Combinatorial Libraries of Bipodal Binders of the Insulin Receptor.

The binding process of insulin to its transmembrane receptor entails a sophisticated interplay between two proteins, each possessing two binding sites. Given the difficulties associated with the use of insulin in the treatment of diabetes, despite its remarkable efficacy, there is interest in smaller and more stable compounds than the native hormone that would effectively activate the receptor. Our study adopts a strategy focused on synthesizing extensive combinatorial libraries of bipodal compounds consisting of two distinct peptides linked to a molecular scaffold. These constructs, evaluated in a resin bead-bound format, were designed to assess their binding to the insulin receptor. Despite notable nonspecific binding, our approach successfully generated and tested millions of compounds. Rigorous evaluations via flow cytometry and specific antibodies revealed peptide sequences with specific interactions at either receptor binding Site 1 or 2. Notably, these sequences bear similarity to peptides discovered through phage display by other researchers. This convergence of chemical and biological methods underscores nature's beauty, revealing general principles in peptide binding to the insulin receptor. Overall, our study deepens the understanding of molecular interactions in ligand binding to the insulin receptor, highlighting the challenges of targeting large proteins with small synthetic peptides.

A viral insulin-like peptide inhibits IGF-1 receptor phosphorylation and regulates IGF1R gene expression.

OBJECTIVE: The insulin/IGF superfamily is conserved across vertebrates and invertebrates. Our team has identified five viruses containing genes encoding viral insulin/IGF-1 like peptides (VILPs) closely resembling human insulin and IGF-1. This study aims to characterize the impact of Mandarin fish ranavirus (MFRV) and Lymphocystis disease virus-Sa (LCDV-Sa) VILPs on the insulin/IGF system for the first time. METHODS: We chemically synthesized single chain (sc, IGF-1 like) and double chain (dc, insulin like) forms of MFRV and LCDV-Sa VILPs. Using cell lines overexpressing either human insulin receptor isoform A (IR-A), isoform B (IR-B) or IGF-1 receptor (IGF1R), and AML12 murine hepatocytes, we characterized receptor binding, insulin/IGF signaling. We further characterized the VILPs' effects of proliferation and IGF1R and IR gene expression, and compared them to native ligands. Additionally, we performed insulin tolerance test in CB57BL/6 J mice to examine in vivo effects of VILPs on blood glucose levels. Finally, we employed cryo-electron microscopy (cryoEM) to analyze the structure of scMFRV-VILP in complex with the IGF1R ectodomain. RESULTS: VILPs can bind to human IR and IGF1R, stimulate receptor autophosphorylation and downstream signaling pathways. Notably, scMFRV-VILP exhibited a particularly strong affinity for IGF1R, with a mere 10-fold decrease compared to human IGF-1. At high concentrations, scMFRV-VILP selectively reduced IGF-1 stimulated IGF1R autophosphorylation and Erk phosphorylation (Ras/MAPK pathway), while leaving Akt phosphorylation (PI3K/Akt pathway) unaffected, indicating a potential biased inhibitory function. Prolonged exposure to MFRV-VILP led to a significant decrease in IGF1R gene expression in IGF1R overexpressing cells and AML12 hepatocytes. Furthermore, insulin tolerance test revealed scMFRV-VILP's sustained glucose-lowering effect compared to insulin and IGF-1. Finally, cryo-EM analysis revealed that scMFRV-VILP engages with IGF1R in a manner closely resembling IGF-1 binding, resulting in a highly analogous structure. CONCLUSIONS: This study introduces MFRV and LCDV-Sa VILPs as novel members of the insulin/IGF superfamily. Particularly, scMFRV-VILP exhibits a biased inhibitory effect on IGF1R signaling at high concentrations, selectively inhibiting IGF-1 stimulated IGF1R autophosphorylation and Erk phosphorylation, without affecting Akt phosphorylation. In addition, MFRV-VILP specifically regulates IGF-1R gene expression and IGF1R protein levels without affecting IR. CryoEM analysis confirms that scMFRV-VILP' binding to IGF1R is mirroring the interaction pattern observed with IGF-1. These findings offer valuable insights into IGF1R action and inhibition, suggesting potential applications in development of IGF1R specific inhibitors and advancing long-lasting insulins.

Insulin-Dendrimer Nanocomplex for Multi-Day Glucose-Responsive Therapy in Mice and Swine.

