Glycine Amidinotransferase Research Articles

Dietary glycine supplementation enhances creatine availability in tissues of pigs with intrauterine growth restriction.

This study tested the hypothesis that dietary supplementation with glycine (Gly) enhances the synthesis and availability of creatine (Cr) in tissues of pigs with intrauterine growth restriction (IUGR). At weaning (21 d of age), IUGR pigs and litter mates with normal birth weights (NBW) were assigned randomly to one of two groups, namely, supplementation with 1% Gly or 1.19% L-alanine (isonitrogenous control) to a corn- and soybean meal-based diet. Blood, kidneys, liver, pancreas, jejunum, longissimus lumborum muscle (LLM), and gastrocnemius muscle (GM) were obtained from the pigs within 1wk after the feeding trial ended at 188 d of age to determine concentrations of guanidinoacetate (GAA), Cr, creatinine, and phosphocreatine (CrP). The organs were also analyzed for activities and mRNA levels for Cr-synthetic enzymes: L-arginine:glycine amidinotransferase (AGAT; forming GAA from Gly and L-arginine) and guanidinoacetate N-methyltransferase (GAMT; converting GAA and L-methionine into Cr). AGAT activity was present in the kidneys, liver, and pancreas, whereas GAMT activity was found in all the organs analyzed. AGAT and GAMT were most active per g of tissue in the kidneys and liver, respectively. Based on tissue mass, the kidneys had the greatest (P < 0.001) AGAT activity per whole organ, followed by the liver, while skeletal muscle had the greatest (P < 0.001) GAMT activity per whole organ, followed by the liver. Thus, the kidneys played a dominant role in forming GAA, whereas skeletal muscle and liver were the major sites for converting GAA into Cr. Dietary supplementation with 1% Gly enhanced AGAT activity in the kidneys and pancreas but reduced GAMT activity in the pancreas and small intestine, therefore directing GAA to the liver and skeletal muscle for Cr production. IUGR selectively reduced the concentration of Cr in LLM among all the organs studied. Except for the GM that had greater mRNA levels for GAMT in IUGR pigs, neither Gly nor IUGR affected mRNA levels for the selected genes in the tissues examined. Collectively, these novel results indicate that dietary Gly intake upregulates the Cr-synthetic pathway in swine.

Targeting AGAT gene expression – a drug screening approach for the treatment of GAMT deficiency

ABSTRACT Background Targeting the enzyme L-Arginine:glycine amidinotransferase (AGAT) to reduce the formation of guanidinoacetate (GAA) in patients with guanidinoacetate methyltransferase (GAMT) deficiency, we attempted to identify drugs for repurposing that reduce the expression of AGAT via transcriptional inhibition. Research Design and Methods The authors applied a HeLa cell line stably expressing AGAT promoter and firefly luciferase reporter for high-content screening and secondary screening. For further assessment, the authors integrated Nanoluc luciferase as a reporter into the endogenous AGAT gene in HAP1 cell lines and used the human immortalized cell line RH30 as model of GAMT deficiency. Results Screening 6,000 drugs and drug-like compounds, the authors identified 43 and 34 high-score candidates as inhibitors and inducers of AGAT promoter-reporter expression, respectively. After further deselection considering dose response, drug toxicity, topical formulations, price, and accessibility, the authors assessed seven candidates and found none of them demonstrating efficacy in HAP1 and RH30 cells and warranting further assessment. Conclusion The selection of the test models is crucial for screening of gene repressor drugs. Almost all drugs with an impact on gene expression had off-target effects. It is unlikely to find drugs that are selective inhibitors of AGAT expression, rendering pharmacological AGAT gene repression a risky approach for the treatment of GAMT deficiency.

Efficient biosynthesis of guanidoacetic acid by a recombinant strain of Bacillus subtilis

Guanidinoacetic acid, as an energetic substance, has a wide range of applications in the food, pharmaceutical, and feed industries. However, the biosynthesis of guanidinoacetic acid has not been applied in industrial production. In this study, we designed the synthetic route of guanidinoacetic acid in a food-grade strain of Bacillus subtilis. By regulating the expression of key enzymes, lifting feedback inhibition, and increasing membrane permeability, we achieved the efficient synthesis of guanidinoacetic acid by whole-cell catalysis. Firstly, the optimal L-arginine:glycine amidinotransferase was screened based on the phylogenetic tree, and the expression of the key enzyme was enhanced by a strategy combining strong promoter and genome integration. Secondly, the ornithine cycle for L-arginine synthesis in Corynebacterium glutamicum was introduced to alleviate the feedback inhibition of the enzyme by the byproduct L-ornithine, and the L-arginine degradation pathway was knocked down to enhance substrate regeneration. Thirdly, the expression of N-acetylmuramoyl-L-alanine amidase (LytC) was up-regulated to increase the cell membrane permeability. Finally, after optimization of whole-cell production conditions, strain Bs-13 achieved guanidinoacetic acid production at a titer of 13.1 g/L after 24 h, with a proudction rate of 0.54 g/(L·h) and a glycine conversion rate of 92.7%. The above strategy improved the production of guanidinoacetic acid and provided a reference for the biosynthesis of guanidinoacetic acid.

