Reduction of alcohol-induced mitochondrial damage with ginsenoside Rg1 studied by atomic force microscopy.

The quantification of mitochondrial morphology and mechanical properties is useful for the diagnosis and treatment of mitochondrial and alcoholic liver disease. In this study, the effects of ginsenoside Rg1 (G-Rg1) on the morphology and mechanical properties of mitochondria that had suffered alcohol-induced damage were investigated under near-physiological conditions. Additionally, the morphological and mechanical properties of mitochondria were quantified through atomic force microscopy. Atomic force microscopy revealed that alcohol-induced significant morphological changes in mitochondria. Compared with that of the mitochondria of normal hepatocytes, the average surface area of the damaged mitochondria was found to have increased significantly under the influence of alcohol. Furthermore, the mitochondrial area tended to be normal under the action of G-Rg1, whilst other parameters (length, width and perimeter) were significantly different from those of the mitochondria with the alcohol-induced damage. Simultaneously, alcohol significantly reduced the adhesion and elastic modulus of mitochondria, whilst the adhesion and elastic modulus of mitochondria in the G-Rg1 treatment group were closer to the values of normal mitochondria. This study overall showed that G-Rg1 could effectively alleviate the swelling and anomalous mechanical properties of mitochondria induced by alcohol.

Detection and classification of hepatocytes and hepatoma cells using atomic force microscopy and machine learning algorithms.

Hepatocellular carcinoma is a high-risk malignant tumor. Hepatoma cells are transformed from normal cells and have unique surface nanofeatures in addition to the characteristics of the original cells. In this paper, atomic force microscopy was used to extract the three-dimensional morphology and mechanical information of HL-7702 human hepatocytes and SMMC-7721 and HepG2 hepatoma cells in culture, such as the elastic modulus and viscoelasticity. The characteristics of different cells were compared and analyzed. Finally, the cell morphology and mechanics information were used for training machine learning algorithms. With the trained model, the detection of cells was realized. The classification accuracy was as high as 94.54%, and the area under the receiver operating characteristic (ROC) curve (AUC) was 0.99. Thus, hepatocytes and hepatoma cells were accurately identified and assessed. We also compared the classification effects of other machine learning algorithms, such as support vector machine and logistic regression. Our method extracts cellular nanofeatures directly from the surface of cells of unknown type for cell classification. Compared with microscope image-based analysis and other methods, this approach can avoid the misjudgment that may occur when different doctors have different levels of experience. Thus, the proposed method provides an objective basis for the early diagnosis of hepatocellular carcinoma. RESEARCH HIGHLIGHTS: The 3D appearance and mechanical characteristics of hepatocellular carcinoma cells are very similar to those of hepatocytes. Application of atomic force microscopy with machine learning algorithm. Collect the data set of nano-characteristic parameters of the cell. The machine learning algorithms is trained by data set, and its classification effect is better than that of a single nano-parameter.

Attenuation of alcohol-induced hepatocyte damage by ginsenoside Rg1 evaluated using atomic force microscopy.

Alcoholic liver disease is an important cause of death worldwide. Hepatocyte apoptosis is commonly observed in alcoholic liver disease. In this study, we investigated the effect of ginsenoside Rg1 (G-Rg1), an organic component of ginseng, on the alcohol-induced morphological and biophysical properties of hepatocytes. Human hepatocytes (HL-7702) were treated in vitro with alcohol and G-Rg1. The cell morphology was observed using scanning electron microscopy. Cell height, roughness, adhesion, and elastic modulus were detected using atomic force microscopy. We found that alcohol significantly induced hepatocyte apoptosis, whereas G-Rg1 attenuated the alcohol-induced hepatocyte damage. Scanning electron microscopy revealed that alcohol-induced significant morphological changes in hepatocytes, including decreased cell contraction, roundness, and pseudopods, whereas G-Rg1 inhibited these negative changes. Atomic force microscopy revealed that alcohol increased the cell height and decreased the adhesion and elastic modulus of hepatocytes. Following treatment with G-Rg1, the cell height, adhesion, and elastic modulus of alcohol-injured hepatocytes were all similar to those of normal cells. Thus, G-Rg1 can attenuate the alcohol-induced damage to hepatocytes by modulating the morphology and biomechanics of the cells. RESEARCH HIGHLIGHTS: In this study, the morphological characteristics of hepatocytes were observed using SEM. The changes in hepatocyte three-dimensional images and biomechanical action caused by alcohol and G-Rg1 were examined at the nanoscale using AFM under near-physiological conditions. Alcohol-induced hepatocytes showed abnormal morphology and biophysical properties. G-Rg1 attenuated the alcohol-induced damage to hepatocytes by modulating the morphology and biomechanics of the cells.

