Circulating extracellular vesicles are associated with lipid and insulin metabolism.

We have reported that hypertrophic adipocytes release extracellular vesicles (EVs) and the number of circulating adipocyte-derived EVs correlated with insulin and homeostasis model assessment-insulin resistance (HOMA-IR) in a pilot study using obese patients. Here, we explored the association between circulating EV level and various metabolic parameters, including obesity and lipid and glucose metabolisms, among 203 subjects (76 men and 127 women; median age, 54 yr) with or without risk factor for metabolic diseases, who received a 75-g oral glucose tolerance test (OGTT). Circulating EV number was significantly higher in men than in women ( P < 0.001). Circulating EV number in individuals with impaired OGTT pattern was significantly higher compared with those with normal OGTT patterns ( P < 0.05). Multiple regression analysis revealed that circulating EV number correlated most strongly and significantly with elevated triglyceride (TG; t = 8.55, P < 0.001). Additionally, circulating EV number correlated significantly with homeostasis model assessment-ß-cell function (HOMA-ß; t = 2.38, P < 0.05). Receiver operating characteristic curve revealed that the cutoff value of EV numbers in individuals with elevated serum TG levels (≧150 mg/dl) was identified (136,738 EVs/µl of plasma, P < 0.001, sensitivity 0.842, false-positive rate of 0.257). Perilipin and asialoglycoprotein receptor 1 were detected on a part of isolated circulating EVs, indicating EV release from adipocytes and hepatocytes, which were related to lipid and glucose metabolism. Circulating EVs represent a promising metabolic biomarker for lipid and glucose metabolism and have potential for monitoring metabolic status in humans, including individuals without metabolic risk factors.

Serum Zinc-α2-glycoprotein Levels Are Associated with the Hepatorenal Function and Predict the Survival in Cases of Chronic Liver Disease.

Objective Zinc-α2-glycoprotein (ZAG) is secreted by various organs, such as liver, kidney and adipose tissue, is involved in lipolysis, and may contribute to the pathogenesis of chronic liver disease (CLD). We therefore assessed whether or not ZAG is a surrogate marker for the hepatorenal function, body composition and all causes of mortality, as well as complications, including ascites, hepatic encephalopathy (HE) and portosystemic shunts (PSS) in CLD. Methods Serum ZAG levels were measured in 180 CLD patients upon hospital admission. The associations of ZAG levels with the liver functional reserve and clinical parameters were investigated using a multiple regression analysis. Kaplan-Meier analyses were performed to evaluate the associations of the ZAG/creatinine ratio (ZAG/Cr) and prognostic factors with mortality. Results High serum ZAG levels were associated with preserving the liver function and renal insufficiency. A multiple regression analysis showed that the estimated glomerular filtration rate (p<0.0001), albumin-bilirubin (ALBI) score (p=0.0018) and subcutaneous fat area (p=0.0023) had a significant independent correlation with serum ZAG levels. Serum ZAG levels were elevated in the absence of HE (p=0.0023) and PSS (p=0.0003). In all patients and those without hepatocellular carcinoma (HCC), the cumulative mortality rate was significantly decreased in patients with a high ZAG/Cr compared with those with a low ZAG/Cr (p=0.0018 and p=0.0002, respectively). The ZAG/Cr, presence of HCC, ALBI score and psoas muscle index were independent predictors of the prognosis in CLD patients. Conclusion Serum ZAG levels are associated with the hepatorenal function and can be used to predict the survival in CLD patients.

Circulating extracellular vesicles are associated with pathophysiological condition including metabolic syndrome-related dysmetabolism in children and adolescents with obesity.

