Microbial production of 5-aminolevulinic acid (ALA) by photosynthetic bacteria compared to other bacteria and algae is reviewed. During aerobic-microaerobic cultivation of Rhodobacter sphaeroides mutant strain CR520, control of the redox potential was effective for producing large amounts of extracellular ALA. ALA has been practically applied in agriculture as an herbicide, an insecticide and a growth-promoting factor for plants. New agricultural applications including salt tolerance and cold temperature tolerance of plants are also described. Finally, recent medical applications for cancer treatment, tumor diagnosis and other clinical uses are discussed.

5-Aminolevulinic acid (ALA) is a key intermediate of heme biosynthesis, which is an essential component of the respiratory chain. Therefore, nutrients that affect ALA biosynthesis eventually affect ATP production, which is the basis of mitochondrial function. Although the effects of various non-nutrient components that affect ALA after biosynthesis have been reported, there are few reports on the effects of dietary amino acids/protein on ALA formation and the effects of dietary vitamins that are involved in amino acid metabolism. In mitochondria, ALA is synthesized from succinyl-CoA and glycine by the pyridoxal phosphate-dependent enzyme ALA synthase [EC 2.3.1.37]. In this study, the effects of dietary amino acids/protein and vitamins on the amount of ALA synthesized were investigated using mice, rats, and cultured cells. Amounts of ALA in plasma and urine, and porphyrins in plasma increased with increasing protein intake. Vitamin B insufficiency did not affect ALA synthesis. Vitamin B insufficiency increased the amount of ALA synthesized, while niacin deficiency markedly reduced ALA synthesis. Thus, for heme synthesis, an essential biological substance for life, the amounts of amino acids, as well as the pathways metabolizing amino acids to glycine and succinyl-CoA are very important. Specifically, it is important that niacin is associated with the formation of glycine and succinyl-CoA from amino acids.

5-Aminolevulinate synthase (ALAS) is a mitochondrial enzyme that catalyzes the first step of the heme biosynthetic pathway. The mitochondrial import, as well as the synthesis, of the nonspecific isoform of ALAS (ALAS1) is regulated by heme through a feedback mechanism. A short amino acid sequence, the heme regulatory motif (HRM), is known to be involved in the regulatory function of heme. To determine the role of the HRM in the heme-regulated transport of the nonspecific and erythroid forms of ALAS in vivo, we constructed a series of mutants of rat ALAS1, in which the cysteine residues in the three putative HRMs in the N-terminal region of the enzyme were converted to serine ones by site-directed mutagenesis. The wild-type and mutant enzymes were expressed in quail QT6 fibroblasts through transient transfection, and the mitochondrial import of these enzymes was examined in the presence of hemin. Hemin inhibited the mitochondrial import of wild-type ALAS1, but this inhibition was reversed on the mutation of all three HRMs in the enzyme, indicating that the HRMs are essential for the heme-mediated inhibition of ALAS1 transport in the cell. By contrast, exogenous hemin did not affect the mitochondrial import of the erythroid-specific ALAS isoform (ALAS2) under the same experimental conditions. These results may reflect the difference in the physiological functions of the two ALAS isoforms.

5-Aminolevulinic acid synthase (ALAS-1) is the first rate controlling enzyme that controls cellular heme biosynthesis. Negative feedback regulation of ALAS-1 by the end product heme is well documented and provides the foundation for heme treatment of acute porphyrias, a group of diseases caused by genetic defects in the heme biosynthesis pathway and exacerbated by controlled up-regulation of ALAS-1. Heme is known to affect ALAS-1 activity by repressing gene transcription, accelerating mRNA degradation, and impeding pre-ALAS-1 mitochondrial translocation. In the current study, we examined the effect of heme on the rate of mature ALAS-1 protein turnover in human cells and tissues and explored the mediator involved in this new regulatory mechanism. We found that heme and other metalloporphyrins such as CoPP and CrPP decreased mitochondrial ALAS-1 protein through proteolysis. This degradative effect cannot be emulated by iron or free protoporphyrin, two major chemical components of the heme ring, and is independent of oxidative stress. Down-regulating the activity of mitochondrial LONP1, an ATP-dependent protease that controls the selective turnover of mitochondrial matrix proteins, with potent inhibitors and specific siRNA diminished the negative effect of heme on mitochondrial ALAS-1. Therefore, our data support the existence of a conserved heme feedback regulatory mechanism that functions on the mature form of ALAS-1 protein through the activity of a mitochondrial proteolytic system.

Inducible hepatic porphyrias are inherited genetic disorders of enzymes of heme biosynthesis. The main clinical manifestations are acute attacks of neuropsychiatric symptoms frequently precipitated by drugs, hormones, or fasting, associated with increased urinary excretion of delta-aminolevulinic acid (ALA). Acute attacks are treated by heme infusion and glucose administration, but the mechanisms underlying the precipitating effects of fasting and the beneficial effects of glucose are unknown. We show that the rate-limiting enzyme in hepatic heme biosynthesis, 5-aminolevulinate synthase (ALAS-1), is regulated by the peroxisome proliferator-activated receptor gamma coactivator 1alpha (PGC-1alpha). Elevation of PGC-1alpha in mice via adenoviral vectors increases the levels of heme precursors in vivo as observed in acute attacks. The induction of ALAS-1 by fasting is lost in liver-specific PGC-1alpha knockout animals, as is the ability of porphyrogenic drugs to dysregulate heme biosynthesis. These data show that PGC-1alpha links nutritional status to heme biosynthesis and acute hepatic porphyria.

