New tusk-analysis techniques reveal surging testosterone in male woolly mammoths

Traces of sex hormones extracted from a woolly mammoth's tusk provide the first direct evidence that adult males experienced musth, a testosterone-driven episode of heightened aggression against rival males, according to a new University of Michigan-led study.

In male elephants, elevated testosterone during musth was previously recognized from blood and urine tests. Musth battles in extinct relatives of modern elephants have been inferred from skeletal injuries, broken tusk tips and other indirect lines of evidence.

But the new study, scheduled for online publication May 3 in the journal Nature, is the first to show that testosterone levels are recorded in the growth layers of mammoth and elephant tusks.

The U-M researchers and their international colleagues report annually recurring testosterone surges -- up to 10 times higher than baseline levels -- within a permafrost-preserved woolly mammoth tusk from Siberia. The adult male mammoth lived more than 33,000 years ago.

The testosterone surges seen in the mammoth tusk are consistent with musth-related testosterone peaks the researchers observed in an African bull elephant tusk, according to the study authors. The word "musth" comes from the Hindi and Urdu word for intoxicated.

"Temporal patterns of testosterone preserved in fossil tusks show that, like modern elephants, mature bull mammoths experienced musth," said study lead author Michael Cherney, a research affiliate at the U-M Museum of Paleontology and a research fellow at the U-M Medical School.

The study demonstrates that both modern and ancient tusks hold traces of testosterone and other steroid hormones. These chemical compounds are incorporated into dentin, the mineralized tissue that makes up the interior portion of all teeth (tusks are elongated upper incisor teeth).

"This study establishes dentin as a useful repository for some hormones and sets the stage for further advances in the developing field of paleoendocrinology," Cherney said. "In addition to broad applications in zoology and paleontology, tooth-hormone records could support medical, forensic and archaeological studies."

Hormones are signaling molecules that help regulate physiology and behavior. Testosterone is the main sex hormone in male vertebrates and is part of the steroid group of hormones. It circulates in the bloodstream and accumulates in various tissues.

Scientists have previously analyzed steroid hormones present in human and animal hair, nails, bones and teeth, in both modern and ancient contexts. But the significance and value of such hormone records have been the subject of ongoing scrutiny and debate.

The authors of the new Nature study say their findings should help change that by demonstrating that steroid records in teeth can provide meaningful biological information that sometimes persists for thousands of years.

"Tusks hold particular promise for reconstructing aspects of mammoth life history because they preserve a record of growth in layers of dentin that form throughout an individual's life," said study co-author Daniel Fisher, a curator at the U-M Museum of Paleontology and professor in the Department of Earth and Environmental Sciences.

"Because musth is associated with dramatically elevated testosterone in modern elephants, it provides a starting point for assessing the feasibility of using hormones preserved in tusk growth records to investigate temporal changes in endocrine physiology," said Fisher, who is also a professor in the U-M Department of Ecology and Evolutionary Biology.

For the study, researchers sampled tusks from one adult African bull elephant and two adult woolly mammoths -- a male and a female -- from Siberia. The samples were obtained in accordance with relevant laws and with appropriate permits.

The researchers used CT scans to identify annual growth increments within the tusks. A tiny drill bit, operated under a microscope and moved across a block of dentin using computer-actuated stepper motors, was used to grind contiguous half-millimeter-wide samples representing approximately monthly intervals of dentin growth.

The powder produced during this milling process was collected and chemically analyzed.

The study required new methods, developed in the laboratory of U-M endocrinologist and study co-author Rich Auchus, to extract steroids from tusk dentin for measurement with a mass spectrometer, an instrument that identifies chemical substances by sorting ions according to their mass and charge.

"We had developed steroid mass spectrometry methods for human blood and saliva samples, and we have used them extensively for clinical research studies. But never in a million years did I imagine that we would be using these techniques to explore 'paleoendocrinology,'" said Auchus, professor of internal medicine and pharmacology at the U-M Medical School.

"We did have to modify the method some, because those tusk powders were the dirtiest samples we ever analyzed. When Mike (Cherney) showed me the data from the elephant tusks, I was flabbergasted. Then we saw the same patterns in the mammoth -- wow!"

The African bull elephant is believed to have been 30 to 40 years old when it was killed by a hunter in Botswana in 1963. According to estimates based on growth layers in its tusk, the male woolly mammoth lived to be about 55 years old. Its right tusk was discovered by a diamond-mining company in Siberia in 2007. Radiocarbon dating revealed that the animal lived 33,291 to 38,866 years ago.

The tusk from the female woolly mammoth was discovered on Wrangel Island, which was connected to northeast Siberia in glacial periods of lower sea level but is now separated from it by the Arctic Ocean. Carbon-dating showed an age of 5,597 to 5,885 years before present. (Wrangel Island is the last known place where woolly mammoths survived, until around 4,000 years ago.)

In contrast to the male tusks, testosterone levels from the female woolly mammoth tusk showed little variation over time -- as expected -- and the average testosterone level was lower than the lowest values in the male mammoth's tusk records.

"With reliable results for some steroids from samples as small as 5 mg of dentin, these methods could be used to investigate records of organisms with smaller teeth, including humans and other hominids," the authors wrote. "Endocrine records in modern and ancient dentin provide a new approach to investigating reproductive ecology, life history, population dynamics, disease, and behavior in modern and prehistoric contexts."

