RESUMEN
Insulin increases muscle microvascular perfusion, which contributes to its metabolic action in muscle, but this action is impaired in obesity. Metformin improves endothelial function beyond its glucose lowering effects. We aim to examine whether metformin could prevent microvascular insulin resistance and endothelial dysfunction during the development of obesity. Adult male rats were fed a high-fat diet (HFD) with or without simultaneous metformin administration for either 2 or 4 wk. Insulin's metabolic and microvascular actions were determined using a combined euglycemic-hyperinsulinemic clamp and contrast-enhanced ultrasound approach. Compared with chow-fed controls, HFD feeding increased body adiposity without excess body weight gain, and this was associated with a marked decrease in insulin-mediated whole body glucose disposal and abolishment of insulin-induced muscle microvascular recruitment. Simultaneous administration of metformin fully rescued insulin-induced muscle microvascular recruitment as early as 2 wk and normalized insulin-mediated whole body glucose disposal at week 4. The divergent responses between insulin's microvascular and metabolic actions seen at week 2 were accompanied with reduced endothelial oxidative stress and vascular inflammation, and improved endothelial function and vascular insulin signaling in metformin-treated rats. In conclusions, metformin could prevent the development of microvascular insulin resistance and endothelial dysfunction by alleviating endothelial oxidative stress and vascular inflammation during obesity development.NEW & NOTEWORTHY Muscle microvascular insulin action contributes to insulin-mediated glucose use. Microvascular insulin resistance is an early event in diet-induced obesity and is associated with vascular inflammation. Metformin effectively reduces endothelial oxidative stress, improves endothelial function, and prevents microvascular insulin resistance during obesity development. These may contribute to metformin's salutary diabetes prevention and cardiovascular protective actions.
Asunto(s)
Resistencia a la Insulina , Metformina , Animales , Glucosa/metabolismo , Inflamación/metabolismo , Insulina/metabolismo , Resistencia a la Insulina/fisiología , Masculino , Metformina/farmacología , Músculo Esquelético/metabolismo , Obesidad/metabolismo , Estrés Oxidativo , RatasRESUMEN
A high-fat diet (HFD) can rapidly recruit neutrophils to insulin target tissues and within days induce microvascular insulin resistance (IR). Myeloperoxidase (MPO) is highly enriched in neutrophils, can inhibit nitric oxide-mediated vasorelaxation in vitro and is associated with increased cardiovascular disease risk. AZD5904 irreversibly inhibits MPO and in human clinical trials. MPO knockout, or chemical inhibition, blunts HFD-induced metabolic IR in mice. Whether MPO affects microvascular IR or muscle metabolic insulin sensitivity in vivo is unknown. We used contrast-enhanced ultrasound and the euglycemic insulin clamp to test whether inhibiting MPO could prevent the development or reverse established HFD-induced metabolic and/or microvascular IR in Sprague-Dawley rats. Two weeks of HFD feeding blocked insulin-mediated skeletal muscle capillary recruitment, inhibited glucose utilization, and insulin signaling to muscle. Continuous subcutaneous AZD5904 infusion during the 2 wk selectively blocked HFD's microvascular effect. Furthermore, AZD5904 infusion during the last 2 of 4 wk of HFD feeding restored microvascular insulin sensitivity but not metabolic IR. We conclude that inhibiting MPO selectively improves vascular IR. This selective microvascular effect may connote a therapeutic potential for MPO inhibition in the prevention of vascular disease/dysfunction seen in IR humans.
Asunto(s)
Aorta/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Resistencia a la Insulina , Microvasos/efectos de los fármacos , Músculo Esquelético/efectos de los fármacos , Peroxidasa/antagonistas & inhibidores , Animales , Aorta/metabolismo , Velocidad del Flujo Sanguíneo/efectos de los fármacos , Dieta Alta en Grasa , Técnica de Clampeo de la Glucosa , Masculino , Microcirculación/efectos de los fármacos , Microvasos/metabolismo , Proteína Quinasa 1 Activada por Mitógenos/efectos de los fármacos , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/efectos de los fármacos , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Músculo Esquelético/irrigación sanguínea , Músculo Esquelético/metabolismo , Óxido Nítrico Sintasa de Tipo III/efectos de los fármacos , Óxido Nítrico Sintasa de Tipo III/metabolismo , Ratas , Ratas Sprague-DawleyRESUMEN
Carbapenemase genes in Enterobacteriaceae are mostly described as being plasmid associated. However, the genetic context of carbapenemase genes is not always confirmed in epidemiological surveys, and the frequency of their chromosomal integration therefore is unknown. A previously sequenced collection of blaKPC-positive Enterobacteriaceae from a single U.S. institution (2007 to 2012; n = 281 isolates from 182 patients) was analyzed to identify chromosomal insertions of Tn4401, the transposon most frequently harboring blaKPC Using a combination of short- and long-read sequencing, we confirmed five independent chromosomal integration events from 6/182 (3%) patients, corresponding to 15/281 (5%) isolates. Three patients had isolates identified by perirectal screening, and three had infections which were all successfully treated. When a single copy of blaKPC was in the chromosome, one or both of the phenotypic carbapenemase tests were negative. All chromosomally integrated blaKPC genes were from Klebsiella spp., predominantly K. pneumoniae clonal group 258 (CG258), even though these represented only a small proportion of the isolates. Integration occurred via IS15-ΔI-mediated transposition of a larger, composite region encompassing Tn4401 at one locus of chromosomal integration, seen in the same strain (K. pneumoniae ST340) in two patients. In summary, we identified five independent chromosomal integrations of blaKPC in a large outbreak, demonstrating that this is not a rare event. blaKPC was more frequently integrated into the chromosome of epidemic CG258 K. pneumoniae lineages (ST11, ST258, and ST340) and was more difficult to detect by routine phenotypic methods in this context. The presence of chromosomally integrated blaKPC within successful, globally disseminated K. pneumoniae strains therefore is likely underestimated.
