Amino Acid Homeostasis Research Articles

GPRC6A Mediates Glucose and Amino Acid Homeostasis in Mice.

GPRC6A, an important member of the G-protein-coupled receptor superfamily, has been widely studied in body health maintenance and related diseases. However, it is still controversial whether GPRC6A plays a vital role in glucose homeostasis, and the role of GPRC6A on amino acid homeostasis has not been reported. In this study, GPRC6A was knocked out in C57BL6 mice, and we found that GPRC6A plays an important role in the glucose metabolism, mainly affecting the glucose clearance capacity and gluconeogenesis in mice. GPRC6A plays an important role in maintaining amino acid homeostasis under dietary restrictions, and this may be realized by participating in the regulation of autophagy. Since a large amount of amino acid is lost from urine in aged GPRC6A−/− mice, it is possible that GPRC6A regulates amino acid homeostasis by affecting the integrity of tissue structure. GPRC6A is involved in the regulation of mTORC1 activation but is not necessary for mTORC1 activation under sufficient nutritional supply. In the absence of exogenous amino acids, the loss of GPRC6A induces the GCN2 pathway activation and excessive autophagy of cells, leading to the overactivation of mTORC1, which may be detrimental to body health and cell survival. In summary, this study provides a theoretical and experimental basis for the metabolic process of GPRC6A in body growth and health.

Metabolic profiling as a powerful tool for the analysis of cellular alterations caused by 20 mycotoxins in HepG2 cells

Mycotoxins are secondary fungal metabolites which exhibit toxic effects in low concentrations. Several mycotoxins are described as carcinogenic or immunosuppressive, but their underlying modes of action especially on molecular level have not yet been entirely elucidated. Metabolic profiling as part of the omics methods is a powerful tool to study the toxicity and the mode of action of xenobiotics. The use of hydrophilic interaction chromatography in combination with targeted mass spectrometric detection enables the selective and sensitive analysis of more than 100 polar and ionic metabolites and allows the evaluation of metabolic alterations caused by xenobiotics such as mycotoxins. For metabolic profiling, the hepato-cellular carcinoma cell line HepG2 was treated with sub-cytotoxic concentrations of 20 mycotoxins. Moniliformin and citrinin significantly affected target elements of the citric acid cycle, but also influenced glycolytic pathways and energy metabolism. Penitrem A, zearalenone, and T2 toxin mainly interfered with the urea cycle and the amino acid homeostasis. The formation of reactive oxygen species seemed to be influenced by T2 toxin and gliotoxin. Glycolysis was altered by ochratoxin A and DNA synthesis was affected by several mycotoxins. The observed effects were not limited to these metabolic reactions as the metabolic pathways are closely interrelated. In general, metabolic profiling proved to be a highly sensitive tool for hazard identification in comparison to single-target cytotoxicity assays as metabolic alterations were already observed at sub-toxic concentrations. Metabolic profiling could therefore be a powerful tool for the overall evaluation of the toxic properties of xenobiotics.

Metabolic indicators of an altered sphingolipid metabolism are biomarkers for non-alcoholic fatty liver disease

Background and Aims : Non-alcoholic fatty liver disease (NAFLD) is the most frequent cause of chronic liver disease in the western world. Steatosis can be accompanied by inflammation and cell damage (non-alcoholic steatohepatitis, NASH) as well as fibrosis. Sphingolipids and in particular an atypical class of 1-deoxysphingolipids have been associated with NAFLD. Increased 1-deoxySL formation is typically related to an altered amino acid homeostasis. We therefore analyzed the metabolic background that is associated with the increased formation of these lipids in NAFLD.

Transcriptome Analysis of Hepatopancreas Provides Insights into Differential Metabolic Mechanisms of Eriocheir sinensis Feeding on Trash Fish and Formulated Feed.

