RESUMO
AIMS/HYPOTHESIS: Sulfonylurea receptor 1 (SUR1) is the regulatory subunit of ATP-sensitive K channels in beta cells. Morphological methods (immunohistochemistry and sulfonylurea binding) were used to establish the cellular and subcellular location of SUR1 in human and rodent islets. RESULTS: In the human, mouse and rat pancreas, all endocrine cells of the islets were immunolabelled with an anti-SUR1 antibody, whereas tissues containing SUR2 were consistently negative, as were those from Sur1 (also known as Abcc8)(-/-) mice. In beta cells of the three species, the plasma membrane was distinctly stained, but SUR1 was mainly present over the cytoplasm, with an intensity that varied between cells. Electron microscopy showed that SUR1 was immunolocalised in insulin, glucagon and somatostatin granules. In rat beta cells degranulated by in vivo treatment with glibenclamide (known as glyburide in the USA and Canada), the insulin and SUR1 staining intensity was similarly decreased by approximately 45%, whereas SUR1 staining was not changed in non-beta cells. In all islet cells, binding of glibenclamide labelled with fluorescent dipyrromethane boron difluoride (BODIPY-FL) was punctate over the cytoplasm, compatible with the labelling of endocrine granules. A faint labelling persisted in Sur1 (-/-) mice, but it was not different from that obtained with BODIPY-FL alone used as negative control. CONCLUSIONS/INTERPRETATION: Our study immunolocalised SUR1 in alpha, beta and delta cells of human, mouse and rat islets, and for the first time visualised it in the plasma membrane. We also show that SUR1 is abundant in endocrine granules, where its function remains to be established. No specific sulfonylurea-binding sites other than SUR1 are identified in islet cells by the glibenclamide-BODIPY-FL technique.
Assuntos
Transportadores de Cassetes de Ligação de ATP/fisiologia , Ilhotas Pancreáticas/fisiologia , Proteínas Associadas à Resistência a Múltiplos Medicamentos/fisiologia , Pâncreas/fisiologia , Canais de Potássio Corretores do Fluxo de Internalização/fisiologia , Canais de Potássio/fisiologia , Receptores de Droga/fisiologia , Transportadores de Cassetes de Ligação de ATP/genética , Animais , Cadáver , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Associadas à Resistência a Múltiplos Medicamentos/deficiência , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Ratos , Ratos Wistar , Receptores de Sulfonilureias , Doadores de TecidosRESUMO
We report on the stimulatory effect of creatine on insulin secretion and ATP concentration in MIN-6 beta-cells. The addition of creatine (5 mM) to MIN-6 cells in the presence of glucose (1-10 mM) elicited a significant (p<0.001) increase in insulin secretion, but no effect was demonstrated in the absence of glucose. The lack of effect of creatine in the absence of glucose suggests that creatine may act as a potentiator of insulin secretion rather than as an initiator. The potentiatory effect of creatine is specific for glucose since no effect was found in the presence of other known initiators of insulin secretion (K(+), 2-ketoisocaproic acid and tolbutamide). Cellular ATP content was markedly increased by glucose (1-15 mM). Creatine (5 and 10 mM) further increased the ATP level at all glucose concentrations, and the effect was observed even in the absence of glucose. The results from this study demonstrate the ability of creatine to increase insulin secretion only in the presence of glucose, while its effect on increased cellular ATP was independent of the presence of glucose. The mechanism whereby creatine potentiates insulin release is yet to be investigated. However, our data suggest possible unique interactions between creatine and the glucose-dependent insulin secretory pathway.
Assuntos
Creatina/farmacologia , Glucose/metabolismo , Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Relação Dose-Resposta a Droga , Glucose/farmacologia , Humanos , Hipoglicemiantes/farmacologia , Secreção de Insulina , Cetoácidos/farmacologia , Tolbutamida/farmacologiaRESUMO
The ATP-sensitive potassium (K(ATP)) channel plays a key role in controlling beta-cell membrane potential and insulin secretion. The channels are composed of two subunits, Kir6.2, which forms the channel pore, and SUR1, which contains binding sites for nucleotides and sulphonylureas and acts as a channel regulator. Our current studies are aimed at delineating the molecular interactions involved in assembly and ligand binding by K(ATP) channel proteins. We have employed a complementation approach in which SUR1 half-molecules are co-expressed in insect cells using a baculovirus system. Together with data from truncated SUR1 molecules and a fusion protein in which SUR1 is linked to Kir6.2, we have interpreted our findings in terms of a model for the structure of the K(ATP) channel. The main features of the model are: (i) the C-terminal end of SUR1 is close to the N-terminus of Kir6.2; (ii) the two nucleotide binding domains (NBDs) of SUR1--NBD1 and NBD2--are in proximity; (iii) transmembrane helix 12 of SUR1 is orientated in such a way that it can make contact with Kir6.2; (iv) formation of the glibenclamide binding site requires that the two cytosolic loops (CLs) CL3 and CL8 are located close to each other; (v) there are homomeric interactions between the NBD1 domains of neighbouring subunits. We suggest that binding of glibenclamide leads to conformational changes in CL3 and CL8 leading to rearrangement of transmembrane helices. These effects are transmitted to Kir6.2 to result in channel closure.