Asunto(s)
Cromosomas Bacterianos/química , ADN Bacteriano/genética , Regulación Bacteriana de la Expresión Génica , Klebsiella pneumoniae/genética , Mutagénesis Insercional , beta-Lactamasas/genética , Mapeo Cromosómico , Cromosomas Bacterianos/metabolismo , Células Clonales , Elementos Transponibles de ADN , ADN Bacteriano/metabolismo , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Infecciones por Klebsiella/epidemiología , Infecciones por Klebsiella/microbiología , Klebsiella pneumoniae/enzimología , Tasa de Mutación , Análisis de Secuencia de ADN , Virginia/epidemiología , beta-Lactamasas/metabolismoRESUMEN
The Klebsiella pneumoniae carbapenemase gene (blaKPC) is typically located within mobile transposon Tn4401 Enhanced KPC expression has been associated with deletions in the putative promoter region upstream of blaKPC Illumina sequences from blaKPC-positive clinical isolates from a single institution were mapped to a Tn4401b reference sequence, which carries no deletions. The novel isoform Tn4401h (188-bp deletion [between istB and blaKPC]) was present in 14% (39/281) of clinical isolates. MICs showed that Escherichia coli strains containing plasmids with Tn4401a and Tn4401h were more resistant to meropenem (≥16 and ≥16, respectively), ertapenem (≥8 and 4, respectively), and cefepime (≥64 and 4, respectively) than E. coli strains with Tn4401b (0.5, ≤0.5, and ≤1, respectively). Quantitative real-time PCR (qRT-PCR) demonstrated that Tn4401a had a 16-fold increase and Tn4401h a 4-fold increase in blaKPC mRNA levels compared to the reference Tn4401b. A lacZ reporter plasmid was used to test the activity of the promoter regions from the different variants, and the results showed that the Tn4401a and Tn4401h promoter sequences generated higher ß-galactosidase activity than the corresponding Tn4401b sequence. Further dissection of the promoter region demonstrated that putative promoter P1 was not functional. The activity of the isolated P2 promoter was greatly enhanced by inclusion of the P1-P2 intervening sequence. These studies indicated that gene expression could be an important consideration in understanding resistance phenotypes predicted by genetic signatures in the context of sequencing-based rapid diagnostics.
Asunto(s)
Antibacterianos/farmacología , Proteínas Bacterianas/genética , Carbapenémicos/farmacología , Elementos Transponibles de ADN/genética , Klebsiella pneumoniae/efectos de los fármacos , Klebsiella pneumoniae/genética , beta-Lactamasas/genética , Proteínas Bacterianas/biosíntesis , Cefepima , Cefalosporinas/farmacología , Ertapenem , Escherichia coli/efectos de los fármacos , Escherichia coli/genética , Humanos , Infecciones por Klebsiella/microbiología , Klebsiella pneumoniae/aislamiento & purificación , Meropenem , Pruebas de Sensibilidad Microbiana , Regiones Promotoras Genéticas/genética , Eliminación de Secuencia/genética , Tienamicinas/farmacología , beta-Galactosidasa/genética , beta-Galactosidasa/metabolismo , beta-Lactamasas/biosíntesis , beta-Lactamas/farmacologíaRESUMEN
There have been an increasing number of reports implicating Gammaproteobacteria as often carrying genes of drug resistance from colonized sink traps to vulnerable hospitalized patients. However, the mechanism of transmission from the wastewater of the sink P-trap to patients remains poorly understood. Herein we report the use of a designated hand-washing sink lab gallery to model dispersion of green fluorescent protein (GFP)-expressing Escherichia coli from sink wastewater to the surrounding environment. We found no dispersion of GFP-expressing E. coli directly from the P-trap to the sink basin or surrounding countertop with coincident water flow from a faucet. However, when the GFP-expressing E. coli cells were allowed to mature in the P-trap under conditions similar to those in a hospital environment, a GFP-expressing E. coli-containing putative biofilm extended upward over 7 days to reach the strainer. This subsequently resulted in droplet dispersion to the surrounding areas (<30 in.) during faucet operation. We also demonstrated that P-trap colonization could occur by retrograde transmission along a common pipe. We postulate that the organisms mobilize up to the strainer from the P-trap, resulting in droplet dispersion rather than dispersion directly from the P-trap. This work helps to further define the mode of transmission of bacteria from a P-trap reservoir to a vulnerable hospitalized patient.IMPORTANCE Many recent reports demonstrate that sink drain pipes become colonized with highly consequential multidrug-resistant bacteria, which then results in hospital-acquired infections. However, the mechanism of dispersal of bacteria from the sink to patients has not been fully elucidated. Through establishment of a unique sink gallery, this work found that a staged mode of transmission involving biofilm growth from the lower pipe to the sink strainer and subsequent splatter to the bowl and surrounding area occurs rather than splatter directly from the water in the lower pipe. We have also demonstrated that bacterial transmission can occur via connections in wastewater plumbing to neighboring sinks. This work helps to more clearly define the mechanism and risk of transmission from a wastewater source to hospitalized patients in a world with increasingly antibiotic-resistant bacteria that can thrive in wastewater environments and cause infections in vulnerable patients.
