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.

Effect of adrenergic agonists on phosphoinositide breakdown in rat skeletal muscle preparations.

Adrenergic regulation of phosphoinositide breakdown in rat skeletal muscle was investigated in 30-min incubations with 10 mM LiCl. In rat hemidiaphragms, prelabelled with D-myo-[2-3H]inositol, addition of alpha-agonists (epinephrine, norepinephrine, phenylephrine) induced a 5-8-fold increase of [3H]inositol monophosphate accumulation. This could be prevented by inclusion of alpha-antagonists (phentolamine, prazosin). beta-Agonists and/or beta-antagonists had no effect. Similar experiments with isolated flexor digitorum brevis muscle fibers yielded confirmatory results. Functional integrity of beta-receptor mediated processes was suggested by the beta-agonist-induced increase of glucose 6-phosphate in hemidiaphragms and cAMP in fiber preparations. The results indicate that phosphoinositide breakdown in differentiated rat skeletal muscle is, at least in part, under alpha-adrenergic control.

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)

Differential down-regulation and induction responses of testicular steroidogenic cytochromes P-450(cscc) and P-450(C17 alpha) to human choriogonadotropin.

Evidence is presented that the regulation of the cytochrome P-450(C17 alpha) of the steroid-17 alpha-monooxygenase and of the cytochrome P-450(cscc) of the cholesterol-monooxygenase by human choriogonadotropin (hCG) in vivo is mediated by differential mechanisms in the adult rat testis. An initial down-regulation of the cytochrome P-450(C17 alpha) but not of the P-450(cscc) can be demonstrated. Furthermore, induction of the cytochrome P-450(cscc) requires exposure to higher hCG doses (32% of the maximal induction rate of 43.7 pmol/(testis x d) are achieved with 4IU hCG/single dose) than induction of the P-450(C17 alpha) (59% of the maximal induction rate of 48.4 pmol/(testis x d) with 4IU hCG/single dose). Finally, induction of cytochrome P-450(cscc) starts faster after initiation of hCG treatment than induction of P-450(C17 alpha).

Biochemical and histochemical studies on the pituitary-testicular axis in obese (C57Bl/6J-ob/ob) mice.

Male C57Bl/6J-ob/ob mice (4 months old) and their homozygous lean controls were compared with respect to pituitary LH secretion and functional parameters of purified Leydig cells in vitro. Compared with controls, obese mice showed reductions in the following parameters: Plasma testosterone levels (reduced by 57%), hCG-stimulated testosterone formation in vitro (by 31%), conversion of progesterone to androgens by Leydig cells (by 39%), and GnRH-stimulated LH secretion (by 26%). Lipid accumulation and a 37% decrease in naphthylesterase activity in the Leydig cells as well as hyperplasia of pituitary gonadotrophs were observed histochemically in obese mice. The changes in testicular endocrine function in obese mice are interpreted as consequences of pituitary dysfunction.

In-vitro studies of the development of pituitary and testicular functions in diabetes (C57Bl/KsJ-db/db) mutant mice.

The hypothesis that male diabetes mutant mice (C57Bl/KsJ-db/db) are suffering from impairment of testicular steroidogenic function and pituitary LH release was tested. A smaller postpubertal increase of testicular weight and a reduction of plasma testosterone and androstenedione levels by 65% at 17 weeks of age were most obvious from the comparison to homozygous lean controls. The ability of constant amounts of Leydig cells, either in crude interstitial cell or in purified Leydig cell suspensions, to respond to maximal doses of hCG or cyclic AMP-was reduced by at least 40% in adult diabetes mice. This defect could be attributed to a 40% decrease of steroid-17 alpha-monooxygenase activity as compared to lean mice. No differences occurred, however, if Leydig cells were submaximally stimulated. GnRH-stimulated pituitary LH release was not significantly changed. The impairment of testicular steroidogenic function in diabetes mutant mice may represent a further aspect of infertility of these animals and of diabetes mellitus.

Influence of insulin and glucose on pyruvate catabolism in perfused rat hindlimbs.

