Non-alcoholic Fatty Liver Disease Phenotype Research Articles

Altered gut microbial profile accompanied by abnormal short chain fatty acid metabolism exacerbates nonalcoholic fatty liver disease progression

Dysregulation of the gut microbiome has associated with the occurrence and progression of non-alcoholic fatty liver disease (NAFLD). To determine the diagnostic capacity of this association, we compared fecal microbiomes across 104 participants including non-NAFLD controls and NAFLD subtypes patients that were distinguished by magnetic resonance imaging. We measured their blood biochemical parameters, 16 S rRNA-based gut microbiota and fecal short-chain fatty acids (SCFAs). Multi-omic analyses revealed that NAFLD patients exhibited specific changes in gut microbiota and fecal SCFAs as compared to non-NAFLD subjects. Four bacterial genera (Faecalibacterium, Subdoligranulum, Haemophilus, and Roseburia) and two fecal SCFAs profiles (acetic acid, and butyric acid) were closely related to NAFLD phenotypes and could accurately distinguish NAFLD patients from healthy non-NAFLD subjects. Twelve genera belonging to Faecalibacterium, Subdoligranulum, Haemophilus, Intestinibacter, Agathobacter, Lachnospiraceae_UCG-004, Roseburia, Butyricicoccus, Actinomycetales_unclassified, [Eubacterium]_ventriosum_group, Rothia, and Rhodococcus were effective to distinguish NAFLD subtypes. Of them, combination of five genera can distinguish effectively mild NAFLD from non-NAFLD with an area under curve (AUC) of 0.84. Seven genera distinguish moderate NAFLD with an AUC of 0.83. Eight genera distinguish severe NAFLD with an AUC of 0.90. In our study, butyric acid distinguished mild-NAFLD from non-NAFLD with AUC value of 0.83. And acetic acid distinguished moderate-NAFLD and severe-NAFLD from non-NAFLD with AUC value of 0.84 and 0.70. In summary, our study and further analysis showed that gut microbiota and fecal SCFAs maybe a method with convenient detection advantages and invasive manner that are not only a good prediction model for early warning of NAFLD occurrence, but also have a strong ability to distinguish NAFLD subtypes.

Causal effects of female reproductive features on nonalcoholic fatty liver disease: A mendelian randomization study.

Epidemiological evidence on the associations between female reproductive features and nonalcoholic fatty liver disease (NAFLD) is conflicting. To explore their causalities, we conducted a Mendelian randomization (MR) study. Summary-level data were obtained, and univariable MR was performed to explore the causalities between female reproductive features and NAFLD. And we performed multivariable MR and MR mediation analysis to explore the mediation effects of educational attainment (EA) and body mass index (BMI) for these associations. Sensitivity analyses were performed to evaluate pleiotropy and heterogeneity. There were causal effects of age at menarche (AAMA) (odds ratio [OR]: 0.817, 95% confidence interval [CI]: 0.736-0.907, per year-increase), age at first birth (AFB) (OR: 0.851, 95%CI: 0.791-0.926, per year-increase) and age at first sexual intercourse (AFS) (OR: 0.676, 95%CI: 0.511-0.896, per standard deviation-increase) on NAFLD risk. Besides, the causal effects were also observed on NAFLD phenotypes including liver fat content (LFC) and alanine aminotransferase (ALT). Further mediation analysis showed that BMI mediated partial proportion of effects of AAMA and AFS on NAFLD/ALT, AFB on NAFLD/LFC/ALT, while EA mediated partial proportion of effects of AFB on NAFLD/LFC/ALT, and AFS on NAFLD/ALT. This study provided convincing evidence that early AAMA, AFB, and AFS were risk factors for NAFLD. Reproductive health education, obesity management, and education spread might be the beneficial strategies for NAFLD prevention.

Serum Metabolomics Uncovers the Mechanisms of Inulin in Preventing Non-Alcoholic Fatty Liver Disease.

