Mouse Liver Samples Research Articles

COMPARATIVE ANALYSIS OF THE TOTAL PROTEOME IN NONALCOHOLIC STEATOHEPATITIS: IDENTIFICATION OF POTENTIAL BIOMARKERS.

Nonalcoholic fatty liver disease (NAFLD) is a hepatic condition characterized excessive fat accumulation in the liver with advanced stage nonalcoholic steatohepatitis (NASH), potentially leading to liver fibrosis, cirrhosis, and cancer. Currently, the identification and classification of NASH require invasive liver biopsy, which has certain limitations. Mass spectrometry-based proteomics can detect crucial proteins and pathways implicated in NASH development and progression. We collected liver and serum samples from choline-deficient, L-amino acid-defined high-fat diet fed NASH C57BL/6J mice and human serum samples to examine proteomic alterations and identify early biomarkers for NASH diagnosis. In-depth targeted multiple reaction monitoring (MRM) scanning and immunoblotting assays were used to verify the biomarker candidates from mouse liver and serum samples, and enzyme-linked immunosorbent assay (ELISA) was employed to analyze human serum samples. The MRM analysis of NASH liver revealed 50 proteins with altered expression (18 up- and 32 downregulated) that are involved in biological processes such as detoxification, fibrosis, inflammation, and fatty acid metabolism. Ingenuity pathway analysis identified impaired protein synthesis, cellular stress and defense, cellular processes and communication, and metabolism in NASH mouse liver. Immunoblotting analysis confirmed that the expression of proteins associated with fatty acid metabolism (Aldo B and Fasn) and urea cycle (Arg1, Cps1, and Otc) was altered in mouse liver and serum. Further analysis on human serum samples using ELISA confirmed the increased expression of multiple proteins, including Aldo B, Asl, and Lgals3, demonstrating values of 0.917, 0.979, and 0.965 of area under the curve in NASH diagnosis. These findings offer valuable insights into the molecular mechanisms of NASH and possible diagnostic biomarkers for early detection.

Sensitive and Deep Coverage Phosphoproteome Detection Method Reveals Spleen as a More Sensitive Organ than Liver in Early Detection of Liver Fibrosis-Related Signaling Protein Dysregulation.

Although cathepsin S is transported from the spleen to the liver, where it cleaves collagen XVIII to produce endostatin and plays a critical role in the onset of early liver fibrosis, the relationship between liver fibrosis and spleen function remains underexplored. Given the roles of phosphorylation in disease, understanding its regulatory mechanism in early liver fibrosis is crucial. Despite advances in mass spectrometry enhancing phosphoproteomics, its application is limited by small clinical samples and subtle protein changes. We optimized a phosphoproteomic workflow, adjusting the protein amounts and using different enrichment beads and varied mass spectrometers, achieving deep phosphoproteomic coverage from minimal samples. We identified over 46,000 phosphosites in HepG2 cells and over 29,000 phosphosites in mouse liver samples using just 500 μg of proteins. Even with as little as 50 μg of 293T proteins, we detected over 11,000 phosphosites, 1.2 times more than the recently reported RUPE-phospho. Using the Sensitive and Deep Coverage Phosphoproteome Detection method, abbreviated as SDC-PhosDet, we demonstrated that in early liver fibrosis, the spleen exhibits more rapid and sensitive phosphorylation changes than the liver, affecting proteins closely linked to signaling and metabolism such as STAT1, JUN, CBL, ATP7B, and PTPN2. These findings highlight the spleen's role and offer new avenues for investigating the molecular mechanisms of early liver fibrosis, diagnosis, and intervention beyond the liver itself. Moreover, this method holds promise for applying phosphoproteomics to early-stage liver fibrosis using clinical microsamples.

Efficacy of Software-Based Analysis of Mouse Liver Samples to Assess Cell Death

Efficacy of Software-Based Analysis of Mouse Liver Samples to Assess Cell Death

Carbohydrate storage in cells: a laboratory activity for the assessment of glycogen stores in biological tissues.

