RESUMO
AIMS/HYPOTHESIS: Glucagon-like peptide 1 (GLP-1) is a major incretin, mainly produced by the intestinal L cells, with beneficial actions on pancreatic beta cells. However, while in vivo only very small amounts of GLP-1 reach the pancreas in bioactive form, some observations indicate that GLP-1 may also be produced in the islets. We performed comprehensive morphological, functional and molecular studies to evaluate the presence and various features of a local GLP-1 system in human pancreatic islet cells, including those from type 2 diabetic patients. METHODS: The presence of insulin, glucagon, GLP-1, proconvertase (PC) 1/3 and PC2 was determined in human pancreas by immunohistochemistry with confocal microscopy. Islets were isolated from non-diabetic and type 2 diabetic donors. GLP-1 protein abundance was evaluated by immunoblotting and matrix-assisted laser desorption-ionisation-time of flight (MALDI-TOF) mass spectrometry. Single alpha and beta cell suspensions were obtained by enzymatic dissociation and FACS sorting. Glucagon and GLP-1 release were measured in response to nutrients. RESULTS: Confocal microscopy showed the presence of GLP-1-like and PC1/3 immunoreactivity in subsets of alpha cells, whereas GLP-1 was not observed in beta cells. The presence of GLP-1 in isolated islets was confirmed by immunoblotting, followed by mass spectrometry. Isolated islets and alpha (but not beta) cell fractions released GLP-1, which was regulated by glucose and arginine. PC1/3 (also known as PCSK1) gene expression was shown in alpha cells. GLP-1 release was significantly higher from type 2 diabetic than from non-diabetic isolated islets. CONCLUSIONS/INTERPRETATION: We have shown the presence of a functionally competent GLP-1 system in human pancreatic islets, which resides in alpha cells and might be modulated by type 2 diabetes.
Assuntos
Diabetes Mellitus Tipo 2/metabolismo , Peptídeo 1 Semelhante ao Glucagon/metabolismo , Células Secretoras de Glucagon/metabolismo , Glucagon/metabolismo , Insulina/metabolismo , Feminino , Humanos , Imuno-Histoquímica , Masculino , Espectrometria de Massas , Pessoa de Meia-Idade , Pâncreas/metabolismoRESUMO
Frataxin (FXN) is a mitochondrial protein involved in iron metabolism and in the modulation of reactive oxygen and/or nitrogen species production. No information is currently available as for the role of frataxin in isolated human pancreatic islets. We studied islets from pancreases of multi-organ donors with (T2DM) and without (Ctrl) Type 2 diabetes mellitus. In these islets, we determined FXN gene and protein expression by qualitative and quantitative Real-Time RT-PCR, nitrotyrosine concentration, and insulin release in response to glucose stimulation (SI). FXN gene and protein were expressed in human islets, though the level of expression was much lower in T2DM islets. The latter also had lower insulin release and higher concentration of nitrotyrosine. A positive correlation was apparent between SI and FXN gene expression, while a negative correlation was found between nitrotyrosine islet concentration and FXN expression. Transfection of Ctrl islets with siRNA FXN caused reduction of FXN expression, increase of nitrotyrosine concentration, and reduction of insulin release. In conclusion, in human pancreatic islets FXN contributes to regulation of oxidative stress and insulin release in response to glucose. In islets from T2DM patients FXN expression is reduced while oxidative stress is increased and insulin release in response to glucose impaired.
Assuntos
Diabetes Mellitus Tipo 2/metabolismo , Proteínas de Ligação ao Ferro/metabolismo , Ilhotas Pancreáticas/metabolismo , Ilhotas Pancreáticas/patologia , Doadores de Tecidos , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/patologia , Feminino , Regulação da Expressão Gênica , Humanos , Proteínas de Ligação ao Ferro/genética , Masculino , Pessoa de Meia-Idade , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/metabolismo , Análise de Regressão , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Tirosina/análogos & derivados , FrataxinaRESUMO
BACKGROUND AND AIMS: GPR40 is a membrane-bound receptor paired with medium and long-chain fatty acids (FFA) as endogenous ligands. Its acute activation potentiates insulin secretion from beta cells, whereas prolonged binding might contribute to the deleterious effects of chronic exposure to FFA. Little information is available on the expression of GPR40 and its regulation in human islets (HI). MATERIAL AND METHODS: HI were prepared by enzymatic digestion and gradient separation from the pancreas of 20 non-diabetic (Ctrl) and 13 type 2 diabetic (T2DM) multiorgan donors, and functional and molecular studies were then performed. RESULTS: By qualitative and quantitative PCR experiments, mRNA expression was shown in HI. Both in T2DM islets and in Ctrl islets pre-exposed for 24 h to 1.0 mmol/l FFA (palmitate:oleate, 2:1), GPR40 mRNA expression was significantly reduced (p<0.01) in the T2DM cells as compared to Ctrl cells. A significant positive correlation was found between glucose-stimulated insulin secretion and GPR40 expression. CONCLUSIONS: These results show the expression of GPR40 in human pancreatic islets which are regulated by FFA. The finding that T2DM islets have a lower GPR40 expression, and the correlation of these genes with insulin secretion, raises the possibility of an involvement of GPR40 in human diabetes beta-cell dysfunction.