Obesity of children and adolescents (OCA) is often accompanied by metabolic syndrome (MetS), which often leads to adult obesity and subsequent complications, yet the entire pathophysiological response is not fully understood. The number and composition of circulating extracellular vesicles (EV) reflect overall patient condition; therefore, we investigated the pathophysiological condition of OCA, including MetS-associated dysmetabolism, using circulating EVs. In total, 107 children and adolescents with or without obesity (boys, n = 69; girls, n = 38; median age, 10 years) were enrolled. Circulating EV number and EV protein composition were assessed via flow cytometry and liquid chromatography tandem-mass spectrometry, respectively. In a multivariate analysis, relative body weight (standardized partial regression coefficient (SPRC) 0.469, P = 0.012) and serum triglyceride level (SPRC 0.548, P < 0.001) were detected as independent parameters correlating with circulating EV number. Proteomic analysis identified 31 upregulated and 45 downregulated EV proteins in OCA. Gene ontology analysis revealed upregulated proteins to be involved in various biological processes, including intracellular protein transport, protein folding, stress response, leukocyte activation, innate immune response, and platelet degranulation, which can modulate lipid and glucose metabolism, skeletal and cardiac muscle development, inflammation, immune response, carcinogenesis, and cancer progression. Notably, several identified EV proteins are involved in neuro-development, neurotransmitter release, and neuro-protective agents in OCA. Circulating EVs were derived from adipocytes, hepatocytes, B cell lymphocytes, and neurons. Circulating EV number is significantly associated with MetS-related dysmetabolism and the EV protein cargo carries a special "signature" that reflects the alteration of various biological processes under the pathophysiological condition of OCA. KEY MESSAGES: Circulating EV number correlates with physical and laboratory parameters for obesity in children and adolescents. Relative body weight and triglyceride are independent factors for increased circulating EVs. EV composition is significantly changed in obesity of children and adolescents. Identified EV composition changes associated with obesity and involves in metabolism, immune response, and cancer progression. Circulating EVs are partially derived from adipocyte, hepatocytes, B cells, and neurons.

Association of lithocholic acid with skeletal muscle hypertrophy through TGR5-IGF-1 and skeletal muscle mass in cultured mouse myotubes, chronic liver disease rats and humans.

Background: Hepatic sarcopenia is one of many complications associated with chronic liver disease (CLD) and has a high mortality rate; however, the liver-muscle axis is not fully understood. Therefore, few effective treatments exist for hepatic sarcopenia, the best of which being branched-chain amino acid (BCAA) supplementation to help increase muscle mass. Our aim was to investigate the molecular mechanism(s) of hepatic sarcopenia focused on bile acid (BA) composition. Methods: The correlation between serum BA levels and psoas muscle mass index (PMI) was examined in 73 CLD patients. Gastrocnemius muscle phenotype and serum BA levels were assessed in CLD rats treated with BCAA. Mouse skeletal muscle cells (C2C12) were incubated with lithocholic acid (LCA), G-protein-coupled receptor 5 (TGR5) agonist or TGR5 antagonist to assess skeletal muscle hypertrophy. Results: In human CLD, serum LCA levels were the sole factor positively correlated with PMI and were significantly decreased in both the low muscle mass group and the deceased group. Serum LCA levels were also shown to predict patient survival. Gastrocnemius muscle weight significantly increased in CLD rats treated with BCAA via suppression of protein degradation pathways, coupled with a significant increase in serum LCA levels. LCA treated C2C12 hypertrophy occurred in a concentration-dependent manner linked with TGR5-Akt pathways based upon inhibition results via a TGR5 antagonist. Conclusions: Our results indicate LCA-mediated skeletal muscle hypertrophy via activation of TGR5-IGF1-Akt signaling pathways. In addition, serum LCA levels were associated with skeletal muscle mass in cirrhotic rats, as well as CLD patients, and predicted overall patient survival. Funding: This research was supported by JSPS KAKENHI Grant Number 22K08011 and 21H02892, and AMED under Grant Number JP21fk0210090 and JP22fk0210115. Maintaining cirrhotic rats were partially supported by Otsuka Pharmaceutical Company.

Complement complex 1 subunit q-mediated hepatic stellate cell activation with connective tissue growth factor elevation is a prognostic factor for survival in rat and human chronic liver diseases.