Heme is an essential component in oxygen transport and metabolism in living systems. In non-erythropoietic cells, 5-aminolevulinate synthase (ALAS1) is the first and rate-limiting enzyme in the heme biosynthesis pathway. ALAS1 expression and heme levels are increased in vivo by drugs and other chemical inducers of cytochrome P450 hemoproteins through mechanisms that are poorly understood. In the present studies, a chicken genomic cosmid library was employed to isolate a major portion of the ALAS1 gene. Two drug-responsive enhancer sequences, 176 and 167 base pairs in length, were identified in the 5'-flanking region of the gene in reporter gene assays in the hepatoma cell line LMH. The relative potency of inducers to activate these enhancers corresponds to induction of ALAS1 mRNA levels in LMH cells. Analysis of putative transcription factor binding sites within the enhancers revealed DR5 and DR4 type recognition sequences for nuclear receptors. Drug activation of the enhancer elements was reduced at least 60% after mutagenesis of individual nuclear receptor binding sites and was virtually eliminated following alteration of both recognition sites within the respective elements. Electrophoretic mobility shift assays and transactivation studies demonstrate direct interactions between the nuclear receptor binding sites and the recently described chicken xenobiotic-sensing receptor, (CXR) implicating drug activation mechanisms for ALAS1 similar to those found in inducible cytochrome(s) P450. This is the first report describing direct transcriptional activation of ALAS1 by drugs via drug-responsive enhancer sequences.

Nuclear receptors have been implicated in the transcriptional regulation of expression of a growing number of genes, including cytochromes P450 and 5-aminolevulinate synthase (ALAS1), the first and rate-limiting enzyme in the heme biosynthesis pathway. Although drugs that induce cytochromes P450 also induce ALAS1, the regulatory mechanisms governing these pathways have not been fully elucidated. We have identified a drug-responsive enhancer in the murine ALAS1 gene. This sequence mediates transcriptional activation by a wide range of compounds including typical cytochrome P450 pan-inducers phenobarbital and metyrapone, as well as specific activators of the pregnane X receptor and the constitutive androstane receptor. ALAS1 drug-responsive enhancer sequences were identified by transient transfection of reporter gene constructs in the drug-responsive leghorn male hepatoma cell line. Using the NUBIScan algorithm, DR4 nuclear receptor binding sites were identified within the elements and their roles in mediating transcriptional activation of ALAS1 were confirmed by site-directed mutagenesis. Electrophoretic mobility shift assays demonstrate clear interactions of mouse pregnane X receptor and constitutive androstane receptor on the ADRES. Transactivation assays in CV-1 cells implicate the nuclear receptors as major contributors to transcriptional activation of ALAS1. Moreover, in vivo studies in knock-out animals confirm the induction of ALAS1 is mediated at least in part by nuclear receptors. These studies are the first to explain drug induction via drug response elements for mammalian ALAS1.

Recently, new genes and molecular mechanisms have been identified in patients with porphyrias and sideroblastic anemias (SA). They all modulate either directly or indirectly the δ-aminolevulinic acid synthase (ALAS) activity. ALAS, is encoded by two genes: the erythroid-specific (ALAS2), and the ubiquitously expressed (ALAS1). In the liver, ALAS1 controls the rate-limiting step in the production of heme and hemoproteins that are rapidly turned over in response to metabolic needs. Several heme regulatory targets have been identified as regulators of ALAS1 activity: 1) transcriptional repression via a heme-responsive element, 2) post-transcriptional destabilization of ALAS1 mRNA, 3) post-translational inhibition via a heme regulatory motif, 4) direct inhibition of the activity of the enzyme and 5) breakdown of ALAS1 protein via heme-mediated induction of the protease Lon peptidase 1. In erythroid cells, ALAS2 is a gatekeeper of production of very large amounts of heme necessary for hemoglobin synthesis. The rate of ALAS2 synthesis is transiently increased during the period of active heme synthesis. Its gene expression is determined by trans-activation of nuclear factor GATA1, CACC box and NF-E2-binding sites in the promoter areas. ALAS2 mRNA translation is also regulated by the iron-responsive element (IRE)/iron regulatory proteins (IRP) binding system. In patients, ALAS enzyme activity is affected in most of the mutations causing non-syndromic SA and in several porphyrias. Decreased ALAS2 activity results either directly from loss-of-function ALAS2 mutations as seen in X-linked sideroblastic anemia (XLSA) or from defect in the availability of one of its two mitochondrial substrates: glycine in SLC25A38 mutations and succinyl CoA in GLRX5 mutations. Moreover, ALAS2 gain of function mutations is responsible for X-linked protoporphyria and increased ALAS1 activity lead to acute attacks of hepatic porphyrias. A missense dominant mutation in the Walker A motif of the ATPase binding site in the gene coding for the mitochondrial protein unfoldase CLPX also contributes to increasing ALAS and subsequently protoporphyrinemia. Altogether, these recent data on human ALAS have informed our understanding of porphyrias and sideroblastic anemias pathogeneses and may contribute to new therapeutic strategies.Copyright © 2019 Elsevier Inc. All rights reserved.

