Changes in the intracellular accumulation and distribution of metallothionein in rat liver and kidney during postnatal development

Metallothionein (MT) bound to zinc and copper was detected in high concentration in fetal and newborn rat livers by a cadmium saturation method. The levels of both hepatic zinc and MT remained high for the first 14 days after birth and decreased to adult levels by 24 days of age. There was a direct linear relationship between hepatic metallothionein and zinc concentrations during the first 31 days after birth. The ratio of MT to zinc levels also decreased with age suggesting a rapid degradation of MT during postnatal development. Immunohistochemical localization of MT by peroxidase-antiperoxidase technique, using a specific antibody to MT, showed intense intranuclear staining for MT in fetal and newborn rat liver which persisted until Day 9. The nuclear MT staining decreased with age; at 11 days it was equal both in nucleus and cytoplasm and at 14 days, MT was localized mainly in the cytoplasm, similar to adult rat liver pattern. The intranuclear localization of MT in neonates could be considered as a typical fetal-neonatal morphological pattern and its subsequent presence in the cytoplasm, an adult pattern.     

Ontogeny and subcellular localization of rat liver mitochondrial branched chain amino-acid aminotransferase.

Branched chain amino-acid aminotransferase (BCAT) activity is present in fetal liver but the developmental pattern of mitochondrial BCAT (BCATm) expression in rat liver has not been studied. The aim of this study was to determine the activity, protein and mRNA concentration of BCATm in fetal and postnatal rat liver, and to localize this enzyme at the cellular and subcellular levels at both developmental stages. Maximal BCAT activity and BCATm mRNA expression occurred at 17 days' gestation in fetal rat liver and then declined significantly immediately after birth. This pattern was observed only in liver; rat heart showed a different developmental pattern. Fetal liver showed intense immunostaining to BCATm in the nuclei and mitochondria of hepatic cells and blood cell precursors; in contrast, adult liver showed mild immunoreactivity located only in the mitochondria of hepatocytes. BCAT activity in isolated fetal liver nuclei was 0.64 mU x mg(-1) protein whereas it was undetectable in adult liver nuclei. By Western blot analysis the BCATm antibody recognized a 41-kDa protein in fetal liver nuclei, and proteins of 41 and 43 kDa in fetal liver supernatant. In adult rat liver supernatant, the BCATm antibody recognized only a 43-kDa protein; however, neither protein was detected in adult rat liver nuclei. The appearance of the 41-kDa protein was associated with the presence of the highly active form of BCATm. These results suggest the existence of active and inactive forms of BCAT in rat liver.     

Analysis of the sinusoidal endothelial cell organization during the developmental stage of the fetal rat liver using a sinusoidal endothelial cell specific antibody, SE-1

The sinusoid organization during the development of fetal rat livers was studied using a SE-1 antibody, which we have previously established as a specific monoclonal antibody against rat sinusoidal endothelial cell (SEC). Expression and localization of the SE-1 antigen in the liver tissues of 13- to 21-day-old fetuses were immunofluorescently and immunoelectron microscopically examined. The first positive fluorescence was observed in the immature liver of 15-day-old fetuses. The initial positive staining was randomly distributed in the liver parenchyma and showed no direct relation to the large vessels which may be derived from the fetal vitelline veins. The positive linear staining increased in number and connected with each other during the course of development. The SE-1 staining pattern and the sinusoidal arrangement became similar to those of the adult liver after 20th day of gestation. Immunoelectron microscopically, the immature SEC showed a weak positive reaction for the SE-1 antigen at their membrane and was observed together with immature hepatocytes and hematopoietic cells in the 15-day-old fetal liver. Along with the liver development, SEC formed a sinusoid structure closely associated with hepatocytes and came to strongly express the SE-1 antigen. These results indicate that the organization of the hepatic sinusoid may start at around 15th day of the gestation and occurs randomly in the fetal liver parenchyma. It is also suggested that the expression of SE-1 antigen is possibly regulated by the intimate association with hepatocytes.     

