The potential role of Omentin-1 in Obesity-Related Metabolic Dysfunction-Associated Steatotic Liver Disease: Evidence from translational studies

The potential role of Omentin-1 in Obesity-Related Metabolic Dysfunction-Associated Steatotic Liver Disease: Evidence from translational studies | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article The potential role of Omentin-1 in Obesity-Related Metabolic Dysfunction-Associated Steatotic Liver Disease: Evidence from translational studies Noel Salvoza, Pablo Giraudi, Silvia Gazzin, Deborah Bonazza, Silvia Palmisano, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3192103/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 11 Dec, 2023 Read the published version in Journal of Translational Medicine → Version 1 posted 4 You are reading this latest preprint version Abstract Background Obesity, characterized by visceral adipose tissue (VAT) expansion, is closely associated with metabolic dysfunction-associated steatotic liver disease (MASLD). Recent research has highlighted the crucial role of the adipose tissue-liver axis in the development of MASLD to its progressive form, metabolic dysfunction-associated steatohepatitis (MASH). In this study, we investigated the potential role of omentin-1, a novel adipokine expressed by VAT, in obesity-related MASLD pathogenesis. Methods Through in silico analysis of differentially expressed genes in VAT from obese patients with and without MASH, we identified omentin-1 as a significant candidate. To validate our findings, we measured omentin-1 levels in VAT and plasma of lean controls and obese patients with biopsy-proven MASLD. Additionally, we assessed omentin-1 expression in the VAT of a juvenile mice MASLD model. In vitro and ex vivo studies were conducted to investigate the effects of omentin-1 on MASLD-related mechanisms, including steatosis, inflammation, ER stress, and oxidative stress. We also analyzed the impact of D-glucose and insulin on VAT omentin-1 levels ex vivo . Results Compared to the lean group, the obese groups exhibited significantly lower VAT and plasma levels of omentin-1. Interestingly, within the MASH group, fibrosis did not affect omentin-1 levels. Likewise, VAT of mice fed with high-fat diet, showing histological signs of MASH showed decreased omentin-1 levels as com-pared to their control diet counterpart. In vitro experiments on fat-laden human hepatocytes revealed that omentin-1 did not affect steatosis but significantly reduced TNF-α levels, ER stress, and oxidative stress. Similar results were obtained using ex vivo VAT explants from obese patients upon omentin-1 supplementation. Furthermore, omentin-1 decreased the expression of NF-κB mRNA, both in vitro and ex vivo . Ex vivo VAT explants showed that D-glucose and insulin significantly reduced omentin-1 mRNA expression and protein levels. Conclusions Collectively, our findings suggest that reduced omentin-1 levels contribute to the development of MASLD. Omentin-1 supplementation mitigates inflammation, ER stress, and oxidative stress, probably via inhibiting the NF-κB pathway and might also play a role in the regulation of glucose and insulin metabolism. Further research is warranted to explore omentin-1 as a potential therapeutic target and/or biomarker for MASLD. MASLD MASH obesity omentin-1 translational models Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Obesity, characterized by adipose tissue (AT) mass expansion, is seen in 51% of MASLD and 81% of MASH patients globally ( 1 ). Fat accumulation in the organs, especially the visceral tissue, leads to their dysfunction, promoting ectopic fat accumulation in the liver, inflammation, ER stress, oxidative stress, and impairment of glucose metabolism, among others ( 2 ). Aside from its role as the regulator of lipid flux to the liver, AT is also recognized as a major endocrine organ producing a large array of mediators, known as adipocytokines ( 3 ). The role of adipocytokines in AT-liver crosstalk has become an important area of MASLD research because of the potential utility of those proteins/mediators, as diagnostic markers and/or therapeutic targets — since no reliable diagnostic marker and pharmacological treatment are currently approved for the disease. Omentin-1 (also known as intelectin-1), a novel adipocytokine, is a peptide of 313 amino acids containing a secretory signal sequence and a fibrinogen-related domain ( 4 ). Omentin-2, a homolog with 83% amino acid identity with omentin-1 is found in the same chromosomal region ( 5 ). Several reports indicated that omentin-1 and − 2 are highly expressed in visceral adipose tissue (VAT), but omentin-1 was shown to be the major circulating isoform in human plasma ( 6 ). Omentin-1 was observed to be secreted exclusively into the culture medium of VAT, not subcutaneous adipose tissue (SAT), with stromal vascular cells playing a primary role in its production over adipocytes within VAT ( 4 ). Plasma omentin-1 level decreases in overweight and obese humans, while it increases after obese patients lose weight or after taking antidiabetic drugs ( 7 , 8 ). Regarding its biological activity, omentin-1 enhances insulin-stimulated glucose uptake via Akt activation in human adipocytes, suggesting its role in type 2 diabetes mellitus (T2DM) susceptibility ( 4 ). Further, omentin-1 exerts anti-inflammatory effect by ameliorating macrophage activation via inhibiting the NF-κB pathway in obese mice ( 9 ). Both insulin resistance and inflammation are associated with MASLD. These two molecular mechanisms, along with steatosis, oxidative stress, ER stress, and fibrosis, are key pathologic drivers in MASLD development. In this study, we conducted a simple in silico analysis identifying omentin-1 and investigated its role in MASLD for the first time, using different translational approaches. We simultaneously determined the expression of omentin-1 in VAT at both mRNA and protein levels in MASLD patients and mice fed with high-fat diet (HFD). Furthermore, we present novel data regarding the plasma levels of omentin-1 in obese subjects with different stages of MASLD. Successively, to elucidate its role in the liver and VAT, we evaluated its beneficial effects in the MASLD-related pathophysiological mechanisms such as steatosis, inflammation, oxidative stress, and ER stress. Materials and Methods In silico strategy The literature review followed PRISMA guidelines, with the paper by du Plessis et al . ( 10 ) being chosen for its similarity to our morbidly obese cohort and the availability of VAT datasets. The gene expression data set GSE58979 was downloaded from GEO, which included 9 obese VAT samples (group 1) and 7 MASH VAT samples (group 3). Differentially expressed genes (DEGs) were identified using GEO2R through limma method (see Additional file 1). The significance of DEGs was calculated by the t-test and was represented by the p-value. The threshold for the DEGs was set as corrected p-value < 0.05 and log 2 fold change (FC) of |1|. In our systematic strategy, the identifiers (IDs) for protein-coding genes in the consulted data resources were standardized, through mapping to the UniProtKB identifiers on UniProt database and only those IDs were further used. Moreover, datasets of our interest collected from Human Protein Atlas (HPA) were used as in silico sieve filters. Dataset comparison and sub-groups selection was performed by applying Venn diagrams using InteractiVenn web-based tool. Venn diagrams were used as in silico filters to identify the interested proteins, those fulfilling the following desired criteria: visceral adipose tissue-enriched, secreted proteins, secreted in plasma or blood, and not included as part of the housekeeping proteome. We finally selected omentin-1 as the most pertinent gene for our subsequent analysis as it fulfils all the criteria, and it is the only adipocytokine on the list (see Additional file 2). Study Participants The assessment of omentin-1 VAT expression and plasma level was performed retrospectively in a morbidly obese (MO) cohort enrolled in a bariatric surgery program. All patients gave their written consent, and the study has been approved by the local Ethical Committee under protocol N. 22979 (Comitato Etico Regionale Unico, FVG, SSN, Italy). The MO cohort was stratified according to obese (Ob) group = 19; obese MASH (Ob-M) group = 20; and obese MASH with fibrosis (Ob-MF) group = 16. The baseline characteristics of the MO cohort are shown in additional file 3. In addition, a total of 17 lean controls with BMI of 18.5–24.9 kg/m 2 were included in the ELISA study. For PCR and western blot, VAT from 5 lean study participants were used as controls. In vitro model of hepatic steatosis Hepatoma cell line Huh7 (JHSRRB, Cat #JCRB0403) was obtained from the Health Science Research Resources Bank (Osaka, Japan) and grown in DMEM-HG with 10% FBS. Huh7 cells were exposed for 24h to 1200 µM of free fatty acids (FFA) (oleic:palmitic ratio 2:1 µmoL/µmoL) as previously described by our group ( 11 ). To determine the experimental concentration, the cytotoxic effect of FFA (1200 µM) and recombinant omentin-1 (Bio Vendor, Candler, NC, USA), alone or in combination was assessed by MTT colorimetric assay after 24 hours. Ex vivo primary explant culture of VAT VAT explants from MO patients (without T2DM) undergoing bariatric surgery were cultured using the modified protocols of Carswell et al ., 2012 ( 12 ) & Tan et al. , 2008 ( 13 ). Within 30 minutes after the surgery, tissue was minced into small pieces, approximately 5–10 mg per piece (∼1–2 mm 3 ) and transferred into six-well plates (∼100 mg/well) containing 3 mL of appropriate medium. VAT explants were cultured for 24 h with or without the addition of insulin (10 − 5 M, 10 − 7 M) or D-glucose (50 mmol/L, 25 mmol/L). Animal Model (In Vivo) C57Bl/6 mice pups were provided by local SPF animal facility (University of Trieste). Immediately after weaning, mice were housed (22°C ± 2°C) in a 12 h light/dark schedule, and fed ad-libitum with control diet (CD, 811900 Special Diets Services, England) or HFD diet (D12331, Research Diets, New Brunswick, NJ, USA) plus 42 g/L fructose/sucrose in drinking water, as previously described ( 14 ). Based on the knowledge of the model and the experimental goals, diet was continued for 3 weeks and 20 weeks. Liver and epididymal fat (a depot of VAT) were dissected for the histologic evaluation and experimental use, respectively. Blood tests and histology were performed as previously described ( 14 ). All experimental protocols were approved by the local OPBA (Organismo Per il Benessere dell’Animale) and by the Competent National Authority (Ministero della Salute-Direzione Generale della Sanità Animale e dei Farmaci Veterinary. Approval 56/2022PR). Fluorometric determination of intracellular fat content Intracellular fat content in vitro was determined by flow cytometry using Nile red staining, a vital lipophilic dye used to label fat accumulation in the cytosol. After 24h of FFA exposure (with or without omentin-1 treatment), intracellular fluorescence was detected using a Becton Dickinson FACSCalibur System on the FL2 emission channel through a 585 ± 21 nm bandpass filter, following excitation with an argon-ion laser source at 488 nm. Data were collected in 10,000 cells and analyzed using FlowJo (Tree Star Inc., Ashland, OR, USA) analysis software. Quantitative PCR Total RNA was extracted from cell culture harvest and homogenized VAT using Tri-reagent kit (Sigma-Aldrich, MO, USA). cDNA was generated with High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Waltham, MA, USA). Quantitative PCR was performed in CFX Connect Real-Time PCR Detection System (Bio-Rad, Hercules, CA USA) in a specific reaction volume containing 25 ng of cDNA, 1X iQ SYBR Green Supermix, and primer pairs. The relative quantification was made using the Pfaffl modification of the ΔΔCt equation, considering the efficiencies of individual genes and housekeeping genes. Western Blot Analysis The following primary antibodies were used: Omentin-1/Intelectin-1 1:100 (Santa Cruz Biotech, Santa Cruz, CA, USA) and the reference α-tubulin 1:2000 (Santa Cruz Biotech, Santa Cruz, CA, USA). Blots were incubated with anti-mouse IgG-HRP-conjugated secondary antibody (1:500 Omentin-1/Intelectin-1 and 1:2000 for α-tubulin). Protein bands were visualized using the ECL immunoblotting detection system (GE Healthcare, Buckinghamshire, UK) and developed on a C-DiGit ® Blot Scanner (LI-COR Biosciences, NE, USA). Results are expressed as the ratio of omentin-1 protein expression to that of a reference protein, α-tubulin. Relative densitometry analyses of the immunoblots were determined using IMAGE STUDIO software. Glutathione Content Assay Superoxide Dismutase (SOD) Activity Assay The simultaneous assay for both GSH (reduced) and GSSG (oxidized) was done using the