Inulin may be a promising therapeutic molecule for treating non-alcoholic fatty liver disease (NAFLD). However, the underlying mechanisms of its therapeutic activity remain unclear. To address this issue, a high-fat-diet-induced NAFLD mouse model was developed and treated with inulin. The NAFLD phenotype was evaluated via histopathological analysis and biochemical parameters, including serum levels of alanine aminotransferase, aspartate aminotransferase, liver triglycerides, etc. A serum metabolomics study was conducted using ultra-performance liquid chromatography coupled with tandem mass spectrometry. The results revealed that inulin mitigated NAFLD symptoms such as histopathological changes and liver cholesterol levels. Through the serum metabolomics study, 347 differential metabolites were identified between the model and control groups, and 139 differential metabolites were identified between the inulin and model groups. Additionally, 48 differential metabolites (such as phosphatidylserine, dihomo-γ-linolenic acid, L-carnitine, and 13-HODE) were identified as candidate targets of inulin and subjected to pathway enrichment analysis. The results revealed that these 48 differential metabolites were enriched in several metabolic pathways such as fatty acid biosynthesis and cardiolipin biosynthesis. Taken together, our results suggest that inulin might attenuate NAFLD partially by modulating 48 differential metabolites and their correlated metabolic pathways, constituting information that might help us find novel therapies for NAFLD.

Ketogenic diet time-dependently prevents NAFLD through upregulating the expression of antioxidant protein metallothionein-2

The past few decades have witnessed a rapid growth in the prevalence of nonalcoholic fatty liver disease (NAFLD). While the ketogenic diet (KD) is considered for managing NAFLD, the safety and efficacy of the KD on NAFLD has been a controversial topic. Here, we aimed to investigate the effect of KD of different durations on metabolic endpoints in mice with NAFLD and explore the underlying mechanisms. NAFLD mice were fed with KD for 1, 2, 4 and 6 weeks, respectively. The blood biochemical indexes (blood lipids, AST, ALT and etc.) and liver fat were measured. The LC-MS/MS based proteomic analysis was performed on liver tissues. Metallothionein-2 (MT2) was knocked down with adeno-associated virus (AAV) or small interfering RNA (siRNA) in NAFLD mice and AML-12cells, respectively. H&E, BODIPY and ROS staining were performed to examine lipid deposition and oxidative stress. Furthermore, MT2 protein levels, nucleus/cytoplasm distribution and DNA binding activity of peroxisome proliferators-activated receptors α (PPARα) were evaluated. KD feeding for 2 weeks showed the best improvement on NAFLD phenotype. Proteomic analysis revealed that MT2 was a key candidate for different metabolic endpoints of NAFLD affected by different durations of KD feeding. MT2 knockdown in NAFLD mice blocked the effects of 2 weeks of KD feeding on HFD-induced steatosis. In mouse primary hepatocytes and AML-12cells, MT2 protein levels were induced by β-hydroxybutyric acid (β-OHB). MT2 Knockdown blunted the effects of β-OHB on alleviating PA-induced lipid deposition. Mechanistically, 2 weeks of KD or β-OHB treatment reduced oxidative stress and upregulated the protein levels of MT2 in nucleus, which subsequently increased its DNA binding activity and PPARα protein expression. Collectively, these findings indicated that KD feeding prevented NAFLD in a time dependent manner and MT2 is a potential target contributing to KD improvement on steatosis.

Tea Polyphenol Epigallocatechin Gallate Protects Against Nonalcoholic Fatty Liver Disease and Associated Endotoxemia in Rats via Modulating Gut Microbiota Dysbiosis and Alleviating Intestinal Barrier Dysfunction and Related Inflammation.

Nonalcoholic fatty liver disease (NAFLD) is characterized by fat accumulation and inflammation. Epigallocatechin gallate (EGCG) has been proven to be effective against NAFLD, but its hepatoprotective mechanisms based on the "gut microbiota-barrier-liver axis" are still not fully understood. Herein, the results demonstrated that EGCG effectively ameliorated NAFLD phenotypes and metabolic disorders in rats fed a high-fat diet (HFD), and inhibited intestinal barrier dysfunction and inflammation, which is also supported in the experiment of Caco-2 cells. Moreover, EGCG could restore gut microbiota diversity and composition, particularly promoting beneficial microbes, including short-chain fatty acids (SCFAs) producers, such as Lactobacillus, and suppressing Gram-negative bacteria, such as Desulfovibrio. The microbial modulation raised SCFA levels, decreased lipopolysaccharide levels, inhibited the TLR4/NF-κB pathway, and strengthened intestinal barrier function via Nrf2 pathway activation, thereby alleviating liver steatosis and inflammation. Spearman's correlation analysis showed that 24 key OTUs, negatively or positively associated with NAFLD and metabolic disorders, were also reshaped by EGCG. Our results suggested that a combinative improvement of EGCG on gut microbiota dysbiosis, intestinal barrier dysfunction, and inflammation might be a potential therapeutic target for NAFLD.

Excessive fatty acids activate PRMT5/MDM2/Drosha pathway to regulate miRNA biogenesis and lipid metabolism.

