RESUMEN
Since glucose stimulates protein biosynthesis in beta cells concomitantly with the stimulation of insulin release, the possible interaction of both processes was explored. The protein biosynthesis was inhibited by 10 µM cycloheximide (CHX) 60 min prior to the stimulation of perifused, freshly isolated or 22 h-cultured NMRI mouse islets. CHX reduced the insulinotropic effect of 25 mM glucose or 500 µM tolbutamide in fresh but not in cultured islets. In cultured islets the second phase of glucose stimulation was even enhanced. In fresh and in cultured islets CHX strongly reduced the content of proinsulin, but not of insulin, and moderately diminished the [Ca2+]i increase during stimulation. The oxygen consumption rate (OCR) of fresh islets was about 50% higher than that of cultured islets at basal glucose and was significantly increased by glucose but not tolbutamide. In fresh, but not in cultured, islets CHX diminished the glucose-induced OCR increase and changes in the NAD(P)H- and FAD-autofluorescence. It is concluded that short-term CHX exposure interferes with the signal function of the mitochondria, which have different working conditions in fresh and in cultured islets. The interference may not be an off-target effect but may result from the inhibited cytosolic synthesis of mitochondrial proteins.
Asunto(s)
Islotes Pancreáticos , Ratones , Femenino , Animales , Secreción de Insulina , Islotes Pancreáticos/metabolismo , Cicloheximida/farmacología , Insulina/metabolismo , Glucosa/metabolismo , Tolbutamida/farmacología , Tolbutamida/metabolismo , NAD/metabolismo , Mitocondrias/metabolismo , Calcio/metabolismoRESUMEN
INTRODUCTION: Pharmacokinetic variability in disease state is common in clinical practice, but its underlying mechanism remains unclear. Recently, gut microbiota has been considered to be pharmacokinetically equivalent to the host liver. Although some studies have explored the roles of gut microbiota and host Cyp450s in drug pharmacokinetics, few have explored their effects on pharmacokinetic variability, especially in disease states. OBJECTIVES: In this study, we aim to investigate the effects of gut microbiota and host Cyp450s on pharmacokinetic variability in mice with non-alcoholic steatohepatitis (NASH), and to elucidate the contribution of gut microbiota and host Cyp450s to pharmacokinetic variability in this setting. METHODS: The pharmacokinetic variability of mice with NASH was explored under intragastric and intravenous administrations of a cocktail mixture of omeprazole, phenacetin, midazolam, tolbutamide, chlorzoxazone, and metoprolol, after which the results were compared with those obtained from the control group. Thereafter, the pharmacokinetic variabilities of all drugs and their relations to the changes in gut microbiota and host Cyp450s were compared and analyzed. RESULTS: The exposures of all drugs, except metoprolol, significantly increased in the NASH group under intragastric administration. However, no significant increase in the exposure of all drugs, except tolbutamide, was observed in the NASH group under intravenous administration. The pharmacokinetic variabilities of phenacetin, midazolam, omeprazole, and chlorzoxazone were mainly associated with decreased elimination activity in the gut microbiota. By contrast, the pharmacokinetic variability of tolbutamide was mainly related to the change in the host Cyp2c65. Notably, gut microbiota and host Cyp450s exerted minimal effects on the pharmacokinetic variability of metoprolol. CONCLUSION: Gut microbiota and host Cyp450s co-contribute to the pharmacokinetic variability in mice with NASH, and the degree of contribution varies from drug to drug. The present findings provide new insights into the explanation of pharmacokinetic variability in disease states.
Asunto(s)
Microbioma Gastrointestinal , Enfermedad del Hígado Graso no Alcohólico , Animales , Clorzoxazona/farmacología , Metoprolol/farmacología , Ratones , Midazolam/farmacología , Enfermedad del Hígado Graso no Alcohólico/tratamiento farmacológico , Omeprazol/farmacología , Preparaciones Farmacéuticas , Fenacetina/farmacología , Tolbutamida/farmacologíaRESUMEN
Natural products embedded crown ethers were prepared by utilizing bioactive natural products including chrysin, tetrahydroisoquinoline (THIQ), and biochanin-A. The prepared crown ether scaffolds were evaluated and compared with their natural product precursors for insulin secretory activity on isolated mice islets and for their fluorescent properties. All the crown adducts were found more active as compared to their natural product precursors. Bischrysin 32-crown-10 (6d), THIQ 15-Crown-5 (6a) and chrysin 16-crown-5 (6c) showed mild, moderate and strong insulin secretory activity, respectively when compared with the standard drug tolbutamide (TB). Particularly crown derivative 6c showed strong activity (31.10 ng/islet/h) that is almost two (02) fold higher than that of standard drug TB (16.82 ng/islet/h). To the best of our knowledge crown ethers based antidiabetic study is being reported for the first time in literature through this work. Furthermore, fluorescence study showed the significant increase in absorption and emission maximum (hypsochromic effect) in crown structures when compared with their natural product precursors. Present optimistic results obtained from this study may be a guided template for developing new effective insulin secretory agents.
