Alcohol-associated liver disease - global epidemiology.

Alcohol-associated liver disease (ALD), as highlighted in this narrative review, is a major public health concern, increasingly impacting global disease burden and premature mortality. In 2019, ALD accounted for the loss of 11 million life-years worldwide. The rising number of deaths and disability-adjusted life years attributed to ALD, particularly pronounced in America, are alarming. Projections suggest that the economic impact of ALD, as seen in the United States, could potentially double by 2040. ALD is increasingly prevalent among younger adults and has become the leading cause for liver transplantation in both America and Europe. During the COVID-19 pandemic, the existing trend was further amplified as high-risk drinking patterns coincided with a rise in hospital admissions for alcohol-associated hepatitis and increased ALD-related mortality. The prevalence of ALD is estimated at 3.5% in the general population, 26.0% among hazardous drinkers, and 55.1% among those with alcohol use disorders. Alarmingly, five-year mortality rates for ALD patients exceed 50%, with even higher rates in more advanced disease stages. Methodological challenges, such as underreporting, diagnostic difficulties, and variability in registry data quality, complicate the accurate assessment of the impact of ALD. Additionally, the contribution of alcohol to the progression of other liver diseases is often underacknowledged in healthcare registries, leading to a significant underestimation of its broader implications for liver health. Addressing the growing ALD concern requires robust public health initiatives, heightened awareness, refined diagnostic techniques, and comprehensive epidemiological studies. These measures are vital to tackle the increasing prevalence of ALD and mitigate its extensive impact on individuals and healthcare systems.

Circulating Citrate Is Associated with Liver Fibrosis in Nonalcoholic Fatty Liver Disease and Nonalcoholic Steatohepatitis.

Nonalcoholic fatty liver disease (NAFLD) is associated with mitochondrial damage. Circulating mitochondrial metabolites may be elevated in NAFLD but their associations with liver damage is not known. This study aimed to assess the association of key mitochondrial metabolites with the degree of liver fibrosis in the context of NAFLD and nonalcoholic steatohepatitis (NASH). Cross-sectional analyses were performed on two cohorts of biopsy-proven NAFLD and/or NASH subjects. The association of circulating mitochondrial metabolite concentrations with liver fibrosis was assessed using linear regression analysis. In the single-center cohort of NAFLD subjects (n = 187), the mean age was 54.9 ±13.0 years, 40.1% were female and 86.1% were White. Type 2 diabetes (51.3%), hypertension (43.9%) and obesity (72.2%) were prevalent. Those with high citrate had a higher proportion of moderate/significant liver fibrosis (stage F ≥ 2) (68.4 vs. 39.6%, p = 0.001) and advanced fibrosis (stage F ≥ 3) (31.6 vs. 13.6%, p = 0.01). Citrate was associated with liver fibrosis independent of age, sex, NAFLD activity score and metabolic syndrome (per 1 SD increase: ß = 0.19, 95% CI: 0.03-0.35, p = 0.02). This association was also observed in a cohort of NASH subjects (n = 176) (ß = 0.21, 95% CI: 0.07-0.36, p = 0.005). The association of citrate with liver fibrosis was observed in males (p = 0.005) but not females (p = 0.41). In conclusion, circulating citrate is elevated and associated with liver fibrosis, particularly in male subjects with NAFLD and NASH. Mitochondrial function may be a target to consider for reducing the progression of liver fibrosis and NASH.

Advancing diagnosis and management of liver disease in adults through exome sequencing.

