RESUMO
Cardiac alteration due to chronic kidney disease is described by tissue fibrosis. This remodeling involves myofibroblasts of various origins, including epithelial or endothelial to mesenchymal transitions. In addition, obesity and insulin resistance together or separately seem to exacerbate cardiovascular risk in chronic kidney disease (CKD). The main objective of this study was to assess if pre-existing metabolic disease exacerbates CKD-induced cardiac alterations. In addition, we hypothesised that endothelial to mesenchymal transition participates in this enhancement of cardiac fibrosis. Rats fed cafeteria type diet for 6 months underwent a subtotal nephrectomy at 4 months. Cardiac fibrosis was evaluated by histology and qRT-PCR. Collagens and macrophages were quantified by immunohistochemistry. Endothelial to mesenchymal transitions were assessed by qRT-PCR (CD31, VE-cadherin, α-SMA, nestin) and also by CD31 immunofluorescence staining. Rats fed with cafeteria type regimen were obese, hypertensive and insulin resistant. Cardiac fibrosis was predominant in CKD rats and was highly majored by cafeteria regimen. Collagen-1 and nestin expressions were higher in CKD rats, independently of regimen. Interestingly, in rats with CKD and cafeteria diet we found an increase of CD31 and α-SMA co-staining with suggest an implication of endothelial to mesenchymal transition during heart fibrosis. We showed that rats already obese and insulin resistant had an enhanced cardiac alteration to a subsequent renal injury. Cardiac fibrosis process could be supported by a involvement of the endothelial to mesenchymal transition phenomenon.
Assuntos
Insulinas , Síndrome Metabólica , Insuficiência Renal Crônica , Ratos , Animais , Nestina , Síndrome Metabólica/patologia , Remodelação Ventricular , Insuficiência Renal Crônica/patologia , Rim/patologia , Fibrose , Obesidade/complicações , Obesidade/patologia , Transição Epitelial-MesenquimalRESUMO
Fibrosis is a common feature of cardiovascular diseases and targets multiple organs, such as the heart and vessels. Endothelial to mesenchymal transition is a complex, vital process that occurs during embryonic formation and plays a crucial role in cardiac development. It is also a fundamental process implicated in cardiac fibrosis and repair, but also in other organs. Indeed, in numerous cardiovascular diseases, the endothelial-to-mesenchymal transition has been shown to be involved in the generation of fibroblasts that are able to produce extracellular matrix proteins such as type I collagen. This massive deposition results in tissue stiffening and organ dysfunction. To advance our understanding of this process for the development of new specific diagnostic and therapeutic strategies, it is essential to develop relevant cellular and animal models of this process. In this review, our aim was to gain an in-depth insight into existing in vitro and in vivo models of endothelial to mesenchymal transition in cardiovascular diseases with a focus on cardiac fibrosis. We discuss important parameters impacting endothelial to mesenchymal transition, and we give perspectives for the development of relevant models to decipher the underlying mechanisms and ultimately find new treatments specific to fibrosis happening in cardiovascular diseases.
Assuntos
Doenças Cardiovasculares , Animais , Coração , Colágeno Tipo I , Fibrose , Modelos TeóricosRESUMO
We previously reported dipeptidomimetic compounds as inhibitors of neuronal and/or inducible NO synthases (n/iNOS) with significant selectivity against endothelial NOS (eNOS). They were composed of an S-ethylisothiocitrullin-like moiety linked to an extension through a peptide bond or a 1,2,4-oxadiazole link. Here, we developed two further series where the extension size was increased to establish more favorable interactions in the NOS substrate access channel. The extension was introduced on the solid phase by the reductive alkylation of an amino-piperidine moiety or an aminoethyl segment in the case of dipeptide-like and 1,2,4-oxadiazole compounds, respectively, with various benzaldehydes. Compared to the previous series, more potent inhibitors were identified with IC50 in the micromolar to the submicromolar range, with significant selectivity toward nNOS. As expected, most compounds did not inhibit eNOS, and molecular modeling was carried out to characterize the reasons for the selectivity toward nNOS over eNOS. Spectral studies showed that compounds were interacting at the heme active site. Finally, selected inhibitors were found to inhibit intra-cellular iNOS and nNOS expressed in RAW264.7 and INS-1 cells, respectively.
