Beta-oxidation as channeled reaction linked to citric acid cycle: evidence from measurements of mitochondrial pyruvate oxidation during fatty acid degradation.

1. The kinetics of mitochondrial mammalian pyruvate dehydrogenase multienzyme complex (PDHC) is studied by the formation of CO2 using tracer amounts of [1-14C]pyruvate. It is found that the Hill plot results in a (pseudo-)cooperativity with a transition of n-1----3 at a pyruvate concentration about Ks. 2. Addition of L-carnitine, octanoate, palmitoyl-CoA or palmitate + L-carnitine + fatty acid-binding protein results in a Hill coefficient of n = 2 following the kinetics of pyruvate oxidation. 3. Addition of fatty acid-binding protein to an assay system oxidizing palmitate in presence of L-carnitine alters the pattern of the kinetics in the Hill plot so that an apparently lower level of L-carnitine is necessary for the reaction course of beta-degradation. 4. It is concluded that beta-degradation is a coordinated, multienzyme-complex based mechanism tightly linked to citric acid cycle and it is proposed that L-carnitine is actively involved into the reaction and not only functioning as carrier-molecule for transmembrane transport.

Tetrahydroisoquinoline alkaloids mimic direct but not receptor-mediated inhibitory effects of estrogens and phytoestrogens on testicular endocrine function. Possible significance for Leydig cell insufficiency in alcohol addiction.

Possible effects of various tetrahydroisoquinolines (TIQs) on rat testicular endocrine function were tested in vitro in order to prove whether these compounds, some of which have been claimed to accumulate in alcoholics, may be mediators of the development of Leydig cell insufficiency, a well-known side-effect of ethanol ingestion. TIQ effects on different levels of regulation of testis function were compared in vitro with estrogen effects, since both classes of compounds have structural similarities. Gonadotropin-stimulated testosterone production by testicular Leydig cells was inhibited by tetrahydropapaveroline and isosalsoline, the IC50 values (30 microM) being comparable to those of estradiol (3 microM), 2-hydroxyestradiol (10 microM), and the phytoestrogens, coumestrol (15 microM) and genistein (7 microM); salsolinol (85 microM) and salsoline (240 microM) were less effective, and salsolidine was ineffective. None of these TIQs interacted significantly with testicular estrogen receptor as analyzed by estradiol displacement. However, tetrahydropapaveroline, isosalsoline and salsolinol competitively inhibited (Ki 130-150 microM) substrate binding to cytochrome P450XVII, one key enzyme of androgen biosynthesis, with similar efficiency as the estrogens did (Ki 50-110 microM); salsoline and salsolidine were again much less effective. Since the efficient TIQ concentrations in this system are identical with those reported to generate central-nervous effects, it is concluded that certain TIQs may amplify peripheral inhibitory effects of ethanol on testicular endocrine function by their interaction with at least one enzyme of the androgen biosynthetic pathway.

Down-regulation of steroidogenic cytochrome P450XVII in cryptorchid rat testes.

The objective of the present study was to investigate the regulation of a key component of testicular androgen biosynthesis, i.e. the cytochrome P450XVII of the steroid-17 alpha-monooxygenase/C17,20-lyase, after surgical induction of bilateral cryptorchidism in vivo. Seven days after induction of cryptorchidism, P450XVII concentrations are diminished (as compared to sham-operated controls) by 64% in isolated purified Leydig cells but only by 44% in the total Leydig cell compartment of the testis, since the Leydig cell yield from cryptorchid testes is by 53% higher than that from control testes. Using microsomal suspensions prepared from testicular homogenates, P450XVII content per testis equivalent is found to be decreased by 36% seven days after incubation of cryptorchidism, whereas the P450XVII concentration per gram testis is not changed due to testicular involution. Fourteen days after induction of cryptorchidism, the induction of the Leydig cell system appears to superimpose on the down-regulation of P450XVII. The study demonstrates both a strong sensitivity of P450XVII to short-term elevation of testicular temperature and a differentiation between effects of cryptorchidism on total testicular content and specific cellular and subcellular concentration of this steroidogenic protein.

