Recombinant Insulin-Like Growth Factor 1 Dimers: Receptor Binding Affinities and Activation Abilities.

Insulin-like growth factor 1 (IGF-1) and its IGF-1 receptor (IGF-1R) belong to an important biological system that is involved in the regulation of normal growth, but that has also been recognized as playing a role in cancer. IGF-1R antagonists could be interesting for the testing of their potential antiproliferative properties as an alternative to IGF-1R tyrosine-kinase inhibitors or anti-IGF-1R monoclonal antibodies. In this study, we were inspired by the successful development of insulin dimers capable of antagonizing insulin effects on the insulin receptor (IR) by simultaneous binding to two separated binding sites and by blocking structural rearrangement of the IR. We designed and produced in Escherichia coli three different IGF-1 dimers in which IGF-1 monomers are interlinked through their N- and C-termini, with linkers having 8, 15 or 25 amino acids. We found that the recombinant products were susceptible to the formation of misfolded or reduced variants, but that some of them were able to bind IGF-1R in low nanomolar affinities and all of them activate IGF-1R proportionally to their binding affinities. Overall, our work can be considered as a pilot study that, although it did not lead to the discovery of new IGF-1R antagonists, explored the possibility of recombinant production of IGF-1 dimers and led to the preparation of active compounds. This work could inspire further studies dealing, for example, with the preparation of IGF-1 conjugates with specific proteins for the study of the hormone and its receptor or for therapeutic applications. Supplementary Information: The online version contains supplementary material available at 10.1007/s10989-023-10499-1.

Characterization of insulin crystalline form in isolated ß-cell secretory granules.

Insulin is stored in vivo inside the pancreatic ß-cell insulin secretory granules. In vitro studies have led to an assumption that high insulin and Zn2+ concentrations inside the pancreatic ß-cell insulin secretory granules should promote insulin crystalline state in the form of Zn2+-stabilized hexamers. Electron microscopic images of thin sections of the pancreatic ß-cells often show a dense, regular pattern core, suggesting the presence of insulin crystals. However, the structural features of the storage forms of insulin in native preparations of secretory granules are unknown, because of their small size, fragile character and difficult handling. We isolated and investigated the secretory granules from MIN6 cells under near-native conditions, using cryo-electron microscopic (Cryo-EM) techniques. The analysis of these data from multiple intra-granular crystals revealed two different rhomboidal crystal lattices. The minor lattice has unit cell parameters (a ≃ b ≃ 84.0 Å, c ≃ 35.2 Å), similar to in vitro crystallized human 4Zn2+-insulin hexamer, whereas the largely prevalent unit cell has more than double c-axis (a ≃ b ≃ c ≃ 96.5 Å) that probably corresponds to two or three insulin hexamers in the asymmetric unit. Our experimental data show that insulin can be present in pancreatic MIN6 cell granules in a microcrystalline form, probably consisting of 4Zn2+-hexamers of this hormone.

A radioligand receptor binding assay for measuring of insulin secreted by MIN6 cells after stimulation with glucose, arginine, ornithine, dopamine, and serotonin.

We adapted a radioligand receptor binding assay for measuring insulin levels in unknown samples. The assay enables rapid and accurate determination of insulin concentrations in experimental samples, such as from insulin-secreting cells. The principle of the method is based on the binding competition of insulin in a measured sample with a radiolabeled insulin for insulin receptor (IR) in IM-9 cells. Both key components, radiolabeled insulin and IM-9 cells, are commercially available. The IR binding assay was used to determine unknown amounts of insulin secreted by MIN6 ß cell line after stimulation with glucose, arginine, ornithine, dopamine, and serotonin. The experimental data obtained by the IR binding assay were compared to the results determined by RIA kits and both methods showed a very good agreement of results. We observed the stimulation of glucose-induced insulin secretion from MIN6 cells by arginine, weaker stimulation by ornithine, but inhibitory effects of dopamine. Serotonin effects were either stimulatory or inhibitory, depending on the concentration of serotonin used. The results will require further investigation. The study also clearly revealed advantages of the IR binding assay that allows the measuring of a higher throughput of measured samples, with a broader range of concentrations than in the case of RIA kits. The IR binding assay can provide an alternative to standard RIA and ELISA assays for the determination of insulin levels in experimental samples and can be especially useful in scientific laboratories studying insulin production and secretion by ß cells and searching for new modulators of insulin secretion.

