Human primary (hpBMEC) and induced pluripotent stem cell (iPSC)-derived brain microvascular endothelial-like cells (hiBMEC) are interchangeably used in blood-brain barrier models to study neurological diseases and drug delivery. Both hpBMEC and hiBMEC use glutamine as a source of carbon and nitrogen to produce metabolites and build proteins essential to cell function and communication. We used metabolomic, transcriptomic, and computational methods to examine how hpBMEC and hiBMEC metabolize glutamine, which may impact their utility in modeling the blood-brain barrier. We found that glutamine metabolism was systemically different between the two cell types. hpBMEC had a higher metabolic rate and produced more glutamate and GABA, while hiBMEC rerouted glutamine to produce more glutathione, fatty acids, and asparagine. Higher glutathione production in hiBMEC correlated with higher oxidative stress compared to hpBMEC. α-ketoglutarate (α-KG) supplementation increased glutamate secretion from hiBMEC to match that of hpBMEC; however, α-KG also decreased hiBMEC glycolytic rate. These fundamental metabolic differences between BMEC types may impact in vitro blood-brain barrier model function, particularly communication between BMEC and surrounding cells, and emphasize the importance of evaluating the metabolic impacts of iPSC-derived cells in disease models.
SeoSNahSYLeeK, et al.
Triculture model of In vitro BBB and its application to study BBB-Associated chemosensitivity and drug delivery in glioblastoma. Adv Funct Materials2022;
32: 2106860. DOI:10.1002/ADFM.202106860.
2.
KimJLeeK-TLeeJS, et al.
Fungal brain infection modelled in a human-neurovascular-unit-on-a-chip with a functional blood–brain barrier. Nat Biomed Eng2021;
5: 830–846.
3.
LyuZParkJKimK-M, et al.
A neurovascular-unit-on-a-chip for the evaluation of the restorative potential of stem cell therapies for ischaemic stroke. Nat Biomed Eng2021;
5: 847–863.
4.
ShinYChoiSHKimE, et al.
Blood–brain barrier dysfunction in a 3D In vitro model of Alzheimer’s disease. Adv Sci (Weinh)2019;
6: 1900962.
5.
LiYSunXLiuH, et al.
Development of human in vitro brain-blood barrier model from induced pluripotent stem cell-derived endothelial cells to predict the in vivo permeability of drugs. Neurosci Bull2019;
35: 996–1010.
6.
WangYIAbaciHEShulerML.Microfluidic blood–brain barrier model provides in vivo-like barrier properties for drug permeability screening. Biotechnol Bioeng2017;
114: 184–194.
7.
VatineGDBarrileRWorkmanMJ, et al.
Human iPSC-derived blood-brain barrier chips enable disease modeling and personalized medicine applications. Cell Stem Cell2019;
24: 995–1005.e6.
8.
ParkT-EMustafaogluNHerlandA, et al.
Hypoxia-enhanced blood-brain barrier chip recapitulates human barrier function and shuttling of drugs and antibodies. Nat Commun2019;
10: 2621.
9.
LippmannESAl-AhmadAAzarinSM, et al.
A retinoic acid-enhanced, multicellular human blood-brain barrier model derived from stem cell sources. Sci Rep February 2014;
4: 4160.
10.
LippmannESAzarinSMKayJE, et al.
Human blood-brain barrier endothelial cells derived from pluripotent stem cells. Nat Biotechnol2012;
30: 783–791.
11.
HollmannEKBaileyAKPotharazuAV, et al.
Accelerated differentiation of human induced pluripotent stem cells to blood-brain barrier endothelial cells. Fluids Barriers CNS2017;
14: 9.
12.
NealEHMarinelliNAShiY, et al.
A simplified, fully defined differentiation scheme for producing Blood-Brain barrier endothelial cells from human iPSCs. Stem Cell Reports2019;
12: 1380–1388.
13.
HeYYaoYTsirkaSE, et al.
Cell-culture models of the blood–brain barrier. Stroke2014;
45: 2514–2526.
14.
LuTMHoughtonSMagdeldinT, et al.
Pluripotent stem cell-derived epithelium misidentified as brain microvascular endothelium requires ETS factors to acquire vascular fate. Proc Natl Acad Sci U S A2021;
118: e2016950118.
15.
LippmannESAzarinSMPalecekSP, et al.
Commentary on human pluripotent stem cell-based blood–brain barrier models. Fluids Barriers CNS2020;
17: 64.
16.
SakamuriSSVPSureVNKolliL, et al.
Glycolytic and oxidative phosphorylation defects precede the development of senescence in primary human brain microvascular endothelial cells. Geroscience2022;
44: 1975–1994.
