Enhanced catabolism of branched-chain amino acids uncouples anti-inflammatory and antioxidant functions in liver macrophages

Enhanced catabolism of branched-chain amino acids uncouples anti-inflammatory and antioxidant functions in liver macrophages | 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 Enhanced catabolism of branched-chain amino acids uncouples anti-inflammatory and antioxidant functions in liver macrophages Magnolia Martínez-Aguilar, Manon Buist-Homan, Hans Blokzijl, Han Moshage This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8077254/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Metabolic dysfunction–associated steatotic liver disease (MASLD) is marked by inflammation mediated by resident liver macrophages (RLMs). Branched-chain amino acids (BCAAs; leucine, isoleucine, valine) are elevated in early MASLD, yet their role in RLM biology is unclear. Aim We investigated whether BCAA exposure and impaired catabolism affect LPS-induced RLMs activation. Methods Primary rat RLMs were treated with high BCAA concentrations (15 mM) and/or LPS stimulation (100 ng/ml). BCAA metabolic enzymes, inflammatory markers, oxidative stress, and metabolic reprogramming were assessed. BCKDK inhibitor BT2 was used to enhance BCAA catabolism: Results RLMs expressed BCAT1, upregulated by LPS but downregulated by BCAAs. BCAAs exerted protective effects by selectively reversing LPS-induced CD11b, MCP-1, HIF-1α, and Arg1 expression, reducing ROS and attenuating NRF2. BCAAs promoted metabolic shift toward oxidative phosphorylation with increased ATP and reduced HK1 expression. BT2 enhanced catabolism restored BCAT1 and maintained anti-inflammatory effects but abolished antioxidant protection. Only leucine-BT2 suppressed NF-κB translocation. HIF-1α stabilization and HMGB1-mediated protection, restoring inflammatory gene expression and ROS levels. Conclusions BCAAs modulate RLM activation by regulating HIF-1α and HMGB1 signaling and redox homeostasis. Their protective effects depend on intact HIF-1α/HMGB1 pathways rather than catabolic flux, revealing a context-dependent role of BCAAs in hepatic inflammation. Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 INTRODUCTION Diet enriched in fat and complex carbohydrates, together with sedentarism, have driven a global increase in metabolic dysfunction–associated steatotic liver disease (MASLD), affecting 38% of adults and is projected to reach 55% by 2040 [ 1 ]. MASLD is defined by hepatic steatosis with cardiometabolic risk factors. Early disease responds to lifestyle changes, yet chronic exposure to risk factors promotes progression to metabolic dysfunction-associated steatohepatitis (MASH), fibrosis, cirrhosis, and hepatocellular carcinoma. Preventing this progression is therefore central to reducing liver-related morbidity and mortality. MASLD pathophysiology follows a “multiple parallel hits” hypothesis [ 2 ], where genetic predisposition and environmental factors synergize [ 3 ]. Lipid accumulation and insulin resistance cause hepatocellular lipotoxicity, mitochondrial dysfunction, and reactive oxygen species (ROS) generation. Concurrently, gut dysbiosis resulting from hypercaloric diets enhances bacterial translocation into the liver [ 4 ], releasing damage- and pathogen-associated molecular patterns (DAMPs/PAMPs), that activate innate immunity [ 5 ]. Resident liver macrophages (RLMs), known as Kupffer cells (KCs), amplify local inflammation, which is central to disease progression [ 6 , 7 ]. Sustained RLMs activation further promotes hepatocyte injury, stellate cell activation, and fibrosis. RLMs constitute ~ 90% of liver macrophages, they mediate debris clearance, iron recycling, cholesterol regulation, immune tolerance, and pathogen defence. Their phenotype is highly plastic, shaped not only by DAMPs and PAMPs but also by lipids, extracellular vesicles and dietary nutrients [ 8 – 11 ], driving polarization toward a pro-inflammatory (M1) or anti-inflammatory (M2) phenotype. Branched-chain amino acids (BCAAs: leucine, isoleucine, and valine) are immunometabolism modulators, which determine how metabolic pathways and their intermediates regulate the energy supply, signaling pathways, and activity of immune cells [ 12 ]. BCAAs regulate glucose homeostasis, mitochondrial energy production, protein synthesis, and autophagy [ 13 ], but their immune effects are context-dependent. In muscle repair, diabetes, cancer, and atherosclerosis BCAA yield contradictory findings, promoting pro-inflammatory phenotypes via such mTORC1–HIF1α–glycolysis, IFNGR1–JAK1–STAT1, or HMGB1–TLR4–NF-κB pathways [ 14 – 17 ], or anti-inflammatory responses, through undefined mechanisms [ 14 , 18 , 19 ] . BCAA catabolism efficiency is critical. Proper catabolism supports anti-inflammatory programs and metabolic homeostasis. However, impaired catabolism, as occurs in metabolic diseases, BCAAs and their ketoacid metabolites accumulate, driving pro-inflammatory polarization and exacerbating tissue dysfunction. MASLD/MASH patients have elevated circulating BCAAs (396–507 µM vs. 350 µM in healthy subjects) [ 20 – 22 ]. Paradoxically, advanced liver disease, shows BCAA depletion, reflecting metabolic collapse. Despite these insights, BCAA effects on RLMs remain unclear. This study investigates how high BCAA exposure affects RLM activation, oxidative stress, and inflammatory signaling, and examines the effects of impaired BCAA catabolism. MATERIAL AND METHODS Materials RPMI medium, heat-inactivated fetal calf serum (FCS) and PBS1x were obtained from Thermo Fisher, Waltham, MA, USA, respectively: 11835030, A5256701, 70011). Penicillin/streptomycin, fungizone, and sodium bicarbonate were purchased from GIBCO (Thermo Fisher). BCAAs purchased from Sigma Aldrich, St. Louis, MO, USA (L-Leucine (L8912-25g), L-Isoleucine (I7403-25g), and L-Valine (V0513-25g)) were dissolved in water to 15 mM. 3,6-Dichloro-benzothiophene-2-carboxylic acid (BT2) (Cayman Chemical, Ann Arbor, MI, USA; 34576-94-8) was dissolved in DMSO to 15 mM. Glycyrrhizic acid (MedChemExpress, Monmouth Junction, NJ, USA; HY-N0184) was dissolved in DMSO to 1 mM (Sigma Aldrich, 276855). DMOG (Sigma-Aldrich, D3695-50MG) was dissolved in DMSO to 1 mM. Animals and cell isolation Male Wistar rats (220–250 g, 5–8 weeks) obtained from Charles River Laboratories (Wilmington, MA, USA) were housed under standard conditions (25 ± 2 °C, 12 h light/dark cycle, with free access to chow and water) at the University Medical Center Groningen. All procedures complied with Dutch legislation (Animal Act 2011) and were approved by the local ethics committee (permit no. 2115139-01-001). Anaesthesia was induced with 5% isoflurane, ketamine (60 mg/kg) and medetomidine (0.5 mg/kg). RLMs were isolated using the two-step collagenase perfusion method. Livers were perfused via the portal vein with Ca²⁺/Mg²⁺-free HBSS, followed by HBSS containing Ca²⁺ (5.7 mM) and collagenase (150 U/ml, Sigma-Aldrich, C0130). Tissue was dissociated and filtered. Hepatocytes were removed by low-speed centrifugation (65 g for 2 min); non-parenchymal cells were enriched by Optiprep (Stemcell Technologies, Vancouver, BC, Canada) gradient centrifugation (17.6% and 8.2%). After washing, cells were resuspended and seeded. After 30 minutes, non-adherent cells were removed by replacing the medium, enriching for adherent RLMs. Cells were identified as CD68⁺/CD163⁺ (Supplemented Figure 2). Treatments and drugs Based on our previous findings that 15 mM BCAA has antifibrotic effects on stellate cells [23], the same concentration was used. RLMs were treated with 15 mM individual BCAAs or combinations (1:2:1 Ile:Leu:Val). To mimic MASLD inflammation, cells were pretreated with BCAAs for 2h, then stimulated with with lipopolysaccharide (LPS, 100 ng/mL, Sigma-Aldrich, L2887) for 4h (total 6h) (Lichtman SN et al., 1994). BT2 (IC₅₀ = 3.19 μM) was applied 30 min before BCAAs to enhance BCAA catabolism. DMOG (IC₅₀ = 0.1 mM; [24]) and glycyrrhizic acid (Gla, Kᴅ = 150 µM; [25]) were administered 30 min before BCAAs. Cell viability Cell viability was assessed using SYTOX Green (125 nmol/L; Thermo Fisher, S7020). Sytox was added after treatment for 15 minutes at 37 °C in an atmosphere containing 5% (v/v) CO₂. Necrotic cells were stained green (SYTOX) and visualized using a Leica fluorescence microscope (Leica Microsystems, Wetzlar, Germany; DMI6000) at 512–542 nm and 585–624 nm, respectively (Supplementary Figure 1A). Additionally, lactate dehydrogenase (LDH) release was calculated as the percentage of the activity of LDH released in the medium vs. the total LDH activity (in both the medium and cell lysates). LDH was measured spectrophotometrically at 340 nm using an Epoch2 microplate reader (Bio-Tek Instruments, Inc., Winooski, VT, USA) (Supplementary Figure 1B). Western blot analysis Protein lysates were isolated by four freezing-thawing cycles followed by centrifugation at 12000 × g for 15 minutes. Protein from supernatant fraction were isolated using a methanol-chloroform extraction protocol [26]. Briefly, 600 µL of a 4:1 methanol: chloroform mixture was added to the sample, vortexed, and centrifuged at 10,000 × g for 5 min at 4°C. The supernatant was discarded, and the protein pellet was washed with 500 µL of methanol, followed by centrifugation. The final pellet was dried, resuspended in 70 µL of 1.5× Laemmli buffer, and denatured at 100°C for 10 min for SDS-PAGE and Western blot analysis. Protein concentration was quantified using the Bio-Rad protein assay kit with BSA as standard (Bio-Rad Laboratories, Hercules, CA, USA; 5000111). For Western blot, 40 µg of protein was loaded on 10% SDS-PAGE gels, electrophoresed at 100 V for 90 min and transferred to nitrocellulose membrane by semi-dry blotting for 30 minutes. Membranes were blocked with 5% BSA, incubated overnight with primary antibodies (Supplementary Table 1), and then with HRP-conjugated secondary antibodies (Goat anti-rabbit horseradish peroxidase-labeled secondary antibody (Agilent Technologies, Santa Clara, CA, USA; P0448)) or polyclonal Rabbit Anti-Mouse Immunoglobulins/HRP (Agilent Technologies, P0260). Bands were visualized using a ChemiDoc XRS system (Bio-Rad). Immunofluorescence RLMs were seeded on glass coverslips in 12-well plates and fixed with 4% paraformaldehyde for 10 min at room temperature. After washing with HBSS, cells were permeabilized with 0.1% Triton X-100 for 30 min at 37 °C and blocked with 2% BSA in PBS for 30 min. Cells were incubated for 1 hour with primary antibody (1:300 in 2% BSA/PBS) against NF-κB p65 (Cell Signaling Technology, Danvers, MA, USA; 8242), CD68 (AbD Serotec, Kidlington, UK; MCA341R) or CD163 (Hycult, Uden, Netherlands; HM3025), followed by Alexa Fluor TM 488 donkey anti-goat (Invitrogen, Carlsbad, CA, USA, A-11055) or Alexa Fluor 568 TM goat anti-mouse (Invitrogen, A-11004) (1:500 in 2% BSA/PBS, 30 min, RT). Nuclei were counterstained with DAPI (1:1000, 30 min) (Roche, 10236276001). Coverslips were mounted using Dako fluorescence mounting medium (Agilent Technologies, S3023) and imaged using a Leica fluorescence microscope (Leica Microsystems, DMI6000). Characterization of RLMs based on the presence of CD68 and CD163 is presented in Supplementary Figure 2. RNA isolation Total RNA was isolated using TRI-reagent (Sigma-Aldrich, T9424) according to the manufacturer's instructions. RNA was separated by chloroform extraction, precipitated with isopropanol, washed with 75% ethanol, air-dried, and resuspended in RNase-free water. RNA concentration and purity were determined using a NanoDrop 2000 spectrophotometer (Thermo Scientific, Waltham, MA, USA; ND-2000). For cDNA synthesis, 2.5 μg of total RNA was reverse transcribed in a 50 μL reaction containing 1X RT buffer (500 mmol/l Tris-HCl[pH 8.3]; 500 mmol/l KCl; 30 mmol/l MgCl₂; 50 mmol/l DTT), 1 mmol/l deoxynucleotides triphosphate (dNTPs, Sigma-Aldrich), 10 ng/μL random nanomers (Thermo Fisher), 0.6 U/μL RNaseOUT™ (Invitrogen), and 4 U/μL M-MLV reverse transcriptase (Invitrogen). The reaction was performed at 25°C for 10 min, 37°C for 60 min, and 95°C for 5 min. The resulting cDNA was diluted 20-fold before qPCR. Real-time qPCR was performed on a StepOnePlus™ system (Applied Biosystems, Thermo Fisher) using TaqMan probes. Each reaction contained a 2X master mix (dNTPs, Hot Gold Star DNA polymerase, 5 mmol/l MgCl₂) (Eurogentec, Liège, Belgium), fluorogenic TaqMan probes, and gene-specific primers (Supplementary Table 2). Reactive oxygen species measurement Intracellular reactive oxygen species (ROS) levels were assessed using the fluorescent probe DCFH‐DA (MedChemExpress, HY-D0940). Cells cultured in 12-well plates were washed twice with 1 mL of warm PBS 1X followed by incubation with 1 mL of 10 µM H₂DCF-DA in PBS 1X at 37°C in 5% CO₂ for 30 minutes in the dark. After incubation, dye was aspirated, and the cells were washed twice with 1 mL of PBS 1X. For nuclear staining, DAPI (1 µg/mL per well) was added for 5 minutes in the dark. Nuclei were counterstained with DAPI (1 µg/mL, 5 min, dark), followed by two PBS 1X washes. Fluorescence was visualized under a fluorescence microscope using FITC (Ex 488 nm / Em 525 nm) and DAPI (Ex 360 nm / Em 460 nm) filter sets. ATP quantification assay Intracellular ATP levels were measured using a luminescence-based ATP assay kit (MedChemExpress, HY-K0314). Cells cultured in 12-well plates were lysed with 200 µL lysis buffer per well. Lysates were centrifuged at 12,000 × g for 5 min at 4 °C. 50 µL of each lysate was mixed with 100 µL of ATP detection working solution in a black 96-well plate, incubated for 3–5 min at room temperature, and luminescence was recorded on a microplate luminometer (endpoint mode; 1 s