Lysine Metabolism Research Articles

Effects of chronic hypoglycemia and euglycemic correction on lysine metabolism in fetal sheep

In this study, we determined rates of lysine metabolism in fetal sheep during chronic hypoglycemia and following euglycemic recovery and compared results with normal, age-matched euglycemic control fetuses to explain the adaptive response of protein metabolism to low glucose concentrations. Restriction of the maternal glucose supply to the fetus lowered the net rates of fetal (umbilical) glucose (42%) and lactate (36%) uptake, causing compensatory alterations in fetal lysine metabolism. The plasma lysine concentration was 1.9-fold greater in hypoglycemic compared with control fetuses, but the rate of fetal (umbilical) lysine uptake was not different. In the hypoglycemic fetuses, the lysine disposal rate also was higher than in control fetuses due to greater rates of lysine flux back into the placenta and into fetal tissue. The rate of CO2 excretion from lysine decarboxylation was 2.4-fold higher in hypoglycemic than control fetuses, indicating greater rates of lysine oxidative metabolism during chronic hypoglycemia. No differences were detected for rates of fetal protein accretion or synthesis between hypoglycemic and control groups, although there was a significant increase in the rate of protein breakdown (P<0.05) in the hypoglycemic fetuses, indicating small changes in each rate. This was supported by elevated muscle specific ubiquitin ligases and greater concentrations of 4E-BP1. Euglycemic recovery after chronic hypoglycemia normalized all fluxes and actually lowered the rate of lysine decarboxylation compared with control fetuses (P<0.05). These results indicate that chronic hypoglycemia increases net protein breakdown and lysine oxidative metabolism, both of which contribute to slower rates of fetal growth over time. Furthermore, euglycemic correction for 5 days returns lysine fluxes to normal and causes an overcorrection of lysine oxidation.

Histoplasma capsulatum proteome response to decreased iron availability

BackgroundA fundamental pathogenic feature of the fungus Histoplasma capsulatum is its ability to evade innate and adaptive immune defenses. Once ingested by macrophages the organism is faced with several hostile environmental conditions including iron limitation. H. capsulatum can establish a persistent state within the macrophage. A gap in knowledge exists because the identities and number of proteins regulated by the organism under host conditions has yet to be defined. Lack of such knowledge is an important problem because until these proteins are identified it is unlikely that they can be targeted as new and innovative treatment for histoplasmosis.ResultsTo investigate the proteomic response by H. capsulatum to decreasing iron availability we have created H. capsulatum protein/genomic databases compatible with current mass spectrometric (MS) search engines. Databases were assembled from the H. capsulatum G217B strain genome using gene prediction programs and expressed sequence tag (EST) libraries. Searching these databases with MS data generated from two dimensional (2D) in-gel digestions of proteins resulted in over 50% more proteins identified compared to searching the publicly available fungal databases alone. Using 2D gel electrophoresis combined with statistical analysis we discovered 42 H. capsulatum proteins whose abundance was significantly modulated when iron concentrations were lowered. Altered proteins were identified by mass spectrometry and database searching to be involved in glycolysis, the tricarboxylic acid cycle, lysine metabolism, protein synthesis, and one protein sequence whose function was unknown.ConclusionWe have created a bioinformatics platform for H. capsulatum and demonstrated the utility of a proteomic approach by identifying a shift in metabolism the organism utilizes to cope with the hostile conditions provided by the host. We have shown that enzyme transcripts regulated by other fungal pathogens in response to lowering iron availability are also regulated in H. capsulatum at the protein level. We also identified H. capsulatum proteins sensitive to iron level reductions which have yet to be connected to iron availability in other pathogens. These data also indicate the complexity of the response by H. capsulatum to nutritional deprivation. Finally, we demonstrate the importance of a strain specific gene/protein database for H. capsulatum proteomic analysis.

