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1.
Mol Metab ; 30: 72-130, 2019 12.
Artículo en Inglés | MEDLINE | ID: mdl-31767182

RESUMEN

BACKGROUND: The glucagon-like peptide-1 (GLP-1) is a multifaceted hormone with broad pharmacological potential. Among the numerous metabolic effects of GLP-1 are the glucose-dependent stimulation of insulin secretion, decrease of gastric emptying, inhibition of food intake, increase of natriuresis and diuresis, and modulation of rodent ß-cell proliferation. GLP-1 also has cardio- and neuroprotective effects, decreases inflammation and apoptosis, and has implications for learning and memory, reward behavior, and palatability. Biochemically modified for enhanced potency and sustained action, GLP-1 receptor agonists are successfully in clinical use for the treatment of type-2 diabetes, and several GLP-1-based pharmacotherapies are in clinical evaluation for the treatment of obesity. SCOPE OF REVIEW: In this review, we provide a detailed overview on the multifaceted nature of GLP-1 and its pharmacology and discuss its therapeutic implications on various diseases. MAJOR CONCLUSIONS: Since its discovery, GLP-1 has emerged as a pleiotropic hormone with a myriad of metabolic functions that go well beyond its classical identification as an incretin hormone. The numerous beneficial effects of GLP-1 render this hormone an interesting candidate for the development of pharmacotherapies to treat obesity, diabetes, and neurodegenerative disorders.


Asunto(s)
Péptido 1 Similar al Glucagón/metabolismo , Péptido 1 Similar al Glucagón/farmacología , Glucemia/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Polipéptido Inhibidor Gástrico/metabolismo , Receptor del Péptido 1 Similar al Glucagón/metabolismo , Glucosa/metabolismo , Humanos , Hipoglucemiantes/uso terapéutico , Insulina/metabolismo , Secreción de Insulina , Células Secretoras de Insulina/metabolismo , Obesidad/metabolismo , Receptores de Glucagón/metabolismo
2.
Diabetologia ; 54(8): 2067-76, 2011 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-21567300

RESUMEN

AIMS/HYPOTHESIS: The endogenous production of stromal cell-derived factor-1 (SDF-1) in beta cells in transgenic mice attenuates the development of diabetes in response to streptozotocin. Here we propose that beta cell injury induces SDF-1 production, and the SDF-1/chemokine (C-X-C motif) receptor 4 (CXCR4) interaction auto-activates Sdf1 expression, resulting in the autocrine production of SDF-1 by beta cells and the paracrine activation of glucagon-like peptide-1 (GLP-1) production by alpha cells. METHODS: SDF-1 production in adult mouse and human islets and rat INS-1 cells was measured in models of beta cell injury. The paracrine actions of SDF-1 on GLP-1 production in alpha cells were explored. The potential synergism between the growth-promoting actions of GLP-1 and the pro-survival actions of SDF-1 on the preservation of cell mass was evaluated by cell viability assays. RESULTS: In adult islets and INS-1 cells, Sdf1 expression was re-induced in response to injury. The interaction of SDF-1 with its receptor on alphaTC1 cells activated protein kinase Akt, stimulated cell proliferation and induced the expression of prohormone convertase 1/3 and the consequent production of GLP-1 in alpha cells. The combination of GLP-1 and SDF-1 additively enhanced both the growth and longevity of INS-1 beta cells. CONCLUSIONS/INTERPRETATION: The results of these studies suggest that in response to beta cell injury and the ensuing induction of SDF-1, the biological function of alpha cells switches from the production of glucagon to the provision of the local growth factor GLP-1 which, in combination with SDF-1, promotes the growth, survival and viability of the beta cells.


Asunto(s)
Quimiocina CXCL12/metabolismo , Quimiocina CXCL12/farmacología , Péptido 1 Similar al Glucagón/metabolismo , Células Secretoras de Insulina/citología , Células Secretoras de Insulina/metabolismo , Islotes Pancreáticos/efectos de los fármacos , Receptores CXCR4/metabolismo , Animales , Línea Celular , Supervivencia Celular/efectos de los fármacos , Quimiocina CXCL12/genética , Humanos , Técnicas In Vitro , Células Secretoras de Insulina/efectos de los fármacos , Islotes Pancreáticos/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Páncreas/citología , Ratas , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa
3.
Diabetes Obes Metab ; 13(1): 26-33, 2011 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-21114600

RESUMEN

AIMS: The metabolic syndrome, a disease arising from the world-wide epidemic of obesity, is manifested as severe insulin resistance, hyperlipidaemia, hepatic steatosis and diabetes. Previously we reported that GLP-1(9-36)amide, derived from the gluco-incretin hormone, glucagon-like peptide-1 (GLP-1), suppresses gluconeogenesis in isolated hepatocytes. The aims of this study were to determine the effects of GLP-1(9-36)amide in diet-induced obese mice that model the development of the metabolic syndrome. METHODS: Mice rendered obese by feeding a very high fat diet were administered GLP-1(9-36)amide via subcutaneous osmopumps for 8 weeks. Body weight, energy intake, plasma insulin and glucose levels (insulin-resistance), and hepatic steatosis were assessed. RESULTS: Eight-week infusions of GLP-1(9-36)amide inhibited weight gain, increased energy intake, prevented the development of fasting hyperinsulinaemia and hyperglycaemia, and curtailed the accumulation of liver triglycerides. The peptide had no effects in mice fed a normal chow diet. Notably, energy intake in the obese mice receiving GLP-1(9-36)amide was 20% greater than obese mice receiving vehicle control. CONCLUSIONS: GLP-1(9-36)amide exerts insulin-like actions in the presence of insulin resistance and prevents the development of metabolic syndrome. Curtailment of weight gain in the face of increased caloric intake suggests that GLP-1(9-36)amide increases energy expenditure. These findings suggest the possibility of the use of GLP-1(9-36)amide, or a peptide mimetic derived there from, for the treatment of obesity, insulin resistance and the metabolic syndrome.


