RESUMO
AIMS/HYPOTHESIS: The pancreatic beta cell ATP-sensitive potassium (K(ATP)) channel, composed of the pore-forming alpha subunit Kir6.2, a member of the inward rectifier K+channel family, and the regulatory beta subunit sulfonylurea receptor 1 (SUR1), a member of the ATP-binding cassette superfamily, couples the metabolic state of the cell to electrical activity. Several endogenous compounds are known to modulate K(ATP) channel activity, including ATP, ADP, phosphatidylinositol diphosphates and long-chain acyl coenzyme A (LC-CoA) esters. LC-CoA esters have been shown to interact with Kir6.2, but the mechanism and binding site(s) have yet to be identified. MATERIALS AND METHODS: Using multiple sequence alignment of known acyl-CoA ester interacting proteins, we were able to identify four conserved amino acid residues that could potentially serve as an acyl-CoA ester-binding motif. The motif was also recognised in the C-terminal region of Kir6.2 (R311-332) but not in SUR1. RESULTS: Oocytes expressing Kir6.2DeltaC26 K332A repeatedly generated K(+)currents in inside-out membrane patches that were sensitive to ATP, but were only weakly activated by 1 mumol/l palmitoyl-CoA ester. Compared with the control channel (Kir6.2DeltaC26), Kir6.2DeltaC26 K332A displayed unaltered ATP sensitivity but significantly decreased sensitivity to palmitoyl-CoA esters. Coexpression of Kir6.2DeltaC26 K332A and SUR1 revealed slightly increased activation by palmitoyl-CoA ester but significantly decreased activation by the acyl-CoA esters compared with the wild-type K(ATP) channel and Kir6.2DeltaC26+SUR1. Computational modelling, using the crystal structure of KirBac1.1, suggested that K332 is located on the intracellular domain of Kir6.2 and is accessible to intracellular modulators such as LC-CoA esters. CONCLUSIONS/INTERPRETATION: These results verify that LC-CoA esters interact at the pore-forming subunit Kir6.2, and on the basis of these data we propose an acyl-CoA ester binding motif located in the C-terminal region.
Assuntos
Acil Coenzima A/farmacologia , Substituição de Aminoácidos , Canais de Potássio Corretores do Fluxo de Internalização/genética , Acil Coenzima A/metabolismo , Difosfato de Adenosina/metabolismo , Difosfato de Adenosina/farmacologia , Trifosfato de Adenosina/metabolismo , Trifosfato de Adenosina/farmacologia , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Diazóxido/farmacologia , Feminino , Humanos , Potenciais da Membrana/efeitos dos fármacos , Camundongos , Camundongos Obesos , Modelos Moleculares , Dados de Sequência Molecular , Oócitos/efeitos dos fármacos , Oócitos/metabolismo , Oócitos/fisiologia , Palmitoil Coenzima A/metabolismo , Palmitoil Coenzima A/farmacologia , Canais de Potássio Corretores do Fluxo de Internalização/química , Canais de Potássio Corretores do Fluxo de Internalização/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Homologia de Sequência de Aminoácidos , XenopusRESUMO
The cells within the intact islet of Langerhans function as a metabolic syncytium, secreting insulin in a coordinated and oscillatory manner in response to external fuel. With increased glucose, the oscillatory amplitude is enhanced, leading to the hypothesis that cells within the islet are secreting with greater synchronization. Consequently, non-insulin-dependent diabetes mellitus (NIDDM; type 2 diabetes)-induced irregularities in insulin secretion oscillations may be attributed to decreased intercellular coordination. The purpose of the present study was to determine whether the degree of metabolic coordination within the intact islet was enhanced by increased glucose and compromised by NIDDM. Experiments were performed with isolated islets from normal and diabetic Psammomys obesus. Using confocal microscopy and the mitochondrial potentiometric dye rhodamine 123, we measured mitochondrial membrane potential oscillations in individual cells within intact islets. When mitochondrial membrane potential was averaged from all the cells in a single islet, the resultant waveform demonstrated clear sinusoidal oscillations. Cells within islets were heterogeneous in terms of cellular synchronicity (similarity in phase and period), sinusoidal regularity, and frequency of oscillation. Cells within normal islets oscillated with greater synchronicity compared with cells within diabetic islets. The range of oscillatory frequencies was unchanged by glucose or diabetes. Cells within diabetic (but not normal) islets increased oscillatory regularity in response to glucose. These data support the hypothesis that glucose enhances metabolic coupling in normal islets and that the dampening of oscillatory insulin secretion in NIDDM may result from disrupted metabolic coupling.