Carbohydrates and fats constitute our primary energy sources. The importance of each of these energy substrates varies across cell types and physiological conditions. For example, the brain normally relies almost exclusively on glucose oxidation, whereas skeletal muscle shifts from lipids toward higher carbohydrate oxidation rates as exercise intensity increases. Understanding how carbohydrates are stored in our cells and which tissues contain significant carbohydrate stores is crucial for health professionals, especially given the role of carbohydrate metabolism in various pathophysiological conditions. This laboratory activity uses a simple and low-cost iodine binding method to quantify glycogen in mouse skeletal muscle and liver samples. By integrating the results of this activity with literature data, students can determine overall glycogen storage in the human body. The primary goal of the activity is to enhance students' understanding of the importance and limitations of glycogen stores in energy metabolism.NEW & NOTEWORTHY Carbohydrates are one of the primary energy sources utilized by our cells. Liver and skeletal muscle glycogen, which are the main carbohydrate reserves in the body, play a central role in energy metabolism, especially during periods of fasting and exercise. In this laboratory activity, students measure glycogen levels in tissues to gain insights into how carbohydrates are stored in our cells and understand the role and limitations of liver and muscle carbohydrate stores.

Linking antibody against apolipoprotein A-1 to metabolic dysfunction-associated steatohepatitis in mice

Abstract Background Metabolic dysfunction-associated fatty liver disease (MASLD) is a common liver condition associated with increased cardiovascular disease (CVD) risk. Cytokeratin 18 (CK-18) is indicative of liver injury and used as a surrogate biomarker covering MASLD to cirrhosis phenotypes. Autoantibodies against apolipoprotein A-1 (AAA-1) are known CVD risk factors inducing MASLD in vitro, but their role in modulating steatohepatitis and fibrosis in vivo is still elusive. We investigated AAA-1's contribution to systemic low-grade inflammation, liver steatosis, and fibrosis using a MASLD mouse model and a validated passive immunization protocol (PIP). Methods 10 weeks-old male C57BL/6J mice were fed a choline-deficient, L-amino acid-defined, high-fat diet (CDAHFD) and immunized with AAA-1 (200ug) or control antibodies for ten days. At sacrifice, plasma samples were collected, and CK-18 levels, and proinflammatory cytokines were measured using the Mesoscale Discovery platform. Hematoxylin and Eosin and Sirius Red Fast Green staining were used to reveal liver steatosis and fibrosis, respectively. RNA were extracted from mouse liver samples and mRNA were then analyzed using a commercial fibrosis-specific genes panel (nCounter Human Fibrosis V2 Panel) and the nCounter Analysis System. Results Compared to control IgG recipients, CDAHFD mice injected with AAA-1 exhibited significantly elevated plasma levels of CK-18 (5.3 vs 2.1, p=0.031), IL-6 (13 vs 6.9, p=0.035), IL-10 (27.3 vs 9.8, p=0.007) and TNF-α (32.1 vs 24.2, p=0.032), and liver steatosis (93.4% vs 73.8%, p=0.007). Nanostring technology experiments revealed upregulation of several pro-fibrotic m-RNAs in liver tissue from AAA-1 immunized mice compared to the IgG immunized control group. These included Serum Amyloid A (Fold Change (FC) AAA-1 vs IgG : 2.5, p=0.02) , G-protein coupled receptors 65 (FC: 2.7, p=0.01), Periostin (FC: 2.6, p=0.001), Phospholipid transfer protein (FC: 2.2, p=0.0004), Vascular Cell Adhesion Molecule 1 (FC: 2.5, p=0.02) and Angiopoietin-like 4 (FC: 2.8, p=0.01). Histologically, the amount of liver fibrosis remained unaffected, probably due to the short time of the study. Conclusions Short AAA-1 PIP induced enhanced liver steatosis and inflammation accompanied by an elevation of the expression of several pro-fibrotic genes in CDAHFD mice, suggesting that AAA-1 may contribute to the transition from MASLD to MASH. Knowing whether increased exposure time to AAA-1 could lead to end stage disease (including cirrhosis and HCC), and if this could apply to humans as well warrant further investigations.

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 (BA) 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/d for 1 wk. 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 BA quantitation analysis, we observed decreases in secondary BAs in BAC-treated mice, which was more pronounced in the female mice. This finding is supported by decreases in bacteria known to metabolize primary BAs into secondary BAs, 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.

High‑fat diet‑induced LCN2 exacerbates myocardial ischemia‑reperfusion injury by enhancing platelet activation.

