Rene A. Frenkel

Peroxisome proliferation and induction of peroxisomal enzymes in mouse and rat liver by dehydroepiandrosterone feeding

Dehydroepiandrosterone (DHEA) treatment is effective in the prevention of various genetic and induced disorders of mice and rats. In studies designed to define some of the basic mechanisms that underline the beneficial chemopreventive effects exerted by the action of this steroid, we found that the liver undergoes profound changes that result in: (i) hepatomegaly; (ii) color change from pink to mahogany; (iii) proliferation of peroxisomes; (iv) increased cross-sectional area and volume density of peroxisomes; (v) increased or decreased number of mitochondria per cell; (vi) decreased mitochondrial cross-sectional area; (vii) marked induction of the peroxisomal bifunctional protein enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase; (viii) increased activities of enoyl-CoA hydratase and other peroxisomal enzymes assayed in this study, viz. catalase, carnitine acetyl-CoA transferase, carnitine octanoyl-CoA transferase, and urate oxidase; and (ix) increased activity of mitochondrial carnitine palmitoyl-CoA transferase. In addition, feeding DHEA to mice resulted in increased plasma cholesterol levels in two strains of mice evaluated in this study, and either slightly decreased or markedly increased plasma triglyceride levels, depending on the strain. Whether liver peroxisome proliferation, induced by DHEA feeding to mice and rats, plays a role in the chemopreventive effects elicited by this steroid remains to be established.

Isolation and some properties of a cytosol and a mitochondrial malic enzyme from bovine brain

Abstract Two distinct isozymes of malic enzyme from bovine brain have been separated and purified partially. By preparing homogenates in solutions of different ionic strength and by extracting isolated mitochondria, it has been established that one isozyme (B I) is located in the cytosol and the other in the mitochondria (B II). The isozymes differ not only in their electrophoretic and chromatographic mobilities, but also in their kinetic properties, since only the mitochondrial variant shows cooperativity at low concentrations of l (−)malate. The isozymes also show a marked difference in their capability to catalyze the reductive carboxylation of pyruvate to malate, since the carboxylation rate approaches 1 5 that of the decarboxylation velocity for the cytosol enzyme, and only 1 100 for the mitochondrial isozyme.

Differential characteristics of the cytosol and mitochondrial isozymes of malic enzyme from bovine brain: Effects of dicarboxylic acids and sulfhydryl reagents☆

Abstract The cytosol and mitochondrial isozymes of bovine brain malic enzyme were studied with respect to their sensitivity towards a series of dicarboxylic acids and sulfhydryl reagents. While no effects were obtained with the dicarboxylic acids in the case of the cytosol enzyme, the activity of the mitochondrial variant was increased considerably when either succinate, 2-mercaptosuccinate, or l -aspartate were tested at low concentrations of l -malate. The activation was associated with a clear decrease in the Hill coefficient for l -malate, and this has been taken as an indication of the presence of an allosteric site on the mitochondrial enzyme. The presence of l -malate or a dicarboxylate anion analog is required at this site in order to achieve optimal velocity. The activators were also effective in increasing the reductive carboxylation of pyruvate by the mitochondrial enzyme and had no effect on the cytosol variant. The two isozymes also showed a clear differential sensitivity to 5,5′-dithiobis(2-nitrobenzoic acid) and Hg2+, since the mitochondrial malic enzyme was inhibited by concentrations of these reagents far below those required in order to achieve an effect on the activity of the malic enzyme found in the cytosol.

The biochemical role of platelet-activating factor in reproduction

The presence of Co-A independent transacylase activity in amnion cells and the preferential transfer of arachidonic acid to acceptor-ethanolamine plasmalogen provide a satisfactory explanation to the questions raised by the observation that arachidonate-enriched ethanolamine plasmalogen increases in amnion late in gestation without alteration in the total amount of ethanolamine glycerophospholipids. The proposed mechanism also serves as a link between the observed changes in glycerophospholipid composition and the generation of PAF. We have emphasized a role for PAF in fetal lung maturation, the initiation and maintenance of parturition, and in certain complications associated with a premature delivery. Although PAF is known to be the most potent lipid mediator yet described and its importance in reproductive biology is well documented, it is our view that these events cannot be attributed solely to PAF and in all likelihood a number of autacoids participate in these processes.

