Mouse Hepatocyte Cell Line Research Articles

FK506-Binding Protein 51 (FKBP51) Suppresses PPARα Activity and Shifts the Insulin Receptor Alternative Splicing Driving Hepatic Insulin Resistance

FK506-binding protein-51 (FKBP51) is a molecular chaperone protein that has great potential to serve as a treatment target for non-alcoholic fatty liver disease (NAFLD) and type II diabetes (T2DM). FKBP51 has been known to regulate nuclear receptors such as glucocorticoid receptor (GR) and peroxisome proliferator-activated receptor γ (PPARγ). However, the connections between FKBP51 and other PPAR family members, such as peroxisome proliferator-activated receptor α (PPARα), are yet to be identified. FKBP51 can also inhibit protein kinase B (AKT) activation, which may impact the insulin receptor signaling pathway. It has been reported that FKBP51 knockout mice presented less adiposity, reduced liver steatosis, and increased insulin sensitivity and insulin clearance, indicating that FKBP51 plays a role in insulin resistance and fatty liver development. However, how FKBP51 regulates insulin resistance, especially in the liver, still needs to be unveiled. It has been shown that insulin receptor (IR) alternative splicing is associated with insulin sensitivity and that PPARα activation is critical in reducing insulin resistance. Thus, we hypothesize that FKBP51 can regulate hepatic insulin resistance through insulin receptor alternative splicing and PPARα suppression. To test this hypothesis, we developed an FKBP51 knockout mouse hepatocyte cell line, 51KO AML12, and treated the cells with insulin for 1hr. We measured RNA and protein expressions in the cells through RT-PCR and western blotting and found that 51KO AML12 had increased phospho-AKT Ser473 (pAKT S473) and IR isoform B (IR-B), indicating increased insulin signaling activity. We also found increased IR isoform A (IR-A) and IR-B expression in 51KO AML12. In addition, we found an increased ratio of IR-B: IR-A in 51KO AML12 treated with insulin. These data indicate a great potential for FKBP51 to be a novel treatment target for hepatic insulin resistance and T2DM. This research is supported by the National Institute of Health R01DK121797 (T.D.H.), R01DA058933 (T.D.H.), and Start-Up funds from the University of Kentucky (T.D.H.). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Identification and validation of cuproptosis-related molecular clusters in non-alcoholic fatty liver disease.

Non-alcoholic fatty liver disease (NAFLD) is a major chronic liver disease worldwide. Cuproptosis has recently been reported as a form of cell death that appears to drive the progression of a variety of diseases. This study aimed to explore cuproptosis-related molecular clusters and construct a prediction model. The gene expression profiles were obtained from the Gene Expression Omnibus (GEO) database. The associations between molecular clusters of cuproptosis-related genes and immune cell infiltration were investigated using 50 NAFLD samples. Furthermore, cluster-specific differentially expressed genes were identified by the WGCNA algorithm. External datasets were used to verify and screen feature genes, and nomograms, calibration curves and decision curve analysis (DCA) were performed to verify the performance of the prediction model. Finally, a NAFLD-diet mouse model was constructed to further verify the predictive analysis, thus providing new insights into the prediction of NAFLD clusters and risks. The role of cuproptosis in the development of non-alcoholic fatty liver disease and immune cell infiltration was explored. Non-alcoholic fatty liver disease was divided into two cuproptosis-related molecular clusters by unsupervised clustering. Three characteristic genes (ENO3, SLC16A1 and LEPR) were selected by machine learning and external data set validation. In addition, the accuracy of the nomogram, calibration curve and decision curve analysis in predicting NAFLD clusters was also verified. Further animal and cell experiments confirmed the difference in their expression in the NAFLD mouse model and Mouse hepatocyte cell line. The present study explored the relationship between non-alcoholic fatty liver disease and cuproptosis, providing new ideas and targets for individual treatment of the disease.

Orthotopic Transplantation of Functional Bioengineered Livers in Rats.