Complement complex 1 subunit q (C1q) has multiple functions, including cell migration, in addition to its traditional complement-activating effect. Research shows C1q is a ligand for frizzled receptors (FZDs). FZD-induced yes-associated protein (YAP)/transcriptional co-activator with PDZ-binding motif (TAZ) alternate Wnt signaling activation induces connective tissue growth factor (CTGF) production and hepatic stellate cell (HSC) activation. However, no study exists in which C1q directly induces CTGF in HSCs. Here, we investigated the role of C1q in HSC activation. Human HSCs (LX2) were incubated with C1q to assess HSC activation. C1q and fibrotic markers were assessed using immunohistochemistry, immunoblotting, and quantitative reverse-transcription polymerase chain reaction in cirrhotic rats administered CCl4 for 21 weeks. Serum C1q, liver function, and fibrosis score were measured in 91 patients with chronic liver disease. The correlations between serum C1q and liver function, fibrosis score, and survival prognosis were examined. C1q-activated LX2s showed morphologic changes, up-regulation of CTGF, tissue inhibitors of metalloproteinases (TIMP-1), and alternate Wnt signal genes FZD2, TAZ, and cysteine-rich angiogenic inducer 61 (Cyr61). Cirrhotic rat liver C1q expression correlated with the Azan-positive area and expression of CTGF, TIMP-1, hyaluronan synthase (HAS)1, HAS3, and CD44. Expression of C1q protein and C1q, CTGF, and TIMP-1 genes were higher in deceased cirrhotic rat livers compared to surviving rats. Human serum C1q levels increased in liver cirrhosis compared to chronic hepatitis and correlated with liver fibrosis and functional markers. Ten patients suffered liver-related death over a 66-month observation period. The C1q cut-off value (11 mg/dl) showed patients with serum values < 11 mg/dl had longer rates of survival compared to C1q ≥ 11 mg/dl. Conclusion: C1q-mediated HSC activation in liver fibrosis is associated with CTGF elevation. Additionally, serum C1q may be diagnostic for survival in human chronic liver diseases.

The prognostic potential of fragmented CK18 serum levels in HCC patients reflecting disease progression and overall hepatocyte damage.

Background: Fragmented cytokeratin 18 (fCK18) is released from damaged hepatocytes undergoing apoptosis and is recognized as a liver condition biomarker. We have developed a highly sensitive serum fCK18 CLEIA and reported that serum levels of this caspase-derived protein were significantly associated with hepatocyte ballooning, thus assisting in the accurate diagnosis of nonalcoholic steatohepatitis (NASH). We aim to investigate serum fCK18 levels in a variety of chronic liver diseases and to explore its potential as a prognostic marker of survival in hepatocellular carcinoma (HCC) patients. Methods: Serum fCK18 levels were measured using a highly sensitive CLEIA in 497 chronic liver disease patients (297 outpatients and 200 hospitalized with HCC). Results: In 497 chronic liver disease patients, serum fCK18 levels were significantly correlated with overall liver condition, including ALT, FIB-4 index and albumin-bilirubin (ALBI) score and were significantly increased in patients with HCC. In 200 HCC patients, serum fCK18 levels were significantly correlated with alpha-fetoprotein (AFP) and des-gamma-carboxy prothrombin (DCP), and were significantly associated with HCC stage, whereas FIB-4 index and ALBI score were not changed based on HCC stage. The Survival group had significantly lower levels of serum fCK18, AFP, DCP, FIB-4 index and ALBI score. A ROC analysis yield area under the curve (AUC) value of 0.728 for serum fCK18 is a significantly high value when compared to AUC measurements for other factors. Notably, AUROC values for serum fCK18 levels were constant in the short- and long-term by time-dependent ROC analysis for the prediction of HCC patient survival. HCC patients with serum fCK18 measured at < 1.15 ng/mL, AFP < 7.7 ng/mL, DCP < 133 mAU/mL, ALBI score < -2.97 or FIB-4 index < 6.4 had significantly longer rates of survival when compared to patients with values exceeding these thresholds. Serum fCK18 (HR, 3.5; P < 0.0001), DCP (HR, 3.2; P < 0.0001) and Barcelona Clinic Liver Cancer (BCLC) (HR, 2.4; P = 0.001) values were independent predictors of patient survival. [Conclusion] Serum fCK18 levels reflect overall liver function, the level of liver fibrosis and the progression of HCC, and are a potential predictor of survival in HCC patients.

Serum Copeptin and Zinc-α2-glycoprotein Levels Are Novel Biomarkers of Tolvaptan Treatment in Decompensated Cirrhotic Patients with Ascites.