Inhibiting membrane rupture with NINJ1 antibodies limits tissue injury.

Plasma membrane rupture (PMR) in dying cells undergoing pyroptosis or apoptosis requires the cell-surface protein NINJ11. PMR releases pro-inflammatory cytoplasmic molecules, collectively called damage-associated molecular patterns (DAMPs), that activate immune cells. Therefore, inhibiting NINJ1 and PMR may limit the inflammation that is associated with excessive cell death. Here we describe an anti-NINJ1 monoclonal antibody that specifically targets mouse NINJ1 and blocks oligomerization of NINJ1, preventing PMR. Electron microscopy studies showed that this antibody prevents NINJ1 from forming oligomeric filaments. In mice, inhibition of NINJ1 or Ninj1 deficiency ameliorated hepatocellular PMR induced with TNF plus D-galactosamine, concanavalin A, Jo2 anti-Fas agonist antibody or ischaemia-reperfusion injury. Accordingly, serum levels of lactate dehydrogenase, the liver enzymes alanine aminotransaminase and aspartate aminotransferase, and the DAMPs interleukin 18 and HMGB1 were reduced. Moreover, in the liver ischaemia-reperfusion injury model, there was an attendant reduction in neutrophil infiltration. These data indicate that NINJ1 mediates PMR and inflammation in diseases driven by aberrant hepatocellular death.

Ultrastructural evidence of four types of lipofuscins in the melanomacrophagic centers in hepatocytes of zebrafish (Denio rerio).

Melanomacrophagic centers (MMCs) were studied in the hepatocytes of zebrafish using transmission electron microscope (TEM). The MMCs with irregular or amoeboid nucleus were located in the hepatocytes adjacent to the bile canaliculi. Several engulfed structures were present in the cytoplasm of MMCs. The most frequent observation was the presence of mitochondria, ranging in size from small to giant, with distorted shape and inconspicuous cristae. Occasionally the fragments of erythrocytes were found. The rough endoplasmic reticulum (rER) showed whirling around the mitochondria and lipid droplets, forming membrane-like structures. The damaged mitochondria were invaded by the lysosomes, and this was covered by a membrane led to the formation of lipofuscin. Four different types of lipofuscins were observed; namely, (1) granular with/without vacuoles of high electron-density, (2) homogenous surrounded by indistinct limiting membrane, (3) lamellated structures similar to inner matrix and cristae of mitochondria, and, (4) compound structure made by the combinations of first 3 types, (granular and homogenous, granular and lamellated, homogenous and lamellated). The present evidence suggests that MMCs in the hepatocytes of zebrafish perform continuous functions of removal of the damaged cellular organelles. The lipofuscin formation work in coordination with the cellular players of immune system and remove pathogens and maintain the internal homeostasis of cells.

Protective Effect of Curcumin against Doxazosin- and Carvedilol-Induced Oxidative Stress in HepG2 Cells.