Glutamyl tRNA(Glu) reductase converts glutamate molecules that are ligated at their alpha-carboxyl groups to tRNA(Glu) into glutamate 1-semialdehyde, an intermediate in the synthesis of 5-aminolevulinate, chlorophyll and haem. The mature plant enzymes contain a highly conserved extension of 31-34 amino acids at the N-terminus not present in bacterial enzymes. It is shown that barley glutamyl tRNAGlu reductases with a deletion of the 30 N-terminal amino acids have the same high specific activity as the untruncated enzymes, but are highly resistant to feed-back inhibition by haem. This peptide domain thus interacts directly or indirectly with haem and the toxicity of the 30 amino acid peptide for Escherichia coli experienced in mutant rescue and overexpression experiments can be explained by extensive haem removal from the metabolic pools that cannot be tolerated by the cell. Induced missense mutations identify nine amino acids in the 451 residue long C-terminal part of the barley glutamyl tRNA(Glu) reductase which upon substitution curtail drastically, but do not eliminate entirely the catalytic activity of the enzyme. These amino acids are thus important for the catalytic reaction or tRNA binding.

The chloroplast glutamyl-tRNA (tRNA) is unique in that it has two entirely different functions. In addition to acting in translation, it serves as the substrate of glutamyl-tRNA reductase (GluTR), the enzyme catalyzing the committed step in the tetrapyrrole biosynthetic pathway. How the tRNA pool is distributed between the two pathways and whether tRNA allocation limits tetrapyrrole biosynthesis and/or protein biosynthesis remains poorly understood. We generated a series of transplastomic tobacco () plants to alter tRNA expression levels and introduced a point mutation into the plastid gene, which has been reported to uncouple protein biosynthesis from tetrapyrrole biosynthesis in chloroplasts of the protist We show that, rather than comparable uncoupling of the two pathways, the mutation is lethal in tobacco because it inhibits tRNA processing, thus preventing translation of Glu codons. Ectopic expression of the mutated gene uncovered an unexpected inhibition of glutamyl-tRNA reductase by immature tRNA We further demonstrate that whereas overexpression of tRNA does not affect tetrapyrrole biosynthesis, reduction of GluTR activity through inhibition by tRNA precursors causes tetrapyrrole synthesis to become limiting in early plant development when active photosystem biogenesis provokes a high demand for de novo chlorophyll biosynthesis. Taken together, our findings provide insight into the roles of tRNA at the intersection of protein biosynthesis and tetrapyrrole biosynthesis.© 2020 American Society of Plant Biologists. All Rights Reserved.

The first committed step of porphyrin synthesis in higher plants is the reduction of glutamyl-tRNA to glutamate 1-semialdehyde. This reaction is catalyzed by glutamyl-tRNA reductase, which is encoded by hemA genes. Two hemA cDNA clones (hemA1 and hemA2) were obtained from cucumber (Cucumis sativus) cotyledons by the PCR and cDNA library screening. They showed significant homology with published hemA sequences. Southern blot analysis of cucumber genomic DNA revealed that these genes are located at different loci and that there is another gene similar to the hemA genes. Accumulation of hemA1 mRNA was detected primarily in cotyledons and hypocotyls of greening cucumber seedlings, whereas that of hemA2 mRNA was detected in all tissues examined. Illumination of cucumber seedlings increased markedly the accumulation of hemA1 mRNA, but it did not induce remarkable changes in that of hemA2 mRNA. These findings suggest that hemA1 mRNA was accumulated in response to the demand of Chl synthesis in photosynthesizing tissues, whereas hemA2 mRNA was expressed in response to the demand of the synthesis of porphyrins other than chlorophylls.

5-aminolevulinic acid (5-ALA) and its ester derivatives are used in photodynamic therapy as precursors for the formation of photosensitizers. This study relates to the mechanisms by which 5-ALA is transported into cells. The transport of 5-ALA has been studied in a human adenocarcinoma cell line (WiDr) by means of [14C]-labeled 5-ALA. The rate of uptake was saturable following Michaelis-Menten kinetics (K(m) = 8-10 mM and Vmax = 18-20 nmol.(mg protein x h)-1), and Arrhenius plot of the temperature-dependent uptake of 5-ALA was characterized by a single discontinuity at 32 degrees C. The activation energy was 112 kJ.mol-1 in the temperature range 15 degrees-32 degrees C and 26 kJ.mol-1 above 32 degrees C. Transport of 5-ALA was Na+ and partly Cl(-)-dependent. Stoichiometric analysis revealed a Na+:5-ALA coupling ratio of 3:1. With the exception of valine, methionine and threonine, zwitterionic and basic amino acids inhibited the transport of 5-ALA. 5-ALA methyl ester was not an inhibitor of 5-ALA uptake. The transport was most efficiently inhibited, i.e. by 65-75%, by the beta-amino acids, beta-alanine and taurine and by gamma-aminobutyric acid (GABA). Accordingly, 5-ALA, but not 5-ALA methyl ester, was found to inhibit cellular uptake of [3H]-GABA and [14C]-beta-alanine. Protoporphyrin IX (PpIX) accumulation in the presence of 5-ALA (0.3 mM) was attenuated 85% in the presence of 10 mM beta-alanine, while PpIX formation in cells treated with 5-ALA methyl ester (0.3 mM) or 5-ALA hexyl ester (4 microM) was not significantly influenced by beta-alanine. Thus, 5-ALA, but not 5-ALA esters, is transported by beta-amino acid and GABA carriers in this cell line.

