RESUMO
Glucose-stimulated insulin secretion is associated with transients of intracellular calcium concentration ([Ca2+]i) in the pancreatic beta-cell. We tested the hypothesis that inositol (1,4,5)-trisphosphate [Ins(1,4,5)P3] [Ca2+]i release is incorporated in glucose-induced [Ca2+]i oscillations in mouse islets and MIN6 cells. We found that depletion of intracellular Ca2+ stores with thapsigargin increased the oscillation frequency by twofold and inhibited the slow recovery phase of [Ca2+]i oscillations. We employed a pleckstrin homology domain-containing fluorescent biosensor, phospholipase C partial differential pleckstrin homology domain-enhanced green fluorescent protein, to visualize Ins(1,4,5)P3 dynamics in insulin-secreting MIN6 cells and mouse islets in real time using a video-rate confocal system. In both types of cells, stimulation with carbamoylcholine (CCh) and depolarization with KCl results in an increase in Ins(1,4,5)P3 accumulation in the cytoplasm. When stimulated with glucose, the Ins(1,4,5)P3 concentration in the cytoplasm oscillates in parallel with oscillations of [Ca2+]i. Maximal accumulation of Ins(1,4,5)P3 in these oscillations coincides with the peak of [Ca2+]i and tracks changes in frequencies induced by the voltage-gated K+ channel blockade. We show that Ins(1,4,5)P3 release in insulin-secreting cells can be stimulated by depolarization-induced Ca2+ flux. We conclude that Ins(1,4,5)P3 concentration oscillates in parallel with [Ca2+]i in response to glucose stimulation, but it is not the driving force for [Ca2+]i oscillations.
Assuntos
Sinalização do Cálcio/fisiologia , Inositol 1,4,5-Trifosfato/metabolismo , Ilhotas Pancreáticas/citologia , Ilhotas Pancreáticas/metabolismo , Animais , Sinalização do Cálcio/efeitos dos fármacos , Carbacol/farmacologia , Linhagem Celular Tumoral , Citoplasma/metabolismo , Retículo Endoplasmático/enzimologia , Ilhotas Pancreáticas/efeitos dos fármacos , Camundongos , Cloreto de Potássio/farmacologia , Tapsigargina/farmacologia , Fosfolipases Tipo C/metabolismoRESUMO
Growth hormone-releasing hormone (GHRH) is produced by the hypothalamus and stimulates growth hormone synthesis and release in the anterior pituitary gland. In addition, GHRH is an important regulator of cellular functions in many cells and organs. Expression of GHRH G-Protein Coupled Receptor (GHRHR) has been demonstrated in different peripheral tissues and cell types, including pancreatic islets. Among the peripheral activities, recent studies demonstrate a novel ability of GHRH analogs to increase and preserve insulin secretion by beta-cells in isolated pancreatic islets, which makes them potentially useful for diabetes treatment. This review considers the role of GHRHR in the beta-cell and addresses the unique engineered GHRH agonists and antagonists for treatment of type 2 diabetes mellitus. We discuss the similarity of signaling pathways activated by GHRHR in pituitary somatotrophs and in pancreatic beta-cells and possible ways as to how the GHRHR pathway can interact with glucose and other secretagogues to stimulate insulin secretion. We also consider the hypothesis that novel GHRHR agonists can improve glucose metabolism in Type 2 diabetes by preserving the function and survival of pancreatic beta-cells. Wound healing and cardioprotective action with new GHRH agonists suggest that they may prove useful in ameliorating certain diabetic complications. These findings highlight the future potential therapeutic effectiveness of modulators of GHRHR activity for the development of new therapeutic approaches in diabetes and its complications.
