RESUMEN
Insulin secretion from glucose-stimulated pancreatic beta-cells is oscillatory, and this is thought to result from oscillations in glucose metabolism. One of the primary metabolic stimulus-secretion coupling factors is the ATP/ADP ratio, which can oscillate as a result of oscillations in glycolysis. Using a novel multiwell culture plate system, we examined oscillations in insulin release and the ATP/ADP ratio in the clonal insulin-secreting cell lines HIT T-15 and INS-1. Insulin secretion from HIT cells grown in multiwell plates oscillated with a period of 4 min, similar to that seen previously in perifusion experiments. Oscillations in the ATP/ADP ratio in cells grown under the same conditions also occurred with a period of 4 min, as did oscillations in [Ca(2+)](i) monitored by fluorescence microscopy. In INS-1 cells oscillations in insulin secretion, the ATP/ADP ratio, and [Ca(2+)](i) were also seen, but with a shorter period of about 1.5 min. These observations of oscillations in the ATP/ADP ratio are consistent with their proposed role in driving the oscillations in [Ca(2+)](i) and insulin secretion. Furthermore, these data show that, at least in the clonal beta-cell lines, cell contact or even circulatory connection is not necessary for synchronous oscillations induced by a rise in glucose.
Asunto(s)
Calcio/metabolismo , Glucosa/farmacología , Insulina/metabolismo , Islotes Pancreáticos/efectos de los fármacos , Adenosina Difosfato/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Cricetinae , Islotes Pancreáticos/metabolismo , Células Tumorales CultivadasRESUMEN
We have shown that intermediate lobe (IL) pituitary cells can be engineered to produce sufficient amounts of insulin (ins) to cure diabetes in nonobese diabetic mice but, unlike transplanted islets, ILins cells evade immune attack. To confer glucose-sensing capabilities into these cells, they were further modified with recombinant adenoviruses to express high levels of GLUT2 and the beta-cell isoform of glucokinase (GK). Although expression of GLUT2 alone had negligible effects on glucose usage and lactate production, expression of GK alone resulted in approximately 2-fold increase in glycolytic flux within the physiological (3-20 mm) glucose range. GLUT2/GK coexpression further increased glycolytic flux at 20 mm glucose but disproportionately increased flux at 3 mm glucose. Despite enhanced glycolytic fluxes, GLUT2/GK-coexpressing cells showed glucose dose-dependent accumulation of hexose phosphates, depletion of intracellular ATP, and severe apoptotic cell death. These studies demonstrate that glucose-sensing properties can be introduced into non-islet cells by the single expression of GK and that glucose responsiveness can be augmented by the coexpression of GLUT2. However, in the metabolic engineering of surrogate beta cells, it is critical that the levels of the components be closely optimized to ensure their physiological function and to avoid the deleterious consequences of glucose-induced toxicity.
Asunto(s)
Glucoquinasa/biosíntesis , Glucosa/toxicidad , Insulina/biosíntesis , Proteínas de Transporte de Monosacáridos/biosíntesis , Hipófisis/metabolismo , Adenosina Trifosfato/metabolismo , Adenoviridae/genética , Animales , Apoptosis , Northern Blotting , Western Blotting , Muerte Celular , Células Cultivadas , Relación Dosis-Respuesta a Droga , Glucosa/metabolismo , Transportador de Glucosa de Tipo 2 , Etiquetado Corte-Fin in Situ , Ratones , Ratones Endogámicos NOD , Ratones Transgénicos , Microscopía Fluorescente , Fosforilación , Isoformas de Proteínas , Distribución TisularRESUMEN
It is generally accepted that endothelial cells generate most of their ATP by anaerobic glycolysis and that very little ATP is derived from the oxidation of fatty acids or glucose. Previously, we have reported that, in cultured human umbilical vein endothelial cells (HUVECs), activation of AMP-activated protein kinase (AMPK) by the cell-permeable activator 5-aminoimidazole-4-carboximide riboside (AICAR) is associated with an increase in the oxidation of (3)H-palmitate. In the present study, experiments carried out with cultured HUVECs revealed the following: (1) AICAR-induced increases in palmitate oxidation during a 2-hour incubation are associated with a decrease in the concentration of malonyl coenzyme A (CoA) (an inhibitor of carnitine palmitoyl transferase 1), which temporally parallels the increase in AMPK activity and a decrease in the activity of acetyl CoA carboxylase (ACC). (2) AICAR does not stimulate either palmitate oxidation when carnitine is omitted from the medium or oxidation of the medium-chain fatty acid octanoate. (3) When intracellular lipid pools are prelabeled with (3)H-palmitate, the measured rate of palmitate oxidation is 3-fold higher, and in the presence of AICAR, it accounts for nearly 40% of calculated ATP generation. (4) Incubation of HUVECs in a glucose-free medium for 2 hours causes the same changes in AMPK, ACC, malonyl CoA, and palmitate oxidation as does AICAR. (5) Under all conditions studied, the contribution of glucose oxidation to ATP production is minimal. The results indicate that the AMPK-ACC-malonyl CoA-carnitine palmitoyl transferase 1 mechanism plays a key role in the physiological regulation of fatty acid oxidation in HUVECs. They also indicate that HUVECs oxidize fatty acids from both intracellular and extracellular sources, and that when this is taken into account, fatty acids can be a major substrate for ATP generation. Finally, they suggest that AMPK is likely to be a major factor in modulating the response of the endothelium to stresses that alter its energy state.
