Hepatocyte Cell Proliferation Research Articles

The dichloromethane fraction from Calotropis gigantea (L.) dryand. Stem bark extract prevents liver cancer in SDT rats with insulin-independent diabetes mellitus

Ethnopharmacological relevanceCalotropis gigantea (L.) Dryand. (C. gigantea) is a traditional medicinal plant, recognized for its effectiveness in managing diabetes, along with its notable antioxidant, anti-inflammatory, and anticancer properties. Type II diabetes mellitus (T2DM) is characterized by chronic metabolic disorders associated with an elevated risk of hepatocellular carcinoma (HCC) due to hyperglycemia and impaired insulin response. The scientific validation of C. gigantea's ethnopharmacological efficacy offers advantages in alleviating cancer progression in T2DM complications, enriching existing knowledge and potentially aiding future clinical cancer treatments. AimThis study aimed to investigate the preventive potential of the dichloromethane fraction of C. gigantea stem bark extract (CGDCM) against diethylnitrosamine (DEN)-induced HCC in T2DM rats, aiming to reduce cancer incidence associated with diabetes while validating C. gigantea's ethnopharmacological efficacy. Materials and methodsSpontaneously Diabetic Torii (SDT) rats were administered DEN to induce HCC (SDT-DEN-VEH), followed by treatment with CGDCM. Metformin was used as a positive control (SDT-DEN-MET). All the treatments were administered for 10 weeks after the initial DEN injection. Diabetes-related parameters, including serum levels of glucose, insulin, and glycosylated hemoglobin (HbA1c), as well as liver function enzymes (aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, and gamma-glutamyl transferase), were quantified. Serum inflammation biomarkers interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) were evaluated. Liver tissue samples were analyzed for inflammation protein expression (IL-6, TNF-α, transforming growth factor-β1 (TGF-β1), and α-smooth muscle actin (α-SMA)). Histopathological evaluation was performed to assess hepatic necrosis, inflammation, and fibrosis. Liver cell proliferation was determined using immunohistochemistry for Ki-67 expression. ResultsRats with SDT-DEN-induced HCC treated with CGDCM exhibited reduced serum glucose levels, elevated insulin levels, and decreased HbA1c levels. CGDCM treatment also reduced elevated hepatic IL-6, TNF-α, TGF-β1, and α-SMA levels in SDT-DEN-VEH rats. Additionally, CGDCM treatment prevented hepatocyte damage, fibrosis, and cell proliferation. No adverse effects on normal organs were observed with CGDCM treatment, suggesting its safety for the treatment of HCC complications associated with diabetes. Additionally, the absence of adverse effects in SD rats treated with CGDCM at 2.5 mg/kg further supports the notion of its safe usage. ConclusionsThese findings suggest that C. gigantea stem bark extract exerts preventive effects against the development of HCC complications in patients with T2DM, expanding the potential benefits of its ethnopharmacological advantages.

Experimental Study on Rapid Hemostasis Using Peptide Hydrogels.

Uncontrolled hemorrhaging resulting from trauma, surgery, and disease-associated or drug-induced blood disorders can cause significant morbidities and mortalities in civilian and military populations. Self-assembling peptide nanofibers are particularly attractive due to their rapid and efficient hemostasis, biocompatibility, and wound-healing properties. In this study, we designed two types of 12-residue peptides by using a strong fishnet-like peptide sequence and a pro-cell adhesion sequence (Arg-Gly-Asp, RGD). The peptides are HN2-X-Ser-Phe-Cys-Phe-Lys-Phe-Glu-X-Arg-Gly-Asp-OH (where X is Pro or Tyr), which dissolve in deionized (DI) water and form stable and transparent functional hydrogels. Transmission electron microscopy and scanning electron microscopy demonstrated that the two peptides self-assemble into nanowebs and nanofibers, forming a fishnet-like and three-dimensional network structure. Circular dichroism and Fourier transform infrared spectroscopy analysis demonstrated that the self-assembled peptides mainly adopt a β-sheet structure with β-turn and α-helix as auxiliary assembly growth. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and SEM analysis showed that the cell survival rates were very good, delivering an obvious promotion of cell proliferation of fibroblasts and hepatocytes. Importantly, in vivo hemostasis delivered that the self-assembled peptide nanowebs and nanofibers had a good hemostatic effect on rat saphenous vein and liver bleeding, achieving 38 s faster hemostasis, which was better than commercial "Instantaneous" hemostatic powder. Accoupling the fast hemostasis and effective promotion of liver defect rapid repair, the peptide self-assembly strategy offers a clinically promising treatment option for life-threatening liver bleeding and serves as a renewed impetus for the development of peptide hydrogels as effective hemostatic agents.

