Emerging Role of Lipids in Metabolism and Disease Printed Edition of the Special Issue Published in International Journal of Molecular Sciences www.mdpi.com/journal/ijms Marco Segatto and Valentina Pallottini Edited by Emerging Role of Lipids in Metabolism and Disease Emerging Role of Lipids in Metabolism and Disease Editors Marco Segatto Valentina Pallottini MDPI • Basel • Beijing • Wuhan • Barcelona • Belgrade • Manchester • Tokyo • Cluj • Tianjin Editors Marco Segatto University of Molise Italy Valentina Pallottini University Roma Tre Italy Editorial Office MDPI St. Alban-Anlage 66 4052 Basel, Switzerland This is a reprint of articles from the Special Issue published online in the open access journal International Journal of Molecular Sciences (ISSN 1422-0067) (available at: https://www.mdpi.com/ journal/ijms/special issues/Lipids Disease). For citation purposes, cite each article independently as indicated on the article page online and as indicated below: LastName, A.A.; LastName, B.B.; LastName, C.C. Article Title. Journal Name Year , Article Number , Page Range. ISBN 978-3-03943-701-6 (Pbk) ISBN 978-3-03943-702-3 (PDF) Cover image courtesy of Marco Segatto. c © 2020 by the authors. Articles in this book are Open Access and distributed under the Creative Commons Attribution (CC BY) license, which allows users to download, copy and build upon published articles, as long as the author and publisher are properly credited, which ensures maximum dissemination and a wider impact of our publications. The book as a whole is distributed by MDPI under the terms and conditions of the Creative Commons license CC BY-NC-ND. Contents About the Editors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vii Marco Segatto and Valentina Pallottini Facts about Fats: New Insights into the Role of Lipids in Metabolism, Disease and Therapy Reprinted from: Int. J. Mol. Sci. 2020 , 21 , 6651, doi:10.3390/ijms21186651 . . . . . . . . . . . . . . 1 Anna Kloska, Magdalena W ę sierska, Marcelina Malinowska, Magdalena Gabig-Cimi ́ nska and Joanna Jak ́ obkiewicz-Banecka Lipophagy and Lipolysis Status in Lipid Storage and Lipid Metabolism Diseases Reprinted from: Int. J. Mol. Sci. 2020 , 21 , 6113, doi:10.3390/ijms21176113 . . . . . . . . . . . . . . 7 Claudia Tonini, Mayra Colardo, Barbara Colella, Sabrina Di Bartolomeo, Francesco Berardinelli, Giuseppina Caretti, Valentina Pallottini and Marco Segatto Inhibition of Bromodomain and Extraterminal Domain (BET) Proteins by JQ1 Unravels a Novel Epigenetic Modulation to Control Lipid Homeostasis Reprinted from: Int. J. Mol. Sci. 2020 , 21 , 1297, doi:10.3390/ijms21041297 . . . . . . . . . . . . . . 39 Pierre Avril, Luciano Vidal, Sophie Barille-Nion, Louis-Rom ́ ee Le Nail, Fran ̧ coise Redini, Pierre Layrolle, Michelle Pinault, St ́ ephane Chevalier, Pierre Perrot and Val ́ erie Trichet Epinephrine Infiltration of Adipose Tissue Impacts MCF7 Breast Cancer Cells and Total Lipid Content Reprinted from: Int. J. Mol. Sci. 2019 , 20 , 5626, doi:10.3390/ijms20225626 . . . . . . . . . . . . . . 55 Anastasia V. Poznyak, Andrey V. Grechko, Reinhard Wetzker and Alexander N. Orekhov In Search for Genes Related to Atherosclerosis and Dyslipidemia Using Animal Models Reprinted from: Int. J. Mol. Sci. 2020 , 21 , 2097, doi:10.3390/ijms21062097 . . . . . . . . . . . . . . 71 Anastasia Poznyak, Andrey V. Grechko, Paolo Poggio, Veronika A. Myasoedova, Valentina Alfieri and Alexander N. Orekhov The Diabetes Mellitus–Atherosclerosis Connection: The Role of Lipid and Glucose Metabolism and Chronic Inflammation Reprinted from: Int. J. Mol. Sci. 2020 , 21 , 1835, doi:10.3390/ijms21051835 . . . . . . . . . . . . . . 85 Khalia R. Primer, Peter J. Psaltis, Joanne T.M. Tan and Christina A. Bursill The Role of High-Density Lipoproteins in Endothelial Cell Metabolism and Diabetes-Impaired Angiogenesis Reprinted from: Int. J. Mol. Sci. 2020 , 21 , 3633, doi:10.3390/ijms21103633 . . . . . . . . . . . . . . 99 Arianna Mazzoli, Maria Stefania Spagnuolo, Cristina Gatto, Martina Nazzaro, Rosa Cancelliere, Raffaella Crescenzo, Susanna Iossa and Luisa Cigliano Adipose Tissue and Brain Metabolic Responses to Western Diet—Is There a Similarity between the Two? Reprinted from: Int. J. Mol. Sci. 2020 , 21 , 786, doi:10.3390/ijms21030786 . . . . . . . . . . . . . . . 