RESUMEN
Insulin plays a significant role in carbohydrate homeostasis as the blood glucose lowering hormone. Glucose-induced insulin secretion (GSIS) is augmented by glucagon-like peptide (GLP-1), a gastrointestinal peptide released in response to ingesting nutriments. The secretion of insulin and GLP-1 is mediated by the binding of nutrients to G protein-coupled receptors (GPCRs) expressed by pancreatic ß-cells and enteroendocrine cells, respectively. Therefore, insulin secretagogues and incretin mimetics currently serve as antidiabetic treatments. This study demonstrates the potency of synthetic isoprenoid derivatives of lysophosphatidylcholines (LPCs) to stimulate GSIS and GLP-1 release. Murine insulinoma cell line (MIN6) and enteroendocrinal L cells (GLUTag) were incubated with LPCs bearing geranic acid (1-GA-LPC), citronellic acid (1-CA-LPC), 3,7-dimethyl-3-vinyloct-6-enoic acid (GERA-LPC), and (E)-3,7,11-trimethyl- 3-vinyldodeca-6,10-dienoic acid (1-FARA-LPC). Respective free terpene acids were also tested for comparison. Besides their insulin- and GLP-1-secreting capabilities, we also investigated the cytotoxicity of tested compounds, the ability to intracellular calcium ion mobilization, and targeted GPCRs involved in maintaining lipid and carbohydrate homeostasis. We observed the high cytotoxicity of 1-GERA-LPC and 1-FARA-LPC in contrast 1-CA-LPC and 1-GA-LPC. Moreover, 1-CA-LPC and 1-GA-LPC demonstrated the stimulatory effect on GSIS and 1-CA-LPC augmented GLP-1 secretion. Insulin and GLP-1 release appeared to be GPR40-, GPR55-, GPR119- and GPR120-dependent.
Asunto(s)
Péptido 1 Similar al Glucagón/biosíntesis , Secreción de Insulina/efectos de los fármacos , Insulina/biosíntesis , Metabolismo de los Lípidos/efectos de los fármacos , Lisofosfatidilcolinas/farmacología , Receptores Acoplados a Proteínas G/metabolismo , Terpenos/farmacología , Calcio/metabolismo , Humanos , Espacio Intracelular/metabolismo , Lisofosfatidilcolinas/química , Estructura Molecular , Terpenos/químicaRESUMEN
GPR119 receptor has been proposed as a metabolic regulator playing a pivotal role in the modulation of glucose homeostasis in type 2 diabetes. GPR119 was identified on pancreatic ß cells and its ligands have the ability to enhance glucose-stimulated insulin secretion (GSIS). Lysophosphatidylcholine (LPC) was shown to potentiate GSIS and our present studies indicate that 2-methoxy-lysophosphatidylcholine (2-OMe-LPC) analogues, unable to undergo 1â2 acyl migration, stimulate GSIS from murine ßTC-3 pancreatic cells even more efficiently. Moreover, biological assays in engineered Tango™ GPR119-bla U2OS cells were carried out to ascertain the agonist activity of 2-OMe-LPC at GPR119. 2-OMe-LPC possessing in sn-1 position the residues of myristic, palmitic, stearic and oleic acid were also evaluated as factors regulating [Ca2+]i mobilization and cAMP levels. Extension of these studies to R- and S-enantiomers of 14:0 2-OMe-LPC revealed that the overall impact on GSIS does not depend on chirality, however, the intracellular calcium mobilization data show that the R enantiomer is significantly more active than S one. Taking into account differences in chemical structure between various native LPCs and their 2-methoxy counterparts the possible binding mode of 2-OMe-LPC to the GPR119 receptor was determined using molecular modeling approach.
