Methylglyoxal and carboxyethyllysine reduce glutamate uptake and S100B secretion in the hippocampus independently of RAGE activation.

Diabetes is a metabolic disease characterized by high fasting-glucose levels. Diabetic complications have been associated with hyperglycemia and high levels of reactive compounds, such as methylglyoxal (MG) and advanced glycation endproducts (AGEs) formation derived from glucose. Diabetic patients have a higher risk of developing neurodegenerative diseases, such as Alzheimer's disease or Parkinson's disease. Herein, we examined the effect of high glucose, MG and carboxyethyllysine (CEL), a MG-derived AGE of lysine, on oxidative, metabolic and astrocyte-specific parameters in acute hippocampal slices, and investigated some of the mechanisms that could mediate these effects. Glucose, MG and CEL did not alter reactive oxygen species (ROS) formation, glucose uptake or glutamine synthetase activity. However, glutamate uptake and S100B secretion were decreased after MG and CEL exposure. RAGE activation and glycation reactions, examined by aminoguanidine and L-lysine co-incubation, did not mediate these changes. Acute MG and CEL exposure, but not glucose, were able to induce similar effects on hippocampal slices, suggesting that conditions of high glucose concentrations are primarily toxic by elevating the rates of these glycation compounds, such as MG, and by generation of protein cross-links. Alterations in the secretion of S100B and the glutamatergic activity mediated by MG and AGEs can contribute to the brain dysfunction observed in diabetic patients.

Alterations of PI3K and Akt signaling pathways in the hippocampus and hypothalamus of Wistar rats treated with highly palatable food.

BACKGROUND/OBJECTIVES: Highly palatable food (HPF), which is enriched in simple sugars and saturated fat, contributes to obesity and insulin resistance in humans. These metabolic changes are associated with serious complications of the central nervous system, including an elevated risk of cognitive dysfunction. We, herein, treated rats with HPF and then examined the insulin-signaling pathway, in particular, the levels of phosphatidylinositol-3 kinase (PI3K), Akt, and insulin receptor substrate-1 (IRS-1) in the hippocampus and hypothalamus. METHODS: Adult Wistar rats fed with HPF (heated or not during preparation) for 4 months and then measured the levels of PI3K, Akt, and IRS-1 in the hippocampus and hypothalamus, by western blotting and quantitative real-time polymerase chain reaction. RESULTS: We observed changes in body weight, glucose intolerance, and lipidemia, confirming that peripheral metabolic alterations were induced using this model. Hippocampal PI3K and hypothalamic Akt were affected in rats that are submitted to chronic exposure to an HPF diet. Moreover, heated HPF caused differentiated alterations in the regulatory subunit of PI3K in the hippocampus. DISCUSSION: Our data suggest that this diet alters insulin signaling differentially in each brain region, and that hippocampal changes induced by this diet could contribute to the understanding of cognitive impairments that are dependent on the hippocampus.

Ascorbate uptake is decreased in the hippocampus of ageing rats.

Ascorbate, an intracellular antioxidant, has been considered critical for neuronal protection against oxidant stress, which is supported especially by in vitro studies. Besides, it has been demonstrated an age-related decrease in brain ascorbate levels. The aims of the present study were to investigate ascorbate uptake in hippocampal slices from old Wistar rats, as well as its neuroprotective effects in in vitro and in vivo assays. Hippocampal slices from male Wistar rats aged 4, 11 and 24 months were incubated with radiolabeled ascorbate and incorporated radioactivity was measured. Hippocampal slices from rats were incubated with different concentrations of ascorbate and submitted to H(2)O(2)-induced injury, cellular damage and S100B protein levels were evaluated. The effect of chronic administration of ascorbate on cellular oxidative state and astrocyte biochemical parameters in the hippocampus from 18-months-old Wistar rats was also studied. The ascorbate uptake was decreased in hippocampal slices from old-aged rats, while supplementation with ascorbate (2 weeks) did not modify any tested oxidative status in the hippocampus and the incubation was unable to protect hippocampal slices submitted to oxidative damage (H(2)O(2)) from old rats. Our data suggest that the decline of ascorbate uptake might be involved in the brain greater susceptibility to oxidative damage with advancing age and both in vitro and vivo assays suggest that ascorbate supplementation did not protect hippocampal cells.

Dietary omega-3 fatty acids attenuate cellular damage after a hippocampal ischemic insult in adult rats.

The role of omega-3 polyunsaturated fatty acids (3PUFAs) on brain function is increasingly demonstrated. Here, the effect of dietary deprivation of essential 3PUFAs on some parameters related to neuroprotection was investigated. Rats were fed with two different diets: omega-3 diet and omega-3-deprived diet. To assess the influence of 3PUFAs on brain responses to ischemic insult, hippocampal slices were subjected to an oxygen and glucose deprivation (OGD) model of in vitro ischemia. The omega-3-deprived group showed higher cell damage and stronger decrease in the [(3)H]glutamate uptake after OGD. Moreover, omega-3 deprivation influenced antiapoptotic cell response after OGD, affecting GSK-3beta and ERK1/2, but not Akt, phosphorylation. Taken together, these results suggest that 3PUFAs are important for cell protection after ischemia and also seem to play an important role in the activation of antiapoptotic signaling pathways.