Asunto(s)
Contaminación de Equipos , Infecciones por Escherichia coli/transmisión , Escherichia coli/aislamiento & purificación , Proteínas Fluorescentes Verdes/análisis , Desinfección de las Manos , Aguas Residuales/microbiología , Biopelículas , Infección Hospitalaria/microbiología , Infección Hospitalaria/prevención & control , Reservorios de Enfermedades/microbiología , Farmacorresistencia Bacteriana , Escherichia coli/química , Infecciones por Escherichia coli/microbiología , Hospitalización , Humanos , Pacientes InternosRESUMEN
Muscle microvasculature critically regulates endothelial exchange surface area to facilitate transendothelial delivery of insulin, nutrients, and oxygen to myocytes. Insulin resistance blunts insulin-mediated microvascular recruitment and decreases muscle capillary density; both contribute to lower microvascular blood volume. Glucagon-like peptide 1 (GLP-1) and its analogs are able to dilate blood vessels and stimulate endothelial cell proliferation. In this study, we aim to determine the effects of sustained stimulation of the GLP-1 receptors on insulin-mediated capillary recruitment and metabolic insulin responses, small arterial endothelial function, and muscle capillary density. Rats were fed a high-fat diet (HFD) for 4 wk with or without simultaneous administration of liraglutide and subjected to a euglycemic hyperinsulinemic clamp for 120 min after an overnight fast. Insulin-mediated muscle microvascular recruitment and muscle oxygenation were determined before and during insulin infusion. Muscle capillary density was determined and distal saphenous artery used for determination of endothelial function and insulin-mediated vasodilation. HFD induced muscle microvascular insulin resistance and small arterial vessel endothelial dysfunction and decreased muscle capillary density. Simultaneous treatment of HFD-fed rats with liraglutide prevented all of these changes and improved insulin-stimulated glucose disposal. These were associated with a significantly increased AMPK phosphorylation and the expressions of VEGF and its receptors. We conclude that GLP-1 receptor agonists may exert their salutary glycemic effect via improving microvascular insulin sensitivity and muscle capillary density during the development of insulin resistance, and early use of GLP-1 receptor agonists may attenuate metabolic insulin resistance as well as prevent cardiovascular complications of diabetes.
Asunto(s)
Capilares/metabolismo , Dieta Alta en Grasa/efectos adversos , Hipoglucemiantes/farmacología , Resistencia a la Insulina , Liraglutida/farmacología , Microvasos/efectos de los fármacos , Músculo Esquelético/irrigación sanguínea , Músculo Esquelético/efectos de los fármacos , Animales , Capilares/efectos de los fármacos , Endotelio Vascular/efectos de los fármacos , Péptido 1 Similar al Glucagón/metabolismo , Receptor del Péptido 1 Similar al Glucagón/biosíntesis , Insulina/farmacología , Masculino , Ratas , Ratas Sprague-Dawley , Flujo Sanguíneo Regional/efectos de los fármacos , Vasodilatación/efectos de los fármacosRESUMEN
RATIONALE: Adiponectin enhances insulin action and induces nitric oxide-dependent vasodilatation. Insulin delivery to muscle microcirculation and transendothelial transport are 2 discrete steps that limit insulin's action. We have shown that expansion of muscle microvascular surface area increases muscle insulin delivery and action. OBJECTIVE: To examine whether adiponectin modulates muscle microvascular recruitment thus insulin delivery and action in vivo. METHODS AND RESULTS: Overnight fasted adult male rats were studied. We determined the effects of adiponectin on muscle microvascular recruitment, using contrast-enhanced ultrasound, on insulin-mediated microvascular recruitment and whole-body glucose disposal, using contrast-enhanced ultrasound and insulin clamp, and on muscle insulin clearance and uptake with (125)I-insulin. Globular adiponectin potently increased muscle microvascular blood volume without altering microvascular blood flow velocity, leading to a significantly increased microvascular blood flow. This was paralleled by a ≈30% to 40% increase in muscle insulin uptake and clearance, and ≈30% increase in insulin-stimulated whole-body glucose disposal. Inhibition of endothelial nitric oxide synthase abolished globular adiponectin-mediated muscle microvascular recruitment and insulin uptake. In cultured endothelial cells, globular adiponectin dose-dependently increased endothelial nitric oxide synthase phosphorylation but had no effect on endothelial cell internalization of insulin. CONCLUSIONS: Globular adiponectin increases muscle insulin uptake by recruiting muscle microvasculature, which contributes to its insulin-sensitizing action.
Asunto(s)
Adiponectina/administración & dosificación , Glucemia/efectos de los fármacos , Hipoglucemiantes/administración & dosificación , Insulina/administración & dosificación , Microcirculación/efectos de los fármacos , Microvasos/efectos de los fármacos , Músculo Esquelético/irrigación sanguínea , Músculo Esquelético/efectos de los fármacos , Adiponectina/química , Animales , Glucemia/metabolismo , Bovinos , Células Cultivadas , Relación Dosis-Respuesta a Droga , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Inhibidores Enzimáticos/farmacología , Ayuno/sangre , Técnica de Clampeo de la Glucosa , Miembro Posterior , Hipoglucemiantes/metabolismo , Infusiones Intravenosas , Inyecciones Intraperitoneales , Insulina/metabolismo , Masculino , Microvasos/diagnóstico por imagen , Músculo Esquelético/metabolismo , Óxido Nítrico/metabolismo , Óxido Nítrico Sintasa de Tipo III/antagonistas & inhibidores , Óxido Nítrico Sintasa de Tipo III/metabolismo , Fosforilación , Ratas , Ratas Sprague-Dawley , Flujo Sanguíneo Regional/efectos de los fármacos , Factores de Tiempo , UltrasonografíaRESUMEN
Muscle microvascular surface area determines substrate and hormonal exchanges between plasma and muscle interstitium. GLP-1 (glucagon-like peptide-1) regulates glucose-dependent insulin secretion and has numerous extrapancreatic effects, including a salutary vascular action. To examine whether GLP-1 recruits skeletal and cardiac muscle microvasculature in healthy humans, 26 overnight-fasted healthy adults received a systemic infusion of GLP-1 (1.2 pmol/kg of body mass per min) for 150 min. Skeletal and cardiac muscle MBV (microvascular blood volume), MFV (microvascular flow velocity) and MBF (microvascular blood flow) were determined at baseline and after 30 and 150 min. Brachial artery diameter and mean flow velocity were measured and total blood flow was calculated before and at the end of the GLP-1 infusion. GLP-1 infusion raised plasma GLP-1 concentrations to the postprandial levels and suppressed plasma glucagon concentrations with a transient increase in plasma insulin concentrations. Skeletal and cardiac muscle MBV and MBF increased significantly at both 30 and 150 min (P<0.05). MFV did not change in skeletal muscle, but decreased slightly in cardiac muscle. GLP-1 infusion significantly increased brachial artery diameter (P<0.005) and flow velocity (P=0.05) at 150 min, resulting in a significant increase in total brachial artery blood flow (P<0.005). We conclude that acute GLP-1 infusion significantly recruits skeletal and cardiac muscle microvasculature in addition to relaxing the conduit artery in healthy humans. This could contribute to increased tissue oxygen, nutrient and insulin delivery and exchange and therefore better prandial glycaemic control and tissue function in humans.