Assuntos
Diabetes Mellitus Tipo 2/metabolismo , Ilhotas Pancreáticas/metabolismo , Mitocôndrias/metabolismo , Animais , Fluorescência , Corantes Fluorescentes , Gerbillinae , Técnicas In Vitro , Ilhotas Pancreáticas/fisiopatologia , Potenciais da Membrana , Microscopia Confocal , Oscilometria , Periodicidade , Rodamina 123RESUMO
AIMS/HYPOTHESIS: The ATP-regulated potassium (KATP) channel in the pancreatic beta cell couples the metabolic state to electrical activity. The primary regulator of the KATP channel is generally accepted to be changes in ATP/ADP ratio, where ATP inhibits and ADP activates channel activity. Recently, we showed that long-chain CoA (LC-CoA) esters form a new class of potent KATP channel activators in rodents, as studied in inside-out patches. METHODS: In this study we have investigated the effects of LC-CoA esters in human pancreatic beta cells using the inside-out and whole-cell configurations of the patch clamp technique. RESULTS: Human KATP channels were potently activated by acyl-CoA esters with a chain length exceeding 12 carbons. Activation by LC-CoA esters did not require the presence of Mg2+ or adenine nucleotides. A detailed characterization of the concentration-dependent relationship showed an EC50 of 0.7+/-0.1 micromol/l. Furthermore, in the presence of an ATP/ADP ratio of 10 (1.1 mmol/l total adenine nucleotides), whole-cell KATP channel currents increased approximately six-fold following addition of 1 micro mol/l LC-CoA ester. The presence of 1 micro mol/l LC-CoA in the recording pipette solution increased beta-cell input conductance, from 0.5+/-0.2 nS to 2.5+/-1.3 nS. CONCLUSION/INTERPRETATION: Taken together, these results show that LC-CoA esters are potent activators of the KATP channel in human pancreatic beta cells. The fact that LC-CoA esters also stimulate KATP channel activity recorded in the whole-cell configuration, points to the ability of these compounds to have an important modulatory role of human beta-cell electrical activity under both physiological and pathophysiological conditions.
Assuntos
Acil Coenzima A/fisiologia , Ilhotas Pancreáticas/fisiologia , Proteínas de Membrana/fisiologia , Acil Coenzima A/química , Acil Coenzima A/farmacologia , Difosfato de Adenosina/farmacologia , Trifosfato de Adenosina/farmacologia , Diabetes Mellitus Tipo 2/fisiopatologia , Diazóxido/farmacologia , Relação Dose-Resposta a Droga , Glucose/farmacologia , Humanos , Ilhotas Pancreáticas/efeitos dos fármacos , Cinética , Cloreto de Magnésio/farmacologia , Potenciais da Membrana/efeitos dos fármacos , Ácido Oleico/farmacologia , Palmitoil Coenzima A/farmacologia , Técnicas de Patch-Clamp , Canais de PotássioRESUMO
Increases in glucose or fatty acids affect metabolism via changes in long-chain acyl-CoA formation and chronically elevated fatty acids increase total cellular CoA. Understanding the response of pancreatic beta cells to increased amounts of fuel and the role that altered insulin secretion plays in the development and maintenance of obesity and Type 2 diabetes is important. Data indicate that the activated form of fatty acids acts as an effector molecule in stimulus-secretion coupling. Glucose increases cytosolic long-chain acyl-CoA because it increases the "switch" compound malonyl-CoA that blocks mitochondrial beta-oxidation, thus implementing a shift from fatty acid to glucose oxidation. We present arguments in support of the following: (i) A source of fatty acid either exogenous or endogenous (derived by lipolysis of triglyceride) is necessary to support normal insulin secretion; (ii) a rapid increase of fatty acids potentiates glucose-stimulated secretion by increasing fatty acyl-CoA or complex lipid concentrations that act distally by modulating key enzymes such as protein kinase C or the exocytotic machinery; (iii) a chronic increase of fatty acids enhances basal secretion by the same mechanism, but promotes obesity and a diminished response to stimulatory glucose; (iv) agents which raise cAMP act as incretins, at least in part, by stimulating lipolysis via beta-cell hormone-sensitive lipase activation. Furthermore, increased triglyceride stores can give higher rates of lipolysis and thus influence both basal and stimulated insulin secretion. These points highlight the important roles of NEFA, LC-CoA, and their esterified derivatives in affecting insulin secretion in both normal and pathological states.