Association between GATM gene polymorphism and progression of chronic kidney disease: a mitochondrial related genome-wide Mendelian randomization study

Chronic Kidney Disease (CKD) stands as a substantial challenge within the global health landscape. The elevated metabolic demands essential for sustaining normal kidney function have propelled an increasing interest in unraveling the intricate relationship between mitochondrial dysfunction and CKD. However, the authentic causal relationship between these two factors remains to be conclusively elucidated. This study endeavors to address this knowledge gap through the Mendelian Randomization (MR) method. We utilized large-scale QTL datasets (including 31,684 eQTLs samples, 1980 mQTLs samples, and 35,559 pQTLs samples) to precisely identify key genes related to mitochondrial function as exposure factors. Subsequently, we employed GWAS datasets (comprising 480,698 CKD samples and 1,004,040 eGFRcrea samples) as outcome factors. Through a comprehensive multi-level analysis (encompassing expression, methylation, and protein quantification loci), we evaluated the causal impact of these genes on CKD and estimated glomerular filtration rate (eGFR). The integration and validation of diverse genetic data, complemented by the application of co-localization analysis, bi-directional MR analysis, and various MR methods, notably including inverse variance weighted, have collectively strengthened our confidence in the robustness of these findings. Lastly, we validate the outcomes through examination in human RNA sequencing datasets encompassing various subtypes of CKD. This study unveils significant associations between the glycine amidinotransferase (GATM) and CKD, as well as eGFR. Notably, an augmentation in GATM gene and protein expression corresponds to a diminished risk of CKD, whereas distinct methylation patterns imply an increased risk. Furthermore, a discernible reduction in GATM expression is observed across diverse pathological subtypes of CKD, exhibiting a noteworthy positive correlation with GFR. These findings establish a causal relationship between GATM and CKD, thereby highlighting its potential as a therapeutic target. This insight lays the foundation for the development of potential therapeutic interventions for CKD, presenting substantial clinical promise.

A novel biomarker GATM suppresses proliferation and malignancy of cholangiocarcinoma cells by modulating the JNK/c-Jun signalling pathways

BackgroundCholangiocarcinoma (CCA) is the second most common primary malignancy of the liver and is associated with poor prognosis. Despite the emerging role of glycine amidinotransferase (GATM) in cancer development, its function in CCA remains elusive. This study investigated the biological significance and molecular mechanisms of GATM in CCA. MethodGATM expression was measured using immunohistochemistry and western blotting. Cell proliferation, migration, and invasion were assessed through CCK-8, EdU, clone formation, wound healing, and Transwell assays. Rescue experiments were performed to determine whether the JNK/c-Jun pathway is involved in GATM-mediated CCA development. Immunoprecipitation and mass spectrometry were performed to screen for proteins that interact with GATM. The role of GATM in vivo was investigated according to the xenograft experiment. ResultGATM expression was downregulated in CCA tissues and cells (p < 0.05) and had a significant suppressive effect on CCA cell proliferation, migration, and invasion in vitro as well as on tumour growth in vivo (p < 0.05); conversely, GATM knockdown promoted these phenotypes (p < 0.05). Notably, GATM inhibited the JNK/c-Jun pathway, and JNK activation abrogated GATM's antitumor effects (p < 0.05). Isocitrate dehydrogenase 1 (IDH1) interacts with GATM, and IDH1 knockdown significantly attenuated GATM protein degradation. Overexpression of IDH1 restored the biological function of CCA by reversing the inhibition of JNK/c-Jun pathway phosphorylation by GATM (p < 0.05). ConclusionGATM acts as a tumour suppressor in CCA by regulating the phosphorylation of the JNK/c-Jun pathway. IDH1 interacted with GATM to regulate CCA progression.

