RESUMO
We evaluated the role of ATP-sensitive K⺠(K(ATP)) channels, somatostatin, and Zn²âº in the control of glucagon secretion from mouse islets. Switching from 1 to 7 mmol/L glucose inhibited glucagon release. Diazoxide did not reverse the glucagonostatic effect of glucose. Tolbutamide decreased glucagon secretion at 1 mmol/L glucose (G1) but stimulated it at 7 mmol/L glucose (G7). The reduced glucagon secretion produced by high concentrations of tolbutamide or diazoxide, or disruption of K(ATP) channels (Sur1(-/-) mice) at G1 could be inhibited further by G7. Removal of the somatostatin paracrine influence (Sst(-/-) mice or pretreatement with pertussis toxin) strongly increased glucagon release, did not prevent the glucagonostatic effect of G7, and unmasked a marked glucagonotropic effect of tolbutamide. Glucose inhibited glucagon release in the absence of functional K(ATP) channels and somatostatin signaling. Knockout of the Zn²âº transporter ZnT8 (ZnT8(-/-) mice) did not prevent the glucagonostatic effect of glucose. In conclusion, glucose can inhibit glucagon release independently of Zn²âº, K(ATP) channels, and somatostatin. Closure of K(ATP) channels controls glucagon secretion by two mechanisms, a direct stimulation of α-cells and an indirect inhibition via somatostatin released from δ-cells. The net effect on glucagon release results from a balance between both effects.
Assuntos
Glucagon/metabolismo , Hipoglicemiantes/farmacologia , Células Secretoras de Insulina/efeitos dos fármacos , Ilhotas Pancreáticas/efeitos dos fármacos , Canais KATP/metabolismo , Células Secretoras de Somatostatina/efeitos dos fármacos , Tolbutamida/farmacologia , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Animais , Proteínas de Transporte de Cátions/genética , Proteínas de Transporte de Cátions/metabolismo , Cruzamentos Genéticos , Diazóxido/farmacologia , Glucose/metabolismo , Células Secretoras de Insulina/metabolismo , Ilhotas Pancreáticas/metabolismo , Canais KATP/agonistas , Canais KATP/antagonistas & inibidores , Moduladores de Transporte de Membrana/farmacologia , Camundongos , Camundongos Knockout , Concentração Osmolar , Bloqueadores dos Canais de Potássio/farmacologia , Canais de Potássio Corretores do Fluxo de Internalização/genética , Canais de Potássio Corretores do Fluxo de Internalização/metabolismo , Receptores de Droga/genética , Receptores de Droga/metabolismo , Somatostatina/genética , Somatostatina/metabolismo , Células Secretoras de Somatostatina/metabolismo , Receptores de Sulfonilureias , Técnicas de Cultura de Tecidos , Transportador 8 de ZincoRESUMO
OBJECTIVE: Sarco-endoplasmic reticulum Ca(2+)-ATPase 2b (SERCA2b) and SERCA3 pump Ca(2+) in the endoplasmic reticulum (ER) of pancreatic ß-cells. We studied their role in the control of the free ER Ca(2+) concentration ([Ca(2+)](ER)) and the role of SERCA3 in the control of insulin secretion and ER stress. RESEARCH DESIGN AND METHODS: ß-Cell [Ca(2+)](ER) of SERCA3(+/+) and SERCA3(-/-) mice was monitored with an adenovirus encoding the low Ca(2+)-affinity sensor D4 addressed to the ER (D4ER) under the control of the insulin promoter. Free cytosolic Ca(2+) concentration ([Ca(2+)](c)) and [Ca(2+)](ER) were simultaneously recorded. Insulin secretion and mRNA levels of ER stress genes were studied. RESULTS: Glucose elicited synchronized [Ca(2+)](ER) and [Ca(2+)](c) oscillations. [Ca(2+)](ER) oscillations were smaller in SERCA3(-/-) than in SERCA3(+/+) ß-cells. Stimulating cell metabolism with various [glucose] in the presence of diazoxide induced a similar dose-dependent [Ca(2+)](ER) rise in SERCA3(+/+) and SERCA3(-/-) ß-cells. In a Ca(2+)-free medium, glucose moderately raised [Ca(2+)](ER) from a highly buffered cytosolic Ca(2+) pool. Increasing [Ca(2+)](c) with high [K] elicited a [Ca(2+)](ER) rise that was larger but more transient in SERCA3(+/+) than SERCA3(-/-) ß-cells because of the activation of a Ca(2+) release from the ER in SERCA3(+/+) ß-cells. Glucose-induced insulin release was larger in SERCA3(-/-) than SERCA3(+/+) islets. SERCA3 ablation did not induce ER stress. CONCLUSIONS: [Ca(2+)](c) and [Ca(2+)](ER) oscillate in phase in response to glucose. Upon [Ca(2+)](c) increase, Ca(2+) is taken up by SERCA2b and SERCA3. Strong Ca(2+) influx triggers a Ca(2+) release from the ER that depends on SERCA3. SERCA3 deficiency neither impairs Ca(2+) uptake by the ER upon cell metabolism acceleration and insulin release nor induces ER stress.