Assuntos
Ilhotas Pancreáticas/metabolismo , Canais de Potássio Corretores do Fluxo de Internalização/química , Canais de Potássio Corretores do Fluxo de Internalização/metabolismo , Canais de Potássio/química , Canais de Potássio/metabolismo , Transportadores de Cassetes de Ligação de ATP , Trifosfato de Adenosina/metabolismo , Animais , Sítios de Ligação , Glibureto/metabolismo , Técnicas In Vitro , Modelos Moleculares , Estrutura Molecular , Receptores de Droga , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Receptores de SulfonilureiasRESUMO
AIMS/HYPOTHESIS: Ca(2+)/calmodulin-dependent protein kinase II, is expressed in the pancreatic beta cells and is activated by glucose and other secretagogues in a manner correlating with insulin secretion. The activation of Ca(2+)/calmodulin-dependent protein kinase II mediates some of the actions of Ca(2+) on the exocytosis of insulin. We therefore investigated the gene encoding the gamma isoform ( CAMK2G) which has been shown to be expressed in human beta cells as a candidate gene for Type II (non-insulin-dependent) diabetes mellitus. METHODS: Human CAMK2G was cloned from a total human P1 artificial chromosome library using a partial Ca(2+)/calmodulin-dependent protein kinase gamma(E) cDNA probe. Positive PAC clones were localised to chromosome 10q22 by fluorescence in situ hybridisation. To obtain structural information and the sequences of the exon-intron boundaries, the published genomic structures of the rat and mouse genes allowed the putative exon-intron boundaries of human CAMK2G to be amplified by vectorette polymerase chain reaction and sequenced. Sequence variants in each exon were identified using single stranded conformational polymorphism analysis. RESULTS: The human CAMK2G gene comprises 22 exons which range in size between 43 to 230 bp. Screening of the exons and exon-intron boundaries identified two single nucleotide polymorphisms. These did not show association with diabetes in 122 patients and 144 control subjects. CONCLUSIONS/INTERPRETATION: We have identified the genomic structure of CAMK2G to enable further study of this potential candidate gene. Variation in this gene is not strongly associated with diabetes in Caucasians in the United Kingdom. We have identified two single nucleotide polymorphisms which, with appropriately large case control studies, can be used to assess the role of CAMK2G in the susceptibility to Type II diabetes.
Assuntos
Proteínas Quinases Dependentes de Cálcio-Calmodulina/genética , Diabetes Mellitus Tipo 2/genética , Variação Genética , Alelos , Processamento Alternativo , Sequência de Bases , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina , Mapeamento Cromossômico , Clonagem Molecular , Diabetes Mellitus Tipo 2/enzimologia , Humanos , Hibridização in Situ Fluorescente , Valores de ReferênciaRESUMO
AIMS/HYPOTHESIS: The neurotransmitters glutamate and gamma-aminobutyric acid (GABA) could participate in the regulation of the endocrine functions of islets of Langerhans. We investigated the role of the metabotropic glutamate (mGluRs) and GABA(B) (GABA(B)Rs) receptors in this process. METHODS: We studied the expression of mGluRs and GABA(B)Rs in rat and human islets of Langerhans and in pancreatic alpha-cell and beta-cell lines using RT-PCR and immunoblot analysis. Effects of mGluR and GABA(B) R agonists on insulin secretion were determined by radioimmunoassays and enzyme-linked immunoadsorbent assays (ELISAs). RESULTS: We detected mGluR3 and mGluR5 (but not mGluR1, 6 and 7) mRNAs in all of the samples examined. Trace amount of mGluR2 was found in MIN6 beta cells; mGluR4 was identified in rat islets; and mGluR8 expression was detected in rat islets, RINm5F and MIN6 cells. GABA(B)R1 a/b and 2 mRNAs were identified in islets of Langerhans and MIN6 cells. The expression of mGluR3, mGluR5, GABA(B)R1 a/b and GABA(B)R2 proteins was confirmed using specific antibodies. Group I (mGluR1/5) and group II (mGluR2/3) specific mGluR agonists increased the release of insulin in the presence of 3 to 10 mmol/l or 3 to 25 mmol/l glucose, respectively, whereas a group III (mGluR4/6-8) specific agonist inhibited insulin release at high (10-25 mmol/l) glucose concentrations. Baclofen, a GABA(B)R agonist, also inhibited the release of insulin but only in the presence of 25 mmol/l glucose. CONCLUSION/INTERPRETATION: These data suggest that mGluRs and GABA(B)Rs play a role in the regulation of the endocrine pancreas with mechanisms probably involving direct activation or inhibition of voltage dependent Ca(2+)-channels, cAMP generation and G-protein-mediated modulation of K(ATP) channels.
