Role of exercise intensity on GLUT4 content, aerobic fitness and fasting plasma glucose in type 2 diabetic mice.

Type 2 diabetes mellitus (T2D) results in several metabolic and cardiovascular dysfunctions, clinically characterized by hyperglycaemia due to lower glucose uptake and oxidation. Physical exercise is an effective intervention for glycaemic control. However, the effects of exercising at different intensities have not yet been addressed. The present study analysed the effects of 8 weeks of training performed at different exercise intensities on type 4 glucose transporters (GLUT4) content and glycaemic control of T2D (ob/ob) and non-diabetic mice (ob/OB). The animals were divided into six groups, with four groups being subjected either to low-intensity (ob/obL and ob/OBL: 3% body weight, three times/week/40 min) or high-intensity (ob/obH and ob/OBH: 6% body weight, three times per week per 20 min) swimming training. An incremental swimming test was performed to measure aerobic fitness. After the training intervention period, glycaemia and the content of GLUT4 were quantified. Although both training intensities were beneficial, the high-intensity regimen induced a more significant improvement in GLUT4 levels and glycaemic profile compared with sedentary controls (p < 0.05). Only animals in the high-intensity exercise group improved aerobic fitness. Thus, our study shows that high-intensity training was more effective for increasing GLUT4 content and glycaemia reduction in insulin-resistant mice, perhaps because of a higher metabolic demand imposed by this form of exercise.

Angiostatic activity of human plasminogen fragments is highly dependent on glycosylation.

To assess the importance of carbohydrate moieties to the anti-angiogenic activity of plasminogen fragments, we cloned the fragment corresponding to amino acids Val(79) to Thr(346) (Kint3-4) that presents the three glycosylation sites. The activity of glycosylated and unglycosylated Kint3-4 was tested in murine sponge implant model. We observed a significant decrease in the neovascularization on the sponge after treatment with Kint3-4 by histological examination and determination of the hemoglobin levels. The effects were more intense with the glycosylated than the unglycosylated protein. (99m)Technecium-labeled red blood cells confirmed the inhibition of cell infiltration in the implanted sponge. Studies using melanoma B16F1 implanted in a mouse demonstrated that treatment with glycosylated Kint3-4 (0.15 nmol/48 h) during 14 days suppresses tumor growth by 80%. The vascular endothelial growth factor mRNA levels on the tumor were reduced after treatment. Kint3-4 is a potent plasminogen fragment that has been found to inhibit tumor growth.

(Pro)renin receptor expression in myocardial infarction in transgenic mice expressing rat tonin.

The (pro)renin receptor [(P)RR] has been implicated as a renin/prorenin receptor, and plays a role in local renin angiotensin system activation. Our goal was to investigate whether a transgenic mouse that expresses rat tonin [TGM'(rTon)] can regulate (P)RR mRNA levels. Control (C) and TGM'(rTon) animals were subdivided into the C sham, C MI, TGM'(rTon) sham, and TGM'(rTon) MI groups. The levels of tonin, (P)RR, and renin were determined using RT-PCR mRNA. Tonin activity as determined by RIE was significantly increased in the TGM'(rTon) sham group as compared to the C sham group in the atrium (AT) and right ventricle (RV), respectively. In most mice, tonin mRNA levels were significantly reduced compared to those in the TGM'(rTon) sham group in the atria. In this structure, the (P)RR mRNA levels were statistically significantly reduced in the TGM'(rTon) sham and TGM'(rTon) MI groups compared to the control groups. However, the (P)RR mRNA values were significantly increased when we compared the TGM'(rTon) MI vs TGM'(rTon) sham groups. In the RV, the renin mRNA levels in the TGM'(rTon) sham group were significantly reduced compared to the C sham group. Tonin overexpression may act in the regulation of (P)RR mRNA levels during MI.

Tonin Overexpression in Mice Diminishes Sympathetic Autonomic Modulation and Alters Angiotensin Type 1 Receptor Response.

