Biomarker For Non-alcoholic Fatty Liver Disease Research Articles

Adipokine/hepatokines profiling of fatty liver in adolescents and young adults: cross-sectional and prospective analyses of the BCAMS study.

The underlying connections between obesity and non-alcoholic fatty liver disease (NAFLD) are not fully understood. One potential link might be the imbalanced adipokines and hepatokines. We aimed to explore the associations between specific adipokines/hepatokines and NAFLD in Chinese youth and to determine how these biomarkers mediate the obesity-NAFLD relationship. We analyzed data from the 10-year follow-up visit of the Beijing Children and Adolescents Metabolic Syndrome (BCAMS) study (n = 509; mean age = 20.2years) for a comprehensive metabolic risk assessment, including liver ultrasound and plasma measurements of adiponectin, leptin, fibroblast growth factor 21 (FGF21), retinol-binding protein 4 (RBP4), and angiopoietin-like protein 8 (ANGPTL8). Longitudinal analysis was performed on a subgroup (n = 307), with complete baseline (mean age = 12.2years) and follow-up data. Mediation models assessed how obesity at baseline and follow-up influence NAFLD through these biomarkers. Participants with NAFLD exhibited a high prevalence of central obesity (90.9%). Both cross-sectional and prospective analyses identified increased RBP4, FGF21, leptin, and decreased adiponectin levels as significant predictors of NAFLD. More adipokine/hepatokine abnormalities were linked to higher NAFLD risk. Furthermore, ratios reflecting adipokine/hepatokine imbalances, including leptin/adiponectin, FGF21/adiponectin, and RBP4/adiponectin, demonstrated stepwise changes correlating with NAFLD severity (all p < 0.05). Mediation analysis indicated that these four adipokines/hepatokines accounted for approximately 72.4% of the central obesity-NAFLD relationship and 80.1% in the subgroup analysis using baseline childhood data. Dysregulated adipokines/hepatokines may explain the onset or progression of obesity-related NAFLD in youths. Higher RBP4, FGF21 and leptin, alongside lower adiponectin, could serve as early biomarkers for NAFLD.

B-063 Changes in nonalcoholic fatty liver disease and M2BPGi due to lifestyle intervention in primary healthcare

Abstract Background A healthy lifestyle is the most important method for managing nonalcoholic fatty liver disease (NAFLD). Mac-2-binding protein glycosylated isomer (M2BPGi) has been suggested as a biomarker for NAFLD. This study aimed to determine the efficacy of personalized lifestyle interventions on NAFLD remission. Methods This single-arm intervention study recruited participants with NAFLD who underwent health checkups at seven health-promotion centers in five South Korean cities. Fatty liver diagnosis was based on ultrasonography (US). The 109 individuals were recruited for personalized lifestyle interventions of hypocaloric diets and exercise. The participants attended the lifestyle intervention programs once per month for the first 3 months, and once every 3 months for the subsequent 6 months. And phone-based self-monitoring were also provided monthly during the 3-month follow-up period. The primary outcome was NAFLD remission at month 12 as measured on US and magnetic resonance elastography. The secondary outcomes were the changes in metabolic factors and M2BPGi. Results The 108 individuals (62 males and 46 females; age 51.1±12.4 years, mean±standard deviation) were finally analyzed after the 12month intervention. Body mass index, waist circumference (WC), blood pressure, blood lipids (total cholesterol, triglycerides, and HDL-C), and fasting blood sugar levels were improved relative to baseline (all P&amp;lt;0.05). Fatty liver at or above the moderate grade according to US was decreased at month 12 relative to baseline (67.6% vs 50.9%) (P=0.002). M2BPGi levels decreased during the 12-month study period (P&amp;lt;0.001). M2BPGi levels were moderately correlated with hepatic fat fraction by magnetic resonance imaging (r=0.33, P=0.05). WC (OR=0.82, 95% CI=0.67-1.00, P=0.05) and HDL-C (OR=1.17, 95% CI=1.03-1.32, P=0.014) were associated with remission of fatty liver in the multivariate analysis. Conclusions The personalized lifestyle intervention was effective in improving fatty liver and metabolic factors, but not hepatic stiffness, in NAFLD.

Identification of common signature genes and pathways underlying the pathogenesis association between nonalcoholic fatty liver disease and heart failure.