The management of diabetes in a manner offering autonomous insulin therapy responsive to glucose-directed need, and moreover with a dosing schedule amenable to facile administration, remains an ongoing goal to improve the standard of care. While basal insulins with reduced dosing frequency, even once-weekly administration, are on the horizon, there is still no approved therapy that offers glucose-responsive insulin function. Herein, a nanoscale complex combining both electrostatic- and dynamic-covalent interactions between a synthetic dendrimer carrier and an insulin analogue modified with a high-affinity glucose-binding motif yields an injectable insulin depot affording both glucose-directed and long-lasting insulin availability. Following a single injection, it is even possible to control blood glucose for at least one week in diabetic swine subjected to daily oral glucose challenges. Measurements of serum insulin concentration in response to challenge show increases in insulin corresponding to elevated blood glucose levels, an uncommon finding even in preclinical work on glucose-responsive insulin. Accordingly, the subcutaneous nanocomplex that results from combining electrostatic- and dynamic-covalent interactions between a modified insulin and a synthetic dendrimer carrier affords a glucose-responsive insulin depot for week-long control following a single routine injection.

Insulin receptor Arg717 and IGF-1 receptor Arg704 play a key role in ligand binding and in receptor activation.

The insulin receptor (IR, with its isoforms IR-A and IR-B) and the insulin-like growth factor 1 receptor (IGF-1R) are related tyrosine kinase receptors. Recently, the portfolio of solved hormone-receptor structures has grown extensively thanks to advancements in cryo-electron microscopy. However, the dynamics of how these receptors transition between their inactive and active state are yet to be fully understood. The C-terminal part of the alpha subunit (αCT) of the receptors is indispensable for the formation of the hormone-binding site. We mutated the αCT residues Arg717 and His710 of IR-A and Arg704 and His697 of IGF-1R. We then measured the saturation binding curves of ligands on the mutated receptors and their ability to become activated. Mutations of Arg704 and His697 to Ala in IGF-1R decreased the binding of IGF-1. Moreover, the number of binding sites for IGF-1 on the His697 IGF-1R mutant was reduced to one-half, demonstrating the presence of two binding sites. Both mutations of Arg717 and His710 to Ala in IR-A inactivated the receptor. We have proved that Arg717 is important for the binding of insulin to its receptor, which suggests that Arg717 is a key residue for the transition to the active conformation.

Mutations at hypothetical binding site 2 in insulin and insulin-like growth factors 1 and 2.

Elucidating how insulin and the related insulin-like growth factors 1 and 2 (IGF-1 and IGF-2) bind to their cellular receptors (IR and IGF-1R) and how the receptors are activated has been the holy grail for generations of scientists. However, deciphering the 3D structure of tyrosine kinase receptors and their hormone-bound complexes has been complicated by the flexible and dimeric nature of the receptors and the dynamic nature of their interaction with hormones. Therefore, mutagenesis of hormones and kinetic studies first became an important tool for studying receptor interactions. It was suggested that hormones could bind to receptors through two binding sites on the hormone surface called site 1 and site 2. A breakthrough in knowledge came with the solution of cryoelectron microscopy (cryoEM) structures of hormone-receptor complexes. In this chapter, we document in detail the mutagenesis of insulin, IGF-1, and IGF-2 with emphasis on modifications of the hypothetical binding site 2 in the hormones, and we discuss the results of structure-activity studies in light of recent cryoEM structures of hormone complexes with IR and IGF-1R.

Non-glycosylated IGF2 prohormones are more mitogenic than native IGF2.

Insulin-like Growth Factor-2 (IGF2) is important for the regulation of human embryonic growth and development, and for adults' physiology. Incorrect processing of the IGF2 precursor, pro-IGF2(156), leads to the formation of two IGF2 proforms, big-IGF2(87) and big-IGF2(104). Unprocessed and mainly non-glycosylated IGF2 proforms are found at abnormally high levels in certain diseases, but their mode of action is still unclear. Here, we found that pro-IGF2(156) has the lowest ability to form its inactivating complexes with IGF-Binding Proteins and has higher proliferative properties in cells than IGF2 and other IGF prohormones. We also showed that big-IGF2(104) has a seven-fold higher binding affinity for the IGF2 receptor than IGF2, and that pro-IGF2(87) binds and activates specific receptors and stimulates cell growth similarly to the mature IGF2. The properties of these pro-IGF2 forms, especially of pro-IGF2(156) and big-IGF2(104), indicate them as hormones that may be associated with human diseases related to the accumulation of IGF-2 proforms in the circulation.