integration, AutoScale gain, normal read speed, 100 ms delay, 1 mm read height, extended dynamic range enabled). ATP concentration was calculated from a standard curve (1–5000 nM ATP) and normalized to total protein content measured by the BCA method. Statistical analysis Statistical analysis was performed using GraphPad Prism 8 (GraphPad Software, San Diego, CA, USA). Data are presented as mean ± standard error of the mean (SEM) of 3 to 4 independent experiments. For multiple group comparisons, one-way analysis of variance (ANOVA) was applied, followed by Tukey's post hoc test for pairwise comparisons or Dunnett's post hoc test for comparisons against the control or LPS. For comparisons between two groups, an unpaired Student's t-test was used. Colocalization analysis was performed using Manders' colocalization coefficient. Differences were considered statistically significant at p < 0.05. RESULTS Activated resident liver macrophages (RLMs) induced the expression of BCAT1 We started by assessing the presence of BCAA catabolic enzymes, BCAT1/2 and BCKDH, enzymes in liver cells (schematic representation of BCAAs metabolism, Figure 1A). Gene expression in hepatocytes, stellate cells, liver sinusoidal endothelial cells (LSEC), and RLMs showed that RLMs express BCAT1 at a higher level than other liver cell types, while BCKDH expression was highest in hepatocytes (Supplementary Figure 3). Next, primary rat RLMs were treated with 15 mM of isoleucine (Ile), leucine (Leu), valine (Val), or their combination (ratio 2:1:1, leucine, valine, isoleucine), with or without (100 ng/mL) (Figure 1B). At the mRNA level (Figure 1C), BCAT1 was significantly induced by LPS, and this increase was abolished by Leu, Val, or the BCAA mixture. BCKDH showed no significant changes after LPS. At the protein level (Figure 1D), BCAT1 and BCKDH remained stable across conditions. These findings suggest RLMs metabolize BCAAs mainly through BCAT1, which is upregulated by LPS but attenuated by BCAA exposure. BCAAs modulate inflammatory gene expression after LPS treatment We next investigated the impact of BCAA treatment on inflammatory gene expression in the absence and presence of LPS (Figure 2). LPS significantly increased the mRNA expression of CD11b, MCP1, IL-1β, IL-6, iNOS, TNFα, HIF-1α, IL-10, and Arg1. Individual BCAAs and the BCAA mixture reversed CD11b, MCP-1, HIF-1α, and Arg-1 induction by LPS. BCAAs did not modify other analysed genes. These results indicate that BCAA exposure selectively downregulates RLMs’ inflammatory response, particularly reversing LPS-induced CD11b, MCP1, HIF-1α, and Arg-1, while other pro-inflammatory mediators remained unaffected. BT2-mediated BCKDK inhibition reverses BCAT1 suppression To model impaired BCAA catabolism in MASLD [27], we used BCKDK inhibitor BT2 (IC50 = 3.19 µM), which enhances BCAA oxidation (Figure 3A). RLMs were pre-treated with BT2, then exposed to BCAAs alone or in combination with LPS (total = 6.5 hours). BCAT1 mRNA expression (Figure 3B) was increased by LPS; BT2 alone did not alter BCAT1, but BCAAs when combined with BT2 in LPS-stimulated RLMs reversed the inhibitory effect previously observed with LPS and BCAA co-treatment (Figure 1B). BCKDH increased only in LPS-stimulated-RLMs and decreased with BT2 (Figure 3B). At the protein level, BCAT1 and BCKDH remained unchanged across all conditions (Figure 3C-D), consistent with the absence of statistical significance shown previously (Figure 1D-E). These findings indicate that the metabolic flux through BCAA oxidation, rather than BCAA substrate availability per se, restores BCAT1 expression in activated-RLMs. BT2-mediated BCKDK inhibition enables leucine to attenuate NF-κB activation while preserving anti-inflammatory effects Regarding inflammatory markers, LPS alone maintained the induction of mRNA expression of CD11b, IL-1β, IL-6, HIF-1α, iNOS, TNF-α, and IL-10 (Figure 4A). BT2 alone did not modify any of these genes. When BT2 was combined with LPS, it partially attenuated LPS-induced upregulation of CD11b, Arg-1, and HIF-1α. This inhibitory effect was maintained and comparable when individual BCAAs or BCAA mixture were added to the BT2+LPS combination on those same genes, recapitulating the anti-inflammatory pattern observed with BCAAs alone (Figure 2). These findings suggest that enhanced BCAA catabolism through BCKDK inhibition preserves the selective anti-inflammatory effects of BCAAs on CD11b, Arg-1, and HIF-1α. To elucidate the mechanism by which BCAAs alone or in combination with BT2 modulate inflammatory responses in LPS-stimulated RLMs, we investigated the NF-κB pathway using immunofluorescence to assess nuclear translocation of activated NF-κB (red fluorescence) from the cytoplasm to the nucleus (blue fluorescence) (Figure 4B). Quantification using Manders' colocalization coefficient (Figure 4C) confirmed NF-κB nuclear translocation upon LPS treatment. Interestingly, leucine, valine, and the BCAA mixture alone increased NF-κB nuclear translocation compared to control conditions. However, when leucine was combined with BT2, NF-κB translocation was attenuated in both basal conditions and LPS-stimulated cells. This inhibitory effect was specific to the leucine plus BT2 combination, suggesting that enhanced leucine catabolism through BCKDK inhibition suppresses NF-κB activation. Enhanced BCAA catabolism abolishes the antioxidant effects of BCAAs in RLMs Oxidative stress is a central mediator of LPS-induced inflammation and is closely linked to BCAA metabolism. Given that NF-κB signaling can both induce and be driven by reactive oxygen species (ROS), we evaluated oxidative stress markers. Antioxidant gene analysis in LPS-stimulated RLMs exposed to BCAAs, either alone or with BT2 showed that NRF2 was upregulated by LPS. Leucine and the BCAA mixture significantly attenuated the LPS-induced NRF2 upregulation (Figure 5A). However, when BT2 enhanced BCAA catabolism, Leu and the BCAA mixture failed to attenuate the LPS-induced NRF2 upregulation (Figure 5B). SOD2 and GPX1 expression were not significantly altered in any treatment condition. At the protein level, neither NRF2 nor MnSOD (SOD2 protein) showed significant changes (Figure 5C–D). ROS generation using the DCFH‐DA probe (Figure 5E–F) showed LPS increased intracellular ROS, which were significantly attenuated by isoleucine, valine, and the BCAA mixture. This protective antioxidant effect of BCAAs was abolished in the presence of BT2. Together, these results demonstrate that BCAAs exert protective antioxidant effects in LPS-stimulated macrophages by reducing ROS and suppressing compensatory NRF2 upregulation. However, enhanced BCAA catabolism through BCKDK inhibition abolishes these protective effects, suggesting BCAA substrate availability, rather than metabolic flux through oxidation, is required for antioxidant protection. BCAAs suppress HK1-mediated glycolysis and enhance mitochondrial ATP production, indicating a mTORC1-independent metabolic shift To investigate the signaling mechanisms modulated by BCAAs, we examined the mTORC1 pathway, a classical target of amino acid signaling. Activation of mTORC1 downstream targets, ribosomal protein S6 kinase (S6K) and 4E-binding protein (4E-BP), was assessed in LPS-stimulated RLMs treated with BCAAs alone or in combination with BT2 (Figure 6A). Only leucine increased S6K mRNA expression in LPS-activated RLMs. No other treatment modified the expression of S6K or 4E-BP. We next evaluated the High-Mobility Group Box 1 (HMGB1) signaling pathway, as BCAAs modulate its redox-dependent translocation [16]. Total HMGB1 protein increased under BT2 treatment (Figure 6B). To assess HMGB1 release, we analysed supernatant fractions. LPS alone did not significantly alter HMGB1 release; however, the addition of individual BCAAs with LPS showed variable effects on total HMGB1 release. Notably, when BCAA catabolism was impaired by BT2 in the presence of LPS and BCAAs, HMGB1 release was markedly enhanced, suggesting BCAA accumulation under impaired catabolism exacerbates inflammatory HMGB1 secretion. Since LPS-induced HIF1α expression was reduced by BCAAs (Figure 2), we analysed its upstream regulator, prolyl hydroxylase domain-containing protein 3 (PHD3), which promotes HIF-α degradation. BCAA did not modulate PHD3 expression with or without BT2(Figure 6C). Finally, to elucidate the impact of BCAAs on cellular metabolism, we assessed energy production by quantifying ATP levels (Figure 6D) and glucose metabolism via the expression of hexokinase 1 (HK1), a key glycolytic enzyme and HIF1α target gene (Figure 6E). BCAA mixture significantly enhanced ATP production both alone and following BT2 supplementation. This synergistic ATP increase is unlikely to be mTORC1-mediated, given the absence of changes in the downstream targets, supporting a model where BCAAs act as oxidative substrates fuelling mitochondrial energy. In parallel, HK1 expression was downregulated in all combinations of BCAA in LPS-activated cells, maintained with BT2 co-treatment, correlating with HIF1α downregulation. This glycolytic enzyme suppression suggests BCAAs drive metabolic shift away from glycolysis, consistent with enhanced oxidative ATP production. HMGB1 upregulation suggests BCAAs influence inflammatory and oxidative signaling context-dependently. HIF-1α stabilization partially reverses BCAA-mediated anti-inflammatory effects but not antioxidant protection Since mTORC1 did not mediate the effects of BCAAs, we investigated the role of HIF1α and HMGB1 using the specific inhibitors dimethyloxalyl glycine (DMOG) and glycyrrhizic acid (Gla), respectively. RLMs were pretreated with BT2 in combination with DMOG or Gla for 30 minutes, then BCAAs for 2 hours and LPS for 4 hours (total treatment = 6.5 hours). Inflammatory gene analysis showed LPS upregulated IL1b, IL6, CD11b, MCP1, and HIF-1α expression as shown previously. Previously we showed that BT2 addition in LPS-activated RLMs did not effect on the BCAA anti-inflammatory effects (Figure 4A). Experiments adding Gla combined with the BCAAs mix and DMOG and Ile reversed the anti-inflammatory effects previously observed in BCAA plus LPS combinations. CD11b in these conditions, while DMOG combined with valine reversed HIF-1α upregulation in BT2-LPS-treated RLMs (Figure 7A and Supplementary Figure 4A). Gla and DMOG treatment did not affect NF-κB activation via p65 nuclear translocation since results are similar to the previous observations using BT and BCAAs in LPS activated RLMS (Supplementary Figure 4B-C). Furthermore, total HMGB1 expression was strongly reduced by both DMOG and Gla treatments (Figure 7B). Importantly, HK-1 suppression previously observed in BCAA-treated, LPS-activated cells was reversed by DMOG, with significant effects for leucine and valine combinations (Figure 7C). Notably, ROS quantification showed that both DMOG and Gla restored the BCAA-induced reduction in ROS production (Figure 7D), indicating that both HIF-1α and HMGB1 pathways contribute to BCAA-mediated antioxidant effects. These findings demonstrate that HIF-1α stabilization and HMGB1 inhibition selectively reverse BCAA suppressive effects on inflammatory genes and metabolic markers (HK-1), indicating that BCAA-mediated anti-inflammatory effects are dependent on both HIF-1α inhibition and HMGB1 modulation. However, the sustained antioxidant protection under conditions of HIF-1α stabilization and HMGB1 inhibition reveals mechanistic independence between BCAA-mediated ROS suppression and these inflammatory signaling pathways. Table 3. Summary of major findings on BCAA-mediated effects in resident liver macrophages. Category LPS BCAA BT2 BCAA + LPS BT2 + BCAA + LPS Metabolic enzymes ↑BCAT1 – Restores BCAT1 ↓BCAT1 (vs LPS) ↑BCAT1 restored Inflammatory genes ( CD11b, MCP1, HIF1α, Arg1) ↑↑ – – ↓ ↓ Antioxidant genes (NRF2) ↑ ↓ (vs LPS) - ↓ ↑ (BT2 reverses) ROS levels ↑↑ ↓ - ↓ ↑ NF- κ B signaling (p65 nuclear translocation) ↑ ↑ (basal) - ↑ ↓ (only Leu+BT2) HMGB1 signaling – – – Ile → disulfide HMGB1 ↑oxidized HMGB1 HIF1α / HK1 / PHD3 ↑HIF1α, ↑HK1 ↓HIF1α, ↓HK1 ↑PHD3 (ns) ↓↓HIF1α, ↓HK1 ↑PHD3, ↓HIF1α, mTORC1 pathway – – ↓S6K – ↓S6K ATP levels – – – ↑ATP (BCAA mix) ↑↑ATP (BCAA mix) Effect of DMOG / Gla – – – – Reversed the proinflammatory effects (Ile (CD11b) and Val (HIF1α)) ROS suppression BCAAs modulated inflammatory and oxidative responses by regulating BCAT1 expression, NF-κB activity, and metabolic reprogramming. BT2 and leucine altered these effects through changes in redox balance and HIF1α/HMGB1 signaling.. BCAAs, branched-chain amino acids; Ile, isoleucine; Leu, leucine; Val, valine; BT2, 3,6-dichlorobenzo-thiophene-2-carboxylic acid; LPS, lipopolysaccharide; HMGB1, high-mobility group box 1; HIF1α, hypoxia-inducible factor 1 alpha; PHD3, prolyl hydroxylase domain-containing protein 3; NFκB, nuclear factor kappa-light-chain-enhancer of activated B cells; ROS, reactive oxygen species; mTORC1, mechanistic target of rapamycin complex 1; ATP, adenosine triphosphate; Gla, glycyrrhizic acid; DMOG, dimethyloxalyl glycine. DISCUSSION This study provides novel insights into the immunometabolic functions of branched-chain amino acids (BCAAs) in resident liver macrophages (RLMs), revealing a complex interplay between amino acid metabolism, inflammatory signaling, and redox homeostasis. Our findings demonstrate that RLMs express the enzymatic machinery for BCAA metabolism, mainly via BCAT1, which is upregulated by LPS and suppressed by BCAA exposure, indicating feedback regulation of catabolism under inflammatory conditions. Functionally, BCAAs selectively attenuated LPS-induced expression of CD11b, MCP1, HIF-1α, and Arg1, while decreasing ROS accumulation and decreasing NRF2 