Journal of Molecular Neuroscience Copyright © 2001 Humana Press Inc. All rights of any nature whatsoever reserved. ISSN0895-8696/01/16:323–331/$12.25
Abstracts of Poster Presentations International Workshop on Brain Uptake and Utilization of Fatty Acids: Applications to Peroxisomal Biogenesis Diseases, Bethesda, MD, March 2–4, 2000 EFFECTS OF DIETARY OMEGA-3 FATTY ACID ON VOLUMETRIC CHANGES IN THE HIPPOCAMPUS OF THE RAT BRAIN Aneeq Ahmad, Rebecca Sheaff Greiner, Toru Moriguchi and Norman Salem Jr., Laboratory of Membrane Biophysics and Biochemistry, National Institute on Alcohol Abuse and Alcoholism, National Institutes of Health, Rockville, MD Omega-3 fatty acids, such as docosahexaenoate (DHA; 22⬊6n3), are major components of phospholipids in the brain. They play an important role in the structural makeup and physiological functioning of nerve cells. Omega-3 fatty acids are localized in neuronal membranes and are accumulated during brain development by placental nutrition, mother’s milk, and dietary sources. Primate studies have shown that deficiency of these fatty acids results in a variety of neuronal dysfunctions that lead to cognitive and behavioral deficits in these animals. Recent studies have shown similar deficits in rats especially associated with their working memory in the Morris water maze as well as olfactory-cued learning tasks. It is known that problems with working memory are related to deficits involving the hippocampus. Therefore, morphological analyses were carried out on the hippocampus of these animals in order to assess the effects of omega3 deficient diets. Female rats were bred on two deficient (low n-3, low n-3+hi n-6), and two adequate (adequate n-3, adequate n-3+DHA) fatty acid diets for three generations to maturity. At 110 d of age, the animals were perfused and unbiased stereological analyses were carried out to assess morphological changes in the volume, density, total number, and the cell body size of neurons in the hippocampus of all dietary groups. Fatty acid analysis of the hippocampus showed that deficient diets lead to high n-6/n-3 ratio and adequate diets to low n-6/n-3 ratio, respectively. There was no difference in the total volume, density, and total number of neurons in the hippocampus of these animals; however, the size of CA1 pyramidal neurons was larger in animals on adequate n-3+DHA diet compared to the low n-3 diet. Infants in North America receive infant formulas that were devoid of DHA, whereas human milk always contains DHA. These data suggest that such formula diets may result in loss of neuronal volume in the hippocampus (and perhaps in other locations in the brain).
DOCOSAHEXAENOIC ACID INHIBITS STAUROSPORINEINDUCED APOPTOSIS IN NEURONAL CELLS Mohammed Akbar and Hee-Yong Kim, Section of Mass Spectrometry, Laboratory of Biochemistry and Biophysics, National Institute on Alcohol Abuse and Alcoholism, National Institutes of Health, 12501 Washington Ave., Rockville, MD 20852 It has been demonstrated that long-chain polyunsaturated fatty acids (PUFA) mediate many physiological functions. Among these, docosahexaenoic acid (DHA) has been shown to be essential for development and maintenance of neuronal functions. Here we demonstrate the role of DHA in apoptosis induced by protein kinase C inhibitor Staurosporine (SP) and Pl3-kinase inhibitor Wortmanin (WT). Enrichment of Neuro 2Acells with DHAinhibited caspase-3 activity induced by SP in a time-dependent manner within 10 h of incubation. How-
Journal of Molecular Neuroscience
ever, DHA supplementation did not affect caspase-3 activity in WT-treated cells. DHA supplementation also inhibited DNA fragmentation in SP-treated cells but not in WT-treated cells. The antiapoptotic effect of DHA in SP-treated cells was abolished by WT, but not by other kinase or phosphatase inhibitors. Phosphorylation of Akt was significantly reduced by treatment with SP, and DHA supplementation prevented this reduction while supplementation with other fatty acids did not. These data suggest that DHA’s protective effect is mediated through the Pl3-Kinase/Akt pathway.
AUDITORY BRAINSTEM EVOKED RESPONSE (ABR) AND BRAIN PHOSPHOLIPID FATTY ACIDS AND MONOAMINES IN RATS FED FORMULA WITHOUT OR WITH ARACHIDONIC ACID (AA) AND/OR DOCOSAHEXAENOIC ACID (DHA) N. Auestad, S.M. Innis, S. de la Presa Owens, J. Stockard, R. Korsak, and J. Edmond, Ross Products Division, Columbus, OH; University of British Columbia, Vancouver, BC; UCLA, Los Angeles, CA; and University of South FL, Tampa, FL Pre- and postnatal maternal dietary DHA(22⬊6n3) in rats increases DHA and lowers AA (20⬊4n6) in cerebrum of 23–311-d-old offspring and slows maturation of ABR. The present study used a formulafeeding rat model to evaluate postnatal dietary DHA. Gastrostomized rats were artificially reared (AR) on rat-milk formulas and diets with (g/100 g FA) 20% 18⬊2n6, 2% 18⬊3n3, and no DHA or AA (control), 0.4% DHA and 0.6% AA (AA + DHA), or 2.3% DHA and no AA (2.3%DHA). ABR and phospholipid fatty acids and monoamine levels in brain were determined on d26-27 and analyzed by ANOVA. ABR was 10% faster in 2.3%-DHA than AA+DHA rats and not different from control or suckling rats. Rats fed 2.3%-DHA had 9–24% higher DHA in whole-brain PE and in phospholipids from inferior (IC) and superior colliculus (SC) and frontal cortex; AA was inversely related to DHA. Dopamine and serotonin levels in IC and SC were not different among AR rats. Norepinephrine was 21% lower in IC from rats fed 2.3%-DHA than AA+DHA but not different in SC among AR rats. In frontal cortex, dopamine was 28% higher in 2.3%-DHA than AA+DHA rats, norepinephrine was not different among AR rats, and serotonin was 26–30% higher in 2.3%-DHA than control and AA+DHA rats. Variable results were also found for comparisons of AR to suckling rats. These results suggest compartmentation of fatty acid pools in brain and region specific effects of postnatal dietary fatty acids on brain fatty acids and monoamines. (Supported by Ross Products Div, Abbott Labs & HD06576.)
LIPID MODIFICATIONS IN OLIGODENDROGLIA CELLS ALTER SUSCEPTIBILITY TO CELL DEATH VIA ERKS ACTIVATION A. Brand and E. Yavin, Department of Neurobiology, Weizmann Institute of Science, Rehovot, 76100, Israel The cellular response to oxidative stress is regulated via a complex network of interacting protein kinases carrying signals from
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324 surface receptors to the nucleus. The main objective of the present work was to clarify the intimate relationship between membrane lipid composition and activation of the ERK signaling cascade in response to oxidative stress. Alterations in phospholipid composition of clonal cells of oligodendroglial origin (OLN 93) was attained by adding docosahexaenoic acid (DHA) 0.05 (M) and/or dimethylethanolamine (dEa, 1 mM) N-base to the cell-culture medium. Within min following 0.1 mM H2O2 addition, dEa-supplemented cultures showed an intense ERK activation detected by using an antibody identifying the phosphorylated form of ERKs. DHA supplements and concomitant treatment with both DHA and dEa caused less activation of ERKs. While ERK activation declined very fast in dEa cultures, it was more persistent in dEa+DHA cultures. By 24 h following stress, neither dEa- nor dEa+DHA-treated cultures showed any activation, but in DHA-treated cells ERKs were still activated. Moreover, while H 2O 2-induced stress caused apoptotic cell death in DHA-enriched cultures, as attested by cell-cycle analysis, cell death was almost completely inhibited by concomitant treatment with dEa even after cells were exposed to 0.5 mM H2O2. Primary rat neuronalcell cultures showed similar patterns of ERK activation and also prevention of cell death by dEa supplements. These data provide grounds to suggest that membrane lipid composition modulates the activation of ERKs following H2O2-induced stress and therefore can promote or prevent apoptotic cell death.