RESUMO
Glucose-stimulated insulin secretion is associated with transients of intracellular Ca(2+) concentration [Ca(2+)](i) in the pancreatic beta-cell. We identified the expression and function of specific small-conductance Ca(2+)-activated K(+) (SK) channel genes in insulin-secreting cells. The presence of mRNA for SK1, -2, -3, and -4 (intermediate-conductance Ca(2+)-activated K(+) 1 [IK1]) channels was demonstrated by RT-PCR in rodent islets and insulinoma cells. SK2 and -3 proteins in mouse islets were detected by immunoblot and immunocytochemistry. In the tTA-SK3 tet-off mouse, a normal amount of SK3 protein was present in islets, but it became undetectable after exposure to doxycycline (DOX), which inhibits the transcription of the tTA-SK3 gene. The SK/IK channel-blockers apamin, dequalinium, and charybdotoxin caused increases in average [Ca(2+)](i) levels and in frequency of [Ca(2+)](i) oscillations in wild-type mouse islets. In SK3-tTA tet-off mice, the addition of apamin with glucose and tetraethylammonium (TEA) caused a similar elevation in [Ca(2+)](i), which was greatly diminished after DOX suppression of SK3 expression. We conclude that SK1, -2, -3, and IK1 (SK4) are expressed in islet cells and insulin-secreting cells and are able to influence glucose-induced calcium responses, thereby regulating insulin secretion.
Assuntos
Glucose/metabolismo , Ilhotas Pancreáticas/metabolismo , Canais de Potássio Cálcio-Ativados , Canais de Potássio/genética , Canais de Potássio/metabolismo , Animais , Anticorpos , Apamina/farmacologia , Sinalização do Cálcio/efeitos dos fármacos , Sinalização do Cálcio/fisiologia , Células Cultivadas , Charibdotoxina/farmacologia , Dequalínio/farmacologia , Expressão Gênica/fisiologia , Canais de Potássio Ativados por Cálcio de Condutância Intermediária , Ilhotas Pancreáticas/citologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Bloqueadores dos Canais de Potássio/farmacologia , Canais de Potássio/imunologia , Canais de Potássio Ativados por Cálcio de Condutância Baixa , Tetraetilamônio/farmacologiaRESUMO
The physiological role of serotonin, or 5-hydroxytryptamine (5-HT), in pancreatic ß-cell function was previously elucidated using a pregnant mouse model. During pregnancy, 5-HT increases ß-cell proliferation and glucose-stimulated insulin secretion (GSIS) through the Gαq-coupled 5-HT2b receptor (Htr2b) and the 5-HT3 receptor (Htr3), a ligand-gated cation channel, respectively. However, the role of 5-HT in ß-cell function in an insulin-resistant state has yet to be elucidated. Here, we characterized the metabolic phenotypes of ß-cell-specific Htr2b(-/-) (Htr2b ßKO), Htr3a(-/-) (Htr3a knock-out [KO]), and ß-cell-specific tryptophan hydroxylase 1 (Tph1)(-/-) (Tph1 ßKO) mice on a high-fat diet (HFD). Htr2b ßKO, Htr3a KO, and Tph1 ßKO mice exhibited normal glucose tolerance on a standard chow diet. After 6 weeks on an HFD, beginning at 4 weeks of age, both Htr3a KO and Tph1 ßKO mice developed glucose intolerance, but Htr2b ßKO mice remained normoglycemic. Pancreas perfusion assays revealed defective first-phase insulin secretion in Htr3a KO mice. GSIS was impaired in islets isolated from HFD-fed Htr3a KO and Tph1 ßKO mice, and 5-HT treatment improved insulin secretion from Tph1 ßKO islets but not from Htr3a KO islets. Tph1 and Htr3a gene expression in pancreatic islets was not affected by an HFD, and immunostaining could not detect 5-HT in pancreatic islets from mice fed an HFD. Taken together, these results demonstrate that basal 5-HT levels in ß-cells play a role in GSIS through Htr3, which becomes more evident in a diet-induced insulin-resistant state.