Assuntos
Ácidos Graxos/metabolismo , Insulina/metabolismo , Ilhotas Pancreáticas/metabolismo , Animais , Humanos , Secreção de InsulinaRESUMO
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.
Assuntos
Cálcio/metabolismo , Glucose/farmacologia , Insulina/metabolismo , Ilhotas Pancreáticas/efeitos dos fármacos , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Cricetinae , Ilhotas Pancreáticas/metabolismo , Células Tumorais CultivadasRESUMO
In phagocytic cells, fMet-Leu-Phe triggers phosphoinositide remodeling, activation of protein kinase C (PKC), release of intracellular Ca(2+) and uptake of extracellular Ca(2+). Uptake of extracellular Ca(2+) can be triggered by store-operated Ca(2+) channels (SOCC) and via a receptor-operated nonselective cation channel(s). In neutrophilic HL60 cells, the PKC activator phorbol myristate acetate (PMA) activates multiple PKC isotypes, PKC-alpha, PKC-beta, and PKC-delta, and inhibits ligand-initiated mobilization of intracellular Ca(2+) and uptake of extracellular Ca(2+). Therefore PKC is a negative regulator at several points in Ca(2+) mobilization. In contrast, selective depletion of PKC-beta in HL60 cells by an antisense strategy enhanced fMet-Leu-Phe-initiated Ca(2+) uptake but not mobilization of intracellular Ca(2+). Thapsigargin-induced Ca(2+) uptake through SOCC was not affected by PKC-beta II depletion. Thus PKC-beta II is a selective negative regulator of Ca(2+) uptake but not release of intracellular Ca(2+) stores. PKC-beta II inhibits a receptor-operated cation or Ca(2+) channel, thus inhibiting ligand-initiated Ca(2+) uptake.
Assuntos
Cálcio/metabolismo , Isoenzimas/fisiologia , Proteína Quinase C/fisiologia , Transporte Biológico/fisiologia , Canais de Cálcio/efeitos dos fármacos , Canais de Cálcio/metabolismo , Diferenciação Celular , Citosol/metabolismo , Células HL-60/patologia , Humanos , Inositol 1,4,5-Trifosfato/biossíntese , Isoenzimas/antagonistas & inibidores , Isoenzimas/genética , Ligantes , Oligonucleotídeos Antissenso/farmacologia , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C/genética , Proteína Quinase C beta , Tapsigargina/farmacologiaRESUMO
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.