Non-alcoholic fatty liver disease (NAFLD) and heart failure (HF) are related conditions with an increasing incidence. However, the mechanism underlying their association remains unclear. This study aimed to explore the shared pathogenic mechanisms and common biomarkers of NAFLD and HF through bioinformatics analyses and experimental validation. NAFLD and HF-related transcriptome data were extracted from the Gene Expression Omnibus (GEO) database (GSE126848 and GSE26887). Differential analysis was performed to identify common differentially expressed genes (co-DEGs) between NAFLD and HF. Gene ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and gene set enrichment analysis (GSEA) were conducted to explore the functions and regulatory pathways of co-DEGs. Protein-protein interaction (PPI) network and support vector machine-recursive feature elimination (SVM-RFE) methods were used to screen common key DEGs. The diagnostic value of common key DEGs was assessed by receiver operating characteristic (ROC) curve and validated with external datasets (GSE89632 and GSE57345). Finally, the expression of biomarkers was validated in mouse models. A total of 161 co-DEGs were screened out in NAFLD and HF patients. GO, KEGG, and GSEA analyses indicated that these co-DEGs were mainly enriched in immune-related pathways. PPI network revealed 14 key DEGs, and SVM-RFE model eventually identified two genes (CD163 and CCR1) as common key DEGs for NAFLD and HF. Expression analysis revealed that the expression levels of CD163 and CCR1 were significantly down-regulated in HF and NAFLD patients. ROC curve analysis showed that CD163 and CCR1 had good diagnostic values for HF and NAFLD. Single-gene GSEA suggested that CD163 and CCR1 were mainly engaged in immune responses and inflammation. Experimental validation indicated unbalanced macrophage polarization in HF and NAFLD mouse models, and the expression of CD163 and CCR1 were significantly down-regulated. This study identified M2 polarization impairment characterized by decreased expression of CD163 and CCR1 as a common pathogenic pathway in NAFLD and HF. The downregulation of CD163 and CCR1 may reflect key pathological changes in the development and progression of NAFLD and HF, suggesting their potential as diagnostic and therapeutic targets.

Association between heavy metal exposure and biomarkers for non-alcoholic fatty liver disease in Korean adolescents

ObjectivesThe global prevalence of non-alcoholic fatty liver disease (NAFLD) in adolescents has increased. In addition to childhood obesity, environmental risk factors, such as heavy metals that are known to be involved in hepatotoxicity, play role in NAFLD occurrence. However, their association with NAFLD remains unclear. This study aimed to investigate the association between heavy metal exposure and NAFLD biomarkers in adolescents. MethodsIn this cross-sectional study, we used the data of a total of 1505 adolescents aged 12–17 years who participated in the Korean National Environmental Health Survey III (2015–2017) and IV (2018–2020). The presence of blood lead (BPb), blood mercury (BHg), urinary mercury (UHg), and urinary cadmium (UCd) were measured. Liver enzymes including serum aspartate aminotransferase (AST), alanine aminotransferase (ALT), and gamma-glutamyl transferase (GGT) were evaluated. For NAFLD biomarkers, the hepatic steatosis index (HSI) was calculated. Multivariate linear regression models, weighted quantile sum (WQS) regression, and Bayesian kernel machine regression (BKMR) model were used to investigate the association between heavy metals and NAFLD biomarkers. ResultsAmong heavy metals, mercury presence showed a significant association with NAFLD biomarkers. Two-fold increases in BHg and UHg were associated with 0.21 points (95 % confidence interval [CI]: 0.08–0.35) and 0.19 points (95 % CI: 0.09–0.30) higher HSI, respectively. In the WQS model, heavy metal mixture was significantly associated with increased HSI (β = 0.06, 95 % CI: 0.01–0.11). Similarly, in the BKMR model, heavy metal mixture was positively associated with NAFLD biomarkers, and BHg was the most important contributor in the association. ConclusionsBHg and UHg were significantly associated with NAFLD biomarkers in adolescents, indicating that organic and inorganic mercury exposure could potentially be a risk factor for NAFLD. To mitigate and address the risk of NAFLD associated with heavy metal exposure, it is imperative to take measure to reduce avoidable mercury exposure is necessary.

Bioinformatics analysis of potential ferroptosis and non-alcoholic fatty liver disease biomarkers.