The effect of dietary lysine and genetics on lysine metabolism and growth in rainbow trout

Since feed cost represents about 70% of production cost, inexpensive protein alternatives are desirable replacements for fish meal. One drawback to the replacement of fish meal with soybean meal is that the latter protein source is first limiting in lysine. To investigate if different genetic lines of fish would respond differently to a lysine‐deficient (LD) diet, five families with high feed efficiency and 5 with low feed efficiency were studied. These 10 families were fed a LD, soybean meal containing, diet. The highest and lowest feed efficiency families also received a lysine‐adequate (LA) diet as a positive control. For each diet x family treatment combination, 5 replicates were used. Feed efficiency and thermal growth coefficient were greater (P&lt;0.05) while hepatic somatic index (HSI) and ammonia excretion were lower (P&lt;0.05) for fish fed the LA diet. There was a family effect on feed efficiency (P&lt;0.0001) and thermal growth coefficient (P&lt;0.0001), but not ammonia excretion, HSI, or indices of lysine catabolism. A single family had a numerically more favorable feed efficiency and thermal growth coefficient, when fed the LD diet, indicating that it may possess an enhanced genetic potential for performance when consuming lysine‐deficient, soybean meal containing diets.Support: USDA/CSREES, WV‐AFMPD

Early Detection of Glutaric Aciduria Type I by Newborn Screening in Taiwan

Glutaric aciduria type 1 (GA1) is an inborn error of lysine and tryptophan metabolism. There is a lack of initial diagnostic signs of the disease, but late treatment often results in severe neurologic impairment. In this study, we analyzed the results of screening for GA1 in a Chinese population. Dry blood spots were obtained at about 3 days of age from 357,307 newborns and tested for elevation of glutaryl (C5DC)-carnitine by tandem mass spectroscopy. A second sample of blood spots was required from those cases with abnormal elevation of C5DC-carnitine (higher than the cut-off value) (recall). If the results remained abnormal, those cases were referred for confirmation of the diagnosis and treatment. Between August 2001 and February 2005, there were 40 cases with C5DC-carnitine more than 0.13 microM (the cut-off value), from whom a second sample of blood spots was obtained (recall rate, 0.02%); two cases were confirmed to be affected by GA1. Because of the low positive prediction rate using this cut-off value, we elevated the cut-off value slightly. Between February 2005 and August 2006, there were eight cases with C5DC-carnitine more than 0.22 microM from whom a second sample of blood spots was obtained (recall rate, 0.01%); three cases were confirmed to be affected by GA1. All five cases with persistent elevation of C5DC-carnitine were referred and diagnosis was confirmed in each, giving an incidence of 1 in 71,461 newborns. There were no false negatives. Magnetic resonance imaging studies obtained from four cases showed frontotemporal atrophy at the time of diagnosis. Two cases were followed for over 1 year, and under treatment with dietary control and carnitine supplementation, both had normal development and neither exhibited a frank episode of encephalopathic crisis. With properly established cut-offs, GA1 can be successfully screened for in populations with a low incidence of the disease. Early treatment is likely to improve the outcome of cases discovered by screening.

Mechanism of age-dependent susceptibility and novel treatment strategy in glutaric acidemia type I

Glutaric acidemia type I (GA-I) is an inherited disorder of lysine and tryptophan metabolism presenting with striatal lesions anatomically and symptomatically similar to Huntington disease. Affected children commonly suffer acute brain injury in the context of a catabolic state associated with nonspecific illness. The mechanisms underlying injury and age-dependent susceptibility have been unknown, and lack of a diagnostic marker heralding brain injury has impeded intervention efforts. Using a mouse model of GA-I, we show that pathologic events began in the neuronal compartment while enhanced lysine accumulation in the immature brain allowed increased glutaric acid production resulting in age-dependent injury. Glutamate and GABA depletion correlated with brain glutaric acid accumulation and could be monitored in vivo by proton nuclear magnetic resonance (1H NMR) spectroscopy as a diagnostic marker. Blocking brain lysine uptake reduced glutaric acid levels and brain injury. These findings provide what we believe are new monitoring and treatment strategies that may translate for use in human GA-I.

Dihydrodipicolinate synthase in opaque and floury maize mutants

Dihydrodipicolinate synthase (DHDPS, EC 4.2.1.52) was isolated and studied in four high-lysine maize mutants (Oh43 o1, Oh43 o2, Oh43 fl1 and Oh43 fl2). The activity of DHDPS was analyzed at 16, 20, and 24 DAP and characterized in the presence of the amino acids, lysine, S-(2-aminoethyl)- l-cysteine (AEC), S-adenosylmethionine (SAM) and calcium. The results indicated that DHDPS was strongly inhibited by lysine, and that there was little variation between the mutants, indicating that lysine accumulation in these mutants may be more dependent on other enzymes involved in lysine metabolism. The higher concentrations of lysine observed in the seeds of the mutants at maturity may be explained by the accumulation of soluble lysine caused by a reduction in lysine degradation, or by changes in the distribution of high lysine containing storage proteins.