Asunto(s)
Aminoimidazol Carboxamida/análogos & derivados , Endotelio Vascular/metabolismo , Ácidos Grasos/metabolismo , Complejos Multienzimáticos/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , 3-O-Metilglucosa/farmacocinética , Proteínas Quinasas Activadas por AMP , Acetil-CoA Carboxilasa/metabolismo , Adenosina Trifosfato/metabolismo , Aminoimidazol Carboxamida/metabolismo , Aminoimidazol Carboxamida/farmacología , Caprilatos/metabolismo , Carnitina/metabolismo , Carnitina/farmacología , Células Cultivadas , Relación Dosis-Respuesta a Droga , Endotelio Vascular/citología , Endotelio Vascular/efectos de los fármacos , Metabolismo Energético/efectos de los fármacos , Metabolismo Energético/fisiología , Activación Enzimática/efectos de los fármacos , Glucosa/metabolismo , Glucosa/farmacocinética , Glucosa/farmacología , Glucólisis/efectos de los fármacos , Humanos , Líquido Intracelular/metabolismo , Malonil Coenzima A/metabolismo , Oxidación-Reducción/efectos de los fármacos , Ácido Palmítico/metabolismo , Ribonucleótidos/metabolismo , Ribonucleótidos/farmacología , Tritio , Venas UmbilicalesRESUMEN
Glucagon-like peptide 1 (GLP-1) is the most potent physiological incretin for insulin secretion from the pancreatic beta-cell, but its mechanism of action has not been established. It interacts with specific cell-surface receptors, generates cAMP, and thereby activates protein kinase A (PKA). Many changes in pancreatic beta-cell function have been attributed to PKA activation, but the contribution of each one to the secretory response is unknown. We show here for the first time that GLP-1 rapidly released free fatty acids (FFAs) from cellular stores, thereby lowering intracellular pH (pHi) and stimulating FFA oxidation in clonal beta-cells (HIT). Similar changes were observed with forskolin, suggesting that stimulation of lipolysis was a function of PKA activation in beta-cells. Triacsin C, which inhibits the conversion of FFAs to long-chain acyl CoA (LC-CoA), enhanced basal FFA efflux as well as GLP-1-induced acidification and efflux of FFAs from the cell. Increasing the concentration of the lipase inhibitor orlistat progressively and largely diminished the increment in secretion caused by forskolin. However, glucose-stimulated secretion was less inhibited by orlistat and only at the highest concentration tested. Because the acute addition of FFAs also increases glucose-stimulated insulin secretion, these data suggest that the incretin function of GLP-1 may involve a major role for lipolysis in cAMP-mediated potentiation of secretion.
Asunto(s)
Glucagón/farmacología , Islotes Pancreáticos/efectos de los fármacos , Islotes Pancreáticos/metabolismo , Lipólisis/efectos de los fármacos , Fragmentos de Péptidos/farmacología , Precursores de Proteínas/farmacología , Calcio/metabolismo , Línea Celular , Inhibidores Enzimáticos/farmacología , Péptido 1 Similar al Glucagón , Péptidos Similares al Glucagón , Concentración de Iones de Hidrógeno , Membranas Intracelulares/metabolismo , Lactonas/farmacología , Lipasa/antagonistas & inhibidores , Orlistat , Fragmentos de Péptidos/antagonistas & inhibidores , Esterol Esterasa/metabolismoRESUMEN
Based on population studies, we have hypothesized that changes in metabolism in pancreatic beta-cells precede changes in Ca2+. It is well known from single-cell Ca2+ studies that variable oscillatory patterns in Ca2+ occur in response to glucose stimulation. The present studies, using the clonal beta-cell line HIT-T-15, were undertaken to evaluate the relationship between glucose concentration, insulin secretion, and O2 consumption and to determine the Ca2+ dependency of glucose-induced changes in O2 consumption. In population studies, an excellent correlation was found between respiration and insulin secretion, with half-maximal values at approximately 1 mmol/l glucose for both respiration and secretion. In the absence of Ca2+, glucose stimulated O2 consumption but not insulin secretion. In single clonal beta-cells, a self-referencing O2 electrode was used to assess O2 consumption. Large-amplitude oscillations were found to occur in response to stimulation by glucose and were blocked by uncoupling respiration with carbonylcyanide p-(trifluoromethoxy)phenylhydrazone (FCCP). They were also blocked and respiration totally inhibited by antimycin A, an inhibitor of complex III of the respiratory chain. Half of the cells sampled (approximately 100 total) exhibited increased oscillatory O2 consumption in response to glucose. Oscillations in O2 occurred in response to glucose even in the absence of Ca2+, and their amplitude increased further on restoration of a normal extracellular Ca2+ level. These studies indicated that oscillatory O2 consumption was not dependent on Ca2+ but that the amplitude of the O2 oscillations increased in the presence of Ca2+, possibly reflecting the additional work involved in insulin secretion and Ca2+ pumping. These studies demonstrated, for the first time, a direct correlation between O2 consumption and insulin secretion, the oscillatory nature of O2 consumption in single cells, and the feasibility of using a highly sensitive noninvasive on-line self-referencing O2 electrode to monitor single beta-cell respiration.
