Long-suffering Lipids Gain Respect

Courtesy of Paul Bertone, Arno Grbac, Heng Zhu, and Michael Snyder Scientists who add detergents to their cell preps, take heed: You might be consigning the most interesting stuff to the trash bin. Not proteins, surely, or nucleic acids, but lipids. A class of molecules united by their common solubility in organic solvents, lipids are like the poor relations among wealthier biological macromolecules. "Researchers are familiar with nucleic acids, RNA, proteins, and maybe even carbohydrates, but usually not with lipids," says Zheng Cui, a biochemist at Wake Forest University School of Medicine. "Lipids are probably the most ignored type of molecules by the entire field of biological sciences, and yet they play a key role in many of the functions of the body." For example, lipids function as both energy stores and signaling molecules, and they literally define cells, separating the intracellular from the extracellular components with an impermeable bilayer. Now, growing recognition of these facts, coupled with technology advances and rising concerns over lipid-associated disorders such as obesity, heart disease, and noninsulin-dependent diabetes, has yielded a new, loosely defined field of research called lipidomics. The term describes diverse research areas, from mapping the entire spectrum of lipids in a biological system--a vast effort that currently no one is known to be attempting--to describing the function and metabolism of individual lipids. "The realization that lipids play pivotal roles in regulating a wide variety of cellular processes has been regarded as one of the major advances in modern biology over the past two decades," says Kansas State University biochemist Xuemin Wang, who runs the Lipidomics Research Center with coinvestigator Ruth Welti. Wang notes that better understanding of lipids is important to addressing many fundamental and applied questions, including why subcellular membranes possess different lipid compositions; the significance of membrane microdomains bearing distinct lipid constituents and functions; and how the compositions of lipid membranes change in response to signals like those from hormones, environmental changes, and pathogenic infections. "Knowing how these distinct compositions and dynamics are generated and what their functions are in the cell will lead to a better understanding of how a cell or organism grows, differentiates, and responds to its environment," Wang predicts. "Identification of the steps regulated by such changes and specific lipid messengers may lead to a better control and manipulation of an organism in its productivity." LIPIDOLOGY 101 Scientists believe that more than a thousand major lipids exist in the human body, along with an unknown number of less prevalent, minor species. Major lipid classes include triacylglycerols, phospholipids, glycolipids, sphingolipids, sterols, waxes, and fatty acids. Courtesy of A. Prokesch, Institute of Biomedical Engineering, Graz University of Technology, Graz, Austria Bone marrow stromal cells (large image) and preadipocytes (inset) induced to differentiate into fat cells. In bone marrow stromal cell differentiation, lipid vacuoles were stained with Oil Red O; cytoplasm was counterstained with light green. In preadip-ocyte differentiation, lipid vacuoles (spherical structures) were made visible using phase-contrast microscopy. In recent years, evidence has emerged of the powerful role of lipids in cellular functioning. Lipids are now recognized as potent players in signal transduction, vesicular trafficking, secretion, and cytoskeletal rearrangements. They are also key activators of specific transcription factors. "Without lipids, DNA would never be decoded into life forms," Cui says. Recently, sphingolipids have emerged as a rich source of molecular bioactivity, according to Yusuf A. Hannun, a molecular biologist at the Medical University of South Carolina. An estimated 300 to 400 distinct species of sphingolipids exist in the human body, with specific enzymes to create and degrade each one, Hannun says. He explains that each enzyme, if subject to regulation, could function as a signaling module, and that these biochemical pathways allow for great versatility in sphingolipid biology, especially when one considers functional integration of these diverse pathways.1 DIVERSITY AND FUNCTION Quantifying and analyzing the many lipids in an organism are complicated by the fact that different tissues and organs contain different lipids, and different cell types and subcellular organelles have unique lipid complements. Adding complexity, these molecules are not evenly distributed; relatively homogeneous membranes are dotted with distinct microdomains called lipid rafts. "In theory, all you need to make a biolayer membrane is one molecular species of phosphatidylcholine," says Cui. "But then why does the cell make so many other lipids? There must be a reason." In his lab, Cui studies the biological relevance of individual lipids using tandem mass spectrometry, which allows him to quickly profile, organize, and analyze known molecules of interest--such as the lipid phosphatidylcholine--directly from crude organic extracts of cells. A similar profiling strategy is applicable to other lipid classes.2 At the Max Planck Institute of Molecular Cell Biology and Genetics, cell biologist Christoph Thiele focuses on the functional implications of lipid diversity. In his functional lipidomics laboratory, as he calls it, Thiele approaches lipids on three levels. First, he characterizes the lipid composition of different cell types and organelles using a combination of liquid chromatography and mass spectrometry. Secondly, Thiele uses this data to establish localization and trafficking information and to identify interaction partners for the lipids. And third, he manipulates the levels of these molecules to reveal their functions. Thiele says his research has begun yielding insights into the different roles specific lipids play in the body, but concedes the process has been slow and technically difficult. A GENOMICS APPROACH The SFB Biomembrane Research Center, a multi-institutional group of European scientists, is leading a discovery-driven effort to understand the mechanisms controlling lipid and energy metabolism, particularly in relationship to lipid-associated disorders. Drawing together scientists from biochemistry and biophysics, electrophysiology, genetics, molecular and cellular biology, genomics, and bioinformatics, the SFB Biomembrane Research Center is attempting to elucidate the mechanisms of membrane assembly, function, and interaction with other cellular components. The center's investigators are developing cDNA microarrays to identify genes that are misregulated in mouse models of obesity and diabetes (an atherosclerosis model is in the works, as well). These mice are deficient for certain enzymes involved in lipid metabolism, so studying their gene expression profiles may provide a