Skip to main content
SpringerLink
Log in

Insulin Exhibits an Antiproliferative and Hypertrophic Effect in First Trimester Human Extravillous Trophoblasts

  • Original Article
  • Published:
Reproductive Sciences Aims and scope Submit manuscript

Abstract

Our aim was to investigate the effect of high levels of glucose, insulin, leptin, and tumor necrosis factor alpha, biomarkers of diabetes in pregnancy, in the process of placentation, using as a cell model a first trimester extravillous human trophoblast cell line (HTR8/SVneo cells). Exposure of HTR8/SVneo cells for 24 hours to either glucose (20 mmol/L) or leptin (25–100 ng/mL) did not cause significant changes in cell proliferation and viability. Tumor necrosis factor alpha (24 hours; 10–100 ng/L) caused a small decrease (10%) in cell proliferation and an increase (9%) in cell viability; however, both effects disappeared when exposure time was increased. Insulin (24 hours; 1–10 nmol/L) caused a concentration- and time-dependent decrease (10%–20%) in cell proliferation; the effect of insulin (10 nmol/L) was more pronounced after a 48 hours exposure (35%). In contrast, exposure to insulin (10 nmol/L; 48 hours) showed no significant effect on cell viability, apoptosis, and migration capacity. Insulin appears to cause hypertrophy of HTR8/SVneo cells as it reduces the cell mitotic index while increasing the culture protein content. The anti-proliferative effect of insulin seems to involve activation of mammalian target of rapamycin, phosphoinositide 3-kinase, and p38 mitogen-activated protein kinase. Finally, simvastatin and the polyphenol quercetin potentiated the antiproliferative effect of insulin; on the contrary, the polyphenol resveratrol, the polyunsaturated fatty acids eicosapentaenoic and docosahexaenoic acids, and folic acid were not able to change it. In conclusion, we show that insulin has an antiproliferative and hypertrophic effect on a first trimester extravillous human trophoblast cell line. So insulin might affect the process of placentation.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  1. Regnault TR, Galan HL, Parker TA, Anthony RV. Placental development in normal and compromised pregnancies—a review. Placenta. 2002;23(suppl A):S119–S129.

    Article  PubMed  Google Scholar 

  2. Fowden AL, Forhead AJ, Coan PM, Burton GJ. The placenta and intrauterine programming. J Neuroendocrinol. 2008;20(4):439–450.

    Article  CAS  PubMed  Google Scholar 

  3. Forbes K, Westwood M. Maternal growth factor regulation of human placental development and fetal growth. J Endocrinol. 2010;207(1):1–16.

    Article  CAS  PubMed  Google Scholar 

  4. Ji L, Brkic J, Liu M, Fu G, Peng C, Wang YL. Placental trophoblast cell differentiation: physiological regulation and pathological relevance to preeclampsia. Mol Aspects Med. 2013;34(5):981–1023.

    Article  CAS  PubMed  Google Scholar 

  5. James JL, Carter AM, Chamley LW. Human placentation from nidation to 5 weeks of gestation. Part I: what do we know about formative placental development following implantation? Placenta. 2012;33(5):327–334.

    CAS  PubMed  Google Scholar 

  6. Sacks DA, Hadden DR, Maresh M, et al. HAPO Study Cooperative Research Group. Frequency of gestational diabetes mellitus at collaborating centers based on IADPSG consensus panel-recommended criteria: the Hyperglycemia and Adverse Pregnancy Outcome (HAPO) Study. Diabetes Care. 2012;35(3):526–528.

    Article  PubMed  PubMed Central  Google Scholar 

  7. Magon N, Chauhan M. Pregnancy in type 1 diabetes mellitus: how special are special issues? N Am J Med Sci. 2012;4(6):250–256.

    Article  PubMed  PubMed Central  Google Scholar 

  8. Lepercq J, Cauzac M, Lahlou N, et al. Overexpression of placental leptin in diabetic pregnancy: a critical role for insulin. Diabetes. 1998;47(5):847–850.