121 Yoon Sun Chun and Sungkwon Chung High-Cholesterol Diet Decreases the Level of Phosphatidylinositol 4,5-Bisphosphate by Enhancing the Expression of Phospholipase C (PLC β 1) in Rat Brain Reprinted from: Int. J. Mol. Sci. 2020 , 21 , 1161, doi:10.3390/ijms21031161 . . . . . . . . . . . . . . 141 v Marina Francis, Alaa Abou Daher, Patrick Azzam, Manal Mroueh and Youssef H. Zeidan Modulation of DNA Damage Response by Sphingolipid Signaling: An Interplay that Shapes Cell Fate Reprinted from: Int. J. Mol. Sci. 2020 , 21 , 4481, doi:10.3390/ijms21124481 . . . . . . . . . . . . . . 151 Samuela Cataldi, Antonella Borrelli, Maria Rachele Ceccarini, Irina Nakashidze, Michela Codini, Oleg Belov, Alexander Ivanov, Eugene Krasavin, Ivana Ferri, Carmela Conte, Federica Filomena Patria, Tommaso Beccari, Aldo Mancini, Francesco Curcio, Francesco Saverio Ambesi-Impiombato and Elisabetta Albi Acid and Neutral Sphingomyelinase Behavior in Radiation-Induced Liver Pyroptosis and in the Protective/Preventive Role of rMnSOD Reprinted from: Int. J. Mol. Sci. 2020 , 21 , 3281, doi:10.3390/ijms21093281 . . . . . . . . . . . . . . 175 Samuela Cataldi, Antonella Borrelli, Maria Rachele Ceccarini, Irina Nakashidze, Michela Codini, Oleg Belov, Alexander Ivanov, Eugene Krasavin, Ivana Ferri, Carmela Conte, Federica Filomena Patria, Giovanna Traina, Tommaso Beccari, Aldo Mancini, Francesco Curcio, Francesco Saverio Ambesi-Impiombato and Elisabetta Albi Neutral Sphingomyelinase Modulation in the Protective/Preventive Role of rMnSOD from Radiation-Induced Damage in the Brain Reprinted from: Int. J. Mol. Sci. 2019 , 20 , 5431, doi:10.3390/ijms20215431 . . . . . . . . . . . . . . 187 Anna Kloska, Marcelina Malinowska, Magdalena Gabig-Cimi ́ nska and Joanna Jak ́ obkiewicz-Banecka Lipids and Lipid Mediators Associated with the Risk and Pathology of Ischemic Stroke Reprinted from: Int. J. Mol. Sci. 2020 , 21 , 3618, doi:10.3390/ijms21103618 . . . . . . . . . . . . . . 197 Vidyani Suryadevara, Ramaswamy Ramchandran, David W. Kamp and Viswanathan Natarajan Lipid Mediators Regulate Pulmonary Fibrosis: Potential Mechanisms and Signaling Pathways Reprinted from: Int. J. Mol. Sci. 2020 , 21 , 4257, doi:10.3390/ijms21124257 . . . . . . . . . . . . . . 223 Federico Carbone, Maria Stefania Lattanzio, Silvia Minetti, Anna Maria Ansaldo, Daniele Ferrara, Emilio Molina-Molina, Anna Belfiore, Edoardo Elia, Stefania Pugliese, Vincenzo Ostilio Palmieri, Fabrizio Montecucco and Piero Portincasa Circulating CRP Levels Are Associated with Epicardial and Visceral Fat Depots in Women with Metabolic Syndrome Criteria Reprinted from: Int. J. Mol. Sci. 2019 , 20 , 5981, doi:10.3390/ijms20235981 . . . . . . . . . . . . . . 269 Milica Markovic, Shimon Ben-Shabat, Aaron Aponick, Ellen M. Zimmermann and Arik Dahan Lipids and Lipid-Processing Pathways in Drug Delivery and Therapeutics Reprinted from: Int. J. Mol. Sci. 2020 , 21 , 3248, doi:10.3390/ijms21093248 . . . . . . . . . . . . . . 283 vi About the Editors Marco Segatto (Assistant Professor) has developed a great interest in cholesterol homeostasis since completing his master’s degree, focusing his attention on the physiopathological role played by lipid homeostasis in different organs and tissues such as liver, muscle, and brain. He continued to acquire new skills and knowledge about cholesterol metabolism in the brain as a Ph.D. student, and as a Postdoctoral Fellow, broadened his experience in the field of metabolism. He is currently Assistant Professor at University of Molise, and his main research interests remain focused on the involvement of lipid metabolism in brain and muscle physiopathology. Valentina Pallottini (Professor), The research activity of Professor Pallottini has been focused on changes in cholesterol metabolism under different physiological and pathological conditions. Her research has been principally carried on HMGCR (3 hydroxy 3-methylglutaryl coenzyme A reductase), the rate-limiting enzyme of cholesterol biosynthetic pathway, and on cholesterol metabolism in different physiological states. Her research on cholesterol metabolism has been principally focused on aged rat livers, models with high concentration of free radicals, and gender differences. During the last 10 years, her research has been focused on the role of cholesterol metabolism in the central nervous system and its involvement in neurodevelopmental and neurodegenerative disorders. vii International Journal of Molecular Sciences Editorial