Assuntos
Glucoquinase/biossíntese , Glucose/toxicidade , Insulina/biossíntese , Proteínas de Transporte de Monossacarídeos/biossíntese , Hipófise/metabolismo , Trifosfato de Adenosina/metabolismo , Adenoviridae/genética , Animais , Apoptose , Northern Blotting , Western Blotting , Morte Celular , Células Cultivadas , Relação Dose-Resposta a Droga , Glucose/metabolismo , Transportador de Glucose Tipo 2 , Marcação In Situ das Extremidades Cortadas , Camundongos , Camundongos Endogâmicos NOD , Camundongos Transgênicos , Microscopia de Fluorescência , Fosforilação , Isoformas de Proteínas , Distribuição TecidualRESUMO
Regional differences in free fatty acid (FFA) handling contribute to diseases associated with particular fat distributions. As cultured rat preadipocytes became differentiated, FFA transfer into preadipocytes increased and was more rapid in single perirenal than in epididymal cells matched for lipid content. Uptake by human omental preadipocytes was greater than uptake by abdominal subcutaneous preadipocytes. Adipose-specific fatty acid binding protein (aP2) and keratinocyte lipid binding protein abundance was higher in differentiated rat perirenal than in epididymal preadipocytes. This interdepot difference in preadipocyte aP2 expression was reflected in fat tissue in older animals. Carnitine palmitoyltransferase 1 activity increased during differentiation and was higher in perirenal than in epididymal preadipocytes, particularly the muscle isoform. Long-chain acyl-CoA levels were higher in perirenal than in epididymal preadipocytes and isolated fat cells. These data are consistent with interdepot differences in fatty acid flux ensuing from differences in fatty acid binding proteins and enzymes of fat metabolism. Heterogeneity among depots results, in part, from distinct intrinsic characteristics of adipose cells. Different depots are effectively separate miniorgans.
Assuntos
Adipócitos/metabolismo , Ácidos Graxos não Esterificados/metabolismo , Proteínas de Neoplasias , Proteínas do Tecido Nervoso , Células-Tronco/metabolismo , Proteínas Supressoras de Tumor , Acil Coenzima A/metabolismo , Adulto , Animais , Carnitina O-Palmitoiltransferase/metabolismo , Proteínas de Transporte/metabolismo , Células Cultivadas , Epididimo , Proteína 7 de Ligação a Ácidos Graxos , Proteínas de Ligação a Ácido Graxo , Feminino , Humanos , Rim , Masculino , Pessoa de Meia-Idade , Omento/citologia , Ratos , Ratos Endogâmicos F344 , Especificidade por SubstratoRESUMO
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.
Assuntos
Glucagon/farmacologia , Ilhotas Pancreáticas/efeitos dos fármacos , Ilhotas Pancreáticas/metabolismo , Lipólise/efeitos dos fármacos , Fragmentos de Peptídeos/farmacologia , Precursores de Proteínas/farmacologia , Cálcio/metabolismo , Linhagem Celular , Inibidores Enzimáticos/farmacologia , Peptídeo 1 Semelhante ao Glucagon , Peptídeos Semelhantes ao Glucagon , Concentração de Íons de Hidrogênio , Membranas Intracelulares/metabolismo , Lactonas/farmacologia , Lipase/antagonistas & inibidores , Orlistate , Fragmentos de Peptídeos/antagonistas & inibidores , Esterol Esterase/metabolismoRESUMO
Administration of dehydroepiandrosterone (DHEA), or its sulfated form (DHEAS), controls hyperglycemia in diabetic rodents without directly altering insulin sensitivity. We show that DHEAS enhanced glucose-stimulated insulin secretion when administered in vivo to rats or in vitro to beta-cell lines, without changing cellular insulin content. Insulin secretion increased from 3 days of steroid exposure in vitro, suggesting that DHEAS did not directly activate the secretory processes. DHEAS selectively increased the beta-cell mRNA expression of acyl CoA synthetase-2 and peroxisomal acyl CoA oxidase in a time-dependent manner. Although DHEAS is a peroxisomal proliferator, it did not alter the mRNA expression of peroxisomal proliferator-activated receptor (PPAR) alpha or beta, or enhance the activity of transfected PPAR alpha, beta, or gamma in vitro. Thus, DHEAS directly affected the beta-cell to enhance glucose-stimulated insulin secretion and increased the mRNA expression of specific beta-cell mitochondrial and peroxisomal lipid metabolic enzymes. This effect of DHEAS on insulin secretion may contribute to the amelioration of hyperglycemia seen in various rodent models of diabetes.
