Ground squirrel microbiomes are neat, unlike human microbiomes

I generally think the microbiome has been overblown. It’s an incredibly appealing target for modern-day drug hunters: millions of species hiding within our guts! Nobody knows which one is the best or what they do! They can all interact with each other! Let’s screen a thousand people, get the genomes of a million bacteria, functionally screen all of them and their interactions, and test them all in high tech “organs on chips”! It’s like the modern day version of exploring the deepest recesses of the rainforest for drugs, but, instead of being brave explorers in the Amazon, we’re whitecoats in Cambridge looking at strangers’ poop!

But, when you actually look into it, the microbiome, in humans, really doesn’t seem to do a whole lot beneficial besides prevent worse bacteria from taking up residence and do a bit of digestion for us¹. Everything else that people have spent hundreds of millions of dollars modulating the microbiome to try to accomplish, like curing ulcerative colitis, cancer, atopic dermatitis, COPD, and even autism, has failed. 

From Flagship Pioneering’s website, a VC/company creation firm best known as the makers of Moderna. Of these 4 companies listed, Evelo and Kaleido shut down, Indigo transitioned to a carbon credit company, and Seres, which IPO’d at $50/share, is now at $1/share. You think Flagship would give up on the microbiome, but hope springs eternal. I appreciate that this page is still up on their website, though.

“What about short chain fatty acids?” I hear you cry. “Surely we need the bacteria in our gut to provide us with short chain fatty acids?” I mean, I guess. Or we can just get them directly from milk or butter. Anyways, it seems a little disappointing that we go from “genetically engineering the microbiome to cure Alzheimer’s” to “if you eat fiber, it will make your bowel movements more regular and maybe also lower cholesterol”.

I’ve gotten so down on the microbiome in humans that I forget that the microbiome in other animals can be really cool. We’ve long known about the fact that ruminants like cows and sheep can only digest cellulose because of the bacteria in their creatively named rumen. Now, this is cool enough already (I mean, it’s a mammal forming a symbiotic relationship with bacteria to unlock an entirely new food source), but now we can add another cool microbiome to the list.

Enter the thirteen-lined ground squirrel, a squirrel that lives on the ground and has 13 lines on its back (guess where its name comes from). These ground squirrels live all across the prairies and grasslands of middle North America, from Texas up through the middle of Alberta. During the bitterly cold North American winter, they hibernate in the ground.

This is a difficult thing to do. Although ground squirrels can and do put on a lot of fat in the summer, they are still small creatures with a large surface area:volume ratio (because of the square:cube law) who have to spend 6 months in close contact with frozen ground, not eating, barely moving, barely breathing (one breath per 5 minutes), not drinking, and barely peeing. Then, when it comes springtime again, they have to hop right back into doing ground squirrel things.

One of the hardest parts about this hibernating ground squirrel lifestyle, besides just keeping enough energy and water to survive the 6 month winter, is maintaining muscle mass. I mean, think of what people look like when they’re in the hospital on bed rest even for a month. They are generally weak and unable to move very well. Ground squirrels are on bed rest for 6 months, and they can’t exit their bed rest as weaklings or else they will get immediately eaten by predators. They have to maintain muscle mass.

Muscle generally breaks down after 2-3 weeks, at least in humans, so ground squirrels have to continually build new muscle even as they’re on bedrest. They especially have to build new muscle at the end of hibernation in order to get ready for spring. But, in order to do so, they need nitrogen, which makes up about 16% of protein. But, the only source of nitrogen is the nitrogen from their broken down muscles.

And that, at last, is where the microbiome comes in. You see, the normal nitrogen pathway in the body, in broad strokes, is amino acid -> ammonia -> urea -> excretion via the kidneys. In hibernating ground squirrels, though, the urea is diverted². After it’s produced in the liver, it’s transported via the blood to the cecum, the part of the large intestine that ruminants use to digest cellulose. There, bacteria living in the cecum of ground squirrels’ can transform the urea back to ammonia, where it can then be redistributed to the muscles. 