Asunto(s)
Vasos Coronarios/metabolismo , Péptido 1 Similar al Glucagón/farmacología , Incretinas/farmacología , Microvasos/metabolismo , Músculo Esquelético/irrigación sanguínea , Adolescente , Adulto , Velocidad del Flujo Sanguíneo/efectos de los fármacos , Volumen Sanguíneo/efectos de los fármacos , Vasos Coronarios/efectos de los fármacos , Glucagón/sangre , Péptido 1 Similar al Glucagón/metabolismo , Péptido 1 Similar al Glucagón/fisiología , Humanos , Microvasos/efectos de los fármacos , Músculo Esquelético/efectos de los fármacos , Flujo Sanguíneo Regional/efectos de los fármacosRESUMEN
BACKGROUND: Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most important infectious agents for the swine industry worldwide. Zinc (Zn) salts, which are widely used as a dietary supplement in swine nutrition, have shown antiviral effects in vitro as well as in vivo. The purpose of this study was to determine the influence of dietary zinc oxide supplementation on vaccination and challenge infection with PRRSV. FINDINGS: The clinical course of PRRS and the success of vaccination with an experimental inactivated vaccine were compared between animals receiving a conventional diet (50 ppm Zn, control group) and diets supplemented with Zn oxide (ZnO) at final Zn concentrations of 150 or 2,500 ppm. Pigs receiving higher dietary Zn levels showed a tendency towards higher neutralizing antibody levels after infection, while dietary Zn levels did not substantially influence the number of antiviral IFN-gamma secreting cells (IFN-gamma-SC) or percentages of blood immune cell subsets after infection. Finally, feeding higher dietary Zn levels reduced neither clinical symptoms nor viral loads. CONCLUSIONS: Our results suggest that higher levels of dietary ZnO do not have the potential to stimulate or modulate systemic immune responses after vaccination and heterologous PRRSV infection to an extent that could improve the clinical and virological outcome.
Asunto(s)
Suplementos Dietéticos , Síndrome Respiratorio y de la Reproducción Porcina/inmunología , Síndrome Respiratorio y de la Reproducción Porcina/virología , Virus del Síndrome Respiratorio y Reproductivo Porcino/inmunología , Óxido de Zinc/administración & dosificación , Animales , Anticuerpos Antivirales/inmunología , Temperatura Corporal , Femenino , Humanos , Inmunoglobulina G/sangre , Inmunoglobulina G/inmunología , Inmunoglobulina M/sangre , Inmunoglobulina M/inmunología , Masculino , Síndrome Respiratorio y de la Reproducción Porcina/prevención & control , Virus del Síndrome Respiratorio y Reproductivo Porcino/genética , Porcinos , Vacunación , Carga Viral , Vacunas Virales/inmunologíaRESUMEN
BACKGROUND: Zinc (Zn) supplementation has been shown to reduce the incidence of diarrhea and to protect animals from intestinal diseases, but the mechanisms of this protective effect against virus infection in vivo have not yet been elucidated. Transmissible gastroenteritis virus (TGEV) causes diarrhea in piglets with an age-dependent decrease of severity. RESULTS: We used 60 weaned piglets that were divided into three groups to evaluate the effect of different Zn levels added to a conventional diet (50 mg Zn/kg diet, Znlow, control group). The other groups received the diet supplemented with ZnO at final concentrations of 150 mg Zn/kg diet (Znmed), or 2,500 mg/kg diet (Znhigh). Oral challenge infection with TGEV was performed when the pigs had been fed for 1 week with the respective diet. Half of the piglets of each group were sacrificed at day 1 and 18 after challenge infection. Fecal consistency was improved and body weights increased in the Znhigh group when compared to the other groups, but no direct effect of Zn concentrations in the diet on fecal TGEV shedding and mucosal immune responses was detectable. However, in the Znhigh group, we found a prevention of villus atrophy and decreased caspase-3-mediated apoptosis of jejunal epithelium. Furthermore, pigs receiving high Zn diet showed a down-regulation of interferon (IFN)-α, oligoadenylate synthetase (OAS), Zn transporter SLC39A4 (ZIP4), but up-regulation of metallothionein-1 (MT1), as well as the Zn transporters SLC30A1 (ZnT1) and SLC30A5 (ZnT5). In addition, forskolin-induced chloride secretion and epithelial resistance were controlled at a physiological level in the Znhigh but not the other groups. Finally, in the Znhigh group, we documented an earlier and higher systemic TGEV-specific serum antibody response. CONCLUSIONS: These results suggest that high dietary Zn could provide enhanced protection in the intestinal tract and stimulate the systemic humoral immune response against TGEV infection.