Doxazosin and carvedilol have been evaluated as an alternative treatment against chronic liver lesions and for their possible role during the regeneration of damage caused by liver fibrosis in a hamster model. However, these drugs have been reported to induce morphological changes in hepatocytes, affecting the recovery of liver parenchyma. The effects of these α/𝛽 adrenoblockers on the viability of hepatocytes are unknown. Herein, we demonstrate the protective effect of curcumin against the possible side effects of doxazosin and carvedilol, drugs with proven antifibrotic activity. After pretreatment with 1 µM curcumin for 1 h, HepG2 cells were exposed to 0.1-25 µM doxazosin or carvedilol for 24, 48, and 72 h. Cell viability was assessed using the MTT assay and SYTOX green staining. Morphological changes were detected using the hematoxylin and eosin (H&E) staining and scanning electron microscopy (SEM). An expression of apoptotic and oxidative stress markers was analyzed using reverse transcription-quantitative PCR (RT-qPCR). The results indicate that doxazosin decreases cell viability in a time- and dose-dependent manner, whereas carvedilol increases cell proliferation; however, curcumin increases or maintains cell viability. SEM and H&E staining provided evidence that doxazosin and carvedilol induced morphological changes in HepG2 cells, and curcumin protected against these effects, maintaining the morphology in 90% of treated cells. Furthermore, curcumin positively regulated the expression of Nrf2, HO-1, and SOD1 mRNAs in cells treated with 0.1 and 0.5 µM doxazosin. Moreover, the Bcl-2/Bax ratio was higher in cells that were treated with curcumin before doxazosin or carvedilol. The present study demonstrates that curcumin controls doxazosin- and carvedilol-induced cytotoxicity and morphological changes in HepG2 cells possibly by overexpression of Nrf2.

Curcumin mitigates aflatoxin B1-induced liver injury via regulating the NLRP3 inflammasome and Nrf2 signaling pathway.

Aflatoxins are produced as secondary metabolites by the toxigenic Aspergillus fungi. Among the aflatoxins, aflatoxin B1 (AFB1) is a common contaminant of global concern in human and animal food products. Prolonged exposure to AFB1 may provoke hepatocyte pyroptosis and oxidative stress, which leads to liver damage. Dietary polyphenols could protect the liver from a wide range of toxins. Curcumin, a polyphenolic substance derived from turmeric, is rich in pharmacological activity. The aim of this study was to systematically investigate the protective effects of curcumin against AFB1-induced liver injury in mice and to explore the possible molecular mechanisms. BALB/c mice received oral gavage of AFB1 (0.75 mg/kg) and curcumin (100 or 200 mg/kg) for 30 days. Our data demonstrated that curcumin attenuated AFB1-induced weight loss in mice and rescued liver injury by mitigating the alterations in pathology and liver function with AFB1 exposure. Curcumin reduced the accumulation of AFB1-DNA adducts in the liver and alleviated hepatotoxicity by inhibiting AFB1-induced oxidative stress and potentiating glutathione S-transferase (GST)-mediated phase II detoxification. In addition, curcumin significantly reduced the characteristic indices of AFB1-induced pyroptosis, such as the expression of mRNAs for genes related to NOD-like receptor protein 3 (NLRP3) inflammasome assembly and activation, the expression of key proteins (NLRP3, Caspase-1 and GSDMD). The release of interleukin-1ß (IL-1ß) and interleukin-18 (IL-18) in the serum detected by ELISA was also significantly decreased. Notably, administration of curcumin upregulated the expression of nuclear factor erythroid 2-related factor 2 (Nrf2) and its related downstream antioxidant molecules (SOD, CAT, HO-1, NQO1) and phase II detoxification enzyme-related molecules (GST, GSH, GSS, GCLC, GCLM) in the presence of AFB1 exposure. To summarize, our results indicated that curcumin could modulate the NLRP3 inflammasome and Nrf2 signaling pathways to attenuate AFB1-induced liver pyroptotic damage and oxidative stress.

Lysophosphatidylcholine disrupts cell adhesion and induces anoikis in hepatocytes.

Lysophosphatidylcholine (LPC), the active metabolite of palmitate, triggers hepatocyte death by activating endoplasmic reticulum stress and JNK signalling-mediated lipoapoptosis. However, LPC-induced cytotoxicity in hepatocytes is not well understood. Here, we found for the first time that LPC-induced cell rounding occurred prior to apoptosis. LPC-induced rounding of cells reduced both cell-extracellular matrix (ECM) adhesion and cell-cell junctions, which promoted detachment-induced apoptosis (defined as anoikis) in hepatocytes. Further study revealed that LPC altered cellular morphology and cell adhesion by inhibiting integrin and cadherin signalling-mediated microfilament polymerization. We also found that ECM supplementation and microfilament cytoskeletal stabilization inhibited LPC-induced hepatocyte death by attenuating anoikis. Our data indicate a novel cytotoxic process and signalling pathway induced by LPC.