Asunto(s)
Productos Biológicos/farmacología , Éteres Corona/farmacología , Hipoglucemiantes/farmacología , Secreción de Insulina/efectos de los fármacos , Islotes Pancreáticos/efectos de los fármacos , Animales , Productos Biológicos/aislamiento & purificación , Éteres Corona/aislamiento & purificación , Hipoglucemiantes/aislamiento & purificación , Islotes Pancreáticos/metabolismo , Masculino , Ratones Endogámicos BALB C , Tolbutamida/farmacologíaRESUMEN
Selenium is an essential trace element of interest for its potential role in glucose homeostasis. The present study investigated the impact of selenium supplementation as selenomethionine (SeMet) on insulin secretion in MIN6-K8 cells, a pancreatic ß-cell model. We found that SeMet enhanced percent glucose-induced insulin secretion, while also increasing tolbutamide- and KCl-induced percent insulin secretion. RNA-sequencing showed that SeMet supplementation altered expression of several selenoproteins, including glutathione peroxidase 3 (Gpx3) and selenoprotein P (SelP). Targeted knockdown of Gpx3 increased both percent and total insulin release, while SelP knockdown increased insulin content and insulin release. Collectively, these studies support a putative role for selenium and selenoproteins in the regulation of insulin secretion, glucose homeostasis, and diabetes risk.
Asunto(s)
Secreción de Insulina/efectos de los fármacos , Insulinoma/metabolismo , Selenometionina/farmacología , Animales , Calcio/metabolismo , Muerte Celular/efectos de los fármacos , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Glutatión Peroxidasa/metabolismo , Insulina/metabolismo , Insulinoma/genética , Insulinoma/patología , Ratones , Potasio/farmacología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Selenoproteínas/genética , Selenoproteínas/metabolismo , Tolbutamida/farmacologíaRESUMEN
OBJECTIVE: Glucagon is secreted by pancreatic α-cells in response to hypoglycemia and its hyperglycemic effect helps to restore normal blood glucose. Insulin and somatostatin (SST) secretions from ß- and δ-cells, respectively, are stimulated by glucose by mechanisms involving an inhibition of their ATP-sensitive K+ (KATP) channels, leading to an increase in [Ca2+]c that triggers exocytosis. Drugs that close KATP channels, such as sulfonylureas, are used to stimulate insulin release in type 2 diabetic patients. α-cells also express KATP channels. However, the mechanisms by which sulfonylureas control glucagon secretion are still largely debated and were addressed in the present study. In particular, we studied the effects of KATP channel blockers on α-cell [Ca2+]c and glucagon secretion in the presence of a low (1 mM) or a high (15 mM) glucose concentration and evaluated the role of SST in these effects. METHODS: Using a transgenic mouse model expressing the Ca2+-sensitive fluorescent protein, GCaMP6f, specifically in α-cells, we measured [Ca2+]c in α-cells either dispersed or within whole islets (by confocal microscopy). By measuring [Ca2+]c in α-cells within islets and glucagon secretion using the same perifusion protocols, we tested whether glucagon secretion correlated with changes in [Ca2+]c in response to sulfonylureas. We studied the role of SST in the effects of sulfonylureas using multiple approaches including genetic ablation of SST, or application of SST-14 and SST receptor antagonists. RESULTS: Application of the sulfonylureas, tolbutamide, or gliclazide, to a medium containing 1 mM or 15 mM glucose increased [Ca2+]c in α-cells by a direct effect as in ß-cells. At low glucose, sulfonylureas inhibited glucagon secretion of islets despite the rise in α-cell [Ca2+]c that they triggered. This glucagonostatic effect was indirect and attributed to SST because, in the islets of SST-knockout mice, sulfonylureas induced a stimulation of glucagon secretion which correlated with an increase in α-cell [Ca2+]c. Experiments with exogenous SST-14 and SST receptor antagonists indicated that the glucagonostatic effect of sulfonylureas mainly resulted from an inhibition of the efficacy of cytosolic Ca2+ on exocytosis. Although SST-14 was also able to inhibit glucagon secretion by decreasing α-cell [Ca2+]c, no decrease in [Ca2+]c occurred during sulfonylurea application because it was largely counterbalanced by the direct stimulatory effect of these drugs on α-cell [Ca2+]c. At high glucose, i.e., in conditions where glucagon release was already low, sulfonylureas stimulated glucagon secretion because their direct stimulatory effect on α-cells exceeded the indirect effect by SST. Our results also indicated that, unexpectedly, SST-14 poorly decreased the efficacy of Ca2+ on exocytosis in ß-cells. CONCLUSIONS: Sulfonylureas exert two opposite actions on α-cells: a direct stimulation as in ß-cells and an indirect inhibition by SST. This suggests that any alteration of SST paracrine influence, as described in diabetes, will modify the effect of sulfonylureas on glucagon release. In addition, we suggest that δ-cells inhibit α-cells more efficiently than ß-cells.