Functional bioengineered livers (FBLs) are promising alternatives to orthotopic liver transplantation. However, orthotopic transplantation of FBLs has not yet been reported. This study aimed to perform the orthotopic transplantation of FBLs in rats subjected to complete hepatectomy. FBLs were developed using rat whole decellularized liver scaffolds (DLSs) with human umbilical vein endothelial cells implanted via the portal vein, and human bone marrow mesenchymal stem cells (hBMSCs) and mouse hepatocyte cell line implanted via the bile duct. FBLs were evaluated in terms of endothelial barrier function, biosynthesis, and metabolism and orthotopically transplanted into rats to determine the survival benefit. The FBLs with well-organized vascular structures exhibited endothelial barrier function, with reduced blood cell leakage. The implanted hBMSCs and hepatocyte cell line were well aligned in the parenchyma of the FBLs. The high levels of urea, albumin, and glycogen in the FBLs indicated biosynthesis and metabolism. Orthotopic transplantation of FBLs achieved a survival time of 81.38 ± 4.263 min in rats (n = 8) subjected to complete hepatectomy, whereas control animals (n = 4) died within 30 min (p < 0.001). After transplantation, CD90-positive hBMSCs and the albumin-positive hepatocyte cell line were scattered throughout the parenchyma, and blood cells were limited within the vascular lumen of the FBLs. In contrast, the parenchyma and vessels were filled with blood cells in the control grafts. Thus, orthotopic transplantation of whole DLS-based FBLs can effectively prolong the survival of rats subjected to complete hepatectomy. In summary, this work was the first to perform the orthotopic transplantation of FBLs, with limited survival benefits, which still has important value for the advancement of bioengineered livers.

Piezo1 alleviates acetaminophen-induced acute liver injury by activating Nrf2 and reducing mitochondrial reactive oxygen species

Acetaminophen (APAP) overdose is the most common cause for acute liver failure (ALF) in the developed countries, with limited treatment options. Piezo1 is a mechanosensitive cation channel. We found that APAP caused upregulation of Piezo1 in both an APAP-induced acute liver injury (ALI) animal model and a mouse hepatocyte cell line AML12. Activation of Piezo1 by its activator Yoda1 reduced APAP-induced hepatotoxicity and ROS level. Mechanistically, activation of Piezo1 led to accumulation of the antioxidant regulator Nrf2 and upregulation of its target genes Nqo1 and Gsta1, while knockdown of Piezo1 downregulated this pathway. Finally, injection of Yoda1 decreased serum AST and ALT levels, reduced cell death and rescued liver injury in the APAP-induced ALI mouse model. Our findings suggested a previously undiscovered protective role of Piezo1 in APAP-induced ALI, which might shed light on a new therapeutic target for this disease.

Changing the phospholipid composition of lipid droplets alters localization of select lipid droplet proteins.

Our experiments aim to determine if decreasing the amount of phosphatidylcholine (PC) relative to phosphatidylethanolamine (PE) at the lipid droplet surface changes the localization of specific lipid droplet proteins. We manipulate lipid droplet phospholipids in both a cultured mouse hepatocyte (AML12) cell line and on synthetic lipid droplets. Decreasing the PC:PE ratio increases perilipin 2, decreases DGAT2, and does not change rab18 or lanosterol synthase levels on lipid droplets. These differences may be explained by the distinct structural motifs that mediate the protein-lipid droplet interactions.

Hepatocyte Rap1a contributes to obesity- and statin-associated hyperglycemia.

SUMMARYExcessive hepatic glucose production contributes to the development of hyperglycemia and is a key feature of type 2 diabetes. Here, we report that activation of hepatocyte Rap1a suppresses gluconeogenic gene expression and glucose production, whereas Rap1a silencing stimulates them. Rap1a activation is suppressed in obese mouse liver, and restoring its activity improves glucose intolerance. As Rap1a′s membrane localization and activation depends on its geranylgeranylation, which is inhibited by statins, we show that statin-treated hepatocytes and the human liver have lower active-Rap1a levels. Similar to Rap1a inhibition, statins stimulate hepatic gluconeogenesis and increase fasting blood glucose in obese mice. Geranylgeraniol treatment, which acts as the precursor for geranylgeranyl isoprenoids, restores Rap1a activity and improves statin-mediated glucose intolerance. Mechanistically, Rap1a activation induces actin polymerization, which suppresses gluconeogenesis by Akt-mediated FoxO1 inhibition. Thus, Rap1a regulates hepatic glucose homeostasis, and blocking its activity, via lowering geranylgeranyl isoprenoids, contributes to statin-induced glucose intolerance.

Cimifugin Ameliorates Lipotoxicity-Induced Hepatocyte Damage and Steatosis through TLR4/p38 MAPK- and SIRT1-Involved Pathways.

