Lysine catabolism reprograms tumour immunity through histone crotonylation.

Cancer cells rewire metabolism to favour the generation of specialized metabolites that support tumour growth and reshape the tumour microenvironment1,2. Lysine functions as a biosynthetic molecule, energy source and antioxidant3-5, but little is known about its pathological role in cancer. Here we show that glioblastoma stem cells (GSCs) reprogram lysine catabolism through the upregulation of lysine transporter SLC7A2 and crotonyl-coenzyme A (crotonyl-CoA)-producing enzyme glutaryl-CoA dehydrogenase (GCDH) with downregulation of the crotonyl-CoA hydratase enoyl-CoA hydratase short chain 1 (ECHS1), leading to accumulation of intracellular crotonyl-CoA and histone H4 lysine crotonylation. A reduction in histone lysine crotonylation by either genetic manipulation or lysine restriction impaired tumour growth. In the nucleus, GCDH interacts with the crotonyltransferase CBP to promote histone lysine crotonylation. Loss of histone lysine crotonylation promotes immunogenic cytosolic double-stranded RNA (dsRNA) and dsDNA generation through enhanced H3K27ac, which stimulates the RNA sensor MDA5 and DNA sensor cyclic GMP-AMP synthase (cGAS) to boost type I interferon signalling, leading to compromised GSC tumorigenic potential and elevated CD8+ T cell infiltration. A lysine-restricted diet synergized with MYC inhibition or anti-PD-1 therapy to slow tumour growth. Collectively, GSCs co-opt lysine uptake and degradation to shunt the production of crotonyl-CoA, remodelling the chromatin landscape to evade interferon-induced intrinsic effects on GSC maintenance and extrinsic effects on immune response.

Dietary lysine restriction induces lipid accumulation in skeletal muscle through an increase in serum threonine levels in rats.

We previously reported that dietary amino acid restriction induces the accumulation of triglycerides (TAG) in the liver of growing rats. However, differences in TAG accumulation in individual cell types or other tissues were not examined. In this study, we show that TAG also accumulates in the muscle and adipose tissues of rats fed a low amino acid (low-AA) diet. In addition, dietary lysine restriction (low-Lys) induces lipid accumulation in muscle and adipose tissues. In adjusting the nitrogen content to that of the control diet, we found that glutamic acid supplementation to the low-AA diet blocked lipid accumulation, but supplementation with the low-Lys diet did not, suggesting that a shortage of nitrogen caused lipids to accumulate in the skeletal muscle in the rats fed a low-AA diet. Serum amino acid measurement revealed that, in rats fed a low-Lys diet, serum lysine levels were decreased, while serum threonine levels were significantly increased compared with the control rats. When the threonine content was restricted in the low-Lys diet, TAG accumulation induced by the low-Lys diet was completely abolished in skeletal muscle. Moreover, in L6 myotubes cultured in medium containing high threonine and low lysine, fatty acid uptake was enhanced compared with that in cells cultured in control medium. These findings suggest that the increased serum threonine in rats fed a low-Lys diet resulted in lipid incorporation into skeletal muscle, leading to the formation of fatty muscle tissue. Collectively, we propose conceptual hypothesis that "amino-acid signal" based on lysine and threonine regulates lipid metabolism.

Consensus guidelines for the diagnosis and management of pyridoxine-dependent epilepsy due to α-aminoadipic semialdehyde dehydrogenase deficiency.

Pyridoxine-dependent epilepsy (PDE-ALDH7A1) is an autosomal recessive condition due to a deficiency of α-aminoadipic semialdehyde dehydrogenase, which is a key enzyme in lysine oxidation. PDE-ALDH7A1 is a developmental and epileptic encephalopathy that was historically and empirically treated with pharmacologic doses of pyridoxine. Despite adequate seizure control, most patients with PDE-ALDH7A1 were reported to have developmental delay and intellectual disability. To improve outcome, a lysine-restricted diet and competitive inhibition of lysine transport through the use of pharmacologic doses of arginine have been recommended as an adjunct therapy. These lysine-reduction therapies have resulted in improved biochemical parameters and cognitive development in many but not all patients. The goal of these consensus guidelines is to re-evaluate and update the two previously published recommendations for diagnosis, treatment, and follow-up of patients with PDE-ALDH7A1. Members of the International PDE Consortium initiated evidence and consensus-based process to review previous recommendations, new research findings, and relevant clinical aspects of PDE-ALDH7A1. The guideline development group included pediatric neurologists, biochemical geneticists, clinical geneticists, laboratory scientists, and metabolic dieticians representing 29 institutions from 16 countries. Consensus guidelines for the diagnosis and management of patients with PDE-ALDH7A1 are provided.