Assuntos
Cálcio/metabolismo , Células Secretoras de Insulina/metabolismo , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Animais , Cálcio/farmacologia , Diazóxido/farmacologia , Retículo Endoplasmático/metabolismo , Deleção de Genes , Regulação da Expressão Gênica , Engenharia Genética , Glucose/farmacologia , Insulina/genética , Insulina/metabolismo , Células Secretoras de Insulina/efeitos dos fármacos , Camundongos , Camundongos Knockout , Regiões Promotoras Genéticas , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/genética , Vasodilatadores/farmacologiaRESUMO
BACKGROUND: The total number of miRNA genes in a genome, expression of which is responsible for the miRNA repertoire of an organism, is not precisely known. Moreover, the question of how new miRNA genes arise during evolution is incompletely understood. Recent data in humans and opossum indicate that retrotranspons of the class of short interspersed nuclear elements have contributed to the growth of microRNA gene clusters. METHOD: We studied a large miRNA gene cluster in intron 10 of the mouse Sfmbt2 gene using bioinformatic tools. RESULTS: Mice and rats are unique to harbor a 55-65 Kb DNA sequence in intron 10 of the Sfmbt2 gene. This intronic region is rich in regularly repeated B1 retrotransposons together with inverted self-complementary CA/TG microsatellites. The smallest repeats unit, called MSHORT1 in the mouse, was duplicated 9 times in a tandem head-to-tail array to form 2.5 Kb MLONG1 units. The center of the mouse miRNA gene cluster consists of 13 copies of MLONG1. BLAST analysis of MSHORT1 in the mouse shows that the repeat unit is unique for intron 10 of the Sfmbt2 gene and suggest a dual phase model for growth of the miRNA gene cluster:arrangement [corrected] of 10 MSHORT1 units into MLONG1 and further duplication of 13 head-to-tail MLONG1 units in the center of the miRNA gene cluster. Rats have a similar arrangement [corrected] of repeat units in intron 10 of the Sfmbt2 gene. The discrepancy between 65 miRNA genes in the mouse cluster as compared to only 1 miRNA gene in the corresponding rat repeat cluster is ascribed to sequence differences between MSHORT1 and RSHORT1 that result in lateral-shifted, less-stable miRNA precursor hairpins for RSHORT1. CONCLUSION: Our data provides new evidence for the emerging concept that lineage-specific retroposons have played an important role in the birth of new miRNA genes during evolution. The large difference in the number of miRNA genes in two closely related species (65 versus 1, mice versus rats) indicates that this species-specific evolution can be a rapid process.
Assuntos
Biologia Computacional , Evolução Molecular , Íntrons/genética , MicroRNAs/genética , Modelos Genéticos , Família Multigênica/genética , Fatores de Transcrição/genética , Animais , Cromossomos de Mamíferos/genética , Humanos , Sequências Repetidas Invertidas/genética , Camundongos , Repetições de Microssatélites/genética , Ratos , Proteínas Repressoras , Retroelementos/genética , Análise de Sequência de RNA , Especificidade da EspécieRESUMO
Pleomorphic adenoma gene-like 1 (PLAGL1) has been linked to transient neonatal diabetes mellitus. Here, we investigated the role of the related pleomorphic adenoma gene 1 (PLAG1) in glucose homeostasis. PLAG1 transgenic mice in which expression of the PLAG1 transgene can be targeted to different organs by Cre-mediated modulation were crossed with Pdx1-Cre or Ngn3-Cre mice, resulting in double transgenic P1-Pdx1Cre or P1-Ngn3Cre mice, respectively. P1-Pdx1Cre and P1-Ngn3Cre mice developed hyperplasia of pancreatic islets due to increased ß- and δ- but not α-cell proliferation. In young P1-Pdx1Cre mice (less than 15 weeks) there was a balanced increase in the pancreatic content of insulin and somatostatin, which was associated with normoglycemia. In older P1-Pdx1Cre mice the pancreatic somatostatin content far exceeded that of insulin, leading to the progressive development of severe hypoglycemia beyond 30 weeks. In contrast, in older P1-Ngn3Cre mice the relative increase of the pancreatic insulin content exceeded that of somatostatin and these mice remained normoglycemic. In conclusion, forced expression of PLAG1 under the control of the Pdx1 or Ngn3 promoter in murine pancreas induces different degrees of endocrine hormone imbalances within the pancreas, which is associated with hypoglycemia in P1-Pdx1Cre mice but not P1-Ngn3Cre mice. These results suggest that once stem cell-derived islet transplantations become possible, the appropriate balance between different hormone-producing cells will need to be preserved to prevent deregulated glucose metabolism.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Proteínas de Ligação a DNA/metabolismo , Glucose/metabolismo , Proteínas de Homeodomínio/metabolismo , Integrases/metabolismo , Ilhotas Pancreáticas/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Hormônios Pancreáticos/metabolismo , Transativadores/metabolismo , Animais , Proliferação de Células , Glucagon , Teste de Tolerância a Glucose , Homeostase , Hiperplasia , Hipoglicemia/patologia , Insulina , Ilhotas Pancreáticas/patologia , Ilhotas Pancreáticas/ultraestrutura , Transplante das Ilhotas Pancreáticas , Camundongos , Camundongos Transgênicos , Tamanho do Órgão , Somatostatina , Fatores de TempoRESUMO