Dual role of cadmium in rat liver: Inducing liver injury and inhibiting the progression of early liver cancer.

The heavy metal cadmium (Cd) can induce damage in liver and liver cancer cells; however, the mechanism underlying its toxicity needs to be further verified in vivo. We daily administered CdCl2 to adult male rats at different dosages via gavage for 12 weeks and established rat liver injury model and liver cancer model to study the dual role of Cd in rat liver. Increased exposure to Cd resulted in abnormal liver function indicators, pathological degeneration, rat liver cell necrosis, and proliferation of collagen fibres. Using immunohistochemistry, we found that the area of GST-P-positive precancerous liver lesions decreased in a dose-dependent manner. Real-time quantitative polymerase chain reaction, western blot, immunohistochemistry, and transmission electron microscopy revealed that Cd induced mitophagy, as well as mitophagy blockade, as evidenced by the downregulation of TOMM20 and upregulation of LC3II and P62 with increasing Cd dose. Next, the expression of PINK1/Parkin, a classic signalling pathway protein that regulates mitophagy, was examined. Cd was found to promote PINK1/Parkin expression, which was proportional to the Cd dose. In conclusion, Cd activates PINK1/Parkin-mediated mitophagy in a dose-dependent manner. Mitophagy blockade likely aggravates Cd toxicity, leading to the dual role of inducing liver injury and inhibiting the progression of early liver cancer.

Factor quinolinone inhibitors alter cell morphology and motility by destabilizing interphase microtubules.

Factor quinolinone inhibitors are promising anti-cancer compounds, initially characterized as specific inhibitors of the oncogenic transcription factor LSF (TFCP2). These compounds exert anti-proliferative activity at least in part by disrupting mitotic spindles. Herein, we report additional interphase consequences of the initial lead compound, FQI1, in two telomerase immortalized cell lines. Within minutes of FQI1 addition, the microtubule network is disrupted, resulting in a substantial, although not complete, depletion of microtubules as evidenced both by microtubule sedimentation assays and microscopy. Surprisingly, this microtubule breakdown is quickly followed by an increase in tubulin acetylation in the remaining microtubules. The sudden breakdown and partial depolymerization of the microtubule network precedes FQI1-induced morphological changes. These involve rapid reduction of cell spreading of interphase fetal hepatocytes and increase in circularity of retinal pigment epithelial cells. Microtubule depolymerization gives rise to FH-B cell compaction, as pretreatment with taxol prevents this morphological change. Finally, FQI1 decreases the rate and range of locomotion of interphase cells, supporting an impact of FQI1-induced microtubule breakdown on cell motility. Taken together, our results show that FQI1 interferes with microtubule-associated functions in interphase, specifically cell morphology and motility.

Role of mitochondrial dysfunction and PINK1/Parkin-mediated mitophagy in Cd-induced hepatic lipid accumulation in chicken embryos.

The present study was performed to investigate the effects of Cd exposure on lipid metabolism and mitochondrial dysfunction and to explore the role of mitophagy in Cd-induced dysregulation of lipid metabolism in chicken embryo liver tissues and hepatocytes. To this end, seven-day-old chicken embryos were exposed to different concentrations of Cd for 7 days, and primary chicken embryo hepatocytes were treated with Cd at four different concentrations for 6 h. Furthermore, the mitophagy inhibitor cyclosporine A (CsA) was used to investigate the role of mitophagy in Cd-induced disruption of lipid metabolism. Lipid accumulation, the expression levels of genes involved in lipid metabolism, mitochondrial dysfunction, and mitophagy were measured. The results demonstrated that Cd exposure increases hepatic triglyceride (TG) accumulation and the expression levels of lipogenic genes while decreasing those of lipolytic genes. Furthermore, Cd exposure was observed to alter mitochondrial morphology in terms of reduced size, excessive mitochondrial damage, and the formation of mitophagosomes. The co-localization of lysosome-associated membrane glycoprotein 2 and LC3 puncta was significantly increased in primary chicken embryo hepatocytes after Cd exposure. Moreover, Cd exposure increased LC3, PINK1, and Parkin protein expression levels. CsA effectively alleviated Cd-induced mitochondrial dysfunction, blocked mitochondrial membrane potential collapse, and suppressed PINK1/Parkin-mediated mitophagy. Furthermore, CsA treatment reversed the Cd-induced TG accumulation in liver tissues but further increased it in hepatocytes. Taken together, our findings demonstrate (for the first time) the importance of mitochondrial dysfunction and mitophagy via the PINK1/Parkin pathway in Cd-induced disruption of lipid metabolism.

