Browsing by Author "Probst, I."

Background-Sufficient ATP concentrations maintain physiological processes and protect tissue from hypoxic damage. With decreasing oxygen concentration, ATP synthesis relies increasingly on the presence of phosphocreatine. Aim-The effect of exogenously applied creatine on phosphocreatine and ATP concentrations was studied under control and anoxic conditions. Methods-Pregnant mice were fed orally with creatine monohydrate (2 g/kg body weight/day), Brainstem slices from these mice pups were compared with those from pups of non-creatine supplemented pregnant mice. Measurements were performed under normoxic and anoxic conditions, In addition, brainstem slices from non-creatine treated mice pups were incubated for 3 hours in control artificial cerebrospinal fluid (CSF) (n = 10) or in artificial CSF containing 200 mu M creatine (n = 10). ATP and phosphocreatine contents were determined enzymatically in single brainstem slices. Results-ATP concentrations were in the same range in all preparations. However, there was a significant increase of phosphocreatine in the brainstems from pups of creatine fed mice when compared with the brainstems of pups from non-creatine treated mice or in non-incubated brainstems of control animals. After 30 minutes anoxia, ATP as well as phosphocreatine concentrations remained significantly higher in creatine pretreated slices compared with controls. Conclusion-The data indicate that exogenous application of creatine is effective in neuroprotection.

Background/Aims: Steroid diabetes is associated with hepatic insulin resistance; in hepatic cell models, however, mainly insulin-permissive effects have been described. Here we investigate modulation by dexamethasone of a larger number of insulin actions. Methods: Adult rat hepatocytes were cultured dexamethasone for 48 h; insulin actions were studied subsequently. Results: Stimulation of glycolysis by insulin but not by glucose required culture with dexamethasone. Activation of glycogen synthesis by insulin or glucose was strongly enhanced by dexamethasone, the insulin effects on glycogenolysis and amino acid uptake were not modulated. When dexamethasone was omitted from the culture, insulin was incapable to activate glycogen synthase, inactivate glycogen phosphorylase or elevate the level of fructose 2,6-bisphosphate. Dexamethasone did not alter insulin binding, insulin receptor number or kinase activity, insulin receptor substrate-1 and Akt protein expression/phosphorylation. Insulin-stimulated association of phosphatidylinositol 3-kinase with insulin receptor substrates-1 and -2 was increased with dexamethasone, the increased association with IRS-2 may, at least partially, be explained by higher IRS-2 protein expression. Conclusions: The steroid does not cause hepatic resistance in vitro. The differential attenuation under steroid deprivation points to defects in branches of the insulin signal chain and/or loss of hormonal regulation at the level of target enzymes. (C) 2002 European Association for the Study of the Liver. Published by Elsevier Science B.V. All rights reserved.

The specific performance of the adult hepatic parenchymal cell is maintained and controlled by factors deriving from the stromal bed; the chemical nature of these factors is unknown. This study aimed to develop a serum-free hierarchical hepatocyte-nonparenchymal (stromal) cell coculture system. Hepatic stromal cells proliferated on crosslinked collagen in serum-free medium with epidermal growth factor, basic fibroblast growth factor, and hepatocyte-conditioned medium; cell type composition changed during the 2-wk culture period. During the first wk, the culture consisted of proliferating sinusoidal endothelial cells with well-preserved sieve plates, proliferating hepatic stellate cells, and partially activated Kupffer cells. The number of endothelial cells declined thereafter; stellate cells and Kupffer cells became the prominent cell types after 8 d. Hepatocytes were seeded onto stromal cells precultured for 4-14 d; they adhered to stellate and Kupffer cells, but spared the islands of endothelial cells. Stellate cells spread out on top of the hepatocytes; Kupffer cell extensions established multiple contacts to hepatocytes and stellate cells. Hepatocyte viability was maintained by coculture; the positive influence of stromal cell signals on hepatocyte differentiation became evident after 48 h; a strong improvement of cell responsiveness toward hormones could he observed in cocultured hepatocytes. Hierarchial hepatocyte coculture enhanced the glucagon-dependent increases in phosphoenolpyruvate carboxykinase activity and messenger ribonucleic acid (mRNA) content three- and twofold, respectively; glucagon-activated urea production was elevated twofold. Coculturing also stimulated glycogen deposition; basal synthesis was increased by 30% and the responsiveness toward insulin and glucose was elevated by 100 and 55%, respectively. The insulin-dependent rise in the glucokinase mRNA content was increased twofold in cocultured hepatocytes. It can be concluded that long-term signals from stromal cells maintain hepatocyte differentiation. This coculture model should, therefore, provide the technical basis for the investigation of stroma-derived differentiation factors.

