Outcomes of Various Classes of Oral Antidiabetic Drugs on Nonalcoholic Fatty Liver Disease.

Importance: Several oral antidiabetic drug (OAD) classes can potentially improve patient outcomes in nonalcoholic fatty liver disease (NAFLD) to varying degrees, but clinical data on which class is favored are lacking. Objective: To investigate which OAD is associated with the best patient outcomes in NAFLD and type 2 diabetes (T2D). Design, Setting, and Participants: This retrospective nonrandomized interventional cohort study used the National Health Information Database, which provided population-level data for Korea. This study involved patients with T2D and concomitant NAFLD. Exposures: Receiving either sodium-glucose cotransporter 2 (SGLT2) inhibitors, thiazolidinediones, dipeptidyl peptidase-4 (DPP-4) inhibitors, or sulfonylureas, each combined with metformin for 80% or more of 90 consecutive days. Main Outcomes and Measures: The main outcomes were NAFLD regression assessed by the fatty liver index and composite liver-related outcome (defined as liver-related hospitalization, liver-related mortality, liver transplant, and hepatocellular carcinoma) using the Fine-Gray model regarding competing risks. Results: In total, 80 178 patients (mean [SD] age, 58.5 [11.9] years; 43 007 [53.6%] male) were followed up for 219 941 person-years, with 4102 patients experiencing NAFLD regression. When compared with sulfonylureas, SGLT2 inhibitors (adjusted subdistribution hazard ratio [ASHR], 1.99 [95% CI, 1.75-2.27]), thiazolidinediones (ASHR, 1.70 [95% CI, 1.41-2.05]), and DPP-4 inhibitors (ASHR, 1.45 [95% CI, 1.31-1.59]) were associated with NAFLD regression. SGLT2 inhibitors were associated with a higher likelihood of NAFLD regression when compared with thiazolidinediones (ASHR, 1.40 [95% CI, 1.12-1.75]) and DPP-4 inhibitors (ASHR, 1.45 [95% CI, 1.30-1.62]). Only SGLT2 inhibitors (ASHR, 0.37 [95% CI, 0.17-0.82]), not thiazolidinediones or DPP-4 inhibitors, were significantly associated with lower incidence rates of adverse liver-related outcomes when compared with sulfonylureas. Conclusions and Relevance: The results of this cohort study suggest that physicians may lean towards prescribing SGLT2 inhibitors as the preferred OAD for individuals with NAFLD and T2D, considering their potential benefits in NAFLD regression and lower incidences of adverse liver-related outcomes. This observational study should prompt future research to determine whether prescribing practices might merit reexamination.

Low-quality muscle mass rather than normal-quality muscle mass determines fibrosis progression in biopsy-proven NAFLD.

BACKGROUND AND AIMS: Sarcopaenia is associated with advanced nonalcoholic fatty liver disease (NAFLD). However, the impact of the muscle mass categorised by muscle quality on fibrosis progression remains unclear. METHODS: A total of 292 patients with biopsy-proven NAFLD who underwent serial vibration-controlled transient elastography assessments at least 1 year from baseline were selected. The skeletal muscle area (SMA) was determined on abdominal computed tomography (CT) at the third lumbar vertebra level and categorised to normal-attenuation muscle area (NAMA), low-attenuation muscle area (LAMA) and intermuscular adipose tissue (IMAT) using a muscle quality map. These SMAs were normalised by the height squared to obtain the skeletal muscle index (SMI). RESULTS: At baseline, as the histological fibrosis stage increased, SMINAMA decreased and SMILAMA increased (p for trend = 0.014 and p for trend <0.001, respectively), which was not significant after adjustment for age, sex and obesity. During a median follow-up of 41 months, fibrosis progression was detected in 48 out of 292 patients, and higher SMILAMA quartiles independently increased the risk of fibrosis progression in a dose-dependent manner (hazard ratio [HR] per quartile: 1.41; 95% confidence interval [CI], 1.04-1.91). The highest quartile of SMILAMA increased the risk of fibrosis progression by 3.25 times compared to the lowest quartile of SMILAMA (95% CI, 1.18-8.90). SMINAMA quartiles were not associated with the risk of fibrosis progression. CONCLUSION: Increased low-quality muscle mass, but not decreased normal-quality muscle mass, as assessed by a muscle quality map in CT, predicts fibrosis progression in patients with NAFLD.

PNPLA3 genotypes modify the adverse effect of the total energy intake on high-risk nonalcoholic steatohepatitis development.

