Dietary soy isoflavones inhibit activation of rat platelets

Isoflavones (isoflavonoids) have been proposed to be the active compounds that contribute to decreased mortality from chronic diseases in populations that consume large amounts of soy products. Diets containing soy protein with and without isoflavones were fed to rats to determine if these compounds could exert in vivo effects on physiologic markers of platelet activation. Three methods were employed to monitor platelet activation: measurement of electronic mean platelet volume, which is an indicator of shape change; monitoring of collagen-induced production of reactive oxygen signals (hydrogen peroxide); and determination of increases in phosphorylation of protein tyrosine residues after collagen stimulation. Apparent volumes were significantly smaller for platelets from rats fed isoflavones, suggesting that these platelets were in a more disc-like, quiescent state compared with platelets from rats fed the isoflavone-reduced diet (means +/- SEM, 5.37 +/- 0.08 vs. 5.70 +/- 0.06 fL, n = 6/group, P < 0.008). Results from the other functional tests were consistent with this finding. Platelet production of hydrogen peroxide was found be significantly lower 1, 3, and 5 minutes after addition of collagen for rats fed isoflavones versus rats fed the isoflavone-reduced diet (n = 6/group, P < 0.004). Phosphorylated tyrosine residues in platelet proteins after stimulation also were shown to be significantly lower in the platelets exposed to dietary isoflavones (n = 5/group, P < 0.047). These combined results indicate that soy isoflavones can alter early-event signaling networks that result in less activated platelets and may partially explain the beneficial effects of dietary soy against human heart disease.     

Up-regulation of multidrug resistance-associated protein 2 (MRP2) expression in rat hepatocytes by dexamethasone.

Regulation of multidrug resistance-associated protein (MRP2) expression in response to dexamethasone (DEX) was analyzed using mainly primary rat hepatocytes. Enhanced levels of MRP2 mRNAs associated with increased amounts of a 190 kDa MRP2 were found in cultured DEX-treated hepatocytes; similarly, administration of DEX to rats (100 mg/kg, i.p.) led to a marked increase of hepatic amounts of MRP2 mRNAs. Maximal induction of MRP2 expression in DEX-treated primary hepatocytes was reached with 10(-5) M DEX, a concentration higher than that (10(-7) M) required for maximal up-regulation of tyrosine aminotransferase (TAT), a typical glucocorticoid receptor-regulated enzyme. In addition, the anti-glucocorticoid compound RU486 failed to inhibit MRP2 induction caused by DEX whereas it fully blocked that of TAT. These findings therefore demonstrate that DEX is a potent inducer of MRP2 expression in rat hepatocytes through a mechanism that seems not to involve the classical glucocorticoid receptor pathway.     

Epidermal growth factor as a new regulator of induction of tyrosine aminotransferase and tryptophan oxygenase by glucocorticoids

Epidermal growth factor (EGF) dose-dependently enhanced the induction of tyrosine aminotransferase and tryptophan oxygenase by glucocorticoids in primary cultures of adult rat hepatocytes without itself having any effect on these enzymes in the absence of glucocorticoids. The amplifications were observed even with dexamethasone at high concentrations (10(-6) M-10(-5) M) that had a maximal effect. EGF had no effect on induction of tyrosine aminotransferase by glucagon or Bt2cAMP. The effect of EGF was also observed in adrenal-ectomized and submaxillary gland-ectomized rats. These results suggest that EGF is an endogenous amplifier of the action of glucocorticoids.     

The effect of sodium butyrate on tyrosine aminotransferase induction in primary cultures of normal adult rat hepatocytes

Treatment of primary cultures of adult rat hepatocytes with 5 mM butyrate inhibited the spontaneous decrease in basal activity and mRNA levels of tyrosine aminotransferase (TAT) that occurred during culture (Staecker et al., submitted). We report here that butyrate treatment of primary cultures of rat hepatocytes initially inhibited the induction of TAT. This inhibition was followed by a period of accelerated TAT induction. TAT induction in butyrate-treated primary cultures of adult rat hepatocytes occurred only after metabolism of butyrate by the cultured hepatocytes. The accelerated induction of TAT in hepatocyte cultures treated with sodium butyrate was reflected by increased TAT activity and mRNA levels. Cultured hepatocytes rapidly metabolized butyrate, but the addition of more butyrate into cultures after its initial metabolism resulted in a rapid reduction in TAT activity. These findings indicate that butyrate treatment can affect the expression of TAT in primary hepatocyte cultures in both a positive (increased basal TAT expression) and a negative (inhibition of the induced expression of TAT) manner.     

