Unraveling Leptin Pathways Identifies New Drug Targets

Four years after its discovery, the weight-controlling protein hormone leptin is still making news. At the American Diabetes Association meeting in Chicago June 14, researchers from Amgen Inc. of Thousand Oaks, Calif., announced promising results of a Phase I clinical trial of leptin. And more than 100 papers and posters featured leptin at the Endocrine Society's annual meeting in New Orleans, June 24 to 27. Much of the excitement, though, lies beyond leptin, with the other players in the signal transduction pathways that control food intake. "We know so much more about the leptin system than we did four years ago. The number of potential drug targets goes up all the time--we're all like kids in the candy store," says Randy Seeley, an associate professor of psychiatry at the University of Cincinnati College of Medicine. Commercial interest in anti-obesity drug development is intense. According to the Institute of Medicine, obesity affects 32 percent of Americans, and the market for anti-obesity drugs is $5 billion. But some researchers worry that pharmaceutical development is proceeding too fast, citing the 1997 recall of fenfluramine and dexfenfluramine due to side effects. "The drug companies are placing bets without appropriate data, because they want to enter early in the game," Seeley says. The desire to find a magic bullet for obesity is being tempered by discoveries of the complexity of the genetic controls of body weight. "At least 17 potential candidate genes regulate body weight. Obese people might have altered function of three or four of these genes," explains Peter Havel, a research faculty member in the nutrition department at the University of California at Davis. Still, gene by gene, researchers are making progress. "Identifying these genes in humans or rodents reveals the normal regulation of body weight, and suggests ways that we can manipulate the system pharmacologically," says Louis Tartaglia, senior director of metabolic diseases and oncology at Millenium Pharmaceuticals Inc. in Cambridge, Mass. A new era in weight control research began in 1994. "The discovery of leptin by Jeff Friedman was the most important single advance over the past 25 years in understanding the basic mechanistic relationships between adipose tissue and the brain," says John Fain, chair of the department of biochemistry at the University of Tennessee in Memphis. While pharmaceutical firms are developing anti-obesity drugs based on the leptin pathways, researchers continue to investigate leptin itself. The following examples illustrate the diversity of these efforts. Richard Londraville, an assistant professor of biology at the University of Akron, was a postdoctoral research fellow at Stanford University, probing heat production in swordfish muscle, when news of leptin's discovery broke. "I said, 'This hormone is for me!' " he recalls. Londraville decided to look for leptin, known only in mammals, in the bony fishes. Last summer he found the hormone in green sunfish and large-mouth bass. Then he turned to sharks. Because these animals lack fat deposits, they should not have leptin, but their body temperatures are high, suggesting that they might indeed have it. To find out, Londraville went in the spring of 1998 to the South African White Shark Institute. But sampling leptin from Jaws proved a bit more challenging than working with chubby mice. "I had no idea how to get blood samples. These are 2,000- to 5,000-pound animals that are not docile. You can't anesthetize them or bring them on board. So, I modified a hypodermic needle and attached it to a powerful fishing line," Londraville relates. Using bait to attract sharks, he leaned over and stabbed them. Londraville got the blood sample, and didn't lose any limbs, but doesn't yet know if he found leptin. When postdoctoral researcher Hao Qian arrived a year ago in the laboratory of Clifton Baile, professor of foods and nutrition and animal science at the University of Georgia in Athens, he suggested that leptin might contribute to weight loss by causing apoptosis in fat cells. To test this hypothesis, the researchers compared rats given leptin with rats on a low-calorie diet, or controls with neither treatment, for the telltale DNA pattern of a cell in the throes of apoptosis. Only the cells of leptin-treated rats died, to be replaced weeks later (Qian, H. et al., Endocrinology, 139:791-4, 1998). But discovering apoptosis raises more questions. "The apoptosis is not due to direct action of leptin on the adipocytes, since leptin treatment in vitro does not cause apoptosis. The factor[s] that cause the apoptosis remained unidentified. Leptin activation of receptors in the central nervous system is required for some cascade of events that leads to apoptosis," explains Baile. Peter Havel, a research faculty member in the nutrition department at the University of California at Davis, is probing the influences of diet on leptin levels. In one study, he and colleagues at the USDA Western Human Nutrition Research Center monitored circulating leptin as people followed a long-term, low-calorie diet, asking the participants about hunger and their sense of fullness. "The decrease in leptin during dieting correlated to [an] increase in hunger, independent of change in body weight," Havel says. In another experiment, Havel and Karen Teff, an investigator at Monell Chemical Senses Center in Philadelphia, assessed leptin levels in women over 24 hours of eating fat-rich or carbohydrate-rich meals. The high-carbo route triggered more leptin release than the high-fat meals, "This result suggests one mechanism by which a high-fat diet can lead to weight gain," explains Havel. Following is a look at three points of intervention into the biochemical circuitry that controls body weight: leptin, leptin receptors, and other signaling proteins. Jeffrey Friedman, a Howard Hughes Medical Institute investigator at Rockefeller University in New York City, and co-workers discovered leptin (Y. Zhang et al., Nature, 372:425-31, 1994) as a cause of obesity in mice. They also pinpointed a human counterpart. Because fat cells (adipocytes) secrete leptin, the hormone's level reflects fat stores. "As leptin increases, it makes the brain think the body has more