RESUMO
OBJECTIVE: This study tested whether galcanezumab, a humanized monoclonal antibody with efficacy against migraine, was superior to placebo for the treatment of mild or moderate osteoarthritis (OA) knee pain. METHOD: In a multicenter, double-blind, placebo- and celecoxib-controlled trial, patients with moderate to severe OA pain were randomized to placebo; celecoxib 200 mg daily for 16 weeks; or galcanezumab 5, 50, 120, and 300 mg subcutaneously every 4 weeks, twice. The primary outcome was change from baseline at Week 8 in Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) pain subscore measured by 100 mm visual analog scale (VAS). The trial was considered positive if ≥1 dose of galcanezumab demonstrated ≥95% Bayesian posterior probability of superiority to placebo and ≥50% posterior probability of superiority to placebo by ≥9 mm. A planned interim analysis allowed termination of the study if posterior probability of superiority to placebo by ≥9 mm was ≤5%. Secondary endpoints included WOMAC function subscore and Patient Global Assessment (PGA) of OA. Safety and tolerability were also assessed. RESULTS: The study was terminated after interim analysis suggested inadequate efficacy. Celecoxib significantly reduced WOMAC pain subscore compared with placebo [-12.0 mm; 95% confidence interval (CI) -23 to -2 mm]. None of the galcanezumab arms demonstrated clinically meaningful improvement (range: 1.5 to -5.0 mm) or met the prespecified success criteria. No improvement in any secondary objective was observed. Galcanezumab was well tolerated by OA patients. CONCLUSIONS: This study failed to demonstrate sufficient statistical evidence that galcanezumab was efficacious for treating OA knee pain. STUDY IDENTIFICATION: NCT02192190.
Assuntos
Anticorpos Monoclonais Humanizados/administração & dosagem , Peptídeo Relacionado com Gene de Calcitonina/antagonistas & inibidores , Osteoartrite do Joelho/tratamento farmacológico , Adulto , Idoso , Anticorpos Monoclonais Humanizados/farmacologia , Anticorpos Monoclonais Humanizados/uso terapêutico , Celecoxib/uso terapêutico , Relação Dose-Resposta a Droga , Método Duplo-Cego , Esquema de Medicação , Feminino , Humanos , Injeções Subcutâneas , Masculino , Pessoa de Meia-Idade , Osteoartrite do Joelho/complicações , Dor/tratamento farmacológico , Dor/etiologia , Manejo da Dor/métodos , Medição da Dor/métodos , Índice de Gravidade de Doença , Resultado do TratamentoRESUMO
AIMS: To compare the immunogenicity profiles and the potential effects on clinical outcomes of LY2963016 insulin glargine (LY IGlar) and Lantus® insulin glargine (IGlar), products with identical primary amino acid sequences, in patients with type 1 or type 2 diabetes mellitus (T1DM or T2DM). METHODS: To assess immunogenicity, anti-insulin glargine antibodies (measured as percent binding) were compared between treatments in 52-week (open-label) and 24-week (double-blind) randomized studies in total study populations of patients with T1DM (N = 535) and T2DM (N = 756), respectively, and two subgroups of patients with T2DM: insulin-naïve patients and those reporting prestudy IGlar treatment (prior IGlar). Relationships between insulin antibody levels and clinical outcomes were assessed using analysis of covariance and partial correlations. Insulin antibody levels were assessed using Wilcoxon rank sum. Treatment comparisons for treatment-emergent antibody response (TEAR) and incidence of detectable antibodies were analysed using Fisher's exact test. RESULTS: No significant treatment differences were observed for insulin antibody levels, incidence of detectable anti-insulin glargine antibodies, or incidence of TEAR [overall and endpoint, by last-observation-carried-forward (LOCF)] in patients with T1DM or patients with T2DM, including the insulin-naïve subgroup. A statistically significant difference was noted in the overall incidence of detectable antibodies but not at endpoint (LOCF) nor in TEAR for the prior IGlar subgroup of patients with T2DM. Insulin antibody levels were low (<5%) in both treatment groups. Insulin antibody levels or developing TEAR was not associated with clinical outcomes. CONCLUSIONS: LY IGlar and IGlar have similar immunogenicity profiles; anti-insulin glargine antibody levels were low for both treatments, with no observed effect on efficacy and safety outcomes.