Clinical and biochemical outcome of a patient with pyridoxine-dependent epilepsy treated by triple therapy (pyridoxine supplementation, lysine-restricted diet, and arginine supplementation).

Pyridoxine-dependent epilepsy (PDE) is a recessive genetic disease characterized by epileptic encephalopathy with therapeutic response to pharmacological doses of pyridoxine and resistance to anti-epileptic treatments. The recent discovery in 2006 of the genetic defect antiquitin (ALDH7A1, OMIM #266100) has helped to understand the underlying mechanism, which is the accumulation of neurotoxic intermediates in the lysine catabolic pathway. The goal of the new therapeutic approach, termed triple therapy (TT) (pyridoxine, lysine-restricted diet and arginine supplementation), is to improve epilepsy control and neurocognitive development in patients with PDE. We present the 3-year treatment outcome for a child with PDE on pyridoxine treatment (started at age 5 months), lysine-restricted diet (started at age 17 months) and arginine supplementation therapy (started at age 19 months). The TT was well-tolerated with good compliance. No adverse events were reported. We observed a neurodevelopmental improvement, significantly fewer seizures, and a reduction of pipecolic acid (PA) as a biomarker of the illness. Our results show an improving clinical evolution, supporting and extending previous studies reporting efficacy of TT.

Lysine is required for growth factor-induced mTORC1 activation.

Mechanistic target of rapamycincomplex 1 (mTORC1) integrates various environmental signals to regulate cell growth and metabolism. mTORC1 activity is sensitive to changes in amino acid levels. Here, we investigated the effect of lysine on mTORC1 activity in non-small cell lung cancer (NSCLC) cells. Lysine deprivation suppressed mTORC1 activity and lysine replenishment restored the decreased mTORC1 activity in lysine-deprived cells. Supplementing growth factors, such as insulin growth factor-1 or insulin restored the decreased mTORC1 activity in serum-deprived cells. However, in serum/lysine-deprived cells, supplementing growth factors was not sufficient to restore mTORC1 activity, suggesting thatgrowth factors could not activate mTORC1 efficiently in the absence of lysine. General control nonderepressible 2 and AMP-activated protein kinase were involved in lysine deprivation-mediated inhibition of mTORC1. Taken together, these results suggest that lysine might play role in the regulation of mTORC1 activation in NSCLC cells.

Review of Lysine Metabolism with a Focus on Humans.

Lysine cannot be synthesized by most higher organisms and, therefore, is an indispensable amino acid (IAA) that must be consumed in adequate amounts to maintain protein synthesis. Although lysine is an abundant amino acid in body proteins, lysine is limited in abundance in many important food sources (e.g. grains). Older observations assigned importance to lysine because animals fed a lysine-deficient diet did not lose weight as fast as animals placed upon other IAA-deficient diets, leading to the theory that there may be a special pool of lysine or metabolites that could be converted to lysine. The first step in the lysine catabolic pathway is the formation of saccharopine and then 2-aminoadipic acid, processes that are mitochondrial. The catabolism of 2-aminoadipic acid proceeds via decarboxylation to a series of CoA esters ending in acetyl-CoA. In mammals, the liver appears to be the primary site of lysine catabolism. In humans, the metabolic and oxidative response of lysine to diets either restricted in protein or in lysine is consistent with what has been measured for other IAAs with isotopically labeled tracers. Intestinal microflora are known to metabolize urea to ammonia and scavenge nitrogen (N) for the synthesis of amino acids. Studies feeding 15N-ammonium chloride or 15N-urea to animals and to humans, demonstrate the appearance of 15N-lysine in gut microbial lysine and in host lysine. However, the amount of 15N-lysine transferred to the host is difficult to assess directly using current methods. It is important to understand the role of the gut microflora in human lysine metabolism, especially in conditions where dietary lysine intake may be limited, but better methods need to be devised.

A novel mouse model for pyridoxine-dependent epilepsy due to antiquitin deficiency.