Asunto(s)
Calcio/fisiología , Glucosa/farmacología , Insulina/metabolismo , Islotes Pancreáticos/fisiología , Consumo de Oxígeno , Animales , Carbonil Cianuro p-Trifluorometoxifenil Hidrazona/farmacología , Células Clonales , Secreción de Insulina , Islotes Pancreáticos/efectos de los fármacos , Cinética , Oscilometría , Consumo de Oxígeno/efectos de los fármacos , Desacopladores/farmacologíaRESUMEN
Alterations in the concentration of malonyl-CoA, an inhibitor of carnitine palmitoyltransferase I, have been linked to the regulation of fatty acid oxidation in skeletal muscle. During contraction decreases in muscle malonyl-CoA concentration have been related to activation of AMP-activated protein kinase (AMPK), which phosphorylates and inhibits acetyl-CoA carboxylase (ACC), the rate-limiting enzyme in malonyl-CoA formation. We report here that the activity of malonyl-CoA decarboxylase (MCD) is increased in contracting muscle. Using either immunopurified enzyme or enzyme partially purified by (NH(4))(2)SO(4) precipitation, 2-3-fold increases in the V(max) of MCD and a 40% decrease in its K(m) for malonyl-CoA (190 versus 119 micrometer) were observed in rat gastrocnemius muscle after 5 min of contraction, induced by electrical stimulation of the sciatic nerve. The increase in MCD activity was markedly diminished when immunopurified enzyme was treated with protein phosphatase 2A or when phosphatase inhibitors were omitted from the homogenizing solution and assay mixture. Incubation of extensor digitorum longus muscle for 1 h with 2 mm 5-aminoimidazole-4-carboxamide-1-beta-d-ribofuranoside, a cell-permeable activator of AMPK, increased MCD activity 2-fold. Here, too, addition of protein phosphatase 2A to the immunopellets reversed the increase of MCD activity. The results strongly suggest that activation of AMPK during muscle contraction leads to phosphorylation of MCD and an increase in its activity. They also suggest a dual control of malonyl-CoA concentration by ACC and MCD, via AMPK, during exercise.
Asunto(s)
Adenilato Quinasa/metabolismo , Aminoimidazol Carboxamida/análogos & derivados , Carboxiliasas/metabolismo , Contracción Muscular/fisiología , Músculo Esquelético/enzimología , Ribonucleótidos/farmacología , Aminoimidazol Carboxamida/farmacología , Animales , Carboxiliasas/aislamiento & purificación , Cinética , Masculino , Músculo Esquelético/inervación , Fosfoproteínas Fosfatasas/metabolismo , Fosforilación , Proteína Fosfatasa 2 , Ratas , Ratas Sprague-Dawley , Nervio Ciático/fisiologíaRESUMEN
Glucose-induced insulin secretion is associated with inhibition of free fatty acid (FFA) oxidation, increased esterification and complex lipid formation by pancreatic beta-cells. Abundant evidence favors a role for cytosolic long-chain acyl-CoA (LC-CoA), including the rapid rise in malonyl CoA, the inhibitory effect of hydroxycitrate or acetyl CoA carboxylase knockout, both of which prevent malonyl CoA formation, and the stimulatory effect of exogenous FFA. On the other hand, some evidence opposes the concept, including the fall in total LC-CoA levels in response to glucose, the stimulatory effect of LC-CoA on K(ATP) channels and the lack of inhibition of glucose-stimulated secretion either by overexpression of malonyl CoA decarboxylase, which markedly lowers malonyl CoA levels, or by triacsin C, which blocks FFA conversion to LC-CoA. Alternative explanations for these data are presented. A revised model of nutrient-stimulated secretion involving two arms of signal transduction that occur simultaneously is proposed. One arm depends on modulation of the K(ATP) channel evoked by changes in the ATP/ADP ratio. The other arm depends upon anaplerotic input into the tricarboxylic acid cycle, generation of excess citrate, and increases in cytosolic malonyl-CoA. Input from this arm is increased LC-CoA. Signaling through both arms would be required for normal secretion. LC-CoA esters and products formed from them are potent regulators of enzymes and channels. It is hypothesized that their elevations directly modulate the activity of enzymes, genes and various beta-cell functions or modify the acylation state of key proteins involved in regulation of ion channels and exocytosis.