Asunto(s)
Calcio/metabolismo , Gliclazida/farmacología , Células Secretoras de Glucagón/efectos de los fármacos , Glucagón/metabolismo , Canales KATP/antagonistas & inhibidores , Bloqueadores de los Canales de Potasio/farmacología , Somatostatina/farmacología , Tolbutamida/farmacología , Animales , Gliclazida/química , Células Secretoras de Glucagón/metabolismo , Canales KATP/metabolismo , Ratones , Ratones Noqueados , Ratones Transgénicos , Bloqueadores de los Canales de Potasio/química , Somatostatina/química , Tolbutamida/químicaRESUMEN
Relacorilant is a selective modulator of the glucocorticoid receptor in development for the treatment of several serious diseases. The widely used cocktail method was employed to assess relacorilant's effect on various cytochrome P450 (CYP) drug metabolizing enzymes in vitro and in vivo. Inhibition of CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP2B6, CYP2C8, CYP3A4, and CYP3A5 as well as induction of CYP1A2, CYP2B6, and CYP3A4 were assessed in vitro (relacorilant concentrations up to 10 µM). A clinical study in healthy subjects (n = 27) evaluated the inhibition of CYP3A4, CYP2C8, and CYP2C9 in vivo by administering single doses of probe CYP substrates (midazolam, pioglitazone, and tolbutamide) alone and in combination with relacorilant (350 mg). Pharmacokinetic sampling was conducted, and safety was assessed throughout the study. Pharmacokinetic parameters were evaluated using 90% confidence intervals of the geometric least squares mean ratios of test (probe substrate with relacorilant) vs reference (probe substrate alone) using boundaries of 80% to 125%. In vitro, relacorilant inhibited CYP3A4, CYP2C8, and CYP2C9 but did not meaningfully affect the activity of the other CYP enzymes evaluated. Consistent with the in vitro data, relacorilant was shown to be a strong CYP3A inhibitor in vivo (>8-fold increase in midazolam area under the concentration versus time curve from time zero to the last quantifiable concentration and area under the concentration versus time curve from time zero extrapolated to infinity). Coadministration of relacorilant with drugs highly dependent on CYP3A for clearance is expected to increase the concentrations of these drugs. Importantly, clinical evaluation of relacorilant showed no inhibition of CYP2C8 or CYP2C9 in vivo. Accordingly, drugs that are substrates of only CYP2C8 and/or CYP2C9 can be coadministered with relacorilant without dose adjustment.
Asunto(s)
Inductores de las Enzimas del Citocromo P-450/farmacología , Inhibidores Enzimáticos del Citocromo P-450/farmacología , Sistema Enzimático del Citocromo P-450/efectos de los fármacos , Isoquinolinas/farmacocinética , Pirazoles/farmacocinética , Piridinas/farmacocinética , Área Bajo la Curva , Estudios Cruzados , Relación Dosis-Respuesta a Droga , Interacciones Farmacológicas , Semivida , Humanos , Midazolam/farmacología , Pioglitazona/farmacología , Tolbutamida/farmacologíaRESUMEN
The role of depolarization in the inverse glucose-dependence of glucagon secretion was investigated by comparing the effects of KATP channel block and of high potassium. The secretion of glucagon and insulin by perifused mouse islets was simultaneously measured. Lowering glucose raised glucagon secretion before it decreased insulin secretion, suggesting an alpha cell-intrinsic signal recognition. Raising glucose affected glucagon and insulin secretion at the same time. However, depolarization by tolbutamide, gliclazide, or 15 mM KCl increased insulin secretion before the glucagon secretion receded. In contrast to the robust depolarizing effect of arginine and KCl (15 and 40 mM) on single alpha cells, tolbutamide was of variable efficacy. Only when applied before other depolarizing agents had tolbutamide a consistent depolarizing effect and regularly increased the cytosolic Ca2+ concentration. When tested on inside-out patches tolbutamide was as effective on alpha cells as on beta cells. In the presence of 1 µM clonidine, to separate insulinotropic from glucagonotropic effects, both 500 µM tolbutamide and 30 µM gliclazide increased glucagon secretion significantly, but transiently. The additional presence of 15 or 40 mM KCl in contrast led to a marked and lasting increase of the glucagon secretion. The glucagon secretion by SUR1 knockout islets was not increased by tolbutamide, whereas 40 mM KCl was of unchanged efficiency. In conclusion a strong and sustained depolarization is compatible with a marked and lasting glucagon secretion. KATP channel closure in alpha cells is less readily achieved than in beta cells, which may explain the moderate and transient glucagonotropic effect.