Excessive fatty acids in the liver lead to the accumulation of lipotoxic lipids and then cellular stress to further evoke the related disease, like non-alcoholic fatty liver disease (NAFLD). As reported, fatty acid stimulation can cause some specific miRNA dysregulation, which caused us to investigate the relationship between miRNA biogenesis and fatty acid overload. Gene expression omnibus (GEO) dataset analysis, miRNA-seq, miRNA cleavage assay, RT-qPCR, western blotting, immunofluorescence and co-immunoprecipitation (co-IP) were used to reveal the change of miRNAs under pathological status and explore the relevant mechanism. High fat, high fructose, high cholesterol (HFHFrHC) diet-fed mice transfected with AAV2/8-shDrosha or AAV2/8-shPRMT5 were established to investigate the invivo effects of Drosha or PRMT5 on NAFLD phenotype. We discovered that the cleavage of miRNAs was inhibited by analysing miRNA contents and detecting some representative pri-miRNAs in multiple mouse and cell models, which was further verified by the reduction of the Microprocessor activity in the presence of palmitic acid (PA). Invitro, PA could induce Drosha, the core RNase III in the Microprocessor complex, degrading through the proteasome-mediated pathway, while invivo, knockdown of Drosha significantly promoted NAFLD to develop to a more serious stage. Mechanistically, our results demonstrated that PA can increase the methyltransferase activity of PRMT5 to degrade Drosha through MDM2, a ubiquitin E3 ligase for Drosha. The above results indicated that PRMT5 may be a critical regulator in lipid metabolism during NAFLD, which was confirmed by the knocking down of PRMT5 improved aberrant lipid metabolism invitro and invivo. We first demonstrated the relationship between miRNA dosage and NAFLD and proved that PA can activate the PRMT5-MDM2-Drosha signalling pathway to regulate miRNA biogenesis.

Abstract MP25: Effect of Marine Omega-3 Fatty Acids on Risk of Incident Cardiovascular Disease Among Individuals With Non-Alcoholic Fatty Liver Disease: Secondary Analysis of a Randomized Clinical Trial

Background: Individuals with non-alcoholic fatty liver disease (NAFLD) are at increased risk for developing cardiovascular disease (CVD), often attributed to adverse metabolic derangements such as insulin resistance and elevated triglycerides. Marine omega-3 (n-3) fatty acids may partially ameliorate these metabolic consequences. Whether supplementation with n-3 fatty acids can reduce the risk of CVD in NAFLD remains unknown. Aims: To investigate the association between a NAFLD phenotype and incident CVD as well as assess whether randomization to n-3 fatty acids can modify this risk. Methods: Among 16,824 participants from the Vitamin D and Omega-3 Trial (VITAL; NCT01169259; a 2x2 factorial randomized trial of 1 g/d of EPA+DHA or 2000 IU/d of vitamin D 3 ) who provided baseline blood samples, we measured liver function tests. NAFLD was defined as baseline alanine aminotransferase (ALT)>40 U/L and/or aspartate aminotransferase (AST)>35 U/L, per guidance from the American Association for the Study of Liver Diseases. Incident CVD was a composite of myocardial infarction (MI), stroke, and CVD death. Multivariable-adjusted Cox models (adjusted for demographics, cardiovascular risk factors, and treatment assignment) were used to estimate hazard ratios (aHRs) and 95% confidence intervals (CI) for NAFLD with incident CVD. Effect of randomization to n-3 fatty acids was assessed among individuals with and without NAFLD. Results: We identified 2,495 participants with NAFLD at baseline, who tended to be younger, male, and non-White compared to individuals without NAFLD. After a median follow-up of 5.3 years, 522 CVD events were ascertained, with 101 occurring among those with NAFLD. Compared to individuals without NAFLD, those with NAFLD were at higher risk of developing CVD, aHR (95%CI): 1.48 (1.19, 1.84), P =0.0004. This association was predominantly driven by a higher incidence of total stroke, 1.53 (1.08, 2.17), P =0.02, compared with MI, 1.15 (0.81, 1.64), P =0.43. Randomization to n-3 fatty acids did not significantly reduce CVD incidence among individuals with, 1.13 (0.77, 1.68) or without NAFLD, 1.02 (0.85,1.24), P interaction =0.70. Conclusion: In a large primary prevention trial of older individuals, NAFLD was associated with a higher risk of CVD, particularly stroke. Randomization to n-3 fatty acids did not significantly reduce CVD outcomes in this high-risk population.