Asunto(s)
Hígado Graso/metabolismo , Péptido 1 Similar al Glucagón/metabolismo , Síndrome Metabólico/metabolismo , Receptores de Glucagón/metabolismo , Aumento de Peso/efectos de los fármacos , Animales , Peso Corporal/efectos de los fármacos , Grasas de la Dieta/administración & dosificación , Ingestión de Energía , Péptido 1 Similar al Glucagón/farmacología , Insulina/biosíntesis , Masculino , Síndrome Metabólico/tratamiento farmacológico , Ratones , Ratones Endogámicos C57BL , Ratones Obesos
4.
Horm Metab Res ; 42(9): 657-62, 2010 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-20645222

RESUMEN

The glucoincretin hormone glucagon-like peptide-1 (GLP-1) augments glucose-stimulated insulin secretion and is in use as an effective treatment for diabetes. However, after its secretion from the intestine, the insulinotropic GLP-1 (7-36) amide hormone is rapidly inactivated by enzymatic cleavage by the diaminopeptidyl peptidase-4 giving rise to GLP-1 (9-36) amide. Inasmuch as most of the circulating GLP-1 is in the form of the metabolite GLP-1 (9-36) amide it has been suggested that it has insulin-like actions on peripheral insulin-sensitive tissues. In earlier studies, infusions of GLP-1 (9-36) amide in obese insulin-resistant subjects showed a marked suppression of hepatic glucose production. However, it remained uncertain whether the effects on glucose production were due to direct effects on hepatocytes, involved central or portal vein-mediated actions, or were mediated by insulin secretion. Here we show that GLP-1 (9-36) amide directly suppresses glucose production in isolated mouse hepatocytes ex vivo independent of the GLP-1 receptor. These findings support direct insulinomimetic actions of the GLP-1 metabolite on gluconeogenesis in hepatocytes that are independent of insulin action and the GLP-1 receptor, and suggest that GLP-1 (9-36) amide-based peptides might present a novel therapy for the treatment of excessive hepatic glucose production in individuals with insulin-resistant diabetes.


Asunto(s)
Amidas/metabolismo , Péptido 1 Similar al Glucagón/metabolismo , Glucosa/biosíntesis , Hepatocitos/metabolismo , Animales , Separación Celular , Regulación de la Expresión Génica/efectos de los fármacos , Glucagón/farmacología , Péptido 1 Similar al Glucagón/farmacología , Receptor del Péptido 1 Similar al Glucagón , Gluconeogénesis/efectos de los fármacos , Gluconeogénesis/genética , Glucosa-6-Fosfatasa/metabolismo , Hepatocitos/efectos de los fármacos , Hepatocitos/enzimología , Ratones , Ratones Endogámicos C57BL , Fosfoenolpiruvato Carboxiquinasa (GTP)/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptores de Glucagón/genética , Receptores de Glucagón/metabolismo
5.
Diabetologia ; 52(8): 1589-98, 2009 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-19468708

RESUMEN

AIMS/HYPOTHESIS: Stromal cell-derived factor-1 (SDF-1) is a chemokine produced in stromal tissues in multiple organs. Earlier we reported on levels of SDF-1 and SDF-1 receptor (CXCR4) in the insulin-producing beta cells of the mouse pancreas and determined that the SDF-1/CXCR4 axis is important for beta cell survival through activation of the prosurvival kinase, protein kinase B (AKT). Since AKT is known to modulate the wingless-type MMTV integration site family (WNT) signalling cascade, we examined the effects of SDF-1/CXCR4 on WNT signalling in beta cells and whether this signalling is important for cell survival. METHODS: Activation of downstream WNT signalling (beta-catenin and transcription factor 7-like 2, [TCF7L2]) in response to SDF-1 was examined in the islets of WNT signalling reporter (Tcf-optimal promoter beta-galactosidase) mice and in INS-1 and MIN6 beta cells. Cytoprotection of beta cells by SDF-1 in response to the induction of apoptosis was assessed by caspase 3 and TUNEL assays. RESULTS: SDF-1 induced WNT signalling in beta cells of isolated islets and in INS-1 cells via CXCR4-mediated activation of Galphai/o-coupled signalling and the phosphatidylinositol 3-kinase/AKT signalling cascade resulting in the inhibition of glycogen synthase kinase 3-beta. The key WNT signalling regulators, beta-catenin and AKT, were activated by SDF-1 at the transcriptional and post-translational levels. Specific inhibition of beta-catenin in the WNT signalling cascade reversed the anti-apoptotic effects of SDF-1. CONCLUSIONS/INTERPRETATION: SDF-1 promotes pancreatic beta cell survival via activation of AKT and downstream WNT signalling via the stabilisation and activation of beta-catenin/TCF7L2 transcriptional activators. These findings suggest a mechanism for SDF-1 based glucose-lowering therapies by enhancing beta cell mass through increasing cell survival.


