What Happens When You Go on Calorie Restriction?

New research pinpoints some very odd metabolites that communicate between the gut and other tissues.

The only surefire way to extend lifespan, at least so far, is to go on a calorie restricted diet. Not just a little restricted- really restricted. The standard protocol is seventy percent of a normal, at-will diet. In most organisms tested, this maintains health and extends lifespan by significant amounts, ten to forty percent. There has naturally been a great deal of interest in the mechanism. Is it due to clearance of senescent cells? Increased autophagy and disposal of sub-optimal cell components? Prevention of cancer? Enhancement or moderation of the immune system? The awesome austerities of the ancient yogis and other religious hermits no longer look so far out.

A recent pair of papers took a meticulous approach to finding one of the secret ingredients that conveys calorie restriction to the body. They find, to begin with, that blood serum from calorie-restricted mice can confer longevity properties to cultured cells. Blood from calorie-restricted mice can also confer relevant properties to other mice. This serum could be heated to 133 degrees without detriment, indicating that the factor involved is not a large protein. On the other hand, dialysis to remove small molecules renders it inactive. Lastly, they put the serum though a lipid-binding purification system, which diminished its activity only slightly. Thus they were looking for a mostly water-soluble metabolite in the blood.

Traditionally, the next step would have been to conduct further fractionation and purification of the various serum components. But times have changed, and their next step was to conduct a vast chemical (mass-spectrometry) analysis of blood from calorie restricted vs control mice. Out of that came over a thousand differential metabolites. Taking the most differential and water-soluble metabolites, they assayed them over their cultured cells for a major hallmark of calorie restriction, activation of the AMPK kinase, a master metabolic regulator. And out of this came only one metabolite that had this effect at concentrations that are found in the calorie restricted serum: lithocholic acid.

Now this is a very odd finding. Lithocholic acid is what is known as a secondary bile acid. It is not natively made by us at all, but is a byproduct of bacterial metabolism in our guts, from a primary bile salt secreted by the liver, chenodeoxycholic acid. These bile salts are detergent-like molecules made from cholesterol that have charged groups stuck on them, and can thus emulsify fats in our digestive tract. Bile salts are extensively recycled through the liver, so that we make a little each day, but use a lot of them in digestion. How such a chemical became the internal signal of starvation is truly curious.

The researchers then demonstrate that lithocholic acid can be administered directly to mice to extend lifespan and have all the expected intermediary effects, including decreased blood glucose levels. Indeed, it did so in flies and worms as well, even though these species do not even see lithocholic acid naturally- their digestion is different from ours. They evidently still have key shared receptors that can recognize this metabolite, perhaps because they use a very similar metabolite internally. 

And what are those receptors and the molecular pathway of this effect? The second paper in this pair shows that lithocholic acid binds to TULP3, a member of the tubby protein family of transcription regulators, which binds to lipids in the plasma membrane and shuttles back and forth to the nucleus to provide feedback on the membrane condition or other events at the cell surface. TULP3 also binds to sirtuins, which are notoriously involved in metabolic regulation, aging, and cell suicide. These latter were all the rage when red wine was thought to extend life span, as sirtuins are activated by resveratrol. At any rate, sirtuins inhibit (by de-acetylation) the lysosomal acidification pump that has been known (curiously enough) to go on to inhibit AMPK, and play basic roles on cell energy balance regulation.

So we are finally at AMPK, a protein kinase that responds to AMP, which is the low-energy converse of ATP, the energy currency of the cell. High levels of AMP indicate the cell is low on energy, and the kinase thus phosphorylates and thus regulates a huge variety of targets, which generally increase energy uptake from the blood, (glucose and fats), reduce internal energy storage, and increase internal recycling of materials and energy, among much else. At any rate, the researchers above show that AMPK activation is absolutely required for the effects of lithocholic acid, and also for life extension more generally from calorie restriction. On the other hand, lithocholic acid does not touch the AMP level in the cell, but, as mentioned above, activates through a much more circuitous route- one that goes through lysosomes, which are sort of the stomach of the cell, and are increasingly recognized as a central player in its energy regulation.

Some beautiful antibody staining to different types of myosin- the key motor protein in muscles- in muscle cells from various places in the mouse body (left to right). The antibodies are to slow-twitch myosin (red), which marks the more efficient slow muscle fibers, and blue and green, which mark fast twitch myosins- generally less efficient and markers of aging. The bottom two rows are from a AMPK knockout mouse, where there is little difference between the two rows. The pairs of rows are from normal (top) and (bottom) a specific mutant of the lysosomal proton pump that has its acetylation sites all mutated to inactivity, thus mimicking in strongest form the action of the sirtuin/lithocholic acid pathway. Here, (second row, especially last frame), there is a higher level of red vs blue, and of blue vs green staining/fibers, suggesting that these muscles have had a durable uptick in efficiency and, implicitly, reduced atrophy and higher longevity.

These are some long and winding roads to get to positive effects from calorie restriction. What is going on? Firstly, biology does not owe it to us to be concise or easy to understand. Lithocholic acid is going to always be around, given that we eat food, but has mostly, heretofore, been regarded as toxic and even carcinogenic. It, along with the other bile acids, bind to dedicated transcription regulators that provide feedback control over their synthesis in the liver. So they are no strangers to our physiology. The authors do not delve into why calorie restriction durably raises the level of lithocholic acid in the gut or the blood, but that is an important place to start. It might well be that our digestive system squeezes every last drop of nutrition out of meagre meals by ramping up bile acid production. It is a recyclable resource, after all. This could have then become a general starvation signal to tissues, in advance of actual energy deficits, which one would rather not face if at all possible. So it is basically a matter of forewarned is forearmed. One is yet again amazed by the depth of biological systems, which have complexity and robustness far beyond our current knowledge, let alone our ability to replicate or model in artificial terms.

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