Tatiana Carolina Alba-Loureiro

Sympathetic hyperactivity differentially affects skeletal muscle mass in developing heart failure : role of exercise training

Sympathetic hyperactivity (SH) is a hallmark of heart failure (HF), and several lines of evidence suggest that SH contributes to HF-induced skeletal myopathy. However, little is known about the influence of SH on skeletal muscle morphology and metabolism in a setting of developing HF, taking into consideration muscles with different fiber compositions. The contribution of SH on exercise tolerance and skeletal muscle morphology and biochemistry was investigated in 3- and 7-mo-old mice lacking both alpha(2A)- and alpha(2C)-adrenergic receptor subtypes (alpha(2A)/alpha(2C)ARKO mice) that present SH with evidence of HF by 7 mo. To verify whether exercise training (ET) would prevent skeletal muscle myopathy in advanced-stage HF, alpha(2A)/alpha(2C)ARKO mice were exercised from 5 to 7 mo of age. At 3 mo, alpha(2A)/alpha(2C)ARKO mice showed no signs of HF and preserved exercise tolerance and muscular norepinephrine with no changes in soleus morphology. In contrast, plantaris muscle of alpha(2A)/alpha(2C)ARKO mice displayed hypertrophy and fiber type shift (IIA --> IIX) paralleled by capillary rarefaction, increased hexokinase activity, and oxidative stress. At 7 mo, alpha(2A)/alpha(2C)ARKO mice displayed exercise intolerance and increased muscular norepinephrine, muscular atrophy, capillary rarefaction, and increased oxidative stress. ET reestablished alpha(2A)/alpha(2C)ARKO mouse exercise tolerance to 7-mo-old wild-type levels and prevented muscular atrophy and capillary rarefaction associated with reduced oxidative stress. Collectively, these data provide direct evidence that SH is a major factor contributing to skeletal muscle morphological changes in a setting of developing HF. ET prevented skeletal muscle myopathy in alpha(2A)/alpha(2C)ARKO mice, which highlights its importance as a therapeutic tool for HF.

Modulation of lipopolysaccharide-induced acute lung inflammation: Role of insulin.

ABSTRACT The present study was undertaken to investigate the influence of insulin on lipopolysaccharide (LPS)-induced acute lung injury. Diabetic male Wistar rats (alloxan, 42 mg/kg, i.v., 30 days) and controls were instilled with saline containing LPS (750 &mgr;g/0.4 mL) or saline alone. The following analyses were performed 6 h there after: (a) total and differential cell counts in bronchoalveolar lavage (BAL) fluid, (b) quantification of tumor necrosis factor &agr;, interleukin (IL) 1&bgr;, IL-10, and cytokine-induced neutrophil chemoattractant 1 in the BAL (enzyme-linked immunosorbent assay), (c)immunohistochemistry for intercellular adhesion molecule 1 and E-selectin on lung vessels, and (d) quantification of metalloproteinases (MMP) 2 and 9 in the BAL (zymography). Relative to controls, diabetic rats exhibited a reduction in the number of neutrophils (80%) and reduced concentrations of tumor necrosis factor &agr; (56%), IL-1&bgr; (66%), and IL-10 (35%) after LPS instillation. Cytokine-induced neutrophil chemoattractant 1 levels did not differ between groups. Increased levels of MMP-2 (90%) and MMP-9 (500%) were observed in diabetic rats compared with controls. Treatment of diabetic rats with neutral protamine Hagedorn insulin (4 IU, s.c.), 2 h before LPS instillation, completely restored the number of neutrophils and concentrations of cytokines in the BAL fluid. Despite no significant differences between diabetic and control groups, there was a remarkable increase in intercellular adhesion molecule 1 and E-selectin expression on lung vessels after insulin treatment. Levels of MMP-2 and MMP-9 did not change after treatment with insulin. Levels of corticosterone were equivalent among groups. Data presented suggest that insulin modulates the production/release of cytokines and the expression of adhesion molecules controlling, therefore, neutrophil migration during the course of LPS-induced acute lung inflammation.

Effect of lipid infusion on metabolism and force of rat skeletal muscles during intense contractions.

The hypothesis that during intense muscle contraction induced by electrical stimulation, long chain fatty acids (LCFA) might reduce mitochondrial ATP/ADP ratio, raising the contribution of glycolysis for ATP production was examined. The effect of a lipid infusion (Lipovenus emulsion) on UCP-3 mRNA level, lactate, glucose-6-phosphate (G-6P) and glycogen content was investigated in rat. Blood samples for determination of free fatty acids and lactate were collected at 0, 30 and 60 min during rest and at 0, 10 and 20 min during muscle contraction. The content of lactate, glycogen and G-6P was also determined in soleus (SO), red gastrocnemius (RG) and white gastrocnemius (WG) muscles collected immediately after muscle contraction period. In addition, the force level was determined during muscle contractions. The effect of Lipovenus emulsion on respiration of mitochondria isolated from rat skeletal muscle, and content of UCP-3 and lactate in cultured skeletal muscle cells was also determined. The in vivo experiments showed that Lipovenus induced a significant increase of UCP-3 mRNA levels. After Lipovenus infusion, lactate level was increased in RG muscle only, whereas the contents of glycogen and G-6P were decreased in both RG and WG muscles (P < 0.05). Lipovenus infusion failed to exert any effect on muscle force performance (P > 0.05). The in vitro experiments showed that Lipovenus infusion induced a significant increase in mitochondrial respiration, but had no effect on UCP-3 content. Lactate concentration was significantly increased in the culture medium of stimulated cells in the control and Lipovenus groups compared with the respective not-stimulated cells (P< 0.05). We concluded that as mitochondrial function becomes limited by the FFA-uncoupling effect, the ATP demand is mainly supplied by anaerobic glucose metabolism preventing an expected decrease in muscle contraction performance.