Asunto(s)
Quimiocina CXCL12/fisiología , Células Secretoras de Insulina/fisiología , Factores de Transcripción TCF/fisiología , beta Catenina/fisiología , Animales , Apoptosis/efectos de los fármacos , Apoptosis/fisiología , División Celular , Supervivencia Celular , Citocinas/farmacología , Genes Reporteros , Secuencias Hélice-Asa-Hélice , Células Secretoras de Insulina/citología , Ratones , Reacción en Cadena de la Polimerasa , Regiones Promotoras Genéticas , ARN Interferente Pequeño/genética , Tapsigargina/farmacología , Proteína 2 Similar al Factor de Transcripción 7 , beta Catenina/genética , beta-Galactosidasa/genética
6.
Diabetes Care ; 24(11): 1951-6, 2001 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-11679463

RESUMEN

An important cause of elevated glucose levels in elderly patients with diabetes is an alteration in non-insulin-mediated glucose uptake (NIMGU). Glucagon-like peptide 1 (GLP-1) is an intestinal insulinotropic hormone. It has been proposed that this hormone also lowers glucose levels by enhancing NIMGU. This study was conducted to determine whether GLP-1 augments NIMGU in elderly patients with diabetes, a group in which NIMGU is known to be impaired. Studies were conducted on 10 elderly patients with type 2 diabetes (aged 75 +/- 2 years, BMI 27 +/- 1 kg/m(2)) who underwent paired 240-min glucose clamp studies. In each study, octreotide was infused to suppress endogenous insulin release, and tritiated glucose methodology was used to measure glucose production and disposal rates. For the first 180 min, no glucose was infused. From 180 to 240 min, glucose was increased to 11 mmol/l using the glucose clamp protocol. In the GLP-1 study, GLP-1 was infused from 30 to 240 min. In a subsequent control study, insulin was infused using the glucose clamp protocol from 30 to 240 min to match the insulin levels that occurred during the GLP-1 infusion study. During hyperglycemia, GLP-1 enhanced glucose disposal (control study: 2.52 +/- 0.19 mg x kg(-1) x min(-1); GLP-1 study: 2.90 +/- 0.17 mg x kg(-1) x min(-1); P < 0.0001). Hepatic glucose output was not different between studies. We conclude that GLP-1 may partially reverse the defect in NIMGU that occurs in elderly patients with diabetes.


Asunto(s)
Glucemia/metabolismo , Diabetes Mellitus/sangre , Hipoglucemiantes/uso terapéutico , Péptidos/administración & dosificación , Administración Oral , Anciano , Análisis de Varianza , Diabetes Mellitus/tratamiento farmacológico , Glucagón/sangre , Péptido 1 Similar al Glucagón , Péptidos Similares al Glucagón , Técnica de Clampeo de la Glucosa , Hemoglobina Glucada/análisis , Humanos , Hipoglucemiantes/administración & dosificación , Insulina/sangre , Selección de Paciente , Fragmentos de Péptidos , Péptidos/sangre
7.
J Gerontol A Biol Sci Med Sci ; 56(11): M681-5, 2001 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-11682575

RESUMEN

BACKGROUND: Glucagon-like peptide-1 (GLP-1) is an intestinal insulinotropic hormone that augments glucose-induced insulin secretion in patients with type 2 diabetes. It has also been proposed that a substantial component of the glucose-lowering effects of GLP-1 occurs because this hormone enhances insulin-mediated glucose disposal. However, interpretations of the studies have been controversial. This study determines the effect of GLP-1 on insulin-mediated glucose disposal in elderly patients with type 2 diabetes. METHODS: Studies were conducted on 8 elderly patients with type 2 diabetes (age range, 76 +/- 1 years; body mass index, 28 +/- 1 kg/m(2)). Each subject underwent two 180-minute euglycemic (insulin infusion rate, 40 mU/m(2)/min) insulin clamps in random order. Glucose production (Ra) and disposal (Rd) rates were measured using tritiated glucose methodology. In one study, glucose and insulin alone were infused. In the other study, a primed-continuous infusion of GLP-1 was administered at a final rate of 1.5 pmol x kg(-1) x min(-1) from 30 to 180 minutes. RESULTS: Glucose values were similar between the control and GLP-1 infusion studies. 120- to 180-minute insulin values appeared to be higher during the GLP-1 infusion study (control, 795 +/- 63 pmol/l; GLP-1, 1140 +/- 275 pmol/l; p = not significant [NS]). The higher insulin values were largely due to 2 subjects who had substantial insulin responses to GLP-1 despite euglycemia and hyperinsulinemia. The 120- to 180-minute insulin values were similar in the other 6 subjects (control, 746 +/- 35 pmol/l; GLP-1, 781 +/- 41 pmol/l; p = NS). Basal (control, 2.08 +/- 0.05 mg/kg/min; GLP-1, 2.13 +/- 0.04 mg/kg/min; p = NS) and 120- to 180-minute (control, 0.50 +/- 0.18 mg/kg/min; GLP-1, 0.45 +/- 0.14 mg/kg/min; p = NS) Ra was similar between studies. The 120- to 180-minute Rd values were higher during the GLP-1 infusion studies (control, 4.73 +/- 0.39 mg/kg/min; GLP-1, 5.52 +/- 0.43 mg/kg/min; p <.01). When the 2 subjects who had significant insulin responses to GLP-1 during the euglycemic clamp were excluded, the 120- to 180-minute Rd values were still higher in the GLP-1 infusion study (control, 5.22 +/- 0.32 mg/kg/min; GLP-1, 6.05 +/- 0.37 mg/kg/min; p <.05). CONCLUSIONS: We conclude that GLP-1 may enhance insulin sensitivity in elderly patients with diabetes.