Assuntos
Sulfato de Desidroepiandrosterona/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Glucose/fisiologia , Insulina/metabolismo , Ilhotas Pancreáticas/efeitos dos fármacos , Ilhotas Pancreáticas/fisiologia , Acil-CoA Oxidase , Animais , Linhagem Celular , Coenzima A Ligases/genética , Secreção de Insulina , Masculino , Proteínas Mitocondriais , Oxirredutases/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Receptores Citoplasmáticos e Nucleares/genética , Receptores Citoplasmáticos e Nucleares/metabolismo , Fatores de Tempo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , TransfecçãoRESUMO
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.
Assuntos
Cálcio/fisiologia , Glucose/farmacologia , Insulina/metabolismo , Ilhotas Pancreáticas/fisiologia , Consumo de Oxigênio , Animais , Carbonil Cianeto p-Trifluormetoxifenil Hidrazona/farmacologia , Células Clonais , Secreção de Insulina , Ilhotas Pancreáticas/efeitos dos fármacos , Cinética , Oscilometria , Consumo de Oxigênio/efeitos dos fármacos , Desacopladores/farmacologiaRESUMO
Glucose-induced insulin secretion is pulsatile. Glucose metabolism generates oscillations in the ATP/ADP ratio which lead to opening and closing of ATP-sensitive K(+)-channels producing subsequent oscillations in membrane potential, cytoplasmic calcium and insulin release. Metabolic signals derived from glucose can also stimulate insulin release independent of their effects on ATP-sensitive K(+)-channels. The ATP/ADP ratio may mediate both ATP-sensitive K(+)-channel-dependent and -independent pathways of secretion. Glucose metabolism also results in an increase in long-chain acyl-CoA, which is proposed to act as an effector molecule in the beta -cell. Long-chain acyl-CoA has a variety of effects in the beta -cell that may effect insulin secretion including opening ATP-sensitive K(+)-channels, activating endoplasmic reticulum Ca(2+)-ATPases and stimulating classical protein kinase C activity. In addition to stimulating insulin release, nutrients also effect gene expression, protein synthesis and beta -cell proliferation. Gene expression is effected by nutrient induction of a variety of immediate early response genes. Glucose stimulates proinsulin biosynthesis both at the translational and transcriptional level. beta -cell proliferation, as a result of insulin-like growth factor and growth hormone mitogenic pathways, is also glucose dependent. Thus, many beta -cell functions in addition to secretion are controlled by nutrient metabolism.
Assuntos
Ilhotas Pancreáticas/fisiologia , Acil Coenzima A/metabolismo , Trifosfato de Adenosina/biossíntese , Animais , Cálcio/metabolismo , Divisão Celular , Regulação da Expressão Gênica , Glucose/farmacologia , Humanos , Insulina/metabolismo , Secreção de Insulina , Ilhotas Pancreáticas/citologia , Ilhotas Pancreáticas/efeitos dos fármacos , Modelos Biológicos , Nucleotídeos/metabolismoRESUMO
Medium-chain triacylglycerols (MCT) are present in milk, coconut oil and other foods, and are used therapeutically in special diets for certain disorders of lipid and glucose utilization. Recently, it has become apparent that MCT are not only oxidized in the liver, but are also present in lymph and fat tissue, particularly after chronic treatment. To evaluate the influence of MCT on metabolism in fat cells, we compared incorporation of octanoate and oleate into cellular triacylglycerols of 3T3-L1 adipocytes as well as their effects on preadipocyte differentiation. We found that less octanoate than oleate was stored and that more octanoate than oleate was oxidized. Octanoate was esterified to a greater extent at the sn-1,3 position of glyceryl carbons than at the sn-2 position, whereas the opposite was true for oleate. Glycerol release from fat cells pre-treated with octanoate was also greater than from cells pre-treated with oleate, presumably related to the preferential release of octanoate from the sn-1,3 position. Octanoate was not incorporated into lipids in undifferentiated cells and did not induce differentiation in these cells, whereas oleate was readily stored and actually induced differentiation. Incorporation of octanoate into lipids increased as cells differentiated, but reached a maximum of about 10% of the total stored fatty acids. If these effects in vitro also occur in vivo, substitution of octanoate for oleate or other long-chain fatty acids could have the beneficial effect of diminishing fat-cell number and lipid content.