Assuntos
Diabetes Mellitus Tipo 2/metabolismo , Ácidos Graxos não Esterificados/toxicidade , Regulação da Expressão Gênica/efeitos dos fármacos , Ilhotas Pancreáticas/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Idoso , Diabetes Mellitus Tipo 2/fisiopatologia , Feminino , Técnica Indireta de Fluorescência para Anticorpo , Glucose/farmacologia , Humanos , Insulina/genética , Insulina/metabolismo , Secreção de Insulina , Ilhotas Pancreáticas/citologia , Ilhotas Pancreáticas/efeitos dos fármacos , Ilhotas Pancreáticas/fisiopatologia , Ligantes , Masculino , Pessoa de Meia-Idade , RNA Mensageiro/metabolismo , RNA Interferente Pequeno , Receptores Acoplados a Proteínas G/antagonistas & inibidores , Receptores Acoplados a Proteínas G/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , TransfecçãoAssuntos
Insulina/genética , Insulina/metabolismo , Ilhotas Pancreáticas/metabolismo , Repetições Minissatélites , Polimorfismo Genético , Idoso , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/metabolismo , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/metabolismo , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Frequência do Gene , Estudos de Associação Genética , Genótipo , Glibureto/farmacologia , Humanos , Hiperglicemia/metabolismo , Hipoglicemiantes/farmacologia , Técnicas In Vitro , Secreção de Insulina , Ilhotas Pancreáticas/efeitos dos fármacos , Masculino , Pessoa de Meia-IdadeRESUMO
A multilaboratory study was conducted to compare the VIDAS LIS immunoassay with the standard cultural methods for the detection of Listeria in foods using an enrichment modification of AOAC Official Method 999.06. The modified enrichment protocol was implemented to harmonize the VIDAS LIS assay with the VIDAS LMO2 assay. Five food types--brie cheese, vanilla ice cream, frozen green beans, frozen raw tilapia fish, and cooked roast beef--at 3 inoculation levels, were analyzed by each method. A total of 15 laboratories representing government and industry participated. In this study, 1206 test portions were tested, of which 1170 were used in the statistical analysis. There were 433 positive by the VIDAS LIS assay and 396 positive by the standard culture methods. A Chi-square analysis of each of the 5 food types, at the 3 inoculation levels tested, was performed. The resulting average Chi square analysis, 0.42, indicated that, overall, there are no statistical differences between the VIDAS LIS assay and the standard methods at the 5% level of significance.
Assuntos
Análise de Alimentos/métodos , Contaminação de Alimentos , Microbiologia de Alimentos , Imunoensaio/métodos , Listeria/metabolismo , Animais , Queijo/microbiologia , Técnicas de Química Analítica/métodos , Fabaceae/microbiologia , Reações Falso-Negativas , Reações Falso-Positivas , Alimentos Congelados/microbiologia , Sorvetes/microbiologia , Carne/microbiologia , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Tilápia/microbiologia , Verduras/microbiologiaRESUMO
A multilaboratory study was conducted to compare the automated BAX system and the standard cultural methods for detection of Listeria monocytogenes in foods. Six food types (frankfurters, soft cheese, smoked salmon, raw, ground beef, fresh radishes, and frozen peas) were analyzed by each method. For each food type, 3 inoculation levels were tested: high (average of 2 CFU/g), low (average of 0.2 CFU/g) and uninoculated controls. A total of 25 laboratories representing government and industry participated. Of the 2335 samples analyzed, 1109 were positive by the BAX system and 1115 were positive by the standard method. A Chi square analysis of each of the 6 food types, at the 3 inoculation levels tested, was performed. For all foods, except radishes, the BAX system performed as well as or better than the standard reference methods based on the Chi square results.
