In many animal species, dietary restriction and reduced reproduction increase lifespans. The biochemical and molecular mechanisms involved in this phenomenon, however, are still poorly understood. Mitochondria are essential for cellular homeostasis, and mitochondrial dysfunction is a hallmark of aging. To understand the role of mitochondrial activity in interventions that increase longevity, researchers led by Professor Fernanda Marques da Cunha, from the Universidade Federal de São Paulo (UNIFESP) and a member of the RIDC Redoxoma, investigated the effects of dietary restriction and inhibition of germline proliferation on mitochondrial respiratory activity in the Caenorhabditis elegans nematode. They found that both interventions increased the efficiency with which mitochondria use lipids as a respiratory substrate, indicating that the ability to oxidize lipids may be determinant in enhanced longevity. The results were published in The FASEB Journal.

“C. elegans is a model widely used in aging studies, mainly by geneticists, who make genetic modifications and check if they interfere with lifespans. We decided to put a biochemical look at this model, and, because of our background, to analyze mitochondrial function, and we found that mitochondrial activity is modulated by these two interventions known to increase the longevity of C. elegans”, Fernanda Cunha said.

The researchers studied C. elegans wild type (WT) and a non-fertile mutant (glp-1). The worms were subjected to total deprivation of food, an extreme situation that increases the life span by up to 50% compared to fed animals. Initially, they did experiments in vivo, with the whole animals, and found a decrease in oxygen consumption, especially when they were deprived of bacteria. “We saw that the amount of oxygen consumed by these animals, even when breathing less, was more devoted to ATP synthesis. These are facts that have already been seen in more complex organisms and C. elegans as well, we corroborated the literature”, said the researcher.

Then, they studied isolated mitochondria, in which chemical modulators of mitochondrial activity can be used. To measure mitochondrial activity, the researchers monitor oxygen consumption and use inhibitors of the electron transport chain and oxidative phosphorylation.

Isolating the mitochondria of an animal as small as C. elegans is not trivial. C. elegans is about 1 millimeter long, non-pathogenic, and lives in the soil, feeding on microbes like bacteria. To isolate their mitochondria, it was necessary to grow one million animals from each experimental group, working under extreme culture conditions, such as very large plates and many animals per plate.

With the experiments, they saw that both wild type animals subjected to bacterial deprivation and animals that did not have the germline, but that were eating, had greater ability to oxidize lipids. They breathed more in the presence of the lipid substrate and were also more efficient at producing ATP from the energy released by lipid oxidation.

In many organisms, including C. elegans, lipids form the main stores of energy and are rapidly mobilized and oxidized during starvation. “It was expected that the animal that does not eat improved the metabolism of lipids to generate ATP, but the same happened with the glp1 mutant kept in the presence of food. If it were just a stimulus dependent on food deprivation, we wouldn’t see that in the fed mutant. And we see it”, the researcher said. Thus, the study demonstrated that inhibition of germline proliferation induces a functional change in mitochondrial metabolism to increase the efficient use of lipids as an energy fuel. And while both food restriction and reproductive inhibition individually increase the efficiency of lipid-based breathing and mitochondrial coupling, they are even more effective in combination.

The study was carried out in collaboration with the groups of Alicia Kowaltowski, from the Instituto de Química at Universidade de São Paulo and a member of the RIDC Redoxoma, and Hugo Aguilaniu, from the Institut de Génomique Fonctionnelle de Lyon, in France, and the Instituto Serrapilheira.

The next step, according to the researcher, will be to study mitochondria isolated from wild-type C. elegans fed and in dietary deprivation using proteomics (the global analysis of proteins in a biological sample), and lipidomics (the global analysis of lipids in biological systems).

Lipid Repertory

Fernanda Cunha’s group already has experience with lipidomics. In another study, recently published in BBA - Molecular and Cell Biology of Lipids, the researchers characterized the lipid profile of C. elegans mutants deficient for lipase LIPL-5 and compared it with the profile of wild type worms. They found major changes in the mutants’ mitochondrial lipids, which were accompanied by significant changes in mitochondrial activity. Lipases are enzymes that have the function of breaking down the fat present in food.

“LIPL-5 mutants, when deprived of food, have an increased lifespan compared to wild type animals under the same conditions. LIPL-5, by homology, was classified as a lipase, but until today its substrate and products are unknown”, said Fernanda Cunha. This made the researchers decide to find out what the lack of this enzyme caused in the repertoire of lipids and the mitochondrial activity of C. elegans. They saw that, in well-fed animals, the mitochondria of the mutants were more efficient than those of the wild type worms, but starvation provoked changes in mitochondrial activity only in the wild type worms.

The lipidomic analysis of the animals showed 409 different lipid species, of which 38 species were modulated differently in the mutants deprived of food. The main changes were related to the ceramide pool, free fatty acids, coenzyme Q-9, and cardiolipins, the last two being specific to the internal mitochondrial membrane. Coenzyme Q-9 plays a role in electron transport in the mitochondrial respiratory chain, while cardiolipins are crucial for mitochondrial functions, being involved in the stabilization of respiratory complexes and supercomplexes, in mitophagy and signaling.

The lipidomic results indicated that the lack of LIPL-5 causes major changes in mitochondrial lipids, and, therefore, that this enzyme is determinant for changes in mitochondrial activity in response to starvation. Lipidomics studies and statistical analyzes were carried out in collaboration with the groups of Professor Sayuri Miyamoto, from Instituto de Química at USP, and Isaias Glezer, from UNIFESP, both members of the RIDC Redoxoma.

According to the researchers, in the C. elegans genome, 471 genes encode proteins involved in lipid metabolism and 70% of them have a human homolog. Lipids have signaling functions and participate in the control of metabolism in health and disease, in addition to having a role in energy storage, which is why lipid metabolism is an important research area. “Despite the increasing number of published papers on the subject, the lipid repertoire of different organisms, their biological functions and their specific remodeling in response to different stimuli are far from being fully characterized,” wrote the authors of the article.

The article Lifespan-Extending Interventions Enhance Lipid-Supported Mitochondrial Respiration in Caenorhabditis elegans, by Felipe Macedo, Talita Romanatto, Carolina Gomes de Assis, Alexia Buis, Alicia J. Kowaltowski, Hugo Aguilaniu, and Fernanda Marques da Cunha, can be accessed here.

The article Lipase-like 5 enzyme controls mitochondrial activity in response to starvation in Caenorhabditis elegans, by Felipe Macedo, Gabriel Loureiro Martins, Luis A. Luévano-Martínez, Gustavo Monteiro Viana, Karin A. Riske, Alex Inague, Marcos Y. Yoshinaga, Hugo Aguilaniu, Sayuri Miyamoto, Isaias Glezer, and Fernanda Marques da Cunha, can be accessed here.