Introduction and Objectives In Latin America, the familial risk of liver fibrosis in steatotic liver disease (SLD) remains poorly characterized. This multinational study aimed to assess the familial risk of liver fibrosis across SLD subtypes in the region. Materials and Methods Cross-sectional analysis of prospectively enrolled adults with SLD (2023 criteria) at 16 tertiary centers in seven countries (2024–2026). We recorded a family history of advanced fibrosis in first-degree relatives. Fibrosis was assessed by liver biopsy or vibration-controlled transient elastography (VCTE) when biopsy was unavailable (significant fibrosis: biopsy F2 or VCTE 8 kPa; advanced fibrosis: biopsy F3 or VCTE 12 kPa). We used logistic regression to model the association of family history of advanced fibrosis with liver fibrosis, adjusting for age, sex, ethnicity, body mass index (BMI), type 2 diabetes mellitus (T2DM), and SLD subtype. Results We included 592 participants (median age 59 [50–66] years; 50.8% women). Among these, 79.7% had MASLD, 8.3% MetALD, and 12.0% ALD. The prevalence of significant and advanced fibrosis was 40.2% and 32.0%, respectively. A total of 20.4% had a first-degree relative with advanced fibrosis. In the models adjusted for demographic, metabolic, and SLD subtype factors, family history was independently associated with higher odds of significant fibrosis (aOR:1.83, 95%CI:1.13–2.96; p=0.014) and advanced fibrosis (aOR:1.76, 95%CI:1.06–2.93; p=0.030)(Figure). Conclusions In this multinational Latin American cohort of adults with SLD, family history of advanced fibrosis in first-degree relatives was frequent and associated with a higher prevalence of both significant and advanced fibrosis (FONDECYT 1241450).

Introduction and Objectives The Mediterranean diet may play a protective role in steatotic liver disease (SLD), but this relationship remains poorly characterized in Latin America. We aimed to identify the association between Mediterranean diet adherence and the risk of liver fibrosis across the SLD subtypes in Latin America. Materials and Methods Cross-sectional analysis of prospectively enrolled adults with SLD (2023 criteria) at 16 tertiary centers in seven countries (2024–2026). We recorded the Chilean Mediterranean Dietary Index (MDI, adapted for regional use). Fibrosis was assessed by liver biopsy and/or vibration-controlled transient elastography (VCTE) (significant fibrosis: biopsy F2 or VCTE 8 kPa if biopsy unavailable). Analysis included a logistic regression, adjusted for age, sex, ethnicity, BMI, type 2 diabetes mellitus (T2DM), MDI, and SLD subtype. Results We included 491 participants (median age 59 years and 52.8% women). A total of 81.6% had MASLD, 9.6% MetALD, and 8.8% ALD. Significant fibrosis prevalence was 40.6%. The mean MDI was 5.5±1.8, and 35.6% had low adherence, 62.7% moderate adherence, and 1.6% high adherence. Mean MDI differed significantly across SLD subtypes (MASLD:5.6±1.7, MetALD:5.2±1.8, and ALD:4.3±1.7; p=0.007) (Figure). Higher MDI adherence was independently associated with lower odds of significant fibrosis (aOR:0.83; 95%CI:0.74–0.94; p=0.003). Conclusions In this well-characterized multinational cohort of adults with SLD, Mediterranean diet adherence was suboptimal, particularly in ALD. Higher MDI was independently associated with lower odds of significant fibrosis, supporting the Mediterranean diet as a potential modifiable factor across the SLD spectrum (FONDECYT 1241450).

