Lipidomics comes of age
By Rebecca Guenard
- The lipidome is the term for the wide range of lipids found in biological systems. For decades, researchers have been identifying and classifying these molecules.
- Lipids participate in most important biological processes. They compose cell membrane structures and organellar membrane structures, where they facilitate intracellular trafficking and cell-signaling.
- More sensitive analytical techniques allow today’s lipidomics researchers to examine the interactions of lipids with other types of biological molecules in a variety of human diseases.
- By studying lipid metabolism and signaling in human cells, researchers are discovering important aspects of human diseases like cancer, cardiovascular disease, neurological disorders, and diabetes that will lead to the development of new treatments.
Scientists spent 30 years focusing their molecular biology experiments on DNA, RNA, and proteins. In the past decade, lipids research has gained more attention. Now, biological lipids are getting the spotlight as potential solutions to medical, nutritional, and cosmetic challenges. As scientists use lipidomics to sift through the thousands of molecules that make up the lipidome, they are increasingly realizing that lipids do much more than just serve as an energy source or a cell barrier for proteins.
“What we have started to realize is that lipids and their chemical diversity are very important to organizing cellular function,” says Anne-Claude Gavin, a biochemistry professor at the University of Geneva in Geneva, Switzerland, who studies lipid metabolism and cellular membrane regulation. Lipids are made up of a limited number of simple chemical building blocks, but they have the potential to generate up to 100,000 different molecular species.
“Lipids are more difficult to study than proteins, which have a genetic code that provides instructions on how the molecules should operate,” says Gavin. Despite this, the past 10 years, have been fruitful for lipidomics researchers. Mass-spectral techniques, including three-dimensional analysis that maps densities, make it possible to evaluate the role of lipids in human cells.
Consequently, researchers are now teasing apart the lipids that serve critical biological roles, identifying their distinctions, and developing targeted products for anti-aging cosmetics and treatments for disease. As lipid-focused consumer products become a reality, lipid scientists are turning their interests to bigger questions, such as: Can lipidomics help us understand the relationship between nutritional and biological lipids? Some seem confident that it can, and that nutritional lipids will be a prominent focus of lipidomics in the coming years.
Biological lipids represent a broad class of compounds involved in a range of cellular roles. Lipids both comprise a cell’s membranes and direct the membrane’s traffic. Proteins attach to a membrane surface or nestle within, but their tasks depend on the lipid composition. Lipids also form clusters, called rafts, that accommodate specific types of proteins and regulate their use. To begin to understand the diversity of lipid functions, scientists needed a way to categorize the corresponding variations in lipid molecular structures.
Genes are mostly constructed from only four nucleic acids, and proteins are made from 20 amino acids. Lipids have comparatively more building blocks, resulting in far more structural options. Lipids are a heterogeneous set of molecules sharing the common property of being insoluble in water. In the early 2010s, a consortium set out to wrangle different classification schemes into one system that would be useful for a bioinformatics approach to lipids (doi:10.1016/j.bbalip.2011.06.009). The consortium redefined lipids as hydrophobic or amphipathic molecules originating from two groups: 1. carbanion condensations of ketoacyl thioesters or 2. carbocation condensations of isoprene. From these two groups, lipids can be divided into eight categories. Six are produced from ketoacyl subunits (fatty acyls, glycerolipids, glycerophospholipds, sphingolipids, saccharolipids, and polyketides), while two (sterols and prenols) are derived from isoprene units (Fig. 1). These categories are further divided to distinguish lipid chemistry and function. Like sorting the edge pieces before starting on a challenging jigsaw puzzle, researchers could begin to build the picture of lipids within eukaryotic cells.
The spectrum of lipids in cells are known to originate mostly from the endoplasmic reticulum, with contributions from a few organelles like mitochondria and peroxisomes. Much of the current lipidomics focus is on understanding how lipids are sorted and distributed after synthesis. Sterols and sphingolipids, for example, concentrate at the cell’s surface, although they are formed deep within the cell.
“Lipids accumulate in very specific areas in cellular systems. This is absolutely key to cell biology,” says Gavin. These lipid gradients generate cellular organization, but researchers still do not know exactly how these gradients are created and maintained, she says. In the past, proteins were believed to be the sole operators in transportation, but it is becoming clear that they work in unison with lipids to perform the task. Gavin maps lipid highways to understand transport schemes. “We can measure lipids inside the cell and follow their metabolic fate,” she says.
