Skip to main content
SpringerLink
Log in

SEAP activity serves for demonstrating ER stress induction by glucolipotoxicity as well as testing ER stress inhibitory potential of therapeutic agents

  • Published:
Molecular and Cellular Biochemistry Aims and scope Submit manuscript

Abstract

Endoplasmic reticulum (ER) stress is emerging as a unifying paradigm and one of the underlying mechanisms in the genesis of diabetes and its complications. While this has prompted the development of ER stress inhibitors, there is a limitation in monitoring of ER stress in vitro and in vivo by reliable methodologies. We validated the secreted alkaline phosphatase (SEAP) activity as a surrogate marker of ER stress in mouse β-TC6 cells exposed to glucolipotoxicity or tunicamycin and studied insulin secretion along with alterations in ER stress markers. SEAP activity assay was measured using the Great EscAPe SEAP kit, insulin levels were determined by Mercodia reagents and mRNA expression of ER stress markers was quantified by real-time PCR. SEAP activity in β-cells was significantly decreased (indicating increased ER stress) on exposure either to glucolipotoxicity or tunicamycin. This was accompanied by an increased mRNA expression of ER stress markers (GRP-78, PERK, IRE1α, ATF6, XBP-1, and CHOP) and decreased insulin secretion. Treating the cells with phenylbutyric acid normalized SEAP activity, decreased mRNA expression of ER stress markers and improved insulin secretion. Interestingly, cells exposed to different classes of anti-diabetes agents or compounds such as resveratrol resisted ER stress. Methylglyoxal also induces ER stress and this was counteracted by aminoguanidine. Out study demonstrates SEAP activity as a novel ER stress monitoring assay to investigate the therapeutic value of agents with ER stress inhibitory potential. Future studies should focus on the exercise of adopting this reporter assay for high-throughput screening mode of drug discovery.

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

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

Similar content being viewed by others

Abbreviations

ATF-6:

Activating transcription factor-6

CHOP:

CCAAT/enhancer-binding homologous protein

GRP-78:

Glucose-regulated protein-78

IRE-1α :

Inositol-requiring enzyme-1α

PERK:

PKR-like ER kinase

XBP-1:

X box binding protein1

PBA:

4-Phenylbutyric acid

SEAP:

Secreted alkaline phosphatase

PDI:

Protein disulfide isomerase

ERO1:

ER oxidoreductase1

INSIG1:

Insulin-induced gene1

SREBP:

Sterol regulatory element-binding transcription factor

AGEs:

Advanced Glycation Endproducts

References

  1. Wang S, Kaufman RJ (2012) The impact of the unfolded protein response on human disease. J Cell Biol 197:857–867

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  2. Anelli T, Sitia R (2008) Protein quality control in the early secretory pathway. EMBO J 27:315–327

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  3. Scheuner D, Kaufman RJ (2008) The unfolded protein response: a pathway that links insulin demand with β-cell failure and diabetes. Endocr Rev 29:317–333

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  4. Tersey SA, NishikiY Templin AT et al (2012) Islet β-cell endoplasmic reticulum stress precedes the onset of type 1 diabetes in the nonobese diabetic mouse model. Diabetes 61:818–827

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  5. Gujral UP, Narayan KM, Kahn SE, Kanaya AM (2014) The relative associations of β-cell function and insulin sensitivity with glycemic status and incident glycemic progression in migrant Asian Indians in the United States: the MASALA study. J Diabetes Complicat 28:45–50

    Article  PubMed  Google Scholar 

  6. Staimez LR, Weber MB, Ranjani H, Ali MK, Echouffo-Tcheugui JB, Phillips LS, Mohan V, Narayan KM (2013) Evidence of reduced β-cell function in Asian Indians with mild dysglycemia. Diabetes Care 36:2772–2778

    Article  PubMed Central  PubMed  Google Scholar 

  7. Thomsen SK, Gloyn AL (2014) The pancreatic β cell: recent insights from human genetics. Trends Endocrinol Metab 25:425–434

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  8. Dimas AS, Lagou V, Barker A et al (2014) Impact of type 2 diabetes susceptibility variants on quantitative glycemic traits reveals mechanistic heterogeneity. Diabetes 63:2158–2171

    Article  CAS  PubMed  Google Scholar 

  9. Pasquali L, Gaulton KJ, Rodríguez-Seguí SA et al (2014) Pancreatic islet enhancer clusters enriched in type 2 diabetes risk-associated variants. Nat Genet 46:136–143

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  10. Lee YS, Kim HK, Chung S, Kim KS, Dutta A (2005) Depletion of human micro-RNA miR-125b reveals that it is critical for the proliferation of differentiated cells but not for the down-regulation of putative targets during differentiation. J Biol Chem 280:16635–16641

