RESUMO
Protein turnover rate is finely regulated through intracellular mechanisms and signals that are still incompletely understood but that are essential for the correct function of cellular processes. Indeed, a dysfunctional proteostasis often impacts the cell's ability to remove unfolded, misfolded, degraded, non-functional, or damaged proteins. Thus, altered cellular mechanisms controlling protein turnover impinge on the pathophysiology of many diseases, making the study of protein synthesis and degradation rates an important step for a more comprehensive understanding of these pathologies. In this manuscript, we describe the application of a dynamic-SILAC approach to study the turnover rate and the abundance of proteins in a cellular model of diabetic nephropathy. We estimated protein half-lives and relative abundance for thousands of proteins, several of which are characterized by either an altered turnover rate or altered abundance between diabetic nephropathic subjects and diabetic controls. Many of these proteins were previously shown to be related to diabetic complications and represent therefore, possible biomarkers or therapeutic targets. Beside the aspects strictly related to the pathological condition, our data also represent a consistent compendium of protein half-lives in human fibroblasts and a rich source of important information related to basic cell biology.
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Diabetes Mellitus , Nefropatias Diabéticas , Humanos , Proteínas/metabolismo , Proteólise , Biossíntese de Proteínas , Fibroblastos/metabolismoRESUMO
Fabry disease (FD) is an X-linked lysosome storage disease that results in the accumulation of globotriaosylceramide (Gb3) throughout the body leading to irreversible target organ damage. As the role of secondary mediators (inflammatory molecules) and their mechanisms has not been fully elucidated, we focused on the interleukin (IL)-6 system in adult FD patients and in matched healthy subjects. To obtain insights into the complex regulation of IL-6 actions, we used a novel approach that integrates information from plasma and exosomes of FD patients (n = 20) and of healthy controls (n = 15). Soluble IL-6 receptor (sIL-6R) levels were measured in plasma with the ELISA method, and membrane-bound IL-6R was quantified in plasma and urinary exosomes using flow cytometry. In FD patients, the levels of soluble IL-6R in plasma were higher than in control subjects (28.0 ± 5.4 ng/mL vs. 18.9 ± 5.4 ng/mL, p < 0.0001); they were also higher in FD subjects with the classical form as compared to those with the late-onset form of the disease (36.0 ± 11.4 ng/mL vs. 26.1 ± 4.5 ng/mL, p < 0.0001). The percentage of urinary exosomes positive for IL-6R was slightly lower in FD (97 ± 1 vs. 100 ± 0% of events positive for IL-6R, p < 0.05); plasma IL-6 levels were not increased. These results suggest a potential role of IL-6 in triggering the inflammatory response in FD. As in FD patients only the levels of sIL-6Rs are consistently higher than in healthy controls, the IL-6 pathogenic signal seems to prevail over the homeostatic one, suggesting a potential mechanism causing multi-systemic damage in FD.
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Human milk contains <50% less protein (casein) than cow milk, but is equally effective in insulin secretion despite lower postingestion hyperaminoacidemia. Such potency of human milk might be modulated either by incretins (glucagon-like polypeptide-1,GLP-1); glucose-inhibitory-polypeptide, GIP), and/or by milk casein content. Healthy volunteers of both sexes were fed iso-lactose loads of two low-protein milks, i.e., human [Hum] (n = 8) and casein-deprived cow milk (Cow [↓Cas]) (n = 10), as well as loads of two high-protein milks, i.e., cow (n = 7), and casein-added human-milk (Hum [↑Cas]) (n = 7). Plasma glucose, insulin, C-peptide, incretins and amino acid concentrations were measured for 240'. All milks induced the same transient hyperglycemia. The early [20'−30'] insulin and C-peptide responses were comparable among all milk types apart from the low-protein (Cow [↓Cas]) milk, where they were reduced by <50% (p < 0.05 vs. others). When comparing the two high-protein milks, GLP-1 and GIP [5'−20'] responses with the (Hum [↑Cas]) milk were lower (by ≈2−3 fold, p < 0.007 and p < 0.03 respectively) than those with cow milk, whereas incretin secretion was substantially similar. Plasma amino acid increments largely reflected the milk protein content. Thus, neither casein milk content, nor incretin or amino acid concentrations, can account for the specific potency of human milk on insulin secretion, which remains as yet unresolved.
