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.

• Klíčová slova
• Biased signaling, IGF-1, IGF1 receptor, IGF1 receptor inhibition, Insulin, Iridoviridae, Viral insulin/IGF-1 like peptides (VILPs),
• MeSH
• elektronová kryomikroskopie MeSH
• exprese genu MeSH
• fosfatidylinositol-3-kinasy metabolismus   MeSH
• fosforylace MeSH
• insulinu podobný růstový faktor I * genetika   metabolismus   MeSH
• inzulin metabolismus   MeSH
• lidé MeSH
• myši MeSH
• protein - isoformy metabolismus   MeSH
• protoonkogenní proteiny c-akt metabolismus   MeSH
• receptor IGF typ 1 * genetika   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• fosfatidylinositol-3-kinasy MeSH
• IGF1R protein, human MeSH Prohlížeč
• insulinu podobný růstový faktor I * MeSH
• inzulin MeSH
• protein - isoformy MeSH
• protoonkogenní proteiny c-akt MeSH
• receptor IGF typ 1 * MeSH

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.

• Klíčová slova
• mutagenesis in vitro, peptide hormone, receptor modification, receptor tyrosine kinase, structure–function,
• MeSH
• elektronová kryomikroskopie MeSH
• insulinu podobný růstový faktor I genetika   chemie   metabolismus   MeSH
• inzulin metabolismus   MeSH
• ligandy MeSH
• receptor IGF typ 1 * genetika   chemie   metabolismus   MeSH
• receptor inzulinu * genetika   chemie   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• insulinu podobný růstový faktor I MeSH
• inzulin MeSH
• ligandy MeSH
• receptor IGF typ 1 * MeSH
• receptor inzulinu * MeSH

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.

• MeSH
• buněčný cyklus MeSH
• dospělí MeSH
• insulinu podobný růstový faktor II * MeSH
• lidé MeSH
• mezibuněčné signální peptidy a proteiny * MeSH
• mitogeny MeSH
• proliferace buněk MeSH
• Check Tag
• dospělí MeSH
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• IGF2 protein, human MeSH Prohlížeč
• insulinu podobný růstový faktor II * MeSH
• mezibuněčné signální peptidy a proteiny * MeSH
• mitogeny MeSH

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.

• Klíčová slova
• antagonism, dicarba, disulfide mimetics, insulin mimetic peptide, insulin receptor, staple, triazole,
• MeSH
• disulfidy chemie   MeSH
• inzulin * metabolismus   MeSH
• peptidy chemie   MeSH
• receptor inzulinu * MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• disulfidy MeSH
• inzulin * MeSH
• peptidy MeSH
• receptor inzulinu * MeSH

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.

• Klíčová slova
• Binding site, Hormone analog, IGF-1, IGF-2, Insulin, Insulin receptor, Mutagenesis, Structure,
• MeSH
• elektronová kryomikroskopie MeSH
• insulinu podobný růstový faktor I genetika   MeSH
• insulinu podobný růstový faktor II * genetika   MeSH
• inzulin * MeSH
• kinetika MeSH
• lidé MeSH
• mutace MeSH
• vazebná místa MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• insulinu podobný růstový faktor I MeSH
• insulinu podobný růstový faktor II * MeSH
• inzulin * MeSH

BACKGROUND AND OBJECTIVES: Declines in stroke admission, IV thrombolysis (IVT), and mechanical thrombectomy volumes were reported during the first wave of the COVID-19 pandemic. There is a paucity of data on the longer-term effect of the pandemic on stroke volumes over the course of a year and through the second wave of the pandemic. We sought to measure the effect of the COVID-19 pandemic on the volumes of stroke admissions, intracranial hemorrhage (ICH), IVT, and mechanical thrombectomy over a 1-year period at the onset of the pandemic (March 1, 2020, to February 28, 2021) compared with the immediately preceding year (March 1, 2019, to February 29, 2020). METHODS: We conducted a longitudinal retrospective study across 6 continents, 56 countries, and 275 stroke centers. We collected volume data for COVID-19 admissions and 4 stroke metrics: ischemic stroke admissions, ICH admissions, IVT treatments, and mechanical thrombectomy procedures. Diagnoses were identified by their ICD-10 codes or classifications in stroke databases. RESULTS: There were 148,895 stroke admissions in the 1 year immediately before compared with 138,453 admissions during the 1-year pandemic, representing a 7% decline (95% CI [95% CI 7.1-6.9]; p < 0.0001). ICH volumes declined from 29,585 to 28,156 (4.8% [5.1-4.6]; p < 0.0001) and IVT volume from 24,584 to 23,077 (6.1% [6.4-5.8]; p < 0.0001). Larger declines were observed at high-volume compared with low-volume centers (all p < 0.0001). There was no significant change in mechanical thrombectomy volumes (0.7% [0.6-0.9]; p = 0.49). Stroke was diagnosed in 1.3% [1.31-1.38] of 406,792 COVID-19 hospitalizations. SARS-CoV-2 infection was present in 2.9% ([2.82-2.97], 5,656/195,539) of all stroke hospitalizations. DISCUSSION: There was a global decline and shift to lower-volume centers of stroke admission volumes, ICH volumes, and IVT volumes during the 1st year of the COVID-19 pandemic compared with the prior year. Mechanical thrombectomy volumes were preserved. These results suggest preservation in the stroke care of higher severity of disease through the first pandemic year. TRIAL REGISTRATION INFORMATION: This study is registered under NCT04934020.