The Chinese mitten crab, Eriocheir sinensis (E. sinensis), is a popular crab species in both domestic and foreign markets. Trash fish are essential for E. sinensis breeding, but have caused serious water pollution. The municipal party committee for the main production areas of E. sinensis implemented a ban on feeding on trash fish since 2020. In this study, we performed a culture experiment of E. sinensis feeding on trash fish and formulated feed, with comparative transcriptome analysis on hepatopancreas of E. sinensis. The results indicate that formulated feed causes no significant difference in growth, survival rate or content of amino acids in the muscles of adult E. sinensis. Formulated feed caused a slight downregulation of pathways involved in amino acid metabolism, development, energy metabolism and homeostasis maintenance. On the whole, formulated feed can serve as an undifferentiated substitution for trash fish. This study provides a theoretical foundation for optimizing research on E. sinensis feed.

Role of the conserved pyridoxal 5'-phosphate-binding protein YggS/PLPBP in vitamin B6 and amino acid homeostasis.

The YggS/PLPBP protein (also called COG0325 or PLPHP) is a conserved pyridoxal 5'-phosphate (PLP)-binding protein present in all 3 domains of life. Recent studies have demonstrated that disruption or mutation of this protein has multifaceted effects in various organisms, including vitamin B6-dependent epilepsy in humans. In Escherichia coli, disruption of this protein-encoded by yggS-perturbs Thr-Ile/Val metabolism, one-carbon metabolism, coenzyme A synthesis, and vitamin B6 homeostasis. This protein is critical for maintaining low levels of pyridoxine 5'-phosphate (PNP) in various organisms. In the yggS-deficient E. coli strain, inhibition of PLP-dependent enzymes, such as the glycine cleavage system by PNP, is the root cause of metabolic perturbation. Our data suggest that the YggS/PLPBP protein may be involved in the balancing of B6 vitamers by mediating efficient turnover of protein-bound B6 vitamers. This paper reviews recent findings on the function of the YggS/PLPBP protein.

Amino Acid Homeostasis and Fatigue in Chronic Hemodialysis Patients.

Patients dependent on chronic hemodialysis treatment are prone to malnutrition, at least in part due to insufficient nutrient intake, metabolic derangements, and chronic inflammation. Losses of amino acids during hemodialysis may be an important additional contributor. In this study, we assessed changes in plasma amino acid concentrations during hemodialysis, quantified intradialytic amino acid losses, and investigated whether plasma amino acid concentrations and amino acid losses by hemodialysis and urinary excretion are associated with fatigue. The study included a total of 59 hemodialysis patients (65 ± 15 years, 63% male) and 33 healthy kidney donors as controls (54 ± 10 years, 45% male). Total plasma essential amino acid concentration before hemodialysis was lower in hemodialysis patients compared with controls (p = 0.006), while total non-essential amino acid concentration did not differ. Daily amino acid losses were 4.0 ± 1.3 g/24 h for hemodialysis patients and 0.6 ± 0.3 g/24 h for controls. Expressed as proportion of protein intake, daily amino acid losses of hemodialysis patients were 6.7 ± 2.4% of the total protein intake, compared to 0.7 ± 0.3% for controls (p < 0.001). Multivariable regression analyses demonstrated that hemodialysis efficacy (Kt/V) was the primary determinant of amino acid losses (Std. β = 0.51; p < 0.001). In logistic regression analyses, higher plasma proline concentrations were associated with higher odds of severe fatigue (OR (95% CI) per SD increment: 3.0 (1.3; 9.3); p = 0.03), while higher taurine concentrations were associated with lower odds of severe fatigue (OR (95% CI) per log2 increment: 0.3 (0.1; 0.7); p = 0.01). Similarly, higher daily taurine losses were also associated with lower odds of severe fatigue (OR (95% CI) per log2 increment: 0.64 (0.42; 0.93); p = 0.03). Lastly, a higher protein intake was associated with lower odds of severe fatigue (OR (95% CI) per SD increment: 0.2 (0.04; 0.5); p = 0.007). Future studies are warranted to investigate the mechanisms underlying these associations and investigate the potential of taurine supplementation.