The effects of insulin and glucose on the oxidative decarboxylation of pyruvate in isolated rat hindlimbs was studied in non-recirculating perfusion with [1-14C]pyruvate. Insulin increased the calculated pyruvate decarboxylation rate in a concentration-dependent manner. At supramaximal insulin concentrations, the calculated pyruvate decarboxylation rate was increased by about 40% in perfusions with 0.15-1.5 mM-pyruvate. Glucose up to 20 mM had no effect. In the presence of insulin and low physiological pyruvate concentrations (0.15 mM), glucose increased the calculated pyruvate oxidation. This effect was abolished by high concentrations of pyruvate (1 mM). The data provide evidence that in resting perfused rat skeletal muscle insulin primarily increased the activity of the pyruvate dehydrogenase complex. The effect of glucose was due to increased intracellular pyruvate supply.

Differential effect of dichloroacetate on branched-chain amino acid catabolism in perfused rat hindlimbs.

The effect of 1 mM and 5 mM dichloroacetate on the catabolism of branched-chain amino acids in isolated rat hindlimbs was investigated in perfusions with 0.5 mM 1-14C-labeled L-leucine or L-valine. The results demonstrate an increasing effect of dichloroacetate on the flux through skeletal muscle branched-chain 2-oxo acid dehydrogenase. A minor effect was observed with the high dichloroacetate concentration. Evidence is presented that this was essentially due to diminished pyruvate supply.

Steroid receptor status of human testicular tumors.

Biopsies of 9 testicular tumors were examined for the presence of cytoplasmic receptor proteins for estrogens, progestins, androgens, and glucocorticoids. Using an arbitrary threshold value of 10 fmol/mg cytosol protein for a postive assay, only 1 of 9 tumors was estrogen-receptor positive. However, no receptor-specific 8S binding could be detected by low salt sucrose gradient centrifugation. 1 tumor was progesterone-receptor positive. No androgen receptors could be demonstrated. In contrast, 8 of 9 neoplasms contained significant quantities of glucocorticoid receptors exclusively sedimenting at 8S. Our findings suggest that androgens, estrogens, and progestins are unlikely to play a major role in the natural history of testicular tumors. The presence of glucocorticoid receptors might offer a chance for endocrine manipulation of the development and growth of these neoplasms by glucocorticoids.

Oxidative determination of 14C-labeled 2-oxo acids.

A simple and rapid assay for the determination of 1-14C- or U-14C-labeled 2-oxo acids is described. It is based on the selective and complete oxidation of the carboxyl group to 14CO2. Preceding purification procedures are not necessary. In rat hindlimb perfusion studies, the procedure was used to develop an indirect method for the estimation of the intracellular dilution of [1-14C]pyruvate and to determine the relationship between the transamination and decarboxylation rates of leucine in the perfused tissue by the use of tracer doses of L-[1-14C]leucine.

Product inhibition of steroid-17 alpha-monooxygenase by endogenous 17 alpha-hydroxyprogesterone in microsomes and isolated Leydig cells from rat testis.

Use of integrated rate equations for analysis of progesterone metabolism by isolated Leydig cells and microsomes from rat testis in presence of several progesterone concentrations within several periods reveals competitive product inhibition by endogenously formed 17 alpha-hydroxyprogesterone (Kpm = 0.1 microM) of steroid-17 alpha-monooxygenase activity (Ksm = 0.8 microM). The discrepancy between this very low interaction constant of endogenous 17 alpha-hydroxyprogesterone with the steroid-17 alpha-monooxygenase, and the respective values (from the literature) for exogenous 17 alpha-hydroxyprogesterone which are about 50-fold higher, may be explained by accumulation of endogenous 17 alpha-hydroxyprogesterone at the catalytic site of the steroid-17 alpha-monooxygenase. This mechanism may be important for intratesticular regulation of androgen biosynthesis from precursor steroids.

In vitro and in situ skeletal muscle pyruvate dehydrogenase activity in adrenalectomized and glucocorticoid treated rats.

Skeletal muscle glucose oxidation is significantly reduced and alanine release enhanced in adrenalectomized rats after short-term glucocorticoid treatment. A possible site of regulation is the pyruvate dehydrogenase complex. Pyruvate dehydrogenase activity was measured in vitro in extracts of the gastrocnemius muscle of normal, adrenalectomized and short-term glucocorticoid treated rats by the p-nitroaniline-arylamine-acetyltransferase method and in situ in non-recirculating perfusions of isolated hindlimbs with physiological pyruvate levels and tracer doses of [1-14C] pyruvate by measuring the off-kinetic of 14CO2 wash out in the effluent. Neither method showed a direct influence of glucocorticoids on skeletal muscle pyruvate dehydrogenase activity.