Assuntos
Microbiologia de Alimentos , Listeria monocytogenes/isolamento & purificação , Reação em Cadeia da Polimerase/métodos , Distribuição de Qui-QuadradoRESUMO
AIMS: Studies suggest that insulin-signaling molecules are present in the pancreatic islets. For this reason, the effects of insulin glulisine, insulin aspart and regular human insulin (RHI) on the function and molecular features of isolated human pancreatic islets were investigated. METHODS: Human pancreatic islets were prepared by collagenase digestion and density-gradient purification of pancreata from multiple organ donors. Islets were then cultured for 48 h in the presence of 5.5 (normal) or 22.2 (high) mmol/L of glucose with and without glulisine, aspart and RHI (10 or 100 nmol/L). Functional (glucose-stimulated insulin secretion) and molecular (quantitative RT-PCR and immunoblot) studies were performed at the end of the different incubation conditions. RESULTS: Glucose-stimulated insulin secretion was blunted in islets cultured in 22.2 mmol/L of glucose, with no significant effects from the exogenous added insulins. In islets maintained at 5.5 mmol/L of glucose, insulin receptor (IR) expression was reduced by low RHI, while phosphatidylinositol-3 kinase p110-alpha (PI3K) was enhanced by both concentrations of glulisine and aspart, and by high RHI. In islets preexposed to high glucose, IR expression was increased by both concentrations of aspart and RHI, but not by glulisine. Glulisine at high concentration significantly (P<0.05) increased PI3K expression. Glulisine and RHI significantly increased IRS-2 phosphorylation compared with control and aspart (P<0.05). CONCLUSION: Insulin analogues have differential effects on the expression of insulin-signaling molecules in human pancreatic islets that are also dependent on the degree of glucose exposure.
Assuntos
Insulina/análogos & derivados , Insulina/metabolismo , Ilhotas Pancreáticas/efeitos dos fármacos , Receptor de Insulina/metabolismo , Idoso , Idoso de 80 Anos ou mais , Western Blotting , Células Cultivadas , Classe Ia de Fosfatidilinositol 3-Quinase/metabolismo , Feminino , Glucose/metabolismo , Humanos , Insulina/farmacologia , Insulina Aspart/farmacologia , Ilhotas Pancreáticas/metabolismo , Masculino , Pessoa de Meia-IdadeRESUMO
GLP-1 and GIP are incretins known to affect beta-cell function and turnover. However, information on the direct actions of these hormones on human islet cells is limited. We tested the effects of acute (45min) or prolonged (2days) exposure to GLP-1 or GIP, alone or in combination, on the function and some molecular features of human islets isolated from non-diabetic and type 2 diabetic multiorgan donors. Acutely, both GLP-1 and, more markedly so, GIP, significantly potentiated glucose-stimulated insulin release, with no apparent synergic action. Some of these effects were observed with type 2 diabetic islets as well. Following prolonged exposure to the incretins, improved insulin secretion was observed, and transcription of insulin, PDX-1 and Bcl-2 was increased in both non-diabetic and diabetic islets, with the combination of GLP-1 and GIP showing more significant effects. Although it is still unclear at what extent these beta-cell direct actions of individual or combined incretins occur in-vivo in humans, nevertheless the results of the present study suggest that enhancing the exposure of pancreatic islets to circulating levels of both incretins may be useful for therapeutical purposes.
Assuntos
Polipeptídeo Inibidor Gástrico/farmacologia , Peptídeo 1 Semelhante ao Glucagon/farmacologia , Incretinas/farmacologia , Insulina/metabolismo , Ilhotas Pancreáticas/efeitos dos fármacos , Ilhotas Pancreáticas/metabolismo , Idoso , Idoso de 80 Anos ou mais , Combinação de Medicamentos , Feminino , Humanos , Secreção de Insulina , Masculino , Pessoa de Meia-Idade , Técnicas de Cultura de Órgãos , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
BACKGROUND AND AIMS: Islet transplantation is an attractive approach to treat type 1 diabetic patients. However, suboptimal islet engraftment still represents an unsolved problem. It has been shown that human islets release monocyte chemoattractant protein-1 (MCP-1), one of the most powerful macrophage chemokines, which may impair the fate of the transplant. The aim of this study was to evaluate the presence and role of MCP-1 in isolated human islets, including genotyping for a common polymorphism. METHODS: Pancreatic islets were isolated by enzymatic digestion and gradient purification from 41 nondiabetic multiorgan donors. We measured MCP-1 mRNA expression by quantitative real- time reverse-transcriptase polymerization chain reaction, analyzed the MCP-1 single nucleotide polymorphism, -2518 G/A (SNP, rs 1024611) and evaluated glucose-stimulated insulin release (IR; microU/islet/min). RESULTS: MCP-1 mRNA expression was found in all studied batches of islets. Overall, IR was significantly higher at 16.7 mmol/L than 3.3 mmol/L glucose. We observed a significant negative correlation between MCP-1 mRNA expression and stimulation index (SI). We found that MCP-1 mRNA expression was significantly higher in CC and CT compared with TT genotype groups. Finally, SI was significant lower in the CC with respect to the TT genotype group. CONCLUSIONS: These data show that MCP-1 gene expression regulated by the -2518 G/A polymorphism, is correlated with glucose-stimulated insulin release. The study of MCP-1 expression and genotype on isolated islets before transplantation may be useful to understand the inflammatory response after infusion of human islets into patients with type 1 diabetes mellitus.
