Low Arginine Research Articles

Metabolic and biochemical analyses reveal heavy metals tolerance mechanisms in Amaranthus retroflexus L.

One of the most prevalent plant species in the contaminated area around a sewage dumping lake at Khulais (Jeddah, Saudi Arabia) in the Arabian desert eco-region was found to be Amaranthus retroflexus L. This plant has the capacity to bioaccumulate and bioremediate heavy metals. The current study, therefore, aimed at investigating the plant's defense mechanisms by conducting metabolic and biochemical assessments. To this end, A. retroflexus plants were collected from the sewage dumping lake of Khulais, across five sites at varying distances from the lake, each exhibiting different level of heavy metal. The results indicated an increase in antioxidant defense system emerged as a protection strategy for A. retroflexus plants against soil contaminations. This, for instance, included the increased synthesis of polyamines (e.g., putrescine, spermidine, and spermine by +9–63%), flavonoids (e.g., naringenin, kaempferol, luteolin, and rutin by +10–146%), and phenolic acids (e.g., chlorogenic acid, gallic acid, caffeic acid, p-coumaric acid, chicoric acid, rosmarinic acid, and protocatechuic acid), increased by 10–254%. Additionally, plants adjusted their metabolic processes by synthesizing various low molecular weight amino acids, including proline (+19–299%), phenylalanine (+98–240%), glutamate (+34–492%), arginine (+10–64%), and ornithine (+51–210%). The activity of the metabolic enzymes involved in metabolism of these amino acids was enhanced accordingly. It can be concluded that the synthesis of polyamines and amino acids can be coordinated and complemented to improve the tolerance of A. retroflexus to cope with heavy metal accumulation in contaminated soils.

ATF4-SLC7A11-GSH axis mediates the acquisition of immunosuppressive properties by activated CD4+ T cells in low arginine condition

The tumor microenvironment (TME) is restricted in metabolic nutrients including the semi-essential amino acid arginine. While complete arginine deprivation causes Tcell dysfunction, it remains unclear how arginine levels fluctuate in the TME to shape Tcell fates. Here, we find that the 20-μM low arginine condition, representing the levels found in the plasma of patients with cancers, confers Treg-like immunosuppressive capacities upon activated Tcells. Invivo mouse tumor models and human single-cell RNA-sequencing datasets reveal positive correlations between low arginine condition and intratumoral Treg accumulation. Mechanistically, low arginine-activated Tcells engage in metabolic and transcriptional reprogramming, using the ATF4-SLC7A11-GSH axis, to preserve their suppressive function. These findings improve our understanding of the role of arginine in human Tcell biology with potential applications for immunotherapy strategies.

Lower Arginine Bioavailability, Increased FeNO Levels, and Airway Resistance on Impulse Oscillometry Are Characteristics of Asthma in Children and Young Adults with Sickle Cell Disease.

Background and Objectives: Data on characteristics of asthma in children with sickle cell disease (SCD) is conflicting. Recently, the L-arginine pathway has gained attention in the pathogenesis of asthma and SCD. This study aimed to determine the distinctive clinical and laboratory features and the role of arginine metabolism in asthmatic children with SCD. Materials and Methods: A total of 52 children and adolescents with SCD, including 24 with asthma (SCD-A) and 28 without asthma (SCD-NA), and 40 healthy controls were included. A questionnaire, atopy tests, fractional exhaled nitric oxide (FeNO), and lung function tests were employed. Serum metabolites of the arginine pathway were measured. The results of the three groups were compared. Results: The demographic characteristics and atopy markers of the three groups were similar. FEV1%, FEV1/FVC, MMEF%, and total lung capacity (TLC%) values of SCD-A patients were not significantly different from the SCD-NA group, but they were significantly lower than the values measured in the controls. FeNO values greater than 35 ppb were present only in the SCD-A group. In impulse oscillometry, median resistance values at 5 Hz (R5)% were higher in both SCD subgroups than in healthy controls (p = 0.001). The (R5-20/R5)% values were higher in the SCD-A group (p = 0.028). Serum arginine levels and arginine bioavailability indices were significantly lower in the SCD-A group than in the SCD-NA group and healthy controls (p = 0.003 and p < 0.001). Conclusions: Asthma in children with SCD was not associated with atopy or low FEV1/FVC levels. However, lower arginine bioavailability and higher FeNO levels differentiated asthma in patients with SCD. High R5% and (R5-20/R5)% values indicated increased airway resistance in SCD, with a predominance of small airway disease in asthmatics.