17.
WilsonCLeeMDBuckleyC, et al.
Mitochondrial ATP production is required for endothelial cell control of vascular tone. Function2023;
4: zqac063.
18.
HongS-G, YXParkJ-Y.Flow pattern-dependent mitochondrial dynamics regulates the metabolic profile and inflammatory state of endothelial cells graphical abstract. JCI Insight2022;
17: e159286.
19.
WeberCMMoizBZicSM, et al.
Induced pluripotent stem cell-derived cells model brain microvascular endothelial cell glucose metabolism. Fluids Barriers CNS2022;
19: 98.
20.
HincaSBSalcedoCWagnerA, et al.
Brain endothelial cells metabolize glutamate via glutamate dehydrogenase to replenish TCA-intermediates and produce ATP under hypoglycemic conditions. J Neurochem2021;
157: 1861–1875.
21.
WuGHaynesTELiH, et al.
Glutamine metabolism in endothelial cells: ornithine synthesis from glutamine via pyrroline-5-carboxylate synthase. Comp Biochem Physiol A Mol Integr Physiol2000;
126: 115–123.
22.
WuGBorbollaAGKnabeDA.The uptake of glutamine and release of arginine, citrulline and proline by the small intestine of developing pigs. J Nutr1994;
124: 2437–2444.
23.
WatfordM.Hepatic glutaminase expression: relationship to kidney‐type glutaminase and to the urea cycle. FASEB J1993;
7: 1468–1474.
24.
NewsholmePGordonSNewsholmeEA.Rates of utilization and fates of glucose, glutamine, pyruvate, fatty acids and ketone bodies by mouse macrophages. Biochem J1987;
242: 631–636.
25.
KimBLiJJangC, et al.
Glutamine fuels proliferation but not migration of endothelial cells. EMBO J2017;
36: 2321–2333.
26.
HuangHVandekeereSKaluckaJ, et al.
Role of glutamine and interlinked asparagine metabolism in vessel formation. EMBO J2017;
36: 2334–2352.
27.
DaiD-FDanovizMEWiczerB, et al.
Mitochondrial maturation in human pluripotent stem cell derived cardiomyocytes. Stem Cells Int2017;
2017: 5153625.
28.
RioDCAresMHannonGJ, et al.
Purification of RNA using TRIzol (TRI reagent). Cold Spring Harb Protoc2010;
2010: pdb.prot5439.
29.
QianTHerndaySEBaoX, et al.
Directed differentiation of human pluripotent stem cells to podocytes under defined conditions. Sci Rep2019;
9: 2765–50.
30.
LeeNGLimMHParkJ, et al.
TGF-β and SHH regulate pluripotent stem cell differentiation into brain microvascular endothelial cells in generating an in vitro blood–brain barrier model. Bioengineering2023;
10: 1132.
31.
SongHWForemanKLGastfriendBD, et al.
Transcriptomic comparison of human and mouse brain microvessels. Sci Rep2020;
10: 12358.
32.
RohaniAKashatusJASessionsDT, et al.
Mito hacker: a set of tools to enable high-throughput analysis of mitochondrial network morphology. Sci Rep December 2020;
10: 18941.
33.
NemkovTHansenKCD'AlessandroA.A three-minute method for high-throughput quantitative metabolomics and quantitative tracing experiments of central carbon and nitrogen pathways. Rapid Commun Mass Spectrom2017;
31: 663–673.
34.
NemkovTReiszJAGehrkeS, et al.
High-throughput metabolomics: Isocratic and gradient mass spectrometry-based methods. Methods Mol Biol2019;
1978: 13–26.
35.
ClasquinMFMelamudERabinowitzJD.LC-MS data processing with MAVEN: a metabolomic analysis and visualization engine. Curr Protoc Bioinformatics2012;
Chapter 14: Unit14.11. DOI:10.1002/0471250953.bi1411s37.
36.
MelamudEVastagLRabinowitzJD.Metabolomic analysis and visualization engine for LC-MS data HHS public access. Anal Chem2010;
82: 9818–9826.
37.
NemkovTD'AlessandroAHansenKC.Three-minute method for amino acid analysis by UHPLC and high-resolution quadrupole orbitrap mass spectrometry. Amino Acids2015;
47: 2345–2357.
38.
MillardPDelépineBGuionnetM, et al.
IsoCor: isotope correction for high-resolution MS labeling experiments. Bioinformatics2019;
35: 4484–4487.
39.
PangZZhouGEwaldJ, et al.
