Cytochrome P-450 heme and the regulation of δ-aminolevulinic acid synthetase in the liver

Hepatic δ-aminolevulinic acid synthetase was induced in rats injected with allylisopropylacetamide. The induction process was studied in relation to experimental perturbation of cytochrome P-450 in the liver. Animals were treated with either administered endotoxin or exogenous heme, both of which accelerate degradation of cytochrome P-450 heme. These manipulations were effective in blocking induction of δ-aminolevulinic acid synthetase, and the effect of each compound was proportional to its ability to stimulate degradation of cytochrome P-450 heme. The findings suggest that the heme moiety of cytochrome P-450 dissociates reversibly from its apoprotein and, prior to its degradation, mixes with endogenously synthesized heme to form a pool that regulates δ-aminolevulinic acid synthetase activity. A similar or identical heme fraction appears to mediate stimulation of heme oxygenase, which suggests that the regulation of δ-aminolevulinic acid synthetase and of heme oxygenase in the liver are closely interrelated.     

Hemin feedback inhibition at reticulocyte δ-aminolevulinic acid synthetase and δ-aminolevulinic acid dehydratase

Addition of hemin (5–200 μM) to a rabbit reticulocyte iron-free incubation medium, resulted in a progressive inhibition of heme synthesis as measured by incorporation of (¹⁴C)-glycine. In contrast when (¹⁴C) δ-aminolevulinic acid incorporation into heme was studied, significant inhibition below that of the (¹⁴C)-glycine control only occurred with hemin concentrations greater than 100 μM. Hemin progressively inhibited cellular and mitochondrialδ-aminolevulinic acid synthetase activity, as well as cellular δ-aminolevulinic acid dehydratase activity. The results indicated that elevated levels of hemin initially control heme synthesis by feedback inhibition at the rate-limiting enzyme of heme synthesis, δ-aminolevulinic acid synthetase. Hemin inhibition of δ-aminolevulinic acid dehydratase is only significant for the entrire heme synthetic pathway when greater than one-third of this enzyme's activity is inhibited.     

Structural requirements in dihydropyridines for ferrochelatase inhibition and δ-aminolevulinic acid synthetase induction

• 1.1. A series of analogues of 3,5-diethoxycarbonyl-1,4-dihydro-2,4,6-trimethylpyridine (DDC) was investigated for hepatic δ-aminolevulinic acid (ALA)-synthetase inducing and ferrochelatase-inhibiting activity in the 17-day-old chick embryo.
• 2.2. A DDC analogue was found which was capable of inducing ALA-synthetase activity without inhibiting ferrochelatase activity.
• 3.3. On the other hand we were unable to find a DDC analogue with the ability to inhibit ferrochelatase activity which was devoid of ALA-synthetase-inducing activity.

     

Inhibition of δ-aminolevulinic acid synthetase and δ-aminolevulinic acid dehydrase by L-2-amino-4-methoxy-trans-3-butenoic acid in the rat

The rate limiting enzyme of heme biosynthesis, δ-aminolevulinic acid synthetase (ALA synthetase), and the second enzyme in the heme biosynthetic pathway, δ-aminolevulinic acid dehydrase (ALA dehydrase), were inhibited by the olefinic amino acid L-2-amino-4-methoxy - $\text{trans}$-3-butenoic acid (AMTB). Administration of AMTB (20 mg/kg; i.p.) to rats inhibited ALA synthetase and ALA dehydrase in control animals and in animals with markedly elevated activity of ALA synthetase which resulted from the administration of 3,5-dicarbethoxy-1,4-dimethyl-collidine (DDC, 200 mg/kg, i.p.) or allylisopropylacetamide (200 mg/kg, s.c.). AMTB also blocked the synthesis of rat hepatic porphyrins and inhibited the increase in the urinary excretion of δ-aminolevulinic acid and porphobilinogen following DDC (150 mg/kg, p.o.) administration. Preincubation of AMTB with liver mitochondria or a soluble fraction of liver decreased the activity of mitochondrial ALA synthetase and soluble ALA dehydrase, respectively.     