Creatine in giant grouper (Epinephelus lanceolatus): Insights into biosynthesis and transporter genes molecular characterization, regulation and impact on muscle metabolism

Creatine is synthesized from glycine, arginine, and methionine with the catalysis of glycine amidinotransferase (GATM) and guanidinoacetate N-methyltransferase (GAMT), playing a crucial role in regulating the cellular metabolism and whole-body energy balance. However, information regarding the molecular characteristics of GATM and GAMT is limited in fish species. In this study, GATM and GAMT cDNA were cloned from giant grouper (Epinephelus lanceolatus). The cloned cDNA sequence of GATM was 1503 bp encoding a 421 aa containing conserved regions of amidinotransferase superfamily. GAMT cDNA was 892 bp encoding a 235 aa containing conserved regions of S-adenosylmethionine-dependent methyltransferases superfamily. The phylogenetic tree indicated that GATM and GAMT were clustered distinctly into teleost and featured common function domains and homology with teleost proteins, but they were distinct from the terrestrial clusters. The mRNA transcripts of GATM and GAMT were detectable mainly in muscle tissue. Additionally, as the creatine synthesis limited enzyme, GATM gene and protein expression were both significantly regulated by supplement of dietary creatine (P < 0.05). Metabolomics analysis showed that dietary creatine significantly affected skeletal muscle nutrients and energy metabolism. Meanwhile, inclusion of dietary creatine led to a significant inhibition of AMPK phosphorylation. What is more, we also recorded that cytochrome oxidase (cox) and mitochondrial fission factor (mff) gene expression were remarkable up-regulated by dietary creatine supplementation. In summary, the results obtained from the present study would help us better understand the creatine synthesis pathway in fish species. We also demonstrated that creatine plays a significant role in fish muscle metabolism.

Integrated transcriptome and metabolome profiles reveal the regulatory molecular mechanisms of meat quality in pasture-fed goose

Goose meat is rich in amino acids with unique taste and aroma traits that are targeted by housing and feeding systems. This study aimed to investigate the impacts of in-house feeding (IHF) and artificial pasture grazing (AGF) systems on nutrient composition, flavor, and taste of thigh muscles of goose. The proximate analysis showed that, compared to IHF group, AGF group contained significantly higher contents of moisture and crude protein than ash and intramuscular fat. The findings of transcriptome analysis revealed that 139 differentially expressed genes (DEGs) displayed differential expression (102 genes downregulated and 37 genes upregulated), and only 13 DEGs were involved in fatty acid metabolic process, protein binding, and calcium ion binding. Together, 79 and 16 significantly differential metabolites (SDMs) were identified through LC-MS/MS and GC-TOF-MS, respectively. Furthermore, we performed KEGG enrichment analysis of DEGs and related them with enriched signaling pathways attained from SDMs. Collectively, we detected 4 common KEGG pathways involved in the development of flavor and taste in AGF geese, namely sphingolipid metabolism, arginine and proline metabolism, alanine, aspartate and glutamate metabolism, and valine, leucine and isoleucine degradation. In these pathways, the upregulated uridine diphosphate (UDP)-glycosyltransferase 8 (UGT8), glycine amidinotransferase (GATM), and aldehyde dehydrogenase (ALDH) and downregulated ceramide synthase 6 (CERS6) genes may regulate the synthesis of metabolites including l-serine, phosphatidylethanolamine, l-arginine, l-glutamate, lysyl-proline, d-ornithine, putrescine, and alpha-ketoisocaproic acid 2 in pasture-fed group. These genes and metabolites may play a key role in regulating protein accumulation, IMF deposition, meat flavor, and taste development in pasture-fed group.

Toxic effects of freshwater grouper (Acrossocheilus fasciatus) eggs on poultry: Morphological and transcriptomic insights into hepatic toxicity

Fish egg poisoning is a serious and neglected public menace that kills hundreds of people and numerous poultry each year. Freshwater groupers (Acrossocheilus fasciatus) are common food fish in the southeastern regions of China. Their toxic eggs are regarded as a significant public health concern. The molecular mechanisms of egg-toxin toxicity in freshwater grouper to poisoned organisms are elusive. In this study, black-boned chicks were exposed to toxic eggs from freshwater grouper at a lethal dose. The hepatic morphology of the intoxicated chick was assessed. An analysis of the liver gene expression profile was conducted by comparing samples exposed to toxic eggs with control samples using RNA-Seq. The result revealed that an increase in vacuolation and congestion was observed in chicks with toxic eggs exposure. The transcriptome analysis revealed 5421 genes with differential expression, comprising 2810 up-regulated and 2611 down-regulated genes. The genes were primarily linked to energy metabolism, cell apoptosis, cell adhesion, exogenous microbial infection, and cell junction. The most strongly upregulated genes were cholecystokinin (CCK), cholecystokinin A receptor (CCKAR), and unc-80 homolog, NALCN activator (UNC80), and the most downregulated genes were glycine amidinotransferase (GATM), fatty acid desaturase 2 (FADS2), and hexokinase 2 (HKDC1). GO term with the highest enrichment of DEGs is nucleosome assembly. According to KEGG pathways, the three most significant metabolic pathways in the liver are DNA replication, retinol metabolism, and steroid biosynthesis. The results could be crucial for comprehending the negative biological impacts of egg-toxin and its toxic mechanisms. The outcome could provide potential biomarkers of egg-toxin exposure in hepatic, which might be useful for manufacturing an antidote to egg-toxin and providing valuable insights for ecotoxicity studies.

