Transepithelial transport of glucose and mRNA of glucose transporters in the small intestine of rats with iron-deficiency anemia

Objective: To evaluate the transepithelial transport of sodium, glucose, potassium, and water and the mRNA level of the sodium-glucose cotransporter (SGLT1) and the facilitated sugar transporter (GLUT2) in the small intestine of iron-deficient rats. Methods: After 6 wk of receiving diets with low or normal iron content, rats (Wistar-EPM) were subjected to two experiments: 1) evaluation of the transepithelial transport of sodium, glucose, potassium, and water by an ""in vivo"" experimental model of intestinal perfusion and 2) determination of relative SGLT1 and GLUT2 mRNA levels in the proximal, intermediate, and distal portions of the small intestine by the northern blotting technique. Results: Hemoglobin and hepatic iron levels were statistically lower in the anemic rats. The mean transepithelial transports of sodium (-33.0 mu Eq . min(-1) . cm(-1)), glucose (426.0 mu M . min(-1) . cm(-1)), and water (0.4 mu L . min(-1) . cm(-1)) in the small intestine of the anemic rats were significantly lower than in the control group (349.1 mu Eq . min(-1) cm(-1), 842.6 mu M . min(-1) . cm(-1), and 4.3 mu l . min(-1) cm(-1), respectively, P < 0.05). The transepithelial transport of potassium was similar for both groups. The relative SGLT1 mRNA levels of the anemic rats in the intermediate (1.796 +/- 0.659 AU) and distal (1.901 +/- 0.766 AU) segments were significantly higher than the values for the control rats (intermediate 1.262 +/- 0.450 AU, distal 1.244 +/- 0.407 AU). No significant difference was observed for the relative SLGT1 mRNA levels in the proximal segment or for the GLUT2 mRNA levels in all segments. Conclusion: Iron deficiency decreases the absorption of glucose, sodium, and water and increases SGLT1 mRNA in the intermediate and distal segments of the small intestine of rats. (C) 2011 Elsevier Inc. All rights reserved.

UPR induces transient burst of apoptosis in islets of early lactating rats through reduced AKT phosphorylation via ATF4/CHOP stimulation of TRB3 expression

Bromati CR, Lellis-Santos C, Yamanaka TS, Nogueira TC, Leonelli M, Caperuto LC, Gorjao R, Leite AR, Anhe GF, Bordin S. UPR induces transient burst of apoptosis in islets of early lactating rats through reduced AKT phosphorylation via ATF4/CHOP stimulation of TRB3 expression. Am J Physiol Regul Integr Comp Physiol 300: R92-R100, 2011. First published November 10, 2010; doi:10.1152/ajpregu.00169.2010.-Endocrine pancreas from pregnant rats undergoes several adaptations that comprise increase in beta-cell number, mass and insulin secretion, and reduction of apoptosis. Lactogens are the main hormones that account for these changes. Maternal pancreas, however, returns to a nonpregnant state just after the delivery. The precise mechanism by which this reversal occurs is not settled but, in spite of high lactogen levels, a transient increase in apoptosis was already reported as early as the 3rd day of lactation (L3). Our results revealed that maternal islets displayed a transient increase in DNA fragmentation at L3, in parallel with decreased RAC-alpha serine/threonine-protein kinase (AKT) phosphorylation (pAKT), a known prosurvival kinase. Wortmannin completely abolished the prosurvival action of prolactin (PRL) in cultured islets. Decreased pAKT in L3-islets correlated with increased Tribble 3 (TRB3) expression, a pseudokinase inhibitor of AKT. PERK and eIF2 alpha phosphorylation transiently increased in islets from rats at the first day after delivery, followed by an increase in immunoglobulin heavy chain-binding protein (BiP), activating transcription factor 4 (ATF4), and C/EBP homologous protein (CHOP) in islets from L3 rats. Chromatin immunoprecipitation (ChIP) and Re-ChIP experiments further confirmed increased binding of the heterodimer ATF4/CHOP to the TRB3 promoter in L3 islets. Treatment with PBA, a chemical chaperone that inhibits UPR, restored pAKT levels and inhibited the increase in apoptosis found in L3. Moreover, PBA reduced CHOP and TRB3 levels in beta-cell from L3 rats. Altogether, our study collects compelling evidence that UPR underlies the physiological and transient increase in beta-cell apoptosis after delivery. The UPR is likely to counteract prosurvival actions of PRL by reducing pAKT through ATF4/CHOP-induced TRB3 expression.

