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CYP2A5 is a major enzyme responsible for nicotine and cotinine metabolism in mice. Nicotine and cotinine enhance alcoholic fatty liver in wild type (WT) mice but not in CYP2A5 knockout (KO) mice, and reactive oxygen species (ROS) generated during the CYP2A5-mediated metabolism contributes to the enhancing effect. In combination with ethanol, nicotine and cotinine increased lipid peroxidation end product 4-hydroxynonenal (HNE) in WT mice but not in KO mice. In ethanol-fed KO mice, only 5 and 10 genes were regulated by nicotine and cotinine, respectively. However, in ethanol-fed WT mice, 59 and 104 genes were regulated by nicotine and cotinine, respectively, and 7 genes were up-regulated by both nicotine and cotinine. Plin 2 and Cdkn1a are among the 7 genes. Plin2 encodes adipose differentiation-related protein (ADRP), a lipid droplet-associated protein, which was confirmed to be increased by nicotine and cotinine in WT mice but not in KO mice. Cdkn1a encodes P21 and elevated P21 in nuclei was also confirmed. HNE can increase P21 and P21 inhibit cell proliferation. Consistently, hepatocyte proliferation markers proliferating cell nuclear antigen (PCNA) and Ki67 were decreased in WT mice but not in KO mice by nicotine/ethanol and cotinine/ethanol, respectively. These results suggest that inhibition of liver proliferation via a ROS-HNE-P21 pathway is involved in nicotine- and cotinine-enhanced alcoholic fatty liver., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Although the regeneration of the adult liver depends on hepatic progenitor cells (HPCs), many uncertainties regarding hepatic regeneration in the injured liver remain. Trefoil factor family 1 (TFF1), a secretory protein predominantly expressed in the gastrointestinal tract, is responsible for mucosal restitution. Here, we investigated the role of TFF1 in liver regeneration using a mouse model of hepatic injury (choline-deficient ethionine-supplemented diet and carbon tetrachloride administration) and genetically engineered mice (TFF1 knockout (TFF1-/-)). Immunohistochemistry analysis of human liver samples revealed TFF1 expression in the hepatocytes close to ductular reaction and the regenerating biliary epithelium in injured liver. The number of cytokeratin 19 (CK19)-positive bile ducts was significantly decreased in the TFF1-/- mice after liver injury. Notch pathway in the TFF1-/- mice was also downregulated. HPCs in the control mice differentiated into biliary cells (CK19 + /SRY HMG box 9 (SOX9) + ) more frequently. In contrast, HPCs in the TFF1-/- mice more frequently differentiated into a hepatic lineage (alpha fetoprotein + /SOX9 + ) after acute liver damage. Hepatocyte proliferation was upregulated, and the liver weight was increased in TFF1-/- mice in response to chronic liver damage. Thus, TFF1 is responsible for liver regeneration after liver injury by promoting HPC differentiation into a biliary lineage and inhibiting HPC differentiation into a hepatic lineage., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Background/aim: Augmenter of liver regeneration (ALR) is a potent growth factor which supports liver regeneration in experimental animals. The aim of this study was to compare proliferation as well as the kinetics of ERK1/2 and Akt/PKB phosphorylation by recombinant human ALR (rhALR) and EGF in human hepatocytes and extrahepatic cells., Methods: Kinetics of ERK1/2 and Akt/PKB phosphorylation were determined in primary human hepatocytes (phh) after stimulation with rhALR and EGF. Induction of proliferation was analyzed in phh and several cell lines of hepatic and extrahepatic origin by the MTT and [(3)H]-thymidine assay., Results: The kinetics of ERK phosphorylation showed clear differences, whereby rhALR caused a transient and EGF a permanent increase during the observation period of 60 min. For both, Akt and ERK phosphorylation, EGF caused a faster effect with maximal levels observed already after 2 min, whereas rhALR caused maximal phosphorylation between 10 and 15 min. Using the EGF receptor inhibitor AG1478 we provide evidence of an EGF receptor independent induction of proliferation by rhALR. Furthermore, rhALR induced proliferation only in phh and the human liver derived cell lines HepG2 and Chang. In contrast, EGF enhanced proliferation in all analyzed cell types including cell lines of colon, bronchial, pancreatic and gastric origin (SW480, BC1, L36PL and GC1)., Conclusion: rhALR and EGF induce different kinetics of ERK and Akt phosphorylation in human hepatocytes. The mitogenic effect of rhALR is liver specific and seems to be at least partially independent from EGF receptor mediated signaling., (2010 Elsevier Inc. All rights reserved.)