modified protocol of Mokrasch and Teschke, 1984 ( 15 ) and were normalized to total µg of proteins. Total SOD activity was also measured (Sigma-Aldrich, MO, USA). Omentin-1 and TNF-α ELISA The plasma level of omentin-1 in patients was measured using Human Omentin-1 ELISA Kit (BioVendor, RD191100200R) and the TNF-α levels of Huh7 and VAT supernatants were quantified by Human TNF alpha ELISA Kit (BioVendor, RAF128R). The levels of protein analytes were normalized to total µg of proteins. Statistical Analysis Unless indicated otherwise, all values are presented as mean ± standard deviation (SD). The normal distribution of variables was evaluated by Kolmogorov–Smirnov test. Differences between two groups were assessed using the Mann-Whitney U test or student’s t-test. Data involving more than two groups were assessed by One-way-ANOVA or Kruskal-Wallis test, followed by post-hoc analysis. Spearman rank correlation was used for the calculation of associations between variables. Specific analysis details are indicated in figure legends. Statistical significance was determined at p < 0.05. All figures and statistical analyses were generated using GraphPad Prism 9 and SPSS 29, respectively. Results In vivo clinical validation Based on in silico analysis, omentin-1 is one of the downregulated genes in VAT of obese MASH. In vivo clinical validation showed decreased omentin-1 mRNA expression in all obese groups as compared to lean controls, independent of the presence of fibrosis (Fig. 1 a). Further, the changes noted at the mRNA level were also reflected at the protein level (Fig. 1 b). Likewise, plasma omentin-1 levels were lower in the obese groups than in the lean control group (Fig. 1 c). Having shown that the plasma level of omentin-1 in obese groups differ, we investigated the relationship of omentin-1 with their clinical and biochemical parameters (n = 55) (Table 1 ). Omentin-1 plasma level had a significant positive correlation with omentin-1 mRNA (ρ = 0.382 p = 0.013) and a significant negative correlation with total cholesterol (ρ = -0.307, p = 0.022). Interestingly, omentin-1 plasma level negatively correlates with ALT (ρ = -0.279, p = 0.039) but not AST. Further, the AST/ALT ratio positively correlates with the omentin-1 plasma level (ρ = 0.285, p = 0.042). Table 1 Correlation of clinical and laboratory parameters with plasma omentin-1 level. Variable rho p value Omentin-1 mRNA 0.382 0.013* BMI (kg/m 2 ) -0.230 0.093 Fasting Glucose (mg/dL) 0.026 0.851 AST (UI/L) -0.206 0.136 ALT (UI/L) -0.279 0.039* AST/ALT Ratio 0.285 0.042* GGT -0.049 0.723 ALP (U/L) -0.034 0.81 Triglycerides (mg/dL) -0.212 0.119 Total Cholesterol (mg/dL) -0.307 0.022* HDL (mg/dL) -0.029 0.836 Insulin (µU/mL) -0.159 0.333 Platelet (x10 9 L) -0.076 0.581 Pearson’s or Spearman’s correlation coefficient (Rho) measures the strength and direction of association between the two variables under study. *p < 0.05. VAT omentin-1 expression in HFD mice Human omentin-1 gene is 80–85% homologous to mice omentin-1 [20]. To investigate the mouse omentin-1 level during diet-induced obesity, we assigned C57Bl/6 littermates to receive either control or high-fat diet, supplemented with fructose/sucrose in drinking water, as described [21]. Mice treated with HFD for 20 weeks developed obesity, dyslipidemia, glycemia, hyperinsulinemia, insulin resistance, and histological signs of MASH as compared to control diet mice (Table 2 ). Real-time PCR showed decreased mRNA expression of omentin-1 in the VAT of HFD versus control diet mice at 20 weeks (Fig. 2a). Similarly, Western blot analysis from representative mice VAT also confirmed the decreased omentin-1 expression in HFD mice versus control (Fig. 2b). Table 2 Anthropometric, biochemical, and histological characteristics of mice. Variable 3 weeks p value 20 weeks p value HFD (n = 8) CD (n = 7) HFD (n = 18) CD (n = 13) Sex (female) 4 (50%) 4 (57.1%) 0.782 11 (61.1%) 7 (53.8%) 0.686 Body Weight (g) 21.3 ± 2.61 18.9 ± 2.25 0.083 39.50 ± 7.38 28.06 ± 3.79 < 0.001*** Body Length (cm) 8.65 ± 0.35 8.36 ± 0.42 0.160 9.27 ± 0.30 8.87 ± 0.32 < 0.001*** BMI (kg/m 2 ) 28.38 ± 2.33 27.14 ± 2.61 0.351 45.82 ± 7.11 35.69 ± 3.40 < 0.001*** Total Cholesterol (mg/dL) 141.50 ± 17.53 86.14 ± 10.75 < 0.001*** 168.22 ± 43.45 85.69 ± 11.71 < 0.001*** HDL (mg/dL) 98.14 ± 13.93 59.43 ± 6.05 < 0.001*** 111.78 ± 25.19 63.69 ± 11.88 < 0.001*** LDL (mg/dL) 82.03 ± 14.42 33.84 ± 9.48 < 0.001*** 110.02 ± 36.82 36.61 ± 10.05 < 0.001*** Triglycerides (mg/dL) 110.00 ± 34.65 87.14 ± 24.31 0.089 74.33 ± 22.47 69.54 ± 15.93 0.516 AST (U/L) 83.60 ± 40.15 73.60 ± 28.04 0.660 247.44 ± 181.57 69.44 ± 20.40 0.008** ALT (U/L) 57.18 ± 33.68 40.20 ± 29.61 0.092 67.35 ± 31.64 46.08 ± 35.41 0.047* Glucose (mg/dL) 329.25 ± 26.00 307.71 ± 34.04 0.188 321.94 ± 79.11 271.00 ± 35.00 0.039* Insulin (µU/mL) 1.33 ± 0.71 0.96 ± 0.25 0.219 2.79 ± 1.88 1.22 ± 0.30 0.042* HOMA-IR 1.08 ± 0.57 0.73 ± 0.22 0.162 2.38 ± 1.78 0.87 ± 0.28 0.010** Steatosis grade (0/1/2/3) 75%/25%/ 0/0 100%/0/ 0/0 0.155 5.6%/66.7%/ 16.7%/11.1% 100%/0/0/0 < 0.001*** Lobular inflammation (0/1/2/3) 62.5%/25%/ 12.5%/0 71.4%/28.6%/ 0/0 0.626 27.8%/27.8%/ 33.3%/11.1% 76.9%/23.1%/ 0/0 0.022* Ballooning (No/Yes) 100%/0 100%/0 - 93.3%/6.7% 100%/0 - Data are shown as mean ± SD for continuous variables, number (%) for binary variables, and frequency for categorical variables. T-test was used to test for significant differences with continuous variables. Chi-Square test was used for categorical variables. ***significant at p < 0.001, **significant at p < 0.01, and *significant at p < 0.05. Figure 2. VAT omentin-1 (a) mRNA expression and (b) protein expression HFD mice and control diet mice. 20 weeks mice fed with HFD (n = 18), showing histological signs of MASH, have significantly lower expression compared to mice fed with a control diet (n = 13). Representative blot and densitometric analysis of omentin-1 normalized to α-tubulin revealed that protein expression is significantly decreased in the VAT of HFD mice as compared to control mice at 20 weeks (n = 3–5 mice/group). * p < 0.05. In vitro effects of omentin-1 on fat-laden hepatocytes Nile red staining through flow cytometry revealed that omentin-1 does not affect steatosis (Fig. 3 a). Interestingly, co-treatment of FFA with omentin-1 decreased the mRNA expression of TNF-α relative to the vehicle control (Fig. 3 b). Consistent with the gene expression results, omentin-1 significantly reduced the release of TNF-α in the cell culture supernatant (Fig. 3 c). Since NF-κB family of inducible transcription factors is activated in response to TNF-α cytokine action, we determined the gene expression of NF-κB p65 in our in vitro model of steatosis. Upon co-treatment with omentin-1, there is a significant reduction of NF-κB expression in fat-laden hepatocytes (Fig. 3 d). To explore the involvement of omentin-1 in hepatocyte ER stress induced by fat overload, we analyzed the expression levels of two ER stress markers ( BiP and CHOP gene markers). The mRNA expression levels of both markers showed a nonsignificant increase upon treatment of FFA (Fig. 3 e). Moreover, both markers showed a nonsignificant decrease upon co-treatment with omentin-1 (Fig. 3 f). The antioxidant role of omentin-1 was evaluated on fat-laden Huh7 cells using glutathione content assay. The reduced glutathione (GSH) and GSH:GSSG ratio showed reduced levels upon FFA treatment and a significant increase upon co-treatment with omentin-1 (Fig. 3 g- 3 i). The exposure to FFA significantly increased the oxidized glutathione (GSSG) content (p < 0.05) vs the vehicle-treated control (Fig. 3 h), indicating a more consistent oxidative stress state. Another important antioxidant defense system is the superoxide dismutase (SOD) enzyme activity. FFA treatment showed increased SOD total activity, probably a compensatory mechanism from oxidative stress. Interestingly, the SOD activity is further enhanced upon co-treatment with omentin-1 (Fig. 3 j). Ex-vivo effects of omentin-1 on VAT explants The addition of recombinant omentin-1 in VAT explants of obese patients significantly reduced the basal TNF-α mRNA expression (Fig. 4 a) and release in the supernatant (Fig. 4 b). Moreover, VAT treated with omentin-1 showed a significant decrease in NF-κB mRNA expression versus control (Fig. 4 c). The mRNA expression level of BiP is significantly reduced upon treatment of omentin-1 (Fig. 4 d) while CHOP mRNA expression is dose-dependently reduced by omentin-1 as compared to the control (Fig. 4 e). The reduced glutathione (GSH) and GSH:GSSG ratio significantly increases upon the addition of omentin-1 300 ng/mL but not omentin-1 150ng/mL (Fig. 4 f, 4 g). On the other hand, the oxidized glutathione (GSSG) almost dose-dependently decreased upon supplementation of omentin-1 (Fig. 4 i). Lastly, omentin-1 supplementation significantly enhanced the SOD activity in both concentrations (Fig. 4 j). Ex vivo effects of D-glucose and insulin on omentin-1 levels Studies revealed that omentin-1 enhances insulin-stimulated glucose uptake in vitro in both omental and subcutaneous adipocytes and its serum levels are reduced in patients with T2DM and glucose intolerance ( 4 , 8 ). We, therefore, hypothesized that omentin-1 level might be affected by glucose and insulin modulation. Using the VAT explants from obese patients (without T2DM), we added either insulin or glucose in the medium and determined the levels of omentin-1. Both glucose and insulin resulted in a significant and almost dose-dependent decrease in omentin-1 mRNA expression levels (Fig. 5 a, 5 b). Likewise, omentin-1 protein levels were also reduced in VAT homogenates showing consistent results with that of mRNA expression (Fig. 5 c, 5 d). Discussion The current study utilized a simple in silico analysis to identify omentin-1 and provided insights regarding its role in MASLD, using a variety of translational approaches. Consistent with previous studies ( 4 , 13 ), we detected omentin-1 mRNA in VAT but not SAT. Both mRNA and protein levels of omentin-1 in VAT are lower in all obese groups than in lean controls. The expression is further decreased in obese groups with MASH (with or without fibrosis) vs the Ob group. Additionally, our in vivo mice results concur with our human validation results. Omentin-1 is reported to be the major circulating form of omentin in human plasma ( 6 ). Interestingly, the results of plasma omentin-1 levels as measured by ELISA are consistent with our VAT mRNA and protein data. Successively, correlation analyses were performed to evaluate the relationship between biochemical parameters and plasma omentin-1 levels in morbidly obese patients. In our study, omentin-1 plasma levels were found to be positively correlated with AST/ALT ratio and negatively correlated with ALT level. These results indicate an association between liver damage and omentin-1 secreted by VAT, supporting the crosstalk theory between the two organs. Our in vivo findings suggest that a reduced level of omentin-1 is associated with MASLD development, probably via VAT–liver crosstalk. Furthermore, when considering only the obese groups in our study, it is unlikely that BMI is solely responsible for their lower omentin-1 levels since all patients had a BMI of > 35 kg/m 2 . Therefore, we also hypothesized that the further decrease in omentin-1 level could be an additive effect of MASLD severity to obesity. As such, the severity of MASLD results from several pathophysiological mechanisms, such as oxidative stress and ER stress, inflammation, and glucose-insulin impairment. To answer our hypotheses, we employed in vitro and ex vivo studies to evaluate the role of omentin-1 in MASLD-related pathophysiological mechanisms. MASLD pathogenesis is complex but the onset of the disease is still represented by the accumulation of fat in the liver ( 16 , 17 ). Our group previously developed an in vitro model of MASH where the exposure of hepatocytes to high concentrations of FFA promotes steatosis, inflammation, oxidative stress, and fibrogenic response, similar to those observed in patients with MASLD ( 18 ). Using this in vitro model to represent the pathologic events in the liver, we evaluated the beneficial effects of recombinant omentin-1. In parallel, we also studied its effects in ex vivo VAT explants obtained from obese patients with MASLD to determine its role in the actual diseased tissue setting. The expression of TNF-α in steatotic hepatocytes and VAT supplemented with omentin-1 has not been examined to date. Here we have demonstrated that omentin-1 reduced the levels of TNF-α in both fat-laden hepatocytes and VAT explants from obese patients. TNF-α is a key mediator in the process of MASLD development by not only promoting inflammatory response, but also mediating insulin resistance, and inducing fibrosis-associated proteins ( 19 ). Hence, the attenuation of TNF-α