BACKGROUND: Whole-exome sequencing (WES) is an effective tool for diagnosis in patients who remain undiagnosed despite a comprehensive clinical work-up. While WES is being used increasingly in pediatrics and oncology, it remains underutilized in non-oncological adult medicine, including in patients with liver disease, in part based on the faulty premise that adults are unlikely to harbor rare genetic variants with large effect size. Here, we aim to assess the burden of rare genetic variants underlying liver disease in adults at two major tertiary referral academic medical centers. METHODS: WES analysis paired with comprehensive clinical evaluation was performed in fifty-two adult patients with liver disease of unknown etiology evaluated at two US tertiary academic health care centers. FINDINGS: Exome analysis uncovered a definitive or presumed diagnosis in 33% of patients (17/52) providing insight into their disease pathogenesis, with most of these patients (12/17) not having a known family history of liver disease. Our data shows that over two-thirds of undiagnosed liver disease patients attaining a genetic diagnosis were being evaluated for cholestasis or hepatic steatosis of unknown etiology. INTERPRETATION: This study reveals an underappreciated incidence and spectrum of genetic diseases presenting in adulthood and underscores the clinical value of incorporating exome sequencing in the evaluation and management of adults with liver disease of unknown etiology. FUNDING: S.V. is supported by the NIH/NIDDK (K08 DK113109 and R01 DK131033-01A1) and the Doris Duke Charitable Foundation Grant #2019081. This work was supported in part by NIH-funded Yale Liver Center, P30 DK34989.

Multicellular Liver Organoids: Generation and Importance of Diverse Specialized Cellular Components.

Over 40,000 patients in the United States are estimated to suffer from end-stage liver disease and acute hepatic failure, for which liver transplantation is the only available therapy. Human primary hepatocytes (HPH) have not been employed as a therapeutic tool due to the difficulty in growing and expanding them in vitro, their sensitivity to cold temperatures, and tendency to dedifferentiate following two-dimensional culture. The differentiation of human-induced pluripotent stem cells (hiPSCs) into liver organoids (LO) has emerged as a potential alternative to orthotropic liver transplantation (OLT). However, several factors limit the efficiency of liver differentiation from hiPSCs, including a low proportion of differentiated cells capable of reaching a mature phenotype, the poor reproducibility of existing differentiation protocols, and insufficient long-term viability in vitro and in vivo. This review will analyze various methodologies being developed to improve hepatic differentiation from hiPSCs into liver organoids, paying particular attention to the use of endothelial cells as supportive cells for their further maturation. Here, we demonstrate why differentiated liver organoids can be used as a research tool for drug testing and disease modeling, or employed as a bridge for liver transplantation following liver failure.

Low vitamin A levels are associated with liver-related mortality: a nationally representative cohort study.

INTRODUCTION: Vitamin A, a fat-soluble vitamin that includes retinol and carotenoids, is implicated in liver fibrosis, whereas its deficiency has been associated with various liver diseases and higher overall mortality. This study aims to determine the relationship between levels of vitamin A species and liver fibrosis, as well as liver-related mortality in the population of the US. METHODS: A total of 12,299 participants from the National Health and Nutrition Examination Survey III (NHANES III) were analyzed to provide nationally representative estimates of the relationship between the levels of vitamin A species and liver fibrosis measured by Fibrosis-4 (FIB-4) index and liver-related mortality. RESULTS: A low blood level of retinol, but not other retinoid derivatives, was associated with significant liver fibrosis after adjustment for demographics, anthropometric measurements, medical history, retinol, and carotene intakes. Compared with vitamin D and E, retinol deficiency demonstrated much stronger associations with a high FIB-4 score. Individuals with known risks of chronic liver disease (CLD) and the lowest pentile of retinol levels had ORs of 3.12 (95% CI, 1.64-5.91) for possible fibrosis and 19.7 (95% CI, 5.71-67.7) for likely fibrosis, and an HR of 7.76 (95% CI, 1.19-50.5) for liver-related mortality compared with those in the highest retinol-level pentile. These relationships were more pronounced among individuals with known risks of chronic liver disease than without. CONCLUSIONS: A low circulating retinol level is associated with liver fibrosis and liver-related mortality in chronic liver disease. This relationship is potentially driven by a mechanistic link rather than the malabsorption of fat-soluble vitamins and may be leveraged for disease prognostication and have therapeutic implications.

Prevalence of High Liver Stiffness and a Screening Strategy Using the SODA-2B Score Among US Adults.