    Article  CAS  PubMed  Google Scholar 

  9. Desoye G, Hauguel-de Mouzon S. The human placenta in gestational diabetes mellitus. the insulin and cytokine network. Diabetes Care. 2007;30(suppl 2):S120–S126.

    Article  CAS  PubMed  Google Scholar 

  10. Vambergue A, Fajardy I. Consequences of gestational and pregestational diabetes on placental function and birth weight. World J Diabetes. 2011;2(11):196–203.

    Article  PubMed  PubMed Central  Google Scholar 

  11. Graham CH, Hawley TS, Hawley RG, et al. Establishment and characterization of first trimester human trophoblast cells with extended lifespan. Exp Cell Res. 1993;206(2):204–211.

    Article  CAS  PubMed  Google Scholar 

  12. Hill JR, Kwon G, Marshall CA, McDaniel ML. Hyperglycemic levels of glucose inhibit interleukin 1 release from RAW 264.7 murine macrophages by activation of protein kinase C. J Biol Chem. 1998;273(6):3308–3313.

    Article  CAS  PubMed  Google Scholar 

  13. Mandl M, Haas J, Bischof P, Nohammer G, Desoye G. Serum-dependent effects of IGF-I and insulin on proliferation and invasion of human first trimester trophoblast cell models. Histochem Cell Biol. 2002;117(5):391–399.

    Article  CAS  PubMed  Google Scholar 

  14. Magarinos MP, Sanchez-Margalet V, Kotler M, Calvo JC, Varone CL. Leptin promotes cell proliferation and survival of trophoblastic cells. Biol Reprod. 2007;76(2):203–210.

    Article  CAS  PubMed  Google Scholar 

  15. Gonzalez E, Flier E, Molle D, Accili D, McGraw TE. Hyperinsulinemia leads to uncoupled insulin regulation of the GLUT4 glucose transporter and the FoxO1 transcription factor. Proc Natl Acad Sci USA. 2011;108(25):10162–10167.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Stubert J, Waldmann K, Dieterich M, Richter DU, Briese V. Progranulin shows cytoprotective effects on trophoblast cells in vitro but does not antagonize TNF-alpha-induced apoptosis. Arch Gynecol Obstet. 2014;290(5):867–873.

    Article  CAS  PubMed  Google Scholar 

  17. Araujo JR, Correia-Branco A, Ramalho C, et al. L-Methionine placental uptake: characterization and modulation in gestational diabetes mellitus. Reprod Sci. 2013;20(12):1492–1507.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Araujo JR, Correia-Branco A, Ramalho C, Keating E, Martel F. Gestational diabetes mellitus decreases placental uptake of long-chain polyunsaturated fatty acids: involvement of long-chain acyl-CoA synthetase. J Nutr Biochem. 2013;24(10):1741–1750.

    Article  CAS  PubMed  Google Scholar 

  19. Araujo JR, Correia-Branco A, Moreira L, Ramalho C, Martel F, Keating E. Folic acid uptake by the human syncytiotrophoblast is affected by gestational diabetes, hyperleptinemia, and TNF-alpha. Pediatr Res. 2013;73(4 pt 1):388–394.

    Article  CAS  PubMed  Google Scholar 

  20. Mosmann T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods. 1983;65(1-2):55–63.

    Article  CAS  PubMed  Google Scholar 

  21. Bergmeyer HU, Bernt E. Lactate dehydrogenase. In: Bergmeyer HU, ed. Methods of Enzymatic Analysis. Vol. 2. New York, NY: Verlag Chemie; 1974:574–579

    Article  Google Scholar 

  22. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976;72(1):248–254.

    Article  CAS  PubMed  Google Scholar 

  23. Ben-Haroush A, Yogev Y, Hod M. Epidemiology of gestational diabetes mellitus and its association with type 2 diabetes. Diabet Med. 2004;21(2):103–113.

    Article  CAS  PubMed  Google Scholar 

  24. Barbour LA, McCurdy CE, Hernandez TL, Kirwan JP, Catalano PM, Friedman JE. Cellular mechanisms for insulin resistance in normal pregnancy and gestational diabetes. Diabetes Care. 2007;30(2):112–119.