• MeSH
• cévní mozková příhoda * epidemiologie   terapie   diagnóza   MeSH
• COVID-19 * epidemiologie   terapie   MeSH
• intrakraniální krvácení MeSH
• ischemie mozku * terapie   MeSH
• lidé MeSH
• následné studie MeSH
• pandemie MeSH
• retrospektivní studie MeSH
• SARS-CoV-2 MeSH
• tepový objem MeSH
• trombektomie MeSH
• trombolytická terapie metody   MeSH
• výsledek terapie MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• klinická studie MeSH

Insulin is stored in vivo inside the pancreatic β-cell insulin secretory granules. In vitro studies have led to an assumption that high insulin and Zn2+ concentrations inside the pancreatic β-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 β-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.

• Klíčová slova
• crystallization in vivo, electron microscopy, insulin secretion, peptide hormone, secretory granules, subcellular vesicle,
• MeSH
• beta-buňky * MeSH
• elektronová mikroskopie MeSH
• inzulin MeSH
• Langerhansovy ostrůvky * MeSH
• lidé MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• inzulin MeSH

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.

• MeSH
• insulinu podobný růstový faktor II * chemie   MeSH
• kosti a kostní tkáň MeSH
• lidé MeSH
• peptidové fragmenty chemie   MeSH
• peptidy MeSH
• vápník * MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• insulinu podobný růstový faktor II * MeSH
• peptidové fragmenty MeSH
• peptidy MeSH
• preptin MeSH Prohlížeč
• vápník * MeSH

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.

• MeSH
• CD antigeny chemie   metabolismus   MeSH
• fosforylace MeSH
• inzulin analogy a deriváty   metabolismus   MeSH
• inzulinová rezistence MeSH
• kalorimetrie metody   MeSH
• lidé MeSH
• myši inbrední C57BL MeSH
• myši MeSH
• protein - isoformy chemie   metabolismus   MeSH
• proteinové agregáty * MeSH
• receptor inzulinu chemie   metabolismus   MeSH
• sekvence aminokyselin MeSH
• vazba proteinů MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• CD antigeny MeSH
• INSR protein, human MeSH Prohlížeč
• inzulin MeSH
• protein - isoformy MeSH
• proteinové agregáty * MeSH
• receptor inzulinu MeSH

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 β 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 β cells and searching for new modulators of insulin secretion.

• Klíčová slova
• Binding assay, Insulin receptor, Insulin secretion, Radioligand, Secretagogue, β Cells,
• MeSH
• arginin metabolismus   MeSH
• beta-buňky metabolismus   MeSH
• buněčné linie MeSH
• dopamin metabolismus   MeSH
• glukosa metabolismus   MeSH
• inzulin analýza   metabolismus   MeSH
• krysa rodu Rattus MeSH
• Langerhansovy ostrůvky metabolismus   MeSH
• lidé MeSH
• myši MeSH
• ornithin metabolismus   MeSH
• potkani Wistar MeSH
• radioimunoanalýza metody   MeSH
• radioligandová zkouška metody   MeSH
• sekrece inzulinu * MeSH
• serotonin metabolismus   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• arginin MeSH
• dopamin MeSH
• glukosa MeSH
• inzulin MeSH
• ornithin MeSH
• serotonin MeSH