How does a little-known enzyme affect inflammatory disease?
A UTMB researcher's novel discoveries may save many lives.
By Michele Rainford
In a darkened lecture hall in UTMB’s Basic Science Building, Satish Srivastava stands beneath the ghostly image of a beating heart.
The gray-and-black ovoid—a video produced from an ultrasonic scan—pulses slowly above a line graph demonstrating the life-and-death importance of aldose reductase, an enzyme that Srivastava, a UTMB professor of biochemistry and molecular biology, has been studying for almost forty years. The graph’s vertical axis measures how well the hearts of laboratory mice pump blood, and its horizontal axis measures how much time has elapsed since researchers injected the mice with lipopolysaccharides (LPS), powerful inflammatory substances found in the outer membranes of dangerous bacteria. The goal of this experiment is to simulate the effects of sepsis—a dangerous over-reaction by the immune system that kills more than 120,000 Americans every year.
“In sepsis, as you know, it doesn’t matter that you give a patient antibiotics and kill the infection,” Srivastava tells his audience. “The LPS from the bacteria are still floating around, along with inflammatory signaling molecules already generated by the immune system. And so the immune system continues to respond, causing more and more inflammation in the heart, so much that it is barely able to pump blood.” He swings his laser pointer back and forth across the bottom-most line on the graph, which indicates a heart pumping about half as efficiently as it should be. These mice, he says, can hardly walk. The bleak next steps—in human beings as well as mice—are kidney and lung failure and death.
But the next line on the graph tells a much happier story. The mice experience a dramatic recovery, with the volume of blood pumped out of their hearts rising quickly to near-normal levels. “With this other group of mice, we used sorbinil to inhibit aldose reductase, and you can see the effect,” he says, a smile spreading across his broad face. “Within hours, the cardiac function is restored. If we can get a similar inhibitor into humans, I think we can save the lives of a lot of patients.”
This biochemist has plenty of reasons to smile. As extraordinary as these results are, they are only a small part of the remarkable outpouring of scientific creativity he has to show his UTMB colleagues today. In the six months before this talk last December, Srivastava and his collaborators had published a flurry of papers linking aldose reductase—a once obscure enzyme previously primarily associated with sugar metabolism and the complications of diabetes—to sepsis and colon cancer. They’ve also pinned down the enzyme’s critical position in the biochemical network that connects oxidative stress and inflammation—the first a natural byproduct of oxygen metabolism and cellular signaling networks, and the second a disease-fighting mechanism that all too often goes haywire and creates disease itself. These two processes are key to a whole range of disorders in addition to sepsis and colon cancer—everything from lung, breast and prostate cancers to allergies and asthma to atherosclerosis and hypertension to Alzheimer’s and Parkinson’s.
“This latest burst of activity is really phenomenal stuff, maybe the most important work he’s done in his career,” says an observer with a unique perspective on Srivastava’s research—his son, Deepak, a UTMB School of Medicine graduate who now directs the Gladstone Institute of Cardiovascular Disease at the University of California, San Francisco. “But the neatest part is the manner in which it’s evolved, which is through very basic discovery of the mechanisms by which the enzyme functions, and the ability to recognize unusual features and then pursue them, using some very out-of-the-box thinking.”
Years earlier, Deepak Srivastava recalls, his father had often spoken of his conviction that aldose reductase, his pet enzyme, was essential to all sorts of critical biochemical reactions with major implications for a multitude of clinical disorders. Deepak reacted skeptically. “You know, it’s your dad, so how much of that do you believe?” the younger man says. “I would just politely listen and nod, and think in my own head, Oh boy, this thing does everything. But it’s held up, both in in vitro and in animal studies. Now I recognize how significant it is.”
The quest to discover the true significance of aldose reductase is one Satish Srivastava has been on for more than four decades. It began in his native India, where, barely out of graduate school, he began looking into the biochemical basis for cataract formation in people with diabetes. It continues today at UTMB, where he leads a group of researchers who see this “master-switch” enzyme as key to a whole range of inflammation-related disorders.
Along the way, Srivastava and his colleagues challenged orthodox ideas of how aldose reductase works, ideas that have led drug companies to spend a quarter century and tens of millions of dollars on research and development programs into thus-far unsuccessful attempts to create aldose reductase–blocking therapies to treat the complications of diabetes. Meanwhile, Srivastava himself has spent his thirty years in Galveston building a smaller-scale but no less impressive research network of his own—what one scientist jokingly calls the “Srivastava empire”—as his team slowly but steadily unravels the basic secrets of the enzyme and maps its connections to biochemical pathways that lead to disease.
