Metabolism of hexacosatetraenoic acid (C26:4,n-6) in immature rat brain.
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ABSTRACT:
SUBMITTER: Robinson BS
PROVIDER: S-EPMC1131329 | biostudies-other | 1990 Apr
REPOSITORIES: biostudies-other
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Project description:Acetyl-L-carnitine (ALCAR) is an endogenous metabolic intermediate that facilitates the influx and efflux of acetyl groups across the mitochondrial inner membrane. Exogenously administered ALCAR has been used as a nutritional supplement and also as an experimental drug with reported neuroprotective properties and effects on brain metabolism. The aim of this study was to determine oxidative metabolism of ALCAR in the immature rat forebrain. Metabolism was studied in 21-22 day-old rat brain at 15, 60 and 120 min after an intraperitoneal injection of [2-(13)C]acetyl-L-carnitine. The amount, pattern, and fractional enrichment of (13)C-labeled metabolites were determined by ex vivo(13)C-NMR spectroscopy. Metabolism of the acetyl moiety from [2-(13)C]ALCAR via the tricarboxylic acid cycle led to incorporation of label into the C4, C3 and C2 positions of glutamate (GLU), glutamine (GLN) and GABA. Labeling patterns indicated that [2-(13)C]ALCAR was metabolized by both neurons and glia; however, the percent enrichment was higher in GLN and GABA than in GLU, demonstrating high metabolism in astrocytes and GABAergic neurons. Incorporation of label into the C3 position of alanine, both C3 and C2 positions of lactate, and the C1 and C5 positions of glutamate and glutamine demonstrated that [2-(13)C]ALCAR was actively metabolized via the pyruvate recycling pathway. The enrichment of metabolites with (13)C from metabolism of ALCAR was highest in alanine C3 (11%) and lactate C3 (10%), with considerable enrichment in GABA C4 (8%), GLN C3 (approximately 4%) and GLN C5 (5%). Overall, our (13)C-NMR studies reveal that the acetyl moiety of ALCAR is metabolized for energy in both astrocytes and neurons and the label incorporated into the neurotransmitters glutamate and GABA. Cycling ratios showed prolonged cycling of carbon from the acetyl moiety of ALCAR in the tricarboxylic acid cycle. Labeling of compounds formed from metabolism of [2-(13)C]ALCAR via the pyruvate recycling pathway was higher than values reported for other precursors and may reflect high activity of this pathway in the developing brain. This is, to our knowledge, the first study to determine the extent and pathways of ALCAR metabolism for energy and neurotransmitter biosynthesis in the brain.
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Project description:Dietary n-6 polyunsaturated fatty acid (PUFA) deprivation in rodents reduces brain arachidonic acid (20:4n-6) concentration and 20:4n-6-preferring cytosolic phospholipase A(2) (cPLA(2) -IVA) and cyclooxygenase (COX)-2 expression, while increasing brain docosahexaenoic acid (DHA, 22:6n-3) concentration and DHA-selective calcium-independent phospholipase A(2) (iPLA(2) )-VIA expression. We hypothesized that these changes are accompanied by up-regulated brain DHA metabolic rates. Using a fatty acid model, brain DHA concentrations and kinetics were measured in unanesthetized male rats fed, for 15 weeks post-weaning, an n-6 PUFA 'adequate' (31.4 wt% linoleic acid) or 'deficient' (2.7 wt% linoleic acid) diet, each lacking 20:4n-6 and DHA. [1-(14) C]DHA was infused intravenously, arterial blood was sampled, and the brain was microwaved at 5 min and analyzed. Rats fed the n-6 PUFA deficient compared with adequate diet had significantly reduced n-6 PUFA concentrations in brain phospholipids but increased eicosapentaenoic acid (EPA, 20:5n-3), docosapentaenoic acid n-3 (DPAn-3, 22:5n-3), and DHA (by 9.4%) concentrations, particularly in ethanolamine glycerophospholipid (EtnGpl). Incorporation rates of unesterified DHA from plasma, which represent DHA metabolic loss from brain, were increased 45% in brain phospholipids, as was DHA turnover. Increased DHA metabolism following dietary n-6 PUFA deprivation may increase brain concentrations of antiinflammatory DHA metabolites, which with a reduced brain n-6 PUFA content, likely promotes neuroprotection and alters neurotransmission.