Facts about Fats: New Insights into the Role of Lipids in Metabolism, Disease and Therapy Marco Segatto 1 and Valentina Pallottini 2, * 1 Department of Biosciences and Territory, University of Molise, Contrada Fonte Lappone, 86090 Pesche (Is), Italy; marco.segatto@unimol.it 2 Department of Science, University Roma Tre, Viale Marconi 446, 00146 Rome, Italy * Correspondence: valentina.pallottinini@uniroma3.it Received: 31 August 2020; Accepted: 7 September 2020; Published: 11 September 2020 Keywords: Cholesterol; Fatty acids; Lipid mediators; Lipids; Lipophagy; Sphingolipids Although initially regarded as a passive system to store energy, lipids are now considered to play crucial, structural and functional roles in almost all the biological processes involved in the regulation of physiological and pathological conditions. For instance, they are pivotal constituents of cell membranes, where they essentially contribute to the assembly of the bilayer configuration. Lipid species are not uniformly distributed in cell membranes, as they are mainly concentrated in specialized sphingolipid- and cholesterol-rich domains called lipid rafts and caveolae, which serve as a matrix for the attachment and the interaction of several proteins implicated in membrane-initiating signal transduction pathways [ 1 ]. Besides the involvement in the organization of membrane domains, lipids may influence numerous cellular processes by directly participating as both primary and secondary messengers. In recent decades, lipids derived from both dietary sources and endogenous biosynthesis gained considerable clinical relevance for their implications in a plethora of human diseases. Hyperlipidemia often results in a premature and increased risk of cardiovascular diseases [ 2 ]. Increasing evidence highlights that the metabolic reprogramming of cancer cells also involves many lipid compounds, such as fatty acids and cholesterol, which contribute to key oncogenic functions [ 3 , 4 ]. Furthermore, a variety of experimental and clinical studies demonstrated that lipids, particularly cholesterol, play essential roles in brain physiology [ 5 ]. Thus, it is not surprising that numerous neurological and neurodegenerative disorders are often accompanied by misbalances in lipid homeostasis [6]. Despite a satisfying level of knowledge being reached in the field of lipid research, several questions still remain unanswered; a deeper comprehension will be important to fully elucidate the molecular mechanisms regulating lipid homeostasis in health and loss of homeostasis in disease, and to design innovative therapeutic strategies. This Special Issue, entitled “Emerging Role of Lipids in Metabolism and Disease”, comprises a collection of seven research articles, seven review articles and one concept paper reporting new insights into the biological role of lipids. Lipid homeostasis is guaranteed by a delicate equilibrium among biosynthesis, uptake and catabolism. Kloska and colleagues [ 7 ] systematically reviewed the current knowledge on lipophagy and cytosolic lipolysis, two selective lipid catabolic processes whose activity is essential for the proper regulation of energy homeostasis in cells. The authors provide an in-depth discussion of the transcriptional modulation of these pathways by the mammalian target of rapamycin complex 1 (mTORC1) and by the transcription factor EB (TFEB). In addition, alterations of both lipophagy and lipolysis in pathological conditions are also documented. Even though basic molecular pathways underlying the maintenance of lipid homeostasis are now well established, several regulatory mechanisms still remain unknown. In this context, the experimental work provided by Tonini and colleagues report a novel epigenetic pathway by which cells physiologically control lipid homeostasis [ 8 ]. Notably, the authors unravel that bromodomain and extraterminal domain (BET) proteins, which Int. J. Mol. Sci. 2020 , 21 , 6651; doi:10.3390 / ijms21186651 www.mdpi.com / journal / ijms 1 Int. J. Mol. Sci. 2020 , 21 , 6651 are epigenetic readers particularly committed to the regulation of cell cycle progression, govern lipid metabolism by modulating the expression of proteins and enzymes implicated in fatty acid and cholesterol biosynthesis, tra ffi cking and uptake. Coherently, BET protein blockade by the small inhibitor JQ1 strongly reduces the amount of intracellular