Organelle function in metabolism and immunity

Mark Li

doi:10.25820/etd.007154

Living organisms must constantly adapt to fluctuations in their environment in order to maintain homeostasis and survive. At the cellular level, many organelles, such as mitochondria, the ER, and lysosomes, orchestrate a myriad of events to regulate various outputs in response to both acute and long-term stressors. The overarching goal of this thesis work was to understand how these subcellular compartments regulate the needs of our body under physiological and pathological conditions. The first part of my work focused on identifying and characterizing a novel micropeptide in controlling pancreatic β-cell function. This cell type primarily secretes insulin in response to elevated glucose, and this response is heavily relied on intracellular levels of calcium in the β-cell. In the context of obesity and type 2 diabetes, β-cells start to lose their capacity to properly secrete insulin in response to elevated levels of blood glucose and it is associated with impaired calcium signaling mediated by the ER. Accordingly, numerous attempts have been made to improve the secretory capacity of the cell. Among key regulators of β-cell function, long non-coding RNAs (lncRNAs) have emerged as promising players. However, with increased technical and analytical power, researchers realized that some of these lncRNAs can encode small-size proteins, termed micropeptides, with significant effects on different aspects of biology. At the time of our study, there was no identified micropeptide in the β-cell; thus, we sought to establish a robust pipeline to identify and study novel micropeptides in the pancreas. By utilizing RNA-sequencing, biochemical and molecular tools, and various functional studies, we were able to identify a micropeptide in the human and mouse β-cell that we named Beta-cell and Neural-cell Regulin (BNLN). We further found that BNLN localized to the ER and interacted with an important calcium pump, termed SERCA3, which in turn regulated intracellular calcium dynamics in response to high glucose levels. In addition, BNLN was both necessary and sufficient for β-cell insulin secretion in rat, mouse, and human cells. Finally, overexpressing BNLN in mouse islets was sufficient to restore obesity-associated insulin secretory defect. The second part of the thesis focused on understanding the role of lysosomes in cardiomyocytes in the setting of heart failure (HF). Cardiomyocytes make up the majority of the heart and their contractile function allows for efficient circulation of blood throughout the body. In HF, cardiomyocytes lose their contractility due to various reasons, resulting in altered immuno-metabolic homeostasis, evident by the presence of defective mitochondrial respiration and aberrant inflammatory responses. Lysosomes play a crucial role in preserving intracellular immuno-metabolic homeostasis by monitoring quality control of mitochondria and suppressing prolonged inflammatory signaling. Notably, mutations in lysosome-resident proteins are known to cause human diseases affecting cardiovascular health, and loss-of-function of some of the lysosomal enzymes negatively contributes to the onset and progression of HF. However, the mechanistic basis of lysosome-dependent preservation of cardiac function in HF remained elusive. Within the lysosome, Gamma-interferon-inducible thiol reductase (GILT) is the only known enzyme with reducing capacity, but its function was exclusively studied in immune cells. We noted that global deletion of GILT caused abnormalities in cardiac function, including pathological hypertrophy and reduced contractility. Therefore, we hypothesized that GILT could have an important protective role in cardiomyocytes. To test this hypothesis, we generated a novel mouse model to conditionally delete GILT in cardiomyocytes. Upon successful deletion of GILT, we observed pathological remodeling and loss of contractile function in the heart in the context of different stressors such as age, high-fat diet, and pressure overload. In addition, intra-cardiac immuno-metabolic homeostasis was altered in GILT-deficient cells as determined by multi-omics, biochemical, imaging, and molecular analyses. Specifically, we noted reduced mitochondrial respiration and increased NLRP3-mediated signaling in GILT-deficient cardiomyocytes. Inhibiting NLRP3 in GILT-deficient cells completely rescued the mitochondrial defect, suggesting GILT-dependent negative regulation of NLRP3, which supports mitochondrial function in the heart. Finally, in the third part of my work, we studied how different organs in our body communicate in times of acute stress, and how organelles contribute to this interaction. The liver is the largest internal organ with numerous functions including lipid metabolism. During episodes of acute infections, the liver outputs large amounts of energy substrates that can be utilized by other peripheral organs, such as the heart. Sepsis is an extreme case of an inflammatory response by our body to acute infection, whereby the liver secretes lipid species to support heart function. However, how this hepatic response is governed is unknown. We focused on the role of a protein called Inositol-requiring enzyme 1 (IRE1) in the major resident cell of the liver, the hepatocyte. IRE1 is the most conserved member of the stress response mechanism within the ER termed the unfolded protein response (UPR), and IRE1 is known to regulate hepatic lipid metabolism among other functions. However, its role in parenchymal cells of the liver in the setting of sepsis was completely unknown. Therefore, we hypothesized that hepatic IRE1 controls the cross-organ interaction between the liver and heart to support host survival in sepsis by mediating lipid metabolism. Deletion of IRE1 specifically in hepatocytes resulted in accelerated mortality as compared to control group. We found that deletion of IRE1 altered heart function without a major impact on other organs. The heart in IRE1 knockout animals became highly inflamed and contractility was altered as revealed by molecular, biochemical, and functional analyses. Moreover, we found that hepatic IRE1 governs the initial metabolic response in the liver during a septic event in part by positively regulating the secretion of lipid particles. As a result, the knockout animals had reduced levels of lipids in their circulation. Notably, the mechanism appears to be of non-canonical nature, as the well-known downstream target of IRE1, sXBP1, did not appear to be relevant in this context. Finally, reconstitution of wild-type lipid particles into the blood of IRE1 knockout animals restored cardiac function. Collectively, the work conducted during my Ph.D. training further supports the importance of different organelles, namely the ER and lysosomes, in maintaining immuno-metabolic balance in various contexts. This work (i) identified previously unknown members of ER-mediated calcium signaling, including the micropeptide BNLN, (ii) established the lysosomal reductase GILT as a platform to studying lysosome-mediated immune and metabolism in the heart, and (iii) identified a major molecular component, IRE1, of the liver-to-heart crosstalk in sepsis. Together, my work laid the foundation for further understanding the mechanistic basis of immuno-metabolic regulation by organelles, which may become of importance for designing therapeutic targets for managing diabetes, heart failure, and sepsis.

Organelle function in metabolism and immunity

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