Assuntos
Inibidores Enzimáticos , Óxido Nítrico Sintase , Óxido Nítrico Sintase/metabolismo , Inibidores Enzimáticos/química , Dipeptídeos/química , Técnicas de Síntese em Fase Sólida , Óxido Nítrico Sintase Tipo I , Óxido Nítrico Sintase Tipo II , Modelos Moleculares , Óxido Nítrico Sintase Tipo IIIRESUMO
In the view of the relationships between excessive sodium intake, immunity and target organ damage, we hypothesized that reduction in dietary sodium would be beneficial in the prevention of cardiac alterations through a restrained local immunity response in a rat model of metabolic syndrome. Sprague-Dawley rats were fed a 60% fructose diet with either a normal sodium (0.64% NaCl) or a low sodium content (<0.01% NaCl) for 8weeks. After 4weeks, rats were infused or not with angiotensin II (200ng·kg-1·min-1, sc) for 4weeks. Tail-cuff blood pressure was determined in conscious rats. Heart and left ventricle weight, cardiomyocyte size, and cardiac fibrosis were evaluated. We performed a transcriptomic analysis in order to identify differentially regulated cardiac mRNAs between normal and low sodium diets. We validated those results using qPCR and immunohistochemistry. Angiotensin II-induced blood pressure rise was blunted (~50%) in the low-sodium fed rats while cardiac hypertrophy and fibrosis were prevented. Transcriptomic analysis revealed 66 differentially regulated genes including 13 downregulated genes under the low sodium diet and implicated in the innate immune response. This was confirmed by reduced cardiac macrophages infiltration under the low sodium diet. Dietary sodium restriction prevents structural alterations of the heart of rats with fructose-induced insulin resistance and angiotensin II-hypertension. The reduction of cardiac inflammation and macrophage infiltration suggests that innate immunity has an important role in the beneficial effect of sodium restriction on cardiac remodeling.
Assuntos
Cardiomegalia , Dieta Hipossódica , Carboidratos da Dieta/efeitos adversos , Frutose/efeitos adversos , Imunidade Inata , Síndrome Metabólica , Animais , Cardiomegalia/dietoterapia , Cardiomegalia/imunologia , Carboidratos da Dieta/farmacologia , Modelos Animais de Doenças , Fibrose , Frutose/farmacologia , Síndrome Metabólica/induzido quimicamente , Síndrome Metabólica/dietoterapia , Síndrome Metabólica/imunologia , Ratos , Ratos Sprague-Dawley , Cloreto de Sódio na Dieta/efeitos adversos , Cloreto de Sódio na Dieta/farmacologiaRESUMO
AIMS/HYPOTHESIS: Insulin-mediated glucose transport and utilisation are decreased in skeletal muscle from type 2 diabetic and glucose-intolerant individuals because of alterations in insulin receptor signalling, GLUT4 translocation to the plasma membrane and microvascular blood flow. Catalytic activity of the muscle-specific isoform of neuronal nitric oxide synthase (nNOS) also participates in the regulation of glucose transport and appears to be decreased in a relevant animal model of drastic insulin resistance, the obese Zucker fa/fa rat. Our objective was to determine the molecular mechanisms involved in this defect. METHODS: Isolated rat muscles and primary cultures of myocytes were used for western blot analysis of protein expression, immunohistochemistry, glucose uptake measurements and GLUT4 translocation assays. RESULTS: nNOS expression was reduced in skeletal muscle from fa/fa rats. This was caused by increased ubiquitination of the enzyme and subsequent degradation by the ubiquitin proteasome pathway. The degradation occurred through a greater interaction of nNOS with the chaperone heat-shock protein 70 and the co-chaperone, carboxyl terminus of Hsc70-interacting protein (CHIP). In addition, an alteration in nNOS sarcolemmal localisation was observed. We confirmed the implication of nNOS breakdown in defective insulin-induced glucose transport by demonstrating that blockade of proteasomal degradation or overexpression of nNOS improved basal and/or insulin-stimulated glucose uptake and GLUT4 translocation in primary cultures of insulin-resistant myocytes. CONCLUSIONS/INTERPRETATION: Recovery of nNOS in insulin-resistant muscles should be considered a potential new approach to address insulin resistance.
