Plasma Gln Research Articles

PSV-29 Plasma Citrulline Is Strongly Correlated with Jejunum and Ileum Citrulline Concentrations in Pigs at Weaning

Abstract Pigs at weaning are abruptly exposed to numerous stressors that negatively affect gut health. While the term gut health lacks a clear definition, it is best measured by its key functions, including nutrient digestion, absorption, and metabolism, mucosal immunity, and barrier function. The gut is the main endogenous source of citrulline, a potential biomarker of gut function. The objective of this study was to determine the relationship between plasma and intestinal concentrations of citrulline and citrulline precursors in pigs. Pigs (initial BW = 7,233 ± 683 g) were either weaned at 21 d age and euthanized at 23 d age (W; n = 9) or were not weaned, remained with the sow, and euthanized at 23 d age (NW; n = 9). At euthanasia, blood, jejunum, and ileum tissue was collected and snap-frozen in liquid nitrogen; segments of mid jejunum and distal ileum were collected for morphological analysis. Plasma and tissue glutamine (Gln), glutamate (Glu), proline (Pro), ornithine (Orn), and citrulline (Cit) concentrations were determined by reverse-phase HPLC following pre-column derivatization with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate. Data were analyzed with the generalized linear mixed model and correlation procedures of SAS (version 9.4). Pearson correlation coefficients between 0.50 and 0.69 were considered as moderate, between 0.70 and 0.89 as strong, and between 0.90 to 1 as very strong. Jejunum villus height was reduced in W compared with NW pigs (370 versus 246 ± 18 µm; P < 0.001), whereas ileum villus height was not different between NW and W pigs (257 versus 215 ± 27 µm; P = 0.27). Among precursors for gut Cit synthesis, plasma Pro was strongly correlated with jejunum Pro (r = 0.83; P < 0.001) and very strongly correlated with ileum Pro (r = 0.91; P < 0.001). Plasma Glu was moderately correlated with ileum Glu (r = 0.58; P = 0.014), but not with jejunum Glu (r = 0.18, P = 0.48), whereas plasma Gln was moderately correlated with ileum Gln (r = 0.57; P = 0.016) and strongly correlated with jejunum Gln (r = 0.80; P < 0.001). Plasma Orn was strongly correlated with both jejunum Orn (r = 0.73; P < 0.001) and ileum Orn (r = 0.78; P < 0.001). Plasma Cit was strongly correlated with jejunum Cit (r = 0.77; P < 0.001) and very strongly correlated with ileum Cit (r = 0.95; P < 0.001). Plasma Cit can be used to estimate gut Cit content and may be predictive of gut function in nursery pigs undergoing weaning stress.

Overcoming Venetoclax (Ven) Resistance through Glutamine (Gln) Depletion: Final Analysis of the Phase 1 Trial of Ven and Pegcrisantaspase (PegC) Combination in Relapsed and Refractory (R/R) Acute Myeloid Leukemia (AML)

Background: Ven-based therapies have been an advancement in the front-line treatment of AML; however development of resistance is common and the efficacy of Ven-containing regimens in R/R AML has been far less promising. We reported in vitro and in vivo depletion of Gln induced by long-acting Erwinia asparaginase, PegC, inhibited proliferation of complex karyotype (CK) AML and synergistically enhanced the antiapoptotic activity of Ven-mediated antagonism of Bcl-2 by decreasing the expression of proteins such as Mcl-1, whose translation is cap-dependent. Here we report the final results of our phase 1 clinical trial (NCT04666649) of VenPegC for treatment of R/R AML including patients (pts) previously treated with Ven. Methods: Pts received Ven 400 mg on D1-28 and PegC (500, 750, 1000 IU/m 2 in cohorts 1-3, IV) D1 & D15 every 4 weeks (wk). Primary study endpoints were incidences of regimen limiting toxicities (RLT) and RP2D. Secondary endpoints included rate of composite complete remissions. Correlative studies included monitoring plasma amino acid levels and asparaginase activity, and expression of proteins regulating cap-dependent mRNA translation in bone marrow (BM) mononuclear cells pre- and post-VenPegC treatment. Results: As of July 31, 2023, 26 pts were enrolled of whom 24 received at least 1 dose of PegC and 17 completed at least 1 cycle of PegC (2 doses). The median age was 61 (range 24-79), 16 (62%) were female; 9 (35%) had CK-AML and 5 (19%) had TP53 mutations; 18 (69%) were previously treated with Ven. Median prior lines of treatment were 2 (range 1-5). No RLT was observed in cohort 1 (n=3). Cohort 2 was expanded to 6 pts due to transient asymptomatic hypertriglyceridemia. Two pts in cohort 3 (n=3) developed grade 3 hyperbilirubinemia determined as RLT. Expansion cohort included 11 pts who were treated at MTD/RP2D (PegC 750 IU/m 2 plus Ven 400 mg) given as out-pt without a need for hospitalization. No pancreatitis, thrombohemorrhagic events, or silent inactivation were observed in the entire study population. Overall response rate (ORR) for pts who received ≥1 cycle of PegC was 47% (8/17), Fig 1. CR+ CRh+CRi rate was 29% (5/17); 1 pt had a partial remission with full count recovery and is currently receiving cycle 6; 2 pts had stable disease. Four pts showed hematologic improvement. Twelve (71%) pts who received ≥1 cycle of PegC were previously treated with Ven and were Ven-refractory. ORR in Ven-refractory pts was 42% (5/12); of the 5 pts who harbored a RAS or PTPN11 mutation and previously treated with Ven, 60% responded. Remarkably, all pts (3/3) who had a RUNX1 mutation achieved a CR; Pt 6 (relapsed AML with NRAS/cKIT/RUNX1 mutations) achieved an MRD-negative CRh and underwent an allogeneic stem cell transplant (alloHSCT); Pt 12 (primary refractory AML with TP53/NRAS/RUNX1 mutations) achieved a clearance of BM blasts after 1 dose of PegC and achieved MRD-negative CR at the end of cycle 1, and Pt 15 (post-alloHSCT relapsed AML with NRAS/PTPN11/CSF3R/RUNX1 mutations) achieved a CRi and received 10 cycles of VenPegC. Pt 31 (post-alloHSCT relapsed AML with FLT3-ITD) who had persistent disease after gilteritinib achieved an MRD-negative CRi, pending 2 nd alloHSCT. Nadir plasma asparaginase activity of >0.1 IU/mL was achieved in cycle 1 for all pts who received ≥1 cycle of VenPegC. All pts with available results achieved undetectable asparagine (Asn) level by day 7 and Asn remained undetectable throughout the study. Plasma Gln reduction from baseline occurred in all pts (except 1) within cycle 1. Pts in cohort 1 reached nadir Gln after initial PegC exposure in an average of 22 days compared to 16 days in cohort 2. In general, pts who achieved CR had the lowest absolute plasma Gln levels. Available Glu and Gln are shown in Fig.1 for pts who achieved a CR/PR. Out of the 8 assessed pts (4 responders & 4 non-responders), VenPegC inhibited cap-dependent translation after 1 cycle in responders, as evidenced by decreased phosphorylated eIF4E (active, pheIF4E) in 4/4 pts and decreased phosphorylated 4EBP1 (inactive, ph4EBP1), a negative regulator of eIF4E, in 2/4 pts. Conversely, pheIF4E and ph4EBP1 expression was increased in 3/4 and 4/4 of the non-responders, respectively (Fig.2). Conclusion: VenPegC is a novel regimen that can induce complete remission in some heavily pretreated R/R AML patients, even with previous exposure to Ven. Pts with RUNX1 mutation, whose translation depends on pheIF4E, may benefit further from this regimen.

