Converting Insulin-like Growth Factors 1 and 2 into High-Affinity Ligands for Insulin Receptor Isoform A by the Introduction of an Evolutionarily Divergent Mutation

Insulin-like growth factors 1 and 2 (IGF-1 and -2, respectively) are protein hormones involved not only in normal growth and development but also in life span regulation and cancer. They exert their functions mainly through the IGF-1R or by binding to isoform A of the insulin receptor (IR-A). The development of IGF-1 and IGF-2 antagonists is of great clinical interest. Mutations of A4 and A8 sites of human insulin lead to disproportionate effects on hormone IR binding and activation. Here, we systematically modified IGF-1 sites 45, 46, and 49 and IGF-2 sites 45 and 48, which correspond, or are close, to insulin sites A4 and A8. The IGF-1R and IR-A binding and autophosphorylation potencies of these analogues were characterized. They retained the main IGF-1R-related properties, but the hormones with His49 in IGF-1 and His48 in IGF-2 showed significantly higher affinities for IR-A and for IR-B, being the strongest IGF-1- and IGF-2-like binders of these receptors ever reported. All analogues activated IR-A and IGF-1R without major discrepancies in their binding affinities. This study revealed that IR-A and IGF-1R contain specific sites, likely parts of their so-called sites 2′, which can interact differently with specifically modified IGF analogues. Moreover, a clear importance of IGF-2 site 44 for effective hormone folding was also observed. These findings may facilitate novel and rational engineering of new hormone analogues for IR-A and IGF-1R studies and for potential medical applications

The efficiency of insulin production and its content in insulin-expressing model β-cells correlate with their Zn 2+ levels : Zn 2+ levels in β-cells

Insulin is produced and stored inside the pancreatic β-cell secretory granules, where it is assumed to form Zn 2+ -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 Zn 2+ levels on the production and storage of insulin in different model β-cells. Here, we systematically compared zinc and insulin contents in the permanent INS-1E and BRIN-BD11 β-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 β-cell lines with the diminished intracellular zinc content. The drop in insulin and Zn 2+ 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 β-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

Příprava a charakterizace prohormonů IGF2

Insulin-like growth factors 1 and 2 are protein hormones that share similar structural and functional features with insulin. They are commonly abbreviated as IGF1 and IGF2. They play important roles in prenatal development and throughout lifespan. Stimulation of growth of all cell types and significant metabolic effects also belong among their known functions. Dysregulation of their activity or elevated levels were observed in a number of diseases such as diabetes mellitus type 2, cancer, hyperthyroidism, amyotrophic lateral sclerosis, muscle dystrophy, acromegaly, Laron dwarfism and other disorders. Functions of IGF2 are less studied than functions of the other members of the family and even less is known about precursors of IGF2. To address these issues we started with production of IGF2 analogues with enhanced specificity to IR for better understanding the mechanism of receptor activation by IGF2. We produced site-specific analogues modified at sites important for receptor binding. IGF2 analogue with Phe48His mutation is the strongest IGF2-derived binder for IR-A reported ever and it is able to activate IR-A to about 60% of insulin ability. We developed new rapid, robust, and sensitive binding assays for testing of ligands of the receptor for IGF2 (IGF2R). First, we immobilized domain 11 of...Insulinu podobné růstové faktory 1 a 2 jsou proteinové hormony, které mají podobné strukturní a funkční vlastnosti s insulinem. Nejčastěji jsou zmiňovány zkratkami jako IGF1 a IGF2. Hrají podstatnou roli jak v prenatálním vývoji, tak i během celé doby života savců. Stimulace růstu všech typů buněk a výrazné metabolické efekty rovněž patří mezi jejich hlavní známé funkce. Deregulace jejich aktivity nebo jejich zvýšené hladiny byly nalezeny u mnoha různých nemocí jako je například diabetes mellitus 2. typu, rakovina, hyperfunkce štítné žlázy, amyotrofická laterální skleróza, svalová dystrofie, akromegalie, Laronův syndrom a další choroby. Funkce IGF2 jsou studovány méně než funkce ostatních členů proteinové rodiny, a ještě méně se ví o funkcích prekurzorů IGF2. Abychom přispěli k lepšímu porozumění funkcím prekurzorů IGF2, začali jsme s vývojem analogů IGF2 se zvýšenou specificitou vůči receptoru insulin (IR) receptoru. Vyprodukovali jsme specifické analogy, modifikované v pozicích důležitých pro vazbu na receptor. IGF2 analog s Phe48His mutací je nejsilnějším známým ligandem pro receptor IR-A zaleženým na molekule IGF2a je schopen aktivovat tento receptoru až do úrovně 60 % aktivity insulinu. Vyvinuli jsme nový, rychlý, robustní a citlivý vazebný protokol pro testování ligandů receptoru pro IGF2...Department of BiochemistryKatedra biochemiePřírodovědecká fakultaFaculty of Scienc

