RESUMEN
Germline mutations in BRCA1 are responsible for most cases of inherited breast and ovarian cancer. However, the function of the BRCA1 protein has remained elusive. We now show that BRCA1 encodes a 190-kD protein with sequence homology and biochemical analogy to the granin protein family. Interestingly, BRCA2 also includes a motif similar to the granin consensus at the C terminus of the protein. Both BRCA1 and the granins localize to secretory vesicles, are secreted by a regulated pathway, are post-translationally glycosylated and are responsive to hormones. As a regulated secretory protein, BRCA1 appears to function by a mechanism not previously described for tumour suppressor gene products.
Asunto(s)
Neoplasias de la Mama/genética , Proteínas de Neoplasias/metabolismo , Neoplasias Ováricas/genética , Factores de Transcripción/metabolismo , Secuencia de Aminoácidos , Animales , Anticuerpos/inmunología , Proteína BRCA1 , Proteína BRCA2 , Mama/metabolismo , Epitelio/metabolismo , Femenino , Genes Supresores de Tumor , Humanos , Proteínas de la Membrana/química , Datos de Secuencia Molecular , Peso Molecular , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/inmunología , Proteínas/química , Conejos , Homología de Secuencia de Aminoácido , Factores de Transcripción/química , Factores de Transcripción/genética , Factores de Transcripción/inmunología , Células Tumorales CultivadasRESUMEN
AIMS/HYPOTHESIS: Prolonged exposure of pancreatic beta cells to excessive levels of glucose and fatty acids, referred to as glucolipotoxicity, is postulated to contribute to impaired glucose homeostasis in patients with type 2 diabetes. However, the relative contribution of defective beta cell function vs diminished beta cell mass under glucolipotoxic conditions in vivo remains a subject of debate. We therefore sought to determine whether glucolipotoxicity in rats is due to impaired beta cell function and/or reduced beta cell mass, and whether older animals are more susceptible to glucolipotoxic condition. METHODS: Wistar rats (2 and 6 months old) received a 72 h infusion of glucose + intravenous fat emulsion or saline control. In vivo insulin secretion and sensitivity were assessed by hyperglycaemic clamps. Ex vivo insulin secretion, insulin biosynthesis and gene expression were measured in isolated islets. Beta cell mass and proliferation were examined by immunohistochemistry. RESULTS: A 72 h infusion of glucose + intravenous fat emulsion in 2-month-old Wistar rats did not affect insulin sensitivity, insulin secretion or beta cell mass. In 6-month-old rats by contrast it led to insulin resistance and reduced insulin secretion in vivo, despite an increase in beta cell mass and proliferation. This was associated with: (1) diminished glucose-stimulated second-phase insulin secretion and proinsulin biosynthesis; (2) lower insulin content; and (3) reduced expression of beta cell genes in isolated islets. CONCLUSIONS/INTERPRETATION: In this in vivo model, glucolipotoxicity is characterised by an age-dependent impairment of glucose-regulated beta cell function despite a marked increase in beta cell mass.
Asunto(s)
Ácidos Grasos/toxicidad , Glucosa/toxicidad , Células Secretoras de Insulina/efectos de los fármacos , Células Secretoras de Insulina/metabolismo , Animales , Apoptosis/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Inmunohistoquímica , Técnicas In Vitro , Insulina/metabolismo , Células Secretoras de Insulina/patología , Masculino , Proinsulina/metabolismo , Ratas , Ratas Wistar , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
AIMS/HYPOTHESIS: The Zucker fatty (ZF) rat subjected to 60% pancreatectomy (Px) develops moderate diabetes by 3 weeks. We determined whether a progressive fall in beta cell mass and/or beta cell dysfunction contribute to beta cell failure in this type 2 diabetes model. METHODS: Partial (60%) or sham Px was performed in ZF and Zucker lean (ZL) rats. At 3 weeks post-surgery, beta cell mass and proliferation, proinsulin biosynthesis, pancreatic insulin content, insulin secretion, and islet glucose and lipid metabolism were measured. RESULTS: ZL-Px rats maintained normal glycaemia and glucose-stimulated insulin secretion (GSIS) despite incomplete recovery of beta cell mass possibly due to compensatory enhanced islet glucose metabolism and lipolysis. ZF-Px rats developed moderate hyperglycaemia (14 mmol/l), hypertriacylglycerolaemia and relative hypoinsulinaemia. Despite beta cell mass recovery and normal arginine-induced insulin secretion, GSIS and pancreatic insulin content were profoundly lowered in ZF-Px rats. Proinsulin biosynthesis was not reduced. Compensatory increases in islet glucose metabolism above those observed in ZF-Sham rats were not seen in ZF-Px rats. Triacylglycerol content was not increased in ZF-Px islets, possibly due to lipodetoxification by enhanced lipolysis and fatty acid oxidation. Fatty acid accumulation into monoacylglycerol and diacylglycerol was increased in ZF-Px islets together with a 4.5-fold elevation in stearoyl-CoA desaturase mRNA expression. CONCLUSIONS/INTERPRETATION: Falling beta cell mass, reduced proinsulin biosynthesis and islet steatosis are not implicated in early beta cell failure and glucolipotoxicity in ZF-Px rats. Rather, severe beta cell dysfunction with a specific reduction in GSIS and marked depletion of beta cell insulin stores with altered lipid partitioning underlie beta cell failure in this animal model of type 2 diabetes.