Oral glutamine supplementation relieves muscle loss in immobilized rats, altering p38MAPK and FOXO3a signaling pathways

BackgroundSkeletal muscle synthesizes, stores, and releases body L-glutamine (GLN). Muscle atrophy due to disabling diseases triggers the activation of proteolytic and pro-apoptotic cell signaling, thus impairing the body's capacity to manage GLN content. This situation has a poor therapeutic prognosis. ObjectiveEvaluating if oral GLN supplementation can attenuate muscle wasting mediated by elevated plasma cortisol and activation of caspase-3, p38MAPK, and FOXO3a signaling pathways in soleus and gastrocnemius muscles of rats submitted to 14-day bilateral hindlimbs immobilization. MethodsAnimals were randomly distributed into six groups: non-immobilized rats (Control), control orally supplemented with GLN (1 g kg−1) in solution with L-alanine (ALA: 0.61 g kg−1; GLN+ALA), control orally supplemented with dipeptide L-alanyl-L-glutamine (DIP; 1.49 g kg−1), hindlimbs immobilized rats (IMOB), IMOB orally GLN+ALA supplemented (GLN+ALA-IMOB), and IMOB orally DIP supplemented (DIP-IMOB). Plasma and muscle GLN concentration, plasma cortisol level, muscle caspase-3 activity, muscle p38MAPK and FOXO3a protein content (total and phosphorylated forms), and muscle cross-sectional area (CSA) were measured. ResultsCompared to controls, IMOB rats presented: a) increased plasma cortisol levels; b) decreased plasma and muscle GLN concentration; c) increased muscle caspase-3 activity; d) increased total and phosphorylated p38MAPK protein content; e) increased FOXO3a and decreased phosphorylated FOXO3a protein content; f) reduced muscle weight and CSA befitting to atrophy. Oral supplementation with GLN+ALA and DIP was able to significantly attenuate these effects. ConclusionsThese findings attest that oral GLN supplementation in GLN+ALA solution or DIP forms attenuates rats' skeletal muscle mass wasting caused by disuse-mediated muscle atrophy.

Bacterial community structure in the rumen and hindgut is associated with nitrogen efficiency in Holstein cows

Nitrogen efficiency (Neff; milk N/N intake) in dairy cows is limited and most of the consumed N is excreted in manure. Despite the crucial role of the gastrointestinal microbiome on N metabolism, associations between bacterial communities at different sections and Neff are not fully elucidated. Enhanced understanding of host-microbiome interactions can provide insights to improve Neff in dairy cows. Twenty-three Holstein cows were selected, and their Neff were determined using a N balance approach. From the cohort of cows, six cows were classified as low Neff and five cows as high Neff and their rumen and fecal bacterial communities were profiled using amplicon sequence variants (ASV) based on 16S rRNA gene sequencing. Then, relationships between differentially abundant bacterial features and Neff were evaluated. Neff in low and high cows averaged 22.8 and 30.3%, respectively. With similar N intake, high Neff cows wasted less N in manure compared to low Neff cows (P < 0.01, 11.0 ± 0.59 vs 14.3 ± 0.54 g of N/kg of milk). Rumen fermentation and plasma profiles were similar between Neff groups, but for plasma Gln which was greater (P = 0.02) in high compared to low Neff cows. In both rumen and feces, the phylogenetic composition of the bacterial communities was similar (P ≥ 0.65) between Neff groups, but differences were observed at the species -level (amplicon sequence variants). In the rumen, differentially abundant species from the genus Prevotella showed strong positive correlations with Neff, whereas in feces, differentially abundant species from the class Clostridia showed strong negative correlations with Neff. Our results revealed that Holstein cows with divergent Neff display distinctive bacterial community structure at the species-level in both the rumen and feces. Strong correlations between differentially abundant species and Neff in both sample sites, support the importance of the rumen bacterial composition on productive responses and suggest a more relevant role of the hindgut microbiome. Targeting both pre- and post-gastric bacterial communities may provide novel opportunities to enhance Neff in dairy cows.

Glutamine and leucine administration attenuates muscle atrophy in sepsis

AimsThis study investigated whether l-glutamine (Gln) and/or l-leucine (Leu) administration could attenuate muscle atrophy in a mouse model of cecal ligation and puncture (CLP)-induced sepsis. Materials and methodsSeptic mice were given a daily intraperitoneal injection of Gln, Leu, or Gln plus Leu, and mice were sacrificed on either day 1 or 4 after CLP. Blood and muscles were collected for analysis of amino acid contents and markers related to protein degradation, muscle regeneration, and protein synthesis. Key findingsLeu treatment alone increased both muscle mass and total muscle protein content on day 4 after CLP. Gln administration reduced muscular Gln contents on day 1 and enhanced plasma Gln levels on day 4. Higher plasma branched-chain amino acid (BCAA) abundances and lower muscular BCAA levels were observed in Leu-treated mice on day 4. Gln and Leu individually suppressed muscle expressions of the E3 ubiquitin ligase genes, Trim63 and Fbxo32, on day 4 after CLP. As to muscle expressions of myogenic genes, both Gln and Leu upregulated Myog expression on day 1, but Leu alone enhanced Myf5 gene expression, whereas Gln plus Leu increased MyoD and Myog expression levels on day 4. Akt/mammalian target of rapamycin (mTOR) signaling was only activated by Gln and Leu when individually administered. SignificanceGln and/or Leu administration reduces sepsis-induced muscle degradation and promotes myogenic gene expressions. Leu treatment alone had more-pronounced effects on maintaining muscle mass during sepsis. A combination of Gln and Leu failed to show synergistic effects on alleviating sepsis-induced muscle atrophy.

Phase 1 Dose Escalation Trial of Pegcrisantaspase in Combination with Venetoclax in Adults with Relapsed or Refractory Acute Myeloid Leukemia

