RESUMO
In this study, we have correlated the translocation of somatostatin (SRIF) receptors from the cell interior to the plasma membrane with the ability of SRIF to inhibit insulin release. Islets were perifused with glucose (30, 100, 165, 200, or 300 mg/dl) in the presence of sodium isethionate. Sodium isethionate inhibits insulin release, but not the recruitment of SRIF receptors. Thus, the recruitment of SRIF receptors to the surface membrane continued without the lysis of secretion vesicles. SRIF binding rose from 3.75 +/- 0.16 to 6.46 +/- 0.28 fmol/10 islets as glucose concentration increased. Sodium isethionate was then removed, islets perifused with low glucose (30 mg/dl), and challenged with 400 microM isobutylmethylxanthine (IBMX) with or without SRIF (5 micrograms/ml). In the islets perifused with high glucose concentration, IBMX lysed a greater number of vesicles and caused enhanced release of insulin. The greater the number of secretion vesicles marginated to the plasma membrane by glucose, the greater the response to IBMX. Colchicine (1 mM) prevented secretion vesicle migration and this potentiation effect of higher concentrations of glucose was eliminated. In experiments with IBMX and SRIF, the degree of inhibition of IBMX-induced insulin release by SRIF was proportional to the magnitude of SRIF binding to these islets. SRIF inhibited insulin release by 20 microU/100 islets initially perifused with low glucose (30 mg/dl) and by 875 microU/100 islets perifused with high glucose (300 mg/dl). The maximal effect of SRIF was observed when its binding reached a level of 5.4 fmol/10 islets. We conclude that inhibition of insulin release by SRIF is proportional to the SRIF receptor concentration, and that translocation of SRIF receptors during exocytosis plays an important role in paracrine regulation of insulin secretion by rendering the islets more sensitive to SRIF.
Assuntos
Insulina/metabolismo , Receptores de Superfície Celular/efeitos dos fármacos , Somatostatina/metabolismo , 1-Metil-3-Isobutilxantina/farmacologia , Animais , Ligação Competitiva , Glucose/farmacologia , Técnicas In Vitro , Secreção de Insulina , Ilhotas Pancreáticas/metabolismo , Masculino , Ratos , Ratos Endogâmicos , Receptores de Superfície Celular/metabolismo , Receptores de SomatostatinaRESUMO
We have used high-pressure freezing techniques to study exocytosis in rat anterior pituitary cells. The cells were either unstimulated or exposed to 1 nM growth hormone releasing factor (GRF) for 10 min before ultrarapid freezing. The magnitude of growth hormone (GH) release was then correlated with the number of exocytotic events observed with freeze-fracture electron microscopy. High-pressure freezing of unfixed and uncryoprotected specimens permits cryofixation of samples up to 1 mm diam (0.5 mm thick) without ice crystal damage, and arrests exocytotic events within 10 ms. Our studies comparing conventionally fixed specimens with those prepared by high-pressure freezing confirm that areas of intramembrane particle clearing at potential exocytotic sites are an artifact of conventional fixation and/or cryoprotection techniques. The cells exposed to 1 nM GRF released approximately fivefold more GH than did unstimulated cells. Morphologically, we have observed a 3.3-fold increase in the number of exocytotic events in GRF-stimulated cells, 33.7 events/100 micron2 compared with 10.4 events/100 micron2 for unstimulated cells. In additional experiments, we studied the effects of two inhibitors of GRF-induced exocytosis, somatostatin and sodium isethionate. Both compounds elicit the same response, a parallel decrease in exocytotic events and in secreted product. We conclude that high-pressure freezing, combined with freeze-fracture and freeze-substitution processing techniques, is an excellent tool for studying the morphological aspects of exocytosis. In the present investigation, it has allowed us to quantitatively relate the biochemistry and morphology of exocytosis in anterior pituitary cells.
Assuntos
Exocitose , Hormônio do Crescimento/metabolismo , Adeno-Hipófise/metabolismo , Animais , Membrana Celular/ultraestrutura , Células Cultivadas , Técnica de Fratura por Congelamento , Congelamento , Hormônio Liberador de Hormônio do Crescimento/farmacologia , Pressão Hidrostática , Ratos , Somatostatina/farmacologiaRESUMO
To study the possible role of the secretion vesicle inligant-receptor interaction, somatostatin binding was measured in islets in the presence of various substances known to promote secretion vesicle migration and fusion with the plasma membrane and insulin release. Rat islets were incubated with glucose, 30 and 300 mg/dl, for 60 min. After inculation, somatostatin binding was measured. In islets preincubated with glucose, 300 mg/dl, somatostatin binding was increased 250% when compared with glucose, 30 mg/dl (P < 0.001). Concomitant with enhanced somatostatin binding, insulin secretion was increased. Galactose, 300 mg/dl, did not stimulate insulin release, and somatostatin binding was unchanged from control levels. The increase in somatostatin binding with glucose was accounted for by a 186% increase in receptor concentration with no change in receptor affinity. Tolbutamide increased somatostatin binding by more than twofold, accompanied by a similar increase in insulin release. Secretion vesicles isolated from the islet exhibited somatostatin binding. We conclude that, first, somatostatin binding is increased concomitantly with the migration and fusion of the secretion vesicle with the plasma membrane and/or the release of insulin; second, enhanced somatostatin binding occurs as a consequence of an increased receptor concentration; and third, augmented somatostatin binding occurring with hormone release may provide a critical constraint in the regulation of secretory events.