The aim of this study was to investigate the gene and protein expression profiles of important drug-transporting proteins in human cell lines commonly used for studies of drug transport mechanisms. Human cell lines used to transiently or stably express single transporters [HeLa, human embryonic kidney (HEK) 293] and leukemia cell lines used to study drug resistance by ATP-binding cassette transporters (HL-60, K562) were investigated and compared with organotypic cell lines (HepG2, Saos-2, Caco-2, and Caco-2 TC7). For gene expression studies, real-time polymerase chain reaction was used, whereas monospecific polyclonal antibodies were generated and used to investigate protein expression by immunohistochemistry. Thirty-six transporters were studied for gene expression, and nine were studied for protein expression. The antibodies were validated using expression patterns in human tissues. Finally, the function of one ubiquitously expressed transporter, MCT1/SLC16A1, was investigated using [(14)C]lactic acid as a substrate. In general, the adherent cell lines (HeLa, HEK293) displayed low transporter expression, and the expression patterns were barely affected by transfection. The leukemia cell lines (K562, HL-60) and Saos-2 also had low endogenous transporter expression, whereas the organotypic cell lines (HepG2 and Caco-2) showed higher expression of some transporters. Comparison of gene and protein expression profiles gave poor correlations, but better agreement was obtained for antibodies with a good validation score, indicating that antibody quality was a significant variable. It is noteworthy that the monocarboxylic acid-transporting protein MCT1 was significantly expressed in all and was functional in most of the cell lines, indicating that MCT1 may be a confounding factor when the transport of small anionic drugs is investigated.

Congenital sideroblastic anemia (CSA) is a hereditary disorder characterized by microcytic anemia and bone marrow sideroblasts. The most common form of CSA is attributed to mutations in the X-linked gene 5-aminolevulinic acid synthase 2 (ALAS2). ALAS2 is a mitochondrial enzyme, which utilizes glycine and succinyl-CoA to form 5-aminolevulinic acid (ALA), a crucial precursor in heme synthesis. Therefore, ALA supplementation could be an effective therapeutic strategy to restore heme synthesis in CSA caused by ALAS2 defects. In a preclinical study, we examined the effects of ALA in human erythroid cells, including K562 cells and human induced pluripotent stem cell-derived erythroid progenitor (HiDEP) cells. ALA treatment resulted in significant dose-dependent accumulation of heme in the K562 cell line. Concomitantly, the treatment substantially induced erythroid differentiation as assessed using benzidine staining. Quantitative reverse transcription polymerase chain reaction (RT-PCR) analysis confirmed significant upregulation of heme-regulated genes, such as the globin genes [hemoglobin alpha (HBA) and hemoglobin gamma (HBG)] and the heme oxygenase 1 (HMOX1) gene, in K562 cells. Next, to investigate the mechanism by which ALA is transported into erythroid cells, quantitative RT-PCR analysis was performed on previously identified ALA transporters, including solute carrier family 15 (oligopeptide transporter), member (SLC15A) 1, SLC15A2, solute carrier family 36 (proton/amino acid symporter), member (SLC36A1), and solute carrier family 6 (neurotransmitter transporter), member 13 (SLC6A13). Our analysis revealed that SLC36A1 was abundantly expressed in erythroid cells. Thus, gamma-aminobutyric acid (GABA) was added to K562 cells to competitively inhibit SLC36A1-mediated transport. GABA treatment significantly impeded the ALA-mediated increase in the number of hemoglobinized cells as well as the induction of HBG, HBA, and HMOX1. Finally, small-interfering RNA-mediated knockdown of ALAS2 in HiDEP cells considerably decreased the expression of HBA, HBG, and HMOX1, and these expression levels were rescued with ALA treatment. In summary, ALA appears to be transported into erythroid cells mainly by SLC36A1 and is utilized to generate heme. ALA may represent a novel therapeutic option for CSA treatment, particularly for cases harboring ALAS2 mutations. Copyright © 2014 Elsevier Inc. All rights reserved.

5-Aminolevulinic acid (5-ALA) has been widely used in photodynamic therapy and immunofluorescence of tumours. In the present study, the intestinal permeability and oral pharmacokinetics of 5-ALA were evaluated to probe the contribution of the proton-coupled oligopeptide transporter 1 (PEPT1) to the oral absorption and systemic exposure of this substrate.In situ single-pass intestinal perfusions and in vivo oral pharmacokinetic studies were performed in wildtype and Pept1 knockout mice. Perfusion studies were performed as a function of concentration dependence, specificity and permeability of 5-ALA in different intestinal segments. Pharmacokinetic studies were performed after 0.2 and 2.0 μmoL·g(-1) doses of 5-ALA.The permeability of 5-ALA was substantial in duodenal, jejunal and ileal regions of wildtype mice, but the residual permeability of 5-ALA in the small intestine from Pept1 knockout mice was only about 10% of that in wildtype animals. The permeability of 5-ALA in jejunum was specific for PEPT1 with no apparent contribution of other transporters, including the proton-coupled amino acid transporter 1 (PAT1). After oral dosing, the systemic exposure of 5-ALA was reduced by about twofold during PEPT1 ablation, and the pharmacokinetics were dose-proportional after the 0.2 and 2.0 µmol·g(-1) doses. PEPT1 had a minor effect on the disposition and peripheral tissue distribution of 5-ALA.Our findings suggested a major role of PEPT1 in the intestinal permeability and oral absorption of 5-ALA. In contrast, another proton-coupled transporter, PAT1, appeared to play a limited role, at best.© 2015 The British Pharmacological Society.

