Role of calcium channels in glucose uptake regulation in the in vitro model of polarized intestinal epithelium

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Abstract

Glucose is the main energy substrate that ensures metabolic processes in the human and animal bodies. Impaired carbohydrate metabolism is often associated with obesity and concomitant diseases, such as cardiovascular diseases, arterial hypertension, insulin resistance, etc. Current data indicate that intestinal glucose absorption is coupled with Ca2+ influx, but additional research is needed to confirm this interaction. We used a cellular model of human intestinal epithelium to elucidate the role of Ca2+ channels in the regulation of glucose absorption. The results of immunofluorescence and immunoelectron microscopy showed that high cellular glucose loading (50 mM) leads to an increase in the density of TRPV6 calcium channels on the apical membrane of the intestinal epithelium. The level of the calcium sensor STIM1, responsible for store-dependent calcium entry (SOCE), on the contrary, showed a decrease when Caco-2 cells were overloaded with glucose, which was accompanied by a decrease in SOCE. Excessive saturation of Caco-2 cells with glucose also led to a decrease in the expression level of the NF-kB transcription factor p65 subunit responsible for the expression of STIM1. The results showed that Ca2+ channels are not only involved in the regulation of glucose uptake, but may themselves be under the control of glucose.

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About the authors

D. E. Bobkov

Institute of Cytology, Russian Academy of Sciences

Email: svsem@incras.ru
Russian Federation, St. Petersburg, 194064

A. V. Lukacheva

Institute of Cytology, Russian Academy of Sciences

Email: svsem@incras.ru
Russian Federation, St. Petersburg, 194064

L. V. Kever

Institute of Cytology, Russian Academy of Sciences

Email: svsem@incras.ru
Russian Federation, St. Petersburg, 194064

V. V. Furman

Institute of Cytology, Russian Academy of Sciences

Email: svsem@incras.ru
Russian Federation, St. Petersburg, 194064

S. B. Semenova

Institute of Cytology, Russian Academy of Sciences

Author for correspondence.
Email: svsem@incras.ru
Russian Federation, St. Petersburg, 194064