    Article  CAS  PubMed  Google Scholar 

  11. Hirota M, Kitagaki M, Itagaki H, Aiba S (2006) Quantitative measurement of spliced XBP1 mRNA as an indicator of endoplasmic reticulum stress. J Toxicol Sci 31:149–156

    Article  CAS  PubMed  Google Scholar 

  12. Li F, Hayashi T, Jin G et al (2005) The protective effect of dantrolene on ischemic neuronal cell death is associated with reduced expression of endoplasmic reticulum stress markers. Brain Res 28:59–68

    Article  Google Scholar 

  13. Mao C, Dong D, Little E, Luo S, Lee AS (2004) Transgenic mouse model for monitoring endoplasmic reticulum stress in vivo. Nat Med 10:1013–1014

    Article  CAS  PubMed  Google Scholar 

  14. Berger J, Hauber J, Hauber R, Geiger R, Cullen BR (1988) Secreted placental alkaline phosphatase: a powerful new quantitative indicator of gene expression in eukaryotic cells. Gene 66:1–10

    Article  CAS  PubMed  Google Scholar 

  15. Cullen BR, Malim MH (1992) Secreted placental alkaline phosphatase as a eukaryotic reporter gene. Methods Enzymol 216:362–368

    Article  CAS  PubMed  Google Scholar 

  16. Taxis C, Vogel F, Wolf DH (2002) ER-golgi traffic is a prerequisite for efficient ER degradation. Mol Biol Cell 13:1806–1818

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  17. Zhang K, Kaufman RJ (2006) The unfolded protein response: a stress signaling pathway critical for health and disease. Neurology 66:102–109

    Article  Google Scholar 

  18. Minami K, Yano H, Miki T et al (2000) Insulin secretion and differential gene expression in glucose-responsive and unresponsive MIN6 sublines. Am J Physiol Endocrinol Metab 2079:773–781

    Google Scholar 

  19. Balasubramanyam M, Aravind S, Gokulakrishnan K, Prabu P, Sathishkumar C, Ranjani H, Mohan V (2011) Impaired miR-146a expression links subclinical inflammation and insulin resistance in Type 2 diabetes. Mol Cell Biochem 351:197–205

    Article  CAS  PubMed  Google Scholar 

  20. Araki E, Oyadomari S, Mori M (2003) Impact of endoplasmic reticulum stress pathway on pancreatic beta-cells and diabetes mellitus. Exp Biol Med 228:1213–1217

    CAS  Google Scholar 

  21. Sparre T, Larsen MR, Heding PE, Karlsen AE, Jensen ON, Pociot F (2005) Unraveling the pathogenesis of type 1 diabetes with proteomics: present and future directions. Mol Cell Proteomics 4:441–457

    Article  CAS  PubMed  Google Scholar 

  22. Sundsten T, Ortsater H (2009) Proteomics in diabetes research. Mol Cell Endocrinol 297:93–103

    Article  CAS  PubMed  Google Scholar 

  23. Dowling P, O’Driscoll L, O’Sullivan F, Dowd A, Henry M, Jeppesen PB, Meleady P, Clynes M (2006) Proteomic screening of glucose-responsive and glucose non-responsive MIN-6 beta cells reveals differential expression of proteins involved in protein folding, secretion and oxidative stress. Proteomics 6:6578–6587

    Article  CAS  PubMed  Google Scholar 

  24. Ahmed M, Bergsten P (2005) Glucose-induced changes of multiple mouse islet proteins analysed by two-dimensional gel electrophoresis and mass spectrometry. Diabetologia 48:477–485

    Article  CAS  PubMed  Google Scholar 

  25. Lu H, Yang Y, Allister EM, Wijesekara N, Wheeler MB (2008) The identification of potential factors associated with the development of type 2 diabetes: a quantitative proteomics approach. Mol Cell Proteomics 7:1434–1451

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  26. Elouil H, Bensellam M, Guiot Y, Vander Mierde D, Pascal SM, Schuit FC, Jonas JC (2007) Acute nutrient regulation of the unfolded protein response and integrated stress response in cultured rat pancreatic islets. Diabetologia 50:1442–1452

    Article  CAS  PubMed  Google Scholar 

  27. Jeffrey KD, Alejandro EU, Luciani DS et al (2008) Carboxypeptidase E mediates palmitate-induced beta-cell ER stress and apoptosis. Proc Natl Acad Sci USA 105:8452–8457

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  28. Pirot P, Naamane N, Libert F, Magnusson NE, Orntoft TF, Cardozo AK, Eizirik DL (2007) Global profiling of genes modified by endoplasmic reticulum stress in pancreatic beta cells reveals the early degradation of insulin mRNAs. Diabetologia 50:1006–1014

    Article  CAS  PubMed  Google Scholar 

  29. Laybutt DR, Preston AM, Akerfeldt MC, Kench JG, Busch AK, Biankin AV, Biden TJ (2007) Endoplasmic reticulum stress contributes to beta cell apoptosis in type 2 diabetes. Diabetologia 50:752–763