Cirrhosis, a rising cause of death in the United States, has an extended preclinical phase characterized by progressive liver fibrosis. Despite the developments in noninvasive fibrosis measurement, there is no recommended screening, in part due to an incomplete understanding of the disease epidemiology on a national scale. Herein, we aim to define the prevalence of liver fibrosis and compare strategies to identify the at-risk population. We analyzed 4,510 US adults with complete liver stiffness measurement (LSM) by vibration-controlled transient elastography (VCTE) in the 2017-2018 National Health and Nutrition Examination Survey to estimate the disease burden of increased liver stiffness. An estimated 11.6 million (95% confidence interval [C.I.], 8.1-15.0 million) US adults had LSM ≥9.5 kPa, indicating advanced fibrosis and representing 1 in every 18 adults. Among them, 7.1 million (95% CI, 5.0-9.1 million) had LSM ≥12.5 kPa, which is concerning for cirrhosis. LSM ≥9.5 kPa is associated with male sex (S), history of other liver diseases (O), diabetes (D), advanced age (A), and an elevated BMI (B). A simple SODA-2B score had a sensitivity of 96.4% in identifying individuals at risk for advanced cirrhosis (LSM ≥9.5 kPa) and a negative predictive value of 99.3% in stratifying more than half of the adult population. When the liver function test (LFT) is available, the inclusion of abnormal LFT and elevated fibrosis-4 index can further increase screening efficiency. Conclusion: Elevated liver stiffness is prevalent among US adults. A SODA-2B score can risk stratify adults for VCTE-based fibrosis screening.

Lipoprotein Z, a hepatotoxic lipoprotein, predicts outcome in alcohol-associated hepatitis.

BACKGROUND AND AIMS: Lipoprotein Z (LP-Z) is an abnormal free cholesterol (FC)-enriched LDL-like particle discovered from patients with cholestatic liver disease. This study aims to define the diagnostic value of LP-Z in alcohol-associated hepatitis (AH) and interrogate the biology behind its formation. APPROACH AND RESULTS: We measured serum levels of LP-Z using nuclear magnetic resonance spectroscopy, a well-established clinical assay. Serum levels of LP-Z were significantly elevated in four AH cohorts compared with control groups, including heavy drinkers and patients with cirrhosis. We defined a Z-index, calculated by the ratio of LP-Z to total apolipoprotein B-containing lipoproteins, representing the degree of deviation from normal VLDL metabolism. A high Z-index was associated with 90-day mortality independent from the Model for End-Stage Liver Disease (MELD) and provided added prognosticative value. Both a Z-index ≤ 0.6 and a decline of Z-index by ≥0.1 in 2 weeks predicted 90-day survival. RNA-sequencing analyses of liver tissues demonstrated an inverse association in the expression of enzymes responsible for the extrahepatic conversion of VLDL to LDL and AH disease severity, which was further confirmed by the measurement of serum enzyme activity. To evaluate whether the FC in LP-Z could contribute to the pathogenesis of AH, we found significantly altered FC levels in liver explant of patients with AH. Furthermore, FC in reconstituted LP-Z particles caused direct toxicity to human hepatocytes in a concentration-dependent manner, supporting a pathogenic role of FC in LP-Z. CONCLUSIONS: Impaired lipoprotein metabolism in AH leads to the accumulation of LP-Z in the circulation, which is hepatotoxic from excessive FC. A Z-index ≤ 0.6 predicts 90-day survival independent from conventional biomarkers for disease prognostication.

Coffee Consumption Is Associated With Lower Liver Stiffness: A Nationally Representative Study.

BACKGROUND & AIMS: Coffee is associated with a reduced risk of liver disease. This association is limited by important sources of confounding such as recall bias, healthy user bias, and indirect measures of liver outcomes or health. We aimed to examine the impact of coffee consumption with liver fibrosis and steatosis in a nationally representative sample. METHODS: We evaluated 4510 subjects 20 years and older from the 2017 to 2018 National Health and Nutrition Examination Survey study who underwent both transient elastography and two 24-hour dietary recall examinations. We tested the associations between liver stiffness measurements (LSM) of 9.5 kpa or greater or controlled attenuation parameter (CAP) and coffee consumption. We used decaffeinated coffee and tea consumption as controls. As a sensitivity analysis, we included all drinks in 1 model, examined the impact of caffeine consumption, and adjusted for the Healthy Eating Index-2015 and sugar-sweetened beverage consumption as separate models. RESULTS: The study sample described was aged 48 ± 0.6 years, 73% were overweight or obese, 10.6% had diabetes, 47.5% reported participation in vigorous physical activity, and 23% drank 2 or more alcoholic drinks per day. After multivariate adjustment, there was no association between coffee and controls with CAP. Subjects who drank more than 3 cups of coffee, but not other drinks, had a 0.9 lower kPa (95% CI, -1.6 to -0.1; P = .03). More than 3 cups of coffee were protective for LSM of 9.5 kpa or higher (odds ratio, 0.4; 95% CI, 0.2-1.0; P = .05). Accounting for all beverages in the same model, only consuming more than 3 cups of coffee remained independently associated with LSM (odds ratio, 0.5; 95% CI, 0.2-0.9; P = .03). Caffeine was not associated significantly with LSM at any dose. Finally, adjusting for sugar-sweetened beverage consumption and Healthy Eating Index-2015, coffee consumption remained associated with a lower LSM. The protective nature of coffee consumption therefore is not attributable to caffeine and persists in participants regardless of their diet quality. CONCLUSIONS: Coffee is associated with lower liver stiffness, but not steatosis, as measured by CAP among US adults.