Asunto(s)
Diabetes Mellitus Tipo 2/tratamiento farmacológico , Diabetes Mellitus Tipo 2/metabolismo , Glucosa/metabolismo , Insulina/metabolismo , Péptidos/farmacología , Anciano , Transporte Biológico Activo/efectos de los fármacos , Glucemia/metabolismo , Péptido C/sangre , Diabetes Mellitus Tipo 2/sangre , Glucagón , Péptido 1 Similar al Glucagón , Péptidos Similares al Glucagón , Técnica de Clampeo de la Glucosa , Humanos , Insulina/sangre , Fragmentos de Péptidos , Péptidos/administración & dosificación , Péptidos/sangre
8.
J Gerontol A Biol Sci Med Sci ; 56(9): M575-9, 2001 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-11524451

RESUMEN

BACKGROUND: The current studies were designed to examine the effect of aging and diabetes on the enteroinsular axis. METHODS: Healthy young control subjects (n = 10 young; age 23 +/- 1 years; body mass index [BMI] 24 +/- 1 kg/m(2)), healthy elderly subjects (n = 10; age 80 +/- 2 years; BMI 26 +/- 1 kg/m(2)), and elderly patients with type 2 diabetes (n = 10; age 76 +/- 2 years; BMI 26 +/- 2 kg/m(2)) underwent a 3-hour oral glucose tolerance test (glucose dose 40 gm/m(2)). RESULTS: Insulin responses were not different between young controls and elderly patients with diabetes but were significantly lower in elderly patients with diabetes and young controls than in elderly controls (young control: 178 +/- 27 pM; elderly control: 355 +/- 57 pM; elderly diabetes: 177 +/- 30 pM; p <.05 elderly control vs young control and elderly diabetes). Total glucagon-like peptide 1 (GLP-1) responses were not significantly different between young and elderly controls and patients with diabetes (young control: 15 +/- 2 pM; old control: 8 +/- 2 pM; elderly diabetes: 12 +/- 3 pM; p = ns). Active GLP-1 responses were also not different between young and elderly controls and patients with diabetes (young control: 5 +/- 1 pM; old control: 6 +/- 1 pM; elderly diabetes: 7 +/- 1 pM; p = ns). However, the difference between total and active GLP levels was significantly greater in the young controls (young control: 10 +/- 2 pM; old control: 2 +/- 2 pM; elderly diabetes: 4 +/- 2 pM; p <.05, young vs elderly). Glucose-dependent insulinotropic polypeptide responses were not different between young and elderly controls and between elderly controls and patients with diabetes but were significantly higher in elderly patients with diabetes than in young controls (young control: 97 +/- 12 pM; elderly control: 121 +/- 16 pM; elderly diabetes: 173 +/- 27 pM; p <.05, young vs elderly diabetes). Glucagon responses were reduced in elderly controls but were similar in young controls and elderly patients with diabetes (young control: 15 +/- 1 pM; elderly control: 9 +/- 1 pM; elderly diabetes: 16 +/- 1 pM; p <.01 elderly control vs young control and elderly diabetes). Dipeptidyl peptidase IV levels were lower in both elderly controls and patients with diabetes when compared with young controls (young control: 0.17 +/- 0.01; elderly control: 0.15 +/- 0.01; elderly diabetes: 0.15 +/- 0.01 DeltaOD/20 minutes; p <.05, elderly vs young). CONCLUSIONS: We conclude that normal aging and diabetes are associated with multiple changes in the enteroinsular axis.


Asunto(s)
Envejecimiento/fisiología , Diabetes Mellitus/fisiopatología , Insulina/metabolismo , Intestinos/fisiología , Islotes Pancreáticos/fisiología , Adulto , Anciano , Anciano de 80 o más Años , Dipeptidil Peptidasa 4/metabolismo , Femenino , Polipéptido Inhibidor Gástrico/metabolismo , Glucagón/metabolismo , Péptido 1 Similar al Glucagón , Humanos , Secreción de Insulina , Masculino , Fragmentos de Péptidos/metabolismo , Inhibidores de Proteasas/uso terapéutico , Precursores de Proteínas/metabolismo
9.
J Clin Invest ; 108(2): 319-29, 2001 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-11457885

RESUMEN

Monogenic forms of diabetes can result from mutations in genes encoding transcription factors. Mutations in the homeodomain transcription factor IDX-1, a critical regulator of pancreas development and insulin gene transcription, confer a strong predisposition to the development of diabetes mellitus in humans. To investigate the role of IDX-1 expression in the pathogenesis of diabetes, we developed a model for the inducible impairment of IDX-1 expression in pancreatic beta cells in vivo by engineering an antisense ribozyme specific for mouse IDX-1 mRNA under control of the reverse tetracycline transactivator (rtTA). Doxycycline-induced impairment of IDX-1 expression reduced activation of the Insulin promoter but activated the Idx-1 promoter, suggesting that pancreatic beta cells regulate IDX-1 transcription to maintain IDX-1 levels within a narrow range. In transgenic mice that express both rtTA and the antisense ribozyme construct, impaired IDX-1 expression elevated glycated hemoglobin levels, diminished glucose tolerance, and decreased insulin/glucose ratios. Metabolic phenotypes induced by IDX-1 deficiency were observed predominantly in male mice over 18 months of age, suggesting that cellular mechanisms to protect IDX-1 levels in pancreatic beta cells decline with aging. We propose that even in the absence of Idx-1 gene mutations, pathophysiological processes that decrease IDX-1 levels are likely to impair glucose tolerance. Therapeutic strategies to attain normal glucose homeostasis by restoring normal IDX-1 levels may be of particular importance for older individuals with diabetes mellitus.


Asunto(s)
Envejecimiento/genética , Diabetes Mellitus Tipo 2/genética , Proteínas de Homeodominio/metabolismo , Páncreas/metabolismo , Transactivadores/deficiencia , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Modelos Animales de Enfermedad , Regulación de la Expresión Génica , Proteínas de Homeodominio/genética , Ratones , Ratones Transgénicos , Datos de Secuencia Molecular , Regiones Promotoras Genéticas , ARN Mensajero/biosíntesis , Transactivadores/biosíntesis , Transactivadores/genética
10.
Diabetes ; 50(7): 1553-61, 2001 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-11423476

RESUMEN

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.