Assuntos
Exocitose , Insulina/metabolismo , Ilhotas Pancreáticas/metabolismo , Somatostatina/metabolismo , Animais , Técnicas de Cultura , Glucose/metabolismo , Secreção de Insulina , Masculino , Ratos , Receptores de Superfície Celular/metabolismoRESUMO
Insulin- and glyburide-stimulated changes in cytosolic free calcium concentrations [( Ca2+]i) were studied in gluteal adipocytes obtained from six obese women (139 +/- 3% ideal body wt) and six healthy, normal weight age- and sex-matched controls. Biopsies were performed after an overnight fast and twice (at 3 and 6 h) during an insulin infusion (40 mU/m2 per min) (euglycemic clamp). In adipocytes obtained from normal subjects before insulin infusion, insulin (10 ng/ml) increased [Ca2+]i from 146 +/- 26 nM to 391 +/- 66 nM. Similar increases were evoked by 2 microM glyburide (329 +/- 41 nM). After 3 h of insulin infusion, basal [Ca2+]i rose to 234 +/- 21 nM, but the responses to insulin and glyburide were completely abolished. In vitro insulin-stimulated 2-deoxyglucose uptake was reduced by insulin and glucose infusion (25% stimulation before infusion, 5.4% at 3 h, and 0.85% at 6 h of infusion). In obese patients, basal adipocyte [Ca2+]i was increased (203 +/- 14 nM, P less than 0.05 vs. normals). The [Ca2+]i response demonstrated resistance to insulin (230 +/- 23 nM) and glyburide (249 +/- 19 nM) stimulation. Continuous insulin infusion increased basal [Ca2+]i (244 +/- 24 nM) and there was no response to either insulin or glyburide at 3 and 6 h of study. Rat adipocytes were preincubated with 1-10 mM glucose and 10 ng/ml insulin for 24 h. Measurements of 2-deoxyglucose uptake demonstrated insulin resistance in these cells. Under these experimental conditions, increased levels of [Ca2+]i that were no longer responsive to insulin were demonstrated. Verapamil in the preincubation medium prevented the development of insulin resistance.
Assuntos
Cálcio/metabolismo , Citosol/metabolismo , Hiperinsulinismo/fisiopatologia , Resistência à Insulina , Obesidade/fisiopatologia , Tecido Adiposo/efeitos dos fármacos , Tecido Adiposo/metabolismo , Adulto , Biópsia , Desoxiglucose/farmacocinética , Feminino , Glibureto/farmacologia , Humanos , Técnicas In Vitro , Insulina/farmacologia , Verapamil/farmacologiaRESUMO
We examined the activities of particulate and cytosolic phosphotyrosine phosphatase (PTPase) and phosphoserine phosphatase (PSPase) in adipocytes and livers of diabetic rats. PTPase activity was assessed with [32P]tyrosine-phosphorylated insulin receptor (IR), whereas PSPase activity was assayed with [32P]serine-phosphorylated glycogen synthase. Diabetes increased adipocyte particulate PTPase activity and enhanced IR dephosphorylation by 75% on the 2nd, 93% on the 14th, and 108% on the 30th day. In contrast, cytosolic PTPase activity decreased by 78% on the 14th and 45% on the 30th day (no change on the 2nd day). Similar changes were observed with PSPase (increased activity in particulate and decreased in cytosolic). Insulin therapy for 14 or 30 days restored PTPase and PSPase activities in both fractions. Vanadate, despite rapid normalization of glycemia, restored these activities only after 30 days of therapy. Diabetes-related changes in liver PTPase activity were observed on the 14th day only. At this time, it was increased in both particulate and cytosolic fractions. There was spontaneous normalization of the liver PTPase activity at 30 days of diabetes. In contrast, liver cytosolic PSPase activity was significantly inhibited and not normalized by the 30th day of disease without therapy. In summary, diabetes appears to induce tissue-specific changes in PTPase and PSPase activities resulting in significant alterations in dephosphorylation of IR and glycogen synthase. Moreover, there appears to be a differential regulation of PTPase and PSPase activities in diabetes, particularly in the liver.