Assuntos
Incretinas , Insulina , Aminoácidos , Animais , Glicemia/metabolismo , Peptídeo C , Caseínas/metabolismo , Bovinos , Feminino , Polipeptídeo Inibidor Gástrico , Peptídeo 1 Semelhante ao Glucagon , Humanos , Lactose/análise , Masculino , Leite/química , Adulto JovemRESUMO
Glycogen storage diseases (GSDs) represent a model of pathological accumulation of glycogen disease in the kidney that, in animal models, results in nephropathy due to abnormal autophagy and mitochondrial function. Patients with Glycogen Storage Disease 1a (GSD1a) accumulate glycogen in the kidneys and suffer a disease resembling diabetic nephropathy that can progress to renal failure. In this study, we addressed whether urine-derived epithelial cells (URECs) from patients with GSD1a maintain their biological features, and whether they can be used as a model to study the renal and metabolic phenotypes of this genetic condition. Studies were performed on cells extracted from urine samples of GSD1a and healthy subjects. URECs were characterized after the fourth passage by transmission electron microscopy and immunofluorescence. Reactive oxygen species (ROS), at different glucose concentrations, were measured by fluorescent staining. We cultured URECs from three patients with GSD1a and three healthy controls. At the fourth passage, URECs from GSD1a patients maintained their massive glycogen content. GSD1a and control cells showed the ciliary structures of renal tubular epithelium and the expression of epithelial (E-cadherin) and renal tubular cells (aquaporin 1 and 2) markers. Moreover, URECs from both groups responded to changes in glucose concentrations by modulating ROS levels. GSD1a cells were featured by a specific response to the low glucose stimulus, which is the condition that more resembles the metabolic derangement of patients with GSD1a. Through this study, we demonstrated that URECs might represent a promising experimental model to study the molecular mechanisms leading to renal damage in GSD1a, due to pathological glycogen storage.
Assuntos
Doença de Depósito de Glicogênio Tipo I , Doença de Depósito de Glicogênio , Células Epiteliais/metabolismo , Glucose , Glicogênio , Doença de Depósito de Glicogênio Tipo I/genética , Rim/metabolismo , Fenótipo , Espécies Reativas de Oxigênio , HumanosRESUMO
Aims: Peripheral artery disease (PAD) is a severe complication of diabetes, characterized by defective traffic of hematopoietic stem/progenitor cells (HSPCs). We examined the hematopoietic versus nonhematopoietic role of p66Shc in regulating HSPC traffic and blood flow recovery after ischemia in diabetic mice. Results: Using streptozotocin-induced diabetes, chimeric mice with green fluorescent protein (GFP)+ bone marrow (BM), and the hind limb ischemia model, we found that the physiologic mobilization and homing of HSPCs were abolished by diabetes, along with impaired vascular recovery. Hematopoietic deletion of p66Shc, obtained by transplanting p66Shc-/- BM cells into wild-type (Wt) recipients, but not nonhematopoietic deletion, constrained hyperglycemia-induced myelopoiesis, rescued postischemic HSPC mobilization, and improved blood flow recovery in diabetic mice. In Wt diabetic mice transplanted with BM cells from GFP+p66Shc-/- mice, the amount of HSPCs homed to ischemic muscles was greater than in mice transplanted with GFP+p66Shc+/+ cells, with recruited cells displaying higher expression of adhesion molecules and Vegf. In 40 patients with diabetes, p66Shc gene expression in mononuclear cells was correlated with myelopoiesis and elevated in the presence of PAD. In 13 patients with diabetes and PAD, p66Shc expression in HSPC-mobilized peripheral blood cells was inversely correlated with VEGF expression. Innovation: For the first time, we dissect the role of hematopoietic versus nonhematopoietic p66Shc in regulating HSPC traffic and ischemic responses. Conclusion: Hematopoietic deletion of p66Shc was sufficient to rescue HSPC mobilization and homing in diabetes after ischemia and improved blood flow recovery. Inhibiting p66Shc in blood cells may be a novel strategy to counter PAD in diabetes. Antioxid. Redox Signal. 36, 593-607. Clinical Trial No.: NCT02790957.
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Diabetes Mellitus Experimental , Animais , Diabetes Mellitus Experimental/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Humanos , Isquemia/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Proteína 1 de Transformação que Contém Domínio 2 de Homologia de Src/genética , Proteína 1 de Transformação que Contém Domínio 2 de Homologia de Src/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
SCOPE: Milk-proteins, besides lactose, stimulate insulin and incretin secretion. Although whey-proteins (WP) are more efficient than casein (Cas) in hormone secretion, the effects of reversal of the (WP/Cas) ratio in whole-milk are poorly known. METHODS AND RESULTS: Healthy volunteers received two different cow-milk drinks, at identical lactose (0.36 g × kg-1 BW) and total-protein (0.18 g × kg1 BW) loads, but at reversed WP/Cas ratio. One is cow-whole milk with a ≈20/80 [WP/Cas] ratio, the other an experimental cow-milk with a ≈70/30 [WP/Cas] ratio ([↑WP↓Cas]-milk). Both milk-types induced the same mild hyperglycemic response. Following [↑WP↓Cas]-milk, the [20'-90'] insulin incremental area (iAUC) (+ ≈44%, p < 0.035), and the [20'-120'] C-peptide iAUC (+ ≈47%, p < 0.015) are greater than those with cow-milk. Similarly, following [↑WP↓Cas]-milk, the GLP-1 [20'-90'] iAUC (+96%, p < 0.025), and the GIP [30'-60'] iAUC (+140%, p < 0.006), were greater than those with cow-milk. Plasma total and branched-chain amino acids are also greater following the [↑WP↓Cas] than cow-milk. CONCLUSIONS: Reversal of the (WP/Cas) ratio in cow-milk enhanced the insulin response, an effect possibly mediated by incretins and/or amino acids(s). These data may be useful in designing specific milk formulas with different effects on insulin and incretin response(s).
