Hepatotrophic Growth Factor Research Articles

Bile acids down-regulate the expression of Augmenter of Liver Regeneration (ALR) via SHP/HNF4α1 and independent of Egr-1

Bile acids (BA) are signaling molecules that activate nuclear factors and g-protein coupled receptors signaling to maintain metabolic homeostasis. However, accumulation of toxic BA promotes liver injury by initiating inflammation, inducing apoptosis and causing oxidative stress leading to cirrhosis and liver failure. Augmenter of Liver Regeneration (ALR) is a hepatotrophic growth factor with anti-apoptotic and anti-oxidative properties that has been shown to improve mitochondrial and hepatic functions in rats after bile duct ligation. In the current study we aimed to analyze the regulation of the pro-survival protein, ALR, under conditions of cytotoxic concentrations of BA. Promoter studies of ALR (−733/+527 bp) revealed potential binding sites for various transcription factors like Egr-1, HNF4α and two bile acid response elements (BARE). Using a full-length and several truncated promoter constructs for ALR we analyzed promoter activity and showed that BA reduce ALR promoter activity whereas Egr-1 transfection induces it. EMSA and supershift analysis confirmed the specific binding of Egr-1 to its response element within ALR promoter and this binding was reduced upon simultaneous stimulation with BA. We also showed that ALR promoter activity and protein expression are induced by HNF4α1 and repressed by SHP. In conclusion, these results indicate that BA negatively regulate ALR expression by SHP activation.

Lipid deposition in liver cells: The influence of short form augmenter of liver regeneration

The short form augmenter of liver regeneration (sfALR) is a novel human hepatotrophic growth factor. The aim of this study was to investigate the potential role of sfALR in NAFLD. The free fatty acids (FFA) induced lipid accumulation in mouse liver parenchymal cells was examined by Oil Red O staining and triglyceride level determination. The cell cycle was determined by flow cytometry and the proliferation was assessed by CCK8. The expression levels of gfer, miR-122, srebp-1c, fas, dgat2, acc1 and Lrp1B were assessed by quantitative real-time PCR. Furthermore, the MAPK pathway was detected by western blot. The results showed that sfALR could alleviate the lipid accumulation in mice both in vivo and in vitro. sfALR relieved the proliferation inhibition and G2 arrest of mouse liver parenchymal cells induced by FFAs. Free fatty acids affected gfer expression in a time-and dose-dependent way. And sfALR suppressed JNK activation, increased miR-122 level and reduced fatty acid synthesis-related gene expression. These findings suggested that sfALR could alleviate the severity of fatty liver in mice.

Hepassocin is required for hepatic outgrowth during zebrafish hepatogenesis

Background & aimsHepassocin (HPS) is a hepatotrophic growth factor that specifically stimulates hepatocyte proliferation and promotes liver regeneration after liver damage. In this paper, zebrafish were used to investigate the role of HPS in liver development. Methods and resultsDuring zebrafish development, HPS expression is enriched in liver throughout hepatogenesis. Knockdown of HPS using its specific morpholino leads to a smaller liver phenotype. Further results showed that the HPS knockdown has no effect on the expression of the early endoderm marker gata6 and early hepatic marker hhex. In addition, results showed that the smaller-liver phenotype in HPS morphants was caused by suppression of cell proliferation, not induction of cell apoptosis. ConclusionsCurrent findings indicated that HPS is essential to the later stages of development in vertebrate liver organogenesis.

Involvement of Hepatic Stimulator Substance in the Regulation of Hepatoblast Maturation into Hepatocytes In Vitro