Background: Tonin, a serine-protease that forms Angiotensin II (AngII) from angiotensinogen, is increased in failing human heart samples. Increased blood pressure (BP) and decreased heart rate (HR) variabilities are associated with higher risk of cardiovascular morbidity. Losartan has been used to reduce hypertension and, therefore, lowers the risk of fatal and non-fatal cardiovascular events. Determination of tonin's impact on BP and HR variabilities as well as the impact of losartan remain questions to be elucidated. Aim: Evaluation of cardiovascular autonomic profile in transgenic mice overexpressing the rat tonin enzyme TGM'(rton) and the impact of AT1 receptor blocker, losartan. Methods: Male C57BL/6 (WT) and TGM'(rTon) mice were cannulated for recording BP (Windaq, 4 MHz) for 30 min at baseline and 30 min after losartan injection (20 mg/kg). BP and HR variabilities were analyzed in time and frequency domain method. Low-frequency (LF) and high-frequency (HF) components were identified for sympathetic and parasympathetic modulations analysis. Ang I, AngII, and Ang1-7 were quantified by high performance liquid chromatography method. The total enzymatic activity for AngI, AngII, and Ang1-7 formation was evaluated in the heart and plasma by Liquid chromatography mass spectrometry (LC-MS/MS). Results: At the baseline TGM'(rTon) exhibited higher BP, lower cardiac LF, higher cardiac HF, lower LF/HF, and lower alpha index than wild type (WT). After losartan injection, TGM'(rTon) mice presented an additional decrease in cardiac LF and increase in HF in relation to baseline and WT. In the vasculature, losartan caused decreased in BP and LF of systolic BP in WT mice in relation to its baseline. A similar effect was observed in the BP of TGM'(rTon) mice; however, LF of systolic BP increased compared to baseline. Our data also indicates that AT1R receptor signaling has been altered in TGM'(rTon)mice. Interestingly, the dynamics of the renin-angiotensin system kinetics change, favoring production of Ang1-7. Conclusion: Autonomic evaluation of TGM'(rTon) mice indicates an unclear prognosis for diseases that affect the heart. HR variability in TGM'(rTon) mice indicates high risk of morbidity, and sympathetic and parasympathetic modulation indicate low risk of morbidity. The low risk of morbidity could be the biased production of Ang1-7 in the heart and circulation; however, the altered response of AT1R in the TGM'(rTon) remains to be elucidated, as well aswhether that signaling is pro-protection or pro-pathology.

Effect of a high-intensity exercise training on the metabolism and function of macrophages and lymphocytes of walker 256 tumor bearing rats.

Epidemiologic studies suggest that moderately intense training promotes augmented immune function, whereas strenuous exercise can cause immunosupression. Because the combat of cancer requires high immune function, high-intensity exercise could negatively affect the host organism; however, despite the epidemiologic data, there is a lack of experimental evidence to show that high-intensity training is harmful to the immune system. Therefore, we tested the influence of high-intensity treadmill training (10 weeks, 5 days/week, 30 mins/day, 85% VO(2)max) on immune system function and tumor development in Walker 256 tumor-bearing Wistar rats. The metabolism of glucose and glutamine in lymphocytes and macrophages was assessed, in addition to some functional parameters such as hydrogen peroxide production, phagocytosis, and lymphocyte proliferative responses. The metabolism of Walker 256 cells was also investigated. Results demonstrated that high-intensity training increased the life span of tumor-bearing rats, promoted a reduction in tumor mass, and prevented indicators of cachexia. Several changes, such as a reduction in body weight and food intake and activation of glutamine metabolism in macrophages and lymphocytes induced by the presence of Walker 256 tumor, were prevented by high intensity training. The reduction in tumor growth was associated with an impairment of tumor cell glucose and glutamine metabolism. These data suggest that high-intensity exercise training may be a viable strategy against tumors.

TRPM8 Channel Activation Induced by Monoterpenoid Rotundifolone Underlies Mesenteric Artery Relaxation.