Different stages of liver regeneration are regulated by a variety of factors such as the liver growth associated protein ALR, augmenter of liver regeneration. Furthermore, small molecules like polyamines were proven to be essential for hepatic growth and regeneration. Therefore, using primary human hepatocytes in vitro we investigated the effect of ALR on the biosynthesis of polyamines. We demonstrated by HPLC analysis that recombinant ALR enhanced intracellular hepatic putrescine, spermidine, and spermine levels within 9-12h. The activation of polyamine biosynthesis was dose dependent with putrescine showing the strongest increase. Additionally, ALR treatment induced mRNA expression of ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase, both key enzymes of polyamine biosynthesis. Further, ALR induced c-myc mRNA expression, a regulator of ODC expression, and therefore we assume that ALR exerts its liver regeneration augmenting effects through stimulation of its signalling pathway leading in part to enhanced polyamine synthesis.

Retroviral vectors are widely used tools for gene therapy. However, in vivo gene transfer is only effective in dividing cells, which, in liver, requires a regenerative stimulus. Follistatin is effective in promoting liver regeneration after 90% and 70% hepatectomy in rats. We studied its efficacy on liver regeneration and retroviral-mediated gene delivery in 50% hepatectomized rats. When human recombinant follistatin was infused into the portal vein immediately after 50% hepatectomy, hepatocyte proliferation was significantly higher than in control 50% hepatectomized rats. A single injection of virus particles administered 23 h after follistatin infusion resulted in more than 20% gene transduction efficiency in hepatocytes compared to 3% in control rats. It is concluded that a single injection of follistatin induces onset of proliferation in 50% hepatectomized rats and allows efficient retroviral-mediated gene transfer to the liver.

The liver has the unique ability to regenerate even in adulthood. While mature hepatocytes can proliferate by themselves, stem cells also play a critical role in liver regeneration and oval cells are considered to be the progeny of activated hepatic stem cells. We herein investigated the gene expression profiles in the conditions inducing oval cells, using microarray analysis. Two approaches were used to induce oval cells. In the first, animals were treated with a combination of an N-2-acetylaminofluorene (AAF)-containing diet and partial hepatectomy (PHx). In the second, animals were supplied with chow containing a 1:1 mixture of choline-deficient and normal diets, as well as 0.075% ethionine in drinking water. Using in-house cDNA microarrays consisting of 2304 cDNA clones from the mouse liver, 69 and 89 genes, respectively, were found to be up-regulated in these two models. Six genes, i.e., those for insulin-like growth factor binding protein-1, CYP4a14, carnitine octanoyltransferase, osteopontin, and two expressed sequence tags (ESTs) were up-regulated in these models but not in ordinary model with PHx alone. They might be specifically activated in the induction of oval cells, and help to clarify the nature of stem cell stimulation that occurs during liver regeneration.

Hepatocyte growth factor (HGF) is a potent mitogen for a variety of cells including hepatocytes. While rat oval cells are supposed to be one of hepatic stem cells, biological effects of HGF on oval cells and their relevant signal transduction pathways remain to be determined. We sought to investigate them on OC/CDE22 rat oval cells, which are established from the liver of rats fed a choline-deficient/DL-ethionine-supplemented diet. The oval cells were cultured on fibronectin-coated dishes and stimulated with recombinant HGF, transforming growth factor-alpha (TGF-alpha), and thrombopoietin (TPO) under the serum-free medium condition. HGF treatment enhanced [3H]thymidine incorporation into oval cells in a dose-dependent manner. On the contrary, treatment with TGF-alpha or TPO had no significant effects on [3H]thymidine incorporation into the oval cells. c-Met protein was phosphorylated at the tyrosine residues after the HGF treatment. AKT, extracellular signal-regulated kinase 1/2 (ERK1/2), and p70(s6k) were simultaneously activated after the HGF stimulation, peaking at 30min after the treatment. The activation of AKT, p70(s6k), and ERK1/2 induced by HGF was abolished by pre-treatment with LY294002, a phosphoinositide 3-OH kinase (PI3K) inhibitor, and U0126, a mitogen-activated protein kinase/ERK kinase (MEK) inhibitor, respectively. When the cells were pre-treated with LY294002 prior to the HGF stimulation, the proliferative action of HGF was completely abrogated, implying that the PI3K/AKT signaling pathway is responsible for the biological effect of HGF. These in vitro data indicate that HGF exerts a proliferative action on hepatic oval cells via activation of the PI3K/AKT signaling pathway.