Assuntos
Diabetes Mellitus Tipo 1/tratamento farmacológico , Diabetes Mellitus Tipo 2/tratamento farmacológico , Hipersensibilidade a Drogas/etiologia , Hipoglicemiantes/efeitos adversos , Anticorpos Anti-Insulina/análise , Insulina Glargina/análogos & derivados , Insulina Glargina/efeitos adversos , Doenças Assintomáticas/epidemiologia , Medicamentos Biossimilares/efeitos adversos , Medicamentos Biossimilares/uso terapêutico , Reações Cruzadas , Diabetes Mellitus Tipo 1/sangue , Diabetes Mellitus Tipo 1/complicações , Diabetes Mellitus Tipo 1/imunologia , Diabetes Mellitus Tipo 2/sangue , Diabetes Mellitus Tipo 2/complicações , Diabetes Mellitus Tipo 2/imunologia , Método Duplo-Cego , Hipersensibilidade a Drogas/complicações , Hipersensibilidade a Drogas/epidemiologia , Hipersensibilidade a Drogas/imunologia , Humanos , Hiperglicemia/prevenção & controle , Hipoglicemia/induzido quimicamente , Hipoglicemia/prevenção & controle , Hipoglicemiantes/uso terapêutico , Fenômenos Imunogenéticos/efeitos dos fármacos , Incidência , Insulina Glargina/uso terapêutico , Insulina Regular Humana/efeitos adversos , Insulina Regular Humana/análogos & derivados , Insulina Regular Humana/genética , Insulina Regular Humana/uso terapêutico , Proteínas Recombinantes/efeitos adversos , Proteínas Recombinantes/uso terapêuticoRESUMO
AIM: To describe the clinical effects of single and multiple doses of a potent, selective, orally administered, small-molecule antagonist of the human glucagon receptor, LY2409021, in healthy subjects and in patients with type 2 diabetes. METHODS: LY2409021 was administered in dose-escalation studies to healthy subjects (n = 23) and patients with type 2 diabetes (n = 9) as single doses (Study 1) and daily to patients with type 2 diabetes (n = 47) for 28 days (Study 2). Safety, tolerability, pharmacokinetic (PK) and pharmacodynamic (PD) assessments were made after single doses and in patients receiving once-daily doses of LY2409021 (5, 30, 60 or 90 mg) for 28 days. RESULTS: LY2409021 was well tolerated at all dose levels in both studies. Fasting and postprandial glucose were reduced and glucagon levels increased after single and multiple dosing, with reductions in fasting serum glucose of up to â¼1.25 mmol/l on day 28. Serum aminotransferases increased in a dose-dependent manner with multiple dosing and reversed after cessation of dosing. Significant glucose-lowering was observed with LY2409021 at dose levels associated with only minor aminotransferase increases. CONCLUSION: Blockade of glucagon signalling in patients with type 2 diabetes is well tolerated and results in substantial reduction of fasting and postprandial glucose with minimal hypoglycaemia, but with reversible increases in aminotransferases. Inhibition of glucagon signalling by LY2409021 is a promising potential treatment for patients with type 2 diabetes and should be evaluated in longer clinical trials to better evaluate benefits and risks.
Assuntos
Compostos de Bifenilo/uso terapêutico , Diabetes Mellitus Tipo 2/tratamento farmacológico , Hiperglicemia/prevenção & controle , Hipoglicemia/prevenção & controle , Hipoglicemiantes/uso terapêutico , Terapia de Alvo Molecular , Receptores de Glucagon/antagonistas & inibidores , Adulto , Idoso , Compostos de Bifenilo/administração & dosagem , Compostos de Bifenilo/sangue , Compostos de Bifenilo/farmacocinética , Estudos de Coortes , Estudos Cross-Over , Diabetes Mellitus Tipo 2/sangue , Diabetes Mellitus Tipo 2/metabolismo , Relação Dose-Resposta a Droga , Método Duplo-Cego , Feminino , Glucagon/agonistas , Glucagon/sangue , Glucagon/metabolismo , Hemoglobinas Glicadas/análise , Meia-Vida , Humanos , Hipoglicemia/induzido quimicamente , Hipoglicemia/epidemiologia , Hipoglicemiantes/administração & dosagem , Hipoglicemiantes/sangue , Hipoglicemiantes/farmacocinética , Masculino , Pessoa de Meia-Idade , Terapia de Alvo Molecular/efeitos adversos , Risco , Método Simples-CegoRESUMO
BACKGROUND: Glycosylphosphatidylinositol-specific phospholipase D (GPI-PLD) may play an important role in inflammation, because it can hydrolyze the GPI anchors of several inflammatory membrane proteins (eg, CD106, CD55, and CD59) and its hydrolytic products upregulate macrophage cytokine expression (eg, interleukin-1 and tumor necrosis factor-alpha). Because of its potential regulatory role in inflammatory reactions, we hypothesized that GPI-PLD might be expressed in atherosclerosis. METHODS AND RESULTS: Immunohistochemistry using human GPI-PLD-specific rabbit polyclonal antiserum was performed on a total of 83 nonatherosclerotic and atherosclerotic human coronary arteries from 23 patients. Macrophages, smooth muscle cells, apoA-I, and oxidation epitopes also were identified immunohistochemically. Cell-associated GPI-PLD was detected in 95% of atherosclerotic segments, primarily on a subset of macrophages. Extracellular GPI-PLD was present in only 30% of atherosclerotic segments and localized to regions with extracellular apoA-I. In contrast, GPI-PLD was not detected in nonatherosclerotic segments. Expression of GPI-PLD mRNA by human macrophages was confirmed in vitro by reverse transcription/polymerase chain reaction. Further studies demonstrated that GPI-PLD-positive plaque macrophages contained oxidation epitopes, suggesting a link between oxidant stress and GPI-PLD expression. This possibility was supported by studies in which exposure of a macrophage cell line to H2O2 led to a 50+/-3% increase in steady-state GPI-PLD mRNA levels. CONCLUSIONS: Collectively, these results suggest that oxidative processes may regulate GPI-PLD expression and suggest a role for GPI-PLD in inflammation and in the pathogenesis of atherosclerosis.