Folate metabolism can be an effective target for cancer treatment. However, standard cell culture conditions utilize folic acid, a non-physiological folate source for most tissues. We find that the enzyme that couples folate and methionine metabolic cycles, methionine synthase, is required for cancer cell proliferation and tumour growth when 5-methyl tetrahydrofolate (THF), the major folate found in circulation, is the extracellular folate source. In such physiological conditions, methionine synthase incorporates 5-methyl THF into the folate cycle to maintain intracellular levels of the folates needed for nucleotide production. 5-methyl THF can sustain intracellular folate metabolism in the absence of folic acid. Therefore, cells exposed to 5-methyl THF are more resistant to methotrexate, an antifolate drug that specifically blocks folic acid incorporation into the folate cycle. Together, these data argue that the environmental folate source has a profound effect on folate metabolism, determining how both folate cycle enzymes and antifolate drugs impact proliferation.© 2021. The Author(s), under exclusive licence to Springer Nature Limited.

The biosynthesis of B, involving up to 30 different enzyme-mediated steps, only occurs in bacteria. Thus, most eukaryotes require an external source of B, and yet the vitamin appears to have only two functions in eukaryotes: as a cofactor for the enzymes methionine synthase and methylmalonylCoA mutase. These two functions are crucial for normal health in humans, and in particular, the formation of methionine is essential for providing methyl groups for over 100 methylation processes. Interference with the methionine synthase reaction not only depletes the body of methyl groups but also leads to the accumulation of homocysteine, a risk factor for many diseases. The syndrome pernicious anemia, characterized by lack of intrinsic factor, leads to a severe, sometimes fatal form of B deficiency. However, there is no sharp cutoff for B deficiency; rather, there is a continuous inverse relationship between serum B and a variety of undesirable outcomes, including neural tube defects, stroke, and dementia. The brain is particularly vulnerable; in children, inadequate B stunts brain and intellectual development. Suboptimal B status (serum B<300pmol/L) is very common, occurring in 30%-60% of the population, in particular in pregnant women and in less-developed countries. Thus, many tens of millions of people in the world may suffer harm from having a poor B status. Public health steps are urgently needed to correct this inadequacy.© 2018 Elsevier Inc. All rights reserved.

Administration of cobaltic protoporphyrin IX (CoPP) into the third ventricle of the brain by intracerebroventricular injection in rodents is known to result in transient hypophagia and remarkably prolonged weight loss. The mechanism of action of CoPP in eliciting these effects is unknown. It is known that nitric oxide plays a role in food intake and that the hyperphagia that results from a wide variety of genetic, physiological, and pharmacological stimuli can be blocked by the administration of inhibitors of the enzyme nitric oxide synthase (NOS). We demonstrate that intracerebroventricular administration of compounds that alter nitrergic tone can also change food ingestion and weight gain patterns in normophagic rats. We also demonstrate that CoPP decreases NOS activity but that it paradoxically increases neuronal NOS transcript expression and increases neuronal NOS protein content on Western blotting.

Heme is a primordial macrocycle upon which most aerobic life on Earth depends. It is essential to the survival and health of nearly all cells, functioning as a prosthetic group for oxygen-carrying proteins and enzymes involved in oxidation/reduction and electron transport reactions. Heme is essential for the function of numerous hemoproteins and has numerous other roles in the biochemistry of life. In mammals, heme is synthesised from glycine, succinyl-CoA, and ferrous iron in a series of eight steps. The first and normally rate-controlling step is catalysed by 5-aminolevulinate synthase (ALAS), which has two forms: ALAS1 is the housekeeping form with highly variable expression, depending upon the supply of the end-product heme, which acts to repress its activity; ALAS2 is the erythroid form, which is regulated chiefly by the adequacy of iron for erythroid haemoglobin synthesis. Abnormalities in the several enzymes of the heme synthetic pathway, most of which are inherited partial enzyme deficiencies, give rise to rare diseases called porphyrias. The existence and role of heme importers and exporters in mammals have been debated. Recent evidence established the presence of heme transporters. Such transporters are important for the transfer of heme from mitochondria, where the penultimate and ultimate steps of heme synthesis occur, and for the transfer of heme from cytoplasm to other cellular organelles. Several chaperones of heme and iron are known and important for cell health. Heme and iron, although promoters of oxidative stress and potentially toxic, are essential cofactors for cellular energy production and oxygenation.© 2024 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Heme is an essential cofactor for numerous cellular functions, but release of free heme during hemolysis results in oxidative tissue damage, vascular dysfunction, and inflammation. Macrophages play a key protective role in heme clearance; however, the mechanisms that regulate metabolic adaptations that are required for effective heme degradation remain unclear. Here we demonstrate that heme loading drives a unique bioenergetic switch in macrophages, which involves a metabolic shift from oxidative phosphorylation toward glucose consumption. Metabolomic and transcriptional analysis of heme-loaded macrophages revealed that glucose is funneled into the pentose phosphate pathway (PPP), which is indispensable for efficient heme detoxification and is required to maintain redox homeostasis. We demonstrate that the metabolic shift to the PPP is controlled by heme oxygenase-dependent generation of carbon monoxide (CO). Finally, we show that PPP upregulation occurs in vivo in organ systems central to heme clearance and that PPP activity correlates with heme levels in mouse sickle cell disease (SCD). Together, our findings demonstrate that metabolic adaptation to heme detoxification in macrophages requires a shift to the PPP that is induced by heme-derived CO, suggesting pharmacologic targeting of macrophage metabolism as a novel therapeutic strategy to improve heme clearance in patients with hemolytic disorders.© 2020 by The American Society of Hematology.