Assuntos
Glucose/farmacologia , Insulina/metabolismo , Ilhotas Pancreáticas/fisiologia , Receptores de GABA-B/fisiologia , Receptores de Glutamato Metabotrópico/fisiologia , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/fisiologia , Linhagem Celular , Membrana Celular/efeitos dos fármacos , Membrana Celular/fisiologia , Primers do DNA , Humanos , Secreção de Insulina , Ilhotas Pancreáticas/efeitos dos fármacos , Ilhotas Pancreáticas/metabolismo , Masculino , Modelos Biológicos , Ratos , Ratos Wistar , Receptor de Glutamato Metabotrópico 5 , Receptores de GABA-B/genética , Receptores de Glutamato Metabotrópico/genética , Reação em Cadeia da Polimerase Via Transcriptase ReversaAssuntos
Proteínas Quinases Dependentes de Cálcio-Calmodulina/genética , Genoma Humano , Ilhotas Pancreáticas/enzimologia , Isoenzimas/genética , Sequências de Repetição em Tandem , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina , Humanos , Dados de Sequência Molecular , Prolina , Estrutura Terciária de Proteína/genéticaRESUMO
We have investigated the structure of the glibenclamide binding site of pancreatic beta-cell ATP-sensitive potassium (K(ATP)) channels. K(ATP) channels are a complex of four pore-forming Kir6.2 subunits and four sulfonylurea receptor (SUR1) subunits. SUR1 (ABCC8) belongs to the ATP binding cassette family of proteins and has two nucleotide binding domains (NBD1 and NBD2) and 17 putative transmembrane (TM) sequences. Co-expression in a baculovirus expression system of two parts of SUR1 between NBD1 and TM12 leads to restoration of glibenclamide binding activity, whereas expression of either individual N- or C-terminal part alone gave no glibenclamide binding activity, confirming a bivalent structure of the glibenclamide binding site. By using N-terminally truncated recombinant proteins we have shown that CL3 - the cytosolic loop between TM5 and TM6 - plays a key role in formation of the N-terminal component of the glibenclamide binding site. Analysis of deletion variants of the C-terminal part of SUR1 showed that CL8 - the cytosolic loop between TM15 and TM16 - is the only determinant for the C-terminal component of the glibenclamide binding site. We suggest that in SUR1 in the native K(ATP) channel close proximity of CL3 and CL8 leads to formation of the glibenclamide binding site.
Assuntos
Transportadores de Cassetes de Ligação de ATP , Glibureto/metabolismo , Ilhotas Pancreáticas/metabolismo , Canais de Potássio Corretores do Fluxo de Internalização , Canais de Potássio/química , Canais de Potássio/metabolismo , Receptores de Droga/química , Receptores de Droga/metabolismo , Animais , Sítios de Ligação , Western Blotting , Linhagem Celular , Membrana Celular/química , Membrana Celular/metabolismo , Glibureto/química , Cinética , Microscopia de Fluorescência , Modelos Moleculares , Canais de Potássio/genética , Estrutura Quaternária de Proteína , Subunidades Proteicas , Ratos , Receptores de Droga/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Spodoptera , Receptores de Sulfonilureias , TermodinâmicaRESUMO
AIMS: The beta-cell ATP-sensitive potassium channel consists of two subunits, SUR1 and Kir6.2. Population association studies have shown that three variants in SUR1 and one in Kir6.2 are associated with Type 2 diabetes. These polymorphisms do not result in a functional change or affect splicing, suggesting that they could be in linkage disequilibrium with a pathogenic mutation. The present study aimed firstly to screen the promoter regions of SUR1 and Kir6.2 to determine whether mutations in linkage disequilibrium with the silent variants lie in regulatory regions, which might lead to changes in gene expression. Secondly, novel and previously described variants associated with Type 2 diabetes (SUR1 exon 16-3t, exon 18 T, and Kir6.2 E23K) were investigated in the UKPDS cohort. METHODS: The promoter sequences of both genes were screened by single-stranded conformational polymorphism analysis for variants associated with Type 2 diabetes. The previously reported variants were evaluated in 364 Type 2 diabetic and 328 normoglycaemic control subjects. RESULTS: Two variants were detected in the SUR1 promoter, a three base insertion (caa) at -522 bp and a single base substitution at - 679 bp (c-->g). Neither of the variants were associated with diabetes, nor were they in a sequence consensus region for transcription factors. No association with diabetes was observed for either SUR1 variant. However, in contrast, analysis of the Kir6.2 E23K variant showed that the KK homozygosity was more frequent in Type 2 diabetic than control subjects. Variants were not associated with clinical characteristics nor did they affect response to sulphonylurea therapy CONCLUSION: There is no support at present for mutations in either Kir6.2 or SUR1 promoter sequences contributing to Type 2 diabetes. However, the minimal promoter region of SUR1 has yet to be investigated. The E23K variant of Kir6.2 is associated with Type 2 diabetes mellitus in the UKPDS cohort.