Pyridoxine-dependent epilepsy (PDE) is a rare autosomal recessive disease caused by mutations in the ALDH7A1 gene leading to blockade of the lysine catabolism pathway. PDE is characterized by recurrent seizures that are resistant to conventional anticonvulsant treatment but are well-controlled by pyridoxine (PN). Most PDE patients also suffer from neurodevelopmental deficits despite adequate seizure control with PN. To investigate potential pathophysiological mechanisms associated with ALDH7A1 deficiency, we generated a transgenic mouse strain with constitutive genetic ablation of Aldh7a1. We undertook extensive biochemical characterization of Aldh7a1-KO mice consuming a low lysine/high PN diet. Results showed that KO mice accumulated high concentrations of upstream lysine metabolites including ∆1-piperideine-6-carboxylic acid (P6C), α-aminoadipic semialdehyde (α-AASA) and pipecolic acid both in brain and liver tissues, similar to the biochemical picture in ALDH7A1-deficient patients. We also observed preliminary evidence of a widely deranged amino acid profile and increased levels of methionine sulfoxide, an oxidative stress biomarker, in the brains of KO mice, suggesting that increased oxidative stress may be a novel pathobiochemical mechanism in ALDH7A1 deficiency. KO mice lacked epileptic seizures when fed a low lysine/high PN diet. Switching mice to a high lysine/low PN diet led to vigorous seizures and a quick death in KO mice. Treatment with PN controlled seizures and improved survival of high-lysine/low PN fed KO mice. This study expands the spectrum of biochemical abnormalities that may be associated with ALDH7A1 deficiency and provides a proof-of-concept for the utility of the model to study PDE pathophysiology and to test new therapeutics.

Metabolic excretion associated with nutrient-growth dysregulation promotes the rapid evolution of an overt metabolic defect.

In eukaryotes, conserved mechanisms ensure that cell growth is coordinated with nutrient availability. Overactive growth during nutrient limitation ("nutrient-growth dysregulation") can lead to rapid cell death. Here, we demonstrate that cells can adapt to nutrient-growth dysregulation by evolving major metabolic defects. Specifically, when yeast lysine-auxotrophic mutant lys- encountered lysine limitation, an evolutionarily novel stress, cells suffered nutrient-growth dysregulation. A subpopulation repeatedly evolved to lose the ability to synthesize organosulfurs (lys-orgS-). Organosulfurs, mainly reduced glutathione (GSH) and GSH conjugates, were released by lys- cells during lysine limitation when growth was dysregulated, but not during glucose limitation when growth was regulated. Limiting organosulfurs conferred a frequency-dependent fitness advantage to lys-orgS- by eliciting a proper slow growth program, including autophagy. Thus, nutrient-growth dysregulation is associated with rapid organosulfur release, which enables the selection of organosulfur auxotrophy to better tune cell growth to the metabolic environment. We speculate that evolutionarily novel stresses can trigger atypical release of certain metabolites, setting the stage for the evolution of new ecological interactions.

Reduction in dietary lysine increases muscle free amino acids through changes in protein metabolism in chickens.

Taste is crucial to meat quality, and free Glu is an important taste-active component in meat. Our recent study showed that the short-term feeding of a low-Lys diet increases the concentration of free Glu and other free amino acids in chicken muscle and improves its taste. Here, we investigated the mechanisms by which the feeding of a low-Lys diet increases free Glu in chicken muscle. Two groups (n = 10 per group) of 28-day-old female Ross strain broiler chickens were fed diets with a graded Lys content of 90% or 100% of the recommended Lys requirement (according to National Research Council [1994] guidelines) for 10 D. Free amino acid concentrations and the mRNA abundance of protein metabolism-related genes were measured in breast muscle, and breast muscle metabolome analysis was conducted. Free Glu in muscle was increased by 51.8% in the Lys 90% group compared with the Lys 100% group (P < 0.01). Free threonine, glutamine, glycine, valine, isoleucine, leucine, tyrosine, phenylalanine, histidine, and 3-methyl-histidine concentrations in breast muscle were also increased in the Lys 90% group (P < 0.05). Metabolome analysis also showed that free amino acids were increased in the Lys 90% group. The mRNA abundance of µ-calpain, caspase-3, and 20S proteasome C2 subunit were increased in the Lys 90% group (P < 0.05). Moreover, the free Glu concentration in muscle was correlated with mRNA abundance of µ-calpain (r = 0.74, P < 0.01), caspase 3 (r = 0.69, P < 0.01), 20S proteasome C2 subunit (r = 0.65, P < 0.01), and cathepsin B (r = 0.52, P < 0.05). Our study suggests that the feeding of a low-Lys diet to chickens increased the free Glu content of breast muscle by promoting protein degradation.

