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Systematic evaluation of the metabolic to mitogenic potency ratio for B10-substituted insulin analogues.

Glendorf T, Knudsen L, Stidsen CE, Hansen BF, Hegelund AC, Sørensen AR, Nishimura E, Kjeldsen T - PLoS ONE (2012)

Bottom Line: Insulin analogues comprising acidic amino acid substitutions at position B10 have previously been shown to display increased mitogenic potencies compared to human insulin and the underlying molecular mechanisms have been subject to much scrutiny and debate.Several B10-substituted insulin analogues devoid of disproportionate increases in mitogenic compared to metabolic potencies were identified.Our results also suggest that the increased mitogenic potency is attributable to both insulin and IGF-I receptor activation.

View Article: PubMed Central - PubMed

Affiliation: Diabetes Research Unit, Novo Nordisk A/S, Maaloev, Denmark. tgle@novonordisk.com

ABSTRACT

Background: Insulin analogues comprising acidic amino acid substitutions at position B10 have previously been shown to display increased mitogenic potencies compared to human insulin and the underlying molecular mechanisms have been subject to much scrutiny and debate. However, B10 is still an attractive position for amino acid substitutions given its important role in hexamer formation. The aim of this study was to investigate the relationships between the receptor binding properties as well as the metabolic and mitogenic potencies of a series of insulin analogues with different amino acid substitutions at position B10 and to identify a B10-substituted insulin analogue without an increased mitogenic to metabolic potency ratio.

Methodology/principal findings: A panel of ten singly-substituted B10 insulin analogues with different amino acid side chain characteristics were prepared and insulin receptor (both isoforms) and IGF-I receptor binding affinities using purified receptors, insulin receptor dissociation rates using BHK cells over-expressing the human insulin receptor, metabolic potencies by lipogenesis in isolated rat adipocytes, and mitogenic potencies using two different cell types predominantly expressing either the insulin or the IGF-I receptor were systematically investigated. Only analogues B10D and B10E with significantly increased insulin and IGF-I receptor affinities as well as decreased insulin receptor dissociation rates displayed enhanced mitogenic potencies in both cell types employed. For the remaining analogues with less pronounced changes in receptor affinities and insulin receptor dissociation rates, no apparent correlation between insulin receptor occupancy time and mitogenicity was observed.

Conclusions/significance: Several B10-substituted insulin analogues devoid of disproportionate increases in mitogenic compared to metabolic potencies were identified. In the present study, receptor binding affinity rather than insulin receptor off-rate appears to be the major determinant of both metabolic and mitogenic potency. Our results also suggest that the increased mitogenic potency is attributable to both insulin and IGF-I receptor activation.

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Representative dissociation curves of [125I]-labelled insulin or analogue from BHK-hIR cells.Dissociation was measured at different time points and the residual binding expressed as a percentage of initial binding. Dissociation of (A) B10D (♦) and B10E (▪); (B) B10W (▴), human insulin (•), and B10R (▾). Data points are means ± SEM (n = 3).
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pone-0029198-g003: Representative dissociation curves of [125I]-labelled insulin or analogue from BHK-hIR cells.Dissociation was measured at different time points and the residual binding expressed as a percentage of initial binding. Dissociation of (A) B10D (♦) and B10E (▪); (B) B10W (▴), human insulin (•), and B10R (▾). Data points are means ± SEM (n = 3).

Mentions: The dissociation rate constants from the insulin receptor were determined using BHK cells overexpressing the human IR-A. Dissociation of [125I]-labelled human insulin or insulin analogue was determined as a function of time in the presence of an excess of unlabelled insulin. Representative dissociation time courses are presented in Figure 3 and the results and the dissociation rate constants relative to human insulin (3.7±0.3×10−2 min−1) are listed in Table 1. Dissociation was measured over a period of 2.5 h; however, analogues B10D and B10E dissociated 5- and 7-fold slower from the receptor, respectively, compared to human insulin and dissociation was therefore measured over a 5 h period for these two analogues in order to get a better curve fit and thereby a more precise determination of the dissociation rate constant.