Assuntos
Diabetes Mellitus Tipo 2/genética , Variação Genética , Canais de Potássio Corretores do Fluxo de Internalização , Canais de Potássio/genética , Regiões Promotoras Genéticas , Transportadores de Cassetes de Ligação de ATP , Adulto , DNA/sangue , Diabetes Mellitus Tipo 2/tratamento farmacológico , Éxons , Genótipo , Humanos , Hipoglicemiantes/uso terapêutico , Pessoa de Meia-Idade , Receptores de Droga , Compostos de Sulfonilureia/uso terapêutico , Receptores de Sulfonilureias , Fatores de Transcrição/genéticaRESUMO
We have investigated the protein interactions involved in the assembly of pancreatic beta-cell ATP-sensitive potassium channels. The channels are a heterooligomeric complex of pore-forming Kir6.2 subunits and sulfonylurea receptor (SUR1) subunits. SUR1 belongs to the ATP binding cassette (ABC) family of proteins and has two nucleotide binding domains (NBD1 and NBD2) and 17 putative transmembrane (TM) sequences. Previously we showed that co-expression in a baculovirus expression system of two parts of SUR1 divided at Pro1042 between TM12 and 13 leads to restoration of glibenclamide binding activity, whereas expression of either individual N- or C-terminal domain alone gave no glibenclamide binding activity [M.V. Mikhailov and S.J.H. Ashcroft (2000) J. Biol. Chem. 275, 3360-3364]. Here we show that the two half-molecules formed by division of SUR1 between NBD1 and TM12 or between TM13 and 14 also self-assemble to give glibenclamide binding activity. However, deletion of NBD1 from the N-part of SUR1 abolished SUR1 assembly, indicating a critical role for NBD1 in SUR1 assembly. We found that differences in glibenclamide binding activity obtained after co-expression of different half-molecules are attributable to different amounts of binding sites, but the binding affinities remained nearly the same. Simultaneous expression of Kir6.2 resulted in enhanced glibenclamide binding activity only when the N-half of SUR1 included TM12. We conclude that TM12 and 13 are not essential for SUR1 assembly whereas TM12 takes part in SUR1 Kir6.2 interaction. This interaction is specific for Kir 6.2 because no enhancement of glibenclamide binding was observed when half-molecules were expressed together with Kir4.1. We propose a model of K(ATP) channel organisation based on these data.
Assuntos
Transportadores de Cassetes de Ligação de ATP , Ilhotas Pancreáticas/fisiologia , Canais de Potássio Corretores do Fluxo de Internalização , Canais de Potássio/fisiologia , Receptores de Droga/fisiologia , Animais , Linhagem Celular , Glibureto/farmacocinética , Camundongos , Modelos Moleculares , Canais de Potássio/química , Canais de Potássio/genética , Estrutura Secundária de Proteína , Subunidades Proteicas , Ratos , Receptores de Droga/química , Receptores de Droga/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Spodoptera , Receptores de Sulfonilureias , TransfecçãoAssuntos
Transportadores de Cassetes de Ligação de ATP , Proteínas de Drosophila , Ilhotas Pancreáticas/fisiologia , Canais de Potássio Corretores do Fluxo de Internalização , Canais de Potássio/fisiologia , Potássio/metabolismo , Receptores de Droga/fisiologia , Trifosfato de Adenosina/farmacologia , Trifosfato de Adenosina/fisiologia , Regulação Alostérica , Motivos de Aminoácidos , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Glicemia/metabolismo , Cromossomos Humanos Par 11/genética , Proteínas de Ligação ao GTP/fisiologia , Humanos , Hiperinsulinismo/congênito , Hiperinsulinismo/genética , Hipoglicemiantes/farmacologia , Recém-Nascido , Insulina/metabolismo , Secreção de Insulina , Peptídeos e Proteínas de Sinalização Intercelular , Ativação do Canal Iônico/fisiologia , Transporte de Íons/fisiologia , Leptina/fisiologia , Potenciais da Membrana , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Peptídeos/farmacologia , Peptídeos/fisiologia , Fosforilação , Canais de Potássio/química , Canais de Potássio/efeitos dos fármacos , Canais de Potássio/genética , Conformação Proteica , Processamento de Proteína Pós-Traducional , Receptores de Droga/química , Receptores de Droga/efeitos dos fármacos , Receptores de Droga/genética , Relação Estrutura-Atividade , Compostos de Sulfonilureia/farmacologia , Receptores de Sulfonilureias , Transcrição GênicaRESUMO
