Grizzly bears spend many months in hibernation, but their muscles do not suffer from the lack of movement. In the journal Scientific Reports, a team led by Michael Gotthardt reports on how they manage to do this.
The grizzly bears’ strategy could help prevent muscle atrophy in humans as well.
A grizzly bear only knows three seasons during the year. Its time of activity starts between March and May.
Around September the bear begins to eat large quantities of food. And sometime between November and January, it falls into hibernation.
From a physiological point of view, this is the strangest time of all. The bear’s metabolism and heart rate drop rapidly.
It excretes neither urine nor feces. The amount of nitrogen in the blood increases drastically and the bear becomes resistant to the hormone insulin.
A person could hardly survive this four-month phase in a healthy state. Afterwards, he or she would most likely have to cope with thromboses or psychological changes.
Above all, the muscles would suffer from this prolonged period of disuse. Anyone who has ever had an arm or leg in a cast for a few weeks or has had to lie in bed for a long time due to an illness has probably experienced this.
A little sluggish, but otherwise fine
Not so the grizzly bear. In the spring, the bear wakes up from hibernation, perhaps still a bit sluggish at first, but otherwise well. Many scientists have long been interested in the bear’s strategies for adapting to its three seasons.
A team led by Professor Michael Gotthardt, head of the Neuromuscular and Cardiovascular Cell Biology group at the Max Delbrueck Center for Molecular Medicine (MDC) in Berlin, has now investigated how the bear’s muscles manage to survive hibernation virtually unharmed.
The scientists from Berlin, Greifswald and the United States were particularly interested in the question of which genes in the bear’s muscle cells are transcribed and converted into proteins, and what effect this has on the cells.
Understanding and copying the tricks of nature
“Muscle atrophy is a real human problem that occurs in many circumstances. We are still not very good at preventing it,” says the lead author of the study, Dr. Douaa Mugahid, once a member of Gotthardt’s research group and now a postdoctoral researcher in the laboratory of Professor Marc Kirschner of the Department of Systems Biology at Harvard Medical School in Boston.
“For me, the beauty of our work was to learn how nature has perfected a way to maintain muscle functions under the difficult conditions of hibernation,” says Mugahid.
“If we can better understand these strategies, we will be able to develop novel and non-intuitive methods to better prevent and treat muscle atrophy in patients.”
Gene sequencing and mass spectrometry
To understand the bears’ tricks, the team led by Mugahid and Gotthardt examined muscle samples from grizzly bears both during and between the times of hibernation, which they had received from Washington State University.
“By combining cutting-edge sequencing techniques with mass spectrometry, we wanted to determine which genes and proteins are upregulated or shut down both during and between the times of hibernation,” explains Gotthardt.
“This task proved to be tricky — because neither the full genome nor the proteome, i.e., the totality of all proteins of the grizzly bear, were known,” says the MDC scientist. In a further step, he and his team compared the findings with observations of humans, mice and nematode worms.
Non-essential amino acids allowed muscle cells to grow
As the researchers reported in the journal “Scientific Reports,” they found proteins in their experiments that strongly influence a bear’s amino acid metabolism during hibernation.
As a result, its muscle cells contain higher amounts of certain non-essential amino acids (NEAAs).
“In experiments with isolated muscle cells of humans and mice that exhibit muscle atrophy, cell growth could also be stimulated by NEAAs,” says Gotthardt, adding that “it is known, however, from earlier clinical studies that the administration of amino acids in the form of pills or powders is not enough to prevent muscle atrophy in elderly or bedridden people.”
“Obviously, it is important for the muscle to produce these amino acids itself — otherwise the amino acids might not reach the places where they are needed,” speculates the MDC scientist.
A therapeutic starting point, he says, could be the attempt to induce the human muscle to produce NEAAs itself by activating corresponding metabolic pathways with suitable agents during longer rest periods.
Tissue samples from bedridden patients
In order to find out which signaling pathways need to be activated in the muscle, Gotthardt and his team compared the activity of genes in grizzly bears, humans and mice.
The required data came from elderly or bedridden patients and from mice suffering from muscle atrophy — for example, as a result of reduced movement after the application of a plaster cast.
“We wanted to find out which genes are regulated differently between animals that hibernate and those that do not,” explains Gotthardt.
However, the scientists came across a whole series of such genes. To narrow down the possible candidates that could prove to be a starting point for muscle atrophy therapy, the team subsequently carried out experiments with nematode worms.
“In worms, individual genes can be deactivated relatively easily and one can quickly see what effects this has on muscle growth,” explains Gotthardt.
A gene for circadian rhythms
With the help of these experiments, his team has now found a handful of genes whose influence they hope to further investigate in future experiments with mice.
These include the genes Pdk4 and Serpinf1, which are involved in glucose and amino acid metabolism, and the gene Rora, which contributes to the development of circadian rhythms.