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Caseínas , Incretinas , Aminoácidos , Animais , Glicemia/metabolismo , Caseínas/metabolismo , Bovinos , Feminino , Polipeptídeo Inibidor Gástrico , Humanos , Incretinas/metabolismo , Insulina , Leite/química , Soro do Leite/metabolismo , Proteínas do Soro do LeiteRESUMO
AIM/HYPOTHESIS: In two large RCTs, fenofibrate reduced the progression of diabetic retinopathy. We investigated whether fenofibrate increases circulating haematopoietic stem/progenitor cells (HSPCs), which have vascular properties and have been shown to protect from retinopathy. METHODS: We conducted a 12 week parallel-group RCT comparing fenofibrate vs placebo. Patients with diabetic retinopathy and without other conditions that would affect HSPCs were enrolled at a tertiary diabetes outpatient clinic and randomised to receive fenofibrate or placebo based on a computer-generated sequence. Patients and study staff assessing the outcomes were blinded to group assignment. The primary endpoint was the change in the levels of circulating HSPCs, defined by expression of the stem cell markers CD34 and/or CD133. Secondary endpoints were the changes in endothelial progenitor cells, lipids, soluble mediators and gene expression. We used historical data on the association between HSPCs and retinopathy outcomes to estimate the effect of fenofibrate on retinopathy progression. RESULTS: Forty-two participants with diabetic retinopathy were randomised and 41 completed treatment and were analysed (20 in the placebo group and 21 in the fenofibrate group). Mean age was 57.4 years, diabetes duration was 18.2 years and baseline HbA1c was 60 mmol/mol (7.6%). When compared with placebo, fenofibrate significantly increased levels of HSPCs expressing CD34 and/or CD133. CD34+ HSPCs non-significantly declined in the placebo group (mean ± SD -44.2 ± 31.6 cells/106) and significantly increased in the fenofibrate group (53.8 ± 31.1 cells/106). The placebo-subtracted increase in CD34+ HSPCs from baseline was 30% (99.3 ± 43.3 cells/106; p = 0.027) which, projected onto the relationship between HSPC levels and retinopathy outcomes, yielded an OR of retinopathy progression of 0.67 for fenofibrate vs placebo. Endothelial differentiation of CD34+ cells, estimated by the %KDR (kinase insert domain receptor) expression, was significantly reduced by fenofibrate. Fenofibrate decreased serum triacylglycerols, but the change in triacylglycerols was unrelated to the change in HSPCs. No effect was observed for endothelial progenitor cells, cytokines/chemokines (stromal-cell derived factor-1, vascular endothelial growth factor, monocyte chemoattractant protein-1) and gene expression in peripheral blood mononuclear cells. CONCLUSIONS/INTERPRETATION: Fenofibrate increased HSPC levels in participants with diabetic retinopathy and this mechanism may explain why fenofibrate reduced retinopathy progression in previous studies. TRIAL REGISTRATION: ClinicalTrials.gov NCT01927315.
Assuntos
Retinopatia Diabética/tratamento farmacológico , Fenofibrato/uso terapêutico , Células-Tronco Hematopoéticas/metabolismo , Hipolipemiantes/uso terapêutico , Antígeno AC133/metabolismo , Adolescente , Adulto , Idoso , Antígenos CD34/metabolismo , Biomarcadores/metabolismo , Glicemia/metabolismo , Retinopatia Diabética/sangue , Feminino , Hemoglobinas Glicadas/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , Método Simples-Cego , Adulto JovemRESUMO
BACKGROUND: Patients with type 2 diabetes (T2DM) have a prothrombotic state that needs to be fully clarified; microparticles (MPs) have emerged as mediators and markers of this condition. Thus, we investigate, in vivo, in T2DM either with good (HbA1c ≤ 7.0%; GGC) or poor (HbA1c > 7.0%; PGC) glycemic control, the circulating levels of MPs, and in vitro, the molecular pathways involved in the release of MPs from platelets (PMP) and tested their pro-inflammatory effects on THP-1 transformed macrophages. METHODS: In 59 T2DM, and 23 control subjects with normal glucose tolerance (NGT), circulating levels of CD62E+, CD62P+, CD142+, CD45+ MPs were determined by flow cytometry, while plasma levels of ICAM-1, VCAM-1, IL-6 by ELISA. In vitro, PMP release and activation of isolated platelets from GGC and PGC were investigated, along with their effect on IL-6 secretion in THP-1 transformed macrophages. RESULTS: We found that MPs CD62P+ (PMP) and CD142+ (tissue factor-bearing MP) were significantly higher in PGC T2DM than GGC T2DM and NGT. Among MPs, PMP were also correlated with HbA1c and IL-6. In vitro, we showed that acute thrombin exposure stimulated a significantly higher PMP release in PGC T2DM than GGC T2DM through a more robust activation of PAR-4 receptor than PAR-1 receptor. Treatment with PAR-4 agonist induced an increased release of PMP in PGC with a Ca2+-calpain dependent mechanism since this effect was blunted by calpain inhibitor. Finally, the uptake of PMP derived from PAR-4 treated PGC platelets into THP-1 transformed macrophages promoted a marked increase of IL-6 release compared to PMP derived from GGC through the activation of the NF-kB pathway. CONCLUSIONS: These results identify PAR-4 as a mediator of platelet activation, microparticle release, and inflammation, in poorly controlled T2DM.