BACKGROUND: The relationship between diet and risk genotypes in nonalcoholic steatohepatitis (NASH) development and fibrosis progression in patients with nonalcoholic fatty liver disease (NAFLD) remains unclear. OBJECTIVE: We aimed to investigate the effects of diet on NASH development and fibrosis progression in patients with NAFLD stratified by the PNPLA3 genotype. METHODS: We performed a prospective study in a cohort of patients with biopsy-confirmed NAFLD. Histologic deterioration was obtained using serial transient elastography at every 1 or 2 y. The primary outcome was fibrosis progression, and the secondary outcome was development of high-risk NASH, defined as FibroScan-aspartate aminotransferase score ≥0.67 during the follow-up of patients with nonalcoholic fatty liver at the baseline. Dietary intake was evaluated using a semiquantitative food frequency questionnaire. RESULTS: The primary outcome was observed in 42 (29.0%) of the 145 patients during a median follow-up of 49 mo; neither the total energy intake nor each macronutrient intake significantly affected the primary outcome occurrence. Conversely, the total energy intake (HR per 1-SD: 3.03; 95% CI: 1.31, 7.01) and the PNPLA3 rs738409 genotype [HR per 1 risk allele (G): 2.06; 95% CI: 1.11, 3.83)] were independent risk factors for high-risk NASH. The significant interaction between the total energy intake and PNPLA3 genotype was noted in developing high-risk NASH (P = 0.044). As the number of PNPLA3 risk alleles decreased, the effect of the total energy intake on high-risk NASH increased; the HR per 1-SD increment in total energy intake was 1.52 (95% CI: 0.42, 5.42), 3.54 (95% CI: 1.23, 10.18), and 8.27 (95% CI: 1.20, 57.23) for the GG, CG, and CC genotypes, respectively. CONCLUSIONS: The total energy intake adversely affected the development of high-risk NASH in patients with biopsy-confirmed NAFLD. The effect was more prominent in patients without the PNPLA3 risk allele, highlighting the importance of personalized dietary interventions in NAFLD treatment.

Metabolic dysfunction-associated fatty liver disease as a risk factor for adverse outcomes in subsequent pregnancy: a nationwide cohort study.

BACKGROUND AND PURPOSE: A recent international expert consensus opinion suggested that metabolic dysfunction-associated fatty liver disease (MAFLD) replaces nonalcoholic fatty liver disease (NAFLD), since MAFLD is a better predictor of cardiovascular disease. We estimated the prevalence of FLD in fertile females and evaluated the clinical impact of either NAFLD or MAFLD on maternal and fetal outcomes during subsequent pregnancy. METHODS: The study population included fertile females who underwent health examinations and became pregnant within 1 year of health examination. Hepatic steatosis was defined as a fatty liver index of ≥ 30. The fertile females were divided into four groups: neither-FLD, NAFLD-only, MAFLD-only, and both-FLDs. During subsequent pregnancy, the risks of adverse pregnancy outcomes, including gestational diabetes, pregnancy-associated hypertension, preterm birth, and low birthweight, were compared among the four groups. RESULTS: The study population comprised 762,401 females, including 720,606 with neither-FLD, 318 with NAFLD-only, 14,371 with MAFLD-only, and 27,106 with both-FLDs. Compared to females with neither-FLD, the risk of adverse pregnancy outcomes was higher in females with any FLD, with an adjusted OR of 1.73 (95% CI 1.25-2.41) in the NALFD-only group, 2.65 (2.53-2.77) in the MAFLD-only group, and 2.39 (2.31-2.48) in the both-FLDs group. Pregnancy outcomes (cesarean delivery, gestational diabetes, and low birthweight) were worse in females with MAFLD compared with NAFLD. CONCLUSION: Any form of FLD is a risk factor for adverse pregnancy outcomes. These data suggest that MAFLD is associated with a higher risk of adverse pregnancy outcomes for both mother and fetus than NAFLD.

Interaction between sarcopenia and nonalcoholic fatty liver disease.

Sarcopenia and nonalcoholic fatty liver disease (NAFLD) are common health problems related to aging. Despite the differences in their diagnostic methods, several cross-sectional and longitudinal studies have revealed the close link between sarcopenia and NAFLD. Sarcopenia and NAFLD are linked by several shared pathogenetic mechanisms, including insulin resistance, hormonal imbalance, systemic inflammation, myostatin and adiponectin dysregulation, nutritional deficiencies, and physical inactivity, thus implicating a bidirectional relationship between sarcopenia and NAFLD. However, there is not sufficient data to support a direct causal relationship between sarcopenia and NAFLD. Moreover, it is currently difficult to conclude whether sarcopenia is a risk factor for nonalcoholic steatohepatitis (NASH) or is a consequence of NASH. Therefore, this review intends to touch on the shared common mechanisms and the bidirectional relationship between sarcopenia and NAFLD.