17β-Hydroxysteroid oxidoreductases of human fetal and adult tissues: Immunological cross-reactivity with an anti-human placental cytosolic 17β-hydroxysteroid oxidoreductase antibody

To determine whether the immunological determinants of human placental 17 beta-hydroxysteroid oxidoreductase (17 beta-HSOR) were present in 17 beta-HSORs of tissues of the human fetus and adult and of various non-human cells maintained in culture, western immunoblot analysis was conducted by use of a polyclonal antibody directed against determinants of the placental cytosolic enzyme. Tissues and cells were evaluated for the presence of immunocross-reactive proteins with a relative molecular mass (Mr) similar to that of placental 17 beta-HSOR (approximately 34,000). By use of homogenates of human fetal tissues, immunostaining of 17 beta-HSORs of Mr approximately 34 kDa was detected in trophoblast, fetal adrenal neocortex, fetal zone of the adrenal gland, liver, intestine, kidney, brain, lung, skin, heart, spleen, pancreas, chorion laeve, and, occasionally, amnion. Immunostaining at Mr approximately 34 kDa also was demonstrated by use of cytosolic preparations of fetal tissues and, in some cases, by use of unwashed microsomal fractions; this protein was either absent or present in almost undetectable amounts in washed microsomes, except for placenta and fetal brain. Immunostaining at approximately 34 kDa was demonstrated occasionally in decidua of pregnant women by use of homogenates, but was not detected in endometrium or myometrium of non-pregnant women, testis of an adult man, mouse and rat Leydig tumour cells, mouse and rat adrenal tumour cells, and normal bovine adrenocortical cells.

Increased “malic enzyme” activity during adaptation to a low protein diet

Abstract Liver “malic enzyme” activity has been observed to increase rapidly starting at an early stage during adaptation to a low protein diet in the young rat. The enzymic activity remains elevated up to 33 days after initiation of a low protein diet. Other NADPH generating enzymes, glucose-6-phosphate dehydrogenase and DL-isocitrate dehydrogenase, do not show increased activities under the same conditions.

The role of PAF in reproductive biology

Abstract Platelet-activating factor (PAF) has been shown to play an important role in both fetal lung maturation and in parturition and it has been demonstrated that part of the PAF present in the amniotic fluid is associated with surfactant and, therefore, of lung origin. PAF receptors are present in type II pneumonocytes and the addition of the autacoid promotes both glycogenolysis and increased surfactant secretion from these cells. Moreover, type II pneumonocytes and a human amnion-derived cell line (WISH) have the capability of metabolizing PAF to ethanolamine phospholipids. It has also been established that PAF will cause intracellular Ca 2+ mobilization and promote myosin phosphorylation and increased contractility in myometrial strips. Increased PAF biosynthesis in fetal tissues takes place during the latter stages of pregnancy, simultaneously with a decrease in the activity of PAF-acetylhydrolase (PAF-AH) in maternal plasma. The activity of PAF-AH is decreased by the administration of estrogens in vivo and is increased by glucocorticoids and progestins. A number of agents lower the secretion of PAF-AH by decidual macrophages, including 1,25-(OH) 2 D 3 , bacterial lipopolysaccharides, and some cytokines (IL1, TNF-α). This inhibitory action may result in higher levels of PAF and increased myometrial contractility as a consequence of infectious processes. PAF causes a profound intestinal necrosis when administered to rats by intravenous injection. Pretreatment with dexamethasone protects against this disease. The presence of PAF-AH in human milk may explain the protective effect of mothers milk against necrotizing enterocolitis.

Dehydroepiandrosterone feeding and protein phosphorylation, phosphatases, and lipogenic enzymes in mouse liver.