Objective Hepatic metabolic disorder induced by lipotoxicity plays a detrimental role in metabolic fatty liver disease pathogenesis. Cimifugin (Cim), a coumarin derivative extracted from the root of Saposhnikovia divaricata, possesses multiple biological properties against inflammation, allergy, and oxidative stress. However, limited study has addressed the hepatoprotective role of Cim. Here, we investigate the protective effect of Cim against lipotoxicity-induced cytotoxicity and steatosis in hepatocytes and clarify its potential mechanisms. Methods AML-12, a nontransformed mouse hepatocyte cell line, was employed in this study. The cells were incubated with palmitate or oleate to imitate hepatotoxicity or steatosis model, respectively. Results Cim significantly reversed palmitate-induced hepatocellular injury in a dose-dependent manner, accompanied by improvements in oxidative stress and mitochondrial damage. Cim pretreatment reversed palmitate-stimulated TLR4/p38 MAPK activation and SIRT1 reduction without affecting JNK, ERK1/2, and AMPK pathways. The hepatoprotective effects of Cim were abolished either through activating TLR4/p38 by their pharmacological agonists or genetical silencing SIRT1 via special siRNA, indicating a mechanistic involvement. Moreover, Cim treatment improved oleate-induced hepatocellular lipid accumulation, which could be blocked by either TLR4 stimulation or SIRT1 knockdown. We observed that SIRT1 was a potential target of TLR4 in palmitate-treated hepatocytes, since TLR4 agonist LPS aggravated, whereas TLR4 antagonist CLI-095 alleviated palmitate-decreased SIRT1 expression. SIRT1 knockdown did not affect palmitate-induced TLR4. In addition, TLR4 activation by LPS significantly abolished Cim-protected SIRT1 reduction induced by palmitate. These results collaboratively indicated that TLR4-regulated SIRT1 pathways was mechanistically involved in the protective effects of Cim against lipotoxicity. Conclusion In brief, we demonstrate the protective effects of Cim against lipotoxicity-induced cell death and steatosis in hepatocytes. TLR4-regulated p38 MAPK and SIRT1 pathways are involved in Cim-protected hepatic lipotoxicity. Cim is a potential candidate for improving hepatic metabolic disorders mediated by lipotoxicity.

The Mechanism of Mitochondrial Injury in Alpha-1 Antitrypsin Deficiency Mediated Liver Disease.

Alpha-1 antitrypsin deficiency (AATD) is caused by a single mutation in the SERPINA1 gene, which culminates in the accumulation of misfolded alpha-1 antitrypsin (ZAAT) within the endoplasmic reticulum (ER) of hepatocytes. AATD is associated with liver disease resulting from hepatocyte injury due to ZAAT-mediated toxic gain-of-function and ER stress. There is evidence of mitochondrial damage in AATD-mediated liver disease; however, the mechanism by which hepatocyte retention of aggregated ZAAT leads to mitochondrial injury is unknown. Previous studies have shown that ER stress is associated with both high concentrations of fatty acids and mitochondrial dysfunction in hepatocytes. Using a human AAT transgenic mouse model and hepatocyte cell lines, we show abnormal mitochondrial morphology and function, and dysregulated lipid metabolism, which are associated with hepatic expression and accumulation of ZAAT. We also describe a novel mechanism of ZAAT-mediated mitochondrial dysfunction. We provide evidence that misfolded ZAAT translocates to the mitochondria for degradation. Furthermore, inhibition of ZAAT expression restores the mitochondrial function in ZAAT-expressing hepatocytes. Altogether, our results show that ZAAT aggregation in hepatocytes leads to mitochondrial dysfunction. Our findings suggest a plausible model for AATD liver injury and the possibility of mechanism-based therapeutic interventions for AATD liver disease.

Inflammation Drives MicroRNAs to Limit Hepatocyte Bile Acid Transport in Murine Biliary Atresia

BackgroundBiliary atresia (BA) is an inflammatory pediatric cholangiopathy with only surgical means for treatment. Many contributors to bile acid synthesis and transport have previously been reported to be downregulated in patients with BA; yet, the driving factors of the abnormal bile acid synthesis and transport in regard to BA have not been previously studied. Materials and methodsWild type or Ig-α-/- mice were injected with salt solution (control) or rotavirus on day of life 0, and analyses were performed on day of life 14. The mRNA levels of bile acid transporters/nuclear receptors and liver microRNAs (miRNAs) were compared between groups. A mouse hepatocyte cell line was used to examine the effects of innate cytokines on miRNA levels and bile acid transporter/nuclear receptor expression and miRNAs on bile acid transporter/nuclear receptor expression. ResultsBA mice had significantly increased mRNA expression of innate cytokines and miRNAs known to bind bile acid transporters/nuclear receptors (miRNAs -22-5p, -34a-5p, and -222-3p) and decreased mRNA expression of bile acid transporters and nuclear receptors. In vitro, TNF-α and IL-1β decreased BSEP and CYP7A1 while increasing miRNA-34a-5p and miRNA 222-3p. LXR, SHP, CYP7A1, NTCP, and MRP2 were decreased by miRNA-34a-5p, whereas miRNA-222-3p decreased NTCP and MRP4. TNF-α and IL-1β increased expression of miRNAs 34a-5p and 222-3p and these miRNAs then decrease expression of multiple bile acid transporters and nuclear receptors. ConclusionsLoss of bile acid transporters increases hepatotoxicity via bile acid retention. Therapeutic agents that increase bile acid transport or nuclear receptor functioning should be investigated in BA.