Assuntos
Arteriosclerose/enzimologia , Glicosilfosfatidilinositóis/metabolismo , Macrófagos/enzimologia , Fosfolipase D/metabolismo , Adulto , Artérias/enzimologia , Linhagem Celular , Vasos Coronários/enzimologia , Epitopos , Homeostase , Humanos , Peróxido de Hidrogênio/farmacologia , Pessoa de Meia-Idade , Monócitos/citologia , Oxirredução , Fosfolipase D/genética , RNA Mensageiro/metabolismo , Valores de Referência , Especificidade por Substrato , Distribuição Tecidual/fisiologiaRESUMO
In this study we examine the hypothesis that an inositol glycan phosphate can act similarly to insulin on intact cells. The inositol glycan phosphate used in this study (glycan alpha) was isolated previously from the glycoinositol phospholipid anchor of human erythrocyte acetylcholinesterase and was shown to have the structure glycine-ethanolamine-PO4-Man-Man-(N,N-dimethylethanolamine-PO4)Man- (N,N-dimethyl)GlcN-inositol-PO4. The cellular response investigated was the glucagon-stimulated activation of glycogen phosphorylase in rat hepatocytes. When hepatocytes were incubated with 20 nM glucagon for 4 min, the ratio of phosphorylase a activity to total phosphorylase increased from a basal value of 0.49 +/- 0.02 to 0.82 +/- 0.03 (mean +/- SE, n = 15). Inclusion of either 100 nM insulin or 3-10 microM glycan alpha during the glucagon incubation significantly decreased the glucagon-stimulated activity ratio to 0.74 +/- 0.03 for either agent. Furthermore, hepatocyte preparations differed in their response to insulin and were divided into insulin-responsive and -resistant groups. Glycan alpha had a significant effect only in the insulin-responsive group for which the observed activity ratio for 10 microM glycan alpha plus glucagon (0.68 +/- 0.05) compared closely with that for insulin plus glucagon (0.70 +/- 0.04). For the insulin-resistant group, the activity ratio in the presence of 10 microM glycan alpha was 0.81 +/- 0.03, unchanged from the control with glucagon alone. Because glycan alpha contains an inositol phosphate group, the effect of inositol cyclic 1,2-phosphate on the glucagon-stimulated activity ratio was determined.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Acetilcolinesterase/química , Eritrócitos/enzimologia , Glicosilfosfatidilinositóis/química , Fosfatos de Inositol/fisiologia , Fosforilases/metabolismo , Animais , Ativação Enzimática/efeitos dos fármacos , Humanos , Fosfatos de Inositol/isolamento & purificação , Fígado/citologia , Masculino , Ratos , Ratos Sprague-DawleyRESUMO
Glycosylphosphatidylinositol-specific phospholipase D (GPI-PLD) is abundant in mammalian serum, but the source of the circulating enzyme is unknown. Pancreatic islets have been reported to contain and secrete GPI-PLD. In this report we examined the regulation of GPI-PLD secretion from beta TC3 cells, a mouse insulinoma cell line. In the absence of glucose, phorbol myristic acid (0.1 microM) stimulated insulin secretion by 2.5-fold and GPI-PLD secretion by 2-fold. Carbachol (5 microM), glucagon-like peptide I-(7-36) amide (0.1 microM), and isobutylmethylxanthine (0.1 mM) had no significant effect on insulin or GPI-PLD secretion in the absence of glucose. Glucose (16.7 mM) stimulated both GPI-PLD and insulin secretion from beta TC3 cells by 55% and 235%, respectively. In addition, glucose potentiated the secretagogue effect of isobutylmethylxanthine, phorbol myristic acid, and glucagon-like peptide I on both insulin and GPI-PLD secretion. By immunohistochemistry and confocal microscopy, beta TC3 cells contain both insulin and GPI-PLD, which generally colocalized intracellularly. However, GPI-PLD secretion differed from insulin secretion by a higher rate of basal release (2.8% vs. 0.23%/h), a lower magnitude of response to secretagogues, and a more prolonged period of increased secretion. These results demonstrate that beta TC3 cells secrete GPI-PLD in response to insulin secretagogues and suggest that GPI-PLD may be secreted via the regulated pathway in these cells.