    Article  CAS  PubMed  Google Scholar 

  30. Karaskov E, Scott C, Zhang L, Teodoro T, Ravazzola M, Volchuk A (2006) Chronic palmitate but not oleate exposure induces endoplasmic reticulum stress, which may contribute to INS-1 pancreatic beta-cell apoptosis. Endocrinology 147:3398–3407

    Article  CAS  PubMed  Google Scholar 

  31. Desai K, Wu L (2007) Methylglyoxal and advanced glycation endproducts: new therapeutic horizons? Recent Patents Cardiovasc Drug Discov 2:89–99

    Article  CAS  Google Scholar 

  32. Vander Jagt DL (2008) Methylglyoxal, diabetes mellitus and diabetic complications. Drug Metab Drug Interact 23:93–124

    CAS  Google Scholar 

  33. Wang H, Meng QH, Gordon JR, Khandwala H, Wu L (2007) Proinflammatory and proapoptotic effects of methylglyoxal on neutrophils from patients with type 2 diabetes mellitus. Clin Biochem 40:1232–1239

    Article  CAS  PubMed  Google Scholar 

  34. McLellan AC, Thornalley PJ, Benn J, Sonksen PH (1994) Glyoxalase system in clinical diabetes mellitus and correlation with diabetic complications. Clin Sci (Lond) 87:21–29

    CAS  Google Scholar 

  35. Dhar A, Dhar I, Jiang B, Desai KM, Wu L (2011) Chronic methylglyoxal infusion by minipump causes pancreatic beta-cell dysfunction and induces type 2 diabetes in Sprague-Dawley rats. Diabetes 60:899–908

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  36. Iwawaki T, Akai R, Kohno K, Miura M (2004) A transgenic mouse model for monitoring endoplasmic reticulum stress. Nat Med 10:98–102

    Article  CAS  PubMed  Google Scholar 

  37. Hiramatsu N, Kasai A, Hayakawa K, Yao J, Kitamura M (2006) Real-time detection and continuous monitoring of ER stress in vitro and in vivo by ESTRAP: evidence for systemic, transient ER stress during endotoxemia. Nucleic Acids Res 34:e93

    Article  PubMed Central  PubMed  Google Scholar 

  38. Ozcan U, Yilmaz E, Özcan L et al (2006) Chemical chaperones reduce ER stress and restore glucose homeostasis in a mouse model of type 2 diabetes. Science 313:1137–1140

    Article  PubMed  Google Scholar 

  39. Ozcan U, CaoQ Yilmaz E et al (2004) Endoplasmic reticulum stress links obesity, insulin action, and type 2 diabetes. Science 306:457–461

    Article  PubMed  Google Scholar 

  40. Kars M, Yang L, Gregor MF et al (2010) Tauroursodeoxycholic acid may improve liver and muscle but not adipose tissue insulin sensitivity in obese men and women. Diabetes 59:1899–1905

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  41. Cadavez L, Montane J, Alcarraz-Vizán G, Visa M, Vidal-Fàbrega Servitja JM, Novials A (2014) Chaperones ameliorate beta cell dysfunction associated with human islet amyloid polypeptide overexpression. PLoS One 9:e101797

    Article  PubMed Central  PubMed  Google Scholar 

  42. Erbay E, Babaev VR, Mayers JR et al (2009) Reducing endoplasmic reticulum stress through a macrophage lipid chaperone alleviates atherosclerosis. Nat Med 15:1383–1391

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  43. Tveten K, HollaOL Ranheim T, Berge KE, Leren TP, Kulseth MA (2007) 4-Phenylbutyrate restores the functionality of a misfolded mutant low-density lipoprotein receptor. FEBS J 274:1881–1893

    Article  CAS  PubMed  Google Scholar 

  44. Ono K, Ikemoto M, Kawarabayashi T et al (2009) A chemical chaperone, sodium 4-phenylbutyric acid, attenuates the pathogenic potency in human α-synuclein A30P + A53T transgenic mice. Parkinsonism Relat Disord 15:649–654

    Article  PubMed  Google Scholar 

  45. Ricobaraza A, Cuadrado-Tejedor M, Pérez-Mediavilla A, Frechilla D, Del Río J, García-Osta A (2009) Phenylbutyrate ameliorates cognitive deficit and reduces tau pathology in an Alzheimer’s disease mouse model. Neuropsychopharmacology 34:1721–1732

    Article  CAS  PubMed  Google Scholar 

  46. Qi W, Mu J, Luo ZF et al (2011) Attenuation of diabetic nephropathy in diabetes rats induced by streptozotocin by regulating the endoplasmic reticulum stress inflammatory response. Metabolism 60:594–603