Regulation of the ontogeny of rat liver metallothionein mRNA by zinc

To investigate the role of metals in the regulation of the ontogenic expression of rat liver metallothionein (MT) mRNA, the concentrations of zinc, MT and MT mRNA were determined in livers of fetal and newborn rats from dams which were fed with a control or zinc-deficient or copper-deficient or iron-deficient diet from day 12 of gestation. The liver samples were analyzed for MT-mRNA levels using a mouse MT-I cRNA probe. Although the newborn hepatic levels of each metal (zinc or copper or iron) was specifically reduced corresponding to the respective mineral deficiencies, the hepatic concentrations of total MT and MT-I mRNA were significantly decreased only in pups born from zinc-deficient dams. Injection of the zinc-deficient newborn pups with 20 mg Zn as ZnSO4/kg restored with MT-I mRNA levels to slightly above control values within 5 h of injection. The hepatic zinc, MT and MT-I mRNA levels were observed to increase significantly in control fetal rat liver on days 17-21 of gestation but there were little changes in either zinc or MT in fetal livers from zinc-deficient dams during the late gestational period. The MT-I mRNA level also did not show an increase on days 18 and 20 of gestation in zinc-deficient fetal liver as compared to controls. These results demonstrate a direct role of zinc in hepatic MT gene expression in rat liver during late gestation. Immunohistochemical localization of MT using a specific antibody to rat liver MT showed that the staining for MT in zinc-deficient pup liver was mainly in the cytosol in contrast to the significant nuclear MT staining observed in control newborn rat liver. The results suggest that maternal zinc deficiency has a marked effect not only in decreasing the levels of hepatic MT and MT-I mRNA but also in the localization of MT in newborn rat liver.     

Cytochrome P-450 localization in normal human adult and foetal liver by immunocytochemistry using a monoclonal antibody against human cytochrome P-450

Immunocytochemical studies with a monoclonal antibody (MAb-HL3), which recognises a major isozyme of human hepatic cytochrome P-450, have demonstrated this cytochrome in both cryostat and formalin-fixed paraffin-embedded sections of normal human adult liver. Prior trypsin digestion of the formalin-fixed sections prevented staining. There was a zonal distribution of immunoreactive cytochrome P-450, with localization predominantly in the hepatocytes of zone 3 of the hepatic acinus (the centrilobular region). Cytochrome P-450 was also demonstrated in foetal liver, but all foetal hepatocytes contained immunoreactive cytochrome P-450 and there was no zonal distribution of the protein. The biliary epithelium of adult liver contained a small amount of immunoreactive cytochrome P-450 whereas there was no immunoreactivity in the epithelium of foetal bile ducts.     

Modulation of mitogen-independent hepatocyte proliferation during the perinatal period in the rat

Summary Late gestation fetal rat hepatocytes can proliferate under defined in vitro conditions in the absence of added mitogens. However, this capacity declines with advancing gestational age of the fetus from which the hepatocytes are derived. The present studies were undertaken to investigate this change in fetal hepatocyte growth regulation. Examination of E19 fetal hepatocyte primary cultures using immunocytochemistry for 5-bromo-2′-deoxyuridine (BrdU) incorporation showed that approximately 80% of these cells traverse S-phase of the cell cycle over the first 48 h in culture. Similarly, 65% of E19 hepatocytes maintained in culture under defined mitogen-free conditions for 24 h showed nuclear expression of proliferating cell nuclear antigen (PCNA). These in vitro findings correlated with a high level of immunoreactive PCNA in immunofluorescent analyses of E19 liver. In contrast, E21 (term) liver showed little immunoreactive PCNA. The in vivo finding was recapitulated by in vitro studies showing that E21 hepatocytes had low levels of BrdU incorporation during the first day in culture and were PCNA negative shortly after isolation. However, within 12 h of plating, E21 hepatocytes showed cytoplasmic staining for PCNA. Although maintained under mitogen-free conditions, PCNA expression progressed synchronously to a nucleolar staining pattern at 24 to 48 h in culture followed by intense, diffuse nuclear staining at 60 h which disappeared by 72 h. This apparently synchronous cell cycle progression was confirmed by studies showing peak BrdU incorporation on the third day in culture. Whereas DNA synthesis by both E19 and E21 hepatocytes was potentiated by transforming growth factor α (TGFα), considerable mitogen-independent DNA synthesis was seen in hepatocytes from both gestational ages. These results may indicate that fetal hepatocytes come under the influence of an exogenous, in vivo growth inhibitory factor as term approaches and that this effect is relieved when term fetal hepatocytes are cultured.     