“After all this time, my approach has not changed—you find one link, and even though in the beginning you don’t know where you’re going, you look for other connections and find that as knowledge improves and people publish, you’re able to link in other things,” Srivastava says. “Thirty years ago we didn’t know about NF kappa B”—a signaling molecule that tells DNA to produce inflammatory proteins—“or any of these other intermediary signaling pathways. Fortunately, other investigators worked these things out, and then we were able to put them together with what we had learned about aldose reductase and solve a real mystery of life— to see how oxidative stress and reactive oxygen species connect with inflammation and cell proliferation. And the key thing is, we should be able to use that knowledge to stop disease. Somehow, we have hit a really good spot.”
Srivastava’s journey to that spot began in the late 1950s at the University of Lucknow in northwestern India, where he made two critical connections. One of them was with a fellow student who would become a lifelong friend and future colleague and collaborator (not to mention father-in-law to Srivastava’s daughter): Yogesh Awasthi, now also a professor of biochemistry and molecular biology at UTMB. The other was with the concept of oxidative stress, which in one way or another would shape the rest of Srivastava’s career.
Oxidative stress is a catch-all term for the damage done to cells by chemical reactions that involve varieties of oxygen and oxygen-containing compounds (particularly those known as reactive oxygen species) stripping electrons away from other molecules. The electron-removal process—crucial to the reactions that supply energy and maintain order within cells—is known to chemists as oxidation, and it’s not limited to biological settings; a rusty nail and a forest on fire are both examples of oxidation reactions happening at dramatically different rates.
“After all this time, my approach has not changed—you find one link, and even though in the beginning you don’t know where you’re going, you look for other connections."
“We are all aerobes, we all require oxygen to live,” Srivastava says, sitting in his office in the Basic Science Building. The wall to his left is papered with photos of his family—images of his son and two daughters and their spouses and children, including pictures of a beaming Srivastava practically buried in grandchildren. “But just as nothing in nature is perfect, so the consumption of oxygen is not 100 percent foolproof—there is a leakage, and that leakage involves these reactive oxygen species and oxidative stress, which for the past forty years I’ve argued is the major cause of a number of diseases.”
In themselves, according to Srivastava, reactive oxygen species are not necessarily bad. In fact, he says, they’re critical to the signaling processes that enable cells to respond to infection, adjust to their environment, and control their rate of reproduction. But just as care has to be taken with a campfire to make sure it doesn’t set the woods ablaze, oxidation inside cells has to be tightly controlled to avoid damage to the cell.
That’s where aldose reductase comes in. But when Srivastava first heard of the enzyme, neither he nor anyone else knew that. Instead, researchers working with aldose reductase were focused on what it did in the lens cells of the eyes of people with diabetes, where it was thought to help generate cataracts by a process called “osmotic stress.”
Enzymes like aldose reductase are molecules made by cells to facilitate particular chemical reactions; they do their catalytic jobs over and over again, ending each reaction cycle ready to repeat a new one. (Like all such molecular machines, enzymes do have a definite half-life, however, eventually breaking down after a certain number of reaction cycles.) Aldose reductase was thought to play its primary role in reactions involving sugar molecules such as glucose and galactose, helping transform them into other molecules, known as sorbitol and galactitol, that were unable to pass back out through the cellular membranes of lens cells. In diabetics and galactosemics (galactosemics are people with a rare genetic disorder that prevents them from metabolizing galactose), the oversupply of sugars was thought to feed such a huge buildup of these molecules that water would be steadily drawn into the cells by osmotic pressure, the same process that plumps up raisins soaked in water. Unlike the raisins, though, the lens cells would never become saturated, because the aldose reductase reactions would keep turning more glucose into sorbitol in diabetics, and more galactose into galactitol in galactosemics. The cells would swell, and the resulting osmotic stress would produce cataracts.
Similar processes involving aldose reductase were also thought to be at work in such other complications of diabetes as damage to cells in the retina, kidneys, and nerves. Naturally, drug companies became interested in the concept, and by the late 1980s they had spent tens of millions of dollars developing “aldose reductase inhibitors”—compounds meant to block the activity of aldose reductase and prevent the disorders that cause diabetics to go blind and suffer kidney and nerve damage.
For Srivastava, it was an interesting concept—he had done some of his earliest postdoctoral work in India on the genesis of diabetic cataracts, and he continued to delve into the subject after coming to the United States in 1966 to work with world-renowned hematologist Ernest Beutler at California’s City of Hope Medical Center. (The NIH has continuously funded Srivastava’s diabetes cataract research since 1967.) But Srivastava’s primary interests—pursued in collaboration with Awasthi, who arrived from India as a postdoc in 1972—were the methods used by cells to get rid of the toxic products of oxidative stress, as well as genetic disorders like Tay-Sachs disease.
Then, around 1980—a few years after he, Awasthi, and lab technician (now professor) Naseem Ansari set up shop at UTMB—Srivastava began to doubt the aldose reductase-osmotic stress hypothesis of diabetes-caused cataracts. That idea was based on experiments with rats, he noted, but his group had found that in human tissues, levels of sorbitol, one of the key products of aldose-reductase interactions with glucose, were only a fraction of those in rat lenses.