Myosteatosis, but not Sarcopenia, Predisposes NAFLD Subjects to Early Steatohepatitis and Fibrosis Progression.

BACKGROUND & AIMS: Sarcopenia and myosteatosis are associated with advanced nonalcoholic fatty liver disease (NAFLD). However, muscle alterations in early stage NAFLD remain unclear. METHODS: Patients with nonalcoholic fatty liver (NAFL) or early nonalcoholic steatohepatitis (NASH) without significant fibrosis were selected from a prospective biopsy-proven NAFLD cohort (N = 338). The skeletal muscle index and mean muscle attenuation (MA) were measured using abdominal fat computed tomography at the third lumbar vertebra level. Severe myosteatosis was defined as the lowest quartile of sex-stratified MA values. RESULTS: Patients with early NASH (n = 87) had lower MA (45.61 ± 6.45 vs 47.48 ± 5.85 HU; P = .028) than patients with NAFL (n = 251) but a similar skeletal muscle index. Patients with more severe lobular inflammation and hepatocellular ballooning had lower MA (P = .003 and P = .041, respectively). The severe myosteatosis prevalence was higher in early NASH than in NAFL (33.3% vs 21.1%; P = .029). Patients with severe myosteatosis were more likely to have early NASH in multivariable analysis adjusted for age, sex, and metabolic factors (odds ratio, 2.45; 95% confidence interval (CI), 1.24-4.86), which was maintained after adjustment for visceral fat amount (odds ratio, 2.44; 95% CI, 1.22-4.89). During a median 29-month follow-up, 170 patients underwent repeated transient elastography. Fibrosis progression-an increase in liver stiffness measurement >2 kPa or second liver stiffness measurement ≥7 kPa-was found in 28 and 31 individuals. Severe myosteatosis was significantly associated with fibrosis progression after adjustment for various confounders (hazard ratio, 2.49; 95% CI, 1.15-5.40 and hazard ratio, 2.09; 95% CI, 1.01-4.34 for different fibrosis progression definitions). CONCLUSIONS: Severe myosteatosis is significantly associated with early NASH and fibrosis progression in early stage NAFLD.

Development of early prediction model for pregnancy-associated hypertension with graph-based semi-supervised learning.

Clinical guidelines recommend several risk factors to identify women in early pregnancy at high risk of developing pregnancy-associated hypertension. However, these variables result in low predictive accuracy. Here, we developed a prediction model for pregnancy-associated hypertension using graph-based semi-supervised learning. This is a secondary analysis of a prospective study of healthy pregnant women. To develop the prediction model, we compared the prediction performances across five machine learning methods (semi-supervised learning with both labeled and unlabeled data, semi-supervised learning with labeled data only, logistic regression, support vector machine, and random forest) using three different variable sets: [a] variables from clinical guidelines, [b] selected important variables from the feature selection, and [c] all routine variables. Additionally, the proposed prediction model was compared with placental growth factor, a predictive biomarker for pregnancy-associated hypertension. The study population consisted of 1404 women, including 1347 women with complete follow-up (labeled data) and 57 women with incomplete follow-up (unlabeled data). Among the 1347 with complete follow-up, 2.4% (33/1347) developed pregnancy-associated HTN. Graph-based semi-supervised learning using top 11 variables achieved the best average prediction performance (mean area under the curve (AUC) of 0.89 in training set and 0.81 in test set), with higher sensitivity (72.7% vs 45.5% in test set) and similar specificity (80.0% vs 80.5% in test set) compared to risk factors from clinical guidelines. In addition, our proposed model with graph-based SSL had a higher performance than that of placental growth factor for total study population (AUC, 0.71 vs. 0.80, p < 0.001). In conclusion, we could accurately predict the development pregnancy-associated hypertension in early pregnancy through the use of routine clinical variables with the help of graph-based SSL.

Interaction effect between NAFLD severity and high carbohydrate diet on gut microbiome alteration and hepatic de novo lipogenesis.