High‐lysine corn generated by endosperm‐specific suppression of lysine catabolism using RNAi

Because of the limited lysine content in corn grain, synthetic lysine supplements are added to corn meal-based rations for animal feed. The development of biotechnology, combined with the understanding of plant lysine metabolism, provides an alternative solution for increasing corn lysine content through genetic engineering. Here, we report that by suppressing lysine catabolism, transgenic maize kernels accumulated a significant amount of lysine. This was achieved by RNA interference (RNAi) through the endosperm-specific expression of an inverted-repeat (IR) sequence targeting the maize bifunctional lysine degradation enzyme, lysine-ketoglutarate reductase/saccharopine dehydrogenase (ZLKR/SDH). Although plant-short interfering RNA (siRNA) were reported to lack tissue specificity due to systemic spreading, we confirmed that the suppression of ZLKR/SDH in developing transgenic kernels was restricted to endosperm tissue. Furthermore, results from our cloning and sequencing of siRNA suggested the absence of transitive RNAi. These results support the practical use of RNAi for plant genetic engineering to specifically target gene suppression in desired tissues without eliciting systemic spreading and the transitive nature of plant RNAi silencing.

Nutritional Consequences of Interspecies Differences in Arginine and Lysine Metabolism

Differences in lysine and arginine requirements among various species such as omnivores (humans, pigs, rats, dogs), carnivores (cats), herbivores (rabbits, horses), ruminants (cattle), poultry, and fish, are covered in detail in this article. Although lysine is classified as an indispensable amino acid across species, the classification of arginine as either an indispensable or dispensable amino acid is more ambiguous because of differences among species in rates of de novo arginine synthesis. Because lysine is most often the limiting amino acid in the diet, its requirement has been extensively studied. By use of the ideal protein concept, the requirements of the other indispensable amino acids can be extrapolated from the lysine requirement. The successful use of this concept in pigs is compared with potential application of the ideal protein concept in humans. The current dietary arginine requirement varies widely among species, with ruminants, rabbits, and rats having relatively low requirements and carnivores, fish, and poultry having high requirements. Interspecies differences in metabolic arginine utilization and reasons for different rates of de novo arginine synthesis are reviewed in detail, as these are the primary determinants of the dietary arginine requirement. There is presently no dietary requirement for humans of any age, although this needs to be reassessed, particularly in neonates. A thorough understanding of the factors contributing to the lysine and arginine requirements in different species will be useful in our understanding of human amino acid requirements.

>Unique Aspects of Lysine Nutrition and Metabolism

Lysine nutrition is unique among indispensable amino acids in that it can be conserved and can be fed 12 h out of phase (delayed supplement) with the other dietary amino acids. In piglets, high levels (2–6%) of l-lysine added to a 10% protein diet can be tolerated without obvious detrimental effects. In both rat and piglet liver preparations, the first enzyme in the saccharopine-dependent pathway of lysine catabolism, lysine α-ketoglutarate reductase (LKR), is found only in the mitochondrial matrix. For Lys catabolism to occur, Lys must first enter the matrix of the mitochondrion. LKR, saccharopine dehydrogenase, mitochondrial lysine uptake, and lysine oxidation (LOX) all increased >3-fold in rats fed high levels of dietary protein (up to 60%). The activities of mitochondrial Lys uptake and LOX were similar when expressed as mmol/(d · 100 g body weight). Thus, LOX can be a proxy for mitochondrial Lys uptake. Piglet liver LKR and LOX increase 5- to 10-fold when piglets are fed high-protein (50 or 75%) diets. In both the rat and piglet, after adapting to the high protein diet, the activity of LKR is 400–500 times that of LOX, suggesting that Lys uptake by a transporter(s) is rate limiting. Quantitative 24-h dietary infusion studies in piglets revealed that >80% of the Lys infused (4% of the diet) could not be recovered in the urine or body or accounted for by calculated Lys oxidation based on liver activity of LOX. Other pathways and tissues may account for the Lys oxidation in piglets.