Assuntos
Insulinoma/enzimologia , Ilhotas Pancreáticas/enzimologia , Neoplasias Pancreáticas/enzimologia , Fosfolipase D/metabolismo , 1-Metil-3-Isobutilxantina/farmacologia , Animais , Carbacol/farmacologia , Cicloeximida/farmacologia , Glucagon , Peptídeo 1 Semelhante ao Glucagon , Peptídeos Semelhantes ao Glucagon , Glucose/farmacologia , Imuno-Histoquímica , Insulina/análise , Insulina/metabolismo , Secreção de Insulina , Ilhotas Pancreáticas/efeitos dos fármacos , Cinética , Camundongos , Fragmentos de Peptídeos/farmacologia , Fosfolipase D/análise , Acetato de Tetradecanoilforbol/farmacologia , Células Tumorais CultivadasRESUMO
Cleavage by the GPI-PLD provides definitive evidence of a minimal GPI structure: glucosamine-phosphatidylinositol. Unlike the case for PI-PLC, cleavage by the GPI-PLD is unaffected by acylation of the inositol ring. Thus the GPI-PLD provides an excellent simple enzymatic tool for analyzing the basic core structure of GPI anchors.
Assuntos
Glicosilfosfatidilinositóis/química , Fosfolipase D/sangue , Fosfolipase D/isolamento & purificação , Acilação , Animais , Configuração de Carboidratos , Sequência de Carboidratos , Cátions Bivalentes/farmacologia , Bovinos , Cromatografia de Afinidade/métodos , Cromatografia em Gel/métodos , Cromatografia por Troca Iônica/métodos , Clonagem Molecular/métodos , DNA Complementar , Detergentes/farmacologia , Eletroforese em Gel de Poliacrilamida/métodos , Escherichia coli , Expressão Gênica , Glucosamina/análise , Humanos , Cinética , Mamíferos , Conformação Molecular , Dados de Sequência Molecular , Peso Molecular , Fosfolipase D/química , Fosforilação , Proteínas Recombinantes/química , Especificidade por Substrato , Ultracentrifugação/métodosRESUMO
Insulin resistance is associated with a compensatory islet hyperactivity to sustain adequate insulin biosynthesis and secretion to maintain near euglycemia. Both glucose and insulin are involved in regulating proteins required for insulin synthesis and secretion within the islet and islet hypertrophy. We have determined that glycosylphosphatidylinositol-specific phospholipase D (GPI-PLD) is present within the secretory granules of islet beta cells. To determine if GPI-PLD is regulated in islet beta cells, we examined the effect of glucose and insulin on GPI-PLD expression in rat islets and murine insulinoma cell lines. Glucose (16.7 mmol/L) increased cellular GPI-PLD activity and mRNA levels 2- to 7-fold in isolated rat islets and betaTC3 and betaTC6-F7 cells. Insulin (10(-7) mol/L) also increased GPI-PLD mRNA levels in rat islets and betaTC6-F7 cells 2- to 4-fold commensurate with an increase in GPI-PLD biosynthesis. To determine if islet GPI-PLD expression is increased in vivo under conditions of islet hyperactivity, we compared GPI-PLD mRNA levels in islets and liver from ob/ob mice and their lean littermates. Islet GPI-PLD mRNA was increased 5-fold while liver mRNA and serum GPI-PLD levels were reduced 30% in ob/ob mice compared with lean littermate controls. These results suggest that glucose and insulin regulate GPI-PLD mRNA levels in isolated islets and beta-cell lines. These regulators may also account for the increased expression of GPI-PLD mRNA in islets from ob/ob mice, a model of insulin resistance and islet hyperactivity.
Assuntos
Expressão Gênica/efeitos dos fármacos , Glucose/farmacologia , Insulina/farmacologia , Fosfolipase D/genética , Animais , Insulinoma , Ilhotas Pancreáticas/efeitos dos fármacos , Ilhotas Pancreáticas/enzimologia , Fígado/enzimologia , Masculino , Camundongos , Camundongos Obesos , Obesidade/enzimologia , Neoplasias Pancreáticas , RNA Mensageiro/análise , Ratos , Ratos Sprague-Dawley , Células Tumorais CultivadasRESUMO
Glycosylphosphatidylinositol-specific phospholipase D (GPI-PLD) has recently been shown to be associated with high-density lipoproteins (HDL) in bovine serum. To determine the distribution of GPI-PLD among lipoproteins and characterize the GPI-PLD-containing lipoproteins in human plasma, we used dextran sulfate and immunoaffinity chromatography to isolate apolipoprotein-specific lipoproteins. This procedure allowed fractionation of lipoprotein particles into those containing apolipoprotein B (Lp B), apolipoproteins AI and AII (Lp AI/AII), or apolipoprotein AI only (Lp AI). In five plasma samples with HDL cholesterol ranging from 40 to 129 mg/dl, 75 +/- 12% (mean +/- SD) of the GPI-PLD activity was associated with Lp AI, 11 +/- 13% with Lp AI/AII, while only 13 +/- 9% was present in plasma devoid of these lipoproteins, suggesting that most of the GPI-PLD in human plasma is associated with apolipoprotein AI. No GPI-PLD activity was detected in Lp B. Further characterization of the GPI-PLD-containing lipoproteins by gel filtration chromatography, nondenaturing poly-acrylamide and agarose gel electrophoresis revealed that GPI-PLD was restricted to an apolipoprotein AI-containing particle or complex that was small (apparent mean Mw of 140 kDa) and distinct from the bulk of HDL. Thus, the majority of plasma GPI-PLD appears to be specifically associated with a small, minor fraction of apolipoprotein AI.
