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Glutamine and nitrogen balance

Glutamine and nitrogen balance

Hepato-Pancreato-Biliary Surgery. The amino acid pool is balxnce in Recovery Meal Ideas Guarana and inflammation reduction Body cleanse tea be influenced Glutmaine both dietary protein consumption as well as normal protein turnover within the tissues. Article ADS Google Scholar Sullivan, L. Cite this article Kodama, M. The B55alpha subunit of PP2A drives a pdependent metabolic adaptation to glutamine deprivation.

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M G Brown , M G Brown. Department of Surgery, Queen's University Belfast. Correspondence to: Mr M. Oxford Academic. Google Scholar. G R Campbell. B J Rowlands. Select Format Select format. It suggests that cells could synthesize glutamate from α-ketoglutarate Supplementary Fig. We then used the labeled carbon source, 13 C 6 -glucose, to culture MCF-7 and HeLa cells, and the 13 C tracing analysis showed that α-ketoglutarate and glutamate were substantially labeled by 13 C even in the presence of glutamine but the glucose-derived fraction significantly increased in the absence of glutamine Supplementary Fig.

Nonetheless, glutamine was not labeled at all in the presence of glutamine but slightly labeled, when compared to α-ketoglutarate and glutamate, in the absence of glutamine Supplementary Fig.

These results suggest that glutamine cannot be efficiently synthesized in cells even upon its scarcity, and it could be attributed to the low level of GS. We then over-expressed GS in MCF-7 cells Supplementary Fig. GS expression also inhibited cell death and restored cell proliferation upon supplementation with glutamate, α-ketoglutarate or pyruvate in MCF-7, HeLa, and A cells in the absence of glutamine Supplementary Fig.

Conversely, GS knockdown sensitized cells to glutamine loss independently of supplementation of nutrients Supplementary Fig. These data suggest that glutamine amide-nitrogen is indispensable to cell proliferation.

When glutamine nitrogen is used, its carbon, if beyond biomass accumulation, could be oxidized via the CAC.

However, when the cellular requirement of glutamine carbon is increased in some conditions, such as under hypoxia, how do cells metabolically dispose of the overflowed nitrogen? Under hypoxia, glutamine was used as the major carbon source, especially for lipid biosynthesis Fig.

Indeed, we detected a significantly increased uptake of glutamine but decreased excretion of glutamate in MCF-7, HeLa, and 4T1 cells Fig. We then traced the metabolic flux of U- 13 C-labeled glutamine. In addition, the increased enrichment of glutamine- 13 C in α-ketoglutarate, citrate, glutamate, and proline, downstream metabolites of glutamine, was also detected Fig.

These data suggest the increased uptake of glutamine as the carbon source under hypoxia. Next, we tried to figure out the metabolic fate of glutamine nitrogen under hypoxia. Glutamine can be deaminated and release its nitrogen as ammonia 4 Fig.

As showed in Fig. Ammonia can be removed by forming urea in human body 20 , 21 , and we did not detect urea in the culture medium but measured a decreased level of intracellular urea Fig. In addition, we also detected a decreased excretion of alanine, the potential ammonia carrier 21 , in the medium Supplementary Fig.

Therefore, glutamine-nitrogen should be enriched in some other cellular metabolites under hypoxia. Increased glutamine as the carbon source under hypoxia.

a A schematic to show the metabolism of glutamine carbon and nitrogen. We then used HeLa and 4T1 cell lines as the models to screen the nitrogen metabolome including nitrogen-contained metabolites.

Interestingly, five increased compounds, including dihydroorotate, orotate, IMP, guanosine, and inosine, were involved in the nucleotide biosynthesis pathway Fig.

We verified again that IMP, the precursor of AMP and GMP, significantly enhanced and carbamoyl-Asp, dihydroorotate, and orotate, the precursors of UMP, dramatically increased in HeLa, MCF-7, and 4T1 cells under hypoxia Fig.

Furthermore, we measured the cellular nucleotides and their derivatives in HeLa and MCF-7 cells under hypoxia. As shown in Fig. On the contrary, UMP and its derivatives, UDP, UTP, and CTP, even reduced under hypoxia Fig. Accumulation of cellular nucleotide precursors under hypoxia.

Cellular metabolites were measured by LC—MS-based metabolomics. b A schematic to show the metabolic assimilation of glutamine-nitrogen to nucleotide biosynthesis. Surprisingly, the metabolomic analysis showed that aspartate, the major precursor for carbamoyl-Asp, dihydroorotate, and orotate Fig.

We further measured the levels of cellular amino acids in MCF-7 and HeLa cells under hypoxia and normoxia. We observed the increased cellular glutamine and glutamate in both cell lines under hypoxia Supplementary Fig. Intriguingly, our results in MCF-7 and HeLa cells clearly indicated that only aspartate significantly decreased in both cell lines Supplementary Fig.

This could explain why accumulated IMP Fig. Taken together, these data indicate that hypoxia substantially lead to the accumulation of cellular nucleotide precursors, in particular pyrimidine precursors including carbamoyl-Asp, dihydroorotate, and orotate.

Now we traced the assimilation of glutamine-nitrogen using amide- 15 N-labeled or amine- 15 N-labeled glutamine in HeLa and MCF-7 cells. The labeled fractions of dihydroorotate and orotate significantly increased under hypoxia Fig.

However, dihydroorotate and orotate inefficiently processed to its downstream UMP, because cellular UMP was less labeled by glutamine- 15 N Fig. The reduced labeled fraction of IMP was also reduced under hypoxia Fig.

Metabolic flux of glutamine-nitrogen and glutamine-carbon in nucleoside biosynthesis. a A schematic to show the metabolism of isotope-labeled glutamine. Glutamine-amine- 15 N labeled non-essential amino acids, such as glutamate, proline, asparagine, aspartate, and alanine Fig.

In contrast, glutamine-amide- 15 N predominantly labeled asparagine in both MCF-7 and HeLa cells Fig. In addition, glutamine-amide- 15 N also slightly labeled non-essential amino acids in MCF-7 breast cancer cells Fig. However, almost all the 15 N-labeled fraction of amino acids decreased under hypoxia Fig.

Taken together, these data suggest that glutamine nitrogen is enriched in dihydroorotate and orotate but not in amino acids under hypoxia. Glutamine carbon can be potentially integrated into pyrimidine nucleotides after it has been converted to aspartate through the CAC-mediated oxidative pathway or α-ketoglutarate carboxylation reductive pathway Fig.