We have investigated protein interactions involved in pancreatic beta-cell ATP-sensitive potassium channel assembly. These channels, which are of key importance for control of insulin release, are a hetero-oligomeric complex of pore-forming Kir6.2 subunits and sulfonylurea receptor (SUR1) subunits with two nucleotide-binding domains (NBD1 and NBD2). We divided SUR1 into two halves at Pro-1042. Expression of either the individual N- or C-terminal domain in a baculovirus expression system did not lead to glibenclamide binding activity, although studies with green fluorescent protein fusion proteins showed that both half-molecules were inserted into the plasma membrane. However, significant glibenclamide binding activity was observed when the half-molecules were co-expressed (even when NBD2 was deleted from the C-terminal half-molecule). Simultaneous expression of Kir6.2 resulted in enhanced glibenclamide binding activity. We conclude that the glibenclamide-binding site includes amino acid residues from both halves of the molecule, that there is strong interaction between different regions of SUR1, that NBD2 is not essential for glibenclamide binding, and that interactions between Kir6.2 and SUR1 participate in ATP-sensitive potassium channel assembly. Investigation of NBD1-green fluorescent protein fusion protein distribution inside insect cells expressing C-terminal halves of SUR1 demonstrated strong interaction between NBD1 and NBD2. We also expressed and purified NBD1 from Escherichia coli. Purified NBD1 was found to exist as a tetramer indicating strong homomeric attractions and a possible role for NBD1 in SUR1 assembly.
Assuntos
Transportadores de Cassetes de Ligação de ATP , Ilhotas Pancreáticas/metabolismo , Canais de Potássio Corretores do Fluxo de Internalização , Canais de Potássio/metabolismo , Receptores de Droga/metabolismo , Animais , Sítios de Ligação , Linhagem Celular , Proteínas de Fluorescência Verde , Proteínas Luminescentes , Canais de Potássio/química , Receptores de Droga/química , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Transdução de Sinais , Compostos de Sulfonilureia/química , Compostos de Sulfonilureia/metabolismo , Receptores de SulfonilureiasRESUMO
AIMS/HYPOTHESIS: Type II (non-insulin-dependent) diabetes mellitus is a common heterogeneous metabolic disorder of largely unknown genetic aetiology. The sarco(endo)plasmic reticulum Ca(2+)-transport ATPase (SERCA) plays an important part in the glucose-activated beta-cell Ca(2+) signalling that regulates insulin secretion. Impaired function and expression of SERCA have been shown in islets of Langerhans from diabetic animal models and have also been associated with beta-cell apoptosis. Thus, the SERCA3 encoding gene is a plausible candidate for a primary pancreatic beta-cell defect. METHODS: In this study, the entire coding and the promoter regions of SERCA3 gene were screened by single-strand conformation polymorphism analysis in white Caucasian Type II diabetic patients. RESULTS: We found four rare missense mutations [Exon 4: Gln(108)-->His (CAG-->CAT), Exon 14: Val(648) -->Met (GTG-->ATG) and Arg(674)-->Cys (CGC--> TGC), and Exon 15: Ile(753)-->Leu (ATC-->CTC)]. The patients with Gln(108)-->His, Val(648)-->Met and Arg(674)-->Cys mutations, which may affect the E1P-E2P transition of SERCA3 during its enzyme cycle, had normal body weight with marked hyperglycaemia and beta-cell dysfunction. That is an unusual phenotype only found in 6 % of the Type II diabetic patients recruited for the UK Prospective Diabetes Study. In addition, five silent polymorphisms, six intron variants and two polymorphisms in the 3' untranslated region of exon 22 were found with similar frequency in diabetic and control subjects. CONCLUSION/INTERPRETATION: Our result suggests that in white Caucasians, the SERCA3 locus possibly contributes to the genetic susceptibility to Type II diabetes [Diabetologia (1999) 42: 1240-1243].
