Recycling of nitrogen in hibernating mammals

Question

Hibernating mammals are required to undergo profound changes in metabolism. In addition to the more studied requirement of providing energy, there are problems in relation to nitrogen metabolism because of the continued turnover of muscle protein and the need to dispose of the urea produced in the absence of urination.

In an abstract written over forty years ago Nelson states that to solve this problem urea is reconverted to amino acids:

“The urea that is formed is hydrolyzed and the nitrogen released is combined with glycerol to form amino acids, which reenter protein synthetic pathways.”

Is this correct and, if so, where does the process take place and what are the precise biochemical reactions involved?

N.B.
I am particularly interested in the hibernating bear.

Answer 1

Summary

The hibernating animal in which the recycling of nitrogen has been best studied is the ground squirrel. Recent studies in this species by Regan et al. (Science (2022) 375, 460–463) have provided strong experimental support to an idea proposed some time ago. This is that the ureases of gut microbiota hydrolyse urea to carbon dioxide and ammonia, the latter being incorporated into amino acids by these symbiotic bacteria. Some of the synthesized amino acids are taken up by the mammalian host and are used for its protein synthesis. It is reasonable to expect that intestinal microbiota may also play a role in other hibernators such as bears, but this remains to be established.

Proposals: Importance and Limitations

R. A. Nelson performed pioneering experiments in hibernating bears. He recognized that muscle protein did turnover, that this entailed disposal of nitrogen as urea, but that the blood concentration of urea was not raised (if it had been it would eventually have reached toxic concentrations) despite the fact that hibernating bears do not urinate. He realized that these two problems could be resolved if the nitrogen in the urea was somehow converted to nitrogen in amino acids, allowing synthesis of muscle protein to replace that which was degraded.

[Simplified diagram showing fate of nitrogen from amino acids resulting from protein breakdown in mammals]

In order to be incorporated into amino acids, nitrogen released from urea (as ammonia) needs a carbon skeleton to be integrated into, and Nelson proposed that this was supplied by glycerol produced from the lipolysis of triglycerides, which after phosphorylation can enter the pathway of glycolysis/gluconeogenesis in liver. Although the major rationale of this is gluconeogenesis to provide glucose for the brain, Nelson performed experiments with ¹⁴C-glycerol which showed that some radioactive label entered alanine, presumably by transamination of pyruvate produced by glycolysis of phosphoglycerate.

There are, in my opinion, two major limitations to this proposal. First, it would require a urease to be present in the liver to produce the ammonia for the transamination of pyruvate. There is no evidence of any mammalian urease, either experimentally or from the predictions of genomic sequences. Second, it would not explain how the degraded essential amino acids — amino acids the carbon skeleton of which mammals cannot synthesize — were replaced.

These difficulties were overcome in the proposal of Riedesel and Steffen for a role of gut microbes — rather than the animal itself — in recycling urea, although these authors studied small hibernating rodents, rather than bears. Their abstract specifically mentions:

…the potential for urea recycling by intestinal microbiota…

They had no experimental evidence that this occurred, but they argued for its feasibility in the context of contemporary knowledge at the time:

“Several monogastric mammalian species, including man, have been shown to harbor microorganisms capable of urease activity (Long et al. Am. J. Clin. Nutr. 31: 1367–1382, 1978).”

[Conversion of urea to ammonia and carbon dioxide by microbial ureases]

Evidence for role of gut symbiots in ground squirrels

The difficulty in performing experiments on either the microbiota or the animals themselves meant that it was over 40 years after the proposal of Riedesel and Steffen before Regan and associates published a paper with confirming evidence in the prestigious journal Science. They injected ground squirrels with ¹³C,¹⁵N-urea and determined its metabolic fate over the hibernation period by detecting the presence of these (stable, non-radioactive) isotopes using modern spectrographic methods. They also examined the abundance of urease gene sequences in ten individual bacterial taxa of the the metagenome. Their findings are conveniently summarized in the figure below, taken from their paper.

…and what happened to Goldilocks?

It remains to be established whether urea recycling through microbiota also occurs in bears, where hibernation has some different features. A 2021 review by Bertile et al. of protein sparing in hibernation includes a consideration of bears in an extensive and comprehensive coverage of this topic. There seems to be some reluctance to the idea of a major role for microbiota in urea cycling in bears, and the authors emphasize other factors that could contribute to protein sparing. It seems to me, as an outsider, that this reluctance will turn out to be unjustified, but, in the absence of experimental evidence, no conclusion is possible at present.