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Project description:HIV-1 transgenic (Tg) rats, a model for human HIV-1 associated neurocognitive disorder (HAND), show upregulated markers of brain arachidonic acid (AA) metabolism with neuroinflammation after 7 months of age. Since lithium decreases AA metabolism in a rat lipopolysaccharide model of neuroinflammation, and may be useful in HAND, we hypothesized that lithium would dampen upregulated brain AA metabolism in HIV-1 Tg rats. Regional brain AA incorporation coefficients k* and rates J ( in ), markers of AA signaling and metabolism, were measured in 81 brain regions using quantitative autoradiography, after intravenous [1-(14) C]AA infusion in unanesthetized 10-month-old HIV-1 Tg and age-matched wildtype rats that had been fed a control or LiCl diet for 6 weeks. k* and J ( in ) for AA were significantly higher in HIV-1 Tg than wildtype rats fed the control diet. Lithium feeding reduced plasma unesterified AA concentration in both groups and J ( in ) in wildtype rats, and blocked increments in k* (19 of 54 regions) and J ( in ) (77 of 81 regions) in HIV-1 Tg rats. These in vivo neuroimaging data indicate that lithium treatment dampened upregulated brain AA metabolism in HIV-1 Tg rats. Lithium may improve cognitive dysfunction and be neuroprotective in HIV-1 patients with HAND through a comparable effect.
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Project description:Hypoxia and ischemia significantly affects perinatal brain development, even worse in preterm infants. However, the details of the mechanism leading to permanent brain damage after hypoxia-ischemia attack have not been fully elucidated. Proteomics could provide insight into the potential mechanism and help to promote the clinical treatment. In this study, quantitative analysis was performed 24 hours after hypoxia-ischemia using liquid-chromatography mass spectrometry coupled to label-free analysis. Compared to control, 193 proteins were present only in hypoxic-ischemic group. In addition, 34 proteins were more than 2 folds up-regulated and 14 proteins were more than 2 folds down-regulated in hypoxia-ischemia group. Gene Ontology database showed that the majority of differentially expressed proteins comprised mitochondrial proteins et al. Molecular function analysis revealed that the majority of proteins were involved in ion binding et al. Biological process analysis showed that the majority of proteins were involved in response to organic substance et al. STRING 9.0 software analysis were used to explore the complex interactions existed among the proteins. Western blot were used to verify the fold changes of some proteins-microtubule-associated protein 2 and microtubule-associated protein tau. This novel study performed a full-scale screening of the proteomics research in hypoxic-ischemic brain damage of immature rat.
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Project description:BackgroundPropofol is commonly used as sedative in newborns and children. Recent experimental studies led to contradictory results, revealing neurodegenerative or neuroprotective properties of propofol on the developing brain. We investigated neurodevelopmental short- and long-term effects of neonatal propofol treatment.Methods6-day-old Wistar rats (P6), randomised in two groups, received repeated intraperitoneal injections (0, 90, 180 min) of 30 mg/kg propofol or normal saline and sacrificed 6, 12 and 24 hrs following the first injection. Cortical and thalamic areas were analysed by Western blot and quantitative real-time PCR (qRT-PCR) for expression of apoptotic and neurotrophin-dependent signalling pathways. Long-term effects were assessed by Open-field and Novel-Object-Recognition at P30 and P120.ResultsWestern blot analyses revealed a transient increase of activated caspase-3 in cortical, and a reduction of active mitogen-activated protein kinases (ERK1/2, AKT) in cortical and thalamic areas. qRT-PCR analyses showed a down-regulation of neurotrophic factors (BDNF, NGF, NT-3) in cortical and thalamic regions. Minor impairment in locomotive activity was observed in propofol treated adolescent animals at P30. Memory or anxiety were not impaired at any time point.ConclusionExposing the neonatal rat brain to propofol induces acute neurotrophic imbalance and neuroapoptosis in a region- and time-specific manner and minor behavioural changes in adolescent animals.
