RESUMO
Pancreatic amyloidosis and loss of α and ß cells have been shown to occur in cats with diabetes mellitus, although the number of studies currently available is very limited. Furthermore, it is not known whether pancreatic islet inflammation is a common feature. The aims of the present study were to characterize islet lesions and to investigate whether diabetic cats have inflammation of the pancreatic islets. Samples of pancreas were collected postmortem from 37 diabetic and 20 control cats matched for age, sex, breed, and body weight. Histologic sections were stained with hematoxylin and eosin and Congo red; double labeled for insulin/CD3, insulin/CD20, insulin/myeloperoxidase, insulin/proliferating cell nuclear antigen, and glucagon/Ki67; and single labeled for amylin and Iba1. Mean insulin-positive cross-sectional area was approximately 65% lower in diabetic than control cats (P = .009), while that of amylin and glucagon was similar. Surprisingly, amyloid deposition was similar between groups (P = .408). Proliferation of insulin- and glucagon-positive cells and the number of neutrophils, macrophages, and T (CD3) and B (CD20) lymphocytes in the islets did not differ. The presence of T and B lymphocytes combined tended to be more frequent in diabetic cats (n = 8 of 37; 21.6%) than control cats (n = 1 of 20; 5.0%). The results confirm previous observations that loss of ß cells but not α cells occurs in diabetic cats. Islet amyloidosis was present in diabetic cats but was not greater than in controls. A subset of diabetic cats had lymphocytic infiltration of the islets, which might be associated with ß-cell loss.
Assuntos
Amiloidose/veterinária , Doenças do Gato/patologia , Diabetes Mellitus/veterinária , Insulina/metabolismo , Ilhotas Pancreáticas/patologia , Amiloidose/metabolismo , Amiloidose/patologia , Animais , Doenças do Gato/metabolismo , Gatos , Diabetes Mellitus/metabolismo , Diabetes Mellitus/patologia , Feminino , Glucagon/metabolismo , Polipeptídeo Amiloide das Ilhotas Pancreáticas/metabolismo , Ilhotas Pancreáticas/metabolismo , Masculino , Pâncreas/metabolismo , Pâncreas/patologiaRESUMO
Pancreatitis has been described in cats with diabetes mellitus, although the number of studies currently available is very limited. In addition, ketoacidosis has been hypothesized to be associated with pancreatitis in diabetic cats. The aims of the present study were to investigate whether diabetic cats have pancreatitis and to determine if pancreatitis is more frequent with ketoacidosis. Samples of pancreas were collected postmortem from 37 diabetic cats, including 15 with ketoacidosis, and 20 control cats matched for age, sex, breed, and body weight. Sections were stained with hematoxylin and eosin, double-labeled for insulin/CD3, insulin/CD20, insulin/myeloperoxidase, insulin/PCNA, and glucagon/Ki67, and single-labeled for Iba1. A previously proposed semiquantitative score was used to characterize pancreatitis, along with counts of inflammatory cells. Scores of pancreatitis and the number of neutrophils, macrophages, and lymphocytes in the exocrine pancreas did not differ between diabetic and control cats or between diabetic cats with and without ketoacidosis. Of note, PCNA-positive acinar cells were increased (P = .002) in diabetic cats, particularly near islets (P < .001). Ki67-positive acinar cells were increased only near islets (P = .038). Ketoacidosis was not linked to proliferation. The results suggest that histopathologic evidence of pancreatitis may not be more frequent in diabetic cats and that ketoacidosis may not be associated with it at the time of death. Augmented PCNA-positive acinar cells might indicate increased proliferation due to chronic pancreatitis. The reason behind the prevalent proliferation of acinar cells surrounding pancreatic islets deserves further investigation.
Assuntos
Doenças do Gato/patologia , Diabetes Mellitus/veterinária , Cetose/veterinária , Pâncreas Exócrino/patologia , Pancreatite/veterinária , Células Acinares/patologia , Animais , Doenças do Gato/metabolismo , Gatos , Diabetes Mellitus/metabolismo , Diabetes Mellitus/patologia , Feminino , Glucagon/metabolismo , Insulina/metabolismo , Cetose/metabolismo , Cetose/patologia , Masculino , Pâncreas/metabolismo , Pâncreas/patologia , Pâncreas Exócrino/metabolismo , Pancreatite/metabolismo , Pancreatite/patologiaRESUMO
BACKGROUND AND PURPOSE: Flexible microstents, such as the closed-cell EN, have facilitated adjunctive coiling of intracranial aneurysms. Little data are available on the ability of the stent struts to maintain vessel-wall apposition once deployed in the tortuous cerebral vasculature and the prevalence of ISA. The purpose of this study was to evaluate the relationship between geometric features of the parent vessel at the stent deployment site and prevalence of ISA. MATERIALS AND METHODS: Postprocedural 3T-MRA was performed in a cohort of 39 patients undergoing EN stent-assisted intracranial aneurysm coiling. 3T-MRA was analyzed for the presence of ISA and supplemented by angiographic C-arm FPCT (DynaCT). Parent vessel diameter, curvature radius, and stent-subtended arc angle were measured at the site of deployment and analyzed for prediction of ISA in the ICA. RESULTS: 3T-MRA uncovered a unique crescent flow pattern (CS) outside the EN struts, which was confirmed by FPCT to indicate ISA resulting from EN crimping. ISA was detected on 3T-MRA in 19/39 patients (49%). Univariate analysis revealed ISA in the ICA to correlate with a large stent-subtended angle, a small curvature radius, and a large diameter but not stent length or jailing versus a sequential technique. Multivariate analysis identified ISA to correlate with vessel-curvature radius (OR, 253; P = .009), stent-subtended angle (OR, 225; P = .005), and parent vessel diameter (OR, 8.49; P = .044). CONCLUSIONS: In this study, ISA was detectable by 3T-MRA in a significant proportion of patients undergoing EN stent-assisted coiling of ICA aneurysms in a vessel geometry- and stent-deployment location-dependent manner. This characteristic of EN coiling at this potentially tortuous location should be taken into account when selecting an endovascular strategy.