Dietary glycine supplementation enhances syntheses of creatine and glutathione by tissues of hybrid striped bass (Morone saxatilis ♀ × Morone chrysops ♂) fed soybean meal-based diets

BackgroundWe recently reported that supplementing glycine to soybean meal-based diets is necessary for the optimum growth of 5- to 40-g (Phase-I) and 110- to 240-g (Phase-II) hybrid striped bass (HSB), as well as their intestinal health. Although glycine serves as an essential substrate for syntheses of creatine and glutathione (GSH) in mammals (e.g., pigs), little is known about these metabolic pathways or their nutritional regulation in fish. This study tested the hypothesis that glycine supplementation enhances the activities of creatine- and GSH-forming enzymes as well as creatine and GSH availabilities in tissues of hybrid striped bass (HSB; Morone saxatilis♀ × Morone chrysops♂).MethodsPhase-I and Phase-II HSB were fed a soybean meal-based diet supplemented with 0%, 1%, or 2% glycine for 8 weeks. At the end of the 56-d feeding, tissues (liver, intestine, skeletal muscle, kidneys, and pancreas) were collected for biochemical analyses.ResultsIn contrast to terrestrial mammals and birds, creatine synthesis occurred primarily in skeletal muscle from all HSB. The liver was most active in GSH synthesis among the HSB tissues studied. In Phase-I HSB, supplementation with 1% or 2% glycine increased (P < 0.05) concentrations of intramuscular creatine (15%–19%) and hepatic GSH (8%–11%), while reducing (P < 0.05) hepatic GSH sulfide (GSSG)/GSH ratios by 14%–15%, compared with the 0-glycine group; there were no differences (P > 0.05) in these variables between the 1% and 2% glycine groups. In Phase-II HSB, supplementation with 1% and 2% glycine increased (P < 0.05) concentrations of creatine and GSH in the muscle (15%–27%) and liver (11%–20%) in a dose-dependent manner, with reduced ratios of hepatic GSSG/GSH in the 1% or 2% glycine group. In all HSB, supplementation with 1% and 2% glycine dose-dependently increased (P < 0.05) activities of intramuscular arginine:glycine amidinotransferase (22%–41%) and hepatic γ-glutamylcysteine synthetase (17%–37%), with elevated activities of intramuscular guanidinoacetate methyltransferase and hepatic GSH synthetase and GSH reductase in the 1% or 2% glycine group. Glycine supplementation also increased (P < 0.05) concentrations of creatine and activities of its synthetic enzymes in tail kidneys and pancreas, and concentrations of GSH and activities of its synthetic enzymes in the proximal intestine.ConclusionsSkeletal muscle and liver are the major organs for creatine and GSH syntheses in HSB, respectively. Dietary glycine intake regulates creatine and GSH syntheses by both Phase-I and Phase-II HSB in a tissue-specific manner. Based on the metabolic data, glycine is a conditionally essential amino acid for the growing fish.

Functional metabolomics reveals arsenic-induced inhibition of linoleic acid metabolism in mice kidney in drinking water