Objective The efficacy of tolvaptan, an orally active vasopressin V2-receptor antagonist, has recently been reported in patients with massive ascites unresponsive to conventional diuretics. However, the effect of tolvaptan varies among patients. Recently, the prognostic role of the tolvaptan response in cases of decompensated liver cirrhosis (LC) has been attracting increasing attention. Using serum copeptin (vasopressin precursor), zinc-α2-glycoprotein (ZAG), cystatin C (renal biomarker), neutrophil gelatinase-associated lipocalin (NGAL) and liver-type fatty acid-binding protein (L-FABP), we explored which factors portend a good response to tolvaptan in LC patients with ascites. Methods We enrolled 113 LC patients and divided them into the tolvaptan treatment group and non-treatment group. Tolvaptan (3.75 or 7.5 mg/day) was administrated to 38 LC patients with ascites, and a follow-up assessment was performed after a 7-day tolvaptan treatment regimen. Results We determined the predictive ability for kidney and/or liver damage of serum copeptin, ZAG, cystatin C, NGAL and L-FABP levels in all patients. After 7-day tolvaptan treatment, 19 patients had lost more than 1.5 kg of body weight (Responders), while 19 showed no marked change in their body weight (Non-responders). Basal blood urea nitrogen (BUN) (p=0.0014), serum copeptin (p=0.0265) and serum ZAG levels (p=0.0142) were significantly higher in the Non-responders than in the Responders. BUN (odds ratio 7.43, p=0.0306), copeptin (odds ratio 9.12, p=0.0136) and ZAG (odds ratio 7.43, p=0.0306) were determined to be predictive factors of drug responsiveness using a multivariate logistic regression analysis. Conclusion Serum BUN, copeptin and ZAG levels predict the patient response to tolvaptan, even when measured prior to treatment.

Branched-chain amino acids protect the liver from cirrhotic injury via suppression of activation of lipopolysaccharide-binding protein, toll-like receptor 4, and signal transducer and activator of transcription 3, as well as Enterococcus faecalis translocation.

OBJECTIVES: Branched-chain amino acids (BCAAs) are used as nutritional support and for improving prognosis in liver cirrhosis. Here we investigate the molecular mechanisms of BCAA treatment and liver damage focused on pathways related to lipopolysaccharide-binding protein (LBP). METHODS: Serum LBP levels were measured in cirrhotic patients and in cirrhotic rats treated with BCAA to examine the correlation between liver function and survival. In cirrhotic rats, liver damage, Enterococcus faecalis translocation, serum capsular polysaccharide, and intestinal tight junction levels were assessed. Damaged HepG2 cells were cultured with BCAA-supplemented, BCAA-deficient, or control amino acid medium, followed by examination of LBP expression. RESULTS: Serum LBP levels were significantly increased in deceased patients individuals with liver cirrhosis. The survival rate in patients with lower serum LBP (<3.48 µg/mL) was significantly improved. In BCAA-treated rat liver samples, protein expression of LBP, toll-like receptor 4 (TLR4), and phosphorylated signal transduction and activator of transcription 3 (STAT3) were significantly reduced. Also in BCAA-treated rats, intestinal zonula occludens gene expression was increased, whereas hepatic translocation of E. faecalis and serum capsular polysaccharide levels were reduced. In damaged HepG2 cells, lipopolysaccharide-induced elevation of LBP expression was rapidly and strongly repressed in BCAA-enriched medium. CONCLUSIONS: Serum LBP level is a prognostic biomarker in liver cirrhosis. BCAA treatment reduced translocation of E. faecalis through intestinal tight junction recovery and reduced LBP expression in the liver, which repressed activation of LBP, toll-like receptor 4, and signal transduction and activator of transcription 3. Our findings suggest that BCAA supplementation protects the liver from damage via multiple pathways.

Protein Composition of Circulating Extracellular Vesicles Immediately Changed by Particular Short Time of High-Intensity Interval Training Exercise.

INTRODUCTION/PURPOSE: High-intensity interval training (HIIT) promotes various biological processes and metabolic effects in multiple organs, but the role of extracellular vesicles (EVs) released from a variety of cells is not fully understood during HIIT exercise (HIIT-Ex). We investigated the changes in circulating number and proteomic profile of EVs to assess the effect of HIIT-Ex. METHODS: Seventeen young men (median age, 20 years) were enrolled in the study. Total duration of the HIIT-Ex was 4 min. Blood samples were collected from before HIIT-Ex (pre-HIIT-Ex), at the immediate conclusion of HIIT-Ex (T0), at 30 min (T30), and at 120 min after HIIT-Ex. The pulse rate and systolic blood pressure were measured. Circulating EVs were characterized, and EV proteins were detected via nano liquid chromatography tandem mass spectrometry. RESULTS: The pulse rate and systolic blood pressure at T0 to pre-HIIT-Ex were significantly higher. Circulating EV number was significantly altered throughout the HIIT-Ex, and the source of circulating EVs included skeletal muscle, hepatocytes, and adipose tissue. Proteomic analysis identified a total of 558 proteins within isolated circulating EVs from pre-HIIT-Ex, T0, and T30. Twenty proteins in total were significantly changed at pre-HIIT-Ex, T0, and T30 and are involved in a variety of pathways, such as activation of coagulation cascades, cellular oxidant detoxification, and correction of acid-base imbalance. Catalase and peroxiredoxin II were increased at T0. CONCLUSION: The circulating EV composition can be immediately changed by particularly a short time of HIIT-Ex, indicating that EVs may intercommunicate across various organs rapidly in response to HIIT-Ex.