action may help prevent or delay the development of MASH. NF-κB transcription factor regulates a cascade of inflammatory responses by TNF-α activation ( 20 , 21 ). Based on this premise, we also investigated the role of omentin-1 in NF-κB expression. Indeed, we found that omentin-1 decreased the expression of NF-κB in both fat-laden hepatocytes and VAT explants, suggesting that the anti-inflammatory effect may act via inhibition of this pathway. Obesity, a state of low-grade systemic inflammation, is associated with ROS overproduction and oxidative stress due to mitochondrial dysfunction ( 22 ). As a result, inflammation and oxidative stress are involved in the induction of ER stress signaling pathways and subsequent unfolded protein response (UPR) activation to restore ER homeostasis ( 23 ). This implies that oxidative stress, ER stress, and inflammatory pathways somewhat converge at different stages of obesity resulting in disease progression. Our group and other authors previously reported that fat-laden hepatocytes increase ROS production and ER stress ( 18 , 24 , 25 ). Similarly, growing evidence suggests that excess energy substrate input associated with obesity enhanced ROS generation and ER stress by VAT ( 26 – 28 ). Interestingly, we presented herein that adding omentin-1 mitigated both the oxidative stress and ER stress in our in vitro and ex vivo setups. Specifically, a significant decrease in oxidized glutathione (GSSG) levels and enhanced SOD enzyme activities were observed in fat-laden hepatocytes and VAT explants. In line with these observations, our results also showed that ER stress markers ( BiP and CHOP ) were also reduced by omentin-1 supplementation. Like omentin-1, vaspin, also exerts the same beneficial effects on ER stress–induced metabolic dysfunctions. However, unlike omentin-1, with no known receptor to date, vaspin binds to BiP , which is recruited from ER to the plasma membrane under ER stress ( 29 ). Further investigations are needed to elucidate the role of omentin-1 in oxidative stress and ER stress, as well as to identify its specific receptor. Nonetheless, our findings suggest that oxidative stress and ER stress, as well as inflammation, all of which increase in parallel with metabolic dysfunctions, could be alleviated by omentin-1. Metabolic disorders like obesity, diabetes, and polycystic ovarian syndrome are all characterized by insulin resistance and impairment of glucose metabolism. It has been shown that in vitro supplementation of recombinant omentin-1 enhances insulin-mediated glucose uptake by adipocytes via GLUT4 translocation and Akt phosphorylation ( 4 , 13 ). Furthermore, as reported herein, omentin-1 decreases the ER stress marker BiP , which is also thought to maintain glucose uptake in glucose storage tissues ( 29 ). Thus, given the role of omentin-1 in glucose homeostasis, we hypothesized that hyperinsulinemia and hyperglycemia decrease its expression. We found that upon increasing the concentration of insulin and glucose in the medium of VAT explants, the expression of omentin-1 is significantly decreased. This finding is in line with several studies showing that the reduced omentin-1 in adipose tissue may contribute to the development of insulin resistance and T2DM. However, it should be noted that our findings relate only to obese patients with MASLD without T2DM. Therefore, it would be of interest if we also determine the levels of omentin-1 in VAT of diabetic or lean patients. Conclusions Obesity is a significant risk factor for MASLD, where the expansion of visceral adipose tissue contributes to numerous pathological events, including the dysregulation of adipocytokines. The present study provides evidence that reduced omentin-1 level is associated with obesity-related MASLD. Although the cause-and-effect relationship is still unclear, we are still able to show that omentin-1 is an adipocytokine that plays a significant role in the VAT-liver crosstalk. As an endocrine factor, we report herein that VAT omentin-1 has a protective role against fat-laden hepatocytes showing MASLD-related pathophysiological mechanisms. Locally, omentin-1 was able to regulate obese VAT mechanisms, especially insulin-glucose impairment. Further studies are required to elucidate the biological activity of omentin-1 in obesity-related MASLD with a focus on specific receptor identification, which could then eventually facilitate new drug development. Abbreviations ALT, alanine aminotransferase; AST, aspartate aminotransferase; AT, adipose tissue; AUF, arbitrary unit of fluorescence; BCA, bicinchoninic assay; BiP , binding of immunoglobulin protein; BMI, body mass index; CD, control diet; CHOP , CCAAT/enhancer-binding protein homologous protein; ER, endoplasmic reticulum; FFA, free fatty acids; GGT, gamma-glutamyl transferase; GSH, reduced glutathione; GSSG, oxidized glutathione; HDL, high-density cholesterol; HFD, high-fat diet; HPRT , hypoxanthine-guanine phosphoribosyltransferase; MASLD, metabolic dysfunction-associated steatotic liver disease; MASH, metabolic-associated steatohepatitis; NF-κB , nuclear factor kappa B; NAFLD, nonalcoholic fatty liver disease; SAT, subcutaneous adipose tissue; SOD, superoxide dismutase; TNF-α, tumor necrosis factor alpha; T2DM, type 2 diabetes mellitus; VAT, visceral adipose tissue. Declarations Acknowledgements The authors would like to express their gratitude to all the study participants of the MO cohort. We would like to thank our colleagues in Fondazione Italiana Fegato, as well as our collaborators from Cattinara Hospital and Burlo Pediatric Institute for the valuable insights and assistance. Author Contributions Conceptualization, N.S., N. R., P.G., and C.T.; methodology, N.S, N.R. and P.G; assisted in animal experiments, S.G., D.B., A.R. and F.S.; provided the clinical samples and data collection, S.P., N.M., and F.Z. All authors have read and agreed to the submitted version of the manuscript. Funding This study was supported by Fondazione Italiana Fegato. Pablo Giraudi reports funding from the HORIZON-HLTH-2022-STAYHLTH-02-01, Proposal number 101095672, PRAESIIDIUM. Noel Salvoza is funded by the Department of Science and Technology - Philippine Council for Health Research and Development (DOST-PCHRD), Philippines. Availability of data and materials The dataset GSE58979 used in this study is available in the GEO repository. Ethics approval and consent to participate All patients gave their written consent, and the study has been approved by the local Ethical Committee under protocol N. 22979 (Comitato Etico Regionale Unico, FVG, SSN, Italy). All animal experimental protocols were approved by the local OPBA (Organismo Per il Benessere dell’Animale) and by the Competent National Authority (Ministero della Salute-Direzione Generale della Sanità Animale e dei Farmaci Veterinary. Approval 56/2022PR). Consent for publication All authors have reviewed the final version of the manuscript and approved it for submission. Competing interests The authors declare that they have no conflict of interest. References Younossi ZM, Koenig AB, Abdelatif D, Fazel Y, Henry L, Wymer M. Global epidemiology of nonalcoholic fatty liver disease-Meta-analytic assessment of prevalence, incidence, and outcomes. 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Liu T, Zhang L, Joo D, Sun SC. NF-κB signaling in inflammation. Sig Transduct Target Ther. 2017 Jul 14;2(1):1–9. Panic A, Stanimirovic J, Sudar-Milovanovic E, Isenovic ER. Oxidative stress in obesity and insulin resistance. Explor Med. 2022 Feb 23;3(1):58–70. Bañuls C, Rovira-Llopis S, Lopez-Domenech S, Diaz-Morales N, Blas-Garcia A, Veses S, et al. Oxidative and endoplasmic reticulum stress is impaired in leukocytes from metabolically unhealthy vs healthy obese individuals. Int J Obes. 2017 Oct;41(10):1556–63. Song MJ, Malhi H. The Unfolded Protein Response and Hepatic Lipid Metabolism in Non-alcoholic fatty liver disease. Pharmacol Ther. 2019 Nov;203:107401. Arroyave-Ospina JC, Wu Z, Geng Y, Moshage H. Role of Oxidative Stress in the Pathogenesis of Non-Alcoholic Fatty Liver Disease: Implications for Prevention and Therapy. Antioxidants (Basel). 2021 Jan 26;10(2):174. Alcalá M, Calderon-Dominguez M, Bustos E, Ramos P, Casals N, Serra D, et al. 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Supplementary Files GraphicalAbstract.png Obesity leads to VAT expansion promoting lipolysis and excessive delivery of fatty acids (FA) to the liver, which is further exacerbated by insulin resistance. Alongside VAT expansion, VAT dysfunction leads to increased inflammation, oxidative stress, and ER stress. In the liver, the presence of increased FA leads to lipotoxicity triggering the same cellular insults, which are all associated with NF-kB activation. Omentin-1, whose receptor is still unknown, exerts its beneficial effects by preventing NF-kB activation, thereby reducing inflammation, oxidative stress, and ER stress in both steatotic hepatocytes and diseased VAT. By preventing these mechanisms, proinflammatory and profibrotic responses that contribute to MASLD development can also be halted. Additionalfile1.jpg Additionalfile2.jpg Additionalfile3.docx Cite Share Download PDF Status: Published Journal Publication published 11 Dec, 2023 Read the published version in Journal of Translational Medicine → Version 1 posted Reviewers agreed at journal 30 Jul, 2023 Reviewers invited by journal 28 Jul, 2023 Editor assigned by journal 26 Jul, 2023 First submitted to journal 24 Jul, 2023 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3192103","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":222330807,"identity":"739886ff-5f09-4bb1-a6c4-59ad3308c73f","order_by":0,"name":"Noel Salvoza","email":"","orcid":"","institution":"Fondazione Italiana Fegato","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Noel","middleName":"","lastName":"Salvoza","suffix":""},{"id":222330808,"identity":"3bf46efb-df34-4110-88e6-d7162fc486c9","order_by":1,"name":"Pablo Giraudi","email":"","orcid":"","institution":"Fondazione Italiana 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Fegato","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Natalia","middleName":"","lastName":"Rosso","suffix":""}],"badges":[],"createdAt":"2023-07-21 13:27:10","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3192103/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3192103/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12967-023-04770-8","type":"published","date":"2023-12-11T15:01:13+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":40966917,"identity":"566c7293-f114-4ba9-9be2-eddfc7730718","added_by":"auto","created_at":"2023-08-02 16:27:01","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":527722,"visible":true,"origin":"","legend":"\u003cp\u003eHuman VAT omentin-1 \u003cstrong\u003e(a)\u003c/strong\u003e mRNA expression, \u003cstrong\u003e(b)\u003c/strong\u003e protein expression, and \u003cstrong\u003e(c)\u003c/strong\u003eplasma levels in obese groups and lean controls. Omentin-1 mRNA expression is significantly decreased in the VAT of all obese groups as compared to the lean controls (N = 60). Representative blot and densitometric analysis of omentin-1 normalized to a-tubulin revealed that protein expression is also significantly decreased in the VAT of all obese groups as compared to the lean controls (n = 3/group). For plasma levels, values presented are the mean ± SD of individual patients (N = 72). Group comparison by Kruskal-Wallis and post hoc Dunn’s test. *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001, ****p \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-3192103/v1/9f08cfeb3734487ae7239bd0.png"},{"id":40966919,"identity":"1aa9744a-784e-4f8c-bd8f-e013ddabcc78","added_by":"auto","created_at":"2023-08-02 16:27:01","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":271031,"visible":true,"origin":"","legend":"\u003cp\u003eVAT omentin-1 \u003cstrong\u003e(a)\u003c/strong\u003e mRNA expression and \u003cstrong\u003e(b)\u003c/strong\u003eprotein expression HFD mice and control diet mice.\u003cstrong\u003e \u003c/strong\u003e20 weeks mice fed with HFD (n = 18), showing histological signs of MASH, have significantly lower expression compared to mice fed with a control diet (n = 13). Representative blot and densitometric analysis of omentin-1 normalized to a-tubulin revealed that protein expression is significantly decreased in the VAT of HFD mice as compared to control mice at 20 weeks (n = 3-5 mice/group). * p\u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-3192103/v1/0020050615b60e5a45784d68.png"},{"id":40966918,"identity":"c7e572ba-40be-432d-8a77-bb770bfbe9d6","added_by":"auto","created_at":"2023-08-02 16:27:01","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":807289,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e(a) \u003c/strong\u003eEffect of omentin-1 on hepatocyte fat accumulation.