Background: Acute myeloid leukemia (AML) cells are sensitive to depletion of extracellular glutamine (Gln) or manipulation of Gln metabolism. We recently reported the synergistic anti-leukemic activity of the combination of the Bcl-2 inhibitor Venetoclax (Ven) and pegcrisantaspase (PegC), a pegylated Erwinia asparaginase, in complex karyotype AML (CK-AML). The potent in vitro and in vivo activity of Ven-PegC regimen is mediated through inhibition of 4EBP1 phosphorylation resulting in decreased mRNA translation and protein synthesis. We hypothesized that safe and tolerable doses of Ven-PegC will result in anti-AML activity in patients (pts) with relapsed/refractory (R/R) AML. Study Design and Methods: The study is an open-label phase 1b clinical trial (NCT04666649) with 3+3 design with 4 cohorts of Ven (400 mg daily in the cohorts 1-3, 600 mg daily in cohort 4) in combination with outpatient intravenous PegC (500, 750, 1000, 1000 IU/m2 in cohorts 1-4, respectively) biweekly in a 28-day cycle. Key eligibility criteria include AML in an adult that has relapsed after, or is refractory to first-line therapy including Ven-containing regimens. The primary endpoints are the incidences of regimen limiting toxicities (RLT) and treatment emergent adverse events of Ven-PegC. Secondary endpoints include rate of composite complete remissions. Exploratory endpoints include the evaluation of plasma amino acid levels of Gln, asparagine (Asn), glutamate (Glu), and serine (Ser), and plasma asparaginase activity post-PegC administration. Results: As of August 1, 2022, 11 pts who received at least 1 dose of PegC were enrolled into the first two cohorts. The median age was 60 (range 24-76) years; 6 (55%) were female; 10 (91%) had adverse-risk AML by European Leukemia Net (ELN) criteria; 7 (64%) had CK-AML, and median prior treatments were 2. Overall response rate was 64% (7 pts) including 3 pts (27%) with CR/CRh/CRi [2 previously Ven-refractory]. In the first cohort, pt2 cleared peripheral blood (PB) blasts within 3 days after the first dose of PegC. Pt5 bone marrow (BM) blasts significantly decreased (78% to 9%) after cycle 1, and pt6 achieved CR with partial hematologic recovery (CRh) with MRD-negativity after 1 cycle and proceeded to alloHSCT after 3 cycles of Ven-PegC. In cohort 2, pt9 cleared PB blasts within 3 days after the first dose of PegC, and pt12 (primary refractory AML with two TP53 mutations) achieved a rapid clearance of PB blasts (41% to 0% after 2 days) and BM blasts after 1 dose of PegC and achieved MRD-negative CR at the end of cycle 1. Pt15 achieved CR with incomplete hematologic recovery (CRi) after 1 cycle of Ven-PegC and is currently receiving cycle 3, and in pt16 absolute PB blasts reduced from 4876/mcL to 192/mcL on Cycle 1 Day 3 and is currently receiving cycle 1 (Fig 1). Eight patients were evaluable for RLT assessment. No RLT was observed in cohort 1. In cohort 2, one transient (<48h) asymptomatic hypertriglyceridemia was observed and resulted in the expansion of cohort 2 to 6 patients. No serious asparaginase-related adverse events of interest have been observed. Pharmacokinetic and pharmacodynamic data are available for 8 patients. All pts achieved a nadir plasma asparaginase activity >0.1 IU/mL during cycle 1 and repetitive doses of PegC did not result in silent inactivation. Mean (range) plasma amino acid levels (µmol/L) at baseline were: Asn 31 (24-38), Gln 412 (323-465), Glu 58 (22-103). All pts achieved undetectable Asn level by day 7 and Asn remained undetectable throughout the study. Plasma Gln reduction from baseline occurred in all pts within cycle 1, with pts in cohort 2 demonstrating a more rapid decrease in Gln levels after initial exposure, confirming a dose-response correlation. Of note, in both cohorts, the pts that responded to Ven-PegC achieved the lowest absolute plasma Gln levels. A concomitant increase in plasma Glu levels was observed in all pts, indicative of the PegC-induced conversion of Gln to Glu. Remarkably, plasma Ser increased ~2-fold at the nadir of plasma Gln, from 75 ± 21 µmol/L at baseline to 114 ± 30 µmol/L after the first dose of PegC (Fig 2). Conclusion: Ven-PegC is a well-tolerated regimen with a novel metabolically-targeted mechanism of action. Early efficacy results in patients with poor prognosis R/R AML, a population with very limited treatment options, has been very promising. A clear correlation between the dose of PegC administered and the reduction in plasma Gln was observed. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Comprehensive Plasma Amino Acid Analysis Following Administration of Short- and Long-Acting Erwinia Asparaginase (Crisantaspase) Demonstrated Significant Increase in Plasma Serine and Glycine Levels

Background: Asparaginases are amidohydrolases that catalyze the conversion of asparagine (Asn) and glutamine (Gln) to aspartate (Asp) and glutamate (Glu), respectively, and can therefore be used therapeutically for cancer, particularly acute lymphoblastic (and myeloid) leukemia, to deplete circulating Asn and Gln from plasma. Clinically available asparaginases are derived from two bacterial sources: Escherichia coli and Erwinia chrysanthemi (also called crisantaspase), with crisantaspases demonstrating higher glutaminase activity. In order to identify any potential contributory pathways or resistance mechanisms to asparaginases, we sought to investigate the impact of short- and long-acting crisantaspases on circulating levels of plasma amino acids (AAs) other than Gln and Asn. Methods: Free AA concentrations were measured via a dedicated high-pressure liquid chromatography AAanalyzer (Biochrom 20 PlusAmino or Biochrom 30 Acid Analyzer). Free AAs were separated with a cation-exchange resin post precipitation of protein with sulfosalicylic acid. The AAs were differentially eluted after increasing the pH and ionic strength of the buffer. The concentration of each AA was measured by reaction of the AA with ninhydrin and detection via a colorimeter. Standard curves were generated for each AA and AAs were quantified and reported as μmol/L (µM) of AAs in plasma. For mouse studies, plasma was isolated from whole blood and delivered on ice to the Biochemical Genetics lab (University of Maryland Baltimore). For human clinical samples, 5-8 mL blood in a sodium or lithium heparin was obtained, kept on ice, and within 40 min of being collected, the plasma was separated by centrifugation (1100 g × 5 min at 4°C) and frozen at -20°C until analysis. Mouse plasma samples were from studies that included treatment with pegcrisantaspase (PegC), a long-acting Erwinia-derived asparaginase, in both immunodeficient and immunocompetent mice. Human plasma samples were from a clinical study (NCT02283190) in patients with acute myeloid leukemia (AML) treated with the short-acting crisantaspase, Erwinaze. Results: As expected, in our in vivo mouse experiments, intravenous pegcrisantaspase significantly decreased plasma Gln and Asn and increased plasma Glu. Interestingly, we observed that post-pegcrisantaspase administration, plasma serine (Ser) and glycine (Gly) increased in a dose-dependent manner. In immunocompromised mice treated with one dose of pegcrisantaspase (125, 250, and 500 IU/kg), three days post-administration, Ser increased from 190 ± 6 µM in the vehicle-treated group to 274 ± 35 µM (p= 0.07) in the 125 IU/kg group, 357 ± 29 µM (p=0.01) in the 250 IU/kg group, and 565 ± 8 µM (p=0.0004) in the 500 IU/kg group. Gly also significantly increased from 295 ± 25 µM in the vehicle-treated group to 438 ± 24 µM (p=0.02) in the 125 IU/kg group, 507 ± 50 µM (p=0.03) in the 250 IU/kg group, and 633 ± 8 µM (p=0.003) in the 500 IU/kg group (Fig 1). Pegcrisantaspase also increased plasma Ser from 143 ± 7.9 µM to 301 ± 73.7 µM (p=0.02) and Gly from 251 ± 45.9 µM to 381 ± 44.4 µM (p=0.02) in immunocompetent C57BL/6 mice. In addition to non-tumor/non-leukemia bearing immunocompetent and immunocompromised mice, this phenomenon was observed in a patient-derived xenograft model of AML (comprehensive plasma AA analysis, Fig 2). Other amino acids significantly increased by pegcrisantaspase in mice include threonine and α-amino-n-butyric acid. In a comprehensive plasma amino acid evaluation in samples from AML patients treated with Erwinaze (NCT02283190), we observed a similar pattern of increase in Ser, from 82 ± 11.2 µM at baseline to 128 ± 8.3 µM on Day 8 (p=0.0007), and Gly, from 191 ± 25.8 µM at baseline to 282 ± 46.6 µM on Day 8 (p=0.006), after 1 dose of Erwinaze (25,000 IU/m2). Systematic literature review of clinical studies with asparaginases revealed that only levels of Gln/Glu and Asn/Asp have been published post-asparaginase administration; demonstrating that this report is the first comprehensive analysis of plasma amino acid levels after asparaginase exposure. Conclusions: We report a comprehensive evaluation of plasma AA alterations in both mice and humans treated with crisantaspase. We observed a reproducible crisantaspase-induced increase in plasma Ser and Gly, which we are currently exploiting for synergism with other AML treatment modalities. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Targeting Serine Biosynthesis in Combination with the Modulation of Glutamine Metabolism Enhances Cellular Cytotoxicity in Acute Myeloid Leukemia

