The effects of hypercaloric diets on glucose homeostasis in the rat: influence of saturated and monounsaturated dietary lipids.

Consumption of energy-dense/high-fat diets is strongly and positively associated with overweight and obesity, which are associated with increase in the prevalence of certain chronic diseases. We evaluated the effect of hypercaloric/fat or normocaloric diets on some biochemical parameters in rats. Seventy-two rats were divided into four groups that were fed for 16 weeks with diets: normocaloric [9.12% soy oil, normocaloric soy oil (NSO)], hypercaloric olive oil [43.8% olive oil, hypercaloric olive oil (HOO)], hypercaloric saturated fat [43.8% saturated fat, hypercaloric saturated fat (HSF)] and normocaloric saturated fat [43.8% saturated fat, normocaloric saturated fat (NSF)]. HSF rats consumed more calories daily than the others and gained more retroperitoneal fat, although HSF and HOO rats had higher body weight. In liver, glycogen synthesis and concentration were higher in rats HSF and NSF. In plasma, total cholesterol (TC) levels were higher in HSF rats than in the others, and triacylglycerol (TAG) levels were lower in HOO and higher in HSF rats in relation to the others. In liver, TC and TAG were elevated in HSF, NSF and HOO rats. Paraoxonase 1 activity, which is related to high-density lipoprotein cholesterol and has anti-atherogenic role was lower in rats HSF. In HOO rats, glucose tolerance test was altered, but insulin tolerance test was normal. These results suggest that consumption of energy-dense/high-fat diets, both saturated or monounsaturated, causes damaging effects. However, more studies are necessary to understand the mechanisms by which these diets cause the metabolic alterations observed.

Na+, K+ ATPase activity is markedly reduced by cis-4-decenoic acid in synaptic plasma membranes from cerebral cortex of rats.

We have previously demonstrated that octanoic (OA) and decanoic acids (DA) inhibit Na+, K+ ATPase activity in synaptic plasma membranes from rat brain. The objective of the present study was to investigate the in vitro effects of the other metabolites that accumulate in tissues of medium-chain acyl-CoA dehydrogenase (MCAD)-deficient patients, namely cis-4-decenoic acid (cDA), octanoylcarnitine (OC), hexanoylcarnitine (HC), hexanoylglycine (HG), phenylpropionylglycine (PPG) and suberoylglycine (SG), on Na+, K+ ATPase activity in synaptic plasma membrane from cerebral cortex of 30-day-old rats. cDA, the pathognomonic compound found in this disorder, provoked the strongest inhibition on this enzyme activity at concentrations as low as 0.25 mM, whereas OC inhibited this activity at 1.0 mM and higher concentrations in a dose-dependent manner. In contrast, HC, HG, PPG and SG did not affect Na+, K+ ATPase activity. Furthermore, pre-treatment of cortical homogenates with the antioxidant enzymes catalase plus superoxide dismutase totally prevented cDA-induced Na+, K+ ATPase inhibition. We also provided evidence that cDA, as well as OA and DA, caused lipid peroxidation, which may explain, at least in part, the inhibitory properties of these compounds towards Na+, K+ ATPase. Considering that Na+, K+ ATPase is a critical enzyme for normal brain development and functioning, it is presumed that these findings, especially those regarding to the marked inhibitory effect of cDA, may be involved in the pathophysiology of the neurological dysfunction of MCAD-deficient patients.

Inhibition of energy metabolism by 2-methylacetoacetate and 2-methyl-3-hydroxybutyrate in cerebral cortex of developing rats.

