The insulin-linked polymorphic region is a variable number of tandem repeats region of DNA in the promoter of the insulin gene that regulates transcription of insulin. This region is known to form the alternative DNA structures, i-motifs and G-quadruplexes. Individuals have different sequence variants of tandem repeats and although previous work investigated the effects of some variants on G-quadruplex formation, there is not a clear picture of the relationship between the sequence diversity, the DNA structures formed, and the functional effects on insulin gene expression. Here we show that different sequence variants of the insulin linked polymorphic region form different DNA structures in vitro. Additionally, reporter genes in cellulo indicate that insulin expression may change depending on which DNA structures form. We report the crystal structure and dynamics of an intramolecular i-motif, which reveal sequences within the loop regions forming additional stabilising interactions that are critical to formation of stable i-motif structures. The outcomes of this work reveal the detail in formation of stable i-motif DNA structures, with potential for rational based drug design for compounds to target i-motif DNA.

• MeSH
• DNA * chemie   genetika   MeSH
• G-kvadruplexy * MeSH
• inzulin * chemie   genetika   MeSH
• konformace nukleové kyseliny MeSH
• krystalografie rentgenová MeSH
• lidé MeSH
• molekulární modely MeSH
• nukleotidové motivy MeSH
• polymorfismus genetický MeSH
• promotorové oblasti (genetika) * MeSH
• reportérové geny MeSH
• sekvence nukleotidů MeSH
• tandemové repetitivní sekvence genetika   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

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.

• MeSH
• bílá tuková tkáň metabolismus   MeSH
• buněčné linie MeSH
• CD antigeny MeSH
• fosforylace MeSH
• glukosa metabolismus   MeSH
• hnědá tuková tkáň metabolismus   MeSH
• insulinu podobný růstový faktor I metabolismus   MeSH
• inzulin genetika   metabolismus   MeSH
• inzuliny metabolismus   MeSH
• Iridovirus genetika   MeSH
• iridoviry genetika   MeSH
• lidé MeSH
• myši inbrední C57BL MeSH
• myši MeSH
• receptor IGF typ 1 genetika   metabolismus   MeSH
• receptor inzulinu metabolismus   MeSH
• signální transdukce MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH

The human zinc transporter ZnT8 provides the granules of pancreatic β-cells with zinc (II) ions for assembly of insulin hexamers for storage. Until recently, the structure and function of human ZnTs have been modelled on the basis of the 3D structures of bacterial zinc exporters, which form homodimers with each monomer having six transmembrane α-helices harbouring the zinc transport site and a cytosolic domain with an α,β structure and additional zinc-binding sites. However, there are important differences in function as the bacterial proteins export an excess of zinc ions from the bacterial cytoplasm, whereas ZnT8 exports zinc ions into subcellular vesicles when there is no apparent excess of cytosolic zinc ions. Indeed, recent structural investigations of human ZnT8 show differences in metal binding in the cytosolic domain when compared to the bacterial proteins. Two common variants, one with tryptophan (W) and the other with arginine (R) at position 325, have generated considerable interest as the R-variant is associated with a higher risk of developing type 2 diabetes. Since the mutation is at the apex of the cytosolic domain facing towards the cytosol, it is not clear how it can affect zinc transport through the transmembrane domain. We expressed the cytosolic domain of both variants of human ZnT8 and have begun structural and functional studies. We found that (i) the metal binding of the human protein is different from that of the bacterial proteins, (ii) the human protein has a C-terminal extension with three cysteine residues that bind a zinc(II) ion, and (iii) there are small differences in stability between the two variants. In this investigation, we employed nickel(II) ions as a probe for the spectroscopically silent Zn(II) ions and utilised colorimetric and fluorimetric indicators for Ni(II) ions to investigate metal binding. We established Ni(II) coordination to the C-terminal cysteines and found differences in metal affinity and coordination in the two ZnT8 variants. These structural differences are thought to be critical for the functional differences regarding the diabetes risk. Further insight into the assembly of the metal centres in the cytosolic domain was gained from potentiometric investigations of zinc binding to synthetic peptides corresponding to N-terminal and C-terminal sequences of ZnT8 bearing the metal-coordinating ligands. Our work suggests the involvement of the C-terminal cysteines, which are part of the cytosolic domain, in a metal chelation and/or acquisition mechanism and, as now supported by the high-resolution structural work, provides the first example of metal-thiolate coordination chemistry in zinc transporters.