Assuntos
Tecido Adiposo/enzimologia , Diabetes Mellitus Experimental/enzimologia , Fígado/enzimologia , Monoéster Fosfórico Hidrolases/metabolismo , Proteínas Tirosina Fosfatases/metabolismo , Tecido Adiposo/efeitos dos fármacos , Animais , Citosol/enzimologia , Diabetes Mellitus Experimental/tratamento farmacológico , Éteres Cíclicos/farmacologia , Glicogênio Sintase/metabolismo , Insulina/uso terapêutico , Cinética , Fígado/efeitos dos fármacos , Substâncias Macromoleculares , Masculino , Ácido Okadáico , Ratos , Ratos Endogâmicos , Receptor de Insulina/metabolismo , Valores de Referência , Vanadatos/farmacologiaRESUMO
We examined Na(+)-K(+)-ATPase activity and the levels of alpha I-, alpha II-, and beta-subunit mRNA and protein in aortic cells of diabetic rats. Diabetes was induced by streptozocin. Na(+)-K(+)-ATPase activity was significantly reduced on the 2nd day of diabetes (9.4 +/- 1.3 vs. 17.5 +/- 2.1 mumol NADH.mg-1 protein.h-1, P less than 0.05) and remained depressed on days 7 and 14. The levels of 5.3-kilobase (kb) mRNA band of the catalytic alpha II-subunit of Na(+)-K(+)-ATPase were also decreased on the 2nd day of diabetes, whereas the second band, 3.4 kb, was not affected. Both bands were significantly decreased on days 7 and 14. This was followed by a reduction in the levels of alpha II-protein (day 14). The levels of alpha I- and beta-subunit mRNA and alpha I- protein were not affected by diabetes. A decrease in Na(+)-K(+)-ATPase activity was accompanied by a significant (P less than 0.001) increase in the cytosolic free Ca2+ concentrations [( Ca2+]i) in diabetic aortic cells (221 +/- 18 nM on the 7th day and 242 +/- 17 nM on the 14th day vs. 153 +/- 7 nM in controls). These findings are consistent with the hypothesis that decreased Na(+)-K(+)-ATPase activity and gene expression in vascular smooth muscle cells with accompanied rises in [Ca2+]i may be an important pathogenetic factor in the development of hypertension and atherosclerosis in diabetes.
Assuntos
Aorta/enzimologia , ATPases Transportadoras de Cálcio/metabolismo , Citosol/enzimologia , Diabetes Mellitus Experimental/metabolismo , ATPase Trocadora de Sódio-Potássio/metabolismo , Animais , Aorta/fisiologia , Aorta/ultraestrutura , Northern Blotting , Western Blotting , ATPases Transportadoras de Cálcio/fisiologia , Densitometria , Diabetes Mellitus Experimental/fisiopatologia , Regulação Enzimológica da Expressão Gênica/genética , Regulação Enzimológica da Expressão Gênica/fisiologia , Masculino , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/fisiologia , RNA Mensageiro/análise , RNA Mensageiro/genética , Ratos , Ratos Endogâmicos , ATPase Trocadora de Sódio-Potássio/genética , ATPase Trocadora de Sódio-Potássio/fisiologia , EstreptozocinaRESUMO
We investigated the effect of insulin and a hypoglycemic sulfonylurea agent glyburide on cytosolic free-Ca2+ concentrations [( Ca2+]i) in isolated rat adipocytes. Both insulin and glyburide increased [Ca2+]i in a dose-dependent manner. Half-maximal effects were seen at 0.5 ng/ml of insulin and 0.5 microM glyburide. Nifedipine (25 microM), a Ca2+-channel blocker, inhibited the effect of both agents. The effect of insulin on [Ca2+]i was 40 and 70% potentiated by ambient glucose concentrations at 180 and 300 mg/dl, respectively. Depolarizing doses of potassium (40 mM) induced an increase in cytosolic Ca2+ that was also inhibited by nifedipine. It is suggested that both insulin and glyburide increase cytosolic free Ca2+ levels at least in part by promoting Ca2+ influx through voltage-dependent Ca2+ channels.