Prognostic implications of the arginine metabolism in patients at nutritional risk: A secondary analysis of the randomized EFFORT trial

BackgroundArginine, a conditionally essential amino acid, is key component in metabolic pathways including immune regulation and protein synthesis. Depletion of arginine contributes to worse outcomes in severely ill and surgical patient populations. We assessed prognostic implications of arginine levels and its metabolites and ratios in polymorbid medical inpatients at nutritional risk regarding clinical outcomes and treatment response. MethodsWithin this secondary analysis of the randomized controlled Effect of early nutritionalsupporton Frailty, Functional Outcomes, and Recovery of malnourished medical inpatients Trial (EFFORT), we investigated the association of arginine, its metabolites and ratios (i.e., ADMA and SDMA, ratios of arginine/ADMA, arginine/ornithine, and global arginine bioavailability ratio) measured on hospital admission with short-term and long-term mortality by means of regression analysis. ResultsAmong the 231 patients with available measurements, low arginine levels ≤90.05 μmol/l (n=86; 37%) were associated with higher all-cause mortality at 30 days (primary endpoint, adjusted HR 3.27, 95% CI 1.86 to 5.75, p<0.001) and at 5 years (adjusted HR 1.50, 95% CI 1.07 to 2.12, p=0.020). Arginine metabolites and ratios were also associated with adverse outcome, but had lower prognostic value. There was, however, no evidence that treatment response was influenced by admission arginine levels. ConclusionThis secondary analysis focusing on medical inpatients at nutritional risk confirms a strong association of low plasma arginine levels and worse clinical courses. The potential effects of arginine-enriched nutritional supplements should be investigated in this population of patients. Clinical Trial Registrationclinicaltrials.gov as NCT02517476 (registered 7 August 2015)

Implication of Low Plasma Arginine among Patients with Diabetic Foot Ulcer (DFU).

&lt;b&gt;Background and Objective:&lt;/b&gt; Diabetic foot ulcer (DFU) is a well-recognized vascular complication of uncontrolled diabetes mellitus. Unless immediate measures are taken some patients end up with amputation. The hallmarks of DFU are hyperglycemia, neuropathy, ischemia and infection (Andrew). Recent studies confirmed the role of low serum arginine in the development of foot ulcer in diabetic patients. The current study aimed to investigate the relationship between low serum magnesium and diabetic foot ulcer and to identify the incidence of low plasma arginine levels in diabetic patients with DFU in Sudan. &lt;b&gt;Materials and Methods:&lt;/b&gt; A total of 120 subjects were recruited in this study and were divided into three groups, namely, diabetic with foot ulcer (group-I), diabetic without foot ulcer (group-II) and non-diabetic healthy subjects (group-III). These subjects were investigated for FBS, HbA1c and serum arginine. Data were analysed using SPSS (28th release). &lt;b&gt;Results:&lt;/b&gt; Analysis of the results obtained showed significantly marked reduced plasma arginine levels in group I than the other two groups where mean plasma arginine level was 12.08, 49.25 and 94.1 micromole/L, respectively. There was no statistical difference between the duration of diabetes and DFU in diabetic patients with DFU and diabetic patients without DFU, as well as levels of HbA1c (p-value 0.457, 0.89) respectively. &lt;b&gt;Conclusion:&lt;/b&gt; The low serum arginine levels were significantly associated with foot ulcers in diabetic patients and play an important role in the development of diabetic foot ulcers.

Low Arginine Bioavailability and Clinical Outcomes in Children with Sickle Cell Disease Hospitalized with Vaso-Occlusive Pain Episode