Introduction and Objectives Alcohol-associated hepatitis (AH) is a severe, cholestatic form of alcohol-associated liver disease with high short-term mortality diagnosed largely on clinical features. We quantified circulating bile acids (BAs), extracellular vesicles (EV)-associated-BA cargo, and synthesis/regulation markers (C4, FGF19) across the ALD spectrum to identify diagnostic biomarkers for AH. Patients and Methods Prospective cohort of patients with ALD in Chile (2020–2024), including alcohol use disorder (AUD), cirrhosis, AH, and healthy controls. Serum BAs were measured by tandem mass spectrometry, EV-associated BA cargo in plasma-derived EVs, and FGF19 by ELISA. Data were log2-transformed and analyzed using left-censored-Tobit models with Tukey-adjusted-contrasts. Results Among 147 participants (AH n=49; cirrhosis n=34; AUD n=31; controls n=33), total BA were significantly higher in AH versus controls (log2FC 2.92, p<0.05). The largest increases in AH were observed for taurocholic-acid (TCA) and taurochenodeoxycholic-acid (TCDCA) (log2FC 5.27 and 5.37, both p<0.0001). In cirrhosis, TCDCA showed the greatest increase (log2FC 4.55, p<0.001), while lithocholic-acid (LCA) was most elevated in AUD (log2FC 2.71, p<0.01). Deoxycholic-acid (DCA) was markedly reduced in AH (log2FC -3.73, p<0.0001). C4 was strongly suppressed in AH (log2FC -3.78, p<0.0001) with concomitant FGF19 elevation (log2FC 1.17, p<0.01). EV counts were increased across ALD, highest in AH and AUD. In ROC analysis, individual BA outperformed EV-associated-BAs; C4 showed the strongest discrimination (AUC=0.83). Conclusions ALD is associated with distinct BA- and EV-profiles, most pronounced in AH. C4 outperformed total BA and EV-associated measures, supporting pathway-specific biomarkers for AH diagnosis.

Introduction and Objectives Latin America is underrepresented in prospective steatotic liver disease (SLD) studies. This multinational study assessed advanced fibrosis risk across SLD subtypes in Latin America. Materials and Methods Cross-sectional analysis of prospectively enrolled adults with SLD (2023 criteria) at 16 tertiary centers in seven countries (2024–2026). We performed standardized interviews and assessed fibrosis using liver biopsy or vibration-controlled transient elastography (VCTE) when biopsy was unavailable (advanced fibrosis: biopsy F3 or VCTE 12 kPa). We used logistic regression adjusted for age, sex, ethnicity, BMI, type 2 diabetes mellitus (T2DM), physical activity, and SLD subtype. Results We included 914 participants; median age was 62 years, 56.6% were women, median BMI was 30.3 kg/m2, and 46.4% had T2DM. Based on historical diagnoses, 86.2% had MASLD, 5.4% MetALD, and 8.4% ALD (Figure A). At enrollment, 87.8% with prior MetALD reported alcohol intake within the MASLD alcohol range, while 50.7% and 46.5% with prior ALD were within the MASLD and MetALD alcohol ranges, respectively. Advanced fibrosis prevalence was 35,5%. Age (aOR:1.05, 95%CI:1.03–1.07; p<0.0001), T2DM (aOR:3.65, 95%CI:2.32–5.74; p<0.0001), MetALD (aOR:2.72, 95%CI:1.32–5.58; p=0.007) and ALD (aOR:21.33, 95%CI:9.02–50.44; p<0.0001) were independently associated with higher odds of advanced fibrosis, whereas those who walked >10,000 daily steps had lower odds (aOR:0.06, 95%CI:0.01–0.35; p=0.002)(Figure B). Conclusions In this multicenter Latin American SLD cohort, advanced fibrosis was common and mainly associated with alcohol-related SLD subtypes, T2DM, and older age, while physical activity was protective (FONDECYT 1241450).