Asunto(s)
Suplementos Dietéticos , Gastroenteritis Porcina Transmisible/tratamiento farmacológico , Virus de la Gastroenteritis Transmisible/genética , Óxido de Zinc/farmacología , Alimentación Animal , Animales , Caspasa 3/metabolismo , Dieta/veterinaria , Femenino , Regulación de la Expresión Génica/fisiología , Mucosa Intestinal/enzimología , Mucosa Intestinal/metabolismo , Yeyuno/enzimología , Masculino , Porcinos , OligoelementosRESUMEN
Ranolazine, an anti-anginal compound, has been shown to significantly improve glycaemic control in large-scale clinical trials, and short-term ranolazine treatment is associated with an improvement in myocardial blood flow. As microvascular perfusion plays critical roles in insulin delivery and action, we aimed to determine if ranolazine could improve muscle microvascular blood flow, thereby increasing muscle insulin delivery and glucose use. Overnight-fasted, anaesthetized Sprague-Dawley rats were used to determine the effects of ranolazine on microvascular recruitment using contrast-enhanced ultrasound, insulin action with euglycaemic hyperinsulinaemic clamp, and muscle insulin uptake using (125)I-insulin. Ranolazine's effects on endothelial nitric oxide synthase (eNOS) phosphorylation, cAMP generation and endothelial insulin uptake were determined in cultured endothelial cells. Ranolazine-induced myographical changes in tension were determined in isolated distal saphenous artery. Ranolazine at therapeutically effective dose significantly recruited muscle microvasculature by increasing muscle microvascular blood volume (â¼2-fold, P < 0.05) and increased insulin-mediated whole body glucose disposal (â¼30%, P = 0.02). These were associated with an increased insulin delivery into the muscle (P < 0.04). In cultured endothelial cells, ranolazine increased eNOS phosphorylation and cAMP production without affecting endothelial insulin uptake. In ex vivo studies, ranolazine exerted a potent vasodilatatory effect on phenylephrine pre-constricted arterial rings, which was partially abolished by endothelium denudement. In conclusion, ranolazine treatment vasodilatates pre-capillary arterioles and increases microvascular perfusion, which are partially mediated by endothelium, leading to expanded microvascular endothelial surface area available for nutrient and hormone exchanges and resulting in increased muscle delivery and action of insulin. Whether these actions contribute to improved glycaemic control in patients with insulin resistance warrants further investigation.
Asunto(s)
Acetanilidas/farmacología , Glucemia/metabolismo , Insulina/sangre , Microvasos/efectos de los fármacos , Músculo Esquelético/irrigación sanguínea , Piperazinas/farmacología , Animales , Bovinos , Células Cultivadas , AMP Cíclico/metabolismo , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Insulina/metabolismo , Sistema de Señalización de MAP Quinasas , Masculino , Microvasos/diagnóstico por imagen , Microvasos/metabolismo , Microvasos/fisiología , Óxido Nítrico Sintasa de Tipo III/metabolismo , Ranolazina , Ratas , Ratas Sprague-Dawley , Flujo Sanguíneo Regional , Ultrasonografía , VasodilataciónRESUMEN
Insulin delivery and transendothelial insulin transport are two discrete steps that limit muscle insulin action. Angiotensin II type 1 receptor (AT1R) blockade recruits microvasculature and increases glucose use in muscle. Increased muscle microvascular perfusion is associated with increased muscle delivery and action of insulin. To examine the effect of acute AT1R blockade on muscle insulin uptake and action, rats were studied after an overnight fast to examine the effects of losartan on muscle insulin uptake (protocol 1), microvascular perfusion (protocol 2), and insulin's microvascular and metabolic actions in the state of insulin resistance (protocol 3). Endothelial cell insulin uptake was assessed, using (125)I-insulin as tracer. Systemic lipid infusion was used to induce insulin resistance. Losartan significantly increased muscle insulin uptake (â¼60%, P < 0.03), which was associated with a two- to threefold increase in muscle microvascular blood volume (MBV; P = 0.002) and flow (MBF; P = 0.002). Losartan ± angiotensin II had no effect on insulin internalization in cultured endothelial cells. Lipid infusion abolished insulin-mediated increases in muscle MBV and MBF and lowered insulin-stimulated whole body glucose disposal (P = 0.0001), which were reversed by losartan administration. Inhibition of nitric oxide synthase abolished losartan-induced muscle insulin uptake and reversal of lipid-induced metabolic insulin resistance. We conclude that AT1R blockade increases muscle insulin uptake mainly via microvascular recruitment and rescues insulin's metabolic action in the insulin-resistant state. This may contribute to the clinical findings of decreased cardiovascular events and new onset of diabetes in patients receiving AT1R blockers.
