In vivo measurement and biological characterisation of the diabetes-associated mutant insulin p.R46Q (GlnB22-insulin)
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.
KeywordsInsulin gene Maturity-onset diabetes of the young Mutant insulin
Baby hamster kidney
Eukaryotic Initiation Factor 2α
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 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.
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.
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