Mouse Liver Research Articles

Effect of primary copper metabolism disturbance on elemental, protein, and lipid composition of the organs in Jackson toxic milk mouse.

Toxic milk (txJ) is an autosomal recessive mutation in the Atp7b gene in the C3H/HeJ strain, observed at The Jackson Laboratory in Maine, USA. TxJ mice exhibit symptoms similar to those of human Wilson's disease (WD). The study aimed to verify organ involvement in a mouse model of WD. TxJ mice and control animals were sacrificed at 2, 4, 8, and 14months of age. Total X-ray Fluorescence Spectroscopy (TXRF) was used to determine the elemental concentration in organs. Tissue chemical composition was measured by Fourier Transform Infrared Spectroscopy (FTIR). Additionally, hybrid mapping of FTIR and microXRF was performed. Elevated concentrations of Cu were observed in the liver, striatum, eye, heart, and duodenum of txJ mice across age groups. In the striatum of the oldest txJ mice, there was lower lipid content and a higher fraction of saturated fats. The secondary structure of striatum proteins was disturbed in txJ mice. In the livers of txJ mice, higher concentrations of saturated fats and disturbances in the secondary structure of proteins were observed. The concentration of neurofilaments was significantly higher in txJ serum. The distribution of Cu deposits in brains was uniform with no prevalence in any anatomic structure in either group, but significant protein structure changes were observed exclusively in the striatum of txJ. In this txJ animal model of WD, pathologic copper accumulation occurs in the duodenum, heart, and eye tissues. Increased copper concentration in the liver and brain results in increased saturated fat content and disturbances in secondary protein structure, leading to hepatic injury and neurodegeneration.

S100A9 regulates M1 macrophage polarization and exacerbates steatotic liver ischemia-reperfusion injury

ObjectiveSteatotic livers exhibit higher susceptibility to ischemia reperfusion (IR) injury, which increase the risk of primary graft non-function following liver transplantation. S100A9 is identified as a pivotal innate immune sensor that regulates the progression of liver diseases. However, its significance in steatotic liver IR injury remains under-investigated. MethodsIn mice model, we generated S100A9 knockout (S100A9 KO) mice to investigate the role of S100A9 in IR-stimulated steatotic livers. In vitro, primary bone marrow-derived macrophages were utilized to explore the effect of S100A9 in regulating macrophage polarization and inflammation. ResultsS100A9 expression was markedly increased in steatotic livers of mice subjected to IR insult. S100A9 deletion significantly attenuated liver inflammatory injury, as evidenced by the diminished infiltration of both monocytes/macrophages and neutrophils (p < 0.05). The expression of proinflammatory factors was reduced (p < 0.05) at the same time. Additionally, S100A9-deficient livers demonstrated M1 polarization decrease and Toll-like receptor 4 (TLR4) suppression (p < 0.05). In vitro, genetic TLR4 inhibition led to nuclear factor kappa B (NF-κB) inactivation and subsequent M1 polarization decrease (p < 0.05) in macrophages treated with recombinant S100A9. ConclusionIn this study, we highlight the pivotal role of TLR4/NF-κB as a critical mediator of S100A9 in inducing M1 macrophage polorization- dependent inflammation in steatotic livers IR injury.

Schisandrin A alleviates non-alcoholic fatty liver disease by improving liver metabolic disorders and suppressing the GSK3β signaling pathway

Non-alcoholic fatty liver disease (NAFLD) is a clinical pathological syndrome characterized by excessive fat deposition in liver cells, posing a new challenge in the field of contemporary medicine. Schisandrin A (SchA) is a lignan derived from the fruit of Schisandra chinensis, a functional food and also a traditional herbal medicine known for its hepatic protective effects. The study aims to identify potential target and regulation mechanism of SchA on NAFLD treatment. Metabolomics analysis revealed that SchA improved the abnormal metabolic profile of the liver in NAFLD mice. In addition to ameliorating lipid metabolism, SchA showed potential in elevating the levels of amino acids such as L-tryptophan, beta-alanine, L-aspartic acid, and L-tyrosine, which were deficient in NAFLD mouse livers, as well as restored the metabolism of abnormal vitamins like vitamin B6. Network pharmacology research and molecular docking suggested that GSK3β was a direct target of SchA, which was experimentally validated as well. These findings highlight the potential value of the active component SchA from Schisandra chinensis in the prevention and treatment of NAFLD.