Mitochondrial beta-ketothiolase and 2-methyl-3-hydroxybutyryl-CoA dehydrogenase (MHBD) deficiencies are inherited neurometabolic disorders affecting isoleucine catabolism. Biochemically, beta-ketothiolase deficiency is characterized by intermittent ketoacidosis and urinary excretion of 2-methyl-acetoacetate (MAA), 2-methyl-3-hydroxybutyrate (MHB) and tiglylglycine (TG), whereas in MHBD deficiency only MHB and tiglylglycine accumulate. Lactic acid accumulation and excretion are also observed in these patients, being more pronounced in MHBD-deficient individuals, particularly during acute episodes of decompensation. Patients affected by MHBD deficiency usually manifest severe mental retardation and convulsions, whereas beta-ketothiolase-deficient patients present encephalopathic crises characterized by metabolic acidosis, vomiting and coma. Considering that the pathophysiological mechanisms responsible for the neurological alterations of these disorders are unknown and that lactic acidosis suggests an impairment of energy production, the objective of the present work was to investigate the in vitro effect of MAA and MHB, at concentrations varying from 0.01 to 1.0 mmol/L, on several parameters of energy metabolism in cerebral cortex from young rats. We observed that MAA markedly inhibited CO2 production from glucose, acetate and citrate at concentrations as low as 0.01 mmol/L. In addition, the activities of the respiratory chain complex II and succinate dehydrogenase were mildly inhibited by MAA. MHB, at 0.01 mmol/L and higher concentrations, strongly inhibited CO2 production from all tested substrates, as well as the respiratory chain complex IV activity. The other activities of the respiratory chain were not affected by these metabolites. The data indicate a marked blockage in the Krebs cycle and a mild inhibition of the respiratory chain caused by MAA and MHB. Furthermore, MHB inhibited total and mitochondrial creatine kinase activities, which was prevented by the use of the nitric-oxide synthase inhibitor L-NAME and glutathione (GSH). These data indicate that the effect of MHB on creatine kinase was probably mediated by oxidation or other modification of essential thiol groups of the enzyme by nitric oxide and other by-products derived from this organic acid. In contrast, MAA did not affect creatine kinase activity. Taken together, these observations indicate that aerobic energy metabolism is inhibited by MAA and to a greater extent by MHB, a fact that may be related to lactic acidaemia occurring in patients affected by MHBD and beta-ketothiolase deficiencies. If the in vitro effects detected in the present study also occur in vivo, it is tempting to speculate that they may contribute, at least in part, to the neurological dysfunction found in these disorders.

Effect of hyperprolinemia on acetylcholinesterase and butyrylcholinesterase activities in rat.

We observed here that acute proline (Pro) administration provoked a decrease (32%) of acetylcholinesterase (AChE) activity in cerebral cortex and an increase (22%) of butyrylcholinesterase (BuChE) activity in the serum of 29-day-old rats. In contrast, chronic administration of Pro did not alter AChE or BuChE activities. Furthermore, pretreatment of rats with vitamins E and C combined or alone, N(omega)-nitro-L-arginine methyl ester or melatonin prevented the reduction of AChE activity caused by acute Pro administration, suggesting the participation of oxidative stress in such effects.

Inhibition of mitochondrial creatine kinase activity from rat cerebral cortex by methylmalonic acid.

Accumulation of methylmalonic acid (MMA) in tissues and biological fluids is the biochemical hallmark of patients affected by the neurometabolic disorder known as methylmalonic acidemia (MMAemia). Although this disease is predominantly characterized by severe neurological findings, the underlying mechanisms of brain injury are not totally established. In the present study, we investigated the effect of MMA, as well as propionic (PA) and tiglic (TA) acids, whose concentrations are also increased but to a lesser extend in MMAemia, on total (tCK), cytosolic (Cy-CK) and mitochondrial (Mi-CK) creatine kinase (CK) activities from cerebral cortex of 30-day-old Wistar rats. Total CK activity (tCK) was measured in whole cell homogenates, whereas Cy-CK and Mi-CK were determined, respectively, in cytosolic and mitochondrial preparations from rat cerebral cortex. We verified that tCK and Mi-CK activities were significantly inhibited by MMA at concentrations as low as 1 mM, in contrast to Cy-CK which was not affected by the presence of the acid in the incubation medium. Furthermore, PA and TA, at concentrations as high as 5 mM, did not alter CK activity. We also observed that the inhibitions provoked by MMA were fully prevented by pre-incubation of the homogenates with reduced glutathione, suggesting that the inhibitory effect of MMA was possibly mediated by oxidation of essential thiol groups of the enzyme. Considering the importance of CK for brain metabolism homeostasis, our results suggest that inhibition of this enzyme by increased levels of MMA may contribute to the neurodegeneration of patients affected by MMAemia and explain previous reports showing an impairment of brain energy metabolism and a reduction of brain phosphocreatine levels caused by MMA.

Effects of histidine and imidazolelactic acid on various parameters of the oxidative stress in cerebral cortex of young rats.