Regulatory mechanism of chicken lysozyme gene expression in oviducts examined using transgenic technology.

The use of promoters that strongly express target genes in the chicken oviduct is beneficial for the production of proteinaceous materials into egg white by transgenic chickens. To examine the regulatory mechanisms of chicken lysozyme gene expression in vivo, genetically manipulated chickens that express human erythropoietin under the control of a lysozyme promoter-enhancer were established. By using several deletion mutants of the promoter-flanking region, we found that a -1.9 kb DNase I hypersensitive site (DHS) was essential for oviduct-specific expression in genetically manipulated chickens. The concentration of human erythropoietin in egg white was 14-75 µg/ml, suggesting that the chicken lysozyme promoter containing -1.9 kb DHS is sufficient for the production of pharmaceuticals using transgenic chickens.

The efficiency of insulin production and its content in insulin-expressing model ß-cells correlate with their Zn2+ levels.

Insulin is produced and stored inside the pancreatic ß-cell secretory granules, where it is assumed to form Zn2+-stabilized oligomers. However, the actual storage forms of this hormone and the impact of zinc ions on insulin production in vivo are not known. Our initial X-ray fluorescence experiment on granules from native Langerhans islets and insulinoma-derived INS-1E cells revealed a considerable difference in the zinc content. This led our further investigation to evaluate the impact of the intra-granular Zn2+ levels on the production and storage of insulin in different model ß-cells. Here, we systematically compared zinc and insulin contents in the permanent INS-1E and BRIN-BD11 ß-cells and in the native rat pancreatic islets by flow cytometry, confocal microscopy, immunoblotting, specific messenger RNA (mRNA) and total insulin analysis. These studies revealed an impaired insulin production in the permanent ß-cell lines with the diminished intracellular zinc content. The drop in insulin and Zn2+ levels was paralleled by a lower expression of ZnT8 zinc transporter mRNA and hampered proinsulin processing/folding in both permanent cell lines. To summarize, we showed that the disruption of zinc homeostasis in the model ß-cells correlated with their impaired insulin and ZnT8 production. This indicates a need for in-depth fundamental research about the role of zinc in insulin production and storage.

Expression of interferon-inducible transmembrane proteins in the chicken and possible role in prevention of viral infections.

In mammals, interferon-inducible transmembrane proteins (IFITMs) prevent infections by various enveloped viruses. The expression of IFITMs in chicken was herein examined in the adult and embryonic organs using a quantitative reverse-transcription-polymerase chain reaction. The results obtained revealed that IFITM3 was expressed at a higher level than IFITM1, 2 and 5, in both embryonic and adult organs. However, the expression levels of IFITMs in embryonic organs were less than 5 % of those in adult lungs. Among the embryonic tissues examined, primordial germ cells (PGCs) at day 2.5 expressed relatively higher levels of IFITM3. IFITM3 expression levels were 1.5-fold higher in the chicken cell line DF-1 than in PGCs. The knockdown of IFITM3 in DF-1 cells by siRNA increased the infectivity of a vesicular stomatitis virus G protein-pseudotyped lentiviral vector, suggesting that lower levels of IFITM3 are still sufficient to restrict this viral vector.

Analyses of chicken sialyltransferases related to O-glycosylation.

The chicken ß-galactoside α2,3-sialyltransferase 1, 2, and 5 (ST3Gal1, 2, and 5) genes were cloned, and their enzymes were expressed in 293FT cells. ST3Gal1 and 2 exhibited enzymatic activities toward galactose-ß1,3-N-acetylgalactosamine and galactose-ß1,3-N-acetylglucosamine. ST3Gal5 only exhibited activity toward lactosylceramide. ST3Gal1 and 2 and previously cloned ST3Gal3 and 6 transferred CMP-sialic acid to asialofetuin. Reverse-transcription-quantitative PCR indicated that ST3Gal1 was expressed at higher levels in the trachea, lung, spleen, and magnum, and the strong expression of ST3Gal5 was observed in the spleen, magnum, and small and large intestines. ST3Gal1, 5, and 6 were also expressed in the tubular gland cells of the magnum, which secretes egg-white proteins. ST3Gal1, 5, and 6 were expressed in the egg chorioallantoic membrane, in which influenza viruses are propagated for the production of vaccines.