Intensive insulin treatment induces insulin resistance in diabetic rats by impairing glucose metabolism-related mechanisms in muscle and liver

Insulin replacement is the only effective therapy to manage hyperglycemia in type 1 diabetes mellitus (T1DM). Nevertheless, intensive insulin therapy has inadvertently led to insulin resistance. This study investigates mechanisms involved in the insulin resistance induced by hyperinsulinization. Wistar rats were rendered diabetic by alloxan injection, and 2 weeks later received saline or different doses of neutral protamine Hagedorn insulin (1.5, 3, 6, and 9 U/day) over 7 days. Insulinopenic-untreated rats and 6U- and 9U-treated rats developed insulin resistance, whereas 3U-treated rats revealed the highest grade of insulin sensitivity, but did not achieve good glycemic control as 6U- and 9U-treated rats did. This insulin sensitivity profile was in agreement with glucose transporter 4 expression and translocation in skeletal muscle, and insulin signaling, phosphoenolpyruvate carboxykinase/glucose-6-phosphatase expression and glycogen storage in the liver. Under the expectation that insulin resistance develops in hyperinsulinized diabetic patients, we believe insulin sensitizer approaches should be considered in treating T1DM. Journal of Endocrinology (2011) 211, 55-64

SGLT1 protein expression in plasma membrane of acinar cells correlates with the sympathetic outflow to salivary glands in diabetic and hypertensive rats

Salivary gland dysfunction is a feature in diabetes and hypertension. We hypothesized that sodium-glucose cotransporter 1 (SGLT1) participates in salivary dysfunctions through a sympathetic- and protein kinase A (PKA)-mediated pathway. In Wistar-Kyoto (WKY), diabetic WKY (WKY-D), spontaneously hypertensive (SHR), and diabetic SHR (SHR-D) rats, PKA/SGLT1 proteins were analyzed in parotid and submandibular glands, and the sympathetic nerve activity (SNA) to the glands was monitored. Basal SNA was threefold higher in SHR (P < 0.001 vs. WKY), and diabetes decreased this activity (similar to 50%, P < 0.05) in both WKY and SHR. The catalytic subunit of PKA and the plasma membrane SGLT1 content in acinar cells were regulated in parallel to the SNA. Electrical stimulation of the sympathetic branch to salivary glands increased (similar to 30%, P < 0.05) PKA and SGLT1 expression. Immunohistochemical analysis confirmed the observed regulations of SGLT1, revealing its location in basolateral membrane of acinar cells. Taken together, our results show highly coordinated regulation of sympathetic activity upon PKA activity and plasma membrane SGLT1 content in salivary glands. Furthermore, the present findings show that diabetic- and/or hypertensive-induced changes in the sympathetic activity correlate with changes in SGLT1 expression in basolateral membrane of acinar cells, which can participate in the salivary glands dysfunctions reported by patients with these pathologies.