Nonalcoholic fatty liver disease (NAFLD) is associated with high carbohydrate (HC) intake. We investigated whether the relationship between carbohydrate intake and NAFLD is mediated by interactions between gut microbial modulation, impaired insulin response, and hepatic de novo lipogenesis (DNL). Stool samples were collected from 204 Korean subjects with biopsy-proven NAFLD (n = 129) and without NAFLD (n = 75). The gut microbiome profiles were analyzed using 16S rRNA amplicon sequencing. Study subjects were grouped by the NAFLD activity score (NAS) and percentage energy intake from dietary carbohydrate. Hepatic DNL-related transcripts were also analyzed (n = 90). Data from the Korean healthy twin cohort (n = 682), a large sample of individuals without NAFLD, were used for comparison and validation. A HC diet rather than a low carbohydrate diet was associated with the altered gut microbiome diversity according to the NAS. Unlike individuals from the twin cohort without NAFLD, the abundances of Enterobacteriaceae and Ruminococcaceae were significantly different among the NAS subgroups in NAFLD subjects who consumed an HC diet. The addition of these two microbial families, along with Veillonellaceae, significantly improved the diagnostic performance of the predictive model, which was based on the body mass index, age, and sex to predict nonalcoholic steatohepatitis in the HC group. In the HC group, two crucial regulators of DNL (SIRT1 and SREBF2) were differentially expressed among the NAS subgroups. In particular, kernel causality analysis revealed a causal effect of the abundance of Enterobacteriaceae on SREBF2 upregulation and of the surrogate markers of insulin resistance on NAFLD activity in the HC group. Consuming an HC diet is associated with alteration in the gut microbiome, impaired glucose homeostasis, and upregulation of hepatic DNL genes, altogether contributing to NAFLD pathogenesis.

Efficacy and Safety of Pregabalin for Muscle Cramps in Liver Cirrhosis: A Double-Blind Randomized Controlled Trial.

BACKGROUND: Muscle cramp is possibly related to peripheral nerve hyperexcitability (PNH), and one of the most debilitating symptoms frequently encountered in patients with liver cirrhosis. We investigated whether pregabalin, a gamma-aminobutyric acid analogue, can suppress neuronal excitability and reduce muscle cramps in cirrhotic patients. METHODS: We conducted a randomized, double-blind, placebo-controlled trial in which study participants with cirrhosis from a single tertiary center were enrolled. Primary endpoint was the relative change in cramp frequency from the run-in to standard dose treatment phase (4 weeks per each). Secondary endpoints included the responder rate, and the changes in cramp frequency during sleep, pain intensity, health-related quality of life (Liver Disease Quality of Life Instrument, Short Form-36) and electrophysiological measures of PNH. RESULTS: This study was terminated early because of insufficient accrual. 80% (n = 56) of the target number of participants (n = 70) were randomized to pregabalin (n = 29) or placebo (n = 27). Median baseline frequency of muscle cramps (interquartile range) was 5.8 (3.5-10) per week in the pregabalin group and 6.5 (4.0-10) in the placebo group (P = 0.970). The primary analysis showed a significant reduction in cramp frequency with pregabalin compared to placebo (-36% vs. 4.5% for the percentage change, P = 0.010). Secondary outcomes did not differ significantly between the two groups. Adverse effects with pregabalin were mainly dizziness and lethargy. CONCLUSION: With multiple problems emerging from premature termination in mind, the results suggested an acceptable safety profile and favorable effect of pregabalin in reducing muscle cramps compared to placebo in cirrhotic patients. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT01271660.

A PNPLA3 Polymorphism Confers Lower Susceptibility to Incident Diabetes Mellitus in Subjects With Nonalcoholic Fatty Liver Disease.

BACKGROUND AND AIMS: We investigated the association between the patatin-like phospholipase domain-containing-3 (PNPLA3) rs738409 genotype and the risk of diabetes mellitus (DM) using a biopsy-confirmed nonalcoholic fatty liver disease (NAFLD) cohort and a longitudinal observational cohort. METHODS: Associations between genotypes and the prevalence of DM were evaluated with stratification according to the histological severity of NAFLD in the Boramae cohort (n = 706). A longitudinal cohort consisting of nondiabetic individuals with ≥2 health checkups was then selected to investigate the risk of incident DM according to the genotype (the GENIE cohort; n = 4998). RESULTS: Among subjects with NAFLD in the Boramae cohort, the G allele was independently associated with a lower prevalence of DM in both NAFL (odds ratio [OR] per 1 allele, 0.66; 95% confidence interval [CI], 0.46-0.97) and nonalcoholic steatohepatitis (OR per 1 allele, 0.59; 95% CI, 0.38-0.92). This result was replicated in the longitudinal GENIE cohort. The G allele was associated with a lower risk of incident DM during the median follow-up of 60 months in subjects with NAFLD (hazard ratio, 0.65; 95% CI, 0.45-0.93). In contrast, G allele carriers without NAFLD showed higher odds for DM in the context of the Boramae cohort (OR, 2.44; 95% CI, 1.00-5.95). CONCLUSIONS: These findings clarify conflicting results regarding the association between the PNPLA3 rs738409 genotype and the risk of DM, demonstrating a clear difference between subjects with and without NAFLD; this difference might be explained by the low metabolic risk in genetic NAFLD.