IL-1Ra Protects Hepatocytes from CCl4-Induced Hepatocellular Apoptosis via Activating the ERK1/2 Pathway

AbstractInterleukin-1 receptor antagonist is an important acute-phase protein and an immune mediator, and its expression is associated with the development of hepatitis or acute liver failure. The aim of this study was to investigate whether recombinant human interleukin-1 receptor antagonist directly targets and improves cell survival in a carbon tetrachloride-induced hepatocyte injury model in vitro. A human hepatoma cell line and a mouse hepatocyte cell line were used to establish carbon tetrachloride-induced cell injury models in vitro, and cell viability, apoptosis, and reactive oxygen species level were determined to assess the degree of hepatocellular damage. Quantitative real-time polymerase chain reaction was used to analyze the level of interleukin-1β, interleukin-6, and tumor necrosis factor-α mRNA in cells; extracellular regulated protein kinases 1/2 phosphorylation in hepatocytes was analyzed using western blotting. Recombinant human interleukin-1 receptor antagonist could directly target hepatocytes, improve cell survival, and decrease carbon tetrachloride-induced cell apoptosis in vitro. In hepatocytes, recombinant human interleukin-1 receptor antagonist remarkably downregulated expression of interleukin-1β, interleukin-6, and tumor necrosis factor-α in hepatocytes exposed to carbon tetrachloride. It also decreased accumulation of reactive oxygen species and abrogated the suppression of extracellular regulated protein kinases 1/2 phosphorylation induced by carbon tetrachloride. However, stimulation of cells with an extracellular regulated protein kinases 1/2 inhibitor blocked the recombinant human interleukin-1 receptor antagonist-induced upregulation of extracellular regulated protein kinase1/2 activation and abrogated the improvement in hepatocyte survival following carbon tetrachloride treatment. Collectively, these findings provide new insights into the hepatocyte-protective mechanism of recombinant human interleukin-1 receptor antagonist.

Enhanced acetylation of ATP-citrate lyase promotes the progression of nonalcoholic fatty liver disease

Hepatic steatosis is a hallmark of nonalcoholic fatty liver disease (NAFLD) and is promoted by dysregulated de novo lipogenesis. ATP-citrate lyase (ACLY) is a crucial lipogenic enzyme that is up-regulated in individuals with NAFLD. A previous study has shown that acetylation of ACLY at Lys-540, Lys-546, and Lys-554 (ACLY-3K) increases ACLY protein stability by antagonizing its ubiquitylation, thereby promoting lipid synthesis and cell proliferation in lung cancer cells. But the functional importance of this regulatory mechanism in other cellular or tissue contexts or under other pathophysiological conditions awaits further investigation. Here, we show that ACLY-3K acetylation also promotes ACLY protein stability in AML12 cells, a mouse hepatocyte cell line, and found that the deacetylase sirtuin 2 (SIRT2) deacetylates ACLY-3K and destabilizes ACLY in these cells. Of note, the livers of mice and humans with NAFLD had increased ACLY protein and ACLY-3K acetylation levels and decreased SIRT2 protein levels. Mimicking ACLY-3K acetylation by replacing the three lysines with three glutamines (ACLY-3KQ variant) promoted lipid accumulation both in high glucose-treated AML12 cells and in the livers of high-fat/high-sucrose (HF/HS) diet-fed mice. Moreover, overexpressing SIRT2 in AML12 cells inhibited lipid accumulation, which was more efficiently reversed by overexpressing the ACLY-3KQ variant than by overexpressing WT ACLY. Additionally, hepatic SIRT2 overexpression decreased ACLY-3K acetylation and its protein level and alleviated hepatic steatosis in HF/HS diet-fed mice. Our findings reveal a posttranscriptional mechanism underlying the up-regulation of hepatic ACLY in NAFLD and suggest that the SIRT2/ACLY axis is involved in NAFLD progression.