Assuntos
Embolização Terapêutica , Aneurisma Intracraniano/epidemiologia , Aneurisma Intracraniano/terapia , Stents/efeitos adversos , Stents/estatística & dados numéricos , Adulto , Idoso , Doenças das Artérias Carótidas/epidemiologia , Doenças das Artérias Carótidas/patologia , Doenças das Artérias Carótidas/terapia , Trombose das Artérias Carótidas/epidemiologia , Trombose das Artérias Carótidas/patologia , Artéria Carótida Interna/patologia , Embolização Terapêutica/efeitos adversos , Embolização Terapêutica/métodos , Embolização Terapêutica/estatística & dados numéricos , Feminino , Humanos , Aneurisma Intracraniano/patologia , Angiografia por Ressonância Magnética/métodos , Masculino , Pessoa de Meia-Idade , Projetos Piloto , Prevalência , Estudos Prospectivos , Fatores de RiscoRESUMO
AIMS/HYPOTHESIS: The hormone glucagon-like peptide 1 (GLP-1) is released in response to a meal from the intestinal L-cells, where it is processed from proglucagon by the proconvertase (PC)1/3. In contrast, in the adult islets proglucagon is processed to glucagon by the PC2 enzyme. The aim of the study was to evaluate if, during the development of diabetes, alpha cells produce GLP-1 that, in turn, might trigger beta cell growth. METHODS: Beta cell mass, GLP-1 and insulin levels were measured in the gerbil Psammomys obesus (P. obesus), a rodent model of nutritionally induced diabetes. Furthermore, the presence of biologically active forms of GLP-1 and PC1/3 in alpha cells was demonstrated by immunofluorescence, and the release of GLP-1 from isolated P. obesus, mouse and human islets was investigated. RESULTS: During the development of diabetes in P. obesus, a significant increase in GLP-1 was detected in the portal vein (9.8 ± 1.5 vs 4.3 ± 0.7 pmol/l, p < 0.05), and in pancreas extracts (11.4 ± 2.2 vs 5.1 ± 1.3 pmol/g tissue, p < 0.05). Freshly isolated islets from hyperglycaemic animals released more GLP-1 following 24 h culture than islets from control animals (28.2 ± 4.4 pmol/l vs 5.8 ± 2.4, p < 0.01). GLP-1 release was increased from healthy P. obesus islets following culture in high glucose for 6 days (91 ± 9.1 pmol/l vs 28.8 ± 6.6, p < 0.01). High levels of GLP-1 were also found to be released from human islets. PC1/3 colocalised weakly with alpha cells. CONCLUSIONS/INTERPRETATION: GLP-1 release from alpha cells is upregulated in P. obesus during the development of diabetes. A similar response is seen in islets exposed to high glucose, which supports the hypothesis that GLP-1 released from alpha cells promotes an increase in beta cell mass and function during metabolic challenge such as diabetes.
Assuntos
Diabetes Mellitus/metabolismo , Gerbillinae/metabolismo , Peptídeo 1 Semelhante ao Glucagon/metabolismo , Células Secretoras de Glucagon/metabolismo , Hiperglicemia/metabolismo , Obesidade/metabolismo , Regulação para Cima/fisiologia , Adaptação Fisiológica/fisiologia , Adulto , Animais , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Diabetes Mellitus/etiologia , Diabetes Mellitus/patologia , Dieta/efeitos adversos , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Feminino , Células Secretoras de Glucagon/efeitos dos fármacos , Células Secretoras de Glucagon/patologia , Glucose/farmacologia , Humanos , Hiperglicemia/patologia , Insulina/metabolismo , Células Secretoras de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/patologia , Ilhotas Pancreáticas/efeitos dos fármacos , Ilhotas Pancreáticas/metabolismo , Ilhotas Pancreáticas/patologia , Masculino , Camundongos , Pessoa de Meia-Idade , Obesidade/genética , Obesidade/patologiaRESUMO
AIMS/HYPOTHESIS: In vitro studies point to a toxic effect of high glucose and non-esterified fatty acids on beta cells. Whether elevated levels of glucose and lipids induce beta cell loss in vivo is less clear. The domestic cat has recently been proposed as a valuable animal model for human type 2 diabetes because feline diabetes shows several similarities with diabetes in humans, including obesity-induced insulin resistance, impaired beta cell function, decreased number of beta cells and pancreatic amyloid deposition. METHODS: We infused healthy cats with glucose or lipids for 10 days to clamp their blood concentrations at the approximate level found in untreated feline diabetes (glucose: 25-30 mmol/l; triacylglycerols: 3-7 mmol/l). RESULTS: Glucose and lipid levels were adequately targeted. Plasma non-esterified fatty acids were increased by lipid infusion 1.7-fold. A dramatic and progressive decline of plasma insulin levels was observed in glucose-infused cats beginning after 2 days of hyperglycaemic clamp. In contrast, plasma insulin concentration and glucose tolerance test were not affected by hyperlipidaemia. Compared with controls, glucose-infused cats had a 50% decrease in beta cells per pancreatic area. Apoptotic islet cells and cleaved caspase-3-positive beta cells were observed in glucose-infused cats only. CONCLUSIONS/INTERPRETATION: Sustained hyperglycaemia but not hyperlipidaemia induces early and severe beta cell dysfunction in cats, and excess glucose causes beta cell loss via apoptosis in vivo. Hyperglycaemic clamps in cats may provide a good model to study the pathogenesis of glucose toxicity in beta cells.