Asunto(s)
Hiperlipidemias/metabolismo , Hiperlipidemias/patología , Células Secretoras de Insulina/patología , Islotes Pancreáticos/patología , Obesidad/metabolismo , Obesidad/patología , Animales , Peso Corporal , Proliferación Celular , Células Cultivadas , Ácidos Grasos no Esterificados/metabolismo , Hiperlipidemias/fisiopatología , Inmunohistoquímica , Insulina/metabolismo , Células Secretoras de Insulina/citología , Células Secretoras de Insulina/metabolismo , Metabolismo de los Lípidos/fisiología , Masculino , Obesidad/fisiopatología , Pancreatectomía , Proinsulina/metabolismo , Ratas , Ratas ZuckerRESUMEN
We report the fortuitous isolation of cDNA clones encoding a novel zinc finger DNA-binding protein termed BZP. The protein encoded is 114 kDa and contains eight zinc finger motifs, seven of which are present in two clusters at opposite ends of the molecule. Both finger clusters bound to the 9-bp sequence AAAGGTGCA with apparent Kds of approximately 2.5 nM. Two of the finger motifs within the amino- and carboxy-terminal finger clusters share 63% amino acid identity. BZP inhibited transcription of the herpes simplex virus thymidine kinase promoter when copies of the 9-bp target motif were linked in cis, suggesting that it functions as a transcriptional repressor. BZP mRNA and immunoreactivity were detected in several established cell lines but were most abundant in hamster insulinoma (HIT) cells, the parental source of the cDNAs. In mouse tissues, BZP mRNA and immunoreactivity were identified in cells of the endocrine pancreas, anterior pituitary, and central nervous system. Interestingly, in HIT cells proliferating in culture, BZP immunoreactivity was predominately nuclear in location, whereas it was usually located in the cytoplasm in most neural and neuroendocrine tissues. Serum deprivation of HIT cells caused BZP immunoreactivity to become predominantly cytoplasmic in location and attenuated its inhibitory effect on transcription, thereby suggesting that the both the subcellular location and the function of this protein are modulated by factors in serum.
Asunto(s)
Proteínas de Unión al ADN/genética , Proteínas Represoras , Factores de Transcripción , Transcripción Genética , Dedos de Zinc , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Compartimento Celular , Sistema Nervioso Central/metabolismo , Clonación Molecular , Medio de Cultivo Libre de Suero , ADN Complementario/genética , Proteínas de Unión al ADN/aislamiento & purificación , Proteínas de Unión al ADN/metabolismo , Regulación de la Expresión Génica , Islotes Pancreáticos/metabolismo , Ratones , Datos de Secuencia Molecular , Fragmentos de Péptidos/metabolismo , Adenohipófisis/metabolismo , ARN Mensajero/análisis , Proteínas Recombinantes/metabolismo , Mapeo Restrictivo , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Distribución TisularRESUMEN
We have analyzed in organ culture the effects of glucose on glucose-induced insulin secretion, glucokinase (GK) activity, and human growth hormone (hGH) expression in pancreatic islets from transgenic mice containing an upstream GK promoter-hGH fusion gene. Freshly isolated islets from these mice had a normal insulin secretory response to glucose but showed subtle defects after culture in low or high glucose for 4 days that may have been due to the accumulation of hGH in the culture media. Islets cultured from both normal and transgenic mice had approximately a fourfold induction of GK activity in response to an increased concentration of glucose in the culture media, whereas no such change in total islet hGH production was observed. Immunocytochemical localization of hGH in islets cultured in 3 mM glucose showed a pattern similar to that in freshly isolated islets. However, after culture in 30 mM glucose, hGH immunostaining became strikingly more heterogeneous. We conclude 1) that GK-hGH transgene expression does not appear to adversely affect glucose-stimulated insulin secretion in vivo or in freshly isolated islets, 2) that glucose does not induce transgene expression, thus providing additional evidence against an effect of glucose on GK gene transcription in the islet, and 3) that glucose stimulates the co-release of hGH with insulin, thereby enhancing the heterogeneous staining pattern seen among pancreatic beta-cells.