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Project description:Injuries to the developing brain due to hypoxia-ischemia (HI) are common causes of neurological disabilities in preterm babies. HI, with oxygen deprivation to the brain or reduced cerebral blood perfusion due to birth asphyxia, often leads to severe brain damage and sequelae. Injury mechanisms include glutamate excitotoxicity, oxidative stress, blood-brain barrier dysfunction, and exacerbated inflammation. Nutritional intervention is emerging as a therapeutic alternative to prevent and rescue brain from HI injury. Lactoferrin (Lf) is an iron-binding protein present in saliva, tears, and breast milk, which has been shown to have antioxidant, anti-inflammatory and anti-apoptotic properties when administered to mothers as a dietary supplement during pregnancy and/or lactation in preclinical studies of developmental brain injuries. However, despite Lf's promising neuroprotective effects, there is no established dose. Here, we tested three different doses of dietary maternal Lf supplementation using the postnatal day 3 HI model and evaluated the acute neurochemical damage profile using 1H Magnetic Resonance Spectroscopy (MRS) and long-term microstructure alterations using advanced diffusion imaging (DTI/NODDI) allied to protein expression and histological analysis. Pregnant Wistar rats were fed either control diet or bovine Lf supplemented chow at 0.1, 1, or 10 g/kg/body weight concentration from the last day of pregnancy (embryonic day 21-E21) to weaning. At postnatal day 3 (P3), pups from both sexes had their right common carotid artery permanently occluded and were exposed to 6% oxygen for 30 min. Sham rats had the incision but neither surgery nor hypoxia episode. At P4, MRS was performed on a 9.4 T scanner to obtain the neurochemical profile in the cortex. At P4 and P25, histological analysis and protein expression were assessed in the cortex and hippocampus. Brain volumes and ex vivo microstructural analysis using DTI/NODDI parameters were performed at P25. Acute metabolic disturbance induced in cortical tissue by HIP3 was reversed with all three doses of Lf. However, data obtained from MRS show that Lf neuroprotective effects were modulated by the dose. Through western blotting analysis, we observed that HI pups supplemented with Lf at 0.1 and 1 g/kg were able to counteract glutamatergic excitotoxicity and prevent metabolic failure. When 10 g/kg was administered, we observed reduced brain volumes, increased astrogliosis, and hypomyelination, pointing to detrimental effects of high Lf dose. In conclusion, Lf supplementation attenuates, in a dose-dependent manner, the acute and long-term cerebral injury caused by HI. Lf reached its optimal effects at a dose of 1 g/kg, which pinpoints the need to better understand effects of Lf, the pathways involved and possible harmful effects. These new data reinforce our knowledge regarding neuroprotection in developmental brain injury using Lf through lactation and provide new insights into lactoferrin's neuroprotection capacities and limitation for immature brains.
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Project description:Glial cells are thought to supply energy for neurotransmission by increasing nonoxidative glycolysis; however, oxidative metabolism in glia may also contribute to increased brain activity. To study glial contribution to cerebral energy metabolism in the unanesthetized state, we measured neuronal and glial metabolic fluxes in the awake rat brain by using a double isotopic-labeling technique and a two-compartment mathematical model of neurotransmitter metabolism. Rats (n = 23) were infused simultaneously with 14C-bicarbonate and [1-13C]glucose for up to 1 hr. The 14C and 13C labeling of glutamate, glutamine, and aspartate was measured at five time points in tissue extracts using scintillation counting and 13C nuclear magnetic resonance of the chromatographically separated amino acids. The isotopic 13C enrichment of glutamate and glutamine was different, suggesting significant rates of glial metabolism compared with the glutamate-glutamine cycle. Modeling the 13C-labeling time courses alone and with 14C confirmed significant glial TCA cycle activity (V(PDH)((g)), approximately 0.5 micromol x gm(-1) x min(-1)) relative to the glutamate-glutamine cycle (V(NT)) (approximately 0.5-0.6 micromol x gm(-1) x min(-1)). The glial TCA cycle rate was approximately 30% of total TCA cycle activity. A high pyruvate carboxylase rate (V(PC), approximately 0.14-0.18 micromol x gm(-1) x min(-1)) contributed to the glial TCA cycle flux. This anaplerotic rate in the awake rat brain was severalfold higher than under deep pentobarbital anesthesia, measured previously in our laboratory using the same 13C-labeling technique. We postulate that the high rate of anaplerosis in awake brain is linked to brain activity by maintaining glial glutamine concentrations during increased neurotransmission.