Metabolic enzymes also offer clues about how lipid gradients form. For a long time, the location of enzymes in different membranes and organelles seemed to indicate a compartmentalization of lipid metabolism, Gavin says. “For example, you take a very simple lipid, phosphatidylserine. It is made in the lipid factory, the endoplasmic reticulum,” she says. “But then this lipid is required in the mitochondria, where it will be decarboxylated to phosphatidylethanolamine.”
Her lipidomics analysis revealed that lipid-transfer proteins bind more than one lipid species. This result indicates a high-level management of lipid metabolism within the cell. “The community’s hypothesis is that the proteins act as a lipid exchanger, taking a lipid in one direction and then a different lipid in the other direction,” says Gavin. “This is a very interesting concept because it would suggest that metabolism is integrated.” In other words, cell organelles coordinate the production and movement of lipids as if they were parts in a manufacturing process. Her team will continue to elaborate on these recent findings.
According to Gavin, lipid-transport proteins represent an emerging lipidomics research area. At least 131 lipid-transfer proteins have been found in humans so far. The complexity of organizing lipids and connecting their metabolic pathways means that lipid-transfer proteins can make mistakes. Researchers now understand that this is the case in Gaucher disease and Farber disease, and they are considering treatments. Gavin also sees lipid-transfer proteins as a potential avenue for drug delivery.
“Some of those lipid transporters are known to bind drugs like ibuprofen,” Gavin, explains. “If we could learn more about how the protein binds hydrophobic drugs, it could be an interesting drug-delivery mechanism.” She says a drug could target the proper cell or even the proper cellular compartment.
Gavin is able to study lipids in detail because of a high-throughput liposome microarray-based assay (LiMA) her group developed that allows researchers to quantify and image protein-lipid interactions. Specialized techniques like this are necessary since the properties of lipids make them difficult to study in a systematic manner.
For example, fatty acids are known to attach to proteins and change their signaling behaviors. To study these lipid-based modifications, researchers developed fluorescence-labeled fatty acid and isoprenoid probes. The probes can be incorporated into the cell through its own biosynthetic processes. Once inside, researchers can track the probe to study how lipids modify certain proteins.
Typical biotechnology tools—like overexpression, which is used to study genes—are not useful for studying lipids, because their population increases non-selectively. To target specific, low-abundance lipids and analyze them without getting confounding signals from other lipids, scientists feed labelled precursors of lipids to cells. Using these techniques, lipidomics is being conducted in live cells, organoids, and tissues that represent an authentic lipid environment.
“Twenty, twenty-five years ago, it was really tough to work with these lipids,” says Besim Ogretmen, a biochemistry professor at the Medical University of South Carolina, in Charleston. “We were mostly dependent on analogs of lipid molecules.” Lipidomics researchers now combine organic and analytical chemistry with genetics. “So instead of looking at lipid analogs, we can actually change the synthesis in the cells,” he says.
Along with new synthesis tools, the field has benefited from advances in mass- spectrometry. Electrospray ionization first made it possible for lipids and biological compounds to be analyzed by mass-spec. Other vaporization methods use high temperatures that decomposed the compounds before they could be analyzed. When electrospray ionization became established, the National Institute of Standard and Technology (NIST) started developing methods to analyze lipids.
“About 15 years ago, we began building a tandem mass-spectral library of ions formed in electrospray which, of course, includes lipids or almost anything that is an organic molecule in a biological system,” says Stephen Stein, director of the Mass-Spectrometry Data Center Group. His colleague and project leader, Xiaoyu Yang, mentioned that this mass-spectral library now contains 1.3 million mass spectra for more than 30,000 compounds, including various lipids such as steroids, phospholipids, and glycolipids. The standards have been collected from commercially available lipids, as well as metabolites from human samples.
Imaging mass-spectrometry has especially contributed to lipidomics analysis, because it reveals the spatial distribution of lipids in tissue. In a complex cellular context, such as liver or muscle tissue, the technique can be used to compare healthy and diseased tissue to reveal how lipid composition differs. This type of analysis would be difficult to do with classic techniques, because one would need to average results from millions of cells.
“Using MALDI (matrix-assisted laser desorption ionization) imaging coupled with liquid chromatography/mass-spectrometry, we are now able to follow lipid-based compounds or their metabolites to identify the tissues where they are localized, and how much they accumulate in a given experiment,” says Ogretmen.
The resolution is not yet subcellular, but it is getting closer. Scientists will soon be able to obtain information on lipid dynamics—like the arrangement of lipid head groups—within cell membranes. Understanding the intricacies of lipids at the cellular level has primed the field for a flood of new studies centered on health and nutrition.