Assuntos
Tecido Adiposo/metabolismo , Cálcio/metabolismo , Glibureto/farmacologia , Insulina/farmacologia , Canais Iônicos/efeitos dos fármacos , Animais , Células Cultivadas , Citosol/metabolismo , Masculino , Ratos , Ratos EndogâmicosRESUMO
We have assessed the effect of somatostatin on glucose-, potassium-, forskolin-, and dibutyryl cAMP-induced changes in cytosolic free [Ca2+] in normal rat pancreatic islet cells with the new Ca2+ indicator fura 2. The cytosolic free [Ca2+] in islet cells incubated with nonstimulatory concentrations of glucose (30 mg/dl) ranged from 54 to 64 nM. In the presence of extracellular Ca2+ (1 mM), glucose (300 mg/dl) rapidly increased the cytosolic free [Ca2+] to a level of 90-110 nM. In the absence of extracellular Ca2+, glucose failed to increase the cytosolic free [Ca2+], which remained at a level of 55-60 nM. Somatostatin inhibited glucose-induced increases in cytosolic free [Ca2+] in a dose-dependent manner (maximal inhibition was 34%). Half-maximal inhibition was observed at 10(-9) M somatostatin, which correlated well with somatostatin binding to islet cells (Kd = 2.6 X 10(-10) M). Potassium (50 mM) rapidly increased the cytosolic free [Ca2+] to 110-120 nM, and its effect was not influenced by the presence of somatostatin. Forskolin (20 microM) and dibutyryl cAMP (1 mM) rapidly increased cytosolic free Ca2+ both in the presence and absence of extracellular Ca2+. More than 80% of the overall increase in cytosolic free-Ca2+ levels could be accounted for by the mobilization of intracellular Ca2+ stores. Somatostatin effectively blocked the forskolin effect (32% inhibition) but not the dibutyryl cAMP-induced effect. Somatostatin appears to inhibit secretagogue-induced increases in cytosolic free [Ca2+] by interfering with cAMP production and probably with Ca2+ transport across the cell membrane.
Assuntos
Cálcio/metabolismo , Citosol/metabolismo , Ilhotas Pancreáticas/metabolismo , Somatostatina/farmacologia , Animais , Benzofuranos , Bucladesina/farmacologia , Cálcio/farmacologia , Colforsina/farmacologia , AMP Cíclico/fisiologia , Ácido Egtázico/farmacologia , Corantes Fluorescentes , Fura-2 , Glucose/farmacologia , Ilhotas Pancreáticas/efeitos dos fármacos , Potássio/farmacologia , Ratos , Espectrometria de FluorescênciaRESUMO
Eighty-seven percent of the total cellular pool of somatostatin (SRIF) receptors in pancreatic islets are located intracellularly. Upon glucose stimulation (300 mg/dl) of insulin release, 8-15% of intracellular SRIF receptors are translocated to the plasma membrane. Affinity of SRIF receptors does not change during their migration and the total cellular pool of receptors remains constant. With prolonged glucose stimulation, surface membrane somatostatin receptor concentration reaches a maximum level at 60 min.
Assuntos
Ilhotas Pancreáticas/metabolismo , Receptores de Superfície Celular/metabolismo , Animais , Membrana Celular/metabolismo , Glucose/farmacologia , Técnicas In Vitro , Insulina/metabolismo , Membranas Intracelulares/metabolismo , Cinética , Masculino , Ratos , Ratos Endogâmicos , Receptores de Superfície Celular/efeitos dos fármacos , Receptores de SomatostatinaRESUMO
We have attempted to define the nature of insulin secretory defect(s) in aged animals. In these studies, pancreatic islets were isolated from 2- and 18-mo-old Fischer 344 rats. Margination of secretion vesicles during exocytosis was assessed by measuring the recruitment of somatostatin (SRIF) receptors to the surface membrane. Section vesicle lysis was studied by measuring insulin release into the incubation media. Submaximal and maximal glucose-induced insulin secretion was significantly greater in islets isolated from younger rats (P less than 0.01). SRIF receptor recruitment was stimulated by glucose in both younger and older Fischer 344 rats. However, an increase in SRIF receptor recruitment was reduced in islets isolated from older animals (from 2.14 +/- 0.4 to 4.6 +/- 0.4 fmol/10 islets) (P less than 0.01) as compared with islets from younger animals (from 2.6 +/- 0.2 to 6.2 +/- 0.4 fmol/10 islets). When secretion vesicle lysis was inhibited by the presence of sodium isethionate in the incubation media, glucose (300 mg/dl) failed to stimulate secretion vesicle margination to the plasma membrane. In contrast, glyburide (0.6 micrograms/ml) continued to stimulate directly secretion vesicle margination in islets from aged animals (from 2.1 +/- 0.3 to 6.0 +/- 0.3 fmol/10 islets). We conclude that glucose-induced margination of secretion vesicles at the plasma membrane is impaired by the aging process. This impairment results in lower submaximal and maximal insulin secretory response to glucose. The fact that glyburide is capable of stimulating secretion vesicle margination suggests that glucose signal recognition and/or stimulus-secretion coupling may be the locus of impairment in the process of insulin secretion in older animals.