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Glicemia/metabolismo , Plaquetas/enzimologia , Cálcio/metabolismo , Calpaína/metabolismo , Micropartículas Derivadas de Células/enzimologia , Diabetes Mellitus Tipo 2/enzimologia , Macrófagos/metabolismo , Ativação Plaquetária , Receptores de Trombina/metabolismo , Biomarcadores/sangue , Plaquetas/efeitos dos fármacos , Estudos de Casos e Controles , Micropartículas Derivadas de Células/efeitos dos fármacos , Diabetes Mellitus Tipo 2/sangue , Feminino , Hemoglobinas Glicadas/metabolismo , Humanos , Interleucina-6/metabolismo , Masculino , Pessoa de Meia-Idade , Ativação Plaquetária/efeitos dos fármacos , Receptores de Trombina/agonistas , Células THP-1 , Trombina/farmacologiaRESUMO
BACKGROUND AND AIMS: Continuous glucose monitoring improves glycemic control in diabetes. This study compared the accuracy of the Dexcom G5 Mobile (Dexcom, San Diego, CA) transcutaneous sensor (DG5) and the first version of Eversense (Senseonics,Inc., Germantown, MD) implantable sensor (EVS). METHODS AND RESULTS: Subjects with type 1 diabetes (T1D) and using EVS wore simultaneously DG5 for seven days. At day 3, patients were admitted to a clinical research center (CRC) to receive breakfast with delayed and increased insulin bolus to induce glucose excursions. At CRC, venous glucose was monitored every 15 min (or 5 min during hypoglycemia) for 6 h by YSI 2300 STAT PLUS™ glucose and lactate analyzer. At home patients were requested to perform 4 fingerstick glucose measurements per day. Eleven patients (9 males, age 47.4 ± 11.3 years, M±SD) were enrolled. During home-stay the median [25th-75th percentile] absolute relative difference (ARD) over all CGM-fingerstick matched-pairs was 11.64% [5.38-20.65]% for the DG5 and 10.75% [5.15-19.74]% for the EVS (p-value = 0.58). At CRC, considering all the CGM-YSI matched-pairs, the DG5 showed overall smaller median ARD than EVS, 7.91% [4.14-14.30]% vs 11.4% [5.04-18.54]% (p-value<0.001). Considering accuracy during blood glucose swings, DG5 performed better than EVS when glucose rate-of-change was -0.5 to -1.5 mg/dL/min, with median ARD of 7.34% [3.71-12.76]% vs 13.59% [4.53-20.78]% (p-value<0.001), and for rate-of-change < -1.5 mg/dl/min, with median ARD of 5.23% [2.09-15.29]% vs 12.73% [4.14-20.82]% (p-value = 0.02). CONCLUSIONS: DG5 was more accurate than EVS at CRC, especially when glucose decreased. No differences were found at home.
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Automonitorização da Glicemia/instrumentação , Glicemia/metabolismo , Diabetes Mellitus Tipo 1/diagnóstico , Transdutores , Tecnologia sem Fio/instrumentação , Adulto , Biomarcadores/sangue , Glicemia/efeitos dos fármacos , Diabetes Mellitus Tipo 1/sangue , Diabetes Mellitus Tipo 1/terapia , Desenho de Equipamento , Feminino , Controle Glicêmico , Humanos , Hipoglicemiantes/uso terapêutico , Insulina/uso terapêutico , Masculino , Pessoa de Meia-Idade , Valor Preditivo dos Testes , Reprodutibilidade dos Testes , Fatores de Tempo , Resultado do TratamentoRESUMO
Metabolic disorders due to over-nutrition are a major global health problem, often associated with obesity and related morbidities. Obesity is peculiar to humans, as it is associated with lifestyle and diet, and so difficult to reproduce in animal models. Here we describe a model of human central adiposity based on a 3-tissue system consisting of a series of interconnected fluidic modules. Given the causal link between obesity and systemic inflammation, we focused primarily on pro-inflammatory markers, examining the similarities and differences between the 3-tissue model and evidence from human studies in the literature. When challenged with high levels of adiposity, the in-vitro system manifests cardiovascular stress through expression of E-selectin and von Willebrand factor as well as systemic inflammation (expressing IL-6 and MCP-1) as observed in humans. Interestingly, most of the responses are dependent on the synergic interaction between adiposity and the presence of multiple tissue types. The set-up has the potential to reduce animal experiments in obesity research and may help unravel specific cellular mechanisms which underlie tissue response to nutritional overload.