Using MetaboAnalyst 5.0 for LC–HRMS spectra processing, multi-omics integration and covariate adjustment of global metabolomics data. Nat Protoc2022;
17: 1735–1761.
AntoniewiczMR.Methods and advances in metabolic flux analysis: a mini-review. J Ind Microbiol Biotechnol2015;
42: 317–325.
42.
DaiZLocasaleJW.Understanding metabolism with flux analysis: from theory to application. Metab Eng2017;
43: 94–102.
43.
RahimMRagavanMDejaS, et al.
INCA 2.0: a tool for integrated, dynamic modeling of NMR-and MS-based isotopomer measurements and rigorous metabolic flux analysis. Metab Eng2022;
69: 1096–7176.
44.
MoizBGarciaJBasehoreS, et al.
13C metabolic flux analysis indicates endothelial cells attenuate metabolic perturbations by modulating TCA activity. Metabolites2021;
11: 226.
45.
VatineGDAl-AhmadABarrigaBK, et al.
Modeling psychomotor retardation using iPSCs from MCT8-Deficient patients indicates a prominent role for the blood-brain barrier. Cell Stem Cell2017;
20: 831–843.e5.
46.
GomesSFMWestermannAJSauerweinT, et al.
Induced pluripotent stem cell-derived brain endothelial cells as a cellular model to study Neisseria meningitidis infection. Front Microbiol2019;
10: 1181.
47.
LinvilleRMDestefanoJGNerenbergRF, et al.
Long-term cryopreservation preserves blood-brain barrier phenotype of iPSC-derived brain microvascular endothelial cells and three-dimensional microvessels HHS public access. Mol Pharm2020;
17: 3425–3434.
48.
LeeCAASeoHSArmienAG, et al.
Modeling and rescue of defective blood-brain barrier function of induced brain microvascular endothelial cells from childhood cerebral adrenoleukodystrophy patients. Fluids Barriers CNS2018;
15: 9.
FaleySLNealEHWangJX, et al.
iPSC-derived brain endothelium exhibits stable, long-term barrier function in perfused hydrogel scaffolds. Stem Cell Reports2019;
12: 474–487.
51.
CouraudP-OGreenwoodJRouxF, et al.
Development and characterization of immortalized cerebral endothelial cell lines. Methods Mol Med2003;
89: 349–364.
52.
LyckRRuderischNMollAG, et al.
Culture-induced changes in blood—brain barrier transcriptome: implications for amino-acid transporters in vivo. J Cereb Blood Flow Metab2009;
29: 1491–1502.
53.
WeberCMMoizBClyneAM.Brain microvascular endothelial cell metabolism and its ties to barrier function. Vitam Horm2024;
126: 25–75.
54.
NicklinPBergmanPZhangB, et al.
Bidirectional transport of amino acids regulates mTOR and autophagy. Cell2009;
136: 521–534.
55.
VerreyF.System L: heteromeric exchangers of large, neutral amino acids involved in directional transport. Pflugers Arch2003;
445: 529–533.
56.
VallerieSNBornfeldtKE.Metabolic flexibility and dysfunction in cardiovascular cells. Arterioscler Thromb Vasc Biol2015;
35: e37–e42.
57.
SongHZhuJLiP, et al.
Metabolic flexibility maintains proliferation and migration of FGFR signaling-deficient lymphatic endothelial cells. J Biol Chem2021;
297: 101149.
58.
PrigioneAFaulerBLurzR, et al.
The senescence-related mitochondrial/oxidative stress pathway is repressed in human induced pluripotent stem cells. Stem Cells2010;
28: 721–733.
59.
VarumSRodriguesASMouraMB, et al.
Energy metabolism in human pluripotent stem cells and their differentiated counterparts. PLoS One2011;
6: e20914.
60.
WaiTLangerT.Mitochondrial dynamics and metabolic regulation. Trends Endocrinol Metab2016;
27: 105–117.
61.
BenadorIYVeliovaMMahdavianiK, et al.
Mitochondria bound to lipid droplets have unique bioenergetics, composition, and dynamics that support lipid droplet expansion. Cell Metab2018;
27: 869–885.e6.
62.
SearsSMHewettSJ.Minireview influence of glutamate and GABA transport on brain excitatory/inhibitory balance. Exp Biol Med (Maywood)2021;
246: 1069–1083.
63.
HuHTakanoNXiangL, et al.
Hypoxia-inducible factors enhance glutamate signaling in cancer cells. Oncotarget2014;
5: 8853–8868.
64.
OnishiYHiraiwaMKamadaH, et al.
Hypoxia affects Slc7a5 expression through HIF‐2α in differentiated neuronal cells. FEBS Open Bio2019;
9: 241–247.