Asunto(s)
Glucemia/fisiología , Proteínas de Homeodominio , Páncreas/metabolismo , Transactivadores/biosíntesis , Tejido Adiposo/metabolismo , Animales , Apoptosis , Regulación hacia Abajo , Regulación de la Expresión Génica , Prueba de Tolerancia a la Glucosa , Inmunohistoquímica , Ratones , Ratones Transgénicos , Microscopía Electrónica , Elastasa Pancreática/genética , Regiones Promotoras Genéticas , Ratas , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transactivadores/genética , Transgenes/genética , Regulación hacia Arriba
11.
Exp Cell Res ; 267(2): 193-204, 2001 Jul 15.
Artículo en Inglés | MEDLINE | ID: mdl-11426938

RESUMEN

CHOP/gadd153 is a transcription factor induced by cellular stresses such as UV light, genotoxic agents, and protein misfolding in the endoplasmic reticulum. The fact that these stresses induce CHOP expression, and at the same time cause cellular apoptosis, suggests that CHOP may be directly involved in apoptosis. However, evidence has been circumstantial. Here, we show that CHOP can directly induce apoptosis. A GFP-tagged CHOP vector, ectopically overexpressed in several cell types (3T3 fibroblasts, keratinocytes, and HeLa cells), caused apoptosis as defined by morphology, DNA fragmentation, and FACS analysis. Apoptosis was quantified using a rapid fluorescence assay that measures the signal from cells collected in culture supernatants. The apoptosis-modulating effects of p38 kinase, previously shown to phosphorylate CHOP, were also examined. Simultaneous overexpression of CHOP and p38 significantly augmented apoptosis. However, although p38 kinase clearly modulated the activity of full-length CHOP, it was not absolutely required. Deletion mapping experiments showed that the bZIP region of CHOP stimulates apoptosis to nearly the same extent as wild-type CHOP. Thus, while the amino-terminal region of CHOP serves an important modulatory role (i.e., regulation by p38), the underlying apoptosis-inducing activity of CHOP resides within the bZIP region of the molecule.


Asunto(s)
Apoptosis/fisiología , Proteínas Potenciadoras de Unión a CCAAT/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Factores de Transcripción/metabolismo , Animales , Antibacterianos/farmacología , Proteínas Potenciadoras de Unión a CCAAT/genética , Línea Celular , Separación Celular , Citometría de Flujo , Proteínas Fluorescentes Verdes , Humanos , Inmunohistoquímica , Etiquetado Corte-Fin in Situ , Indicadores y Reactivos/metabolismo , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Factor de Transcripción CHOP , Factores de Transcripción/genética , Tunicamicina/farmacología , Proteínas Quinasas p38 Activadas por Mitógenos
13.
Diabetes ; 50(3): 521-33, 2001 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-11246871

RESUMEN

The endocrine cells of the rat pancreatic islets of Langerhans, including insulin-producing beta-cells, turn over every 40-50 days by processes of apoptosis and the proliferation and differentiation of new islet cells (neogenesis) from progenitor epithelial cells located in the pancreatic ducts. However, the administration to rats of islet trophic factors such as glucose or glucagon-like peptide 1 for 48 h results in a doubling of islet cell mass, suggesting that islet progenitor cells may reside within the islets themselves. Here we show that rat and human pancreatic islets contain a heretofore unrecognized distinct population of cells that express the neural stem cell-specific marker nestin. Nestin-positive cells within pancreatic islets express neither the hormones insulin, glucagon, somatostatin, or pancreatic polypeptide nor the markers of vascular endothelium or neurons, such as collagen IV and galanin. Focal regions of nestin-positive cells are also identified in large, small, and centrolobular ducts of the rat pancreas. Nestin-positive cells in the islets and in pancreatic ducts are distinct from ductal epithelium because they do not express the ductal marker cytokeratin 19 (CK19). After their isolation, these nestin-positive cells have an unusually extended proliferative capacity when cultured in vitro (approximately 8 months), can be cloned repeatedly, and appear to be multipotential. Upon confluence, they are able to differentiate into cells that express liver and exocrine pancreas markers, such as alpha-fetoprotein and pancreatic amylase, and display a ductal/endocrine phenotype with expression of CK19, neural-specific cell adhesion molecule, insulin, glucagon, and the pancreas/duodenum specific homeodomain transcription factor, IDX-1. We propose that these nestin-positive islet-derived progenitor (NIP) cells are a distinct population of cells that reside within pancreatic islets and may participate in the neogenesis of islet endocrine cells. The NIP cells that also reside in the pancreatic ducts may be contributors to the established location of islet progenitor cells. The identification of NIP cells within the pancreatic islets themselves suggest possibilities for treatment of diabetes, whereby NIP cells isolated from pancreas biopsies could be expanded ex vivo and transplanted into the donor/recipient.


