Maristela Cesquini

t-BOOH-induced oxidative damage in sickle red blood cells and the role of flavonoids.

Sickle cell anemia is a genetic disease characterized byan increase in generation of reactive oxygen species, abnormal iron release and low antioxidant activity which can lead to cell injury. Several therapies have been used to decrease the oxidative damage in these patients. In this study, we investigated the effect of flavonoids (quercetin and rutin) on the oxidation of red blood cells (RBC) from sickle cell anemia patients following exposure of the cells to tert-butyl hydroperoxide (t-BOOH). Quercetin provided greater protection against Hb oxidation, the binding of Hb to membrane and lipid peroxidation than did rutin. Quercetin (150 microM) reduced Hb oxidation by 30% and increased the level of oxyHb from 17.5 to 29 microM. Rutin prevented Hb oxidation only at concentrations higher than 200 microM and did not prevent the binding of Hb to RBC membrane. These distinct effects of the flavonoids probably reflect their structural characteristics. Thus, quercetin, which possesses a suitable structure for free-radical scavenging and ion quelation, was a more effective antioxidant than rutin. The presence of rutinose at position C(3) in rutin may impair its antioxidant effect. The presence of ascorbic acid enhanced the protective effect of quercetin and rutin against oxidative stress in sickle Hb and lipid peroxidation. This synergistic action helped to maintain a constant supply of flavonoids and thus, rescue the cells from the injury caused by free radicals and iron ions.

TNF-α acts in the hypothalamus inhibiting food intake and increasing the respiratory quotient—Effects on leptin and insulin signaling pathways

Acting in the hypothalamus, tumor necrosis factor-alpha (TNF-alpha) produces a potent anorexigenic effect. However, the molecular mechanisms involved in this phenomenon are poorly characterized. In this study, we investigate the capacity of TNF-alpha to activate signal transduction in the hypothalamus through elements of the pathways employed by the anorexigenic hormones insulin and leptin. High dose TNF-alpha promotes a reduction of 25% in 12h food intake, which is an inhibitory effect that is marginally inferior to that produced by insulin and leptin. In addition, high dose TNF-alpha increases body temperature and respiratory quotient, effects not reproduced by insulin or leptin. TNF-alpha, predominantly at the high dose, is also capable of activating canonical pro-inflammatory signal transduction in the hypothalamus, inducing JNK, p38, and NFkappaB, which results in the transcription of early responsive genes and expression of proteins of the SOCS family. Also, TNF-alpha activates signal transduction through JAK-2 and STAT-3, but does not activate signal transduction through early and intermediary elements of the insulin/leptin signaling pathways such as IRS-2, Akt, ERK and FOXO1. When co-injected with insulin or leptin, TNF-alpha, at both high and low doses, partially impairs signal transduction through IRS-2, Akt, ERK and FOXO1 but not through JAK-2 and STAT-3. This effect is accompanied by the partial inhibition of the anorexigenic effects of insulin and leptin, when the low, but not the high dose of TNF-alpha is employed. In conclusion, TNF-alpha, on a dose-dependent way, modulates insulin and leptin signaling and action in the hypothalamus.

Intracerebroventricular injection of citrate inhibits hypothalamic AMPK and modulates feeding behavior and peripheral insulin signaling

We hypothesized that citrate might modulate the AMP-activated protein kinase/acetyl-CoA carboxylase (AMPK)/(ACC) pathway and participate in neuronal feeding control and glucose homeostasis. To address this issue, we injected citrate into the lateral ventricle of rats. Intracerebroventricular (ICV) injection of citrate diminished the phosphorylation of hypothalamic AMPK/ACC, increased the expression of anorexigenic neuropeptide (pro-opiomelanocortin and corticotropin-releasing hormone), elevated the level of malonyl-CoA in the hypothalamus, and reduced food intake. No change was observed in the concentration of blood insulin after the injection of citrate. With a euglycemic-hyperinsulinemic clamp, the glucose infusion rate was higher in the citrate group than in the control group (28.6+/-0.8 vs 19.3+/-0.2 mU/kg body weight/min respectively), and so was glucose uptake in skeletal muscle and the epididymal fat pad. Concordantly, insulin receptor (IR), IR substrate type 1 (IRS1), IRS2, and protein kinase B (AKT) phosphorylation in adipose tissue and skeletal muscle was improved by citrate ICV treatment. Moreover, the treatment with citrate for 7 days promoted body weight loss and decreased the adipose tissue. Our results suggest that citrate and glucose may serve as signals of energy and nutrient availability to hypothalamic cells.