Assuntos
Envelhecimento , Insulina/metabolismo , 1-Metil-3-Isobutilxantina/farmacologia , Animais , Exocitose/efeitos dos fármacos , Glucose/farmacologia , Glibureto/farmacologia , Gliceraldeído/farmacologia , Secreção de Insulina , Ilhotas Pancreáticas/efeitos dos fármacos , Ilhotas Pancreáticas/metabolismo , Ratos , Ratos Endogâmicos F344 , Ratos Endogâmicos , Receptores de Superfície Celular/metabolismo , Receptores de SomatostatinaRESUMO
It may now be possible to identify certain intracellular events that impact specifically on secretion-granule fusion to the plasma membrane or on granule lysis. Secretion vesicles in isolated rat islets appear to translocate somatostatin (SRIF) receptors from the Golgi apparatus to the plasma membrane. We have proposed that secretion granule fusion to the plasma membrane can be determined by measuring recruitment of SRIF receptors to the surface membrane. Granule lysis can be assessed by measuring insulin release. To activate cyclic AMP (cAMP)-dependent pathways, we employed isobutylmethylxanthine (IBMX, 400 microM), glucagon (10 microM), and forskolin (20 microM), a diterpene activator of adenylate cyclase. These agents evoked rapid release of insulin (from 0.41 +/- 0.02 to 1.88 +/- 0.02; 0.41 +/- 0.02 to 1.93 +/- 0.08; and 0.41 +/- 0.02 to 1.66 +/- 0.03 microU/islet/min, respectively, P less than 0.001). There was no concomitant recruitment of SRIF receptors. Somatostatin (10 micrograms/ml), which inhibits cAMP-stimulated protein phosphorylation, suppresses insulin release evoked by IBMX, glucagon, or forskolin (inhibition: 80, 75, or 82%, respectively). In contrast, trifluoperazine (10 microM), an inhibitor of calmodulin, did not suppress insulin release induced through cAMP-dependent pathways. Trifluoperazine suppresses glucose-induced insulin release and the recruitment of SRIF receptors to the surface membrane, suggesting the possible role of calmodulin in promoting secretion-granule fusion with the plasma membrane.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Calmodulina/fisiologia , AMP Cíclico/fisiologia , Exocitose , Hormônios/metabolismo , 1-Metil-3-Isobutilxantina/farmacologia , Animais , Colforsina , AMP Cíclico/análise , Diterpenos/farmacologia , Glucagon/farmacologia , Glucose/farmacologia , Insulina/metabolismo , Secreção de Insulina , Ilhotas Pancreáticas/análise , Ilhotas Pancreáticas/efeitos dos fármacos , Masculino , Ratos , Ratos Endogâmicos , Receptores de Superfície Celular/metabolismo , Receptores de Somatostatina , Somatostatina/farmacologiaRESUMO
The effect of metabolic inhibitors, 2,4-dinitrophenol (DNP) and NaF, on insulin binding and degradation has been studied in cultured Buffalo rat liver (BRL) cells. In control studies, 1.8 fmol of 125I-insulin binds to 1.2 x 10(6) cells, possessing approximately 40,000 receptor sites per cell with binding affinity of 5.52 x 10(-8) M. When the cells were preincubated with increasing concentrations of either DNP or NaF, a dose- and time-dependent decrease in both insulin binding and degradation was observed. The total amount of 125I-insulin bound to BRL cells preincubated with metabolic inhibitors was reduced to 1.2 fmol per 1.2 x 10(6) cells. The point of 1/2 B max was achieved in the presence of 50 ng/ml of native insulin, 1.7 times that of the control level. The number of receptor sites was unaffected by either DNP or NaF, but an average affinity profile revealed a decrease in the affinity of the ATP-depleted cells for insulin (KD: 7.31 x 10(-8) M and 7.06 x 10(-8) M in DNP- and NaF-treated cells, respectively). The decrease in insulin binding and degradation following the exposure of the BRL cells to the metabolic inhibitors was associated with a 20% reduction in intracellular ATP and adenylate energy charge. DNP and NaF did not affect the equilibrium constant for the myokinase catalyzed reaction and the intracellular concentration of hypoxanthine was stable, confirming the integrity of the cells during the experiments. It is suggested that ATP levels must remain intact to maintain normal insulin receptor affinity. Furthermore, the rate of insulin degradation by ATP-depleted cells is slower than that of intact cells. It is conceivable that the depression of insulin degradation by partially ATP-depleted cells results from either diminished binding or decreased endocytosis and lysosomal activity, all of which appear to be energy dependent.
Assuntos
Trifosfato de Adenosina/metabolismo , Insulina/metabolismo , Fígado/metabolismo , Receptor de Insulina/efeitos dos fármacos , Difosfato de Adenosina/metabolismo , Monofosfato de Adenosina/metabolismo , Animais , Células Cultivadas , Dinitrofenóis/farmacologia , Ligação Proteica/efeitos dos fármacos , Ratos , Fluoreto de Sódio/farmacologia , Estatística como Assunto , Fatores de TempoRESUMO
Treatment of adipocytes with depolarizing concentrations of K+ (40 mM) for 60 min increased [Ca2+]i from 158 +/- 28 nM to 328 +/- 38 nM. This significantly reduced (up to 80% inhibition) dephosphorylation of insulin receptor (IR), EGF receptor (EGF-R) and glycogen synthase (GS). The calcium channel blocker, nitrendipine (30 microM), or Ca2+ free medium completely prevented K(+)-induced inhibition of phosphoprotein phosphatase (PPTase). This effect of high [Ca2+]i was completely reversible when the cells were returned into the non-depolarizing medium. Trypsin treatment (4 micrograms/ml) of the membrane fraction containing inhibited PPTase activity, restored dephosphorylation activity to normal suggesting that elevated [Ca2+]i may inhibit PPTase by promoting its association with the inhibitors. These observations indicate that dephosphorylation of IR and GS can be regulated by [Ca2+]i.
