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Betaine critically contributes to the control of hepatocellular hydration and provides protection of the liver from different kinds of stress. To investigate how the hepatocellular hydration state affects gene expression of enzymes involved in the metabolism of betaine and related organic osmolytes, we used quantitative RT-PCR gene expression studies in rat hepatoma cells as well as metabolic and gene expression profiling in primary hepatocytes of both wild-type and 5,10- methylenetetrahydrofolate reductase (MTHFR)-deficient mice. Anisotonic incubation caused coordinated adaptive changes in the expression of various genes involved in betaine metabolism, in particular of betaine homocysteine methyltransferase, dimethylglycine dehydrogenase, and sarcosine dehydrogenase. The expression of betaine- degrading enzymes was downregulated by cell shrinking and strongly induced by an increase in cell volume under hypotonic conditions. Metabolite concentrations in the culture system changed accordingly. Expression changes were mediated through tyrosine kinases, cyclic nucleotide-dependent protein kinases, and JNKdependent signaling. Assessment of hepatic gene expression using a customized microarray chip showed that hepatic betaine depletion in MTHFR-/- mice was associated with alterations that were comparable to those induced by cell swelling in hepatocytes. In conclusion, the adaptation of hepatocytes to changes in cell volume involves the coordinated regulation of betaine synthesis and degradation and concomitant changes in intracellular osmolyte concentrations. The existence of such a well-orchestrated response underlines the importance of cell volume homeostasis for liver function and of methylamine osmolytes such as betaine as hepatic osmolytes. [ABSTRACT FROM AUTHOR]

We studied the downregulation of hepatobiliary transport systems and the effect of pharmacological heme oxygenase-1 (HO-1) preinduction by Hemoglobin-Glutamer 200 (HbG200) in cold ischemia-reperfused rat liver (I/R). Cold I/R reduced bile flow in the reperfusion period from 3.10±0.10 ml/3 h to 0.54±0.20 ml/3 h ( p<0.05) and biliary taurocholate excretion from 45.9±13.81 μmol/3 h to 1.87±0.46 μmol/3 h ( p<0.05). Mrp2, Bsep and Ntcp peak immunofluorescence in pericentral hepatocytes decreased to 79.0±2.6% ( p<0.001), 80.6±8.4% ( p<0.05) and 65.8±5.0% ( p<0.01), respectively. Pre-induction of HO-1 by HbG200 was largely confined to pericentral hepatocytes. HO-1 induction attenuated the decreased bile flow (0.91±0.16 ml/3 h, p<0.05) and canalicular taurocholate secretion (4.33±1.71 μmol/3 h, p<0.05). Bsep and Mrp2 peak immunofluorescence in pericentral hepatocytes was largely restored. Activation of JNK and Fyn by cold I/R was significantly attenuated by HO-1. Inhibiting HO activity by tin protoporphyrin IX after HbG200 administration reversed the effect on bile flow and canalicular transporter expression. In conclusion, pericentral downregulation of Bsep and Mrp2 following cold I/R is ameliorated by inducing HO-1 and was associated with diminished hepatocellular JNK and Fyn signaling. HO-1 may serve as a therapeutic target to attenuate hepatocellular cholestasis following I/R injury. [ABSTRACT FROM AUTHOR]