Assuntos
Resistência à Insulina , Células Secretoras de Insulina/fisiologia , Insulina/metabolismo , Receptores de Serotonina/metabolismo , Serotonina/fisiologia , Animais , Dieta Hiperlipídica , Secreção de Insulina , Masculino , Camundongos Knockout , Receptores de Serotonina/genética , Triptofano Hidroxilase/genética , Triptofano Hidroxilase/metabolismoRESUMO
Gene manipulation using Cre-loxP recombination has proven to be an important approach for studying the impact of gene expression on pancreatic ß-cell biology. We report the generation of a transgenic mouse line that enables a highly specific system for conditional gene manipulation within ß-cells and achieve tissue specific and temporally regulated deletion of the Ctnnb1 (ß-catenin) gene in pancreatic ß-cells. cDNA encoding Cre recombinase fused to modified estrogen receptor (CreERT) under control of mouse insulin 1 gene promoter (Ins1) was used to construct the mouse line Tg(Ins1-Cre/ERT)1Lphi, also termed MIP1-CreERT. In a cross of MIP1-CreERT with a ROSA26/LacZ reporter strain, tamoxifen [Tmx] - dependent ß-galactosidase expression occurred within pancreatic ß-cells but not in other organ systems. Intraperitoneal glucose tolerance tests and glucose-stimulated changes in ß-cell cytoplasmic calcium concentration were not adversely affected in adult MIP1-CreERT. A mouse line with floxed Ctnnb1 gene (Ctnnb1f/f) was crossed with the MIP1-CreERT line to generate a mouse model for inducible ß-cell specific deletion of ß-catenin gene (Ctnnb1f/f:MIP1-CreERT). Ctnnb1f/f:MIP1-CreERT mice and Ctnnb1f/f littermate controls, were injected with Tmx as adults to knock down ß-catenin production in the majority of pancreatic ß-cells. These mice showed normal glucose tolerance, islet cyto-architecture and insulin secretion. A novel protein fraction of 50Kd, immunoreactive with anti-ß-catenin was observed in islet extracts from Ctnnb1f/f:MIP1-CreERT[Tmx] mice but not MIP1-CreERT-negative Ctnnb1f/f[Tmx] controls, indicating possible presence of a cryptic protein product of recombined Ctnnb1 gene. The MIP1-CreERT mouse line is a powerful tool for conditional manipulation of gene expression in ß-cells.
Assuntos
Células Secretoras de Insulina/metabolismo , Insulina/genética , Integrases/genética , Regiões Promotoras Genéticas , Receptores de Estrogênio/genética , Animais , Feminino , Deleção de Genes , Marcação de Genes , Técnicas de Transferência de Genes , Insulina/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , beta Catenina/genética , beta Catenina/metabolismoRESUMO
Drosophila melanogaster has been widely used as a model of human Mendelian disease, but its value in modeling complex disease has received little attention. Fly models of complex disease would enable high-resolution mapping of disease-modifying loci and the identification of novel targets for therapeutic intervention. Here, we describe a fly model of permanent neonatal diabetes mellitus and explore the complexity of this model. The approach involves the transgenic expression of a misfolded mutant of human preproinsulin, hINS(C96Y), which is a cause of permanent neonatal diabetes. When expressed in fly imaginal discs, hINS(C96Y) causes a reduction of adult structures, including the eye, wing, and notum. Eye imaginal discs exhibit defects in both the structure and the arrangement of ommatidia. In the wing, expression of hINS(C96Y) leads to ectopic expression of veins and mechano-sensory organs, indicating disruption of wild-type signaling processes regulating cell fates. These readily measurable "disease" phenotypes are sensitive to temperature, gene dose, and sex. Mutant (but not wild-type) proinsulin expression in the eye imaginal disc induces IRE1-mediated XBP1 alternative splicing, a signal for endoplasmic reticulum stress response activation, and produces global change in gene expression. Mutant hINS transgene tester strains, when crossed to stocks from the Drosophila Genetic Reference Panel, produce F1 adults with a continuous range of disease phenotypes and large broad-sense heritability. Surprisingly, the severity of mutant hINS-induced disease in the eye is not correlated with that in the notum in these crosses, nor with eye reduction phenotypes caused by the expression of two dominant eye mutants acting in two different eye development pathways, Drop (Dr) or Lobe (L), when crossed into the same genetic backgrounds. The tissue specificity of genetic variability for mutant hINS-induced disease has, therefore, its own distinct signature. The genetic dominance of disease-specific phenotypic variability in our model of misfolded human proinsulin makes this approach amenable to genome-wide association study in a simple F1 screen of natural variation.