Introduction: Global arginine bioavailability ratio (GABR) (Arginine/[Citrulline+Ornithine]) is associated with clinical outcomes including pain, pulmonary hypertension, and mortality in patients with sickle cell disease (SCD). Arginine replacement therapy has demonstrated opioid-sparing effects, improved blood pressure and cardiovascular function, and shorten length of hospital stay. Arginine is a conditionally essential amino acid synthesized from citrulline in the kidneys. During a vaso-occlusive pain episode (VOE), hemolysis leads to the release of erythrocyte-arginase (arginine-metabolizing enzyme) that metabolizes arginine into ornithine. Arginine-to-ornithine ratio (arg/orn) is a biomarker of arginase activity and GABR incorporates the impact of kidney function on global arginine bioavailability. The association between arginine bioavailability and clinical outcomes has not been sufficiently explored in children with SCD-VOE. Objective: To evaluate associations between arginine bioavailability and clinical outcomes in children with SCD-VOE who received intravenous (IV) arginine replacement therapy. Methods: We conducted a secondary analysis of a prospective, single-center, double-blinded randomized controlled trial of IV arginine therapy (TID, up to 7 days) in children with SCD age 3-21 years hospitalized for VOE requiring IV opioids. Patients with significant liver/renal dysfunction or those previously enrolled were excluded. Subjects were randomized into 1 of 3 arms: 1) 100 mg/kg/dose arginine (standard dose), 2) loading dose: 200 mg/kg followed by standard dose or 3) placebo. Demographics, total parenteral opioid (TPO) use (morphine equivalents, mg/kg) time to crisis resolution (time of study drug delivery to last IV opioid in hours), pain scores, and targeted amino acids were obtained before treatment and at discharge. Mean±SD, paired t-tests, and Pearson correlation analyses between groups were performed. This trial was registered at www.clinicaltrials.goc as #NCT02536170 Results: 1,548 patients were screened, 266 were eligible, 114 consented, and 108 were randomized (54.4% were female, mean age 12.44 ± 3.90). All participants identified as African American and 75% had genotype Hb-SS. 69.1% were on hydroxyurea. Safety results of this RCT have been previously reported ( Reyes et al, Am J Hematol 2022). Plasma arginine levels were low at VOE presentation (mean 50±28 μM), with low arginine levels (&amp;lt;60 μM) found in 74% of patients. Arginine replacement therapy increased arginine concentration by 182% (p&amp;lt;0.0001) compared to 26% (p=0.24) in placebo. Arg/orn correlated to arginase concentration ( r= - 0.38, p&amp;lt;0.0001). Time-to-crisis-resolution strongly correlated with TPO ( r=0.72, p&amp;lt;0.0001). Arginine bioavailability at presentation was inversely correlated to time-to-crisis-resolution for both arg/orn and GABR ( r= -0.37 p=0.02) in the placebo arm only but not in those participants who received arginine therapy (GABR: r= - 0.05, p=0.6; arg/orn: r= - 0.07, p=0.55) (Fig1A,1B). Similar trends were observed for TPO. Pain scores at presentation positively correlated with TPO ( r= 0.33, p=0.04) in the placebo arm only, but not in the arm that received arginine therapy ( r= 0.03, p=0.76). Time-to-crisis-resolution positively correlated with age ( r= 0.2, p=0.03). Conclusion: In the absence of arginine replacement therapy, low arginine bioavailability predicted longer time-to-crisis resolution and a higher use of IV opioids in patients with SCD-VOE. Arginine bioavailability may represent a novel biomarker of SCD-pain severity. This data further confirms arg/orn as a surrogate for arginase activity reflective of hemolytic rate as previously described. Arginine replacement therapy ameliorates the influence of arginine deficiency on clinical outcomes related to pain in SCD. An NHLBI/ Pediatric Emergency Care Applied Research Network (PECARN) supported Phase-3 multi-center trial of arginine replacement therapy enrolling 360 children with SCD (SCD Treatment with Arginine Therapy - STArT) is ongoing.

Low Arginine Suggesting the Diagnosis of Mitochondrial Cardiomyopathy.

Low Arginine Suggesting the Diagnosis of Mitochondrial Cardiomyopathy.

Metabolic signatures of cardiorenal dysfunction in plasma from sickle cell patients as a function of therapeutic transfusion and hydroxyurea treatment.

Metabolomics studies in sickle cell disease (SCD) have been so far limited to tens of samples, owing to technical and experimental limitations. To overcome these limitations, we performed plasma metabolomics analyses on 596 samples from patients with SCD enrolled in the WALK-PHaSST study (clinicaltrials gov. Identifier: NCT00492531). Clinical covariates informed the biological interpretation of metabolomics data, including genotypes (hemoglobin [Hb] SS, hemoglobin SC), history of recent transfusion (HbA%), response to hydroxyurea treatment (fetal Hb%). We investigated metabolic correlates to the degree of intravascular hemolysis, cardiorenal function, as determined by tricuspid regurgitation velocity (TRV), estimated glomerular filtration rate (eGFR), and overall hazard ratio (unadjusted or adjusted by age). Recent transfusion events or hydroxyurea treatment were associated with elevation in plasma-free fatty acids and decreases in acyl-carnitines, urate, kynurenine, indoles, carboxylic acids, and glycine- or taurine-conjugated bile acids. High levels of these metabolites, along with low levels of plasma S1P and L-arginine were identified as top markers of hemolysis, cardiorenal function (TRV, eGFR), and overall hazard ratio. We thus uploaded all omics and clinical data on a novel online portal that we used to identify a potential mechanism of dysregulated red cell S1P synthesis and export as a contributor to the more severe clinical manifestations in patients with the SS genotype compared to SC. In conclusion, plasma metabolic signatures - including low S1P, arginine and elevated kynurenine, acyl-carnitines and bile acids - are associated with clinical manifestation and therapeutic efficacy in SCD patients, suggesting new avenues for metabolic interventions in this patient population.