In this study, our aims were to investigate transient receptor potential melastatin-8 channels (TRPM8) involvement in rotundifolone induced relaxation in the mesenteric artery and to increase the understanding of the role of these thermosensitive TRP channels in vascular tissue. Thus, message and protein levels of TRPM8 were measured by semi-quantitative PCR and western blotting in superior mesenteric arteries from 12 week-old Spague-Dawley (SD) rats. Isometric tension recordings evaluated the relaxant response in mesenteric rings were also performed. Additionally, the intracellular Ca2+ changes in mesenteric artery myocytes were measured using confocal microscopy. Using PCR and western blotting, both TRPM8 channel mRNA and protein expression was measured in SD rat mesenteric artery. Rotundifolone and menthol induced relaxation in the isolated superior mesenteric artery from SD rats and improved the relaxant response induced by cool temperatures. Also, this monoterpene induced an increase in transient intracellular Ca2+. These responses were significantly attenuated by pretreatment with capsazepine or BCTC, both TRPM8 channels blockers. The response induced by rotundifolone was not significantly attenuated by ruthenium red, a non-selective TRP channels blocker, or following capsaicin-mediated desensitization of TRPV1. Our findings suggest that rotundifolone induces relaxation by activating TRPM8 channels in rat superior mesenteric artery, more selectively than menthol, the classic TRPM8 agonist, and TRPM8 channels participates in vasodilatory pathways in isolated rat mesenteric arteries.

Antinociceptive response in transgenic mice expressing rat tonin.

Angiotensin II (Ang II) may be produced directly from angiotensinogen by tonin. Studies have demonstrated that Ang II and its metabolite Ang-(1-7) produce antinociception in pain animal models. The aim of the present study was to determine whether the transgenic mice that express rat tonin (TGM(rTon)) show altered nociceptive behavior and investigate the possible involvement of angiotensin metabolites. Nociception was evaluated using the thermal tail-flick and chemical acetic acid writhing tests, and the drugs were administered by intracerebroventricular and subcutaneous pathways, respectively. Probabilities less than 5% (P<0.05) were considered to be statistically significant (t test; ANOVA/Bonferroni's test). The results demonstrate that the transgenic mice showed an antinociceptive effect in the tail-flick and acetic acid writhing tests. In addition, it was observed that losartan, an AT1 receptor antagonist and A-779 (D-Ala7-Ang-(1-7)), a Mas receptor antagonist attenuated the antinociceptive behavior. Our data suggest that the Ang II produced in TGM(rTon) induces antinociception via the AT1 receptor, while the Ang-(1-7) produced from Ang II induced antinociception via the Mas receptor.

Estrogen therapy attenuates adiposity markers in spontaneously hypertensive rats.

Ovarian hormones modulate the metabolism of adipose cells and present a protective effect against hypertension. The aim of this study was to compare the effect of estradiol on adiposity markers in spontaneously hypertensive rats. Ovariectomized spontaneously hypertensive rats treated with estradiol (5 µg/100 g/day), three weeks after ovariectomy, presented decreased blood pressure and insulin levels and increased hepatic glycogen content. Periuterine or mesenteric adipocytes from treated animals were smaller as compared to vehicle treated group, whereas no differences were observed in relation to the number of cells. Basal rates of glycerol release were higher only in periuterine adipocytes of treated rats. The increment of glycerol release over basal values in response to isoproterenol was 400% and 440%, 283% and 330% for vehicle and estradiol treated periuterine and mesenteric adipocytes, respectively. The estradiol treated group was more sensitive to insulin inhibition of isoproterenol-stimulated lipolysis than the control animals. The lipoprotein lipase activity decreased after treatment, only in periuterine adipose tissue. Estradiol administration increased basal and insulin-stimulated rates of glucose transport in adipocytes of both sites, although the values obtained by periuterine were higher than those observed for mesenteric adipocytes. Both adipose tissues from treated animals exhibited a decreased expression of the peroxisome proliferator-activated receptor-γ, but an increased expression of peroxisome proliferator-activated receptor-α in liver. These findings suggest that estrogen administration attenuates adiposity markers of spontaneously hypertensive rats as a result of the decreased expression levels of peroxisome proliferator-activated receptor-γ in adipose tissue and increased expression of peroxisome proliferator-activated receptor-α in liver.

Investigation of the cardiomyocyte dysfunction in bradykinin type 2 receptor knockout mice.

