Na+-glucose transporter-2 messenger ribonucleic acid expression in kidney of diabetic rats correlates with glycemic levels: Involvement of hepatocyte nuclear factor-1 alpha expression and activity

Mutations in Na+-glucose transporters (SGLT)-2 and hepatocyte nuclear factor (HNF)-1 alpha genes have been related to renal glycosuria and maturity-onset diabetes of the young 3, respectively. However, the expression of these genes have not been investigated in type 1 and type 2 diabetes. Here in kidney of diabetic rats, we tested the hypotheses that SGLT2 mRNA expression is altered; HNF-1 alpha is involved in this regulation; and glycemic homeostasis is a related mechanism. The in vivo binding of HNF-1 alpha into the SGLT2 promoter region in renal cortex was confirmed by chromatin immunoprecipitation assay. SGLT2 and HNF-1 alpha mRNA expression (by Northern and RT-PCR analysis) and HNF-1 binding activity of nuclear proteins (by EMSA) were investigated in diabetic rats and treated or not with insulin or phlorizin (an inhibitor of SGLT2). Results showed that diabetes increases SGLT2 and HNF-1 alpha mRNA expression (similar to 50%) and binding of nuclear proteins to a HNF-1 consensus motif (similar to 100%). Six days of insulin or phlorizin treatment restores these parameters to nondiabetic-rat levels. Moreover, both treatments similarly reduced glycemia, despite the differences in plasma insulin and urinary glucose concentrations, highlighting the plasma glucose levels as involved in the observed modulations. This study shows that SGLT2 mRNA expression and HNF-1 alpha expression and activity correlate positively in kidney of diabetic rats. It also shows that diabetes-induced changes are reversed by lowering glycemia, independently of insulinemia. Our demonstration that HNF-1 alpha binds DNA that encodes SGLT2 supports the hypothesis that HNF-1 alpha, as a modulator of SGLT2 expression, may be involved in diabetic kidney disease.

Ventrolateral medulla mechanisms involved in cardiorespiratory responses to central chemoreceptor activation in rats

A rise in arterial PCO(2) stimulates breathing and sympathetic activity to the heart and blood vessels. In the present study, we investigated the involvement of the retrotrapezoid nucleus (RTN) and glutamatergic mechanisms in the Botzinger/C1 region (Botz/C1) in these responses. Splanchnic sympathetic nerve discharge (sSND) and phrenic nerve discharge (PND) were recorded in urethane-anesthetized, sino-aortic-denervated, vagotomized, and artificially ventilated rats subjected to hypercapnia (end-expiratory CO(2) from 5% to 10%). Phrenic activity was absent at end-expiratory CO(2) of 4%, and strongly increased when end-expiratory CO(2) reached 10%. Hypercapnia also increased sSND by 103 +/- 7%. Bilateral injections of the GABA-A agonist muscimol (2 mM) into the RTN eliminated the PND and blunted the sSND activation (Delta = +56 +8%) elicited by hypercapnia. Injections of NMDA receptor antagonist AP-5 (100 mM), non-NMDA receptor antagonist 6,7-dinitro-quinoxaline-2,3-dione (DNQX; 100 mM) or metabotropic glutamate receptor antagonist (+/-)-alpha-methyl-4-carboxyphenylglycine (MCPG; 100 mM) bilaterally into the Botz/C1 reduced PND (Delta = +43 +/- 7%, +52 +/- 6% or +56 +/- 11%, respectively). MCPG also reduced sSND (Delta = +41 +/- 7%), whereas AP-5 and DNQX had no effect. In conclusion, the increase in sSND caused by hypercapnia depends on increased activity of the RTN and on metabotropic receptors in the Botz/C1, whereas PND depends on increased RTN activity and both ionotropic and metabotropic receptors in the Botz/C1.