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Inflamação/fisiopatologia , Modelos Biológicos , Obesidade Abdominal/fisiopatologia , Vasculite/fisiopatologia , Adiposidade , Albuminas/biossíntese , Animais , Biomarcadores/metabolismo , Reatores Biológicos , Técnicas de Cocultura/métodos , Células Hep G2 , Células Endoteliais da Veia Umbilical Humana , Humanos , Técnicas In Vitro , Inflamação/complicações , Mediadores da Inflamação/fisiologia , Gordura Intra-Abdominal/fisiopatologia , Dispositivos Lab-On-A-Chip , Lipídeos/biossíntese , Obesidade Abdominal/complicações , Vasculite/complicaçõesRESUMO
AIMS: Atherosclerosis is an inflammatory disease wherein cholesterol-loaded macrophages play a major role. MicroRNAs and microparticles propagate inflammatory pathways and are involved in cardiovascular disease. We aimed to screen and validate circulating microRNAs correlated with atherosclerosis development in humans, and to dissect the molecular mechanisms associated with atherogenesis using in vitro and in vivo approaches. METHODS AND RESULTS: A panel of 179 secreted microRNAs was screened in plasma samples of patients with and without atherosclerosis, and validated cross-sectionally and prospectively in patients followed for up to 11 years. miR-30c-5p was inversely correlated with total and LDL cholesterol, carotid intimal media thickness (CIMT), presence and future development of plaques. Using a human macrophage line and in vitro gene silencing strategies, we found that miR-30c-5p was downregulated by oxidized LDL (oxLDL) via the scavenger receptor CD36 and inhibition miR processing by Dicer. In turn, miR-30c-5p downregulation was responsible for the effects of oxLDL on macrophage IL-1ß release, caspase-3 expression, and apoptosis. miR-30c-5p loaded into microparticles was uptaken by macrophages and regulated target genes, like caspase-3, at transcriptional level. To establish the relevance of this pathway on endothelial damage as the earliest step of atherogenesis, we show that systemic miR-30c-5p knockdown induced caspase-3 and impaired endothelial healing after carotid injury in C57Bl/6 J mice. CONCLUSIONS: With an unbiased screening of secreted microRNAs, we identify reduction of miR-30c-5p in microparticles as a promoter of early atherosclerosis, by conveying pro-inflammatory pro-apoptotic signals and impairing endothelial healing. Therefore, stimulation of miR-30c-5p is a candidate direct anti-atherosclerotic therapy.
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Doenças das Artérias Carótidas/metabolismo , MicroRNA Circulante/metabolismo , Inflamação/metabolismo , Macrófagos/metabolismo , MicroRNAs/metabolismo , Placa Aterosclerótica , Animais , Apoptose , Antígenos CD36/metabolismo , Doenças das Artérias Carótidas/diagnóstico por imagem , Doenças das Artérias Carótidas/genética , Doenças das Artérias Carótidas/patologia , Lesões das Artérias Carótidas/genética , Lesões das Artérias Carótidas/metabolismo , Lesões das Artérias Carótidas/patologia , Espessura Intima-Media Carotídea , Estudos de Casos e Controles , Caspase 3/metabolismo , LDL-Colesterol/sangue , MicroRNA Circulante/sangue , MicroRNA Circulante/genética , Estudos Transversais , RNA Helicases DEAD-box/metabolismo , Modelos Animais de Doenças , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Humanos , Inflamação/genética , Interleucina-1beta/metabolismo , Lipoproteínas LDL/metabolismo , Macrófagos/patologia , Camundongos Endogâmicos C57BL , MicroRNAs/sangue , MicroRNAs/genética , Estudos Prospectivos , Ribonuclease III/metabolismo , Células THP-1 , Fatores de TempoRESUMO
BACKGROUND: Sodium-glucose co-transporter-2 inhibitors (SGLT2i) reduce glucose levels, body weight, and blood pressure, possibly resulting in cardiovascular protection. In phase III trials, SGLT2i were shown to increase HDL cholesterol. We aimed to evaluate whether the SGLT2i dapagliflozin affects HDL function in a randomized placebo-controlled trial. METHODS: Thirty-three type 2 diabetic patients were randomized to receive dapagliflozin 10 mg or placebo for 12 weeks on top of their glucose lowering medications. The primary end-point was the change in cholesterol efflux capacity (CEC) from macrophages at study end versus baseline. Secondary endpoints were changes in: distribution of HDL subfractions, lipid profile, activity of enzymes that mediate HDL antioxidant properties (PON1 and ARE) and cholesterol metabolism (CETP), HbA1c, body weight and composition. RESULTS: Thirty-one patients completed the study, n = 16 in the placebo group and n = 15 in the dapagliflozin group. Patients randomized to dapagliflozin were older and had lower adiposity indexes, although these differences disappeared after correction for multiple testing. Therapy with dapagliflozin reduced HbA1c by 0.9% and body weight by 3.1 kg, mainly attributable to reduction of body water and lean mass. As compared to placebo, dapagliflozin reduced CEC (-6.7 ± 2.4 versus 0.3 ± 1.8%; p = 0.043), but this effect was no longer significant after adjusting for age and BMI. No change was detected in HDL cholesterol, HDL subfractions, activity of PON1, ARE, and CETP. CONCLUSIONS: Despite improvements in glucose control and reduction in body weight, therapy with dapagliflozin exerted no significant effect on HDL cholesterol levels and HDL functionality. Trial registration EudraCT 2014-004270-42; NCT02327039.