Not only does this allow ground squirrels to build muscle without having to eat any more food, but it also saves them from having to pee so often. In both humans and squirrels, one of the big reasons that we have to pee is to get rid of waste products from muscle catabolism, which this diversion tactic avoids.

So, ground squirrels’ microbiome keeps them buff and prevents them from having to get up from sleep to pee. That, to me, is finally a good use for a microbiome³.

1. And yes, I am talking about every microbiome when I say this: skin, vagina, lung, mouth. Literally every healthy human microbiome outside of the stomach just prevents worse microbiomes from taking hold, and all of them are “good” insofar as they don’t harbor bad bacteria. There’s no point in this mass screening.

2. All of this ground squirrel nitrogen recycling stuff is based on this paper. One of the interesting things about this paper is how they work to put together all the evidence to actually make a persuasive case that this is happening. The trouble they faced is that they can’t actually observe the bacteria working in vivo (they can’t see inside the squirrel), and I don’t think they figured out how to get the bacteria working in vitro, either.

So, they have the evidence that ground squirrels do, in fact, put on muscle mass during hibernation, and that nitrogen has to come from somewhere, and the squirrels aren’t eating anything, so it’s not coming from their food. So what do you do to prove it’s gut bacteria that are recycling the nitrogen?

Well, the first and most obvious thing is just to compare ground squirrels with microbiomes to ground squirrels without microbiomes. So, do the obvious thing, and carpet bomb half the squirrels’ guts with antibiotics. This is a little tricky to do without messing with the squirrels’ hibernation, but apparently they managed.

So, now you can compare the urea concentration in the plasma of ground squirrels with microbiomes and see that it’s way higher than the plasma of ground squirrels who have depleted microbiomes, although weirdly enough only in the summer. What else?

Well, try to see if the urea is actually being recycled. One way to do that is to inject the squirrels with radioactive carbon. Squirrels who perform ureolysis (the breakdown of urea to transform it into protein again) will end up using that radioactive carbon and exhale it as carbon dioxide, which can be measured. Once again, you see that squirrels with microbiomes do way more ureolysis than squirrels without.

Last, you can inject the squirrels with radioactive nitrogen and trace everywhere that the nitrogen goes. Sure enough, you find a lot more of the radioactive nitrogen in the cecum and the liver in the squirrels with microbiomes, suggesting that they’re actively recycling them (although, weirdly, not in the skeletal muscle). Depressingly, you have to kill the squirrels for this last step :(.

As you can probably tell by my overuse of weirdly, I think there are some major unexplained issues with the data in this paper, and I’m a little suspicious of how much you can mess with a ground squirrel without messing with its hibernation. I think the story they tell is plausible enough and their data is ok, but there are definitely some parts missing.

If I were to put some probabilities on all of this, here’s what I’d say:

Probability that ground squirrels recycle nitrogen during hibernation - 95%. The nitrogen for building their muscles has to come from somewhere, and the only other possibility is that ground squirrels have figured out some symbiotic relationship with fungi or bacteria to fix nitrogen, like termites apparently have

Probability that ground squirrels use gut bacteria to recycle at least some nitrogen (assuming they do in, fact, recycle nitrogen) - 75%. The antibiotics definitely seemed to do something to the urea concentration, and ruminants, at least, can recycle nitrogen.

Probability that ground squirrels use gut bacteria to recycle all nitrogen - 40%. There’s something weird going on with their data, especially with the lack of radioactive nitrogen in skeletal muscle. I think there must be some other way for ground squirrels to recycle or conserve nitrogen that’s not being accounted for here.

3. The paper that I took this from suggests in the final paragraph that this could be a treatment for sarcopenia, or muscle wasting, arguing that human beings might also have the ability to salvage urea, at least according to a pretty sparse paper from 1979. While I appreciate the thought and the biohacking potential, I’m pretty sure sarcopenia isn’t caused by lack of nitrogen. It’s not like people on bed rest or old people lack nitrogen, it’s that their muscles waste away.

Sadly, as with the butyrate example, most microbiome stuff is kind of pointless for humans that live in an era with basically unlimited food/protein. Fun food for thought, though.