Molecular Neurobiology

, Volume 55, Issue 2, pp 1112–1122 | Cite as

Time-Dependent Lactate Production and Amino Acid Utilization in Cultured Astrocytes Under High Glucose Exposure

  • Dan Wang
  • Liangcai Zhao
  • Hong Zheng
  • Minjian Dong
  • Linlin Pan
  • Xi Zhang
  • Huajie ZhangEmail author
  • Hongchang GaoEmail author


Accumulating investigations have focused on the severity of central nervous system (CNS) complications in diabetic patients. The effects of the high glucose (HG) probably attribute to the metabolic disturbances in CNS. Astrocytes, with powerful ability of metabolic regulation, play crucial roles in physiological and pathological processes in CNS. Hence, an in-depth analysis as to metabolic alterations of astrocytes exposure to HG would facilitate to explore the underlying pathogenesis. In this study, the 1H NMR-based metabonomic approach was performed to characterize the metabolic variations of intracellular metabolites and corresponding culture media in a time-dependent manner. Our results revealed a significant elevation in lactate production and release. Four amino acids, leucine, isoleucine, methionine and tyrosine, were the most important metabolites for utilization. Also, profound disturbances of several metabolic pathways, including osmoregulation, energy metabolism, and cellular biosynthesis were observed. In that sense, the detailed information of astrocyte metabolism under HG exposure provides us a comprehensive understanding of the intrinsic metabolic disorders in CNS during hyperglycemia or diabetes.


Astrocytes Central neural system High glucose 1H NMR Metabonomics 



Astrocyte-neuron lactate shuttle


Adenosine triphosphate


Central nervous system


Dimethyl sulfoxide


Dulbecco’s modified Eagle’s medium


Deuterium oxide


Fetal bovine serum


Glial fibrillary acidic protein


Glucose transporter


High glucose


Hank’s balanced salt solution


Kyoto encyclopedia of genes and genomes


Nuclear magnetic resonance


Monocarboxylate transporters


Nicotinamide adenine dinucleotide (oxidation state)


Principal component


Partial least squares-discriminate analysis


Small molecule pathway database

TCA cycle

Tricarboxylic acid cycle




Variable importance in the projection



This work was supported by the National Natural Science Foundation of China (Nos.: 21575105, 21175099, 21605115), Research Fund for the Doctoral Program of Higher Education of China (No. 20133321120006), Zhejiang Provincial Natural Science Foundation (Nos.:LY14H090014, LY17H160049), and Opening Project of Zhejiang Provincial Top Key Discipline of Pharmaceutical Sciences (No. YKFJ2-003).

Compliance with Ethical Standards

All animals received care, and research procedures were in accordance with the ARRIVE Guidelines [20], which were also approved by the Institutional Animal Care and Use Committee of Wenzhou Medical University.ᅟ

Conflict of Interest

The authors declare that they have no conflict of interest.