Abstract 4373: Lack of FGF21 promotes NASH-HCC transition via exosome-mediated carcinogenetic signaling

Abstract Background: Nonalcoholic steatohepatitis (NASH) is the most severe form of non-alcoholic fatty liver disease (NAFLD) and a potential precursor of hepatocellular carcinoma (HCC). FGF21, with its metabolic benefits, was reported as the liver safeguard to protect from obesity, diabetes, and NAFLD. Recent study showed an exosome based FGF21 inhibition via organs communication to regulate hepatic metabolism. However, the role of the exosome-FGF21 axis in NASH-HCC has never been studied. Aim: To study the exosome based FGF21 metabolic regulation during NASH-HCC carcinogenetic transformation. Methods: NASH-HCC model was induced in FGF21 knockout (KO) mice and wild type (WT) control using methionine-choline-deficient diets (MCD) and diethylnitrosamine (DEN). The exosomes releasing, miRNA and protein contents were analyzed in the primary cultured hepatocytes from the liver tissues of NASH-HCC mice (KO and WT) as well as benign controls. FGF21 was knockdown (KD) by shRNA in a mouse hepatoma cell line (Hepa1-6) and a benign mouse hepatocyte cell line (FL83B) to investigate the potential carcinogenetic signaling related to exosome-FGF21 axis. Result: NASH-HCC model was successfully established with confirmation of the metabolic disorders and liver cancer development. Early HCC detection and increased HCC incidence were found in the KO-MCD mice (P&amp;lt;0.05 versus KO-CD and WT controls). The collected exosomes from primary cultured hepatocytes and cell lines were identified using the antibodies of Tsg101, CD63 and cytochrome C by western blotting and immunofluorescent staining. In the KO-MCD mice, exosomes retained in the cytosol of benign hepatocyte but released increasingly from cancer cell. Similar results were showed in the Hepa1-6KD cells and the FL83BKD cells. Aberrant EMT, and p53 and Wnt signaling was found in the KO-MCD mice as well as Hepa1-6KD cells challenged with free fatty acid. Conclusion: lack of FGF21 plays critical role for the exosome retention in hepatocytes of NASH mice but not in HCC mice. Abnormal traffic of exosomes contribute to the carcinogenetic signaling. This work was supported by an Institutional Development Award (IDeA) from the NIGMS of the National Institutes of Health under grant number P20GM113226. Citation Format: Youxi Yu, Robert C. Martin, Qianqian Zheng, Xiaoju Shi, Xingkai Liu, Wei Guo, Suping Li, Ping Zhang, Yan Li. Lack of FGF21 promotes NASH-HCC transition via exosome-mediated carcinogenetic signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4373.

Abstract 985: Development and validation of cell-based TFEB translocation assay in a high-content and high-throughput screening format

Abstract Transcription factor EB (TFEB) is a master regulator of lysosomal gene expression and mitochondrial biogenesis. Autophagy is known to act as a double edged sword in cancer. Autophagy acts as a tumor suppressor during the tumorigenesis process but it also helps to sustain the survival of already transformed cancer cells. Lysosomes sit at the end of the autophagy pathway, which is critical to meet the needs of autophagy. Therefore, small molecules that inhibit TFEB-mediated lysosomal biogenesis and autophagic degradation may be helpful for inhibiting cancer cell growth and limiting cancer progression. On the other hand, small molecules that enhance TFEB-mediated lysosomal degradation may be used to prevent tumorigenesis. In order to identify compounds that modulate TFEB translocation, an image-based TFEB translocation assay was developed in a 1536-well plate format with a GFP-TFEB stable AML12 cell line, which is an immortalized mouse hepatocyte cell line. Torin 1, a known TFEB translocation inducer, was used as positive control in the study. Torin 1 increased TFEB nuclear translocation in a concentration dependent manner with an EC50 of 150nM. To optimize the assay condition, various cell densities per well and time courses were tested. To validate this assay, a group of 384 known anticancer compounds were screened at 11 concentrations ranging from 0.8 nM to 46 μM. From this screening, we identified 25 compounds that enhanced TFEB nuclear translocation with EC50s ranging from 2 nM to 12 μM. Several known and novel TFEB inducers were among these compounds. These results demonstrate that this high-content and high-throughput assay can be used to screen large numbers of chemicals and provide a robust in vitro system to identify TFEB agonists and antagonists, which may be used to either prevent (agonists) or treat (antagonist) cancer. Citation Format: Li Zhang, Ruili Huang, Wen Xing Ding, Menghang Xia. Development and validation of cell-based TFEB translocation assay in a high-content and high-throughput screening format [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 985.