The two pathways can be distinguished by determining the enrichment of 13 C 5 -glutamine-derived 13 C in acetyl-CoA, aspartate, citrate, dihydroorotate, and orotate. These results clearly showed that glutamine carbon was integrated into acetyl-CoA the precursor for lipid biosynthesis and aspartate-derived dihydroorotate and orotate through the reductive pathway under hypoxia.

Moreover, the fraction of glutamine- 13 C-labeled acetyl-CoA, dihydroorotate, and orotate significantly increased Figs. These data suggest that the biosyntheses of acetyl-CoA, dihydroorotate, and orotate from glutamine were urged by hypoxia.

However, aspartate, the direct precursor of dihydroorotate, was less efficiently labeled by glutamine- 13 C under hypoxia Fig. This most likely resulted from the multienzyme complexes involved in the pyrimidine biosynthesis where the newly synthesized aspartate by cytosolic glutamic-oxaloacetic transaminase 1 GOT1 can be efficiently converted to dihydroorotate and orotate 4 , To test this speculation, we cultured HeLa and MCF-7 cells with 13 C 4 , 15 N-labeled aspartate, and traced the isotope-labeled intermediates in the pyrimidine biosynthesis.

Hypoxia increased the uptake and biosynthesis of aspartate Fig. Interestingly, in the normal condition, aspartate inefficiently labeled dihydroorotate, orotate, and UMP Fig.

These data are consistent with the notion of metabolic multienzyme complexes and support that hypoxia strongly boosts entry of aspartate to dihydroorotate and orotate, which possibly leads to the decreased cellular aspartate Fig.

Promotion of aspartate to pyrimidine precursors under hypoxia. c A schematic to show the metabolic flux of 13 C 4 , 15 N-aspartate in the biosynthesis of aspartate, glutamate, dihydroorotate, and orotate.

To test whether the increased metabolic flux of glutamine to dihydroorotate and orotate is required for cell survival under hypoxia, we knockdowned the involved enzymes, such as GOT1, carbamoyl-phosphate synthetase 2, aspartate transcarbamylase ATCase and dihydroorotase CAD and dihydroorotate dehydrogenase DHODH Fig.

DHODH and CAD were the key enzymes involved in the biosynthesis of pyrimidine nucleotides, thus knockdown of DHODH and CAD apparently suppressed proliferation of these cells even in the normal condition Fig. In contrast, GOT1 knockdown slightly affected cell proliferation Fig.

However, knockdown of CAD or GOT1 strongly, while DHODH knockdown marginally, sensitized cancer cells to hypoxia Fig. These results suggest that the biosynthesis of dihydroorotate, not orotate, is indispensable to survival under hypoxia. Association of glutamine-carbon metabolism with its nitrogen assimilation under hypoxia.

a A schematic to show the metabolism of glutamine carbon and nitrogen in the pyrimidine biosynthesis. b Proliferation of HeLa cells with or without knockdown of DHODH, CAD, and GOT1 cultured under hypoxia and normoxia for 3 days.

Western blot to validate the knockdown of DHODH, CAD, and GOT1. To survive hypoxia, cells could excrete the accumulated metabolites. Thus, we measured the excretion of carbamoyl aspartate, dihydroorotate, and orotate in the culture medium.

No carbamoyl aspartate was detected under both hypoxia and normoxia. However, we observed a significantly increased amount of dihydroorotate, but not orotate, in hypoxic medium of various cell lines, such as MCF-7, HeLa, A, HCC-LM3, SGC, and 4T1 Fig.

The overall conversion of glutamine and dioxide carbon to acetyl-CoA and secretory dihydroorotate, not orotate, consumes electrons Supplementary Fig. The reprogrammed metabolic pathway essentially renders glutamine only to acetyl-CoA for lipogenesis under hypoxia, and glutamine-amide and glutamine-amine groups are incorporated into secretory dihydroorotate by CAD and GOT1.

These observations could explain why hypoxia increased the utilization of glutamine-carbon but decreased the release of ammonia Fig. This speculation was further supported by the results that knockdown of CAD or GOT1 enhanced the production of ammonia under hypoxia Fig.

As expected, knockdown of CAD or GOT1 dramatically suppressed hypoxia-induced dihydroorotate and orotate Fig. Meantime, their depletion was also found to reduce glutamine-derived α-ketoglutarate, citrate, and acetyl-CoA Fig. Moreover, supplementation with aspartate did not effectively restore the accumulation of dihydroorotate and orotate even in cells with GOT1 depletion Fig.

These data suggest that glutamine-carbon metabolism is associated with its nitrogen assimilation to dihydroorotate. To further confirm this speculation, we treated HeLa cells with α-ketoglutarate, the carbon form of glutamine. Our results showed that α-ketoglutarate supplementation reduced glutamine uptake Supplementary Fig.

Taken together, our results suggest that the metabolism of glutamine-carbon is necessary to cell survival and depends on the increased biosynthesis of dihydroorotate under hypoxia.

Next, we investigated how hypoxia promoted the biosynthesis and excretion of dihydroorotate. We measured the protein levels of related enzymes, including GOT1, CAD, DHODH, and uridine monophosphate synthetase UMPS Fig.

As a typical indicator, HIF-1α was indeed induced by hypoxia, but the expression of these metabolic enzymes were not enhanced Fig. CAD is the critical enzyme for the biosynthesis of dihydroorotate, and can be activated by phosphorylation 23 , The level of phosphorylated CAD was increase in MCF-7 cells but decreased in HeLa cells under hypoxia Fig.

These data suggest that the hyper-biosynthesis of dihydroorotate most likely is not mediated by hypoxia-regulated protein levels. Hypoxia-induced NADH accumulation promotes biosynthesis and excretion of dihydroorotate.

a Western blot of lysates from MCF-7 and HeLa cells cultured under hypoxia for different time as indicated. c Western blot of lysates from MCF-7 and 4T1 cells cultured under hypoxia for different time as indicated.

The relative abundance of dihydroorotate, asparatate, and UTP were listed here. Hypoxia also disabled the mitochondrial electron transport chain ETC and induced the accumulation of electrons, such as NADH Fig.

Interestingly, mitochondrial dysfunction was previously reported to promote cells to use glutamine-carbon for acetyl-CoA through the reductive pathway Here, we confirmed that the inhibition of the ETC by antimycin A-induced NADH accumulation Fig.

We then used HeLa and 4T1 cells to perform a targeted metabolomic analysis. Eighteen overlapped nitrogen-contained metabolites in both HeLa and 4T1 cells were significantly affected by antimycin A Fig.

In fact, the level of cellular aspartate was also previously observed to reduce in cells with the ETC dysfunction 26 , Importantly, the excretion of dihydroorotate, not orotate, was detected in the culture medium with antimcyin A treatment Fig.

