Bruno Christ

Sub‐compartmentation of the ‘cytosolic’ glucose 6‐phosphate pool in cultured rat hepatocytes

[14C]Glucose release either from endogenous 14C‐prelabelled glycogen or from added 14C‐labelled glucose 6‐phosphate was measured in filipin‐treated, permeabilized hepatocytes in 48 h culture. [14C]Glucose output from prelabelled glycogen was not altered by the addition of 5 mM glucose 6‐phospate to the incubation medium. Conversely, [14C]glucose release from 5 mM labelled glucose 6‐phosphate was not influenced by different glycogen concentrations in the cells. Moreover, in the permeabilized cells the anion transport inhibitor DIDS (4,4′‐diisothiocyanatostilbene‐2,2′‐disulfonic acid) inhibited only the liberation of [14C]glucose from labelled glucose 6‐phosphate but not from glycogen. It is therefore concluded that there exist at least 2 separate, mutually non‐accessible glucose 6‐phosphate pools in cultured rat hepatocytes, one linked to glycogenolysis and the other to gluconeogenesis.

Elevated expression of hormone-regulated rat hepatocyte functions in a new serum-free hepatocyte-stromal cell coculture model.

SummaryThe 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 be 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.

Inhibition by PGE2 of glucagon‐induced increase in phosphoenolpyruvate carboxykinase mRNA and acceleration of mRNA degradation in cultured rat hepatocytes

In cultured rat hepatocytes the key gluconeogenic enzyme phosphoenolpyruvate carboxykinase (PCK) is known to be induced by glucagon via an elevation of cAMP. Prostaglandin E2 has been shown to antagonize the glucagon‐activated cAMP formation, glycogen phosphorylase activity and glucose output in hepatocytes. It was the purpose of the current investigation to study the potential of PGE2 to inhibit the glucagon‐induced expression of PCK on the level of mRNA and enzyme activity. PCK mRNA and enzyme activity were increased by 0.1 nM glucagon to a maximum after 2 h and 4 h, respectively. This increase was completely inhibited if 10 μM PGE2 was added concomitantly with glucagon. This inhibition by PGE2 of glucagon‐induced PCK activity was abolished by pertussis toxin treatment. When added at the maximum of PCK mRNA at 2 h, PGE2 accelerated the decay of mRNA and reduced enzyme activity. This effect was not reversed by pertussis toxin treatment. Since in liver PGE2 is derived from Kupffer cells, which play a key role in the local inflammatory response, the present data imply that during inflammation PGE2 may reduce the hepatic gluconeogenic capacity via a Gi‐linked signal chain.

Purification of a RNA-binding Protein from Rat Liver IDENTIFICATION AS FERRITIN L CHAIN AND DETERMINATION OF THE RNA/PROTEIN BINDING CHARACTERISTICS

In cultured rat hepatocytes the degradation of phosphoenolpyruvate carboxykinase mRNA might be regulated by protein(s), which by binding to the mRNA alter its stability. The 3′-untranslated region of phosphoenolpyruvate carboxykinase mRNA as a potential target was used to select RNA-binding protein(s) from rat liver by the use of gel retardation assays. A cytosolic protein was isolated, which bound to the phosphoenolpyruvate carboxykinase mRNA 3′-untranslated region and other in vitro synthesized RNAs. The protein was purified to homogeneity; it had an apparent molecular mass of 400 kDa and consisted of identical subunits with an apparent size of 24.5 kDa. Sequence analysis of a tryptic peptide from the 24.5-kDa protein revealed its identity with rat ferritin light chain. Binding of ferritin to RNA was abolished after phosphorylation with cAMP-dependent protein kinase and was augmented after dephosphorylation with alkaline phosphatase. Weak binding was observed in extracts from okadaic acid-treated cultured hepatocytes compared with untreated cells. Preincubation of ferritin with an anti-phosphoserine or an anti-phosphothreonine antibody attenuated binding to RNA, while an anti-phosphotyrosine antibody generated a supershift indicating that phosphoserine and phosphothreonine but not phosphotyrosine residues were in close proximity to the RNA-binding region. Ferritin is the iron storage protein in the liver. Binding of ferritin to RNA was diminished in the presence of increasing iron concentrations, whereas the iron chelator desferal was without effect. It is concluded that ferritin might function as RNA-binding protein and that it may have important functions in the general regulation of cellular RNA metabolism.

Interactions of nuclear protein from cultured rat hepatocytes with the cyclic AMP responsive elements and the NF1-CTF site in the promotor of the rat phosphoenolpyruvate carboxykinase gene

Nuclear extracts from cultured rat hepatocytes were analyzed by gel mobility shift assay for protein binding to the cyclic AMP responsive elements CRE1 (-96/-77) and CRE2 (-152/-132) and the NF1-CTF binding site (-121/-99) of the phosphoenolpyruvate carboxykinase (PCK) promotor. Binding was very weak to the CRE2 and CRE1. The NF1-CTF site formed two complexes with nuclear protein. Protein binding was increased, when the NF1-CTF site was coupled to the CRE1, and further, when it was coupled to both the CRE1 and the CRE2. Complex formation was not altered by treatment of the hepatocytes with glucagon or with glucagon and insulin. Thus, protein binding was most efficient when all three elements were in context, which might be necessary for full transcriptional activation of the PCK gene.

Regulation of the Glucose/Glucose-6-Phosphate Cycle in Cultured Hepatocytes by Insulin and Glucagon

Die Regulation des Glucose/Glucose-6-Phosphat-Zyklus durch Insulin und Glucagon wurde in Leberzellen in Primarkultur untersucht. Zur Abschatzung der Fluxraten des Zyklus wurde einerseits der Verbrauch von (u14c, 23H)-Glucose, andererseits der Verbrauch von (23H)-Glucose, die Bildung von (u14c)-Glucose aus (u14c)-Glykogen bestimmt.