Biosynthesis of δ-aminolevulinic acid in Rhodopseudomonas sphaeroides

The biosynthesis of δ-aminolevulinic acid was investigated in three strains of Rhodopseudomonas sphaeroides. A wild-type strain (NCIB 8253) possessed both δ-aminolevulinic acid synthetase and γ,δ-dioxovaleric acid transaminase in the cytoplasmic and membrane cell fractions. δ-Aminolevulinic acid synthetase activities were not detected in extracts of mutant strains H5 and H5D. However, γ,δ-dioxovaleric acid transaminase was found in the cytoplasmic and membrane fractions of these latter two strains. Strain H5 required exogenously added δ-aminolevulinic acid for growth and bacteriochlorophyll synthesis. Strain H5D did not require this compound for growth and bacteriochlorophyll synthesis. γ,δ-Dioxovaleric acid added in the growth medium did not support the growth of H5, although it was actively transported into the cells. Addition of γ,δ-dioxovaleric acid to the growth medium did not enhance the growth of either the wild-type or H5D strains. These results indicate that ALA synthetase is not required for growth and bacteriochlorophyll synthesis in H5D and that γ,δ-dioxovaleric acid is probably not an intermediate in the formation of δ-aminolevulinic acid in the strains of Rhodopseudomonas sphaeroides studied. In strain H5D another pathway may function in the formation of δ-aminolevulinic acid other than that catalyzed by δ-aminolevulinic acid synthetase or γ,δ-dioxovaleric acid transaminase.     

A model for the regulation of δ-aminolaevulinate synthetase induction in rat liver

A reciprocal relationship exists between the cytochrome P-450 content and delta-aminolaevulinate synthetase activity in adult rats. In young rats the basal delta-aminolaevulinate synthetase activity is higher and the cytochrome P-450 content is lower compared with the adult rat liver. Administration of allylisopropylacetamide neither induces the enzyme nor causes degradation of cytochrome P-450 in the young rat liver, unlike adult rat liver. Allylisopropylacetamide fails to induce delta-aminolaevulinate synthetase in adrenalectomized-ovariectomized animals or intact animals pretreated with successive doses of the drug, in the absence of cortisol. The cortisol-mediated induction of the enzyme is sensitive to actinomycin D. Allylisopropylacetamide administration degrades microsomal haem but not nuclear haem. Haem does not counteract the decrease in cytochrome P-450 content caused by allylisopropylacetamide administration, but there is evidence for the formation of drug-resistant protein-bound haem in liver microsomal material under these conditions. Phenobarbital induces delta-aminolaevulinate synthetase under conditions when there is no breakdown of cytochrome P-450. On the basis of these results and those already published, a model is proposed for the regulation of delta-aminolaevulinate synthetase induction in rat liver.     

Nutritional regulation and role of peroxisome proliferator-activated receptor δ in fatty acid catabolism in skeletal muscle

Peroxisome proliferator-activated receptors (PPARs) are nuclear receptors primarily involved in lipid homeostasis. PPARδ displays strong expression in tissues with high lipid metabolism, such as adipose, intestine and muscle. Its role in skeletal muscle remains largely unknown. After a 24-h starvation period, PPARδ mRNA levels are dramatically up-regulated in gastrocnemius muscle of mice and restored to control level upon refeeding. The rise of PPARδ is accompanied by parallel up-regulations of fatty acid translocase/CD36 (FAT/CD36) and heart fatty acid binding protein (H-FABP), while refeeding promotes down-regulation of both genes. To directly access the role of PPARδ in muscle cells, we forced its expression and that of a dominant-negative PPARδ mutant in C2C12 myogenic cells. Differentiated C2C12 cells responds to 2-bromopalmitate or synthetic PPARδ agonist by induction of genes involved in lipid metabolism and increment of fatty acid oxidation. Overexpression of PPARδ enhanced these cellular responses, whereas expression of the dominant-negative mutant exerts opposite effects. These data strongly support a role for PPARδ in the regulation of fatty acid oxidation in skeletal muscle and in adaptive response of this tissue to lipid catabolism.     

Is regulation of proteolysis associated with redox-state changes in rat skeletal muscle?

In isolated rat diaphragms, only those substrates that increased the tissue NADH/NAD+ ratio lowered the rate of proteolysis. However, direct inhibition of proteinase activity by leupeptin promoted oxidation of the NAD couple of the muscles. These results suggest that changes in muscle reduction-oxidation state may be important in the regulation of proteolysis.     