Assuntos
Glucose/metabolismo , Resistência à Insulina/fisiologia , Músculo Esquelético/metabolismo , Óxido Nítrico Sintase Tipo I/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Animais , Western Blotting , Células Cultivadas , Transportador de Glucose Tipo 4/metabolismo , Imunoprecipitação , Masculino , Células Musculares/metabolismo , Óxido Nítrico Sintase Tipo I/genética , Ratos , Ratos Zucker , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Sodium depletion has a protective effect on target-organ damage in hypertension independent of blood pressure. Here we tested whether chronic dietary sodium restriction may prevent the development of renal alterations associated with insulin resistance by reducing the inflammatory and oxidant state. Rats were fed normal-salt-60% fructose, low-salt-60% fructose, or control normal-salt diet for 12 weeks. Insulin resistance induced by high-fructose diet was associated with an increase in albuminuria, tubular and glomerular hypertrophy, and inflammation of kidney and adipose tissue. The low-salt diet improved insulin sensitivity and prevented kidney damage. These beneficial effects of sodium depletion were associated with a decrease in renal inflammation (macrophage infiltration, IL-6, TNF-α) and oxidative stress (NADPH oxidase activity), and a prevention of histologic changes in retroperitoneal fat induced by high fructose. Thus, dietary salt depletion has beneficial effects on renal and metabolic alterations associated with a high-fructose diet in rats.
Assuntos
Albuminúria/prevenção & controle , Dieta Hipossódica , Frutose , Resistência à Insulina , Rim/metabolismo , Albuminúria/sangue , Albuminúria/induzido quimicamente , Albuminúria/imunologia , Albuminúria/patologia , Animais , Glicemia/metabolismo , Desmina/metabolismo , Modelos Animais de Doenças , Fibrose , Mediadores da Inflamação/metabolismo , Interleucina-6/metabolismo , Gordura Intra-Abdominal/metabolismo , Gordura Intra-Abdominal/patologia , Rim/imunologia , Rim/ultraestrutura , Macrófagos/imunologia , Masculino , NADPH Oxidases/metabolismo , Estresse Oxidativo , Ratos , Ratos Sprague-Dawley , Fatores de Tempo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Obesity is a significant risk factor for chronic kidney disease (CKD). This study aimed to evaluate the impact of obesity on the development of kidney fibrosis in a model of cafeteria diet rats undergoing 5/6th nephrectomy (SNx). Collagen 1, 3, and 4 expression, adipocyte size, macrophage number, and the expression of 30 adipokines were determined. Collagen 1 expression in kidney tissue was increased in Standard-SNx and Cafeteria-SNx (7.1 ± 0.6% and 8.9 ± 0.9 tissue area, respectively). Renal expression of collagen 3 and 4 was significantly increased (p < 0.05) in Cafeteria-SNx (8.6 ± 1.5 and 10.9 ± 1.9% tissue area, respectively) compared to Cafeteria (5.2 ± 0.5 and 6.3 ± 0.6% tissue area, respectively). Adipocyte size in eWAT was significantly increased by the cafeteria diet. In Cafeteria-SNx, we observed a significant increase in macrophage number in the kidney (p = 0.01) and a consistent tendency in eWAT. The adipokine level was higher in the Cafeteria groups. Interleukin 11, dipeptidyl peptidase 4, and serpin 1 were increased in Cafeteria-SNx. In the kidney, collagen 3 and 4 expressions and the number of macrophages were increased in Cafeteria-SNx, suggesting an exacerbation by preexisting obesity of CKD-induced renal inflammation and fibrosis. IL11, DPP4, and serpin 1 can act directly on fibrosis and participate in the observed worsening CKD.
Assuntos
Insuficiência Renal Crônica , Serpinas , Ratos , Animais , Insuficiência Renal Crônica/etiologia , Insuficiência Renal Crônica/metabolismo , Nefrectomia/efeitos adversos , Fibrose , Obesidade/complicações , Dieta/efeitos adversos , ColágenoRESUMO
Increased senescent cell burden and dysregulation of the nuclear factor erythroid 2-related factor 2 (NRF2) pathway have been associated with numerous age-related pathologies; however, their role in promoting vascular calcification (VC) in chronic kidney disease (CKD) has yet to be determined. We investigated whether senescence and NRF2 pathways may serve as drivers of uremia-induced VC using three complementary approaches: a novel model of induced VC in 5/6-nephrectomized rats supplemented with high phosphate and vitamin D; epigastric arteries from CKD patients with established medial calcification; and vascular smooth muscle cells (VSMCs) incubated with uremic serum. Expression of p16Ink4a and p21Cip1, as well as γ-H2A-positive cells, confirmed increased senescent cell burden at the site of calcium deposits in aortic sections in rats, and was similarly observed in calcified epigastric arteries from CKD patients through increased p16Ink4a expression. However, uremic serum-induced VSMC calcification was not accompanied by senescence. Expression of NRF2 and downstream genes, Nqo1 and Sod1, was associated with calcification in uremic rats, while no difference was observed between calcified and non-calcified EAs. Conversely, in vitro uremic serum-driven VC was associated with depleted NRF2 expression. Together, our data strengthen the importance of senescence and NRF2 pathways as potential therapeutic options to combat VC in CKD.
