Einleitung. Lipopolysaccharide (LPS) gehören zu den wichtigsten Entzündungsauslösern im Säugerorganismus. Eigene Vorarbeiten haben gezeigt, dass Hepatozyten der Maus den LPS-Rezeptor-Komplex aus membranständigem CD14 (mCD14) und Toll-like-Rezeptor 4 (TLR4) nicht exprimieren und dementsprechend auch nicht direkt durch LPS stimulierbar sind. Durch IL-6 oder IL-1β sind in Ratten-Hepatozyten Rezeptoren für Anaphylatoxin C5a als Endprodukt der durch LPS ausgelösten Komplementaktivierung de novo induzierbar. Es sollte daher untersucht werden, ob auch mCD14/TLR4 in Hepatozyten induzierbar ist und ob dieser Rezeptor-Komplex funktionell aktiv ist. Methodik. Hepatozyten wurden aus Mäuselebern isoliert, mit IL-6, IL-1β oder TNFα stimuliert und die Expression von CD14-und TLR4-mRNA durch quantitative Real-Time-PCR, von CD14- und TLR4-Protein durch Western-Blots von Triton X114-Lysaten analysiert. Zur Überprüfung der Funktionalität der de novo exprimierten Rezeptoren wurden mit IL-1β vorstimulierte Hepatozyten mit LPS inkubiert und die α2-Macroglobulin-Synthese mittels Northern- und Western-Blot analysiert. Ergebnisse. Hepatozyten exprimierten konstitutiv geringe Mengen an CD14-mRNA aber kein mCD14-Protein und keine TLR4-mRNA. Die Stimulation mit proinflammatorischen Cyto? kinen verstärkte die Expression von CD14-mRNA und induzierte die Expression von TLR-4-mRNA und von mCD14-Protein in der Membran. Alle 3 Parameter wurden durch IL-1β am stärksten, durch IL-6 etwas weniger und durch TNFα am wenigsten stark beeinflusst. In mit IL-1β vorstimulierten Hepatozyten induzierte LPS – anders als in unbehandelten Hepatozyten – die mRNA- und Protein-Expression des Typ-2-Akut-Phase-Proteins α2-Macroglobulin. Schlussfolgerung: Durch Stimulation mit proinflammatorischen Cytokinen wird die Expression des LPS-Rezeptor-Komplexes aus mCD14 und TLR4 in Hepatozyten der Maus induziert. Durch diese Behandlung erlangen die Zellen eine direkte Reaktivität für LPS.

Prostanoids, i.e. prostaglandins and thromboxane, regulate liver-specific functions both in homeostasis and during defense reactions. For example, prostanoids are released from Kupffer cells, the resident liver macrophages, in response to the inflammatory mediator anaphylatoxin C5a, and mediate an enhanced glucose output from hepatocytes as energy supply. In perfused rat livers, the thromboxane receptor antagonist daltroban enhanced C5a-induced prostanoid overflow and reduced glucose output. It was the aim of this study to elucidate whether daltroban interfered with prostanoid release from Kupffer cells or prostanoid clearance by hepatocytes, and/or whether it directly influenced prostanoid-dependent glucose metabolism in these cells. In perfused rat livers, daltroban enhanced prostaglandin (PG)D(2) overflow not only after infusion of C5a (15-fold), but also after PGD(2) (10-fold). Neither daltroban nor another receptor antagonist, ifetroban, or the thromboxane synthase inhibitor furegrelate enhanced prostanoid release from Kupffer cells. In contrast, all inhibitors reduced clearance, i.e. uptake and degradation, of PGD(2) by hepatocytes: within 5 min uptake of 1 nmol/L PGD(2) was reduced from 43+/-5 fmol (controls) to 22+/-6 fmol (daltroban), 24+/-6 fmol (ifetroban) and 21+/-6 fmol (furegrelate). PGD(2) in the medium was reduced to 39+/-7% in the controls, but remained at 93+/-9%, 93+/-11% and 60+/-3% in the presence of the inhibitors. PGD(2)-dependent glucose output in the perfused liver or activation of glycogen phosphorylase in isolated hepatocytes remained unaffected by daltroban. These data clearly demonstrate that the thromboxane-inhibitors reduced PGD(2) clearance by hepatocytes, presumably by inhibition of prostanoid transport into the cells. In contrast, they did not interfere with PGD(2)-dependent glucose metabolism, suggesting an independent mechanism for the inhibition of glucose output from the liver.

Since protein binding to the 3' end of mRNA is believed to be involved in the control of mRNA stability, the time course of alterations in glucagon-induced phosphoenolpyruvate-carboxykinase-mRNA (PCK) levels, in the absence and presence of insulin, was correlated with the time course of changes in the binding of cytosolic protein from 24-h cultured rat hepatocytes to the 3' end of PCK mRNA. PCK-mRNA levels were monitored by Northern blot analysis and protein binding was analyzed by an electrophoretic mobility-shift assay. In 24-h cultured rat hepatocytes, binding of cytosolic protein to the PCK-mRNA 3' end and PCK-mRNA levels were increased to a transient maximum at 2 h and 2-4 h, respectively, by a 1-nM glucagon treatment, added with a change of medium. 100 nM insulin, added simultaneously with glucagon, reduced the glucagon-induced maximum of protein binding by 80% and the increase of PCK mRNA by about 30%. In controls without hormonal treatment protein binding at 1 h was also increased; this increase was prevented by insulin. 100 nM insulin, added 1 h after glucagon, reversed protein binding to the 3' end of PCK mRNA to nearly initial levels within 1 h and impaired the glucagon-induced increase in PCK-mRNA levels by 30%. The transcriptional inhibitor cordycepin, added 1 h after glucagon, did not prevent the further increase in glucagon-enhanced protein binding nor its reversal by insulin. It did, however, prevent a further significant increase in PCK mRNA. Hormonally regulated protein binding could be localized to the 256 distal bases of the PCK-mRNA 3' end. The proximal 466 bases of the PCK-mRNA 3' end as well as the 1050 bases of the histone-H1(0)-mRNA 3' end and the 1200 bases of the arylsulfatase-A-mRNA 3' end also bound cytosolic protein(s), but this protein binding was not altered by treatment with glucagon or insulin. The 3' end of PCK, arylsulfatase A and H1(0) mRNA exhibited strong binding of cytosolic protein(s) from diverse rat tissues such as heart, liver and lung as well as Fao rat hepatoma cells. Cytosolic protein(s) from spleen showed weak binding and proteins from HeLa and U937 tumor cells did not bind. Protein binding was most prominent with the 3' end of PCK mRNA and cytosolic extracts from liver.(ABSTRACT TRUNCATED AT 400 WORDS)

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.