Assuntos
Adipócitos/metabolismo , Caprilatos/metabolismo , Ácidos Graxos não Esterificados/metabolismo , Ácido Oleico/metabolismo , Células 3T3 , Acetilcoenzima A/metabolismo , Adipócitos/citologia , Adipócitos/efeitos dos fármacos , Animais , Caprilatos/farmacologia , Isótopos de Carbono , Diferenciação Celular/efeitos dos fármacos , Esterificação , Glicerol/metabolismo , Humanos , Lipólise/fisiologia , Camundongos , Ácido Oleico/farmacologia , Oxirredução , Triglicerídeos/metabolismo , Células Tumorais CultivadasRESUMO
Pancreatic beta-cells contain protein kinase C (PKC) isoforms that may play a role in insulin secretion. Activity of PKC classes (cPKC, nPKC, aPKC) and their regulation by acyl-CoA derivatives was examined in extracts of clonal pancreatic beta-cells (HIT) by protein phosphorylation. PKC classes were distinguished based on their previously defined cofactor requirements. Down-regulation of PKC by phorbol esters was confirmed by Western blotting and resulted in the complete loss of cPKC activity, partial loss of nPKC activity and preservation of aPKC activity and glucose-stimulated insulin secretion. aPKC activity was potentiated 4- to 8-fold by the CoA esters of myristate, palmitate, and oleate with a half-maximal value of 3 microM. Both oleoyl- and myristol-CoA, but not palmitoyl-CoA, caused inhibition of nPKC activity. Oleoyl-CoA inhibited nPKC activity up to 75% with a half-maximal effect at 10 microM. This value was independent of the concentration of diacylglycerol used. The addition of exogenous oleate or palmitate potentiated glucose-stimulated insulin secretion 2-fold and was unaffected by PMA-induced down-regulation. Stimulation by glucose or glucose and oleate also increased the mass of PKC-zeta found in the particulate fraction. These data are consistent with increased cytosolic long-chain acylCoA-activating aPKC isoforms resulting in stimulation and/or potentiation of glucose-induced insulin secretion.
Assuntos
Acil Coenzima A/farmacologia , Ácidos Graxos/farmacologia , Glucose/farmacologia , Insulina/metabolismo , Ilhotas Pancreáticas/metabolismo , Proteína Quinase C/metabolismo , Western Blotting , Linhagem Celular , Células Clonais , Sinergismo Farmacológico , Inibidores Enzimáticos/farmacologia , Secreção de Insulina , Ilhotas Pancreáticas/efeitos dos fármacos , Ácido Mirístico/farmacologia , Ácido Oleico/farmacologia , Ácido Palmítico/farmacologia , Palmitoil Coenzima A/farmacologia , Proteína Quinase C/antagonistas & inibidoresRESUMO
Synaptotagmin is involved in Ca2+-regulated secretion and has been suggested to serve as a general Ca2+ sensor on the membrane of secretory vesicles in neuronal cells. Insulin exocytosis from the pancreatic beta-cell is an example of a Ca2+-dependent secretory process. Previous studies of pancreatic beta-cells were unable to show presence of synaptotagmin I. We now present biochemical and immunohistochemical data showing that synaptotagmin III is present in pancreatic beta-cells as well as in the insulin-secreting cell line HIT-T15 and in rat insulinoma. By subcellular fractionation, we found synaptotagmin III in high-density fractions together with insulin and secretogranin I, indicating colocalization of synaptotagmin III and insulin in secretory granules. We could also show that blockade of synaptotagmin III by a specific antibody inhibited Ca2+-induced changes in beta-cell membrane capacitance, suggesting that synaptotagmin III is part of the functional protein complex regulating beta-cell exocytosis. The synaptotagmin III antibody did not affect the activity of the voltage-gated L-type Ca2+-channel. These findings are compatible with the view that synaptotagmin III, because of its distinct localization in the pancreatic beta-cell, functionally modulates insulin exocytosis. This indicates that synaptotagmin may have a general role in the regulation of exocytosis not only in neuronal cells but also in endocrine cells.