65.
HorieTFukasawaKIezakiT, et al.
Hypoxic stress upregulates the expression of SLC38A1 in brown adipocytes via hypoxia-inducible factor-1α. Pharmacology2018;
101: 64–71.
66.
EzzeddiniRTaghikhaniMSomiMH, et al.
Clinical importance of FASN in relation to HIF-1α and SREBP-1c in gastric adenocarcinoma. Life Sci2019;
224: 169–176.
67.
SelakMAArmourSMMacKenzieED, EDet al.
Succinate links TCA cycle dysfunction to oncogenesis by inhibiting HIF-α prolyl hydroxylase. Cancer Cell2005;
7: 77–85.
68.
FraserTTaylerHLoveS.Fatty acid composition of frontal, temporal and parietal neocortex in the normal human brain and in Alzheimer’s disease. Neurochem Res2010;
35: 503–513.
69.
WangZJLiGMTangWL, et al.
Neuroprotective effects of stearic acid against toxicity of oxygen/glucose deprivation or glutamate on rat cortical or hippocampal slices. Acta Pharmacol Sin2006;
27: 145–150.
70.
ShangQChenGZhangP, et al.
Myristic acid alleviates hippocampal aging correlated with GABAergic signaling. Front Nutr2022;
9: 907526.
71.
RuzzoEKCapo-ChichiJ-MBen-ZeevB, et al.
Deficiency of asparagine synthetase causes congenital microcephaly and a progressive form of encephalopathy. Neuron2013;
80: 429–441.
72.
ZhangYZhangMZhuW, et al.
Succinate accumulation induces mitochondrial reactive oxygen species generation and promotes status epilepticus in the kainic acid rat model. Redox Biol2020;
28: 101365.
73.
ChenJJThiyagarajahMSongJ, et al.
Altered Central and blood glutathione in Alzheimer’s disease and mild cognitive impairment: a meta-analysis. Alzheimers Res Ther December 2022;
14: 23.
74.
ChoiI-YLeePAdanyP, et al.
In vivo evidence of oxidative stress in brains of patients with progressive multiple sclerosis. Mult Scler2018;
24: 1029–1038.
75.
ShuklaDGoelAMandalPK, et al.
Glutathione depletion and concomitant elevation of susceptibility in patients with parkinson’s disease: State-of-the-art MR spectroscopy and neuropsychological study. ACS Chem Neurosci2023;
14: 4383–4394.
76.
DuranCMartínAS.Do endothelial cells eat tryptophan to die?Circ Res2014;
114: 406–408.
77.
WangYLiuHMckenzieG, et al.
Kynurenine is a novel endothelium-derived relaxing factor produced during inflammation. Nat Med2010;
16: 279–285.
WangXLiuRQuX, et al.
α-Ketoglutarate-activated NF-κB signaling promotes compensatory glucose uptake and brain tumor development. Mol Cell2019;
76: 148–162.e7.
80.
SabbaghMFNathansJ.A genome-wide view of the de-differentiation of Central nervous system endothelial cells in culture. Elife January 2020;
9: e51276.
81.
BasehoreSEBohlmanSWeberC, et al.
Laminar flow on endothelial cells suppresses eNOS O-GlcNAcylation to promote eNOS activity. Circ Res2021;
129: 1054–1066.
82.
DoddaballapurAMichalikKMManavskiY, et al.
Laminar shear stress inhibits endothelial cell metabolism via KLF2-mediated repression of PFKFB3. Arterioscler Thromb Vasc Biol2015;
35: 137–145.
83.
WangYLiuJLiuH, et al.
Slow flow induces endothelial dysfunction by regulating thioredoxin-interacting protein-mediated oxidative metabolism and vascular inflammation. Front Cardiovasc Med2022;
9: 1064375.
84.
WuLHChangHCTingPC, et al.
Laminar shear stress promotes mitochondrial homeostasis in endothelial cells. J Cell Physiol2018;
233: 5058–5069.
85.
BerteroTOldhamWMCottrillKA, et al.
Vascular stiffness mechanoactivates YAP/TAZ-dependent glutaminolysis to drive pulmonary hypertension. J Clin Invest2016;
126: 3313–3335.
Supplementary Material
Please find the following supplemental material available below.
For Open Access articles published under a Creative Commons License, all supplemental material carries the same license as the article it is associated with.
For non-Open Access articles published, all supplemental material carries a non-exclusive license, and permission requests for re-use of supplemental material or any part of supplemental material shall be sent directly to the copyright owner as specified in the copyright notice associated with the article.