Glucagon resistance of hepatoma cells. Evidence for receptor and post-receptor defects.

Of all available liver cells in culture, only primary cultured hepatocytes are known to respond to glucagon in vitro. In the present study we investigated whether glucagon could stimulate amino acid transport and tyrosine aminotransferase (TAT;EC 2.6.1.5) activity (two well-characterized glucagon effects in the liver) in Fao cells, a highly differentiated rat hepatoma cell line. We found that glucagon had no effect on transport of alpha-aminoisobutyric acid (AIB; a non-metabolizable alanine analogue) nor on TAT activity, even though both activities could be fully induced by insulin [2-fold and 3-fold effects for AIB transport and TAT activity, respectively, after 6h; EC50 (median effective concentration) = 0.3 nM], or by dexamethasone (5-8-fold effects after 20 h; EC50 = 2 nM). Analysis of [125I]iodoglucagon binding revealed that Fao cells bind less than 1% as much glucagon as do hepatocytes, whereas insulin binding in Fao cells was 50% higher than in hepatocytes. The addition of dibutyryl cyclic AMP, which fully mimics the glucagon stimulation of both AIB transport and TAT activity in hepatocytes, induced TAT activity in Fao cells (a 2-fold effect at 0.1 mM-dibutyryl cyclic AMP) but had no effect on AIB transport. Cholera toxin stimulated TAT activity to the same extent as did dibutyryl cyclic AMP. These results indicate that the lack of glucagon responsiveness in cultured hepatoma cells results from both a receptor defect and, for amino acid transport, an additional post-receptor defect. Moreover, the results show that amino acid transport and TAT activity, which appeared to be co-induced by insulin or by dexamethasone in these cells, respond differently to cyclic AMP. This suggests that different mechanisms are involved in the induction of these activities by glucagon in liver.     

Lack of glucose effect on the induction of 5-aminolevulinate synthetase and tyrosine aminotransferase in the isolated perfused rat liver.

In the isolated perfused rat liver, both 5-aminolevulinate synthetase and tyrosine aminotransferase were induced by the addition of 3.5 mmol/l allylisopropylacetamide and 58 mumol/l dexamethasone to the perfusion medium. Glucose (40 mmol/l) did not affect either the induction of these enzymes or the intrahepatic level of cyclic AMP. The results suggest that the glucose effect on the induction of 5-aminolevulinate synthetase and tyrosine aminotransferase in vivo is mediated by extrahepatic factors.     

Induction of cytochrome P-450 by glucocorticoids in rat liver. II. Evidence that glucocorticoids regulate induction of cytochrome P-450 by a nonclassical receptor mechanism

In the companion report we used primary cultures of adult rat hepatocytes to demonstrate that glucocorticoids comprise a "class" of compounds that stimulate de novo synthesis of a form of cytochrome P-450 (P450PCN) indistinguishable from that induced by the nonhormonal steroid pregnenolone 16 alpha-carbonitrile (PCN). Because induction of P450PCN is stereospecific for glucocorticoids and is dependent on the concentration of and the length of exposure to steroids it seemed possible that P450PCN represented another of the many genes whose expression is coordinately regulated by glucocorticoids bound to their specific cytoplasmic receptor and translocated into the nucleus. However, in cultured hepatocytes treated with glucocorticoids, synthesis of P450PCN failed to parallel synthesis of a typical glucocorticoid-responsive liver function, tyrosine aminotransferase, in the time course of induction, in the concentrations of glucocorticoids required for half-maximal induction, and in the order of effective steroids ranked by potency. Indeed, two moderately potent inducers of P450PCN either failed to induce tyrosine aminotransferase (spironolactone) or actually antagonized induction of tyrosine aminotransferase synthesis by glucocorticoids (PCN). Moreover, in the same cultures in which glucocorticoid induction of tyrosine aminotransferase was blocked by the presence of PCN or other previously identified antiglucocorticoids, synthesis of P450PCN was actually enhanced. We conclude that synthesis of P450PCN is a specific glucocorticoid-responsive liver function evoked by a novel mechanism readily distinguishable from the classic glucocorticoid receptor pathway.     