Histidinemia is an inherited metabolic disorder caused by deficiency of histidase activity, which leads to tissue accumulation of histidine and its derivatives. Affected patients usually present with speech delay and mental retardation, although asymptomatic patients have been reported. Considering that the pathophysiology of the neurological dysfunction of histidinemia is not yet understood and since histidine has been considered a pro-oxidant agent, in the present study we investigated the effect of histidine and one of its derivatives, l-beta-imidazolelactic acid, at concentrations ranging from 0.1 to 10 mM, on various parameters of oxidative stress in cerebral cortex of 30-day-old Wistar rats. Chemiluminescence, total radical-trapping antioxidant potential (TRAP), thiobarbituric acid reactive substances (TBA-RS), and the activities of the antioxidant enzymes superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px) were measured in tissue homogenates in the presence of l-histidine or l-beta-imidazolelactic acid. We observed that l-histidine provoked an increase of chemiluminescence and a reduction of TRAP at concentrations of 2.5 mM and higher, while TBA-RS measurement, GSH-Px, CAT and SOD activities were not affected. Furthermore, l-beta-imidazolelactic acid provoked antioxidant effects at high concentrations (5-10 mM) as observed by the reduction of chemiluminescence, although this compound enhanced chemiluminescence at low concentrations (0.5-1 mM). These results suggest that in vitro oxidative stress is elicited by histidine but only at supraphysiological concentrations.

D-2-hydroxyglutaric acid inhibits creatine kinase activity from cardiac and skeletal muscle of young rats.

BACKGROUND: Tissue accumulation of high amounts of D-2-hydroxyglutaric acid (DGA) is the biochemical hallmark of the inherited neurometabolic disorder D-2-hydroxyglutaric aciduria (DHGA). Patients affected by this disease usually present hypotonia, muscular weakness, hypertrophy and cardiomyopathy, besides severe neurological findings. However, the underlying mechanisms of muscle injury in this disorder are virtually unknown. MATERIALS AND METHODS: In the present study we have evaluated the in vitro role of DGA, at concentrations ranging from 0.25 to 5.0 mM, on total, cytosolic and mitochondrial creatine kinase activities from skeletal and cardiac muscle of 30-day-old Wistar rats. We also tested the effects of various antioxidants on the effects elicited by DGA. RESULTS: We first verified that total creatine kinase (CK) activity from homogenates was significantly inhibited by DGA (22-24% inhibition) in skeletal and cardiac muscle, and that this activity was approximately threefold higher in skeletal muscle than in cardiac muscle. We also observed that CK activities from mitochondrial (Mi-CK) and cytosolic (Cy-CK) preparations from skeletal muscle and cardiac muscle were also inhibited (12-35% inhibition) by DGA at concentrations as low as 0.25 mm, with the effect being more pronounced in cardiac muscle preparations. Finally, we verified that the DGA-inhibitory effect was fully prevented by preincubation of the homogenates with reduced glutathione and cysteine, suggesting that this effect is possibly mediated by modification of essential thiol groups of the enzyme. Furthermore, alpha-tocopherol, melatonin and the inhibitor of nitric oxide synthase L-NAME were unable to prevent this effect, indicating that the most common reactive oxygen and nitrogen species were not involved in the inhibition of CK provoked by DGA. CONCLUSION: Considering the importance of creatine kinase activity for cellular energy homeostasis, our results suggest that inhibition of this enzyme by increased levels of DGA might be an important mechanism involved in the myopathy and cardiomyopathy of patients affected by DHGA.

Ganglioside alterations in the central nervous system of rats chronically injected with methylmalonic and propionic acids.