Assuntos
Diabetes Mellitus/genética , Proinsulina/genética , Animais , Animais Geneticamente Modificados , Análise por Conglomerados , Modelos Animais de Doenças , Drosophila melanogaster , Olho/metabolismo , Feminino , Dosagem de Genes , Perfilação da Expressão Gênica , Humanos , Masculino , Mutação , Fenótipo , Proinsulina/química , Dobramento de Proteína , Característica Quantitativa Herdável , Transcriptoma , Transgenes , Asas de Animais/metabolismoRESUMO
The identification and validation of gene-gene interactions is a major challenge in human studies. Here, we explore an approach for studying epistasis in humans using a Drosophila melanogaster model of neonatal diabetes mellitus. Expression of the mutant preproinsulin (hINS(C96Y)) in the eye imaginal disc mimics the human disease: it activates conserved stress-response pathways and leads to cell death (reduction in eye area). Dominant-acting variants in wild-derived inbred lines from the Drosophila Genetics Reference Panel produce a continuous, highly heritable distribution of eye-degeneration phenotypes in a hINS(C96Y) background. A genome-wide association study (GWAS) in 154 sequenced lines identified a sharp peak on chromosome 3L, which mapped to a 400-bp linkage block within an intron of the gene sulfateless (sfl). RNAi knockdown of sfl enhanced the eye-degeneration phenotype in a mutant-hINS-dependent manner. RNAi against two additional genes in the heparan sulfate (HS) biosynthetic pathway (ttv and botv), in which sfl acts, also modified the eye phenotype in a hINS(C96Y)-dependent manner, strongly suggesting a novel link between HS-modified proteins and cellular responses to misfolded proteins. Finally, we evaluated allele-specific expression difference between the two major sfl-intronic haplotypes in heterozygtes. The results showed significant heterogeneity in marker-associated gene expression, thereby leaving the causal mutation(s) and its mechanism unidentified. In conclusion, the ability to create a model of human genetic disease, map a QTL by GWAS to a specific gene, and validate its contribution to disease with available genetic resources and the potential to experimentally link the variant to a molecular mechanism demonstrate the many advantages Drosophila holds in determining the genetic underpinnings of human disease.
Assuntos
Diabetes Mellitus/genética , Variação Genética , Proinsulina/genética , Alelos , Animais , Animais Geneticamente Modificados , Diabetes Mellitus/metabolismo , Modelos Animais de Doenças , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Epistasia Genética , Olho/metabolismo , Olho/patologia , Feminino , Expressão Gênica , Técnicas de Silenciamento de Genes , Estudo de Associação Genômica Ampla , Heparitina Sulfato/biossíntese , Humanos , Íntrons , Masculino , Mutação , Fenótipo , Proinsulina/química , Dobramento de Proteína , Interferência de RNA , Sulfotransferases/química , Sulfotransferases/genética , Sulfotransferases/metabolismoRESUMO
OBJECTIVE: Conditional gene targeting has been extensively used for in vivo analysis of gene function in ß-cell biology. The objective of this study was to examine whether mouse transgenic Cre lines, used to mediate ß-cell- or pancreas-specific recombination, also drive Cre expression in the brain. RESEARCH DESIGN AND METHODS: Transgenic Cre lines driven by Ins1, Ins2, and Pdx1 promoters were bred to R26R reporter strains. Cre activity was assessed by ß-galactosidase or yellow fluorescent protein expression in the pancreas and the brain. Endogenous Pdx1 gene expression was monitored using Pdx1(tm1Cvw) lacZ knock-in mice. Cre expression in ß-cells and co-localization of Cre activity with orexin-expressing and leptin-responsive neurons within the brain was assessed by immunohistochemistry. RESULTS: All transgenic Cre lines examined that used the Ins2 promoter to drive Cre expression showed widespread Cre activity in the brain, whereas Cre lines that used Pdx1 promoter fragments showed more restricted Cre activity primarily within the hypothalamus. Immunohistochemical analysis of the hypothalamus from Tg(Pdx1-cre)(89.1Dam) mice revealed Cre activity in neurons expressing orexin and in neurons activated by leptin. Tg(Ins1-Cre/ERT)(1Lphi) mice were the only line that lacked Cre activity in the brain. CONCLUSIONS: Cre-mediated gene manipulation using transgenic lines that express Cre under the control of the Ins2 and Pdx1 promoters are likely to alter gene expression in nutrient-sensing neurons. Therefore, data arising from the use of these transgenic Cre lines must be interpreted carefully to assess whether the resultant phenotype is solely attributable to alterations in the islet ß-cells.