Asunto(s)
Proteínas de Filamentos Intermediarios/metabolismo , Islotes Pancreáticos/citología , Islotes Pancreáticos/metabolismo , Hígado/citología , Proteínas del Tejido Nervioso , Páncreas/citología , Células Madre/citología , Células Madre/metabolismo , Animales , Biomarcadores , Diferenciación Celular , División Celular , Separación Celular , Células Cultivadas , Femenino , Humanos , Islotes Pancreáticos/fisiología , Hígado/fisiología , Masculino , Nestina , Páncreas/fisiología , Conductos Pancreáticos/metabolismo , Fenotipo , Ratas , Ratas Sprague-Dawley , Células Madre/fisiología , Distribución Tisular
14.
J Biol Chem ; 276(16): 12938-44, 2001 Apr 20.
Artículo en Inglés | MEDLINE | ID: mdl-11278585

RESUMEN

The expression of the paracrine signaling hormone pituitary adenylate cyclase-activating polypeptide (PACAP) is regulated in a cyclical fashion during the 12-day spermatogenic cycle of the adult rat testis. The precise functions of PACAP in the development of germ cells are uncertain, but cycle- and stage-specific expression may augment cAMP-regulated gene expression in germ cells and associated Sertoli cells. Here we report the existence of a heretofore unrecognized exon in the extracellular domain of the PACAP type 1 receptor (PAC1R) that is alternatively spliced during the spermatogenic cycle in the rat testis. This splice variant encodes a full-length receptor with the insertion of an additional 72 base pairs encoding 24 amino acids (exon 3a) between coding exons 3 and 4. The PAC1R(3a) mRNA is preferentially detected in seminiferous tubules and is expressed at the highest levels in round spermatids and Sertoli cells. Analyses of ligand binding and signaling functions in stably transfected HEK293 cells expressing the two receptor isoforms reveals a 6-fold increase in the affinity of the PAC1R(3a) to bind PACAP-38, and alterations in its coupling to both cAMP and inositol phosphate signaling pathways relative to the wild type PAC1R. These findings suggest that the extracellular region between coding exons 3 and 6 of PAC1R may play an important role in the regulation of the relative ligand affinities and the relative coupling to G(s) (cAMP) and G(q) (inositol phosphates) signal transduction pathways during spermatogenesis.


Asunto(s)
Empalme Alternativo , Exones , Receptores de la Hormona Hipofisaria/genética , Receptores de la Hormona Hipofisaria/fisiología , Espermatogénesis/fisiología , Testículo/fisiología , Transcripción Genética , Animales , Sitios de Unión , Encéfalo/fisiología , Calcio/metabolismo , Línea Celular , AMP Cíclico/metabolismo , Proteínas de Unión al GTP/metabolismo , Variación Genética , Humanos , Fosfatos de Inositol/metabolismo , Islotes Pancreáticos/fisiología , Ligandos , Masculino , Neuropéptidos/química , Neuropéptidos/metabolismo , Polipéptido Hipofisario Activador de la Adenilato-Ciclasa , Hipófisis/fisiología , Reacción en Cadena de la Polimerasa , Subunidades de Proteína , ARN Mensajero/análisis , Ratas , Receptores del Polipéptido Activador de la Adenilato-Ciclasa Hipofisaria , Receptores del Polipéptido Activador de la Adenilato-Ciclasa Hipofisaria , Receptores de la Hormona Hipofisaria/química , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Túbulos Seminíferos/fisiología , Transducción de Señal/fisiología , Testículo/citología , Transfección
15.
J Clin Endocrinol Metab ; 86(1): 396-404, 2001 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-11232031

RESUMEN

Calcitonin precursors (CTpr), including procalcitonin, are important markers and also potentially harmful mediators in response to microbial infections. The source and function of CTpr production in sepsis, however, remains an enigma. In the classical view, the transcription of the CT-I gene is restricted to neuroendocrine cells, in particular the C cells of the thyroid. To better understand the pathophysiology of CTpr induction in sepsis, we used an animal model analog to human sepsis, in which bacterial infection is induced in hamsters by implanting Escherichia coli pellets ip. Compared with control hamsters, levels of CTpr were elevated several fold in septic plasma and in nearly all septic hamster tissues analyzed. Unexpectedly, CT-messenger RNA was ubiquitously and uniformly expressed in multiple tissues throughout the body in response to sepsis. Notably, the transcriptional expression of CT-messenger RNA seemed more widely up-regulated in sepsis than were classical cytokines (e.g. tumor necrosis factor-alpha and interleukin-6). Our findings, which describe a potentially new mechanism of host response to a microbial infection mediated by CTpr, introduce a new pathophysiological role for the CT-I gene.


Asunto(s)
Calcitonina/genética , Infecciones por Escherichia coli/genética , Expresión Génica , Animales , Calcitonina/sangre , Calcitonina/metabolismo , Cricetinae , Infecciones por Escherichia coli/metabolismo , Masculino , Mesocricetus , Profármacos/metabolismo , Isoformas de Proteínas/sangre , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , ARN Mensajero/metabolismo , Valores de Referencia , Distribución Tisular
16.
Endocrinology ; 142(3): 1033-40, 2001 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-11181516

RESUMEN

Insulin gene expression in pancreatic beta-cells is regulated by signals from developmental morphogen proteins known as hedgehogs (Hhs). By analyzing 5'-deletion insulin promoter-reporter constructs in transient transfections of clonal INS-1 beta-cells, we located activating Hh-responsive regions within the rat insulin I promoter that include the glucose-response elements Far (E2) and Flat (A2/A3). Activation of Hh signaling in INS-1 cells by ectopic Hh expression increased (and inhibition of Hh signaling with the Hh-specific inhibitor cyclopamine decreased) transcriptional activation of a multimerized FarFlat enhancer-reporter construct. In DNA-binding studies, nuclear extracts from INS-1 cells activated by ectopic Hh expression increased (and extracts from INS-1 cells treated with cyclopamine decreased) protein binding to a radiolabeled FarFlat oligonucleotide probe. An antiserum directed against the transcription factor islet duodenum homeobox-1 (IDX-1), a regulator of pancreas development and activator of the insulin gene promoter, attenuated the binding activity of Hh-responsive protein complexes. Nuclear IDX-1 protein levels on Western blots were increased by ectopic Hh expression, thereby providing a mechanism for Hh-mediated regulation of the insulin promoter. Addition of cyclopamine to INS-1 cells decreased IDX-1 messenger RNA expression. In transient transfections of a -4.5-kb mouse IDX-1 promoter-reporter construct, ectopic Hh expression increased (and cyclopamine administration decreased) transcriptional activation of the IDX-1 promoter in a dose-dependent manner. Thus, the IDX-1 gene is a direct regulatory target of Hh signaling in insulin-producing pancreatic beta-cells. We propose that Hh signaling activates the insulin gene promoter indirectly via the direct activation of IDX-1 expression. Because IDX-1 gene expression is essential for insulin gene expression, pancreatic beta-cell development, and normal glucose homeostasis, our findings that Hh signaling regulates IDX-1 expression in the endocrine pancreas suggest possible novel therapeutic approaches for diabetes mellitus.


