Metabolic Regulation Of Transcription Factor Binding To IGHM Enhancer 3 (TFE3) In Obesity And Diabetes-Related Heart Disease

Abstract

Data from the Pulinilkunnil laboratory show that inactivation of TFEB, a MiTF/TFE transcription factor, and lysosomal dysfunction predispose cardiomyocytes to lipid accumulation, mitochondrial defects, and cardiac injury in obesity and diabetes. While TFEB is a key regulator of lysosomal biogenesis and autophagy, transcription factor E3 (TFE3), another MiTF/TFE family member, also regulates lysosome-autophagy genes. TFEB and TFE3 regulate the transcription of genes in the lysosome-autophagy pathway and, in turn, are regulated by the mechanistic target of rapamycin complex 1 (mTORC1)- dependent or independent pathways. However, the role of TFE3 in the heart and its regulation in health and nutrient stress remains unclear. Using rodent and cardiac cell lines models of metabolic maladaptation, this thesis examined the impact of nutrient stress and lipotoxicity on TFE3 protein content and ascertained if the loss of function of TFE3 renders cardiomyocytes susceptible or resistant to mitochondrial dysfunction, insulin resistance, and metabolic maladaptation. Twenty-week high-fat fed C57BL/6J male and female mice showed increased TFE3 protein content, accompanied by a concomitant decline in TFEB, when compared to chow diet-fed animals. Myocardial TFE3 content was also significantly increased in 7-week post Streptozotocin (STZ)-treated type 1 diabetic Wistar rats and in C57BL/6J mice fed a high-fat diet and treated with STZ, mimicking type 2 diabetes. Cardiomyocyte-specific TFEB knockout mice on a high-fat diet showed further upregulation of TFE3, suggesting that nutrient overload augments TFE3 in a TFEB-independent manner. Indeed, saturated fatty acid palmitate treatment ex vivo, increased total TFE3 and either short (TFE3S) of long (TFE3L) isoforms, while reducing TFEB protein in human AC16 cardiomyocyte-like cells, mouse C2C12 myotubes, rat H9c2 cells, and primary neonatal rat (NRCM) and adult rat (ARCM) and mouse (AMCM) cardiomyocytes. Unsaturated fatty acid, Oleate treatment had a variable effect on the protein content of total TFE3 or its isoforms in different cell lines. Efficient TFE3 knockdown (>70%) was achieved in AC16, H9c2, C2C12, and NRCMs. TFE3 silencing in AC16 cells increased lipid droplet (LD) accumulation and triglyceride (TAG) content under oleate loading. High-resolution respirometry revealed reduced glucose- and fatty acid-linked oxygen consumption in TFE3-deficient NRCMs, C2C12, and H9c2 cells, despite increased OXPHOS complex proteins indicating flux of lipids away from oxidation towards LD-TAG formation. Additionally, in AC16 and C2C12 cells, TFE3 silencing blunted insulin-induced phosphorylation of p70S6K (Thr389) and Akt (Ser473), a downstream effector of insulin’s metabolic and mitogenic action. Collectively, this thesis examined the previously unexplored role of cardiomyocyte TFE3 and uncovered the critical role of TFE3 in altering lipid handling, mitochondrial respiration, and insulin signaling. This research advances the understanding of nutrient regulation of TFE3 and clarifies the role of TFE3 in cardiac energy metabolism and insulin signalling, plausibly facilitating functional outcomes and therapeutic targeting in obesity and diabetes related heart disease. It remains to be determined whether TFE3 action depends on transcription factor cooperativity with TFEB, governing metabolic adaptation and cardiac function.