3^rd International Conference on Lipid Science & Technology December 11-12, 2017 Rome, Italy

Jingwen Liu is a Principal Investigator at the Palo Alto VA Hospital where she directs a research program seeking new therapeutic options to treat cardiometabolic diseases through studies conducted in liver cells and various animal models. Among numerous accomplishments, her team has characterized a new cellular pathway that upregulates the liver LDL receptor transcription through a cholesterol-independent mechanism and identified a novel post-transcriptional mechanism of LDLR gene that mediates the cholesterol-lowering effects of herbal medicine berberine. In the last decade, PCSK9 has emerged as a new therapeutic target for treating hypercholesterolemia. Her research in characterizing the transcriptional mechanism of PCSK9 has brought new insight for developing novel therapeutic approach to inhibit PCSK9 biosynthesis with a potential to improve the efficacy of current cholesterol- lowering drug statins. Her recent study in obeticholic acid has led to new understandings of lipid regulatory effects of FXR agonists in hyperlipidemic patients.

The farnesoid X receptor (FXR) and liver X receptor (LXR) are known to regulate distinct biological pathways in BA synthesis and cholesterol metabolism. Obeticholic acid (OCA) is an FXR agonist being developed for treating various chronic liver diseases. Previous studies reported inconsistent effects of OCA on regulating plasma cholesterol levels in different animal models and in different patient populations. The mechanisms underlying its divergent effects yet have not been thoroughly investigated. The scavenger receptor class B type I (SR-BI) is an FXR modulated gene and the major receptor for HDL-C. We recently showed that OCA treatment effectively lowered plasma HDL-C levels and increased hepatic SR-BI expression in hypercholesterolemic hamsters but not in normolipidemic hamsters, suggesting that hepatic cholesterol might play a role in OCA-induced SR-BI transcription. In this current study, by conducting genomic sequence analysis, reporter assays and direct DNA binding assays, we have identified a highly conserved regulatory region in the first intron of hamster SR-BI gene that contains a functional FXRE motif and an LXRE site separated by 57 base pairs. Promoter reporter activity assays demonstrated the functional involvement of this critical regulatory region in SR-BI gene transcription upon activations of FXR and LXR in a synergistic fashion. In vivo studies of normolipidemic hamsters showed that hepatic SR-BI gene expression is not affected by separate treatment of OCA or LXR agonist GW3965 but the co-activation of FXR and LXR by the combined treatment of OCA and GW3965 results in significant increases in hepatic SR-BI mRNA and protein levels. Taken together, we have discovered an unprecedented cross-talk between FXR and LXR that leads to a synergistic activation of SR-BI gene transcription in liver tissue and consequently affected HDL-C metabolism. Our novel findings shed new light for a better understanding of lipid regulatory effects of OCA in hyperlipidemic patients.

 

Charles Chalfant received his PhD from the University of South Florida, College of Medicine and was an NRSA Postdoctoral fellow at both Duke Medical Center and The Medical University of South Carolina under Dr Yusuf Hannun. He is currently a GS15 Research Career Scientist with the Richmond Veterans Administration Medical Center. He is also a Tenured Professor and Vice Chair of the Department of Biochemistry and Molecular Biology at Virginia Commonwealth University, School of Medicine. He currently holds the Paul M Corman, MD Endowed Chair in Cancer Research for the VCU Massey Cancer Center and has published more than 100 peer-reviewed manuscripts. The Chalfant laboratory has more than 20 years of experience in Lipid Biology, Cell Signaling, and RNA Splicing.