    Article  CAS  PubMed  Google Scholar 

  47. Zode GS, Kuehn MH, Nishimura DY et al (2011) Reduction of ER stress via a chemical chaperone prevents disease phenotypes in a mouse model of primary open angle glaucoma. J Clin Invest 121:3542–3553

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  48. Xiao C, Giacca A, Lewis GF (2011) Sodium phenylbutyrate, a drug with known capacity to reduce endoplasmic reticulum stress, partially alleviates lipid-induced insulin resistance and β-cell dysfunction in humans. Diabetes 60:918–924

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  49. Lily M, Godwin M (2009) Treating prediabetes with metformin systematic review and meta-analysis. Can Fam Phys 55:363–369

    Google Scholar 

  50. Balasubramanyam M (2014) Metformin—Nature’s gift that keeps on giving more. J Obes Metab Res 1:118–120

    Article  Google Scholar 

  51. Simon-Szabó L, Kokas M, Mandl J, Kéri G, Csala M (2014) Metformin attenuates palmitate-induced endoplasmic reticulum stress, serine phosphorylation of IRS-1and apoptosis in rat insulinoma cells. PLoS One 9:97868

    Article  Google Scholar 

  52. Oh YS, Lee YJ, Kang Y, Han J, Lim OK, Jun HS (2013) Exendin-4 inhibits glucolipotoxic ER stress in pancreatic β cells via regulation of SREBP1c and C/EBPβ transcription factors. J Endocrinol 216(3):343–352

    Article  CAS  PubMed  Google Scholar 

  53. Liang CP, Han S, Li G, Tabas I, Tall AR (2012) Impaired MEK signaling and SERCA expression promote ER stress and apoptosis in insulin-resistant macrophages and are reversed by exenatide treatment. Diabetes 61:2609–2620

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  54. Shimizu S, Hosooka T, Matsuda T et al (2012) DPP4 inhibitor vildagliptin preserves β-cell mass through amelioration of endoplasmic reticulum stress in C/EBPB transgenic mice. J Mol Endocrinol 49:125–135

    Article  CAS  PubMed  Google Scholar 

  55. Wu YJ, Guo X, Li CJ, Li DQ, Zhang J, Yang Y, Kong Y, Guo H, Liu DM, Chen LM (2015) Dipeptidyl peptidase-4 inhibitor, vildagliptin, inhibits pancreatic beta cell apoptosis in association with its effects suppressing endoplasmic reticulum stress in db/db mice. Metabolism. 64:226–235

    Article  CAS  PubMed  Google Scholar 

  56. Szkudelski T, Szkudelska K (2011) Anti-diabetic effects of resveratrol. Ann N Y Acad Sci 1215:34–39

    Article  CAS  PubMed  Google Scholar 

  57. Gertz M, Nguyen GT, Fischer F, Suenkel B, Schlicker C, Franzel B, Tomaschewski J, Aladini F, Becker C, Wolters D, Steegborn C (2012) A molecular mechanism for direct sirtuin activation by resveratrol. PLoS One 7:e49761

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  58. Yamabe S, Hirose J, Uehara Y, Okada T, Okamoto N, Oka K, Taniwaki T, Mizuta H (2013) Intracellular accumulation of advanced glycation end products induces apoptosis via endoplasmic reticulum stress in chondrocytes. FEBS J 280:1617–1629

    Article  CAS  PubMed  Google Scholar 

  59. Bronstein I, Fortin J, Stanley PE, Stewart GS, Kricka LJ (1994) Chemiluminescent and bioluminescent reporter gene assays. Anal Biochem 219:169–181

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

This work was supported by research Grants from the Department of Biotechnology (DBT) and the Department of Science and Technology (DST), Government of India, New Delhi, India. RL acknowledges the financial assistance (Senior Research Fellowship) from the Council of Scientific and Industrial Research (CSIR), New Delhi, India.

Conflict of interest

None.

Author information

Authors and Affiliations

  1. Department of Cell and Molecular Biology, Madras Diabetes Research Foundation & Dr. Mohan’s Diabetes Specialties Centre, Gopalapuram, Chennai, 600086, India

    Raji Lenin, Viswanathan Mohan & Muthuswamy Balasubramanyam

Authors
  1. Raji Lenin
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Viswanathan Mohan
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Muthuswamy Balasubramanyam
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Muthuswamy Balasubramanyam.

Additional information

M. Balasubramanyam is Dean of Research Studies and Senior Scientist in Madras Diabetes Research Foundation.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Lenin, R., Mohan, V. & Balasubramanyam, M. SEAP activity serves for demonstrating ER stress induction by glucolipotoxicity as well as testing ER stress inhibitory potential of therapeutic agents. Mol Cell Biochem 404, 271–279 (2015). https://doi.org/10.1007/s11010-015-2387-1

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11010-015-2387-1

Keywords

Search

Navigation