Asunto(s)
Bloqueadores del Receptor Tipo 1 de Angiotensina II/farmacología , Capilares/efectos de los fármacos , Hipoglucemiantes/farmacología , Insulina/metabolismo , Insulina/farmacología , Lípidos/farmacología , Losartán/farmacología , Músculo Esquelético/metabolismo , Animales , Presión Sanguínea/efectos de los fármacos , Células Cultivadas , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Inhibidores Enzimáticos/farmacología , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Masculino , Microcirculación/efectos de los fármacos , NG-Nitroarginina Metil Éster/farmacología , Óxido Nítrico/sangre , Óxido Nítrico/fisiología , Óxido Nítrico Sintasa/antagonistas & inhibidores , Fosforilación/efectos de los fármacos , Proteínas Proto-Oncogénicas c-akt/biosíntesis , Ratas , Ratas Sprague-Dawley , Transducción de Señal/efectos de los fármacosRESUMEN
Glucagon-like peptide-1 (GLP-1) causes vasodilation and increases muscle glucose uptake independent of insulin. Recently, we have shown that GLP-1 recruits muscle microvasculature and increases muscle glucose use via a nitric oxide (NO)-dependent mechanism. Protein kinase A (PKA) is a major signaling intermediate downstream of GLP-1 receptors. To examine whether PKA mediates GLP-1's microvascular action in muscle, GLP-1 was infused to overnight-fasted male rats for 120 min in the presence or absence of H89, a PKA inhibitor. Hindleg muscle microvascular recruitment and glucose use were determined. GLP-1 infusion acutely increased muscle microvascular blood volume within 30 min without altering microvascular blood flow velocity or blood pressure. This effect persisted throughout the 120-min infusion period, leading to a significant increase in muscle microvascular blood flow. These changes were paralleled with an approximately twofold increase in plasma NO levels and hindleg glucose extraction. Systemic infusion of H89 completely blocked GLP-1-mediated muscle microvascular recruitment and increases in NO production and muscle glucose extraction. In cultured endothelial cells, GLP-1 acutely increased PKA activity and stimulated endothelial NO synthase phosphorylation at Ser(1177) and NO production. PKA inhibition abolished these effects. In ex vivo studies, perfusion of the distal saphenous artery with GLP-1 induced significant vasorelaxation that was also abolished by pretreatment of the vessels with PKA inhibitor H89. We conclude that GLP-1 recruits muscle microvasculature by expanding microvascular volume and increases glucose extraction in muscle via a PKA/NO-dependent pathway in the vascular endothelium. This may contribute to postprandial glycemic control and complication prevention in diabetes.
Asunto(s)
Proteínas Quinasas Dependientes de AMP Cíclico/fisiología , Péptido 1 Similar al Glucagón/farmacología , Músculo Esquelético/irrigación sanguínea , Músculo Esquelético/efectos de los fármacos , Óxido Nítrico/metabolismo , Animales , Presión Sanguínea/efectos de los fármacos , Presión Sanguínea/fisiología , Bovinos , Células Cultivadas , Proteínas Quinasas Dependientes de AMP Cíclico/antagonistas & inhibidores , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Péptido 1 Similar al Glucagón/fisiología , Insulina/sangre , Masculino , Microvasos/efectos de los fármacos , Microvasos/fisiología , Músculo Esquelético/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Ratas , Ratas Sprague-Dawley , Flujo Sanguíneo Regional/efectos de los fármacosRESUMEN
The enteropathogenic coronavirus transmissible gastroenteritis virus (TGEV) causes severe disease in young piglets. We have studied the protective effects of the probiotic Enterococcus faecium NCIMB 10415 (E. faecium), which is approved as a feed additive in the European Union, against TGEV infection. E. faecium was added to swine testicle (ST) cells before, concomitantly with, or after TGEV infection. Viability assays revealed that E. faecium led to a dose-dependent rescue of viability of TGEV-infected cells reaching nearly to complete protection. Virus yields of the E. faecium-treated cultures were reduced by up to three log10 units. Western blot analysis of purified TGEV revealed that the levels of all viral structural proteins were reduced after E. faecium treatment. Using transmission electron microscopy, we observed attachment of TGEV particles to the surface of E. faecium which might be a means to trap virus and to prevent infection. Increased production of nitric oxide in the cells treated with E. faecium and elevated expression of interleukin 6 and 8 pointed to stimulated cellular defense as a mechanism to fight TGEV infection.
Asunto(s)
Enterococcus faecium/fisiología , Gastroenteritis Porcina Transmisible/prevención & control , Probióticos , Virus de la Gastroenteritis Transmisible , Alimentación Animal , Animales , Línea Celular , Supervivencia Celular , Citocinas/genética , Citocinas/metabolismo , Dieta/veterinaria , Regulación de la Expresión Génica , Masculino , Óxido Nítrico/metabolismo , Porcinos , Testículo/citología , Cultivo de VirusRESUMEN
Resveratrol, a polyphenol found in many plants, has antioxidant and anti-inflammatory actions. It also improves endothelial function and may be cardioprotective. Tumor necrosis factor-α (TNFα) causes oxidative stress and microvascular endothelial dysfunction. Whether resveratrol affects microvascular function in vivo and, if so, whether inflammatory cytokines antagonize its microvascular action are not clear. In cultured bovine aortic endothelial cells (BAECs), resveratrol (100 nM) increased the phosphorylation of protein kinase B (Akt), endothelial nitric oxide (NO) synthase (eNOS), and ERK1/2 within 15 min by more than twofold, and this effect lasted for at least 2 h. Treatment of BAECs with TNFα (10 ng/ml) significantly increased the NADPH oxidase activity and the production of hydrogen peroxide and superoxide. Pretreatment of cells with resveratrol (100 nM) prevented each of these. Injection (ip) of resveratrol in rats potently increased muscle microvascular blood volume (MBV; P = 0.007) and flow (MBF; P < 0.02) within 30 min, and this was sustained for at least 2 h. The phosphorylation of Akt in liver or muscle was unchanged. Superimposed systemic infusion of L-NAME (NOS inhibitor) completely abolished resveratrol-induced increases in MBV and MBF. Similarly, systemic infusion of TNFα prevented resveratrol-induced muscle microvascular recruitment. In conclusion, resveratrol activates eNOS and increases muscle microvascular recruitment via an NO-dependent mechanism. Despite the potent antioxidant effect of resveratrol, TNFα at concentrations that block insulin-mediated muscle microvascular recruitment completely neutralized resveratrol's microvascular action. Thus, chronic inflammation, as seen in type 2 diabetes, may limit resveratrol's vasodilatory actions on muscle microvasculature.