Assuntos
Tecido Adiposo/metabolismo , Cálcio/metabolismo , Glicogênio Sintase/metabolismo , Fosfoproteínas Fosfatases/antagonistas & inibidores , Receptor de Insulina/metabolismo , Tecido Adiposo/citologia , Animais , Cálcio/análise , Eletroforese em Gel de Poliacrilamida , Receptores ErbB/análise , Receptores ErbB/química , Receptores ErbB/metabolismo , Fura-2 , Glicogênio Sintase/análise , Fígado/química , Masculino , Potenciais da Membrana , Nitrendipino/farmacologia , Fosfoproteínas Fosfatases/metabolismo , Fosforilação , Potássio/química , Potássio/metabolismo , Ratos , Ratos Endogâmicos , Tripsina/farmacologia , Aglutininas do Germe de TrigoRESUMO
Sustained elevations in cytosolic calcium concentrations ([Ca2+]i) have been shown to render insulin target cells resistant to insulin action. In this study we examined the mechanisms of the detrimental effect of high levels of [Ca2+]i on insulin-induced 2-deoxyglucose (2-DOG) uptake. To elevate [Ca2+]i, we incubated rat adipocytes with either 40 mM potassium (K+) or 20 ng/ml PTH for 1 h for in vitro experiments and injected rats with PTH (injections of 50 micrograms, ip, every hour for 3 h) for in vivo studies. Adipocytes with elevated [Ca2+]i demonstrated a 30% decrease in insulin-stimulated 2-DOG uptake. A calcium channel blocker (nitrendipine) and a cAMP antagonist (RpcAMP) each partially restored insulin-stimulated glucose transport, but together they completely restored 2-DOG uptake. Concomitantly, we found a significant increase in phosphorylation of GLUT-4 in adipocytes with elevated [Ca2+]i. This change in GLUT-4 phosphorylation was also attenuated by nitrendipine and RpcAMP. These observations confirm that elevated [Ca2+]i diminishes insulin-stimulated glucose transport and suggest that increased phosphorylation of GLUT-4 in adipocytes with high [Ca2+]i may alter its intrinsic activity.
Assuntos
Tecido Adiposo/metabolismo , Cálcio/metabolismo , Proteínas de Transporte de Monossacarídeos/metabolismo , Tecido Adiposo/efeitos dos fármacos , Animais , AMP Cíclico/antagonistas & inibidores , Desoxiglucose/metabolismo , Insulina/farmacologia , Masculino , Nitrendipino/farmacologia , Hormônio Paratireóideo/farmacologia , Fosforilação , Potássio/farmacologia , Ratos , Ratos EndogâmicosRESUMO
We have examined the relationship between insulin-stimulated 2-deoxyglucose uptake and cytosolic free calcium concentrations, [( Ca2+]i), in adipocytes isolated from 2- and 12-month-old rats. The basal rates of glucose uptake and the levels of cytosolic Ca2+ were only minimally reduced in 12-month-old animals. In contrast, insulin-stimulated glucose up-take and [Ca2+]i were significantly decreased in older adipocytes at all insulin concentrations (P less than 0.01). When the rate of glucose uptake was plotted as a function of [Ca2+]i, insulin-stimulated glucose uptake was almost identical in older and younger animals at any given level of [Ca2+]i. Similar to insulin, glyburide and K+ increased [Ca2+]i in both younger and older adipocytes. However, glyburide- and K+-elicited responses were lower in older rats (P less than 0.01). The effects of insulin, glyburide, and K+ on [Ca2+]i are mediated via voltage-dependent Ca2+ channels. Thus, the present observations suggest an impairment in either function and/or availability of the voltage-dependent Ca2+ channels in older animals. This was supported by the finding of reduced [3H]nitrendipine binding in adipocytes isolated from older animals (6.5% vs. 3.3% in 2- and 12-month-old rats, respectively; P less than 0.01). The results of these experiments indicate that the postreceptor changes in adipocyte responsiveness to insulin in aging may involve inadequate increases in [Ca2+]i. The latter probably occurs as a result of decreased availability and/or function of the voltage-dependent calcium channels.