Introduction and Objectives: Severe alcohol-associated hepatitis (sAH) is an acute liver disease with high mortality. While plasma exchange (TPE) improves survival in ACLF, its role in sAH is unknown. This pilot study evaluated TPE’s effect on bile acid (BA) profiles and clinical outcomes. Materials and Methods: We retrospectively analyzed 11 patients with sAH treated with five TPE sessions at a center in Banská Bystrica, Slovakia. Serum and effluent BA concentrations were quantified by liquid chromatography-tandem mass spectrometry. Clinical and laboratory data were collected pre- and post-TPE. Results: The median age was 48.7 years; two (18%) were women. Median MELD decreased from 34.3 to 24.6 post-TPE. Five patients (45.5%) died. BA composition between serum and effluent was similar (p=0.689), but UDCA concentrations differed significantly (399.6 vs. 669.4 ng/mL; p=0.04). Before TPE, deceased patients had higher levels of total BA (75,213 vs. 44,736 ng/mL; p=0.026), glycine-conjugated BA (49,769 vs. 28,350 ng/mL; p=0.013), total conjugated BA (73,837 vs. 43,881 ng/mL; p=0.026), G-UDCA (19,038 vs. 7,819 ng/mL; p=0.021), LCA (6.34 vs. 4.62 ng/mL; p=0.048), and G-CDCA (21,132 vs. 11,370 ng/mL; p=0.012). In the effluent, deceased patients had higher total unconjugated BA (3,512 vs. 871 ng/mL; p=0.032), UDCA (3,353 vs. 520 ng/mL; p=0.026), and DCA (25.9 vs. 17.5 ng/mL; p=0.047). Conclusions: Patients who died had distinct BA profiles before and after TPE. These findings suggest TPE modifies circulating BA, and specific profiles may predict poor outcomes. Larger studies are needed to clarify their clinical relevance.

Introduction and Objectives: Metabolic Dysfunction-Associated Steatotic Liver Disease (MASLD) is a leading cause of chronic liver disease with rising global prevalence. Bile acids (BAs), beyond their role in lipid digestion, act as key metabolic regulators. Alterations in BA composition have been implicated in MASLD pathogenesis and may serve as biomarkers for disease progression. Previous studies have reported stage-specific changes in BA profiles; however, their association with histological severity remains to be fully elucidated. Objectives: To assess serum BA concentrations in a liver biopsy-characterized MASLD cohort and to investigate their relationship with histological severity, distinguishing between isolated steatosis and Metabolic Dysfunction-Associated Steatohepatitis (MASH) Materials and Methods: A total of 127 patients with MASLD were included, comprising 38 with isolated steatosis and 89 with MASH. Plasma BA levels were quantified using High-Performance Liquid Chromatography (HPLC). Results: Patients with MASH showed significantly higher total serum BA levels compared to those with steatosis. Eight individual BAs were markedly elevated in the MASH group, including deoxycholic acid, chenodeoxycholic acid, their glycine conjugates, glycocholic acid and its glycine conjugate, as well as ursodeoxycholic acid and its taurine conjugate. Conclusions: Elevated plasma BA levels in MASH suggest a potential role for BAs as non-invasive markers of disease severity in MASLD. These findings support further investigation into BA profiling as a diagnostic and prognostic tool in the clinical management of MASLD.

Prostate cancer (PCa) is the second leading cause of cancer-related death among men in most Western countries. Current therapies for PCa are limited, often ineffective, and associated with significant side effects. As a result, there is a growing interest in exploring new therapeutic agents, particularly from the polyphyletic group of algae, which offers a promising source of compounds with anticancer properties. Our research group has focused on investigating the effects of a novel oleoresin from Gracilaria chilensis, known as Gracilex®, as a potential therapeutic agent against PCa using both in vitro and in vivo models. Our findings indicate that Gracilex® exhibits a time- and dose-dependent inhibitory effect on cell survival in LNCaP and PC-3 PCa, reducing viability by over 50% and inducing apoptosis, as evidenced by a significant increase in activated caspase-3 expression in both cell lines. Moreover, Gracilex® significantly reduces the proliferation rate of both LNCaP and PC-3 prostate cancer cell lines, as evidenced by a marked decrease in the growth curve slope (p = 0.0034 for LNCaP; p < 0.0001 for PC-3) and a 40–50% reduction in the proportion of Ki-67-positive PCa cells. In addition, Gracilex® significantly reduces in vitro cell migration and invasion in LNCaP and PC-3 cell lines. Lastly, Gracilex® inhibits tumor growth in an in vivo xenograft model, an effect that correlates with the reduced PCa cell proliferation observed in tumor tissue sections. Collectively, our data strongly support the broad antitumoral effects of Gracilex® on PCa cells in vitro and in vivo. These findings advance our understanding of its potential therapeutic role in PCa and highlight the relevance of further investigating algae-derived compounds for cancer treatment.