Asunto(s)
Acilcoenzima A/fisiología , Malonil Coenzima A/fisiología , Transducción de Señal/fisiología , Citosol/metabolismo , Ácidos Grasos no Esterificados/metabolismo , Glucosa/metabolismo , Humanos , Insulina/metabolismo , Secreción de Insulina , Mitocondrias/metabolismo , Páncreas/metabolismo , Páncreas/fisiología , Canales de Potasio/metabolismoRESUMEN
In several non-vascular tissues in which it has been studied, AMP-activated protein kinase (AMPK) appears to modulate the cellular response to stresses such as ischemia. In liver and muscle, it phosphorylates and inhibits acetyl CoA carboxylase (ACC), leading to an increase in fatty acid oxidation; and in muscle, its activation is associated with an increase in glucose transport. Here we report the presence of both AMPK and ACC in human umbilical vein endothelial cells (HUVEC). Incubation of HUVEC with 2 mM AICAR, an AMPK activator, caused a 5-fold activation of AMPK, which was accompanied by a 70% decrease in ACC activity and a 2-fold increase in fatty acid oxidation. Surprisingly, glucose uptake and glycolysis, the dominant energy-producing pathway in HUVEC, were diminished by 40-60%. Despite this, cellular ATP levels were increased by 35%. Thus activation of AMPK by AICAR is associated with major alterations in endothelial cell energy balance. Whether these alterations protect the endothelium during ischemia or other stresses remains to be determined.
Asunto(s)
Aminoimidazol Carboxamida/análogos & derivados , Endotelio Vascular/metabolismo , Hipoglucemiantes/farmacología , Complejos Multienzimáticos/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Ribonucleótidos/farmacología , Proteínas Quinasas Activadas por AMP , Acetil-CoA Carboxilasa/metabolismo , Adenosina Trifosfato/metabolismo , Aminoimidazol Carboxamida/farmacología , Células Cultivadas , Endotelio Vascular/efectos de los fármacos , Activación Enzimática , Glucosa/metabolismo , Glucólisis/efectos de los fármacos , Humanos , Cinética , Ácido Palmítico/metabolismo , Venas UmbilicalesRESUMEN
The glucose-fatty acid cycle of Randle entails two elements: decreased pyruvate dehydrogenase (PDH) activity, which inhibits glucose oxidation, and inhibition of phosphofructokinase (PFK) by a rise in citrate so that glucose-6-phosphate (G-6-P) levels increase, thereby inhibiting hexokinase activity and hence glucose utilization. Chronic exposure of islets to long-chain fatty acids (FA) is reported to lower PDH activity, but the effect on glucose oxidation and glucose-induced insulin secretion is uncertain. We investigated rat islets that were cultured for 4 days with 0.25 mmol/l oleate/5.5 mmol/l glucose. Glucose oxidation was doubled at 2.8 mmol/l glucose and unchanged at 27.7 mmol/l glucose in the FA-cultured islets despite a 35% decrease in assayed PDH activity. Pyruvate content was increased 60%, which may well compensate for the decreased PDH activity and maintain flux through the citric acid cycle. However, a greater diversion of pyruvate metabolism through the pyruvate-malate shuttle is suggested by unchanged pyruvate carboxylase Vmax and a fourfold higher release of malate from isolated mitochondria. The FA-cultured islets also showed increased basal glucose usage and insulin secretion together with a lowered level of G-6-P and 50% reductions in citrate synthase Vmax and the citrate content. Thus, the effects of chronic FA exposure on islet glucose metabolism differ from the glucose-fatty acid interactions reported in some other tissues.
Asunto(s)
Glucemia/metabolismo , Ácidos Grasos/sangre , Islotes Pancreáticos/metabolismo , Animales , Células Cultivadas , Glicerolfosfato Deshidrogenasa/metabolismo , Insulina/metabolismo , Secreción de Insulina , Mitocondrias/metabolismo , Oxidación-Reducción , Fosfofructoquinasa-1/metabolismo , Complejo Piruvato Deshidrogenasa/metabolismo , Ácido Pirúvico/metabolismo , Ratas , Ratas Sprague-DawleyRESUMEN
The synthesis and in vitro enzyme inhibition profile of a series of novel trifluoromethylketone (TFMK) inhibitors of human plasma kallikrein (PK) are described. We have developed an efficient method for the construction of peptide TFMKs that provides the final product devoid of compromised stereochemical integrity. Many of these compounds are potent inhibitors of PK and exhibit reduced inhibition of tissue kallikrein (TK) and plasmin (HP).