Abstract 5824: Fully-humanized, bioengineered miR-1291 modulates key nutrient transport and metabolism to control tumor metabolism

Abstract Tumor progression is accompanied by metabolic reprogramming to allow carcinoma cells to access essential nutrients under the tumor microenvironment. To meet energetic and material needs for high-rate proliferation and tumorigenesis, cancer cells generally rely more heavily on vital nutrients (e.g., glucose, amino acids, and vitamins) than normal cells. Therefore, controlling nutrient transport and metabolism in cancer cells is a promising therapeutic strategy. Our previous studies have suggested that microRNA-1291-5p (miR-1291-5p or miR-1291) is downregulated in pancreatic cancer (PC) cells and acts as a tumor suppressor. The current study is to delineate the role of miR-1291 in the control of PC cell metabolism and develop miR-1291 therapy. We first employed our newly-invented RNA bioengineering technology to achieve high-level and large-scale, in vivo fermentation production of a fully-humanized biologic miR-1291 agent at a high degree of homogeneity (&amp;gt; 4 mg BioRNA per liter of culture and &amp;gt; 97% purity). RNA sequencing studies revealed that BioRNA/miR-1291 was selectively processed to target miR-1291-5p to modulate the transcriptome in PC cells. Proteomics study showed that a number of proteins were reduced remarkably in AsPC-1 cells by biologic miR-1291-5p, among which PNPO, an enzyme that mediates the synthesis of essential nutrient vitamin B6 (VB6), was revealed as a leading target of miR-1291-5p. Besides PNPO and the previously-reported glucose transporter SLC2A1/GLUT1, amino acid transporter SLC7A5/LAT1 was verified as a direct target for miR-1291-5p. Downregulation of PNPO, LAT1, GLUT1 protein expression by biological miR-1291 led to sharp alteration of homeostasis of glucose, amino acids, and VB6 in human PC cells, and subsequent suppression of glycolysis capacity and mitochondrial function as well as the increase of oxidative stress. Combination treatment with bioengineered miR-1291 and 5-FU exerted strong synergism in the inhibition of PC cell and organoid proliferation in vitro. In addition, miR-1291 mono- and combination therapy were effective to control tumor growth in pancreatic cancer patient-derived xenograft (PDX) mouse models. These results demonstrate a critical role for miR-1291 in the regulation of PC cell metabolism which provides insight in developing new therapeutic strategies for PC. Citation Format: Mei-Juan Tu, Frank J. Gonzalez, Ai-Ming Yu. Fully-humanized, bioengineered miR-1291 modulates key nutrient transport and metabolism to control tumor metabolism [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5824.

Short-Term High-Fat Diet Fuels Colitis Progression in Mice Associated With Changes in Blood Metabolome and Intestinal Gene Expression.

Clinical cases and animal experiments show that high-fat (HF) diet is involved in inflammatory bowel disease (IBD), but the specific mechanism is not fully clear. A close association between long-term HF-induced obesity and IBD has been well-documented. However, there has been limited evaluation of the impact of short-term HF feeding on the risk of intestinal inflammation, particularly on the risk of disrupted metabolic homeostasis. In this study, we analyzed the metabolic profile and tested the vulnerability of 2,4,6-trinitrobenzenesulfonic acid (TNBS)-induced colitis after short-term HF feeding in mice. The results showed that compared with the control diet (CD), the fatty acid (FA), amino acid (AA), and bile acid (BA) metabolisms of mice in the HF group were significantly changed. HF-fed mice showed an increase in the content of saturated and unsaturated FAs and a decrease in the content of tryptophan (Trp). Furthermore, the disturbed spatial distribution of taurocholic acid (TCA) in the ileum and colon was identified in the HF group using matrix-assisted laser desorption/ionization-mass spectrometry imaging (MALDI-MSI). After HF priming, mice on TNBS induction were subjected to more severe colonic ulceration and histological damage compared with their CD counterparts. In addition, TNBS enema induced higher gene expressions of mucosal pro-inflammatory cytokines under HF priming conditions. Overall, our results show that HF may promote colitis by disturbing lipid, AA, and BA metabolic homeostasis and inflammatory gene expressions.