Functions and Clinical Relevance of Liver-Derived Immunoglobulins.

Liver is the largest internal organ of the body with vital functions. In addition to its endocrine and exocrine activities, liver also plays a pivotal role in the immune system, including haematopoietic functions. Liver parenchymal cells, which are epithelial cells, have been found to possess innate immune functions by expressing pattern-recognition receptors (PRRs), producing complement components, and secreting cytokines. Intriguingly, in recent years, it has been discovered that liver epithelial cells also produce immunoglobulins (Igs), which have long been thought to be produced exclusively by B cells. Notably, even liver epithelial cells from B lymphocyte-deficient mice, including SCID mice and μMT mice, could also produce Igs. Compelling evidence has revealed both the physiological and pathological functions of liver-derived Igs. For instance, liver epithelial cells-derived IgM can serve as a source of natural and specific antibodies that contribute to innate immune responses, while liver-produced IgG can act as a growth factor to promote cell proliferation and survival in normal hepatocytes and hepatocarcinoma. Similar to that in B cells, the toll-like receptor 9 (TLR9)-MyD88 signaling pathway is also actively involved in promoting liver epithelial cells to secrete IgM. Liver-derived Igs could potentially serve as biomarkers, prognostic indicators, and therapeutic targets in the clinical setting, particularly for liver cancers and liver injury. Nevertheless, despite significant advances, much remains unknown about the mechanisms governing Ig transcription in liver cells, as well as the detailed functions of liver-derived Igs and their involvement in diseases and adaptive immunity. Further studies are still needed to reveal these underlying, undefined issues related to the role of liver-derived Igs in both immunity and diseases.

Abstract 4847: Investigating the transcriptional regulation by estrogen-related receptor alpha (ERRα) under different metabolic conditions

Abstract Estrogen-related receptors (ERRs) are orphan nuclear receptors identified based on their high sequence similarity to estrogen receptors (ERs), but ERRs do not have a known endogenous ligand. ERRs play a primary role in regulating the transcription of genes involved in mitochondrial and lipid metabolism and are abundantly expressed across tissues. Because of ERR’s role in metabolism, it is suggested that they may also play a role in tumor metabolism, where dysfunction in lipid metabolism promotes tumor cell growth. The ERR subfamily is comprised of three isoforms: ERRα, ERRβ, and ERRγ. Studies targeting the ERR isoforms found that the absence of ERRα presents obesity and insulin resistance with an increase in bone mass, deletion of ERRβ causes placental abnormalities and embryonic lethality, and deletion of ERRγ leads to mitochondrial dysfunction. Together, these studies strongly suggest that ERRs, particularly ERR α and γ, function primarily as metabolic regulators, with ERRα being the predominant isoform expressed in the liver. In addition, the mechanisms leading to lipid accumulation vary under different feeding conditions. Previous studies from our lab showed that PTEN and PI3K/AKT signaling regulates ERRa expression and its function. Furthermore, we demonstrated that inhibiting ERRα blocks liver steatosis and steatohepatitis developed in a mouse model where loss of tumor suppressor PTEN drives both steatosis and cancer development. In addition, we found that ERR-PA, a small molecule inhibitor for ERR, attenuated cancer cell growth and proliferation in both mouse hepatocytes and human cancer cell lines. Here, we report transcriptome network regulated by ERRα under different metabolic conditions and further explored its regulation by the PI3K/AKT pathway. A better understanding of the role ERRα plays in physiology will allow further development of ERR-PA as a potential therapy for liver steatohepatitis, which progresses to end stage cirrhosis and ultimately liver cancer. Citation Format: Brittney A. Hua, Chien-yu Chen, Yang Li, Lina He, Bangyan Stiles, Ielyzaveta Slarve. Investigating the transcriptional regulation by estrogen-related receptor alpha (ERRα) under different metabolic conditions. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4847.