Glucose homeostasis is critically dependent on insulin release from pancreatic beta-cells, which is strictly regulated by glucose-induced oscillations in membrane potential (V(m)) and the cytosolic calcium level ([Ca(2+)](cyt)). We propose that TRPM5, a Ca(2+)-activated monovalent cation channel, is a positive regulator of glucose-induced insulin release. Immunofluorescence revealed expression of TRPM5 in pancreatic islets. A Ca(2+)-activated nonselective cation current with TRPM5-like properties is significantly reduced in Trpm5(-/-) cells. Ca(2+)-imaging and electrophysiological analysis show that glucose-induced oscillations of V(m) and [Ca(2+)](cyt) have on average a reduced frequency in Trpm5(-/-) islets, specifically due to a lack of fast oscillations. As a consequence, glucose-induced insulin release from Trpm5(-/-) pancreatic islets is significantly reduced, resulting in an impaired glucose tolerance in Trpm5(-/-) mice.
Assuntos
Sinalização do Cálcio/efeitos dos fármacos , Glucose/farmacologia , Ilhotas Pancreáticas/efeitos dos fármacos , Ilhotas Pancreáticas/metabolismo , Canais de Cátion TRPM/deficiência , Animais , Cátions , Regulação da Expressão Gênica/efeitos dos fármacos , Teste de Tolerância a Glucose , Insulina/metabolismo , Secreção de Insulina , Espaço Intracelular/efeitos dos fármacos , Espaço Intracelular/metabolismo , Ativação do Canal Iônico/efeitos dos fármacos , Ilhotas Pancreáticas/citologia , Potenciais da Membrana/efeitos dos fármacos , Camundongos , Fenótipo , Canais de Cátion TRPM/genética , Canais de Cátion TRPM/metabolismoRESUMO
MicroRNAs (miRNAs) are a class of 19-25 nt long non-coding RNAs that regulate gene expression post-transcriptionally by binding with partially complementary sequences in the 3'-untranslated region (3'-UTR) and inhibiting mRNA translation or by affecting mRNA stability. We have characterized the structures and the equilibrium between hairpin and homo-duplex form of the mature strand of hsa-mir-520h by various concentration and temperature dependent 1D, 2D NMR experiments and those structures correspond well with Mfold-folded and UNAFold-aligned secondary structures. A detailed folding and alignment analysis in physiological conditions of all mature miRNA strands from the complete database of known miRNAs (miRBase) was performed. The statistical analysis of the resulting folding and alignment data showed for the first time the potential of a large number of mature miRNAs to form significant hairpin and/or homo-duplex structures in solution. The self-complementarity of mature miRNAs can provide a mechanistic tuning and a regulatory sophistication to the process of miRNA mediated gene regulation.
Assuntos
MicroRNAs/química , Conformação de Ácido Nucleico , Estabilidade de RNA , RNA Complementar/química , Sequência de Bases , Temperatura Alta , Humanos , MicroRNAs/genética , Ressonância Magnética Nuclear Biomolecular , RNA Complementar/genética , Alinhamento de SequênciaRESUMO
Islets of patients with type 2 diabetes mellitus (T2DM) display features of an inflammatory process including elevated levels of the cytokine IL-1beta, various chemokines, and macrophages. IL-1beta is a master regulator of inflammation, and IL-1 receptor type I (IL-1RI) blockage improves glycemia and insulin secretion in humans with T2DM and in high-fat-fed mice pointing to a pivotal role of IL-1RI activity in intra-islet inflammation. Given the association of dyslipidemia and T2DM, we tested whether free fatty acids (FFA) promote the expression of proinflammatory factors in human and mouse islets and investigated a role for the IL-1RI in this response. A comparison of 22 mouse tissues revealed the highest IL-1RI expression levels in islets and MIN6 beta-cells. FFA induced IL-1beta, IL-6, and IL-8 in human islets and IL-1beta and KC in mouse islets. Elevated glucose concentrations enhanced FFA-induced proinflammatory factors in human islets. Blocking the IL-1RI with the IL-1R antagonist (IL-1Ra) strongly inhibited FFA-mediated expression of proinflammatory factors in human and mouse islets. Antibody inhibition of IL-1beta revealed that FFA stimulated IL-1RI activity via the induction of the receptor ligand. FFA-induced IL-1beta and KC expression in mouse islets was completely dependent on the IL-1R/Toll-like receptor (TLR) docking protein Myd88 and partly dependent on TLR2 and -4. Activation of TLR2 in purified human beta-cells and islets stimulated the expression of proinflammatory factors, and IL-1RI activity increased the TLR2 response in human islets. We conclude that FFA and TLR stimulation induce proinflammatory factors in islets and that IL-1RI engagement results in signal amplification.