Effect of high- and low-protein diets on the regulation of rat liver tyrosine aminotransferase and tryptophan oxygenase activities by l-tyrosine and l-tryptophan

Induction of rat liver tyrosine aminotransferase by l-tyrosine and tryptophan oxygenase by l-tryptophan was studied in groups of rats fed on diets containing 18 or 5% protein. The basal activity of hepatic tyrosine aminotransferase of rats receiving 5% protein gradually increased with the age of the animals but that of rats receiving 18% protein did not. l-Tyrosine induced hepatic tyrosine aminotransferase in rats receiving 18% protein when tested at ages from 4 to 20 weeks. When induction by l-tyrosine was carried out in rats receiving the 5% protein diet, significant induction of tyrosine aminotransferase occurred only in 4- or 6-week-old rats. Induction by l-tryptophan of tryptophan oxygenase in liver or the basal activity of this enzyme in liver did not differ between the groups fed on 5 and 18% protein. On changing the diet from 0 to 18% protein, the above-mentioned effects on the induction of hepatic tyrosine aminotransferase were reversed.     

Involvement of G(i) proteins and Src tyrosine kinase in TNFalpha production induced by lipopolysaccharide,group B Streptococci and Staphylococcus aureus

Previous studies have suggested that heterotrimeric G(i) proteins, Src tyrosine kinase and phosphatidylinositol-3 kinase (PI3 Kinase) are involved in signaling events induced by lipopolysaccharide (LPS) leading to pro-inflammatory cytokines gene expression. To investigate the involvement of these mediators in Gram-positive bacteria induced pro-inflammatory cytokine expression, LPS (10 ng/ml), heat killed group B Streptococci (GBS 1 microg/ml) and Staphylococcus aureus (SA 10 microg/ml) were used to induce TNFalpha production in the murine J774A.1 macrophage (M?) cell line and human promonocytic THP-1 cell line. Pertussis toxin (PTx, 1 microg/ml), an inhibitor of G(i) protein; pyrazolopyrimidine-2 (PP2, 1 or 25 microM), a Src tyrosine kinase inhibitor; and LY294002 (100 nM), an inhibitor of PI3 Kinase were used to examine the involvement of G(i), Src tyrosine kinase and PI3 Kinase, respectively, in TNFalpha production. In J774A.1 cells, pretreatment with PTx and PP2 attenuated TNFalpha production induced by LPS (60+/-9% and 81+/-11% inhibition, n=3, p<0.05, respectively), GBS (95+/-1% and 80+/-6% inhibition, n=3, p<0.05, respectively) and SA (51+/-18% and 68+/-16% inhibition, n=4, p<0.05, respectively). However, pretreatment with LY 294002 inhibited LPS induced TNFalpha production (82+/-13% inhibition, n=3, p<0.05), but did not inhibit GBS or SA induced TNFalpha production. In THP-1 cells, pretreatment with PTx, PP2 and LY 294002 inhibited TNFalpha production induced by LPS (84+/-3%, 59+/-12% and 84+/-4% inhibition, n=3, p<0.05, respectively) and SA (56+/-7%, 87+/-1% and 35+/-6% inhibition, n=3, p<0.05, respectively). These data support our hypothesis that G(i)-coupled and Src tyrosine kinase-coupled signaling pathways are involved in both Gram-negative and Gram-positive bacteria induced pro-inflammatory cytokine expression. However, unlike LPS, involvement of PI3 Kinase in Gram-positive bacteria induced signaling pathways are species dependent.     

Dibutyryl cyclic AMP increases the amount of functional messenger RNA coding for tyrosine aminotransferase in rat liver.

The administration of N6,O2-dibutyryl cyclic AMP and theophylline to adrenalectomized rats results in an increase in the amount of functional mRNA coding for tyrosine aminotransferase that can be isolated from liver. The induction of this specific mRNA, as quantitated in a mRNA-dependent reticulocyte lysate system, and using poly(A)+ mRNA extracted from total tissue and polysomes, is very rapid. Within an hour after the intraperitoneal injection of the cyclic AMP derivative there is a 5- to 7-fold elevation of functional mRNA coding for tyrosine aminotransferase (mRNATAT), and by 3 h this has returned to basal levels. In contrast, the 4- to 5-fold induction of tyrosine aminotransferase catalytic activity is maximal at 2 h and is still significantly greater than the basal level at 5 h. In the basal state, tyrosine aminotransferase mRNA codes for 0.019 +/- 0.003% of the protein synthesized in the in vitro system, whereas after cyclic nucleotide treatment this value 0.115 +/- 0.015%, hence the increase in mRNATAT activity is relatively specific. Cordycepin, at a concentration which prevents the accumulation in cytoplasm of poly(A)+ mRNA, completely blocks the increase in both the catalytic and mRNA activity of this enzyme. The marked increase in functional mRNA, the requirement for continued synthesis of poly(A)+ RNA, and the rapid induction and deinduction suggest that the cyclic nucleotide is enhancing specific mRNA synthesis and/or, processing, however an effect on mRNA degradation cannot be excluded.     