Assuntos
ATPases Transportadoras de Cálcio/genética , Diabetes Mellitus Tipo 2/genética , Análise Mutacional de DNA , Testes Genéticos , Humanos , Isoenzimas/genética , Mutação de Sentido Incorreto , Reação em Cadeia da Polimerase , Polimorfismo Genético , Polimorfismo Conformacional de Fita Simples , Estudos Prospectivos , Distribuição Aleatória , Reino Unido , População Branca/genéticaRESUMO
BACKGROUND: There is considerable evidence that calcium/calmodulin-dependent protein kinase II (CaM kinase II) plays a key role in insulin secretion and the enzyme provides a candidate gene for Type 2 diabetes. Since several isoforms of the enzyme exist, it is essential to define which are expressed by the beta-cell. METHODS: A human islet cDNA library in lambdaZAPII was screened with a probe for the 5'-end of human gamma CaM kinase II. Since this region is very homologous between the different isoforms, it is expected that isoforms other than gamma would be detected. From each of the six positive clones obtained, DNA was prepared and subjected to PCR using primers spanning the variable region in which the main variability of CaM kinase II isoforms resides. PCR products were purified and sequenced in both directions. The beta-cell line MIN6 was screened for CaM kinase II delta by reverse transcriptase-polymerase chain reaction (RT-PCR) and by Western blotting. RESULTS: The sequences of five of the human islet PCR products indicated that the clones corresponded to the gamma(B) isoform whose expression in human islets we have previously documented. The other PCR product, however, gave a sequence containing the variable domains II and VII characteristic of CaM kinase II delta. This sequence and the absence of other domains in this region identified the clone as CaM kinase II delta(C). The expression of CaM kinase II delta in MIN6 beta-cells was confirmed by RT-PCR and by Western blotting. CONCLUSIONS: Human islets of Langerhans express the delta(C) isoform of CaM kinase II.
Assuntos
Proteínas Quinases Dependentes de Cálcio-Calmodulina/genética , Expressão Gênica , Ilhotas Pancreáticas/enzimologia , Isoenzimas/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Western Blotting , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina , Proteínas Quinases Dependentes de Cálcio-Calmodulina/química , Linhagem Celular , Biblioteca Gênica , Humanos , Isoenzimas/química , Camundongos , Dados de Sequência Molecular , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
ATP-sensitive potassium channels (K(ATP)) are formed from four pore-forming Kir6.2 subunits complexed with four regulatory sulfonylurea receptor subunits (SUR1 in pancreatic beta-cells, SUR2A in heart). The sensitivity of the channel to different sulfonylureas depends on the SUR isoform. In particular, Kir6.2-SUR1 but not Kir6.2-SUR2A channels are blocked by tolbutamide with high affinity. We made chimeras between SUR1 and SUR2A to identify the region of the protein involved in high-affinity tolbutamide block. Chimeric SURs were coexpressed with Kir6.2 in Xenopus oocytes, and macroscopic currents were measured in inside-out membrane patches. High-affinity tolbutamide inhibition could be conferred on SUR2A by replacing transmembrane domains (TMs) 14-16 with the corresponding region of SUR1. Conversely, high-affinity tolbutamide inhibition of SUR1 was abolished by replacing TMs 13-16 with the corresponding SUR2A sequence, or by mutating a single serine residue within this region to tyrosine (S1237Y). Binding of [3H]glibenclamide to membranes expressing SUR1 was abolished concomitantly with the loss of high-affinity tolbutamide block. These results suggest that a site in the COOH-terminal set of TMs of the SUR1 subunit of the K(ATP) channel is involved in the binding of tolbutamide and glibenclamide.
Assuntos
Transportadores de Cassetes de Ligação de ATP , Hipoglicemiantes/metabolismo , Canais de Potássio Corretores do Fluxo de Internalização , Canais de Potássio/metabolismo , Receptores de Droga/metabolismo , Compostos de Sulfonilureia/metabolismo , Tolbutamida/metabolismo , Difosfato de Adenosina/metabolismo , Substituição de Aminoácidos , Animais , Sítios de Ligação , Células COS , Glibureto/metabolismo , Canais de Potássio/genética , Ratos , Receptores de Droga/genética , Receptores de Sulfonilureias , Transfecção , XenopusRESUMO