Assuntos
Doenças do Gato/fisiopatologia , Hiperglicemia/veterinária , Hiperlipidemias/veterinária , Células Secretoras de Insulina/fisiologia , Animais , Animais Domésticos , Glicemia/metabolismo , Gatos , Ácidos Graxos não Esterificados/sangue , Ácidos Graxos não Esterificados/toxicidade , Hiperglicemia/fisiopatologia , Hiperlipidemias/fisiopatologia , Insulina/sangue , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/patologia , MasculinoRESUMO
It is currently believed that pancreatic progenitor or stem cells exist in the ductal cell population and that these cells have the ability to be grown and differentiated into endocrine cells for the treatment of diabetes. In this study, we have examined this potential in IMPAN (Immortalized Pancreatic) cells. These cells are derived from the adult H-2K(b)-tsA58 transgenic mouse. We observed an increased mRNA expression of insulin, proendocrine gene neurogenin 3, and beta-cell transcription factor Pdx1 when the cells were grown on bovine collagen I gels. The induction profile of these three genes was similar under the tested conditions. No glucagon or other endocrine-specific transcription factors were detectable. Application of GIP, GLP-1 derivative NN2211, and activin-A/betacellulin to IMPAN cells in normal culture did not lead to endocrine differentiation. In conclusion, it appears that the ability of IMPAN cells to mature to endocrine cells is limited.
Assuntos
Linhagem Celular , Glândulas Endócrinas/citologia , Proteínas de Homeodomínio , Ativinas/farmacologia , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos , Bovinos , Diferenciação Celular/efeitos dos fármacos , Colágeno/metabolismo , Colágeno Tipo I/metabolismo , Glucagon/análogos & derivados , Glucagon/farmacologia , Peptídeo 1 Semelhante ao Glucagon/análogos & derivados , Subunidades beta de Inibinas/farmacologia , Insulina/biossíntese , Liraglutida , Camundongos , Camundongos Transgênicos , Proteínas do Tecido Nervoso/biossíntese , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células-Tronco/citologia , Temperatura , Transativadores/biossínteseRESUMO
The homeodomain protein PDX-1 is critical for pancreas development and is a key regulator of insulin gene expression. PDX-1 nullizygosity and haploinsufficiency in mice and humans results in pancreatic agenesis and diabetes, respectively. At embryonic day (e) 10.5, PDX-1 is expressed in all pluripotential gut-derived epithelial cells destined to differentiate into the exocrine and endocrine pancreas. At e15, PDX-1 expression is downregulated in exocrine cells, but remains high in endocrine cells. The aim of this study was to determine whether targeted overexpression of PDX-1 to the exocrine compartment of the developing pancreas at e15 would allow for respecification of the exocrine cells. Transgenic (TG) mice were generated in which PDX-1 was expressed in the exocrine pancreas using the exocrine-specific elastase-1 promoter. These mice exhibited a marked dysmorphogenesis of the exocrine pancreas, manifested by increased rates of replication and apoptosis in acinar cells and a progressive fatty infiltration of the exocrine pancreas with age. Interestingly, the TG mice exhibited improved glucose tolerance, but absolute beta-cell mass was not increased. These findings indicate that downregulation of PDX-1 is required for the proper maintenance of the exocrine cell phenotype and that upregulation of PDX-1 in acinar cells affects beta-cell function. The mechanisms underlying these observations remain to be elucidated.