Asunto(s)
Células Secretoras de Glucagón/efectos de los fármacos , Células Secretoras de Glucagón/metabolismo , Glucagón/metabolismo , Insulina/metabolismo , Canales KATP/metabolismo , Potasio/metabolismo , Animales , Arginina/farmacología , Calcio/metabolismo , Membrana Celular , Femenino , Gliclazida/farmacología , Glucosa/farmacología , Hipoglucemiantes/farmacología , Secreción de Insulina/efectos de los fármacos , Células Secretoras de Insulina , Potenciales de la Membrana , Ratones , Cloruro de Potasio/farmacología , Tolbutamida/farmacologíaRESUMEN
AIMS: To study the effects of blood glucose regulating compounds on human and rat sulfotransferases (SULTs) expressions. BACKGROUND: Phase-II enzymes, sulfotransferases catalyze the sulfuryl-group-transfer to endogenous/exogenous compounds. The alteration of expressions of SULTs may have influence on the sulfation of its substrate and other biomolecules. OBJECTIVES: The influence of the altered biotransformation might alter different biochemical events, drug-drug interactions and bioaccumulation or excretion pattern of certain drug. METHODS: In this brief study, diabetes-inducing drug streptozotocin (STZ; 10 or 50 mg/kg to male Sprague Dawley rat for 2 weeks) or hyperglycemia controlling drug tolbutamide (TLB 0.1 or 10µM to human hepato-carcinoma cells, HepG2 for 10 days) was applied and the SULTs expressions were verified. Extensive protein-protein (STa, SULT2A1/DHEAST) interactions were studied by the STRING (Search-Tool-for-the-Retrieval-of-Interacting Genes/Proteins) Bioinformatics-software. RESULTS: Present result suggests that while STZ increased the STa (in rat) (dehydroepiandrosterone catalyzing SULT; DHEAST in human HepG2), tolbutamide decreased PPST (phenol catalyzing SULT) and DHEAST activity in human HepG2 cells. Moderate decreases of MPST (monoamine catalyzing SULT) and EST (estrogen catalyzing) activities are noticed in this case. STa/DHEAST was found to be highly interactive to SHBG/- sex-hormone-binding-globulin; PPARα/lipid-metabolism-regulator; FABP1/fatty-acid-binding-protein. CONCLUSION: Streptozotocin and tolbutamide, these two glycaemia-modifying drugs demonstrated regulation of rat and human SULTs activities. The reciprocal nature of these two drugs on SULTs expression may be associated with their contrasting abilities in influencing glucose-homeostasis. Possible association of certain SULT-isoform with hepatic fat-regulations may indicate an unfocused link between calorie-metabolism and the glycemic-state of an individual. Explorations of this work may uncover the role of sulfation metabolism of specific biomolecule on cellular glycemic regulation.
Asunto(s)
Hipoglucemiantes/farmacología , Mapas de Interacción de Proteínas/efectos de los fármacos , Estreptozocina/administración & dosificación , Sulfotransferasas/metabolismo , Tolbutamida/farmacología , Animales , Biotransformación , Glucemia/efectos de los fármacos , Glucemia/metabolismo , Diabetes Mellitus Experimental/sangre , Diabetes Mellitus Experimental/inducido químicamente , Diabetes Mellitus Experimental/metabolismo , Interacciones Farmacológicas , Células Hep G2 , Humanos , Hipoglucemiantes/uso terapéutico , Masculino , Mapeo de Interacción de Proteínas , Ratas , Ratas Sprague-Dawley , Tolbutamida/uso terapéuticoRESUMEN
The effective concentration of a drug in the blood, i.e. the concentration of a free drug in the blood, is influenced by the strength of drug binding onto plasma proteins. Besides its efficacy, these interactions subsequently influence the liberation, absorption, distribution, metabolism, excretion, and toxicological properties of the drug. It is important to not only determine the binding strength and stoichiometry, but also the binding site of a drug on the plasma protein molecule, because the co-administration of drugs with the same binding site can affect the above-mentioned concentration and as a result the pharmacological behavior of the drugs and lead to side effects caused by the change in free drug concentration, its toxicity. In this study, the binding characteristics of six drugs with human serum albumin, the most abundant protein in human plasma, were determined by capillary electrophoresis-frontal analysis, and the obtained values of binding parameters were compared with the literature data. The effect of several drugs and site markers on the binding of l-tryptophan and lidocaine to human serum albumin was investigated in subsequent displacement studies which thus demonstrated the usability of capillary electrophoresis as an automated high-throughput screening method for drug-protein binding studies.
Asunto(s)
Clorpropamida/análisis , Diclofenaco/análisis , Flurbiprofeno/análisis , Ibuprofeno/análisis , Fenilbutazona/análisis , Tolbutamida/análisis , Sitios de Unión/efectos de los fármacos , Clorpropamida/farmacología , Diclofenaco/farmacología , Electroforesis Capilar , Flurbiprofeno/farmacología , Humanos , Ibuprofeno/farmacología , Lidocaína/antagonistas & inhibidores , Lidocaína/química , Fenilbutazona/farmacología , Albúmina Sérica Humana/química , Tolbutamida/farmacología , Triptófano/antagonistas & inhibidores , Triptófano/químicaRESUMEN
Insulin is released from pancreatic islets in a biphasic and pulsatile manner in response to elevated glucose levels. This highly dynamic insulin release can be studied in vitro with islet perifusion assays. Herein, a novel platform to perform glucose-stimulated insulin secretion (GSIS) assays with single islets is presented for studying the dynamics of insulin release at high temporal resolution. A standardized human islet model is developed and a microfluidic hanging-drop-based perifusion system is engineered, which facilitates rapid glucose switching, minimal sample dilution, low analyte dispersion, and short sampling intervals. Human islet microtissues feature robust and long-term glucose responsiveness and demonstrate reproducible dynamic GSIS with a prominent first phase and a sustained, pulsatile second phase. Perifusion of single islet microtissues produces a higher peak secretion rate, higher secretion during the first and second phases of insulin release, as well as more defined pulsations during the second phase in comparison to perifusion of pooled islets. The developed platform enables to study compound effects on both phases of insulin secretion as shown with two classes of insulin secretagogs. It provides a new tool for studying physiologically relevant dynamic insulin secretion at comparably low sample-to-sample variation and high temporal resolution.
