RESUMEN
Beta-cell mass, hexokinase/glucokinase (HK/GK) activity, glucose metabolism and insulin secretion were studied in the islets of rats with fructose-induced insulin resistance (IR). Normal male Wistar rats were fed a standard commercial diet and water without (control, C) or with 10% fructose-rich diet (FRD) for 3 weeks. Blood glucose (strips), triglyceride (commercial kit), and insulin (RIA) levels were measured at the time of death. Glucose-induced insulin release, glucose metabolism ((14)CO(2) and (3)H(2)O production from D-[U-(14)C]- and D-[5-(3)H]-glucose) and HK/GK activity (G-6-P production), transcription (RT-PCR), protein expression (Western blot), and cellular compartmentalization were measured in isolated islets (collagenase digestion). FRD rats presented normoglycemia but impaired glucose tolerance, hypertriglyceridemia, hyperinsulinemia, and increased HOMA-IR index. In these rats, beta-cell mass decreased significantly by 33%, with a 44% increase in the percentage of apoptotic cells. Glucose-induced insulin release and islet glucose metabolism were higher in FRD rats. While GK activity (total and cytosolic fraction) and protein expression were significantly higher in FRD islets, HK showed no change in any of these parameters. Our results demonstrate that the changes induced by dietary-induced IR upon beta-cell function and mass are strongly conditional on the nutrient model used. In our model (intact animals with impaired glucose tolerance), GK activity increases through mechanisms previously shown only in vitro or under highly hyperglycemic conditions. Such an increase plays a pivotal role in the adaptive increased release of insulin in response to IR, even in the presence of marked beta-cell mass reduction.
Asunto(s)
Fructosa/farmacología , Glucosa/metabolismo , Células Secretoras de Insulina/efectos de los fármacos , Células Secretoras de Insulina/enzimología , Insulina/metabolismo , Animales , Glucemia/análisis , Expresión Génica , Glucoquinasa/metabolismo , Hexoquinasa/metabolismo , Insulina/sangre , Resistencia a la Insulina , Secreción de Insulina , Masculino , Ratas , Ratas Wistar , Triglicéridos/sangreRESUMEN
Administration of a sucrose-rich diet (SRD) to normal hamsters induces an insulin-resistant state and a significant increase of insulin secretion and beta-cell mass. Islets isolated from these animals had a marked increase in glucose metabolism and glucose-induced insulin secretion, at both low and high glucose concentrations. They also presented increased hexokinase (HK) activity, without measurable changes in glucokinase (GK) activity. In this study we measured HK and GK activity in homogenates of islets isolated from normal control and SRD-fed hamsters, as well as in their particulate and cytosolic fractions. We also measured transcription rate (mRNA by reverse transcriptase PCR) and expression levels (Western blotting) of both enzymes in these islets. We found an increase in HK activity and expression levels, without measurable changes in HK mRNA level in SRD-fed animals. Whereas a similar GK activity was measured in homogenates of islets isolated from both groups, such activity was significantly higher in the cytosolic fraction of SRD islets. On the other hand, GK transcription rate and expression level were similar in both experimental groups. Our results suggest that the increased beta-cell secretory response to low glucose can be partly ascribed to an increased activity of islet HK consecutive to an enhanced expression of the enzyme, while the enhanced response to high glucose could be due to changes in GK compartmentalization.
Asunto(s)
Glucoquinasa/metabolismo , Hexoquinasa/metabolismo , Islotes Pancreáticos/metabolismo , Sacarosa/administración & dosificación , Animales , Glucemia/análisis , Western Blotting/métodos , Peso Corporal/fisiología , Cricetinae , Citosol/metabolismo , Dieta , Ingestión de Líquidos/fisiología , Expresión Génica/genética , Glucoquinasa/análisis , Glucoquinasa/genética , Hexoquinasa/análisis , Hexoquinasa/genética , Insulina/sangre , Insulina/metabolismo , Secreción de Insulina , Células Secretoras de Insulina/metabolismo , Masculino , Mesocricetus , Fosforilación , ARN Mensajero/análisis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Transcripción Genética/genéticaRESUMEN
The aim of this study was to quantify the glucose modulation of the plasma membrane calcium pump (PMCA) function in rat pancreatic islets. Ca2+-ATPase activity and levels of phosphorylated PMCA intermediates both transiently declined to a minimum in response to stimulation by glucose. Strictly dependent on Ca2+ concentration, this inhibitory effect was fully expressed at physiological concentrations of the cation (less than 0.5 muM), then progressively diminished at higher concentrations. These results, together with those previously reported on the effects of insulin secretagogues and blockers on the activity, expression and cellular distribution of the PMCA, support the concept that the PMCA plays a key role in the regulation of Ca2+ signaling and insulin secretion in pancreatic islets.