Introduction and Objectives Prospective data on liver-fibrosis risk among Latin Americans with MASLD remain scarce, although genetic susceptibility and lifestyle behaviors may heighten vulnerability. This multinational, prospective study aims to define the principal determinants of fibrosis in this high-risk population across Latin America. Materials and Methods We performed a cross-sectional baseline analysis of the STELLA study, which is prospectively enrolling adults with MASLD (2023 criteria) at 10 centers (Argentina 15.1%, Brazil 66.2%, Chile 5.9%, Colombia 1.9%, Mexico 0.3%, Peru 10.6%). Alcohol intake, dietary patterns, and vibration-controlled transient elastography (VCTE) were assessed in all participants. When biopsy was unavailable, fibrosis was staged by liver stiffness measurements (LSMs) on VCTE cut-offs (advanced ≥ 8.8 kPa, cirrhosis ≥ 11.8 kPa). Factors associated with liver stiffness were examined with multivariable linear regression adjusted for age, sex, body mass index (BMI), type 2 diabetes mellitus (T2DM), hypertension, and dyslipidemia. Results A total of 370 participants were analyzed (median age 66 [58–73] years; 66.7% women; median BMI 30.9 [27.5–34.8] kg/m2). The prevalence of T2DM was 55.8%, hypertension 38.3%, and dyslipidemia 39.4%. The median alcohol intake was 0 [0–28] grams/week. Median liver stiffness was 9.2 [6.1–16.6] kPa, with advanced fibrosis present in 53.2% and cirrhosis in 18.8%. In the adjusted model, female sex (β = +3.0 kPa; 95%CI 0.2–5.8; p=0.034), T2DM (β = +4.9 kPa; 95%CI 2.2–7.6; p<0.001), and dyslipidemia (β = +3.9 kPa; 95%CI 1.2–6.5; p=0.005) were independently associated with higher LSM values, with T2DM showing the strongest effect (Figure). Conclusions In this well-characterized cohort of Latin-American adults with MASLD, female sex, T2DM, and dyslipidemia emerged as leading risk factors for liver fibrosis. The STELLA project, including a larger sample and longitudinal follow-up, may further clarify the natural history of MASLD in Latin America (FONDECYT 1241450).

Introduction and Objectives Insulin-like growth factor 1 (IGF-1) is a hepatokine that has a protective effect on fatty liver. Previous studies in healthy subjects suggest that isocaloric exercise (with neutral caloric balance) would increase serum levels of IGF-1. This study aimed to evaluate the effect of moderate isocaloric exercise (ICBE) and negative caloric balance exercise (NCBE) on serum levels of IGF-1 in subjects with initial and advanced (F3-4) MAFLD (Metabolic Associated Fatty Liver Disease). Materials and Methods Prospective trial in postmenopausal women undergoing supervised and standardized exercise at moderate intensity (1 hour, 3 times per week). The study includes subjects with initial MAFLD (F0-2 Fibroscan <8 kPa) and advanced MAFLD (F3-4, Fibroscan >8 kPa). The protocol consisted of an initial two-week period of ICBE (with nutritional supplement) followed by two weeks of NCBE (without supplement). Using the t-student test for paired samples, the change was analyzed pre vs. post-protocol, and the comparison between groups used the analysis for unpaired samples. Results We recruited 27 subjects (20 non-advanced MAFLD and 7 advanced MAFLD). We demonstrated that: (1) Exercise did not significantly increase IGF-1 levels in MAFLD; (2) There was a tendency for subjects with initial MAFLD to have higher IGF-1 levels than subjects with advanced MAFLD before and after exercise, which became significant after 2 weeks of exercise (F0-2 67.9 + 6.4 (ng/mL) versus F3-4 41.2 + 5.3 (ng/mL), p 0.047); and (3) There were no significant differences in IGF-1 levels between ICBE and NCBE (figure 1). Conclusions Subjects with advanced MAFLD tend to have lower IGF-1 levels than subjects with initial MAFLD, which becomes significant after 2 weeks of exercise. This suggests that the response to exercise in terms of changes in hepatokines (IGF-1) varies depending on the stage of the disease.