fat; therefore, the brain calls for a decrease in food intake and an increase in metabolism," explains Robert Frederick, an assistant professor of internal medicine at the University of Kentucky in Lexington. Leptin likely helped our ancestors survive famine. "Probably leptin evolved to signal low energy stores, not to keep people from getting fat. It is very important if leptin levels are low to trigger hunger," says Havel. Today, most obese people actually have too much leptin--deficiency is extremely rare (Carl T. Montague et al, Nature, 387:903-6, 1997). It seems illogical to give obese people who apparently cannot recognize their own leptin additional hormone, but the Amgen trial results suggest that this approach may work. Mark McCamish, director of clinical research for leptin at Amgen, says, "Perhaps leptin is in a relative deficiency, rather than an absolute one." In the Amgen trials, 53 lean and 70 obese individuals received daily injections of placebo or one of four doses of recombinant leptin for four weeks to assess safety and pharmacokinetics. Then 47 of the obese participants continued for 20 additional weeks to examine dose-response relationships. Results were variable, but promising. "The average loss was 16 pounds in the highest dose group, and 1.5 pounds in the lowest group. But in each group were individuals who lost a large amount of weight, and some who didn't lose. In the highest cohort, one individual actually gained weight," reports McCamish. The high leptin levels in obese people refocused research efforts on leptin receptors. The receptors are found primarily in the hypothalamus and peripherally in the ovaries, testes, and pancreas. Defective receptors in the hypothalamus might explain inability to recognize abundant leptin, causing overeating. Cloning and patenting the leptin receptor developed into an interesting corporate race. Millenium announced cloning the leptin receptor, which the company calls OB-R, in December 1995 (L. Tartaglia, Cell, 83:1263-71, 1995), an outgrowth of their work on the obese mouse. But Progenitor Inc., then of Columbus, Ohio, and now of Menlo Park, Calif., had beaten them to it. Progenitor filed in September 1994 for a partial gene sequence and in December 1994 for the complete sequence of "a novel human hematopoietin." The abstracts for both patents refer to the gene as Hu-B1.219, not the leptin receptor. "Progenitor was identifying random sequences with homologies to cytokine receptors. They had no idea it was involved in body weight regulation, obesity, or that it was the leptin receptor. They filed the patent on random sequences, which happens all the time in the genomics field," explains Tartaglia. Millenium holds patents on several other genes that control body weight. Progenitor licensed exclusive rights for development and commercialization of products using the leptin receptor to Amgen in December 1996. And in March 1998, Progenitor began collaborating with Cambridge Antibody Technology Ltd. in Cambridge, England "to develop human monoclonal antibodies that function as agonists against the leptin receptor," says Douglass Given, president and CEO of Progenitor. The effort will target peripheral leptin receptors thought to trigger apoptosis of adipocytes. People with defective leptin receptors, like people with deficient leptin, are extremely rare, suggesting that drug developers probe even farther down the pathways (K. Clement et al, Nature, 392:398-401, 1998). "There's not a lot of data to support a receptor defect. Most likely, elevated leptin results from a pre- or post-receptor event," says Frederick. A pre-receptor defect may compromise transport of leptin across the blood-brain barrier to its receptors in the hypothalamus. "High leptin in the blood would predict high leptin in cerebrospinal fluid too; however, there is less than expected for the high blood level (M.W. Schwartz et al., Nature Medicine, 2:589-93, 1996). This suggests impediment in leptin transport from blood to brain. There's probably a receptor-mediated transport process across the blood-brain barrier," says Havel. Prime post-receptor drug targets are neuropeptide Y and the melanocortin-4 receptor (MC4-R). Neuropeptide Y, produced in the hypothalamus when leptin levels drop due to weight loss, stimulates response to starvation--increased appetite, infertility, and decreased energy use (J.C. Erickson et al, Science, 274:1704-7, 1996). MC4-R is activated when melanocyte-stimulating hormone binds it, which happens when the leptin level rises due to weight gain. MC4-R binding dampens appetite and ups energy use (R.J. Seeley et al, Nature, 390:349, 1997). "Neuropeptide Y is the accelerator and MC4-R is the brake. If leptin levels are low, the body turns on the accelerator, and turns off the brake. The opposite occurs when leptin levels are high," explains Seeley. Drug company efforts seek to antagonize neuropeptide Y binding, or promote MC4-R binding. Seeley calls the pharmaceutical effort to jump-start MC4-R "monstrous." But developing drugs based on neuropeptide Y or MC4-R may not be straightforward. Researchers at Synaptic Pharmaceuticals Corp., in Paramus, N.J., for example, are developing antagonists to a neuropeptide Y receptor called Y5. But Christoph Gerard, head of molecular biology at the company, warns that so far the receptors seem to be distributed in different parts of rodent brains than human brains. "Proof of the concept of activity of Y5 antagonists can come only after human trials. It is in preclinical research now, so that will be at least two years from now," he says. New understanding of the signaling pathways that control body weight means a more targeted and rational approach to anti-obesity drug development, even as basic research continues. Says Tartaglia, "If you want a safe drug for body weight regulation, the right approach is not to manipulate in a broad sweep a neurotransmitter that regulates everything, but to deal with specific pathways that regulate body weight-- even though it might take a little longer." Ricki Lewis, a freelance science writer based in Scotia, N.Y., is the author of several biology textbooks. She can be reached online at rickilewis@nasw.org.

Unraveling Leptin Pathways Identifies New Drug Targets

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