The blood‐brain barrier, formed by microvessel endothelial cells, is the restrictive barrier between the brain parenchyma and the circulating blood. Arachidonic acid (ARA; 5,8,11,14‐cis‐eicosatetraenoic acid) is a conditionally essential polyunsaturated fatty acid [20:4(n‐6)] and is a major constituent of brain lipids. The current study examined the transport processes for ARA in confluent monolayers of human brain microvessel endothelial cells (HBMEC). Addition of radioactive ARA to the apical compartment of HBMEC cultured on Transwell® inserts resulted in rapid incorporation of radioactivity into the basolateral medium. Knock down of fatty acid transport proteins did not alter ARA passage into the basolateral medium due to the rapid generation of prostaglandin E2 (PGE2), an eicosanoid known to facilitate opening of the blood‐brain barrier. Permeability following ARA or PGE2 exposure was confirmed by an increased movement of fluorescein‐labeled dextran from apical to basolateral medium. ARA‐mediated permeability was attenuated by specific cyclooxygenase‐2 inhibitors. EP3 and EP4 receptor antagonists attenuated the ARA mediated permeability of HBMEC. The results indicate that ARA increases permeability of HBMEC monolayers likely via increased production of PGE2 which acts upon EP3 and EP4 receptors to mediate permeability. These observations may explain the rapid influx of ARA into the brain previously observed upon plasma infusion with ARA.

The present study evaluates, both functionally and biochemically, brain tumor-induced alterations in brain capillary endothelial cells. Brain tumors were induced in Balb/c mice via intracranial injection of Lewis Lung carcinoma cells into the right hemisphere of the mouse brain using stereotaxic apparatus. Blood–brain barrier (BBB) permeability was assessed at various stages of tumor development, using both radiolabeled tracer permeability and magnetic resonance imaging with gadolinium diethylene-triamine-pentaacetate contrast enhancement (Gad-DTPA). The expression of the drug efflux transporter, P-glycoprotein (P-gp), in the BBB at various stages of tumor development was also evaluated by Western blot and immunohistochemistry. Median mouse survival following tumor cell injection was 17 days. The permeability of the BBB to 3 H-mannitol was similar in both brain hemispheres at 7 and 10 days post-injection. By day 15, there was a twofold increase in 3 H-mannitol permeability in the tumor bearing hemispheres compared to the non-tumor hemispheres. Examination of BBB permeability with Gad-DTPA contrast enhanced MRI indicated cerebral vascular permeability changes were confined to the tumor area. The permeability increase observed at the later stages of tumor development correlated with an increase in cerebral vascular volume suggesting angiogenesis within the tumor bearing hemisphere. Furthermore, the Gad-DPTA enhancement observed within the tumor area was significantly less than Gad-DPTA enhancement within the circumventricular organs not protected by the BBB. Expression of P-gp in both the tumor bearing and non-tumor bearing portions of the brain appeared similar at all time points examined. These studies suggest that although BBB integrity is altered within the tumor site at later stages of development, the BBB is still functional and limiting in terms of solute and drug permeability in and around the tumor.

The blood‐brain barrier, formed by microvessel endothelial cells, is the restrictive barrier between the brain parenchyma and the circulating blood. Arachidonic acid (AA) is a conditionally essential polyunsaturated fatty acid [20:4(n‐6)] and a major constituent of brain lipids. In this study transport of AA in confluent monolayers of human brain microvessel endothelial cells (HBMEC) was examined. Addition of [3H]AA to the apical compartment of HBMEC confluent monolayers cultured on Transwell® inserts resulted in rapid incorporation of [3H]AA into the basolateral medium. Knock down of FATP‐1 or CD36 did not alter [3H]AA movement into the basolateral medium and this was due to a rapid generation of prostaglandin E2 (PGE2), an eicosanoid known to facilitate opening of the blood‐brain barrier. Disruption of HBMEC monolayers following AA exposure was confirmed by an increased movement of fluorescein‐labeled dextran from the apical to basolateral medium. HBMECs expressed PGE2 synthase, cyclooxygenase‐1 and ‐2, PGE2 receptors, tight junction proteins and prostaglandin transporters. Specific cyclooxygenase‐2 inhibitors attenuated the AA‐mediated increase in membrane permeability. The results indicate that AA increases the permeability of HBMEC monolayers via increased production of PGE2. [Funded by NSERC Canada; GMH is a Canada Research Chair in Molecular Cardiolipin Metabolism]