Asunto(s)
Proteínas de Homeodominio , Islotes Pancreáticos/metabolismo , Proteínas/fisiología , Transducción de Señal/fisiología , Transactivadores/metabolismo , Animales , Línea Celular , ADN/metabolismo , Glucosa/fisiología , Proteínas Hedgehog , Insulina/genética , Regiones Promotoras Genéticas/fisiología , Ratas , Elementos de Respuesta/fisiología , Transactivadores/genética
17.
Endocrinology ; 142(3): 1179-87, 2001 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-11181533

RESUMEN

The insulin gene promoter contains many transcriptional response elements that predispose the gene to a wide range of regulatory signals. Glucagon-like peptide 1 (GLP-1) stimulates insulin gene transcription by intracellular second messenger cascades leading to direct transcription factor activation or to the up-regulation of insulin promoter specific transcription factors. In these studies, we have identified a novel regulatory signaling mechanism acting on the rat insulin 1 promoter (rINS1) in the INS-1 beta-cell line. In the presence of stimulatory concentrations of GLP-1 (0.1--100 nM) on rINS1 activity, inhibition of p38 mitogen-activated protein kinase (p38 MAPK) using SB 203580 resulted in a marked increase in promoter activity (maximum 3-fold) over GLP-1 alone, as determined by rINS1 promoter-luciferase reporter gene expression. This effect was revealed to be mediated via the cAMP response element (CRE) of rINS1, because site directed mutagenesis of the CRE motif in rINS1 abolished the increased response to SB 203580. Furthermore, inhibition of p38 MAPK uncovered a similar, more pronounced, response in the expression of a generic CRE promoter driven reporter gene. Time course dose-response studies indicate that the p38 MAPK induced inhibitory response may involve expression of immediate early genes (IEGs); maximum repression of rINS1 activity occurred after 4 h of treatment, comparable with regulatory responses by IEGs. In conclusion, these results demonstrate a novel signaling mechanism whereby p38 MAPK represses rINS1 promoter activity in response to GLP-1, suggesting the involvement of a robust regulatory control by p38 MAPK in insulin gene expression. The relevance of this mechanism may be most apparent during periods of cellular stress in which p38 MAPK activity is stimulated. In this regard, reduced insulin expression levels caused by chronic hyperglycemia (glucotoxicity) and/or hyperlipidemia (lipotoxicity) may be a direct consequence of this mechanism.


Asunto(s)
Glucagón/farmacología , Insulina/genética , Proteínas Quinasas Activadas por Mitógenos/fisiología , Fragmentos de Péptidos/farmacología , Regiones Promotoras Genéticas/efectos de los fármacos , Regiones Promotoras Genéticas/fisiología , Precursores de Proteínas/farmacología , Animales , Células Cultivadas , AMP Cíclico/fisiología , Proteínas Quinasas Dependientes de AMP Cíclico/fisiología , Activación Enzimática , Inhibidores Enzimáticos/farmacología , Expresión Génica/efectos de los fármacos , Péptido 1 Similar al Glucagón , Imidazoles/farmacología , Proteínas Quinasas Activadas por Mitógenos/antagonistas & inhibidores , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Piridinas/farmacología , Ratas , Elementos de Respuesta/fisiología , Transducción de Señal , Factores de Tiempo , Proteínas Quinasas p38 Activadas por Mitógenos
18.
Endocrinology ; 142(1): 129-38, 2001 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-11145575

RESUMEN

The regulation of glucose-dependent insulin secretion in pancreatic beta-cells is linked to the expression and function of the ATP-sensitive potassium channel (K(ATP)), which is composed of a sulfonylurea receptor (SUR1) and an inwardly rectifying potassium channel (Kir6.2). Previous animal and human genetic studies have demonstrated that disruption or defective expression of K(ATP) subunit genes has a profound impact on the regulation of insulin secretion. Little is known about how SUR1 and Kir6.2 gene expression is regulated. Here we show that high glucose concentrations lead to a marked decrease (approximately 70%) in Kir6.2 messenger RNA (mRNA) levels in isolated rat pancreatic islets as well as in the INS-1 beta-cell line. This effect is reversible, because exposure to low glucose reinduces Kir6.2 transcript levels. The cognate K(ATP) channel subunit SUR1 showed similar down-regulation at high glucose concentration. The K(ATP) channel activity of INS-1 cells cultivated at high glucose was reduced by 33-51%. In contrast, glucagon-like peptide-1 (GLP-1) induced Kir6.2 mRNA steady state levels and was able to prevent glucose-dependent inhibition of Kir6.2 mRNA and K(ATP) channel activity. To provide further insight into the mechanisms by which glucose and GLP-1 regulate beta-cell K(ATP) channel genes, we have cloned and initiated the characterization of the Kir6.2 gene transcriptional regulatory regions contained within the entire 4.5 kb flanked by the SUR1 and Kir6.2 genes. Transient transfection experiments with five deletion constructs in a pancreatic beta-cell line (INS-1) showed that the proximal 988 bp of the Kir6.2 promoter sequence contributes only 25-30% to the total basal promoter activity. The minimal promoter region -67/+140, also encompassing parts of the 5'-untranslated region, confers sensitivity to GLP-1, which stimulates transcriptional activity of the Kir6.2 minigene by about 2-fold. We propose that glucose- and GLP-1-dependent regulation of K(ATP) subunit genes may be important in the adaptation of beta-cells to changes in secretory demands in physiological and diseased states.