Asunto(s)
Células Secretoras de Insulina/efectos de los fármacos , Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Lisofosfatidilcolinas/farmacología , Receptores Acoplados a Proteínas G/agonistas , Secuencia de Aminoácidos , Animales , Sitios de Unión , Línea Celular , Hipoglucemiantes/química , Hipoglucemiantes/farmacología , Secreción de Insulina , Ligandos , Lisofosfatidilcolinas/química , Ratones , Modelos Moleculares , Receptores Acoplados a Proteínas G/química , Relación Estructura-ActividadRESUMEN
The GPR55 signaling is fertile ground for drug discovery, however despite considerable research progress during the past 10 years, many open questions remain. The GPR55 pharmacology remains controversial, as many ligands have been reported with inconsistent results. Here, we show that various molecular species of lysophosphatidylcholine (LPC) elicit intracellular Ca2+ mobilization in GPR55-expressing PC-3 human prostate carcinoma cells. The response was even stronger than [Ca2+]i flux evoked by endogenous (OEA) and synthetic (Abn-CBD) agonists. Treatment with GPR55 antagonists CID16020046 and ML193 as well as the lipid raft disrupter methyl-ß-cyclodextrin strongly blunted LPC-induced calcium signal. Additionally, molecular modeling analysis revealed that LPC 16:0 and LPC 18:1 interact stronger with the receptor than to OEA. Identified electrostatic interactions between GPR55 residues and the ligands overlap with the binding site identified previously for lysophosphatidylinositol. Therefore, we prove that LPC is another GPR55-sensitive ligand. This finding is relevant in understanding lysophospolipids-mediated signaling and opens new avenues to develop therapeutic approach based on GPR55 targeting.
Asunto(s)
Señalización del Calcio/efectos de los fármacos , Lisofosfatidilcolinas/farmacología , Receptores Acoplados a Proteínas G/metabolismo , Fosfatasa Alcalina/aislamiento & purificación , Fosfatasa Alcalina/metabolismo , Animales , Bovinos , Relación Dosis-Respuesta a Droga , Humanos , Mucosa Intestinal/enzimología , Ligandos , Lisofosfatidilcolinas/química , Lisofosfatidilcolinas/metabolismo , Modelos Moleculares , Receptores de Cannabinoides , Receptores Acoplados a Proteínas G/agonistas , Relación Estructura-Actividad , Células Tumorales CultivadasRESUMEN
The chemical synthesis of phosphorothioate/phosphodiester analogues of 2-methoxy-lysophosphatidylethanolamine has been described. For the preparation of phosphorothioate derivatives oxathiaphospholane approach has been employed. The phosphodiester compounds were prepared by OXONE® oxidation of corresponding phosphorothioates. Each lysophospholipid analogue was synthesized as a series of four compounds, bearing different fatty acid residues both saturated (14:0, 16:0, 18:0) and unsaturated (18:1). The methylation of glycerol 2-hydroxyl function was applied in order to increase the stability of prepared analogues by preventing 1â2 acyl migration. The cytotoxicity of newly synthesized 2-methoxy-lysophosphatidylethanolamine derivatives was evaluated with resazurin-based method in prostate cancer PC3 cell line. The highest reduction of cell viability was noted for LPE analogues containing myristoyl acyl chain.
Asunto(s)
Ésteres/farmacología , Lisofosfolípidos/farmacología , Compuestos de Sulfhidrilo/farmacología , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Ésteres/síntesis química , Ésteres/química , Humanos , Lisofosfolípidos/química , Estructura Molecular , Relación Estructura-Actividad , Compuestos de Sulfhidrilo/síntesis química , Compuestos de Sulfhidrilo/químicaRESUMEN
Four new carbocyanines containing symmetric and asymmetric heterocyclic moieties and N-carboxyalkyl groups have been synthesized and characterized. The binding mechanism established between these cyanines and several proteins was evaluated using saturation transfer difference (STD) NMR. The results obtained for the different dyes revealed a specific interaction to the standard proteins lysozyme, α-chymotrypsin, ribonuclease (RNase), bovine serum albumin (BSA), and gamma globulin. For instance, the two un-substituted symmetrical dyes (cyanines 1 and 3) interacted preferentially through its benzopyrrole and dibenzopyrrole units with lysozyme, α-chymotrypsin, and RNase, whereas the symmetric disulfocyanine dye (cyanine 2) bound BSA and gamma globulin through its carboxyalkyl chains. On the other hand, the asymmetric dye (cyanine 4) interacts with lysozyme and α-chymotrypsin through benzothiazole moiety and with RNase through dibenzopyrrole unit. Thus, STD-NMR technique was successfully used to screen cyanine-protein interactions and determine potential binding sites of the cyanines for posterior use as ligands in affinity chromatography.
