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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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Fatty Acid Uptake by and Transport in the Brain

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

Co-chairs: Jacques H. Veerkamp and James A. Hamilton

Discussion Moderator: Alexander Leaf

Prepared by: James A. Hamilton and Paul A. Watkins

Fatty acids (FA) must enter the brain to remodel membrane phospholipids and even provide energy for certain cells (e.g., endothelial cells). Knowledge of the barriers between FA in the blood and brain cells is fundamental for understanding FA uptake in the brain. Lester Drewes presented an overview of the brain microvascular endothelium and the blood-brain barrier (BBB). The major barrier between the capillaries and the brain is provided by endothelial cells, which are similar to other endothelial cells except for tight junctions between adjacent cells. This morphology assures that molecules entering the brain must pass through the plasma membrane on each side of the endothelial cell. There is known micro-heterogeneity of transporters, both with respect to adjacent cells and to the sides of the cell. While transporters for ions and some small molecules have been characterized, potential transporters for FA are poorly characterized.

The model of free diffusion of FA in membranes as presented by Jim Hamilton is based on biophysical properties of FA in protein-free phospholipid model membranes. Key tenets are that FA partition very favorably into phospholipids, they equilibrate across the phospholipid bilayer spontaneously via flip-flop of the un-ionized FA, and they spontaneously desorb from a bilayer with rates highly dependent on the FA chain length and unsaturation. In new studies of adipocytes it was shown that binding of oleic acid occurs very rapidly, as measured by a fluorescent probe in the extracellular medium, and that flip-flop follows immediately and rapidly, as measured simultaneously by an intracellular pH fluorophore. The implication of the studies of model systems and certain cells to data is that FA can move through membranes without catalysis by a protein transporter. Although FA uptake into cells of the nervous system have not been studied to date by biophysical methods as above, the diffusion model may also be applicable to the brain, as brought out in the discussion. Rapoport and co-workers have demonstrated rapid uptake of FA (5% of the total FA within 1-2 s) into rodent brains in a manner quantitatively explicable by free diffusion.

Uptake of FA into cells can be influenced greatly by metabolism, which can also complicate interpretations of the mechanisms of uptake, as illustrated by Henry Pownall. As measured by a conventional radioisotope assay, FA uptake was enhanced by expression of a protein, much in the same fashion as found for putative membrane FA transporters. By analogy, this protein was initially described as an unknown FA transporter. However, the protein was identified as an intracellular enzyme in the pathway of triglyceride synthesis, AGAT (1-acylglycerol-3-phosphate acyltransferase), showing that an enzyme can mimic some of the properties of a "FA transporter". Pownall’s evaluation of the putative membrane transporters such as CD36 and FATP is that they might enhance the diffusion of FA through the phospholipid bilayer but do not provide an independent mechanism of FA transport through the membrane.

Jan Glatz and Nada Amburad presented evidence in support of the hypothesis that FAT/CD36 enhances FA uptake from albumin complexes. The studies have focused mainly on cells (adipocytes), including those from genetically engineered mice and have shown evidence, for example, that at low FA/albumin ratios the CD36 null mouse adipocytes exhibit lower uptake of radio-labeled FA than wild type. One interesting feature of the CD36 protein is that a region of the extracellular domain has significant homology to intracellular FABP. Whether this domain binds FA and participates in membrane transport of FA, perhaps by enhancing the adsorption of FA to the plasma membrane, remains to be determined. To remove the metabolic component of FA uptake, Glatz focused on giant vesicles made from muscle cells and encapsulating intracellular fatty acid binding protein (FABP), but not organelles. Many of the features attributed to protein-mediated uptake in cells (e.g., inhibition by trypsin and chemical agents) were observed in the vesicles. In contracting skeletal muscle, CD36 appears to be translocated from intracellular stores to the plasma membrane.

Inside a cell, FA can bind to FABP, small, soluble proteins that have a high specificity for FA. As discussed by Jacques Veerkamp, nervous tissue contains 4 types of FABP: myelin FABP in peripheral nerves, brain FABP and epidermal FABP in glial cells and neurons, and heart FABP in adult brain. The FABP were named according to the original tissue from which they were isolated, and epidermal and heart forms have a very wide tissue distribution. All types found in nervous tissue have similar binding specificity and affinity for various FA, including PUFA, suggesting that binding of PUFA to FABP is not a mechanism for preferential uptake or enrichment.

In spite of the abundance of FABP, FA bind mainly to the membranes under normal conditions. There is thus a large reserve binding capacity of FABP. The functions of FABP remain to be established. One possible function in brain cells is the protection of cell membranes against high concentrations of FA generated during ischemia.

The mechanisms of transfer of FA between membranes and FABP are under active investigation. In simple model membranes, FA can desorb spontaneously from the membrane. Judy Storch studied the transfer of a fluorescent long chain FA from brain FABP to phospholipid bilayers. Previous studies form the same group have concluded that certain types of FABP transfer the FA spontaneously while others require a collision mechanism for rapid transfer. The new studies of brain FABP showed an enhancement of transfer in the presence of anionic phospholipids and suggested a collision mechanism.

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Fatty Acid Uptake by and Transport in the Brain

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

1. Except for the studies of uptake of radiolabeled FA into brains of rodents, most studies of FA uptake have been carried out in non-neural cells, notably the adipocyte. Conventional radioisotope uptake studies should be extended to brain endothelial cells and other brain and neural tissue cells. A wide range of FA need to be studied with controls for metabolism to determine whether the membrane transport is different for different FA species. The possible influences of putative transporters present in brain cells, such as FATP, on FA uptake could be evaluated by analogy to studies with other cell types. The fluorescent approach measuring intracellular pH can be extended to cells of the nervous system, with appropriate adaptations of the instrumental methods to detect fluorescence in cell monolayers. This method has an important advantage that all types of FA can be tested without isotopic labeling or addition of a fluorescent probe to the FA molecule. Investigations of fatty acid transport and expression levels of putative transporter proteins in different brain regions (macroheterogeneity) and different cell types within these regions (microheterogeneity) should also be carried out.
   
2. Animal models for over-expressing and knocking out putative membrane FA transporters and FABP should be applied to cells of the nervous system. With the approaches of points 1 and 2, the contributions of diffusion and protein-mediated transport can be differentiated and quantified.
   
3. The first appearance of tight junctions in the cerebrovascular capillary endothelium during development and the functions of these structures, particularly with respect to fatty acid transport, are important areas that require additional investigation.
   
4. Collaboration between basic and applied scientists is essential for these new studies which require expertise in multiple disciplines, including biophysics, physiology, and molecular biology. Enhanced communication between different disciplines interested in brain lipids must be encouraged.

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