Assuntos
Ácidos Graxos não Esterificados/farmacologia , Mediadores da Inflamação/metabolismo , Ilhotas Pancreáticas/efeitos dos fármacos , Receptores de Interleucina-1/metabolismo , Adulto , Idoso , Animais , Western Blotting , Linhagem Celular Tumoral , Células Cultivadas , Feminino , Humanos , Proteína Antagonista do Receptor de Interleucina 1/genética , Proteína Antagonista do Receptor de Interleucina 1/metabolismo , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Interleucina-8/genética , Interleucina-8/metabolismo , Ilhotas Pancreáticas/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pessoa de Meia-Idade , Fator 88 de Diferenciação Mieloide/genética , Fator 88 de Diferenciação Mieloide/metabolismo , Receptores de Interleucina-1/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Receptor 2 Toll-Like/genética , Receptor 2 Toll-Like/metabolismo , Receptor 4 Toll-Like/genética , Receptor 4 Toll-Like/metabolismo , Adulto JovemRESUMO
OBJECTIVE: Zinc ions are essential for the formation of hexameric insulin and hormone crystallization. A nonsynonymous single nucleotide polymorphism rs13266634 in the SLC30A8 gene, encoding the secretory granule zinc transporter ZnT8, is associated with type 2 diabetes. We describe the effects of deleting the ZnT8 gene in mice and explore the action of the at-risk allele. RESEARCH DESIGN AND METHODS: Slc30a8 null mice were generated and backcrossed at least twice onto a C57BL/6J background. Glucose and insulin tolerance were measured by intraperitoneal injection or euglycemic clamp, respectively. Insulin secretion, electrophysiology, imaging, and the generation of adenoviruses encoding the low- (W325) or elevated- (R325) risk ZnT8 alleles were undertaken using standard protocols. RESULTS: ZnT8(-/-) mice displayed age-, sex-, and diet-dependent abnormalities in glucose tolerance, insulin secretion, and body weight. Islets isolated from null mice had reduced granule zinc content and showed age-dependent changes in granule morphology, with markedly fewer dense cores but more rod-like crystals. Glucose-stimulated insulin secretion, granule fusion, and insulin crystal dissolution, assessed by total internal reflection fluorescence microscopy, were unchanged or enhanced in ZnT8(-/-) islets. Insulin processing was normal. Molecular modeling revealed that residue-325 was located at the interface between ZnT8 monomers. Correspondingly, the R325 variant displayed lower apparent Zn(2+) transport activity than W325 ZnT8 by fluorescence-based assay. CONCLUSIONS: ZnT8 is required for normal insulin crystallization and insulin release in vivo but not, remarkably, in vitro. Defects in the former processes in carriers of the R allele may increase type 2 diabetes risks.
Assuntos
Glicemia/metabolismo , Proteínas de Transporte de Cátions/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Insulina/metabolismo , Zinco/metabolismo , Animais , Proteínas de Transporte de Cátions/genética , Grânulos Citoplasmáticos/metabolismo , Diabetes Mellitus Tipo 2/epidemiologia , Diabetes Mellitus Tipo 2/genética , Exocitose/fisiologia , Feminino , Expressão Gênica/fisiologia , Células HeLa , Homeostase/fisiologia , Humanos , Secreção de Insulina , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/patologia , Células Secretoras de Insulina/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Polimorfismo Genético , Fatores de Risco , Transportador 8 de ZincoRESUMO
This paper connects Alu repeats, the most abundant repetitive elements in the human genome and microRNAs, small RNAs that alter gene expression at the post-transcriptional level. Base-pair complementarity could be demonstrated between the seed sequence of a subset of human microRNAs and Alu repeats that are integrated parallel (sense) in mRNAs. The most common target site coincides with the evolutionary most conserved part of Alu. A primate-specific gene cluster on chromosome 19 encodes the majority of miRNAs that target the most conserved sense Alu site. The individual miRNA genes within this cluster are flanked by an Alu-LINE signature, which has been duplicated with the clustered miRNA genes. Gene duplication events in this locus are supported by comparing repeat length variations of the LINE elements within the cluster with those in the rest of the chromosome. Thus, a dual relationship exists between an evolutionary young miRNA cluster and their Alu targets that may have evolved in the same time window. One hypothesis for this dual relationship is that these miRNAs could protect against too high rates of duplicative transposition, which would destroy the genome.
