Dr. Brian Kennedy’s lab at the Buck Institute recently came out with a publication in the Journal of Clinical Investigation titled, “Muscle-specific 4E-BP1 signaling activation improves metabolic parameters during aging and obesity“. This study elegantly shows how blocking mTOR activity specifically in the skeletal muscles of mice can protect against age and diet-induced declines in metabolic function.
SAGE sat down with the publication’s first author, postdoc Shihyin Tsai, to get her thoughts and major insights about this exciting study.
1. What are the main findings of this paper?
For a long time, scientists thought that mTOR signaling was important for skeletal muscle function, and that mTOR activation is required for building muscle mass and repairing injured skeletal muscle. However, chronic activation of mTOR in skeletal muscle, at least in mice, leads to severe late-onset myopathy (muscle disease) and shortens lifespan.
In our recent publication, we report that blocking mTOR signaling (by turning on the activity of its downstream translational repressor 4E-BP1) in skeletal muscle results in increased metabolic rate and resistance to obesity. Ultimately, these transgenic mice are protected from metabolic decline induced by overnutrition or aging.
Therefore, our finding suggests that reducing mTOR signaling in skeletal muscle is beneficial for long-term systemic metabolic protection. Interventions that activate 4E-BP1 may have therapeutic potential on diet- or aging-induced metabolic declines, especially for the elderly or people with disabilities.
2. What’s the most significant finding from this publication?
We found that selective activation of 4E-BP1, which is resistant to mTOR regulation, in mouse skeletal muscles instead of adipose (fat) tissue, is protective against high fat diet-induced type II diabetes and aging-induced metabolic dysfunction. Mechanistically, 4E-BP1-mediated metabolic protection is cell autonomous (only occurring in skeletal muscle, not all cells) and leads to an increase in type I muscle fiber, which is the slow twitch oxidative fiber. This mechanism likely occurs through increased translation of skeletal muscle peroxisome-proliferator-activated receptor-γ coactivator-1α (PGC-1α). 4E-BP1-mediated metabolic protection can also occur in a non-cell autonomous manner through the preservation of brown adipose tissue metabolism and reduction of white adipose accumulation.
3. Describe the technological or conceptual innovations from this study?
We developed an assay in collaboration with Dr. Martin Brand at the Buck Institute to measure mitochondrial activity in skeletal muscle cells cultured in a microplate. This was a challenge due to the difficulty of isolating mitochondria from muscle.
Another challenge was the multiple transgenic mouse lines that we generated to fully characterize 4E-BP1 function cell autonomously vs non-cell autonomously and chronically vs acutely. Since we observed that activation of 4E-BP1 in skeletal muscles results in white adipose atrophy, we further characterized the mice with activated 4E-BP1 in adipose tissues. These mice were not protected from obesity, and this result confirms that the adipose tissue phenotype is indeed a result of muscular 4E-BP1 activities.
Transgenic activation of muscular 4E-BP1 results in small mice, therefore we concluded that the beneficial metabolic protection in these mice is due to developmental compensation. To clarify this, we generated another mouse line with inducible muscular 4E-BP1 activity, and we confirmed that acute activation of 4E-BP1 in adult skeletal muscle is able to recapitulate most of metabolic protection observed from the other mouse models.
4. How did you start this project?
I have had a long-term interest in the communication between different organ systems in mammals. I am interested in understanding how processes like exercise and muscle cell activity influence nutrient storage in fat cells, protect neuron cells from stress, and induce brown adipose tissues to burn extra energy.
I was astonished to know that activation of 4E-BP1 in skeletal muscle results in a “couch potato” mouse (these mice did not like to be forced to run on the treadmill!). None-the-less, these mice are lean and resistant to metabolic dysfunction caused by both overnutrition and aging. This is a great example of how mammals live in that the functions of each of their systems are dependent on the others.