Hypoglycemic sulfonylureas stimulate insulin release by binding to a regulatory subunit of plasma membrane ATP-sensitive K+ (K(ATP)) channels. The consequent closure of K(ATP) channels leads to depolarization, opening of voltage-dependent Ca2+ channels, Ca2+ influx, and a rise in intracellular [Ca2+]. Recently, however, it has been suggested that sulfonylureas may have an additional action on secretion, independent of changes in intracellular [Ca2+] but dependent on the activity of protein kinase C (PKC). We have investigated the mechanisms involved in the PKC-dependent effect of sulfonylureas on the secretion machinery in beta-cells. In MIN6 beta-cells permeabilized by streptolysin O, insulin release was stimulated by elevation of [Ca2+] from 10(-8) to 10(-5) mol/l. At a [Ca2+] of 10(-8) mol/l, insulin release from permeabilized beta-cells was stimulated by addition of GTP-gamma-S, or by addition of a phorbol ester, 12-O-tetradecanoylphorbol 13-acetate (TPA). TPA, but not GTP-gamma-S, also increased insulin release when [Ca2+] was 10(-5) mol/l. Insulin release from permeabilized beta-cells was stimulated by tolbutamide (0.1-1 mmol/l) at 10(-8) but not at 10(-5) mol/l Ca2+. The effect of tolbutamide was blocked either by inhibition of PKC or when phorbol ester-sensitive PKC isoforms were maximally stimulated by TPA. Meglitinide and glibenclamide also stimulated insulin release from permeabilized beta-cells. To assess the possibility that direct activation of PKC mediates the exocytotic response to sulfonylureas, we studied the effect of tolbutamide and glibenclamide on PKC activity. Purified brain PKC was not activated by tolbutamide or glibenclamide, whether tested in the absence or presence of phosphatidylserine or TPA, or at low or high [Ca2+]; nor was the total PKC activity in extracts of MIN6 beta-cells affected by tolbutamide. Neither tolbutamide nor glibenclamide elicited translocation of any isoform of PKC in intact or permeabilized beta-cells under conditions in which TPA evoked a marked redistribution of PKC alpha- and epsilon-isoforms. We conclude that although the plasma membrane K(ATP) channel-independent stimulation of exocytosis by sulfonylureas may require functional PKC, the mechanism does not involve a direct activation of the enzyme.
Assuntos
Exocitose/efeitos dos fármacos , Hipoglicemiantes/farmacologia , Ilhotas Pancreáticas/metabolismo , Proteína Quinase C/metabolismo , Compostos de Sulfonilureia/farmacologia , Animais , Cálcio/metabolismo , Cálcio/farmacologia , Ativação Enzimática/efeitos dos fármacos , Glibureto/farmacologia , Guanosina 5'-O-(3-Tiotrifosfato)/farmacologia , Insulinoma , Ilhotas Pancreáticas/efeitos dos fármacos , Isoenzimas/metabolismo , Camundongos , Neoplasias Pancreáticas , Fosfatidilserinas/farmacologia , Canais de Potássio/fisiologia , Acetato de Tetradecanoilforbol/farmacologia , Tolbutamida/farmacologia , Células Tumorais CultivadasRESUMO
The role of the AMP-activated protein kinase (AMPK) cascade in the glucose-sensitive pancreatic beta cell lines HIT-T15 and INS-1 was addressed. In both cell types, removal of glucose leads to a >5-fold activation of AMPK activity. Activation of AMPK was due to phosphorylation, since the effect was reversed by protein phosphatase treatment of the extracts, and was restored by re-addition of MgATP and the purified upstream kinase. When the effects of different concentrations of medium glucose were examined, insulin secretion and AMPK activity were inversely related, and varied over the same concentration range. The activation in response to glucose removal appeared to be due to changes in the concentration of the known regulators of the cascade, i.e. AMP and ATP, since AMPK activation was associated with a large increase in the cellular AMP/ATP ratio, and the two parameters varied over the same range of glucose concentrations. In late-passage HIT-T15 cells that had lost the glucose-dependent insulin secretion response, both AMPK activity and the AMP/ATP ratio also became insensitive to the extracellular glucose concentration. Treatment of INS-1 cells, but not HIT-T15 cells, with AICA riboside (5-aminoimidazole-4-carboxamide riboside) results in accumulation of the ribotide, ZMP (AICA riboside monophosphate), and activation of AMPK. AICA riboside treatment of INS-1 cells, and of isolated rat islets, had both inhibitory and stimulatory effects on insulin secretion. These results show that in beta cell lines the AMP-activated protein kinase, like its yeast homologue the SNF1 complex, can respond to the level of glucose in the medium, and may be involved in regulating insulin release.