Severe Hepatic Steatosis by Controlled Attenuation Parameter Predicts Quality of Life Independent of Fibrosis.

BACKGROUND & AIM: Liver fibrosis is associated with poor patient-reported outcomes (PROs), but the impact of steatosis is unknown. We aimed to evaluate the impact of steatosis on PROs independent of liver fibrosis. METHODS: We evaluated the impact of steatosis, measured by Controlled-Attenuation Parameter (CAP) on transient elastography, and PROs using the 2017-2018 National Health and Nutrition Examination Survey (NHANES) database. We used univariate and multivariate logistic and ordinal regression to evaluate categorical CAP score with PROs measuring physical disability, general health and depression. RESULTS: Of 4,509 participants included, 38% had severe steatosis (> 280 dB/m). Those with severe steatosis were older and more likely to be male (56% vs. 43% and 51%). On univariate analysis, severe steatosis was associated with more difficulty walking (P = 0.01), dressing (P = 0.005), lifting objects (P = 0.02), bending (P < 0.001), and moving large objects (P = 0.0006). After multivariate adjustment, severe steatosis remained associated with difficulty lifting objects (odds ratio [OR]: 1.7, 95% confidence interval [CI]: 1.2-2.4, P = 0.01) and difficulty bending (OR: 1.8, 95% CI: 1.2-2.7, P = 0.006). Severe steatosis increased risk of having any of the disabilities (OR: 1.7, 95% CI: 1.2-2.4, P = 0.008) and had higher ordinal disability index (OR: 1.6, 95% CI: 1.2-2.2, P = 0.007). Lastly, severe steatosis was also associated with worse self-perceived health status (OR: 1.5, 95% CI: 1.2-1.9, P = 0.002), while general health compared to one year ago and depression trended toward significance. CONCLUSION: Patients with severe steatosis are at increased risk of physical disability and have worse self-perceived health status independent of liver fibrosis.

Circulating high density lipoprotein distinguishes alcoholic hepatitis from heavy drinkers and predicts 90-day outcome: lipoproteins in alcoholic hepatitis.

BACKGROUND: Alcohol-associated liver disease (ALD) and alcoholic hepatitis (AH) significantly impact the liver, an organ central to the lipid and lipoprotein metabolism. OBJECTIVE: To define changes in the lipid and lipoprotein profiles in subjects with alcoholic hepatitis (AH) versus heavy drinkers with normal liver function and to determine the association of the AH-mediated lipoprotein phenotype with AH severity and outcomes. METHODS: AH cases (n=196) and a heavy drinker control group (n=169) were identified in a multicenter, prospective cohort. The relationships between lipid panels and lipoprotein profiles among AH and heavy drinkers were interrogated using three common measurements: the conventional lipid panel, extended lipid panel by NMR, and NMR-based direct lipoprotein profiling. Predictive values for AH severity and mortality were determined using Harrell's C-Index. RESULTS: Lipid and lipoprotein profiles were significantly different in AH compared to heavy drinkers. Among them, high density lipoprotein (HDL) particle concentration exhibited the most significant reduction in AH compared to heavy drinkers (5.3 ± 3.4 vs 22.3 ± 5.4 µmol/L, p < 0.001). Within AH patients, HDL particle concentration was inversely associated with Maddrey's Discriminant Function (DF) (p < 0.001), and independently associated with mortality at both 90 and 365 days even after adjustment for DF (p = 0.02, p = 0.05 respectively). HDL particle concentration less than 3.5 µmol/L and total cholesterol ≤ 96 mg/dL identified AH patients with higher 90-day mortality. CONCLUSION: Lipid and lipoprotein profiles are profoundly altered in AH and can help in prognosticating disease severity and mortality.