Assuntos
Elementos Alu/genética , Evolução Molecular , MicroRNAs/genética , Sequência de Bases , Cromossomos Humanos Par 19/genética , DNA Antissenso/genética , Duplicação Gênica , Humanos , Elementos Nucleotídeos Longos e Dispersos/genética , Dados de Sequência Molecular , Família MultigênicaRESUMO
Interleukin-6 (IL-6) is systemically elevated in obesity and is a predictive factor to develop type 2 diabetes. Pancreatic islet pathology in type 2 diabetes is characterized by reduced beta-cell function and mass, an increased proportion of alpha-cells relative to beta-cells, and alpha-cell dysfunction. Here we show that the alpha cell is a primary target of IL-6 actions. Beginning with investigating the tissue-specific expression pattern of the IL-6 receptor (IL-6R) in both mice and rats, we find the highest expression of the IL-6R in the endocrine pancreas, with highest expression on the alpha-cell. The islet IL-6R is functional, and IL-6 acutely regulates both pro-glucagon mRNA and glucagon secretion in mouse and human islets, with no acute effect on insulin secretion. Furthermore, IL-6 stimulates alpha-cell proliferation, prevents apoptosis due to metabolic stress, and regulates alpha-cell mass in vivo. Using IL-6 KO mice fed a high-fat diet, we find that IL-6 is necessary for high-fat diet-induced increased alpha-cell mass, an effect that occurs early in response to diet change. Further, after high-fat diet feeding, IL-6 KO mice without expansion of alpha-cell mass display decreased fasting glucagon levels. However, despite these alpha-cell effects, high-fat feeding of IL-6 KO mice results in increased fed glycemia due to impaired insulin secretion, with unchanged insulin sensitivity and similar body weights. Thus, we conclude that IL-6 is necessary for the expansion of pancreatic alpha-cell mass in response to high-fat diet feeding, and we suggest that this expansion may be needed for functional beta-cell compensation to increased metabolic demand.
Assuntos
Células Secretoras de Glucagon/citologia , Interleucina-6/metabolismo , Animais , Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Gorduras na Dieta/farmacologia , Comportamento Alimentar/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Glucagon/genética , Glucagon/metabolismo , Células Secretoras de Glucagon/efeitos dos fármacos , Teste de Tolerância a Glucose , Humanos , Células Secretoras de Insulina/citologia , Células Secretoras de Insulina/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fosforilação/efeitos dos fármacos , Precursores de Proteínas/genética , Precursores de Proteínas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Receptores de Interleucina-6/genética , Receptores de Interleucina-6/metabolismo , Fator de Transcrição STAT3/metabolismoAssuntos
Diabetes Mellitus/metabolismo , Peptídeo 1 Semelhante ao Glucagon/metabolismo , Hipoglicemiantes/farmacologia , Células Secretoras de Insulina/citologia , Receptores de Glucagon/metabolismo , Transdução de Sinais/fisiologia , Proliferação de Células , Receptor do Peptídeo Semelhante ao Glucagon 1 , Humanos , Células Secretoras de Insulina/efeitos dos fármacos , Interferência de RNA , RNA Mensageiro/metabolismoRESUMO
Both the rate of overall translation and the specific acceleration of proinsulin synthesis are known to be glucose-regulated processes in the beta-cell. In this study, we propose that glucose-induced stimulation of overall translation in beta-cells depends on a protein phosphatase-1-mediated decrease in serine-51 phosphorylation of eukaryotic translation initiation factor 2alpha (eIF2alpha), a pivotal translation initiation factor. The decrease was rapid and detectable within 15 min and proportional to the range of glucose concentrations that also stimulate translation. Lowered net eIF2alpha phosphorylation was not associated with a detectable decrease in activity of any eIF2alpha kinase. Moreover, okadaic acid blocked glucose-induced eIF2alpha dephosphorylation, suggesting that the net effect was mediated by a protein phosphatase. Experiments with salubrinal on intact cells and nuclear inhibitor of protein phosphatase-1 (PP1) on cell extracts suggested that this phosphatase was PP1. The net effect contained, however, a component of glucose-induced folding load in the endoplasmic reticulum because coincubation with cycloheximide further amplified the effect of glucose on eIF2alpha dephosphorylation. Thus, the steady-state level of eIF2alpha phosphorylation in beta-cells is the result of a balance between folding-load-induced phosphorylation and PP1-dependent dephosphorylation. Because defects in the pancreatic endoplasmic reticulum kinase-eIF2alpha signaling system lead to beta-cell failure and diabetes, deregulation of the PP1 system could likewise lead to cellular dysfunction and disease.