Asunto(s)
Carbocianinas/química , Cromatografía de Afinidad , Colorantes Fluorescentes/química , Albúmina Sérica Bovina/química , Animales , Benzotiazoles/química , Sitios de Unión , Bovinos , Ligandos , Espectroscopía de Resonancia Magnética , Muramidasa/química , Unión Proteica , Proteínas/químicaRESUMEN
For many years the role of lysophospholipids (LPLs) was associated only with structural and storage components of the cell without any informational function. Today, based on many research projects performed during the last decades, it is clear that some of the LPLs act as hormone-like signaling molecules and thus are very important inter- and intracellular lipid mediators. They can activate specific membrane receptors and/or nuclear receptors regulating many crucial physiological and pathophysiological processes. The LPLs were iden- tified as involved in a majority of cellular processes, including modulation of disease-related mechanisms observed, for instance, in case of diabetes, obesity, atherosclerosis and cancer. Among LPLs, lysophosphatidylcholine (LPC) and lysophosphatidylinositol (LPI) are becoming attractive research topics. Their recently revealed activities as novel ligands of orphan G protein-coupled receptors (i.e., GPR55 and GPR119) involved in modulation of tumor physiology and insulin secretion seem to be one of the most interesting aspects of these compounds. Moreover, the most recent scientific reports emphasize the significance of the acyl chain structure bound to the glycerol basis of LPL, as it entails different biological properties and activities of the compounds.
Asunto(s)
Lisofosfatidilcolinas/metabolismo , Lisofosfatidilcolinas/farmacología , Lisofosfolípidos/metabolismo , Lisofosfolípidos/farmacología , Receptores Acoplados a Proteínas G/metabolismo , Transducción de Señal/fisiología , Animales , Diabetes Mellitus/tratamiento farmacológico , Diabetes Mellitus/metabolismo , Humanos , Ligandos , Lisofosfatidilcolinas/química , Lisofosfolípidos/química , Neoplasias/tratamiento farmacológico , Neoplasias/metabolismo , Receptores de Cannabinoides , Receptores Acoplados a Proteínas G/genéticaRESUMEN
BACKGROUND: We previously reported that, among all the naturally occurring amino acids, L-valine is the most powerful luminal stimulator of glucagon-like peptide 1 (GLP-1) release from the upper part of the rat small intestine. This makes L-valine an interesting target for nutritional-based modulation of GLP-1 secretion. However, the molecular mechanism of L-valine-induced secretion remains unknown. METHODS: We aimed to investigate the effect of orally given L-valine in mice and to identify the molecular details of L-valine stimulated GLP-1 release using the isolated perfused rat small intestine and GLUTag cells. In addition, the effect of L-valine on hormone secretion from the distal intestine was investigated using a perfused rat colon. RESULTS: Orally given L-valine (1 g/kg) increased plasma levels of active GLP-1 comparably to orally given glucose (2 g/kg) in male mice, supporting that L-valine is a powerful stimulator of GLP-1 release in vivo (P > 0.05). Luminal L-valine (50 mM) strongly stimulated GLP-1 release from the perfused rat small intestine (P < 0.0001), and inhibition of voltage-gated Ca2+-channels with nifedipine (10 µM) inhibited the GLP-1 response (P < 0.01). Depletion of luminal Na+ did not affect L-valine-induced GLP-1 secretion (P > 0.05), suggesting that co-transport of L-valine and Na+ is not important for the depolarization necessary to activate the voltage-gated Ca2+-channels. Administration of the KATP-channel opener diazoxide (250 µM) completely blocked the L-valine induced GLP-1 response (P < 0.05), suggesting that L-valine induced depolarization arises from metabolism and opening of KATP-channels. Similar to the perfused rat small intestine, L-valine tended to stimulate peptide tyrosine-tyrosine (PYY) and GLP-1 release from the perfused rat colon. CONCLUSIONS: L-valine is a powerful stimulator of GLP-1 release in rodents. We propose that intracellular metabolism of L-valine leading to closure of KATP-channels and opening of voltage-gated Ca2+-channels are involved in L-valine induced GLP-1 secretion.