Cell hydration changes play a key role in the regulation of cell function and critically affect insulin sensitivity of carbohydrate- and protein metabolism. Here, the modulation of gene expression profiles by hyperosmolarity and insulin was examined in H4IIE rat hepatoma cells by cDNA/oligonucleotiode array-, Northern- and Western blot analysis. Osmosensitive expression of the insulin-like growth factor binding protein Igfbp1, the multidrug resistance protein Mrp5 (Abcc5a) and cyclin D1 (Ccnd1) was established at the mRNA and protein level. Despite a hyperosmotic increase of cyclin D1 mRNA induction by insulin, the cyclin D1 protein expression was decreased by hyperosmolarity, suggesting a hyperosmotic interference with cyclin D1 mRNA translation. Hyperosmolarity at the mRNA level blunted the insulin response of betaine homocysteine-S-methyl transferase, the multidrug resistance proteins Mdr1a (Abcb1a) and 2 (Abcb4), the Igfbp 2 and 5, cyclin G1, dual specificity phosphatase Dusp1, signal transducers and activators of transcription Stat3 and 5, catalase and the bile salt export pump Bsep (Abcb11), whereas the insulin response was increased for Mrp5, cyclin D1 and the phosphoenolpyruvate carboxykinase. Insulin effects on the mRNA expression of the eukaryotic initiation factor 4E binding protein 4e-bp1, tubulin, gene 33, growth hormone receptor, keratin18, ornithine decarboxylase and heme oxygenase 1 were largely insensitive to hyperosmolarity. The data indicate that hyperosmolarity differentially modulates insulin sensitivity at the level of gene expression. Copyright © 2007 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Cell hydration changes critically affect liver metabolism and gene expression. In the course of gene expression studies using nylon cDNA-arrays we found that hyperosmolarity (405 mosmol/l) suppressed the betaine-homocysteine methyltransferase (Bhmt) mRNA expression in H4IIE rat hepatoma cells. This was confirmed by Northern blot and real-time quantitative RT-PCR analysis, which in addition unraveled a pronounced induction of Bhmt mRNA expression by hypoosmotic (205 mosmol/l) swelling. Osmotic regulation of Bhmt mRNA expression was largely paralleled at the levels of Bhmt protein and enzymatic activity. Like hyperosmotic NaCl, hyperosmotic raffinose but not hyperosmotic urea suppressed Bhmt mRNA expression, suggesting that cell shrinkage rather than increased ionic strength or hyperosmolarity per se is the trigger. Hypoosmolarity increased the expression of a reporter gene driven by the entire human BHMT promoter, whereas destabilization of BHMT mRNA was observed under hyperosmotic conditions. Osmosensitivity of Bhmt mRNA expression was impaired by inhibitors of tyrosine kinases and cyclic nucleotide-dependent kinases. The osmotic regulation of BHMT may be part of a cell volume-regulatory response and additionally lead to metabolic alterations that depend on the availability of betaine-derived methyl groups. [ABSTRACT FROM AUTHOR]

The aim of this study was to investigate the regulation of hepatic transport systems during liver regeneration. Methods: A DNA oligonucleotide microarray was developed with probes for 400 transcripts. Data were confirmed using real-time PCR and on a functional level in the perfused rat liver. Liver homogenates were taken 3–48 h following 2/3-hepatectomy in rats and compared with sham-operated and non-operated controls. Results: A more than two-fold increase or decrease of expression was obtained in 183 genes following partial hepatectomy and in 16 genes in sham-operated rats. A strong induction during liver regeneration was detected for the amino acid transporters LAT4, SN2 and sodium-dependent neutral amino acid transporter (ASCT)2, whereas amino acid transport system (ATA)2 and ATA3 expressions remained unchanged. The upregulation of ASCT2 may be responsible for the increase in sodium-dependent neutral amino acid influx important for liver cell proliferation. Expression of the osmolyte transporters Smit, TauT and Bgt1 was almost unchanged indicating that osmolytes are not involved in the cell volume increase during liver regeneration. The basolateral bile salt transporter Ntcp messenger RNA (mRNA) was significantly downregulated, whereas bile salt export pump (Bsep) and multidrug resistance protein (Mrp)2 expressions remained almost unchanged. An increased mRNA expression following partial hepatectomy was detected for organic anion transporting polypeptide (Oatp)5, Octn1, Octn2 and SGLT2. In contrast, Mrp6, Oatp 2, Oatp 3, Oatp 4 and Oatp 7 were downregulated. A five-fold upregulation at the protein level was shown for the Na+–K+–2Cl− cotransporter sodium-potassium-2-chloride cotransporter (NKCC1). Conclusions: The data show a differential regulation of hepatic transport systems during liver regeneration. [ABSTRACT FROM AUTHOR]