Neuromuscular electrical stimulation improves GLUT-4 and morphological characteristics of skeletal muscle in rats with heart failure

Aim: Changes in skeletal muscle morphology and metabolism are associated with limited functional capacity in heart failure, which can be attenuated by neuromuscular electrical stimulation (ES). The purpose of the present study was to analyse the effects of ES upon GLUT-4 protein content, fibre structure and vessel density of the skeletal muscle in a rat model of HF subsequent to myocardial infarction. Methods: Forty-four male Wistar rats were assigned to one of four groups: sham (S), sham submitted to ES (S+ES), heart failure (HF) and heart failure submitted to ES (HF+ES). The rats in the ES groups were submitted to ES of the left leg during 20 days (2.5 kHz, once a day, 30 min, duty cycle 50%- 15 s contraction/15 s rest). After this period, the left tibialis anterior muscle was collected from all the rats for analysis. Results: HF+ES rats showed lower values of lung congestion when compared with HF rats (P = 0.0001). Although muscle weight was lower in HF rats than in the S group, thus indicating hypotrophy, 20 days of ES led to their recovery (P < 0.0001). In both groups submitted to ES, there was an increase in muscle vessel density (P < 0.04). Additionally, heart failure determined a 49% reduction in GLUT-4 protein content (P < 0.03), which was recovered by ES (P < 0.01). Conclusion: In heart failure, ES improves morphological changes and raises GLUT-4 content in skeletal muscle.

The Antiapoptotic Effect of Granulocyte Colony-stimulating Factor Reduces Infarct Size and Prevents Heart Failure Development in Rats

Background/Aim. Granulocyte colony-stimulating factor (G-CSF) reduces myocardial injury and improves cardiac function after myocardial infarction (MI). We investigated the early alterations provided by G-CSF and the chronic repercussions in infarcted rats. Methods. Male Wistar rats (200-250g) received vehicle (MI) or G-CSF (MI-GCSF) (50 mu g/kg, sc) at 7, 3 and 1 days before MI surgery. Afterwards MI was produced and infarct size was measured 1 and 15 days after surgery. Expression of anti-and proapoptotic proteins was evaluated immediately before surgery. 24 hours after surgery, apoptotic nuclei were evaluated. Two weeks after MI, left ventricular (LV) function was evaluated, followed by in situ LV diastolic pressure-volume evaluation. Results. Infarct size was decreased by 1 day pretreatment before occlusion (36 +/- 2.8 vs. 44 +/- 2.1% in MI; P<0.05) and remained reduced at 15 days after infarction (28 +/- 2.2 vs. 36 +/- 1.4% in MI; P<0.05). G-CSF pretreatment increased Bcl-2 and Bcl-xL protein expression, but did not alter Bax in LV. Apoptotic nuclei were reduced by treatment (Sham: 0.46 +/- 0.42, MI: 15.5 +/- 2.43, MI-GCSF: 5.34 +/- 3.34%; P<0.05). Fifteen days after MI, cardiac function remained preserved in G-CSF pretreated rats. The LV dilation was reduced in MI-G-CSF group as compared to MI rats, being closely associated with infarct size. Conclusion. The early beneficial effects of G-CSF were essentials to preserve cardiac function at a chronic stage of myocardial infarction. Copyright (C) 2011 S. Karger AG, Basel

Hyperglycaemia protects the heart after myocardial infarction: aspects of programmed cell survival and cell death

Exposure to a high glucose medium or diabetes has been found to protect the heart against ischaemia. The activation of antiapoptotic and proliferative factors seems to be involved in this cardioprotection. This study was designed to evaluate the role of hyperglycaemia in cardiac function, programmed cell survival, and cell death in diabetic rats after myocardial infarction (MI). Male Wistar rats were divided into four groups (n = 8): control (C), diabetic (D), myocardial infarcted (MI), and diabetic myocardial infarcted (DI). The following measures were assessed in the left ventricle: size of MI, systolic and diastolic function by echocardiography, cytokines by ELISA (TNF-alpha, IL-1 beta, IL-6, and IL-10), gene expression by real-time PCR (Bax, Fas, p53, Bcl-2, HIF1-alpha, VEGF, and IL8r), caspase-3 activity by spectrofluorometric assay, glucose transporter type 1 and 4 (GLUT-1 and GLUT-4) protein expression by western blotting, and capillary density and fibrosis by histological analysis. Systolic function was improved by hyperglycaemia in the DI group, and this was accompanied by no improvement in diastolic dysfunction, a reduction of 36% in MI size, reduced proinflammatory cytokines, apoptosis activation, and an increase in cell survival factors (HIF1-alpha, VEGFa and IL8r) assessed 15 days post-MI. Moreover, hyperglycaemia resulted in angiogenesis (increased capillary density) before and after MI, accompanied by a reduction in fibrosis. Together, these results suggest that greater plasticity and cellular resistance to ischaemic injury result from chronic diabetic hyperglycaemia in rat hearts.

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.

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.