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.

Modulation of the antagonistic properties of an insulin mimetic peptide by disulfide bridge modifications.

Insulin is a peptide responsible for regulating the metabolic homeostasis of the organism; it elicits its effects through binding to the transmembrane insulin receptor (IR). Insulin mimetics with agonistic or antagonistic effects toward the receptor are an exciting field of research and could find applications in treating diabetes or malignant diseases. We prepared five variants of a previously reported 20-amino acid insulin-mimicking peptide. These peptides differ from each other by the structure of the covalent bridge connecting positions 11 and 18. In addition to the peptide with a disulfide bridge, a derivative with a dicarba bridge and three derivatives with a 1,2,3-triazole differing from each other by the presence of sulfur or oxygen in their staples were prepared. The strongest binding to IR was exhibited by the peptide with a disulfide bridge. All other derivatives only weakly bound to IR, and a relationship between increasing bridge length and lower binding affinity can be inferred. Despite their nanomolar affinities, none of the prepared peptide mimetics was able to activate the insulin receptor even at high concentrations, but all mimetics were able to inhibit insulin-induced receptor activation. However, the receptor remained approximately 30% active even at the highest concentration of the agents; thus, the agents behave as partial antagonists. An interesting observation is that these mimetic peptides do not antagonize insulin action in proportion to their binding affinities. The compounds characterized in this study show that it is possible to modulate the functional properties of insulin receptor peptide ligands using disulfide mimetics.

Targeting the insulin receptor with hormone and peptide dimers.

Insulin is a key hormone involved in the regulation of overall energetic homeostasis of the organism. The dimeric character of the receptor for insulin evokes ideas about its activation or inhibition with peptide dimers that could either trigger or block the structural transition of the insulin receptor, leading to its activation. Herewith, we present the chemical engineering and biological characterization of several series of insulin dimers or dimers of specific peptides that should be able to bind receptors for insulin or insulin growth factor 1. The hormones or peptides in the dimers were interconnected with different linkers, consisting of triazole moieties and 3, 6, 8, 11, or 23 polyethylene glycol units. The prepared dimers were weaker in binding to insulin receptors than human insulin. However, some of the insulin dimers showed preferential binding specificity toward the isoform A of the insulin receptor, and the insulin dimers also stimulated the insulin receptor more strongly than would be consistent with their binding affinities. Our results suggest that designing insulin dimers may be a promising strategy for modulating the ability of the hormone to activate the receptor or to alter its specificity toward insulin receptor isoforms.

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.

Chiral analysis of ß-alanyl-d,l-tyrosine and its derivatives and estimation of binding constants of their complexes with 2-hydroxypropyl-ß-cyclodextrin by capillary electrophoresis.

Chiral CE methods were developed for the elucidation of l- or d-configuration of tyrosine residue in antimicrobial dipeptide ß-alanyl-tyrosine (ß-Ala-Tyr) isolated from the hemolymph of larvae of fleshfly Neobellieria bullata and for the evaluation of enantiopurity of its synthetic isomers (ß-Ala-d-Tyr and ß-Ala-l-Tyr), and enantiomers of their amidated and acetylated derivatives, ß-Ala-d,l-Tyr-NH2 and N-Ac-ß-Ala-d,l-Tyr, respectively. Baseline separations were achieved for all three pairs of enantiomers: (i) for ß-Ala-d,l-Tyr in acidic background electrolyte composed of 32/50 mM tris(hydroxymethyl)aminomethane/H3 PO4 , pH 2.5, and 20 mg/mL 2-hydroxypropyl-ß-cyclodextrin as chiral selector; (ii) for ß-Ala-d,l-Tyr-NH2 enantiomers in acidic background electrolyte consisting of 48/50 mM tris(hydroxymethyl)aminomethane/H3 PO4 , pH 3.5, and 30 mg/mL 2-hydroxypropyl-ß-cyclodextrin; and (iii) for enantiomers of N-Ac-ß-Ala-d,l-Tyr in alkaline background electrolyte composed of 50/49 mM Na2 B4 O7 /NaOH, pH 10.5, and 60 mg/mL 2-hydroxypropyl-ß-cyclodextrin. From CE analyses of mixed samples of isolated ß-Ala-Tyr and synthetic standards ß-Ala-l-Tyr and ß-Ala-d-Tyr, it turned out that isolated ß-Ala-Tyr was pure l-enantiomer. In addition, the average apparent binding constants, Kb , and average actual ionic mobilities of the complexes of ß-Ala-d,l-Tyr and its above derivatives with 2-hydroxypropyl-ß-cyclodextrin were determined. These complexes were weak, with Kb values ranging from 11.2 to 79.1 L/mol. Their cationic mobilities were equal to (5.6-9.2) × 10-9 m2 /V/s, and anionic mobilities to (-1.3-1.6) × 10-9 m2 /V/s.