Neurological dysfunction and structural cerebral abnormalities are commonly found in patients with methylmalonic and propionic acidemia. However, the mechanisms underlying the neuropathology of these disorders are poorly understood. We have previously demonstrated that methylmalonic and propionic acids induce a significant reduction of ganglioside N-acetylneuraminic acid in the brain of rats subjected to chronic administration of these metabolites. In the present study, we investigated the in vivo effects of chronic administration of methylmalonic (MMA) and propionic (PA) acids (from the 6th to the 28th day of life) on the distribution and composition of gangliosides in the cerebellum and cerebral cortex of rats. Control rats were treated with the same volumes of saline. It was first verified that MMA and PA treatment did not modify body, cerebellum, or cortical weight, nor the ganglioside concentration in the cerebral cortex of the animals. In contrast, a significant reduction in total ganglioside content in the cerebellum of approximately 20-30% and 50% of control levels occurred in rats injected with MMA and PA, respectively. Moreover, chronic MMA and PA administration did not interfere with the ganglioside pattern in the cerebral cortex, whereas the distribution of individual gangliosides was altered in the cerebellum of MMA- and PA-treated animals. Rats injected with MMA demonstrated a marked decrease in GM1 and GD3, whereas chronic PA treatment provoked a significant reduction of all ganglioside species, with the exception of an increase in GM2. Since gangliosides are closely related to the dendritic surface and other neural membranes, indirectly reflecting synaptogenesis, these ganglioside abnormalities may be associated with the brain damage found in methylmalonic and propionic acidemias.

Inhibition of the mitochondrial respiratory chain complex activities in rat cerebral cortex by methylmalonic acid.

Propionic and methylmalonic acidemic patients have severe neurologic symptoms whose etiopathogeny is still obscure. Since increase of lactic acid is detected in the urine of these patients, especially during metabolic decompensation when high concentrations of methylmalonate (MMA) and propionate (PA) are produced, it is possible that cellular respiration may be impaired in these individuals. Therefore, we investigated the effects of MMA and PA (1, 2.5 and 5mM), the principal metabolites which accumulate in these conditions, on the mitochondrial respiratory chain complex activities succinate: 2,6-dichloroindophenol (DCIP) oxireductase (complex II); succinate: cytochrome c oxireductase (complexII+CoQ+III); NADH: cytochrome c oxireductase (complex I+CoQ+complex III); and cytochrome c oxidase (COX) (complex IV) from cerebral cortex homogenates of young rats. The effect of MMA on ubiquinol: cytochrome c oxireductase (complex III) and NADH: ubiquinone oxireductase (complex I) activities was also tested. Control groups did not contain MMA and PA in the incubation medium. MMA significantly inhibited complex I+III (32-46%), complex I (61-72%), and complex II+III (15-26%), without affecting significantly the activities of complexes II, III and IV. However, by using 1mM succinate in the assay instead of the usual 16mM concentration, MMA was able to significantly inhibit complex II activity in the brain homogenates. In contrast, PA did not affect any of these mitochondrial enzyme activities. The effect of MMA and PA on succinate: phenazine oxireductase (soluble succinate dehydrogenase (SDH)) was also measured in mitochondrial preparations. The results showed significant inhibition of the soluble SDH activity by MMA (11-27%) in purified mitochondrial fractions. Thus, if the in vitro inhibition of the oxidative phosphorylation system is also expressed under in vivo conditions, a deficit of brain energy production might explain some of the neurological abnormalities found in patients with methylmalonic acidemia (MMAemia) and be responsible for the lactic acidemia/aciduria identified in some of them.

Inhibition of in vitro CO2 production and lipid synthesis by 2-hydroxybutyric acid in rat brain

2-Hydroxybutyric acid appears at high concentrations in situations related to deficient energy metabolism (e.g., birth asphyxia) and also in inherited metabolic diseases affecting the central nervous system during neonatal development, such as "cerebral" lactic acidosis, glutaric aciduria type II, dihydrolipoyl dehydrogenase (E3) deficiency, and propionic acidemia. The present study was carried out to determine the effect of 2-hydroxybutyric acid at various concentrations (1-10 mM) on CO2 production and lipid synthesis from labeled substrates in cerebral cortex of 30-day-old Wistar rats in vitro. CO2 production was significantly inhibited (30-70 percent) by 2-hydroxybutyric acid in cerebral cortex prisms, in total homogenates and in the mitochondrial fraction. We also demonstrated a significant inhibition of lipid synthesis (20-45 percent) in cerebral cortex prisms and total homogenates in the presence of 2-hydroxybutyric acid. However, no inhibition of lipid synthesis occurred in homogenates free of nuclei and mitochondria. The results indicate an impairment of mitochondrial energy metabolism caused by 2-hydroxybutyric acid, a fact that may secondarily lead to reduction of lipid synthesis. It is possible that these findings may be associated with the neuropathophysiology of the situations where 2-hydroxybutyric acid is accumulated