Asunto(s)
Expresión Génica/efectos de los fármacos , Glucoquinasa/genética , Glucoquinasa/metabolismo , Glucosa/farmacología , Hormona del Crecimiento/genética , Insulina/metabolismo , Islotes Pancreáticos/fisiología , Regiones Promotoras Genéticas , Animales , Glucemia/metabolismo , Células Cultivadas , Hormona del Crecimiento/análisis , Humanos , Inmunohistoquímica , Insulina/análisis , Insulina/sangre , Secreción de Insulina , Islotes Pancreáticos/efectos de los fármacos , Islotes Pancreáticos/enzimología , Cinética , Ratones , Ratones TransgénicosRESUMEN
Transgenic mice that overexpress the entire glucokinase (GK) gene locus have been previously shown to be mildly hypoglycemic and to have improved tolerance to glucose. To determine whether increased GK might also prevent or diminish diabetes in diet-induced obese animals, we examined the effect of feeding these mice a high-fat high-simple carbohydrate low-fiber diet (HF diet) for 30 weeks. In response to this diet, both normal and transgenic mice became obese and had similar BMIs (5.3 +/- 0.1 and 5.0 +/- 0.1 kg/m2 in transgenic and non-transgenic mice, respectively). The blood glucose concentration of the control mice increased linearly with time and reached 17.0 +/- 1.3 mmol/l at the 30th week. In contrast, the blood glucose of GK transgenic mice rose to only 9.7 +/- 1.2 mmol/l at the 15th week, after which it returned to 7.6 +/- 1.0 mmol/l by the 30th week. The plasma insulin concentration was also lower in the GK transgenic animals (232 +/- 79 pmol/l) than in the controls (595 +/- 77 pmol/l), but there was no difference in plasma glucagon concentrations. Together, these data indicate that increased GK levels dramatically lessen the development of both hyperglycemia and hyperinsulinemia associated with the feeding of an HF diet.
Asunto(s)
Mapeo Cromosómico , Diabetes Mellitus Tipo 2/etiología , Diabetes Mellitus Tipo 2/genética , Predisposición Genética a la Enfermedad , Glucoquinasa/genética , Obesidad/complicaciones , Transgenes/fisiología , Animales , Glucemia/análisis , Grasas de la Dieta/administración & dosificación , Glucagón/sangre , Glucoquinasa/metabolismo , Insulina/sangre , Hígado/enzimología , Ratones , Ratones Transgénicos/genética , Obesidad/sangre , Obesidad/etiología , ARN Mensajero/metabolismo , Valores de ReferenciaRESUMEN
Sympatholytic dopamine agonist treatment utilizing bromocriptine and SKF38393 (BC/SKF) significantly lowers basal plasma insulin levels and normalizes basal and glucose-induced insulin secretion of the pancreatic beta cell in ob/ob mice. While BC/SKF has no significant effect on pancreatic islet cells directly, drug action is mediated via alterations in the hypothalamic-neuroendocrine axis, which drives metabolic changes in peripheral tissues leading to a marked reduction in hyperglycemia and hyperlipidemia and corrects autonomic control of islet function. To elucidate the nature of the functional response of islets to systemic BC/SKF treatment in ob/ob mice, we investigated the relative changes in the levels of functionally important beta-cell proteins in situ, as well as differences in the beta-cell turnover rate, following a 2-week drug treatment. Isolated islets from treated mice exhibit a 3.5-fold increase in insulin content (P <.01) that correlated with a 51% reduction in basal plasma insulin levels (P <.01) compared with vehicle-treated controls. Using quantitative immunofluorescence microscopy on pancreatic tissue sections, insulin and GLUT2 immunoreactivity of islet beta cells of BC/SKF-treated mice were significantly increased (approximately 2.3-fold and approximately 4.4-fold, respectively; P <.002) to the levels observed in islets of their lean littermates. Glucokinase (GK) immunoreactivity was greatly (75%) reduced in beta cells from ob/ob versus lean mice (P <.0001). A modest increase in GK immunoreactivity in beta cells of drug-treated mice was observed (approximately 1.6-fold; P <.05). Isolated islets from BC/SKF-treated mice exhibit a 42% reduction in DNA content compared with vehicle-treated controls (P <.01) to levels observed in lean mice, but without notable differences in islet size. In situ assays for mitosis and apoptosis, using 5-bromodeoxyuridine (BrdU) and terminal deoxyribotransferase (TdT)-UTP nick end labeling (TUNEL) staining techniques, respectively, were performed in pancreas of these mice to determine if beta cells show a reduction in hyperplasia following BC/SKF treatment. Accordingly, a pronounced decrease in replicating, BrdU-positive beta cells in the drug-treated mice compared with the control group was observed, but without differences in their TUNEL-staining patterns. Collectively, these data suggest that systemic sympatholytic dopaminergic therapy that attenuates hyperglycemia and hyperlipidemia improves islet function in ob/ob mice by improving aberrations in the beta cell's glucose-sensing apparatus, enhancing insulin storage and/or retention, and stabilizing hyperplasia, thus reducing basal insulin levels.