Ferroptosis plays a crucial role in the development of non-alcoholic fatty liver disease (NAFLD). In this study, we aimed to use a comprehensive bioinformatics approach and experimental validation to identify and verify potential ferroptosis-related genes in NAFLD. We downloaded the microarray datasets for screening differentially expressed genes (DEGs) and identified the intersection of these datasets with ferroptosis-related DEGs from the Ferroptosis database. Subsequently, ferroptosis-related DEGs were obtained using SVM analysis; the LASSO algorithm was then used to identify six marker genes. Furthermore, the CIBERSORT algorithm was used to estimate the proportion of different types of immune cells. Subsequently, we constructed drug regulatory networks and ceRNA regulatory networks. We identified six genes as marker genes for NAFLD, demonstrating their robust diagnostic abilities. Subsequent functional enrichment analysis results revealed that these marker genes were associated with multiple diseases and play a key role in NAFLD via the regulation of immune response and amino acid metabolism, among other pathways. The expression of hepatic EGR1, IL-6, SOCS1, and NR4A1 was significantly downregulated in the NAFLD model. Our findings provide new insights and molecular clues for understanding and treating NAFLD. Further studies are needed to assess the diagnostic potential of these markers for NAFLD.

A flavonoid-rich diet is associated with lower risk and improved imaging biomarkers of nonalcoholic fatty liver disease: a prospective cohort study

BackgroundMechanistic studies and short-term randomized trials suggest higher intakes of dietary flavonoids may protect against nonalcoholic fatty liver disease (NAFLD). ObjectivesWe aimed to perform the first population-based study with long-term follow-up on flavonoid consumption, incident NAFLD, and validated NAFLD biomarkers. MethodsIn a prospective study, we assessed the associations between flavonoid intake based on ≥2 24-h dietary assessments and NAFLD risk among 121,064 adults aged 40–69 y by multivariable Cox regression analyses. We further assessed the associations between flavonoid intake and magnetic resonance imaging-derived liver fat (a subset of n = 11,435) and liver-corrected T1 values (cT1; a subset of n = 9570), a marker of steatosis, more sensitive to inflammatory pathology. ResultsOver 10 y of follow-up, 1081 cases of NAFLD were identified. Participants in the highest quartile (Q4) of the flavodiet score reflecting the consumption of foods high in flavonoids, had a 19% lower risk of NAFLD compared to the lowest quartile (Q1) [hazard ratio (HR) (95% confidence interval (CI): 0.81 (0.67, 0.97), P-trend = 0.02)]. Moreover, participants in the Q4 of the flavodiet score had lower liver fat and cT1 values than those in Q1 (liver fat: relative difference Q1 compared with Q4: –5.28%, P-trend = <0.001; cT1: relative difference Q1 compared with Q4: –1.73%, P-trend = <0.001). When compared to low intakes, high intakes of apples and tea were associated with lower NAFLD risk [apples: HR (95% CI): 0.78 (0.67, 0.92), P-trend = <0.01; tea: HR (95% CI): 0.86 (0.72, 1.02), P-trend = 0.03)]. Additionally, when compared to low intakes, high apple, tea, and dark chocolate intakes were significantly associated with lower liver fat values, whereas high tea and red pepper intakes were significantly associated with lower cT1 values. ConclusionsThe consumption of flavonoid-rich foods was associated with a reduced risk of NAFLD among middle-aged adults.

Analysis of machine learning based integration to identify the crosslink between inflammation and immune response in non-alcoholic fatty liver disease through bioinformatic analysis

BackgroundThe prevalence of nonalcoholic fatty liver disease (NAFLD) is a major form of chronic liver disease. This study aimed to scrutinize the diagnostic biomarkers of NAFLD and their correlation with the immune microenvironment through bioinformatic analysis. MethodsTo identify genes associated with nonalcoholic fatty liver disease (NAFLD), we obtained microarray datasets (GSE63067 and GSE89632) from the Gene Expression Omnibus (GEO) database. Machine learning techniques such as Support Vector Machine (SVM), Least Absolute Shrinkage and Selection Operator (LASSO) and Random Forest (RF) were used to identify key genes. We performed gene ontology analysis to identify the driver pathways of NAFLD. External datasets (merging GSE48452, GSE66676 and GSE135251) were used to validate the identified genes and confirm protein levels by Western blotting. The CIBERSORT algorithm and immune-related techniques, such as ssGSEA, were used to assess the level of infiltration of different immune cell types and their functions. Finally, Spearman's analysis confirmed the relationship between pivotal genes and immune cells. ResultsHub genes (BBOX1, FOSB, NR4A2, RAB26 and SOCS2) were identified as potential biomarkers. This study demonstrates that these hub genes are significantly dysregulated in NAFLD, suggesting that they may be useful as diagnostic indicators and possible targets for treatment. Also covered are their possible effects on inflammation, immune cell activation, and liver damage in NAFLD. A better understanding of the intricate relationship between metabolic inefficiency, immunological response, and liver pathology in NAFLD may be gained from this work, which can lead to the development of new diagnostic tools and clinical treatments. ConclusionThe current study identified BBOX1, FOSB, NR4A2, RAB26 and SOCS2 as important diagnostic biomarkers for NAFLD. The study highlights the important function of immune cell infiltration in developing NAFLD. Their findings provide valuable molecular biological insights into the development of NAFLD and may lead to novel therapeutic strategies for treating this disease.