Asunto(s)
Péptido 1 Similar al Glucagón , Intestino Delgado , Canales KATP , Valina , Animales , Péptido 1 Similar al Glucagón/metabolismo , Masculino , Valina/farmacología , Ratas , Ratones , Intestino Delgado/metabolismo , Intestino Delgado/efectos de los fármacos , Canales KATP/metabolismo , Canales de Calcio/metabolismo , Colon/metabolismo , Colon/efectos de los fármacos , Ratones Endogámicos C57BL , Ratas WistarRESUMEN
Alpha-cyclodextrin (α-CD) is a non-absorbable and soluble fiber that causes weight loss. We studied whether this is due to an effect on GLP-1 secretion. In GLUTag cells, α-CD increased GLP-1 secretion up to 170% via adenylyl cyclase, phospholipase C, and L-type calcium channels dependent processes. In rat isolated colon perfusions, luminal α-CD increased GLP-1 secretion with 20%. In lean mice, once daily α-CD versus saline caused weight loss and lowered the peak in glucose after an oral glucose tolerance test (OGTT). In obese mice, α-CD added to high-fat diet caused weight loss similar to the control group (receiving cellulose). However, compared to cellulose, the α-CD group ate less. During an OGTT, no differences were observed in glucose, insulin and GLP-1. Thus, α-CD increases GLP-1 secretion in a dose-dependent manner and could be a safe and easy addition to food products to help reduce body weight.
Asunto(s)
Péptido 1 Similar al Glucagón , Ratones Endogámicos C57BL , alfa-Ciclodextrinas , Animales , Ratones , Péptido 1 Similar al Glucagón/metabolismo , alfa-Ciclodextrinas/metabolismo , alfa-Ciclodextrinas/química , alfa-Ciclodextrinas/farmacología , Masculino , Ratas , Humanos , Prueba de Tolerancia a la Glucosa , Glucemia/metabolismo , Obesidad/metabolismo , Obesidad/tratamiento farmacológico , Obesidad/fisiopatología , Insulina/metabolismo , Dieta Alta en Grasa/efectos adversosRESUMEN
Objective: Fiber-free diet impairs intestinal and colonic health in mice, in parallel with a reduction in glucagon like peptide-1 (GLP-1) levels. Endogenous GLP-1 is important for intestinal growth and maintenance of the intestinal integrity. We aimed to investigate whether fiber-free diet reduces luminal content of metabolites which, upon supplementation, could increase GLP-1 secretion and restore the adverse effects of fiber-free diet. Methods: Untargeted metabolomics (LC-MS) was performed on colonic content of mice fed a fiber-free diet, identifying a metabolite of particular interest: indole-3-carboxyaldehyde (I3A). We exposed cultured GLUTag cells to I3A, and measured cumulative GLP-1 secretion. Isolated colon perfusions were performed in male C57BL/6JRj mice and Wistar rats. I3A was administered luminally or vascularly, and GLP-1 was measured in portal vein effluent. Finally, female C57BL/6JRJ mice were fed chow or fiber-free diet, with I3A or vehicle by oral gavage. After 10 days, plasma GLP-1 (ELISA) and intestinal permeability (FITC-dextran) were measured, animals were sacrificed and organs removed for histology. Results: Mice fed a fiber-free diet had significantly lower I3A in their colonic content compared to a control diet (7883 ± 3375 AU, p=0.04). GLP-1 secretion from GLUTag cells was unchanged after five minutes of exposure to I3A. However, GLP-1 levels increased after 120 minutes of exposure to 1 mM (60% increase, p=0.016) and 5 mM (89% increase, p=0.0025) I3A. In contrast, 48 h exposure to 1 mM decreased GLP-1 secretion (51% decrease, p<0.001) and viability. In isolated perfused mouse and rat colon, I3A applied into the luminal or vascular