Asunto(s)
Agonistas de Dopamina/farmacología , Glucoquinasa/metabolismo , Insulina/metabolismo , Islotes Pancreáticos/efectos de los fármacos , Proteínas de Transporte de Monosacáridos/metabolismo , Simpaticolíticos/farmacología , Animales , Apoptosis/efectos de los fármacos , División Celular/efectos de los fármacos , ADN/metabolismo , Femenino , Glucagón/metabolismo , Transportador de Glucosa de Tipo 2 , Hiperplasia/patología , Hiperplasia/prevención & control , Inmunohistoquímica , Etiquetado Corte-Fin in Situ , Islotes Pancreáticos/metabolismo , Islotes Pancreáticos/patología , Ratones , Ratones Endogámicos C57BL , Ratones Obesos , Somatostatina/metabolismoAsunto(s)
Diabetes Mellitus Tipo 2/genética , Glucoquinasa/genética , Islotes Pancreáticos/enzimología , Receptores de Superficie Celular/genética , Diabetes Mellitus Tipo 2/enzimología , Diabetes Mellitus Tipo 2/metabolismo , Glucoquinasa/metabolismo , Glucosa/metabolismo , Homeostasis , Humanos , Islotes Pancreáticos/metabolismo , Hígado/metabolismo , Mutación , Receptores de Superficie Celular/metabolismoRESUMEN
Topiramate (TPM) is a novel neurotherapeutic agent. Clinical studies reported that TPM treatment reduced body weight and decreased fasting blood glucose levels in obese patients with or without type 2 diabetes. It is unclear whether the blood glucose-normalizing phenomenon observed during TPM treatment is a primary effect or the consequence of reduced food intake and weight loss. In the present studies, we chronically treated female Zucker diabetic fatty (ZDF) rats (fed with a diabetogenic diet) and db/db mice with TPM (30-300 mg/kg/day) to examine the effect of TPM on hyperglycaemia and its relationship with food intake and body weight gain. Our data showed that TPM treatment markedly reduced blood glucose levels in both ZDF rats and db/db mice without a significant reduction in body weight gain. Pair-fed db/db mice treated with the vehicle alone did not exhibit a significant decrease in blood glucose levels compared with mice fed ad libitum. TPM treatment increased glucose-stimulated insulin release by 2-3-fold during an oral glucose tolerance test in both ZDF rats and db/db mice. We also observed a 1.4-fold increase of pancreatic insulin content and heightened insulin immunostaining in pancreatic beta cells in db/db mice treated with TPM. Our data suggest that the antidiabetic effect of TPM is independent of the changes in body weight gain and food intake. Improved glucose-induced insulin release may, in part, underlie the mechanisms by which TPM ameliorates the hyperglycaemia.
Asunto(s)
Fructosa/análogos & derivados , Hiperglucemia/tratamiento farmacológico , Hipoglucemiantes/uso terapéutico , Insulina/metabolismo , Abdomen , Tejido Adiposo/efectos de los fármacos , Animales , Glucemia/efectos de los fármacos , Ingestión de Alimentos/efectos de los fármacos , Femenino , Fructosa/uso terapéutico , Prueba de Tolerancia a la Glucosa/métodos , Hiperglucemia/metabolismo , Hiperglucemia/patología , Islotes Pancreáticos/patología , Ratones , Ratones Endogámicos C57BL , Fármacos Neuroprotectores/uso terapéutico , Páncreas/metabolismo , Ratas , Ratas Zucker , Topiramato , Triglicéridos/sangre , Aumento de Peso/efectos de los fármacosRESUMEN
The cellular location of glucokinase (GK), a key component of the glucose-sensing mechanism of the pancreatic islet, was determined using immunocytochemical techniques. In rat islets, GK immunoreactivity was detected only in beta cells with no immunoreactivity detected in alpha, delta, or pancreatic polypeptide-containing (PP) cells. However, within various beta cells, GK immunoreactivity varied considerably. Most beta cells displayed relatively low levels of cytoplasmic immunoreactivity whereas other beta cells stained intensely for this enzyme. Colocalization studies of GK and GLUT2, the high Km glucose transporter of beta cells, confirmed that these proteins are located in different subcellular domains of beta cells. The lack of GK immunoreactivity in glucagon- and somatostatin-secreting cells in islets suggests that these cells are not directly responsive to glucose or utilize a fundamentally different mechanism for sensing glucose fluctuations. Moreover, the differential expression of GK among pancreatic beta cells suggests that glucose phosphorylation is the probable enzymatic control point for the functional diversity of these cells.