Evaluation of the diagnostic role of circulating miR-16, miR-10b, and miR-21 expression in patients with nonalcoholic fatty liver disease

Non-alcoholic fatty liver disease (NAFLD) is a growing global health concern and is characterized by fat accumulation in the liver in the absence of significant alcohol consumption. However, current diagnostic tools, including liver biopsy and imaging techniques, have limitations in terms of accessibility, invasiveness, and sensitivity. Recently, microRNAs (miRNAs) have emerged as non-invasive biomarkers for the diagnosis of NAFLD. In this study, we investigated the expression of three microRNAs (miR-16, miR-10b, and miR-21) in patients with NAFLD across different disease stages compared with healthy subjects. First, miRNAs were extracted from serum samples collected from 34 healthy controls and 38 patients with NAFLD, including 20 with grade 1 and 18 with grade 2 of the disease. Subsequently, cDNA was synthesized from RNA, and miR-21, miR-16, and miR-10b expression was measured using RT-qPCR. The results revealed the downregulation of miR-16 in the early and advanced stages of NAFLD. The serum expression of miR-21 (p < 0 0.01) and miR-10b (p < 0.05) increased in the total NAFLD samples compared with the control group. Moreover, miR-10b expression was significantly higher in patients with stage 2 of NAFLD than in those with stage1 of NAFLD (p < 0.05), suggesting its potential as a biomarker to distinguish between the different grades of the disease. Our results revealed the clinical value of these miRNAs as non-invasive, sensitive, and stage-specific biomarkers for NAFLD. These findings suggest that the assessment of miR-16, miR-21, and miR-10b expression levels could serve as a potentially useful tool for the diagnosis of NAFLD presence and severity.

Identification of key markers for the stages of nonalcoholic fatty liver disease: An integrated bioinformatics analysis and experimental validation

BackgroundThe identification of biomarkers for the early diagnosis of nonalcoholic fatty liver disease (NAFLD) is urgently needed. Here, we aimed to identify NAFLD biomarkers in the early stages of steatosis (SS) and nonalcoholic steatohepatitis (NASH) based on differential gene expression from bioinformatics data. MethodsA meta-analysis was performed from transcriptomic databases retrieved from public repositories containing data from biopsies of patients at various stages of NAFLD development. The status of the selected molecules was validated in the serum of patients with NAFLD by ELISA. Results: We identified 121 differentially expressed genes (DEGs) associated with SS and 402 associated with NASH. Gene Ontology (GO) enrichment revealed that the altered genes were primarily associated with dysfunction of primary cellular processes, and pathway analyses were mainly related to cholesterol metabolism. We identified ACSS2, PCSK9, and CYP7A1 as candidate biomarkers for SS and ANGPTL3, CD36, CYP51A1, FASN, FAS, FDFT1, and LSS as candidate biomarkers for NASH. ConclusionsBy experimental validation of bioinformatics data from patients with NAFLD, we identified promising biomarkers for detecting SS and NASH that might be useful for screening and diagnosing early NAFLD stages in humans.

A compact breath breathalyzer for identifying the non-alcoholic fatty liver disease biomarker

Non-alcoholic fatty liver disease (NAFLD) is a prevalent chronic liver disease worldwide. Currently, its diagnosis relies primarily on imaging and histological examinations, which are invasive and prone to misdiagnosis in the early stage. To address these limitations, detection and analysis of volatile organic compounds (VOCs) in human breath can be a rapid and non-invasive screening method for NAFLD. In this study, a compact breath breathalyzer was developed, utilizing a miniaturized gas chromatography chip with the STM32 microcontroller as the main control chip to manage airflow, temperature, and receive terminal signals from the photoionization detector. In the experiment, a gas mixture comprising five VOCs (pentane, acetone, toluene, octane, and decane) was selected as the simulated typical disease biomarkers in human breath to investigate the breathalyzer's performance and optimize testing conditions for multi-polar and wide-boiling-range breath samples. Results show that the breathalyzer can detect low-boiling components (< 100°C) such as the isoprene and acetone, with a detection limit less than 50 ppb which are two commonly biomarkers of NAFLD. Furthermore, breath samples were collected from 35 non-diseased individuals, and NAFLD early-stage patient samples were simulated by increasing the isoprene concentration by 10 ppb. Convolutional neural network (CNN) were used to identify the VOC signatures in gas chromatograms with predictive accuracy of 85% for the classification model. Therefore, the compact breath breathalyzer has potential application in the rapid and early screening of NAFLD.