activation, suggesting direct antioxidant effects that do not rely on compensatory stress responses. Inhibition of BCKDK with BT2 restored BCAT1 expression and preserved the anti-inflammatory effects of BCAAs but abolished their antioxidant protection, revealing that these two beneficial functions are mechanistically separable and governed by BCAA substrate availability versus metabolic flux, respectively. Mechanistically, only the leucine–BT2 combination suppressed NF-κB p65 nuclear translocation, implying that leucine catabolism produces specific intermediates or metabolic conditions that uniquely restrain this pathway. BCAA also promoted a metabolic shift from glycolysis toward oxidative phosphorylation, evidenced by increased ATP production and decreased HK1 expression, an effect independent of mTORC1 signaling. Given that mTORC1 did not mediate BCAA effects, we investigated the involvement of HIF-1α and HMGB1 using the specific modulators DMOG and glycyrrhizic acid (Gla). Modulation of the HIF1α and HMGB1 axes revealed that BCAA anti-inflammatory effects depend partly on HIF1α inhibition and HMGB1 modulation, whereas antioxidant actions require coordinated regulation through both pathways. DMOG also reversed the suppression of HK1 by BCAAs, especially with leucine and valine, confirming that BCAA-dependent repression of glycolysis is driven by HIF-1α downregulation. These findings extend prior evidence that LPS alters systemic BCAA metabolism by lowering circulating BCAA concentrations and leucine oxidation [ 28 , 29 ]. We demonstrate that macrophages themselves respond through BCAT1-dependent feedback, potentially preventing over-catabolism during inflammation. Consistent with earlier reports, BCAT1 enrichment in activated tumor-associated macrophages promotes immunosuppressive polarization [ 30 , 31 ], whereas its inhibition enhances anti-inflammatory phenotypes in models of arthritis [ 32 ]. Our data refine this concept by showing that while BT2 restores BCAT1 and maintains anti-inflammatory signaling, it disrupts redox homeostasis, suggesting that the balance between BCAA availability and metabolic capacity determines the immune outcome. This aligns with studies showing that impaired BCAA catabolism leads to BCKA accumulation, perturbs central carbon metabolism, and drives oxidative stress [ 33 ]. The selective modulation of inflammatory mediators by BCAAs which suppresses CD11b, MCP1, HIF-1α, and Arg1 while leaving IL-1β, IL-6, iNOS, and TNFα unaffected suggests targeted pathway regulation rather than global immunosuppression. Clinical data support this, showing that BCAAs reduce LPS-binding protein and TLR4 activation [ 34 ]. Importantly, our data demonstrate that the anti-inflammatory effects of BCAAs persist even when BCAA catabolism is enhanced by BT2, indicating that metabolic acceleration alone does not abolish their protective signaling. However, disruption of HIF-1α or HMGB1 signaling, through DMOG + Ile or Gla + BCAA mix, respectively, reversed these protective effects, restoring MCP1 and HIF-1α expression. These findings suggest that intact HIF-1α and HMGB1 pathways are essential mediators of BCAA-induced immunomodulation. In contrast to our results, BCAA supplementation enhanced M1 activation through mTORC1-HIF-1α-glycolysis signaling in exercise-induced muscle injury [ 14 ], underscoring tissue-specific responses. When BCAA catabolism is impaired, as in atherosclerotic macrophages with reduced BCAT2 and BCKDHA expression, excess BCAAs increase mitochondrial H₂O₂ and activate TLR4/NF-κB signaling [ 16 ]. Similarly, disrupted BCAA metabolism in obesity promotes pro-inflammatory polarization via IFNGR1/JAK1/STAT1 pathways [ 17 ]. These studies, together with our findings, reinforce the concept that context and metabolic balance determine whether BCAAs are protective or pathogenic. A remarkable observation is the interplay between BCAA metabolism, HIF-1α/HMGB1 signaling, and oxidative stress. BCAAs effectively attenuated LPS-induced ROS accumulation while reducing NRF2 gene expression, consistent with direct ROS-scavenging or prevention of mitochondrial overload. This is consistent with observations on itaconate metabolism, as BCAT1 inhibition reduces IRG1 expression and itaconate levels required for Nrf2 activation by alkylating cysteine residues [ 32 , 35 ]. BT2 treatment reversed this effect, increasing ROS, while both DMOG and Gla restored ROS reduction. This suggests that BCAA-mediated antioxidant protection relies on HIF-1α and HMGB1 pathways acting in concert. DMOG experiments further showed that HIF-1α stabilization reversed HK1 suppression, especially for leucine and valine, linking glycolytic control to HIF-1α regulation. This agrees with reports that excessive BCAA concentrations downregulate HIF-1α and GLUT1, reducing glycolytic flux [ 36 ] and that HIF-1α deletion enhances amino acid catabolism [ 37 ]. Because HK1-dependent glycolysis promotes inflammasome activation [ 38 ], its downregulation likely contributes to the anti-inflammatory phenotype observed here. HMGB1 functions as a redox-sensitive DAMP molecule [ 39 ]. Its redox status determines biological activity, with the disulfide form promoting inflammation and the fully reduced form acting as a chemoattractant, while terminal oxidation (sulfonation) abolishes these immune functions [ 40 ]. Zhao S. et al. (2023) [ 16 ] demonstrated that excessive BCAAs in atherosclerotic macrophages increase mitochondrial H₂O₂ production, promoting disulfide HMGB1 release and activating the TLR4/NF-κB pathway. In our study, BCAA exposure suppressed HMGB1-associated inflammation and reduced ROS, but addition of Gla with the BCAA mix reversed these effects, restoring MCP1 and HIF-1 expression. This indicates that BCAA-mediated protection requires functional HMGB1 signaling, and that excessive inhibition or interference of this pathway disrupts redox and inflammatory control. Moreover, increased extracellular HMGB1 detected under BT2 + LPS conditions suggests that when catabolism is dysregulated, BCAAs can no longer restrain HMGB1-driven inflammation. Although we could not determine specific HMGB1 redox isoforms, future work employing non-reducing electrophoresis and redox-specific antibodies should clarify whether altered BCAA metabolism induces distinct oxidative HMGB1 modifications. At the metabolic level, BCAAs increased ATP production and suppressed HK1, consistent with enhanced oxidative phosphorylation and reduced glycolytic flux, features characteristic of anti-inflammatory macrophages dependent on the TCA cycle. Interestingly, while oxidative metabolism and reduced HIF-1α expression reflected M2-like features, the simultaneous downregulation of Arg1 indicates that this reprogramming is independent of the canonical arginine–Arg1 axis, possibly mediated by BCAA-derived glutamine or TCA intermediates. The accumulation of BCKAs during accelerated catabolism likely underlies the loss of antioxidant capacity, as BCKAs inhibit BCKDH activity in a feedback loop [ 41 ] and interfere with PHD2, stabilizing HIF-1α under aerobic conditions [ 42 ]. We propose that α-ketoglutarate (αKG) availability represents a central node linking BCAA flux and HIF-1α regulation. When BCAT1 is overexpressed, it can consume too much αKG, reducing its availability for these enzymes and thereby stabilizing HIF-1α [ 43 ]. Moderate BCAA metabolism generates αKG, supporting prolyl hydroxylase activity and HIF-1α degradation. In contrast, excessive BCAT1 activity or BT2-driven catabolism depletes αKG and accumulates BCKAs, impairing hydroxylase function and stabilizing HIF-1α. This dual effect reconciles the paradox whereby both BCAA supplementation and excessive catabolism produce opposing HIF-1α outcomes. The clinical relevance of our study is supported by the therapeutic benefits of BCAAs in liver diseases, with protective effects mediated via LPS-binding protein and TLR4 suppression [ 34 ]. However, in metabolic liver diseases such as MASLD, where BCAA catabolism is impaired, BCAA accumulation and defective oxidation could drive inflammation via ROS- and HMGB1-dependent mechanisms. Therapeutic strategies should therefore focus on restoring balanced BCAA metabolism, rather than simple supplementation or restriction, to avoid the harmful buildup of catabolic intermediates. Finally, our study has limitations. The experiments were performed in vitro, under short-term exposure (6.5 h) and with single LPS doses, which cannot fully replicate the hepatic microenvironment. Future studies should address time-dependent effects, integration with glucose and lipid metabolism, BCAA transport regulation, and direct quantification of BCKAs to delineate mechanisms controlling the balance between anti-inflammatory and antioxidant responses. CONCLUSIONS This study establishes BCAA metabolism as a central checkpoint in liver macrophage activation, integrating inflammatory and redox pathways through distinct but interconnected mechanisms. BCAAs suppress HIF-1α-driven glycolysis and selected inflammatory mediators while promoting oxidative metabolism. These anti-inflammatory effects remain intact under enhanced BCAA catabolism (BT2 treatment) but are abolished when HIF-1α or HMGB1 signaling is perturbed, demonstrating that BCAA-mediated protection depends on the integrity of these pathways. BCAAs thus emerge as dual regulators integrating metabolism and immune signaling; when metabolized appropriately, they maintain oxidative balance and limit inflammation; when catabolic or signaling control is lost, the system shifts toward oxidative and inflammatory stress. These context-dependent effects emphasize that substrate availability and signaling integrity, rather than flux alone, dictate macrophage responses. Clinically, this work links systemic inflammation to BCAA dysregulation in metabolic liver disease. Therapeutic strategies should aim to optimize BCAA metabolism, maintaining adequate substrate to support coordinated HIF-1α/HMGB1-mediated defense while avoiding BCKA accumulation or excessive pathway interference. Assessing BCAT1/BCKDH activity or BCAA/BCKA ratios may help identify metabolic states most likely to benefit from BCAA-targeted therapy. Balancing catabolic efficiency with pathway stability may restore integrated control of inflammation and oxidative stress, providing a rational framework for BCAA-based interventions in MASLD and related disorders. Abbreviations Arg-1, Arginase-1; ATP, Adenosine triphosphate; BCAA, Branched-chain amino acid; BCAT1/2, Branched-chain amino acid transaminase 1/2; BCKDH, Branched-chain α-ketoacid dehydrogenase; BCKDK, Branched-chain α-ketoacid dehydrogenase kinase; DAMP, Damage-associated molecular pattern; DMOG, Dimethyloxalylglycine; Gla, Glycyrrhizic acid; HBSS, Hank’s balanced salt solution; HIF-1α, Hypoxia-inducible factor 1α; HMGB1, High-mobility group box 1; HK1, Hexokinase 1; IL, Interleukin; iNOS, Inducible nitric oxide synthase; KCs, Kupffer cells; LPS, Lipopolysaccharide; MASLD, Metabolic dysfunction–associated steatotic liver disease; MASH, Metabolic dysfunction–associated steatohepatitis; MAPK, Mitogen-activated protein kinase; MCP1, Monocyte chemoattractant protein-1; mTORC1, Mechanistic target of rapamycin complex 1; NF-κB, Nuclear factor kappa B; NRF2, Nuclear factor erythroid 2–related factor 2; PAMP, Pathogen-associated molecular pattern; PARP1, Poly(ADP-ribose) polymerase 1; PHD3, Prolyl hydroxylase domain protein 3; qPCR, Quantitative polymerase chain reaction; RLMs, Resident liver macrophages; ROS, Reactive oxygen species; S6K, Ribosomal protein S6 kinase; SOD2, Superoxide dismutase 2; TLR4, Toll-like receptor 4; TNF-α, Tumor necrosis factor alpha. Declarations Authors’ contributions: H.M and H.B provided supervision and project concept. M. M.A designed the study, performed experiments, and analysed results. M. B.H. provided technical support. M. M.A. drafted the manuscript. All authors critically revised the manuscript and approved the final version. Address for correspondence: Magnolia Martinez, [email protected] , or Han Moshage, [email protected] ; Department of Gastroenterology and Hepatology, University Medical Center Groningen, University of Groningen, P.O. Box 30.001, 9700 RB Groningen, The Netherlands. Conflict of interest: The authors have no conflicts of interest to report. Financial support and sponsorship: The author(s) gratefully acknowledge the financial support provided by CONACYT (now SECIHTI) through the PhD grant (no. 795389). 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Nature . 2017;551(7680):384-388. doi:10.1038/nature24294 Additional Declarations No competing interests reported. Supplementary Files Supplementedfigures.BCAAmodulateresidentlivermacrophage.MM.4thversionHM.docx Cite Share Download PDF Status: Posted Version 1 posted 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. 