side did not affect GLP-1 secretion. Mice fed a fiber-free diet tended to weigh less compared to chow fed mice; and the small intestine and colon were significantly smaller. No differences were seen in crypt depth, villus length, mucosal area, and intestinal permeability. Supplementing I3A did not affect body weight, morphology or plasma GLP-1 levels. Conclusions: Fiber-free diet lowered colonic content of I3A in mice. I3A stimulates GLP-1 secretion in vitro, but not in animal studies. Moreover, it has no evident beneficial effect on intestinal health when administered in vivo.
Asunto(s)
Péptido 1 Similar al Glucagón , Intestino Delgado , Ratas , Ratones , Animales , Masculino , Femenino , Ratas Wistar , Ratones Endogámicos C57BL , Intestino Delgado/metabolismo , Péptido 1 Similar al Glucagón/metabolismo , DietaRESUMEN
The chemical synthesis of phosphorothioate/phosphorodithioate analogues of 2-methoxy-lysophosphatidylcholine has been described. For the preparation of new sulfur derivatives of lysophosphatidylcholine both oxathiaphospholane and dithiaphospholane approaches have been employed. Each lysophospholipid analogue was synthesized as a series of five compounds, bearing different fatty acid residues both saturated (12:0, 14:0, 16:0, 18:0) and unsaturated (18:1). The methylation of glycerol 2-hydroxyl function was applied in order to increase the stability of prepared analogues by preventing 1 â 2 acyl migration. The cellular toxicity of newly synthesized 2-methoxy-lysophosphatidylcholine derivatives was measured using MTT viability assay and lactate dehydrogenase release method.
Asunto(s)
Lisofosfatidilcolinas/química , Lisofosfatidilcolinas/toxicidad , Azufre/química , Animales , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Ácidos Grasos/química , Lisofosfatidilcolinas/síntesis química , Ratones , Relación Estructura-ActividadRESUMEN
Dietary trans-palmitoleic acid (trans 16:1n-7, tPOA), a biomarker for high-fat dairy product intake, has been associated with a lower risk of type 2 diabetes mellitus (T2DM) in some cross-sectional and prospective epidemiological studies. Here, we investigated the insulin secretion-promoting activity of tPOA and compared them with the effects evoked by the cis-POA isomer (cPOA), an endogenous lipokine biosynthesized in the liver and adipose tissue, and found in some natural food sources. The debate about the positive and negative relationships of those two POA isomers with metabolic risk factors and the underlying mechanisms is still going on. Therefore, we examined the potency of both POA isomers to potentiate insulin secretion in murine and human pancreatic ß cell lines. We also investigated whether POA isomers activate G protein-coupled receptors proposed as potential targets for T2DM treatment. We show that tPOA and cPOA augment glucose-stimulated insulin secretion (GSIS) to a similar extent; however, their insulin secretagogue activity is associated with different signaling pathways. We also performed ligand docking and molecular dynamics simulations to predict the preferred orientation of POA isomers and the strength of association between those two fatty acids and GPR40, GPR55, GPR119, and GPR120 receptors. Overall, this study provides insight into the bioactivity of tPOA and cPOA toward selected GPCR functions, indicating them as targets responsible for the insulin secretagogue action of POA isomers. It reveals that both tPOA and cPOA may promote insulin secretion and subsequently regulate glucose homeostasis.