Assuntos
Glicemia/fisiologia , Proteínas de Homeodomínio , Pâncreas/metabolismo , Transativadores/biossíntese , Tecido Adiposo/metabolismo , Animais , Apoptose , Regulação para Baixo , Regulação da Expressão Gênica , Teste de Tolerância a Glucose , Imuno-Histoquímica , Camundongos , Camundongos Transgênicos , Microscopia Eletrônica , Elastase Pancreática/genética , Regiões Promotoras Genéticas , Ratos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transativadores/genética , Transgenes/genética , Regulação para CimaRESUMO
The nature and identity of the pancreatic beta-cell precursor has remained elusive for many years. One model envisions an early multihormonal precursor that gives rise to both alpha- and beta-cells and the other endocrine cell types. Alternatively, beta-cells have been suggested to arise late, directly from the GLUT2- and pancreatic duodenal homeobox factor-1 (PDX1)-expressing epithelium, which gives rise also to the acinar cells during this stage. In this study, we have identified a subset of the PDX1+ epithelial cells that are marked by expression of Neurogenin3 (Ngn3). Ngn3, a member of the basic helix-loop-helix (bHLH) family of transcription factors, is suggested to act upstream of NeuroD in a bHLH cascade. Detailed analysis of Ngn3/paired box factor 6 (PAX6) and NeuroD/PAX6 co-expression shows that the two bHLH factors are expressed in a largely nonoverlapping set of cells, but such analysis also suggests that the NeuroD+ cells arise from cells expressing Ngn3 transiently. NeuroD+ cells do not express Ki-67, a marker of proliferating cells, which shows that these cells are postmitotic. In contrast, Ki-67 is readily detected in Ngn3+ cells. Thus, Ngn3+ cells fulfill the criteria for an endocrine precursor cell. These expression patterns support the notion that both alpha- and beta-cells develop independently from PDX1+/Ngn3+ epithelial cells, rather than from GLU+/INS+ intermediate stages. The earliest sign of alpha-cell development appears to be Brain4 expression, which apparently precedes Islet-1 (ISL1) expression. Based on our expression analysis, we propose a temporal sequence of gene activation and inactivation for developing alpha- and beta-cells beginning with activation of NeuroD expression. Endocrine cells leave the cell cycle before NeuroD activation, but re-enter the cell cycle at perinatal stages. Dynamic expression of Notch1 in PDX+ epithelial cells suggests that Notch signaling could inhibit a Ngn-NeuroD cascade as seen in the nervous system and thus prevent premature differentiation of endocrine cells.
Assuntos
Proteínas de Homeodomínio , Ilhotas Pancreáticas/citologia , Proteínas do Tecido Nervoso/metabolismo , Células-Tronco/metabolismo , Fatores de Transcrição , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos , Biomarcadores , Diferenciação Celular/fisiologia , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Glucagon/metabolismo , Antígeno Ki-67/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Membrana/fisiologia , Camundongos , Camundongos Endogâmicos , Pâncreas/embriologia , Pâncreas/metabolismo , Ratos , Ratos Endogâmicos WF , Receptor Notch1 , Receptor Notch2 , Receptores de Superfície Celular/metabolismo , Receptores Notch , Células-Tronco/citologia , Transativadores/metabolismoRESUMO
Development of endocrine cells in the endoderm involves Atonal and Achaete/Scute-related basic helix-loop-helix (bHLH) proteins. These proteins also serve as neuronal determination and differentiation factors, and are antagonized by the Notch pathway partly acting through Hairy and Enhancer-of-split (HES)-type proteins. Here we show that mice deficient in Hes1 (encoding Hes-1) display severe pancreatic hypoplasia caused by depletion of pancreatic epithelial precursors due to accelerated differentiation of post-mitotic endocrine cells expressing glucagon. Moreover, upregulation of several bHLH components is associated with precocious and excessive differentiation of multiple endocrine cell types in the developing stomach and gut, showing that Hes-1 operates as a general negative regulator of endodermal endocrine differentiation.
Assuntos
Proteínas de Drosophila , Glândulas Endócrinas/embriologia , Endoderma , Sequências Hélice-Alça-Hélice , Proteínas de Homeodomínio/fisiologia , Proteínas Repressoras , Sequência de Aminoácidos , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos , Diferenciação Celular , Proteínas de Ligação a DNA/metabolismo , Glândulas Endócrinas/citologia , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/genética , Proteínas de Insetos/metabolismo , Intestinos/patologia , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Knockout , Dados de Sequência Molecular , Pâncreas/embriologia , Pâncreas/patologia , Pâncreas/fisiopatologia , Proteínas/metabolismo , Receptores Notch , Transdução de Sinais , Estômago/patologia , Fatores de Transcrição HES-1RESUMO
Expression of the homeodomain transcription factor IDX-1 (also known as IPF-1, STF-1, and PDX-1) is required for pancreas development, because disruption of the gene in mice and humans results in pancreatic agenesis. During embryonic development the idx-1 gene is first expressed in a localized region of foregut endoderm from which the duodenum and pancreas later develop. To more fully understand the role of IDX-1 in pancreas development, transgenic mice expressing the Escherichia coli lacZ gene under control of the 5'-proximal 4.6 kb of the idx-1 promoter were created as a reporter for the developmental expression of IDX-1. Here we show that the determinants for the developmental and tissue-specific expression of the endogenous idx-1 gene are faithfully reproduced by the 4.6-kb region of the idx-1 promoter. Expression of lacZ is detected in the development of the exocrine and endocrine pancreas in pancreatic ducts, common bile and cystic ducts, pyloric glands of the distal stomach, Brunner's glands, the intestinal epithelium of the duodenum, and the spleen. The observed spatial and temporal pattern of lacZ expression directed by the IDX-1 promoter further supports an important role of IDX-1 in specifying the development of several endodermal structures within the midsegment of the body. An unexpected finding is that IDX-1 promoter-driven (transcriptional) lacZ activity does not always coincide with the localization of IDX-1 messenger RNA by in situ hybridization and IDX-1 protein by immunocytochemistry in adult rat duodenum, suggesting the existence of regulation of IDX-1 expression at the posttranscriptional level of expression of the idx-1 gene.
