Periodic Reporting for period 3 - MetaboSENS (Metabolic integration by nutrient SENSing)
Reporting period: 2022-11-01 to 2024-04-30
We are captivated by the question of how living organisms adapt their metabolism while experiencing a daily roller-coaster of circulating nutrients caused by feeding-fasting cycles. These homeostatic mechanisms represent the essential characteristics of life, as they dictate how energy is utilized to drive growth and reproduction. Consequently, the responsiveness of cells to metabolic stress (changes in nutrient levels) and their metabolic flexibility (response to nutrient types) defines our health span and drives our lifespan. Fundamentally, the activity of nutrient sensing signaling pathways together with effector gene transcription networks, assure response and adaptation to available energy. Inevitably, defects in nutrient sensing are either causative or associated with aging and virtually every disease state (e.g. cancer, metabolic and infectious diseases). The understanding of this fundamental process holds the therapeutic keys to improving cellular resilience to biotic and abiotic stresses, an approach that could be universally applied to human diseases. These complex mechanisms are far from being understood, urging for more basic research. The current field of metabolic homeostasis is largely shaped by the works that focused on ON-OFF states of growth permissive nutrient sensing pathways. A nutrient rich environment and high insulin levels promote anabolism through class 1 phosphoinositol-3 kinase (PI3K) and protein kinase mammalian target of rapamycin (mTOR) signaling. They co-activate nutrient uptake and macromolecule synthesis while blocking lysosomal degradation by autophagy. We challenge this paradigm by proposing that pro-catabolic class 3 PI3K nutrient sensor play a role of an essential counterbalance force to the pro-anabolic signaling in establishing metabolic homeostasis. In the MetaboSENS project we investigate biology of class 3 PI3K, the only ubiquitous PI3K in all eukaryotes. Class 3 PI3K engages in distinct multi-protein complexes for its canonical lipid kinase function to generate the second messenger PI3P; assuring endocytosis, autophagy, and lysosomal activity. The findings of our lab proposed that it might also have non-canonical cellular activities unrelated to its known role as a lipid kinase.
The specific objectives of the MetaboSENS project are:
1) to establish the functional significance of both canonical and non-canonical activities of class 3 PI3K;
2) to discover its temporal regulation and its implication in metabolic function at different biological scales from cellular organelles to governing whole-body energy utilization;
3) to gain the molecular mechanistic understanding of class 3 PI3K functions in metabolic control over lifespan, its input into health-span, and its contribution to human pathologies.
From translational point, we address an unmet translational need to understand the implication of class 3 PI3K in human diseases with focus on rare pediatric diseases with manifestations in liver.
The specific objectives of the MetaboSENS project are:
1) to establish the functional significance of both canonical and non-canonical activities of class 3 PI3K;
2) to discover its temporal regulation and its implication in metabolic function at different biological scales from cellular organelles to governing whole-body energy utilization;
3) to gain the molecular mechanistic understanding of class 3 PI3K functions in metabolic control over lifespan, its input into health-span, and its contribution to human pathologies.
From translational point, we address an unmet translational need to understand the implication of class 3 PI3K in human diseases with focus on rare pediatric diseases with manifestations in liver.
In this reporting period of the project, we have 1) developed and characterized new cell and animal models of class 3 PI3K activation and inhibition; 2) progressed on detailing the molecular mechanisms of the non-canonical functions of class 3 PI3K and functionally interrogated their input in the cellular metabolic activities both in acute fasting and during physiological feeding cycles and 3) obtained first evidence on specific metabolic rearrangements in human rare liver diseases.