Integrating RNA-sequencing and untargeted LC-MS metabolomics to evaluate the effect of lysine deficiency on hepatic functions in Holstein calves.

Lysine (Lys) is majorly metabolized in the liver. The liver functional consequences of a dietary Lys deficiency in young Holstein calves are unknown. This study aimed to investigate the effects of Lys deficiency in Holstein calf livers using RNA-sequencing and untargeted LC-MS metabolomics. Calves (n = 36; initial body weight 101.2 ± 10.8 kg; 90-day-old) were fed restricted diets, for 90 days, containing 19.2% crude protein that varied in Lys content (PC group 1.21%; PC-Lys group 0.85%; dry matter basis) for 90 days. Body weight, average daily gain, gain/feed, and Lys intake were significantly decreased in response to Lys deficiency (P < 0.05). Dry matter intake was not altered (P > 0.05). Network and pathway analyses revealed that noradrenaline, adenosine 5'-monophosphate, acetyl-CoA, and coenzyme A were significantly decreased. Regulating of lipolysis in adipocytes pathway and fatty acid degradation pathway were downregulated. We also identified eight significantly differentially expressed genes (SDEGs), among which adrenoceptor beta 2 (ADRB2), WAP four-disulfide core domain 2 (WFDC2), and claudin-4 (CLDN4) were associated with inhibition of lipolysis, and carbon catabolite repression 4-like (CCRN4L), FOS like 2 (FOSL2), and arginase 2 (ARG2) were associated with inhibiting lipid synthesis. Correlation tests showed that coenzyme A was strongly correlated with SDEGs (0.82 ≤|r|≤ 0.96). Acetyl-CoA and adenosine 5'-monophosphate were strongly correlated with CCRN4L (0.90 ≤|r|≤ 0.92), indicating a strong correlation between the changes in SDEGs and these metabolites. In conclusion, Lys deficiency caused dysplasia and affected lipid metabolism in the liver by inhibiting lipolysis and lipid synthesis in calves.

Genotype and dietary lysine deficiency affect carcass and muscle amino acid composition of pigs growing from 10 to 25 kg body weight.

Amino acid (AA) composition of body protein is considered constant although there are evidences that AA pattern in pigs may be altered by different factors. Pigs with different body composition and protein deposition rates-like fatty and lean pigs-may differ in AA composition, with possible consequences on their AA requirements. This work investigates effects of genotype and dietary lysine deficiency on AA composition of carcass and muscles of Iberian and Landrace × Large White pigs. Twenty-eight barrows (10 kg body weight [BW]), 14 from each breed, were used. They were randomly assigned to two experimental diets according to a factorial arrangement (two breeds × two diets). Diets were isonitrogenous and isoenergetic (200 ± 1 g CP/kg dry matter (DM); 14.7 ± 0.1 MJ ME/kg DM) and with identical chemical composition except for lysine concentration (10.9 and 5.20 g lysine/kg DM, for lysine-adequate (AL) diet and lysine-deficient (DL) diet respectively). Pigs were individually housed, and daily feed allowance was adjusted on a weekly basis according to BW. Pigs were slaughtered at 25 kg BW. Isoleucine, valine and phenylalanine concentration were higher in carcass protein of Iberian pigs (p < .01). In longissimus muscle, higher concentration of arginine, isoleucine, phenylalanine, lysine and valine (p < .001-p < .05), and lower of methionine (p < .001) were detected in Iberian pigs, whereas phenylalanine, leucine, lysine, threonine and methionine concentration decreased and arginine increased (p < .001-p < .05) when pigs were fed DL diet. Genotype and lysine deficiency effects were moderate in the AA composition of protein of biceps femoris muscle. The results show that AA proportions in protein of carcass and longissimus muscle can be influenced by pig genotype and conditions of lysine shortage. The biceps femoris muscle, with different functional and metabolic properties, shows more constant AA composition than longissimus, which seem to prevail independent from genotype or nutritional challenges.