LEARNING DEFICITS IN RATS WITH LOW BRAIN DOCOSAHEXAENOIC ACID Janice N. Catalan,1 Toru Moriguchi,1 Burton Slotnick2 and Norman Salem, Jr.,1 1Laboratory of Membrane Biochemistry and Biophysics, National Institute on Alcohol Abuse and Alcoholism, Rockville MD; and 2The American University, Department of Psychology, Washington, DC The ability to develop a strategy for solving problems is fundamental for efficient learning. Learning set tasks are commonly used to assist in measuring higher cognitive function by assessing whether humans or animals can abstract a rule to help solve a series of similar problems. Our objective was to determine if a decrease in brain docosahexaenoic acid (DHA) would alter learning ability in n-3 deficient rats. Second-generation males and their dams were maintained on either an n-3 deficient or an n-3 adequate diet containing alphalinolenic acid and DHA. Fifteen males per dietary group were placed on partial water deprivation and at 10 wk of life were trained by operant conditioning, in a multi-channel olfactometer, on a series of “go, no-go” discrimination tasks. Each task consisted of 60 trials where responding to the presence of the positive odor was reinforced with water while responding to the negative odor was punished by an extended inter-trial interval. Brains of eight-wk old littermates were analyzed for fatty acid composition. There was an 85% decrease of DHA in the brains of the n-3 deficient rats. Over the first 15 problems, both dietary groups demonstrated acquisition of the task. In problems 16–20, the n-3 adequate rats were able to improve performance by achieving near errorless performance, while the n-3 deficient animals did not improve. As measured by olfactory learning-set performance, our results point to a mild cognitive deficit in n-3 deficient animals in their ability to acquire a response strategy.
MATERNAL DOCOSAHEXAENOIC ACID (DHA) DURING PREGNANCY ENHANCES INFANT NEUROBEHAVIORAL DEVELOPMENT S. Cheruku, H. Montgomery-Downs, S. Farkas, E. B. Thoman, C. J. Lammi-Keefe, Department of Nutritional Sciences, Department of Biobehavioral Sciences, University of Connecticut, Storrs, CT 06269 Functional outcome for the infant with respect to the effect of DHAon the developing brain remains unexplored. Sleep/wake states for infants are uniquely appropriate for assessing the functional integrity of the central nervous system (CNS). Women (n = 17) were recruited at delivery. Maternal venous blood was collected, and plasma phospholipid (PL) fatty acids were ana-
Journal of Molecular Neuroscience
Abstracts of Poster Presentations lyzed by gas chromatography (GC). In the hospital the infants’ sleep states were recorded nonintrusively using the Motility Monitoring System (MMS): a pressure-sensitive pad placed under the infants’ bedding in the crib. Infants’ body movements and respiratory patterns were logged and scored for sleep/wake states on the first (P1) and second (P2) postnatal days. Plasma PL DHA (wt%) ranged from 1.91–4.51. Based on previously published data, and the median DHA (wt%), the women were categorized into two groups: high (H) (>3.0 wt%), and low (L) (≤3.0 wt%). On P1, infants of women in the H group had a lower Active/Quiet Sleep Ratio (AS/QS) (H = 2.7 ± 0.35; L = 4.9 ± 0.92, p < 0.05), less Active Sleep (AS) (H = 44.7 ± 2.31; L = 52.4 ± 2.36, p < 0.05), and more Quiet Sleep (QS) (H = 18.2 ± 1.78; L = 12.5 ± 1.92, p < 0.05). On P2, the findings were similar. Further, regression analyses of maternal DHA values with the sleep/wake states are consistent with these results. In the months following birth, the developmental trend is towards a decreased ratio of Active Sleep to Quiet Sleep. The lower AS/QS in infants of mothers with the higher DHA suggests a developmental advantage. (Supported in part by Donaghue Med. Res. Fdn, NIH#HD32903, USDA#93-37200-8876, and UCONN Res. Fdn.)
MUTATIONS IN THE GENE ENCODING PEROXISOMAL α-METHYLACYL-COA RACEMASE CAUSE ADULT-ONSET MOTOR NEUROPATHY S. Ferdinandusse,1 S. Denis,1 P. T. Clayton,3 A. Graham,4 J. E. Rees,4 J. T. Allen,5 B. N. McLean,6 A. Y. Brown,5 P. Vreken,1 H. R. Waterham,2 and R. J. A. Wanders,1,2 Departments of 1Clinical Chemistry and 2Pediatrics, Emma Children’s Hospital, Academic Medical Center, University of Amsterdam, The Netherlands; 3Biochemistry Unit, Institute of Child Health, London, UK; 4Hurstwood Park Neurological Center, The Princess Royal Hospital, West Sussex, UK; 5 Biochemical Genetics Unit, Southmead Hospital, Bristol, UK; 6 Royal Cornwall Hospital, Treliske, Truro, UK Sensory-motor neuropathy is associated with several inherited disorders including Charcot-Marie Tooth disease, X-linked adrenoleukodystrophy/adrenomyeloneuropathy, and Refsum disease. In the latter two, the neuropathy is thought to result from the accumulation of specific fatty acids. We describe three patients with elevated plasma concentrations of 1) pristanic acid and 2) di- and trihydroxycholestanoic acid (DHCA and THCA) but normal very long-chain fatty acids (VLCFA). Two of the patients suffered from adult-onset sensory-motor neuropathy. One patient also had pigmentary retinopathy, suggesting Refsum disease, whereas the other patient had upper motor neuron signs in the legs, suggesting adrenomyeloneuropathy. The profile in plasma pointed to a specific defect in the peroxisomal β-oxidation of 2-methyl branched-chain fatty acids. To study this, we measured the activity of the enzymes involved including: 1) branched-chain acyl-CoA oxidase, 2) D-bifunctional protein, and 3) branched-chain ketothiolase (SCPx). To our surprise, all enzymes were normally active in the fibroblasts from the 3 patients. This focused our attention on another enzyme involved in the oxidation of branched-chain fatty acids called α-methylacyl-CoA racemase. This enzyme is responsible for the conversion of (2R)-pristanoylCoA and (2R)-DHCA- and THCA-CoA into their (2S)-stereoisomers, which are the true substrates for β-oxidation. We have set up a method to measure racemase activity in fibroblasts and found a complete deficiency in all 3 patients. Furthermore, we cloned the human racemase cDNA and identified two mutations that produced an inactive enzyme as shown by expression studies in Escherichia coli. Our findings have major implications for the diagnosis of adultonset neuropathies of unknown aetiology.
WHICH PROPERTIES OF BIOMEMBRANES CHANGE WHEN HYDROCARBON CHAINS ARE MOSTLY POLYUNSATURATED? Klaus Gawrisch, Laura L. Holte, Bernd W. Koenig, Daniel Huster, Ivan V. Polozov, Judith A. Barry, Frances Separovic, Anne Michele
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Abstracts of Poster Presentations Safley, Walter E. Teague, and Edward Sternin, Laboratory of Membrane Biochemistry and Biophysics, National Institute on Alcohol Abuse and Alcoholism, National Institutes of Health, 12420 Parklawn Dr., Rm. 158, Rockville, MD 20852 The membranes of brain synaptosomes and retinal rod outer segments contain 30–50 mol% of the sixfold unsaturated docosahexaenoic acid (DHA, 22⬊6 n-3) as lipid hydrocarbon chains. It is evident that, particularly in certain neural membranes, the inclusion of double bonds is critical for proper function. Using a variety of nuclear magnetic resonance (NMR) methods, we determined differences in biophysical properties as a function of hydrocarbon-chain unsaturation. Polyunsaturated lipids have significantly larger area per molecule in membranes. When under lateral tension, the polyunsaturated chain undergoes larger changes in length than neighboring saturated chains. The order of polyunsaturated chains is less sensitive to changes in temperature and changes in lipid headgroups, and they are less influenced by membrane-perturbing agents like ethanol. Cholesterol avoids interaction with polyunsaturated chains. Ca2+ binding constants to polyunsaturated membranes are significantly smaller than those for monounsaturated bilayers. Polyunsaturated membranes are much more permeable for water. Polyunsaturated membranes are under curvature stress, in particular if the polyunsaturated chains are paired with ethanolamine headgroups. A common perception of polyunsaturated chains is that they are stiff and inflexible due to the presence of motionally restricted double bonds. In contrast, our order parameter studies indicate exceptionally high deformability of DHAchains in biomembranes. The alteration of elasticity of neural membranes by the number of double bonds per fatty acid is one possible role of lipid polyunsaturation. With NMR spectroscopy and X-ray diffraction, we are able to follow the elastic deformation of membranes under tension, including the changes in structure and motions of lipid hydrocarbon chains. The results suggest that polyunsaturated chains in membranes prefer flexible, looped and helical structures, providing increased flexibility to receptor-rich neural membranes.