AIMS: Bradykinin type 2 receptor (B(2)R) is the key component to trigger the intracellular signaling pathway in response to bradykinin under physiological conditions. The present study sought to investigate whether the B(2)R gene deletion will have an impact on myocardial function. MAIN METHODS: Isolated cell shortening, patch-clamp technique, Western blot and confocal microscopy. KEY FINDINGS: Isolated cell shortening measurements showed significant reduction in B(2)R knockout (B(2)R(-/-)) left ventricular cardiac myocytes' shortening. Whole-cell recordings were used to study the electrophysiological aspects of the left ventricular B(2)R(-/-) cardiomyocytes. Results showed: 1) action potential lengthening; 2) unchanged inwardly rectifying K(+) current; 3) reduced transient outward K(+) (I(to)) and L-type Ca(2+) current densities; 5) changes in kinetic properties related to I(to) and I(Ca,L). In addition, transient sarcoplasmic reticulum (SR) Ca(2+) release was found to be smaller in B(2)R(-/-) cardiomyocytes. Importantly, evidence is provided that NO constitutive production is, at least in part, responsible for the reported electrophysiological modifications observed in cardiomyocytes from B(2)R(-/-) mice. Surprisingly, NO is not involved in the SR Ca(2+) release reduction as demonstrated in the present study. SIGNIFICANCE: Taken together, our findings indicate that B(2)R plays a fundamental role in the regulation of cardiac function and Ca(2+) homeostasis, probably through a NO dependent pathway. These results may contribute to our understanding of the kinins participation in the control of cardiac function.

Cytokine profile of rats fed a diet containing shrimp / Perfil das citocinas de ratos alimentados com dieta de camarão

OBJECTIVE: Studies have shown that shrimps reduced the tensile strength of scars in rat skin. The aim of the present study was to assess the cytokine profile of rats fed shrimp. METHODS: Group 1 (control) received a regular diet and Group 2 (experimental) received a diet containing 33% shrimp for nine days. The two diets contained the same amounts of proteins, fats and carbohydrates. Serum cytokine levels were determined by ELISA and a segment of the jejunum was taken to investigate its histological morphology and eosinophil infiltrate. RESULTS: The experimental group had lower serum levels of interleukin-4 (IL-4) (14.4±1.9 versus 18.11±2.6pg/mL; p<0.05) and IL-10 (5.0±0.98 versus 7.5±1.2pg/mL; p<0.05) and higher levels of IL-6 (17.8±2.3 versus 3.2±0.4pg/mL, p<0.001) than controls. Morphologically, the shrimp-based diet caused an architectural disorganization of the intestinal mucosa and a greater amount of eosinophils in the jejunal villus. CONCLUSION: Our data suggests that shrimp consumption leads to a significant increase in the cytokine IL-6, a decrease in the immunomodulatory cytokine IL-10 in the serum of rats, and high eosinophil infiltration in the jejunum. The cytokine profile typical of inflammation and the histological aspect of the jejunum are compatible with food allergy.

OBJETIVO: Estudos mostraram que a dieta suplementada com camarão reduziu a resistência cicatricial na pele de ratos. Nesse contexto, o objetivo do presente estudo foi avaliar o perfil das citocinas de ratos que receberam dieta adicionada com camarão. MÉTODOS: Foram comparados um grupo controle e um grupo experimental, que receberam uma dieta enriquecida com camarão (33%) durante nove dias. As duas dietas continham quantidades semelhantes de proteínas, lipídeos, e carboidratos. Os níveis séricos de citocinas foram avaliados por ELISA, assim como um segmento de jejuno foi obtido para exame histológico da morfologia e infiltrado de eosinófilos. RESULTADOS: A dieta adicionada com camarão diminuiu os níveis séricos de IL-4 (14,4±1,9 versus 18,11±2,6pg/mL, p<0,05) e IL-10 (5,0±0,98 versus 7,5±1,2pg/mL, p<0,05 e aumentou os níveis séricos de IL-6 (3,2±0,4 versus 17,8±2,3pg/mL, p<0,001) quando comparada com os animais controle. Morfologicamente, a dieta adicionada com camarão causou uma desorganização da arquitetura da mucosa intestinal, juntamente com uma abundância de eosinófilos nas vilosidades jejunais. CONCLUSÃO: Os dados sugerem que a ingestão de dieta adicionada com camarão leva a um aumento significativo da citocina IL-6, juntamente com uma diminuição da citocina imunomoduladora IL-10 no soro de ratos e um infiltrado de eosinófilos no jejuno. O padrão inflamatório das citocinas e o aspecto histológico do jejuno são compatíveis com alergia alimentar.