Asunto(s)
Transportadoras de Casetes de Unión a ATP , Regulación de la Expresión Génica , Islotes Pancreáticos/metabolismo , Canales de Potasio de Rectificación Interna , Canales de Potasio/genética , Receptores de Droga/genética , Transcripción Genética , Animales , Línea Celular , Células Cultivadas , Clonación Molecular , Regulación de la Expresión Génica/efectos de los fármacos , Glucagón/farmacología , Péptido 1 Similar al Glucagón , Glucosa/farmacología , Islotes Pancreáticos/citología , Masculino , Fragmentos de Péptidos/farmacología , Canales de Potasio/efectos de los fármacos , Canales de Potasio/fisiología , Precursores de Proteínas/farmacología , Subunidades de Proteína , ARN Mensajero/genética , Ratas , Ratas Wistar , Receptores de Droga/efectos de los fármacos , Receptores de Droga/fisiología , Proteínas Recombinantes/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Eliminación de Secuencia , Receptores de Sulfonilureas , Transcripción Genética/efectos de los fármacos , Transfección
19.
Diabetes ; 49(12): 2039-47, 2000 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-11118005

RESUMEN

Hedgehogs (Hhs) are intercellular signaling molecules that regulate tissue patterning in mammalian development. Mammalian Hhs include Sonic hedgehog (Shh), Indian hedgehog (Ihh), and Desert hedgehog (Dhh). The absence of Shh expression is required for the early development of the endocrine and exocrine pancreas, but whether Hh signaling functions in the fully developed adult endocrine pancreas is unknown. Here we report that Hhs Ihh and Dhh and their receptors patched (Ptc) and smoothened are expressed in the endocrine islets of Langerhans of the fully developed rat pancreas and in the clonal gamma-cell line INS-1. We demonstrate the coexpression of Ptc with insulin in beta-cells of mouse pancreatic islets, indicating that beta-cells are targets of active Hh signaling. The administration of cyclopamine, a Hh signaling inhibitor, decreases both insulin secretion from and insulin content of INS-1 cells. The effects of Hh signaling on insulin production occur at the transcriptional level because activation of Hh signal transduction by ectopic expression of Shh increases rat insulin I promoter activation in a dose-dependent manner in transient transfections of INS-1 and MIN6 beta-cell lines. In contrast, inhibition of Hh signaling with increasing concentrations of cyclopamine progressively reduces insulin promoter activity. Furthermore, the treatment of INS-1 cells with cyclopamine diminishes endogenous insulin mRNA expression. We propose that Hh signaling is not restricted to patterning in early pancreas development but also continues to signal in differentiated beta-cells of the endocrine pancreas in regulating insulin production. Thus, defective Hh signaling in the pancreas should be considered as a potential factor in the pathogenesis of type 2 diabetes.


Asunto(s)
Insulina/biosíntesis , Islotes Pancreáticos/metabolismo , Proteínas/fisiología , Transducción de Señal/fisiología , Transactivadores , Animales , Línea Celular , Proteínas Hedgehog , Insulina/genética , Islotes Pancreáticos/citología , Ratones , Proteínas/antagonistas & inhibidores , Proteínas/genética , ARN Mensajero/metabolismo , Transducción de Señal/efectos de los fármacos , Transcripción Genética/fisiología , Alcaloides de Veratrum/farmacología
20.
Endocrinology ; 141(11): 3923-30, 2000 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-11089521

RESUMEN

cAMP signaling contributes to the control of the developmental progression of germ cells during the spermatogenic cycle. Genes regulated by cAMP include those encoding transcription factors such as the cAMP-responsive element modulator (CREM). The disruption of CREM gene expression in crem null mice results in arrest of spermatogenesis and infertility. The transcriptional control of the CREM gene is attributed to two promoters, P1 and P2. The P1 promoter constitutively activates the synthesis of messenger RNAs encoding activator (tau) and repressor (alpha) forms of CREM, whereas the cAMP-responsive P2 promoter activates the formation of messenger RNAs encoding the inducible cAMP early repressor. Here we report the identification of two additional promoters in the CREM gene, P3 and P4, that in the rat testis encode two novel transcriptional activator CREM isoforms, termed CREM theta1 and CREM theta2, respectively. Notably, the P3 and P4 promoters are activated by cAMP-dependent protein kinase, thereby providing cAMP-regulated transcription of CREM activators in addition to the established cAMP-regulated inducible cAMP early repressor. Analysis ex vivo of CREM gene expression in temporally staged segments of the seminiferous tubule during the spermatogenic cycle shows that the activities of the P1, P3, and P4 promoters are independently regulated. Our identification of the cAMP-activated P3 and P4 promoters that direct expression of the novel theta1 and theta2 activator isoforms of CREM brings further insight into the complex expression of the CREM gene during germ cell development and may have implications in understanding the control of fertility.


Asunto(s)
AMP Cíclico/farmacología , Proteínas de Unión al ADN/genética , Regiones Promotoras Genéticas , Proteínas Represoras , Elementos de Respuesta , Testículo/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Modulador del Elemento de Respuesta al AMP Cíclico , Proteínas de Unión al ADN/química , Expresión Génica , Humanos , Masculino , Ratones , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , ARN Mensajero/análisis , Ratas , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Túbulos Seminíferos/metabolismo , Transducción de Señal , Espermatogénesis
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