Hepatic epidermal growth factor-regulated mitogen-activated protein kinase kinase kinase activity in the rat: lack of identity with known forms of raf and MEKK

Mitogenic signaling involves protein kinases that phosphorylate the mitogen-activated protein kinase (MAPK) activator, MEK. In rats, basal hepatic MEK kinase activity is low in vivo in both adult rats and late gestation fetal rats, and is markedly stimulated by intraperitoneal administration of epidermal growth factor (EGF). The level of stimulated MEK phosphorylating activity is approximately 15 times higher in fetal liver than in adult liver. To identify regulated forms of the two categories of MEK kinase, Raf and MEKK, Western immunoblotting, immunoprecipitation kinase assays and immunodepletion studies were performed. Western immunoblotting confirmed that Raf-1, A-Raf, B-Raf, MEKK1 and MEKK2 were present at similar levels in E19 and adult liver. However, specific immunoprecipitation kinase assays did not detect any kinases that could account for marked EGF sensitivity or the higher level of activity in E19 fetuses. Immunodepletion studies produced a marked reduction in immunoreactive Raf/MEKK content and activity, but a minimal decrease in the ability of chromatography fractions to phosphorylate and activate recombinant MEK-1. Our results indicate that hepatic, EGF-sensitive MEK kinase activity may reside with a previously unidentified and physiologically relevant form of Raf and/or MEKK.     

The senescence marker protein (SMP-2) of the rat liver: purification, immunochemical characterization and age-dependent regulation

In vitro translation of total rat hepatic mRNAs has identified a 31 kilodalton senescence marker protein (SMP-2) which is present in higher amounts in prepubertal and senescent males than in the post-pubertal adult male (more than 10-fold). SMP-2 is an androgen-repressible protein. The negative regulation of the SMP-2 gene activity by androgen accounts for its increased expression during the androgen insensitive states of the prepubertal and senescent livers, and its constitutive expression in the female liver. A combination of separation procedures including salt fractionation, chromatofocusing, ion-exchange chromatography and preparative gel electrophoresis have led to the purification of SMP-2 to apparent homogeneity. The purified protein showed the same electrophoretic mobility as the sex- and age-specific in vitro translation product of hepatic mRNAs. The polyclonal antibody to SMP-2 was produced in the rabbit. The antibody selectively reacted with the 31 kDa sex- and age-specific translation product of hepatic mRNAs. Western blot analysis of the liver cytosol confirms monospecificity of the antiserum, as well as age- and sex-dependent changes in the tissue level of SMP-2. Histochemical staining of liver sections with the antiserum reveals a preferential periportal localization of SMP-2 in the hepatocytes. This finding is in marked contrast to the androgen-inducible alpha 2u globulin which is preferentially synthesized and localized in the pericentral hepatocytes. Thus, the zonal distribution of SMP-2 correlates with polarized androgen sensitivity of the hepatocytes within the liver lobule.     

Changes in parameters of lipoprotein metabolism during rat hepatic development

Maintenance of whole body cholesterol homeostasis is determined in part by the liver. Thus, changes in expression of hepatic parameters important in the regulation of cholesterol metabolism may play key roles in determining how homeostasis is maintained. The expression of hepatic lipoprotein uptake systems was studied during development using as a ligand very-low density lipoproteins rich in apolipoprotein E that had been obtained from hypercholesterolemic adult rats. These lipoproteins can serve as ligands for cell surface receptors recognizing apolipoproteins B and/or E. Uptake was lowest in freshly isolated fetal rat hepatocytes, increased substantially in hepatocytes from neonates and was intermediate in those from adults. Binding of these lipoproteins to liver membranes prepared from fetal, neonatal, suckling, weaned and adult rats was lowest in fetal preparations, while those from suckling, weaned and adult livers behaved similarly. Numbers of binding sites in neonatal liver membranes were similar to those in adult, but showed a different affinity. On the basis of this data, the ability of hepatocytes to recognize and remove apolipoprotein B/E-containing lipoproteins from the plasma appears to be a function of the differential expression or regulation of lipoprotein-uptake systems during development.     

