Mitochondrial biogenesis in the liver during development and oncogenesis.

The analysis of the expression of oxidative phosphorylation genes in the liver during development reveals the existence of two biological programs involved in the biogenesis of mitochondria. Differentiation is a short-term program of biogenesis that is controlled at post-transcriptional levels of gene expression and is responsible for the rapid changes in the bioenergetic phenotype of mitochondria. In contrast, proliferation is a long-term program controlled both at the transcriptional and post-transcriptional levels of gene expression and is responsible for the increase in mitochondrial mass in the hepatocyte. Recently, a specific subcellular structure involved in the localization and control of the translation of the mRNA encoding the beta-catalytic subunit of the H(+)-ATP synthase (beta-mRNA) has been identified. It is suggested that this structure plays a prominent role in the control of mitochondrial biogenesis at post-transcriptional levels. The fetal liver has many phenotypic manifestations in common with highly glycolytic tumor cells. In addition, both have a low mitochondrial content despite a paradoxical increase in the cellular representation of oxidative phosphorylation transcripts. Based on the paradigm provided by the fetal liver we hypothesize that the aberrant mitochondrial phenotype of fast-growing hepatomas represents a reversion to a fetal program of expression of oxidative phosphorylation genes by the activation, or increased expression, of an inhibitor of beta-mRNA translation.

Protein phosphatase 1 and 2A inhibitors activate acyl-CoA:cholesterol acyltransferase and cholesterol ester formation in isolated rat hepatocytes.

Okadaic acid, calyculin A and cantharidin, potent and specific inhibitors of protein phosphatases 1 (PP1) and 2A (PP2A), stimulated both acyl-CoA:cholesterol acyltransferase (ACAT) activity and cholesterol ester formation in suspension cultures of isolated rat hepatocytes. The activation of microsomal ACAT was marked (up to 14-fold the basal values), fast in onset (within 5 min), persistent in duration (up to 45 min) and concentration-dependent. Concentrations of okadaic acid (OA) or calyculin A > or = 100 nM or of cantharidin > or = 1 microM were required to stimulate enzyme activity, which specifically points to a dominant contribution of PP1. No effects were seen with up to 1 microM nor-okadaone, an inactive OA analogue. Rises in [3H]oleate incorporation into cell cholesteryl esters closely paralleled those in ACAT activity, though were somewhat less accentuated. The increases in microsomal ACAT activity seen in OA-, calyculin A- or cantharidin-treated hepatocytes were not linked to changes in bulk microsomal unesterified cholesterol or in the de novo cholesterol synthesis. The findings firmly indicate a role for protein phosphatase activity, probably that of PP1, in controlling the cholesterol esterification rate and ACAT activity in intact rat hepatocytes, which is not secondary to an alteration of the steady-state distribution of cholesterol mass between cell membranes. However, as the OA-induced stimulation of ACAT was not abrogated by addition of purified PP1 or PP2A to microsomes, it is unlikely that the phosphatase inhibitors here used act directly on the phosphorylation degree of the ACAT enzyme.

The integrity of thiol groups is essential for catalytic efficiency of rat liver cholesterol ester hydrolase either in microsomal membranes or after solubilization.

Neutral cholesterol ester hydrolase from rat liver microsomes was inactivated in a dose and time-dependent manner by classical sulphydryl-reacting reagents such as p-hydroxymercuribenzoic acid, 5,5'-dithio-bis-(2-nitrobenzoic acid), N-ethylmaleimide, or iodoacetate. The concentrations at which half-maximal inhibition of the native microsomal cholesterol ester hydrolase occurred (IC50) were 15, 68, and 370 mumol/l and 68 mmol/l, respectively. Only partial reactivation of the enzyme was observed under excess dithiothreitol or mercaptoethanol treatment. The stimulation of cholesterol ester hydrolase by the metal ions Ca2+ and Mg2+ was dependent on the integrity of the thiol groups. Solubilization of cholesterol ester hydrolase from membranes preserved its sensitivity towards sulphydryl reagents and thiols, as well as its ability to be activated by Ca2+ and Mg2+. Dithiothreitol, mercaptoethanol, and Ca2+ and Mg2+ provided total protection of the enzyme against inactivation by thiol-reacting reagents. The results indicate that one or more thiol groups are either at the active centre of the native and solubilized forms of rat liver microsomal cholesterol ester hydrolase or are sufficiently near, to interfere with the catalysis when they are reacted.