Functional stapled fragments of human preptin of minimised length.

Preptin is a 34-amino-acid-long peptide derived from the E-domain of a precursor of insulin-like growth factor 2 (pro-IGF2) with bone-anabolic and insulin secretion amplifying properties. Here, we describe the synthesis, structures, and biological activities of six shortened analogues of human preptin. Eight- and nine-amino-acid-long peptide amides corresponding to the C-terminal part of human preptin were stabilised by two types of staples to induce a higher proportion of helicity in their secondary structure. We monitored the secondary structure of the stapled peptides using circular dichroism. The biological effect of the structural changes was determined afterwards by the ability of peptides to stimulate the release of intracellular calcium ions. We confirmed the previous observation that the stabilisation of the disordered conformation of human preptin has a deleterious effect on biological potency. However, surprisingly, one of our preptin analogues, a nonapeptide stabilised by olefin metathesis between positions 3 and 7 of the amino acid chain, had a similar ability to stimulate calcium ions' release to the full-length human preptin. Our findings could open up new ways to design new preptin analogues, which may have potential as drugs for the treatment of diabetes and osteoporosis.

Application of Capillary and Free-Flow Zone Electrophoresis for Analysis and Purification of Antimicrobial ß-Alanyl-Tyrosine from Hemolymph of Fleshfly Neobellieria bullata.

The problem of a growing resistance of bacteria and other microorganisms to conventional antibiotics gave rise to a search for new potent antimicrobial agents. Insect antimicrobial peptides (AMPs) seem to be promising novel potential anti-infective therapeutics. The dipeptide ß-alanyl-tyrosine (ß-Ala-Tyr) is one of the endogenous insect toxins exhibiting antibacterial activity against both Gram-negative and Gram-positive bacteria. Prior to testing its other antimicrobial activities, it has to be prepared in a pure form. In this study, we have developed a capillary zone electrophoresis (CZE) method for analysis of ß-Ala-Tyr isolated from the extract of the hemolymph of larvae of the fleshfly Neobellieria bullata by reversed-phase high-performance liquid chromatography (RP-HPLC). Based on our previously described correlation between CZE and free-flow zone electrophoresis (FFZE), analytical CZE separation of ß-Ala-Tyr and its admixtures have been converted into preparative purification of ß-Ala-Tyr by FFZE with preparative capacity of 45.5 mg per hour. The high purity degree of the ß-Ala-Tyr obtained by FFZE fractionation was confirmed by its subsequent CZE analysis.

Insulin Analogues with Altered Insulin Receptor Isoform Binding Specificities and Enhanced Aggregation Stabilities.

Insulin is a lifesaver for millions of diabetic patients. There is a need for new insulin analogues with more physiological profiles and analogues that will be thermally more stable than human insulin. Here, we describe the chemical engineering of 48 insulin analogues that were designed to have changed binding specificities toward isoforms A and B of the insulin receptor (IR-A and IR-B). We systematically modified insulin at the C-terminus of the B-chain, at the N-terminus of the A-chain, and at A14 and A18 positions. We discovered an insulin analogue that has Cα-carboxyamidated Glu at B31 and Ala at B29 and that has a more than 3-fold-enhanced binding specificity in favor of the "metabolic" IR-B isoform. The analogue is more resistant to the formation of insulin fibrils at 37 °C and is also more efficient in mice than human insulin. Therefore, [AlaB29,GluB31,amideB31]-insulin may be interesting for further clinical evaluation.

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.

Characterization of viral insulins reveals white adipose tissue-specific effects in mice.