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13:31:20","extension":"xml","order_by":21,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":137799,"visible":true,"origin":"","legend":"","description":"","filename":"1dceedfbdc3c461caa7b8e26b0e80a311structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-8077254/v1/5f16b618213e8a888b7cca08.xml"},{"id":98425318,"identity":"18a8f5df-6ab5-40a7-9957-d33f8d74d49e","added_by":"auto","created_at":"2025-12-17 16:34:38","extension":"html","order_by":22,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":146650,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8077254/v1/43d7a1e324e6449ba57889ef.html"},{"id":97984692,"identity":"98a172fc-6984-4a6a-b1e9-0610e4307639","added_by":"auto","created_at":"2025-12-11 13:31:18","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":264199,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eActivated resident liver macrophages (RLMs) induce the expression of BCAT1. A)\u003c/strong\u003eSchematic representation of BCAA catabolism. \u003cstrong\u003eB)\u003c/strong\u003e Primary rat RLMs were pre-treated with BCAAs (Ile, leu, val) alone or as a mixture (1:2:1 ratio) (15 mM) for 2 h and then co-treated with LPS (100 ng/mL) for 4 h. \u003cstrong\u003eC)\u003c/strong\u003e BCAT1 and BCKDH mRNA expression (fold-change, 2^–ΔΔCt). \u003cstrong\u003eD-E)\u003c/strong\u003e Representative immunoblots and quantification (adjusted band volume) of BCKDH and BCAT1 protein, normalized to GAPDH. Statistical analysis was performed using one-way ANOVA followed by Tukey’s (for multiple comparisons) or Dunnett’s (vs control) post hoc test. Significance is indicated as \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05 (*\u003cem\u003e), p \u0026lt; 0.01 (**), p \u0026lt; 0.001 (***\u003c/em\u003e). BCAA, branched-chain amino acids; BCAT1/2, branched-chain amino acid transaminase 1/2; BCKDH, branched-chain α-keto acid dehydrogenase; Ile, isoleucine; Leu, leucine; Val, valine; LPS, lipopolysaccharide.\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8077254/v1/53a68ee9d055948b09359319.jpeg"},{"id":98424617,"identity":"e181a1fe-a11d-4557-87e4-46038b1d3d02","added_by":"auto","created_at":"2025-12-17 16:33:34","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":240984,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBCAAs attenuate inflammatory gene expression in RLMs following LPS stimulation. \u003c/strong\u003eRLMs were treated with isoleucine (Ile), leucine (Leu), valine (Val), or a BCAA mixture (2:1:1 ratio) in the presence or absence of LPS (100 ng/mL). mRNA expression (fold-change, 2^–ΔΔCt) of M1 (CD11b, MCP1, IL-1β, IL-6, iNOS, TNFα, HIF-1α) and M2 (IL-10, Arginase-1) phenotype-related genes. Data are presented as mean ± SEM (n = 4). Statistical analysis was performed using one-way ANOVA followed by Tukey’s (for multiple comparisons) or Dunnett’s (vs. control) post hoc test. Significance is indicated as p \u0026lt; 0.05 (*), p \u0026lt; 0.01 (**), p \u0026lt; 0.001 (***), and \u003cem\u003ep\u003c/em\u003e\u0026lt; 0.0001 (****). \u0026nbsp;Abbreviations: BCAA, branched-chain amino acids; Ile, isoleucine; Leu, leucine; Val, valine; LPS, lipopolysaccharide; TNFα, tumor necrosis factor alpha; MCP1, monocyte chemoattractant protein-1; iNOS, inducible nitric oxide synthase; HIF-1α, hypoxia-inducible factor 1 alpha; IL, interleukin.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-8077254/v1/d24ba1df958a2488b617caaf.png"},{"id":98423735,"identity":"fde0b818-a5b3-4371-8ad2-2960e06c4699","added_by":"auto","created_at":"2025-12-17 16:32:34","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":201453,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBT2-mediated BCKDK inhibition reverses BCAT1 suppression.\u003c/strong\u003e RLMs treated with BT2 and BCAAs ± LPS (100 ng/mL)\u003cstrong\u003e. A)\u003c/strong\u003e Schematic of the BCKDK inhibitor BT2 and its role in enhancing BCAA oxidation. \u003cstrong\u003eB)\u003c/strong\u003e Relative mRNA expression (fold-change, 2^–ΔΔCt) of BCAT1 and BCKDH. \u003cstrong\u003eC–D)\u003c/strong\u003eRepresentative blots (C) and protein quantification (adjusted band volume) normalized to GAPDH (D). Data are mean ± SEM (n = 4). Statistical analysis was performed using one-way ANOVA with Tukey’s (multiple comparisons) or Dunnett’s (vs. control) post hoc test. Significance is indicated as \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05 (*\u003cem\u003e), p \u0026lt; 0.01 (**), p \u0026lt; 0.001 (***\u003c/em\u003e). BCAA, branched-chain amino acid; BCAT1/2, branched-chain amino acid transaminase 1/2; BT2, 3,6-dichlorobenzothiophene-2-carboxylic acid; BCKDH, branched-chain α-ketoacid dehydrogenase; BCKDK, branched-chain α-ketoacid dehydrogenase kinase; LPS, lipopolysaccharide.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-8077254/v1/08cafb5f24f5010aaa5c2dc9.png"},{"id":98423777,"identity":"12a4c8ff-9a5f-484d-866b-f6d7037cff84","added_by":"auto","created_at":"2025-12-17 16:32:35","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":260587,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBT2-mediated BCKDK inhibition enables leucine to attenuate NF-κB activation while preserving the anti-inflammatory effect.\u003c/strong\u003e RLMs treated with BT2 and BCAAs ± LPS (100 ng/mL).\u003cstrong\u003e \u0026nbsp;A) \u003c/strong\u003emRNA expression (fold-change, 2^–ΔΔCt) of M1 (CD11b, MCP1, IL-1β, IL-6, iNOS, TNFα, HIF-1α) and M2 (IL-10, Arginase-1) phenotype-related genes.\u003cstrong\u003e B-C) \u003c/strong\u003eNF-κB nuclear translocation. Immunofluorescence staining of NF-κB (red) and nuclei (DAPI, blue), magnification 40x (C) and quantification of colocalization using Manders’ coefficient (D). Data are mean ± SEM of n = 4. Statistical analysis was performed using one-way ANOVA followed by Tukey’s post hoc test. Significance compared with LPS alone is indicated as \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05 (*\u003cem\u003e), p \u0026lt; 0.01 (**)\u003c/em\u003e. BCAA, branched-chain amino acid; BT2, 3,6-dichlorobenzothiophene-2-carboxylic acid; LPS, lipopolysaccharide; NF-κB, nuclear factor kappa B; MAPK, mitogen-activated protein kinase; p-p65, phosphorylated p65 subunit.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-8077254/v1/9bc44a94ca2ffc72ba5a92d6.png"},{"id":98423989,"identity":"85c8461b-18fd-47cb-8953-11606e16991f","added_by":"auto","created_at":"2025-12-17 16:32:50","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":423474,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEnhanced BCAA catabolism abolishes the antioxidant effects of BCAAs in RLMs. \u003c/strong\u003eRLMs treated with BT2 and BCAAs ± LPS (100 ng/mL).\u003cstrong\u003e A–B)\u003c/strong\u003e mRNA expression (fold-change, 2^–ΔΔCt) of NRF2, SOD2, and GPX1 after treatment with BCAAs alone (A) or in combination with BT2 (B). \u003cstrong\u003eC)\u003c/strong\u003eQuantification (adjusted band volume) of NRF2 and MnSOD protein levels in RLMs treated with BCAAs and/or BT2 in the presence or absence of LPS.\u003cstrong\u003e D) \u003c/strong\u003eRepresentative immunoblots of NRF2 and MnSOD. \u003cstrong\u003eE-F)\u003c/strong\u003e ROS production assessed with the DCFH‐DA probe, representative images (E) and quantification (average raw intensity) (F). Data are mean ± SEM of \u003cem\u003en\u003c/em\u003e = 4. Statistical analysis was performed using one-way ANOVA followed by Tukey’s post hoc test. Significance compared with LPS alone is indicated as p \u0026lt; 0.05 (*), p \u0026lt; 0.01 (**), p \u0026lt; 0.001 (***), and p \u0026lt; 0.0001 (****). BCAA, branched-chain amino acid; LPS, lipopolysaccharide; NRF2, nuclear factor erythroid 2– related factor 2; SOD2, superoxide dismutase 2; GPX1, glutathione peroxidase 1; ROS, reactive oxygen species.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-8077254/v1/3491ef35ef661671c5266c00.png"},{"id":98424974,"identity":"d9d187bc-5b0e-4037-9fe1-9c278dbfc78e","added_by":"auto","created_at":"2025-12-17 16:34:08","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":253943,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBCAAs drive a glycolytic-to-oxidative metabolic shift while redox-modulating HMGB1 in a mTORC1-independent pathway.\u003c/strong\u003e RLMs treated with BT2 and BCAAs ± LPS (100 ng/mL).\u003cstrong\u003e \u003c/strong\u003eRLMs treated with individual or a mixture of BCAAs with or without BT2, in the presence of LPS. Representative immunoblots (up) and quantification (adjusted band volume) of A) mTORC1 downstream targets S6K and 4E-BP, B) HMGB1 \u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u003cbr\u003e\n protein expression in whole cell lysates and supernatants, and C) PHD3 expression. D) ATP quantification (pM) using a luminescence kit. E) mRNA expression (fold-change, 2^–ΔΔCt) of hexokinase-1 (HK1). Data are mean ± SEM of \u003cem\u003en\u003c/em\u003e = 3. Statistical analysis was performed using one-way ANOVA followed by Tukey’s post hoc test. Significance compared with LPS alone is indicated by p \u0026lt; 0.05 (*), p \u0026lt; 0.01 (**), p \u0026lt; 0.001 (***), and p \u0026lt; 0.0001 (****). BCAA, branched-chain amino acids; BT2, BCKDK inhibitor; HMGB1, High-Mobility Group Box 1; LPS, lipopolysaccharide; PHD3, prolyl hydroxylase domain-containing protein 3.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-8077254/v1/577c7f3ad3319d5e9f335cd6.png"},{"id":98424711,"identity":"f2fe495e-0fa7-4f48-a77e-408f4c4967e0","added_by":"auto","created_at":"2025-12-17 16:33:43","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":346744,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eInhibition of HIF1αand HMGB1 modulates the effects of BCAAs in BT2-LPS-stimulated RLMs.\u003cbr\u003e\n \u003c/strong\u003eRLMs\u003cstrong\u003e \u003c/strong\u003etreated with BCAAs (alone or as a mixture), LPS (100 ng/mL), and BT2 with the HMGB1 (Gla) or the HIF1α (DMOG) inhibitor.\u003cstrong\u003e A) \u003c/strong\u003emRNA expression (fold-change, 2^–ΔΔCt) of HIF1α, Arg1, MCP-1, and CD11b. \u003cstrong\u003eB) \u003c/strong\u003eRepresentative immunoblot (left) and quantification (adjusted band volume) of total HMGB1.\u003cstrong\u003e C) \u003c/strong\u003emRNA expression (fold-change, 2^–ΔΔCt) of hexokinase-1 (HK1). \u003cstrong\u003e\u0026nbsp;D) \u003c/strong\u003eROS production assessed with the DCFH‐DA probe, representative images (right), and quantification (average raw intensity). Data are mean ± SEM of n = 3. Statistical analysis was performed using one-way ANOVA followed by Tukey’s post hoc test. Significance compared with LPS alone is indicated as p \u0026lt; 0.05 (*), p \u0026lt; 0.01 (**),. BCAA, branched-chain amino acids; BT2, BCKDK inhibitor; DMOG, dimethyloxallyl glycine; Gla,\u003cstrong\u003e \u003c/strong\u003eglycyrrhizic acid; HMGB1, High-Mobility Group Box 1; LPS, lipopolysaccharide; ROS, reactive oxygen species.\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-8077254/v1/3c70576e03093dd4c9c1c307.png"},{"id":98425354,"identity":"7d80dcb4-7d28-4438-8ab3-91dbda8ab7f0","added_by":"auto","created_at":"2025-12-17 16:34:40","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":380978,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eImmunometabolic reprogramming of resident liver macrophages by BCAAs. \u003c/strong\u003eLPS induced BCAT1 expression and pro-inflammatory and oxidative response. BCAA attenuated LPS-induced activation of CD11b, MCP1, HIF1α, and Arg1, while reducing ROS and NRF2 upregulation. BT2 reversed BCAA-mediated BCAT1 suppression and preserved the anti-inflammatory profile but abolished the antioxidant protection. Leucine + BT2 combination attenuated NF-κB activation. BCAAs promoted a glycolytic-to-oxidative phosphorylation shift (↑ ATP, ↓ HK1), while modulating HMGB1 redox status. Stabilization of HIF1α (DMOG) or inhibition of HMGB1 (glycyrrhizin acid, Gla) reversed BCAA/BT2-mediated inflammatory effects and promote ROS suppression. BCAAs, branched-chain amino acids; Ile, isoleucine; Leu, leucine; Val, valine; BT2, 3,6-dichlorobenzo-thiophene-2-carboxylic acid; LPS, lipopolysaccharide; HMGB1, high-mobility group box 1; HIF1α, hypoxia-inducible factor 1 alpha; PHD3, prolyl hydroxylase domain-containing protein 3; NFκB, nuclear factor kappa-light-chain-enhancer of activated B cells; ROS, reactive oxygen species; mTORC1, mechanistic target of rapamycin complex 1; ATP, adenosine triphosphate; Gla, glycyrrhizic acid; DMOG, dimethyloxalyl glycine.\u003c/p\u003e","description":"","filename":"floatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-8077254/v1/441fba577b756c29a9e65df4.png"},{"id":102855778,"identity":"5fe1f077-9d13-4522-ad36-eb79a53a5d8c","added_by":"auto","created_at":"2026-02-17 14:57:34","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3574350,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8077254/v1/25f316dd-5d55-485f-9337-f9a5c5ee7901.pdf"},{"id":97984715,"identity":"3c4e0ead-f187-459d-a0e7-1847db6e12cd","added_by":"auto","created_at":"2025-12-11 13:31:20","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":19786181,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementedfigures.BCAAmodulateresidentlivermacrophage.MM.4thversionHM.docx","url":"https://assets-eu.researchsquare.com/files/rs-8077254/v1/150b72329341548de37634d4.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Enhanced catabolism of branched-chain amino acids uncouples anti-inflammatory and antioxidant functions in liver macrophages","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eDiet enriched in fat and complex carbohydrates, together with sedentarism, have driven a global increase in metabolic dysfunction\u0026ndash;associated steatotic liver disease (MASLD), affecting 38% of adults and is projected to reach 55% by 2040 [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. MASLD is defined by hepatic steatosis with cardiometabolic risk factors. Early disease responds to lifestyle changes, yet chronic exposure to risk factors promotes progression to metabolic dysfunction-associated steatohepatitis (MASH), fibrosis, cirrhosis, and hepatocellular carcinoma. Preventing this progression is therefore central to reducing liver-related morbidity and mortality.