The objective of this study was to investigate the effect of 5-aminolevulinic acid (5-ALA) as a dietary supplement on milk yield and composition as well as iron status and immune response in lactating dairy cows. In this study 13 lactating Holstein cows were randomly assigned to either a control group or a treatment group supplemented with 10 mg of 5-ALA per kilogram of dry matter. During feeding, 5-ALA was mixed with a small amount of the total mixed ration and top-dressed. The experiments followed a crossover design with 2 periods. Each period consisted of an adaptation period of 12 d and a test period of 2 d. Dairy cows fed the diet supplemented with 5-ALA exhibited increased counts of white blood cells and granulocytes compared with the control group. The rate of phagocytosis and mitogen-induced proliferation of peripheral blood mononuclear cells in cows fed 5-ALA were higher than in cows fed a basal diet. However, 5-ALA did not affect iron status or plasma biochemical composition. Supplementation with 5-ALA improved milk protein and milk casein contents; however, it had no effect on milk production, milk fat, lactose, total solids, or solids-not-fat, compared with the control. We conclude that dietary supplementation of 5-ALA to lactating dairy cows may have a positive effect on milk protein synthesis and the immune response.Copyright © 2019 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

This study was conducted to evaluate the effect of dietary administered delta-aminolevulinic acid (ALA) and vitamin C on the hematological profiles and performance of sows and sucking pigs and to determine if iron status could be improved by dietary administration of ALA. Seven days before parturition, 120 sows (Landrace x Yorkshire) were randomly assigned within parities to 1 of 4 dietary treatments. The lactation treatments were arranged as a 2 x 2 factorial with 2 ALA concentrations (0 or 10 mg/kg of feed, as fed) and 2 vitamin C concentrations (0 or 500 mg/kg of feed, as fed). After farrowing, daily feed allowance increased gradually, and sows had ad libitum access to feed by wk 2. Sows provided diets with ALA tended to consume more feed (P < 0.10) than sows fed diets that did not contain ALA. Conversely, backfat losses were less in sows that received the diets with ALA compared with those received the nonsupplemented diets (P < 0.05). Supplementation with ALA and vitamin C resulted in improved hematological profiles (total protein, albumin, and IgG) in sows (P < 0.05). The iron status in the blood and milk were also improved by ALA supplementation (P < 0.05). Moreover, the BW at weaning and the ADG during the sucking period increased in response to supplementation of ALA and vitamin C (P < 0.05). The blood iron status of piglets was also improved by ALA supplementation (P < 0.05). In conclusion, dietary supplementation of ALA can increase the blood iron status of sows and sucking pigs, as well as the iron transfer efficiency from sows to piglets through elevated milk iron concentrations.

The effect of by-products of oriental medicinal plants (OMP; T1) containing 0.03% herb extracts (T2) or 0.1% aminolevulinic acid (T3) on the production performance of swine during the finishing period and on its meat quality were investigated. No significant differences were found in the weight gain, feed intake and feed conversion rate among the tested groups (P > 0.05). But the treated group showed higher (P < 0.05) moisture and ash and lower protein than the control group. The T3 group showed a lower meat cholesterol content (38.42 mg/100 g) compared to the other groups (P < 0.05). The vitamin E content of the muscle in the treated groups was higher compared to the control group. No antibiotic content was detected in all treated and control samples. The values of the volatile basic nitrogen (VBN) and thiobarbituric acid reactive substance (TBARS) of the treated groups were significantly lower (P < 0.01) than the control group. The treated groups had significantly better (P < 0.05) sensory-test scores for color, flavor, off-flavor and total acceptability compared to the control group.© 2011 The Authors. Animal Science Journal © 2011 Japanese Society of Animal Science.

The objective of this study was to investigate the effect of dietary supplementation with 5-aminolevulinic acid (5-ALA) on the immune system, inflammatory response, and growth performance of broiler chickens. The levels of cluster of differentiation 3 (CD3) mRNA in the spleens of chickens gradually increased with dietary 5-ALA concentration, while the expression levels of interleukin (IL)-2 decreased. Mitogen-induced proliferation of splenic mononuclear cells and blood mononuclear cell phagocytosis in chickens fed 0.001 and 0.01% 5-ALA-supplemented diets were significantly greater than in chickens fed a basal diet (control). Plasma thiobarbituric acid reactive substance (TBARS) concentration gradually increased along with 5-ALA supplement concentration. These results provide the first evidence that the use of dietary 0.001 and 0.01% 5-ALA supplementation induces the T-cell immune system via mild oxidative stress in chickens. Three hours after Escherichia coli lipopolysaccharide-induced immune stimulation, the levels of mRNA encoding pro-inflammatory cytokines, such as IL-6 and tumor necrosis factor-like ligand 1A (TL1A), in chickens fed a 0.001% 5-ALA-supplemented diet were significantly lower than those in chickens exposed to other treatments. The plasma caeruloplasmin concentration in chickens fed a 0.001% 5-ALA-supplemented diet was significantly lower than in controls or in chickens fed diets supplemented with other concentrations of 5-ALA 24 h after injection of LPS. In addition, BW at 21 and 50 d of age was significantly higher in chickens fed a 0.001% 5-ALA-supplemented diet than in control chickens. The findings suggest that supplementation of diets with 0.001% 5-ALA could prevent the catabolic changes induced by immunological stimulation. These results show that 5-ALA might be useful as an immunomodulator to stimulate T-cells via mild oxidative stress in growing broiler chickens, thereby improving the growth performance.