Role of Hepatocyte Growth Regulators in Liver Regeneration

We have studied whether growth factors, cytokines, hormones, neurotransmitters, and local hormones (autacoids) promote the proliferation of hepatic parenchymal cells (i.e., hepatocytes) using in vitro primary cultured hepatocytes. The indicators used for this purpose include changes in DNA synthesis activity, nuclear number, cell number, cell cycle, and gene expression. In addition, the intracellular signaling pathways from the plasma membrane receptors to the nucleus have been examined in detail for representative growth-promoting factors that have been found to promote DNA synthesis and cell proliferation of hepatocytes. In examining intracellular signaling pathways, the effects of specific inhibitors of presumed signaling factors involved have been pharmacologically confirmed, and the phosphorylation activities of the signaling factors (e.g., RTK, ERK, mTOR, and p70 S6K) have been evaluated. As a result, it has been found that there are many factors that promote the proliferation of hepatocytes (e.g., HGF, EGF, TGF-α, IL-1β, TNF-α, insulin, growth hormone (GH), prostaglandin (PG)), and serotonin (5-HT)), while there are very few factors (e.g., TGF-β1 and glucocorticoids) that inhibit the effects of growth-promoting factors. We have also found that 5-HT and GH promote the proliferation of hepatocytes via different autocrine factors (e.g., TGF-α and IGF-I, respectively). Using primary cultured hepatocytes, it will be possible to further study the molecular and cellular aspects of liver regeneration.

Comparative Transcriptome Analysis Provides Novel Molecular Events for the Differentiation and Maturation of Hepatocytes during the Liver Development of Zebrafish.

The liver plays an essential role in multiple biological functions including metabolism, detoxification, digestion, coagulation, and homeostasis in vertebrates. The specification and differentiation of embryonic hepatoblasts, the proliferation of hepatocytes, and the hepatic tissue architecture are well documented, but molecular events governing the maturation of hepatocytes during liver development remain largely unclear. In this study, we performed a comparative transcriptome analysis of hepatocytes that were sorted by flow cytometry from developing zebrafish embryos at 60, 72, and 96 hpf. We identified 667 up-regulated and 3640 down-regulated genes in hepatocytes between 60 and 72 hpf, 606 up-regulated and 3924 down-regulated genes between 60 and 96 hpf, and 1693 up-regulated genes and 1508 down-regulated genes between 72 and 96 hpf. GO enrichment analysis revealed that key biological processes, cellular components, and molecular functions in hepatocytes between 60 to 72 hpf, such as cell cycle, DNA replication, DNA repair, RNA processing, and transcription regulation, are mainly associated with the proliferation of hepatocytes. In addition to biological processes, cellular components, and molecular functions for cell proliferation, molecular functions for carbohydrate metabolism were enriched in hepatocytes during 72 to 96 hpf. KEGG enrichment analysis identified key signaling pathways, such as cell cycle, RNA degradation, ubiquitin-mediated proteolysis, ErbB and Hedgehog signaling, basal transcription factors, Wnt signaling, and glycan degradation, which are closely associated with cell proliferation or carbohydrate metabolism in hepatocytes between 60 to 72 hpf. Newly enriched signaling pathways in hepatocytes during 72 to 96 hpf include metabolisms of pyrimidine, purine, nicotinate and nicotinamide, caffeine, glycine, serine and threonine, ABC transporters, and p53 signaling that function in metabolisms of lipid, protein and energy, cellular secretion, or detoxification, indicating the functional maturation of hepatocytes between 72 to 96 hpf. These findings provide novel clues for further understanding the functional differentiation and maturation of hepatocytes during liver development.