Following acute myocardial infarction, the recovery of blood flow leads to myocardial ischemia‑reperfusion (MI/R) injury, which is primarily characterized by the activation of inflammatory signals, microvascular obstruction, increased oxidative stress and excessive Ca2+ overload. It has also been demonstrated that platelets can exacerbate MI/R injury by releasing reactive oxygen species, inflammatory factors and chemokines, while also obstructing microvessels through thrombus formation. As a bioactive molecule with proinflammatory and chemotactic properties, lipocalin 2 (LCN2) exhibits a positive correlation with obesity, hyperglycemia, hypertriglyceridemia and insulin resistance index, which are all significant risk factors for ischemic cardiomyopathy. Notably, the potential role of LCN2 in promoting atherosclerosis may be related to its influence on the function of macrophages, smooth muscle cells and endothelial cells, but its effect on platelet function has not yet been reported. In the present study, the effect of a high‑fat diet (HFD) on LCN2 expression was determined by detecting LCN2 expression levels in the liver and serum samples of mice through reverse transcription‑quantitative PCR and enzyme linked immunosorbent assay, respectively. The effect of LCN2 on platelet function was evaluated by examining whether LCN2 affected platelet activation, aggregation, adhesion, clot retraction and P‑selectin expression. To determine whether LCN2 aggravated MI/R injury in HFD‑fed mice by affecting platelet and inflammatory cell recruitment, wild‑type and LCN2 knockout mice fed a HFD were subjected to MI/R injury, then hearts were collected for hematoxylin and eosin staining and 2,3,5‑triphenyltetrazolium chloride staining, and immunohistochemistry was employed to detect the expression of CD42b, Ly6G, CD3 and B220. Based on observing the upregulation of LCN2 expression in mice fed a HFD, the present study further confirmed that LCN2 could accelerate platelet activation, aggregation and adhesion. Moreover, in vivo studies validated that knockout of LCN2 not only mitigated MI/R injury, but also inhibited the recruitment of platelets and inflammatory cells in myocardial tissue following ischemia‑reperfusion. In conclusion, the current findings suggested that the effect of HFD‑induced LCN2 on aggravating MI/R injury may totally or partially dependent on its promotion of platelet function.

CDK4/6 inhibitor PD-0332991 suppresses hepatocarcinogenesis by inducing senescence of hepatic tumor-initiating cells

IntroductionOwing to the limited treatment options for hepatocellular carcinoma (HCC), interventions targeting pre-HCC stages have attracted increasing attention. In the pre-HCC stage, hepatic tumor-initiating cells (hTICs) proliferate abnormally and contribute to hepatocarcinogenesis. Numerous studies have investigated targeted senescence induction as an HCC intervention. However, it remains to be clarified whether senescence induction of hTICs could serve as a pre-HCC intervention. ObjectivesThis study was designed to investigate whether senescence induction of hTICs in the precancerous stage inhibit HCC initiation. Methods and ResultsHCC models developed from chronic liver injury (CLI) were established by using Fah-/- mice and N-Ras + AKT mice. PD-0332991, a selective CDK4/6 inhibitor that blocks the G1/S transition in proliferating cells, was used to induce senescence during the pre-HCC stage. Upon administration of PD-0332991, we observed a significant reduction in HCC incidence following selective senescence induction in hTICs, and an alleviation liver injury in the CLI-HCC models. PD-0332991 also induced senescence in vitro in cultured hTICs isolated from CLI-HCC models. Moreover, RNA sequencing (RNA-seq) analysis delineated that the “Cyclin D-CDK4/6-INK4-Rb” pathway was activated in both mouse and human liver samples during the pre-HCC stage, while PD-0332991 exhibited substantial inhibition of this pathway, thereby inducing cellular senescence in hTICs. Regarding the immune microenvironment, we demonstrated that senescent hTICs secrete key senescence-associated secretory phenotypic (SASP) factors, CXCL10 and CCL2, to activate and recruit macrophages, and contribute to immune surveillance. ConclusionWe found that hTICs can be targeted and induced into a senescent state during the pre-HCC stage. The SASP factors released by senescent hTICs further activate the immune response, facilitating the clearance of hTICs, and consequently suppressing HCC occurrence. We highlight the importance of pre-HCC interventions and propose that senescence-inducing drugs hold promise for preventing HCC initiation under CLI.

