DIET AND LIVER DETERMINE BRAIN ARACHIDONIC AND DOCOSAHEXAENOIC ACID METABOLISM Dietary requirements for maintaining brain and heart docosahexaenoic acid (DHA, 22:6n-3) homeostasis are not certain, because rates of liver DHA synthesis from circulating alpha-linolenic acid (alpha-LNA, 18:3n-3) have not been quantified. These rates can be estimated using intravenous radiotracer- or heavy isotope-labeled alpha-LNA infusion. In adult unanesthetized male rats, such infusion shows that liver synthesis-secretion rates of DHA from alpha-LNA markedly exceed brain and heart DHA synthesis rates and the brain DHA consumption rate, and that liver but not heart or brain synthesis is upregulated when dietary n-3 ply unsaturated fatty acid (PUFA) content is reduced. These rate differences reflect much higher expression of DHA-synthesizing enzymes in liver, and upregulation of liver but not heart or brain enzyme expression by n-3 PUFAs. The intravenous U-13Calpha-LNA infusion method could be extended for human studies (Rapoport et al., 2010). DIETARY N-6 PUFA DEPRIVATION DOWNREGULATES ARACHIDONATE BUT UPREGULATES DOCOSAHEXAENOATE METABOLIZING ENZYMES IN RAT BRAIN. For maintaining metabolic homeostasis, we hypothesized that dietary n-6 PUFA deprivation would decrease expression of arachidonic acid (AA 20:4n-6)-selective cytosolic phospholipase A2 (cPLA2) IVA and cyclooxygenase (COX)-2 in rat brain, while increasing expression of docosahexaenoic acid (DHA 22:6n-3)-selective calcium-independent iPLA2 VIA. Brain expression of these enzymes and of their transcription factors was quantified in male rats fed an n-6 PUFA adequate or deficient diet for 15 weeks post-weaning. The deficient compared with adequate diet increased mRNA, protein and activity of iPLA2 VIA and 15-lipoxygenase (LOX), but decreased cPLA2 IVA and COX-2 expression. The protein level of the iPLA2 transcription factor SREBP-1 was elevated, while protein levels were decreased for AP-2alpha and NF-kappaB p65, cPLA2 and COX-2 transcription factors, respectively. These changes would be expected to homeostatically dampen reductions in brain n-6 PUFA concentrations and metabolism, while increasing n-3 PUFA metabolism (Kim et al., 2011). REGULATION OF RAT BRAIN POLYUNSATURATED FATTY ACID (PUFA) METABOLISM DURING GRADED DIETARY N-3 PUFA DEPRIVATION. Knowing threshold changes in brain lipids and lipid enzymes during dietary n-3 polyunsaturated fatty acid deprivation can elucidate regulation processes of brain lipid metabolism. To determine thresholds, rats were fed for 15 weeks DHA-free diets having graded reductions of α-linolenic acid (alpha-LNA). Compared with control diet (4.6% alpha-LNA), plasma DHA fell significantly at <1.7% dietary alpha-LNA. Brain DHA remained unchanged down to 0.8% alpha-LNA, when plasma and brain docosapentaenoic acid (DPAn-6) were increased and DHA-selective iPLA2 and COX-1 activities were downregulated. Brain AA was unchanged by deprivation, but AA selective-cPLA2, sPLA2 and COX-2 activities were increased at or below 0.8% dietary α-LNA, likely in response to elevated brain DPAn-6. Homeostatic mechanisms appear to maintain a control brain DHA concentration down to 0.8% dietary alpha-LNA, despite reduced plasma DHA, to when DPAn-6 replaces DHA. At extreme deprivation, decreased brain iPLA2 and COX-1 activities likely reduce brain DHA loss (Kim et al., In Press). BRAIN PROTECTION BY RAPESEED OIL IN MAGNESIUM-DEFICIENT MICE. Diets given for 30 days having various mono-(MUFA) and poly-(PUFA) unsaturated fatty acid contents were evaluated for brain protection in magnesium-deficient mice: a commercial and three