Arsenic (As) is a heavy metal known for its detrimental effects on the kidneys, but the precise mechanisms underlying its toxicity remain unclear. In this study, we employed an integrated approach combining traditional toxicology methods with functional metabolomics to explore the nephrotoxicity induced by As in mice. Our findings demonstrated that after 28 days of exposure to sodium arsenite, blood urea nitrogen, serum creatinine levels were significantly increased, and pathological examination of the kidneys revealed dilation of renal tubules and glomerular injury. Additionally, uric acid, total cholesterol, and low-density lipoprotein cholesterol levels were significant increased while triglyceride level was decreased, resulting in renal insufficiency and lipid disorders. Subsequently, the kidney metabolomics analysis revealed that As exposure disrupted 24 differential metabolites, including 14 up-regulated and 10 down-regulated differential metabolites. Ten metabolic pathways including linoleic acid and glycerophospholipid metabolism were significantly enriched. Then, 80 metabolic targets and 168 predicted targets were identified using metabolite network pharmacology analysis. Of particular importance, potential toxicity targets, such as glycine amidinotransferase, mitochondrial (GATM), and nitric oxide synthase, and endothelial (NOS3), were prioritized through the “metabolite-target-pathway” network. Receiver operating characteristics curve and molecular docking analyses suggested that 1-palmitoyl-2-myristoyl-sn-glycero-3-PC, linoleic acid, and L-hydroxyarginine might be functional metabolites associated with GATM and NOS3. Moreover, targeted verification result showed that the level of linoleic acid in As group was 0.4951 μg/mL, which was significantly decreased compared with the control group. And in vivo and in vitro protein expression experiments confirmed that As exposure inhibited the expression of GATM and NOS3. In conclusion, these results suggest that As-induced renal injury may be associated with the inhibition of linoleic acid metabolism through the down-regulation of GATM and NOS3, resulting in decreased levels of linoleic acid, 1-palmitoyl-2-myristoyl-sn-glycero-3-PC, and L-hydroxyarginine metabolites.

A18 EARLY LIFE EXPOSURE TO PARENTAL CROHN'S DISEASE IS ASSOCIATED WITH GUT MICROBIAL COMPOSITION AND FUNCTION, GUT PERMEABILITY AND INCREASED RISK OF FUTURE CROHN'S DISEASE

Abstract Background Previous studies show that offspring of persons with Crohn’s disease (CD) have a 7-fold higher risk of incident CD than the general population. Newborns of women with CD (vs healthy women) have altered stool microbiome composition and elevated fecal calprotectin (FCP). The perinatal period (pregnancy and first year postpartum) is critical for the development of the gut microbiome and immune system. Aims We investigated whether perinatal exposure to parental CD (compared to exposure later in life) has an impact on offspring’s gut barrier function, microbiome composition, and risk of future CD. Methods We assessed 1252 healthy offspring of persons with CD who were recruited in the CCC-GEM Project. We classified offspring into "perinatally exposed (pregnancy and first year postpartum)" vs "exposed later in life" to parental CD diagnosis. We measured baseline FCP and urinary fractional excretion ratio of lactulose to mannitol (marker of intestinal permeability, LMR). Fecal microbiome composition was determined by 16S rRNA gene sequencing. Microbiome function was imputed by PICRUSt2 (q value defined as false discovery rate-adjusted p&amp;lt;0.05). Multivariable regression and Cox proportional-hazards analyses were used to assess if peri-natal exposure to parental CD is associated with LMR, FCP, altered microbial composition, and future CD onset. Results Offspring exposed to a CD parent perinatally had a 4.7-fold higher risk (aHR 95%CI 1.3-17.5) of developing CD compared to those exposed to a parent who developed CD later in life (11/586 vs 3/666). LMR was significantly higher in the offspring exposed to parental CD perinatally (p=0.003), while no significant difference was observed for FCP at recruitment (p=0.94). Five bacterial taxa were significantly increased in the perinatally exposed group (q&amp;lt;0.05). The microbial functions such as glycine amidinotransferase, serine protein kinase and cyanophycin synthase were significantly increased in the perinatally exposed group (q&amp;lt;0.05). Conclusions Offspring exposed to parental CD perinatally had a higher risk of developing CD than those exposed to parental CD later in life. Early life exposure to parental CD was associated with higher baseline LMR, altered bacterial taxa and functional capacities. Environmental exposure during the perinatal period may determine the offspring’s risk of developing CD. Funding Agencies CAG, CCC, CIHRCanada Research Chair

Beyond protein synthesis: the emerging role of arginine in poultry nutrition and host-microbe interactions.