Inactivation of tyrosine aminotransferase in neutral homogenates and rat liver slices

Inactivation of tyrosine aminotransferase induced in vivo by triamcinolone was studied in a homogenate incubated at neutral pH values. The integrity and the presence of subcellular particles together with a compartment of acidic pH are necessary for inactivation of tyrosine aminotransferase. It is suggested that tyrosine aminotransferase is inactivated inside lysosomes. The system responsible for inactivation of tyrosine aminotransferase was partially purified and identified with lysosomal cathepsins B and B(1). Inactivation of tyrosine aminotransferase in liver slices is controlled by the amino acid concentration and strongly stimulated by cysteine. 3,3',5-Tri-iodo-l-thyronine reversibly and strongly decreases the rate of inactivation of tyrosine aminotransferase. The effect is not due to an increased rate of tyrosine aminotransferase synthesis.     

Evidence for epithelial-mesenchymal interactions mediating glucocorticoid effects in developing chick liver

When trypsin-dissociated liver cells from 17-day chick embryos were grown in regular minimum essential medium, mixed hepatocyte-fibroblast cultures resulted. When D-valine was substituted for L-valine in this medium, fibroblast growth was suppressed, leaving virtually pure hepatocyte cultures. Tyrosine aminotransferase activity is induced by cortisol in mixed cultures. No induction of enzyme activity is observed with cortisol exposure to hepatocytes, grown in D-valine. However, when cortisol-containing medium is conditioned by pre-incubation with mixed cells and then transferred to hepatocytes, tyrosine aminotransferase activity is induced. Enzyme activity is also induced in mixed cells incubated in D-valine medium in the presence of cortisol. It appears that a substance produced in the presence of fibroblasts exposed to cortisol is capable of inducing tyrosine aminotransferase activity in hepatocytes. This activity, which we have termed fibroblast hepatocyte factor, is heat stable, of low molecular weight, and antigenically different from fibroblast pneumonocyte factor, a factor similar to that produced by lung fibroblasts exposed to cortisol.     

Phosphatase activity in rat adipocytes: effects of insulin and insulin resistance

Insulin regulates the activity of both protein kinases and phosphatases. Little is known concerning the subcellular effects of insulin on phosphatase activity and how it is affected by insulin resistance. The purpose of this study was to determine insulin-stimulated subcellular changes in phosphatase activity and how they are affected by insulin resistance. We used an in vitro fatty acid (palmitate) induced insulin resistance model, differential centrifugation to fractionate rat adipocytes, and a malachite green phosphatase assay using peptide substrates to measure enzyme activity. Overall, insulin alone had no effect on adipocyte tyrosine phosphatase activity; however, subcellularly, insulin increased plasma membrane adipocyte tyrosine phosphatase activity 78 +/- 26% (n = 4, P < 0.007), and decreased high-density microsome adipocyte tyrosine phosphatase activity 42 +/- 13% (n = 4, P < 0.005). Although insulin resistance induced specific changes in basal tyrosine phosphatase activity, insulin-stimulated changes were not significantly altered by insulin resistance. Insulin-stimulated overall serine/threonine phosphatase activity by 16 +/- 5% (n = 4, P < 0.005), which was blocked in insulin resistance. Subcellularly, insulin increased plasma membrane and crude nuclear fraction serine/threonine phosphatase activities by 59 +/- 19% (n = 4, P < 0. 005) and 21 +/- 7% (n = 4, P < 0.007), respectively. This increase in plasma membrane fractions was inhibited 23 +/- 7% (n = 4, P < 0. 05) by palmitate. Furthermore, insulin increased cytosolic protein phosphatase-1 (PP-1) activity 160 +/- 50% (n = 3, P < 0.015), and palmitate did not significantly reduce this activity. However, palmitate did reduce insulin-treated low-density microsome protein phosphatase-1 activity by 28 +/- 6% (n = 3, P < 0.04). Insulin completely inhibited protein phosphatase-2A activity in the cytosol and increased crude nuclear fraction protein phosphatase-2A activity 70 +/- 29% (n = 3, P < 0.038). Thus, the major effects of insulin on phosphatase activity in adipocytes are to increase plasma membrane tyrosine and serine/threonine phosphatase, crude nuclear fraction protein phosphatase-2A, and cytosolic protein phosphatase-1 activities, while inhibiting cytosolic protein phosphatase-2A. Insulin resistance was characterized by reduced insulin-stimulated serine/threonine phosphatase activity in the plasma membrane and low-density microsomes. Specific changes in phosphatase activity may be related to the development of insulin resistance.     