Ultrastructural Profile Combined with Immunohistochemistry of a Hepatic Progenitor Cell Line in Pediatric Autoimmune Hepatitis: New Insights into the Morphological Pattern of the Disease.

Considering that the heterogenic population of a hepatic progenitor cell line (HPCL) can play a vital role in autoimmune hepatitis (AIH), we decided to conduct pioneering retrospective evaluation of these cells in pediatric AIH by means of transmission electron microscopy (TEM). The aim of the study was to assess the ultrastructure of the HPCL in children with untreated AIH. Ultrastructural analysis of the HPCL population, preceded by immunohistochemical staining for cytokeratin 7 (CK7), was performed using pretreatment liver biopsies from 23 children with clinicopathologically diagnosed AIH. Immunohistochemical assessment for CK7 allowed detection of proliferating immature epithelial cells differentiating towards periportal and intralobular intermediate hepatocytes without marked formation of ductular reactions in AIH children. Using TEM, we distinguished three morphological types of HPCs: I-the most undifferentiated progenitor cells; III-intermediate hepatocyte-like cells; II-intermediate bile duct cells. Most frequent were the cells differentiating towards hepatocytes, most rare-those differentiating towards cholangiocytes. The results indicate that an HPCL may be an important source of hepatocyte regeneration. Ultrastructural analyses of the HPCL population, combined with immunohistochemistry for CK7, might be a useful tool to evaluate liver cell regeneration, including fibrogenesis, and may help better understand the morphological pattern of the disease, in pediatric AIH. Frequent appearance of an HPCL in the vicinity of fibrotic foci, often accompanied by hyperactive Kupffer cells and transitional hepatic stellate cells, may indicate their significant involvement in liver fibrogenesis.

Hepatic and Extrahepatic Sources and Manifestations in Endoplasmic Reticulum Storage Diseases.

Alpha-1-antitrypsin (AAT) and fibrinogen are secretory acute phase reactant proteins. Circulating AAT and fibrinogen are synthesized exclusively in the liver. Mutations in the encoding genes result in conformational abnormalities of the two molecules that aggregate within the rough endoplasmic reticulum (RER) instead of being regularly exported. That results in AAT-deficiency (AATD) and in hereditary hypofibrinogenemia with hepatic storage (HHHS). The association of plasma deficiency and liver storage identifies a new group of pathologies: endoplasmic reticulum storage disease (ERSD).

Ground-glass hepatocellular inclusions are associated with polypharmacy.

Ground-glass (GG) hepatocytes are classically associated with chronic hepatitis B (HBV) infection, storage disorders, or cyanamide therapy. In a subset of cases, an exact etiology cannot be identified. In this study, we sought to characterize the clinical, histological, and ultrastructural findings associated with HBV-negative GG hepatocytes. Our institutional laboratory information system was searched from 2000 to 2019 for all cases of ground-glass hepatocytes. Ten liver biopsies with GG hepatocellular inclusions and negative HBV serology, no known history of storage disorders, or cyanamide therapy were reviewed. Half of the patients had history of organ transplantation and/or malignancy. These patients took on average 8.1 medications (range: 3-14) with the most common medications being immunosuppressive and health supplements. Histologically, GG hepatocytes show either peri-portal or centrizonal distribution. The inclusions are PAS-positive and diastase sensitive. Electron microscopy showed intracytoplasmic granular inclusions with low electron density, consistent with unstructured glycogen. In summary, GG hepatocytes are a rare finding in liver biopsies, but are more common in patients with hepatitis B. They can also be seen in HBV-negative patients who have polypharmacy. In these cases, they are the result of unstructured glycogen accumulation putatively due to altered cell metabolism.