Assuntos
Fator de Iniciação 2 em Eucariotos/metabolismo , Glucose/farmacologia , Células Secretoras de Insulina/metabolismo , Fosfoproteínas Fosfatases/metabolismo , Biossíntese de Proteínas/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Animais , Linhagem Celular , Cicloeximida/farmacologia , Sinergismo Farmacológico , Retículo Endoplasmático/metabolismo , Ativação Enzimática , Homeostase/fisiologia , Técnicas In Vitro , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fosforilação/efeitos dos fármacos , Dobramento de Proteína , Proteína Fosfatase 1 , Inibidores da Síntese de Proteínas/farmacologia , Proteínas/metabolismo , eIF-2 Quinase/metabolismoRESUMO
Task division in multicellular organisms ensures that differentiated cell types produce cell-specific proteins that fulfill tasks for the whole organism. In some cases, the encoded mRNA species is so abundant that it represents a sizeable fraction of total mRNA in the cell. In this study, we have used a probe- and primer-free technique to quantify such abundant mRNA species in order to assess regulatory effects of in vitro and in vivo conditions. As a first example, we were able to quantify the regulation of proinsulin mRNA abundance in beta-cells by food intake or by the glucose concentration in tissue culture. The second example of application of this technique is the effect of corticosteroids on growth hormone mRNA in enriched somatrotrophs. It is anticipated that other examples exist in which measurement of very abundant mRNAs in dedicated cells will help to understand biological processes, monitor disease states, or assist biotechnological manufacturing procedures.
Assuntos
Insulina/genética , RNA Mensageiro/genética , Actinas/genética , Animais , Biotecnologia/métodos , Ingestão de Energia , Ilhotas Pancreáticas/fisiologia , Cinética , Proinsulina/genética , RNA Complementar/genética , Ratos , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Type 2 diabetes is a disorder of hyperglycemia resulting from failure of beta cells to produce adequate insulin to accommodate an increased metabolic demand. Here we show that regulation of mRNA translation through phosphorylation of eukaryotic initiation factor 2 (eIF2alpha) is essential to preserve the integrity of the endoplasmic reticulum (ER) and to increase insulin production to meet the demand imposed by a high-fat diet. Accumulation of unfolded proteins in the ER activates phosphorylation of eIF2alpha at Ser51 and inhibits translation. To elucidate the role of this pathway in beta-cell function we studied glucose homeostasis in Eif2s1(tm1Rjk) mutant mice, which have an alanine substitution at Ser51. Heterozygous (Eif2s1(+/tm1Rjk)) mice became obese and diabetic on a high-fat diet. Profound glucose intolerance resulted from reduced insulin secretion accompanied by abnormal distension of the ER lumen, defective trafficking of proinsulin, and a reduced number of insulin granules in beta cells. We propose that translational control couples insulin synthesis with folding capacity to maintain ER integrity and that this signal is essential to prevent diet-induced type 2 diabetes.
Assuntos
Retículo Endoplasmático/metabolismo , Fator de Iniciação 2 em Eucariotos/metabolismo , Glucose/metabolismo , Ilhotas Pancreáticas/metabolismo , Biossíntese de Proteínas/genética , Substituição de Aminoácidos , Animais , Células Cultivadas , Diabetes Mellitus Tipo 2/etiologia , Dietas da Moda , Dieta com Restrição de Gorduras , Retículo Endoplasmático/genética , Retículo Endoplasmático/patologia , Chaperona BiP do Retículo Endoplasmático , Fator de Iniciação 2 em Eucariotos/genética , Intolerância à Glucose/genética , Proteínas de Choque Térmico/metabolismo , Homeostase/genética , Insulina/metabolismo , Secreção de Insulina , Ilhotas Pancreáticas/efeitos dos fármacos , Ilhotas Pancreáticas/patologia , Camundongos , Camundongos Mutantes , Chaperonas Moleculares/metabolismo , Obesidade/genética , Fosforilação , Proinsulina/metabolismoRESUMO
Cellular redox state is an important metabolic variable, influencing many aspects of cell function like growth, apoptosis, and reductive biosynthesis. In this report, we identify NADPH as a candidate signaling molecule for exocytosis in neuroendocrine cells. In pancreatic beta-cells, glucose acutely raised the NADPH-to-NADP+ ratio and stimulated insulin release in parallel. Furthermore, intracellular addition of NADPH directly stimulated exocytosis of insulin granules. Effects of NADPH on exocytosis are proposed to be mediated by the redox proteins glutaredoxin (GRX) and thioredoxin (TRX) on the basis of the following evidence: 1) Expression of GRX mRNA is very high in beta-cells compared with other studied tissues, and GRX protein expression is high in islets and in brain; 2) GRX and TRX are localized in distinct microdomains in the cytosol of beta-cells; and 3) microinjection of recombinant GRX potentiated effects of NADPH on exocytosis, whereas TRX antagonized the NADPH effect. We propose that the NADPH/GRX/TRX redox regulation mediates a novel signaling pathway of nutrient-induced insulin secretion.
