Activation of peroxisome proliferator-activated receptors (PPARs) by their ligands and protein kinase A activators.

The nuclear peroxisome proliferator-activated receptors (PPARs) alpha, beta, and gamma activate the transcription of multiple genes involved in lipid metabolism. Several natural and synthetic ligands have been identified for each PPAR isotype but little is known about the phosphorylation state of these receptors. We show here that activators of protein kinase A (PKA) can enhance mouse PPAR activity in the absence and the presence of exogenous ligands in transient transfection experiments. Activation function 1 (AF-1) of PPARs was dispensable for transcriptional enhancement, whereas activation function 2 (AF-2) was required for this effect. We also show that several domains of PPAR can be phosphorylated by PKA in vitro. Moreover, gel retardation experiments suggest that PKA stabilizes binding of the liganded PPAR to DNA. PKA inhibitors decreased not only the kinase-dependent induction of PPARs but also their ligand-dependent induction, suggesting an interaction between both pathways that leads to maximal transcriptional induction by PPARs. Moreover, comparing PPAR alpha knockout (KO) with PPAR alpha WT mice, we show that the expression of the acyl CoA oxidase (ACO) gene can be regulated by PKA-activated PPAR alpha in liver. These data demonstrate that the PKA pathway is an important modulator of PPAR activity, and we propose a model associating this pathway in the control of fatty acid beta-oxidation under conditions of fasting, stress, and exercise.

Maintenance of primary murine hepatocyte functions in multicomponent polymer capsules--in vitro cryopreservation studies.

BACKGROUND/AIMS: The potential of a new encapsulation system has been evaluated as an artificial housing for liver cells. METHODS: Murine hepatocytes were encapsulated in specially designed multicomponent capsules formed by polyelectrolyte complexation of sodium alginate, cellulose sulphate and poly(methylene-co-guanidine) hydrochloride, the permeability of which has previously been characterised. RESULTS: We demonstrate here the absence of cytotoxicity and the excellent biocompatibility of these capsules towards primary culture of murine hepatocytes. Experimental results demonstrated that the encapsulated hepatocytes retained their specific functions--transaminase activity, urea synthesis and protein secretion--over the first 4 days of culture in minimum medium. The cryopreservation of encapsulated hepatocytes, for periods of up to 4 months, did not alter their functional capacities, as no major differences were observed between unfrozen and frozen encapsulated cells for the functions tested. CONCLUSIONS: Because of the absence of cytotoxicity, and the ease of handling and cryopreservation, while maintaining liver specific functions, the described system appears to be valuable for murine liver cell encapsulation. It is also a promising tool for fundamental research into drug metabolism, intercellular regulation, metabolic pathways, and the establishment of banks for the supply and storage of murine hepatocytes.