Asunto(s)
Glucoquinasa/metabolismo , Islotes Pancreáticos/enzimología , Secuencia de Aminoácidos , Animales , Técnica del Anticuerpo Fluorescente , Expresión Génica , Glucoquinasa/genética , Glucoquinasa/inmunología , Insulina/metabolismo , Masculino , Datos de Secuencia Molecular , Proteínas de Transporte de Monosacáridos/metabolismo , ARN Mensajero/genética , Ratas , Ratas EndogámicasRESUMEN
The ryanodine and inositol 1,4,5-trisphosphate (IP3) receptors have previously been found to be intracellular Ca2+ release channels characterized by their large size and 4-fold symmetry. In this study, cardiomyocytes are found to have a different intracellular localization for the two receptors. At the level of light microscopy, the IP3 receptor is immunolocalized in rat ventricular cardiomyocytes at the region of the intercalated discs. By contrast, immunoreactivity of the ryanodine receptor is observed as transverse bands throughout the length of the cardiomyocyte, coincident with the triad junction at the I-bands. At the level of electron microscopy, immunogold particles directed to the IP3 receptor specifically decorate the intercalated discs of rat ventricular and atrial cardiomyocytes, preferentially at the fascia adherens. Binding of [3H] IP3 and [3H]ryanodine were measured in cardiac subcellular fractions. IP3 binding is enriched in a fraction containing intercalated discs. Little or no IP3 binding was detected in longitudinal sarcoplasmic reticulum (SR), junctional SR, sarcolemma, mitochondria, and submitochondrial vesicles. Ryanodine binding is the highest in junctional SR. We conclude that the IP3 receptor is present in ventricular and atrial cardiomyocytes and localized at the region of the intercalated discs. These results suggest a possible role of the IP3 receptor in Ca2+ entry through intercalated discs and/or intercellular signaling between cardiomyocytes.
Asunto(s)
Canales de Calcio , Inositol 1,4,5-Trifosfato/metabolismo , Miocardio/metabolismo , Receptores de Superficie Celular/análisis , Receptores Colinérgicos/análisis , Receptores Citoplasmáticos y Nucleares , Rianodina/metabolismo , Animales , Western Blotting , Células Cultivadas , Perros , Ventrículos Cardíacos , Inmunohistoquímica , Inositol 1,4,5-Trifosfato/aislamiento & purificación , Receptores de Inositol 1,4,5-Trifosfato , Masculino , Microscopía Inmunoelectrónica , Peso Molecular , Miocardio/citología , Miocardio/ultraestructura , Ratas , Ratas Sprague-Dawley , Receptores de Superficie Celular/aislamiento & purificación , Receptores de Superficie Celular/metabolismo , Receptores Colinérgicos/metabolismo , Canal Liberador de Calcio Receptor de Rianodina , Fracciones Subcelulares/metabolismo , Fracciones Subcelulares/ultraestructuraRESUMEN
Transgenic mice containing an upstream glucokinase (betaGK) promoter- simian virus 40 T antigen (Tag) fusion gene develop neuroendocrine tumors primarily in the pancreas, gut, and pituitary. Pancreatic tumors from a line with delayed tumorigenesis were of two different types: insulinomas and noninsulinomas. The noninsulinomas are often periductal in location, express none of the four major islet peptide hormones, Glut-2, Pdx1, tyrosine hydroxylase, Pax4, Pax6, or Nkx6.1, but do express glucokinase, Sur1, Isl1, Hnf3beta, Hnf6, Beta2/NeuroD, and Nkx2.2. Cells from two different noninsulinoma tumors, when adapted to culture, began to express either insulin, glucagon, or somatostatin. Given the partial gene expression repertoire of the noninsulinoma tumors, their apparent periductal origin, and the ability of these cells to partially cytodifferentiate in culture, we suggest that these tumors are derived from islet progenitor cells. Thus, betaGK-Tag transgenic mice provide a new model system for studying the events that occur during both islet cell neogenesis and normal embryonic development.
Asunto(s)
Insulina/metabolismo , Islotes Pancreáticos/patología , Neoplasias Pancreáticas/patología , Animales , Genes Reporteros , Glucoquinasa/genética , Proteína Homeobox Nkx-2.2 , Inmunohistoquímica , Islotes Pancreáticos/enzimología , Islotes Pancreáticos/metabolismo , Ratones , Ratones Transgénicos , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismoRESUMEN
Studies of dispersed beta cells have been used to infer their behavior in the intact pancreatic islet. When dispersed, beta cells exhibit multiple metabolic glucose-response populations with different insulin secretion properties. This has led to a model for glucose-dependent insulin secretion from the islet based on a step-wise recruitment of individual beta cells. However, previously reported synchronous and uniform Ca2+ activity and electrical responses indicate that beta cell behavior within intact islets is more uniform. Therefore, uncertainty remains whether beta cell metabolic heterogeneity is functionally important in intact islets. We have used two-photon excitation microscopy to measure and compare the glucose-induced NAD(P)H autofluorescence response in dispersed beta cells and within intact islets. Over 90% of beta cells in intact islets responded to glucose with significantly elevated NAD(P)H levels, compared with less than 70% of dispersed beta cells. In addition, all responding beta cells within intact islets exhibited a sigmoidal glucose dose response behavior with inflection points of approximately 8 mm glucose. These results suggest that beta cell heterogeneity may be functionally less important in the intact islet than has been predicted from studies of dispersed beta cells and support the role of glucokinase as the rate-limiting enzyme in the beta cell glucose response.