Asunto(s)
Calcio/fisiología , Membrana Celular/enzimología , Islotes Pancreáticos/enzimología , ATPasas Transportadoras de Calcio de la Membrana Plasmática/fisiología , Animales , Membrana Celular/efectos de los fármacos , Glucosa/farmacología , Técnicas In Vitro , Activación del Canal Iónico , Islotes Pancreáticos/efectos de los fármacos , Isoenzimas/fisiología , Masculino , Fosforilación , Ratas , Ratas WistarRESUMEN
This study aimed to determine the relative importance of different functional and morphological pancreatic changes induced by the chronic administration of a sucrose-rich diet (SRD) to maintain normal glucose homeostasis. Male Wistar rats were fed either sucrose (SRD) or starch (CD) for 6 and 12 months. At both periods, serum glucose and triacylglycerol levels were significantly higher (P<0.05; paired and unpaired Student's t-test) in SRD rats. Serum insulin levels were significantly lower in SRD only at 12 months. At 6 months, the insulin secretion dose-response curve in SRD rats showed a shift to the left that was no longer observed at 12 months, when SRD islets decreased their response to 16 mM glucose. At 6 months, SRD rats showed a significant increase in beta-cell volume density (Vvi) and islet cell replication rate, together with a decrease in beta-cell apoptotic rate. Changes were not detected in the percentage of PDX-1- and islet neogenesis associated protein (INGAP)-positive cells. Conversely, at 12 months, there was a significant decrease in beta-cell Vvi and in the percentage of PDX-1-positive cells; the islet cell replication rate was not modified, and the number of apoptotic beta-cells increased significantly. No signs of increased neogenesis or INGAP-positive cells were recorded at any period in SRD rats. Our results show that SRD rats are unable to develop functional and morphological pancreatic reactive changes sufficient to maintain normal glucose and triacylglycerol levels for a long period. Such failure could be ascribed to their inability to increase the rate of neogenesis and of INGAP production.
Asunto(s)
Carbohidratos de la Dieta/administración & dosificación , Insulina/metabolismo , Islotes Pancreáticos/fisiología , Adaptación Fisiológica , Animales , Apoptosis/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Tamaño de la Célula/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Secreción de Insulina , Masculino , Proteínas Asociadas a Pancreatitis , Ratas , Ratas Wistar , Almidón/administración & dosificación , Sacarosa/administración & dosificación , Factores de TiempoRESUMEN
The aim of the present study was to test the possible presence and expression of islet neogenesis-associated protein (INGAP) in islet cells of normal adult hamsters. Pancreata from normal male Syrian hamsters were removed to perform the following studies. (i) Western blot analysis using the cytosolic fraction from homogenates of isolated islets, exocrine tIssue and whole pancreas, and rabbit INGAP-specific antibody. (ii) Immunohistochemical identification of INGAP-positive cells in fixed sections of intact pancreata, fresh and 72 h cultured islets (isolated by collagenase digestion), and smears of exocrine pancreatic cells, using the same INGAP-specific antibody and streptavidin-biotin complex. (iii) RT-PCR using total RNA extracted from isolated islets and from exocrine tIssue as template, and a specific pair of primers. (iv) Control of the sequence of the PCR products. INGAP protein was identified by Western blot in the cytosolic fraction of homogenates from fresh isolated islets, exocrine cells and whole fresh pancreas. INGAP-immunopositive cells were observed in duct, exocrine and islet cells in either fixed intact or digested pancreatic tIssue. INGAP mRNA was identified in samples of total RNA from fresh and cultured isolated islets and from exocrine cells. Our data demonstrate that INGAP is present and expressed in islets and in exocrine pancreatic cells of normal hamsters. The ubiquitous localization of INGAP suggests its possible role in the physiological process of islet growth and its protective effect upon streptozotocin-induced diabetes.