lipids. In addition, Tonini and colleagues also showed that the decreased proliferation of HepG2 cancer cells induced by JQ1 is dependent on the modulation of cholesterol metabolism. The connection between lipid metabolism and the biology of cancer cells is also investigated by Avril et al., who studied the impact of epinephrine-infiltrated adipose tissue (AT) on MCF-7 breast cancer cells [ 9 ]. They observed that epinephrine-infiltrated AT secretes di ff erent factors, including lipids, which may act as signaling molecules. Indeed, both epinephrine-infiltrated AT and its corresponding conditioned medium (CM) enhance the proliferation of MCF-7 breast cancer cells in vitro . The secreted factors contained in CM also appear to increment the in vivo growth of MCF7 cells in mice. However, injection of whole epinephrine-infiltrated AT did not induce any change in the progression of MCF-7- tumor in mice, suggesting that the employment of CM to mimic the secretome of cells or tissues may explain some divergences observed among in vitro , pre-clinical and clinical data using AT samples. Besides cancer biology, alterations of lipid metabolism are a major public health concern because of their association to cardiovascular diseases. Atherosclerosis, a complex process characterized by progressive inflammation and build-up of cholesterol and other lipids in the artery walls, is the leading cause of cardiovascular diseases. The exact etiopathogenesis of atherosclerosis is not fully elucidated, however, several risk factors have been identified, such as high blood pressure, hypercholesterolemia, obesity and diabetes. In their review article, Poznyak and colleagues provided up-to-date information about the use of knockout mice as experimental models to investigate genes involved in atherosclerosis and dyslipidemia [ 10 ]. In addition, Poznyak et al. discuss the current knowledge concerning the mechanisms by which diabetes mellitus promotes the atherogenic process, and summarize the physiopathological hallmarks linking atherosclerosis and diabetes mellitus, such as protein kinase signaling, oxidative stress, miRNA alterations and epigenetic changes [ 11 ]. The breakdown of homeostatic regulation of lipids occurring during diabetes has also been examined by Primer and collaborators [ 12 ]. In particular, they summarize the current understanding of endothelial cell metabolism and its dysregulation during diabetes and discuss the di ff erent mechanisms by which high-density lipoproteins (HDL) modulate endothelial cell metabolic reprogramming and counteract diabetes-impaired angiogenesis. Lipid metabolism is also finely regulated within the brain: it maintains neuronal functionality and signals the nutrient status to regulate the whole-body metabolism by modulating key peripheral organs and tissues, such as the liver and adipose tissue. Alterations of lipid metabolism have been frequently associated with disturbances in brain functioning, such as neurodegeneration, neuroinflammation, cognitive alterations and neurodevelopmental problems. It is becoming increasingly clear that high levels of dietary fats and sugars, which are typically comprised in a western diet, may be detrimental to brain health. Mazzoli and colleagues provided an experimental work aimed at analyzing the putative e ff ects of a western diet on the metabolic response of nervous and adipose tissue [ 13 ]. For instance, they reported that the expression of specific cyto / adipokines, such as TNF α and adiponectin, are significantly a ff ected in both brain and adipose tissue of rats fed with a diet high in saturated fats and fructose (HFF). The observed changes are accompanied by a reduction in brain-derived neurotrophic factor (BDNF) and synaptotagmin I levels, and by an increase in the expression of the post-synaptic density protein, PSD95, in HFF-fed animals. When evaluated as a whole, these results underline that a western diet may induce similar metabolic alterations in adipose tissue and brain. Chun and Chung further confirmed the involvement of dietary lipids in brain physiology [ 14 ]. Specifically, they show experimental evidence that a high-cholesterol diet significantly decreases the expression levels of phospholipase C β 1 (PLC β 1) and of phosphatidylinositol 4,5-bisphosphate (PIP 2 ). Interestingly, there is no direct correlation between the amount of cholesterol and of PIP 2 , suggesting that PIP 2 levels are modulated by cholesterol through changes in the expression of PLC β 1. Since 2 Int. J. Mol. Sci. 2020 , 21 , 6651 the reduction in PIP 2 levels has been associated with β -amyloid production, these results indicate that a high cholesterol diet may influence brain cholesterol, which reflects in PIP 2 changes that could contribute to the pathogenesis of neurodegenerative conditions. Considering that the blood brain barrier prevents the uptake of lipoprotein-bound cholesterol from the bloodstream, it will be a stimulating challenge to comprehend how diet-derived cholesterol can influence neuronal processes. Among lipids, sphingolipids represent a major subcategory. These molecules are not only structural components, but also act as bioactive compounds in the mediation of physiological processes involved in cell proliferation, survival, inflammation, senescence and death. A number of experimental evidence sustains that the metabolic pathway, which governs sphingolipid metabolism, exerts a pivotal role in the regulation of the response to DNA damage. Francis and colleagues analytically reviewed how sphingolipid signaling influences the DNA damage response (DDR) induced by metabolic stress, ionizing radiation or other genotoxic stimuli [ 15 ]. In particular, they illustrate how di ff erent sphingolipid metabolites interact with the mediators of DDR to define cell fate. In the field of sphingolipid research, Cataldi and colleagues provided new interesting insights about the involvement of sphingomyelinases in the e ff ects of ionizing radiations. Notably, experimental data highlighted that ionizing radiations cause altered hepatic cell structure and increased caspase-1 expression in mice. These e ff ects are attenuated by the administration of recombinant manganese superoxide dismutase (rMnSOD). Importantly, rMnSOD counteracts the radiation-induced liver damage exerting a protective role via acid sphingomyelinase (aSMase), and a preventive role via neutral sphingomyelinase (nSMase) [ 16 ]. Cataldi et al. also demonstrated that nSMase is responsible for the preventive and protective e ff ect elicited by rMnSOD against radiation-induced damage in the brain [17]. As already mentioned above, lipids not only serve as structural components, but also exert crucial biological roles as signaling molecules. This aspect is extensively discussed in the review article proposed by Kloska and collaborators, who focused their attention on the role of lipid mediators in the risk and pathology of ischemic stroke [ 18 ]. Notably, a lively metabolism of polyunsaturated fatty acid has been documented in ischemic brain, and di ff erent lipid mediators are implicated in the neuroprotective or neurodegenerative e ff ects occurring in the post-stroke brain tissue. Among signaling molecules, eicosanoids seem to play crucial roles in the disease pathology, and a variety of reports suggest them as useful molecular targets for innovative therapeutic interventions. The involvement of lipid molecules as signaling mediators is further examined in the comprehensive review by Suryadevara et al., which collects up-to-date knowledge about the pathways mediated by lipid mediators in pulmonary fibrosis [ 19 ]. Indeed, the metabolism of phospholipids, sphingolipids, and polyunsaturated fatty acids may generate key molecules capable of signaling properties, which exhibit pro- and anti-fibrotic e ff ects in patients and preclinical models of idiopathic pulmonary fibrosis (IPF). In light of this evidence, it is not surprising that prostanoids, lysophospholipids, sphingolipids and their metabolizing enzymes are currently under active investigation as potential pharmacological targets to treat IPF. Finally, Carbone and collaborators contributed to this Special Issue with a research article aimed at assessing the association of circulating C-reactive protein (CRP) levels with epicardial and visceral fat depots in women with one or more defining criteria for metabolic syndrome [ 20 ]. The main findings highlight that men and women have a di ff erent epicardial fat deposition and systemic inflammation. Intriguingly, a correlation between visceral / epicardial fat depots and chronic low-grade inflammation was also noted, suggesting that sex may play an essential role in the stratification of obese individuals and dysmetabolic patients. In conclusion, the