, Volume 60, Issue 8, pp 1423–1431 | Cite as

In vivo measurement and biological characterisation of the diabetes-associated mutant insulin p.R46Q (GlnB22-insulin)

  • Julie Støy
  • Jørgen Olsen
  • Soo-Young Park
  • Søren Gregersen
  • Claudia U. Hjørringgaard
  • Graeme I. Bell



Heterozygous mutations in the insulin gene that affect proinsulin biosynthesis and folding are associated with a spectrum of diabetes phenotypes, from permanent neonatal diabetes to MODY. In vivo studies of these mutations may lead to a better understanding of insulin mutation-associated diabetes and point to the best treatment strategy. We studied an 18-year-old woman with MODY heterozygous for the insulin mutation p.R46Q (GlnB22-insulin), measuring the secretion of mutant and wild-type insulin by LC-MS. The clinical study was combined with in vitro studies of the synthesis and secretion of p.R46Q-insulin in rat INS-1 insulinoma cells.


We performed a standard 75 g OGTT in the 18-year-old woman and measured plasma glucose and serum insulin (wild-type insulin and GlnB22-insulin), C-peptide, proinsulin, glucagon and amylin. The affinity of GlnB22-insulin was tested on human insulin receptors expressed in baby hamster kidney (BHK) cells. We also examined the subcellular localisation, secretion and impact on cellular stress markers of p.R46Q-insulin in INS-1 cells.


Plasma GlnB22-insulin concentrations were 1.5 times higher than wild-type insulin at all time points during the OGTT. The insulin-receptor affinity of GlnB22-insulin was 57% of that of wild-type insulin. Expression of p.R46Q-insulin in INS-1 cells was associated with decreased insulin secretion, but not induction of endoplasmic reticulum stress.


The results show that beta cells can process and secrete GlnB22-insulin both in vivo and in vitro. Our combined approach of immunoprecipitation and LC-MS to measure mutant and wild-type insulin may be useful for the study of other mutant insulin proteins. The ability to process and secrete a mutant protein may predict a more benign course of insulin mutation-related diabetes. Diabetes develops when the beta cell is stressed because of increased demand for insulin, as observed in individuals with other insulin mutations that affect the processing of proinsulin to insulin or mutations that reduce the affinity for the insulin receptor.


Insulin gene Maturity-onset diabetes of the young Mutant insulin 



Baby hamster kidney


Eukaryotic Initiation Factor 2α


Endoplasmic reticulum


Neonatal diabetes mellitus



We are grateful to the individual in this study for her participation. The authors of the study acknowledge laboratory technicians A. Mengel (Medical Research Laboratories, Aarhus University, Aarhus, Denmark) and K. Meyhoff-Madsen (ADME Department, Novo Nordisk, Måløv, Denmark) for invaluable assistance with the biochemical analyses and during the study day. Senior principal scientist T. Børglum Kjeldsen and principal laboratory technician A. Frost Bjerre (Recombinant Protein Technology, Novo Nordisk, Måløv, Denmark) are thanked for data from the insulin-receptor binding assay.

Data availability

Data from the study are available from the corresponding author on request.


The in vitro studies were supported by NIDDK grants P30 DK020595 and R01 DK10494 and by a gift from the Kovler Family Foundation.

Duality of interest

JO and CUH are employees of, and own stocks in, Novo Nordisk. JS, S-YP, SG and GIB declare that there is no duality of interest associated with this manuscript.

Contribution statement

All authors conceived and designed the study. JS, JO, CUH and S-YP collected and analysed the data. JS and JO wrote the first draft of the manuscript. All authors revised the manuscript critically and gave final approval of the submitted version. JS is the guarantor of the work.

Supplementary material

125_2017_4295_MOESM1_ESM.pdf (926 kb)
ESM 1 (PDF 925 kb)