Systematic evaluation of the metabolic to mitogenic potency ratio for B10-substituted insulin analogues.

Glendorf T, Knudsen L, Stidsen CE, Hansen BF, Hegelund AC, Sørensen AR, Nishimura E, Kjeldsen T - PLoS ONE (2012)

Representative dissociation curves of [125I]-labelled insulin or analogue from BHK-hIR cells.Dissociation was measured at different time points and the residual binding expressed as a percentage of initial binding. Dissociation of (A) B10D (♦) and B10E (▪); (B) B10W (▴), human insulin (•), and B10R (▾). Data points are means ± SEM (n = 3).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3285154&req=5

pone-0029198-g003: Representative dissociation curves of [125I]-labelled insulin or analogue from BHK-hIR cells.Dissociation was measured at different time points and the residual binding expressed as a percentage of initial binding. Dissociation of (A) B10D (♦) and B10E (▪); (B) B10W (▴), human insulin (•), and B10R (▾). Data points are means ± SEM (n = 3).
Mentions: The dissociation rate constants from the insulin receptor were determined using BHK cells overexpressing the human IR-A. Dissociation of [125I]-labelled human insulin or insulin analogue was determined as a function of time in the presence of an excess of unlabelled insulin. Representative dissociation time courses are presented in Figure 3 and the results and the dissociation rate constants relative to human insulin (3.7±0.3×10−2 min−1) are listed in Table 1. Dissociation was measured over a period of 2.5 h; however, analogues B10D and B10E dissociated 5- and 7-fold slower from the receptor, respectively, compared to human insulin and dissociation was therefore measured over a 5 h period for these two analogues in order to get a better curve fit and thereby a more precise determination of the dissociation rate constant.

Bottom Line: Insulin analogues comprising acidic amino acid substitutions at position B10 have previously been shown to display increased mitogenic potencies compared to human insulin and the underlying molecular mechanisms have been subject to much scrutiny and debate.Several B10-substituted insulin analogues devoid of disproportionate increases in mitogenic compared to metabolic potencies were identified.Our results also suggest that the increased mitogenic potency is attributable to both insulin and IGF-I receptor activation.

View Article: PubMed Central - PubMed

Affiliation: Diabetes Research Unit, Novo Nordisk A/S, Maaloev, Denmark. tgle@novonordisk.com

ABSTRACT

Background: Insulin analogues comprising acidic amino acid substitutions at position B10 have previously been shown to display increased mitogenic potencies compared to human insulin and the underlying molecular mechanisms have been subject to much scrutiny and debate. However, B10 is still an attractive position for amino acid substitutions given its important role in hexamer formation. The aim of this study was to investigate the relationships between the receptor binding properties as well as the metabolic and mitogenic potencies of a series of insulin analogues with different amino acid substitutions at position B10 and to identify a B10-substituted insulin analogue without an increased mitogenic to metabolic potency ratio.

Methodology/principal findings: A panel of ten singly-substituted B10 insulin analogues with different amino acid side chain characteristics were prepared and insulin receptor (both isoforms) and IGF-I receptor binding affinities using purified receptors, insulin receptor dissociation rates using BHK cells over-expressing the human insulin receptor, metabolic potencies by lipogenesis in isolated rat adipocytes, and mitogenic potencies using two different cell types predominantly expressing either the insulin or the IGF-I receptor were systematically investigated. Only analogues B10D and B10E with significantly increased insulin and IGF-I receptor affinities as well as decreased insulin receptor dissociation rates displayed enhanced mitogenic potencies in both cell types employed. For the remaining analogues with less pronounced changes in receptor affinities and insulin receptor dissociation rates, no apparent correlation between insulin receptor occupancy time and mitogenicity was observed.

Conclusions/significance: Several B10-substituted insulin analogues devoid of disproportionate increases in mitogenic compared to metabolic potencies were identified. In the present study, receptor binding affinity rather than insulin receptor off-rate appears to be the major determinant of both metabolic and mitogenic potency. Our results also suggest that the increased mitogenic potency is attributable to both insulin and IGF-I receptor activation.

Show MeSH
Related in: MedlinePlus