Asunto(s)
Abatacept/uso terapéutico , Antirreumáticos/uso terapéutico , Artritis Reumatoide/tratamiento farmacológico , Abatacept/efectos adversos , Antirreumáticos/efectos adversos , Artritis Reumatoide/diagnóstico , Artritis Reumatoide/epidemiología , Artritis Reumatoide/inmunología , Humanos , Italia/epidemiología , Estudios Prospectivos , Inducción de Remisión , Estudios Retrospectivos , Factores de Tiempo , Resultado del TratamientoRESUMEN
Adiponectin influences insulin sensitivity and lipid metabolism, but it is not clear whether these effects are correlated with fat mass or distribution. We studied the relationship between plasma adiponectin and leptin levels, insulin sensitivity, and serum lipids by a cross-sectional study (n = 242 subjects) and by an intervention study (95 of 242) to evaluate the effect of weight loss (WL). Considering all subjects both together and subdivided into nonobese (n = 107) and obese (n = 135) groups, plasma adiponectin, but not plasma leptin, was significantly (P < 0.01) correlated with insulin sensitivity [homeostasis model assessment of insulin-resistance index (HOMAIR), insulin sensitivity index (ISI) at oral glucose tolerance test, and clamp in 115 of 242 individuals], high-density lipoprotein cholesterol, and triglycerides. These relationships were still significant (P < 0.01) after adjusting for age, gender, body mass index (BMI), and ISI. After WL (-16.8 +/- 0.8%), plasma adiponectin increased, and plasma leptin decreased (P < 0.0001 for both). Their changes (Delta) were significantly correlated with Delta-BMI (P < 0.05 for both). Delta-Adiponectin, but not Delta-leptin, significantly (P < 0.001) correlated with Delta-high-density lipoprotein cholesterol and Delta-triglycerides; these correlations were independent of age, gender, Delta-BMI, and Delta-ISI (P < 0.005). In conclusion, both cross-sectional and intervention studies indicate that plasma adiponectin level correlates with serum lipids independently of fat mass. The intervention study also suggests that adiponectin increase after WL is correlated with serum lipid improvement independently of insulin sensitivity changes.
Asunto(s)
Composición Corporal/fisiología , HDL-Colesterol/sangre , Péptidos y Proteínas de Señalización Intercelular , Obesidad/metabolismo , Obesidad/terapia , Proteínas/metabolismo , Triglicéridos/sangre , Adiponectina , Tejido Adiposo/anatomía & histología , Tejido Adiposo/metabolismo , Adolescente , Adulto , Factores de Edad , Anciano , Peso Corporal/fisiología , Estudios Transversales , Femenino , Técnica de Clampeo de la Glucosa , Humanos , Resistencia a la Insulina/fisiología , Leptina/sangre , Masculino , Persona de Mediana Edad , Pérdida de Peso/fisiologíaRESUMEN
Insulin gene transcription is critical for the maintenance of pancreatic beta-cell differentiation and insulin production. In this study, we found that the basic helix-loop-helix transcription factor Mad, which usually acts as a repressor to c-Myc, enhances insulin gene transcription. In isolated rat islets adenoviral overexpression of Mad augmented insulin mRNA expression and insulin protein content, as well as glucokinase and GLUT2 mRNA expression. Also, Mad overexpression upregulated insulin promoter activity in beta-cell-derived cell lines, MIN6 and betaTC1, as well as in non-insulin producing liver cell line, HepG2. Mad overexpression in rat islets enhanced PDX-1 expression and its DNA binding activity. We found that Mad mediated increased PDX-1 expression by an E-box dependent transcriptional regulation of the PDX-1 gene. That the effects of Mad on insulin expression were mediated through PDX-1 was further substantiated by studies showing inhibition of insulin promoter activation by Mad in the presence of mutated PDX-1 binding site. Although Mad functions as a negative regulatory factor for multiple target genes, these studies establish the fact that Mad can also function as a positive regulatory factor for insulin gene transcription. Such regulation of insulin expression by Mad with modulation of PDX-1 expression and DNA binding activity could offer useful therapeutic and/or experimental tools to promote insulin production in appropriate cell types.
