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BRAIN UPTAKE AND UTILIZATION OF FATTY ACIDS: 

Applications to Peroxisomal Biogenesis Diseases

(An International Workshop)

March 2-4, 2000, Holiday Inn Bethesda, Bethesda, Maryland

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Brain Uptake, Transport and Metabolism of PUFA: in vivo and in vitro studies

(Friday, March 3, 2000, Afternoon Session Summary)

Co-chairs: Steven A. Moore and Arthur A. Spector

Discussion moderator: Howard W. Sprecher    
Prepared by: Steven A. Moore and Arthur A. Spector

Arachidonic acid (20:4n-6, AA) and docosatetraenoic acid (22:4n-6, DTA) are the most abundant n-6 polyunsaturated fatty acids (PUFA) present in the human brain, and docosahexaenoic acid (22:6n-3, DHA) is the most abundant n-3 PUFA. These PUFA are necessary for normal brain function, but humans and animals cannot synthesize n-6 or n-3 PUFA de novo. They are derived from plants and microorganisms that enter the food chain and are then supplied by the circulation to all tissues, including the brain. To fully understand PUFA metabolism in the brain, it is essential to know what PUFA are available in the plasma and whether they are present as FFA or in the triglycerides (TG), phospholipids (PL) and cholesterol esters (CE) of lipoproteins. 

Dr. Arthur A. Spector discussed the sources of n-6 and n-3 PUFA in human plasma. When humans consume a Western diet, the plasma contains large amounts of n-6 PUFA. Linoleic acid (18:2n-6, LA), the precursor of AA and DTA, comprises about 15% of the plasma FFA. LA also is the most abundant n-6 PUFA in the PL, TG and CE contained in plasma lipoproteins. Although substantial amounts of AA are present in PL and CE, AA accounts for only 1-2% of the plasma FFA and TG, the fractions that ordinarily supply fatty acid to the tissues. Very little DTA is present in human plasma. Thus, it appears that LA probably is a major source of n-6 PUFA for the brain. However, enough AA may be present to satisfy the needs of the brain, especially if lipoprotein lipids can be utilized.

The amount of PUFA in the plasma depends on the dietary intake. Because the level of n-6 PUFA usually is high, the increment produced by increasing the dietary intake is relatively small. By contrast, n-3 PUFA usually account for only about 2-4% of the plasma fatty acids in individuals consuming a Western diet, and increases in dietary intake have a profound effect on the plasma n-3 PUFA content because the basal levels are so low. This suggests the possibility that the n-3 PUFA supply to the brain may be episodic and may depend heavily on the recent dietary intake.

The most abundant n-3 PUFA in the plasma FFA is a-linolenic acid (18:3n-3, ALA). Thus, if the brain obtains fatty acid primarily in the form of FFA, ALA probably is its major source of n-3 PUFA.

Most of the DHA in plasma is present in PL, a lipoprotein lipid that does not ordinarily deliver large amounts of fatty acid to the tissues. However, if the brain is able to utilize lipoprotein lipids, PL would be a ready source of DHA.

Dr. Michel Lagarde presented data indicating how this may take place. His experimental results demonstrate that lysophosphatidylcholine (lysoPC) containing DHA can cross the blood-brain barrier and be utilized by brain-derived cells. Although the heart and red blood cells utilize unesterified DHA more readily, brain-derived cells take up much more DHA in the form of lysoPC. LysoPC containing DHA also can be transported through a monolayer of cultured brain microvessel endothelial cells grown on micropore filters, and brain-derived cells present in the basolateral compartment of the culture chamber incorporate the transported lysoPC. Conversion to lysoPC by hydrolysis may explain how plasma DHA contained in lipoprotein PL becomes available for utilization by the brain.

In vivo studies of brain PUFA uptake and utilization

Although sources of PUFA in the plasma range from FFA to the TG, PL and CE in the plasma lipoproteins, the uptake and utilization of PUFA by brain from these sources are incompletely understood.  Models of brain fatty acid uptake have utilized brain perfusion with radiolabeled fatty acids, feeding studies with U-¹³C- or fully deuterated-fatty acids, or the lysoPC studies presented by Dr. Lagarde that are summarized above.