Peripheral glucose metabolism is altered by epileptic seizures.

The aim of the present study was to investigate the status of jejunal absorption and peripheral metabolism of glucose in Wistar Audiogenic Rats (WAR), a genetic model of epilepsy, after seizures induced by intensive sound exposure. The jejunal loop of rats was isolated and infused (0.5 mL min(-1)) with Tyrode solution containing twice the normal concentrations of glucose, sodium, and potassium. Samples were taken at 5 or 10-min intervals over a 40-min period. At the end of the experiment, samples of liver and gastrocnemius muscle were taken to measure the levels of glycogen, glucose-6-phosphate, fructose-6-phosphate and glucose transporter-4 (GLUT4). Hepatic glucose-6-phosphate increased in WAR submitted to audiogenic seizure (21.90 +/- 3.08) as compared to non-susceptible Wistar rats (8.12 +/- 0.87) and to WAR not submitted to audiogenic stimulation (5.17 +/- 0.97). In addition, an increase in hepatic fructose-6-phosphate, an intermediate metabolite of the glycolytic pathway, was observed in WAR submitted to audiogenic seizure (5.98 +/- 0.99) compared to non-susceptible Wistar rats (2.38 +/- 0.53). According to the present results, jejunal absorption of glucose was not changed by seizures. However, generalized tonic-clonic seizures produced by sound stimulation resulted in a decrease in muscle glycogen content. In addition, our results demonstrated that the concentration of GLUT4 in the gastrocnemius muscle of WAR was 1.6-fold higher than that observed in resistant rats and that the audiogenic stimulus led to decreased concentration of this receptor in the muscle of WAR animals.

Efeito da proteína Kint3-4 do plasminogênio humano no crescimento do tumor de Ehrlich / Kint3-4 protein from human plasminogen delays Ehrlich tumor growth in mice

INTRODUCTION AND OBJECTIVE: Kint3-4 protein, originated from a genetic recombination of K1-3 and K1-4 human plasminogen segments, is recognized for its antiangiogenic and anti-inflammatory potential. This study aimed to evaluate the effect of Kint3-4 protein on tumor development in Swiss mice previously inoculated with Ehrlich tumor cells. METHODS: The protein fragment was obtained from Pichia pastoris cloning and transformation. After tumor cell inoculation three different protocols were used to assess tumor growth: beginning (0-6 days), peak (0-12 days) and after peak (0-18 days). We analyzed tumor growth, histomorphological characteristics and immunohistochemistry by use of CDC47 (cellular proliferation marker) and CD31 (blood vessel marker). RESULTS: Animals treated with Kint3-4 protein (150 µg/kg/48 h) showed lower tumor growth in all protocols. Based on histological assessment, inflammation and tumor areas were also reduced. Moreover, both the lowest rate of tumor cell proliferation and low microvessel density were observed in animals treated with Kint3-4 protein compared with the untreated control group. CONCLUSION: The effect of Kint3-4 recombinant protein on tumor angiogenesis and control of malignant cell proliferation enhances the prospects of its use in clinical and antiangiogenic treatment.