Asunto(s)
Glucosa/metabolismo , Islotes Pancreáticos/metabolismo , NADP/metabolismo , NAD/metabolismo , Animales , Células Cultivadas , Técnica del Anticuerpo Fluorescente , Glucoquinasa/metabolismo , Islotes Pancreáticos/citología , Cinética , Microscopía Confocal/métodos , NAD/análisis , NADP/análisis , Ratas , Fracciones Subcelulares/metabolismo , Fracciones Subcelulares/ultraestructuraRESUMEN
Dysfunction of pancreatic islets plays a crucial role in the etiology of type II diabetes. Chronic hyperglycaemia or hyperlipidaemia may impair islet function. Previous studies by our laboratory have demonstrated that dopaminergic agonists ameliorated hyperglycaemia and hyperlipidaemia in obese and diabetic rodents. In the present study, we investigated the effect of a treatment with the dopamine D2/D1 receptor agonists (bromocriptine/SKF38393, BC/SKF) on islet dysfunction in db/db mice. Our results show that a 2-week BC/SKF treatment markedly reduced hyperglycaemia and hyperlipidaemia, and significantly improved islet dysfunction demonstrated by an increase of secretagogue-stimulated insulin release from islets of db/db mice to levels observed in islets from lean mice. There was also a fourfold increase of insulin content in the pancreas of BC/SKF-treated db/db mice compared with that in untreated controls. The effect of BC/SKF on islet function cannot be mimicked in pair-fed animals. BC/SKF had no direct stimulatory effect on islet insulin secretion, suggesting BC/SKF treatment improved islet function via an indirect mechanism. This treatment markedly improved the abnormally elevated daily levels of corticosterone, blood glucose and plasma lipids, supporting the view that BC/SKF may affect the neuroendocrine system that in turn regulates peripheral metabolism and thereby improves islet function.
Asunto(s)
Bromocriptina/farmacología , Islotes Pancreáticos/efectos de los fármacos , Animales , Glucemia/efectos de los fármacos , Corticosterona/sangre , Diabetes Mellitus Tipo 2 , Modelos Animales de Enfermedad , Agonistas de Dopamina/farmacología , Técnica del Anticuerpo Fluorescente , Inmunohistoquímica , Insulina/sangre , Islotes Pancreáticos/patología , Lípidos/sangre , Ratones , Ratones Obesos , Páncreas/efectos de los fármacos , Páncreas/patologíaRESUMEN
Hepatic glucokinase (GK) is acutely regulated by binding to its nuclear-anchored regulatory protein (GKRP). Although GK release by GKRP is tightly coupled to the rate of glycogen synthesis, the nature of this association is obscure. To gain insight into this coupling mechanism under physiological stimulating conditions in primary rat hepatocytes, we analyzed the subcellular distribution of GK and GKRP with immunofluorescence, and glycogen deposition with glycogen cytochemical fluorescence, using confocal microscopy and quantitative image analysis. Following stimulation, a fraction of the GK signal translocated from the nucleus to the cytoplasm. The reduction in the nuclear to cytoplasmic ratio of GK, an index of nuclear export, correlated with a >50% increase in glycogen cytochemical fluorescence over a 60 min stimulation period. Furthermore, glycogen accumulation was initially deposited in a peripheral pattern in hepatocytes similar to that of GK. These data suggest that a compartmentalization exists of both active GK and the initial sites of glycogen deposition at the hepatocyte surface.
Asunto(s)
Proteínas Portadoras , Núcleo Celular/enzimología , Glucoquinasa/metabolismo , Glucógeno Hepático/biosíntesis , Hígado/enzimología , Transporte Activo de Núcleo Celular , Animales , Células Cultivadas , Péptidos y Proteínas de Señalización Intracelular , Cinética , Perfusión , Proteínas/metabolismo , Ratas , Ratas Sprague-DawleyRESUMEN
Different glucokinase isoforms are produced by tissue-specific alternative RNA splicing in the liver and pancreatic islet, the only tissues in which glucokinase activity has been detected. To determine whether differences in protein structure brought about by alternative RNA splicing have an effect on glucose phosphorylating activity, we expressed cDNAs encoding four different hepatic and islet glucokinase isoforms and determined the Km and Vmax of each. When the glucokinase B1 and L1 isoforms were expressed in eukaryotic cells, both high Km glucose phosphorylating activity and immunoreactive protein were detected. However, when the glucokinase B2 and L2 isoforms were expressed, both of which differ by deletion of 17 amino acids in a region between the putative glucose and ATP-binding domains, no high Km glucose phosphorylating activity and much less immunoreactive protein were detected. When the glucokinase B1 and B2 isoforms were expressed in Escherichia coli as fusion proteins with glutathione S-transferase, affinity-purified B1 fusion protein was able to phosphorylate glucose whereas the B2 fusion protein was not, thus indicating that the lack of glucose phosphorylating activity from both the B2 and L2 isoforms is due to lack of intrinsic activity in addition to accumulation of less protein. The Km values of the B1 and L1 isoforms, which differ from each other by 15 amino acids at the NH2 terminus, were similar, but the Vmax of the B1 isoform was 2.8-fold higher than that of the L1 isoform. Mutagenesis of the first two potential initiation codons in the glucokinase B1 cDNA from ATG to GTC (methionine to valine) indicated that the first ATG was crucial for activity and is, therefore, the likely translation initiation codon. Messenger RNAs encoding both the B2 and L2 isoforms of glucokinase were detected in islet and liver by polymerase chain reaction amplification of total cDNA, indicating that mRNAs utilizing this weak alternate splice acceptor site in the fourth exon are normally present in both the liver and islet but as minor components. A regulatory role for weak alternate splice acceptor and donor sites in the glucokinase gene was suggested by examining the expression of the gene in the pituitary and in AtT-20 cells. Interestingly, although glucokinase mRNAs of appropriate sizes were detected in both the AtT-20 cells and rat pituitaries, neither exhibited any detectable high Km glucose phosphorylating activity.(ABSTRACT TRUNCATED AT 400 WORDS)