    Article  CAS  Google Scholar 

  25. Pollock KE, Stevens D, Pennington KA, et al. Hyperleptinemia during pregnancy decreases adult weight of offspring and is associated with increased offspring locomotor activity in mice. Endocrinology. 2015;156(10):3777–3790.

    Article  CAS  PubMed  Google Scholar 

  26. Mayama R, Izawa T, Sakai K, Suciu N, Iwashita M. Improvement of insulin sensitivity promotes extravillous trophoblast cell migration stimulated by insulin-like growth factor-I. Endocr J. 2013;60(3):359–368.

    Article  CAS  PubMed  Google Scholar 

  27. Westgate JA, Lindsay RS, Beattie J, et al. Hyperinsulinemia in cord blood in mothers with type 2 diabetes and gestational diabetes mellitus in New Zealand. Diabetes Care. 2006;29(6):1345–1350.

    Article  PubMed  Google Scholar 

  28. Lassance L, Miedl H, Absenger M, et al. Hyperinsulinemia stimulates angiogenesis of human fetoplacental endothelial cells: a possible role of insulin in placental hypervascularization in diabetes mellitus. J Clin Endocrinol Metab. 2013;98(9):E1438–E1447.

    Article  CAS  PubMed  Google Scholar 

  29. Perez-Perez A, Guadix P, Maymo J, et al. Insulin and leptin signaling in placenta from gestational diabetic subjects. Horm Metab Res. 2016;48(1):62–69.

    CAS  PubMed  Google Scholar 

  30. Hiden U, Glitzner E, Hartmann M, Desoye G. Insulin and the IGF system in the human placenta of normal and diabetic pregnancies. J Anat. 2009;215(1):60–68.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Hiden U, Maier A, Bilban M, et al. Insulin control of placental gene expression shifts from mother to foetus over the course of pregnancy. Diabetologia. 2006;49(1):123–131.

    Article  CAS  PubMed  Google Scholar 

  32. Knofler M. Critical growth factors and signalling pathways controlling human trophoblast invasion. Int J Dev Biol. 2010;54(2-3): 269–280.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  33. Blagosklonny MV. TOR-centric view on insulin resistance and diabetic complications: perspective for endocrinologists and gerontologists. Cell Death Dis. 2013;4(12):964–972.

    Article  CAS  Google Scholar 

  34. Murakami M, Ichisaka T, Maeda M, et al. mTOR is essential for growth and proliferation in early mouse embryos and embryonic stem cells. Mol Cell Biol. 2004;24(15):6710–6718.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Ferretti C, Bruni L, Dangles-Marie V, Pecking AP, Bellet D. Molecular circuits shared by placental and cancer cells, and their implications in the proliferative, invasive an migratory capacities of trophoblasts. Hum Reprod Update. 2007;13(2):121–141.

    Article  CAS  PubMed  Google Scholar 

  36. Ambrosino C, Nebreda AR. Cell cycle regulation by p38 MAP kinases. Bio cell. 2001;93(1-2):47–51.

    Article  CAS  Google Scholar 

  37. Singh CK, Kumar A, Lavoie HA, Dipette DJ, Singh US. Diabetic complications in pregnancy: is resveratrol a solution? Exp Biol Med (Maywood). 2013;238(5):482–490.

    Article  CAS  Google Scholar 

  38. Zou Y, Zuo Q, Huang S, et al. Resveratrol inhibits trophoblast apoptosis through oxidative stress in preeclampsia-model rats. Molecules. 2014;19(12):20570–20579.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  39. Chen B, Tuuli MG, Longtine MS, et al. Pomegranate juice and punicalagin attenuate oxidative stress and apoptosis in human placenta and in human placental trophoblasts. Am J Physiol Endocrinol Metab. 2012;302(9):E1142–E1152.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Klingler M, Blaschitz A, Campoy C, et al. The effect of docosa-hexaenoic acid and folic acid supplementation on placental apop-tosis and proliferation. Br J Nutr. 2006;96(1):182–190.