Mettl14-Mediated m6A Modification Facilitates Liver Regeneration by Maintaining Endoplasmic Reticulum Homeostasis.

BACKGROUND & AIMS: N6-methyladenosine (m6A), the most prevalent and dynamic posttranscriptional methylation modification of mammalian mRNA, is involved in various biological processes, but its role in liver regeneration has not been characterized. METHODS: We first conducted transcriptome-wide m6A mRNA sequencing and characterized the expression pattern of m6A in regenerating mouse liver. Next, we generated hepatocyte-specific Mettl3- or Mettl14-deficient mice and investigated their role in liver regeneration. A series of biochemical experiments in vitro and in vivo was further performed to investigate potential mechanisms. RESULTS: We identified an overwhelming proportion of m6A-modified genes with initially up-regulated and subsequently down-regulated m6A levels as liver regeneration progressed. Loss of Mettl14 but not of Mettl3 resulted in markedly disrupted liver regeneration, and Mettl14-ablated hepatocytes were arrested in the G1 phase of the cell cycle. Most strikingly, the Mettl14-ablated regenerating liver exhibited extensive parenchymal necrosis. mRNA transcripts, such as Hsp90b1, Erp29, Stt3a, P4hb, and Lman1, encoding proteins involved in polypeptide processing and the endoplasmic reticulum (ER) stress response, were m6A-hypomethylated, and their mRNA and protein levels were subsequently decreased, resulting in unresolved ER stress, hepatocyte death, and inhibited proliferation. CONCLUSIONS: We demonstrate the essential role of Mettl14 in facilitating liver regeneration by modulating polypeptide-processing proteins in the ER in an m6A-dependent manner.

3D bioprinting of hepatocytes: core-shell structured co-cultures with fibroblasts for enhanced functionality.

With the aim of understanding and recapitulating cellular interactions of hepatocytes in their physiological microenvironment and to generate an artificial 3D in vitro model, a co-culture system using 3D extrusion bioprinting was developed. A bioink based on alginate and methylcellulose (algMC) was first shown to be suitable for bioprinting of hepatocytes; the addition of Matrigel to algMC enhanced proliferation and morphology of them in monophasic scaffolds. Towards a more complex system that allows studying cellular interactions, we applied core-shell bioprinting to establish tailored 3D co-culture models for hepatocytes. The bioinks were specifically functionalized with natural matrix components (based on human plasma, fibrin or Matrigel) and used to co-print fibroblasts and hepatocytes in a spatially defined, coaxial manner. Fibroblasts acted as supportive cells for co-cultured hepatocytes, stimulating the expression of certain biomarkers of hepatocytes like albumin. Furthermore, matrix functionalization positively influenced both cell types in their respective compartments by enhancing their adhesion, viability, proliferation and function. In conclusion, we established a functional co-culture model with independently tunable compartments for different cell types via core-shell bioprinting. This provides the basis for more complex in vitro models allowing co-cultivation of hepatocytes with other liver-specific cell types to closely resemble the liver microenvironment.

Aging-Related Changes in the Ultrastructure of Hepatocytes and Cardiomyocytes of Elderly Mice Are Enhanced in ApoE-Deficient Animals.

Biological aging is associated with various morphological and functional changes, yet the mechanisms of these phenomena remain unclear in many tissues and organs. Hyperlipidemia is among the factors putatively involved in the aging of the liver and heart. Here, we analyzed morphological, ultrastructural, and biochemical features in adult (7-month-old) and elderly (17-month-old) mice, and then compared age-related features between wild type (C57Bl/6 strain) and ApoE-deficient (transgenic ApoE-/-) animals. Increased numbers of damaged mitochondria, lysosomes, and lipid depositions were observed in the hepatocytes of elderly animals. Importantly, these aging-related changes were significantly stronger in hepatocytes from ApoE-deficient animals. An increased number of damaged mitochondria was observed in the cardiomyocytes of elderly animals. However, the difference between wild type and ApoE-deficient mice was expressed in the larger size of mitochondria detected in the transgenic animals. Moreover, a few aging-related differences were noted between wild type and ApoE-deficient mice at the level of plasma biochemical markers. Levels of cholesterol and HDL increased in the plasma of elderly ApoE-/- mice and were markedly higher than in the plasma of elderly wild type animals. On the other hand, the activity of alanine transaminase (ALT) decreased in the plasma of elderly ApoE-/- mice and was markedly lower than in the plasma of elderly wild type animals.