The preparation and characterization of IGF2 prohormones

Insulin-like growth factors 1 and 2 are protein hormones that share similar structural and functional features with insulin. They are commonly abbreviated as IGF1 and IGF2. They play important roles in prenatal development and throughout lifespan. Stimulation of growth of all cell types and significant metabolic effects also belong among their known functions. Dysregulation of their activity or elevated levels were observed in a number of diseases such as diabetes mellitus type 2, cancer, hyperthyroidism, amyotrophic lateral sclerosis, muscle dystrophy, acromegaly, Laron dwarfism and other disorders. Functions of IGF2 are less studied than functions of the other members of the family and even less is known about precursors of IGF2. To address these issues we started with production of IGF2 analogues with enhanced specificity to IR for better understanding the mechanism of receptor activation by IGF2. We produced site-specific analogues modified at sites important for receptor binding. IGF2 analogue with Phe48His mutation is the strongest IGF2-derived binder for IR-A reported ever and it is able to activate IR-A to about 60% of insulin ability. We developed new rapid, robust, and sensitive binding assays for testing of ligands of the receptor for IGF2 (IGF2R). First, we immobilized domain 11 of..

Prevention of diet-induced obesity in rats by oral application of collagen fragments

The aim of the present study was to determine whether orally applied collagen fragments (CFs) could affect the development of obesity in obese rats. To this end, experimental rats that were exposed to a high-calorie diet (HCD) for four weeks were randomly divided into two groups: HCD and HCD+CFs, with both groups continuing to receive the HCD. However, rats from the HCD+CFs group were also provided with CFs in a 0.05-M citrate buffer (pH 5.0) (1 g·kg-1 of body weight) by intragastric administration, every other day for the next six weeks. Selected parameters associated with obesity development and insulin resistance, as well as serum markers of oxidative stress and the cytokine profile were assessed at the end of the 10th week. Supplementation with CFs resulted in a decrease in body weight and body mass index when compared to animals exposed to a HCD. The observed changes were assumed to be caused by a lower food intake and increased water intake by obese rats treated with CFs. Enhanced activity of superoxide dismutase (SOD), catalase (CAT) and decreased malondialdehyde (MDA) concentration were detected in the HCD+CF group of animals when compared to untreated HCD-fed rats. Administration of CFs also lowered the serum concentrations of the proinflammatory cytokines IL-1β and IL-12, whereas the concentration of the anti-inflammatory cytokine IL-10 was significantly increased and the concentration of cytokine IL-4 was near the control value. Decreased concentrations of fasting blood glucose, glycated hemoglobin (GHbA1c) and serum insulin and increased tolerance to glucose in the oral glucose tolerance test (OGTT) were observed in the HCD+CF group of animals when compared to rats in the HCD group. We concluded that CFs mediated a therapeutic effect on obesity development in rats exposed to a HCD by affecting pathways involved in obesity pathogenesis

Non-glycosylated IGF2 prohormones are more mitogenic than native IGF2

Abstract 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