References Gawrisch K. and Holte L. L. (1996) Chemistry and Physics of Lipids 81, 105–116. Holte L. L., Peter S. A., Sinnwell T. M., and Gawrisch K. (1995) Biophys. J. 68, 2396–2403. Holte L. L., Separovic F., and Gawrisch K (1996) Lipids 31, S199–S203. Holte L. L. and Gawrisch K. (1997) Biochemistry 36, 4669–4674. Huster D., Arnold K., and Gawrisch K. (1998) Biochemistry 37, 17,299–17,308. Huster D., Jin A. J., Arnold K., and Gawrisch K. (1997) Biophys. J. 73, 855–864. Koenig B. W., Strey, H. H. and Gawrisch K. (1997) Biophys. J. 73, 1954–1966. Separovic F. and Gawrisch K. (1996) Biophys. J. 71, 274–282.≠–
DOCOSAHEXAENOIC ACID DEFICIENT RATS SHOW IMPAIRED ACQUISITION IN AN OLFACTORY DISCRIMINATION TASK Rebecca S. Greiner, Toru Moriguchi, Burton Slotnick, and Norman Salem Jr., Laboratory of Membrane Biophysics and Biochemistry, National Institute on Alcohol Abuse and Alcoholism, National Institutes of Health, Rockville, MD Docosahexaenoic acid (DHA), an omega-3 fatty acid highly concentrated in the central nervous system (CNS), is thought to be involved in cognitive processes such as learning and memory. A chronic DHA deficiency has been linked to impaired visual acuity and altered electroretinograms in human infants and altered learning in rats. To further examine the role of DHA in the CNS and its impact on cognitive processes, we studied DHA deficient rats using a novel technique based on olfaction. Rats are macrosomatic; therefore employing the olfactory modality is more appropriate because rats use primarily their sense of smell to learn about their environment. An n-3 fatty acid-deficient diet was used to deplete DHA in the deficient group. The addition of flaxseed oil, as a source of 18⬊3n3, to the adequate diet was the only difference between the two exper-
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325 imental diets. Rats were fed these diets for two generations. After testing male rats with seven, two-odor discrimination problems (1/d), animals were euthanized, the whole brains were extracted, and the fatty acid profiles were determined by standard gas chromatography (GC) methods. F2 generation n-3 deficient rats had 81 and 82% less DHA in whole brain and olfactory bulb, respectively, in comparison with n-3 adequate rats. The n-3 deficient rats made significantly more total errors on the two-odor discrimination task and also made significantly more errors on several individual problems. These results indicate that rats perform more poorly on an olfactory discrimination task when brain levels of DHAare depleted.
DOCOSAHEXAENOIC ACID MODULATES PHOSPHATIDYLSERINE ACCUMULATION SPECIFICALLY IN NEURONAL TISSUES Jillonne Hamilton,1 Rebbeca Greiner, Norman Salem, Jr., and HeeYong Kim,1 1Section of Mass Spectrometry, Laboratory of Membrane Biophysics and Biochemistry, National Institute on Alcohol Abuse and Alcoholism, National Institutes of Health, Rockville, MD Docosahexaenoic acid (22⬊6n-3) is the major polyunsaturated fatty acid (PUFA) in neuronal tissues. We have previously shown that the 22⬊6n-3 status influences biosynthesis and accumulation of phosphatidylserine (PS) in brain microsomes. In this study, we aim to determine whether modulation of PS accumulation by 22⬊6n-3 is a general phenomenon or occurs specifically in neuronal cells. The phospholipid composition of various tissues from rats reared on diets that were adequate or deficient in n-3 fatty acids were examined using electrospray LC/MS. We observed that rat brain cortex, mitochondria, and olfactory bulb, where 22⬊6n-3 is highly concentrated, contain significantly higher levels of PS in comparison to liver and adrenal where 22⬊6n-3 is a minor component. Phospholipid molecular-species analysis revealed that in brain cortex, mitochondria, and olfactory bulb, 18⬊0,22⬊6n-3 was the most abundant PS species, representing 45–65% of the total PS. In liver and adrenal, 18⬊0,20⬊4n-6 was the major PS species. Dietary depletion of 22⬊6n-3 resulted in approx 30–40% reduction in total PS from the brain cortex, mitochondria, and olfactory bulb, with the most dramatic reduction occurring in 18⬊0,22⬊6n-3 PS. Total PS levels in liver or adrenal, however, remained unchanged. These results establish that variations in the membrane 22⬊6n-3 fatty acid composition have a profound influence on PS accumulation in neuronal cells where 22⬊6n-3 is abundant. These data have implications in neuronal cellular events, such as translocation of protein kinases to the plasma membrane where PS is believed to play a major role.
DEVELOPMENT OF PSYCHOTIC ILLNESSES AMONG CHILDREN AND MATERNAL PLASMA EFA COMPOSITION SAMPLED AT BIRTH J. R. Hibbeln,1 S. Bulka,3 R. Yolken,2 M. Klebanoff,4 S. Majchrzak,1 and N. Salem, Jr.1, 1Laboratory of Membrane Biophysics and Biochemistry, National Institute on Alcohol Abuse and Alcoholism, National Institutes of Health, Rockville MD; 2Johns Hopkins University, Baltimore, MD; 3Harvard School of Public Health, Boston, MA; and 4National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD The Collaborative Perinatal Project was conducted from 1959–1966 to assess developmental factors in the etiology of mental retardation and neuropsychiatric outcomes in childhood. Plasma was collected from the mothers on the day of birth and was frozen at –20°C for more than three decades. Offspring were located during 1987–91 and lifetime psychiatric diagnoses were determined using structured interviews and hospital records. The final study sample included 27 subjects with a psychotic illness and 51 unaffected control subjects, matched for date of birth, gender, and ethnicity. Maternal plasma was separated into phospholipid (PL), triglyceride (TG), and cholesterol ester (CE) fractions. Fatty acid concentrations were quantified in each fraction using internal standards. Total plasma fatty acid composition was estimated by summing lipid fractions (PL/TG/CE sum). Group differences were compared using Mann-
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Abstracts of Poster Presentations TABLE 1 Control
SD
Psychotic
SD
p value
TG/PL/CE Total FA’s ug/mL 6311.9 TG/PL/CE 18 2n6 % 27
2192.5 3.1
6637.8 29
1911 4.3
ns 0.05
CE Total FA’S (ug/mL)
2281.1
844.4
2593.9
863.8
ns
CE n-3 total (ug/mL)
19.3
9.9
25.8
16.9
ns
CE 18:3 n3, (ug/mL)
7.2
3.7
9.5
5.1
0.04
CE 20.5 n3, (ug/mL)
3.6
2.2
5.3
4.7
ns
CE 22:6 n3, (ug/mL)
8.4
4.9
11
8.1
ns
CE 18:2 N6, (ug/mL)
1065.3
418.5
1260.5
460
0.04
CE 20:4 n6, (ug/mL)
121.8
67.0
143.5
81.4
ns
Whitney nonparametric testing. Results: The groups did not differ comparing concentrations of total fatty acids, total saturates, total monounsaturates, total polyunsaturates or 22⬊5n6 in any fraction or in the PL/TG/CE sum. Conclusion: Among mothers who gave birth to children who developed psychosis, linoleic acid was elevated in a sum of the TG, PL, and CE fractions. The increase in linolenic acid in the CE fraction was consistent with a trend towards an increase in 20⬊5n3, 22⬊5n3, and 22⬊6n3 in all fractions. These data are not consistent with the hypothesis that mothers of children with psychotic illnesses were deficient in any omega-3 or omega-6 essential fatty acid.
DOCOSAHEXAENOIC ACID IS NOT A MAJOR PATHOGENIC FACTOR IN PEROXISOME-DEFICIENT MICE A. Janssen, M. Baes, P. Gressens, G. P. Mannaerts, P. Declercq, and P. P. Van Veldhoven, Laboratory of Clinical Chemistry and Department of Pharmacology, K.U. Leuven, Leuven, Belgium; and Laboratoire de Neurologie du Développement, Hôpital RobertDebré, Paris, France Docosahexaenoic acid (DHA), a major component of membrane phospholipids in brain and retina, is profoundly reduced in patients with peroxisome biogenesis disorders (Zellweger syndrome). Supplementing the patients with DHA resulted in improved muscular tone and visual functions. The purpose of this study was to investigate: 1) whether DHA levels were also reduced in newborn PEX5 knockout mice, the mouse model of Zellweger syndrome that we recently generated; 2) whether these levels could be normalized by supplying DHA; and 3) whether this results in longer survival. The DHA concentration in brains of newborn PEX5–/– mice was reduced by 40% as compared to levels in normal littermates. The daily administration of 10 mg DHAethylester to pregnant heterozygous mothers during the last 8 d of gestation resulted in a normalization of DHA levels in Zellweger pups. However, no clinical improvement was observed in these pups and the neuronal migration defect was unaltered. These data suggest that the accretion of DHA in the brain at the end of embryonal development is not only supported by the maternal supply but also depends on synthesis in the fetal brain. Furthermore, the DHA deficit does not seem to be a pathogenic factor in the Zellweger mice.