Dr. Quentin Smith presented data in adult rats, (also discussed extensively by Dr. Stanley I. Rapoport  next session's summary) that indicate rapid and complete uptake of palmitate and PUFAs (such as AA and DHA) from the vascular space by the brain.  When introduced into the blood, tracer quantities of radiolabeled FFA quickly reach equilibrium with brain.  In short term experiments these fatty acids are incorporated intact into a variety of glycerolipids in the brain.

Feeding studies presented by Dr. Stephen C. Cunnane, on the other hand, suggest that LA and ALA are extensively catabolized in the suckling rat model and that their beta-oxidation products are reutilized by brain for local synthesis of saturated fatty acids and cholesterol.

Dr. John Edmond presented data on brain uptake of perdeuterated palmitate or deuterated (D7) cholesterol fed to postnatal rats in an artificial rearing system. Since neither were found intact in the brain, Dr. Edmond concluded that the brain synthesizes all of these compounds that it requires and is independent of outside sources of saturated fatty acids and cholesterol .  Perdeuterated LA, on the other hand, is found intact in brain following feeding in this rat pup rearing system.  This data led him to suggest that selective mechanisms are likely to be in place for the uptake and/or retention of n-3 and n-6 PUFA in his animal model system.

While on the surface these perfusion and feeding studies appear to be contradictory, the time frame and methods by which each was carried out do not lend themselves readily to direct comparison.  Thus, FFA may be taken up by the blood-brain barrier and rapidly incorporated into brain glycerolipids, then subsequently reutilized through a combination of glycerolipid remodeling, beta oxidation, and fatty acid synthesis.

Local synthesis of n-3 and n-6 PUFA by brain and retina-derived cells in vitro

The ability of brain to locally synthesize longer chain n-3 and n-6 from 18-carbon precursors has been strongly suggested by cell culture studies utilizing endothelium derived from cerebral or retinal microvessels and astrocytes derived from the brain. 

Dr. Robert E. Anderson and Dr. Steven A. Moore reviewed published data indicating that the microvascular endothelial cells readily form AA from LA and eicosapentaenoic (EPA) and docosapentaenoic (DPA) acids from ALA.  While endothelia can also synthesize DHA from these shorter chain precursors, they have a limited capacity to do so.  Astrocytes much more readily convert n-3 precursor PUFA to DHA.  Neuronal cultures, on the other hand, can only carry out limited elongation of n-3 or n-6 precursor PUFA and do not synthesize DHA.  Neurons, therefore, appear dependent on an external source of DHA.

The synthetic pathway in brain- and retina-derived cells follows the elongation/desaturation scheme put forward by Dr. Howard Sprecher and co-workers.  This pathway for n-3 PUFA is:  18:3 18:4 20:4 20:5   22:5 24:5 24:6 22:6.  In a series of studies performed with [3-¹⁴C]-labeled 22:5, 24:5, and 24:6, Dr. Moore obtained additional evidence for this synthetic pathway in cerebral endothelial cells and astrocytes.  Astrocytes readily convert all these fatty acids to DHA, while cerebral endothelial cells produce substantial amounts of DHA only when incubated with the 24-carbon precursors.  The endothelial cell data is particularly instructive.  First, the observation that DHA is readily formed from 24:5 but not 22:5 brings further doubt to the existence of a 4-desaturase enzyme that would be necessary to directly convert 22:5 to DHA.  Second, the relative inability of endothelial cells to elongate 22:5 compared to their ready elongation of 18- and 20-carbon n-3 PUFA suggests that there may be two different elongation enzymes.

Dr. Robert E. Anderson reported new  studies that investigated the role of de novo synthesis in the enrichment of DHA in molecular species of retinal glycerolipids. Species differences (between frog and rat) were observed in the synthesis of glycerolipid molecular species containing two PUFA (one which was DHA) in larger amounts than predictable by their steady state mass levels. Di-DHA glycerolipid species were also found at relatively high levels. These findings indicate that besides de novo synthesis, remodeling and reutilization of products of fatty acid beta-oxidation must play a role in maintaining higher levels of DHA in rod outer segment membranes than in whole retina or retinal pigment epithelium.

Thus, studies in both brain- and retina-derived cells indicate that limited beta-oxidation (retroconversion) of n-3 PUFA occurs.