Tangshen formula targets the gut microbiota to treat non-alcoholic fatty liver disease in HFD mice: A 16S rRNA and non-targeted metabolomics analyses

BackgroundTangshen formula (TSF) has an ameliorative effect on hepatic lipid metabolism in non-alcoholic fatty liver disease (NAFLD), but the role played by the gut microbiota in this process is unknown. MethodWe conducted three batches of experiments to explore the role played by the gut microbiota: TSF administration, antibiotic treatment, and fecal microbial transplantation. NAFLD mice were induced with a high-fat diet to investigate the ameliorative effects of TSF on NAFLD features and intestinal barrier function. 16S rRNA sequencing and serum untargeted metabolomics were performed to further investigate the modulatory effects of TSF on the gut microbiota and metabolic dysregulation in the body. ResultsTSF ameliorated insulin resistance, hypercholesterolemia, lipid metabolism disorders, inflammation, and impairment of intestinal barrier function. 16S rRNA sequencing analysis revealed that TSF regulated the composition of the gut microbiota and increased the abundance of beneficial bacteria. Antibiotic treatment and fecal microbiota transplantation confirmed the importance of the gut microbiota in the treatment of NAFLD with TSF. Subsequently, untargeted metabolomics identified 172 differential metabolites due to the treatment of TSF. Functional predictions suggest that metabolisms of choline, glycerophospholipid, linoleic acid, alpha-linolenic acid, and arachidonic acid are the key metabolic pathways by which TSF ameliorates NAFLD and this may be influenced by the gut microbiota. ConclusionTSF treats the NAFLD phenotype by remodeling the gut microbiota and improving metabolic profile, suggesting that TSF is a functional gut microbial and metabolic modulator for the treatment of NAFLD.

A regulatory element associated to NAFLD in the promoter of DIO1 controls LDL-C, HDL-C and triglycerides in hepatic cells

BackgroundGenome-wide association studies (GWAS) have identified genetic variants linked to fat metabolism and related traits, but rarely pinpoint causative variants. This limitation arises from GWAS not considering functional implications of noncoding variants that can affect transcription factor binding and potentially regulate gene expression. The aim of this study is to investigate a candidate noncoding functional variant within a genetic locus flagged by a GWAS SNP associated with non-alcoholic fatty liver disease (NAFLD), a condition characterized by liver fat accumulation in non-alcohol consumers.MethodsCRISPR-Cas9 gene editing in HepG2 cells was used to modify the regulatory element containing the candidate functional variant linked to NAFLD. Global gene expression in mutant cells was assessed through RT-qPCR and targeted transcriptomics. A phenotypic assay measured lipid droplet accumulation in the CRISPR-Cas9 mutants.ResultsThe candidate functional variant, rs2294510, closely linked to the NAFLD-associated GWAS SNP rs11206226, resided in a regulatory element within the DIO1 gene's promoter region. Altering this element resulted in changes in transcription factor binding sites and differential expression of candidate target genes like DIO1, TMEM59, DHCR24, and LDLRAD1, potentially influencing the NAFLD phenotype. Mutant HepG2 cells exhibited increased lipid accumulation, a hallmark of NAFLD, along with reduced LDL-C, HDL-C and elevated triglycerides.ConclusionsThis comprehensive approach, that combines genome editing, transcriptomics, and phenotypic assays identified the DIO1 promoter region as a potential enhancer. Its activity could regulate multiple genes involved in the NAFLD phenotype or contribute to defining a polygenic risk score for enhanced risk assessment in NAFLD patients.

BMAL1 deletion protects against obesity and non-alcoholic fatty liver disease induced by a high-fat diet.

Obesity and non-alcoholic fatty liver disease (NAFLD) are major health concerns. The circadian rhythm is an autonomous and intrinsic timekeeping system closely associated with energy metabolism and obesity. Thus, this study explored the role of brain and muscle aryl hydrocarbon receptor nuclear translocator-like1 (BMAL1), a circadian clock regulator, in the development of obesity and NAFLD. We generated BMAL1 knockout (BMAL1 KO) mice to imitate circadian rhythm disruption. The study comprised three groups from the same litter: BMAL1 KO mice fed a high-fat diet (to establish obesity and NAFLD phenotypes), wild-type mice fed normal chow, and wild-type mice fed a high-fat diet. The metabolic and NAFLD phenotypes were assessed via physiological measurements and histological examinations. Quantitative polymerase chain reaction and western blotting were used to identify and validate changes in the signaling pathways responsible for the altered NAFLD phenotypes in the wild-type and BMAL1 KO mice. BMAL1 depletion protected against obesity and metabolic disorders induced by a high-fat diet. BMAL1 depletion also prevented hepatic steatosis and inhibited cluster of differentiation 36 and peroxisome proliferator-activated receptor gamma (i.e., PPARγ) expression. BMAL1 plays an important role in the development of obesity and NAFLD and, thus, is a potential therapeutic target for these conditions.