“We will now examine the effects of deactivating these genes,” says Gotthardt. “After all, they are only suitable as therapeutic targets if there are either limited side effects or none at all.”
Grizzly bears (Ursus arctos horribilis) have an annual cycle that includes hyperphagia and fat accumulation followed by winter-time hibernation in response to periods of food scarcity1–4 (Fig. 1).
Hibernation is characterized by lowered body temperature, inactivity, metabolic depression, and insulin resistance5–8.
Yet, despite its annual occurrence, bears avoid the long-term detrimental effects that occur in obese, inactive, or fasting humans, such as bone loss, elevated blood nitrogen (azotemia), ketosis, hyperglycemia, and muscle protein catabolism and atrophy7,9–13.
Thus, a series of carefully orchestrated and potentially unique mechanisms have evolved to maintain proper energy balance and metabolic health in bears throughout the year.
Importantly, since these physiological changes in hibernators occur on an annual basis, mechanisms have evolved to naturally reverse these processes and thereby avoid any secondary complications9.
Major annual physiological cycles of grizzly (brown) bears. a Three major annual metabolic stages—hibernation, active, and hyperphagia—are indicated along with the letter abbreviation for the corresponding months of the year. Blue line along the perimeter indicates the approximate period of insulin resistance. Brown bear subcutaneous adipose, liver, and muscle samples were collected from six captive bears at the Washington State University Bear Research, Education, and Conservation Center during the metabolic stages. White arrows indicate sampling points. Photo credit to Donald Montemorra. b Body weight for each of the bears at the three sampling points indicated in a. *p < 0.05 (raw data in Supplementary Table 5). c Body temperature for each of the bears at the three sampling points indicated in a. **p < 0.01. N = 6 animals. Panels b and c include individual values, mean, and standard deviation (raw data in Supplementary Table 6). d Photograph of the same individual at the Washington State University Bear Research, Education, and Conservation Center coming out of hibernation (April) and entering hibernation (October)
Numerous studies of hibernating rodents have revealed tissue- and season-specific changes in gene expression5,14,15. Hibernating rodents and other small mammals share a common characteristic during hibernation, namely the ability to periodically arouse from deep torpor, which is an energetically expensive process16,17.
Using patterns of expression in liver of arctic ground squirrels (Urocitellus parryii) compared to those of calorie-restricted, cold-exposed, and PPARα knockout mice, Xu et al.18 revealed a signature of torpor and arousal. However, unlike the small-bodied hibernators, bears of the genus Ursus exhibit a continuous hibernation characterized by metabolic suppression and much smaller reduction in body temperature of 4–7 °C2,19.
Therefore, by gaining a greater understanding of the cellular adaptations in different hibernating species one can envision new treatments being developed for human ailments7,9,20.
Our understanding of the precise mechanisms of these reversible phenotypes in bears remains incomplete. Earlier studies in bears have identified some molecular changes in liver, heart, adipose, and skeletal muscle at different times of the year using targeted approaches such as microarray and PCR21–23.
However, none have used unbiased methods, which could lead to the discovery of novel mediators. Thus, to provide a more complete picture of the molecular events involved, we performed RNA-sequencing (RNA-seq) on metabolically active tissues24 obtained from the same six captive bears of both sexes across seasons, and in two bears over two consecutive hibernation seasons.
The unbiased methods employed in the present study reveal that hibernation is characterized by dynamic changes in gene expression. These changes are largely tissue specific; however, a subset of genes is DE in the same direction in all three tissues. The latter highlights the possible role of shared regulatory pathways and tissue crosstalk in hibernation.
A more complete understanding of these changes during hibernation challenges the long-standing belief that hibernation is a static process and suggests a more nuanced regulation occurring on an on-demand basis.
Our use of ribosomal-depleted RNA-seq allows us to develop a more holistic understanding of the role of long-non-coding RNAs.
A greater knowledge of the reversible, and highly regulated, expression of the hibernation phenotype may facilitate the development of new treatments for human disease.
Story Source:
Materials provided by Max Delbrück Center for Molecular Medicine in the Helmholtz Association. Note: Content may be edited for style and length.
Journal Reference:
- D. A. Mugahid, T. G. Sengul, X. You, Y. Wang, L. Steil, N. Bergmann, M. H. Radke, A. Ofenbauer, M. Gesell-Salazar, A. Balogh, S. Kempa, B. Tursun, C. T. Robbins, U. Völker, W. Chen, L. Nelson, M. Gotthardt. Proteomic and Transcriptomic Changes in Hibernating Grizzly Bears Reveal Metabolic and Signaling Pathways that Protect against Muscle Atrophy. Scientific Reports, 2019; 9 (1) DOI: 10.1038/s41598-019-56007-8