\u003c/p\u003e\u003cp\u003eMASLD pathophysiology follows a \u0026ldquo;multiple parallel hits\u0026rdquo; hypothesis [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e], where genetic predisposition and environmental factors synergize [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Lipid accumulation and insulin resistance cause hepatocellular lipotoxicity, mitochondrial dysfunction, and reactive oxygen species (ROS) generation. Concurrently, gut dysbiosis resulting from hypercaloric diets enhances bacterial translocation into the liver [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e], releasing damage- and pathogen-associated molecular patterns (DAMPs/PAMPs), that activate innate immunity [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Resident liver macrophages (RLMs), known as Kupffer cells (KCs), amplify local inflammation, which is central to disease progression [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Sustained RLMs activation further promotes hepatocyte injury, stellate cell activation, and fibrosis.\u003c/p\u003e\u003cp\u003eRLMs constitute\u0026thinsp;~\u0026thinsp;90% of liver macrophages, they mediate debris clearance, iron recycling, cholesterol regulation, immune tolerance, and pathogen defence. Their phenotype is highly plastic, shaped not only by DAMPs and PAMPs but also by lipids, extracellular vesicles and dietary nutrients [\u003cspan additionalcitationids=\"CR9 CR10\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e], driving polarization toward a pro-inflammatory (M1) or anti-inflammatory (M2) phenotype.\u003c/p\u003e\u003cp\u003eBranched-chain amino acids (BCAAs: leucine, isoleucine, and valine) are immunometabolism modulators, which determine how metabolic pathways and their intermediates regulate the energy supply, signaling pathways, and activity of immune cells [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. BCAAs regulate glucose homeostasis, mitochondrial energy production, protein synthesis, and autophagy [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], but their immune effects are context-dependent. In muscle repair, diabetes, cancer, and atherosclerosis BCAA yield contradictory findings, promoting pro-inflammatory phenotypes via such mTORC1\u0026ndash;HIF1α\u0026ndash;glycolysis, IFNGR1\u0026ndash;JAK1\u0026ndash;STAT1, or HMGB1\u0026ndash;TLR4\u0026ndash;NF-κB pathways [\u003cspan additionalcitationids=\"CR15 CR16\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e], or anti-inflammatory responses, through undefined mechanisms [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e] .\u003c/p\u003e\u003cp\u003eBCAA catabolism efficiency is critical. Proper catabolism supports anti-inflammatory programs and metabolic homeostasis. However, impaired catabolism, as occurs in metabolic diseases, BCAAs and their ketoacid metabolites accumulate, driving pro-inflammatory polarization and exacerbating tissue dysfunction. MASLD/MASH patients have elevated circulating BCAAs (396\u0026ndash;507 \u0026micro;M vs. 350 \u0026micro;M in healthy subjects) [\u003cspan additionalcitationids=\"CR21\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Paradoxically, advanced liver disease, shows BCAA depletion, reflecting metabolic collapse.\u003c/p\u003e\u003cp\u003eDespite these insights, BCAA effects on RLMs remain unclear. This study investigates how high BCAA exposure affects RLM activation, oxidative stress, and inflammatory signaling, and examines the effects of impaired BCAA catabolism.\u003c/p\u003e"},{"header":"MATERIAL AND METHODS","content":"\u003cp\u003e\u003cstrong\u003eMaterials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRPMI medium, heat-inactivated fetal calf serum (FCS) and PBS1x were obtained from Thermo Fisher, Waltham, MA, USA, respectively: 11835030, A5256701, 70011). \u0026nbsp; \u0026nbsp;Penicillin/streptomycin, fungizone, and sodium bicarbonate were purchased from GIBCO (Thermo Fisher). BCAAs purchased from Sigma Aldrich, St. Louis, MO, USA (L-Leucine (L8912-25g), L-Isoleucine (I7403-25g), and L-Valine (V0513-25g)) were dissolved in water to 15 mM. 3,6-Dichloro-benzothiophene-2-carboxylic acid (BT2) (Cayman Chemical, Ann Arbor, MI, USA; 34576-94-8) was dissolved in DMSO to 15 mM. Glycyrrhizic acid (MedChemExpress, Monmouth Junction, NJ, USA; HY-N0184) was dissolved in DMSO to 1 mM (Sigma Aldrich, 276855). DMOG (Sigma-Aldrich, D3695-50MG) was dissolved in DMSO to 1 mM.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnimals and cell isolation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMale Wistar rats (220\u0026ndash;250 g, 5\u0026ndash;8 weeks) obtained from Charles River Laboratories (Wilmington, MA, USA) were housed under standard conditions (25 \u0026plusmn; 2 \u0026deg;C, 12 h light/dark cycle, with free access to chow and water) at the University Medical Center Groningen. All procedures complied with Dutch legislation (Animal Act 2011) and were approved by the local ethics committee (permit no. 2115139-01-001). Anaesthesia was induced with 5% isoflurane, ketamine (60 mg/kg) and medetomidine (0.5 mg/kg). RLMs were isolated using the two-step collagenase perfusion method. Livers were perfused via the portal vein with Ca\u0026sup2;⁺/Mg\u0026sup2;⁺-free HBSS, followed by HBSS containing Ca\u0026sup2;⁺\u0026nbsp;(5.7 mM) and collagenase (150 U/ml, Sigma-Aldrich, C0130). Tissue was dissociated and filtered. Hepatocytes were removed by low-speed centrifugation (65 g for 2 min); non-parenchymal cells were enriched by Optiprep (Stemcell Technologies, Vancouver, BC, Canada) gradient centrifugation (17.6% and 8.2%). After washing, cells were resuspended and seeded. After 30 minutes, non-adherent cells were removed by replacing the medium, enriching for adherent RLMs. Cells were identified as CD68⁺/CD163⁺\u0026nbsp;(Supplemented Figure 2).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTreatments and drugs\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBased on our previous findings that 15 mM BCAA has antifibrotic effects on stellate cells [23], the same concentration was used. RLMs were treated with 15 mM individual BCAAs or combinations (1:2:1 Ile:Leu:Val). To mimic MASLD inflammation, cells were pretreated with BCAAs for 2h, then stimulated with with lipopolysaccharide (LPS, 100 ng/mL, Sigma-Aldrich, L2887) for 4h (total 6h) (Lichtman SN et al., 1994). BT2 (IC₅₀ = 3.19 \u0026mu;M) was applied 30 min before BCAAs to enhance BCAA catabolism. DMOG (IC₅₀ = 0.1 mM; [24]) and glycyrrhizic acid (Gla, Kᴅ\u0026nbsp;= 150 \u0026micro;M; [25]) were administered 30 min before BCAAs.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCell viability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCell viability was assessed using SYTOX Green (125 nmol/L; Thermo Fisher, S7020). Sytox was added after treatment for 15 minutes at 37 \u0026deg;C in an atmosphere containing 5% (v/v) CO₂. Necrotic cells were stained green (SYTOX) and visualized using a Leica fluorescence microscope (Leica Microsystems, Wetzlar, Germany; DMI6000) at 512\u0026ndash;542 nm and 585\u0026ndash;624 nm, respectively (Supplementary Figure 1A).\u003c/p\u003e\n\u003cp\u003eAdditionally, lactate dehydrogenase (LDH) release was calculated as the percentage of the activity of LDH released in the medium vs. the total LDH activity (in both the medium and cell lysates). LDH was measured spectrophotometrically at 340 nm using an Epoch2 microplate reader (Bio-Tek Instruments, Inc., Winooski, VT, USA) (Supplementary Figure 1B).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWestern blot analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eProtein lysates were isolated by four freezing-thawing cycles followed by centrifugation at 12000 \u0026times; g for 15 minutes. Protein from supernatant fraction were isolated using a methanol-chloroform extraction protocol [26]. Briefly, 600 \u0026micro;L of a 4:1 methanol: chloroform mixture was added to the sample, vortexed, and centrifuged at 10,000 \u0026times; g for 5 min at 4\u0026deg;C. The supernatant was discarded, and the protein pellet was washed with 500 \u0026micro;L of methanol, followed by centrifugation. The final pellet was dried, resuspended in 70 \u0026micro;L of 1.5\u0026times; Laemmli buffer, and denatured at 100\u0026deg;C for 10 min for SDS-PAGE and Western blot analysis. Protein concentration was quantified using the Bio-Rad protein assay kit with BSA as standard (Bio-Rad Laboratories, Hercules, CA, USA; 5000111). For Western blot, 40 \u0026micro;g of protein was loaded on 10% SDS-PAGE gels, electrophoresed at 100 V for 90 min and transferred to nitrocellulose membrane by semi-dry blotting for 30 minutes. Membranes were blocked with 5% BSA, incubated overnight with primary antibodies (Supplementary Table 1), and then with HRP-conjugated secondary antibodies (Goat anti-rabbit horseradish peroxidase-labeled secondary antibody (Agilent Technologies, Santa Clara, CA, USA; P0448)) or polyclonal Rabbit Anti-Mouse Immunoglobulins/HRP (Agilent Technologies, P0260). Bands were visualized using a ChemiDoc XRS system (Bio-Rad).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImmunofluorescence\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRLMs were seeded on glass coverslips in 12-well plates and fixed with 4% paraformaldehyde for 10 min at room temperature. After washing with HBSS, cells were permeabilized with 0.1% Triton X-100 for 30 min at 37 \u0026deg;C and blocked with 2% BSA in PBS for 30 min. Cells were incubated for 1 hour with primary antibody (1:300 in 2% BSA/PBS) against NF-\u0026kappa;B p65 (Cell Signaling Technology, Danvers, MA, USA; 8242), CD68 (AbD Serotec, Kidlington, UK; MCA341R) or CD163 (Hycult, Uden, Netherlands; HM3025), followed by Alexa Fluor TM 488 donkey anti-goat (Invitrogen, Carlsbad, CA, USA, \u0026nbsp;A-11055) or Alexa Fluor 568 \u003csup\u003eTM\u003c/sup\u003e goat anti-mouse (Invitrogen, A-11004) (1:500 in 2% BSA/PBS, 30 min, RT). Nuclei were counterstained with DAPI (1:1000, 30 min) (Roche, 10236276001). Coverslips were mounted using Dako fluorescence mounting medium (Agilent Technologies, S3023) and imaged using a Leica fluorescence microscope (Leica Microsystems, DMI6000). Characterization of RLMs based on the presence of CD68 and CD163 is presented in Supplementary Figure 2.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRNA isolation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTotal RNA was isolated using TRI-reagent (Sigma-Aldrich, T9424) according to the manufacturer\u0026apos;s instructions. RNA was separated by chloroform extraction, precipitated with isopropanol, washed with 75% ethanol, air-dried, and resuspended in RNase-free water. RNA concentration and purity were determined using a NanoDrop 2000 spectrophotometer (Thermo Scientific, Waltham, MA, USA; ND-2000).\u003c/p\u003e\n\u003cp\u003eFor cDNA synthesis, 2.5 \u0026mu;g of total RNA was reverse transcribed in a 50 \u0026mu;L reaction containing 1X RT buffer (500 mmol/l Tris-HCl[pH 8.3]; 500 mmol/l KCl; 30 mmol/l MgCl₂; 50 mmol/l DTT), 1 mmol/l deoxynucleotides triphosphate (dNTPs, Sigma-Aldrich), 10 ng/\u0026mu;L random nanomers (Thermo Fisher), 0.6 U/\u0026mu;L RNaseOUT\u0026trade; (Invitrogen), and 4 U/\u0026mu;L M-MLV reverse transcriptase (Invitrogen). The reaction was performed at 25\u0026deg;C for 10 min, 37\u0026deg;C for 60 min, and 95\u0026deg;C for 5 min. The resulting cDNA was diluted 20-fold before qPCR.\u003c/p\u003e\n\u003cp\u003eReal-time qPCR was performed on a StepOnePlus\u0026trade; system (Applied Biosystems, Thermo Fisher) using TaqMan probes. Each reaction contained a 2X master mix (dNTPs, Hot Gold Star DNA polymerase, 5 mmol/l MgCl₂) (Eurogentec, Li\u0026egrave;ge, Belgium), fluorogenic TaqMan probes, and gene-specific primers (Supplementary Table 2).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eReactive oxygen species measurement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIntracellular reactive oxygen species (ROS) levels were assessed using the fluorescent probe DCFH‐DA (MedChemExpress, HY-D0940). Cells cultured in 12-well plates were washed twice with 1 mL of warm PBS 1X followed by incubation with 1 mL of 10 \u0026micro;M H₂DCF-DA in PBS 1X at 37\u0026deg;C in 5% CO₂ for 30 minutes in the dark. After incubation, dye was aspirated, and the cells were washed twice with 1 mL of PBS 1X. For nuclear staining, DAPI (1 \u0026micro;g/mL per well) was added for 5 minutes in the dark. Nuclei were counterstained with DAPI (1 \u0026micro;g/mL, 5 min, dark), followed by two PBS 1X washes. Fluorescence was visualized under a fluorescence microscope using FITC (Ex 488 nm / Em 525 nm) and DAPI (Ex 360 nm / Em 460 nm) filter sets.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eATP quantification assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIntracellular ATP levels were measured using a luminescence-based ATP assay kit (MedChemExpress, HY-K0314). Cells cultured in 12-well plates were lysed with 200 \u0026micro;L lysis buffer per well. Lysates were centrifuged at 12,000 \u0026times; g for 5 min at 4 \u0026deg;C. 50 \u0026micro;L of each lysate was mixed with 100 \u0026micro;L of ATP detection working solution in a black 96-well plate, incubated for 3\u0026ndash;5 min at room temperature, and luminescence was recorded on a microplate luminometer (endpoint mode; 1 s integration, AutoScale gain, normal read speed, 100 ms delay, 1 mm read height, extended dynamic range enabled). \u0026nbsp;ATP concentration was calculated from a standard curve (1\u0026ndash;5000 nM ATP) and normalized to total protein content measured by the BCA method.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eStatistical analysis was performed using GraphPad Prism 8 (GraphPad Software, San Diego, CA, USA). Data are presented as mean \u0026plusmn; standard error of the mean (SEM) of 3 to 4 independent experiments. For multiple group comparisons, one-way analysis of variance (ANOVA) was applied, followed by Tukey\u0026apos;s post hoc test for pairwise comparisons or Dunnett\u0026apos;s post hoc test for comparisons against the control or LPS. For comparisons between two groups, an unpaired Student\u0026apos;s t-test was used. Colocalization analysis was performed using Manders\u0026apos; colocalization coefficient. Differences were considered statistically significant at p \u0026lt; 0.05.