191-OR: Hepatic Expression of the Branched-Chain Keto Acid Dehydrogenase Kinase Is Sufficient to Cause Glucose Intolerance in Lean Mice

Perturbed branched-chain amino acid homeostasis is a feature of obesity, insulin-resistance and type 2 diabetes. Induction of the branched-chain keto acid dehydrogenase kinase (BDK) in liver is one mechanism that is thought to contribute to elevated BCAA levels in these conditions. Whether higher levels of BDK in liver are sufficient to disturb systemic BCAA homeostasis and promote glucose intolerance in the absence of obesity or insulin resistance has never been directly tested. We treated 12 week-old C57BL6/J mice with adeno-associated virus, serotype 8 (AAV8) -BDK or control AAV8-GFP to promote long term expression of BDK specifically in liver of lean mice. Mice were then maintained on a standard low-fat chow diet for 8 weeks (n= 9 AAV8-GFP and n=AAV8-BDK) . AAV8-BDK significantly raised BDK protein and mRNA expression in liver but not adipose, skeletal muscle, or cardiac tissue compared to AAV8-GFP control mice. The higher expression of BDK in liver was associated with robust inhibitory phosphorylation of the branched-chain keto acid dehydrogenase (BCKDH) on ser293. AAV8-mediated induction of BDK expression in liver in our study was sufficient to promote significant elevations in circulating levels of the direct substrate of BCKDH, the branched chain α-keto acids as well as their cognate BCAA. There was no effect of AAV8-BDK to alter food intake, weight gain or adiposity. However, glucose tolerance measured by a 1g/kg intraperitoneal glucose tolerance test at week 7 was significantly impaired in mice receiving AAV8-BDK compared to AAV8-GFP control. These data demonstrate for the first time that induction of BDK in liver, as occurs with obesity, is sufficient to disturb systemic BCAA and glucose homeostasis in lean mice. Disclosure J.Walejko: None. R.W.Mcgarrah: None. P.J.White: None. Funding American Diabetes Association (1-16-INI-17)

1353-P: Activation of Brown Adipose Tissue by Low-Protein Diet Ameliorates Hyperglycemia in Lipodystrophic Diabetic Mice Model

Long-term ad libitum dietary restriction such as low-protein diet (LPD) improves metabolic health and extends the life span of mice and possibly humans. However, most studies conducted thus far have focused on the preventive, but not therapeutic, potential of LPDs. Previously we shown that LPD (5.1% kcal from protein) was able to improve postprandial blood glucose values of the lipodystrophic IRFKO (adipose-specific insulin receptor knockout) mice after 14 days with strong activation of interscapular brown adipose tissue (iBAT) and increase energy expenditure. To determine whether iBAT activation was responsible for the hyperglycemia normalization after the LPD treatment, we surgically denervated iBAT of IRFKO mice before LPD treatment. Blood glucose were measured twice a week for up to 4 weeks, at the end of the monitoring mice were subjected to indirect calorimetry assessment (Promethion, Sable Systems) . Our data showed that surgical denervation of iBAT abolished the hyperglycemia normalization effect of LPD in the IRFKO mice. Morphologically, surgical denervation also prevented the ‘browning’ effect of LPD on the whitening iBAT observed in IRFKO mice. In line with the morphological data, LPD failed to induce thermogenic markers including UCP1 and BMP8b in denervated iBAT. In contrast, iBAT denervation has lesser effects on the LPD-induced energy expenditure and circulating FGF21 level, possibly due to compensation by beige fat formation in the inguinal WAT. Together, our data confirms that hyperglycemia normalization caused by LPD on IRFKO is iBAT activation dependent. Our observation suggests that blood glucose could be utilized to fuel LPD-induced iBAT activation as a mechanism for amino acid homeostasis. Further experimentation needs to be done in order to narrow down the mechanism driving this phenomenon. Taken together, our data suggest that short-term LPD could be a potential strategy for the treatment of metabolic syndrome. Disclosure M.D.Munoz: None. A.Zamudio: None. M.A.Mccann: None. C.Liew: None. Funding NIH RDK109015A1

The Drosophila melanogaster enzyme glycerol-3-phosphate dehydrogenase 1 is required for oogenesis, embryonic development, and amino acid homeostasis