Assuntos
Insuficiência Renal Crônica , Calcificação Vascular , Ratos , Animais , Fator 2 Relacionado a NF-E2/metabolismo , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Músculo Liso Vascular/metabolismo , Calcificação Vascular/genética , Insuficiência Renal Crônica/patologia , Senescência CelularRESUMO
BACKGROUND: The objective of the COMET (COllection of MEtabolic Tissues) biobank project is to create a high-quality collection of insulin-sensitive tissues (liver, muscle, adipose tissues, and epiploic artery) and blood sample derivatives (plasma, serum, DNA and RNA), collected from 270 grade 2-3 obese patients undergoing bariatric surgery. Relevant data on patient such as clinical/biological characteristics and sample handling are also collected. For this, our aim was to establish a Quality Management System (QMS) to meet the reliability and quality requirements necessary for its scientific exploitation. MATERIALS AND METHODS: The COMET QMS includes: (1) Quality Assurance to standardize all stages of the biobanking process, (2) Quality Controls on samples from the first patients included in order to validate the sample management process and ensure reproducible quality; and 3) "in process" Quality Controls to ensure the reliability of the storage procedures and the stability of the samples over time. RESULTS: For serum and plasma, several corrective actions, such as temperature handling and centrifugation conditions, were made to the protocol and led to improvement of the volume and quality of samples. Regarding DNA, all samples evaluated achieved a satisfactory level of purity and integrity and most of them yielded the required DNA quantity. All frozen tissue samples had RNAs of good purity. RNA quality was confirmed by RIN, achieving values in most cases over 7 and efficient amplification of housekeeping genes by RT-qPCR, with no significant differences among samples from the same tissue type. In the "in process" Quality Controls, DNA, RNA, and histological integrity of tissues showed no differences among samples after different preservation times. CONCLUSION: Quality Control results have made it possible to validate the entire biobank process and confirm the utility of implementing QMS to guarantee the quality of a biospecimen collection.
Assuntos
Bancos de Espécimes Biológicos , RNA , Humanos , Reprodutibilidade dos Testes , Preservação Biológica , Manejo de Espécimes/métodos , DNARESUMO
During early stages of type 2 diabetes, named prediabetes, pancreatic ß-cells compensate for insulin resistance through increased insulin secretion in order to maintain normoglycemia. Obesity leads to the development of ectopic fat deposits, among which peri-pancreatic white adipose tissue (pWAT) can communicate with ß-cells through soluble mediators. Thus we investigated whether pWAT produced oxygenated lipids, namely isoprostanes and neuroprostanes and whether they can influence ß-cell function in obesity. In the Zucker fa/fa rat model, pWAT and epididymal white adipose tissue (eWAT) displayed different inflammatory profiles. In obese rats, pWAT, but not eWAT, released less amounts of 5-F2t-isoprostanes, 15-F2t-isoprostanes, 4-F4t-neuroprostanes and 10-F4t-neuroprostane compared to lean animals. These differences could be explained by a greater induction of antioxidant defenses enzymes such as SOD-1, SOD-2, and catalase in pWAT of obese animals compared to eWAT. In addition, sPLA2 IIA, involved in the release of isoprostanoids from cellular membranes, was decreased in pWAT of obese animals, but not in eWAT, and may also account for the reduced release of oxidized lipids by this tissue. At a functional level, 15-F2t-isoprostane epimers, but not 5-F2t-isoprostanes, were able to decrease glucose-induced insulin secretion in pancreatic islets from Wistar rats. This effect appeared to be mediated through activation of the thromboxane A2 receptor and reduction of cAMP signaling in pancreatic islets. In conclusion, through the removal of an inhibitory tone exerted by isoprostanes, we have shown, for the first time, a new mechanism allowing ß-cells to compensate for insulin resistance in obesity that is linked to a biocommunication between adipose tissue and ß-cells.