    Article  CAS  PubMed  Google Scholar 

  41. Wietrak E, Kaminski K, Leszczynska-Gorzelak B, Oleszczuk J. Effect of docosahexaenoic acid on apoptosis and proliferation in the placenta: preliminary report. Biomed Res Int. 2015;2015: 482875.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  42. Jones ML, Mark PJ, Waddell BJ. Maternal dietary omega-3 fatty acids and placental function. Reproduction. 2014;147(5):143–152.

    Article  CAS  Google Scholar 

  43. Basak S, Das MK, Duttaroy AK. Fatty acid-induced angiogenesis in first trimester placental trophoblast cells: possible roles of cellular fatty acid-binding proteins. Life Sci. 2013;93(21):755–762.

    Article  CAS  PubMed  Google Scholar 

  44. Antony AC. In utero physiology: role of folic acid in nutrient delivery and fetal development. Am J Clin Nutr. 2007;85(2):598–603.

    Article  Google Scholar 

  45. Ahmed T, Fellus I, Gaudet J, MacFarlane AJ, Fontaine-Bisson B, Bainbridge SA. Effect of folic acid on human trophoblast health and function in vitro. Placenta. 2016;37:7–15.

    Article  CAS  PubMed  Google Scholar 

  46. Forbes K, Shah VK, Siddals K, Gibson JM, Aplin JD, Westwood M. Statins inhibit insulin-like growth factor action in first trimester placenta by altering insulin-like growth factor 1 receptor glycosylation. Mol Hum Reprod. 2015;21(1):105–114.

    Article  CAS  PubMed  Google Scholar 

  47. Kenis I, Tartakover-Matalon S, Cherepnin N, et al. Simvastatin has deleterious effects on human first trimester placental explants. Hum Reprod. 2005;20(10):2866–2872.

    Article  CAS  PubMed  Google Scholar 

  48. Godfrey LM, Erramouspe J, Cleveland KW. Teratogenic risk of statins in pregnancy. Ann Pharmacother. 2012;46(10):1419–1424.

    Article  PubMed  Google Scholar 

  49. Zarek J, Koren G. The fetal safety of statins: a systematic review and meta-analysis. J Obstet Gynaecol Can. 2014;36(6):506–509.

    Article  PubMed  Google Scholar 

  50. Andrade SE, Raebel MA, Brown J, et al. Outpatient use of cardiovascular drugs during pregnancy. Pharmacoepidemiol Drug Saf. 2008;17(3):240–247.

    Article  PubMed  Google Scholar 

  51. Egert S, Wolffram S, Bosy-Westphal A, et al. Daily quercetin supplementation dose-dependently increases plasma quercetin concentrations in healthy humans. J Nutr. 2008;138(9):1615–1621.

    Article  CAS  PubMed  Google Scholar 

  52. Brull V, Burak C, Stoffel-Wagner B, et al. Effects of a quercetinrich onion skin extract on 24 h ambulatory blood pressure and endothelial function in overweight-to-obese patients with (pre-)-hypertension: a randomised double-blinded placebo-controlled cross-over trial. Br J Nutr. 2015;114(8):1263–1277.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  53. Araujo JR, Goncalves P, Martel F. Chemopreventive effect of dietary polyphenols in colorectal cancer cell lines. Nutr Res. 2011;31(2):77–87.

    Article  CAS  PubMed  Google Scholar 

  54. Nomura M, Takahashi T, Nagata N, et al. Inhibitory mechanisms of flavonoids on insulin-stimulated glucose uptake in MC3T3-G2/ PA6 adipose cells. Biol Pharm Bull. 2008;31(7):1403–1409.

    Article  CAS  PubMed  Google Scholar 

  55. Johnsen GM, Basak S, Weedon-Fekjær MS, et al. Docosahexaenoic acid stimulates tube formation in first trimester trophoblast cells, HTR8/SVneo. Placenta. 2011;32(9):626–632.

    Article  CAS  PubMed  Google Scholar 

  56. Bjorkhem-Bergman L, Lindh JD, Bergman P. What is a relevant statin concentration in cell experiments claiming pleiotropic effects? Br J Clin Pharmacol. 2011;72(1):164–165.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  57. Bellosta S, Ferri N, Bernini F, Paoletti R, Corsini A. Non-lipid-related effects of statins. Ann Med. 2000;32(3):164–176.