Asunto(s)
Diabetes Mellitus Tipo 2 , Humanos , Ratones , Animales , Secreción de Insulina , Diabetes Mellitus Tipo 2/metabolismo , Estudios Prospectivos , Estudios Transversales , Secretagogos de Insulina , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Glucosa/metabolismo , Biomarcadores/metabolismo , Insulina/metabolismo , Receptores de Cannabinoides/metabolismoRESUMEN
The impaired spatial arrangement and connections between cells creating islets of Langerhans as well as altered expression of G protein-coupled receptors (GPCRs) often lead to dysfunction of insulin-secreting pancreatic ß cells and can significantly contribute to the development of diabetes. Differences in glucose-stimulated insulin secretion (GSIS) are noticeable not only in diabetic individuals but also in model pancreatic ß cells, e.g., ßTC3 and MIN6 ß cell lines with impaired and normal insulin secretion, respectively. Now, we compare the ability of GPCR agonists (lysophosphatidylcholines bearing fatty acid chains of different lengths) to potentiate GSIS in ßTC3 and MIN6 ß cell models, cultured as adherent monolayers and in a form of pseudoislets (PIs) with pancreatic MS1 endothelial cells. Our aim was also to investigate differences in expression of the GPCRs responsive to LPCs in these experimental systems. Aggregation of ß cells into islet-like structures greatly enhanced the expression of Gpr40, Gpr55, and Gpr119 receptors. In contrast, the co-culture of ßTC3 cells with endothelial cells converted the GPCR expression pattern closer to the pattern observed in MIN6 cells. Additionally, the efficiencies of various LPC species in ßTC3-MS1 PIs also shifted toward the MIN6 cell model.
Asunto(s)
Betacelulina/metabolismo , Glucosa/metabolismo , Secreción de Insulina/fisiología , Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Lisofosfatidilcolinas/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , HumanosRESUMEN
In this study, the influence of guelder rose (Viburnum opulus) fruit fresh juice (FJ) and a phenolic-rich fraction (PRF) isolated from juice on mice insulinoma MIN6 cells activities was investigated. Extracts were able to decrease intracellular oxidative stress at the highest non-cytotoxic concentrations. They induced glucagon-like peptide-1 (GLP-1) secretion in the presence of an elevated glucose concentration, and they inhibited in vitro activity of the dipeptidyl peptidase-4 (DPP4) enzyme. Nonetheless, inhibition of glucose-stimulated insulin secretion was detected, which was accompanied by a decrease of cellular membrane fluidity and hyperpolarization effect. In addition, the increase of free fatty acid uptake and accumulation of lipid droplets in MIN6 cells were observed. Elevated extract concentrations induced cell apoptosis through the intrinsic mitochondrial pathway with activation of initiatory caspase-9 and downstream caspases-3/7. The fluorescence-quenching studies indicated that PRF extract has binding affinity to human serum albumin, which is one of the factors determining drug bioavailability. Taken together, despite the cytoprotective activity against generated intracellular oxidative stress, V. opulus revealed potential toxic effects as well as decreased insulin secretion from MIN6 cells. These findings are relevant in understanding V. opulus limitations in developing diet supplements designed for the prevention and treatment of postprandial glucose elevation.