As the fruit fly, Drosophila melanogaster, progresses from one life stage to the next, many of the enzymes that compose intermediary metabolism undergo substantial changes in both expression and activity. These predictable shifts in metabolic flux allow the fly meet stage-specific requirements for energy production and biosynthesis. In this regard, the enzyme glycerol-3-phosphate dehydrogenase 1 (GPDH1) has been the focus of biochemical genetics studies for several decades and, as a result, is one of the most well-characterized Drosophila enzymes. Among the findings of these earlier studies is that GPDH1 acts throughout the fly lifecycle to promote mitochondrial energy production and triglyceride accumulation while also serving a key role in maintaining redox balance. Here, we expand upon the known roles of GPDH1 during fly development by examining how depletion of both the maternal and zygotic pools of this enzyme influences development, metabolism, and viability. Our findings not only confirm previous observations that Gpdh1 mutants exhibit defects in larval development, lifespan, and fat storage but also reveal that GPDH1 serves essential roles in oogenesis and embryogenesis. Moreover, metabolomics analysis reveals that a Gpdh1 mutant stock maintained in a homozygous state exhibits larval metabolic defects that significantly differ from those observed in the F1 mutant generation. Overall, our findings highlight unappreciated roles for GPDH1 in early development and uncover previously undescribed metabolic adaptations that could allow flies to survive the loss of this key enzyme.

Amino acid balance of microbial-tissue complex in the small intestinal and liver under administration of lead acetate

It is known that the initiating event of liver damage during lead intoxication is the effect of lead on the intestinal microbiome and the metabolic profile of enterocytes. The aim of the study was to reveal the dependence of the concentrations of free amino acids and their derivatives in the liver on the amino acid pool of the microbial-tissue complex of the small intestine. We used rats weighing 120‒140 g, which were injected intragastrically for three weeks with drinking water with lead acetate. Free amino acids and their nitrogen-containing metabolites in the microbial-tissue complex of the intestine and liver were determined by HPLC. Enteral intake of lead acetate with drinking water changes the profile of free amino acids and their derivatives in the microbial-tissue complex of the small intestine, which correlates with a violation of amino acid homeostasis in the liver. The analysis of the correlations of the indicators we determined indicates the essential role of nitrogen-containing metabolites of amino acids ‒ ethanolamine and phosphoethanolamine, taurine, and cystathionine. The direction of the correlation relationship between various nitrogen-containing metabolites of the microbial-tissue complex and the liver can be considered as marker of discoordination of homeostasis.

Elucidating the cellular mechanism of methionine toxicity

Cells possess several mechanisms to maintain amino acid homeostasis including regulation of amino acid permease trafficking, protein synthesis, metabolic reactions, and storage within the lysosome or vacuole. With aging and in certain diseases these processes are impaired, leading to a toxic accumulation of amino acids. While it is well‐established that excess amino acids are problematic for cells, it is poorly understood how individual amino acids drive cellular dysfunction and how cells combat toxic levels of these metabolites. To begin to elucidate cellular mechanisms of amino acid toxicity, we focused our analysis on methionine, because of its unique role in translation, metabolism, and as a precursor for the methyl donor, s‐adenosylmethionine. Additionally, methionine restriction has been shown to extend lifespan and delay progression or improve disease phenotypes, suggesting that methionine itself can be problematic to organisms.To study methionine toxicity, we first identified yeast strains that were sensitive to the amino acid. We genetically modulated pathways cells use to traffic two amino acid permeases – the high‐affinity methionine permease (Mup1) and the general amino acid permease (Gap1). These permeases are targeted for removal from the plasma membrane by ubiquitination of N‐terminal lysine residues, which is facilitated by an α‐arrestin protein. Mup1 internalization was inhibited by knocking out the α‐arrestin, LDB19, and Gap1 internalization was blocked usingGAP1K9R,K16R. Compared to wildtype, these mutants were greatly sensitized to exogenous methionine, measured by growth inhibition, highlighting the importance of modulating methionine levels to maintain cellular health. To explore mechanisms of methionine toxicity, we conducted RNA‐sequencing and found that excess methionine leads to an upregulation of genes under transcriptional regulation of Hsf1 and Gcn4. These transcription factors are activated in response to disruptions in protein homeostasis and tRNA charging, respectively. In addition to Hsf1 activation, we observed aggregation of heat shock proteins, Hsp104 and Hsp42, via fluorescent microscopy. Genetic depletion of Hsf1 using a tet‐repressible allele led to diminished growth on methionine compared to control cells, suggesting there is an increased requirement for Hsf1 when cells are exposed to excess methionine. Finally, we found a decrease in total protein when cells are exposed to excess methionine. This could be explained by the activation of Gcn4 which is activated in response to alternations in tRNA homeostasis and would reduce translation rates.The reasons for methionine toxicity are currently unknown, but our data suggests that excess methionine drives proteotoxicity and may perturb tRNA charging homeostasis. This work provides us with preliminary evidence for the mechanisms through which methionine imparts toxicity and how cells respond to excess levels of this amino acid.