Assuntos
Apolipoproteína A-II/análise , Apolipoproteína A-I/análise , Apolipoproteínas B/análise , Fosfolipase D/análise , Animais , Glicosilfosfatidilinositóis/metabolismo , Humanos , Lipoproteínas HDL/sangue , Especificidade por SubstratoAssuntos
Hiperlipidemias/tratamento farmacológico , Hiperlipidemias/enfermagem , Hipolipemiantes/uso terapêutico , Anticolesterolemiantes/uso terapêutico , Humanos , Hiperlipidemias/etiologia , Hiperlipidemias/prevenção & controle , Lipídeos/sangue , Avaliação em Enfermagem , Educação de Pacientes como Assunto , Medição de RiscoRESUMO
Seizures are common in acute exacerbations of hepatic porphyria, even though the etiology is not identified in most cases. We have reported a case of normeperidine-induced seizures in a patient with hereditary coproporphyria. Although meperidine is commonly used for pain control during acute attacks in these patients, this report suggests that meperidine is not a good analgesic choice in porphyria. Normeperidine-induced seizures in patients with porphyria may be treated by withdrawal of meperidine therapy and selective use of anticonvulsants.
Assuntos
Inibidores da Colinesterase/efeitos adversos , Coproporfirinas/metabolismo , Hepatopatias/genética , Meperidina/análogos & derivados , Porfirias/genética , Convulsões/induzido quimicamente , Adulto , Feminino , Humanos , Lactente , Hepatopatias/complicações , Hepatopatias/metabolismo , Meperidina/efeitos adversos , Porfirias/complicações , Porfirias/metabolismo , Convulsões/metabolismoRESUMO
A protective effect of habitual fish consumption on the development of impaired glucose tolerance and diabetes is once again suggested in a recent trial. In nondiabetic individuals with essential hypertension, a group known to be insulin resistant, fish oil was not associated with a negative impact on glycemic control, insulin secretion or peripheral insulin sensitivity, even in a subgroup who had impaired glucose tolerance. Furthermore, more recent, long term, placebo-controlled trials in type II patients have failed to demonstrate a significant impact of fish oil supplementation on glycemic control. Additional information is available regarding qualitative changes in VLDL- and LDL-lipoproteins in type II diabetes patients in response to dietary fish oil supplementation. The impact of fish oil on LDL oxidation is the focus of two recent trials. Vitamin E supplementation may mitigate much of the enhanced oxidation of LDL that is potentially seen with dietary fish oil supplementation.
Assuntos
Diabetes Mellitus Tipo 2/dietoterapia , Ácidos Graxos Ômega-3/uso terapêutico , Teste de Tolerância a Glucose , Hiperlipidemias/dietoterapia , Animais , Glicemia/análise , Diabetes Mellitus Tipo 2/sangue , Diabetes Mellitus Tipo 2/complicações , Dieta , Óleos de Peixe/administração & dosagem , Peixes , Hiperlipidemias/sangue , Hiperlipidemias/complicaçõesRESUMO
Limited information is known regarding the regulation, structural features, and functional domains of glycosylphosphatidylinositol-specific phospholipase D (GPI-PLD, EC 3. 1.4.50). Previous studies demonstrated that trypsin cleavage of GPI-PLD at or near Arg325 and/or Arg589 in bovine serum GPI-PLD was associated with an increase in enzymatic activity. Since the Arg325 is predicted to be in a region between the catalytic domain and predicted beta-propeller structure in the C-terminal portion of GPI-PLD (T. A. Springer, Proc. Natl. Acad. Sci. USA 94, 65-72, 1997), we hypothesized that this connecting region is important for catalytic activity. Trypsin cleavage of human serum GPI-PLD, which has an Arg325 but lacks the Arg589 present in bovine serum GPI-PLD, also increased GPI-PLD activity. Peptide-specific antibodies to residues 275-296 (anti-GPI-PLD(275)) and a monoclonal antibody, 191, with an epitope encompassing Arg325, also stimulated GPI-PLD activity. Pretreating human GPI-PLD with trypsin demonstrated that anti-GPI-PLD(275) only stimulated the activity of intact GPI-PLD. These results suggest that trypsin activation and anti-GPI-PPLD(275) may have similar effects on GPI-PLD. Consistent with this is the observation that both manipulations decreased the affinity of GPI-PLD for mixed micelle substrates. These results indicate that the midportion region of GPI-PLD is important in regulating enzymatic activity.
