Ethanolamine modulates the rate of rat hepatocyte proliferation in vitro and in vivo

A low molecular weight, heat-resistant hepatotrophic factor in an extract from the bovine intestinal mucosa was purified and identified as ethanolamine by structural analyses. The mode of action of ethanolamine in vitro and in vivo coincided with that of the crude extract of the tissue, indicating that ethanolamine is the active component. Ethanolamine synergistically elevated the stimulation of DNA synthesis in hepatocytes in primary culture when added together with a growth factor, such as epidermal growth factor, with the ED50 being 20 μM, although it showed little stimulatory effect by itself. Contrary to these in vitro results, the intraperitoneal administration of ethanolamine hydrochloride (24 mg of ethanolamine per kg of body weight) enhanced hepatocyte proliferation in regenerating rat livers after two-thirds hepatectomy without the administration of any growth factors. In the regenerating liver, hepatocyte proliferation may be initiated by an endogenous growth factor, but the supply of ethanolamine in circulation may not be sufficient for optimal hepatocyte proliferation; thus, the exogenous administration of ethanolamine may further enhance hepatocyte proliferation. Ethanolamine in circulation may be a humoral hepatotrophic factor.

Mice mutant for glucokinase regulatory protein exhibit decreased liver glucokinase: A sequestration mechanism in metabolic regulation

The importance of glucokinase (GK; EC 2.7.1.12) in glucose homeostasis has been demonstrated by the association of GK mutations with diabetes mellitus in humans and by alterations in glucose metabolism in transgenic and gene knockout mice. Liver GK activity in humans and rodents is allosterically inhibited by GK regulatory protein (GKRP). To further understand the role of GKRP in GK regulation, the mouse GKRP gene was inactivated. With the knockout of the GKRP gene, there was a parallel loss of GK protein and activity in mutant mouse liver. The loss was primarily because of posttranscriptional regulation of GK, indicating a positive regulatory role for GKRP in maintaining GK levels and activity. As in rat hepatocytes, both GK and GKRP were localized in the nuclei of mouse hepatocytes cultured in low-glucose-containing medium. In the presence of fructose or high concentrations of glucose, conditions known to relieve GK inhibition by GKRP in vitro, only GK was translocated into the cytoplasm. In the GKRP-mutant hepatocytes, GK was not found in the nucleus under any tested conditions. We propose that GKRP functions as an anchor to sequester and inhibit GK in the hepatocyte nucleus, where it is protected from degradation. This ensures that glucose phosphorylation is minimal when the liver is in the fasting, glucose-producing phase. This also enables the hepatocytes to rapidly mobilize GK into the cytoplasm to phosphorylate and store or metabolize glucose after the ingestion of dietary glucose. In GKRP-mutant mice, the disruption of this regulation and the subsequent decrease in GK activity leads to altered glucose metabolism and impaired glycemic control.

A Specific Light Chain of Kinesin Associates with Mitochondria in Cultured Cells

The motor protein kinesin is implicated in the intracellular transport of organelles along microtubules. Kinesin light chains (KLCs) have been suggested to mediate the selective binding of kinesin to its cargo. To test this hypothesis, we isolated KLC cDNA clones from a CHO-K1 expression library. Using sequence analysis, they were found to encode five distinct isoforms of KLCs. The primary region of variability lies at the carboxyl termini, which were identical or highly homologous to carboxyl-terminal regions of rat KLC B and C, human KLCs, sea urchin KLC isoforms 1–3, and squid KLCs. To examine whether the KLC isoforms associate with different cytoplasmic organelles, we made an antibody specific for a 10-amino acid sequence unique to B and C isoforms. In an indirect immunofluorescence assay, this antibody specifically labeled mitochondria in cultured CV-1 cells and human skin fibroblasts. On Western blots of total cell homogenates, it recognized a single KLC isoform, which copurified with mitochondria. Taken together, these data indicate a specific association of a particular KLC (B type) with mitochondria, revealing that different KLC isoforms can target kinesin to different cargoes.