Assuntos
Marcação de Genes/métodos , Células Secretoras de Insulina/fisiologia , Integrases/genética , Tamoxifeno/farmacologia , Animais , Encéfalo/fisiologia , Cruzamentos Genéticos , Antagonistas de Estrogênios/farmacologia , Feminino , Galactosídeos/metabolismo , Genes Reporter/genética , Imunoglobulina G , Imuno-Histoquímica , Insulina/imunologia , Leptina/farmacologia , Masculino , Camundongos , Camundongos Transgênicos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , SuínosRESUMO
Calcium (Ca(2+)) signaling regulates insulin secretion in pancreatic beta-cells. STIM1 has been proposed to function as an endoplasmic reticulum (ER) Ca(2+) sensor regulating store-operated Ca(2+) entry (SOCE). Here we studied the translocation of EYFP-STIM1 in response to ER calcium depletion in mouse insulinoma MIN6 cells by fluorescent microscopy. While in resting cells EYFP-STIM1 is co-localized with an ER marker, in thapsigargin (Tg)-stimulated cells it occupied highly defined areas of the peri-PM space in punctae adjacent to, but not entirely coincident with the ER. Co-staining with fluorescent phalloidin revealed that EYFP-STIM1 punctae was located in actin-poor areas. Use of the SOCE blocker in MIN6 cells, 2-aminoethoxy diphenylborate (2-APB), prevented store depletion-dependent translocation of EYFP-STIM1 to the PM in a concentration-dependent (3.75-100muM) and reversible manner. TIRF microscopy revealed that 2-APB treatment led to the reversible disappearance of peri-PM EYFP-STIM1 punctae, while the ER structure in this compartment remained grossly unaffected. We conclude from this data that in these cells EYFP-STIM1 is delivered to a peri-PM location from the ER upon store depletion and this trafficking is reversibly blocked by 2-APB.
Assuntos
Proteínas de Bactérias/metabolismo , Sinalização do Cálcio , Células Secretoras de Insulina/metabolismo , Proteínas Luminescentes/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Animais , Compostos de Boro/farmacologia , Cálcio/farmacologia , Canais de Cálcio , Sinalização do Cálcio/efeitos dos fármacos , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Células Secretoras de Insulina/citologia , Células Secretoras de Insulina/efeitos dos fármacos , Camundongos , Transporte Proteico/efeitos dos fármacos , Molécula 1 de Interação EstromalRESUMO
Calcium-binding proteins regulate transcription and secretion of pancreatic islet hormones. Here, we demonstrate neuroendocrine expression of the calcium-binding downstream regulatory element antagonistic modulator (DREAM) and its role in glucose-dependent regulation of prodynorphin (PDN) expression. DREAM is distributed throughout beta- and alpha-cells in both the nucleus and cytoplasm. As DREAM regulates neuronal dynorphin expression, we determined whether this pathway is affected in DREAM(-/-) islets. Under low glucose conditions, with intracellular calcium concentrations of <100 nM, DREAM(-/-) islets had an 80% increase in PDN message compared with controls. Accordingly, DREAM interacts with the PDN promoter downstream regulatory element (DRE) under low calcium (<100 nM) conditions, inhibiting PDN transcription in beta-cells. Furthermore, beta-cells treated with high glucose (20 mM) show increased cytoplasmic calcium (approximately 200 nM), which eliminates DREAM's interaction with the DRE, causing increased PDN promoter activity. As PDN is cleaved into dynorphin peptides, which stimulate kappa-opioid receptors expressed predominantly in alpha-cells of the islet, we determined the role of dynorphin A-(1-17) in glucagon secretion from the alpha-cell. Stimulation with dynorphin A-(1-17) caused alpha-cell calcium fluctuations and a significant increase in glucagon release. DREAM(-/-) islets also show elevated glucagon secretion in low glucose compared with controls. These results demonstrate that PDN transcription is regulated by DREAM in a calcium-dependent manner and suggest a role for dynorphin regulation of alpha-cell glucagon secretion. The data provide a molecular basis for opiate stimulation of glucagon secretion first observed over 25 years ago.