Gene Variants Implicated in Steatotic Liver Disease: Opportunities for Diagnostics and Therapeutics.

Non-alcoholic fatty liver disease (NAFLD) describes a steatotic (or fatty) liver occurring as a consequence of a combination of metabolic, environmental, and genetic factors, in the absence of significant alcohol consumption and other liver diseases. NAFLD is a spectrum of conditions. Steatosis in the absence of inflammation is relatively benign, but the disease can progress into more severe forms like non-alcoholic steatohepatitis (NASH), liver cirrhosis, and hepatocellular carcinoma. NAFLD onset and progression are complex, as it is affected by many risk factors. The interaction between genetic predisposition and other factors partially explains the large variability of NAFLD phenotype and natural history. Numerous genes and variants have been identified through large-scale genome-wide association studies (GWAS) that are associated with NAFLD and one or more subtypes of the disease. Among them, the largest effect size and most consistent association have been patatin-like phospholipase domain-containing protein 3 (PNPLA3), transmembrane 6 superfamily member 2 (TM6SF2), and membrane-bound O-acyltransferase domain containing 7 (MBOAT7) genes. Extensive in vitro and in vivo studies have been conducted on these variants to validate these associations. The focus of this review is to highlight the genetics underpinning the molecular mechanisms driving the onset and progression of NAFLD and how they could potentially be used to improve genetic-based diagnostic testing of the disease and develop personalized, targeted therapeutics.

Metabolic health phenotype better predicts subclinical atherosclerosis than body mass index-based obesity phenotype in the non-alcoholic fatty liver disease population.

Non-alcoholic fatty liver disease (NAFLD), especially lean NAFLD is associated with an increased risk of atherosclerotic cardiovascular disease (CVD). It is not currently known which clinical phenotypes of NAFLD contribute most to individual subclinical atherosclerosis risk. We examined the relationship between body mass index (BMI), the metabolically healthy status, and subclinical atherosclerosis in the NAFLD population. Data from asymptomatic NAFLD subjects who participated in a routine health check-up examination were collected. Participants were stratified by BMI (cutoff values: 24.0-27.9 kg/m2 for overweight and ≥28.0 kg/m2 for obesity) and metabolic status, which was defined by Adult Treatment Panel III criteria. Subclinical atherosclerosis was evaluated by brachial-ankle pulse wave velocity (baPWV) in 27,738 participants and by carotid plaque in 14,323 participants. Within each BMI strata, metabolically unhealthy subjects had a significantly higher prevalence of subclinical atherosclerosis than metabolically healthy subjects, whereas fewer differences were observed across subjects within the same metabolic category. When BMI and metabolic status were assessed together, a metabolically unhealthy status was the main contributor to the association of clinical phenotypes with the subclinical atherosclerosis burden (all p < 0.001). When BMI and metabolic abnormalities were assessed separately, the incidence of subclinical disease did not increase across BMI categories; however, it increased with an increase in the number of metabolic abnormalities (0, 1, 2 and ≥3). A metabolically healthy status in NAFLD patients was closely correlated with subclinical atherosclerosis, beyond that of the BMI-based obesity phenotype. The application of metabolic phenotyping strategies could enable more precise classification in evaluating cardiovascular risk in NAFLD.

Toxicity mechanisms of biomass burning aerosols in in vitro hepatic steatosis models