Assuntos
Envelhecimento/metabolismo , Cálcio/metabolismo , Desoxiaçúcares/metabolismo , Desoxiglucose/metabolismo , Tecido Adiposo/efeitos dos fármacos , Tecido Adiposo/metabolismo , Animais , Citosol/metabolismo , Glibureto/farmacologia , Insulina/farmacologia , Canais Iônicos/fisiologia , Cinética , Masculino , Nitrendipino/metabolismo , Potássio/farmacologia , Ratos , Ratos Endogâmicos , Verapamil/farmacologiaRESUMO
A new sequential gating perifusion technique was employed to investigate secretion vesicle margination and granule lysis in islets isolated from 2- and 18-month-old Fischer 344 rats. The technique is based on sequential perifusion (periods A, B, and C) of isolated islets with glucose (30, 165, or 300 mg/dl) in the presence of sodium isethionate, an inhibitor of granule lysis, followed thereafter by trifluoperazine, an inhibitor of secretion vesicle margination, and glucose (300 mg/dl) or isobutylmethylxanthine (IBMX; 400 microM). When glucose was employed during period A to marginate secretion vesicles to the plasma membrane, subsequent glucose- and IBMX-induced insulin release (period C) was depressed in islets from 18-month-old rats [maximal increase above the basal rate of release (delta max), 9 +/- 2 nU/micron X min] compared to that in the 2-month-old animals (delta max, -19 +/- 3 nU/micron X min). With glyburide (400 microM) used to induce secretion vesicle margination, glucose- and IBMX-induced insulin release was the same in young and old animals (delta max, 14 +/- 3 and 15 +/- 3 nU/micron X min, respectively). Insulin release was then studied as a function of secretion vesicle margination at the plasma membrane by measuring somatostatin (SRIF) receptor recruitment. The islets from older animals must be stimulated with 300 mg/dl glucose to attain the same level of SRIF binding as in islets isolated from younger animals stimulated with 150-165 mg/dl glucose. Insulin release per unit SRIF binding was identical in young and old animals (65 and 69 nU/liter fmol SRIF binding), indicating normal lysis of marginated secretion granules. These studies implicate glucose-induced secretion vesicle margination as the site of impairment in age-related insulin release.
Assuntos
Envelhecimento , Grânulos Citoplasmáticos/fisiologia , Insulina/metabolismo , Ilhotas Pancreáticas/metabolismo , 1-Metil-3-Isobutilxantina/farmacologia , Animais , Grânulos Citoplasmáticos/efeitos dos fármacos , Glucose/farmacologia , Técnicas In Vitro , Secreção de Insulina , Ilhotas Pancreáticas/efeitos dos fármacos , Masculino , Ratos , Ratos Endogâmicos F344 , Somatostatina/metabolismoRESUMO
We have examined the effect of exogenous insulin on secretion vesicle margination and secretion vesicle lysis in isolated perifused rat pancreatic islets. Recruitment of somatostatin (SRIF) receptors to the plasma membrane was used as a marker of secretion vesicle margination, whereas insulin release reflected the process of secretion vesicle lysis. A newly designed perifusion protocol allows one to interrupt intermittently either secretion vesicle margination or secretion vesicle lysis. Islets were initially perifused with glucose (30, 100, 165, 200, or 300 mg/dl) in the presence of sodium isethionate. Sodium isethionate inhibits secretion vesicle lysis, but not the recruitment of SRIF receptors. Thus, the margination of secretion vesicles to the surface membrane continued without their lysis. Sodium isethionate was then removed, and islets were challenged with 400 microM isobutylmethylxanthine (IBMX). In the islets perifused with high glucose concentrations, IBMX lysed a greater number of vesicles and caused enhanced release of insulin. The presence of exogenous insulin during the initial phase of secretion vesicle margination did not affect subsequent IBMX-induced insulin secretion from the islets perifused with low glucose concentrations (30 or even 100 mg/dl). When the glucose concentration was increased to 165, 200, or 300 mg/dl, insulin significantly diminished IBMX-induced insulin release. In separate experiments, increasing concentrations of insulin (50, 100, and 200 microU/ml) reduced glucose-induced recruitment of SRIF receptors in a dose-dependent manner. Our observations strongly suggest the existence of a well balanced relationship between ambient glucose and insulin concentrations in terms of their positive and negative feedback actions on insulin release. Their influences seem to be exerted at the level of secretion vesicle margination at the plasma membrane.