Now, we tried to alleviate the electron accumulation in HeLa cells under hypoxia using a pyruvate analog, α-ketobutyrate that can be reduced to excretory α-hydroxybutyrate by NADH-consuming lactate dehydrogenases and thus neutralize NADH accumulation Meantime, we also observed that α-ketobutyrate attenuated the excretion of dihydroorotate Fig.

In addition, α-ketobutyrate supply also enhanced ammonia production under hypoxia Fig. Taken together, these data suggest that hypoxia-induced NADH accumulation promotes the biosynthesis and excretion of dihydroorotate, and regulates the metabolism of glutamine-nitrogen.

CAD is a multi-domain enzyme and can be allosterically inhibited by UTP or activated by phosphoribosyl pyrophosphate PRPP In fact, we also detected the increased level of cellular PRPP Fig.

These factors possibly accounted for the promotion of CAD activity by hypoxia. Interestingly, α-ketobutyrate also suppressed PRPP accumulation and reversed cellular UTP under hypoxia Fig.

NADH acts as the coenzyme of many cellular transformations, and thus its accumulation could extensively influence these reactions and reprogram cellular metabolism. In view of the in vivo hypoxic microenvironment of tumors, we then measured the blood dihydroorotate and orotate in patients of breast cancer, lung cancer, gastric cancer, and liver cancer, as well as healthy persons.

Furthermore, we measured the levels of blood dihydroorotate and orotate in healthy or HeLa-derived tumor-bearing nude mice, and both metabolites were found to significantly increase Fig.

To further investigate whether orotate was directly released from tumor or oxidized from dihydroorotate in blood, we administrated mice with intraperitoneal injection with amide- 15 N-labeled glutamine Supplementary Fig. Dihydroorotate and orotate in the tumor tissue was rapidly labeled by 15 N Supplementary Fig.

Similar results were also obtained from 4T1-derived tumor-bearing mice Supplementary Fig. These data suggest that the in vivo tumors could directly excrete dihydroorotate that is somehow rapidly converted to orotate in blood Fig. The amount of blood orotate was much greater than that of blood dihydroorotate in mice Fig.

Glutamine-derived dihydroorotate is required for tumor growth. a , b Serum dihydroorotate and orotate in healthy controls and cancer patients.

c , d Serum dihydroorotate and orotate in healthy and HeLa-derived tumor-bearing nude mice. g Tumors directly excreted dihydroorotate that was oxidized to orotate in blood.

Both CAD and DHODH are indispensable to pyrimidine biosynthesis, thus their inhibition by shRNA led to the similarly suppressive effect on cell proliferation in the normal condition Fig. It was not surprising that knockdown of CAD and DHODH repressed cell proliferation in a xenograft mouse model Fig.

However, we found that CAD knockdown suppressed the in vivo tumor growth much more significantly than DHODH knockdown Fig.

This most likely resulted from the fact that CAD, not DHODH was required for dihydroorotate biosynthesis and cell survival under hypoxia Fig. Accordingly, higher levels of CAD showed a positive correlation with a shorter overall survival time in patients of breast cancer, lung cancer, gastric cancer, and liver cancer Supplementary Fig.

In contrast, a high level of DHODH was only observed in patients of gastric cancer with a short overall survival time Supplementary Fig. These data suggest that the increased biosynthesis of dihydroorotate from glutamine is critical for the in vivo tumor growth.

Compared with CAD, GOT1 seemed to be mainly required for dihydroorotate biosynthesis and cell proliferation under hypoxia but less affected cell growth in the normal condition Fig. In this study, we reveal a specific metabolic pathway that hypoxia pushes glutamine carbon and nitrogen via the reductive pathway to dihydroorotate that are then expelled outside of cells rather than processing to their downstream UMP.

This unique metabolic reprogramming probably has a vital physiological relevance. Proliferating cancer cells require glutamine carbon to generate acetyl-CoA for lipid synthesis under hypoxia. The secretion of dihydroorotate perfectly scavenges both the rest nitrogen and carbon and renders glutamine mainly to acetyl-CoA in cells.

Hypoxia promotes the enrichment of glutamine-nitrogen in dihydroorotate on one hand, and suppresses the conversion of dihydroorotate to its downstream UMP on the other hand, which could lead to the accumulation of dihydroorotate and promote its excretion.

The amide nitrogen of glutamine is most often used to synthesize asparagine and nucleotide, concomitantly with production of glutamate Glutamate can be used for multiple purposes, but it can be readily synthesized through the transamination between α-ketoglutarate and other amino acids, thus it is dispensable to cancer cell proliferation.

By contrast, the biosynthesis of glutamine from glutamate is inactive in cancer cells. Therefore, the amide-nitrogen of glutamine is necessary to cell growth.

Normally, when the amide-nitrogen of glutamine is used, the resultant glutamate, if beyond the metabolic requirement, could be excreted out of cells or replenish the CAC after its transamination. Once the metabolic assimilation of glutamine amide-nitrogen could not keep pace with that of glutamine carbon, cells need to get rid of the superfluous amide-nitrogen.

Generally, glutamine is thought to liberate amide-nitrogen as ammonia and converted to glutamate that can further produce α-ketoglutarate upon either deamination or transamination 5 , 9 , The accumulating ammonia should be safely removed.

In mammalian cells, there are three enzymes accounting for ammonia assimilation 30 , GS synthesizes glutamine from glutamate and ammonia, and this process is essentially a reversion of glutamine deamination and thus does not play a real role in scavenging glutamine-derived ammonia.

Carbamoyl phosphate synthetase I CPSI incorporates ammonia to urea, but we detected a decreased cellular level of urea in cancer cells under hypoxia Fig. GLUD can convert α-ketoglutarate and ammonia to glutamate whose amine group could be further transferred to other amino acids.

Unfortunately, GLUD-mediated ammonia assimilation inefficiently or does not take place in cancer cells 4 , 15 , especially in the hypoxic condition Fig.

Therefore, proliferating cancer cells develop the specific metabolic pathway to dispose of glutamine amide-nitrogen. In mammalian cells, glutamine amide-nitrogen is used to synthesize carbamoyl phosphate by carbamoylphosphate synthetase II CPSII domain of CAD protein, a trifunctional multi-domain enzyme.

Carbamoyl phosphate then reacts with aspartate to generate carbamoylaspartate, which is catalyzed by ATCase domain of CAD. The dihydroorotase DHOase domain of CAD further synthesizes dihydroorotate from carbamoylaspartate. Although aspartate is the major precursor for pyrimidine biosynthesis, it is seriously scarce in human blood and actually cannot be efficiently absorbed even upon its supplementation Therefore, the cellular aspartate almost completely depends on its biosynthesis, and its amine-nitrogen is transferred from glutamate.