References

  1. Грефнер Н.М., Громова Л.В., Груздков А.А., Комиссарчик Я.Ю. 2010. Сравнительный анализ распределения переносчиков SGLT1 и GLUT2 в энтероцитах тонкой кишки крысы и клетках Сасо2 при всасывании гексоз. Цитология. Т. 52. C. 580. (Grefner N.M., Gromova L.V., Gruzdkov A.A., Komissarchik Ya.Yu. 2010. Comparative analysis of SGLT1 and GLUT2 transporter distribution in rat small intestine enterocytes and Caco2 cells during hexose absorption. Tsitologiya. V. 52. P. 580.)
  2. Грефнер Н.М., Громова Л.В., Груздков А.А., Комиссарчик Я.Ю. 2014. Взаимодействие транспортеров глюкозы SGLT1 и GLUT2 и цитоскелета в энтероцитах и клетках Сасо2 при транспорте сахаров. Цитология. Т. 56. C. 749–757. (Grefner L.V. Gromova A.A. Gruzdkov, Komissarchik Ya.Yu. 2014. The interaction between SGLT1 or GLUT2 glucose transporter and the cytoskeleton in the enterocyte as well as Caco2 cell during hexose absorbtion. Tsitologiya. V. 56. P. 749.)
  3. Affleck J.A., Helliwell P.A., Kellett G.L. 2003. Immunocytochemical detection of GLUT2 at the rat intestinal brush-border membrane. J. Histochem. Cytochem. V. 51. P. 1567. https://doi.org/10.1177/002215540305101116
  4. Alexander A.N., Carey H.V. 2001. Involvement of PI 3-kinase in IGF-I stimulation of jejunal Na+-K+-ATPase activity and nutrient absorption. Am. J. Physiol. Gastrointest. Liver Physiol. V. 280. P. G222. https://doi.org/10.1152/ajpgi.2001.280.2.G222.
  5. Blais A., Bissonnette P., Berteloot A. Common characteristics for Na+-dependent sugar transport in Caco-2 cells and human fetal colon. 1987. J. Membr. Biol. V. 99. P. 113.
  6. Bourzac J.F., L’eriger K., Larrivée J.F., Arguin G., Bilodeau M.S., Stankova J., Gendron F.P. 2013. Glucose transporter 2 expression is down regulated following P2X7 activation in enterocytes. J. Cell. Physiol. V. 228. P. 120. https://doi.org/10.1002/jcp.24111
  7. Brown E.M. 2013. Role of the calcium-sensing receptor in extracellular calcium homeostasis. Best Pract. Res. Clin. Endocrinol. Metab. V. 27. P. 333. https://doi.org/10.1016/j.beem.2013.02.006
  8. Chung H.K., Rathor N., Wang S.R., Wang J.Y., Rao J.N. 2015. RhoA enhances store-operated Ca2+ entry and intestinal epithelial restitution by interacting with TRPC1 after wounding. Am. J. Physiol. Gastrointest. Liver Physiol. V. 309. P. G759. https://doi.org/10.1152/ajpgi.00185.2015
  9. DebRoy A., Vogel S.M., Soni D., Sundivakkam P.C., Malik A.B., Tiruppathi C. 2014. Cooperative signaling via transcription factors NF-κB and AP1/c-Fos mediates endothelial cell STIM1 expression and hyperpermeability in response to endotoxin. J. Biol. Chem. V. 289. P. 24188. https://doi.org/10.1074/jbc.M114.570051
  10. Diaz R., Hurwitz S., Chattopadhyay N., Pines M., Yan, Y., Kifor O., Brown E.M. 1997. Cloning, expression, and tissue localization of the calcium-sensing receptor in chicken (Gallus domesticus). Am. J. Physiol. Regul. Integr. Comp. Physiol. V. 273. P. R1008. https://doi.org/10.1152/ajpregu.1997.273.3.R1008
  11. Eylenstein A., Schmidt S., Gu S., Yang W., Schmid E., Schmidt E.M., Alesutan I., Szteyn K., Regel I., Shumilina E., Lang F. 2012. Transcription factor NF-κB regulates expression of pore-forming Ca2+ channel unit, Orai1, and its activator, STIM1, to control Ca2+ entry and affect cellular functions. J. Biol. Chem. V. 287. P. 2719. https://doi.org/10.1074/jbc.M111.275925
  12. Gorboulev V., Schürmann A., Vallon V., Kipp H., Jaschke A., Klessen D., Friedrich A., Scherneck S., Rieg T., Cunard R., Veyhl-Wichmann M., Srinivasan A., Balen D., Breljak D., Rexhepaj R., et al. 2012. Na+-D-glucose cotransporter SGLT1 is pivotal for intestinal glucose absorption and glucose-dependent incretin secretion. Diabetes. V. 61. P. 187. https://doi.org/10.2337/db11-1029
  13. Hall E., Nitert M.D., Volkov P., Malmgren S., Mulder H., Bacos K., Ling C. 2018. The effects of high glucose exposure on global gene expression and DNA methylation in human pancreatic islets. Mol. Cell. Endocrinol. V. 472. P. 67. https://doi.org/10.1016/j.mce.2017.11.019
  14. Helliwell P.A., Rumsby M.G., Kellett G.L. 2003. Intestinal sugar absorption is regulated by phosphorylation and turnover of protein kinase C βII mediated by phosphatidylinositol 3-kinase-and mammalian target of rapamycin-dependent pathways. J. Biol. Chem. V. 278. P. 28644. https://doi.org/10.1074/jbc.M301479200
  15. Hoenderop J.G., Nilius B., Bindels R.J. 2002. ECaC: the gatekeeper of transepithelial Ca2+ transport. Biochim. Biophys. Acta–Proteins Proteom. V. 1600. P. 6. https://doi.org/10.1016/s1570-9639(02)00438-7
  16. Kellett G.L. 2001. The facilitated component of intestinal glucose absorption. Physiol. J. V. 531. P. 585. https://doi.org/10.1111/j.1469-7793.2001.0585h.x
  17. Kellett G.L., Helliwell P.A. 2000. The diffusive component of intestinal glucose absorption is mediated by the glucose-induced recruitment of GLUT2 to the brush-border membrane. Biochem. J. V. 350. P. 155.
  18. Koster H.P.G., Hartog A., Bindels R.J.M. 1995. Calbindin-D28K facilitates cytosolic calcium diffusion without interfering with calcium signaling. Cell Calcium. V. 18. P. 187. https://doi.org/10.1016/0143-4160(95)90063-2
  19. Kuhre R.E., Christiansen C.B., Saltiel M.Y., Wewer Albrechtsen N.J., Holst J.J. 2017. On the relationship between glucose absorption and glucose‐stimulated secretion of GLP‐1, neurotensin, and PYY from different intestinal segments in the rat. Physiol. Rep. V. 5. P. e13507. https://doi.org/10.14814/phy2.13507
  20. Mace O.J., Morgan E.L., Affleck J.A., Lister N., Kellett G.L. 2007. Calcium absorption by Cav1. 3 induces terminal web myosin II phosphorylation and apical GLUT2 insertion in rat intestine. Physiol. J. V. 580. P. 605. https://doi.org/10.1113/jphysiol.2006.124784
  21. Mace O.J., Morgan E.L., Affleck J.A., Lister N., Kellett G.L. 2007. Calcium absorption by Cav1. 3 induces terminal web myosin II phosphorylation and apical GLUT2 insertion in rat intestine. Physiol. J. V. 580. P. 605. https://doi.org/10.1113/jphysiol.2006.124784
  22. Morgan E.L., Mace O.J., Affleck J., Kellett G.L. 2007. Apical GLUT2 and Cav1. 3: regulation of rat intestinal glucose and calcium absorption. Physiol. J. V. 580. P. 593. https://doi.org/10.1113/jphysiol.2006.124768
  23. Nijenhuis T., Hoenderop J.G., Nilius B., Bindels R.J. 2003. (Patho) physiological implications of the novel epithelial Ca2+ channels TRPV5 and TRPV6. Pflügers Arch. V. 446. P. 401. https://doi.org/10.1007/s00424-003-1038-7
  24. Peng J.B., Chen X.Z., Berger U.V., Vassilev P.M., Tsukaguchi H., Brown E.M., Hediger M.A. 1999. Molecular cloning and characterization of a channel-like transporter mediating intestinal calcium absorption. J. Biol. Chem. V. 274. P. 22739. https://doi.org/10.1074/jbc.274.32.22739
  25. Röder P.V., Geillinger K.E., Zietek T.S., Thorens B., Koepsell H., Daniel H. 2014. The role of SGLT1 and GLUT2 in intestinal glucose transport and sensing. PloS one. V. 9. P. e89977. https://doi.org/10.1371/journal.pone.0089977
  26. Tharabenjasin P., Douard V., Patel C., Krishnamra N., Johnson R.J., Zuo J., Ferraris R.P. 2014. Acute interactions between intestinal sugar and calcium transport in vitro. Am. J. Physiol. Gastrointest. Liver Physiol. V. 306. P. G1–G12.
  27. Westerhout J., van de Steeg E., Grossouw D., Zeijdner E.E., Krul C.A.M., Verwei M., Wortelboer H.M. 2014. A new approach to predict human intestinal absorption using porcine intestinal tissue and biorelevant matrices. Eur. J. Pharm. Sci. V. 63. P. 167. https://doi.org/10.1016/j.ejps.2014.07.003
  28. Yee S. 1997. In vitro permeability across Caco-2 cells (colonic) can predict in vivo (small intestinal) absorption in man-fact or myth. Pharm. Res. V. 14. P. 763. https://doi.org/10.1023/a:1012102522787
  29. Zheng Y., Scow J.S., Duenes J.A., Sarr M.G. 2012. Mechanisms of glucose uptake in intestinal cell lines: role of GLUT2. Surgery. V. 151. P. 13. https://doi.org/10.1016/j.surg.2011.07.010