Nonalcoholic fatty liver disease (NAFLD) is a common chronic liver disease that contributes to the global rise in liver-related morbidity and mortality. Wood tar (WT) aerosols are a significant fraction of carbonaceous aerosol originating from biomass smoldering, contributing to air pollution particles smaller than 2.5 mm (PM2.5). Mechanistic biological associations exist between exposure to PM2.5 and increased NAFLD phenotypes in both cell and animal models. Therefore, this study examines whether an existing NAFLD-like condition can enhance the biological susceptibility of liver cells exposed to air pollution in the form of WT material. Liver cells were incubated with lauric or oleic acid (LA, OA, respectively) for 24 h to accumulate lipids and served as an in vitro hepatic steatosis model. When exposed to 0.02 or 0.2 g/L water-soluble WT aerosols, both steatosis model cells showed increased cell death compared to the control cells (blank-treated cells with or without pre-incubation with LA or OA) or compared to WT-treated cells without pre-incubation with LA or OA. Furthermore, alterations in oxidative status included variations in reactive oxygen species (ROS) levels, elevated levels of lipid peroxidation adducts, and decreased expression of antioxidant genes associated with the NRF2 transcription factor. In addition, steatosis model cells exposed to WT had a higher degree of DNA damage than the control cells (blank-treated cells with or without pre-incubation with LA or OA). These results support a possible systemic effect through the direct inflammatory and oxidative stress response following exposure to water-soluble WT on liver cells, especially those predisposed to fatty liver. Furthermore, the liver steatosis model can be influenced by the type of fatty acid used; increased adverse effects of WT on metabolic dysregulation were observed in the LA model to a higher extent compared to the OA model.

Duration of intermittent hypoxia impacts metabolic outcomes and severity of murine NAFLD.

Obstructive sleep apnea (OSA) is associated with metabolic dysfunction, including progression of nonalcoholic fatty liver disease (NAFLD). Chronic intermittent hypoxia (IH) as a model of OSA worsens hepatic steatosis and fibrosis in rodents with diet induced obesity. However, IH also causes weight loss, thus complicating attempts to co-model OSA and NAFLD. We sought to determine the effect of various durations of IH exposure on metabolic and liver-related outcomes in a murine NAFLD model. We hypothesized that longer IH duration would worsen the NAFLD phenotype. Male C57BL/6J mice (n = 32) were fed a high trans-fat diet for 24 weeks, to induce NAFLD with severe steatohepatitis. Mice were exposed to an IH profile modeling severe OSA, for variable durations (0, 6, 12, or 18 weeks). Intraperitoneal glucose tolerance test was measured at baseline and at six-week intervals. Liver triglycerides, collagen and other markers of NAFLD were measured at sacrifice. Mice exposed to IH for 12 weeks gained less weight (p = 0.023), and had lower liver weight (p = 0.008) relative to room air controls. These effects were not observed in the other IH groups. IH of longer duration transiently worsened glucose tolerance, but this effect was not seen in the groups exposed to shorter durations of IH. IH exposure for 12 or 18 weeks exacerbated liver fibrosis, with the largest increase in hepatic collagen observed in mice exposed to IH for 12 weeks. Duration of IH significantly impacts clinically relevant outcomes in a NAFLD model, including body weight, fasting glucose, glucose tolerance, and liver fibrosis.

Identifying a distinct fibrosis subset of NAFLD via molecular profiling and the involvement of profibrotic macrophages

BackgroundThere are emerging studies suggesting that non-alcoholic fatty liver disease (NAFLD) is a heterogeneous disease with multiple etiologies and molecular phenotypes. Fibrosis is the key process in NAFLD progression. In this study, we aimed to explore molecular phenotypes of NAFLD with a particular focus on the fibrosis phenotype and also aimed to explore the changes of macrophage subsets in the fibrosis subset of NAFLD.MethodsTo assess the transcriptomic alterations of key factors in NAFLD and fibrosis progression, we included 14 different transcriptomic datasets of liver tissues. In addition, two single-cell RNA sequencing (scRNA-seq) datasets were included to construct transcriptomic signatures that could represent specific cells. To explore the molecular subsets of fibrosis in NAFLD based on the transcriptomic features, we used a high-quality RNA-sequencing (RNA-seq) dataset of liver tissues from patients with NAFLD. Non-negative matrix factorization (NMF) was used to analyze the molecular subsets of NAFLD based on the gene set variation analysis (GSVA) enrichment scores of key molecule features in liver tissues.ResultsThe key transcriptomic signatures on NAFLD including non-alcoholic steatohepatitis (NASH) signature, fibrosis signature, non-alcoholic fatty liver (NAFL) signature, liver aging signature and TGF-β signature were constructed by liver transcriptome datasets. We analyzed two liver scRNA-seq datasets and constructed cell type-specific transcriptomic signatures based on the genes that were highly expressed in each cell subset. We analyzed the molecular subsets of NAFLD by NMF and categorized four main subsets of NAFLD. Cluster 4 subset is mainly characterized by liver fibrosis. Patients with Cluster 4 subset have more advanced liver fibrosis than patients with other subsets, or may have a high risk of liver fibrosis progression. Furthermore, we identified two key monocyte-macrophage subsets which were both significantly correlated with the progression of liver fibrosis in NAFLD patients.ConclusionOur study revealed the molecular subtypes of NAFLD by integrating key information from transcriptomic expression profiling and liver microenvironment, and identified a novel and distinct fibrosis subset of NAFLD. The fibrosis subset is significantly correlated with the profibrotic macrophages and M2 macrophage subset. These two liver macrophage subsets may be important players in the progression of liver fibrosis of NAFLD patients.