Oral exposure to benzalkonium chlorides in male and female mice reveals alteration of the gut microbiome and bile acid profile.

Benzalkonium chlorides (BACs) are commonly used disinfectants in a variety of consumer and food-processing settings, and the COVID-19 pandemic has led to increased usage of BACs. The prevalence of BACs raises the concern that BAC exposure could disrupt the gastrointestinal microbiota, thus interfering with the beneficial functions of the microbes. We hypothesize that BAC exposure can alter the gut microbiome diversity and composition, which will disrupt bile acid homeostasis along the gut-liver axis. In this study, male and female mice were exposed orally to d7-C12- and d7-C16-BACs at 120 µg/g/day for one week. UPLC-MS/MS analysis of liver, blood, and fecal samples of BAC-treated mice demonstrated the absorption and metabolism of BACs. Both parent BACs and their metabolites were detected in all exposed samples. Additionally, 16S rRNA sequencing was carried out on the bacterial DNA isolated from the cecum intestinal content. For female mice, and to a lesser extent in males, we found that treatment with either d7-C12- or d7-C16-BAC led to decreased alpha diversity and differential composition of gut bacteria with notably decreased actinobacteria phylum. Lastly, through a targeted bile acid quantitation analysis, we observed decreases in secondary bile acids in BAC-treated mice, which was more pronounced in the female mice. This finding is supported by decreases in bacteria known to metabolize primary bile acids into secondary bile acids, such as the families of Ruminococcaceae and Lachnospiraceae. Together, these data signify the potential impact of BACs on human health through disturbance of the gut microbiome and gut-liver interactions.

Hepatic retinol dehydrogenase 11 dampens stress associated with the maintenance of cellular cholesterol levels

ObjectiveDysregulation of hepatic cholesterol metabolism can contribute to elevated circulating cholesterol levels, which is a significant risk factor for cardiovascular disease. Cholesterol homeostasis in mammalian cells is tightly regulated by an integrated network of transcriptional and post-transcriptional signalling pathways. Whilst prior studies have identified many of the central regulators of these pathways, the extended supporting networks remain to be fully elucidated. MethodsHere, we leveraged an integrated discovery platform, combining multi-omics data from 107 strains of mice to investigate these supporting networks. We identified retinol dehydrogenase 11 (RDH11; also known as SCALD) as a novel protein associated with cholesterol metabolism. Prior studies have suggested that RDH11 may be regulated by alterations in cellular cholesterol status, but its specific roles in this pathway are mostly unknown. ResultsHere, we show that mice fed a Western diet (high fat, high cholesterol) exhibited a significant reduction in hepatic Rdh11 mRNA expression. Conversely, mice treated with a statin (3-hydroxy-3-methyl-glutaryl-coenzyme A reductase (HMGCR) inhibitor) exhibited a 2-fold increase in hepatic Rdh11 mRNA expression. Studies in human and mouse hepatocytes demonstrated that RDH11 expression was regulated by altered cellular cholesterol conditions in a manner consistent with SREBP2 target genes HMGCR and LDLR. Modulation of RDH11 in vitro and in vivo demonstrated modulation of pathways associated with cholesterol metabolism, inflammation and cellular stress. Finally, RDH11 silencing in mouse liver was associated with a reduction in hepatic cardiolipin abundance and a concomitant reduction in the abundance of proteins of the mitochondrial electron transport chain. ConclusionTaken together, these findings suggest that RDH11 likely plays a role in protecting cells against the cellular toxicity that can arise as a by-product of endogenous cellular cholesterol synthesis.