Asunto(s)
Antígenos de Neoplasias , Biomarcadores de Tumor , Islotes Pancreáticos/química , Lectinas Tipo C , Proteínas/análisis , Animales , Western Blotting/métodos , Células Cultivadas , Cricetinae , Citosol/química , Inmunohistoquímica/métodos , Masculino , Mesocricetus , Páncreas/química , Páncreas/citología , Proteínas Asociadas a Pancreatitis , Proteínas/genética , ARN Mensajero/análisis , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
The aim of this work was to study the possible relationship between pancreatic duodenal homeobox-1 (Pdx-1) and islet neogenesis-associated protein (INGAP) during induced islet neogenesis. Pregnant hamsters were fed with (S) and without (C) sucrose, and glycemia, insulin secretion in vitro, and pancreas immunomorphometric parameters were measured in their 7-day-old offspring. S offspring had significantly lower glycemic levels than C animals. Insulin release in response to increasing glucose concentrations in the incubation medium (2-16 mM glucose) did not increase in pancreata from either C or S offspring. However, pancreata from S offspring released more insulin than those from C animals. In S offspring, beta-cell mass, beta-cell replication rate and islet neogenesis increased significantly, with a simultaneous decrease in beta-cell apoptotic rate. INGAP- and Pdx-1-positive cell mass also increased in the islets and among acinar and duct cells. We found two subpopulations of Pdx-1 cells: INGAP-positive and INGAP-negative. Pdx-1/INGAP-positive cells did not stain with insulin, glucagon, somatostatin, pancreatic polypeptide, or neurogenin 3 antibodies. The increment of Pdx-1/INGAP-positive cells represented the major contribution to the Pdx-1 cell mass increase. Such increments varied among pancreas subsectors: ductal>insular>extrainsular. Our results suggested that INGAP participates in the regulation of islet neogenesis, and Pdx-1/INGAP-positive cells represent a new stem cell subpopulation at an early stage of development, highly activateable in neogenesis.
Asunto(s)
Antígenos de Neoplasias , Biomarcadores de Tumor , Proteínas de Homeodominio , Lectinas Tipo C , Páncreas/metabolismo , Proteínas/análisis , Células Madre/metabolismo , Transactivadores/análisis , Animales , Animales Recién Nacidos , Apoptosis , Biomarcadores/análisis , Peso Corporal , Cricetinae , Femenino , Inmunohistoquímica/métodos , Insulina/metabolismo , Secreción de Insulina , Islotes Pancreáticos/citología , Islotes Pancreáticos/metabolismo , Páncreas/citología , Proteínas Asociadas a Pancreatitis , EmbarazoRESUMEN
We correlated the changes in glucose-induced insulin secretion with those observed in glucose metabolism and hexokinase/glucokinase activity in islets from normal sucrose-fed hamsters. Blood glucose and insulin levels were measured in normal male hamsters fed with (S5) or without (C5) 10% sucrose in the drinking water for 5 weeks. Isolated islets (collagenase digestion) from both groups of animals were used to study insulin secretion, (14)CO(2) and (3)H(2)O production from D-[U-(14)C]-glucose and D-[5-(3)H]-glucose respectively, with 3.3 or 16.7 mM glucose in the medium, and hexokinase/glucokinase activity (fluorometric assay) in islet homogenates. Whereas S5 and C5 animals had comparable normal blood glucose levels, S5 showed higher insulin levels than C5 hamsters (2.3+/-0.1 vs 0.6+/-0.03 ng/ml, P<0.001). Islets from S5 hamsters released significantly more insulin than C5 islets in the presence of low and high glucose (3.3 mM glucose: 0.77+/-0.04 vs 0.20+/-0.06 pg/ng DNA/min, P<0.001; 16.7 mM glucose: 2.77+/-0.12 vs 0.85+/-0.06 pg/ng DNA/min, P<0.001) and produced significantly higher amounts of (14)CO(2) and (3)H(2)O at both glucose concentrations ((14)CO(2): 3.3 mM glucose: 0.27+/-0.01 vs 0.18+/-0.01, P<0.001; 