experimental data summarized and presented in this Special Issue further strengthen the centrality of lipids in a plethora of biological processes and underline the importance of lipid research in physiopathology. Indeed, lipids may represent useful biomarkers for a number of diseases, and alterations in their metabolism may concur to the development of di ff erent disorders. Lipids can also be combined or conjugated to drug compounds, determining several benefits in terms of treatment e ff ect. In this context, further basic, translational, and clinical research are imperative to 3 Int. J. Mol. Sci. 2020 , 21 , 6651 discover novel mechanisms controlling lipid metabolism in health and disease, and to set up optimal drug design. All these concepts are clearly debated in the concept paper by Markovic et al. [21]. Author Contributions: Writing—original draft preparation, M.S. and V.P.; writing—review and editing, M.S. and V.P. All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. Conflicts of Interest: The authors declare no conflict of interest. References 1. Escrib á , P.V.; Gonz á lez-Ros, J.M.; Goñi, F.M.; Kinnunen, P.K.; Vigh, L.; S á nchez-Magraner, L.; Fern á ndez, A.M.; Busquets, X.; Horv á th, I.; Barcel ó -Coblijn, G. Membranes: A meeting point for lipids, proteins and therapies. J. Cell Mol. Med. 2008 , 12 , 829–875. [CrossRef] [PubMed] 2. Kuller, L.H. Nutrition, lipids, and cardiovascular disease. Nutr. Rev. 2006 , 64 , S15–S26. [CrossRef] 3. Butler, L.M.; Perone, Y.; Dehairs, J.; Lupien, L.E.; de Laat, V.; Talebi, A.; Loda, M.; Kinlaw, W.B.; Swinnen, J.V. Lipids and cancer: Emerging roles in pathogenesis, diagnosis and therapeutic intervention. Adv. Drug Deliv. Rev. 2020 . [CrossRef] [PubMed] 4. Pesiri, V.; Totta, P.; Segatto, M.; Bianchi, F.; Pallottini, V.; Marino, M.; Acconcia, F. 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This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http: // creativecommons.org / licenses / by / 4.0 / ). 5 International Journal of Molecular Sciences Review Lipophagy and Lipolysis Status in Lipid Storage and Lipid Metabolism Diseases Anna Kloska 1 , Magdalena W ̨ esierska 1 , Marcelina Malinowska 1 , Magdalena Gabig-Cimi ́ nska 1,2, * and Joanna Jak ó bkiewicz-Banecka 1, * 1 Department of Medical Biology and Genetics, Faculty of Biology, University of Gda ́ nsk, Wita Stwosza 59, 80-308 Gda ́ nsk, Poland; anna.kloska@ug.edu.pl (A.K.); magdalena.wesierska@phdstud.ug.edu.pl (M.W.); marcelina.malinowska@ug.edu.pl (M.M.) 2 Laboratory of Molecular Biology, Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Kładki 24, 80-822 Gda ́ nsk, Poland * Correspondence: magdalena.gabig-ciminska@ug.edu.pl (M.G.-C.); joanna.jakobkiewicz-banecka@ug.edu.pl (J.J.-B.); Tel.: + 48-585-236-046 (M.G.-C.); + 48-585-236-043 (J.J.-B.) Received: 6 July 2020; Accepted: 21 August 2020; Published: 25 August 2020 Abstract: This review discusses how lipophagy and cytosolic lipolysis degrade cellular lipids, as well as how these pathway ys communicate, how they a ff ect lipid metabolism and energy homeostasis in cells and how their dysfunction a ff ects the pathogenesis of lipid storage and lipid metabolism diseases. Answers to these questions will likely uncover novel strategies for the treatment of aforementioned human diseases, but, above all, will avoid destructive e ff ects of high concentrations of lipids—referred to as lipotoxicity—resulting in cellular dysfunction and cell death. Keywords: lipophagy; lipolysis; lipid metabolism; lipid droplets; lipid storage diseases; lipid metabolism diseases; mTORC1; TFEB 1. Introduction Lipids are water-insoluble biological macromolecules that are essential for maintaining cellular structure, function, signaling and energy storage. They are basic components of all cellular membranes which separate cell compartments in eukaryotic cells and provide a permeability barrier. These membrane boundaries are necessary for maintaining cellular homeostasis [ 1 , 2 ]. Moreover, lipids a ff ect the function of membrane proteins. Lipid rafts play a specific role in protein segregation; membrane proteins can interact with lipids, which serve as cofactors [ 3 , 4 ]. Finally, changes in lipid organization influence signal transduction and membrane tra ffi cking [ 2 ]. Cholesterol serves as a precursor for steroid hormones and bile acid biosynthesis [ 5 ]. Lipids are also molecules that serve