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Project description:Aristolochic acids are natural nitro-compounds found globally in the plant genus Aristolochia that have been implicated in the severe illness in humans termed aristolochic acid nephropathy (AAN). Aristolochic acids undergo nitroreduction, among other metabolic reactions, and active intermediates arise that are carcinogenic. Previous experiments with rats showed that aristolochic acid I (AA-I), after oral administration or injection, is subjected to detoxication reactions to give aristolochic acid Ia, aristolactam Ia, aristolactam I, and their glucuronide and sulfate conjugates that can be found in urine and feces. Results obtained with whole rats do not clearly define the role of liver and kidney in such metabolic transformation. In this study, in order to determine the specific role of the kidney on the renal disposition of AA-I and to study the biotransformations suffered by AA-I in this organ, isolated kidneys of rats were perfused with AA-I. AA-I and metabolite concentrations were determined in perfusates and urine using HPLC procedures. The isolated perfused rat kidney model showed that AA-I distributes rapidly and extensively in kidney tissues by uptake from the peritubular capillaries and the tubules. It was also established that the kidney is able to metabolize AA-I into aristolochic acid Ia, aristolochic acid Ia O-sulfate, aristolactam Ia, aristolactam I, and aristolactam Ia O-glucuronide. Rapid demethylation and sulfation of AA-I in the kidney generate aristolochic acid Ia and its sulfate conjugate that are voided to the urine. Reduction reactions to give the aristolactam metabolites occur to a slower rate. Renal clearances showed that filtered AA-I is reabsorbed at the tubules, whereas the metabolites are secreted. The unconjugated metabolites produced in the renal tissues are transported to both urine and perfusate, whereas the conjugated metabolites are almost exclusively secreted to the urine.
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Project description:To investigate the effect of docosahexaenoic acid (DHA) without other highly unsaturated fatty acids (HUFA) on n-3 and n-6 essential fatty acid (EFA) metabolism and fatty acid composition in mammals, a stable isotope tracer technique was used in adult rats fed diets with or without 1.3% of algal DHA in a base diet containing 15% of linoleic acid and 3% of alpha-linolenic acid over 8 weeks. The rats were administered orally a mixed oil containing 48 mg/kg body weight of deuterated linoleic and alpha-linolenic acids and euthanized at 4, 8, 24, 96, 168, 240, 360 and 600 h after administration of the isotopes. Fatty acid compositions and the concentrations of deuterated precursors and their respective metabolites were determined in rat liver, plasma, heart and brain as a function of time. DHA, docosapentaenoic acid and eicosapentaenoic acid in the n-3 EFA family were significantly increased in all organs tested in the DHA-fed group, ranging from 5% to 200% greater in comparison with the control group. The accumulation of the metabolites, deuterated-DHA and deuterated-docosapentaenoic acid n-6 was greatly decreased by 1.5- to 2.5-fold in the dietary DHA group. In summary, feeding preformed DHA led to a marked increase in n-3 HUFA content of rat organs at the expense of n-6 HUFA and also prevented the accumulation of newly synthesized deuterated end products. This is the first study which has isolated the effects of DHA on the de novo metabolism on both the n-6 and n-3 EFA pathways.
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