Arginine is a functional amino acid essential for various physiological processes in poultry. The dietary essentiality of arginine in poultry stems from the absence of the enzyme carbamoyl phosphate synthase-I. The specific requirement for arginine in poultry varies based on several factors, such as age, dietary factors, and physiological status. Additionally, arginine absorption and utilization are also influenced by the presence of antagonists. However, dietary interventions can mitigate the effect of these factors affecting arginine utilization. In poultry, arginine is utilized by four enzymes, namely, inducible nitric oxide synthase arginase, arginine decarboxylase and arginine: glycine amidinotransferase (AGAT). The intermediates and products of arginine metabolism by these enzymes mediate the different physiological functions of arginine in poultry. The most studied function of arginine in humans, as well as poultry, is its role in immune response. Arginine exerts immunomodulatory functions primarily through the metabolites nitric oxide (NO), ornithine, citrulline, and polyamines, which take part in inflammation or the resolution of inflammation. These properties of arginine and arginine metabolites potentiate its use as a nutraceutical to prevent the incidence of enteric diseases in poultry. Furthermore, arginine is utilized by the poultry gut microbiota, the metabolites of which might have important implications for gut microbial composition, immune regulation, metabolism, and overall host health. This comprehensive review provides insights into the multifaceted roles of arginine and arginine metabolites in poultry nutrition and wellbeing, with particular emphasis on the potential of arginine in immune regulation and microbial homeostasis in poultry.

Suggestion of creatine as a new neurotransmitter by approaches ranging from chemical analysis and biochemistry to electrophysiology

The discovery of a new neurotransmitter, especially one in the central nervous system, is both important and difficult. We have been searching for new neurotransmitters for 12 y. We detected creatine (Cr) in synaptic vesicles (SVs) at a level lower than glutamate and gamma-aminobutyric acid but higher than acetylcholine and 5-hydroxytryptamine. SV Cr was reduced in mice lacking either arginine:glycine amidinotransferase (a Cr synthetase) or SLC6A8, a Cr transporter with mutations among the most common causes of intellectual disability in men. Calcium-dependent release of Cr was detected after stimulation in brain slices. Cr release was reduced in Slc6a8 and Agat mutants. Cr inhibited neocortical pyramidal neurons. SLC6A8 was necessary for Cr uptake into synaptosomes. Cr was found by us to be taken up into SVs in an ATP-dependent manner. Our biochemical, chemical, genetic, and electrophysiological results are consistent with the possibility of Cr as a neurotransmitter, though not yet reaching the level of proof for the now classic transmitters. Our novel approach to discover neurotransmitters is to begin with analysis of contents in SVs before defining their function and physiology.

Suggestion of creatine as a new neurotransmitter by approaches ranging from chemical analysis and biochemistry to electrophysiology.

The discovery of a new neurotransmitter, especially one in the central nervous system, is both important and difficult. We have been searching for new neurotransmitters for 12 y. We detected creatine (Cr) in synaptic vesicles (SVs) at a level lower than glutamate and gamma-aminobutyric acid but higher than acetylcholine and 5-hydroxytryptamine. SV Cr was reduced in mice lacking either arginine:glycine amidinotransferase (a Cr synthetase) or SLC6A8, a Cr transporter with mutations among the most common causes of intellectual disability in men. Calcium-dependent release of Cr was detected after stimulation in brain slices. Cr release was reduced in Slc6a8 and Agat mutants. Cr inhibited neocortical pyramidal neurons. SLC6A8 was necessary for Cr uptake into synaptosomes. Cr was found by us to be taken up into SVs in an ATP-dependent manner. Our biochemical, chemical, genetic, and electrophysiological results are consistent with the possibility of Cr as a neurotransmitter, though not yet reaching the level of proof for the now classic transmitters. Our novel approach to discover neurotransmitters is to begin with analysis of contents in SVs before defining their function and physiology.

Myeloid cell-derived creatine in the hypoxic niche promotes glioblastoma growth

Glioblastoma (GBM) is a malignancy dominated by the infiltration of tumor-associated myeloid cells (TAMCs). Examination of TAMC metabolic phenotypes in mouse models and patients with GBM identified the de novo creatine metabolic pathway as a hallmark of TAMCs. Multi-omics analyses revealed that TAMCs surround the hypoxic peri-necrotic regions of GBM and express the creatine metabolic enzyme glycine amidinotransferase (GATM). Conversely, GBM cells located within these same regions are uniquely specific in expressing the creatine transporter (SLC6A8). We hypothesized that TAMCs provide creatine to tumors, promoting GBM progression. Isotopic tracing demonstrated that TAMC-secreted creatine is taken up by tumor cells. Creatine supplementation protected tumors from hypoxia-induced stress, which was abrogated with genetic ablation or pharmacologic inhibition of SLC6A8. Lastly, inhibition of creatine transport using the clinically relevant compound, RGX-202-01, blunted tumor growth and enhanced radiation therapy invivo. This work highlights that myeloid-to-tumor transfer of creatine promotes tumor growth in the hypoxic niche.