Abstract 6278: Per- polyfluoroalkyl substance (PFAS) modulates steatosis and fibrogenic signaling in hepatocellular carcinoma cells

Abstract Perfluorooctanoic acid (PFOA), heptafluorobutyric acid (HFBA), and perfluorotetradecanoic acid (PFTA) are the per- and polyfluoroalkyl substances (PFAS) and organic pollutants that predominantly exist in the environment. Exposure to PFAS affects immune, thyroid and kidney function, lipid and insulin dysregulation, liver disease, and cancer. Mechanisms underlying the impact of low and physiologically relevant concentration of PFAS in liver diseases remain elusive. In the current study, we investigated the effect of PFOA, HFBA, and PFTA on cell proliferation, steatosis, and fibrogenic signaling in hepatocytes and liver cancer cell models. Exposure of PFOA and PFBA (10 to 1000 nM) specifically promoted cell proliferation in non-tumor derived HepaRG liver cells and in hepatocellular carcinoma HepG2 cells. PFASs increased the expression of TNFα and IL6 inflammatory markers and produced endogenous reactive oxygen species (ROS) production. Further, PFAS enhanced cell steatosis in liver (HepaRG and HepG2) cells which was accompanied by upregulation of steatotic, and fibrogenic markers as well as activated Unfolded Protein Response (UPR) signaling. RNA-seq data suggested that chronic exposures of PFOA modulated the expression of fatty acid/lipid metabolic gene expression that is involved in the development of fatty liver disease. Collectively our data suggests that chronic exposure to low concentration of PFAS may modulate cell steatosis and fibrogenic signaling in hepatocytes and HCC cells by activation of UPR signaling. Citation Format: Qi Qi, Suryakant Niture, Sashi Gadi, Deepak Kumar. Per- polyfluoroalkyl substance (PFAS) modulates steatosis and fibrogenic signaling in hepatocellular carcinoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6278.

Quantification of Hepatocellular Mitoses in a Toxicological Study in Rats Using a Convolutional Neural Network.

Convolutional neural networks (CNNs) have been recognized as valuable tools for rapid quantitative analysis of morphological changes in toxicologic histopathology. We have assessed the performance of CNN-based (Halo-AI) mitotic figure detection in hepatocytes in comparison with detection by pathologists. In addition, we compared with Ki-67 and 5-bromodesoxyuridin (BrdU) immunohistochemistry labeling indices (LIs) obtained by image analysis. Tissues were from an exploratory toxicity study with a glycogen synthase kinase-3 (GSK-3) inhibitor. Our investigations revealed that (1) the CNN achieved similarly accurate but faster results than pathologists, (2) results of mitotic figure detection were comparable to Ki-67 and BrdU LIs, and (3) data from different methods were only moderately correlated. The latter is likely related to differences in the cell cycle component captured by each method. This highlights the importance of considering the differences of the available methods upon selection. Also, the pharmacology of our test item acting as a GSK-3 inhibitor potentially reduced the correlation. We conclude that hepatocyte cell proliferation assessment by CNNs can have several advantages when compared with the current gold standard: it relieves the pathologist of tedious routine tasks and contributes to standardization of results; the CNN algorithm can be shared and iteratively improved; it can be performed on routine histological slides; it does not require an additional animal experiment and in this way can contribute to animal welfare according to the 3R principles.

Glucose and insulin regulate cell proliferation and glucose transporter (GLUT) synthesis in the liver of overfed geese

Abstract In order to detect the role of glucose and insulin in cell proliferation of primary goose hepatocytes, Tianfu Meat Geese were overfed and the primary goose hepatocytes were treated with single insulin (50 nmol/l), single glucose (5 mmol/l), glucose (5 mmol/l) and insulin (50 nmol/l), respectively. The related parameters of cell proliferation and glucose transporter expression were measured. The results showed that after overfeeding, the serum concentration of insulin and glucose increased ( P<Â 0.05), the mRNA level of Cyclin D1 ( CCND1), Glucose transporter 1 ( GLUT1) and Glucose transporter 2 ( GLUT2) increased and the mRNA level of P21 decreased ( P<Â 0.05). Compared with the control group and the single treatment groups of glucose or insulin, the co-treatment of glucose and insulin had more dyed green bromodeoxyuridine (BrdU)-positive cells, higher G2/M phase and proliferative index (PI), indicating that the cell proliferation of primary hepatocytes was more evident. Secondly, compared with the control group, the co-treatment of glucose and insulin significantly increased the mRNA expression levels of CCND1, Cyclin D2 ( CCND2) and Cyclin D3 ( CCND3) ( P<Â 0.05). Compared with the control group, single treatments of glucose or insulin, and the co-treatment with glucose and insulin significantly increased the Cyclin D1 protein content ( P<Â 0.05). Also, compared with the control group, the co-treatment of glucose and insulin increased significantly ( P<Â 0.05) the mRNA levels of GLUT1 and GLUT2. In conclusion, the combined treatment of glucose and insulin had a stronger effect on cell proliferation, and GLUT1 and GLUT2 could indirectly induce cell proliferation of goose hepatocytes by regulating glucose uptake and insulin secretion of hepatocytes.