Evidence for the Presence of 5S rRNA in Mammalian Mitochondria

Mammalian mitochondrial ribosomes contain two prokaryotic-like rRNAs, 12S and 16S, both encoded by mitochondrial DNA. As opposed to cytosolic ribosomes, however, these ribosomes are not thought to contain 5S rRNA. For this reason, it has been unclear whether 5S rRNA, which can be detected in mitochondrial preparations, is an authentic organellar species imported from the cytosol or is merely a copurifying cytosol-derived contaminant. We now show that 5S rRNA is tightly associated with highly purified mitochondrial fractions of human and rat cells and that 5S rRNA transcripts derived from a synthetic gene transfected transiently into human cells are both expressed in vivo and present in highly purified mitochondria and mitoplasts. We conclude that 5S rRNA is imported into mammalian mitochondria, but its function there still remains to be clarified.

Localization of the Wilson’s disease protein product to mitochondria

Wilson’s disease (WND) is an inherited disorder of copper homeostasis characterized by abnormal accumulation of copper in several tissues, particularly in the liver, brain, and kidney. The disease-associated gene encodes a copper-transporting P-type ATPase, the WND protein, the subcellular location of which could be regulated by copper. We demonstrate that the WND protein is present in cells in two forms, the 160-kDa and the 140-kDa products. The 160-kDa product was earlier shown to be targeted to trans-Golgi network. The 140-kDa product identified herein is located in mitochondria as evidenced by the immunofluorescent staining of HepG2 cells with specific mitochondria markers and polyclonal antibody directed against the C terminus of the WND molecule. The mitochondrial location for the 140-kDa WND product was confirmed by membrane fractionation and by analysis of purified human mitochondria. The antibody raised against a repetitive sequence in the N-terminal portion of the WND molecule detects an additional 16-kDa protein, suggesting that the 140-kDa product was formed after proteolytic cleavage of the full-length WND protein at the N terminus. Thus, the WND protein is a P-type ATPase with an unusual subcellular localization. The mitochondria targeting of the WND protein suggests its important role for copper-dependent processes taking place in this organelle.

Nonsense-mediated Decay of mRNA for the Selenoprotein Phospholipid Hydroperoxide Glutathione Peroxidase Is Detectable in Cultured Cells but Masked or Inhibited in Rat Tissues

Previous studies of mRNA for classical glutathione peroxidase 1 (GPx1) demonstrated that hepatocytes of rats fed a selenium-deficient diet have less cytoplasmic GPx1 mRNA than hepatocytes of rats fed a selenium-adequate diet. This is because GPx1 mRNA is degraded by the surveillance pathway called nonsense-mediated mRNA decay (NMD) when the selenocysteine codon is recognized as nonsense. Here, we examine the mechanism by which the abundance of phospholipid hydroperoxide glutathione peroxidase (PHGPx) mRNA, another selenocysteine-encoding mRNA, fails to decrease in the hepatocytes and testicular cells of rats fed a selenium-deficient diet. We demonstrate with cultured NIH3T3 fibroblasts or H35 hepatocytes transiently transfected with PHGPx gene variants under selenium-supplemented or selenium-deficient conditions that PHGPx mRNA is, in fact, a substrate for NMD when the selenocysteine codon is recognized as nonsense. We also demonstrate that the endogenous PHGPx mRNA of untransfected H35 cells is subject to NMD. The failure of previous reports to detect the NMD of PHGPx mRNA in cultured cells is likely attributable to the expression of PHGPx cDNA rather than the PHGPx gene. We conclude that 1) the sequence of the PHGPx gene is adequate to support the NMD of product mRNA, and 2) there is a mechanism in liver and testis but not cultured fibroblasts and hepatocytes that precludes or masks the NMD of PHGPx mRNA.

Circular RNAs from transcripts of the rat cytochrome P450 2C24 gene: correlation with exon skipping.