Glutamine Supplementation Stimulates Protein-Synthetic and Inhibits Protein-Degradative Signaling Pathways in Skeletal Muscle of Diabetic Rats

In this study, we investigated the effect of glutamine (Gln) supplementation on the signaling pathways regulating protein synthesis and protein degradation in the skeletal muscle of rats with streptozotocin (STZ)-induced diabetes. The expression levels of key regulatory proteins in the synthetic pathways (Akt, mTOR, GSK3 and 4E-BP1) and the degradation pathways (MuRF-1 and MAFbx) were determined using real-time PCR and Western blotting in four groups of male Wistar rats; 1) control, non-supplemented with glutamine; 2) control, supplemented with glutamine; 3) diabetic, non-supplemented with glutamine; and 4) diabetic, supplemented with glutamine. Diabetes was induced by the intravenous injection of 65 mg/kg bw STZ in citrate buffer (pH 4.2); the non-diabetic controls received only citrate buffer. After 48 hours, diabetes was confirmed in the STZ-treated animals by the determination of blood glucose levels above 200 mg/dL. Starting on that day, a solution of 1 g/kg bw Gln in phosphate buffered saline (PBS) was administered daily via gavage for 15 days to groups 2 and 4. Groups 1 and 3 received only PBS for the same duration. The rats were euthanized, and the soleus muscles were removed and homogenized in extraction buffer for the subsequent measurement of protein and mRNA levels. The results demonstrated a significant decrease in the muscle Gln content in the diabetic rats, and this level increased toward the control value in the diabetic rats receiving Gln. In addition, the diabetic rats exhibited a reduced mRNA expression of regulatory proteins in the protein synthesis pathway and increased expression of those associated with protein degradation. A reduction in the skeletal muscle mass in the diabetic rats was observed and was alleviated partially with Gln supplementation. The data suggest that glutamine supplementation is potentially useful for slowing the progression of muscle atrophy in patients with diabetes.

Stress-Induced Downregulation of Macrophage Phagocytic Function Is Attenuated by Exercise Training in Rats

Background/Aims: Acute restraint stress may induce impaired macrophage phagocytic function. Moderate physical training is associated with beneficial effects on immunological functions. We investigated the effects of moderate physical training on phagocytic function of alveolar macrophages in rats submitted to acute restraint stress. Methods: Thirty male Wistar rats weighing 210–226 g were randomly divided into 4 groups: nontrained rats (n = 7), nontrained rats submitted to stress (n = 8), trained rats (n = 7) and trained rats submitted to stress (n = 8). Trained rats were submitted to a program of moderate running training over a period of 8 weeks. Rats subjected to restraint stress were kept immobilized in glass cylinders (8 cm in diameter and 24 cm long) during 60 min. Phagocytosis capacity of macrophages was evaluated by either Escherichia coli orzymosan stimuli. Results: Restraint stress induced a decrease in phagocytosis of E. coli and zymosan particle stimulation by macrophages. Neither of these alterations was observed in trained animals submitted to acute restraint stress. Conclusions: Our data confirm that acute restraint stress is associated with impaired function of macrophages. Moreover, moderate physical training attenuates the effects of acute stress by a mechanism that involves an increase in tolerance of macrophages.

NADPH Oxidase-Dependent Production of Reactive Oxygen Species Induces Endoplasmatic Reticulum Stress in Neutrophil-Like HL60 Cells

Reactive oxygen species (ROS) primarily produced via NADPH oxidase play an important role for killing microorganisms in neutrophils. In this study we examined if ROS production in Human promyelocytic leukemia cells (HL60) differentiated into neutrophil-like cells (dHL60) induces ER stress and activates the unfolded protein response (UPR). To cause ROS production cells were treated with PMA or by chronic hyperglycemia. Chronic hyperglycemia failed to induce ROS production and did not cause activation of the UPR in dHL60 cells. PMA, a pharmacologic NADPH oxidase activator, induced ER stress in dHL60 cells as monitored by IRE-1 and PERK pathway activation, and this was independent of calcium signaling. The NADPH oxidase inhibitor, DPI, abolished both ROS production and UPR activation. These results show that ROS produced by NADPH oxidase induces ER stress and suggests a close association between the redox state of the cell and the activation of the UPR in neutrophil-like HL60 cells.