\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cp\u003e\u003cstrong\u003eActivated resident liver macrophages (RLMs) induced the expression of BCAT1\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe started by assessing the presence of BCAA catabolic enzymes, BCAT1/2 and BCKDH, enzymes in liver cells (schematic representation of BCAAs metabolism, Figure 1A). \u0026nbsp;Gene expression in hepatocytes, stellate cells, liver sinusoidal endothelial cells (LSEC), and RLMs showed that RLMs express BCAT1 at a higher level than other liver cell types, while BCKDH expression was highest in hepatocytes (Supplementary Figure 3). \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eNext, primary rat RLMs were treated with 15 mM of isoleucine (Ile), leucine (Leu), valine (Val), or their combination (ratio 2:1:1, leucine, valine, isoleucine), with or without (100 ng/mL) (Figure 1B). At the mRNA level (Figure 1C), BCAT1 was significantly induced by LPS, and this increase was abolished by Leu, Val, or the BCAA mixture. BCKDH showed no significant changes after LPS. At the protein level (Figure 1D), BCAT1 and BCKDH remained stable across conditions. These findings suggest RLMs metabolize BCAAs mainly through BCAT1, which is upregulated by LPS but attenuated by BCAA exposure.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBCAAs modulate inflammatory gene expression after LPS treatment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe next investigated the impact of BCAA treatment on inflammatory gene expression in the absence and presence of LPS (Figure 2). LPS significantly increased the mRNA expression of CD11b, MCP1, IL-1\u0026beta;, IL-6, iNOS, TNF\u0026alpha;, HIF-1\u0026alpha;, IL-10, and Arg1. Individual BCAAs and the BCAA mixture reversed CD11b, MCP-1, HIF-1\u0026alpha;, and Arg-1 induction by LPS. \u0026nbsp;BCAAs did not modify other analysed genes.\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003eThese results indicate that BCAA exposure selectively downregulates RLMs\u0026rsquo; inflammatory response, particularly reversing LPS-induced CD11b, MCP1, HIF-1\u0026alpha;, and Arg-1, while other pro-inflammatory mediators remained unaffected.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBT2-mediated BCKDK inhibition reverses BCAT1 suppression\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo model impaired BCAA catabolism in MASLD [27], we used BCKDK inhibitor BT2 (IC50 = 3.19 \u0026micro;M), which enhances BCAA oxidation (Figure 3A). RLMs were pre-treated with BT2, then exposed to BCAAs alone or in combination with LPS (total = 6.5 hours). BCAT1 mRNA expression (Figure 3B) was increased by LPS; BT2 alone did not alter BCAT1, but BCAAs when combined with BT2 in LPS-stimulated RLMs reversed the inhibitory effect previously observed with LPS and BCAA co-treatment (Figure 1B). BCKDH increased only in LPS-stimulated-RLMs and decreased with BT2 (Figure 3B).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAt the protein level, BCAT1 and BCKDH remained unchanged across all conditions (Figure 3C-D), consistent with the absence of statistical significance shown previously (Figure 1D-E). These findings indicate that the metabolic flux through BCAA oxidation, rather than BCAA substrate availability per se, restores BCAT1 expression in activated-RLMs.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBT2-mediated BCKDK inhibition enables leucine to attenuate NF-\u0026kappa;B activation\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003ewhile preserving anti-inflammatory effects\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRegarding inflammatory markers, LPS alone maintained the induction of mRNA expression of CD11b, IL-1\u0026beta;, IL-6, HIF-1\u0026alpha;, iNOS, TNF-\u0026alpha;, and IL-10 (Figure 4A). BT2 alone did not modify any of these genes. When BT2 was combined with LPS, it partially attenuated LPS-induced upregulation of CD11b, Arg-1, and HIF-1\u0026alpha;. This inhibitory effect was maintained and comparable when individual BCAAs or BCAA mixture were added to the BT2+LPS combination on those same genes, recapitulating the anti-inflammatory pattern observed with BCAAs alone (Figure 2). These findings suggest that enhanced BCAA catabolism through BCKDK inhibition preserves the selective anti-inflammatory effects of BCAAs on CD11b, Arg-1, and HIF-1\u0026alpha;.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo elucidate the mechanism by which BCAAs alone or in combination with BT2 modulate inflammatory responses in LPS-stimulated RLMs, we investigated the NF-\u0026kappa;B pathway using immunofluorescence to assess nuclear translocation of activated NF-\u0026kappa;B (red fluorescence) from the cytoplasm to the nucleus (blue fluorescence) (Figure 4B). Quantification using Manders\u0026apos; colocalization coefficient (Figure 4C) confirmed NF-\u0026kappa;B nuclear translocation upon LPS treatment. Interestingly, leucine, valine, and the BCAA mixture alone increased NF-\u0026kappa;B nuclear translocation compared to control conditions. However, when leucine was combined with BT2, NF-\u0026kappa;B translocation was attenuated in both basal conditions and LPS-stimulated cells. This inhibitory effect was specific to the leucine plus BT2 combination, suggesting that enhanced leucine catabolism through BCKDK inhibition suppresses NF-\u0026kappa;B activation.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEnhanced BCAA catabolism abolishes the antioxidant effects of BCAAs in RLMs\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOxidative stress is a central mediator of LPS-induced inflammation and is closely linked to BCAA metabolism. Given that NF-\u0026kappa;B signaling can both induce and be driven by reactive oxygen species (ROS), we evaluated oxidative stress markers.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAntioxidant gene analysis in LPS-stimulated RLMs exposed to BCAAs, either alone or with BT2 showed that NRF2 was upregulated by LPS. Leucine and the BCAA mixture significantly attenuated the LPS-induced NRF2 upregulation (Figure 5A). However, when BT2 enhanced BCAA catabolism, Leu and the BCAA mixture failed to attenuate the LPS-induced NRF2 upregulation (Figure 5B). SOD2 and GPX1 expression were not significantly altered in any treatment condition. At the protein level, neither NRF2 nor MnSOD (SOD2 protein) showed significant changes (Figure 5C\u0026ndash;D).\u003c/p\u003e\n\u003cp\u003eROS generation using the DCFH‐DA probe (Figure 5E\u0026ndash;F) showed LPS increased intracellular ROS, which were significantly attenuated by isoleucine, valine, and the BCAA mixture. This protective antioxidant effect of BCAAs was abolished in the presence of BT2.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTogether, these results demonstrate that BCAAs exert protective antioxidant effects in LPS-stimulated macrophages by reducing ROS and suppressing compensatory NRF2 upregulation. However, enhanced BCAA catabolism through BCKDK inhibition abolishes these protective effects, suggesting BCAA substrate availability, rather than metabolic flux through oxidation, is required for antioxidant protection.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBCAAs suppress HK1-mediated glycolysis and enhance mitochondrial ATP production, indicating a mTORC1-independent metabolic shift\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo investigate the signaling mechanisms modulated by BCAAs, we examined the mTORC1 pathway, a classical target of amino acid signaling. Activation of mTORC1 downstream targets, ribosomal protein S6 kinase (S6K) and 4E-binding protein (4E-BP), was assessed in LPS-stimulated RLMs treated with BCAAs alone or in combination with BT2 (Figure 6A). Only leucine increased S6K mRNA expression in LPS-activated RLMs. No other treatment modified the expression of S6K or 4E-BP.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWe next evaluated the High-Mobility Group Box 1 (HMGB1) signaling pathway, as BCAAs modulate its redox-dependent translocation [16]. Total HMGB1 protein increased under BT2 treatment (Figure 6B). To assess HMGB1 release, we analysed supernatant fractions. LPS alone did not significantly alter HMGB1 release; however, the addition of individual BCAAs with LPS showed variable effects on total HMGB1 release. Notably, when BCAA catabolism was impaired by BT2 in the presence of LPS and BCAAs, HMGB1 release was markedly enhanced, suggesting BCAA accumulation under impaired catabolism exacerbates inflammatory HMGB1 secretion.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSince LPS-induced HIF1\u0026alpha; expression was reduced by BCAAs (Figure 2), we analysed its upstream regulator, prolyl hydroxylase domain-containing protein 3 (PHD3), which promotes HIF-\u0026alpha; degradation. BCAA did not modulate PHD3 expression with or without BT2(Figure 6C).\u003c/p\u003e\n\u003cp\u003eFinally, to elucidate the impact of BCAAs on cellular metabolism, we assessed energy production by quantifying ATP levels (Figure 6D) and glucose metabolism via the expression of hexokinase 1 (HK1), a key glycolytic enzyme and HIF1\u0026alpha; target gene (Figure 6E). BCAA mixture significantly enhanced ATP production both alone and following BT2 supplementation. This synergistic ATP increase is unlikely to be mTORC1-mediated, given the absence of changes in the downstream targets, supporting a model where BCAAs act as oxidative substrates fuelling mitochondrial energy. In parallel, HK1\u003cem\u003e\u0026nbsp;\u003c/em\u003eexpression was downregulated in all combinations of BCAA in LPS-activated cells, maintained with BT2 co-treatment, correlating with HIF1\u0026alpha; downregulation.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThis glycolytic enzyme suppression suggests BCAAs drive metabolic shift away from glycolysis, consistent with enhanced oxidative ATP production. HMGB1 upregulation suggests BCAAs influence inflammatory and oxidative signaling context-dependently.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHIF-1\u0026alpha; stabilization partially reverses BCAA-mediated anti-inflammatory effects but not antioxidant protection\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSince mTORC1 did not mediate the effects of BCAAs, we investigated the role of HIF1\u0026alpha; and HMGB1 using the specific inhibitors dimethyloxalyl glycine (DMOG) and glycyrrhizic acid (Gla), respectively. RLMs were pretreated with BT2 in combination with DMOG or Gla for 30 minutes, then BCAAs for 2 hours and LPS for 4 hours (total treatment = 6.5 hours).\u003c/p\u003e\n\u003cp\u003eInflammatory gene analysis showed LPS upregulated IL1b, IL6, CD11b, MCP1, and HIF-1\u0026alpha; expression as shown previously. Previously we showed that BT2 addition in LPS-activated RLMs did not effect on the BCAA anti-inflammatory effects (Figure 4A). Experiments adding Gla combined with the BCAAs mix and DMOG and Ile reversed the anti-inflammatory effects previously observed in BCAA plus LPS combinations. CD11b in these conditions, while DMOG combined with valine reversed HIF-1\u0026alpha; upregulation in BT2-LPS-treated RLMs (Figure 7A and Supplementary Figure 4A).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eGla and DMOG treatment did not affect NF-\u0026kappa;B activation via p65 nuclear translocation since results are similar to the previous observations using BT and BCAAs in LPS activated RLMS (Supplementary Figure 4B-C). Furthermore, total HMGB1 expression was strongly reduced by both DMOG and Gla treatments (Figure 7B).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eImportantly, HK-1 suppression previously observed in BCAA-treated, LPS-activated cells was reversed by DMOG, with significant effects for leucine and valine combinations (Figure 7C). Notably, ROS quantification showed that both DMOG and Gla restored the BCAA-induced reduction in ROS production (Figure 7D), indicating that both HIF-1\u0026alpha; and HMGB1 pathways contribute to BCAA-mediated antioxidant effects.\u003c/p\u003e\n\u003cp\u003eThese findings demonstrate that HIF-1\u0026alpha; stabilization and HMGB1 inhibition selectively reverse BCAA suppressive effects on inflammatory genes and metabolic markers (HK-1), indicating that BCAA-mediated anti-inflammatory effects are dependent on both HIF-1\u0026alpha; inhibition and HMGB1 modulation. However, the sustained antioxidant protection under conditions of HIF-1\u0026alpha; stabilization and HMGB1 inhibition reveals mechanistic independence between BCAA-mediated ROS suppression and these inflammatory signaling pathways.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eTable 3. Summary of major findings on BCAA-mediated effects in resident liver macrophages.