Ammonium bicarbonate buffers combined with hybrid surface technology columns improve the peak shape of strongly tailing lipids

BackgroundLipids such as phosphatidic acids (PAs) and cardiolipins (CLs) present strongly tailing peaks in reversed phase liquid chromatography, which entails low detectability. They are usually analyzed by hydrophilic interaction liquid chromatography (HILIC), which hampers high-throughput lipidomics. Thus, there is a great need for improved analytical methods in order to obtain a broader coverage of the lipidome in a single chromatographic method. We investigated the effect of ammonium bicarbonate (ABC) on peak asymmetry and detectability, in comparison with ammonium formate (AFO) on both a conventional BEH C18 column and an HST-CSH C18 column. ResultsThe combination of 2.5 mM ABC buffer pH 8 with an HST-CSH C18 column produced significantly improved results, reducing the asymmetry factor at 10 % peak height of PA 16:0/18:1 from 8.4 to 1.6. Furthermore, on average, there was up to a 54-fold enhancement in the peak height of its [M − H]- ion compared to AFO and the BEH C18 column. We confirmed this beneficial effect on other strongly tailing lipids, with accessible phosphate moieties e.g., cardiolipins, phosphatidylinositol phosphate, phosphatidylinositol bisphosphate, phosphorylated ceramide and phosphorylated sphingosine. Furthermore, we found an increased detectability of phospho- and sphingolipids up to 28 times in negative mode when using an HST-CSH C18 column. The method was successfully applied to mouse liver samples, where previously undetected endogenous phospholipids could be analyzed with improved chromatographic separation. SignificanceIn conclusion, the use of 2.5 mM ABC substantially improved the peak shape of PAs and enhanced the detectability of the lipidome in negative mode on an RPLC-ESI-Q-TOF-MS system on both BEH C18 and HST-CSH C18 columns. This method provides a wider coverage of the lipidome with one single injection for future lipidomic applications in negative mode.

Identification of a chromatin-bound ERRα interactome network in mouse liver

ObjectivesEstrogen-related-receptor α (ERRα) plays a critical role in the transcriptional regulation of cellular bioenergetics and metabolism, and perturbations in its activity have been associated with metabolic diseases. While several coactivators and corepressors of ERRα have been identified to date, a knowledge gap remains in understanding the extent to which ERRα cooperates with coregulators in the control of gene expression. Herein, we mapped the primary chromatin-bound ERRα interactome in mouse liver. MethodsRIME (Rapid Immuno-precipitation Mass spectrometry of Endogenous proteins) analysis using mouse liver samples from two circadian time points was used to catalog ERRα-interacting proteins on chromatin. The genomic crosstalk between ERRα and its identified cofactors in the transcriptional control of precise gene programs was explored through cross-examination of genome-wide binding profiles from chromatin immunoprecipitation-sequencing (ChIP-seq) studies. The dynamic interplay between ERRα and its newly uncovered cofactor Host cell factor C1 (HCFC1) was further investigated by loss-of-function studies in hepatocytes. ResultsCharacterization of the hepatic ERRα chromatin interactome led to the identification of 48 transcriptional interactors of which 42 were previously unknown including HCFC1. Interrogation of available ChIP-seq binding profiles highlighted oxidative phosphorylation (OXPHOS) under the control of a complex regulatory network between ERRα and multiple cofactors. While ERRα and HCFC1 were found to bind to a large set of common genes, only a small fraction showed their colocalization, found predominately near the transcriptional start sites of genes particularly enriched for components of the mitochondrial respiratory chain. Knockdown studies demonstrated inverse regulatory actions of ERRα and HCFC1 on OXPHOS gene expression ultimately dictating the impact of their loss-of-function on mitochondrial respiration. ConclusionsOur work unveils a repertoire of previously unknown transcriptional partners of ERRα comprised of chromatin modifiers and transcription factors thus advancing our knowledge of how ERRα regulates metabolic transcriptional programs.

An integrated strategy to discover the quality control markers of herbal formulae of Danning tablet with anti-cholestasis applications

Quality makers (Q-markers) are pivotal for the standardization of quality control for traditional Chinese medicine (TCM) drugs. However, it’s a great challenge to identify the Q-markers of TCM drugs especially for TCM formulas. This study was designed to provide a method to discover Q-markers of TCM formulas and applied to identify the Q-markers responsible for the anti-cholestatic effect of Danning tablet (DNT), a TCM formulae consisting with seven herbal ingredients. In this study, the anti-cholestatic effect of DNT was evaluated in mice with cholestasis induced by α-naphthyl isothiocyanate (ANIT). An untargeted metabolomic profiling were performed using serum and liver samples of mice. Xenobiotics derived from DNT and endogenous compounds responsible for the anti-cholestatic effect of DNT were screened by statistical analysis and the Q-markers of DNT were illustrated, followed by predicting the potential targets and mechanisms via network pharmacology study. DNT exhibited a protective effect against liver injury in mice with ANIT-induced cholestasis. Three endogenous compounds, namely taurochenodeoxycholic acid (TCDCA), taurocholic acid (TCA), and glycocholic acid (GCA), were identified as the endogenous markers responsible for the anti-cholestatic effect of DNT. By setting a cutoff value of the correlation co-efficient at 0.8, 6 promising Q-markers, including luteolin, kaempferol, apigenin, emodin, luteolin-7-glucoside, and 5,4′-dihydroxy-3,6,7,8,3′-pentamethoxyflavone, were screened out. These Q-markers were stably existed in DNT and predicted to be highly active in treating cholestasis. This study established a pipeline to screen out the potential Q-markers of TCM formulas and 6 key Q-markers responsible for the anti-cholestatic effect of DNT were identified.