Assuntos
Sistema Digestório/crescimento & desenvolvimento , Expressão Gênica , Genes Reporter , Pâncreas/crescimento & desenvolvimento , Regiões Promotoras Genéticas , Transativadores/genética , Animais , Sistema Digestório/embriologia , Sistema Digestório/metabolismo , Duodeno/embriologia , Duodeno/crescimento & desenvolvimento , Duodeno/metabolismo , Mucosa Gástrica/metabolismo , Idade Gestacional , Proteínas de Homeodomínio/genética , Hibridização In Situ , Óperon Lac , Camundongos , Camundongos Transgênicos , Pâncreas/embriologia , Pâncreas/metabolismo , RNA Mensageiro/análise , Proteínas Recombinantes de Fusão , Baço/crescimento & desenvolvimento , Baço/metabolismo , Estômago/embriologia , Estômago/crescimento & desenvolvimentoRESUMO
Glucagon-like peptide-1 (GLP-1) is an intestinally derived insulinotropic hormone currently under investigation for use as a novel therapeutic agent in the treatment of type 2 diabetes mellitus. In vitro studies of pancreatic islets of Langerhans demonstrated that GLP-1 interacts with specific beta-cell G protein-coupled receptors, thereby facilitating insulin exocytosis by raising intracellular levels of cAMP and Ca2+. Here we report that the stimulatory influence of GLP-1 on Ca2+ signaling results, in part, from cAMP-dependent mobilization of ryanodine-sensitive Ca2+ stores. Studies of human, rat, and mouse beta-cells demonstrate that the binding of a fluorescent derivative of ryanodine (BODIPY FL-X ryanodine) to its receptors is specific, reversible, and of high affinity. Rat islets and BTC3 insulinoma cells are shown by reverse transcriptase polymerase chain reaction analyses to express mRNA corresponding to the type 2 isoform of ryanodine receptor-intracellular Ca2+ release channel (RYR2). Single-cell measurements of [Ca2+]i using primary cultures of rat and human beta-cells indicate that GLP-1 facilitates Ca2+-induced Ca2+ release (CICR), whereby mobilization of Ca2+ stores is triggered by influx of Ca2+ through L-type Ca2+ channels. In these cells, GLP-1 is shown to interact with metabolism of D-glucose to produce a fast transient increase of [Ca2+]i. This effect is reproduced by 8-Br-cAMP, but is blocked by a GLP-1 receptor antagonist (exendin-(9-39)), a cAMP antagonist ((Rp)-cAMPS), an L-type Ca2+ channel antagonist (nimodipine), an antagonist of the sarco(endo)plasmic reticulum Ca2+ ATPase (thapsigargin), or by ryanodine. Characterization of the CICR mechanism by voltage clamp analysis also demonstrates a stimulation of Ca2+ release by caffeine. These findings provide new support for a model of beta-cell signal transduction whereby GLP-1 promotes CICR by sensitizing intracellular Ca2+ release channels to the stimulatory influence of cytosolic Ca2+.
Assuntos
Cálcio/metabolismo , AMP Cíclico/metabolismo , Glucagon/fisiologia , Ilhotas Pancreáticas/metabolismo , Fragmentos de Peptídeos/fisiologia , Precursores de Proteínas/fisiologia , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Transdução de Sinais , Animais , Compostos de Boro , Cafeína/farmacologia , Corantes Fluorescentes , Peptídeo 1 Semelhante ao Glucagon , Humanos , Ilhotas Pancreáticas/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Reação em Cadeia da Polimerase , RNA Mensageiro/metabolismo , Ratos , Rianodina/metabolismo , Células Tumorais CultivadasRESUMO
Alpha-cell specific transcription of the glucagon gene is mainly conferred by the glucagon promoter G1-element, while additional elements G2, G3, and G4 have broad islet cell specificity. Transcription of the glucagon gene has been shown to be stimulated by Pax6 through binding to the glucagon gene promoter G3-element. In this report, we show that Pax6 additionally binds the glucagon gene promoter G1-element and forms a transcriptionally active complex with another homeodomain protein, Cdx2/3. Two distinct mutations in the G1-element, that both reduce promoter activity by 85-90%, is shown to eliminate binding of either Pax6 or Cdx2/3. Additionally, Pax6 enhanced Cdx2/3 mediated activation of a glucagon reporter in heterologous cells. We discuss how Pax6 may contribute to cell-type specific transcription in the pancreatic islets by complex formation with different transcription factors.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Glucagon/genética , Proteínas de Homeodomínio/metabolismo , Regiões Promotoras Genéticas , Células 3T3 , Sequência de Aminoácidos , Animais , Fator de Transcrição CDX2 , Cricetinae , Proteínas do Olho , Regulação da Expressão Gênica , Genes Reporter , Camundongos , Dados de Sequência Molecular , Mutagênese , Fator de Transcrição PAX6 , Fatores de Transcrição Box Pareados , Ratos , Proteínas Repressoras , TransativadoresRESUMO