Comprehensive characterization of hepatocyte-derived extracellular vesicles identifies direct miRNA-based regulation of hepatic stellate cells and DAMP-based hepatic macrophage IL-1ß and IL-17 upregulation in alcoholic hepatitis mice.

Extracellular vesicles (EVs) have been growingly recognized as biomarkers and mediators of alcoholic liver disease (ALD) in human and mice. Here we characterized hepatocyte-derived EVs (HC-EVs) and their cargo for their biological functions in a novel murine model that closely resembles liver pathology observed in patients with alcoholic hepatitis (AH), the most severe spectrum of ALD. The numbers of circulating EVs and HC-EVs were significantly increased by 10-fold in AH mice compared with control mice. The miRNA (miR)-seq analysis detected 20 upregulated and 4 downregulated miRNAs (P < 0.001-0.05) in AH-HC-EVs. Treatment of murine primary hepatic stellate cells (HSCs) with AH-HC-EVs induced α-SMA (P < 0.05) and Col1a1 (P < 0.001). Smad7 and Nr1d2 genes, which were downregulated in HSCs from the AH mice, were predicted targets of 20 miRs upregulated in AH-HC-EVs. Among them were miR-27a and miR-181 which upon transfection in HSCs, indeed repressed Nr1d2, the quiescent HSC marker. AH-HC-EVs were also enriched with organelle proteins and mitochondrial DNA (10-fold, P < 0.05) and upregulated IL-1ß and IL-17 production by hepatic macrophages (HMs) from AH mice in a TLR9-dependent manner. These results demonstrate HC-EV release is intensified in AH and suggest that AH-HC-EVs orchestrate liver fibrogenesis by directly targeting the quiescent HSC transcripts via a unique set of miRNAs and by amplifying HSC activation via DAMP-based induction of profibrogenic IL-1ß and IL-17 by HMs. KEY MESSAGES: • Circulating EVs and HC-EVs were increased in AH mice compared with control mice • AH-HC-EVs were enriched in miRNAs, organelle proteins, and mitochondrial DNA • AH-HC-EVs increased cytokine production by AH-HMs in a TLR9-dependent manner.

Neutrophil gelatinase-associated lipocalin level is a prognostic factor for survival in rat and human chronic liver diseases.

Chronic liver disease patients often have complications, such as hepatocellular carcinoma (HCC) and acute bacterial infection. Model for end-stage liver disease and Child-Pugh scores are useful prognostic factors for chronic liver diseases but not for all chronic conditions, such as HCC. Our investigative aim targeted the prognostic abilities of neutrophil gelatinase-associated lipocalin (NGAL) in rat and human chronic liver diseases. Blood NGAL levels were measured by enzyme-linked immunosorbent assay in rats with cirrhosis and 96 patients with chronic liver disease and HCC. We examined the correlation between blood NGAL levels and liver functions as well as survival. In our rat model, liver NGAL expression was assessed by immunostaining, real-time quantitative polymerase chain reaction, and immunoblot. In rats with cirrhosis, blood NGAL levels were continuously and significantly elevated in the deceased group and were significantly correlated with liver functions. Liver NGAL, toll-like receptor 4, and interleukin-6 levels were increased in the deceased group compared to the survival group. Blood NGAL levels were significantly correlated with liver NGAL levels, indicating blood NGAL was derived from the liver. In patients with chronic liver disease, blood NGAL levels were associated with liver function and renal function. Blood NGAL levels were significantly increased in patients with chronic liver disease with HCC compared to without HCC. For the survival group, 38 out of 96 patients were dead in the average follow-up period of 9.9 months. The patients with blood NGAL ≤119 ng/mL had significantly longer rates of survival compared to patients with blood NGAL >119 ng/mL. Conclusion: Blood NGAL predicts the survival rate in rat and human chronic liver diseases. Our findings suggest blood NGAL may be prognostic of survival in chronic liver diseases complicated by HCC. (Hepatology Communications 2017;1:946-956).