Nutritional and hormonal regulation of mRNA levels of lipogenic enzymes in primary cultures of rat hepatocytes.

The effects of nutrients and hormones on the mRNA levels of acetyl-CoA carboxylase, fatty acid synthase, malic enzyme, and glucose 6-phosphate dehydrogenase were examined in primary cultures of rat hepatocytes during the process of induction. The addition of both glucose and insulin to the culture medium markedly enhanced the lipogenic enzyme mRNA induction due to either of them, in 16 h. Fructose or glycerol proved to be an effective substitute for glucose, suggesting that glycolytic metabolites were involved in the mRNA induction. It is remarkable that mRNA induction of acetyl-CoA carboxylase was the most sensitive to glucose and also to insulin among the lipogenic enzymes. Polyunsaturated fatty acids markedly reduced the mRNA induction of lipogenic enzymes. Dexamethasone enhanced all the lipogenic enzyme mRNA induction by insulin. On the other hand, triiodothyronine addition greatly increased the mRNA concentrations of lipogenic enzymes, but dexamethasone decreased rather than increased the mRNA induction by triiodothyronine. The effects of insulin on the induction of the lipogenic enzyme mRNAs were similar, but those of triiodothyronine were not. Triiodothyronine markedly enhanced malic enzyme mRNA induction by insulin with dexamethasone, and tended to enhance the induction of the acetyl-CoA carboxylase and fatty acid synthase mRNAs, but not that of glucose 6-phosphate dehydrogenase mRNA. It appeared that insulin and triiodothyronine synergistically enhanced lipogenic enzyme mRNA induction by glucose, but the mechanisms were different.     

Continuous spectrophotometric assays for three regulatory enzymes of the arginine biosynthetic pathway

N-Acetylglutamate synthase (AGS), N-acetylglutamate kinase (AGK), and glutamate N-acetyltransferase (GAT) are the key enzymes in the synthesis of arginine that serves as an important precursor for the synthesis of protein, polyamines, urea, and nitric oxide. Current assays available for these three enzymes are laborious and time-consuming and do not allow continuous monitoring of enzyme activities. Here we established continuous enzyme assays for AGS, AGK, and GAT based on the coupling of AGS and GAT reactions to AGK followed by coupling of the AGK reaction to N-acetylglutamate 5-phosphate reductase (AGPR). The rate of AGPR-dependent oxidation of reduced nicotinamide adenine dinucleotide phosphate was monitored continuously as a change in absorbance at 340 nm using spectrophotometry. These methods were applied to kinetic analyses for Escherichia coli AGK, E. coli AGS, and Saccharomyces cerevisiae GAT, and the kinetic parameters obtained in the coupling assays showed nearly the same values as those obtained previously using discontinuous assays. The specificity of these coupled assays was confirmed by the lack of enzyme activity from extracts of E. coli AGS-, E. coli AGK-, and S. cerevisiae GAT-deletion mutants. Moreover, the coupled assay enabled us to measure AGS activity from mammalian liver mitochondrial extracts, known to be an important regulatory enzyme for the urea cycle. These coupled enzyme assays are rapid, highly sensitive, and reproducible.     

Interrelated regulation of sulphur-containing amino-acid biosynthetic enzymes and folate-metabolizing enzymes in Aspergillus nidulans

In Aspergillus nidulans homocysteine can be metabolized both to cysteine and methionine. Mutants impaired in the main pathway of cysteine synthesis or in the sulphate assimilation pathway show a low pool of glutathione and elevated levels of homocysteine synthase and of the homocysteine-to-cysteine pathway enzymes. On the other hand, the level of methionine synthase and other enzymes of folate metabolism is depressed in these mutants. This anticoordinated regulation provides a mechanism controlling the partition of homocysteine between the two diverging pathways. Homocysteine synthase was found derepressed, along with folate enzymes, in a strain carrying a mutation which suppresses mutations in metA, metB and metG genes. These results indicate that homocysteine synthase can be regarded as the enzyme of an alternative pathway of methionine synthesis and strongly suggest that the regulatory mechanisms governing sulphur-containing amino acid and folate metabolisms are interrelated.     

Steroid hormonal regulation of calcium-binding protein regucalcin mRNA expression in the kidney cortex of rats

Ethylenediamine tetraacetate ( EDTA ) inhibits lactoperoxidase (LPO)-catalyzed rate of iodide oxidation in concentration and pH-dependent manner. A plot of log Ki_app values against various pH yields a sigmoidal curve from which an ionisable group of pK_a value 6.0 could be ascertained for controlling the inhibition of catalytically active LPO by EDTA. Kinetic studies indicate that EDTA competitively inhibits iodide oxidation by acting as an electron donor. EDTA al so reduces LPO-compound-11 to the native ferric state by one-electron transfer as evidenced by the spectral shift from 428 to 412 nm. Optical difference spectroscopic studies indicate that EDTA binds to LPO with the apparent equilibrium dissociation constant (KD) of 12 ± 2 mM at pH 6.5. A plot of log K_D values against various pH produces a sigmoidal curve from which an ionisable group of LPO having pk_a = 5.47 could be calculated, deprotonation of which favours EDTA binding. EDTA also binds to LPO-CN- complex indicating its binding site away from heme iron centre. The K_D of LPO-EDTA complex is significantly increased (62 ± 5 mM) by iodide suggesting that EDTA binds close to the iodide binding site. EDTA also increases the K_D value of LPO-hydroquinone complex from 62 ± 5 mM to 200 ± 21 mM indicating that EDTA and aromatic donor binding sites are also close. We suggest that EDTA inhibits iodide oxidation competitively as an electron donor by interacting at or near the iodide binding site and these sites are close to the aromatic donor binding site.     