Assuntos
Diabetes Mellitus Tipo 2 , Resistência à Insulina , Tecido Adiposo , Animais , Insulina , Isoprostanos , Obesidade , Ratos , Ratos Wistar , Ratos ZuckerRESUMO
AIM: Diagnosis of nonalcoholic steatohepatitis (NASH) relies on liver biopsy. Noninvasive tools would be useful to target patients to refer for a biopsy. We aimed to determine the diagnostic value of the triglycerides and glucose (TyG) index, an insulin-resistance indicator, to predict NASH. METHODS: Our study included grade II-III obese patients aged 18-65 years undergoing bariatric surgery and included in the COMET (COllection of MEtabolic Tissues) biobank (NCT02861781). Liver biopsies performed during bariatric surgery were collected from the biobank along with blood derivatives. Biopsies were analysed according to the steatosis, activity and fibrosis (SAF) scoring system to diagnose NASH, nonalcoholic fatty liver disease (NAFLD), and fibrosis. Logistic regression models were performed to identify factors predicting NASH, NAFLD, and fibrosis. RESULTS: Of 238 analysed subjects (mean age 43±12 years, 33.6% men), 29% had type 2 diabetes. Steatosis was present in 67.2%, while NASH and advanced fibrosis (stage F3) were diagnosed in 18.1% and 2.9% respectively. TyG index was independently associated with NASH (odds ratio (OR): 4.7 [95% confidence interval: 2.3;9.5] P < 0.0001), NAFLD (OR: 2.0 [1.1;3.7] P = 0.03) and stages 2-3 fibrosis (OR: 4.0 [1.5;10.8] P = 0.007). NASH was also predicted by gamma-glutamyl transferase (GGT) with an area under the ROC curve: 0.79 [0.71;0.87 P = 0.04] for GGT and TyG index combined. CONCLUSION: In our cohort of severely obese patients, TyG index, when associated with GGT level, exhibited high diagnostic performance to predict NASH. Although validation in larger populations is needed, this result may be of considerable clinical value to predict need for liver biopsy.
Assuntos
Diabetes Mellitus Tipo 2 , Hepatopatia Gordurosa não Alcoólica , Adolescente , Adulto , Idoso , Biomarcadores , Biópsia , Diabetes Mellitus Tipo 2/complicações , Diabetes Mellitus Tipo 2/epidemiologia , Diabetes Mellitus Tipo 2/patologia , Feminino , Fibrose , Glucose , Humanos , Fígado/patologia , Masculino , Pessoa de Meia-Idade , Hepatopatia Gordurosa não Alcoólica/complicações , Hepatopatia Gordurosa não Alcoólica/epidemiologia , Obesidade/complicações , Obesidade/epidemiologia , Obesidade/patologia , Triglicerídeos , Adulto JovemRESUMO
Constitution of biobank of human tissues requires careful handling and storage of biological material, to guarantee the quality of samples. Tissue preparation is also critical for further applications such as transcriptomic profiling. In this study, our aim was to evaluate the impact of different disruption techniques (FastPrep-24 instrument, GentleMACS dissociator, and syringe/needle) and homogenizing buffers (RLT versus QIAzol) on RNA purity and quality of metabolic tissues (adipose tissues, liver and skeletal muscle) present in the COMET Biobank. For all homogenization methods used and tissue types, the A260/280 ratios reached values ≥ 1.8, which are in the range of what is found in human tissues and cell lines, while the A260/230 ratios were however ≤ 1.8, with the lowest value obtained with GentleMACS Dissociator. In addition, GentleMACS Dissociator combined with QIAzol reagent gave the highest RIN value and 28S/18S ratio for all tissues tested, except for muscle. Performing RT-qPCR, Ct values for different housekeeping genes can be influenced by extraction methods and RNA quality of samples. In conclusion, we have demonstrated that different disruption techniques and homogenizing buffers impact the purity and some quality markers of RNA, and can also impact quantification of mRNAs by RT-qPCR in human metabolic tissues.
Assuntos
Tecido Adiposo/química , Fígado/química , Músculo Esquelético/química , RNA Mensageiro/isolamento & purificação , Bancos de Espécimes Biológicos , Perfilação da Expressão Gênica , Técnicas Genéticas , Humanos , Reação em Cadeia da Polimerase em Tempo Real , Manejo de EspécimesRESUMO
Excessive fat consumption leads to the development of ectopic adipose tissues, affecting the organs they surround. Peripancreatic adipose tissue is implicated in glucose homeostasis regulation and can be impaired in obesity. High palm oil consumption's effects on health are still debated. We hypothesised that crude and refined palm oil high-fat feeding may have contrasting effects on peripancreatic adipocyte hypertrophy, inflammation and lipid oxidation compound production in obese rats. In Wistar rats, morphological changes, inflammation and isoprostanoid production following oxidative stress were assessed in peripancreatic adipose tissue after 12 weeks of diets enriched in crude or refined palm oil or lard (56% energy from fat in each case) versus a standard chow diet (11% energy from fat). Epididymal white and periaortic brown adipose tissues were also included in the study. A refined palm oil diet disturbed glucose homeostasis and promoted lipid deposition in periaortic locations, as well as adipocyte hypertrophy, macrophage infiltration and isoprostanoid (5-F2c-isoprostane and 7(RS)-ST-Δ8-11-dihomo-isofuran) production in peripancreatic adipose tissue. Crude palm oil induced a lower impact on adipose deposits than its refined form and lard. Our results show that the antioxidant composition of crude palm oil may have a protective effect on ectopic adipose tissues under the condition of excessive fat intake.