INTRODUÇÃO E OBJETIVO: A proteína Kint3-4 originou-se a partir de uma recombinação genética dos segmentos K1-3 e K1--4 do plasminogênio humano e é reconhecida por seu potencial anti-inflamatório e antiangiogênico. Este estudo teve como objetivo avaliar o efeito da proteína Kint3-4 no desenvolvimento de tumores em camundongos inoculados com células do tumor de Ehrlich. MÉTODOS: O fragmento de proteína foi obtido por uma técnica de clonagem e transformação de Pichia pastoris. Três diferentes protocolos foram avaliados após a inoculação das células tumorais: no início (0-6 dias), no pico (0-12 dias) e após o pico (0-18 dias) de crescimento do tumor. Foram analisados o crescimento do tumor e as características histomorfológica e imuno-histoquímica com CDC47 (marcador de proliferação celular) e CD31 (marcador de vasos sanguíneos). RESULTADOS: Os animais tratados com a proteína Kint3-4 (150 µg/kg/48 h) nos três diferentes protocolos apresentaram menor crescimento do tumor. Áreas de inflamação e tumor também foram reduzidas, avaliadas por exame histológico. Além disso, a menor taxa de proliferação das células tumorais e a baixa densidade de microvasos foram observadas nos animais tratados com proteína Kint3-4 em comparação com o grupocontrole. CONCLUSÃO: A participação da proteína recombinante Kint3-4 na angiogênese tumoral e no controle da proliferação de células malignas abre perspectivas para seu uso no tratamento clínico como antiangiogênico.

Tonin in rat heart with experimental hypertrophy.

The present study was undertaken to determine tonin expression and activity in rat heart presenting isoproterenol-induced hypertrophy. Renin, angiotensin-converting enzyme (ACE), and angiotensinogen (AG) expression were also determined. Wistar rats were treated with isoproterenol for 7 days (5 mg x kg(-1) x day(-1) sc). For untreated animals, the levels of tonin-specific activity in the atrium were 2.6- and 5.5-fold higher than those of the left and right ventricle, respectively. After treatment, the levels of tonin-specific activity increased twofold in the atrium but did not change in the ventricles. Renin expression was not detectable in these structures, and ACE expression levels did not change with treatment. AG expression was detected in the left ventricle at very low levels compared with the atrium and increased significantly only in the hypertrophied atrium (1.8-fold). Tonin mRNA was not detected in the ventricle but was found at low levels in the atrium, which increased after isoproterenol treatment. Our results permit us to conclude that tonin may play a role in the process of heart hypertrophy in the rat.

Diagnóstico de resistência do Mycobacterium tuberculosis à rifampicina utilizando-se da reação em cadeia da polimerase / Diagnostic of rifampicin-resistance of Mycobacterium tuberculosis by polymerase chain reaction

A resistência do Mycobacterium tuberculosis aos tuberculostáticos tem surgido como grande ameaça à Saúde Pública. A resistência à rifampicina pode ser considerada como um marcador para a multi-resistência a fármacos e tem sido atribuída a mudanças estruturais da RNA polimerase, produto de expressão do gene rpoB. Os pacientes portadores dessas cepas têm baixa perspectiva frente ao tratamento. Os testes convencionais de sensibilidade aos fármacos realizados em cultura do Mtb requerem várias semanas para o crescimento. Por este motivo, a Reação em Cadeia da Polimerase (PCR), método de baixo custo e que pode reduzir o tempo para o diagnóstico, representa alternativa viável e promissora. Neste artigo estão descritos os métodos mais comumente empregados na detecção de mutantes resistentes à rifampicina baseados na PCR, como análise de Polimorfismo Conformacional de Fita Simples (Single-Strand Conformation Polymorphism, SSCP), PCR Heteroduplex e INNO-LIPA. Recentemente, padronizou-se a técnica de PCR em baixa estringência, usando um único iniciador (Low Stringency Single Specific Primer, LSSP), que se mostrou um método rápido e sensível na detecção de mutações no gene rpoB.

The resistance of Mycobacterium tuberculosis to tuberculostatic drugs has emerged as a major public health threat. The resistance to rifampicin which has been attributed to structural changes in RNA polymerase can be considered as a marker for multi-drug-resistance to tuberculosis (MDR-TB). Patients bearing rifampicin-resistant strains have poor diagnosis even with treatment. Conventional culture-based drug sensibility testing can require several weeks due to the growth. In this paper we describe the most common PCR-based methods for detection of mutations that lead to rifampicin resistance, such as Single-Strand Conformation Polymorphism (SSCP), PCR Heteroduplex and INNO-LIPA. Recently, by Low Stringency using a Single Specific Primer (LSSP) assay, it was standardized a protocol that showed to be rapid and sensitive for the detection of mutations in the rpoB gene.