Assuntos
Proteínas de Ligação ao Cálcio , Exocitose/fisiologia , Ilhotas Pancreáticas/metabolismo , Glicoproteínas de Membrana/fisiologia , Proteínas do Tecido Nervoso/fisiologia , Animais , Anticorpos/farmacologia , Canais de Cálcio Tipo L/metabolismo , Linhagem Celular , Membrana Celular/efeitos dos fármacos , Membrana Celular/fisiologia , Grânulos Citoplasmáticos/metabolismo , Condutividade Elétrica , Imuno-Histoquímica , Insulina/metabolismo , Secreção de Insulina , Insulinoma/metabolismo , Ilhotas Pancreáticas/efeitos dos fármacos , Ilhotas Pancreáticas/fisiologia , Masculino , Glicoproteínas de Membrana/imunologia , Glicoproteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/imunologia , Proteínas do Tecido Nervoso/metabolismo , Neoplasias Pancreáticas/metabolismo , Ratos , Ratos Sprague-Dawley , Frações Subcelulares/metabolismo , Sinaptotagmina I , SinaptotagminasRESUMO
Non-insulin-dependent diabetes mellitus is associated with, in addition to impaired insulin release, elevated levels of free fatty acids (FFA) in the blood. Insulin release is stimulated when beta-cells are acutely exposed to FFA, whereas chronic exposure may inhibit glucose-induced insulin secretion. In the present study we investigated the direct effects of long chain acyl-CoA (LC-CoA), the active intracellular form of FFA, on insulin exocytosis. Palmitoyl-CoA stimulated both insulin release from streptolysin-O-permeabilized HIT cells and fusion of secretory granules to the plasma membrane of mouse pancreatic beta-cells, as measured by cell capacitance. The LC-CoA effect was chain length-dependent, requiring chain lengths of at least 14 carbons. LC-CoA needed to be present to stimulate insulin release, and consequently there was no effect following its removal. The stimulatory effect was observed after inhibition of protein kinase activity and in the absence of ATP, even though both kinases and ATP, themselves, modulate exocytosis. The effect of LC-CoA was inhibited by cerulenin, which has been shown to block protein acylation. The data suggest that altered LC-CoA levels, resulting from FFA or glucose metabolism, may act directly on the exocytotic machinery to stimulate insulin release by a mechanism involving LC-CoA protein binding.
Assuntos
Exocitose/efeitos dos fármacos , Insulina/metabolismo , Ilhotas Pancreáticas/efeitos dos fármacos , Palmitoil Coenzima A/farmacologia , Animais , Linhagem Celular , Secreção de Insulina , Ilhotas Pancreáticas/metabolismo , Cinética , CamundongosRESUMO
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.
Assuntos
Acil Coenzima A/fisiologia , Malonil Coenzima A/fisiologia , Transdução de Sinais/fisiologia , Citosol/metabolismo , Ácidos Graxos não Esterificados/metabolismo , Glucose/metabolismo , Humanos , Insulina/metabolismo , Secreção de Insulina , Mitocôndrias/metabolismo , Pâncreas/metabolismo , Pâncreas/fisiologia , Canais de Potássio/metabolismoRESUMO
Oleate is one of the most abundant dietary fatty acids, and much remains to be learned about its metabolism in fat cells. We studied the incorporation of exogenous [1-13C]oleate into triglycerides (TG) in differentiating 3T3L1 preadipocytes using 13C NMR spectroscopy. The quantity of oleate incorporated into TG was found to increase as preadipocytes differentiated into fat cells. The ratio of unesterified [1-13C]oleate to total stored fatty acids was higher in less differentiated cells, and declined at later stages of differentiation as cells accumulated fatty acids through de novo synthesis. When added as the only exogenous fatty acid, oleate was largely esterified at the sn-2 position. When equimolar unlabeled linoleate was co-provided at the same time, the ratio of [1-13C]oleate esterified at the sn-1,3 position increased, implying competition between linoleate and oleate for esterification, especially at the sn-2 position. When cells pre-enriched with [1-13C]oleate (esterified to TG) were treated with isoproterenol, a lipolytic agent, most of the [1-13C]oleate was still found in TG, despite a high rate of lipolysis determined by measuring glycerol release. This implies extensive re-esterification of the oleate released by lipolysis.