Plasma amino acid signatures define types of pediatric diabetes

Metabolic biomarkers with pathophysiological relevance is lacking in pediatric diabetes. We aimed to identify novel metabolic biomarkers in pediatric type 1 (T1D) and type 2 diabetes (T2D). We hypothesized that (1) targeted plasma metabolomics, focused on plasma amino acid concentrations, could identify distinctively altered patterns in children with T1D or T2D, and (2) there are specific changes in concentrations of metabolites related to branch chain amino acids (BCAA) and arginine metabolism in children with T2D. In a pilot study, we enrolled children with T1D (n=15) and T2D (n=13), and healthy controls (n=15). Fasting plasma amino acid concentrations were measured by ultra-performance liquid chromatography, and compared between the groups after adjustment for confounding factors. The mean age (SD) of participants was 16.4 (0.9) years. There were no group differences in age, gender, race/ethnicity, or 24-h protein intake. Mean BMI percentile was higher in the T2D than the T1D group or controls (p<0.001). The T2D group had lower arginine, citrulline, glutamine, glycine, phenylalanine, methionine, threonine, asparagine and symmetric dimethylarginine (SDMA) but higher aspartate than controls, after adjusting for BMI percentiles (all p<0.05). Children with T2D also had lower glycine but higher ornithine, proline, leucine, isoleucine, valine, total BCAA, lysine and tyrosine than those with T1D after adjusting for confounding factors (all p<0.05). Children with T1D had lower phenylalanine, methionine, threonine, glutamine, tyrosine, asymmetric dimethylarginine (ADMA) and SDMA than controls (all p<0.05). Children with T2D and T1D have distinct fasting plasma amino acid signatures that suggest varying pathogenic mechanisms and could serve as biomarkers for these conditions.

Composition Analysis and Nutritional Value Evaluation of Amino Acids in the Fruit of 161 Jujube Cultivars.

Amino acids are an essential group of compounds involved in protein synthesis and various metabolic and immune reactions in the human body. Chinese jujubes (Ziziphus jujuba Mill.) are an important fruit and medicinal plant which are native to China and have been introduced into around 50 countries. However, systematic research on the composition and content diversity of amino acids in the jujube is still lacking. In this experiment, the amino acid composition and the contents of the dominant amino acids in the fruit of 161 cultivars of jujube were determined by HPLC. Of the twenty-one kinds of amino acids detected, a total of fourteen kinds of amino acids were detected, of which eight kinds of amino acids were relatively high, including five essential amino acids (threonine, valine, isoleucine, leucine, and phenylalanine) and three nonessential amino acids (glycine, alanine, and proline). However, the contents of the remaining six amino acids were relatively low (aspartic acid, glutamic acid, histidine, serine, arginine, and tryptophan). Therefore, the eight primary amino acids were used as the index to evaluate the amino acids of 161 jujube varieties. Proline accounts for 56.8% of the total amino acid content among the eight amino acids. The total content of the eight primary amino acids in most jujube varieties was 1-1.5 g/100 g, and the highest content of 'Zaoqiangmalianzao' was 2.356 g/100 g. The average content of proline was 6.01-14.84 times that of the other seven amino acids. According to the WHO/FAO revised model spectrum of ideal essential amino acids for humans, 19 cultivars met the E/T (essential amino acids/total amino acids) standard, and their values ranged from 35% to 45%; 12 cultivars meet E/NE (non-essential amino acids) ≥ 60%. All cultivars reached the requirement of BC (branched-chain amino acids)/E ≥ 40% with 15 cultivars over 68%. One hundred and fifty-seven cultivars reach the standard of BC/A (aromatic amino acids) ≈ 3.0~3.5. The amino acid ratio coefficient analysis showed that phenylalanine was the first limiting amino acid of all the jujube cultivars. The SRC (the score of amino acid ratio coefficient) values of 134 cultivars were between 50% and 70%, with 12 cultivars over 70%, indicating that jujube fruits are of high nutritional value in terms of amino acids. Based on the principal component analysis and comprehensive ranking of amino acid nutritional value, the top five cultivars were screened from the 161 ones tested, i.e., 'Tengzhouchanghongzao', 'Xinzhengxiaoyuanzao', 'Hanguowudeng', 'Xuputiansuanzao', and 'Lichengxiaozao'. This study established, firstly, a complete basic data analysis of amino acid content in jujube fruit which could be used to select germplasm resources suitable for developing functional amino acid food, and provide theoretical support for the high value utilization of amino acids in jujubes.

Association of High and Low Molecular Weight Glutenin Subunits with Gluten Strength in Tetraploid Durum Wheat (Triticum turgidum spp. Durum L.)