  1. 1.
    Scholz J, Rathmell JP, David WS, Chad DA, Broderick AC, Perros SG, Shin NS, Wells JL et al (2016) A standardized clinical evaluation of phenotypic diversity in diabetic polyneuropathy. Pain. doi: 10.1097/j.pain.0000000000000648 Google Scholar
  2. 2.
    Reagan LP (2012) Diabetes as a chronic metabolic stressor: causes, consequences and clinical complications. Exp Neurol 233(1):68–78. doi: 10.1016/j.expneurol.2011.02.004 CrossRefPubMedGoogle Scholar
  3. 3.
    Hsieh HL, Chi PL, Lin CC, Yang CC, Yang CM (2014) Up-regulation of ROS-dependent matrix metalloproteinase-9 from high-glucose-challenged astrocytes contributes to the neuronal apoptosis. Mol Neurobiol 50(2):520–533. doi: 10.1007/s12035-013-8628-y CrossRefPubMedGoogle Scholar
  4. 4.
    Lyoo IK, Yoon SJ, Musen G, Simonson DC, Weinger K, Bolo N, Ryan CM, Kim JE et al (2009) Altered prefrontal glutamate-glutamine-gamma-aminobutyric acid levels and relation to low cognitive performance and depressive symptoms in type 1 diabetes mellitus. Arch Gen Psychiat 66(8):878–887. doi: 10.1001/archgenpsychiatry.2009.86 CrossRefPubMedGoogle Scholar
  5. 5.
    Sims-Robinson C, Zhao S, Hur J, Feldman EL (2012) Central nervous system endoplasmic reticulum stress in a murine model of type 2 diabetes. Diabetologia 55(8):2276–2284. doi: 10.1007/s00125-012-2573-6 CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Roberts RO, Knopman DS, Przybelski SA, Mielke MM, Kantarci K, Preboske GM, Senjem ML, Pankratz VS et al (2014) Association of type 2 diabetes with brain atrophy and cognitive impairment. Neurology 82(13):1132–1141. doi: 10.1212/WNL.0000000000000269 CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Umegaki H (2015) Dementia: type 2 diabetes has a slow and insidious effect on cognition. Nature Rev Neurol 11(3):127–128. doi: 10.1038/nrneurol.2015.17 CrossRefGoogle Scholar
  8. 8.
    Kodl CT, Seaquist ER (2008) Cognitive dysfunction and diabetes mellitus. Endocr Rev 29(4):494–511. doi: 10.1210/er.2007-0034 CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Dallerac G, Rouach N (2016) Astrocytes as new targets to improve cognitive functions. Prog Neurobiol. doi: 10.1016/j.pneurobio.2016.01.003 PubMedGoogle Scholar
  10. 10.
    Haskew-Layton RE, Payappilly JB, Smirnova NA, Ma TC, Chan KK, Murphy TH, Guo H, Langley B et al (2010) Controlled enzymatic production of astrocytic hydrogen peroxide protects neurons from oxidative stress via an Nrf2-independent pathway. Proc Natl Acad Sci U S A 107(40):17385–17390. doi: 10.1073/pnas.1003996107 CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Rodriguez JJ, Olabarria M, Chvatal A, Verkhratsky A (2009) Astroglia in dementia and Alzheimer’s disease. Cell Death Differ 16(3):378–385. doi: 10.1038/cdd.2008.172 CrossRefPubMedGoogle Scholar
  12. 12.
    Wang J, Li G, Wang Z, Zhang X, Yao L, Wang F, Liu S, Yin J et al (2012) High glucose-induced expression of inflammatory cytokines and reactive oxygen species in cultured astrocytes. Neuroscience 202:58–68. doi: 10.1016/j.neuroscience.2011.11.062 CrossRefPubMedGoogle Scholar
  13. 13.
    Bradley SA, Ouyang A, Purdie J, Smitka TA, Wang T, Kaerner A (2010) Fermentanomics: monitoring mammalian cell cultures with NMR spectroscopy. J Am Chem Soc 132(28):9531–9533. doi: 10.1021/ja101962c CrossRefPubMedGoogle Scholar
  14. 14.
    Gao H, Dong B, Jia J, Zhu H, Diao C, Yan Z, Huang Y, Li X (2012) Application of ex vivo 1H NMR metabonomics to the characterization and possible detection of renal cell carcinoma metastases. J Cancer Res Clini 138(5):753–761. doi: 10.1007/s00432-011-1134-6 CrossRefGoogle Scholar
  15. 15.