Assuntos
Fosfolipase D/imunologia , Fosfolipase D/metabolismo , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/farmacologia , Domínio Catalítico , Bovinos , Ativação Enzimática/efeitos dos fármacos , Epitopos/química , Humanos , Imunoquímica , Técnicas In Vitro , Cinética , Camundongos , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/imunologia , Fosfolipase D/química , Coelhos , Especificidade por Substrato , Tripsina/farmacologiaRESUMO
The identification of free glycoinositol phospholipids (GPIs) following biosynthetic labeling with [3H]glucosamine in cultured cells has been reported by several laboratories. We applied this procedure to two of the cell types used in these studies, H4IIE hepatoma cells and isolated hepatocytes, but were unable to detect a [3H]glucosamine-containing lipid that met any of the criteria for GPIs, including sensitivity to phosphatidylinositol-specific phospholipase C (PIPLC) or GPI-specific phospholipase D. Part of the difficulty in radiolabeling a GPI by this procedure was the rapid metabolic conversion of [3H]glucosamine to galactosamine and neutral or anionic derivatives. A PIPLC-sensitive radiolabeled lipid was detected only after 16 h of labeling. The water-soluble fragments released from this lipid by PIPLC corresponded largely to myo-inositol 1,2-cyclic phosphate and myo-inositol 1-phosphate, products expected from PIPLC cleavage of phosphatidylinositol or lyso-phosphatidylinositol. In an alternative approach that we introduce here, free GPIs in lipid extracts from rat liver plasma membranes were labeled by reductive radiomethylation. This procedure, which radiomethylates primary and secondary amines, has been shown to label a glucosamine residue adjacent to inositol in all GPIs characterized to date. The labeled extracts were fractionated by two-dimensional thin-layer chromatography, and a cluster of polar labeled lipids were assigned as GPIs based upon the following observations. 1) They were cleaved by PIPLC, 2) after hydrolysis in 6 N HCl, both radiomethylated glucosamine and a glucosamine-inositol conjugate were identified by cation exchange chromatography, and 3) hydrolysis in 4 M trifluoroacetic acid generated a fragment consistent with glucosamine-inositol-phosphate. These results illustrate new criteria for the identification of GPIs. The labeled GPIs also contained radiomethylated ethanolamine, another component found in GPI anchors of proteins and in mature lipid precursors of GPI anchors, suggesting that the liver plasma membrane GPIs retained considerable structural homology to GPI anchors.
Assuntos
Aminas/química , Glucosamina/química , Fígado/química , Animais , Membrana Celular/química , Células Cultivadas , Cromatografia em Camada Fina , Hidrólise , Cinética , Fígado/citologia , Neoplasias Hepáticas Experimentais/química , Masculino , Metilação , Oxirredução , Fosfatidilinositol Diacilglicerol-Liase , Fosfoinositídeo Fosfolipase C , Diester Fosfórico Hidrolases/metabolismo , Ratos , Ratos Endogâmicos , Células Tumorais CultivadasRESUMO
Glycosylphosphatidylinositol-specific phospholipase D (GPI-PLD) is abundant in serum and associates with high density lipoproteins (HDL). We have characterized the distribution of GPI-PLD among lipoproteins in human plasma. Apolipoprotein (apo)-specific lipoproteins containing apoB (Lp[B]), apoA-I and A-II (Lp[A-I, A-II]), or apoA-I only (Lp[A-I]) were isolated using dextran sulfate and immunoaffinity chromatography. In six human plasma samples with HDL cholesterol ranging from 39 to 129 mg/dl, 79 +/- 14% (mean +/- SD) of the total plasma GPI-PLD activity was associated with Lp[A-I], 9 +/- 12% with Lp[A-I, A-II], and 1 +/- 1% with Lp[B]; and 11 +/- 10% was present in plasma devoid of these lipoproteins. Further characterization of the GPI-PLD-containing lipoproteins by gel-filtration chromatography and nondenaturing polyacrylamide and agarose gel electrophoresis revealed that these apoA-I-containing particles/complexes were small (8 nm) and migrated with pre-beta particles on agarose electrophoresis. Immunoprecipitation of GPI-PLD with a monoclonal antibody to GPI-PLD co-precipitated apoA-I and apoA-IV but little or no apoA-II, apoC-II, apoC-III, apoD, or apoE. In vitro, apoA-I but not apoA-IV or bovine serum albumin interacted directly with GPI-PLD, but did not stimulate GPI-PLD-mediated cleavage of a cell surface GPI-anchored protein. Thus, the majority of plasma GPI-PLD appears to be specifically associated with a small, discrete, and minor fraction of lipoproteins containing apoA-I and apoA-IV. -- Deeg, M. A., E. L. Bierman, and M. C. Cheung. GPI-specific phospholipase D associates with an apoA-I- and apoA-IV-containing complex. J. Lipid Res. 2001. 42: 442--451.