Cardiolipin (CL) is a mitochondrial membrane phospholipid which plays a key role in apoptosis and supports mitochondrial respiratory chain complexes involved in the generation of ATP. In order to facilitate its role CL must be remodeled with appropriate fatty acids. We previously identified a human monolysocardiolipin acyltransferase activity which remodels CL via acylation of monolysocardiolipin (MLCL) to CL and was identical to the alpha subunit of trifunctional protein (αTFP) lacking the first 227 amino acids. Full length αTFP is an enzyme that plays a prominent role in mitochondrial β-oxidation, and in this study we assessed the role, if any, which this metabolic enzyme plays in the remodeling of CL. Purified human recombinant αTFP exhibited acyl-CoA acyltransferase activity in the acylation of MLCL to CL with linoleoyl-CoA, oleoyl-CoA and palmitoyl-CoA as substrates. Expression of αTFP increased radioactive linoleate or oleate or palmitate incorporation into CL in HeLa cells. Expression of αTFP in Barth Syndrome lymphoblasts, which exhibit reduced tetralinoleoyl-CL, elevated linoleoyl-CoA acylation of MLCL to CL in vitro, increased mitochondrial respiratory Complex proteins and increased linoleate-containing species of CL. Knock down of αTFP in Barth Syndrome lymphoblasts resulted in greater accumulation of MLCL than those with normal αTFP levels. The results clearly indicate that the human αTFP exhibits MLCL acyltransferase activity for the resynthesis of CL from MLCL and directly links an enzyme of mitochondrial β-oxidation to CL remodeling.

As the specific composition of lipids is essential for the maintenance of membrane integrity, enzyme function, ion channels, and membrane receptors, an alteration in lipid composition or metabolism may be one of the crucial changes occurring during skeletal and cardiac myopathies. Although the inheritance (autosomal dominant, autosomal recessive, and X-linked traits) and underlying/defining mutations causing these myopathies are known, the contribution of lipid homeostasis in the progression of these diseases needs to be established. The purpose of this review is to present the current knowledge relating to lipid changes in inherited skeletal muscle disorders, such as Duchenne/Becker muscular dystrophy, myotonic muscular dystrophy, limb-girdle myopathic dystrophies, desminopathies, rostrocaudal muscular dystrophy, and Dunnigan-type familial lipodystrophy. The lipid modifications in familial hypertrophic and dilated cardiomyopathies, as well as Barth syndrome and several other cardiac disorders associated with abnormal lipid storage, are discussed. Information on lipid alterations occurring in these myopathies will aid in the design of improved methods of screening and therapy in children and young adults with or without a family history of genetic diseases.

The blood-brain barrier (BBB), formed by the brain capillary endothelial cells, provides a protective barrier between the systemic blood and the extracellular environment of the CNS. Passage of fatty acids from the blood to the brain may occur either by diffusion or by proteins that facilitate their transport. Currently several protein families have been implicated in fatty acid transport. The focus of the present study was to identify the fatty acid transport proteins (FATPs) expressed in the brain microvessel endothelial cells and characterize their involvement in fatty acid transport across an in vitro BBB model. The major fatty acid transport proteins expressed in human brain microvessel endothelial cells (HBMEC), mouse capillaries and human grey matter were FATP-1, -4 and fatty acid binding protein 5 and fatty acid translocase/CD36. The passage of various radiolabeled fatty acids across confluent HBMEC monolayers was examined over a 30-min period in the presence of fatty acid free albumin in a 1 : 1 molar ratio. The apical to basolateral permeability of radiolabeled fatty acids was dependent upon both saturation and chain length of the fatty acid. Knockdown of various fatty acid transport proteins using siRNA significantly decreased radiolabeled fatty acid transport across the HBMEC monolayer. Our findings indicate that FATP-1 and FATP-4 are the predominant fatty acid transport proteins expressed in the BBB based on human and mouse expression studies. While transport studies in HBMEC monolayers support their involvement in fatty acid permeability, fatty acid translocase/CD36 also appears to play a prominent role in transport of fatty acids across HBMEC.

The blood-brain barrier formed by the brain capillary endothelial cells provides a protective barrier between the systemic blood and the extracellular environment of the central nervous system. Brain capillaries are a continuous layer of endothelial cells with highly developed tight junctional complexes and a lack of fenestrations. The presence of these tight junctions in the cerebral microvessel endothelial cells aids in the restriction of movement of molecules and solutes into the brain. Fatty acids are important components of biological membranes, are precursors for the biosynthesis of phospholipids and sphingolipids and are utilized for mitochondrial β-oxidation. The brain is capable of synthesizing only a few fatty acids. Hence, most fatty acids must enter into the brain from the blood. Here we review current mechanisms of transport of free fatty acids into cells and describe how free fatty acids move from the blood into the brain. We discuss both diffusional as well as protein-mediated movement of fatty acids across biological membranes.