The antianginal ranolazine fails to improve glycaemia in obese liver-specific pyruvate dehydrogenase deficient male mice.

Recent studies have demonstrated that stimulating pyruvate dehydrogenase (PDH, gene Pdha1), the rate-limiting enzyme of glucose oxidation, can reverse obesity-induced non-alcoholic fatty liver disease (NAFLD), which can be achieved via treatment with the antianginal ranolazine. Accordingly, our aim was to determine whether ranolazine's ability to mitigate obesity-induced NAFLD and hyperglycaemia requires increases in hepatic PDH activity. We generated liver-specific PDH-deficient (Pdha1Liver-/- ) mice, which were provided a high-fat diet for 12 weeks to induce obesity. Pdha1Liver-/- mice and their albumin-Cre (AlbCre ) littermates were randomized to treatment with either vehicle control or ranolazine (50 mg/kg) once daily via oral gavage during the final 5weeks, following which we assessed glucose and pyruvate tolerance. Pdha1Liver-/- mice exhibited no overt phenotypic differences (e.g. adiposity, glucose tolerance) when compared to their AlbCre littermates. Of interest, ranolazine treatment improved glucose tolerance and mildly reduced hepatic triacylglycerol content in obese AlbCre mice but not in obese Pdha1Liver-/- mice. The latter was independent of changes in hepatic mRNA expression of genes involved in regulating lipogenesis. Liver-specific PDH deficiency is insufficient to promote an NAFLD phenotype. Nonetheless, hepatic PDH activity partially contributes to how the antianginal ranolazine improves glucose tolerance and alleviates hepatic steatosis in obesity.

Histological features of non-alcoholic fatty liver disease revealed in response to mixed vehicle emission exposure and consumption of a high-fat diet in wildtype C57Bl/6 male mice

Non-alcoholic fatty liver disease (NAFLD) is currently plaguing the population at pandemic proportions and is expected to become more prevalent over the next decade. Recent epidemiological studies have demonstrated a correlation between the manifestation of NAFLD and ambient air pollution levels, which is exacerbated by other risk factors, such as diabetes, dyslipidemia, obesity, and hypertension. Exposure to airborne particulate matter has also been associated with inflammation, hepatic lipid accumulation, oxidative stress, fibrosis, and hepatocyte injury. While prolonged consumption of a high-fat (HF) diet is associated with NAFLD, little is known regarding the effects of inhaled traffic-generated air pollution, a ubiquitous environmental pollutant, on the pathogenesis of NAFLD. Therefore, we investigated the hypothesis that exposure to a mixture of gasoline and diesel engine emissions (MVE), coupled with the concurrent consumption of a HF diet, promotes the development of a NAFLD phenotype within the liver. Three-month-old male C57Bl/6 mice were placed on either a low-fat or HF diet and exposed via whole-body inhalation to either filtered (FA) air or MVE (30 µg PM/m3 gasoline engine emissions + 70 µg PM/m3 diesel engine emissions) 6 hr/day for 30 days. Histology revealed mild microvesicular steatosis and hepatocyte hypertrophy in response to MVE exposure alone, compared to FA controls, yielding a classification of "borderline NASH" under the criteria of the modified NAFLD active score (NAS) system. As anticipated, animals on a HF diet exhibited moderate steatosis; however, we also observed inflammatory infiltrates, hepatocyte hypertrophy, and increased lipid accumulation, with the combined effect of HF diet and MVE exposure. Our results indicate that inhalation exposure to traffic-generated air pollution initiates hepatocyte injury and further exacerbates lipid accumulation and hepatocyte injury induced by the consumption of a HF diet, thereby contributing to the progression of NAFLD-related pathologies.

Prospects of evolution-based artificial intelligence models in genome-wide studies to stratify genetic risk variants in nonalcoholic fatty liver disease.