SUPPRESSION OF PEROXISOMAL RESPIRATION IN CHILDREN WITH AUTISTIC SPECTRUM DISORDER: PATTERN RECOGNITION AND NEUROBIOLOGICAL ROLE OF A TREATMENT PROTOCOL Patricia Kane, BodyBio, 45 Reese Road, Millville, NJ 08332 Purpose: Exploration of the systemic presentation of Autistic Spectrum Disorder (ASD) through examination of fatty acid aberration examined through red cell lipid biopsy with development of a therapeutic approach towards metabolic stability of lipid metabolism and detoxification within cell structures.
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Objectives: 1. Discuss the potential role of disordered fatty-acid metabolism in ASD. 2. Compare and contrast metabolic interventions. 3. Appreciate the profound influence essential fatty acids have upon cellular and brain metabolism. Conclusion: Characteristic findings in ASD as the accumulation of very longchain fatty acids (VLCFAs), complex omega-3 and omega-6 imbalances, blocked prostaglandin-1 synthesis, demyelination, poor cellular integrity, and aberrant lipid production may be managed by opening a gateway into cellular metabolism with metabolic intervention (cofactors, substrates) through manipulation of cellmembrane lipids and thereby impacting the systemic nature of the disorder.
TEMPORAL AND SPATIAL PATTERNS OF EXPRESSION OF FATTY ACID TRANSPORT REGULATORY PROTEINS IN THE DEVELOPING RAT PLACENTA Gregory T. Knipp,1 Bing Liu,2 Kenneth L. Audus,3 Hiroshi Fuji,4 Teruo Ono,4 and Michael J. Soares,2 1Department of Pharmaceutics, Rutgers University, 160 Frelinghuysen Road, Piscataway, NJ 088548022; 2Department of Molecular and Integrative Physiology, The University of Kansas Medical Center, 39011 Rainbow Boulevard, Kansas City, KS 66160; 3Department of Pharmaceutical Chemistry, University of Kansas, 2095 Constant Ave, Lawrence, KS 66045; and 4Department of Biochemistry, Niigata University School of Medicine, Niigata 951, Japan The placenta forms a selective barrier that functions to transport nutrients that are of critical use to the fetus. Delivery of essential fatty acids to the fetus is dependent on transplacental transport and provides the chemical backbone for the biosynthesis of biological membranes, myelin, and various signaling molecules. The primary objective of this research was to elucidate the expression patterns of genes that regulate fatty acid transport across the placenta. Several fatty acid transport regulatory genes have been identified in the rat including; cytoplasmic heart fatty acid binding protein (hFABP), plasma membrane fatty acid binding protein (FABPpm), fatty acid translocase (FAT), and fatty acid transport protein (FATP). In this study, we have elucidated temporal and spatial expression patterns for these genes in the rat placenta and in cell-culture models of the rat placenta. We have utilized several molecular biological techniques including Northern-blot analysis, reverse transcriptionpolymerase chain reaction (RT-PCR), and Southern-blot analysis, Western-blot analysis, and in situ hybridization analysis to elucidate these expression patterns. Expression of hFABP was specific to the labyrinth zone, the main barrier and site of transplacental transport in the rat placenta. In addition, the levels of hFABP expression increased with gestational age, suggesting a growing requirement for fatty acid transport with advancing stages of pregnancy. FABPpm, FAT, and FATP are expressed in the junctional and labyrinth zones of the rat placenta. FAT was predominantly localized to the labyrinth zone by in situ hybridization analysis. The placental cell expression patterns of the genes involved in fatty acid transport were supported by our observations HRP-1 (labyrinth zone) and Rcho-l (junctional zone) trophoblast cell-culture models. The cell-surface location of FABPpm, FAT, and FATP suggest that they participate in placental fatty acid uptake. The predominant expression of hFABP and FAT in the labyrinth zone of the chorioallantoic placenta implicates hFABP and FAT in the transplacental movement of fatty acids from maternal to fetal compartments. The results of these studies provide a template in which insights into the regulation of these transporters can be explored. In addition, insights into the mechanisms by which essential fatty acids are transported across the placenta during disease states and implications of aberrant transfer can be investigated.
TEMPORAL AND SPATIAL PATTERNS OF EXPRESSION OF PPAR AND RXR ISOFORMS IN THE RAT PLACENTA Qing Wang,1 Qi Li,1 Dea Herrera-Ruiz,1 Michael J. Soares,2 Hiroshi Fuji,3 and Gregory T. Knipp,1 1Department of Pharmaceutics, Rutgers University, 160 Frelinghuysen Road, Piscataway, NJ 088548022; 2Department of Molecular and Integrative Physiology, The
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Abstracts of Poster Presentations University of Kansas Medical Center, 39011 Rainbow Boulevard, Kansas City, KS 66160; and 3Department of Biochemistry, School of Medicine, Niigata University, Niigata 951, Japan The ability of fatty acids to permeate the placenta is critical to proper fetal development. For example, essential fatty acids (n-3 and n-6 series) are critical components in brain and central nervous system (CNS) formation and growth of the fetus. Currently, it is unclear how fatty acid transfer across the placenta is mediated and regulated. Insights into the regulation of placental fatty acid transfer may lie in identifying and elucidating the expression of nuclear steroid hormones in the placenta. Peroxisome proliferator-activated receptors (PPAR) and retinoid-X-receptor (RXR) families are the members of the nuclear steroid hormone receptors super-family and have been demonstrated to be critical elements as transcription regulators of genes involved in fatty acid transport and metabolism. For example, the PPARγ/RXRα heterodimer has been demonstrated to be critical in the transcription regulation of expression of fatty acid translocase and the heart cytoplasmic fatty acid binding protein (FABP) in the rat heart. This study has been designed to elucidate the patterns of expressions of PPAR and RXR isoforms in the rat-placenta and cellculture models of the rat placenta. The results of these studies indicate that PPARα, PPARγ, RXRα, and RXRβ exhibit ubiquitous expression in both the junctional and labyrinthine zones of the rat placenta, from d 13 of gestation until term and in cell-culture models of rat placenta, HRP-1, and Rcho-l. RXRγ was indicated to exhibit a site-specific pattern of expression in rat placenta: with predominant expression witnessed in the junctional zone from d 13 of gestation until term. PPARβ was not observed in these tissues by reverse transcription-polymerase chain reaction (RT-PCR), suggesting that it may not play a role in regulating transplacental fatty acid transfer or metabolism. The concomitant expression of PPARγ and RXRα suggests that placental accumulation and transport of fatty acids from the maternal circulation to the fetus may be highly regulated through these receptors. The continued research into the intrinsic link between the activity of PPAR and RXR isoforms and their ability to regulate fatty acid transport and metabolism across the placenta will be critical to develop relationships between placental fatty acid transfer and fetal development. In addition, these studies will hopefully provide new therapeutic windows for antagonistic and agonistic medical intervention where placental fatty acid transfer disorders may act to adversely influence proper fetal development.
A NOVEL ACYL-COA SYNTHETASE IN HUMAN BRAIN Frans A. Kuypers and Kiran T. Malhotra, Children’s Hospital Oakland Research Institute, Oakland, CA 94609 Long-chain acyl-CoA synthetase (LACS, EC 6.2.1.3) plays a keyrole in fatty acid utilization. We have cloned, sequenced, and expressed full-length cDNAs encoding two forms of acyl-CoA synthetase from a K- 562 human erythroleukemic cell line (Malhotra et al., 1999). The first form, named long-chain acyl-CoA synthetase 5 (LACS5), was found to be a novel, unreported, human acyl-CoA synthetase, whereas the second form (65% identical to LACS5) was 97% identical to human liver LACS1. The LACS5 gene encodes a highly expressed 2.9 kilobase and 9.6 kb mRNA transcript in human brain. A 2.9 and 6.3 kb transcript of LACS5 is found in human red-cell precursors, but transcripts are virtually absent in human heart, kidney, liver, lung, pancreas, spleen, and skeletal muscle. LACS5 is distinctly different (30% amino acid-sequence identity) from LACS4, another form reported to be present in human brain. Antibodies directed against LACS5 cross-reacted with red cell membranes, indicating that LACS5 plays an important role in plasma-membrane fatty acid utilization. The 78 kDa expressed LACS5 protein used long-chain fatty acids such as palmitic-, oleic-, and arachidonic acid (AA) as substrates. We hypothesize that an altered LACS5 activity may be responsible for the reported differences in red-cell fatty acid utilization in patients with psychiatric disorders such as schizophrenia (Horrobin et al., 1999). Studies in blood samples to characterize red cell acyl CoA synthetase activity in combination with LACS5 sequence analysis may shed light on the altered utilization of fatty acids in the brain of these patients.