Asunto(s)
Glucosa/metabolismo , Insulina/metabolismo , Islotes Pancreáticos , Modelos Biológicos , Análisis de Matrices Tisulares/métodos , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/fisiopatología , Descubrimiento de Drogas/métodos , Exenatida/farmacología , Humanos , Hipoglucemiantes/farmacología , Islotes Pancreáticos/efectos de los fármacos , Islotes Pancreáticos/metabolismo , Islotes Pancreáticos/fisiología , Técnicas Analíticas Microfluídicas/métodos , Tolbutamida/farmacologíaRESUMEN
Insulin pulsatility is important to hepatic response in regulating blood glucose. Growing evidence suggests that insulin-secreting pancreatic ß-cells can adapt to chronic disruptions of pulsatility to rescue this physiologically important behavior. We determined the time scale for adaptation and examined potential ion channels underlying it. We induced the adaptation both by chronic application of the ATP-sensitive K+ [K(ATP)] channel blocker tolbutamide and by application of the depolarizing agent potassium chloride (KCl). Acute application of tolbutamide without pretreatment results in elevated Ca2+ as measured by fura-2AM and the loss of endogenous pulsatility. We show that after chronic exposure to tolbutamide (12-24 h), Ca2+ oscillations occur with subsequent acute tolbutamide application. The same experiment was conducted with potassium chloride (KCl) to directly depolarize the ß-cells. Once again, following chronic exposure to the cell stimulator, the islets produced Ca2+ oscillations when subsequently exposed to tolbutamide. These experiments suggest that it is the chronic stimulation, and not tolbutamide desensitization, that is responsible for the adaptation that rescues oscillatory ß-cell activity. This compensatory response also causes islet glucose sensitivity to shift rightward following chronic tolbutamide treatment. Mathematical modeling shows that a small increase in the number of K(ATP) channels in the membrane is one adaptation mechanism that is compatible with the data. To examine other compensatory mechanisms, pharmacological studies provide support that Kir2.1 and TEA-sensitive channels play some role. Overall, this investigation demonstrates ß-cell adaptability to overstimulation, which is likely an important mechanism for maintaining glucose homeostasis in the face of chronic stimulation.
Asunto(s)
Adaptación Fisiológica , Señalización del Calcio , Islotes Pancreáticos/metabolismo , Canales de Potasio/metabolismo , Animales , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Hiperinsulinismo Congénito/metabolismo , Humanos , Células Secretoras de Insulina/efectos de los fármacos , Células Secretoras de Insulina/metabolismo , Canales KATP/metabolismo , Masculino , Ratones , Modelos Teóricos , Bloqueadores de los Canales de Potasio/farmacología , Canales de Potasio de Rectificación Interna/metabolismo , Cloruro de Potasio , Estimulación Química , Tolbutamida/farmacologíaRESUMEN
Binding strength of the anti-diabetic drugs chlorpropamide (CPM) and tolbutamide (TBM) with model protein bovine serum albumin (BSA) shows strong modulation in presence of colloidal gold nanoparticles (AuNP). Intrinsic tryptophan fluorescence of both the native BSA and BSA-AuNP conjugate quenched in presence of the drugs. Stern-Volmer quenching constant (KSV) of CPM binding to BSA-AuNP conjugate at different temperatures is almost twice (6.76~14.76 × 103 M-1) than the corresponding values in native BSA (3.21~5.72 × 103 M-1). However, the calculated KSV values with TBM show certain degree of reduction in presence of AuNP (6.46× 103 M-1), while comparing with native BSA (8.83 × 103 M-1). The binding mode of CPM towards BSA-AuNP conjugate is mainly through hydrophobic forces; whereas, TBM binding is identified to be Van der Waal's and hydrogen bonding type of interaction. Fluorescence lifetime analysis confirms static type of quenching for the intrinsic tryptophan fluorescence of BSA as well as BSA-AuNP conjugate with addition of CPM and TBM at different concentrations. The α-helical content in the secondary structure of BSA is decreased to 48.32% and 45. 28% in presence of AuNP, when the concentration of CPM is 0.08 mM and 0.16 mM in comparison with that of native protein (50.13%). On the other hand, the intensity of sugar induced advanced glycated end (AGE) product fluorescence is decreased by 55% and 80% at 0.13 nM and 0.68 nM AuNP, respectively. Change in the binding strength of the drugs with transport protein and reduced AGE product formation in presence of AuNP could lead to a major development in the field of nanomedicine and associated drug delivery techniques. Graphical Abstract Modulated drug binding ability and AGE product formation of serum proteins in presence of AuNP.