Barth Syndrome (BTHS) is a rare X‐linked genetic disorder caused by a mutation in the Tafazzin gene (taz), an enzyme involved in the remodelling pathway of cardiolipin (CL). Mutation in taz results in a biochemical deficiency of CL and accumulation of monolysocardiolipin (MLCL). We investigated the contribution of another enzyme, monolysocardiolipin acyltransferase‐1 (MLCL AT‐1), to the remodelling pathway of CL in human lymphoblasts. MLCL AT‐1 or taz or both were knocked down using RNAi in normal or BTHS lymphoblasts and MLCL AT‐1 enzyme activity examined. MLCL AT‐1 enzyme activity was reduced approximately 50% (p<0.05) when MLCL AT‐1 was knocked down in normal human lymphoblasts and unaltered when taz was knocked down. Knock down of both MLCL AT‐1 and taz simultaneously did not result in a further reduction in MLCL AT‐1 activity compared to knock down of MLCL AT‐1 alone. Expression of MLCL AT‐1 in BTHS lymphoblasts with different mutations in taz elevated CL synthesis and mass over 2‐fold (p<0.05) in these cells compared to controls. These studies indicate that MLCL AT‐1 may act independently of taz. This work was supported by grants from the Manitoba Health Research Council and the Barth Syndrome Foundation (USA & Canada).

ATP binding cassette A1 (ABCA1) transports cholesterol, phospholipids and lipophilic molecules to and across cellular membranes. We examined if ABCA1 expression altered cellular de novo glycerolipid biosynthesis in growing Baby hamster kidney (BHK) cells. Mock BHK cells or cells expressing a mifepristone-inducible ABCA1 (ABCA1) were incubated plus or minus mifepristone and then with [ 3 H]serine or [ 3 H]inositol or [ 3 H]ethanolamine or [ methyl - 3 H]choline or [ 3 H]glycerol or [ 14 C]oleate and radioactivity incorporated into glycerolipids determined. Mifepristone did not affect [1,3- 3 H]glycerol or [ 14 C]oleate or [ 3 H]ethanolamine or [ methyl - 3 H]choline uptake in BHK cells. In contrast, [ 3 H]glycerol and [ 14 C]oleate incorporated into phosphatidylserine (PtdSer) were elevated 2.4-fold ( p < 0.05) and 54% ( p < 0.05), respectively, upon ABCA1 induction confirming increased PtdSer biosynthesis from these precursors. However, mifepristone inhibited [ 3 H]serine uptake and incorporation into PtdSer indicating that PtdSer synthesis from serine in BHK cells is dependent on serine uptake. Mifepristone stimulated [ 3 H]inositol uptake in mock and ABCA1 cells but not its incorporation into phosphatidylinositol indicating that its synthesis from inositol is independent of inositol uptake in BHK cells. [ 3 H]glycerol and [ 14 C]oleate incorporated into triacylglycerol were reduced and into diacylglycerol elevated only in mifepristone-induced ABCA1 expressing cells due to a decrease in diacylglycerol acyltransferase-1 (DGAT-1) activity. The presence of trichostatin A, a class I and II histone deacetylase inhibitor, reversed the ABCA1-mediated reduction in DGAT-1 activity but did not affect DGAT-1 mRNA expression. Thus, mifepristone has diverse effects on de novo glycerolipid synthesis. We suggest that caution should be exercised when using mifepristone-inducible systems for studies of glycerolipid metabolism in cells expressing glucocorticoid responsive receptors.

Endothelial cells lining the blood capillaries of the Blood Brain Barrier (BBB) are tightly packed thus regulating the transport of substances from the blood into the brain, including fatty acids. Fatty acids are essential for both the developing and adult mammalian brain. Since most fatty acids in the brain enter from the blood, we examined the mechanism of their transport across Human Brain Microvessel Endothelial Cells (HBMECs). HBMECs were plated onto transwell plate inserts and then incubated for up to 4 h with [1‐14C]oleate in the apical medium. Radioactivity in the basolateral medium was temporally examined. There was a near linear increase in [1‐14C]oleate incorporation into the basolateral media in the presence of albumin indicating a protein acceptor is required for oleate transport. The presence of phloretin, a non‐specific fatty acid uptake inhibitor, significantly decreased [1‐14C]oleate transport into the basolateral medium. In addition, siRNA knockdown of fatty acid transport protein‐1 (FATP‐1) or fatty acid translocase (FAT/CD36) significantly decreased [1‐14C]oleate incorporation into the basolateral media. In summary, transport of oleate across HBMECs is, in part, a transcellular process. Oleate transport across HBMECs is mediated by both diffusion and carrier mediated processes. (Supported by the Manitoba Health Research Council and the Canadian Institutes of Health Research)

There are many anatomical variations in and around the carpal tunnel that affect the nerves, tendons and arteries in this area. Awareness of these variations is important both during the clinical examination and during carpal tunnel release. The purpose of the present review is to highlight recognized anatomical variations within the carpal tunnel including variation in nerve anatomy, tendon anatomical variants, vascular anatomical variations and muscle anatomical variations.