Evidence of an intracellular creatine-sensing mechanism that modulates creatine biosynthesis via AGAT expression in human HAP1 cells

Cellular homeostasis of creatine (CT), integral part of the energy buffering and transducing system connecting intracellular sites of ATP production and utilization, comprises of mechanisms that increase CT, i.e., biosynthesis and cellular uptake, and CT-lowering processes, such as export and non-enzymatic conversion to creatinine. The biosynthesis of CT is controlled by negative feedback loop via suppression of the rate-limiting enzyme arginine:glycine amidinotransferase (AGAT). Although the regulatory mechanism involved is not well understood, AGAT suppression is successfully used in patients with guanidinoacetate methyltransferase (GAMT) deficiency to reduce the neurotoxic accumulation of the AGAT-mediated guanidinoacetate production by supplementing patients with CT. Utilizing the CT-dependent feedback loop for the upregulation of AGAT expression may well represent a therapeutic target for an additional CT deficiency syndrome, the CT transporter (CrT) defect, for which no effective treatment option is available so far. We have used CRISPR to tag the C-terminus of AGAT with a nanoluc luciferase (NLuc) reporter in HAP1 cells. A biphasic decay of AGAT-NLuc in response to increasing extracellular CT was observed, whereas the decrease in AGAT-NLuc expression was directly proportional to the rise in intracellular CT levels with an approximate IC50 of 1–2 mM. CRISPR generated HAP1 CrT null cells and HAP1 CrT null cells stably expressing a CrT-GFP fusion protein further demonstrated that the biphasic response to extracellular CT is mediated by a high-affinity (Km 9–10 µM) CrT dependent, saturable mechanism and a CrT independent, unsaturable uptake process. The direct response to intracellular CT suggests the existence of an intracellular CT sensing system enabling a dynamic cell response to changing CT concentration that is relevant for cellular CT homeostasis.

Molecular Mechanism of ε-Polylysine Treatment of Animal-Derived Foods: Glycine Amidinotransferase Activity Implicates Upregulation of l-Arginine and Creatine.

ε-Polylysine is a novel food preservative approved by the U.S. Food and Drug Administration (FDA), yet the mechanism of its effect on animal-derived foods remains unclear. Assessment of the effect of preservatives on goat meat products is necessary. Herein, metabolite accumulation and protein expression of ε-polylysine (0.025%, w/w) spiked with goat meat were investigated by nontarget metabolomics and proteomics combined with ultrahigh performance liquid chromatography quadrupole-Orbitrap high-resolution-mass spectrometry (UHPLC-Q-Orbitrap HRMS) in a simulated in vitro digestion model. The amino side chain of ε-polylysine increased the activity of glycine aminotransferase due to its nucleophilic nature, inducing a significant upregulation of l-arginine (0.43-0.72 mg kg-1) and creatine (3.98-6.89 mg kg-1), with an improvement in muscle quality of goat meat. Downregulation of enzyme phenylalanine hydroxylase expression led to upregulation of l-phenylalanine (2.26-3.25 mg kg-1) and l-tyrosine (0.98-1.29 mg kg-1). Collectively, this study first revealed the biochemical mechanism of ε-polylysine in goat meat products, which makes available new prospects for more accurate use of ε-polylysine in animal-derived foods.

Systemic Effects of Homoarginine Supplementation on Arginine Metabolizing Enzymes in Rats with Heart Failure with Preserved Ejection Fraction.

A restoration of low homoarginine (hArg) levels in obese ZSF1 rats (O-ZSF1) before (S1-ZSF1) and after (S2-ZSF1) the manifestation of heart failure with preserved ejection fraction (HFpEF) did not affect the worsening of cardiac HFpEF characteristics. Here, potential regulation of key enzymes of arginine metabolism in other organs was analyzed. Arginase 2 (ARG2) was reduced >35% in the kidney and small intestine of hArg-supplemented rats compared to O-ZSF1. Glycine amidinotransferase (GATM) was 29% upregulated in the kidneys of S1-ZSF1. Dimethylarginine dimethylaminohydrolase 1 (DDAH1) levels were reduced >50% in the livers of O-ZSF1 but restored in S2-ZSF1 compared to healthy rats (L-ZSF1). In the skeletal muscle, iNOS was lower in O-ZSF1 and further decreased in S1-ZSF1 and S2-ZSF1 compared to L-ZSF1. iNOS levels were lower in the liver of the S2-ZSF1 group but higher in the kidneys of S1-ZSF1 compared to L-ZSF1. Supplementation with hArg in an in vivo HFpEF model resulted in the inhibition of renal ARG2 and an increase in GATM expression. This supplementation might contribute to the stabilization of intestinal iNOS and ARG2 imbalances, thereby enhancing barrier function. Additionally, it may offer protective effects in skeletal muscle by downregulating iNOS. In the conceptualization of hArg supplementation studies, the current disease progression stage as well as organ-specific enzyme regulation should be considered.