Assuntos
Encefalinas/genética , Regulação da Expressão Gênica , Ilhotas Pancreáticas/metabolismo , Proteínas Interatuantes com Canais de Kv/metabolismo , Precursores de Proteínas/genética , Proteínas Repressoras/metabolismo , (trans)-Isômero de 3,4-dicloro-N-metil-N-(2-(1-pirrolidinil)-ciclo-hexil)-benzenoacetamida/farmacologia , Animais , Cálcio/metabolismo , Linhagem Celular , Núcleo Celular/metabolismo , DNA/metabolismo , Dinorfinas/farmacologia , Ensaio de Desvio de Mobilidade Eletroforética , Glucagon/metabolismo , Células Secretoras de Glucagon/química , Células Secretoras de Glucagon/efeitos dos fármacos , Células Secretoras de Glucagon/metabolismo , Glucose/farmacologia , Humanos , Células Secretoras de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/metabolismo , Ilhotas Pancreáticas/efeitos dos fármacos , Proteínas Interatuantes com Canais de Kv/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Naltrexona/análogos & derivados , Naltrexona/farmacologia , Ligação Proteica/fisiologia , Receptores Opioides kappa/análise , Receptores Opioides kappa/antagonistas & inibidores , Proteínas Repressoras/genéticaRESUMO
The delayed-rectifier (voltage-activated) K(+) conductance (K(V)) in pancreatic islet beta-cells has been proposed to regulate plasma membrane repolarization during responses to glucose, thereby determining bursting and Ca(2+) oscillations. Here, we verified the expression of K(V)2.1 channel protein in mouse and human islets of Langerhans. We then probed the function of K(V)2.1 channels in islet glucose responses by comparing the effect of hanatoxin (HaTx), a specific blocker of K(V)2.1 channels, with a nonspecific K(+) channel blocker, tetraethylammonium (TEA). Application of HaTx (1 microM) blocked delayed-rectifier currents in mouse beta-cells, resulting in a 40-mV rightward shift in threshold of activation of the voltage-dependent outward current. In the presence of HaTx, there was negligible voltage-activated outward current below 0 mV, suggesting that K(V)2.1 channels form the predominant part of this current in the physiologically relevant range. We then employed HaTx to study the role of K(V)2.1 in the beta-cell Ca(2+) responses to elevated glucose in comparison with TEA. Only HaTx was able to induce slow intracellular Ca(2+) concentration ([Ca(2+)](i)) oscillations in cells stimulated with 20 mM glucose, whereas TEA induced an immediate rise in [Ca(2+)](i) followed by rapid oscillations. In human islets, HaTx acted in a similar fashion. The data were analyzed using a detailed mathematical model of ionic flux and Ca(2+) regulation in beta-cells. The results can be explained by a specific HaTx effect on the K(V) current, whereas TEA affects multiple K(+) conductances. The results underscore the importance of K(V)2.1 channel in repolarization of the pancreatic beta-cell plasma membrane and its role in regulating insulin secretion.