Assay of δ-aminolevulinic acid synthetase in homogenates of mouse,rat, and human liver: Species differences in requirement for an exogenous succinyl-CoA-generating system

Conditions required for optimal assay of low levels of activity of hepatic δ-aminolevulinic acid synthetase have been studied, comparing dilute homogenates of mouse, rat, and human livers. The assay method used was a modification of that described by Ebert et al. (Biochim. Biophys. Acta (1970)208, 236–250), and livers were studied from both untreated animal and human subjects and subjects pretreated with porphyrinogenic compounds. In homogenates of mouse and human but not rat liver, maximal rates of δ-aminolevulinic acid formation required addition to the incubation mixture of an exogenous system for succinyl-CoA generation. The requirement for this generating system was increased if livers from pretreated subjects were frozen and stored prior to assay, suggesting that the endogenous capacity for succinyl-CoA generation was more labile than δ-aminolevulinic acid synthetase under these conditions. Of the metabolic inhibitors tested (F^?, malonate, and arsenite), only F^? (100 mm final concentration) enhanced activity. Increasing the permeability of mitochondria by quick freezethawing of fresh homogenates just before assay did not increase the rate of δ-aminolevulinic acid formation.     

Control of δ-aminolaevulate synthetase activity in Rhodopseudomonas spheroides

1. delta-Aminolaevulate synthetase from Rhodopseudomonas spheroides grown semi-anaerobically undergoes a spontaneous activation during the first hour after the disruption of cells when homogenates are stored at 4 degrees . 2. After cultures of R. spheroides growing semi-anaerobically are oxygenated no activation of delta-aminolaevulate synthetase occurs in cell extracts. Cessation of activation in extracts is almost complete 10min. after oxygenation of cells has begun. 3. A heat-stable fraction of low molecular weight from semi-anaerobic cells reactivates delta-aminolaevulate synthetase in extracts of oxygenated cells and appears to contain a compound responsible for the spontaneous activation. 4. A heat-stable fraction of low molecular weight from oxygenated cells inhibits the spontaneous activation in extracts of semi-anaerobic cells. 5. The effect of oxygen on the rate of bacteriochlorophyll synthesis in R. spheroides may be mediated through alterations in the concentrations of a low-molecular-weight activator and inhibitor of delta-aminolaevulate synthetase.     

Rapid loss of δ-aminolevulinic acid dehydratase activity in primary cultures of adult rat hepatocytes: A new model of zinc deficiency

Nonproliferating cultures of adult rat hepatocytes were found to lose 60–70% of cell-associated zinc during their first 24 h of incubation in standard, serum-free medium. The loss of zinc was accompanied by a profound loss (95%) in the activity of the zinc metalloenzyme, δ-aminolevulinic acid dehydratase, as well as a loss (>85%) in the cellular content of immunoreactive δ-aminolevulinic acid dehydratase protein. Restoration of cellular zinc content by the addition of zinc to the culture medium partially prevented the losses of both δ-aminolevulinic acid dehydratase activity and immunoreactive protein. Since the spontaneous, selective loss of cellular zinc appears to have specific effects on a relevant hepatic function, this culture system constitutes a novel $\text{in}$$\text{vitro}$ model of zinc deficiency in mature liver.     

The biosynthesis of δ-aminolevulinic acid in greening maize leaves

In greening maize leaves δ-aminolevulinic acid (ALA) was not formed from succinyl-CoA and glycine as shown by the incorporation of [¹⁴C]-labeled     

Inhibition of Na+, K+-ATPase activity by δ-aminolevulinic acid

-Aminolaevulinic acid (ALA) has been shown to be toxic to cultured neurons and glia at concentrations as low as 10 M. In an attempt to elucidate the mechanism of toxicity, the effects of ALA on membrane ATPase activity were investigated. Exposure of neuron cultures to 1 mM ALA for 7 days caused a substantial decrease in both Na⁺, K⁺-ATPase and Mg²⁺-ATPase activities. At lower concentrations, ALA affected only the Na⁺, K⁺-component. ALA appeared to act directly, inhibiting Na⁺, K⁺-ATPase activity in rat brain cortex membrane preparations at 10 M Although this effect was slight, it may well represent the mechanism of action of ALA, since ouabain, a potent inhibitor of Na⁺, K⁺-ATPase activity, proved to be more toxic to cultured neurons than ALA. Furthermore, cardiac glycoside overdosage causes neurological disturbances which are very similar to those observed in the acute attack of porphyria.     