New roles for sphingolipids such as ceramide, ceramide-1-phosphate (C1P), and sphingosine-1-phosphate continue to emerge. My research, for example, has implicated C1P as a major regulator of eicosanoid synthesis, and despite the importance of eicosanoids in the inflammatory process, the regulation of eicosanoid synthesis proximal to the activation of Group IVA phospholipase A₂ (cPLA₂α) is still an enigma. In this regard, my laboratory demonstrated that C1P is a direct and required lipid co-factor for cPLA₂α activation in cellular models. In further studies, one interaction site for C1P was localized to the calcium-lipid binding domain (C2 domain) of the enzyme allowing for the genetic ablation of the site in vivo via the generation of a cPLA₂α knock-in (KI) mouse. In this lecture, the characterization of this new mouse model in comparison to the full genetic ablation of the enzyme will be presented. Specifically, the loss of the C1P/cPLA₂α interaction induced a class-switch in the production specific eicosanoids and specialized lipid mediators driving accelerated wound repair and regeneration, both in acute and chronic murine models. Cellular studies demonstrated that loss of this lipid, protein interaction led to enhanced dermal fibroblast and neutrophil migration, which was mimicked in vivo. In further mechanistic studies, C1P was found to modulate the substrate specificity of cPLA₂α in opposition to another lipid mediator of the enzyme, PIP2, explaining the class switch as to bioactive lipid mediators observed in the cPLA₂α KI mouse. Using lipidomic analyses, these specific lipid fingerprints were linked to human wound healing outcomes, which suggests that modulation of specific lipid mediators could be explored to promote wound healing and regeneration in a number of contexts.

Figure 1.The current mechanistic hypothesis, “shifting function hypothesis, to explain the loss of pro-inflammatory lipid mediators and concomitant increase in pro-resolution lipid mediators in response to loss of the C1P/cPLA₂α interaction.

Giuseppe Maulucci attended La Sapienza University in Rome, where he obtained his Bachelor’s degree in Biophysics. He gained his PhD degree at Roma Tre University and worked as Teacher and Researcher at the Institute of Physics and at the center of light and electron microscopy (LABCEMI) of the Catholic University of the Sacred Heart (UCSC). He is an expert in microscopy techniques (University of Florence, University of Genoa, Hebrew University of Jerusalem, University of Patras). His research activity is focused on Metabolic Imaging, a discipline that unites Molecular Biology and In vivo Imaging. It enables the visualization of endogenous molecules and supramolecular properties of major importance to maintain energy homeostasis in the cells, and provides a window to several important metabolic processes essential to cell survival.

Although numerous investigators have studied lipid droplets formation through several imaging techniques, they can’t draw conclusions regarding the overall process of lipid metabolism in live cells. Here, a method for studying lipid metabolism with high spatial and temporal resolution is presented, based on the measurement of intracellular polarity through the solvatochromic and lipophilic probe Nile red, which undergoes to a red shift upon a change of polarity of the neighboring lipids. A confocal spectral imaging approach captures in detail polarity variations by acquiring the fluorescence emission spectra at pixel resolution. The analysis of the spectra trough the technique of spectral phasors allowed semi-blind spectral unmixing of the contribution of different classes of lipids in the image, namely hyper polar, polar and non-polar lipids. The method allows a fine-tuned, real-time monitoring of fatty acid metabolism in live cells with submicrometric resolution.

Figure 1: Overview of fatty acid metabolism, and relative change of polarity of the Nile Red probe.

Chryssostomos Chatgilialoglu is a Research Director at the Italian National Research Council (CNR) in Bologna, Italy. He is also a Co-founder and President of the spin-off company Lipinutragen. His research interests lie in free radical reactivity applied to biosciences and biomarker discovery. He is an author of more than 250 publications in peer-reviewed journals, 34 book chapters and six patents and author or editor of several books, including Membrane Lipidomics for Personalized Health, Wiley 2015.