Background/Aims: A major problem in rat liver endothelial cell culture is the rapid loss of cells after 48 h, This study aimed to develop a protocol that allowed easy maintenance and proliferation of sinusoidal endothelial cells in serum-free culture for 5-6 days. Methods: Cells isolated from adult rat liver by collagenase digestion were purified by centrifugal elutriation and cultured on glutaraldehyde-crosslinked collagen, Results: At high plating densities cells could be maintained serum-free for 6 days in the presence of hydrocortisone and basic fibroblast growth factor; at lower plating densities medium had to be supplemented with additional growth-promoting factors. Conditioned medium of adult rat hepatocytes proved to be the most effective growth stimulus; it increased thymidine incorporation, DNA content and cell number per dish with a half-maximal effect at 20% (v/v), Cell proliferation was also observed with either vascular endothelial growth factor, phorbol ester or conditioned media from FAO or HEPG2 liver cell lines provided the cultures were additionally supplemented with 1% newborn calf serum. Vascular endothelial growth factor was detected in all conditioned media. In the absence of hepatocyte-conditioned medium, 1% serum helped to maintain cultures; it itself exerted a low proliferative effect. Higher serum concentrations (>5%), however, led to cell loss after 48 h, The numerous sieve plates of 6-h-old cells progressively disappeared during culture and were replaced by randomly distributed pores, which later grouped together at cell-cell borders. More than 90% of the cells endocytosed acetylated low-density lipoprotein, Conclusions: The study shows that cultured hepatocytes secrete growth-promoting substances that stimulate in vitro endothelial cell proliferation in the absence of serum; this effect could be mimicked by the combined addition of vascular endothelial growth factor and 1% serum. The new media formulations should facilitate future research on liver endothelial cells in mono- or coculture.

Diacylglycerol (DAG) and protein kinase C (PKC) isoforms have been implicated in insulin signalling in muscle and fat cells. We evaluated the involvement of DAG and PKC in the action of insulin in adult rat hepatocytes cultured with dexamethasone, but in the absence of serum, for 48 h. Our results show that although insulin stimulated glycolysis and glycogen synthesis, it had no effect on DAG mass or molecular species composition. Epidermal growth factor showed the expected insulin-mimetic effect on glycolysis, whereas ATP and exogenous phospholipase C acted as antagonists and abolished the insulin signal. Similarly to insulin, epidermal growth factor had no effect on DAG mass or molecular species composition. In contrast, both ATP and phospholipase C induced a prominent increase in several DAG molecular species, including 18:0/20:4, 18:0/20:5, 18:0/22:5 and a decrease in 18:1/18:1. These changes were paralleled by an increase in phospholipase D activity, which was absent in insulin-treated cells. By immunoblotting or by measuring PKC activity, we found that neither insulin nor ATP translocated the PKCalpha, -delta, -epsilon or -zeta isoforms from the cytosol to the membrane in cells cultured for six or 48 h. Similarly, insulin had no effect on immunoprecipitable PKCzeta. Suppression of the glycogenic insulin signal by phorbol 12-myristate 13-acetate, but not by ATP, could be completely alleviated by bisindolylmaleimide. Finally, insulin showed no effect on DAG mass or translocation of PKC isoforms in the perfused liver, although it reduced the glucagon-stimulated glucose output by 75%. Together these results indicate that phospholipases C and D or multiple PKC isoforms are not involved in the hepatic insulin signal chain.

Background/Aims: Cultured adult hepatocytes may be stimulated into clonal expansion. We raise the question whether adult hepatocytes proliferating in vitro recapitulate the early process of hepatic development. Methods: A non-enzymatic method was used to isolate hepatocytes free of contamination with non-parenchymal cells. Hepatocytes were stimulated into proliferation in the presence of mitogens and conditioned media from nonparenchymal cell and hepatocyte culture supernatants. Immunofluorescence methods and PCR analysis were used to demonstrate immunophenotypical characteristics and gene expression profiles similar to those of progenitor cells. Results: Rapid growth occurred during the first 7 days of culture. Cells continued to express hepatic markers (phosphoenolpyruvate carboxykinase, cytokeratin 18, transferrin and dipeptidylpeptidase IV), but the gap junction protein connexin 32 was down-regulated. In the early stage of proliferation, cells started to express biliary and extrahepatic progenitor markers (cytokeratin 19, CD49b, CD49f, nestin, vimentin, Thyl and c-kit), followed by cytokeratin 7, connexin 43, and neural cell adhesion molecule. Co-expression of the epithelial liver progenitor marker alpha-foetoprotein with either nestin (neural marker) or Thyl (mesenchymal marker) was also demonstrated. Conclusions: Mature, hepatocytes reveal their potential to regain a spectrum of progenitor markers from different germ layers, suggesting, enormous plasticity and differentiation potential of adult liver cells. (c) 2006 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.