Effects of CdSe/ZnS-QD525 on oxidative stress in the liver and kidneys of mice

Nanotechnology is widely applied in biomedicine and pharmacy. It is necessary to systematic analysis the potential hazardous of QDs s to ensure their safe usage. Here, we analyzed the in vivo oxidative stress of CdSe/ZnS-QD525 at different time via intraperitoneal injection of CdSe/ZnS-QD525 in male KM mice. The results showed that in 30 nmol/kg dose group, the GSH levels of liver and kidneys were significantly decreased at day 11 (p < 0.05), while the GSH levels in liver on day 33 and in kidneys on day 22 were significantly increased (p < 0.01). The SOD activity in liver was significantly reduced (p < 0.01) of the 30 nmol/kg dose group on day 22, and the SOD activity in liver of low, middle and high dose groups treated for 33 days was significantly increased (p < 0.01). The CAT activity in liver of 30 nmol/kg and 150 nmol/kg dose groups on day 33 was significantly lower (p < 0.05) than the control group, the CAT activity was significantly increased (p < 0.05) of 30 nmol/kg dose group in kidneys. Therefore, this study indicated that CdSe/ZnS-QD525 induced oxidative damage, and caused related toxicity, which provided valuable information in evaluating the biological safety of QDs.

Using Spectral Flow Cytometry to Characterize Anti-Tumor Immunity in Orthotopic and Subcutaneous Mouse Models of Cancer.

Mouse models remain at the forefront of immuno-oncology research, providing invaluable insights into the complex interactions between the immune system and developing tumors. While several flow cytometry panels have been developed to study cancer immunity in mice, most are limited in their capacity to address the complexity of anti-cancer immune responses. For example, many of the panels developed to date focus on a restricted number of leukocyte populations (T cells or antigen-presenting cells), failing to include the multitude of other subsets that participate in anti-cancer immunity. In addition, these panels were developed using blood or splenic leukocytes. While the immune composition of the blood or spleen can provide information on systemic immune responses to cancer, it is in the tumor microenvironment (TME) that local immunity takes place. Therefore, we optimized this spectral flow cytometry panel to identify the chief cell types that take part in cancer immunity using immune cells from cancer tissue. We used pancreatic tumors implanted both orthotopically and subcutaneously to demonstrate the panel's flexibility and suitability in diverse mouse models. The panel was also validated in peripheral immune districts (the blood, spleen, and liver of tumor-bearing mice) to allow comparisons between local and systemic anti-tumor immunity. © 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Tumor induction-Orthotopic Alternate Protocol: Tumor induction-Subcutaneous Basic Protocol 2: Preparation of single-cell suspensions from the tumor, spleen, liver, and blood of tumor-bearing mice Basic Protocol 3: Staining single-cell suspensions from the tumor, spleen, liver, and blood of tumor-bearing mice.

Neoastilbin ameliorates sepsis-induced liver and kidney injury by blocking the TLR4/NF-κB pathway.

Sepsis frequently causes systemic inflammatory response syndrome and multiple organ failure in patients. Neoastilbin (NAS) is a flavonoid that plays vital functions in inflammation. This work aims to investigate the protective effects of NAS against sepsis-induced liver and kidney injury and elucidate its underlying mechanisms. The mouse model was established using cecal ligation puncture (CLP) induction. NAS was given to mice by gavage for 7 consecutive days before surgery. Liver and kidney function, oxidative stress, and inflammatory factors in serum or tissues were examined by ELISA or related kits. The expression of relevant proteins was assessed by Western blot. Hematoxylin and eosin and/or periodic acid-Schiff staining revealed that NAS ameliorated the pathological damage in liver and kidney tissues of CLP-induced mice. NAS improved liver and kidney functions, as evidenced by elevated levels of blood urea nitrogen, Creatinine, ALT, and AST in the serum of septic mice. TUNEL assay and the expression of Bcl-2 and Bax showed that NAS dramatically reduced apoptosis in liver and renal tissues. NAS treatment lowered the levels of myeloperoxidase and malondialdehyde, while elevated the superoxide dismutase content in liver and kidney tissues of CLP-induced mice. The levels of inflammatory cytokines (IL-6, TNF-α, and IL-1β) in the serum and both tissues of CLP-injured mice were markedly decreased by NAS. Mechanically, NAS downregulated TLR4 expression and inhibited NF-κB activation, and overexpression of TLR4 reversed the protective effects of NAS against liver and kidney injury. Collectively, NAS attenuated CLP-induced apoptosis, oxidative stress, inflammation, and dysfunction in the liver and kidney by restraining the TLR4/NF-κB pathway.