Hepatic stimulator substance (HSS), also known as augmenter of liver regeneration (ALR), acts as a hepatotrophic growth factor to promote liver regeneration after liver damage or partial hepatectomy. However, the expression and function of HSS during liver development in mammals remain largely unknown. In this work, the hepatoblasts were isolated from mice at embryonic day 13.5 (E13.5), and HSS expression and its role during hepatoblast maturation were investigated. The results showed that HSS expression was enhanced in the hepatoblasts compared with mouse primary hepatocytes. HSS expression (23 kDa) was significantly decreased if the hepatoblast maturation was induced by a combination of oncostatin M (OSM), dexamethasone (DEX), and hepatocyte growth factor (HGF). We also found that knockdown of HSS expression (mainly 23-kDa isoform) by siRNA promoted hepatoblast maturation and also activated the signal transducer and activator of transcription 3 (STAT3) phosphorylation levels. However, if STAT3 activity was blocked by a small-molecule inhibitor Stattic, then hepatocyte maturation could be abolished, suggesting that STAT3 was most likely a potential molecule responsible for HSS signaling. In summary, our results demonstrated for the first time that HSS might be an active factor participating in the regulation of liver development and hepatocyte maturation.

Augmenter of Liver Regeneration (alr) Promotes Liver Outgrowth during Zebrafish Hepatogenesis

Augmenter of Liver Regeneration (ALR) is a sulfhydryl oxidase carrying out fundamental functions facilitating protein disulfide bond formation. In mammals, it also functions as a hepatotrophic growth factor that specifically stimulates hepatocyte proliferation and promotes liver regeneration after liver damage or partial hepatectomy. Whether ALR also plays a role during vertebrate hepatogenesis is unknown. In this work, we investigated the function of alr in liver organogenesis in zebrafish model. We showed that alr is expressed in liver throughout hepatogenesis. Knockdown of alr through morpholino antisense oligonucleotide (MO) leads to suppression of liver outgrowth while overexpression of alr promotes liver growth. The small-liver phenotype in alr morphants results from a reduction of hepatocyte proliferation without affecting apoptosis. When expressed in cultured cells, zebrafish Alr exists as dimer and is localized in mitochondria as well as cytosol but not in nucleus or secreted outside of the cell. Similar to mammalian ALR, zebrafish Alr is a flavin-linked sulfhydryl oxidase and mutation of the conserved cysteine in the CxxC motif abolishes its enzymatic activity. Interestingly, overexpression of either wild type Alr or enzyme-inactive AlrC131S mutant promoted liver growth and rescued the liver growth defect of alr morphants. Nevertheless, alr C131S is less efficacious in both functions. Meantime, high doses of alr MOs lead to widespread developmental defects and early embryonic death in an alr sequence-dependent manner. These results suggest that alr promotes zebrafish liver outgrowth using mechanisms that are dependent as well as independent of its sulfhydryl oxidase activity. This is the first demonstration of a developmental role of alr in vertebrate. It exemplifies that a low-level sulfhydryl oxidase activity of Alr is essential for embryonic development and cellular survival. The dose-dependent and partial suppression of alr expression through MO-mediated knockdown allows the identification of its late developmental role in vertebrate liver organogenesis.

Augmenter of liver regeneration (ALR) protects human hepatocytes against apoptosis

Augmenter of liver regeneration (ALR) is known to support liver regeneration and to stimulate proliferation of hepatocytes. However, it is not known if ALR exerts anti-apoptotic effects in human hepatocytes and whether this protective effect is cell type specific. This is relevant, because compounds that protect the liver against apoptosis without undesired effects, such as protection of metastatic tumour cells, would be appreciated in several clinical settings. Primary human hepatocytes (phH) and organotypic cancer cell lines were exposed to different concentrations of apoptosis inducers (ethanol, TRAIL, anti-Apo, TGF-β, actinomycin D) and cultured with or without recombinant human ALR (rhALR). Apoptosis was evaluated by the release of cytochrome c from mitochondria and by FACS with propidium iodide (PI) staining.ALR significantly decreased apoptosis induced by ethanol, TRAIL, anti-Apo, TGF-β and actinomycin D. Further, the anti-apoptotic effect of ALR was observed in primary human hepatocytes and in HepG2 cells but not in bronchial (BC1), colonic (SW480), gastric (GC1) and pancreatic (L3.6PL) cell lines.Therefore, the hepatotrophic growth factor ALR acts in a liver specific manner with regards to both its mitogenic and its anti-apoptotic effect. Unlike the growth factors HGF and EGF, rhALR acts in a liver specific manner. Therefore, ALR is a promising candidate for further evaluation as a possible hepatoprotective factor in clinical settings.