\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 19.7432%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eCategory\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18.138%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eLPS\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13.6437%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eBCAA\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13.6437%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eBT2\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 16.6934%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eBCAA + LPS\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18.138%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eBT2 + BCAA + LPS\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 19.7432%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMetabolic enzymes\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18.138%;\"\u003e\n \u003cp\u003e\u0026uarr;BCAT1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13.6437%;\"\u003e\n \u003cp\u003e\u0026ndash;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13.6437%;\"\u003e\n \u003cp\u003eRestores BCAT1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 16.6934%;\"\u003e\n \u003cp\u003e\u0026darr;BCAT1 (vs LPS)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18.138%;\"\u003e\n \u003cp\u003e\u0026uarr;BCAT1 restored\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 19.7432%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eInflammatory genes (\u003c/strong\u003e\u003cstrong\u003eCD11b, MCP1, HIF1\u0026alpha;, Arg1)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18.138%;\"\u003e\n \u003cp\u003e\u0026uarr;\u0026uarr;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13.6437%;\"\u003e\n \u003cp\u003e\u0026ndash;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13.6437%;\"\u003e\n \u003cp\u003e\u0026ndash;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 16.6934%;\"\u003e\n \u003cp\u003e\u0026darr;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18.138%;\"\u003e\n \u003cp\u003e\u0026darr;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 19.7432%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eAntioxidant genes (NRF2)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18.138%;\"\u003e\n \u003cp\u003e\u0026uarr;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13.6437%;\"\u003e\n \u003cp\u003e\u0026darr; (vs LPS)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13.6437%;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 16.6934%;\"\u003e\n \u003cp\u003e\u0026darr;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18.138%;\"\u003e\n \u003cp\u003e\u0026uarr;\u0026nbsp;(BT2 reverses)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 19.7432%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eROS levels\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18.138%;\"\u003e\n \u003cp\u003e\u0026uarr;\u0026uarr;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13.6437%;\"\u003e\n \u003cp\u003e\u0026darr;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13.6437%;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 16.6934%;\"\u003e\n \u003cp\u003e\u0026darr;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18.138%;\"\u003e\n \u003cp\u003e\u0026uarr;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 19.7432%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eNF-\u003c/strong\u003e\u003cstrong\u003e\u0026kappa;\u003c/strong\u003e\u003cstrong\u003eB signaling (p65 nuclear translocation)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18.138%;\"\u003e\n \u003cp\u003e\u0026uarr;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13.6437%;\"\u003e\n \u003cp\u003e\u0026uarr; (basal)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13.6437%;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 16.6934%;\"\u003e\n \u003cp\u003e\u0026uarr;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18.138%;\"\u003e\n \u003cp\u003e\u0026darr; (only Leu+BT2)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 19.7432%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eHMGB1 signaling\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18.138%;\"\u003e\n \u003cp\u003e\u0026ndash;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13.6437%;\"\u003e\n \u003cp\u003e\u0026ndash;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13.6437%;\"\u003e\n \u003cp\u003e\u0026ndash;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 16.6934%;\"\u003e\n \u003cp\u003eIle \u0026rarr; disulfide HMGB1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18.138%;\"\u003e\n \u003cp\u003e\u0026uarr;oxidized HMGB1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 19.7432%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eHIF1\u0026alpha; / HK1 / PHD3\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18.138%;\"\u003e\n \u003cp\u003e\u0026uarr;HIF1\u0026alpha;, \u0026uarr;HK1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13.6437%;\"\u003e\n \u003cp\u003e\u0026darr;HIF1\u0026alpha;, \u0026darr;HK1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13.6437%;\"\u003e\n \u003cp\u003e\u0026uarr;PHD3 (ns)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 16.6934%;\"\u003e\n \u003cp\u003e\u0026darr;\u0026darr;HIF1\u0026alpha;, \u0026darr;HK1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18.138%;\"\u003e\n \u003cp\u003e\u0026uarr;PHD3, \u0026darr;HIF1\u0026alpha;,\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 19.7432%;\"\u003e\n \u003cp\u003e\u003cstrong\u003emTORC1 pathway\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18.138%;\"\u003e\n \u003cp\u003e\u0026ndash;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13.6437%;\"\u003e\n \u003cp\u003e\u0026ndash;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13.6437%;\"\u003e\n \u003cp\u003e\u0026darr;S6K\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 16.6934%;\"\u003e\n \u003cp\u003e\u0026ndash;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18.138%;\"\u003e\n \u003cp\u003e\u0026darr;S6K\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 19.7432%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eATP levels\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18.138%;\"\u003e\n \u003cp\u003e\u0026ndash;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13.6437%;\"\u003e\n \u003cp\u003e\u0026ndash;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13.6437%;\"\u003e\n \u003cp\u003e\u0026ndash;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 16.6934%;\"\u003e\n \u003cp\u003e\u0026uarr;ATP (BCAA mix)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18.138%;\"\u003e\n \u003cp\u003e\u0026uarr;\u0026uarr;ATP (BCAA mix)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 19.7432%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eEffect of DMOG / Gla\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18.138%;\"\u003e\n \u003cp\u003e\u0026ndash;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13.6437%;\"\u003e\n \u003cp\u003e\u0026ndash;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13.6437%;\"\u003e\n \u003cp\u003e\u0026ndash;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 16.6934%;\"\u003e\n \u003cp\u003e\u0026ndash;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18.138%;\"\u003e\n \u003cp\u003eReversed the proinflammatory effects (Ile (CD11b) and Val (HIF1\u0026alpha;))\u003c/p\u003e\n \u003cp\u003eROS suppression\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eBCAAs modulated inflammatory and oxidative responses by regulating BCAT1 expression, NF-\u0026kappa;B activity, and metabolic reprogramming. BT2 and leucine altered these effects through changes in redox balance and HIF1\u0026alpha;/HMGB1 signaling.. BCAAs, branched-chain amino acids; Ile, isoleucine; Leu, leucine; Val, valine; BT2, 3,6-dichlorobenzo-thiophene-2-carboxylic acid; LPS, lipopolysaccharide; HMGB1, high-mobility group box 1; HIF1\u0026alpha;, hypoxia-inducible factor 1 alpha; PHD3, prolyl hydroxylase domain-containing protein 3; NF\u0026kappa;B, nuclear factor kappa-light-chain-enhancer of activated B cells; ROS, reactive oxygen species; mTORC1, mechanistic target of rapamycin complex 1; ATP, adenosine triphosphate; Gla, glycyrrhizic acid; DMOG, dimethyloxalyl glycine.\u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eThis study provides novel insights into the immunometabolic functions of branched-chain amino acids (BCAAs) in resident liver macrophages (RLMs), revealing a complex interplay between amino acid metabolism, inflammatory signaling, and redox homeostasis. Our findings demonstrate that RLMs express the enzymatic machinery for BCAA metabolism, mainly via BCAT1, which is upregulated by LPS and suppressed by BCAA exposure, indicating feedback regulation of catabolism under inflammatory conditions. Functionally, BCAAs selectively attenuated LPS-induced expression of CD11b, MCP1, HIF-1α, and Arg1, while decreasing ROS accumulation and decreasing NRF2 activation, suggesting direct antioxidant effects that do not rely on compensatory stress responses.\u003c/p\u003e\u003cp\u003eInhibition of BCKDK with BT2 restored BCAT1 expression and preserved the anti-inflammatory effects of BCAAs but abolished their antioxidant protection, revealing that these two beneficial functions are mechanistically separable and governed by BCAA substrate availability versus metabolic flux, respectively. Mechanistically, only the leucine\u0026ndash;BT2 combination suppressed NF-κB p65 nuclear translocation, implying that leucine catabolism produces specific intermediates or metabolic conditions that uniquely restrain this pathway. BCAA also promoted a metabolic shift from glycolysis toward oxidative phosphorylation, evidenced by increased ATP production and decreased HK1 expression, an effect independent of mTORC1 signaling.\u003c/p\u003e\u003cp\u003eGiven that mTORC1 did not mediate BCAA effects, we investigated the involvement of HIF-1α and HMGB1 using the specific modulators DMOG and glycyrrhizic acid (Gla). Modulation of the HIF1α and HMGB1 axes revealed that BCAA anti-inflammatory effects depend partly on HIF1α inhibition and HMGB1 modulation, whereas antioxidant actions require coordinated regulation through both pathways. DMOG also reversed the suppression of HK1 by BCAAs, especially with leucine and valine, confirming that BCAA-dependent repression of glycolysis is driven by HIF-1α downregulation.\u003c/p\u003e\u003cp\u003eThese findings extend prior evidence that LPS alters systemic BCAA metabolism by lowering circulating BCAA concentrations and leucine oxidation [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. We demonstrate that macrophages themselves respond through BCAT1-dependent feedback, potentially preventing over-catabolism during inflammation. Consistent with earlier reports, BCAT1 enrichment in activated tumor-associated macrophages promotes immunosuppressive polarization [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e], whereas its inhibition enhances anti-inflammatory phenotypes in models of arthritis [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Our data refine this concept by showing that while BT2 restores BCAT1 and maintains anti-inflammatory signaling, it disrupts redox homeostasis, suggesting that the balance between BCAA availability and metabolic capacity determines the immune outcome. This aligns with studies showing that impaired BCAA catabolism leads to BCKA accumulation, perturbs central carbon metabolism, and drives oxidative stress [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe selective modulation of inflammatory mediators by BCAAs which suppresses CD11b, MCP1, HIF-1α, and Arg1 while leaving IL-1β, IL-6, iNOS, and TNFα unaffected suggests targeted pathway regulation rather than global immunosuppression. Clinical data support this, showing that BCAAs reduce LPS-binding protein and TLR4 activation [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Importantly, our data demonstrate that the anti-inflammatory effects of BCAAs persist even when BCAA catabolism is enhanced by BT2, indicating that metabolic acceleration alone does not abolish their protective signaling. However, disruption of HIF-1α or HMGB1 signaling, through DMOG\u0026thinsp;+\u0026thinsp;Ile or Gla\u0026thinsp;+\u0026thinsp;BCAA mix, respectively, reversed these protective effects, restoring MCP1 and HIF-1α expression. These findings suggest that intact HIF-1α and HMGB1 pathways are essential mediators of BCAA-induced immunomodulation.\u003c/p\u003e\u003cp\u003eIn contrast to our results, BCAA supplementation enhanced M1 activation through mTORC1-HIF-1α-glycolysis signaling in exercise-induced muscle injury [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], underscoring tissue-specific responses. When BCAA catabolism is impaired, as in atherosclerotic macrophages with reduced BCAT2 and BCKDHA expression, excess BCAAs increase mitochondrial H₂O₂ and activate TLR4/NF-κB signaling [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Similarly, disrupted BCAA metabolism in obesity promotes pro-inflammatory polarization via IFNGR1/JAK1/STAT1 pathways [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. These studies, together with our findings, reinforce the concept that context and metabolic balance determine whether BCAAs are protective or pathogenic.\u003c/p\u003e\u003cp\u003eA remarkable observation is the interplay between BCAA metabolism, HIF-1α/HMGB1 signaling, and oxidative stress. BCAAs effectively attenuated LPS-induced ROS accumulation while reducing NRF2 gene expression, consistent with direct ROS-scavenging or prevention of mitochondrial overload. This is consistent with observations on itaconate metabolism, as BCAT1 inhibition reduces IRG1 expression and itaconate levels required for Nrf2 activation by alkylating cysteine residues [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eBT2 treatment reversed this effect, increasing ROS, while both DMOG and Gla restored ROS reduction. This suggests that BCAA-mediated antioxidant protection relies on HIF-1α and HMGB1 pathways acting in concert. DMOG experiments further showed that HIF-1α stabilization reversed HK1 suppression, especially for leucine and valine, linking glycolytic control to HIF-1α regulation. This agrees with reports that excessive BCAA concentrations downregulate HIF-1α and GLUT1, reducing glycolytic flux [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e] and that HIF-1α deletion enhances amino acid catabolism [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Because HK1-dependent glycolysis promotes inflammasome activation [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e], its downregulation likely contributes to the anti-inflammatory phenotype observed here.