Assuntos
Tecido Adiposo/efeitos dos fármacos , Gorduras na Dieta/administração & dosagem , Inflamação/induzido quimicamente , Óleo de Palmeira/administração & dosagem , Tecido Adiposo/patologia , Animais , Glicemia , Peso Corporal , Dieta Hiperlipídica/efeitos adversos , Glucose/metabolismo , Lipídeos/sangue , Macrófagos/efeitos dos fármacos , Macrófagos/fisiologia , Masculino , Ratos , Ratos WistarRESUMO
BACKGROUND: Due to the shortage of multi-organ donors, human pancreatic islet transplantation has now been extended to islets originating from obese subjects. In this study, our aim is to compare the respective sensitivity of human islets from lean vs obese donors to chronic high glucose or high palmitate. METHODS: Human islets were isolated from pancreases harvested from brain-dead multi-organ donors. Islets were cultured during 72 hours in the presence of moderate (16.7 mmol/L) or high (28 mmoL/L) glucose concentrations, or glucose (5.6 mmoL/L) and palmitate (0.4 mmoL/L), before measurement of their response to glucose. RESULTS: We first observed a greater insulin response in islets from obese donors under both basal and high-glucose conditions, confirming their hyperresponsiveness to glucose. When islets from obese donors were cultured in the presence of moderate or high glucose concentrations, insulin response to glucose remained unchanged or was slightly reduced, as opposed to that observed in lean subjects. Moreover, culturing islets from obese donors with high palmitate also induced less reduction in insulin response to glucose than in lean subjects. This partial protection of obese islets is associated with less induction of inducible nitric oxide synthase in islets, together with a greater expression of the transcription factor forkhead box O1 (FOXO1). CONCLUSIONS: Our data suggest that in addition to an increased sensitivity to glucose, islets from obese subjects can be considered as more resistant to glucose and fatty acid excursions and are thus valuable candidates for transplantation.
Assuntos
Glucose/farmacologia , Secreção de Insulina/efeitos dos fármacos , Ilhotas Pancreáticas/efeitos dos fármacos , Obesidade/metabolismo , Palmitatos/farmacologia , Idoso , Humanos , Ilhotas Pancreáticas/metabolismo , Masculino , Pessoa de Meia-IdadeRESUMO
More than 160 arginine analogues modified on the C-terminus via either an amide bond or a heterocyclic moiety (1,2,4-oxadiazole, 1,3,4-oxadiazole and 1,2,4-triazole) were prepared as potential inhibitors of NO synthases (NOS). A methodology involving formation of a thiocitrulline intermediate linked through its side-chain on a solid support followed by modification of its carboxylate group was developed. Finally, the side-chain thiourea group was either let unchanged, S-alkylated (Me, Et) or guanidinylated (Me, Et) to yield respectively after TFA treatment the corresponding thiocitrulline, S-Me/Et-isothiocitrulline and N-Me/Et-arginine substrate analogues. They all were tested against three recombinant NOS isoforms. Several compounds containing a S-Et- or a S-Me-Itc moiety and mainly belonging to both the dipeptide-like and 1,2,4-oxadiazole series were shown to inhibit nNOS and iNOS with IC50 in the 1-50â µM range. Spectral studies confirmed that these new compounds interacted at the heme active site. The more active compounds were found to inhibit intra-cellular iNOS expressed in RAW264.7 and INS-1 cells with similar efficiency than the reference compounds L-NIL and SEIT.