Purification and characterization of choline/ethanolamine kinase from rat liver

Choline kinase, the first enzyme in the CDP-choline pathway for phosphatidylcholine biosynthesis, was purified 26,000-fold from rat liver to a specific activity of 143,000 nmol.min-1.mg-1 protein. The subunit molecular mass was 47 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, while the apparent native molecular mass was 160 kDa by size exclusion chromatography, suggesting a tetrameric structure. Two peaks of choline kinase activity were obtained by chromatofocusing. These isoforms eluted at pH 4.7 (CKI) and 4.5 (CKII). CKII appeared to be homogeneous by sodium dodecyl sulfate gel electrophoresis. Peptide mapping of two isoforms indicated a high degree of similarity, although there were peptides not common to both. Ethanolamine kinase activity copurified with both isoforms. The ratio of choline to ethanolamine kinase activity was 3.7 +/- 0.7 throughout the purification procedure. Choline and ethanolamine were mutually competitive inhibitors. The respective Km values, 0.013 and 1.2 mM, were similar to the Ki values of 0.014 and 2.2 mM. An antibody raised against CKII immunoprecipitated both choline and ethanolamine kinase activities from liver cytosol at the same titer. These data suggest that both activities reside on the same protein and occur at the same active site. Similarly, both activities were immunoprecipitated from brain, lung, and kidney cytosols. Western blot analysis showed both purified liver isoforms, as well as brain, lung and kidney enzymes, to have a molecular mass of 47 kDa.     

Ribosomal protein S6 phosphorylation and function during late gestation liver development in the rat

The phosphorylation of ribosomal protein S6 is thought to be required for biosynthesis of the cell's translational apparatus, a critical component of cell growth and proliferation. We have studied the signal transduction pathways involved in hepatic S6 phosphorylation during late gestation in the rat. This is a period during which hepatocytes show a high rate of proliferation that is, at least in part, independent of mitogenic signaling pathways that are operative in mature hepatocytes. Our initial studies demonstrated that there was low basal activity of two S6 kinases in liver, S6K1 and S6K2, on embryonic day 19 (2 days preterm). In addition, insulin- and growth factor-mediated S6K1 and S6K2 activation was markedly attenuated compared with that in adult liver. Nonetheless, two-dimensional gel electrophoresis demonstrated that fetal liver S6 itself was highly phosphorylated. To characterize the fetal hepatocyte pathway for S6 phosphorylation, we went on to study the sensitivity of hepatocyte proliferation to the S6 kinase inhibitor rapamycin. Unexpectedly, administration of rapamycin to embryonic day 19 fetuses in situ did not affect hepatocyte DNA synthesis. This resistance to the growth inhibitory effect of rapamycin occurred even though S6K1 and S6K2 were inhibited. Furthermore, fetal hepatocyte proliferation was sustained even though rapamycin administration resulted in the dephosphorylation of ribosomal protein S6. In contrast, rapamycin blocked hepatic DNA synthesis in adult rats following partial hepatectomy coincident with S6 dephosphorylation. We conclude that hepatocyte proliferation in the late gestation fetus is supported by a rapamycin-resistant mechanism that can function independently of ribosomal protein S6 phosphorylation.     

Biosynthesis of rat liver pyruvate kinase. Measurement of enzyme lifetime and the rate of synthesis at weaning.

Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of immunoprecipitates of liver cytosol with anti-(L-type pyruvate kinase) serum revealed proteins of mol.wt. 56 000 and 42 000 in addition to the heavy and light chains. The ratio of the 56 000 mol.wt. to the 42 000 mol.wt. protein increased under dietary conditions that resulted in an increase in the apparent specific activity of hepatic pyruvate kinase. The 42 000 mol.wt. protein was removed from immunoprecipitates if the liver cytosol was partially purified by pH precipitation and (NH4)2SO4 fractionation before addition of the antiserum. This technique may be used to analyse the formation of pure L-type pyruvate kinase in liver. By using H14CO3-labelling, the t1/2 of L-type pyruvate kinase was estimated as 75 +/- 1.7 h in post-weaned high-carbohydrate-diet-fed rats. Before weaning there was little immunoreactive pyruvate kinase in rat liver cytosol. Induction began between 6 and 24 h after weaning and reached a maximum value 120 h after weaning. When clearly enhanced total pyruvate kinase activity was first observed at 24 h post-weaning, the apparent specific activity of hepatic pyruvate kinase was considerably lower than the specific activity of the pure isolated enzyme. When the induction of L-type pyruvate kinase was monitored by the incorporation of L-[4,5-3H]leucine, the maximum rate of synthesis occurred 24--48 h after weaning. After this period synthesis declined, indicating a relatively slow turnover of the enzyme once the enzyme concentration was established in the liver.     