Effects of lysine deficiency on carcass composition and activity and gene expression of lipogenic enzymes in muscles and backfat adipose tissue of fatty and lean piglets.

The purpose of this study was to investigate potential mechanisms involved in fat deposition promoted by dietary lysine deficiency, particularly intramuscular fat (IMF), and differential responses between fatty and lean pigs. Carcass traits and lipogenic enzyme activities and gene expression levels in muscles and adipose tissue were investigated in Iberian (fatty) and Landrace × Large White (LDW) pigs under identical feeding level (based on body weight (BW)) and management conditions. Twenty-eight barrows of 10 kg initial BW, 14 per breed, were fed two isoproteic (200 g CP /kg DM) and isocaloric (14.7 MJ metabolizable energy/kg DM) diets with identical composition except for the lysine content (1.09% for diet adequate in lysine and 0.52% for diet deficient in lysine). At a BW of 25 kg, pigs were slaughtered. Compared with pigs fed the lysine-adequate diet, in both genotypes lysine-deficient diet led to lower carcass protein concentration, lower relative proportions of leaner components (loin, ham and shoulder; P < 0.01), and higher carcass fatty components and carcass lipid concentration (P < 0.001). Irrespective of diet, the activity and gene expression of lipogenic enzymes (fatty acid synthase (FAS), malic enzyme (ME) and glucose-6-phosphate dehydrogenase (G6PDH)) were greater in Iberian than in LDW pigs, particularly in adipose tissue where transcriptional regulators involved in the control of adipogenesis and lipogenesis were also upregulated in Iberian animals. In backfat tissue, there was a small decrease induced by or no effects of lysine-deficient diet on the activity and gene expression of lipogenic enzymes, nor in gene expression levels of upstream regulators of lipogenesis and adipogenesis. In longissimus muscle, the activity of FAS, G6PDH and ME increased with lysine deficiency in both genotypes (P < 0.01) and an upregulation of gene expression of lipogenic enzymes was specifically observed in Iberian pigs (P < 0.05 to P < 0.001). In biceps femoris muscle of lysine-deficient pigs, the activity of FAS and ME enzymes increased, ME1 gene was upregulated (added to FASN gene in the case of Iberian pigs; P < 0.01 to P < 0.001) and PPARA gene was downregulated (P < 0.05). The results show that in both fatty and lean pigs, the effect of lysine deficiency on lipid metabolism was tissue-specific, with an activation of lipogenesis in longissimus and biceps femoris muscle but no apparent stimulation in backfat adipose tissue. Suitable feeding protocols including lysine-deficient diets should be designed for each pig type in order to increase intramuscular lipids without penalizing the growth of lean carcass components.

Effects of lysine deficiency or excess on growth and the expression of lipid metabolism genes in slow-growing broilers.

This experiment was conducted to evaluate the effects of lysine deficiency or excess on growth and the expression of lipid metabolism genes in slow-growing birds. A total of 360 one-day-old chicks were randomly divided into 3 groups, with 6 replicates of 20 birds each. The birds fed the basal diet with a total lysine 0.60% (LL), 1.00% (ML), or 1.40% (HL). The amount of lysine (ML) as the control group, LL and HL as the experimental group, the trial period last 3 wk. The results showed that compared with ML, LL significantly decreased average daily gain and average daily feed intake and remarkably increased feed conversion ratio of birds at 21 day old (P < 0.01), while the above indices in HL had no significant effects (P > 0.05). Besides, LL reduced the pectoral muscle rate (P < 0.01) and decreased the percentage of abdominal fat significantly (P < 0.05). In addition, compared with ML, the expression of fatty acid binding protein 1 (FABP1), acetyl-CoA carboxylase (ACC), malic enzyme (ME), and sterol regulatory element binding protein 1 (SREBP1c) mRNA of liver in LL was significantly decreased (P < 0.05), and the expression of cholesteryl ester transfer protein (CETP) mRNA was significantly increased (P < 0.01), whereas LL had no significant effects on the expression of peroxisome proliferator activated receptor alpha (PPARα) mRNA (P > 0.05). Moreover, compared with ML, HL significantly reduced the expression of FABP1, ACC, ME, SREBP-1c, and PPARα mRNA in the liver (P < 0.05), and had no significant effects on the expression of CETP mRNA (P > 0.05). The results of current research suggest that dietary lysine deficiency could reduce the growth and fat deposition of slow-growing broilers mainly by downregulating the expression of lipid synthesis genes.