Nonalcoholic fatty liver disease and early prediction of gestational diabetes mellitus using machine learning methods.

BACKGROUND/AIMS: To develop an early prediction model for gestational diabetes mellitus (GDM) using machine learning and to evaluate whether the inclusion of nonalcoholic fatty liver disease (NAFLD)-associated variables increases the performance of model. METHODS: This prospective cohort study evaluated pregnant women for NAFLD using ultrasound at 10-14 weeks and screened them for GDM at 24-28 weeks of gestation. The clinical variables before 14 weeks were used to develop prediction models for GDM (setting 1, conventional risk factors; setting 2, addition of new risk factors in recent guidelines; setting 3, addition of routine clinical variables; setting 4, addition of NALFD-associated variables, including the presence of NAFLD and laboratory results; and setting 5, top 11 variables identified from a stepwise variable selection method). The predictive models were constructed using machine learning methods, including logistic regression, random forest, support vector machine, and deep neural networks. RESULTS: Among 1,443 women, 86 (6.0%) were diagnosed with GDM. The highest performing prediction model among settings 1-4 was setting 4, which included both clinical and NAFLD-associated variables (area under the receiver operating characteristic curve [AUC] 0.563-0.697 in settings 1-3 vs. 0.740-0.781 in setting 4). Setting 5, with top 11 variables (which included NAFLD and hepatic steatosis index), showed similar predictive power to setting 4 (AUC 0.719-0.819 in setting 5, P=not significant between settings 4 and 5). CONCLUSION: We developed an early prediction model for GDM using machine learning. The inclusion of NAFLDassociated variables significantly improved the performance of GDM prediction. (ClinicalTrials.gov Identifier: NCT02276144).

Metabolic Dysfunction-Associated Fatty Liver Disease and Subsequent Development of Adverse Pregnancy Outcomes.

BACKGROUND & AIMS: Recently, metabolic dysfunction-associated fatty liver disease (MAFLD), rather than nonalcoholic fatty liver disease (NAFLD), was proposed to better describe liver disease associated with metabolic dysfunction (MD). In this study, we attempted to investigate the impact of MAFLD on pregnancy complications. METHODS: The current study is a secondary analysis of a multicenter prospective cohort designed to examine the risk of NAFLD during pregnancy. In the first trimester, enrolled pregnant women were evaluated for hepatic steatosis by liver ultrasonography, and blood samples were collected for biochemical measurements. The study population was divided into 3 groups: no NAFLD, hepatic steatosis but without metabolic dysfunction (non-MD NAFLD), and MAFLD. The primary outcome was the subsequent development of adverse pregnancy outcomes, including gestational diabetes mellitus, pregnancy-associated hypertension, preterm birth, and fetal growth abnormalities. RESULTS: The study population consisted of 1744 pregnant women, including 1523 with no NAFLD, 43 with non-MD NAFLD, and 178 with MAFLD. The risk of subsequent development of adverse pregnancy outcomes was higher in MAFLD than in non-MD NAFLD (adjusted odds ratio, 4.03; 95% CI, 1.68-9.67), whereas the risk was not significantly different between no NAFLD and non-MD NAFLD. Among women with no NAFLD, the presence of MD increased the risk of adverse pregnancy outcomes. However, women with MAFLD were at higher risk for adverse pregnancy outcomes than women with no NAFLD without MD or those with no NAFLD with MD. CONCLUSIONS: In pregnant women, MAFLD may be associated with an increased risk of subsequent adverse pregnancy outcomes.

Gut microbiome signatures distinguish type 2 diabetes mellitus from non-alcoholic fatty liver disease.

