The effect of Smallanthus sonchifolius leaf extracts on rat hepatic metabolism.

Smallanthus sonchifolius (yacon), originating from South America, has become popular in Japan and in New Zealand for its tubers which contain beta-1,2-oligofructans as the main saccharides. The plant is also successfully cultivated in Central Europe in the Czech Republic in particular. Its aerial part is used in Japan and in Brazil as a component in medicinal teas; while aqueous leaf extracts have been studied for their hypoglycemic activity in normal and diabetic rats. We have already demonstrated the high content of phenolic compounds in yacon leaf extracts and their in vitro antioxidant activity. In this paper, we present the effects of two organic fractions and two aqueous extracts from the leaves of S. sonchifolius on rat hepatocyte viability, on oxidative damage induced by tert-butyl hydroperoxide (t-BH) and allyl alcohol (AA), and on glucose metabolism and their insulin-like effect on the expression of cytochrome P450 (CYP) mRNA. All the extracts tested exhibited strong protective effect against oxidative damage to rat hepatocyte primary cultures in concentrations ranging from 1 to 1000 microg/ml, reduced hepatic glucose production via gluconeogenesis and glycogenolysis at 1000 microg/ml. Moreover, the effects of the organic fractions (200 and 250 microg/ml) and to a lesser extent, the tea infusion (500 microg/ml) on rat CYP2B and CYP2E mRNA expression, were comparable to those observed with insulin. The combination of radical scavenging, cytoprotective and anti-hyperglycemic activity predetermine S. sonchifolius leaves for use in prevention and treatment of chronic diseases involving oxidative stress, particularly diabetes.

Regulation of adenylyl cyclase activity in human peripheral blood mononuclear cells: effects of protein kinase inhibitors and of a calcium ionophore.

In a previous paper we presented evidence for a negative regulation of adenylyl cyclase activity by tyrosine protein kinase(s) in the human leukemic T cell line Jurkat. In order to examine this point in non malignant cells, we conducted the present study in human peripheral blood mononuclear cells (PBMC). In these cells, staurosporine, a broad spectrum protein kinase inhibitor, enhanced not only the receptor-mediated, induced by prostaglandin E2 (PGE2), but also the direct (forskolin-induced) stimulation of adenylyl cyclase activity. Herbimycin A, a specific protein tyrosine kinase inhibitor, reproduced only in part the effect of staurosporine, whereas bisindolylmaleimide, the most specific protein kinase C (PKC) inhibitor known at present time, was ineffective. All these observations were made both in the absence and presence of isobutylmethylxanthine, a phosphodiesterase inhibitor, indicating that the effects of staurosporine and herbimycin A on cAMP accumulation were not due to phosphodiesterase inhibition. The calcium ionophore A 23187 also enhanced the PGE2-induced cAMP accumulation, and this effect was not additive to that of staurosporine, but additive to that of herbimycin A. These results confirm and extend those obtained in Jurkat cells. Taken together, they indicate that in human PBMC the adenylyl cyclase activity is negatively regulated by tyrosine kinase(s) and not by PKC, and positively regulated by Ca2+. They also suggest that the major enhancement by staurosporine of the PGE2-induced cAMP accumulation, although chiefly mediated by protein tyrosine kinase inhibition, also depends on another, presently undetermined, effect of the drug simulating that of Ca2+.

Expression of CYP3A in the human liver--evidence that the shift between CYP3A7 and CYP3A4 occurs immediately after birth.

CYP3A isoforms are responsible for the biotransformation of a wide variety of exogenous chemicals and endogenous steroids in human tissues. Two members of the CYP3A subfamily display developmentally regulated expression in the liver; CYP3A7 is expressed in the fetal liver, whereas CYP3A4 is the major cyrochrome P-450 isoform present in the adult liver. To gain insight into the descriptive ontogenesis of CYP3A isoforms during the neonatal period, we have developed several approaches to explore a neonatal liver bank. Although CYP3A4 and CYP3A7 are structurally closely related, they differ in their capacity to carry out monooxygenase reactions. We have cloned CYP3A4 and CYP3A7 and established stable transfectants in Ad293 cells to investigate their substrate specificities. The 16alpha hydroxylation of dehydroepiandrosterone is catalyzed by both proteins, but CYP3A7 has a higher affinity and maximal velocity than CYP3A4. Conversely, the conversion of testosterone into its 6beta derivative is essentially supported by CYP3A4. We used these two probes to determine the ontogenic evolution at the protein level; CYP3A7 was very active in the fetal liver and its activity was maximal during the first week following birth before to progressively decline and reached a very low level in adult livers. Conversely, the activity of CYP3A4 was extremely weak in the fetus and began to raise after birth to reach 30-40% of the adult activity after one month. CYP3A4 RNA accumulation displays a similar pattern of evolution; when probed with an oligonucleotide, its concentration increased rapidly after birth to reach a plateau as soon as the first week of age. These data supports the assumption that CYP3A4 expression is transcriptionally activated during the first week after birth and is accompanied by a simultaneous decrease of CYP3A7 expression, in such a way that the overall CYP3A protein content and the level of pentoxyresorufin dealkylase catalyzed by the two proteins remain nearly constant.