References Horrobin D. F. and Bennet C. N. (1999) New gene targets related to schizophrenia and other psychiatric disorders: enzymes, bind-
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327 ing proteins and transport proteins involved in phospholipid and fatty acid metabolism. Prostaglandins, Leukotrienes Essential Fatty Acids 60, 141. Malhotra K. T., Malhotra K., Lubin B. H., and Kuypers F. A. (1999) identification and molecular characterization of Acyl-CoA synthetase in human erythrocytes and erythroid precursors. Biochem. J. 344, 135.
DETERMINATION OF THE LATENCY FOR REPLACING ESSENTIAL FATTY ACIDS LOST FROM THE NERVOUS SYSTEM DUE TO DIETARY RESTRICTION Toru Moriguchi, James Loewke, Megan E. Garrison, and Norman Salem, Jr., Laboratory of Membrane Biochemistry and Biophysics, National Institute on Alcohol Abuse and Alcoholism, National Institutes of Health, Rockville, MD Previously, we have found that n-3 fatty acid deficiency produces behavioral deficits in learning and memory in rats. These results suggested that docosahexaenoic acid (DHA) supplementation may improve several adverse health and behavioral alterations attributed to n-3 fatty acid deficiency. We investigated the latency in replacing DHA in the n-3 fatty acid deficient adult when an n-3 adequate diet was introduced. Rats were fed an n-3 fatty acid adequate (n-3 Adq) or n-3 fatty acid deficient diet (n-3 Def) for two generations. The n-3 Adq diet included 1.3% DHA. At 7 wk age in second-generation males, the n-3 Def group was changed to an n-3 Adq diet from an n-3 Def diet. The brain, retina, liver, and serum were collected at 0, 1, 2, 4, and 8 wk after diet reversal. The DHA level in the brain at 1 and 2 wk after diet reversal was only partially recovered, rising to approx 20 and 35% of the n-3 Adq group level, respectively. The latency to recover DHA to the n-3 Adq group level was about 8 wk after diet reversal. On the other hand, the level of DHA in the serum and liver was approx 90% replaced in 2 wk after diet reversal. These results indicate that the DHA replacement in the adult nervous system is markedly slower compared with the recovery of other tissues.
BEHAVIORAL EFFECTS OF DIETARY N-3 FATTY ACID DEFICIENCY IN SECOND AND THIRD GENERATION RATS Toru Moriguchi, Rebecca S. Greiner, and Norman Salem, Jr., Laboratory of Membrane Biochemistry and Biophysics, National Institute on Alcohol Abuse and Alcoholism, National Institutes of Health, Rockville, MD Omega-3 (n-3) fatty acids, such as docosahexaenoic acid (DHA), are deposited primarily during the period of rapid brain development and play an important role in the nervous system. We investigated the influence of n-3 fatty acid deficiency on learning performance in adult rats over two generations. Rats were fed either an n-3 fatty acid adequate (n-3 Adq) or deficient diet (n-3 Def) for three generations (F1-F3). In the n-3 Def group, total n-3 fatty acids in the frontal cortex were reduced by 83% in the F2 generation and by 87% in the F3 generation compared to the n-3 Adq group. In the Morris water maze, the n-3 Def group showed a markedly longer escape latency and delayed acquisition of this task as compared with the n-3 Adq group in both generations. The acquisition and memory levels of the n-3 Def group in the F3 generation was generally worse than that of the F2 generation. The ratio of 22⬊5n-6/22⬊6n-3 ratio in the frontal cortex and dams’ milk was markedly increased in the n-3 Def group and this ratio was significantly higher in the F3 generation compared to the F2 generation. These results suggest that dietary n-3 deficiency negatively affected spatial learning and memory related behaviors in rats via changes in the long-chain polyunsaturated distribution in the central nervous system which was markedly influenced not only by the weanling diet but also by the milk received from the mother during lactation.
THE EFFECTS OF DIETARY OMEGA-3 FATTY ACIDS ON THE FATTY ACID AND MOLECULAR SPECIES COMPOSITIONS OF PHOSPHOLIPIDS IN THE RAT HIPPOCAMPUS Mahadev Murthy, Aneeq Ahmad, Jillonne Hamilton, Rebecca Sheaff Greiner, Toru Moriguchi, Hee-Yong Kim, and Norman Salem Jr., Laboratory of Membrane Biophysics and Biochemistry, National
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328 Institute on Alcohol Abuse and Alcoholism, National Institutes of Health, Rockville, MD Docosahexaenoic acid (DHA; 22–6, n-3), a major component of phospholipids in the brain, is known to have a significant role in the pathophysiology of the central nervous system (CNS). Both animal and human studies have demonstrated a strong association between n-3 fatty acids and physiological functions such as cognitive, behavioral, and visual functions. Furthermore, studies with rats have shown similar associations between n-3 fatty acids and memory, specifically with their working memory in the Morris water maze and the olfactory-based learning. In this regard, the hippocampus is hypothesized to be important, particularly in DHA-associated memory changes. The present study evaluated the effects of the diets that were deficient and adequate in n-3 fatty acids on changes in fatty acid and molecular species compositions in hippocampus phospholipids (PL). For this purpose, female rats were bred to maturity on two n-3 fatty acid deficient (low n-3, low n-3 + high n-6), and two n-3 fatty acid adequate (adequate n-3, adequate n-3 + DHA) diets for three generations. The hippocampus from each of these rats was removed at 110 d of age and lipids were extracted and analyzed for their fatty acid compositions, including total fatty acids, phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), and phosphatidylinositol (PI) and their corresponding molecular species compositions. The results on total fatty acids indicate a significant difference between deficient and adequate groups in the DHA content of the hippocampus. This difference is also apparent across different PL classes with the exception of PI. However, there is a greater enrichment of DHA percentage wise in both PS and PE compared to PC in the hippocampus from groups that were adequate in n-3 fatty acids. The compensatory increase in docosapentaenoic acid (DPA; 22⬊5, n-6) in both groups that were deficient in n-3 fatty acids is observed in PC, PS, and PE. Again greater increases were seen in PS and PE. However, the PI contained very small amounts of DHA or DPA in the respective groups. The changes observed in PLfatty acids and molecular species may have a significant bearing on hippocampus functions, in relation to the regulation of memory changes.
INFLUENCE OF DHA-CONTAINING PHOSPHOLIPIDS ON THE KINETICS AND EQUILIBRIUM OF G-PROTEIN BINDING Shui-Lin Niu, Drake C. Mitchell, and Burton J. Litman, Laboratory of Membrane Biophysics and Biochemistry, National Institute on Alcohol Abuse and Alcoholism, National Institutes of Health, 12420 Parklawn Dr., Rockville, MD 20852 The disk membrane contains high level of DHA-containing phospholipids which has been found to be essential for metarhodopsin II (MII) formation. Yet the role of DHA-containing lipids on the extent and kinetics of MII-G formation, which is the initial step in the signaltransduction cascade, remains unknown. In this study, the kinetics and equilibrium of MII formation and MII-G formation were characterized in reconstituted rhodopsin-containing vesicles with welldefined acyl-chain composition and cholesterol content using flash photolysis and UV/Vis spectroscopic methods. The kinetics of G-protein binding can be resolved into two components of MII formation and MII-G formation. The later process occurs via diffusion in the plane of the lipid membrane. The presence of 30 mol% cholesterol in the lipid bilayer slows both processes, with a greater reduction in the rate of MII-G formation (by ~40%). These results indicate that lipid-membrane composition modulates the diffusionally controlled MII-G formation. Previous studies, using diphenylhexatriene (DPH) as a probe, found that acyl-chain dynamics were less affected by cholesterol in the presence of DHA-containing lipids. Because the visual-transduction system is a prototype of the G-protein coupled signal-transduction systems, our findings of lipid modulation of the coupling of receptor and G-protein likely extend to other members of this receptor superfamily.