SGLT2 Inhibitors Attenuate Protein Abundance of Collectin Kidney 1 and Mannose-Binding Lectin in the Kidney and Liver in Mice with Experimental Diabetes

SGLT2 Inhibitors Attenuate Protein Abundance of Collectin Kidney 1 and Mannose-Binding Lectin in the Kidney and Liver in Mice with Experimental Diabetes

Palmitoylation licenses RIPK1 kinase activity and cytotoxicity in the TNF pathway.

Tumor necrosis factor (TNF)-induced receptor-interacting serine/threonine protein kinase 1 (RIPK1)-mediated cell death, including apoptosis and necroptosis, is increasingly recognized as a major driver of inflammatory diseases. Cell death checkpoints normally suppress RIPK1 kinase to safeguard the organism from its detrimental consequences. However, the mechanisms licensing RIPK1 kinase activity when a protective checkpoint is disabled remain unclear. Here, we identified S-palmitoylation as a licensing modification for RIPK1 kinase. TNF induces RIPK1 palmitoylation, mediated by DHHC5 and dependent on K63-linked ubiquitination of RIPK1, which enhances RIPK1 kinase activity by promoting the homo-interaction of its kinase domain and promotes cell death upon cell death checkpoint blockade. Furthermore, DHHC5 is amplified by fatty acid in the livers of mice with metabolic dysfunction-associated steatohepatitis, contributing to increased RIPK1 cytotoxicity observed in this condition. Our findings reveal that ubiquitination-dependent palmitoylation licenses RIPK1 kinase activity to induce downstream cell death signaling and suggest RIPK1 palmitoylation as a feasible target for inflammatory diseases.

Clam-derived exosome-like nanovesicles alleviate alcohol-induced liver injury by improving mitochondrial function

Alcoholic liver disease (ALD) is a common chronic liver disease worldwide that urgently needs safe and effective strategies. Clams are popular seafood with hepatoprotective effects, while the effective components and mechanisms remain unclear. Herein, we demonstrated that clam-derived exosome-like nanovesicles (CENs) ameliorated alcohol-induced liver injury by preventing mitochondrial dysfunction and inhibiting mitochondrial apoptotic pathway. Results showed that CENs exhibited exosome-like morphology with a particle size of 120.08 ± 1.60 nm. Mass spectrometry revealed that CENs naturally carry multiple functional lipids and proteins. Furthermore, CENs were found to survive in the gastrointestinal environment and accumulate in the liver of mice following oral administration. Importantly, administration of CENs effectively alleviated alcohol-induced liver injury and hepatocyte death in mice by restoring mitochondrial function, as indicated by increased ATP levels, enhanced mitochondrial complex I activity, and reduced mitochondrial ROS production. Further cell experiments indicated that CENs could be phagocytosed by HepG2 cells and effectively alleviated alcohol-induced hepatocyte apoptosis by inhibiting mitochondrial apoptotic pathway. Collectively, these results indicate that CENs possess potent hepatoprotective properties and may serve as a novel functional ingredient for managing alcoholic liver disease.

Leaf Extract of Kentut (Paederia foetida L.) as a Preventive Measure Against Interleukin-6 Expression in the Liver of Mice in a Sepsis Model Injected with Escherichia coli

Leaf Extract of Kentut (Paederia foetida L.) as a Preventive Measure Against Interleukin-6 Expression in the Liver of Mice in a Sepsis Model Injected with Escherichia coli

Inhibition of Selenium Supply Function of Selenoprotein P through Adduct Formation by Sulforaphane

Selenium is a potent nucleophile essential for selenoenzymes, such as glutathione peroxidase (also known as GSH-Px; GPX; GPx) and selenoprotein P (also known as SelP; SEPP1; SELENOP; SeP). SeP is predominantly secreted from the liver and functions as a selenium carrier in plasma. We previously found that sulforaphane, an electrophilic phytochemical, reduces SeP production in cultured hepatocytes and mouse liver, however, the effect of electrophilic modification of SeP by SFN on selenium transport and metabolism remains unclear. In the present study, we demonstrate that sulforaphane covalently modifies selenocysteine/cysteine residues of SeP using an acidic biotin PAEC5 maleimide labeling assay, which allows for focused-labeling of selenocysteine residues. Although the SFN-SeP adduct can be taken up by HepG2 cells and degraded by the lysosome, it was less effective in inducing GPx expression. Our findings indicate that SFN disrupts the selenium supply pathway through the formation of the SeP-SFN adduct.