• MeSH
• beta-buňky metabolismus   MeSH
• diabetes mellitus 2. typu genetika   patologie   MeSH
• inzulin genetika   metabolismus   MeSH
• konformace proteinů, alfa-helix genetika   MeSH
• lidé MeSH
• molekulární konformace MeSH
• nikl chemie   MeSH
• proteinové domény genetika   MeSH
• transportní proteiny chemie   genetika   ultrastruktura   MeSH
• vztahy mezi strukturou a aktivitou * MeSH
• zinek chemie   MeSH
• zinkový transportér 8 chemie   genetika   ultrastruktura   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

Ticks are blood-feeding arachnids transmitting a variety of pathogens to humans and animals. A unique trait in tick physiology is their ability to engorge and digest large amounts of host blood, ensuring their high reproductive potential. Activation of the blood digestive machinery in the tick gut, as well as processes controlling maturation of ovaries, are triggered upon blood meal uptake by still largely unknown mechanisms. Sensing of the nutritional status in metazoan organisms is facilitated by the evolutionarily conserved Insulin Signaling Pathway (ISP) and the interlinked Target of Rapamycin (TOR) pathway. Recently, we have identified three components of these pathways in the hard tick Ixodes ricinus midgut transcriptome, namely a putative insulin receptor (InR), and the downstream intracellular serine/threonine kinases AKT and TOR. In this study, we primarily focus on the molecular and functional characterization of the I. ricinus insulin receptor (IrInR), the first InR characterized in Chelicerates. A phylogenetic analysis across the major Arthropod lineages demonstrated that ticks possess only one gene encoding an InR-related molecule. Tissue expression profiling by quantitative PCR in semi-engorged I. ricinus females revealed that the IrInR, as well as AKT (IrAKT) and TOR (IrTOR) are expressed in various organs, with the highest expression being detected in ovaries. We have further evaluated the impact of RNAi-mediated knock-down (KD) of IrInR, IrAKT, and IrTOR on tick blood-feeding and reproductive capacity. Weights of engorged IrInR KD females and laid egg clutches were reduced compared to the control group, and these quantitative parameters clearly correlated with the efficiency of RNAi-KD achieved in individual ticks. The most striking phenotype was observed for IrAKT KD that impaired tick feeding and completely aborted egg production. A recombinant extracellular fragment of the IrInR α-subunit was used to produce antibodies in experimental rabbits to assess its potential as a protective antigen against tick feeding and reproduction. Our data clearly indicate the functionality of the ISP in ticks and demonstrate the need for further investigation of specific roles played by the endogenous insulin-like peptides in tick physiological processes.

• MeSH
• inzulin genetika   metabolismus   MeSH
• klíště genetika   metabolismus   MeSH
• proteiny členovců analýza   MeSH
• receptor inzulinu analýza   MeSH
• signální transdukce * MeSH
• zvířata MeSH
• Check Tag
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Insulin is produced and stored inside the pancreatic β-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 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 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 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.