\u003cstrong\u003e (b – c) \u003c/strong\u003eEffect of omentin-1 and FFA co-treatment on the mRNA expression and supernatant release of TNF-α in Huh7 cells with FFA.\u003cstrong\u003e(d) \u003c/strong\u003eEffect of omentin-1 treatment on the \u003cem\u003eNF-kB\u003c/em\u003e mRNA expression in Huh7 cells with FFA.\u003cstrong\u003e (e – f) \u003c/strong\u003eEffect of omentin-1 treatment on ER stress markers \u003cem\u003eBiP\u003c/em\u003e and \u003cem\u003eCHOP\u003c/em\u003e in Huh7 cells with FFA.\u003cstrong\u003e (g – j)\u003c/strong\u003e Effects of omentin-1 treatment on the production of ROS in Huh7 with FFA. Values presented are the mean ± SD of three biological replicates. GSH and GSSG contents were normalized by the total proteins present in the cell lysates (μg) assessed using BCA.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-3192103/v1/a16073b883ed54fbbd359ec7.png"},{"id":40966925,"identity":"a3d1558e-8bdf-483b-95a9-6688f9a08a96","added_by":"auto","created_at":"2023-08-02 16:27:01","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":791540,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e(a\u003c/strong\u003e \u003cstrong\u003e– b)\u003c/strong\u003e Effect of omentin-1 and FFA co-treatment on the mRNA expression and supernatant release of TNF-α in VAT explants. \u003cstrong\u003e(c)\u003c/strong\u003e Effect of omentin-1 treatment on the \u003cem\u003eNF-kB\u003c/em\u003e mRNA expression in VAT explants. \u003cstrong\u003e(d – e)\u003c/strong\u003eEffect of omentin-1 treatment on ER stress markers \u003cem\u003eBiP\u003c/em\u003e and \u003cem\u003eCHOP \u003c/em\u003ein VAT explants. \u003cstrong\u003e(f– i)\u003c/strong\u003e Effects of omentin-1 treatment on the production of ROS in VAT explants. Values presented are the mean ± SD of three biological replicates. GSH and GSSG contents were normalized by the total proteins present in the cell lysates (μg) assessed using BCA. Values presented are the mean ± SD of three patients. * p \u0026lt; 0.05, ** p \u0026lt; 0.01, *** p \u0026lt; 0.001, **** p \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-3192103/v1/c726f3d2060ead5d024a17ae.png"},{"id":40966923,"identity":"15db8128-9a4b-4b65-a57f-c97f2b7954e8","added_by":"auto","created_at":"2023-08-02 16:27:01","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":531204,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of\u003cstrong\u003e (a) \u003c/strong\u003einsulin and\u003cstrong\u003e(b) \u003c/strong\u003eglucose on the mRNA expression of omentin-1 in VAT of obese patients; effects of\u003cstrong\u003e (c) \u003c/strong\u003einsulin and\u003cstrong\u003e (d) \u003c/strong\u003eglucose on the protein level of omentin-1 in VAT of obese patients. Omentin-1 protein level was normalized by the total proteins present in the tissue homogenates (μg) assessed using BCA. Values presented are the mean ± SD of 3-4 patients. * p \u0026lt; 0.05, **p \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-3192103/v1/5cd5053dba272e2e1f0ad4c0.png"},{"id":48402186,"identity":"a8243634-ab21-4cb6-8231-9948e69ce3cd","added_by":"auto","created_at":"2023-12-18 15:12:28","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1427114,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3192103/v1/57e2a637-a36e-41ba-85a5-2fd47d1adf66.pdf"},{"id":40966921,"identity":"2005611b-4947-4b3a-bb76-f40a5728476e","added_by":"auto","created_at":"2023-08-02 16:27:01","extension":"png","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":268660,"visible":true,"origin":"","legend":"\u003cp\u003eObesity leads to VAT expansion promoting lipolysis and excessive delivery of fatty acids (FA) to the liver, which is further exacerbated by insulin resistance. Alongside VAT expansion, VAT dysfunction leads to increased inflammation, oxidative stress, and ER stress. In the liver, the presence of increased FA leads to lipotoxicity triggering the same cellular insults, which are all associated with NF-kB activation. Omentin-1, whose receptor is still unknown, exerts its beneficial effects by preventing NF-kB activation, thereby reducing inflammation, oxidative stress, and ER stress in both steatotic hepatocytes and diseased VAT. By preventing these mechanisms, proinflammatory and profibrotic responses that contribute to MASLD development can also be halted.\u003c/p\u003e","description":"","filename":"GraphicalAbstract.png","url":"https://assets-eu.researchsquare.com/files/rs-3192103/v1/263c4606621039b2a6e2e2db.png"},{"id":40967713,"identity":"e3ac6602-9ef4-430f-83d3-9680408def88","added_by":"auto","created_at":"2023-08-02 16:35:01","extension":"jpg","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":57931,"visible":true,"origin":"","legend":"","description":"","filename":"Additionalfile1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3192103/v1/a487345bbcd04b41da17e473.jpg"},{"id":40968152,"identity":"ec6a6b1d-3285-4120-a67d-6d0dd6ef94df","added_by":"auto","created_at":"2023-08-02 16:43:01","extension":"jpg","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":118737,"visible":true,"origin":"","legend":"","description":"","filename":"Additionalfile2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3192103/v1/1008dca4678514b81e217bd0.jpg"},{"id":40967714,"identity":"fce3a6d7-63bb-4760-8ea2-bf486348da79","added_by":"auto","created_at":"2023-08-02 16:35:01","extension":"docx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":17122,"visible":true,"origin":"","legend":"","description":"","filename":"Additionalfile3.docx","url":"https://assets-eu.researchsquare.com/files/rs-3192103/v1/87f537973fb9a1f7ac1a2545.docx"}],"financialInterests":"","formattedTitle":"The potential role of Omentin-1 in Obesity-Related Metabolic Dysfunction-Associated Steatotic Liver Disease: Evidence from translational studies","fulltext":[{"header":"Introduction","content":"\u003cp\u003eObesity, characterized by adipose tissue (AT) mass expansion, is seen in 51% of MASLD and 81% of MASH patients globally (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). Fat accumulation in the organs, especially the visceral tissue, leads to their dysfunction, promoting ectopic fat accumulation in the liver, inflammation, ER stress, oxidative stress, and impairment of glucose metabolism, among others (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). Aside from its role as the regulator of lipid flux to the liver, AT is also recognized as a major endocrine organ producing a large array of mediators, known as adipocytokines (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). The role of adipocytokines in AT-liver crosstalk has become an important area of MASLD research because of the potential utility of those proteins/mediators, as diagnostic markers and/or therapeutic targets \u0026mdash; since no reliable diagnostic marker and pharmacological treatment are currently approved for the disease.\u003c/p\u003e \u003cp\u003eOmentin-1 (also known as intelectin-1), a novel adipocytokine, is a peptide of 313 amino acids containing a secretory signal sequence and a fibrinogen-related domain (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e). Omentin-2, a homolog with 83% amino acid identity with omentin-1 is found in the same chromosomal region (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e). Several reports indicated that omentin-1 and \u0026minus;\u0026thinsp;2 are highly expressed in visceral adipose tissue (VAT), but omentin-1 was shown to be the major circulating isoform in human plasma (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e). Omentin-1 was observed to be secreted exclusively into the culture medium of VAT, not subcutaneous adipose tissue (SAT), with stromal vascular cells playing a primary role in its production over adipocytes within VAT (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003ePlasma omentin-1 level decreases in overweight and obese humans, while it increases after obese patients lose weight or after taking antidiabetic drugs (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e). Regarding its biological activity, omentin-1 enhances insulin-stimulated glucose uptake via Akt activation in human adipocytes, suggesting its role in type 2 diabetes mellitus (T2DM) susceptibility (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e). Further, omentin-1 exerts anti-inflammatory effect by ameliorating macrophage activation via inhibiting the NF-κB pathway in obese mice (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e). Both insulin resistance and inflammation are associated with MASLD. These two molecular mechanisms, along with steatosis, oxidative stress, ER stress, and fibrosis, are key pathologic drivers in MASLD development.\u003c/p\u003e \u003cp\u003eIn this study, we conducted a simple \u003cem\u003ein silico\u003c/em\u003e analysis identifying omentin-1 and investigated its role in MASLD for the first time, using different translational approaches. We simultaneously determined the expression of omentin-1 in VAT at both mRNA and protein levels in MASLD patients and mice fed with high-fat diet (HFD). Furthermore, we present novel data regarding the plasma levels of omentin-1 in obese subjects with different stages of MASLD. Successively, to elucidate its role in the liver and VAT, we evaluated its beneficial effects in the MASLD-related pathophysiological mechanisms such as steatosis, inflammation, oxidative stress, and ER stress.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003e \u003cem\u003eIn silico strategy\u003c/em\u003e \u003c/p\u003e \u003cp\u003eThe literature review followed PRISMA guidelines, with the paper by du Plessis \u003cem\u003eet al\u003c/em\u003e. (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e) being chosen for its similarity to our morbidly obese cohort and the availability of VAT datasets. The gene expression data set GSE58979 was downloaded from GEO, which included 9 obese VAT samples (group 1) and 7 MASH VAT samples (group 3). Differentially expressed genes (DEGs) were identified using GEO2R through limma method (see Additional file 1). The significance of DEGs was calculated by the t-test and was represented by the p-value. The threshold for the DEGs was set as corrected p-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 and log\u003csub\u003e2\u003c/sub\u003e fold change (FC) of |1|.\u003c/p\u003e \u003cp\u003eIn our systematic strategy, the identifiers (IDs) for protein-coding genes in the consulted data resources were standardized, through mapping to the UniProtKB identifiers on UniProt database and only those IDs were further used. Moreover, datasets of our interest collected from Human Protein Atlas (HPA) were used as \u003cem\u003ein silico\u003c/em\u003e sieve filters. Dataset comparison and sub-groups selection was performed by applying Venn diagrams using InteractiVenn web-based tool. Venn diagrams were used as \u003cem\u003ein silico\u003c/em\u003e filters to identify the interested proteins, those fulfilling the following desired criteria: visceral adipose tissue-enriched, secreted proteins, secreted in plasma or blood, and not included as part of the housekeeping proteome. We finally selected omentin-1 as the most pertinent gene for our subsequent analysis as it fulfils all the criteria, and it is the only adipocytokine on the list (see Additional file 2).\u003c/p\u003e \u003cp\u003e \u003cem\u003eStudy Participants\u003c/em\u003e \u003c/p\u003e \u003cp\u003eThe assessment of omentin-1 VAT expression and plasma level was performed retrospectively in a morbidly obese (MO) cohort enrolled in a bariatric surgery program. All patients gave their written consent, and the study has been approved by the local Ethical Committee under protocol N. 22979 (Comitato Etico Regionale Unico, FVG, SSN, Italy). The MO cohort was stratified according to obese (Ob) group\u0026thinsp;=\u0026thinsp;19; obese MASH (Ob-M) group\u0026thinsp;=\u0026thinsp;20; and obese MASH with fibrosis (Ob-MF) group\u0026thinsp;=\u0026thinsp;16. The baseline characteristics of the MO cohort are shown in additional file 3. In addition, a total of 17 lean controls with BMI of 18.5\u0026ndash;24.9 kg/m\u003csup\u003e2\u003c/sup\u003e were included in the ELISA study. For PCR and western blot, VAT from 5 lean study participants were used as controls.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cem\u003eIn vitro model of hepatic steatosis\u003c/em\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eHepatoma cell line Huh7 (JHSRRB, Cat #JCRB0403) was obtained from the Health Science Research Resources Bank (Osaka, Japan) and grown in DMEM-HG with 10% FBS. Huh7 cells were exposed for 24h to 1200 \u0026micro;M of free fatty acids (FFA) (oleic:palmitic ratio 2:1 \u0026micro;moL/\u0026micro;moL) as previously described by our group (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e). To determine the experimental concentration, the cytotoxic effect of FFA (1200 \u0026micro;M) and recombinant omentin-1 (Bio Vendor, Candler, NC, USA), alone or in combination was assessed by MTT colorimetric assay after 24 hours.