Assuntos
Dipeptídeos/farmacologia , Inibidores Enzimáticos/farmacologia , Compostos Heterocíclicos/farmacologia , Óxido Nítrico Sintase/antagonistas & inibidores , Técnicas de Síntese em Fase Sólida , Animais , Bovinos , Linhagem Celular , Dipeptídeos/síntese química , Dipeptídeos/química , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Compostos Heterocíclicos/síntese química , Compostos Heterocíclicos/química , Camundongos , Óxido Nítrico Sintase/metabolismo , RatosRESUMO
Stimulation of numerous G protein-coupled receptors leads to the elevation of intracellular concentrations of cAMP, which subsequently activates the PKA pathway. Specificity of the PKA signaling module is determined by a sophisticated subcellular targeting network that directs the spatiotemporal activation of the kinase. This specific compartmentalization mechanism occurs through high-affinity interactions of PKA with A-kinase anchoring proteins (AKAPs), the role of which is to target the kinase to discrete subcellular microdomains. Recently, a peptide designated "AKAPis" has been proposed to competitively inhibit PKA-AKAP interactions in vitro. We therefore sought to characterize a cell-permeable construct of the AKAPis inhibitor and use it as a tool to characterize the impact of PKA compartmentalization by AKAPs. Using insulin-secreting pancreatic beta-cells (INS-1 cells), we showed that TAT-AKAPis (at a micromolar range) dose dependently disrupted a significant fraction of endogenous PKA-AKAP interactions. Immunoflurescent analysis also indicated that TAT-AKAPis significantly affected PKA subcellular localization. Furthermore, TAT-AKAPis markedly attenuated glucagon-induced phosphorylations of p44/p42 MAPKs and cAMP response element binding protein, which are downstream effectors of PKA. In parallel, TAT-AKAPis dose dependently inhibited the glucagon-induced potentiation of insulin release. Therefore, AKAP-mediated subcellular compartmentalization of PKA represents a key mechanism for PKA-dependent phosphorylation events and potentiation of insulin secretion in intact pancreatic beta-cells. More interestingly, our data highlight the effectiveness of the cell-permeable peptide-mediated approach to monitoring in cellulo PKA-AKAP interactions and delineating PKA-dependent phosphorylation events underlying specific cellular responses.
Assuntos
Proteínas de Ancoragem à Quinase A/metabolismo , Permeabilidade da Membrana Celular , Subunidade RIIalfa da Proteína Quinase Dependente de AMP Cíclico/metabolismo , Células Secretoras de Insulina/enzimologia , Proteínas Nucleares/metabolismo , Fragmentos de Peptídeos/metabolismo , Transdução de Sinais , Animais , Linhagem Celular , AMP Cíclico/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Imunofluorescência , Glucagon/metabolismo , Glucose/metabolismo , Imunoprecipitação , Insulina/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Fosforilação , Ligação Proteica , Transporte Proteico , Ratos , Fatores de Tempo , Produtos do Gene tat do Vírus da Imunodeficiência Humana/metabolismoRESUMO
BACKGROUND: Zika virus (ZIKV) has recently re-emerged as a pathogenic agent with epidemic capacities as was well illustrated in South America. Because of the extent of this health crisis, a number of more serious symptoms have become associated with ZIKV infection than what was initially described. In particular, neuronal and ocular disorders have been characterized, both in infants and in adults. Notably, the macula and the retina can be strongly affected by ZIKV, possibly by a direct effect of the virus. This is supported by the detection of replicative and infectious virus in lachrimal fluid in human patients and mouse models. METHODS: Here, we used an innovative, state-of-the-art iPSC-derived human retinal pigment epithelium (RPE) model to study ZIKV retinal impairment. FINDINGS: We showed that the human RPE is highly susceptible to ZIKV infection and that a ZIKV African strain was more virulent and led to a more potent epithelium disruption and stronger anti-viral response than an Asian strain, suggesting lineage differences. Moreover, ZIKV infection led to impaired membrane dynamics involved in endocytosis, organelle biogenesis and potentially secretion, key mechanisms of RPE homeostasis and function. INTERPRETATION: Taken together, our results suggest that ZIKV has a highly efficient ocular tropism, which creates a strong inflammatory environment that could have acute or chronic adverse effects. FUND: This work was funded by Retina France, REACTing and La Région Languedoc-Roussillon.
Assuntos
Interferons/metabolismo , Epitélio Pigmentado da Retina/virologia , Infecção por Zika virus/imunologia , Zika virus/patogenicidade , Células Cultivadas , Homeostase , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/imunologia , Células-Tronco Pluripotentes Induzidas/virologia , Interferons/genética , Modelos Biológicos , Fagocitose , Epitélio Pigmentado da Retina/citologia , Epitélio Pigmentado da Retina/imunologia , Tropismo Viral , Replicação Viral , Zika virus/classificação , Zika virus/fisiologia , Infecção por Zika virus/genética , Infecção por Zika virus/virologiaRESUMO
Caffeic acid and chlorogenic acid (CGA), a mono-caffeoyl ester, have been described as potential antidiabetic agents. Using in vitro studies, we report the effects of a dicaffeoyl ester, chicoric acid (CRA) purified from Cichorium intybus, on glucose uptake and insulin secretion. Our results show that CRA and CGA increased glucose uptake in L6 muscular cells, an effect only observed in the presence of stimulating concentrations of insulin. Moreover, we found that both CRA and CGA were able to stimulate insulin secretion from the INS-1E insulin-secreting cell line and rat islets of Langerhans. In the later case, the effect of CRA is only observed in the presence of subnormal glucose levels. Patch clamps studies show that the mechanism of CRA and CGA was different from that of sulfonylureas, as they did not close K(ATP) channels. Chicoric acid is a new potential antidiabetic agent carrying both insulin sensitizing and insulin-secreting properties.