as a source of energy when tissue energy is depleted [ 6 ]. Despite their role in essential cellular functions, incorrect lipid distribution or metabolism can result in abnormal concentrations of lipids being toxic because of their limited solubility and amphipathic nature, their adverse impact on cellular homeostasis and their ready transformation into highly bioactive, cytotoxic lipid species. These e ff ects have serious consequences for cellular function and homeostasis and may even lead to cell death [2]. In this review, we provide information about lipid metabolism in health and disease, focusing on lipid storage diseases and lipid metabolism diseases. We summarize the current knowledge about the role of two cytosolic pathways designed for lipid selective catabolism—lipophagy and lipolysis—and discuss the transcriptional regulation of these processes by the mechanistic target of rapamycin kinase complex 1 (mTORC1)—transcription factor EB (TFEB) signaling. We also characterize lipid storage and lipid metabolism diseases, highlighting the latest research on the contribution of mTORC1-TFEB signaling in the regulation of lipophagy, a subtype of macroautophagy, and lipolysis, an enzymatic hydrolysis process, in the selected human dysfunctions. Int. J. Mol. Sci. 2020 , 21 , 6113; doi:10.3390 / ijms21176113 www.mdpi.com / journal / ijms 7 Int. J. Mol. Sci. 2020 , 21 , 6113 2. Lipids in Eukaryotic Cells Based on the chemical origin (i.e., whether ketoacyl groups or isoprene groups serve as fundamental “building blocks”), lipids are divided into eight categories: fatty acids, glycerolipids, glycerophospholipids, sphingolipids, saccharolipids, polyketides, sterols and prenols [7]. 2.1. Fatty Acids and Cholesterol—Essential and Toxic Fatty acids (FAs) are hydrophobic molecules consisting of an aliphatic hydrocarbon chain terminating in a carboxylic acid moiety. FAs usually contain 16–18 carbons, and the chain can be fully saturated (saturated FA) or may contain one or more double bonds (unsaturated FAs). The main source of FAs for humans and other animals are dietary fats and oils, but they can also be synthesized de novo from metabolites of sugar and protein catabolic pathways [ 8 ]. Fatty acids can be harmful to cells because of lipotoxicity; thus, cells convert FAs into neutral lipids for storage in organelles called lipid droplets (LDs). Biogenesis of LDs is stimulated upon the increase in cellular free FA levels. Di ff erent cell types have LDs of various sizes and numbers, potentially reflecting the capacity of the cell for managing lipid storage. Moreover, these organelles are often heterogeneous within a population of a single cell type. It is believed that LDs not only serve as lipid storage organelles, but also interact with most, if not all, cellular organelles, mediating lipid transfer via direct contact [9]. Cholesterol is an elementary component of mammalian cell membranes. It interacts with phospholipids and sphingolipid fatty acyl chains in order to maintain appropriate membrane fluidity. Interactions between these lipids also regulate water and ion membrane permeability [ 10 ]. Cholesterol is required for normal prenatal development, as embryonic and fetal cells demonstrate high membrane formation rates [ 11 , 12 ]. At both fetal and adult stages of development, cholesterol is the precursor for biosynthesis of five major classes of steroid hormones (i.e., androgens, estrogens, glucocorticoids, mineralocorticoids and gestagens), vitamin D and bile acids [ 10 , 11 , 13 ]. Mammalian cells require cholesterol for proliferation. Moreover, cholesterol is specifically required for the transition from G1 to S during cell cycle progression [ 14 , 15 ]. Additionally, cholesterol is essential for mitosis progression and its deficiency leading to aberrant mitosis and polyploid cell formation [15,16]. The cell synthesizes cholesterol de novo or internalizes it from exogenous sources. Interestingly, cells do not have any enzymes to break down the sterol core to acetyl-CoA units; thus, cholesterol cannot be used as an energy source [ 10 ]. Regardless of its source, free cholesterol must be esterified; otherwise, it has a toxic e ff ect on cellular membranes and induces cell death. Esterified cholesterol is stored in cells in cytoplasmic lipid droplets [2,5]. 