Asunto(s)
Clorpropamida/farmacología , Productos Finales de Glicación Avanzada/antagonistas & inhibidores , Hipoglucemiantes/farmacología , Albúmina Sérica Bovina/química , Tolbutamida/farmacología , Adsorción , Animales , Sitios de Unión/efectos de los fármacos , Bovinos , Clorpropamida/química , Coloides/química , Productos Finales de Glicación Avanzada/metabolismo , Oro/química , Oro/farmacología , Interacciones Hidrofóbicas e Hidrofílicas , Hipoglucemiantes/química , Nanopartículas del Metal/química , Tamaño de la Partícula , Albúmina Sérica Bovina/antagonistas & inhibidores , Albúmina Sérica Bovina/metabolismo , Espectrometría de Fluorescencia , Propiedades de Superficie , Temperatura , Tolbutamida/químicaRESUMEN
Tambulin, a flavonol isolated from Zanthoxylum armatum, showed potent insulin secretory activity in our preliminary anti-diabetic screening. Here, we explored the insulin secretory mechanism(s) of tambulin focusing in glucose-dependent, KATP â and Ca2+âchannels dependent, and cAMP-PKA pathways. Mice islets and MIN6 cells were incubated with tambulin in the presence of pharmacological agonists/antagonists and the secreted insulin was measured using mouse insulin ELISA kit. The intracellular cAMP was measured by an acetylation cAMP ELISA kit. Tambulin (200⯵M) showed potent insulin secretory activity only at stimulatory glucose (11-25â¯mM) concentrations; however, no change in insulin release was observed at basal glucose both in mice islets and MIN6 cells. Notably, in the presence of diazoxide, a KATP channel opener; the incomplete inhibition of tambulin-induced insulin secretion was observed whereas, complete inhibition was found using verapamil, an L-type Ca2+ channel blocker. Furthermore, the insulinotropic potential of tambulin was amplified in tolbutamide treated, and depolarized islets suggest tambulin's target other than tolbutamide. Tambulin showed no additive effect in the IBMX-induced intracellular cAMP; whereas, exerted an additive effect in the IBMX-induced insulin secretion. Furthermore, tambulin-induced insulin secretion was dramatically inhibited by PKA inhibitor (H-89), while moderate inhibition was found by using PKC inhibitor (calphostin C). Molecular docking studies also showed the best binding affinities of tambulin with PKA suggest the PKA dependent signaling cascade is involved more in tambulin-induced insulin secretion. Based on these findings, it is concluded that tambulin stimulates insulin secretion in a Ca2+ channel-dependent but KATP channel-independent manner, most likely by activating the cAMP-PKA pathway.
Asunto(s)
Benzopiranos/farmacología , Señalización del Calcio/efectos de los fármacos , Glucosa/farmacología , Hipoglucemiantes/farmacología , Insulina/metabolismo , Islotes Pancreáticos/efectos de los fármacos , Canales KATP/metabolismo , Zanthoxylum , Animales , Benzopiranos/aislamiento & purificación , Línea Celular , AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Activación del Canal Iónico/efectos de los fármacos , Islotes Pancreáticos/metabolismo , Masculino , Ratones , Ratones Endogámicos BALB C , Vías Secretoras , Tolbutamida/farmacología , Zanthoxylum/químicaRESUMEN
A drug-drug anhydrous pharmaceutical salt containing tolbutamide {systematic name: 3-butyl-1-[(4-methylbenzene)sulfonyl]urea, TOL, C12H18N2O3S} and metformin (systematic name: 1-carbamimidamido-N,N-dimethylmethanimidamide, MET, C4H11N5) was created based on antidiabetic drug combinations to overcome the poor pharmaceutical properties of the parent drugs. Proton transfer and the proportion of the two components were confirmed by 1H NMR spectroscopy and single-crystal X-ray diffraction analysis. Comprehensive characterization of the new pharmaceutical salt crystal, 2-[(dimethylamino)(iminiumyl)methyl]guanidine (butylcarbamoyl)[(4-methylbenzene)sulfonyl]azanide, C4H12N5+·C12H17N2O3S-, was performed and showed enhancement of the pharmaceutical properties, such as lower hygroscopicity and greater accelerated stability than the parent drug MET, and higher solubility and dissolution rate than TOL. The property alterations were correlated with the crystal packing features and potential hydrogen-bonding sites through observed changes in the crystal structures.