Influence of gabaculine on growth,chlorophyll synthesis,and δ-aminolevulinic acid synthase activity in Euglena gracilis

Euglena gracilis is capable of forming the heme and chlorophyll precursor δ-aminolevulinic acid (ALA) by two routes: from glutamate via the five-carbon path in the chloroplasts, and by ALA synthase-mediated condensation of succinyl-CoA and glycine, probably in the mitochondrion. 5-Amino-1,3-cyclohexadienyl carboxylic acid (gabaculine), a powerful inhibitor of ALA formation via the five-carbon path, was administered to E. gracilis Klebs strain Z Pringsheim cells growing in the light or dark, and its effects on growth, chlorophyll accumulation and extractable ALA synthase activity were measured. Gabaculine had no effect in vitro on ALA synthase or ALA dehydratase, even at 100 μM. Administration of 100 μM gabaculine to wild-type cells growing in the light slowed growth, inhibited chlorophyll accumulation, and induced an increase in extractable ALA synthase activity. Chlorophyll accumulation in the light was abolished by prior administration of the compound to growing cells for 6 h in the dark, whereas chlorophyll accumulation in cells without gabaculine began immediately after transfer to light. Extractable ALA synthase activity from gabaculine-pretreated dark-grown cells was initially lower than the activity from untreated cells, but it did not undergo a further decline after transfer of the cells to the light, whereas the activity from untreated cells dropped to less than one eighth the dark level after 2 h in the light, and by 4 h had fallen to a level five times lower than that extractable from gabaculine-treated cells. These results suggest that suppression of ALA synthase activity by light in untreated cells is related to light-induced activation of the five-carbon ALA biosynthetic pathway in the plastids, and may result from a contribution by a product of the five-carbon pathway to non-plastid tetrapyrrole pools in the light.     

Modulation of δ-aminolevulinic acid dehydratase activity by the sorbitol-induced osmotic stress in maize leaf segments

Osmotic stress induced with 1 M sorbitol inhibited δ-aminolevulinic acid dehydratase (ALAD) and aminolevulinic acid (ALA) synthesizing activities in etiolated maize leaf segments during greening; the ALAD activity was inhibited to a greater extent than the ALA synthesis. When the leaves were exposed to light, the ALAD activity increased for the first 8 h, followed by a decrease observed at 16 and 24 h in both sorbitol-treated and untreated leaf tissues. The maximum inhibition of the enzyme activity was observed in the leaf segments incubated with sorbitol for 4 to 8 h. Glutamate increased the ALAD activity in the in vitro enzymatic preparations obtained from the sorbitol-treated leaf segments; sorbitol inhibited the ALAD activity in the preparations from both sorbitol-treated and untreated leaves. It was suggested that sorbitol-induced osmotic stress inhibits the enzyme activity by affecting the ALAD induction during greening and regulating the ALAD steady-state level of ALAD in leaf cells. The protective effect of glutamate on ALAD in the preparations from the sorbitol-treated leaves might be due to its stimulatory effect on the enzyme.     

Identification, expression and tissue distribution of cytidine 5′-monophosphate N-acetylneuraminic acid synthetase activity in the rat

We report the postnatal developmental profiles of N-acetylneuraminic acid cytidylyltransferase (EC 2.7.7.43) (CMP-Neu5Ac synthetase) in different rat tissues. This enzyme, which catalyses the activation of NeuAc to CMP-Neu5Ac, was detected in brain, kidney, heart, spleen, liver, stomach, intestine, lung, thymus, prostate and urinary bladder but not in skeletal muscle. Comparative analysis of the different specific activity profiles obtained shows that the expression of CMP Neu5Ac synthetase is tissue-dependent and does not seem to be embryologically determined. Changes in the level of sialylation during development were also found to be intimately related to variations in the expression of this enzyme, at least in brain, heart, kidney, stomach, intestine and lung.     