The cytochrome P450 2C24 gene is characterized by the capability to generate, in rat kidney, a transcript containing exons 2 and 4 spliced at correct sites but having the donor site of exon 4 directly joined to the acceptor site of exon 2 (exon scrambling). By reverse transcriptase-PCR analysis, it is now shown that the only exons present in the scrambled transcript are exons 2, 3, and 4 and that this molecule lacks a poly(A)+ tail. Furthermore, the use of PCR primers in both orientations of either exon 2 or exon 4 revealed that the orders of the exons in the scrambled transcript are 2-3-4-2 and 4-2-3-4, respectively. These results, combined with the observation that P450 2C24 is a single-copy gene, with no duplication of the exon 2 to exon 4 segment, suggest that the scrambled transcript has properties consistent with that of a circular molecule. In line with this is the observation of an increased resistance of the transcript to phosphodiesterase I, a 3'-exonuclease. Moreover, an alternatively processed cytochrome P450 2C24 mRNA, lacking the three scrambled exons and having exon 1 directly joined to exon 5, has been identified in kidney and liver, tissues that express the scrambled transcript. This complete identity of the exons that are absent in the alternatively processed mRNA but present in the scrambled transcript is interpreted as indicative of the possibility that exon scrambling and exon skipping might be interrelated phenomena. It is therefore proposed that alternative pre-mRNA processing has the potential to generate not only mRNAs lacking one or more exons but also circular RNA molecules.

Targeted overexpression of androgen receptor with a liver-specific promoter in transgenic mice.

The rodent liver displays marked age- and sex-dependent changes in androgen sensitivity due to the sexually dimorphic and temporally programmed expression of the androgen receptor (AR) gene. We have altered this normal phenotype by constitutive overexpression of the rat AR transgene in the mouse liver by targeting it via the human phenylalanine hydroxylase (hPAH) gene promoter. These transgenic animals in their heterozygous state produce an approximately 30-fold higher level of the AR in the liver as compared with the nontransgenic control. Androgen inactivation via sulfonation of the hormone by dehydroepiandrosterone sulfotransferase (DST), an androgen-repressible enzyme, also contributes to the age- and sex-dependent regulation of hepatic androgen sensitivity. DST has a broad range of substrate specificity and is responsible for the age- and sex-specific activation of certain polycyclic aromatic hepatocarcinogens as well, by converting them to electrophilic sulfonated derivatives. In the transgenic female, the hepatic expression of DST was approximately 4-fold lower than in normal females, a level comparable to that in normal males. The hPAH-AR mice will serve as a valuable model for studying the sex- and age-invariant expression of liver-specific genes, particularly those involved in the activation of environmental hepatocarcinogens such as the aromatic hydrocarbons.

Selenophosphate synthetase: detection in extracts of rat tissues by immunoblot assay and partial purification of the enzyme from the archaean Methanococcus vannielii.

In Escherichia coli and Salmonella typhimurium it has been shown that selenophosphate serves as the selenium donor for the conversion of seryl-tRNA to selenocysteyl-tRNA and for the synthesis of 2-selenouridine, a modified nucleoside present in tRNAs. Although selenocysteyl-tRNA also is formed in eukaryotes and is used for the specific insertion of selenocysteine into proteins, the precise mechanism of its biosynthesis from seryl-tRNA in these systems is not known. Because selenophosphate is extremely oxygen labile and difficult to identify in biological systems, we used an immunological approach to detect the possible presence of selenophosphate synthetase in mammalian tissues. With antibodies elicited to E. coli selenophosphate synthetase the enzyme was detected in extracts of rat brain, liver, kidney, and lung by immunoblotting. Especially high levels were detected in Methanococcus vannielii, a member of the domain Archaea, and the enzyme was partially purified from this source. It seems likely that the use of selenophosphate as a selenium donor is widespread in biological systems.

Rotenone induces degeneration of photoreceptors and impairs the dopaminergic system in the rat retina

Rotenone is a widely used pesticide and a potent inhibitor of mitochondrial complex I (NADH-quinone reductase) that elicits the degeneration of dopaminergic neurons and thereby the appearance of a parkinsonian syndrome. Here we have addressed the alterations induced by rotenone at the functional, morphological and molecular levels in the retina, including those involving both dopaminergic and non-dopaminergic retinal neurons. Rotenone-treated rats showed abnormalities in equilibrium, postural instability and involuntary movements. In their outer retina we observed a loss of photoreceptors, and a reduced synaptic connectivity between those remaining and their postsynaptic neurons. A dramatic loss of mitochondria was observed in the inner segments, as well as in the axon terminals of photoreceptors. In the inner retina we observed a decrease in the expression of dopaminergic cell molecular markers, including loss of tyrosine hydroxylase immunoreactivity, associated with a reduction of the dopaminergic plexus and cell bodies. An increase in immunoreactivity of AII amacrine cells for parvalbumin, a Ca2+-scavenging protein, was also detected. These abnormalities were accompanied by a decrease in the amplitude of scotopic and photopic a- and b-waves and an increase in the b-wave implicit time, as well as by a lower amplitude and greater latency in oscillatory potentials. These results indicate that rotenone induces loss of vision by promoting photoreceptor cell death and impairment of the dopaminergic retinal system.