Introduction and Objectives The liver is considered an important immunological site where natural killer (NK) cells mediate the immune response and tumoral immune surveillance. Understanding the involvement of NK cell function at different stages of metabolic fatty liver disease (MAFLD) is crucial to understand the oncogenic risk of MAFLD and hepatocellular carcinoma (HCC) carcinogenesis. This study aims to characterize the phenotype and function of peripheral NK cells in subjects with different stages of MAFLD. Materials and Methods We recruited 15 patients with non-cirrhotic MAFLD (NC-MAFLD), 18 with cirrhosis (CR-MAFLD), and 7 with HCC and compared them with 10 control subjects (HD). Peripheral blood NK cell analysis was performed using multiparametric flow cytometry to characterize NK cells in terms of maturation and function. LDH (lactate dehydrogenase) release assay was used to assess cytotoxicity against tumor cells in isolated NK cells. The results are expressed in percentages in Figure 1 with statistical analysis. Results NK cells from patients with NC-MAFLD have a significantly lower cytotoxic capacity than controls (HD=89.7% vs. MAFLD=54.06%, p=0.0115).CD107a, a marker of NK cell degranulation, was significantly reduced in NC-MAFLD and CR-MAFLD after exposure to tumor cells. IFN- (cytotoxicity marker) is reduced in all three MAFLD groups compared to HD. Activation of NKG2D and CXCR3 receptors is significantly decreased in all three MAFLD groups compared to controls, while CD69 is decreased in NC-MAFLD and CR-MAFLD. In addition, the NKG2A (inhibitory receptor) is also decreased (Figure N°1, attached). Markers known to be involved in the NK cell apoptosis process, TRAIL, and FASL, are increased in the cirrhosis and HCC group. Conclusions These results suggest that early stages in patients with MASLD (particularly NC-MAFLD) have NK cell dysfunction with reduced cytotoxic capacity and activating receptors. These findings suggest NK cells exhaustion could be present in early stages of MASLD.

Conflict of interest: No Introduction and Objectives: Liver fibrosis is an important prognostic factor in alcohol-associated liver disease (ALD) and metabolic dysfunction-associated steatohepatitis liver disease (MASLD). New drugs in steatotic liver disease (SLD), such as Resmetirom, are indicated in individuals with at least significant fibrosis. Cytokeratin-18 is a hepatocyte cytoskeleton protein that is released during apoptosis in its cleaved form by caspases (M30) and can be used as a non-invasive test (NIT) to stratify liver fibrosis. However, data on its performance is scarce in the Hispanic population. We aim to evaluate the diagnostic performance and additive value of M30 to identify significant fibrosis in a cohort of patients with ALD and MASLD. Patients / Materials and Methods: We conducted a cross-sectional cohort study of patients with ALD and MASLD who underwent liver biopsy or transient elastography between 2014–2023. The cutoff points for significant fibrosis (F2) and cirrhosis by transient elastography were ≥7.8 and ≥12.5 kPa, respectively. A receiver operator characteristic (ROC) was used to assess the performance of M30 and FIB-4. Results and Discussion: We included 55 ALD and 43 MASLD patients. The median age was 51 [42–60] years and 70.4% were male. Median liver stiffness was 6.8 [4.6–27.9] kPa and median M30 190.4 [146-274.8] U/l. Around 41.8% had F2 and 33.6% had cirrhosis. FIB-4 outperformed M30 in predicting significant fibrosis (AUROC 0.88 vs. 0.66, p-value=0.007) and cirrhosis (AUROC 0.93 vs. 0.56, p-value<0.001) (Figure 1). Five out of 29 (17.2%) patients had a low FIB-4 (<1.3) but significant fibrosis; in this scenario, M30 correctly identified F2 in 4 (80%) of them. Thus, the misclassification of significant fibrosis was reduced from 5.1% to 1.0% using a stepwise assessment with FIB-4 and then M30. Conclusions: M30 had limited diagnostic value in detecting liver fibrosis in the Hispanic population, but its use in combination with FIB-4 can identify more patients with significant fibrosis than FIB-4 alone.

