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Neurochemical Research

, Volume 44, Issue 1, pp 188–199 | Cite as

Metformin Accelerates Glycolytic Lactate Production in Cultured Primary Cerebellar Granule Neurons

  • Eva-Maria Blumrich
  • Ralf DringenEmail author
Original Paper

Abstract

Metformin is the most frequently used drug for the treatment of type-II diabetes. As metformin has been reported to cross the blood–brain barrier, brain cells will encounter this drug. To test whether metformin may affect the metabolism of neurons, we exposed cultured rat cerebellar granule neurons to metformin. Treatment with metformin caused a time- and concentration-dependent increase in glycolytic lactate release from viable neurons as demonstrated by the three-to fivefold increase in extracellular lactate concentration determined after exposure to metformin. Half-maximal stimulation of lactate production was found after incubation of neurons for 4 h with around 2 mM or for 24 h with around 0.5 mM metformin. Neuronal cell viability was not affected by millimolar concentrations of metformin during acute incubations in the hour range nor during prolonged incubations, although alterations in cell morphology were observed during treatment with 10 mM metformin for days. The acute stimulation of neuronal lactate release by metformin was persistent upon removal of metformin from the medium and was not affected by the presence of modulators of adenosine monophosphate activated kinase activity. In contrast, rabeprazole, an inhibitor of the organic cation transporter 3, completely prevented metformin-mediated stimulation of neuronal lactate production. In summary, the data presented identify metformin as a potent stimulator of glycolytic lactate production in viable cultured neurons and suggest that organic cation transporter 3 mediates the uptake of metformin into neurons.

Keywords

Glycolysis Lactate Metformin Neurons 

Notes

Compliance with Ethical Standards

Conflict of interest

The authors have no conflict of interest to declare.