2.2. Lipid Droplets—Storage of Neutral Lipids LDs are highly dynamic cellular organelles responsible for the storage of neutral lipids. They are found in most eukaryotic cell types. The size of LDs varies within the range 0.4–100 μ m in di ff erent cell types or within the same cells, depending on physiological conditions. Lipid droplets originate from the endoplasmic reticulum (ER) and have a unique architecture consisting of a hydrophobic core of neutral lipids which is enclosed by a phospholipid monolayer with hundreds of resident and transient proteins that influence LD metabolism and signaling, known generically as perilipins (PLINs) (Figure 1A). Organization of these organelles is quite di ff erent than any other because the core of a LD is hydrophobic, the hydrophobic acyl chains of the monolayer’s phospholipids are in contact with neutral lipids and the polar head groups face the aqueous cytosol [ 17 ]. Furthermore, LDs can also be found in the nuclei, where they are thought to regulate nuclear lipid homeostasis and modulate signaling through lipid molecules [ 18 ]. Cells preserve lipids by converting them into neutral lipids such as triacylglycerols (TAGs) and sterol esters (ESs), which are in various ratio deposit in LDs. Depending on the cell type, many other endogenous neutral lipids, such as retinyl esters, ether lipids and free cholesterol, can be stored in the LD core. Defects in LD biogenesis lead to insu ffi cient or excess storage. Beyond the main function in energy metabolism, LDs play an important role in various cellular 8 Int. J. Mol. Sci. 2020 , 21 , 6113 events, such as protein degradation, sequestration of transcription factors and chromatin components, generation of lipid ligands for certain nuclear receptors and serving as fatty acid tra ffi cking nodes [ 19 ]. Figure 1. Structure and catabolism of a lipid droplet (LD). ( A ) LD is surrounded by the phospholipid monolayer enclosing a core filled with neutral lipids, e.g., triacylglycerol (TAG) and sterol esters. Polar heads of phospholipids are oriented toward the cytosol, whereas their acyl chains contact the hydrophobic lipid core. The LD surface is associated with various proteins, e.g., members of the perilipin (PLIN) family. There are two major types of LD catabolism: lipolysis—an enzymatic hydrolysis of lipids in cytosol, and lipophagy—an autophagic / lysosomal pathway in the form of macroautophagy or chaperone-mediated autophagy (CMA). ( B ) In lipolysis, protein kinase A (PKA) phosphorylates PLIN1 proteins, leading to their proteasomal degradation and activating adipose triglyceride lipase (ATGL), which then initiates TAG hydrolysis to generate diacylglycerols (DAGs) and free fatty acids (FAs). Further degradation of DAGs occurs through activation of the hormone sensitive lipase (HSL), leading to monoacylglycerol (MAG) and FAs production. MAGs are released to the cytosol and cleaved by monoacylglycerol lipase (MGL) to generate glycerol and FAs. ( C ) In macroautophagy, the phagophore is formed and LC3 positive membranes engulf small LD or sequester portions of a large LD to form the autophagosome, which later fuses with lysosome where LD degradation and neutral lipid catabolism occur. ( D ) In chaperone-mediated autophagy, lipid droplet-coat proteins—PLIN2 and PLIN3—are degraded through a coordinated action of Hsc70 protein and lysosome-associated membrane protein 2A (LAMP2A) receptor; this makes the LD surface accessible to cytosolic lipases, which hydrolyze LD cargo to generate FAs, which next are released to the cytosol and undergo subsequent mitochondrial β -oxidation. In mammalian cells, the phospholipid composition of LD membranes di ff ers from that of the ER and other organelles. The main constituent is phosphatidylcholine (PC), followed by phosphatidylethanolamine (PE), phosphatidylinositol (PI), phosphatidylserine (PS) and sphingomyelin (SM), as well as free cholesterol and phosphatidic acid in minor amounts. The unique phospholipid membrane composition a ff ects LD synthesis, size and catabolism. The homeostasis of these organelles under physiological conditions is maintained through changes in membrane phospholipid ratios in various cell types [20]. In addition to the composition of phospholipids, LD membrane surface proteins are another key factor that is important for their homeostasis and intracellular interactions. Each LD has many 9 Int. J. Mol. Sci. 2020 , 21 , 6113 di ff erent structural and functional protein