Effects of glyoxal or methylglyoxal on the metabolism of amino acids, lactate, glucose and acetate in the cerebral cortex of young and adult rats.

The in vitro effects of glyoxal and methylglyoxal on the metabolism of glycine, alanine, leucine, glutamate, glutamine, glucose, lactate and acetate were evaluated in cortico-cerebral slices from young (10-day-old) or adult (3-month-old) rats. In a first set of experiments with cortico-cerebral slices from young animals, the compounds glyoxal or methylglyoxal at 400 microM, increased the oxidation of alanine, leucine and glycine to CO(2) and decreased the protein synthesis from these amino acids. Lipid synthesis from alanine, leucine and glycine was not changed in the cortico-cerebral slices from young rats after glyoxals exposure. Moreover, glutamine oxidation to CO(2) decreased by glyoxals exposure, but glutamate oxidation was not affected. In a second set of experiments with brain slices from adult animals, glycine metabolism (oxidation to CO(2), conversion to lipids or incorporation into proteins) was not changed by glyoxals exposure. In addition, the oxidation rates of glucose, lactate, acetate, glutamine and glutamate to CO(2) were also not modified. Taken together, these results indicate that glyoxal disrupts the energetic metabolism of the rat cerebral cortex in vitro. However, only young animals were susceptible to such events, suggesting that the immature cerebral cortex is less capable of dealing with glyoxal than the mature one.

High fat and highly thermolyzed fat diets promote insulin resistance and increase DNA damage in rats.

Many studies have demonstrated that DNA damage may be associated with type 2 diabetes mellitus (T2DM) and its complications. The goal of this study was to evaluate the effects of the potential relationship between fat (thermolyzed) intake, glucose dyshomeostasis and DNA injury in rats. Biochemical parameters related to glucose metabolism (i.e., blood glucose levels, insulin tolerance tests, glucose tolerance tests and fat cell glucose oxidation) and general health parameters (i.e., body weight, retroperitoneal and epididymal adipose tissue) were evaluated in rats after a 12-month treatment with either a high fat or a high thermolyzed fat diet. The high fat diet (HFD) and high fat thermolyzed diet (HFTD) showed increased body weight and impaired insulin sensitivity at the studied time-points in insulin tolerance test (ITT) and glucose tolerance test (GTT). Interestingly, only animals subjected to the HFTD diet showed decreased epididymal fat cell glucose oxidation. We show which high fat diets have the capacity to reduce glycogen synthesis by direct and indirect pathways. HFTD promoted an increase in lipid peroxidation in the liver, demonstrating significant damage in lipids in relation to other groups. Blood and hippocampus DNA damage was significantly higher in animals subjected to HFDs, and the highest damage was observed in animals from the HFTD group. Striatum DNA damage was significantly higher in animals subjected to HFDs, compared with the control group. These results show a positive correlation between high fat diet, glucose dyshomeostasis, oxidative stress and DNA damage.

Resveratrol protects against oxidative injury induced by H2O2 in acute hippocampal slice preparations from Wistar rats.

There is a current interest in dietary compounds (such as trans-resveratrol) that can inhibit or reverse oxidative stress, the common pathway for a variety of brain disorders, including Alzheimer's disease and stroke. The objective of the present study was to investigate the effects of resveratrol, under conditions of oxidative stress induced by H(2)O(2), on acute hippocampal slices from Wistar rats. Here, we evaluated cell viability, extracellular lactate, glutathione content, ERK(MAPK) activity, glutamate uptake and S100B secretion. Resveratrol did not change the decrease in lactate levels and in cell viability (by MTT assay) induced by 1mM H(2)O(2), but prevented the increase in cell permeability to Trypan blue induced by H(2)O(2). Moreover, resveratrol per se increased total glutathione levels and prevented the decrease in glutathione induced by 1mM H(2)O(2). The reduction of S100B secretion induced by H(2)O(2) was not changed by resveratrol. Glutamate uptake was decreased in the presence of 1mM H(2)O(2) and this effect was not prevented by resveratrol. There was also a significant activation of ERK1/2 by 1mM H(2)O(2) and resveratrol was able to completely prevent this activation, leading to activity values lower than control levels. The impairments in astrocyte activities, induced by H(2)O(2), confirmed the importance of these cells as targets for therapeutic strategy in brain disorders involving oxidative stress. This study reinforces the protective role of resveratrol and indicates some possible molecular sites of activity of this compound on glial cells, in the acute damage of brain tissue during oxidative stress.

Profile of glutamate uptake and cellular viability in hippocampal slices exposed to oxygen and glucose deprivation: developmental aspects and protection by guanosine.