Neutrophil to albumin ratio: a biomarker in non-alcoholic fatty liver disease and with liver fibrosis.

Given the high prevalence of non-alcoholic fatty liver disease (NAFLD) and its potential to progress to liver fibrosis, it is crucial to identify the presence of NAFLD in patients to guide their subsequent management. However, the current availability of non-invasive biomarkers for NAFLD remains limited. Therefore, further investigation is needed to identify and develop non-invasive biomarkers for NAFLD. A retrospective analysis was conducted on 11,883 patients admitted to the Healthcare Center, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, from January 2016 to December 2019 and divided into NAFLD and non-NAFLD groups. Anthropometric and laboratory examination data were collected. The correlations between variables and NAFLD were evaluated using the student's t-test or Mann-Whitney U test and binary logistic regression analysis. The predictive ability of these variables for NAFLD was assessed using the areas under the curves (AUCs) of receiver operating characteristics. Among the included patients, 3,872 (32.58%) were diagnosed with NAFLD, with 386 (9.97%) individuals having liver fibrosis. Patients with NAFLD exhibited a higher proportion of males, elevated body mass index (BMI), and increased likelihood of hypertension, diabetes mellitus, and atherosclerosis. Logistic regression analysis identified the neutrophil to albumin ratio (NAR) as the most promising novel inflammation biomarkers, with the highest AUC value of 0.701, a cut-off value of 0.797, sensitivity of 69.40%, and specificity of 66.00% in identifying the risk of NAFLD. Moreover, NAR demonstrated superior predictive value in identifying NAFLD patients at risk of liver fibrosis, with an AUC value of 0.795, sensitivity of 71.30%, and specificity of 73.60% when NAR reached 1.285. These findings highlight that the novel inflammatory biomarker, NAR, is a convenient and easily accessible non-invasive predictor for NAFLD and NAFLD with liver fibrosis.

Immune cell balance as potential biomarker of progressing non-alcoholic fatty liver disease

Nonalcoholic fatty liver disease (NAFLD) is a widespread chronic, slowly progressive metabolic multifactorial disease. It is represented by several clinical and morphological forms: steatosis, nonalcoholic steatohepatitis (NASH) (with or without fibrosis), and liver cirrhosis. The search for minimally invasive and cost-effective biomarkers of NAFLD is a key task in the diagnosis, staging of progression, and long-term monitoring of NAFLD. This article discusses the possibility of using immune cell balance as potential minimally invasive peripheral markers of NAFLD progression. In the progression of NASH from steatosis to fibrosis and cirrhosis, inflammation plays an important role because of the activation of Kupffer cells and increased migration of monocytes, dendritic cells, neutrophils, and activated T lymphocytes into the tissues. Macrophages originating from monocytes, with NASH progression, gradually begin to prevail over the pool of resident macrophages. The risk of NASH and fibrosis development in patients with NAFLD increases with the ratio of neutrophils/lymphocytes in the liver. An increase in the Th17 cell count and a decrease in T-regulatory cell count can contribute to increased hepatic steatosis and inflammation development in NAFLD and accelerate the transition from simple steatosis to steatohepatitis and fibrosis. Information on the participation of noncoding RNAs in the regulation of the balance of immune cells in NAFLD is presented, which also allows us to consider them as additional, along with cellular, markers of disease progression.

Changes in nonalcoholic fatty liver disease and M2BPGi due to lifestyle intervention in primary healthcare.