Asunto(s)
Calicreínas/antagonistas & inhibidores , Cetonas/síntesis química , Inhibidores de Serina Proteinasa/síntesis química , Fibrinolisina/antagonistas & inhibidores , Humanos , Cetonas/farmacología , Inhibidores de Serina Proteinasa/farmacologíaRESUMEN
Diabetic states are characterized by a raised serum/islet level of triglycerides and a lowered EC50 (concentration at half-maximal stimulation) for glucose-induced insulin secretion. Culturing islets with long-chain fatty acids (FAs) replicates the basal insulin hypersecretion. In a previous study, we showed that the mechanism involved deinhibition of hexokinase by a 60% decrease in glucose-6-phosphate (G-6-P). The key event was proposed to be an increased phosphofructokinase (PFK) Vmax secondary to an upregulatory effect of the FA metabolite, long-chain acyl-coenzyme A (LC-CoA). We now show another contributory factor, a lowered content of the PFK inhibitor citrate. Citrate synthase Vmax and citrate levels were lowered 45% in rat islets cultured with 250 micromol/l oleate for 24 h. Both effects were reversed by triacsin C, an inhibitor of fatty acyl-CoA synthetase, the enzyme that generates LC-CoA. Culturing islets with high doses of glucose (16.7 mmol/l) for 48 h should also raise cytosolic LC-CoA. As predicted, citrate synthase Vmax was lowered and PFK Vmax was increased, both in a triacsin C-reversible fashion. These results show shared selected functional and biochemical properties in beta-cells of so-called glucotoxicity and lipotoxicity.
Asunto(s)
Citrato (si)-Sintasa/efectos de los fármacos , Ácidos Grasos/farmacología , Glucosa/farmacología , Islotes Pancreáticos/efectos de los fármacos , Fosfofructoquinasa-1/efectos de los fármacos , Animales , Citrato (si)-Sintasa/metabolismo , Citratos/metabolismo , Relación Dosis-Respuesta a Droga , Técnicas In Vitro , Islotes Pancreáticos/enzimología , Cinética , Ácido Oléico/administración & dosificación , Ácido Oléico/farmacología , Excipientes Farmacéuticos/administración & dosificación , Excipientes Farmacéuticos/farmacología , Fosfofructoquinasa-1/metabolismo , Ratas , Ratas Sprague-Dawley , Factores de TiempoRESUMEN
A series of tripeptide aldehyde derivatives containing variations at the P3 subsite and the amino terminus has been prepared and evaluated for trypsin-like serine protease inhibition. These compounds exhibit strong in vitro inhibition of human plasma kallikrein (HPK), porcine pancreatic kallikrein (PPK) and human plasmin (HP). As suspected from an examination of a related crystal structure, the presence of a hydrophobic residue (adamantyl) at the amino terminus dramatically improves the binding to PPK. The adamantyl group, however, represents a peak in binding; larger residues cause the binding to be reduced, and thus are less well accommodated in this subsite. Although both HP and HPK also can accept large molecular volume at the amino terminus, they do not exhibit the same preference for large residues at this subsite that is demonstrated by PPK. Selectivity differences also are observed with P3 subsite substitution; with PPK preferring a bulky, but compact side-chain (t-butyl) and HP and HPK preferring a more extended (e.g. benzyl) group.
Asunto(s)
Aldehídos/farmacología , Calicreínas/antagonistas & inhibidores , Péptidos/química , Inhibidores de Serina Proteinasa/síntesis química , Animales , Sitios de Unión/fisiología , Proteínas Sanguíneas/metabolismo , Humanos , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Conformación Molecular , Estructura Molecular , Páncreas/enzimología , PorcinosRESUMEN
Diabetic states are characterized by a raised serum/islet level of long chain fatty acids and a lowered ED50 for glucose-induced insulin secretion. Prolonged culture (> 6 h) of islets with long chain fatty acids replicates the basal insulin hypersecretion. We examined this effect in rat islets cultured for 24 h with 0.25 mM oleate. Insulin secretion at 2.8 mM glucose was doubled in combination with a 60% lowered islet content of glucose-6-phosphate (G6P). Investigation of the lowered G6P showed: (a) increased glucose usage from 0.5 to 100 mM glucose with identical values measured by [2-3H]glucose and [5-3H]glucose, (c) indicating little glucose- 6-phosphatase activity, (b) unchanged low pentose phosphate shunt activity, (c) 50% increased phosphofructokinase (PFK) Vmax, (d) a normal ATP/ADP ratio, and (e) unchanged fructose 2,6 bisphosphate content. Triacsin C, an inhibitor of fatty acyl-CoA synthetase, prevented the increase in PFK activity and the lowered G6P content. These results suggest that long chain acyl-CoA mediates the rise in PFK activity, which in turn lowers the G6P level. We speculate that the inhibition of hexokinase by G6P is thus attenuated, thereby causing the basal insulin hypersecretion.