Further evidence for direct interaction of pyruvate dehydrogenase multienzyme complex with citric acid cycle based on nonlinearity of hill plots in presence of C2 and C4 substrate analogues.

1. Mammalian pyruvate dehydrogenase multienzyme complex (PDC) is measured with two different optical assays: (i) formation of p-nitro-acetanilid with arylamine-acetyltransferase and (ii) NAD reduction. 2. It is found that in contrast to the NAD assay system (ii) the coupled system (i) exhibits cooperativity with a Hill coefficient n = 3 over the whole range of substrate concentration. 3. The cooperative behaviour can be modified by presence of dichloroacetate (n = 2) and acetoin (n = 1----3). From additional measurements of PDC activity with toluene permeabilized mitochondria of fed and starved rats it is concluded that PDC activity in vivo is modified by changes in enzyme enzyme aggregation and interaction beside the known phosphorylation dephosphorylation mechanism.

Transamination and oxidative decarboxylation of L-isoleucine, L-alloisoleucine and related 2-oxo acids in perfused rat hind limb muscle.

Metabolism of L-isoleucine, L-alloisoleucine and corresponding 2-oxo acids in rat hind limb muscle was comparatively studied under steady-state perfusion conditions. At 0.5 mM L-[1-14C]isoleucine, apparent transamination and 2-oxo acid decarboxylation rates amounted to about 17 and 4 nmol/min per g of muscle, respectively. With L-allo[1-14C]isoleucine, the corresponding rates were about 5- and 10-fold lower, respectively. After addition of dichloroacetate (1-5 mM), the portion of (S)- and (R)-methyl-2-oxopentanoate undergoing further oxidative decarboxylation within the tissue was similarly increased by over 40%. In perfusions with 0.5 mM (R,S)-3-methyl-2-oxopentanoate and tracer doses of 1-14C-labeled (S)- or (R)-enantiomer, the 14CO2 production was comparable (about 0.5 nmol/min per g of muscle). Dichloroacetate caused a several-fold increase in 14CO2 release from either enantiomer, apparent 2-oxo acid transamination rates remaining unaffected. Indications for a racemization of 2-oxo acid were not obtained in the experiments. The results are discussed with respect to the appearance/disappearance of L-alloisoleucine in vivo and to the fact that (R)-3-methyl-2-oxopentanoate, but not L-alloisoleucine, can support growth of rats on a diet deficient in L-isoleucine.

Specificity of steroid binding to testicular microsomal cytochrome P-450. Relation of steroid structure to type-I spectral responses after correction for hydrophobic association with the membrane.

On the basis of the concept that steroids accumulate in the lipid phase of endoplasmic reticulum membranes and approach the active sites of steroidogenic cytochromes P-450 from a hydrophobic environment, we describe a procedure that allows calculation of spectral dissociation constants Ks for steroid interaction with testicular microsomal cytochrome P-450 after correction for hydrophobic association of ligand with the membrane. Maximal type-I spectral responses, apparent Ks, and partition into microsomal lipids were determined for 36 steroids, and corrected Ks values were derived from these primary data. Partition coefficients range from 60 to 62,000, and corrected Ks range from 60 microM to 25 mM steroid concentration in the lipid phase. Full spectral properties depend on a side-chain (1-3 carbon atoms) at the C17-position which may be hydrophobic or may bear a 20-oxo or 20 beta-hydroxy, but not a 20 alpha-hydroxy group. Binding constants are especially sensitive towards modifications of ring A structure (aromatization or 5 beta-, but not 5 alpha-reduction) and of the side-chain length. Androgens, with the exception of those bearing a 17 beta-acetoxy or 17 beta-propionyloxy group, are poorly accommodated by this cytochrome P-450.

Specific accumulation of 17 alpha-hydroxyprogesterone in microsomal membranes during the process of cytochrome P-450(C-17)-catalysed androgen biosynthesis. A dynamic study of intermediate formation and turnover.