Abstract Dehydroepiandrosterone (DHEA) treatment is effective in preventing or delaying the onset of various genetic and induced disorders of mice and rats. Associated with the beneficial therapeutic effects exerted by action of this steroid is the development of hepatomegaly. To determine whether the changes associated with hepatomegaly also involve alterations in activities of tissue enzymes, we evaluated the effects of DHEA (0.45% in food, w/w) on hepatic protein kinases, phosphatases, and lipogenic enzymes in mice of various strains. The rates of fatty acid and cholesterol syntheses also were evaluated. DHEA administration resulted in profound changes in the sodium dodecylsulfate-polyacrylamide gel electrophoresis patterns of endogenous radiophosphorylated proteins obtained by incubation of liver homogenates with (γ-32P]ATP. These changes were dependent upon the medium used for homogenization. Thus, when homogenates of liver tissue of DHEA-treated mice were prepared in Tris buffer containing sucrose (0.25 M) there was a marked decrease in phosphorylation of the proteins of relative molecular weight ~116,000 (Mr ~116,000), ~82,000, ~80,000, ~58,000, ~56,000, ~48,000, ~34,000, ~32,000, and ~31,000 compared with controls. With liver homogenates of DHEA-treated mice prepared in Tris buffer alone, there was a marked increase in phosphorylation of the proteins of Mr ~70,000, ~49,000, ~34,000, ~31,000, and 28,000 compared with controls. Moreover, the specific activity of kinases for endogenous protein acceptors in liver of control mice was higher than that in liver of DHEA-treated animals. The specific activities of casein kinase, cAMP-dependent protein kinase, and cGMP-dependent protein kinase remained unchanged with DHEA treatment, but the specific activity of histone kinase was increased approximately 30%. Long-term administration of DHEA also was associated with increases in the specific activities of liver AMPase and GTPase (approximately two times), but not of other nucleotidases, alkaline phosphatase, acid phosphatase, glucose-6-phosphatase, or phosphotyrosine phosphatase. The activity of hepatic NADP-linked malic enzyme was increased significantly (two to three times) by DHEA treatment of female mice of three different strains, but was unchanged in male C57BL/6 mice. The specific activities of hepatic glucose-6-phosphate dehydrogenase, NADP-linked isocitrate dehydrogenase, and ATP-citrate lyase were not affected significantly by DHEA treatment of mice. The rate of hepatic lipogenesis, determined by incorporation of tritium from 3H2O into fatty acids, was decreased approximately 70% in DHEA-treated mice, while the rate of cholesterol synthesis was increased approximately 44% compared with controls.

Dietary induction of hepatic malic enzyme activity: Differentiation of the induction process☆

Abstract The activity of rat liver malic enzyme shows a marked increase when the animals are maintained on a restricted protein diet. Of the NADP-linked dehydrogenases tested (malic enzyme, glucose-6-phosphate dehydrogenase, and isocitrate dehydrogenase), the response is confined only to malic enzyme. Dietary sucrose is not required for the increase in activity, but elevated dietary levels of this disaccharide increase hepatic malic enzyme regardless of dietary protein. Glucose-6-phosphate dehydrogenase activity is increased by dietary sucrose provided adequate dietary protein is supplied. The specificity of the response to lowered dietary protein shown by malic enzyme suggests that the control of the hepatic enzyme is mediated by processes different from those controlling the activity of glucose-6-phosphate dehydrogenase.

Induction of murine hepatic glutathione S-transferase by dietary dehydroepiandrosterone

The naturally occurring steroid dehydroepiandrosterone (DHEA), when administered as a supplement to the diet of mice and rats, produces alterations in the relative concentrations of specific liver proteins; among these, a protein of Mr approximately 28 K is markedly induced by DHEA action. In the present study we identified the murine hepatic approximately 28 kDa protein as glutathione S-transferase subtype GT-8.7. Glutathione S-transferases belong to a gene superfamily that encode closely related proteins which are induced in liver and other tissues by various chemicals, including carcinogens and chemoprotective agents such as dietary antioxidants. Based on the above finding, we evaluated glutathione S-transferase activity in cytosols and microsomes prepared from liver tissue of mice fed either a control diet or a DHEA-containing diet (0.45%, by weight). The specific activity of hepatic cytosolic glutathione S-transferase in mice treated with DHEA up to 7 days was either unchanged or slightly decreased when compared to controls; however, treatment for 14 days or longer resulted in significant increases in activity. The specific activity of microsomal glutathione S-transferase also was increased by long-term DHEA treatment; however, its activity was approximately one-tenth of that in corresponding cytosols.