Asunto(s)
Glucosa/farmacología , Insulina/metabolismo , Islotes Pancreáticos/efectos de los fármacos , Ácido Oléico/farmacología , Proteínas Represoras , Proteínas de Saccharomyces cerevisiae , Regulación Alostérica , Animales , Coenzima A Ligasas/antagonistas & inhibidores , Diabetes Mellitus/metabolismo , Interacciones Farmacológicas , Fructosadifosfatos/análisis , Glucosa-6-Fosfato/análisis , Secreción de Insulina , Islotes Pancreáticos/citología , Oxidación-Reducción , Vía de Pentosa Fosfato , Fosfofructoquinasa-1/metabolismo , Ratas , Ratas Sprague-Dawley , Triazenos/farmacologíaRESUMEN
Normal insulin secretion is oscillatory in vivo and in vitro, with a period of approximately 5-10 min. The mechanism of generating these oscillations is not yet established, but a metabolic basis seems most likely for glucose-stimulated secretion. The rationale is that 1) spontaneous oscillatory operation of glycolysis is a well-established phenomenon; 2) oscillatory behavior of glycolysis involves oscillations in the ATP/ADP ratio, which can cause alternating opening and closing of ATP-sensitive K+ channels, leading to the observed oscillations in membrane potential and Ca2+ influx in pancreatic beta-cells, and may also have downstream effects on exocytosis; 3) spontaneous Ca2+ oscillations are an unlikely basis in this case, since intracellular stores are not of primary importance in the stimulus-secretion coupling, and furthermore, insulin oscillations occur under conditions when intracellular Ca2+ levels are not changing; 4) a neural basis cannot account for insulin oscillations from perifused islets and clonal beta-cells or from transplanted islets or pancreas in vivo; 5) observed oscillations in metabolite levels and fluxes further support a metabolic basis, as does the presence in beta-cells of the oscillatory isoform of phosphofructokinase (PFK-M). The fact that normal oscillatory secretion is impaired in patients with NIDDM and in their near relatives suggests that such derangement may be involved in the development of the disease; furthermore, this probably reflects an early defect in the regulation and operation of the fuel metabolizing/sensing pathways of the pancreatic beta-cell.
Asunto(s)
Glucólisis , Insulina/metabolismo , Islotes Pancreáticos/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Calcio/fisiología , Metabolismo Energético , Glucosa/fisiología , Humanos , Secreción de Insulina , Mitocondrias/metabolismo , Periodicidad , Fosfofructoquinasa-1/metabolismo , Transducción de SeñalRESUMEN
The knowledge of the mechanism whereby glucose and other fuel stimuli promote the release of insulin by the pancreatic beta cell remains fragmentary. The closure of metabolically sensitive K+ channels and a rise in cytosolic free Ca2+ are key features of beta-cell metabolic signal transduction. However, these two signalling events do not account for the dose dependence of glucose-induced insulin secretion. In fact, recent evidence indicates that there are KATP channel and Ca2+ independent pathway(s) of beta-cell activation which remain to be defined. In this review, we have limited our attention to the recent developments in our understanding of the mode of action of nutrient secretagogues. A particular emphasis is placed in summarising the evidence in support of two new concepts: 1) oscillations in the glycolytic pathway and beta-cell metabolism contribute to the oscillatory nature of beta-cell ionic events and insulin secretion; 2) malonyl-CoA and long chain acyl-CoA esters may act as metabolic coupling factors in beta-cell signalling. Finally, we propose that the altered expression of genes encoding enzymes in the pathway of malonyl-CoA formation and fatty acid oxidation contributes to the beta-cell insensitivity to glucose in some patients with non-insulin-dependent diabetes mellitus.