The role of protein tyrosine kinases in CYP1A1 induction by omeprazole and thiabendazole in rat hepatocytes

Benzimidazoles compounds like omeprazole (OME) and thiabendazole (TBZ) mediate CYP1A1 induction differently from classical aryl hydrocarbon receptor (AhR) ligands, 3-methylcholanthrene (3-MC) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). To clarify the involvement of an intracellular signal pathway in CYP1A1 induction by OME and TBZ, the TBZ, OME and 3-MC signal-transducing pathways were compared by using specific protein tyrosine kinase inhibitors in primary culture of rat hepatocytes. The effect of OME and TBZ (75-250 microM) on cytochrome P450 1A1 (CYP1A1) expression was therefore studied in primary cultures of rat hepatocytes after 24 h, 48 h and 72 h of exposure. Both compounds provoked a dose- and time-dependent increase in CYP1A1 (EROD activity, protein and mRNA levels), but OME was less effective at all the concentrations and times tested. The mechanism of benzimidazole-mediated induction of CYP1A1 was investigated by comparison with 3-MC, a prototypical AhR ligand. As expected, OME and TBZ were unable to displace [(3)H]-TCDD from its binding sites to the AhR in competitive binding studies. Moreover, classic tyrosine kinase inhibitor herbimycin A (HA) inhibited the two benzimidazoles-mediated CYP1A1 inductions, but only partially inhibited the 3-MC-mediated one. Another two tyrosine kinase inhibitors, Lavendustin A (LA) and genistein (GEN), had no effect on CYP1A1 induction by benzimidazoles and 3-MC. These results are consistent with the implication of a tyrosine kinase, most probably the Src tyrosine kinase, in the mechanism of CYP1A1 induction in rat hepatocytes.     

Sphingosine inhibition of tyrosine aminotransferase and tryptophan oxygenase induction by dexamethasone in primary culture of rat hepatocytes

The effect of sphingosine, a known selective inhibitor of protein kinase C, on the induction of tyrosine aminotransferase (TAT) and tryptophan oxygenase (TO) by dexamethasone was studied in the primary culture of rat hepatocytes to determine the possible involvement of protein kinase C in the expression of glucocorticoid action. Sphingosine inhibits the induction of TAT by dexamethasone in a concentration- and time-dependent manner in primary culture of rat hepatocytes. It does not inhibit the induction of TAT by Bt2cAMP. Sphingosine inhibits also the induction of TO by dexamethasone in a manner similar to TAT inhibition. It has no effect on the activity of lactate dehydrogenase, a cytosolic marker enzyme and on the protein content of the cultured hepatocytes. These findings indicate that endogenous modulator of protein kinase C, such as sphingosine, may influence the expression of glucocorticoid action in rat hepatocytes.     

Possible involvement of cGMP in the control of tyrosine aminotransferase degradation in rat hepatocytes

Addition of theophylline to primary cultures of rat hepatocytes in which tyrosine aminotransferase had been preinduced with dexamethasone caused a further increase in specific activity of the enzyme. This increase was due in part to a reduction in the rate of tyrosine aminotransferase degradation that began about 2 hr after theophylline was added. The level of cGMP also increased with a similar time lag following the addition of theophylline. The concentration of theophylline which produced the above effects (1 mM) did not alter the rate of general protein degradation in hepatocytes. Addition of 8-bromo-cGMP (0.5 mM) resulted in an immediate reduction in the rate of tyrosine aminotransferase degradation and in an increase in the activity of the enzyme. Treating hepatocytes with MnCl2 (0.9 mM) caused an elevation of cGMP and a concomitant slowing of tyrosine aminotransferase degradation without changing the level of cAMP significantly. These results suggest an inverse relationship between the level of cGMP and the rate of tyrosine aminotransferase degradation in hepatocytes.