Assuntos
Exocitose/fisiologia , Insulina/metabolismo , Ilhotas Pancreáticas/fisiologia , NADP/metabolismo , Oxirredutases/genética , Tiorredoxinas/genética , Animais , Sequência de Bases , Primers do DNA , Exocitose/efeitos dos fármacos , Glucose/farmacologia , Glutarredoxinas , Homeostase , Secreção de Insulina , Ilhotas Pancreáticas/efeitos dos fármacos , Ilhotas Pancreáticas/metabolismo , RNA Mensageiro/genética , RatosRESUMO
It is well established that the acute rise in plasma glucose and in the incretin hormones glucose-dependent insulinotropic peptide (GIP) and glucagon-like peptide-1 (7-36) amide (GLP-1), as occurs during a meal, is of pivotal importance in regulating the minute-to-minute output of insulin from pancreatic beta cells. In addition to this well studied acute effect, both glucose and incretin hormones have been recently observed to determine the future secretory responsiveness of the cells. Such plasticity of the insulin secretory competence would imply that glucose and incretins not only act during the present meal, but also help to prepare the beta cells to function during the subsequent meal. Evidence supporting this hypothesis is growing as a result of physiological studies of cultured beta cells (either primary cells or beta cell lines), as well as from an increasing number of large-scale gene expression studies, exploring transcriptional and post-transcriptional events in genes regulated by glucose and incretins. On the basis of this hypothesis, one can speculate that genetic or environmental disturbances of plasticity of the insulin secretory competence is one aspect of beta cell dysfunction that can contribute to the aetiology of type 2 diabetes.
Assuntos
Ingestão de Alimentos/fisiologia , Ilhotas Pancreáticas/metabolismo , Ilhotas Pancreáticas/fisiologia , Animais , HumanosRESUMO
AMP-activated protein kinase (AMPK) is viewed as a fuel sensor for glucose and lipid metabolism. To better understand the physiological role of AMPK, we generated a knockout mouse model in which the AMPKalpha2 catalytic subunit gene was inactivated. AMPKalpha2(-/-) mice presented high glucose levels in the fed period and during an oral glucose challenge associated with low insulin plasma levels. However, in isolated AMPKalpha2(-/-) pancreatic islets, glucose- and L-arginine-stimulated insulin secretion were not affected. AMPKalpha2(-/-) mice have reduced insulin-stimulated whole-body glucose utilization and muscle glycogen synthesis rates assessed in vivo by the hyperinsulinemic euglycemic clamp technique. Surprisingly, both parameters were not altered in mice expressing a dominant-negative mutant of AMPK in skeletal muscle. Furthermore, glucose transport was normal in incubated isolated AMPKalpha2(-/-) muscles. These data indicate that AMPKalpha2 in tissues other than skeletal muscles regulates insulin action. Concordantly, we found an increased daily urinary catecholamine excretion in AMPKalpha2(-/-) mice, suggesting altered function of the autonomic nervous system that could explain both the impaired insulin secretion and insulin sensitivity observed in vivo. Therefore, extramuscular AMPKalpha2 catalytic subunit is important for whole-body insulin action in vivo, probably through modulation of sympathetic nervous activity.
Assuntos
Insulina/metabolismo , Complexos Multienzimáticos/química , Complexos Multienzimáticos/metabolismo , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Quinases Ativadas por AMP , Alelos , Animais , Transporte Biológico , Southern Blotting , Peso Corporal , Domínio Catalítico , Relação Dose-Resposta a Droga , Genótipo , Glucose/farmacologia , Teste de Tolerância a Glucose , Glicogênio/metabolismo , Insulina/farmacologia , Fígado/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Músculo Esquelético/citologia , Músculo Esquelético/metabolismo , Músculos/metabolismo , Estrutura Terciária de Proteína , Fatores de TempoRESUMO
We investigated inaugural disease phenotype in relation to the presence or absence of diabetes-associated autoantibodies and human leukocyte antigen (HLA) DQ risk genotypes in adult-onset diabetic patients. Blood samples and questionnaires were obtained from 1584 recent-onset Belgian Caucasian patients (age 15-39 yr at diagnosis of primary diabetes) who were recruited by the Belgian Diabetes Registry over an 11-yr period. At clinical diagnosis, antibody-positive patients (n = 1198) were on average younger and had more symptoms, a more acute disease onset, lower body mass index, and random C-peptide levels, but higher insulin needs, glycemia, and prevalence of ketonuria, HLA-DQ, and 5' insulin gene susceptibility genotypes (P < 0.001 vs. antibody-negative patients; n = 386). In antibody-positive patients, these characteristics did not differ according to HLA-DQ genotype. However, in antibody-negative subjects, we found that patients were younger (P = 0.001); had a lower body mass index (P < 0.001), higher insulin needs (P = 0.014), and amylasemia (P = 0.001); and tended to have a higher glycemia and lower C-peptide in the presence of susceptible HLA-DQ genotypes. Differences according to HLA-DQ genotype subsisted after careful age-matching. In conclusion, we found no relation between initial disease phenotype and HLA-DQ genotype in antibody-positive diabetic young adults. In contrast, antibody-negative patients displayed more type 1-like features when carrying susceptible HLA-DQ genotypes known to promote the development of antibody-positive diabetes. The overrepresentation of these susceptibility genotypes in antibody-negative patients suggests the existence of an immune-mediated disease process with as yet unidentified immune markers in a subgroup of seronegative patients.