The blood–brain barrier formed by the brain capillary endothelial cells provides a protective barrier between the systemic blood and the extracellular environment of the CNS. As most fatty acids in the brain enter from the blood, we examined the mechanism of oleate (C18:1) transport across primary human brain microvessel endothelial cells (HBMEC). The permeability of [1‐ 14 C]oleate was determined using confluent cells grown on Transwell® inserts in both the absence or presence of bovine serum albumin in the basolateral media, and following inhibition of various fatty acid transporters. The passage of [1‐ 14 C]oleate across confluent HBMEC monolayers was significantly enhanced when fatty acid free albumin was present in the basolateral media. The presence of the non‐specific fatty acid uptake inhibitor phloretin significantly decreased [1‐ 14 C]oleate uptake by HBMEC and the subsequent release of [1‐ 14 C]oleate into the basolateral medium. Knockdown of fatty acid transport protein‐1 or fatty acid translocase/CD36 significantly decreased [1‐ 14 C]oleate transport across the HBMEC monolayer from either apical as well as basolateral sides. The findings indicate that a fatty acid acceptor is a requirement for oleate transport across HBMEC monolayers. In addition, transport of oleate across HBMEC is, in part, a transcellular process mediated by fatty acid transport proteins.

Cardiolipin (CL) is a major phospholipid involved in energy metabolism mammalian mitochondria and fatty acid transport protein-1 (FATP-1) is a fatty acid transport protein that may regulate the intracellular level of fatty acyl-Coenzyme A's. Since fatty acids are required for oxidative phosphorylation via mitochondrial oxidation, we examined the effect of altering FATP-1 levels on CL biosynthesis. HEK-293 mock- and FATP-1 siRNA transfected cells or mock and FATP-1 expressing cells were incubated for 24 h with 0.1 mM oleic acid bound to albumin (1:1 molar ratio) then incubated for 24 h with 0.1 mM [1,3-(3)H]glycerol and radioactivity incorporated into CL determined. FATP-1 siRNA transfected cells exhibited reduced FATP-1 mRNA and increased incorporation of [1,3-(3)H]glycerol into CL (2-fold, p<0.05) compared to controls indicating elevation in de novo CL biosynthesis. The reason for this was an increase in [1,3-(3)H]glycerol uptake and increase in activity and mRNA expression of the CL biosynthetic enzymes. In contrast, expression of FATP-1 resulted a reduction in incorporation of [1,3-(3)H]glycerol into CL (65%, p<0.05) indicating reduced CL synthesis. [1,3-(3)H]Glycerol uptake was unaltered whereas activity of cytidine-5'-diphosphate-1,2-diacyl-sn-glycerol synthetase (CDS) and CDS-2 mRNA expression were reduced in FATP-1 expressing cells compared to control. In addition, in vitro CDS activity was reduced by exogenous addition of oleoyl-Coenzyme A. The data indicate that CL de novo biosynthesis may be regulated by FATP-1 through CDS-2 expression in HEK 293 cells.

The Blood Brain Barrier (BBB) is the physical protective barrier between the cerebral blood vessels and parts of the central nervous system. Endothelial cells lining the blood capillaries are packed tightly thus regulating the transport of substances from the blood into the brain, including fatty acids. Fatty acids are essential for both the developing and adult mammalian brain. Since most of the fatty acids in the brain enter from the blood, we examined the mechanism of transport across the BBB. Rat Brain Microvessel Endothelial cells (RBMEC) were plated onto 12 transwell plate inserts for these permeability studies. RBMECs were then incubated for up to 4 h with 0.1 μM [14C]oleic acid in the apical medium and the radioactivity was temporally determined in the basolateral medium in the absence or presence of bovine serum albumin. There was a near linear increase in [14C]oleic acid incorporation into the basolateral medium with time in the presence of either regular bovine serum albumin or fatty acid free bovine serum albumin. In contrast, no [14C]oleic acid incorporation into the basolateral media was observed in the absence of albumin, indicating that the permeability layer was intact and that a fatty acid acceptor is a requirement for fatty acid transport across the permeability layer of these cells. (Supported by grants from the Manitoba Health Research Council and the Canada Research Chair program)