Staurosporine and herbimycin A augment agonist-induced elevation of cAMP in Jurkat T-lymphoblasts.

Cross-talk between signalling pathways appears to play an important role in T-lymphocyte activation. In the present work, we have studied the effects of different inhibitors of protein tyrosine kinases or protein serine/ threonine kinases on the agonist-induced cAMP accumulation in the human T-lymphoblast cell line Jurkat. Staurosporine, a potent but nonspecific inhibitor of protein kinases, produced a ten-fold enhancement of the response to PGE2. No significant effect was obtained with two specific protein kinase C inhibitors (GF 109203X and H7), whereas herbimycin A, a specific protein tyrosine kinase inhibitor, markedly enhanced the PGE2-induced cAMP accumulation: its effect was approximately 60% that of staurosporine. It was confirmed that both staurosporine and herbimycin A inhibited by more than 90% the release of IP3 induced by ligation of the T-cell receptor, a known protein tyrosine kinase-dependent mechanism. To our knowledge, this study provides the first indication of a protein tyrosine kinase-mediated inhibition of agonist-induced cAMP accumulation. The possible targets of this inhibition are discussed.

T-cell antigen receptor-mediated enhancement of the adenylate cyclase pathway depends on tyrosine protein kinases.

We have examined in the human T-cell line Jurkat the interaction between the activation through the T-cell receptor/CD3 complex and the adenylate cyclase pathway. OKT3, an anti-CD3 monoclonal antibody, did not activate by itself adenylate cyclase but produced a 3-7-fold increase of the cAMP accumulation induced by indirect (chloroadenosine, PGE2) or direct (forskolin) agonists of adenylate cyclase. A more detailed study with forskolin showed that OKT3 enhanced the effect of low concentrations of the agonist without affecting the maximal capacity of cAMP synthesis of the cells. The same concentrations of OKT3 produced both the enhancement of the adenylate cyclase pathway and the activation of phospholipase C. The enhancement by OKT3 of the adenylate cyclase pathway was inhibited by 0.5 microM staurosporine, a potent inhibitor of protein kinases, including tyrosine kinases and protein kinase C, whereas it was not inhibited by H7, a specific inhibitor of PKC. Staurosporine, at the same concentration, also inhibited the OKT3-induced activation of phospholipase C, a tyrosine kinase-dependent process. Taken together, these data indicate that activation of T-cell through the T-cell receptor enhances the adenylate cyclase pathway by a tyrosine protein kinase-dependent mechanism.

Influence of adenosine deaminase inhibition on the phosphoinositide turnover in the initial stages of human T cell activation.

An experimental model of adenosine deaminase deficiency was established on the human T cell line Jurkat by using 2'-deoxycoformycin, a strong specific inhibitor of the enzyme. When deoxyadenosine was added to the inhibited cells, the nucleotide profile was modified reproducing that found in lymphocytes from adenosine deaminase-deficient children. The metabolism of phosphoinositides, analyzed by either the release of [3H]inositol phosphates or the breakdown of 32P-prelabeled phosphatidyl inositides, was compared in normal and modified cells where dATP was accumulated. No modification in 32P labeling of phosphoinositides was detectable within the 32P-loading period. However, when the cells were stimulated by phytohemagglutinin or anti-CD3 monoclonal antibody, the phosphoinositide hydrolysis was strongly reduced in the dATP-containing lymphoblasts. This decrease was correlated with the intracellular dATP concentration.

The effect of adenosine and chloroadenosine on sex differences in the energy metabolism of rat hepatocytes.

The intensity and regulation of metabolic pathways are different depending on the sex of the source animal for hepatocytes isolated from mature rats. In cells from fed animals incubated without exogenous substrate, ATP level and ketone body production are higher in males (+25% and +100%) and lactate production is higher (+64%) in females; oleate enhances mitochondrial pyruvate oxidation in hepatocytes from fed male rats but not from fed females; in cells from starved animals oleate increases gluconeogenesis in both sexes at saturating levels of gluconeogenic substrates. However, at physiological levels (1 mM lactate and 0.1 mM pyruvate), this activation can only be detected in cells from males. In both sexes, oleate activation is abolished by adenosine which reduces in parallel the mitochondrial oxidation of pyruvate; chloroadenosine, an A2-receptor agonist, increases glycogenolysis strongly in hepatocytes from male animals (+80%) but only very slightly in female cells (+12%).

Energy metabolism in adenosine deaminase-inhibited human erythrocytes.

The metabolic changes induced by the deoxycoformycin inhibition of adenosine deaminase were studied in human erythrocytes incubated with nucleosides. 1 Adenosine nucleotide levels and glycolytic rate were increased by adenosine. 2 With deoxyadenosine, the cellular ATP level was reduced when dATP increased and the glycolytic rate was similarly enhanced. 3 The hypoxanthine production was equivalent in both cases. Our data demonstrate that human red cells are able to catabolize adenine deoxynucleotides into hypoxanthine, and the control of energy metabolism is not impaired by adenosine deaminase inhibition when PO identical to 4 and NAD+ are not limiting.