FITM2 deficiency results in ER lipid accumulation, ER stress, and reduced apolipoprotein B lipidation and VLDL triglyceride secretion in vitro and in mouse liver

Triglycerides (TGs) associate with apolipoprotein B100 (apoB100) to form very low density lipoproteins (VLDLs) in the liver. The repertoire of factors that facilitate this association is incompletely understood. FITM2, an integral endoplasmic reticulum (ER) protein, was originally discovered as a factor participating in cytosolic lipid droplet (LD) biogenesis in tissues that do not form VLDL. We hypothesized that in the liver, in addition to promoting cytosolic LD formation, FITM2 would also transfer TG from its site of synthesis in the ER membrane to nascent VLDL particles within the ER lumen. Experiments were conducted using a rat hepatic cell line (McArdle-RH7777, or McA cells), an established model of mammalian lipoprotein metabolism, and mice. FITM2 expression was reduced using siRNA in cells and by liver specific cre-recombinase mediated deletion of the Fitm2 gene in mice. Effects of FITM2 deficiency on VLDL assembly and secretion invitro and invivo were measured by multiple methods, including density gradient ultracentrifugation, chromatography, mass spectrometry, stimulated Raman scattering (SRS) microscopy, sub-cellular fractionation, immunoprecipitation, immunofluorescence, and electron microscopy. 1) FITM2-deficient hepatic cells invitro and invivo secrete TG-depleted VLDL particles, but the number of particles is unchanged compared to controls; 2) FITM2 deficiency in mice on a high fat diet (HFD) results in decreased plasma TG levels. The number of apoB100-containing lipoproteins remains similar, but shift from VLDL to low density lipoprotein (LDL) density; 3) Both invitro and invivo, when TG synthesis is stimulated and FITM2 is deficient, TG accumulates in the ER, and despite its availability this pool is unable to fully lipidate apoB100 particles; 4) FITM2 deficiency disrupts ER morphology and results in ER stress. The results suggest that FITM2 contributes to VLDL lipidation, especially when newly synthesized hepatic TG is in abundance. In addition to its fundamental importance in VLDL assembly, the results also suggest that under dysmetabolic conditions, FITM2 may be an important factor in the partitioning of TG between cytosolic LDs and VLDL particles.

Evaluation of the correlation between nuclear localization levels and genome editing efficiencies of Cas12a fused with nuclear localization signals

Genome editing technology using the CRISPR-Cas system is attracting much attention not only as a promising experimental tool for analysis of genome functions, but also as a novel therapeutic approach for genetic disorders. Among the various types of Cas proteins, Cas12a is expected to be a promising gene editing tool due to its unique properties, including low off-target effects. As Cas proteins are of prokaryotic origin, they need to be fused with appropriate localization signals to perform their function in eukaryotic cells. Cas12a proteins fused with a nuclear localization signal (NLS) have been developed so far, but the relation between the nuclear localization activity and the genome editing efficiency has not been fully elucidated. Here, utilizing two Cas12a orthologs, AsCas12a and LbCas12a, with various number of NLSs derived from various origins, we revealed that the improved nuclear localization resulted in increased genome editing efficiencies when expressed using adenovirus (Ad) vector in cultured cells. However, when they were expressed in mouse liver, the improvement of the nuclear localization activity was not necessarily required to achieve the maximum genome editing efficiency four weeks after Ad vector administration. These data indicated that the optimized NLS modification of Cas12a proteins in in vitro situations differed from that in in vivo.