Epidermal growth factor down-regulates the expression of human hepatic stimulator substance via CCAAT/enhancer-binding protein β in HepG2 cells

hHSS (human hepatic stimulator substance), acting as a hepatotrophic growth factor, promotes liver regeneration. However, the regulatory mechanisms for hHSS transcription are still poorly understood. In the present study, we investigated transcription of hHSS triggered by EGF (epidermal growth factor) and the role of C/EBPβ (CCAAT/enhancer-binding protein β) as a potential core factor responsible for hHSS transcription in HepG2 cells. The results show that EGF suppresses hHSS mRNA expression at early time points. Using a promoter deletion assay, we identified a proximal region (-358/-212) that is required for EGF suppression. Overexpression of C/EBPβ enhances EGF suppression of hHSS, and mutation of the C/EBPβ-binding site at -292/-279 or siRNA (short interfering RNA) interference abolishes EGF suppression. Furthermore, using an electrophoretic mobility-shift assay and chromatin immunoprecipitation analysis, we found that C/EBPβ specifically binds to the -292/-279 site that is responsible for EGF inhibition. Moreover, using a knockin (overexpression) and knockdown strategy (siRNA), we confirmed that C/EBPβ is a key factor responsible for inhibition of hHSS mRNA expression. Pre-treatment with an inhibitor of JNK (c-Jun N-terminal kinase) or down-regulation of JNK1 with specific siRNA reverses EGF-inhibited hHSS expression. Our results provide a crucial regulatory mechanism for EGF in hHSS transcription within the promoter proximal region.

Foxa2 (HNF-3β) regulates expression of hepatotrophic factor ALR in liver cells

Liver regeneration is a multistep and well-orchestrated process which is initiated by injuries such as tissue loss, infectious or toxic insults. Augmenter of liver regeneration (ALR) is a hepatotrophic growth factor which has been shown to stimulate hepatic regeneration after partial hepatectomy and therefore seems to be regulated during the regenerative process in the liver. Our aim was to analyze how ALR is regulated in hepatic tissues and which transcription factors might regulate its tissue-specific expression. Promoter studies of ALR (−733/+527bp) revealed potential regulatory elements for various transcription factors like Foxa2, IL-6 RE-BP and C/EBPβ. Analysis of the promoter activity by performing luciferase assays revealed that co-transfection with Foxa2 significantly induced the activity of ALR promoter in HepG2 cells. EMSA and Supershift analysis using anti-Foxa2 antibody confirmed the specific binding of Foxa2 to ALR promoter and this binding was inducible when the cells were simultaneously stimulated with IL-6. The increased binding after activation with IL-6 and/or Foxa2 was confirmed by elevated ALR protein levels using Western blot technique. In addition, we could not detect any binding of C/EBPβ and IL-6 RE-BP to the promoter of ALR. In conclusion, these results indicate that ALR is regulated by Foxa2, and this regulation may be amplified by IL-6.

Enhanced differentiation of embryonic stem cells using co‐cultivation with hepatocytes