Biomarkers Identified in Inborn Errors for Lysine, Arginine, and Ornithine

Inborn errors of lysine, arginine, and ornithine metabolism are very rare: only a few patients affected with these disorders have been carefully investigated, and very few reports on long-term outcome are available. These rare data make it difficult to define safety limits of these amino acids and useful biomarkers from these disorders. Only 4 disorders give rise to an important increase of the plasma amino acid concentration proximal to the metabolic block: lysine in 2-aminoadipic semialdehyde synthase deficiency, arginine in arginase deficiency, ornithine in ornithine amino transferase deficiency, and hyperammonemia hyperornithinemia homocitrullinuria syndrome. There is an obvious discrepancy between the important physiological role of these amino acids in cell metabolism and nutrition and the clinical consequences that are actually observed in these disorders.

Pyrroloquinoline quinone nutritional status and parameters important to mitochondrial function

Pyrroloquinoline quinone (PQQ) deficiency influences the mitochondrial content of heart and liver (Stites T et al., J Nutr., 2006) and impacts the metabolism of lysine and other amino acids oxidized via mitochondrial and peroxisomal pathways (Bauerly K et al., Biochim Biophys Acta, 2006). Because of mitochondria’s central role in metabolism, we investigated the effect of PQQ deficiency on mitochondriogenesis. Herein are data for serum glucose and fatty acids (FA) in rats fed amino-acid based diets with (2 mg PQQ/Kg diet) or devoid of PQQ for 3–5 weeks in post-weanling rats (fed and/or fasted states). Serum glucose, free fatty acids (FFA), RQ, triglycerides (TG), sphingomyelin (SM), total energy expenditure (EE) per metabolic body size (Wt3/4), relative mitochondrial amount, and expression of serine palmitoyl transferase (SPT) were estimated. Mitochondria content decreased 20% in PQQ deficient rats. Although no differences in serum glucose and FFA levels were observed (PQQ+ vs. PQQ−, fed or fasted), EE was significantly lower in PQQ deficiency. In the fed state, EE was positively correlated with mitochondrial amount and less in PQQ deficiency. Composition of FFA, TG, and SM were altered with PQQ deficiency, suggesting disturbances in long-chain FA metabolic pathways; serum TG were elevated while serum SM tended to be lower with corresponding decreases in liver SPT activity and mRNA levels in deficient rats. These data are consistent with our previous observations regarding PQQ status, mitochondrial function and associated activities. Funded in part by Mitsubishi, Inc., CA Vitamin Settlement Fund, and NDC.

In Vivo 15N-Enrichment of Metabolites in Suspension Cultured Cells and Its Application to Metabolomics

The incorporation of stable isotopes in suspension cultured cells is very simple and useful as a preliminary experimental method in the experimental scene of plant metabolomics to elucidate the metabolic profiles of mutants and transformants. Stable isotope methods would afford a dynamic explanation of turnover speed that would concern the metabolic flux. Utilization of suspension cultured cells allows genes to be easily induced or suppressed, culture conditions to be controlled, and samples to be easily prepared. Stable isotope tracing allows an index of metabolic flux to be obtained. Here we present an experiment feeding (15)N-labeled inorganic salts to Arabidopsis (cell line T87) and Coptis cultured cells. Results of a comparison of (15)N labeling ratios of amino acids derived from T87 cells cultured under light with those cultured in the dark corresponded to transcriptional expressions revealed by microarray experiments published previously, demonstrating the validity of this procedure. Furthermore, (15)N labeling ratios of Coptis cultured cells revealed arginine and lysine metabolism inhibition, which should result in inhibition of polyamine biosynthesis and cell division. This very simple experiment allowed us to uncover metabolic dynamic features of the plant cell. Therefore this method is very useful for forming working hypotheses and experimental design.