Abstract: Little is known about the regulation of signal transducer and activator of transcription (STAT) stability. Here the osmolarity-dependence of STAT3 stability, ubiquitination, Tyr705 phosphorylation, STAT3 transactivation and γ-fibrinogen (γ-FBG) expression was studied in hepatoma cells. Hyper-osmolarity accelerated STAT3 degradation which was prevented by proteasome inhibitors. Hypo-osmolarity stabilized STAT3, most likely due to a decrease in STAT3 ubiquitination. Accordingly, STAT3 Tyr705 phosphorylation, α2-macroglobulin promoter activity and γ-FBG expression were osmosensitive. Modulation of STAT3 stability may contribute to a hydration dependence of acute phase protein expression. [Copyright &y& Elsevier]

The distribution and cellular localisation of the phosphatidylinositol 4-kinase isoforms, PI4K230 and PI4K92, that are believed to play important roles in the intracellular signalling mechanisms were studied in the rat brain (cortex, cerebellum, hippocampus and spinal cord) using immunocytochemistry with light and electron microscopy. PI4K230 was detected with a specific antibody purified by affinity chromatography from the egg yolk of chicken immunised with a 33-kDa fragment of bovine PI4K230, comprising amino acids 873–1175 of the native protein. PI4K92 was immunostained with a commercially available antibody raised in rabbit against amino acid residues 410–537 of human PI4K92. At the light microscopic level, the immunostaining of PI4K230 and PI4K92 showed a very similar distribution throughout the neurons and appeared as dense punctate labelling in the cytoplasm of perikarya and stem dendrites of various neurons. In addition to neurons, a strongly stained cell population was observed in the molecular layer of the cerebellar cortex that resembled Bergmann glia cells. Electron microscopy of neurons in the ventral horn of the spinal cord showed dense granular immunoprecipitates for both PI4K230 and PI4K92, mostly associated with the outer membrane of mitochondria and membranes of the rough endoplasmic reticulum. In addition, immunostaining of PI4K92 was also frequently found on the outer surface of cisterns and vesicles of Golgi complexes, whereas PI4K230 immunoreactivity was colocalised with some multivesicular bodies. Neither nuclear localisation nor a regular attachment to the cell membrane of these enzymes were observed. Our findings indicate that PI4K230 and PI4K92 are not involved directly in the ligand-stimulated turnover of phosphoinositides at the plasma membrane of neurons. However, they may provide regulatory phosphoinositides for intracellular vesicular traffic being associated with various organelles. [ABSTRACT FROM AUTHOR]

Polyphosphoinositides are involved in many signal transduction pathways in eukaryotic cells. The first committed step is catalysed by phosphatidylinositol 4-kinase leading to the formation of phosphatidylinositol 4-phosphate. In the last four years, ten cDNA molecules have been cloned which code isoforms of phosphatidylinositol 4-kinase; some of which are highly related. Characteristically, they contain a C-terminal catalytic domain which is similar to that of (poly)phosphoinositide 3-kinases and to that of more distantly related lipid/protein kinases. Alignment has characterised cDNAs from Chaenorabditis, Dictyostelium and Schizostaphyloccus pombe as those of phosphatidylinositol 4-kinases also. All these lipid kinases are related to the superfamily of protein kinases. Several amino acids are highly conserved in catalytic domains of lipid and protein kinases. Employing the catalytic subunit of the cAMP-dependent protein kinase as template, these residues can be assigned functionally. On the basis of the alignment, a phylogenetic tree of the superfamily of phosphatidylinositol kinases has been constructed. Three families, the phosphatidylinositol 4-kinases, phosphoinositide 3-kinases, and the phosphatidylinositol related lipid/protein kinases, can be recognised. Each family comprises two subfamilies. The involvement of the phosphatidylinositol 4-kinases in signal transduction processes is summarised and a new hypothesis for the function of their isoforms in polyphosphoinositide signalling is presented. The involvement of phosphatidylinositol 4-kinases in formation of lipid-protein interactions with cytoskeleton proteins and the metabolism of polyphosphoinositide in the nucleus is discussed. [ABSTRACT FROM AUTHOR]