Assuntos
Ácidos Cafeicos/farmacologia , Glucose/metabolismo , Hipoglicemiantes/farmacologia , Insulina/metabolismo , Succinatos/farmacologia , Animais , Transporte Biológico/efeitos dos fármacos , Linhagem Celular , Cichorium intybus/química , Ácido Clorogênico/farmacologia , Secreção de Insulina , Ilhotas Pancreáticas/efeitos dos fármacos , Ilhotas Pancreáticas/metabolismo , Canais KATP/antagonistas & inibidores , Células Musculares/efeitos dos fármacos , Células Musculares/metabolismo , Ratos , XenopusRESUMO
Central serotonin systems have long been associated with the control of feeding behavior and the modulation of behavioral effects of psychostimulants. 5-HT2C receptors are present in hypothalamic centers such as the arcuate nucleus (ARC), controlling homeostatic regulation of food intake, as well as in the ventral tegmental area (VTA), a region involved in motivation aspects in multiple behaviors, including feeding. In the present study, we investigated whether the 5-HT2C receptors control amphetamine-evoked locomotor activity and regulate food consumption. Localized microinjections into the VTA or the ARC were used to assess the effects of a highly selective 5-HT2C receptor agonist, AR231630, on the locomotor stimulant effect of amphetamine as well as on food intake. AR231630 injected into the VTA, but not into the ARC, dose-dependently reduced locomotor activity elicited by amphetamine. Unexpectedly, intra-ARC injection of AR231630 did not reduce food intake even at the dose of 10⯵g, whereas intra-VTA injection of the same dose of AR231630 did. In addition, we showed that pretreatment with the selective 5-HT2C receptor antagonist SB242084 infused into the VTA partially prevented hypophagia induced by peripheral administration of AR231630. We can conclude that 5-HT2C receptor in the VTA, but not in the ARC, participates in both homeostatic and hedonic food intake and brain reward function.
Assuntos
Núcleo Arqueado do Hipotálamo/metabolismo , Comportamento Alimentar/fisiologia , Atividade Motora/fisiologia , Piperazinas/farmacologia , Receptor 5-HT2C de Serotonina/metabolismo , Recompensa , Área Tegmentar Ventral/metabolismo , Aminopiridinas/farmacologia , Anfetamina/farmacologia , Animais , Núcleo Arqueado do Hipotálamo/efeitos dos fármacos , Estimulantes do Sistema Nervoso Central/farmacologia , Ingestão de Alimentos/efeitos dos fármacos , Ingestão de Alimentos/fisiologia , Comportamento Alimentar/efeitos dos fármacos , Privação de Alimentos , Indóis/farmacologia , Masculino , Motivação/efeitos dos fármacos , Motivação/fisiologia , Atividade Motora/efeitos dos fármacos , Ratos Sprague-Dawley , Agonistas do Receptor 5-HT2 de Serotonina/farmacologia , Antagonistas do Receptor 5-HT2 de Serotonina/farmacologia , Área Tegmentar Ventral/efeitos dos fármacosRESUMO
As of 2018, Alzheimer's disease (AD) is the most common form of neurodegenerative dementia. It contributes to a progressive neuron loss, deterioration of memory, and cognitive impairment. Current therapies may provide a symptomatic benefit, but do not treat the underlying process. Ongoing researches focus on understanding the causal mechanisms and finding neuropathological hallmarks of AD. Therapeutic approaches targeting senile plaques or neurofibrillary tangles have not yet resulted in a significant cognitive improvement. However, recent data according to the analysis of AD clinical trials (clinicaltrials.gov database) show promising results. This literature review aims at summarizing the recent advances and at highlighting the most promising results of the ongoing researches. It compares the merits of small-molecules, antibodies, cell, and gene-based therapies and emphasizes the need for treatment at earlier stages of the disease.