Adenosine deaminase 2 produced by infiltrative monocytes promotes liver fibrosis in nonalcoholic fatty liver disease.

Elevated circulating activity of adenosine deaminase 2 (ADA2) is associated with liver fibrosis in nonalcoholic fatty liver disease (NAFLD). In the liver of NAFLD patients, ADA2-positive portal macrophages are significantly associated with the degree of liver fibrosis. These liver macrophages are CD14- and CD16-positive and co-express chemokine receptors CCR2, CCR5, and CXCR3, indicating infiltrative monocyte origin. Human circulatory monocytes release ADA2 upon macrophage differentiation in vitro. When stimulated by recombinant human ADA2 (rhADA2), human monocyte-derived macrophages demonstrate upregulation of pro-inflammatory and pro-fibrotic genes, including PDGF-B, a key pro-fibrotic cytokine. This PDGF-B upregulation is reproduced by inosine, the enzymatic product of ADA2, but not adenosine, and is abolished by E359N, a loss-of-function mutation in ADA2. Finally, rhADA2 also stimulates PDGF-B production from Kupffer cells in primary human liver spheroids. Together, these data suggest that infiltrative monocytes promote fibrogenesis in NAFLD via ADA2-mediated autocrine/paracrine signaling culminating in enhanced PDGF-B production.

Heterozygosity of the Alpha 1-Antitrypsin Pi*Z Allele and Risk of Liver Disease.

The serpin family A member 1 (SERPINA1) Z allele is present in approximately one in 25 individuals of European ancestry. Z allele homozygosity (Pi*ZZ) is the most common cause of alpha 1-antitrypsin deficiency and is a proven risk factor for cirrhosis. We examined whether heterozygous Z allele (Pi*Z) carriers in United Kingdom (UK) Biobank, a population-based cohort, are at increased risk of liver disease. We replicated findings in Massachusetts General Brigham Biobank, a hospital-based cohort. We also examined variants associated with liver disease and assessed for gene-gene and gene-environment interactions. In UK Biobank, we identified 1,493 cases of cirrhosis, 12,603 Z allele heterozygotes, and 129 Z allele homozygotes among 312,671 unrelated white British participants. Heterozygous carriage of the Z allele was associated with cirrhosis compared to noncarriage (odds ratio [OR], 1.53; P = 1.1×10-04); homozygosity of the Z allele also increased the risk of cirrhosis (OR, 11.8; P = 1.8 × 10-09). The OR for cirrhosis of the Z allele was comparable to that of well-established genetic variants, including patatin-like phospholipase domain containing 3 (PNPLA3) I148M (OR, 1.48; P = 1.1 × 10-22) and transmembrane 6 superfamily member 2 (TM6SF2) E167K (OR, 1.34; P = 2.6 × 10-06). In heterozygotes compared to noncarriers, the Z allele was associated with higher alanine aminotransferase (ALT; P = = 4.6 × 10-46), aspartate aminotransferase (AST; P = 2.2 × 10-27), alkaline phosphatase (P = 3.3 × 10-43), gamma-glutamyltransferase (P = 1.2 × 10-05), and total bilirubin (P = 6.4 × 10-06); Z allele homozygotes had even greater elevations in liver biochemistries. Body mass index (BMI) amplified the association of the Z allele for ALT (P interaction = 0.021) and AST (P interaction = 0.0040), suggesting a gene-environment interaction. Finally, we demonstrated genetic interactions between variants in PNPLA3, TM6SF2, and hydroxysteroid 17-beta dehydrogenase 13 (HSD17B13); there was no evidence of epistasis between the Z allele and these variants. Conclusion: SERPINA1 Z allele heterozygosity is an important risk factor for liver disease; this risk is amplified by increasing BMI.