"The establishment of aldose reductase as an enzyme that protects against oxidative stress was purely Satish’s idea. He has this talent for setting his research priorities to take him right to the heart of the matter, and then picking up on things that others miss."
As time went on and Srivastava continued to investigate the role of aldose reductase in causing cataracts, his doubts increased. The compounds developed by the drug companies to block aldose reductase, he found, were “non-specific”—that is, they did more than just keep aldose reductase from interacting with glucose and could possibly give rise to unwanted side effects with long-term use (as subsequent clinical testing revealed they did, much to the drug-makers’ disappointment). Finally, his team discovered that aldose reductase also was involved in reactions involving lipid aldehydes, destructive molecules produced when reactive oxygen species encounter the fatty cellular membrane. Not only that: it acted more powerfully on lipid aldehydes, and lipid aldehydes linked to glutathione (a key molecule involved in relieving oxidative stress) than it did on glucose. Aldose reductase, it turned out, was actually a major part of the cell’s oxidative stress-control machinery.
“The ideas on what roles different enzymes play in negating oxidative stress evolved slowly from our studies and those of others,” Yogesh Awasthi says. “But the establishment of aldose reductase as an enzyme that protects against oxidative stress was purely Satish’s idea. He has this talent for setting his research priorities to take him right to the heart of the matter, and then picking up on things that others miss—and this aldose reductase discovery was really something.”
By this time—the early Nineties—Awasthi was well established as an independent researcher. He had specialized in studying an enzyme called glutathione-s transferase, developing his program as an offshoot of the Srivastava lab’s demonstration that glutathione was one of the most important parts of the cell’s oxidative-stress control apparatus. Many of Srivastava’s former postdoctoral fellows and graduate students comment on the unusual freedom they felt to pursue their own interests within Srivastava’s lab—a freedom followed by a continuing closeness and collaborative productivity with their former mentor after they had struck out on their own. Awasthi is a special example: he’s gone from nightly tea-drinking discussions with Srivastava in college back in the Fifties, to side-by-side lab work in California and at UTMB, to watching his son Sanjay marry Srivastava’s daughter Sangeeta. The two men’s friendship remained constant even as their research interests drifted apart, but remain linked by oxidative stress and glutathione.
Now, Awasthi says, like other current and former Srivastava collaborators, he found himself drawn in by the excitement of a discovery that went far beyond overturning the decades-old dogma on how diabetic cataracts develop. It held the promise—for those who could see it—of making sense of a whole web of intracellular connections, and in the process opening up new areas of exploration that could lead to any number of new therapies.
The picture that has emerged draws on work done by Srivastava, Awasthi, Ansari, and UTMB assistant professor (and former Srivastava postdoc) Kota Venkata Ramana, as well as Mark Petrash of the University of Washington in St. Louis and Aruni Bhatnagar of the University of Louisville (two more Srivastava lab veterans) and Srivastava’s cardiologist son Deepak, with significant contributions from UTMB structural biology professors Stanley J. Watowich and Mark White.
In this new version of events, aldose reductase performs an essential first step in the process of cleaning up damaging lipid aldehydes. But when a wider disorder causes a cell to produce an overload of reactive oxygen species, those molecules generate a huge quantity of lipid aldehydes—so many that they overtax the cleanup process, generating what Srivastava calls “leakage.”
Aldose reductase enzyme still interacts with the toxic lipid aldehydes, but the products of that interaction, which are normally picked up by other enzymes and transformed into substances that can be easily pumped out of the cell, grow so numerous that they spill over and become available to activate other reaction pathways. These, in turn, lead to the activation of “transcription factors” called AP-1 and NF-kappa B: molecules that act directly on DNA, encouraging cell proliferation and the production of inflammatory signals that generate still more reactive oxygen species, thus continuing the cycle.
It doesn’t matter whether the original external stimulus is an explosion of immune signaling molecules produced in response to a severe bacterial infection (sepsis), the flood of growth factors generated by tumor cells (colon cancer), or a diabetes-caused jump in glucose concentrations (diabetic complications like cataracts and cardiovascular disorders). The results are similar, and the one essential link in the process leading to those results is aldose reductase.
“Nature has made reactive oxygen species a major signaling process in our cells,” Srivastava says. “What our experiments have shown is that aldose reductase is the main mediator in oxidative species signaling. That means that if there’s no aldose reductase activity, the signals will be severely impaired.” The implication is both simple and powerful. If researchers can block aldose reductase in the right way and at the right time, they can shut down the signals that cause sepsis, colon cancer, and many complications of diabetes; in addition, Srivastava has reason to believe that aldose reductase may be crucial to other cancers and such inflammation-driven disorders as asthma, allergies, and atherosclerosis. “It makes sense, looking at how the body maintains certain functions, that a number of those functions would be correlated,” Srivastava continues. “If you can reach to the bottom of the signaling pathways that start with something like reactive oxygen species, you can do a lot of things.”