本试验旨在探究饲粮中添加5-氨基乙酰丙酸(5-ALA)对肉鸡生长性能、血常规指标、免疫功能和肠道形态的影响。选取300只1日龄爱拔益加(AA)肉公鸡随机分成5组,每组6个重复,每个重复10只鸡。各组分别饲喂5-ALA添加水平为0(对照)、15、30、45和60 mg/kg的饲粮,试验期为42 d。结果表明:1)与对照组相比,饲粮中添加5-ALA对肉鸡平均日增重(ADG)和平均日采食量(ADFI)无显著影响(P&gt;0.05),但添加60 mg/kg的5-ALA能显著降低肉鸡料重比(F/G)(P&lt;0.05);且随饲粮中5-ALA添加水平的增加,F/G呈线性降低趋势(P&lt;0.05)。2)与对照组相比,饲粮中添加30、45和60 mg/kg的5-ALA能显著提高21日龄肉鸡血液中血红蛋白(HGB)含量;随饲粮中5-ALA添加水平的增加,21和42日龄肉鸡血液中HGB含量和红细胞数(RBC)呈线性升高趋势(P&lt;0.05)。3)与对照组相比,饲粮中添加45 mg/kg 5-ALA显著提高了21日龄时肉鸡血清中免疫球蛋白M(IgM)和免疫球蛋白G(IgG)含量(P&lt;0.05),饲粮中添加45和60 mg/kg 5-ALA显著提高了21日龄时肉鸡血清中补体3(C3)和补体4(C4)含量(P&lt;0.05);且21日龄肉鸡血清中C3和C4含量呈线性升高趋势(P&lt;0.05)。与对照组相比,饲粮中添加30和45 mg/kg 5-ALA显著提高了42日龄时肉鸡血清中免疫球蛋白A(IgA)、IgM和IgG含量(P&lt;0.05);饲粮中添加5-ALA显著提高了21日龄时肉鸡血清中干扰素-γ(INF-γ)含量(P&lt;0.05)。4)与对照组相比,饲粮中添加5-ALA显著提高了21日龄肉鸡肝脏鸡源Cathelicidins抗菌肽1(CATH1)、鸡源Cathelicidins抗菌肽2(CATH2)、鸡源Cathelicidins抗菌肽3(CATH3)、禽β-防御素1(AvBD1)、禽β-防御素4(AvBD4)和禽β-防御素7(AvBD7)的mRNA相对表达量(P&lt;0.05)。5)与对照组相比,饲粮中添加30和60 mg/kg 5-ALA显著提高回肠绒毛高度/隐窝深度比值(V/C)(P&lt;0.05);且随饲粮中5-ALA添加水平的增加,42日龄肉鸡十二指肠绒毛高度呈线性升高趋势(P&lt;0.05)。综上所述,饲粮中添加适宜水平的5-ALA能促进肉鸡HGB的形成,提高肉鸡的免疫功能,改善肠道形态结构。在本试验条件下,肉鸡饲粮中5-ALA的适宜添加水平为45~60 mg/kg。

5-aminolevulinic acid (ALA) modulates various defense systems in plants and confers abiotic stress tolerance. Enhancement of crop production is a challenge due to numerous abiotic stresses such as, salinity, drought, temperature, heavy metals, and UV. Plants often face one or more abiotic stresses in their life cycle because of the challenging growing environment which results in reduction of growth and yield. Diverse studies have been conducted to discern suitable mitigation strategies to enhance crop production by minimizing abiotic stress. Exogenous application of different plant growth regulators is a well-renowned approach to ameliorate adverse effects of abiotic stresses on crop plants. Among the numerous plant growth regulators, 5-aminolevulinic acid (ALA) is a novel plant growth regulator, also well-known to alleviate the injurious effects of abiotic stresses in plants. ALA enhances abiotic stress tolerance as well as growth and yield by regulating photosynthetic and antioxidant machineries and nutrient uptake in plants. However, the regulatory roles of ALA in plants under different stresses have not been studied and assembled systematically. Also, ALA-mediated abiotic stress tolerance mechanisms have not been fully elucidated yet. Therefore, this review discusses the role of ALA in crop growth enhancement as well as its ameliorative role in abiotic stress mitigation and also discusses the ALA-mediated abiotic stress tolerance mechanisms and its limitation and future promises for sustainable crop production.