Lipid research in life sciences has renewed its attractiveness in the beginning of the 21st century linking chemical, analytical, and biological subjects, with health and nutrition, as well as with the increasing societal relevance of diseases related to inflammation and lipid metabolism. Fatty acid-based membrane lipidomics examines phospholipid components related to individual metabolic and nutritional status. Indeed, fatty acid-based nutrition and nutraceuticals are essential to life and occupy a leader positions in the healthcare market. Unsaturated fatty acids are involved in oxidative and free radical processes, which also naturally occur during metabolic functions and signaling activities. The transformations due to peroxidation processes have been considered for many decades to be by far the most crucial events to natural lipids. In the last decade, the cis to trans isomerization of the double bond, due to the reversible addition of sulfur-centered radicals, has attracted considerable attention. On the top of these transformations, fatty acids are affected by stress conditions that activate the remodeling by phospholipase enzymes (PLA2 response), starting the cascade of lipid signaling, so decisive for inflammatory and apoptotic consequences. Fatty acid-based membrane lipidomics represents a powerful diagnostic tool for assessing the quantity and quality of fatty acid constituents of the lipid pool in individuals, and also for the follow-up of the membrane fatty acid remodeling, that are associated with different physiological and pathological conditions. We contributed in this area of lipidomics studying the chemical and biological responses to various metabolic and environmental conditions of free radical and metabolic stress, modeling the effects of a liposome, cellular and animal models. Our analytical methodology is able to evidence positional and geometrical fatty acid isomers. For example: sapienic acid, a member of the hexadecenoic family, has been recognized as biomarker of obesity and; the reference membrane lipidomic cluster, formed by the cohort of ten fatty acids in phospholipids of red blood cell membranes, has been clearly connected to metabolic and nutritional status in healthy and diseased subjects.

Figure 1: Fatty acid-based membrane lipidomics (membrane profile) at the crossroad of personalized health.

Calvano Cosima Damiana is currently working as Assistant Professor at University of Bari in Department of Chemistry and she received the eligibility to become Associate Professor in Analytical Chemistry. She graduated in Chemistry at the University of Bari in 2003. In 2007, she accomplished her PhD studies in Microbiology, Health and Food Chemistry. In 2012, she received the Best Young Researcher Award from the Analytical Chemistry Division of the Italian Chemical Society (SCI). Her research interests focus on the development of new analytical protocols for biomolecules study, on the use of mass spectrometry in proteomics and lipidomics fields for applications in molecular science, food, clinical and cultural heritage. Her scientific activity is documented by 75 papers divided as peerto- peer reviewing publications in international journals, book chapters and conference proceedings. She is the author/co-author of more than 100 communications to national/international conferences.

Parkinson’s disease (PD) is a progressive neurodegenerative disease involving the nigrostriatal pathway; patients’ manifest motor symptoms dysfunction when more than 50% of neurons are lost. Though it is well recognized that alterations of lipid signaling, and metabolism plays a significant role in many human diseases, little is known about the role of lipids during this specific disease. Recently, it has been reported that altered lipid pathways in the primary visual cortex and the anterior cingulate are possible in neurological disorders such as PD by analyzing post-mortem tissues from patients in advanced neuronal degeneration stage. Such an approach, however, hinders the identification of the first neuronal changes. Thus, understanding the mechanisms of PD and recognizing neuronal changes in the early phase of PD represents a crucial task. According to their polygenic predisposition and environmental etiopathology skin fibroblasts are today widely recognized as a useful model of primary human cells, capable of reflecting the chronological and biological aging of the patients. A lipidomics study of easily accessible primary human fibroblasts is presented here based on hydrophilic interaction liquid chromatography coupled to electrospray ionization-Fourier transform mass spectrometry, using both positive and negative polarities. Phospholipids (PL) from dermal fibroblasts of two unrelated PD patients with different parkin mutations and two controls were characterized by recurring to single and tandem MS measurements on a hybrid quadrupole-Orbitrap mass spectrometer. This untargeted approach enabled the identification of various PL classes as phosphatidylethanolamines (PE), phosphatidylcholines (PC), sphingomyelins, lysoPC, lysoPE, phosphatidylinositols, phosphatidylserines, mono-, di- and tri-hexosylceramides  and ganglioside GM1, GM2 and GM3. To identify the main lipids involved in the pathological condition of PD, lipidomics data on a higher number of samples need to be collected and processed by multivariate statistical analyses. In this communication, an interesting set of preliminary findings will be reported and discussed.

 

Figure 1. HILIC-ESI (+) MS total ion current chromatogram of a lipid extract from human fibroblasts.