Contribution of excitatory amino acid receptors of the retrotrapezoid nucleus to the sympathetic chemoreflex in rats

In the present study, we evaluated the role of glutamatergic mechanisms in the retrotrapezoid nucleus (RTN) in changes of splanchnic sympathetic nerve discharge (sSND) and phrenic nerve discharge (PND) elicited by central and peripheral chemoreceptor activation. Mean arterial pressure (MAP), sSND and PND were recorded in urethane-anaesthetized, vagotomized, sino-aortic denervated and artificially ventilated male Wistar rats. Hypercapnia (10% CO(2)) increased MAP by 32 +/- 4 mmHg, sSND by 104 +/- 4% and PND amplitude by 101 +/- 5%. Responses to hypercapnia were reduced after bilateral injection of the NMDA receptor antagonist D,L-2-amino-5-phosphonovalerate (AP-5; 100mm in 50 nl) in the RTN (MAP increased by 16 +/- 3 mmHg, sSNDby 82 +/- 3% and PND amplitudeby 63 +/- 7%). Bilateral injection of the non-NMDA receptor antagonist 6,7-dinitro-quinoxaline-2,3-dione(DNQX; 100 mm in 50 nl) and the metabotropic receptor antagonist (+/-)-alpha-methyl-4-carboxyphenylglycine (MCPG; 100mm in 50 nl) in the RTN did not affect sympathoexcitatory responses induced by hypercapnia. Injection of DNQX reduced hypercapnia-induced phrenic activation, whereas MCPG did not. In animals with intact carotid chemoreceptors, bilateral injections of AP-5 and DNQX in the RTN reduced increases in MAP, sSND and PND amplitude produced by intravenous injection of NaCN (50 mu g kg(-1)). Injection of MCPG in the RTN did not change responses produced by NaCN. These data indicate that RTN ionotropic glutamatergic receptors are involved in the sympathetic and respiratory responses produced by central and peripheral chemoreceptor activation.

Dietary Free Oleic and Linoleic Acid Enhances Neutrophil Function and Modulates the Inflammatory Response in Rats

The high ingestion of oleic (OLA) and linoleic (LNA) acids by Western populations, the presence of inflammatory diseases in these populations, and the importance of neutrophils in the inflammatory process led us to investigate the effects of oral ingestion of unesterified OLA and LNA on rat neutrophil function. Pure OLA and LNA were administered by gavage over 10 days. The doses used (0.11, 0.22 and 0.44 g/kg of body weight) were based on the Western consumption of OLA and LNA. Neither fatty acid affected food, calorie or water intake. The fatty acids were not toxic to neutrophils as evaluated by cytometry using propidium iodide (membrane integrity and DNA fragmentation). Neutrophil migration in response to intraperitoneal injection of glycogen and in the air pouch assay, was elevated after administration of either OLA or LNA. This effect was associated with enhancement of rolling and increased release of the chemokine CINC-2 alpha beta. Both fatty acids elevated l-selectin expression, whereas no effect on beta(2)-integrin expression was observed, as evaluated by flow cytometry. LNA increased the production of proinflammatory cytokines (IL-1 beta and CINC-2 alpha beta) by neutrophils after 4 h in culture and both fatty acids decreased the release of the same cytokines after 18 h. In conclusion, OLA and LNA modulate several functions of neutrophils and can influence the inflammatory process.

Exercise changes regional vascular control by commissural NTS in spontaneously hypertensive rats

Ogihara CA, Schoorlemmer GHM, Levada AC, Pithon-Curi TC, Curi R, Lopes OU, Colombari E, Sato MA. Exercise changes regional vascular control by commissural NTS in spontaneously hypertensive rats. Am J Physiol Regul Integr Comp Physiol 299: R291-R297, 2010. First published April 21, 2010; doi: 10.1152/ajpregu.00055.2009.-Inhibition of the commissural nucleus of the solitary tract (commNTS) induces a fall in sympathetic nerve activity and blood pressure in spontaneously hypertensive rats (SHR), which suggests that this subnucleus of the NTS is a source of sympathoexcitation. Exercise training reduces sympathetic activity and arterial pressure. The purpose of the present study was to investigate whether the swimming exercise can modify the regional vascular responses evoked by inhibition of the commNTS neurons in SHR and normotensive Wistar-Kyoto (WKY) rats. Exercise consisted of swimming, 1 h/day, 5 days/wk for 6 wks, with a load of 2% of the body weight. The day after the last exercise session, the rats were anesthetized with intravenous alpha-chloralose, tracheostomized, and artificially ventilated. The femoral artery was cannulated for mean arterial pressure (MAP) and heart rate recordings, and Doppler flow probes were placed around the lower abdominal aorta and superior mesenteric artery. Microinjection of 50 mM GABA into the commNTS caused similar reductions in MAP in swimming and sedentary SHR (-25 +/- 6 and -30 +/- 5 mmHg, respectively), but hindlimb vascular conductance increased twofold in exercised vs. sedentary SHR (54 +/- 8 vs. 24 +/- 5%). GABA into the commNTS caused smaller reductions in MAP in swimming and sedentary WKY rats (-20 +/- 4 and -16 +/- 2 mmHg). Hindlimb conductance increased fourfold in exercised vs. sedentary WKY rats (75 +/- 2% vs. 19 +/- 3%). Therefore, our data suggest that the swimming exercise induced changes in commNTS neurons, as shown by a greater enhancement of hindlimb vasodilatation in WKY vs. SHR rats in response to GABAergic inhibition of these neurons.