The emergence of genome-wide association studies (GWAS) has identified genetic traits and polymorphisms that are associated with the progression of nonalcoholic fatty liver disease (NAFLD). Phospholipase domain-containing 3 and transmembrane 6 superfamily member 2 are genes commonly associated with NAFLD phenotypes. However, there are fewer studies and replicability in lesser-known genes such as LYPLAL1 and glucokinase regulator (GCKR). With the advent of artificial intelligence (AI) in clinical genetics, studies have utilized AI algorithms to identify phenotypes through electronic health records and utilize convolution neural networks to improve the accuracy of variant identification, predict the deleterious effects of variants, and conduct phenotype-to-genotype mapping. Natural language processing (NLP) and machine-learning (ML) algorithms are popular tools in GWAS studies and connect electronic health record phenotypes to genetic diagnoses using a combination of international classification disease (ICD)-based approaches. However, there are still limitations to machine-learning - and NLP-based models, such as the lack of replicability in larger cohorts and underpowered sample sizes, which prevent the accurate prediction of genetic variants that may increase the risk of NAFLD and its progression to advanced-stage liver fibrosis. This may be largely due to the lack of understanding of the clinical consequence in the majority of pathogenic variants. Though the concept of evolution-based AI models and evolutionary algorithms is relatively new, combining current international classification disease -based NLP models with phylogenetic and evolutionary data can improve prediction accuracy and create valuable connections between variants and their pathogenicity in NAFLD. Such developments can improve risk stratification within clinical genetics and research while overcoming limitations in GWAS studies that prevent community-wide interpretations.

Characterization of hepatic fatty acids using magnetic resonance spectroscopy for the assessment of treatment response to metformin in an eNOS-/- mouse model of metabolic nonalcoholic fatty liver disease/nonalcoholic steatohepatitis.

Nonalcoholic fatty liver disease (NAFLD) is the leading cause of chronic liver disease worldwide. Liver biopsy remains the gold standard for diagnosis and staging of disease. There is a clinical need for noninvasive diagnostic tools for risk stratification, follow-up, and monitoring treatment response that are currently lacking, as well as preclinical models that recapitulate the etiology of the human condition. We have characterized the progression of NAFLD in eNOS-/- mice fed a high fat diet (HFD) using noninvasive Dixon-based magnetic resonance imaging and single voxel STEAM spectroscopy-based protocols to measure liver fat fraction at 3T. After 8weeks of diet intervention, eNOS-/- mice exhibited significant accumulation of intra-abdominal and liver fat compared with control mice. Liver fat fraction measured by 1 H-MRS in vivo showed a good correlation with the NAFLD activity score measured by histology. Treatment of HFD-fed NOS3-/- mice with metformin showed significantly reduced liver fat fraction and altered hepatic lipidomic profile compared with untreated mice. Our results show the potential of in vivo liver MRI and 1 H-MRS to noninvasively diagnose and stage the progression of NAFLD and to monitor treatment response in an eNOS-/- murine model that represents the classic NAFLD phenotype associated with metabolic syndrome.

Diet-induced induction of hepatic serine/threonine kinase STK38 triggers proinflammation and hepatic lipid accumulation

Nonalcoholic fatty liver disease (NAFLD) is one of the most common liver diseases worldwide. Although the involvement of chronic overnutrition, systemic inflammation, and insulin resistance in the development of NAFLD is well-established, however, the associations among these remain to be elucidated. Several studies have reported that chronic overnutrition, such as excessive consumption of fats (high-fat diet, HFD), can cause insulin resistance and inflammation. However, the mechanisms by which HFD exerts inflammation and thereby promotes insulin resistance and intrahepatic fat accumulation remain poorly understood. Here, we show that HFD induces the expression of hepatic serine/threonine kinase 38 (STK38), which further induces systemic inflammation leading to insulin resistance. Notably, ectopic expression of STK38 in mouse liver leads to lean NAFLD phenotype with hepatic inflammation, insulin resistance, intrahepatic lipid accumulation, and hypertriglyceridemia in mice fed on a regular chow diet. Further, depletion of hepatic STK38 in HFD-fed mice remarkably reduces proinflammation, improves hepatic insulin sensitivity, and decreases hepatic fat accumulation. Mechanistically, two critical stimuli are elicited by STK38 action. For one stimulus, STK38 binds to Tank-Binding protein Kinase 1 and induces Tank-Binding protein Kinase 1 phosphorylation to promote NF-κβ nuclear translocation that mobilizes the release of proinflammatory cytokines and eventually leads to insulin resistance. The second, stimulus involves intrahepatic lipid accumulation by enhanced de novo lipogenesis via reducing the AMPK-ACC signaling axis. These findings identify STK38 as a novel nutrient-sensitive proinflammatory and lipogenic factor in maintaining hepatic energy homeostasis, and it provides a promising target for hepatic and immune health.