THE CHEMOTHERAPEUTIC ROLE OF p-MCA IN AVERTING NDEA INDUCED HEPATIC CARCINOGENESIS IN EXPERIMENTAL WISTAR RATS

OBJECTIVES: Hepatocellular carcinoma (HCC) is the primary liver cancer and second leading cause of cancer related deaths worldwide. The aim of this present study was to evaluate the biochemical, histopathological and chemotherapeutic efcacy of p-methoxycinnamic acid (p-MCA) against N- nitrosodiethylamine (NDEA) in a rat model of hepatocellular carcinoma. MATERIALS AND METHODS: Approximately thirty male wistar rats weighing 150-200 g were designated for this study. The rats were arbitrarily separated into ve groups and each group comprised of six rats. Group 1 served as control; Group 2 rats received p-MCA at the dose of 80 mg/kg b.w. Group 3, 4 and 5 rats were induced HCC using NDEA. 2-acetylaminouorene (AAF) was used as a promotor. Group 4 and 5 rats received p-MCA at the doses of 40 and 80 mg/kg b.w. throughout the 12 week experimental period. At the end of the experimental period, liver tissues from all the rats were collected and liver specic enzymes, lipid peroxidation, markers, xenobiotic metabolizing enzymes, antioxidant status and brotic markers were evaluated.RESULTS: NDEA administration induced hepatocyte damage, oxidative stress, cell proliferation, inammation and brosis. The liver sections from NDEA induced group 3 rats showed loss of lobular architecture, morphological changes in the nuclei and DNA damage. Administration of p-MCA to NDEA treated rats restored the hepatic architecture, enzyme activities, cell proliferation, inammation and brosis.CONCLUSION: We conclude that oral administration of p-MCA for 12 weeks exerts a signicant therapeutic effect against HCC by regulating the concentration of specic hepatic and xenobiotic enzymes, suppressing oxidative stress, inhibiting cell proliferation and reducing the inammatory response.

Role of forkhead box protein O1 and insulin on cell proliferation mediated by sirtuin 1 in goose primary hepatocytes

Summary To explore the role of insulin and forkhead box protein O1 (FoxO1) on cell proliferation regulated by sirtuin 1 (Sirt1) in goose primary hepatocyte, goose primary hepatocytes were isolated and incubated with Sirt1 inhibitor and nicotinamide, and then, some hepatocytes were transfected with FoxO1 interference plasmid or treated with insulin. The indexes related with cell proliferation were measured. The results first showed Sirt1 inhibition induced by nicotinamide increased cell proliferation in goose primary hepatocytes (P 0.05), protein content of p21 (P > 0.05), and cell proliferative index (PI) (67.40 vs. 69.00%). Finally, the PI of cotreatment with nicotinamide and insulin (56.65%) was lower than the single nicotinamide treatment (67.40%); the DNA synthesis rate in cotreatment of nicotinamide and insulin was lower than the single nicotinamide treatment (P

Luteoloside Ameliorates Palmitic Acid-Induced in Vitro Model of Non-alcoholic Fatty Liver Disease via Activating STAT3-Triggered Hepatocyte Regeneration

Luteoloside (Lute), a bioactive natural ingredient, widely exists in nature and possesses hepatoprotective and hepatocyte proliferation-promoting properties. This study aimed to investigate whether Lute could counteract non-alcoholic fatty liver disease (NAFLD)-caused hepatocyte damage via its stimulation of hepatocyte regeneration efficacy and to explore the involved mechanism. LO2 cells and primary hepatocytes were used to examine the hepatocyte proliferation effects of Lute under physiological conditions and in the palmitic acid (PA)- induced in vitro model of NAFLD. STAT3 and cell cycle-related proteins (cyclin D1, c-myc and p21) were evaluated by Western blot. Under physiological conditions, LO2 cells and primary hepatocytes treated with various concentration of Lute for 12 and 24 h showed increased hepatocyte proliferation, especially with 20 μM treatment for 24 h. More notably, under the model conditions, co-incubation with 20 μM of Lute also markedly reversed PA-induced inhibition of cell proliferation and viability in primary hepatocytes. Mechanistically, Lute could activate STAT3 and subsequently increase cyclin D1 and cmyc expression, which positively regulates cell cycle progression, and decrease expression of p21, an inhibitor of cell cycle progression. Furthermore, Luteinduced hepatocyte proliferation-promoting efficacy was abolished by STAT3 inhibitor stattic. Collectively, Lute can alleviate PA-induced hepatocyte damage via activating STAT3-mediated hepatocyte regeneration.