Mitochondrial dysfunction is associated with type 2 diabetes mellitus (T2DM). 5-Aminolevulinic acid (ALA), a natural amino acid produced only in the mitochondria, is a precursor of heme. Cytochromes that contain heme play an important role in aerobic energy metabolism. Thus, ALA may help reduce T2DM-associated hyperglycemia. In this study, we investigated the effect of ALA combined with sodium ferrous citrate (SFC) on hyperglycemia in Zucker diabetic fatty (ZDF) rats. We found that the gavage administration of ALA combined with SFC (ALA/SFC) for 6 weeks reduced plasma glucose and hemoglobin A1c (HbA1c) levels in rats without affecting plasma insulin levels. The glucose-lowering effect depended on the amount of ALA/SFC administered per day. Furthermore, the glucose tolerance was also significantly improved by ALA/SFC administration. Although food intake was slightly reduced in the rats administered ALA/SFC, there was no effect on their body weight. Importantly, ALA/SFC administration induced heme oxygenase-1 (HO-1) expression in white adipose tissue and liver, and the induced expression levels of HO-1 correlated with the glucose-lowering effects of ALA/SFC. Taken together, these results suggest that ALA combined with ferrous ion is effective in reducing hyperglycemia of T2DM without affecting plasma insulin levels. HO-1 induction may be involved in the mechanisms underlying the glucose-lowering effect of ALA/SFC.

Recently, sarcopenia has attracted attention as therapeutic target because it constitutes a risk factor for metabolic and cardiovascular diseases. We focused 5-aminolevulinic acid (ALA) which act as electron carriers in the mitochondrial electron transport system. The mice that received ALA for 8 weeks gained muscle strength and endurance, and exhibited increased muscle mass and mitochondrial amount. Administration of ALA to sarcopenia mice aged 100 weeks and chronic kidney disease (CKD) model mice also increased muscle mass and improved physical performance. Metabolome analysis revealed increased branched-chain amino acids (BCAAs) levels in the skeletal muscle of ALA-treated mice. Quantitative PCR analysis revealed decreased expression levels in branched-chain amino acid transaminases (BCATs) that degrade BCAAs and other muscle-degrading factors, and increased levels of mitochondria-activating factors. We also studied in cultured myocytes and obtained compatible results. ALA-treated mice tended to increase body weight, but reduced blood glucose level. These suggested that ALA treatment not only activated muscle mitochondria but also enhanced muscle mass through an increase in BCAAs contents, as to improve muscle strength, endurance and glucose tolerance in mice. In these ways, muscle mitochondrial activation with ALA is suggested to be useful for the treatment of sarcopenia and glucose intolerance.

Alpha-thalassemia X-linked intellectual disability (ATR-X) syndrome is caused by mutations in ATRX, which encodes a chromatin-remodeling protein. Genome-wide analyses in mouse and human cells indicate that ATRX tends to bind to G-rich sequences with a high potential to form G-quadruplexes. Here, we report that Atrx mutation induces aberrant upregulation of Xlr3b expression in the mouse brain, an outcome associated with neuronal pathogenesis displayed by ATR-X model mice. We show that ATRX normally binds to G-quadruplexes in CpG islands of the imprinted Xlr3b gene, regulating its expression by recruiting DNA methyltransferases. Xlr3b binds to dendritic mRNAs, and its overexpression inhibits dendritic transport of the mRNA encoding CaMKII-α, promoting synaptic dysfunction. Notably, treatment with 5-ALA, which is converted into G-quadruplex-binding metabolites, reduces RNA polymerase II recruitment and represses Xlr3b transcription in ATR-X model mice. 5-ALA treatment also rescues decreased synaptic plasticity and cognitive deficits seen in ATR-X model mice. Our findings suggest a potential therapeutic strategy to target G-quadruplexes and decrease cognitive impairment associated with ATR-X syndrome.

Oxidative stress is associated with the progression of the neurodegenerative diseases Parkinson's disease (PD) and cerebral ischemia. Recently, 5-aminolevulinic acid (5-ALA), an intermediate in the porphyrin synthesis pathway, was reported to exert antioxidative effects on macrophages and cardiomyocytes. Here, we demonstrated the neuroprotective effects of 5-ALA using rat models of PD and ischemia as well as in vitro in SH-SY5Y cells. 5-ALA partially prevented neurodegeneration in each condition. These results suggest that 5-ALA has a potential for promising therapeutic agent to protect against neurodegeneration exacerbated by oxidative stress.Copyright © 2020 The Authors. Production and hosting by Elsevier B.V. All rights reserved.

To explore whether 5-Aminolevulinic acid combined with ferrous iron (5-ALA/Fe) could protect testicular tissues damage of mice subjected to heat stress (HS) and provide its underlying mechanisms.5-ALA/Fe was administered intragastrically to mice for 10 days, then exposed to a scrotal heat stress at 43°C for 20 min on third day. Testes were harvested for morphologic and histopathological examination, oxidative stress, apoptosis, heme oxygenase-1 (HO-1) and inflammation detection. The mitogen-activated protein kinases (MAPK) signaling pathway in testis and CD4FoxP3regulatory T (Treg) cells in spleen were also investigated.Compared to control group, the testis weight decreased and histological damage severed in HS group. Besides, HS also increased the oxidative stress, apoptosis and inflammation in testis. However, these indicators were ameliorated after 5-ALA/Fe treatment but deteriorated after receiving ZnPPIX. The expression of HO-1 was increased both in HS group and 5-ALA/Fe group. The protein expression levels of MAPK proteins were activated by HS and inhibited by 5-ALA/Fe. The CD4FoxP3 Treg generation was reduced by HS and increased by 5-ALA/Fe.In this study, we have demonstrated that 5-ALA/Fe ameliorated the spermatogenic damage induced by scrotal heat stress via up-regulating the expression of HO-1 and inhibiting MAPK mediated oxidative stress and apoptosis and inducing CD4Foxp3 Tregs to inhibit the inflammation induced by HS in mice.© 2023. The Author(s), under exclusive licence to Springer Nature B.V.