Liver architecture remodeling following partial hepatectomy (PHx) involves the formation of a complex network of liver sinusoids through which the blood flows. The present study examines the involvement of vascular endothelial growth factor (VEGF) and angiopoietin-1 (ang-1) during liver regeneration. Following PHx, VEGF and ang-1 mRNA levels increase, followed by gradual return to baseline levels. RT-PCR analysis of VEGF mRNA reveals three isoforms, VEGF120, VEGF164 and VEGF188. Of the three, VEGF188 is the predominant isoform, VEGF120 being the less abundant. Although VEGF mRNA fluctuates following PHx, the relative expression of each isoform remains the same throughout the recovery process. The level of neuropilin-1, an accessory receptor of VEGF to main receptor corresponds with that of VEGF and ang-1. We have previously demonstrated the capacity of exogenous VEGF165 to stimulate liver cell proliferation following PHx. We now report similar effect using VEGF121, further demonstrating the benefit of manipulating growth factors where such an intervention is required., (Copyright 2001 Academic Press.)

The aim of our study was to detect four P450s (CYP1A, CYP2B, CYP2E1, CYP3A) on the basis of selective enzyme activities and protein amount, and to investigate the effect of dexamethasone treatment during liver regeneration. Partial hepatectomy of rats resulted in the loss of CYP1A, CYP2B, CYP2E1, and CYP3A activities. The reduction of enzyme activities and the loss of enzyme protein of CYP2B1/2, CYP2E1, and CYP3A1/2 were the most pronounced. In the case of CYP1A1, only slight decrease was observed. Dexamethasone treatment seems to counteract this loss mainly in the first 12 h., (Copyright 2001 Academic Press.)

In fulminant hepatic failure, survival is not possible without recovery of sufficient hepatocyte mass. Remarkably, only a few studies exist that provide insight into the mechanisms that control proliferation of residual hepatocytes after extensive hepatocyte loss. In this regard, the role of growth-regulatory factors, including pro-inflammatory cytokines such as interleukin-6 (IL-6), is not well understood. In the present study we show that in rats with critically low (10%) hepatocyte mass, whether with or without ongoing liver cell necrosis, inhibition of liver regeneration is associated with early and sustained increase in blood IL-6 levels. Under these conditions, the signal transducer and activator of transcription (Stat3) DNA binding activity was lowered at the time of G1/S cell-cycle transition. We further demonstrate that the protein inhibitor of activated Stat3 (PIAS3) and the suppressor of cytokine signaling (SOCS-1) were up-regulated early after induction of liver failure (6-12 h). In vitro, IL-6 induced PIAS3 expression in HGF stimulated rat hepatocytes. These findings suggest that after massive hepatocyte loss, an early and rapid rise in blood IL-6 levels may weaken the hepatic regenerative response through up-regulation of Stat3 inhibitors PIAS3 and SOCS-1., (Copyright 2000 Academic Press.)

Regulation in expression and activation of proteinases is one of the most important mechanisms in organ morphogenesis. In this study, we investigated the expression of MMPs in primary hepatocytes and their roles in liver remodeling. A hepatocyte proliferation initiating cytokine, TNFalpha, induced MMP-9 expression in these cells while the expression of MMP-2 did not change by zymography analysis. Interestingly, both the induced MMP-9 expression and hepatocyte proliferation by TNFalpha were synergistically enhanced by HGF in vitro. The increased proliferation was suppressed by MMP inhibitor TIMP-1, suggesting that cytokine-induced MMP regulates proliferation. The increased expression of MMP-9 by the cytokines was inhibited by cytochalasin D or colchicine but not by PI3 kinase inhibitor wortmannin. In addition, co-stimulation by TNFalpha and HGF of spheroidal hepatocytes cultured in 3-dimensional collagen gel drastically induced morphological changes by cell extension and migration in the gel, which was in parallel with the induced expression of MMP-9 and was inhibited by TIMP-1 and -2. The MMP activity was also detected in vivo in the regenerating liver after partial hepatectomy by in situ zymography. These results suggest the roles of MMPs produced by parenchymal cells in liver remodeling., (Copyright 2000 Academic Press.)