Non-alcoholic fatty liver disease (NAFLD) is closely associated with type 2 diabetes mellitus (T2D), and these two metabolic diseases demonstrate bidirectional influences. The identification of microbiome profiles that are specific to liver injury or impaired glucose metabolism may assist understanding of the role of the gut microbiota in the relationship between NAFLD and T2D. Here, we studied a biopsy-proven Asian NAFLD cohort (n = 329; 187 participants with NAFLD, 101 with NAFLD and T2D, and 41 with neither) and identified Enterobacter, Romboutsia, and Clostridium sensu stricto as the principal taxa associated with the severity of NAFLD and T2D, whereas Ruminococcus and Megamonas were specific to NAFLD. In particular, the taxa that were associated with both severe liver pathology and T2D were also significantly associated with markers of diabetes, such as fasting blood glucose and Hb1Ac. Enterotype analysis demonstrated that participants with NAFLD had a significantly higher proportion of Bacteroides and a lower proportion of Ruminococcus than a Korean healthy twin cohort (n = 756). However, T2D could not be clearly distinguished from NAFLD. Analysis of an independent T2D cohort (n = 185) permitted us to validate the T2D-specific bacterial signature identified in the NAFLD cohort. Functional inference analysis revealed that endotoxin biosynthesis pathways were significantly enriched in participants with NAFLD and T2D, compared with those with NAFLD alone. These findings may assist with the development of effective therapeutic approaches for metabolic diseases that are associated with specific bacterial signatures.

Maternal dyslipidemia and altered cholesterol metabolism in early pregnancy as a risk factor for small for gestational age neonates.

We evaluated the relationship between maternal cholesterol levels and its biologically active precursors and metabolites in the first trimester and subsequent risk for small-for-gestational-age birthweight (SGA). This is a secondary analysis of a prospective cohort study which enrolled healthy singleton pregnancies (n = 1337). Maternal fasting blood was taken in the first trimester and followed up till delivery. The lipid parameters were compared between women who delivered SGA neonates (SGA-group, birthweight < 10th percentile, n = 107) and women who did not (non-SGA-group, n = 1230). In addition, metabolic signatures of cholesterol were evaluated in a subset consisting of propensity-score matched SGA (n = 56) and control group (n = 56). Among lipid parameters, maternal high-density lipoprotein cholesterol (HDL-C) levels were significantly lower in SGA-group than in non-SGA-group (p = 0.022). The risk for SGA was negatively correlated with maternal serum HDL-C quartiles (p = 0.003), and this association remained significant after adjustment for confounding variables. In metabolic signatures of cholesterol, the cholesterol/lathosterol ratio in SGA-group was significantly higher than non-SGA-group [(2.7 (1.6-3.7) vs. 2.1 (1.5-2.9), respectively; p = 0.034)], suggesting increased endogenous cholesterol biosynthesis. We demonstrated that dyslipidemia and increased cholesterol biosynthesis led to delivery of SGA neonates even in early pregnancy.

Association between circulating bile acid alterations and nonalcoholic steatohepatitis independent of obesity and diabetes mellitus.

BACKGROUND AND AIMS: Bile acid (BA) dysregulation is related to not only metabolic diseases but also nonalcoholic fatty liver disease (NAFLD). We investigated whether circulating BA levels are altered according to the histological severity of NAFLD independent of metabolic derangements. METHODS: Global metabolic profiling and targeted BA analysis using sera collected from biopsy-proven no-NAFLD (n = 67), nonalcoholic fatty liver (NAFL) (n = 99), and nonalcoholic steatohepatitis (NASH, n = 75) subjects were performed sequentially. Circulating metabolome analysis integrated with the hepatic transcriptome was performed to elucidate the mechanistic basis of altered circulating BA profiles after stratification by obesity (body mass index ≤ 25 kg/m2 ). Circulating BA alterations were also validated in an independent validation cohort (29 no-NAFLD, 70 NAFL and 37 NASH). RESULTS: Global profiling analysis showed that BA was the metabolite significantly altered in NASH compared to NAFL. Targeted BA analysis demonstrated that glyco-/tauro-conjugated primary BAs were commonly increased in nonobese and obese NASH, while unconjugated primary BAs increased only in nonobese NASH. These characteristic primary BA level changes were maintained even after stratification according to diabetes status and were replicated in the independent validation cohort. Compared to nonobese NAFL patients, nonobese NASH patients exhibited upregulated hepatic expression of CYP8B1. CONCLUSIONS: BA metabolism is dysregulated as the histological severity of NAFLD worsens, independent of obesity and diabetes status; dysregulation is more prominent in nonobese NAFLD patients. Metabolome-driven omics approach provides new insight into our understanding of altered BA metabolism associated with individual phenotypes of NAFLD.

Disease-specific eQTL screening reveals an anti-fibrotic effect of AGXT2 in non-alcoholic fatty liver disease.