Assuntos
Glucose/fisiologia , Insulina/metabolismo , Ilhotas Pancreáticas/enzimologia , Complexos Multienzimáticos/metabolismo , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases , Proteínas Quinases Ativadas por AMP , Difosfato de Adenosina/metabolismo , Monofosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Trifosfato de Adenosina/farmacologia , Sequência de Aminoácidos , Aminoimidazol Carboxamida/análogos & derivados , Aminoimidazol Carboxamida/metabolismo , Aminoimidazol Carboxamida/farmacologia , Animais , Domínio Catalítico , Linhagem Celular , Ativação Enzimática , Glucose/farmacologia , Técnicas In Vitro , Secreção de Insulina , Ilhotas Pancreáticas/efeitos dos fármacos , Ilhotas Pancreáticas/metabolismo , Cinética , Masculino , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Fosforilação , Proteínas Tirosina Fosfatases/química , Proteínas Tirosina Fosfatases/metabolismo , Ratos , Ratos Wistar , Ribonucleotídeos/metabolismo , Ribonucleotídeos/farmacologiaRESUMO
The beta-cell ATP-sensitive potassium channel (K-ATP channel), which regulates insulin secretion, is composed of two types of subunits: 1) a sulfonylurea receptor (SUR1) and 2) an inwardly rectifying potassium channel (Kir6.2). We have isolated clones containing 5'-flanking DNA for both genes by hybridization screening of a human genomic library. Sequencing of over one kilobase of each upstream region has revealed that the putative promoters are G + C rich, with no TATA box. Several E-boxes and potential Sp1 sites are present in both promoters, and the Kir6.2 upstream region contains an Alu repeat. Using a luciferase reporter gene in transient transfection assays, we demonstrate that the upstream DNA contains promoters that are active in the beta-cell lines HIT T15 and MIN6. The promoters are completely inactive in the fibroblast cell line COS7 but show some activity in HepG2 (liver) and HEK293 (epithelial) cell lines. Deletion analysis suggests that a short (173-base pair [bp]) fragment of SUR1 5'-flanking sequence is sufficient for maximal promoter activity. In contrast, over 900 bp of Kir6.2 5' sequence are required for similar high level expression, and deletion of the Alu repeat results in an increase in promoter activity.
Assuntos
Transportadores de Cassetes de Ligação de ATP , Clonagem Molecular , Ilhotas Pancreáticas/metabolismo , Canais de Potássio Corretores do Fluxo de Internalização , Canais de Potássio/genética , Regiões Promotoras Genéticas/genética , Receptores de Droga/genética , Sequência de Bases , Mapeamento Cromossômico , Deleção de Genes , Humanos , Dados de Sequência Molecular , Receptores de Sulfonilureias , Transcrição Gênica/genéticaRESUMO
The efficacy of Tinospora crispa (Menispermaceae) extract for the treatment of diabetes has previously been verified in animal models. In order to substantiate the antidiabetic effect, we characterised the antihyperglycaemic properties by studying its effect on intestinal glucose absorption and glucose uptake into adipocytes. We also performed experiments to characterise in more detail the mechanism of T. crispa-evoked insulin release by challenging it with insulin secretory antagonists viz. adrenaline, somatostatin, verapamil and nifedipine. In addition, we also performed experiments to determine the effect of the extract on cAMP content. The results clearly showed that the antihyperglycaemic effect is not due to interference with intestinal glucose uptake or uptake of the sugar into the peripheral cells. Rather, the antihyperglycaemic effect of T. crispa is probably due to stimulation of insulin release via modulation of beta-cell Ca2+ concentration. That the insulinotropic effect of T. crispa is physiological suggests that the extract contains compounds which could be purified for use in the treatment of type II diabetes.
Assuntos
Hipoglicemiantes/farmacologia , Ilhotas Pancreáticas/efeitos dos fármacos , Extratos Vegetais/farmacologia , Plantas Medicinais/química , Adipócitos/metabolismo , Linhagem Celular , AMP Cíclico/metabolismo , Glucose/metabolismo , Insulina/metabolismo , Antagonistas da Insulina/farmacologia , Secreção de Insulina , Mucosa Intestinal/metabolismo , Ilhotas Pancreáticas/citologia , Ilhotas Pancreáticas/metabolismo , Rubídio/metabolismoRESUMO
One type of maturity-onset diabetes of the young (MODY2) is caused by mutations in the glucokinase gene, a key glycolytic enzyme in the beta cell and liver. Glucose fails to stimulate insulin secretion in mice in which the glucokinase gene has been selectively knocked out in the beta cell. We tested the hypothesis that this effect results from defective metabolic regulation of beta cell ATP-sensitive potassium (K(ATP)) channels. Glucose had little effect on K(ATP) currents in homozygous (-/-) mice but inhibited K(ATP) currents in wild-type (+/+) and heterozygous (+/-) mice with EC50 of 3.2 mM and 5.5 mM, respectively, in newborn animals, and of 4.7 mM and 9.9 mM, respectively, in 1.5-year-old mice. Glucose (20 mmol/l) did not affect the resting membrane potential of -/- beta cells but depolarised wild-type and + /- beta cells and induced electrical activity. In contrast, 20 mmol/l ketoisocaproic acid or 0.5 mmol/ l tolbutamide depolarised all three types of beta-cell. These results support the idea that defective glycolytic metabolism, produced by a loss (-/- mice) or reduction (+/- mice) of glucokinase activity, leads to defective K(ATP) channel regulation and thereby to the selective loss, or reduction, of glucose-induced insulin secretion.