Assuntos
Células 3T3/metabolismo , Divisão Celular/efeitos dos fármacos , Ácido Oleico/farmacocinética , Triglicerídeos/metabolismo , Animais , Isótopos de Carbono , Divisão Celular/fisiologia , Ácidos Linoleicos/farmacologia , Lipídeos/química , Lipólise , Espectroscopia de Ressonância Magnética , Camundongos , Fatores de TempoRESUMO
A comprehensive metabolic study was carried out to understand how chronic exposure of pancreatic beta-cells to fatty acids causes high basal secretion and impairs glucose-induced insulin release. INS-1 beta-cells were exposed to 0.4 mM oleate for 3 days and subsequently incubated at 5 or 25 mM glucose, after which various parameters were measured. Chronic oleate promoted triglyceride deposition, increased fatty acid oxidation and esterification, and reduced malonyl-CoA at low glucose in association with elevated basal O(2) consumption and redox state. Oleate caused a modest (25%) reduction in glucose oxidation but did not affect glucose usage, the glucose 6-phosphate and citrate contents, and the activity of pyruvate dehydrogenase of INS-1 cells. Thus changes in glucose metabolism and a Randle-glucose/fatty acid cycle do not explain the altered secretory properties of beta-cells exposed to fatty acids. The main response of INS-1 cells to chronic oleate, which is to increase the oxidation and esterification of fatty acids, may contribute to cause high basal insulin secretion via increased production of reducing equivalents and/or the generation of complex lipid messenger molecule(s).
Assuntos
Glucose/metabolismo , Insulina/metabolismo , Ilhotas Pancreáticas/efeitos dos fármacos , Ilhotas Pancreáticas/metabolismo , Metabolismo dos Lipídeos , Ácido Oleico/farmacologia , Linhagem Celular , Metabolismo Energético/efeitos dos fármacos , Secreção de Insulina , Oxirredução/efeitos dos fármacos , Fatores de TempoRESUMO
The ATP-dependent potassium (KATP) channel in the pancreatic beta-cell is a complex of two proteins, the pore-forming Kir6.2 and the sulfonylurea receptor type 1 (SUR1). Both subunits are required for functional KATP channels because expression of Kir6.2 alone does not result in measurable currents. However, truncation of the last 26 or 36 amino acids of the C terminus of Kir6.2 enables functional expression of the pore-forming protein in the absence of SUR1. Thus, by using the truncated form of Kir6.2, expressed in the absence and presence of SUR1, it has been shown that the site at which ATP mediates channel inhibition is likely to be situated on Kir6.2. We have now examined the effects of long chain acyl-CoA (LC-CoA) esters on the C-terminally truncated mouse Kir6.2Delta365-390 (Kir6. 2DeltaC26) in inside-out patches isolated from Xenopus laevis oocytes. LC-CoA esters, saturated (C14:0, C16:0) and unsaturated (C18:1), increased Kir6.2DeltaC26 currents, whereas short and medium chain CoA esters (C3:0, C8:0, C12:0) were unable to affect channel activity. The LC-CoA esters were also able to counteract the blocking effect of ATP on Kir6.2DeltaC26. The stimulatory effect of the esters could be explained by the induction of a prolonged open state of Kir6.2DeltaC26. In the presence of the esters, channel open time was increased approximately 3-fold, which is identical to what was obtained in the native mouse KATP channel. Coexpression of SUR1 together with Kir6.2DeltaC26 did not further increase the ability of LC-CoA esters to stimulate channel activity. We conclude that Kir6.2 is the primary target for LC-CoA esters to activate the KATP channel and that the esters are likely to induce a conformational change by a direct interference with the pore-forming subunit, leading to openings of long duration.