Mesenchymal stromal cells protect hepatocytes from lipotoxicity through alleviation of endoplasmic reticulum stress by restoring SERCA activity.

The aim of this study was to investigate how mesenchymal stromal cells (MSCs) modulate metabolic balance and attenuate hepatic lipotoxicity in the context of non-alcoholic fatty liver disease (NAFLD). In vivo, male SD rats were fed with high-fat diet (HFD) to develop NAFLD; then, they were treated twice by intravenous injections of rat bone marrow MSCs. In vitro, HepG2 cells were cocultured with MSCs by transwell and exposed to palmitic acid (PA) for 24 hours. The endoplasmic reticulum (ER) stressor thapsigargin and sarco/ER Ca2+ -ATPase (SERCA2)-specific siRNA were used to explore the regulation of ER stress by MSCs. We found that MSC administration improved hepatic steatosis, restored systemic hepatic lipid and glucose homeostasis, and inhibited hepatic ER stress in HFD-fed rats. In hepatocytes, MSCs effectively alleviated the cellular lipotoxicity. Particularly, MSCs remarkably ameliorated the ER stress and intracellular calcium homeostasis induced by either PA or thapsigargin in HepG2 cells. Additionally, long-term HFD or PA stimulation would activate pyroptosis in hepatocytes, which may contribute to the cell death and liver dysfunction during the process of NAFLD, and MSC treatment effectively ameliorates these deleterious effects. SERCA2 silencing obviously abolished the ability of MSCs against the PA-induced lipotoxicity. Conclusively, our study demonstrated that MSCs were able to ameliorate liver lipotoxicity and metabolic disturbance in the context of NAFLD, in which the regulation of ER stress and the calcium homeostasis via SERCA has played a key role.

Lysosomal SLC46A3 modulates hepatic cytosolic copper homeostasis.

The environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) causes hepatic toxicity associated with prominent lipid accumulation in humans. Here, the authors report that the lysosomal copper transporter SLC46A3 is induced by TCDD and underlies the hepatic lipid accumulation in mice, potentially via effects on mitochondrial function. SLC46A3 was localized to the lysosome where it modulated intracellular copper levels. Forced expression of hepatic SLC46A3 resulted in decreased mitochondrial membrane potential and abnormal mitochondria morphology consistent with lower copper levels. SLC46A3 expression increased hepatic lipid accumulation similar to the known effects of TCDD exposure in mice and humans. The TCDD-induced hepatic triglyceride accumulation was significantly decreased in Slc46a3-/- mice and was more pronounced when these mice were fed a high-fat diet, as compared to wild-type mice. These data are consistent with a model where lysosomal SLC46A3 induction by TCDD leads to cytosolic copper deficiency resulting in mitochondrial dysfunction leading to lower lipid catabolism, thus linking copper status to mitochondrial function, lipid metabolism and TCDD-induced liver toxicity.

p62/SQSTM1-droplet serves as a platform for autophagosome formation and anti-oxidative stress response.

Autophagy contributes to the selective degradation of liquid droplets, including the P-Granule, Ape1-complex and p62/SQSTM1-body, although the molecular mechanisms and physiological relevance of selective degradation remain unclear. In this report, we describe the properties of endogenous p62-bodies, the effect of autophagosome biogenesis on these bodies, and the in vivo significance of their turnover. p62-bodies are low-liquidity gels containing ubiquitin and core autophagy-related proteins. Multiple autophagosomes form on the p62-gels, and the interaction of autophagosome-localizing Atg8-proteins with p62 directs autophagosome formation toward the p62-gel. Keap1 also reversibly translocates to the p62-gels in a p62-binding dependent fashion to activate the transcription factor Nrf2. Mice deficient for Atg8-interaction-dependent selective autophagy show that impaired turnover of p62-gels leads to Nrf2 hyperactivation in vivo. These results indicate that p62-gels are not simple substrates for autophagy but serve as platforms for both autophagosome formation and anti-oxidative stress.