Background: Metabolic reprogramming contributes to tumor development and introduces metabolic liabilities that can be exploited to treat cancer. Glutamine (Gln) is the most abundant amino acid intracellularly and in the plasma. Gln depletion has emerged as a therapeutic approach for cancers that have exhibited a dependence on Gln for survival, including acute myeloid leukemia (AML). Crisantaspase, an Erwinia asparaginase, has demonstrated promising anti-AML activity in preclinical/clinical models through interference with plasma Gln supply; however, residual leukemia burden was shown in vivo following Gln depletion by a crisantaspase. Following treatment with asparaginases in vitro, we observed a consistent increase in serine (Ser). Therefore, we hypothesized that in response to Gln depletion, AML cells upregulate de novo Ser biosynthesis as a compensatory pathway, and the inhibition of this pathway will synergize with Gln depletion to increase AML cell death. To test this, we knocked-out phosphoglycerate dehydrogenase (PHGDH), a key enzyme in Ser biosynthesis, in AML cell lines (MOLM-14 and U937) by CRISPR-cas9 and examined the response of the knockout lines to Gln depletion either through Gln withdrawal from the media or crisantaspase treatment. Methods: Single-cell cloning methods were used on bulk populations of PHGDH CRISPR-cas9 knockout cells to characterize positive knockouts and clones were further confirmed by Sangar sequencing. The impact of Gln depletion and PHGDH knockout on AML cell proliferation/viability was assessed using water soluble tetrazolium (WST-1), trypan blue, and measurement of Lactate Dehydrogenase (LDH) release. Immunoblotting was used to examine the expression of the Ser biosynthesis proteins, PHGDH and phosphoserine aminotransferase (PSAT), and glutathione (GSH) levels were measured via a luminescent-based assay. Results: We determined that short-acting crisantaspase (Rylaze) inhibited the proliferation of several AML cell lines with pharmacologically relevant IC50 values ranging from 0.001 to 0.08 µg/mL. Similarly, decreasing the concentration of Gln in the media resulted in a dose-dependent decrease in cell proliferation/growth, confirming AML sensitivity to Gln depletion. Protein expression of PHGDH and PSAT was upregulated following Gln withdrawal from the media (Fig. 1A) as well as following crisantaspase treatment (Fig. 1B). PHGDH knockout AML cells were significantly more sensitive to crisantaspase, yielding IC50 values ranging from 1.34 to 6.59 picogram/mL, ~250-fold lower compared to the parental lines (Fig. 1C). Gln depletion through crisantaspase treatment or withdrawal from the media decreased GSH levels and increased LDH release, both of which were further enhanced by PHGDH knockout. Taken together, these results support dual targeting of Gln and Ser metabolism in AML. Preliminary testing of an NAD-dependent cell permeable PHGDH competitive inhibitor prodrug (Ethyl 2-{4-[(1S)-1-[(4,5-dichloro-1,6-dimethyl-1H-indol-2-yl)formamido]-2-hydroxyethyl]benzenesulfonyl}acetate) demonstrated an inhibition of AML cell proliferation in multiple cell lines, with IC50 concentrations ranging between 1.2 to 2.3 µM. Conclusion: Simultaneous targeting of Ser and Gln metabolism for AML treatment shows promising results in vitro with great potential for translation into clinical practice with clinically available asparaginases, and other glutamine modulators. Ongoing work is focused on the design of a more potent pharmacologic inhibitor of PHGDH to combine with crisantaspase as a novel treatment approach for AML. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

P527: PHASE 1 TRIAL OF PEGCRISANTASPASE IN COMBINATION WITH VENETOCLAX IN ADULTS WITH RELAPSED OR REFRACTORY ACUTE MYELOID LEUKEMIA (R/R AML) - SAFETY, EFFICACY AND PK/PD IN THE FIRST TWO COHORTS

Background: Glutamine depletion induced by Erwinia asparaginases including pegcrisantaspase (PegC) downregulates the mTOR/p70S6K pathway and inhibits 4EBP1 phosphorylation. We recently found that the combination of PegC with the Bcl-2 inhibitor venetoclax (Ven) has strong in vitro and in vivo activity in poor-prognosis AML, mediated by enhancement of eIF4E-4EBP1 interaction on the cap-binding complex and decreasing protein synthesis. We hypothesized that Ven-PegC treatment will be safe and effective in the treatment of R/R AML. Here we report the results from the first two cohorts of a phase 1 trial of Ven-PegC in adults with R/R AML. Aims: The primary objective is to estimate the maximum tolerated dose and to determine the regimen-limiting toxicities (RLT) of Ven-PegC. The exploratory endpoints include plasma amino acids levels 1- and 2-weeks post-PegC administration. Methods: This is an open-label phase 1b clinical trial (NCT04666649) of daily oral Ven in combination with biweekly out-patient IV PegC in 28-day cycles that will be administered to 4 cohorts (Table 1A) based on a standard 3 + 3 design. Results: Eight patients (pts) were enrolled into the first two cohorts; 7 received ≥ 1 dose of PegC and one dropped out before receiving study drugs. Pts’ characteristics are shown in Table 1B. Median age was 66 (59-76) years; two (29%) were female; six (86%) had adverse-risk AML by ELN classification; and four (57%) had complex karyotypes. Pharmacokinetic (PK) and pharmacodynamic (PD) data are available for 5 pts; all pts achieved a nadir plasma asparaginase activity > 0.1 IU/mL during cycle 1 (3/3 in cohort 1 within 3 weeks and 2/2 in cohort 2 within 10 days). Mean plasma amino acid levels (µmol/L) at baseline were: asparagine (Asn) 29 (17-38), glutamine (Gln) 421 (384-465), glutamate (Glu) 59 (22-103), serine (Ser) 66 (49-80) and glycine (Gly) 203 (166-207). All pts achieved an Asn level of 0 by day 7; Asn remained undetectable throughout the study. Plasma Gln (µmol/L) reduction from baseline occurred in 4/5 (80%) pts within cycle 1: pt4 423→273 (Day15), pt5 446→86 (Day28), pt6 467→216 (Day22), pt7 388→156 (Day8), pt10 420→395 (Day10). Plasma Glu levels increased in all pts, indicating conversion of Gln to Glu by PegC. Interestingly, plasma levels of Ser increased 1.5-2-fold at the nadir of plasma Gln (Figure 1A-C, maximum amino acid changes after Ven-PegC). Seven pts from cohorts 1 (4 pts) and 2 (3 pts) who received at least one dose of Ven-PegC were included in analysis of toxicity and efficacy, with data cutoff February 26, 2022 (Table 1B). No RLT has been observed. In cohort 1, pt2 died before completing cycle 1 due to septic shock and delay in seeking medical care, pt4 died of AML progression. In cohort 2, pt7 had progressive disease after cycle 1, pt9 opted for comfort care before evaluation of response, and pt10 died of AML progression, In cohort 1, pt6 achieved measurable residual disease (MRD)-negative complete remission with partial hematologic recovery (CRh) and proceeded to allogeneic hematopoietic stem cell transplant (allo-HSCT) after 3 cycles of Ven-PegC, and bone marrow blasts in pt5 decreased from 78% to 9% after cycle 1. These two pts had the lowest plasma Gln levels (103 [pt6, cycle2 day29] and 86 [pt5, cycle1 day28]). Image:Summary/Conclusion: No RLT has been observed following Ven-PegC in cohorts 1 or 2. The Ven-PegC combination at the lowest dose of PegC administered, produced an MRD negative CRh in one patient and a transient substantial reduction in bone marrow blast count in another, both associated with decreases in plasma Gln levels. The study is ongoing.

