Systematic Evaluation of the Metabolic to Mitogenic Potency Ratio for B10-Substituted Insulin Analogues

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 i

Engineering of Insulin Receptor Isoform-Selective Insulin Analogues

BACKGROUND: The insulin receptor (IR) exists in two isoforms, A and B, and the isoform expression pattern is tissue-specific. The C-terminus of the insulin B chain is important for receptor binding and has been shown to contact the IR just adjacent to the region where the A and B isoforms differ. The aim of this study was to investigate the importance of the C-terminus of the B chain in IR isoform binding in order to explore the possibility of engineering tissue-specific/liver-specific insulin analogues. METHODOLOGY/PRINCIPAL FINDINGS: Insulin analogue libraries were constructed by total amino acid scanning mutagenesis. The relative binding affinities for the A and B isoform of the IR were determined by competition assays using scintillation proximity assay technology. Structural information was obtained by X-ray crystallography. Introduction of B25A or B25N mutations resulted in analogues with a 2-fold preference for the B compared to the A isoform, whereas the opposite was observed with a B25Y substitution. An acidic amino acid residue at position B27 caused an additional 2-fold selective increase in affinity for the receptor B isoform for analogues bearing a B25N mutation. Furthermore, the combination of B25H with either B27D or B27E also resulted in B isoform-preferential analogues (2-fold preference) even though the corresponding single mutation analogues displayed no differences in relative isoform binding affinity. CONCLUSIONS/SIGNIFICANCE: We have discovered a new class of IR isoform-selective insulin analogues with 2-4-fold differences in relative binding affinities for either the A or the B isoform of the IR compared to human insulin. Our results demonstrate that a mutation at position B25 alone or in combination with a mutation at position B27 in the insulin molecule confers IR isoform selectivity. Isoform-preferential analogues may provide new opportunities for developing insulin analogues with improved clinical benefits

Elective course planning

Efficient planning increasingly becomes an indispensable tool for management of both companies and public organizations. This is also the case for high school management in Denmark, because the growing individual freedom of the students to choose courses makes planning much more complex. Due to reforms, elective courses are today an important part of the curriculum, and elective courses are a good way to make high school education more attractive for the students. In this article, the problem of planning the elective courses is modeled using integer programming and three different solution approaches are suggested, including a Branch-and-Price framework using partial Dantzig-Wolfe decomposition. Explicit Constraint Branching is used to enhance the solution process, both on the original IP model and in the Branch-and-Price algorithm. To the best of our knowledge, no exact algorithm for the Elective Course Planning Problem has been described in the literature before. The proposed algorithms are tested on data sets from 98 of the 150 high schools in Denmark. The tests show that for the majority of the problems, the optimal solution can be obtained within the one hour time bound. Furthermore the suggested algorithms achieve better results than the currently applied meta-heuristic.Elective course planning High school planning Integer programming Dantzig-Wolfe decomposition Branch and Price Explicit Constraint Branching

Receptor-isoform-selective insulin analogues give tissue-preferential effects

International audienceThe relative expression patterns of the two insulin receptor isoforms, +/- exon11 (IR-B/A respectively), are tissue dependent. Therefore we have developed insulin analogues with different binding affinities for the two isoforms, to test whether tissue-preferential biological effects can be attained. In rats and mice, IR-B is the most prominent isoform in liver (<95%) and fat (<90%), whereas in muscles IR-A is the dominant isoform (<95%). As a consequence, insulin analogue INS-A, which has a higher relative affinity for human IR-A, had a higher relative potency (compared to human insulin, HI) for glycogen synthesis in rat muscle strips (26%) than for glycogen accumulation in rat hepatocytes (5%) and for lipogenesis in rat adipocytes (4%). In contrast, the INS-B analogue, which has an increased affinity for human IR-B, had higher relative potencies (compared to HI) for inducing glycogen accumulation (75%) and lipogenesis (130%) than for affecting muscle (45%). For the same blood glucose lowering effect upon acute i.v. dosing to mice, INS-B gave a significantly higher degree of IR phosphorylation in liver than HI. These in vitro and in vivo results indicate that insulin analogues with IR isoform-preferential binding affinity are able to elicit tissue-selective biological responses, depending on the IR-A/B expression

Relative receptor affinities, metabolic and mitogenic potencies, and IR off-rates [% of human insulin].

<p>All assays were performed in at least three independent experiments. Data are means ± SD and presented relative to human insulin. For human insulin, IR assay IC₅₀ values were in the picomolar affinity range, IGF-IR assay IC₅₀ values were in the nanomolar affinity range, rFFC assay EC₅₀ values were in the picomolar range, and mitogenic assay EC₅₀ values were in the nanomolar range (HMECs) and low nanomolar range (L6-hIR). The dissociation rate constant for human insulin was (3.7±0.3×10⁻² min⁻¹).</p