A Highly Bioactive Lys-Deficient IFN Leads to a Site-Specific Di-PEGylated IFN with Equivalent Bioactivity to That of Unmodified IFN-α2b.

Although conjugation with polyethylene glycol (PEGylation) improves the pharmacokinetics of therapeutic proteins, it drastically decreases their bioactivity. Site-specific PEGylation counters the reduction in bioactivity, but developing PEGylated proteins with equivalent bioactivity to that of their unmodified counterparts remains challenging. This study aimed to generate PEGylated proteins with equivalent bioactivity to that of unmodified counterparts. Using interferon (IFN) as a model protein, a highly bioactive Lys-deficient protein variant generated using our unique directed evolution methods enables the design of a site-specific di-PEGylated protein. Antiviral activity of our di-PEGylated IFN was similar to that of unmodified IFN-α2b. The di-PEGylated IFN exhibited 3.0-fold greater antiviral activity than that of a commercial PEGylated IFN. Moreover, our di-PEGylated IFN showed higher in vitro and in vivo stability than those of unmodified IFN-α2b. Hence, we propose that highly bioactive Lys-deficient proteins solve the limitation of conventional PEGylation with respect to the reduction in bioactivity of PEGylated proteins.

Effects of dietary lysine restriction on inflammatory responses in piglets.

The aim of this study was to investigate the effects of lysine restriction on inflammatory responses in piglets. 38 male piglets with similar body weight of 9.62 kg were randomly divided into control group (basal diet) and lysine-restricted group (diet containing 70% lysine of the control diet). The results showed that lysine restriction increased the serum concentration of IgG an IgM. Piglets fed the lysine-restricted diet exhibited overexpression of interleukin-8 (IL-8) in the kidney (P < 0.05) and IL-6 and IL-4 in the spleen (P < 0.05). The mRNA abundances of IL-4 in the kidney (P < 0.05) and IL-10 in the liver (P < 0.05) were significantly lower in the lysine-restricted group compared with the control group. Meanwhile, lysine restriction increased the mRNA level of Tlr8 in the kidney (P < 0.05) but decreased the mRNA level of Tlr8 in the liver (P < 0.05). Finally, lysine restriction markedly enhanced extracellular signal regulated kinases 1/2 (ERK1/2) phosphorylation in the kidney and liver and nuclear transcription factor kappa B (NF-κB) was activated in the liver and spleen in response to dietary lysine restriction. In conclusion, lysine restriction affected inflammatory responses in the kidney, liver, and spleen via mediating serum antibody volume, inflammatory cytokines, Tlrs system, and ERK1/2 and NF-κB signals in piglets.

Lysine Restriction Affects Feed Intake and Amino Acid Metabolism via Gut Microbiome in Piglets.

BACKGROUND/AIMS: Our previous reports suggested that dietary supplementation with lysine influenced intestinal absorption and metabolism of amino acids. In this study, we further investigated the effect of lysine restriction (30%) on feed intake and we also tested the hypothesis that gut microbiome contributed to the potential mechanism of lysine restriction-mediated feeding behavior. Here, we profiled gut microbial communities by sequencing 16S ribosomal ribonucleic acid (rRNA) genes from gut samples as well as growth performance, serum hormones, and intestinal lysine transport in a piglet model. RESULTS: Piglets preferred to the lysine restricted diet when giving three diets and the feed intake was markedly higher in the lysine-restricted group than that in the control group. Altered hormones (leptin, CCK, and ghrelin) might contribute to the feeding behavior caused by lysine restriction. Meanwhile, lysine transporting ability (SLC7A1 and SLC7A2 expression, intestinal electrophysiological changes, and amino acid pool in mesenteric vein) was decreased in response to lysine restriction. Through deep sequencing of bacterial rRNA markers, we observed that bacterial diversity was enhanced in the lysine-restricted group (Shannon H, PD, and Chao1). At the phylum level, lysine restriction enhanced gut Actinobacteria, Saccharibacteria, and Synergistetes abundances. At the family level, Moraxellaceae, Halomonadaceae, Shewanellaceae, Corynebacteriaceae, Bacillaceae, Comamonadaceae, Microbacteriaceae, Caulobacteraceae, and Synergistaceae abundances were increased in response to lysine restriction. Predictive functional profiling of microbial communities by PICRUSt also confirmed that dietary lysine restriction affected gut microbiome, which might further mediate amino acid metabolism, membrane transport, and endocrine system. CONCLUSION: Our results indicated that lysine restriction inhibited intestinal lysine transport and promoted feed intake, which might be associated with gut microbiome.