We have investigated the expression of several growth-related genes in the liver after partial hepatectomy in three experimental models: normal, Dexamethasone-pretreated, and hypophysectomized rats. Dexamethasone and hypophysectomy resulted in a delay in the peak of cell replication in 6 and 18 h, respectively, when compared to the normal animals. TGFalpha mRNA expression was shifted together with the DNA synthesis, but the expression of c-myc, c-fos, c-jun, HGF, TGFbeta1, IL1beta did not delay. This result suggests that liver-derived TGFalpha but not the other factors are important in the timing of the proliferative response after partial hepatectomy., (Copyright 2000 Academic Press.)

Metallothioneins (MTs) are low molecular weight, zinc-binding proteins that by activating zinc metalloenzymes participate in the regulation of growth and development. The present study was designed to examine the roles of MTs in cell proliferation using an in vivo model of liver regeneration following partial hepatectomy (PH) in rats. The levels of MT-I and MT-II were studied with respect to regulation of proliferative potential, cell cycle checkpoint activity, and oxidative stress in the rat PH model. We synthesized a 17-mer antisense phosphorothioate oligodeoxynucleotide (S-ODN), named aMT, complimentary to the start site of the MT-I mRNA sequence and an appropriate control. Both S-ODNs were administered intraperitoneally at the dose of 5 mg/kg following 70% PH. MT became induced 57.4 +/- 9.8-fold following PH and the said effect became attenuated dramatically following administration of aMT. In addition, PH rats treated with aMT exhibited decreased rate of liver regeneration as measured by expression of proliferating cell nuclear antigen and elevated cell cycle checkpoint activity as determined by expression of p53. The results of these studies suggest that MT isoforms with their high thiol contents do play an important role in cellular functions and especially during stressful states induced by a broad range of mediators generating free radicals.

Interleukin-6 (IL-6) induced activation of Signal Transducer and Activator Transcription Factor 3 (Stat3) is a critical step in liver regeneration. Chronic ethanol consumption is known to increase the plasma concentration of IL-6, yet the ability of the liver to regenerate and the regenerative induction of several IL-6 initiated events are impaired in chronic alcoholic liver disease. We hypothesized that chronic ethanol consumption inhibits IL-6 dependent signal transduction. To test this hypothesis, the effect of ethanol on the Stat3 signal transduction pathway was studied in the adult rat liver. In vitro treatment of freshly isolated normal adult rat hepatocytes with 50-100 mM ethanol for 30 min blocked IL-6-induced Stat3 activation. Long-term ethanol intake in vivo significantly attenuated the activation of Stat3 induced either in vivo by partial hepatectomy or in vitro by IL-6. In contrast, short-term ethanol consumption enhanced the regenerative induction of Stat3 but inhibited IL-6 induced Stat3 activation. These data suggest that the inhibition of liver regeneration by chronic ethanol consumption is, at least in part, mediated by modulating the activation of Stat3.

The effect of human hepatocyte growth factor on the proliferation of intrasplenically transplanted hepatocytes was investigated. A human hepatocyte growth factor (360 micrograms/kg BW) was administered intravenously every 24 hr for 5 days following hepatocyte transplantation in rats. A significant increase in hepatocyte proliferation was observed morphologically and immunohistochemically (p < 0.05). The bromodexyuridine labeling index was significantly greater in human hepatocyte growth factor treated rats than in the control rats (p < 0.05). Hepatocyte growth factor may promote the proliferation of intrasplenically transplanted hepatocytes.