Protein kinase D2 modulates hepatic insulin sensitivity in male mice

ObjectivesProtein kinase D (PKD) family is emerging as relevant regulator of metabolic homeostasis. However, the precise role of PKD2 in modulating hepatic insulin signaling has not been fully elucidated and it is the aim of this study. MethodsPKD inhibition was analyzed for insulin signaling in mouse and human hepatocytes. PKD2 was overexpressed in Huh7 hepatocytes and mouse liver, and insulin responses were evaluated. Mice with hepatocyte-specific PKD2 depletion (PKD2ΔHep) and PKD2fl/fl mice were fed a chow (CHD) or high fat diet (HFD) and glucose homeostasis and lipid metabolism were investigated. ResultsPKD2 silencing enhanced insulin signaling in hepatocytes, an effect also found in primary hepatocytes from PKD2ΔHep mice. Conversely, a constitutively active PKD2 mutant reduced insulin-stimulated AKT phosphorylation. A more in-depth analysis revealed reduced IRS1 serine phosphorylation under basal conditions and increased IRS1 tyrosine phosphorylation in PKD2ΔHep primary hepatocytes upon insulin stimulation and, importantly PKD co-immunoprecipitates with IRS1. In vivo constitutively active PKD2 overexpression resulted in a moderate impairment of glucose homeostasis and reduced insulin signaling in the liver. On the contrary, HFD-fed PKD2ΔHep male mice displayed improved glucose and pyruvate tolerance, as well as higher peripheral insulin tolerance and enhanced hepatic insulin signaling compared to control PKD2fl/fl mice. Despite of a remodeling of hepatic lipid metabolism in HFD-fed PKD2ΔHep mice, similar steatosis grade was found in both genotypes. ConclusionsResults herein have unveiled an unknown role of PKD2 in the control of insulin signaling in the liver at the level of IRS1 and point PKD2 as a therapeutic target for hepatic insulin resistance.

AKR7A5 knockout promote acute liver injury by inducing inflammatory response, oxidative stress and apoptosis in mice.

Alcohol liver disease has become a worldwide critical health problem. The ingested alcohol could be converted into acetaldehyde or combined with free fatty acids to induce the endoplasmic reticulum oxidative stress in the liver. Coincidentally, AKR7A5 has both aldehyde detoxification and antioxidant effects. Therefore, we discuss the possible role and mechanism of AKR7A5 in the acute alcohol injury of mice liver. There were four experiment groups, the C57BL/6 mice of wild-type mice (WT) or AKR7A5-/- mice (KO) were intragastrically administrated with saline or 50% ethanol at 14 mL/kg, respectively. Compared to the WT + alcohol group, abnormal liver function, disordered hepatic cord, severe congestion in the hepatic sinus and the space of the hepatic cord, occurrence of oxidative stress, DNA damage and different expressions of apoptosis-related proteins were detected in the KO + alcohol group. Meanwhile, the biological process enrichment analysis showed that the down-regulated proteins were related to the metabolism of fatty acid, the up-regulated proteins positive regulation of reactive oxygen species metabolic process, negative regulation of coagulation and haemostasis. In conclusion, single ethanol binge combined with the absence of AKR7A5 caused more severe inflammatory response, oxidative stress, apoptosis of endogenous pathways, abnormal lipids metabolism and disordered coagulation in mice liver.

Elongation factor 1A1 inhibition elicits changes in lipid droplet size, the bulk transcriptome, and cell type-associated gene expression in MASLD mouse liver.

Eukaryotic elongation factor 1A1 (EEF1A1), originally identified for its role in protein synthesis, has additional functions in diverse cellular processes. Of note, we previously discovered a role for EEF1A1 in hepatocyte lipotoxicity. We also demonstrated that a 2-wk intervention with the EEF1A1 inhibitor didemnin B (DB) (50 µg/kg) decreased liver steatosis in a mouse model of obesity and metabolic dysfunction-associated steatotic liver disease (MASLD) [129S6/SvEvTac mice fed Western diet (42% fat) for 26 wk]. Here, we further characterized the hepatic changes occurring in these mice by assessing lipid droplet (LD) size, bulk differential expression, and cell type-associated alterations in gene expression. Consistent with the previously demonstrated decrease in hepatic steatosis, we observed decreased median LD size in response to DB. Bulk RNA sequencing (RNA-Seq) followed by gene set enrichment analysis revealed alterations in pathways related to energy metabolism and proteostasis in DB-treated mouse livers. Deconvolution of bulk data identified decreased cell type association scores for cholangiocytes, mononuclear phagocytes, and mesenchymal cells in response to DB. Overrepresentation analyses of bulk data using cell type marker gene sets further identified hepatocytes and cholangiocytes as the primary contributors to bulk differential expression in response to DB. Thus, we show that chemical inhibition of EEF1A1 decreases hepatic LD size and decreases gene expression signatures associated with several liver cell types implicated in MASLD progression. Furthermore, changes in hepatic gene expression were primarily attributable to hepatocytes and cholangiocytes. This work demonstrates that EEF1A1 inhibition may be a viable strategy to target aspects of liver biology implicated in MASLD progression.NEW & NOTEWORTHY Chemical inhibition of EEF1A1 decreases hepatic lipid droplet size and decreases gene expression signatures associated with liver cell types that contribute to MASLD progression. Furthermore, changes in hepatic gene expression are primarily attributable to hepatocytes and cholangiocytes. This work highlights the therapeutic potential of targeting EEF1A1 in the setting of MASLD, and the utility of RNA-Seq deconvolution to reveal valuable information about tissue cell type composition and cell type-associated gene expression from bulk RNA-Seq data.