\u003c/p\u003e\u003cp\u003eHMGB1 functions as a redox-sensitive DAMP molecule [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Its redox status determines biological activity, with the disulfide form promoting inflammation and the fully reduced form acting as a chemoattractant, while terminal oxidation (sulfonation) abolishes these immune functions [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Zhao S. et al. (2023) [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e] demonstrated that excessive BCAAs in atherosclerotic macrophages increase mitochondrial H₂O₂ production, promoting disulfide HMGB1 release and activating the TLR4/NF-κB pathway. In our study, BCAA exposure suppressed HMGB1-associated inflammation and reduced ROS, but addition of Gla with the BCAA mix reversed these effects, restoring MCP1 and HIF-1 expression. This indicates that BCAA-mediated protection requires functional HMGB1 signaling, and that excessive inhibition or interference of this pathway disrupts redox and inflammatory control. Moreover, increased extracellular HMGB1 detected under BT2\u0026thinsp;+\u0026thinsp;LPS conditions suggests that when catabolism is dysregulated, BCAAs can no longer restrain HMGB1-driven inflammation. Although we could not determine specific HMGB1 redox isoforms, future work employing non-reducing electrophoresis and redox-specific antibodies should clarify whether altered BCAA metabolism induces distinct oxidative HMGB1 modifications.\u003c/p\u003e\u003cp\u003eAt the metabolic level, BCAAs increased ATP production and suppressed HK1, consistent with enhanced oxidative phosphorylation and reduced glycolytic flux, features characteristic of anti-inflammatory macrophages dependent on the TCA cycle. Interestingly, while oxidative metabolism and reduced HIF-1α expression reflected M2-like features, the simultaneous downregulation of Arg1 indicates that this reprogramming is independent of the canonical arginine\u0026ndash;Arg1 axis, possibly mediated by BCAA-derived glutamine or TCA intermediates. The accumulation of BCKAs during accelerated catabolism likely underlies the loss of antioxidant capacity, as BCKAs inhibit BCKDH activity in a feedback loop [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e] and interfere with PHD2, stabilizing HIF-1α under aerobic conditions [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eWe propose that α-ketoglutarate (αKG) availability represents a central node linking BCAA flux and HIF-1α regulation. When BCAT1 is overexpressed, it can consume too much αKG, reducing its availability for these enzymes and thereby stabilizing HIF-1α [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Moderate BCAA metabolism generates αKG, supporting prolyl hydroxylase activity and HIF-1α degradation. In contrast, excessive BCAT1 activity or BT2-driven catabolism depletes αKG and accumulates BCKAs, impairing hydroxylase function and stabilizing HIF-1α. This dual effect reconciles the paradox whereby both BCAA supplementation and excessive catabolism produce opposing HIF-1α outcomes.\u003c/p\u003e\u003cp\u003eThe clinical relevance of our study is supported by the therapeutic benefits of BCAAs in liver diseases, with protective effects mediated via LPS-binding protein and TLR4 suppression [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. However, in metabolic liver diseases such as MASLD, where BCAA catabolism is impaired, BCAA accumulation and defective oxidation could drive inflammation via ROS- and HMGB1-dependent mechanisms. Therapeutic strategies should therefore focus on restoring balanced BCAA metabolism, rather than simple supplementation or restriction, to avoid the harmful buildup of catabolic intermediates.\u003c/p\u003e\u003cp\u003eFinally, our study has limitations. The experiments were performed in vitro, under short-term exposure (6.5 h) and with single LPS doses, which cannot fully replicate the hepatic microenvironment. Future studies should address time-dependent effects, integration with glucose and lipid metabolism, BCAA transport regulation, and direct quantification of BCKAs to delineate mechanisms controlling the balance between anti-inflammatory and antioxidant responses.\u003c/p\u003e"},{"header":"CONCLUSIONS","content":"\u003cp\u003eThis study establishes BCAA metabolism as a central checkpoint in liver macrophage activation, integrating inflammatory and redox pathways through distinct but interconnected mechanisms. BCAAs suppress HIF-1α-driven glycolysis and selected inflammatory mediators while promoting oxidative metabolism. These anti-inflammatory effects remain intact under enhanced BCAA catabolism (BT2 treatment) but are abolished when HIF-1α or HMGB1 signaling is perturbed, demonstrating that BCAA-mediated protection depends on the integrity of these pathways. BCAAs thus emerge as dual regulators integrating metabolism and immune signaling; when metabolized appropriately, they maintain oxidative balance and limit inflammation; when catabolic or signaling control is lost, the system shifts toward oxidative and inflammatory stress. These context-dependent effects emphasize that substrate availability and signaling integrity, rather than flux alone, dictate macrophage responses.\u003c/p\u003e\u003cp\u003eClinically, this work links systemic inflammation to BCAA dysregulation in metabolic liver disease. Therapeutic strategies should aim to optimize BCAA metabolism, maintaining adequate substrate to support coordinated HIF-1α/HMGB1-mediated defense while avoiding BCKA accumulation or excessive pathway interference. Assessing BCAT1/BCKDH activity or BCAA/BCKA ratios may help identify metabolic states most likely to benefit from BCAA-targeted therapy. Balancing catabolic efficiency with pathway stability may restore integrated control of inflammation and oxidative stress, providing a rational framework for BCAA-based interventions in MASLD and related disorders.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eArg-1, Arginase-1; ATP, Adenosine triphosphate; BCAA, Branched-chain amino acid; BCAT1/2, Branched-chain amino acid transaminase 1/2; BCKDH, Branched-chain \u0026alpha;-ketoacid dehydrogenase; BCKDK, Branched-chain \u0026alpha;-ketoacid dehydrogenase kinase; DAMP, Damage-associated molecular pattern; DMOG, Dimethyloxalylglycine; Gla, Glycyrrhizic acid; HBSS, Hank\u0026rsquo;s balanced salt solution; HIF-1\u0026alpha;, Hypoxia-inducible factor 1\u0026alpha;; HMGB1, High-mobility group box 1; HK1, Hexokinase 1; IL, Interleukin; iNOS, Inducible nitric oxide synthase; KCs, Kupffer cells; LPS, Lipopolysaccharide; MASLD, Metabolic dysfunction\u0026ndash;associated steatotic liver disease; MASH, Metabolic dysfunction\u0026ndash;associated steatohepatitis; MAPK, Mitogen-activated protein kinase; MCP1, Monocyte chemoattractant protein-1; mTORC1, Mechanistic target of rapamycin complex 1; NF-\u0026kappa;B, Nuclear factor kappa B; NRF2, Nuclear factor erythroid 2\u0026ndash;related factor 2; PAMP, Pathogen-associated molecular pattern; PARP1, Poly(ADP-ribose) polymerase 1; PHD3, Prolyl hydroxylase domain protein 3; qPCR, Quantitative polymerase chain reaction; RLMs, Resident liver macrophages; ROS, Reactive oxygen species; S6K, Ribosomal protein S6 kinase; SOD2, Superoxide dismutase 2; TLR4, Toll-like receptor 4; TNF-\u0026alpha;, Tumor necrosis factor alpha.\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contributions:\u003c/strong\u003e H.M and H.B provided supervision and project concept. M. M.A designed the study, performed experiments, and analysed results. M. B.H. provided technical support. \u0026nbsp;M. M.A. drafted the manuscript. All authors critically revised the manuscript and approved the final version.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAddress for correspondence:\u003c/strong\u003e Magnolia Martinez, [email protected], or Han Moshage, [email protected]; Department of Gastroenterology and Hepatology, University Medical Center Groningen, University of Groningen, P.O. Box 30.001, 9700 RB Groningen, The Netherlands.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest:\u003c/strong\u003e The authors have no conflicts of interest to report.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFinancial support and sponsorship:\u003c/strong\u003e The author(s) gratefully acknowledge the financial support provided by CONACYT (now SECIHTI) through the PhD grant (no. 795389).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAddress for correspondence:\u003c/strong\u003e Magnolia Martinez-Aguilar, [email protected]; Department of Gastroenterology and Hepatology, University Medical Center Groningen, University of Groningen, P.O. Box 30.001, 9700RB Groningen, The Netherlands.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data supporting the findings of this study are available within the paper and its Supplementary Information.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eLe MH, Yeo YH, Zou B, Barnet S, Henry L, Cheung R, et al. Forecasted 2040 global prevalence of nonalcoholic fatty liver disease using hierarchical bayesian approach. Clin. Mol Hepatol. 2022;28:841\u0026ndash;850.\u003c/li\u003e\n\u003cli\u003eBuzzetti, E., Pinzani, M., \u0026amp; Tsochatzis, E. A. (2016). The multiple-hit pathogenesis of non-alcoholic fatty liver disease (NAFLD). Metabolism, 65(8), 1038\u0026ndash;1048. doi: 10.1016/j.metabol.2015.12.012\u003c/li\u003e\n\u003cli\u003eJuanola O, Mart\u0026iacute;nez-L\u0026oacute;pez S, Franc\u0026eacute;s R, G\u0026oacute;mez-Hurtado I. 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Redox-sensitive high-mobility group box-1 isoforms contribute to liver fibrosis progression and resolution in mice. \u003cem\u003eJournal of hepatology\u003c/em\u003e, \u003cem\u003e80\u003c/em\u003e(3), 482\u0026ndash;494. https://doi.org/10.1016/j.jhep.2023.11.005\u003c/li\u003e\n\u003cli\u003eHonda T, Fukuda Y, Nakano I, et al. Effects of liver failure on branched-chain alpha-keto acid dehydrogenase complex in rat liver and muscle: comparison between acute and chronic liver failure. \u003cem\u003eJ Hepatol\u003c/em\u003e. 2004;40(3):439-445. doi:10.1016/j.jhep.2003.11.003\u003c/li\u003e\n\u003cli\u003eXiao W, Shrimali N, Vigder N, et al. Branched-chain \u0026alpha;-ketoacids aerobically activate HIF1\u0026alpha; signalling in vascular cells. Nat Metab. 2024;6(11):2138-2156. doi:10.1038/s42255-024-01150-4\u003c/li\u003e\n\u003cli\u003eRaffel S, Falcone M, Kneisel N, et al. BCAT1 restricts \u0026alpha;KG levels in AML stem cells leading to IDHmut-like DNA hypermethylation. \u003cem\u003eNature\u003c/em\u003e. 2017;551(7680):384-388. doi:10.1038/nature24294\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-8077254/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8077254/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e\u003cp\u003eMetabolic dysfunction\u0026ndash;associated steatotic liver disease (MASLD) is marked by inflammation mediated by resident liver macrophages (RLMs). Branched-chain amino acids (BCAAs; leucine, isoleucine, valine) are elevated in early MASLD, yet their role in RLM biology is unclear.\u003c/p\u003e\u003ch2\u003eAim\u003c/h2\u003e\u003cp\u003eWe investigated whether BCAA exposure and impaired catabolism affect LPS-induced RLMs activation.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e\u003cp\u003ePrimary rat RLMs were treated with high BCAA concentrations (15 mM) and/or LPS stimulation (100 ng/ml). BCAA metabolic enzymes, inflammatory markers, oxidative stress, and metabolic reprogramming were assessed. BCKDK inhibitor BT2 was used to enhance BCAA catabolism:\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003eRLMs expressed BCAT1, upregulated by LPS but downregulated by BCAAs. BCAAs exerted protective effects by selectively reversing LPS-induced CD11b, MCP-1, HIF-1α, and Arg1 expression, reducing ROS and attenuating NRF2. BCAAs promoted metabolic shift toward oxidative phosphorylation with increased ATP and reduced HK1 expression. BT2 enhanced catabolism restored BCAT1 and maintained anti-inflammatory effects but abolished antioxidant protection. Only leucine-BT2 suppressed NF-κB translocation. HIF-1α stabilization and HMGB1-mediated protection, restoring inflammatory gene expression and ROS levels.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e\u003cp\u003eBCAAs modulate RLM activation by regulating HIF-1α and HMGB1 signaling and redox homeostasis. Their protective effects depend on intact HIF-1α/HMGB1 pathways rather than catabolic flux, revealing a context-dependent role of BCAAs in hepatic inflammation.\u003c/p\u003e","manuscriptTitle":"Enhanced catabolism of branched-chain amino acids uncouples anti-inflammatory and antioxidant functions in liver macrophages","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-11 13:31:13","doi":"10.21203/rs.3.rs-8077254/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"7564b624-7da2-4cdc-96bf-3311d0ea9450","owner":[],"postedDate":"December 11th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-02-17T14:56:19+00:00","versionOfRecord":[],"versionCreatedAt":"2025-12-11 13:31:13","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8077254","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8077254","identity":"rs-8077254","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}