Introduction and Objectives: Alcohol consumption is among the five main factors responsible for the burden of disease and mortality. It is associated with changes in serum bile acids, and in recent years, extracellular vesicles (EV´s) and their Cargo have shown special interest in various lines of research due to their role in intercellular communication. This study aimed to characterize the serum levels of bile acids, changes in their composition, extracellular vesicles and their cargo in alcohol-associated liver disease (ALD). Materials and Methods: Prospective cohort, years 2019-2021. They were divided into four groups; control group, alcohol-associated hepatitis (AH), alcohol-related cirrhosis, and alcohol use disorder (AUD). Measurement of serum bile acids and C4 was performed through Liquid Chromatography /Tandem Mass Spectrometry, serum measurement of FGF19 and the serum concentration of extracellular vesicles through NTA (nanoparticle tracking analysis) and their cargo of bile acids. Results: A greater concentration of total bile acids was measured in the AUD group (1366.28 ng/ml) compared to the control group (552.42 ng/ml) (p = 0.003). The concentration of chenodeoxycholic acid is higher in the group of patients with AH (734.23 ng/ml) (p=0.04). The EV´s concentration is higher in the HA groups (1.292 E^11 ± 6.4E^10 particles/ml) and in AUD (9.9E^10+ 4.9E^ particles/ml) (p=0.005). It was possible to analyze the Cargo of BA in exosomes with proportional differences between the groups. Conclusions: Serum bile acids, both in concentration and composition, are modified in patients with AUD and HA, respectively; both present a higher concentration of exosomes, which could be a hepato-specific, dynamic and potentially prognostic biomarker in subjects with ALD.

Introduction and Objectives: Nonalcoholic-fatty-liver disease (NAFLD) is considered the hepatic manifestation of metabolic syndrome (MetS). Mineralocorticoid receptor (MR) activation is associated with increased risk of MetS but few studies have assessed the role of liver MR on NAFLD. We aimed to evaluate the effect of MR modulation by sodium intake in liver injury in experimental models of NAFLD. Materials and Methods: C57BL/6J mice were fed either a high-fat-diet (HFD) or a choline/methionine deficient (MCD) diet with different sodium concentrations. Hepatic concentration of lipid species, serum aldosterone levels, expression of MR, proinflammatory and profibrotic markers and liver histology were assessed. Results: Mice fed with High-Na+/HFD showed a lower MR expression in liver (p = 0.01) and less steatosis on histology (p = 0.04). Consistently, animals from this group exhibited lower levels of serum aldosterone (p = 0.028) and lower hepatic triglyceride content (p = 0.008). This associated to a reduced expression of lipogenic genes, significant changes in lipid subspecies, lower HOMA-IR (p < 0.05), and lower expression of pro-inflammatory and profibrotic markers compared to those mice fed a Low-Na+/HFD. Additionally, mice fed a High-Na+/HFD showed higher expression of salt-inducible kinase (SIK)-1 and lower expression of serum-and-glucocorticoid-inducible kinase (SGK)-1. Similar results were observed with the MCD diet model. Conclusion: We identified in two experimental models of NAFLD that High-Na+ diet content is associated to lower serum aldosterone levels and hepatic MR downregulation, associated to decreased steatosis and reduced de novo hepatic lipogenesis, proinflammatory and profibrotic markers. Decreased activation of hepatic MR seems to generate beneficial downstream inhibition of lipogenesis in experimental NAFLD. (C) 2021 Fundacion Clinica Medica Sur, A.C. Published by Elsevier Espa?a, S.L.U.