\u003c/p\u003e \u003cp\u003e \u003cem\u003eEx vivo primary explant culture of VAT\u003c/em\u003e \u003c/p\u003e \u003cp\u003eVAT explants from MO patients (without T2DM) undergoing bariatric surgery were cultured using the modified protocols of Carswell \u003cem\u003eet al\u003c/em\u003e., 2012 (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e) \u0026amp; Tan \u003cem\u003eet al.\u003c/em\u003e, 2008 (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e). Within 30 minutes after the surgery, tissue was minced into small pieces, approximately 5\u0026ndash;10 mg per piece (\u0026sim;1\u0026ndash;2 mm\u003csup\u003e3\u003c/sup\u003e) and transferred into six-well plates (\u0026sim;100 mg/well) containing 3 mL of appropriate medium. VAT explants were cultured for 24 h with or without the addition of insulin (10\u003csup\u003e\u0026minus;\u0026thinsp;5\u003c/sup\u003e M, 10\u003csup\u003e\u0026minus;\u0026thinsp;7\u003c/sup\u003e M) or D-glucose (50 mmol/L, 25 mmol/L).\u003c/p\u003e \u003cp\u003e \u003cem\u003eAnimal Model (In Vivo)\u003c/em\u003e \u003c/p\u003e \u003cp\u003eC57Bl/6 mice pups were provided by local SPF animal facility (University of Trieste). Immediately after weaning, mice were housed (22\u0026deg;C\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C) in a 12 h light/dark schedule, and fed ad-libitum with control diet (CD, 811900 Special Diets Services, England) or HFD diet (D12331, Research Diets, New Brunswick, NJ, USA) plus 42 g/L fructose/sucrose in drinking water, as previously described (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e). Based on the knowledge of the model and the experimental goals, diet was continued for 3 weeks and 20 weeks. Liver and epididymal fat (a depot of VAT) were dissected for the histologic evaluation and experimental use, respectively. Blood tests and histology were performed as previously described (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e). All experimental protocols were approved by the local OPBA (Organismo Per il Benessere dell\u0026rsquo;Animale) and by the Competent National Authority (Ministero della Salute-Direzione Generale della Sanit\u0026agrave; Animale e dei Farmaci Veterinary. Approval 56/2022PR).\u003c/p\u003e \u003cp\u003e \u003cem\u003eFluorometric determination of intracellular fat content\u003c/em\u003e \u003c/p\u003e \u003cp\u003eIntracellular fat content \u003cem\u003ein vitro\u003c/em\u003e was determined by flow cytometry using Nile red staining, a vital lipophilic dye used to label fat accumulation in the cytosol. After 24h of FFA exposure (with or without omentin-1 treatment), intracellular fluorescence was detected using a Becton Dickinson FACSCalibur System on the FL2 emission channel through a 585\u0026thinsp;\u0026plusmn;\u0026thinsp;21 nm bandpass filter, following excitation with an argon-ion laser source at 488 nm. Data were collected in 10,000 cells and analyzed using FlowJo (Tree Star Inc., Ashland, OR, USA) analysis software.\u003c/p\u003e \u003cp\u003e \u003cem\u003eQuantitative PCR\u003c/em\u003e \u003c/p\u003e \u003cp\u003eTotal RNA was extracted from cell culture harvest and homogenized VAT using Tri-reagent kit (Sigma-Aldrich, MO, USA). cDNA was generated with High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Waltham, MA, USA). Quantitative PCR was performed in CFX Connect Real-Time PCR Detection System (Bio-Rad, Hercules, CA USA) in a specific reaction volume containing 25 ng of cDNA, 1X iQ SYBR Green Supermix, and primer pairs. The relative quantification was made using the Pfaffl modification of the ΔΔCt equation, considering the efficiencies of individual genes and housekeeping genes.\u003c/p\u003e \u003cp\u003e \u003cem\u003eWestern Blot Analysis\u003c/em\u003e \u003c/p\u003e \u003cp\u003eThe following primary antibodies were used: Omentin-1/Intelectin-1 1:100 (Santa Cruz Biotech, Santa Cruz, CA, USA) and the reference α-tubulin 1:2000 (Santa Cruz Biotech, Santa Cruz, CA, USA). Blots were incubated with anti-mouse IgG-HRP-conjugated secondary antibody (1:500 Omentin-1/Intelectin-1 and 1:2000 for α-tubulin). Protein bands were visualized using the ECL immunoblotting detection system (GE Healthcare, Buckinghamshire, UK) and developed on a C-DiGit \u0026reg; Blot Scanner (LI-COR Biosciences, NE, USA). Results are expressed as the ratio of omentin-1 protein expression to that of a reference protein, α-tubulin. Relative densitometry analyses of the immunoblots were determined using IMAGE STUDIO software.\u003c/p\u003e \u003cp\u003e \u003cem\u003eGlutathione Content Assay Superoxide Dismutase (SOD) Activity Assay\u003c/em\u003e \u003c/p\u003e \u003cp\u003eThe simultaneous assay for both GSH (reduced) and GSSG (oxidized) was done using the modified protocol of Mokrasch and Teschke, 1984 (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e) and were normalized to total \u0026micro;g of proteins. Total SOD activity was also measured (Sigma-Aldrich, MO, USA).\u003c/p\u003e \u003cp\u003e \u003cem\u003eOmentin-1 and TNF-α ELISA\u003c/em\u003e \u003c/p\u003e \u003cp\u003eThe plasma level of omentin-1 in patients was measured using Human Omentin-1 ELISA Kit (BioVendor, RD191100200R) and the TNF-α levels of Huh7 and VAT supernatants were quantified by Human TNF alpha ELISA Kit (BioVendor, RAF128R). The levels of protein analytes were normalized to total \u0026micro;g of proteins.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003eUnless indicated otherwise, all values are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD). The normal distribution of variables was evaluated by Kolmogorov\u0026ndash;Smirnov test. Differences between two groups were assessed using the Mann-Whitney U test or student\u0026rsquo;s t-test. Data involving more than two groups were assessed by One-way-ANOVA or Kruskal-Wallis test, followed by post-hoc analysis. Spearman rank correlation was used for the calculation of associations between variables. Specific analysis details are indicated in figure legends. Statistical significance was determined at p\u0026thinsp;\u0026lt;\u0026thinsp;0.05. All figures and statistical analyses were generated using GraphPad Prism 9 and SPSS 29, respectively.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003e \u003cem\u003eIn vivo clinical validation\u003c/em\u003e \u003c/p\u003e \u003cp\u003eBased on \u003cem\u003ein silico\u003c/em\u003e analysis, omentin-1 is one of the downregulated genes in VAT of obese MASH. \u003cem\u003eIn vivo\u003c/em\u003e clinical validation showed decreased omentin-1 mRNA expression in all obese groups as compared to lean controls, independent of the presence of fibrosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). Further, the changes noted at the mRNA level were also reflected at the protein level (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). Likewise, plasma omentin-1 levels were lower in the obese groups than in the lean control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec).\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003eHaving shown that the plasma level of omentin-1 in obese groups differ, we investigated the relationship of omentin-1 with their clinical and biochemical parameters (n\u0026thinsp;=\u0026thinsp;55) (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Omentin-1 plasma level had a significant positive correlation with omentin-1 mRNA (ρ\u0026thinsp;=\u0026thinsp;0.382 p\u0026thinsp;=\u0026thinsp;0.013) and a significant negative correlation with total cholesterol (ρ = -0.307, p\u0026thinsp;=\u0026thinsp;0.022). Interestingly, omentin-1 plasma level negatively correlates with ALT (ρ = -0.279, p\u0026thinsp;=\u0026thinsp;0.039) but not AST. Further, the AST/ALT ratio positively correlates with the omentin-1 plasma level (ρ\u0026thinsp;=\u0026thinsp;0.285, p\u0026thinsp;=\u0026thinsp;0.042).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eCorrelation of clinical and laboratory parameters with plasma omentin-1 level.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVariable\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003erho\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003ep value\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOmentin-1 mRNA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.382\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.013*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBMI (kg/m\u003csup\u003e2\u003c/sup\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e-0.230\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.093\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFasting Glucose (mg/dL)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.026\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.851\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAST (UI/L)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e-0.206\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.136\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eALT (UI/L)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e-0.279\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.039*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAST/ALT Ratio\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.285\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.042*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGGT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e-0.049\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.723\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eALP (U/L)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e-0.034\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.81\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTriglycerides (mg/dL)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e-0.212\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.119\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTotal Cholesterol (mg/dL)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e-0.307\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.022*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHDL (mg/dL)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e-0.029\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.836\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eInsulin (\u0026micro;U/mL)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e-0.159\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.333\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePlatelet (x10\u003csup\u003e9\u003c/sup\u003e L)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e-0.076\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.581\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003ePearson\u0026rsquo;s or Spearman\u0026rsquo;s correlation coefficient (Rho) measures the strength and direction of association between the two variables under study. *p\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003cp\u003e \u003cem\u003eVAT omentin-1 expression in HFD mice\u003c/em\u003e \u003c/p\u003e \u003cp\u003eHuman omentin-1 gene is 80\u0026ndash;85% homologous to mice omentin-1 [20]. To investigate the mouse omentin-1 level during diet-induced obesity, we assigned C57Bl/6 littermates to receive either control or high-fat diet, supplemented with fructose/sucrose in drinking water, as described [21].\u003c/p\u003e \u003cp\u003eMice treated with HFD for 20 weeks developed obesity, dyslipidemia, glycemia, hyperinsulinemia, insulin resistance, and histological signs of MASH as compared to control diet mice (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Real-time PCR showed decreased mRNA expression of omentin-1 in the VAT of HFD versus control diet mice at 20 weeks (Fig.\u0026nbsp;2a). Similarly, Western blot analysis from representative mice VAT also confirmed the decreased omentin-1 expression in HFD mice versus control (Fig.\u0026nbsp;2b).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eAnthropometric, biochemical, and histological characteristics of mice.