References

  1. 1.
    Bailey CJ, Day C (2004) Metformin: its botanical background. Pract Diab Int 21:115–117Google Scholar
  2. 2.
    Pryor R, Cabreiro F (2015) Repurposing metformin: an old drug with new tricks in its binding pockets. Biochem J 471:307–322Google Scholar
  3. 3.
    Bao B, Azmi AS, Ali S, Zaiem F, Sarkar FH (2014) Metformin may function as anti-cancer agent via targeting cancer stem cells: the potential biological significance of tumor-associated miRNAs in breast and pancreatic cancers. Ann Transl Med 2:59Google Scholar
  4. 4.
    Lv WS, Wen JP, Li L, Sun RX, Wang J, Xian YX, Cao CX, Wang YL, Gao YY (2012) The effect of metformin on food intake and its potential role in hypothalamic regulation in obese diabetic rats. Brain Res 1444:11–19Google Scholar
  5. 5.
    Mitchell PL, Nachbar R, Lachance D, St-Pierre P, Trottier J, Barbier O, Marette A (2017) Treatment with a novel agent combining docosahexaenoate and metformin increases protectin DX and IL-6 production in skeletal muscle and reduces insulin resistance in obese diabetic db/db mice. Diabetes Obes Metab 3:313–319Google Scholar
  6. 6.
    Nagi DK, Yudkin JS (1993) Effects of metformin on insulin resistance, risk factors for cardiovascular disease, and plasminogen activator inhibitor in NIDDM subjects: a study of two ethnic groups. Diabetes Care 16:621–629Google Scholar
  7. 7.
    Rojas LB, Gomes MB (2013) Metformin: an old but still the best treatment for type 2 diabetes. Diabetol Metab Syndr 5:6Google Scholar
  8. 8.
    Marin-Penalver JJ, Martin-Timon I, Sevillano-Collantes C, Del Canizo-Gomez FJ (2016) Update on the treatment of type 2 diabetes mellitus. World J Diabetes 7:354–395Google Scholar
  9. 9.
    DeFronzo R, Fleming GA, Chen K, Bicsak TA (2016) Metformin-associated lactic acidosis: current perspectives on causes and risk. Metabolism 65:20–29Google Scholar
  10. 10.
    Kajbaf F, Lalau J-D (2013) The prognostic value of blood pH and lactate and metformin concentrations in severe metformin-associated lactic acidosis. BMC Pharmacol Toxicol 14:22–22Google Scholar
  11. 11.
    Gong L, Goswami S, Giacomini KM, Altman RB, Klein TE (2012) Metformin pathways: pharmacokinetics and pharmacodynamics. Pharmacogenet Genom 22:820–827Google Scholar
  12. 12.
    Kajbaf F, Bennis Y, Hurtel-Lemaire AS, Andrejak M, Lalau JD (2015) Unexpectedly long half-life of metformin elimination in cases of metformin accumulation. Diabet Med 33:105–110Google Scholar
  13. 13.
    Wilcock C, Bailey CJ (1994) Accumulation of metformin by tissues of the normal and diabetic mouse. Xenobiotica 24:49–57Google Scholar
  14. 14.
    Labuzek K, Suchy D, Gabryel B, Bielecka A, Liber S, Okopien B (2010) Quantification of metformin by the HPLC method in brain regions, cerebrospinal fluid and plasma of rats treated with lipopolysaccharide. Pharmacol Rep 62:956–965Google Scholar
  15. 15.
    Oshima R, Yamada M, Kurogi E, Ogino Y, Serizawa Y, Tsuda S, Ma X, Egawa T, Hayashi T (2015) Evidence for organic cation transporter-mediated metformin transport and 5′-adenosine monophosphate-activated protein kinase activation in rat skeletal muscles. Metabolism 64:296–304Google Scholar
  16. 16.
    Chen EC, Liang X, Yee SW, Geier EG, Stocker SL, Chen L, Giacomini KM (2015) Targeted disruption of organic cation transporter 3 attenuates the pharmacologic response to metformin. Mol Pharmacol 88:75–83Google Scholar
  17. 17.
    Segal ED, Yasmeen A, Beauchamp MC, Rosenblatt J, Pollak M, Gotlieb WH (2011) Relevance of the OCT1 transporter to the antineoplastic effect of biguanides. Biochem Biophys Res Commun 414:694–699Google Scholar