Targeting GCN2 Regulation of Amino Acid Homeostasis in Prostate Cancer

The Integrated Stress Response (ISR) plays a critical role in the adaptation and survival of tumor cells to exogenous and endogenous stresses. The ISR features four protein kinases (PERK, GCN2, PKR, and HRI), each activated by different stresses, that phosphorylate the eukaryotic translation initiation factor eIF2, resulting in repression of global protein synthesis. Paradoxically, eIF2 phosphorylation also enhances translation of select gene transcripts, including the transcription factor ATF4, which is central for ISR‐directed gene transcription. Therefore, the ISR directs translational and transcriptional control that is critical for cancer stress adaptation. Moreover, eIF2 phosphorylation and ATF4 have recently been suggested to play a role in prostate cancer (PCa) growth and survival; however, the specific function of ISR kinases, their mode of activation, and the mechanisms by which the ISR facilitate PCa progression are unknown.We discovered that GCN2 is activated in a range of PCa cell lines, contributing to enhanced eIF2 phosphorylation and ATF4 expression. Genetic or pharmacological inhibition of GCN2 reduces growth in androgen‐sensitive and castration‐resistant PCa cell lines in culture and cell line‐derived and patient‐derived xenograft mouse models in vivo. Induction of GCN2 is accompanied by limitations of select amino acids and accumulation of cognate tRNAs that are reported to be activators of GCN2.A transcriptome analysis of PCa cells treated with a specific GCN2 small molecular inhibitor indicates that GCN2 is critical for expression of SLCgenes involved in metabolite transport. We found that GCN2 inhibition decreases intracellular amino acid levels accounting for reduced growth in PCa cells. Using CRISPR‐based phenotypic screens and genome‐wide gene expression analyses of wild‐type and GCN2‐depleted PCa cells, we confirmed the importance of the transporter genes in PCa fitness. One transporter, SLC3A2 (4F2), is induced by GCN2 and is essential for PCa proliferation. SLC3A2 engages with many nutrient transporters, allowing for their localization to the plasma membrane. Importantly, expression of SLC3A2 reduced GCN2 activation and rescued decreased amino acid levels and growth inhibition due to loss of GCN2.Our results indicate that select amino acid limitations activate GCN2 in PCa, resulting in the upregulation of key amino acid transporters, including 4F2 (SLC3A2), which provide for nutrient import to facilitate protein synthesis and metabolism required for PCa progression. We conclude that GCN2 and the ISR are promising therapeutic targets for both androgen‐sensitive and castration‐resistant prostate cancer.

Pilot Study on Acute Effects of Pharmacological Intraperitoneal L-Homoarginine on Homeostasis of Lysine and Other Amino Acids in a Rat Model of Isoprenaline-Induced Takotsubo Cardiomyopathy.