    Wang N, Zhao LC, Zheng YQ, Dong MJ, Su Y, Chen WJ, Hu ZL, Yang YJ et al (2015) Alteration of interaction between astrocytes and neurons in different stages of diabetes: a nuclear magnetic resonance study using [1-13C]glucose and [2-13C]acetate. Mol Neurobiol 51(3):843–852. doi: 10.1007/s12035-014-8808-4 CrossRefPubMedGoogle Scholar
  16. 16.
    Zheng H, Zheng Y, Wang D, Cai A, Lin Q, Zhao L, Chen M, Deng M et al (2016) Analysis of neuron-astrocyte metabolic cooperation in the brain of db/db mice with cognitive decline using 13C NMR spectroscopy. J Cerebr Blood F Met. doi: 10.1177/0271678X15626154 Google Scholar
  17. 17.
    Zheng Y, Yang Y, Dong B, Zheng H, Lin X, Du Y, Li X, Zhao L et al (2016) Metabonomic profiles delineate potential role of glutamate-glutamine cycle in db/db mice with diabetes-associated cognitive decline. Mol Brain 9:40. doi: 10.1186/s13041-016-0223-5 CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Du F, Qian ZM, Zhu L, Wu XM, Qian C, Chan R, Ke Y (2010) Purity, cell viability, expression of GFAP and bystin in astrocytes cultured by different procedures. J Cell Biochem 109(1):30–37. doi: 10.1002/jcb.22375 PubMedGoogle Scholar
  19. 19.
    Petters C, Dringen R (2014) Comparison of primary and secondary rat astrocyte cultures regarding glucose and glutathione metabolism and the accumulation of iron oxide nanoparticles. Neurochem Res 39(1):46–58. doi: 10.1007/s11064-013-1189-7 CrossRefPubMedGoogle Scholar
  20. 20.
    Kilkenny C, Browne WJ, Cuthill IC, Emerson M, Altman DG (2010) Improving bioscience research reporting: the ARRIVE guidelines for reporting animal research. PLoS Biol 8(6):e1000412. doi: 10.1371/journal.pbio.1000412 CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    McCarthy KD, de Vellis J (1980) Preparation of separate astroglial and oligodendroglial cell cultures from rat cerebral tissue. J Cell Biol 85(3):890–902CrossRefPubMedGoogle Scholar
  22. 22.
    Rivera-Aponte DE, Mendez-Gonzalez MP, Rivera-Pagan AF, Kucheryavykh YV, Kucheryavykh LY, Skatchkov SN, Eaton MJ (2015) Hyperglycemia reduces functional expression of astrocytic Kir4.1 channels and glial glutamate uptake. Neuroscience 310:216–223. doi: 10.1016/j.neuroscience.2015.09.044 CrossRefPubMedPubMedCentralGoogle Scholar
  23. 23.
    Liu Y, Biarnes Costa M, Gerhardinger C (2012) IL-1β is upregulated in the diabetic retina and retinal vessels: cell-specific effect of high glucose and IL-1β autostimulation. PLoS One 7(5):e36949. doi: 10.1371/journal.pone.0036949 CrossRefPubMedPubMedCentralGoogle Scholar
  24. 24.
    Orellana JA, Hernandez DE, Ezan P, Velarde V, Bennett MV, Giaume C, Saez JC (2010) Hypoxia in high glucose followed by reoxygenation in normal glucose reduces the viability of cortical astrocytes through increased permeability of connexin 43 hemichannels. Glia 58(3):329–343. doi: 10.1002/glia.20926 PubMedPubMedCentralGoogle Scholar
  25. 25.
    Pang A, Hu Y, Zhou P, Long G, Tian X, Men L, Shen Y, Liu Y et al (2015) Corin is down-regulated and exerts cardioprotective action via activating pro-atrial natriuretic peptide pathway in diabetic cardiomyopathy. Cardiovasc Diabetol 14:134. doi: 10.1186/s12933-015-0298-9 CrossRefPubMedPubMedCentralGoogle Scholar
  26. 26.
    Teng Q, Huang WL, Collette TW, Ekman DR, Tan C (2009) A direct cell quenching method for cell-culture based metabolomics. Metabolomics 5(2):199–208. doi: 10.1007/s11306-008-0137-z CrossRefGoogle Scholar
  27. 27.
    Dromms RA, Styczynski MP (2012) Systematic applications of metabolomics in metabolic engineering. Metabolites 2(4):1090–1122. doi: 10.3390/metabo2041090 CrossRefPubMedPubMedCentralGoogle Scholar
  28. 28.
    Shao W, Gu J, Huang C, Liu D, Huang H, Huang Z, Lin Z, Yang W et al (2014) Malignancy-associated metabolic profiling of human glioma cell lines using 1H NMR spectroscopy. Mol Cancer 13:197. doi: 10.1186/1476-4598-13-197 CrossRefPubMedPubMedCentralGoogle Scholar