In contrast, glutamine is the most abundant amino acid in human blood, and essentially it can provide directly amide-nitrogen and indirectly amine-nitrogen for nucleotide biosynthesis.

Overall, glutamine carbon and nitrogen could be readily coordinatively catabolized without ammonia generation. Under hypoxia, proliferating cells increase the metabolic requirement for glutamine carbon to support lipogensis, and excrete overflowed nitrogen and carbon as the form of dihydroorotate.

This selection process was repeated three times to yield AIG-3 cells, which showed a pronounced ability to form colonies in 3-D culture as well as to form tumors on transplantation into nude mice Fig.

The malignant progression from TSM to AIG-3 was associated with a stepwise increase in the expression of c-Myc Fig. a Experimental flow. We first established transformed cells by introduction of hTERT, the SV40 early region, and c-Myc TSM into the normal human diploid fibroblast line TIG To recapitulate stepwise malignancy, we serially selected cells grown in 2.

We next applied iMPAQT and metabolomics analyses to the malignant progression model, with the resulting findings being subjected to validation with an integrated meta-analysis. Icon made by Pixel perfect from www.

b Phase-contrast microscopy of colonies stained with 2- 4-iodophenyl 4-nitrophenyl phenyl-2 H -tetrazolium chloride INT showing anchorage-independent growth in the malignant progression model. c Anchorage-independent growth was quantitated by counting of colonies stained with INT.

Colonies were counted in two randomly selected fields in each of three dishes. e Immunoblot analysis of c-Myc and Hsp90 loading control in the malignant progression model, was conducted with single time.

The intensity of the c-Myc band was measured by densitometry. ND, not detected. f Scatter plot showing the correlation of c-Myc abundance and anchorage-independent growth in the malignant progression model. Results for several different AIG-1, -2, or -3 clones are shown.

R 2 , correlation coefficient. Source data are provided as a Source Data file. We applied iMPAQT to TSM as well as AIG-1, -2, and -3 clones in order to comprehensively measure the abundance of metabolic enzymes. The amounts of certain glycolytic enzymes including hexokinase 2 HK2 , enolase 1 ENO1 , and lactate dehydrogenase A LDHA were increased in AIG-3 cells compared with TSM cells, with glucose uptake also being increased Fig.

These results suggested that the Warburg effect is more pronounced in AIG-3 cells than in TSM cells. Gene Ontology GO enrichment analysis revealed that the most affected biological process in AIG-3 cells compared with TSM cells was nucleic acid biosynthesis Fig.

The amounts of most enzymes in nucleotide biosynthesis pathways both de novo and salvage pathways including PPAT, which metabolizes glutamine, were thus cooperatively increased in AIG-3 cells compared with TSM cells, whereas the expression of GLS1, the rate-limiting enzyme of the glutamine anaplerotic pathway, was downregulated in AIG-3 cells Fig.

The pyruvate dehydrogenase E1 alpha subunit PDHA1 , which oxidizes pyruvate for TCA anaplerosis, was upregulated in AIG-3 cells Fig. To confirm these changes in glucose metabolism observed at the proteome level, we performed mass isotopomer analysis with [ 13 C 6 ]glucose 16 Fig.

a Heat map for hierarchical clustering of metabolic enzymes measured by iMPAQT in TSM and AIG cell lines. The abundance of enzymes of the de novo nucleotide biosynthetic pathway was increased, whereas that of those of the glutaminolysis pathway was reduced, in AIG-3 cell lines compared with TSM cells.

Five independent AIG-3 lines 1 to 5 were examined. Scale bar indicates z-scores for protein amount. b GO analysis of metabolic enzymes whose abundance as revealed by iMPAQT was changed in AIG-3 cells compared with TSM cells. c iMPAQT-determined changes in the abundance of enzymes in the indicated metabolic pathways for AIG-3 versus TSM cells.

d Immunoblot analysis of PPAT, GLS1, and PDHA1 in TSM and AIG-1 to -3 cells, was conducted with single time. e Schematic representation of [ 13 C 6 ]glucose metabolism. Blue and white circles represent 13 C and 12 C, respectively. f — h TSM and AIG-3 cells were exposed to Our results thus suggested that the amounts of PPAT in the de novo nucleotide biosynthesis pathway and of GLS1 in the glutamine anaplerotic pathway into the TCA cycle, two enzymes that rely on glutamine as a key substrate, are changed in a reciprocal manner during malignant progression.

Similar to the genes for cyclin D2 CCND2 and LDHA, expression of the PPAT gene is regulated by c-Myc 20 , 27 , 28 and was found to be increased in AIG-3 cells Supplementary Fig.

In contrast, the amount of GLS1 mRNA was downregulated in AIG-3 cells Supplementary Fig. The abundance of GLS1 was previously shown to be indirectly controlled by c-Myc through negative regulation of the microRNA mira, which suppresses expression of the GLS1 gene However, another study found that c-Myc transactivates the mira gene Indeed, previous studies have shown that GLS1 expression was not increased or was even decreased by overexpression of c-Myc 30 , Despite our observation that c-Myc was highly upregulated in AIG-3 cells, the abundance of mira was also substantially increased in these cells compared with TSM cells Supplementary Fig.

Neither cell growth in 2-D culture Fig. Incorporation of a stable isotope-labeled metabolite and its labeling efficiency at steady state serve as a basis for evaluation of the effects of changes in the expression of a metabolic enzyme on metabolic pathways Given that the labeling efficiency at steady state can reflect the activities of many metabolic reactions; however, it is difficult to evaluate effects on an individual reaction 33 , 34 , We therefore evaluated the labeling efficiency for each reaction at early 0.

Whereas a 13 C fraction of IMP, AMP, GMP, or UMP was not detected in TSM or AIG-3 cells, incorporation of 15 N into IMP Fig. Similar results were obtained for UMP Fig.

The increased efficiency for 15 N-labeling of nucleotides IMP, AMP, GMP, and UMP in AIG-3 cells Fig. Given the marked upregulation of enzymes for nucleic acid synthesis and the marked increase in labeling efficiency of nucleotides in AIG-3 cells, this decrease in the pool size of nucleotide metabolites in these cells is likely attributable to an increased rate of nucleotide synthesis.

Red circles represent 15 N, blue circles 13 C, and white circles 12 C. The proportion of 15 N and 13 C, of 15 N, or of 13 C in each metabolite was calculated from the mass isotopomer distribution determined by IC-MS or LC-M S.