BACKGROUND & AIMS: Non-alcoholic fatty liver disease (NAFLD) poses an increasing clinical burden. Genome-wide association studies have revealed a limited contribution of genomic variants to the disease, requiring alternative but robust approaches to identify disease-associated variants and genes. We carried out a disease-specific expression quantitative trait loci (eQTL) screen to identify novel genetic factors that specifically act on NAFLD progression on the basis of genotype. METHODS: We recruited 125 Korean patients (83 with biopsy-proven NAFLD and 42 without NAFLD) and performed eQTL analyses using 21,272 transcripts and 3,234,941 genotyped and imputed single nucleotide polymorphisms. We then selected eQTLs that were detected only in the NAFLD group, but not in the control group (i.e., NAFLD-eQTLs). An additional cohort of 162 Korean individuals with NAFLD was used for replication. The function of the selected eQTL toward NAFLD development was validated using HepG2, primary hepatocytes and NAFLD mouse models. RESULTS: The NAFLD-specific eQTL screening yielded 242 loci. Among them, AGXT2, encoding alanine-glyoxylate aminotransferase 2, displayed decreased expression in patients with NAFLD homozygous for the non-reference allele of rs2291702, compared to no-NAFLD individuals with the same genotype (p = 4.79 × 10-6). This change was replicated in an additional 162 individuals, yielding a combined p value of 8.05 × 10-8 from a total of 245 patients with NAFLD and 42 controls. Knockdown of AGXT2 induced palmitate-overloaded hepatocyte death by increasing endoplasmic reticulum stress, and exacerbated NAFLD diet-induced liver fibrosis in mice, while overexpression of AGXT2 attenuated liver fibrosis and steatosis. CONCLUSIONS: We identified a new molecular role for AGXT2 in NAFLD. Our overall approach will serve as an efficient tool for uncovering novel genetic factors that contribute to liver steatosis and fibrosis in patients with NAFLD. LAY SUMMARY: Elucidating causal genes for non-alcoholic fatty liver disease (NAFLD) has been challenging due to limited tissue availability and the polygenic nature of the disease. Using liver and blood samples from 125 Korean individuals (83 with NAFLD and 42 without NAFLD), we devised a new analytic method to identify causal genes. Among the candidates, we found that AGXT2-rs2291702 protects against liver fibrosis in a genotype-dependent manner with the potential for therapeutic interventions. Our approach enables the discovery of causal genes that act on the basis of genotype.

Muscle alterations are independently associated with significant fibrosis in patients with nonalcoholic fatty liver disease.

BACKGROUND & AIM: Anthropometric data are associated with nonalcoholic fatty liver disease (NAFLD) development and progression. We investigated whether the quantity and quality of muscle and visceral fat assessed by computed tomography (CT) are associated with fibrosis severity in NAFLD. METHODS: In a prospective biopsy-confirmed NAFLD cohort of 521 patients, we measured skeletal muscle index (SMI), muscle attenuation (MA) and visceral adipose tissue index (VATI) via CT. Low skeletal muscle mass (LSMM) was defined using previously validated cut-offs. Myosteatosis and visceral adiposity were defined as the lowest and highest quartile, respectively. Significant fibrosis was defined as F2-F4 in liver histology. RESULTS: Patients with significant fibrosis had lower SMI and MA and higher VATI than those without. The significant fibrosis prevalence was significantly higher in subjects with LSMM (45.1% vs 30.8%, P = .005), myosteatosis (46.1% vs 29.7%, P = .001) and visceral adiposity (46.9% vs 29.9%, P = .001) than those without. The significant fibrosis risk increased with increasing numbers of body composition components (24.5%, 35.6%, 53.0% and 72.7% in patients with 0, 1, 2 and 3 components respectively). Multivariable analysis revealed that LSMM (OR, 1.72; 95% CI, 1.05-2.84), myosteatosis (OR, 1.65; 95% CI, 1.01-2.68) and visceral adiposity (OR, 1.75; 95% CI, 1.09-2.83) were independent predictors of significant fibrosis. Subjects with sarcopenia had a higher risk of significant fibrosis (OR, 2.17; 95% CI, 1.03-4.56). CONCLUSION: Muscle alterations and visceral adiposity assessed by CT are associated with significant fibrosis in NAFLD. LSMM and myosteatosis have additive values in prediction of significant fibrosis.

Association of arterial stiffness with the histological severity of nonalcoholic fatty liver disease.