L-Arginine:glycine amidinotransferase (AGAT) catalyzes the formation of L-homoarginine (hArg) and L-ornithine (Orn) from L-arginine (Arg) and L-lysine (Lys): Arg + Lys ↔ hArg + Orn; equilibrium constant KhArg. AGAT also catalyzes the formation of guanidinoacetate (GAA) and Orn from Arg and glycine (Gly): Arg + Gly ↔ GAA + Orn; equilibrium constant KGAA. In humans, pharmacological hArg is metabolized to Lys. Low circulating and low excretory concentrations of hArg are associated with worse outcomes and mortality in the renal and cardiovascular systems. The metabolism and pharmacology of hArg have been little investigated. In the present study, we investigated the effects of pharmacological hArg (i.p., 0, 20, 220, 440 mg/kg at time point 0 min) on amino acids homeostasis in a rat model of isoprenaline-induced takotsubo cardiomyopathy (i.p., 50 mg/kg at time point 15 min). We measured by gas chromatography-mass spectrometry free and proteinic amino acids, as well as the polyamines putrescine and spermidine in the heart, lung, kidney, and liver of ten rats sacrificed at various time points (range, 0 to 126 min). hArg administration resulted in multiple changes in the tissue contents of several free and proteinic amino acids, as well as in the putrescine-spermidine molar ratio, an indicator of polyamines catabolism. Our results suggest that Lys and Arg are major metabolites of pharmacological hArg. Kidneys and heart seem to play a major metabolic role for hArg. Circulating Lys does not change over time, yet there is a considerable interchange of free Lys between organs, notably kidney and heart, during the presence of isoprenaline in the rats (time range, 15 to 90 min). Antidromic changes were observed for KhArg and KGAA, notably in the heart in this time window. Our study shows for the first time that free hArg and sarcosine (N-methylglycine) are positively associated with each other. The acute effects of high-dosed hArg administration and isoprenaline on various amino acids and on AGAT-catalyzed reaction in the heart, lung, kidney, and liver are detailed and discussed.

Untargeted metabolomics identifies the potential role of monocarboxylate transporter 6 (MCT6/SLC16A5) in lipid and amino acid metabolism pathways.

Monocarboxylate transporter 6 (MCT6; SLC16A5) is an orphan transporter protein with expression in multiple tissues. The endogenous function of MCT6 related to human health and disease remains unknown. Our previous transcriptomic and proteomic analyses in Mct6 knockout (KO) mice suggested that MCT6 may play a role in lipid and glucose homeostasis, but additional evidence is required. Thus, the objective of this study was to further explore the impact of MCT6 on metabolic function using untargeted metabolomic analysis in Mct6 KO mice. The plasma from male and female mice and livers from male mice were submitted for global metabolomics analysis to assess the relative changes in endogenous small molecules across the liver and systemic circulation associated with absence of Mct6. More than 782 compounds were detected with 101 and 51 metabolites significantly changed in plasma of male and female mice, respectively, and 100 metabolites significantly changed in the livers of male mice (p < .05). Significant perturbations in lipid metabolism were annotated in the plasma and liver metabolome, with additional alterations in the amino acid metabolism pathway in plasma samples from male and female mice. Elevated lipid diacylglycerol and altered fatty acid metabolite concentrations were found in liver and plasma samples of male Mct6 KO mice. Significant reduction of N‐terminal acetylated amino acids was found in plasma samples of male and female Mct6 KO mice. In summary, the present study confirmed the significant role of MCT6 in lipid and amino acid homeostasis, suggesting its contribution in metabolic diseases.

The Role of Amino Acids in Endothelial Biology and Function.

The vascular endothelium acts as an important component of the vascular system. It is a barrier between the blood and vessel wall. It plays an important role in regulating blood vessel tone, permeability, angiogenesis, and platelet functions. Several studies have shown that amino acids (AA) are key regulators in maintaining vascular homeostasis by modulating endothelial cell (EC) proliferation, migration, survival, and function. This review summarizes the metabolic and signaling pathways of AAs in ECs and discusses the importance of AA homeostasis in the functioning of ECs and vascular homeostasis. It also discusses the challenges in understanding the role of AA in the development of cardiovascular pathophysiology and possible directions for future research.

Proteomics Approach Highlights Early Changes in Human Fibroblasts-Pancreatic Ductal Adenocarcinoma Cells Crosstalk.