A healthy lifestyle is the most important method for managing nonalcoholic fatty liver disease (NAFLD). Mac-2-binding protein glycosylated isomer (M2BPGi) has been suggested as a biomarker for NAFLD. This study aimed to determine the efficacy of personalized lifestyle interventions on NAFLD remission. This single-arm intervention study recruited participants with NAFLD who underwent health checkups at seven health-promotion centers in five South Korean cities. Fatty liver diagnosis was based on ultrasonography (US). The 109 individuals were recruited for personalized lifestyle interventions of hypocaloric diets and exercise. The participants attended the lifestyle intervention programs once per month for the first 3 months, and once every 3 months for the subsequent 6 months. In addition to sessions through center visits, phone-based intervention and self-monitoring at 4-, 5-, 7-, and 8-month were provided during the 9-month intervention period. And phone-based self-monitoring were also provided monthly during the 3-month follow-up period. The primary outcome was NAFLD remission at month 12 as measured on US and magnetic resonance elastography. The secondary outcomes were the changes in metabolic factors and M2BPGi. The 108 individuals (62 males and 46 females; age 51.1±12.4 years, mean±standard deviation) were finally analyzed after the 12month intervention. Body mass index, waist circumference (WC), blood pressure, blood lipids (total cholesterol, triglycerides, and HDL-C), and fasting blood sugar levels were improved relative to baseline (all P<0.05). Fatty liver at or above the moderate grade according to US was decreased at month 12 relative to baseline (67.6% vs 50.9%) (P = 0.002). M2BPGi levels decreased during the 12-month study period (P<0.001). M2BPGi levels were moderately correlated with hepatic fat fraction by magnetic resonance imaging (r = 0.33, P = 0.05). WC (OR = 0.82, 95% CI = 0.67-1.00, P = 0.05) and HDL-C (OR = 1.17, 95% CI = 1.03-1.32, P = 0.014) were associated with remission of fatty liver in the multivariate analysis. The personalized lifestyle intervention was effective in improving fatty liver and metabolic factors, but not hepatic stiffness, in NAFLD. ICTRP, cris.nih.go.kr (KCT0006380).

CCN3/NOV Serum Levels in Non-Alcoholic Fatty Liver Disease (NAFLD) Patients in Comparison with the Healthy Group and their Correlation with TNF-α and IL-6.

Adipokine irregularity leads to inflammation, endothelial dysfunction, insulin resistance (IR), and Non-Alcoholic Fatty Liver Disease (NAFLD). Previous studies linked NOV/CCN3 to obesity, IR, and inflammation, but no research has explored the connection between CCN3 serum levels and NAFLD. This case-control study assessed CCN3, IL-6, adiponectin, and TNF-α serum levels in 80 NAFLD patients and 80 controls using ELISA kits. Biochemical parameters were measured with commercial kits and an auto analyzer. NAFLD patients exhibited significantly higher CCN3 (2399.85 ± 744.53 vs. 1712.84 ± 478.19 ng/ml), TNF-α, and IL-6 levels, and lower adiponectin levels compared to controls (P<0.0001). In the NAFLD group, CCN3 showed positive correlations with FBG, insulin, HOMA-IR, and TNF-α. Binary logistic regression analysis revealed increased NAFLD risk in the adjusted model (OR [95% CI] = 1.220 [1.315 -1.131]). A CCN3 cut-off value of 1898.0050 pg/mL differentiated NAFLD patients from controls with 78.8% sensitivity and 73.2% specificity. It was found that elevated CCN3 serum levels directly correlate with NAFLD incidence and inflammation markers (IL-6 and TNF-α). CCN3 could serve as a potential biomarker for NAFLD, but further research is needed to validate this finding and assess its clinical utility.

Non-alcoholic fatty liver disease biomarkers estimate cardiovascular risk based on coronary artery calcium score in type 2 diabetes: a cross-sectional study with two independent cohorts