BACKGROUND: Although arterial stiffness has been reported to be associated with nonalcoholic fatty liver disease (NAFLD), previous studies have relied only on noninvasive assessments for the diagnosis of NAFLD. This study attempted to investigate the association of arterial stiffness with the histological severity of NAFLD. METHODS: We analyzed data from a biopsy-proven prospective NAFLD cohort. Augmentation index corrected for a heart rate of 75 bpm (AI@75) was obtained using applanation tonometry of the radial artery. RESULTS: A total of 154 patients (mean age 55.2 years; females 55.8%) with biopsy-proven NAFLD were analyzed. Patients with higher AI@75 (≥ 76%) showed more severe grades of lobular and portal inflammation and hepatocellular ballooning, and more advanced stages of fibrosis compared to those with lower AI@75 (< 76%) (p < 0.05 for each). The presence of nonalcoholic steatohepatitis (NASH) (adjusted odds ratio [aOR] 2.48; 95% confidence interval [CI] 1.31-6.16; p = 0.008), lobular inflammation (aOR 2.03; 95% CI 1.09-3.78; p = 0.025) hepatocellular ballooning (aOR 2.82; 95% CI 1.23-6.43; p = 0.014), and significant fibrosis (≥ F2) (aOR 3.42; 95% CI 1.50-7.79; p = 0.003) were independently associated with higher AI@75 (≥ 76%) even after adjustment for confounders. CONCLUSION: Arterial stiffness as indicated by higher AI@75 was associated with more severe NAFLD histology. This adds to the evidence for the association between increased arterial stiffness and NAFLD.

Distinct signatures of gut microbiome and metabolites associated with significant fibrosis in non-obese NAFLD.

Nonalcoholic fatty liver disease (NAFLD) is associated with obesity but also found in non-obese individuals. Gut microbiome profiles of 171 Asians with biopsy-proven NAFLD and 31 non-NAFLD controls are analyzed using 16S rRNA sequencing; an independent Western cohort is used for external validation. Subjects are classified into three subgroups according to histological spectra of NAFLD or fibrosis severity. Significant alterations in microbiome diversity are observed according to fibrosis severity in non-obese, but not obese, subjects. Ruminococcaceae and Veillonellaceae are the main microbiota associated with fibrosis severity in non-obese subjects. Furthermore, stool bile acids and propionate are elevated, especially in non-obese subjects with significant fibrosis. Fibrosis-related Ruminococcaceae and Veillonellaceae species undergo metagenome sequencing, and four representative species are administered in three mouse NAFLD models to evaluate their effects on liver damage. This study provides the evidence for the role of the microbiome in the liver fibrosis pathogenesis, especially in non-obese subjects.

Circulating lipidomic alterations in obese and non-obese subjects with non-alcoholic fatty liver disease.

BACKGROUND: Non-alcoholic fatty liver disease (NAFLD) affects obese and non-obese individuals. However, mechanisms underlying non-obese non-alcoholic steatohepatitis (NASH) remain unclear. AIMS: To attempt to identify metabolic perturbations associated with non-obese and obese NAFLD using a lipidomics approach. METHODS: A cross-sectional analysis of 361 subjects with biopsy-proven NAFLD (157 NAFL and 138 NASH) and healthy controls (n = 66) was performed. Individuals were categorised as obese or non-obese based on the Asian cut-off for body mass index. Circulating lipidomic profiling of sera was performed based on the histological severity of NAFLD. Circulating lipidomic alterations were validated with an independent validation set (154 NAFLD subjects [93 NAFL and 61 NASH] and 21 healthy controls). RESULTS: Saturated sphingomyelin (SM) species were significantly associated with visceral adiposity in non-obese NAFLD (SM d38:0; P < 0.001) but not in obese NAFLD. Additionally, SM levels were significantly associated with systemic and adipose tissue insulin resistance (SM d38:0; P = 0.002 and <0.001, respectively). Five potential lipid metabolites for non-obese subjects and seven potential lipids for obese subjects were selected to predict NAFLD and NASH. These lipid combinations showed good diagnostic performance for non-obese (area under the curve [AUC] for NAFLD/NASH = 0.916/0.813) and obese (AUC for NAFLD/NASH = 0.967/0.812) subjects. Moreover, distinctly altered patterns of diacylglycerol (DAG), triacylglycerol (TAG) and SM levels were confirmed in the validation set depending on the histological severity of NAFLD. CONCLUSION: Non-obese and obese NAFLD subjects exhibit unique circulating lipidomic signatures, including DAGs, TAGs and SMs. These lipid combinations may be useful biomarkers for non-obese and obese NAFLD patients.