Actions of L-Glutamine vs. COVID-19 Suggest Additional Benefit in Sickle Cell Disease

Background:The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection associated with coronavirus disease 2019 (COVID-19) causes a 3- to 9-fold higher age-adjusted mortality in African American and Hispanic populations, the major US racial groups affected by sickle cell disease (SCD). The Centers for Disease Control designates SCD as a condition at increased risk for severe COVID-19. An urgent need for repurposing of available and safe therapeutics has been cited for such high-risk populations until vaccines are widely available.L-glutamine (GLN) ameliorates clinical pathology of SCD related to elements of COVID-19. Multiple systemic complications of COVID-19 are increasingly attributed to oxidative damage, a target which GLN regulates. Prescription-grade L-glutamine (PGLG) (Endari®, Emmaus Medical) decreases oxidative stress by increasing the ratio of reduced nicotinamide adenine dinucleotide (NAD) to total NAD, which may increase availability of reduced glutathione. PGLG also decreases red cell endothelial adhesion in patients with SCD. Of note, additional analysis of the phase 3 trial demonstrated a 63% lower occurrence of acute chest syndrome (ACS) in PGLG-treated SCD patients compared to control, which has important relevance in the pandemic. A recent report of two computational screens of FDA-approved therapeutics, directed to protein and chemistry targets and to gene expression changes induced by SARS-CoV-2, predicts glutathione and GLN are highly likely to confer benefit in COVID-19 (Kim, J Translat Med, 2020).Methods:We therefore reviewed reports of multi-system effects of GLN in experimental respiratory distress animal models and in ICU and COVID-19 patients. We focused on contributors to cytokine storm and acute respiratory distress syndrome (ARDS), the leading causes of mortality in COVID-19 (Huang, Lancet, 2020). We also conducted a clinical trial in hospitalized COVID-19 patients on ESPEN-recommended nutrition +/- GLN.Results:In experimental ARDS, sepsis, and endotoxin-induced lung injury, GLN decreases consolidation, pulmonary edema, and neutrophil infiltration and increases lung compliance, oxygen saturation, heat shock protein activation, and survival by 2.5-fold over saline controls (Perng WC, Clin Exp Pharmacol Physiol, 2010; Singleton, Crit Care Med, 2005). Patients with severe COVID-19 have increased proinflammatory cytokines; interleukin 6 (IL-6) levels predict and contribute to severity of COVID-19 (Yuki, Clin Immunol, 2020). GLN modulates inflammatory responses by suppressing C-reactive protein, IL-6, and TNF-α release; it also reduces IL-6 in murine studies (37% decrease,p< 0.05; Chuang, BMC Pulm Med, 2014), which could benefit COVID-19 patients.Myocardial injury occurs in up to 12% of COVID-19 patients directly with viral entry through ACE-2 receptors, microvascular damage, endothelial shedding, and inflammation-mediated damage, which GLN protects against (Shi, Eur Heart J, 2020; Shi, JAMA Cardiol, 2020; Shao, Pak J Med Sci, 2015). Inflammatory states lead to GLN consumption and negative GLN balance (Santos, Amino Acids, 2019). Deficient plasma GLN (< 420 µmol/L) is a defined risk for higher mortality in ICU and COVID-19 patients (Shen, Cell, 2020). Glutathione deficiency contributes to SARS-CoV-2 oxidative lung damage and severe disease (Polonikov, ACS Infect Dis, 2020).In a recent clinical trial, patients were confirmed to have SARS-CoV-2 by RT-PCR, had positive CT scans, and were admitted from a COVID-19 clinic. Both arms received ESPEN-recommended nutrition for COVID-19 alone or with GLN (10 grams, 3 times/day; see Table).Conclusions:GLN and glutathione deficiency contribute to COVID-19 severity, and GLN has salutary biologic actions on reducing lung pathology, mediators of cytokine storm, and myocardial injury in animal models, SCD, and ICU patients. GLN reduces severity in standard risk COVID-19 patientsafterinfection has occurred. These findings, combined with computational prediction of GLN benefit vs. COVID-19, support the hypothesis that PGLG treatmentprior toSARS-CoV-2 infection mayreducethe development of severe COVID-19 in SCD and perhaps other high-risk populations. The data as a whole provides a strong rationale for a controlled clinical trial of PGLG toreducesevere COVID-19 in high-risk SCD patients and improve outcomes if infection occurs.DisclosuresCengiz:Biruni University Medical Faculty:Current Employment;Istanbul University-Cerrahpasa Medical Faculty:Ended employment in the past 24 months.Yavuzer:Istanbul University-Cerrahpasa, Cerrahpasa School of Medicine, Department of Internal Medicine, Division of Geriatrics:Current Employment.Yavuzer:Biruni University Medical Faculty:Current Employment;Istanbul University-Cerrahpasa Mediacl Faculty:Ended employment in the past 24 months.Tang:Kaiser Permanente:Current Employment.Ward:Emmaus Medical, Inc.:Current Employment.Goodrow:Emmaus Medical, Inc.:Current Employment;Emmaus Life Sciences Shareholder:Current equity holder in publicly-traded company.Ludlum:Emmaus Life Sciences, Inc.:Consultancy, Current equity holder in publicly-traded company.Stark:Emmaus Life Sciences Shareholder:Current equity holder in publicly-traded company;Emmaus Medical, Inc:Current Employment.Perrine:Cetya Inc.:Membership on an entity’s Board of Directors or advisory committees;Phoenicia Bioscience:Current Employment, Current equity holder in private company, Membership on an entity’s Board of Directors or advisory committees;Phoenicia Therapeutics:Membership on an entity’s Board of Directors or advisory committees, Patents & Royalties;Boston University School of Medicine:Current Employment, Patents & Royalties;Viracta Therapeutics:Patents & Royalties.

Glutamine depletion in patients with Crimean-Congo hemorrhagic fever.

Crimean-Congo hemorrhagic fever (CCHF) is a viral disease. There is not enough knowledge about plasma amino acid levels in CCHF. Therefore, we investigated plasma amino acid levels in patients with CCHF and the association between the levels of these amino acids and disease severity. The plasma amino acid levels (including glutamate [Glu], aspartate [Asp], glutamine [Gln], asparagine [Asn] and gamma-aminobutyric acid [GABA]) in CCHF patients and controls were measured by using liquid chromatography-mass spectrometry. Plasma levels of Gln were lower while Asp, Glu, and GABA levels were higher in patients. In fatal CCHF patients, we found the plasma level of Asn was increased whereas the plasma level of GABA was decreased. This study is the first in the literature to evaluate the plasma Gln, Glu, Asn, Asp, and GABA levels in CCHF patients. We found that the plasma Gln levels were significantly lower in CCHF patients while Asp, Glu, and GABA levels were elevated. Considering that these amino acids are important for immune cells, the plasma amino acid levels of CCHF patients may contribute to the understanding of the pathophysiology of disease and it can be important for supportive treatment of CCHF.