Hypoxia causes sex-specific hepatic toxicity at the transcriptome level in marine medaka (Oryzias melastigma)

Hypoxia, a low environmental oxygen level, is a common problem in the ocean globally. Hypoxia has been known to cause disruption to the endocrine system of marine organisms in both laboratory and field studies. Our previous studies have demonstrated the sex-specific response to hypoxia in the neural and reproductive systems of marine fish. In the current report, we aim to study the sex-specific hepatic response of fish at the transcriptome level to hypoxic stress. By using a comparative transcriptome analysis, followed by a systematic bioinformatics analysis including Database for Annotation, Visualization and Integrated Discovery (DAVID) and Ingenuity Pathway Analysis (IPA), we found that hypoxia altered expression of genes related to cell proliferation and apoptosis of hepatocytes, which are associated with human pathologies, such as liver inflammation hepatic steatosis and steatohepatitis. Furthermore, we observed sex-specific responses in the livers of fish through different cell signaling pathways. In female fish, hypoxia causes dysregulation of expression of genes related to impairment in endoplasmic reticulum structure and liver metabolism. In male fish, genes associated with redox homeostasis and fatty acid metabolism were altered by hypoxic stress. The findings of this study support the notion that hypoxia could cause sex-specific changes (hepatic toxicity and changes) in marine fish.

The concentration of tumor necrosis factor-\u03b1 determines its protective or damaging effect on liver injury by regulating Yap activity

Previous studies have shown that tumor necrosis factor (TNF)-α is a mediator of hepatotoxicity in liver injury. Moreover, TNF-α has also been reported to have a protective effect in liver regeneration, yet the function of TNF-α during liver injury remains controversial. Here, we report that the concentration of TNF-α determines its functions. High concentrations of TNF-α could aggravate LPS-induced liver injury. However, the TNF-α level was unchanged during APAP-induced liver injury, which exerted a protective effect. We expected that the concentration of TNF-α may affect its function. To test this hypothesis, TNF-α−/− rats or hepatocyte cells were treated with different concentrations of TNF-α. We found low TNF-α could reduce the levels of ALT and AST in the plasma of TNF-α−/− rats and promote the proliferation of hepatocyte cells. However, the levels of ALT and AST increased gradually with increasing TNF-α concentration after reaching the lowest value. Moreover, we showed that TNF-α affects the cell proliferation and cell death of hepatocytes by regulating Yap activity. Low TNF-α promoted Yap1 nuclear translocation, triggering the proliferation of hepatocytes. However, high TNF-α triggered the phosphorylation and inactivation of Yap1, preventing its nuclear import and consequently promoting cell death. Collectively, our findings provide novel evidence that the concentration of TNF-α is an important factor affecting its function in liver injury, which may provide a reference for the clinical treatment of liver injury.

1874-P: A Novel Role for SerpinB1 in the Regulation of Insulin Signaling in Hepatocytes