Identification and validation of cuproptosis-related genes in acetaminophen-induced liver injury using bioinformatics analysis and machine learning.

Acetaminophen (APAP) is commonly used as an antipyretic analgesic. However, acetaminophen overdose may contribute to liver injury and even liver failure. Acetaminophen-induced liver injury (AILI) is closely related to mitochondrial oxidative stress and dysfunction, which play critical roles in cuproptosis. Here, we explored the potential role of cuproptosis-related genes (CRGs) in AILI. The gene expression profiles were obtained from the Gene Expression Omnibus database. The differential expression of CRGs was determined between the AILI and control samples. Protein protein interaction, correlation, and functional enrichment analyses were performed. Machine learning was used to identify hub genes. Immune infiltration was evaluated. The AILI mouse model was established by intraperitoneal injection of APAP solution. Quantitative real-time PCR and western blotting were used to validate hub gene expression in the AILI mouse model. The copper content in the mouse liver samples and AML12 cells were quantified using a colorimetric assay kit. Ammonium tetrathiomolybdate (ATTM), was administered to mouse models and AML12 cells in order to investigate the effects of copper chelator on AILI. The analysis identified 7,809 differentially expressed genes, 4,245 of which were downregulated and 3,564 of which were upregulated. Four optimal feature genes (OFGs; SDHB, PDHA1, NDUFB2, and NDUFB6) were identified through the intersection of two machine learning algorithms. Further nomogram, decision curve, and calibration curve analyses confirmed the diagnostic predictive efficacy of the four OFGs. Enrichment analysis indicated that the OFGs were involved in multiple pathways, such as IL-17 pathway and chemokine signaling pathway, that are related to AILI progression. Immune infiltration analysis revealed that macrophages were more abundant in AILI than in control samples, whereas eosinophils and endothelial cells were less abundant. Subsequently, the AILI mouse model was successfully established, and histopathological analysis using hematoxylin-eosin staining along with liver function tests revealed a significant induction of liver injury in the APAP group. Consistent with expectations, both mRNA and protein levels of the four OFGs exhibited a substantial decrease. The administration of ATTAM effectively mitigates copper elevation induced by APAP in both mouse model and AML12 cells. However, systemic administration of ATTM did not significantly alleviate AILI in the mouse model. This study first revealed the potential role of CRGs in the pathological process of AILI and offered novel insights into its underlying pathogenesis.

PPARα is one of the key targets for dendrobine to improve hepatic steatosis in NAFLD

Ethnopharmacological relevanceDendrobium nobile Lindl. (DNL) is a traditional Chinese ethnobotanical herb. Dendrobine (DNE) has been designated as a quality indicator for DNL in the Chinese Pharmacopoeia. DNE exhibits various pharmacological activities, including the reduction of blood lipids, regulation of blood sugar levels, as well as anti-inflammatory and antioxidant properties. Aim of the studyThe objective of this study is to explore the impact of DNE on lipid degeneration in nonalcoholic fatty liver disease (NAFLD) liver cells and elucidate its specific mechanism. The findings aim to offer theoretical support for the development of drugs related to DNL. Materials and methodsWe utilized male C57BL/6J mice, aged 6 weeks old, to establish a NAFLD model. This model allowed us to assess the impact of DNE on liver pathology and lipid levels in NAFLD mice. We investigated the mechanism of DNE's regulation of lipid metabolism through RNA-seq analysis. Furthermore, a NAFLD model was established using HepG2 cells to further evaluate the impact of DNE on the pathological changes of NAFLD liver cells. The potential mechanism of DNE's improvement was rapidly elucidated using HT-qPCR technology. These results were subsequently validated using mouse liver samples. Following the in vitro activation or inhibition of PPARα function, we observed changes in DNE's ability to ameliorate pathological changes in NAFLD hepatocytes. This mechanism was further verified through RT-qPCR and Western blot analysis. ResultsDNE demonstrated a capacity to enhance serum TC, TG, and liver TG levels in mice, concurrently mitigating liver lipid degeneration. RNA-seq analysis unveiled that DNE primarily modulates the expression of genes related to metabolic pathways in mouse liver. Utilizing HT-qPCR technology, it was observed that DNE markedly regulates the expression of genes associated with the PPAR signaling pathway in liver cells. Consistency was observed in the in vivo data, where DNE significantly up-regulated the expression of PPARα mRNA and its protein level in mouse liver. Additionally, the expression of fatty acid metabolism-related genes (ACOX1, CPT2, HMGCS2, LPL), regulated by PPARα, was significantly elevated following DNE treatment. In vitro experiments further demonstrated that DNE notably ameliorated lipid deposition, peroxidation, and inflammation levels in NAFLD hepatocytes, particularly when administered in conjunction with fenofibrate. Notably, the PPARα inhibitor GW6471 attenuated these effects of DNE. ConclusionsIn summary, DNE exerts its influence on the expression of genes associated with downstream fat metabolism by regulating PPARα. This regulatory mechanism enhances liver lipid metabolism, mitigates lipid degeneration in hepatocytes, and ultimately ameliorates the pathological changes in NAFLD hepatocytes.