Asunto(s)
Proteínas de Unión al ADN/fisiología , Proteínas de Homeodominio , Insulina/genética , Islotes Pancreáticos/metabolismo , Proteínas Represoras , Activación Transcripcional , Adenoviridae/genética , Animales , Línea Celular , Células Cultivadas , Proteínas de Unión al ADN/biosíntesis , Proteínas de Unión al ADN/genética , Vectores Genéticos , Insulina/biosíntesis , Islotes Pancreáticos/citología , Masculino , Regiones Promotoras Genéticas , Ratas , Ratas Sprague-Dawley , Transactivadores/metabolismoRESUMEN
We investigated the effect of a chronic exposure to high levels of free fatty acid (FFA; 2 mmol/L oleate/palmitate 2:1) or glucose (16.7 mmol/L) on islet cell apoptosis. Apoptosis was detected using 4 different methods: (1) cell staining with annexin-V fluorescien isothiocyanate (FITC) conjugate and propidium iodide (PI); (2) quantification of cytoplasmatic DNA fragments by an enzyme-linked immunosorbent assay (ELISA); (3) assay of caspase 3 activity; and (4) TdT-mediated dUTP nick-end labeling (TUNEL). Islet cells were also costained with an anti-insulin antibody to identify apoptotic beta cells. We also evaluated by reverse-transcriptase polymerase chain reaction (RT-PCR) the expression of bax, bcl-2, and caspas 3, genes involved in apoptosis. In islets cultured for 7 days in the presence of high FFA or for 3 days in the presence of high glucose levels, we observed: (1) a 2- to 3-fold increase of apoptotic cells conjugated with annexin-V FITC and PI; (2) a 4- to 6-fold increase of cytoplasmatic DNA fragments; (3) a 3- to 4-fold increase of caspase 3 activity; and (4) a significant increase of insulin positive apoptotic cells as detected with the TUNEL method. RT-PCR analysis indicated in islets exposed to high FFA or glucose levels an increase of bax (proapoptotic gene), a reduction of bcl-2 (antiapoptotic gene), and a slight (although not significant) increase in caspase 3 expression. Western blot analysis also showed an increase of Bax protein levels in islets exposed to high FFA or glucose. The simultaneous presence of both metabolic abnormalities did not further increase the amount of apoptotic cells, although the time-course of the cellular damage induced by FFA was accelerated by the contemporary presence of high glucose. To elucidate the mechanism by which FFA and glucose may induce pancreatic beta-cell damage, we examined whether nicotinamide prevents apoptosis in pancreatic islets cultured for 7 days with high FFA or for 3 days with high glucose. Nicotinamide was able to prevent beta-cell damage by significantly reducing apoptosis in both experimental conditions. Also, the increase of Bax protein level was prevented by nicotinamide. These data indicate that chronic exposure to elevated FFA or glucose levels increases apoptosis in rat pancreatic islets and these cytotoxic effects could be mediated by oxidative stress. This may contribute to the beta-cell failure that occurs in most in type 2 diabetic patients few years after clinical diabetes onset.
