Loss of a specific enzyme increases fat metabolism and endurance exercise in mice

Sugars and fats are the primary fuels that power every cell, tissue and organ. For most cells, sugar is the energy source of choice, but when nutrients are scarce, such as during starvation or extreme exertion, cells will switch to fat breakdown.

The mechanisms by which cells regenerate their metabolism in response to changes in the availability of resources are not yet fully understood, but new research reveals a surprising consequence of disabling such a mechanism: an increased capacity to exercise endurance.

In a study published in the August 4th issue. Issue of Cell Metabolism, Harvard Medical School researchers identified a critical role of the enzyme, prolyl hydroxylase 3 (PHD3), in sensing the availability of nutrients and regulating the ability of muscle cells to break down fats. When nutrients are in abundance, PHD3 acts as a inhibitor that impedes unnecessary fat metabolism. This brake is released when fuel is low and more energy is needed, such as during exercises.

Notably, blocking PHD3 production in mice leads to dramatic improvements in certain measures of fitness, the study found. Compared to their normal litter sizes, mice without PHD3 enzyme ran 40 percent longer and 50 percent further on treadmills and had higher VO2 max, a marker of aerobic endurance that measures maximum oxygen uptake during exercise.

The findings shed light on a key mechanism for how cells metabolize fuels and provide clues to a better understanding of muscle function and fitness, the authors said.

“Our results suggest that whole-body PHD3 inhibition as skeletal muscle is beneficial for fitness in terms of endurance for exercise, running time and running distance,” said former study author Marcia Haigis, professor of self-biology at the Blavatnik Institute at HMS. “Understanding this pathway and how our cells metabolize energy and fuels has the potential to have broad applications in biology, ranging from cancer management to physiological exercise.”

Furthermore, further studies are needed to explain whether this pathway in humans can be manipulated to improve muscle function in disease settings, the authors said.

Haigis and colleagues set out to investigate the function of PHD3, an enzyme they found to play a role in regulating fat metabolism in certain cancers in previous studies. Their work showed that PHD3, under normal circumstances, chemically modifies another enzyme, ACC2, which in turn prevents fatty acids from entering the mitochondria to break down energy.

In the present study, the researchers’ experiments revealed that PHD3 and another enzyme called AMPK simultaneously control the activity of ACC2 to regulate fat metabolism, depending on the availability of energy.

In isolated mouse cells grown under sugary conditions, the team found that PHD3 chemically modifies the ACC2 to inhibit fat metabolism. However, under low sugar conditions, AMPK activates and places another, opposite chemical modification on ACC2, which suppresses PHD3 activity and fatty acids entering the mitochondria can be broken down for energy.

These observations were confirmed in live mice that were fixed to induce energy-deficient conditions. In solid mice, the PHD3-dependent chemical modification to ACC2 was significantly reduced in skeletal and cardiac muscles, compared to fed mice. In contrast, the AMPK-dependent modification to ACC2 increased.

Longer and further

Next, the researchers examined the effects when PHD3 activity was inhibited, using genetically modified mice that did not express PHD3. Because PHD3 is most expressed in skeletal muscle cells and AMPK has previously been shown to increase energy expenditure and exercise tolerance, the team performed a series of endurance experiments.

“The question we asked was if we excluded PHD3,” Haigis said, “would that increase fat burning capacity and energy production and have a beneficial effect in skeletal muscle, requiring energy for muscle function and exercise capacity?”

To investigate, the team trained young, PHD3-deficient mice to run on an inclined treadmill. They found that these mice ran significantly longer and longer before reaching the point of exhaustion, compared to mice with normal PHD3. These PHD3-deficient mice also had higher oxygen consumption rates, as reflected by increased VO2 and VO2 max.

After the endurance exercise, the muscles of PHD3-deficient mice had increased rates of fat metabolism and an altered fatty acid composition and metabolic profile. The PHD3-dependent modification to ACC2 was almost undetectable, but the AMPK-dependent modification increased, suggesting that changes in fat metabolism play a role in improving exercise capacity.

These observations were indeed kept in mice genetically modified to specifically prevent PHD3 production in skeletal muscle, proving that PHD3 loss in muscle tissue is sufficient to increase exercise capacity, according to the authors.

“It was exciting to see this huge, dramatic effect on exercise capacity that could be replicated with a muscle-specific PHD3 knockout,” Haigis said. “The effect of PHD3 loss was very robust and reproducible.”

The research team also performed a series of molecular analyzes to detail the exact molecular interactions that PHD3 can adapt to ACC2, as well as how its activity is suppressed by AMPK.

The research results suggest a new potential approach for improving exercise performance by inhibiting PHD3. While the findings are interesting, the authors note that further studies are needed to better understand exactly how blocking PHD3 causes a beneficial effect on exercise capacity.

In addition, Haigis and colleagues found in previous studies that in certain cancers, such as some forms of leukemia, mutated cells express significantly lower levels of PHD3 and consume fats to induce abnormal growth and proliferation. Attempts to control this pathway as a potential strategy for treating such cancers may inform research in other areas, such as muscle disorders.

It remains unclear if there are any negative effects of PHD3 loss. To know whether PHD3 can be manipulated in humans – to improve performance in athletic activities or as a treatment for certain diseases – additional research will be needed in a variety of contexts, the authors said.

It also remains unclear if PHD3 loss triggers other changes, such as weight loss, blood sugar and other metabolic markers, which are now being investigated by the team.

“A better understanding of these processes and the underlying mechanisms of PHD3 function may one day help to unlock new applications in humans, as well as new strategies for treating muscle disorders,” Haigis said.

Additional authors on the study include Haejin Yoon, Jessica Spinelli, Elma Zaganjor, Samantha Wong, Natalie German, Elizabeth Randall, Afsah Dean, Allen Clermont, Joao Paulo, Daniel Garcia, Hao Li, Olivia Rombold, Nathalie Agar, Laurie Goodyear, Reuben Shaw, Steven Gygi and Johan Auwerx.