• MeSH
• beta-buňky metabolismus   ultrastruktura   MeSH
• chemická frakcionace MeSH
• cytoplazmatická granula metabolismus   MeSH
• exprese genu * MeSH
• glukosa metabolismus   MeSH
• inzulin genetika   metabolismus   MeSH
• krysa rodu rattus MeSH
• Langerhansovy ostrůvky metabolismus   MeSH
• messenger RNA genetika   metabolismus   MeSH
• průtoková cytometrie metody   MeSH
• zinek metabolismus   MeSH
• zinkový transportér 8 MeSH
• zvířata MeSH
• Check Tag
• krysa rodu rattus MeSH
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Information on how insulin and insulin-like growth factors 1 and 2 (IGF-1 and -2) activate insulin receptors (IR-A and -B) and the IGF-1 receptor (IGF-1R) is crucial for understanding the difference in the biological activities of these peptide hormones. Cryo-EM studies have revealed that insulin uses its binding sites 1 and 2 to interact with IR-A and have identified several critical residues in binding site 2. However, mutagenesis studies suggest that Ile-A10, Ser-A12, Leu-A13, and Glu-A17 also belong to insulin's site 2. Here, to resolve this discrepancy, we mutated these insulin residues and the equivalent residues in IGFs. Our findings revealed that equivalent mutations in the hormones can result in differential biological effects and that these effects can be receptor-specific. We noted that the insulin positions A10 and A17 are important for its binding to IR-A and IR-B and IGF-1R and that A13 is important only for IR-A and IR-B binding. The IGF-1/IGF-2 positions 51/50 and 54/53 did not appear to play critical roles in receptor binding, but mutations at IGF-1 position 58 and IGF-2 position 57 affected the binding. We propose that IGF-1 Glu-58 interacts with IGF-1R Arg-704 and belongs to IGF-1 site 1, a finding supported by the NMR structure of the less active Asp-58-IGF-1 variant. Computational analyses indicated that the aforementioned mutations can affect internal insulin dynamics and inhibit adoption of a receptor-bound conformation, important for binding to receptor site 1. We provide a molecular model and alternative hypotheses for how the mutated insulin residues affect activity.

• MeSH
• insulinu podobný růstový faktor I chemie   genetika   MeSH
• insulinu podobný růstový faktor II chemie   genetika   MeSH
• inzulin analogy a deriváty   chemická syntéza   chemie   genetika   MeSH
• lidé MeSH
• mnohočetné abnormality genetika   MeSH
• mutace genetika   MeSH
• nukleární magnetická rezonance biomolekulární MeSH
• poruchy růstu genetika   MeSH
• proteinové domény genetika   MeSH
• receptor IGF typ 1 chemie   genetika   MeSH
• receptor inzulinu chemie   genetika   MeSH
• sekvence aminokyselin genetika   MeSH
• vazba proteinů genetika   MeSH
• vazebná místa genetika   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Detection and quantification of unmethylated circulating insulin (INS) DNA presumably released from β cells has been previously used for assessing their destruction. As the targets within the INS gene suffer from suboptimal specificity, we sought to improve the assay parameters by using the glucokinase gene (GCK) tissue-specific pancreatic promoter. The amount of methylated and unmethylated GCK DNA was measured using a droplet polymerase chain reaction assay and compared with the previously published INS-targeted assay. The method was tested using synthetic target sequences and DNA from pancreatic islets, blood, brain, kidney, large intestine, liver, lung, small intestine, and stomach. Circulating serum DNA was obtained from children with recent-onset type 1 diabetes (T1D) (n = 25), autoantibody-positive first-degree relatives of T1D patients (n = 14), and healthy controls (n = 20). The unmethylated GCK DNA was found to be more islet specific than unmethylated INS DNA. The proportion of the unmethylated GCK DNA was lower than INS in all tested extrapancreatic tissues, except kidney. Although the amounts of methylated DNA measured by the two assays were similar, the INS assay detected considerably more unmethylated DNA. Whereas none of the assays showed significant increase in the amount of unmethylated DNA, the ratio of unmethylated/methylated GCK DNA was borderline significantly increased in autoantibody-positive relatives compared with T1D patients (P = 0.04) and controls (P = 0.06). Targeting the assay into the GCK gene improved analytical parameters of the assay. As the amount of unmethylated target DNA in properly treated samples is very low, the clinical utility of this method remains to be evaluated.