Pancreatic ductal adenocarcinoma (PDAC) is a leading cause of cancer mortality worldwide. Non-specific symptoms, lack of biomarkers in the early stages, and drug resistance due to the presence of a dense fibrous stroma all contribute to the poor outcome of this disease. The extracellular matrix secreted by activated fibroblasts contributes to the desmoplastic tumor microenvironment formation. Given the importance of fibroblast activation in PDAC pathology, it is critical to recognize the mechanisms involved in the transformation of normal fibroblasts in the early stages of tumorigenesis. To this aim, we first identified the proteins released from the pancreatic cancer cell line MIA-PaCa2 by proteomic analysis of their conditioned medium (CM). Second, normal fibroblasts were treated with MIA-PaCa2 CM for 24 h and 48 h and their proteostatic changes were detected by proteomics. Pathway analysis indicated that treated fibroblasts undergo changes compatible with the activation of migration, vasculogenesis, cellular homeostasis and metabolism of amino acids and reduced apoptosis. These biological activities are possibly regulated by ITGB3 and TGFB1/2 followed by SMAD3, STAT3 and BAG3 activation. In conclusion, this study sheds light on the crosstalk between PDAC cells and associated fibroblasts. Data are available via ProteomeXchange with identifier PXD030974.

The Rice Aspartyl-tRNA Synthetase YLC3 Regulates Amino Acid Homeostasis and Chloroplast Development Under Low Temperature.

Aminoacyl tRNA synthetases primarily function to attach specific amino acids to the corresponding tRNAs during protein translation. However, their roles in regulating plant growth and development still remain elusive. Here we reported a rice thermo-sensitive mutant yellow leaf chlorosis3 (ylc3) with reduced chlorophyll content, altered thylakoid structure, and substantially elevated levels of free aspartate, asparagine and glutamine in leaves under low temperature condition. Map-based cloning identified that YLC3 encodes an aspartyl-tRNA synthetase which is localized in cytosol and mitochondria. In addition, quantitative proteomics analysis revealed that both nuclear and chloroplast-encoded thylakoid proteins were significantly down-regulated in the mutant. On the other hand, proteins involved in amino acid metabolism and the process of protein synthesis were up-regulated in ylc3, particularly for key enzymes that convert aspartate to asparagine. Moreover, uncharged tRNA-Asp accumulation and phosphorylation of the translation initiation factor eIF2α was detected in the mutant, suggesting that YLC3 regulates the homeostasis of amino acid metabolism and chloroplast thylakoid development through modulation of processes during protein synthesis.

NPRL2 Inhibition of mTORC1 Controls Sodium Channel Expression and Brain Amino Acid Homeostasis.

Genetic mutations in nitrogen permease regulator-like 2 (NPRL2) are associated with a wide spectrum of familial focal epilepsies, autism, and sudden unexpected death of epileptics (SUDEP), but the mechanisms by which NPRL2 contributes to these effects are not well known. NPRL2 is a requisite subunit of the GAP activity toward Rags 1 (GATOR1) complex, which functions as a negative regulator of mammalian target of rapamycin complex 1 (mTORC1) kinase when intracellular amino acids are low. Here, we show that loss of NPRL2 expression in mouse excitatory glutamatergic neurons causes seizures before death, consistent with SUDEP in humans with epilepsy. Additionally, the absence of NPRL2 expression increases mTORC1-dependent signal transduction and significantly alters amino acid homeostasis in the brain. Loss of NPRL2 reduces dendritic branching and increases the strength of electrically stimulated action potentials (APs) in neurons. The increased AP strength is consistent with elevated expression of epilepsy-linked, voltage-gated sodium channels in the NPRL2-deficient brain. Targeted deletion of NPRL2 in primary neurons increases the expression of sodium channel Scn1A, whereas treatment with the pharmacological mTORC1 inhibitor called rapamycin prevents Scn1A upregulation. These studies demonstrate a novel role of NPRL2 and mTORC1 signaling in the regulation of sodium channels, which can contribute to seizures and early lethality.