We examined the effects of co-cultivated hepatocytes on the hepatospecific differentiation of murine embryonic stem (ES) cells. Utilizing an established mouse ES cell line expressing high or low levels of E-cadherin, that we have previously shown to be responsive to hepatotrophic growth factor stimulation (Dasgupta et al., 2005. Biotechnol Bioeng 92(3):257-266), we compared co-cultures of cadherin-expressing ES (CE-ES) cells with cultured rat hepatocytes, allowing for either paracrine interactions (indirect co-cultures) or both juxtacrine and paracrine interactions (direct co-cultures, random and patterned). Hepatospecific differentiation of ES cells was evaluated in terms of hepatic-like cuboidal morphology, heightened gene expression of late maturation marker, glucose-6-phosphatase in relation to early marker, alpha-fetoprotein (AFP), and the intracellular localization of albumin. Hepatocytes co-cultured with growth factor primed CE-ES cells markedly enhanced ES cell differentiation toward the hepatic lineage, an effect that was reversed through E-cadherin blockage and inhibited in control ES cells with reduced cadherin expression. Comparison of single ES cell cultures versus co-cultures show that direct contact co-cultures of hepatocytes and CE-ES cells maximally promoted ES cell commitment towards hepatodifferentiation, suggesting cooperative effects of cadherin-based juxtacrine and paracrine interactions. In contrast, E-cadherin deficient mouse ES (CD-ES) cells co-cultured with hepatocytes failed to show increased G6P expression, confirming the role of E-cadherin expression. To establish whether albumin expression in CE-ES cells was spatially regulated by co-cultured hepatocytes, we co-cultivated CE-ES cells around micropatterned, pre-differentiated rat hepatocytes. Albumin localization was enhanced "globally" within CE-ES cell colonies and was inhibited through E-cadherin antibody blockage in all but an interfacial band of ES cells. Thus, stem cell based cadherin presentation may be an effective tool to induce hepatotrophic differentiation by leveraging both distal/paracrine and contact/juxtacrine interactions with primary cells of the liver.

Construction of High Expression Plasmid of Human Augmenter of Liver Regeneration (hALR), Expression and Purification of hALR

Experimental evidence has been presented to suggest that the human augmenter of liver regeneration (hALR) serves as a hepatotrophic growth factor during liver regeneration and as a generalized growth factor during pancreas transplant/regeneration. A prokaryotic expression plasmid, pRSET/6his-c-myc-hALR was constructed, by cloning synthesized hALR cDNA into pRSET/6his-c-myc that was improved on the basis of pRSET B by the group. As a result, the protein was highly expressed in E. coli BL21. The recombinant hALR was over 60% of the total protein in E. coli. Its validity was confirmed by means of Western Blotting. The protein was purified by Ni-NTA affinity chromatography and this FAD-dependent sulfhydryl oxidase activity was measured.

Insights on augmenter of liver regeneration cloning and function.

Hepatic stimulator substance (HSS) has been referred to as a liver-specific but species non-specific growth factor. Gradient purification and sequence analysis of HSS protein indicated that it contained the augmenter of liver regeneration (ALR), also known as hepatopoietin (HPO). ALR, acting as a hepatotrophic growth factor, specifically stimulated proliferation of cultured hepatocytes as well as hepatoma cells in vitro, promoted liver regeneration and recovery of damaged hepatocytes and rescued acute hepatic failure in vivo. ALR belongs to the new Erv1/Alr protein family, members of which are found in lower and higher eukaryotes from yeast to man and even in some double-stranded DNA viruses. The present review article focuses on the molecular biology of ALR, examining the ALR gene and its expression from yeast to man and the biological function of ALR protein. ALR protein seems to be non-liver-specific as was previously believed, increasing the necessity to extend research on mammalian ALR protein in different tissues, organs and developmental stages in conditions of normal and abnormal cellular growth.

Repression of Cytochrome P450 Activity in Human Hepatocytes in Vitro by a Novel Hepatotrophic Factor, Augmenter of Liver Regeneration

Pathological disorders of the liver were shown to be associated with an impairment of hepatic drug metabolism mediated in part by growth factors. Augmenter of liver regeneration (ALR) is a novel liver-specific hepatotrophic growth factor, whereas its action on cytochrome P450 (P450) metabolism is completely unknown. Application of ALR to primary human hepatocytes in vitro reduced P450 isoenzyme activities (1A2 and 2A6) in a dose-dependent manner. Time-course analysis revealed that the maximal inhibitory effect was reached after 24 to 72 h of exposure with 50 nM ALR. The reduction of basal activities upon ALR treatment was 35% for CYP1A2, 56% for CYP2A6, 18% for CYP2B6, and 45% for CYP2E1. Additionally, after induction of P450 with specific inducers, ALR revealed an inhibitory effect on the isoenzyme activities (CYP1A2, 41%; CYP2B6, 35%). Investigations of protein and mRNA expression of basal and induced CYP1A2 and CYP3A4 after ALR treatment by Western blotting and real-time reverse transcriptase-polymerase chain reaction, respectively, suggest a regulation on the transcriptional level. Furthermore, ALR treatment increased nuclear factor kB activity and reduced constitutive androstane receptor but not pregnane X receptor or aryl hydrocarbon receptor expression. In contrast, ALR revealed no effects on phase II reactions (glutathione/oxidized glutathione, UDP-glucuronyltransferase conjugation). Our results indicate that ALR, as a member of hepatotrophic factors, down-regulates basal and induced P450 in human liver and therefore cross-links growth signals to regulation of hepatic metabolism. These findings further imply a possible role of ALR in drug interactions during impaired hepatic function, whereas liver regeneration is triggered.