The somatostatin upstream enhancer (SMS-UE) is a highly complex enhancer element. The distal A-element contains overlapping Pdx1 and Pbx binding sites. However, a point mutation in the A-element that abolishes both Pdxl and Pbx binding does not impair promoter activity. In contrast, a point mutation that selectively eliminates Pdx1 binding to a proximal B-element reduces the promoter activity. The B-element completely overlaps with a Pax6 binding site, the C-element. A point mutation in the C-element demonstrates that Pax6 binding is essential for promoter activity. Interestingly, a block mutation in the A-element reduces both Pax6 binding and promoter activity. In heterologous cells, Pdx1 potentiated Pax6 mediated activation of a somatostatin reporter. We conclude that the beta/delta-cell-specific activity of the SMS-UE is achieved through simultaneous binding of Pdx1 and Pax6 to the B- and C-elements, respectively. Furthermore, the A-element appears to stabilise Pax6 binding.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Elementos Facilitadores Genéticos , Proteínas de Saccharomyces cerevisiae , Somatostatina/genética , Transativadores/metabolismo , Células 3T3 , Animais , Fusão Gênica Artificial , Sequência de Bases , Sítios de Ligação , Fator de Transcrição CDX2 , Proteínas de Ligação a DNA/genética , Proteínas do Olho , Proteínas Fúngicas/genética , Regulação da Expressão Gênica , Genes Reporter , Proteínas de Homeodomínio/genética , Camundongos , Dados de Sequência Molecular , Mutagênese , Fator de Transcrição PAX6 , Fatores de Transcrição Box Pareados , Ratos , Proteínas Repressoras , Relação Estrutura-Atividade , Transativadores/genética , Fatores de Transcrição/genéticaRESUMO
BACKGROUND & AIMS: The endoderm-specific homeodomain transcription factor IDX-1 is critical for pancreas development and for the regulation of islet cell-specific genes. During development, IDX-1 is expressed in the epithelial cells of the endoderm in the pancreatic anlage of the foregut. The aim of this study was to determine whether IDX-1 may have potential properties of a master homeotic determinant of pancreas and/or gut development. METHODS: Transgenic mice were generated in which the expression of IDX-1 was misdirected by a promoter of the mesoderm-specific homeodomain protein Hoxa-4 known to express in the stomach and hindgut during development. The expectation was the formation of ectopic pancreatic tissue or alterations of gut patterning or morphology. RESULTS: Although no ectopic induction of pancreatic markers was found in these transgenic mice, they manifested an altered midgut-hindgut union and agenesis of the cecum. Further, IDX-1 binds to the gut-specific homeodomain protein Cdx-2 and inhibits transactivation of the sucrase-isomaltase promoter by Cdx-2. CONCLUSIONS: These findings further support the emerging understanding that interactions among different classes of homeodomain proteins, expressed in a spatially and temporally restricted manner during development, determine the pattern of organogenesis. A possible mechanism for the dysmorphogenesis of the proximal colon may be an inhibition of Cdx-2 actions by IDX-1.
Assuntos
Ceco/anormalidades , Proteínas de Ligação a DNA , Expressão Gênica , Proteínas de Homeodomínio/genética , Regiões Promotoras Genéticas/fisiologia , Transativadores/genética , Animais , Fator de Transcrição CDX2 , Colo/patologia , Expressão Gênica/fisiologia , Proteínas de Homeodomínio/metabolismo , Proteínas de Homeodomínio/fisiologia , Mucosa Intestinal/patologia , Camundongos , Camundongos Transgênicos/genética , Oligo-1,6-Glucosidase/genética , Sacarase/genética , Transativadores/metabolismo , Fatores de Transcrição , Transcrição Gênica/fisiologiaRESUMO
In the genetic mutant mouse models ob/ob or db/db, leptin deficiency or resistance, respectively, results in severe obesity and the development of a syndrome resembling NIDDM. One of the earliest manifestations in these mutant mice is hyperinsulinemia, suggesting that leptin may normally directly suppress the secretion of insulin. Here, we show that pancreatic islets express a long (signal-transducing) form of leptin-receptor mRNA and that beta-cells bind a fluorescent derivative of leptin (Cy3-leptin). The expression of leptin receptors on insulin-secreting beta-cells was also visualized utilizing antisera generated against an extracellular epitope of the receptor. A functional role for the beta-cell leptin receptor is indicated by our observation that leptin (100 ng/ml) suppressed the secretion of insulin from islets isolated from ob/ob mice. Furthermore, leptin produced a marked lowering of [Ca2+]i in ob/ob beta-cells, which was accompanied by cellular hyperpolarization and increased membrane conductance. Cell-attached patch measurements of ob/ob beta-cells demonstrated that leptin activated ATP-sensitive potassium channels (K(ATP)) by increasing the open channel probability, while exerting no effect on mean open time. These effects were reversed by the sulfonylurea tolbutamide, a specific inhibitor of K(ATP). Taken together, these observations indicate an important physiological role for leptin as an inhibitor of insulin secretion and lead us to propose that the failure of leptin to inhibit insulin secretion from the beta-cells of ob/ob and db/db mice may explain, in part, the development of hyperinsulinemia, insulin resistance, and the progression to NIDDM.
