A little-studied enzyme has been discovered to play a crucial role in adding fat to the body, scientists at the University of California, San Francisco report. The enzyme makes a promising target for fat-reducing drugs, the researchers said, since blocking its action causes people no harm.
The research, based on studies with human plasma, live mice and cell cultures, is being published in the March issue of Nature Cell Biology.
The enzyme, known as plasma kallikrein, triggers a physical breakdown of the environment immediately surrounding immature, or precursor, fat cells - the zone known as the extracellular matrix. The process frees the cells to balloon into mature fat cells or adipocytes. It is this transformation into full-blown orbs of fat, rather than an increase in the total number of cells, that is primarily responsible for adding body fat.
Underscoring the view that a cell's genes alone do not control its fate, the study shows that this key enzyme performs different tasks in different environments. It is produced in the liver and migrates continuously through the bloodstream. Until now, it was thought to be primarily involved in blood clotting and blood pressure maintenance. But when it enters tissues, it prepares the way for fat cells to develop.
Plasma kallikrein turns out to be a member of the "plasminogen activator" family, the researchers report, a small group of enzymes that unleash the potent bond-cleaving enzyme plasmin -- the prime mover in breaking down connective tissue and also clotting blood.
The better-known plasminogen activators, tPA and uPA, have been the subject of keen interest because they may facilitate invasion of tissues by cancer cells and metastasis to distant tissue sites. Yet efforts by other researchers to develop a cancer therapy by blocking these activators have so far proved disappointing, said Zena Werb, PhD, UCSF professor of anatomy, a member of the UCSF Comprehensive Cancer Center, and senior author on the new paper.
"Proteins are often implicated in a disease process because they are at the right place at the right time," Werb says. "But what we are finding is that despite the circumstantial evidence, another protein is often the real culprit."
Indeed plasma kallikrein had been thought to be important only for blood clotting based on how it was discovered, yet this turns out not to be true, she said: "Its real job seems to be in making plasmin active. We've shown it is certainly crucial for allowing fat cells to develop, and it may be important in the cancer process."
In the UCSF study, plasma kallikrein was shown to be sufficient to launch plasmin on a natural course of tearing down the scaffolding that supports precursor fat cells, physically freeing the cells to "round up." This swelling in turn prompts a gene-driven program to create a new environment for the mature fat cells and produce fat in them. The new microenvironment, called a basement membrane, holds the fat cells in place, yet allows them to enlarge with fat more than 100-fold, Werb explained.
The research not only demonstrated that plasma kallikrein alone can trigger plasmin to remodel the environment for fat cell development but, much to the scientists' surprise, it showed too that plasminogen activators tPA and uPA -- thought to be crucial to this process -- are in fact unnecessary.
The scientists found that even though the development of fat cells absolutely requires plasmin -- and therefore the presence of some plasminogen activator to process the plasmin -- fat cells actually generate a molecule that inhibits the action of tPA and uPA. The inhibitor does not seem to affect plasma kallikrein at all, they report, further supporting the conclusion that plasma kallikrein plays the primary role among the three in the fat development process.
To tease apart the players from the non-players in the remodeling steps that prepare for fat cell development, the research team used both genetic techniques and approaches that block specific enzymes.
Charles Craik, PhD, UCSF professor of pharmaceutical chemistry and co-author on the paper, is an authority on proteases, the family of enzymes that includes the plasminogen activators. He and his colleagues have developed a novel technique to pick out the key proteases active in a biological process from among many proteases that may be present. They adapted a protease inhibitor called ecotin so that it could selectively block specific members of an entire class of proteases. Using this tool they discovered that the expected players, tPA and uPA, were not central to preparing for fat development, but that plasma kallikrein was a prime mover.
The research traced the action of plasminogen activators in mouse mammary glands during the first few days after lactation when the glands undergo radical changes from milk production to fat deposition. Plasma from people who lack the gene for plasma kallikrein was also introduced into mouse cell cultures, where the researchers discovered that this plasma kallikrein "knockout" plasma failed to support fat cell development.
"We are excited by this observation," Craik said, "because we believe that other fat deposits may also decrease when there is no plasmin action. Plasma kallikrein may give us a drug target for modifying fat deposition."
In a hopeful sign for potential anti-fat drug development, people who lack the gene for plasma kallikrein appear to be perfectly healthy, Werb said.
"This suggests that if a drug can be developed to reduce fat buildup by blocking or inhibiting plasma kallikrein, it is unlikely to have any serious side effect."
"We also suspect that kallikrein may be essential in remodeling the zone around other types of cells," she said. "Changes in the environment around cells lead to changes in cell behavior and recruitment of different cell types, such as development of new blood vessels. For cancer to spread, for example, the tumor cells' ecological niche must change from quiescent and stable to dynamic. As cells begin to pick up mutations, they recruit the neighboring normal cells to make enzymes that then change the microenvironment. This leads to further recruitment of inflammatory cells and blood vessels, fostering growth, invasion and eventually metastasis of the tumor.
"What we are really hoping is that drugs against this enzyme may also modify cancer. That is where our research is heading," she added.
First author on the paper is Sushma Selvarajan, graduate student in pharmaceutical chemistry and in anatomy at UCSF. Co-authors are Leif R. Lunda, PhD, a former UCSF postdoctoral researcher now at the Finsen Laboratory in Denmark; and Toshihiko Takeuchi, a PhD, a pharmaceutical chemistry postdoctoral researcher at UCSF.
The research was supported by the National Cancer Institute and the National Science Foundation.
Materials provided by University Of California, San Francisco. Note: Content may be edited for style and length.
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