Stroke syndromes are a major cause of disability in middle and later life resulting in severe neuronal degeneration and loss of brain functions. In situations with energy failure, glutamate transport is impaired and high levels of this amino acid accumulate on the synaptic cleft. Our group has showed that guanosine exerts neuroprotection against neurotoxicity situations. The aim of this work is draw a post-ischemic profile of glutamate uptake and cell damage using an oxygen and glucose deprivation model (OGD) in hippocampal slices from young (P10) and adult (P60) rats, analyzing guanosine effect. OGD decreases glutamate uptake in both ages and recovery times, although decrease in cell viability was only observed 1 and 3 h after OGD in young and adult animals, respectively. Guanosine partially protected cell damage from 1 h in P10 and at 3 h in P60 rats and avoided glutamate uptake decrease from P10 rats at 3 h. The impairment of glutamate transporters since immediately after the insult observed here is probably due to an energetic failure; loss of cell viability was only observed from 1 h after OGD. The mechanism by which guanosine acts in the 'ischemic' model used here is still unknown, but evidence leads to its antiapoptotic effect.

Brain glutathione content and glutamate uptake are reduced in rats exposed to pre- and postnatal protein malnutrition.

The brain is particularly susceptible to oxidative insults and its antioxidant defense is dependent on its glutathione content. Protein malnutrition (PMN) is an important and very common insult during development and compromises antioxidant defenses in the body, particularly glutathione levels. We investigated whether brain glutathione content and related metabolic pathways, predominantly regulated by astrocytes (particularly glutamate uptake and glutamine synthesis), are altered by pre- and postnatal PMN in rats. Thus, we measured the glutathione content, glutamine synthetase (GS) activity, and glutamate uptake activity in the cerebral cortex (Cx) and hippocampus of rats subjected to pre- and postnatal PMN and in nourished controls. Although malnourished rats exhibited an ontogenetic profile of glutathione levels in both brain regions similar to that of controls, they had lower levels on postnatal d 2 (P2); in Cx this decrease persisted until postnatal d 15. In addition, we found other changes, such as reduced total antioxidant reactivity and glutathione peroxidase activity on P2, and these were not accompanied by alterations in free radical levels or lipoperoxidation in either brain region. Moreover, malnourished rats had elevated GS and reduced glutamate uptake. Taken together, these alterations indicate specific changes in astrocyte metabolism, possibly responsible for the higher vulnerability to excitotoxic/oxidative damage in malnourished rats. The lower antioxidant defense appears to be the main alteration that causes oxidative imbalance, rather than an increase in reactive oxygen species. Moreover, a recovery of altered metabolic variables may occur during adulthood, despite persistent PMN.

Effect of 2-deoxy-D-glucose on aminoacids metabolism in rats' cerebral cortex slices.

We studied the effect of different concentrations of 2-deoxy-D-glucose on the L-[U-14C]leucine, L-[1-14C]leucine and [1-14C]glycine metabolism in slices of cerebral cortex of 10-day-old rats. 2-deoxy-D-glucose since 0.5 mM concentration has inhibited significantly the protein synthesis from L-[U-14C]leucine and from [1-14C]glycine in relation to the medium containing only Krebs Ringer bicarbonate. Potassium 8.0 mM in incubation medium did not stimulate the protein synthesis compared to the medium containing 2.7 mM, and at 50 mM diminishes more than 2.5 times the protein synthesis compared to the other concentration. Only at the concentration of 5.0 mM, 2-deoxy-D-glucose inhibited the CO2 production and lipid synthesis from L-[U-14C] leucine. This compound did not inhibit either CO2 production, or lipid synthesis from [1-14C]glycine. Lactate at 10 mM and glucose 5.0 mM did not revert the inhibitory effect of 2-deoxy-D-glucose on the protein synthesis from L-[U-14C]leucine. 2-deoxy-D-glucose at 2.0 mM did not show any effect either on CO2 production, or on lipid synthesis from L-[U-14C]lactate 10 mM and glucose 5.0 mM.

Ontogenetic changes in glial fibrillary acid protein phosphorylation, glutamate uptake and glutamine synthetase activity in olfactory bulb of rats.

Phosphorylation of the glial fibrillary acidic protein (GFAP) in hippocampal and cerebellar slices from immature rats is stimulated by glutamate. This effect occurs via a group II metabotropic glutamate receptor in the hippocampus and an NMDA ionotropic receptor in the cerebellum. We investigated the glutamate modulation of GFAP phosphorylation in the olfactory bulb slices of Wistar rats of different ages (post-natal day 15 = P15, post-natal day 21 = P21 and post-natal day 60 = P60). Our results showed that glutamate stimulates GFAP phosphorylation in young animals and this is mediated by NMDA receptors. We also observed a decrease in glutamate uptake at P60 compared to P15, a finding similar to that found in the hippocampus. The activity of glutamine synthetase was elevated after birth, but was found to decrease with development from P21 to P60. Together, these data confirm the importance of glutamatergic transmission in the olfactory bulb, its developmental regulation in this brain structure and extends the concept of glial involvement in glutamatergic neuron-glial communication.