Neutrophil fatty acid composition: effect of a single session of exercise and glutamine supplementation.

Summary.The fatty acid composition of immune cells appears to contribute to variations of cell function. The independent and combined effects of a single session of exercise (SSE) and glutamine supplementation (GS) on neutrophil fatty acid composition were investigated. Compared to control (no treatment given – i.e. neither SSE or GS), single session of exercise decreased myristic, palmitic and eicosapentaenoic (EPA) acids, and increased lauric, oleic, linoleic, arachidonic (AA) and docosahexaenoic (DHA) acids whereas glutamine supplementation combined with SSE (GS+SSE) increased oleic acid. Polyunsaturated/saturated fatty acid ratio and Unsaturation index were higher in neutrophils from the SSE and GS groups as compared with control. These findings support the proposition that SSE and GS may modulate neutrophil function through alterations in fatty acid composition.

Reduced cytokine production by glycogen-elicited peritoneal cells from diabetic rats.

IL-1&bgr;, TNF-&agr;, cytokine-induced neutrophil chemoattractant-2&agr;/&bgr;, and IL-10 measurements were performed in elicited peritoneal cells from control, diabetic, and insulin-treated diabetic rats. Production/liberation of these cytokines was decreased in elicited peritoneal cells from diabetic rats. These changes were abolished by insulin treatment of diabetic rats. The alterations observed might be involved in the impaired inflammatory response and high occurrence of apoptosis observed in neutrophils under diabetic states.

Attenuation of obesity and insulin resistance by fish oil supplementation is associated with improved skeletal muscle mitochondrial function in mice fed a high-fat diet.

Omega-3 polyunsaturated fatty acids (n-3 PUFAs) have been reported to improve insulin sensitivity and glucose homeostasis in animal models of insulin resistance, but the involved mechanisms still remain unresolved. In this study, we evaluated the effects of fish oil (FO), a source of n-3 PUFAs, on obesity, insulin resistance and muscle mitochondrial function in mice fed a high-fat diet (HFD). C57Bl/6 male mice, 8 weeks old, were divided into four groups: control diet (C), high-fat diet (H), C+FO (CFO) and H+FO (HFO). FO was administered by oral gavage (2 g/kg b.w.), three times a week, starting 4 weeks before diet administration until the end of the experimental protocol. HFD-induced obesity and insulin resistance associated with impaired skeletal muscle mitochondrial function, as indicated by decreased oxygen consumption, tricarboxylic acid cycle intermediate (TCAi) contents (citrate, α-ketoglutarate, malate and oxaloacetate), oxidative phosphorylation protein content and mitochondrial biogenesis. These effects were associated with elevated reactive oxygen species production, decreased PGC1-a transcription and reduced Akt phosphorylation. The changes induced by the HFD were partially attenuated by FO, which decreased obesity and insulin resistance and increased mitochondrial function. In the H group, FO supplementation also improved oxygen consumption; increased TCAi content, and Akt and AMPK phosphorylation; and up-regulated mRNA expression of Gpat1, Pepck, catalase and mitochondrial proteins (Pgc1α, Pparα, Cpt1 and Ucp3). These results suggest that dietary FO attenuates the deleterious effects of the HFD (obesity and insulin resistance) by improving skeletal muscle mitochondrial function.

Autophagy Is Impaired in Neutrophils from Streptozotocin-Induced Diabetic Rats

We tested the hypothesis that changes reported on functions of neutrophils from streptozotocin-induced diabetic rats involve autophagy impairment. Wistar rats were rendered diabetic by streptozotocin injection (65 mg/kg, i.v.), and the measurements were carried out 2 weeks afterward. Neutrophils were collected through intraperitoneal cavity lavage after 4 h of i.p. oyster glycogen type 2 injection. Neutrophils cultured with PMA (20 nM) for 1 h were used for analysis of plasma membrane integrity, DNA fragmentation, and mitochondrial depolarization by flow cytometry; expression of Atg5, Atg14, Beclin1, LC3BII, and Rab9 by RT-PCR; the contents of caspase 3, LC3BII/LC3BI, and pS6 by western blotting; ATP content by fluorescence essay; reactive oxygen species production by chemiluminescence (Luminol), and autophagy by immunofluorescence tracking LC3B cleavage. Herein, neutrophils from diabetic rats had high DNA fragmentation, depolarization of mitochondrial membrane, low content of ATP, and high content of cleaved caspase 3 after PMA stimulation. Neutrophils from diabetic rats also had low expression of LC3B, failed to increase the expression of Rab9 and Atg14 induced by PMA stimulation. Neutrophils from diabetic animals also had low cleavage of LC3BI to LC3BII and do not present punctate structures that label autophagosomal membranes after stimulus. The changes of neutrophil function reported in diabetic rats do involve impaired autophagy. The suppression of autophagy in neutrophils from diabetic rats may be associated with the activation of the mTOR signaling as indicated by the high content of pS6.