Beneficial Effects of Physical Training on the Cardio-Inflammatory Disorder Induced by Lung Ischemia/Reperfusion in Rats

Our laboratory demonstrated that training program attenuated the inflammatory responses in lung ischemia/reperfusion (IR). Considering the importance of the inflammatory responses on the cardiovascular system, we evaluate the effect of physical training on the vascular responsiveness and its underlying mechanism after lung IR. Male Wistar rats were submitted to run training and lung IR. Concentration-response curves for relaxing and contracting agents were obtained. Protein expressions of SOD-1 and p47(phox), plasma nitritre/nitrate (NO (x) (-) ) and interleukin 6 (IL-6) were evaluated. A decreased in the potency for acetylcholine and phenylephrine associated with an upregulation of the p47(phox) expression were found after Lung IR as well as an increase in IL-6 and NO (x) (-) levels. Run training attenuated the vascular dysfunction that was accompanied by reduction of the p47(phox) protein expression and IL-6 levels. Our findings show the beneficial effect of training on the vascular function that was associated with reduction in inflammatory response in lung IR.

Augmentation of insulin secretion by leucine supplementation in malnourished rats: possible involvement of the phosphatidylinositol 3-phosphate kinase/mammalian target protein of rapamycin pathway

A regimen of low-protein diet induces a reduction of pancreatic islet function that is associated with development of metabolic disorders including diabetes and obesity afterward. In the present study, the influence of leucine supplementation on metabolic parameters, insulin secretion to glucose and to amino acids, as well as the levels of proteins that participate in the phosphatidylinositol 3-phosphate kinase (PI3K) pathway was investigated in malnourished rats. Four groups were fed with different diets for 12 weeks: a normal protein diet (17%) without (NP) or with leucine supplementation (NPL) or a low (6%)-protein diet without (LP) or with leucine supplementation (LPL). Leucine was given in the drinking water during the last 4 weeks. As indicated by the intraperitoneal glucose tolerance test, LPL rats exhibited increased glucose tolerance as compared with NPL group. Both NPL and LPL rats had higher circulating insulin levels than controls. The LPL rats also showed increased insulin secretion by pancreatic islets in response to glucose or arginine compared with those observed in islets from LP animals. Glucose oxidation was significantly reduced in NPL, LP, and LPL isolated islets as compared with NP; but no alteration was observed for leucine and glutamate oxidation among the 4 groups. Western blotting analysis demonstrated increased PI3K and mammalian target protein of rapamycin protein contents in LPL compared with LP islets. A significant increase in insulin-induced insulin receptor substrate I associated PI3K activation was also observed in LPL compared with LP islets. These findings indicate that leucine supplementation can augment islet function in malnourished rats and that activation of the PI3K/maminalian target protein of rapamycin pathway may play a role in this process. (C) 2010 Elsevier Inc. All rights reserved.