Glutamine Depletion By Addicted Myeloma Cells Inhibits Osteoblastic Differentiation of Bone Marrow Mesenchymal Stromal Cells Limiting Asparagine Availability: A Possible New Mechanism for Myeloma Bone Disease

Metabolic alterations of cancer cells, aimed at sustaining their growth, may also influence the behavior of the tumor microenvironment. Our group has recently demonstrated that multiple myeloma (MM) is a highly glutamine(Gln)-addicted tumor that utilize huge amounts of Gln to fuel its metabolism through the enzyme glutaminase (GLS). For this reason, MM cells exhibits increased Gln uptake, mainly through the ASCT2 transporter. Interestingly, lower bone marrow (BM) plasma Gln concentration (down to a median value of 0.4 mM vs. a median value of 0.6 mM) was found in MM patients as compared with smoldering MM (SMM) and Monoclonal Gammopathy of Uncertain Significance (MGUS). The main feature of MM BM microenvironment is the suppression of osteoblastic (OB) differentiation leading to the development of osteolytic bone lesions, the hallmark of MM. Most recently, it has been demonstrated that Gln metabolism is needed to sustain bone mass formation in murine models and that GLS inhibition decreases OB differentiation of human mesenchymal stromal cells (hMSCs). However, no information is yet available on the role of Gln depletion imposed by MM cell metabolism on OB differentiation into the BM. This topic has been investigated in the present study. Firstly, human MM cells were co-cultured with BM hMSCs, and Gln medium concentration was evaluated with mass spectrometry (MS), demonstrating a MM-induced depletion of the amino acid. Upon Gln depletion, MSC exhibited a sustained induction of Glutamine Synthetase (GS). On the contrary, when differentiated in osteogenic medium (D-MEM + 5% Fetal Bovine Serum, supplemented with 2 mM Gln, ascorbic acid and dexamethasone), GS was suppressed. Conversely, GLS (both KGA and GAC isoforms) and SLC38A2, the gene for the concentrative Gln transporter SNAT2, were induced. These data suggest that hMSCs differentiation in OBs is associated with an increased dependence upon extracellular Gln. Consistent with this conclusion, the activity of SNAT2 was absent in undifferentiated hMSCs but well detectable after 14 days of OB differentiation, when total Gln uptake was also increased. Under the same conditions, OB differentiation markers (RUNX2, COL1A1, ALPL expression and ALPL activity or staining) were significantly induced but their expression was blunted by incubation in low-Gln (0.4 mM) medium or in the presence of the SNAT2 inhibitor MeAIB. The incubation in Gln-free D-MEM suppressed the induction of GLS and SLC38A2 along with OB differentiation, which was restored by the supplementation of Non-Essential Amino Acids (NEAA). Among NEAA, only asparagine (Asn) was able to rescue OB differentiation in the absence of Gln. The determination of intracellular amino acids with MS indicated that OB differentiation was associated with the increase of cell Asn, without significant changes of Gln, glutamate (Glu) or aspartate (Asp). Asparagine Synthetase (ASNS), the Gln-dependent enzyme that accounts for Asn synthesis, was also found induced during OB differentiation of hMSCs. Gene Expression Profiles of primary BM hMSCs and OBs from bone biopsies of both healthy donors (n=7) and MM patients (n=16) indicated that GLS, ASNS, and SLC38A2 are more expressed in OBs, while the expression of GLUL, the gene for GS, is higher in undifferentiated hMSCs from healthy donors. Overall, these results indicate that (1) OB differentiation of hMSCs is Gln-dependent; (2) the partial Gln depletion, imposed by Gln-addicted MM cells in the BM microenvironment, contributes to the impairment of osteoblastic differentiation of hMSCs; (3) hindrance of differentiation may depend on the limited availability of intracellular Asn derived from Gln-dependent ASNS. These results support the evidence that Gln addiction of MM cells affects bone microenvironment leading to the inhibition of OB differentiation and, consequently, to the development of MM bone disease. Disclosures Giuliani: Janssen: Research Funding.

Oral glutamine supplementation attenuates inflammation and oxidative stress-mediated skeletal muscle protein content degradation in immobilized rats: Role of 70 kDa heat shock protein

Skeletal muscle disuse results in myofibrillar atrophy and protein degradation, via inflammatory and oxidative stress-mediated NF-kB signaling pathway activation. Nutritional interventions, such as l-glutamine (GLN) supplementation have shown antioxidant properties and cytoprotective effects through the modulation on the 70-kDa heat shock protein (HSP70) expression. However, these GLN-mediated effects on cell signaling pathways and biochemical mechanisms that control the myofibrillar protein content degradation in muscle disuse situations are poorly known yet. This study investigated the effects of oral GLN plus l-alanine (ALA; GLN ​+ ​ALA-solution) supplementation, either in their free or dipeptide (L-alanyl-l-glutamine-DIP) form, on GLN-glutathione (GSH) axis and cytoprotection mediated by HSP70 protein expression in the slow-twitch soleus and fast-twitch gastrocnemius skeletal muscle of rats submitted to 14-days of hindlimb immobilization-induced disuse muscle atrophy. Forty-eight Wistar rats were distributed into 6 groups: hindlimb immobilized (IMOB group) and hindlimb immobilized orally supplemented with either GLN (1 g kg−1) plus ALA (0.61 g kg−1) ​(GLN ​+ ​ALA-IMOB group) or 1.49 ​g ​kg−1 of DIP (DIP-IMOB group) and; no-immobilized (CTRL) and no-immobilized supplemented GLN ​+ ​ALA and DIP baselines groups. All animals, including CTRL and IMOB rats (water), were supplemented via intragastric gavage for 14 days, concomitantly to immobilization period. Plasma and muscle GLN levels, lipid (thiobarbituric acid reactive substances-TBARS) and protein (carbonyl) peroxidation, erythrocyte concentration of reduced GSH and GSH disulfide (GSSG), plasma and muscle pro-inflammatory TNF-α levels, muscle IKKα/β-NF-kB signaling pathway and, the myofibrillar protein content (MPC) were measured. The MPC was significantly lower in IMOB rats, compared to CTRL, GLN ​+ ​ALA, and DIP animals (p ​< ​0.05). This finding was associated with reduced plasma and muscle GLN concentration, equally in IMOB animals. Conversely, both GLN ​+ ​ALA and DIP supplementation restored plasma and muscle GLN levels, which equilibrated GSH and intracellular redox status (GSSG/GSH ratio) in erythrocytes and skeletal muscle even as, increased muscle HSP70 protein expression; attenuating oxidative stress and TNF-α-mediated NF-kB pathway activation, fact that reverberated on reduction of MPC degradation in GLN ​+ ​ALA-IMOB and DIP-IMOB animals (p ​< ​0.05). In conclusion, the findings shown herein support the oral GLN ​+ ​ALA and DIP supplementations as a therapeutic and effective nutritional alternative to attenuate the deleterious effects of the skeletal muscle protein degradation induced by muscle disuse.

S834 PEGCRISANTASPASE AND VENETOCLAX COMBINATION IS HIGHLY EFFECTIVE AGAINST ACUTE MYELOID LEUKEMIA (AML), MECHANISTICALLY DRIVEN SYNERGISM BETWEEN GLUTAMINE DEPLETION AND BCL‐2 INHIBITION

