oxidative Stress and Metabolic Derangements in Non-Alcoholic Fatty Liver Disease: The Role of Selenoprotein P.

Background: Pathogenetic mechanisms involved in the progression of non-alcoholic fatty liver disease (NAFLD) are complex and multifactorial. We investigated oxidative stress through the measurement of selenoprotein P (SeP) in serum and we explored its relation to metabolic derangements and liver damage in a group of non-diabetic NAFLD subjects. Methods: 57 NAFLD patients underwent a double-tracer oral glucose tolerance test (OGTT). Insulin resistance (IR) components were calculated at baseline as follows: hepatic-IR = (endogenous glucose production*insulin); peripheral-IR = (glucose rate of disappearance(Rd)); adipose-tissue(AT)-IR as Lipo-IR = (glycerol rate of appearance (Ra)*insulin) or AT-IR = (free fatty acids (FFAs)*insulin). The lipid and amino acid (AA) profiles were assessed by gas chromatography-mass spectrometry. SeP levels were measured by enzyme immunosorbent assay. Results: Circulating SeP correlated with insulin (rS = 0.28), FFAs (rS = 0.42), glucose Rd (rS = -0.33) and glycerol Ra (rS = -0.34); consistently, SeP levels correlated with Lipo-IR and AT-IR (rS > 0.4). Among the AA and lipid profiles, SeP inversely correlated with serine (rS = -0.31), glycine (rS = -0.44) and branched chain AA (rS = -0.32), and directly correlated with saturated (rS = 0.41) and monounsaturated FFAs (rS = 0.40). Hepatic steatosis and fibrosis increased in subjects with higher levels of SeP. In multivariable regression analysis, SeP was associated with the degree of hepatic fibrosis (t = 2.4, p = 0.022). Conclusions: SeP levels were associated with an altered metabolic profile and to the degree of hepatic fibrosis, suggesting a role in the pathogenesis of NAFLD.

Crosstalk between adipose tissue insulin resistance and liver macrophages in non-alcoholic fatty liver disease.

BACKGROUND & AIMS: The pathogenesis of non-alcoholic fatty liver disease (NAFLD) and steatohepatitis (NASH) is likely due to the interaction between a deranged metabolic milieu and local mediators of hepatic inflammation and fibrosis. We undertook this study to elucidate the interplay between macrophage activation, insulin resistance (IR) in target organs/tissues and hepatic damage. METHODS: In 40 non-diabetic patients with biopsy-proven NAFLD we assessed: i) endogenous glucose production (EGP), glucose clearance and indexes of IR in the adipose tissue (Adipo-IR and Lipo-IR) and in the liver (Hep-IR) by tracer infusion ([6,6-2H2]glucose and [2H5]glycerol); ii) macrophage activity (by soluble sCD163) and iii) hepatic expression of CD163 (hCD163). RESULTS: We found that sCD163 levels paralleled both the plasma free fatty acid (FFA) levels and lipolysis from adipose tissue. Consistently, sCD163 significantly correlated with adipose tissue IR (Adipo-IR: r = 0.32, p = 0.042; Lipo-IR: r = 0.39, p = 0.012). At multiple regression analysis, sCD163 levels were associated with FFA levels (rp = 0.35, p = 0.026). In vitro exposure of human monocyte-derived macrophages to palmitate enhanced sCD163 secretion. Conversely, sCD163 did not correlate with EGP or with Hep-IR. In the liver, hCD163 positively correlated with sCD163 (r = 0.58, p = 0.007) and the degree of steatosis (r = 0.34, p = 0.048), but not with EGP or Hep-IR (r = -0.27 and r = 0.11, respectively, p >0.10, both). CONCLUSIONS: Our findings suggest a link between deranged metabolism in the adipose tissue and activation of hepatic macrophages in patients with NAFLD, possibly in response to FFA overflow and independent of obesity and diabetes. Conversely, our findings do not support a link between activated hepatic macrophages and glucose metabolism (EGP or Hep-IR). The relationship between adipose tissue IR and hepatic macrophages should be considered to define therapeutic targets for NAFLD. LAY SUMMARY: The pathogenesis of non-alcoholic fatty liver disease (NAFLD) and steatohepatitis (NASH) is likely due to the interaction between a deranged metabolic milieu and local mediators of hepatic inflammation and fibrosis in the insulin resistant state. This study provides in vivo support for a possible link between deranged metabolism in the adipose tissue and activation of hepatic macrophages in patients with NAFLD, most likely in response to free fatty acid overflow and independent of obesity and diabetes.

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.

Altered amino acid concentrations in NAFLD: Impact of obesity and insulin resistance.