Site-Specific Identification of Protein S-Acylation by IodoTMT0 Labeling and Immobilized Anti-TMT Antibody Resin Enrichment.

Protein S-acylation is a reversible post-translational modification (PTM). It is present on diverse proteins and has important roles in regulating protein function. Aminolysis with hydroxylamine is widely used in the global identification of the PTM. However, the identification is indirect. Distinct criteria have been used for identification, and the false discovery rate has not been addressed. Here, we report a site-specific method for S-acylation identification based on tagging of S-acylation sites with iodoTMT0. Efforts to improve the performance of the method and confidence of identification are discussed, highlighting the importance of reducing contaminant peptides and keeping the recovery rate consistent between aliquots with or without hydroxylamine treatment. With very stringent criteria, presumptive S-acylation sites of 269, 684, 695, and 780 were identified from HK2 cells, HK11 cells, mouse brain, and mouse liver samples, respectively. Among them, the newly identified protein S-acylation sites are equivalent to 34% of human and 24% of mouse S-acylation sites reported previously. In addition, false-positive rates for S-acylation identification and S-acylation abundances were estimated. Significant differences in S-acylation abundance were found from different samples (from 0.08% in HK2 cells to 0.76% in mouse brain), and the false-positive rates were significantly higher for samples with a low abundance of S-acylation.

Visualization of hydrocarbon chain length and degree of saturation of fatty acids in mouse livers by combining near-infrared hyperspectral imaging and machine learning

Fatty acids play various physiological roles owing to their diverse structural characteristics, such as hydrocarbon chain length (HCL) and degree of saturation (DS). Although the distribution of fatty acids in biological tissues is associated with lipid metabolism, in situ imaging tools are still lacking for HCL and DS. Here, we introduce a framework of near-infrared (1000–1400 nm) hyperspectral label-free imaging with machine learning analysis of the fatty acid HCL and DS distribution in the liver at each pixel, in addition to the previously reported total lipid content. The training data of 16 typical fatty acids were obtained by gas chromatography from liver samples of mice fed with various diets. A two-dimensional mapping of these two parameters was successfully performed. Furthermore, the HCL/DS plot exhibited characteristic clustering among the different diet groups. Visualization of fatty acid distribution would provide insights for revealing the pathophysiological conditions of liver diseases and metabolism.

Nonalcoholic steatohepatitis-associated hepatocarcinogenesis in mice fed a modified choline-deficient, methionine-lowered, L-amino acid-defined diet and the role of signal changes.