  18. 18.
    Nies AT, Koepsell H, Damme K, Schwab M (2011) Organic cation transporters (OCTs, MATEs), in vitro and in vivo evidence for the importance in drug therapy. In: Fromm MF, Kim RB (eds) Drug transporters. Springer, Heidelberg, pp 105–167Google Scholar
  19. 19.
    Perdan-Pirkmajer K, Pirkmajer S, Cerne K, Krzan M (2012) Molecular and kinetic characterization of histamine transport into adult rat cultured astrocytes. Neurochem Int 61:415–422Google Scholar
  20. 20.
    Shang T, Uihlein AV, Van Asten J, Kalyanaraman B, Hillard CJ (2003) 1-Methyl-4-phenylpyridinium accumulates in cerebellar granule neurons via organic cation transporter 3. J Neurochem 85:358–367Google Scholar
  21. 21.
    Xie Z, Dong Y, Scholz R, Neumann D, Zou MH (2008) Phosphorylation of LKB1 at serine 428 by protein kinase C-zeta is required for metformin-enhanced activation of the AMP-activated protein kinase in endothelial cells. Circulation 117:952–962Google Scholar
  22. 22.
    Zhou G, Myers R, Li Y, Chen Y, Shen X, Fenyk-Melody J, Wu M, Ventre J, Doebber T, Fujii N, Musi N, Hirshman MF, Goodyear LJ, Moller DE (2001) Role of AMP-activated protein kinase in mechanism of metformin action. J Clin Invest 108:1167–1174Google Scholar
  23. 23.
    Wheaton WW, Weinberg SE, Hamanaka RB, Soberanes S, Sullivan LB, Anso E, Glasauer A, Dufour E, Mutlu GM, Budigner GS, Chandel NS (2014) Metformin inhibits mitochondrial complex I of cancer cells to reduce tumorigenesis. eLife 3:e02242Google Scholar
  24. 24.
    El-Mir MY, Detaille D, G RV, Delgado-Esteban M, Guigas B, Attia S, Fontaine E, Almeida A, Leverve X (2008) Neuroprotective role of antidiabetic drug metformin against apoptotic cell death in primary cortical neurons. J Mol Neurosci 34:77–87Google Scholar
  25. 25.
    Owen MR, Doran E, Halestrap AP (2000) Evidence that metformin exerts its anti-diabetic effects through inhibition of complex 1 of the mitochondrial respiratory chain. Biochem J 348:607–614Google Scholar
  26. 26.
    Madiraju AK, Erion DM, Rahimi Y, Zhang XM, Braddock DT, Albright RA, Prigaro BJ, Wood JL, Bhanot S, MacDonald MJ, Jurczak MJ, Camporez JP, Lee HY, Cline GW, Samuel VT, Kibbey RG, Shulman GI (2014) Metformin suppresses gluconeogenesis by inhibiting mitochondrial glycerophosphate dehydrogenase. Nature 510:542–546Google Scholar
  27. 27.
    Jin Q, Cheng J, Liu Y, Wu J, Wang X, Wei S, Zhou X, Qin Z, Jia J, Zhen X (2014) Improvement of functional recovery by chronic metformin treatment is associated with enhanced alternative activation of microglia/macrophages and increased angiogenesis and neurogenesis following experimental stroke. Brain Behav Immun 40:131–142Google Scholar
  28. 28.
    Pintana H, Apaijai N, Pratchayasakul W, Chattipakorn N, Chattipakorn SC (2012) Effects of metformin on learning and memory behaviors and brain mitochondrial functions in high fat diet induced insulin resistant rats. Life Sci 91:409–414Google Scholar
  29. 29.
    Zhao RR, Xu XC, Xu F, Zhang WL, Zhang WL, Liu LM, Wang WP (2014) Metformin protects against seizures, learning and memory impairments and oxidative damage induced by pentylenetetrazole-induced kindling in mice. Biochem Biophys Res Commun 448:414–417Google Scholar
  30. 30.
    Chung M-M, Chen Y-L, Pei D, Cheng Y-C, Sun B, Nicol CJ, Yen C-H, Chen H-M, Liang Y-J, Chiang M-C (2015) The neuroprotective role of metformin in advanced glycation end product treated human neural stem cells is AMPK-dependent. Biochem Biophys Acta-Mol Basis Dis 5:720–731Google Scholar
  31. 31.
    Zhou C, Sun R, Zhuang S, Sun C, Jiang Y, Cui Y, Li S, Xiao Y, Du Y, Gu H, Liu Q (2016) Metformin prevents cerebellar granule neurons against glutamate-induced neurotoxicity. Brain Res Bull 121:241–245Google Scholar