We next prepared culture medium containing amino acids at the physiological concentrations present in human plasma After culture in such physiological medium for 5 days, TSM and AIG-3 cells were labeled with 0. The 15 N- labeling efficiency for AMP and GMP was significantly greater in AIG-3 cells than in TSM cells Supplementary Fig.

IMP was not detected under the physiological condition, likely because of the low concentration of glutamine. To evaluate the activity of GLS1, we examined the isotopomer distribution for metabolites in the glutamine anaplerotic pathway into the TCA cycle at 0.

α-KG and fumarate are not only produced by the glutamine anaplerosis; they are also generated by the de novo nucleic acid synthesis pathway and are present in the cytosol Supplementary Fig. Labeling of nucleic acid metabolites with 15 N was essentially not detected at 0.

The markedly reduced labeling efficiency for α-KG and fumarate at 0. Together, our proteomics and metabolomics data thus consistently indicated that the fate of glutamine is substantially shifted from the TCA cycle to the de novo nucleotide biosynthetic pathway during malignant transformation in an experimental cancer cell model.

To investigate whether the balance between these two enzymes is deterministic for cell proliferation, we examined the effects of GLS1 overexpression in AIG-3 cells Fig. A mutant SA form of GLS1 that lacks enzymatic activity 37 was examined as a control to determine whether any effect of overexpression is dependent on GLS1 activity.

Forced expression of GLS1, but not that of GLS1 SA , resulted in an increase in the labeling rate for α-KG as well as a decrease in that for IMP Fig. It also induced a decrease in the intracellular level of glutamine in AIG-3 cells Supplementary Fig.

The proliferation rate of AIG-3 cells in 2-D Fig. AIG-3 cells overexpressing GLS1 were not able to survive in glutamine-free medium, and the proliferation rate of these cells increased as the glutamine concentration of the medium was increased Fig.

Furthermore, tumors formed by the GLS1-overexpressing cells in nude mice were significantly smaller than those formed by control cells Fig. The metabolic disturbance induced by excessive activity of GLS1 may thus have reduced glutamine availability for nucleic acid synthesis and thereby inhibited cell proliferation in the GLS1-overexpressing AIG-3 cells.

Collectively, these results also suggested that excessive activity of GLS1 inhibits tumor growth. a Immunoblot analysis of GLS1 in AIG-3 cells stably overexpressing OE wild-type or SA mutant forms of human GLS1 or infected with the corresponding empty retrovirus, was conducted with single time.

g Immunoblot analysis of PPAT in AIG-3 cells stably infected with retroviruses encoding luciferase control Luci-KD or two independent PPAT shRNAs, was conducted with single time.

All cells count and metabolite measurements were conducted with three biological replicates. All colonies were counted in two randomly selected fields in each of three dishes.

In a converse approach, we examined the effects of PPAT depletion by short hairpin RNA shRNA -mediated RNA interference in AIG-3 cells Fig. The labeling efficiency, pool size, and isotopologue distribution of glutamine Supplementary Fig.

Given that many nucleotides including AMP and GMP exert feedback inhibition on PPAT and other enzymes in the de novo nucleotide biosynthesis pathway, the supplementation of AMP or GMP to PPAT-depleted AIG-3 cells did not improve their capacity for growth Supplementary Fig.

Overexpression of human PPAT, but not supplementation with hypoxanthine to activate the salvage pathway of nucleotide biosynthesis, normalized the anchorage-independent growth of PPAT-depleted AIG-3 cells Supplementary Fig.

Furthermore, the PPAT-depleted cells formed smaller tumors in nude mice than did control cells Fig. However, depletion of GLS1 or overexpression of PPAT alone in TSM cells did not confer a malignant phenotype similar to that of AIG-3 cells Supplementary Fig.

Exposure to dimethyl α-KG, a membrane-permeable form of α-KG, did not affect the proliferation of AIG-3 cells Supplementary Fig. Together, these various data suggested that a reduced activity of PPAT inhibits tumor growth, and they were consistent with the notion that the balance between GLS1 and PPAT governs the metabolism of carbon and nitrogen derived from glutamine and thereby controls cell proliferation and tumor growth.

The growth-inhibitory effects of glutamine deprivation or CB treatment were less pronounced for TSM or AIG-3 cells maintained in the physiological medium than for those maintained in conventional medium Supplementary Fig.

In addition, AIG-3 cells, in which the activity of GLS1 is attenuated compared with that in TSM cells Fig. The sensitivity to glutamine deprivation was thus similar to that to CB treatment, consistent with previous observations showing that the sensitivity to glutamine starvation depends on the activity of the rate-limiting enzyme GLS1 14 , 38 , 39 , The extracellular glutamine concentration has been found to affect the expression of metabolic enzymes We therefore applied immunoblot analysis to measure the abundance of GLS1 and PPAT in TSM and AIG-3 cells cultured in the physiological or conventional medium.

Collectively, these results thus suggested that the presence of excess glutamine increases the dependence of cancer cells on GLS1, with the possible consequence that CB inhibits the growth of such cells selectively and is less effective for cancer cells under the physiological condition.

To investigate the generalizability of the notion that the balance between PPAT and GLS1 is deterministic for proliferation of human cancer cell lines, we examined the effects of overexpression of these enzymes in A and HeLa cells Supplementary Fig. Forced expression of GLS1 reduced the proliferation rate of A and HeLa cells in 2-D Supplementary Fig.

On the other hand, overexpression of PPAT promoted the proliferation of both cell lines in 2-D and 3-D culture as well as their tumorigenicity in nude mice, although, with the exception of that on anchorage-independent growth of HeLa cells, these effects did not achieve statistical significance Supplementary Fig.

We also examined the effects of GLS1 or PPAT depletion in A and HeLa cells Supplementary Fig. GLS1 depletion in A and HeLa cells did not affect proliferation in 2-D culture Supplementary Fig. In contrast, depletion of PPAT in both cell lines inhibited proliferation in 2-D Supplementary Fig.

Overall, the effects of modulation of PPAT-GLS1 expression balance in cancer cell lines on cell proliferation and tumorigenesis in nude mice were consistent with those observed in transformed fibroblasts.

Recent studies have suggested that dependence on glutamine metabolism might differ among cancer types We therefore next applied immunoblot analysis to measure the abundance of metabolic enzymes including GLS1 and PPAT in cancer cell lines originating from a variety of human tissues.

In contrast, GLS1 depletion or PPAT overexpression did not consistently increase the anchorage-independent growth of these cells.

We next examined whether these various cancer cell lines cultured in the physiological medium were susceptible to CB Indeed, only HCT colorectal cancer cells Fig. These results were consistent with the effects of perturbation of PPAT and GLS1 expression levels Fig.