Hepatopoietin interacts directly with COP9 signalosome and regulates AP-1 activity

Hepatopoietin (HPO)/augmenter of liver regeneration (ALR) is a specific hepatotrophic growth factor, which plays a key role in liver regeneration. Our previous study indicated that HPO executes its function by an inter-reactive network of the autocrine, paracrine and endocrine pathways. Recently, we have demonstrated that intracellular HPO interacts with Jun activation domain-binding protein 1 (JAB1) and leads to potentiation of activating protein-1 (AP-1) activity in a MAPK independent fashion. JAB1 is the fifth subunit of the COP9 signalosome (CSN), which is first identified as a suppressor of plant morphogenesis. A protein complex kinase activity associated with the CSN has been reported but not identified yet. In this report, we investigated further the association of HPO with the whole CSN. HPO exists in a complex with the eight-component CSN, both when purified from glycerol gradient centrifugation and when reciprocal immunoprecipitated from the lysates of transfected COS-7 cells. Intracellular HPO colocalizes with endogenous CSN in nucleus of hepatic cells. In addition, intracellular function of HPO that increases the phosphorylation of c-Jun leading to potentiate the AP-1 activity is inhibited by curcumin, a potent inhibitor of CSN-associated kinase. Taken together, these results elucidate a novel relationship of intracellular growth factor, HPO with large protein complex, CSN, which suggests a possible linkage between CSN and liver regeneration.

The Potentiation Role of Hepatopoietin on Activator Protein-1 Is Dependent on Its Sulfhydryl Oxidase Activity

Hepatopoietin (HPO) is a novel hepatotrophic growth factor that stimulates hepatocyte proliferation by two pathways. In the first, intracellular HPO specifically modulates the activator protein-1 (AP-1) pathway through JAB1 (Jun activation domain-binding protein 1), whereas in the second, extracellular HPO triggers the mitogen-activated protein kinase pathway by binding its specific receptor on the cell surface. In this report we demonstrate that HPO is a flavin-linked sulfhydryl oxidase, and the invariant CXXC (Cys-Xaa-Xaa-Cys) motif in HPO is essential for the enzyme activity of HPO but not for its dimerization nor for its binding ability with JAB1. Two intramolecular disulfides were identified in HPO by mass spectrometry, one of which is formed by the redox CXXC cysteine residues. HPO site-directed mutants (Cys/Ser) at active sites, which lost sulfhydryl oxidase activity, could not increase c-Jun phosphorylation and failed to potentiate JAB1-mediated AP-1 activation. However, the mutants still have mitogenic stimulation and mitogen-activated protein kinase activation effects on HepG2 cells. Thus, it can be concluded that the potentiation role of HPO on AP-1 is dependent on its sulfhydryl oxidase activity.