  29. 29.
    Northam EA, Rankins D, Cameron FJ (2006) Therapy insight: the impact of type 1 diabetes on brain development and function. Nat Clini Pract Neuro 2(2):78–86. doi: 10.1038/ncpneuro0097 CrossRefGoogle Scholar
  30. 30.
    Baydas G, Nedzvetskii VS, Tuzcu M, Yasar A, Kirichenko SV (2003) Increase of glial fibrillary acidic protein and S-100B in hippocampus and cortex of diabetic rats: effects of vitamin E. Eur J Pharmacol 462(1–3):67–71. doi: 10.1016/S0014-2999(03)01294-9 CrossRefPubMedGoogle Scholar
  31. 31.
    Duarte JM, Agostinho PM, Carvalho RA, Cunha RA (2012) Caffeine consumption prevents diabetes-induced memory impairment and synaptotoxicity in the hippocampus of NONcZNO10/LTJ mice. PLoS One 7(4):e21899. doi: 10.1371/journal.pone.0021899 CrossRefPubMedPubMedCentralGoogle Scholar
  32. 32.
    An J, Haile WB, Wu F, Torre E, Yepes M (2014) Tissue-type plasminogen activator mediates neuroglial coupling in the central nervous system. Neuroscience 257:41–48. doi: 10.1016/j.neuroscience.2013.10.060 CrossRefPubMedGoogle Scholar
  33. 33.
    Yu T, Robotham JL, Yoon Y (2006) Increased production of reactive oxygen species in hyperglycemic conditions requires dynamic change of mitochondrial morphology. Proc Natl Acad Sci U S A 103(8):2653–2658. doi: 10.1073/pnas.0511154103 CrossRefPubMedPubMedCentralGoogle Scholar
  34. 34.
    Pascente R, Frigerio F, Rizzi M, Porcu L, Boido M, Davids J, Zaben M, Tolomeo D et al (2016) Cognitive deficits and brain myo-Inositol are early biomarkers of epileptogenesis in a rat model of epilepsy. Neurobiol Dis 93:146–155. doi: 10.1016/j.nbd.2016.05.001 CrossRefPubMedGoogle Scholar
  35. 35.
    Carroll B, Maetzel D, Maddocks OD, Otten G, Ratcliff M, Smith GR, Dunlop EA, Passos JF, Davies OR, Jaenisch R, Tee AR, Sarkar S, Korolchuk VI (2016) Control of TSC2-Rheb signaling axis by arginine regulates mTORC1 activity. eLife 5. doi: 10.7554/eLife.11058
  36. 36.
    Pellerin L, Magistretti PJ (1994) Glutamate uptake into astrocytes stimulates aerobic glycolysis: a mechanism coupling neuronal activity to glucose utilization. Proc Natl Acad Sci U S A 91(22):10625–10629CrossRefPubMedPubMedCentralGoogle Scholar
  37. 37.
    Magistretti PJ, Allaman I (2015) A cellular perspective on brain energy metabolism and functional imaging. Neuron 86(4):883–901. doi: 10.1016/j.neuron.2015.03.035 CrossRefPubMedGoogle Scholar
  38. 38.
    Kasischke KA, Vishwasrao HD, Fisher PJ, Zipfel WR, Webb WW (2004) Neural activity triggers neuronal oxidative metabolism followed by astrocytic glycolysis. Science 305(5680):99–103. doi: 10.1126/science.1096485 CrossRefPubMedGoogle Scholar
  39. 39.
    Bitoun M, Tappaz M (2000) Gene expression of the transporters and biosynthetic enzymes of the osmolytes in astrocyte primary cultures exposed to hyperosmotic conditions. Glia 32(2):165–176CrossRefPubMedGoogle Scholar
  40. 40.
    Brand A, Richter-Landsberg C, Leibfritz D (1993) Multinuclear NMR studies on the energy metabolism of glial and neuronal cells. Dev Neurosci-Basel 15(3–5):289–298CrossRefGoogle Scholar
  41. 41.
    Teahan O, Bevan CL, Waxman J, Keun HC (2011) Metabolic signatures of malignant progression in prostate epithelial cells. Int J Biochem Cell Biol 43(7):1002–1009. doi: 10.1016/j.biocel.2010.07.003 CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2017

Authors and Affiliations

  • Dan Wang
    • 1
  • Liangcai Zhao
    • 1
  • Hong Zheng
    • 1
  • Minjian Dong
    • 1
  • Linlin Pan
    • 1
  • Xi Zhang
    • 1
  • Huajie Zhang
    • 1
    Email author
  • Hongchang Gao
    • 1
    Email author
  1. 1.School of Pharmaceutical SciencesWenzhou Medical UniversityWenzhouChina

Personalised recommendations