THE BRAIN IS THE SITE OF DHA PRODUCTION IN THE DEVELOPING FELINE Robert J. Pawlosky1 and Norman Salem Jr.,2 1Beltsville Human Nutrition Research Center, USDA; and 2National Institute on
Journal of Molecular Neuroscience
Abstracts of Poster Presentations Alcohol Abuse and Alcoholism, National Institutes of Health, Rockville, MD A series of studies in felines using stable isotopically labeled fatty acids and mass spectrometry have shown that the final steps of docosahexaenoic acid (DHA) production takes place in the brain and not in the liver in the domestic cat. The results from several studies on the metabolism of essential fatty acid in adult and juvenile animals from this species will be presented. Adult felines were able to elaborate the 18-carbon fatty acids to the 20-carbon polyunsaturated fatty acids (PUFAs) producing 20⬊4n6 from 18⬊2n6 and 20⬊5n3 from 18⬊3n3 in their livers. However, based on the isotopic tracer profiles the production of the 22- and 24-carbon polyenoic fatty acids took place in the brain and not in the liver. Juvenile animals could also synthesize the 20-carbon polyenoic fatty acids in their livers but depended on the brain metabolism to produce 22⬊6n3. In addition, young animals that were reared to mothers fed diets deficient in long-chain PUFAs (corn oil diets) had markedly lower concentrations of DHA in their nervous tissue (brain and retina) and showed deficiencies in their electroretinograms. Ultimately, it was shown that dietary DHA was necessary to obtain optimal levels of brain DHA in felines during development.
TREATMENT OF SCHIZOPHRENIA WITH ETHYL-EICOSAPENTAENOATE (EPA): A RANDOMIZED, PLACEBOCONTROLLED TRIAL M. Peet,1 D. F. Horrobin,2 A. I. M. Glen,3 P. Ward,3 P. Rowlands,3 B. Puri,3 R. Murray,3 C. de Wet,3 K. Kendall,3 R. Abed,3 H. Inch,3 P. Driscoll,3 G. Lloyd,3 M. Barber,3 A. Hay,3 A. McGregor,3 J. Hellewell,3 A. Mackenzie,3 and F. Glen,3 1Department of Psychiatry, University of Sheffield, Northern General Hospital Sheffield S5; 7AU, England; 2Laxdale Research, Stirling, Scotland FK7; and 3EPA Multicentre Trial Group Schizophrenia is a serious psychiatric disorder with a neurodevelopmental basis and evidence of neurodegeneration in many patients. At least two and possibly many more genes may be involved. There is evidence of excess activity of phospholipase (PL) A2, coupled with impaired incorporation of long-chain fatty acids into phospholipids. Existing drugs have only modest effects: they work by blocking dopamine and serotonin receptors, which are known to activate PLA2. EPAis known to inhibit PLA2. Two placebo-controlled pilot studies have shown that EPA-enriched oil but not docosahexaenoic acid (DHA)-enriched oil, had beneficial effects in schizophrenia. In order to explore this further, a multicenter, randomized, placebo-controlled trial of 97% pure ethyl-EPA was set up. Patients who entered the trial had a DSM-IV diagnosis of schizophrenia, were partially stabilized on existing neuroleptic drugs, but in spite of this had substantial remaining symptoms as shown by a score of 50 or more on the Positive and Negative Schizophrenia Symptom scale (PANSS). Patients were randomized to receive placebo or 1, 2, or 4 g of ethyl-EPA per day on a double-blind basis for a period of 12 wk. Patients were assessed at baseline and at 4, 8, and 12 wk on the PANSS, the Montgomery-Asberg Depression Scale, the Abnormal Involuntary Movement Scale, the Simpson-Angus scale for abnormal movements, the Barnes Akathisia scale, and the Liverpool University Neuroleptic Side Effect Rating Scale. 122 patients entered the trial, 114 had a rating while on treatment, and 90 completed the full 12-wk study. The database has been locked and the code will be broken early in 2000. Results will be available by the time of the meeting.
TREATMENT OF HUNTINGTON’S DISEASE WITH ETHYL-EICOSAPENTAENOIC ACID: A RANDOMIZED, PLACEBO-CONTROLLED TRIAL B. K. Puri,1 G. M. Bydder,1 S. J. Counsell,1 A. J. Richardson,1 J. V. Hajnal,1 C. Appel,2 B. Corridan,2 J. Binder,2 C. J. Higgins,2 M. Lennon,2 J. A. M. Wilson,2 T. Easton,2 D. Oyewole,2 H. M. McKee,2 K. S. Vaddadi,3 and D. F. Horrobin,4 1MRI Unit, Hammersmith Hospital, London W12 OHS; 2Ealing, Hammersmith and Fulham Mental Health NHS Trust and Imperial College School of Medicine, London; 3Monash University Medical School, Melbourne; and 4 Laxdale Research, Stirling, Scotland FK7 9JQ Huntington’s disease (HD) is a devastating illness caused by overexpression of the protein huntingtin, apparently due to excess num-
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Abstracts of Poster Presentations bers of CAG repeats in association with the gene on chromosome 4. The normal role of huntingtin and the mechanism of the damage are unknown. However, there is evidence of increased membrane breakdown and oxidative damage to neurons. This may be associated with excess activity of phospholipase (PL) A2. The disease causes psychotic behavior, movement disorders, memory loss, and dementia. There are no known treatments. Eicosapentaenoic acid (EPA) is an inhibitor of PLA2. A randomized, placebo-controlled double-blind trial of 2 g/d of the 97% pure ethyl ester of EPA was performed over a 6-mo period in patients with end-stage HD requiring 24-h nursing care. The overall condition of each patient was assessed on the Unified Huntington’s Disease Rating Scale (UHDRS) and the brain of each patient at the beginning and end of the study was assessed by magnetic resonance imaging (MRI). Eight patients entered the study but one died prior to randomization. At the end of the study, four patients were rated as deteriorated and three as improved on the HDRS. Improvements were particularly apparent in relation to oculomotor function and cognitive function. On breaking the treatment code, it was found that the four patients who deteriorated were on placebo while the three who improved were on ethyl EPA( p < 0.04, Fisher’s exact test). All patients had MRI scans at the beginning and end of the study but, because of the movement disorders, the before and after pairs were evaluable in only four patients. Two of these were on placebo and two on ethyl-EPA. In the two patients on placebo, ventricular size had increased, indicating disease progression. In contrast, in the two patients on ethyl-EPA ventricular size decreased consistent with some recovery of brain mass. Ethyl-EPA is the first treatment shown to be beneficial in HD in a placebo-controlled study.
VERY LONG-CHAIN FATTY ACIDS IN ADRENOLEUKODYSTROPHIC FIBROBLASTS: LEVELS AND β-OXIDATION ARE MODULATED BY ANDROGENS A. Petroni,1 M. Blasevich,1 C. Galli,1 and R. C. Melcangi,2 1Institute of Pharmacological Sciences; and 2Department of Endocrinology, University of Milan, via Balzaretti 9, 20133 Milan, Italy Adrenoleukodystrophy (ALD) is associated with the accumulation, in plasma and fibroblasts, of saturated very long-chain fatty acids (VLCFA) such as lignoceric acid (24:0). Singh et al. demonstrated that the pathological accumulation of VLCFA was due to the impaired β-oxidation, which normally takes place in the peroxisomes (Singh et al., 1981). The aim of our study was to evaluate, in skin fibroblasts of adrenoleukodystrophic and control subjects, the levels of VLCFA, the β-oxidation of labeled lignoceric and the effect of steroids on these parameters. Levels of VLCFA were measured by gas chromatography (GC). The metabolism of lignoceric acid was evaluated by incubating fibroblasts, for different times, with the labeled fatty acid dissolved in a solution containing α-cyclodextrin. The measurements of labeled CO2 and the catabolic products derived from lignoceric acid-β-oxidation were evaluated according to the standard procedures. Levels of VLCFA were markedly elevated in plasma and fibroblasts at adrenoleukodystrophic patients, when compared to controls. The β-oxidation of labeled lignoceric acid was markedly reduced in adrenoleukodystrophic fibroblasts. Selected androgens, which appeared to have an altered metabolism in ALD towards control fibroblasts, were able to markedly reduce VLCFA levels by enhancing their β-oxidation.