• MeSH
• beta-buňky patologie   fyziologie   MeSH
• buněčná smrt genetika   MeSH
• diabetes mellitus 1. typu diagnóza   genetika   patologie   MeSH
• dítě MeSH
• dospělí MeSH
• glukokinasa analýza   genetika   MeSH
• inzulin analýza   genetika   MeSH
• kojenec MeSH
• lidé MeSH
• metylace DNA MeSH
• mladiství MeSH
• mladý dospělý MeSH
• mutační analýza DNA metody   MeSH
• polymerázová řetězová reakce MeSH
• prediktivní hodnota testů MeSH
• předškolní dítě MeSH
• studie případů a kontrol MeSH
• Check Tag
• dítě MeSH
• dospělí MeSH
• kojenec MeSH
• lidé MeSH
• mladiství MeSH
• mladý dospělý MeSH
• mužské pohlaví MeSH
• předškolní dítě MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH

BACKGROUND: In models extensively used in studies of aging and extended lifespan, such as C. elegans and Drosophila, adult senescence is regulated by gene networks that are likely to be similar to ones that underlie lifespan extension during dormancy. These include the evolutionarily conserved insulin/IGF, TOR and germ line-signaling pathways. Dormancy, also known as dauer stage in the larval worm or adult diapause in the fly, is triggered by adverse environmental conditions, and results in drastically extended lifespan with negligible senescence. It is furthermore characterized by increased stress resistance and somatic maintenance, developmental arrest and reallocated energy resources. In the fly Drosophila melanogaster adult reproductive diapause is additionally manifested in arrested ovary development, improved immune defense and altered metabolism. However, the molecular mechanisms behind this adaptive lifespan extension are not well understood. RESULTS: A genome wide analysis of transcript changes in diapausing D. melanogaster revealed a differential regulation of more than 4600 genes. Gene ontology (GO) and KEGG pathway analysis reveal that many of these genes are part of signaling pathways that regulate metabolism, stress responses, detoxification, immunity, protein synthesis and processes during aging. More specifically, gene readouts and detailed mapping of the pathways indicate downregulation of insulin-IGF (IIS), target of rapamycin (TOR) and MAP kinase signaling, whereas Toll-dependent immune signaling, Jun-N-terminal kinase (JNK) and Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathways are upregulated during diapause. Furthermore, we detected transcriptional regulation of a large number of genes specifically associated with aging and longevity. CONCLUSIONS: We find that many affected genes and signal pathways are shared between dormancy, aging and lifespan extension, including IIS, TOR, JAK/STAT and JNK. A substantial fraction of the genes affected by diapause have also been found to alter their expression in response to starvation and cold exposure in D. melanogaster, and the pathways overlap those reported in GO analysis of other invertebrates in dormancy or even hibernating mammals. Our study, thus, shows that D. melanogaster is a genetically tractable model for dormancy in other organisms and effects of dormancy on aging and lifespan.

• MeSH
• dlouhověkost genetika   MeSH
• Drosophila melanogaster genetika   fyziologie   MeSH
• genom hmyzu MeSH
• genová ontologie MeSH
• inzulin genetika   MeSH
• regulace genové exprese * MeSH
• rozmnožování genetika   MeSH
• signální transdukce MeSH
• stárnutí genetika   fyziologie   MeSH
• transkriptom genetika   MeSH
• zárodečné buňky metabolismus   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

Diabetes mellitus 1. typu je choroba podmienená auto-imunitnou alebo idiopatickou deštrukciou pankreatických ostrovčekov, ktorá vyústi do absolútneho chýbania inzulínu. Vývoj choroby je podmienený genetickými a environmentálnymi faktormi. Všeobecne akceptovaným názorom je, že chorobu u geneticky predisponovaných jedincov spúšťajú environmentálne faktory, ako sú vírusová infekcia, diétne faktory v skorom detstve alebo klimatické vplyvy. Existuje niekoľko kandidátskych génov, z ktorých najvýznamnejšie sú gény hlavného histokompatibilného systému. Najčastejšími autoantigénmi sú inzulín, dekarboxyláza kyseliny glutámovej 65 a zinkový transportér ZnT8. Deštrukcia b-buniek pankreasu je sprostredkovaná bunkovými mechanizmami, protilátky sú pravdepodobne len odrazom prebiehajúcich autoimunitných procesov a do poškodenia tkaniva sa priamo nezapájajú. Objavujú sa však už pred nástupom klinických príznakov, čo umožňuje ich praktické využitie v prevencii choroby.