Assuntos
Compostos Benzidrílicos/administração & dosagem , Glicemia/efeitos dos fármacos , HDL-Colesterol/sangue , Diabetes Mellitus Tipo 2/sangue , Diabetes Mellitus Tipo 2/tratamento farmacológico , Glucosídeos/administração & dosagem , Inibidores do Transportador 2 de Sódio-Glicose , Idoso , Glicemia/metabolismo , Peso Corporal/efeitos dos fármacos , Peso Corporal/fisiologia , HDL-Colesterol/antagonistas & inibidores , Quimioterapia Combinada , Feminino , Seguimentos , Humanos , Masculino , Pessoa de Meia-Idade , Tamanho da Partícula , Transportador 2 de Glucose-Sódio , Resultado do TratamentoRESUMO
CONTEXT: Circulating cells, including endothelial progenitor cells (EPCs) and monocyte subtypes, are involved in diabetic complications. Modulation of these cells may mediate additional benefits of glucose-lowering medications. OBJECTIVE: We assessed whether the dipeptidyl peptidase-4 (DPP-4) inhibitor linagliptin acutely modifies EPCs and monocyte subsets in patients with type 2 diabetes. DESIGN: This was a randomized, crossover, placebo-controlled trial. SETTING: The study was conducted at a tertiary referral diabetes outpatient clinic. PATIENTS: Forty-six type 2 diabetes patients with (n = 18) or without (n = 28) chronic kidney disease (CKD) participated in the study. INTERVENTION: Intervention included a 4-day treatment with linagliptin 5 mg or placebo during two arms separated by a 2-week washout. MAIN OUTCOME MEASURES: Before and after each treatment, we determined the levels of circulating progenitor cells (CD34, CD133, KDR) and monocyte subtypes (CD14/CD16, chemokine and scavenger receptors) and the concentrations of soluble mediators. RESULTS: Compared with placebo, linagliptin increased CD34(+)CD133(+) progenitor cells (placebo subtracted effect 40.4 ± 18.7/10(6); P = .036), CD34(+)KDR(+) EPCs (placebo subtracted effect 22.1 ± 10.2/10(6); P = .036), and CX3CR1(bright) monocytes (placebo subtracted effect 1.7 ± 0.8%; P = .032). Linagliptin abated DPP-4 activity by greater than 50%, significantly increased active glucagon-like peptide-1 and stromal cell-derived factor-1α, and reduced monocyte chemotactic protein-1, CCL22, and IL-12. Patients with CKD, as compared with those without, had lower baseline CD133(+) and CD34(+)CD133(+) cells and had borderline reduced CD34(+) and CD34(+)KDR(+) cells. The effects of linagliptin on progenitor cells and monocyte subtypes were similar in patients with or without CKD. Fasting plasma glucose, triglycerides and free fatty acids were unaffected. CONCLUSIONS: DPP-4 inhibition with linagliptin acutely increases putative vasculoregenerative and antiinflammatory cells. Direct effects of DPP-4 inhibition may be important to lower vascular risk in diabetes, especially in the presence of CKD.
Assuntos
Diabetes Mellitus Tipo 2/tratamento farmacológico , Hipoglicemiantes/uso terapêutico , Linagliptina/uso terapêutico , Monócitos/efeitos dos fármacos , Células-Tronco/efeitos dos fármacos , Idoso , Estudos Cross-Over , Dipeptidil Peptidase 4/metabolismo , Inibidores da Dipeptidil Peptidase IV/uso terapêutico , Células Endoteliais/efeitos dos fármacos , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Fenótipo , Insuficiência Renal Crônica/complicações , Insuficiência Renal Crônica/metabolismoRESUMO
CK2 is a multifunctional, pleiotropic protein kinase involved in the regulation of cell proliferation and survival. Since fibroblasts from Type 1 Diabetes patients (T1DM) with Nephropathy exhibit increased proliferation, we studied cell viability, basal CK2 expression and activity, and response to specific CK2 inhibitors TBB (4,5,6,7-tetrabenzotriazole) and CX4945, in fibroblasts from T1DM patients either with (T1DM+) or without (T1DM-) Nephropathy, and from healthy controls (N). We tested expression and phosphorylation of CK2-specific molecular targets. In untreated fibroblasts from T1DM+, the cell viability was higher than in both N and T1DM-. CK2 inhibitors significantly reduced cell viability in all groups, but more promptly and with a larger effect in T1DM+. Differences in CK2-dependent phosphorylation sites were detected. In conclusion, our results unveil a higher dependence of T1DM+ cells on CK2 for their survival, despite a similar expression and a lower activity of this kinase compared with those of normal cells.