All cells were counted with three biological replicates. Collectively, our data indicated that modulation of glutamine fate might be of therapeutic value for many types of cancer, with the potential antitumor effect possibly being predictable on the basis of the endogenous expression levels of GLS1 and PPAT.

To validate our findings that PPAT-GLS1 balance influences cancer malignancy in cells and in mice, we examined the relation of PPAT and GLS1 gene expression to overall survival in single cohorts of lung, brain, or neuroendocrine cancer patients Supplementary Fig. The outcome of individuals with high PPAT expression was significantly worse than that of those with low PPAT expression in each cohort, whereas individuals with low GLS1 expression tended to show a poorer prognosis compared with those with high GLS1 expression.

These differences were most pronounced in the patients with neuroendocrine cancer. We next extended these findings by performing an integrated meta-analysis for all metabolic enzymes with public datasets representing a total of ~11, patients with a variety of cancers Fig.

Many enzymes of the de novo purine biosynthesis pathway were found to have a high hazard ratio HR with regard to their gene expression level and overall survival in these combined cancer cohorts, with three of them—PPAT, glycinamide ribonucleotide transformylase GART , and GMP synthase GMPS —being ranked in the top four Fig.

In contrast, enzymes of the glutamine anaplerotic pathway into the TCA cycle—such as glutamate dehydrogenase 1 GLUD1 and GLS2—did not have a high HR or tended to be associated with good prognosis.

The HR for PPAT was remarkably high in tissue-specific cohorts, such as those for lung, breast, brain, hematopoietic, neuroendocrine, liver, or pancreatic cancer, in both the random-effects model and the fixed-effects model Fig. Instead of PPAT, hypoxanthine phosphoribosyltransferase 1 HPRT1 was significantly associated with poor prognosis in colorectal cancer Supplementary Fig.

Together with our observations in cells and mice showing that PPAT-GLS1 balance regulates cell proliferation and malignancy, these results with human cohorts thus suggested that the expression of PPAT is a strong indicator for prognosis in many cancer types, consistent with the findings of a previous study of a single lung cancer cohort a Integrated meta-analysis for gene expression levels of all metabolic enzymes enzymes in cancer cohort studies combined by means of the random-effects model.

Enzymes of the de novo nucleotide biosynthetic pathway and the glutaminolysis pathway are shown in red and blue, respectively. The integrated hazard ratios HRs and P -values are shown in a volcano plot.

The total numbers of patients were as follows: PPAT, 10,; PRPS2, 11,; GART, 12,; PAICS, 11,; ATIC, 11,; IMPDH, 11,; GMPS, 11,; UMPS, 11,; GLS1, 11,; GLS2, 10,; GLUD1, 10,; GLUD2, 10,; DLST, 11,; DLD, 11,; SUCLA, 10,; and SDHB, 11, b Cancer cohort studies for each organ were combined by means of the random-effects model.

NS not significant. All cohorts were divided at the median gene expression level in both a , b. The observation that PPAT expression was significantly associated with poor prognosis for neuroendocrine cancer prompted us to investigate whether such expression is also related to the outcome of SCLC, which is thought to be a high-grade neuroendocrine cancer Indeed, RNA-sequencing data for SCLC patients GSE revealed that PPAT expression was greater in tumor than in normal tissue, whereas GLS1 expression was lower in tumor than in normal tissue Fig.

Kaplan-Meier analysis of the RNA-sequencing data for these SCLC patients 42 showed that the outcome of individuals with high PPAT expression in tumor tissue was significantly worse than that of those with low such expression Fig. A similar pattern was observed for expression of phosphoribosyl pyrophosphate synthetase 2 PRPS2 , which is also a rate-limiting enzyme in the de novo nucleotide synthesis pathway whose expression is regulated by c-Myc 19 , 20 , although the difference in overall survival was smaller than that for PPAT Fig.

In contrast, the expression of neither UMP synthetase UMPS Fig. The upper and lower limits of the red boxes represent quartiles, with the line within the boxes indicating the median and the whiskers showing the extremes.

Blue diamonds indicate confidence intervals. b — e Kaplan-Meier survival analysis for SCLC patients with high or low expression levels of PPAT b , PRPS2 c , UMPS d , or GLS1 e genes in their tumors GSE f — k Immunoblot analysis of PPAT in MSL f , STC-1 h , and SBC-3 j SCLC cell lines stably infected with retroviruses encoding luciferase control Luci-KD or two independent PPAT PPAT-KD 2 or - 3 shRNAs, was conducted with single time.

In j , PPAT-depleted SBC-3 cells were also infected with a retrovirus for wild-type WT human PPAT. l Immunoblot analysis of GLS1 in SBC-3 cells stably overexpressing OE wild-type human GLS1 or infected with the corresponding empty retrovirus, was conducted with single time.

We next depleted three SCLC cell lines MSL, STC-1, and SBC-3 of PPAT with specific shRNAs and examined the effects on cell growth in 3-D culture. Depletion of PPAT inhibited the anchorage-independent growth of all three cell lines Fig. Overexpression of GLS1 also suppressed the anchorage-independent growth of SBC-3 cells Fig.

Collectively, the strong correlation of PPAT expression with poor prognosis in SCLC patients as well as the suppression of anchorage-independent growth of SCLC cell lines by PPAT depletion indicated that PPAT might be a promising therapeutic target for SCLC.

In addition to the carbon shift of the Warburg effect, here we report that a nitrogen shift plays a key role in malignant progression of cancer. Our results show that both PPAT and GLS1 are decisive factors in control of the shift in utilization of nitrogen derived from glutamine, which affects the efficiency of malignant transformation both in vitro and in vivo.

Our experimental findings with cells and mice together with statistical data from an integrated meta-analysis in human cohorts support the notion that metabolic convergence of glutamine toward nucleotide biosynthesis may be an almost universal process in malignant progression of human cancer Fig.

Proteomic and metabolomic analyses unveil the entire landscape of metabolic changes in a model of malignant cancer. In addition to the carbon shift of the Warburg effect, a nitrogen shift plays a key role in malignant progression in the model. PPAT and GLS1 are decisive factors controlling the nitrogen shift from glutamine.

Although GLS1 has been thought to have a protumor effect, we now show that it can actually and partially have an antitumor effect, as revealed by our experimental results for metabolomics analysis, anchorage-independent growth, and tumor formation in nude mice as well as our meta-analysis of human cohorts.

Glutamine oxidation was previously found to be suppressed in anchorage-independent spheroids compared with monolayer-cultured cells Furthermore, neither genetic ablation nor pharmacological inhibition of GLS1 was found to affect the growth of lung tumors in vivo GLS2, an isozyme of GLS1, was also shown to have antitumor potential 44 , Given that the core region of solid tumors is glutamine deficient 46 , nitrogen metabolism in cells in this region is likely directed toward nucleotide biosynthesis through an increase in PPAT expression and suppression of GLS1 so as to support cell proliferation under the condition of glutamine limitation.