FoxK1/K2 Are New, Important Components of IR and IGF1R Signaling and Control of Cell Proliferation and Metabolism

Insulin and IGF-1 are regulators of cell growth and metabolism. Despite considerable progress in understanding the molecular mechanisms underlying these effects, except for members of the FoxO family, many regulators of the transcriptional effects of these hormones remain poorly understood. To identify new components of IR/IGF1R signaling, we created brown preadipocyte lines in which both endogenous IR and IGF1R genes had been deleted and then reconstituted these cells with normal IR, IGF1R or chimeric receptors containing the extracellular domain of IR fused to intracellular domain of IGF1R and vice versa. Mass spectroscopic proteomic analysis revealed a number of proteins co-precipitated with each receptor construct, in both ligand stimulation-dependent and -independent manners. Among the proteins that associated with both receptors and chimeric receptors in a ligand-dependent manner was FoxK1. In contrast to FoxO1, the transcription factor FoxK1 was translocated from the cytoplasm to the nucleus after insulin stimulation, a pattern that is reciprocal to that of FoxO1 after insulin stimulation. We show that FoxK1 and FoxK2 are phosphorylated in two major domains and that translocation to the nucleus is dependent on the Akt-mTOR mediated phosphorylation, while their localization to the cytoplasm in the basal state is dependent on GSK3 phosphorylation. Knockdown of FoxK1 and FoxK2 in a mouse hepatocyte cell line causes marked alteration of the transcription of genes associated with apoptotic pathway and down-regulation of genes involved in control of cell cycle and metabolism. This resulted in decreased cell proliferation and altered mitochondrial fatty acid metabolism in these cells. Thus, insulin stimulates the reciprocal translocation of FoxKs and FoxO1 between the cytoplasm and nucleus turning off FoxO1-regulated genes and turning on FoxK-regulated genes resulting in an elaborate balance in insulin regulation of metabolism, growth and cell survival. Disclosure M. Sakaguchi: None. W. Cai: None. C. Wang: None. T.M. Batista: None. E. Araki: Speaker's Bureau; Self; Astellas Pharma US, Inc., MSD K.K., Kowa Pharmaceuticals America, Inc., Sanofi, Novo Nordisk Inc.. Research Support; Self; Astellas Pharma US, Inc., MSD K.K., Ono Pharmaceutical Co., Ltd., Shionogi &amp; Co., Ltd., Takeda Pharmaceuticals U.S.A., Inc., Daiichi Sankyo Company, Limited, Nippon Boehringer Ingelheim Co. Ltd., Novartis Pharma K.K., Novo Nordisk Inc., Sanofi, Mitsubishi Tanabe Pharma Corporation, Sumitomo Dainippon Pharma Co., Ltd., Taisho Toyama Pharmaceutical Co. C. Kahn: Advisory Panel; Self; CohBar, ERX Therapeutics, AntriaBio, Inc.. Board Member; Self; Kaleio Biosciences.

Detection of Necroptosis in Ligand-Mediated and Hypoxia-Induced Injury of Hepatocytes Using a Novel Optic Probe-Detecting Receptor-Interacting Protein (RIP)1/RIP3 Binding.

Liver injury is often observed in various pathological conditions including posthepatectomy state and cancer chemotherapy. It occurs mainly as a consequence of the combined necrotic and apoptotic types of cell death. In order to study liver/hepatocyte injury by the necrotic type of cell death, we studied signal-regulated necrosis (necroptosis) by developing a new optic probe for detecting receptor-interacting protein kinase 1 (RIP)/RIP3 binding, an essential process for necroptosis induction. In the mouse hepatocyte cell line, TIB-73 cells, TNF-α/cycloheximide (T/C) induced RIP1/3 binding only when caspase activity was suppressed by the caspase-specific inhibitor z-VAD-fmk (zVAD). T/C/zVAD-induced RIP1/3 binding was inhibited by necrostatin-1 (Nec-1), an allosteric inhibitor of RIP1. The reduced cell survival by T/C/zVAD was improved by Nec-1. These facts indicate that T/C induces necroptosis of hepatocytes when the apoptotic pathway is inhibited/unavailable. FasL also induced cell death, which was only partially inhibited by zVAD, indicating the possible involvement of necroptosis rather than apoptosis. FasL activated caspase 3 and, similarly, induced RIP1/3 binding when the caspases were inactivated. Interestingly, FasL-induced RIP1/3 binding was significantly suppressed by the antioxidants Trolox and N-acetyl cysteine (NAC), suggesting the involvement of reactive oxygen species (ROS) in FasL-induced necroptotic cellular processes. H2O2, by itself, induced RIP1/3 binding that was suppressed by Nec-1, but not by zVAD. Hypoxia induced RIP1/3 binding after reoxygenation, which was suppressed by Nec-1 or by the antioxidants. Cell death induced by hypoxia/reoxygenation (H/R) was also improved by Nec-1. Similar to H2O2, H/R did not require caspase inhibition for RIP1/3 binding, suggesting the involvement of a caspase-independent mechanism for non-ligand-induced and/or redox-mediated necroptosis. These data indicate that ROS can induce necroptosis and mediate the FasL- and hypoxia-induced necroptosis via a molecular mechanism that differs from a conventional caspase-dependent pathway. In conclusion, necroptosis is potentially involved in liver/hepatocyte injury induced by oxidative stress and FasL in the absence of apoptosis.