  32. 32.
    Chen B, Teng Y, Zhang X, Lv X, Yin Y (2016) Metformin alleviated Aβ-Induced apoptosis via the suppression of JNK MAPK signaling pathway in cultured hippocampal neurons. Biomed Res Int 2016:1421430Google Scholar
  33. 33.
    Takata F, Dohgu S, Matsumoto J, Machida T, Kaneshima S, Matsuo M, Sakaguchi S, Takeshige Y, Yamauchi A, Kataoka Y (2013) Metformin induces up-regulation of blood–brain barrier functions by activating AMP-activated protein kinase in rat brain microvascular endothelial cells. Biochem Biophys Res Commun 433:586–590Google Scholar
  34. 34.
    Westhaus A, Blumrich EM, Dringen R (2017) The antidiabetic drug metformin stimulates glycolytic lactate production in cultured primary rat astrocytes. Neurochem Res 42:294–305Google Scholar
  35. 35.
    Hohnholt M, Blumrich E, Waagepetersen H, Dringen R (2017) The anti-diabetic drug metformin decreases mitochondrial respiration and tricarboxylic acid cycle activity in cultured primary rat astrocytes. J Neurosci Res. doi: 10.1002/jnr.24050 Google Scholar
  36. 36.
    Tulpule K, Hohnholt MC, Hirrlinger J, Dringen R (2014) Primary cultures of rat astrocytes and neurons as model systems to study metabolism and metabolite export from brain cells. In: Hirrlinger J, Waagepetersen H (eds) Neuromethods 90: brain energy metabolism. Springer, New York, pp 45–72Google Scholar
  37. 37.
    Dringen R, Kussmaul L, Hamprecht B (1998) Detoxification of exogenous hydrogen peroxide and organic hydroperoxides by cultured astroglial cells assessed by microtiter plate assay. Brain Res Brain Res Protoc 2:223–228Google Scholar
  38. 38.
    Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193:265–275Google Scholar
  39. 39.
    Courousse T, Gautron S (2015) Role of organic cation transporters (OCTs) in the brain. Pharmacol Ther 146:94–103Google Scholar
  40. 40.
    Nies AT, Hofmann U, Resch C, Schaeffeler E, Rius M, Schwab M (2011) Proton pump inhibitors inhibit metformin uptake by organic cation transporters (OCTs). PLoS ONE 6:e22163Google Scholar
  41. 41.
    Russell RR, Bergeron R, Shulman GI, Young LH (1999) Translocation of myocardial GLUT-4 and increased glucose uptake through activation of AMPK by AICAR. Am J Physiol 277:H643–H649Google Scholar
  42. 42.
    Hardie DG (2008) AMPK: a key regulator of energy balance in the single cell and the whole organism. Int J Obes 32 Suppl 4:S7–S12Google Scholar
  43. 43.
    Liu X, Chhipa RR, Nakano I, Dasgupta B (2014) The AMPK inhibitor Compound C is a potent AMPK-independent anti-glioma agent. Mol Cancer Ther 13:596–605Google Scholar
  44. 44.
    Sun Y, Tian T, Gao J, Liu X, Hou H, Cao R, Li B, Quan M, Guo L (2016) Metformin ameliorates the development of experimental autoimmune encephalomyelitis by regulating T helper 17 and regulatory T cells in mice. J Neuroimmunol 292:58–67Google Scholar
  45. 45.
    Gupta A, Bisht B, Dey CS (2011) Peripheral insulin-sensitizer drug metformin ameliorates neuronal insulin resistance and Alzheimer’s-like changes. Neuropharmacology 60:910–920Google Scholar
  46. 46.
    Potter WB, O’Riordan KJ, Barnett D, Osting SM, Wagoner M, Burger C, Roopra A (2010) Metabolic regulation of neuronal plasticity by the energy sensor AMPK. PLoS ONE 5:e8996Google Scholar
  47. 47.
    Bikas A, Jensen K, Patel A, Costello J Jr, McDaniel D, Klubo-Gwiezdzinska J, Larin O, Hoperia V, Burman KD, Boyle L, Wartofsky L, Vasko V (2015) Glucose-deprivation increases thyroid cancer cells sensitivity to metformin. Endocr Relat Cancer 22:919–932Google Scholar