Plasma concentrations of amino acids (AAs), in particular, branched chain AAs (BCAAs), are often found increased in nonalcoholic fatty liver disease (NAFLD); however, if this is due to increased muscular protein catabolism, obesity, and/or increased insulin resistance (IR) or impaired tissue metabolism is unknown. Thus, we evaluated a) if subjects with NAFLD without obesity (NAFLD-NO) compared to those with obesity (NAFLD-Ob) display altered plasma AAs compared to controls (CTs); and b) if AA concentrations are associated with IR and liver histology. Glutamic acid, serine, and glycine concentrations are known to be altered in NAFLD. Because these AAs are involved in glutathione synthesis, we hypothesized they might be related to the severity of NAFLD. We therefore measured the AA profile of 44 subjects with NAFLD without diabetes and who had a liver biopsy (29 NAFLD-NO and 15 NAFLD-Ob) and 20 CTs without obesity, by gas chromatography-mass spectrometry, homeostasis model assessment of insulin resistance, hepatic IR (Hep-IR; Hep-IR = endogenous glucose production × insulin), and the new glutamate-serine-glycine (GSG) index (glutamate/[serine + glycine]) and tested for an association with liver histology. Most AAs were increased only in NAFLD-Ob subjects. Only alanine, glutamate, isoleucine, and valine, but not leucine, were increased in NAFLD-NO subjects compared to CTs. Glutamate, tyrosine, and the GSG-index were correlated with Hep-IR. The GSG-index correlated with liver enzymes, in particular, gamma-glutamyltransferase (R = 0.70), independent of body mass index. Ballooning and/or inflammation at liver biopsy were associated with increased plasma BCAAs and aromatic AAs and were mildly associated with the GSG-index, while only the new GSG-index was able to discriminate fibrosis F3-4 from F0-2 in this cohort. CONCLUSION: Increased plasma AA concentrations were observed mainly in subjects with obesity and NAFLD, likely as a consequence of increased IR and protein catabolism. The GSG-index is a possible marker of severity of liver disease independent of body mass index. (Hepatology 2018;67:145-158).

Hypoxia-inducible factor 2α drives nonalcoholic fatty liver progression by triggering hepatocyte release of histidine-rich glycoprotein.

Mechanisms underlying progression of nonalcoholic fatty liver disease (NAFLD) are still incompletely characterized. Hypoxia and hypoxia-inducible factors (HIFs) have been implicated in the pathogenesis of chronic liver diseases, but the actual role of HIF-2α in the evolution of NAFLD has never been investigated in detail. In this study, we show that HIF-2α is selectively overexpressed in the cytosol and the nuclei of hepatocytes in a very high percentage (>90%) of liver biopsies from a cohort of NAFLD patients at different stages of the disease evolution. Similar features were also observed in mice with steatohepatitis induced by feeding a methionine/choline-deficient diet. Experiments performed in mice carrying hepatocyte-specific deletion of HIF-2α and related control littermates fed either a choline-deficient L-amino acid-defined or a methionine/choline-deficient diet showed that HIF-2α deletion ameliorated the evolution of NAFLD by decreasing parenchymal injury, fatty liver, lobular inflammation, and the development of liver fibrosis. The improvement in NAFLD progression in HIF-2α-deficient mice was related to a selective down-regulation in the hepatocyte production of histidine-rich glycoprotein (HRGP), recently proposed to sustain macrophage M1 polarization. In vitro experiments confirmed that the up-regulation of hepatocyte HRGP expression was hypoxia-dependent and HIF-2α-dependent. Finally, analyses performed on specimens from NAFLD patients indicated that HRGP was overexpressed in all patients showing hepatocyte nuclear staining for HIF-2α and revealed a significant positive correlation between HIF-2α and HRGP liver transcript levels in these patients. CONCLUSIONS: These results indicate that hepatocyte HIF-2α activation is a key feature in both human and experimental NAFLD and significantly contributes to the disease progression through the up-regulation of HRGP production. (Hepatology 2018;67:2196-2214).

Altered Metabolic Profile and Adipocyte Insulin Resistance Mark Severe Liver Fibrosis in Patients with Chronic Liver Disease.

Metabolomics/lipidomics are important tools to identify novel biomarkers associated with liver damage. Patients with chronic liver disease (CLD) and hepatitis C virus (HCV) infection often have alterations in glucose, lipid and protein metabolism. The aim of this study was to evaluate if dysfunctional lipid and amino acid metabolism was associated with fibrosis severity and insulin resistance in CLD/HCV patients. We analyzed the baseline sera of 75 subjects with CLD/HCV infection HCV genotype-1, with proven liver biopsy prior to antiviral treatment. We measured amino acid (AA) and lipid concentration by gas and liquid chromatography-mass spectrometry respectively. Alterations in peripheral glucose metabolism due to insulin resistance (IR) were assesed by HOMA-IR (Glucose x Insulin/22.5), while adipose tissue IR was estimated as (Adipo-IR = Free Fatty Acids x Insulin). Baseline HOMA-IR and Adipo-IR were related to the degree of liver fibrosis. Reduction in ceramides 18:1/22:0, 18:1/24:0, diacylglycerol 42:6 and increased phosphocholine 40:6 were associated with higher fibrosis. Adipo-IR was related to lower levels of lysophosphatidylcholine 14:0 and 18:2 and with higher levels of sphingomyelin 18:2/24:0 and 18:2/24:1. Almost all AA were positively associated with Adipo-IR but not with HOMA-IR. We further confirmed the potential use of metabolomics and lipidomics in CLD/HCV subjects finding novel biomarkers of hepatic fibrosis and show that the adipose tissue IR is associated with more severe liver disease and is an important marker not only of altered lipid but also AA metabolism.