Background: The transition from steatosis to non-alcoholic steatohepatitis (NASH) is a key issue in non-alcoholic fatty liver disease (NAFLD). Observations in patients with obstructive sleep apnea syndrome (OSAS) suggest that hypoxia contributes to progression to NASH and liver fibrosis, and the release of extracellular vesicles (EVs) by injured hepatocytes has been implicated in NAFLD progression. Aim: To evaluate the effects of hypoxia on hepatic pro-fibrotic response and EV release in experimental NAFLD and to assess cellular crosstalk between hepatocytes and human hepatic stellate cells (LX-2). Methods: HepG2 cells were treated with fatty acids and subjected to chemically induced hypoxia using the hypoxia-inducible factor 1 alpha (HIF-1 alpha) stabilizer cobalt chloride (CoCl2). Lipid droplets, oxidative stress, apoptosis and pro-inflammatory and pro-fibrotic-associated genes were assessed. EVs were isolated by ultracentrifugation. LX-2 cells were treated with EVs from hepatocytes. The CDAA-fed mouse model was used to assess the effects of intermittent hypoxia (IH) in experimental NASH. Results: Chemical hypoxia increased steatosis, oxidative stress, apoptosis and pro-inflammatory and pro-fibrotic gene expressions in fat-laden HepG2 cells. Chemical hypoxia also increased the release of EVs from HepG2 cells. Treatment of LX2 cells with EVs from fat-laden HepG2 cells undergoing chemical hypoxia increased expression pro-fibrotic markers. CDAA-fed animals exposed to IH exhibited increased portal inflammation and fibrosis that correlated with an increase in circulating EVs. Conclusion: Chemical hypoxia promotes hepatocellular damage and pro-inflammatory and pro-fibrotic signaling in steatotic hepatocytes both in vitro and in vivo. EVs from fat-laden hepatocytes undergoing chemical hypoxia evoke pro-fibrotic responses in LX-2 cells.

Background: Obstructive sleep apnea syndrome (OSAS) is associated to intermittent hypoxia (IH) and is an aggravating factor of non-alcoholic fatty liver disease (NAFLD). We investigated the effects of hypoxia in both in vitro and in vivo models of NAFLD. Methods: Primary rat hepatocytes treated with free fatty acids (FFA) were subjected to chemically induced hypoxia (CH) using the hypoxia-inducible factor-1 alpha (HIF-1 alpha) stabilizer cobalt chloride (CoCl2). Triglyceride (TG) content, mitochondrial superoxide production, cell death rates, cytokine and inflammasome components gene expression and protein levels of cleaved caspase-1 were assessed. Also, Kupffer cells (KC) were treated with conditioned medium (CM) and extracellular vehicles (EVs) from hypoxic fat-laden hepatic cells. The choline deficient L-amino acid defined (CDAA)-feeding model used to assess the effects of IH on experimental NAFLD in vivo. Results: Hypoxia induced HIF-1 alpha in cells and animals. Hepatocytes exposed to FFA and CoCl2 exhibited increased TG content and higher cell death rates as well as increased mitochondrial superoxide production and mRNA levels of pro-inflammatory cytokines and of inflammasome-components interleukin-1 beta, NLRP3 and ASC. Protein levels of cleaved caspase-1 increased in CH-exposed hepatocytes. CM and EVs from hypoxic fat-laden hepatic cells evoked a pro-inflammatory phenotype in KC. Livers from CDAA-fed mice exposed to IH exhibited increased mRNA levels of pro-inflammatory and inflammasome genes and increased levels of cleaved caspase-1. Conclusion: Hypoxia promotes inflammatory signals including inflammasome/caspase-1 activation in fat-laden hepatocytes and contributes to cellular crosstalk with KC by release of EVs. These mechanisms may underlie the aggravating effect of OSAS on NAFLD.