Release and transfer of PUFA by brain- and retina-derived cells in vitro

Cell culture studies using microvascular endothelium, astrocytes, retinal pigment epithelial cells, and neurons have also suggested mechanisms for the accretion and retention of DHA by the CNS and for the transfer of DHA to neurons or photoreceptor cells.

Dr. Steven Moore reported that endothelial cells grown on filter chambers are able to enrich the basolateral compartment with both PUFA and prostaglandins suggesting there is polar release, relative absence of basolateral uptake, or both.  Either mechanism would contribute to the brain’s accretion and retention of PUFA.  Co-cultures of endothelium and astrocytes cooperate to synthesize more 20- and 22-carbon PUFA from precursors than either cell type grown alone. Neurons co-cultured with astrocytes also acquire substantially more DHA and other n-3 PUFA from 18- and 20-carbon precursors than neurons cultured alone.  Thus, astrocytes and endothelium may carry out a supportive role for neurons by taking up n-3 PUFA precursors from the circulation and providing DHA for neurons.

Although release of these PUFAs from cultured endothelium or astrocytes occurs predominantly as FFA, small amounts of phospholipid have been detected in the medium of astrocyte cultures.  Since astrocytes are capable of apolipoprotein synthesis, lipoproteins may mediate some portion of the intercellular transfer of PUFA in the brain.

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Brain Uptake, Transport and Metabolism of PUFA: in vivo and in vitro studies

(Friday, March 3, 2000, Afternoon Session Recommendations for Future Research)

Plasma sources of PUFA

Although data from serum lipid composition suggest that a potential major source of brain PUFA is lipoprotein-associated complex lipids, few studies have as yet addressed specific details of PUFA transport and uptake by this mechanism.  Numerous lipoprotein classes and glycerolipid pools may be involved.  Thus, investigations directed at the identification and functional characterization of PUFA-selective lipoprotein classes and PUFA-enriched glycerolipid pools would be enlightening.  It may be possible to utilize stable PUFA isotopes (¹³C or perdeuterated) to identify and follow these lipoproteins/glycerolipids in feeding studies.

Role of barrier cells and astrocytes in the uptake and processing of PUFA or lipoprotein-associated PUFA

Studies that parallel the focus on serum lipid composition (particularly lipoproteins) should also be directed at cells carrying out barrier functions between blood and neurons.  Foremost among these barrier cells are cerebral endothelium of the blood-brain barrier, choroid plexus epithelium that produce the bulk of cerebrospinal fluid, and astrocytes that closely associate with both cerebral blood vessels and neurons.  The distribution, molecular nature, and function of lipoprotein lipase, lipoprotein receptors, and intracellular mechanisms for lipoprotein processing or trafficking in these cells are some of the major areas that require attention.

Once PUFAs are free of their carrier proteins and/or glycerolipids, a different group of binding and transport proteins are likely to be important in directing the flow of PUFA across barrier cells or within the brain.  In this regard molecular and cell biological approaches should be aimed at understanding how fatty acid binding proteins (FABP) and fatty acid transport proteins (FATP) are distributed and how they function.  Attention should also be directed at the specificity of enzymes that esterify PUFA, since the selectivity observed in membrane lipids may be regulated significantly at the esterification level.

Finally, additional carrier proteins or glycerolipids are likely to be involved in the intercellular distribution of PUFA within the brain parenchyma.  For example, astrocytes are known to be a source of apolipoprotein synthesis in the brain, but little is known about the function of brain apolipoproteins in the intercellular exchange of PUFA.  The fact that the E4 allele of apolipoprotein E is a major risk factor for Alzheimer’s disease provides a significant allure to this area of study.

Local synthesis of PUFA

Although there is strong evidence for local synthesis of long-chain PUFA by brain-derived cells, recent advances in the  molecular and cell biology of desaturase enzymes and peroxisomal assembly have yet to be applied systematically to the brain.  The application of this new knowledge, particularly using genetic engineering approaches, should provide the clearest evidence yet on the role of local synthesis in the accretion of PUFA by brain.  It might also provide a basis for developing molecular approaches to therapy in neurological disorders where long-chain PUFAs are deficient.

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