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVariable\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e3 weeks\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cem\u003ep\u003c/em\u003e value\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003e20 weeks\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cem\u003ep\u003c/em\u003e value\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHFD\u003c/p\u003e \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;8)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCD\u003c/p\u003e \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;7)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eHFD\u003c/p\u003e \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;18)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eCD\u003c/p\u003e \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;13)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSex (female)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4 (50%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4 (57.1%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.782\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e11 (61.1%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e7 (53.8%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.686\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBody Weight (g)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e21.3\u0026thinsp;\u0026plusmn;\u0026thinsp;2.61\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e18.9\u0026thinsp;\u0026plusmn;\u0026thinsp;2.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.083\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e39.50\u0026thinsp;\u0026plusmn;\u0026thinsp;7.38\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e28.06\u0026thinsp;\u0026plusmn;\u0026thinsp;3.79\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001***\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBody Length (cm)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e8.65\u0026thinsp;\u0026plusmn;\u0026thinsp;0.35\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e8.36\u0026thinsp;\u0026plusmn;\u0026thinsp;0.42\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.160\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e9.27\u0026thinsp;\u0026plusmn;\u0026thinsp;0.30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e8.87\u0026thinsp;\u0026plusmn;\u0026thinsp;0.32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001***\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBMI (kg/m\u003csup\u003e2\u003c/sup\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e28.38\u0026thinsp;\u0026plusmn;\u0026thinsp;2.33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e27.14\u0026thinsp;\u0026plusmn;\u0026thinsp;2.61\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.351\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e45.82\u0026thinsp;\u0026plusmn;\u0026thinsp;7.11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e35.69\u0026thinsp;\u0026plusmn;\u0026thinsp;3.40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001***\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTotal Cholesterol (mg/dL)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e141.50\u0026thinsp;\u0026plusmn;\u0026thinsp;17.53\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e86.14\u0026thinsp;\u0026plusmn;\u0026thinsp;10.75\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e168.22\u0026thinsp;\u0026plusmn;\u0026thinsp;43.45\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e85.69\u0026thinsp;\u0026plusmn;\u0026thinsp;11.71\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001***\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHDL (mg/dL)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e98.14\u0026thinsp;\u0026plusmn;\u0026thinsp;13.93\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e59.43\u0026thinsp;\u0026plusmn;\u0026thinsp;6.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e111.78\u0026thinsp;\u0026plusmn;\u0026thinsp;25.19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e63.69\u0026thinsp;\u0026plusmn;\u0026thinsp;11.88\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001***\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLDL (mg/dL)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e82.03\u0026thinsp;\u0026plusmn;\u0026thinsp;14.42\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e33.84\u0026thinsp;\u0026plusmn;\u0026thinsp;9.48\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e110.02\u0026thinsp;\u0026plusmn;\u0026thinsp;36.82\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e36.61\u0026thinsp;\u0026plusmn;\u0026thinsp;10.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001***\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTriglycerides (mg/dL)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e110.00\u0026thinsp;\u0026plusmn;\u0026thinsp;34.65\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e87.14\u0026thinsp;\u0026plusmn;\u0026thinsp;24.31\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.089\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e74.33\u0026thinsp;\u0026plusmn;\u0026thinsp;22.47\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e69.54\u0026thinsp;\u0026plusmn;\u0026thinsp;15.93\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.516\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAST (U/L)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e83.60\u0026thinsp;\u0026plusmn;\u0026thinsp;40.15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e73.60\u0026thinsp;\u0026plusmn;\u0026thinsp;28.04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.660\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e247.44\u0026thinsp;\u0026plusmn;\u0026thinsp;181.57\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e69.44\u0026thinsp;\u0026plusmn;\u0026thinsp;20.40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.008**\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eALT (U/L)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e57.18\u0026thinsp;\u0026plusmn;\u0026thinsp;33.68\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e40.20\u0026thinsp;\u0026plusmn;\u0026thinsp;29.61\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.092\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e67.35\u0026thinsp;\u0026plusmn;\u0026thinsp;31.64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e46.08\u0026thinsp;\u0026plusmn;\u0026thinsp;35.41\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.047*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGlucose (mg/dL)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e329.25\u0026thinsp;\u0026plusmn;\u0026thinsp;26.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e307.71\u0026thinsp;\u0026plusmn;\u0026thinsp;34.04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.188\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e321.94\u0026thinsp;\u0026plusmn;\u0026thinsp;79.11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e271.00\u0026thinsp;\u0026plusmn;\u0026thinsp;35.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.039*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eInsulin (\u0026micro;U/mL)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.33\u0026thinsp;\u0026plusmn;\u0026thinsp;0.71\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.96\u0026thinsp;\u0026plusmn;\u0026thinsp;0.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.219\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e2.79\u0026thinsp;\u0026plusmn;\u0026thinsp;1.88\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.22\u0026thinsp;\u0026plusmn;\u0026thinsp;0.30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.042*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHOMA-IR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.08\u0026thinsp;\u0026plusmn;\u0026thinsp;0.57\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.73\u0026thinsp;\u0026plusmn;\u0026thinsp;0.22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.162\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e2.38\u0026thinsp;\u0026plusmn;\u0026thinsp;1.78\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.87\u0026thinsp;\u0026plusmn;\u0026thinsp;0.28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.010**\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSteatosis grade (0/1/2/3)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e75%/25%/\u003c/p\u003e \u003cp\u003e0/0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e100%/0/\u003c/p\u003e \u003cp\u003e0/0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.155\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e5.6%/66.7%/\u003c/p\u003e \u003cp\u003e16.7%/11.1%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e100%/0/0/0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001***\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLobular inflammation (0/1/2/3)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e62.5%/25%/\u003c/p\u003e \u003cp\u003e12.5%/0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e71.4%/28.6%/\u003c/p\u003e \u003cp\u003e0/0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.626\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e27.8%/27.8%/\u003c/p\u003e \u003cp\u003e33.3%/11.1%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e76.9%/23.1%/\u003c/p\u003e \u003cp\u003e0/0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.022*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBallooning (No/Yes)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e100%/0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e100%/0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e93.3%/6.7%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e100%/0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eData are shown as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD for continuous variables, number (%) for binary variables, and frequency for categorical variables. T-test was used to test for significant differences with continuous variables. Chi-Square test was used for categorical variables. ***significant at p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, **significant at p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, and *significant at p\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eFigure 2.\u003c/b\u003e VAT omentin-1 \u003cb\u003e(a)\u003c/b\u003e mRNA expression and \u003cb\u003e(b)\u003c/b\u003e protein expression HFD mice and control diet mice. 20 weeks mice fed with HFD (n\u0026thinsp;=\u0026thinsp;18), showing histological signs of MASH, have significantly lower expression compared to mice fed with a control diet (n\u0026thinsp;=\u0026thinsp;13). Representative blot and densitometric analysis of omentin-1 normalized to α-tubulin revealed that protein expression is significantly decreased in the VAT of HFD mice as compared to control mice at 20 weeks (n\u0026thinsp;=\u0026thinsp;3\u0026ndash;5 mice/group). * p\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003cp\u003e \u003cem\u003eIn vitro effects of omentin-1 on fat-laden hepatocytes\u003c/em\u003e \u003c/p\u003e \u003cp\u003eNile red staining through flow cytometry revealed that omentin-1 does not affect steatosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). Interestingly, co-treatment of FFA with omentin-1 decreased the mRNA expression of \u003cem\u003eTNF-α\u003c/em\u003e relative to the vehicle control (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). Consistent with the gene expression results, omentin-1 significantly reduced the release of TNF-α in the cell culture supernatant (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003ec).\u003c/p\u003e \u003cp\u003eSince \u003cem\u003eNF-κB\u003c/em\u003e family of inducible transcription factors is activated in response to TNF-α cytokine action, we determined the gene expression of \u003cem\u003eNF-κB p65\u003c/em\u003e in our \u003cem\u003ein vitro\u003c/em\u003e model of steatosis. Upon co-treatment with omentin-1, there is a significant reduction of \u003cem\u003eNF-κB\u003c/em\u003e expression in fat-laden hepatocytes (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003ed).\u003c/p\u003e \u003cp\u003eTo explore the involvement of omentin-1 in hepatocyte ER stress induced by fat overload, we analyzed the expression levels of two ER stress markers (\u003cem\u003eBiP\u003c/em\u003e and \u003cem\u003eCHOP\u003c/em\u003e gene markers). The mRNA expression levels of both markers showed a nonsignificant increase upon treatment of FFA (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003ee). Moreover, both markers showed a nonsignificant decrease upon co-treatment with omentin-1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003ef).\u003c/p\u003e \u003cp\u003eThe antioxidant role of omentin-1 was evaluated on fat-laden Huh7 cells using glutathione content assay. The reduced glutathione (GSH) and GSH:GSSG ratio showed reduced levels upon FFA treatment and a significant increase upon co-treatment with omentin-1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003eg-\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003ei). The exposure to FFA significantly increased the oxidized glutathione (GSSG) content (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) \u003cem\u003evs\u003c/em\u003e the vehicle-treated control (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003eh), indicating a more consistent oxidative stress state. Another important antioxidant defense system is the superoxide dismutase (SOD) enzyme activity. FFA treatment showed increased SOD total activity, probably a compensatory mechanism from oxidative stress. Interestingly, the SOD activity is further enhanced upon co-treatment with omentin-1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003ej).