Hormone replacement therapy one hour at a time

Rodent study suggests approach to keep good and lose bad effects of standard hormone treatments

Giving hormone doses in pulses, rather than as a steady exposure, may maximize the benefits and limit the side effects now associated with hormone therapies. This is one implication of the findings scientists at Rockefeller University report in the August 17 issue of the Proceedings of the National Academy of Sciences.

The Rockefeller scientists, Lee-Ming Kow, Ph.D., and Donald W. Pfaff, Ph.D., show that two distinct biochemical pathways interact in brain cells of female rats to trigger mating behavior. They studied rats that, similar to post-menopausal women, were not naturally producing estrogen. By giving estrogen replacement to the rats, the scientists studied the actions of the hormone at the level of the brain cell’s protective outer membrane, and inside the nucleus where the cell’s DNA is housed.

They found that both the membrane and the DNA pathways are crucial, with one facilitating the other, in triggering female rat mating behavior, as measured by the animal’s arousal and physical readiness to copulate.

By limiting the estrogen exposure of to short pulses, the total dose can be kept much smaller than with steady delivery, and therefore some of the negative effects will be reduced. “You want the good effects of estrogen on the brain, on the bone — to avoid osteoporosis — and in the blood vessels,” Pfaff says, “but, we do not want to allow breast cells or uterine cells grow and divide because of the risk of cancer.”

“This finding shows that one can engineer a temporal schedule of hormone administration to optimize its effects for anybody who needs hormones for anything, including women on hormone replacement therapy,” says Pfaff, professor and head of the Laboratory of Neurobiology and Behavior at Rockefeller University. “The best hormone therapy would be an exact mimic of a person’s normal pattern of hormone release.”

Natural levels of hormones such as estrogen normally vary over time, often in cycles that can be measured over hours, days, months, or even years. Today’s typical therapies, however, give a steady dosage of hormone without accounting for these time variations.

Trouble with testosterone

Pfaff also predicts that pulsed therapies with androgen hormones, such as testosterone, may lessen their harmful effects for men.

“Consider an 80-year old man,” Pfaff says. “When giving testosterone, you’d like to support muscle development, libido, and a general feeling of well being. Androgens help people feel better. But you do not want to upset the liver and cause an abnormal lipoprotein distribution, and you do not want to give him prostate cancer.”

Even some cases of infertility have been reversed by time-dependent hormone therapy, Pfaff says. He describes a group of patients treated at Massachusetts General Hospital, whose brains failed to deliver the hormone GnRH. Without sufficient GnRH, their pituitary glands did not put out another hormone which is needed for women to ovulate and for men to make sperm. With a pump that released GnRH under the skin every 45 minutes, these patients regained their fertility. By contrast, long-term implants that released GnRH at a steady rate had no effect at all.

Estrogens function in two ways, both essential to its effects on brain cells and behavior. In the first of these pathways, estrogen binds to a specific protein on the cell’s outer surface, the plasma membrane. Pfaff calls this a “membrane pathway.” In the second pathway, estrogen molecules that have entered the cell bind to estrogen receptors that move directly to the cell nucleus, where they control the activation of specific target genes, and thereby the production of key proteins. This, Pfaff calls the “genomic pathway” of estrogen signaling.

Besides its potential for use in medical practice, time-varied hormone delivery also makes new kinds of scientific research possible. Just as Kow and Pfaff have discovered the difference between the membrane and genomic effects of estrogen, hormone pulses also allow scientists to tease apart cellular events that cannot be distinguished during constant hormone exposure.

Brief history of timed doses

Since the 1960s, most molecular biologists in the field have believed that the genomic pathway alone causes the effects of estrogen, Pfaff says. However, a few opposing voices argued that some of the effects of estrogen happen so quickly that the genomic pathway cannot be totally responsible, and have additionally considered actions on the cell surface, the membrane pathway. In the new PNAS article, Pfaff and Kow prove that both routes are used in the rat brain, and that one pathway facilitates the other.

In 1981, Pfaff, together with Bruce McEwen, Ph.D., another Rockefeller head of laboratory, showed that a steady supply of estrogen can be replaced by short pulses of the hormone and still give rise to mating behavior and gene expression in rats. “You can substitute very long and constant hormone exposure by two brief pulses,” Pfaff says. But exactly how the short pulses of estrogen could mimic the effects of a steady supply was not yet clear.

In 2001, Nandini Vasudevan, Ph.D., then a postdoctoral fellow in Pfaff’s laboratory, studied the effects of estrogens on nerve cells in a test tube. She gave the cells two pulses of estrogen — one pulse of about one hour followed by a waiting period of four hours and then a second one-hour pulse. Vasudevan found that the cells produced a “reporter protein,” a strong indication that estrogen had turned on the genomic pathway.

To determine whether the membrane pathway was also involved in the reaction to estrogen, Vasudevan used a trick. She attached estrogen molecules to a very large protein. This way the estrogen could still bind to receptors on the membrane surrounding the cell. But because of its increased bulk, the estrogen “package” was prevented from entering the cells, and therefore could not act along the genomic pathway. By contrast, free estrogen molecules easily cross the cell membrane and enter the cell nucleus, where they can activate gene expression.

Using this technique, the Rockefeller scientists found that if they only used the membrane-limited estrogens in the first pulse and free estrogen in the second pulse, the genomic pathway was activated. But if the order of the pulses was reversed, the genomic pathway was never activated. Pfaff and his colleagues concluded that the membrane-limited pathway actually enhances the genomic pathway for turning on gene expression.

Surprise finding

In the new PNAS paper, Kow and Pfaff report studies of female rats whose ovaries had been removed so that they no longer produced estrogen. To simulate arousal, they injected estrogen directly into the rat’s hypothalamus, the part of the brain that controls mating and many other types of behavior. They then observed the rats’ behavior, focusing on lordosis, a hormone-triggered receptive posture that prepares the female rat for mating.

When Kow and Pfaff injected the membrane-limited estrogen in a first pulse and free hormone in the second pulse, they saw a strong reaction: the rats showed lordosis behavior. This was expected based on the results from their earlier experiments in nerve cells. They then reversed the order of the pulses, starting with free estrogen and giving membrane-limited hormone in the second pulse. Based on their earlier nerve-cell results, they did not expect to see mating behavior in this experiment. But to their surprise, this also caused lordosis.

“The puzzle is that you can reverse the order and it still works,” Pfaff says. He speculates that the effects of estrogen at the membrane make the estrogen receptor in the cell nucleus a better transcription factor. “The import of the membrane effect is to make the necessary transcriptional effect more efficient,” Pfaff says.

Based on all of this research, Pfaff believes that the results of hormone therapy can be improved by individually tailoring the timing of the treatments.

These studies were supported by the National Institutes of Health.