RESUMEN
Diabetes mellitus is a worldwide health problem with high rates of mortality and morbidity. Management of diabetes mellitus by dietary components is achievable especially at the initial stage of the disease. Several studies confirmed the antidiabetic activities of simple phenolic acids and lysophosphatidylcholine (LPC). The main goal of this study was to identify new potential insulin secretion modulators obtained by combining the structures of two natural compounds, namely O-methyl derivatives of phenolic acids and phospholipids. LPC and phosphatidylcholine bearing methoxylated aromatic carboxylic acids were tested as potential agents able to improve glucose-stimulated insulin secretion (GSIS) and intracellular calcium mobilization in MIN6 ß pancreatic cell line. Our results show that LPC with covalently bonded molecule of p-anisic acid at the sn-1 position was able to induce GSIS and intracellular calcium flux. Notably, 1-anisoyl-2-hydroxy-sn-glycero-3-phosphocholine did not affect the viability of MIN6 cells, suggesting its potential safe use. Furthermore, we have shown that three G protein coupled receptors, namely GPR40, GPR55, and GPR119, are targeted by this LPC derivative.
Asunto(s)
Ácido Benzoico/farmacología , Ácidos Carboxílicos/farmacología , Secreción de Insulina/efectos de los fármacos , Células Secretoras de Insulina/metabolismo , Lisofosfatidilcolinas/farmacología , Fosfatidilcolinas/farmacología , Receptores Acoplados a Proteínas G/metabolismo , Animales , Calcio/metabolismo , Células Cultivadas , Glucosa/farmacología , Ratones , Estimulación QuímicaRESUMEN
G protein-coupled receptors (GPCRs) are the most intensively studied drug targets, because of their diversity, cell-specific expression, and druggable sites accessible at the cell surface. Preclinical and clinical studies suggest that targeting GPCRs activated by fatty acid-derived lipids may have potential to improve glucose homeostasis and reduce complications in patients with type 2 diabetes (T2D). Despite the discontinued development of fasiglifam (TAK-875), the first FFA1 agonist to reach late-stage clinical trials, lipid-sensing receptors remain a viable target, albeit with a need for further characterization of their binding mode, intracellular signaling, and toxicity. Herein, we analyze general discovery trends, various signaling pathways, as well as possible challenges following activation of GPCRs that have been validated clinically to control blood glucose levels.
Asunto(s)
Diabetes Mellitus Tipo 2/metabolismo , Ácidos Grasos/metabolismo , Receptores Acoplados a Proteínas G/antagonistas & inhibidores , Animales , Benzofuranos/administración & dosificación , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Diabetes Mellitus Tipo 2/genética , Humanos , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Sulfonas/administración & dosificaciónRESUMEN
Lysophosphatidylcholine (LPC) is an endogenous ligand for GPR119 receptor, mediating glucose-stimulated insulin secretion (GSIS). We demonstrate that LPC facilitates GSIS in MIN6 pancreatic ß-cell line and murine islets of Langerhans by recognizing not only GPR119 but also GPR40 (free fatty acid receptor 1) and GPR55 activated by lysophosphatidylinositol. Natural LPCs are unstable when administered in vivo limiting their therapeutic value and therefore, we present phosphorothioate LPC analogues with increased stability. All the modified LPCs under study (12:0, 14:0, 16:0, 18:0, and 18:1) significantly enhanced GSIS. The 16:0 sulfur analogue was the most potent, evoking 2-fold accentuated GSIS compared to the native counterpart. Interestingly, LPC analogues evoked GPR40-, GPR55-and GPR119-dependent [Ca2+]i signaling, but did not stimulate cAMP accumulation as in the case of unmodified molecules. Thus, introduction of a phosphorothioate function not only increases LPC stability but also modulates affinity towards receptor targets and evokes different signaling pathways.