Asunto(s)
Insulina/metabolismo , Islotes Pancreáticos/metabolismo , Malonil Coenzima A/metabolismo , Transducción de Señal , Animales , Humanos , Secreción de InsulinaRESUMEN
To determine whether oscillations in glycolysis could underlie the oscillations in O2 consumption observed in intact islets, we evaluated the capacity of an islet extract to exhibit spontaneous oscillations in glycolysis. When a cell-free extract obtained from approximately 1,000 islets was supplied with glucose and glycolytic cofactors, oscillations in NADH fluorescence were obtained. After this demonstration of spontaneous oscillations in islet extracts, we bathed permeabilized clonal beta-cells in the more plentiful spontaneously oscillating glycolytic muscle extract that generates pulses of alpha-glycerophosphate and pyruvate and induces oscillations in free Ca2+ and the ATP/ADP ratio. This preparation was used to investigate whether changes in Ca2+ and possibly alpha-glycerophosphate or pyruvate supply could underlie observed oscillations in O2 consumption and explain coordination between cytosolic and mitochondrial metabolism. We found that oscillations of O2 consumption and Ca2+ of a similar period were induced. Removal of medium Ca2+ with EGTA did not prevent the oscillations in O2 consumption nor were they greatly affected by the substantial rise in medium Ca2+ on treatment with thapsigargin to inhibit sequestration into the endoplasmic reticulum. The 02 oscillations were also not eliminated by the addition of relatively high concentrations of pyruvate or alpha-glycerophosphate. However, they were lost on addition of fructose-2,6-P2 at concentrations that prevent oscillations of glycolysis and the ATP/ADP ratio. Addition of a high concentration of ADP increased 02 consumption and also prevented 02 oscillations. These results suggest that the changes in respiration reflected in the 02 oscillations occur in response to the oscillations in the ATP/ADP ratio or ADP concentration and that this parameter is a primary regulator of 02 consumption in the pancreatic beta-cell.
Asunto(s)
Glucólisis , Islotes Pancreáticos/metabolismo , Músculo Esquelético/fisiología , NAD/metabolismo , Consumo de Oxígeno , Adenosina Difosfato/farmacología , Adenosina Trifosfato/metabolismo , Animales , Calcio/metabolismo , Permeabilidad de la Membrana Celular , Células Clonales , Ácido Egtácico/farmacología , Fructosadifosfatos/farmacología , Glicerofosfatos/farmacología , Islotes Pancreáticos/efectos de los fármacos , Cinética , Oscilometría , Consumo de Oxígeno/efectos de los fármacos , Ácido Pirúvico/farmacología , Ratas , Espectrometría de Fluorescencia , Tapsigargina/farmacologíaRESUMEN
Normal insulin secretion is oscillatory in vivo and from groups of perifused islets. Stimulation of rat islets with different glucose concentrations gave insulin oscillations of similar period (5-8 min) but increasing amplitude. It has been assumed that oscillatory secretion is due to oscillations in intracellular free Ca2+, as seen in single islets and single pancreatic beta-cells. However, when islets were perifused with diazoxide and high KCl to maintain high intracellular free Ca2+, insulin oscillations of similar amplitude and period still occurred on glucose stimulation, although superimposed on elevated basal secretion. Several likely possibilities for a diffusible synchronizing factor were tested, including pyruvate, lactate, ATP, and insulin itself; nevertheless, perifusion with high concentrations of these did not prevent insulin oscillations. Clonal pancreatic beta-cells (HIT) and dissociated islets also exhibited oscillatory insulin secretion, but with the 5- to 8-min period oscillations superimposed on 15- to 20-min period oscillations. These results indicate that the mechanisms for generating and synchronizing insulin oscillations reside in the beta-cell, although the structure of the islet may modulate the oscillation pattern.
Asunto(s)
Glucosa/fisiología , Insulina/metabolismo , Islotes Pancreáticos/metabolismo , Animales , Calcio/fisiología , Comunicación Celular , Células Cultivadas , Células Clonales , Glucosa/administración & dosificación , Secreción de Insulina , Islotes Pancreáticos/citología , Lactatos/metabolismo , Masculino , Perfusión , Periodicidad , Piruvatos/metabolismo , Ratas , Ratas Sprague-DawleyRESUMEN
The main function of white adipose tissue is to store nutrient energy in the form of triglycerides. The mechanism by which free fatty acids (FFA) move into and out of the adipocyte has not been resolved. We show here that changes in intracellular pH (pH1) in adipocytes correlate with the movement of FFA across cellular membranes as predicted by the Kamp and Hamilton model of passive diffusion of FFA. Exposure of fat cells to lipolytic agents or external FFA results is a rapid intracellular acidification that is reversed by metabolism of the FFA or its removal by albumin. In contrast, insulin causes an alkalinization of the cell, consistent with its main function to promote esterification. Inhibition of Na+/H+ exchange in adipocytes does not prevent the changes in pHi caused by FFA, lipolytic agents, or insulin. A fatty acid dimer, which diffuses into the cell but is not metabolized, causes an irreversible acidification. Taken together, the data suggest that changes in pHi occur in adipocytes in response to the passive diffusion of un-ionized FFA (flip-flop) into and out of the cell and in response to their metabolism and production within the cell. These changes in pHi may, in turn, modulate hormonal signaling and metabolism with significant impact on cell function.