Genetic Variation in the Mitochondrial Glycerol-3-Phosphate Acyltransferase Is Associated With Liver Injury.

BACKGROUND AND AIMS: Most of the genetic basis of chronic liver disease remains undiscovered. APPROACH AND RESULTS: To identify genetic loci that modulate the risk of liver injury, we performed genome-wide association studies on circulating levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), and total bilirubin across 312,671 White British participants in the UK Biobank. We focused on variants associated with elevations in all four liver biochemistries at genome-wide significance (P < 5 × 10-8 ) and that replicated using Mass General Brigham Biobank in 19,323 European ancestry individuals. We identified a genetic locus in mitochondrial glycerol-3-phosphate acyltransferase (GPAM rs10787429) associated with increased levels of ALT (P = 1.4 × 10-30 ), AST (P = 3.6 × 10-10 ), ALP (P = 9.5 × 10-30 ), and total bilirubin (P = 2.9 × 10-12 ). This common genetic variant was also associated with an allele dose-dependent risk of alcohol-associated liver disease (odd ratio [OR] = 1.34, P = 2.6 × 10-5 ) and fatty liver disease (OR = 1.18, P = 5.8 × 10-4 ) by International Classification of Diseases, 10th Revision codes. We identified significant interactions between GPAM rs10787429 and elevated body mass index in association with ALT and AST (P = 7.1 × 10-9 and 3.95 × 10-8 , respectively), as well as between GPAM rs10787429 and weekly alcohol consumption in association with ALT, AST, and alcohol-associated liver disease (P = 4.0 × 10-2 , 1.6 × 10-2 , and 1.3 × 10-2 , respectively). Unlike previously described genetic variants that are associated with an increased risk of liver injury but confer a protective effect on circulating lipids, GPAM rs10787429 was associated with an increase in total cholesterol (P = 2.0 × 10-17 ), LDL cholesterol (P = 2.0 × 10-10 ), and HDL cholesterol (P = 6.6 × 10-37 ). Single-cell RNA-sequencing data demonstrated hepatocyte-predominant expression of GPAM in cells that co-express genes related to VLDL production (P = 9.4 × 10-103 ). CONCLUSIONS: Genetic variation in GPAM is associated with susceptibility to liver injury. GPAM may represent a therapeutic target in chronic liver disease.

A Dynamic Aspartate-to-Alanine Aminotransferase Ratio Provides Valid Predictions of Incident Severe Liver Disease.

The aspartate-to-alanine aminotransferase ratio (AAR) is associated with liver fibrosis, but its predictive performance is suboptimal. We hypothesized that the association between AAR and liver disease depends on absolute transaminase levels and developed and validated a model to predict liver-related outcomes in the general population. A Cox regression model based on age, AAR, and alanine aminotransferase (ALT) level (dynamic AAR [dAAR]) using restricted cubic splines was developed in Finnish population-based health-examination surveys (FINRISK, 2002-2012; n = 18,067) with linked registry data for incident liver-related hospitalizations, hepatocellular carcinoma, or liver death. The model was externally validated for liver-related outcomes in a Swedish population cohort (Swedish Apolipoprotein Mortality Risk [AMORIS] subcohort; n = 126,941) and for predicting outcomes and/or prevalent fibrosis/cirrhosis in biopsied patients with nonalcoholic fatty liver disease (NAFLD), chronic hepatitis C, or alcohol-related liver disease (ALD). The dynamic AAR model predicted liver-related outcomes both overall (optimism-corrected C-statistic, 0.81) and in subgroup analyses of the FINRISK cohort and identified persons with >10% risk for liver-related outcomes within 10 years. In independent cohorts, the C-statistic for predicting liver-related outcomes up to a 10-year follow-up was 0.72 in the AMORIS cohort, 0.81 in NAFLD, and 0.75 in ALD. Area-under-the-curve (AUC) for detecting prevalent cirrhosis was 0.80-0.83 in NAFLD, 0.80 in hepatitis C, but only 0.71 in ALD. In ALD, model performance improved when using aspartate aminotransferase instead of ALT in the model (C-statistic, 0.84 for outcome; AUC, 0.82 for prevalent cirrhosis). Conclusion: A dAAR score provides prospective predictions for the risk of incident severe liver outcomes in the general population and helps detect advanced liver fibrosis/cirrhosis. The dAAR score could potentially be used for screening the unselected general population and as a trigger for further liver evaluations.