Assuntos
Diabetes Mellitus/genética , Diabetes Mellitus/imunologia , Antígenos HLA-DQ/genética , Adolescente , Adulto , Autoanticorpos/análise , Diabetes Mellitus/fisiopatologia , Feminino , Genótipo , Humanos , Masculino , FenótipoRESUMO
The molecular mechanisms mediating acute regulation of insulin release by glucose are partially known. The process involves at least two pathways that can be discriminated on basis of their (in)dependence of closure of ATP-sensitive potassium (K+(ATP)) channels. The mechanism of the K+(ATP) channel-independent pathway was proposed to involve cataplerosis, the export of mitochondrial intermediates into the cytosol and in the induction of fatty acid-derived signaling molecules. In the present article, we have explored in fluorescence-activated cell sorter (FACS)-purified rat beta-cells the molecular steps involved in chronic glucose regulation of the insulin secretory response. When compared with culture in 10 mmol/l glucose, 24 h culture in 3 mmol/l glucose shifts the phenotype of the cells into a state with low further secretory responsiveness to glucose, lower rates of glucose oxidation, and lower rates of cataplerosis. Microarray mRNA analysis indicates that this shift can be attributed to differences in expression of genes involved in the K+(ATP) channel-dependent pathway, in cataplerosis and in fatty acid/cholesterol biosynthesis. This response was paralleled by glucose upregulation of the transcription factor sterol regulatory element binding protein 1c (SREBP1c) (ADD1) and downregulation of peroxisome proliferator-activated receptor (PPAR)-alpha and PPAR-beta (PPARdelta). The functional importance of cataplerosis via citrate for glucose-induced insulin release was further supported by the observation that two ATP-citrate lyase inhibitors, radicicol and (-)-hydroxycitrate, block part of glucose-stimulated release in beta-cells. In conclusion, chronic glucose regulation of the glucose-responsive secretory phenotype is associated with coordinated changes in gene expression involved in the K+(ATP) channel-dependent pathway, in cataplerosis via citrate and in acyl CoA/cholesterol biosynthesis.
Assuntos
Citratos/fisiologia , Glucose/farmacologia , Insulina/metabolismo , Ilhotas Pancreáticas/metabolismo , Mitocôndrias/fisiologia , Animais , Células Cultivadas , Citosol/metabolismo , Homeostase , Secreção de Insulina , Canais Iônicos/fisiologia , Ilhotas Pancreáticas/efeitos dos fármacos , Cinética , Fígado/efeitos dos fármacos , Fígado/fisiologia , Proteínas de Membrana/fisiologia , Modelos Biológicos , Canais de Potássio , RNA Mensageiro/genética , Ratos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/fisiologia , Transcrição GênicaRESUMO
HLA-DQ genotyping remains the cornerstone of genetic risk stratification in type I diabetes prediction and prevention studies. We developed a genetic screening strategy for predisposition to type I diabetes in the Belgian population based upon HLA-DQA1-DQB1 typing and taking into account the age at clinical onset. A group of 1866 autoantibody-positive type I patients below age 40 years recruited by the Belgian Diabetes Registry and a group of 750 control subjects were DQA1-DQB1 genotyped. In the total study population 16 different DQA1-DQB1 haplotypes were revealed, allowing the stratification of 81 genotypes in ten different genotype groups. Apart from the highest risk DQA1*-DQB1* genotype 0301-0302/0501-0201 (odds ratio 21; absolute risk 6%), three other genotype groups conferred a highly significant disease risk (p < 10(-6)). Altogether, these susceptibility genotypes were carried by 9% of the control subjects versus 60% of the patients diagnosed before age 40 years and up to 70% of those under age 5 years. All other genotypes were protective, neutral, infrequent or associated with a moderate protection or susceptibility. A strong, although not absolute protection was conferred by DQB1*0602-positive haplotypes (odds ratio = 0.03). This study in a large cohort of autoantibody-positive patients shows that a DQA1-DQB1-based genotyping strategy allows the identification of a subgroup representing less than 10% of the Belgian population but harbouring the majority of future type I patients arising in childhood or early adulthood. Future prediction and prevention studies should take into account the age dependency of this HLA-DQ associated risk.