Pro-fibrotic polymorphisms predictive of advanced liver fibrosis in the severely obese

Background/Aims: Insulin resistance and systemic hypertension are predictors of advanced fibrosis in obese patients with non-alcoholic fatty liver disease (NAFLD). Genetic factors may also be important. We hypothesize that high angiotensinogen (AT) and transforming growth factor-beta1 (TGF-beta1) producing genotypes increase the risk of liver fibrosis in obese subjects with NAFLD. Methods: One hundred and five of 130 consecutive severely obese patients having a liver biopsy at the time of laparoscopic obesity surgery agreed to have genotype analysis. Influence of specific genotype or combination of genotypes on the stage of hepatic fibrosis was assessed after controlling for known risk factors. Results: There was no fibrosis in 70 (67%), stages 1-2 in 21 (20%) and stages 3-4 fibrosis in 14 (13%) of subjects. There was no relationship between either high AT or TGF-beta1 producing genotypes alone and hepatic fibrosis after controlling for confounding factors. However, advanced hepatic fibrosis occurred in five of 13 subjects (odds ratio 5.7, 95% confidence interval 1.5-21.2, P = 0.005) who inherited both high AT and TGF-beta1 producing polymorphisms. Conclusions: The combination of high AT and TGF-beta1 producing polymorphisms is associated with advanced hepatic fibrosis in obese patients with NAFLD. These findings support the hypothesis that angiotensin II stimulated TGF-beta1 production may promote hepatic fibrosis. (C) 2003 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

Increased mononuclear cell activation and apoptosis early after human liver transplantation is associated with a reduced frequency of acute rejection

Experimental models of orthotopic liver transplantation (OLT) have shown that the very early events post-OLT are critical in distinguishing immunogenic and tolerogenic reactions. In rodents, increased leukocyte apoptosis and cytokine expression have been demonstrated in tolerogenic strain combinations. Information from human OLT recipients is less abundant. The aim of this study was to determine the amount of early leukocyte activation and apoptosis following human OLT, and to correlate this with subsequent rejection status. Peripheral blood mononuclear cells (PBMC) were isolated from 76 patients undergoing OLT - on the day prior, 5 hrs after reperfusion (day 0), and 18-24 hrs post-OLT (day 1). The mean level of apoptotic PBMCs on post OLT day 1 was higher than healthy recipients (0.9% +/- 0.2 vs. 0.2% +/- 0.1, p = 0.013). Apoptosis was greater in nonrejecting (NR) (1.1% +/- 0.3) compared with acutely-rejecting (R) (0.3% +/- 0.1, p = 0.021) patients. On day 1, PBMC from NR patients had increased expression of IFN-gamma (p = 0.006), IL-10 (p = 0.016), and CD40 ligand (p = 0.02) compared with R. Donor cell chimerism on day 1 did not differ between the groups indicating that this was unlikely to account for increased PBMC apoptosis in the NR group. Interestingly, the level of chimerism on day 0 was significantly higher in NR (3.8% +/- 0.6) compared with R (1.2% +/- 0.4, p = 0.004) patients and there was a close correlation between chimerism on day 0 and cytokine expression on day 1. These results imply that similar mechanisms are occurring in the human liver to promote graft acceptance as in the experimental models of liver transplantation and suggest that strategies that promote liver transplant acceptance in rodents might be applicable to humans.