Global deletion of NTPDase3 protects against diet-induced obesity by increasing basal energy metabolism.

BACKGROUND: Ecto-nucleoside triphosphate diphosphohydrolase 3 (NTPDase3), also known as CD39L3, is the dominant ectonucleotidase expressed by beta cells in the islet of Langerhans and on nerves. NTPDase3 catalyzes the conversion of extracellular ATP and ADP to AMP and modulates purinergic signaling. Previous studies have shown that NTPDase3 decreases insulin release from beta-cells in vitro. This study aims to determine the impact of NTPDase3 in diet-induced obesity (DIO) and metabolism in vivo. METHODS: We developed global NTPDase3 deficient (Entpd3-/-) and islet beta-cell-specific NTPDase-3 deficient mice (Entpd3flox/flox,InsCre) using Ins1-Cre targeted gene editing to compare metabolic phenotypes with wildtype (WT) mice on a high-fat diet (HFD). RESULTS: Entpd3-/- mice exhibited similar growth rates compared to WT on chow diet. When fed HFD, Entpd3-/- mice demonstrated significant resistance to DIO. Entpd3-/- mice consumed more calories daily and exhibited less fecal calorie loss. Although Entpd3-/- mice had no increases in locomotor activity, the mice exhibited a significant increase in basal metabolic rate when on the HFD. This beneficial phenotype was associated with improved glucose tolerance, but not higher insulin secretion. In fact, Entpd3flox/flox,InsCre mice demonstrated similar metabolic phenotypes and insulin secretion compared to matched controls, suggesting that the expression of NTPDase3 in beta-cells was not the primary protective factor. Instead, we observed a higher expression of uncoupling protein 1 (UCP-1) in brown adipose tissue and an augmented browning in inguinal white adipose tissue with upregulation of UCP-1 and related genes involved in thermogenesis in Entpd3-/- mice. CONCLUSIONS: Global NTPDase3 deletion in mice is associated with resistance to DIO and obesity-associated glucose intolerance. This outcome is not driven by the expression of NTPDase3 in pancreatic beta-cells, but rather likely mediated through metabolic changes in adipocytes.

Characterization of LP-Z Lipoprotein Particles and Quantification in Subjects with Liver Disease Using a Newly Developed NMR-Based Assay.

BACKGROUND: Lipoprotein particles with abnormal compositions, such as lipoprotein X (LP-X) and lipoprotein Z (LP-Z), have been described in cases of obstructive jaundice and cholestasis. The study objectives were to: (1) develop an NMR-based assay for quantification of plasma/serum LP-Z particles, (2) evaluate the assay performance, (3) isolate LP-Z particles and characterize them by lipidomic and proteomic analysis, and (4) quantify LP-Z in subjects with various liver diseases. METHODS: Assay performance was assessed for linearity, sensitivity, and precision. Mass spectroscopy was used to characterize the protein and lipid content of isolated LP-Z particles. RESULTS: The assay showed good linearity and precision (2.5-6.3%). Lipid analyses revealed that LP-Z particles exhibit lower cholesteryl esters and higher free cholesterol, bile acids, acylcarnitines, diacylglycerides, dihexosylceramides, lysophosphatidylcholines, phosphatidylcholines, triacylglycerides, and fatty acids than low-density lipoprotein (LDL) particles. A proteomic analysis revealed that LP-Z have one copy of apolipoprotein B per particle such as LDL, but less apolipoprotein (apo)A-I, apoC3, apoA-IV and apoC2 and more complement C3. LP-Z were not detected in healthy volunteers or subjects with primary biliary cholangitis, primary sclerosing cholangitis, autoimmune hepatitis, or type 2 diabetes. LP-Z were detected in some, but not all, subjects with hypertriglyceridemia, and were high in some subjects with alcoholic liver disease. CONCLUSIONS: LP-Z differ significantly in their lipid and protein content from LDL. Further studies are needed to fully understand the pathophysiological reason for the enhanced presence of LP-Z particles in patients with cholestasis and alcoholic liver disease.