An initiator and its flanking elements function as a core promoter driving transcription of the Hepatopoietin gene

Hepatopoietin (HPO)/ALR (augmenter of liver regeneration), as a versatile hepatotrophic growth factor and a cellular thiol oxidase, is involved in a wide variety of basic processes of various tissues, especially in liver and testis. Here, we studied the regulation of HPO gene expression. By sequential deletion of the HPO 5′-flanking region, the minimal promoter of the HPO gene was shown to span positions −22 to +42 relative to the transcriptional start point. Further transfection assay and mutation analysis showed that the core promoter contains a functional initiator. Interestingly, three tandem repeats of a CTGGAGGC element, surrounding the transcription start site and bound by specific nuclear factors, were found to be pivotal for the promoter activity. This initiator flanking element functions in an initiator-dependent fashion and is present in many initiator-containing genes. Taken together, our findings revealed that the initiator-like element and its flanking repeat sequence comprise a core promoter and drive the transcriptional initiation of the HPO gene in a combinatorial manner. The HPO gene promoter might represent a novel architecture for core promoters.

Identification of hepatopoietin dimerization, its interacting regions and alternative splicing of its transcription.

Hepatopoietin (HPO) is a novel human hepatotrophic growth factor. Recently, we demonstrated that the extracellular ligand form of HPO can stimulate proliferation of hepatic cells via its specific receptor, which is on the surface of these cells. Also, the intracellular form of HPO potentiates the transcriptional factor AP-1. Intriguingly, a variety of HPO complexes with different molecular masses were detected in hepatocytes. In this study, we screened a human fetal liver cDNA library using the yeast two hybrid system with HPO as bait, hoping to find an HPO-binding protein. Surprisingly, HPO, itself, and a previously unidentified alternatively spliced form of HPO (named HPO23) were identified as interacting with HPO, indicating that HPO may exist as a homodimer or heterodimer. These results were further confirmed in vitro and in vivo. First, mass spectrometry analysis demonstrated that recombinant human HPO exist as a homodimer and that disulfide bonds were involved in the formation of rhHPO dimer. Secondly, a pull-down experiment confirmed that GST-HPO and HA-HPO, could bind together in vitro. Using HPO and various HPO deletion mutants, we identified the extreme 30 amino acids at both N- and C-termini are essential for HPO dimerization. Finally, Western blotting analysis demonstrated that HPO exists as two isoforms at 15 kDa and 23 kDa (HPO23) in liver cells, the 15 kDa species is located in nucleus, and the 23 kDa species mainly in the cytoplasm. These results implicated that the capacity of HPO to form both homodimers and heterodimers with the alternatively spliced forms might contribute to the existence of various HPO complexes in hepatic cells.

An essential function of the mitochondrial sulfhydryl oxidase Erv1p/ALR in the maturation of cytosolic Fe/S proteins.

Biogenesis of Fe/S clusters involves a number of essential mitochondrial proteins. Here, we identify the essential Erv1p of Saccharomyces cerevisia mitochondria as a novel component that is specifically required for the maturation of Fe/S proteins in the cytosol, but not in mitochondria. Furthermore, Erv1p was found to be important for cellular iron homeostasis. The homologous mammalian protein ALR ('augmenter of liver regeneration'), also termed hepatopoietin, can functionally replace defects in Erv1p and thus represents the mammalian orthologue of yeast Erv1p. Previously, a fragment of ALR was reported to exhibit an activity as an extracellular hepatotrophic growth factor. Both Erv1p and full-length ALR are located in the mitochondrial intermembrane space and represent the first components of this compartment with a role in the biogenesis of cytosolic Fe/S proteins. It is likely that Erv1p/ALR operates downstream of the mitochondrial ABC transporter Atm1p/ABC7/Sta1, which also executes a specific task in this essential biochemical process.

Stimulation of the Mitogen-activated Protein Kinase Cascade and Tyrosine Phosphorylation of the Epidermal Growth Factor Receptor by Hepatopoietin