Elena A Goun has been appointed tenure track Assistant Professor of Bio- Organic Chemistry at the School of Basic Sciences (FSB). She received her MS degree from University of Central Florida under supervision of D Howard Miles in the field of Medicinal Chemistry of Natural Products. She then continued her PhD studies in the field of Medicinal Chemistry and Drug Delivery in the group of Professor Paul Wender at Stanford University. After graduation with a PhD degree in 2008, she did her Postdoctoral studies in the field of Chemical Biology at the group of Carolyn Bertozzi University of California at Berkeley. She is an Advocate of an Interdisciplinary Approach, Combining Synthetic Chemistry, Optical Imaging, and an understanding of cellular functions at molecular level to find solutions to fundamental problems in biology and medicine. She has developed several new non-invasive imaging techniques that allow studies of molecular signatures of cancer and metabolic diseases. She will perform her research work in the context of the new Chair in Biological Chemistry, at EPFL’s Institute of Chemical Sciences and Engineering.

Triglycerides (TG) are the main form of fat in the human diet, but increased consumption of TG may lead to the development of obesity and diseases such as type 2 diabetes, cardiac lipotoxicity, hepatic steatosis and cancer. Despite the important role of TG in human health and nutrition the role of this important metabolite still remains elusive due to the lack of real-time noninvasive imaging tools. To address this unmet need we developed a novel optical probe that is based on sensitive bioluminescent readout. This reagent allows quantification and imaging of TG uptake in live cells and living mice non- invasively in real time. Using this new reagent, we have investigated the role of  gut microbiota on the rate/amount of absorption of TG in live mice.

 

Sergey Korolev has his expertise in Protein Crystallography and Biochemistry. He is an Associate Professor of Biochemistry and Molecular Biology at Saint Louis University School of Medicine. He has published structural and functional studies of medically relevant proteins including ubiquitination systems, DNA recombination and repair proteins and enzymes. His current projects in lab include structure-function studies of tumor suppressors and proteins involved in neurodegeneration.

Statement of the Problem: Calcium-independent phospholipase PLA₂G6A (also known as iPLA₂ β or PNPLA9) is a signaling enzyme which hydrolyzes phospholipids to generate potent lipid second messengers in response to stress or injury1,2. The enzyme is a product of the PARK14 gene with strong genetic link to a spectrum of neurodegenerative disorders including Parkinson’s disease (PD)3,4,5. It is also linked to idiopathic PD and represents one of the major phospholipase activities in the brain. Alterations in iPLA₂β function have demonstrated its role in other human pathologies including cardiovascular disease, cancer and diabetes. Correspondingly, novel inhibitors of PLA₂G6A have been sought for therapeutic applications. Mechanisms of its activation and tissue-specific functions remain poorly understood. This contrasts with known enzymatic activity and several well-characterized downstream signaling cascades implicated in agonist-induced arachidonic acid release, insulin secretion, vascular constriction/relaxation, store-operated calcium-entry, cellular proliferation, migration and autophagy.

Methodology & Theoretical Orientation: We have solved a crystal structure of the full-length mammalian PLA₂G6A and investigated mechanisms of the protein activity and interaction with calmodulin.

Findings: The first crystal structure of PLA₂G6A significantly revises existing mechanistic models6. It demonstrated unexpected oligomeric structure and the conformation of catalytic and auxiliary protein-interaction domains. The structure suggests the mechanisms of inhibition by calmodulin, activation through the autoacylation reaction and the potential role of ATP in stabilizing ankyrin repeats.

Conclusion & Significance: The novel crystal structure together with biochemical studies has immediate implications for the mechanisms of the phospholipase activity, of the inhibition and activation as well as of the potential mechanism of tissue specific cellular localization. It provides a well-defined framework to investigate the role of neurodegenerative mutations and the function of PLA₂G6A in the brain as well as its role in other diseases.

Figure 1: The mechanism of PLA₂G6 activation and interaction with membrane and membrane proteins.