Assuntos
Apolipoproteína A-I/sangue , Apolipoproteínas A/sangue , Fosfolipase D/sangue , Adulto , Anticorpos Monoclonais , Apolipoproteína A-I/farmacologia , HDL-Colesterol/sangue , Cromatografia em Gel , Eletroforese em Gel de Ágar , Eletroforese em Gel de Poliacrilamida , Feminino , Glicosilfosfatidilinositóis/metabolismo , Humanos , Técnicas de Imunoadsorção , Masculino , Pessoa de Meia-Idade , Tamanho da PartículaRESUMO
The dynamics and compartmental characteristics of cAMP metabolism were examined by 18O labeling of cellular adenine nucleotide alpha phosphoryls in rat parotid gland stimulated to secrete with beta-adrenergic and cholinergic agents. The secretory response occurred in association with a rapidly increased rate of cAMP hydrolysis apparently coordinated with an equivalent increase in the rate of cAMP synthesis, since the cellular concentration of cAMP remained unchanged. The magnitude of this metabolic response was equivalent to the metabolism of 10-75 times the cellular content of cAMP within the first minute of stimulation. This increased metabolic rate occurred only during the early (1-3 min) period of stimulation, in what appeared to be an exclusive cellular compartment distinguished by a unique distribution of 18O among adenine nucleotide alpha phosphoryls. This 18O distribution contrasted with that produced by forskolin, which increased cellular cAMP concentration and elicited only a delayed response missing the early secretory component. The early acceleration of cAMP metabolism appeared linked to a stimulus-induced increase in intracellular Ca2+ concentration, since the Ca2+ ionophore ionomycin produced the same metabolic response in association with secretion. These observations suggest that cAMP metabolism is involved in stimulus-secretion coupling by a Ca2+-linked mechanism different from that in which cAMP plays the role of a second messenger.
Assuntos
Agonistas Adrenérgicos beta/farmacologia , Cálcio/metabolismo , AMP Cíclico/biossíntese , Parassimpatomiméticos/farmacologia , Glândula Parótida/metabolismo , Amilases/metabolismo , Animais , Carbacol/farmacologia , Colforsina/farmacologia , Éteres/farmacologia , Ionomicina , Isoproterenol/farmacologia , Masculino , Octopamina/análogos & derivados , Octopamina/farmacologia , Potássio/metabolismo , Ratos , Ratos EndogâmicosRESUMO
Protein kinase C (PKC) seems to play an important role in many of HDL effects on cells, including removal of excess cholesterol. HDL removes cholesterol by at least two mechanisms. One mechanism involves desorption/diffusion of cholesterol from the plasma membrane onto the acceptor particle, whereas the second is mediated by apolipoproteins and may involve intracellular translocation of cholesterol to the plasma membrane for subsequent efflux. In this report, we examined the possibility that mitogen-activated protein (MAP) kinase is one of the downstream events from HDL activation of PKC. Using a gel kinase assay with myelin basic protein incorporated into the gel, HDL (50 micrograms protein/mL) stimulated multiple kinases of 42, 50, 52, 58, and 60 kDa. The 42-kDa protein kinase, corresponding to the unresolved MAP kinases ERK1 and ERK2 based on immunoblotting, was activated over 2-fold by HDL. HDL activated all identified kinases in a concentration- and time-dependent manner, which became maximal within 5 to 10 minutes and remained activated for at least 60 minutes. HDL activation of MAP kinase seems to be partially mediated by PKC, because down-regulation of PKC and known PKC inhibitors inhibited the HDL effect by 40 to 50%. Free apolipoproteins A-I (10 micrograms/mL) and A-II (10 micrograms/mL) had no significant effect on MAP kinase activation. Moreover, modifying HDL with trypsin or tetranitromethane, which abolishes apolipoprotein-mediated cholesterol efflux, had no effect on HDL activation of MAP kinase. These results suggest that HDL activates MAP kinase via multiple signal transduction pathways that are likely involved in an HDL effect unrelated to apolipoprotein-mediated cholesterol translocation and efflux.