Pyridoxine-Dependent Epilepsy in Zebrafish Caused by Aldh7a1 Deficiency.

Pyridoxine-dependent epilepsy (PDE) is a rare disease characterized by mutations in the lysine degradation gene ALDH7A1 leading to recurrent neonatal seizures, which are uniquely alleviated by high doses of pyridoxine or pyridoxal 5'-phosphate (vitamin B6 vitamers). Despite treatment, neurodevelopmental disabilities are still observed in most PDE patients underlining the need for adjunct therapies. Over 60 years after the initial description of PDE, we report the first animal model for this disease: an aldh7a1-null zebrafish (Danio rerio) displaying deficient lysine metabolism and spontaneous and recurrent seizures in the larval stage (10 days postfertilization). Epileptiform electrographic activity was observed uniquely in mutants as a series of population bursts in tectal recordings. Remarkably, as is the case in human PDE, the seizures show an almost immediate sensitivity to pyridoxine and pyridoxal 5'-phosphate, with a resulting extension of the life span. Lysine supplementation aggravates the phenotype, inducing earlier seizure onset and death. By using mass spectrometry techniques, we further explored the metabolic effect of aldh7a1 knockout. Impaired lysine degradation with accumulation of PDE biomarkers, B6 deficiency, and low γ-aminobutyric acid levels were observed in the aldh7a1-/- larvae, which may play a significant role in the seizure phenotype and PDE pathogenesis. This novel model provides valuable insights into PDE pathophysiology; further research may offer new opportunities for drug discovery to control seizure activity and improve neurodevelopmental outcomes for PDE.

Similar effects of lysine deficiency in muscle biochemical characteristics of fatty and lean piglets.

The main objective of this work was to investigate the effects of feeding Lys-deficient diets on muscle biochemical characteristics, particularly intramuscular fat concentration and fatty acid profile, in a fatty (Iberian) and a conventional pig genotype (Landrace × Large White [LDW]) maintained in identical experimental conditions. Performance and plasma metabolite changes were also monitored. Twenty-eight barrows of 10 kg initial BW, 14 of Iberian and 14 of LDW breed, were randomly assigned to each of 2 experimental diets in a 2 × 2 factorial arrangement (2 breeds × 2 diets). Seven pigs were allocated to each treatment combination. Diets (isonitrogenous and isoenergetic; 200 g CP/kg DM and 14.7 MJ ME) based on barley, corn, corn gluten meal, and soybean meal, with identical composition, except for their Lys content (10.9 g/kg for the diet adequate in Lys and 5.2 g/kg for the diet deficient in Lys), were assayed. Pigs were housed in individual 2 m pens and fed at 85% of ad libitum intake of the Iberian genotype, of greater intake capacity. Daily feed allowance was based on BW individually measured each week. At 25 kg BW, pigs were slaughtered by exsanguination after electrical stunning. Blood samples were taken and longissimus dorsi and biceps femoris muscles were rapidly dissected and stored frozen prior to analysis. Performance was reduced in both pig breeds when fed Lys-deficient diets, particularly in LDW pigs (breed × diet interaction, < 0.05). Intramuscular fat content increased in longissimus dorsi of Iberian ( < 0.05) and in biceps femoris of both pig genotypes ( < 0.01) when fed Lys-deficient diets. Oleic acid increased ( < 0.05) and PUFA acid decreased ( < 0.01) in longissiumus dorsi and biceps femoris of pigs of both genotypes fed Lys-deficient diets. The proportion of oxidative fibers ( < 0.001) and free carnitine content ( < 0.05) increased in longissimus dorsi of both pigs types fed Lys-deficient diets. Plasma creatinine was greater in LDW pigs compared with Iberian pigs ( < 0.01). Urea and total cholesterol increased in pigs consuming Lys-deficient diets ( < 0.01). The plasma free carnitine concentration was higher in Iberian pigs than in LDW pigs ( < 0.059). No changes in plasma carnitine status due to dietary Lys supply were detected. Feeding Lys-deficient diets could be a suitable strategy for increasing intramuscular fat content in fatty and lean pigs.