The potential for the formation of the arginine biosynthetic enzymes and its masking during evolution

The present account spans the history of arginine regulation from its discovery in 1955 until the present. In 1957 I demonstrated that not only added arginine but also internally produced arginine represses enzyme formation and that the potential for enzyme synthesis is in excess of what is required for growth. In 1959 I located the regulatory gene argR encoding the arginine repressor. An unusual feature of this research was the finding that in E. coli B, in contrast to E. coli K12, arginine synthesis is permanently repressed, independent of arginine. This was due to a single amino acid difference between the two repressors. Recent studies showed that, in natural populations of E. coli, K12-type regulation is much more frequent than B-type regulation, and that E. coli B evolved from a strain with K12-type regulation. In competition experiments, E. coli K12 was found to be favored in the presence of arginine and E. coli B in its absence, showing that contrary to expectations permanently turned off regulation is favored over negative regulation in some environments.     

Genes, enzymes and regulation of arginine biosynthesis in plants.

Arabidopsis genes encoding enzymes for each of the eight steps in L-arginine (Arg) synthesis were identified, based upon sequence homologies with orthologs from other organisms. Except for N-acetylglutamate synthase (NAGS; EC 2.3.1.1), which is encoded by two genes, all remaining enzymes are encoded by single genes. Targeting predictions for these enzymes, based upon their deduced sequences, and subcellular fractionation studies, suggest that most enzymes of Arg synthesis reside within the plastid. Synthesis of the L-ornthine (Orn) intermediate in this pathway from L-glutamate occurs as a series of acetylated intermediates, as in most other organisms. An N-acetylornithine:glutamate acetyltransferase (NAOGAcT; EC 2.3.1.35) facilitates recycling of the acetyl moiety during Orn formation (cyclic pathway). A putative N-acetylornithine deacetylase (NAOD; EC 3.5.1.16), which participates in the "linear" pathway for Orn synthesis in some organisms, was also identified. Previous biochemical studies have indicated that allosteric regulation of the first and, especially, the second steps in Orn synthesis (NAGS; N-acetylglutamate kinase (NAGK), EC 2.7.2.8) by the Arg end-product are the major sites of metabolic control of the pathway in organisms using the cyclic pathway. Gene expression profiling for pathway enzymes further suggests that NAGS, NAGK, NAOGAcT and NAOD are coordinately regulated in response to changes in Arg demand during plant growth and development. Synthesis of Arg from Orn is further coordinated with pyrimidine nucleotide synthesis, at the level of allocation of the common carbamoyl-P intermediate.     

Estrogen regulation of progestin biosynthetic enzymes in cultured rat granulosa cells

The mechanism by which estrogens enhance gonadotropin-stimulated ovarian progestin production was investigated by studying the modulation of pregnenolone biosynthesis as well as the activities of 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) and 20 alpha-hydroxysteroid dehydrogenase (20 alpha-HSD) in cultured rat granulosa cells. Cells from immature hypophysectomized, estrogen-treated rats were cultured for 3 days with follicle-stimulating hormone (FSH) and/or estrogens. Pregnenolone production was measured in the presence of cyanoketone which inhibits 3 beta-HSD activity. Activities of 3 beta-HSD and 20 alpha-HSD were determined in cell homogenates by direct enzyme assays. Some cells were also primed with FSH to induce luteinizing hormone (LH) receptors for studies on the effects of estrogens on LH-modulated parameters. Pregnenolone production by cultured granulosa cells was stimulated by FSH, while treatment with diethylstilbestrol (DES) or estradiol further enhanced the gonadotropin action. Treatment with FSH increased 3 beta-HSD activity. Similarly, concomitant treatment with DES further enhanced 3 beta-HSD activity in a dose-dependent manner with an apparent ED50 of 10(-8) M. Also, treatment with estrogens alone increased 3 beta-HSD activity. The increases in enzyme activity induced by estrogen alone or in combination with FSH were not associated with changes in the apparent Km values. FSH also stimulated 20 alpha-HSD activity by 2-fold in these cells, while concomitant treatment with DES did not affect the FSH action. In FSH-primed cells, LH stimulated pregnenolone production while the LH action was enhanced by concomitant treatment with the estrogens. Likewise, LH stimulated the activity of 3 beta-HSD, while concomitant DES treatment further augmented LH action. LH did not stimulate 20 alpha-HSD activity when added alone or in combination with DES. Thus, the estrogen enhancement of the gonadotropin-stimulated progesterone production in cultured rat granulosa cells is associated with increases in pregnenolone biosynthesis and the activity of the 3 beta-HSD enzyme, without affecting the 20 alpha-HSD activity.     