Hepatocyte growth factor (HGF), originally identified as a potent mitogen for mature parenchymal hepatocytes, is a hepatotrophic factor involved in liver regeneration and is even essential for development of the liver. We report here that human recombinant HGF at a very low concentration and given intraportaly stimulated liver regeneration in dogs. In vitro, HGF dose-dependently stimulated DNA synthesis of primary cultured hepatocytes isolated from a dog. The maximal activity was twofold higher than that of epidermal growth factor, and insulin potentiated the mitogenic activity of HGF. When human recombinant HGF was infused through the portal vein into 30% partially hepatectomized dogs at 0.25 microg/kg body weight in order to directly target the liver, HGF stimulated DNA synthesis of hepatocytes and liver weight at 72 h after the operation; labeling indices in saline- and HGF-injected groups were 0.75 and 1.82%, respectively, and the liver weights in saline- and HGF-injected groups were 302 and 374 g, respectively. Since HGF exerts potent antihepatitis activity as well as mitogenic activity, these results indicate that intraportal administration of HGF may be particularly important to enhance liver regeneration and prevent the severe hepatic insufficiently after hepatic surgery.

Chronic ethanol exposure inhibits the capacity of the liver to regenerate. Insulin is a potent hepatotrophic factor and it was determined if ethanol interferes with insulin receptor substrate (IRS-1)-protein mediated signal transduction during liver regeneration. Tyrosyl phosphorylation of IRS-1 was strikingly increased prior to the major wave of DNA synthesis in isocaloric pair-fed control rats; a blunted and delayed response was found in ethanol-fed rats. Enzymatic activity of phosphatidylinositol 3-kinase, a Src homology 2 (SH2) domain containing signal transduction molecule was enhanced by the association with tyrosyl phosphorylated IRS-1, whereas in ethanol-fed rats, this activity was greatly diminished and delayed. These results indicate that one potential molecular mechanism whereby ethanol inhibits hepatocyte DNA synthesis is through its action on the IRS-1-mediated signal transduction cascade.

Gene expression of liver facilitated glucose transporter was rapidly induced during the liver regenerating process in rats. It reached maximum of 2.7 times at 8 hr of the regenerating course and returned to normal by 48 hr. The protein synthesis inhibitor, cycloheximide, did not interfere with the increased gene expression of liver facilitated glucose transporter. By contrast, erythrocyte/brain-type glucose transporter mRNA could not be detected in the livers of partially hepatectomized rats and sham-operated rats. The plasma glucose levels were transiently increased within 2 hr of the regenerative course and then decreased to a nadir at 4 hr. These results suggest that the increased gene expression of liver facilitated glucose transporter contributes to the decrease in plasma glucose levels.

In an attempt to elucidate the mechanisms that control the hepatic DNA replication, effect of low doses of actinomycin D on DNA synthesis and c-myc expression during the early stage of liver regeneration was investigated. Small amounts of actinomycin D, in amounts that had no effect on the rate of RNA synthesis in normal rats, were multiple injected at 0, 2 and 4 hours after partial hepatectomy. DNA synthesis and c-myc expression in these rats were compared with those in untreated rats. Hepatic DNA synthesis in the treated rats was delayed about 4 to 6 hours in comparison with control rats. In contrast, time course of c-myc expression in inhibitor treated rats was very similar to that in control rats.

The inhibitory action of transforming growth factor beta (TGF beta) on DNA synthesis in hepatocytes isolated from the liver of normal rats or from the liver remnant of rats 18 h following partial hepatectomy was compared. Continuous exposure to TGF beta inhibited DNA synthesis of cultured hepatocytes to a similar degree in both groups when labelled with 3H thymidine from 24-48 h or 48-72 h. At 20 pM TGF beta, 3H-thymidine incorporation was reduced by 64-78% in hepatocytes from normal liver and by 60-73% in cells from 18 h regenerating liver. The nuclear labelling index was reduced by 70-80% in all cells. Exposure to TGF beta at concentrations up to 500 pM from 0-24 h had no effect on 3H-thymidine incorporation, but exposure at 20 pM for 24 h periods thereafter was uniformally effective. These results indicate that there is no change in sensitivity of hepatocytes from 18 h regenerating liver to TGF beta, compared with normal cells, and that TGF beta may act at some point in the G1 phase of the cell cycle to inhibit hepatocyte growth.