Assuntos
Insulina/metabolismo , Ilhotas Pancreáticas/metabolismo , 1-Metil-3-Isobutilxantina/farmacologia , Animais , Técnicas de Cultura , Retroalimentação , Glucose/fisiologia , Insulina/fisiologia , Secreção de Insulina , Masculino , Ratos , Receptores de Superfície Celular/metabolismo , Receptores de Somatostatina , Taxa Secretória/efeitos dos fármacosRESUMO
Concomitant with glucose-induced insulin release, there occurred an increase of ATP from 4.40 plus or minus 0.21 to 23.16 plus or minus 0.52 pmol/100 islets/min (P less than 0.001) in the effluent from perifused rat islets. There is a linear relationship between circulating ATP and insulin levels both in the stimulated and basal state (r = 0.689, P less than 0.01). Islets incubated with labeled adenine for a short period of time (37.5 min) showed no release of radioactivity upon subsequent glucose-induced insulin release. Islets incubated for a prolonged interval with labeled adenine (150 min) showed an increase in acid soluble radioactivity in the effluent during glucose-induced insulin release. Following incubation of the islets with labeled adenine for 150 min, approximately 5% of the homogenate radioactivity was found in the secretory granules. Using column chromatography to separate the adenine nucleotides, the distribution of radioactivity among the various nucleotides in the secretory granule fraction was found to be: AMP 54.42 plus or minus 4.96%, ADP 14.20 plus or minus 1.63%, ATP 15.39 plus or minus 3.84%, and cAMP 16.07 plus or minus 2.11%. The distribution of radioactivity in the effluent adenine nucleotides after glucose-induced insulin release was: AMP 32.83 plus or minus 4.62%, ADP 24.52 plus or minus 2.77%, ATP 28.13 plus or minus 5.45%, and cAMP 26.01 plus or minus 3.34%. The absolute levels of adenine nucleotides in the secretory granules were ATP 4.19 plus or minus 0.88, ATP madp 7.94 plus or minus 2.20 and cAMP 4.46 plus or minus 1.74 pmol/ug prot. The levels in the islet effluent were ATP, 15.30 plus or minus 2.70, ATP qDP, 29.43 plus or minus 3.49 and cAMP 7.66 plus or minus 1.93 pmol/100 islets/min for the first ten min of glucose-stimulated insulin release. Thereafter there was a rapid decline in effluent cAMP while ATP and ADP remained in essentially equivalent amounts. The distribution of radioactivity and absolute levels of the adenine nucleotides in the effluent reflects that found in the secretory granules, confirming previous observations that insulin release is occurring by exocytosis.
Assuntos
Nucleotídeos de Adenina/metabolismo , Ilhotas Pancreáticas/metabolismo , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Fracionamento Celular , Cromatografia por Troca Iônica , AMP Cíclico/metabolismo , Exocitose , Glucose/farmacologia , Insulina/metabolismo , Secreção de Insulina , Masculino , Ratos , Fatores de TempoRESUMO
Considerable progress has been made in the acquisition of knowledge relative to diabetes and its complications. However, areas exist wherein even greater progress can be anticipated. These include: (1) defining the genetics of diabetes; (2) the development of suitable markers for early identification of those persons at risk for developing diabetes; (3) the formulation of intervention strategies for preventing or ameliorating the phenotypic expression of diabetes; (4) improving the treatment of diabetes to achieve better control; (5) the possible use of adjunct agents to prevent the chronic complications associated with long-term diabetes; and (6) the development of possible approaches to achieving a cure for diabetes including application of the new knowledge in molecular biology to gene therapy.
Assuntos
Diabetes Mellitus/etiologia , Diabetes Mellitus/terapia , Complicações do Diabetes , Previsões , Marcadores Genéticos , Antígenos HLA , HumanosRESUMO
The exposure of normal pancreatic islets to cyclosporin-A (1 microgram/ml) for 24 hr resulted in significant inhibition of glucose-induced (16.7 mM) insulin release from 197 +/- 14 microU/10 islets/15 min (control) to 103 +/- 14 microU/10 islets/15 min (Cy-A-treated islets; P less than 0.001). Cy-A did not alter insulin release in the presence of non-stimulatory (1.7 mM) or submaximally effective glucose concentrations (9.2 mM). In parallel experiments, Cy-A reduced glucose-stimulated increases in cytosolic free calcium concentrations, [Ca2+]i (217 +/- 15 nM without and 137 +/- 3 nM with Cy-A in the presence of 16.7 mM glucose, P less than 0.01). To better define the site of Cy-A action, we studied its effect on insulin release and increases in [Ca2+]i induced by either K+ (50 mM), which promotes Ca2+ influx via voltage-dependent Ca2+ channels, or by forskolin (20 microM), dibutyryl cyclic AMP (1 mM) or arachidonic acid (49 microM), all of which stimulate mobilization of intracellular Ca2+ stores. Cy-A significantly inhibited K+-induced changes (203 +/- 13 nM without and 77 +/- 6 nM with Cy-A, respectively, P less than 0.001), but not those induced by forskolin, dibutyryl cyclic AMP or arachidonic acid. These observations suggest that Cy-A inhibits insulin release by interfering with Ca2+ influx via voltage-dependent calcium channels.