Klaus Gawrisch , PhD, Chief
National Institute on Alcohol Abuse and Alcoholism
National Institutes of Health

5625 Fishers Lane, Room 3N07:MSC 9410
Bethesda MD 20892-9410
telephone: +1 301.594.3750

fax: +1 301 594.0035
e-mail: klausg@mail.nih.gov

 

The mission of the Laboratory of Membrane Biochemistry & Biophysics (LMBB), established in 1992, is to study the alterations in cell membrane structure and function caused by alcohol abuse with a focus upon polyunsaturated lipids. The lab has a particular emphasis on the most highly unsaturated essential fatty acid found in mammalian tissues, docosahexaenoic acid (DHA). This fatty acid typically occurs as a phospholipid in brain and retinal membranes where it is highly enriched, thus these systems are of great interest to those within the laboratory. A nutritional approach to membrane structure and composition is often taken both with respect to essential fatty acid profiles and alcohol content since these are powerful modulators of lipid content and membrane properties.

Methodology

The LMBB is truly multi-disciplinary in its methodological approaches which include psychiatry, nutritional neuroscience, biochemistry and spectroscopy. Clinical studies are performed with the cooperation of the clinical laboratory within our division. These currently involve studies of PET imaging using the novel ligand 11C-DHA as well as a randomized, placebo controlled trial of the effects of EPA/DHA feeding on craving, impulsivity and relapse in aggressive alcoholics. Recent metabolic trials have included the use of orally administered stable isotopically labeled essential fatty acids in adult alcoholics and controls as well as in newborn infants. Biochemical studies include molecular biological techniques as well as studies of essential fatty acid metabolism using sophisticated tracer techniques and NCI GC/MS as the detection tool, methods developed within our laboratory. Artificial membranes are formed using traditional liposomes where proteins like rhodopsin and the peripheral cannabinoid receptor, CB2, are incorporated, as well as newer technology developed within the NMR Section using flow-through lipid nanotube arrays made up of anodic aluminum oxide. Receptor activation can be then studied using ligand binding and G-protein activation assays as well as various spectroscopic techniques. A range of biophysical studies are conducted using solid state NMR, including magic angle spinning in combination with multidimensional NMR techniques, NMR diffusion measurements with application of pulsed magnetic field gradients. Our principal equipment includes 500 and 800 MHz nuclear magnetic resonance spectrometers. The laboratory is also equipped with differential scanning and isothermal titration calorimetric equipment, time resolved and steady state fluorimeters and circular dichroism spectrometers. The LMBB is also pioneering the application of robotic technology for chemical derivatization as part of the development of high throughput methods for fatty acid analysis. It can be seen then that a full spectrum of approaches is taken to study the functions of polyunsaturated lipids ranging from the clinical to NMR studies at the molecular level.

LMBB consists of three sections: the Section on Nutritional Neuroscience which is led by CAPT Joseph R. Hibbeln, M.D; the Section on Nuclear Magnetic Resonance, led by Klaus Gawrisch; and the Section on Molecular Biology, led by B. J. Song. Laboratory administrative personnel are within an Office of the Chief.


Accomplishments

The activities of the individual sections are described separately in greater detail. Major contributions of the LMBB include:

1. Developed new mass spectrometric methods for tracer labeling and detection of essential fatty acid metabolic pathways. These methods, developed in rats, were rapidly translated to the clinic for human application. This technology has been greatly expanded recently to include simultaneous labeling of four segments of the essential fatty acid metabolic network of pathways so that they can be simultaneously monitored. Mathematical modeling of the pathways has also been pioneered.

2. Demonstrated for the first time that human infants can elongate and desaturate alpha-linolenic acid to DHA in vivo. This was important for policy making in terms of infant nutritional requirements for optimal brain development as infant formulas contain only the DHA precursor, though the brain structure uses only the DHA, itself. The demonstration that even premature infants are competent for the in vivo metabolism of essential fatty acids was received with great interest, though the low level of such activity indicated that metabolism was inadequate to provide the DHA necessary for the nervous system. Quantitative studies of metabolism vs. incorporation of preformed DHA indicate that the latter is the overriding source of brain and liver DHA and thus is a dietary requirement.

3. It has been demonstrated that alcoholics as well as animals exposed to alcohol lose DHA and other polyunsaturated lipids from their brains, livers and other tissues. These studies have also been extended to pregnant women who drink alcohol, as their blood has been observed to contain lower levels of DHA when they drink on a daily basis.

4. An animal model has been developed whereby brain DHA can be manipulated using dietary means. A deficiency in brain DHA has been shown to lead to deficits in learning in, for example, spatial tasks, olfactory discriminations and reversal learning. This may in part be related to a greater stress response in n-3 deficient animals.

5. A robotic system has been devised that automates the rather laborious transmethylation and extraction procedures for the analysis of fatty acids in plasma samples. Fast GC methodology has also been devised so that these two procedures together form the basis of a high throughput system that can potentially increase our productivity by 30-fold.