In first objective, we discovered that class 3 PI3K is important for metabolic adaptation to fasting. We found that its acute inactivation in liver, results in massive defects in gene transcription in response to fasting. We reported that this response is partially linked to activity of key transcription factor activated in fasting, a Glucocorticoid Receptor (Y. Shibayama et al., Acta Physiologica, 2022; highlighted in the editorial). We expanded these analyses by performing bulk transcriptomic, proteomics, and metabolomics analyses in liver of mice that were challenged by fasting. These analyses were fructuous and provided several candidates. We are now performing molecular validation studies in order to dissect how class 3 PI3K controls specific cellular metabolic activities in fasting in different ages of young and old male and female animals. Moreover, from cell to the organism scale, the metabolic demands fluctuate rhythmically relying on coordination between the circadian clock and nutrient sensing signaling pathways. Thus, in second objective, we progressed in understanding how class 3 PI3K expression and activity is changing around the clock. We also demonstrated its requirement for metabolic rhythmicity in liver. We are now investigating in details the molecular mechanisms of its potential crosstalk with the clock in different organs. We also apply our expertise of basic science in the field of nutrient sensing signaling and metabolism to investigate novel metabolic signatures on rare pediatric diseases with manifestations in liver such as biliary atresia (BA). BA is an uncurable disease that manifests as obliteration of the extrahepatic bile ducts and alteration of the intrahepatic biliary tree in neonates. Left untreated, it quickly progresses to cirrhosis and liver failure leading to death in the first 2 years of life. While rare (a worldwide incidence of ~1:20,000 live births), BA remains the most frequent cause of liver transplantation in children. In this funding period, we have established the biobank of patient derived material, generated patient derived cell models and animal models of BA that were instrumental in performing molecular analyses. Our findings in this direction have indicated potential therapeutic targets that we are in process of validation.
In first objective, we discovered that class 3 PI3K is important for metabolic adaptation to fasting. We found that its acute inactivation in liver, results in massive defects in gene transcription in response to fasting. We reported that this response is partially linked to activity of key transcription factor activated in fasting, a Glucocorticoid Receptor (Y. Shibayama et al., Acta Physiologica, 2022; highlighted in the editorial). We expanded these analyses by performing bulk transcriptomic, proteomics, and metabolomics analyses in liver of mice that were challenged by fasting. These analyses were fructuous and provided several candidates. We are now performing molecular validation studies in order to dissect how class 3 PI3K controls specific cellular metabolic activities in fasting in different ages of young and old male and female animals. Moreover, from cell to the organism scale, the metabolic demands fluctuate rhythmically relying on coordination between the circadian clock and nutrient sensing signaling pathways. Thus, in second objective, we progressed in understanding how class 3 PI3K expression and activity is changing around the clock. We also demonstrated its requirement for metabolic rhythmicity in liver. We are now investigating in details the molecular mechanisms of its potential crosstalk with the clock in different organs. We also apply our expertise of basic science in the field of nutrient sensing signaling and metabolism to investigate novel metabolic signatures on rare pediatric diseases with manifestations in liver such as biliary atresia (BA). BA is an uncurable disease that manifests as obliteration of the extrahepatic bile ducts and alteration of the intrahepatic biliary tree in neonates. Left untreated, it quickly progresses to cirrhosis and liver failure leading to death in the first 2 years of life. While rare (a worldwide incidence of ~1:20,000 live births), BA remains the most frequent cause of liver transplantation in children. In this funding period, we have established the biobank of patient derived material, generated patient derived cell models and animal models of BA that were instrumental in performing molecular analyses. Our findings in this direction have indicated potential therapeutic targets that we are in process of validation.
Using gene deletions and manipulating nutrient levels in cells and in vivo in mice we have discovered novel mechanisms of class 3 PI3K involvement in response to lack or presence of food. In the next funding period, reassured by our success, we will develop further the objectives as outlined above. Our work on the first objective will focus on mechanistic studies that will dissect the functional interactions with novel partners of class 3 PI3K that we have identified as well as characterizing the upstream signals that control it. In second objective, we will expand our analyses of class 3 PI3K function in metabolic rhythmicity. Lastly, in third objective, we will focus on validation mechanistic studies in newly generated patient derived cell models and in animal models of disease that might lead to future therapies of biliary atresia.