Differential Associations of Circulating MicroRNAs With Pathogenic Factors in NAFLD.

Nonalcoholic fatty liver disease (NAFLD) is a heterogeneous disease driven by genetic and environmental factors. MicroRNAs (miRNAs) serve as pleiotropic post-transcriptional regulators of cellular pathways. Although several miRNAs have been associated with NAFLD and fibrosis, there are limited studies in humans examining their differential association with pathogenic factors or histological features of NAFLD. We examined the differential relationships of five of the best-described circulating microRNAs (miR-34a, miR-122, miR-191, miR-192, and miR-200a) with histological features and pathogenic factors of NAFLD. A cross-sectional study was conducted to examine the relationship between relative levels of circulating microRNAs standardized by z-scores and histological features of NAFLD, common NAFLD genetic polymorphisms, and insulin resistance measured by the enhanced lipoprotein insulin resistance index in 132 subjects with biopsy-proven NAFLD. We found that miR-34a, miR-122, miR-192, miR-200a, but not miR-191, strongly correlate with fibrosis in NAFLD by increases of 0.20 to 0.40 SD (P < 0.005) with each stage of fibrosis. In multivariate analysis, miR-34a, miR-122, and miR-192 levels are independently associated with hepatic steatosis and fibrosis, but not lobular inflammation or ballooning degeneration, whereas miR-200a is only associated with fibrosis. Among the four miRNAs, miR-34a, miR-122, and miR-192 are associated with pathogenic factors of NAFLD, including insulin resistance measured by eLP-IR, patatin-like phospholipase domain containing 3 I148M, and transmembrane 6 superfamily 2 (TM6SF2) E167K polymorphisms. In contrast, miR-200a is only associated with the TM6SF2 E167K variant. Finally, miR-34a has the strongest predictive value for various stages of fibrosis, with C-statistic approximates-combined predictive score for miRNAs. Conclusion: miR-34a, miR-122, miR-192, and miR-200a demonstrate strong associations with NAFLD severity by histology, but differential associations with pathogenic factors.

Provider Attitudes and Practice Patterns for Direct-Acting Antiviral Therapy for Patients With Hepatocellular Carcinoma.

BACKGROUND & AIMS: Direct-acting antivirals (DAAs) are effective against hepatitis C virus and sustained virologic response is associated with reduced incidence of hepatocellular carcinoma (HCC). However, there is controversy over the use of DAAs in patients with active or treated HCC and uncertainty about optimal management of these patients. We aimed to characterize attitudes and practice patterns of hepatology practitioners in the United States regarding the use of DAAs in patients with HCC. METHODS: We conducted a survey of hepatology providers at 47 tertiary care centers in 25 states. Surveys were sent to 476 providers and we received 279 responses (58.6%). RESULTS: Provider beliefs about risk of HCC recurrence after DAA therapy varied: 48% responded that DAAs reduce risk, 36% responded that DAAs do not change risk, and 16% responded that DAAs increase risk of HCC recurrence. However, most providers believed DAAs to be beneficial to and reduce mortality of patients with complete response to HCC treatment. Accordingly, nearly all providers (94.9%) reported recommending DAA therapy to patients with early-stage HCC who received curative treatment. However, fewer providers recommended DAA therapy for patients with intermediate (72.9%) or advanced (57.5%) HCC undergoing palliative therapies. Timing of DAA initiation varied among providers based on HCC treatment modality: 49.1% of providers reported they would initiate DAA therapy within 3 months of surgical resection whereas 45.9% and 5.0% would delay DAA initiation for 3-12 months and >1 year post-surgery, respectively. For patients undergoing transarterial chemoembolization (TACE), 42.0% of providers would provide DAAs within 3 months of the procedure, 46.7% would delay DAAs until 3-12 months afterward, and 11.3% would delay DAAs more than 1 year after TACE. CONCLUSIONS: Based on a survey sent to hepatology providers, there is variation in provider attitudes and practice patterns regarding use and timing of DAAs for patients with HCC. Further studies are needed to characterize the risks and benefits of DAA therapy in this patient population.