Chronic postnatal administration of methylmalonic acid provokes a decrease of myelin content and ganglioside N-acetylneuraminic acid concentration in cerebrum of young rats

Levels of methylmalonic acid (MMA) comparable to those of human methylmalonic acidemia were achieved in blood (2-2.5 mmol/l) and brain (1.35 æmol/g) of rats by administering buffered MMA, pH 7.4, subcutaneously twice a day from the 5th to the 28th day of life. MMA doses ranged from 0.76 to 1.67 æmol/g as a function of animal age. Control rats were treated with saline in the same volumes. The animals were sacrificed by decapitation on the 28th day of age. Blood was taken and the brain was rapidly removed. Medulla, pons, the olfactory lobes and cerebellum were discarded and the rest of the brain ("cerebrum") was isolated. Body and "cerebrum" weight were measured, as well as the cholesterol and triglyceride concentrations in blood and the content of myelin, total lipids, and the concentrations of the lipid fractions (cholesterol, glycerolipids, phospholipids and ganglioside N-acetylneuraminic acid (ganglioside-NANA)) in the "cerebrum". Chronic MMA administration had no effect on body or "cerebrum" weight, suggesting that the metabolites per se neither affect the appetite of the rats nor cause malnutrition. In contrast, MMA caused a significant reduction of plasma triglycerides, but not of plasma cholesterol levels. A significant diminution of myelin content and of ganglioside-NANA concentration was also observed in the "cerebrum". We propose that the reduction of myelin content and ganglioside-NANA caused by MMA may be related to the delayed myelination/cerebral atrophy and neurological dysfunction found in methylmalonic acidemic children

ATP diphosphohydrolase activity in synaptosomes from cerebral cortex of rats subjected to chemically induced phenylketonuria

ATP diphosphohydrolase (apyrase)(EC3.6.1.5) activity was measured in synaptosomes from cerebral cortex of Wistar rats of both sexes subjected to experimental phenylketonuria, i.e., chemical hyperphenylaninemia induced by subcutaneous administration of 5.2 µmol phenylalanine/g body weight (twice a day) plus 0.9 µmol p-chlorophenylalanine/g body weight (once a day). ATP diphosphohydrolase specific activity (nmol Pi min-1 mg protein-1) of synaptosomes was significantly decreased compared to controls for both ATp (from 147.6 to 129.9) and ADP (from 70.2 to 63.1) hydrolysis one hour after single administration of the drugs to 35-day old rats. Chronic treatment was performed from the 6th to the 28th postpartum day. The enzyme specific activity of synaptosomes was measured one week after the last administration of the drugs and was significantly reduced compared to controls for both ATP (from 164.1 to 150.2) and ADP (from 76.3 to 62.1) hydrolysis. The in vitro effects of the drugs on the synaptosome enzyme specific activity were also investigated. Phenylalnine alone or associated with p-chlorophenylalanine significantly reduced enzyme specific activity for both ATP (from 150.2 to 136.0) and ADP (from 70.5 to 59.3) nucleotides as substrates. Since ATP diphosphohrolase seems to play an important role in neurotransmission, these findings may be related to the neurological dysfunction characteristic of human phenylketonuria

Effect of postnatal methylmalonate administration on adult rat behavior

Methylmalonate (MMA) levels (2.0-2.5 mM) comparable to those of human methylmalonic acidemia were achieved in blood of young rats from the 5th to the 25th day of life by of life by injecting the drug subcutaneously twice a day with an interval of 8h. MMA doses ranged from 0.76 to 1.69 *mol/g body weight as a function of animal age. MMA-treated rats had normal body and brain weights. Behavioral studies using aversive and nonaversive tasks were performaed at 60 days of life. Motor activity was similar in MMA-treated and saline-treated controls. No differences in performance between these groups were identified in the shuttle-avoidance responses and in the inhibitory avoidance tasks. However, MMA-injected rats escaped footshock faster than the controls (1.22 ñ 0.11 vs 1.76 ñ 0.14 (mean ñ SEM) for 24 rats in each group (P<0.01)) suggesting that they may be hyperreactive to this stimulus. In the open field, a nonaversive behavior task, MMA-injected rats, in contrast to control rats, presented no habituation. Our results suggest that MMA by itself may impair central nervous system function, causing minor disabilities which result in specific learning deficiencies