T3 acutely increases GH mRNA translation rate and GH secretion in hypothyroid rats

Cytoskeleton controls the stability of transcripts, by mechanisms that involve mRNAs and eEF1A attachment to it. Besides, it plays a key role in protein synthesis and secretion, which seems to be impaired in somatotrophs of hypothyroid rats, whose cytoskeleton is disarranged. This study investigated the: eEF1A and GH mRNA binding to cytoskeleton plus GH mRNA translation rate and GH secretion, in sham-operated and thyroidectomized rats treated with T3 or saline, and killed 30 min thereafter. Thyroidectomy reduced: (a) pituitary F-actin content, and eEF1A plus GH mRNA binding to it; (b) GH mRNA recruitment to polysome; and (c) liver IGF-1 mRNA expression, indicating that GH mRNA stability and translation rate, as well as GH secretion were impaired. T3 acutely reversed all these changes, which points toward a nongenomic action of T3 on cytoskeleton rearrangement, which might contribute to the increase on GH mRNA translation rate and GH secretion. (C) 2009 Elsevier Ireland Ltd. All rights reserved.

Effect of chronic supplementation with shark liver oil on immune responses of exercise-trained rats

Previous studies have reported that chronic supplementation with shark liver oil (SLO) improves immune response of lymphocyte, macrophage and neutrophil in animal models and humans. In a similar manner, exercise training also stimulates the immune system. However, we are not aware of any study about the association of exercise and SLO supplementation on immune response. Thus, our main goal was to investigate the effect of chronic supplementation with SLO on immune responses of exercise-trained rats. Male Wistar rats were divided into four groups: sedentary with no supplementation (SED, n = 20), sedentary with SLO supplementation (SEDslo, n = 20), exercised (EX, n = 17) and exercised supplemented with SLO (EXslo, n = 19). Rats swam for 6 weeks, 1.5 h/day, in water at 32 +/- A 1A degrees C, with a load of 6.0% body weight attached to the thorax of rat. Animals were killed 48 h after the last exercise session. SLO supplementation did not change phagocytosis, lysosomal volume, superoxide anion and hydrogen peroxide production by peritoneal macrophages and blood neutrophils. Thymus and spleen lymphocyte proliferation were significantly higher in SEDslo, EX, and EXslo groups compared with SED group (P < 0.05). Gut-associated lymphocyte proliferation, on the other hand, was similar between the four experimental groups. Our findings show that SLO and EX indeed are able to increase lymphocyte proliferation, but their association did not induce further stimulation in the adaptive immune response and also did not modify innate immunity.

The sleep-wake cycle in adult rats following pilocarpine-induced temporal lobe epilepsy

The relationship between sleep and epilepsy is both complex and clinically significant. Temporal lobe epilepsy (TLE) influences sleep architecture, while sleep plays an important role in facilitating and/or inhibiting possible epileptic seizures. The pilocarpine experimental model reproduces several features of human temporal lobe epilepsy and is one of the most widely used models in basic research. The aim of the present study was to characterize, behaviorally and electrophysiologically, the phases of sleep-wake cycles (SWC) in male rats with pilocarpine-induced epilepsy. Epileptic rats presented spikes in all phases of the SWC as well as atypical cortical synchronization during attentive wakefulness and paradoxical sleep. The architecture of the sleep-wake phases was altered in epileptic rats, as was the integrity of the SWC. Because our findings reproduce many relevant features observed in patients with epilepsy, this model is suitable to study sleep dysfunction in epilepsy. (C) 2009 Elsevier Inc. All rights reserved.