Hepatopoietin (HPO) is a novel human hepatotrophic growth factor, which specifically stimulates proliferation of cultured primary hepatocytes in vitro and liver regeneration after liver partial hepatectomy in vivo. Recently, the identification of the mitogenic effect of HPO on hepatoma cell lines and the existence of HPO-specific receptors indicate that HPO acts via its specific cell surface receptor. However, the molecular mechanism of HPO action is not fully elucidated. In this report, we examined the signal transduction events induced by HPO in hepatoma cell line (HepG2). Our results demonstrated that HPO induces phosphorylation of mitogen-activated protein kinase kinase and mitogen-activated protein kinase (MAPK) in a rapid and transient manner. HPO stimulates tyrosine phosphorylation of epidermal growth factor receptor (EGFR). Furthermore, we observed that both MAPK activation and the mitogenic effect of HPO on HepG2 cells were completely blocked by AG1478, a specific inhibitor of EGFR tyrosine kinase activity. However, the effects of HPO were not antagonized by an EGFR-blocking antibody, mAb528, which blocks the interaction between epidermal growth factor and EGFR, indicating that stimulation of tyrosine phosphorylation of EGFR by HPO was not mediated by epidermal growth factor. In contrast, genistein, a general tyrosine kinase inhibitor, significantly attenuated the tyrosine phosphorylation of EGFR in response to HPO. In conclusion, our results suggest that tyrosine phosphorylation of EGFR may play a critical role in MAPK activation and mitogenic stimulation by HPO.

Clinical Relevance of Antibiotic‐induced Endotoxin Release in Patients Undergoing Hepatic Resection RID="" ID="" This International Society of Surgery (ISS)/Société Internationale de Chirurgie (SIC) article was presented at the 37th World Congress of Surgery International Surgical Week (ISW97), Acapulco, Mexico, August 24–30, 1997.

It has been proved that antibiotics binding to penicillin-binding protein 3 (PBP3) are associated with the greater release of endotoxin than those that bind to PBP2 in both in vitro and animal models. The aim of this study is to evaluate the potential clinical implications of antibiotic-induced endotoxin release after hepatic resection. Forty-five patients who underwent hepatic resection in our clinic were enrolled. The patients were divided into two groups. Group A (n = 26): antibiotics that bind primarily to PBP3, including cefmetazole (CMZ), latamoxef (LMOX), flomoxef (FMOX), were used. Group B (n = 19); antibiotics that bind to both PBP2 and PBP3, including cefazolin (CEZ), cefoperazone (CPZ), cefotiam (CTM). Postoperative complications, liver functional tests, and chemical mediators [endotoxin, interleukins (IL-6, IL-8), tumor necrosis factor alpha (TNFalpha), granulocyte colony-stimulating factor (G-CSF), hepatotrophic growth factor (HGF) were examined after hepatic resection. There were no significant differences in the backgrounds of the two groups. Eight patients in each group developed postoperative complications; in particular, 9 of 13 patients with biliary tract carcinoma developed postoperative complications. No significant elevation of peripheral blood endotoxin was noted by the endospecy method, in any of the patients, although six died following sepsis. Pre- and postoperative levels of cytokines showed no significant difference between the two groups. Our data suggest that clinical antibiotic-induced endotoxin release would not occur after hepatic resection regardless of the antibiotic, probably owing to continuous scavenging of endotoxin from peripheral blood.

Stimulation of rat hepatocyte proliferation in vitro and in vivo by factors derived from the bovine small intestinal mucosa.

A factor with a molecular weight of less than 1 kDa in the mucosa of the bovine small intestine (low molecular weight factor or LMW factor) stimulated DNA synthesis in rat hepatocytes in primary culture. This factor only showed its activity when it was added with a larger factor with a molecular weight of 30 kDa that was also found in the same tissue (high molecular weight factor or HMW factor). The LMW factor probably acts to enhance the action of a hepatotrophic growth factor, since EGF and HGF can substitute for the HMW factor. The action of the LMW factor was not due to the actions of low molecular weight substances such as norepinephrine, estradiol, triiodothyronine, and putrescine, which enhance the action of EGF or HGF, since substantial amounts of these substances were not found in the extract. When intraperitoneally administered into rats, after two-thirds hepatectomy, the LMW factor enhanced hepatocyte proliferation without the administration of the HMW factor. In the regenerating liver, a hepatotrophic growth factor(s), which acts synergistically with the LMW factor, might be properly provided, but the supply of the LMW factor might be below the level that maximally stimulates hepatocyte proliferation.