A Facile LC-MS Method for Profiling Cholesterol and Cholesteryl Esters in Mammalian Cells and Tissues.

Cholesterol is central to mammalian lipid metabolism and serves many critical functions in the regulation of diverse physiological processes. Dysregulation in cholesterol metabolism is causally linked to numerous human diseases, and therefore, in vivo, the concentrations and flux of cholesterol and cholesteryl esters (fatty acid esters of cholesterol) are tightly regulated. While mass spectrometry has been an analytical method of choice for detecting cholesterol and cholesteryl esters in biological samples, the hydrophobicity, chemically inert nature, and poor ionization of these neutral lipids have often proved a challenge in developing lipidomics compatible liquid chromatography-mass spectrometry (LC-MS) methods to study them. To overcome this problem, here, we report a reverse-phase LC-MS method that is compatible with existing high-throughput lipidomics strategies and capable of identifying and quantifying cholesterol and cholesteryl esters from mammalian cells and tissues. Using this sensitive yet robust LC-MS method, we profiled different mammalian cell lines and tissues and provide a comprehensive picture of cholesterol and cholesteryl esters content in them. Specifically, among cholesteryl esters, we find that mammalian cells and tissues largely possess monounsaturated and polyunsaturated variants. Taken together, our lipidomics compatible LC-MS method to study this lipid class opens new avenues in understanding systemic and tissue-level cholesterol metabolism under various physiological conditions.

Lysophosphatidylserine: A Signaling Lipid with Implications in Human Diseases.

Lysophosphatidylserine (lyso-PS) has emerged as yet another important signaling lysophospholipid in mammals, and deregulation in its metabolism has been directly linked to an array of human autoimmune and neurological disorders. It has an indispensable role in several biological processes in humans, and therefore, cellular concentrations of lyso-PS are tightly regulated to ensure optimal signaling and functioning in physiological settings. Given its biological importance, the past two decades have seen an explosion in the available literature toward our understanding of diverse aspects of lyso-PS metabolism and signaling and its association with human diseases. In this Review, we aim to comprehensively summarize different aspects of lyso-PS, such as its structure, biodistribution, chemical synthesis, and SAR studies with some synthetic analogs. From a biochemical perspective, we provide an exhaustive coverage of the diverse biological activities modulated by lyso-PSs, such as its metabolism and the receptors that respond to them in humans. We also briefly discuss the human diseases associated with aberrant lyso-PS metabolism and signaling and posit some future directions that may advance our understanding of lyso-PS-mediated mammalian physiology.

Metabolic transitions regulate global protein fatty acylation.

Intermediary metabolites and flux through various pathways have emerged as key determinants of post-translational modifications. Independently, dynamic fluctuations in their concentrations are known to drive cellular energetics in a bi-directional manner. Notably, intracellular fatty acid pools that drastically change during fed and fasted states act as precursors for both ATP production and fatty acylation of proteins. Protein fatty acylation is well regarded for its role in regulating structure and functions of diverse proteins; however, the effect of intracellular concentrations of fatty acids on protein modification is less understood. In this regard, we unequivocally demonstrate that metabolic contexts, viz. fed and fasted states, dictate the extent of global fatty acylation. Moreover, we show that presence or absence of glucose that influences cellular and mitochondrial uptake/utilization of fatty acids and affects palmitoylation and oleoylation, which is consistent with their intracellular abundance in fed and fasted states. Employing complementary approaches including click-chemistry, lipidomics, and imaging, we show the top-down control of cellular metabolic state. Importantly, our results establish the crucial role of mitochondria and retrograde signaling components like SIRT4, AMPK, and mTOR in orchestrating protein fatty acylation at a whole cell level. Specifically, pharmacogenetic perturbations that alter either mitochondrial functions and/or retrograde signaling affect protein fatty acylation. Besides illustrating the cross-talk between carbohydrate and lipid metabolism in mediating bulk post-translational modification, our findings also highlight the involvement of mitochondrial energetics.