    Article  CAS  PubMed  Google Scholar 

  58. Nordstrand A, Lundholm M, Larsson A, Lerner UH, Widmark A, Wikstrom P. Inhibition of the insulin-like growth factor-1 receptor enhances effects of simvastatin on prostate cancer cells in co-culture with bone. Cancer Microenviron. 2013;6(3):231–240.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  59. Knofler M, Mosl B, Bauer S, Griesinger G, Husslein P. TNF-alpha/TNFRI in primary and immortalized first trimester cytotro-phoblasts. Placenta. 2000;21(5-6):525–535.

    Article  CAS  PubMed  Google Scholar 

  60. Basak S, Das MK, Srinivas V, Duttaroy AK. The interplay between glucose and fatty acids on tube formation and fatty acid uptake in the first trimester trophoblast cells, HTR8/SVneo. Mol Cell Biochem. 2015;401(1-2):11–19.

    Article  CAS  PubMed  Google Scholar 

  61. Weiss U, Cervar M, Puerstner P, et al. Hyperglycaemia in vitro alters the proliferation and mitochondrial activity of the choriocarcinoma cell lines BeWo, JAR an JEG-3 as models for human first-trimester trophoblast. Diabetologia. 2001;44(2):209–219.

    Article  CAS  PubMed  Google Scholar 

  62. Masumoto A, Takamoto N, Masuyama H, Akahori Y, Inoue S, Hiramatsu Y. Effects of intermittent high glucose on BeWo choriocarcinoma cells in culture. J Obstet Gynaecol Res. 2011;37(10):1365–1375.

    Article  PubMed  Google Scholar 

  63. Chen H, Wu Y, Liu B, Li Z, Wang Z. Adiponectin exerts anti-proliferative effect on human placenta via modulation of the JNK/ c-Jun pathway. Int J Clin Exp Pathol. 2014;7(6):2894–2904.

    CAS  PubMed  PubMed Central  Google Scholar 

  64. Belkacemi L, Lash GE, Macdonald-Goodfellow SK, Caldwell JD, Graham CH. Inhibition of human trophoblast invasiveness by high glucose concentrations. J Clin Endocrinol Metab. 2005;90(8):4846–4851.

    Article  CAS  PubMed  Google Scholar 

  65. Cauzac M, Czuba D, Girard J, Hauguel-de Mouzon S. Transduction of leptin growth signals in placental cells is independent of JAK-STAT activation. Placenta. 2003;24(4):378–384.

    Article  CAS  PubMed  Google Scholar 

  66. Seki H, Matuoka K, Inooku H, Takeda S. TNF-alpha from monocyte of patients with pre-eclampsia-induced apoptosis in human trophoblast cell line. J Obstet Gynaecol Res. 2007;33(4):408–416.

    Article  CAS  PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Biochemistry, Faculty of Medicine, University of Porto, Al. Prof. Hernâni Monteiro, 4200-319, Porto, Portugal

    Cláudia Silva MSc, Catarina Nunes MSc, Ana Correia-Branco MSc & Fátima Martel PhD

  2. Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Portugal

    Cláudia Silva MSc, Catarina Nunes MSc, Ana Correia-Branco MSc & Fátima Martel PhD

  3. Unité de Pathogénie Microbienne Moléculaire, INSERM U1202, Institut Pasteur, Paris, France

    João R. Araújo PhD

Authors
  1. Cláudia Silva MSc
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Catarina Nunes MSc
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Ana Correia-Branco MSc
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. João R. Araújo PhD
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Fátima Martel PhD
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Fátima Martel PhD.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Silva, C., Nunes, C., Correia-Branco, A. et al. Insulin Exhibits an Antiproliferative and Hypertrophic Effect in First Trimester Human Extravillous Trophoblasts. Reprod. Sci. 24, 582–594 (2017). https://doi.org/10.1177/1933719116667220

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1177/1933719116667220

Keywords

Search

Navigation