Assuntos
Caseína Quinase II/antagonistas & inibidores , Nefropatias Diabéticas/metabolismo , Fibroblastos/metabolismo , Adulto , Caseína Quinase II/metabolismo , Sobrevivência Celular , Células Cultivadas , Feminino , Fibroblastos/efeitos dos fármacos , Humanos , Masculino , Pessoa de Meia-Idade , Naftiridinas/farmacologia , Fenazinas , Inibidores de Proteínas Quinases/farmacologiaRESUMO
Adenocarcinoma (AdC) is the most common lung cancer subtype and is often associated with pleural effusion (PE). Its poor prognosis is attributable to diagnostic delay and lack of effective treatments and there is a pressing need in discovering new biomarkers for early diagnosis or targeted therapies. To date, little is known about lung AdC proteome. We investigated protein expression of lung AdC in PE using the isobaric Tags for Relative and Absolute Quantification (iTRAQ) approach to identify possible novel diagnostic/prognostic biomarkers. This provided the identification of 109 of lung AdC-related proteins. We further analyzed lumican, one of the overexpressed proteins, in 88 resected lung AdCs and in 23 malignant PE cell-blocks (13 lung AdCs and 10 non-lung cancers) using immunohistochemistry. In AdC surgical samples, lumican expression was low in cancer cells, whereas it was strong and diffuse in the stroma surrounding the tumor. However, lumican expression was not associated with tumor grade, stage, and vascular/pleural invasion. None of the lung cancer cell-blocks showed lumican immunoreaction, whereas those of all the other tumors were strongly positive. Finally, immunoblotting analysis showed lumican expression in both cell lysate and conditioned medium of a fibroblast culture but not in those of A549 lung cancer cell line. PE is a valid source of information for proteomic analysis without many of the restrictions of plasma. The high lumican levels characterizing AdC PEs are probably due to its release by the fibroblasts surrounding the tumor. Despite the role of lumican in lung AdC is still elusive, it could be of diagnostic value.
Assuntos
Adenocarcinoma/metabolismo , Proteoglicanas de Sulfatos de Condroitina/metabolismo , Sulfato de Queratano/metabolismo , Neoplasias Pulmonares/metabolismo , Derrame Pleural Maligno/metabolismo , Adenocarcinoma/genética , Adenocarcinoma/patologia , Adenocarcinoma de Pulmão , Linhagem Celular Tumoral , Feminino , Humanos , Imuno-Histoquímica , Lumicana , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Masculino , Gradação de Tumores , Estadiamento de Neoplasias , Proteoma , Proteômica/métodosRESUMO
Type 2 diabetes (T2D) is characterized by impaired vascular regeneration owing to reduced endothelial progenitor cells (EPCs). While statins are known to increase EPCs, the effects of statin withdrawal on EPCs are unknown. Herein, we evaluated the effects of statin discontinuation on EPCs, inflammation and in vivo angiogenesis. Thirty-four T2D patients were randomized to 5-day discontinuation or continuation of statin treatment. At baseline and at day 5, we determined lipid profile, EPC levels, monocyte-macrophage polarization, and concentrations of hsCRP, VEGF, SDF-1α, and G-CSF. Angiogenesis by human circulating cells was assessed in vivo. At day 5, patients who stopped statins showed raised total and LDL cholesterol and EPCs compared to baseline, while no changes were observed in patients who continued statins. No changes were observed in hsCRP, VEGF, SDF-1α, G-CSF, M1 and M2 macrophages and classical, intermediate and nonclassical monocytes in both groups. In vivo angiogenesis by circulating cells was increased in patients who stopped statin treatment. In vitro, cholesterol supplementation stimulated mobilizing signals in human bone marrow mesenchymal stem cells. In conclusion, a brief statin withdrawal increases circulating EPCs and functional proangiogenic cells in T2D. These findings identify statin-sensitive pathways as reverse target mechanisms to stimulate vascular repair in diabetes.