Walnut consumption and gut microbial metabolism: Results of an exploratory analysis from a randomized, crossover, controlled-feeding study

The effect of walnut-related modulation of gut microbiota composition on microbiota functionality is unknown. The aim was to characterize the effect of a walnut-enriched diet (WD), compared to a fatty acid-matched diet devoid of walnuts (WFMD) and a diet where oleic acid replaces alpha-linolenic acid (ORAD), on bacterial gene expression. A 3-period, randomized, crossover, controlled-feeding study was conducted. Participants were provided a 2-week run-in standard western diet (SWD; 50% kcal carbohydrate, 16% protein, 34% fat, 12% SFA). Following the SWD in random sequence order, participants were provided the WD, WFMD, and ORAD (48% carbohydrate; 17% protein; fat 35%; 7% SFA). The WD contained 18% of energy from walnuts (57g/d/2100kcal). The WFMD and ORAD were devoid of walnuts; liquid non-tropical plant oils were included in these diets. Metatranscriptomic analyses were performed as an exploratory outcome. The analytical sample included 35 participants (40% female) with a mean±SD age of 43±10y and BMI of 30.3±4.9kg/m2. The ⍺-diversity of taxa actively expressing genes, assessed by observed species (p=0.27) and Pielou's Evenness (p=0.09), did not differ among the diets. The ⍺-diversity of actively expressed genes was greater following the WD compared to the WFMD and ORAD as assessed by the observed genes and Pielou's Evenness metrics (p<0.05). β-Diversity of the actively expressed genes differed following the WD compared to the WFMD (p=0.001) and ORAD (p=0.001); β-diversity did not differ between the WFMD and ORAD. Active composition analyses showed increased Gordonibacter (p<0.001) activity following the WD vs. the ORAD. Greater expression of many genes was observed following the WD compared to the WFMD and ORAD. Following the WD, greater expression of metabolism-related genes encoding glycine amidinotransferase (GATM; K00613) and arginine deiminase (K01478) was observed compared to the WFMD. Greater expression of glycine amidinotransferase (GATM; K00613) by Gordonibacter was also observed following the WD vs. the WFMD and ORAD. Our results suggest walnut intake may increase endogenous production of homoarginine through gut microbiota-mediated upregulation of GATM, which is a novel mechanism by which walnuts may lower cardiovascular disease risk. However, given the exploratory nature replication is needed. Clinicaltrials.gov (NCT02210767).

Aspects of human uterine creatine metabolism during the menstrual cycle and at term pregnancy†.

Creatine metabolism likely contributes to energy homeostasis in the human uterus, but whether this organ synthesizes creatine and whether creatine metabolism is adjusted throughout the menstrual cycle and with pregnancy are largely unknown. This study determined endometrial protein expression of creatine-synthesizing enzymes arginine:glycine amidinotransferase (AGAT) and guanidinoacetate methyltransferase (GAMT), creatine kinase (CKBB), and the creatine transporter (SLC6A8) throughout the menstrual cycle in fertile and primary infertile women. It also characterized creatine metabolism at term pregnancy, measuring aspects of creatine metabolism in myometrial and decidual tissue. In endometrial samples, AGAT, GAMT, SLC6A8, and CKBB were expressed in glandular and luminal epithelial cells. Except for SLC6A8, the other proteins were also located in stromal cells. Irrespective of fertility, AGAT, GAMT, and SLC6A8 high-intensity immunohistochemical staining was greatest in the early secretory phase of the menstrual cycle. During the proliferative phase, staining for SLC6A8 protein was greater (P = 0.01) in the primary infertile compared with the fertile group. Both layers of the term pregnant uterus contained creatine, phosphocreatine, guanidinoacetic acid, arginine, glycine, and methionine; detectable gene and protein expression of AGAT, GAMT, CKBB, and ubiquitous mitochondrial CK (uMt-CK); and gene expression of SLC6A8. The proteins AGAT, GAMT, CKBB, and SLC6A8 were uniformly distributed in the myometrium and localized to the decidual glands. In conclusion, endometrial tissue has the capacity to produce creatine and its capacity is highest around the time of fertilization and implantation. Both layers of the term pregnant uterus also contained all the enzymatic machinery and substrates of creatine metabolism.