16.7 mM glucose: 1.44+/-0.15 vs 0.96+/-0.08, P<0.02; (3)H(2)O: 3.3 mM glucose: 0.31+/-0.02 vs 0.15+/-0.01, P<0.001; 16.7 mM glucose: 1.46+/-0.20 vs 0.76+/-0.05 pmol glucose/ng DNA/min, P<0.005). The hexokinase K(m) and V(max) values from S5 animals were significantly higher than those from C5 ones (K(m): 100.14+/-7.01 vs 59.90+/- 3.95 microM, P<0.001; V(max): 0.010+/-0.0005 vs 0.008+/- 0.0006 pmol glucose/ng DNA/min, P<0.02). Conversely, the glucokinase K(m) value from S5 animals was significantly lower than in C5 animals (K(m): 15.31+/-2.64 vs 35.01+/-1.65 mM, P<0.001), whereas V(max) figures were within a comparable range in both groups (V(max): 0.048+/-0.009 vs 0.094+/-0.035 pmol glucose/ng DNA/min, not significant). The glucose phosphorylation ratio measured at 1 and 100 mM (hexokinase/glucokinase ratio) was significantly higher in S5 (0.26+/-0.02) than in C5 animals (0.11+/-0.01, P<0.005), and it was attributable to an increase in the hexokinase activity in S5 animals. In conclusion, sucrose administration increased the hexokinase/glucokinase activity ratio in the islets, which would condition the increase in glucose metabolism by beta-cells, and in beta-cell sensitivity and responsiveness to glucose. These results support the concept that increased hexokinase rather than glucokinase activity causes the beta-cell hypersensitivity to glucose, hexokinase being metabolically more active than glucokinase to up-regulate beta-cell function.
Asunto(s)
Glucosa/metabolismo , Islotes Pancreáticos/efectos de los fármacos , Sacarosa/farmacología , Animales , Glucemia/efectos de los fármacos , Glucemia/metabolismo , Peso Corporal/efectos de los fármacos , Técnicas de Cultivo de Célula , Cricetinae , ADN/análisis , Ingestión de Líquidos/efectos de los fármacos , Glucoquinasa/metabolismo , Hexoquinasa/metabolismo , Insulina/sangre , Insulina/metabolismo , Secreción de Insulina , Islotes Pancreáticos/enzimología , Islotes Pancreáticos/metabolismo , Masculino , MesocricetusRESUMEN
The possible action of 2-hydroxyoestradiol (2-OHE2) on glucose-induced insulin secretion was evaluated in pancreatic islets isolated from normal rats by collagenase digestion and incubated in KRB buffer. Insulin output in response to either 3.3 or 16.6 mM glucose was measured by radioimmunoassay in the absence or presence of different concentrations of 2-OHE2, norepinephrine (NE), or oestradiol. Islets were also incubated with 2-OHE2, NE, or oestradiol plus a fixed concentration (1 microM) of the alpha 2-adrenergic-receptor blocking agent yohimbine. The results showed that 2-OHE2, oestradiol and NE within a range of 0.1 to 20 microM inhibited glucose-induced insulin secretion in a dose-dependent manner: Ki (microM): 0.04 +/- 0.0001, 0.04 +/- 0.0002, and 0.01 +/- 9.1 E-6 respectively. This suppression was significantly reversed by yohimbine. Contrary to NE and 2-OHE2, oestradiol at lower concentrations (increasing within a range of 0.001 to 0.05 microM) in incubation medium in the same experimental conditions had a significant stimulatory effect on insulin secretion. Thus, it would appear that catecholoestrogens suppress islet insulin release via alpha 2-adrenergic receptors, which suggests that oestrogens may exert a dual modulatory effect on insulin secretion by enhancing release via direct interaction with the cytosolic-oestrogen receptor and inhibiting release after their local hydroxylation and the interaction of their new catechol moiety with alpha 2-adrenergic receptors. Our results suggest that these compounds may play a complementary role to CAs as negative modulators, and they also provide a broader scope for understanding the effect of oestrogens and/or their metabolites in the control of endocrine functions other than those related to reproduction.