“We have started our focus on intercellular transporters,” says Gavin. “But, of course, there is an entire world of secreted transporters that deliver lipids from peripheral tissues to the liver or, in the brain, between astrocytes and nerves.”
A variety of research facilities are focused on understanding the impact of different types of lipids: glycolipids, phospholipids, and cholesterol, for example. However, studies indicate that sphingolipids, found in cell membranes, play an important role in aging and its related diseases. These lipids are converted into sphingosine-1-phospate and ceramide, which perform the opposing duties of cell proliferation and cell death (Fig. 2). Biological development and homeostasis require the critical process of balancing these lipids. It is now clear to researchers that a shift can lead to cancer and other illnesses.
“One important aspect of these sphingolipids is that they are stress-response molecules,” says Ogretmen. “They signal the production of lipids in response to aging.” Ogretmen employed lipidomics to follow the metabolic processes of sphingolipids and identify the enzymes that are responsible for ceramide production. He then genetically altered mice so the machinery that manufactures the enzymes was turned on or off. “Instead of targeting the lipids, we went after the enzymes that metabolize them,” he says.
By this means, Ogretmen developed anticancer therapies that regulate ceramides through the enzymes that build or destroy them. “We are conducting a clinical trial for one of these enzyme inhibitors for prostate cancer patients,” he says. “The idea is to inhibit the enzymes that eliminate ceramide signaling to improve cell death pathways in cancer cells.” His clinic is currently recruiting patients for a phase 2 trial of this therapy. In addition, they are investigating Alzheimer’s disease, immunotherapy, and aging (Fig. 2).
On the horizon
Building the lipidomics knowledge base to begin answering questions surrounding human health and disease has been a slow process. But now that libraries are being compiled and the necessary analytical tools are being established, researchers are eager to use what they have learned.
“We have really struggled to understand these lipid molecules, to identify enzymes, their metabolic sites, and how they are regulated,” says Ogretmen. “Now that we have all these tools, people will start asking those important questions about diet and environmental factors that affect lipid metabolism and signaling.” He says, these include a myriad of age-related diseases, such as, cancer, neurodegeneration, cardiovascular dysfunction, diabetes, and obesity.
Aging is accompanied by changes in lipid levels and in their fatty acid composition, notably desaturation. Specific chemical properties, like solubility and fluidity, are determined by the level of saturation along the lipid chain. Unsaturation also makes lipids more susceptible to oxidative damage. Hence, polyunsaturated fatty acids, which contain multiple double bonds in their carbon chains, are more susceptible to oxidation than monounsaturated fatty acids. Oxidized lipids are particularly detrimental to cellular function. Studies show that these effects have an influence on cell membranes and that longevity coincides with unaltered cell membranes. What dietary choices or environmental precautions should consumers make to protect their cellular lipids? Are there products that can reverse aging effects? These are questions that will be answered by lipidomics in the coming years.
For example, a diet rich in omega-3 fatty acids has the known benefit of lowering cardiovascular disease; however, little is known about how the lipid content of the diet affects the lipid composition of cell membranes. So far, researchers have studied the most common lipid classes, such as triacylglycerols, cholesterol, cholesterol esters, major glycerophospholipids, and ceramides. Profiling of the molecular composition of the plasma lipidome suggests that a few species of relatively low-abundant lipid classes may be involved in diet-related disorders. To really know, Gavin says it is time to put together the equivalent of a genetic code for lipids. “It is absolutely critical now that we have a comprehensive list of all the body’s lipids. That we really start to compile the human lipidome,” she says.
After building its foundation for decades, the field of lipidomics is posed to answer long-standing questions surrounding human aging and disease.
Rebecca Guenard is the associate editor of INFORM at AOCS. She can be contacted at email@example.com.
Identification of metabolites from tandem mass spectra with a machine learning approach utilizing structural features, Li, Y., et al., Bioinformatics 36: 1213–1218, 2020.
Dietary lipids, gut microbiota, and lipid metabolism, Schoeler, M. and R. Caesar, Rev. Endocr. Metab. Disord. 20: 461–472, 2019.
Linking lipid metabolism to chromatin regulation in aging, Papsdorf, K. and A. Brunet, Trends Cell Biol. 29: 97–116, 2019.
Chemical biology: fats as research subjects, Marx, V., Nat. Methods 15: 35–38, 2018.
The effect of altered sphingolipid acyl chain length on various disease models, Park, W. and J. Park, Biol. Chem. 396: 693–705, 2015.
Lipid classification, structures, and tools, Fahy, E., et al., Biochim Biophys Acta. 1811: 637–647, 2011.