Pyrroloquinoline quinone nutritional status alters lysine metabolism and modulates mitochondrial DNA content in the mouse and rat

Pyrroloquinoline quinone (PQQ) added to purified diets devoid of PQQ improves indices of perinatal development in rats and mice. Herein, PQQ nutritional status and lysine metabolism are described, prompted by a report that PQQ functions as a vitamin-like enzymatic cofactor important in lysine metabolism (Nature 422 [2003] 832). Alternatively, we propose that PQQ influences lysine metabolism, but by mechanisms that more likely involve changes in mitochondrial content. PQQ deprivation in both rats and mice resulted in a decrease in mitochondrial content. In rats, α-aminoadipic acid (αAA), which is derived from α-aminoadipic semialdehyde (αAAS) and made from lysine in mitochondria, and the plasma levels of amino acids known to be oxidized in mitochondria (e.g., Thr, Ser, and Gly) were correlated with changes in the liver mitochondrial content of PQQ-deprived rats, but not PQQ-supplemented rats. In contrast, the levels of NAD dependent α-aminoadipate-δ-semialdehyde dehydrogenase (AASDH), a cytosolic enzyme important to αAA production from αAAS, was not influenced by PQQ dietary status. Moreover, the levels of U26 mRNA were not significantly changed even when diets differed markedly in PQQ and dietary lysine content. U26 mRNA levels were measured, because of U26's proposed, albeit questionable role as a PQQ-dependent enzyme involved in αAA formation.

Glutaric acidemia type 1

Glutaric acidemias comprise different disorders resulting in an increased urinary excretion of glutaric acid. Glutaric acidemia type 1 (GA-1) is an autosomal recessive disorder of lysine, hydroxylysine, and tryptophan metabolism caused by deficiency of glutaryl-CoA dehydrogenase. It results in the accumulation of 3-hydroxyglutaric and glutaric acid. Affected patients can present with brain atrophy and macrocephaly and with acute dystonia secondary to striatal degeneration in most cases triggered by an intercurrent childhood infection with fever between 6 and 18 months of age. This disorder can be identified by increased glutaryl (C5DC) carnitine on newborn screening. Urine organic acid analysis indicates the presence of excess 3-OH-glutaric acid, and urine acylcarnitine profile shows glutaryl carnitine as the major peak. Therapy consists in carnitine supplementation to remove glutaric acid, a diet restricted in amino acids capable of producing glutaric acid, and prompt treatment of intercurrent illnesses. Early diagnosis and therapy reduce the risk of acute dystonia in patients with GA-1.

Ss-LrpB from Sulfolobus solfataricus Condenses about 100 Base Pairs of Its Own Operator DNA into Globular Nucleoprotein Complexes

Ss-LrpB from the hyperthermoacidophilic crenarchaeote Sulfolobus solfataricus P2 is a member of the Lrp-like family of Bacterial/Archaeal transcription regulators that binds its own control region at three regularly spaced and partially conserved 15-bp-long imperfect palindromes. We have used atomic force microscopy to analyze the architecture of Ss-LrpB.DNA complexes with a different stoichiometry formed with the wild type operator and with an operator mutant. Binding of dimeric Ss-LrpB to all three target sites is accompanied by the formation of globular complexes, in which the protein induces strong DNA deformations. Furthermore, DNA contour length foreshortening of these complexes indicates DNA wrapping, with about 100 bp being condensed. The average bending angle is 260 degrees . The establishment of protein-protein contacts between Ss-LrpB dimers in these globular complexes will contribute to the cooperativity of the binding. The profound remodeling of the control region is expected to have a strong impact on gene expression and might constitute the key element in the autoregulatory process.

Lysine catabolism, an effective versatile regulator of lysine level in plants

Lysine is a nutritionally important essential amino acid, whose synthesis in plants is strongly regulated by the rate of its synthesis. Yet, lysine level in plants is also finely controlled by a super-regulated catabolic pathway that catabolizes lysine into glutamate and acetyl Co-A. The first two enzymes of lysine catabolism are synthesized from a single LKR/SDH gene. Expression of this gene is subject to compound developmental, hormonal and stress-associated regulation. Moreover, the LKR/SDH gene of different plant species encodes up to three distinct polypeptides: (i) a bifunctional enzyme containing the linked lysine-ketoglutarate (LKR) and saccharopine dehydrogenase (SDH) whose LKR activity is regulated by its linked SDH enzyme; (ii) a monofunctional SDH encoded by an internal promoter, which is a part of the coding DNA region of the LKR/SDH gene; and (iii) a monofunctional, highly potent LKR that is formed by polyadenylation within an intron. LKR activity in the bifunctional LKR/SDH polypeptide is also post-translationally regulated by phosphorylation by casein kinase-2 (CK2), but the consequence of this regulation is still unknown. Why is lysine metabolism super-regulated by synthesis and catabolism? A hypothesis addressing this important question is presented, suggesting that lysine may serve as a regulator of plant growth and interaction with the environment.