Nonalcoholic steatohepatitis (NASH) can progress to cirrhosis and even hepatocellular carcinoma (HCC). The incidence of NASH-associated HCC is increasing, posing a serious public health threat. Unfortunately, the underlying pathological mechanisms, including the possible differences between neoplastic and non-neoplastic lesions, remain largely unknown. Previously, we reported a dietary mouse NASH model with a choline-deficient, methionine-lowered, L-amino-acid-defined, high-fat diet containing shortening without trans fatty acids (CDAA-HF-T[-]), which rapidly induces fibrosis and proliferative lesions in the liver. This study aimed to develop a mouse CDAA-HF-T(-) model capable of assessing NASH-associated hepatocarcinogenesis and identifying key signaling factors involved in its underlying mechanisms. Multiple large masses, histopathologically hepatocellular adenomas and carcinomas, and hemangiosarcomas were detected in the liver samples of mice fed CDAA-HF-T(-) for 52 or 63 weeks, along with highly advanced fibrosis and numerous foamy, phagocytic macrophages in the adjacent nontumoral area. Multiple metastatic nodules were found in the lungs of one of the animals, and lymphoid clusters were found in all CDAA-HF-T(-) group mice. In the Ingenuity Pathways Analysis of RNA expression data, the CDAA-HF-T(-) feeding revealed common signal changes in nontumoral and tumoral liver tissues, including increased IL-8 and RhoGTPases signaling and decreased lipid metabolism. Meanwhile, macrophage inflammatory protein 2 (MIP-2) expression levels were upregulated in nontumoral liver tissue from the end of Week 13 of CDAA-HF-T(-) feeding to the end of Week 63. On the other hand, MIP-2 was expressed on macrophages in non-tumor areas and hepatocytes in tumor areas. Therefore, the CDAA-HF-T(-) mouse model is useful for assessing NASH and NASH-associated hepatocarcinogenesis, and IL-8 signaling plays important roles in NASH-associated carcinogenesis and cirrhosis, but it may also play different roles in nontumoral liver tissue and tumorigenesis.

Anti-PCSK9 Treatment Attenuates Liver Fibrosis via Inhibiting Hypoxia-Induced Autophagy in Hepatocytes

Hypoxia and its induced autophagy are involved in the initiation and progression of liver fibrosis. Proprotein convertase subtilisin/kexin type 9 (PCSK9) has been recognized as a potential regulator of autophagy. Our previously reported study found that PCSK9 expression increased in liver fibrosis and that anti-PCSK9 treatment alleviated liver injury. This study aimed to investigate the mechanism of anti-PCSK9 treatment on liver fibrosis by inhibiting hypoxia-induced autophagy. Carbon tetrachloride-induced mouse liver fibrosis and mouse hepatocyte line AML12, cultured under the hypoxic condition, were established to undergo PCSK9 inhibition. The degree of liver fibrosis was shown with histological staining. The reactive oxygen species (ROS) generation was detected by flow cytometry. The expression of PCSK9, hypoxia-inducible factor-1α (HIF-1α), and autophagy-related proteins was examined using Western blot. The autophagic flux was assessed under immunofluorescence and transmission electron microscope. The mouse liver samples were investigated via RNA-sequencing to explore the underlying signaling pathway. The results showed that PCSK9 expression was upregulated with the development of liver fibrosis, which was accompanied by enhanced autophagy. In vitro data verified that PCSK9 increased via hypoxia and inflammation, accompanied by the hypoxia-induced autophagy increased. Then, the validation was acquired of the bidirectional interaction of hypoxia-ROS and PCSK9. The hypoxia reversal attenuated PCSK9 expression and autophagy. Additionally, anti-PCSK9 treatment alleviated liver inflammation and fibrosis, reducing hypoxia and autophagy in vivo. In mechanism, the AMPK/mTOR/ULK1 signaling pathway was identified as a target for anti-PCSK9 therapy. In conclusion, anti-PCSK9 treatment could alleviate liver inflammation and fibrosis by regulating AMPK/mTOR/ULK1 signaling pathway to reduce hypoxia-induced autophagy in hepatocytes.