Asunto(s)
Microvasos/efectos de los fármacos , Músculo Esquelético/irrigación sanguínea , Músculo Esquelético/efectos de los fármacos , Óxido Nítrico/metabolismo , Estilbenos/farmacología , Factor de Necrosis Tumoral alfa/metabolismo , Vasodilatadores/farmacología , Animales , Antioxidantes/farmacología , Bovinos , Células Cultivadas , Endotelio Vascular/citología , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/metabolismo , Inhibidores Enzimáticos/farmacología , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Masculino , Microvasos/metabolismo , Músculo Esquelético/metabolismo , Óxido Nítrico Sintasa de Tipo III/antagonistas & inhibidores , Óxido Nítrico Sintasa de Tipo III/metabolismo , Estrés Oxidativo/efectos de los fármacos , Fosforilación/efectos de los fármacos , Proteínas Proto-Oncogénicas c-akt/metabolismo , Ratas , Ratas Sprague-Dawley , Flujo Sanguíneo Regional/efectos de los fármacos , ResveratrolRESUMEN
BACKGROUND: Type 2 diabetes mellitus (T2DM) is associated with an increased risk of atherosclerotic vascular disease, but it is unknown whether the other way around is true too. C57BL/6 (B6) and BALB/cJ (BALB) are two mouse strains that differ markedly in their susceptibility to atherosclerosis. In this study we investigated the development of diet-induced T2DM in these two strains. METHODS AND RESULTS: When deficient in apolipoprotein E (apoE(-/-)) and fed a Western diet for 12 weeks, atherosclerosis-susceptible B6 mice developed significant hyperglycemia. In contrast, atherosclerosis-resistant BALB apoE(-/-) mice had much lower plasma glucose levels than B6.apoE(-/-) mice on either chow or Western diet and during an intraperitoneal glucose tolerance test. In response to glucose BALB.apoE(-/-) mice displayed both the first and second phases of insulin secretion but the second phase of insulin secretion was absent in B6.apoE(-/-) mice. In response to insulin B6.apoE(-/-) mice showed a deeper and longer-lasting fall in blood glucose levels while BALB.apoE(-/-) mice showed little reduction in glucose levels. Pancreatic islet area of BALB.apoE(-/-) mice on light microscopy nearly doubled the area of B6.apoE(-/-) mice. Most circulating proinflammatory cytokines were lower in BALB.apoE(-/-) than in B6.apoE(-/-) mice on the Western diet, as determined by protein arrays. Increased macrophage infiltration in islets was observed in B6.apoE(-/-) mice by immunostaining for Mac2 and also by flow cytometry. CONCLUSION: This study demonstrates that defects in insulin secretion rather than defects in insulin resistance explain the marketed difference in susceptibility to T2DM in the B6.apoE(-/-) and BALB.apoE(-/-) mouse model. A smaller islet mass and more prominent islet inflammation may explain the vulnerability of B6.apoE(-/-) mice to diet-induced diabetes.
Asunto(s)
Enfermedades de la Aorta/metabolismo , Apolipoproteínas E/deficiencia , Aterosclerosis/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Islotes Pancreáticos/metabolismo , Animales , Enfermedades de la Aorta/etiología , Enfermedades de la Aorta/genética , Enfermedades de la Aorta/patología , Apolipoproteínas E/genética , Aterosclerosis/etiología , Aterosclerosis/genética , Aterosclerosis/patología , Biomarcadores/sangre , Glucemia/metabolismo , Citocinas/sangre , Diabetes Mellitus Tipo 2/etiología , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/patología , Dieta Alta en Grasa , Modelos Animales de Enfermedad , Femenino , Citometría de Flujo , Galectina 3/metabolismo , Prueba de Tolerancia a la Glucosa , Inmunohistoquímica , Mediadores de Inflamación/sangre , Insulina/sangre , Resistencia a la Insulina , Islotes Pancreáticos/patología , Macrófagos/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Análisis por Matrices de Proteínas , Especificidad de la Especie , Factores de TiempoRESUMEN
The emergence of novel and variant porcine reproductive and respiratory syndrome virus (PRRSV) strains has made controlling this disease a challenge in China. Several NADC30-like PRRSV outbreaks have occurred in mainland China since 2013. The objective of the present study was to evaluate the cross-protection efficacy of two commercial PRRS modified-live virus (MLV) vaccines, derived from classical PRRSV (VR2332) and highly pathogenic (HP) PRRSV (TJM-F92), against an increasingly circulating NADC30-like lineage in pigs. Thirty-five PRRSV- and antibody-free pigs were randomly divided into the following four groups: strict control (SC), negative control (NC), Boehringer control (BC), and Zoetis control (ZC) groups. The NADC30-like PRRSV used in this study caused fever, clinical respiratory signs, and gross and microscopic lung lesions in inoculated pigs in the NC group. Vaccination with the VR2332 vaccine significantly reduced the percentage of viremic pigs as well as gross lung lesions and improved average daily weight gain compared to the ZC and NC groups, suggesting that this MLV vaccine provides cross-protection against the NADC30-like virus. There were no significant differences in the efficacy of the two MLV vaccines based on clinical scores, immunological responses, or pathological outcomes. This study demonstrated that VR2332 MLV was effective against circulating NADC30-like PRRSV and could be used to control NADC30-like virus infections in the field.