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cem\u003eEx-vivo effects of omentin-1 on VAT explants\u003c/em\u003e \u003c/p\u003e \u003cp\u003eThe addition of recombinant omentin-1 in VAT explants of obese patients significantly reduced the basal \u003cem\u003eTNF-α\u003c/em\u003e mRNA expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003ea) and release in the supernatant (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). Moreover, VAT treated with omentin-1 showed a significant decrease in \u003cem\u003eNF-κB\u003c/em\u003e mRNA expression versus control (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003ec). The mRNA expression level of \u003cem\u003eBiP\u003c/em\u003e is significantly reduced upon treatment of omentin-1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003ed) while \u003cem\u003eCHOP\u003c/em\u003e mRNA expression is dose-dependently reduced by omentin-1 as compared to the control (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003ee). The reduced glutathione (GSH) and GSH:GSSG ratio significantly increases upon the addition of omentin-1 300 ng/mL but not omentin-1 150ng/mL (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003ef, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003eg). On the other hand, the oxidized glutathione (GSSG) almost dose-dependently decreased upon supplementation of omentin-1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003ei). Lastly, omentin-1 supplementation significantly enhanced the SOD activity in both concentrations (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003ej).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cem\u003eEx vivo effects of D-glucose and insulin on omentin-1 levels\u003c/em\u003e \u003c/p\u003e \u003cp\u003eStudies revealed that omentin-1 enhances insulin-stimulated glucose uptake \u003cem\u003ein vitro\u003c/em\u003e in both omental and subcutaneous adipocytes and its serum levels are reduced in patients with T2DM and glucose intolerance (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e). We, therefore, hypothesized that omentin-1 level might be affected by glucose and insulin modulation.\u003c/p\u003e \u003cp\u003eUsing the VAT explants from obese patients (without T2DM), we added either insulin or glucose in the medium and determined the levels of omentin-1. Both glucose and insulin resulted in a significant and almost dose-dependent decrease in omentin-1 mRNA expression levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003ea, \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003eb). Likewise, omentin-1 protein levels were also reduced in VAT homogenates showing consistent results with that of mRNA expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003ec, \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003ed).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe current study utilized a simple \u003cem\u003ein silico\u003c/em\u003e analysis to identify omentin-1 and provided insights regarding its role in MASLD, using a variety of translational approaches. Consistent with previous studies (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e), we detected omentin-1 mRNA in VAT but not SAT. Both mRNA and protein levels of omentin-1 in VAT are lower in all obese groups than in lean controls. The expression is further decreased in obese groups with MASH (with or without fibrosis) \u003cem\u003evs\u003c/em\u003e the Ob group. Additionally, our \u003cem\u003ein vivo\u003c/em\u003e mice results concur with our human validation results.\u003c/p\u003e \u003cp\u003eOmentin-1 is reported to be the major circulating form of omentin in human plasma (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e). Interestingly, the results of plasma omentin-1 levels as measured by ELISA are consistent with our VAT mRNA and protein data. Successively, correlation analyses were performed to evaluate the relationship between biochemical parameters and plasma omentin-1 levels in morbidly obese patients. In our study, omentin-1 plasma levels were found to be positively correlated with AST/ALT ratio and negatively correlated with ALT level. These results indicate an association between liver damage and omentin-1 secreted by VAT, supporting the crosstalk theory between the two organs.\u003c/p\u003e \u003cp\u003eOur \u003cem\u003ein vivo\u003c/em\u003e findings suggest that a reduced level of omentin-1 is associated with MASLD development, probably via VAT\u0026ndash;liver crosstalk. Furthermore, when considering only the obese groups in our study, it is unlikely that BMI is solely responsible for their lower omentin-1 levels since all patients had a BMI of \u0026gt;\u0026thinsp;35 kg/m\u003csup\u003e2\u003c/sup\u003e. Therefore, we also hypothesized that the further decrease in omentin-1 level could be an additive effect of MASLD severity to obesity. As such, the severity of MASLD results from several pathophysiological mechanisms, such as oxidative stress and ER stress, inflammation, and glucose-insulin impairment. To answer our hypotheses, we employed \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003eex vivo\u003c/em\u003e studies to evaluate the role of omentin-1 in MASLD-related pathophysiological mechanisms.\u003c/p\u003e \u003cp\u003eMASLD pathogenesis is complex but the onset of the disease is still represented by the accumulation of fat in the liver (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). Our group previously developed an \u003cem\u003ein vitro\u003c/em\u003e model of MASH where the exposure of hepatocytes to high concentrations of FFA promotes steatosis, inflammation, oxidative stress, and fibrogenic response, similar to those observed in patients with MASLD (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e). Using this \u003cem\u003ein vitro\u003c/em\u003e model to represent the pathologic events in the liver, we evaluated the beneficial effects of recombinant omentin-1. In parallel, we also studied its effects in \u003cem\u003eex vivo\u003c/em\u003e VAT explants obtained from obese patients with MASLD to determine its role in the actual diseased tissue setting.\u003c/p\u003e \u003cp\u003eThe expression of \u003cem\u003eTNF-α\u003c/em\u003e in steatotic hepatocytes and VAT supplemented with omentin-1 has not been examined to date. Here we have demonstrated that omentin-1 reduced the levels of \u003cem\u003eTNF-α\u003c/em\u003e in both fat-laden hepatocytes and VAT explants from obese patients. \u003cem\u003eTNF-α\u003c/em\u003e is a key mediator in the process of MASLD development by not only promoting inflammatory response, but also mediating insulin resistance, and inducing fibrosis-associated proteins (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e). Hence, the attenuation of \u003cem\u003eTNF-α\u003c/em\u003e action may help prevent or delay the development of MASH. \u003cem\u003eNF-κB\u003c/em\u003e transcription factor regulates a cascade of inflammatory responses by TNF-α activation (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e). Based on this premise, we also investigated the role of omentin-1 in \u003cem\u003eNF-κB\u003c/em\u003e expression. Indeed, we found that omentin-1 decreased the expression of \u003cem\u003eNF-κB\u003c/em\u003e in both fat-laden hepatocytes and VAT explants, suggesting that the anti-inflammatory effect may act via inhibition of this pathway.\u003c/p\u003e \u003cp\u003eObesity, a state of low-grade systemic inflammation, is associated with ROS overproduction and oxidative stress due to mitochondrial dysfunction (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e). As a result, inflammation and oxidative stress are involved in the induction of ER stress signaling pathways and subsequent unfolded protein response (UPR) activation to restore ER homeostasis (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e). This implies that oxidative stress, ER stress, and inflammatory pathways somewhat converge at different stages of obesity resulting in disease progression. Our group and other authors previously reported that fat-laden hepatocytes increase ROS production and ER stress (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e). Similarly, growing evidence suggests that excess energy substrate input associated with obesity enhanced ROS generation and ER stress by VAT (\u003cspan additionalcitationids=\"CR27\" citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e). Interestingly, we presented herein that adding omentin-1 mitigated both the oxidative stress and ER stress in our \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003eex vivo\u003c/em\u003e setups. Specifically, a significant decrease in oxidized glutathione (GSSG) levels and enhanced SOD enzyme activities were observed in fat-laden hepatocytes and VAT explants. In line with these observations, our results also showed that ER stress markers (\u003cem\u003eBiP\u003c/em\u003e and \u003cem\u003eCHOP\u003c/em\u003e) were also reduced by omentin-1 supplementation. Like omentin-1, vaspin, also exerts the same beneficial effects on ER stress\u0026ndash;induced metabolic dysfunctions. However, unlike omentin-1, with no known receptor to date, vaspin binds to \u003cem\u003eBiP\u003c/em\u003e, which is recruited from ER to the plasma membrane under ER stress (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e). Further investigations are needed to elucidate the role of omentin-1 in oxidative stress and ER stress, as well as to identify its specific receptor. Nonetheless, our findings suggest that oxidative stress and ER stress, as well as inflammation, all of which increase in parallel with metabolic dysfunctions, could be alleviated by omentin-1.\u003c/p\u003e \u003cp\u003eMetabolic disorders like obesity, diabetes, and polycystic ovarian syndrome are all characterized by insulin resistance and impairment of glucose metabolism. It has been shown that \u003cem\u003ein vitro\u003c/em\u003e supplementation of recombinant omentin-1 enhances insulin-mediated glucose uptake by adipocytes via GLUT4 translocation and Akt phosphorylation (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e). Furthermore, as reported herein, omentin-1 decreases the ER stress marker \u003cem\u003eBiP\u003c/em\u003e, which is also thought to maintain glucose uptake in glucose storage tissues (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e). Thus, given the role of omentin-1 in glucose homeostasis, we hypothesized that hyperinsulinemia and hyperglycemia decrease its expression. We found that upon increasing the concentration of insulin and glucose in the medium of VAT explants, the expression of omentin-1 is significantly decreased. This finding is in line with several studies showing that the reduced omentin-1 in adipose tissue may contribute to the development of insulin resistance and T2DM. However, it should be noted that our findings relate only to obese patients with MASLD without T2DM. Therefore, it would be of interest if we also determine the levels of omentin-1 in VAT of diabetic or lean patients.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eObesity is a significant risk factor for MASLD, where the expansion of visceral adipose tissue contributes to numerous pathological events, including the dysregulation of adipocytokines. The present study provides evidence that reduced omentin-1 level is associated with obesity-related MASLD. Although the cause-and-effect relationship is still unclear, we are still able to show that omentin-1 is an adipocytokine that plays a significant role in the VAT-liver crosstalk. As an endocrine factor, we report herein that VAT omentin-1 has a protective role against fat-laden hepatocytes showing MASLD-related pathophysiological mechanisms. Locally, omentin-1 was able to regulate obese VAT mechanisms, especially insulin-glucose impairment. Further studies are required to elucidate the biological activity of omentin-1 in obesity-related MASLD with a focus on specific receptor identification, which could then eventually facilitate new drug development.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eALT, alanine aminotransferase; AST, aspartate aminotransferase; AT, adipose tissue; AUF, arbitrary unit of fluorescence; BCA, bicinchoninic assay;\u003cem\u003e\u0026nbsp;BiP\u003c/em\u003e, binding of immunoglobulin protein; BMI, body mass index; CD, control diet; \u003cem\u003eCHOP\u003c/em\u003e, CCAAT/enhancer-binding protein homologous protein; ER, endoplasmic reticulum; FFA, free fatty acids; GGT, gamma-glutamyl transferase; GSH, reduced glutathione; GSSG, oxidized glutathione; HDL, high-density cholesterol; HFD, high-fat diet; \u003cem\u003eHPRT\u003c/em\u003e, hypoxanthine-guanine phosphoribosyltransferase; MASLD, metabolic dysfunction-associated steatotic liver disease; MASH, metabolic-associated steatohepatitis; \u003cem\u003eNF-\u0026kappa;B\u003c/em\u003e, nuclear factor kappa B; NAFLD, nonalcoholic fatty liver disease; SAT, subcutaneous adipose tissue; SOD, superoxide dismutase; TNF-\u0026alpha;, tumor necrosis factor alpha; T2DM, type 2 diabetes mellitus; VAT, visceral adipose tissue.