OBJECTIVE: Members of the insulin/insulin-like growth factor (IGF) superfamily are well conserved across the evolutionary tree. We recently showed that four viruses in the Iridoviridae family possess genes that encode proteins highly homologous to human insulin/IGF-1. Using chemically synthesized single-chain (sc), i.e., IGF-1-like, forms of the viral insulin/IGF-1-like peptides (VILPs), we previously showed that they can stimulate human receptors. Because these peptides possess potential cleavage sites to form double chain (dc), i.e., more insulin-like, VILPs, in this study, we have characterized dc forms of VILPs for Grouper iridovirus (GIV), Singapore grouper iridovirus (SGIV) and Lymphocystis disease virus-1 (LCDV-1) for the first time. METHODS: The dcVILPs were chemically synthesized. Using murine fibroblast cell lines overexpressing insulin receptor (IR-A or IR-B) or IGF1R, we first determined the binding affinity of dcVILPs to the receptors and characterized post-receptor signaling. Further, we used C57BL/6J mice to study the effect of dcVILPs on lowering blood glucose. We designed a 3-h dcVILP in vivo infusion experiment to determine the glucose uptake in different tissues. RESULTS: GIV and SGIV dcVILPs bind to both isoforms of human insulin receptor (IR-A and IR-B) and to the IGF1R, and for the latter, show higher affinity than human insulin. These dcVILPs stimulate IR and IGF1R phosphorylation and post-receptor signaling in vitro and in vivo. Both GIV and SGIV dcVILPs stimulate glucose uptake in mice. In vivo infusion experiments revealed that while insulin (0.015 nmol/kg/min) and GIV dcVILP (0.75 nmol/kg/min) stimulated a comparable glucose uptake in heart and skeletal muscle and brown adipose tissue, GIV dcVILP stimulated 2-fold higher glucose uptake in white adipose tissue (WAT) compared to insulin. This was associated with increased Akt phosphorylation and glucose transporter type 4 (GLUT4) gene expression compared to insulin in WAT. CONCLUSIONS: Our results show that GIV and SGIV dcVILPs are active members of the insulin superfamily with unique characteristics. Elucidating the mechanism of tissue specificity for GIV dcVILP will help us to better understand insulin action, design new analogs that specifically target the tissues and provide new insights into their potential role in disease.

Multipodal insulin mimetics built on adamantane or proline scaffolds.

Multi-orthogonal molecular scaffolds can be applied as core structures of bioactive compounds. Here, we prepared four tri-orthogonal scaffolds based on adamantane or proline skeletons. The scaffolds were used for the solid-phase synthesis of model insulin mimetics bearing two different peptides on the scaffolds. We found that adamantane-derived compounds bind to the insulin receptor more effectively (Kd value of 0.5 µM) than proline-derived compounds (Kd values of 15-38 µM) bearing the same peptides. Molecular dynamics simulations suggest that spacers between peptides and central scaffolds can provide greater flexibility that can contribute to increased binding affinity. Molecular modeling showed possible binding modes of mimetics to the insulin receptor. Our data show that the structure of the central scaffold and flexibility of attached peptides in this type of compound are important and that different scaffolds should be considered when designing peptide hormone mimetics.

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.

A radioligand binding assay for the insulin-like growth factor 2 receptor.

Insulin-like growth factors 2 and 1 (IGF2 and IGF1) and insulin are closely related hormones that are responsible for the regulation of metabolic homeostasis, development and growth of the organism. Physiological functions of insulin and IGF1 are relatively well-studied, but information about the role of IGF2 in the body is still sparse. Recent discoveries called attention to emerging functions of IGF2 in the brain, where it could be involved in processes of learning and memory consolidation. It was also proposed that these functions could be mediated by the receptor for IGF2 (IGF2R). Nevertheless, little is known about the mechanism of signal transduction through this receptor. Here we produced His-tagged domain 11 (D11), an IGF2-binding element of IGF2R; we immobilized it on the solid support through a well-defined sandwich, consisting of neutravidin, biotin and synthetic anti-His-tag antibodies. Next, we prepared specifically radiolabeled [125I]-monoiodotyrosyl-Tyr2-IGF2 and optimized a sensitive and robust competitive radioligand binding assay for determination of the nanomolar binding affinities of hormones for D11 of IGF2. The assay will be helpful for the characterization of new IGF2 mutants to study the functions of IGF2R and the development of new compounds for the treatment of neurological disorders.

Radiolabeled hormones in insulin research, a minireview.

Preparation of both 125 I-labeled insulin and insulin-like growth factor 1 (IGF-1) was critical because it enabled a detailed characterization of binding properties of these important hormones towards their cognate transmembrane receptors. Binding modes of hundreds of hormone derivatives were analyzed using competition radioligand binding assays. This effort has resulted in development of six insulin analogs that are today clinically used for the treatment of diabetes. Here, we will briefly summarize a history of insulin research employing iodinated hormones.