Ube4A maintains metabolic homeostasis and facilitates insulin signaling in vivo

ObjectiveDefining the regulators of cell metabolism and signaling is essential to design new therapeutic strategies in obesity and NAFLD/NASH. E3 ubiquitin ligases control diverse cellular functions by ubiquitination-mediated regulation of protein targets, and thus their functional aberration is associated with many diseases. The E3 ligase Ube4A has been implicated in human obesity, inflammation, and cancer. However, its in vivo function is unknown, and no animal models are available to study this novel protein. MethodsA whole-body Ube4A knockout (UKO) mouse model was generated, and various metabolic parameters were compared in chow- and high fat diet (HFD)-fed WT and UKO mice, and in their liver, adipose tissue, and serum. Lipidomics and RNA-Seq studies were performed in the liver samples of HFD-fed WT and UKO mice. Proteomic studies were conducted to identify Ube4A's targets in metabolism. Furthermore, a mechanism by which Ube4A regulates metabolism was identified. ResultsAlthough the body weight and composition of young, chow-fed WT and UKO mice are similar, the knockouts exhibit mild hyperinsulinemia and insulin resistance. HFD feeding substantially augments obesity, hyperinsulinemia, and insulin resistance in both sexes of UKO mice. HFD-fed white and brown adipose tissue depots of UKO mice have increased insulin resistance and inflammation and reduced energy metabolism. Moreover, Ube4A deletion exacerbates hepatic steatosis, inflammation, and liver injury in HFD-fed mice with increased lipid uptake and lipogenesis in hepatocytes. Acute insulin treatment resulted in impaired activation of the insulin effector protein kinase Akt in liver and adipose tissue of chow-fed UKO mice. We identified the Akt activator protein APPL1 as a Ube4A interactor. The K63-linked ubiquitination (K63-Ub) of Akt and APPL1, known to facilitate insulin-induced Akt activation, is impaired in UKO mice. Furthermore, Ube4A K63-ubiquitinates Akt in vitro. ConclusionUbe4A is a novel regulator of obesity, insulin resistance, adipose tissue dysfunction and NAFLD, and preventing its downregulation may ameliorate these diseases.

Dihydrosphingolipids are associated with steatosis and increased fibrosis damage in non-alcoholic fatty liver disease

Dihydrosphingolipids are lipids biosynthetically related to ceramides. An increase in ceramides is associated with enhanced fat storage in the liver, and inhibition of their synthesis is reported to prevent the appearance of steatosis in animal models. However, the precise association of dihydrosphingolipids with non-alcoholic fatty liver disease (NAFLD) is yet to be established. We employed a diet induced NAFLD mouse model to study the association between this class of compounds and disease progression. Mice fed a high-fat diet were sacrificed at 22, 30 and 40 weeks to reproduce the full spectrum of histological damage found in human disease, steatosis (NAFL) and steatohepatitis (NASH) with and without significant fibrosis. Blood and liver tissue samples were obtained from patients whose NAFLD severity was assessed histologically. To demonstrate the effect of dihydroceramides over NAFLD progression we treated mice with fenretinide an inhibitor of dihydroceramide desaturase-1 (DEGS1). Lipidomic analyses were performed using liquid chromatography-tandem mass spectrometry. Triglycerides, cholesteryl esters and dihydrosphingolipids were increased in the liver of model mice in association with the degree of steatosis and fibrosis. Dihydroceramides increased with the histological severity observed in liver samples of mice (0.024 ± 0.003 nmol/mg vs 0.049 ± 0.005 nmol/mg, non-NAFLD vs NASH-fibrosis, p < 0.0001) and patients (0.105 ± 0.011 nmol/mg vs 0.165 ± 0.021 nmol/mg, p = 0.0221). Inhibition of DEGS1 induce a four-fold increase in dihydroceramides improving steatosis but increasing the inflammatory activity and fibrosis. In conclusion, the degree of histological damage in NAFLD correlate with dihydroceramide and dihydrosphingolipid accumulation. Lay summaryAccumulation of triglyceride and cholesteryl ester lipids is the hallmark of non-alcoholic fatty liver disease. Using lipidomics, we examined the role of dihydrosphingolipids in NAFLD progression. Our results demonstrate that de novo dihydrosphingolipid synthesis is an early event in NAFLD and the concentrations of these lipids are correlated with histological severity in both mouse and human disease.

Multi-omics analysis identifies drivers of protein phosphorylation

BackgroundPhosphorylation of proteins is a key step in the regulation of many cellular processes including activation of enzymes and signaling cascades. The abundance of a phosphorylated peptide (phosphopeptide) is determined by the abundance of its parent protein and the proportion of target sites that are phosphorylated.ResultsWe quantified phosphopeptides, proteins, and transcripts in heart, liver, and kidney tissue samples of mice from 58 strains of the Collaborative Cross strain panel. We mapped ~700 phosphorylation quantitative trait loci (phQTL) across the three tissues and applied genetic mediation analysis to identify causal drivers of phosphorylation. We identified kinases, phosphatases, cytokines, and other factors, including both known and potentially novel interactions between target proteins and genes that regulate site-specific phosphorylation. Our analysis highlights multiple targets of pyruvate dehydrogenase kinase 1 (PDK1), a regulator of mitochondrial function that shows reduced activity in the NZO/HILtJ mouse, a polygenic model of obesity and type 2 diabetes.ConclusionsTogether, this integrative multi-omics analysis in genetically diverse CC strains provides a powerful tool to identify regulators of protein phosphorylation. The data generated in this study provides a resource for further exploration.