Asunto(s)
Adipocitos/metabolismo , Ácidos Grasos no Esterificados/metabolismo , Ácidos Grasos no Esterificados/farmacología , Concentración de Iones de Hidrógeno , Insulina/farmacología , Adipocitos/efectos de los fármacos , Amilorida/análogos & derivados , Amilorida/farmacología , Animales , Membrana Celular/metabolismo , Células Cultivadas , Colforsina/farmacología , Difusión , Fluoresceínas , Colorantes Fluorescentes , Isoproterenol/farmacología , Cinética , Lipólisis/efectos de los fármacos , Masculino , Modelos Biológicos , Norepinefrina/farmacología , Ratas , Ratas Sprague-Dawley , Albúmina Sérica Bovina/farmacología , Sodio/metabolismo , Factores de TiempoRESUMEN
To gain insight into the regulation of pancreatic beta-cell mitochondrial metabolism, the direct effects on respiration of different mitochondrial substrates, variations in the ATP/ADP ratio and free Ca2+ were examined using isolated mitochondria and permeabilized clonal pancreatic beta-cells (HIT). Respiration from pyruvate was high and not influenced by Ca2+ in State 3 or under various redox states and fixed values of the ATP/ADP ratio; nevertheless, high Ca2+ elevated pyridine nucleotide fluorescence, indicating activation of pyruvate dehydrogenase by Ca2+. Furthermore, in the presence of pyruvate, elevated Ca2+ stimulated CO2 production from pyruvate, increased citrate production and efflux from the mitochondria and inhibited CO2 production from palmitate. The latter observation suggests that beta-cell fatty acid oxidation is not regulated exclusively by malonyl-CoA but also by the mitochondrial redox state. alpha-Glycerophosphate (alpha-GP) oxidation was Ca(2+)-dependent with a half-maximal rate observed at around 300 nM Ca2+. We have recently demonstrated that increases in respiration precede increases in Ca2+ in glucose-stimulated clonal pancreatic beta-cells (HIT), indicating that Ca2+ is not responsible for the initial stimulation of respiration [Civelek, Deeney, Kubik, Schultz, Tornheim and Corkey (1996) Biochem. J. 315, 1015-1019]. It is suggested that respiration is stimulated by increased substrate (alpha-GP and pyruvate) supply together with oscillatory increases in ADP [Nilsson, Schultz, Berggren, Corkey and Tornheim (1996) Biochem. J. 314, 91-94]. The rise in Ca2+, which in itself may not significantly increase net respiration, could have the important functions of (1) activating the alpha-GP shuttle, to maintain an oxidized cytosol and high glycolytic flux; (2) activating pyruvate dehydrogenase, and indirectly pyruvate carboxylase, to sustain production of citrate and hence the putative signal coupling factors, malonyl-CoA and acyl-CoA; and (3) increasing mitochondrial redox state to implement the switch from fatty acid to pyruvate oxidation.
Asunto(s)
Adenosina Difosfato/metabolismo , Adenosina Difosfato/farmacología , Calcio/farmacología , Islotes Pancreáticos/metabolismo , Mitocondrias/metabolismo , Consumo de Oxígeno , Adenosina Trifosfato/metabolismo , Animales , Células Clonales , Ácido Egtácico/farmacología , Glicerofosfatos/metabolismo , Insulinoma , Ácidos Cetoglutáricos/farmacología , Cinética , Malonil Coenzima A/metabolismo , Mitocondrias/efectos de los fármacos , Oxidación-Reducción , Consumo de Oxígeno/efectos de los fármacos , Neoplasias Pancreáticas , Complejo Piruvato Deshidrogenasa/metabolismo , RatasRESUMEN
Stimulation of insulin release by glucose requires increased metabolism of glucose and a rise in cytosolic free Ca2+ in the pancreatic beta-cell. It is accompanied by increases in respiratory rate, pyridine and flavin nucleotide reduction state, intracellular pH and the ATP/ADP ratio. To test alternative proposals of the regulatory relationships among free Ca2+, mitochondrial metabolism and cellular energy state, we determined the temporal sequence of these metabolic and ionic changes following addition of glucose to clonal pancreatic beta-cells (HIT). Combined measurements of the native fluorescence of reduced pyridine nucleotides and oxidized flavin, intracellular pH, and free Ca2+ were performed together with simultaneous measurement of O2 tension or removal of samples for assay of the ATP/ADP ratio. The initial changes were detected in three phases. First, decreases occurred in the ATP/ADP ratio (<3 s) and increases in pyridine (2 +/- 1 s) and flavin (2 +/- 1 s) nucleotide reduction. Next, increases in the O2 consumption rate (20 +/- 5 s), the ATP/ADP ratio (29 +/- 12 s) and internal pH (48 +/- 5 s) were observed. Finally, cytosolic free Ca2+ rose (114 +/- 10 s). Maximal changes in the ATP/ADP ratio, O2 consumption and pyridine and flavin nucleotide fluorescence preceded the beginning of the Ca2+ change. These relationships are consistent with a model in which phosphorylation of glucose is the initial event which generates the signals that lead to an increase in respiration, a rise in the ATP/ADP ratio and finally influx of Ca2+. Our results indicate that Ca2+ does not function as the initiator of increased mitochondrial respiration.