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors would like to express their gratitude to all the study participants of the MO cohort. We would like to thank our colleagues in Fondazione Italiana Fegato, as well as our collaborators from Cattinara Hospital and Burlo Pediatric Institute for the valuable insights and assistance.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eConceptualization, N.S., N. R., P.G., and C.T.; methodology, N.S, N.R. and P.G; assisted in animal experiments, S.G., D.B., A.R. and F.S.; provided the clinical samples and data collection, S.P., N.M., and F.Z. All authors have read and agreed to the submitted version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by Fondazione Italiana Fegato. Pablo Giraudi reports funding from the HORIZON-HLTH-2022-STAYHLTH-02-01, Proposal number 101095672, PRAESIIDIUM. Noel Salvoza is funded by the Department of Science and Technology - Philippine Council for Health Research and Development (DOST-PCHRD), Philippines.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe dataset\u0026nbsp;GSE58979\u0026nbsp;used in this study is available in the GEO repository.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll patients gave their written consent, and the study has been approved by the local Ethical Committee under protocol N. 22979 (Comitato Etico Regionale Unico, FVG, SSN, Italy).\u0026nbsp;All animal experimental protocols were approved by the local OPBA (Organismo Per il Benessere dell\u0026rsquo;Animale) and by the Competent National Authority (Ministero della Salute-Direzione Generale della Sanit\u0026agrave; Animale e dei Farmaci Veterinary. Approval 56/2022PR).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors have reviewed the final version of the manuscript and approved it for submission.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eYounossi ZM, Koenig AB, Abdelatif D, Fazel Y, Henry L, Wymer M. Global epidemiology of nonalcoholic fatty liver disease-Meta-analytic assessment of prevalence, incidence, and outcomes. Hepatology. 2016;64(1):73\u0026ndash;84. \u003c/li\u003e\n\u003cli\u003eGodoy-Matos AF, Silva J\u0026uacute;nior WS, Valerio CM. NAFLD as a continuum: from obesity to metabolic syndrome and diabetes. 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Association of Adipose Tissue Inflammation With Histologic Severity of Nonalcoholic Fatty Liver Disease. Gastroenterology. 2015 Sep;149(3):635-648.e14. \u003c/li\u003e\n\u003cli\u003eSalvoza N, Bedin C, Saccani A, Tiribelli C, Rosso N. The Beneficial Effects of Triterpenic Acid and Acteoside in an In Vitro Model of Nonalcoholic Steatohepatitis (NASH). International Journal of Molecular Sciences. 2022 Jan;23(7):3562. \u003c/li\u003e\n\u003cli\u003eCarswell KA, Lee MJ, Fried SK. Culture of Isolated Human Adipocytes and Isolated Adipose Tissue. Methods Mol Biol. 2012;806:203\u0026ndash;14. \u003c/li\u003e\n\u003cli\u003eTan BK, Adya R, Farhatullah S, Lewandowski KC, O\u0026rsquo;Hare P, Lehnert H, et al. Omentin-1, a novel adipokine, is decreased in overweight insulin-resistant women with polycystic ovary syndrome: ex vivo and in vivo regulation of omentin-1 by insulin and glucose. Diabetes. 2008 Apr;57(4):801\u0026ndash;8. \u003c/li\u003e\n\u003cli\u003eMarin V, Rosso N, Dal Ben M, Raseni A, Boschelle M, Degrassi C, et al. An Animal Model for the Juvenile Non-Alcoholic Fatty Liver Disease and Non-Alcoholic Steatohepatitis. PLoS One [Internet]. 2016 Jul 8 [cited 2020 Mar 11];11(7). Available from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4938400/\u003c/li\u003e\n\u003cli\u003eMokrasch LC, Teschke EJ. Glutathione content of cultured cells and rodent brain regions: A specific fluorometric assay. Analytical Biochemistry. 1984 Aug 1;140(2):506\u0026ndash;9. \u003c/li\u003e\n\u003cli\u003eBuzzetti E, Pinzani M, Tsochatzis EA. The multiple-hit pathogenesis of non-alcoholic fatty liver disease (NAFLD). Metab Clin Exp. 2016 Aug;65(8):1038\u0026ndash;48. \u003c/li\u003e\n\u003cli\u003eRosso N, Chavez-Tapia NC, Tiribelli C, Bellentani S. Translational approaches: From fatty liver to non-alcoholic steatohepatitis. World J Gastroenterol. 2014 Jul 21;20(27):9038\u0026ndash;49. \u003c/li\u003e\n\u003cli\u003eChavez-Tapia NC, Rosso N, Tiribelli C. Effect of intracellular lipid accumulation in a new model of non-alcoholic fatty liver disease. BMC Gastroenterol. 2012 Mar 1;12:20. \u003c/li\u003e\n\u003cli\u003eKakino S, Ohki T, Nakayama H, Yuan X, Otabe S, Hashinaga T, et al. Pivotal Role of TNF-\u0026alpha; in the Development and Progression of Nonalcoholic Fatty Liver Disease in a Murine Model. Horm Metab Res. 2018 Jan;50(1):80\u0026ndash;7. \u003c/li\u003e\n\u003cli\u003eHayden MS, Ghosh S. Regulation of NF-\u0026kappa;B by TNF Family Cytokines. Semin Immunol. 2014 Jun;26(3):253\u0026ndash;66. \u003c/li\u003e\n\u003cli\u003eLiu T, Zhang L, Joo D, Sun SC. NF-\u0026kappa;B signaling in inflammation. Sig Transduct Target Ther. 2017 Jul 14;2(1):1\u0026ndash;9. \u003c/li\u003e\n\u003cli\u003ePanic A, Stanimirovic J, Sudar-Milovanovic E, Isenovic ER. Oxidative stress in obesity and insulin resistance. Explor Med. 2022 Feb 23;3(1):58\u0026ndash;70. \u003c/li\u003e\n\u003cli\u003eBa\u0026ntilde;uls C, Rovira-Llopis S, Lopez-Domenech S, Diaz-Morales N, Blas-Garcia A, Veses S, et al. Oxidative and endoplasmic reticulum stress is impaired in leukocytes from metabolically unhealthy vs healthy obese individuals. Int J Obes. 2017 Oct;41(10):1556\u0026ndash;63. \u003c/li\u003e\n\u003cli\u003eSong MJ, Malhi H. The Unfolded Protein Response and Hepatic Lipid Metabolism in Non-alcoholic fatty liver disease. Pharmacol Ther. 2019 Nov;203:107401. \u003c/li\u003e\n\u003cli\u003eArroyave-Ospina JC, Wu Z, Geng Y, Moshage H. Role of Oxidative Stress in the Pathogenesis of Non-Alcoholic Fatty Liver Disease: Implications for Prevention and Therapy. Antioxidants (Basel). 2021 Jan 26;10(2):174. \u003c/li\u003e\n\u003cli\u003eAlcal\u0026aacute; M, Calderon-Dominguez M, Bustos E, Ramos P, Casals N, Serra D, et al. Increased inflammation, oxidative stress and mitochondrial respiration in brown adipose tissue from obese mice. Sci Rep. 2017 Nov 22;7(1):16082. \u003c/li\u003e\n\u003cli\u003eDelli Bovi AP, Marciano F, Mandato C, Siano MA, Savoia M, Vajro P. Oxidative Stress in Non-alcoholic Fatty Liver Disease. An Updated Mini Review. Frontiers in Medicine [Internet]. 2021 [cited 2022 Feb 9];8. Available from: https://www.frontiersin.org/article/10.3389/fmed.2021.595371\u003c/li\u003e\n\u003cli\u003eMasarone M, Rosato V, Dallio M, Gravina AG, Aglitti A, Loguercio C, et al. Role of Oxidative Stress in Pathophysiology of Nonalcoholic Fatty Liver Disease. Oxid Med Cell Longev [Internet]. 2018 Jun 11 [cited 2021 May 3];2018. Available from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6016172/\u003c/li\u003e\n\u003cli\u003eNakatsuka A, Wada J, Iseda I, Teshigawara S, Higashio K, Murakami K, et al. Vaspin Is an Adipokine Ameliorating ER Stress in Obesity as a Ligand for Cell-Surface GRP78/MTJ-1 Complex. Diabetes. 2012 Nov;61(11):2823\u0026ndash;32. \u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"journal-of-translational-medicine","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jtrm","sideBox":"Learn more about [Journal of Translational Medicine](http://translational-medicine.biomedcentral.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/jtrm/default.aspx","title":"Journal of Translational Medicine","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"MASLD, MASH, obesity, omentin-1, translational models","lastPublishedDoi":"10.21203/rs.3.rs-3192103/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3192103/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eObesity, characterized by visceral adipose tissue (VAT) expansion, is closely associated with metabolic dysfunction-associated steatotic liver disease (MASLD). Recent research has highlighted the crucial role of the adipose tissue-liver axis in the development of MASLD to its progressive form, metabolic dysfunction-associated steatohepatitis (MASH). In this study, we investigated the potential role of omentin-1, a novel adipokine expressed by VAT, in obesity-related MASLD pathogenesis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThrough \u003cem\u003ein silico\u003c/em\u003e analysis of differentially expressed genes in VAT from obese patients with and without MASH, we identified omentin-1 as a significant candidate. To validate our findings, we measured omentin-1 levels in VAT and plasma of lean controls and obese patients with biopsy-proven MASLD. Additionally, we assessed omentin-1 expression in the VAT of a juvenile mice MASLD model. \u003cem\u003eIn vitro\u003c/em\u003e and \u003cem\u003eex vivo\u003c/em\u003e studies were conducted to investigate the effects of omentin-1 on MASLD-related mechanisms, including steatosis, inflammation, ER stress, and oxidative stress. We also analyzed the impact of D-glucose and insulin on VAT omentin-1 levels \u003cem\u003eex vivo\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCompared to the lean group, the obese groups exhibited significantly lower VAT and plasma levels of omentin-1. Interestingly, within the MASH group, fibrosis did not affect omentin-1 levels. Likewise, VAT of mice fed with high-fat diet, showing histological signs of MASH showed decreased omentin-1 levels as com-pared to their control diet counterpart. \u003cem\u003eIn vitro\u003c/em\u003e experiments on fat-laden human hepatocytes revealed that omentin-1 did not affect steatosis but significantly reduced TNF-α levels, ER stress, and oxidative stress. Similar results were obtained using \u003cem\u003eex vivo\u003c/em\u003e VAT explants from obese patients upon omentin-1 supplementation. Furthermore, omentin-1 decreased the expression of \u003cem\u003eNF-κB\u003c/em\u003e mRNA, both \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003eex vivo\u003c/em\u003e. \u003cem\u003eEx vivo\u003c/em\u003e VAT explants showed that D-glucose and insulin significantly reduced omentin-1 mRNA expression and protein levels.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCollectively, our findings suggest that reduced omentin-1 levels contribute to the development of MASLD. Omentin-1 supplementation mitigates inflammation, ER stress, and oxidative stress, probably via inhibiting the NF-κB pathway and might also play a role in the regulation of glucose and insulin metabolism. Further research is warranted to explore omentin-1 as a potential therapeutic target and/or biomarker for MASLD.\u003c/p\u003e","manuscriptTitle":"The potential role of Omentin-1 in Obesity-Related Metabolic Dysfunction-Associated Steatotic Liver Disease: Evidence from translational studies","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-08-02 16:26:56","doi":"10.21203/rs.3.rs-3192103/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2023-07-30T15:17:12+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2023-07-28T13:15:17+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2023-07-26T04:50:00+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Translational Medicine","date":"2023-07-24T04:57:20+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"journal-of-translational-medicine","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jtrm","sideBox":"Learn more about [Journal of Translational Medicine](http://translational-medicine.biomedcentral.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/jtrm/default.aspx","title":"Journal of Translational Medicine","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"331b6f27-247b-4a4b-97c3-957b856c7b7d","owner":[],"postedDate":"August 2nd, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2023-12-18T15:11:40+00:00","versionOfRecord":{"articleIdentity":"rs-3192103","link":"https://doi.org/10.1186/s12967-023-04770-8","journal":{"identity":"journal-of-translational-medicine","isVorOnly":false,"title":"Journal of Translational Medicine"},"publishedOn":"2023-12-11 15:01:13","publishedOnDateReadable":"December 11th, 2023"},"versionCreatedAt":"2023-08-02 16:26:56","video":"","vorDoi":"10.1186/s12967-023-04770-8","vorDoiUrl":"https://doi.org/10.1186/s12967-023-04770-8","workflowStages":[]},"version":"v1","identity":"rs-3192103","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3192103","identity":"rs-3192103","version":["v1"]},"buildId":"WrCJVZZCHTDjtuVLN7oU0","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}