Asunto(s)
Anticuerpos Antivirales/sangre , Protección Cruzada , Síndrome Respiratorio y de la Reproducción Porcina/prevención & control , Enfermedades de los Porcinos/prevención & control , Vacunas Virales/inmunología , Animales , Filogenia , Síndrome Respiratorio y de la Reproducción Porcina/inmunología , Virus del Síndrome Respiratorio y Reproductivo Porcino , Porcinos , Enfermedades de los Porcinos/inmunología , Vacunación , Vacunas Atenuadas/inmunología , Viremia/prevención & control , VirulenciaRESUMEN
CONTEXT: Glucagon-like peptide-1 (GLP-1) and insulin increase muscle microvascular perfusion, thereby increasing tissue endothelial surface area and nutrient delivery. OBJECTIVE: To examine whether GLP-1 and insulin act additively on skeletal and cardiac microvasculature and conduit artery. DESIGN: Healthy adults underwent three study protocols in random order. SETTING: Clinical Research Unit at the University of Virginia. METHODS: Overnight-fasted participants received an intravenous infusion of GLP-1 (1.2 pmol/kg/min) or normal saline for 150 minutes with or without a 2-hour euglycemic insulin clamp (1 mU/kg/min) superimposed from 30 minutes onward. Skeletal and cardiac muscle microvascular blood volume (MBV), flow velocity, and flow; brachial artery diameter, flow velocity, and blood flow; and pulse wave velocity (PWV) were measured. RESULTS: GLP-1 significantly increased skeletal and cardiac muscle MBV and microvascular blood flow (MBF) after 30 minutes; these remained elevated at 150 minutes. Insulin also increased skeletal and cardiac muscle MBV and MBF. Addition of insulin to GLP-1 did not further increase skeletal and cardiac muscle MBV and MBF. GLP-1 and insulin increased brachial artery diameter and blood flow, but this effect was not additive. Neither GLP-1, insulin, nor GLP-1 and insulin altered PWV. Combined GLP-1 and insulin infusion did not result in higher whole-body glucose disposal. CONCLUSION: GLP-1 and insulin at physiological concentrations acutely increase skeletal and cardiac muscle microvascular perfusion and dilate conduit artery in healthy adults; these effects are not additive. Thus, GLP-1 and insulin may regulate skeletal and cardiac muscle endothelial surface area and nutrient delivery under physiological conditions.
RESUMEN
Plasma free fatty acids are elevated in patients with type 2 diabetes and contribute to the pathogenesis of insulin resistance and endothelial dysfunction. The p38 MAPK mediates stress, inflammation, and apoptosis. Whether free fatty acids induce apoptosis and/or activate nuclear factor-kappaB inflammatory pathway in human coronary artery endothelial cells (hCAECs) and, if so, whether this involves the p38 MAPK pathway is unknown. hCAECs (passages 4-6) were grown to 70% confluence and then incubated with palmitate at concentrations of 0-300 microm for 6-48 h. Palmitate at 100, 200, or 300 microm markedly increased apoptosis after 12 h of incubation. This apoptotic effect was time (P=0.008) and dose (P=0.006) dependent. Palmitate (100 microm for 24 h) induced a greater than 2-fold increase in apoptosis, which was accompanied with a 4-fold increase in p38 MAPK activity (P<0.001). Palmitate did not affect the phosphorylation of Akt1 or ERK1/2. SB203580 (a specific inhibitor of p38 MAPK) alone did not affect cellular apoptosis; however, it abolished palmitate-induced apoptosis and p38 MAPK activation. Palmitate significantly reduced the level of inhibitor of nuclear factor-kappaB (IkappaB). However, treatment of cells with SB203580 did not restore IkappaB to baseline. We conclude that palmitate induces hCAEC apoptosis via a p38 MAPK-dependent mechanism and may participate in coronary endothelial injury in diabetes. However, palmitate-mediated IkappaB degradation in hCAECs is independent of p38 MAPK activity.
Asunto(s)
Apoptosis/efectos de los fármacos , Vasos Coronarios/metabolismo , Células Endoteliales/metabolismo , Proteínas I-kappa B/metabolismo , Ácido Palmítico/farmacología , Procesamiento Proteico-Postraduccional , Proteínas Quinasas p38 Activadas por Mitógenos/fisiología , Células Cultivadas , Activación Enzimática/efectos de los fármacos , Humanos , Proteína Oncogénica v-akt/metabolismo , FosforilaciónRESUMEN
In muscle, physiologic hyperinsulinemia, presumably acting on endothelial cells (ECs), dilates arterioles and regulates both total blood flow and capillary recruitment, which in turn influences glucose disposal. In cultured ECs, however, supraphysiological (e.g. >or=10 nM) insulin concentrations are typically used to study insulin receptor (IR) signaling pathways and nitric oxide generation. IGF-I receptors (IGF-IRs) are more abundant than IR in ECs, and they also respond to high concentrations of insulin. To address whether IR mediates responses to physiologic insulin stimuli, we examined the insulin concentration dependence of IR and IGF-IR-mediated insulin signaling in bovine aortic ECs (bAECs). We also assessed whether insulin/IGF-I hybrid receptors were present in bAECs. Insulin, at 100-500 pM, significantly stimulated the phosphorylation of IRbeta, Akt1, endothelial isoform of nitric oxide synthase, and ERK 1/2 but not the IGF-IRbeta subunit. At concentrations 1-5 nm or greater, insulin dose-dependently enhanced the tyrosine phosphorylation of IGF-IRbeta, and this was inhibited by IGF-IR neutralizing antibody. In addition, immunoprecipitation of IRbeta pulled down the IGF-IRbeta, and the IRbeta immunocytochemically colocalized with IGF-IRbeta, suggesting that ECs have insulin/IGF-I hybrid receptors. We conclude that: 1) insulin at physiological concentrations selectively activates IR signaling in bAECs; 2) bAECs express IGF-IR and insulin/IGF-I hybrid receptors in addition to IR; 3) high concentrations of insulin (>or=1-5 nM) activate IGF-IR and hybrid receptors as well as IR; and 4) this crossover activation can confound interpretation of studies of insulin action in ECs when high insulin concentrations are used.