Ketones in Mitochondrial Energy Metabolism: A brief update

Gee, has it been a long time since I was able to write on Medium. I’ve been swamped with lots of stuff that I will hopefully be able to post on here eventually. A lot of what I’ve been up to involves learning R, writing a few manuscripts, collecting data, and progressing with my degree.

In any case, I’d like to discuss the latest research in ketone metabolism as published by Petrick et al. in the Journal of Physiology. This project appears to be some low-hanging fruit that I’ve wanted to do for some time (ah, the joys of being young, inexperienced, and at the mercy of my mentors — not that I’m necessarily complaining; my mentor is great) and essentially covers a topic of ketone research that has inexplicably never been performed: analyzing mitochondrial respiration (i.e. oxygen consumption) in the presence of ketone bodies.

But first, some background

If we remember way back to our high school biology, we should be able to remember the renowned saying: “Mitochondria are the powerhouse of the cell.” This concept (which, after all, is true), is the primary mechanism behind multicellular energy creation: mitochondria contain a series of enzymes that pass electrons to create ATP, the universal form of cellular energy. Without getting into too much detail, the mitochondria contain a series of 4 enzyme complexes that pass electrons down this line. With each movement of electrons, protons are shuttled against their gradient into the intermembrane space. Once electrons pass through the 4th enzyme, they are combined with oxygen (O₂) and some protons to make water, as shown below.

Some substrates and inhibitors we use to test mitochondrial respiration. Glutamate (G), malate (M), succinate (S), and octanoylcarnitine (Oct; a fatty acid) are commonly used as electron donors. Rotenone, Antimycin A, and Oligomycin are inhibitors, while FCCP shuttles protons out of the mitochondria; all which reduce ATP production

The protons that are shuttled into the intermembrane space create an electrochemical gradient (now we are talking physics), which is used to spin the 5th complex, ATP synthase, like a hydroelectric turbine, thus creating ATP.

We know this is the case because if we place tissue into a liquid containing oxygen, the oxygen levels of the liquid decrease if we add certain substrates. We can also look at other things, such as the preference of mitochondria to use different fuel sources (fats, ketones, carbohydrates, etc.) and the “efficiency” of the mitochondria (ATP produced per O₂ consumed).

Traditionally only “carbohydrate derived” substrates (glutamate, pyruvate, malate, and succinate) are used to stimulate mitochondrial respiration. Lately, there has been a growing interest in fatty acid oxidation, but that trend is still fairly uncommon. No other study, to my knowledge, has looked at alternative fuels (ketones or lactate) as mitochondrial fuels in this type of experiment.

Why do we care?

To make this simple, several studies have argued that ketone oxidation is more efficient than carbohydrate or fatty acid oxidation. This was shown first by a couple of works done in Richard Veech’s lab (1, 2). Work by Benjamin Bikman’s lab showed that there are plenty of [beneficial] changes to skeletal muscle enzyme content and oxidative stress if the muscles are exposed to β‐hydroxybutyrate. More recently, it has been shown that certain tissues (such as the heart, kidney, and brain) prefer ketones to glucose, however, these studies only show rates of tissue uptake, not of substrate utilization. So, this paper looks at whether we see the same thing at the mitochondrial level as we do the whole body level; or, more simply, whether the preferential ketone uptake is due to the mitochondria using the ketones as a primary fuel.

The Study

This study took muscle and heart samples from rats, and muscle samples from human legs to measure the effects of the ketones β‐hydroxybutyrate and acetoacetate on mitochondrial respiration. To sum this up short and sweet, neither ketone seems to contribute to a meaningful increase in mitochondrial respiration for a couple of reasons:

1. The amount of ketones needed to induce meaningfully O₂ consumption was much, much higher (8x higher) than normal muscle ketone levels
2. The oxidation of ketones was minimized with the addition of another substrate (in this case, pyruvate, succinate, and palmitoylcarnitine).

Therefore, the authors conclude:

Altogether, the ability of KBs to drive mitochondrial respiration is minimal and they are likely outcompeted by other substrates, compromising their use as an important energy source.

That much is pretty evident in this paper. It’s unclear why that is — the authors speculate that there may be some substrate competition governed by enzyme inhibition. They do mention that the long-term effects of a ketogenic diet or fasting regimen may alter oxidation rates, but I would somehow doubt this would be the case.

So, what gives?

If ketones aren’t used for fuel, why are they so readily picked up by numerous tissues when they’re available?

Well, I’d say there are many possibilities. My biggest hunch is that ketones have a more important purpose than energy metabolism — signaling.

I would say that ketones (and most macronutrients, really) serve a much larger purpose than simply providing energy; they also serve to indicate some physiological state that requires change. Ketones, for example, are produced endogenously from fatty acids when fatty acid metabolism is increased due to nutrient deficit, thus exceeding the capacity for the liver to oxidize acetyl-CoA. The ketones are then released from the liver. Conventional thinking tells us that ketones are converted back to acetyl-CoA, which can be further oxidized and used as fuel. However, this paper provides evidence against that line of thinking, thus indicating that ketones may actually serve a much more robust role as interorgan signaling molecules.

Ketones could signal to other tissues that the organism is under nutrient stress — there appears to be low food availability (or the appearance of which) so the body must conserve glucose and other nutrients as much as possible. This could be done by several mechanisms such as butyrylation or direct regulation of fatty acid metabolism. One interesting signaling effect ketones have is the activation of the hydroxycarboxylic acid receptor (HCA2) in endothelial cells, which may play a role in preventing hypertension.

My hunch is that we need to look at energy nutrients more as signaling molecules, rather than just sources of energy.