Hepatocyte-specific deletion of LASS2 protects against diet-induced hepatic steatosis and insulin resistance.

Homo sapienslongevity assurance homolog 2 of yeast LAG1 (LASS2) is expressed mostly in human liver. Here, we explored roles of LASS2 in pathogenesis of hepatic steatosis. Hepatocyte-specific LASS2 knockout (LASS2-/-) mice were generated using Cre-LoxP system. LASS2-/- and wild-type (WT) mice were fed with chow or high-fat diet (HFD). We found LASS2-/- mice were resistant to HFD-induced hepatic steatosis and insulin resistance. In HFD-fed mice, LASS2 deficiency significantly inhibited p38 MAPK and ERK1/ERK2 signaling in mouse liver. This effect was mediated by a significant increase of V-ATPase activity and a decrease of ROS level. We also observed that elevated expression of LASS2 in mouse hepatocyte cell line AML12 obviously decreased V-ATPase activity and increased ROS level by activation of p38 MAPK and ERK1/ERK2 signaling. Our findings indicate that LASS2 plays an important role in the pathogenesis of diet-induced hepatic steatosis and is a potential novel target for prevention and intervention of liver diseases.

Long-term administration of tacrolimus and everolimus prevents high cholesterol-high fructose-induced steatosis in C57BL/6J mice by inhibiting de-novo lipogenesis

AimTo investigate the effects of tacrolimus (TC) and everolimus (EV) on non-alcoholic steatohepatitis (NASH) induced by high fat, high cholesterol and fructose (fast food) diet in C57BL/6J mice.Materials and MethodsC57BL/6J mice were divided into four groups (n=8). 1) Standard Chow (SC); 2) Fast food (FF) diet; 3) FF + Tacrolimus (TC, 1mg/kg) and; 4) FF + Everolimus (EV, 1mg/kg) and treated for 16 weeks. Serum and tissue samples were analyzed for evidence of inflammation, fibrosis, lipogenesis, and apoptosis.ResultsTC and EV treatments significantly reduced the hepatic lipid accumulation, improved liver-body weight ratio, blood biochemistry, and insulin resistance in mice fed with FF diet. However, inflammation, enlarged portal tracts, and fibrosis were pronounced in EV treated group. The lipogenic parameters, Peroxisome proliferator-activated receptor gamma (PPAR-γ), Sterol regulatory element-binding protein 1(SREBP-1), mammalian target of rapamycin (m-TOR), Stearoyl-CoA desaturase-1 (SCD-1) and fatty acid translocase (CD36) were significantly down-regulated in livers of TC and EV treated groups as compared to FF group. TC improved Bcl2/Bax ratio, decreased apoptosis, CYP2E1 protein expression and liver fibrosis levels, however, EV offered no such protection. Further, in an In-vitro model of lipotoxicity using the mouse hepatocyte (AML-12) cell line, treatment with TC and EV significantly reduced lipid accumulation and lipogenic and apoptotic markers induced with palmitic acid.ConclusionIn FF diet induced model of NASH, both TC and EV inhibited hepatic lipid accumulation and improved metabolic parameters such as insulin resistance and dyslipidemia. However, mice administered with EV exhibited inflammatory and fibrotic responses despite reduced hepatic steatosis.

Role of glycoprotein 78 and cidec in hepatic steatosis.