Asunto(s)
Glucoquinasa/genética , Islotes Pancreáticos/enzimología , Isoenzimas/genética , Hígado/enzimología , Hipófisis/enzimología , Empalme del ARN , ARN Mensajero/genética , Animales , Secuencia de Bases , Línea Celular , Exones , Glucoquinasa/metabolismo , Hexoquinasa/metabolismo , Isoenzimas/metabolismo , Ratones , Datos de Secuencia Molecular , Mutagénesis Insercional , Mutagénesis Sitio-Dirigida , Sondas de Oligonucleótidos , Reacción en Cadena de la Polimerasa/métodos , Ratas , Mapeo Restrictivo , Homología de Secuencia de Ácido Nucleico , TransfecciónRESUMEN
AIMS/HYPOTHESIS: Early compensatory mechanisms of regeneration following partial pancreatectomy involve ductal proliferation and, subsequently, differentiation into acinar and endocrine cell types, although it is not clear how these processes are regulated. We investigated the expression and roles of insulin receptor substrate-2 (IRS-2) and protein kinase B/Akt (Akt) in pancreatic regeneration that starts with the common duct epithelium using a non-diabetic model of beta cell adaptation and mass expansion, 60 %-pancreatectomy rats. METHODS: We used confocal immunofluorescence microscopy to study IRS-2 and Akt expression and activation in pancreatic common ducts at intervals after surgery. These proteins were studied in relation to proliferation markers and insulin immunostaining. RESULTS: In pancreatectomized rats, a short-term increase in proliferation was observed in the common duct epithelial lining ( approximately 4-fold) compared with sham-operated control rats which correlated with about a 1.8-fold increase in IRS-2 immunoreactivity 2 days after surgery. Interspersed with proliferating cells of the common duct, evaginations were rare single and clustered insulin immunopositive cells which expressed high levels of IRS-2 immunoreactivity. Epithelium of duct evaginations from 2-day post-Px rats exhibited striking phospho-Akt staining ( approximately 3.5-fold above control rats) without any detectable changes in total Akt staining. CONCLUSION/INTERPRETATION: Our data suggest that IRS-2 plays an important role in pancreatic regeneration and growth by mediating duct proliferation and by maintaining the differentiated beta cell. The restricted staining pattern of phospho-Akt to cells of the common duct evaginations suggests that it has a role in regulating post-mitotic events related to cell-specific gene expression or survival or both.
Asunto(s)
Regulación de la Expresión Génica , Pancreatectomía , Conductos Pancreáticos/fisiología , Fosfoproteínas/genética , Proteínas Serina-Treonina Quinasas , Proteínas Proto-Oncogénicas/genética , Regeneración , Animales , Células CHO , División Celular , Cricetinae , Células Epiteliales/citología , Células Epiteliales/fisiología , Técnica del Anticuerpo Fluorescente , Proteínas Sustrato del Receptor de Insulina , Péptidos y Proteínas de Señalización Intracelular , Cinética , Masculino , Microscopía Confocal , Conductos Pancreáticos/citología , Proteínas Proto-Oncogénicas c-akt , Ratas , Ratas Sprague-Dawley , Receptor de Insulina/genética , Factores de Tiempo , TransfecciónRESUMEN
Increases in ventromedial hypothalamic (VMH) norepinephrine (NE) levels and/or activities have been observed in a variety of animal models of the obese insulin-resistant condition. This study examined the metabolic effects of chronic NE infusion (25 nmol/h) into the unilateral VMH of normal rats. Within 4 days, VMH NE infusion significantly increased plasma insulin (140%), glucagon (45%), leptin (300%), triglyceride (100%), abdominal fat pad weight (50%), and white adipocyte lipogenic (100%) and lipolytic (100%) activities relative to vehicle-infused rats. Furthermore, isolated islet insulin secretory response to glucose (15 mM) within 4 days of such treatment was increased over twofold (P < 0.05). Among treated animals, fat stores continued to increase over time and plateaued at approximately 2 wk (3-fold increase), remaining elevated to the end of the study (5 wk). By week 4 of treatment, NE infusion induced glucose intolerance as evidenced by a 32% increase in plasma glucose total area under the glucose tolerance test curve (P < 0.01). Whole body fat oxidation rate measured after 5 wk of infusion was significantly increased among treated animals as evidenced by a reduced respiratory quotient (0.87 +/- 0.01) relative to controls (0. 90 +/- 0.01). VMH NE infusion induced hyperphagia (30%) only during the first week and did not affect body weight over the 5-wk period. Increases in VMH NE activity that are common among obese insulin-resistant animal models can cause the development of this obese glucose-intolerant (metabolic) syndrome.