Assuntos
Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Lipoproteínas HDL/farmacologia , Pele/enzimologia , Apolipoproteínas/fisiologia , Colesterol/metabolismo , Ativação Enzimática/efeitos dos fármacos , Fibroblastos/enzimologia , Fibroblastos/metabolismo , Quinase 3 da Glicogênio Sintase , Humanos , Concentração Osmolar , Proteína Quinase C/fisiologia , Pele/citologia , Fatores de TempoRESUMO
The hydrolytic rates and metabolic pool sizes of ATP were determined in intact cells by monitoring the time courses of 18O incorporation from 18O-water into the gamma-phosphoryl of ATP and orthophosphate. To calculate the rate of ATP hydrolysis, a kinetic model is used to fit the time course of the 18O labeling. The size of the metabolic pool of ATP is calculated from the 18O distribution after isotopic equilibrium has been achieved. Metabolic pools have a binomial distribution of 18O whereas nonmetabolic pools exhibit negligible 18O labeling. The application and limitations of this approach are illustrated with data from isolated toad retinas and human platelets. At 22 degrees C, the time constant of ATP hydrolysis in the dark-adapted toad retina is about 30 s. Under these conditions, over 80% of the retinal ATP is involved in high-energy phosphate metabolism. It is calculated that when cGMP metabolic flux in the photoreceptors is maximally stimulated by light, it accounts for 10% of the ATP utilization by the entire retina. The time constant of ATP hydrolysis in human platelets at 37 degrees C is approximately 1 s, and 60% of the platelet ATP is involved in energy metabolism.
Assuntos
Trifosfato de Adenosina/metabolismo , Plaquetas/metabolismo , Retina/metabolismo , Difosfato de Adenosina/metabolismo , Animais , Humanos , Cinética , Matemática , Modelos Teóricos , Isótopos de Oxigênio , Fosfatos/metabolismoRESUMO
Glycosylphosphatidylinositol-specific phospholipase D (GPI-PLD) is a high-density lipoprotein-associated protein. However, the tissue source(s) for circulating GPI-PLD and whether serum levels are regulated are unknown. Because the diabetic state alters lipoprotein metabolism, and liver and pancreatic islets are possible sources of GPI-PLD, we hypothesized that GPI-PLD levels would be altered in diabetes. GPI-PLD serum activity and liver mRNA were examined in two mouse models of type 1 diabetes, a nonobese diabetic (NOD) mouse model and low-dose streptozotocin-induced diabetes in CD-1 mice. With the onset of hyperglycemia (2- to 5-fold increase over nondiabetic levels), GPI-PLD serum activity and liver mRNA increased 2- to 4-fold in both models. Conversely, islet expression of GPI-PLD was absent as determined by immunofluorescence. Insulin may regulate GPI-PLD expression, because insulin treatment of diabetic NOD mice corrected the hyperglycemia along with reducing serum GPI-PLD activity and liver mRNA. Our data demonstrate that serum GPI-PLD levels are altered in the diabetic state and are consistent with liver as a contributor to circulating GPI-PLD.
Assuntos
Diabetes Mellitus Tipo 1/enzimologia , Regulação Enzimológica da Expressão Gênica/genética , Fosfolipase D/biossíntese , Animais , Cromatografia Líquida de Alta Pressão , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/patologia , Diabetes Mellitus Tipo 1/patologia , Imunofluorescência , Fígado/enzimologia , Masculino , Camundongos , Camundongos Endogâmicos NOD , Estado Nutricional , Pâncreas/patologia , Fenótipo , RNA Mensageiro/biossínteseRESUMO
Inositol glycans were prepared from reductively radiomethylated human erythrocyte acetylcholinesterase by sequential treatment with Proteinase K, methanolic KOH, and phosphatidylinositol-specific phospholipase C. Four glycans denoted alpha-delta were resolved by anion exchange high performance liquid chromatography (HPLC). Each glycan was subjected to hydrolysis in 4 M trifluoroacetic acid, and their hexose and hexose phosphate compositions were determined by anion exchange HPLC. The predominant glycan alpha showed a relative stoichiometry of 2 mannoses, 1 mannose 6-phosphate, 1 radiomethylated glucosamine, 1 radiomethylated ethanolamine, and 1 inositol. In contrast, the stoichiometry of glycan beta was 1 mannose, 2 mannose 6-phosphates, 1 radiomethylated glucosamine, 2 radiomethylated ethanolamines, and 1 inositol. Glycans alpha and beta were analyzed by electrospray ionization-mass spectrometry, and respective parent ions of m/z 1266 and 1417 were observed. The fragmentation pattern produced by collision-induced dissociation mass spectrometry of these parent ions was consistent with a common linear core glycan sequence prior to radiomethylation of ethanolamine-phosphate-mannose - mannose - mannose - glucosamine - inositol. Glycan alpha contained a single additional radiomethylated phosphoethanolamine branching from the mannose adjacent to glucosamine, whereas glycan beta contained two additional radiomethylated phosphoethanolamines, one branching from each of the mannoses nearest to glucosamine. Trifluoroacetic acid hydrolysis did not cleave within the N,N-dimethylglucosamine-inositol-phosphate moiety in these glycans, and this component was resolved by anion exchange HPLC and structurally confirmed by mass spectrometry. Dephosphorylation of this component by treatment with 50% HF produced N,N-dimethylglucosamine-inositol, and this conjugate was shown to have a characteristic elution time on cation exchange chromatography in an amino acid analyzer. Both of these fragments involving an intact radiomethylated glucosamine-inositol bond are proposed as new diagnostic indicators in the search for minor glycoinositol phospholipids in cells and tissues.