Compensatory expansion of β cell mass has been pursued as a potential therapeutic strategy to cure diabetes. We previously identified SerpinB1 as a hepatocyte-derived circulating factor that contributes to adaptive beta cell expansion in the liver-specific insulin receptor knockout (LIRKO) mouse. To understand how SerpinB1 is regulated and secreted, we began by first investigating the mechanism(s) and signaling pathways that modulate the gene and protein expression of SerpinB1 in hepatocytes. Hepatic SerpinB1 expression was elevated in insulin resistant mouse models such as db/db and ob/ob, and circulating interleukin-6 (IL-6) levels were higher in these mice. Indeed, IL-6 induced SerpinB1 expression in HepG2 cells and human primary hepatocytes via the JAK/STAT3 pathway, indicating an important role for this cytokine in inducing the expression of the protease inhibitor in insulin resistant states. Conversely, silencing of SERPINB1 gene in hepatocytes decreased INSR gene and protein expression with a consequent alteration of expression of several downstream signaling genes related to gluconeogenesis and cell proliferation in hepatocytes. Ribosomal protein S3 (RPS3), identified as a binding partner of SerpinB1 in HepG2 cells, exhibited reduced expression in db/db and ob/ob mice liver along with reduced insulin receptor (IR) expression. Silencing of RPS3 gene in HepG2 cells, phenocopied the effects of SerpinB1 gene silencing, and decreased IR protein expression with consequent alterations in downstream signaling proteins. Additional studies are required to examine the precise regulation of IR expression by SerpinB1 and how the latter is secreted from insulin resistant hepatocytes. These results indicate that SerpinB1 expression is upregulated in insulin resistance by IL-6 and the SerpinB1/RPS3 complex plays a role in regulating insulin signaling in hepatocytes. Disclosure K. Orime: None. D.F. De Jesus: None. U. Jhala: None. R. Kulkarni: None.

Retraction notice to: "Role of Mammalian sirtuin 1 (SIRT1) in lipids metabolism and cell proliferation of goose primary hepatocytes" MCE Volume 382, Issue 1, 25 January 2014, Pages 282-291.

Retraction notice to: "Role of Mammalian sirtuin 1 (SIRT1) in lipids metabolism and cell proliferation of goose primary hepatocytes" MCE Volume 382, Issue 1, 25 January 2014, Pages 282-291.

A Novel Role for SerpinB1, a Protease Inhibitor, in Hepatic Biology

SerpinB1, a protease inhibitor that is secreted from the liver in response to insulin resistance, was recently reported to promote pancreatic β cell proliferation. We focused on investigating the significance of SerpinB1 in the liver to gain insights into its role in regulating whole body metabolism. We determined SerpinB1 gene expression in multiple metabolic tissues harvested under fasting or fed states from ∼34-week-old male Liver-specific insulin receptor knockout (LIRKO) mice and littermate controls. While in controls SerpinB1 expression was reduced by ∼50% after feeding when compared to the fasting state, (P=0.02, n=4-5), the LIRKOs, in contrast, exhibited a 2.3-fold higher (P=0.01, n=5-6) expression in the fed vs. fasting states and the higher levels were consistent with earlier observations in the LIRKO livers. We next took advantage of the availability of liver samples from 18 to 24-week-old male SerpinB1 KO mice and littermate controls and subjected them to RNA-sequencing. As expected, SerpinB1 gene expression was absent in the KO samples, and interestingly, was associated with a significant decrease in gene expression related to cell cycle, NF-kappa B signaling, and inflammatory pathways. Independent validation of these data by RT-qPCR confirmed a decrease in cell cycle-related genes such as Ccna2 (-60%, P=0.04), Ccnb2 (-54%, P=0.04), Ccnd1 (-46%, P=0.03), and Ccne1 (-51%, P=0.07), and the cell proliferation marker, Mki67 (-60%, P=0.00) (n=5). Furthermore, knockdown of SerpinB1 in the human hepatocyte cell line, HepG2, exhibited significantly decreased expression of Ccnb1 (-36%, P=0.01), Ccnb2 (-44%, P=0.00), Ccne1 (-21%, P=0.00), MKi67 (-30%, P=0.00), and MCM2 (-29%, P=0.00) genes, and increased expression of P21 (+165%, P=0.00) (n=3 for control vs. knockdown groups). These novel data implicate endogenous SerpinB1 in cell proliferation in hepatocytes and warrant further investigation of its role in hepatocyte function and the overall impact on glucose homeostasis. Disclosure K. Orime: None. D.F. De Jesus: None. R. Kulkarni: None.