5. What were the roles of your collaborators in the project?
I was very grateful that many scientists from the Buck helped to establish all the assays used in this publication. We relocated from the University of Washington to the Buck just a year after I started my project. Many people at the Buck helped me optimize the assays, but they are very humble and refused authorship on this publication. Dr. Martin Brand and his lab helped with all the mitochondrial assays. Dr. Pankaj Kapahi and Dr. Subhash Katewa provided me with ideas and preliminary data from their 4E-BP1 Drosophila models. Dr. Gary Scott helped me trouble shoot an immunoprecipitation experiment that our reviewers asked for. Dr. Monique O’Leary and Emmeline Academia are from metabolic morphological core at the Buck, and they helped me run the metabolic cages. The majority of the other coauthors are my talented interns including Joanna Sitzmann, Ariana Rodriguez, Stephanie Vu and Circe McDonald. Without them, I could not have finished analyzing the metabolicprofiles of 500 mice! And of course, Dr. Brian Kennedy fully supported this project and did back away when he saw the bill from the vivarium!
As for outside collaborators, Dr. Albert La Spada, a Professor at the University of California, San Diego, along with Dr. Somasish Dastidar and Dr. Travis Ashe shared the 4E-BP1 transgenic mice with us.
6. What’s next after this paper for this field?
Recent data suggests that skeletal muscle can be newly defined as an endocrine tissue. Our study found that the hormone FGF-21 was secreted from 4E-BP1 activated skeletal muscle and mediated the adipose tissue phenotype observed in the 4E-BP1 transgenic mice. An exciting follow-up study would be to identify other myokines (hormones specifically secreted by skeletal muscle) secreted from 4E-BP1 activated skeletal muscle, which could be the potential therapeutic candidates for treating metabolic dysfunction diseases.
7. What obstacles did you face in completing this study?
Simplifying the study was the major difficulty we had when we submitted our manuscript. Since we have generated more transgenic 4E-BP1 mice, which have different responses to metabolic challenges, we had lots of discussions between the reviewers and Brian in order to make the data more intriguing and precise. We took out data from the other two transgenic mouse lines to strengthen our main point that mTOR-dependent regulated 4E-BP1 activity in skeletal muscle is critical for systemic metabolic homeostasis. It took over a year, and I am happy that it finally worked out!
8. What are your plans post-publication?
Get my other papers published and have a cup of tea! But scientifically, Brian and I have initiated a collaboration to test our findings in human studies. Hopefully we could obtain clinical samples to see whether the protective effects of 4E-BP1 is conserved in humans.
9. How does your study relate to other findings from the Kennedy lab or the other labs at the Buck?
Our findings are related to work by Dr. Subhash Katewa from Dr. Pankaj Kapahi’s lab. He also observed that activation of 4E-BP1 in skeletal muscles alters fat metabolism in Drosophila. More excitingly, they and Dr. Norbert Perrimon at Harvard Medical School independently showed that activation of 4E-BP1 in skeletal muscles is sufficient to extend lifespan in Drosophila. Taken together, our findings provide further evidence that the longevity pathway might be evolutionary conserved.
10. What is the “big picture” of your study, and how will it impact the field of aging research?
Our study provides genetic and molecular mechanisms for how mTOR differentially regulates metabolism in different metabolic tissues. So when we evaluate functions of a “gene” or “signaling pathway” in the regulation of longevity, it’s critical to consider where they contribution their effects on longevity, how they effect the health of the entire organism, and the “timing” of these effects (e.g. when their modification is beneficial for promoting longevity).
I received my Ph.D. at Ohio State University and worked in the laboratory of Dr. Gustavo Leone, who is well known for his work on Rb-E2F signaling in the regulation of cell cycle progression. In his lab, I was thoroughly trained in mouse genetics, and I generated multiple E2F knockout and knockin mice for a project that distinguished the normal growth and the oncogenic roles of E2Fs factors in postnatal development. My work was eventually published in Nature and Proceedings of the National Academy of Sciences.
I am originally from Taiwan, and I always wondered what the world outside the island would be like. To become a skilled scientist, I decided to pursue a Ph.D. in United States. I had an oncology background, but I realized that cancer is a very complex disease to study. I liked the strategy that Brian’s lab proposed: to target and understand aging, which will ultimately shed light on other aging-related diseases including cancer. That’s why I joined Brian’s lab.