Liver Cancer: Connections with Obesity, Fatty Liver, and Cirrhosis.

The burden of hepatocellular carcinoma (HCC), the most common form of liver cancer, is steadily growing because obesity, type 2 diabetes, and nonalcoholic fatty liver disease (NAFLD) are replacing viral- and alcohol-related liver disease as major pathogenic promoters. The most worrisome aspects of these new risk factors are their large spread in the general population and their link with HCC arising in noncirrhotic livers. HCC may be the presenting feature of an asymptomatic nonalcoholic steatohepatitis (NASH), the progressive form of NAFLD. The HCC risk connected to metabolic factors has been underestimated so far, and a poorer surveillance has prevented an adequate treatment. Systemic and hepatic molecular mechanisms involved in obesity- and NAFLD-induced hepatocarcinogenesis as well as potential early markers of HCC are being extensively investigated. This review summarizes current evidence linking obesity, NAFLD and liver cancer, discusses its clinical impact and describes the main mechanisms underlying this complex relationship.

Usefulness of the index of NASH - ION for the diagnosis of steatohepatitis in patients with non-alcoholic fatty liver: An external validation study.

BACKGROUND & AIMS: The non-invasive identification of steatohepatitis (NASH) in patients with Non-Alcoholic Fatty Liver Disease is an unmet need in clinical practice. Index of NASH (ION) is a new tool for the prediction of NASH. We aimed to externally validate ION and to compare it with CK-18. Since necroinflammation precedes fibrosis, we also tested ION in combination with non-invasive tools for fibrosis. METHODS: We analysed data from 292 Italian patients (169 Southern cohort, and 123 Northern cohort) with an histological diagnosis of NAFLD. The ION, FIB-4 and NFS scores were calculated according to published algorithms. Serum cytokeratin18-Aspartate396 levels and liver stiffness (LS) by Fibroscan were assessed within three months from liver biopsy. RESULTS: The diagnostic accuracy of ION for the identification of NASH was not as satisfactory as reported (area under the ROC curve, AUROC = 0.687 [95% CI = 0.62-0.75]). The proposed cut-off value ≥50 showed a poor sensitivity (Se) (28%) and a good specificity (Sp) (92%), with a positive predictive value (PPV) of 91% and a negative predictive value (NPV) of 30%. A new cut-off value >26 improved Se (73%) but decreased Sp (60%) (PPV of 84% and a NPV of 43%). ION performed slightly better in obese NAFLD (AUROC = 0.700). The combination of ION and markers of fibrosis did not improve the identification of advanced liver disease. CONCLUSIONS: ION is not feasible for the non-invasive diagnosis of NASH across different populations of NAFLD patients, mainly because its limited reproducibility in non-obese subjects.

Low Birthweight Increases the Likelihood of Severe Steatosis in Pediatric Non-Alcoholic Fatty Liver Disease.

OBJECTIVES: Small for gestational age (SGA) is associated with an increased risk of non-alcoholic fatty liver disease (NAFLD). Our aim was to investigate the correlation of birthweight with the severity of liver damage in a large cohort of children with NAFLD. METHODS: Two hundred and eighty-eight consecutive Caucasian Italian overweight/obese children with biopsy-proven NAFLD were included in the study. We examined the relative association of each histological feature of NAFLD with metabolic alterations, insulin-resistance, I148M polymorphism in the patatin-like phospholipase domain-containing protein 3 (PNPLA3) gene, and birthweight relative to gestational age. RESULTS: In the whole NAFLD cohort, 12.2% of patients were SGA, 62.8% appropriate for gestational age (AGA), and 25% large for gestational age (LGA). SGA children had a higher prevalence of severe steatosis (69%) and severe portal inflammation (14%) compared with the AGA and LGA groups. Notably, severe steatosis (>66%) was decreasing from SGA to AGA and LGA, whereas the prevalence of moderate steatosis (33-66%) was similar in three groups. The prevalence of type 1 NAFLD is higher in the LGA group with respect to the other two groups (25% vs.5.2% vs.9.4%), whereas the SGA group shows a higher prevalence of overlap type (85.8%) with respect to the LGA group (51.4%) but not compared with the AGA group (75%). At multivariable regression analysis, SGA at birth increased fourfold the likelihood of severe steatosis (odds ratio (OR) 4.0, 95% confidence interval (CI) 1.43-10.9, P=0.008) and threefold the likelihood of NAFLD Activity Score (NAS)≥5 (OR 2.98, 95% CI 1.06-8.33, P=0.037) independently of homeostasis model assessment of insulin resistance and PNPLA3 genotype. The PNPLA3-CC wild-type genotype was the strongest independent predictor of the absence of significant fibrosis (OR 0.26, 95% CI 0.13-0.52, P=<0.001). CONCLUSIONS: In children with NAFLD, the risk of severe steatosis is increased by SGA at birth, independent of and in addition to other powerful risk factors (insulin-resistance and I148M variant of the PNPLA3 gene).