Insulin signaling during perinatal liver development in the rat

Insulin has long been assigned a key role in the regulation of growth and metabolism during fetal life. Our prior observations indicated that hepatic insulin signaling is attenuated in the late-gestation fetal rat. Therefore, we studied the perinatal ontogeny of hepatic insulin signaling extending from phosphatidylinositol 3-kinase (PI3K) to the ribosome. Initial studies demonstrated markedly decreased insulin-mediated activation of ribosomal protein S6 kinase 1 (S6K1) in the fetus. We found a similar pattern in the regulation of Akt, a kinase upstream from S6K1. Insulin produced minimal activation of insulin receptor substrate (IRS)-1-associated PI3K activity in fetal liver. A modest IRS-2-associated response was seen in the fetus. However, levels of both IRS-1 and IRS-2 were very low in fetal liver relative to adult liver. IRS-1 content and insulin responsiveness of PI3K, Akt, and S6K1 showed a transition to the adult phenotype during the first several postnatal weeks. Examination of downstream insulin signaling to the translational apparatus showed marked attenuation, relative to the adult, of fetal hepatic insulin-mediated phosphorylation of 4E-BP1, the regulatory protein for the eukaryotic initiation factor eIF4E, and ribosomal protein S6. The mammalian target of rapamycin (mTOR), a key integrator of nutritional and metabolic regulation of translation, was present in low amounts, was hypophosphorylated, and was not insulin sensitive in the fetus. Our results indicate that protein synthesis during late-gestation liver development may be mTOR and insulin independent. Reexamination of the role of insulin in fetal liver physiology may be warranted.     

A serine residue in the N-terminal acidic region of rat RPB6, one of the common subunits of RNA polymerases, is exclusively phosphorylated by casein kinase II in vitro.

RPB6 is one of the common subunits of all eukaryotic RNA polymerases and is indispensable for the enzyme function. Here, we isolated a rat cDNA encoding RPB6. It contained 127 amino acid (a.a.) residues. From alignment of RPB6 homologues of various eukaryotes, we defined two conserved regions, i.e. an N-terminal acidic region and a C-terminal core. In this study, we investigated in vitro phosphorylation of rat RPB6 by casein kinase II (CKII), a pleiotropic regulator of numerous cellular proteins. Three putative CKII-phosphorylated a.a. within rat RPB6 were assigned. We found that serines were phosphorylated by CKII in vitro. Mutagenesis studies provided evidence that a serine at a.a. position 2 was exclusively phosphorylated. Finally, an RPB6-engaged in-gel kinase assay clarified that CKII was a prominent protein kinase in rat liver nuclear extract that phosphorylates RPB6. Therefore, RPB6 was implied to be phosphorylated by CKII in the nucleus. We postulate that the N-terminal acidic region of the RPB6 subunit has some phosphorylation-coupled regulatory functions.     

Purification and immunochemical characterization of the cytoplasmic androgen-binding protein of rat liver

The cytoplasmic androgen-binding (CAB) protein of the male rat liver has been implicated to play a role in the androgen-dependent regulation of alpha 2u-globulin synthesis. The liver of the adult male rat contains about 50 fmol of specific high-affinity androgen-binding activity per milligram of total cytosolic protein. Photoaffinity labeling with [3H]R-1881 followed by SDS-polyacrylamide gel electrophoresis and autoradiography shows that the CAB is a 31-kilodalton protein. By means of DEAE-cellulose chromatography and preparative SDS-polyacrylamide gel electrophoresis, we have purified the CAB protein to electrophoretic homogeneity and have raised polyclonal rabbit antiserum that is monospecific to this protein. In the sucrose density gradient, the antiserum reacted with the androgen-binding component of the male liver cytosol prelabeled with tritiated dihydrotestosterone. Western blot analysis of the liver cytosol showed that the antiserum recognizes only the 31-kDa androgen-binding component. Such immunoblotting also showed that unlike the young adult, the androgen-insensitive states during prepuberty and senescence are associated with a marked reduction in the hepatic concentration of the immunoreactive CAB protein. No immuno-chemical cross-reactivity between CAB and another androgen-binding component of Mr 29K (which is associated with androgen insensitivity during prepuberty and senescence) was observed. The latter finding favors the possibility that 31- and 29-kDa androgen-binding components may have distinct sequence structure.