synthetic diets (n-6PUFA, n-3PUFA and MUFA-based chows enriched with 5% corn/sunflower oils 1:3, with 5% rapeseed oil and with 5% high oleic acid sunflower oil/sunflower oil 7:3, respectively). The n-3PUFA but not other diets protected magnesium-deficient mice against hyperactivity and electroshock- and NMDA-induced seizures. This diet also inhibited audiogenic seizures by 50%. Because, matched control MUFA diet failed to provide protection, alpha-linolenate (ALA) rather than reduced n-6 PUFA diet content likely caused n-3PUFA neuroprotection. Our data ALA support supplementation for neuroprotection in epilepsy (Pages et al., 2011). CONVERSION OF LINOLEIC TO ARACHIDONIC AND OTHER N-6 POLYUNSATURATED FATTY ACIDS IN UNANESTHETIZED RATS. Isotope feeding studies report a wide range of conversion fractions of shorter-chain to long-chain PUFAs, which limits assessing nutritional requirements and organ effects of arachidonic (AA, 20:4n-6) or docosahexaenoic (DHA, 22:6n-3) acid. Knowing conversion rates can help to assess daily nutritional requirements. Using our in vivo infusion method, we determined rates and coefficients of conversion of circulating unesterified linoleic acid (LA, 18:2n-6) to esterified AA and to other n-6 PUFAs in unanesthetized adult rats on a high DHA but AA-free diet. The conversion rate of LA to AA equaled 16 micromol/day, exceeding the brain AA consumption rate 27-fold. The heavy-isotope intravenous infusion model could be used to quantify e liver synthesis-secretion of AA from LA under different conditions in rodents or humans (Gao et al., 2010). LIVER CONVERSION OF DOCOSAHEXAENOIC AND ARACHIDONIC ACIDS FROM THEIR 18-CARBON PRECURSORS IN RATS ON A DHA-FREE ALPHA-LNA-CONTAINING N-3 PUFA ADEQUATE DIET. The long-chain polyunsaturated fatty acids (PUFAs), eicosapentaenoic acid (EPA, 20:5n-3), docosahexaenoic acid (DHA, 22:6n-3), and arachidonic acid (AA, 20:4n-6), are critical for health. These PUFAs can be synthesized in liver from their plant-derived precursors, alpha-linolenic acid (alpha-LNA, 18:3n-3) and linoleic acid (LA, 18:2n-6). Vegetarians and vegans may have a suboptimal n-3 PUFA status, and the extent of the conversion of alpha-LNA to EPA and DHA by the liver is debatable. We quantified liver conversion to DHA and other n-3 PUFAs of circulating alpha-LNA in rats fed a DHA-free alpha-LNA adequate diet, and compared results to conversion of LA to AA. U-13CLA or U-13Calpha-LNA was infused intravenously for 2 hours at a constant rate into these unanesthetized rats. Using our published equations to calculate kinetic parameters, the conversion coefficient k* of DHA from alpha-LNA was shown to be much higher than of AA from LA, showing elongation-desaturation selectivity for n-3 PUFA biosynthesis. The net daily secretion rate of DHA exceeded the re brain DHA consumption rate by 50-fold, suggesting that the liver can maintain brain DHA metabolism with an adequate dietary supply solely of alpha-LNA. This infusion method could be used to determine minimal daily requirements of EPA and DHA in humans (Gao et al., 2011). WHOLE-BODY SYNTHESIS OF DOCOSAHEXAENOIC ACID IN HUMAN SUBJECTS. We are writing a clinical protocol with members of the NIAAA to characterize whole-body synthesis-secretion rates of long-chain n-3 polyunsaturated fatty acids (PUFAs) using infusion of deuterated alpha-linolenic acid (d5-LNA) in humans, to evaluate effects of lowering the dietary nutrient linoleic acid) as a controlled variable on synthesis-secretion rates. This protocol extends our method for animal studies (Gao et al., 2010).
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