The gluten strength and the composition of high- and low-molecular-weight glutenin subunits (HMWGSs and LMWGSs) of fifty-one durum wheat genotypes were evaluated using sodium dodecyl sulfate (SDS) sedimentation testing and SDS polyacrylamide gel electrophoresis (SDS-PAGE). This study examined the allelic variability and the composition of HMWGSs and LMWGSs in T. durum wheat genotypes. SDS-PAGE was proven to be a successful method for identifying HMWGS and LMWGS alleles and their importance in determining the dough quality. The evaluated durum wheat genotypes with HMWGS alleles 7+8, 7+9, 13+16, and 17+18 were highly correlated with improved dough strength. The genotypes containing the LMW-2 allele displayed stronger gluten than those with the LMW-1 allele. The comparative in silico analysis indicated that Glu-A1, Glu-B1, and Glu-B3 possessed a typical primary structure. The study also revealed that the lower content of glutamine, proline, glycine, and tyrosineand the higher content of serine and valine in the Glu-A1 and Glu-B1 glutenin subunits, and the higher cysteine residues in Glu-B1 and lower arginine, isoleucine, and leucine in the Glu-B3 glutenin, are associated with the suitability of durum wheat for pasta making and the suitability of bread wheat with good bread-making quality. The phylogeny analysis reported that both Glu-B1 and Glu-B3 had a closer evolutionary relationship in bread and durum wheat, while the Glu-A1 was highly distinct. The results of the current research may help breeders to manage the quality of durum wheat genotypes by exploiting the allelic variation in glutenin. Computational analysis showed the presence of higher proportions of glutamine, glycine, proline, serine, and tyrosine than the other residues in both HMWGSs and LMWGSs. Thus, durum wheat genotype selection according to the presence of a few protein components effectively distinguishes the strongest from the weakest types of gluten.

Associations of sleep quality with the skeletal muscle strength in patients with type 2 diabetes with poor glycemic control

Patients with type 2 diabetes mellitus are reported to be at a high risk for sarcopenia, and are known to have a poorer sleep quality. However, the association between sleep quality and skeletal muscle in patients with type 2 diabetes mellitus is not yet precisely understood. A total of 110 inpatients with type 2 diabetes mellitus aged 40-90 years were enrolled. The sleep quality was assessed using the Pittsburgh sleep quality index (PSQI). Skeletal muscle mass was measured using bioelectrical impedance analysis. Muscle strength was evaluated by measuring the grip strength. We also performed dietary surveys and measurements of the plasma amino acid levels. A high total score on the PSQI was significantly associated with reduced muscle strength, and the association persisted even after adjustments for confounders. On the other hand, adjusted analysis did not reveal any significant associations between the PSQI total score and the skeletal muscle mass. In regard to the associations with subscores of the PSQI, the scores for sleep latency, sleep efficiency, and daytime dysfunction were significantly negatively associated with the muscle strength. Although poor sleep quality was associated with a high confectionery intake and low plasma arginine, citrulline, and ornithine levels, neither confectionery intake levels nor the plasma levels of these amino acids was associated with the muscle strength. Our study revealed a significant association between the sleep quality and muscle strength in patients with type 2 diabetes mellitus. These results suggest that poor sleep quality is an important risk factor for sarcopenia in patients with type 2 diabetes mellitus.

Abstract 9482: Coronary Spasm in a Patient With Argininosuccinic Aciduria

Case presentation: A 39-year-old male patient was referred for the treatment of hypertension. The patient had been treated for argininosuccinic aciduria (ASA) since eight months of age. Therapeutic drugs including, L-arginine, sodium phenylbutyrate and antiepileptic drugs had been administered. A detailed medical history taking revealed that the patient sometimes complained of chest discomfort under psychological stress. A 12-lead electrocardiogram showed abnormal q waves in lead III and aVF. Transthoracic echocardiography showed hypokinesis of left ventricular posterior wall. Endothelial function was quantified with brachial artery flow-mediated dilatation (FMD). The patient’s FMD value was 2.4% (normal cut off value 7.0%). Coronary computed tomography angiography showed no significant coronary stenosis. Cardiac magnetic resonance revealed transmural late gadolinium enhancement in the mid-ventricular level of the left ventricular posterior wall. The patient was suspected to have prior myocardial infarction. The patient was started on amlodipine and irbesartan, however, he continued to complain of chest discomfort at rest. Cardiac catheterization was performed to make a definitive diagnosis. The acetylcholine spasm provocation test revealed diffuse left and right coronary vasospasm. Transient ST-segment elevation with chest discomfort was observed. The patient was diagnosed as having coronary spastic angina. Chest discomfort was disappeared after additional administration of isosorbide mononitrate. Discussion: ASA is a genetic disorder of the urea cycle, caused by deficiency of argininosuccinate lyase (ASL). Specific laboratory findings are hyperammonemia, argininosuccinic acidemia and low plasma arginine levels. ASL is required to utilize extracellular arginine for NOS-dependent NO synthesis in endothelial cell. Reduction of ASL enzymatic activity leads to decline in NO production even if arginine is supplemented. Efficacy of NO supplementation for treatment of long-term complications in ASA patients has been reported. The precise mechanisms of coronary spasm remain to be elucidated. Our case report might support the significance of endothelial function in the pathogenesis of coronary spasm.