  1. 1.
    Carmody D, Støy J, Greeley SA, Bell GI, Philipson LH (2016) A clinical guide to monogenic diabetes. In: Weiss RE, Refetoff S (eds) Genetic diagnosis of endocrine disorders, 2nd edn. Academic Press, Cambridge, pp 21–78CrossRefGoogle Scholar
  2. 2.
    Støy J, Edghill EL, Flanagan SE et al (2007) Insulin gene mutations as a cause of permanent neonatal diabetes. Proc Natl Acad Sci U S A 104:15040–15044CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Edghill EL, Flanagan SE, Patch AM et al (2008) Insulin mutation screening in 1,044 patients with diabetes: mutations in the INS gene are a common cause of neonatal diabetes but a rare cause of diabetes diagnosed in childhood or adulthood. Diabetes 57:1034–1042CrossRefPubMedGoogle Scholar
  4. 4.
    Colombo C, Porzio O, Liu M et al (2008) Seven mutations in the human insulin gene linked to permanent neonatal/infancy-onset diabetes mellitus. J Clin Invest 118:2148–2156PubMedPubMedCentralGoogle Scholar
  5. 5.
    Steiner DF, Tager HS, Nanjo K, Chan SJ, Rubenstein AH (1995) Familial syndromes of hyperproinsulinemia and hyperinsulinemia with mild diabetes. In: Scriver CR, Beaudet AL, Sly AS, Valle D (eds) The metabolic and molecular bases of inherited disease. McGraw-Hill, New York, pp 897–904Google Scholar
  6. 6.
    Molven A, Ringdal M, Nordbo AM et al (2008) Mutations in the insulin gene can cause MODY and autoantibody-negative type 1 diabetes. Diabetes 57:1131–1135CrossRefPubMedGoogle Scholar
  7. 7.
    Boesgaard TW, Pruhova S, Andersson EA et al (2010) Further evidence that mutations in INS can be a rare cause of maturity-onset diabetes of the young (MODY). BMC Med Genet 11:42CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Meur G, Simon A, Harun N et al (2010) Insulin gene mutations resulting in early-onset diabetes: marked differences in clinical presentation, metabolic status, and pathogenic effect through endoplasmic reticulum retention. Diabetes 59:653–661CrossRefPubMedGoogle Scholar
  9. 9.
    Dusatkova L, Dusatkova P, Vosahlo J et al (2015) Frameshift mutations in the insulin gene leading to prolonged molecule of insulin in two families with maturity-onset diabetes of the young. Eur J Med Genet 58:230–234CrossRefPubMedGoogle Scholar
  10. 10.
    Polak M, Dechaume A, Cave H et al (2008) Heterozygous missense mutations in the insulin gene are linked to permanent diabetes appearing in the neonatal period or in early infancy: a report from the French ND (Neonatal Diabetes) Study Group. Diabetes 57:1115–1119CrossRefPubMedGoogle Scholar
  11. 11.
    Rajan S, Eames SC, Park SY et al (2010) In vitro processing and secretion of mutant insulin proteins that cause permanent neonatal diabetes. Am J Physiol Endocrinol Metab 298:E403–E410CrossRefPubMedGoogle Scholar
  12. 12.
    Park SY, Ye H, Steiner DF, Bell GI (2010) Mutant proinsulin proteins associated with neonatal diabetes are retained in the endoplasmic reticulum and not efficiently secreted. Biochem Biophys Res Commun 391:1449–1454CrossRefPubMedGoogle Scholar
  13. 13.
    Izumi T, Yokota-Hashimoto H, Zhao S, Wang J, Halban PA, Takeuchi T (2003) Dominant negative pathogenesis by mutant proinsulin in the Akita diabetic mouse. Diabetes 52:409–416CrossRefPubMedGoogle Scholar
  14. 14.
    Yoshioka M, Kayo T, Ikeda T, Koizumi A (1997) A novel locus, Mody4, distal to D7Mit189 on chromosome 7 determines early-onset NIDDM in nonobese C57BL/6 (Akita) mutant mice. Diabetes 46:887–894CrossRefPubMedGoogle Scholar
  15. 15.
    Liu M, Sun J, Cui J et al (2014) INS-gene mutations: from genetics and beta cell biology to clinical disease. Mol Asp Med 42:3–18CrossRefGoogle Scholar
  16. 16.
    Liu M, Hodish I, Haataja L et al (2010) Proinsulin misfolding and diabetes: mutant INS gene-induced diabetes of youth. Trends Endocrinol Metab: TEM 21:652–659CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Johansen A, Ek J, Mortensen HB, Pedersen O, Hansen T (2005) Half of clinically defined maturity-onset diabetes of the young patients in Denmark do not have mutations in HNF4A, GCK, and TCF1. J Clin Endocrinol Metab 90:4607–4614CrossRefPubMedGoogle Scholar
  18. 18.
    Glendorf T, Sorensen AR, Nishimura E, Pettersson I, Kjeldsen T (2008) Importance of the solvent-exposed residues of the insulin B chain alpha-helix for receptor binding. Biochemistry 47:4743–4751CrossRefPubMedGoogle Scholar
  19. 19.
    Andersen L, Dinesen B, Jorgensen PN, Poulsen F, Roder ME (1993) Enzyme immunoassay for intact human insulin in serum or plasma. Clin Chem 39:578–582PubMedGoogle Scholar
  20. 20.
    Dimas AS, Lagou V, Barker A et al (2014) Impact of type 2 diabetes susceptibility variants on quantitative glycemic traits reveals mechanistic heterogeneity. Diabetes 63:2158–2171CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Krizkova K, Veverka V, Maletinska L et al (2014) Structural and functional study of the GlnB22-insulin mutant responsible for maturity-onset diabetes of the young. PLoS One 9:e112883CrossRefPubMedPubMedCentralGoogle Scholar
  22. 22.
    Menting JG, Yang Y, Chan SJ, et al. (2014) Protective hinge in insulin opens to enable its receptor engagement. Proc Natl Acad Sci USAGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2017

Authors and Affiliations

  • Julie Støy
    • 1
  • Jørgen Olsen
    • 2
  • Soo-Young Park
    • 3
    • 4
  • Søren Gregersen
    • 5
  • Claudia U. Hjørringgaard
    • 6
  • Graeme I. Bell
    • 3
    • 4
  1. 1.Department of Internal Medicine and EndocrinologyAarhus University HospitalAarhus CDenmark
  2. 2.ADME DepartmentNovo NordiskMåløvDenmark
  3. 3.Department of MedicineUniversity of ChicagoChicagoUSA
  4. 4.Department of Human GeneticsUniversity of ChicagoChicagoUSA
  5. 5.Department of Internal Medicine and EndocrinologyAarhus University HospitalAarhusDenmark
  6. 6.Protein & Peptide ChemistryNovo NordiskMåløvDenmark

Personalised recommendations