Asunto(s)
Apoptosis/efectos de los fármacos , Ácidos Grasos no Esterificados/farmacología , Glucosa/farmacología , Islotes Pancreáticos/citología , Estrés Oxidativo/fisiología , Proteínas Proto-Oncogénicas c-bcl-2 , Animales , Anexina A5 , Antioxidantes/farmacología , Western Blotting , Caspasas/metabolismo , Células Cultivadas , Fragmentación del ADN/efectos de los fármacos , Fluoresceína-5-Isotiocianato , Colorantes Fluorescentes , Genes bcl-2/genética , Etiquetado Corte-Fin in Situ , Islotes Pancreáticos/efectos de los fármacos , Masculino , Necrosis , Niacinamida/farmacología , Propidio , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Ratas , Ratas Wistar , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteína X Asociada a bcl-2RESUMEN
In rat pancreatic islets chronically exposed to high glucose or high free fatty acid (FFA) levels, glucose-induced insulin release and mitochondrial glucose oxidation are impaired. These abnormalities are associated with high basal ATP levels but a decreased glucose-induced ATP production (Delta of increment over baseline 0.7 +/- 0.5 or 0.5 +/- 0.3 pmol/islet in islets exposed to glucose or FFA vs. 12.0 +/- 0.6 in control islets, n = 3; P < 0.01) and, as a consequence, with an altered ATP/ADP ratio. To investigate further the mechanism of the impaired ATP formation, we measured in rat pancreatic islets glucose-stimulated pyruvate dehydrogenase (PDH) activity, a key enzyme for pyruvate metabolism and for the subsequent glucose oxidation through the Krebs cycle, and also the uncoupling protein-2 (UCP-2) content by Western blot. In islets exposed to high glucose or FFA, glucose-stimulated PDH activity was impaired and UCP-2 was overexpressed. Because UCP-2 expression is modulated by a peroxisome proliferator- activated receptor (PPAR)-dependent pathway, we measured PPAR-gamma contents by Western blot and the effects of a PPAR-gamma antagonist. PPAR-gamma levels were overexpressed in islets cultured with high FFA levels but unaffected in islets exposed to high glucose. In islets exposed to high FFA concentration, a PPAR-gamma antagonist was able to prevent UCP-2 overexpression and to restore insulin secretion and the ATP/ADP ratio. These data indicate that in rat pancreatic islets chronically exposed to high glucose or FFA, glucose-induced impairment of insulin secretion is associated with (and might be due to) altered mitochondrial function, which results in impaired glucose oxidation, overexpression of the UCP-2 protein, and a consequent decrease of ATP production. This alteration in FFA cultured islets is mediated by the PPAR-gamma pathway.
Asunto(s)
Adenosina Trifosfato/biosíntesis , Ácidos Grasos no Esterificados/administración & dosificación , Glucosa/administración & dosificación , Insulina/metabolismo , Islotes Pancreáticos/metabolismo , Proteínas de Transporte de Membrana , Proteínas Mitocondriales , Proteínas/fisiología , Receptores Citoplasmáticos y Nucleares/antagonistas & inhibidores , Factores de Transcripción/antagonistas & inhibidores , Adenosina Difosfato/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Relación Dosis-Respuesta a Droga , Ácidos Grasos no Esterificados/farmacología , Glucosa/metabolismo , Glucosa/farmacología , Técnicas In Vitro , Secreción de Insulina , Canales Iónicos , Islotes Pancreáticos/efectos de los fármacos , Ligandos , Masculino , Oxidación-Reducción , Complejo Piruvato Deshidrogenasa/metabolismo , Ratas , Ratas Wistar , Receptores Citoplasmáticos y Nucleares/metabolismo , Factores de Transcripción/metabolismo , Proteína Desacopladora 2RESUMEN
Type 2 (non-insulin-dependent) diabetes results from decreased insulin action in peripheral target tissues (insulin resistance) and impaired pancreatic beta-cell function. These defects reflect both genetic components and environmental risk factors. Recently, the common Gly(972)-->Arg amino acid polymorphism of insulin receptor substrate 1 (Arg(972) IRS-1) has been associated with human type 2 diabetes. In this study, we report on some functional and morphological properties of isolated human islets carrying the Arg(972) IRS-1 polymorphism. Insulin content was lower in variant than control islets (94 +/- 47 vs. 133 +/- 56 microU/islet; P < 0.05). Stepwise glucose increase (1.7 to 16.7 mmol/l) significantly potentiated insulin secretion from control islets, but not Arg(972) IRS-1 islets, with the latter also showing a relatively lower response to glyburide and a significantly higher response to arginine. Proinsulin release mirrored insulin secretion, and the insulin-to-proinsulin ratio in response to arginine was significantly lower from Arg(972) IRS-1 islets than from control islets. Glucose utilization and oxidation did not differ in variant and wild-type islets at both low and high glucose levels. Electron microscopy showed that Arg(972) IRS-1 beta-cells had a severalfold greater number of immature secretory granules and a lower number of mature granules than control beta-cells. In conclusion, Arg(972) IRS-1 islets have reduced insulin content, impaired insulin secretion, and a lower amount of mature secretory granules. These alterations may account for the increased predisposition to type 2 diabetes in individuals carrying the Gly(972)-->Arg amino acid polymorphism of IRS-1.