A complete dynamic analysis of cytochrome P-450(C-17)-catalysed androgen biosynthesis from a single dose of progesterone and 17 alpha-hydroxyprogesterone in a double-label double-substrate experiment was performed in order to elucidate the controversial intermediacy of 17 alpha-hydroxyprogesterone. Label distribution within the steroid fractions as well as in the membrane and buffer compartments yields direct evidence that the endogenously formed 17 alpha-hydroxyprogesterone (which is in an 'intermediate state') accumulates to a higher degree in microsomal membranes than does the exogenously added 17 alpha-hydroxyprogesterone (which is in a 'substrate state') under certain conditions. It is also demonstrated that endogenously formed 17 alpha-hydroxyprogesterone may partly leave the membrane compartment (in terms of a 'leakage' or 'overflow' phenomenon) and is then able to equilibrate with the pool of exogenously added 17 alpha-hydroxyprogesterone. Since only the label distribution in the membrane-associated (but not always in the aqueous) 17 alpha-hydroxyprogesterone pool corresponds to the label distribution in the androgen fraction, it is concluded that only the membrane-associated 17 alpha-hydroxyprogesterone pool is directly accessible to cytochrome P-450(C-17)-catalysed conversion into androgens.

14CO2 fixation in incubated rat diaphragms.

The time course (0-60 min) of label incorporation from NaH14 CO3 into citric-acid-cycle intermediates and amino acids was investigated in incubations of isolated rat diaphragms. On the basis of these results, 14CO2 exchange by isocitrate dehydrogenase and 14CO2 fixation by propionyl-CoA carboxylation and pyruvate carboxylation could be estimated. Apparent rates amounted to about 30-40, 2, and 35 nmol/min per g of muscle, respectively. About 90 percent of C4-carbon compounds originating from 14CO2 fixation were subsequently removed by decarboxylation. 2-Cyano-4-hydroxycinnamate, an inhibitor of mitochondrial pyruvate transport, effectively reduced 14CO2 production from [1-14C]pyruvate but did not affect incorporation of radioactive label from NaH14CO3. In cell-free muscle extracts, 14CO2 fixation was demonstrable under assay conditions suitable for NADP -dependent 'malic' enzyme(s). Addition of hydroxymalonate, an inhibitor of the latter enzyme(s), significantly reduced 14CO2 incorporation. The results provide evidence for a continuous cytosolic replenishment and mitochondrial depletion of citric-acid-cycle carbon skeletons in resting skeletal muscle tissue. The functional role of malic (iso)enzyme activities in these processes is discussed.

Age-dependence of the rat Leydig cell and Sertoli cell function. Development of the peripheral testosterone level and its relation to mitochondrial and microsomal cytochromes P-450 and to androgen-binding protein.

The first part of this study compares peripheral testosterone levels with intratesticular levels of the cytochromes P-450 of two key enzymes of androgen biosynthesis, i.e. mitochondrial cholesterol monooxygenase (P-450(cscc)) and microsomal steroid-17 alpha-monooxygenase (P-450(C17 alpha)) during puberty and early adulthood of male Wistar rats. From 4 to 10 weeks of age, the testosterone level increases 8.7-fold, the P-450(C17 alpha) level 8.3-fold, but the P-450(cscc) level 24.5-fold as an indication of specific induction of this protein. From 13 to 50 weeks of age, the testosterone level remains constant, the P-450(cscc) level increases continuously by a factor of 1.4, but 62% of the P-450(C17 alpha) content are lost. This discrepancy is explained by a divergent regulation of the cytochromes P-450 of the two steroid monooxygenases: a persisting induction of P-450(cscc) and a concurrent down-regulation of P-450(C17 alpha) that may be a consequence of the high rate of Leydig cell steroid hydroxylation after puberty. Overlapping of both processes may (probably besides other developmental factors) result in a constant testosterone concentration in blood. The second part of the study compares testicular and epididymal levels of androgen-binding protein (ABP) with the peripheral testosterone level. The peripubertal increase in testicular ABP content is shown to be related only to the increase in testicular mass, whereas a specific accumulation of ABP occurs in the epididymis from 4 to 13 weeks of age. This pattern indicates an increasing secretory activity of the Sertoli cells that remains high during adulthood up to the 50th week.(ABSTRACT TRUNCATED AT 250 WORDS)