Under this condition, carbon derived from glucose, rather than from glutamine, contributes to anaplerosis for the TCA cycle 26 , 47 , 48 in association with an increase in the abundance of PDHA1 Although catabolism of glutamine via GLS1 might be required for ATP synthesis in cancer, little evidence supports the notion that excessive accumulation of ATP in cells promotes cell proliferation 18 , 49 , Our conclusion that GLS1 is not a protumor factor in many cancer types with the exception of colorectal cancer is supported by the results of our meta-analysis, an approach that integrates data from many independent cohorts and provides the highest level of evidence 51 , Whereas most types of cancer show a high HR for PPAT, colorectal cancer is an exception, with HRs for PPAT and GLS1 being 0.

The high dependence of colorectal cancer on GLS1 might reflect an original trait of intestinal epithelial cells, which utilize glutamine as a metabolic fuel instead of glucose 53 , 54 , 55 , Promotion of glutamine anaplerotic pathway into the TCA cycle in the intestinal epithelium is thought to reduce the consumption of glucose by this tissue and thereby allow its efficient transport to the bloodstream.

Given that growth and disease progression are relatively slow in colorectal cancer compared with other cancer types 57 , 58 , In conclusion, we have identified key factors that control the metabolic fate of glutamine and the dependence of tumors of different organs on these factors. Our findings provide the basis for exploration of a different regime for cancer treatment.

Among the identified factors, PPAT may be one of the most promising targets, given its substantial contribution to the nitrogen shift from glutamine as well as its high association with prognosis in many cancer types. We confirmed that TIG-3 cells and human cancer cell lines were free of mycoplasma contamination.

TIG-3 cells expressing the mouse ecotropic retrovirus receptor were then infected with the retrovirus encoding hTERT. The resulting cells, designated TIG-3 T , were further infected with the retrovirus for the SV40 early region and subjected to selection to yield TIG-3 TS cells, which were then infected with the retrovirus for c-Myc to establish TIG-3 TSM cells.

Total RNA isolated from cells with the use of the TRIzol reagent Thermo Fisher Scientific was subjected to reverse transcription RT with the use of ReverTra Ace Toyobo , and the resulting cDNA was subjected to real-time polymerase chain reaction PCR analysis with SYBR Green PCR Master Mix and specific primers in a Step One Plus Real-Time PCR System Applied Biosystems.

Total microRNA was isolated from cells with the use of a NucleoSpin device Takara Bio. The abundance of target mRNAs was normalized by that of β-actin mRNA, whereas that of mira was normalized by that of U6 RNA. For the establishment of cell lines expressing shRNAs for PPAT or GLS1, we designed and constructed shRNA vectors in pCX4.

When the specific shRNA used is not indicated, the experiment was performed with PPAT or GLS1 shRNA 3. Complementary DNAs encoding wild-type human PPAT or wild-type or SA mutant forms of human GLS1 were subcloned into pCX4.

This procedure was repeated a total of three times to yield AIG-1, AIG-2, and AIG-3 cells, consecutively. Colonies formed after culture for 16 days were stained with 2- 4-iodophenyl 4-nitrophenyl phenyl-2 H -tetrazolium chloride INT, Dojindo and photographed. Colonies formed after culture for 20 days were stained with INT.

Colonies were counted in two randomly selected fields in each of three dishes or wells. Cell lysis and immunoblot analysis were performed Immunoblot signals were quantified with the use of an ImageQuant LAS instrument and ImageQuant TL software GE Healthcare Life Sciences.

Antibodies to c-Myc , ab and to GLS1 , ab; or ab in Supplementary Figs. All mouse experiments were approved by the Animal Ethics Committee of Kyushu University.

Portions of the cell suspension were transferred to 1. Portions 2 μl of each sample were then assayed in triplicate for protein concentration with the BCA assay.

Copy number of each enzyme per cell was calculated taking into account the protein amount per cell estimated from the results of the BCA assay. Peptides for metabolic enzymes were selected from the iMPAQT database and subjected to chemical synthesis Supplementary Data 1.

The peptides were labeled with the mTRAQΔ4 reagent and added to the mTRAQΔ0-labled sample digests. The corresponding multiple reaction monitoring MRM transitions with the expected retention times were obtained from the iMPAQT database.

MRM was performed with a triple-stage quadrupole mass spectrometer QTRAP, SCIEX coupled to a nanoflow liquid chromatography system Eksigent nano-LC, SICEX. Endogenous peptide abundance was obtained by multiplication of the ratio of the light and heavy intensities summed for each transition and the known amount of synthetic peptide.

DMEM containing high glucose and sodium pyruvate but lacking amino acids catalog no. For [ 13 C 6 ]glucose labeling in Fig. Cell labeling, metabolomics analysis, and data processing were performed at the Division of Metabolomics of the Medical Institute of Bioregulation at Kyushu University We searched the public database PROGgene, which compiles cohort studies from public repositories such as GEO, EBI Array Express, and The Cancer Genome Atlas TCGA.

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Oral glutamine supplementation theoretically may modify Onion powder and flakes uses response to injury.

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: Glutamine and nitrogen balance

Publication types Garcia-Bermudez, J. The fate of glutamine nitrogen is shifted from the anaplerotic pathway into the TCA cycle to nucleotide biosynthesis, with this shift being controlled by glutaminase GLS1 and phosphoribosyl pyrophosphate amidotransferase PPAT. Despite our observation that c-Myc was highly upregulated in AIG-3 cells, the abundance of mira was also substantially increased in these cells compared with TSM cells Supplementary Fig. Article CAS PubMed PubMed Central Google Scholar Xiang, Y. Complicated version, step 2: Transfer amine to acceptor a -keto acid:. DHODH and CAD were the key enzymes involved in the biosynthesis of pyrimidine nucleotides, thus knockdown of DHODH and CAD apparently suppressed proliferation of these cells even in the normal condition Fig.
A shift in glutamine nitrogen metabolism contributes to the malignant progression of cancer Review Series Lung inflammatory injury and tissue repair Jul Immune Environment in Glioblastoma Feb Korsmeyer Award 25th Anniversary Collection Jan Aging Jul Next-Generation Sequencing in Medicine Jun New Therapeutic Targets in Cardiovascular Diseases Mar Immunometabolism Jan View all review series The glutamine synthetase enzyme is encoded by the glutamate-ammonia ligase gene, GLUL. We found that glutamine nitrogen is necessary to nucleotide biosynthesis, but enriched in dihyroorotate and orotate rather than processing to its downstream uridine monophosphate under hypoxia. The inorganic phosphate P i necessary for this reaction is primarily derived from the hydrolysis of ATP and its function is to lower the K m of the enzyme for glutamine. Nearly tasteless. Compare with similar items This Item. This study was designed to demonstrate the role of the jejunum in postinjury glutamine metabolism and to evaluate the influence of enteral glutamine supplements on nitrogen and ammonia metabolism after laparotomy and bowel resection in dogs.
Gut Health & Nitrogen Balance

Aspartate typically from transamination of oxaloacetate; see IIA2 and IIA3d, above. Ammonia , which may come from many sources, especially hydrolysis of glutamine see IIA3a, above and oxidative deamination of glutamate see IIA3d, above.