The effect of controlled fermentation on the fate of synthetic lysine in liquid diets for pigs

Crystalline lysine is frequently added to pig diets to provide the correct amino acid balance. Recent studies have shown that free lysine can be lost when liquid pig feed is fermented with lactic acid bacteria (LAB). The metabolism of lysine was examined, over 72 h, in uninoculated liquid feed (Control) and in liquid feed inoculated with lactic acid bacteria (LAB) ( Lactobacillus plantarum and Pediococcus acidilactici); or Escherichia coli and combinations of LAB and E. coli. In the Control and the fermentations that included E. coli, lysine was rapidly metabolised, with ca. 90% degraded in 21 h. The production of cadaverine accounted for ca. half of the lysine lost. There was no loss of lysine in the LAB fermentations and no cadaverine was produced. Combining E. coli and LAB resulted in a loss of ca. 20% lysine in the first 7 h with no further metabolism thereafter. Cadaverine produced in these fermentations accounted for all of the lysine lost. The rate of lysine metabolism was inhibited by the increase in lactic acid present in the fermentations. Fermenting E. coli and LAB with the addition of 50 mM lactic acid at 0 h resulted in the lysine levels remaining unaltered throughout the fermentation period. These results imply that the loss of lysine from fermented liquid pig diets is due to the metabolism of lysine by E. coli before they are eliminated from the diet by the increasing acidity resulting from LAB fermentation.

Saccharopine Dehydrogenase Activity in the High-Lysine Opaque and Floury Maize Mutants

Lysine is an essential amino acid normally present in very low concentration in cereal seeds. In previous reports we have studied the metabolism of lysine in several distinct high-lysine maize mutants and observed drastic variations in the activity of saccharopine dehydrogenase (SDH), a key enzyme involved in lysine degradation. We have now analyzed the activity of SDH using non-denaturing polyacrylamide gel electrophoresis (PAGE) to identify possible isoenzymes that could explain the patterns of activity previously observed. The results indicated the presence of at least two SDH isoenzymes, one contributing to approximately 90% of the total enzyme activity and a minor form only present in the wild type lines and the opaque-1 mutant. The results suggest that the differences in total SDH activity among the genotypes tested are due to alterations in the predominant SDH isoenzymic form, which is likely to be the bifunctional polypeptide containing lysine 2-oxoglutarate reductase.

Isolation and Characterization of Enzymes Involved in Lysine Catabolism from Sorghum Seeds

Lysine is an essential amino acid synthesized in plants via the aspartic acid pathway. The catabolism of lysine is performed by the action of two consecutive enzymes, lysine 2-oxoglutarate reductase (LOR, EC 1.5.1.8) and saccharopine dehydrogenase (SDH, EC 1.5.1.9). The final soluble lysine concentration in cereal seeds is controlled by both synthesis and catabolism rates. The production and characterization of high-lysine plants species depends on knowledge of the regulatory aspects of lysine metabolism and manipulation of the key enzymes. We have for the first time isolated, partially purified, and characterized LOR and SDH from developing sorghum seeds, which exhibited low levels of activity. LOR and SDH were only located in the endosperm and were very unstable during the isolation and purification procedures. LOR and SDH exhibited some distinct properties when compared to the enzymes isolated from other plant species, including a low salt concentration required to elute the enzymes during anion-exchange chromatography and the presence of multimeric forms with distinct molecular masses.

Is pyrroloquinoline quinone a vitamin?

The announcement by Kasahara and Kato of pyrroloquinoline quinone (PQQ) as a 'new' vitamin has received considerable attention. We have since attempted to reproduce the findings on which their conclusion is based, namely that defects in lysine metabolism occur in PQQ-deprived rodents. However, we find that the activity of alpha-aminoadipic acid-delta-semialdehyde (AAS) dehydrogenase in liver and plasma levels of alpha-aminoadipic acid (AAA), both of which act as indicators of lysine degradation in mammals, are not affected by changes in PQQ dietary status. Our results call into question the identification of PQQ as a new vitamin.