  48. 48.
    Orecchioni S, Reggiani F, Talarico G, Mancuso P, Calleri A, Gregato G, Labanca V, Noonan DM, Dallaglio K, Albini A, Bertolini F (2015) The biguanides metformin and phenformin inhibit angiogenesis, local and metastatic growth of breast cancer by targeting both neoplastic and microenvironment cells. Int J Cancer 136:E534–E544Google Scholar
  49. 49.
    Ming M, Sinnett-Smith J, Wang J, Soares HP, Young SH, Eibl G, Rozengurt E (2014) Dose-dependent AMPK-dependent and independent mechanisms of berberine and metformin inhibition of mTORC1, ERK, DNA synthesis and proliferation in pancreatic cancer cells. PLoS ONE 9:e114573Google Scholar
  50. 50.
    Allaman I, Grenningloh G, Magistretti P (2015) Modulation of astrocytic glucose metabolism by the antidiabetic drug metformin. J Neurochem 134(Suppl 1):260Google Scholar
  51. 51.
    Hohnholt MC, Blumrich EM, Dringen R (2015) Multiassay analysis of the toxic potential of hydrogen peroxide on cultured neurons. J Neurosci Res 93:1127–1137Google Scholar
  52. 52.
    Tulpule K, Hohnholt MC, Dringen R (2013) Formaldehyde metabolism and formaldehyde-induced stimulation of lactate production and glutathione export in cultured neurons. J Neurochem 125:260–272Google Scholar
  53. 53.
    Itoh Y, Esaki T, Shimoji K, Cook M, Law MJ, Kaufman E, Sokoloff L (2003) Dichloroacetate effects on glucose and lactate oxidation by neurons and astroglia in vitro and on glucose utilization by brain in vivo. Proc Natl Acad Sci USA 100:4879–4884Google Scholar
  54. 54.
    Walz W, Mukerji S (1988) Lactate release from cultured astrocytes and neurons: a comparison. Glia 1:366–370Google Scholar
  55. 55.
    Biffi E, Regalia G, Menegon A, Ferrigno G, Pedrocchi A (2013) The influence of neuronal density and maturation on network activity of hippocampal cell cultures: a methodological study. PLoS ONE 8:e83899Google Scholar
  56. 56.
    Smieszek A, Czyrek A, Basinska K, Trynda J, Skaradzinska A, Siudzinska A, Maredziak M, Marycz K (2015) Effect of metformin on viability, morphology, and ultrastructure of mouse bone marrow-derived multipotent mesenchymal stromal cells and Balb/3T3 embryonic fibroblast cell line. BioMed Res Int 2015:769402Google Scholar
  57. 57.
    Brunmair B, Staniek K, Gras F, Scharf N, Althaym A, Clara R, Roden M, Gnaiger E, Nohl H, Waldhausl W, Furnsinn C (2004) Thiazolidinediones, like metformin, inhibit respiratory complex I: a common mechanism contributing to their antidiabetic actions? Diabetes 53:1052–1059Google Scholar
  58. 58.
    Kinaan M, Ding H, Triggle CR (2015) Metformin: an old drug for the treatment of diabetes but a new drug for the protection of the endothelium. Med Princ Pract 24:401–415Google Scholar
  59. 59.
    Lee JY, Lee SH, Chang JW, Song JJ, Jung HH, Im GJ (2014) Protective effect of metformin on gentamicin-induced vestibulotoxicity in rat primary cell culture. Clin Exp Otorhinolaryngol 7:286–294Google Scholar
  60. 60.
    Yonezawa A, Masuda S, Nishihara K, Yano I, Katsura T, Inui K-i (2005) Association between tubular toxicity of cisplatin and expression of organic cation transporter rOCT2 (Slc22a2) in the rat. Biochem Pharmacol 70:1823–1831Google Scholar
  61. 61.
    Chen L, Pawlikowski B, Schlessinger A, More SS, Stryke D, Johns SJ, Portman MA, Chen E, Ferrin TE, Sali A, Giacomini KM (2010) Role of organic cation transporter 3 (SLC22A3) and its missense variants in the pharmacologic action of metformin. Pharmacogenet Genom 20:687–699Google Scholar
  62. 62.
    Lee N, Duan H, Hebert MF, Liang CJ, Rice KM, Wang J (2014) Taste of a pill: organic cation transporter-3 (OCT3) mediates metformin accumulation and secretion in salivary glands. J Biol Chem 289:27055–27064Google Scholar