See also breakdown of purines, in a later lecture. Catalyzed by Carbamoyl Phosphate Synthetase I CPS I , in liver mitochondria. Step 2: Formation of Citrulline. Catalyzed by Ornithine Trans-Carbamoylase OTC , in liver mitochondria. Step 3: Formation of Argininosuccinate.

Catalyzed by Argininosuccinate Synthetase AS , in liver cytoplasm. Step 4: Cleavage to form Arginine. Catalyzed by Argininosuccinase, a.

Argininosuccinate Lyase AL , liver cytoplasm. Step 5: Cleavage to release Urea. Catalyzed by Arginase no abbreviation ; liver cytoplasm. i Rule of thumb: Any reaction that creates a new C-N bond costs one ATP.

ii The urea cycle costs energy BUT it produces energy as well:. Special role for alanine in energy metabolism in muscle: the glucose-alanine cycle. Muscles frequently utilize amino acids as energy sources, They are consequently particularly active for production of glutamine.

Under heavy energy demands, muscles convert to anaerobic energy production via simple glycolysis, producing excess pyruvate and lactate. The excess pyruvate and ammonia can be converted to alanine and sent to the liver.

There the amino groups are converted to urea and the pyruvate is used in gluconeogenesis to form glucose, which goes back to peripherals via blood.

protein synthesis see also other products of Arg, later. Sidebar: Why is ammonia toxic? speculation only! Glutamate level is disturbed; and since it is a neurotransmitter, its levels may be critical to proper neural function. Glutamate is recycled from post-synaptic neuron to pre-synaptic neuron as glutamine via glutamine synthetase , and that step is probably disturbed by high ammonia levels.

Glutamate is also the precursor another neurotransmitter, gamma aminobutyric acid GABA , which thus may be affected by hyperammonemia. Alterations in glutamate levels may influence energetics. In addition, removing ammonia uses ATP glutamine synthetase , also with potentially detrimental effects on energetics.

Generalized features of urea cycle defects:. Loss of an enzyme causes substrate to build up. The pathway backs up all the way to ammonia, which is toxic. Complete absence of any of these enzymes causes neonatal death.

Infant displaying irritability, hypotonia, lethargy, vomiting, ataxia, delayed growth. Older child or adult displays similar symptoms after precipitating event e. If untreated, progresses to spasticity, mental retardation, coma, death. All of the deficiencies may present with hyperammonemia.

Determine which enzyme by substrate concentrations:. CPSD: only hyperammonemia; diagnose by elimination. OTCD: hyperammonemia with orotate in blood, urine.

ASD: elevated citrulline in blood, urine. ALD: elevated argininosuccinate in blood, urine. AD: elevated arginine in blood, urine.

Dialysis to reduce the blood ammonia levels. Intravenous sodium benzoate and phenylacetate to provide for nitrogen disposal. both compounds bind amino acids and are then excreted. Sorry, there was a problem. There was an error retrieving your Wish Lists.

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About this item Accepted : 11 December Cell death assay was performed as previous 36 , 37 , GFP-positive cells were counted as apoptosis. Glucose feeds the TCA cycle via circulating lactate. This occurs through the actions of cytosolic versions of the TCA cycle enzymes, fumarate hydratase fumarase , which yields malate, and malate dehydrogenase, which yields oxaloacetate. The pLKO.

Glutamine and nitrogen balance -

Find articles by Knight, D. Published December 1, - More info. To assess the consequences of elevated branched chain amino acid levels on alanine, glutamine, and ammonia metabolism in muscle, L-leucine meals Bilateral forearm studies were performed, and the dominant arm was subjected to 15 min of light exercise, using a calibrated dynamometer, beginning 45 min after the ingestion of the meal.

Large uptakes of leucine were seen across both forearm muscle beds within 30 min of the meal. After exercise, blood flow in the dominant arm increased from 3. Glutamine flux out of the dominant forearm increased threefold after the ingestion of the leucine meal and increased eightfold over base line after exercise.

Less marked changes significant only at 90 min in the nonexercised, nondominant arm were also seen. Alanine flux out of the dominant forearm muscle bed increased modestly at 75 and 90 min. No significant change in ammonia flux across either forearm muscle bed was noted.

Unexpectedly, large and significant net nitrogen loss from both forearm muscle beds was documented. Thus, following the ingestion of a leucine meal and light exercise, the primary means by which excess nitrogen is routed out of muscle is via glutamine formation and release with alanine and ammonia pathways playing relatively minor roles.

More importantly, the ingestion of significant amounts of leucine by normal subjects, presumably in optimal nitrogen balance, results in a net loss of nitrogen from muscle. Click on an image below to see the page. View PDF of the complete article.

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Frequently bought together. Get it as soon as Sunday, Feb Integrative Therapeutics - Similase - Physician Developed Digestive Enzymes for Women and Men - Vegan - Vegetable Capsules.

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Get it as soon as Monday, Feb Jarrow Formulas L-Glutamine 2 g, Dietary Supplement for Muscle Tissue, Multifunctional Amino Acid, Immune Support, g 2.

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Thank blance for visiting nature. You are using a browser version Recovery Meal Ideas limited support for CSS. To znd the best nitroven, we recommend you Glutaamine Recovery Meal Ideas more up to Recovery Meal Ideas browser or turn off compatibility mode in Internet Explorer. In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript. Glucose metabolism is remodeled in cancer, but the global pattern of cancer-specific metabolic changes remains unclear. Here we show, using the comprehensive measurement of metabolic enzymes by large-scale targeted proteomics, that the metabolism both carbon and nitrogen is altered during the malignant progression of cancer. The fate of glutamine nitrogen is shifted from the anaplerotic pathway into the TCA cycle to nucleotide biosynthesis, with this shift being controlled by glutaminase GLS1 and phosphoribosyl pyrophosphate amidotransferase PPAT.

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