Asunto(s)
Hipoglucemiantes/farmacología , Metformina/farmacología , Tolbutamida/farmacología , Cristalografía por Rayos X , Combinación de Medicamentos , Enlace de Hidrógeno , Hipoglucemiantes/síntesis química , Hipoglucemiantes/química , Metformina/síntesis química , Metformina/química , Estructura Molecular , Solubilidad , Tolbutamida/síntesis química , Tolbutamida/químicaRESUMEN
Beta-arrestin-1 and -2 (Barr1 and Barr2, respectively) are intracellular signaling molecules that regulate many important metabolic functions. We previously demonstrated that mice lacking Barr2 selectively in pancreatic beta-cells showed pronounced metabolic impairments. Here we investigated whether Barr1 plays a similar role in regulating beta-cell function and whole body glucose homeostasis. Initially, we inactivated the Barr1 gene in beta-cells of adult mice (beta-barr1-KO mice). Beta-barr1-KO mice did not display any obvious phenotypes in a series of in vivo and in vitro metabolic tests. However, glibenclamide and tolbutamide, two widely used antidiabetic drugs of the sulfonylurea (SU) family, showed greatly reduced efficacy in stimulating insulin secretion in the KO mice in vivo and in perifused KO islets in vitro. Additional in vivo and in vitro studies demonstrated that Barr1 enhanced SU-stimulated insulin secretion by promoting SU-mediated activation of Epac2. Pull-down and co-immunoprecipitation experiments showed that Barr1 can directly interact with Epac2 and that SUs such as glibenclamide promote Barr1/Epac2 complex formation, triggering enhanced Rap1 signaling and insulin secretion. These findings suggest that strategies aimed at promoting Barr1 signaling in beta-cells may prove useful for the development of efficacious antidiabetic drugs.
Asunto(s)
Secreción de Insulina , Células Secretoras de Insulina/metabolismo , Compuestos de Sulfonilurea/química , beta-Arrestina 1/metabolismo , Animales , Genotipo , Gliburida/farmacología , Factores de Intercambio de Guanina Nucleótido/metabolismo , Hipoglucemiantes/farmacología , Masculino , Ratones , Ratones Noqueados , Ratones Transgénicos , Fenotipo , Transducción de Señal , Tolbutamida/farmacología , Arrestina beta 2/metabolismoRESUMEN
Corydalis decumbens, a Traditional Chinese Medicine, has been widely used for the alternative and/or complementary therapy of hypertension, arrhythmias rheumatoid arthritis, sciatica, stroke, hemiplegia, paraplegia, and vascular embolism. The aim of this study was to determinate the potential effects of Corydalis decumbens on the five cytochrome P450 (CYP) enzyme activities (CYP1A2, CYP3A4, CYP2C9, CYP2C19, and CYP2D6) by cocktail approach. To evaluate whether concurrent use of Corydalis decumbens interferes with the effect of several prescription drugs, saline (control group) or Corydalis decumbens (XTW group) were administrated via gavage for 7 successive days. A probe cocktail solution (phenacetin, omeprazole, metoprolol, tolbutamide, and midazolam) was given 24 h after the last dose of saline or Corydalis decumbens. A specific and sensitive UHPLC-MS/MS method was validated for the determination of five substrates and their metabolites in control group and XTW group. Our results indicated that Corydalis decumbens could have inductive effects of CYP2C19 and inhibit the activities of CYP1A2 and CYP3A4. However, Corydalis decumbens had no significant influence on CYP2C9 and CYP2D6. The herb-drug interaction should require more attention by careful monitoring and appropriate drug dosing adjustments to the concurrent use of western medications which were metabolized by CYP1A2, CYP2C19, and CYP3A4 in human-Corydalis decumbens, Cytochrome P450, Cocktail, Pharmacokinetics, herb-drug interactions.
Asunto(s)
Corydalis/química , Inhibidores Enzimáticos del Citocromo P-450/farmacología , Sistema Enzimático del Citocromo P-450/metabolismo , Medicamentos Herbarios Chinos/farmacología , Animales , Interacciones de Hierba-Droga/fisiología , Masculino , Midazolam/farmacología , Omeprazol/farmacología , Fenacetina/farmacología , Ratas , Ratas Sprague-Dawley , Tolbutamida/farmacologíaRESUMEN
Many cellular processes, including pulsatile release of insulin, are triggered by increase of cytoplasmic Ca2+. This study examines how somatostatin affects glucose generation of cytoplasmic Ca2+ oscillations in mouse islets in absence and presence of tolbutamide blockade of the KATP channels. Ca2+ was measured with dual wavelength microflurometry in isolated islets loaded with the indicator Fura-2. Rise of glucose from 3 to 20â¯mM evoked introductory lowering of Ca2+ prolonged by activation of somatostatin receptors. During continued superfusion exposure to somatostatin triggered oscillations mediated by periodic increase from the basal level (absence of tolbutamide) or by periodic interruption of an elevated level (presence of tolbutamide). In the latter situation the oscillations were transformed into sustained elevation by activation of muscarinic receptors (acetylcholine) or increase of cyclic AMP (IBMX, 8-bromo-cyclic AMP, forskolin). The observed effect of cyclic AMP raises the question whether high proportions of the glucagon-producing α-cells promote steady-state elevation of Ca2+. In support for this idea somatostatin was found to trigger glucose-induced Ca2+ oscillations essentially in small islets that contain very few α-cells. The results indicate that somatostatin promotes glucose generation of Ca2+oscillations with similar characteristics both in the absence and presence of functional KATP channels.