Effects of graded removal of lysine from an intravenously infused amino acid mixture on lactation performance and mammary amino acid metabolism in lactating goats.

To investigate responses of milk protein synthesis and mammary AA metabolism to a graded decrease of postruminal Lys supply, 4 lactating goats fitted with jugular vein, mammary vein, and carotid artery catheters and transonic blood flow detectors on the external pudic artery were used in a 4 × 4 Latin square experiment. Goats were fasted for 24 h and then received a 9-h intravenous infusion of an AA mixture plus glucose. Milk yield was recorded and samples were taken in h 2 to 8 of the infusion period; a mammary biopsy was performed in the last hour. Treatments were graded decrease of lysine content in the infusate to 100 (complete), 60, 30, or 0% as in casein. Lysine-removed infusions linearly decreased milk yield, tended to decrease lactose yield, and tended to increase milk fat to protein ratio. Milk protein content and yield were linearly decreased by graded Lys deficiency. Mammary Lys uptake was concomitantly decreased, but linear regression analysis found no significant relationship between mammary Lys uptake and milk protein yield. Treatments had no effects on phosphorylation levels of the downstream proteins measured in the mammalian target or rapamycin pathway except for a tended quadratic effect on that of eukaryotic initiation factor 2, which was increased and then decreased by graded Lys deficiency. Removal of Lys from the infusate linearly increased circulating glucagon and glucose. Removal of Lys from the infusate linearly decreased arterial and venous concentrations of Lys. Treatments also had a significant quadratic effect on venous Lys, suggesting mechanisms to stabilize circulating Lys at a certain range. The 2 infusions partially removing Lys resulted in a similar 20% decrease, whereas the 0% Lys infusion resulted in an abrupt 70% decrease in mammary Lys uptake compared with that of the full-AA mixture infusion. Consistent with the abrupt decrease, mammary Lys uptake-to-output ratio decreased from 2.2 to 0.92, suggesting catabolism of Lys in the mammary gland could be completely prevented when the animal faced severe Lys deficiency. Mammary blood flow was linearly increased, consistent with the linearly increased circulating nitric oxide by graded Lys deficiency, indicating mechanisms to ensure the priority of the mammary gland in acquiring AA for milk protein synthesis. Infusions with Lys removed increased mammary clearance rate of Lys numerically by 2 to 3 fold. In conclusion, the decreased milk protein yield by graded Lys deficiency was mainly a result of the varied physiological status, as indicated by the elevated circulating glucagon and glucose, rather than a result of the decreased mammary Lys uptake or depressed signals in the mTOR pathway. Mechanisms of Lys deficiency to promote glucagon secretion and mammary blood flow and glucagon to depress milk protein synthesis need to be clarified by future studies.

Effects of Lysine deficiency and Lys-Lys dipeptide on cellular apoptosis and amino acids metabolism.

SCOPE: Lysine (Lys) is a common limiting amino acids (AA) for humans and animals and plays an important role in cell proliferation and metabolism, while metabolism of Lys deficiency and its dipeptide is still obscure. Thus, this study mainly investigated the effects of Lys deficiency and Lys-Lys dipeptide on apoptosis and AA metabolism in vitro and in vivo models. METHODS AND RESULTS: Lys deficiency induced cell-cycle arrest and apoptosis and upregulated Lys transporters in vitro and in vivo. SLC7A11, a cystine-glutamate antiporter, was markedly upregulated by Lys deficiency and then further mediated cystine uptake and glutamate release, which was negatively regulated by cystine and glutamate transporters. Meanwhile, Lys deprivation upregulated pept1 expression, which might improve Lys-Lys dipeptide absorption to compensate for the reduced Lys availability. Lys-Lys dipeptide alleviated Lys deficiency induced cell-cycle arrest and apoptosis and influenced AA metabolism. Furthermore, the mammalian target of rapamycin signal might be involved in sensing cellular Lys starvation and Lys-Lys dipeptide. CONCLUSIONS: Altogether, these studies suggest that Lys deficiency impairs AA metabolism and causes apoptosis. Lys-Lys dipeptide serves as a Lys source and alleviates Lys deficiency induced cellular imbalance.