Beatriz Pereira da Silva

Background.: Activation of de novo lipogenesis (DNL) is a major hallmark of steatotic liver disease (SLD), a highly prevalent group of diseases ranging from a harmless steatosis to more severe steatohepatitis, cirrhosis, and hepatocellular carcinoma. We interrogated herein the early alterations in liver proteome, transcriptome, lipidome, inflammation, fibrosis, and oxidative status in a mouse model of progressive SLD induced by constitutive activation of PI3K-AKT signaling, the major inducer of DNL. Methods.: Male, 8 weeks-old mice bearing phosphatase and tensin homolog ( Pten ) deletion and therefore constitutive activation of PI3K-AKT in hepatocytes (albumin-Cre) and littermate controls were evaluated for liver steatosis, injury, lipid peroxidation, metabolism, proteome, lipidome and transcriptome either upon feeding with a chow or high-fat diets supplemented or not with linoleic acid or after treatment with vehicle or acetyl-CoA carboxylase (ACC) inhibitor (ND-630, 20 mg/kg/day, i.p.) Results.: Mice bearing Pten deletion and therefore constitutive activation of PI3K-AKT in hepatocytes display steatohepatitis characterized by enhanced DNL, severe steatosis, hepatocyte ballooning, inflammation, fibrosis and hepatomegaly, which, unexpectedly, was associated with reduced lipid peroxidation, increased GSH content and enhanced rates of triacylglycerol turnover, fatty acid β-oxidation, tricarboxylic acid cycle flux, and mitochondrial respiration, altogether promoting a robust lipidome remodeling mainly defined by enrichment of DNL-derived fatty acids in detriment of a broad depletion of essential fatty acids (linoleic and α-linolenic acids) and cardiolipins. Dietary restoration of liver linoleic acid content mildly attenuated liver inflammation, without however affecting lipid peroxidation and improving steatosis and fibrosis, whereas pharmacological acetyl-CoA carboxylase (ACC) and DNL inhibition markedly reduced steatosis, inflammation and fibrosis, despite further lowering hepatic linoleic acid content. Conclusions.: Collectively, these findings support a harmful role of essential fatty acids depletion fomenting liver inflammation and identify DNL as a major culprit of steatohepatitis induced by constitutive PI3K-AKT activation. What is already known on this topic: Pten inactivation in hepatocytes leading to constitutive PI3K-AKT signaling and de novo lipogenesis activation (DNL) is a recurrent liver signature found in steatotic liver disease (SLD)-bearing patients, promoting disease progression through not completely defined molecular mechanisms. What this study adds: Pten inactivation in hepatocytes promotes liver steatosis, injury, inflammation and fibrosis, in spite of depleting essential fatty acids and cardiolipin, reducing lipid peroxidation, enhancing antioxidant capacity and mitochondrial mass and respiration, and eliciting lipid-related futile metabolic cycles. Dietary restoration of liver essential linoleic acid partially attenuates inflammation, but not steatosis and fibrosis induced by Pten inactivation in hepatocytes Pharmacological ACC and DNL inhibition mitigate the steatosis, inflammation and fibrosis induced by Pten inactivation in hepatocytes, but further deplete liver essential fatty acids. How this study might affect research, practice or policy: This study raises evidence supporting a harmful role of essential fatty acid depletion exacerbating liver inflammation. Pharmacological strategies that increase fatty acid oxidation including ACC inhibition may enhance, while dietary supplementation may mitigate essential fatty acid depletion. Despite protecting healthy cells from oxidative damage, the enhanced antioxidant capacity induced by Pten inactivation in hepatocytes may favor survival of hepatocytes bearing DNA mutations and tumorigenesis.

Peroxidasin (PXDN) catalyzes collagen IV sulfilimine crosslinking through the generation of hypobromous acid (HOBr), a reaction that requires hydrogen peroxide (H2O2). However, the physiological source of H₂O₂ that sustains PXDN activity in the extracellular matrix (ECM) remains unclear. Here, we show that ECM derived from HEK293 cells overexpressing PXDN exhibits markedly elevated peroxidase activity even in the absence of exogenous H₂O₂. This intrinsic activity is completely abolished by catalase or by the PXDN inhibitor phloroglucinol (PHG), indicating in situ peroxide generation. Importantly, H2O2 formation in the ECM was dependent on a fully active PXDN, supporting the existence of endogenous H2O2 production in this system. Proteomic profiling of ECM-enriched fractions does not reveal enrichment of known H2O2-source enzymes, arguing against canonical extracellular oxidases as the main peroxide source. Using ECM derived from PFHR9 cells, we further demonstrate collagen IV crosslinking activity in the absence of exogenous H2O2, which is inhibited by catalase or PHG. Finally, we identified the formation of L-DOPA and L-DOPA-quinone in basement membrane proteins, including collagen IV within the crosslinking region. These oxidative modifications were enhanced by H2O2 and bromide treatment and were inhibited by a PXDN inhibitor. Together, these results support a model in which PXDN-driven oxidants production, e.g. HOBr, and/or direct protein residues oxidation generates redox-active DOPA residues that promote local radical cycling and generation of superoxide anion radical (O2•⁻) and H2O2, establishing a confined ECM redox microenvironment capable of sustaining collagen IV crosslinking under low-peroxide conditions.

Background and Aims: Alcohol‐related liver disease (ALD) is associated with elevated blood immunoglobulin‐type M (IgM) levels and hepatic lipid peroxidation. Nevertheless, the functional relevance of systemic IgM targeting lipid peroxidation products during ALD is incompletely understood. Methods: Levels of IgM and IgG recognising malondialdehyde–acetaldehyde (MAA), as a hallmark epitope of lipid peroxidation, as well as complement factors were assessed in the serum of patients with ALD. The influence of alcohol abstinence on anti‐MAA IgM and IgG levels was determined in AUD patients. A chronic‐binge ethanol diet was given to mice deficient in sialic acid‐binding immunoglobulin‐like lectin G ( Siglec‐G ^−/− ), with specifically increased systemic IgM, and mice lacking soluble IgM ( sIgM ^−/− ), and their corresponding littermates. Furthermore, wildtype mice were injected with MAA‐binding IgM antibodies (LR04) or isotype control IgM during chronic‐binge ethanol feeding. Siglec‐G ^−/− bone marrow transplantation into wildtype or complement C3 deficient mice ( C3 ^−/− ) was performed to investigate the involvement of complement activation by elevated IgM in ALD. Results: Serum levels of anti‐MAA IgM positively correlated with ALD severity in humans. While mice lacking soluble IgM had less pronounced liver injury, increased circulating anti‐MAA IgM titers in Siglec‐G ^−/− mice associated with more hepatocellular damage after ethanol feeding than wildtypes. Similarly, mice receiving LR04 displayed elevated liver injury compared to control‐injected mice. Moreover, besides less hepatic neutrophil and macrophage content, and stellate cell activation than wildtypes, ethanol‐fed Siglec‐G ^−/− and LR04‐treated mice had increased hepatic C3b deposition. Mice deficient in C3 displayed ameliorated ethanol‐induced liver injury compared with controls, despite similarly high anti‐MAA IgM levels after Siglec‐G ^−/− bone marrow transplantation, suggesting complement‐dependent liver injury upon high anti‐MAA IgM. In line, levels of MAA‐binding IgM inversely correlated with C3c and C4 in serum of patients with ALD. Conclusion: Elevated systemic IgM titres that recognise MDA facilitate complement recruitment, which enhances hepatocyte injury, thereby promoting alcohol‐associated liver disease.