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Newly identified hormone may be a critical driver of type 1 and type 2 diabetes

A newly discovered hormone named fabkin helps regulate metabolism and may play an important role in the development of both type 1 and type 2 diabetes, according to research led by the Sabri Ülker Center for Metabolic Research at Harvard T.H. Chan School of Public Health.

The study showed blood levels of fabkin were abnormally high in mice and human patients with either type 1 or type 2 diabetes. The researchers found that blocking the activity of fabkin prevented the development of both forms of diabetes in the animals. Fabkin likely plays a similar role in humans and the hormone complex could be a promising therapeutic target, according to the researchers.

"For many decades, we have been searching for the signal that communicates the status of energy reserves in adipocytes to generate appropriate endocrine responses, such as the insulin production from pancreatic beta cells," said senior author Gökhan S. Hotamisligil, director of the Sabri Ülker Center. "We now have identified fabkin as a novel hormone that controls this critical function through a very unusual molecular mechanism."

The findings will be published online in Nature on December 8, 2021.

Many hormones are involved in the regulation of metabolism, such as insulin and leptin. Fabkin is different from traditional hormones in that it is not a single molecule with a single defined receptor. Instead, fabkin is composed of a functional protein complex consisting of multiple proteins, including fatty acid binding protein 4 (FABP4), adenosine kinase (ADK) and nucleoside diphosphate kinase (NDPK). Through a series of experiments, the researchers determined that fabkin regulates energy signals outside of cells. These signals then act through a family of receptors to control target cell function. In the case of diabetes, fabkin controls the function of beta cells in the pancreas that are responsible for insulin production.

More than a decade ago, Hotamisligil and colleagues discovered that a protein known as FABP4 is secreted from fat cells during lipolysis, the process in which lipids stored within fat cells are broken down, typically in response to starvation. Numerous studies have since shown correlations between circulating FABP4 and metabolic diseases including obesity, diabetes, cardiovascular disease, and cancer. However, the mechanism of action was unknown.

In the new study, the researchers showed that when FABP4 is secreted from fat cells and enters the blood stream, it binds with the enzymes NDPK and ADK to form the protein complex now identified as fabkin. In this protein complex, FABP4 modifies the activity of NDPK and ADK to regulate levels of molecules known as ATP and ADP, which are the essential units of energy in biology. The researchers discovered that surface receptors on nearby cells sense the changing ratio of ATP to ADP, triggering the cells to respond to the changing energy status. As such, fabkin is able to regulate the function of these target cells.

The authors showed that the insulin-producing beta cells of the pancreas are a target of fabkin and that the hormone is a driving force behind the development of diabetes. When the researchers used an antibody to neutralize fabkin in mice, the animals did not develop diabetes. When the antibody was given to obese, diabetic mice, they reverted to a healthy state.

"The discovery of fabkin required us to take a step back and reconsider our fundamental understanding of how hormones work." said lead author Kacey Prentice, research associate in the Sabri Ülker Center and Department of Molecular Metabolism. "I am extremely excited to find a new hormone, but even more so about seeing the long-term implications of this discovery."

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Hormone found to switch off hunger could help tackle obesity

 A hormone that can suppress food intake and increase the feeling of fullness in mice has shown similar results in humans and non-human primates, says a new study published today in eLife.

The hormone, called Lipocalin-2 (LCN2), could be used as a potential treatment in people with obesity whose natural signals for feeling full no longer work.

LCN2 is mainly produced by bone cells and is found naturally in mice and humans. Studies in mice have shown that giving LCN2 to the animals long term reduces their food intake and prevents weight gain, without leading to a slow-down in their metabolism.

"LCN2 acts as a signal for satiety after a meal, leading mice to limit their food intake, and it does this by acting on the hypothalamus within the brain," explains lead author Peristera-Ioanna Petropoulou, who was a Postdoctoral Research Scientist at Columbia University Irving Medical Center, New York, US, at the time the study was carried out, and is now at the Helmholtz Diabetes Center, Helmholtz Zentrum München, Munich, Germany. "We wanted to see whether LCN2 has similar effects in humans, and whether a dose of it would be able to cross the blood-brain barrier."

The team first analysed data from four different studies of people in the US and Europe who were either normal weight, overweight or living with obesity. The people in each study were given a meal after an overnight fast, and the amount of LCN2 in their blood before and after the meal was studied. The researchers found that in those who were of normal weight, there was an increase in LCN2 levels after the meal, which coincided with how satisfied they felt after eating.

By contrast, in people who were overweight or had obesity, LCN2 levels decreased after a meal. Based on this post-meal response, the researchers grouped people as non-responders or responders. Non-responders, who showed no increase in LCN2 after a meal, tended to have a larger waist circumference and higher markers of metabolic disease -- including BMI, body fat, increased blood pressure and increased blood glucose. Remarkably, however, people who had lost weight after gastric bypass surgery were found to have a restored sensitivity to LCN2 -- changing their status from non-responders before their surgery, to responders afterwards.

Taken together, these results mirror those seen in mice, and suggest that this loss of post-meal LCN2 regulation is a new mechanism contributing to obesity and could be a potential target for weight-loss treatments.

After verifying that LCN2 can cross into the brain, the team explored whether treatment with the hormone might reduce food intake and prevent weight gain. To do this, they treated monkeys with LCN2 for a week. They saw a 28% decrease in food intake compared with that before treatment within a week, and the monkeys also ate 21% less than their counterparts who were treated only with saline. Moreover, after only one week of treatment, measurements of body weight, body fat and blood fat levels showed a declining trend in treated animals.

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