better understanding of lipid function in disease, says Zlatko Trajanoski, a biomedical engineer at the Graz University of Technology who is in charge of the center's microarrays. Yale University cell biologist Michael Snyder also uses microarrays to answer biological questions about lipids. Adopting a proteomics approach, he developed protein chips for yeast to dissect biochemical activities involving certain lipids. "We decided that it would be a high priority to determine ... all the lipid-binding proteins in yeast," Snyder says, "because lipids are involved in a huge range of processes." They have known structural and signaling roles, for example, but lipids are also involved in many other processes, and identifying these can be challenging, given the relative dearth of information. Snyder and his colleagues over-produced nearly all the yeast proteins and screened for phosphoinositide (PI)-binding proteins.3 Key components of cell membranes also function as second-messengers, regulating such processes as growth, differentiation, and membrane trafficking. The results, says Snyder, were unanticipated. "One of the big surprises was that most of the protein kinases showed specificity for the phosphoinositides, suggesting that many of these protein kinases are regulated by specific phosphoinositides. If that is the case, that means that these lipids are controlling lots of different signaling pathways," he says. The team was further surprised to learn that many glycolytic proteins, such as hexokinase, bind lipids. Extrapolating from these findings, Snyder sees two possible biological explanations: phospholipids either regulate these enzymes to coordinate lipid and glucose metabolism, or lipids serve as a structural scaffold for many steps of glycolysis. Snyder says he would like to follow up this work to find out whether lipids stimulate or accelerate glycolysis by acting as a solid surface. "At the end of the day, the hope is that we would know all of the lipid-binding proteins for all of the different phospholipids proteins in yeast. It's very possible. It's simply a matter of probing with all the lipids that you can get your hands on," he says. Courtesy of Yusuf A. Hannun DON'T TRIM THAT FAT! Scientists are only just beginning to recognize the complexity of sphingolipid signaling and function that arises from the topology of lipid metabolism. DATA DELUGE The research is slowly revealing clues about lipids, but it is also yielding reams of unanalyzed data that can be produced literally overnight. This data deluge is daunting to Snyder and others in the field. "The hard part now is not the probing of the microarray chips, it's analyzing and confirming the data," Snyder says. Cui agrees. "The pages after pages of data with all these [mass spectrometry] peaks: What do you do with it? The next challenge is how are we going to organize this data in a meaningful way so that people can use it?" The SFB Biomembrane Research Center has more than 10 informaticians working on this problem, says Trajanoski. "We need some system that will organize the data and allow us to associate different data sources," he says. The center's researchers are producing piles of lipid data that need to be stored, analyzed, and integrated with other information such as phenotypic, metabolic, and microscopy data, he says. "Someone knowledgeable can look at the data and extract some information just by looking at it, but these people are not available all the time and they cannot look at tens of thousands of genes at the same time," says Trajanoski. LIPIDOME MAPPING? Large-scale study of lipids and mapping an organism's entire lipidome is now theoretically possible owing to recent technology advances, researchers say. But even today, it would be labor intensive and time-consuming, and it is a low priority for most lipid researchers who want to do more than just profile various lipids, notes Cui. Scientists also want to make sense of the importance of individual lipids to an organism's overall health, he explains. All the same, researchers are intrigued by the idea of a lipidome. "If we view everything together as a whole picture, we may be surprised at how powerful lipids are," Cui says. "A logical way to find out how lipids work and how they influence our health is to map all the lipids and organize the profiles so that lipids can be analyzed in the context of biology, physiology, and other equally important biomolecules, such as proteins and carbohydrates. 'Lipidomics' or 'lipid informatics' would be a good step toward answering in a systematic way the questions that remain," Cui says. And as KSU's Wang notes, "It probably isn't possible right now to measure every lipid. New lipids, presumably involved in cellular regulation such as signal transduction, are being discovered regularly in plants and other organisms." His research group is focusing first on mapping polar (membrane) lipid species and their metabolites. Nevertheless, some commercial companies are moving towards full-fledged lipidomics. West Sacramento, Calif.-based Lipomics Technologies recently published a study that examined lipid metabolite profiles in plasma and tissues from diabetic mice treated with the drug rosiglitazone.4 Researchers separated lipid classes using thin-layer chromatography and identified individual metabolites with gas chromatography. At Cytochroma, a Markham, Ont.-based biotech firm, researchers focus on lipids that are formed from P450 enzyme activity, but they plan to sequentially move on to mapping all lipids in humans, according to Alvin Berger, manager of LIPIDOMICS™. The company uses high performance liquid chromatography and existing mass spectrometry techniques to obtain lipid maps and structural information as well as molecular weight information. "Our final goal is to map all lipids. With this and related techniques, we believe that we can eventually accomplish this goal," says Ma'an Amad, Cytochroma's mass spectrometry expert. There is still much to learn, but much has already been revealed. "We're already starting to make enormous progress in understanding how lipids affect diverse functions," Berger says. "We're beginning to understand how lipids work and why they're able to affect so many different signaling cascades in the body." Jennifer Fisher Wilson (jfwilson@snip.net) is a contributing editor. References 1. Y.A. Hannun et al., "Enzymes of sphingolipid metabolism: From modular to integrative signaling," Biochemistry, 40:4893-903, 2001. 2. C.J. DeLong et al., "Molecular distinction of phosphatidylcholine synthesis between the CDP-choline pathway and phosphatidylethanolamine methylation pathway," J Biol Chem, 274:29683-8, 1999. 3. H. Zhu et al., "Global analysis of protein activities using proteome chips," Science, 293:2101-5, 2001. 4. S.M. Watkins et al., "Lipid metabolome-wide effects of the PPAR* agonist rosiglitazone," J Lipid Res, 43:1809-17, 2002.

Long-suffering Lipids Gain Respect

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