Asunto(s)
Estradiol/análogos & derivados , Estrógenos de Catecol/farmacología , Insulina/metabolismo , Islotes Pancreáticos/efectos de los fármacos , Islotes Pancreáticos/metabolismo , Antagonistas Adrenérgicos alfa , Animales , Relación Dosis-Respuesta a Droga , Estradiol/administración & dosificación , Estradiol/farmacología , Glucosa/farmacología , Secreción de Insulina , Masculino , Norepinefrina/administración & dosificación , Norepinefrina/farmacología , Ratas , Ratas Wistar , Yohimbina/farmacologíaRESUMEN
The aim of this work was to investigate the possible presence of DOPA decarboxylase (DDC) in endocrine cells of adult rat pancreas. Islet peptide hormones (insulin, glucagon, and somatostatin), as well as DDC, were detected immunohistochemically using the double-immunofluorescence technique and specific antibodies. DDC-like immunoreactivity was present in cytoplasmic granules within endocrine cells located at islet peripheries in a distribution consistent with islet localisation of A cells. Moreover, these same cells stained positively with glucagon antibody. As DDC is an enzyme specifically involved in catecholamine synthesis, insular cells must possess the capacity to elaborate this class of hormone at least up to the dopamine-decarboxylation step. Thus, after further metabolic processing either in A cells or elsewhere, endogenously-synthesised islet catecholamines may be released and participate in paracrine regulation of insulin secretion.
Asunto(s)
Dopa-Decarboxilasa/análisis , Islotes Pancreáticos/enzimología , Animales , Técnica del Anticuerpo Fluorescente , Glucagón/análisis , Inmunohistoquímica , Islotes Pancreáticos/citología , Ratas , Ratas Sprague-Dawley , Somatostatina/análisisAsunto(s)
Hormona Adrenocorticotrópica/análogos & derivados , Hormona Adrenocorticotrópica/farmacología , Insulina/metabolismo , Islotes Pancreáticos/fisiología , Animales , Calcio/metabolismo , ATPasas Transportadoras de Calcio/metabolismo , Separación Celular , Células Cultivadas , Citosol/metabolismo , Electrofisiología , Ayuno , Glucosa/farmacología , Secreción de Insulina , Islotes Pancreáticos/citología , Islotes Pancreáticos/efectos de los fármacos , Ratones , RatasRESUMEN
In view of recent evidence for the endogenous synthesis of proopiomelanocortin (POMC) by pancreatic islets, we have assessed (1) the release of POMC-derived corticotropin (ACTH)-like peptides (ACTH-LP) from isolated perifused rat islets, and (2) the potential paracrine modulatory effect on insulin output of these putative secretagogues. Islets perifused at a glucose concentration of 3.3 mmol/L secreted ACTH-LP at 0.15 +/- 0.005 ng/islet/10 min, which was increased by 17-fold at 16.7 mmol/L glucose. Islets statically incubated with different concentrations of medium glucose plus synthetic 1-39ACTH at 55 pmol/L showed a significant increase of insulin release at 8 (by 79%) and 16 (by 119%) mmol/L glucose, but not at 4 mmol/L. To determine the possible cis-directed effects of these endogenously released islet ACTH-LP on insulin secretion, we either blocked their biological action by immunoneutralization with an ACTH-specific antiserum or prevented their receptor interaction by addition of the ACTH-inhibiting polypeptide (CIP) to the incubation medium. In the presence of 16.7 mmol/L glucose, the rate of insulin output decreased by approximately 25% upon exposure to the antiserum and by approximately 50% in the presence of CIP. The foregoing observations would therefore suggest that both (1) the elaboration of ACTH-LP by isolated perifused islets and (2) the stimulation of islet insulin release by exogenous 1-39ACTH in static incubation occur as a function of glucose concentration in the incubation medium, and that (3) the newly-secreted endogenous ACTH-LP operate in a cis mode to enhance islet insulin output in a manner analogous to that of exogenously added ACTH species. These results strongly support the view that islet-elaborated ACTH-LP are important physiological paracrine modulators of insulin secretion.
Asunto(s)
Hormona Adrenocorticotrópica/metabolismo , Insulina/metabolismo , Islotes Pancreáticos/metabolismo , Péptidos/metabolismo , Proopiomelanocortina/metabolismo , Hormona Adrenocorticotrópica/antagonistas & inhibidores , Animales , Masculino , Pruebas de Neutralización , Péptidos/farmacología , Ratas , Ratas WistarRESUMEN
The aim of this work was to get some insight into the mechanism by which ACTH produces its enhancing effect on glucose-induced insulin secretion. For this purpose we have determined: a) the release of insulin by isolated rat islets incubated with 3.3 or 16.6 mM glucose with or without the addition of 500 pg/ml ACTH, together with the changes induced by ACTH on b) cytosolic [Ca2+] of isolated B cells, c) islet plasma membrane Ca(2+)-ATPase activity and d) changes in membrane potential of single mouse islets. ACTH significantly enhanced the release of insulin elicited by either 3.3 or 16.6 mM glucose. This hormone concentration also induced a significant increase in the cytosolic [Ca2+] in isolated B cells. ACTH did not produce B cell membrane depolarization. Conversely, ACTH produced a significant decrease in islet plasma membrane Ca(2+)-ATPase activity. These results suggest that ACTH in concentrations similar to those attained by the endogenous peptide at the islet interstitium exerts its positive modulation on glucose-induced secretion of insulin, at least partly through its increasing effect on cytosolic [Ca2+] of B cells. The latter might be the consequence of the decreasing effect of ACTH on Ca(2+)-ATPase activity rather than to stimulation of voltage-dependent Ca(2+)-channels.