Asunto(s)
Señalización del Calcio/efectos de los fármacos , Glucosa/farmacología , Hipoglucemiantes/farmacología , Islotes Pancreáticos/efectos de los fármacos , Somatostatina/farmacología , Tolbutamida/farmacología , Animales , Señalización del Calcio/fisiología , Células Cultivadas , Sinergismo Farmacológico , Femenino , Hormonas/farmacología , Islotes Pancreáticos/fisiología , Ratones , Ratones Endogámicos C57BLRESUMEN
Glucose homeostasis depends critically on insulin that is secreted by pancreatic ß-cells. Serum glucose, which is directly sensed by ß-cells, stimulates depolarization- and Ca2+-dependent exocytosis of insulin granules. Here we show that pancreatic islets prominently express LRRC8A and LRRC8D, subunits of volume-regulated VRAC anion channels. Hypotonicity- or glucose-induced ß-cell swelling elicits canonical LRRC8A-dependent VRAC currents that depolarize ß-cells to an extent that causes electrical excitation. Glucose-induced excitation and Ca2+ responses are delayed in onset, but not abolished, in ß-cells lacking the essential VRAC subunit LRRC8A. Whereas Lrrc8a disruption does not affect tolbutamide- or high-K+-induced insulin secretion from pancreatic islets, it reduces first-phase glucose-induced insulin secretion. Mice lacking VRAC in ß-cells have normal resting serum glucose levels but impaired glucose tolerance. We propose that opening of LRRC8/VRAC channels increases glucose sensitivity and insulin secretion of ß-cells synergistically with KATP closure. Neurotransmitter-permeable LRRC8D-containing VRACs might have additional roles in autocrine/paracrine signaling within islets.
Asunto(s)
Glucosa/metabolismo , Insulina/metabolismo , Canales Iónicos/metabolismo , Proteínas de la Membrana/metabolismo , Animales , Aniones/metabolismo , Glucemia/efectos de los fármacos , Glucemia/genética , Femenino , Hipoglucemiantes/farmacología , Células Secretoras de Insulina/efectos de los fármacos , Células Secretoras de Insulina/metabolismo , Masculino , Proteínas de la Membrana/genética , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Modelos Animales , Canales de Potasio de Rectificación Interna/metabolismo , Cultivo Primario de Células , Multimerización de Proteína , Tolbutamida/farmacologíaRESUMEN
In vitro studies of human pancreatic islets are critical for understanding normal insulin secretion and its perturbations in diabetic ß-cells, but the influence of islet preparation characteristics and organ donor attributes in such experiments is poorly documented. Preparations from normal donors were tested with a standardized protocol evaluating dynamic insulin secretion induced by glucose, tolbutamide, and cAMP (forskolin). Secretion rates, normalized to insulin content (fractional insulin secretion), were analyzed as a function of preparation and donor characteristics. Low purity (25-45%) of the preparation (n = 8) blunted the first phase of insulin secretion induced by glucose or tolbutamide and increased basal secretion, resulting in threefold lower stimulation index than in more pure (55-95%) preparations (n = 43). In these more pure preparations, cold ischemia time (1-13 h) before pancreas digestion did not impact insulin secretion. Islet size (estimated by the islet size index) did not influence the dynamics of secretion, but fractional insulin secretion rates were greater in large than small islets, and positively correlated with islet size. Age of the donors (20-68 years) had no influence on islet size and insulin content or on dynamics and amplitude of insulin secretion, which were also similar in islets from male and female donors. In contrast, islet size and islet insulin content (normalized for size), and basal or stimulated insulin secretion positively correlated with Body-Mass Index (19-33). These results contradict previous reports on the impact of donor age and islet size and point to possible confounding effects of donor BMI in insulin secretion studies with isolated human islets.
Asunto(s)
Secreción de Insulina , Células Secretoras de Insulina/metabolismo , Recolección de Tejidos y Órganos/normas , Adulto , Factores de Edad , Anciano , Células Cultivadas , Colforsina/farmacología , Glucosa/farmacología , Humanos , Células Secretoras de Insulina/citología , Células Secretoras de Insulina/efectos de los fármacos , Persona de Mediana Edad , Donantes de Tejidos , Tolbutamida/farmacologíaRESUMEN
CYP2C11 is involved in the metabolism of many drugs in rats. To assess the roles of CYP2C11 in physiology and drug metabolism, a CYP2C11-null rat model was generated using the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9method. A 2-base pair insertion was added to exon 6 of CYP2C11 in Sprague-Dawley rats. CYP2C11 was not detected by western blotting in liver microsomes of CYP2C11-null rats. No off-target effects were found at 11 predicted sites of the knockout model. The CYP2C11-null rats were viable and had no obvious abnormalities, with the exception of reduced fertility. Puberty in CYP2C11-null rats appeared to be delayed by â¼20 days, and the average litter size fell by 43%. Tolbutamide was used as a probe in this drug metabolism study. In the liver microsomes of CYP2C11-null rats, the Vmax and intrinsicclearance values decreased by 22% and 47%, respectively, compared with those of wild-type rats. The Km values increased by 47% compared with that of wild types. However, our pharmacokinetics study showed no major differences in any parameters between the two strains, in both males and females. In conclusion, a CYP2C11-null rat model was successfully generated and is a valuable tool to study the in vivo function of CYP2C11.