The role and control of arginine levels in arginase 1 deficiency.

Arginase 1 Deficiency (ARG1-D) is a rare urea cycle disorder that results in persistent hyperargininemia and a distinct, progressive neurologic phenotype involving developmental delay, intellectual disability, and spasticity, predominantly affecting the lower limbs and leading to mobility impairment. Unlike the typical presentation of other urea cycle disorders, individuals with ARG1-D usually appear healthy at birth and hyperammonemia is comparatively less severe and less common. Clinical manifestations typically begin to develop in early childhood in association with high plasma arginine levels, with hyperargininemia (and not hyperammonemia) considered to be the primary driver of disease sequelae. Nearly five decades of clinical experience with ARG1-D and empirical studies in genetically manipulated models have generated a large body of evidence that, when considered in aggregate, implicates arginine directly in disease pathophysiology. Severe dietary protein restriction to minimize arginine intake and diversion of ammonia from the urea cycle are the mainstay of care. Although this approach does reduce plasma arginine and improve patients' cognitive and motor/mobility manifestations, it is inadequate to achieve and maintain sufficiently low arginine levels and prevent progression in the long term. This review presents a comprehensive discussion of the clinical and scientific literature, the effects and limitations of the current standard of care, and the authors' perspectives regarding the past, current, and future management of ARG1-D.

Preclinical evidence of a direct pro-survival role of arginine deprivation in multiple myeloma.

Multiple myeloma grows by establishing multiple interactions with bone marrow cells. These include expansion of myeloid-derived suppressor cells, which drive immunoevasion via mechanisms that include arginase-1-driven depletion of L-arginine, thus indirectly promoting myeloma cell survival and tumor progression. The peculiar biology of malignant plasma cells postulates that arginine depletion may benefit their fitness also directly, e.g., by engaging the integrated stress response, or by stimulating autophagy through mTORC1 inhibition. We thus investigated the direct impact of arginine deprivation on myeloma cells and challenged its pathophysiological relevance in vitro and in vivo. First, we found that partial arginine depletion spared proliferation of human multiple myeloma cells at concentrations that arrest human T cells. Next, we asked if arginine shortage activates putative adaptive pathways in myeloma cells. Low arginine failed to activate the integrated stress response, as indicated by unmodified phosphorylation of the eukaryotic initiation factor 2α, but sizably inhibited mTORC1, as revealed by reduced phosphorylation of ribosomal protein S6. Notably, depressed mTORC1 activity was not sufficient to increase autophagy, as assessed by the lysosomal digestion rate of the autophagosome-associated protein, LC3-II. Rather, it stimulated mTORC2, resulting in increased phosphatidylinositol-3 kinase-dependent AKT phosphorylation and activity, leading to heightened inhibitory phosphorylation of the pro-apoptotic BAD protein. We then tested whether arginine depletion-activated AKT may protect malignant plasma cells from cell death. Indeed, culturing myeloma cells in low arginine medium significantly reduced the apoptotic effect of the first-in-class proteasome inhibitor, bortezomib, an outcome prevented by pharmacological inhibition of AKT phosphorylation. Finally, we challenged the relevance of the identified circuit in vivo. To gauge the pathophysiologic relevance of low arginine to myeloma growth independently of immunoevasion, we xenotransplanted human myeloma cells subcutaneously into T cell-deficient Rag2–/–γc–/– recipient mice and treated palpable tumor-bearing mice with the clinical-grade arginase inhibitor CB1158. Arginase inhibition significantly raised serum arginine concentration, reduced tumor growth by caliper assessment, and decreased intra-tumor AKT phosphorylation in vivo. Altogether, our results reveal a novel direct pro-survival effect of arginine deprivation on myeloma cells, with potential therapeutic implications.

Invariant NKT cells metabolically adapt to the acute myeloid leukaemia environment

Acute myeloid leukaemia (AML) creates an immunosuppressive environment to conventional T cells through Arginase 2 (ARG2)-induced arginine depletion. We identify that AML blasts release the acute phase protein serum amyloid A (SAA), which acts in an autocrine manner to upregulate ARG2 expression and activity, and promote AML blast viability. Following in vitro cross-talk invariant natural killer T (iNKT) cells become activated, upregulate mitochondrial capacity, and release IFN-γ. iNKT retain their ability to proliferate and be activated despite the low arginine AML environment, due to the upregulation of Large Neutral Amino Acid Transporter-1 (LAT-1) and Argininosuccinate Synthetase 1 (ASS)-dependent amino acid pathways, resulting in AML cell death. T cell proliferation is restored in vitro and in vivo. The capacity of iNKT cells to restore antigen-specific T cell immunity was similarly demonstrated against myeloid-derived suppressor cells (MDSCs) in wild-type and Jα18−/− syngeneic lymphoma-bearing models in vivo. Thus, stimulation of iNKT cell activity has the potential as an immunotherapy against AML or as an adjunct to boost antigen-specific T cell immunotherapies in haematological or solid cancers.