The rat Na+-sulfate cotransporter rNaS2: functional characterization, tissue distribution, and gene (slc13a4) structure

Inorganic sulfate is essential for numerous functions in mammalian physiology. In the present study, we characterized the functional properties of the rat Na+-sulfate cotransporter NaS2 (rNaS2), determined its tissue distribution, and identified its gene (slc13a4) structure. Expression of rNaS2 protein in Xenopus oocytes led to a Na+-dependent transport of sulfate that was inhibited by phosphate, thiosulfate, tungstate, selenate, oxalate, and molybdate, but not by citrate, succinate, or DIDS. Transport kinetics of rNaS2 determined a K-M for sulfate of 1.26 mM. Na+ kinetics determined a Hill coefficient of n=3.0 +/- 0.7, suggesting a Na+:SO42- stoichiometry of 3:1. rNaS2 mRNA was highly expressed in placenta, with lower levels found in the brain and liver. slc13a4 maps to rat chromosome 4 and contains 17 exons, spanning over 46 kb in length. This gene produces two alternatively spliced transcripts, of which the transcript lacking exon 2 is the most abundant form. Its 5' flanking region contains CAAT- and GC-box motifs and a number of putative transcription factor binding sites, including GATA-1, SP1, and AP-2 consensus sequences. This is the first study to characterize rNaS2 transport kinetics, define its tissue distribution, and resolve its gene (slc13a4) structure and 5' flanking region.

Mitochondrial dysmorphology in the neuroepithelium of rat embryos following a single dose of maternal hyperthermia during gestation

Hyperthermia is teratogenic to human and animal embryos and induces mainly anomalies of the nervous system. However, the teratogenic mechanism is poorly understood. Mammalian embryos are known to switch from anaerobic to aerobic metabolism around the time of neural tube closure. This critical event might be sensitive to hyperthermia. The objective of the present study was to evaluate the ultrastructural changes of the mitochondria of the neuroepithelium (NE) of rat embryos following maternal exposure to hyperthermia. Pregnant rats were heat stressed for an hour on gestation day (GD) 9 and embryos were examined by electron microscopy on GD 10. NE presented extensive apoptosis. Intercellular junctions were weakened and copious cellular debris projected into the ventricle. The mitochondria were of diverse size and shape. Most of them were swollen and had short cristae and electron dense matrix. Hydropic changes were also observed in numerous mitochondria. Lipid-laden mitochondria were found in the apical portions of neuroblasts. The mesenchyme (ME) of heat-treated embryos showed paucity of cells and only as frequent apoptosis as the controls. Their mitochondria also showed changes similar to those of the NE. Additionally extensive lipid accumulation was observed in and in the vicinity of mitochondria, often surrounded by short strands of endoplasmic reticulum. Whereas mitochondrial pathology was associated with profound apoptosis in the NE, growth restriction and lipid accumulation accompanied mitochondrial changes in the ME. The results of this study indicate that the embryonic response to maternal heat shock is tissue-specific and morphologically distinct in this species.

Tetrahydrobiopterin metabolism, neurotransmitters and behaviour in the rat

Various neurotoxins were investigated to assess their suitability for developing an animal model to study partial brain BH4 deficiency, neurotransmitters and behavioural alterations. Acute dosing with lead, diethylstilboestrol (DES), amphetamine and scopolamine produced no significant changes in rat brain BH4 metabolism though total biopterins in the liver were significantly reduced by lead and DES. Acute starvation of adult rats decreased brain biopterins. This loss of biopterins may be due to enhanced oxidative catabolism of the active cofactor caused by glutathione depletion. Dietary administration of a BH4 biosynthesis inhibitor, DAHP, consistently decreased brain total biopterins in weaner rats but did not alter the levels of DA, NA, 5-HT or metabolites. However the DAHP diet also induced a marked reduction in food intake. Rats subjected to an equivalent degree of food restriction without inhibitor showed significant but less severe reductions in brain biopterins and again no effect on transmitter levels. DAHP produced a significant decrease in locomotor activity and rearing. This could not be ascribed to reduction in food intake as animals subjected to just dietary restriction showed an increase in these activities. As gross brain levels of DA, NA and 5-HT were unaltered by DAHP the behavioural changes associated with the induced deficiency in brain total biopterins might not have been mediated through the action of these compounds. Although localised changes in neurotransmitter levels may have been obscured by gross analysis it is also possible that the behaviour changes were mediated by a role of BH4 not yet elucidated. Long-term administration of a high aluminium low calcium diet to mice produced no effect on gross brain total biopterins, catecholamines, serotonin or choline acetyltransferase activity though significant behavioural changes were observed.