Reference Singh I., Moser H. W., Moser A. B., and Kishimoto Y. (1981) Biochem. Biophys. Res. Commun. 102, 1229–1229.
RHODOPSIN’S PREFERENCE FOR POLYUNSATURATED LIPIDS: DEUTERIUM-NMR STUDY I. V. Polozov, B. J. Litman, and K. Gawrisch, Laboratory of Membrane Biochemistry and Biophysics, National Institute on Alcohol Abuse and Alcoholism, National Institutes of Health, 12420 Parklawn Dr., Rockville, MD, 20852 Rhodopsin, the primary visual receptor, a member of G-proteincoupled receptor family, in vivo is located in retina membranes that are rich in polyunsaturated fatty acids (PUFAs), particularly in
Journal of Molecular Neuroscience
329 docosahexaenoic acid (DHA). We studied rhodopsin-lipid interactions to get an insight on how membrane properties change in the presence of the membrane proteins. Quadrupolar splittings of deuterium-labeled lipids provide important information about molecular order in lipid membranes. We used the spin-dry technique for preparation of oriented, solid-supported, multi-bilayer samples. This method reduced curvature artifacts in the 2H-nuclear magnetic resonance (NMR) spectra and significantly increased sensitivity and resolution, enabling work with milligram-size samples. In this study, we compared rhodopsin effects on the deuterium order parameter profile in mono- and polyunsaturated lipid systems including: l-stearoyl(d35)-2-oleoyl-PC, 1-stearoyl(d35)-2-docosohexaenoyl-PC, and mixtures of both where only one lipid was deuterated at a time. Protein incorporation decreased the order parameters of polyunsaturated PC, while not affecting that of monounsaturated one. Our results are consistent with a limited preference of rhodopsin for polyunsaturated lipids. However, we have no evidence of largescale, lateral-phase separation.
ROLE OF DHA IN MEMBRANE ORGANIZATION: CHOLESTEROL-DEPENDENT ASSOCIATION OF G PROTEINCOUPLED RECEPTOR WITH POLYUNSATURATED LIPID Alla Polozova, Burton J. Litman, Laboratory of Membrane Biochemistry and Biophysics, National Institute on Alcohol Abuse and Alcoholism, National Institutes of Health, Rockville, MD Lipid-protein interaction is one of the key elements in organization, regulation, and functioning of biological membranes. A significant portion of these function-modulating interactions occurs in the hydrophobic core of the membranes, and is believed to be driven by lipid acyl-chain composition. The visual signal-transduction pathway is extremely sensitive to lipid unsaturation level. Formation of the G-protein activating conformation of rhodopsin was shown to be promoted by lipid unsaturation. The lateral segregation of rhodopsin with polyunsaturated lipids was proposed as a possible native mechanism regulating rhodopsin activation. To examine this hypothesis, we employed fluorescence-resonance energy transfer (FRET) technique, using phosphatidylethanolamines (PEs) labeled at the headgroup with pyrene as donors and rhodopsin’s retinal group as acceptor. FRET efficiency from different acyl chain-specific probes was compared in mixed liposomes containing rhodopsin, cholesterol, saturated (16⬊0), and polyunsaturated (22⬊6) phosphatidylcholines (PCs). The higher FRET efficiencies detected for polyunsaturated probes point to the preferential segregation of rhodopsin with these lipids. In similar mixed bilayers containing no cholesterol, no cluster formation was observed at temperatures above lipid-phase transition, indicating a crucial role of cholesterol in microdomains formation.
INCREASING GLIOMA-CELL POLYUNSATURATED FATTY ACID CONTENT LEADS TO INCREASED LIPID PEROXIDATION AND GLIOMA-CELL KILL Meredith Preuss,1 Terry L. Kaduce,1 Arthur A. Spector,2 and Michael E. C. Robbins,1 1Free Radical and Radiation Biology Program, Department of Radiology, and 2Department of Biochemistry, University of Iowa, Iowa City, IA 52242 Glioma-cell resistance to radiation and other anticancer therapies is proposed to reflect a lack of reactive oxygen species (ROS) generation after treatment. The latter may reflect low polyunsaturated fatty acid (PUFA) content required to initiate ROS generation and propagate lipid peroxidation. We hypothesize that increasing glioma-cell PUFA content will lead to increased ROS generation and lipid peroxidation with a resultant decrease in glioma-cell survival. Comparison of the fatty acid composition of rat 36B10 glioma cells with primary rat astrocytes revealed that the glioma cells contained less PUFA, primarily arachidonic acid (AA) and docosahexaenoic acid (DHA). This deficiency was overcome by incubating the 36B10 rat glioma cells with 20 µM γ-linolenic acid (GLA). This led to a 2.3-fold increase in the PUFAcontent of the glioma cells but only a minor increase in the astrocytes. The rat glioma cells incorporated significantly more ( p < 0.001) radiolabeled GLA than the astrocytes, but there was little difference in the particular fatty acid metabolites produced. Analysis of the media
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330 indicated that the astrocytes formed nearly twofold more chainshortened products as compared to the glioma cells. The increase in PUFAcontent was associated with selective 36B10 glioma-cell kill and increased levels of 8-isoprostane, a marker of lipid peroxidation. Preliminary studies with U87 human glioma cells have shown that 48-h incubations with 30 µM GLA can increase PUFA content from 23.8 to 32.4%. These results suggest that increase in PUFA content may make glioma cells more sensitive to oxidative stress.
ORDER PARAMETER PROFILE OF DOCOSAHEXAENOIC ACID AS DETERMINED FROM DEUTERIUM-NMR EXPERIMENTS A. M. Safley, I. V. Polozov, and K. Gawrisch, Laboratory of Membrane Biochemistry and Biophysics, National Institute on Alcohol Abuse and Alcoholism, National Institutes of Health, 12420 Parklawn Dr., Rockville, MD, 20852 Neural membranes contain high levels of docosahexaenoic acid (DHA; 22⬊6n3), a highly unsaturated ω3-fatty acid. Numerous biochemical, clinical, and behavioral studies have shown the importance of DHAfor brain function. To get an insight into the biophysical basis of its importance, we synthesized deuterium-enriched DHA. The 2H-labeled DHA was incorporated into a lipid matrix and the polyunsaturated chain order parameters were investigated by 2 H-nuclear magnetic resonance (NMR). Six quadrupolar splittings and their intensities were determined. Partial-order parameter assignments were made using magic angle-spinning NMR techniques. The results agree qualitatively with results from DROSS experiment that were previously conducted at our laboratory. In general, order parameters of the polyunsaturated chains are much lower, reflecting both a different bond geometry and increased amplitudes of angle fluctuations for some of the bond angles. We compared DHA order parameter profiles in the presence and in the absence of the membrane protein rhodopsin, a member of G-protein-coupled receptor family. In vivo rhodopsin, a visual receptor, is located in retina membranes, which are very rich in DHA. Observed changes in order parameters suggest some conformational change of the DHA molecule in the vicinity of rhodopsin.
CARDIAC FATTY ACID IMPORT/UTILIZATION IMBALANCE IS ASSOCIATED WITH CARDIOMYOPATHY Hsiu-Chiang Chiu, Attila Kovacs, and Jean E. Schaffer, Center for Cardiovascular Research, Washington University School of Medicine, St. Louis, MO 63110 Fatty acid import into mammalian tissues is highly regulated in accordance with metabolic needs. Inherited disorders of mitochondrial β-oxidation are associated with cardiomyopathy characterized by marked intracellular lipid accumulation in the heart. To test the hypothesis that mismatch between fatty acid uptake and utilization leads to the accumulation of toxic lipid species, and to establish a mouse model of metabolic cardiomyopathy, we generated transgenic mouse lines with cardiac over-expression of long-chain acyl CoA synthetase using the alpha myosin heavy-chain gene promoter (MHC-ACS1). Initial characterization of three independent lines demonstrates cardiac-restricted expression of the transgene. Histologic studies reveal marked cardiomyocyte lipid accumulation in transgenic animals. Accumulation of myocardial lipid in the transgenic animals is associated with initial cardiac hypertrophy beginning at 3 wk of age, development of left ventricular dysfunction and clinical heart failure by 4–6 wk of age, and premature death. Taken together, our data suggest that ACS1 overexpression causes significant increases of fatty acid import into cardiac myocytes and that fatty acid uptake/utilization mismatch leads to accumulation of lipid species toxic to cardiac myocytes.