Asunto(s)
Secreción de Insulina/efectos de los fármacos , Lisofosfatidilcolinas/farmacología , Fosfatos/farmacología , Receptores de Cannabinoides/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Animales , Señalización del Calcio/efectos de los fármacos , Línea Celular , AMP Cíclico/metabolismo , Endocannabinoides/farmacología , Glucosa/farmacología , Lisofosfatidilcolinas/química , Masculino , Ratones Endogámicos C57BL , Ácidos Oléicos/farmacología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptores de Cannabinoides/genética , Receptores Acoplados a Proteínas G/genéticaRESUMEN
Nutrient-induced increase in intracellular Ca(2+) concentration ([Ca(2+)]i) is one of the key mechanisms responsible for insulin release from pancreatic islet ß cells. Lysophosphatidylcholine (LPC) was demonstrated to induce insulin secretion from ß cells, activate glucose uptake and effectively lower blood glucose levels in mouse models of type 1 and 2 diabetes mellitus. The article hereby presents the results of a characterization of 2-OMe-LPC sulfur analogues with defined acyl residues in terms of their effect on intracellular Ca(2+) concentration and cellular membrane integrity in the murine ßTC-3 cell model. Active LPC series that could induce calcium flux in ßTC-3 cell model include unmodified LPC 12:0, 14:0, 16:0, and 18:0 as well as phosphorothioate analogues of LPC 12:0, 14:0 and 16:0. However, in the case of species bearing mirystoyl and palmitoyl residues [Ca(2+)]i was associated with membrane permeabilization as demonstrated by propidium iodide incorporation and lactate dehydrogenase release. LPC 12:0 (both unmodified and a sulfurcontaining counterpart) and unmodified LPC 18:0 did not demonstrate membrane disruption but acted as calcium inducers. Interestingly, no stimulation of calcium flux or membrane disruption was observed in the case of LPC analogues with two sulfur atoms introduced into a phosphate group. Experiments with nitrendipine and NiCl2 blocking voltage-dependent calcium channels and the general calcium influx, respectively, revealed remarkably that the compounds studied were involved in different signaling mechanisms while administered to the cell culture, which is clearly related to their chemical structure, both acyl chain and modification dependently.
Asunto(s)
Antineoplásicos/química , Células Secretoras de Insulina/efectos de los fármacos , Insulina/metabolismo , Lisofosfatidilcolinas/química , Compuestos de Azufre/química , Animales , Antineoplásicos/farmacología , Calcio/metabolismo , Canales de Calcio/metabolismo , Técnicas de Cultivo de Célula , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Secreción de Insulina , Células Secretoras de Insulina/metabolismo , Lisofosfatidilcolinas/farmacología , Fluidez de la Membrana/efectos de los fármacos , Ratones , Estructura Molecular , Compuestos de Azufre/farmacologíaRESUMEN
A method has been developed for laser desorption/ionization of catecholamines from porous silicon. This methodology is particularly attractive for analysis of small molecules. MALDI TOF mass spectrometry, although a very sensitive technique, utilizes matrices that need to be mixed with the sample prior to their analysis. Each matrix produces its own background, particularly in the low-molecular mass region. Therefore, detection and identification of molecules below 400 Da can be difficult. Desorption/ionization of samples deposited on porous silicon does not require addition of a matrix, thus, spectra in the low-molecular mass region can be clearly readable. Here, we describe a method for the analysis of catecholamines. While MALDI TOF is superior for proteomics/peptidomics, desorption/ionization from porous silicon can extend the operating range of a mass spectrometer for studies on metabolomics (small organic molecules and their metabolites, such as chemical neurotransmitters, prostaglandins, steroids, etc.).
Asunto(s)
Catecolaminas/química , Silicio/química , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Biotecnología , Espectrometría de Masas , Propiedades de SuperficieRESUMEN
Desorption/ionization from porous silicon dioxide (DIOSD), in combination with a standard matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) instrument, was used for the identification of catecholamines in the human peripheral blood lymphocytes. A routine MALDI-TOF analysis does not allow for sensitive detection of low molecular mass compounds (i.e. below 400 Da) due to the pronounced background ions arising from the matrix. Therefore, we have tested DIOSD methodology for the identification of catecholamines in the immune system. Using DIOSD, catecholamines were unambiguously identified in the cell extract of peripheral blood lymphocytes at the femtomolar level. The DIOSD extends the possible use of MALDI-TOF mass spectrometry towards small molecules that were previously detected by other methods.