Abstract 1251: Low plasma Arginine level is associated with resistance to immune checkpoint blockers in patients with advanced cancer

Abstract Background: The discovery of immune checkpoint blockers (ICB) has revolutionized the systemic approach of the treatment of cancer. However, most patients receiving ICB do not derive benefit. Therefore, there is a crucial need to identify reliable predictive biomarkers of response to anti-PD-1/PD-L1 agents, both to develop precision medicine in cancer immunotherapy and to better understand mechanisms of sensitivity and resistance. One pathway that plays an important role in the regulation of immune cell reactivity is L-Arginine (Arg) metabolism, which is essential to T-cell activation. We therefore aimed at evaluating the association of baseline plasmatic level of Arg - serving as a surrogate of Arginase (Arg1) activity - and clinical benefit to ICB. Methods: Correlation with Arg levels and efficacy of ICB in the pre-clinical setting was assessed by using a syngeneic mouse model of colorectal cancer (MC38) known to be responsive to ICB. Correlation of Arg levels and clinical activity of ICB was assessed by analyzing the plasma samples obtained before treatment onset in two independent cohorts of patient with advanced cancer and included in two institutional molecular profiling programs (discovery cohort: BIP, NCT02534649, n=77; validation cohort: PREMIS, n=295, NCT03984318). In addition, using matched PBMCs-plasma samples, we analyzed the correlation between Arg level and features of PBMCs that were captured through multiplexed-flow cytometry analysis. Results: As expected, treatment of MC38-tumor bearing mice with anti-PD(L)1 antibodies demonstrated a strong anti-tumor effect with tumor rejection observed for app. 40% of mice (11 out of 28). The tumor rejection rate was significantly higher in mice with high baseline Arg level than in mice with low Arg level: 85.7% versus 23.8%, p=0.004. In both discovery and validation cohorts, low Arg level at baseline (42 &amp;lt;µmol/L) was significantly associated with worse clinical benefit rate, progression-free survival (PFS) and overall survival (OS). Multivariate analysis showed that low baseline Arg level isd an independent prognostic factor for both PFS and OS. Finally, PBMCs immunophenotyping showed that low Arg level was significantly associated with increased PDL1 expression in several immune cell subsets from the myeloid lineage. Conclusions: Altogether, our results demonstrate that baseline Arg levels are highly predictive of ICB efficacy. Increase in PDL1 expression in myeloid cells upon Arg deprivation could partly underly its suppressive activity. Plasmatic Arg quantification can therefore represent an attractive biomarker to tailor novel therapeutic regimens targeting the Arginase pathway in combination with ICB. Citation Format: Jean-Philippe Guegan, Florent Peyraud, Aurelien Marabelle, Nathalie Chaput, Dominique Bodet, Laure Fontan, Anthony Gaultier, Imane NAFIA, Francois-Xavier Danlos, David Planchard, Caroline Robert, Caroline Even, Mohamed Khettab, Lambros Tselikas, Luc Friboulet, Jean-Charles Soria, Fabrice Andre, Fabrice Barlesi, Alban Bessede, Antoine Italiano. Low plasma Arginine level is associated with resistance to immune checkpoint blockers in patients with advanced cancer [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 1251.

Abstract 2177: Myeloid-derived arginase depletes microenvironmental arginine in PDAC tumors and leads to activation of arginine de novo biosynthesis in cancer cells

Abstract Arginine is an amino acid critical for various cellular processes, not only protein synthesis but also metabolism of other essential metabolites, like polyamines, as well as a signaling factor for pathways such as the growth regulator mTOR. Previously, our group measured arginine levels in the interstitial fluid of tumors (TIF) of pancreatic ductal adenocarcinoma (PDAC) murine models and found extremely low arginine levels (2-5 uM) in the tumor microenvironment (TME). Despite near complete absence of this critical nutrient in the TME, pancreatic tumors exhibit aggressive growth. We have sought to understand both how the PDAC TME becomes arginine limited and how PDAC cells adapt to proliferate in the absence of arginine. Using genetically engineered mice, we find that arginase activity in the myeloid compartment of PDAC tumors is responsible for arginine depletion in the TME. Staining of Arg1+ myeloid populations in human PDAC samples suggest a similar mechanism reduces arginine availability in human PDAC tumors as well. We then leveraged our newfound knowledge of PDAC TIF composition to develop a novel ex vivo cell culture media formulation with physiologically relevant nutrient levels and monitored arginine acquisition pathways using isotope tracing and metabolomics assays to determine how PDAC cells cope with arginine deprivation. Under TME nutrient conditions, PDAC cells consume available citrulline and use it to produce arginine by de novo synthesis. Starving cells of citrulline or genetically perturbing arginosuccinate synthase (ASS1), key enzyme in arginine biosynthesis, significantly reduces PDAC cellular arginine and proliferative capacity. Immunohistochemical analysis of both human and mouse PDAC tumors indicates that the de novo arginine synthesis pathway is highly expressed in PDAC but not in untransformed pancreas, suggesting a key role for this pathway in PDAC progression. Altogether, we find that myeloid-derived arginase challenges PDAC cells by limiting arginine availability and suggest that de novo arginine synthesis may be a critical metabolic pathway that enables PDAC tumors to cope with this metabolic challenge. Citation Format: Juan Apiz-Saab, Alex Muir. Myeloid-derived arginase depletes microenvironmental arginine in PDAC tumors and leads to activation of arginine de novo biosynthesis in cancer cells [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 2177.