Assuntos
Trifosfato de Adenosina/farmacologia , Proteínas de Transporte/genética , Insulina/metabolismo , Ilhotas Pancreáticas/fisiologia , Canais de Potássio/metabolismo , Proteínas/fisiologia , Receptores de Superfície Celular , Animais , Proteínas de Transporte/análise , Proteínas de Transporte/biossíntese , Técnicas de Cultura , Primers do DNA/química , Relação Dose-Resposta a Droga , Feminino , Expressão Gênica , Secreção de Insulina , Ilhotas Pancreáticas/química , Ilhotas Pancreáticas/ultraestrutura , Leptina , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Camundongos , Camundongos Obesos , Reação em Cadeia da Polimerase , RNA Mensageiro/análise , RNA Mensageiro/genética , Receptores para Leptina , Transdução de Sinais , Somatostatina/efeitos dos fármacos , Somatostatina/metabolismo , Células Tumorais CultivadasRESUMO
Glucagon-like peptide I (GLP-I), an intestine-derived incretin hormone, is a potent stimulator of insulin and somatostatin secretion. In some studies, GLP-I is an inhibitor of glucagon secretion. It remains uncertain, however, whether the effect of GLP-I on the inhibition of glucagon secretion is direct, owing to interactions with GLP-I receptors on alpha-cells, or indirect, via paracrine suppression by insulin or somatostatin. The localization of the GLP-I receptor on insulin and somatostatin-producing cells in the islets is well established. Whether the GLP-I receptor also resides on the glucagon-producing alpha-cells remains controversial and is reported to be absent on rat alpha-cells. To investigate the distribution of the GLP-I receptor on islet cells, we examined the expression of GLP-I receptor mRNA in phenotypically distinct islet cell lines and islets, and the presence of immunoreactive GLP-I receptor in dispersed rat islet cells using a specific antiserum. GLP-I receptor mRNA was readily detected by reverse transcription-polymerase chain reaction (RT-PCR) in both rat islets and in established islet cell lines representing distinct alpha-, beta-, and delta-cell phenotypes. In addition, GLP-I receptor expression was detected in single rat alpha-cells by single-cell RT-PCR. In dispersed rat islet cells analyzed by double immunofluorescent staining, 90% of the insulin, 76% of the somatostatin, and 20% of the glucagon positive cells colocalized with the GLP-I receptor immunoreactivity. Thus, a substantial population of glucagon immunoreactive a-cells express the GLP-I receptor. These findings imply that GLP-I may have a direct receptor-mediated action in the regulation of the physiological functions on a substantial subpopulation of alpha-cells. We suggest that a possible role for GLP-I receptors on alpha-cells may be to provide positive autocrine feedback control on glucagon secretion during fasting and/or to dampen the potent paracrine suppression of glucagon secretion by insulin during feeding.
Assuntos
Glucagon/metabolismo , Insulina/metabolismo , Ilhotas Pancreáticas/química , Receptores de Glucagon/biossíntese , Animais , Linhagem Celular , Feminino , Receptor do Peptídeo Semelhante ao Glucagon 1 , Soros Imunes/farmacologia , Secreção de Insulina , Ilhotas Pancreáticas/metabolismo , Masculino , Reação em Cadeia da Polimerase , DNA Polimerase Dirigida por RNA , Ratos , Ratos Sprague-Dawley , Receptores de Glucagon/imunologiaRESUMO
The effectiveness of heart failure management in clinical practice is limited by physicians' suboptimal utilization of effective medications, patients' poor adherence to dietary sodium limitation and optimal drug therapy, and the lack of systematic monitoring of patients after hospitalization. The present study evaluated the feasibility and safety of MULTIFIT, a physician-supervised, nurse-mediated, home-based system for heart failure management that implements consensus guidelines for pharmacologic and dietary therapy using a nurse manager to enhance dietary and pharmacologic adherence and to monitor clinical status by frequent telephone contact. Fifty-one patients with the clinical diagnosis of heart failure were followed for 138 +/- 44 days. Daily dietary sodium intake fell by 38%, from 3,393 to 2,088 mg (p = 0.0001); average daily medication doses increased significantly (lisinopril: 17 to 23 mg, p <0.001; hydralazine: 140 to 252 mg, p = 0.01). Functional status and exercise capacity improved significantly (p = 0.01). Compared with the 6 months before enrollment and normalized for variable follow-up, the frequency of general medical and cardiology visits declined by 23% and 31%, respectively (both p <0.03); emergency room visits for heart failure and for all causes declined 67% and 53%, respectively (both p <0.001). Hospitalization rates for heart failure and for all causes declined 87% and 74%, respectively (p = 0.001), compared with the year before enrollment. The MULTIFIT system enhanced the effectiveness of pharmacologic and dietary therapy for heart failure in clinical practice, improving clinical outcomes and reducing medical resource utilization.