Changes of glycogen content in liver, skeletal muscle, and heart from fasted rats

Glycogen content of white and red skeletal muscles, cardiac muscle, and liver was investigated in conditions where changes in plasma levels of non-esterified fatty acids (NEFA) occur. The experiments were performed in fed and 12 and 48 h-fasted rats. The animals were also submitted to swimming for 10 and 30 min. Glycogen content was also investigated in both pharmacologically induced low plasma NEFA levels fasted rats and pharmacologically induced high plasma NEFA levels fed rats. The participation of Akt and glycogen synthase kinase-3 (GSK-3) in the changes observed was investigated. Plasma levels of NEFA, glucose, and insulin were determined in all conditions. Fasting increased plasma NEFA levels and reduced glycogen content in the liver and skeletal muscles. However, an increase of glycogen content was observed in the heart under this condition. Akt and GSK-3 phosphorylation was reduced during fasting in the liver and skeletal muscles but it remained unchanged in the heart. Our results suggest that in conditions of increased plasma NEFA levels, changes in insulin-stimulated phosphorylation of Akt and GSK-3 and glycogen content vary differently in liver, skeletal muscles, and heart. Akt and GSK-3 phosphorylation and glycogen content are decreased in liver and skeletal Muscles, but in the heart it remain unchanged (Akt and GSK-3 phosphorylation) or increased (glycogen content) due to consistent increase of plasma NEFA levels. Copyright (C) 2009 John Wiley & Sons, Ltd.

Acute exhaustive exercise regulates IL-2, IL-4 and MyoD in skeletal muscle but not adipose tissue in rats

Background: The purpose of this study was to evaluate the effect of exhaustive exercise on proteins associated with muscle damage and regeneration, including IL-2, IL-4 and MyoD, in extensor digitorum longus (EDL) and soleus muscles and mesenteric (MEAT) and retroperitoneal adipose tissues (RPAT). Methods: Rats were killed by decapitation immediately (E0 group, n = 6), 2 (E2 group, n = 6) or 6 (E6 group, n = 6) hours after the exhaustion protocol, which consisted of running on a treadmill at approximately 70% of VO(2max) for fifty minutes and then at an elevated rate that increased at one m/min every minute, until exhaustion. Results: The control group (C group, n = 6) was not subjected to exercise. IL-2 protein expression increased at E0 in the soleus and EDL; at E2, this cytokine returned to control levels in both tissues. In the soleus, IL-2 protein expression was lower than that in the control at E6. IL-4 protein levels increased in EDL at E6, but the opposite result was observed in the soleus. MyoD expression increased at E6 in EDL. Conclusion: Exhaustive exercise was unable to modify IL-2 and IL-4 levels in MEAT and RPAT. The results show that exhaustive exercise has different effects depending on which muscle is analysed.

Opposite effects of fasting on TGF-beta 3 and T beta RI distribution in the gastric mucosa of suckling and early weanling rats

Objective: Our aim was to evaluate the effects of a dietary regimen (suckling or early weaning) and feeding status (fed or fasted) on the distribution of transforming growth factor-beta 3 (TGF-beta 3) and TGF receptor-I (T beta RI) in the gastric epithelium of pups Methods: Wistar rats were used At 15 d, half of the pups were separated from dams and fed with hydrated powered chow On day 17, suckling and early weanling rats were subjected to fasting (17 h). Four different conditions were established. suckling fed and fasted and early weanling fed and fasted At 18 d stomachs were collected under anesthesia and were fixed in 4% formaldehyde for immunohistochemistry The number of immunostained epithelial cells per microscopic field was determined for TGF-beta 3 and T beta RI in longitudinal sections from the gastric mucosa Results: We found that during suckling, fasting reduced the number of immunolabeled cells per field of both molecules when compared with the fed group (P < 0.05), whereas in early weaning, food restriction increased TGF-beta 3 and T beta RI distributions (P < 0.05) We also observed that TGF-beta 3 and T beta RI were more concentrated in parietal cells in the upper gland in suckling pups, whereas after early weaning these were displaced to parietal and chief cells at the bottom of the gland Conclusion: Suckling and early weaning directly influence TGF-beta 3 and T beta RI distributions in the gastric epithelium in response to fasting, such that early weaning anticipates the effects observed in adult rats. Furthermore, the differential concentrations of TGF-beta 3 and T beta RI indicate that they might be important for cell proliferation events in growth control (C) 2010 Elsevier Inc. All rights reserved