Type 1 diabetes mellitus (T1 DM) is caused by autoimmune-mediated and idiopathic beta-cell destruction of the pancreatic islets of Langerhans resulting in absolute insulin deficiency. Susceptibility to T1 DM is influenced by both genetic and environmental factors. It is generally believed that in genetically susceptible individuals, the disease is triggered by environmental agents, such as viral infections, dietary factors in early infancy, or climatic influences. Many candidate genes for diabetes have been reported; those within the Major Histocompatibility Complex being among the most important. The most common autoantigens are insulin, glutamic acid decarboxylase 65, insuloma-associated antigen 2, and zinc transporter ZnT8. The destruction of b-cells is mediated mainly by cellular mechanisms; antibodies only seem to reflect the ongoing autoimmune processes and are not directly involved in the tissue damage. They, however, appear prior to the onset of insulin deficiency which makes them suitable for use in the prevention of the disease.

• Klíčová slova
• HLA-alely,
• MeSH
• antigen CTLA-4 genetika   MeSH
• autoantigeny imunologie   MeSH
• autoimunita imunologie   MeSH
• autoimunitní nemoci MeSH
• autoprotilátky krev   MeSH
• B-lymfocyty MeSH
• diabetes mellitus 1. typu * etiologie   genetika   MeSH
• haplotypy MeSH
• hlavní histokompatibilní komplex genetika   MeSH
• inzulin genetika   MeSH
• lidé MeSH
• náchylnost k nemoci imunologie   MeSH
• NKT buňky MeSH
• T-lymfocyty MeSH
• transkripční faktor STAT4 genetika   MeSH
• tyrosinfosfatasa nereceptorového typu 22 genetika   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• přehledy MeSH

Mutations in the insulin (INS) gene rarely occur in patients with Maturity-Onset Diabetes of the Young (MODY). We aimed to describe in detail two MODY families with INS mutations. The INS gene was screened by direct sequencing. The probands and their affected relatives underwent a mixed-meal test. Mutation predictions were modeled using I-TASSER and were visualized by Swiss-PdbViewer. A novel heterozygous frameshift mutation p.Gln78fs in the INS gene was found in three generations of patients with clinically distinct diabetes. The single nucleotide deletion (c.233delA) is predicted to change and prolong amino acid sequence, resulting in aberrant proinsulin without native structures of C-peptide and A-chain. In the second family, the heterozygous mutation c.188-31G>A within the terminal intron was detected. The mother and her daughter were misdiagnosed as having type 1 diabetes since the ages of 6 and 2 years, respectively. This result is in contrast to the previously described carrier of the same mutation who was diagnosed with permanent neonatal diabetes. We identified a novel coding frameshift mutation and an intronic mutation in the INS gene leading to childhood-onset diabetes. INS mutations may result in various phenotypes, suggesting that additional mechanisms may be involved in the pathogenesis and clinical manifestation of diabetes.

• MeSH
• C-peptid genetika   MeSH
• diabetes mellitus 2. typu genetika   MeSH
• dospělí MeSH
• inzulin genetika   MeSH
• lidé středního věku MeSH
• lidé MeSH
• molekulární sekvence - údaje MeSH
• posunová mutace genetika   MeSH
• rodina MeSH
• rodokmen MeSH
• sekvence aminokyselin MeSH
• sekvence nukleotidů MeSH
• sekvenční analýza DNA MeSH
• senioři MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• kazuistiky MeSH
• práce podpořená grantem MeSH