Supplementary files

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2. Fig. 1. Schematic showing a culture insert with cells on a semipermeable membrane. The insert is located in a well of a plastic cell culture plate.

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3. Fig. 2. Ultrathin section of two adjacent epithelial cells in the area of intercellular contacts. Root filaments of microvilli, intercellular contacts, mitochondria and membranes of the endoplasmic reticulum are visible. Control drug. Arrows indicate microvilli (m), desmosomes (d) at the sites of intercellular contacts and ribosomes (ri) on the surface of the endoplasmic recticulum. Scale bar: 400 nm.

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4. Fig. 3. Model of polarized intestinal epithelium formed by Caco-2 cells on semi-permeable membranes in a culture medium. Sections in the X–Y (a, b) and Y–Z (c) directions. Rhodamine-phalloidin staining (red) demonstrates actin-rich microvilli on the apical surface of cells, and DAPI nuclear staining (blue) indicates the division of cells into apical and basal regions. Control drug. Scale bar: 10 µm.

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5. Fig. 4. Polarized intestinal epithelium formed by Caco-2 cells under 3D cultivation conditions. Section at the level of the middle of the cell in the X–Y direction (a, b). Staining with antibodies against the glucose transporter GLUT2 (green) demonstrates its higher concentration when cells are loaded with 50 mM glucose; scale bar: 10 µm; c – fluorescence intensity (FI) of GLUT2 in cells incubated in the presence of 5 mM or 50 mM glucose. Vertical bars – errors of the mean; differences are significant (*) at P < 0.05.

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6. Fig. 5. Polarized intestinal epithelium formed by Caco-2 cells. Section at the level of the apical (a) and basal (b) membrane of cells in the X–Y direction. Anti-TRPV6 staining (red) demonstrates a higher concentration of TRPV6 at the apical surface when cells are loaded with 50 mM glucose; c, d – fluorescence intensity (FI) of TRPV6; scale bar: 10 µm. Vertical bars are standard errors of the mean; differences are significant (*) at P < 0.05.

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7. Fig. 6. Localization of TRPV6 channels in intestinal epithelial cells depending on glucose load. Immunoelectron microscopy. Micrographs of the distribution of colloidal gold particles (10 nm, arrows) corresponding to TRPV6 channels in cells loaded with 5 nM and 50 mM glucose; scale bar: 100 nm; The histogram shows the numerical distribution of gold particles in cells incubated in the presence of 5 and 50 mM glucose. Mean values and their errors are shown; differences are significant (*) at P < 0.05.

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8. Fig. 7. The effect of glucose on the store-dependent calcium entry into Caco-2 cells: a – Western blotting: the effect of various glucose concentrations on the content of proteins STIM1, ORAI1 and the p65 subunit of the NF-kB factor in Caco-2 cells; b – lower store-dependent calcium entry (in response to the action of thapsigargin Tg) into Caco-2 cells after loading them with 25 mM glucose (compared to that after loading the cells with 2.5 mM glucose). The dependence of fluorescence intensity (FI) of the calcium-sensitive probe Fluo-4 (OY axis) on time (OX axis) according to intravital confocal microscopy data is shown. The colored lines in the graphs show the signal from different cells located in the same field of view.

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