Aging-induced short-chain acyl-CoA dehydrogenase promotes age-related hepatic steatosis by suppressing lipophagy.

Hepatic steatosis, the first step in the development of nonalcoholic fatty liver disease (NAFLD), is frequently observed in the aging population. However, the underlying molecular mechanism remains largely unknown. In this study, we first employed GSEA enrichment analysis to identify short-chain acyl-CoA dehydrogenase (SCAD), which participates in the mitochondrial β-oxidation of fatty acids and may be associated with hepatic steatosis in elderly individuals. Subsequently, we examined SCAD expression and hepatic triglyceride content in various aged humans and mice and found that triglycerides were markedly increased and that SCAD was upregulated in aged livers. Our further evidence in SCAD-ablated mice suggested that SCAD deletion was able to slow liver aging and ameliorate aging-associated fatty liver. Examination of the molecular pathways by which the deletion of SCAD attenuates steatosis revealed that the autophagic degradation of lipid droplets, which was not detected in elderly wild-type mice, was maintained in SCAD-deficient old mice. This was due to the decrease in the production of acetyl-coenzyme A (acetyl-CoA), which is abundant in the livers of old wild-type mice. In conclusion, our findings demonstrate that the suppression of SCAD may prevent age-associated hepatic steatosis by promoting lipophagy and that SCAD could be a promising therapeutic target for liver aging and associated steatosis.

Notch-Driven Cholangiocarcinogenesis Involves the Hippo Pathway Effector TAZ via METTL3-m6A-YTHDF1

Background and AimsNotch and TAZ are implicated in cholangiocarcinogenesis, but whether and how these oncogenic molecules interact remain unknown. MethodsThe development of CCA was induced by hydrodynamic tail vein (HDTV) injection of oncogenes (NICD/AKT) to the FVB/NJ mice. CCA xenograft was developed by inoculation of human CCA cells into the livers of SCID mice. Tissues and cells were analyzed using qRT-PCR, Western blotting analyses, Immunohistochemistry, ChIP-qPCR and WST-1 cell proliferation Assay. ResultsOur experimental findings show that TAZ is indispensable in NICD-driven cholangiocarcinogenesis. Notch activation induces the expression of METTL3 (Methyltransferase like-3) which catalyzes N6-methyladenosine (m6A) modification of TAZ mRNA and that this mechanism plays a central role in the crosstalk between Notch and TAZ in CCA cells. Mechanistically, Notch regulates the expression of METTL3 through the binding of NICD to its downstream transcription factor CSL in the promoter region of METTL3. METTL3 in turn mediates m6A modification of TAZ mRNA which is recognized by the m6A reader YTHDF1 to enhance TAZ protein translation. We observed that inhibition of Notch signaling decreased the protein levels of both MELLT3 and TAZ. Depletion of METTL3 by shRNAs or by the next generation GapmeR antisense oligonucleotides (ASOs) decreased the level of TAZ protein and inhibited the growth of human CCA cells in vitro and in mice. ConclusionThis study describes a novel Notch-METTL3-TAZ signaling cascade which is important in CCA development and progression. Our experimental results provide new insight into how the Notch pathway cooperates with TAZ signaling in CCA, and the findings may have important therapeutic implications.