Assuntos
Diabetes Mellitus Tipo 2/sangue , Diabetes Mellitus Tipo 2/tratamento farmacológico , Células Progenitoras Endoteliais/efeitos dos fármacos , Células Progenitoras Endoteliais/metabolismo , Inibidores de Hidroximetilglutaril-CoA Redutases/administração & dosagem , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Células Cultivadas , LDL-Colesterol/sangue , Esquema de Medicação , Feminino , Humanos , Inflamação/sangue , Inflamação/diagnóstico , Masculino , Camundongos , Camundongos Knockout , Pessoa de Meia-Idade , Fatores de TempoRESUMO
CK2 is an extremely pleiotropic Ser/Thr protein kinase, responsible for the generation of a large proportion of the human phosphoproteome and implicated in a wide variety of biological functions. CK2 plays a global role as an anti-apoptotic agent, a property which is believed to partially account for the addiction of many cancer cells to high CK2 levels. To gain information about the CK2 targets whose phosphorylation is primarily implicated in its pro-survival signaling advantage has been taken of quinalizarin (QZ) a cell permeable fairly specific CK2 inhibitor, previously shown to be able to block endogenous CK2 triggering an apoptotic response. HEK-293T cells either treated or not for 3h with 50µM QZ were exploited to perform a quantitative SILAC phosphoproteomic analysis of phosphosites readily responsive to QZ treatment. Our analysis led to the identification of 4883 phosphosites, belonging to 1693 phosphoproteins. 71 phosphosites (belonging to 47 proteins) underwent a 50% or more decreased occupancy upon QZ treatment. Almost 50% of these fulfilled the typical consensus sequence recognized by CK2 (S/T-x-x-E/D/pS) and in several cases were validated as bona fide substrates of CK2 either based on data in the literature or by performing in vitro phosphorylation experiments with purified proteins. The majority of the remaining phosphosites drastically decreased upon QZ treatment display the pS/T-P motif typical of proline directed protein kinases and a web logo extracted from them differentiates from the web logo extracted from all the proline directed phosphosites quantified during our analysis (1151 altogether). A paradoxical outcome of our study was the detection of 116 phosphosites (belonging to 92 proteins altogether) whose occupancy is substantially increased (50% or more), rather than decreased by QZ treatment: 40% of these display the typical motif recognized by proline directed kinases, while about 25% fulfill the CK2 consensus. Collectively taken our data on one side have led to the disclosure of a subset of CK2 targets which are likely to be implicated in the early steps of CK2 signaling counteracting apoptosis, on the other they provide evidence for the existence of side and off-target effects of the CK2 inhibitor quinalizarin, paving the road toward the detection of other kinases susceptible to this compound. This article is part of a Special Issue entitled: Medical Proteomics.
Assuntos
Antraquinonas/farmacologia , Caseína Quinase II/antagonistas & inibidores , Fosfoproteínas/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Proteoma/metabolismo , Células HEK293 , HumanosRESUMO
Type 2 diabetes (T2D) is characterized by low HDL cholesterol (HDL-C) and HDL dysfunction. We herein tested whether lowering HbA1c affects HDL-C and reverse cholesterol transport (RCT). Forty-two uncontrolled T2D patients initiating basal insulin were included. HbA1c, HDL-C and RCT were assessed at baseline and after 6 months. At baseline, HDL-C and RCT were directly correlated (r = 0.50; p < 0.001). After 6 months of insulin therapy, HbA1c dropped from 8.8 ± 0.16% to 7.1 ± 0.1%, while average HDL-C and RCT did not change. Follow-up HDL-C and RCT were still correlated (r = 0.31; p = 0.033) and ΔHDL-C correlated with ΔRCT (r = 0.32; p = 0.029). ΔHbA1c correlated with ΔHDL-C (r = 0.43, p = 0.001), but not with ΔRCT. In patients with ΔHbA1c above the median value (1.3%), HDL-C (but not RCT) increased significantly. In conclusion, glucose control correlates with increased HDL-C, but not with improved RCT. Thus, persistent HDL dysfunction despite improved HbA1c and HDL-C can contribute to residual cardiovascular risk in T2D.
Assuntos
Transporte Biológico/efeitos dos fármacos , HDL-Colesterol/metabolismo , Colesterol/metabolismo , Diabetes Mellitus Tipo 2/fisiopatologia , Hemoglobinas Glicadas/metabolismo , Insulina/uso terapêutico , Idoso , Diabetes Mellitus Tipo 2/tratamento farmacológico , Feminino , Humanos , MasculinoRESUMO
IEF is often used in multidimensional shotgun proteomics and the narrow range of 3.5-4.5 is the recommended pH interval for the fractionation of tryptic peptides. Usually, even if IEF is performed in IPG strip with a narrow range pH, the entire sample must be loaded onto the strip, including the "out of IPG range" peptides. We describe a simple protocol to recover at least a part of these missing peptides and show that this recovery significantly influences the overall fractionation result, increasing the number of the identified proteins and the protein coverage.
Assuntos
Focalização Isoelétrica/métodos , Peptídeos/química , Concentração de Íons de Hidrogênio , Focalização Isoelétrica/instrumentação , Proteômica/instrumentação , Proteômica/métodosRESUMO
Nutrient balance in the human body is maintained through systemic signaling between different cells and tissues. Breaking down this circuitry to its most basic elements and reconstructing the metabolic network in-vitro provides a systematic method to gain a better understanding of how cross-talk between the organs contributes to the whole body metabolic profile and of the specific role of each different cell type. To this end, a 3-way connected culture of hepatocytes, adipose tissue and endothelial cells representing a simplified model of energetic substrate metabolism in the visceral region was developed. The 3-way culture was shown to maintain glucose and fatty acid homeostasis in-vitro. Subsequently it was challenged with insulin and high glucose concentrations to simulate hyperglycaemia. The aim was to study the capacity of the 3-way culture to maintain or restore normal circulating glucose concentrations in response to insulin and to investigate the effects these conditions on other metabolites involved in glucose and lipid metabolism. The results show that the system's metabolic profile changes dramatically in the presence of high concentrations of glucose, and that these changes are modulated by the presence of insulin. Furthermore, we observed an increase in E-selectin levels in hyperglycaemic conditions and increased IL-6 concentrations in insulin-free-hyperglycaemic conditions, indicating, respectively, endothelial injury and proinflammatory stress in the challenged 3-way system.