Finding genes that regulate fat storage by special label-free microscopy
Two lasers are better than one in a new technique called label-free stimulated Raman scattering microscopy that allows researchers at Baylor College of Medicine and Harvard University to identify the genes that regulate fat storage in the body.
In a report in a recent issue of the journal Nature Methods, Dr. Meng C. Wang, assistant professor in the Huffington Center on Aging at BCM, and collaborators at Harvard University and Harvard Medical School used this new technique to identify eight new genes that regulate fat storage.
With a growing concern about obesity and the increases in type 2 diabetes world-wide, the ability to understand lipid and its accumulation is increasingly important, Wang said.
"If you cannot understand on the basic level how lipids are regulated and how their storage is regulated, it will be impossible to move forward to address important medical questions surrounding obesity," she said.
"My primary research is on lipid (or fat) metabolism in aging," she said. As people get older, they complain that they gain weight and they are eating the same things they did in younger years. Even though some may argue that older people exercise less, Wang said that even those who maintain their activity levels still accumulate fat in different ways.
Unsaturated fats vs. saturated fats
Perhaps some lipids are more important than others, she said. Unsaturated fats are one class of lipids; saturated fats are another. Her question is, which one is most important?
She has found the new microscopy technique combined with important properties of the transparent worm Caenorhabditis elegans (C. elegans) valuable in studying the problem.
First, she wanted to see how the lipids are stored in living animals.
Determining how best to do that started with the laboratory in which Wang did her post-doctoral studies at Harvard. There, she and her collaborators in the laboratory led by Dr. Sunney Xie of Harvard Medical School, applied a new technique called stimulated Raman scattering microscopy on lipid imaging. It is based on the use of a laser to excite molecules. The technique was developed in Xie’s laboratory.
"When any chemical molecules interact with photons from a laser, most of the photons pass through without any exchange," said Wang. "A small fraction of the photons exchange energy with the chemical. When the photon gives energy to the chemical molecules, the molecules vibrate and the color of the laser (source of the photon) changes, indicating the loss of energy."
Making something invisible visible
However, a single laser is inefficient because it affects no more than 1 in 10 million photons. Using two lasers give the chemical molecules two photons at the same time, and that enhances the frequency of this energy-exchanging event, she said.
The technique uses the vibrations of the chemical bonds in molecules to detect specific molecules. The microscope uses two lasers to excite a target. The beams are tuned to the chemical bond. The difference in the frequency of their light equals the frequency of the vibration in the molecule.
Other imaging techniques have their place, said Wang. Fluorescent is valuable in many contexts, but it is a small signal in a huge background.
This technique – abbreviated label-free SRS – eliminates the problem in labeling lipids, which are chemically inactive and do not react with dyes or other labeling materials. Using a lot of dye is a problem in itself because it can change the chemical composition of the lipid, she said.
"Before this technique, it was impossible to directly visualize lipids in vivo (in live animals)," said Wang. "This is the first technique to make something invisible visible."
All lipids contain long-chain fatty acids -- long chains of hydrogen and carbon (more than 8 to 10 carbon atoms) with a final carboxyl molecule consisting of a carbon atom attached with two bonds to an oxygen atom and a single bond to a hydroxyl (OH) group. The carbon-hydrogen bonds in these fatty acid chains are specific and Wang and her colleagues could tune the laser to match the energy in those bonds. They are imaging the carbon-hydrogen bonds, she said.
Creating an image
Using the lasers and determining the energy loss and gain between the two, enables Wang and her colleagues to make an image in a living animal. In this case, they focused on the transparent worm C. elegans. Previous attempts to quantify lipid molecule accumulation in these worms had serious limitations. Biochemical assays require many worms and time-consuming tests that are difficult to complete on a genomic scale.
Using organic solvents can change the location of lipids in the animals, she said. Other microscopy techniques lack the background contrast needed to see the lipid droplets and are difficult to use to quantify concentration.
Wang and her colleagues used RNA interference to screen the worm for genes that allowed fat storage. RNA interference can turn off or down regulate genes in the worm, some of which causes an alteration in fat storage. Using the special microscopy technique, they could actually see and quantify these changes in live animals.
She plans to follow the worms and the fat accumulation as they age. She hopes to identify different kinds of genes involved in fat storage.
Others who took part in this work include Wei Min, Christian W Freudiger, Gary Ruvkun and X Sunney Xie, all of Harvard University and/or Harvard Medical School.
Funding for this research came from the National Institutes of Health and the Boehringer Ingelheim Fonds.