Asunto(s)
Hormona Adrenocorticotrópica/farmacología , ATPasas Transportadoras de Calcio/metabolismo , Calcio/metabolismo , Insulina/metabolismo , Islotes Pancreáticos/efectos de los fármacos , Potenciales de la Membrana , Animales , Citosol/metabolismo , Glucosa/farmacología , Secreción de Insulina , Islotes Pancreáticos/fisiología , Islotes Pancreáticos/ultraestructura , Masculino , Ratas , Ratas WistarRESUMEN
This work was performed to study the release of proopiomelanocortin (POMC)-derived peptides from isolated pancreatic islets and the effect of ACTH--a member of that peptide family--on insulin secretion. Islets were incubated with 3,3 and 16.6 mM glucose and insulin and ACTH-like products (ACTH-LP) were measured by radioimmunoassay. Glucose stimulated the simultaneous release of insulin and ACTH-LP, the ACTH-LP concentration being higher when assayed with an antibody reacting with the N-terminus of ACTH. However, the increment in this release in the presence of the higher glucose concentration was larger when measured with an antibody against the ACTH mid-portion. Thus, although the islets would release more of a smaller ACTH-LP, 16.6 mM glucose would selectively increase the release of peptides of larger molecular size. Islets incubated with different concentrations of synthetic ACTH (50-500 pg/ml) increased the release of insulin in a dose-dependent manner. These results suggest that the release of endogenous ACTH-LP could contribute to the paracrine regulation of insulin secretion.
Asunto(s)
Hormona Adrenocorticotrópica/metabolismo , Glucosa/farmacología , Islotes Pancreáticos/metabolismo , Hormona Adrenocorticotrópica/farmacología , Animales , Técnicas In Vitro , Insulina/metabolismo , Secreción de Insulina , Islotes Pancreáticos/efectos de los fármacos , Ratas , Ratas WistarRESUMEN
The aim of this work was to determine the possible rapid modulatory effect of glucose on the activity of pancreatic islet lysosomal enzymes. For this purpose, beta-N-acetylglucosaminidase and beta-galactosidase activities were measured in homogenates of isolated rat islets after a 5, 15, 30 or 60-min exposure to either 3.3 or 16.6 mM glucose. The enzyme activities were determined spectrofluorometrically by means of their respective 4-methylumbelliferyl derivatives as substrates. beta-N-acetylglucosaminidase activity measured in freshly isolated non-incubated islets was 5.482 +/- 0.281 mumol/mg protein/h at 37 degrees C. In islets incubated with 3.3 mM glucose, this activity dropped significantly after 5 min and remained almost constant until the end of the incubation period. In islets incubated with 16.6 mM glucose, beta-N-acetylglucosaminidase activity also decreased significantly at 5 min, and attained its lowest value after 15 min of incubation. After this interval, the activity began to recover and thereafter gained a value close to that measured in non-incubated islets by 60 minutes' time. Despite this ultimate recovery, the enzyme activities measured were significantly lower than those found in islets incubated with 3.3 mM glucose. beta-galactose activity in freshly isolated non-incubated islets was 0.515 +/- 0.094 mumol/mg protein/h at 37 degrees C. This value remained almost unchanged throughout the incubation period in the presence of either 3.3 or 16.6 mM glucose. These results show that beta-N-acetylglucosaminidase activity, a lysosomal hydrolase of pancreatic rat islets,--and only this enzyme--is modulated by glucose.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Acetilglucosaminidasa/metabolismo , Glucosa/farmacología , Islotes Pancreáticos/enzimología , Animales , Insulina/metabolismo , Secreción de Insulina , Islotes Pancreáticos/efectos de los fármacos , Islotes Pancreáticos/metabolismo , Cinética , Lisosomas/enzimología , Masculino , Ratas , Ratas Wistar , Espectrometría de Fluorescencia , beta-Galactosidasa/metabolismoRESUMEN
The effect of tunicamycin, a glycoprotein biosynthesis-blocker, on insulin secretion was studied using rat isolated pancreatic islets. Islets cultured with tunicamycin during 24 hr released significantly less insulin in response to 11 mM glucose than the corresponding controls. This inhibitory effect of tunicamycin was not significant in the presence of 3.3 mM glucose. The culture of these islets for a second 24 hr period in a medium devoid of tunicamycin did not remove the blocking effect of the drug on the glucose-induced insulin secretion, as demonstrated by measuring the concentration of insulin in the second culture medium or its release during a 60 min incubation period. Fresh isolated islets incubated for 60 min only with tunicamycin also released during this period less insulin in response to 16.6 mM glucose than the corresponding controls. These results might suggest that some islet glycoproteins participate in the mechanism of insulin secretion.