The Role of Fecal Microbiota in Liver Toxicity Induced by Perfluorooctane Sulfonate in Male and Female Mice.

Background:Perfluorooctane sulfonate (PFOS) is a persistent organic pollutant that can cause hepatotoxicity. The underlying toxicological mechanism remains to be investigated. Given the critical role of fecal microbiota in liver function, it is possible that fecal microbiota may contribute to the liver toxicity induced by PFOS.Objectives:We aimed to investigate the role of liver-fecal microbiota axis in modulating PFOS-induced liver injury in mice.Methods:Male and female mice were exposed to PFOS or vehicle for 14 d. In this investigation, 16S rDNA sequencing and metabolomic profiling were performed to identify the perturbed fecal microbiota and altered metabolites with PFOS exposure. In addition, antibiotic treatment, fecal microbiota transplantation, and bacterial administration were conducted to validate the causal role of fecal microbiota in mediating PFOS-induced liver injury and explore the potential underlying mechanisms.Results:Both male and female mice exposed to PFOS exhibited liver inflammation and steatosis, which were accompanied by fecal microbiota dysbiosis and the disturbance of amino acid metabolism in comparison with control groups. The hepatic lesions were fecal microbiota-dependent, as supported by antibiotic treatment and fecal microbiota transplantation. Mice with altered fecal microbiota in antibiotic treatment or fecal microbiota transplantation experiments exhibited altered arginine concentrations in the liver and feces. Notably, we observed sex-specific lower levels of key microbiota, including Lactobacillus, Enterococcus, and Akkermansia. Mice treated with specific bacteria showed lower arginine levels and lower expression of the phosphorylated mTOR and P70S6K, suggesting lower activity of the related pathway and mitigation of the pathological differences observed in PFOS-exposed mice.Conclusions:Our study demonstrated the critical role of the fecal microbiota in PFOS-induced liver injury in mice. We also identified several critical bacteria that could protect against liver injury induced by PFOS in male and female mice. Our present research provided novel insights into the mechanism of PFOS-induced liver injury in mice. https://doi.org/10.1289/EHP10281

Donor Simvastatin Treatment is More Effective in Donors with Low Plasma Levels of Arginine

<h3>Purpose</h3> Brain death may lead to a severe impairment of endothelial function and affect graft function. Recent study shows that simvastatin treatment to the organ donor decreases plasma biomarker levels of myocardial injury and heart failure and reduces the number of acute rejection episodes with hemodynamic compromise early after heart transplantation. However, the mechanisms of donor simvastatin treatment remain unknown. Therefore, we analyzed the plasma metabolomics of brain-dead heart transplant donors with or without simvastatin treatment to examine the molecular background of the beneficial effects. <h3>Methods</h3> In a prospective, double-blinded randomized controlled trial, 84 multiorgan donors were randomly assigned to receive either 80 mg of simvastatin (n=42) via nasogastric tube or to receive no simvastatin (n=42). Plasma samples from 84 human brain-dead multi-organ donors were collected immediately before organ procurement. We profiled over 100 targeted metabolites using mass spectrometry. <h3>Results</h3> We found that arginine and tryptophan were weakly negatively correlated with simvastatin treatment. Univariate Cox regression analysis showed that low levels of arginine were significantly associated with acute rejection and graft-related mortality (Figure 1 A,B). Additionally, we saw that donor simvastatin treatment significantly reduced the number of acute rejections by 41% (p=0.012) in low arginine group within the first year (Figure 1C). There was no difference in donor demographics between the groups. However, there was a statistically significantly higher need for mechanical circulatory support for hearts from donors with low arginine levels. <h3>Conclusion</h3> This study shows that donor simvastatin treatment could be more efficient in brain-dead heart transplant donors with low arginine. Further analysis will be conducted by exploring the interplay between arginine and simvastatin in detail. The complete results will be presented at the ISHLT2022 congress.