Asunto(s)
Diabetes Mellitus Tipo 2/genética , Insulina/metabolismo , Islotes Pancreáticos/metabolismo , Fosfoproteínas/genética , Polimorfismo de Nucleótido Simple , Adolescente , Adulto , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/patología , Femenino , Humanos , Técnicas In Vitro , Proteínas Sustrato del Receptor de Insulina , Secreción de Insulina , Islotes Pancreáticos/patología , Masculino , Microscopía Electrónica , Persona de Mediana Edad , Vesículas Secretoras/patología , Vesículas Secretoras/ultraestructuraRESUMEN
In an effort to better understand the phenomenon of lipotoxicity in human beta-cells, we evaluated the effects of 48-h preculture with 1.0 or 2.0 mmol/l free fatty acid (FFA) (2:1 oleate to palmitate) on the function and survival of isolated human islets and investigated some of the possible mechanisms. Compared with control islets, triglyceride content was significantly increased and insulin content and glucose-stimulated insulin release were significantly reduced in islets precultured with increased FFA concentrations. These changes were accompanied by a significant reduction of glucose utilization and oxidation. By cell death detection techniques, it was observed that exposure to FFAs induced a significant increase of the amount of dead cells. Electron microscopy showed the involvement of beta-cells, with morphological appearance compatible with the presence of apoptotic phenomena. FFA-induced islet cell death was blocked by inhibition of upstream caspases and partially prevented by inhibiton of ceramide synthesis or serine protease activity, whereas inhibition of nitric oxide synthesis had no effect. RT-PCR studies revealed no major change of iNOS and Bax mRNA expression and a marked decrease of Bcl-2 mRNA expression in the islets cultured with FFA. Thus, prolonged exposure to FFAs has cytostatic and pro-apoptotic effects on human pancreatic beta-cells. The cytostatic action is likely to be due to the FFA-induced reduction of intraislet glucose metabolism, and the proapoptotic effects are mostly caspase mediated, partially dependent on ceramide pathway, and possibly Bcl-2 regulated.
Asunto(s)
Apoptosis/efectos de los fármacos , Ácidos Grasos no Esterificados/farmacología , Islotes Pancreáticos/citología , Islotes Pancreáticos/efectos de los fármacos , Anciano , Caspasas/metabolismo , Supervivencia Celular/efectos de los fármacos , Ceramidas/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Femenino , Glucosa/metabolismo , Humanos , Técnicas In Vitro , Insulina/metabolismo , Secreción de Insulina , Islotes Pancreáticos/enzimología , Masculino , Persona de Mediana Edad , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Triglicéridos/metabolismoRESUMEN
Human pancreatic islets from eight donors were incubated for 48 h in the presence of 2.0 mmol/l free fatty acid (FFA) (oleate to palmitate, 2 to 1). Insulin secretion was then assessed in response to glucose (16.7 mmol/l), arginine (20 mmol/l), and glyburide (200 micromol/l) during static incubation or by perifusion. Glucose oxidation and utilization and intra-islet triglyceride content were measured. The effect of metformin (2.4 microg/ml) was studied because it protects rat islets from lipotoxicity. Glucose-stimulated but not arginine- or glyburide-stimulated insulin release was significantly lower from FFA-exposed islets. Impairment of insulin secretion after exposure to FFAs was mainly accounted for by defective early-phase release. In control islets, increasing glucose concentration was associated with an increase in glucose utilization and oxidation. FFA incubation reduced both glucose utilization and oxidation at maximal glucose concentration. Islet triglyceride content increased significantly after FFA exposure. Addition of metformin to high-FFA media prevented impairment in glucose-mediated insulin release, decline of first-phase insulin secretion, and reduction of glucose utilization and oxidation without significantly affecting islet triglyceride accumulation. These results show that lipotoxicity in human islets is characterized by selective loss of glucose responsiveness and impaired glucose metabolism, with a clear defect in early-phase insulin release. Metformin prevents these deleterious effects, supporting a direct protective action on human beta-cells.