6. Dietary intakes of long chain omega-3 fatty acids that meet criteria for putative Dietary Reference Intakes (DRI’S) have been calculated from cross-national and epidemiological data sets. Large differences in the mortality rates from cardiovascular disease, stroke and all cause mortality prevalence as well as several psychiatric disorders amongst populations with high or low measures of seafood consumption.

7. The relative risks and benefits of fish consumption during pregnancy were evaluated in a large epidemiological study. Women whose seafood consumption during pregnancy were in concordance with the limits advised in 2004 by the FDA and EPA, create nutritional inadequacies for their children during early development which were associated with low verbal IQ and deviant prosocial and peer behaviors.

8. A key mechanism of action for DHA in the nervous system has been discovered – the regulation of rhodopsin signaling by DHA species of phospholipids. Moreover, a general mechanism for the regulation of G-protein coupled receptors (GPCRs) has been proposed based on this work as an explanation for brain functions of DHA. In addition it has been shown that the reduced rhodopsin signaling in membranes high in cholesterol is exacerbated by the replacement of DHA with omega-6 polyunsaturates, as occurs during omega-3 deficiency.

9. NMR experiments indicate that polyunsaturated fatty acids like DHA are highly flexible molecules that convert rapidly between a multitude of conformations. It appears that flexibility and adaptability of polyunsaturated fatty acids impart unique elastic properties on lipid bilayers which are likely to ease conformational transitions of GPCR upon their activation.

10. We have recently shown that lipids with polyunsaturated hydrocarbon chains bind to particular sites on bovine rhodopsin. Such interactions could allow lipids to serve as cofactors in rhodopsin activation. The GPCR rhodopsin adjusts its structure to the properties of the lipid matrix far more nimbly than generally assumed. With increasing hydrophobic thickness of bilayers an increase of helical content was observed. Photoactivation of the receptor did not alter membrane thickness near the protein.

11. Human peripheral-type cannabinoid receptor, CB2, was expressed in E. coli, purified, and successfully reconstituted into lipid bilayers. The recombinant CB2 retained its ligand binding properties. Expression, purification, and functional reconstitution of milligram quantities of the recombinant receptor for structural studies was achieved.

12. The properties of the liquid ordered phase state of lipids that have been implicated in formation of so-called rafts were characterized by NMR. We obtained evidence for critical behavior in cholesterol-containing membranes at physiological temperatures and concentrations of cholesterol in membranes. It may explain the small size and transient nature of cholesterol- induced domain formation. The feasibility of detection of liquid ordered states in the plasma membrane of cells by 1H NMR with magic angle spinning was demonstrated. The method was successfully applied to study ordered phase formation in the membranes of flu viruses.

13. Using novel NMR techniques, the ethanol-binding sites on membranes with atomic- scale resolution were resolved and the dynamics of lipid-ethanol associations were studied. Interaction of ethanol with membranes is primarily controlled by polar interactions, like hydrogen bonding to lipid phosphate groups.

14. Signaling mechanisms underlying mitochondria-dependent apoptosis caused by CYP2E1 substrates such as ethanol, acetaminophen and anti-cancer agents have been elucidated. These agents strongly activated JNK and/or p38 kinase-related cell death pathways and translocation of pro-apoptotic Bax to mitochondria. We recently identified a causal relationship between activation of JNK or p38 kinase and phosphorylation of Bax prior to its translocation to mitochondria and apoptosis. This approach with systematic analysis of JNK/p38 kinase activation and translocation of Bax to mitochondria has been successfully applied to study the mechanism of ethanol-induced apoptosis of human colon cells.

15. An efficient method to identify and characterize oxidatively-modified proteins was developed. This method has been used to identify such proteins in animal models of alcoholic fatty liver as well as non-alcoholic liver diseases caused by ischemia-reperfusion or MDMA (3 ,4-methylenedioxymethamphetamine, ecstasy) a frequently abused drug. We also demonstrated the beneficial effects of polyunsaturated fatty acids, including physiologically relevant concentrations of docosahexaenoic acid and arachidonic acid, for reducing alcohol- mediated oxidative stress, mitochondrial dysfunction and fat accumulation (fatty liver). Furthermore, our sensitive method is being utilized to identify oxidatively-modified proteins (cytosolic and mitochondrial fractions) in autopsied brain samples from human alcoholics and Alzheimer’s disease patients compared to age- and gender-matched controls.

16. In collaboration with Dr. Frank J. Gonzalez, NCI, NIH, we use peroxisomal proliferator activated receptor a (PPARa)-null mice and CYP2E1-null mice, respectively, to delineate the molecular mechanisms of alcohol and drug-induced mitochondrial dysfunction, fat accumulation, inflammation, and subsequent organ damage (hepatitis with fibrosis/cirrhosis potentials) as new mouse models of alcoholic and non-alcoholic fatty liver diseases.