Citrate diminishes hypothalamic acetyl-CoA carboxylase phosphorylation and modulates satiety signals and hepatic mechanisms involved in glucose homeostasis in rats

The hypothalamic AMP-activated protein kinase (AMPK)/acetyl-CoA carboxylase (ACC) pathway is known to play an important role in the control of food intake and energy expenditure. Here, we hypothesize that citrate, an intermediate metabolite, activates hypothalamic ACC and is involved in the control of energy mobilization. Initially, we showed that ICV citrate injection decreased food intake and diminished weight gain significantly when compared to control and pair-fed group results. In addition, we showed that intracerebroventricular (ICV) injection of citrate diminished (80% of control) the phosphorylation of ACC, an important AMPK substrate. Furthermore, citrate treatment inhibited (75% of control) hypothalamic AMPK phosphorylation during fasting. In addition to its central effect, ICV citrate injection led to low blood glucose levels during glucose tolerance test (GTT) and high glucose uptake during hyperglycemic-euglycemic clamp. Accordingly, liver glycogen content was higher in animals given citrate (ICV) than in the control group (23.3+/-2.5 vs. 2.7+/-0.5 microg mL(-1) mg(-1), respectively). Interestingly, liver AMPK phosphorylation was reduced (80%) by the citrate treatment. The pharmacological blockade of beta3-adrenergic receptor (SR 59230A) blocked the effect of ICV citrate and citrate plus insulin on liver AMPK phosphorylation. Consistently with these results, rats treated with citrate (ICV) presented improved insulin signal transduction in liver, skeletal muscle, and epididymal fat pad. Similar results were obtained by hypothalamic administration of ARA-A, a competitive inhibitor of AMPK. Our results suggest that the citrate produced by mitochondria may modulate ACC phosphorylation in the hypothalamus, controlling food intake and coordinating a multiorgan network that controls glucose homeostasis and energy uptake through the adrenergic system.

Activation of AMPK in rat hypothalamus participates in cold‐induced resistance to nutrient‐dependent anorexigenic signals

The exposure of homeothermic animals to a cold environment leads to a powerful activation of orexigenic signalling which is accompanied by molecular and functional resistance to insulin‐induced inhibition of feeding. Recent evidence suggests that AMPK participates in nutrient‐dependent control of satiety and adiposity. The objective of the present study was to evaluate the effect of cold exposure upon the molecular activation of AMPK signalling in the hypothalamus of rats. Immunoblotting demonstrated that cold exposure per se is sufficient for inducing, on a time‐dependent basis, the molecular activation of the serine/threonine kinase AMP‐activated protein kinase (AMPK) and inactivation of the acetyl‐CoA carboxylase (ACC). These molecular phenomena were accompanied by resistance to nutrient‐induced inactivation of AMPK and activation of ACC. Moreover, cold‐exposure led to a partial inhibition of a feeding‐induced anorexigenic response, which was paralleled by resistance to insulin‐induced suppression of feeding. Finally, cold exposure significantly impaired insulin‐induced inhibition of AMPK through a mechanism dependent on the molecular cross‐talk between phosphatidylinositol‐3(PI3)‐kinase/Akt and AMPK. In conclusion, increased feeding during cold exposure results, at least in part, from resistance to insulin‐ and nutrient‐dependent anorexigenic signalling in the hypothalamus.

Effect of thiol reagents on functional properties and heme oxidation in the hemoglobin of Geochelone carbonaria.

The reaction of thiol reagents with G. carbonaria hemoglobin was studied, and the oxygen equilibrium and kinetic of oxidation of derivatives determined. The oxygen affinity and kinetic of oxidation of hemoglobin derivatives were modified to various extents depending on the nature of thiol reagents used. Diamide yielded approximately 80% polymeric hemoglobin, although the oxidation kinetic, and the functional properties, were practically invariant (T1/2=10.0 min.; P50 = 5.0 mm Hg at pH 7.4; alkaline Bohr effect = ‐0.64). Iodoacetamide did not modify the electrophoretic pattern significantly, although all the free SH groups of hemoglobin were alkylated. A P50 of 2.5 mmHg at pH 7.4 and the Bohr effect of ‐0.15 were obtained; the T1/2 of about 6.4 min. was shorter than that for un‐modified Hb. Similar T1/2 were obtained for Hb treated with oxidized glutathione, which produced polymeric Hb and glutathionyl‐Hb. The oxygen binding characteristics showed that both of Hb derivatives, glutathionyl‐Hb and polymeric Hb, maintain the capacity to transport the gas.

OXYGEN-BINDING PROPERTIES OF TOTAL HEMOGLOBIN AND ISOLATED COMPONENTS OF THE TERRESTRIAL TORTOISE GEOCHELONE CARBONARIA

Abstract The hemoglobin of the terrestrial tortoise Geochelone carbonaria was separated into two major components by ion exchange chromatography. The oxygen binding properties of both the unfractionated hemolysate and the separated components were investigated. The stripped hemoglobin of G. carbonaria displayed an oxygen affinity of approximately 6.3 mm Hg at pH 7.4 with a Bohr effect of about −0.55. The Hb-O 2 binding properties were also studied in the presence of the organic polyphosphates ATP and IHP. Inositol hexaphosphate (IHP) was more effective in lowering oxygen affinity, yielding a P 50 of 20.1 mm Hg at pH 7.4. The n 50 value were quite low in the stripped form of the protein (1.0–1.9) but increased in the presence of both organic phosphates, attaining a value of approximately 2.9 at pH 7.9. The oxygen binding properties of the major hemoglobin components were also investigated in the presence and absence of organic phosphates, and the values were compared with those obtained for the total hemolysate.