Hepatic glycoprotein (gp78), a membrane-anchored E3 ubiquitin ligase, has been reported to be involved in regulating lipid and energy metabolism in animals, and cell death-inducing DFFA-like effector c (cidec) has emerged as an important regulator of metabolism, which has been implicated in the process of fat differentiation. Nonalcoholic fatty liver disease is a metabolic disorder associated with hepatic steatosis. In the present study, to investigate the role of gp78 and cidec in hepatic steatosis, an in vitro cell culture model of hepatic steatosis was established, using the AML12 mouse hepatocyte cell line to assess the protein expression of gp78. The results of Oil Red O staining, phase contrast microscopy and triglyceride content detection experiments indicated that the overexpression of gp78 induced lipid accumulation, whereas gp78-knockdown led to a reduction in lipid accumulation in the AML12 cells. The increased expression of gp78 was associated with steatosis. The expression of cidec was consistent with gp78, and the colocalization of gp78 and cidec was observed on the surface of lipid droplets using immunofluorescence analysis. Furthermore, an interaction between gp78 and cidec was detected using coimmunoprecipitation analysis, and this interaction promoted lipid accumulation. Based on these data, it was hypothesized that gp78 is a regulator of hepatic steatosis, and that it may be a putative molecular mediator in metabolic diseases.

Exosomes derived from human menstrual blood-derived stem cells alleviate fulminant hepatic failure

BackgroundHuman menstrual blood-derived stem cells (MenSCs) are a novel source of MSCs that provide the advantage of being easy to collect and isolate. Exosomes contain some mRNAs and adhesion molecules that can potentially impact cellular and animal physiology. This study aimed to investigate the therapeutic potential of MenSC-derived exosomes (MenSC-Ex) on AML12 cells (in vitro) and D-GalN/LPS-induced FHF mice (in vivo).MethodsTransmission electron microscopy and Western blot were used to identify MenSC-Ex. Antibody array was used to examine cytokine levels on MenSC-Ex. MenSC-Ex were treated in D-GalN/LPS-induced AML12 in vitro. Cell proliferation and apoptosis were measured. MenSC-Ex were injected into the tail veins of mice 24 h before treatment with D-GalN/LPS. Blood and liver tissues served as physiological and biochemical indexes. The number of liver mononuclear cells (MNCs) and the amount of the active apoptotic protein caspase-3 were determined to elaborate the mechanism of hepatoprotective activity.ResultsHuman menstrual blood-derived stem cell-derived exosomes (MenSC-Ex) are bi-lipid membrane vesicles that have a round, ball-like shape with a diameter of approximately 30–100 nm. Cytokine arrays have shown that MenSC-Ex expressed cytokines, including ICAM-1, angiopoietin-2, Axl, angiogenin, IGFBP-6, osteoprotegerin, IL-6, and IL-8. MenSC-Ex markedly improved liver function, enhanced survival rates, and inhibited liver cell apoptosis at 6 h after transplantation. MenSC-Ex migrated to sites of injury and to AML12 cells (a mouse hepatocyte cell line), respectively. Moreover, MenSC-Ex reduced the number of liver mononuclear cells (MNCs) and the amount of the active apoptotic protein caspase-3 in injured livers.ConclusionsIn conclusion, our results provide preliminary evidence for the anti-apoptotic capacity of MenSC-Ex in FHF and suggest that MenSC-Ex may be an alternative therapeutic approach to treat FHF.

Elk-3 Contributes to the Progression of Liver Fibrosis by Regulating the Epithelial-Mesenchymal Transition.

Background/AimsThe role of Elk-3 in the epithelial-mesenchymal transition (EMT) during liver fibrogenesis remains unclear. Here, we determined the expression of Elk-3 in in vitro and in vivo models and in human liver fibrotic tissues. We also investigated the molecular relationships among Elk-3, early growth response-1 (Egr-1), and the mitogen activated protein kinases (MAPK) pathway during EMT in hepatocytes.MethodsWe established anin vitro EMT model in which normal mouse hepatocyte cell lines were treated with transforming growth factor (TGF)-β1 and a CCl4-induced liver fibrosis model. Characteristics of EMT were determined by evaluating the expression levels of related markers. The expression of Elk-3 and its target Egr-1 were analyzed using Western blotting. Gene silencing of Elk-3 was performed using an siRNA knockdown system.ResultsThe expression levels of mesenchymal markers were increased during TGF-β1-induced EMT of hepatocytes. The expression levels of Elk-3 and Egr-1 were significantly (p<0.05) increased during the EMT of hepatocytes, in CCl4-induced mouse liver fibrotic tissues, and in human liver cirrhotic tissues. Silencing of Elk-3 and inhibition of the Ras-Elk-3 pathway with an inhibitor suppressed the expression of EMT-related markers. Moreover, Elk-3 expression was regulated by p38 MAPK phosphorylation during EMT.ConclusionsElk-3 contributes to the progression of liver fibrosis by modulating the EMT via the regulation of Egr-1 under MAPK signaling.