Asunto(s)
Insulina/metabolismo , Islotes Pancreáticos/metabolismo , Tunicamicina/farmacología , Animales , Células Cultivadas , Glucosa/farmacología , Glicoproteínas/antagonistas & inhibidores , Secreción de Insulina , Islotes Pancreáticos/efectos de los fármacos , Cinética , Masculino , Ratas , Ratas EndogámicasRESUMEN
Insulin secretion and glucose metabolism were simultaneously studied in isolated islets obtained from normal, endocrine-deficient (adrenalectomized, ovariectomized and radiothyroidectomized) rats as well as from this latter group of rats previously submitted to specific substitutive therapy. The islets from all the endocrine-deficient rats showed clear and similar changes in both the insulin secretion and the metabolism of glucose, being the correlation between the degree of these two alterations highly significant. All the above mentioned abnormalities were corrected by the administration of the corresponding substitutive therapy. These results suggest that the hormones involved in this study could modify the B cell insulin secretion by changing their capacity to metabolize glucose.
Asunto(s)
Glucosa/metabolismo , Hormonas/fisiología , Insulina/metabolismo , Islotes Pancreáticos/metabolismo , Corticoesteroides/fisiología , Adrenalectomía , Animales , Estrógenos/farmacología , Femenino , Técnicas In Vitro , Secreción de Insulina , Masculino , Ovariectomía , Progesterona/farmacología , Ratas , Ratas Endogámicas , Hormonas Tiroideas/fisiología , Tiroidectomía , Tiroxina/farmacologíaRESUMEN
Insulin secretion and glucose metabolism were simultaneously studied in isolated islets obtained from normal, endocrine-deficient (adrenalectomized, ovariectomized and radiothyroidectomized) rats as well as from this latter group of rats previously submitted to specific substitutive therapy. The islets from all the endocrine-deficient rats showed clear and similar changes in both the insulin secretion and the metabolism of glucose, being the correlation between the degree of these two alterations highly significant. All the above mentioned abnormalities were corrected by the administration of the corresponding substitutive therapy. These results suggest that the hormones involved in this study could modify the B cell insulin secretion by changing their capacity to metabolize glucose.
RESUMEN
Glucose-induced insulin secretion and net calcium uptake were simultaneously studied in isolated islets obtained from normal, adrenalectomized, ovariectomized and radiothyroidectomized rats, as well as from the corresponding hormone deprived rats following the administration of specific substitutive therapy. Both parameters were also studied in islets from normal rats incubated in the presence of Trifluoperazine (TFP). In all these unrelated experimental conditions simultaneous changes were obtained, observed in the release of insulin and the net calcium uptake elicited by glucose. Otherwise, the modifications of these two parameters obtained in the hormone deprived states were brought back to normal when the animals received the specific substitutive hormonal treatment. On the other hand, TFP also induces simultaneous diminution in both glucose-induced insulin release and net calcium uptake by isolated islets. On account of our results, we could suggest that the mechanism involved in the control of the glucose-induced net calcium uptake is actively modulated by adrenal and ovarian steroids and thyroid hormones as well as by calmodulin. Therefore, changes induced either in the level or activity of these modulators will modify the rate of influx and efflux of Ca2+ across the plasma membrane, with the consequent alteration in the mechanism of stimulus: secretion coupling of insulin.