Assuntos
Baixo Débito Cardíaco/terapia , Serviços de Assistência Domiciliar , Idoso , California , Baixo Débito Cardíaco/complicações , Aconselhamento , Estudos de Viabilidade , Sistemas Pré-Pagos de Saúde , Humanos , Pessoa de Meia-Idade , Cooperação do Paciente , TelefoneRESUMO
Glucagon is insulinotropic, but it remains uncertain whether the insulinotropic action is mediated directly by glucagon receptors expressed on beta-cells or by cross-binding to the insulinotropic glucagon-like peptide-1 (GLP-1) receptor known to be expressed on beta-cells. Binding of [125I]glucagon to GLP-1 receptors and not to glucagon receptors has been reported in tumor-derived beta-cells (15). The objectives of the current study were to use receptor-binding techniques and a glucagon receptor-specific antiserum to determine whether glucagon receptors are present on beta-cells. Specific binding (7.2 +/- 0.8%) of [125I]GLP-1 to beta TC-3 cells was displaced equivalently with GLP-1 and exendin-(9-39) )Kd = 0.9 and 0.4 nM. respectively), whereas approximately 700-fold higher concentrations of glucagon were required for equal displacement (Kd = 400 nM). Binding of [125I]glucagon to beta TC-3 cells (approximately 1%) was displaced equivalently with 1 microM glucagon, GLP-1, or exendin-(9-39). These observations support earlier findings that beta TC-3 cells do not express functional glucagon receptors. However, specific binding of [125I]glucagon was observed on INS-1 cells (2.3 +/- 0.2%); this was displaced with glucagon (Kd = 1 nM), but not 1 microM GLP-1 or exendin-(9-39). To examine the distribution of glucagon receptors on native beta-cells, dispersed cultured rat islets were immunostained for glucagon, somatostatin, or insulin in combination with a polyclonal rabbit antiserum raised to an extracellular portion of the glucagon receptor (KD-14). The glucagon receptor antiserum colocalized staining with approximately 97% of immunoreactive insulin cells, 9% of immunoreactive glucagon cells, and 11% of immunoreactive somatostatin cells. Perfusion of the rat pancreas with concentrations of glucagon as low as 10(-12) M resulted in significant insulin release. These results suggest that whereas the tumor-derived beta-cell line beta TC-3 does not express functional glucagon receptors, INS-1 cells and isolated rat pancreatic beta-cells have specific glucagon receptors, as do a subpopulation of alpha- and delta-cells. A model is proposed for the role of glucagon in islet hormone secretion during feeding and fasting.
Assuntos
Glucagon/metabolismo , Ilhotas Pancreáticas/metabolismo , Fragmentos de Peptídeos/metabolismo , Precursores de Proteínas/metabolismo , Receptores de Glucagon/metabolismo , Animais , Ligação Competitiva , Linhagem Celular , Células Cultivadas , Ingestão de Alimentos , Jejum , Feminino , Peptídeo 1 Semelhante ao Glucagon , Receptor do Peptídeo Semelhante ao Glucagon 1 , Imuno-Histoquímica , Insulina/metabolismo , Ilhotas Pancreáticas/citologia , Cinética , Masculino , Modelos Biológicos , Coelhos , Ratos , Ratos Sprague-Dawley , Receptores de Glucagon/análise , Somatostatina/metabolismoRESUMO
Leptin (Ob protein) is a recently isolated hormone produced by adipocytes and is a powerful regulator of satiety centers in the brain. A defect in either leptin production or transmission of the leptin signal in animal models, i.e. ob/ob and db/db mice, respectively, results in a syndrome of obesity and diabetes which closely resembles that which occurs in humans. Leptin release is regulated in part by nutritional status and its expression in adipose tissue is up-regulated by insulin. Since hyperinsulinemia is a primary defect in ob/ob and db/db mice which manifests early in the disease, we postulated that leptin may also regulate insulin release as part of a "adipoinsular' feedback loop. We demonstrate the expression of leptin receptor mRNA in primary rat pancreatic islets and in the insulinoma cell line beta TC-3. Furthermore, we find binding of 125I-leptin to beta TC-3 cells which is significantly displaced by leptin. These findings suggest the possibility that the binding of leptin to its receptor in beta-cells may modulate insulin expression in a negative feedback loop, and thereby may have an anti-obesity effect.
Assuntos
Proteínas de Transporte/biossíntese , Ilhotas Pancreáticas/metabolismo , Proteínas/metabolismo , Receptores de Superfície Celular , Animais , Sequência de Bases , Northern Blotting , Encéfalo/metabolismo , Proteínas de Transporte/metabolismo , Células Cultivadas , Primers do DNA , Diabetes Mellitus/genética , Diabetes Mellitus Tipo 2/genética , Cinética , Leptina , Pulmão/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos , Modelos Biológicos , Dados de Sequência Molecular , Obesidade , Reação em Cadeia da Polimerase , RNA Mensageiro/análise , RNA Mensageiro/biossíntese , Ratos , Ratos Sprague-Dawley , Receptores para LeptinaRESUMO
The incretin hormone glucagon-like peptide-1 (GLP-1) is an important regulator of postprandial insulin secretion. In addition to its insulinotropic actions on pancreatic beta-cells, GLP-1 enhances glucose disposal by insulin-independent mechanisms, suggesting that GLP-1 receptors are located on extrapancreatic tissues. In this study, we examined the tissue distribution of GLP-1 receptor (GLP-lR) messenger RNA (mRNA) in rat by RNAse protection, RT-PCR, and in situ hybridization. We identified GLP-1R mRNA in the lung, pancreatic islets, stomach, and kidney by the RNAse protection assay. RT-PCR analysis also detected GLP-1R mRNA in the hypothalamus and heart. In situ hybridization experiments identified receptor mRNA in the gastric pits of the stomach, large nucleated cells in the lung, crypts of the duodenum, and pancreatic islets. No localized specific grains were found in kidney, skeletal muscle, heart, liver, or adipocytes. These results indicate that sequences corresponding to the cloned rat islet GLP-1 receptor are expressed in the pancreatic islets, lung, hypothalamus, stomach, heart, and kidney but not in adipose, liver, and skeletal muscle. Further, the GLP-1 receptor expressed in the kidney and heart may be structural variants of the known receptor. Therefore, the observed extrapancreatic actions of GLP-1 may not be strictly confined to interactions with the defined GLP-1 receptor.