Background:We reported that Erwinaze could effectively deplete plasma glutamine (Gln) to undetectable levels in patients (pts) with relapsed/refractory (R/R) AML and these pts had higher probability of clinical response. We hypothesized that Gln depletion (pegcrisantaspase, a recombinant pegylated Erwinia crisantaspase, PegC [Jazz Pharmaceuticals]) leads to activation of mitochondrial apoptotic pathways and synergizes with Bcl‐2 inhibition (venetoclax, Ven). In vitro, we demonstrated that anti‐AML activity of Ven was potentiated 10–44 fold by PegC; presence of PegC (0.01 IU/mL) decreased Ven's IC50 (5.3→0.12 μM in MOLM‐14 &amp; 7.5→0.76 μM in MonoMac‐6 cells). Ven‐PegC demonstrated synergy in killing AML cells. Here, we report the mechanism of action (MOA), and in vivo safety and efficacy of Ven‐PegC in AML.Aims:(1) To determine toxicity profile of Ven‐PegC in mice (2) To analyze effect of Ven‐PegC on plasma Gln in vivo (3) To determine efficacy of Ven‐PegC in orthotopic pt‐derived xenograft (PDX), and (4) To assess changes in the transcriptome/translatome of AML cells treated with Ven‐PegC.Methods:Toxicity profile of Ven‐PegC was investigated in immune competent (CD1) and incompetent (NRG/NSG) mice. To measure pharmacodynamic (PD) effect of PegC, NSG mice were dosed once IV at 3 different doses [125, 250 and 500 IU/kg (n = 4 mice/dose)]. We euthanized 2 mice on days 3 and 10 and measured plasma amino acids by LCMS. Efficacy of Ven‐PegC was assessed in an AML pt‐derived xenograft model (AML‐45) that expresses luciferase. We injected 1x106 AML‐45‐luc cells IV into NRG mice, and after engraftment mice were treated with vehicle, Ven (75 mg/kg, PO, 5x/week), PegC (200 IU/kg, IV, 1x/week), or Ven‐PegC for 2 weeks (wks), 1 wk off, then 2 more wks. Mice were imaged weekly and survival monitored. For MOA, MOLM‐14 cells were treated with each agent alone and combined followed by RNA isolation, RNA‐seq and transcriptome analysis. For translatome, cells were treated similarly and subjected to western blotting, cap enrichment and polysomal fractionation.Results:For safety, Ven‐PegC resulted in mild weight loss with full recovery at PD active doses. Amylase, lipase, creatinine, and transaminases were unchanged after 3 wks of Ven‐PegC treatment. Ven‐PegC induced myelosuppression as expected (7.4 ± 1.0x103/μL control vs 1.9 ± 0.3x103/μL combination, p&lt;0.003). For PD, asparagine was not detectable on days 3 &amp; 10 post dosing. No plasma Gln was detected on day 3 and on day 10 was undetectable at the highest dose. For efficacy, Ven‐PegC significantly lowered leukemia burden compared to monotherapies (p &lt; 0.03). On Day 36, AML was virtually undetectable in mice treated with Ven‐PegC (Fig. 1A‐1B). For MOA, immunoblot analysis showed decreased phosphorylation of mTOR substrates (p70S6K &amp; 4E‐BP1) with Ven‐PegC. A significant decrease in cap driven translation complexes and in eIF4E phosphorylation at Ser‐209 followed by enhanced interaction with 4E‐BP1 were observed with Ven‐PegC (Fig. 1C). Transcriptome analysis showed differential gene expression pattern between groups (RT‐qPCR validated; Fig. 1D). p90RSK, a key gene in ribosomal biogenesis and cell proliferation, was reduced upon Ven‐PegC treatment in MOLM‐14 transcriptome profiling (RNA‐qPCR/immunoblot analysis confirmed). Analysis of altered translational profile is ongoing.Summary/Conclusion:Ven‐PegC is a mechanistically‐driven, highly active treatment against AML in the PDX model. Ven‐PegC appears to broadly regulate the ribosomal biosynthetic pathway, a major driver for chemo‐resistance. The Phase 1 clinical trial in pts with R/R AML is planned.image

Glutamine and Glutamate Supplementation Increases the Levels of These Amino Acids in the Milk of Pasture-fed Mares

Background: L-Glutamine (Gln), the most abundant free alpha amino acid in the body, plays a major role in the transport of nitrogen and carbon between tissues, and is an important source of respiratory energy for intestinal and immune system cells. Mares lose lean body mass during lactation, when plasma and milk Gln levels change significantly. However, supplementation with Gln combined with other amino acids may not alter equine plasma Gln levels. The work reported here was designed to test the hypothesis that supplementation with a mixture of glutamine and glutamate (AminoGut) alters blood and milk free glutamine and glutamate levels in pasture-fed lactating mares.Materials, Methods &amp; Results: This study involved 31 multiparous Quarter Horse mares, which were divided into three groups immediately postpartum, as follows: G-CON (n = 19); G-50 g supplemented with 50 g of Gln + Glu plus 200 g of concentrate (n = 6); and G-100 g, supplemented with 100 g of Gln + Glu plus 200 g of concentrate (n = 6). Blood and milk samples were collected on the day of parturition prior to supplementation, and monthly until weaning. The milk samples were used to analyze the Gln, Glu composition and levels, while the blood samples were used for further analysis of blood biomarkers. The results were analyzed by ANOVA and by Tukey’s test and the P value was set at 5%. The G-CON group showed a significant reduction of 11-35% in the mean blood glutamine levels from the first month postpartum and throughout lactation. In contrast, blood glutamine levels in groups G-50 g and G-100 g did not change significantly from parturition through 5 months of lactation. The supplemented groups showed no significant differences in blood variables such as protein, albumin, urea, creatinine, cholesterol, triglycerides and minerals. Free glutamine levels in milk did not change from parturition through the end of lactation in the G-CON group, but groups G-50 g and the G-100 g showed a marked rise in milk glutamine levels throughout the first three months of lactation (~3x), which remained high (~2x) until the foals were weaned (P &gt; 0.05).Discussion: The results of this study indicate that Gln + Glu supplementation successfully increased Gln levels in mare milk in the first three months of lactation, and Glu levels in G-100 g in the first four months, without affecting the levels of these amino acids in the animals’ blood, which remained similar to data obtained at parturition. In fact, the Gln levels in both supplemented groups exceeded 1,000 mmol/mL throughout lactation, unlike those of the control group and of the samples obtained at parturition. Moreover, supplementation did not produce significant changes in blood biomarkers, including those pertaining to protein metabolism (urea, creatinine, uric acid, albumin and total proteins), indicating that the product used for supplementation did not interfere in these biomarkers, which remained within the normal physiological variations for the species. It was concluded that daily dietary supplementation with 50 g of a mixture of glutamine and glutamate produced an effect similar to supplementation with 100 g/day. Both supplementation protocols succeeded in raising glutamine levels in mare milk in the first three months of lactation, without interfering in blood biomarkers or milk composition. In view of the cost of the product, we recommend that the daily diet of mares during lactation be supplemented with 50 g of a mixture of Gln + Glu in order to produce the desired nutritional effects.

Dependence on glutamine uptake and glutamine addiction characterize myeloma cells: a new attractive target

AbstractThe importance of glutamine (Gln) metabolism in multiple myeloma (MM) cells and its potential role as a therapeutic target are still unknown, although it has been reported that human myeloma cell lines (HMCLs) are highly sensitive to Gln depletion. In this study, we found that both HMCLs and primary bone marrow (BM) CD138+ cells produced large amounts of ammonium in the presence of Gln. MM patients have lower BM plasma Gln with higher ammonium and glutamate than patients with indolent monoclonal gammopathies. Interestingly, HMCLs expressed glutaminase (GLS1) and were sensitive to its inhibition, whereas they exhibited negligible expression of glutamine synthetase (GS). High GLS1 and low GS expression were also observed in primary CD138+ cells. Gln-free incubation or treatment with the glutaminolytic enzyme l-asparaginase depleted the cell contents of Gln, glutamate, and the anaplerotic substrate 2-oxoglutarate, inhibiting MM cell growth. Consistent with the dependence of MM cells on extracellular Gln, a gene expression profile analysis, on both proprietary and published datasets, showed an increased expression of the Gln transporters SNAT1, ASCT2, and LAT1 by CD138+ cells across the progression of monoclonal gammopathies. Among these transporters, only ASCT2 inhibition in HMCLs caused a marked decrease in Gln uptake and a significant fall in cell growth. Consistently, stable ASCT2 downregulation by a lentiviral approach inhibited HMCL growth in vitro and in a murine model. In conclusion, MM cells strictly depend on extracellular Gln and show features of Gln addiction. Therefore, the inhibition of Gln uptake is a new attractive therapeutic strategy for MM.