Effect of an exogenous succinyl-CoA-generating system on the measurement of δ-aminolevulinic acid synthase activity in rat liver tissue by a radiochemical assay

Rat liver tissue was used to examine the effect of an exogenous succinyl-CoA-generating system on the radiochemical assay for δ-aminolevulinic acid synthase (succinyl-CoA:glycine C-succinyltransferase (decarboxylating), EC 2.3.1.37) activity developed by Ebert et al. (Ebert, P.S., Tschudy, D.P., Choudry, J.N. and Chirigos, M.A. (1970) Biochim. Biophys. Acta 208, 236–250). In the absence of exogenous succinate thiokinase, 34–62% (average 55%) of the radioactivity in the final column eluate could be attributed to δ-amino-[4-¹⁴C]levulinic acid, as assessed by conversion of δ-aminolevulinic acid in the eluate to a pyrrole. The addition of succinate thiokinase markedly enhanced the formation of the contaminant(s), as succinyl-CoA was metabolized to a compound or compounds that eluted chromatographically with δ-amino-levulinic acid. This effect was abolished by 10 mM EDTA, probably because the generation of succinyl-CoA was suppressed due to the chelation of Mg²⁺. These observations indicate that this radiochemical assay should be carefully examined for each set of assay conditions employed.     

The influence of melatonin and N-acetylcysteine in δ-aminolevulinic acid and lead induced genotoxicity in lymphocytes in vitro

As is well known from earlier studies, the genotoxic effect of lead exposure was partly attributed to the formation of the highly reactive oxygen metabolites (ROMs) in the blood. However, lead ions have no ability to generate ROMs. Therefore, the recently published studies paid more attention to the role of δ-aminolevulinic acid (ALA) accumulation in lead-induced DNA damage. If the above-mentioned assumptions were taken into consideration, it seemed a reasonable approach to study the possible protective effects of antioxidants against genotoxic effects of lead. According to our results, N-acetylcysteine (NAC) and melatonin (MEL) were able to reduce significantly (p < 0.05) the lead- and ALA-induced sister chromatid exchange frequencies in human lymphocytes in vitro. In spite of a relative reduction in the lead- and ALA-induced micronucleus formation in human lymphocytes, the reduction was not statistically significant (p > 0.05). These results could be evaluated as supportive evidence for the hypothesis that increased antioxidant capacity of cells might fortify the efficiency of protective pathways against cytogenetic damage in lead exposure.     

Role of fatty acid uptake and fatty acid β-oxidation in mediating insulin resistance in heart and skeletal muscle

Fatty acids are a major fuel source used to sustain contractile function in heart and oxidative skeletal muscle. To meet the energy demands of these muscles, the uptake and β-oxidation of fatty acids must be coordinately regulated in order to ensure an adequate, but not excessive, supply for mitochondrial β-oxidation. However, imbalance between fatty acid uptake and β-oxidation has the potential to contribute to muscle insulin resistance. The action of insulin is initiated by binding to its receptor and activation of the intrinsic protein tyrosine kinase activity of the receptor, resulting in the initiation of an intracellular signaling cascade that eventually leads to insulin-mediated alterations in a number of cellular processes, including an increase in glucose transport. Accumulation of fatty acids and lipid metabolites (such as long chain acyl CoA, diacylglycerol, triacylglycerol, and/or ceramide) can lead to alterations in this insulin signaling pathway. An imbalance between fatty acid uptake and oxidation is believed to be responsible for this lipid accumulation, and is thought to be a major cause of insulin resistance in obesity and diabetes, due to lipid accumulation and inhibition of one or more steps in the insulin-signaling cascade. As a result, decreasing muscle fatty acid uptake can improve insulin sensitivity. However, the potential role of increasing fatty acid β-oxidation in the heart or skeletal muscle in order to prevent cytoplasmic lipid accumulation and decrease insulin resistance is controversial. While increased fatty acid β-oxidation may lower cytoplasmic lipid accumulation, increasing fatty acid β-oxidation can decrease muscle glucose metabolism, and incomplete fatty acid oxidation has the potential to also contribute to insulin resistance. In this review, we discuss the proposed mechanisms by which alterations in fatty acid uptake and oxidation contribute to insulin resistance, and how targeting fatty acid uptake and oxidation is a potential therapeutic approach to treat insulin resistance.