Progestin regulation of progesterone biosynthetic enzymes in cultured rat granulosa cells

Progestins have recently been shown to augment gonadotropin-stimulated progesterone and 20 alpha-hydroxypregn-4-en-3-one (20 alpha-OH-P) biosynthesis in cultured rat granulosa cells. The mechanism by which progestins autoregulate ovarian progestin biosynthesis was investigated by studying the modulation of pregnenolone biosynthesis as well as the activities of the enzymes 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) and 20 alpha-hydroxysteroid dehydrogenase (20 alpha-HSD). Granulosa cells obtained from immature hypophysectomized, estrogen-treated rats were cultured with FSH and/or progestins. Pregnenolone production was measured in the presence of cyanoketone (10(-6) M) to inhibit 3 beta-HSD activity. Enzymatic activities of 3 beta-HSD and 20 alpha-HSD were determined in cell homogenates by direct enzyme assays. FSH stimulated pregnenolone production, while treatment with progesterone or R5020 alone was ineffective. Concomitant treatment with the progestins further enhanced FSH-stimulated pregnenolone production in a dose-dependent manner with minimal effective doses of 10(-8) and 10(-7) M for R5020 and progesterone, respectively. In FSH-primed cells, LH increased pregnenolone accumulation, and concomitant treatment with R5020 also enhanced the LH action. Furthermore, the gonadotropins stimulated the activity of 3 beta-HSD, and this effect was further enhanced by concomitant treatment with either R5020 or progesterone in a dose-dependent manner. In addition, the 20 alpha-HSD activities were enhanced by progestins in cells treated with FSH but not with LH. Thus, both natural and synthetic progestins stimulate the gonadotropin-induced progesterone production in cultured granulosa cells via enhancing the 3 beta-HSD enzyme as well as pregnenolone biosynthesis.     

Nutritional and hormonal regulation of malic enzyme synthesis in rat mammary gland.

Cytosolic malic enzyme was purified from rat mammary gland by L-malate affinity chromatography. The pure enzyme obtained was used to produce a specific antiserum in a rabbit. Relative synthesis of malic enzyme in the mammary gland of mid-lactating rats was 0.097%, measured by labelling the enzyme in isolated acini. When food was removed, malic enzyme synthesis decreased to 35% and 20% of the control value at 4 and 6 h respectively. Incorporation of [3H]leucine into soluble proteins was constant during the first 6 h of starvation. When lactating rats (maintained with their pups) were starved for 24 h and then re-fed, the relative rate of enzyme synthesis increased 2.5-, 4-, and 4.5-fold at 3 h, 6 h and 18 h respectively after initiation of re-feeding. The relative rate of malic enzyme synthesis was about 50% of normal at 15 h after weaning, whereas the rate of synthesis of soluble proteins did not change. Administration of bromocriptine or adrenalectomy of lactating rats decreased the relative rate of synthesis of malic enzyme by 40% or 30% respectively; these effects were counteracted by hormone supplementation. Hormone therapy also caused an increase in the rate of incorporation of [3H]leucine into soluble proteins and in malic enzyme activity.     

Androgen regulation of progestin biosynthetic enzymes in FSH-treated rat granulosa cells in vitro

The influence of androgens on the FSH modulation of progestin biosynthetic enzymes was studied $\text{in vitro}$. Granulosa cells obtained from immature, hypophysectomized, estrogen-treated rats were cultured for 3 days in a serum-free medium containing FSH (20 ng/ml) with or without increasing concentrations (10^?9?10^?6 M) of 17β-hydroxy-5α-androstan-3-one (dihydrotestosterone; DHT), 5α-androstane-3α, 17β-diol (3α-diol), or the synthetic androgen 17β-hydroxy-17-methyl-4,9,11-estratrien-3-one (methyltrienolone; R1881). FSH treatment increased progesterone and 20α-hydroxy-4-pregnen-3-one(20α-OH-P) production by 10.2- and 11-fold, respectively. Concurrent androgen treatment augmented FSH-stimulated progesterone and 20α-OH-P production in a dose-related manner (R1881 > 3α-diol > DHT). In the presence of an inhibitor of 3β-hydroxysteroid dehydrogenase (3β-HSD), the FSH-stimulated pregnenolone (3β-hydroxy-5-pregnen-20-one) production (a 20-fold increase) was further enhanced by co-treatment with R1881, 3α-diol or DHT. Furthermore, FSH treatment increased 4.4-fold the activity of 3β-HSD, which converts pregnenolone to progesterone. This stimulatory action of FSH was further augmented by concurrent androgen treatment. In contrast, androgen treatment did not affect FSH-stimulated activity of a progesterone breakdown enzyme, 20α-hydroxysteroid dehydrogenase(20α-HSD). These results demonstrate that the augmenting effect of androgens upon FSH-stimulated progesterone biosynthesis is not due to changes in the conversion of progesterone to 20α-OH-P, but involves an enhancing action upon 3β-HSDΔ⁵, Δ⁴-isomerase complexes and additional enzymes prior to pregnenolone biosynthesis.