Brain interstitial fluid glutamine homeostasis is controlled by blood–brain barrier SLC7A5/LAT1 amino acid transporter

L-glutamine (Gln) is the most abundant amino acid in plasma and cerebrospinal fluid and a precursor for the main central nervous system excitatory (L-glutamate) and inhibitory (γ-aminobutyric acid (GABA)) neurotransmitters. Concentrations of Gln and 13 other brain interstitial fluid amino acids were measured in awake, freely moving mice by hippocampal microdialysis using an extrapolation to zero flow rate method. Interstitial fluid levels for all amino acids including Gln were ∼5–10 times lower than in cerebrospinal fluid. Although the large increase in plasma Gln by intraperitoneal (IP) injection of 15N2-labeled Gln (hGln) did not increase total interstitial fluid Gln, low levels of hGln were detected in microdialysis samples. Competitive inhibition of system A (SLC38A1&2; SNAT1&2) or system L (SLC7A5&8; LAT1&2) transporters in brain by perfusion with α-(methylamino)-isobutyric acid (MeAIB) or 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid (BCH) respectively, was tested. The data showed a significantly greater increase in interstitial fluid Gln upon BCH than MeAIB treatment. Furthermore, brain BCH perfusion also strongly increased the influx of hGln into interstitial fluid following IP injection consistent with transstimulation of LAT1-mediated transendothelial transport. Taken together, the data support the independent homeostatic regulation of amino acids in interstitial fluid vs. cerebrospinal fluid and the role of the blood–brain barrier expressed SLC7A5/LAT1 as a key interstitial fluid gatekeeper.

Effects of Eimeria acervulina infection severity on growth performance, apparent ileal amino acid digestibility, and plasma concentrations of amino acids, carotenoids, and α1-acid glycoprotein in broilers

An experiment was conducted to evaluate growth performance, apparent ileal digestibility (AID) of amino acids, and plasma concentrations of amino acids, carotenoids, and α1-acid glycoprotein, an acute-phase protein, in broilers inoculated with graded doses of E. acervulina oocysts. Ross 308 male broilers (400 total) were housed in battery cages from 1 to 21 d post-hatch and received common corn-soybean meal-based diets throughout the experiment. At 9 d post-hatch, birds were individually weighed and allotted to 4 treatment groups with 10 replicate cages of 10 birds per cage. At 15 d post-hatch, all birds were inoculated with 1 mL of distilled water that contained 0, 2.5 × 105, 5.0 × 105, or 1.0 × 106 sporulated E. acervulina oocysts. At 21 d, birds were euthanized for collection of blood and ileal digesta. Body weight gain and feed efficiency decreased linearly (P < 0.05) with increasing E. acervulina dose. With the exception of Trp and Gly, AID values decreased (P < 0.05) linearly or quadratically for all amino acids by an average of 2.6 percentage units for birds inoculated with 1.0 × 106 oocysts compared with uninfected birds. Infection with E. acervulina caused a quadratic decrease (P < 0.05) in plasma carotenoid concentrations. Plasma concentrations of Arg and Tyr decreased linearly (P < 0.05) with increasing E. acervulina inoculation dose and plasma Gln and Asn decreased quadratically (P < 0.01). Linear increases (P < 0.05) were observed for plasma Lys, Leu, Ile, Val, Pro, and Orn as E. acervulina inoculation dose increased. Plasma α1-acid glycoprotein of broilers was not influenced (P > 0.05) by E. acervulina infection. In conclusion, E. acervulina challenge adversely impacted growth performance, plasma carotenoids, and AID of amino acids in a dose-dependent manner. However, plasma amino acid responses to graded E. acervulina inoculation doses varied considerably among amino acids. Thus, these results indicated that alterations in amino acid metabolism caused by E. acervulina infection extended beyond reduced amino acid digestibility.

The role of skeletal muscle in liver glutathione metabolism during acetaminophen overdose

Marked alterations in systemic glutamate–glutamine metabolism characterize the catabolic state, in which there is an increased breakdown and decreased synthesis of skeletal muscle protein. Among these alterations are a greatly increased net release of glutamine (Gln) from skeletal muscle into blood plasma and a dramatic depletion of intramuscular Gln. Understanding the catabolic state is important because a number of pathological conditions with very different etiologies are characterized by its presence; these include major surgery, sepsis, trauma, and some cancers. Acetaminophen (APAP) overdose is also accompanied by dramatic changes in systemic glutamate–glutamine metabolism including large drops in liver glutathione (for which glutamate is a precursor) and plasma Gln. We have constructed a mathematical model of glutamate and glutamine metabolism in rat which includes liver, blood plasma and skeletal muscle. We show that for the normal rat, the model solutions fit experimental data including the diurnal variation in liver glutathione (GSH). We show that for the rat chronically dosed with dexamethasone (an artificial glucocorticoid which induces a catabolic state) the model can be used to explain empirically observed facts such as the linear decline in intramuscular Gln and the drop in plasma glutamine. We show that for the Wistar rat undergoing APAP overdose the model reproduces the experimentally observed rebound of liver GSH to normal levels by the 24-h mark. We show that this rebound is achieved in part by the action of the cystine–glutamate antiporter, an amino acid transporter not normally expressed in liver but induced under conditions of oxidative stress. Finally, we explain why supplementation with Gln, a Glu precursor, assists in the preservation of liver GSH during APAP overdose despite the fact that under normal conditions only Cys is rate-limiting for GSH formation.

Lactation performance of dairy cows fed increasing concentrations of wheat dried distillers grains with solubles

In Western Canada, dried distillers grains with solubles (DDGS) is produced from mixtures of corn and wheat at variable ratios, and used as a source of dietary crude protein (CP) in diets of lactating dairy cows. The objective of this study was to determine the effect of increasing dietary allocation of wheat DDGS on dry matter intake, milk production, milk composition, feed efficiency, plasma metabolites, and ruminal fermentation of dairy cows in midlactation. Sixteen multiparous and 16 primiparous lactating Holstein cows were used in a replicated 4×4 Latin square design with 3-wk periods. Dietary treatments were a control diet containing canola meal as the primary protein source (CON) and diets containing increasing concentrations of wheat DDGS in place of corn DDGS (0, 50, and 100% of dietary DDGS allocation). The treatment protein sources supplied approximately 35% of dietary CP. Yields of milk, milk fat, lactose, and energy-corrected milk were greater for diets containing DDGS compared with the CON diet. Although cows fed the DDGS diets tended to have lower CP digestibility compared with those fed the CON diet, concentrations of ruminal ammonia nitrogen, plasma urea nitrogen, and milk urea nitrogen were higher, but milk protein concentration was lower for cows fed the DDGS diets. Although dry matter intake increased linearly as the dietary allocation of wheat DDGS increased, milk yield was not affected, thus decreasing feed efficiency linearly. Feeding increasing levels of wheat DDGS tended to decrease plasma glucose concentration linearly. Plasma Leu concentration decreased linearly and plasma Gln concentration increased linearly as dietary inclusion of wheat DDGS increased. Apparent total-tract digestibility of nutrients except for CP was not affected by dietary treatments. A mixture of wheat and corn DDGS seems to have similar feeding values to both DDGS sources and it can be used as an alternative protein source in diets for lactating dairy cows. However, replacing corn DDGS with wheat DDGS might decrease feed efficiency.