Asunto(s)
Intolerancia a la Glucosa , Norepinefrina/administración & dosificación , Obesidad/fisiopatología , Núcleo Hipotalámico Ventromedial/fisiología , Tejido Adiposo/patología , Animales , Metabolismo de los Hidratos de Carbono , Glándulas Endocrinas/fisiopatología , Femenino , Hormonas/sangre , Inyecciones , Isoproterenol/farmacología , Leptina/sangre , Metabolismo de los Lípidos , Lipólisis/efectos de los fármacos , Lipólisis/fisiología , Norepinefrina/farmacología , Obesidad/patología , Ratas , Ratas Sprague-Dawley , Valores de Referencia , Síndrome , Factores de TiempoRESUMEN
The mouse Mf3 gene, also known as Fkh5 and HFH-e5.1, encodes a winged helix/forkhead transcription factor. In the early embryo, transcripts for Mf3 are restricted to the presomitic mesoderm and anterior neurectoderm and mesoderm. By 9.5 days post coitum, expression in the nervous system is predominantly in the diencephalon, midbrain and neural tube. After midgestation, the highest level of mRNA is in the mammillary bodies, the posterior-most part of the hypothalamus. Mice homozygous for a deletion of the mf3 locus on a [129 x Black Swiss] background display variable phenotypes consistent with a requirement for the gene at several stages of embryonic and postnatal development. Approximately six percent of the mf3-/- embryos show an open neural tube in the diencephalon and midbrain region, and another five percent show a severe reduction of the posterior body axis; both these classes of affected embryos die in utero. Surviving homozygotes have an apparently normal phenotype at birth. Postnatally, however, mf3-/- pups are severely growth retarded and approximately one third die before weaning. This growth defect is not a direct result of lack of circulating growth hormone or thyrotropin. Mice that survive to weaning are healthy, but they show an abnormal clasping of the hindfeet when suspended by the tail. Although much smaller than normal, the mice are fertile. However, mf3-/- females cannot eject their milk supply to feed their pups. This nursing defect can be corrected with interperitoneal injections of oxytocin. These results provide evidence that Mf3 is required for normal hypothalamus development and suggest that Mf3 may play a role in postnatal growth and lactation.
Asunto(s)
Tipificación del Cuerpo/genética , Encéfalo/embriología , Encéfalo/crecimiento & desarrollo , Proteínas de Unión al ADN/genética , Eyección Láctea/fisiología , Factores de Transcripción/genética , Animales , Conducta Animal/fisiología , Quimera , Cruzamientos Genéticos , Diencéfalo/embriología , Diencéfalo/crecimiento & desarrollo , Femenino , Factores de Transcripción Forkhead , Heterocigoto , Miembro Posterior/fisiología , Homocigoto , Hipotálamo/anatomía & histología , Inmunohistoquímica , Masculino , Glándulas Mamarias Animales/anomalías , Mesencéfalo/embriología , Mesencéfalo/crecimiento & desarrollo , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Ratones Mutantes , Fenotipo , Hipófisis/fisiología , Radioinmunoensayo , Reflejo/genéticaRESUMEN
Phosphorylation of glucose to glucose 6-phosphate by glucokinase (GK; EC 2.7.1.2) serves as a glucose-sensing mechanism for regulating insulin secretion in beta cells. Recent findings of heterozygous GK gene mutations in patients with maturity-onset diabetes of the young (MODY), a form of type II (non-insulin-dependent) diabetes characterized by autosomal dominant inheritance, have raised the possibility that a decrease in beta-cell GK activity may impair the insulin secretory response of these cells to glucose. To generate an animal model for MODY we have expressed in transgenic mice a GK antisense RNA with a ribozyme element under control of the insulin promoter. Mice in two independent lineages had about 30% of the normal islet GK activity. Insulin release in response to glucose from in situ-perfused pancreas was impaired; however, the plasma glucose and insulin levels of the mice remained normal. These mice are likely to be predisposed to type II diabetes and may manifest increased susceptibility to genetic and environmental diabetogenic factors. They provide an animal model for studying the interaction of such factors with the reduced islet GK activity.