BackgroundStudies have demonstrated that coronary artery calcification on one hand and non-alcoholic fatty liver disease (NAFLD) on the other hand are strongly associated with cardiovascular events. However, it remains unclear whether NAFLD biomarkers could help estimate cardiovascular risk in individuals with type 2 diabetes (T2D). The primary objective of the present study was to investigate whether the biomarkers of NAFLD included in the FibroMax® panels are associated with the degree of coronary artery calcification in patients with T2D.MethodsA total of 157 and 460 patients with T2D were included from the DIACART and ACCoDiab cohorts, respectively. The coronary artery calcium score (CACS) was measured in both cohorts using computed tomography. FibroMax® panels (i.e., SteatoTest®, FibroTest®, NashTest®, and ActiTest®) were determined from blood samples as scores and stages in the DIACART cohort and as stages in the ACCoDiab cohort.ResultsCACS significantly increased with the FibroTest® stages in both the DIACART and ACCoDiab cohorts (p-value for trend = 0.0009 and 0.0001, respectively). In DIACART, the FibroTest® score was positively correlated with CACS in univariate analysis (r = 0.293, p = 0.0002) and remained associated with CACS independently of the traditional cardiovascular risk factors included in the SCORE2-Diabetes model [β = 941 ± 425 (estimate ± standard error), p = 0.028]. In the ACCoDiab cohort, the FibroTest® F3-F4 stage was positively correlated with CACS in point-biserial analysis (rpbi = 0.104, p = 0.024) and remained associated with CACS after adjustment for the traditional cardiovascular risk factors included in the SCORE2-Diabetes model (β = 234 ± 97, p = 0.016). Finally, the prediction of CACS was improved by adding FibroTest® to the traditional cardiovascular risk factors included in the SCORE2-Diabetes model (goodness-of-fit of prediction models multiplied by 4.1 and 6.7 in the DIACART and ACCoDiab cohorts, respectively). In contrast, no significant relationship was found between FibroMax® panels other than FibroTest® and CACS in either cohort.ConclusionsFibroTest® is independently and positively associated with the degree of coronary artery calcification in patients with T2D, suggesting that FibroTest® could be a relevant biomarker of coronary calcification and cardiovascular risk.Trial registration: ClinicalTrials.gov identifiers NCT02431234 and NCT03920683.

Liver-derived Exosomal miRNA in NAFLD: Mechanisms of Action, Biomarkers, and Therapeutic Applications.

Nonalcoholic fatty liver disease (NAFLD) is of global concern due to its high prevalence worldwide. NAFLD, as one of the most common causes of liver function abnormalities, is associated with obesity, insulin resistance, and type 2 diabetes mellitus, and there are no medications available to treat NAFLD. Extracellular vesicles (EVs) are nanosized, membrane-bound vesicles that deliver biomolecules between cells. Exosomes are a subtype of EVs that mediate intercellular communication by delivering proteins and RNAs. MicroRNAs (miRNAs) are a highly conserved class of small tissue-specific non-coding RNAs that influence the expression of many functionally interacting genes. Hepatic-derived exosomal miRNAs are tightly associated with NAFLD occurrence and progression through multiple mechanisms. In addition, the characterization of miRNAs suggests that they may serve as multifunctional biomarkers for NAFLD, be used as clinical therapeutic targets for NAFLD, and be significant predictors of patient prognosis. Here, we review recent advances in the regulation and function of exosome-derived miRNAs in NAFLD, focusing on miRNAs specifically expressed or enriched in hepatocytes (HCs), hepatic macrophages, hepatic stellate cells (HSCs), and other immune cells in the liver. Finally, we discuss future research directions on exosomal miRNAs as biomarkers for NAFLD's diagnosis and clinical therapeutic targets.

Benzorhodol derived far-red/near-infrared fluorescent probes for selective and sensitive detection of butyrylcholinesterase activity in living cells and the non-alcoholic fatty liver of zebrafish.

Liver diseases are a growing public health concern and the development of non-alcoholic fatty liver disease (NAFLD) has a significant impact on human metabolism. Butyrylcholinesterase (BChE) is a vital biomarker for NAFLD, making it crucial to monitor BChE activity with high sensitivity and selectivity. In this study, we designed and synthesized a range of benzorhodol-derived far-red/near-infrared fluorescent probes, FRBN-B, NF-SB, and NF-B, for the quantitative detection and imaging of BChE. These probes differed in the size of their conjugated systems and in the number of incorporated cyclopropanecarboxylates, acting as the recognition site for BChE. Comprehensive characterization showed that FRBN-B and NF-SB fluorescence was triggered by BChE-mediated hydrolysis, while an additional cyclopropanecarboxylate in NF-B impeded the fluorescence release. High selectivity towards BChE was observed for FRBN-B and NF-SB, with a detection limit of 7.2 × 10-3 U mL-1 for FRBN-B and 1.9 × 10-3 U mL-1 for NF-SB. The probes were further employed in the evaluation of BChE inhibitor efficacy and imaging of intracellular BChE activity. Additionally, FRBN-B was utilized for imaging the BChE activity level in liver tissues in zebrafish, demonstrating its potential as a diagnostic tool for NAFLD.

Machine learning identifies ferroptosis-related gene ANXA2 as potential diagnostic biomarkers for NAFLD.