MiR-1224 inhibits cell proliferation in acute liver failure by targeting the antiapoptotic gene Nfib

Patient outcome in acute liver failure (ALF) is crucially determined by the appropriate balance between cell death and compensatory cell proliferation. MicroRNAs (miRNAs) - small non-coding RNAs that function as guide molecules in RNA silencing - have evolved as crucial mediators of nearly all developmental and pathological processes, including the physiology and pathology of the liver. We investigated the role of miR-1224 during ALF. We measured miR-1224 in livers of mice in various acute liver disease murine models and in, patients with ALF, using quantitative real-time PCR. We studied the regulation of miR-1224 in AML12 cells and primary hepatocytes upon H2O2 stimulation. Cell proliferation and cell death were analysed by 5-bromo-2'-deoxyuridine and terminal deoxynucleotide transferase nick end labelling stainings, respectively. We found that miR-1224 was up-regulated in hepatocytes upon ischaemia-reperfusion in vivo and in vitro. This was accompanied by impaired proliferation and elevated apoptosis. This function of miR-1224 was mediated by repressing the anti-apoptotic gene Nfib in hepatocytes. Strikingly, miR-1224 was also up-regulated in human livers and the serum of patients with ALF and indicated an unfavourable prognosis with an excellent prognostic value compared to other known serum markers in this clinical setting. miR-1224 is a previously unrecognised regulator of proliferation after ALF in hepatocytes and represents a novel and specific biomarker of liver injury with prognostic value in ALF. Thus, miR-1224 may represent a target for novel therapeutic and diagnostic strategies in the context of ALF and warrants further testing as a biomarker in prospective trials. Lay summary: In acute liver failure, miR-1224 expression is modulated by oxidative stress. This leads to a decrease in hepatocyte cell proliferation and increase in apoptosis. Increased serum levels of miR-1224 could be a useful diagnostic marker in patients with acute liver failure.

Prohibitin 1 Regulates the H19-Igf2 Axis and Proliferation in Hepatocytes

Prohibitin 1 (PHB1) is a mitochondrial chaperone that regulates cell growth. Phb1 knock-out mice exhibit liver injury and hepatocellular carcinoma (HCC). Phb1 knock-out livers show induction of tumor growth-associated genes, H19 and insulin-like growth factor 2 (Igf2). These genes are controlled by the imprinting control region (ICR) containing CCCTC-binding transcription factor (CTCF)-binding sites. Because Phb1 knock-out mice exhibited induction of H19 and Igf2, we hypothesized that PHB1-mediated regulation of the H19-Igf2 axis might control cell proliferation in normal hepatocytes. H19 and Igf2 were induced (8-20-fold) in 3-week-old Phb1 knock-out livers, in Phb1 siRNA-treated AML12 hepatocytes (2-fold), and HCC cell lines when compared with control. Phb1 knockdown lowered CTCF protein in AML12 by ∼30% when compared with control. CTCF overexpression lowered basal H19 and Igf2 expression by 30% and suppressed Phb1 knockdown-mediated induction of these genes. CTCF and PHB1 co-immunoprecipitated and co-localized on the ICR element, and Phb1 knockdown lowered CTCF ICR binding activity. The results suggest that PHB1 and CTCF cooperation may control the H19-Igf2 axis. Human HCC tissues with high levels of H19 and IGF2 exhibited a 40-50% reduction in PHB1 and CTCF expression and their ICR binding activity. Silencing Phb1 or overexpressing H19 in the mouse HCC cell line, SAMe-D, induced cell growth. Blocking H19 induction prevented Phb1 knockdown-mediated growth, whereas H19 overexpression had the reverse effect. Interestingly H19 silencing induced PHB1 expression. Taken together, our results demonstrate that the H19-Igf2 axis is negatively regulated by CTCF-PHB1 cooperation and that H19 is involved in modulating the growth-suppressive effect of PHB1 in the liver.

Hypoxia-driven Hif2a coordinates mouse liver regeneration by coupling parenchymal growth to vascular expansion.

Oxygen is a key regulator of liver regeneration. Hypoxia-inherent to the expansion of parenchyme-activates Hif2a to couple hepatocyte mitosis with the angiogenic phase. Hif2a acts as a safeguard to initiate sinusoidal reconstruction only upon successful hepatocyte mitosis, thereby enforcing a timely order onto cell type-specific regeneration patterns. These findings portray the hypoxia-driven Hif2a-Vegf axis as a prime node in coordinating sinusoidal endothelial cell-hepatocyte crosstalk during liver regeneration. (Hepatology 2016;64:2198-2209).