Journal of Molecular Neuroscience
Abstracts of Poster Presentations EFFECT OF FATTY ACID-BINDING PROTEINS ON INTERMEMBRANE FATTY ACID TRANSPORT: STUDIES ON DIFFERENT TYPES AND MUTANTS J. H. Veerkamp, H. T. B. Van Moerkerk, and A. W. Zimmerman, Department of Biochemistry, University Nijmegen, Nijmegen, 6500 HB, The Netherlands Intracellular fatty acid binding proteins (FABPs) are postulated to be involved in FA uptake and targeting and in modulation of FA concentration and their role in signal transduction, gene transcription, and metabolism. Nine FABP types have been isolated with a characteristic tissue and cellular distribution. We compared the function of 8 FABP types and some heart (H-)FABP mutants in intermembrane FA transport in a system of immobilized liposomes with different charge and isolated rat heart or liver mitochondria. [14C]palmitate oxidation was used as parameter. All FABP preparations were recombinant proteins. The charge of liposomes did not influence the solubilization and mitochondrial oxidation of palmitate without FABP and the solubilized amount in the presence of FABP. Mitochondria did not show preference for oxidation of FABP-bound palmitate from their tissuespecific FABP type. All FABP types increased palmitate oxidation by heart and liver mitochondria with neutral, positive, and negative liposomes 2.5-, 3.2-, and 2-fold, respectively. Ileal lipid-binding protein and inactive H-FABP mutants had no effect. Other H-FABP mutants had different effects, dependent on the site of mutation. In conclusion, the transfer of palmitate from liposomal membranes to mitochondria was increased by all FABP types to a similar extent. The solubilization rate of palmitate from the liposomes to FABP was influenced by their charge.
COMPARATIVE STUDIES ON EIGHT DIFFERENT TYPES OF HUMAN FATTY ACID-BINDING PROTEIN A. W. Zimmerman and J. H. Veerkamp, Department of Biochemistry, University of Nijmegen, Nijmegen, 6500 HB, The Netherlands Fatty acid binding proteins (FABPs) are approx 15kDa cytosolic proteins that facilitate the solubility and intracellular transport of fatty acids. Although the amino acid sequences of the nine FABP family members differ, their backbone structures are virtually identical. We prepared eight recombinant human FABP types: heart (H), brain (B), myelin (M), adipocyte (A), epidermal (E), intestinal (I), liver (L), and ileal lipid-binding protein (I-LBP). These FABP types were tested on their binding of different fatty acids and fluorescent probes, their stability under denaturating conditions, and on crossreactivity of their antisera. All FABP types except B-FABP have a higher affinity for oleic acid than palmitic acid. B-FABP shows about the same affinity for oleic acid and palmitic acid. H-FABP, M-FABP, I-FABP, and B-FABP show a higher affinity for both oleic acid and palmitic acid than L-FABP and A-FABP. I-LBP does not bind oleic acid or palmitic acid. A-FABP and I-FABP show a relatively weak binding of arachidonic acid (AA). The fatty acid binding center is differentially stable under denaturating conditions. Oleic acid binding to H-FABP and I-FABP appeared to be less sensitive to urea compared to other FABP types. All FABP types, except I-FABP and I-LBP, interact with 1-anilinonaphtalene-8-sulphonic acid (ANS), as indicated by a high fluorescence emission. Only L-FABP, I-FABP and, to a less extent, M-FABP show a high fluorescence enhancement with 11-((5-dimethylaminonaphtalene-1-sulfonyl)amino)undecanoic acid (DAUDA), indicating an interaction between FABP and DAUDA. FABP types are differentially affected upon urea denaturation. H-FABP, A-FABP, and I-FABP have a more stable conformation than the other FABP types. Cross-reactivity of antisera raised against different FABP types was determined to elucidate surface properties of
Volume 16, 2001
Abstracts of Poster Presentations the FABPs. H-FABP shows interaction with anti B-FABP antiserum, and A-FABP reacts with anti-H-FABP and anti B-FABP antisera. These results suggest that H-FABP shares epitopes with B-FABP, and A-FABP with H-FABP and B-FABP.
DIFFERENTIATED ASTROCYTES RETAIN THE CAPACITY TO FORM DOCOSAHEXAENOIC ACID FROM N-3 FATTY ACID PRECURSORS Deborah E. Williard,1 Shawn D. Harmon,1 Meredith Preuss,2 Terry Kaduce,1 Steven A. Moore,3 and Arthur A. Spector,1 1Departments of Biochemistry, 2Radiology, and 3Pathology, College of Medicine, University of Iowa, Iowa City, IA 52242 Previous studies indicate that primary cultures of rat brain astrocytes synthesize docosahexaenoic acid (DHA) from n-3 fatty acid (FA) precursors. To determine whether differentiated astrocytes retain this capability, primary astrocytes cultured from the brains of 2-d-old rat pups were exposed to 1 mM dibutyryl cyclic AMP(dBcAMP) for 48 h. GFAP immunofluorescence showed that the dBcAMP treatment induced morphologic changes in the astrocytes that are associated with differentiation. Differentiated cultureswere incubated with 5 µM [1-14C]-18⬊3n-3, [3-14C]-22⬊5n-3, [3-14C]-24⬊5n-3, or [3-14C]-24⬊6n-3 for 48 h and the radioactive cell and medium lipids assayed by highperformance liquid chromatography (HPLC). Much of the DHA formed was retained in the astrocyte lipids, but anywhere from 12.1 to 55.0% was released to the medium. Differentiated astrocytes produced primarily 22⬊5 and 22⬊6 from 18⬊3n-3, although some radiolabeled 16⬊0 and 18⬊0 was also detected. The major product released to the medium was DHA. 22:5n-3 was also converted to DHA, although a substantial amount was retroconverted to 20⬊5. Preformed DHA was readily taken up by the differentiated astrocytes, and when DHA was available to the cells, the conversion of 18⬊3 to 22⬊6 was decreased. These results indicate that differentiated astrocytes retain the capacity to synthesize DHA from n-3 FA precursors, but the amount of 18⬊3 converted to DHA is affected by the availability of preformed DHA. While the differentiated astrocytes retain much of the newly formed DHA, a significant amount is released into the extracellular fluid. Pre-
Journal of Molecular Neuroscience
331 sumably, the released DHA is available for uptake by other cells of the brain, including neurons. (Supported by NM HL 49264.)
INTRAVENOUSLY INJECTED [1-14C]ARACHIDONIC ACID TARGETS PHOSPHOLIPIDS AND [1-14C]PALMITIC ACID TARGETS NEUTRAL LIPIDS IN HEARTS OF AWAKE RATS Eric J. Murphy, Thad A. Rosenberger, and Stanley I. Rapoport, Section on Brain Physiology and Metabolism, National Institute on Aging, National Institutes of Health, Bethesda, MD 20892-1582 The differential uptake and targeting of intravenously infused [1- 14 C]palmitic ([1- 14 C]16⬊ 0) and [1- 14 C]arachidonic ([1- 14 C] 20⬊4 n-6) acids into heart lipid pools was determined in awake adult male rats. The fatty acid tracers were infused (l70 µCi/kg) through the femoral vein at a constant rate of 0.4 mL/min over 5 min. At 10 min post-infusion, the rats were killed using pentobarbital. The hearts were rapidly removed, washed free of exogenous blood, and frozen in dry ice. Arterial blood was withdrawn over the course of the experiment to determine plasma radiotracer levels. Lipids were extracted from heart tissue using a two-phase system and total radioactivity was measured in the nonvolatile aqueous and organic fractions. Both fatty acid tracers had similar plasma curves, but were differentially distributed into heart lipid compartments. The extent of [1- 14C]20⬊4 n-6 esterification into heart phospholipids, primarily choline glycerophospholipids, was elevated 3.5-fold compared to [1-14C]16⬊0. The unilateral incorporation coefficient, k*, which represents tissue radioactivity divided by the integrated plasma radioactivity, for heart phospholipid was 7-fold greater for [1-14C]20⬊4 n-6 than for [1-14C]16⬊0. In contrast, [1-14C]16⬊0 was esterified mainly into heart neutral lipids, primarily triacylglycerols and was also found in the nonvolatile aqueous compartment. Thus, in rat heart, [1-14C]20⬊4 n-6 was primarily targeted for esterification into phospholipids, while [1-14C]16⬊0 was targeted for esterification into triacylglycerols or metabolized into nonvolatile aqueous components.
Volume 16, 2001