Non-alcoholic fatty liver disease (NAFLD), a major cause of chronic liver disease, still lacks effective therapeutic targets today. Ferroptosis, a type of cell death characterized by lipid peroxidation, has been linked to NAFLD in certain preclinical trials, yet the exact molecular mechanism remains unclear. Thus, we analyzed the relationship between ferroptosis genes and NAFLD using high-throughput data. We utilized a total of 282 samples from five datasets, including two mouse ones, one human one, one single nucleus dataset and one single cell dataset from Gene Expression Omnibus (GEO), as the data basis of our study. To filter robust treatment targets, we employed four machine learning methods (LASSO, SVM, RF and Boruta). In addition, we used an unsupervised consensus clustering algorithm to establish a typing scheme for NAFLD based on the expression of ferroptosis related genes (FRGs). Our study is also the first to investigate the dynamics of FRGs throughout the disease process by time series analysis. Finally, we validated the relationship between core gene and ferroptosis by in vitro experiments on HepG2 cells. We discovered ANXA2 as a central focus in NAFLD and indicated its potential to boost ferroptosis in HepG2 cells. Additionally, based on the results obtained from time series analysis, ANXA2 was observed to significantly define the disease course of NAFLD. Our results demonstrate that implementing a ferroptosis-based staging method may hold promise for the diagnosis and treatment of NAFLD. Our findings suggest that ANXA2 may be a useful biomarker for the diagnosis and characterization of NAFLD.

Bioinformatics analysis of ferroptosis-related genes and immune cell infiltration in non-alcoholic fatty liver disease

BackgroundThe morbidity and mortality rates of patients with non-alcoholic fatty liver disease (NAFLD) have been steadily increasing in recent years. Previous studies have confirmed the important role of ferroptosis in NAFLD development; however, the precise mechanism through which ferroptosis influences NAFLD occurrence remains unclear. The present study aimed to identify and validate ferroptosis-related genes involved in NAFLD pathogenesis and to investigate the underlying molecular mechanisms of NAFLD.MethodsWe downloaded microarray datasets GSE72756 and GSE24807 to identify differentially expressed genes (DEGs) between samples from healthy individuals and patients with NAFLD. From these DEGs, we extracted ferroptosis-related DEGs. GSE89632, another microarray dataset, was used to validate the expression of ferroptosis-related genes. A protein–protein interaction (PPI) network of ferroptosis-related genes was then constructed. The target genes were also subjected to Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses. Finally, competing endogenous RNA networks were constructed. We used the CIBERSORT package to evaluate the infiltration of immune cells infiltration in NAFLD.ResultsFive ferroptosis-related genes (SCP2, MUC1, DPP4, SLC1A4, and TF) were identified as promising diagnostic biomarkers for NAFLD. Enrichment analyses revealed that these genes are mainly involved in metabolic processes. NEAT1-miR-1224-5p-SCP2, NEAT1-miR-485-5p-MUC1, MALAT1-miR-485-5p-MUC1, and CNOT6-miR-145-5p-SLC1A4 are likely to be the potential RNA regulatory pathways that affect NAFLD development. Principal component analysis indicated significant differences in immune cell infiltration between the two groups.ConclusionsThis study identified five ferroptosis-related genes as potential biomarkers for diagnosing NAFLD. The correlations between the expression of ferroptosis-related genes and immune cell infiltration might shed light on the study of the molecular mechanism underlying NAFLD development.

Endotoxin biomarkers, hepatic fat fraction, liver volume and liver stiffness among adolescents at high-risk for non-alcoholic fatty liver disease: The HEROES study.

Non-alcoholic fatty liver disease (NAFLD) is on the rise among youth. Identifying biomarkers of NAFLD progression/risk can aid in prevention efforts. This pilot study investigated associations of two endotoxin biomarkers-lipopolysaccharide-binding protein (LBP) and anti-endotoxin core immunoglobulin G (EndoCab)-with markers of NAFLD among 99 Latino/Latina adolescents (11-19 years) with obesity. We used linear regression to examine associations of each endotoxin biomarker (per 1-SD) with hepatic fat fraction (HFF), liver volume, and liver stiffness. We found positive associations of LBP with HFF and liver volume. Each 1-SD increment in LBP corresponded with 2.35% (95% CI: 0.46%, 4.23%) higher HFF and 0.14 (0.06, 0.23) L greater liver volume after adjusting for age, sex, and maternal education. Accounting for abdominal adiposity and Tanner stage did not change results. Excluding 72 participants with NAFLD attenuated associations of LBP with HFF but associations with liver volume persisted (0.11 [0.01, 0.21] L). EndoCab was not associated with any liver outcomes. Neither endotoxin biomarker predicted liver stiffness. While additional research is warranted, our results support LBP as a biomarker of NAFLD risk/progression in high-risk youth.