  63. 63.
    Ouyang J, Parakhia RA, Ochs RS (2011) Metformin activates AMP kinase through inhibition of AMP deaminase. J Biol Chem 286:1–11Google Scholar
  64. 64.
    Miller RA, Chu Q, Xie J, Foretz M, Viollet B, Birnbaum MJ (2013) Biguanides suppress hepatic glucagon signalling by decreasing production of cyclic AMP. Nature 494:256–260Google Scholar
  65. 65.
    Labuzek K, Liber S, Gabryel B, Okopien B (2010) Metformin has adenosine-monophosphate activated protein kinase (AMPK)-independent effects on LPS-stimulated rat primary microglial cultures. Pharmacol Rep 62:827–848Google Scholar
  66. 66.
    Zhang Y, Ye J (2012) Mitochondrial inhibitor as a new class of insulin sensitizer. Acta Pharm Sin B 2:341–349Google Scholar
  67. 67.
    Liemburg-Apers DC, Schirris TJ, Russel FG, Willems PH, Koopman WJ (2015) Mitoenergetic dysfunction triggers a rapid compensatory increase in steady-state glucose flux. Biophys J 109:1372–1386Google Scholar
  68. 68.
    Sokolov SS, Balakireva AV, Markova OV, Severin FF (2015) Negative feedback of glycolysis and oxidative phosphorylation: mechanisms of and reasons for it. BioChemistry 80:559–564Google Scholar
  69. 69.
    Warburg O (1956) On the origin of cancer cells. Science 123:309–314Google Scholar
  70. 70.
    Jia Y, Ma Z, Liu X, Zhou W, He S, Xu X, Ren G, Xu G, Tian K (2015) Metformin prevents DMH-induced colorectal cancer in diabetic rats by reversing the Warburg effect. Cancer Med 4:1730–1741Google Scholar
  71. 71.
    Guimaraes TA, Farias LC, Santos ES, de Carvalho Fraga CA, Orsini LA, de Freitas Teles L, Feltenberger JD, de Jesus SF, de Souza MG, Santos SH, de Paula AM, Gomez RS, Guimaraes AL (2016) Metformin increases PDH and suppresses HIF-1α under hypoxic conditions and induces cell death in oral squamous cell carcinoma. Oncotarget 7:55057–55068Google Scholar
  72. 72.
    Crabtree HG (1928) The carbohydrate metabolism of certain pathological overgrowths. Biochem J 22:1289–1298Google Scholar
  73. 73.
    Diaz-Ruiz R, Rigoulet M, Devin A (2011) The Warburg and Crabtree effects: on the origin of cancer cell energy metabolism and of yeast glucose repression. Biochem Biophys Acta 1807:568–576Google Scholar
  74. 74.
    Chen M, Zhang J, Hu F, Liu S, Zhou Z (2015) Metformin affects the features of a human hepatocellular cell line (HepG2) by regulating macrophage polarization in a co-culture microenviroment. Diabetes Metab Res Rev 31:781–789Google Scholar
  75. 75.
    Kajbaf F, De Broe ME, Lalau JD (2016) Therapeutic concentrations of metformin: a systematic review. Clin Pharmacokinet 55:439–459Google Scholar
  76. 76.
    He L, Wondisford FE (2015) Metformin action: concentrations matter. Cell Metab 21:159–162Google Scholar
  77. 77.
    Song JZ, Chen HF, Tian SJ, Sun ZP (1998) Determination of metformin in plasma by capillary electrophoresis using field-amplified sample stacking technique. J Chromatogr B Biomed Sci Appl 708:277–283Google Scholar
  78. 78.
    Tucker GT, Casey C, Phillips PJ, Connor H, Ward JD, Woods HF (1981) Metformin kinetics in healthy subjects and in patients with diabetes mellitus. Br J Clin Pharmacol 12:235–246Google Scholar

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© Springer Science+Business Media, LLC 2017

Authors and Affiliations

  1. 1.Centre for Biomolecular Interactions Bremen, Faculty 2 (Biology/Chemistry)University of BremenBremenGermany
  2. 2.Centre for Environmental Research and Sustainable TechnologyUniversity of BremenBremenGermany

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