Assuntos
Quimiocina CCL2/genética , Ilhotas Pancreáticas/fisiologia , Polimorfismo de Nucleotídeo Único , Adenina/análise , Diabetes Mellitus Tipo 1/cirurgia , Regulação da Expressão Gênica , Glucose/farmacologia , Guanina/análise , Humanos , Inflamação/etiologia , Inflamação/genética , Insulina/metabolismo , Secreção de Insulina , Ilhotas Pancreáticas/efeitos dos fármacos , Ilhotas Pancreáticas/metabolismo , Transplante das Ilhotas Pancreáticas/efeitos adversos , Polimorfismo Genético , RNA Mensageiro/genética , Sequências Reguladoras de Ácido Nucleico/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Doadores de TecidosRESUMO
BACKGROUND AND AIMS: Pancreatic islet transplantation has become one of the potential treatments for type 1 diabetes. We evaluated functional and viability parameters of isolated islets in relation to donors clinical characteristics and preparation variables. METHODS: Islets were isolated from 70 nondiabetic multiorgan donors of overall age of 62.5 +/- 15.9 years. There were 41 men and 29 women. Their mean body mass index (BMI) was 25.62 +/- 3.09 kg/m(2). We evaluated the islet number (IEQ/g pancreatic tissue) insulin release (IR; microU/islet/min) in response to 3.3 (g) or 16.7 (G) mmol/L glucose; calcium flux concentration (CFC); and islet cell viability. RESULTS: IEQ was 5249 +/- 1505, with 73.7 +/- 14.96% viable islet cells. IR was 0.03 +/- 0.01 at g and 0.11 +/- 0.06 at G (stimulation index [S] = 3.24 +/- 1.96). CFC was 1.95 +/- 1.03 DeltaRFU. We observed positive correlations between viable cells and IR at g (R(2) = 0.260; P = .013), IR at G (R(2) = 0.165; P = .013), and CFC (R(2) = 0.175; P = .047). A positive correlation was documented between BMI and g (R(2) = 0.245; P = .016) and negative correlations between age with SI (R(2) = 0.188; P = .052) and cold ischemia time with IEQ (R(2) = 0.865; P = .0061). CONCLUSIONS: These results showed that quality control of isolated human pancreatic islets allowed assessment of beta-cell function and survival before transplantation, revealing several important variables.
Assuntos
Células Secretoras de Insulina/citologia , Células Secretoras de Insulina/fisiologia , Ilhotas Pancreáticas/citologia , Ilhotas Pancreáticas/fisiologia , Idoso , Índice de Massa Corporal , Contagem de Células , Separação Celular/métodos , Sobrevivência Celular , Diabetes Mellitus Tipo 1/cirurgia , Feminino , Humanos , Transplante das Ilhotas Pancreáticas/métodos , Masculino , Pessoa de Meia-Idade , Transplante de Pâncreas/métodos , Doadores de TecidosRESUMO
AIM: To evaluate the effects of exposure to high glucose (HG) levels and sulphonylurea on isolated human islet-cell function, and to investigate some of the mechanisms that might be involved. METHODS: Islet cells were isolated, using collagenase digestion and gradient purification, from 13 pancreata from non-diabetic multiorgan donors (age: 61.2+/-11.5 years; gender: 7 men/6 women; body mass index: 25.1+/-2.8kg/m(2)). The cells were then cultured for 5 days with normal glucose (NG) concentrations (5.5mmol/L), or NG and HG (16.7mmol/L) levels (alternating every 24h), with or without the addition of therapeutic concentrations of gliclazide (10micromol/L) or glibenclamide (1.0micromol/L). At the end of incubation, functional (glucose-stimulated insulin secretion), morphological (electron microscopy) and molecular (gene and protein expression) studies were performed. RESULTS: Insulin secretion differed significantly between study groups, with marked decreases in the presence of HG plus glibenclamide. Compared with NG, insulin expression decreased significantly with HG, and increased similarly with gliclazide as with glibenclamide. However, exposure to gliclazide, but not glibenclamide, significantly induced expression (at both gene and protein levels) of PDX-1, a fundamental beta-cell differentiation transcription factor, and Ki67, a marker of proliferation. However, gliclazide and glibenclamide did not differ in terms of effects on gene expression of the antiapoptotic molecule Bcl2 (increased significantly with both) and the proapoptotic molecule Bax (decreased significantly with both). CONCLUSION: Gliclazide and glibenclamide have different effects on the changes induced by prolonged exposure of human islet cells to high levels of glucose.